Post on 02-Jun-2022
Aus dem Institut fuumlr Tierzucht und Vererbungsforschung der
Tieraumlrztlichen Hochschule Hannover
Molecular genetic analysis of canine congenital sensorineural deafness
in Dalmatian dogs
INAUGURAL-DISSERTATION
zur Erlangung des Grades einer
DOKTORIN DER VETERINAumlRMEDIZIN
(Dr med vet)
durch die Tieraumlrztliche Hochschule Hannover
Vorgelegt von
Katharina Mieskes aus Goumlttingen
Hannover 2006
Scientific supervisor Univ-Prof Dr Dr O Distl
Examiner Univ-Prof Dr Dr O Distl
Co-examiner Univ-Prof Dr H Y Naim
Oral examination 18 Mai 2006
This work was supported by a grant from the Gesellschaft zur Foumlrderung
Kynologischer Forschung (GKF) eV Bonn Germany
To my family
Parts of this work have been submitted for publication in the following journals
1 Gene
2 Journal of Heredity
3 Animal Genetics
Contents
1 Introduction 1
2 A comparative overview of the molecular genetics of non-syndromic deafness in dogs and humans 5
Abstract 7
The structure of the ear 7
Deafness in man 8
Deafness in dogs 9
The canine genome project 11
3 Linkage analysis of gene-associated microsatellite markers with congenital sensorineural deafness in Dalmatian dogs 23
Abstract 25
Introduction 25
Material and methods 26
Results and discussion 28
4 Evaluation of eight candidate genes for canine congenital sensorineural deafness in Dalmatian dogs 41
Abstract 43
Introduction 43
Material and methods 44
Results and discussion 46
5 Molecular characterization of the canine myosin heavy polypeptide 9 non-muscle (MYH9) gene on dog chromosome 10q232 63
Abstract 65
Introduction 65
Materials and Methods 67
Results and Discussion 69
6 Identification of a 5 Mb region on canine chromosome 10 harbouring a causative gene responsible for congenital sensorineural deafness in German Dalmatian dogs 81
Abstract 83
Introduction 83
Material and methods 84
Results 86
Discussion 87
7 Analysis of canine chromosome 1 and the Gap junction protein alpha 1 (GJA1) gene in Dalmatian dogs segregating for congenital sensorineural deafness 97
Abstract 99
Introduction 99
Material and methods 100
Results and discussion 102
8 Analysis of canine chromosome 31 and the claudin-14 (CLDN14) gene in Dalmatian dogs segregating for congenital sensorineural deafness 107
Abstract 109
Introduction 109
Material and methods 110
Results and discussion 112
9 General Discussion 117
The candidate gene approach 119
Linkage and association analysis 120
CFA1 122
CFA31 123
CFA10 123
10 Summary 125
11 Erweiterte Zusammenfassung 129
12 References 145
13 Appendix I 14 List of publications XIII
Abbreviations
List of abbreviations
A adenine
Acc No accession number
ACTG1 actin gamma 1
AEP akustisch evozierte Potentiale (acoustically evoked potentials)
APS ammonium persulphate
AT annealing temperature
BAC bacterial artificial chromosome
BAER brain stem auditory evoked response
BLAST Basic Local Alignment Search Tool
bp base pair
C cytosine
CCSD canine congenital sensorineural deafness
CDH23 cadherin related 23
cDNA copy desoxyribonucleic acid
CFA chromosome of Canis familiaris
CLDN14 claudin-14
cM centiMorgan
COCH coagulation factor C homolog cochlin
COL11A2 collagen type XI alpha 2
CRYM crystallin mu
CSD cochleosaccular degeneration
CX connexin
DFN x-linked deafness locus
DFNA autosomal dominant deafness locus
DFNA5 deafness autosomal dominant 5
DFNB autosomal recessive deafness locus
DIAPH1 diaphanous homolog 1 (Drosophila)
DMSO dimethyl sulfoxide
Abbreviations
DNA deoxyribonucleic acid
dNTPs deoxy nucleoside 5rsquotriphosphates (N is ACG or T)
EDN3 endothelin 3
EDNRB endothelin receptor type B
EDTA ethylenediamine tetraaceticacid
EMBL European Molecular Biology Laboratory
ESPN espin
EST expressed sequence tag
EYA4 eyes absent homolog 4 (Drosophila)
F forward
FISH fluorescence in situ hybridisation
G guanine
GJA1 gap junction protein alpha 1 43kD (connexin 43)
GJB2 gap junction protein beta 2 26k (connexin 26)
GJB3 gap junction protein beta 3 31kDa (connexin 31)
GJB6 gap junction protein beta 6 (connexin 30)
GKF Gesellschaft zur Foumlrderung Kynologischer Forschung (Society for the
Advancement of Cynological Research)
HET observed heterozygocity
HE expected heterozygosity value
HSA chromosome of Homo sapiens
IBD identical by descent
IRD infrared dye
KCNQ4 potassium voltage-gated channel KQT-like subfamily member 4
Kb kilobase
LD linkage disequilibrium
LINE long interspersed nuclear element
LOD logarithm of the odds
M molar
Mb megabase
Merlin multipoint engine for rapid likelihood inference
MITF microphthalmia-associated transcription factor
Abbreviations
MS microsatellite
MTRNR1 mitochondrially encoded 12S RNA
MTTS1 mitochondrially encoded tRNA serine 1 (UCN)
MYH9 myosin heavy polypeptide 9 non-muscle
MYH14 myosin heavy polypeptide 14
MYO1A myosin IA
MYO3A myosin IIIA
MYO6 myosin VI
MYO7A myosin VIIA
MYO15A myosin XVA
NCBI National Center for Biotechnology Information
NMMHC-A nonmuscle myosin heavy chain-A
ODDD oculodentodigital dysplasia
OMIM Online Mendelian Inheritance in Man
OTOA Otoancorin
OTOF otoferlin
P error probability
PAX3 paired box gene 3 (Waardenburg syndrome 1)
PCDH15 Protocadherin-15
PCR polymerase chain reaction
PIC polymorphism information content
POU3F4 POU domain class 3 transcription factor 4
POU4F3 POU domain class 4 transcription factor 3
PRES solute carrier family 26 member 5 (prestin)
QTL quantitative trait locus
R reverse
RACE rapid amplification of cDNA ends
RH radiation-hybrid
RLM RNA ligase-mediated
RNA ribonucleic acid
RT-PCR reverse transcription polymerase chain reaction
SAS Statistical Analysis System
Abbreviations
SH1 Src homology 1
SINE short interspersed nuclear element
SLC26A4 solute carrier family 26 member 4 (pendrin)
SLC26A5 solute carrier family 26 member 5 (prestin)
SNP single nucleotide polymorphism
SOX10 SRY (sex determining region Y)-box 10
STRC stereocilin
STS sequence-tagged site
T thymine
TBE tris-borate-ethylenediamine tetraacetic acid
TECTA tectorin alpha
TEMED NNNrsquoNrsquo-tetramethylenediamine
TFCP2L3 transcription factor CP2-like 3
TJ tight juncions
TMC1 transmembrane channel-like gene 1
TMIE transmembran inner ear gene
TMPRSS3 transmembrane protease serine 3
U unit
USH1C Usher syndrome 1C
UTR untranslated region
WFS1 Wolfram syndrome 1 (wolframin)
wgs whole genome shotgun
WHRN whirlin
WS Waardenburg syndrome
Chapter 1
Introduction
Introduction 3
Introduction
Canine congenital sensorineural deafness (CCSD) has often been reported in the
literature and occurs in more than 54 different breeds of dogs with the Dalmatian dog
showing the highest incidence The inheritance and segregation of a major gene in
CCSD has been demonstrated in different Dalmatian dog populations But although
several studies have demonstrated the mode of inheritance in Dalmatian dogs no
universally accepted mode of inheritance for the other dog breeds affected by CCSD
has yet been identified
Breeding with blue-eyed Dalmatian dogs and unilaterally or bilaterally deaf dams and
sires is forbidden by paragraph 11b of the German animal welfare laws and thus the
hearing status of all Dalmatian dogs has to be tested as a puppy at about six to eight
weeks old using the brain stem auditory evoked response (BAER) test As deaf dogs
are very difficult to raise and often become aggressive and snappish from fear most
puppies suffering from bilateral hearing loss are euthanized However it has been
shown in recent years that auditory testing does not seem to be an effective way of
clearly reducing the high incidence of deafness in this breed Thus prevention of
CCSD cannot be achieved alone by exclusion of affected animals from breeding
Consequently a molecular genetic approach toward unravelling the responsible
genes in carriers is urgently needed
Many genetic disorders in humans and domestic dogs (Canis familiaris) demonstrate
a high level of clinical and molecular similarity Therefore the mutated genes in
human hereditary deafness seemed to be appropriate candidates for canine
congenital sensorineural deafness
The objective of the present study is to localize the gene that is involved in the
development of CCSD in Dalmatian dogs In order to achieve this goal successively
32 canidate genes were evaluated by means of linkage analyses using microsatellite
markers and single nucleotide polymorphisms (SNPs) This candidate gene
approach using gene-associated markers for linkage studies in families segregating
for deafness turned out to be little effective Therefore the canine chromosomes
(CFA) 1 CFA10 and CFA31 were scanned entirely with microsatellite markers
Additionally single nucleotide polymorphisms (SNPs) were developed for fine
mapping the identified CCSD regions
Introduction 4
Overview of chapter contents Chapter 2 reviews the identified 39 mutated genes causing non-syndromic hereditary
hearing impairment in humans Parallels and differences in canine and human
deafness are shown including the clinical signs inheritance patterns and
histopathology We located the humane deafness genes in the canine genome and
discussed the advantages of comparative genomics and different molecular genetic
approaches
In Chapter 3 an existing set of 43 microsatellite markers for in total 24 candidate
genes were used for a non-parametric linkage analysis with congenital sensorineural
deafness (CCSD) in Dalmatian dog families segregating for deafness
In Chapter 4 newly developed SNP markers associated with in total eight candidate
genes were evaluated for CCSD in Dalmatian dogs
In Chapter 5 the molecular characterization of the canine myosin heavy polypeptide
9 non-muscle (MYH9) gene on dog chromosome 10q232 is described
Chapter 6 7 and 8 present linkage analyses performed in Dalmatian dog families
segregating for congenital sensorineural deafness using microsatellite markers on
canine chromosome (CFA) 1 CFA10 and CFA31 and the results of fine mapping
regions linked with the CCSD phenotype using newly developed SNPs
Chapter 9 provides a general discussion and conclusions referring to Chapters 1-8
Chapter 10 is a concise English summary of this thesis while Chapter 11 is an
expanded detailed German summary which takes into consideration the overall
research context
Chapter 2
A comparative overview of the molecular genetics of non-syndromic deafness
in dogs and humans
Non-syndromic deafness in dogs and humans 7
A comparative overview of the molecular genetics of non-syndromic deafness in dogs and humans
Abstract
In man as in different dog breeds deafness is an often diagnosed disorder with the
Dalmatian dog showing the highest incidence Deafness in Dalmatian dogs is clearly
heritable and the presence of a recessive major gene affecting the disorder was
shown in several Dalmatian dog populations
This Chapter provides an overview of the identified 39 mutated genes causing
human non-syndromic hereditary hearing impairment as well as of the five genes
responsible for Waardenburg syndrome in humans We point out their cytogenetic
and genomic localisations in man and dog and compare the genomic and mRNA
sequences of these genes between man and dog Moreover an overview is given on
deafness genes-associated markers identified in Dalmatian dogs and on candidate
genes characterized in dogs
The structure of the ear The inner ear consists of the semicircular canals the vestibule and the cochlea
whereas the vestibule and the semicircular canals are concerned with vestibular
function (balance) and the cocnlea is concerned with hearing Reissneracutes membrane
and the basilar membrane divide the cochlea longitudinally into three scalae the
scala vestibule the scala media and the scala tympani The process of transduction
occurs in the structures within scala media sitting on the basilar membrane and
comprising the organ of Corti Cutting the cochlea tube cross sectionally the scala
media is more or less triangular formed by Reissneracutes membrane basilar
membrane and a structure called the stria vascularis The fluid that fills scala
tympani and scala vestibule is called perilymph the fluid that fills scala media is
called endolymph The organ of Corti rests on the basilar membrane within scala
media The cochlea contains an array of highly specialized cells arranged in a highly
Non-syndromic deafness in dogs and humans 8
specialized manner Two types of cells in the organ of Corti are support cells and
hair cells The hair cells are the receptor cells that trancsduce sound
When a sound wave brings physical displacement of the membranes separating the
perilymph from the endolymph they cause the organ of Corti to move and the hair
cells on it are scraped along the bottom of the tectorial membrane The tectorial
membrane is firmly anchored to the bone Relative movement of the organ of Corti
and its hair cells with respect to the tectorial membrane is the source of the
deformation of the hair cells microvilli The hair cells are so constructed that any
deformation of their microvilli will cause a change in the overall membrane potential
of the cell This signal is detected by the fibers from the cells in the spiral ganglion
These fibers are neural elements and they carry their own depolarization wave into
the auditory region of the brain
Deafness in man There are various ways to categorise deafness The two main types of deafness are
classified based on which portions of the auditory system are affected conductive
hearing loss occurs when when sound is not conducted efficiently through the outer
andor middle part of the ear Much more common is the sensorineural hearing loss
Sensorineural hearing loss occurs when there is damage to the inner ear (cochlea)
or to the nerve pathways from the inner ear (retrocochlear) to the brain Most cases
of sensorineural hearing loss are due to cochlear defects (Petit et al 2001)
Hearing loss can be present at birth (congenital) or become evident later in life
(acquired) Congenital deafness similarly may or may not be genetic In fact more
than half of congenital hearing loss is inherited Alternatively congenital deafness
may be due to a condition or infection to which the mother was exposed during
pregnancy Furthermore congenital hereditary deafness may occur as part of a
multisystem disease (syndromic) or as a disorder restricted to the ear and vestibular
system (non-syndromic) As non-syndromic hereditary hearing impairment is almost
exclusively caused by cochlear defects affected patients suffer from sensorineural
hearing loss In Table 1 and 2 the genes underlying human hereditary non-
syndromic deafness as a result of cochlear defects in consequence of primary
defects in hair cells non-sensory cells and the tectorial membrane or unknown cell
type are shown Non-syndromic deafness is estimated to cause 60-70 of cases of
congenital hereditary deafness in man (Morton 1991) In 70-80 of all cases this
Non-syndromic deafness in dogs and humans 9
non-syndromic form of deafness shows an autosomal recessive inheritance followed
by an autosomal dominant inheritance in 10-20 of all cases and 1-2 of all cases
are X-linked A maternally inherited form may also occur (Van Camp and Smith
2003)
Non-syndromic forms of hereditary deafness are classified by their mode of
inheritance DFN DFNA and DFNB refer to deafness forms inherited on the X
chromosome-linked autosomal dominant and autosomal recessive modes of
transmission respectively
Human hereditary isolated hearing loss is genetically heterogeneous (Petit et al
2001) Up to 1 of the human genes are estimated to be necessary for hearing
(Friedmann and Griffith 2003) Today approximately 120 genes for human
hereditary deafness have been identified approximately 80 for syndromic and 39 for
non-syndromic hereditary deafness which is suspected to be one-third of the total
(Nance 2003) In Table 1 the identified 39 mutated genes causing non-syndromic
hereditary hearing impairment in humans are shown Out of the 39 genes 15 genes
cause autosomal recessive and 15 genes cause autosomal dominant forms six
genes are involved in both recessive and dominant forms one gene causes X-linked
and two a maternally inherited form (The Hereditary Hearing Loss Hompage
httpwebhostuaacbehhh)
Furthermore several hundred forms of syndromes with hearing loss have been
documented in humans (Van Camp and Smith 2003) One is the human
Waardenburg syndrome (WS) which manifests itself with sensorineural deafness
and pigmentation defects in the iris hair and skin The WS is classified into four
types depending on the presence or absence of additional symptoms which are
caused by mutations in the five genes EDN3 EDNRB MITF PAX3 and SOX10
respectively These genes are shown in Table 3 They are known to be expressed in
the neural crest (EDN3 EDNRB PAX3 SOX10 ) or directly in the melanocytes
(MITF) and are inter alia involved in migration differentiation or survival of
melanocytes respectively (Bondurand et al 2000)
Deafness in dogs Congenital sensorineural deafness (CSD) has been reported in a variety of mammal
species other than humans ranging from mice to dogs guinea pigs and mink
Canine congenital deafness has often been reported in the literature and occurs in
Non-syndromic deafness in dogs and humans 10
more than 54 different breeds of dogs according to Strain (1996 and 2004) The
breeds with the highest incidence include Dalmatian dogs Bull Terrier English
Cocker Spaniel English Setter Australian Cattle Dog Australian Shepherd West-
Highland-White-Terrier Dobermann and Dogo Argentino The incidence of canine
congenital deafness is highest in Dalmatian dogs of which 165 to 30 exhibit
unilateral or bilateral hearing loss (Famula et al 1996 Wood and Lakhani 1997
Muhle et al 2002 Juraschko et al 2003a Rak and Distl 2005) The inheritance
and segregation of a major gene in canine congenital sensorineural deafness
(CCSD) has been demonstrated in different Dalmatian dog populations (Famula et
al 2000 Muhle et al 2002 Juraschko et al 2003b) But although several studies
have demonstrated the mode of inheritance in Dalmatian dogs no universally
accepted mode of inheritance for the other dog breeds affected by CCSD has yet
been identified
Congenital sensorineural hearing impairment can be recognised in dogs at four to
eight weeks of age (Strain 1996) while histological studies of deaf Dalmatian dogs
have shown that the degeneration of the inner ear structures begins as early as one
day after birth and is histologically clearly evident by four weeks of age (Johnsson et
al 1973) In 70 of the cases with human hereditary deafness the histological
pattern is known as cochleo-saccular degeneration (CSD) (Lalwani et al 1997)
commonly known as Scheibe dysplasia with preservation of the pars superior of the
membranous labyrinth and an unremarkable bony labyrinth As in man also in many
affected dog breeds the histological pattern of congenital sensorineural deafness is
known as cochleo-saccular degeneration
Breeding with blue-eyed Dalmatian dogs and unilaterally or bilaterally deaf dams and
sires is forbidden by paragraph 11b of the German animal welfare laws and thus the
hearing status of all Dalmatian dogs has to be tested as a puppy at about six to eight
weeks old using the brainstem auditory evoked response (BAER) test that detects
electrical activity in the cochlea and auditory pathways in the brain Although the
BAER test is a reliable method for identifying unilaterally and bilaterally deaf dogs it
does not seem to be an effective way of clearly reducing the incidence of deafness in
affected breeds particularly in a recessive mode of inheritance so that hearing dogs
can still be genetic carriers Furthermore deaf dogs are very difficult to raise and
often become aggressive and snappish from fear consequently most puppies
Non-syndromic deafness in dogs and humans 11
suffering from bilateral hearing loss are euthanized Thus prevention of CCSD
cannot be achieved alone by exclusion of affected animals from breeding and
consequently a molecular genetic approach toward unravelling the responsible
genes in carriers is urgently needed
Over the past decade it has become increasingly clear how far structural and
functional homologies at the gene level extend across even distantly related species
Much is known about deafness-causing gene mutations in humans and mice
including the fact that the clinical and histopathological findings are often very similar
to those of deafness in Dalmatian dogs Thus despite the genetical heterogeneity of
human non-syndromic deafness the genes that are responsible for non-syndromic
congenital hereditary deafness in humans (Table 1) seemed to be appropriate
candidate genes for CCSD especially in Dalmatian dogs (Rak and Distl 2005) The
genes that are mutated in the human WS (Table 2) were selected as candidates
because the WS phenotype where the deafness is associated with pigmentation
defects seems to be similar to the phenotype of most affected dog breeds (Strain
and Tedford 1996) Both Juraschko et al (2003a) and Strain et al (1992) have
demonstrated that patched Dalmatians are less likely to be deaf than unpatched
animals and blue-eyed Dalmatians are more likely to be affected from hearing
impairment than brown-eyed animals
In an attempt to achieve the aim of molecular genetic diagnosis of CCSD carriers
Rak et al (2003) and co-workers (Droumlgemuumlller et al 2002 Kuiper et al 2002 Rak
et al 2002a 2002b 2003) already mapped 24 potential candidate genes for
sensorineural deafness in dogs by fluorescence in situ hybridization and a radiation
hybrid panel to 16 different canine chromosomes
The canine genome project In December 2005 an international research team led by scientists at the Broad
Institute of MIT and Harvard achieved the completion of a high-quality genome
sequence of the domestic dog together with a catalog of 25 million specific genetic
differences across several dog breeds (Lindblad-Toh et al 2005) The authors found
that humans share more of their ancestral DNA with dogs than with mice confirming
the utility of dog genetics for understanding human disease Furthermore the
physiology disease presentation and clinical response of dogs often mimic human
Non-syndromic deafness in dogs and humans 12
diseases closely As indicated above hearing impairment seemed to be no
exception
In the last years most projects have exploited canine traits for which either direct
candidate genes could be proposed and evaluated or for which large informative
pedigrees were available to enable linkage mapping to identify candidate regions A
major component of such research efforts comprised the cloning sequencing and
mapping of individual canine homologs of genes either proposed as candidate
genes or expected to be located in candidate regions This was necessary to
identify new informative polymorphisms (eg single nucleotide polymorphisms
(SNPs) microsatellites) for high resolution mapping of candidate regions and to
examine each exon and exonintron boundary for positional candidates Availability
of the second version of the dog genome assembly (build 21) of the NCBI database
shortcut this effort and increase the investigators efficency
The current RH map with 3200 markers provides a good estimate of the order and
physical spacing (ie in base pairs) of markers along canine chromosomes (Guyon
et al 2003) and was recently complemented by the construction of a 4249-marker
integrated canine genome RH map that consists of 900 genes 1589 microsatellites
and 1760 BAC end markers (Breen et al 2004) all included and available in the
NCBI database The second version 1 of the NCBIs genome annotation consists of
large contigs covering all canine chromosomes given with their located markers and
genes The great majority of genes are derived by automated computational analysis
using the gene prediction method GNOMON
With this help either additional candidate genes for canine CSD can be found directly
by its gene symbol in the 21 of the NCBIs genome annotation or if a candidate gene
is yet not annotated a BLAST (Basic Local Alignment Search Tool) search versus
the canine whole genome shotgun (wgs) sequence resource can be used to obtain
the sequence of the canine genomic contigs containing the human homologous
gene The localisation of all 39 known human non-syndromic hereditary deafness
genes in the canine genome with the corresponding accession numbers of the contig
and if available the accession number of the genomic sequence and mRNA of the
canine gene are shown in Tables 4 and 5 Furthermore the identity of canine and
human or mouse mRNA is shown in Table 5 The average identity of canine and
human mRNA is with 088 percent higher than the average identity of canine and
mouse mRNA with 084 percent Canine sequences that correspond to the human
Non-syndromic deafness in dogs and humans 13
candidate gene can now be used to find microsatellite or SNP markers associated to
the respective canine gene These markers can be used for linkage and haplotype
studies in dog families segregating for deafness
Table 7 shows the microsatellite and SNP markers developed for in total 32
candidate genes for CCSD
The candidate genes for which a set of in total 43 microsatellite marker were
designed by Rak (2003) included the following 24 genes CDH23 CLDN14 COCH
COL11A2 DFNA5 DIAPH1 EDN3 EDNRB EYA4 GJA1 GJB2 GJB6 MITF
MYH9 MYO6 MYO7A MYO15A OTOF PAX3 POU4F3 SLC26A4 SOX10
TECTA and TMPRSS3 This existing set of 43 microsatellite markers for 24
candidate genes were used for linkage and haplotype studies in Dalmatian dog
families segregating for deafness (Chapter 3) These 24 genes are known to be
involved either in human non-syndromic deafness or in the human Waardenburg
syndrome For another eight candidate genes including TMC1 TMIE USH1C
MYH14 MYO3A PRES WHRN and ESPN SNP markers were newly developed
(Chapter 4) and subsequently used for linkage and association analyses in
Dalmatian dog families segregating for deafness These genes are also involved in
human non-syndromic deafness
Non-syndromic deafness in dogs and humans 14
Tabele 1 Genes responsible for non-syndromic congenital hereditary deafness in
humans
Inheritance Gene Gene product Type of molecule Locus namea
ACTG1 γ-Actin cytoskeletal protein DFNA20 DFNA26
COCH Cochlin extracellular matrix component DFNA9
COL11A2 Collagen XI (α2-chain) extracellular matrix component DFNA13
CRYM micro-Cristallin thyroid hormone-binding protein DFNAb DFNA5 Unidentified Unidentified DFNA5 DIAPH1 Diaphanous-1 cytoskeleton regulatory protein DFNA1 EYA4 EYA4 transcriptional coactivator DFNA10 GJB3 Connexin-31 gap junction protein DFNA2 KCNQ4 KCNQ4 K+ channel subunit DFNA2 MYH14 Myosin IIC motor protein DFNA4 MYH9 Myosin IIA motor protein DFNA17 MYO1A Myosin IA motor protein DFNA48 POU4F3 POU4F3 transcription factor DFNA15 TFCP2L3 TFCP2L3 transcription factor DFNA28
Autosomal dominant
WFS1 Wolframin endoplasmic-reticulum membrane protein DFNA6 DFNA14
CDH23 Cadherin-23 cell-adhesion protein DFNB12 CLDN14 Claudin-14 tight-junction protein DFNB29 ESPN Espin actin-bundling protein DFNB36 DFNAb MYO15 Myosin XV motor protein DFNB3 MYO3A Myosin IIIA motor protein DFNB30 OTOA Otoancorin cell-surface protein DFNB22 OTOF Otoferlin putative vesicle traffic protein DFNB9 PCDH15 Protocadherin-15 cell-adhesion protein DFNB23 SLC26A4 Pendrin IminusndashClminus transporter DFNB4 SLC26A5 Prestin anion transporter DFNB61 STRC Stereocilin DFNB16
TMIE TMIE transmembrane domain- containing protein DFNB6
TMPRSS3 TMPRSS3 transmembrane serine protease DFNB8 DFNB10 USH1C Harmonin PDZ domain-containing protein DFNB18
Autosomal recessive
WHRN Whirlin PDZ domain-containing protein DFNB31 GJB2 Connexin-26 gap junction protein DFNB1 DFNA3 GJB6 Connexin-30 gap junction protein DFNB1 DFNA3 MYO6 Myosin VI motor protein DFNA22DFNB37MYO7A Myosin VIIA motor protein DFNB2DFNA11
TECTA α-Tectorin extracellular matrix component DFNA8 DFNA12DFNB21
Autosomal dominant and autosomal recessive
TMC1 TMC1 transmembrane channel-like protein
DFNB7 DFNB11DFNA36
X-linked POU3F4 POU3F4 transcription factor DFN3
MTRNR1 Mitochondrial 12S rRNA not defined
nomenclature Mitochondrial
MTTS1 Mitochondrial 12S rRNA not defined
nomenclature a Autosomal recessive loci are designated DFNB autosomal dominant loci DFNA
Non-syndromic deafness in dogs and humans 15
Table 2 Genes underlying hereditary non-syndromic deafness as a result of primary
defects in hair cells non-sensory cells and the tectorial membrane or unknown cell
type
Primary defect
Gene
Hair cells MYO7A MYO15 MYO6 MYO3A MYO1A ACTG1 USH1C
WHRN CDH23 PCDH15 TMIE STRC SLC26A5 ESPN
KCNQ4 TMC1 OTOF POU4F3
Non-sensory cells
GJB2 GJB6 GJB3 SLC26A4 CRYM OTOA CLDN14
COCH TMPRSS3 MYH9 MYH14 EYA4 POU3F4
Tectorial membrane
COL11A2 TECTA
Unknown
DIAPH1 DFNA5 WFS1 TFCP2L3 MTRNR1 MTTS1
Table 3 Genes involved in the human Waardenburg syndrome
Inheritance Gene Gene product Type of molecule Type
EDN3 endothelin 3 vasoconstricted peptide WS type IV4
EDNRBendothelin
receptor type B receptor protein WS type IV4
MITF
microphthalmia-
associated
transcription
factor
transcription factor WS type II2
PAX3 paired box 3 DNA-binding protein WS type I1and III 2
SOX10 SRY-box 10 transcription factor WS type IV4 1Type I Dystopia canthorum 2Type II No dystopia canthorum 3Klein-Waardenburg syndrome (type III) Type I and upper limb abnormalities 4Waardenburg-Shah syndrome (type IV) Type II and Hirschsprung disease
(autosomal recessive inheritance)
Non-syndromic deafness in dogs and humans 16
Leng
th o
f co
ntig
(bp)
7799
0652
3821
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7077
2986
7251
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5989
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8239
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326
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ine
gene
al
iase
s
none
none
LOC
4877
51
LOC
4906
40
LOC
4817
34
LOC
4798
18
none
LOC
6112
23
LOC
4896
31
EYA4
GJB
2
LOC
4824
86
Acc
No
hu
man
m
RN
A
NM
_001
614
NM
_022
124
NM
_144
492
NM
_004
086
NM
_080
680
NM
_001
888
NM
_005
219
NM
_004
403
NM
_031
475
NM
_172
105
NM
_004
004
NM
_024
009
Gen
e lo
catio
n on
H
SA
17
10
21
14
6 16
5 7 1 6 13
1
Tabl
e 4
Loca
lisat
ion
of h
uman
non
-syn
drom
ic h
ered
itary
dea
fnes
s ge
nes
in th
e ca
nine
gen
ome
the
cani
ne
gene
loc
alis
atio
n in
meg
abas
es (
Mb)
th
e ac
cess
ion
num
ber
(Acc
No
) of
the
who
le g
enom
e sh
otgu
n (W
GS
) con
tig c
onta
inin
g th
e hu
man
hom
olog
ous
gene
and
the
corr
espo
ndin
g E
-val
ue s
core
and
leng
th o
f th
e co
ntig
Hum
an
deaf
ness
ge
ne
AC
TG1
CD
H23
CLD
N14
CO
CH
CO
L11A
2
CR
YM
DIA
PH1
DFN
A5
ESPN
EYA
4
GJB
2
GJB
3
der
ived
from
the
NC
BIs
can
ine
geno
me
anno
tatio
n ve
rsio
n 2
1
Non-syndromic deafness in dogs and humans 17
Leng
th o
f co
ntig
6421
1953
5300
4996
5294
2087
2607
3285
1654
5469
1249
9463
7251
5492
5102
4781
5989
7527
5159
1990
1284
7264
7521
5785
Scor
e of
co
ntig
525
521
973
2256
348
567
1236
404
337
383
604
1715
E-va
lue
of
cont
ig
3 e-
146
1 e-
144
0 0
9 e-
93
2 e-
158
0
3 e-
109
3 e-
89
7 e-
103
2 e-
169
0
Acc
No
of
WG
S co
ntig
unkn
own
NW
_876
259
NW
_876
270
NW
_876
251
NW
_876
313
NW
_876
250
NW
_876
290
NW
_876
254
NW
_876
273
NW
_876
321
NW
_876
263
NW
_876
283
NW
_879
563
Mb
from
to
unkn
own
521
52
3
109
24
109
35
311
33
119
443
64
441
415
41
7
103
41
056
404
14
050
245
42
460
261
32
619
235
02
359
371
43
769
674
86
748
Gen
e lo
catio
n on
C
FA
25
15
1 10
5 10
2 12
21
6 17
26
X
Can
ine
gene
al
iase
s
none
LOC
4824
51
none
LOC
4812
80
LOC
4795
22
LOC
4744
10
LOC
4871
06
LOC
4818
84
LOC
4851
74
LOC
6086
55
LOC
6079
61
none
LOC
4919
88
Acc
No
hu
man
m
RN
A
NM
_006
783
NM
_004
700
NM
_024
729
NM
_002
473
NM
_016
239
NM
_005
379
NM
_017
433
XM
_376
516
NM
_000
260
NM
_144
672
NM
_194
248
NM
_033
056
NM
_000
307
Gen
e lo
catio
n on
H
SA
13
1 19
22
17
12
10
6 11
16
2 10
X
Tabl
e 4
(con
tinue
d)
Hum
an
deaf
ness
ge
ne
GJB
6
KC
NQ
4
MYH
14
MYH
9
MYO
15
MYO
1A
MYO
3A
MYO
6
MYO
7A
OTO
A
OTO
F
PCD
H15
POU
3F4
der
ived
from
the
NC
BIs
can
ine
geno
me
anno
tatio
n ve
rsio
n 2
1
Non-syndromic deafness in dogs and humans 18
Leng
th o
f co
ntig
4533
7677
2532
0482
2532
0482
4020
6070
3002
9677
2968
9717
5300
4996
3309
7591
3821
0901
5102
4781
1104
8438
6535
5756
Scor
e of
co
ntig
1404
283
283
1683
883
529
354
216
198
354
2238
689
E-va
lue
of
cont
ig
0
6 e-
73
2 E
-73
0 0
5 e-
147
2 e-
94
4 e-
53
9 e-
48
2 E
-94
0 0
Acc
No
of
WG
S co
ntig
NW
_876
292
NW
_876
265
NW
_876
265
NW
_876
294
NW
_876
312
NW
_876
255
NW
_876
270
NW
_876
272
NW
_876
295
NW
_876
273
NW
_876
256
NW
_876
253
Mb
from
to
436
14
361
158
61
592
197
91
982
134
31
345
158
81
595
622
63
7
880
88
822
450
54
505
390
33
905
432
44
328
414
94
151
716
47
172
Gen
e lo
catio
n on
C
FA
2 18
18
30
5 13
1 20
31
21
13
11
Can
ine
gene
al
iase
s
LOC
4872
00
LOC
4832
63
LOC
4832
74
LOC
4782
78
LOC
4893
57
LOC
4819
85
LOC
4841
68
LOC
6093
50
LO
C61
0987
LOC
6108
50
LOC
4821
13
LOC
6125
88
Acc
No
hu
man
m
RN
A
NM
_002
700
NM
_000
441
NM
_206
883
NM
_153
700
NM
_005
422
NM
_024
915
NM
_138
691
NM
_147
196
NM
_024
022
NM
_153
676
NM
_006
005
NM
_015
404
Gen
e lo
catio
n on
H
SA
5 7 7 15
11
8 9 3 21
11
4 9
Tabl
e 4
(con
tinue
d)
Hum
an
deaf
ness
ge
ne
POU
4F3
SLC
26A
4
SLC
26A
5
STR
C
TEC
TA
TFC
P2L3
TMC
1
TMIE
TMPR
SS3
USH
1C
WFS
1
WH
RN
der
ived
from
the
NC
BIs
can
ine
geno
me
anno
tatio
n ve
rsio
n 2
1
Non-syndromic deafness in dogs and humans 19
Leng
th o
f co
ntig
(bp)
4776
3139
5561
1003
2518
2130
3091
5115
5294
2087
Scor
e of
co
ntig
262
721
2927
967
1179
E-va
lue
of
cont
ig
8e-6
7
0 0 0 0
Acc
No
of
WG
S co
ntig
NW
_876
277
NW
_876
274
NW
_876
271
NW
_876
304
NW
_876
251
Mb
from
to
470
14
703
343
63
438
248
52
488
313
43
144
297
52
976
Gen
e lo
catio
n on
C
FA
24
22
20
37
10
Can
ine
gene
al
iase
s
ED
N3
ED
NR
B
MIT
F
PAX3
LOC
4812
58
Acc
No
hu
man
m
RN
A
NM
_207
032
NM
_000
115
NM
_198
159
NM
_181
457
NM
_006
941
Gen
e lo
catio
n on
H
SA
20
13
3 2 22
Tabl
e 5
Loca
lisat
ion
of g
enes
invo
lved
in th
e hu
man
Waa
rden
burg
syn
drom
e (W
S)
in th
e ca
nine
gen
ome
th
e ca
nine
gen
e lo
calis
atio
n in
meg
abas
es (
Mb)
th
e ac
cess
ion
num
ber
(Acc
No
) of
the
who
le g
enom
e sh
otgu
n (W
GS
) con
tig c
onta
inin
g th
e hu
man
hom
olog
ous
gene
and
the
corr
espo
ndin
g E
-val
ue s
core
and
le
ngth
of t
he c
ontig
Hum
an
deaf
ness
ge
ne
EDN
3
EDN
RB
MIT
F
PAX3
SOX1
0
der
ived
from
the
NC
BIs
can
ine
geno
me
anno
tatio
n ve
rsio
n 2
1
Non-syndromic deafness in dogs and humans 20
Table 6 Canine candidate genes for CCSD with their accession number (AccNo) of
the genomic sequence and mRNA and if available the percent identity of canine and
human or mouse mRNA Canine candidate gene
Acc No canine genomic sequence
Acc No canine mRNA (predicted)
Canine mRNA (bp)
Identity () of canine and human mRNA
Identity () of canine and mouse mRNA
ACTG1 none1 none unknown unknown unknown CDH23 none1 none unknown 8800 8690 CLDN14 NC_006613 XM_544876 714 8880 8460 COCH NC_006590 XM_547762 3050 8770 8170
COL11A2 NC_006594 XM_857285 5202 8970 8740 CRYM NC_006588 XM_845361 1279 8720 7890
DIAPH1 none1 none unknown 8630 unknown DFNA5 NC_006596 XM_848863 1512 8320 7680 ESPN NC_006587 XM_546751 2565 8930 8270 EYA4 NC_006583 XM_541108 3969 9240 8120 GJB2 NC_006607 XM_543177 2208 7860 6720 GJB3 NC_006597 XM_539603 1305 8550 8090 GJB6 none none unknown unknown unknown
KCNQ4 NC_006597 XM_539568 1836 9350 8760 MYH14 none1 none unknown 8770 8220 MYH9 NC_006592 XM_538401 6201 9130 8940
MYO15 NC_006587 XM_536660 10128 8500 7960 MYO1A NC_006592 XM_531642 3388 8600 8350 MYO3A NC_006584 XM_544234 5589 8510 8910 MYO6 NC_006594 XM_862465 3983 9250 8610
MYO7A NC_006603 XM_542292 6519 9190 8880 OTOA NC_006588 XM_845746 3558 8740 7990 OTOF NC_006599 XM_844665 5994 9020 8780
PCDH15 none1 none unknown 8210 7390 POU3F4 NC_006621 XM_549108 1086 9190 9060 POU4F3 NC_006584 XM_544328 1017 9380 9160 SLC26A4 NC_006600 XM_540382 2382 7970 8240 SLC26A5 NC_006600 XM_540393 2235 9250 8740
STRC NC_006612 XM_535452 5301 8980 8520 TECTA NC_006587 XM_546475 7136 9110 8510
TFCP2L3 NC_006595 XM_539106 2127 9030 8760 TMC1 NC_006583 XM_541284 2580 8860 8740 TMIE NC_006602 XM_846596 396 9110 8910
TMPRSS3 NC_006613 XM_848589 1542 8500 8340 USH1C NC_006603 XM_860072 1730 9010 8670 WFS1 NC_006595 XM_539234 2667 8510 8420 WHRN NC_006593 XM_850321 2817 8440 8110 EDN3 NC_006606 NM_001002942 1976 747 716
EDNRB NC_006604 NM_001010943 1329 889 827 MITF NC_006602 XM_850501 1590 934 862 PAX3 NC_006619 XM_545664 1474 869 861
SOX10 NC_006592 XM_538379 1987 926 900 derived from the NCBIs canine genome annotation version 21 1 the definite region in the whole genome sequence (WGS) contig was used to get the percent identity of mRNAs
Non-syndromic deafness in dogs and humans 21
Table 7 Microsatellite markers and single nucleotide polymorphisms (SNPs)
of canine candidate genes for canine congenital sensorineural deafness in
Dalmatian dogs
Canine candidate gene
Number of gene-associated
microsatellites
Number of gene-associated
SNPs
CDH23 2 0 CLDN14 3 8 COCH 2 0
COL11A2 2 0 DIAPH1 2 0 DFNA5 2 0 ESPN 0 5 EYA4 2 0 GJB2 3 0 GJB3 1 0 GJB6 1 0
MYH14 0 2 MYH9 2 22
MYO15 2 0 MYO3A 0 3 MYO6 1 0
MYO7A 3 0 OTOF 1 0 PAX3 1 0
POU4F3 1 0 SLC26A4 1 0 SLC26A5 0 2 TECTA 2 0 TMC1 1 1 TMIE 1 3
TMPRSS3 2 0 USH1C 0 2 WHRN 0 3
Chapter 3
Linkage analysis of gene-associated microsatellite markers with
congenital sensorineural deafness in Dalmatian dogs
Linkage analysis of gene-associated microsatellites 25
Linkage analysis of gene-associated microsatellite markers with congenital sensorineural deafness in Dalmatian dogs
Abstract Deafness is a disorder often diagnosed in different dog breeds In this Chapter an
existing set of 43 microsatellite markers associated with in total 24 candidate genes
for canine congenital sensorineural deafness (CCSD) were used for linkage and
haplotype analyses in a large Dalmatian dog population with frequent occurrence of
CCSD We found significant linkage for the genes GJA1 MYH9 and CLDN14 As
linkage was found for different candidate genes in different families the results of
these test statistics indicate that the inheritance of non-syndromic deafness in
Dalmatian dogs is heterogenic in origin
Introduction
Canine congenital sensorineural deafness (CCSD) has been reported to occur in
more than 54 different breeds of dogs (Strain 1996) As in man also in dog breeds
the most commonly observed histological pattern of degenerative inner ear changes
is known as the cochleo-saccular or Scheibe type of end organ degeneration
The incidence of this congenital anomaly is highest in Dalmatian dogs of which 165-
30 exhibit unilateral or bilateral hearing loss (Famula et al 1996 Wood and
Lakhani 1997 Muhle et al 2002 Juraschko et al 2003a Rak and Distl 2005) The
inheritance and segregation of a major gene in CCSD has been demonstrated in
different Dalmatian dog populations (Famula et al 2000 Muhle et al 2002 Juraschko et al 2003b) Moreover deafness in Dalmatian dogs seems to be
pigment-associated (Greibrokk 1994 Holliday et al 1992 Juraschko et al 2003a
2003b Mair 1976 Strain et al 1992 Strain 1996)
No gene mutation has yet been identified that is responsible for CCSD in Dalmatian
dogs or in one of the various other dog breeds that suffer from inherited hearing
impairment Since mutations in various genes have already been found to be the
cause of sensorineural hearing impairment in humans or mice 24 of these genes
Linkage analysis of gene-associated microsatellites
26
were considered as candidates for CCSD in Dalmatian dogs (Rak et al 2005)
Details of the 24 candidate genes are given in Table 1
Rak et al (2003) and co-workers (Droumlgemuumlller et al 2002 Kuiper et al 2002 Rak et
al 2002a 2002b 2003) mapped this 24 potential candidate genes for sensorineural
deafness in dogs by fluorescence in situ hybridization and a radiation hybrid panel
Subsequently Rak (2003) developed altogether 43 new highly polymorphic DNA
markers for the 24 candidate genes including CDH23 CLDN14 COCH COL11A2
DFNA5 DIAPH1 EDN3 EDNRB EYA4 GJA1 GJB2 GJB6 MITF MYH9 MYO6
MYO7A MYO15A OTOF PAX3 POU4F3 SLC26A4 SOX10 TECTA and
TMPRSS3 (Table 2)
Among the 24 candidate genes seven genes cause autosomal dominant non-
syndromic forms of deafness seven cause autosomal recessive forms and five
genes cause both recessive and dominant forms of non-syndromic deafness in
different human families segregating for either forms
The functions of these 19 deafness-causing genes are diverse and include gap
junctions and tight junctions (GJA1 GJB2 GJB6 CLDN14) ion channels (SLC26A4)
and ion channel activators (TMPRSS3) Included are also unconventional myosins
(MYO6 MYO7A MYH9 MYO15A) transcription factors (POU4F3 EYA4) as well as
extracellular matrix components (COCH COL11A2 TECTA) a cytoskeleton
regulatory protein (DIAPH1) a cell-adhesion protein (CDH13) and genes with
unknown or only suspected functions (DFNA5 OTOF) The 24 candidates also
include five genes which are mutated in the human Waardenburg syndrome (WS)
The WS is classified into four types depending on the presence or absence of
additional symptoms which are caused by mutations in the five genes EDN3
EDNRB MITF PAX3 and SOX10 respectively The objective of the current study
was to use this set of markers developed by Rak (2003) for a non-parametric linkage
analysis with CCSD in a German and French Dalmatian dog population
Material and methods Pedigree material
For the linkage analysis we used DNA from altogether 215 animals belonging to a
total of 24 Dalmatian dog families The families included 22 full-sib families and one
large paternal half-sib family of German Dalmatian dogs as well as 46 animals of a
Linkage analysis of gene-associated microsatellites 27
large paternal half-sib family of French Dalmatian dogs All families were segregating
for CCSD The genotyped families included all affected dogs (unilaterally and
bilaterally deaf) their parents if available and one to four unaffected animals At least
two of the full sibs of each family were unilaterally deaf
In total these 24 families included 402 individuals with an average family size of 168
ranging from 5 to 116 animals and covering two to four generations The hearing
status of 344 dogs was examined by veterinarians using the BAER (brain stem
auditory evoked response) test and the other animals included in the pedigree being
not BAER tested were used to construct relationships among CSD affected dogs
The prevalence of CSD in this pedigree was 285
Microsatellite marker analysis An existing marker set consisting of 43 microsatellite markers (Table 2) was used for
linkage analysis This set included 36 markers developed by Rak (2003) and 7
markers of the RH map available at httpwww-recomgenuniv-rennes1frdoggyhtml
For most of the 24 candidate genes two markers were available for two of the
candidates three markers were available but for seven candidate genes the set
contains only one marker The marker set is composed of 33 perfect repeats two
imperfect six compound-perfect and two compound-imperfect repeats
The majority (674) of the 43 markers in the set was represented by dinucleotide
repeats 209 were tetranucleotide repeats 47 trinucleotide repeats and 23
pentanucleotide repeats In addition one marker (23) was a compound di-
tetranucleotide and another one (23) was a compound tetra-pentanucleotide
repeat The average number of alleles was 35 with a minimum of 2 and a maximum
of 8 different alleles per marker
PCR reactions for microsatellites were carried out in 12 microl reaction mixtures
containing 2 microl genomic DNA 12 microl 10x PCR buffer (Qbiogene Heidelberg
Germany) 024 microl dimethyl sulfoxide (DMSO) 02 microl dNTPs (100 microM) 01 microl Taq
Polymerase (5Umicrol) (Qbiogene) 06 microl (10 microM) 5rsquo-IRD700 or IRD800 labelled forward
primer 06 microl (10 microM) unlabelled reverse primer The PCR reactions were carried out
in MJ Research thermocyclers with the following program 4 min at 94 degC followed
by 32 cycles of 30 sec at 94degC 30 sec at maximum annealing temperature and a
final extension of 45 sec at 72degC PCR-products were diluted with formamide loading
buffer in ratios from 110 to 140 and then size-fractionated by gel electrophoresis on
Linkage analysis of gene-associated microsatellites
28
automated LI-COR 42004300 sequencers (LI-COR inc Lincoln NE USA) using
denaturing 4 and 6 polyacrylamide gels (RotiphoreseregGel 40 Roth Karlsruhe
Germany)
To localize the 24 candidate genes and their associated microsatellites exactly the
canine candidate gene sequences were derived from sequences deposited in the
current dog genome assembly (Boxer genome assembly 21) of the NCBI GenBank
by BLAST (Basic Local Alignment Search Tool) search
(httpwwwncbinlmnihgovBLAST) using the human reference mRNA sequence
(Table 3)
Linkage analysis
Multipoint linkage and haplotype analyses were performed using the MERLIN
software version 0102 (multipoint engine for rapid likelihood inference Center for
Statistical Genetics University of Michigan MI USA Abecasis et al 2002) The test
statistics Z-mean and Lod score were used to test for the proportion of alleles shared
by affected individuals identical by descent (IBD) for the considered marker loci
Linkage analyses were performed regarding the marker set consisting of 43 gene-
associated microsatellite markers Linkage analysis was at first carried out for all 24
families conjoined After this the families were scanned separately
The data of the genotypes was additionally analyzed using SASGenetics (Statistical
Analysis System Version 913 SAS Institute Inc Cary NC USA 2005) to specify
the number of alleles of each marker the allele frequency the observed (HET) and
expected (HE) heterozygosity and the polymorphism information content (PIC)
(Table 4 and 5)
Results and discussion
Test statistics for all families conjoined are given in Table 6 Significant CCSD loci
were located on different chromosomes The loci were located on canine
chromosome (CFA) 1 10 12 20 and 31 Linkage analyses per family indicated even
higher test statistics for subgroups of families (Table 7) Scanning only families with
Zmeans gt1 test statistics for linkage increased for the genes GJA1 on CFA1 MYH9
on CFA10 and CLDN14 on CFA31 with congenital sensorineural deafness in different
Dalmatian dog families (Table 7) Therefore it is probable that these genes or genes
Linkage analysis of gene-associated microsatellites 29
in their flanking regions are involved in the development of the disease in the
respective familes The results of this test statistics indicate that the inheritance of
non-syndromic hearing loss in Dalmatian dogs is probably as heterogenic in origin as
it is in humans Genetic heterogeneity means that different mutations cause the same
phenotype or disease the different mutations can either be found at the same locus
(allelic heterogeneity) or even at different loci (non-allelic heterogeneity)
The extreme heterogeneity of human deafness often hampered genetic studies
because many different genetic forms of hearing loss give rise to similar clinical
phenotypes and conversely mutations in the same gene can result in a variety of
clinical phenotypes In man genes that transport ions across membranes to maintain
appropriate solute concentration and pH as well as regulatory genes mainly
transcription factors and genes that play a part in structural integrity are essential for
the hearing process
However this study was a first step in identifying genes responsible for CCSD in
Dalmatian dogs The genes GJA1 MYH9 and CLDN14 and their flanking regions will
be further analyzed with a combined approach using microsatellites and single
nucleotide polymorphisms (SNPs) (Chapter 6-9) As linkage was found for different
candidate genes in different families subsequently only the families indicating
linkage will be chosen for further molecular analyses of the respective gene
To confirm the result of this study the density of the intragenic markers has to be
increased The current RH map with 3200 markers provides a good estimation of the
order and physical spacing (ie in base pairs) of markers along canine
chromosomes (Guyon et al 2003) and was recently complemented by the
construction of a 4249-marker integrated canine genome RH map which consists of
900 genes 1589 microsatellites and 1760 BAC end markers (Breen et al 2004) all
included and available in the NCBI database (httpwebncbinlmnihgov)
Thus microsatellites derived from the NCBI database could be used to confirm the
linkage Alternatively BLAST searches versus the canine whole genome shotgun
(wgs) sequence resource were perfomed to localize the genes exactly and to obtain
the sequence of the canine genomic contigs containing the human homologous gene
The results of the BLAST searches of the 24 candidate genes against the Boxer
genome assembly 21 are shown in Table 3 The genomic sequence of the
respective candidate gene can now be used to search for intragenic SNPs as these
polymorphisms are the most abundant and useful markers for fine mapping
Linkage analysis of gene-associated microsatellites
30
Development of SNPs requires sequencing of DNA for the respective genomic
regions of the parents with the aim to identify heterozygous base pair exchanges
After a heterozygous base pair is found the whole family can be genotyped for this
informative SNP marker These polymorphisms can than be used for linkage
analyses as well as association studies
Fine mapping using SNP markers for all genes indicating linkage with CCSD
identified by this study should enable us to detect mutations responsible for CCSD in
parts of the Dalmatian dog population
Linkage analysis of gene-associated microsatellites 31
Table 1 Details of the 24 selected human candidate genes
Symbol Gene name Locus name1 Most important reference
CDH23 cadherin related 23 DFNB12 Bork et al 2001
CLDN14 claudin 14 DFNB29 Wilcox et al 2001
COCH coagulation factor C homolog cochlin DFNA9 Robertson et al 1998
COL11A2 collagen type XI alpha 2 DFNA13 McGuirt et al 1999
DFNA5 deafness autosomal dominant 5 DFNA5 Van Laer et al 1998
DIAPH1 diaphanous homolog 1 (Drosophila) DFNA1 Lynch et al 1997
EDN3 endothelin 3 WS type IV Edery et al 1996
EDNRB endothelin receptor type B WS type IV Attie et al 1995
EYA4 eyes absent homolog 4 (Drosophila) DFNA10 Wayne et al 2001
GJA1 gap junction protein alpha 1 43kD (connexin 43)
2 Liu et al 2001
GJB2 gap junction protein beta 2 26k (connexin 26) DFNA3 DFNB1 Kelsell et al 1997
GJB6 gap junction protein beta 6 (connexin 30) DFNA3 DFNB1 Grifa et al 1999
Del Castillo et al 2002
MITF microphthalmia-associated transcription factor WS type II Tassabehji et al 1994
MYH9 myosin heavy polypeptide 9 non-muscle DFNA17 Lalwani et al 2000
MYO6 myosin VI DFNA22 DFNB37 Melchionda et al 2001 Ahmed et al 2003
MYO7A myosin VIIA DFNA11 DFNB2 Liu et al 1997a Liu et al 1997b Weil et al 1997
MYO15A myosin XVA DFNB3 Wang et al 1998
OTOF otoferlin DFNB9 Yasunaga et al 1999
PAX3 paired box gene 3 (Waardenburg syndrome 1) WS type I WS type III Hoth et al 1993 Tassabehji
et al 1992
POU4F3 POU domain class 4 transcription factor 3 DFNA15 Vahava et al 1998
SLC26A4 solute carrier family 26 member 4 DFNB4 Li et al 1998
SOX10 SRY (sex determining region Y)-box 10 WS type IV Pingault et al 1998
TECTA tectorin alpha DFNB21 DFNA8DFNA12
Mustapha et al 1999 Verhoeven et al 1998
TMPRSS3 transmembrane protease serine 3 DFNB8DFNB10 Scott et al 2001
1 Autosomal recessive loci are designated DFNB autosomal dominant loci DFNA 2 To our knowledge no deafness locus has been determined for this autosomal recessive gene
Linkage analysis of gene-associated microsatellites
32
HET
()
638
338
366
544
616
632
456
656
433
614
458
586
529
PIC
()
752
548
336
484
775
70
63
9
74
6
61
6
83
7
62
762
621
No
of
alle
les
6 4 4 4 9 8 6 6 6 13 5 11 5
PCR
pr
oduc
t (b
p)
175
109
123
156
146
179
259
151
241
219
186
214
191
AT
(degC
)
62 60 56 60 60 62 58 60 62 58 58 60 58
Prim
er s
eque
nces
(5rsquorarr
3rsquo)
F A
ATC
AG
GA
GG
GG
TGAG
TGTG
R
CC
CC
CAG
CTC
ATA
CA
TTC
TC
F C
CTG
TAC
TGA
ATG
CTT
GA
GG
R
CA
TCTC
TAG
AA
GA
AG
CC
TCC
F
TC
AC
ATA
GC
ATT
ATA
TATG
GA
C
R T
TGA
GAT
GG
CTC
TTAC
TGA
G
F T
CG
ATG
ATG
CTT
TCTG
GTT
G
R A
GG
CTG
TGA
AA
TGG
ATG
GA
G
F G
AG
AA
GC
AC
CA
GG
CA
TAG
G
R T
GG
TTTA
GC
AA
GG
CTG
TTC
C
F T
GA
CA
TAC
GG
AG
GAC
CA
AG
AG
R C
CC
CTC
CC
TTG
CTC
TATC
TC
F T
GC
CC
CTC
AG
AG
ATA
ATC
AC
R
CTT
CA
ATTA
TAC
AC
ACA
GG
TAC
F
TG
AA
TATG
GG
GC
TGAG
GA
AG
R
TTC
TCC
CTC
TGC
CTG
TGTC
F
GG
TTTA
GC
AC
TGC
CTT
CA
GC
R
CA
TTA
AG
CA
TCTG
GC
ATG
TGG
F
GA
AA
AC
TCA
GA
TTA
GC
CTG
G
R A
TCTT
GA
GA
GC
AA
AGG
TTG
TG
F T
GG
TTA
GG
GC
ATG
ATTC
CA
G
R C
ATG
TATA
AA
GA
GTA
ATG
CC
AG
F
CG
GG
AG
AGG
GTT
TGAC
TAC
R
CTC
CG
TATT
GC
TCA
TCTT
TCC
F
AG
CTT
CC
CTT
CTC
TGA
GA
C
R G
AG
AA
TAG
AG
TTTG
TGC
TCA
G
Rep
eat
(TTT
A)1
5 (A
CC
)9(A
TC)3
(A
TTT)
7(G
TTT)
5 (C
A)2
1 (G
A)2
0 (C
TTT)
~20
(GA
)13
(AC
)20(
AG)9
(C
T)16
TT
(CT)
5 (A
AA
AT)
~25
(CT)
11(G
T)4(
CT)
2 (A
TTT(
T))2
3 (A
C)2
2
Mar
ker n
ame
CD
H23
_MS1
C
DH
23_M
S2_F
2 C
LDN
14_M
S1
CLD
N14
_MS
2 C
LDN
14_M
S3
CO
CH
_MS1
C
OC
H_M
S2
CO
L11A
2_M
S1
CO
L11A
2_M
S3
DFN
A5_
MS
1 D
FNA
5 _M
S2
DIA
PH
1 _M
S1
DIA
PH
1_M
S2
Mar
ker o
rigin
RP
CI8
1-99
C20
R
PC
I81-
99C
20
RP
CI8
1-97
L17
RP
CI8
1-97
L17
RP
CI8
1-97
L17
RP
CI8
1-32
1I4
RP
CI8
1-32
1I4
RP
CI8
1-24
M6
RP
CI8
1-24
M6
RP
CI8
1-66
C12
R
PC
I81-
66C
12
RP
CI8
1-36
2I5
RP
CI8
1-36
2I5
Tabl
e 2
Can
dida
te g
ene-
asso
ciat
ed m
arke
rs f
or c
anin
e de
afne
ss d
evel
oped
by
Rak
(20
03)
For
each
gen
e th
e m
icro
sate
llite-
base
d m
arke
r th
e m
arke
r or
igin
rep
eat t
ype
PC
R p
rimer
s w
ith o
ptim
ised
con
ditio
ns a
nd th
e nu
mbe
r of
alle
les
pol
ymor
phis
m
info
rmat
ion
cont
ent (
PIC
) and
obs
erve
d he
tero
zygo
sity
(HE
T) fo
r all
dogs
gen
otyp
ed a
re s
how
n
Can
dida
te
gene
CD
H23
C
LDN
14
CO
CH
C
OL1
1A2
DFN
A5
DIA
PH
1
Linkage analysis of gene-associated microsatellites 33
HET
()
815
345
695
578
562
508
583
441
75
63
7
33
414
30
76
7
44
8
PIC
()
853
679
798
774
652
63
72
3
59
5
83
2
77
9
50
5
54
1
40
4
88
9
59
1
No
of
alle
les
14 6 7 6 9 5 9 6 13 13 4 6 4 14 6
PCR
pr
oduc
t (b
p)
147
131
258
227
134
190
148
195
218
260
181
164 96
227 94
AT
(degC
)
60 58 58 58 60 60 58 62 58 58 62 56 58 58 62
Prim
er s
eque
nces
(5rsquorarr
3rsquo)
F G
CTA
GG
AA
AA
ATC
CG
CA
ATG
R
GAC
CC
CC
TAG
GAC
ACC
AAC
F
GA
GA
ATT
GG
GC
ATG
GG
CAG
A
R T
GA
CTT
TATC
AC
TGG
TCTT
TG
F T
TATG
CAG
CC
CA
TGAC
AA
TC
R C
AA
GG
GA
AC
TCA
AAG
GC
TTG
F
TG
GA
CC
AG
GTC
AGTT
TGTG
R
TC
TGC
CTG
TGTC
TCTG
CC
F
ATG
GC
ATG
AA
GA
GG
ATA
CC
G
R A
GG
AC
AGG
TGAC
GG
CTC
TAC
F
GC
TAG
TAC
TCG
ATT
GTG
GTC
R
TC
ATG
GG
TTG
TGA
GA
TCC
AG
F T
TAA
TTTG
CTC
GTC
TTC
CTG
R
TG
TAA
GC
TCC
ACG
GA
TCA
CC
F
CTC
TCTT
GG
TCTC
CC
TCTG
C
R G
GG
AG
TAG
GG
GTG
GAG
TAG
G
F G
GTG
TTTC
CTT
TCC
TTTT
CT
R G
GTG
TTC
TCTC
CC
TTTC
TCT
F C
TCTA
TGA
AA
GG
TGA
TTG
CC
R
CA
GC
CAT
AC
AA
ATG
AGA
ATT
G
F C
TAC
AG
TGA
ATC
AG
CA
CA
GAC
R
CA
GC
CTT
GA
CTG
TTTC
TTTG
G
F T
GA
TTG
AC
TCTA
CTT
TAC
AC
A
R T
ATA
TTA
GG
CG
GTT
TTC
TTC
T F
AC
CC
AG
GTG
GC
CTG
ATTG
R
GC
AC
GC
AC
GTT
CTC
TCTT
TC
F G
AA
TGC
CC
TTC
ACC
TTG
AA
A
R G
GA
AA
AG
GA
GA
GA
TGA
TGC
C
F T
CTT
CC
TTG
GA
AA
GG
GA
AC
TC
R T
GC
CC
TAA
CA
CTT
GG
AA
TGG
Rep
eat
(TA
GA
)12
(GA
)25
(GT)
10(A
T)13
(A
G)2
1 (G
T)16
(A
G)1
2 (C
A)1
2 (G
A)8
(C
TTT)
~20
(GA
AA
)~25
(G
T)12
(C
A)1
5 (A
G)1
1 (G
AA
A)~
43
(AC
)13
Mar
ker n
ame
ED
N3_
MS
1 E
DN
RB
_M
S1
EY
A4_
MS
1 E
YA
4_M
S2
GJA
1_M
S1
GJA
1_M
S2
GJB
2_M
S1
GJB
2_M
S2
GJB
6_M
S2
GJB
2+6_
MS1
M
ITF
_MS
2 M
ITF
_MS
3 M
YH
9_M
S2
MY
H9_
MS
3 M
YO
6_M
S2
Mar
ker o
rigin
RP
CI8
1-36
6E14
Z
emke
et a
l (1
999)
R
PC
I81-
301N
19
RP
CI8
1-30
1N19
R
PC
I81-
370A
16
RP
CI8
1-37
0A16
R
PC
I81-
133O
22
RP
CI8
1-13
3O22
R
PC
I81-
343C
15
FH
2324
R
PC
I81-
119P
24
RE
N10
0J13
R
PC
I81-
374A
12
FH
2293
R
PC
I81-
156P
14
Tabl
e 2
(con
tinue
d)
Can
dida
te g
ene
ED
N3
ED
NR
B
EY
A4
GJA
1 G
JB2
GJB
6 M
ITF
MY
H9
MY
O6
Linkage analysis of gene-associated microsatellites
34
HET
()
359
366
431
566
51
50
5
28
3
35
8
79
9
70
4
69
2
23
9
68 71
698
PIC
()
496
623
627
765
635
756
502
575
756
76
70
1
44
2
75
4
84
5
80
6
No
of
alle
les
5 4 6 13 7 8 5 3 10 11 8 8 8 10 11
PCR
pr
oduc
t (b
p)
293
166
100
137
174
197
164
201
160
297
267
140
276
228
260
AT
(degC
)
56 60 56 62 62 62 60 60 58 58 56 58 58 62 60
Prim
er s
eque
nces
(5rsquorarr
3rsquo)
F T
GG
TTA
AA
AC
ATT
AA
AC
TTA
TAG
R
TA
GTA
TATA
GA
GA
TGC
AA
TGG
F
CA
TTG
GG
TGC
TTTC
CTG
TTC
R
TG
GA
GC
TGC
AG
GTA
TAG
CC
F
CC
AG
GC
ATT
CG
AG
GG
TG
R C
AG
AA
CTT
GA
GG
AAC
CA
TAG
F
CC
ATG
AAC
TTTG
TGG
AA
CTG
C
R A
AA
GG
GTT
GC
TGTG
GA
GA
TG
F A
GG
CA
GG
TTC
ATC
TGTG
TCC
R
TC
CC
AG
AC
CC
AG
CTA
CA
TTC
F
CA
GC
CAA
CTG
TATT
CTC
CTT
G
R A
TCTT
GA
GC
CC
TGC
ATT
AG
G
F G
AA
GC
GA
GG
AG
AG
ACA
GTC
C
R A
AG
GA
AG
CC
TCC
TGAC
AA
CC
F
CA
GG
GTC
AG
GC
TCTA
TGC
TC
R T
CC
TATC
ATC
CG
GC
TTTG
AC
F
TC
TGG
ATTG
TGG
TCA
CA
AC
C
R A
CTG
GAC
AC
TTC
TTTT
CA
GAC
G
F A
AA
GTG
GC
TGG
TTC
GG
AA
G
R A
GC
AG
CA
GC
ATA
CA
TTC
CTC
F
AA
GTA
GA
TCC
TATT
ATC
GTG
G
R A
GTT
TCA
GTG
TCTG
TTA
AA
TAG
F
CC
GG
ATT
TCTG
AG
GAG
GC
R
CA
TGC
TCTT
CA
CC
AGA
AC
C
F T
CA
GC
ATG
GA
TTTT
GTA
AA
ATC
R
GG
AC
TGC
GTG
GA
CAT
CTG
F
AC
AC
GG
TTC
TCG
CTG
ATG
TG
R T
GA
AG
GG
GA
TTG
AAC
AG
AG
G
F T
TCA
TTC
CG
AG
GTT
CTA
AC
TG
R C
AC
CA
TCTC
GTA
GC
CTT
TATC
Rep
eat
(TC
)13
(AC
)12
(GT)
12
(AC
)18
(GT)
12
(TA
)3(T
G)9
(TA
)2(C
A)2
C
(TA
AA
)5
(AA
T)18
(C
T)13
(T
G)1
3 (C
T)17
(T
AA
A)1
4 (G
T)3
CC
(GT)
19
(ATT
T)12
(T
C)8
(CA)
5(C
G)2
(CA)
9 (G
T)16
Mar
ker n
ame
MY
O7A
_M
S1
MY
O7A
_M
S2
MY
O7A
_M
S3
MY
O15
A _
MS
1 M
YO
15A
_M
S2
OTO
F _M
S1
PA
X3_
MS
1 P
AX
3_M
S2
PO
U4F
3_M
S4
SLC
26A
4_M
S2
SO
X10
_MS
2 T
EC
TA _
MS
1 T
EC
TA _
MS
2 T
MP
RS
S3_
MS
1 T
MP
RS
S3_
MS
2
Mar
ker o
rigin
RP
CI8
1-19
3deg2
RP
CI8
1-19
3deg2
AH
TH29
8 R
PC
I81-
362deg
13
RP
CI8
1-36
2deg13
R
PC
I81-
198L
15
RP
CI8
1-25
7H23
R
PC
I81-
257H
23
G2C
024
66
RP
CI8
1-47
P17
RP
CI8
1-50
5H2
RP
CI8
1-59
C2
RP
CI8
1-59
C2
RP
CI8
1-12
5P17
A
HTH
246
Tabl
e 2
(con
tinue
d)
Can
dida
te
gene
MY
O7A
M
YO
15A
O
TOF
PA
X3
PO
U4F
3 S
LC26
A4
SO
X10
TE
CTA
TM
PR
SS
3
Linkage analysis of gene-associated microsatellites 35
Table 3 The gene-associated microsatellites (MS) for 24 candidate genes of canine
congenital sensorineural deafness were localized using BLAST searches against the
Boxer genome assembly 21 The accession numbers (AccNo) of the whole
genome shotgun (WGS) contigs containing the genes as well as their associated
microsatellites are given
Microsatellite Acc No of WGS contig CFA Location of
Gene (Mb) Location of
MS (bp) Location of
MS in relation to gene
CDH23_MS1 NW_876311 4 2541025780 25510 intragenic
CDH23_MS2 NW_876311 4 2541025780 25630 intragenic
CLDN14_MS1 NW_876295 31 3379533796 33790 proximal
CLDN14_MS2 NW_876295 31 3379533796 33950 distal
CLDN14_MS3 NW_876295 31 3379533796 33790 proximal
COCH_MS1 NW_876327 8 1321513232 13225 intragenic
COCH_MS2 NW_876327 8 1321513232 13290 distal
COL11A2_MS1 NW_876254 12 56315659 5608 proximal
COL11A2_MS3 NW_876254 12 56315659 5578 proximal
DFNA5_MS1 NW_876258 14 4116941237 41135 proximal
DFNA5_MS2 NW_876258 14 4116941237 41250 distal
DIAPH1_MS1 NW_876292 2 3933039430 39382 intragenic
DIAPH1_MS2 NW_876292 2 3933039430 39552 distal
EDN3_MS1 NW_876277 24 4701347032 47057 distal
EDNRB_MS1 Zemke et al (1999) 22 3436334383 34354 proximal
EYA4_MS1 NW_876269 1 2928029550 29531 intragenic
EYA4_MS2 NW_876269 1 2928029550 29500 intragenic
GJA1_MS1 NW_876269 1 6399463996 64150 distal
GJA1_MS2 NW_876269 1 6399463996 64160 distal
GJB2_MS1 NW_8762781 25 2093620942 20938 intragenic
GJB2_MS2 NW_8762781 25 2093620942 20863 proximal
GJB2+6_MS1 FH2324 25 2093620942 17543 proximal
GJB3_MS2 NW_876259 15 1018310194 4530 proximal
GJB6_MS2 NW_876278 25 2090420906 20953 distal
Linkage analysis of gene-associated microsatellites
36
Table 3 (continued)
Microsatellite Acc No of WGS contig CFA Location of
Gene (Mb) Location of
MS (bp) Location of
MS in relation to gene
MITF_MS2 NW_876271 20 2485324884 24844 proximal
MITF_MS3 REN100J13 20 2485324884 25668 distal
MYH9_MS2 NW_876251 10 3113531193 31244 distal
MYH9_MS3 FH2293 10 3113531193 31696 distal
MYO15A_MS1 NW_876313 5 4436944419 44330 proximal
MYO15A_MS2 NW_876313 5 4436944419 44409 intragenic
MYO6_MS2 NW_876254 12 4041740504 40534 distal MYO7A_MS1 NW_876273 21 2454324609 24465 proximal
MYO7A_MS2 NW_876273 21 2454324609 24566 intragenic
MYO7A_MS3 AHTH298 21 2454324609 24594 distal
OTOF_MS1 NW_876263 17 2350223595 23463 proximal
PAX3_MS1 NW_876304 37 3134831445 31426 intragenic
PAX3_MS2 NW_876304 37 3134831445 31481 distal
POU4F3_MS4 G2C02466 2 4361043612 - -
SLC26A4_MS2 NW_876265 18 1586715927 15960 distal
SOX10_MS2 NW_876251 10 2975129762 29740 proximal
TECTA_MS1 NW_876312 5 1588515954 15910 intragenic
TECTA_MS2 NW_876312 5 1588515954 15930 intragenic
TMPRSS3_MS1 NW_876295 31 3903539054 38942 proximal
TMPRSS3_MS2 AHTH246 31 3903539054 38508 proximal
Linkage analysis of gene-associated microsatellites 37
Table 4 Number of alleles observed (HET) and expected (HE) heterozygosity and
polymorphism information content (PIC) for the developed marker-set
Feature Mean SD Min Max
No of alleles 75 31 3 14
hO () 703 122 370 898
hE () 532 151 239 815
PIC () 667 130 336 889
Table 5 Number of alleles per microsatellite locus and their PIC () values of the
developed marker-set
No of alleles per
microsatellite
Number of marker
loci PIC ()
3 1 575
4 6 483
5 5 574
6 11 652
7 2 716
8 5 671
9 3 717
10 2 800
11 3 776
13 4 803
14 2 871
Linkage analysis of gene-associated microsatellites
38
Table 6 Significant test statistics for linkage analysis carried out for all 24 genotyped
families conjoined Zmeans and LOD scores are given with their respective error
probabilities for the gene-associated markers of the candidate genes CLDN14
COL11A2 GJA1 MITF MYH9 and SOX10
Marker Location on canine chromosome (CFA)
Zmean pZmean LOD score pLOD
CLDN14_MS1 31q15 134 009 086 002
CLDN14_MS2 31q15 168 005 105 001
CLDN14_MS3 31q15 108 014 049 007
COL11A2_MS1 12q11-q12 166 005 085 002
COL11A2_MS3 12q11-q12 167 005 078 003
GJA1_MS1 1q24-q25 151 007 118 001
GJA1_MS2 1q24-q25 151 007 118 001
MITF_MS2 20q13 101 02 080 003
MITF_MS3 20q13 121 011 104 001
MYH9_MS2 10q232 080 02 018 02
MYH9_MS3 10q232 175 004 097 002
SOX10_MS2 10q21-q23 146 007 110 001
Linkage analysis of gene-associated microsatellites 39
Table 7 Significant test statistics for linkage analyses carried out each family
separately Zmeans and LOD scores are given with their respective error probabilities
for the gene-associated markers of the candidate genes CLDN14 MYH9 and GJA1
Gene-associated
marker
Number of families with significant linkage to
CCSD
Number of corresponding
family members
Zmean pZmean LOD-score pLOD
CLDN14_MS1 51 40 278 0003 112 0011
CLDN14_MS2 383 000007 170 0003
CLDN14_MS3 281 0002 113 0011
MYH9_MS2 32 21 081 02 023 02
MYH9_MS3
(=FH2293) 156 006 058 005
GJA1_MS1 13 46 295 0002 052 006
GJA1_MS2 286 0002 051 006 1 families with significant linkage include four German full-sib families and one
German half-sib family 2 families with significant linkage include three German full-sib families 3 families with significant linkage include one French half-sib family
Chapter 4
Evaluation of eight candidate genes for canine congenital sensorineural deafness
in Dalmatian dogs
Evaluation of eight candidate genes for CCSD 43
Evaluation of eight candidate genes for canine congenital sensorineural deafness in Dalmatian dogs Abstract
In this study we have been focusing on genomic loci that encode various enzymes
and transporters involved in the hearing process in humans We developed intragenic
markers for the canine genes ESPN MYH14 MYO3A PRES TMC1 TMIE USH1C
and WHRN on canine chromosomes (CFA) 1 2 5 11 18 20 and 21 which have
been shown to be responsible for human hereditary deafness and to employ these
newly developed markers for non-parametric linkage analyses with canine congenital
sensorineural deafness (CCSD) We screened DNA from altogether six Dalmatian
dogs which represent the parents of four families for single nucleotide polymorphisms
(SNPs) in the eight candidate genes by means of direct sequencing combined with a
polymerase chain reaction method for amplifying genomic DNA We characterized 20
SNPs and one insertion-deletion polymorphism For the TMC1 and TMIE genes we
additionally genotyped one microsatellite marker each The families used for
subsequent genotyping of the markers included 39 members from four full-sib
families with frequent occurrence of CCSD We concluded that mutations in ESPN
MYH14 MYO3A PRES TMC1 TMIE USH1C and WHRN do not play a role in
CCSD of the Dalmatian dog population investigated here
Introduction Over the past ten years significant progress has been made in the identification of
genes causing different forms of human deafness Currently 39 of the genes
responsible for non-syndromic hearing impairment have been identified in different
human populations (The Hereditary Hearing Loss Hompage
httpwebhostuaacbehhh Van Camp and Smith 2003)
Since non-syndromic hereditary hearing impairment is almost exclusively caused by
cochlear defects affected patients suffer from sensorineural hearing loss
Evaluation of eight candidate genes for CCSD
44
The most common histopathologic finding in cases of profound congenital deafness
in humans is the cochleosaccular degeneration (CSD) It is estimated to occur in
approximately 70 of cases in man and also in dog breeds the histological pattern is
known as cochleosaccular degeneration
Many genetic disorders in humans and domestic dogs (Canis familiaris) demonstrate
a high level of clinical and molecular similarity Therefore genes responsible for
human hereditary deafness seem to be appropriate candidate genes for CCSD
especially in Dalmatian dogs
In this report we provide 21 single-nucleotide polymorphisms (SNPs) and two
microsatellite markers in altogether eight selected human candidate genes (Table 1)
This eight candidate genes were only recently identified for being responsible for
different form of human non-syndromic deafness In order to evaluate whether any of
this candidate genes is responsible for congenital sensorineural deafness in
Dalmatian dogs we analyzed the association of the ESPN MYH14 MYO3A PRES
TMC1 TMIE USH1C and WHRN haplotypes with the CCSD phenotype in four
families of Dalmatian dogs with frequent occurrence of CCSD
Material and methods Pedigree structure and sampling
For the linkage analysis we used blood samples from 39 Dalmatian dogs They
belong to four full-sib families segregating for CCSD At least two of the full sibs of
each family were unilaterally deaf The phenotype of the affected animals had been
confirmed by brainstem auditory evoked response (BAER) that detects electrical
activity in the cochlea and auditory pathways in the brain
The families consisted of eight to 12 individuals In two families a blood sample of the
sire and dam respectively was not available Screening for SNPs was performed by
comparative sequencing of genomic DNA from the parents of the families used for
linkage analyses
SNP and microsatellite marker identification for genotyping
The canine ESPN MYH14 MYO3A PRES TMC1 TMIE USH1C and WHRN gene
sequences was derived from sequences deposited in the current dog genome
assembly (Boxer genome assembly 21) of the NCBI GenBank (Table 2) by BLAST
Evaluation of eight candidate genes for CCSD 45
(Basic Local Alignment Search Tool) search (httpwwwncbinlmnihgovBLAST)
using the human ESPN MYH14 MYO3A PRES TMC1 TMIE USH1C and WHRN
reference mRNA sequence
We compared the canine genomic DNA sequence from the eight candidate genes to
canine cDNA fragments in the canine EST database using the BLASTN program
For the localization of the exonintron boundaries canine or alternatively human
mRNA sequences were used for the mRNA-to-genomic alignment program Spidey
(httpwwwncbinlmnihgovIEBResearchOstellSpideyindexhtml) The human
and canine mRNA sequences which were used to determine the exon organization of
the candidate genes are given in Table 2
For each of the eight candidate genes we designed intragenic primer pairs to amplifly
intronic sequences yielding products with a length of 560 to 670 bp PCR primers
were developed with the Primer3 program (httpfrodowimiteducgi-
binprimer3primer3_wwwcgi) The PCR reactions were performed in a total of 50 microl
containing 125 microM dNTPs 25 pmol of each primer the reaction buffer supplied by
the manufacturer (Qbiogene Heidelberg Germany) and 1 U Taq polymerase After
a 4 min initial denaturation at 95degC 35 cycles of 30 sec at 94degC 45 sec at 58degC and
80 sec at 72degC were performed in a MJ Research thermocycler (Biozym Hessisch
Oldendorf Germany) The obtained PCR products were directly sequenced with the
DYEnamic ET Terminator kit (AmershamBiosciences Freiburg Germany) and a
MegaBACE 1000 capillary sequencer (AmershamBiosciences) using the PCR
primers as sequencing primers Sequence data were analyzed with Sequencher 42
(GeneCodes Ann Arbor MI)
In the first step sequence analyses were performed for PCR products of the parents
of four full-sib families If a heterozygous SNP was found for one or both parents all
progeny of the respective families were analyzed for that SNP Additionally to the
SNPs we used two microsatellite markers for linkage analyses We identified one
intragenic microsatelllite within the TMC1 gene As we could develop only one SNP
for the TMIE gene we additionally genotyped one microsatellite marker derived from
the NCBI database (httpwebncbinlmnihgov) located very closely to the TMIE
gene (Table 3)
Evaluation of eight candidate genes for CCSD
46
SNP marker analysis
A total of 21 SNPs were identified within ESPN MYH14 MYO3A PRES TMC1
TMIE USH1C and WHRN (Table 3) and used for linkage analysis Of all SNPs six
out of the observed 21 SNPs were polymorphic in all four examined families Out of
the 21 SNPs eleven SNPs were present in family 1 18 SNPs were heterozygous for
one or both parents in family 2 and 15 SNPs could be used for linkage analysis in
family 3 and 4 respectively (Table 4)
The most frequent form of SNPs with a frequency of 238 was the CT transition
motif The scarcest one was the CG transversion motif with a frequency of 48
respectively
Linkage and haplotype analysis Multipoint linkage and haplotype analysis were performed using MERLIN version
0102 (multipoint engine for rapid likelihood inference Center for Statistical Genetics
University of Michigan MI USA Abecasis et al 2002) The test statistics Z-mean
and Lod score were used to test for the proportion of alleles shared by affected
individuals identical by descent (IBD) for the considered marker loci The data of the
genotypes was additionally computed using the software package SAS Genetics
(Statistical Analysis System Version 913 SAS Institute Inc Cary NC USA 2005)
to specify the observed heterozygocity values (HET) and the polymorphism
information content (PIC)
Furthermore linkage disequilibrium (LD) and association of haplotypes with CCSD
was tested using the procedures CASECONTROL and HAPLOTYPE of
SASGenetics (Statistical Analysis System version 913 Cary NC USA)
Results and discussion The non-parametric multipoint linkage analysis in four full-sib families did not show
significant test statistics The highest Z-mean value was 046 the highest LOD Score
was 008 while the error probabilities ranged from 03 to 08 (Table 5) The maximum
achievable Z-mean was 448 and the corresponding value for the LOD score was
160 Marker-trait association tests for haplotypes of the candidate gene markers
were not significant Obviously no haplotype was associated with CCSD in these
Dalmatian dog families as demonstrated (Figure 1 2 3 4 5 6 7 and 8) The
Evaluation of eight candidate genes for CCSD 47
paternal and maternal haplotypes as far as they could be estimated were distributed
among the affected progeny closely to the expected proportion of 50 and thus no
excess of a certain haplotype could be observed in the affected dogs
Due to the fact that both animals with unilateral or bilaterally hearing loss and
bilateral hearing animals shared identical haplotypes it is unlikely that the ESPN
MYH14 MYO3A PRES TMC1 TMIE USH1C and WHRN genes are involved in the
pathogenesis of CCSD in Dalmatian dogs However the ESPN MYH14 MYO3A
PRES TMC1 TMIE USH1C and WHRN gene-associated marker may serve for
further linkage studies in other Dalmatian dog populations and dog breeds other than
Dalmatians
Evaluation of eight candidate genes for CCSD
48
Table 1 Details of the six selected human candidate genes
Gene symbol
Gene name Locus name1 Most important reference
ESPN espin DFNB36 Naz et al (2004)
MYH14 myosin heavy polypeptide 14 DFNA4 Donaudy et al (2004)
MYO3A myosin IIIA DFNB30 Walsh et al (2002)
PRES solute carrier family 26
member 5 (prestin) DFNB61
Zheng et al (2000) Liu et
al (2003)
TMC1 transmembrane channel-like
gene 1
DFNB7
DFNA36 Kurima et al (2002)
TMIE transmembran inner ear DFNB6 Naz et al 2002
USH1C Usher syndrome 1C DFNB18 Verpy et al (2000) Ouyang
et al (2002)
WHRN whirlin DFNB31 Mburu et al (2003)
1 Autosomal recessive loci are designated DFNB autosomal dominant loci DFNA
Evaluation of eight candidate genes for CCSD 49
Table 2 Selected human candidate genes with their location on HSA and CFA and
corresponding accession numbers
Gene symbol Gene
location on HSA1
Acc No 3 human mRNA
Gene location
on CFA2
Acc No 3 canine
genomic sequence
Acc No 3 canine mRNA
ESPN 1 NM_031475 5 NC_006587 XM_546751
MYH14 19 NM_024729 1 NW_876270 -
MYO3A 10 NM_017433 2 NC_006584 XM_544234
PRES 7 NM_206883 18 NC_006600 XM_540393
TMC1 9 NM_138691 1 NC_006583 XM_541284
TMIE 3 NM_147196 20 NC_006602 XM_846596
USH1C 11 NM_153676 21 NC_006603 XM_860072
WHRN 9 NM_015404 11 NC_006593 XM_850321
1 Homo sapiens autosome 2 Canis familiaris autosome 3 Accession number
Evaluation of eight candidate genes for CCSD
50
Table 3 The 21 newly developed intragenic SNPs and two microsatellite markers for
Dalmatian dogs in the candidate genes ESPN MYH14 MYO3A PRES TMC1
TMIE USH1C and WHRN with their corresponding primers the SNP motif the
product size and the annealing temperature the observed heterozygosity (HET) and
polymorphism information content (PIC)
SNP Primer F1 (5acute -gt 3acute) SNP motif primer R2 (5acute -gt 3acute)
Bp3 AT4 PIC ()
HET()
ESPN_SNP1
ACCAGCACCCTCTCCAACTA
AGGAATTCACAA (CT)CACACATACA
ACTCAAGCTCAGGGTGTGGT
565 60 9 10
ESPN_SNP2
ACCAGCACCCTCTCCAACTA
ATGGCTGGCGCT (AG)GAGGCTGCCC
ACTCAAGCTCAGGGTGTGGT
565 60 27 41
ESPN_SNP3
ACCAGCACCCTCTCCAACTA
ACACTCTTCCCA (CT)GGCTGGCGCT
ACTCAAGCTCAGGGTGTGGT
565 60 23 32
ESPN_SNP4
ACCAGCACCCTCTCCAACTA
TGGGAAGAGGGA (AG)GGGGGAGCAT
ACTCAAGCTCAGGGTGTGGT
565 60 23 32
ESPN_SNP5
ACCAGCACCCTCTCCAACTA
GAGTGGGCCAGG (CT)TGGGAAGAGG
ACTCAAGCTCAGGGTGTGGT
565 60 28 42
MYH14_SNP1
CTCTCCCCAACTCAGTTCCA
ACGTGTATTCGG (GT)CGCTTTTATT
GTGATAGGGACGAGCAGCAT
670 60 35 42
MYH14_SNP2
CATGGGACCGTTCCTACACT
AGCCTCGTTTAA (CT)CTAAAAGGAA
GCTCAATAGGCACGACATCA
640 60 34 39
Evaluation of eight candidate genes for CCSD 51
Table 3 (continued)
SNP Primer F1 (5acute -gt 3acute) SNP motif primer R2 (5acute -gt 3acute)
Bp3 AT4 PIC ()
HET()
MYO3A_SNP1
AATGCTTGAGTTTGGGATGC
GGCAGTCCCATG (GT)CCCTTATAAG
ACCTAATTGCCCAGATGCAG
650 60 37 77
MYO3A_SNP2
AATGCTTGAGTTTGGGATGC
GTGGAGAGCCAC (CG)TTGGGAGAGG
ACCTAATTGCCCAGATGCAG
650 60 37 70
MYO3A_SNP3
AACCTCCTGGCGTAGTATTCC
CATTACCTATTT (AT)GATCCTTATA
TTTTCCACTTCAGGCACACA
650 60 25 36
PRES_SNP1
CCCTTACCCCATACCATTCC
GATAGACTTCCT (AG)CCCTCAGACT
TTCAGGACAGCATCATCTGC
560 60 37 64
PRES_SNP2
CCCTTACCCCATACCATTCC
TGATGTCTGCTG (AT)TAACCCATTC
TTCAGGACAGCATCATCTGC
560 60 37 66
TMC1_SNP1
GCAACCTCTCGGTTTATCCA
CGTGAAGTGCCC (AT)TTGATGGAAA
AAGCTGGGGAAGTGGATATGT
610 60 57 37
TMC1_SNP2
GCAACCTCTCGGTTTATCCA
GGAGACATTACC (AG)TGAAGTGCCC
AAGCTGGGGAAGTGGATATGT
610 60 47 29
TMC1_SNP3
GGAAGCAAGACTGAGGTTGG
AGGCTTTTTAAA (AG)CTGTTCTGGG
CTGCTGCATTTGCCTGTAAG
650 60 48 30
TMIE_SNP1
AGAACACCACCGTCTCCTTG
CAAGGCGACGCC (AT)GTGCTGTCCT
GCCTCTGGTCAGAAGAGGTG
625 60 59 36
Evaluation of eight candidate genes for CCSD
52
Table 3 (continued)
SNP Primer F1 (5acute -gt 3acute) SNP motif primer R2 (5acute -gt 3acute)
Bp3 AT4 PIC()
HET()
USH1C_SNP2
CTCCCGGTCTGTCAGGAAC
GGCCTGGGGGGA (AC)AAGCGGACGG
ATGGCATCGACTTCTCCAAC
560 60 37 35
USH1C_SNP4
CTCCCGGTCTGTCAGGAAC
GGTCTCAGACCG (AC)GGCAGGGAGA
ATGGCATCGACTTCTCCAAC
560 60 37 37
WHRN_SNP1
TTCACCTCCAGGATCTGGTC
CCTGAGCCCGAG (CT)CCACGCTGCT
GGCTACTTTTCTTCCCCCTTT
600 60 25 37
WHRN_SNP2
TTCACCTCCAGGATCTGGTC
GGTCACGGGGGC (CT)CCGGGAGGTT
GGCTACTTTTCTTCCCCCTTT
600 59 24 33
WHRN_SNP3
TTCACCTCCAGGATCTGGTC
GTCCGAGTCCCG (AG)CCCCAGCCTG
GGCTACTTTTCTTCCCCCTTT
600 60 34 55
Microsatellite marker
Primers (forward reverse ) 5acute -gt 3acute Bp AT PIC()
HET()
TMC1_MS1
GCCCCCAGCTAAAAAGAGAA
TTCTCTTCCTCCCTCCTGTTC
220-220 60 76 57
FH2158 ATGGCCACATCACCCTAGTC
CTCTCTCTGCATCTCTCATGAA
274-302 58 57 66
Microsatllite marker derived from the NCBI database (httpwebncbinlmnihgov) 1 Forward 2 Reverse 3 Product size (basepairs) 4 Annealing temperature (degC)
Evaluation of eight candidate genes for CCSD 53
Table 4 The 21 newly developed intragenic SNPs for Dalmatian dogs in the
candidate genes ESPN MYH14 MYO3A PRES TMC1 TMIE USH1C and WHRN
with their nucleotide polymorphism allele and genotype frequencies
Gene symbol Fam1 Nucleotide polymorphism
Allele frequencies
Genotype frequencies2
ESPN_SNP1 4 CgtT 083017 840
ESPN_SNP2 2 3 4 AgtG 074026 15160
ESPN_SNP3 2 3 TgtC 068032 07120
ESPN_SNP4 2 3 GgtA 068032 07120
ESPN_SNP5 2 3 4 CgtT 074026 15160
MYH14_SNP1 2 3 4 GgtT 058041 51610
MYH14_SNP2 2 3 4 CgtT 058041 51610
MYO3A_SNP1 1 2 GgtT 062038 5111
MYO3A_SNP2 1 2 CgtG 062038 5111
MYO3A_SNP3 1 2 4 TgtA 076024 15140
PRES_SNP1 1 2 3 4 AgtG 058042 10254
PRES_SNP2 1 2 3 4 TgtA 058042 10254
TMC1_SNP1 1 2 3 4 AgtT 056044 11226
TMC1_SNP2 1 2 3 4 AgtG 076024 20190
TMC1_SNP3 1 2 3 4 AgtG 074026 19200
TMIE_SNP1 1 2 3 4 AgtT 058042 12216
USH1C_SNP2 1 3 4 AgtC 053047 9147
USH1C_SNP4 1 3 4 AgtC 053047 9147
WHRN_SNP1 2 CgtT 075025 360
WHRN_SNP2 2 CgtT 075025 360
WHRN_SNP3 2 3 4 InDel3 069031 13171 1 Number of families which were informative for the respective SNPs 2 Frequence of genotypes (first number number of animals homozygous for allele 1
second number heterozygous third number homozygous for allele 2) 3 Insertiondeletion of one base-pair (adenine)
Evaluation of eight candidate genes for CCSD
54
Table 5 Linkage analyses for families 1 2 3 and 4 for the 21 SNP and two
microsatellite markers within the eight candidate genes regarding Zmean LOD score
and error probabilities (p-values)
Gene symbol Marker Zmean pz-value1 LOD score pL-value2
ESPN ESPN_SNP1 014 04 002 04
ESPN_SNP2 014 04 002 04
ESPN_SNP3 014 04 002 04
ESPN_SNP4 014 04 002 04
ESPN_SNP5 014 04 002 04
MYH14 MYH14_SNP1 -089 08 -019 08
MYH14_SNP1 -089 08 -019 08
MYO3A MYO3A_SNP1 -049 07 -011 08
MYO3A_SNP2 -049 07 -011 08
MYO3A_SNP3 -049 07 -011 08
PRES PRES_SNP1 -094 08 -019 08
PRES_SNP2 -094 08 -019 08
TMC1 TMC1_SNP1 -034 06 -008 07
TMC1_SNP2 -034 06 -008 07
TMC1_SNP3 -034 06 -008 07
TMC1_MS1 -035 06 -008 07
TMIE TMIE_SNP1 013 04 003 03
FH2158 -056 07 -013 08
USH1C USH1C_SNP2 018 04 04 03
USH1C_SNP4 018 04 04 03
WHRN WHRN_SNP1 046 03 008 03
WHRN_SNP2 046 03 008 03
WHRN_SNP3 046 03 008 03 1 Error probability regarding Zmean 2 Error probability regarding LOD score
Microsatellite marker
Evaluation of eight candidate genes for CCSD 55
Figu
re 1
SN
P-h
aplo
type
s of
the
ES
PN
gen
e in
all
anal
yzed
Dal
mat
ian
dog
fam
ilies
The
hapl
otyp
es b
elon
ging
to F
amily
2 3
and
4 re
gard
ing
the
gene
ES
PN
sho
w n
o as
soci
atio
n w
ith th
e C
CS
D p
heno
type
In
the
thre
e fa
milie
s th
e ha
plot
ype
21
43
2 is
the
mos
t com
mon
one
bei
ng p
rese
nt in
72
o
f all
anim
als
It is
follo
wed
by
the
hapl
otyp
e 2
32
14
with
18
a
nd 4
34
34
with
6
Nor
mal
hea
ring
anim
als
as
wel
l as
thei
r de
af s
iblin
gs s
how
all
thes
e th
ree
hapl
otyp
es T
here
is n
o re
com
bina
tion
of th
e ha
plot
ypes
of t
he E
SP
N g
ene
in th
e th
ree
fam
ilies
Evaluation of eight candidate genes for CCSD 56
Figu
re 2
SN
P-h
aplo
type
s of
the
MY
H14
gen
e in
all
anal
yzed
Dal
mat
ian
dog
fam
ilies
Evaluation of eight candidate genes for CCSD 57
Figu
re 3
SN
P-h
aplo
type
s of
the
MY
O3A
gen
e in
all
anal
yzed
Dal
mat
ian
dog
fam
ilies
Evaluation of eight candidate genes for CCSD 58
Figu
re 4
SN
P-h
aplo
type
s of
the
PR
ES
gen
e in
all
anal
yzed
Dal
mat
ian
dog
fam
ilies
Evaluation of eight candidate genes for CCSD 59
Figu
re 5
SN
P- a
nd m
icro
sate
llite-
hapl
otyp
es o
f the
TM
C1
gene
in a
ll an
alyz
ed D
alm
atia
n
Evaluation of eight candidate genes for CCSD 60
Figu
re 6
SN
P- a
nd m
icro
sate
llite-
hapl
otyp
es o
f the
TM
IE g
ene
in a
ll an
alyz
ed D
alm
atia
n do
g fa
milie
s
Evaluation of eight candidate genes for CCSD 61
Figu
re 7
SN
P-h
aplo
type
s of
the
US
H1C
gen
e in
all
anal
yzed
Dal
mat
ian
dog
fam
ilies
Evaluation of eight candidate genes for CCSD 62
Figu
re 8
SN
P-h
aplo
type
s of
the
WH
RN
gen
e in
all
anal
yzed
Dal
mat
ian
dog
fam
ilies
Chapter 5
Molecular characterization of the canine myosin heavy polypeptide 9
non-muscle (MYH9) gene on dog chromosome 10q232
Canine MYH9 gene 65
Molecular characterization of the canine myosin heavy polypeptide 9 non-muscle (MYH9) gene on dog chromosome 10q232 Abstract A missense mutation of the Myosin heavy polypeptide 9 (MYH9) gene which
encodes the nonmuscle myosin heavy chain-A caused non-syndromic sensorineural
deafness in a human family which was characterized by cochleosaccular
degeneration In the present study we evaluated whether MYH9 gene mutations are
responsible for canine congenital sensorineural deafness (CCSD) in Dalmatian dogs
As described in Chapter 3 two MYH9 gene-associated microsatellites were
genotyped in 25 Dalmatian dog families segregating for CCSD We could find
significant linkage with CCSD for the microsatellite marker MYH9_MS3 (=FH2293)
We used data deposited in the NCBI to assemble the canine MYH9 gene DNA
sequence Characterization of the canine MYH9 gene revealed that the canine gene
consists of 41 exons spanning approximately 90 kb
We detected 22 single nucleotide polymorphisms (SNPs) in a mutation scan of
altogether 16 Dalmatian dogs from three families which showed significant linkage
between the deafness phenotype and the MYH9 gene-associated microsatellite
None of the SNPs affects the amino acid sequence of MYH9 We concluded that the
exons of the canine MYH9 gene do not play a role in Dalmatian CCSD Availability of
the microsatellite marker SNPs and DNA sequence reported in this study enhance
evaluation of the MYH9 gene as a CCSD candidate gene in other Dalmatian dog
populations and other dog breeds affected by CCSD
Introduction Myosin is a functional protein associated with cellular movement cell division muscle
contraction and other functions Members of the myosin super-family are
distinguished from the myosin heavy chains that play crucial roles in cellular
processes The human MYH9 gene consists of 40 exons and spans 67959 bp
Canine MYH9 gene
66
Toothaker et al (1991) mapped a nonmuscle myosin heavy chain gene to human
chromosome (HSA) 22q123-q131 Simons et al (1991) likewise mapped the gene
to HSA22q112 which they designated nonmuscle myosin heavy chain-A (NMMHC-
A)
The similarities between the autosomal dominant giant-platelet disorders May-
Hegglin anomaly Fechtner syndrome and Sebastian syndrome and refinement of the
disease loci for May-Hegglin anomaly and Fechtner syndrome to an overlapping
region of 480 kb on human chromosome 22 suggested that all these three disorders
may be allelic Among the identified candidate genes was the gene encoding
nonmuscle myosin heavy-chain A (MYH9) The May-HegglinFechtner Syndrome
Consortium (2000) demonstrated that mutations in MYH9 result in one of the three
disorders mentioned above The same Consortium also speculated that mutations in
MYH9 may also play a role in another autosomal dominant disorder a form of
nonsyndromic deafness characterized by progressive hearing impairment and
cochleosaccular degeneration This autosomal dominant form of human
nonsyndromic hereditary deafness (DFNA17) was described by Lalwani et al (1999)
They studied a five-generation American family previously reported by Lalwani et al
(1997) with deafness caused by cochleosaccular degeneration (CSD) CSD is the
most common histopathologic finding in cases of profound congenital deafness and
is estimated to occur in approximately 70 of cases in man
DFNA17 maps to the same region as MYH9 Because of the importance of myosins
in hearing Lalwani et al (2000) tested MYH9 as a candidate gene for DFNA17 and
demonstrated a missense mutation in the MYH9 gene in affected members of a
kindred with DFNA17 They found a G-to-A transition at nucleotide 2114 in the MYH9
gene This missense mutation changed codon 705 from an invariant arginine to a
histidine within a highly conserved Src homology 1 (SH1) linker region Previous
studies had shown that modification of amino acid residues within the SH1 helix
causes dysfunction of the ATPase activity of the motor domain in myosin
Many genetic disorders in humans and domestic dogs (Canis familiaris) demonstrate
a high level of clinical and molecular similarity Rak et al (2003) mapped 20 potential
candidate genes for sensorineural deafness in dogs by fluorescence in situ
hybridization and a radiation hybrid panel among them the MYH9 gene that was
assigned to canine chromosome (CFA) 10q232 Congenital sensorineural deafness
has been reported for approximately 54 different breeds of dogs (Strain 1996)
Canine MYH9 gene 67
The incidence of this congenital anomaly is highest in Dalmatian dogs of which 165-
30 exhibit unilateral or bilateral hearing loss (Famula et al 1996 Wood and
Lakhani 1997 Muhle et al 2002 Juraschko et al 2003a Rak and Distl 2005) The
inheritance and segregation of a major gene in CCSD has been demonstrated in
different Dalmatian dog populations (Famula et al 2000 Muhle et al 2002
Juraschko et al 2003b)
In dog breeds the histological pattern is known as cochleosaccular degeneration
commonly known as Scheibe dysplasia as it is described in approximately 70 of
cases with human hereditary deafness (Strain 1996) Therefore the MYH9 gene
seems to be a perfect candidate gene for CCSD especially in Dalmatian dogs
In this report we provide the genomic organization and the complete sequence of the
canine MYH9 gene A mutation analysis was performed to identify single nucleotide
polymorphisms (SNPs) in this gene In order to evaluate whether the MYH9 gene is
responsible for congenital sensorineural deafness in Dalmatian dogs we analyzed
the association of the MYH9 haplotypes with the CCSD phenotype in three families of
Dalmatian dogs with frequent occurrence of CCSD and significant linkage to the
gene-associated microsatellite MYH9_MS3 (Chapter 3)
Materials and Methods
Cloning and sequencing of canine MYH9 cDNA
The canine MYH9 gene sequence was derived from sequences deposited in the
current dog genome assembly (Boxer genome assembly 21) of the NCBI GenBank
(Genbank Acc No NW_1398701) by BLAST (Basic Local Alignment Search Tool)
search (httpwwwncbinlmnihgovBLAST) using the human MYH9 reference
mRNA sequence (Genbank Acc No NM_002473)
The distance between MYH9 and the microsatellite MYH9_MS2 is about 181 kb
whereas the microsatellite MYH9_MS3 (=FH2293) places about 511 kb from MYH9
The isolation of full length cDNAs was achieved by a modified rapid amplification of
cDNA ends (RACE) protocol Total RNA from dog lung of a normal female Beagle
(Biocat Heidelberg Germany) was used for the overlapping 5rsquo- and 3rsquo-RACE
products with the FirstChoiceTM RNA ligase-mediated (RLM)-RACE kit (Ambion
Europe Huntingdon UK) according to the protocols of the manufacturer Reverse
transcription polymerase chain reaction (RT-PCR) using two pairs of nested
Canine MYH9 gene
68
gene-specific and supplied 5rsquo-adaptor-specific primers were used to amplify the
complete 5rsquo-end of the investigated cDNA Similarly the 3rsquo-end were amplified using
two pairs of nested gene-specific primers in combination with supplied 3rsquo-adaptor-
specific primers Additionally RT-PCR using six pairs of gene-specific primers were
used to amplify the complete sequence of the investigated cDNAs
RACE products were cloned into pCRR21 vector using the Invitrogen TA cloning kit
(Invitrogen Karlsruhe Germany) Several clones for each cDNA were sequenced
with the ThermoSequenase kit (AmershamBiosciences Freiburg Germany) and a
LI-COR 4300 DNA Sequencer (LICOR inc Bad Homburg Germany) Sequence
data were analyzed with Sequencher 42 (GeneCodes Ann Arbor MI)
Sequence analysis of canine MYH9 gene For the localization of the exonintron boundaries after cloning and sequencing of full
length canine cDNAs (as described above) the mRNA-to-genomic alignment program
Spidey (httpwwwncbinlmnihgovIEBResearchOstellSpideyindexhtml) was
used Repetitive elements were analyzed with Repeatmasker 2
(httprepeatmaskergenomewashingtonedu) The GC content was calculated with
the EBI toolbox CpG PlotCpGreport (httpwwwebiacukToolssequencehtml)
Mutation analysis To identify polymorphisms within the canine MYH9 sequence exons with flanking
regions were PCR amplified and sequenced from 16 Dalmatian dogs which
represent three families consisting of three to six full sibs and at the best of both
parents At least two of the full sibs of each family were unilaterally deaf The
phenotype of the affected animals had been confirmed by brainstem auditory evoked
response (BAER) that detects electrical activity in the cochlea and auditory pathways
in the brain PCR primers and conditions for the amplification of MYH9 exons with
flanking sequences and microsatellite flanking primers for PCR reactions are given in
Table 1 PCR primers were developed with the Primer3 program
(httpfrodowimiteducgi-binprimer3primer3_wwwcgi) The PCR reactions for
exons and their flanking sequences were performed in a total of 50 microl containing 125
microM dNTPs 25 pmol of each primer the reaction buffer supplied by the manufacturer
(Qiagen Hilden Germany) and 1 U Taq polymerase After a 4 min initial
denaturation at 95 degC 35 cycles of 30 sec at 94 degC 45 sec at 58 degC and 80 sec
Canine MYH9 gene 69
at 72 degC were performed in a MJ Research thermocycler (Biozym Hessisch
Oldendorf Germany) The obtained PCR products were directly sequenced with the
DYEnamic ET Terminator kit (AmershamBiosciences Freiburg Germany) and a
MegaBACE 1000 capillary sequencer (AmershamBiosciences) using the PCR
primers as sequencing primers Sequence data were analyzed with Sequencher 42
(GeneCodes Ann Arbor MI)
Multipoint linkage and haplotype analysis were performed using MERLIN version
0102 (multipoint engine for rapid likelihood inference Center for Statistical Genetics
University of Michigan MI USA Abecasis et al 2002) The test statistics Z-mean
and Lod score were used to test for the proportion of alleles shared by affected
individuals identical by descent (IBD) for the considered marker loci
Linkage means that a haplotype characterized by microsatellites or SNPs is
significantly more often present in family members with the phenotype under study
than expected by random assortment For construction of haplotypes sets of closely
linked genetic markers on the same chromosome are needed which tend to be
inherited together as they are not likely to be separated by recombination
Association analysis can be carried out as a method of genetic analysis that
compares the frequency of alleles between affected and unaffected individuals
across all families A given allele is considered to be associated with the disease if
the presence of that allele explains a significant proportion of the phenotypic trait
variation
Results and Discussion
Analysis of the genomic organization and cDNA of the canine MYH9 gene
A full-length cDNA sequence of 7484 bp of the canine MYH9 gene was obtained by
using the RACE protocol The obtained RT-PCR products were sequenced and the
generated sequence data were submitted to the EMBL nucleotide database (Acc No
AM086385)
Using the RT-PCR analyses for cDNA-genomic sequence comparisons we detected
that the canine MYH9 gene consists of 41 exons with an untranslated exon 1 and
exonintron boundaries that conform perfectly to the GTAG rule (Table 2)
The 5rsquo-UTR consists of 181 bp while the 3rsquo-UTR consists of 1432 bp Assuming that
the homologous ATG start codon as in man is used the canine MYH9 cDNA
Canine MYH9 gene
70
contains an open reading frame of 5889 bp encoding a protein of 1963 amino acids
A canonical polyadenylation signal AAUAAA is located approximately 14 kb
downstream of the stop codon
The exon sizes range from 28 to 1556 bp the introns between these exons span
between 86 and 13493 bp the total size of the canine MYH9 gene is approximately
90 kb However the sequence homology between the human murine and canine
MYH9 gene is limited to the coding sequence of exon 2 to 41 The coding sequence
of canine MYH9 displays 926 and 899 similarities to the human and murine
MYH9 gene respectively In the untranslated regions the sequence similarity
between dog and human and mouse is rather low The canine MYH9 protein shows
982 and 968 identities to the orthologous human and murine protein
respectively (Fig 2)
The repeat content in the 90 kb region of the canine MYH9 gene is 1458 Most of
the repetitive elements belong to the SINE family (892) followed by the fraction of
the LINEs (306) other repetitive elements constitute 165 respectively The
entire canine MYH9 gene has an overall GC-content of 54 The canine MYH9 gene
contains 12 CpG islands (Gardiner-Garden and Frommer 1987) distributed over the
entire gene whereas by far the longest one with a length of 789 bp can be found in
the region of the second exon or in the first translated exon respectively (GC content
of 50 over 200 bp)
Mutation and haplotype analysis
All coding exons with flanking intronic regions of MYH9 could be amplified from the
examined 16 dogs and the sequences were compared to the Boxer genome
assembly 21 of the NCBI Gen Bank (Genbank Acc No NW_1398701)
The search for sequence variations within the MYH9 gene revealed a total of 22
SNPs shown in Table 3 Most of the polymorphisms were found in the flanking
regions of exons only 3 were within exons Only five out of the observed 22 SNPs
were polymorphic in all three examined families
None of the observed polymorphism did alter the predicted amino acid sequence of
MYH9 nor the identified hapltypes showed an association with the CCSD phenotype
Multipoint test statistics for MYH9 alleles identical by descent were close to zero (Z-
mean 035 to -035 Lod score 013 to -007) and not significant (p= 04 to 07)
However the microsatellite MYH9_MS3 (=FH2293) showed a Z-mean of 156
Canine MYH9 gene 71
(p= 006) and a Lod score of 058 (p= 005) indicating linkage The reason for this
result was heterogeneity among these three families For family 1 and 2 the test
statistics indicated linkage (Z-mean 191 with p= 003 Lod score 058 with p= 005)
whereas for familiy 3 the Z-mean and Lod score was -050 (p= 07) and -008 (p=
07)
Thus these silent point mutations found in affected and unaffected Dalmatian dogs
seem not responsible for the CCSD phenotype in these three families
Conclusions
The characterization of the transcript and genomic sequences of canine MYH9 gene
revealed a conserved organization with respect to the human orthologs In general
the gene size in dog is bigger compared to the human sequence due to the
untranlated first exon in the 5rsquo-UTR Several single nucleotide polymorphisms in the
canine MYH9 gene were identified However because of the fact that both animals
with unilateral hearing loss and bilateral hearing animals shared identical
haplotypes these polymorphisms are obviously not associated with CCSD in these
Dalmatian dog families Furthermore the present study revealed no functional
mutations of the complete coding region of MYH9 We can exclude the MYH9 gene
as a candidate gene for congenital sensorineunal deafness in Dalmatian dogs
However the MYH9 gene sequence SNPs and microsatellite markers reported in
this study enhance evaluation of the MYH9 gene in other Dalmatian dog populations
and dog breeds other than Dalmatians
Canine MYH9 gene
72
Table 1 PCR primers for the amplification of all MYH9 exons
Exon Primer Sequence (5rsquo ndash 3rsquo) Product
size (bp)
Annealing temperature
(degC) exon 1 MYH9_Ex1_F GCC TGG TTT GAC AAG TTT TG MYH9_Ex1_R TTC CTC AAG TCC CCT ACT CC 560 58 exon 2 MYH9_Ex2_F AGG ACC CTC ACC CAA CAC MYH9_Ex2_R GCA CAG AGT CTG GAC AAG AAT G 621 58 exon 3 MYH9_Ex3_F CAC CCC TCA ATA CTC AGT GC MYH9_Ex3_R GAG AAG ACT CCT GCC TCT CC 588 58 exon 4 MYH9_Ex4_F GGC AAT GTG TTT GAT TGT ACT G MYH9_Ex4_R TTT CCA AAG CAA CTC AGA AAG 542 58 exon 5 -6 MYH9_Ex56_F ACA GAG CAG GCG TGA CAG MYH9_Ex56_R GAG GAC AAG AGC TGG AAG C 724 58 exon 7 -8 MYH9_Ex78_F AAG CGG AAA GTT GCC TTC MYH9_Ex78_R ATG GCA CAA TGA CCA GTC TC 699 58 exon 9 MYH9_Ex9_F TGG GCA CAG TTT TTG TCT G MYH9_Ex9_R CAC ACA ATC AAG GGC TTT TC 590 58 exon 10 MYH9_Ex10_F GGA GCC AGT TGT CCT AGT AGT G MYH9_Ex10_R GCC AGG TGC CTA TCA CTG 540 58 exon 11 MYH9_Ex11_F TGT TTC GAA GCG TTT TTA CC MYH9_Ex11_R GGT CCC ATT GTT TCT AGC AC 551 58 exon 12 MYH9_Ex12_F TTT CCC AAG CAG CTA GAA AG MYH9_Ex12_R ACT GGG TGG TCT TGA GAC TG 586 58 exon 13 MYH9_Ex13_F CTG CCT TTG GTG ACA AGT G MYH9_Ex13_R GCA AGC CCT AAG TAC AAT GG 548 58 exon 14 MYH9_Ex14_F ATA CCA GGT CAA GGG GTG TC MYH9_Ex14_R TCA CCT GGT TCT AGG TGG AG 524 58 exon 15 MYH9_Ex15_F CGT TTG GAG TTG AT CCT AGC MYH9_Ex15_R AGC CAA GTT TCC TGA CTG TG 566 58 exon 16 MYH9_Ex16_F AGA TAG GCT GCA GTG GTA GC MYH9_Ex16_R CAG TAG CAA ACA GGG CTA GG 568 58 exon 17 MYH9_Ex17_F AAC GGT CCA ATG TGA GTT TC MYH9_Ex17_R ACC AGC TCA CAA CAG TAC CC 559 58 exon 18 MYH9_Ex18_F TGT AGG CTG TGA TTG CAG AG MYH9_Ex18_R CCC TCT CTC AAA CAA ATT GC 586 58 exon 19 MYH9_Ex19_F CCT TGG TGA AGG AAA GTG G MYH9_Ex19_R GCT GTG TGC AGG TTC TGA C 657 58 exon 20 MYH9_Ex20_F AGC AGC ATA GCA CTG AAT CC MYH9_Ex20_R TTC CCA TCT CTC ATG TTC TTG 579 58
Canine MYH9 gene 73
Table 1 (continued)
Exon Primer Sequence (5rsquo ndash 3rsquo) Product
size (bp)
Annealing temperature
(degC) exons 21 MYH9_Ex21_F CTC CTG AGT GTT GGG TTA GC MYH9_Ex21_R CTC TAA CAG GCA GCC CAA G 555 58 exon 22 MYH9_Ex22_F GGA GGA AAC GCA GAA ATC C MYH9_Ex22_R ACG GAC ACC CAG GAA AAC 660 58 exon 23 MYH9_Ex23_F CAG GGG TGC TAG TGA TCT TTC MYH9_Ex23_R AGA GCT GGG CTC AGA CCT AC 630 58 exon 24 MYH9_Ex24_F TCA GGG ATG TGC TTT CTT TC MYH9_Ex24_R CCC CAA GCA CAT CTG TAG TC 563 58 exon 24 MYH9_Ex24_F TCA GGG ATG TGC TTT CTT TC MYH9_Ex24_R CCC CAA GCA CAT CTG TAG TC 563 58 exon 25 -26 MYH9_Ex2526F GGC TGC TCT TCT CTG AAG C MYH9_Ex2526R GGA GTC AGG CTT CCA GTA AAG 851 58 exon 27 -28 MYH9_Ex2728F AGC ACA TTC TGG ATC TGG AG MYH9_Ex2728R GCG ACA GCT GAA TTC CTG 752 58 exon 29 MYH9_Ex29_F CAG CTT CAG GAC ACA CAG G MYH9_Ex29_R CTC TGG GTC TCC CCT GTC 601 58 exon 30 MYH9_Ex30_F GAG GCT TGG TGT ATG TCT CG MYH9_Ex30_R TGG TTC CAT AGA AAC GAT CTG 592 58 exon 31 - 32 MYH9_Ex3132F CAT TCC CCA AGG AGT TTT G MYH9_Ex3132R AAC ACA GCC CAG GGA AAG 770 58 exon 33 MYH9_Ex33_F CCT GGA ACA AGG CTG AGA AC MYH9_Ex33_R TCT CAG AGC AGC TTT GCT TC 600 58 exon 34 MYH9_Ex34_F GGT GTG CTT GGT AAA TCC TG MYH9_Ex34_R CAC AAA GCC TCA TTT CAC G 611 58 exon 34 - 36 MYH9_Ex3536F AGG TCA CGG TCT GGG TTC T MYH9_Ex3536R CAA GGG AGG CTG TTG CAG 401 60 exon 37 MYH9_Ex37_F CTC CCT TGG TTT CCC TCT CT MYH9_Ex37_R CAG GTC AGG AGG GGA CAT 488 60 exon 38 -39 MYH9_Ex3839F AGG CCT AAG GAC AGC TCA TC MYH9_Ex3839R CCA GGG CAC AGA TAG AAC C 545 58 exon 40 MYH9_Ex40_F1 GTC CCT CTA AAG ATG GCT TTG MYH9_Ex40_R1 TGG GGA GGA ACG ACT CTC 844 58 MYH9_Ex40_F2 TGG GTG ATT GGG GAA GAG MYH9_Ex40_R2 CCA GAA GAT GCA CAA AGT AGG 981 58
Canine MYH9 gene
74
Table 2 Exonintron boundaries of the canine MYH9 gene
3rsquo-Splice site Exon 5rsquo-Splice site
Intron phase
Intron size
-20 (exon 1 162 bp) GTTCAGgtgagcgagccccgc
gt30000 bp
-19 +333 gctgtagGTCCTGGCTGTGA (exon 2 352 bp) CGTAAgtggggcctggggcc
0
4922 bp
+334 +490 tctttagACCTATTCAGGCC (exon 3 157 bp) GCAAGgtgatgcctcggcctt
1
13493 bp
+491 +518 gtttcagACCGAGAGGACCA (exon 4 28 bp) TGCACgtaagtgtttctgcc
2
803 bp
+519 +612 tcggcagGGGTGAGTCTGGA (exon 5 94 bp) ACCAGgtgagtgcgggcctt
0
4077 bp
+613 +705 cttgtagGGTGAGTTGGAGA (exon 6 93 bp) GATTTgtgagtagcggccag
0
427 bp
+706 +769 tttttagGGCAAATTCATTC (exon 7 64 bp) GACTTgtatccttggttgat
1
738 bp
+770 +868 tgcaaagATCTTCTGGAGAA (exon 8 99 bp) GAAGAgtgagtagtgagccc
1
343 bp
+869 +1012 ctttcagCTGATCTCCTGTT (exon 9 144 bp) AATGGgtacagagctggccc
1
749 bp
+1013 +1108 cccgaagGCTTGTTGCGAGT (exon 10 96 bp) CACAGgtaacgcctgtggtcc
1
1041 bp
+1109 +1227 atcgcagCTGCCCAGAAAGT (exon 11 119 bp) AACAGgtaggctctctggcc
0
1877 bp
+1228 +1380 accccagGCTGACTTTGCCA (exon 12 153 bp) TTGATgtgagctcctctcct
0
922 bp
+1381 +1554 cctgcagCTGAACTCCTTCG (exon 13 174 bp) AGCCAgtaagagccagacct
0
1801 bp
+1555 +1728 ttcccagGCAGGGCCCCCTG (exon 14 174 bp) GCAAGgtgaggcaagcacgt
0
2049 bp
+1729 +1843 cttgcagGTGGACTACAAAG (exon 15 115 bp) GGATGgtacgtgccccctgg
1
1877 bp
+1844 +2037 tttgcagTGGACCGCATCAT (exon 16 194 bp) AGAAGgtgcgggtgctgggc
0
343 bp
+2038 +2159 tctccagGCTGGCAAACTGG (exon 17 122 bp) CAGAGgtgagcggggcctgt
1
835 bp
+2160 +2229 ccctcagATATGAGATCTTG (exon 18 70 bp) TCATGgtgagtagagctgct
2
1418 bp
+2230 +2390 cgtctagATCAAAGCCCTGG (exon 19 161 bp) AGGAAgtgagttcctaaagg
1
851 bp
+2391 +2499 tccccagGGCCTTTGCCAAG (exon 20 109 bp) CCAAGgtgggttctggaagg
2
1428 bp
+2500 +2631 cttctagGTCAAGCCACTGC (exon 21 132 bp) CCCAGgtaggtgtcagcagg
2
398 bp
+2632 +2838 ttggcagCTCATGGCAGAGA (exon 22 207 bp) TCCAAgtgagccccttccct
2
488 bp
Canine MYH9 gene 75
Table 2 (continued)
3rsquo-Splice site Exon 5rsquo-Splice site
Intron phase
Intron size
+2839 +2976 atggtagGAACTGGAGGAGC (exon 23 138 bp) CCAAGgtgaggcccgtggtg
2
971 bp
+2977 +3100 cccccagGAGAAGAAGCTTC (exon 24 124 bp) GGAAGgtaagagggtaacag
0
1603 bp
+3101 +3272 tcctcagAGCGCCTCCGAAG (exon 25 172 bp) GCCAGgtgagggcctcgctt
1
719 bp
+3273 +3485 ggcagagTGGAGGAGGAAGC (exon 26 213 bp) CTCAGgtgtgccatgcggcc
1
270 bp
+3486 +3630 tctccagGTCCAAACGCGAA (exon 27 145 bp) AACGGgtgcgtgggctcccc
2
480 bp
+3631 +3837 ccaccagGTGAAAGCCAACT (exon 28 207 bp) TGCAGgtgaggcctccatgg
2
232 bp
+3838 +3941 gccacagGTTGAGCTGGACA (exon 29 104 bp) ACACAgtaaaggtggcagcg
1
204 bp
+3942 +4094 ggtgtagGAGCTGCTCCAGG (exon 30 153 bp) CCCAGgttcttatcggcatg
1
1083 bp
+4095 +4343 tgcccagGTGACGGACATGA (exon 31 249 bp) ACCAGgtgtgggccccaggc
1
1298 bp
+4344 +4556 tccccagCTACTGGCCGAGG (exon 32 213 bp) AGAGCgtgagtaccatgtca
1
150 bp
+4557 +4769 gtggcagGTCCACGAGCTGG (exon 33 213 bp) GACAGgtgtggaggccctcc
1
303 bp
+4770 +4931 gccccagGTGCGGGAGATGG (exon 34 162 bp) TGCAGgtgaggtcggctggg
1
1173 bp
+4932 +5060 accccagGCCCAGATGAAGG (exon 35 129 bp) AGGAGgtgggccgggcctca
1
941 bp
+5061 +5149 atggcagGAACTGGCAGCTG (exon 36 89 bp) AAGGGgtgagccgtggcgtg
0
92 bp
+5150 +5273 cacccagAGCTCTGGCATTG (exon 37 124 bp) TGCAGgtgggcggggtcggg
2
224 bp
+5274 +5482 cccacagATCGACCAGATCA (exon 38 209 bp) ACCAAgtaggtgttcctgcc
1
563 bp
+5483 +5591 ctcgcagGGAGCGCCAGGCA (exon 39 109 bp) ACCAGgtgtgtgtgcacacc
2
86 bp
+5592 +5763 tctgcagGCCGACAAGGCAT (exon 40 172 bp) CTCAGgtgagccctgcctcc
0
739 bp
+5764 +7295 tgcaaagATCTTCTGGAGAA (exon 41 1532 bp) CGTCACAATAAAGAGAACCA
Exon sequences are shown in uppercase letters and intron sequences in lowercase
letters Untranslated regions are shown in italics The conserved GTAG exonintron
junctions are shown in boldface type For the last exon the polyadenylation signal is
shown underlined instead of an exonintron junction Position +1 corresponds to the
adenine of the translation initiation codon ATG
Canine MYH9 gene
76
16
TT
TC
G
A
TC
A
A C
A
CT
T
C
GC
G
G
AG
C
C
AG
G
A
CT
C
T
GA
A
G
CC
C
T
CC
T
C
96
96
229
23
7
15
TT
TC
G
A
TC
A
A C
A
CT
T
C
GC
G
G
AG
C
C
AG
G
A
CT
C
T
GA
A
G
CC
C
T
CC
T
C
96
96
229
22
9
14
deaf
T
T T
C
GA
T
C
AA
CA
C
T
TC
G
C
GG
A
G
CC
A
G
GA
C
T
CT
G
A
AG
C
C
CT
C
C
TC
96
96
22
9
229
13
deaf
T
T T
C
GA
T
C
AA
CA
C
T
TC
G
C
GG
A
G
CC
A
G
GA
C
T
CT
G
A
AG
C
C
CT
C
C
TC
96
96
22
9
229
12
deaf
T
T C
C
AA
CC
A
A A
A T
T C
C
CC
G
G
GG
C
C
GG
A
A C
C
CC
A
A G
G
CC
T
T C
C
CC
96
10
0 24
1
229
11
deaf
T
T C
C
AA
CC
A
A A
A T
T C
C
CC
G
G
GG
C
C
GG
A
A C
C
CC
A
A G
G
CC
T
T C
C
CC
96
10
0 24
1
229
10
TT
TC
G
A
TC
A
A C
A
CT
T
C
GC
G
G
AG
C
C
AG
G
A
CT
C
T
GA
A
G
CC
C
T
CC
T
C
96
96
237
22
9
Fam
ily 3
9 T
T C
C
AA
CC
A
A A
A T
T C
C
CC
G
G
GG
C
C
- - CC
C
C
AA
GG
C
C
TT
CC
C
C
96
100
229
24
1
8 T
T C
T
AG
C
T
AA
AA
TT
CC
C
C
GG
G
G
CT
G
G
AA
CC
C
C
AA
GG
C
C
TT
CC
C
C
96
96
225
25
3
7 de
af
TC
C
C
AG
C
T
AT
A
A T
T C
C
CC
G
A
GG
C
T
GG
A
G
CC
C
C
AA
GG
C
T
TC
C
T
CC
96
89
22
1
237
6 de
af
TC
C
C
AG
C
T
AT
A
A T
T C
C
CC
G
A
GG
C
T
GG
A
G
CC
C
C
AA
GG
C
T
TC
C
T
CC
96
89
22
1
237 Fa
mily
2
5 T
C
CC
A
G
CT
A
T
AA
TT
CC
C
C
GA
G
G
CT
G
G
AG
C
C
CC
A
A G
G
CT
T
C
CT
C
C
96
89
221
23
7
4 de
af
TT
CC
A
A C
C
AA
AA
TT
CC
C
C
GG
G
G
CC
G
G
AG
C
C
CC
A
A G
G
CC
T
T C
C
CC
96
96
23
7
257
3 de
af
TC
C
C
AG
C
T
AT
A
A T
T C
C
CC
G
A
GG
C
T
GG
G
A
CC
C
C
AA
GG
C
T
TC
C
T
CC
96
96
23
7
257
2 T
C
CC
A
G
CT
A
T
AA
TT
CC
C
C
GA
G
G
CT
G
G
AA
CC
C
C
AA
GG
C
T
TC
C
T
CC
96
96
23
7
257 Fa
mily
1
1 C
T
CC
G
A
TC
T
A A
A T
T C
C
CC
A
G
GG
T
C
GG
A
G
CC
C
C
AA
GG
T
C
CT
T
C
CC
96
96
25
7
257
Ref
eren
ce
se
quen
ce
NW
_139
870
T C
A T A A T C
C
G
G
C
G
A C
C
A G
C
T C
C
posi
tion1
212
9
1 4
4 2
39
125
1
32
316
3
78
42
218
1
8 1
75
24
13
8
207
2
63
287
9
22
40
59
64
Pos
ition
with
in M
YH
9 In
tron
34
Exo
n 4
Intro
n 14
15
Intro
n 15
16
Intro
n 16
17
Intro
n 18
19
Intro
n 20
21
Intro
n 20
21
Intro
n 25
26
Intro
n 25
26
Intro
n 26
27
Intro
n 27
28
Exo
n 29
In
tron
293
0 In
tron
303
1 E
xon
32
Intro
n 32
33
Intro
n 32
33
Intro
n 34
35
Intro
n 38
39
Intro
n 38
39
Intro
n 38
39
Mic
rosa
tellit
e M
YH
9_M
S2
Mic
rosa
tellit
e M
YH
9_M
S3
(=FH
2293
)
1 N
umbe
ring
refe
rs to
the
posi
tion
of th
e po
lym
orph
ic n
ucle
otid
e w
ithin
the
give
n ex
on
or in
tron
Tabl
e 3
Hap
loty
pes
of th
e ca
nine
MY
H9
gene
in16
Dal
mat
ian
dogs
Canine MYH9 gene 77
Figure 1 Family structure of the analyzed Dalmatian dog families Hearing status was tested by brain auditory evoked response (BAER)
Canine MYH9 gene
78
Figure 2 Alignment of the canine MYH9 protein with orthologous human and murine
MYH9 protein sequences The sequences where derived from GenBank entries with
the accessions NP_002464 (human MYH9) and NP_071855 (mouse MYH9)
Identical residues are indicated by asterisk beneath the alignment while dots and
colons represent similar or very similar amino acids respectively
dog MAQQAADKYLYVDKNFINNPLAQADWAAKKLVWVPSDKSGFEPASLKEEVGEEAIVELVE 60 human MAQQAADKYLYVDKNFINNPLAQADWAAKKLVWVPSDKSGFEPASLKEEVGEEAIVELVE 60 mouse MAQQAADKYLYVDKNFINNPLAQADWAAKKLVWVPSSKNGFEPASLKEEVGEEAIVELVE 60 dog NGKKVKVNKDDIQKMNPPKFSKVEDMAELTCLNEASVLHNLKERYYSGLIYTYSGLFCVV 120 human NGKKVKVNKDDIQKMNPPKFSKVEDMAELTCLNEASVLHNLKERYYSGLIYTYSGLFCVV 120 mouse NGKKVKVNKDDIQKMNPPKFSKVEDMAELTCLNEASVLHNLKERYYSGLIYTYSGLFCVV 120 dog INPYKNLPIYSEEIVEMYKGKKRHEMPPHIYAITDTAYRSMMQDREDQSILCTGESGAGK 180 human INPYKNLPIYSEEIVEMYKGKKRHEMPPHIYAITDTAYRSMMQDREDQSILCTGESGAGK 180 mouse INPYKNLPIYSEEIVEMYKGKKRHEMPPHIYAITDTAYRSMMQDREDQSILCTGESGAGK 180 dog TENTKKVIQYLAHVASSHKSKKDQGELERQLLQANPILEAFGNAKTVKNDNSSRFGKFIR 240 human TENTKKVIQYLAYVASSHKSKKDQGELERQLLQANPILEAFGNAKTVKNDNSSRFGKFIR 240 mouse TENTKKVIQYLAHVASSHKSKKDQGELERQLLQANPILEAFGNAKTVKNDNSSRFGKFIR 240 dog INFDVNGYIVGANIETYLLEKSRAIRQAKEERTFHIFYYLLSGAGEHLKTDLLLEPYNKY 300 human INFDVNGYIVGANIETYLLEKSRAIRQAKEERTFHIFYYLLSGAGEHLKTDLLLEPYNKY 300 mouse INFDVNGYIVGANIETYLLEKSRAIRQAKEERTFHIFYYLLSGAGEHLKTDLLLEPYNKY 300 dog RFLSNGHVTIPGQQDKDMFQETMEAMRIMGIPEEEQMGLLRVISGVLQLGNIVFKKERNT 360 human RFLSNGHVTIPGQQDKDMFQETMEAMRIMGIPEEEQMGLLRVISGVLQLGNIVFKKERNT 360 mouse RFLSNGHVTIPGQQDKDMFQETMEAMRIMGIPEDEQMGLLRVISGVLQLGNIAFKKERNT 360 dog DQASMPDNTGNASAQKVSHLLGINVTDFTRGILTPRIKVGRDYVQKAQTKEQADFAIEAL 420 human DQASMPDNT---AAQKVSHLLGINVTDFTRGILTPRIKVGRDYVQKAQTKEQADFAIEAL 417 mouse DQASMPDNT---AAQKVSHLLGINVTDFTRGILTPRIKVGRDYVQKAQTKEQADFAIEAL 417 dog AKATYERMFRWLVLRINKALDKTKRQGASFIGILDIAGFEIFDLNSFEQLCINYTNEKLQ 480 human AKATYERMFRWLVLRINKALDKTKRQGASFIGILDIAGFEIFDLNSFEQLCINYTNEKLQ 477 mouse AKATYERMFRWLVLRINKALDKTKRQGASFIGILDIAGFEIFDLNSFEQLCINYTNEKLQ 477 dog QLFNHTMFILEQEEYQREGIEWNFIDFGLDLQPCIDLIEKPAGPPGILALLDEECWFPKA 540 human QLFNHTMFILEQEEYQREGIEWNFIDFGLDLQPCIDLIEKPAGPPGILALLDEECWFPKA 537 mouse QLFNHTMFILEQEEYQREGIEWNFIDFGLDLQPCIDLIEKPAGPPGILALLDEECWFPKA 537 dog TDKSFVEKVVQEQGTHPKFQKPKQLKDKADFCIIHYAGKVDYKADEWLMKNMDPLNDNIA 600 human TDKSFVEKVMQEQGTHPKFQKPKQLKDKADFCIIHYAGKVDYKADEWLMKNMDPLNDNIA 597 mouse TDKSFVEKVVQEQGTHPKFQKPKQLKDKADFCIIHYAGKVDYKADEWLMKNMDPLNDNIA 597
Canine MYH9 gene 79
dog TLLHQSSDKFVSELWKDVDRIIGLDQVAGMSETALPGAFKTRKGMFRTVGQLYKEQLAKL 660 human TLLHQSSDKFVSELWKDVDRIIGLDQVAGMSETALPGAFKTRKGMFRTVGQLYKEQLAKL 657 mouse TLLHQSSDKFVSELWKDVDRIIGLDQVAGMSETALPGAFKTRKGMFRTVGQLYKEQLAKL 657 dog MATLRNTNPNFVRCIIPNHEKKAGKLDPHLVLDQLRCNGVLEGIRICRQGFPNRVVFQEF 720 human MATLRNTNPNFVRCIIPNHEKKAGKLDPHLVLDQLRCNGVLEGIRICRQGFPNRVVFQEF 717 mouse MATLRNTNPNFVRCIIPNHEKKAGKLDPHLVLDQLRCNGVLEGIRICRQGFPNRVVFQEF 717 dog RQRYEILTPNSIPKGFMDGKQACVLMIKALELDSNLYRIGQSKVFFRAGVLAHLEEERDL 780 human RQRYEILTPNSIPKGFMDGKQACVLMIKALELDSNLYRIGQSKVFFRAGVLAHLEEERDL 777 mouse RQRYEILTPNSIPKGFMDGKQACVLMIKALELDSNLYRIGQSKVFFRAGVLAHLEEERDL 777 dog KITDVIIGFQACCRGYLARKAFAKRQQQLTAMKVLQRNCAAYLKLRNWQWWRLFTKVKPL 840 human KITDVIIGFQACCRGYLARKAFAKRQQQLTAMKVLQRNCAAYLKLRNWQWWRLFTKVKPL 837 mouse KITDVIIGFQACCRGYLARKAFAKRQQQLTAMKVLQRNCAAYLRLRNWQWWRLFTKVKPL 837 dog LQVSRQEEEMMAKEEELVKVREKQLAAENRLTEMETLQSQLMAEKLQLQEQLQAETELCA 900 human LQVSRQEEEMMAKEEELVKVREKQLAAENRLTEMETLQSQLMAEKLQLQEQLQAETELCA 897 mouse LNSIRHEDELLAKEAELTKVREKHLAAENRLTEMETMQSQLMAEKLQLQEQLQAETELCA 897 dog EAEELRARLTAKKQELEEICHDLEARVEEEEERCQHLQAEKKKMQQNIQELEEQLEEEES 960 human EAEELRARLTAKKQELEEICHDLEARVEEEEERCQHLQAEKKKMQQNIQELEEQLEEEES 957 mouse EAEELRARLTAKKQELEEICHDLEARVEEEEERCQYLQAEKKKMQQNIQELEEQLEEEES 957 dog ARQKLQLEKVTTEAKLKKLEEDQIIMEDQNCKLAKEKKLLEDRIAEFTTNLMEEEEKSKS 1020 human ARQKLQLEKVTTEAKLKKLEEEQIILEDQNCKLAKEKKLLEDRIAEFTTNLTEEEEKSKS 1017 mouse ARQKLQLEKVTTEAKLKKLEEDQIIMEDQNCKLAKEKKLLEDRVAEFTTNLMEEEEKSKS 1017 dog LAKLKNKHEAMITDLEERLRREEKQRQELEKTRRKLEGDSTDLNDQIAELQAQIAELKMQ 1080 human LAKLKNKHEAMITDLEERLRREEKQRQELEKTRRKLEGDSTDLSDQIAELQAQIAELKMQ 1077 mouse LAKLKNKHEAMITDLEERLRREEKQRQELEKTRRKLEGDSTDLSDQIAELQAQIAELKMQ 1077 dog LAKKEEELQAALARVEEEATQKNMALKKIRELESQISELQEDLESERASRNKAEKQKRDL 1140 human LAKKEEELQAALARVEEEAAQKNMALKKIRELESQISELQEDLESERASRNKAEKQKRDL 1137 mouse LAKKEEELQAALARVEEEAAQKNMALKKIRELETQISELQEDLESERASRNKAEKQKRDL 1137 dog GEELEALKTELEDTLDSTAAQQELRSKREQEVNILKKTLEEEARTHEAQIQEMRQKHSQA 1200 human GEELEALKTELEDTLDSTAAQQELRSKREQEVNILKKTLEEEAKTHEAQIQEMRQKHSQA 1197 mouse GEELEALKTELEDTLDSTAAQQELRSKREQEVSILKKTLEDEAKTHEAQIQEMRQKHSQA 1197 dog VEELAEQLEQTKRVKANLEKAKQTLENERGELANEVKVLQQGKGDSEHKRKKAEAQLQEL 1260 human VEELAEQLEQTKRVKANLEKAKQTLENERGELANEVKVLLQGKGDSEHKRKKVEAQLQEL 1257 mouse VEELADQLEQTKRVKATLEKAKQTLENERGELANEVKALLQGKGDSEHKRKKVEAQLQEL 1257 dog QVKFTEGERVRTELADKVTKLQVELDNVMGLLTQSDSKSSKLTKDFSALESQLQDTQELL 1320 human QVKFNEGERVRTELADKVTKLQVELDNVTGLLSQSDSKSSKLTKDFSALESQLQDTQELL 1317 mouse QVKFSEGERVRTELADKVTKLQVELDSVTGLLSQSDSKSSKLTKDFSALESQLQDTQELL 1317
Canine MYH9 gene
80
dog QEENRQKLSLSTKLKQMEDEKNSFKEQLEEEEEAKRNLEKQIATLHAQVTDMKKKMEDGV 1380 human QEENRQKLSLSTKLKQVEDEKNSFREQLEEEEEAKHNLEKQIATLHAQVADMKKKMEDSV 1377 mouse QEENRQKLSLSTKLKQMEDEKNSFREQLEEEEEAKRNLEKQIATLHAQVTDMKKKMEDGV 1377 dog GCLETAEEAKRKLQKDLEGLGQRYEEKVAAYDKLEKTKTRLQQELDDLLVDLDHQRQTAS 1440 human GCLETAEEVKRKLQKDLEGLSQRHEEKVAAYDKLEKTKTRLQQELDDLLVDLDHQRQSAC 1437 mouse GCLETAEEAKRRLQKDLEGLSQRLEEKVAAYDKLEKTKTRLQQELDDLLVDLDHQRQSVS 1437 dog NLEKKQKKFDQLLAEEKTISAKYAEERDRAEAEAREKETKALSLARALEEAMEQKAELER 1500 human NLEKKQKKFDQLLAEEKTISAKYAEERDRAEAEAREKETKALSLARALEEAMEQKAELER 1497 mouse NLEKKQKKFDQLLAEEKTISAKYAEERDRAEAEAREKETKALSLARALEEAMEQKAELER 1497 dog LNKQFRTEMEDLMSSKDDVGKSVHELEKSKRALEQQVEEMKTQLEELEDELQATEDAKLR 1560 human LNKQFRTEMEDLMSSKDDVGKSVHELEKSKRALEQQVEEMKTQLEELEDELQATEDAKLR 1557 mouse LNKQFRTEMEDLMSSKDDVGKSVHELEKSKRALEQQVEEMKTQLEELEDELQATEDAKLR 1557 dog LEVNLQAMKAQFERDLQGRDEQSEEKKKQLVRQVREMEAELEDEKKQRSMAVAARKKLEM 1620 human LEVNLQAMKAQFERDLQGRDEQSEEKKKQLVRQVREMEAELEDERKQRSMAVAARKKLEM 1617 mouse LEVNLQAMKAQFERDLQGRDEQSEEKKKQLVRQVREMEAELEDERKQRSMAMAARKKLEM 1617 dog DLKDLEAHIDSANKNRDEAIKQLRKLQAQMKDCVRELDDTRASREEILAQAKENEKKMKS 1680 human DLKDLEAHIDSANKNRDEAIKQLRKLQAQMKDCMRELDDTRASREEILAQAKENEKKLKS 1677 mouse DLKDLEAHIDTANKNREEAIKQLRKLQAQMKDCMRELDDTRASREEILAQAKENEKKLKS 1677 dog MEAEMIQLQEELAAAERAKRQAQQERDELADEIANSSGKGALALEEKRRLEARIAQLEEE 1740 human MEAEMIQLQEELAAAERAKRQAQQERDELADEIANSSGKGALALEEKRRLEARIAQLEEE 1737 mouse MEAEMIQLQEELAAAERAKRQAQQERDELADEIANSSGKGALALEEKRRLEARIALLEEE 1737 dog LEEEQGNTELVNDRLKKANLQIDQINTDLNLERSHAQKNENARQQLERQNKELKVKLQEM 1800 human LEEEQGNTELINDRLKKANLQIDQINTDLNLERSHAQKNENARQQLERQNKELKVKLQEM 1797 mouse LEEEQGNTELINDRLKKANLQIDQINTDLNLERSHAQKNENARQQLERQNKELKAKLQEM 1797 dog EGTVKSKYKASITALEAKIAQLEEQLDNETKERQAACKQVRRAEKKLKDVLLQVDDERRN 1860 human EGTVKSKYKASITALEAKIAQLEEQLDNETKERQAACKQVRRTEKKLKDVLLQVDDERRN 1857 mouse ESAVKSKYKASIAALEAKIAQLEEQLDNETKERQAASKQVRRTEKKLKDVLLQVEDERRN 1857 dog AEQFKDQADKASTRLKQLKRQLEEAEEEAQRANASRRKLQRELEDATETADAMNREVSSL 1920 human AEQYKDQADKASTRLKQLKRQLEEAEEEAQRANASRRKLQRELEDATETADAMNREVSSL 1917 mouse AEQFKDQADKASTRLKQLKRQLEEAEEEAQRANASRRKLQRELEDATETADAMNREVSSL 1917 dog KNKLRRGDLPFVVPRRVARKGAGDCSDEEVDGKADGAEAKAAE 1963 human KNKLRRGDLPFVVPRRMARKGAGDGSDEEVDGKADGAEAKPAE 1960 mouse KNKLRRGDLPFVVTRRIVRKGTGDCSDEEVDGKADGADAKAAE 1960
Chapter 6
Identification of a 5 Mb region on canine chromosome 10
harbouring a causative gene responsible for congenital sensorineural deafness
in German Dalmatian dogs
Fine mapping of CFA10 83
Identification of a 5 Mb region on canine chromosome 10 harbouring a causative gene responsible for congenital sensorineural deafness in German Dalmatian dogs
Abstract
In the present study we evaluated whether the canine chromosome (CFA) 10
harbours a gene responsible for canine congenital sensorineural deafness (CCSD) in
Dalmatian dogs Altogether 27 microsatellite markers covering the whole CFA10
were genotyped in 176 Dalmatian dogs with frequent occurrence of CCSD
Significant linkage between the deafness phenotype and microsatellites located in a
region of 36 Mb to 48 Mb on CFA10 was found In order to refine the location of the
causative canine congenital sensorineural deafness (CCSD) gene we used data
deposited in the NCBI to search for single nucleotide polymorphisms (SNPs) in the
intronic sequences of the canine genes located on CFA10 in this region We
characterized 26 SNPs and used them for non-parametric linkage and association
analyses with CCSD of altogether 34 Dalmatian dogs from five full-sib families We
could find significant linkage to SNPs located in the region spanning 39 Mb to 44 Mb
and significant haplotype-trait association for SNPs in this region These results
enforce further evaluation of this 5 Mb region with the aim to detect the gene
responsible for CCSD in Dalmatian dogs
Introduction
Of the identified genes responsible for different forms of sensorineural non-syndromic
deafness in humans Rak et al (2003) and co-workers (Droumlgemuumlller et al 2002
Kuiper et al 2002 Rak et al 2002a 2002b 2003) considered 24 genes as
candidates for sensorineural deafness in dogs among them the MYH9 gene on
CFA10 Subsequently Rak (2003) developed a set of microsatellite markers for the
respective 24 candidate genes
As described in chapter 3 we could show significant linkage of CCSD with the MYH9
associated microsaellite MYH9_MS3 (=FH2293) in different German Dalmatian dog
Fine mapping of CFA10
84
families However as described in Chapter 5 we already excluded MYH9 for being
responsible for the CCSD phenotype in German Dalmatian dog families segregating
for CCSD by comparative sequencing of genomic sequences from deaf and normal
hearing Dalmatian dogs and additionally by sequencing of full length canine cDNA
The reason for the MYH9_MS3 (=FH2293) indicating linkage may be caused by a
closely linked gene involved in CCSD Thus the objective of the present study was to
perform a scan of canine chromosome 10 using microsatellite markers and single
nucleotide polymorphisms (SNPs) in order to evaluate whether CFA10 harbours a
gene responsible for the deafness phenotype in Dalmatian dogs Additionally we
analyzed the association of the CCSD phenotype with a large number of newly
developed SNPs located in the genomic deafness region on CFA10
Material and methods Pedigree material
For the linkage analysis of the microsatellite marker on CFA10 we used DNA from
altogether 176 animals belonging to 22 full-sib families and one large paternal half-
sib family of German Dalmatian dogs All families were segregating for CCSD The
genotyped families included all the affected dogs (unilaterally and bilaterally deaf)
their parents if available and one to four unaffected full-sibs At least two of the full
sibs of each family were unilaterally deaf The phenotype of the affected animals had
been confirmed by brainstem auditory evoked response (BAER) that detects
electrical activity in the cochlea and auditory pathways in the brain
Screening for SNPs was performed by comparative sequencing of DNA from parents
of five families with significant linkage of microsatellites located in the region
spanning 36 Mb to 48 Mb to CCSD For the linkage analysis concerning the SNPs we
then used blood samples from 34 Dalmatian dogs consisting of the progeny and their
parents of the abovementioned five full-sib families of Dalmatian dogs with frequent
occurrence of CCSD The families consisted of five to nine individuals and their
parents
Microsatellite marker analysis
We used 27 microsatellite marker derived from the NCBI database
(httpwebncbinlmnihgov) to cover CFA10 completely (Appendix Table 1)
Fine mapping of CFA10 85
PCR reactions for microsatellites were carried out in 12 microl reaction mixtures
containing 2 microl genomic DNA 12 microl 10x PCR buffer (Qbiogene Heidelberg
Germany) 024 microl dimethyl sulfoxide (DMSO) 02 microl dNTPs (100 microM) 01 microl Taq
Polymerase (5Umicrol) (Qbiogene) 06 microl (10 microM) 5rsquo-IRD700 or IRD800 labelled forward
primer 06 microl (10 microM) unlabelled reverse primer The PCR reactions were carried out
in MJ Research thermocyclers with the following program 4 min at 94 degC followed
by 32 cycles of 30 sec at 94degC 30 sec at maximum annealing temperature and a
final extension of 45 sec at 72degC PCR-products were diluted with formamide loading
buffer in ratios from 110 to 140 and then size-fractionated by gel electrophoresis on
automated LI-COR 42004300 sequencers (LI-COR inc Lincoln NE USA) using
denaturing 4 and 6 polyacrylamide gels (RotiphoreseregGel 40 Roth Karlsruhe
Germany)
Development of single nucleotide polymorphisms (SNPs)
We developed SNPs for the region between 36 Mb to 48 Mb on CFA10 previously
proven to be linked to the CCSD phenotype The SNPs for this region were derived
from one 5294 Mb large whole genome shotgun sequence (NW_876251) deposited
in the current dog genome assembly (Boxer genome assembly 21) of the NCBI
GenBank The canine genomic sequences and mRNA of the genes that were used
for the analysis were also derived from sequences deposited in the current dog
genome assembly (Boxer genome assembly 21) of the NCBI GenBank (Table 1)
In total 72 primer pairs were designed most of them located intragenic in intronic
sequences or alternatively in flanking regions of the 5rsquo- or 3rsquondashends of the respective
gene yielding products with a length of around 600 bp PCR primers were developed
with the Primer3 program (httpfrodowimiteducgi-binprimer3primer3_wwwcgi)
The PCR reactions were performed in a total of 50 microl containing 125 microM dNTPs 25
pmol of each primer the reaction buffer supplied by the manufacturer (Qiagen
Hilden Germany) and 1 U Taq polymerase After a 4 min initial denaturation at
95degC 35 cycles of 30 sec at 94degC 45 sec at 58degC and 80 sec at 72degC were
performed in a MJ Research thermocycler (Biozym Hessisch Oldendorf Germany)
The obtained PCR products were directly sequenced with the DYEnamic ET
Terminator kit (AmershamBiosciences Freiburg Germany) and a MegaBACE 1000
capillary sequencer (AmershamBiosciences) using the PCR primers as sequencing
primers Sequence data were analyzed with Sequencher 42
Fine mapping of CFA10
86
(GeneCodes Ann Arbor MI USA) If a heterozygous SNP was found for one or both
parents all progeny of the respective families were analyzed for that SNP
We developed altogether 26 SNPs with 20 SNPs located intragenic in intronic
sequences and 6 SNPs located very closely at the 5rsquo- or 3rsquo-ends respectively with
one to four SNPs per gene (Table 3)
The newly identified 26 SNPs are shown in Tabele 2 Only SNPs are listed and
chosen for linkage analyses that were heterozygous for one or both parents of at
least two of the five families Of all SNPs only one was heterozygous in all families
(Table 3) The most frequent form of SNPs with a frequency of 423 was the AG
transition motif The scarcest one with a frequency of 385 was the CG and the
AC transversion motif respectively
Linkage analysis
Multipoint non-parametric linkage and haplotype analysis were performed using the
MERLIN software version 0102 (multipoint engine for rapid likelihood inference
Center for Statistical Genetics University of Michigan MI USA Abecasis et al
2002) The test statistics Z-mean and Lod score were used to test for the proportion
of alleles shared by affected individuals identical by descent (IBD) for the considered
marker loci
In a first step linkage analysis was performed regarding the 27 marker covering the
whole CFA10 In a second step a linkage analysis was performed including the newly
added SNPs spanning the region 36 Mb to 48 Mb on CFA10
The observed heterozygosity (HET) and the polymorphism information content (PIC)
were calculated using the software package SASGenetics (Statistical Analysis
System Version 913 SAS Institute Inc Cary NC USA 2005)
Linkage disequilibrium (LD) and association of haplotypes with CCSD were tested
using the procedures CASECONTROL and HAPLOTYPE of SASGenetics
(Statistical Analysis System version 913 Cary NC USA)
Results
A linkage analysis was at first carried out for the 176 animals that were analyzed with
27 microsatellite markers covering the whole CFA10
Fine mapping of CFA10 87
The microsatellite FH3302 positioned at 3887 Mb showed the highest Zmean with a
value of 234 (p= 001) and a Lod score of 121 (p= 0009) at 3887 Mb A Zmean
value of 217 (p= 0015) and a Lod score of 12 (p= 0009) was reached for the
microsatellite REN181G20 positioned at 3936 Mb indicating linkage but all Zmean
values and error probabilities of eight microsatellite markers in the interval from 3666
Mb up to 488 Mb were almost as high Consequently we screened the region
spanning from 36 Mb to 48 Mb for SNPs Out of the 23 analyzed Dalmatian dog
families five full-sib families were chosen to screen for SNPs because of their
significant contribution to the test statistics
Twenty-six new SNP markers covering the region of 36 Mb to 48 Mb equally were
developed and added to the linkage analysis These SNPs had PIC values ranging
from 013 to 037
Figure 1 shows the Zmeans and LOD scores for all 27 microsatellite markers on
CFA10 harbouring a genomic deafness region Figure 2 shows the corresponding p-
values Adding the SNPs in the interval spanning 36 Mb to 48 Mb we could narrow
the significant region down to 5 Mb spanning 39 Mb to 44 Mb (Table 4 Figures 3 and
4)
Haplotype-trait association test statistics for the SNPs 16-23 located in the interval
from 39 Mb to 44 Mb were significant However the marker-trait association test
failed the 5 threshold of the error probability (p= 007) but was lowest for all
possible haplotype-trait combinations The χ2ndashtests of the procedure
CASECONTROL were not significant indicating that a SNP for the causative
deafness gene was not yet found
Discussion
We used 27 microsatellite marker and 26 newly developed SNP markers with the
intention to create a dense map for linkage analysis of CFA10 especially the region
spanning 36 Mb to 48 Mb proven to be linked to the CCSD phenotype All SNP
markers were chosen due to their heterozygosity in one or both parents of at least
two families
The significant Zmeans on CFA10 reported for the chromosome scan using only
microsatellites was confirmed by adding the SNP markers Furthermore with the use
of SNPs the apparently linked region from 36 Mb to 48 Mb could be narrowed down
Fine mapping of CFA10
88
to 5 Mb spanning 39 Mb to 44 Mb For this reason the use of SNPs in addition to
informative microsatellites appears highly recommendable
The identified CCSD region spanning 5 Mb might be participating in the development
of CCSD in the analyzed Dalmatian dog families
However genes that are known to be involved in the hearing process or known to be
expressed in the inner ear are not annotated at the syntenic human region on homo
sapiens autosome (HSA) 2 spanning from 100 Mb to 108 Mb Therefore a well-
defined candidate gene for canine hereditary deafness in the abovementioned 5 Mb
does not exist This means that for all genes in this region informative SNPs have to
be developed and tested for linkage disequilibrium and association with CCSD
Despite the remarkable progress that has been made in the past few years in
identifying deafness genes in man and mouse it is suspected that only one-third of all
deafness causing genes have been identified so far Therefore more SNPs have to
be developed within the identified region on CFA10 to localize the deafness causing
gene or to find unambiguously associated SNP markers which could be used for a
population-wide genetic test for CCSD
Fine mapping of CFA10 89
Table 1 Canine genes where gene-associated SNPs could be developed with their
exact location on CFA10 and their corresponding accession numbers (Acc No)
Gene symbol Gene description Position in
Mb
Acc No canine genomic sequence
Acc No canine mRNA
LOC474535 similar to hypothetical protein MGC40397 36493650 NC_006592 XM_531764
LOC474536 similar to KM-102-derived reductase-like factor
37023714 NC_006592 XM_531765
LOC610953 similar to NHP2-like protein 1 37293729 NC_006592 XM_848546
LOC481302 similar to C2orf26 protein 37363737 NC_006592 XM_848552
LOC610991 hypothetical protein LOC610991 38103820 NC_006592 XM_848591
LOC611007 similar to eukaryotic translation elongation factor 1 alpha 2
38353835 NC_006592 XM_848614
LOC474541 similar to GRIP coiled-coil protein GCC185 isoform a
38503857 NC_006592 XM_531770
LOC474542 similar to Sulfotransferase 1C2 38613862 NC_006592 XM_531771
LOC481308 similar to Keratin type I cytoskeletal 18 38653865 NC_006592 XM_538429
LOC474543 similar to Sulfotransferase K1 38683870 NC_006592 XM_857994
LOC609217 similar to family with sequence similarity 32 member A like
39453945 NC_006592 XM_858065
LOC611115 similar to D-PCa-2 protein isoform c 39843984 NC_006592 XM_848756
LOC481325 similar to ubiquitin-conjugating enzyme E2C
42564272 NC_006592 XM_538446
LOC481330 similar to Interleukin-1 receptor type II precursor
44084413 NC_006592 XM_538451
LOC611493 hypothetical protein LOC611493 44384440 NC_006592 XM_863243
LOC481337 similar to DNA repair protein REV1
46264637 NC_006592 XM_538458
LOC611728 similar to 3-hydroxyanthranilate 34-dioxygenase
48494852 NC_006592 XM_849433
Predicted gene derived from the dog genome assembly (build 21) that used gene
prediction method GNOMON supported by EST evidence
Fine mapping of CFA10
90
Table 2 The 26 newly developed SNPs for Dalmatian dogs located in the region
spanning 36 Mb to 48 Mb on CFA10 with their corresponding primers the SNP motif
the product size and the annealing temperature
Gene
description SNP
Location
(intron or
5rsquo3rsquo-end)
Primer F1 (5acute -gt 3acute)
SNP motif
Primer R2 (5acute -gt 3acute)
Bp3 AT4
LOC474535
SNP_1
intron
ACCCAAGCCTAACTGCAGAA
ACCCCAGTCT(CG)GCCAGAGCTGTT
590 60
SNP_2 intron TGTGTGCATA(AG)TGTGCTGAGCCT
TCATCTGTTAAAACAGGGGTGAT
LOC474536
SNP_3
intron
CCAGTTAATGATTGTTTCGTTGA
AAGCTGCTTT(AC)CACCCCCATCAG
TCATTCCTGCTGTTGTGCTC
590 60
LOC610953
SNP_4
intron
CTGTCTTGGGGACTGTTTGC
AAGGCAGACG(CT)AATGACTGAGGC
600 60
SNP_5 intron GACTTGAAGC(CT)ACCACAGATACT
GCCATCACGATGAACTCAGA
LOC481302
SNP_6
3rsquo-end
AATTGAGGCCGAAGTCCAAT
CTTTTCCCCA(GT)GCCACCCCTCTG
GAGCACTATTTACGATACAAACAGGA
610 60
LOC610991
SNP_7
intron
CATGCATGATGCCCAGAGTA
CCCAAAGCAC(AG)CTGTGATTTAAT
AGGGCTTCCTGGGAAAAGT
600 60
LOC611007
SNP_8
intron
CAGACCAACAGTGACCCAGA
TAGGCATACC(GT)TCAGTCCTAAAG
GCCTGTTGTGGGCAGAGTAT
480 60
LOC474541
SNP_9
intron
ACTGAGCCAAAGGTGGATTG
AGAGAATAGC(AG)CTGTGTTTTACA
ACCTGCACATCGGGATTTAG
575 60
Fine mapping of CFA10 91
Table 2 (continued)
Gene-
symbol SNP
Location
(intron or
5rsquo3rsquo-end)
Primer F1 (5acute -gt 3acute)
SNP motif
Primer R2 (5acute -gt 3acute)
Bp3 AT4
LOC474542
SNP_10
intron
CTTCCCCAGGAGAGAGTGAC
AATATGATCA(CT)ATTTAAAGAAAT
CTTTTGTCAACATCCCCTTCA
560 60
LOC481308
SNP_11
3rsquo-end
ACCCATTGTCTCTCCAGCAC
CCACATAACT(GT)AGCATCCCTAGC
600 60
TGATGATGTAAGTTGGCCTCA
LOC474543
SNP_12
intron
TTGAAGTTGTGTGAGTAAATGAAAGA
CAATATATAA(AG)CATTTGCTACAA 600 60
SNP_13 intron TTGAGATGAA(AT)ATAATAGAGCTG
SNP_14 intron CGATTTTCCA(AG)GACATGGAGATG
SNP_15 intron ACTGAACCAA(AG)TGTGCAAAGCAT
GGAAACCATGCAGTCTTTGG
LOC609217
SNP_16
5rsquo-end
TGGCCTCATTTTCCAGTATG
GAAGGAGTTA(CT)ACAGTGAAGATA
580 60
SNP_17 5rsquo-end AAGTGATCAA(AG)CCAGTGCATTCT
GGCAATTACCCTGAGTGGTG
LOC611115
SNP_18
3rsquo-end
GGGCTGTCTTAGAGGTGCTG
TGTGGTCTCA(CT)ACACTTCCTGAG
590 60
SNP_19 3rsquo-end ATTGCAAGTG(GT)CCTGTGATGGCC
CTTCTTTGGGCAGGAAAGTG
LOC481325
SNP_20
intron
AAAATGATTGATCGCAAAAGAAA
AATTACTGTA(CT)AACAGTATCAGA
600 60
SNP_21 intron TAACAGTATC(AG)GAATAAAAGTTT
TTCTGTGATTGCACTGACCG
Fine mapping of CFA10
92
Table 2 (continued)
Gene-
symbol SNP
Location
(intron
or 5rsquo3rsquo-
end)
Primer F1 (5acute -gt 3acute)
SNP motif
Primer R2 (5acute -gt 3acute)
Bp3 AT4
LOC481330
SNP_22
intron
GAAAGGCCTGGGTTCAAAA
GGCAGGGAGG(AG)TCACCATCGTTC
AATTTCCCCAAATGCCTCAC
575 60
LOC611493
SNP_23
intron
GCATGAAGGAGCCCTATGTC
CCAAGAGTCC(AT)GCCCAACACCCT
GGAGGGATGGCATTCTATGA
590 60
LOC481337
SNP_24
intron
GGCTGAGGAGATTGTGTTTCA
GCTGATATTT(AG)GCCTTCTGAGAT
620 60
SNP_25 intron AAAGCCAGAC(AG)CTTTGCCATGGT
CAGCTCCCTGTAATGGGAAA
LOC611728
SNP_26
intron
TCCTACTCCCATCACTTCCAA
CCACACTGGG(GT)CCTGGGATGAGG
CACAGCTCCATGTAGGTCCA
620 60
hypothetical gene found in the 2 version 1 of the NCBIs genome annotation 1 Forward 2 Reverse 3 Product size (basepairs) 4 Annealing temperature (degC)
Fine mapping of CFA10 93
Table 3 The 26 newly developed intragenic SNPs for Dalmatian dogs with their
nucleotide polymorphism allele and genotype frequencies observed heterozygosity
(HET) and polymorphism information content (PIC)
SNP Fam1 Nucleotide
polymorphism
Allele
frequencies
Genotype
frequencies2 PIC HET
SNP_1 125 CgtG 042054 3135 036 055
SNP_2 124 AgtG 064039 91013 037 044
SNP_3 2345 AgtC 041049 2156 033 047
SNP_4 345 CgtT 065035 6140 035 067
SNP_5 34 CgtT 065035 490 028 042
SNP_6 345 CgtT 066034 6130 035 039
SNP_7 1345 AgtG 054046 6174 037 053
SNP_8 145 GgtT 075025 01010 029 045
SNP_9 1345 AgtG 052054 6145 037 047
SNP_10 1234 CgtT 057043 7173 037 068
SNP_11 14 GgtT 065035 5120 035 062
SNP_12 15 AgtG 028072 097 030 044
SNP_13 45 AgtT 077023 870 017 021
SNP_14 5 AgtG 036064 052 013 015
SNP_15 134 AgtG 034066 0157 034 059
SNP_16 145 CgtT 030070 3812 033 036
SNP_17 245 AgtG 058042 6113 030 032
SNP_18 123 CgtT 037063 1126 027 035
SNP_19 123 GgtT 045055 3115 030 032
SNP_20 2345 CgtT 032068 1129 029 039
SNP_21 1235 AgtG 063037 883 037 052
SNP_22 1234 AgtG 068032 10102 030 034
SNP_23 12345 AgtT 030070 21313 033 046
SNP_24 124 AgtG 047053 667 033 019
SNP_25 234 AgtG 053047 3132 035 053
SNP_26 234 GgtT 060040 3120 026 0391 Number of families which were informative for the respective SNPs 2 Genotypes are given as number of animals [homozygous for allele 1heterozygous
homozygous for allele 2]
Fine mapping of CFA10
94
Table 4 Linkage analysis for canine chromosome 10 regarding the region spanning
39 Mb to 44 Mb with the significant Zmean LOD score and error probabilities (p-
values)
Marker Location (Mb) Zmean pz-value1 LOD-score pL-value2
SNP_16 39453 262 0004 123 0009
SNP_17 39455 262 0004 123 0009
SNP_18 39840 261 0004 123 0009
SNP_19 39843 261 0004 123 0009
SNP_20 4260 255 0005 118 0010
SNP_21 4270 255 0005 117 0010
SNP_22 4405 317 00008 131 0007
SNP_23 4439 337 00004 134 0007 1 Error probability regarding Zmean 2 Error probability regarding LOD score
Fine mapping of CFA10 95
Figure 1 Zmeans and LOD scores for 27 microsatellite markers on CFA10
harbouring a congenital sensorineural deafness region (number of families 23
number of genotyped dogs 176)
Figure 2 P-values for Zmeans and LOD scores of 27 microsatellite markers on
CFA10 harbouring a congenital sensorineural deafness region (number of families
23 number of genotyped dogs 176)
Fine mapping of CFA10
96
Figure 3 Zmeans and LOD scores of 53 markers on CFA10 harbouring a congenital
sensorineural deafness region (number of families 5 number of genotyped dogs 34)
Figure 4 P-values for Zmeans and LOD scores of 53 markers on CFA10 for the
region between 30 to 60 Mb harbouring a congenital sensorineural deafness region
(number of families 5 number of genotyped dogs 34)
CCSD region (SNP 16-23)
Chapter 7
Analysis of canine chromosome 1 and the Gap junction protein alpha 1 (GJA1) gene
in Dalmatian dogs segregating for congenital sensorineural deafness
CFA1 and the GJA1 gene 99
Analysis of canine chromosome 1 and the Gap junction protein alpha 1 (GJA1) gene in Dalmatian dogs segregating for congenital sensorineural deafness Abstract Two microsatellite markers associated to the GJA1 (alternatively connexin 43) gene
showed significant linkage with canine congenital deafness (CCSD) in a large French
half-sib family (Chapter 3) PCR products of this half-sib family were used to perform
a sequence analysis in order to evaluate whether GJA1 is responsible for CCSD As
the linkage could not be confirmed we used altogether 27 microsatellite markers for
a scan of canine chromosome (CFA) 1 with 23 Dalmatian dog families segregating
for CCSD A non-parametric linkage analysis was performed to see whether
significant test statistics for other genomic regions on CFA1 and for more families can
be shown As a result we could not find linkage to any microsatellite in the analyzed
families
Introduction Rak (2003) considered in total 24 genes as candidates for sensorineural deafness in
dogs among them the Gap junction protein alpha 1 (GJA1) or connexin 43 gene on
canine chromosome (CFA) 1 A marker set of altogether 43 microsatellites were
developed by Rak (2003) among them two microsatellite marker associated to the
GJA1 gene
GJA1 or connexin 43 is a member of the connexin gene family and a component of
gap junctions Mutations in 4 members of the connexin gene family have been shown
to underlie distinct genetic forms of deafness including GJB2 (connexin [CX] 26)
GJB3 (CX31) GJB6 (CX30) and GJB1 (CX32)
Liu et al (2001) reported mutations in GJA1 in association with sensorineural
recessive deafness in man However these mutations have recently been shown to
involve the pseudogene of GJA1 on human chromosome (HSA) 5 and not the
CFA1 and the GJA1 gene
100
GJA1 gene on HSA 6 (WA Paznekas cited a personal communication from the
senior author (W E Nance) of the paper by Liu et al 2001)
In previously performed studies (Chapter 3) one large French Dalmatian dog family
with frequent occurrence of CCSD showed linkage to microsatellites associated to
the GJA1 gene
In this report we performed a mutation analysis of the GJA1 gene sequence to
identify polymorphisms In order to evaluate whether the GJA1 gene is responsible
for congenital sensorineural deafness in Dalmatian dogs we analyzed the
association of the GJA1 haplotypes with the CCSD phenotype Furthermore we
employed 27 microsatellite markers covering the entire CFA1 and used them for a
non-parametric linkage analysis with CCSD in a Dalmatian dog population of 176
animals with frequent occurrence of CCSD
Material and methods Pedigree material
For the linkage analysis of the microsatellite marker we used DNA from altogether
176 animals belonging to 22 full-sib families and one large paternal half-sib family of
German Dalmatian dogs All families were segregating for CCSD The genotyped
families included all affected dogs (unilaterally and bilaterally deaf) their parents if
available and one to four unaffected animals At least two of the full sibs of each
family were unilaterally or bilaterally deaf The phenotype of the affected animals had
been confirmed by brainstem auditory evoked response (BAER) that detects
electrical activity in the cochlea and auditory pathways in the brain
Mutation analysis and screening for SNPs was performed by comparative
sequencing of DNA from 16 animals consisting of the parents and their progenies of
one large half-sib family of French Dalmatian dogs which showed significant linkage
to GJA1-associated microsatellites (Chapter 3)
Sequencing of canine genomic DNA and mutation analysis
The canine GJA1 gene sequence was derived from sequences deposited in the
current dog genome assembly (Boxer genome assembly 21) of the NCBI GenBank
(Genbank Acc No NC_006583) We compared the canine genomic DNA sequence
to canine cDNA fragments in the canine EST database using the
CFA1 and the GJA1 gene 101
BLASTN (Basic Local Alignment Search Tool Nucleotide) program Three canine
ESTs (Acc No DN880776 BU744925 and DN369900) were used to confirm the
assembly of the GJA1 gene
To identify polymorphisms within the canine GJA1 sequence the gene consisting of
one 1251 bp spanning exon were PCR amplified and sequenced from 16 French
Dalmatian dogs Additionally large sequences from the 3acute-end were amplified Primer
pairs were designed yielding products with a length of around 600 bp PCR primers
were developed with the Primer3 program (httpfrodowimiteducgi-
binprimer3primer3_wwwcgi) The PCR reactions were performed in a total of 50 microl
containing 125 microM dNTPs 25 pmol of each primer the reaction buffer supplied by
the manufacturer (Qbiogene Heidelberg Germany) and 1 U Taq polymerase After
a 4 min initial denaturation at 95degC 35 cycles of 30 sec at 94degC 45 sec at 58degC and
80 sec at 72degC were performed in a MJ Research thermocycler (Biozym Hessisch
Oldendorf Germany)
The obtained PCR products were directly sequenced with the DYEnamic ET
Terminator kit (AmershamBiosciences Freiburg Germany) and a MegaBACE 1000
capillary sequencer (AmershamBiosciences) using the PCR primers as sequencing
primers Sequence data were analyzed with Sequencher 42
(GeneCodes Ann Arbor MI USA)
Microsatellite marker analysis
We used 27 microsatellite marker derived from the NCBI database
(httpwebncbinlmnihgov) to cover CFA1 completely (Appendix Table 1)
PCR reactions for microsatellites were carried out in 12 microl reaction mixtures
containing 2 microl genomic DNA 12 microl 10x PCR buffer (Qbiogene) 024 microl dimethyl
sulfoxide (DMSO) 02 microl dNTPs (100 microM) 01 microl Taq Polymerase (5Umicrol)
(Qbiogene) 06 microl (10 microM) 5rsquo-IRD700 or IRD800 labelled forward primer 06 microl (10
microM) unlabelled reverse primer The PCR reactions were carried out in MJ Research
thermocyclers with the following program 4 min at 94 degC followed by 32 cycles of 30
sec at 94degC 30 sec at maximum annealing temperature and a final extension of 45
sec at 72degC PCR-products were diluted with formamide loading buffer in ratios from
110 to 140 and then size-fractionated by gel electrophoresis on automated LI-COR
42004300 sequencers (LI-COR inc Lincoln NE USA) using denaturing 4 and 6
polyacrylamide gels (RotiphoreseregGel 40 Roth Karlsruhe Germany)
CFA1 and the GJA1 gene
102
Linkage and haplotype analysis
Multipoint non-parametric linkage and haplotype analyses were performed using the
MERLIN software version 0102 (multipoint engine for rapid likelihood inference
Center for Statistical Genetics University of Michigan MI USA Abecasis et al
2002) The test statistics Z-mean and Lod score were used to test for the proportion
of alleles shared by affected individuals identical by descent (IBD) for the considered
marker loci
A non-parametric linkage analysis was performed with 27 microsatellite markers in 23
Dalmatian dog families consisting of 176 Dalmatian dogs The results were added to
the linkage analysis performed in Chapter 3
Results and discussion
In previously performed studies (Chapter 3) one French Dalmatian dog family
reached a Zmean value of 295 (plt0002) for GJA1_MS1 and a Zmean value of 286
(plt0002) for GJA1_MS2 indicating linkage Both markers were associated with the
GJA1 gene
By sequence analysis we revealed a total of 3 SNPs found in sequences of the 3acute-
UTR shown in Table 1 None of the polymorphisms were polymorphic in all parents of
the examined French half-sib family Neither of the observed polymorphism did alter
the predicted amino acid sequence of GJA1 nor showed the identified haplotypes an
association with the CCSD phenotype (Figure 1)
There is no recombination of the haplotypes of the GJA1 gene in this family The
paternal and maternal haplotypes as far as they could be estimated were distributed
among the affected progeny closely to the expected proportion of 50 and therefore
no excess of a certain haplotype could be observed in the affected dogs
Thus evaluation of the SNP markers debilitates the linkage to CCSD for the French
half-sib family Because of the fact that both animals with unilateral hearing loss and
bilateral hearing animals shared identical haplotypes these polymorphisms are
obviously not associated with CCSD in these Dalmatian dog families Furthermore
the present study revealed no functional mutations of the complete coding region of
GJA1 It is therefore unlikely that the GJA1 gene is involved in the pathogenesis of
CCSD in the analyzed French Dalmatian dogs The reason for GJA1_MS1 and
GJA1_MS2 indicating linkage may be caused by a closely linked gene involved in
CFA1 and the GJA1 gene 103
CCSD among the half-sib family of French Dalmatian dogs or by a false positive
result of the microsatellite study performed in Chapter 3
To clarify whether significant test statistics for other genomic regions on CFA1 and
for more families can be shown we used 27 microsatellite markers derived from the
NCBI database (httpwebncbinlmnihgov) to cover CFA1 completely A linkage
analysis was carried out after genotyping 176 German Dalmatian dogs with a set of
27 microsatellite markers The results of this linkage analysis were added to the
results of the test statistics for the microsatellites GJA1_MS1 and GJA1_MS2
(Chapter 3)
As a result we could not find linkage to any microsatellite in the analyzed families
(Figure 2 and 3) It is unlikely that the canine chromosome 1 harbours genomic
regions that are involved in the development of CCSD in the analyzed Dalmatian dog
families
With hindsight it was revealed that GJA1 is not responsible for sensorineural non-
syndromic deafness in humans as Liu et al (2001) has published GJA1 is
participating in a human syndrome called oculodentodigital dysplasia (ODDD) that
can be accompanied with hearing impairment (Paznekas et al 2003) But the type of
deafness in human ODDD differs from the typical hearing loss associated with other
connexin mutations because it is conductive rather than sensorineural
As deafness in dogs especially in Dalmatians is almost exclusively caused by
sensorineural non-syndromic forms also known as cochleosaccular degeneration
the GJA1 gene should not be considered as a candidate gene for CCSD anymore
CFA1 and the GJA1 gene
104
Table 1 Three newly developed intragenic SNPs and two microsatellite markers for
Dalmatian dogs in the candidate gene GJA1 with their corresponding primers the
SNP motif the product size and the annealing temperature
SNP
Primer F (5acute -gt 3acute)
SNP motif
Primer R (5acute -gt 3acute)
Product
size (bp)
Annealing
temperatur
GJA1_SNP1+2
CACCTTAGGCGTTCATTTTG
CCGGGGAG(AG)AAAA(AG)AAAAATACTT
TGGCTTGATTCCCTGACTC
650 58
GJA1_SNP3
TCTGAAATGTAATCATGGATGC
CAGAACTTGTAT(AT)CTGTTAAGAG
AATCACACAGGATATAGAGGCTATC
600 58
Microsatellite
marker Primers (forward reverse ) 5acute -gt 3acute
Product
size (bp)
Annealing
temperatur
GJA1_MS1
ATGGCATGAAGAGGATACCG
AGGACAGGTGACGGCTCTAC
134 60
GJA1_MS2
GCTAGTACTCGATTGTGGTC
TCATGGGTTGTGAGATCCAG
190 60
CFA1 and the GJA1 gene 105
Figu
re 1
Hap
loty
pes
of th
e G
JA1
gene
in th
e an
alyz
ed D
alm
atia
n do
g fa
mily
CFA1 and the GJA1 gene
106
Figure 2 Zmeans and LOD scores for 29 microsatellite markers on CFA1 (number of
families 23 number of genotyped dogs 176)
Figure 3 P-values for Zmeans and LOD scores of 29 microsatellite markers on CFA1
(number of families 23 number of genotyped dogs 176)
Chapter 8
Analysis of canine chromosome 31 and the claudin-14 (CLDN14) gene
in Dalmatian dogs segregating for congenital sensorineural deafness
CFA31 and the CLDN14 gene 109
Analysis of canine chromosome 31 and the claudin-14 (CLDN14) gene in Dalmatian dogs segregating for congenital sensorineural deafness Abstract A previously accomplished linkage analysis revealed significant linkage of
microsatellite markers associated with the CLDN14 gene on canine chromosome
(CFA) 31 with the phenotype of canine sensorineural deafness (Chapter 3) The
objective of the present study was to perform a sequence analysis in order to find
single nucleotide polymorphisms (SNPs) for the CLDN14 gene and additionally to
use a set of six microsatellite markers evenly distributed on CFA31 for non-
parametric linkage analysis with the aim to verify the significant test statistics shown
in Chapter 3
Introduction
Of the genes responsible for different forms of sensorineural non-syndromic deafness
in humans Rak (2003) considered 24 genes as candidates for sensorineural
deafness in dogs among them the CLDN14 gene on canine chromosome (CFA) 31 CLDN14 encodes a member of the claudin family which comprises the major
components of tight juncions (TJ) The human CLDN14 gene consists of one
tranlatete exon and six exons in the 5rsquountranlated region Five splice isoformes are
identified so far (Wilcox et al 2001 Wattenhofer et al 2005)
For the compartmentalization of perilymph and endolymph in the inner ear the
leakage of solutes through a paracellular pathway must be prevented by tight
junctions TJ are an intercellular junction found at the most apical region of polarised
epithelial and endothelial cells at which adjacent plasma membranes are joined
tightly together separating apical membranes and basolateral menbranes They are
specialised membrane domains containing branching strands of integral proteins and
create a primary barrier preventing paracellular transport of solutes and restricting
lateral diffusion of membrane lipids and proteins (Schneeberger and Lynch 2004)
CFA31 and the CLDN14 gene
110
The important role of the CLDN14 gene in the TJ of the inner ear was demonstrated
by Wilcox et al (2001) when he identified mutations in the CLDN14 gene responsible
for a hereditary human deafness in families segregating for congenital recessive
deafness (DFNB29)
In previously performed studies one large half-sib family as well as several full-sib
families of German Dalmatian dog indicated linkage to CLDN14 gene-associated
markers (Chapter 3)
A sequence analysis was performed to identify single nucleotide polymorphisms
(SNPs) either intergenic located or alternatively in the flanking 5rsquo- and 3rsquo-Regions
Furthermore we employed microsatellite markers covering CFA31 and used them for
a non-parametric linkage analysis with CCSD in a German Dalmatian dog population
of 176 animals with frequent occurrence of CCSD
Material and methods Pedigree material
For the linkage analysis of the microsatellite marker we used DNA from altogether
176 animals belonging to 22 full-sib families and one large paternal half-sib family of
German Dalmatian dogs All families were segregating for CCSD The genotyped
families included all the affected dogs (unilaterally and bilaterally deaf) their parents
if available and one to four unaffected animals At least two of the full sibs of each
family were unilaterally or bilaterally deaf The phenotype of the affected animals had
been confirmed by brainstem auditory evoked response (BAER) that detects
electrical activity in the cochlea and auditory pathways in the brain
Mutation analysis and screening for SNPs was performed by comparative
sequencing of DNA from 36 animals consisting of the parents and their progenies of
one half-sib family and four full-sib families of Dalmatian dogs which showed
significant linkage to a CLDN14-associated microsatellite (Chapter 3)
Sequencing of canine genomic DNA and development of single nucleotide
polymorphisms (SNPs)
The canine CLDN14 gene sequence was derived from sequences deposited in the
current dog genome assembly (Boxer genome assembly 21) of the NCBI GenBank
(Genbank Acc No NW_876295) by BLAST (Basic Local Alignment Search Tool)
CFA31 and the CLDN14 gene 111
search (httpwwwncbinlmnihgovBLAST) using the human CLDN14 reference
mRNA sequence (Genbank Acc No NM_144492)
We compared the canine genomic DNA sequence to canine cDNA fragments in the
canine EST database using the BLASTN program As no ESTs could be found
human mRNA sequences were used for the localization of the exonintron
boundaries using the mRNA-to-genomic alignment program Spidey
(httpwwwncbinlmnihgovIEBResearchOstellSpideyindexhtml)
We screened exon three for mutations as this exon is the only translated one in man
Additionally we screened large intronic sequences and sequences located in the 5rsquo-
and 3rsquo-UTR of the CLDN14 gene for Polymorphisms For this purpose primer pairs
were designed yielding products with a length of around 600 bp
PCR primers were developed with the Primer3 program (httpfrodowimiteducgi-
binprimer3primer3_wwwcgi) The PCR reactions were performed in a total of 50 microl
containing 125 microM dNTPs 25 pmol of each primer the reaction buffer supplied by
the manufacturer (Qbiogene Heidelberg Germany) and 1 U Taq polymerase After
a 4 min initial denaturation at 95degC 35 cycles of 30 sec at 94degC 45 sec at 58degC and
80 sec at 72degC were performed in a MJ Research thermocycler (Biozym Hessisch
Oldendorf Germany)
The obtained PCR products were directly sequenced with the DYEnamic ET
Terminator kit (AmershamBiosciences Freiburg Germany) and a MegaBACE 1000
capillary sequencer (AmershamBiosciences) using the PCR primers as sequencing
primers Sequence data were analyzed with Sequencher 42 (GeneCodes Ann
Arbor MI USA) The eight newly developed SNPs are shown in Table 1
Microsatellite marker analysis
In total six microsatellite marker were derived from the NCBI database
(httpwebncbinlmnihgov) to cover CFA31 completely (Appendix Table 1)
PCR reactions for microsatellites were carried out in 12 microl reaction mixtures
containing 2 microl genomic DNA 12 microl 10x PCR buffer (Qbiogene) 024 microl dimethyl
sulfoxide (DMSO) 02 microl dNTPs (100 microM) 01 microl Taq Polymerase (5Umicrol)
(Qbiogene) 06 microl (10 microM) 5rsquo-IRD700 or IRD800 labelled forward primer 06 microl (10
microM) unlabelled reverse primer The PCR reactions were carried out in MJ Research
thermocyclers with the following program 4 min at 94 degC followed by 32 cycles of 30
sec at 94degC 30 sec at maximum annealing temperature and a final extension of
CFA31 and the CLDN14 gene
112
45 sec at 72degC PCR-products were diluted with formamide loading buffer in ratios
from 110 to 140 and then size-fractionated by gel electrophoresis on automated LI-
COR 42004300 sequencers (LI-COR inc Lincoln NE USA) using denaturing 4
and 6 polyacrylamide gels (RotiphoreseregGel 40 Roth Karlsruhe Germany)
Linkage and haplotype analysis
Multipoint non-parametric linkage and haplotype analysis were performed using
MERLIN version 0102 (multipoint engine for rapid likelihood inference Center for
Statistical Genetics University of Michigan MI USA Abecasis et al 2002) The test
statistics Z-mean and Lod score were used to test for the proportion of alleles shared
by affected individuals identical by descent (IBD) for the considered marker loci A
linkage analysis was performed with 6 microsatellite markers on 176 Dalmatian dogs
The results were added to the linkage analysis performed in Chapter 3
Results and discussion CLDN14-associted microsatellites were significantly linked with CCSD in a scan of
candidate genes using 24 families with 215 Dalmatian dogs (Chapter 3) Selection of
four full-sib families and one half-sib family with the highest tests statistics lead to a
Zmean value of 383 (plt000007) for the CLDN14 gene-associated marker
CLDN14_MS2
To substantiate the linkage to the CLDN14 gene we searched for sequence
variations within the CLDN14 gene in four full-sib families and one half-sib family with
the highest contribution to the test statistics shown in Chapter 3 Most of the identified
polymorphisms were found in intronic sequences none were within exon three None
of the observed polymorphism did alter the predicted amino acid sequence of exon
three We revealed a total of eight SNPs (Table 1) but only two (SNP_4 and SNP_5)
out of the identified eight SNPs were polymorphic in the examined families Both
unilaterally and bilaterally deaf animals as well as normal hearing animals showed
identical haplotypes for these two polymorphic SNPs and thus no co-segregation with
the deaf Dalmatian dogs could be revealed (Figure 1 and 2) In this study we did not
identify any causal mutations for CCSD in the analyzed Dalmatian dogs Despite this
results a mutation outside of the translated genomic regions analyzed here may exist
that would affect CLDN14 expression
CFA31 and the CLDN14 gene 113
To clarify if other regions on CFA31 are responsible for the CCSD phenotype
additionally six microsatellite markers covering CFA31 were tested in 23 Dalmatian
dog families consisting of 176 individuals A non-parametric linkage analysis was
performed regarding these 23 families The results of this linkage analysis were
added to the results of the test statistics for the CLDN14 gene-associated
microsatellites (Chapter 3)
We could only find significant linkage with CCSD for CLDN14-associted
microsatellites in the abovementioned five families The two microsatellites located
most closely were proximal and distal 4 Mb off the CLDN14 gene REN110K04 with a
Zmean value of -009 (plt05) and FH2712 with a Zmean value of 022 (plt03) not
indicating linkage
It can not be excluded that CLDN14 or genes in its flanking regions are involved in
the development of CCSD in the analyzed Dalmatian dog families Other genes than
the CLDN14 that are known to be involved in the hearing process or known to be
expressed in the inner ear are not annotated at the syntenic human region of homo
sapiens autosome (HSA) 21q223 Therefore despite the CLDN14 gene no other
well-defined candidate gene for canine hereditary deafness exists in the linked
region Despite the remarkable progress that has been made in the past few years in
identifying deafness genes in man and mouse it is suspected that only one-third of all
deafness causing genes have been identified so far and thus it seems possible that
other genes in the flanking region of the CLDN14 gene are involved in the
development of the disease Anyway to clarify the importance of CLDN14 in the
CCSD phenotype more SNPs have to be developed within the CLDN14 gene as well
as in its flanking regions with the aim to find significant linkage disequilibrium of SNP
markers with CCSD
CFA31 and the CLDN14 gene
114
Table 1 The 8 newly developed SNPs for Dalmatian dogs in the CLDN14 gene on
CFA31 with their corresponding primers the SNP motif the product size and the
annealing temperature
SNP
Location
(intron or
5rsquo3rsquo-UTR)
Primer F1 (5acute -gt 3acute)
SNP motif
Primer R2 (5acute -gt 3acute)
Bp3 AT4
CLDN14_SNP1
intron
GACCATATGTTTGTGGCC
CTTCCAGGGAAA(AT)TGTCGTAGCC
CGTCAGGATGTTGGTGCC
580 60
CLDN14_SNP2
inton
GACCATATGTTTGTGGCC
GAAATTGTCGTA(AG)CCCGGCCGCT
CGTCAGGATGTTGGTGCC
580 60
CLDN14_SNP3
3rsquo-UTR
CTGCCTTCAAGGACAACC
CCAGAGGAATAA(CT)ATGATCGTGA
GATGAGTATCAGCCCAGC
550 60
CLDN14_SNP4
3rsquo-UTR
CTGCCTTCAAGGACAACC
ACCACCGCACAC(CT)GTCACAGCTT
GATGAGTATCAGCCCAGC
550 60
CLDN14_SNP5 3rsquo-UTR
CATGCCTTTGTCCCAAACTT
GAGACCCTCTGG(CT)TCCTTTTGGC
GTACCTGTTGCCTGGGTTGT
610 60
CLDN14_SNP6 5rsquo-UTR
CCTTCATCCTTTCTGGTTGA
GCTCACAGTCCC(AC)ATGGGGACAT
GGGGAGCATAATGTGGTCAT
585 60
CLDN14_SNP7 5rsquo-UTR
TGAACTGGTCCCAAGGAAAG
GCACGACCGAGC(CT)CTGGCTTTAC
GGGATGAGAGGGAGGTTTTT
580 60
CLDN14_SNP8 3rsquo-UTR
AATGCCTATCCCTTCTTTGGA
CACGTTACTGTG(AG)ACCTCTCTAC
GCAGGCTTTCCTCAAGTGTC
680 60
1 Forward 2 Reverse 3 Product size (basepairs) 4 Annealing temperature (degC)
CFA31 and the CLDN14 gene 115
Figure 1 Haplotypes of the CLDN14 gene-associated markers SNP_4 and SNP_5 in
the analyzed four Dalmatian dog full-sib families
CFA31 and the CLDN14 gene
116
Figu
re 2
Hap
loty
pes
of th
e C
LDN
14 g
ene-
asso
ciat
ed m
arke
rs S
NP
_4 a
nd S
NP
_5 in
the
anal
yzed
Dal
mat
ian
dog
half-
sib
fam
ily
Chapter 9
General discussion
General discussion 119
General discussion
The candidate gene approach In the first step of this thesis candidate genes for canine congenital sensorineural
deafness (CCSD) in Dalmatian dogs were analyzed by means of microsatellite
markers or alternatively by single nucleotide polymorphisms (SNPs)
The candidate genes for which a set of in total 43 microsatellites was available
included the following 24 genes CDH23 CLDN14 COCH COL11A2 DFNA5
DIAPH1 EDN3 EDNRB EYA4 GJA1 GJB2 GJB6 MITF MYH9 MYO6 MYO7A
MYO15A OTOF PAX3 POU4F3 SLC26A4 SOX10 TECTA and TMPRSS3
(Rak 2003) These genes are known to be involved either in human non-syndromic
deafness or in the human Waardenburg syndrome The Waardenburg syndrome
(WS) manifests with sensorineural deafness and pigmentation defects in iris hair and
skin WS is classified into four types depending on the presence or absence of
additional symptoms which are caused by mutations in the five genes EDN3
EDNRB MITF PAX3 and SOX respectively
For another eight recently identified genes responsible for different forms of human
non-syndromic deafness including TMC1 TMIE USH1C MYH14 MYO3A PRES
WHRN and ESPN linkage and association analyses were performed using newly
developed SNPs
In the last years most projects have exploited canine traits for which either direct
candidate genes could be proposed and evaluated or for which large informative
pedigrees were available to enable linkage mapping to identify candidate regions A
major component of such research efforts comprised the cloning sequencing and
mapping of individual canine homologs of genes either proposed as candidate
genes or expected to be located in candidate regions This was necessary to identify
new informative polymorphisms (eg SNPs microsatellites) for high resolution
mapping of candidate regions and to examine each exon and exonintron boundary
for positional candidates Availability of the second version of the dog genome
assembly (build 21) of the NCBI database shortcuts this effort and increases the
investigators efficiency Now either additional candidate genes for canine congenital
sensorineural deafness can be found directly by its gene symbol in the 21 of the
General discussion
120
NCBIs genome annotation or if a candidate gene is not yet annotated a BLAST
(Basic Local Alignment Search Tool) search against the canine whole genome
shotgun (wgs) sequence resource can be used to obtain the sequence of the canine
genomic contigs containing the human homologous gene and thus intragenic
markers can be developed for subsequent linkage and association analyses
Over the past decade it has become increasingly clear how far structural and
functional homologies at the gene level extend across even distantly related species
Much is known about deafness-causing gene mutations in humans and mice
including the fact that the clinical and histopathological findings are often very similar
to those of deafness in Dalmatian dogs Thus genes responsible for non-syndromic
congenital hereditary deafness in humans seem to be appropriate candidate genes
for CCSD (Rak and Distl 2005) In this thesis we first concentrated on the candidate
gene approach combined with linkage analysis method using affected pedigree
members Once a significant linkage was found only the linked genes with the
required low error probability values were used for further molecular genetic analysis
The method of candidate gene approach using either gene-associated microsatellite
or alternatively SNP markers was applied for in total 32 candidate genes which
comprise nearly all of the identified mutated genes causing non-syndromic hereditary
hearing impairment in humans
Linkage and association analysis In principle linkage and association are totally different phenomena Linkage is a
relation between loci and association is a relation between alleles
Linkage means that a haplotype characterised by microsatellites or SNPs is
significantly more often present in family members with the phenotype under study
than expected by random assortment For construction of haplotypes sets of closely
linked genetic markers on the same chromosome are needed which tend to be
inherited together as they are not likely to be separated by recombination Linkage
creates associations within families but not among unrelated induviduals
Association is a statistical statement about the co-occurrence of alleles or
phenotypes Association analysis can be carried out as a method of genetic analysis
that compares the frequency of alleles between affected and unaffected individuals
across all families Thus for association family structures are not necessary A given
allele is considered to be associated with the disease if the presence of that allele
General discussion 121
explains a significant proportion of the phenotypic trait variation For association
studies the developing of a marker set consisting of SNPs rather than microsatellites
is needed
In this thesis a total of 32 candidate genes for canine congenital deafness were
analyzed which showed an appropriate clinical and histological disease pattern in
comparison to deafness in Dalmatian dogs Rak (2003) developed a set of 43
microsatellites for in total 24 candidate genes among them the CLDN14 gene on
canine chromosome (CFA) 31 and the MYH9 gene on CFA10 The GJA1 on CFA1
was also considered as a candidate gene for CCSD (Rak 2003) and therefore two
gene-associated microsatellites have been developed Recently it turned out that
GJA1 is not responsible for human sensorineural non-syndromic deafness but for a
human syndromic disorder that can be related with conductive deafness
By the use of a non-parametric linkage analysis using the existing set of 43
microsatellites associated to 24 candidate genes we found linkage to markers
associated to CLDN14 on CFA31 MYH9 on CFA10 and GJA1 on CFA1
For another another eight candidate genes it was possible to develop SNPs
Performing linkage analyses as well as association and haplotype studies it was
possible to exclude these eight candidate genes from being responsible for the
CCSD phenotype
Over the past ten years significant progress has been made in the identification of
deafness gene localisations Up to now approximately 120 loci have been reported
for both autosomal dominant and recessive forms of non-syndromic hereditary
deafness in humans and only for one third the responsible gene mutation could be
detected Thus it can be expected that additional potential human candidates for
CCSD in Dalmatian dogs will become available in future (Van Camp and Smith
2003)
The extreme heterogeneity of human deafness often hampered genetic studies
because many different genetic forms of hearing loss give rise to similar clinical
phenotypes and conversely mutations in the same gene can result in a variety of
clinical phenotypes In man genes that transport ions across membranes to
maintain appropriate solute concentration and pH as well as regulatory genes mainly
transcription factors and genes that play a part in structural integrity are essential
for the hearing process
General discussion
122
The results of this thesis indicate that the inheritance of hearing loss in Dalmatian
dogs is probably as heterogenic in origin as it is in humans Genetic heterogeneity
means that different mutations cause a similar phenotype the different mutations
can either be found at the same locus (allelic heterogeneity) or even at different loci
(non-allelic heterogeneity) As linkage was found for different candidate genes in
different families subsequently only the families indicating linkage were chosen for
further molecular analyses
GJA1 on CFA1 MYH9 on CFA10 and CLDN14 on CFA31 and their flanking regions
are further analyzed with a combined approach using microsatellite and SNP
markers
CFA1 By the use of GJA1-associated microsatellites one large French Dalmatian dog
family reached a Zmean value of 295 (plt0002) for GJA1_MS1 and a Zmean value
of 286 (plt0002) for GJA1_MS2 indicating linkage Subsequently a sequence
analysis of the GJA1 gene using the above mentioned French Dalmatian dog family
was performed None of the observed polymorphism did alter the predicted amino
acid sequence of GJA1 nor showed the identified haplotypes an association with the
CCSD phenotype Thus evaluation of the SNP markers debilitates the linkage to
CCSD for the French half-sib family It is unlikely that the GJA1 gene is involved in
the pathogenesis of CCSD in Dalmatian dogs To see whether significant test
statistics for other genomic regions on CFA1 and for more families can be shown a
non-parametric linkage analysis was performed with 27 microsatellite markers
covering CFA1 completely In total 176 animals were genotyped We could not find
linkage to any microsatellite in the analyzed families Furthermore it was revealed
that GJA1 is not a candidate gene for sensorineural non-syndromic deafness in
humans (WA Paznekas cited a personal communication from the senior author (W
E Nance) of the paper by Liu et al 2001) GJA1 is participating in a human
syndrome called oculodentodigital dysplasia (ODDD) that can be accompanied with
hearing impairment (Paznekas et al 2003) But the type of deafness in human
ODDD is conductive rather than sensorineural As deafness in dogs especially in
Dalmatians is almost exclusively caused by sensorineural non-syndromic forms also
known as cochleosaccular degeneration the GJA1 gene should not be considered
as a candidate gene for CCSD anymore
General discussion 123
CFA31 By the use of microsatellites as well as SNPs we found significant linkage with CCSD
for CLDN14-associated microsatellites in four full-sib and one half-sib Dalmatian dog
familiy with a Zmean value of 383 (plt000007) A mutation analysis was performed
for exon three as this is the only translated one in man None of the observed
polymorphisms did alter the predicted amino acid sequence However to clarify the
importance of the CLDN14 gene and its flanking regions in the CCSD phenotype
more SNPs have to be developed within the CLDN14 gene as well as in its flanking
regions with the aim to find significant linkage disequilibrium of SNP markers
CFA10 A significant co-segregation of markers alleles and the phenotypic expression of
deafness in a large sample of German Dalmatian dog families was determined for
one marker (Z-mean of 156 [p= 006] and a Lod score of 058 [p= 005]) associated
to the MYH9 gene Subsequently we evaluated whether MYH9 gene mutations are
responsible for CCSD in these Dalmatian dog families An initial priority in defining
gene structure is to obtain a full-length cDNA sequence and identify translational
initiation and termination sites and polyadenylation site(s) Exonintron structure can
then be determined by referencing the cDNA sequence against sequences of
cognate genomic DNA One popular method of obtaining full-length cDNA sequences
is the RACE (rapid amplification of cDNA ends) technique RACE-PCR is an anchor
PCR modification of RT-PCR The rationale is to amplify sequences between a single
previously characterised region in the mRNA (cDNA) and an anchor sequence that is
coupled to the 5 or the 3 end A primer is designed from the known internal
sequence and the second primer is selected from the relevant anchor sequence
To provide the genomic organization and the complete sequence of the canine
MYH9 gene the isolation of full length cDNAs was achieved with the help of a
modified rapid amplification of cDNA ends (RACE) protocol A mutation analysis was
performed to identify single nucleotide polymorphisms (SNPs) in this gene We
analyzed the association of the MYH9 haplotypes with the CCSD phenotype in three
families of Dalmatian dogs with frequent occurrence of CCSD and significant linkage
to gene-associated microsatellites Using the RT-PCR analyses for cDNA-genomic
sequence comparisons we detected that the canine MYH9 gene is bigger compared
to the human sequence due to the untranlated first exon in the 5rsquo-UTR
General discussion
124
The canine MYH9 gene consists of 41 exons with an untranslated exon 1 and
exonintron boundaries that conform perfectly to the GTAG rule
None of the observed polymorphisms did alter the predicted amino acid sequence of
MYH9 nor showed the identified haplotypes an association with the CCSD
phenotype
Thus these silent point mutations found in affected and unaffected Dalmatian dogs
do not seem to be responsible for the CCSD phenotype in these three families
To clarify if other regions on CFA10 are responsible for the CCSD phenotype we
added in a second step 27 microsatellite markers derived from the NCBI database to
cover CFA10 with a high density in the flanking region of the MYH9 gene A linkage
analysis was carried out for 23 Dalmatian dog families consisting of 176 animals that
were genotyped with the marker set of 27 microsatellite markers
We found significant linkage to microsatellites in the region spanning 36 Mb to 48 Mb
Consequently we screened this 12 Mb spanning region for SNPs Out of the 23
analyzed Dalmatian dog families five full-sib families were chosen to screen for
SNPs because of their obviously significant values at the above mentioned region
Twenty-six new SNP markers covering the region of 36 Mb to 48 Mb equally were
developed and added to the linkage analysis The significant Zmeans on CFA10 was
confirmed after adding the SNP markers Furthermore with the use of SNPs the
apparently linked region spanning 36 Mb to 48 Mb could be narrowed down to 5 Mb
spanning from 39 Mb to 44 Mb For this reason the use of SNPs in addition to
informative microsatellites appears highly recommendable
In further studies more SNPs have to be developed within the identified CCSD region
on CFA10 to localize the deafness causing gene or to find unambiguously associsted
SNP markers which could be used for a population-wide genetic test for CCSD
Chapter 10
Summary
Summary 127
Summary
Molecular genetic analysis of canine congenital sensorineural deafness in Dalmatian dogs
Katharina Mieskes (2006) The objective of the present study was to localize the gene or genomic region that is
involved in the development of canine congenital sensorineural deafness (CCSD) in
Dalmatian dogs
In man as in different dog breeds deafness is an often diagnosed disorder with the
Dalmatian dog showing the highest incidence Many genetic disorders in humans
and domestic dogs (Canis familiaris) demonstrate a high level of clinical and
molecular similarity
Altogether 39 genes have already been found causative for sensorineural non-
syndromic hearing impairment in humans Out of this 39 deafness causing genes a
total of 32 candidate genes were selected for canine congenital deafness which
showed an appropriate clinical and histological disease pattern in comparison to
deafness in Dalmatians dogs
On the one hand an existing set of 43 microsatllite markers for in total 24 candidate
genes were used for a non-parametric linkage analysis among them the claudin-14
(CLDN14) gene on canine chromosome (CFA) 31 and the myosin heavy polypeptide
9 (MYH9) gene on CFA10 The gap junction protein alpha 1 (GJA1) gene on CFA1
was also considered as a candidate gene for CCSD and thus GJA1-associated
microsatellites were part of the existing set Recently it turned out that GJA1 is not
responsible for human sensorineural non-syndromic deafness but for a human
syndromic disorder that can be related with conductive deafness In the last few
years more human deafness genes have been identified among them eight genes
that were considered as appropriate candidates for CCSD For these eight genes a
total of 21 SNPs were newly developed and used for non-parametric linkage and
association analyses
Summary
128
The used microsatellite and SNP markers derived either from a partial sequence
analysis of BAC clones each containing a different candidate gene or from
sequences deposited in the current dog genome assembly (Boxer genome assembly
21) of the NCBI GenBank We found significant linkage to markers associated to
CLDN14 on CFA31 MYH9 on CFA10 and GJA1 on CFA1 To substantiate the
linkage we searched for sequence variations within these three genes SNPs found
in intronic sequences of either gene were included in the linkage analyses Sequence
analysis neither revealed a causative mutation nor significant linkage disequilibrium
of SNP markers with CCSD Subsequently CFA1 10 and 31 were scanned
completely with microsatellite markers derived from the NCBI database with the
purpose to see if other regions on this three chromosomes harbour a gene that is
involved in the development of CCSD
The analyses of SNPs and more microsatellite markers on CFA1 revealed no
significant linkage to CCSD Thus it is unlikely that the canine chromosome 1 and
the GJA1 gene are responsible for the CCSD phenoptype As deafness in dogs
especially in Dalmatians is almost exclusively caused by sensorineural non-
syndromic forms the GJA1 gene should not be considered as a candidate gene for
CCSD anymore
On CFA10 we could exclude MYH9 for being causal for deafness but by adding
more microsatellites covering CFA10 completely we found significant linkage to the
CCSD phenotype in the region distal of MYH9 Hence new SNP-markers for fine
mapping the region spanning 36 to 48 Mb were developed by sequence analyses of
different Dalmatian dogs The search for SNPs was carried out on genomic
sequences of genes located in the significantly linked region The sequences of
these genomic sequences were derived from the NCBI GenBank The SNPs
confirmed the linkage and narrowed the region harbouring a causative CCSD gene
down to 5 Mb spanning from 39 to 44 Mb
After scanning CFA31 we could not exclude CLDN14 for being responsible for the
CCSD phenotype as microsatellite markers associated to CLDN14 indicated linkage
However to clarify the importance of CLDN14 in the CCSD phenotype more SNPs
have to be developed within the CLDN14 gene as well as in its flanking regions with
the aim to find linkage disequilibrium for SNP markers
Chapter 11
Erweiterte Zusammenfassung
Erweiterte Zusammenfassung 131
Erweiterte Zusammenfassung
Katharina Mieskes
Molekulargenetische Untersuchung der kongenitalen sensorineuralen Taubheit beim Dalmatiner
Einleitung Es gibt mehrere Moumlglichkeiten Taubheit zu klassifizieren Je nach den betroffenen
Strukturen des Gehoumlrorgans lassen sich konduktive und sensorineurale Formen
unterscheiden Erstere entstehen durch Stoumlrungen des Konduktionsmechanismus im
aumluszligeren Gehoumlrgang und im Mittelohr Ursachen fuumlr konduktive Houmlrstoumlrungen koumlnnen
zum Beispiel chronische Otitis externa oder media Frakturen von Mittelohranteilen
oder Tumoren sein Sehr viel oumlfter tritt die sensorineurale Taubheit auf bei der ein
Houmlrverlust als Folge von Transduktions- und Transmissionsstoumlrungen in der Cochlea
(Schnecke) sowie in peripheren und zentralen Anteilen des Nervus cochlearis auftritt
Neben der erblichen Form koumlnnen auch eine Otitis interna Tumoren oder
ototoxische Medikamente zur sensorineuralen Taubheit fuumlhren
Der Houmlrverlust kann bereits seit der Geburt bestehen (kongenital) oder er kann erst
im Laufe der weiteren Lebenszeit auftreten
Beim Menschen ist mehr als die Haumllfte der kongenitalen Taubheit erblich bedingt Die
erbliche kongenitale Taubheit kann als Teil einer multisystemischen Krankheit
(syndromisch) oder als Funktionsstoumlrung auftreten die auf das Ohr und das
vestibulaumlre System (nicht-syndromisch) begrenzt ist Da die nicht-syndromische
ererbte Taubheit fast ausschlieszliglich durch Cochleadefekte verursacht wird leiden die
Patienten unter sensorineuraler Taubheit Es wird geschaumltzt dass die nicht-
syndromische erbliche Taubheit 60-70 aller menschlichen Taubheitsfaumllle
verursacht Weiterhin sind 70-80 der Faumllle der nicht-syndromischen Taubheit auf
einen autosomal rezessiven Erbgang zuruumlck zu fuumlhren gefolgt von einem autosomal
dominanten Erbgang mit 10-20 der Faumllle und 1-2 der Faumllle sind X- gekoppelt Ein
noch geringerer Prozentsatz wird durch einen maternalen Erbgang verursacht
Von den ca 25000 bis 30000 menschlichen Genen ist nach Schaumltzungen bis zu 1
wichtig fuumlr den Houmlrprozess Annaumlhernd 120 Gene sind inzwischen identifiziert die fuumlr
Erweiterte Zusammenfassung
132
verschiedene Formen der menschlichen vererbten Taubheit verantwortlich sind
Darunter sind ungefaumlhr 80 Gene fuumlr die syndromische und 39 Gene fuumlr die nicht
syndromische vererbte Taubheit verantwortlich Insgesamt stellen die 120
identifizierten Gene vermutlich ein Drittel aller taubheitsverursachenden Gene dar
Die kongenitale sensorineurale Taubheit ist eine Erkrankung von der viele
Saumlugetierspezies betroffen sind Beim Hund werden uumlber 54 verschiedene Rassen
beschrieben bei welchen ein kongenitaler Houmlrverlust gehaumluft auftritt In zahlreichen
europaumlischen und amerikanischen Studien weisen hierbei die Dalmatiner mit 165-
30 die houmlchste Taubheitsinzidenz auf
Die histologischen Veraumlnderungen im Innenohr werden im Allgemeinen aumlhnlich wie
beim Menschen als cochleosacculaumlr beschrieben Die Degenerationen im Innenohr
schreiten bei betroffenen Tieren mit zunehmendem Alter fort und fuumlhren
normalerweise in einem Alter von drei bis vier Wochen spaumltestens aber in einem
Alter von etwa vier Monaten zu einem vollstaumlndigen ein- oder beidseitigen
Houmlrverlust
Eine objektive Diagnose der Taubheit besonders wenn sie nur einseitig ist stuumltzt
sich auf die brainstem auditory evoked response (BAER in Deutschland
audiometrischer Test) ein elektro-diagnostischer Test bei dem durch einen
bestimmten Reiz ausgeloumlste elektrische Aktivitaumlten (akustisch evozierte Potentiale) in
der Cochlea und den Nervenbahnen zur auditorischen Hirnrinde detektiert werden
Obwohl fuumlr den Dalmatiner die Erblichkeit der Erkrankung wiederholt nachgewiesen
werden konnte wurden fuumlr die verschiedenen untersuchten Dalmatinerpopulationen
auch sehr viele unterschiedliche Erbgaumlnge in Betracht gezogen Es ist bisher nicht
gelungen die genaue Anzahl der beteiligten Gene zu bestimmen Auch war es
bislang fuumlr keine der betroffenen Hunderassen moumlglich ein taubheitsverursachendes
Gen zu identifizieren
Bei deutschen Dalmatinern ergab eine Auswertung mittels komplexer
Segregationsanalysen dass ein Modell mit einem rezessiven Hauptgen und einer
polygenen Komponente das Auftreten der kongenitalen Taubheit in der untersuchten
Population am besten erklaumlrte Zusaumltzlich konnte ein Zusammenhang zu
Pigmentierungsgenorten nachgewiesen werden da Tiere mit blauer Augenfarbe
signifikant haumlufiger von Taubheit betroffen waren und gleichzeitig Tiere mit
Plattenzeichnung seltener kongenitalen Houmlrverlust zeigten
Erweiterte Zusammenfassung 133
Das Zuumlchten ein- oder beidseitig tauber Dalmatiner sowie Dalmatinern mit blauen
Augen ist in Deutschland schon laumlnger verboten und es wird von den
Zuchtverbaumlnden vorgeschrieben dass alle Dalmatinerwelpen in einem Alter von etwa
6 bis 8 Wochen audiometrisch untersucht werden Des Weiteren ist der Defekt
tierschutzrelevant nach sect 11b des Deutschen Tierschutzgesetzes da der artgemaumlszlige
Gebrauch eines Organs eingeschraumlnkt wird und eine Erblichkeit nachgewiesen
wurde Somit duumlrfen Tiere die Defektgentraumlger sind oder bei denen damit zu
rechnen ist nicht zur Zucht verwendet werden Waumlhrend einseitig taube Dalmatiner
als ganz normale Familienhunde gehalten werden koumlnnen sind beidseitig taube
Dalmatiner schwierig zu erziehen schreckhafter und daraus resultierend auch
oftmals aggressiv und bissig Aus diesen Gruumlnden werden fast alle Welpen mit
beidseitigem Houmlrverlust euthanasiert
Es hat sich gezeigt dass allein der Zuchtausschluss betroffener Tiere nicht genuumlgt
um die hohe Taubheitsinzidenz beim Dalmatiner deutlich zu reduzieren Aus diesen
Gruumlnden ist es von erheblicher Bedeutung die kongenitale sensorineurale Taubheit
beim Dalmatiner molekulargenetisch zu untersuchen um so ein
molekulargenetisches Testverfahren entwickeln zu koumlnnen welches auch die
Identifizierung von Anlagetraumlgern ermoumlglicht
Innerhalb des letzten Jahrzehnts wurden auch zwischen relativ unverwandten
Spezies die strukturellen und funktionellen Homologien auf genetischer Ebene
zunehmend verdeutlicht
Es wurde inzwischen eine groszlige Anzahl von taubheitsverursachenden
Genmutationen beim Menschen und der Maus aufgeklaumlrt Da auch die
histopathologischen Befunde beim Menschen und der Maus oftmals sehr aumlhnlich
denen beim Dalmatiner sind scheinen Gene die beim Menschen verantwortlich fuumlr
die congenitale sensorineurale Taubheit sind geeignete Kandidatengene fuumlr die
canine congenitale sensorineurale Taubheit zu sein
Das Ziel dieser Dissertation ist es die fuumlr die Taubheit kausalen Gene bzw Genorte
zu lokalisieren Um die kongenitale sensorineurale Taubheit beim Dalmatiner
moumlglichst effizient molekulargenetisch aufzuklaumlren wurden 32 Kandidatengene mit
genassoziierten Markern auf eine potenzielle Kopplung mit der caninen kongenitalen
sensorineuralen Taubheit untersucht
Die Genombereiche mit signifikanter Kopplung zur kongenitalen sensorineuralen
Taubheit wurden weitergehend molekulargenetisch analysiert
Erweiterte Zusammenfassung
134
Kopplungsanalyse fuumlr 24 Kandidatengene der caninen kongenitalen sensorineuralen Taubheit (CCSD) mit 43 genassoziierten Mikrosatelliten Markern
Material und Methoden
Fuumlr 24 Kandidatengene stand ein Mikrosatelliten Marker Set zur Verfuumlgung Das
Marker Set beinhaltete Mikrosatelliten fuumlr folgende Kandidatengene CDH23
CLDN14 COCH COL11A2 DFNA5 DIAPH1 EDN3 EDNRB EYA4 GJA1 GJB2
GJB6 MITF MYH9 MYO6 MYO7A MYO15A OTOF PAX3 POU4F3 SLC26A4
SOX10 TECTA und TMPRSS3 Als Familienmaterial fuumlr die Mikrosatellitenstudie
wurden 215 Dalmatiner verwendet Die Tiere verteilten sich auf 22 unverwandte
Vollgeschwisterfamilien und zwei paternale Halbgeschwistergruppen Die
genotypisierten Familien bestanden aus saumlmtlichen tauben Nachkommen (uni- oder
bilateral taub) ihren Eltern und einem bis vier nicht betroffenen Voll- bzw
Halbgeschwistern Mindestens zwei der Vollgeschwister einer Familie waren uni-
oder bilateral taub Der Phaumlnotyp der verwendeten Tiere wurde durch einen
audiometrischen Test bestimmt
Alle Mikrosatellitenmarker wurden uumlber PCR und Polyacrylamidgelelektrophorese
ausgewertet
Kopplungsanalyse
Die nicht-parametrische Kopplungsanalyse wurde unter Verwendung der Software
MERLIN (multipoint engine for rapid likelihood inference Version 0102)
durchgefuumlhrt und basierte auf dem identical-by-descent (IBD) Verfahren Dabei
wurden die Markerallele auf Kosegregation mit der phaumlnotypischen Auspraumlgung der
Erkrankung getestet Darauf folgend wurde die einer Normalverteilung folgende
Teststatistik fuumlr den Anteil von IBD-Markerallelen (Zmean) und ein daraus
abgeleiteter LOD-Score berechnet Als signifikant fuumlr die Kosegregation eines
Markerallels mit dem Phaumlnotyp der caninen kongenitalen sensorineuralen Taubheit
(CCSD) gelten Irrtumswahrscheinlichkeiten (p) von 005 oder kleiner
Die Daten wurden mit dem Softwarepaket SAS (Statistical Analysis System Version
913) bearbeitet um die Marker bezuumlglich Allelfrequenzen beobachteten und
erwarteten Heterozygotiegrad und PIC (Polymorphism information content)
charakterisieren zu koumlnnen
Erweiterte Zusammenfassung 135
Ergebnisse und Diskussion Das Mikrosatelliten Set bestand insgesamt aus 43 Markern mit einem bis drei
Markern pro Kandidatengen Zunaumlchst wurde fuumlr alle 24 Familien zusammen eine
Kopplungsanalyse durchgefuumlhrt Vermeintliche CCSD Loci wurden auf den caninen
Chromosomen (CFA) 1 10 12 20 und 31 lokalisiert Im Anschluss wurden die
Ergebnisse der Kopplungsanalyse auf Heterogenie zwischen den Familien uumlberpruumlft
Fuumlr einzelne Familien konnten houmlhere Teststatistiken und somit engere
Kopplungsbeziehungen als bei der Analyse aller Familien nachgewiesen werden
Eine signifikante Kopplung mit der kongenitalen sensorineuralen Taubheit wurde fuumlr
die Gene GJA1 auf CFA1 MYH9 auf CFA10 und CLDN14 auf CFA31 sowohl im
Gesamtmaterial wie in jeweils unterschiedlichen Gruppen von Dalmatinerfamilien
gefunden Es ist daher wahrscheinlich dass diese Gene oder Gene in ihrer naumlheren
Umgebung an der Entwicklung der Taubheit in den jeweiligen Familien involviert
sind Fuumlr die restlichen Kandidatengene lagen die Multipoint Teststatistiken fuumlr den
Anteil von IBD-Markerallelen (Zmean) und daraus abgeleiteten LOD-Scores bei
annaumlhernd Null und waren nicht signifikant
Die Ergebnisse dieser Teststatistiken zeigen dass der Erbgang der nicht-
syndromischen Taubheit bei Dalmatinern vermutlich ebenso heterogen ist wie er
sich beim Menschen darstellt
Dennoch stellt diese Studie den ersten Schritt dar um die CCSD verursachenden
Gene beim Dalmatiner zu identifizieren Die Gene GJA1 MYH9 und CLDN14 sowie
ihre naumlhere Umgebungen werden im weiteren Verlauf der Studie weitergehend
molekulargenetisch mit Hilfe von SNPs und Mikrosatelliten untersucht Aufgrund der
Heterogenitaumlt zwischen den Familien wurden fuumlr die weitere molekulargenetische
Analyse bestimmter Gene oder Genomregionen nur die Familien mit dem houmlchsten
Beitrag zu den Teststatistiken der Kopplungsanalyse (Zmean gt 1) ausgewaumlhlt
Erweiterte Zusammenfassung
136
Evaluierung von neu entwickelten SNP Markern assoziiert mit Kandidatengenen fuumlr die canine kongenitale sensorineurale Taubheit (CCSD) Material und Methoden Fuumlr acht Kandidatengene (TMC1 TMIE USH1C MYH14 MYO3A PRES WHRN
und ESPN) wurden intragenische Single Nucleotide Polymorphism (SNP) Marker neu
entwickelt
Als Familienmaterial fuumlr die Genotypisierung der SNPs wurde DNA von insgesamt 39
Dalmatinern verwendet die vier Vollgeschwisterfamilien angehoumlrten Mindestens
zwei der Vollgeschwister einer Familie waren uni- oder bilateral taub Der Phaumlnotyp
der verwendeten Tiere wurde durch einen audiometrischen Test bestimmt Die vier
Familien bestanden aus sechs bis zehn Vollgeschwistern und den dazugehoumlrenden
Elterntieren
Identifizierung von SNPs (Single Nucleotide Polymorphisms)
Die caninen Sequenzen der Kandidatengene wurden mit Hilfe der humanen mRNA
mittels BLAST (Basic Local Alignment Search Tool) Suche aus veroumlffentlichten
Sequenzen der NCBI Datenbank (Boxer genome assembly 21) entnommen Die
Exon Intron Grenzen wurden mit Hilfe des mRNA-to-genomic alignment Programms
Spidey bestimmt und Primer mit Hilfe des Primer3 Programms in die intronischen
Sequenzen gelegt Die SNPs wurden an den DNA-Sequenzen von Elterntieren der
betroffenen Dalmatinerfamilien identifiziert Die Amplifikate wurden mit Hilfe des
MegaBACE 1000 sequenziert und auf SNPs untersucht Bei Vorliegen eines oder
mehrerer SNPs wurde die entsprechende DNA-Sequenz der zugehoumlrigen
Nachkommen ebenfalls amplifiziert und sequenziert Die Auswertung erfolgte mit
Hilfe des Sequencher 42 Programms
Kopplungsanalyse
Die Genotypen der neuen SNP Marker wurden unter Verwendung des Programms
MERLIN ausgewertet Die Daten wurden mit dem Softwarepaket SAS (Statistical
Analysis System Version 913) bearbeitet um das Markerset bezuumlglich
Allelfrequenzen beobachteten Heterozygotiegrad und PIC (Polymorphism
information content) charakterisieren zu koumlnnen Des Weiteren wurde auf
Kopplungsungleichgewicht und die Assoziation der Haplotypen mit CCSD mittels
Erweiterte Zusammenfassung 137
des Programms CASECONTROL und HAPLOTYPE von SAS Genetics (Statistical
Analysis System Version 913 Cary NC USA) getestet
Ergebnisse und Diskussion Fuumlr acht Kandidatengene wurden mindestens zwei heterozygote SNPs entwickelt
Die nicht-parametrische Kopplungsanalyse zeigte keine signifikanten Teststatistiken
Offensichtlich war kein Haplotyp mit der caninen kongenitalen sensorineuralen
Taubheit (CCSD) in den untersuchten Familien assoziiert Da sowohl uni- und
bilateral taube Tiere als auch beidseitig houmlrende Tiere uumlber identische Haplotypen
verfuumlgen ist es unwahrscheinlich dass die Gene TMC1 TMIE USH1C MYH14
MYO3A PRES WHRN und ESPN an der Entstehung der CCSD beim Dalmatiner
beteiligt sind
Molekulargenetische Analyse des caninen myosin heavy polypeptide 9 non-muscle (MYH9) auf dem caninen Chromosom 10q232
Material und Methoden Klonierung und Sequenzierung von caniner MYH9 cDNA
Die canine MYH9 Gensequenz wurde mit Hilfe der BLAST Suche aus
veroumlffentlichten Sequenzen der NCBI Datenbank Die Isolierung der vollstaumlndigen
cDNA wurde durch ein Protokoll zur Amplifizierung von cDNA-Enden (rapid
amplification of cDNA ends [RACE]) erreicht Nach Klonierung und Sequenzierung
der RACE-Produkte wurden die Sequenzen mit Hilfe des Sequencher 42
Programms ausgewertet
Sequenzanalyse des caninen MYH9 Gens
Nach Fertigstellung des RACE Protokolls und Auswertung der Sequenzen wurden
die Exon Introngrenzen mit Hilfe des mRNA-to-genomic alignment Programms
Spidey lokalisiert Weiterhin wurden repetitive Elemente mit dem Programm
Repeatmaster 2 sowie der GC Gehalt mit der EBI toolbox CpG PlotCpGreport
ermittelt
Erweiterte Zusammenfassung
138
Mutatiosanalyse
Zur Identifizierung von Polymorphismen innerhalb der caninen MYH9 Sequenz
wurden die Exons mit ihren flankierenden intronischen Sequenzen mit Hilfe der PCR
amplifiziert und sequenziert Verwendet wurden hierfuumlr die DNA von insgesamt 16
Dalmatinern aus drei Familien Mindestens zwei der Vollgeschwister einer Familie
waren unilateral taub Der Phaumlnotyp der verwendeten Tiere wurde durch einen
audiometrischen Test bestimmt
PCR Primer wurden mit Hilfe des Primer3 Programms entwickelt PCR Reaktionen
fuumlr saumlmtliche Exons des MYH9 Gens mit ihren flankierenden Sequenzen wurden
durchgefuumlhrt die PCR Produkte sequenziert und die Sequenzen mit Hilfe des
Sequencher 42 Programms ausgewertet
Unter Verwendung der Software MERLIN (multipoint engine for rapid likelihood
inference Version 0910) wurde nach signifikanten nicht parametrischen LOD-
Scores fuumlr die Kosegregation von Markerallelen mit der phaumlnotypischen Auspraumlgung
der sensorineuralen Taubheit beim Dalmatiner gesucht
Ergebnisse und Diskussion
Analyse der genomischen Organisation und der cDNA des caninen MYH9 Gens
Eine vollstaumlndige cDNA Sequenz des caninen MYH9 Gens von 7484 bp konnte uumlber
die Reverse Transkription-PCR erhalten und bei der EMBL Nukleotid Datenbank
eingereicht werden Das canine MYH9 Gen besteht aus 41 Exons einschlieszliglich
einem untranslatierten ersten Exon und Exon Intron Grenzen die stets der GT AG
Regel folgen Der 5rsquo-UTR Bereich besteht aus 181 bp waumlhrend der 3rsquo-UTR aus 1432
bp besteht Weiterhin besitzt die canine MYH9 cDNA Sequenz einen offenen
Leserahmen von 5889 bp welcher ein Protein kodiert bestehend aus 1963
Aminosaumluren Die Groumlszlige der Exons variiert von 28 bis 1556 bp waumlhrend das
gesamte MYH9 Gens ungefaumlhr 90 kb umfasst Die Homologie der Sequenzen
zwischen dem humanen murinen und caninen MYH9 Gen ist limitiert auf die
kodierende Sequenz von Exon 2 bis 41
Mutations- und Haplotypenanalyse
Alle kodierenden Exons mit ihren flankierenden intronischen Sequenzen des MYH9
Gens konnten von den 16 Dalmatinern amplifiziert werden Die Sequenzen wurden
mit den entsprechenden Sequenzen vom Boxer aus der NCBI Genbank verglichen
Erweiterte Zusammenfassung 139
Die Suche nach Sequenzvariationen im MYH9 Gen lieszlig 22 SNPs erkennen wobei
die meisten Polymorphismen in den flankierenden Regionen der Exons gefunden
wurden Keiner der beobachteten Polymorphismen in den Exons aumlnderte die
Aminosaumluresequenz von MYH9 Auch zeigten die identifizierten Haplotypen keine
Assoziation mit dem CCSD Phaumlnotyp Multipoint Teststatistiken fuumlr den Anteil von
IBD-Markerallelen (Zmean) und ein daraus abgeleiteter LOD-Score lagen bei
annaumlhernd Null und waren nicht signifikant Zwei genassoziierte Mikrosatelliten
zeigten eine Kopplung diese kann jedoch mit Heterogenitaumlt zwischen den Familien
erklaumlrt werden
Fazit
Die Charakterisierung des Transkriptes und der genomischen Sequenz des caninen
MYH9 Gens zeigten einen konservierten Aufbau des Gens im Bezug auf das
humane orthologe Gen Aufgrund eines untranslatierten Exons im 5rsquo-UTR Bereich ist
das canine Gen groumlszliger als das humane Gen
Sowohl uni- oder bilateral taube und bilateral houmlrende Dalmatiner weisen identische
Haplotypen auf Daher sind in den untersuchten Dalmatinerfamilien die gefundenen
Polymorphismen offensichtlich nicht mit CCSD assoziiert Des Weiteren zeigt diese
Studie dass keine funktionellen Mutationen der vollstaumlndigen kodierenden Region
von MYH9 vorliegen Wir koumlnnen also abschlieszligend das MYH9 Gen als
Kandidatengen fuumlr die congenitale sensorineurale Taubheit beim Dalmatiner
ausschlieszligen
Erweiterte Zusammenfassung
140
Evaluierung von Mikrosatelliten und SNP Markern der caninen Chromosomen 1 10 und 31 auf Kopplung und Assoziation mit der caninen kongenitalen Taubheit Material und Methoden Pedigreematerial
Insgesamt wurden 176 Dalmatiner in die Untersuchung einbezogen Die Tiere
verteilten sich auf 23 unverwandte Vollgeschwisterfamilien und eine paternale
Halbgeschwistergruppe Die genotypisierten Familien bestanden aus saumlmtlichen
tauben Nachkommen (unilateral oder bilateral taub) ihren Eltern und einen bis vier
nicht betroffenen Voll- bzw Halbgeschwistern Mindestens zwei der Vollgeschwister
einer Familie waren unilateral oder bilateral taub Der Phaumlnotyp der verwendeten
Tiere wurde durch einen audiometrischen Test bestimmt
Alle 176 Dalmatiner wurden fuumlr die Mikrosatellitenstudie auf den caninen
Chromosomen 1 10 und 31 einbezogen Fuumlr die Feinkartierung mit Hilfe der SNPs
wurden dagegen nur noch die Dalmatinerfamilien mit einer eindeutigen Kopplung zu
der entsprechenden genomischen Region untersucht Fuumlr die Region auf CFA10
wurden dafuumlr 34 Tiere aus fuumlnf Vollgeschwisterfamilien ausgewaumlhlt und zur
Untersuchung des CLDN14 Gens auf CFA31 wurden 24 Tiere aus vier
Vollgeschwisterfamilien und 12 Tiere aus einer groszligen Halbgeschwisterfamilie
verwendet Fuumlr die Suche nach Polymorphismen im GJA1 Gen wurden 16 Tiere aus
einer groszligen Halbgeschwisterfamilie verwendet
Mikrosatellitenstudie
Fuumlr die Mikrosatellitenstudie von CFA1 10 und 31 wurden insgesamt 60 Marker
verwendet Mit jeweils 27 Mikrosatelliten wurde CFA1 und mit 27 Mikrosatelliten
wurde CFA10 vollstaumlndig abgedeckt Insgesamt 6 Marker wurden fuumlr CFA31
verwendet
Alle hier verwendeten Mikrosatelliten sind der NCBI Datenbank entnommen und
wurden uumlber PCR und Polyacrylamidgelelektrophorese ausgewertet
Identifizierung von SNPs (Single Nucleotide Polymorphisms)
Zur weiteren Abklaumlrung und zum Ausschluss falsch positiver Ergebnisse wurden
zusaumltzlich zu den Mikrosatelliten SNPs in den signifikanten Regionen auf CFA1 10
Erweiterte Zusammenfassung 141
und CFA 31 entwickelt und an den Familien die eine Kopplung zeigten
genotypisiert
Die caninen genomischen Sequenzen der untersuchten Gene wurden der NCBI
Datenbank (Boxer genome assembly 21) entnommen Exon Intron Grenzen wurden
bestimmt und Primer entwickelt Nach Durchfuumlhrung der PCR wurden die Amplifikate
mit Hilfe des MegaBACE 1000 sequenziert und auf SNPs untersucht Die
Auswertung erfolgte mit Hilfe des Sequencher 42 Programms
Die Genotypen der neuen SNP Marker wurden zu den Genotypen der fuumlr die
Kopplungsanalyse verwendeten Mikrosatelliten hinzugefuumlgt und gemeinsam unter
Verwendung des Programms MERLIN ausgewertet
Kopplungsanalyse
Unter Verwendung der Software MERLIN (multipoint engine for rapid likelihood
inference Version 0910) wurde nach signifikanten nicht parametrischen LOD-
Scores fuumlr die Kosegregation von Markerallelen mit der phaumlnotypischen Auspraumlgung
der sensorineuralen Taubheit beim Dalmatiner gesucht
Die Daten wurden mit dem Softwarepaket SAS (Statistical Analysis System Version
913) bearbeitet um die Marker bezuumlglich Allelfrequenzen beobachteten
Heterozygotiegrad und PIC (Polymorphism information content) charakterisieren zu
koumlnnen Des Weiteren wurde das Kopplungsungleichgewicht und die Assoziation der
Haplotypen mit CCSD mittles des Programms CASECONTROL und HAPLOTYPE
von SAS Genetics getestet
Ergebnisse und Diskussion CFA1
In einer groszligen Halbgeschwisterfamilie wurde eine signifikante Kopplung zu einem
GJA1 genassozierten Marker festgestellt Mit Hilfe der DNA von Tieren aus dieser
Halbgeschwisterfamilie wurde eine Sequenzanalyse des GJA1 Gens durchgefuumlhrt
Es wurden 3 SNPs im 3acute-untranslatierten Bereich gefunden Die identifizierten
Haplotypen zeigten keine Assoziation mit dem CCSD Phaumlnotyp Sowohl uni- oder
bilateral taube und bilateral houmlrende Dalmatiner wiesen identische Haplotypen auf
Daher sind in den untersuchten Dalmatinerfamilien die gefunden Polymorphismen
offensichtlich nicht mit CCSD assoziiert Auch wurde keine funktionelle Mutation in
der vollstaumlndigen kodierenden Region von GJA1 gefunden wurde Es ist daher
Erweiterte Zusammenfassung
142
unwahrscheinlich dass das GJA1 Gen an der Entwicklung der CCSD in der
untersuchten Halbgeschwisterfamilie beteiligt ist Die signifikante Kopplung der
GJA1-benachbarten Mikrosatelliten mit CCSD kann durch ein eng gekoppeltes Gen
hervorgerufen worden sein welches fuumlr die Entstehung der Taubheit in der
untersuchten Halbgeschwisterfamilie verantwortlich ist oder durch ein falsch-
positives Ergebnis der vorhergehend durchgefuumlhrten Mikrosatellitenstudie
Eine nicht-parametrische Kopplungsanalyse mit Hilfe von 27 Mikrosatelliten Marker
an 24 deutschen Dalmatinerfmilien wurde durchgefuumlhrt um abzuklaumlren ob andere
Genombereiche auf CFA1 signifikante Teststatistiken in mehr Familien zeigen Die
Kopplungsanalyse zeigte keine signifikanten Teststatistiken Es ist daher
unwahrscheinlich dass auf CFA1 Gene fuumlr die Entwicklung von CCSD in den
untersuchten Familien der aus Deutschland stammenden Dalmatiner verantwortlich
sind
Da kuumlrzlich festgestellt wurde dass das GJA1 Gen auch beim Menschen nicht fuumlr
eine Form der sensorineuralen nicht-syndromischen Taubheit verantwortlich ist
sondern an einer syndromischen Krankheit beteiligt ist die mit konduktiver Taubheit
einhergehen kann sollte das GJA1 Gen nicht laumlnger als ein Kandidatengen fuumlr
CCSD angesehen werden
CFA31
Auf CFA31 wurde durch eine nicht-parametrische Kopplungsanalyse von
Mikrosatelliten eine Kopplung eines CLDN14-benachbarten Markers mit der
sensorineuralen Taubheit beim Dalmatiner festgestellt Daraufhin wurden zusaumltzlich
zu den Mikrosatelliten acht SNPs entwickelt indem das einzige beim Menschen
translatierte Exon weite intronische Sequenzen sowie 5rsquo- und 3rsquo- untranslatierte
Bereiche des CLDN14 Gens einer Sequenzanalyse unterzogen wurden Nur zwei der
identifizierten SNPs waren polymorph in den untersuchten fuumlnf Dalmatinerfamilien
Sowohl uni- als auch bilateral taube und bilateral houmlrende Dalmatiner teilten
identische Haplotypen daher konnte keine Kosegregation mit tauben Dalmatinern
festgestellt werden
Da bislang keine funktionelle Mutation in der kodierenden Region von CLDN14
gefunden wurde ist es fuumlr eine eindeutige Aussage uumlber die Rolle des CLDN14
Gens im Entstehungsprozess der kongenitalen sensorineuralen Taubheit
Erweiterte Zusammenfassung 143
beim Dalmatiner notwendig noch weitere Marker im CLDN14 Gen sowie seiner
naumlheren Umgebung zu entwickeln und zu analysieren Da keine weitere Kopplung zu
einem Mikrosatelliten distal oder proximal des CLDN14 Gen festgestellt werden
konnte ist es wahrscheinlich dass entweder das CLDN14 Gen oder ein Gen in
seiner naumlheren Umgebung fuumlr die Entwicklung der sensorineuralen Taubheit in den
untersuchten Dalmatinerfamilien verantwortlich ist
CFA10
Auf CFA10 wurde durch eine nicht-parametrische Kopplungsanalyse von 27
Mikrosatelliten eine 12 Mb umfassende genomische Region mit einer signifikanten
Kopplung zur CCSD identifiziert Zur weiteren Feinkartierung wurden 26 neue SNPs
mit einem durchschnittlichen Abstand von 03 Mb fuumlr diesen Bereich entwickelt
Die SNPs waren groumlszligtenteils informativ im vorliegenden Familienmaterial und
bestaumltigten in einer folgenden Kopplungsanalyse die Lokalisation auf CFA10
deutlich Weiterhin konnte die gekoppelte Region auf einem 5 Mb umfassenden
Bereich von 39 bis 44 Mb eingegrenzt werden Es ist wahrscheinlich dass die
identifizierte CCSD Region ein Gen beinhaltet welches auf die Entstehung der
caninen kongenitalen Taubheit einwirkt
Es ist notwendig mehr SNPs zu entwickeln um das taubheitsverursachende Gen zu
lokalisieren oder einen eindeutig assoziierten SNP zu identifizieren
Chapter 12
References
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MELDRIM J MELNIKOV A MENEUS L MIHALEV A MIHOVA T MILLER K
MITTELMAN R MLENGA V MULRAIN L MUNSON G NAVIDI A
NAYLOR J NGUYEN T NGUYEN N NGUYEN C NGUYEN T NICOL R
NORBU N NORBU C NOVOD N NYIMA T OLANDT P ONEILL B
ONEILL K OSMAN S OYONO L PATTI C PERRIN D PHUNKHANG P
PIERRE F PRIEST M RACHUPKA A RAGHURAMAN S RAMEAU R
RAY V RAYMOND C REGE F RISE C ROGERS J ROGOV P SAHALIE J
SETTIPALLI S SHARPE T SHEA T SHEEHAN M SHERPA N SHI J
SHIH D SLOAN J SMITH C SPARROW T STALKER J STANGE-
THOMANN N STAVROPOULOS S STONE C STONE S SYKES S
TCHUINGA P TENZING P TESFAYE S THOULUTSANG D
THOULUTSANG Y TOPHAM K TOPPING I TSAMLA T VASSILIEV H
VENKATARAMAN V VO A WANGCHUK T WANGDI T WEIAND M
WILKINSON J WILSON A YADAV S YANG S YANG X YOUNG G YU Q
ZAINOUN J ZEMBEK L ZIMMER A LANDER ES (2005) Genome sequence
comparative analysis and haplotype structure of the domestic dog Nature
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GANGAROSSA S CARIDI G BORDO D LO NIGRO C GHIGGERI GM
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Chapter 13
Appendix
Appendix microsatellite marker III
HE
Te
79
5 7
98
76
9 5
12
71
3 8
33
70
2 4
35
72
7 7
00
54
6 4
77
48
8 3
08
44
8 5
40
67
4 8
05
76
2 6
54
70
8 7
73
78
1 6
87
65
4 6
26
51
9 5
89
76
4 1
60
PIC
d
72
2 8
41
67
0 4
85
67
1 7
85
57
2 4
07
61
4 6
29
35
8 3
30
40
0 2
90
35
0 4
60
70
0 7
90
76
2 6
21
63
8 7
26
77
1 6
26
60
1 6
13
38
7 5
52
80
0 1
40
Prim
er (r
ever
se)
5acute -gt
3acute
AA
CTG
TTTG
ATT
TTG
ATG
AG
GC
C
TTTG
ACC
TAC
ATA
TAA
AA
CA
AG
C
CA
CG
AA
GAA
AG
CC
ATG
GTT
T C
AA
CA
ATT
CC
TTTG
TTG
GTG
C
CTG
CC
AG
CTT
CC
TAC
AGC
T
GTG
GTC
ACA
AG
AC
TTTA
GC
C
CTG
CA
CC
AAA
GA
AA
CTC
C
GG
AC
CTA
TTC
TGA
AG
CC
TAA
C
TG
GC
CTT
TAA
TTTA
TCAT
GG
AG
A
TCA
GG
CC
CA
GA
TAG
TATG
C
TC
CTT
TGG
TTTT
TAG
CAG
GG
T
CTG
TGC
CC
AC
CTG
TGG
AG
C
AA
GA
GG
ATA
CC
GG
TGG
CA
G
CG
TGC
TTTG
TTA
TGG
CTT
GA
C
AG
CA
TGA
AG
GA
TCTC
TGA
CTA
A
CTA
AC
TCG
CC
AG
AC
TATT
C
CC
CTC
CAC
CA
ATC
TCTT
CA
C
GC
AA
AG
GTG
TATT
TAC
ATG
ATG
G
GG
CA
GC
CTT
ATT
ATT
CA
TGG
A
AC
AC
AG
GC
AC
AG
GA
GC
ATC
C
AG
CC
ATC
CA
GG
AA
ATC
G
CTG
AA
CTG
GTC
CC
TCAA
GC
A
GTC
ATG
CTG
ATT
TCTG
TGC
C
GA
CC
TATC
CA
TAC
ATG
CC
C
GAA
GG
AAG
GAA
GG
AAG
AAAA
GA
TTA
GTA
AA
GA
GA
CA
GC
TGA
TTG
CC
A
TCTA
CC
CTG
CA
ACC
CTG
TG
ATT
GA
GA
CC
CA
AG
ACTG
TTA
GTG
G
GTA
AC
AAC
CA
GAG
TGTG
TGTG
A
CG
TCG
AG
CTC
CTG
GC
AT
Prim
er (f
orw
ard)
5acute -
gt 3acute
GG
GTA
GA
TTTC
AA
CA
AAT
AG
TAC
TGG
T
AG
AA
AG
GC
CTG
GA
TGTC
G
AG
TAG
AA
GA
GG
CC
AC
GC
AA
A
TTT
TCC
AA
AA
TGA
AA
AC
CTG
C
AG
ATT
GTT
TATG
CA
GG
CA
TTG
A
GA
GTT
GAA
AG
GTT
GAA
AA
TGG
G
CTC
AG
AG
GC
AG
ATA
GA
AA
A
ATT
GA
TTC
ATG
ACC
CA
CTA
A
TTT
TGTC
AAG
CA
GC
CC
TATA
TC
TG
AA
ATG
GTA
CTT
CA
GC
ATC
G
AG
CTT
AG
CTT
AC
TGC
CC
TGG
T
CC
CC
GG
CC
CC
TGC
CC
GG
AG
T
AC
CAC
TGTC
ATT
TTTC
CA
TGC
C
CC
TTA
GG
AGG
AG
GC
AAG
AC
C
CA
AG
GG
GTA
TGTT
GTC
TATT
ACT
GG
GTG
GG
AA
ATG
TGA
CTC
TG
GG
GC
TGC
CC
ATT
TCTT
TAA
T T
TGC
CA
CAA
ATC
AC
TTAA
AG
G
GA
CTG
AG
TTC
TTTC
AG
CA
CA
GTG
A
AAAT
AAC
ACAG
CAT
CAG
G
GA
ATC
CC
CA
ATG
TAC
ATG
GC
A
AC
AG
GC
GG
ATT
CTG
TAG
C
AC
TGG
CC
AAA
GA
GTA
CA
AG
G
CC
TTG
AC
TCA
GC
AGC
CC
TAC
A
AA
GA
TTG
TCTT
GA
CAC
GC
TG
GG
GTC
CTG
GG
ATC
AA
GC
C
AC
TGA
CTG
ATG
TCC
TGTG
CG
A
AA
AA
GTG
TAG
AG
CTT
TCTT
CA
AA
T
AA
ATT
CA
ATA
AG
CC
ATG
GA
GG
A
CC
TCC
AAG
ATG
GC
TCTT
GA
ATc
(degC
) 60
58
58
56
58
60
54
56
60
60
58
60
60
60
60
60
60
60
60
52
56
58
54
62
60
60
62
62
60
60
Pro
duct
si
ze (b
p)
380-
400
337-
409
267-
304
225-
235
312-
336
350-
394
155-
161
126-
156
510-
570
436-
468
189-
191
72-8
0 13
0-13
6 34
0-35
0 14
5-15
3 98
-102
22
0-24
0 21
2-24
0 34
7-40
3 32
5-34
1 25
1-27
9 32
5-36
1 19
7-24
5 30
9-40
9 34
1-37
5 21
5-23
1 30
8-31
6 16
2-17
4 46
8-50
2 18
2-19
0
Alle
les
6 14
8 4 7 11
3 6 8 7 2 2 3 4 2 3 5 8 12
7 8 7 14
10
9 7 3 3 15
2
Mbb
54
1
49
19
8 2
18
23
5 2
47
26
0 3
02
35
3 3
98
58
8 6
26
63
9 7
61
77
5 7
78
78
6 7
98
85
4 1
060
1
090
1
100
1
100
1
110
1
130
1
150
1
170
3
5
12
4 1
63
CFA
a
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10
10
10
Tabl
e 1
Cha
ract
eris
tics
of m
icro
sate
llite
mar
kers
sel
ecte
d fo
r CFA
1 1
0 an
d 31
com
pris
ing
in to
tal 6
0 m
arke
rs
(Con
tinue
d on
nex
t pag
e) T
he m
icro
sate
llite
mar
kers
wer
e al
l der
ived
from
the
NC
BI d
atab
ase
(http
w
ebn
cbin
lmn
ihg
ov)
Mar
ker
FH33
25
FH36
03
FH23
13
RE
N18
9I14
FH
3219
FH
3413
R
EN
136G
19
RE
N13
5K06
FH
3922
FH
3300
C
014
24
AH
T107
C
012
51
RE
N17
2C02
D
0150
5 R
EN
04H
08
RE
N15
9F24
FH
3894
FH
2309
R
EN
06N
11
FH23
26
FH33
22
FH35
05
FH25
98
FH26
34
FH22
94
RE
N22
9P15
FH
2537
FH
4081
C
107
81
Appendix microsatellite marker
IV
H
ETe
64
0 6
80
63
0 6
51
54
6 7
06
81
0 6
14
30
2 7
42
34
4 7
40
63
0 8
67
82
2 6
70
75
0 6
80
60
0 4
70
31
0 5
70
80
1 5
80
83
1 5
10
64
0 6
90
19
0 8
22
PIC
d
53
0 6
90
52
2 5
89
80
0 6
70
75
9 5
24
24
7 7
09
26
9 6
90
50
0 7
98
75
5 5
50
69
0 5
30
56
0 3
40
26
0 5
00
80
1 5
30
74
0 3
70
64
0 6
70
25
0 7
21
Prim
er (r
ever
se)
5acute -gt
3acute
AG
GC
CA
GC
AC
CA
TAA
CTC
AG
C
CTC
TCC
TCC
TTC
CC
CTT
CTC
A
GTG
CTG
TCC
ATT
GC
AA
GTC
T
GC
TCC
CC
TGTG
TTC
TGAG
G
CTG
GTG
ACTC
AGA
GAA
GTC
C
GG
AG
TTG
ATA
GTA
AA
TCTT
TGC
GG
T
GC
CC
TATT
CA
TTC
AA
CTG
G
AC
AC
TCTG
GTG
CA
AG
CG
AC
TC
TTC
TAC
TGA
AG
CA
TCC
GC
CT
GA
TTTT
CTC
TCTG
TCC
AC
TT
AA
CA
GC
ATT
TCA
GAC
AGA
GG
G
GG
TTTT
GG
CTG
TGA
GG
ATA
G
AG
TTC
CAG
ATC
GAG
TCC
CA
C
CA
CAT
GAT
TTC
ACTT
GTA
TATG
G
CC
TGA
GG
GTT
TTTA
CAG
TATG
G
TG
AA
ATC
TGG
GA
TTC
TGA
TAC
C
CC
AA
GC
TTTC
AC
CTA
ATC
AC
C
GA
TATT
TTTC
TCTC
CC
AC
CC
C
AC
ATT
TCTA
GG
CAG
TGG
CA
C
TGTC
CC
ATC
TAA
AG
GA
TAG
G
AG
TAA
TGC
TGC
CC
ACG
GA
AC
G
GC
TGG
AA
CC
TTC
CC
CTT
TAG
A
GC
TCTT
ACA
CG
CA
TTG
AG
G
AA
AC
GG
AG
TTC
CA
TCTC
TGG
C
AC
AG
TGA
GG
AG
GG
GTG
G
TG
TAA
CC
CTC
CTG
TGA
TATG
A
TGTG
AA
CC
CC
GC
CC
AATA
G
AA
GTG
AC
TGA
GTA
CC
TGA
AA
TCG
G
CC
TCTG
CC
TCTG
CC
TCTG
T G
AG
CC
CTG
TTC
TCA
GG
TTG
Prim
er (f
orw
ard)
5acute -
gt 3acute
AG
AC
CC
CC
CC
ACTA
CC
CC
AC
GC
GTT
AGC
TGC
CA
TCA
TCTT
G
CTG
GTT
GG
AG
CA
ATA
CC
AA
G
CC
ATT
CA
TGTT
GTT
GC
AG
ATG
T
AA
GG
AG
GG
CA
CTT
GAT
GG
C
TTA
TGG
CA
TTTG
GC
CTG
TC
AA
GG
AG
CAT
CTT
CC
AG
AAC
C
CG
CTC
ATG
CA
AG
TCA
TCA
CA
T C
TGG
CTT
AAA
CC
AC
TGG
TCA
C
CTT
CC
ATC
CC
GTT
GTG
TGT
ATT
CC
CC
AGC
GAT
ACC
A C
TTG
AG
TGG
CTT
GA
GTG
GC
TAC
T
TCTT
GC
TTC
TTG
AA
GTA
AG
CC
T
TGC
CC
GTC
CTA
TAC
TCC
TG
GA
AG
GTG
GTT
ATT
GTC
CTT
GG
G
AA
CC
ACA
GA
AG
AG
CTG
GA
A
GC
ATT
TGA
TGA
AA
TAA
AG
GG
A
GA
ATG
AA
AA
CG
GA
GC
AG
CA
G
CC
TTC
AAC
AC
CC
ATA
GC
TC
TC
AG
CA
ACTA
TAC
ATT
TAA
GA
GC
A
ATG
GA
CA
AATG
AA
CA
AA
AG
T
GTG
AC
TTTC
TTA
TCC
GC
CC
C
CC
AG
AA
AC
TCA
ACTG
ATG
C
TG
GA
CG
CTA
AG
CC
TGAC
TTT
CC
ATA
AG
ATA
CTC
AG
AA
AC
ATG
CA
C
AC
CAT
AA
ATG
GA
TGG
ATA
G
CA
GTG
AG
CA
AA
GC
AA
ATG
AA
C
CC
ATT
AG
CA
AA
TGA
CTG
GG
A
AA
GA
ATG
GG
AA
AA
CTG
ATA
A
AA
GG
TAG
TCC
CA
CG
ATC
CTC
ATc
(degC
) 60
60
60
60
60
60
60
60
60
60
60
60
60
62
60
60
60
60
60
60
60
60
60
60
60
60
60
60
60
60
Pro
duct
si
ze (b
p)
100-
110
112-
124
194-
206
210-
230
309-
341
350-
362
276-
300
96-1
30
130-
138
120-
138
200-
204
278-
296
157-
165
178-
254
358-
368
327-
337
380-
404
160-
170
320-
330
165-
169
230-
232
300-
310
276-
380
255-
263
258-
278
106-
120
164-
180
568-
608
223-
239
172-
186
Alle
les
3 5 3 5 8 4 7 4 2 5 3 7 5 9 6 4 7 3 4 2 2 3 11
5 6 2 4 7 4 8
Mbb
24
9 3
23
34
9 3
67
38
9 3
94
40
1 4
10
45
8 4
64
48
0 4
88
51
5 5
64
56
8 5
97
62
7 6
33
63
7 6
53
67
3 6
85
69
1 7
08
0
66
9
7
28
1 2
96
37
5
CFA
a
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
31
31
31
31
31
31
Tabl
e 1
(con
tinue
d)
Mar
ker
RE
N06
H21
R
EN
68B
08
FH30
55
C10
769
FH
3302
R
EN
181G
20
FH34
03
G02
512
RE
N15
0B12
A
HT1
01
RE
N73
F08
ZUB
EC
A1
C10
16
FH24
22
FH34
48
FH26
65
DTR
105
C
106
02
RE
N16
1L12
C
0410
7 R
EN
91H
07
RE
N15
4O19
FH
3381
R
EN
154G
10
FH21
89
RV
C11
R
EN
43H
24
FH22
39
RE
N11
0K04
FH
2712
a Can
ine
chro
mos
ome
b Pos
ition
of m
arke
r (M
b) c A
nnea
ling
tem
pera
ture
d P
olym
orph
ism
info
rmat
ion
cont
ent (
)
e obse
rved
Het
eroz
ygos
ity
Appendix itemisation of alleles V
Table 2 Itemisation of alleles to bases for SNPs in the pedigrees
Code no 1 2 3 4
Base Adenine Cytosine Guanine Thymine
Appendix laboratory paraphernalia
VI
Laboratory paraphernalia Equipment Thermocycler
PTC-100trade Programmable Thermal Controller (MJ Research Watertown USA)
PTC-100trade Peltier Thermal Cycler (MJ Research Watertown USA)
PTC-200trade Peltier Thermal Cycler (MJ Research Watertown USA)
Automated sequencers
LI-COR Gene Read IR 4200 DNA Analyzer (LI-COR Inc Lincoln NE USA)
LI-COR Gene Read IR 4300 DNA Analyzer (LI-COR Inc Lincoln NE USA)
MegaBACE 1000 (Amersham Biosciences Freiburg)
Centrifuges
Sigma centrifuge 4-15 (Sigma Laborzentrifugen GmbH Osterode)
Desk-centrifuge 5415D (Eppendorf Hamburg)
Biofuge stratos (Heraeus Osterode)
Centrifuge Centrikon H 401 (Kontron Gosheim)
Megafuge 1OR (Heraeus Osterode)
Speed Vacreg Plus (Savant Instruments Farmingdale NY USA)
Agarose gel electrophoresis and pulsed field gel electrophoresis
Electrophoresis chambers OWL Separation Systems Portsmouth NH USA
Biometra Goumlttingen
BioRad Muumlnchen
Generators 2301 Macrodrive 1 (LKB Bromma Sweden)
Power Pac 3000 (BioRad Muumlnchen)
Gel documentation system BioDocAnalyze 312 nm Goumlttingen
Appendix laboratory paraphernalia VII
Others
Milli-Qreg biocel water purification system (Millipore GmbH Eschborn)
Incubator VT 5042 (Heraeus Osterode)
UV-Illuminator 312 nm (Bachhofer Reutlingen)
Centomatreg R Desk-Shaker (B Braun Melsungen AG Melsungen)
Biophotometer (Eppendorf AG Hamburg)
Kits
DNA purification
Montage PCR96 Cleanup Kit (Millipore GmbH Eschborn)
Cloning
Invitrogen TA cloningreg kit (Invitrogen Karlsruhe Germany)
Isolation of DNA
QIAamp 96 DNA Blood Kit (QIAGEN Hilden)
Plasmid Mini Prep 96 Kit (Millipore GmbH Eschborn)
Sequencing
ThermoSequenase Sequencing Kit (Amersham Biosciences Freiburg)
DYEnamic-ET-Terminator Cycle Sequencing Kit (Amersham Biosciences Freiburg
Germany)
RACE
FirstChoiceTM RNA ligase-mediated (RLM)-RACE kit (Ambion Europe Huntingdon
UK)
Appendix laboratory paraphernalia
VIII
RNA Total RNA (Biocat Heidelberg Germany)
Size standards
100 bp Ladder (New England Biolabs Schwalbach Taunus)
1 kb Ladder (New England Biolabs Schwalbach Taunus)
IRDyetrade 700 or 800 (LI-COR Inc Lincoln NE USA)
Reagents and buffers APS solution (10 )
1 g APS
10 ml H2O
Bromophenol blue solution
05 g bromophenol blue
10 ml 05 M EDTA solution
H2O ad 50 ml
dNTP solution
100 microl dATP [100 mM]
100 microl dCTP [100 mM]
100 microl dGTP [100 mM]
100 microl dTTP [100 mM]
1600 microl H2O
the concentration of each dNTP in the ready-to-use solution is 5 mM
Gel solution
1275 ml UreaTBE solution (Roth Karlsruhe)
225 ml Rotiphoresereg Gel 40 (38 acrylamide and 2 bisacrylamide)
95 microl APS solution (10 )
95 microl TEMED
Appendix laboratory paraphernalia IX
Loading buffer for agarose gels
EDTA pH 8 100 mM
Ficoll 400 20 (wv)
Bromophenol blue 025 (wv)
Xylencyanol 025 (wv)
Loading buffer for gel electrophoresis
2 ml bromophenol blue solution
20 ml formamide
TBE-buffer (1x)
100 ml TBE-buffer (10x)
900 ml H2O
TBE-buffer (10x)
108 g Tris [12114 M]
55 g boric acid [6183 M]
744 g EDTA [37224 M]
H2O ad 1000 ml
pH 80
UreaTBE solution (6 )
425 g urea [6006 M]
250 ml H2O
100 ml TBE-buffer (10x)
solubilise in a water bath at 65degC
H2O ad 850 ml
Appendix laboratory paraphernalia
X
Chemicals Agarose (Invitrogen Paisley UK)
Ammonium persulfate (APS) ge 98 (Sigma-Aldrich Chemie GmbH Steinheim)
Ampicillin (Serva Heidelberg)
Boric acid ge 998 pa (Carl Roth GmbH amp Co Karlsruhe)
Bromophenol blue (Merck KgaA Darmstadt)
dATP dCTP dGTP dTTP gt 98 (Carl Roth GmbH amp Co Karlsruhe)
Chloramphenicol (Serva Heidelberg)
DMSO ge 995 pa (Carl Roth GmbH amp Co Karlsruhe)
dNTP-Mix (Qbiogene GmbH Heidelberg)
EDTA ge 99 pa (Carl Roth GmbH amp Co Karlsruhe)
Ethidium bromide (Carl Roth GmbH amp Co Karlsruhe)
Ethyl alcohol (AppliChem Darmstadt)
Formamide ge 995 pa (Carl Roth GmbH amp Co Karlsruhe)
LB (Luria Bertani) agar (Scharlau Microbiology Barcelona Spain)
Paraffin (Merck KgaA Darmstadt)
RotiphoreseregGel40 (Carl Roth GmbH amp Co Karlsruhe)
SephadexTM G-50 Superfine (Amersham Biosciences Freiburg)
TEMED 99 pa (Carl Roth GmbH amp Co Karlsruhe)
Tris PUFFERANreg ge 999 pa (Carl Roth GmbH amp Co Karlsruhe)
Urea ge 995 pa (Carl Roth GmbH amp Co Karlsruhe)
Water was taken from the water purification system Milli-Qreg
X-Gal (AppliChem Darmstadt)
Enzymes Taq-DNA-Polymerase 5 Umicrol (Qbiogene GmbH Heidelberg)
Taq-DNA-Polymerase 5 U microl (Qiagen Hilden Germany)
Incubation Mix (10x) TPol with MgCl2 [15 mM] (Qbiogene GmbH Heidelberg)
The polymerase was always used in the presence of incubation Mix TPol 10x buffer
The encyme ECO RI (New England Biolabs Schwalbach Taunus) were used with
the adequate 10x encyme buffer
Appendix laboratory paraphernalia XI
Consumables
96 Well Multiply PCR plates skirted (Sarstedt Nuumlmbrecht)
Combitipsreg plus (Eppendorf AG Hamburg)
Pipette tips 01 ndash 10 microl 01 ndash 10 microl 5 ndash 200 microl (Carl Roth GmbH amp Co Karlsruhe)
Reaction tubes 15 and 20 ml (nerbe plus GmbH WinsenLuhe)
Reaction tubes 10 und 50 ml (Falcon) (Renner Darmstadt)
Thermo-fast 96 well plate skirted (ABgene Hamburg)
Software BLAST trace archive httpwwwncbinlmnihgov
httpwwwensemblorg
EBI toolbox httpwwwebiacukToolssequencehtml
MERLIN 0102 package httpwwwsphumicheducsgabecasisMerlin
Order of primers MWG Biotech-AG Ebersberg (httpsecom
mwgdnacomregisterindextcl)
biomersnet GmbH Ulm (orderbiomersnet)
PED50 Dr H Plendl et al (2005) Institute for Human Genetics
Kiel
Primer design httpfrodowimiteducgi-binprimer3primer3_ wwwcgi
Repeat Masker httpwwwrepeatmaskergenome washingtonedu
Sequencher 42 GeneCodes Ann Arbor MI USA
Spidey httpwwwncbinlmnihgovIEBResearch
OstellSpideyindexhtml
SUN Ultra Enterprise 450 Sun microsystems
XIII
List of publications Journal articles 1 MIESKES K WOumlHLKE A DROumlGEMUumlLLER C DISTL O 2006 Molecular
characterization of the canine myosin heavy polypeptide 9 non-muscle (MYH9)
gene on dog chromosome 10q232 Submitted for publication in Gene
2 MIESKES K DISTL O 2006 Evaluation of 17 single nucleotide
polymorphisms (SNPs) in six candidate genes for hereditary non-syndromic deafness
in Dalmatian dogs Submitted for publication in Journal of Heredity
3 MIESKES K DISTL O 2006 Evaluation of the TMC1 and TMIE genes as
candidates for hereditary non-syndromic deafness in Dalmatian dogs Submitted for
publication in Animal Genetics
XV
Acknowledgements
First of all I wish to thank Prof Dr Dr habil Ottmar Distl the supervisor of my
doctoral thesis for his academic guidance and support of this work
I am very thankful to Heike Klippert-Hasberg and Stefan Neander for technical
expertise and assistance
I am very grateful to Joumlrn Wrede for his support during the statistical analyses and his
help with computer problems
I wish to express my appreciation to the Gesellschaft zur Foumlrderung Kynologischer
Forschung (GKF) eV Germany for funding this work with a grant
I am appreciative to all Dalmatian breeders and owners for providing me blood
samples and the results of the BAER tests
My special thanks go to all colleagues and friends of the Institute for Animal Breeding
and Genetics of the University of Veterinary Medicine Hannover for their support
humour and the friendly atmosphere in the laboratory
Last but not least I wish to thank my family for their support during the work on this
thesis
Scientific supervisor Univ-Prof Dr Dr O Distl
Examiner Univ-Prof Dr Dr O Distl
Co-examiner Univ-Prof Dr H Y Naim
Oral examination 18 Mai 2006
This work was supported by a grant from the Gesellschaft zur Foumlrderung
Kynologischer Forschung (GKF) eV Bonn Germany
To my family
Parts of this work have been submitted for publication in the following journals
1 Gene
2 Journal of Heredity
3 Animal Genetics
Contents
1 Introduction 1
2 A comparative overview of the molecular genetics of non-syndromic deafness in dogs and humans 5
Abstract 7
The structure of the ear 7
Deafness in man 8
Deafness in dogs 9
The canine genome project 11
3 Linkage analysis of gene-associated microsatellite markers with congenital sensorineural deafness in Dalmatian dogs 23
Abstract 25
Introduction 25
Material and methods 26
Results and discussion 28
4 Evaluation of eight candidate genes for canine congenital sensorineural deafness in Dalmatian dogs 41
Abstract 43
Introduction 43
Material and methods 44
Results and discussion 46
5 Molecular characterization of the canine myosin heavy polypeptide 9 non-muscle (MYH9) gene on dog chromosome 10q232 63
Abstract 65
Introduction 65
Materials and Methods 67
Results and Discussion 69
6 Identification of a 5 Mb region on canine chromosome 10 harbouring a causative gene responsible for congenital sensorineural deafness in German Dalmatian dogs 81
Abstract 83
Introduction 83
Material and methods 84
Results 86
Discussion 87
7 Analysis of canine chromosome 1 and the Gap junction protein alpha 1 (GJA1) gene in Dalmatian dogs segregating for congenital sensorineural deafness 97
Abstract 99
Introduction 99
Material and methods 100
Results and discussion 102
8 Analysis of canine chromosome 31 and the claudin-14 (CLDN14) gene in Dalmatian dogs segregating for congenital sensorineural deafness 107
Abstract 109
Introduction 109
Material and methods 110
Results and discussion 112
9 General Discussion 117
The candidate gene approach 119
Linkage and association analysis 120
CFA1 122
CFA31 123
CFA10 123
10 Summary 125
11 Erweiterte Zusammenfassung 129
12 References 145
13 Appendix I 14 List of publications XIII
Abbreviations
List of abbreviations
A adenine
Acc No accession number
ACTG1 actin gamma 1
AEP akustisch evozierte Potentiale (acoustically evoked potentials)
APS ammonium persulphate
AT annealing temperature
BAC bacterial artificial chromosome
BAER brain stem auditory evoked response
BLAST Basic Local Alignment Search Tool
bp base pair
C cytosine
CCSD canine congenital sensorineural deafness
CDH23 cadherin related 23
cDNA copy desoxyribonucleic acid
CFA chromosome of Canis familiaris
CLDN14 claudin-14
cM centiMorgan
COCH coagulation factor C homolog cochlin
COL11A2 collagen type XI alpha 2
CRYM crystallin mu
CSD cochleosaccular degeneration
CX connexin
DFN x-linked deafness locus
DFNA autosomal dominant deafness locus
DFNA5 deafness autosomal dominant 5
DFNB autosomal recessive deafness locus
DIAPH1 diaphanous homolog 1 (Drosophila)
DMSO dimethyl sulfoxide
Abbreviations
DNA deoxyribonucleic acid
dNTPs deoxy nucleoside 5rsquotriphosphates (N is ACG or T)
EDN3 endothelin 3
EDNRB endothelin receptor type B
EDTA ethylenediamine tetraaceticacid
EMBL European Molecular Biology Laboratory
ESPN espin
EST expressed sequence tag
EYA4 eyes absent homolog 4 (Drosophila)
F forward
FISH fluorescence in situ hybridisation
G guanine
GJA1 gap junction protein alpha 1 43kD (connexin 43)
GJB2 gap junction protein beta 2 26k (connexin 26)
GJB3 gap junction protein beta 3 31kDa (connexin 31)
GJB6 gap junction protein beta 6 (connexin 30)
GKF Gesellschaft zur Foumlrderung Kynologischer Forschung (Society for the
Advancement of Cynological Research)
HET observed heterozygocity
HE expected heterozygosity value
HSA chromosome of Homo sapiens
IBD identical by descent
IRD infrared dye
KCNQ4 potassium voltage-gated channel KQT-like subfamily member 4
Kb kilobase
LD linkage disequilibrium
LINE long interspersed nuclear element
LOD logarithm of the odds
M molar
Mb megabase
Merlin multipoint engine for rapid likelihood inference
MITF microphthalmia-associated transcription factor
Abbreviations
MS microsatellite
MTRNR1 mitochondrially encoded 12S RNA
MTTS1 mitochondrially encoded tRNA serine 1 (UCN)
MYH9 myosin heavy polypeptide 9 non-muscle
MYH14 myosin heavy polypeptide 14
MYO1A myosin IA
MYO3A myosin IIIA
MYO6 myosin VI
MYO7A myosin VIIA
MYO15A myosin XVA
NCBI National Center for Biotechnology Information
NMMHC-A nonmuscle myosin heavy chain-A
ODDD oculodentodigital dysplasia
OMIM Online Mendelian Inheritance in Man
OTOA Otoancorin
OTOF otoferlin
P error probability
PAX3 paired box gene 3 (Waardenburg syndrome 1)
PCDH15 Protocadherin-15
PCR polymerase chain reaction
PIC polymorphism information content
POU3F4 POU domain class 3 transcription factor 4
POU4F3 POU domain class 4 transcription factor 3
PRES solute carrier family 26 member 5 (prestin)
QTL quantitative trait locus
R reverse
RACE rapid amplification of cDNA ends
RH radiation-hybrid
RLM RNA ligase-mediated
RNA ribonucleic acid
RT-PCR reverse transcription polymerase chain reaction
SAS Statistical Analysis System
Abbreviations
SH1 Src homology 1
SINE short interspersed nuclear element
SLC26A4 solute carrier family 26 member 4 (pendrin)
SLC26A5 solute carrier family 26 member 5 (prestin)
SNP single nucleotide polymorphism
SOX10 SRY (sex determining region Y)-box 10
STRC stereocilin
STS sequence-tagged site
T thymine
TBE tris-borate-ethylenediamine tetraacetic acid
TECTA tectorin alpha
TEMED NNNrsquoNrsquo-tetramethylenediamine
TFCP2L3 transcription factor CP2-like 3
TJ tight juncions
TMC1 transmembrane channel-like gene 1
TMIE transmembran inner ear gene
TMPRSS3 transmembrane protease serine 3
U unit
USH1C Usher syndrome 1C
UTR untranslated region
WFS1 Wolfram syndrome 1 (wolframin)
wgs whole genome shotgun
WHRN whirlin
WS Waardenburg syndrome
Chapter 1
Introduction
Introduction 3
Introduction
Canine congenital sensorineural deafness (CCSD) has often been reported in the
literature and occurs in more than 54 different breeds of dogs with the Dalmatian dog
showing the highest incidence The inheritance and segregation of a major gene in
CCSD has been demonstrated in different Dalmatian dog populations But although
several studies have demonstrated the mode of inheritance in Dalmatian dogs no
universally accepted mode of inheritance for the other dog breeds affected by CCSD
has yet been identified
Breeding with blue-eyed Dalmatian dogs and unilaterally or bilaterally deaf dams and
sires is forbidden by paragraph 11b of the German animal welfare laws and thus the
hearing status of all Dalmatian dogs has to be tested as a puppy at about six to eight
weeks old using the brain stem auditory evoked response (BAER) test As deaf dogs
are very difficult to raise and often become aggressive and snappish from fear most
puppies suffering from bilateral hearing loss are euthanized However it has been
shown in recent years that auditory testing does not seem to be an effective way of
clearly reducing the high incidence of deafness in this breed Thus prevention of
CCSD cannot be achieved alone by exclusion of affected animals from breeding
Consequently a molecular genetic approach toward unravelling the responsible
genes in carriers is urgently needed
Many genetic disorders in humans and domestic dogs (Canis familiaris) demonstrate
a high level of clinical and molecular similarity Therefore the mutated genes in
human hereditary deafness seemed to be appropriate candidates for canine
congenital sensorineural deafness
The objective of the present study is to localize the gene that is involved in the
development of CCSD in Dalmatian dogs In order to achieve this goal successively
32 canidate genes were evaluated by means of linkage analyses using microsatellite
markers and single nucleotide polymorphisms (SNPs) This candidate gene
approach using gene-associated markers for linkage studies in families segregating
for deafness turned out to be little effective Therefore the canine chromosomes
(CFA) 1 CFA10 and CFA31 were scanned entirely with microsatellite markers
Additionally single nucleotide polymorphisms (SNPs) were developed for fine
mapping the identified CCSD regions
Introduction 4
Overview of chapter contents Chapter 2 reviews the identified 39 mutated genes causing non-syndromic hereditary
hearing impairment in humans Parallels and differences in canine and human
deafness are shown including the clinical signs inheritance patterns and
histopathology We located the humane deafness genes in the canine genome and
discussed the advantages of comparative genomics and different molecular genetic
approaches
In Chapter 3 an existing set of 43 microsatellite markers for in total 24 candidate
genes were used for a non-parametric linkage analysis with congenital sensorineural
deafness (CCSD) in Dalmatian dog families segregating for deafness
In Chapter 4 newly developed SNP markers associated with in total eight candidate
genes were evaluated for CCSD in Dalmatian dogs
In Chapter 5 the molecular characterization of the canine myosin heavy polypeptide
9 non-muscle (MYH9) gene on dog chromosome 10q232 is described
Chapter 6 7 and 8 present linkage analyses performed in Dalmatian dog families
segregating for congenital sensorineural deafness using microsatellite markers on
canine chromosome (CFA) 1 CFA10 and CFA31 and the results of fine mapping
regions linked with the CCSD phenotype using newly developed SNPs
Chapter 9 provides a general discussion and conclusions referring to Chapters 1-8
Chapter 10 is a concise English summary of this thesis while Chapter 11 is an
expanded detailed German summary which takes into consideration the overall
research context
Chapter 2
A comparative overview of the molecular genetics of non-syndromic deafness
in dogs and humans
Non-syndromic deafness in dogs and humans 7
A comparative overview of the molecular genetics of non-syndromic deafness in dogs and humans
Abstract
In man as in different dog breeds deafness is an often diagnosed disorder with the
Dalmatian dog showing the highest incidence Deafness in Dalmatian dogs is clearly
heritable and the presence of a recessive major gene affecting the disorder was
shown in several Dalmatian dog populations
This Chapter provides an overview of the identified 39 mutated genes causing
human non-syndromic hereditary hearing impairment as well as of the five genes
responsible for Waardenburg syndrome in humans We point out their cytogenetic
and genomic localisations in man and dog and compare the genomic and mRNA
sequences of these genes between man and dog Moreover an overview is given on
deafness genes-associated markers identified in Dalmatian dogs and on candidate
genes characterized in dogs
The structure of the ear The inner ear consists of the semicircular canals the vestibule and the cochlea
whereas the vestibule and the semicircular canals are concerned with vestibular
function (balance) and the cocnlea is concerned with hearing Reissneracutes membrane
and the basilar membrane divide the cochlea longitudinally into three scalae the
scala vestibule the scala media and the scala tympani The process of transduction
occurs in the structures within scala media sitting on the basilar membrane and
comprising the organ of Corti Cutting the cochlea tube cross sectionally the scala
media is more or less triangular formed by Reissneracutes membrane basilar
membrane and a structure called the stria vascularis The fluid that fills scala
tympani and scala vestibule is called perilymph the fluid that fills scala media is
called endolymph The organ of Corti rests on the basilar membrane within scala
media The cochlea contains an array of highly specialized cells arranged in a highly
Non-syndromic deafness in dogs and humans 8
specialized manner Two types of cells in the organ of Corti are support cells and
hair cells The hair cells are the receptor cells that trancsduce sound
When a sound wave brings physical displacement of the membranes separating the
perilymph from the endolymph they cause the organ of Corti to move and the hair
cells on it are scraped along the bottom of the tectorial membrane The tectorial
membrane is firmly anchored to the bone Relative movement of the organ of Corti
and its hair cells with respect to the tectorial membrane is the source of the
deformation of the hair cells microvilli The hair cells are so constructed that any
deformation of their microvilli will cause a change in the overall membrane potential
of the cell This signal is detected by the fibers from the cells in the spiral ganglion
These fibers are neural elements and they carry their own depolarization wave into
the auditory region of the brain
Deafness in man There are various ways to categorise deafness The two main types of deafness are
classified based on which portions of the auditory system are affected conductive
hearing loss occurs when when sound is not conducted efficiently through the outer
andor middle part of the ear Much more common is the sensorineural hearing loss
Sensorineural hearing loss occurs when there is damage to the inner ear (cochlea)
or to the nerve pathways from the inner ear (retrocochlear) to the brain Most cases
of sensorineural hearing loss are due to cochlear defects (Petit et al 2001)
Hearing loss can be present at birth (congenital) or become evident later in life
(acquired) Congenital deafness similarly may or may not be genetic In fact more
than half of congenital hearing loss is inherited Alternatively congenital deafness
may be due to a condition or infection to which the mother was exposed during
pregnancy Furthermore congenital hereditary deafness may occur as part of a
multisystem disease (syndromic) or as a disorder restricted to the ear and vestibular
system (non-syndromic) As non-syndromic hereditary hearing impairment is almost
exclusively caused by cochlear defects affected patients suffer from sensorineural
hearing loss In Table 1 and 2 the genes underlying human hereditary non-
syndromic deafness as a result of cochlear defects in consequence of primary
defects in hair cells non-sensory cells and the tectorial membrane or unknown cell
type are shown Non-syndromic deafness is estimated to cause 60-70 of cases of
congenital hereditary deafness in man (Morton 1991) In 70-80 of all cases this
Non-syndromic deafness in dogs and humans 9
non-syndromic form of deafness shows an autosomal recessive inheritance followed
by an autosomal dominant inheritance in 10-20 of all cases and 1-2 of all cases
are X-linked A maternally inherited form may also occur (Van Camp and Smith
2003)
Non-syndromic forms of hereditary deafness are classified by their mode of
inheritance DFN DFNA and DFNB refer to deafness forms inherited on the X
chromosome-linked autosomal dominant and autosomal recessive modes of
transmission respectively
Human hereditary isolated hearing loss is genetically heterogeneous (Petit et al
2001) Up to 1 of the human genes are estimated to be necessary for hearing
(Friedmann and Griffith 2003) Today approximately 120 genes for human
hereditary deafness have been identified approximately 80 for syndromic and 39 for
non-syndromic hereditary deafness which is suspected to be one-third of the total
(Nance 2003) In Table 1 the identified 39 mutated genes causing non-syndromic
hereditary hearing impairment in humans are shown Out of the 39 genes 15 genes
cause autosomal recessive and 15 genes cause autosomal dominant forms six
genes are involved in both recessive and dominant forms one gene causes X-linked
and two a maternally inherited form (The Hereditary Hearing Loss Hompage
httpwebhostuaacbehhh)
Furthermore several hundred forms of syndromes with hearing loss have been
documented in humans (Van Camp and Smith 2003) One is the human
Waardenburg syndrome (WS) which manifests itself with sensorineural deafness
and pigmentation defects in the iris hair and skin The WS is classified into four
types depending on the presence or absence of additional symptoms which are
caused by mutations in the five genes EDN3 EDNRB MITF PAX3 and SOX10
respectively These genes are shown in Table 3 They are known to be expressed in
the neural crest (EDN3 EDNRB PAX3 SOX10 ) or directly in the melanocytes
(MITF) and are inter alia involved in migration differentiation or survival of
melanocytes respectively (Bondurand et al 2000)
Deafness in dogs Congenital sensorineural deafness (CSD) has been reported in a variety of mammal
species other than humans ranging from mice to dogs guinea pigs and mink
Canine congenital deafness has often been reported in the literature and occurs in
Non-syndromic deafness in dogs and humans 10
more than 54 different breeds of dogs according to Strain (1996 and 2004) The
breeds with the highest incidence include Dalmatian dogs Bull Terrier English
Cocker Spaniel English Setter Australian Cattle Dog Australian Shepherd West-
Highland-White-Terrier Dobermann and Dogo Argentino The incidence of canine
congenital deafness is highest in Dalmatian dogs of which 165 to 30 exhibit
unilateral or bilateral hearing loss (Famula et al 1996 Wood and Lakhani 1997
Muhle et al 2002 Juraschko et al 2003a Rak and Distl 2005) The inheritance
and segregation of a major gene in canine congenital sensorineural deafness
(CCSD) has been demonstrated in different Dalmatian dog populations (Famula et
al 2000 Muhle et al 2002 Juraschko et al 2003b) But although several studies
have demonstrated the mode of inheritance in Dalmatian dogs no universally
accepted mode of inheritance for the other dog breeds affected by CCSD has yet
been identified
Congenital sensorineural hearing impairment can be recognised in dogs at four to
eight weeks of age (Strain 1996) while histological studies of deaf Dalmatian dogs
have shown that the degeneration of the inner ear structures begins as early as one
day after birth and is histologically clearly evident by four weeks of age (Johnsson et
al 1973) In 70 of the cases with human hereditary deafness the histological
pattern is known as cochleo-saccular degeneration (CSD) (Lalwani et al 1997)
commonly known as Scheibe dysplasia with preservation of the pars superior of the
membranous labyrinth and an unremarkable bony labyrinth As in man also in many
affected dog breeds the histological pattern of congenital sensorineural deafness is
known as cochleo-saccular degeneration
Breeding with blue-eyed Dalmatian dogs and unilaterally or bilaterally deaf dams and
sires is forbidden by paragraph 11b of the German animal welfare laws and thus the
hearing status of all Dalmatian dogs has to be tested as a puppy at about six to eight
weeks old using the brainstem auditory evoked response (BAER) test that detects
electrical activity in the cochlea and auditory pathways in the brain Although the
BAER test is a reliable method for identifying unilaterally and bilaterally deaf dogs it
does not seem to be an effective way of clearly reducing the incidence of deafness in
affected breeds particularly in a recessive mode of inheritance so that hearing dogs
can still be genetic carriers Furthermore deaf dogs are very difficult to raise and
often become aggressive and snappish from fear consequently most puppies
Non-syndromic deafness in dogs and humans 11
suffering from bilateral hearing loss are euthanized Thus prevention of CCSD
cannot be achieved alone by exclusion of affected animals from breeding and
consequently a molecular genetic approach toward unravelling the responsible
genes in carriers is urgently needed
Over the past decade it has become increasingly clear how far structural and
functional homologies at the gene level extend across even distantly related species
Much is known about deafness-causing gene mutations in humans and mice
including the fact that the clinical and histopathological findings are often very similar
to those of deafness in Dalmatian dogs Thus despite the genetical heterogeneity of
human non-syndromic deafness the genes that are responsible for non-syndromic
congenital hereditary deafness in humans (Table 1) seemed to be appropriate
candidate genes for CCSD especially in Dalmatian dogs (Rak and Distl 2005) The
genes that are mutated in the human WS (Table 2) were selected as candidates
because the WS phenotype where the deafness is associated with pigmentation
defects seems to be similar to the phenotype of most affected dog breeds (Strain
and Tedford 1996) Both Juraschko et al (2003a) and Strain et al (1992) have
demonstrated that patched Dalmatians are less likely to be deaf than unpatched
animals and blue-eyed Dalmatians are more likely to be affected from hearing
impairment than brown-eyed animals
In an attempt to achieve the aim of molecular genetic diagnosis of CCSD carriers
Rak et al (2003) and co-workers (Droumlgemuumlller et al 2002 Kuiper et al 2002 Rak
et al 2002a 2002b 2003) already mapped 24 potential candidate genes for
sensorineural deafness in dogs by fluorescence in situ hybridization and a radiation
hybrid panel to 16 different canine chromosomes
The canine genome project In December 2005 an international research team led by scientists at the Broad
Institute of MIT and Harvard achieved the completion of a high-quality genome
sequence of the domestic dog together with a catalog of 25 million specific genetic
differences across several dog breeds (Lindblad-Toh et al 2005) The authors found
that humans share more of their ancestral DNA with dogs than with mice confirming
the utility of dog genetics for understanding human disease Furthermore the
physiology disease presentation and clinical response of dogs often mimic human
Non-syndromic deafness in dogs and humans 12
diseases closely As indicated above hearing impairment seemed to be no
exception
In the last years most projects have exploited canine traits for which either direct
candidate genes could be proposed and evaluated or for which large informative
pedigrees were available to enable linkage mapping to identify candidate regions A
major component of such research efforts comprised the cloning sequencing and
mapping of individual canine homologs of genes either proposed as candidate
genes or expected to be located in candidate regions This was necessary to
identify new informative polymorphisms (eg single nucleotide polymorphisms
(SNPs) microsatellites) for high resolution mapping of candidate regions and to
examine each exon and exonintron boundary for positional candidates Availability
of the second version of the dog genome assembly (build 21) of the NCBI database
shortcut this effort and increase the investigators efficency
The current RH map with 3200 markers provides a good estimate of the order and
physical spacing (ie in base pairs) of markers along canine chromosomes (Guyon
et al 2003) and was recently complemented by the construction of a 4249-marker
integrated canine genome RH map that consists of 900 genes 1589 microsatellites
and 1760 BAC end markers (Breen et al 2004) all included and available in the
NCBI database The second version 1 of the NCBIs genome annotation consists of
large contigs covering all canine chromosomes given with their located markers and
genes The great majority of genes are derived by automated computational analysis
using the gene prediction method GNOMON
With this help either additional candidate genes for canine CSD can be found directly
by its gene symbol in the 21 of the NCBIs genome annotation or if a candidate gene
is yet not annotated a BLAST (Basic Local Alignment Search Tool) search versus
the canine whole genome shotgun (wgs) sequence resource can be used to obtain
the sequence of the canine genomic contigs containing the human homologous
gene The localisation of all 39 known human non-syndromic hereditary deafness
genes in the canine genome with the corresponding accession numbers of the contig
and if available the accession number of the genomic sequence and mRNA of the
canine gene are shown in Tables 4 and 5 Furthermore the identity of canine and
human or mouse mRNA is shown in Table 5 The average identity of canine and
human mRNA is with 088 percent higher than the average identity of canine and
mouse mRNA with 084 percent Canine sequences that correspond to the human
Non-syndromic deafness in dogs and humans 13
candidate gene can now be used to find microsatellite or SNP markers associated to
the respective canine gene These markers can be used for linkage and haplotype
studies in dog families segregating for deafness
Table 7 shows the microsatellite and SNP markers developed for in total 32
candidate genes for CCSD
The candidate genes for which a set of in total 43 microsatellite marker were
designed by Rak (2003) included the following 24 genes CDH23 CLDN14 COCH
COL11A2 DFNA5 DIAPH1 EDN3 EDNRB EYA4 GJA1 GJB2 GJB6 MITF
MYH9 MYO6 MYO7A MYO15A OTOF PAX3 POU4F3 SLC26A4 SOX10
TECTA and TMPRSS3 This existing set of 43 microsatellite markers for 24
candidate genes were used for linkage and haplotype studies in Dalmatian dog
families segregating for deafness (Chapter 3) These 24 genes are known to be
involved either in human non-syndromic deafness or in the human Waardenburg
syndrome For another eight candidate genes including TMC1 TMIE USH1C
MYH14 MYO3A PRES WHRN and ESPN SNP markers were newly developed
(Chapter 4) and subsequently used for linkage and association analyses in
Dalmatian dog families segregating for deafness These genes are also involved in
human non-syndromic deafness
Non-syndromic deafness in dogs and humans 14
Tabele 1 Genes responsible for non-syndromic congenital hereditary deafness in
humans
Inheritance Gene Gene product Type of molecule Locus namea
ACTG1 γ-Actin cytoskeletal protein DFNA20 DFNA26
COCH Cochlin extracellular matrix component DFNA9
COL11A2 Collagen XI (α2-chain) extracellular matrix component DFNA13
CRYM micro-Cristallin thyroid hormone-binding protein DFNAb DFNA5 Unidentified Unidentified DFNA5 DIAPH1 Diaphanous-1 cytoskeleton regulatory protein DFNA1 EYA4 EYA4 transcriptional coactivator DFNA10 GJB3 Connexin-31 gap junction protein DFNA2 KCNQ4 KCNQ4 K+ channel subunit DFNA2 MYH14 Myosin IIC motor protein DFNA4 MYH9 Myosin IIA motor protein DFNA17 MYO1A Myosin IA motor protein DFNA48 POU4F3 POU4F3 transcription factor DFNA15 TFCP2L3 TFCP2L3 transcription factor DFNA28
Autosomal dominant
WFS1 Wolframin endoplasmic-reticulum membrane protein DFNA6 DFNA14
CDH23 Cadherin-23 cell-adhesion protein DFNB12 CLDN14 Claudin-14 tight-junction protein DFNB29 ESPN Espin actin-bundling protein DFNB36 DFNAb MYO15 Myosin XV motor protein DFNB3 MYO3A Myosin IIIA motor protein DFNB30 OTOA Otoancorin cell-surface protein DFNB22 OTOF Otoferlin putative vesicle traffic protein DFNB9 PCDH15 Protocadherin-15 cell-adhesion protein DFNB23 SLC26A4 Pendrin IminusndashClminus transporter DFNB4 SLC26A5 Prestin anion transporter DFNB61 STRC Stereocilin DFNB16
TMIE TMIE transmembrane domain- containing protein DFNB6
TMPRSS3 TMPRSS3 transmembrane serine protease DFNB8 DFNB10 USH1C Harmonin PDZ domain-containing protein DFNB18
Autosomal recessive
WHRN Whirlin PDZ domain-containing protein DFNB31 GJB2 Connexin-26 gap junction protein DFNB1 DFNA3 GJB6 Connexin-30 gap junction protein DFNB1 DFNA3 MYO6 Myosin VI motor protein DFNA22DFNB37MYO7A Myosin VIIA motor protein DFNB2DFNA11
TECTA α-Tectorin extracellular matrix component DFNA8 DFNA12DFNB21
Autosomal dominant and autosomal recessive
TMC1 TMC1 transmembrane channel-like protein
DFNB7 DFNB11DFNA36
X-linked POU3F4 POU3F4 transcription factor DFN3
MTRNR1 Mitochondrial 12S rRNA not defined
nomenclature Mitochondrial
MTTS1 Mitochondrial 12S rRNA not defined
nomenclature a Autosomal recessive loci are designated DFNB autosomal dominant loci DFNA
Non-syndromic deafness in dogs and humans 15
Table 2 Genes underlying hereditary non-syndromic deafness as a result of primary
defects in hair cells non-sensory cells and the tectorial membrane or unknown cell
type
Primary defect
Gene
Hair cells MYO7A MYO15 MYO6 MYO3A MYO1A ACTG1 USH1C
WHRN CDH23 PCDH15 TMIE STRC SLC26A5 ESPN
KCNQ4 TMC1 OTOF POU4F3
Non-sensory cells
GJB2 GJB6 GJB3 SLC26A4 CRYM OTOA CLDN14
COCH TMPRSS3 MYH9 MYH14 EYA4 POU3F4
Tectorial membrane
COL11A2 TECTA
Unknown
DIAPH1 DFNA5 WFS1 TFCP2L3 MTRNR1 MTTS1
Table 3 Genes involved in the human Waardenburg syndrome
Inheritance Gene Gene product Type of molecule Type
EDN3 endothelin 3 vasoconstricted peptide WS type IV4
EDNRBendothelin
receptor type B receptor protein WS type IV4
MITF
microphthalmia-
associated
transcription
factor
transcription factor WS type II2
PAX3 paired box 3 DNA-binding protein WS type I1and III 2
SOX10 SRY-box 10 transcription factor WS type IV4 1Type I Dystopia canthorum 2Type II No dystopia canthorum 3Klein-Waardenburg syndrome (type III) Type I and upper limb abnormalities 4Waardenburg-Shah syndrome (type IV) Type II and Hirschsprung disease
(autosomal recessive inheritance)
Non-syndromic deafness in dogs and humans 16
Leng
th o
f co
ntig
(bp)
7799
0652
3821
0901
7077
2986
7251
5492
5989
7527
4533
7677
6093
8239
9498
326
6961
0260
1859
5814
6421
1953
Scor
e of
co
ntig
614
938
792
567
300
715
323
535
1208
1634
923
E-va
lue
of
cont
ig
4 E
-172
0 0
2 E
-158
1 e-
78
0
2 e-
85
7 E
-149
0 0 0
Acc
No
of
WG
S co
ntig
unkn
own
NW
_876
311
NW
_876
295
NW
_876
327
NW
_876
254
NW
_876
321
NW
_876
292
NW
_876
258
NW
_876
315
NW
_876
269
NW
_876
278
NW
_876
259
Mb
from
to
unkn
own
254
12
578
337
93
379
132
11
323
563
56
5
272
12
723
393
33
943
411
64
123
632
86
331
292
82
955
209
32
094
101
81
019
Gen
e lo
catio
n on
C
FA
unkn
own
4 31
8 12
6 2 14
5 1 25
15
Can
ine
gene
al
iase
s
none
none
LOC
4877
51
LOC
4906
40
LOC
4817
34
LOC
4798
18
none
LOC
6112
23
LOC
4896
31
EYA4
GJB
2
LOC
4824
86
Acc
No
hu
man
m
RN
A
NM
_001
614
NM
_022
124
NM
_144
492
NM
_004
086
NM
_080
680
NM
_001
888
NM
_005
219
NM
_004
403
NM
_031
475
NM
_172
105
NM
_004
004
NM
_024
009
Gen
e lo
catio
n on
H
SA
17
10
21
14
6 16
5 7 1 6 13
1
Tabl
e 4
Loca
lisat
ion
of h
uman
non
-syn
drom
ic h
ered
itary
dea
fnes
s ge
nes
in th
e ca
nine
gen
ome
the
cani
ne
gene
loc
alis
atio
n in
meg
abas
es (
Mb)
th
e ac
cess
ion
num
ber
(Acc
No
) of
the
who
le g
enom
e sh
otgu
n (W
GS
) con
tig c
onta
inin
g th
e hu
man
hom
olog
ous
gene
and
the
corr
espo
ndin
g E
-val
ue s
core
and
leng
th o
f th
e co
ntig
Hum
an
deaf
ness
ge
ne
AC
TG1
CD
H23
CLD
N14
CO
CH
CO
L11A
2
CR
YM
DIA
PH1
DFN
A5
ESPN
EYA
4
GJB
2
GJB
3
der
ived
from
the
NC
BIs
can
ine
geno
me
anno
tatio
n ve
rsio
n 2
1
Non-syndromic deafness in dogs and humans 17
Leng
th o
f co
ntig
6421
1953
5300
4996
5294
2087
2607
3285
1654
5469
1249
9463
7251
5492
5102
4781
5989
7527
5159
1990
1284
7264
7521
5785
Scor
e of
co
ntig
525
521
973
2256
348
567
1236
404
337
383
604
1715
E-va
lue
of
cont
ig
3 e-
146
1 e-
144
0 0
9 e-
93
2 e-
158
0
3 e-
109
3 e-
89
7 e-
103
2 e-
169
0
Acc
No
of
WG
S co
ntig
unkn
own
NW
_876
259
NW
_876
270
NW
_876
251
NW
_876
313
NW
_876
250
NW
_876
290
NW
_876
254
NW
_876
273
NW
_876
321
NW
_876
263
NW
_876
283
NW
_879
563
Mb
from
to
unkn
own
521
52
3
109
24
109
35
311
33
119
443
64
441
415
41
7
103
41
056
404
14
050
245
42
460
261
32
619
235
02
359
371
43
769
674
86
748
Gen
e lo
catio
n on
C
FA
25
15
1 10
5 10
2 12
21
6 17
26
X
Can
ine
gene
al
iase
s
none
LOC
4824
51
none
LOC
4812
80
LOC
4795
22
LOC
4744
10
LOC
4871
06
LOC
4818
84
LOC
4851
74
LOC
6086
55
LOC
6079
61
none
LOC
4919
88
Acc
No
hu
man
m
RN
A
NM
_006
783
NM
_004
700
NM
_024
729
NM
_002
473
NM
_016
239
NM
_005
379
NM
_017
433
XM
_376
516
NM
_000
260
NM
_144
672
NM
_194
248
NM
_033
056
NM
_000
307
Gen
e lo
catio
n on
H
SA
13
1 19
22
17
12
10
6 11
16
2 10
X
Tabl
e 4
(con
tinue
d)
Hum
an
deaf
ness
ge
ne
GJB
6
KC
NQ
4
MYH
14
MYH
9
MYO
15
MYO
1A
MYO
3A
MYO
6
MYO
7A
OTO
A
OTO
F
PCD
H15
POU
3F4
der
ived
from
the
NC
BIs
can
ine
geno
me
anno
tatio
n ve
rsio
n 2
1
Non-syndromic deafness in dogs and humans 18
Leng
th o
f co
ntig
4533
7677
2532
0482
2532
0482
4020
6070
3002
9677
2968
9717
5300
4996
3309
7591
3821
0901
5102
4781
1104
8438
6535
5756
Scor
e of
co
ntig
1404
283
283
1683
883
529
354
216
198
354
2238
689
E-va
lue
of
cont
ig
0
6 e-
73
2 E
-73
0 0
5 e-
147
2 e-
94
4 e-
53
9 e-
48
2 E
-94
0 0
Acc
No
of
WG
S co
ntig
NW
_876
292
NW
_876
265
NW
_876
265
NW
_876
294
NW
_876
312
NW
_876
255
NW
_876
270
NW
_876
272
NW
_876
295
NW
_876
273
NW
_876
256
NW
_876
253
Mb
from
to
436
14
361
158
61
592
197
91
982
134
31
345
158
81
595
622
63
7
880
88
822
450
54
505
390
33
905
432
44
328
414
94
151
716
47
172
Gen
e lo
catio
n on
C
FA
2 18
18
30
5 13
1 20
31
21
13
11
Can
ine
gene
al
iase
s
LOC
4872
00
LOC
4832
63
LOC
4832
74
LOC
4782
78
LOC
4893
57
LOC
4819
85
LOC
4841
68
LOC
6093
50
LO
C61
0987
LOC
6108
50
LOC
4821
13
LOC
6125
88
Acc
No
hu
man
m
RN
A
NM
_002
700
NM
_000
441
NM
_206
883
NM
_153
700
NM
_005
422
NM
_024
915
NM
_138
691
NM
_147
196
NM
_024
022
NM
_153
676
NM
_006
005
NM
_015
404
Gen
e lo
catio
n on
H
SA
5 7 7 15
11
8 9 3 21
11
4 9
Tabl
e 4
(con
tinue
d)
Hum
an
deaf
ness
ge
ne
POU
4F3
SLC
26A
4
SLC
26A
5
STR
C
TEC
TA
TFC
P2L3
TMC
1
TMIE
TMPR
SS3
USH
1C
WFS
1
WH
RN
der
ived
from
the
NC
BIs
can
ine
geno
me
anno
tatio
n ve
rsio
n 2
1
Non-syndromic deafness in dogs and humans 19
Leng
th o
f co
ntig
(bp)
4776
3139
5561
1003
2518
2130
3091
5115
5294
2087
Scor
e of
co
ntig
262
721
2927
967
1179
E-va
lue
of
cont
ig
8e-6
7
0 0 0 0
Acc
No
of
WG
S co
ntig
NW
_876
277
NW
_876
274
NW
_876
271
NW
_876
304
NW
_876
251
Mb
from
to
470
14
703
343
63
438
248
52
488
313
43
144
297
52
976
Gen
e lo
catio
n on
C
FA
24
22
20
37
10
Can
ine
gene
al
iase
s
ED
N3
ED
NR
B
MIT
F
PAX3
LOC
4812
58
Acc
No
hu
man
m
RN
A
NM
_207
032
NM
_000
115
NM
_198
159
NM
_181
457
NM
_006
941
Gen
e lo
catio
n on
H
SA
20
13
3 2 22
Tabl
e 5
Loca
lisat
ion
of g
enes
invo
lved
in th
e hu
man
Waa
rden
burg
syn
drom
e (W
S)
in th
e ca
nine
gen
ome
th
e ca
nine
gen
e lo
calis
atio
n in
meg
abas
es (
Mb)
th
e ac
cess
ion
num
ber
(Acc
No
) of
the
who
le g
enom
e sh
otgu
n (W
GS
) con
tig c
onta
inin
g th
e hu
man
hom
olog
ous
gene
and
the
corr
espo
ndin
g E
-val
ue s
core
and
le
ngth
of t
he c
ontig
Hum
an
deaf
ness
ge
ne
EDN
3
EDN
RB
MIT
F
PAX3
SOX1
0
der
ived
from
the
NC
BIs
can
ine
geno
me
anno
tatio
n ve
rsio
n 2
1
Non-syndromic deafness in dogs and humans 20
Table 6 Canine candidate genes for CCSD with their accession number (AccNo) of
the genomic sequence and mRNA and if available the percent identity of canine and
human or mouse mRNA Canine candidate gene
Acc No canine genomic sequence
Acc No canine mRNA (predicted)
Canine mRNA (bp)
Identity () of canine and human mRNA
Identity () of canine and mouse mRNA
ACTG1 none1 none unknown unknown unknown CDH23 none1 none unknown 8800 8690 CLDN14 NC_006613 XM_544876 714 8880 8460 COCH NC_006590 XM_547762 3050 8770 8170
COL11A2 NC_006594 XM_857285 5202 8970 8740 CRYM NC_006588 XM_845361 1279 8720 7890
DIAPH1 none1 none unknown 8630 unknown DFNA5 NC_006596 XM_848863 1512 8320 7680 ESPN NC_006587 XM_546751 2565 8930 8270 EYA4 NC_006583 XM_541108 3969 9240 8120 GJB2 NC_006607 XM_543177 2208 7860 6720 GJB3 NC_006597 XM_539603 1305 8550 8090 GJB6 none none unknown unknown unknown
KCNQ4 NC_006597 XM_539568 1836 9350 8760 MYH14 none1 none unknown 8770 8220 MYH9 NC_006592 XM_538401 6201 9130 8940
MYO15 NC_006587 XM_536660 10128 8500 7960 MYO1A NC_006592 XM_531642 3388 8600 8350 MYO3A NC_006584 XM_544234 5589 8510 8910 MYO6 NC_006594 XM_862465 3983 9250 8610
MYO7A NC_006603 XM_542292 6519 9190 8880 OTOA NC_006588 XM_845746 3558 8740 7990 OTOF NC_006599 XM_844665 5994 9020 8780
PCDH15 none1 none unknown 8210 7390 POU3F4 NC_006621 XM_549108 1086 9190 9060 POU4F3 NC_006584 XM_544328 1017 9380 9160 SLC26A4 NC_006600 XM_540382 2382 7970 8240 SLC26A5 NC_006600 XM_540393 2235 9250 8740
STRC NC_006612 XM_535452 5301 8980 8520 TECTA NC_006587 XM_546475 7136 9110 8510
TFCP2L3 NC_006595 XM_539106 2127 9030 8760 TMC1 NC_006583 XM_541284 2580 8860 8740 TMIE NC_006602 XM_846596 396 9110 8910
TMPRSS3 NC_006613 XM_848589 1542 8500 8340 USH1C NC_006603 XM_860072 1730 9010 8670 WFS1 NC_006595 XM_539234 2667 8510 8420 WHRN NC_006593 XM_850321 2817 8440 8110 EDN3 NC_006606 NM_001002942 1976 747 716
EDNRB NC_006604 NM_001010943 1329 889 827 MITF NC_006602 XM_850501 1590 934 862 PAX3 NC_006619 XM_545664 1474 869 861
SOX10 NC_006592 XM_538379 1987 926 900 derived from the NCBIs canine genome annotation version 21 1 the definite region in the whole genome sequence (WGS) contig was used to get the percent identity of mRNAs
Non-syndromic deafness in dogs and humans 21
Table 7 Microsatellite markers and single nucleotide polymorphisms (SNPs)
of canine candidate genes for canine congenital sensorineural deafness in
Dalmatian dogs
Canine candidate gene
Number of gene-associated
microsatellites
Number of gene-associated
SNPs
CDH23 2 0 CLDN14 3 8 COCH 2 0
COL11A2 2 0 DIAPH1 2 0 DFNA5 2 0 ESPN 0 5 EYA4 2 0 GJB2 3 0 GJB3 1 0 GJB6 1 0
MYH14 0 2 MYH9 2 22
MYO15 2 0 MYO3A 0 3 MYO6 1 0
MYO7A 3 0 OTOF 1 0 PAX3 1 0
POU4F3 1 0 SLC26A4 1 0 SLC26A5 0 2 TECTA 2 0 TMC1 1 1 TMIE 1 3
TMPRSS3 2 0 USH1C 0 2 WHRN 0 3
Chapter 3
Linkage analysis of gene-associated microsatellite markers with
congenital sensorineural deafness in Dalmatian dogs
Linkage analysis of gene-associated microsatellites 25
Linkage analysis of gene-associated microsatellite markers with congenital sensorineural deafness in Dalmatian dogs
Abstract Deafness is a disorder often diagnosed in different dog breeds In this Chapter an
existing set of 43 microsatellite markers associated with in total 24 candidate genes
for canine congenital sensorineural deafness (CCSD) were used for linkage and
haplotype analyses in a large Dalmatian dog population with frequent occurrence of
CCSD We found significant linkage for the genes GJA1 MYH9 and CLDN14 As
linkage was found for different candidate genes in different families the results of
these test statistics indicate that the inheritance of non-syndromic deafness in
Dalmatian dogs is heterogenic in origin
Introduction
Canine congenital sensorineural deafness (CCSD) has been reported to occur in
more than 54 different breeds of dogs (Strain 1996) As in man also in dog breeds
the most commonly observed histological pattern of degenerative inner ear changes
is known as the cochleo-saccular or Scheibe type of end organ degeneration
The incidence of this congenital anomaly is highest in Dalmatian dogs of which 165-
30 exhibit unilateral or bilateral hearing loss (Famula et al 1996 Wood and
Lakhani 1997 Muhle et al 2002 Juraschko et al 2003a Rak and Distl 2005) The
inheritance and segregation of a major gene in CCSD has been demonstrated in
different Dalmatian dog populations (Famula et al 2000 Muhle et al 2002 Juraschko et al 2003b) Moreover deafness in Dalmatian dogs seems to be
pigment-associated (Greibrokk 1994 Holliday et al 1992 Juraschko et al 2003a
2003b Mair 1976 Strain et al 1992 Strain 1996)
No gene mutation has yet been identified that is responsible for CCSD in Dalmatian
dogs or in one of the various other dog breeds that suffer from inherited hearing
impairment Since mutations in various genes have already been found to be the
cause of sensorineural hearing impairment in humans or mice 24 of these genes
Linkage analysis of gene-associated microsatellites
26
were considered as candidates for CCSD in Dalmatian dogs (Rak et al 2005)
Details of the 24 candidate genes are given in Table 1
Rak et al (2003) and co-workers (Droumlgemuumlller et al 2002 Kuiper et al 2002 Rak et
al 2002a 2002b 2003) mapped this 24 potential candidate genes for sensorineural
deafness in dogs by fluorescence in situ hybridization and a radiation hybrid panel
Subsequently Rak (2003) developed altogether 43 new highly polymorphic DNA
markers for the 24 candidate genes including CDH23 CLDN14 COCH COL11A2
DFNA5 DIAPH1 EDN3 EDNRB EYA4 GJA1 GJB2 GJB6 MITF MYH9 MYO6
MYO7A MYO15A OTOF PAX3 POU4F3 SLC26A4 SOX10 TECTA and
TMPRSS3 (Table 2)
Among the 24 candidate genes seven genes cause autosomal dominant non-
syndromic forms of deafness seven cause autosomal recessive forms and five
genes cause both recessive and dominant forms of non-syndromic deafness in
different human families segregating for either forms
The functions of these 19 deafness-causing genes are diverse and include gap
junctions and tight junctions (GJA1 GJB2 GJB6 CLDN14) ion channels (SLC26A4)
and ion channel activators (TMPRSS3) Included are also unconventional myosins
(MYO6 MYO7A MYH9 MYO15A) transcription factors (POU4F3 EYA4) as well as
extracellular matrix components (COCH COL11A2 TECTA) a cytoskeleton
regulatory protein (DIAPH1) a cell-adhesion protein (CDH13) and genes with
unknown or only suspected functions (DFNA5 OTOF) The 24 candidates also
include five genes which are mutated in the human Waardenburg syndrome (WS)
The WS is classified into four types depending on the presence or absence of
additional symptoms which are caused by mutations in the five genes EDN3
EDNRB MITF PAX3 and SOX10 respectively The objective of the current study
was to use this set of markers developed by Rak (2003) for a non-parametric linkage
analysis with CCSD in a German and French Dalmatian dog population
Material and methods Pedigree material
For the linkage analysis we used DNA from altogether 215 animals belonging to a
total of 24 Dalmatian dog families The families included 22 full-sib families and one
large paternal half-sib family of German Dalmatian dogs as well as 46 animals of a
Linkage analysis of gene-associated microsatellites 27
large paternal half-sib family of French Dalmatian dogs All families were segregating
for CCSD The genotyped families included all affected dogs (unilaterally and
bilaterally deaf) their parents if available and one to four unaffected animals At least
two of the full sibs of each family were unilaterally deaf
In total these 24 families included 402 individuals with an average family size of 168
ranging from 5 to 116 animals and covering two to four generations The hearing
status of 344 dogs was examined by veterinarians using the BAER (brain stem
auditory evoked response) test and the other animals included in the pedigree being
not BAER tested were used to construct relationships among CSD affected dogs
The prevalence of CSD in this pedigree was 285
Microsatellite marker analysis An existing marker set consisting of 43 microsatellite markers (Table 2) was used for
linkage analysis This set included 36 markers developed by Rak (2003) and 7
markers of the RH map available at httpwww-recomgenuniv-rennes1frdoggyhtml
For most of the 24 candidate genes two markers were available for two of the
candidates three markers were available but for seven candidate genes the set
contains only one marker The marker set is composed of 33 perfect repeats two
imperfect six compound-perfect and two compound-imperfect repeats
The majority (674) of the 43 markers in the set was represented by dinucleotide
repeats 209 were tetranucleotide repeats 47 trinucleotide repeats and 23
pentanucleotide repeats In addition one marker (23) was a compound di-
tetranucleotide and another one (23) was a compound tetra-pentanucleotide
repeat The average number of alleles was 35 with a minimum of 2 and a maximum
of 8 different alleles per marker
PCR reactions for microsatellites were carried out in 12 microl reaction mixtures
containing 2 microl genomic DNA 12 microl 10x PCR buffer (Qbiogene Heidelberg
Germany) 024 microl dimethyl sulfoxide (DMSO) 02 microl dNTPs (100 microM) 01 microl Taq
Polymerase (5Umicrol) (Qbiogene) 06 microl (10 microM) 5rsquo-IRD700 or IRD800 labelled forward
primer 06 microl (10 microM) unlabelled reverse primer The PCR reactions were carried out
in MJ Research thermocyclers with the following program 4 min at 94 degC followed
by 32 cycles of 30 sec at 94degC 30 sec at maximum annealing temperature and a
final extension of 45 sec at 72degC PCR-products were diluted with formamide loading
buffer in ratios from 110 to 140 and then size-fractionated by gel electrophoresis on
Linkage analysis of gene-associated microsatellites
28
automated LI-COR 42004300 sequencers (LI-COR inc Lincoln NE USA) using
denaturing 4 and 6 polyacrylamide gels (RotiphoreseregGel 40 Roth Karlsruhe
Germany)
To localize the 24 candidate genes and their associated microsatellites exactly the
canine candidate gene sequences were derived from sequences deposited in the
current dog genome assembly (Boxer genome assembly 21) of the NCBI GenBank
by BLAST (Basic Local Alignment Search Tool) search
(httpwwwncbinlmnihgovBLAST) using the human reference mRNA sequence
(Table 3)
Linkage analysis
Multipoint linkage and haplotype analyses were performed using the MERLIN
software version 0102 (multipoint engine for rapid likelihood inference Center for
Statistical Genetics University of Michigan MI USA Abecasis et al 2002) The test
statistics Z-mean and Lod score were used to test for the proportion of alleles shared
by affected individuals identical by descent (IBD) for the considered marker loci
Linkage analyses were performed regarding the marker set consisting of 43 gene-
associated microsatellite markers Linkage analysis was at first carried out for all 24
families conjoined After this the families were scanned separately
The data of the genotypes was additionally analyzed using SASGenetics (Statistical
Analysis System Version 913 SAS Institute Inc Cary NC USA 2005) to specify
the number of alleles of each marker the allele frequency the observed (HET) and
expected (HE) heterozygosity and the polymorphism information content (PIC)
(Table 4 and 5)
Results and discussion
Test statistics for all families conjoined are given in Table 6 Significant CCSD loci
were located on different chromosomes The loci were located on canine
chromosome (CFA) 1 10 12 20 and 31 Linkage analyses per family indicated even
higher test statistics for subgroups of families (Table 7) Scanning only families with
Zmeans gt1 test statistics for linkage increased for the genes GJA1 on CFA1 MYH9
on CFA10 and CLDN14 on CFA31 with congenital sensorineural deafness in different
Dalmatian dog families (Table 7) Therefore it is probable that these genes or genes
Linkage analysis of gene-associated microsatellites 29
in their flanking regions are involved in the development of the disease in the
respective familes The results of this test statistics indicate that the inheritance of
non-syndromic hearing loss in Dalmatian dogs is probably as heterogenic in origin as
it is in humans Genetic heterogeneity means that different mutations cause the same
phenotype or disease the different mutations can either be found at the same locus
(allelic heterogeneity) or even at different loci (non-allelic heterogeneity)
The extreme heterogeneity of human deafness often hampered genetic studies
because many different genetic forms of hearing loss give rise to similar clinical
phenotypes and conversely mutations in the same gene can result in a variety of
clinical phenotypes In man genes that transport ions across membranes to maintain
appropriate solute concentration and pH as well as regulatory genes mainly
transcription factors and genes that play a part in structural integrity are essential for
the hearing process
However this study was a first step in identifying genes responsible for CCSD in
Dalmatian dogs The genes GJA1 MYH9 and CLDN14 and their flanking regions will
be further analyzed with a combined approach using microsatellites and single
nucleotide polymorphisms (SNPs) (Chapter 6-9) As linkage was found for different
candidate genes in different families subsequently only the families indicating
linkage will be chosen for further molecular analyses of the respective gene
To confirm the result of this study the density of the intragenic markers has to be
increased The current RH map with 3200 markers provides a good estimation of the
order and physical spacing (ie in base pairs) of markers along canine
chromosomes (Guyon et al 2003) and was recently complemented by the
construction of a 4249-marker integrated canine genome RH map which consists of
900 genes 1589 microsatellites and 1760 BAC end markers (Breen et al 2004) all
included and available in the NCBI database (httpwebncbinlmnihgov)
Thus microsatellites derived from the NCBI database could be used to confirm the
linkage Alternatively BLAST searches versus the canine whole genome shotgun
(wgs) sequence resource were perfomed to localize the genes exactly and to obtain
the sequence of the canine genomic contigs containing the human homologous gene
The results of the BLAST searches of the 24 candidate genes against the Boxer
genome assembly 21 are shown in Table 3 The genomic sequence of the
respective candidate gene can now be used to search for intragenic SNPs as these
polymorphisms are the most abundant and useful markers for fine mapping
Linkage analysis of gene-associated microsatellites
30
Development of SNPs requires sequencing of DNA for the respective genomic
regions of the parents with the aim to identify heterozygous base pair exchanges
After a heterozygous base pair is found the whole family can be genotyped for this
informative SNP marker These polymorphisms can than be used for linkage
analyses as well as association studies
Fine mapping using SNP markers for all genes indicating linkage with CCSD
identified by this study should enable us to detect mutations responsible for CCSD in
parts of the Dalmatian dog population
Linkage analysis of gene-associated microsatellites 31
Table 1 Details of the 24 selected human candidate genes
Symbol Gene name Locus name1 Most important reference
CDH23 cadherin related 23 DFNB12 Bork et al 2001
CLDN14 claudin 14 DFNB29 Wilcox et al 2001
COCH coagulation factor C homolog cochlin DFNA9 Robertson et al 1998
COL11A2 collagen type XI alpha 2 DFNA13 McGuirt et al 1999
DFNA5 deafness autosomal dominant 5 DFNA5 Van Laer et al 1998
DIAPH1 diaphanous homolog 1 (Drosophila) DFNA1 Lynch et al 1997
EDN3 endothelin 3 WS type IV Edery et al 1996
EDNRB endothelin receptor type B WS type IV Attie et al 1995
EYA4 eyes absent homolog 4 (Drosophila) DFNA10 Wayne et al 2001
GJA1 gap junction protein alpha 1 43kD (connexin 43)
2 Liu et al 2001
GJB2 gap junction protein beta 2 26k (connexin 26) DFNA3 DFNB1 Kelsell et al 1997
GJB6 gap junction protein beta 6 (connexin 30) DFNA3 DFNB1 Grifa et al 1999
Del Castillo et al 2002
MITF microphthalmia-associated transcription factor WS type II Tassabehji et al 1994
MYH9 myosin heavy polypeptide 9 non-muscle DFNA17 Lalwani et al 2000
MYO6 myosin VI DFNA22 DFNB37 Melchionda et al 2001 Ahmed et al 2003
MYO7A myosin VIIA DFNA11 DFNB2 Liu et al 1997a Liu et al 1997b Weil et al 1997
MYO15A myosin XVA DFNB3 Wang et al 1998
OTOF otoferlin DFNB9 Yasunaga et al 1999
PAX3 paired box gene 3 (Waardenburg syndrome 1) WS type I WS type III Hoth et al 1993 Tassabehji
et al 1992
POU4F3 POU domain class 4 transcription factor 3 DFNA15 Vahava et al 1998
SLC26A4 solute carrier family 26 member 4 DFNB4 Li et al 1998
SOX10 SRY (sex determining region Y)-box 10 WS type IV Pingault et al 1998
TECTA tectorin alpha DFNB21 DFNA8DFNA12
Mustapha et al 1999 Verhoeven et al 1998
TMPRSS3 transmembrane protease serine 3 DFNB8DFNB10 Scott et al 2001
1 Autosomal recessive loci are designated DFNB autosomal dominant loci DFNA 2 To our knowledge no deafness locus has been determined for this autosomal recessive gene
Linkage analysis of gene-associated microsatellites
32
HET
()
638
338
366
544
616
632
456
656
433
614
458
586
529
PIC
()
752
548
336
484
775
70
63
9
74
6
61
6
83
7
62
762
621
No
of
alle
les
6 4 4 4 9 8 6 6 6 13 5 11 5
PCR
pr
oduc
t (b
p)
175
109
123
156
146
179
259
151
241
219
186
214
191
AT
(degC
)
62 60 56 60 60 62 58 60 62 58 58 60 58
Prim
er s
eque
nces
(5rsquorarr
3rsquo)
F A
ATC
AG
GA
GG
GG
TGAG
TGTG
R
CC
CC
CAG
CTC
ATA
CA
TTC
TC
F C
CTG
TAC
TGA
ATG
CTT
GA
GG
R
CA
TCTC
TAG
AA
GA
AG
CC
TCC
F
TC
AC
ATA
GC
ATT
ATA
TATG
GA
C
R T
TGA
GAT
GG
CTC
TTAC
TGA
G
F T
CG
ATG
ATG
CTT
TCTG
GTT
G
R A
GG
CTG
TGA
AA
TGG
ATG
GA
G
F G
AG
AA
GC
AC
CA
GG
CA
TAG
G
R T
GG
TTTA
GC
AA
GG
CTG
TTC
C
F T
GA
CA
TAC
GG
AG
GAC
CA
AG
AG
R C
CC
CTC
CC
TTG
CTC
TATC
TC
F T
GC
CC
CTC
AG
AG
ATA
ATC
AC
R
CTT
CA
ATTA
TAC
AC
ACA
GG
TAC
F
TG
AA
TATG
GG
GC
TGAG
GA
AG
R
TTC
TCC
CTC
TGC
CTG
TGTC
F
GG
TTTA
GC
AC
TGC
CTT
CA
GC
R
CA
TTA
AG
CA
TCTG
GC
ATG
TGG
F
GA
AA
AC
TCA
GA
TTA
GC
CTG
G
R A
TCTT
GA
GA
GC
AA
AGG
TTG
TG
F T
GG
TTA
GG
GC
ATG
ATTC
CA
G
R C
ATG
TATA
AA
GA
GTA
ATG
CC
AG
F
CG
GG
AG
AGG
GTT
TGAC
TAC
R
CTC
CG
TATT
GC
TCA
TCTT
TCC
F
AG
CTT
CC
CTT
CTC
TGA
GA
C
R G
AG
AA
TAG
AG
TTTG
TGC
TCA
G
Rep
eat
(TTT
A)1
5 (A
CC
)9(A
TC)3
(A
TTT)
7(G
TTT)
5 (C
A)2
1 (G
A)2
0 (C
TTT)
~20
(GA
)13
(AC
)20(
AG)9
(C
T)16
TT
(CT)
5 (A
AA
AT)
~25
(CT)
11(G
T)4(
CT)
2 (A
TTT(
T))2
3 (A
C)2
2
Mar
ker n
ame
CD
H23
_MS1
C
DH
23_M
S2_F
2 C
LDN
14_M
S1
CLD
N14
_MS
2 C
LDN
14_M
S3
CO
CH
_MS1
C
OC
H_M
S2
CO
L11A
2_M
S1
CO
L11A
2_M
S3
DFN
A5_
MS
1 D
FNA
5 _M
S2
DIA
PH
1 _M
S1
DIA
PH
1_M
S2
Mar
ker o
rigin
RP
CI8
1-99
C20
R
PC
I81-
99C
20
RP
CI8
1-97
L17
RP
CI8
1-97
L17
RP
CI8
1-97
L17
RP
CI8
1-32
1I4
RP
CI8
1-32
1I4
RP
CI8
1-24
M6
RP
CI8
1-24
M6
RP
CI8
1-66
C12
R
PC
I81-
66C
12
RP
CI8
1-36
2I5
RP
CI8
1-36
2I5
Tabl
e 2
Can
dida
te g
ene-
asso
ciat
ed m
arke
rs f
or c
anin
e de
afne
ss d
evel
oped
by
Rak
(20
03)
For
each
gen
e th
e m
icro
sate
llite-
base
d m
arke
r th
e m
arke
r or
igin
rep
eat t
ype
PC
R p
rimer
s w
ith o
ptim
ised
con
ditio
ns a
nd th
e nu
mbe
r of
alle
les
pol
ymor
phis
m
info
rmat
ion
cont
ent (
PIC
) and
obs
erve
d he
tero
zygo
sity
(HE
T) fo
r all
dogs
gen
otyp
ed a
re s
how
n
Can
dida
te
gene
CD
H23
C
LDN
14
CO
CH
C
OL1
1A2
DFN
A5
DIA
PH
1
Linkage analysis of gene-associated microsatellites 33
HET
()
815
345
695
578
562
508
583
441
75
63
7
33
414
30
76
7
44
8
PIC
()
853
679
798
774
652
63
72
3
59
5
83
2
77
9
50
5
54
1
40
4
88
9
59
1
No
of
alle
les
14 6 7 6 9 5 9 6 13 13 4 6 4 14 6
PCR
pr
oduc
t (b
p)
147
131
258
227
134
190
148
195
218
260
181
164 96
227 94
AT
(degC
)
60 58 58 58 60 60 58 62 58 58 62 56 58 58 62
Prim
er s
eque
nces
(5rsquorarr
3rsquo)
F G
CTA
GG
AA
AA
ATC
CG
CA
ATG
R
GAC
CC
CC
TAG
GAC
ACC
AAC
F
GA
GA
ATT
GG
GC
ATG
GG
CAG
A
R T
GA
CTT
TATC
AC
TGG
TCTT
TG
F T
TATG
CAG
CC
CA
TGAC
AA
TC
R C
AA
GG
GA
AC
TCA
AAG
GC
TTG
F
TG
GA
CC
AG
GTC
AGTT
TGTG
R
TC
TGC
CTG
TGTC
TCTG
CC
F
ATG
GC
ATG
AA
GA
GG
ATA
CC
G
R A
GG
AC
AGG
TGAC
GG
CTC
TAC
F
GC
TAG
TAC
TCG
ATT
GTG
GTC
R
TC
ATG
GG
TTG
TGA
GA
TCC
AG
F T
TAA
TTTG
CTC
GTC
TTC
CTG
R
TG
TAA
GC
TCC
ACG
GA
TCA
CC
F
CTC
TCTT
GG
TCTC
CC
TCTG
C
R G
GG
AG
TAG
GG
GTG
GAG
TAG
G
F G
GTG
TTTC
CTT
TCC
TTTT
CT
R G
GTG
TTC
TCTC
CC
TTTC
TCT
F C
TCTA
TGA
AA
GG
TGA
TTG
CC
R
CA
GC
CAT
AC
AA
ATG
AGA
ATT
G
F C
TAC
AG
TGA
ATC
AG
CA
CA
GAC
R
CA
GC
CTT
GA
CTG
TTTC
TTTG
G
F T
GA
TTG
AC
TCTA
CTT
TAC
AC
A
R T
ATA
TTA
GG
CG
GTT
TTC
TTC
T F
AC
CC
AG
GTG
GC
CTG
ATTG
R
GC
AC
GC
AC
GTT
CTC
TCTT
TC
F G
AA
TGC
CC
TTC
ACC
TTG
AA
A
R G
GA
AA
AG
GA
GA
GA
TGA
TGC
C
F T
CTT
CC
TTG
GA
AA
GG
GA
AC
TC
R T
GC
CC
TAA
CA
CTT
GG
AA
TGG
Rep
eat
(TA
GA
)12
(GA
)25
(GT)
10(A
T)13
(A
G)2
1 (G
T)16
(A
G)1
2 (C
A)1
2 (G
A)8
(C
TTT)
~20
(GA
AA
)~25
(G
T)12
(C
A)1
5 (A
G)1
1 (G
AA
A)~
43
(AC
)13
Mar
ker n
ame
ED
N3_
MS
1 E
DN
RB
_M
S1
EY
A4_
MS
1 E
YA
4_M
S2
GJA
1_M
S1
GJA
1_M
S2
GJB
2_M
S1
GJB
2_M
S2
GJB
6_M
S2
GJB
2+6_
MS1
M
ITF
_MS
2 M
ITF
_MS
3 M
YH
9_M
S2
MY
H9_
MS
3 M
YO
6_M
S2
Mar
ker o
rigin
RP
CI8
1-36
6E14
Z
emke
et a
l (1
999)
R
PC
I81-
301N
19
RP
CI8
1-30
1N19
R
PC
I81-
370A
16
RP
CI8
1-37
0A16
R
PC
I81-
133O
22
RP
CI8
1-13
3O22
R
PC
I81-
343C
15
FH
2324
R
PC
I81-
119P
24
RE
N10
0J13
R
PC
I81-
374A
12
FH
2293
R
PC
I81-
156P
14
Tabl
e 2
(con
tinue
d)
Can
dida
te g
ene
ED
N3
ED
NR
B
EY
A4
GJA
1 G
JB2
GJB
6 M
ITF
MY
H9
MY
O6
Linkage analysis of gene-associated microsatellites
34
HET
()
359
366
431
566
51
50
5
28
3
35
8
79
9
70
4
69
2
23
9
68 71
698
PIC
()
496
623
627
765
635
756
502
575
756
76
70
1
44
2
75
4
84
5
80
6
No
of
alle
les
5 4 6 13 7 8 5 3 10 11 8 8 8 10 11
PCR
pr
oduc
t (b
p)
293
166
100
137
174
197
164
201
160
297
267
140
276
228
260
AT
(degC
)
56 60 56 62 62 62 60 60 58 58 56 58 58 62 60
Prim
er s
eque
nces
(5rsquorarr
3rsquo)
F T
GG
TTA
AA
AC
ATT
AA
AC
TTA
TAG
R
TA
GTA
TATA
GA
GA
TGC
AA
TGG
F
CA
TTG
GG
TGC
TTTC
CTG
TTC
R
TG
GA
GC
TGC
AG
GTA
TAG
CC
F
CC
AG
GC
ATT
CG
AG
GG
TG
R C
AG
AA
CTT
GA
GG
AAC
CA
TAG
F
CC
ATG
AAC
TTTG
TGG
AA
CTG
C
R A
AA
GG
GTT
GC
TGTG
GA
GA
TG
F A
GG
CA
GG
TTC
ATC
TGTG
TCC
R
TC
CC
AG
AC
CC
AG
CTA
CA
TTC
F
CA
GC
CAA
CTG
TATT
CTC
CTT
G
R A
TCTT
GA
GC
CC
TGC
ATT
AG
G
F G
AA
GC
GA
GG
AG
AG
ACA
GTC
C
R A
AG
GA
AG
CC
TCC
TGAC
AA
CC
F
CA
GG
GTC
AG
GC
TCTA
TGC
TC
R T
CC
TATC
ATC
CG
GC
TTTG
AC
F
TC
TGG
ATTG
TGG
TCA
CA
AC
C
R A
CTG
GAC
AC
TTC
TTTT
CA
GAC
G
F A
AA
GTG
GC
TGG
TTC
GG
AA
G
R A
GC
AG
CA
GC
ATA
CA
TTC
CTC
F
AA
GTA
GA
TCC
TATT
ATC
GTG
G
R A
GTT
TCA
GTG
TCTG
TTA
AA
TAG
F
CC
GG
ATT
TCTG
AG
GAG
GC
R
CA
TGC
TCTT
CA
CC
AGA
AC
C
F T
CA
GC
ATG
GA
TTTT
GTA
AA
ATC
R
GG
AC
TGC
GTG
GA
CAT
CTG
F
AC
AC
GG
TTC
TCG
CTG
ATG
TG
R T
GA
AG
GG
GA
TTG
AAC
AG
AG
G
F T
TCA
TTC
CG
AG
GTT
CTA
AC
TG
R C
AC
CA
TCTC
GTA
GC
CTT
TATC
Rep
eat
(TC
)13
(AC
)12
(GT)
12
(AC
)18
(GT)
12
(TA
)3(T
G)9
(TA
)2(C
A)2
C
(TA
AA
)5
(AA
T)18
(C
T)13
(T
G)1
3 (C
T)17
(T
AA
A)1
4 (G
T)3
CC
(GT)
19
(ATT
T)12
(T
C)8
(CA)
5(C
G)2
(CA)
9 (G
T)16
Mar
ker n
ame
MY
O7A
_M
S1
MY
O7A
_M
S2
MY
O7A
_M
S3
MY
O15
A _
MS
1 M
YO
15A
_M
S2
OTO
F _M
S1
PA
X3_
MS
1 P
AX
3_M
S2
PO
U4F
3_M
S4
SLC
26A
4_M
S2
SO
X10
_MS
2 T
EC
TA _
MS
1 T
EC
TA _
MS
2 T
MP
RS
S3_
MS
1 T
MP
RS
S3_
MS
2
Mar
ker o
rigin
RP
CI8
1-19
3deg2
RP
CI8
1-19
3deg2
AH
TH29
8 R
PC
I81-
362deg
13
RP
CI8
1-36
2deg13
R
PC
I81-
198L
15
RP
CI8
1-25
7H23
R
PC
I81-
257H
23
G2C
024
66
RP
CI8
1-47
P17
RP
CI8
1-50
5H2
RP
CI8
1-59
C2
RP
CI8
1-59
C2
RP
CI8
1-12
5P17
A
HTH
246
Tabl
e 2
(con
tinue
d)
Can
dida
te
gene
MY
O7A
M
YO
15A
O
TOF
PA
X3
PO
U4F
3 S
LC26
A4
SO
X10
TE
CTA
TM
PR
SS
3
Linkage analysis of gene-associated microsatellites 35
Table 3 The gene-associated microsatellites (MS) for 24 candidate genes of canine
congenital sensorineural deafness were localized using BLAST searches against the
Boxer genome assembly 21 The accession numbers (AccNo) of the whole
genome shotgun (WGS) contigs containing the genes as well as their associated
microsatellites are given
Microsatellite Acc No of WGS contig CFA Location of
Gene (Mb) Location of
MS (bp) Location of
MS in relation to gene
CDH23_MS1 NW_876311 4 2541025780 25510 intragenic
CDH23_MS2 NW_876311 4 2541025780 25630 intragenic
CLDN14_MS1 NW_876295 31 3379533796 33790 proximal
CLDN14_MS2 NW_876295 31 3379533796 33950 distal
CLDN14_MS3 NW_876295 31 3379533796 33790 proximal
COCH_MS1 NW_876327 8 1321513232 13225 intragenic
COCH_MS2 NW_876327 8 1321513232 13290 distal
COL11A2_MS1 NW_876254 12 56315659 5608 proximal
COL11A2_MS3 NW_876254 12 56315659 5578 proximal
DFNA5_MS1 NW_876258 14 4116941237 41135 proximal
DFNA5_MS2 NW_876258 14 4116941237 41250 distal
DIAPH1_MS1 NW_876292 2 3933039430 39382 intragenic
DIAPH1_MS2 NW_876292 2 3933039430 39552 distal
EDN3_MS1 NW_876277 24 4701347032 47057 distal
EDNRB_MS1 Zemke et al (1999) 22 3436334383 34354 proximal
EYA4_MS1 NW_876269 1 2928029550 29531 intragenic
EYA4_MS2 NW_876269 1 2928029550 29500 intragenic
GJA1_MS1 NW_876269 1 6399463996 64150 distal
GJA1_MS2 NW_876269 1 6399463996 64160 distal
GJB2_MS1 NW_8762781 25 2093620942 20938 intragenic
GJB2_MS2 NW_8762781 25 2093620942 20863 proximal
GJB2+6_MS1 FH2324 25 2093620942 17543 proximal
GJB3_MS2 NW_876259 15 1018310194 4530 proximal
GJB6_MS2 NW_876278 25 2090420906 20953 distal
Linkage analysis of gene-associated microsatellites
36
Table 3 (continued)
Microsatellite Acc No of WGS contig CFA Location of
Gene (Mb) Location of
MS (bp) Location of
MS in relation to gene
MITF_MS2 NW_876271 20 2485324884 24844 proximal
MITF_MS3 REN100J13 20 2485324884 25668 distal
MYH9_MS2 NW_876251 10 3113531193 31244 distal
MYH9_MS3 FH2293 10 3113531193 31696 distal
MYO15A_MS1 NW_876313 5 4436944419 44330 proximal
MYO15A_MS2 NW_876313 5 4436944419 44409 intragenic
MYO6_MS2 NW_876254 12 4041740504 40534 distal MYO7A_MS1 NW_876273 21 2454324609 24465 proximal
MYO7A_MS2 NW_876273 21 2454324609 24566 intragenic
MYO7A_MS3 AHTH298 21 2454324609 24594 distal
OTOF_MS1 NW_876263 17 2350223595 23463 proximal
PAX3_MS1 NW_876304 37 3134831445 31426 intragenic
PAX3_MS2 NW_876304 37 3134831445 31481 distal
POU4F3_MS4 G2C02466 2 4361043612 - -
SLC26A4_MS2 NW_876265 18 1586715927 15960 distal
SOX10_MS2 NW_876251 10 2975129762 29740 proximal
TECTA_MS1 NW_876312 5 1588515954 15910 intragenic
TECTA_MS2 NW_876312 5 1588515954 15930 intragenic
TMPRSS3_MS1 NW_876295 31 3903539054 38942 proximal
TMPRSS3_MS2 AHTH246 31 3903539054 38508 proximal
Linkage analysis of gene-associated microsatellites 37
Table 4 Number of alleles observed (HET) and expected (HE) heterozygosity and
polymorphism information content (PIC) for the developed marker-set
Feature Mean SD Min Max
No of alleles 75 31 3 14
hO () 703 122 370 898
hE () 532 151 239 815
PIC () 667 130 336 889
Table 5 Number of alleles per microsatellite locus and their PIC () values of the
developed marker-set
No of alleles per
microsatellite
Number of marker
loci PIC ()
3 1 575
4 6 483
5 5 574
6 11 652
7 2 716
8 5 671
9 3 717
10 2 800
11 3 776
13 4 803
14 2 871
Linkage analysis of gene-associated microsatellites
38
Table 6 Significant test statistics for linkage analysis carried out for all 24 genotyped
families conjoined Zmeans and LOD scores are given with their respective error
probabilities for the gene-associated markers of the candidate genes CLDN14
COL11A2 GJA1 MITF MYH9 and SOX10
Marker Location on canine chromosome (CFA)
Zmean pZmean LOD score pLOD
CLDN14_MS1 31q15 134 009 086 002
CLDN14_MS2 31q15 168 005 105 001
CLDN14_MS3 31q15 108 014 049 007
COL11A2_MS1 12q11-q12 166 005 085 002
COL11A2_MS3 12q11-q12 167 005 078 003
GJA1_MS1 1q24-q25 151 007 118 001
GJA1_MS2 1q24-q25 151 007 118 001
MITF_MS2 20q13 101 02 080 003
MITF_MS3 20q13 121 011 104 001
MYH9_MS2 10q232 080 02 018 02
MYH9_MS3 10q232 175 004 097 002
SOX10_MS2 10q21-q23 146 007 110 001
Linkage analysis of gene-associated microsatellites 39
Table 7 Significant test statistics for linkage analyses carried out each family
separately Zmeans and LOD scores are given with their respective error probabilities
for the gene-associated markers of the candidate genes CLDN14 MYH9 and GJA1
Gene-associated
marker
Number of families with significant linkage to
CCSD
Number of corresponding
family members
Zmean pZmean LOD-score pLOD
CLDN14_MS1 51 40 278 0003 112 0011
CLDN14_MS2 383 000007 170 0003
CLDN14_MS3 281 0002 113 0011
MYH9_MS2 32 21 081 02 023 02
MYH9_MS3
(=FH2293) 156 006 058 005
GJA1_MS1 13 46 295 0002 052 006
GJA1_MS2 286 0002 051 006 1 families with significant linkage include four German full-sib families and one
German half-sib family 2 families with significant linkage include three German full-sib families 3 families with significant linkage include one French half-sib family
Chapter 4
Evaluation of eight candidate genes for canine congenital sensorineural deafness
in Dalmatian dogs
Evaluation of eight candidate genes for CCSD 43
Evaluation of eight candidate genes for canine congenital sensorineural deafness in Dalmatian dogs Abstract
In this study we have been focusing on genomic loci that encode various enzymes
and transporters involved in the hearing process in humans We developed intragenic
markers for the canine genes ESPN MYH14 MYO3A PRES TMC1 TMIE USH1C
and WHRN on canine chromosomes (CFA) 1 2 5 11 18 20 and 21 which have
been shown to be responsible for human hereditary deafness and to employ these
newly developed markers for non-parametric linkage analyses with canine congenital
sensorineural deafness (CCSD) We screened DNA from altogether six Dalmatian
dogs which represent the parents of four families for single nucleotide polymorphisms
(SNPs) in the eight candidate genes by means of direct sequencing combined with a
polymerase chain reaction method for amplifying genomic DNA We characterized 20
SNPs and one insertion-deletion polymorphism For the TMC1 and TMIE genes we
additionally genotyped one microsatellite marker each The families used for
subsequent genotyping of the markers included 39 members from four full-sib
families with frequent occurrence of CCSD We concluded that mutations in ESPN
MYH14 MYO3A PRES TMC1 TMIE USH1C and WHRN do not play a role in
CCSD of the Dalmatian dog population investigated here
Introduction Over the past ten years significant progress has been made in the identification of
genes causing different forms of human deafness Currently 39 of the genes
responsible for non-syndromic hearing impairment have been identified in different
human populations (The Hereditary Hearing Loss Hompage
httpwebhostuaacbehhh Van Camp and Smith 2003)
Since non-syndromic hereditary hearing impairment is almost exclusively caused by
cochlear defects affected patients suffer from sensorineural hearing loss
Evaluation of eight candidate genes for CCSD
44
The most common histopathologic finding in cases of profound congenital deafness
in humans is the cochleosaccular degeneration (CSD) It is estimated to occur in
approximately 70 of cases in man and also in dog breeds the histological pattern is
known as cochleosaccular degeneration
Many genetic disorders in humans and domestic dogs (Canis familiaris) demonstrate
a high level of clinical and molecular similarity Therefore genes responsible for
human hereditary deafness seem to be appropriate candidate genes for CCSD
especially in Dalmatian dogs
In this report we provide 21 single-nucleotide polymorphisms (SNPs) and two
microsatellite markers in altogether eight selected human candidate genes (Table 1)
This eight candidate genes were only recently identified for being responsible for
different form of human non-syndromic deafness In order to evaluate whether any of
this candidate genes is responsible for congenital sensorineural deafness in
Dalmatian dogs we analyzed the association of the ESPN MYH14 MYO3A PRES
TMC1 TMIE USH1C and WHRN haplotypes with the CCSD phenotype in four
families of Dalmatian dogs with frequent occurrence of CCSD
Material and methods Pedigree structure and sampling
For the linkage analysis we used blood samples from 39 Dalmatian dogs They
belong to four full-sib families segregating for CCSD At least two of the full sibs of
each family were unilaterally deaf The phenotype of the affected animals had been
confirmed by brainstem auditory evoked response (BAER) that detects electrical
activity in the cochlea and auditory pathways in the brain
The families consisted of eight to 12 individuals In two families a blood sample of the
sire and dam respectively was not available Screening for SNPs was performed by
comparative sequencing of genomic DNA from the parents of the families used for
linkage analyses
SNP and microsatellite marker identification for genotyping
The canine ESPN MYH14 MYO3A PRES TMC1 TMIE USH1C and WHRN gene
sequences was derived from sequences deposited in the current dog genome
assembly (Boxer genome assembly 21) of the NCBI GenBank (Table 2) by BLAST
Evaluation of eight candidate genes for CCSD 45
(Basic Local Alignment Search Tool) search (httpwwwncbinlmnihgovBLAST)
using the human ESPN MYH14 MYO3A PRES TMC1 TMIE USH1C and WHRN
reference mRNA sequence
We compared the canine genomic DNA sequence from the eight candidate genes to
canine cDNA fragments in the canine EST database using the BLASTN program
For the localization of the exonintron boundaries canine or alternatively human
mRNA sequences were used for the mRNA-to-genomic alignment program Spidey
(httpwwwncbinlmnihgovIEBResearchOstellSpideyindexhtml) The human
and canine mRNA sequences which were used to determine the exon organization of
the candidate genes are given in Table 2
For each of the eight candidate genes we designed intragenic primer pairs to amplifly
intronic sequences yielding products with a length of 560 to 670 bp PCR primers
were developed with the Primer3 program (httpfrodowimiteducgi-
binprimer3primer3_wwwcgi) The PCR reactions were performed in a total of 50 microl
containing 125 microM dNTPs 25 pmol of each primer the reaction buffer supplied by
the manufacturer (Qbiogene Heidelberg Germany) and 1 U Taq polymerase After
a 4 min initial denaturation at 95degC 35 cycles of 30 sec at 94degC 45 sec at 58degC and
80 sec at 72degC were performed in a MJ Research thermocycler (Biozym Hessisch
Oldendorf Germany) The obtained PCR products were directly sequenced with the
DYEnamic ET Terminator kit (AmershamBiosciences Freiburg Germany) and a
MegaBACE 1000 capillary sequencer (AmershamBiosciences) using the PCR
primers as sequencing primers Sequence data were analyzed with Sequencher 42
(GeneCodes Ann Arbor MI)
In the first step sequence analyses were performed for PCR products of the parents
of four full-sib families If a heterozygous SNP was found for one or both parents all
progeny of the respective families were analyzed for that SNP Additionally to the
SNPs we used two microsatellite markers for linkage analyses We identified one
intragenic microsatelllite within the TMC1 gene As we could develop only one SNP
for the TMIE gene we additionally genotyped one microsatellite marker derived from
the NCBI database (httpwebncbinlmnihgov) located very closely to the TMIE
gene (Table 3)
Evaluation of eight candidate genes for CCSD
46
SNP marker analysis
A total of 21 SNPs were identified within ESPN MYH14 MYO3A PRES TMC1
TMIE USH1C and WHRN (Table 3) and used for linkage analysis Of all SNPs six
out of the observed 21 SNPs were polymorphic in all four examined families Out of
the 21 SNPs eleven SNPs were present in family 1 18 SNPs were heterozygous for
one or both parents in family 2 and 15 SNPs could be used for linkage analysis in
family 3 and 4 respectively (Table 4)
The most frequent form of SNPs with a frequency of 238 was the CT transition
motif The scarcest one was the CG transversion motif with a frequency of 48
respectively
Linkage and haplotype analysis Multipoint linkage and haplotype analysis were performed using MERLIN version
0102 (multipoint engine for rapid likelihood inference Center for Statistical Genetics
University of Michigan MI USA Abecasis et al 2002) The test statistics Z-mean
and Lod score were used to test for the proportion of alleles shared by affected
individuals identical by descent (IBD) for the considered marker loci The data of the
genotypes was additionally computed using the software package SAS Genetics
(Statistical Analysis System Version 913 SAS Institute Inc Cary NC USA 2005)
to specify the observed heterozygocity values (HET) and the polymorphism
information content (PIC)
Furthermore linkage disequilibrium (LD) and association of haplotypes with CCSD
was tested using the procedures CASECONTROL and HAPLOTYPE of
SASGenetics (Statistical Analysis System version 913 Cary NC USA)
Results and discussion The non-parametric multipoint linkage analysis in four full-sib families did not show
significant test statistics The highest Z-mean value was 046 the highest LOD Score
was 008 while the error probabilities ranged from 03 to 08 (Table 5) The maximum
achievable Z-mean was 448 and the corresponding value for the LOD score was
160 Marker-trait association tests for haplotypes of the candidate gene markers
were not significant Obviously no haplotype was associated with CCSD in these
Dalmatian dog families as demonstrated (Figure 1 2 3 4 5 6 7 and 8) The
Evaluation of eight candidate genes for CCSD 47
paternal and maternal haplotypes as far as they could be estimated were distributed
among the affected progeny closely to the expected proportion of 50 and thus no
excess of a certain haplotype could be observed in the affected dogs
Due to the fact that both animals with unilateral or bilaterally hearing loss and
bilateral hearing animals shared identical haplotypes it is unlikely that the ESPN
MYH14 MYO3A PRES TMC1 TMIE USH1C and WHRN genes are involved in the
pathogenesis of CCSD in Dalmatian dogs However the ESPN MYH14 MYO3A
PRES TMC1 TMIE USH1C and WHRN gene-associated marker may serve for
further linkage studies in other Dalmatian dog populations and dog breeds other than
Dalmatians
Evaluation of eight candidate genes for CCSD
48
Table 1 Details of the six selected human candidate genes
Gene symbol
Gene name Locus name1 Most important reference
ESPN espin DFNB36 Naz et al (2004)
MYH14 myosin heavy polypeptide 14 DFNA4 Donaudy et al (2004)
MYO3A myosin IIIA DFNB30 Walsh et al (2002)
PRES solute carrier family 26
member 5 (prestin) DFNB61
Zheng et al (2000) Liu et
al (2003)
TMC1 transmembrane channel-like
gene 1
DFNB7
DFNA36 Kurima et al (2002)
TMIE transmembran inner ear DFNB6 Naz et al 2002
USH1C Usher syndrome 1C DFNB18 Verpy et al (2000) Ouyang
et al (2002)
WHRN whirlin DFNB31 Mburu et al (2003)
1 Autosomal recessive loci are designated DFNB autosomal dominant loci DFNA
Evaluation of eight candidate genes for CCSD 49
Table 2 Selected human candidate genes with their location on HSA and CFA and
corresponding accession numbers
Gene symbol Gene
location on HSA1
Acc No 3 human mRNA
Gene location
on CFA2
Acc No 3 canine
genomic sequence
Acc No 3 canine mRNA
ESPN 1 NM_031475 5 NC_006587 XM_546751
MYH14 19 NM_024729 1 NW_876270 -
MYO3A 10 NM_017433 2 NC_006584 XM_544234
PRES 7 NM_206883 18 NC_006600 XM_540393
TMC1 9 NM_138691 1 NC_006583 XM_541284
TMIE 3 NM_147196 20 NC_006602 XM_846596
USH1C 11 NM_153676 21 NC_006603 XM_860072
WHRN 9 NM_015404 11 NC_006593 XM_850321
1 Homo sapiens autosome 2 Canis familiaris autosome 3 Accession number
Evaluation of eight candidate genes for CCSD
50
Table 3 The 21 newly developed intragenic SNPs and two microsatellite markers for
Dalmatian dogs in the candidate genes ESPN MYH14 MYO3A PRES TMC1
TMIE USH1C and WHRN with their corresponding primers the SNP motif the
product size and the annealing temperature the observed heterozygosity (HET) and
polymorphism information content (PIC)
SNP Primer F1 (5acute -gt 3acute) SNP motif primer R2 (5acute -gt 3acute)
Bp3 AT4 PIC ()
HET()
ESPN_SNP1
ACCAGCACCCTCTCCAACTA
AGGAATTCACAA (CT)CACACATACA
ACTCAAGCTCAGGGTGTGGT
565 60 9 10
ESPN_SNP2
ACCAGCACCCTCTCCAACTA
ATGGCTGGCGCT (AG)GAGGCTGCCC
ACTCAAGCTCAGGGTGTGGT
565 60 27 41
ESPN_SNP3
ACCAGCACCCTCTCCAACTA
ACACTCTTCCCA (CT)GGCTGGCGCT
ACTCAAGCTCAGGGTGTGGT
565 60 23 32
ESPN_SNP4
ACCAGCACCCTCTCCAACTA
TGGGAAGAGGGA (AG)GGGGGAGCAT
ACTCAAGCTCAGGGTGTGGT
565 60 23 32
ESPN_SNP5
ACCAGCACCCTCTCCAACTA
GAGTGGGCCAGG (CT)TGGGAAGAGG
ACTCAAGCTCAGGGTGTGGT
565 60 28 42
MYH14_SNP1
CTCTCCCCAACTCAGTTCCA
ACGTGTATTCGG (GT)CGCTTTTATT
GTGATAGGGACGAGCAGCAT
670 60 35 42
MYH14_SNP2
CATGGGACCGTTCCTACACT
AGCCTCGTTTAA (CT)CTAAAAGGAA
GCTCAATAGGCACGACATCA
640 60 34 39
Evaluation of eight candidate genes for CCSD 51
Table 3 (continued)
SNP Primer F1 (5acute -gt 3acute) SNP motif primer R2 (5acute -gt 3acute)
Bp3 AT4 PIC ()
HET()
MYO3A_SNP1
AATGCTTGAGTTTGGGATGC
GGCAGTCCCATG (GT)CCCTTATAAG
ACCTAATTGCCCAGATGCAG
650 60 37 77
MYO3A_SNP2
AATGCTTGAGTTTGGGATGC
GTGGAGAGCCAC (CG)TTGGGAGAGG
ACCTAATTGCCCAGATGCAG
650 60 37 70
MYO3A_SNP3
AACCTCCTGGCGTAGTATTCC
CATTACCTATTT (AT)GATCCTTATA
TTTTCCACTTCAGGCACACA
650 60 25 36
PRES_SNP1
CCCTTACCCCATACCATTCC
GATAGACTTCCT (AG)CCCTCAGACT
TTCAGGACAGCATCATCTGC
560 60 37 64
PRES_SNP2
CCCTTACCCCATACCATTCC
TGATGTCTGCTG (AT)TAACCCATTC
TTCAGGACAGCATCATCTGC
560 60 37 66
TMC1_SNP1
GCAACCTCTCGGTTTATCCA
CGTGAAGTGCCC (AT)TTGATGGAAA
AAGCTGGGGAAGTGGATATGT
610 60 57 37
TMC1_SNP2
GCAACCTCTCGGTTTATCCA
GGAGACATTACC (AG)TGAAGTGCCC
AAGCTGGGGAAGTGGATATGT
610 60 47 29
TMC1_SNP3
GGAAGCAAGACTGAGGTTGG
AGGCTTTTTAAA (AG)CTGTTCTGGG
CTGCTGCATTTGCCTGTAAG
650 60 48 30
TMIE_SNP1
AGAACACCACCGTCTCCTTG
CAAGGCGACGCC (AT)GTGCTGTCCT
GCCTCTGGTCAGAAGAGGTG
625 60 59 36
Evaluation of eight candidate genes for CCSD
52
Table 3 (continued)
SNP Primer F1 (5acute -gt 3acute) SNP motif primer R2 (5acute -gt 3acute)
Bp3 AT4 PIC()
HET()
USH1C_SNP2
CTCCCGGTCTGTCAGGAAC
GGCCTGGGGGGA (AC)AAGCGGACGG
ATGGCATCGACTTCTCCAAC
560 60 37 35
USH1C_SNP4
CTCCCGGTCTGTCAGGAAC
GGTCTCAGACCG (AC)GGCAGGGAGA
ATGGCATCGACTTCTCCAAC
560 60 37 37
WHRN_SNP1
TTCACCTCCAGGATCTGGTC
CCTGAGCCCGAG (CT)CCACGCTGCT
GGCTACTTTTCTTCCCCCTTT
600 60 25 37
WHRN_SNP2
TTCACCTCCAGGATCTGGTC
GGTCACGGGGGC (CT)CCGGGAGGTT
GGCTACTTTTCTTCCCCCTTT
600 59 24 33
WHRN_SNP3
TTCACCTCCAGGATCTGGTC
GTCCGAGTCCCG (AG)CCCCAGCCTG
GGCTACTTTTCTTCCCCCTTT
600 60 34 55
Microsatellite marker
Primers (forward reverse ) 5acute -gt 3acute Bp AT PIC()
HET()
TMC1_MS1
GCCCCCAGCTAAAAAGAGAA
TTCTCTTCCTCCCTCCTGTTC
220-220 60 76 57
FH2158 ATGGCCACATCACCCTAGTC
CTCTCTCTGCATCTCTCATGAA
274-302 58 57 66
Microsatllite marker derived from the NCBI database (httpwebncbinlmnihgov) 1 Forward 2 Reverse 3 Product size (basepairs) 4 Annealing temperature (degC)
Evaluation of eight candidate genes for CCSD 53
Table 4 The 21 newly developed intragenic SNPs for Dalmatian dogs in the
candidate genes ESPN MYH14 MYO3A PRES TMC1 TMIE USH1C and WHRN
with their nucleotide polymorphism allele and genotype frequencies
Gene symbol Fam1 Nucleotide polymorphism
Allele frequencies
Genotype frequencies2
ESPN_SNP1 4 CgtT 083017 840
ESPN_SNP2 2 3 4 AgtG 074026 15160
ESPN_SNP3 2 3 TgtC 068032 07120
ESPN_SNP4 2 3 GgtA 068032 07120
ESPN_SNP5 2 3 4 CgtT 074026 15160
MYH14_SNP1 2 3 4 GgtT 058041 51610
MYH14_SNP2 2 3 4 CgtT 058041 51610
MYO3A_SNP1 1 2 GgtT 062038 5111
MYO3A_SNP2 1 2 CgtG 062038 5111
MYO3A_SNP3 1 2 4 TgtA 076024 15140
PRES_SNP1 1 2 3 4 AgtG 058042 10254
PRES_SNP2 1 2 3 4 TgtA 058042 10254
TMC1_SNP1 1 2 3 4 AgtT 056044 11226
TMC1_SNP2 1 2 3 4 AgtG 076024 20190
TMC1_SNP3 1 2 3 4 AgtG 074026 19200
TMIE_SNP1 1 2 3 4 AgtT 058042 12216
USH1C_SNP2 1 3 4 AgtC 053047 9147
USH1C_SNP4 1 3 4 AgtC 053047 9147
WHRN_SNP1 2 CgtT 075025 360
WHRN_SNP2 2 CgtT 075025 360
WHRN_SNP3 2 3 4 InDel3 069031 13171 1 Number of families which were informative for the respective SNPs 2 Frequence of genotypes (first number number of animals homozygous for allele 1
second number heterozygous third number homozygous for allele 2) 3 Insertiondeletion of one base-pair (adenine)
Evaluation of eight candidate genes for CCSD
54
Table 5 Linkage analyses for families 1 2 3 and 4 for the 21 SNP and two
microsatellite markers within the eight candidate genes regarding Zmean LOD score
and error probabilities (p-values)
Gene symbol Marker Zmean pz-value1 LOD score pL-value2
ESPN ESPN_SNP1 014 04 002 04
ESPN_SNP2 014 04 002 04
ESPN_SNP3 014 04 002 04
ESPN_SNP4 014 04 002 04
ESPN_SNP5 014 04 002 04
MYH14 MYH14_SNP1 -089 08 -019 08
MYH14_SNP1 -089 08 -019 08
MYO3A MYO3A_SNP1 -049 07 -011 08
MYO3A_SNP2 -049 07 -011 08
MYO3A_SNP3 -049 07 -011 08
PRES PRES_SNP1 -094 08 -019 08
PRES_SNP2 -094 08 -019 08
TMC1 TMC1_SNP1 -034 06 -008 07
TMC1_SNP2 -034 06 -008 07
TMC1_SNP3 -034 06 -008 07
TMC1_MS1 -035 06 -008 07
TMIE TMIE_SNP1 013 04 003 03
FH2158 -056 07 -013 08
USH1C USH1C_SNP2 018 04 04 03
USH1C_SNP4 018 04 04 03
WHRN WHRN_SNP1 046 03 008 03
WHRN_SNP2 046 03 008 03
WHRN_SNP3 046 03 008 03 1 Error probability regarding Zmean 2 Error probability regarding LOD score
Microsatellite marker
Evaluation of eight candidate genes for CCSD 55
Figu
re 1
SN
P-h
aplo
type
s of
the
ES
PN
gen
e in
all
anal
yzed
Dal
mat
ian
dog
fam
ilies
The
hapl
otyp
es b
elon
ging
to F
amily
2 3
and
4 re
gard
ing
the
gene
ES
PN
sho
w n
o as
soci
atio
n w
ith th
e C
CS
D p
heno
type
In
the
thre
e fa
milie
s th
e ha
plot
ype
21
43
2 is
the
mos
t com
mon
one
bei
ng p
rese
nt in
72
o
f all
anim
als
It is
follo
wed
by
the
hapl
otyp
e 2
32
14
with
18
a
nd 4
34
34
with
6
Nor
mal
hea
ring
anim
als
as
wel
l as
thei
r de
af s
iblin
gs s
how
all
thes
e th
ree
hapl
otyp
es T
here
is n
o re
com
bina
tion
of th
e ha
plot
ypes
of t
he E
SP
N g
ene
in th
e th
ree
fam
ilies
Evaluation of eight candidate genes for CCSD 56
Figu
re 2
SN
P-h
aplo
type
s of
the
MY
H14
gen
e in
all
anal
yzed
Dal
mat
ian
dog
fam
ilies
Evaluation of eight candidate genes for CCSD 57
Figu
re 3
SN
P-h
aplo
type
s of
the
MY
O3A
gen
e in
all
anal
yzed
Dal
mat
ian
dog
fam
ilies
Evaluation of eight candidate genes for CCSD 58
Figu
re 4
SN
P-h
aplo
type
s of
the
PR
ES
gen
e in
all
anal
yzed
Dal
mat
ian
dog
fam
ilies
Evaluation of eight candidate genes for CCSD 59
Figu
re 5
SN
P- a
nd m
icro
sate
llite-
hapl
otyp
es o
f the
TM
C1
gene
in a
ll an
alyz
ed D
alm
atia
n
Evaluation of eight candidate genes for CCSD 60
Figu
re 6
SN
P- a
nd m
icro
sate
llite-
hapl
otyp
es o
f the
TM
IE g
ene
in a
ll an
alyz
ed D
alm
atia
n do
g fa
milie
s
Evaluation of eight candidate genes for CCSD 61
Figu
re 7
SN
P-h
aplo
type
s of
the
US
H1C
gen
e in
all
anal
yzed
Dal
mat
ian
dog
fam
ilies
Evaluation of eight candidate genes for CCSD 62
Figu
re 8
SN
P-h
aplo
type
s of
the
WH
RN
gen
e in
all
anal
yzed
Dal
mat
ian
dog
fam
ilies
Chapter 5
Molecular characterization of the canine myosin heavy polypeptide 9
non-muscle (MYH9) gene on dog chromosome 10q232
Canine MYH9 gene 65
Molecular characterization of the canine myosin heavy polypeptide 9 non-muscle (MYH9) gene on dog chromosome 10q232 Abstract A missense mutation of the Myosin heavy polypeptide 9 (MYH9) gene which
encodes the nonmuscle myosin heavy chain-A caused non-syndromic sensorineural
deafness in a human family which was characterized by cochleosaccular
degeneration In the present study we evaluated whether MYH9 gene mutations are
responsible for canine congenital sensorineural deafness (CCSD) in Dalmatian dogs
As described in Chapter 3 two MYH9 gene-associated microsatellites were
genotyped in 25 Dalmatian dog families segregating for CCSD We could find
significant linkage with CCSD for the microsatellite marker MYH9_MS3 (=FH2293)
We used data deposited in the NCBI to assemble the canine MYH9 gene DNA
sequence Characterization of the canine MYH9 gene revealed that the canine gene
consists of 41 exons spanning approximately 90 kb
We detected 22 single nucleotide polymorphisms (SNPs) in a mutation scan of
altogether 16 Dalmatian dogs from three families which showed significant linkage
between the deafness phenotype and the MYH9 gene-associated microsatellite
None of the SNPs affects the amino acid sequence of MYH9 We concluded that the
exons of the canine MYH9 gene do not play a role in Dalmatian CCSD Availability of
the microsatellite marker SNPs and DNA sequence reported in this study enhance
evaluation of the MYH9 gene as a CCSD candidate gene in other Dalmatian dog
populations and other dog breeds affected by CCSD
Introduction Myosin is a functional protein associated with cellular movement cell division muscle
contraction and other functions Members of the myosin super-family are
distinguished from the myosin heavy chains that play crucial roles in cellular
processes The human MYH9 gene consists of 40 exons and spans 67959 bp
Canine MYH9 gene
66
Toothaker et al (1991) mapped a nonmuscle myosin heavy chain gene to human
chromosome (HSA) 22q123-q131 Simons et al (1991) likewise mapped the gene
to HSA22q112 which they designated nonmuscle myosin heavy chain-A (NMMHC-
A)
The similarities between the autosomal dominant giant-platelet disorders May-
Hegglin anomaly Fechtner syndrome and Sebastian syndrome and refinement of the
disease loci for May-Hegglin anomaly and Fechtner syndrome to an overlapping
region of 480 kb on human chromosome 22 suggested that all these three disorders
may be allelic Among the identified candidate genes was the gene encoding
nonmuscle myosin heavy-chain A (MYH9) The May-HegglinFechtner Syndrome
Consortium (2000) demonstrated that mutations in MYH9 result in one of the three
disorders mentioned above The same Consortium also speculated that mutations in
MYH9 may also play a role in another autosomal dominant disorder a form of
nonsyndromic deafness characterized by progressive hearing impairment and
cochleosaccular degeneration This autosomal dominant form of human
nonsyndromic hereditary deafness (DFNA17) was described by Lalwani et al (1999)
They studied a five-generation American family previously reported by Lalwani et al
(1997) with deafness caused by cochleosaccular degeneration (CSD) CSD is the
most common histopathologic finding in cases of profound congenital deafness and
is estimated to occur in approximately 70 of cases in man
DFNA17 maps to the same region as MYH9 Because of the importance of myosins
in hearing Lalwani et al (2000) tested MYH9 as a candidate gene for DFNA17 and
demonstrated a missense mutation in the MYH9 gene in affected members of a
kindred with DFNA17 They found a G-to-A transition at nucleotide 2114 in the MYH9
gene This missense mutation changed codon 705 from an invariant arginine to a
histidine within a highly conserved Src homology 1 (SH1) linker region Previous
studies had shown that modification of amino acid residues within the SH1 helix
causes dysfunction of the ATPase activity of the motor domain in myosin
Many genetic disorders in humans and domestic dogs (Canis familiaris) demonstrate
a high level of clinical and molecular similarity Rak et al (2003) mapped 20 potential
candidate genes for sensorineural deafness in dogs by fluorescence in situ
hybridization and a radiation hybrid panel among them the MYH9 gene that was
assigned to canine chromosome (CFA) 10q232 Congenital sensorineural deafness
has been reported for approximately 54 different breeds of dogs (Strain 1996)
Canine MYH9 gene 67
The incidence of this congenital anomaly is highest in Dalmatian dogs of which 165-
30 exhibit unilateral or bilateral hearing loss (Famula et al 1996 Wood and
Lakhani 1997 Muhle et al 2002 Juraschko et al 2003a Rak and Distl 2005) The
inheritance and segregation of a major gene in CCSD has been demonstrated in
different Dalmatian dog populations (Famula et al 2000 Muhle et al 2002
Juraschko et al 2003b)
In dog breeds the histological pattern is known as cochleosaccular degeneration
commonly known as Scheibe dysplasia as it is described in approximately 70 of
cases with human hereditary deafness (Strain 1996) Therefore the MYH9 gene
seems to be a perfect candidate gene for CCSD especially in Dalmatian dogs
In this report we provide the genomic organization and the complete sequence of the
canine MYH9 gene A mutation analysis was performed to identify single nucleotide
polymorphisms (SNPs) in this gene In order to evaluate whether the MYH9 gene is
responsible for congenital sensorineural deafness in Dalmatian dogs we analyzed
the association of the MYH9 haplotypes with the CCSD phenotype in three families of
Dalmatian dogs with frequent occurrence of CCSD and significant linkage to the
gene-associated microsatellite MYH9_MS3 (Chapter 3)
Materials and Methods
Cloning and sequencing of canine MYH9 cDNA
The canine MYH9 gene sequence was derived from sequences deposited in the
current dog genome assembly (Boxer genome assembly 21) of the NCBI GenBank
(Genbank Acc No NW_1398701) by BLAST (Basic Local Alignment Search Tool)
search (httpwwwncbinlmnihgovBLAST) using the human MYH9 reference
mRNA sequence (Genbank Acc No NM_002473)
The distance between MYH9 and the microsatellite MYH9_MS2 is about 181 kb
whereas the microsatellite MYH9_MS3 (=FH2293) places about 511 kb from MYH9
The isolation of full length cDNAs was achieved by a modified rapid amplification of
cDNA ends (RACE) protocol Total RNA from dog lung of a normal female Beagle
(Biocat Heidelberg Germany) was used for the overlapping 5rsquo- and 3rsquo-RACE
products with the FirstChoiceTM RNA ligase-mediated (RLM)-RACE kit (Ambion
Europe Huntingdon UK) according to the protocols of the manufacturer Reverse
transcription polymerase chain reaction (RT-PCR) using two pairs of nested
Canine MYH9 gene
68
gene-specific and supplied 5rsquo-adaptor-specific primers were used to amplify the
complete 5rsquo-end of the investigated cDNA Similarly the 3rsquo-end were amplified using
two pairs of nested gene-specific primers in combination with supplied 3rsquo-adaptor-
specific primers Additionally RT-PCR using six pairs of gene-specific primers were
used to amplify the complete sequence of the investigated cDNAs
RACE products were cloned into pCRR21 vector using the Invitrogen TA cloning kit
(Invitrogen Karlsruhe Germany) Several clones for each cDNA were sequenced
with the ThermoSequenase kit (AmershamBiosciences Freiburg Germany) and a
LI-COR 4300 DNA Sequencer (LICOR inc Bad Homburg Germany) Sequence
data were analyzed with Sequencher 42 (GeneCodes Ann Arbor MI)
Sequence analysis of canine MYH9 gene For the localization of the exonintron boundaries after cloning and sequencing of full
length canine cDNAs (as described above) the mRNA-to-genomic alignment program
Spidey (httpwwwncbinlmnihgovIEBResearchOstellSpideyindexhtml) was
used Repetitive elements were analyzed with Repeatmasker 2
(httprepeatmaskergenomewashingtonedu) The GC content was calculated with
the EBI toolbox CpG PlotCpGreport (httpwwwebiacukToolssequencehtml)
Mutation analysis To identify polymorphisms within the canine MYH9 sequence exons with flanking
regions were PCR amplified and sequenced from 16 Dalmatian dogs which
represent three families consisting of three to six full sibs and at the best of both
parents At least two of the full sibs of each family were unilaterally deaf The
phenotype of the affected animals had been confirmed by brainstem auditory evoked
response (BAER) that detects electrical activity in the cochlea and auditory pathways
in the brain PCR primers and conditions for the amplification of MYH9 exons with
flanking sequences and microsatellite flanking primers for PCR reactions are given in
Table 1 PCR primers were developed with the Primer3 program
(httpfrodowimiteducgi-binprimer3primer3_wwwcgi) The PCR reactions for
exons and their flanking sequences were performed in a total of 50 microl containing 125
microM dNTPs 25 pmol of each primer the reaction buffer supplied by the manufacturer
(Qiagen Hilden Germany) and 1 U Taq polymerase After a 4 min initial
denaturation at 95 degC 35 cycles of 30 sec at 94 degC 45 sec at 58 degC and 80 sec
Canine MYH9 gene 69
at 72 degC were performed in a MJ Research thermocycler (Biozym Hessisch
Oldendorf Germany) The obtained PCR products were directly sequenced with the
DYEnamic ET Terminator kit (AmershamBiosciences Freiburg Germany) and a
MegaBACE 1000 capillary sequencer (AmershamBiosciences) using the PCR
primers as sequencing primers Sequence data were analyzed with Sequencher 42
(GeneCodes Ann Arbor MI)
Multipoint linkage and haplotype analysis were performed using MERLIN version
0102 (multipoint engine for rapid likelihood inference Center for Statistical Genetics
University of Michigan MI USA Abecasis et al 2002) The test statistics Z-mean
and Lod score were used to test for the proportion of alleles shared by affected
individuals identical by descent (IBD) for the considered marker loci
Linkage means that a haplotype characterized by microsatellites or SNPs is
significantly more often present in family members with the phenotype under study
than expected by random assortment For construction of haplotypes sets of closely
linked genetic markers on the same chromosome are needed which tend to be
inherited together as they are not likely to be separated by recombination
Association analysis can be carried out as a method of genetic analysis that
compares the frequency of alleles between affected and unaffected individuals
across all families A given allele is considered to be associated with the disease if
the presence of that allele explains a significant proportion of the phenotypic trait
variation
Results and Discussion
Analysis of the genomic organization and cDNA of the canine MYH9 gene
A full-length cDNA sequence of 7484 bp of the canine MYH9 gene was obtained by
using the RACE protocol The obtained RT-PCR products were sequenced and the
generated sequence data were submitted to the EMBL nucleotide database (Acc No
AM086385)
Using the RT-PCR analyses for cDNA-genomic sequence comparisons we detected
that the canine MYH9 gene consists of 41 exons with an untranslated exon 1 and
exonintron boundaries that conform perfectly to the GTAG rule (Table 2)
The 5rsquo-UTR consists of 181 bp while the 3rsquo-UTR consists of 1432 bp Assuming that
the homologous ATG start codon as in man is used the canine MYH9 cDNA
Canine MYH9 gene
70
contains an open reading frame of 5889 bp encoding a protein of 1963 amino acids
A canonical polyadenylation signal AAUAAA is located approximately 14 kb
downstream of the stop codon
The exon sizes range from 28 to 1556 bp the introns between these exons span
between 86 and 13493 bp the total size of the canine MYH9 gene is approximately
90 kb However the sequence homology between the human murine and canine
MYH9 gene is limited to the coding sequence of exon 2 to 41 The coding sequence
of canine MYH9 displays 926 and 899 similarities to the human and murine
MYH9 gene respectively In the untranslated regions the sequence similarity
between dog and human and mouse is rather low The canine MYH9 protein shows
982 and 968 identities to the orthologous human and murine protein
respectively (Fig 2)
The repeat content in the 90 kb region of the canine MYH9 gene is 1458 Most of
the repetitive elements belong to the SINE family (892) followed by the fraction of
the LINEs (306) other repetitive elements constitute 165 respectively The
entire canine MYH9 gene has an overall GC-content of 54 The canine MYH9 gene
contains 12 CpG islands (Gardiner-Garden and Frommer 1987) distributed over the
entire gene whereas by far the longest one with a length of 789 bp can be found in
the region of the second exon or in the first translated exon respectively (GC content
of 50 over 200 bp)
Mutation and haplotype analysis
All coding exons with flanking intronic regions of MYH9 could be amplified from the
examined 16 dogs and the sequences were compared to the Boxer genome
assembly 21 of the NCBI Gen Bank (Genbank Acc No NW_1398701)
The search for sequence variations within the MYH9 gene revealed a total of 22
SNPs shown in Table 3 Most of the polymorphisms were found in the flanking
regions of exons only 3 were within exons Only five out of the observed 22 SNPs
were polymorphic in all three examined families
None of the observed polymorphism did alter the predicted amino acid sequence of
MYH9 nor the identified hapltypes showed an association with the CCSD phenotype
Multipoint test statistics for MYH9 alleles identical by descent were close to zero (Z-
mean 035 to -035 Lod score 013 to -007) and not significant (p= 04 to 07)
However the microsatellite MYH9_MS3 (=FH2293) showed a Z-mean of 156
Canine MYH9 gene 71
(p= 006) and a Lod score of 058 (p= 005) indicating linkage The reason for this
result was heterogeneity among these three families For family 1 and 2 the test
statistics indicated linkage (Z-mean 191 with p= 003 Lod score 058 with p= 005)
whereas for familiy 3 the Z-mean and Lod score was -050 (p= 07) and -008 (p=
07)
Thus these silent point mutations found in affected and unaffected Dalmatian dogs
seem not responsible for the CCSD phenotype in these three families
Conclusions
The characterization of the transcript and genomic sequences of canine MYH9 gene
revealed a conserved organization with respect to the human orthologs In general
the gene size in dog is bigger compared to the human sequence due to the
untranlated first exon in the 5rsquo-UTR Several single nucleotide polymorphisms in the
canine MYH9 gene were identified However because of the fact that both animals
with unilateral hearing loss and bilateral hearing animals shared identical
haplotypes these polymorphisms are obviously not associated with CCSD in these
Dalmatian dog families Furthermore the present study revealed no functional
mutations of the complete coding region of MYH9 We can exclude the MYH9 gene
as a candidate gene for congenital sensorineunal deafness in Dalmatian dogs
However the MYH9 gene sequence SNPs and microsatellite markers reported in
this study enhance evaluation of the MYH9 gene in other Dalmatian dog populations
and dog breeds other than Dalmatians
Canine MYH9 gene
72
Table 1 PCR primers for the amplification of all MYH9 exons
Exon Primer Sequence (5rsquo ndash 3rsquo) Product
size (bp)
Annealing temperature
(degC) exon 1 MYH9_Ex1_F GCC TGG TTT GAC AAG TTT TG MYH9_Ex1_R TTC CTC AAG TCC CCT ACT CC 560 58 exon 2 MYH9_Ex2_F AGG ACC CTC ACC CAA CAC MYH9_Ex2_R GCA CAG AGT CTG GAC AAG AAT G 621 58 exon 3 MYH9_Ex3_F CAC CCC TCA ATA CTC AGT GC MYH9_Ex3_R GAG AAG ACT CCT GCC TCT CC 588 58 exon 4 MYH9_Ex4_F GGC AAT GTG TTT GAT TGT ACT G MYH9_Ex4_R TTT CCA AAG CAA CTC AGA AAG 542 58 exon 5 -6 MYH9_Ex56_F ACA GAG CAG GCG TGA CAG MYH9_Ex56_R GAG GAC AAG AGC TGG AAG C 724 58 exon 7 -8 MYH9_Ex78_F AAG CGG AAA GTT GCC TTC MYH9_Ex78_R ATG GCA CAA TGA CCA GTC TC 699 58 exon 9 MYH9_Ex9_F TGG GCA CAG TTT TTG TCT G MYH9_Ex9_R CAC ACA ATC AAG GGC TTT TC 590 58 exon 10 MYH9_Ex10_F GGA GCC AGT TGT CCT AGT AGT G MYH9_Ex10_R GCC AGG TGC CTA TCA CTG 540 58 exon 11 MYH9_Ex11_F TGT TTC GAA GCG TTT TTA CC MYH9_Ex11_R GGT CCC ATT GTT TCT AGC AC 551 58 exon 12 MYH9_Ex12_F TTT CCC AAG CAG CTA GAA AG MYH9_Ex12_R ACT GGG TGG TCT TGA GAC TG 586 58 exon 13 MYH9_Ex13_F CTG CCT TTG GTG ACA AGT G MYH9_Ex13_R GCA AGC CCT AAG TAC AAT GG 548 58 exon 14 MYH9_Ex14_F ATA CCA GGT CAA GGG GTG TC MYH9_Ex14_R TCA CCT GGT TCT AGG TGG AG 524 58 exon 15 MYH9_Ex15_F CGT TTG GAG TTG AT CCT AGC MYH9_Ex15_R AGC CAA GTT TCC TGA CTG TG 566 58 exon 16 MYH9_Ex16_F AGA TAG GCT GCA GTG GTA GC MYH9_Ex16_R CAG TAG CAA ACA GGG CTA GG 568 58 exon 17 MYH9_Ex17_F AAC GGT CCA ATG TGA GTT TC MYH9_Ex17_R ACC AGC TCA CAA CAG TAC CC 559 58 exon 18 MYH9_Ex18_F TGT AGG CTG TGA TTG CAG AG MYH9_Ex18_R CCC TCT CTC AAA CAA ATT GC 586 58 exon 19 MYH9_Ex19_F CCT TGG TGA AGG AAA GTG G MYH9_Ex19_R GCT GTG TGC AGG TTC TGA C 657 58 exon 20 MYH9_Ex20_F AGC AGC ATA GCA CTG AAT CC MYH9_Ex20_R TTC CCA TCT CTC ATG TTC TTG 579 58
Canine MYH9 gene 73
Table 1 (continued)
Exon Primer Sequence (5rsquo ndash 3rsquo) Product
size (bp)
Annealing temperature
(degC) exons 21 MYH9_Ex21_F CTC CTG AGT GTT GGG TTA GC MYH9_Ex21_R CTC TAA CAG GCA GCC CAA G 555 58 exon 22 MYH9_Ex22_F GGA GGA AAC GCA GAA ATC C MYH9_Ex22_R ACG GAC ACC CAG GAA AAC 660 58 exon 23 MYH9_Ex23_F CAG GGG TGC TAG TGA TCT TTC MYH9_Ex23_R AGA GCT GGG CTC AGA CCT AC 630 58 exon 24 MYH9_Ex24_F TCA GGG ATG TGC TTT CTT TC MYH9_Ex24_R CCC CAA GCA CAT CTG TAG TC 563 58 exon 24 MYH9_Ex24_F TCA GGG ATG TGC TTT CTT TC MYH9_Ex24_R CCC CAA GCA CAT CTG TAG TC 563 58 exon 25 -26 MYH9_Ex2526F GGC TGC TCT TCT CTG AAG C MYH9_Ex2526R GGA GTC AGG CTT CCA GTA AAG 851 58 exon 27 -28 MYH9_Ex2728F AGC ACA TTC TGG ATC TGG AG MYH9_Ex2728R GCG ACA GCT GAA TTC CTG 752 58 exon 29 MYH9_Ex29_F CAG CTT CAG GAC ACA CAG G MYH9_Ex29_R CTC TGG GTC TCC CCT GTC 601 58 exon 30 MYH9_Ex30_F GAG GCT TGG TGT ATG TCT CG MYH9_Ex30_R TGG TTC CAT AGA AAC GAT CTG 592 58 exon 31 - 32 MYH9_Ex3132F CAT TCC CCA AGG AGT TTT G MYH9_Ex3132R AAC ACA GCC CAG GGA AAG 770 58 exon 33 MYH9_Ex33_F CCT GGA ACA AGG CTG AGA AC MYH9_Ex33_R TCT CAG AGC AGC TTT GCT TC 600 58 exon 34 MYH9_Ex34_F GGT GTG CTT GGT AAA TCC TG MYH9_Ex34_R CAC AAA GCC TCA TTT CAC G 611 58 exon 34 - 36 MYH9_Ex3536F AGG TCA CGG TCT GGG TTC T MYH9_Ex3536R CAA GGG AGG CTG TTG CAG 401 60 exon 37 MYH9_Ex37_F CTC CCT TGG TTT CCC TCT CT MYH9_Ex37_R CAG GTC AGG AGG GGA CAT 488 60 exon 38 -39 MYH9_Ex3839F AGG CCT AAG GAC AGC TCA TC MYH9_Ex3839R CCA GGG CAC AGA TAG AAC C 545 58 exon 40 MYH9_Ex40_F1 GTC CCT CTA AAG ATG GCT TTG MYH9_Ex40_R1 TGG GGA GGA ACG ACT CTC 844 58 MYH9_Ex40_F2 TGG GTG ATT GGG GAA GAG MYH9_Ex40_R2 CCA GAA GAT GCA CAA AGT AGG 981 58
Canine MYH9 gene
74
Table 2 Exonintron boundaries of the canine MYH9 gene
3rsquo-Splice site Exon 5rsquo-Splice site
Intron phase
Intron size
-20 (exon 1 162 bp) GTTCAGgtgagcgagccccgc
gt30000 bp
-19 +333 gctgtagGTCCTGGCTGTGA (exon 2 352 bp) CGTAAgtggggcctggggcc
0
4922 bp
+334 +490 tctttagACCTATTCAGGCC (exon 3 157 bp) GCAAGgtgatgcctcggcctt
1
13493 bp
+491 +518 gtttcagACCGAGAGGACCA (exon 4 28 bp) TGCACgtaagtgtttctgcc
2
803 bp
+519 +612 tcggcagGGGTGAGTCTGGA (exon 5 94 bp) ACCAGgtgagtgcgggcctt
0
4077 bp
+613 +705 cttgtagGGTGAGTTGGAGA (exon 6 93 bp) GATTTgtgagtagcggccag
0
427 bp
+706 +769 tttttagGGCAAATTCATTC (exon 7 64 bp) GACTTgtatccttggttgat
1
738 bp
+770 +868 tgcaaagATCTTCTGGAGAA (exon 8 99 bp) GAAGAgtgagtagtgagccc
1
343 bp
+869 +1012 ctttcagCTGATCTCCTGTT (exon 9 144 bp) AATGGgtacagagctggccc
1
749 bp
+1013 +1108 cccgaagGCTTGTTGCGAGT (exon 10 96 bp) CACAGgtaacgcctgtggtcc
1
1041 bp
+1109 +1227 atcgcagCTGCCCAGAAAGT (exon 11 119 bp) AACAGgtaggctctctggcc
0
1877 bp
+1228 +1380 accccagGCTGACTTTGCCA (exon 12 153 bp) TTGATgtgagctcctctcct
0
922 bp
+1381 +1554 cctgcagCTGAACTCCTTCG (exon 13 174 bp) AGCCAgtaagagccagacct
0
1801 bp
+1555 +1728 ttcccagGCAGGGCCCCCTG (exon 14 174 bp) GCAAGgtgaggcaagcacgt
0
2049 bp
+1729 +1843 cttgcagGTGGACTACAAAG (exon 15 115 bp) GGATGgtacgtgccccctgg
1
1877 bp
+1844 +2037 tttgcagTGGACCGCATCAT (exon 16 194 bp) AGAAGgtgcgggtgctgggc
0
343 bp
+2038 +2159 tctccagGCTGGCAAACTGG (exon 17 122 bp) CAGAGgtgagcggggcctgt
1
835 bp
+2160 +2229 ccctcagATATGAGATCTTG (exon 18 70 bp) TCATGgtgagtagagctgct
2
1418 bp
+2230 +2390 cgtctagATCAAAGCCCTGG (exon 19 161 bp) AGGAAgtgagttcctaaagg
1
851 bp
+2391 +2499 tccccagGGCCTTTGCCAAG (exon 20 109 bp) CCAAGgtgggttctggaagg
2
1428 bp
+2500 +2631 cttctagGTCAAGCCACTGC (exon 21 132 bp) CCCAGgtaggtgtcagcagg
2
398 bp
+2632 +2838 ttggcagCTCATGGCAGAGA (exon 22 207 bp) TCCAAgtgagccccttccct
2
488 bp
Canine MYH9 gene 75
Table 2 (continued)
3rsquo-Splice site Exon 5rsquo-Splice site
Intron phase
Intron size
+2839 +2976 atggtagGAACTGGAGGAGC (exon 23 138 bp) CCAAGgtgaggcccgtggtg
2
971 bp
+2977 +3100 cccccagGAGAAGAAGCTTC (exon 24 124 bp) GGAAGgtaagagggtaacag
0
1603 bp
+3101 +3272 tcctcagAGCGCCTCCGAAG (exon 25 172 bp) GCCAGgtgagggcctcgctt
1
719 bp
+3273 +3485 ggcagagTGGAGGAGGAAGC (exon 26 213 bp) CTCAGgtgtgccatgcggcc
1
270 bp
+3486 +3630 tctccagGTCCAAACGCGAA (exon 27 145 bp) AACGGgtgcgtgggctcccc
2
480 bp
+3631 +3837 ccaccagGTGAAAGCCAACT (exon 28 207 bp) TGCAGgtgaggcctccatgg
2
232 bp
+3838 +3941 gccacagGTTGAGCTGGACA (exon 29 104 bp) ACACAgtaaaggtggcagcg
1
204 bp
+3942 +4094 ggtgtagGAGCTGCTCCAGG (exon 30 153 bp) CCCAGgttcttatcggcatg
1
1083 bp
+4095 +4343 tgcccagGTGACGGACATGA (exon 31 249 bp) ACCAGgtgtgggccccaggc
1
1298 bp
+4344 +4556 tccccagCTACTGGCCGAGG (exon 32 213 bp) AGAGCgtgagtaccatgtca
1
150 bp
+4557 +4769 gtggcagGTCCACGAGCTGG (exon 33 213 bp) GACAGgtgtggaggccctcc
1
303 bp
+4770 +4931 gccccagGTGCGGGAGATGG (exon 34 162 bp) TGCAGgtgaggtcggctggg
1
1173 bp
+4932 +5060 accccagGCCCAGATGAAGG (exon 35 129 bp) AGGAGgtgggccgggcctca
1
941 bp
+5061 +5149 atggcagGAACTGGCAGCTG (exon 36 89 bp) AAGGGgtgagccgtggcgtg
0
92 bp
+5150 +5273 cacccagAGCTCTGGCATTG (exon 37 124 bp) TGCAGgtgggcggggtcggg
2
224 bp
+5274 +5482 cccacagATCGACCAGATCA (exon 38 209 bp) ACCAAgtaggtgttcctgcc
1
563 bp
+5483 +5591 ctcgcagGGAGCGCCAGGCA (exon 39 109 bp) ACCAGgtgtgtgtgcacacc
2
86 bp
+5592 +5763 tctgcagGCCGACAAGGCAT (exon 40 172 bp) CTCAGgtgagccctgcctcc
0
739 bp
+5764 +7295 tgcaaagATCTTCTGGAGAA (exon 41 1532 bp) CGTCACAATAAAGAGAACCA
Exon sequences are shown in uppercase letters and intron sequences in lowercase
letters Untranslated regions are shown in italics The conserved GTAG exonintron
junctions are shown in boldface type For the last exon the polyadenylation signal is
shown underlined instead of an exonintron junction Position +1 corresponds to the
adenine of the translation initiation codon ATG
Canine MYH9 gene
76
16
TT
TC
G
A
TC
A
A C
A
CT
T
C
GC
G
G
AG
C
C
AG
G
A
CT
C
T
GA
A
G
CC
C
T
CC
T
C
96
96
229
23
7
15
TT
TC
G
A
TC
A
A C
A
CT
T
C
GC
G
G
AG
C
C
AG
G
A
CT
C
T
GA
A
G
CC
C
T
CC
T
C
96
96
229
22
9
14
deaf
T
T T
C
GA
T
C
AA
CA
C
T
TC
G
C
GG
A
G
CC
A
G
GA
C
T
CT
G
A
AG
C
C
CT
C
C
TC
96
96
22
9
229
13
deaf
T
T T
C
GA
T
C
AA
CA
C
T
TC
G
C
GG
A
G
CC
A
G
GA
C
T
CT
G
A
AG
C
C
CT
C
C
TC
96
96
22
9
229
12
deaf
T
T C
C
AA
CC
A
A A
A T
T C
C
CC
G
G
GG
C
C
GG
A
A C
C
CC
A
A G
G
CC
T
T C
C
CC
96
10
0 24
1
229
11
deaf
T
T C
C
AA
CC
A
A A
A T
T C
C
CC
G
G
GG
C
C
GG
A
A C
C
CC
A
A G
G
CC
T
T C
C
CC
96
10
0 24
1
229
10
TT
TC
G
A
TC
A
A C
A
CT
T
C
GC
G
G
AG
C
C
AG
G
A
CT
C
T
GA
A
G
CC
C
T
CC
T
C
96
96
237
22
9
Fam
ily 3
9 T
T C
C
AA
CC
A
A A
A T
T C
C
CC
G
G
GG
C
C
- - CC
C
C
AA
GG
C
C
TT
CC
C
C
96
100
229
24
1
8 T
T C
T
AG
C
T
AA
AA
TT
CC
C
C
GG
G
G
CT
G
G
AA
CC
C
C
AA
GG
C
C
TT
CC
C
C
96
96
225
25
3
7 de
af
TC
C
C
AG
C
T
AT
A
A T
T C
C
CC
G
A
GG
C
T
GG
A
G
CC
C
C
AA
GG
C
T
TC
C
T
CC
96
89
22
1
237
6 de
af
TC
C
C
AG
C
T
AT
A
A T
T C
C
CC
G
A
GG
C
T
GG
A
G
CC
C
C
AA
GG
C
T
TC
C
T
CC
96
89
22
1
237 Fa
mily
2
5 T
C
CC
A
G
CT
A
T
AA
TT
CC
C
C
GA
G
G
CT
G
G
AG
C
C
CC
A
A G
G
CT
T
C
CT
C
C
96
89
221
23
7
4 de
af
TT
CC
A
A C
C
AA
AA
TT
CC
C
C
GG
G
G
CC
G
G
AG
C
C
CC
A
A G
G
CC
T
T C
C
CC
96
96
23
7
257
3 de
af
TC
C
C
AG
C
T
AT
A
A T
T C
C
CC
G
A
GG
C
T
GG
G
A
CC
C
C
AA
GG
C
T
TC
C
T
CC
96
96
23
7
257
2 T
C
CC
A
G
CT
A
T
AA
TT
CC
C
C
GA
G
G
CT
G
G
AA
CC
C
C
AA
GG
C
T
TC
C
T
CC
96
96
23
7
257 Fa
mily
1
1 C
T
CC
G
A
TC
T
A A
A T
T C
C
CC
A
G
GG
T
C
GG
A
G
CC
C
C
AA
GG
T
C
CT
T
C
CC
96
96
25
7
257
Ref
eren
ce
se
quen
ce
NW
_139
870
T C
A T A A T C
C
G
G
C
G
A C
C
A G
C
T C
C
posi
tion1
212
9
1 4
4 2
39
125
1
32
316
3
78
42
218
1
8 1
75
24
13
8
207
2
63
287
9
22
40
59
64
Pos
ition
with
in M
YH
9 In
tron
34
Exo
n 4
Intro
n 14
15
Intro
n 15
16
Intro
n 16
17
Intro
n 18
19
Intro
n 20
21
Intro
n 20
21
Intro
n 25
26
Intro
n 25
26
Intro
n 26
27
Intro
n 27
28
Exo
n 29
In
tron
293
0 In
tron
303
1 E
xon
32
Intro
n 32
33
Intro
n 32
33
Intro
n 34
35
Intro
n 38
39
Intro
n 38
39
Intro
n 38
39
Mic
rosa
tellit
e M
YH
9_M
S2
Mic
rosa
tellit
e M
YH
9_M
S3
(=FH
2293
)
1 N
umbe
ring
refe
rs to
the
posi
tion
of th
e po
lym
orph
ic n
ucle
otid
e w
ithin
the
give
n ex
on
or in
tron
Tabl
e 3
Hap
loty
pes
of th
e ca
nine
MY
H9
gene
in16
Dal
mat
ian
dogs
Canine MYH9 gene 77
Figure 1 Family structure of the analyzed Dalmatian dog families Hearing status was tested by brain auditory evoked response (BAER)
Canine MYH9 gene
78
Figure 2 Alignment of the canine MYH9 protein with orthologous human and murine
MYH9 protein sequences The sequences where derived from GenBank entries with
the accessions NP_002464 (human MYH9) and NP_071855 (mouse MYH9)
Identical residues are indicated by asterisk beneath the alignment while dots and
colons represent similar or very similar amino acids respectively
dog MAQQAADKYLYVDKNFINNPLAQADWAAKKLVWVPSDKSGFEPASLKEEVGEEAIVELVE 60 human MAQQAADKYLYVDKNFINNPLAQADWAAKKLVWVPSDKSGFEPASLKEEVGEEAIVELVE 60 mouse MAQQAADKYLYVDKNFINNPLAQADWAAKKLVWVPSSKNGFEPASLKEEVGEEAIVELVE 60 dog NGKKVKVNKDDIQKMNPPKFSKVEDMAELTCLNEASVLHNLKERYYSGLIYTYSGLFCVV 120 human NGKKVKVNKDDIQKMNPPKFSKVEDMAELTCLNEASVLHNLKERYYSGLIYTYSGLFCVV 120 mouse NGKKVKVNKDDIQKMNPPKFSKVEDMAELTCLNEASVLHNLKERYYSGLIYTYSGLFCVV 120 dog INPYKNLPIYSEEIVEMYKGKKRHEMPPHIYAITDTAYRSMMQDREDQSILCTGESGAGK 180 human INPYKNLPIYSEEIVEMYKGKKRHEMPPHIYAITDTAYRSMMQDREDQSILCTGESGAGK 180 mouse INPYKNLPIYSEEIVEMYKGKKRHEMPPHIYAITDTAYRSMMQDREDQSILCTGESGAGK 180 dog TENTKKVIQYLAHVASSHKSKKDQGELERQLLQANPILEAFGNAKTVKNDNSSRFGKFIR 240 human TENTKKVIQYLAYVASSHKSKKDQGELERQLLQANPILEAFGNAKTVKNDNSSRFGKFIR 240 mouse TENTKKVIQYLAHVASSHKSKKDQGELERQLLQANPILEAFGNAKTVKNDNSSRFGKFIR 240 dog INFDVNGYIVGANIETYLLEKSRAIRQAKEERTFHIFYYLLSGAGEHLKTDLLLEPYNKY 300 human INFDVNGYIVGANIETYLLEKSRAIRQAKEERTFHIFYYLLSGAGEHLKTDLLLEPYNKY 300 mouse INFDVNGYIVGANIETYLLEKSRAIRQAKEERTFHIFYYLLSGAGEHLKTDLLLEPYNKY 300 dog RFLSNGHVTIPGQQDKDMFQETMEAMRIMGIPEEEQMGLLRVISGVLQLGNIVFKKERNT 360 human RFLSNGHVTIPGQQDKDMFQETMEAMRIMGIPEEEQMGLLRVISGVLQLGNIVFKKERNT 360 mouse RFLSNGHVTIPGQQDKDMFQETMEAMRIMGIPEDEQMGLLRVISGVLQLGNIAFKKERNT 360 dog DQASMPDNTGNASAQKVSHLLGINVTDFTRGILTPRIKVGRDYVQKAQTKEQADFAIEAL 420 human DQASMPDNT---AAQKVSHLLGINVTDFTRGILTPRIKVGRDYVQKAQTKEQADFAIEAL 417 mouse DQASMPDNT---AAQKVSHLLGINVTDFTRGILTPRIKVGRDYVQKAQTKEQADFAIEAL 417 dog AKATYERMFRWLVLRINKALDKTKRQGASFIGILDIAGFEIFDLNSFEQLCINYTNEKLQ 480 human AKATYERMFRWLVLRINKALDKTKRQGASFIGILDIAGFEIFDLNSFEQLCINYTNEKLQ 477 mouse AKATYERMFRWLVLRINKALDKTKRQGASFIGILDIAGFEIFDLNSFEQLCINYTNEKLQ 477 dog QLFNHTMFILEQEEYQREGIEWNFIDFGLDLQPCIDLIEKPAGPPGILALLDEECWFPKA 540 human QLFNHTMFILEQEEYQREGIEWNFIDFGLDLQPCIDLIEKPAGPPGILALLDEECWFPKA 537 mouse QLFNHTMFILEQEEYQREGIEWNFIDFGLDLQPCIDLIEKPAGPPGILALLDEECWFPKA 537 dog TDKSFVEKVVQEQGTHPKFQKPKQLKDKADFCIIHYAGKVDYKADEWLMKNMDPLNDNIA 600 human TDKSFVEKVMQEQGTHPKFQKPKQLKDKADFCIIHYAGKVDYKADEWLMKNMDPLNDNIA 597 mouse TDKSFVEKVVQEQGTHPKFQKPKQLKDKADFCIIHYAGKVDYKADEWLMKNMDPLNDNIA 597
Canine MYH9 gene 79
dog TLLHQSSDKFVSELWKDVDRIIGLDQVAGMSETALPGAFKTRKGMFRTVGQLYKEQLAKL 660 human TLLHQSSDKFVSELWKDVDRIIGLDQVAGMSETALPGAFKTRKGMFRTVGQLYKEQLAKL 657 mouse TLLHQSSDKFVSELWKDVDRIIGLDQVAGMSETALPGAFKTRKGMFRTVGQLYKEQLAKL 657 dog MATLRNTNPNFVRCIIPNHEKKAGKLDPHLVLDQLRCNGVLEGIRICRQGFPNRVVFQEF 720 human MATLRNTNPNFVRCIIPNHEKKAGKLDPHLVLDQLRCNGVLEGIRICRQGFPNRVVFQEF 717 mouse MATLRNTNPNFVRCIIPNHEKKAGKLDPHLVLDQLRCNGVLEGIRICRQGFPNRVVFQEF 717 dog RQRYEILTPNSIPKGFMDGKQACVLMIKALELDSNLYRIGQSKVFFRAGVLAHLEEERDL 780 human RQRYEILTPNSIPKGFMDGKQACVLMIKALELDSNLYRIGQSKVFFRAGVLAHLEEERDL 777 mouse RQRYEILTPNSIPKGFMDGKQACVLMIKALELDSNLYRIGQSKVFFRAGVLAHLEEERDL 777 dog KITDVIIGFQACCRGYLARKAFAKRQQQLTAMKVLQRNCAAYLKLRNWQWWRLFTKVKPL 840 human KITDVIIGFQACCRGYLARKAFAKRQQQLTAMKVLQRNCAAYLKLRNWQWWRLFTKVKPL 837 mouse KITDVIIGFQACCRGYLARKAFAKRQQQLTAMKVLQRNCAAYLRLRNWQWWRLFTKVKPL 837 dog LQVSRQEEEMMAKEEELVKVREKQLAAENRLTEMETLQSQLMAEKLQLQEQLQAETELCA 900 human LQVSRQEEEMMAKEEELVKVREKQLAAENRLTEMETLQSQLMAEKLQLQEQLQAETELCA 897 mouse LNSIRHEDELLAKEAELTKVREKHLAAENRLTEMETMQSQLMAEKLQLQEQLQAETELCA 897 dog EAEELRARLTAKKQELEEICHDLEARVEEEEERCQHLQAEKKKMQQNIQELEEQLEEEES 960 human EAEELRARLTAKKQELEEICHDLEARVEEEEERCQHLQAEKKKMQQNIQELEEQLEEEES 957 mouse EAEELRARLTAKKQELEEICHDLEARVEEEEERCQYLQAEKKKMQQNIQELEEQLEEEES 957 dog ARQKLQLEKVTTEAKLKKLEEDQIIMEDQNCKLAKEKKLLEDRIAEFTTNLMEEEEKSKS 1020 human ARQKLQLEKVTTEAKLKKLEEEQIILEDQNCKLAKEKKLLEDRIAEFTTNLTEEEEKSKS 1017 mouse ARQKLQLEKVTTEAKLKKLEEDQIIMEDQNCKLAKEKKLLEDRVAEFTTNLMEEEEKSKS 1017 dog LAKLKNKHEAMITDLEERLRREEKQRQELEKTRRKLEGDSTDLNDQIAELQAQIAELKMQ 1080 human LAKLKNKHEAMITDLEERLRREEKQRQELEKTRRKLEGDSTDLSDQIAELQAQIAELKMQ 1077 mouse LAKLKNKHEAMITDLEERLRREEKQRQELEKTRRKLEGDSTDLSDQIAELQAQIAELKMQ 1077 dog LAKKEEELQAALARVEEEATQKNMALKKIRELESQISELQEDLESERASRNKAEKQKRDL 1140 human LAKKEEELQAALARVEEEAAQKNMALKKIRELESQISELQEDLESERASRNKAEKQKRDL 1137 mouse LAKKEEELQAALARVEEEAAQKNMALKKIRELETQISELQEDLESERASRNKAEKQKRDL 1137 dog GEELEALKTELEDTLDSTAAQQELRSKREQEVNILKKTLEEEARTHEAQIQEMRQKHSQA 1200 human GEELEALKTELEDTLDSTAAQQELRSKREQEVNILKKTLEEEAKTHEAQIQEMRQKHSQA 1197 mouse GEELEALKTELEDTLDSTAAQQELRSKREQEVSILKKTLEDEAKTHEAQIQEMRQKHSQA 1197 dog VEELAEQLEQTKRVKANLEKAKQTLENERGELANEVKVLQQGKGDSEHKRKKAEAQLQEL 1260 human VEELAEQLEQTKRVKANLEKAKQTLENERGELANEVKVLLQGKGDSEHKRKKVEAQLQEL 1257 mouse VEELADQLEQTKRVKATLEKAKQTLENERGELANEVKALLQGKGDSEHKRKKVEAQLQEL 1257 dog QVKFTEGERVRTELADKVTKLQVELDNVMGLLTQSDSKSSKLTKDFSALESQLQDTQELL 1320 human QVKFNEGERVRTELADKVTKLQVELDNVTGLLSQSDSKSSKLTKDFSALESQLQDTQELL 1317 mouse QVKFSEGERVRTELADKVTKLQVELDSVTGLLSQSDSKSSKLTKDFSALESQLQDTQELL 1317
Canine MYH9 gene
80
dog QEENRQKLSLSTKLKQMEDEKNSFKEQLEEEEEAKRNLEKQIATLHAQVTDMKKKMEDGV 1380 human QEENRQKLSLSTKLKQVEDEKNSFREQLEEEEEAKHNLEKQIATLHAQVADMKKKMEDSV 1377 mouse QEENRQKLSLSTKLKQMEDEKNSFREQLEEEEEAKRNLEKQIATLHAQVTDMKKKMEDGV 1377 dog GCLETAEEAKRKLQKDLEGLGQRYEEKVAAYDKLEKTKTRLQQELDDLLVDLDHQRQTAS 1440 human GCLETAEEVKRKLQKDLEGLSQRHEEKVAAYDKLEKTKTRLQQELDDLLVDLDHQRQSAC 1437 mouse GCLETAEEAKRRLQKDLEGLSQRLEEKVAAYDKLEKTKTRLQQELDDLLVDLDHQRQSVS 1437 dog NLEKKQKKFDQLLAEEKTISAKYAEERDRAEAEAREKETKALSLARALEEAMEQKAELER 1500 human NLEKKQKKFDQLLAEEKTISAKYAEERDRAEAEAREKETKALSLARALEEAMEQKAELER 1497 mouse NLEKKQKKFDQLLAEEKTISAKYAEERDRAEAEAREKETKALSLARALEEAMEQKAELER 1497 dog LNKQFRTEMEDLMSSKDDVGKSVHELEKSKRALEQQVEEMKTQLEELEDELQATEDAKLR 1560 human LNKQFRTEMEDLMSSKDDVGKSVHELEKSKRALEQQVEEMKTQLEELEDELQATEDAKLR 1557 mouse LNKQFRTEMEDLMSSKDDVGKSVHELEKSKRALEQQVEEMKTQLEELEDELQATEDAKLR 1557 dog LEVNLQAMKAQFERDLQGRDEQSEEKKKQLVRQVREMEAELEDEKKQRSMAVAARKKLEM 1620 human LEVNLQAMKAQFERDLQGRDEQSEEKKKQLVRQVREMEAELEDERKQRSMAVAARKKLEM 1617 mouse LEVNLQAMKAQFERDLQGRDEQSEEKKKQLVRQVREMEAELEDERKQRSMAMAARKKLEM 1617 dog DLKDLEAHIDSANKNRDEAIKQLRKLQAQMKDCVRELDDTRASREEILAQAKENEKKMKS 1680 human DLKDLEAHIDSANKNRDEAIKQLRKLQAQMKDCMRELDDTRASREEILAQAKENEKKLKS 1677 mouse DLKDLEAHIDTANKNREEAIKQLRKLQAQMKDCMRELDDTRASREEILAQAKENEKKLKS 1677 dog MEAEMIQLQEELAAAERAKRQAQQERDELADEIANSSGKGALALEEKRRLEARIAQLEEE 1740 human MEAEMIQLQEELAAAERAKRQAQQERDELADEIANSSGKGALALEEKRRLEARIAQLEEE 1737 mouse MEAEMIQLQEELAAAERAKRQAQQERDELADEIANSSGKGALALEEKRRLEARIALLEEE 1737 dog LEEEQGNTELVNDRLKKANLQIDQINTDLNLERSHAQKNENARQQLERQNKELKVKLQEM 1800 human LEEEQGNTELINDRLKKANLQIDQINTDLNLERSHAQKNENARQQLERQNKELKVKLQEM 1797 mouse LEEEQGNTELINDRLKKANLQIDQINTDLNLERSHAQKNENARQQLERQNKELKAKLQEM 1797 dog EGTVKSKYKASITALEAKIAQLEEQLDNETKERQAACKQVRRAEKKLKDVLLQVDDERRN 1860 human EGTVKSKYKASITALEAKIAQLEEQLDNETKERQAACKQVRRTEKKLKDVLLQVDDERRN 1857 mouse ESAVKSKYKASIAALEAKIAQLEEQLDNETKERQAASKQVRRTEKKLKDVLLQVEDERRN 1857 dog AEQFKDQADKASTRLKQLKRQLEEAEEEAQRANASRRKLQRELEDATETADAMNREVSSL 1920 human AEQYKDQADKASTRLKQLKRQLEEAEEEAQRANASRRKLQRELEDATETADAMNREVSSL 1917 mouse AEQFKDQADKASTRLKQLKRQLEEAEEEAQRANASRRKLQRELEDATETADAMNREVSSL 1917 dog KNKLRRGDLPFVVPRRVARKGAGDCSDEEVDGKADGAEAKAAE 1963 human KNKLRRGDLPFVVPRRMARKGAGDGSDEEVDGKADGAEAKPAE 1960 mouse KNKLRRGDLPFVVTRRIVRKGTGDCSDEEVDGKADGADAKAAE 1960
Chapter 6
Identification of a 5 Mb region on canine chromosome 10
harbouring a causative gene responsible for congenital sensorineural deafness
in German Dalmatian dogs
Fine mapping of CFA10 83
Identification of a 5 Mb region on canine chromosome 10 harbouring a causative gene responsible for congenital sensorineural deafness in German Dalmatian dogs
Abstract
In the present study we evaluated whether the canine chromosome (CFA) 10
harbours a gene responsible for canine congenital sensorineural deafness (CCSD) in
Dalmatian dogs Altogether 27 microsatellite markers covering the whole CFA10
were genotyped in 176 Dalmatian dogs with frequent occurrence of CCSD
Significant linkage between the deafness phenotype and microsatellites located in a
region of 36 Mb to 48 Mb on CFA10 was found In order to refine the location of the
causative canine congenital sensorineural deafness (CCSD) gene we used data
deposited in the NCBI to search for single nucleotide polymorphisms (SNPs) in the
intronic sequences of the canine genes located on CFA10 in this region We
characterized 26 SNPs and used them for non-parametric linkage and association
analyses with CCSD of altogether 34 Dalmatian dogs from five full-sib families We
could find significant linkage to SNPs located in the region spanning 39 Mb to 44 Mb
and significant haplotype-trait association for SNPs in this region These results
enforce further evaluation of this 5 Mb region with the aim to detect the gene
responsible for CCSD in Dalmatian dogs
Introduction
Of the identified genes responsible for different forms of sensorineural non-syndromic
deafness in humans Rak et al (2003) and co-workers (Droumlgemuumlller et al 2002
Kuiper et al 2002 Rak et al 2002a 2002b 2003) considered 24 genes as
candidates for sensorineural deafness in dogs among them the MYH9 gene on
CFA10 Subsequently Rak (2003) developed a set of microsatellite markers for the
respective 24 candidate genes
As described in chapter 3 we could show significant linkage of CCSD with the MYH9
associated microsaellite MYH9_MS3 (=FH2293) in different German Dalmatian dog
Fine mapping of CFA10
84
families However as described in Chapter 5 we already excluded MYH9 for being
responsible for the CCSD phenotype in German Dalmatian dog families segregating
for CCSD by comparative sequencing of genomic sequences from deaf and normal
hearing Dalmatian dogs and additionally by sequencing of full length canine cDNA
The reason for the MYH9_MS3 (=FH2293) indicating linkage may be caused by a
closely linked gene involved in CCSD Thus the objective of the present study was to
perform a scan of canine chromosome 10 using microsatellite markers and single
nucleotide polymorphisms (SNPs) in order to evaluate whether CFA10 harbours a
gene responsible for the deafness phenotype in Dalmatian dogs Additionally we
analyzed the association of the CCSD phenotype with a large number of newly
developed SNPs located in the genomic deafness region on CFA10
Material and methods Pedigree material
For the linkage analysis of the microsatellite marker on CFA10 we used DNA from
altogether 176 animals belonging to 22 full-sib families and one large paternal half-
sib family of German Dalmatian dogs All families were segregating for CCSD The
genotyped families included all the affected dogs (unilaterally and bilaterally deaf)
their parents if available and one to four unaffected full-sibs At least two of the full
sibs of each family were unilaterally deaf The phenotype of the affected animals had
been confirmed by brainstem auditory evoked response (BAER) that detects
electrical activity in the cochlea and auditory pathways in the brain
Screening for SNPs was performed by comparative sequencing of DNA from parents
of five families with significant linkage of microsatellites located in the region
spanning 36 Mb to 48 Mb to CCSD For the linkage analysis concerning the SNPs we
then used blood samples from 34 Dalmatian dogs consisting of the progeny and their
parents of the abovementioned five full-sib families of Dalmatian dogs with frequent
occurrence of CCSD The families consisted of five to nine individuals and their
parents
Microsatellite marker analysis
We used 27 microsatellite marker derived from the NCBI database
(httpwebncbinlmnihgov) to cover CFA10 completely (Appendix Table 1)
Fine mapping of CFA10 85
PCR reactions for microsatellites were carried out in 12 microl reaction mixtures
containing 2 microl genomic DNA 12 microl 10x PCR buffer (Qbiogene Heidelberg
Germany) 024 microl dimethyl sulfoxide (DMSO) 02 microl dNTPs (100 microM) 01 microl Taq
Polymerase (5Umicrol) (Qbiogene) 06 microl (10 microM) 5rsquo-IRD700 or IRD800 labelled forward
primer 06 microl (10 microM) unlabelled reverse primer The PCR reactions were carried out
in MJ Research thermocyclers with the following program 4 min at 94 degC followed
by 32 cycles of 30 sec at 94degC 30 sec at maximum annealing temperature and a
final extension of 45 sec at 72degC PCR-products were diluted with formamide loading
buffer in ratios from 110 to 140 and then size-fractionated by gel electrophoresis on
automated LI-COR 42004300 sequencers (LI-COR inc Lincoln NE USA) using
denaturing 4 and 6 polyacrylamide gels (RotiphoreseregGel 40 Roth Karlsruhe
Germany)
Development of single nucleotide polymorphisms (SNPs)
We developed SNPs for the region between 36 Mb to 48 Mb on CFA10 previously
proven to be linked to the CCSD phenotype The SNPs for this region were derived
from one 5294 Mb large whole genome shotgun sequence (NW_876251) deposited
in the current dog genome assembly (Boxer genome assembly 21) of the NCBI
GenBank The canine genomic sequences and mRNA of the genes that were used
for the analysis were also derived from sequences deposited in the current dog
genome assembly (Boxer genome assembly 21) of the NCBI GenBank (Table 1)
In total 72 primer pairs were designed most of them located intragenic in intronic
sequences or alternatively in flanking regions of the 5rsquo- or 3rsquondashends of the respective
gene yielding products with a length of around 600 bp PCR primers were developed
with the Primer3 program (httpfrodowimiteducgi-binprimer3primer3_wwwcgi)
The PCR reactions were performed in a total of 50 microl containing 125 microM dNTPs 25
pmol of each primer the reaction buffer supplied by the manufacturer (Qiagen
Hilden Germany) and 1 U Taq polymerase After a 4 min initial denaturation at
95degC 35 cycles of 30 sec at 94degC 45 sec at 58degC and 80 sec at 72degC were
performed in a MJ Research thermocycler (Biozym Hessisch Oldendorf Germany)
The obtained PCR products were directly sequenced with the DYEnamic ET
Terminator kit (AmershamBiosciences Freiburg Germany) and a MegaBACE 1000
capillary sequencer (AmershamBiosciences) using the PCR primers as sequencing
primers Sequence data were analyzed with Sequencher 42
Fine mapping of CFA10
86
(GeneCodes Ann Arbor MI USA) If a heterozygous SNP was found for one or both
parents all progeny of the respective families were analyzed for that SNP
We developed altogether 26 SNPs with 20 SNPs located intragenic in intronic
sequences and 6 SNPs located very closely at the 5rsquo- or 3rsquo-ends respectively with
one to four SNPs per gene (Table 3)
The newly identified 26 SNPs are shown in Tabele 2 Only SNPs are listed and
chosen for linkage analyses that were heterozygous for one or both parents of at
least two of the five families Of all SNPs only one was heterozygous in all families
(Table 3) The most frequent form of SNPs with a frequency of 423 was the AG
transition motif The scarcest one with a frequency of 385 was the CG and the
AC transversion motif respectively
Linkage analysis
Multipoint non-parametric linkage and haplotype analysis were performed using the
MERLIN software version 0102 (multipoint engine for rapid likelihood inference
Center for Statistical Genetics University of Michigan MI USA Abecasis et al
2002) The test statistics Z-mean and Lod score were used to test for the proportion
of alleles shared by affected individuals identical by descent (IBD) for the considered
marker loci
In a first step linkage analysis was performed regarding the 27 marker covering the
whole CFA10 In a second step a linkage analysis was performed including the newly
added SNPs spanning the region 36 Mb to 48 Mb on CFA10
The observed heterozygosity (HET) and the polymorphism information content (PIC)
were calculated using the software package SASGenetics (Statistical Analysis
System Version 913 SAS Institute Inc Cary NC USA 2005)
Linkage disequilibrium (LD) and association of haplotypes with CCSD were tested
using the procedures CASECONTROL and HAPLOTYPE of SASGenetics
(Statistical Analysis System version 913 Cary NC USA)
Results
A linkage analysis was at first carried out for the 176 animals that were analyzed with
27 microsatellite markers covering the whole CFA10
Fine mapping of CFA10 87
The microsatellite FH3302 positioned at 3887 Mb showed the highest Zmean with a
value of 234 (p= 001) and a Lod score of 121 (p= 0009) at 3887 Mb A Zmean
value of 217 (p= 0015) and a Lod score of 12 (p= 0009) was reached for the
microsatellite REN181G20 positioned at 3936 Mb indicating linkage but all Zmean
values and error probabilities of eight microsatellite markers in the interval from 3666
Mb up to 488 Mb were almost as high Consequently we screened the region
spanning from 36 Mb to 48 Mb for SNPs Out of the 23 analyzed Dalmatian dog
families five full-sib families were chosen to screen for SNPs because of their
significant contribution to the test statistics
Twenty-six new SNP markers covering the region of 36 Mb to 48 Mb equally were
developed and added to the linkage analysis These SNPs had PIC values ranging
from 013 to 037
Figure 1 shows the Zmeans and LOD scores for all 27 microsatellite markers on
CFA10 harbouring a genomic deafness region Figure 2 shows the corresponding p-
values Adding the SNPs in the interval spanning 36 Mb to 48 Mb we could narrow
the significant region down to 5 Mb spanning 39 Mb to 44 Mb (Table 4 Figures 3 and
4)
Haplotype-trait association test statistics for the SNPs 16-23 located in the interval
from 39 Mb to 44 Mb were significant However the marker-trait association test
failed the 5 threshold of the error probability (p= 007) but was lowest for all
possible haplotype-trait combinations The χ2ndashtests of the procedure
CASECONTROL were not significant indicating that a SNP for the causative
deafness gene was not yet found
Discussion
We used 27 microsatellite marker and 26 newly developed SNP markers with the
intention to create a dense map for linkage analysis of CFA10 especially the region
spanning 36 Mb to 48 Mb proven to be linked to the CCSD phenotype All SNP
markers were chosen due to their heterozygosity in one or both parents of at least
two families
The significant Zmeans on CFA10 reported for the chromosome scan using only
microsatellites was confirmed by adding the SNP markers Furthermore with the use
of SNPs the apparently linked region from 36 Mb to 48 Mb could be narrowed down
Fine mapping of CFA10
88
to 5 Mb spanning 39 Mb to 44 Mb For this reason the use of SNPs in addition to
informative microsatellites appears highly recommendable
The identified CCSD region spanning 5 Mb might be participating in the development
of CCSD in the analyzed Dalmatian dog families
However genes that are known to be involved in the hearing process or known to be
expressed in the inner ear are not annotated at the syntenic human region on homo
sapiens autosome (HSA) 2 spanning from 100 Mb to 108 Mb Therefore a well-
defined candidate gene for canine hereditary deafness in the abovementioned 5 Mb
does not exist This means that for all genes in this region informative SNPs have to
be developed and tested for linkage disequilibrium and association with CCSD
Despite the remarkable progress that has been made in the past few years in
identifying deafness genes in man and mouse it is suspected that only one-third of all
deafness causing genes have been identified so far Therefore more SNPs have to
be developed within the identified region on CFA10 to localize the deafness causing
gene or to find unambiguously associated SNP markers which could be used for a
population-wide genetic test for CCSD
Fine mapping of CFA10 89
Table 1 Canine genes where gene-associated SNPs could be developed with their
exact location on CFA10 and their corresponding accession numbers (Acc No)
Gene symbol Gene description Position in
Mb
Acc No canine genomic sequence
Acc No canine mRNA
LOC474535 similar to hypothetical protein MGC40397 36493650 NC_006592 XM_531764
LOC474536 similar to KM-102-derived reductase-like factor
37023714 NC_006592 XM_531765
LOC610953 similar to NHP2-like protein 1 37293729 NC_006592 XM_848546
LOC481302 similar to C2orf26 protein 37363737 NC_006592 XM_848552
LOC610991 hypothetical protein LOC610991 38103820 NC_006592 XM_848591
LOC611007 similar to eukaryotic translation elongation factor 1 alpha 2
38353835 NC_006592 XM_848614
LOC474541 similar to GRIP coiled-coil protein GCC185 isoform a
38503857 NC_006592 XM_531770
LOC474542 similar to Sulfotransferase 1C2 38613862 NC_006592 XM_531771
LOC481308 similar to Keratin type I cytoskeletal 18 38653865 NC_006592 XM_538429
LOC474543 similar to Sulfotransferase K1 38683870 NC_006592 XM_857994
LOC609217 similar to family with sequence similarity 32 member A like
39453945 NC_006592 XM_858065
LOC611115 similar to D-PCa-2 protein isoform c 39843984 NC_006592 XM_848756
LOC481325 similar to ubiquitin-conjugating enzyme E2C
42564272 NC_006592 XM_538446
LOC481330 similar to Interleukin-1 receptor type II precursor
44084413 NC_006592 XM_538451
LOC611493 hypothetical protein LOC611493 44384440 NC_006592 XM_863243
LOC481337 similar to DNA repair protein REV1
46264637 NC_006592 XM_538458
LOC611728 similar to 3-hydroxyanthranilate 34-dioxygenase
48494852 NC_006592 XM_849433
Predicted gene derived from the dog genome assembly (build 21) that used gene
prediction method GNOMON supported by EST evidence
Fine mapping of CFA10
90
Table 2 The 26 newly developed SNPs for Dalmatian dogs located in the region
spanning 36 Mb to 48 Mb on CFA10 with their corresponding primers the SNP motif
the product size and the annealing temperature
Gene
description SNP
Location
(intron or
5rsquo3rsquo-end)
Primer F1 (5acute -gt 3acute)
SNP motif
Primer R2 (5acute -gt 3acute)
Bp3 AT4
LOC474535
SNP_1
intron
ACCCAAGCCTAACTGCAGAA
ACCCCAGTCT(CG)GCCAGAGCTGTT
590 60
SNP_2 intron TGTGTGCATA(AG)TGTGCTGAGCCT
TCATCTGTTAAAACAGGGGTGAT
LOC474536
SNP_3
intron
CCAGTTAATGATTGTTTCGTTGA
AAGCTGCTTT(AC)CACCCCCATCAG
TCATTCCTGCTGTTGTGCTC
590 60
LOC610953
SNP_4
intron
CTGTCTTGGGGACTGTTTGC
AAGGCAGACG(CT)AATGACTGAGGC
600 60
SNP_5 intron GACTTGAAGC(CT)ACCACAGATACT
GCCATCACGATGAACTCAGA
LOC481302
SNP_6
3rsquo-end
AATTGAGGCCGAAGTCCAAT
CTTTTCCCCA(GT)GCCACCCCTCTG
GAGCACTATTTACGATACAAACAGGA
610 60
LOC610991
SNP_7
intron
CATGCATGATGCCCAGAGTA
CCCAAAGCAC(AG)CTGTGATTTAAT
AGGGCTTCCTGGGAAAAGT
600 60
LOC611007
SNP_8
intron
CAGACCAACAGTGACCCAGA
TAGGCATACC(GT)TCAGTCCTAAAG
GCCTGTTGTGGGCAGAGTAT
480 60
LOC474541
SNP_9
intron
ACTGAGCCAAAGGTGGATTG
AGAGAATAGC(AG)CTGTGTTTTACA
ACCTGCACATCGGGATTTAG
575 60
Fine mapping of CFA10 91
Table 2 (continued)
Gene-
symbol SNP
Location
(intron or
5rsquo3rsquo-end)
Primer F1 (5acute -gt 3acute)
SNP motif
Primer R2 (5acute -gt 3acute)
Bp3 AT4
LOC474542
SNP_10
intron
CTTCCCCAGGAGAGAGTGAC
AATATGATCA(CT)ATTTAAAGAAAT
CTTTTGTCAACATCCCCTTCA
560 60
LOC481308
SNP_11
3rsquo-end
ACCCATTGTCTCTCCAGCAC
CCACATAACT(GT)AGCATCCCTAGC
600 60
TGATGATGTAAGTTGGCCTCA
LOC474543
SNP_12
intron
TTGAAGTTGTGTGAGTAAATGAAAGA
CAATATATAA(AG)CATTTGCTACAA 600 60
SNP_13 intron TTGAGATGAA(AT)ATAATAGAGCTG
SNP_14 intron CGATTTTCCA(AG)GACATGGAGATG
SNP_15 intron ACTGAACCAA(AG)TGTGCAAAGCAT
GGAAACCATGCAGTCTTTGG
LOC609217
SNP_16
5rsquo-end
TGGCCTCATTTTCCAGTATG
GAAGGAGTTA(CT)ACAGTGAAGATA
580 60
SNP_17 5rsquo-end AAGTGATCAA(AG)CCAGTGCATTCT
GGCAATTACCCTGAGTGGTG
LOC611115
SNP_18
3rsquo-end
GGGCTGTCTTAGAGGTGCTG
TGTGGTCTCA(CT)ACACTTCCTGAG
590 60
SNP_19 3rsquo-end ATTGCAAGTG(GT)CCTGTGATGGCC
CTTCTTTGGGCAGGAAAGTG
LOC481325
SNP_20
intron
AAAATGATTGATCGCAAAAGAAA
AATTACTGTA(CT)AACAGTATCAGA
600 60
SNP_21 intron TAACAGTATC(AG)GAATAAAAGTTT
TTCTGTGATTGCACTGACCG
Fine mapping of CFA10
92
Table 2 (continued)
Gene-
symbol SNP
Location
(intron
or 5rsquo3rsquo-
end)
Primer F1 (5acute -gt 3acute)
SNP motif
Primer R2 (5acute -gt 3acute)
Bp3 AT4
LOC481330
SNP_22
intron
GAAAGGCCTGGGTTCAAAA
GGCAGGGAGG(AG)TCACCATCGTTC
AATTTCCCCAAATGCCTCAC
575 60
LOC611493
SNP_23
intron
GCATGAAGGAGCCCTATGTC
CCAAGAGTCC(AT)GCCCAACACCCT
GGAGGGATGGCATTCTATGA
590 60
LOC481337
SNP_24
intron
GGCTGAGGAGATTGTGTTTCA
GCTGATATTT(AG)GCCTTCTGAGAT
620 60
SNP_25 intron AAAGCCAGAC(AG)CTTTGCCATGGT
CAGCTCCCTGTAATGGGAAA
LOC611728
SNP_26
intron
TCCTACTCCCATCACTTCCAA
CCACACTGGG(GT)CCTGGGATGAGG
CACAGCTCCATGTAGGTCCA
620 60
hypothetical gene found in the 2 version 1 of the NCBIs genome annotation 1 Forward 2 Reverse 3 Product size (basepairs) 4 Annealing temperature (degC)
Fine mapping of CFA10 93
Table 3 The 26 newly developed intragenic SNPs for Dalmatian dogs with their
nucleotide polymorphism allele and genotype frequencies observed heterozygosity
(HET) and polymorphism information content (PIC)
SNP Fam1 Nucleotide
polymorphism
Allele
frequencies
Genotype
frequencies2 PIC HET
SNP_1 125 CgtG 042054 3135 036 055
SNP_2 124 AgtG 064039 91013 037 044
SNP_3 2345 AgtC 041049 2156 033 047
SNP_4 345 CgtT 065035 6140 035 067
SNP_5 34 CgtT 065035 490 028 042
SNP_6 345 CgtT 066034 6130 035 039
SNP_7 1345 AgtG 054046 6174 037 053
SNP_8 145 GgtT 075025 01010 029 045
SNP_9 1345 AgtG 052054 6145 037 047
SNP_10 1234 CgtT 057043 7173 037 068
SNP_11 14 GgtT 065035 5120 035 062
SNP_12 15 AgtG 028072 097 030 044
SNP_13 45 AgtT 077023 870 017 021
SNP_14 5 AgtG 036064 052 013 015
SNP_15 134 AgtG 034066 0157 034 059
SNP_16 145 CgtT 030070 3812 033 036
SNP_17 245 AgtG 058042 6113 030 032
SNP_18 123 CgtT 037063 1126 027 035
SNP_19 123 GgtT 045055 3115 030 032
SNP_20 2345 CgtT 032068 1129 029 039
SNP_21 1235 AgtG 063037 883 037 052
SNP_22 1234 AgtG 068032 10102 030 034
SNP_23 12345 AgtT 030070 21313 033 046
SNP_24 124 AgtG 047053 667 033 019
SNP_25 234 AgtG 053047 3132 035 053
SNP_26 234 GgtT 060040 3120 026 0391 Number of families which were informative for the respective SNPs 2 Genotypes are given as number of animals [homozygous for allele 1heterozygous
homozygous for allele 2]
Fine mapping of CFA10
94
Table 4 Linkage analysis for canine chromosome 10 regarding the region spanning
39 Mb to 44 Mb with the significant Zmean LOD score and error probabilities (p-
values)
Marker Location (Mb) Zmean pz-value1 LOD-score pL-value2
SNP_16 39453 262 0004 123 0009
SNP_17 39455 262 0004 123 0009
SNP_18 39840 261 0004 123 0009
SNP_19 39843 261 0004 123 0009
SNP_20 4260 255 0005 118 0010
SNP_21 4270 255 0005 117 0010
SNP_22 4405 317 00008 131 0007
SNP_23 4439 337 00004 134 0007 1 Error probability regarding Zmean 2 Error probability regarding LOD score
Fine mapping of CFA10 95
Figure 1 Zmeans and LOD scores for 27 microsatellite markers on CFA10
harbouring a congenital sensorineural deafness region (number of families 23
number of genotyped dogs 176)
Figure 2 P-values for Zmeans and LOD scores of 27 microsatellite markers on
CFA10 harbouring a congenital sensorineural deafness region (number of families
23 number of genotyped dogs 176)
Fine mapping of CFA10
96
Figure 3 Zmeans and LOD scores of 53 markers on CFA10 harbouring a congenital
sensorineural deafness region (number of families 5 number of genotyped dogs 34)
Figure 4 P-values for Zmeans and LOD scores of 53 markers on CFA10 for the
region between 30 to 60 Mb harbouring a congenital sensorineural deafness region
(number of families 5 number of genotyped dogs 34)
CCSD region (SNP 16-23)
Chapter 7
Analysis of canine chromosome 1 and the Gap junction protein alpha 1 (GJA1) gene
in Dalmatian dogs segregating for congenital sensorineural deafness
CFA1 and the GJA1 gene 99
Analysis of canine chromosome 1 and the Gap junction protein alpha 1 (GJA1) gene in Dalmatian dogs segregating for congenital sensorineural deafness Abstract Two microsatellite markers associated to the GJA1 (alternatively connexin 43) gene
showed significant linkage with canine congenital deafness (CCSD) in a large French
half-sib family (Chapter 3) PCR products of this half-sib family were used to perform
a sequence analysis in order to evaluate whether GJA1 is responsible for CCSD As
the linkage could not be confirmed we used altogether 27 microsatellite markers for
a scan of canine chromosome (CFA) 1 with 23 Dalmatian dog families segregating
for CCSD A non-parametric linkage analysis was performed to see whether
significant test statistics for other genomic regions on CFA1 and for more families can
be shown As a result we could not find linkage to any microsatellite in the analyzed
families
Introduction Rak (2003) considered in total 24 genes as candidates for sensorineural deafness in
dogs among them the Gap junction protein alpha 1 (GJA1) or connexin 43 gene on
canine chromosome (CFA) 1 A marker set of altogether 43 microsatellites were
developed by Rak (2003) among them two microsatellite marker associated to the
GJA1 gene
GJA1 or connexin 43 is a member of the connexin gene family and a component of
gap junctions Mutations in 4 members of the connexin gene family have been shown
to underlie distinct genetic forms of deafness including GJB2 (connexin [CX] 26)
GJB3 (CX31) GJB6 (CX30) and GJB1 (CX32)
Liu et al (2001) reported mutations in GJA1 in association with sensorineural
recessive deafness in man However these mutations have recently been shown to
involve the pseudogene of GJA1 on human chromosome (HSA) 5 and not the
CFA1 and the GJA1 gene
100
GJA1 gene on HSA 6 (WA Paznekas cited a personal communication from the
senior author (W E Nance) of the paper by Liu et al 2001)
In previously performed studies (Chapter 3) one large French Dalmatian dog family
with frequent occurrence of CCSD showed linkage to microsatellites associated to
the GJA1 gene
In this report we performed a mutation analysis of the GJA1 gene sequence to
identify polymorphisms In order to evaluate whether the GJA1 gene is responsible
for congenital sensorineural deafness in Dalmatian dogs we analyzed the
association of the GJA1 haplotypes with the CCSD phenotype Furthermore we
employed 27 microsatellite markers covering the entire CFA1 and used them for a
non-parametric linkage analysis with CCSD in a Dalmatian dog population of 176
animals with frequent occurrence of CCSD
Material and methods Pedigree material
For the linkage analysis of the microsatellite marker we used DNA from altogether
176 animals belonging to 22 full-sib families and one large paternal half-sib family of
German Dalmatian dogs All families were segregating for CCSD The genotyped
families included all affected dogs (unilaterally and bilaterally deaf) their parents if
available and one to four unaffected animals At least two of the full sibs of each
family were unilaterally or bilaterally deaf The phenotype of the affected animals had
been confirmed by brainstem auditory evoked response (BAER) that detects
electrical activity in the cochlea and auditory pathways in the brain
Mutation analysis and screening for SNPs was performed by comparative
sequencing of DNA from 16 animals consisting of the parents and their progenies of
one large half-sib family of French Dalmatian dogs which showed significant linkage
to GJA1-associated microsatellites (Chapter 3)
Sequencing of canine genomic DNA and mutation analysis
The canine GJA1 gene sequence was derived from sequences deposited in the
current dog genome assembly (Boxer genome assembly 21) of the NCBI GenBank
(Genbank Acc No NC_006583) We compared the canine genomic DNA sequence
to canine cDNA fragments in the canine EST database using the
CFA1 and the GJA1 gene 101
BLASTN (Basic Local Alignment Search Tool Nucleotide) program Three canine
ESTs (Acc No DN880776 BU744925 and DN369900) were used to confirm the
assembly of the GJA1 gene
To identify polymorphisms within the canine GJA1 sequence the gene consisting of
one 1251 bp spanning exon were PCR amplified and sequenced from 16 French
Dalmatian dogs Additionally large sequences from the 3acute-end were amplified Primer
pairs were designed yielding products with a length of around 600 bp PCR primers
were developed with the Primer3 program (httpfrodowimiteducgi-
binprimer3primer3_wwwcgi) The PCR reactions were performed in a total of 50 microl
containing 125 microM dNTPs 25 pmol of each primer the reaction buffer supplied by
the manufacturer (Qbiogene Heidelberg Germany) and 1 U Taq polymerase After
a 4 min initial denaturation at 95degC 35 cycles of 30 sec at 94degC 45 sec at 58degC and
80 sec at 72degC were performed in a MJ Research thermocycler (Biozym Hessisch
Oldendorf Germany)
The obtained PCR products were directly sequenced with the DYEnamic ET
Terminator kit (AmershamBiosciences Freiburg Germany) and a MegaBACE 1000
capillary sequencer (AmershamBiosciences) using the PCR primers as sequencing
primers Sequence data were analyzed with Sequencher 42
(GeneCodes Ann Arbor MI USA)
Microsatellite marker analysis
We used 27 microsatellite marker derived from the NCBI database
(httpwebncbinlmnihgov) to cover CFA1 completely (Appendix Table 1)
PCR reactions for microsatellites were carried out in 12 microl reaction mixtures
containing 2 microl genomic DNA 12 microl 10x PCR buffer (Qbiogene) 024 microl dimethyl
sulfoxide (DMSO) 02 microl dNTPs (100 microM) 01 microl Taq Polymerase (5Umicrol)
(Qbiogene) 06 microl (10 microM) 5rsquo-IRD700 or IRD800 labelled forward primer 06 microl (10
microM) unlabelled reverse primer The PCR reactions were carried out in MJ Research
thermocyclers with the following program 4 min at 94 degC followed by 32 cycles of 30
sec at 94degC 30 sec at maximum annealing temperature and a final extension of 45
sec at 72degC PCR-products were diluted with formamide loading buffer in ratios from
110 to 140 and then size-fractionated by gel electrophoresis on automated LI-COR
42004300 sequencers (LI-COR inc Lincoln NE USA) using denaturing 4 and 6
polyacrylamide gels (RotiphoreseregGel 40 Roth Karlsruhe Germany)
CFA1 and the GJA1 gene
102
Linkage and haplotype analysis
Multipoint non-parametric linkage and haplotype analyses were performed using the
MERLIN software version 0102 (multipoint engine for rapid likelihood inference
Center for Statistical Genetics University of Michigan MI USA Abecasis et al
2002) The test statistics Z-mean and Lod score were used to test for the proportion
of alleles shared by affected individuals identical by descent (IBD) for the considered
marker loci
A non-parametric linkage analysis was performed with 27 microsatellite markers in 23
Dalmatian dog families consisting of 176 Dalmatian dogs The results were added to
the linkage analysis performed in Chapter 3
Results and discussion
In previously performed studies (Chapter 3) one French Dalmatian dog family
reached a Zmean value of 295 (plt0002) for GJA1_MS1 and a Zmean value of 286
(plt0002) for GJA1_MS2 indicating linkage Both markers were associated with the
GJA1 gene
By sequence analysis we revealed a total of 3 SNPs found in sequences of the 3acute-
UTR shown in Table 1 None of the polymorphisms were polymorphic in all parents of
the examined French half-sib family Neither of the observed polymorphism did alter
the predicted amino acid sequence of GJA1 nor showed the identified haplotypes an
association with the CCSD phenotype (Figure 1)
There is no recombination of the haplotypes of the GJA1 gene in this family The
paternal and maternal haplotypes as far as they could be estimated were distributed
among the affected progeny closely to the expected proportion of 50 and therefore
no excess of a certain haplotype could be observed in the affected dogs
Thus evaluation of the SNP markers debilitates the linkage to CCSD for the French
half-sib family Because of the fact that both animals with unilateral hearing loss and
bilateral hearing animals shared identical haplotypes these polymorphisms are
obviously not associated with CCSD in these Dalmatian dog families Furthermore
the present study revealed no functional mutations of the complete coding region of
GJA1 It is therefore unlikely that the GJA1 gene is involved in the pathogenesis of
CCSD in the analyzed French Dalmatian dogs The reason for GJA1_MS1 and
GJA1_MS2 indicating linkage may be caused by a closely linked gene involved in
CFA1 and the GJA1 gene 103
CCSD among the half-sib family of French Dalmatian dogs or by a false positive
result of the microsatellite study performed in Chapter 3
To clarify whether significant test statistics for other genomic regions on CFA1 and
for more families can be shown we used 27 microsatellite markers derived from the
NCBI database (httpwebncbinlmnihgov) to cover CFA1 completely A linkage
analysis was carried out after genotyping 176 German Dalmatian dogs with a set of
27 microsatellite markers The results of this linkage analysis were added to the
results of the test statistics for the microsatellites GJA1_MS1 and GJA1_MS2
(Chapter 3)
As a result we could not find linkage to any microsatellite in the analyzed families
(Figure 2 and 3) It is unlikely that the canine chromosome 1 harbours genomic
regions that are involved in the development of CCSD in the analyzed Dalmatian dog
families
With hindsight it was revealed that GJA1 is not responsible for sensorineural non-
syndromic deafness in humans as Liu et al (2001) has published GJA1 is
participating in a human syndrome called oculodentodigital dysplasia (ODDD) that
can be accompanied with hearing impairment (Paznekas et al 2003) But the type of
deafness in human ODDD differs from the typical hearing loss associated with other
connexin mutations because it is conductive rather than sensorineural
As deafness in dogs especially in Dalmatians is almost exclusively caused by
sensorineural non-syndromic forms also known as cochleosaccular degeneration
the GJA1 gene should not be considered as a candidate gene for CCSD anymore
CFA1 and the GJA1 gene
104
Table 1 Three newly developed intragenic SNPs and two microsatellite markers for
Dalmatian dogs in the candidate gene GJA1 with their corresponding primers the
SNP motif the product size and the annealing temperature
SNP
Primer F (5acute -gt 3acute)
SNP motif
Primer R (5acute -gt 3acute)
Product
size (bp)
Annealing
temperatur
GJA1_SNP1+2
CACCTTAGGCGTTCATTTTG
CCGGGGAG(AG)AAAA(AG)AAAAATACTT
TGGCTTGATTCCCTGACTC
650 58
GJA1_SNP3
TCTGAAATGTAATCATGGATGC
CAGAACTTGTAT(AT)CTGTTAAGAG
AATCACACAGGATATAGAGGCTATC
600 58
Microsatellite
marker Primers (forward reverse ) 5acute -gt 3acute
Product
size (bp)
Annealing
temperatur
GJA1_MS1
ATGGCATGAAGAGGATACCG
AGGACAGGTGACGGCTCTAC
134 60
GJA1_MS2
GCTAGTACTCGATTGTGGTC
TCATGGGTTGTGAGATCCAG
190 60
CFA1 and the GJA1 gene 105
Figu
re 1
Hap
loty
pes
of th
e G
JA1
gene
in th
e an
alyz
ed D
alm
atia
n do
g fa
mily
CFA1 and the GJA1 gene
106
Figure 2 Zmeans and LOD scores for 29 microsatellite markers on CFA1 (number of
families 23 number of genotyped dogs 176)
Figure 3 P-values for Zmeans and LOD scores of 29 microsatellite markers on CFA1
(number of families 23 number of genotyped dogs 176)
Chapter 8
Analysis of canine chromosome 31 and the claudin-14 (CLDN14) gene
in Dalmatian dogs segregating for congenital sensorineural deafness
CFA31 and the CLDN14 gene 109
Analysis of canine chromosome 31 and the claudin-14 (CLDN14) gene in Dalmatian dogs segregating for congenital sensorineural deafness Abstract A previously accomplished linkage analysis revealed significant linkage of
microsatellite markers associated with the CLDN14 gene on canine chromosome
(CFA) 31 with the phenotype of canine sensorineural deafness (Chapter 3) The
objective of the present study was to perform a sequence analysis in order to find
single nucleotide polymorphisms (SNPs) for the CLDN14 gene and additionally to
use a set of six microsatellite markers evenly distributed on CFA31 for non-
parametric linkage analysis with the aim to verify the significant test statistics shown
in Chapter 3
Introduction
Of the genes responsible for different forms of sensorineural non-syndromic deafness
in humans Rak (2003) considered 24 genes as candidates for sensorineural
deafness in dogs among them the CLDN14 gene on canine chromosome (CFA) 31 CLDN14 encodes a member of the claudin family which comprises the major
components of tight juncions (TJ) The human CLDN14 gene consists of one
tranlatete exon and six exons in the 5rsquountranlated region Five splice isoformes are
identified so far (Wilcox et al 2001 Wattenhofer et al 2005)
For the compartmentalization of perilymph and endolymph in the inner ear the
leakage of solutes through a paracellular pathway must be prevented by tight
junctions TJ are an intercellular junction found at the most apical region of polarised
epithelial and endothelial cells at which adjacent plasma membranes are joined
tightly together separating apical membranes and basolateral menbranes They are
specialised membrane domains containing branching strands of integral proteins and
create a primary barrier preventing paracellular transport of solutes and restricting
lateral diffusion of membrane lipids and proteins (Schneeberger and Lynch 2004)
CFA31 and the CLDN14 gene
110
The important role of the CLDN14 gene in the TJ of the inner ear was demonstrated
by Wilcox et al (2001) when he identified mutations in the CLDN14 gene responsible
for a hereditary human deafness in families segregating for congenital recessive
deafness (DFNB29)
In previously performed studies one large half-sib family as well as several full-sib
families of German Dalmatian dog indicated linkage to CLDN14 gene-associated
markers (Chapter 3)
A sequence analysis was performed to identify single nucleotide polymorphisms
(SNPs) either intergenic located or alternatively in the flanking 5rsquo- and 3rsquo-Regions
Furthermore we employed microsatellite markers covering CFA31 and used them for
a non-parametric linkage analysis with CCSD in a German Dalmatian dog population
of 176 animals with frequent occurrence of CCSD
Material and methods Pedigree material
For the linkage analysis of the microsatellite marker we used DNA from altogether
176 animals belonging to 22 full-sib families and one large paternal half-sib family of
German Dalmatian dogs All families were segregating for CCSD The genotyped
families included all the affected dogs (unilaterally and bilaterally deaf) their parents
if available and one to four unaffected animals At least two of the full sibs of each
family were unilaterally or bilaterally deaf The phenotype of the affected animals had
been confirmed by brainstem auditory evoked response (BAER) that detects
electrical activity in the cochlea and auditory pathways in the brain
Mutation analysis and screening for SNPs was performed by comparative
sequencing of DNA from 36 animals consisting of the parents and their progenies of
one half-sib family and four full-sib families of Dalmatian dogs which showed
significant linkage to a CLDN14-associated microsatellite (Chapter 3)
Sequencing of canine genomic DNA and development of single nucleotide
polymorphisms (SNPs)
The canine CLDN14 gene sequence was derived from sequences deposited in the
current dog genome assembly (Boxer genome assembly 21) of the NCBI GenBank
(Genbank Acc No NW_876295) by BLAST (Basic Local Alignment Search Tool)
CFA31 and the CLDN14 gene 111
search (httpwwwncbinlmnihgovBLAST) using the human CLDN14 reference
mRNA sequence (Genbank Acc No NM_144492)
We compared the canine genomic DNA sequence to canine cDNA fragments in the
canine EST database using the BLASTN program As no ESTs could be found
human mRNA sequences were used for the localization of the exonintron
boundaries using the mRNA-to-genomic alignment program Spidey
(httpwwwncbinlmnihgovIEBResearchOstellSpideyindexhtml)
We screened exon three for mutations as this exon is the only translated one in man
Additionally we screened large intronic sequences and sequences located in the 5rsquo-
and 3rsquo-UTR of the CLDN14 gene for Polymorphisms For this purpose primer pairs
were designed yielding products with a length of around 600 bp
PCR primers were developed with the Primer3 program (httpfrodowimiteducgi-
binprimer3primer3_wwwcgi) The PCR reactions were performed in a total of 50 microl
containing 125 microM dNTPs 25 pmol of each primer the reaction buffer supplied by
the manufacturer (Qbiogene Heidelberg Germany) and 1 U Taq polymerase After
a 4 min initial denaturation at 95degC 35 cycles of 30 sec at 94degC 45 sec at 58degC and
80 sec at 72degC were performed in a MJ Research thermocycler (Biozym Hessisch
Oldendorf Germany)
The obtained PCR products were directly sequenced with the DYEnamic ET
Terminator kit (AmershamBiosciences Freiburg Germany) and a MegaBACE 1000
capillary sequencer (AmershamBiosciences) using the PCR primers as sequencing
primers Sequence data were analyzed with Sequencher 42 (GeneCodes Ann
Arbor MI USA) The eight newly developed SNPs are shown in Table 1
Microsatellite marker analysis
In total six microsatellite marker were derived from the NCBI database
(httpwebncbinlmnihgov) to cover CFA31 completely (Appendix Table 1)
PCR reactions for microsatellites were carried out in 12 microl reaction mixtures
containing 2 microl genomic DNA 12 microl 10x PCR buffer (Qbiogene) 024 microl dimethyl
sulfoxide (DMSO) 02 microl dNTPs (100 microM) 01 microl Taq Polymerase (5Umicrol)
(Qbiogene) 06 microl (10 microM) 5rsquo-IRD700 or IRD800 labelled forward primer 06 microl (10
microM) unlabelled reverse primer The PCR reactions were carried out in MJ Research
thermocyclers with the following program 4 min at 94 degC followed by 32 cycles of 30
sec at 94degC 30 sec at maximum annealing temperature and a final extension of
CFA31 and the CLDN14 gene
112
45 sec at 72degC PCR-products were diluted with formamide loading buffer in ratios
from 110 to 140 and then size-fractionated by gel electrophoresis on automated LI-
COR 42004300 sequencers (LI-COR inc Lincoln NE USA) using denaturing 4
and 6 polyacrylamide gels (RotiphoreseregGel 40 Roth Karlsruhe Germany)
Linkage and haplotype analysis
Multipoint non-parametric linkage and haplotype analysis were performed using
MERLIN version 0102 (multipoint engine for rapid likelihood inference Center for
Statistical Genetics University of Michigan MI USA Abecasis et al 2002) The test
statistics Z-mean and Lod score were used to test for the proportion of alleles shared
by affected individuals identical by descent (IBD) for the considered marker loci A
linkage analysis was performed with 6 microsatellite markers on 176 Dalmatian dogs
The results were added to the linkage analysis performed in Chapter 3
Results and discussion CLDN14-associted microsatellites were significantly linked with CCSD in a scan of
candidate genes using 24 families with 215 Dalmatian dogs (Chapter 3) Selection of
four full-sib families and one half-sib family with the highest tests statistics lead to a
Zmean value of 383 (plt000007) for the CLDN14 gene-associated marker
CLDN14_MS2
To substantiate the linkage to the CLDN14 gene we searched for sequence
variations within the CLDN14 gene in four full-sib families and one half-sib family with
the highest contribution to the test statistics shown in Chapter 3 Most of the identified
polymorphisms were found in intronic sequences none were within exon three None
of the observed polymorphism did alter the predicted amino acid sequence of exon
three We revealed a total of eight SNPs (Table 1) but only two (SNP_4 and SNP_5)
out of the identified eight SNPs were polymorphic in the examined families Both
unilaterally and bilaterally deaf animals as well as normal hearing animals showed
identical haplotypes for these two polymorphic SNPs and thus no co-segregation with
the deaf Dalmatian dogs could be revealed (Figure 1 and 2) In this study we did not
identify any causal mutations for CCSD in the analyzed Dalmatian dogs Despite this
results a mutation outside of the translated genomic regions analyzed here may exist
that would affect CLDN14 expression
CFA31 and the CLDN14 gene 113
To clarify if other regions on CFA31 are responsible for the CCSD phenotype
additionally six microsatellite markers covering CFA31 were tested in 23 Dalmatian
dog families consisting of 176 individuals A non-parametric linkage analysis was
performed regarding these 23 families The results of this linkage analysis were
added to the results of the test statistics for the CLDN14 gene-associated
microsatellites (Chapter 3)
We could only find significant linkage with CCSD for CLDN14-associted
microsatellites in the abovementioned five families The two microsatellites located
most closely were proximal and distal 4 Mb off the CLDN14 gene REN110K04 with a
Zmean value of -009 (plt05) and FH2712 with a Zmean value of 022 (plt03) not
indicating linkage
It can not be excluded that CLDN14 or genes in its flanking regions are involved in
the development of CCSD in the analyzed Dalmatian dog families Other genes than
the CLDN14 that are known to be involved in the hearing process or known to be
expressed in the inner ear are not annotated at the syntenic human region of homo
sapiens autosome (HSA) 21q223 Therefore despite the CLDN14 gene no other
well-defined candidate gene for canine hereditary deafness exists in the linked
region Despite the remarkable progress that has been made in the past few years in
identifying deafness genes in man and mouse it is suspected that only one-third of all
deafness causing genes have been identified so far and thus it seems possible that
other genes in the flanking region of the CLDN14 gene are involved in the
development of the disease Anyway to clarify the importance of CLDN14 in the
CCSD phenotype more SNPs have to be developed within the CLDN14 gene as well
as in its flanking regions with the aim to find significant linkage disequilibrium of SNP
markers with CCSD
CFA31 and the CLDN14 gene
114
Table 1 The 8 newly developed SNPs for Dalmatian dogs in the CLDN14 gene on
CFA31 with their corresponding primers the SNP motif the product size and the
annealing temperature
SNP
Location
(intron or
5rsquo3rsquo-UTR)
Primer F1 (5acute -gt 3acute)
SNP motif
Primer R2 (5acute -gt 3acute)
Bp3 AT4
CLDN14_SNP1
intron
GACCATATGTTTGTGGCC
CTTCCAGGGAAA(AT)TGTCGTAGCC
CGTCAGGATGTTGGTGCC
580 60
CLDN14_SNP2
inton
GACCATATGTTTGTGGCC
GAAATTGTCGTA(AG)CCCGGCCGCT
CGTCAGGATGTTGGTGCC
580 60
CLDN14_SNP3
3rsquo-UTR
CTGCCTTCAAGGACAACC
CCAGAGGAATAA(CT)ATGATCGTGA
GATGAGTATCAGCCCAGC
550 60
CLDN14_SNP4
3rsquo-UTR
CTGCCTTCAAGGACAACC
ACCACCGCACAC(CT)GTCACAGCTT
GATGAGTATCAGCCCAGC
550 60
CLDN14_SNP5 3rsquo-UTR
CATGCCTTTGTCCCAAACTT
GAGACCCTCTGG(CT)TCCTTTTGGC
GTACCTGTTGCCTGGGTTGT
610 60
CLDN14_SNP6 5rsquo-UTR
CCTTCATCCTTTCTGGTTGA
GCTCACAGTCCC(AC)ATGGGGACAT
GGGGAGCATAATGTGGTCAT
585 60
CLDN14_SNP7 5rsquo-UTR
TGAACTGGTCCCAAGGAAAG
GCACGACCGAGC(CT)CTGGCTTTAC
GGGATGAGAGGGAGGTTTTT
580 60
CLDN14_SNP8 3rsquo-UTR
AATGCCTATCCCTTCTTTGGA
CACGTTACTGTG(AG)ACCTCTCTAC
GCAGGCTTTCCTCAAGTGTC
680 60
1 Forward 2 Reverse 3 Product size (basepairs) 4 Annealing temperature (degC)
CFA31 and the CLDN14 gene 115
Figure 1 Haplotypes of the CLDN14 gene-associated markers SNP_4 and SNP_5 in
the analyzed four Dalmatian dog full-sib families
CFA31 and the CLDN14 gene
116
Figu
re 2
Hap
loty
pes
of th
e C
LDN
14 g
ene-
asso
ciat
ed m
arke
rs S
NP
_4 a
nd S
NP
_5 in
the
anal
yzed
Dal
mat
ian
dog
half-
sib
fam
ily
Chapter 9
General discussion
General discussion 119
General discussion
The candidate gene approach In the first step of this thesis candidate genes for canine congenital sensorineural
deafness (CCSD) in Dalmatian dogs were analyzed by means of microsatellite
markers or alternatively by single nucleotide polymorphisms (SNPs)
The candidate genes for which a set of in total 43 microsatellites was available
included the following 24 genes CDH23 CLDN14 COCH COL11A2 DFNA5
DIAPH1 EDN3 EDNRB EYA4 GJA1 GJB2 GJB6 MITF MYH9 MYO6 MYO7A
MYO15A OTOF PAX3 POU4F3 SLC26A4 SOX10 TECTA and TMPRSS3
(Rak 2003) These genes are known to be involved either in human non-syndromic
deafness or in the human Waardenburg syndrome The Waardenburg syndrome
(WS) manifests with sensorineural deafness and pigmentation defects in iris hair and
skin WS is classified into four types depending on the presence or absence of
additional symptoms which are caused by mutations in the five genes EDN3
EDNRB MITF PAX3 and SOX respectively
For another eight recently identified genes responsible for different forms of human
non-syndromic deafness including TMC1 TMIE USH1C MYH14 MYO3A PRES
WHRN and ESPN linkage and association analyses were performed using newly
developed SNPs
In the last years most projects have exploited canine traits for which either direct
candidate genes could be proposed and evaluated or for which large informative
pedigrees were available to enable linkage mapping to identify candidate regions A
major component of such research efforts comprised the cloning sequencing and
mapping of individual canine homologs of genes either proposed as candidate
genes or expected to be located in candidate regions This was necessary to identify
new informative polymorphisms (eg SNPs microsatellites) for high resolution
mapping of candidate regions and to examine each exon and exonintron boundary
for positional candidates Availability of the second version of the dog genome
assembly (build 21) of the NCBI database shortcuts this effort and increases the
investigators efficiency Now either additional candidate genes for canine congenital
sensorineural deafness can be found directly by its gene symbol in the 21 of the
General discussion
120
NCBIs genome annotation or if a candidate gene is not yet annotated a BLAST
(Basic Local Alignment Search Tool) search against the canine whole genome
shotgun (wgs) sequence resource can be used to obtain the sequence of the canine
genomic contigs containing the human homologous gene and thus intragenic
markers can be developed for subsequent linkage and association analyses
Over the past decade it has become increasingly clear how far structural and
functional homologies at the gene level extend across even distantly related species
Much is known about deafness-causing gene mutations in humans and mice
including the fact that the clinical and histopathological findings are often very similar
to those of deafness in Dalmatian dogs Thus genes responsible for non-syndromic
congenital hereditary deafness in humans seem to be appropriate candidate genes
for CCSD (Rak and Distl 2005) In this thesis we first concentrated on the candidate
gene approach combined with linkage analysis method using affected pedigree
members Once a significant linkage was found only the linked genes with the
required low error probability values were used for further molecular genetic analysis
The method of candidate gene approach using either gene-associated microsatellite
or alternatively SNP markers was applied for in total 32 candidate genes which
comprise nearly all of the identified mutated genes causing non-syndromic hereditary
hearing impairment in humans
Linkage and association analysis In principle linkage and association are totally different phenomena Linkage is a
relation between loci and association is a relation between alleles
Linkage means that a haplotype characterised by microsatellites or SNPs is
significantly more often present in family members with the phenotype under study
than expected by random assortment For construction of haplotypes sets of closely
linked genetic markers on the same chromosome are needed which tend to be
inherited together as they are not likely to be separated by recombination Linkage
creates associations within families but not among unrelated induviduals
Association is a statistical statement about the co-occurrence of alleles or
phenotypes Association analysis can be carried out as a method of genetic analysis
that compares the frequency of alleles between affected and unaffected individuals
across all families Thus for association family structures are not necessary A given
allele is considered to be associated with the disease if the presence of that allele
General discussion 121
explains a significant proportion of the phenotypic trait variation For association
studies the developing of a marker set consisting of SNPs rather than microsatellites
is needed
In this thesis a total of 32 candidate genes for canine congenital deafness were
analyzed which showed an appropriate clinical and histological disease pattern in
comparison to deafness in Dalmatian dogs Rak (2003) developed a set of 43
microsatellites for in total 24 candidate genes among them the CLDN14 gene on
canine chromosome (CFA) 31 and the MYH9 gene on CFA10 The GJA1 on CFA1
was also considered as a candidate gene for CCSD (Rak 2003) and therefore two
gene-associated microsatellites have been developed Recently it turned out that
GJA1 is not responsible for human sensorineural non-syndromic deafness but for a
human syndromic disorder that can be related with conductive deafness
By the use of a non-parametric linkage analysis using the existing set of 43
microsatellites associated to 24 candidate genes we found linkage to markers
associated to CLDN14 on CFA31 MYH9 on CFA10 and GJA1 on CFA1
For another another eight candidate genes it was possible to develop SNPs
Performing linkage analyses as well as association and haplotype studies it was
possible to exclude these eight candidate genes from being responsible for the
CCSD phenotype
Over the past ten years significant progress has been made in the identification of
deafness gene localisations Up to now approximately 120 loci have been reported
for both autosomal dominant and recessive forms of non-syndromic hereditary
deafness in humans and only for one third the responsible gene mutation could be
detected Thus it can be expected that additional potential human candidates for
CCSD in Dalmatian dogs will become available in future (Van Camp and Smith
2003)
The extreme heterogeneity of human deafness often hampered genetic studies
because many different genetic forms of hearing loss give rise to similar clinical
phenotypes and conversely mutations in the same gene can result in a variety of
clinical phenotypes In man genes that transport ions across membranes to
maintain appropriate solute concentration and pH as well as regulatory genes mainly
transcription factors and genes that play a part in structural integrity are essential
for the hearing process
General discussion
122
The results of this thesis indicate that the inheritance of hearing loss in Dalmatian
dogs is probably as heterogenic in origin as it is in humans Genetic heterogeneity
means that different mutations cause a similar phenotype the different mutations
can either be found at the same locus (allelic heterogeneity) or even at different loci
(non-allelic heterogeneity) As linkage was found for different candidate genes in
different families subsequently only the families indicating linkage were chosen for
further molecular analyses
GJA1 on CFA1 MYH9 on CFA10 and CLDN14 on CFA31 and their flanking regions
are further analyzed with a combined approach using microsatellite and SNP
markers
CFA1 By the use of GJA1-associated microsatellites one large French Dalmatian dog
family reached a Zmean value of 295 (plt0002) for GJA1_MS1 and a Zmean value
of 286 (plt0002) for GJA1_MS2 indicating linkage Subsequently a sequence
analysis of the GJA1 gene using the above mentioned French Dalmatian dog family
was performed None of the observed polymorphism did alter the predicted amino
acid sequence of GJA1 nor showed the identified haplotypes an association with the
CCSD phenotype Thus evaluation of the SNP markers debilitates the linkage to
CCSD for the French half-sib family It is unlikely that the GJA1 gene is involved in
the pathogenesis of CCSD in Dalmatian dogs To see whether significant test
statistics for other genomic regions on CFA1 and for more families can be shown a
non-parametric linkage analysis was performed with 27 microsatellite markers
covering CFA1 completely In total 176 animals were genotyped We could not find
linkage to any microsatellite in the analyzed families Furthermore it was revealed
that GJA1 is not a candidate gene for sensorineural non-syndromic deafness in
humans (WA Paznekas cited a personal communication from the senior author (W
E Nance) of the paper by Liu et al 2001) GJA1 is participating in a human
syndrome called oculodentodigital dysplasia (ODDD) that can be accompanied with
hearing impairment (Paznekas et al 2003) But the type of deafness in human
ODDD is conductive rather than sensorineural As deafness in dogs especially in
Dalmatians is almost exclusively caused by sensorineural non-syndromic forms also
known as cochleosaccular degeneration the GJA1 gene should not be considered
as a candidate gene for CCSD anymore
General discussion 123
CFA31 By the use of microsatellites as well as SNPs we found significant linkage with CCSD
for CLDN14-associated microsatellites in four full-sib and one half-sib Dalmatian dog
familiy with a Zmean value of 383 (plt000007) A mutation analysis was performed
for exon three as this is the only translated one in man None of the observed
polymorphisms did alter the predicted amino acid sequence However to clarify the
importance of the CLDN14 gene and its flanking regions in the CCSD phenotype
more SNPs have to be developed within the CLDN14 gene as well as in its flanking
regions with the aim to find significant linkage disequilibrium of SNP markers
CFA10 A significant co-segregation of markers alleles and the phenotypic expression of
deafness in a large sample of German Dalmatian dog families was determined for
one marker (Z-mean of 156 [p= 006] and a Lod score of 058 [p= 005]) associated
to the MYH9 gene Subsequently we evaluated whether MYH9 gene mutations are
responsible for CCSD in these Dalmatian dog families An initial priority in defining
gene structure is to obtain a full-length cDNA sequence and identify translational
initiation and termination sites and polyadenylation site(s) Exonintron structure can
then be determined by referencing the cDNA sequence against sequences of
cognate genomic DNA One popular method of obtaining full-length cDNA sequences
is the RACE (rapid amplification of cDNA ends) technique RACE-PCR is an anchor
PCR modification of RT-PCR The rationale is to amplify sequences between a single
previously characterised region in the mRNA (cDNA) and an anchor sequence that is
coupled to the 5 or the 3 end A primer is designed from the known internal
sequence and the second primer is selected from the relevant anchor sequence
To provide the genomic organization and the complete sequence of the canine
MYH9 gene the isolation of full length cDNAs was achieved with the help of a
modified rapid amplification of cDNA ends (RACE) protocol A mutation analysis was
performed to identify single nucleotide polymorphisms (SNPs) in this gene We
analyzed the association of the MYH9 haplotypes with the CCSD phenotype in three
families of Dalmatian dogs with frequent occurrence of CCSD and significant linkage
to gene-associated microsatellites Using the RT-PCR analyses for cDNA-genomic
sequence comparisons we detected that the canine MYH9 gene is bigger compared
to the human sequence due to the untranlated first exon in the 5rsquo-UTR
General discussion
124
The canine MYH9 gene consists of 41 exons with an untranslated exon 1 and
exonintron boundaries that conform perfectly to the GTAG rule
None of the observed polymorphisms did alter the predicted amino acid sequence of
MYH9 nor showed the identified haplotypes an association with the CCSD
phenotype
Thus these silent point mutations found in affected and unaffected Dalmatian dogs
do not seem to be responsible for the CCSD phenotype in these three families
To clarify if other regions on CFA10 are responsible for the CCSD phenotype we
added in a second step 27 microsatellite markers derived from the NCBI database to
cover CFA10 with a high density in the flanking region of the MYH9 gene A linkage
analysis was carried out for 23 Dalmatian dog families consisting of 176 animals that
were genotyped with the marker set of 27 microsatellite markers
We found significant linkage to microsatellites in the region spanning 36 Mb to 48 Mb
Consequently we screened this 12 Mb spanning region for SNPs Out of the 23
analyzed Dalmatian dog families five full-sib families were chosen to screen for
SNPs because of their obviously significant values at the above mentioned region
Twenty-six new SNP markers covering the region of 36 Mb to 48 Mb equally were
developed and added to the linkage analysis The significant Zmeans on CFA10 was
confirmed after adding the SNP markers Furthermore with the use of SNPs the
apparently linked region spanning 36 Mb to 48 Mb could be narrowed down to 5 Mb
spanning from 39 Mb to 44 Mb For this reason the use of SNPs in addition to
informative microsatellites appears highly recommendable
In further studies more SNPs have to be developed within the identified CCSD region
on CFA10 to localize the deafness causing gene or to find unambiguously associsted
SNP markers which could be used for a population-wide genetic test for CCSD
Chapter 10
Summary
Summary 127
Summary
Molecular genetic analysis of canine congenital sensorineural deafness in Dalmatian dogs
Katharina Mieskes (2006) The objective of the present study was to localize the gene or genomic region that is
involved in the development of canine congenital sensorineural deafness (CCSD) in
Dalmatian dogs
In man as in different dog breeds deafness is an often diagnosed disorder with the
Dalmatian dog showing the highest incidence Many genetic disorders in humans
and domestic dogs (Canis familiaris) demonstrate a high level of clinical and
molecular similarity
Altogether 39 genes have already been found causative for sensorineural non-
syndromic hearing impairment in humans Out of this 39 deafness causing genes a
total of 32 candidate genes were selected for canine congenital deafness which
showed an appropriate clinical and histological disease pattern in comparison to
deafness in Dalmatians dogs
On the one hand an existing set of 43 microsatllite markers for in total 24 candidate
genes were used for a non-parametric linkage analysis among them the claudin-14
(CLDN14) gene on canine chromosome (CFA) 31 and the myosin heavy polypeptide
9 (MYH9) gene on CFA10 The gap junction protein alpha 1 (GJA1) gene on CFA1
was also considered as a candidate gene for CCSD and thus GJA1-associated
microsatellites were part of the existing set Recently it turned out that GJA1 is not
responsible for human sensorineural non-syndromic deafness but for a human
syndromic disorder that can be related with conductive deafness In the last few
years more human deafness genes have been identified among them eight genes
that were considered as appropriate candidates for CCSD For these eight genes a
total of 21 SNPs were newly developed and used for non-parametric linkage and
association analyses
Summary
128
The used microsatellite and SNP markers derived either from a partial sequence
analysis of BAC clones each containing a different candidate gene or from
sequences deposited in the current dog genome assembly (Boxer genome assembly
21) of the NCBI GenBank We found significant linkage to markers associated to
CLDN14 on CFA31 MYH9 on CFA10 and GJA1 on CFA1 To substantiate the
linkage we searched for sequence variations within these three genes SNPs found
in intronic sequences of either gene were included in the linkage analyses Sequence
analysis neither revealed a causative mutation nor significant linkage disequilibrium
of SNP markers with CCSD Subsequently CFA1 10 and 31 were scanned
completely with microsatellite markers derived from the NCBI database with the
purpose to see if other regions on this three chromosomes harbour a gene that is
involved in the development of CCSD
The analyses of SNPs and more microsatellite markers on CFA1 revealed no
significant linkage to CCSD Thus it is unlikely that the canine chromosome 1 and
the GJA1 gene are responsible for the CCSD phenoptype As deafness in dogs
especially in Dalmatians is almost exclusively caused by sensorineural non-
syndromic forms the GJA1 gene should not be considered as a candidate gene for
CCSD anymore
On CFA10 we could exclude MYH9 for being causal for deafness but by adding
more microsatellites covering CFA10 completely we found significant linkage to the
CCSD phenotype in the region distal of MYH9 Hence new SNP-markers for fine
mapping the region spanning 36 to 48 Mb were developed by sequence analyses of
different Dalmatian dogs The search for SNPs was carried out on genomic
sequences of genes located in the significantly linked region The sequences of
these genomic sequences were derived from the NCBI GenBank The SNPs
confirmed the linkage and narrowed the region harbouring a causative CCSD gene
down to 5 Mb spanning from 39 to 44 Mb
After scanning CFA31 we could not exclude CLDN14 for being responsible for the
CCSD phenotype as microsatellite markers associated to CLDN14 indicated linkage
However to clarify the importance of CLDN14 in the CCSD phenotype more SNPs
have to be developed within the CLDN14 gene as well as in its flanking regions with
the aim to find linkage disequilibrium for SNP markers
Chapter 11
Erweiterte Zusammenfassung
Erweiterte Zusammenfassung 131
Erweiterte Zusammenfassung
Katharina Mieskes
Molekulargenetische Untersuchung der kongenitalen sensorineuralen Taubheit beim Dalmatiner
Einleitung Es gibt mehrere Moumlglichkeiten Taubheit zu klassifizieren Je nach den betroffenen
Strukturen des Gehoumlrorgans lassen sich konduktive und sensorineurale Formen
unterscheiden Erstere entstehen durch Stoumlrungen des Konduktionsmechanismus im
aumluszligeren Gehoumlrgang und im Mittelohr Ursachen fuumlr konduktive Houmlrstoumlrungen koumlnnen
zum Beispiel chronische Otitis externa oder media Frakturen von Mittelohranteilen
oder Tumoren sein Sehr viel oumlfter tritt die sensorineurale Taubheit auf bei der ein
Houmlrverlust als Folge von Transduktions- und Transmissionsstoumlrungen in der Cochlea
(Schnecke) sowie in peripheren und zentralen Anteilen des Nervus cochlearis auftritt
Neben der erblichen Form koumlnnen auch eine Otitis interna Tumoren oder
ototoxische Medikamente zur sensorineuralen Taubheit fuumlhren
Der Houmlrverlust kann bereits seit der Geburt bestehen (kongenital) oder er kann erst
im Laufe der weiteren Lebenszeit auftreten
Beim Menschen ist mehr als die Haumllfte der kongenitalen Taubheit erblich bedingt Die
erbliche kongenitale Taubheit kann als Teil einer multisystemischen Krankheit
(syndromisch) oder als Funktionsstoumlrung auftreten die auf das Ohr und das
vestibulaumlre System (nicht-syndromisch) begrenzt ist Da die nicht-syndromische
ererbte Taubheit fast ausschlieszliglich durch Cochleadefekte verursacht wird leiden die
Patienten unter sensorineuraler Taubheit Es wird geschaumltzt dass die nicht-
syndromische erbliche Taubheit 60-70 aller menschlichen Taubheitsfaumllle
verursacht Weiterhin sind 70-80 der Faumllle der nicht-syndromischen Taubheit auf
einen autosomal rezessiven Erbgang zuruumlck zu fuumlhren gefolgt von einem autosomal
dominanten Erbgang mit 10-20 der Faumllle und 1-2 der Faumllle sind X- gekoppelt Ein
noch geringerer Prozentsatz wird durch einen maternalen Erbgang verursacht
Von den ca 25000 bis 30000 menschlichen Genen ist nach Schaumltzungen bis zu 1
wichtig fuumlr den Houmlrprozess Annaumlhernd 120 Gene sind inzwischen identifiziert die fuumlr
Erweiterte Zusammenfassung
132
verschiedene Formen der menschlichen vererbten Taubheit verantwortlich sind
Darunter sind ungefaumlhr 80 Gene fuumlr die syndromische und 39 Gene fuumlr die nicht
syndromische vererbte Taubheit verantwortlich Insgesamt stellen die 120
identifizierten Gene vermutlich ein Drittel aller taubheitsverursachenden Gene dar
Die kongenitale sensorineurale Taubheit ist eine Erkrankung von der viele
Saumlugetierspezies betroffen sind Beim Hund werden uumlber 54 verschiedene Rassen
beschrieben bei welchen ein kongenitaler Houmlrverlust gehaumluft auftritt In zahlreichen
europaumlischen und amerikanischen Studien weisen hierbei die Dalmatiner mit 165-
30 die houmlchste Taubheitsinzidenz auf
Die histologischen Veraumlnderungen im Innenohr werden im Allgemeinen aumlhnlich wie
beim Menschen als cochleosacculaumlr beschrieben Die Degenerationen im Innenohr
schreiten bei betroffenen Tieren mit zunehmendem Alter fort und fuumlhren
normalerweise in einem Alter von drei bis vier Wochen spaumltestens aber in einem
Alter von etwa vier Monaten zu einem vollstaumlndigen ein- oder beidseitigen
Houmlrverlust
Eine objektive Diagnose der Taubheit besonders wenn sie nur einseitig ist stuumltzt
sich auf die brainstem auditory evoked response (BAER in Deutschland
audiometrischer Test) ein elektro-diagnostischer Test bei dem durch einen
bestimmten Reiz ausgeloumlste elektrische Aktivitaumlten (akustisch evozierte Potentiale) in
der Cochlea und den Nervenbahnen zur auditorischen Hirnrinde detektiert werden
Obwohl fuumlr den Dalmatiner die Erblichkeit der Erkrankung wiederholt nachgewiesen
werden konnte wurden fuumlr die verschiedenen untersuchten Dalmatinerpopulationen
auch sehr viele unterschiedliche Erbgaumlnge in Betracht gezogen Es ist bisher nicht
gelungen die genaue Anzahl der beteiligten Gene zu bestimmen Auch war es
bislang fuumlr keine der betroffenen Hunderassen moumlglich ein taubheitsverursachendes
Gen zu identifizieren
Bei deutschen Dalmatinern ergab eine Auswertung mittels komplexer
Segregationsanalysen dass ein Modell mit einem rezessiven Hauptgen und einer
polygenen Komponente das Auftreten der kongenitalen Taubheit in der untersuchten
Population am besten erklaumlrte Zusaumltzlich konnte ein Zusammenhang zu
Pigmentierungsgenorten nachgewiesen werden da Tiere mit blauer Augenfarbe
signifikant haumlufiger von Taubheit betroffen waren und gleichzeitig Tiere mit
Plattenzeichnung seltener kongenitalen Houmlrverlust zeigten
Erweiterte Zusammenfassung 133
Das Zuumlchten ein- oder beidseitig tauber Dalmatiner sowie Dalmatinern mit blauen
Augen ist in Deutschland schon laumlnger verboten und es wird von den
Zuchtverbaumlnden vorgeschrieben dass alle Dalmatinerwelpen in einem Alter von etwa
6 bis 8 Wochen audiometrisch untersucht werden Des Weiteren ist der Defekt
tierschutzrelevant nach sect 11b des Deutschen Tierschutzgesetzes da der artgemaumlszlige
Gebrauch eines Organs eingeschraumlnkt wird und eine Erblichkeit nachgewiesen
wurde Somit duumlrfen Tiere die Defektgentraumlger sind oder bei denen damit zu
rechnen ist nicht zur Zucht verwendet werden Waumlhrend einseitig taube Dalmatiner
als ganz normale Familienhunde gehalten werden koumlnnen sind beidseitig taube
Dalmatiner schwierig zu erziehen schreckhafter und daraus resultierend auch
oftmals aggressiv und bissig Aus diesen Gruumlnden werden fast alle Welpen mit
beidseitigem Houmlrverlust euthanasiert
Es hat sich gezeigt dass allein der Zuchtausschluss betroffener Tiere nicht genuumlgt
um die hohe Taubheitsinzidenz beim Dalmatiner deutlich zu reduzieren Aus diesen
Gruumlnden ist es von erheblicher Bedeutung die kongenitale sensorineurale Taubheit
beim Dalmatiner molekulargenetisch zu untersuchen um so ein
molekulargenetisches Testverfahren entwickeln zu koumlnnen welches auch die
Identifizierung von Anlagetraumlgern ermoumlglicht
Innerhalb des letzten Jahrzehnts wurden auch zwischen relativ unverwandten
Spezies die strukturellen und funktionellen Homologien auf genetischer Ebene
zunehmend verdeutlicht
Es wurde inzwischen eine groszlige Anzahl von taubheitsverursachenden
Genmutationen beim Menschen und der Maus aufgeklaumlrt Da auch die
histopathologischen Befunde beim Menschen und der Maus oftmals sehr aumlhnlich
denen beim Dalmatiner sind scheinen Gene die beim Menschen verantwortlich fuumlr
die congenitale sensorineurale Taubheit sind geeignete Kandidatengene fuumlr die
canine congenitale sensorineurale Taubheit zu sein
Das Ziel dieser Dissertation ist es die fuumlr die Taubheit kausalen Gene bzw Genorte
zu lokalisieren Um die kongenitale sensorineurale Taubheit beim Dalmatiner
moumlglichst effizient molekulargenetisch aufzuklaumlren wurden 32 Kandidatengene mit
genassoziierten Markern auf eine potenzielle Kopplung mit der caninen kongenitalen
sensorineuralen Taubheit untersucht
Die Genombereiche mit signifikanter Kopplung zur kongenitalen sensorineuralen
Taubheit wurden weitergehend molekulargenetisch analysiert
Erweiterte Zusammenfassung
134
Kopplungsanalyse fuumlr 24 Kandidatengene der caninen kongenitalen sensorineuralen Taubheit (CCSD) mit 43 genassoziierten Mikrosatelliten Markern
Material und Methoden
Fuumlr 24 Kandidatengene stand ein Mikrosatelliten Marker Set zur Verfuumlgung Das
Marker Set beinhaltete Mikrosatelliten fuumlr folgende Kandidatengene CDH23
CLDN14 COCH COL11A2 DFNA5 DIAPH1 EDN3 EDNRB EYA4 GJA1 GJB2
GJB6 MITF MYH9 MYO6 MYO7A MYO15A OTOF PAX3 POU4F3 SLC26A4
SOX10 TECTA und TMPRSS3 Als Familienmaterial fuumlr die Mikrosatellitenstudie
wurden 215 Dalmatiner verwendet Die Tiere verteilten sich auf 22 unverwandte
Vollgeschwisterfamilien und zwei paternale Halbgeschwistergruppen Die
genotypisierten Familien bestanden aus saumlmtlichen tauben Nachkommen (uni- oder
bilateral taub) ihren Eltern und einem bis vier nicht betroffenen Voll- bzw
Halbgeschwistern Mindestens zwei der Vollgeschwister einer Familie waren uni-
oder bilateral taub Der Phaumlnotyp der verwendeten Tiere wurde durch einen
audiometrischen Test bestimmt
Alle Mikrosatellitenmarker wurden uumlber PCR und Polyacrylamidgelelektrophorese
ausgewertet
Kopplungsanalyse
Die nicht-parametrische Kopplungsanalyse wurde unter Verwendung der Software
MERLIN (multipoint engine for rapid likelihood inference Version 0102)
durchgefuumlhrt und basierte auf dem identical-by-descent (IBD) Verfahren Dabei
wurden die Markerallele auf Kosegregation mit der phaumlnotypischen Auspraumlgung der
Erkrankung getestet Darauf folgend wurde die einer Normalverteilung folgende
Teststatistik fuumlr den Anteil von IBD-Markerallelen (Zmean) und ein daraus
abgeleiteter LOD-Score berechnet Als signifikant fuumlr die Kosegregation eines
Markerallels mit dem Phaumlnotyp der caninen kongenitalen sensorineuralen Taubheit
(CCSD) gelten Irrtumswahrscheinlichkeiten (p) von 005 oder kleiner
Die Daten wurden mit dem Softwarepaket SAS (Statistical Analysis System Version
913) bearbeitet um die Marker bezuumlglich Allelfrequenzen beobachteten und
erwarteten Heterozygotiegrad und PIC (Polymorphism information content)
charakterisieren zu koumlnnen
Erweiterte Zusammenfassung 135
Ergebnisse und Diskussion Das Mikrosatelliten Set bestand insgesamt aus 43 Markern mit einem bis drei
Markern pro Kandidatengen Zunaumlchst wurde fuumlr alle 24 Familien zusammen eine
Kopplungsanalyse durchgefuumlhrt Vermeintliche CCSD Loci wurden auf den caninen
Chromosomen (CFA) 1 10 12 20 und 31 lokalisiert Im Anschluss wurden die
Ergebnisse der Kopplungsanalyse auf Heterogenie zwischen den Familien uumlberpruumlft
Fuumlr einzelne Familien konnten houmlhere Teststatistiken und somit engere
Kopplungsbeziehungen als bei der Analyse aller Familien nachgewiesen werden
Eine signifikante Kopplung mit der kongenitalen sensorineuralen Taubheit wurde fuumlr
die Gene GJA1 auf CFA1 MYH9 auf CFA10 und CLDN14 auf CFA31 sowohl im
Gesamtmaterial wie in jeweils unterschiedlichen Gruppen von Dalmatinerfamilien
gefunden Es ist daher wahrscheinlich dass diese Gene oder Gene in ihrer naumlheren
Umgebung an der Entwicklung der Taubheit in den jeweiligen Familien involviert
sind Fuumlr die restlichen Kandidatengene lagen die Multipoint Teststatistiken fuumlr den
Anteil von IBD-Markerallelen (Zmean) und daraus abgeleiteten LOD-Scores bei
annaumlhernd Null und waren nicht signifikant
Die Ergebnisse dieser Teststatistiken zeigen dass der Erbgang der nicht-
syndromischen Taubheit bei Dalmatinern vermutlich ebenso heterogen ist wie er
sich beim Menschen darstellt
Dennoch stellt diese Studie den ersten Schritt dar um die CCSD verursachenden
Gene beim Dalmatiner zu identifizieren Die Gene GJA1 MYH9 und CLDN14 sowie
ihre naumlhere Umgebungen werden im weiteren Verlauf der Studie weitergehend
molekulargenetisch mit Hilfe von SNPs und Mikrosatelliten untersucht Aufgrund der
Heterogenitaumlt zwischen den Familien wurden fuumlr die weitere molekulargenetische
Analyse bestimmter Gene oder Genomregionen nur die Familien mit dem houmlchsten
Beitrag zu den Teststatistiken der Kopplungsanalyse (Zmean gt 1) ausgewaumlhlt
Erweiterte Zusammenfassung
136
Evaluierung von neu entwickelten SNP Markern assoziiert mit Kandidatengenen fuumlr die canine kongenitale sensorineurale Taubheit (CCSD) Material und Methoden Fuumlr acht Kandidatengene (TMC1 TMIE USH1C MYH14 MYO3A PRES WHRN
und ESPN) wurden intragenische Single Nucleotide Polymorphism (SNP) Marker neu
entwickelt
Als Familienmaterial fuumlr die Genotypisierung der SNPs wurde DNA von insgesamt 39
Dalmatinern verwendet die vier Vollgeschwisterfamilien angehoumlrten Mindestens
zwei der Vollgeschwister einer Familie waren uni- oder bilateral taub Der Phaumlnotyp
der verwendeten Tiere wurde durch einen audiometrischen Test bestimmt Die vier
Familien bestanden aus sechs bis zehn Vollgeschwistern und den dazugehoumlrenden
Elterntieren
Identifizierung von SNPs (Single Nucleotide Polymorphisms)
Die caninen Sequenzen der Kandidatengene wurden mit Hilfe der humanen mRNA
mittels BLAST (Basic Local Alignment Search Tool) Suche aus veroumlffentlichten
Sequenzen der NCBI Datenbank (Boxer genome assembly 21) entnommen Die
Exon Intron Grenzen wurden mit Hilfe des mRNA-to-genomic alignment Programms
Spidey bestimmt und Primer mit Hilfe des Primer3 Programms in die intronischen
Sequenzen gelegt Die SNPs wurden an den DNA-Sequenzen von Elterntieren der
betroffenen Dalmatinerfamilien identifiziert Die Amplifikate wurden mit Hilfe des
MegaBACE 1000 sequenziert und auf SNPs untersucht Bei Vorliegen eines oder
mehrerer SNPs wurde die entsprechende DNA-Sequenz der zugehoumlrigen
Nachkommen ebenfalls amplifiziert und sequenziert Die Auswertung erfolgte mit
Hilfe des Sequencher 42 Programms
Kopplungsanalyse
Die Genotypen der neuen SNP Marker wurden unter Verwendung des Programms
MERLIN ausgewertet Die Daten wurden mit dem Softwarepaket SAS (Statistical
Analysis System Version 913) bearbeitet um das Markerset bezuumlglich
Allelfrequenzen beobachteten Heterozygotiegrad und PIC (Polymorphism
information content) charakterisieren zu koumlnnen Des Weiteren wurde auf
Kopplungsungleichgewicht und die Assoziation der Haplotypen mit CCSD mittels
Erweiterte Zusammenfassung 137
des Programms CASECONTROL und HAPLOTYPE von SAS Genetics (Statistical
Analysis System Version 913 Cary NC USA) getestet
Ergebnisse und Diskussion Fuumlr acht Kandidatengene wurden mindestens zwei heterozygote SNPs entwickelt
Die nicht-parametrische Kopplungsanalyse zeigte keine signifikanten Teststatistiken
Offensichtlich war kein Haplotyp mit der caninen kongenitalen sensorineuralen
Taubheit (CCSD) in den untersuchten Familien assoziiert Da sowohl uni- und
bilateral taube Tiere als auch beidseitig houmlrende Tiere uumlber identische Haplotypen
verfuumlgen ist es unwahrscheinlich dass die Gene TMC1 TMIE USH1C MYH14
MYO3A PRES WHRN und ESPN an der Entstehung der CCSD beim Dalmatiner
beteiligt sind
Molekulargenetische Analyse des caninen myosin heavy polypeptide 9 non-muscle (MYH9) auf dem caninen Chromosom 10q232
Material und Methoden Klonierung und Sequenzierung von caniner MYH9 cDNA
Die canine MYH9 Gensequenz wurde mit Hilfe der BLAST Suche aus
veroumlffentlichten Sequenzen der NCBI Datenbank Die Isolierung der vollstaumlndigen
cDNA wurde durch ein Protokoll zur Amplifizierung von cDNA-Enden (rapid
amplification of cDNA ends [RACE]) erreicht Nach Klonierung und Sequenzierung
der RACE-Produkte wurden die Sequenzen mit Hilfe des Sequencher 42
Programms ausgewertet
Sequenzanalyse des caninen MYH9 Gens
Nach Fertigstellung des RACE Protokolls und Auswertung der Sequenzen wurden
die Exon Introngrenzen mit Hilfe des mRNA-to-genomic alignment Programms
Spidey lokalisiert Weiterhin wurden repetitive Elemente mit dem Programm
Repeatmaster 2 sowie der GC Gehalt mit der EBI toolbox CpG PlotCpGreport
ermittelt
Erweiterte Zusammenfassung
138
Mutatiosanalyse
Zur Identifizierung von Polymorphismen innerhalb der caninen MYH9 Sequenz
wurden die Exons mit ihren flankierenden intronischen Sequenzen mit Hilfe der PCR
amplifiziert und sequenziert Verwendet wurden hierfuumlr die DNA von insgesamt 16
Dalmatinern aus drei Familien Mindestens zwei der Vollgeschwister einer Familie
waren unilateral taub Der Phaumlnotyp der verwendeten Tiere wurde durch einen
audiometrischen Test bestimmt
PCR Primer wurden mit Hilfe des Primer3 Programms entwickelt PCR Reaktionen
fuumlr saumlmtliche Exons des MYH9 Gens mit ihren flankierenden Sequenzen wurden
durchgefuumlhrt die PCR Produkte sequenziert und die Sequenzen mit Hilfe des
Sequencher 42 Programms ausgewertet
Unter Verwendung der Software MERLIN (multipoint engine for rapid likelihood
inference Version 0910) wurde nach signifikanten nicht parametrischen LOD-
Scores fuumlr die Kosegregation von Markerallelen mit der phaumlnotypischen Auspraumlgung
der sensorineuralen Taubheit beim Dalmatiner gesucht
Ergebnisse und Diskussion
Analyse der genomischen Organisation und der cDNA des caninen MYH9 Gens
Eine vollstaumlndige cDNA Sequenz des caninen MYH9 Gens von 7484 bp konnte uumlber
die Reverse Transkription-PCR erhalten und bei der EMBL Nukleotid Datenbank
eingereicht werden Das canine MYH9 Gen besteht aus 41 Exons einschlieszliglich
einem untranslatierten ersten Exon und Exon Intron Grenzen die stets der GT AG
Regel folgen Der 5rsquo-UTR Bereich besteht aus 181 bp waumlhrend der 3rsquo-UTR aus 1432
bp besteht Weiterhin besitzt die canine MYH9 cDNA Sequenz einen offenen
Leserahmen von 5889 bp welcher ein Protein kodiert bestehend aus 1963
Aminosaumluren Die Groumlszlige der Exons variiert von 28 bis 1556 bp waumlhrend das
gesamte MYH9 Gens ungefaumlhr 90 kb umfasst Die Homologie der Sequenzen
zwischen dem humanen murinen und caninen MYH9 Gen ist limitiert auf die
kodierende Sequenz von Exon 2 bis 41
Mutations- und Haplotypenanalyse
Alle kodierenden Exons mit ihren flankierenden intronischen Sequenzen des MYH9
Gens konnten von den 16 Dalmatinern amplifiziert werden Die Sequenzen wurden
mit den entsprechenden Sequenzen vom Boxer aus der NCBI Genbank verglichen
Erweiterte Zusammenfassung 139
Die Suche nach Sequenzvariationen im MYH9 Gen lieszlig 22 SNPs erkennen wobei
die meisten Polymorphismen in den flankierenden Regionen der Exons gefunden
wurden Keiner der beobachteten Polymorphismen in den Exons aumlnderte die
Aminosaumluresequenz von MYH9 Auch zeigten die identifizierten Haplotypen keine
Assoziation mit dem CCSD Phaumlnotyp Multipoint Teststatistiken fuumlr den Anteil von
IBD-Markerallelen (Zmean) und ein daraus abgeleiteter LOD-Score lagen bei
annaumlhernd Null und waren nicht signifikant Zwei genassoziierte Mikrosatelliten
zeigten eine Kopplung diese kann jedoch mit Heterogenitaumlt zwischen den Familien
erklaumlrt werden
Fazit
Die Charakterisierung des Transkriptes und der genomischen Sequenz des caninen
MYH9 Gens zeigten einen konservierten Aufbau des Gens im Bezug auf das
humane orthologe Gen Aufgrund eines untranslatierten Exons im 5rsquo-UTR Bereich ist
das canine Gen groumlszliger als das humane Gen
Sowohl uni- oder bilateral taube und bilateral houmlrende Dalmatiner weisen identische
Haplotypen auf Daher sind in den untersuchten Dalmatinerfamilien die gefundenen
Polymorphismen offensichtlich nicht mit CCSD assoziiert Des Weiteren zeigt diese
Studie dass keine funktionellen Mutationen der vollstaumlndigen kodierenden Region
von MYH9 vorliegen Wir koumlnnen also abschlieszligend das MYH9 Gen als
Kandidatengen fuumlr die congenitale sensorineurale Taubheit beim Dalmatiner
ausschlieszligen
Erweiterte Zusammenfassung
140
Evaluierung von Mikrosatelliten und SNP Markern der caninen Chromosomen 1 10 und 31 auf Kopplung und Assoziation mit der caninen kongenitalen Taubheit Material und Methoden Pedigreematerial
Insgesamt wurden 176 Dalmatiner in die Untersuchung einbezogen Die Tiere
verteilten sich auf 23 unverwandte Vollgeschwisterfamilien und eine paternale
Halbgeschwistergruppe Die genotypisierten Familien bestanden aus saumlmtlichen
tauben Nachkommen (unilateral oder bilateral taub) ihren Eltern und einen bis vier
nicht betroffenen Voll- bzw Halbgeschwistern Mindestens zwei der Vollgeschwister
einer Familie waren unilateral oder bilateral taub Der Phaumlnotyp der verwendeten
Tiere wurde durch einen audiometrischen Test bestimmt
Alle 176 Dalmatiner wurden fuumlr die Mikrosatellitenstudie auf den caninen
Chromosomen 1 10 und 31 einbezogen Fuumlr die Feinkartierung mit Hilfe der SNPs
wurden dagegen nur noch die Dalmatinerfamilien mit einer eindeutigen Kopplung zu
der entsprechenden genomischen Region untersucht Fuumlr die Region auf CFA10
wurden dafuumlr 34 Tiere aus fuumlnf Vollgeschwisterfamilien ausgewaumlhlt und zur
Untersuchung des CLDN14 Gens auf CFA31 wurden 24 Tiere aus vier
Vollgeschwisterfamilien und 12 Tiere aus einer groszligen Halbgeschwisterfamilie
verwendet Fuumlr die Suche nach Polymorphismen im GJA1 Gen wurden 16 Tiere aus
einer groszligen Halbgeschwisterfamilie verwendet
Mikrosatellitenstudie
Fuumlr die Mikrosatellitenstudie von CFA1 10 und 31 wurden insgesamt 60 Marker
verwendet Mit jeweils 27 Mikrosatelliten wurde CFA1 und mit 27 Mikrosatelliten
wurde CFA10 vollstaumlndig abgedeckt Insgesamt 6 Marker wurden fuumlr CFA31
verwendet
Alle hier verwendeten Mikrosatelliten sind der NCBI Datenbank entnommen und
wurden uumlber PCR und Polyacrylamidgelelektrophorese ausgewertet
Identifizierung von SNPs (Single Nucleotide Polymorphisms)
Zur weiteren Abklaumlrung und zum Ausschluss falsch positiver Ergebnisse wurden
zusaumltzlich zu den Mikrosatelliten SNPs in den signifikanten Regionen auf CFA1 10
Erweiterte Zusammenfassung 141
und CFA 31 entwickelt und an den Familien die eine Kopplung zeigten
genotypisiert
Die caninen genomischen Sequenzen der untersuchten Gene wurden der NCBI
Datenbank (Boxer genome assembly 21) entnommen Exon Intron Grenzen wurden
bestimmt und Primer entwickelt Nach Durchfuumlhrung der PCR wurden die Amplifikate
mit Hilfe des MegaBACE 1000 sequenziert und auf SNPs untersucht Die
Auswertung erfolgte mit Hilfe des Sequencher 42 Programms
Die Genotypen der neuen SNP Marker wurden zu den Genotypen der fuumlr die
Kopplungsanalyse verwendeten Mikrosatelliten hinzugefuumlgt und gemeinsam unter
Verwendung des Programms MERLIN ausgewertet
Kopplungsanalyse
Unter Verwendung der Software MERLIN (multipoint engine for rapid likelihood
inference Version 0910) wurde nach signifikanten nicht parametrischen LOD-
Scores fuumlr die Kosegregation von Markerallelen mit der phaumlnotypischen Auspraumlgung
der sensorineuralen Taubheit beim Dalmatiner gesucht
Die Daten wurden mit dem Softwarepaket SAS (Statistical Analysis System Version
913) bearbeitet um die Marker bezuumlglich Allelfrequenzen beobachteten
Heterozygotiegrad und PIC (Polymorphism information content) charakterisieren zu
koumlnnen Des Weiteren wurde das Kopplungsungleichgewicht und die Assoziation der
Haplotypen mit CCSD mittles des Programms CASECONTROL und HAPLOTYPE
von SAS Genetics getestet
Ergebnisse und Diskussion CFA1
In einer groszligen Halbgeschwisterfamilie wurde eine signifikante Kopplung zu einem
GJA1 genassozierten Marker festgestellt Mit Hilfe der DNA von Tieren aus dieser
Halbgeschwisterfamilie wurde eine Sequenzanalyse des GJA1 Gens durchgefuumlhrt
Es wurden 3 SNPs im 3acute-untranslatierten Bereich gefunden Die identifizierten
Haplotypen zeigten keine Assoziation mit dem CCSD Phaumlnotyp Sowohl uni- oder
bilateral taube und bilateral houmlrende Dalmatiner wiesen identische Haplotypen auf
Daher sind in den untersuchten Dalmatinerfamilien die gefunden Polymorphismen
offensichtlich nicht mit CCSD assoziiert Auch wurde keine funktionelle Mutation in
der vollstaumlndigen kodierenden Region von GJA1 gefunden wurde Es ist daher
Erweiterte Zusammenfassung
142
unwahrscheinlich dass das GJA1 Gen an der Entwicklung der CCSD in der
untersuchten Halbgeschwisterfamilie beteiligt ist Die signifikante Kopplung der
GJA1-benachbarten Mikrosatelliten mit CCSD kann durch ein eng gekoppeltes Gen
hervorgerufen worden sein welches fuumlr die Entstehung der Taubheit in der
untersuchten Halbgeschwisterfamilie verantwortlich ist oder durch ein falsch-
positives Ergebnis der vorhergehend durchgefuumlhrten Mikrosatellitenstudie
Eine nicht-parametrische Kopplungsanalyse mit Hilfe von 27 Mikrosatelliten Marker
an 24 deutschen Dalmatinerfmilien wurde durchgefuumlhrt um abzuklaumlren ob andere
Genombereiche auf CFA1 signifikante Teststatistiken in mehr Familien zeigen Die
Kopplungsanalyse zeigte keine signifikanten Teststatistiken Es ist daher
unwahrscheinlich dass auf CFA1 Gene fuumlr die Entwicklung von CCSD in den
untersuchten Familien der aus Deutschland stammenden Dalmatiner verantwortlich
sind
Da kuumlrzlich festgestellt wurde dass das GJA1 Gen auch beim Menschen nicht fuumlr
eine Form der sensorineuralen nicht-syndromischen Taubheit verantwortlich ist
sondern an einer syndromischen Krankheit beteiligt ist die mit konduktiver Taubheit
einhergehen kann sollte das GJA1 Gen nicht laumlnger als ein Kandidatengen fuumlr
CCSD angesehen werden
CFA31
Auf CFA31 wurde durch eine nicht-parametrische Kopplungsanalyse von
Mikrosatelliten eine Kopplung eines CLDN14-benachbarten Markers mit der
sensorineuralen Taubheit beim Dalmatiner festgestellt Daraufhin wurden zusaumltzlich
zu den Mikrosatelliten acht SNPs entwickelt indem das einzige beim Menschen
translatierte Exon weite intronische Sequenzen sowie 5rsquo- und 3rsquo- untranslatierte
Bereiche des CLDN14 Gens einer Sequenzanalyse unterzogen wurden Nur zwei der
identifizierten SNPs waren polymorph in den untersuchten fuumlnf Dalmatinerfamilien
Sowohl uni- als auch bilateral taube und bilateral houmlrende Dalmatiner teilten
identische Haplotypen daher konnte keine Kosegregation mit tauben Dalmatinern
festgestellt werden
Da bislang keine funktionelle Mutation in der kodierenden Region von CLDN14
gefunden wurde ist es fuumlr eine eindeutige Aussage uumlber die Rolle des CLDN14
Gens im Entstehungsprozess der kongenitalen sensorineuralen Taubheit
Erweiterte Zusammenfassung 143
beim Dalmatiner notwendig noch weitere Marker im CLDN14 Gen sowie seiner
naumlheren Umgebung zu entwickeln und zu analysieren Da keine weitere Kopplung zu
einem Mikrosatelliten distal oder proximal des CLDN14 Gen festgestellt werden
konnte ist es wahrscheinlich dass entweder das CLDN14 Gen oder ein Gen in
seiner naumlheren Umgebung fuumlr die Entwicklung der sensorineuralen Taubheit in den
untersuchten Dalmatinerfamilien verantwortlich ist
CFA10
Auf CFA10 wurde durch eine nicht-parametrische Kopplungsanalyse von 27
Mikrosatelliten eine 12 Mb umfassende genomische Region mit einer signifikanten
Kopplung zur CCSD identifiziert Zur weiteren Feinkartierung wurden 26 neue SNPs
mit einem durchschnittlichen Abstand von 03 Mb fuumlr diesen Bereich entwickelt
Die SNPs waren groumlszligtenteils informativ im vorliegenden Familienmaterial und
bestaumltigten in einer folgenden Kopplungsanalyse die Lokalisation auf CFA10
deutlich Weiterhin konnte die gekoppelte Region auf einem 5 Mb umfassenden
Bereich von 39 bis 44 Mb eingegrenzt werden Es ist wahrscheinlich dass die
identifizierte CCSD Region ein Gen beinhaltet welches auf die Entstehung der
caninen kongenitalen Taubheit einwirkt
Es ist notwendig mehr SNPs zu entwickeln um das taubheitsverursachende Gen zu
lokalisieren oder einen eindeutig assoziierten SNP zu identifizieren
Chapter 12
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ROBERTSON NG LU L HELLER S MERCHANT SN EAVEY RD MCKENNA M
NADOL JB JR MIYAMOTO RT LINTHICUM FH JR LUBIANCA NETO JF
HUDSPETH AJ SEIDMAN CE MORTON CC SEIDMAN JG (1998)
Mutations in a novel cochlear gene cause DFNA9 a human nonsyndromic
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SCHNEEBERGER EE LYNCH RD (2004) The tight junction a multifunctional
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GUIPPONI M WANG J KAWASAKI K ASAKAWA S MINOSHIMA S
YOUNUS F MEHDI SQ RADHAKRISHNA U PAPASAVVAS MP
GEHRIG C ROSSIER C KOROSTISHEVSKY M GAL A SHIMIZU N
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SIMONS M WANG M MCBRIDE OW KAWAMOTO S YAMAKAWA K GDULA D
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539
STRAIN GM KEARNEY MT GIGNAC IJ LEVESQUE DC NELSON HJ
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TASSABEHJI M READ AP NEWTON VE HARRIS R BALLING R GRUSS P
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VAHAVA O MORELL R LYNCH ED WEISS S KAGAN ME AHITUV N
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VAN LAER L HUIZING EH VERSTREKEN M VAN ZUIJLEN D WAUTERS JG
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VERPY E LEIBOVICI M ZWAENEPOEL I LIU XZ GAL A SALEM N
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WANG A LIANG Y FRIDELL RA PROBST FJ WILCOX ER TOUCHMAN JW
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WATTENHOFER M REYMOND A FALCIOLA V CHAROLLAIS A CAILLE D
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Chapter 13
Appendix
Appendix microsatellite marker III
HE
Te
79
5 7
98
76
9 5
12
71
3 8
33
70
2 4
35
72
7 7
00
54
6 4
77
48
8 3
08
44
8 5
40
67
4 8
05
76
2 6
54
70
8 7
73
78
1 6
87
65
4 6
26
51
9 5
89
76
4 1
60
PIC
d
72
2 8
41
67
0 4
85
67
1 7
85
57
2 4
07
61
4 6
29
35
8 3
30
40
0 2
90
35
0 4
60
70
0 7
90
76
2 6
21
63
8 7
26
77
1 6
26
60
1 6
13
38
7 5
52
80
0 1
40
Prim
er (r
ever
se)
5acute -gt
3acute
AA
CTG
TTTG
ATT
TTG
ATG
AG
GC
C
TTTG
ACC
TAC
ATA
TAA
AA
CA
AG
C
CA
CG
AA
GAA
AG
CC
ATG
GTT
T C
AA
CA
ATT
CC
TTTG
TTG
GTG
C
CTG
CC
AG
CTT
CC
TAC
AGC
T
GTG
GTC
ACA
AG
AC
TTTA
GC
C
CTG
CA
CC
AAA
GA
AA
CTC
C
GG
AC
CTA
TTC
TGA
AG
CC
TAA
C
TG
GC
CTT
TAA
TTTA
TCAT
GG
AG
A
TCA
GG
CC
CA
GA
TAG
TATG
C
TC
CTT
TGG
TTTT
TAG
CAG
GG
T
CTG
TGC
CC
AC
CTG
TGG
AG
C
AA
GA
GG
ATA
CC
GG
TGG
CA
G
CG
TGC
TTTG
TTA
TGG
CTT
GA
C
AG
CA
TGA
AG
GA
TCTC
TGA
CTA
A
CTA
AC
TCG
CC
AG
AC
TATT
C
CC
CTC
CAC
CA
ATC
TCTT
CA
C
GC
AA
AG
GTG
TATT
TAC
ATG
ATG
G
GG
CA
GC
CTT
ATT
ATT
CA
TGG
A
AC
AC
AG
GC
AC
AG
GA
GC
ATC
C
AG
CC
ATC
CA
GG
AA
ATC
G
CTG
AA
CTG
GTC
CC
TCAA
GC
A
GTC
ATG
CTG
ATT
TCTG
TGC
C
GA
CC
TATC
CA
TAC
ATG
CC
C
GAA
GG
AAG
GAA
GG
AAG
AAAA
GA
TTA
GTA
AA
GA
GA
CA
GC
TGA
TTG
CC
A
TCTA
CC
CTG
CA
ACC
CTG
TG
ATT
GA
GA
CC
CA
AG
ACTG
TTA
GTG
G
GTA
AC
AAC
CA
GAG
TGTG
TGTG
A
CG
TCG
AG
CTC
CTG
GC
AT
Prim
er (f
orw
ard)
5acute -
gt 3acute
GG
GTA
GA
TTTC
AA
CA
AAT
AG
TAC
TGG
T
AG
AA
AG
GC
CTG
GA
TGTC
G
AG
TAG
AA
GA
GG
CC
AC
GC
AA
A
TTT
TCC
AA
AA
TGA
AA
AC
CTG
C
AG
ATT
GTT
TATG
CA
GG
CA
TTG
A
GA
GTT
GAA
AG
GTT
GAA
AA
TGG
G
CTC
AG
AG
GC
AG
ATA
GA
AA
A
ATT
GA
TTC
ATG
ACC
CA
CTA
A
TTT
TGTC
AAG
CA
GC
CC
TATA
TC
TG
AA
ATG
GTA
CTT
CA
GC
ATC
G
AG
CTT
AG
CTT
AC
TGC
CC
TGG
T
CC
CC
GG
CC
CC
TGC
CC
GG
AG
T
AC
CAC
TGTC
ATT
TTTC
CA
TGC
C
CC
TTA
GG
AGG
AG
GC
AAG
AC
C
CA
AG
GG
GTA
TGTT
GTC
TATT
ACT
GG
GTG
GG
AA
ATG
TGA
CTC
TG
GG
GC
TGC
CC
ATT
TCTT
TAA
T T
TGC
CA
CAA
ATC
AC
TTAA
AG
G
GA
CTG
AG
TTC
TTTC
AG
CA
CA
GTG
A
AAAT
AAC
ACAG
CAT
CAG
G
GA
ATC
CC
CA
ATG
TAC
ATG
GC
A
AC
AG
GC
GG
ATT
CTG
TAG
C
AC
TGG
CC
AAA
GA
GTA
CA
AG
G
CC
TTG
AC
TCA
GC
AGC
CC
TAC
A
AA
GA
TTG
TCTT
GA
CAC
GC
TG
GG
GTC
CTG
GG
ATC
AA
GC
C
AC
TGA
CTG
ATG
TCC
TGTG
CG
A
AA
AA
GTG
TAG
AG
CTT
TCTT
CA
AA
T
AA
ATT
CA
ATA
AG
CC
ATG
GA
GG
A
CC
TCC
AAG
ATG
GC
TCTT
GA
ATc
(degC
) 60
58
58
56
58
60
54
56
60
60
58
60
60
60
60
60
60
60
60
52
56
58
54
62
60
60
62
62
60
60
Pro
duct
si
ze (b
p)
380-
400
337-
409
267-
304
225-
235
312-
336
350-
394
155-
161
126-
156
510-
570
436-
468
189-
191
72-8
0 13
0-13
6 34
0-35
0 14
5-15
3 98
-102
22
0-24
0 21
2-24
0 34
7-40
3 32
5-34
1 25
1-27
9 32
5-36
1 19
7-24
5 30
9-40
9 34
1-37
5 21
5-23
1 30
8-31
6 16
2-17
4 46
8-50
2 18
2-19
0
Alle
les
6 14
8 4 7 11
3 6 8 7 2 2 3 4 2 3 5 8 12
7 8 7 14
10
9 7 3 3 15
2
Mbb
54
1
49
19
8 2
18
23
5 2
47
26
0 3
02
35
3 3
98
58
8 6
26
63
9 7
61
77
5 7
78
78
6 7
98
85
4 1
060
1
090
1
100
1
100
1
110
1
130
1
150
1
170
3
5
12
4 1
63
CFA
a
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10
10
10
Tabl
e 1
Cha
ract
eris
tics
of m
icro
sate
llite
mar
kers
sel
ecte
d fo
r CFA
1 1
0 an
d 31
com
pris
ing
in to
tal 6
0 m
arke
rs
(Con
tinue
d on
nex
t pag
e) T
he m
icro
sate
llite
mar
kers
wer
e al
l der
ived
from
the
NC
BI d
atab
ase
(http
w
ebn
cbin
lmn
ihg
ov)
Mar
ker
FH33
25
FH36
03
FH23
13
RE
N18
9I14
FH
3219
FH
3413
R
EN
136G
19
RE
N13
5K06
FH
3922
FH
3300
C
014
24
AH
T107
C
012
51
RE
N17
2C02
D
0150
5 R
EN
04H
08
RE
N15
9F24
FH
3894
FH
2309
R
EN
06N
11
FH23
26
FH33
22
FH35
05
FH25
98
FH26
34
FH22
94
RE
N22
9P15
FH
2537
FH
4081
C
107
81
Appendix microsatellite marker
IV
H
ETe
64
0 6
80
63
0 6
51
54
6 7
06
81
0 6
14
30
2 7
42
34
4 7
40
63
0 8
67
82
2 6
70
75
0 6
80
60
0 4
70
31
0 5
70
80
1 5
80
83
1 5
10
64
0 6
90
19
0 8
22
PIC
d
53
0 6
90
52
2 5
89
80
0 6
70
75
9 5
24
24
7 7
09
26
9 6
90
50
0 7
98
75
5 5
50
69
0 5
30
56
0 3
40
26
0 5
00
80
1 5
30
74
0 3
70
64
0 6
70
25
0 7
21
Prim
er (r
ever
se)
5acute -gt
3acute
AG
GC
CA
GC
AC
CA
TAA
CTC
AG
C
CTC
TCC
TCC
TTC
CC
CTT
CTC
A
GTG
CTG
TCC
ATT
GC
AA
GTC
T
GC
TCC
CC
TGTG
TTC
TGAG
G
CTG
GTG
ACTC
AGA
GAA
GTC
C
GG
AG
TTG
ATA
GTA
AA
TCTT
TGC
GG
T
GC
CC
TATT
CA
TTC
AA
CTG
G
AC
AC
TCTG
GTG
CA
AG
CG
AC
TC
TTC
TAC
TGA
AG
CA
TCC
GC
CT
GA
TTTT
CTC
TCTG
TCC
AC
TT
AA
CA
GC
ATT
TCA
GAC
AGA
GG
G
GG
TTTT
GG
CTG
TGA
GG
ATA
G
AG
TTC
CAG
ATC
GAG
TCC
CA
C
CA
CAT
GAT
TTC
ACTT
GTA
TATG
G
CC
TGA
GG
GTT
TTTA
CAG
TATG
G
TG
AA
ATC
TGG
GA
TTC
TGA
TAC
C
CC
AA
GC
TTTC
AC
CTA
ATC
AC
C
GA
TATT
TTTC
TCTC
CC
AC
CC
C
AC
ATT
TCTA
GG
CAG
TGG
CA
C
TGTC
CC
ATC
TAA
AG
GA
TAG
G
AG
TAA
TGC
TGC
CC
ACG
GA
AC
G
GC
TGG
AA
CC
TTC
CC
CTT
TAG
A
GC
TCTT
ACA
CG
CA
TTG
AG
G
AA
AC
GG
AG
TTC
CA
TCTC
TGG
C
AC
AG
TGA
GG
AG
GG
GTG
G
TG
TAA
CC
CTC
CTG
TGA
TATG
A
TGTG
AA
CC
CC
GC
CC
AATA
G
AA
GTG
AC
TGA
GTA
CC
TGA
AA
TCG
G
CC
TCTG
CC
TCTG
CC
TCTG
T G
AG
CC
CTG
TTC
TCA
GG
TTG
Prim
er (f
orw
ard)
5acute -
gt 3acute
AG
AC
CC
CC
CC
ACTA
CC
CC
AC
GC
GTT
AGC
TGC
CA
TCA
TCTT
G
CTG
GTT
GG
AG
CA
ATA
CC
AA
G
CC
ATT
CA
TGTT
GTT
GC
AG
ATG
T
AA
GG
AG
GG
CA
CTT
GAT
GG
C
TTA
TGG
CA
TTTG
GC
CTG
TC
AA
GG
AG
CAT
CTT
CC
AG
AAC
C
CG
CTC
ATG
CA
AG
TCA
TCA
CA
T C
TGG
CTT
AAA
CC
AC
TGG
TCA
C
CTT
CC
ATC
CC
GTT
GTG
TGT
ATT
CC
CC
AGC
GAT
ACC
A C
TTG
AG
TGG
CTT
GA
GTG
GC
TAC
T
TCTT
GC
TTC
TTG
AA
GTA
AG
CC
T
TGC
CC
GTC
CTA
TAC
TCC
TG
GA
AG
GTG
GTT
ATT
GTC
CTT
GG
G
AA
CC
ACA
GA
AG
AG
CTG
GA
A
GC
ATT
TGA
TGA
AA
TAA
AG
GG
A
GA
ATG
AA
AA
CG
GA
GC
AG
CA
G
CC
TTC
AAC
AC
CC
ATA
GC
TC
TC
AG
CA
ACTA
TAC
ATT
TAA
GA
GC
A
ATG
GA
CA
AATG
AA
CA
AA
AG
T
GTG
AC
TTTC
TTA
TCC
GC
CC
C
CC
AG
AA
AC
TCA
ACTG
ATG
C
TG
GA
CG
CTA
AG
CC
TGAC
TTT
CC
ATA
AG
ATA
CTC
AG
AA
AC
ATG
CA
C
AC
CAT
AA
ATG
GA
TGG
ATA
G
CA
GTG
AG
CA
AA
GC
AA
ATG
AA
C
CC
ATT
AG
CA
AA
TGA
CTG
GG
A
AA
GA
ATG
GG
AA
AA
CTG
ATA
A
AA
GG
TAG
TCC
CA
CG
ATC
CTC
ATc
(degC
) 60
60
60
60
60
60
60
60
60
60
60
60
60
62
60
60
60
60
60
60
60
60
60
60
60
60
60
60
60
60
Pro
duct
si
ze (b
p)
100-
110
112-
124
194-
206
210-
230
309-
341
350-
362
276-
300
96-1
30
130-
138
120-
138
200-
204
278-
296
157-
165
178-
254
358-
368
327-
337
380-
404
160-
170
320-
330
165-
169
230-
232
300-
310
276-
380
255-
263
258-
278
106-
120
164-
180
568-
608
223-
239
172-
186
Alle
les
3 5 3 5 8 4 7 4 2 5 3 7 5 9 6 4 7 3 4 2 2 3 11
5 6 2 4 7 4 8
Mbb
24
9 3
23
34
9 3
67
38
9 3
94
40
1 4
10
45
8 4
64
48
0 4
88
51
5 5
64
56
8 5
97
62
7 6
33
63
7 6
53
67
3 6
85
69
1 7
08
0
66
9
7
28
1 2
96
37
5
CFA
a
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
31
31
31
31
31
31
Tabl
e 1
(con
tinue
d)
Mar
ker
RE
N06
H21
R
EN
68B
08
FH30
55
C10
769
FH
3302
R
EN
181G
20
FH34
03
G02
512
RE
N15
0B12
A
HT1
01
RE
N73
F08
ZUB
EC
A1
C10
16
FH24
22
FH34
48
FH26
65
DTR
105
C
106
02
RE
N16
1L12
C
0410
7 R
EN
91H
07
RE
N15
4O19
FH
3381
R
EN
154G
10
FH21
89
RV
C11
R
EN
43H
24
FH22
39
RE
N11
0K04
FH
2712
a Can
ine
chro
mos
ome
b Pos
ition
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Appendix itemisation of alleles V
Table 2 Itemisation of alleles to bases for SNPs in the pedigrees
Code no 1 2 3 4
Base Adenine Cytosine Guanine Thymine
Appendix laboratory paraphernalia
VI
Laboratory paraphernalia Equipment Thermocycler
PTC-100trade Programmable Thermal Controller (MJ Research Watertown USA)
PTC-100trade Peltier Thermal Cycler (MJ Research Watertown USA)
PTC-200trade Peltier Thermal Cycler (MJ Research Watertown USA)
Automated sequencers
LI-COR Gene Read IR 4200 DNA Analyzer (LI-COR Inc Lincoln NE USA)
LI-COR Gene Read IR 4300 DNA Analyzer (LI-COR Inc Lincoln NE USA)
MegaBACE 1000 (Amersham Biosciences Freiburg)
Centrifuges
Sigma centrifuge 4-15 (Sigma Laborzentrifugen GmbH Osterode)
Desk-centrifuge 5415D (Eppendorf Hamburg)
Biofuge stratos (Heraeus Osterode)
Centrifuge Centrikon H 401 (Kontron Gosheim)
Megafuge 1OR (Heraeus Osterode)
Speed Vacreg Plus (Savant Instruments Farmingdale NY USA)
Agarose gel electrophoresis and pulsed field gel electrophoresis
Electrophoresis chambers OWL Separation Systems Portsmouth NH USA
Biometra Goumlttingen
BioRad Muumlnchen
Generators 2301 Macrodrive 1 (LKB Bromma Sweden)
Power Pac 3000 (BioRad Muumlnchen)
Gel documentation system BioDocAnalyze 312 nm Goumlttingen
Appendix laboratory paraphernalia VII
Others
Milli-Qreg biocel water purification system (Millipore GmbH Eschborn)
Incubator VT 5042 (Heraeus Osterode)
UV-Illuminator 312 nm (Bachhofer Reutlingen)
Centomatreg R Desk-Shaker (B Braun Melsungen AG Melsungen)
Biophotometer (Eppendorf AG Hamburg)
Kits
DNA purification
Montage PCR96 Cleanup Kit (Millipore GmbH Eschborn)
Cloning
Invitrogen TA cloningreg kit (Invitrogen Karlsruhe Germany)
Isolation of DNA
QIAamp 96 DNA Blood Kit (QIAGEN Hilden)
Plasmid Mini Prep 96 Kit (Millipore GmbH Eschborn)
Sequencing
ThermoSequenase Sequencing Kit (Amersham Biosciences Freiburg)
DYEnamic-ET-Terminator Cycle Sequencing Kit (Amersham Biosciences Freiburg
Germany)
RACE
FirstChoiceTM RNA ligase-mediated (RLM)-RACE kit (Ambion Europe Huntingdon
UK)
Appendix laboratory paraphernalia
VIII
RNA Total RNA (Biocat Heidelberg Germany)
Size standards
100 bp Ladder (New England Biolabs Schwalbach Taunus)
1 kb Ladder (New England Biolabs Schwalbach Taunus)
IRDyetrade 700 or 800 (LI-COR Inc Lincoln NE USA)
Reagents and buffers APS solution (10 )
1 g APS
10 ml H2O
Bromophenol blue solution
05 g bromophenol blue
10 ml 05 M EDTA solution
H2O ad 50 ml
dNTP solution
100 microl dATP [100 mM]
100 microl dCTP [100 mM]
100 microl dGTP [100 mM]
100 microl dTTP [100 mM]
1600 microl H2O
the concentration of each dNTP in the ready-to-use solution is 5 mM
Gel solution
1275 ml UreaTBE solution (Roth Karlsruhe)
225 ml Rotiphoresereg Gel 40 (38 acrylamide and 2 bisacrylamide)
95 microl APS solution (10 )
95 microl TEMED
Appendix laboratory paraphernalia IX
Loading buffer for agarose gels
EDTA pH 8 100 mM
Ficoll 400 20 (wv)
Bromophenol blue 025 (wv)
Xylencyanol 025 (wv)
Loading buffer for gel electrophoresis
2 ml bromophenol blue solution
20 ml formamide
TBE-buffer (1x)
100 ml TBE-buffer (10x)
900 ml H2O
TBE-buffer (10x)
108 g Tris [12114 M]
55 g boric acid [6183 M]
744 g EDTA [37224 M]
H2O ad 1000 ml
pH 80
UreaTBE solution (6 )
425 g urea [6006 M]
250 ml H2O
100 ml TBE-buffer (10x)
solubilise in a water bath at 65degC
H2O ad 850 ml
Appendix laboratory paraphernalia
X
Chemicals Agarose (Invitrogen Paisley UK)
Ammonium persulfate (APS) ge 98 (Sigma-Aldrich Chemie GmbH Steinheim)
Ampicillin (Serva Heidelberg)
Boric acid ge 998 pa (Carl Roth GmbH amp Co Karlsruhe)
Bromophenol blue (Merck KgaA Darmstadt)
dATP dCTP dGTP dTTP gt 98 (Carl Roth GmbH amp Co Karlsruhe)
Chloramphenicol (Serva Heidelberg)
DMSO ge 995 pa (Carl Roth GmbH amp Co Karlsruhe)
dNTP-Mix (Qbiogene GmbH Heidelberg)
EDTA ge 99 pa (Carl Roth GmbH amp Co Karlsruhe)
Ethidium bromide (Carl Roth GmbH amp Co Karlsruhe)
Ethyl alcohol (AppliChem Darmstadt)
Formamide ge 995 pa (Carl Roth GmbH amp Co Karlsruhe)
LB (Luria Bertani) agar (Scharlau Microbiology Barcelona Spain)
Paraffin (Merck KgaA Darmstadt)
RotiphoreseregGel40 (Carl Roth GmbH amp Co Karlsruhe)
SephadexTM G-50 Superfine (Amersham Biosciences Freiburg)
TEMED 99 pa (Carl Roth GmbH amp Co Karlsruhe)
Tris PUFFERANreg ge 999 pa (Carl Roth GmbH amp Co Karlsruhe)
Urea ge 995 pa (Carl Roth GmbH amp Co Karlsruhe)
Water was taken from the water purification system Milli-Qreg
X-Gal (AppliChem Darmstadt)
Enzymes Taq-DNA-Polymerase 5 Umicrol (Qbiogene GmbH Heidelberg)
Taq-DNA-Polymerase 5 U microl (Qiagen Hilden Germany)
Incubation Mix (10x) TPol with MgCl2 [15 mM] (Qbiogene GmbH Heidelberg)
The polymerase was always used in the presence of incubation Mix TPol 10x buffer
The encyme ECO RI (New England Biolabs Schwalbach Taunus) were used with
the adequate 10x encyme buffer
Appendix laboratory paraphernalia XI
Consumables
96 Well Multiply PCR plates skirted (Sarstedt Nuumlmbrecht)
Combitipsreg plus (Eppendorf AG Hamburg)
Pipette tips 01 ndash 10 microl 01 ndash 10 microl 5 ndash 200 microl (Carl Roth GmbH amp Co Karlsruhe)
Reaction tubes 15 and 20 ml (nerbe plus GmbH WinsenLuhe)
Reaction tubes 10 und 50 ml (Falcon) (Renner Darmstadt)
Thermo-fast 96 well plate skirted (ABgene Hamburg)
Software BLAST trace archive httpwwwncbinlmnihgov
httpwwwensemblorg
EBI toolbox httpwwwebiacukToolssequencehtml
MERLIN 0102 package httpwwwsphumicheducsgabecasisMerlin
Order of primers MWG Biotech-AG Ebersberg (httpsecom
mwgdnacomregisterindextcl)
biomersnet GmbH Ulm (orderbiomersnet)
PED50 Dr H Plendl et al (2005) Institute for Human Genetics
Kiel
Primer design httpfrodowimiteducgi-binprimer3primer3_ wwwcgi
Repeat Masker httpwwwrepeatmaskergenome washingtonedu
Sequencher 42 GeneCodes Ann Arbor MI USA
Spidey httpwwwncbinlmnihgovIEBResearch
OstellSpideyindexhtml
SUN Ultra Enterprise 450 Sun microsystems
XIII
List of publications Journal articles 1 MIESKES K WOumlHLKE A DROumlGEMUumlLLER C DISTL O 2006 Molecular
characterization of the canine myosin heavy polypeptide 9 non-muscle (MYH9)
gene on dog chromosome 10q232 Submitted for publication in Gene
2 MIESKES K DISTL O 2006 Evaluation of 17 single nucleotide
polymorphisms (SNPs) in six candidate genes for hereditary non-syndromic deafness
in Dalmatian dogs Submitted for publication in Journal of Heredity
3 MIESKES K DISTL O 2006 Evaluation of the TMC1 and TMIE genes as
candidates for hereditary non-syndromic deafness in Dalmatian dogs Submitted for
publication in Animal Genetics
XV
Acknowledgements
First of all I wish to thank Prof Dr Dr habil Ottmar Distl the supervisor of my
doctoral thesis for his academic guidance and support of this work
I am very thankful to Heike Klippert-Hasberg and Stefan Neander for technical
expertise and assistance
I am very grateful to Joumlrn Wrede for his support during the statistical analyses and his
help with computer problems
I wish to express my appreciation to the Gesellschaft zur Foumlrderung Kynologischer
Forschung (GKF) eV Germany for funding this work with a grant
I am appreciative to all Dalmatian breeders and owners for providing me blood
samples and the results of the BAER tests
My special thanks go to all colleagues and friends of the Institute for Animal Breeding
and Genetics of the University of Veterinary Medicine Hannover for their support
humour and the friendly atmosphere in the laboratory
Last but not least I wish to thank my family for their support during the work on this
thesis
To my family
Parts of this work have been submitted for publication in the following journals
1 Gene
2 Journal of Heredity
3 Animal Genetics
Contents
1 Introduction 1
2 A comparative overview of the molecular genetics of non-syndromic deafness in dogs and humans 5
Abstract 7
The structure of the ear 7
Deafness in man 8
Deafness in dogs 9
The canine genome project 11
3 Linkage analysis of gene-associated microsatellite markers with congenital sensorineural deafness in Dalmatian dogs 23
Abstract 25
Introduction 25
Material and methods 26
Results and discussion 28
4 Evaluation of eight candidate genes for canine congenital sensorineural deafness in Dalmatian dogs 41
Abstract 43
Introduction 43
Material and methods 44
Results and discussion 46
5 Molecular characterization of the canine myosin heavy polypeptide 9 non-muscle (MYH9) gene on dog chromosome 10q232 63
Abstract 65
Introduction 65
Materials and Methods 67
Results and Discussion 69
6 Identification of a 5 Mb region on canine chromosome 10 harbouring a causative gene responsible for congenital sensorineural deafness in German Dalmatian dogs 81
Abstract 83
Introduction 83
Material and methods 84
Results 86
Discussion 87
7 Analysis of canine chromosome 1 and the Gap junction protein alpha 1 (GJA1) gene in Dalmatian dogs segregating for congenital sensorineural deafness 97
Abstract 99
Introduction 99
Material and methods 100
Results and discussion 102
8 Analysis of canine chromosome 31 and the claudin-14 (CLDN14) gene in Dalmatian dogs segregating for congenital sensorineural deafness 107
Abstract 109
Introduction 109
Material and methods 110
Results and discussion 112
9 General Discussion 117
The candidate gene approach 119
Linkage and association analysis 120
CFA1 122
CFA31 123
CFA10 123
10 Summary 125
11 Erweiterte Zusammenfassung 129
12 References 145
13 Appendix I 14 List of publications XIII
Abbreviations
List of abbreviations
A adenine
Acc No accession number
ACTG1 actin gamma 1
AEP akustisch evozierte Potentiale (acoustically evoked potentials)
APS ammonium persulphate
AT annealing temperature
BAC bacterial artificial chromosome
BAER brain stem auditory evoked response
BLAST Basic Local Alignment Search Tool
bp base pair
C cytosine
CCSD canine congenital sensorineural deafness
CDH23 cadherin related 23
cDNA copy desoxyribonucleic acid
CFA chromosome of Canis familiaris
CLDN14 claudin-14
cM centiMorgan
COCH coagulation factor C homolog cochlin
COL11A2 collagen type XI alpha 2
CRYM crystallin mu
CSD cochleosaccular degeneration
CX connexin
DFN x-linked deafness locus
DFNA autosomal dominant deafness locus
DFNA5 deafness autosomal dominant 5
DFNB autosomal recessive deafness locus
DIAPH1 diaphanous homolog 1 (Drosophila)
DMSO dimethyl sulfoxide
Abbreviations
DNA deoxyribonucleic acid
dNTPs deoxy nucleoside 5rsquotriphosphates (N is ACG or T)
EDN3 endothelin 3
EDNRB endothelin receptor type B
EDTA ethylenediamine tetraaceticacid
EMBL European Molecular Biology Laboratory
ESPN espin
EST expressed sequence tag
EYA4 eyes absent homolog 4 (Drosophila)
F forward
FISH fluorescence in situ hybridisation
G guanine
GJA1 gap junction protein alpha 1 43kD (connexin 43)
GJB2 gap junction protein beta 2 26k (connexin 26)
GJB3 gap junction protein beta 3 31kDa (connexin 31)
GJB6 gap junction protein beta 6 (connexin 30)
GKF Gesellschaft zur Foumlrderung Kynologischer Forschung (Society for the
Advancement of Cynological Research)
HET observed heterozygocity
HE expected heterozygosity value
HSA chromosome of Homo sapiens
IBD identical by descent
IRD infrared dye
KCNQ4 potassium voltage-gated channel KQT-like subfamily member 4
Kb kilobase
LD linkage disequilibrium
LINE long interspersed nuclear element
LOD logarithm of the odds
M molar
Mb megabase
Merlin multipoint engine for rapid likelihood inference
MITF microphthalmia-associated transcription factor
Abbreviations
MS microsatellite
MTRNR1 mitochondrially encoded 12S RNA
MTTS1 mitochondrially encoded tRNA serine 1 (UCN)
MYH9 myosin heavy polypeptide 9 non-muscle
MYH14 myosin heavy polypeptide 14
MYO1A myosin IA
MYO3A myosin IIIA
MYO6 myosin VI
MYO7A myosin VIIA
MYO15A myosin XVA
NCBI National Center for Biotechnology Information
NMMHC-A nonmuscle myosin heavy chain-A
ODDD oculodentodigital dysplasia
OMIM Online Mendelian Inheritance in Man
OTOA Otoancorin
OTOF otoferlin
P error probability
PAX3 paired box gene 3 (Waardenburg syndrome 1)
PCDH15 Protocadherin-15
PCR polymerase chain reaction
PIC polymorphism information content
POU3F4 POU domain class 3 transcription factor 4
POU4F3 POU domain class 4 transcription factor 3
PRES solute carrier family 26 member 5 (prestin)
QTL quantitative trait locus
R reverse
RACE rapid amplification of cDNA ends
RH radiation-hybrid
RLM RNA ligase-mediated
RNA ribonucleic acid
RT-PCR reverse transcription polymerase chain reaction
SAS Statistical Analysis System
Abbreviations
SH1 Src homology 1
SINE short interspersed nuclear element
SLC26A4 solute carrier family 26 member 4 (pendrin)
SLC26A5 solute carrier family 26 member 5 (prestin)
SNP single nucleotide polymorphism
SOX10 SRY (sex determining region Y)-box 10
STRC stereocilin
STS sequence-tagged site
T thymine
TBE tris-borate-ethylenediamine tetraacetic acid
TECTA tectorin alpha
TEMED NNNrsquoNrsquo-tetramethylenediamine
TFCP2L3 transcription factor CP2-like 3
TJ tight juncions
TMC1 transmembrane channel-like gene 1
TMIE transmembran inner ear gene
TMPRSS3 transmembrane protease serine 3
U unit
USH1C Usher syndrome 1C
UTR untranslated region
WFS1 Wolfram syndrome 1 (wolframin)
wgs whole genome shotgun
WHRN whirlin
WS Waardenburg syndrome
Chapter 1
Introduction
Introduction 3
Introduction
Canine congenital sensorineural deafness (CCSD) has often been reported in the
literature and occurs in more than 54 different breeds of dogs with the Dalmatian dog
showing the highest incidence The inheritance and segregation of a major gene in
CCSD has been demonstrated in different Dalmatian dog populations But although
several studies have demonstrated the mode of inheritance in Dalmatian dogs no
universally accepted mode of inheritance for the other dog breeds affected by CCSD
has yet been identified
Breeding with blue-eyed Dalmatian dogs and unilaterally or bilaterally deaf dams and
sires is forbidden by paragraph 11b of the German animal welfare laws and thus the
hearing status of all Dalmatian dogs has to be tested as a puppy at about six to eight
weeks old using the brain stem auditory evoked response (BAER) test As deaf dogs
are very difficult to raise and often become aggressive and snappish from fear most
puppies suffering from bilateral hearing loss are euthanized However it has been
shown in recent years that auditory testing does not seem to be an effective way of
clearly reducing the high incidence of deafness in this breed Thus prevention of
CCSD cannot be achieved alone by exclusion of affected animals from breeding
Consequently a molecular genetic approach toward unravelling the responsible
genes in carriers is urgently needed
Many genetic disorders in humans and domestic dogs (Canis familiaris) demonstrate
a high level of clinical and molecular similarity Therefore the mutated genes in
human hereditary deafness seemed to be appropriate candidates for canine
congenital sensorineural deafness
The objective of the present study is to localize the gene that is involved in the
development of CCSD in Dalmatian dogs In order to achieve this goal successively
32 canidate genes were evaluated by means of linkage analyses using microsatellite
markers and single nucleotide polymorphisms (SNPs) This candidate gene
approach using gene-associated markers for linkage studies in families segregating
for deafness turned out to be little effective Therefore the canine chromosomes
(CFA) 1 CFA10 and CFA31 were scanned entirely with microsatellite markers
Additionally single nucleotide polymorphisms (SNPs) were developed for fine
mapping the identified CCSD regions
Introduction 4
Overview of chapter contents Chapter 2 reviews the identified 39 mutated genes causing non-syndromic hereditary
hearing impairment in humans Parallels and differences in canine and human
deafness are shown including the clinical signs inheritance patterns and
histopathology We located the humane deafness genes in the canine genome and
discussed the advantages of comparative genomics and different molecular genetic
approaches
In Chapter 3 an existing set of 43 microsatellite markers for in total 24 candidate
genes were used for a non-parametric linkage analysis with congenital sensorineural
deafness (CCSD) in Dalmatian dog families segregating for deafness
In Chapter 4 newly developed SNP markers associated with in total eight candidate
genes were evaluated for CCSD in Dalmatian dogs
In Chapter 5 the molecular characterization of the canine myosin heavy polypeptide
9 non-muscle (MYH9) gene on dog chromosome 10q232 is described
Chapter 6 7 and 8 present linkage analyses performed in Dalmatian dog families
segregating for congenital sensorineural deafness using microsatellite markers on
canine chromosome (CFA) 1 CFA10 and CFA31 and the results of fine mapping
regions linked with the CCSD phenotype using newly developed SNPs
Chapter 9 provides a general discussion and conclusions referring to Chapters 1-8
Chapter 10 is a concise English summary of this thesis while Chapter 11 is an
expanded detailed German summary which takes into consideration the overall
research context
Chapter 2
A comparative overview of the molecular genetics of non-syndromic deafness
in dogs and humans
Non-syndromic deafness in dogs and humans 7
A comparative overview of the molecular genetics of non-syndromic deafness in dogs and humans
Abstract
In man as in different dog breeds deafness is an often diagnosed disorder with the
Dalmatian dog showing the highest incidence Deafness in Dalmatian dogs is clearly
heritable and the presence of a recessive major gene affecting the disorder was
shown in several Dalmatian dog populations
This Chapter provides an overview of the identified 39 mutated genes causing
human non-syndromic hereditary hearing impairment as well as of the five genes
responsible for Waardenburg syndrome in humans We point out their cytogenetic
and genomic localisations in man and dog and compare the genomic and mRNA
sequences of these genes between man and dog Moreover an overview is given on
deafness genes-associated markers identified in Dalmatian dogs and on candidate
genes characterized in dogs
The structure of the ear The inner ear consists of the semicircular canals the vestibule and the cochlea
whereas the vestibule and the semicircular canals are concerned with vestibular
function (balance) and the cocnlea is concerned with hearing Reissneracutes membrane
and the basilar membrane divide the cochlea longitudinally into three scalae the
scala vestibule the scala media and the scala tympani The process of transduction
occurs in the structures within scala media sitting on the basilar membrane and
comprising the organ of Corti Cutting the cochlea tube cross sectionally the scala
media is more or less triangular formed by Reissneracutes membrane basilar
membrane and a structure called the stria vascularis The fluid that fills scala
tympani and scala vestibule is called perilymph the fluid that fills scala media is
called endolymph The organ of Corti rests on the basilar membrane within scala
media The cochlea contains an array of highly specialized cells arranged in a highly
Non-syndromic deafness in dogs and humans 8
specialized manner Two types of cells in the organ of Corti are support cells and
hair cells The hair cells are the receptor cells that trancsduce sound
When a sound wave brings physical displacement of the membranes separating the
perilymph from the endolymph they cause the organ of Corti to move and the hair
cells on it are scraped along the bottom of the tectorial membrane The tectorial
membrane is firmly anchored to the bone Relative movement of the organ of Corti
and its hair cells with respect to the tectorial membrane is the source of the
deformation of the hair cells microvilli The hair cells are so constructed that any
deformation of their microvilli will cause a change in the overall membrane potential
of the cell This signal is detected by the fibers from the cells in the spiral ganglion
These fibers are neural elements and they carry their own depolarization wave into
the auditory region of the brain
Deafness in man There are various ways to categorise deafness The two main types of deafness are
classified based on which portions of the auditory system are affected conductive
hearing loss occurs when when sound is not conducted efficiently through the outer
andor middle part of the ear Much more common is the sensorineural hearing loss
Sensorineural hearing loss occurs when there is damage to the inner ear (cochlea)
or to the nerve pathways from the inner ear (retrocochlear) to the brain Most cases
of sensorineural hearing loss are due to cochlear defects (Petit et al 2001)
Hearing loss can be present at birth (congenital) or become evident later in life
(acquired) Congenital deafness similarly may or may not be genetic In fact more
than half of congenital hearing loss is inherited Alternatively congenital deafness
may be due to a condition or infection to which the mother was exposed during
pregnancy Furthermore congenital hereditary deafness may occur as part of a
multisystem disease (syndromic) or as a disorder restricted to the ear and vestibular
system (non-syndromic) As non-syndromic hereditary hearing impairment is almost
exclusively caused by cochlear defects affected patients suffer from sensorineural
hearing loss In Table 1 and 2 the genes underlying human hereditary non-
syndromic deafness as a result of cochlear defects in consequence of primary
defects in hair cells non-sensory cells and the tectorial membrane or unknown cell
type are shown Non-syndromic deafness is estimated to cause 60-70 of cases of
congenital hereditary deafness in man (Morton 1991) In 70-80 of all cases this
Non-syndromic deafness in dogs and humans 9
non-syndromic form of deafness shows an autosomal recessive inheritance followed
by an autosomal dominant inheritance in 10-20 of all cases and 1-2 of all cases
are X-linked A maternally inherited form may also occur (Van Camp and Smith
2003)
Non-syndromic forms of hereditary deafness are classified by their mode of
inheritance DFN DFNA and DFNB refer to deafness forms inherited on the X
chromosome-linked autosomal dominant and autosomal recessive modes of
transmission respectively
Human hereditary isolated hearing loss is genetically heterogeneous (Petit et al
2001) Up to 1 of the human genes are estimated to be necessary for hearing
(Friedmann and Griffith 2003) Today approximately 120 genes for human
hereditary deafness have been identified approximately 80 for syndromic and 39 for
non-syndromic hereditary deafness which is suspected to be one-third of the total
(Nance 2003) In Table 1 the identified 39 mutated genes causing non-syndromic
hereditary hearing impairment in humans are shown Out of the 39 genes 15 genes
cause autosomal recessive and 15 genes cause autosomal dominant forms six
genes are involved in both recessive and dominant forms one gene causes X-linked
and two a maternally inherited form (The Hereditary Hearing Loss Hompage
httpwebhostuaacbehhh)
Furthermore several hundred forms of syndromes with hearing loss have been
documented in humans (Van Camp and Smith 2003) One is the human
Waardenburg syndrome (WS) which manifests itself with sensorineural deafness
and pigmentation defects in the iris hair and skin The WS is classified into four
types depending on the presence or absence of additional symptoms which are
caused by mutations in the five genes EDN3 EDNRB MITF PAX3 and SOX10
respectively These genes are shown in Table 3 They are known to be expressed in
the neural crest (EDN3 EDNRB PAX3 SOX10 ) or directly in the melanocytes
(MITF) and are inter alia involved in migration differentiation or survival of
melanocytes respectively (Bondurand et al 2000)
Deafness in dogs Congenital sensorineural deafness (CSD) has been reported in a variety of mammal
species other than humans ranging from mice to dogs guinea pigs and mink
Canine congenital deafness has often been reported in the literature and occurs in
Non-syndromic deafness in dogs and humans 10
more than 54 different breeds of dogs according to Strain (1996 and 2004) The
breeds with the highest incidence include Dalmatian dogs Bull Terrier English
Cocker Spaniel English Setter Australian Cattle Dog Australian Shepherd West-
Highland-White-Terrier Dobermann and Dogo Argentino The incidence of canine
congenital deafness is highest in Dalmatian dogs of which 165 to 30 exhibit
unilateral or bilateral hearing loss (Famula et al 1996 Wood and Lakhani 1997
Muhle et al 2002 Juraschko et al 2003a Rak and Distl 2005) The inheritance
and segregation of a major gene in canine congenital sensorineural deafness
(CCSD) has been demonstrated in different Dalmatian dog populations (Famula et
al 2000 Muhle et al 2002 Juraschko et al 2003b) But although several studies
have demonstrated the mode of inheritance in Dalmatian dogs no universally
accepted mode of inheritance for the other dog breeds affected by CCSD has yet
been identified
Congenital sensorineural hearing impairment can be recognised in dogs at four to
eight weeks of age (Strain 1996) while histological studies of deaf Dalmatian dogs
have shown that the degeneration of the inner ear structures begins as early as one
day after birth and is histologically clearly evident by four weeks of age (Johnsson et
al 1973) In 70 of the cases with human hereditary deafness the histological
pattern is known as cochleo-saccular degeneration (CSD) (Lalwani et al 1997)
commonly known as Scheibe dysplasia with preservation of the pars superior of the
membranous labyrinth and an unremarkable bony labyrinth As in man also in many
affected dog breeds the histological pattern of congenital sensorineural deafness is
known as cochleo-saccular degeneration
Breeding with blue-eyed Dalmatian dogs and unilaterally or bilaterally deaf dams and
sires is forbidden by paragraph 11b of the German animal welfare laws and thus the
hearing status of all Dalmatian dogs has to be tested as a puppy at about six to eight
weeks old using the brainstem auditory evoked response (BAER) test that detects
electrical activity in the cochlea and auditory pathways in the brain Although the
BAER test is a reliable method for identifying unilaterally and bilaterally deaf dogs it
does not seem to be an effective way of clearly reducing the incidence of deafness in
affected breeds particularly in a recessive mode of inheritance so that hearing dogs
can still be genetic carriers Furthermore deaf dogs are very difficult to raise and
often become aggressive and snappish from fear consequently most puppies
Non-syndromic deafness in dogs and humans 11
suffering from bilateral hearing loss are euthanized Thus prevention of CCSD
cannot be achieved alone by exclusion of affected animals from breeding and
consequently a molecular genetic approach toward unravelling the responsible
genes in carriers is urgently needed
Over the past decade it has become increasingly clear how far structural and
functional homologies at the gene level extend across even distantly related species
Much is known about deafness-causing gene mutations in humans and mice
including the fact that the clinical and histopathological findings are often very similar
to those of deafness in Dalmatian dogs Thus despite the genetical heterogeneity of
human non-syndromic deafness the genes that are responsible for non-syndromic
congenital hereditary deafness in humans (Table 1) seemed to be appropriate
candidate genes for CCSD especially in Dalmatian dogs (Rak and Distl 2005) The
genes that are mutated in the human WS (Table 2) were selected as candidates
because the WS phenotype where the deafness is associated with pigmentation
defects seems to be similar to the phenotype of most affected dog breeds (Strain
and Tedford 1996) Both Juraschko et al (2003a) and Strain et al (1992) have
demonstrated that patched Dalmatians are less likely to be deaf than unpatched
animals and blue-eyed Dalmatians are more likely to be affected from hearing
impairment than brown-eyed animals
In an attempt to achieve the aim of molecular genetic diagnosis of CCSD carriers
Rak et al (2003) and co-workers (Droumlgemuumlller et al 2002 Kuiper et al 2002 Rak
et al 2002a 2002b 2003) already mapped 24 potential candidate genes for
sensorineural deafness in dogs by fluorescence in situ hybridization and a radiation
hybrid panel to 16 different canine chromosomes
The canine genome project In December 2005 an international research team led by scientists at the Broad
Institute of MIT and Harvard achieved the completion of a high-quality genome
sequence of the domestic dog together with a catalog of 25 million specific genetic
differences across several dog breeds (Lindblad-Toh et al 2005) The authors found
that humans share more of their ancestral DNA with dogs than with mice confirming
the utility of dog genetics for understanding human disease Furthermore the
physiology disease presentation and clinical response of dogs often mimic human
Non-syndromic deafness in dogs and humans 12
diseases closely As indicated above hearing impairment seemed to be no
exception
In the last years most projects have exploited canine traits for which either direct
candidate genes could be proposed and evaluated or for which large informative
pedigrees were available to enable linkage mapping to identify candidate regions A
major component of such research efforts comprised the cloning sequencing and
mapping of individual canine homologs of genes either proposed as candidate
genes or expected to be located in candidate regions This was necessary to
identify new informative polymorphisms (eg single nucleotide polymorphisms
(SNPs) microsatellites) for high resolution mapping of candidate regions and to
examine each exon and exonintron boundary for positional candidates Availability
of the second version of the dog genome assembly (build 21) of the NCBI database
shortcut this effort and increase the investigators efficency
The current RH map with 3200 markers provides a good estimate of the order and
physical spacing (ie in base pairs) of markers along canine chromosomes (Guyon
et al 2003) and was recently complemented by the construction of a 4249-marker
integrated canine genome RH map that consists of 900 genes 1589 microsatellites
and 1760 BAC end markers (Breen et al 2004) all included and available in the
NCBI database The second version 1 of the NCBIs genome annotation consists of
large contigs covering all canine chromosomes given with their located markers and
genes The great majority of genes are derived by automated computational analysis
using the gene prediction method GNOMON
With this help either additional candidate genes for canine CSD can be found directly
by its gene symbol in the 21 of the NCBIs genome annotation or if a candidate gene
is yet not annotated a BLAST (Basic Local Alignment Search Tool) search versus
the canine whole genome shotgun (wgs) sequence resource can be used to obtain
the sequence of the canine genomic contigs containing the human homologous
gene The localisation of all 39 known human non-syndromic hereditary deafness
genes in the canine genome with the corresponding accession numbers of the contig
and if available the accession number of the genomic sequence and mRNA of the
canine gene are shown in Tables 4 and 5 Furthermore the identity of canine and
human or mouse mRNA is shown in Table 5 The average identity of canine and
human mRNA is with 088 percent higher than the average identity of canine and
mouse mRNA with 084 percent Canine sequences that correspond to the human
Non-syndromic deafness in dogs and humans 13
candidate gene can now be used to find microsatellite or SNP markers associated to
the respective canine gene These markers can be used for linkage and haplotype
studies in dog families segregating for deafness
Table 7 shows the microsatellite and SNP markers developed for in total 32
candidate genes for CCSD
The candidate genes for which a set of in total 43 microsatellite marker were
designed by Rak (2003) included the following 24 genes CDH23 CLDN14 COCH
COL11A2 DFNA5 DIAPH1 EDN3 EDNRB EYA4 GJA1 GJB2 GJB6 MITF
MYH9 MYO6 MYO7A MYO15A OTOF PAX3 POU4F3 SLC26A4 SOX10
TECTA and TMPRSS3 This existing set of 43 microsatellite markers for 24
candidate genes were used for linkage and haplotype studies in Dalmatian dog
families segregating for deafness (Chapter 3) These 24 genes are known to be
involved either in human non-syndromic deafness or in the human Waardenburg
syndrome For another eight candidate genes including TMC1 TMIE USH1C
MYH14 MYO3A PRES WHRN and ESPN SNP markers were newly developed
(Chapter 4) and subsequently used for linkage and association analyses in
Dalmatian dog families segregating for deafness These genes are also involved in
human non-syndromic deafness
Non-syndromic deafness in dogs and humans 14
Tabele 1 Genes responsible for non-syndromic congenital hereditary deafness in
humans
Inheritance Gene Gene product Type of molecule Locus namea
ACTG1 γ-Actin cytoskeletal protein DFNA20 DFNA26
COCH Cochlin extracellular matrix component DFNA9
COL11A2 Collagen XI (α2-chain) extracellular matrix component DFNA13
CRYM micro-Cristallin thyroid hormone-binding protein DFNAb DFNA5 Unidentified Unidentified DFNA5 DIAPH1 Diaphanous-1 cytoskeleton regulatory protein DFNA1 EYA4 EYA4 transcriptional coactivator DFNA10 GJB3 Connexin-31 gap junction protein DFNA2 KCNQ4 KCNQ4 K+ channel subunit DFNA2 MYH14 Myosin IIC motor protein DFNA4 MYH9 Myosin IIA motor protein DFNA17 MYO1A Myosin IA motor protein DFNA48 POU4F3 POU4F3 transcription factor DFNA15 TFCP2L3 TFCP2L3 transcription factor DFNA28
Autosomal dominant
WFS1 Wolframin endoplasmic-reticulum membrane protein DFNA6 DFNA14
CDH23 Cadherin-23 cell-adhesion protein DFNB12 CLDN14 Claudin-14 tight-junction protein DFNB29 ESPN Espin actin-bundling protein DFNB36 DFNAb MYO15 Myosin XV motor protein DFNB3 MYO3A Myosin IIIA motor protein DFNB30 OTOA Otoancorin cell-surface protein DFNB22 OTOF Otoferlin putative vesicle traffic protein DFNB9 PCDH15 Protocadherin-15 cell-adhesion protein DFNB23 SLC26A4 Pendrin IminusndashClminus transporter DFNB4 SLC26A5 Prestin anion transporter DFNB61 STRC Stereocilin DFNB16
TMIE TMIE transmembrane domain- containing protein DFNB6
TMPRSS3 TMPRSS3 transmembrane serine protease DFNB8 DFNB10 USH1C Harmonin PDZ domain-containing protein DFNB18
Autosomal recessive
WHRN Whirlin PDZ domain-containing protein DFNB31 GJB2 Connexin-26 gap junction protein DFNB1 DFNA3 GJB6 Connexin-30 gap junction protein DFNB1 DFNA3 MYO6 Myosin VI motor protein DFNA22DFNB37MYO7A Myosin VIIA motor protein DFNB2DFNA11
TECTA α-Tectorin extracellular matrix component DFNA8 DFNA12DFNB21
Autosomal dominant and autosomal recessive
TMC1 TMC1 transmembrane channel-like protein
DFNB7 DFNB11DFNA36
X-linked POU3F4 POU3F4 transcription factor DFN3
MTRNR1 Mitochondrial 12S rRNA not defined
nomenclature Mitochondrial
MTTS1 Mitochondrial 12S rRNA not defined
nomenclature a Autosomal recessive loci are designated DFNB autosomal dominant loci DFNA
Non-syndromic deafness in dogs and humans 15
Table 2 Genes underlying hereditary non-syndromic deafness as a result of primary
defects in hair cells non-sensory cells and the tectorial membrane or unknown cell
type
Primary defect
Gene
Hair cells MYO7A MYO15 MYO6 MYO3A MYO1A ACTG1 USH1C
WHRN CDH23 PCDH15 TMIE STRC SLC26A5 ESPN
KCNQ4 TMC1 OTOF POU4F3
Non-sensory cells
GJB2 GJB6 GJB3 SLC26A4 CRYM OTOA CLDN14
COCH TMPRSS3 MYH9 MYH14 EYA4 POU3F4
Tectorial membrane
COL11A2 TECTA
Unknown
DIAPH1 DFNA5 WFS1 TFCP2L3 MTRNR1 MTTS1
Table 3 Genes involved in the human Waardenburg syndrome
Inheritance Gene Gene product Type of molecule Type
EDN3 endothelin 3 vasoconstricted peptide WS type IV4
EDNRBendothelin
receptor type B receptor protein WS type IV4
MITF
microphthalmia-
associated
transcription
factor
transcription factor WS type II2
PAX3 paired box 3 DNA-binding protein WS type I1and III 2
SOX10 SRY-box 10 transcription factor WS type IV4 1Type I Dystopia canthorum 2Type II No dystopia canthorum 3Klein-Waardenburg syndrome (type III) Type I and upper limb abnormalities 4Waardenburg-Shah syndrome (type IV) Type II and Hirschsprung disease
(autosomal recessive inheritance)
Non-syndromic deafness in dogs and humans 16
Leng
th o
f co
ntig
(bp)
7799
0652
3821
0901
7077
2986
7251
5492
5989
7527
4533
7677
6093
8239
9498
326
6961
0260
1859
5814
6421
1953
Scor
e of
co
ntig
614
938
792
567
300
715
323
535
1208
1634
923
E-va
lue
of
cont
ig
4 E
-172
0 0
2 E
-158
1 e-
78
0
2 e-
85
7 E
-149
0 0 0
Acc
No
of
WG
S co
ntig
unkn
own
NW
_876
311
NW
_876
295
NW
_876
327
NW
_876
254
NW
_876
321
NW
_876
292
NW
_876
258
NW
_876
315
NW
_876
269
NW
_876
278
NW
_876
259
Mb
from
to
unkn
own
254
12
578
337
93
379
132
11
323
563
56
5
272
12
723
393
33
943
411
64
123
632
86
331
292
82
955
209
32
094
101
81
019
Gen
e lo
catio
n on
C
FA
unkn
own
4 31
8 12
6 2 14
5 1 25
15
Can
ine
gene
al
iase
s
none
none
LOC
4877
51
LOC
4906
40
LOC
4817
34
LOC
4798
18
none
LOC
6112
23
LOC
4896
31
EYA4
GJB
2
LOC
4824
86
Acc
No
hu
man
m
RN
A
NM
_001
614
NM
_022
124
NM
_144
492
NM
_004
086
NM
_080
680
NM
_001
888
NM
_005
219
NM
_004
403
NM
_031
475
NM
_172
105
NM
_004
004
NM
_024
009
Gen
e lo
catio
n on
H
SA
17
10
21
14
6 16
5 7 1 6 13
1
Tabl
e 4
Loca
lisat
ion
of h
uman
non
-syn
drom
ic h
ered
itary
dea
fnes
s ge
nes
in th
e ca
nine
gen
ome
the
cani
ne
gene
loc
alis
atio
n in
meg
abas
es (
Mb)
th
e ac
cess
ion
num
ber
(Acc
No
) of
the
who
le g
enom
e sh
otgu
n (W
GS
) con
tig c
onta
inin
g th
e hu
man
hom
olog
ous
gene
and
the
corr
espo
ndin
g E
-val
ue s
core
and
leng
th o
f th
e co
ntig
Hum
an
deaf
ness
ge
ne
AC
TG1
CD
H23
CLD
N14
CO
CH
CO
L11A
2
CR
YM
DIA
PH1
DFN
A5
ESPN
EYA
4
GJB
2
GJB
3
der
ived
from
the
NC
BIs
can
ine
geno
me
anno
tatio
n ve
rsio
n 2
1
Non-syndromic deafness in dogs and humans 17
Leng
th o
f co
ntig
6421
1953
5300
4996
5294
2087
2607
3285
1654
5469
1249
9463
7251
5492
5102
4781
5989
7527
5159
1990
1284
7264
7521
5785
Scor
e of
co
ntig
525
521
973
2256
348
567
1236
404
337
383
604
1715
E-va
lue
of
cont
ig
3 e-
146
1 e-
144
0 0
9 e-
93
2 e-
158
0
3 e-
109
3 e-
89
7 e-
103
2 e-
169
0
Acc
No
of
WG
S co
ntig
unkn
own
NW
_876
259
NW
_876
270
NW
_876
251
NW
_876
313
NW
_876
250
NW
_876
290
NW
_876
254
NW
_876
273
NW
_876
321
NW
_876
263
NW
_876
283
NW
_879
563
Mb
from
to
unkn
own
521
52
3
109
24
109
35
311
33
119
443
64
441
415
41
7
103
41
056
404
14
050
245
42
460
261
32
619
235
02
359
371
43
769
674
86
748
Gen
e lo
catio
n on
C
FA
25
15
1 10
5 10
2 12
21
6 17
26
X
Can
ine
gene
al
iase
s
none
LOC
4824
51
none
LOC
4812
80
LOC
4795
22
LOC
4744
10
LOC
4871
06
LOC
4818
84
LOC
4851
74
LOC
6086
55
LOC
6079
61
none
LOC
4919
88
Acc
No
hu
man
m
RN
A
NM
_006
783
NM
_004
700
NM
_024
729
NM
_002
473
NM
_016
239
NM
_005
379
NM
_017
433
XM
_376
516
NM
_000
260
NM
_144
672
NM
_194
248
NM
_033
056
NM
_000
307
Gen
e lo
catio
n on
H
SA
13
1 19
22
17
12
10
6 11
16
2 10
X
Tabl
e 4
(con
tinue
d)
Hum
an
deaf
ness
ge
ne
GJB
6
KC
NQ
4
MYH
14
MYH
9
MYO
15
MYO
1A
MYO
3A
MYO
6
MYO
7A
OTO
A
OTO
F
PCD
H15
POU
3F4
der
ived
from
the
NC
BIs
can
ine
geno
me
anno
tatio
n ve
rsio
n 2
1
Non-syndromic deafness in dogs and humans 18
Leng
th o
f co
ntig
4533
7677
2532
0482
2532
0482
4020
6070
3002
9677
2968
9717
5300
4996
3309
7591
3821
0901
5102
4781
1104
8438
6535
5756
Scor
e of
co
ntig
1404
283
283
1683
883
529
354
216
198
354
2238
689
E-va
lue
of
cont
ig
0
6 e-
73
2 E
-73
0 0
5 e-
147
2 e-
94
4 e-
53
9 e-
48
2 E
-94
0 0
Acc
No
of
WG
S co
ntig
NW
_876
292
NW
_876
265
NW
_876
265
NW
_876
294
NW
_876
312
NW
_876
255
NW
_876
270
NW
_876
272
NW
_876
295
NW
_876
273
NW
_876
256
NW
_876
253
Mb
from
to
436
14
361
158
61
592
197
91
982
134
31
345
158
81
595
622
63
7
880
88
822
450
54
505
390
33
905
432
44
328
414
94
151
716
47
172
Gen
e lo
catio
n on
C
FA
2 18
18
30
5 13
1 20
31
21
13
11
Can
ine
gene
al
iase
s
LOC
4872
00
LOC
4832
63
LOC
4832
74
LOC
4782
78
LOC
4893
57
LOC
4819
85
LOC
4841
68
LOC
6093
50
LO
C61
0987
LOC
6108
50
LOC
4821
13
LOC
6125
88
Acc
No
hu
man
m
RN
A
NM
_002
700
NM
_000
441
NM
_206
883
NM
_153
700
NM
_005
422
NM
_024
915
NM
_138
691
NM
_147
196
NM
_024
022
NM
_153
676
NM
_006
005
NM
_015
404
Gen
e lo
catio
n on
H
SA
5 7 7 15
11
8 9 3 21
11
4 9
Tabl
e 4
(con
tinue
d)
Hum
an
deaf
ness
ge
ne
POU
4F3
SLC
26A
4
SLC
26A
5
STR
C
TEC
TA
TFC
P2L3
TMC
1
TMIE
TMPR
SS3
USH
1C
WFS
1
WH
RN
der
ived
from
the
NC
BIs
can
ine
geno
me
anno
tatio
n ve
rsio
n 2
1
Non-syndromic deafness in dogs and humans 19
Leng
th o
f co
ntig
(bp)
4776
3139
5561
1003
2518
2130
3091
5115
5294
2087
Scor
e of
co
ntig
262
721
2927
967
1179
E-va
lue
of
cont
ig
8e-6
7
0 0 0 0
Acc
No
of
WG
S co
ntig
NW
_876
277
NW
_876
274
NW
_876
271
NW
_876
304
NW
_876
251
Mb
from
to
470
14
703
343
63
438
248
52
488
313
43
144
297
52
976
Gen
e lo
catio
n on
C
FA
24
22
20
37
10
Can
ine
gene
al
iase
s
ED
N3
ED
NR
B
MIT
F
PAX3
LOC
4812
58
Acc
No
hu
man
m
RN
A
NM
_207
032
NM
_000
115
NM
_198
159
NM
_181
457
NM
_006
941
Gen
e lo
catio
n on
H
SA
20
13
3 2 22
Tabl
e 5
Loca
lisat
ion
of g
enes
invo
lved
in th
e hu
man
Waa
rden
burg
syn
drom
e (W
S)
in th
e ca
nine
gen
ome
th
e ca
nine
gen
e lo
calis
atio
n in
meg
abas
es (
Mb)
th
e ac
cess
ion
num
ber
(Acc
No
) of
the
who
le g
enom
e sh
otgu
n (W
GS
) con
tig c
onta
inin
g th
e hu
man
hom
olog
ous
gene
and
the
corr
espo
ndin
g E
-val
ue s
core
and
le
ngth
of t
he c
ontig
Hum
an
deaf
ness
ge
ne
EDN
3
EDN
RB
MIT
F
PAX3
SOX1
0
der
ived
from
the
NC
BIs
can
ine
geno
me
anno
tatio
n ve
rsio
n 2
1
Non-syndromic deafness in dogs and humans 20
Table 6 Canine candidate genes for CCSD with their accession number (AccNo) of
the genomic sequence and mRNA and if available the percent identity of canine and
human or mouse mRNA Canine candidate gene
Acc No canine genomic sequence
Acc No canine mRNA (predicted)
Canine mRNA (bp)
Identity () of canine and human mRNA
Identity () of canine and mouse mRNA
ACTG1 none1 none unknown unknown unknown CDH23 none1 none unknown 8800 8690 CLDN14 NC_006613 XM_544876 714 8880 8460 COCH NC_006590 XM_547762 3050 8770 8170
COL11A2 NC_006594 XM_857285 5202 8970 8740 CRYM NC_006588 XM_845361 1279 8720 7890
DIAPH1 none1 none unknown 8630 unknown DFNA5 NC_006596 XM_848863 1512 8320 7680 ESPN NC_006587 XM_546751 2565 8930 8270 EYA4 NC_006583 XM_541108 3969 9240 8120 GJB2 NC_006607 XM_543177 2208 7860 6720 GJB3 NC_006597 XM_539603 1305 8550 8090 GJB6 none none unknown unknown unknown
KCNQ4 NC_006597 XM_539568 1836 9350 8760 MYH14 none1 none unknown 8770 8220 MYH9 NC_006592 XM_538401 6201 9130 8940
MYO15 NC_006587 XM_536660 10128 8500 7960 MYO1A NC_006592 XM_531642 3388 8600 8350 MYO3A NC_006584 XM_544234 5589 8510 8910 MYO6 NC_006594 XM_862465 3983 9250 8610
MYO7A NC_006603 XM_542292 6519 9190 8880 OTOA NC_006588 XM_845746 3558 8740 7990 OTOF NC_006599 XM_844665 5994 9020 8780
PCDH15 none1 none unknown 8210 7390 POU3F4 NC_006621 XM_549108 1086 9190 9060 POU4F3 NC_006584 XM_544328 1017 9380 9160 SLC26A4 NC_006600 XM_540382 2382 7970 8240 SLC26A5 NC_006600 XM_540393 2235 9250 8740
STRC NC_006612 XM_535452 5301 8980 8520 TECTA NC_006587 XM_546475 7136 9110 8510
TFCP2L3 NC_006595 XM_539106 2127 9030 8760 TMC1 NC_006583 XM_541284 2580 8860 8740 TMIE NC_006602 XM_846596 396 9110 8910
TMPRSS3 NC_006613 XM_848589 1542 8500 8340 USH1C NC_006603 XM_860072 1730 9010 8670 WFS1 NC_006595 XM_539234 2667 8510 8420 WHRN NC_006593 XM_850321 2817 8440 8110 EDN3 NC_006606 NM_001002942 1976 747 716
EDNRB NC_006604 NM_001010943 1329 889 827 MITF NC_006602 XM_850501 1590 934 862 PAX3 NC_006619 XM_545664 1474 869 861
SOX10 NC_006592 XM_538379 1987 926 900 derived from the NCBIs canine genome annotation version 21 1 the definite region in the whole genome sequence (WGS) contig was used to get the percent identity of mRNAs
Non-syndromic deafness in dogs and humans 21
Table 7 Microsatellite markers and single nucleotide polymorphisms (SNPs)
of canine candidate genes for canine congenital sensorineural deafness in
Dalmatian dogs
Canine candidate gene
Number of gene-associated
microsatellites
Number of gene-associated
SNPs
CDH23 2 0 CLDN14 3 8 COCH 2 0
COL11A2 2 0 DIAPH1 2 0 DFNA5 2 0 ESPN 0 5 EYA4 2 0 GJB2 3 0 GJB3 1 0 GJB6 1 0
MYH14 0 2 MYH9 2 22
MYO15 2 0 MYO3A 0 3 MYO6 1 0
MYO7A 3 0 OTOF 1 0 PAX3 1 0
POU4F3 1 0 SLC26A4 1 0 SLC26A5 0 2 TECTA 2 0 TMC1 1 1 TMIE 1 3
TMPRSS3 2 0 USH1C 0 2 WHRN 0 3
Chapter 3
Linkage analysis of gene-associated microsatellite markers with
congenital sensorineural deafness in Dalmatian dogs
Linkage analysis of gene-associated microsatellites 25
Linkage analysis of gene-associated microsatellite markers with congenital sensorineural deafness in Dalmatian dogs
Abstract Deafness is a disorder often diagnosed in different dog breeds In this Chapter an
existing set of 43 microsatellite markers associated with in total 24 candidate genes
for canine congenital sensorineural deafness (CCSD) were used for linkage and
haplotype analyses in a large Dalmatian dog population with frequent occurrence of
CCSD We found significant linkage for the genes GJA1 MYH9 and CLDN14 As
linkage was found for different candidate genes in different families the results of
these test statistics indicate that the inheritance of non-syndromic deafness in
Dalmatian dogs is heterogenic in origin
Introduction
Canine congenital sensorineural deafness (CCSD) has been reported to occur in
more than 54 different breeds of dogs (Strain 1996) As in man also in dog breeds
the most commonly observed histological pattern of degenerative inner ear changes
is known as the cochleo-saccular or Scheibe type of end organ degeneration
The incidence of this congenital anomaly is highest in Dalmatian dogs of which 165-
30 exhibit unilateral or bilateral hearing loss (Famula et al 1996 Wood and
Lakhani 1997 Muhle et al 2002 Juraschko et al 2003a Rak and Distl 2005) The
inheritance and segregation of a major gene in CCSD has been demonstrated in
different Dalmatian dog populations (Famula et al 2000 Muhle et al 2002 Juraschko et al 2003b) Moreover deafness in Dalmatian dogs seems to be
pigment-associated (Greibrokk 1994 Holliday et al 1992 Juraschko et al 2003a
2003b Mair 1976 Strain et al 1992 Strain 1996)
No gene mutation has yet been identified that is responsible for CCSD in Dalmatian
dogs or in one of the various other dog breeds that suffer from inherited hearing
impairment Since mutations in various genes have already been found to be the
cause of sensorineural hearing impairment in humans or mice 24 of these genes
Linkage analysis of gene-associated microsatellites
26
were considered as candidates for CCSD in Dalmatian dogs (Rak et al 2005)
Details of the 24 candidate genes are given in Table 1
Rak et al (2003) and co-workers (Droumlgemuumlller et al 2002 Kuiper et al 2002 Rak et
al 2002a 2002b 2003) mapped this 24 potential candidate genes for sensorineural
deafness in dogs by fluorescence in situ hybridization and a radiation hybrid panel
Subsequently Rak (2003) developed altogether 43 new highly polymorphic DNA
markers for the 24 candidate genes including CDH23 CLDN14 COCH COL11A2
DFNA5 DIAPH1 EDN3 EDNRB EYA4 GJA1 GJB2 GJB6 MITF MYH9 MYO6
MYO7A MYO15A OTOF PAX3 POU4F3 SLC26A4 SOX10 TECTA and
TMPRSS3 (Table 2)
Among the 24 candidate genes seven genes cause autosomal dominant non-
syndromic forms of deafness seven cause autosomal recessive forms and five
genes cause both recessive and dominant forms of non-syndromic deafness in
different human families segregating for either forms
The functions of these 19 deafness-causing genes are diverse and include gap
junctions and tight junctions (GJA1 GJB2 GJB6 CLDN14) ion channels (SLC26A4)
and ion channel activators (TMPRSS3) Included are also unconventional myosins
(MYO6 MYO7A MYH9 MYO15A) transcription factors (POU4F3 EYA4) as well as
extracellular matrix components (COCH COL11A2 TECTA) a cytoskeleton
regulatory protein (DIAPH1) a cell-adhesion protein (CDH13) and genes with
unknown or only suspected functions (DFNA5 OTOF) The 24 candidates also
include five genes which are mutated in the human Waardenburg syndrome (WS)
The WS is classified into four types depending on the presence or absence of
additional symptoms which are caused by mutations in the five genes EDN3
EDNRB MITF PAX3 and SOX10 respectively The objective of the current study
was to use this set of markers developed by Rak (2003) for a non-parametric linkage
analysis with CCSD in a German and French Dalmatian dog population
Material and methods Pedigree material
For the linkage analysis we used DNA from altogether 215 animals belonging to a
total of 24 Dalmatian dog families The families included 22 full-sib families and one
large paternal half-sib family of German Dalmatian dogs as well as 46 animals of a
Linkage analysis of gene-associated microsatellites 27
large paternal half-sib family of French Dalmatian dogs All families were segregating
for CCSD The genotyped families included all affected dogs (unilaterally and
bilaterally deaf) their parents if available and one to four unaffected animals At least
two of the full sibs of each family were unilaterally deaf
In total these 24 families included 402 individuals with an average family size of 168
ranging from 5 to 116 animals and covering two to four generations The hearing
status of 344 dogs was examined by veterinarians using the BAER (brain stem
auditory evoked response) test and the other animals included in the pedigree being
not BAER tested were used to construct relationships among CSD affected dogs
The prevalence of CSD in this pedigree was 285
Microsatellite marker analysis An existing marker set consisting of 43 microsatellite markers (Table 2) was used for
linkage analysis This set included 36 markers developed by Rak (2003) and 7
markers of the RH map available at httpwww-recomgenuniv-rennes1frdoggyhtml
For most of the 24 candidate genes two markers were available for two of the
candidates three markers were available but for seven candidate genes the set
contains only one marker The marker set is composed of 33 perfect repeats two
imperfect six compound-perfect and two compound-imperfect repeats
The majority (674) of the 43 markers in the set was represented by dinucleotide
repeats 209 were tetranucleotide repeats 47 trinucleotide repeats and 23
pentanucleotide repeats In addition one marker (23) was a compound di-
tetranucleotide and another one (23) was a compound tetra-pentanucleotide
repeat The average number of alleles was 35 with a minimum of 2 and a maximum
of 8 different alleles per marker
PCR reactions for microsatellites were carried out in 12 microl reaction mixtures
containing 2 microl genomic DNA 12 microl 10x PCR buffer (Qbiogene Heidelberg
Germany) 024 microl dimethyl sulfoxide (DMSO) 02 microl dNTPs (100 microM) 01 microl Taq
Polymerase (5Umicrol) (Qbiogene) 06 microl (10 microM) 5rsquo-IRD700 or IRD800 labelled forward
primer 06 microl (10 microM) unlabelled reverse primer The PCR reactions were carried out
in MJ Research thermocyclers with the following program 4 min at 94 degC followed
by 32 cycles of 30 sec at 94degC 30 sec at maximum annealing temperature and a
final extension of 45 sec at 72degC PCR-products were diluted with formamide loading
buffer in ratios from 110 to 140 and then size-fractionated by gel electrophoresis on
Linkage analysis of gene-associated microsatellites
28
automated LI-COR 42004300 sequencers (LI-COR inc Lincoln NE USA) using
denaturing 4 and 6 polyacrylamide gels (RotiphoreseregGel 40 Roth Karlsruhe
Germany)
To localize the 24 candidate genes and their associated microsatellites exactly the
canine candidate gene sequences were derived from sequences deposited in the
current dog genome assembly (Boxer genome assembly 21) of the NCBI GenBank
by BLAST (Basic Local Alignment Search Tool) search
(httpwwwncbinlmnihgovBLAST) using the human reference mRNA sequence
(Table 3)
Linkage analysis
Multipoint linkage and haplotype analyses were performed using the MERLIN
software version 0102 (multipoint engine for rapid likelihood inference Center for
Statistical Genetics University of Michigan MI USA Abecasis et al 2002) The test
statistics Z-mean and Lod score were used to test for the proportion of alleles shared
by affected individuals identical by descent (IBD) for the considered marker loci
Linkage analyses were performed regarding the marker set consisting of 43 gene-
associated microsatellite markers Linkage analysis was at first carried out for all 24
families conjoined After this the families were scanned separately
The data of the genotypes was additionally analyzed using SASGenetics (Statistical
Analysis System Version 913 SAS Institute Inc Cary NC USA 2005) to specify
the number of alleles of each marker the allele frequency the observed (HET) and
expected (HE) heterozygosity and the polymorphism information content (PIC)
(Table 4 and 5)
Results and discussion
Test statistics for all families conjoined are given in Table 6 Significant CCSD loci
were located on different chromosomes The loci were located on canine
chromosome (CFA) 1 10 12 20 and 31 Linkage analyses per family indicated even
higher test statistics for subgroups of families (Table 7) Scanning only families with
Zmeans gt1 test statistics for linkage increased for the genes GJA1 on CFA1 MYH9
on CFA10 and CLDN14 on CFA31 with congenital sensorineural deafness in different
Dalmatian dog families (Table 7) Therefore it is probable that these genes or genes
Linkage analysis of gene-associated microsatellites 29
in their flanking regions are involved in the development of the disease in the
respective familes The results of this test statistics indicate that the inheritance of
non-syndromic hearing loss in Dalmatian dogs is probably as heterogenic in origin as
it is in humans Genetic heterogeneity means that different mutations cause the same
phenotype or disease the different mutations can either be found at the same locus
(allelic heterogeneity) or even at different loci (non-allelic heterogeneity)
The extreme heterogeneity of human deafness often hampered genetic studies
because many different genetic forms of hearing loss give rise to similar clinical
phenotypes and conversely mutations in the same gene can result in a variety of
clinical phenotypes In man genes that transport ions across membranes to maintain
appropriate solute concentration and pH as well as regulatory genes mainly
transcription factors and genes that play a part in structural integrity are essential for
the hearing process
However this study was a first step in identifying genes responsible for CCSD in
Dalmatian dogs The genes GJA1 MYH9 and CLDN14 and their flanking regions will
be further analyzed with a combined approach using microsatellites and single
nucleotide polymorphisms (SNPs) (Chapter 6-9) As linkage was found for different
candidate genes in different families subsequently only the families indicating
linkage will be chosen for further molecular analyses of the respective gene
To confirm the result of this study the density of the intragenic markers has to be
increased The current RH map with 3200 markers provides a good estimation of the
order and physical spacing (ie in base pairs) of markers along canine
chromosomes (Guyon et al 2003) and was recently complemented by the
construction of a 4249-marker integrated canine genome RH map which consists of
900 genes 1589 microsatellites and 1760 BAC end markers (Breen et al 2004) all
included and available in the NCBI database (httpwebncbinlmnihgov)
Thus microsatellites derived from the NCBI database could be used to confirm the
linkage Alternatively BLAST searches versus the canine whole genome shotgun
(wgs) sequence resource were perfomed to localize the genes exactly and to obtain
the sequence of the canine genomic contigs containing the human homologous gene
The results of the BLAST searches of the 24 candidate genes against the Boxer
genome assembly 21 are shown in Table 3 The genomic sequence of the
respective candidate gene can now be used to search for intragenic SNPs as these
polymorphisms are the most abundant and useful markers for fine mapping
Linkage analysis of gene-associated microsatellites
30
Development of SNPs requires sequencing of DNA for the respective genomic
regions of the parents with the aim to identify heterozygous base pair exchanges
After a heterozygous base pair is found the whole family can be genotyped for this
informative SNP marker These polymorphisms can than be used for linkage
analyses as well as association studies
Fine mapping using SNP markers for all genes indicating linkage with CCSD
identified by this study should enable us to detect mutations responsible for CCSD in
parts of the Dalmatian dog population
Linkage analysis of gene-associated microsatellites 31
Table 1 Details of the 24 selected human candidate genes
Symbol Gene name Locus name1 Most important reference
CDH23 cadherin related 23 DFNB12 Bork et al 2001
CLDN14 claudin 14 DFNB29 Wilcox et al 2001
COCH coagulation factor C homolog cochlin DFNA9 Robertson et al 1998
COL11A2 collagen type XI alpha 2 DFNA13 McGuirt et al 1999
DFNA5 deafness autosomal dominant 5 DFNA5 Van Laer et al 1998
DIAPH1 diaphanous homolog 1 (Drosophila) DFNA1 Lynch et al 1997
EDN3 endothelin 3 WS type IV Edery et al 1996
EDNRB endothelin receptor type B WS type IV Attie et al 1995
EYA4 eyes absent homolog 4 (Drosophila) DFNA10 Wayne et al 2001
GJA1 gap junction protein alpha 1 43kD (connexin 43)
2 Liu et al 2001
GJB2 gap junction protein beta 2 26k (connexin 26) DFNA3 DFNB1 Kelsell et al 1997
GJB6 gap junction protein beta 6 (connexin 30) DFNA3 DFNB1 Grifa et al 1999
Del Castillo et al 2002
MITF microphthalmia-associated transcription factor WS type II Tassabehji et al 1994
MYH9 myosin heavy polypeptide 9 non-muscle DFNA17 Lalwani et al 2000
MYO6 myosin VI DFNA22 DFNB37 Melchionda et al 2001 Ahmed et al 2003
MYO7A myosin VIIA DFNA11 DFNB2 Liu et al 1997a Liu et al 1997b Weil et al 1997
MYO15A myosin XVA DFNB3 Wang et al 1998
OTOF otoferlin DFNB9 Yasunaga et al 1999
PAX3 paired box gene 3 (Waardenburg syndrome 1) WS type I WS type III Hoth et al 1993 Tassabehji
et al 1992
POU4F3 POU domain class 4 transcription factor 3 DFNA15 Vahava et al 1998
SLC26A4 solute carrier family 26 member 4 DFNB4 Li et al 1998
SOX10 SRY (sex determining region Y)-box 10 WS type IV Pingault et al 1998
TECTA tectorin alpha DFNB21 DFNA8DFNA12
Mustapha et al 1999 Verhoeven et al 1998
TMPRSS3 transmembrane protease serine 3 DFNB8DFNB10 Scott et al 2001
1 Autosomal recessive loci are designated DFNB autosomal dominant loci DFNA 2 To our knowledge no deafness locus has been determined for this autosomal recessive gene
Linkage analysis of gene-associated microsatellites
32
HET
()
638
338
366
544
616
632
456
656
433
614
458
586
529
PIC
()
752
548
336
484
775
70
63
9
74
6
61
6
83
7
62
762
621
No
of
alle
les
6 4 4 4 9 8 6 6 6 13 5 11 5
PCR
pr
oduc
t (b
p)
175
109
123
156
146
179
259
151
241
219
186
214
191
AT
(degC
)
62 60 56 60 60 62 58 60 62 58 58 60 58
Prim
er s
eque
nces
(5rsquorarr
3rsquo)
F A
ATC
AG
GA
GG
GG
TGAG
TGTG
R
CC
CC
CAG
CTC
ATA
CA
TTC
TC
F C
CTG
TAC
TGA
ATG
CTT
GA
GG
R
CA
TCTC
TAG
AA
GA
AG
CC
TCC
F
TC
AC
ATA
GC
ATT
ATA
TATG
GA
C
R T
TGA
GAT
GG
CTC
TTAC
TGA
G
F T
CG
ATG
ATG
CTT
TCTG
GTT
G
R A
GG
CTG
TGA
AA
TGG
ATG
GA
G
F G
AG
AA
GC
AC
CA
GG
CA
TAG
G
R T
GG
TTTA
GC
AA
GG
CTG
TTC
C
F T
GA
CA
TAC
GG
AG
GAC
CA
AG
AG
R C
CC
CTC
CC
TTG
CTC
TATC
TC
F T
GC
CC
CTC
AG
AG
ATA
ATC
AC
R
CTT
CA
ATTA
TAC
AC
ACA
GG
TAC
F
TG
AA
TATG
GG
GC
TGAG
GA
AG
R
TTC
TCC
CTC
TGC
CTG
TGTC
F
GG
TTTA
GC
AC
TGC
CTT
CA
GC
R
CA
TTA
AG
CA
TCTG
GC
ATG
TGG
F
GA
AA
AC
TCA
GA
TTA
GC
CTG
G
R A
TCTT
GA
GA
GC
AA
AGG
TTG
TG
F T
GG
TTA
GG
GC
ATG
ATTC
CA
G
R C
ATG
TATA
AA
GA
GTA
ATG
CC
AG
F
CG
GG
AG
AGG
GTT
TGAC
TAC
R
CTC
CG
TATT
GC
TCA
TCTT
TCC
F
AG
CTT
CC
CTT
CTC
TGA
GA
C
R G
AG
AA
TAG
AG
TTTG
TGC
TCA
G
Rep
eat
(TTT
A)1
5 (A
CC
)9(A
TC)3
(A
TTT)
7(G
TTT)
5 (C
A)2
1 (G
A)2
0 (C
TTT)
~20
(GA
)13
(AC
)20(
AG)9
(C
T)16
TT
(CT)
5 (A
AA
AT)
~25
(CT)
11(G
T)4(
CT)
2 (A
TTT(
T))2
3 (A
C)2
2
Mar
ker n
ame
CD
H23
_MS1
C
DH
23_M
S2_F
2 C
LDN
14_M
S1
CLD
N14
_MS
2 C
LDN
14_M
S3
CO
CH
_MS1
C
OC
H_M
S2
CO
L11A
2_M
S1
CO
L11A
2_M
S3
DFN
A5_
MS
1 D
FNA
5 _M
S2
DIA
PH
1 _M
S1
DIA
PH
1_M
S2
Mar
ker o
rigin
RP
CI8
1-99
C20
R
PC
I81-
99C
20
RP
CI8
1-97
L17
RP
CI8
1-97
L17
RP
CI8
1-97
L17
RP
CI8
1-32
1I4
RP
CI8
1-32
1I4
RP
CI8
1-24
M6
RP
CI8
1-24
M6
RP
CI8
1-66
C12
R
PC
I81-
66C
12
RP
CI8
1-36
2I5
RP
CI8
1-36
2I5
Tabl
e 2
Can
dida
te g
ene-
asso
ciat
ed m
arke
rs f
or c
anin
e de
afne
ss d
evel
oped
by
Rak
(20
03)
For
each
gen
e th
e m
icro
sate
llite-
base
d m
arke
r th
e m
arke
r or
igin
rep
eat t
ype
PC
R p
rimer
s w
ith o
ptim
ised
con
ditio
ns a
nd th
e nu
mbe
r of
alle
les
pol
ymor
phis
m
info
rmat
ion
cont
ent (
PIC
) and
obs
erve
d he
tero
zygo
sity
(HE
T) fo
r all
dogs
gen
otyp
ed a
re s
how
n
Can
dida
te
gene
CD
H23
C
LDN
14
CO
CH
C
OL1
1A2
DFN
A5
DIA
PH
1
Linkage analysis of gene-associated microsatellites 33
HET
()
815
345
695
578
562
508
583
441
75
63
7
33
414
30
76
7
44
8
PIC
()
853
679
798
774
652
63
72
3
59
5
83
2
77
9
50
5
54
1
40
4
88
9
59
1
No
of
alle
les
14 6 7 6 9 5 9 6 13 13 4 6 4 14 6
PCR
pr
oduc
t (b
p)
147
131
258
227
134
190
148
195
218
260
181
164 96
227 94
AT
(degC
)
60 58 58 58 60 60 58 62 58 58 62 56 58 58 62
Prim
er s
eque
nces
(5rsquorarr
3rsquo)
F G
CTA
GG
AA
AA
ATC
CG
CA
ATG
R
GAC
CC
CC
TAG
GAC
ACC
AAC
F
GA
GA
ATT
GG
GC
ATG
GG
CAG
A
R T
GA
CTT
TATC
AC
TGG
TCTT
TG
F T
TATG
CAG
CC
CA
TGAC
AA
TC
R C
AA
GG
GA
AC
TCA
AAG
GC
TTG
F
TG
GA
CC
AG
GTC
AGTT
TGTG
R
TC
TGC
CTG
TGTC
TCTG
CC
F
ATG
GC
ATG
AA
GA
GG
ATA
CC
G
R A
GG
AC
AGG
TGAC
GG
CTC
TAC
F
GC
TAG
TAC
TCG
ATT
GTG
GTC
R
TC
ATG
GG
TTG
TGA
GA
TCC
AG
F T
TAA
TTTG
CTC
GTC
TTC
CTG
R
TG
TAA
GC
TCC
ACG
GA
TCA
CC
F
CTC
TCTT
GG
TCTC
CC
TCTG
C
R G
GG
AG
TAG
GG
GTG
GAG
TAG
G
F G
GTG
TTTC
CTT
TCC
TTTT
CT
R G
GTG
TTC
TCTC
CC
TTTC
TCT
F C
TCTA
TGA
AA
GG
TGA
TTG
CC
R
CA
GC
CAT
AC
AA
ATG
AGA
ATT
G
F C
TAC
AG
TGA
ATC
AG
CA
CA
GAC
R
CA
GC
CTT
GA
CTG
TTTC
TTTG
G
F T
GA
TTG
AC
TCTA
CTT
TAC
AC
A
R T
ATA
TTA
GG
CG
GTT
TTC
TTC
T F
AC
CC
AG
GTG
GC
CTG
ATTG
R
GC
AC
GC
AC
GTT
CTC
TCTT
TC
F G
AA
TGC
CC
TTC
ACC
TTG
AA
A
R G
GA
AA
AG
GA
GA
GA
TGA
TGC
C
F T
CTT
CC
TTG
GA
AA
GG
GA
AC
TC
R T
GC
CC
TAA
CA
CTT
GG
AA
TGG
Rep
eat
(TA
GA
)12
(GA
)25
(GT)
10(A
T)13
(A
G)2
1 (G
T)16
(A
G)1
2 (C
A)1
2 (G
A)8
(C
TTT)
~20
(GA
AA
)~25
(G
T)12
(C
A)1
5 (A
G)1
1 (G
AA
A)~
43
(AC
)13
Mar
ker n
ame
ED
N3_
MS
1 E
DN
RB
_M
S1
EY
A4_
MS
1 E
YA
4_M
S2
GJA
1_M
S1
GJA
1_M
S2
GJB
2_M
S1
GJB
2_M
S2
GJB
6_M
S2
GJB
2+6_
MS1
M
ITF
_MS
2 M
ITF
_MS
3 M
YH
9_M
S2
MY
H9_
MS
3 M
YO
6_M
S2
Mar
ker o
rigin
RP
CI8
1-36
6E14
Z
emke
et a
l (1
999)
R
PC
I81-
301N
19
RP
CI8
1-30
1N19
R
PC
I81-
370A
16
RP
CI8
1-37
0A16
R
PC
I81-
133O
22
RP
CI8
1-13
3O22
R
PC
I81-
343C
15
FH
2324
R
PC
I81-
119P
24
RE
N10
0J13
R
PC
I81-
374A
12
FH
2293
R
PC
I81-
156P
14
Tabl
e 2
(con
tinue
d)
Can
dida
te g
ene
ED
N3
ED
NR
B
EY
A4
GJA
1 G
JB2
GJB
6 M
ITF
MY
H9
MY
O6
Linkage analysis of gene-associated microsatellites
34
HET
()
359
366
431
566
51
50
5
28
3
35
8
79
9
70
4
69
2
23
9
68 71
698
PIC
()
496
623
627
765
635
756
502
575
756
76
70
1
44
2
75
4
84
5
80
6
No
of
alle
les
5 4 6 13 7 8 5 3 10 11 8 8 8 10 11
PCR
pr
oduc
t (b
p)
293
166
100
137
174
197
164
201
160
297
267
140
276
228
260
AT
(degC
)
56 60 56 62 62 62 60 60 58 58 56 58 58 62 60
Prim
er s
eque
nces
(5rsquorarr
3rsquo)
F T
GG
TTA
AA
AC
ATT
AA
AC
TTA
TAG
R
TA
GTA
TATA
GA
GA
TGC
AA
TGG
F
CA
TTG
GG
TGC
TTTC
CTG
TTC
R
TG
GA
GC
TGC
AG
GTA
TAG
CC
F
CC
AG
GC
ATT
CG
AG
GG
TG
R C
AG
AA
CTT
GA
GG
AAC
CA
TAG
F
CC
ATG
AAC
TTTG
TGG
AA
CTG
C
R A
AA
GG
GTT
GC
TGTG
GA
GA
TG
F A
GG
CA
GG
TTC
ATC
TGTG
TCC
R
TC
CC
AG
AC
CC
AG
CTA
CA
TTC
F
CA
GC
CAA
CTG
TATT
CTC
CTT
G
R A
TCTT
GA
GC
CC
TGC
ATT
AG
G
F G
AA
GC
GA
GG
AG
AG
ACA
GTC
C
R A
AG
GA
AG
CC
TCC
TGAC
AA
CC
F
CA
GG
GTC
AG
GC
TCTA
TGC
TC
R T
CC
TATC
ATC
CG
GC
TTTG
AC
F
TC
TGG
ATTG
TGG
TCA
CA
AC
C
R A
CTG
GAC
AC
TTC
TTTT
CA
GAC
G
F A
AA
GTG
GC
TGG
TTC
GG
AA
G
R A
GC
AG
CA
GC
ATA
CA
TTC
CTC
F
AA
GTA
GA
TCC
TATT
ATC
GTG
G
R A
GTT
TCA
GTG
TCTG
TTA
AA
TAG
F
CC
GG
ATT
TCTG
AG
GAG
GC
R
CA
TGC
TCTT
CA
CC
AGA
AC
C
F T
CA
GC
ATG
GA
TTTT
GTA
AA
ATC
R
GG
AC
TGC
GTG
GA
CAT
CTG
F
AC
AC
GG
TTC
TCG
CTG
ATG
TG
R T
GA
AG
GG
GA
TTG
AAC
AG
AG
G
F T
TCA
TTC
CG
AG
GTT
CTA
AC
TG
R C
AC
CA
TCTC
GTA
GC
CTT
TATC
Rep
eat
(TC
)13
(AC
)12
(GT)
12
(AC
)18
(GT)
12
(TA
)3(T
G)9
(TA
)2(C
A)2
C
(TA
AA
)5
(AA
T)18
(C
T)13
(T
G)1
3 (C
T)17
(T
AA
A)1
4 (G
T)3
CC
(GT)
19
(ATT
T)12
(T
C)8
(CA)
5(C
G)2
(CA)
9 (G
T)16
Mar
ker n
ame
MY
O7A
_M
S1
MY
O7A
_M
S2
MY
O7A
_M
S3
MY
O15
A _
MS
1 M
YO
15A
_M
S2
OTO
F _M
S1
PA
X3_
MS
1 P
AX
3_M
S2
PO
U4F
3_M
S4
SLC
26A
4_M
S2
SO
X10
_MS
2 T
EC
TA _
MS
1 T
EC
TA _
MS
2 T
MP
RS
S3_
MS
1 T
MP
RS
S3_
MS
2
Mar
ker o
rigin
RP
CI8
1-19
3deg2
RP
CI8
1-19
3deg2
AH
TH29
8 R
PC
I81-
362deg
13
RP
CI8
1-36
2deg13
R
PC
I81-
198L
15
RP
CI8
1-25
7H23
R
PC
I81-
257H
23
G2C
024
66
RP
CI8
1-47
P17
RP
CI8
1-50
5H2
RP
CI8
1-59
C2
RP
CI8
1-59
C2
RP
CI8
1-12
5P17
A
HTH
246
Tabl
e 2
(con
tinue
d)
Can
dida
te
gene
MY
O7A
M
YO
15A
O
TOF
PA
X3
PO
U4F
3 S
LC26
A4
SO
X10
TE
CTA
TM
PR
SS
3
Linkage analysis of gene-associated microsatellites 35
Table 3 The gene-associated microsatellites (MS) for 24 candidate genes of canine
congenital sensorineural deafness were localized using BLAST searches against the
Boxer genome assembly 21 The accession numbers (AccNo) of the whole
genome shotgun (WGS) contigs containing the genes as well as their associated
microsatellites are given
Microsatellite Acc No of WGS contig CFA Location of
Gene (Mb) Location of
MS (bp) Location of
MS in relation to gene
CDH23_MS1 NW_876311 4 2541025780 25510 intragenic
CDH23_MS2 NW_876311 4 2541025780 25630 intragenic
CLDN14_MS1 NW_876295 31 3379533796 33790 proximal
CLDN14_MS2 NW_876295 31 3379533796 33950 distal
CLDN14_MS3 NW_876295 31 3379533796 33790 proximal
COCH_MS1 NW_876327 8 1321513232 13225 intragenic
COCH_MS2 NW_876327 8 1321513232 13290 distal
COL11A2_MS1 NW_876254 12 56315659 5608 proximal
COL11A2_MS3 NW_876254 12 56315659 5578 proximal
DFNA5_MS1 NW_876258 14 4116941237 41135 proximal
DFNA5_MS2 NW_876258 14 4116941237 41250 distal
DIAPH1_MS1 NW_876292 2 3933039430 39382 intragenic
DIAPH1_MS2 NW_876292 2 3933039430 39552 distal
EDN3_MS1 NW_876277 24 4701347032 47057 distal
EDNRB_MS1 Zemke et al (1999) 22 3436334383 34354 proximal
EYA4_MS1 NW_876269 1 2928029550 29531 intragenic
EYA4_MS2 NW_876269 1 2928029550 29500 intragenic
GJA1_MS1 NW_876269 1 6399463996 64150 distal
GJA1_MS2 NW_876269 1 6399463996 64160 distal
GJB2_MS1 NW_8762781 25 2093620942 20938 intragenic
GJB2_MS2 NW_8762781 25 2093620942 20863 proximal
GJB2+6_MS1 FH2324 25 2093620942 17543 proximal
GJB3_MS2 NW_876259 15 1018310194 4530 proximal
GJB6_MS2 NW_876278 25 2090420906 20953 distal
Linkage analysis of gene-associated microsatellites
36
Table 3 (continued)
Microsatellite Acc No of WGS contig CFA Location of
Gene (Mb) Location of
MS (bp) Location of
MS in relation to gene
MITF_MS2 NW_876271 20 2485324884 24844 proximal
MITF_MS3 REN100J13 20 2485324884 25668 distal
MYH9_MS2 NW_876251 10 3113531193 31244 distal
MYH9_MS3 FH2293 10 3113531193 31696 distal
MYO15A_MS1 NW_876313 5 4436944419 44330 proximal
MYO15A_MS2 NW_876313 5 4436944419 44409 intragenic
MYO6_MS2 NW_876254 12 4041740504 40534 distal MYO7A_MS1 NW_876273 21 2454324609 24465 proximal
MYO7A_MS2 NW_876273 21 2454324609 24566 intragenic
MYO7A_MS3 AHTH298 21 2454324609 24594 distal
OTOF_MS1 NW_876263 17 2350223595 23463 proximal
PAX3_MS1 NW_876304 37 3134831445 31426 intragenic
PAX3_MS2 NW_876304 37 3134831445 31481 distal
POU4F3_MS4 G2C02466 2 4361043612 - -
SLC26A4_MS2 NW_876265 18 1586715927 15960 distal
SOX10_MS2 NW_876251 10 2975129762 29740 proximal
TECTA_MS1 NW_876312 5 1588515954 15910 intragenic
TECTA_MS2 NW_876312 5 1588515954 15930 intragenic
TMPRSS3_MS1 NW_876295 31 3903539054 38942 proximal
TMPRSS3_MS2 AHTH246 31 3903539054 38508 proximal
Linkage analysis of gene-associated microsatellites 37
Table 4 Number of alleles observed (HET) and expected (HE) heterozygosity and
polymorphism information content (PIC) for the developed marker-set
Feature Mean SD Min Max
No of alleles 75 31 3 14
hO () 703 122 370 898
hE () 532 151 239 815
PIC () 667 130 336 889
Table 5 Number of alleles per microsatellite locus and their PIC () values of the
developed marker-set
No of alleles per
microsatellite
Number of marker
loci PIC ()
3 1 575
4 6 483
5 5 574
6 11 652
7 2 716
8 5 671
9 3 717
10 2 800
11 3 776
13 4 803
14 2 871
Linkage analysis of gene-associated microsatellites
38
Table 6 Significant test statistics for linkage analysis carried out for all 24 genotyped
families conjoined Zmeans and LOD scores are given with their respective error
probabilities for the gene-associated markers of the candidate genes CLDN14
COL11A2 GJA1 MITF MYH9 and SOX10
Marker Location on canine chromosome (CFA)
Zmean pZmean LOD score pLOD
CLDN14_MS1 31q15 134 009 086 002
CLDN14_MS2 31q15 168 005 105 001
CLDN14_MS3 31q15 108 014 049 007
COL11A2_MS1 12q11-q12 166 005 085 002
COL11A2_MS3 12q11-q12 167 005 078 003
GJA1_MS1 1q24-q25 151 007 118 001
GJA1_MS2 1q24-q25 151 007 118 001
MITF_MS2 20q13 101 02 080 003
MITF_MS3 20q13 121 011 104 001
MYH9_MS2 10q232 080 02 018 02
MYH9_MS3 10q232 175 004 097 002
SOX10_MS2 10q21-q23 146 007 110 001
Linkage analysis of gene-associated microsatellites 39
Table 7 Significant test statistics for linkage analyses carried out each family
separately Zmeans and LOD scores are given with their respective error probabilities
for the gene-associated markers of the candidate genes CLDN14 MYH9 and GJA1
Gene-associated
marker
Number of families with significant linkage to
CCSD
Number of corresponding
family members
Zmean pZmean LOD-score pLOD
CLDN14_MS1 51 40 278 0003 112 0011
CLDN14_MS2 383 000007 170 0003
CLDN14_MS3 281 0002 113 0011
MYH9_MS2 32 21 081 02 023 02
MYH9_MS3
(=FH2293) 156 006 058 005
GJA1_MS1 13 46 295 0002 052 006
GJA1_MS2 286 0002 051 006 1 families with significant linkage include four German full-sib families and one
German half-sib family 2 families with significant linkage include three German full-sib families 3 families with significant linkage include one French half-sib family
Chapter 4
Evaluation of eight candidate genes for canine congenital sensorineural deafness
in Dalmatian dogs
Evaluation of eight candidate genes for CCSD 43
Evaluation of eight candidate genes for canine congenital sensorineural deafness in Dalmatian dogs Abstract
In this study we have been focusing on genomic loci that encode various enzymes
and transporters involved in the hearing process in humans We developed intragenic
markers for the canine genes ESPN MYH14 MYO3A PRES TMC1 TMIE USH1C
and WHRN on canine chromosomes (CFA) 1 2 5 11 18 20 and 21 which have
been shown to be responsible for human hereditary deafness and to employ these
newly developed markers for non-parametric linkage analyses with canine congenital
sensorineural deafness (CCSD) We screened DNA from altogether six Dalmatian
dogs which represent the parents of four families for single nucleotide polymorphisms
(SNPs) in the eight candidate genes by means of direct sequencing combined with a
polymerase chain reaction method for amplifying genomic DNA We characterized 20
SNPs and one insertion-deletion polymorphism For the TMC1 and TMIE genes we
additionally genotyped one microsatellite marker each The families used for
subsequent genotyping of the markers included 39 members from four full-sib
families with frequent occurrence of CCSD We concluded that mutations in ESPN
MYH14 MYO3A PRES TMC1 TMIE USH1C and WHRN do not play a role in
CCSD of the Dalmatian dog population investigated here
Introduction Over the past ten years significant progress has been made in the identification of
genes causing different forms of human deafness Currently 39 of the genes
responsible for non-syndromic hearing impairment have been identified in different
human populations (The Hereditary Hearing Loss Hompage
httpwebhostuaacbehhh Van Camp and Smith 2003)
Since non-syndromic hereditary hearing impairment is almost exclusively caused by
cochlear defects affected patients suffer from sensorineural hearing loss
Evaluation of eight candidate genes for CCSD
44
The most common histopathologic finding in cases of profound congenital deafness
in humans is the cochleosaccular degeneration (CSD) It is estimated to occur in
approximately 70 of cases in man and also in dog breeds the histological pattern is
known as cochleosaccular degeneration
Many genetic disorders in humans and domestic dogs (Canis familiaris) demonstrate
a high level of clinical and molecular similarity Therefore genes responsible for
human hereditary deafness seem to be appropriate candidate genes for CCSD
especially in Dalmatian dogs
In this report we provide 21 single-nucleotide polymorphisms (SNPs) and two
microsatellite markers in altogether eight selected human candidate genes (Table 1)
This eight candidate genes were only recently identified for being responsible for
different form of human non-syndromic deafness In order to evaluate whether any of
this candidate genes is responsible for congenital sensorineural deafness in
Dalmatian dogs we analyzed the association of the ESPN MYH14 MYO3A PRES
TMC1 TMIE USH1C and WHRN haplotypes with the CCSD phenotype in four
families of Dalmatian dogs with frequent occurrence of CCSD
Material and methods Pedigree structure and sampling
For the linkage analysis we used blood samples from 39 Dalmatian dogs They
belong to four full-sib families segregating for CCSD At least two of the full sibs of
each family were unilaterally deaf The phenotype of the affected animals had been
confirmed by brainstem auditory evoked response (BAER) that detects electrical
activity in the cochlea and auditory pathways in the brain
The families consisted of eight to 12 individuals In two families a blood sample of the
sire and dam respectively was not available Screening for SNPs was performed by
comparative sequencing of genomic DNA from the parents of the families used for
linkage analyses
SNP and microsatellite marker identification for genotyping
The canine ESPN MYH14 MYO3A PRES TMC1 TMIE USH1C and WHRN gene
sequences was derived from sequences deposited in the current dog genome
assembly (Boxer genome assembly 21) of the NCBI GenBank (Table 2) by BLAST
Evaluation of eight candidate genes for CCSD 45
(Basic Local Alignment Search Tool) search (httpwwwncbinlmnihgovBLAST)
using the human ESPN MYH14 MYO3A PRES TMC1 TMIE USH1C and WHRN
reference mRNA sequence
We compared the canine genomic DNA sequence from the eight candidate genes to
canine cDNA fragments in the canine EST database using the BLASTN program
For the localization of the exonintron boundaries canine or alternatively human
mRNA sequences were used for the mRNA-to-genomic alignment program Spidey
(httpwwwncbinlmnihgovIEBResearchOstellSpideyindexhtml) The human
and canine mRNA sequences which were used to determine the exon organization of
the candidate genes are given in Table 2
For each of the eight candidate genes we designed intragenic primer pairs to amplifly
intronic sequences yielding products with a length of 560 to 670 bp PCR primers
were developed with the Primer3 program (httpfrodowimiteducgi-
binprimer3primer3_wwwcgi) The PCR reactions were performed in a total of 50 microl
containing 125 microM dNTPs 25 pmol of each primer the reaction buffer supplied by
the manufacturer (Qbiogene Heidelberg Germany) and 1 U Taq polymerase After
a 4 min initial denaturation at 95degC 35 cycles of 30 sec at 94degC 45 sec at 58degC and
80 sec at 72degC were performed in a MJ Research thermocycler (Biozym Hessisch
Oldendorf Germany) The obtained PCR products were directly sequenced with the
DYEnamic ET Terminator kit (AmershamBiosciences Freiburg Germany) and a
MegaBACE 1000 capillary sequencer (AmershamBiosciences) using the PCR
primers as sequencing primers Sequence data were analyzed with Sequencher 42
(GeneCodes Ann Arbor MI)
In the first step sequence analyses were performed for PCR products of the parents
of four full-sib families If a heterozygous SNP was found for one or both parents all
progeny of the respective families were analyzed for that SNP Additionally to the
SNPs we used two microsatellite markers for linkage analyses We identified one
intragenic microsatelllite within the TMC1 gene As we could develop only one SNP
for the TMIE gene we additionally genotyped one microsatellite marker derived from
the NCBI database (httpwebncbinlmnihgov) located very closely to the TMIE
gene (Table 3)
Evaluation of eight candidate genes for CCSD
46
SNP marker analysis
A total of 21 SNPs were identified within ESPN MYH14 MYO3A PRES TMC1
TMIE USH1C and WHRN (Table 3) and used for linkage analysis Of all SNPs six
out of the observed 21 SNPs were polymorphic in all four examined families Out of
the 21 SNPs eleven SNPs were present in family 1 18 SNPs were heterozygous for
one or both parents in family 2 and 15 SNPs could be used for linkage analysis in
family 3 and 4 respectively (Table 4)
The most frequent form of SNPs with a frequency of 238 was the CT transition
motif The scarcest one was the CG transversion motif with a frequency of 48
respectively
Linkage and haplotype analysis Multipoint linkage and haplotype analysis were performed using MERLIN version
0102 (multipoint engine for rapid likelihood inference Center for Statistical Genetics
University of Michigan MI USA Abecasis et al 2002) The test statistics Z-mean
and Lod score were used to test for the proportion of alleles shared by affected
individuals identical by descent (IBD) for the considered marker loci The data of the
genotypes was additionally computed using the software package SAS Genetics
(Statistical Analysis System Version 913 SAS Institute Inc Cary NC USA 2005)
to specify the observed heterozygocity values (HET) and the polymorphism
information content (PIC)
Furthermore linkage disequilibrium (LD) and association of haplotypes with CCSD
was tested using the procedures CASECONTROL and HAPLOTYPE of
SASGenetics (Statistical Analysis System version 913 Cary NC USA)
Results and discussion The non-parametric multipoint linkage analysis in four full-sib families did not show
significant test statistics The highest Z-mean value was 046 the highest LOD Score
was 008 while the error probabilities ranged from 03 to 08 (Table 5) The maximum
achievable Z-mean was 448 and the corresponding value for the LOD score was
160 Marker-trait association tests for haplotypes of the candidate gene markers
were not significant Obviously no haplotype was associated with CCSD in these
Dalmatian dog families as demonstrated (Figure 1 2 3 4 5 6 7 and 8) The
Evaluation of eight candidate genes for CCSD 47
paternal and maternal haplotypes as far as they could be estimated were distributed
among the affected progeny closely to the expected proportion of 50 and thus no
excess of a certain haplotype could be observed in the affected dogs
Due to the fact that both animals with unilateral or bilaterally hearing loss and
bilateral hearing animals shared identical haplotypes it is unlikely that the ESPN
MYH14 MYO3A PRES TMC1 TMIE USH1C and WHRN genes are involved in the
pathogenesis of CCSD in Dalmatian dogs However the ESPN MYH14 MYO3A
PRES TMC1 TMIE USH1C and WHRN gene-associated marker may serve for
further linkage studies in other Dalmatian dog populations and dog breeds other than
Dalmatians
Evaluation of eight candidate genes for CCSD
48
Table 1 Details of the six selected human candidate genes
Gene symbol
Gene name Locus name1 Most important reference
ESPN espin DFNB36 Naz et al (2004)
MYH14 myosin heavy polypeptide 14 DFNA4 Donaudy et al (2004)
MYO3A myosin IIIA DFNB30 Walsh et al (2002)
PRES solute carrier family 26
member 5 (prestin) DFNB61
Zheng et al (2000) Liu et
al (2003)
TMC1 transmembrane channel-like
gene 1
DFNB7
DFNA36 Kurima et al (2002)
TMIE transmembran inner ear DFNB6 Naz et al 2002
USH1C Usher syndrome 1C DFNB18 Verpy et al (2000) Ouyang
et al (2002)
WHRN whirlin DFNB31 Mburu et al (2003)
1 Autosomal recessive loci are designated DFNB autosomal dominant loci DFNA
Evaluation of eight candidate genes for CCSD 49
Table 2 Selected human candidate genes with their location on HSA and CFA and
corresponding accession numbers
Gene symbol Gene
location on HSA1
Acc No 3 human mRNA
Gene location
on CFA2
Acc No 3 canine
genomic sequence
Acc No 3 canine mRNA
ESPN 1 NM_031475 5 NC_006587 XM_546751
MYH14 19 NM_024729 1 NW_876270 -
MYO3A 10 NM_017433 2 NC_006584 XM_544234
PRES 7 NM_206883 18 NC_006600 XM_540393
TMC1 9 NM_138691 1 NC_006583 XM_541284
TMIE 3 NM_147196 20 NC_006602 XM_846596
USH1C 11 NM_153676 21 NC_006603 XM_860072
WHRN 9 NM_015404 11 NC_006593 XM_850321
1 Homo sapiens autosome 2 Canis familiaris autosome 3 Accession number
Evaluation of eight candidate genes for CCSD
50
Table 3 The 21 newly developed intragenic SNPs and two microsatellite markers for
Dalmatian dogs in the candidate genes ESPN MYH14 MYO3A PRES TMC1
TMIE USH1C and WHRN with their corresponding primers the SNP motif the
product size and the annealing temperature the observed heterozygosity (HET) and
polymorphism information content (PIC)
SNP Primer F1 (5acute -gt 3acute) SNP motif primer R2 (5acute -gt 3acute)
Bp3 AT4 PIC ()
HET()
ESPN_SNP1
ACCAGCACCCTCTCCAACTA
AGGAATTCACAA (CT)CACACATACA
ACTCAAGCTCAGGGTGTGGT
565 60 9 10
ESPN_SNP2
ACCAGCACCCTCTCCAACTA
ATGGCTGGCGCT (AG)GAGGCTGCCC
ACTCAAGCTCAGGGTGTGGT
565 60 27 41
ESPN_SNP3
ACCAGCACCCTCTCCAACTA
ACACTCTTCCCA (CT)GGCTGGCGCT
ACTCAAGCTCAGGGTGTGGT
565 60 23 32
ESPN_SNP4
ACCAGCACCCTCTCCAACTA
TGGGAAGAGGGA (AG)GGGGGAGCAT
ACTCAAGCTCAGGGTGTGGT
565 60 23 32
ESPN_SNP5
ACCAGCACCCTCTCCAACTA
GAGTGGGCCAGG (CT)TGGGAAGAGG
ACTCAAGCTCAGGGTGTGGT
565 60 28 42
MYH14_SNP1
CTCTCCCCAACTCAGTTCCA
ACGTGTATTCGG (GT)CGCTTTTATT
GTGATAGGGACGAGCAGCAT
670 60 35 42
MYH14_SNP2
CATGGGACCGTTCCTACACT
AGCCTCGTTTAA (CT)CTAAAAGGAA
GCTCAATAGGCACGACATCA
640 60 34 39
Evaluation of eight candidate genes for CCSD 51
Table 3 (continued)
SNP Primer F1 (5acute -gt 3acute) SNP motif primer R2 (5acute -gt 3acute)
Bp3 AT4 PIC ()
HET()
MYO3A_SNP1
AATGCTTGAGTTTGGGATGC
GGCAGTCCCATG (GT)CCCTTATAAG
ACCTAATTGCCCAGATGCAG
650 60 37 77
MYO3A_SNP2
AATGCTTGAGTTTGGGATGC
GTGGAGAGCCAC (CG)TTGGGAGAGG
ACCTAATTGCCCAGATGCAG
650 60 37 70
MYO3A_SNP3
AACCTCCTGGCGTAGTATTCC
CATTACCTATTT (AT)GATCCTTATA
TTTTCCACTTCAGGCACACA
650 60 25 36
PRES_SNP1
CCCTTACCCCATACCATTCC
GATAGACTTCCT (AG)CCCTCAGACT
TTCAGGACAGCATCATCTGC
560 60 37 64
PRES_SNP2
CCCTTACCCCATACCATTCC
TGATGTCTGCTG (AT)TAACCCATTC
TTCAGGACAGCATCATCTGC
560 60 37 66
TMC1_SNP1
GCAACCTCTCGGTTTATCCA
CGTGAAGTGCCC (AT)TTGATGGAAA
AAGCTGGGGAAGTGGATATGT
610 60 57 37
TMC1_SNP2
GCAACCTCTCGGTTTATCCA
GGAGACATTACC (AG)TGAAGTGCCC
AAGCTGGGGAAGTGGATATGT
610 60 47 29
TMC1_SNP3
GGAAGCAAGACTGAGGTTGG
AGGCTTTTTAAA (AG)CTGTTCTGGG
CTGCTGCATTTGCCTGTAAG
650 60 48 30
TMIE_SNP1
AGAACACCACCGTCTCCTTG
CAAGGCGACGCC (AT)GTGCTGTCCT
GCCTCTGGTCAGAAGAGGTG
625 60 59 36
Evaluation of eight candidate genes for CCSD
52
Table 3 (continued)
SNP Primer F1 (5acute -gt 3acute) SNP motif primer R2 (5acute -gt 3acute)
Bp3 AT4 PIC()
HET()
USH1C_SNP2
CTCCCGGTCTGTCAGGAAC
GGCCTGGGGGGA (AC)AAGCGGACGG
ATGGCATCGACTTCTCCAAC
560 60 37 35
USH1C_SNP4
CTCCCGGTCTGTCAGGAAC
GGTCTCAGACCG (AC)GGCAGGGAGA
ATGGCATCGACTTCTCCAAC
560 60 37 37
WHRN_SNP1
TTCACCTCCAGGATCTGGTC
CCTGAGCCCGAG (CT)CCACGCTGCT
GGCTACTTTTCTTCCCCCTTT
600 60 25 37
WHRN_SNP2
TTCACCTCCAGGATCTGGTC
GGTCACGGGGGC (CT)CCGGGAGGTT
GGCTACTTTTCTTCCCCCTTT
600 59 24 33
WHRN_SNP3
TTCACCTCCAGGATCTGGTC
GTCCGAGTCCCG (AG)CCCCAGCCTG
GGCTACTTTTCTTCCCCCTTT
600 60 34 55
Microsatellite marker
Primers (forward reverse ) 5acute -gt 3acute Bp AT PIC()
HET()
TMC1_MS1
GCCCCCAGCTAAAAAGAGAA
TTCTCTTCCTCCCTCCTGTTC
220-220 60 76 57
FH2158 ATGGCCACATCACCCTAGTC
CTCTCTCTGCATCTCTCATGAA
274-302 58 57 66
Microsatllite marker derived from the NCBI database (httpwebncbinlmnihgov) 1 Forward 2 Reverse 3 Product size (basepairs) 4 Annealing temperature (degC)
Evaluation of eight candidate genes for CCSD 53
Table 4 The 21 newly developed intragenic SNPs for Dalmatian dogs in the
candidate genes ESPN MYH14 MYO3A PRES TMC1 TMIE USH1C and WHRN
with their nucleotide polymorphism allele and genotype frequencies
Gene symbol Fam1 Nucleotide polymorphism
Allele frequencies
Genotype frequencies2
ESPN_SNP1 4 CgtT 083017 840
ESPN_SNP2 2 3 4 AgtG 074026 15160
ESPN_SNP3 2 3 TgtC 068032 07120
ESPN_SNP4 2 3 GgtA 068032 07120
ESPN_SNP5 2 3 4 CgtT 074026 15160
MYH14_SNP1 2 3 4 GgtT 058041 51610
MYH14_SNP2 2 3 4 CgtT 058041 51610
MYO3A_SNP1 1 2 GgtT 062038 5111
MYO3A_SNP2 1 2 CgtG 062038 5111
MYO3A_SNP3 1 2 4 TgtA 076024 15140
PRES_SNP1 1 2 3 4 AgtG 058042 10254
PRES_SNP2 1 2 3 4 TgtA 058042 10254
TMC1_SNP1 1 2 3 4 AgtT 056044 11226
TMC1_SNP2 1 2 3 4 AgtG 076024 20190
TMC1_SNP3 1 2 3 4 AgtG 074026 19200
TMIE_SNP1 1 2 3 4 AgtT 058042 12216
USH1C_SNP2 1 3 4 AgtC 053047 9147
USH1C_SNP4 1 3 4 AgtC 053047 9147
WHRN_SNP1 2 CgtT 075025 360
WHRN_SNP2 2 CgtT 075025 360
WHRN_SNP3 2 3 4 InDel3 069031 13171 1 Number of families which were informative for the respective SNPs 2 Frequence of genotypes (first number number of animals homozygous for allele 1
second number heterozygous third number homozygous for allele 2) 3 Insertiondeletion of one base-pair (adenine)
Evaluation of eight candidate genes for CCSD
54
Table 5 Linkage analyses for families 1 2 3 and 4 for the 21 SNP and two
microsatellite markers within the eight candidate genes regarding Zmean LOD score
and error probabilities (p-values)
Gene symbol Marker Zmean pz-value1 LOD score pL-value2
ESPN ESPN_SNP1 014 04 002 04
ESPN_SNP2 014 04 002 04
ESPN_SNP3 014 04 002 04
ESPN_SNP4 014 04 002 04
ESPN_SNP5 014 04 002 04
MYH14 MYH14_SNP1 -089 08 -019 08
MYH14_SNP1 -089 08 -019 08
MYO3A MYO3A_SNP1 -049 07 -011 08
MYO3A_SNP2 -049 07 -011 08
MYO3A_SNP3 -049 07 -011 08
PRES PRES_SNP1 -094 08 -019 08
PRES_SNP2 -094 08 -019 08
TMC1 TMC1_SNP1 -034 06 -008 07
TMC1_SNP2 -034 06 -008 07
TMC1_SNP3 -034 06 -008 07
TMC1_MS1 -035 06 -008 07
TMIE TMIE_SNP1 013 04 003 03
FH2158 -056 07 -013 08
USH1C USH1C_SNP2 018 04 04 03
USH1C_SNP4 018 04 04 03
WHRN WHRN_SNP1 046 03 008 03
WHRN_SNP2 046 03 008 03
WHRN_SNP3 046 03 008 03 1 Error probability regarding Zmean 2 Error probability regarding LOD score
Microsatellite marker
Evaluation of eight candidate genes for CCSD 55
Figu
re 1
SN
P-h
aplo
type
s of
the
ES
PN
gen
e in
all
anal
yzed
Dal
mat
ian
dog
fam
ilies
The
hapl
otyp
es b
elon
ging
to F
amily
2 3
and
4 re
gard
ing
the
gene
ES
PN
sho
w n
o as
soci
atio
n w
ith th
e C
CS
D p
heno
type
In
the
thre
e fa
milie
s th
e ha
plot
ype
21
43
2 is
the
mos
t com
mon
one
bei
ng p
rese
nt in
72
o
f all
anim
als
It is
follo
wed
by
the
hapl
otyp
e 2
32
14
with
18
a
nd 4
34
34
with
6
Nor
mal
hea
ring
anim
als
as
wel
l as
thei
r de
af s
iblin
gs s
how
all
thes
e th
ree
hapl
otyp
es T
here
is n
o re
com
bina
tion
of th
e ha
plot
ypes
of t
he E
SP
N g
ene
in th
e th
ree
fam
ilies
Evaluation of eight candidate genes for CCSD 56
Figu
re 2
SN
P-h
aplo
type
s of
the
MY
H14
gen
e in
all
anal
yzed
Dal
mat
ian
dog
fam
ilies
Evaluation of eight candidate genes for CCSD 57
Figu
re 3
SN
P-h
aplo
type
s of
the
MY
O3A
gen
e in
all
anal
yzed
Dal
mat
ian
dog
fam
ilies
Evaluation of eight candidate genes for CCSD 58
Figu
re 4
SN
P-h
aplo
type
s of
the
PR
ES
gen
e in
all
anal
yzed
Dal
mat
ian
dog
fam
ilies
Evaluation of eight candidate genes for CCSD 59
Figu
re 5
SN
P- a
nd m
icro
sate
llite-
hapl
otyp
es o
f the
TM
C1
gene
in a
ll an
alyz
ed D
alm
atia
n
Evaluation of eight candidate genes for CCSD 60
Figu
re 6
SN
P- a
nd m
icro
sate
llite-
hapl
otyp
es o
f the
TM
IE g
ene
in a
ll an
alyz
ed D
alm
atia
n do
g fa
milie
s
Evaluation of eight candidate genes for CCSD 61
Figu
re 7
SN
P-h
aplo
type
s of
the
US
H1C
gen
e in
all
anal
yzed
Dal
mat
ian
dog
fam
ilies
Evaluation of eight candidate genes for CCSD 62
Figu
re 8
SN
P-h
aplo
type
s of
the
WH
RN
gen
e in
all
anal
yzed
Dal
mat
ian
dog
fam
ilies
Chapter 5
Molecular characterization of the canine myosin heavy polypeptide 9
non-muscle (MYH9) gene on dog chromosome 10q232
Canine MYH9 gene 65
Molecular characterization of the canine myosin heavy polypeptide 9 non-muscle (MYH9) gene on dog chromosome 10q232 Abstract A missense mutation of the Myosin heavy polypeptide 9 (MYH9) gene which
encodes the nonmuscle myosin heavy chain-A caused non-syndromic sensorineural
deafness in a human family which was characterized by cochleosaccular
degeneration In the present study we evaluated whether MYH9 gene mutations are
responsible for canine congenital sensorineural deafness (CCSD) in Dalmatian dogs
As described in Chapter 3 two MYH9 gene-associated microsatellites were
genotyped in 25 Dalmatian dog families segregating for CCSD We could find
significant linkage with CCSD for the microsatellite marker MYH9_MS3 (=FH2293)
We used data deposited in the NCBI to assemble the canine MYH9 gene DNA
sequence Characterization of the canine MYH9 gene revealed that the canine gene
consists of 41 exons spanning approximately 90 kb
We detected 22 single nucleotide polymorphisms (SNPs) in a mutation scan of
altogether 16 Dalmatian dogs from three families which showed significant linkage
between the deafness phenotype and the MYH9 gene-associated microsatellite
None of the SNPs affects the amino acid sequence of MYH9 We concluded that the
exons of the canine MYH9 gene do not play a role in Dalmatian CCSD Availability of
the microsatellite marker SNPs and DNA sequence reported in this study enhance
evaluation of the MYH9 gene as a CCSD candidate gene in other Dalmatian dog
populations and other dog breeds affected by CCSD
Introduction Myosin is a functional protein associated with cellular movement cell division muscle
contraction and other functions Members of the myosin super-family are
distinguished from the myosin heavy chains that play crucial roles in cellular
processes The human MYH9 gene consists of 40 exons and spans 67959 bp
Canine MYH9 gene
66
Toothaker et al (1991) mapped a nonmuscle myosin heavy chain gene to human
chromosome (HSA) 22q123-q131 Simons et al (1991) likewise mapped the gene
to HSA22q112 which they designated nonmuscle myosin heavy chain-A (NMMHC-
A)
The similarities between the autosomal dominant giant-platelet disorders May-
Hegglin anomaly Fechtner syndrome and Sebastian syndrome and refinement of the
disease loci for May-Hegglin anomaly and Fechtner syndrome to an overlapping
region of 480 kb on human chromosome 22 suggested that all these three disorders
may be allelic Among the identified candidate genes was the gene encoding
nonmuscle myosin heavy-chain A (MYH9) The May-HegglinFechtner Syndrome
Consortium (2000) demonstrated that mutations in MYH9 result in one of the three
disorders mentioned above The same Consortium also speculated that mutations in
MYH9 may also play a role in another autosomal dominant disorder a form of
nonsyndromic deafness characterized by progressive hearing impairment and
cochleosaccular degeneration This autosomal dominant form of human
nonsyndromic hereditary deafness (DFNA17) was described by Lalwani et al (1999)
They studied a five-generation American family previously reported by Lalwani et al
(1997) with deafness caused by cochleosaccular degeneration (CSD) CSD is the
most common histopathologic finding in cases of profound congenital deafness and
is estimated to occur in approximately 70 of cases in man
DFNA17 maps to the same region as MYH9 Because of the importance of myosins
in hearing Lalwani et al (2000) tested MYH9 as a candidate gene for DFNA17 and
demonstrated a missense mutation in the MYH9 gene in affected members of a
kindred with DFNA17 They found a G-to-A transition at nucleotide 2114 in the MYH9
gene This missense mutation changed codon 705 from an invariant arginine to a
histidine within a highly conserved Src homology 1 (SH1) linker region Previous
studies had shown that modification of amino acid residues within the SH1 helix
causes dysfunction of the ATPase activity of the motor domain in myosin
Many genetic disorders in humans and domestic dogs (Canis familiaris) demonstrate
a high level of clinical and molecular similarity Rak et al (2003) mapped 20 potential
candidate genes for sensorineural deafness in dogs by fluorescence in situ
hybridization and a radiation hybrid panel among them the MYH9 gene that was
assigned to canine chromosome (CFA) 10q232 Congenital sensorineural deafness
has been reported for approximately 54 different breeds of dogs (Strain 1996)
Canine MYH9 gene 67
The incidence of this congenital anomaly is highest in Dalmatian dogs of which 165-
30 exhibit unilateral or bilateral hearing loss (Famula et al 1996 Wood and
Lakhani 1997 Muhle et al 2002 Juraschko et al 2003a Rak and Distl 2005) The
inheritance and segregation of a major gene in CCSD has been demonstrated in
different Dalmatian dog populations (Famula et al 2000 Muhle et al 2002
Juraschko et al 2003b)
In dog breeds the histological pattern is known as cochleosaccular degeneration
commonly known as Scheibe dysplasia as it is described in approximately 70 of
cases with human hereditary deafness (Strain 1996) Therefore the MYH9 gene
seems to be a perfect candidate gene for CCSD especially in Dalmatian dogs
In this report we provide the genomic organization and the complete sequence of the
canine MYH9 gene A mutation analysis was performed to identify single nucleotide
polymorphisms (SNPs) in this gene In order to evaluate whether the MYH9 gene is
responsible for congenital sensorineural deafness in Dalmatian dogs we analyzed
the association of the MYH9 haplotypes with the CCSD phenotype in three families of
Dalmatian dogs with frequent occurrence of CCSD and significant linkage to the
gene-associated microsatellite MYH9_MS3 (Chapter 3)
Materials and Methods
Cloning and sequencing of canine MYH9 cDNA
The canine MYH9 gene sequence was derived from sequences deposited in the
current dog genome assembly (Boxer genome assembly 21) of the NCBI GenBank
(Genbank Acc No NW_1398701) by BLAST (Basic Local Alignment Search Tool)
search (httpwwwncbinlmnihgovBLAST) using the human MYH9 reference
mRNA sequence (Genbank Acc No NM_002473)
The distance between MYH9 and the microsatellite MYH9_MS2 is about 181 kb
whereas the microsatellite MYH9_MS3 (=FH2293) places about 511 kb from MYH9
The isolation of full length cDNAs was achieved by a modified rapid amplification of
cDNA ends (RACE) protocol Total RNA from dog lung of a normal female Beagle
(Biocat Heidelberg Germany) was used for the overlapping 5rsquo- and 3rsquo-RACE
products with the FirstChoiceTM RNA ligase-mediated (RLM)-RACE kit (Ambion
Europe Huntingdon UK) according to the protocols of the manufacturer Reverse
transcription polymerase chain reaction (RT-PCR) using two pairs of nested
Canine MYH9 gene
68
gene-specific and supplied 5rsquo-adaptor-specific primers were used to amplify the
complete 5rsquo-end of the investigated cDNA Similarly the 3rsquo-end were amplified using
two pairs of nested gene-specific primers in combination with supplied 3rsquo-adaptor-
specific primers Additionally RT-PCR using six pairs of gene-specific primers were
used to amplify the complete sequence of the investigated cDNAs
RACE products were cloned into pCRR21 vector using the Invitrogen TA cloning kit
(Invitrogen Karlsruhe Germany) Several clones for each cDNA were sequenced
with the ThermoSequenase kit (AmershamBiosciences Freiburg Germany) and a
LI-COR 4300 DNA Sequencer (LICOR inc Bad Homburg Germany) Sequence
data were analyzed with Sequencher 42 (GeneCodes Ann Arbor MI)
Sequence analysis of canine MYH9 gene For the localization of the exonintron boundaries after cloning and sequencing of full
length canine cDNAs (as described above) the mRNA-to-genomic alignment program
Spidey (httpwwwncbinlmnihgovIEBResearchOstellSpideyindexhtml) was
used Repetitive elements were analyzed with Repeatmasker 2
(httprepeatmaskergenomewashingtonedu) The GC content was calculated with
the EBI toolbox CpG PlotCpGreport (httpwwwebiacukToolssequencehtml)
Mutation analysis To identify polymorphisms within the canine MYH9 sequence exons with flanking
regions were PCR amplified and sequenced from 16 Dalmatian dogs which
represent three families consisting of three to six full sibs and at the best of both
parents At least two of the full sibs of each family were unilaterally deaf The
phenotype of the affected animals had been confirmed by brainstem auditory evoked
response (BAER) that detects electrical activity in the cochlea and auditory pathways
in the brain PCR primers and conditions for the amplification of MYH9 exons with
flanking sequences and microsatellite flanking primers for PCR reactions are given in
Table 1 PCR primers were developed with the Primer3 program
(httpfrodowimiteducgi-binprimer3primer3_wwwcgi) The PCR reactions for
exons and their flanking sequences were performed in a total of 50 microl containing 125
microM dNTPs 25 pmol of each primer the reaction buffer supplied by the manufacturer
(Qiagen Hilden Germany) and 1 U Taq polymerase After a 4 min initial
denaturation at 95 degC 35 cycles of 30 sec at 94 degC 45 sec at 58 degC and 80 sec
Canine MYH9 gene 69
at 72 degC were performed in a MJ Research thermocycler (Biozym Hessisch
Oldendorf Germany) The obtained PCR products were directly sequenced with the
DYEnamic ET Terminator kit (AmershamBiosciences Freiburg Germany) and a
MegaBACE 1000 capillary sequencer (AmershamBiosciences) using the PCR
primers as sequencing primers Sequence data were analyzed with Sequencher 42
(GeneCodes Ann Arbor MI)
Multipoint linkage and haplotype analysis were performed using MERLIN version
0102 (multipoint engine for rapid likelihood inference Center for Statistical Genetics
University of Michigan MI USA Abecasis et al 2002) The test statistics Z-mean
and Lod score were used to test for the proportion of alleles shared by affected
individuals identical by descent (IBD) for the considered marker loci
Linkage means that a haplotype characterized by microsatellites or SNPs is
significantly more often present in family members with the phenotype under study
than expected by random assortment For construction of haplotypes sets of closely
linked genetic markers on the same chromosome are needed which tend to be
inherited together as they are not likely to be separated by recombination
Association analysis can be carried out as a method of genetic analysis that
compares the frequency of alleles between affected and unaffected individuals
across all families A given allele is considered to be associated with the disease if
the presence of that allele explains a significant proportion of the phenotypic trait
variation
Results and Discussion
Analysis of the genomic organization and cDNA of the canine MYH9 gene
A full-length cDNA sequence of 7484 bp of the canine MYH9 gene was obtained by
using the RACE protocol The obtained RT-PCR products were sequenced and the
generated sequence data were submitted to the EMBL nucleotide database (Acc No
AM086385)
Using the RT-PCR analyses for cDNA-genomic sequence comparisons we detected
that the canine MYH9 gene consists of 41 exons with an untranslated exon 1 and
exonintron boundaries that conform perfectly to the GTAG rule (Table 2)
The 5rsquo-UTR consists of 181 bp while the 3rsquo-UTR consists of 1432 bp Assuming that
the homologous ATG start codon as in man is used the canine MYH9 cDNA
Canine MYH9 gene
70
contains an open reading frame of 5889 bp encoding a protein of 1963 amino acids
A canonical polyadenylation signal AAUAAA is located approximately 14 kb
downstream of the stop codon
The exon sizes range from 28 to 1556 bp the introns between these exons span
between 86 and 13493 bp the total size of the canine MYH9 gene is approximately
90 kb However the sequence homology between the human murine and canine
MYH9 gene is limited to the coding sequence of exon 2 to 41 The coding sequence
of canine MYH9 displays 926 and 899 similarities to the human and murine
MYH9 gene respectively In the untranslated regions the sequence similarity
between dog and human and mouse is rather low The canine MYH9 protein shows
982 and 968 identities to the orthologous human and murine protein
respectively (Fig 2)
The repeat content in the 90 kb region of the canine MYH9 gene is 1458 Most of
the repetitive elements belong to the SINE family (892) followed by the fraction of
the LINEs (306) other repetitive elements constitute 165 respectively The
entire canine MYH9 gene has an overall GC-content of 54 The canine MYH9 gene
contains 12 CpG islands (Gardiner-Garden and Frommer 1987) distributed over the
entire gene whereas by far the longest one with a length of 789 bp can be found in
the region of the second exon or in the first translated exon respectively (GC content
of 50 over 200 bp)
Mutation and haplotype analysis
All coding exons with flanking intronic regions of MYH9 could be amplified from the
examined 16 dogs and the sequences were compared to the Boxer genome
assembly 21 of the NCBI Gen Bank (Genbank Acc No NW_1398701)
The search for sequence variations within the MYH9 gene revealed a total of 22
SNPs shown in Table 3 Most of the polymorphisms were found in the flanking
regions of exons only 3 were within exons Only five out of the observed 22 SNPs
were polymorphic in all three examined families
None of the observed polymorphism did alter the predicted amino acid sequence of
MYH9 nor the identified hapltypes showed an association with the CCSD phenotype
Multipoint test statistics for MYH9 alleles identical by descent were close to zero (Z-
mean 035 to -035 Lod score 013 to -007) and not significant (p= 04 to 07)
However the microsatellite MYH9_MS3 (=FH2293) showed a Z-mean of 156
Canine MYH9 gene 71
(p= 006) and a Lod score of 058 (p= 005) indicating linkage The reason for this
result was heterogeneity among these three families For family 1 and 2 the test
statistics indicated linkage (Z-mean 191 with p= 003 Lod score 058 with p= 005)
whereas for familiy 3 the Z-mean and Lod score was -050 (p= 07) and -008 (p=
07)
Thus these silent point mutations found in affected and unaffected Dalmatian dogs
seem not responsible for the CCSD phenotype in these three families
Conclusions
The characterization of the transcript and genomic sequences of canine MYH9 gene
revealed a conserved organization with respect to the human orthologs In general
the gene size in dog is bigger compared to the human sequence due to the
untranlated first exon in the 5rsquo-UTR Several single nucleotide polymorphisms in the
canine MYH9 gene were identified However because of the fact that both animals
with unilateral hearing loss and bilateral hearing animals shared identical
haplotypes these polymorphisms are obviously not associated with CCSD in these
Dalmatian dog families Furthermore the present study revealed no functional
mutations of the complete coding region of MYH9 We can exclude the MYH9 gene
as a candidate gene for congenital sensorineunal deafness in Dalmatian dogs
However the MYH9 gene sequence SNPs and microsatellite markers reported in
this study enhance evaluation of the MYH9 gene in other Dalmatian dog populations
and dog breeds other than Dalmatians
Canine MYH9 gene
72
Table 1 PCR primers for the amplification of all MYH9 exons
Exon Primer Sequence (5rsquo ndash 3rsquo) Product
size (bp)
Annealing temperature
(degC) exon 1 MYH9_Ex1_F GCC TGG TTT GAC AAG TTT TG MYH9_Ex1_R TTC CTC AAG TCC CCT ACT CC 560 58 exon 2 MYH9_Ex2_F AGG ACC CTC ACC CAA CAC MYH9_Ex2_R GCA CAG AGT CTG GAC AAG AAT G 621 58 exon 3 MYH9_Ex3_F CAC CCC TCA ATA CTC AGT GC MYH9_Ex3_R GAG AAG ACT CCT GCC TCT CC 588 58 exon 4 MYH9_Ex4_F GGC AAT GTG TTT GAT TGT ACT G MYH9_Ex4_R TTT CCA AAG CAA CTC AGA AAG 542 58 exon 5 -6 MYH9_Ex56_F ACA GAG CAG GCG TGA CAG MYH9_Ex56_R GAG GAC AAG AGC TGG AAG C 724 58 exon 7 -8 MYH9_Ex78_F AAG CGG AAA GTT GCC TTC MYH9_Ex78_R ATG GCA CAA TGA CCA GTC TC 699 58 exon 9 MYH9_Ex9_F TGG GCA CAG TTT TTG TCT G MYH9_Ex9_R CAC ACA ATC AAG GGC TTT TC 590 58 exon 10 MYH9_Ex10_F GGA GCC AGT TGT CCT AGT AGT G MYH9_Ex10_R GCC AGG TGC CTA TCA CTG 540 58 exon 11 MYH9_Ex11_F TGT TTC GAA GCG TTT TTA CC MYH9_Ex11_R GGT CCC ATT GTT TCT AGC AC 551 58 exon 12 MYH9_Ex12_F TTT CCC AAG CAG CTA GAA AG MYH9_Ex12_R ACT GGG TGG TCT TGA GAC TG 586 58 exon 13 MYH9_Ex13_F CTG CCT TTG GTG ACA AGT G MYH9_Ex13_R GCA AGC CCT AAG TAC AAT GG 548 58 exon 14 MYH9_Ex14_F ATA CCA GGT CAA GGG GTG TC MYH9_Ex14_R TCA CCT GGT TCT AGG TGG AG 524 58 exon 15 MYH9_Ex15_F CGT TTG GAG TTG AT CCT AGC MYH9_Ex15_R AGC CAA GTT TCC TGA CTG TG 566 58 exon 16 MYH9_Ex16_F AGA TAG GCT GCA GTG GTA GC MYH9_Ex16_R CAG TAG CAA ACA GGG CTA GG 568 58 exon 17 MYH9_Ex17_F AAC GGT CCA ATG TGA GTT TC MYH9_Ex17_R ACC AGC TCA CAA CAG TAC CC 559 58 exon 18 MYH9_Ex18_F TGT AGG CTG TGA TTG CAG AG MYH9_Ex18_R CCC TCT CTC AAA CAA ATT GC 586 58 exon 19 MYH9_Ex19_F CCT TGG TGA AGG AAA GTG G MYH9_Ex19_R GCT GTG TGC AGG TTC TGA C 657 58 exon 20 MYH9_Ex20_F AGC AGC ATA GCA CTG AAT CC MYH9_Ex20_R TTC CCA TCT CTC ATG TTC TTG 579 58
Canine MYH9 gene 73
Table 1 (continued)
Exon Primer Sequence (5rsquo ndash 3rsquo) Product
size (bp)
Annealing temperature
(degC) exons 21 MYH9_Ex21_F CTC CTG AGT GTT GGG TTA GC MYH9_Ex21_R CTC TAA CAG GCA GCC CAA G 555 58 exon 22 MYH9_Ex22_F GGA GGA AAC GCA GAA ATC C MYH9_Ex22_R ACG GAC ACC CAG GAA AAC 660 58 exon 23 MYH9_Ex23_F CAG GGG TGC TAG TGA TCT TTC MYH9_Ex23_R AGA GCT GGG CTC AGA CCT AC 630 58 exon 24 MYH9_Ex24_F TCA GGG ATG TGC TTT CTT TC MYH9_Ex24_R CCC CAA GCA CAT CTG TAG TC 563 58 exon 24 MYH9_Ex24_F TCA GGG ATG TGC TTT CTT TC MYH9_Ex24_R CCC CAA GCA CAT CTG TAG TC 563 58 exon 25 -26 MYH9_Ex2526F GGC TGC TCT TCT CTG AAG C MYH9_Ex2526R GGA GTC AGG CTT CCA GTA AAG 851 58 exon 27 -28 MYH9_Ex2728F AGC ACA TTC TGG ATC TGG AG MYH9_Ex2728R GCG ACA GCT GAA TTC CTG 752 58 exon 29 MYH9_Ex29_F CAG CTT CAG GAC ACA CAG G MYH9_Ex29_R CTC TGG GTC TCC CCT GTC 601 58 exon 30 MYH9_Ex30_F GAG GCT TGG TGT ATG TCT CG MYH9_Ex30_R TGG TTC CAT AGA AAC GAT CTG 592 58 exon 31 - 32 MYH9_Ex3132F CAT TCC CCA AGG AGT TTT G MYH9_Ex3132R AAC ACA GCC CAG GGA AAG 770 58 exon 33 MYH9_Ex33_F CCT GGA ACA AGG CTG AGA AC MYH9_Ex33_R TCT CAG AGC AGC TTT GCT TC 600 58 exon 34 MYH9_Ex34_F GGT GTG CTT GGT AAA TCC TG MYH9_Ex34_R CAC AAA GCC TCA TTT CAC G 611 58 exon 34 - 36 MYH9_Ex3536F AGG TCA CGG TCT GGG TTC T MYH9_Ex3536R CAA GGG AGG CTG TTG CAG 401 60 exon 37 MYH9_Ex37_F CTC CCT TGG TTT CCC TCT CT MYH9_Ex37_R CAG GTC AGG AGG GGA CAT 488 60 exon 38 -39 MYH9_Ex3839F AGG CCT AAG GAC AGC TCA TC MYH9_Ex3839R CCA GGG CAC AGA TAG AAC C 545 58 exon 40 MYH9_Ex40_F1 GTC CCT CTA AAG ATG GCT TTG MYH9_Ex40_R1 TGG GGA GGA ACG ACT CTC 844 58 MYH9_Ex40_F2 TGG GTG ATT GGG GAA GAG MYH9_Ex40_R2 CCA GAA GAT GCA CAA AGT AGG 981 58
Canine MYH9 gene
74
Table 2 Exonintron boundaries of the canine MYH9 gene
3rsquo-Splice site Exon 5rsquo-Splice site
Intron phase
Intron size
-20 (exon 1 162 bp) GTTCAGgtgagcgagccccgc
gt30000 bp
-19 +333 gctgtagGTCCTGGCTGTGA (exon 2 352 bp) CGTAAgtggggcctggggcc
0
4922 bp
+334 +490 tctttagACCTATTCAGGCC (exon 3 157 bp) GCAAGgtgatgcctcggcctt
1
13493 bp
+491 +518 gtttcagACCGAGAGGACCA (exon 4 28 bp) TGCACgtaagtgtttctgcc
2
803 bp
+519 +612 tcggcagGGGTGAGTCTGGA (exon 5 94 bp) ACCAGgtgagtgcgggcctt
0
4077 bp
+613 +705 cttgtagGGTGAGTTGGAGA (exon 6 93 bp) GATTTgtgagtagcggccag
0
427 bp
+706 +769 tttttagGGCAAATTCATTC (exon 7 64 bp) GACTTgtatccttggttgat
1
738 bp
+770 +868 tgcaaagATCTTCTGGAGAA (exon 8 99 bp) GAAGAgtgagtagtgagccc
1
343 bp
+869 +1012 ctttcagCTGATCTCCTGTT (exon 9 144 bp) AATGGgtacagagctggccc
1
749 bp
+1013 +1108 cccgaagGCTTGTTGCGAGT (exon 10 96 bp) CACAGgtaacgcctgtggtcc
1
1041 bp
+1109 +1227 atcgcagCTGCCCAGAAAGT (exon 11 119 bp) AACAGgtaggctctctggcc
0
1877 bp
+1228 +1380 accccagGCTGACTTTGCCA (exon 12 153 bp) TTGATgtgagctcctctcct
0
922 bp
+1381 +1554 cctgcagCTGAACTCCTTCG (exon 13 174 bp) AGCCAgtaagagccagacct
0
1801 bp
+1555 +1728 ttcccagGCAGGGCCCCCTG (exon 14 174 bp) GCAAGgtgaggcaagcacgt
0
2049 bp
+1729 +1843 cttgcagGTGGACTACAAAG (exon 15 115 bp) GGATGgtacgtgccccctgg
1
1877 bp
+1844 +2037 tttgcagTGGACCGCATCAT (exon 16 194 bp) AGAAGgtgcgggtgctgggc
0
343 bp
+2038 +2159 tctccagGCTGGCAAACTGG (exon 17 122 bp) CAGAGgtgagcggggcctgt
1
835 bp
+2160 +2229 ccctcagATATGAGATCTTG (exon 18 70 bp) TCATGgtgagtagagctgct
2
1418 bp
+2230 +2390 cgtctagATCAAAGCCCTGG (exon 19 161 bp) AGGAAgtgagttcctaaagg
1
851 bp
+2391 +2499 tccccagGGCCTTTGCCAAG (exon 20 109 bp) CCAAGgtgggttctggaagg
2
1428 bp
+2500 +2631 cttctagGTCAAGCCACTGC (exon 21 132 bp) CCCAGgtaggtgtcagcagg
2
398 bp
+2632 +2838 ttggcagCTCATGGCAGAGA (exon 22 207 bp) TCCAAgtgagccccttccct
2
488 bp
Canine MYH9 gene 75
Table 2 (continued)
3rsquo-Splice site Exon 5rsquo-Splice site
Intron phase
Intron size
+2839 +2976 atggtagGAACTGGAGGAGC (exon 23 138 bp) CCAAGgtgaggcccgtggtg
2
971 bp
+2977 +3100 cccccagGAGAAGAAGCTTC (exon 24 124 bp) GGAAGgtaagagggtaacag
0
1603 bp
+3101 +3272 tcctcagAGCGCCTCCGAAG (exon 25 172 bp) GCCAGgtgagggcctcgctt
1
719 bp
+3273 +3485 ggcagagTGGAGGAGGAAGC (exon 26 213 bp) CTCAGgtgtgccatgcggcc
1
270 bp
+3486 +3630 tctccagGTCCAAACGCGAA (exon 27 145 bp) AACGGgtgcgtgggctcccc
2
480 bp
+3631 +3837 ccaccagGTGAAAGCCAACT (exon 28 207 bp) TGCAGgtgaggcctccatgg
2
232 bp
+3838 +3941 gccacagGTTGAGCTGGACA (exon 29 104 bp) ACACAgtaaaggtggcagcg
1
204 bp
+3942 +4094 ggtgtagGAGCTGCTCCAGG (exon 30 153 bp) CCCAGgttcttatcggcatg
1
1083 bp
+4095 +4343 tgcccagGTGACGGACATGA (exon 31 249 bp) ACCAGgtgtgggccccaggc
1
1298 bp
+4344 +4556 tccccagCTACTGGCCGAGG (exon 32 213 bp) AGAGCgtgagtaccatgtca
1
150 bp
+4557 +4769 gtggcagGTCCACGAGCTGG (exon 33 213 bp) GACAGgtgtggaggccctcc
1
303 bp
+4770 +4931 gccccagGTGCGGGAGATGG (exon 34 162 bp) TGCAGgtgaggtcggctggg
1
1173 bp
+4932 +5060 accccagGCCCAGATGAAGG (exon 35 129 bp) AGGAGgtgggccgggcctca
1
941 bp
+5061 +5149 atggcagGAACTGGCAGCTG (exon 36 89 bp) AAGGGgtgagccgtggcgtg
0
92 bp
+5150 +5273 cacccagAGCTCTGGCATTG (exon 37 124 bp) TGCAGgtgggcggggtcggg
2
224 bp
+5274 +5482 cccacagATCGACCAGATCA (exon 38 209 bp) ACCAAgtaggtgttcctgcc
1
563 bp
+5483 +5591 ctcgcagGGAGCGCCAGGCA (exon 39 109 bp) ACCAGgtgtgtgtgcacacc
2
86 bp
+5592 +5763 tctgcagGCCGACAAGGCAT (exon 40 172 bp) CTCAGgtgagccctgcctcc
0
739 bp
+5764 +7295 tgcaaagATCTTCTGGAGAA (exon 41 1532 bp) CGTCACAATAAAGAGAACCA
Exon sequences are shown in uppercase letters and intron sequences in lowercase
letters Untranslated regions are shown in italics The conserved GTAG exonintron
junctions are shown in boldface type For the last exon the polyadenylation signal is
shown underlined instead of an exonintron junction Position +1 corresponds to the
adenine of the translation initiation codon ATG
Canine MYH9 gene
76
16
TT
TC
G
A
TC
A
A C
A
CT
T
C
GC
G
G
AG
C
C
AG
G
A
CT
C
T
GA
A
G
CC
C
T
CC
T
C
96
96
229
23
7
15
TT
TC
G
A
TC
A
A C
A
CT
T
C
GC
G
G
AG
C
C
AG
G
A
CT
C
T
GA
A
G
CC
C
T
CC
T
C
96
96
229
22
9
14
deaf
T
T T
C
GA
T
C
AA
CA
C
T
TC
G
C
GG
A
G
CC
A
G
GA
C
T
CT
G
A
AG
C
C
CT
C
C
TC
96
96
22
9
229
13
deaf
T
T T
C
GA
T
C
AA
CA
C
T
TC
G
C
GG
A
G
CC
A
G
GA
C
T
CT
G
A
AG
C
C
CT
C
C
TC
96
96
22
9
229
12
deaf
T
T C
C
AA
CC
A
A A
A T
T C
C
CC
G
G
GG
C
C
GG
A
A C
C
CC
A
A G
G
CC
T
T C
C
CC
96
10
0 24
1
229
11
deaf
T
T C
C
AA
CC
A
A A
A T
T C
C
CC
G
G
GG
C
C
GG
A
A C
C
CC
A
A G
G
CC
T
T C
C
CC
96
10
0 24
1
229
10
TT
TC
G
A
TC
A
A C
A
CT
T
C
GC
G
G
AG
C
C
AG
G
A
CT
C
T
GA
A
G
CC
C
T
CC
T
C
96
96
237
22
9
Fam
ily 3
9 T
T C
C
AA
CC
A
A A
A T
T C
C
CC
G
G
GG
C
C
- - CC
C
C
AA
GG
C
C
TT
CC
C
C
96
100
229
24
1
8 T
T C
T
AG
C
T
AA
AA
TT
CC
C
C
GG
G
G
CT
G
G
AA
CC
C
C
AA
GG
C
C
TT
CC
C
C
96
96
225
25
3
7 de
af
TC
C
C
AG
C
T
AT
A
A T
T C
C
CC
G
A
GG
C
T
GG
A
G
CC
C
C
AA
GG
C
T
TC
C
T
CC
96
89
22
1
237
6 de
af
TC
C
C
AG
C
T
AT
A
A T
T C
C
CC
G
A
GG
C
T
GG
A
G
CC
C
C
AA
GG
C
T
TC
C
T
CC
96
89
22
1
237 Fa
mily
2
5 T
C
CC
A
G
CT
A
T
AA
TT
CC
C
C
GA
G
G
CT
G
G
AG
C
C
CC
A
A G
G
CT
T
C
CT
C
C
96
89
221
23
7
4 de
af
TT
CC
A
A C
C
AA
AA
TT
CC
C
C
GG
G
G
CC
G
G
AG
C
C
CC
A
A G
G
CC
T
T C
C
CC
96
96
23
7
257
3 de
af
TC
C
C
AG
C
T
AT
A
A T
T C
C
CC
G
A
GG
C
T
GG
G
A
CC
C
C
AA
GG
C
T
TC
C
T
CC
96
96
23
7
257
2 T
C
CC
A
G
CT
A
T
AA
TT
CC
C
C
GA
G
G
CT
G
G
AA
CC
C
C
AA
GG
C
T
TC
C
T
CC
96
96
23
7
257 Fa
mily
1
1 C
T
CC
G
A
TC
T
A A
A T
T C
C
CC
A
G
GG
T
C
GG
A
G
CC
C
C
AA
GG
T
C
CT
T
C
CC
96
96
25
7
257
Ref
eren
ce
se
quen
ce
NW
_139
870
T C
A T A A T C
C
G
G
C
G
A C
C
A G
C
T C
C
posi
tion1
212
9
1 4
4 2
39
125
1
32
316
3
78
42
218
1
8 1
75
24
13
8
207
2
63
287
9
22
40
59
64
Pos
ition
with
in M
YH
9 In
tron
34
Exo
n 4
Intro
n 14
15
Intro
n 15
16
Intro
n 16
17
Intro
n 18
19
Intro
n 20
21
Intro
n 20
21
Intro
n 25
26
Intro
n 25
26
Intro
n 26
27
Intro
n 27
28
Exo
n 29
In
tron
293
0 In
tron
303
1 E
xon
32
Intro
n 32
33
Intro
n 32
33
Intro
n 34
35
Intro
n 38
39
Intro
n 38
39
Intro
n 38
39
Mic
rosa
tellit
e M
YH
9_M
S2
Mic
rosa
tellit
e M
YH
9_M
S3
(=FH
2293
)
1 N
umbe
ring
refe
rs to
the
posi
tion
of th
e po
lym
orph
ic n
ucle
otid
e w
ithin
the
give
n ex
on
or in
tron
Tabl
e 3
Hap
loty
pes
of th
e ca
nine
MY
H9
gene
in16
Dal
mat
ian
dogs
Canine MYH9 gene 77
Figure 1 Family structure of the analyzed Dalmatian dog families Hearing status was tested by brain auditory evoked response (BAER)
Canine MYH9 gene
78
Figure 2 Alignment of the canine MYH9 protein with orthologous human and murine
MYH9 protein sequences The sequences where derived from GenBank entries with
the accessions NP_002464 (human MYH9) and NP_071855 (mouse MYH9)
Identical residues are indicated by asterisk beneath the alignment while dots and
colons represent similar or very similar amino acids respectively
dog MAQQAADKYLYVDKNFINNPLAQADWAAKKLVWVPSDKSGFEPASLKEEVGEEAIVELVE 60 human MAQQAADKYLYVDKNFINNPLAQADWAAKKLVWVPSDKSGFEPASLKEEVGEEAIVELVE 60 mouse MAQQAADKYLYVDKNFINNPLAQADWAAKKLVWVPSSKNGFEPASLKEEVGEEAIVELVE 60 dog NGKKVKVNKDDIQKMNPPKFSKVEDMAELTCLNEASVLHNLKERYYSGLIYTYSGLFCVV 120 human NGKKVKVNKDDIQKMNPPKFSKVEDMAELTCLNEASVLHNLKERYYSGLIYTYSGLFCVV 120 mouse NGKKVKVNKDDIQKMNPPKFSKVEDMAELTCLNEASVLHNLKERYYSGLIYTYSGLFCVV 120 dog INPYKNLPIYSEEIVEMYKGKKRHEMPPHIYAITDTAYRSMMQDREDQSILCTGESGAGK 180 human INPYKNLPIYSEEIVEMYKGKKRHEMPPHIYAITDTAYRSMMQDREDQSILCTGESGAGK 180 mouse INPYKNLPIYSEEIVEMYKGKKRHEMPPHIYAITDTAYRSMMQDREDQSILCTGESGAGK 180 dog TENTKKVIQYLAHVASSHKSKKDQGELERQLLQANPILEAFGNAKTVKNDNSSRFGKFIR 240 human TENTKKVIQYLAYVASSHKSKKDQGELERQLLQANPILEAFGNAKTVKNDNSSRFGKFIR 240 mouse TENTKKVIQYLAHVASSHKSKKDQGELERQLLQANPILEAFGNAKTVKNDNSSRFGKFIR 240 dog INFDVNGYIVGANIETYLLEKSRAIRQAKEERTFHIFYYLLSGAGEHLKTDLLLEPYNKY 300 human INFDVNGYIVGANIETYLLEKSRAIRQAKEERTFHIFYYLLSGAGEHLKTDLLLEPYNKY 300 mouse INFDVNGYIVGANIETYLLEKSRAIRQAKEERTFHIFYYLLSGAGEHLKTDLLLEPYNKY 300 dog RFLSNGHVTIPGQQDKDMFQETMEAMRIMGIPEEEQMGLLRVISGVLQLGNIVFKKERNT 360 human RFLSNGHVTIPGQQDKDMFQETMEAMRIMGIPEEEQMGLLRVISGVLQLGNIVFKKERNT 360 mouse RFLSNGHVTIPGQQDKDMFQETMEAMRIMGIPEDEQMGLLRVISGVLQLGNIAFKKERNT 360 dog DQASMPDNTGNASAQKVSHLLGINVTDFTRGILTPRIKVGRDYVQKAQTKEQADFAIEAL 420 human DQASMPDNT---AAQKVSHLLGINVTDFTRGILTPRIKVGRDYVQKAQTKEQADFAIEAL 417 mouse DQASMPDNT---AAQKVSHLLGINVTDFTRGILTPRIKVGRDYVQKAQTKEQADFAIEAL 417 dog AKATYERMFRWLVLRINKALDKTKRQGASFIGILDIAGFEIFDLNSFEQLCINYTNEKLQ 480 human AKATYERMFRWLVLRINKALDKTKRQGASFIGILDIAGFEIFDLNSFEQLCINYTNEKLQ 477 mouse AKATYERMFRWLVLRINKALDKTKRQGASFIGILDIAGFEIFDLNSFEQLCINYTNEKLQ 477 dog QLFNHTMFILEQEEYQREGIEWNFIDFGLDLQPCIDLIEKPAGPPGILALLDEECWFPKA 540 human QLFNHTMFILEQEEYQREGIEWNFIDFGLDLQPCIDLIEKPAGPPGILALLDEECWFPKA 537 mouse QLFNHTMFILEQEEYQREGIEWNFIDFGLDLQPCIDLIEKPAGPPGILALLDEECWFPKA 537 dog TDKSFVEKVVQEQGTHPKFQKPKQLKDKADFCIIHYAGKVDYKADEWLMKNMDPLNDNIA 600 human TDKSFVEKVMQEQGTHPKFQKPKQLKDKADFCIIHYAGKVDYKADEWLMKNMDPLNDNIA 597 mouse TDKSFVEKVVQEQGTHPKFQKPKQLKDKADFCIIHYAGKVDYKADEWLMKNMDPLNDNIA 597
Canine MYH9 gene 79
dog TLLHQSSDKFVSELWKDVDRIIGLDQVAGMSETALPGAFKTRKGMFRTVGQLYKEQLAKL 660 human TLLHQSSDKFVSELWKDVDRIIGLDQVAGMSETALPGAFKTRKGMFRTVGQLYKEQLAKL 657 mouse TLLHQSSDKFVSELWKDVDRIIGLDQVAGMSETALPGAFKTRKGMFRTVGQLYKEQLAKL 657 dog MATLRNTNPNFVRCIIPNHEKKAGKLDPHLVLDQLRCNGVLEGIRICRQGFPNRVVFQEF 720 human MATLRNTNPNFVRCIIPNHEKKAGKLDPHLVLDQLRCNGVLEGIRICRQGFPNRVVFQEF 717 mouse MATLRNTNPNFVRCIIPNHEKKAGKLDPHLVLDQLRCNGVLEGIRICRQGFPNRVVFQEF 717 dog RQRYEILTPNSIPKGFMDGKQACVLMIKALELDSNLYRIGQSKVFFRAGVLAHLEEERDL 780 human RQRYEILTPNSIPKGFMDGKQACVLMIKALELDSNLYRIGQSKVFFRAGVLAHLEEERDL 777 mouse RQRYEILTPNSIPKGFMDGKQACVLMIKALELDSNLYRIGQSKVFFRAGVLAHLEEERDL 777 dog KITDVIIGFQACCRGYLARKAFAKRQQQLTAMKVLQRNCAAYLKLRNWQWWRLFTKVKPL 840 human KITDVIIGFQACCRGYLARKAFAKRQQQLTAMKVLQRNCAAYLKLRNWQWWRLFTKVKPL 837 mouse KITDVIIGFQACCRGYLARKAFAKRQQQLTAMKVLQRNCAAYLRLRNWQWWRLFTKVKPL 837 dog LQVSRQEEEMMAKEEELVKVREKQLAAENRLTEMETLQSQLMAEKLQLQEQLQAETELCA 900 human LQVSRQEEEMMAKEEELVKVREKQLAAENRLTEMETLQSQLMAEKLQLQEQLQAETELCA 897 mouse LNSIRHEDELLAKEAELTKVREKHLAAENRLTEMETMQSQLMAEKLQLQEQLQAETELCA 897 dog EAEELRARLTAKKQELEEICHDLEARVEEEEERCQHLQAEKKKMQQNIQELEEQLEEEES 960 human EAEELRARLTAKKQELEEICHDLEARVEEEEERCQHLQAEKKKMQQNIQELEEQLEEEES 957 mouse EAEELRARLTAKKQELEEICHDLEARVEEEEERCQYLQAEKKKMQQNIQELEEQLEEEES 957 dog ARQKLQLEKVTTEAKLKKLEEDQIIMEDQNCKLAKEKKLLEDRIAEFTTNLMEEEEKSKS 1020 human ARQKLQLEKVTTEAKLKKLEEEQIILEDQNCKLAKEKKLLEDRIAEFTTNLTEEEEKSKS 1017 mouse ARQKLQLEKVTTEAKLKKLEEDQIIMEDQNCKLAKEKKLLEDRVAEFTTNLMEEEEKSKS 1017 dog LAKLKNKHEAMITDLEERLRREEKQRQELEKTRRKLEGDSTDLNDQIAELQAQIAELKMQ 1080 human LAKLKNKHEAMITDLEERLRREEKQRQELEKTRRKLEGDSTDLSDQIAELQAQIAELKMQ 1077 mouse LAKLKNKHEAMITDLEERLRREEKQRQELEKTRRKLEGDSTDLSDQIAELQAQIAELKMQ 1077 dog LAKKEEELQAALARVEEEATQKNMALKKIRELESQISELQEDLESERASRNKAEKQKRDL 1140 human LAKKEEELQAALARVEEEAAQKNMALKKIRELESQISELQEDLESERASRNKAEKQKRDL 1137 mouse LAKKEEELQAALARVEEEAAQKNMALKKIRELETQISELQEDLESERASRNKAEKQKRDL 1137 dog GEELEALKTELEDTLDSTAAQQELRSKREQEVNILKKTLEEEARTHEAQIQEMRQKHSQA 1200 human GEELEALKTELEDTLDSTAAQQELRSKREQEVNILKKTLEEEAKTHEAQIQEMRQKHSQA 1197 mouse GEELEALKTELEDTLDSTAAQQELRSKREQEVSILKKTLEDEAKTHEAQIQEMRQKHSQA 1197 dog VEELAEQLEQTKRVKANLEKAKQTLENERGELANEVKVLQQGKGDSEHKRKKAEAQLQEL 1260 human VEELAEQLEQTKRVKANLEKAKQTLENERGELANEVKVLLQGKGDSEHKRKKVEAQLQEL 1257 mouse VEELADQLEQTKRVKATLEKAKQTLENERGELANEVKALLQGKGDSEHKRKKVEAQLQEL 1257 dog QVKFTEGERVRTELADKVTKLQVELDNVMGLLTQSDSKSSKLTKDFSALESQLQDTQELL 1320 human QVKFNEGERVRTELADKVTKLQVELDNVTGLLSQSDSKSSKLTKDFSALESQLQDTQELL 1317 mouse QVKFSEGERVRTELADKVTKLQVELDSVTGLLSQSDSKSSKLTKDFSALESQLQDTQELL 1317
Canine MYH9 gene
80
dog QEENRQKLSLSTKLKQMEDEKNSFKEQLEEEEEAKRNLEKQIATLHAQVTDMKKKMEDGV 1380 human QEENRQKLSLSTKLKQVEDEKNSFREQLEEEEEAKHNLEKQIATLHAQVADMKKKMEDSV 1377 mouse QEENRQKLSLSTKLKQMEDEKNSFREQLEEEEEAKRNLEKQIATLHAQVTDMKKKMEDGV 1377 dog GCLETAEEAKRKLQKDLEGLGQRYEEKVAAYDKLEKTKTRLQQELDDLLVDLDHQRQTAS 1440 human GCLETAEEVKRKLQKDLEGLSQRHEEKVAAYDKLEKTKTRLQQELDDLLVDLDHQRQSAC 1437 mouse GCLETAEEAKRRLQKDLEGLSQRLEEKVAAYDKLEKTKTRLQQELDDLLVDLDHQRQSVS 1437 dog NLEKKQKKFDQLLAEEKTISAKYAEERDRAEAEAREKETKALSLARALEEAMEQKAELER 1500 human NLEKKQKKFDQLLAEEKTISAKYAEERDRAEAEAREKETKALSLARALEEAMEQKAELER 1497 mouse NLEKKQKKFDQLLAEEKTISAKYAEERDRAEAEAREKETKALSLARALEEAMEQKAELER 1497 dog LNKQFRTEMEDLMSSKDDVGKSVHELEKSKRALEQQVEEMKTQLEELEDELQATEDAKLR 1560 human LNKQFRTEMEDLMSSKDDVGKSVHELEKSKRALEQQVEEMKTQLEELEDELQATEDAKLR 1557 mouse LNKQFRTEMEDLMSSKDDVGKSVHELEKSKRALEQQVEEMKTQLEELEDELQATEDAKLR 1557 dog LEVNLQAMKAQFERDLQGRDEQSEEKKKQLVRQVREMEAELEDEKKQRSMAVAARKKLEM 1620 human LEVNLQAMKAQFERDLQGRDEQSEEKKKQLVRQVREMEAELEDERKQRSMAVAARKKLEM 1617 mouse LEVNLQAMKAQFERDLQGRDEQSEEKKKQLVRQVREMEAELEDERKQRSMAMAARKKLEM 1617 dog DLKDLEAHIDSANKNRDEAIKQLRKLQAQMKDCVRELDDTRASREEILAQAKENEKKMKS 1680 human DLKDLEAHIDSANKNRDEAIKQLRKLQAQMKDCMRELDDTRASREEILAQAKENEKKLKS 1677 mouse DLKDLEAHIDTANKNREEAIKQLRKLQAQMKDCMRELDDTRASREEILAQAKENEKKLKS 1677 dog MEAEMIQLQEELAAAERAKRQAQQERDELADEIANSSGKGALALEEKRRLEARIAQLEEE 1740 human MEAEMIQLQEELAAAERAKRQAQQERDELADEIANSSGKGALALEEKRRLEARIAQLEEE 1737 mouse MEAEMIQLQEELAAAERAKRQAQQERDELADEIANSSGKGALALEEKRRLEARIALLEEE 1737 dog LEEEQGNTELVNDRLKKANLQIDQINTDLNLERSHAQKNENARQQLERQNKELKVKLQEM 1800 human LEEEQGNTELINDRLKKANLQIDQINTDLNLERSHAQKNENARQQLERQNKELKVKLQEM 1797 mouse LEEEQGNTELINDRLKKANLQIDQINTDLNLERSHAQKNENARQQLERQNKELKAKLQEM 1797 dog EGTVKSKYKASITALEAKIAQLEEQLDNETKERQAACKQVRRAEKKLKDVLLQVDDERRN 1860 human EGTVKSKYKASITALEAKIAQLEEQLDNETKERQAACKQVRRTEKKLKDVLLQVDDERRN 1857 mouse ESAVKSKYKASIAALEAKIAQLEEQLDNETKERQAASKQVRRTEKKLKDVLLQVEDERRN 1857 dog AEQFKDQADKASTRLKQLKRQLEEAEEEAQRANASRRKLQRELEDATETADAMNREVSSL 1920 human AEQYKDQADKASTRLKQLKRQLEEAEEEAQRANASRRKLQRELEDATETADAMNREVSSL 1917 mouse AEQFKDQADKASTRLKQLKRQLEEAEEEAQRANASRRKLQRELEDATETADAMNREVSSL 1917 dog KNKLRRGDLPFVVPRRVARKGAGDCSDEEVDGKADGAEAKAAE 1963 human KNKLRRGDLPFVVPRRMARKGAGDGSDEEVDGKADGAEAKPAE 1960 mouse KNKLRRGDLPFVVTRRIVRKGTGDCSDEEVDGKADGADAKAAE 1960
Chapter 6
Identification of a 5 Mb region on canine chromosome 10
harbouring a causative gene responsible for congenital sensorineural deafness
in German Dalmatian dogs
Fine mapping of CFA10 83
Identification of a 5 Mb region on canine chromosome 10 harbouring a causative gene responsible for congenital sensorineural deafness in German Dalmatian dogs
Abstract
In the present study we evaluated whether the canine chromosome (CFA) 10
harbours a gene responsible for canine congenital sensorineural deafness (CCSD) in
Dalmatian dogs Altogether 27 microsatellite markers covering the whole CFA10
were genotyped in 176 Dalmatian dogs with frequent occurrence of CCSD
Significant linkage between the deafness phenotype and microsatellites located in a
region of 36 Mb to 48 Mb on CFA10 was found In order to refine the location of the
causative canine congenital sensorineural deafness (CCSD) gene we used data
deposited in the NCBI to search for single nucleotide polymorphisms (SNPs) in the
intronic sequences of the canine genes located on CFA10 in this region We
characterized 26 SNPs and used them for non-parametric linkage and association
analyses with CCSD of altogether 34 Dalmatian dogs from five full-sib families We
could find significant linkage to SNPs located in the region spanning 39 Mb to 44 Mb
and significant haplotype-trait association for SNPs in this region These results
enforce further evaluation of this 5 Mb region with the aim to detect the gene
responsible for CCSD in Dalmatian dogs
Introduction
Of the identified genes responsible for different forms of sensorineural non-syndromic
deafness in humans Rak et al (2003) and co-workers (Droumlgemuumlller et al 2002
Kuiper et al 2002 Rak et al 2002a 2002b 2003) considered 24 genes as
candidates for sensorineural deafness in dogs among them the MYH9 gene on
CFA10 Subsequently Rak (2003) developed a set of microsatellite markers for the
respective 24 candidate genes
As described in chapter 3 we could show significant linkage of CCSD with the MYH9
associated microsaellite MYH9_MS3 (=FH2293) in different German Dalmatian dog
Fine mapping of CFA10
84
families However as described in Chapter 5 we already excluded MYH9 for being
responsible for the CCSD phenotype in German Dalmatian dog families segregating
for CCSD by comparative sequencing of genomic sequences from deaf and normal
hearing Dalmatian dogs and additionally by sequencing of full length canine cDNA
The reason for the MYH9_MS3 (=FH2293) indicating linkage may be caused by a
closely linked gene involved in CCSD Thus the objective of the present study was to
perform a scan of canine chromosome 10 using microsatellite markers and single
nucleotide polymorphisms (SNPs) in order to evaluate whether CFA10 harbours a
gene responsible for the deafness phenotype in Dalmatian dogs Additionally we
analyzed the association of the CCSD phenotype with a large number of newly
developed SNPs located in the genomic deafness region on CFA10
Material and methods Pedigree material
For the linkage analysis of the microsatellite marker on CFA10 we used DNA from
altogether 176 animals belonging to 22 full-sib families and one large paternal half-
sib family of German Dalmatian dogs All families were segregating for CCSD The
genotyped families included all the affected dogs (unilaterally and bilaterally deaf)
their parents if available and one to four unaffected full-sibs At least two of the full
sibs of each family were unilaterally deaf The phenotype of the affected animals had
been confirmed by brainstem auditory evoked response (BAER) that detects
electrical activity in the cochlea and auditory pathways in the brain
Screening for SNPs was performed by comparative sequencing of DNA from parents
of five families with significant linkage of microsatellites located in the region
spanning 36 Mb to 48 Mb to CCSD For the linkage analysis concerning the SNPs we
then used blood samples from 34 Dalmatian dogs consisting of the progeny and their
parents of the abovementioned five full-sib families of Dalmatian dogs with frequent
occurrence of CCSD The families consisted of five to nine individuals and their
parents
Microsatellite marker analysis
We used 27 microsatellite marker derived from the NCBI database
(httpwebncbinlmnihgov) to cover CFA10 completely (Appendix Table 1)
Fine mapping of CFA10 85
PCR reactions for microsatellites were carried out in 12 microl reaction mixtures
containing 2 microl genomic DNA 12 microl 10x PCR buffer (Qbiogene Heidelberg
Germany) 024 microl dimethyl sulfoxide (DMSO) 02 microl dNTPs (100 microM) 01 microl Taq
Polymerase (5Umicrol) (Qbiogene) 06 microl (10 microM) 5rsquo-IRD700 or IRD800 labelled forward
primer 06 microl (10 microM) unlabelled reverse primer The PCR reactions were carried out
in MJ Research thermocyclers with the following program 4 min at 94 degC followed
by 32 cycles of 30 sec at 94degC 30 sec at maximum annealing temperature and a
final extension of 45 sec at 72degC PCR-products were diluted with formamide loading
buffer in ratios from 110 to 140 and then size-fractionated by gel electrophoresis on
automated LI-COR 42004300 sequencers (LI-COR inc Lincoln NE USA) using
denaturing 4 and 6 polyacrylamide gels (RotiphoreseregGel 40 Roth Karlsruhe
Germany)
Development of single nucleotide polymorphisms (SNPs)
We developed SNPs for the region between 36 Mb to 48 Mb on CFA10 previously
proven to be linked to the CCSD phenotype The SNPs for this region were derived
from one 5294 Mb large whole genome shotgun sequence (NW_876251) deposited
in the current dog genome assembly (Boxer genome assembly 21) of the NCBI
GenBank The canine genomic sequences and mRNA of the genes that were used
for the analysis were also derived from sequences deposited in the current dog
genome assembly (Boxer genome assembly 21) of the NCBI GenBank (Table 1)
In total 72 primer pairs were designed most of them located intragenic in intronic
sequences or alternatively in flanking regions of the 5rsquo- or 3rsquondashends of the respective
gene yielding products with a length of around 600 bp PCR primers were developed
with the Primer3 program (httpfrodowimiteducgi-binprimer3primer3_wwwcgi)
The PCR reactions were performed in a total of 50 microl containing 125 microM dNTPs 25
pmol of each primer the reaction buffer supplied by the manufacturer (Qiagen
Hilden Germany) and 1 U Taq polymerase After a 4 min initial denaturation at
95degC 35 cycles of 30 sec at 94degC 45 sec at 58degC and 80 sec at 72degC were
performed in a MJ Research thermocycler (Biozym Hessisch Oldendorf Germany)
The obtained PCR products were directly sequenced with the DYEnamic ET
Terminator kit (AmershamBiosciences Freiburg Germany) and a MegaBACE 1000
capillary sequencer (AmershamBiosciences) using the PCR primers as sequencing
primers Sequence data were analyzed with Sequencher 42
Fine mapping of CFA10
86
(GeneCodes Ann Arbor MI USA) If a heterozygous SNP was found for one or both
parents all progeny of the respective families were analyzed for that SNP
We developed altogether 26 SNPs with 20 SNPs located intragenic in intronic
sequences and 6 SNPs located very closely at the 5rsquo- or 3rsquo-ends respectively with
one to four SNPs per gene (Table 3)
The newly identified 26 SNPs are shown in Tabele 2 Only SNPs are listed and
chosen for linkage analyses that were heterozygous for one or both parents of at
least two of the five families Of all SNPs only one was heterozygous in all families
(Table 3) The most frequent form of SNPs with a frequency of 423 was the AG
transition motif The scarcest one with a frequency of 385 was the CG and the
AC transversion motif respectively
Linkage analysis
Multipoint non-parametric linkage and haplotype analysis were performed using the
MERLIN software version 0102 (multipoint engine for rapid likelihood inference
Center for Statistical Genetics University of Michigan MI USA Abecasis et al
2002) The test statistics Z-mean and Lod score were used to test for the proportion
of alleles shared by affected individuals identical by descent (IBD) for the considered
marker loci
In a first step linkage analysis was performed regarding the 27 marker covering the
whole CFA10 In a second step a linkage analysis was performed including the newly
added SNPs spanning the region 36 Mb to 48 Mb on CFA10
The observed heterozygosity (HET) and the polymorphism information content (PIC)
were calculated using the software package SASGenetics (Statistical Analysis
System Version 913 SAS Institute Inc Cary NC USA 2005)
Linkage disequilibrium (LD) and association of haplotypes with CCSD were tested
using the procedures CASECONTROL and HAPLOTYPE of SASGenetics
(Statistical Analysis System version 913 Cary NC USA)
Results
A linkage analysis was at first carried out for the 176 animals that were analyzed with
27 microsatellite markers covering the whole CFA10
Fine mapping of CFA10 87
The microsatellite FH3302 positioned at 3887 Mb showed the highest Zmean with a
value of 234 (p= 001) and a Lod score of 121 (p= 0009) at 3887 Mb A Zmean
value of 217 (p= 0015) and a Lod score of 12 (p= 0009) was reached for the
microsatellite REN181G20 positioned at 3936 Mb indicating linkage but all Zmean
values and error probabilities of eight microsatellite markers in the interval from 3666
Mb up to 488 Mb were almost as high Consequently we screened the region
spanning from 36 Mb to 48 Mb for SNPs Out of the 23 analyzed Dalmatian dog
families five full-sib families were chosen to screen for SNPs because of their
significant contribution to the test statistics
Twenty-six new SNP markers covering the region of 36 Mb to 48 Mb equally were
developed and added to the linkage analysis These SNPs had PIC values ranging
from 013 to 037
Figure 1 shows the Zmeans and LOD scores for all 27 microsatellite markers on
CFA10 harbouring a genomic deafness region Figure 2 shows the corresponding p-
values Adding the SNPs in the interval spanning 36 Mb to 48 Mb we could narrow
the significant region down to 5 Mb spanning 39 Mb to 44 Mb (Table 4 Figures 3 and
4)
Haplotype-trait association test statistics for the SNPs 16-23 located in the interval
from 39 Mb to 44 Mb were significant However the marker-trait association test
failed the 5 threshold of the error probability (p= 007) but was lowest for all
possible haplotype-trait combinations The χ2ndashtests of the procedure
CASECONTROL were not significant indicating that a SNP for the causative
deafness gene was not yet found
Discussion
We used 27 microsatellite marker and 26 newly developed SNP markers with the
intention to create a dense map for linkage analysis of CFA10 especially the region
spanning 36 Mb to 48 Mb proven to be linked to the CCSD phenotype All SNP
markers were chosen due to their heterozygosity in one or both parents of at least
two families
The significant Zmeans on CFA10 reported for the chromosome scan using only
microsatellites was confirmed by adding the SNP markers Furthermore with the use
of SNPs the apparently linked region from 36 Mb to 48 Mb could be narrowed down
Fine mapping of CFA10
88
to 5 Mb spanning 39 Mb to 44 Mb For this reason the use of SNPs in addition to
informative microsatellites appears highly recommendable
The identified CCSD region spanning 5 Mb might be participating in the development
of CCSD in the analyzed Dalmatian dog families
However genes that are known to be involved in the hearing process or known to be
expressed in the inner ear are not annotated at the syntenic human region on homo
sapiens autosome (HSA) 2 spanning from 100 Mb to 108 Mb Therefore a well-
defined candidate gene for canine hereditary deafness in the abovementioned 5 Mb
does not exist This means that for all genes in this region informative SNPs have to
be developed and tested for linkage disequilibrium and association with CCSD
Despite the remarkable progress that has been made in the past few years in
identifying deafness genes in man and mouse it is suspected that only one-third of all
deafness causing genes have been identified so far Therefore more SNPs have to
be developed within the identified region on CFA10 to localize the deafness causing
gene or to find unambiguously associated SNP markers which could be used for a
population-wide genetic test for CCSD
Fine mapping of CFA10 89
Table 1 Canine genes where gene-associated SNPs could be developed with their
exact location on CFA10 and their corresponding accession numbers (Acc No)
Gene symbol Gene description Position in
Mb
Acc No canine genomic sequence
Acc No canine mRNA
LOC474535 similar to hypothetical protein MGC40397 36493650 NC_006592 XM_531764
LOC474536 similar to KM-102-derived reductase-like factor
37023714 NC_006592 XM_531765
LOC610953 similar to NHP2-like protein 1 37293729 NC_006592 XM_848546
LOC481302 similar to C2orf26 protein 37363737 NC_006592 XM_848552
LOC610991 hypothetical protein LOC610991 38103820 NC_006592 XM_848591
LOC611007 similar to eukaryotic translation elongation factor 1 alpha 2
38353835 NC_006592 XM_848614
LOC474541 similar to GRIP coiled-coil protein GCC185 isoform a
38503857 NC_006592 XM_531770
LOC474542 similar to Sulfotransferase 1C2 38613862 NC_006592 XM_531771
LOC481308 similar to Keratin type I cytoskeletal 18 38653865 NC_006592 XM_538429
LOC474543 similar to Sulfotransferase K1 38683870 NC_006592 XM_857994
LOC609217 similar to family with sequence similarity 32 member A like
39453945 NC_006592 XM_858065
LOC611115 similar to D-PCa-2 protein isoform c 39843984 NC_006592 XM_848756
LOC481325 similar to ubiquitin-conjugating enzyme E2C
42564272 NC_006592 XM_538446
LOC481330 similar to Interleukin-1 receptor type II precursor
44084413 NC_006592 XM_538451
LOC611493 hypothetical protein LOC611493 44384440 NC_006592 XM_863243
LOC481337 similar to DNA repair protein REV1
46264637 NC_006592 XM_538458
LOC611728 similar to 3-hydroxyanthranilate 34-dioxygenase
48494852 NC_006592 XM_849433
Predicted gene derived from the dog genome assembly (build 21) that used gene
prediction method GNOMON supported by EST evidence
Fine mapping of CFA10
90
Table 2 The 26 newly developed SNPs for Dalmatian dogs located in the region
spanning 36 Mb to 48 Mb on CFA10 with their corresponding primers the SNP motif
the product size and the annealing temperature
Gene
description SNP
Location
(intron or
5rsquo3rsquo-end)
Primer F1 (5acute -gt 3acute)
SNP motif
Primer R2 (5acute -gt 3acute)
Bp3 AT4
LOC474535
SNP_1
intron
ACCCAAGCCTAACTGCAGAA
ACCCCAGTCT(CG)GCCAGAGCTGTT
590 60
SNP_2 intron TGTGTGCATA(AG)TGTGCTGAGCCT
TCATCTGTTAAAACAGGGGTGAT
LOC474536
SNP_3
intron
CCAGTTAATGATTGTTTCGTTGA
AAGCTGCTTT(AC)CACCCCCATCAG
TCATTCCTGCTGTTGTGCTC
590 60
LOC610953
SNP_4
intron
CTGTCTTGGGGACTGTTTGC
AAGGCAGACG(CT)AATGACTGAGGC
600 60
SNP_5 intron GACTTGAAGC(CT)ACCACAGATACT
GCCATCACGATGAACTCAGA
LOC481302
SNP_6
3rsquo-end
AATTGAGGCCGAAGTCCAAT
CTTTTCCCCA(GT)GCCACCCCTCTG
GAGCACTATTTACGATACAAACAGGA
610 60
LOC610991
SNP_7
intron
CATGCATGATGCCCAGAGTA
CCCAAAGCAC(AG)CTGTGATTTAAT
AGGGCTTCCTGGGAAAAGT
600 60
LOC611007
SNP_8
intron
CAGACCAACAGTGACCCAGA
TAGGCATACC(GT)TCAGTCCTAAAG
GCCTGTTGTGGGCAGAGTAT
480 60
LOC474541
SNP_9
intron
ACTGAGCCAAAGGTGGATTG
AGAGAATAGC(AG)CTGTGTTTTACA
ACCTGCACATCGGGATTTAG
575 60
Fine mapping of CFA10 91
Table 2 (continued)
Gene-
symbol SNP
Location
(intron or
5rsquo3rsquo-end)
Primer F1 (5acute -gt 3acute)
SNP motif
Primer R2 (5acute -gt 3acute)
Bp3 AT4
LOC474542
SNP_10
intron
CTTCCCCAGGAGAGAGTGAC
AATATGATCA(CT)ATTTAAAGAAAT
CTTTTGTCAACATCCCCTTCA
560 60
LOC481308
SNP_11
3rsquo-end
ACCCATTGTCTCTCCAGCAC
CCACATAACT(GT)AGCATCCCTAGC
600 60
TGATGATGTAAGTTGGCCTCA
LOC474543
SNP_12
intron
TTGAAGTTGTGTGAGTAAATGAAAGA
CAATATATAA(AG)CATTTGCTACAA 600 60
SNP_13 intron TTGAGATGAA(AT)ATAATAGAGCTG
SNP_14 intron CGATTTTCCA(AG)GACATGGAGATG
SNP_15 intron ACTGAACCAA(AG)TGTGCAAAGCAT
GGAAACCATGCAGTCTTTGG
LOC609217
SNP_16
5rsquo-end
TGGCCTCATTTTCCAGTATG
GAAGGAGTTA(CT)ACAGTGAAGATA
580 60
SNP_17 5rsquo-end AAGTGATCAA(AG)CCAGTGCATTCT
GGCAATTACCCTGAGTGGTG
LOC611115
SNP_18
3rsquo-end
GGGCTGTCTTAGAGGTGCTG
TGTGGTCTCA(CT)ACACTTCCTGAG
590 60
SNP_19 3rsquo-end ATTGCAAGTG(GT)CCTGTGATGGCC
CTTCTTTGGGCAGGAAAGTG
LOC481325
SNP_20
intron
AAAATGATTGATCGCAAAAGAAA
AATTACTGTA(CT)AACAGTATCAGA
600 60
SNP_21 intron TAACAGTATC(AG)GAATAAAAGTTT
TTCTGTGATTGCACTGACCG
Fine mapping of CFA10
92
Table 2 (continued)
Gene-
symbol SNP
Location
(intron
or 5rsquo3rsquo-
end)
Primer F1 (5acute -gt 3acute)
SNP motif
Primer R2 (5acute -gt 3acute)
Bp3 AT4
LOC481330
SNP_22
intron
GAAAGGCCTGGGTTCAAAA
GGCAGGGAGG(AG)TCACCATCGTTC
AATTTCCCCAAATGCCTCAC
575 60
LOC611493
SNP_23
intron
GCATGAAGGAGCCCTATGTC
CCAAGAGTCC(AT)GCCCAACACCCT
GGAGGGATGGCATTCTATGA
590 60
LOC481337
SNP_24
intron
GGCTGAGGAGATTGTGTTTCA
GCTGATATTT(AG)GCCTTCTGAGAT
620 60
SNP_25 intron AAAGCCAGAC(AG)CTTTGCCATGGT
CAGCTCCCTGTAATGGGAAA
LOC611728
SNP_26
intron
TCCTACTCCCATCACTTCCAA
CCACACTGGG(GT)CCTGGGATGAGG
CACAGCTCCATGTAGGTCCA
620 60
hypothetical gene found in the 2 version 1 of the NCBIs genome annotation 1 Forward 2 Reverse 3 Product size (basepairs) 4 Annealing temperature (degC)
Fine mapping of CFA10 93
Table 3 The 26 newly developed intragenic SNPs for Dalmatian dogs with their
nucleotide polymorphism allele and genotype frequencies observed heterozygosity
(HET) and polymorphism information content (PIC)
SNP Fam1 Nucleotide
polymorphism
Allele
frequencies
Genotype
frequencies2 PIC HET
SNP_1 125 CgtG 042054 3135 036 055
SNP_2 124 AgtG 064039 91013 037 044
SNP_3 2345 AgtC 041049 2156 033 047
SNP_4 345 CgtT 065035 6140 035 067
SNP_5 34 CgtT 065035 490 028 042
SNP_6 345 CgtT 066034 6130 035 039
SNP_7 1345 AgtG 054046 6174 037 053
SNP_8 145 GgtT 075025 01010 029 045
SNP_9 1345 AgtG 052054 6145 037 047
SNP_10 1234 CgtT 057043 7173 037 068
SNP_11 14 GgtT 065035 5120 035 062
SNP_12 15 AgtG 028072 097 030 044
SNP_13 45 AgtT 077023 870 017 021
SNP_14 5 AgtG 036064 052 013 015
SNP_15 134 AgtG 034066 0157 034 059
SNP_16 145 CgtT 030070 3812 033 036
SNP_17 245 AgtG 058042 6113 030 032
SNP_18 123 CgtT 037063 1126 027 035
SNP_19 123 GgtT 045055 3115 030 032
SNP_20 2345 CgtT 032068 1129 029 039
SNP_21 1235 AgtG 063037 883 037 052
SNP_22 1234 AgtG 068032 10102 030 034
SNP_23 12345 AgtT 030070 21313 033 046
SNP_24 124 AgtG 047053 667 033 019
SNP_25 234 AgtG 053047 3132 035 053
SNP_26 234 GgtT 060040 3120 026 0391 Number of families which were informative for the respective SNPs 2 Genotypes are given as number of animals [homozygous for allele 1heterozygous
homozygous for allele 2]
Fine mapping of CFA10
94
Table 4 Linkage analysis for canine chromosome 10 regarding the region spanning
39 Mb to 44 Mb with the significant Zmean LOD score and error probabilities (p-
values)
Marker Location (Mb) Zmean pz-value1 LOD-score pL-value2
SNP_16 39453 262 0004 123 0009
SNP_17 39455 262 0004 123 0009
SNP_18 39840 261 0004 123 0009
SNP_19 39843 261 0004 123 0009
SNP_20 4260 255 0005 118 0010
SNP_21 4270 255 0005 117 0010
SNP_22 4405 317 00008 131 0007
SNP_23 4439 337 00004 134 0007 1 Error probability regarding Zmean 2 Error probability regarding LOD score
Fine mapping of CFA10 95
Figure 1 Zmeans and LOD scores for 27 microsatellite markers on CFA10
harbouring a congenital sensorineural deafness region (number of families 23
number of genotyped dogs 176)
Figure 2 P-values for Zmeans and LOD scores of 27 microsatellite markers on
CFA10 harbouring a congenital sensorineural deafness region (number of families
23 number of genotyped dogs 176)
Fine mapping of CFA10
96
Figure 3 Zmeans and LOD scores of 53 markers on CFA10 harbouring a congenital
sensorineural deafness region (number of families 5 number of genotyped dogs 34)
Figure 4 P-values for Zmeans and LOD scores of 53 markers on CFA10 for the
region between 30 to 60 Mb harbouring a congenital sensorineural deafness region
(number of families 5 number of genotyped dogs 34)
CCSD region (SNP 16-23)
Chapter 7
Analysis of canine chromosome 1 and the Gap junction protein alpha 1 (GJA1) gene
in Dalmatian dogs segregating for congenital sensorineural deafness
CFA1 and the GJA1 gene 99
Analysis of canine chromosome 1 and the Gap junction protein alpha 1 (GJA1) gene in Dalmatian dogs segregating for congenital sensorineural deafness Abstract Two microsatellite markers associated to the GJA1 (alternatively connexin 43) gene
showed significant linkage with canine congenital deafness (CCSD) in a large French
half-sib family (Chapter 3) PCR products of this half-sib family were used to perform
a sequence analysis in order to evaluate whether GJA1 is responsible for CCSD As
the linkage could not be confirmed we used altogether 27 microsatellite markers for
a scan of canine chromosome (CFA) 1 with 23 Dalmatian dog families segregating
for CCSD A non-parametric linkage analysis was performed to see whether
significant test statistics for other genomic regions on CFA1 and for more families can
be shown As a result we could not find linkage to any microsatellite in the analyzed
families
Introduction Rak (2003) considered in total 24 genes as candidates for sensorineural deafness in
dogs among them the Gap junction protein alpha 1 (GJA1) or connexin 43 gene on
canine chromosome (CFA) 1 A marker set of altogether 43 microsatellites were
developed by Rak (2003) among them two microsatellite marker associated to the
GJA1 gene
GJA1 or connexin 43 is a member of the connexin gene family and a component of
gap junctions Mutations in 4 members of the connexin gene family have been shown
to underlie distinct genetic forms of deafness including GJB2 (connexin [CX] 26)
GJB3 (CX31) GJB6 (CX30) and GJB1 (CX32)
Liu et al (2001) reported mutations in GJA1 in association with sensorineural
recessive deafness in man However these mutations have recently been shown to
involve the pseudogene of GJA1 on human chromosome (HSA) 5 and not the
CFA1 and the GJA1 gene
100
GJA1 gene on HSA 6 (WA Paznekas cited a personal communication from the
senior author (W E Nance) of the paper by Liu et al 2001)
In previously performed studies (Chapter 3) one large French Dalmatian dog family
with frequent occurrence of CCSD showed linkage to microsatellites associated to
the GJA1 gene
In this report we performed a mutation analysis of the GJA1 gene sequence to
identify polymorphisms In order to evaluate whether the GJA1 gene is responsible
for congenital sensorineural deafness in Dalmatian dogs we analyzed the
association of the GJA1 haplotypes with the CCSD phenotype Furthermore we
employed 27 microsatellite markers covering the entire CFA1 and used them for a
non-parametric linkage analysis with CCSD in a Dalmatian dog population of 176
animals with frequent occurrence of CCSD
Material and methods Pedigree material
For the linkage analysis of the microsatellite marker we used DNA from altogether
176 animals belonging to 22 full-sib families and one large paternal half-sib family of
German Dalmatian dogs All families were segregating for CCSD The genotyped
families included all affected dogs (unilaterally and bilaterally deaf) their parents if
available and one to four unaffected animals At least two of the full sibs of each
family were unilaterally or bilaterally deaf The phenotype of the affected animals had
been confirmed by brainstem auditory evoked response (BAER) that detects
electrical activity in the cochlea and auditory pathways in the brain
Mutation analysis and screening for SNPs was performed by comparative
sequencing of DNA from 16 animals consisting of the parents and their progenies of
one large half-sib family of French Dalmatian dogs which showed significant linkage
to GJA1-associated microsatellites (Chapter 3)
Sequencing of canine genomic DNA and mutation analysis
The canine GJA1 gene sequence was derived from sequences deposited in the
current dog genome assembly (Boxer genome assembly 21) of the NCBI GenBank
(Genbank Acc No NC_006583) We compared the canine genomic DNA sequence
to canine cDNA fragments in the canine EST database using the
CFA1 and the GJA1 gene 101
BLASTN (Basic Local Alignment Search Tool Nucleotide) program Three canine
ESTs (Acc No DN880776 BU744925 and DN369900) were used to confirm the
assembly of the GJA1 gene
To identify polymorphisms within the canine GJA1 sequence the gene consisting of
one 1251 bp spanning exon were PCR amplified and sequenced from 16 French
Dalmatian dogs Additionally large sequences from the 3acute-end were amplified Primer
pairs were designed yielding products with a length of around 600 bp PCR primers
were developed with the Primer3 program (httpfrodowimiteducgi-
binprimer3primer3_wwwcgi) The PCR reactions were performed in a total of 50 microl
containing 125 microM dNTPs 25 pmol of each primer the reaction buffer supplied by
the manufacturer (Qbiogene Heidelberg Germany) and 1 U Taq polymerase After
a 4 min initial denaturation at 95degC 35 cycles of 30 sec at 94degC 45 sec at 58degC and
80 sec at 72degC were performed in a MJ Research thermocycler (Biozym Hessisch
Oldendorf Germany)
The obtained PCR products were directly sequenced with the DYEnamic ET
Terminator kit (AmershamBiosciences Freiburg Germany) and a MegaBACE 1000
capillary sequencer (AmershamBiosciences) using the PCR primers as sequencing
primers Sequence data were analyzed with Sequencher 42
(GeneCodes Ann Arbor MI USA)
Microsatellite marker analysis
We used 27 microsatellite marker derived from the NCBI database
(httpwebncbinlmnihgov) to cover CFA1 completely (Appendix Table 1)
PCR reactions for microsatellites were carried out in 12 microl reaction mixtures
containing 2 microl genomic DNA 12 microl 10x PCR buffer (Qbiogene) 024 microl dimethyl
sulfoxide (DMSO) 02 microl dNTPs (100 microM) 01 microl Taq Polymerase (5Umicrol)
(Qbiogene) 06 microl (10 microM) 5rsquo-IRD700 or IRD800 labelled forward primer 06 microl (10
microM) unlabelled reverse primer The PCR reactions were carried out in MJ Research
thermocyclers with the following program 4 min at 94 degC followed by 32 cycles of 30
sec at 94degC 30 sec at maximum annealing temperature and a final extension of 45
sec at 72degC PCR-products were diluted with formamide loading buffer in ratios from
110 to 140 and then size-fractionated by gel electrophoresis on automated LI-COR
42004300 sequencers (LI-COR inc Lincoln NE USA) using denaturing 4 and 6
polyacrylamide gels (RotiphoreseregGel 40 Roth Karlsruhe Germany)
CFA1 and the GJA1 gene
102
Linkage and haplotype analysis
Multipoint non-parametric linkage and haplotype analyses were performed using the
MERLIN software version 0102 (multipoint engine for rapid likelihood inference
Center for Statistical Genetics University of Michigan MI USA Abecasis et al
2002) The test statistics Z-mean and Lod score were used to test for the proportion
of alleles shared by affected individuals identical by descent (IBD) for the considered
marker loci
A non-parametric linkage analysis was performed with 27 microsatellite markers in 23
Dalmatian dog families consisting of 176 Dalmatian dogs The results were added to
the linkage analysis performed in Chapter 3
Results and discussion
In previously performed studies (Chapter 3) one French Dalmatian dog family
reached a Zmean value of 295 (plt0002) for GJA1_MS1 and a Zmean value of 286
(plt0002) for GJA1_MS2 indicating linkage Both markers were associated with the
GJA1 gene
By sequence analysis we revealed a total of 3 SNPs found in sequences of the 3acute-
UTR shown in Table 1 None of the polymorphisms were polymorphic in all parents of
the examined French half-sib family Neither of the observed polymorphism did alter
the predicted amino acid sequence of GJA1 nor showed the identified haplotypes an
association with the CCSD phenotype (Figure 1)
There is no recombination of the haplotypes of the GJA1 gene in this family The
paternal and maternal haplotypes as far as they could be estimated were distributed
among the affected progeny closely to the expected proportion of 50 and therefore
no excess of a certain haplotype could be observed in the affected dogs
Thus evaluation of the SNP markers debilitates the linkage to CCSD for the French
half-sib family Because of the fact that both animals with unilateral hearing loss and
bilateral hearing animals shared identical haplotypes these polymorphisms are
obviously not associated with CCSD in these Dalmatian dog families Furthermore
the present study revealed no functional mutations of the complete coding region of
GJA1 It is therefore unlikely that the GJA1 gene is involved in the pathogenesis of
CCSD in the analyzed French Dalmatian dogs The reason for GJA1_MS1 and
GJA1_MS2 indicating linkage may be caused by a closely linked gene involved in
CFA1 and the GJA1 gene 103
CCSD among the half-sib family of French Dalmatian dogs or by a false positive
result of the microsatellite study performed in Chapter 3
To clarify whether significant test statistics for other genomic regions on CFA1 and
for more families can be shown we used 27 microsatellite markers derived from the
NCBI database (httpwebncbinlmnihgov) to cover CFA1 completely A linkage
analysis was carried out after genotyping 176 German Dalmatian dogs with a set of
27 microsatellite markers The results of this linkage analysis were added to the
results of the test statistics for the microsatellites GJA1_MS1 and GJA1_MS2
(Chapter 3)
As a result we could not find linkage to any microsatellite in the analyzed families
(Figure 2 and 3) It is unlikely that the canine chromosome 1 harbours genomic
regions that are involved in the development of CCSD in the analyzed Dalmatian dog
families
With hindsight it was revealed that GJA1 is not responsible for sensorineural non-
syndromic deafness in humans as Liu et al (2001) has published GJA1 is
participating in a human syndrome called oculodentodigital dysplasia (ODDD) that
can be accompanied with hearing impairment (Paznekas et al 2003) But the type of
deafness in human ODDD differs from the typical hearing loss associated with other
connexin mutations because it is conductive rather than sensorineural
As deafness in dogs especially in Dalmatians is almost exclusively caused by
sensorineural non-syndromic forms also known as cochleosaccular degeneration
the GJA1 gene should not be considered as a candidate gene for CCSD anymore
CFA1 and the GJA1 gene
104
Table 1 Three newly developed intragenic SNPs and two microsatellite markers for
Dalmatian dogs in the candidate gene GJA1 with their corresponding primers the
SNP motif the product size and the annealing temperature
SNP
Primer F (5acute -gt 3acute)
SNP motif
Primer R (5acute -gt 3acute)
Product
size (bp)
Annealing
temperatur
GJA1_SNP1+2
CACCTTAGGCGTTCATTTTG
CCGGGGAG(AG)AAAA(AG)AAAAATACTT
TGGCTTGATTCCCTGACTC
650 58
GJA1_SNP3
TCTGAAATGTAATCATGGATGC
CAGAACTTGTAT(AT)CTGTTAAGAG
AATCACACAGGATATAGAGGCTATC
600 58
Microsatellite
marker Primers (forward reverse ) 5acute -gt 3acute
Product
size (bp)
Annealing
temperatur
GJA1_MS1
ATGGCATGAAGAGGATACCG
AGGACAGGTGACGGCTCTAC
134 60
GJA1_MS2
GCTAGTACTCGATTGTGGTC
TCATGGGTTGTGAGATCCAG
190 60
CFA1 and the GJA1 gene 105
Figu
re 1
Hap
loty
pes
of th
e G
JA1
gene
in th
e an
alyz
ed D
alm
atia
n do
g fa
mily
CFA1 and the GJA1 gene
106
Figure 2 Zmeans and LOD scores for 29 microsatellite markers on CFA1 (number of
families 23 number of genotyped dogs 176)
Figure 3 P-values for Zmeans and LOD scores of 29 microsatellite markers on CFA1
(number of families 23 number of genotyped dogs 176)
Chapter 8
Analysis of canine chromosome 31 and the claudin-14 (CLDN14) gene
in Dalmatian dogs segregating for congenital sensorineural deafness
CFA31 and the CLDN14 gene 109
Analysis of canine chromosome 31 and the claudin-14 (CLDN14) gene in Dalmatian dogs segregating for congenital sensorineural deafness Abstract A previously accomplished linkage analysis revealed significant linkage of
microsatellite markers associated with the CLDN14 gene on canine chromosome
(CFA) 31 with the phenotype of canine sensorineural deafness (Chapter 3) The
objective of the present study was to perform a sequence analysis in order to find
single nucleotide polymorphisms (SNPs) for the CLDN14 gene and additionally to
use a set of six microsatellite markers evenly distributed on CFA31 for non-
parametric linkage analysis with the aim to verify the significant test statistics shown
in Chapter 3
Introduction
Of the genes responsible for different forms of sensorineural non-syndromic deafness
in humans Rak (2003) considered 24 genes as candidates for sensorineural
deafness in dogs among them the CLDN14 gene on canine chromosome (CFA) 31 CLDN14 encodes a member of the claudin family which comprises the major
components of tight juncions (TJ) The human CLDN14 gene consists of one
tranlatete exon and six exons in the 5rsquountranlated region Five splice isoformes are
identified so far (Wilcox et al 2001 Wattenhofer et al 2005)
For the compartmentalization of perilymph and endolymph in the inner ear the
leakage of solutes through a paracellular pathway must be prevented by tight
junctions TJ are an intercellular junction found at the most apical region of polarised
epithelial and endothelial cells at which adjacent plasma membranes are joined
tightly together separating apical membranes and basolateral menbranes They are
specialised membrane domains containing branching strands of integral proteins and
create a primary barrier preventing paracellular transport of solutes and restricting
lateral diffusion of membrane lipids and proteins (Schneeberger and Lynch 2004)
CFA31 and the CLDN14 gene
110
The important role of the CLDN14 gene in the TJ of the inner ear was demonstrated
by Wilcox et al (2001) when he identified mutations in the CLDN14 gene responsible
for a hereditary human deafness in families segregating for congenital recessive
deafness (DFNB29)
In previously performed studies one large half-sib family as well as several full-sib
families of German Dalmatian dog indicated linkage to CLDN14 gene-associated
markers (Chapter 3)
A sequence analysis was performed to identify single nucleotide polymorphisms
(SNPs) either intergenic located or alternatively in the flanking 5rsquo- and 3rsquo-Regions
Furthermore we employed microsatellite markers covering CFA31 and used them for
a non-parametric linkage analysis with CCSD in a German Dalmatian dog population
of 176 animals with frequent occurrence of CCSD
Material and methods Pedigree material
For the linkage analysis of the microsatellite marker we used DNA from altogether
176 animals belonging to 22 full-sib families and one large paternal half-sib family of
German Dalmatian dogs All families were segregating for CCSD The genotyped
families included all the affected dogs (unilaterally and bilaterally deaf) their parents
if available and one to four unaffected animals At least two of the full sibs of each
family were unilaterally or bilaterally deaf The phenotype of the affected animals had
been confirmed by brainstem auditory evoked response (BAER) that detects
electrical activity in the cochlea and auditory pathways in the brain
Mutation analysis and screening for SNPs was performed by comparative
sequencing of DNA from 36 animals consisting of the parents and their progenies of
one half-sib family and four full-sib families of Dalmatian dogs which showed
significant linkage to a CLDN14-associated microsatellite (Chapter 3)
Sequencing of canine genomic DNA and development of single nucleotide
polymorphisms (SNPs)
The canine CLDN14 gene sequence was derived from sequences deposited in the
current dog genome assembly (Boxer genome assembly 21) of the NCBI GenBank
(Genbank Acc No NW_876295) by BLAST (Basic Local Alignment Search Tool)
CFA31 and the CLDN14 gene 111
search (httpwwwncbinlmnihgovBLAST) using the human CLDN14 reference
mRNA sequence (Genbank Acc No NM_144492)
We compared the canine genomic DNA sequence to canine cDNA fragments in the
canine EST database using the BLASTN program As no ESTs could be found
human mRNA sequences were used for the localization of the exonintron
boundaries using the mRNA-to-genomic alignment program Spidey
(httpwwwncbinlmnihgovIEBResearchOstellSpideyindexhtml)
We screened exon three for mutations as this exon is the only translated one in man
Additionally we screened large intronic sequences and sequences located in the 5rsquo-
and 3rsquo-UTR of the CLDN14 gene for Polymorphisms For this purpose primer pairs
were designed yielding products with a length of around 600 bp
PCR primers were developed with the Primer3 program (httpfrodowimiteducgi-
binprimer3primer3_wwwcgi) The PCR reactions were performed in a total of 50 microl
containing 125 microM dNTPs 25 pmol of each primer the reaction buffer supplied by
the manufacturer (Qbiogene Heidelberg Germany) and 1 U Taq polymerase After
a 4 min initial denaturation at 95degC 35 cycles of 30 sec at 94degC 45 sec at 58degC and
80 sec at 72degC were performed in a MJ Research thermocycler (Biozym Hessisch
Oldendorf Germany)
The obtained PCR products were directly sequenced with the DYEnamic ET
Terminator kit (AmershamBiosciences Freiburg Germany) and a MegaBACE 1000
capillary sequencer (AmershamBiosciences) using the PCR primers as sequencing
primers Sequence data were analyzed with Sequencher 42 (GeneCodes Ann
Arbor MI USA) The eight newly developed SNPs are shown in Table 1
Microsatellite marker analysis
In total six microsatellite marker were derived from the NCBI database
(httpwebncbinlmnihgov) to cover CFA31 completely (Appendix Table 1)
PCR reactions for microsatellites were carried out in 12 microl reaction mixtures
containing 2 microl genomic DNA 12 microl 10x PCR buffer (Qbiogene) 024 microl dimethyl
sulfoxide (DMSO) 02 microl dNTPs (100 microM) 01 microl Taq Polymerase (5Umicrol)
(Qbiogene) 06 microl (10 microM) 5rsquo-IRD700 or IRD800 labelled forward primer 06 microl (10
microM) unlabelled reverse primer The PCR reactions were carried out in MJ Research
thermocyclers with the following program 4 min at 94 degC followed by 32 cycles of 30
sec at 94degC 30 sec at maximum annealing temperature and a final extension of
CFA31 and the CLDN14 gene
112
45 sec at 72degC PCR-products were diluted with formamide loading buffer in ratios
from 110 to 140 and then size-fractionated by gel electrophoresis on automated LI-
COR 42004300 sequencers (LI-COR inc Lincoln NE USA) using denaturing 4
and 6 polyacrylamide gels (RotiphoreseregGel 40 Roth Karlsruhe Germany)
Linkage and haplotype analysis
Multipoint non-parametric linkage and haplotype analysis were performed using
MERLIN version 0102 (multipoint engine for rapid likelihood inference Center for
Statistical Genetics University of Michigan MI USA Abecasis et al 2002) The test
statistics Z-mean and Lod score were used to test for the proportion of alleles shared
by affected individuals identical by descent (IBD) for the considered marker loci A
linkage analysis was performed with 6 microsatellite markers on 176 Dalmatian dogs
The results were added to the linkage analysis performed in Chapter 3
Results and discussion CLDN14-associted microsatellites were significantly linked with CCSD in a scan of
candidate genes using 24 families with 215 Dalmatian dogs (Chapter 3) Selection of
four full-sib families and one half-sib family with the highest tests statistics lead to a
Zmean value of 383 (plt000007) for the CLDN14 gene-associated marker
CLDN14_MS2
To substantiate the linkage to the CLDN14 gene we searched for sequence
variations within the CLDN14 gene in four full-sib families and one half-sib family with
the highest contribution to the test statistics shown in Chapter 3 Most of the identified
polymorphisms were found in intronic sequences none were within exon three None
of the observed polymorphism did alter the predicted amino acid sequence of exon
three We revealed a total of eight SNPs (Table 1) but only two (SNP_4 and SNP_5)
out of the identified eight SNPs were polymorphic in the examined families Both
unilaterally and bilaterally deaf animals as well as normal hearing animals showed
identical haplotypes for these two polymorphic SNPs and thus no co-segregation with
the deaf Dalmatian dogs could be revealed (Figure 1 and 2) In this study we did not
identify any causal mutations for CCSD in the analyzed Dalmatian dogs Despite this
results a mutation outside of the translated genomic regions analyzed here may exist
that would affect CLDN14 expression
CFA31 and the CLDN14 gene 113
To clarify if other regions on CFA31 are responsible for the CCSD phenotype
additionally six microsatellite markers covering CFA31 were tested in 23 Dalmatian
dog families consisting of 176 individuals A non-parametric linkage analysis was
performed regarding these 23 families The results of this linkage analysis were
added to the results of the test statistics for the CLDN14 gene-associated
microsatellites (Chapter 3)
We could only find significant linkage with CCSD for CLDN14-associted
microsatellites in the abovementioned five families The two microsatellites located
most closely were proximal and distal 4 Mb off the CLDN14 gene REN110K04 with a
Zmean value of -009 (plt05) and FH2712 with a Zmean value of 022 (plt03) not
indicating linkage
It can not be excluded that CLDN14 or genes in its flanking regions are involved in
the development of CCSD in the analyzed Dalmatian dog families Other genes than
the CLDN14 that are known to be involved in the hearing process or known to be
expressed in the inner ear are not annotated at the syntenic human region of homo
sapiens autosome (HSA) 21q223 Therefore despite the CLDN14 gene no other
well-defined candidate gene for canine hereditary deafness exists in the linked
region Despite the remarkable progress that has been made in the past few years in
identifying deafness genes in man and mouse it is suspected that only one-third of all
deafness causing genes have been identified so far and thus it seems possible that
other genes in the flanking region of the CLDN14 gene are involved in the
development of the disease Anyway to clarify the importance of CLDN14 in the
CCSD phenotype more SNPs have to be developed within the CLDN14 gene as well
as in its flanking regions with the aim to find significant linkage disequilibrium of SNP
markers with CCSD
CFA31 and the CLDN14 gene
114
Table 1 The 8 newly developed SNPs for Dalmatian dogs in the CLDN14 gene on
CFA31 with their corresponding primers the SNP motif the product size and the
annealing temperature
SNP
Location
(intron or
5rsquo3rsquo-UTR)
Primer F1 (5acute -gt 3acute)
SNP motif
Primer R2 (5acute -gt 3acute)
Bp3 AT4
CLDN14_SNP1
intron
GACCATATGTTTGTGGCC
CTTCCAGGGAAA(AT)TGTCGTAGCC
CGTCAGGATGTTGGTGCC
580 60
CLDN14_SNP2
inton
GACCATATGTTTGTGGCC
GAAATTGTCGTA(AG)CCCGGCCGCT
CGTCAGGATGTTGGTGCC
580 60
CLDN14_SNP3
3rsquo-UTR
CTGCCTTCAAGGACAACC
CCAGAGGAATAA(CT)ATGATCGTGA
GATGAGTATCAGCCCAGC
550 60
CLDN14_SNP4
3rsquo-UTR
CTGCCTTCAAGGACAACC
ACCACCGCACAC(CT)GTCACAGCTT
GATGAGTATCAGCCCAGC
550 60
CLDN14_SNP5 3rsquo-UTR
CATGCCTTTGTCCCAAACTT
GAGACCCTCTGG(CT)TCCTTTTGGC
GTACCTGTTGCCTGGGTTGT
610 60
CLDN14_SNP6 5rsquo-UTR
CCTTCATCCTTTCTGGTTGA
GCTCACAGTCCC(AC)ATGGGGACAT
GGGGAGCATAATGTGGTCAT
585 60
CLDN14_SNP7 5rsquo-UTR
TGAACTGGTCCCAAGGAAAG
GCACGACCGAGC(CT)CTGGCTTTAC
GGGATGAGAGGGAGGTTTTT
580 60
CLDN14_SNP8 3rsquo-UTR
AATGCCTATCCCTTCTTTGGA
CACGTTACTGTG(AG)ACCTCTCTAC
GCAGGCTTTCCTCAAGTGTC
680 60
1 Forward 2 Reverse 3 Product size (basepairs) 4 Annealing temperature (degC)
CFA31 and the CLDN14 gene 115
Figure 1 Haplotypes of the CLDN14 gene-associated markers SNP_4 and SNP_5 in
the analyzed four Dalmatian dog full-sib families
CFA31 and the CLDN14 gene
116
Figu
re 2
Hap
loty
pes
of th
e C
LDN
14 g
ene-
asso
ciat
ed m
arke
rs S
NP
_4 a
nd S
NP
_5 in
the
anal
yzed
Dal
mat
ian
dog
half-
sib
fam
ily
Chapter 9
General discussion
General discussion 119
General discussion
The candidate gene approach In the first step of this thesis candidate genes for canine congenital sensorineural
deafness (CCSD) in Dalmatian dogs were analyzed by means of microsatellite
markers or alternatively by single nucleotide polymorphisms (SNPs)
The candidate genes for which a set of in total 43 microsatellites was available
included the following 24 genes CDH23 CLDN14 COCH COL11A2 DFNA5
DIAPH1 EDN3 EDNRB EYA4 GJA1 GJB2 GJB6 MITF MYH9 MYO6 MYO7A
MYO15A OTOF PAX3 POU4F3 SLC26A4 SOX10 TECTA and TMPRSS3
(Rak 2003) These genes are known to be involved either in human non-syndromic
deafness or in the human Waardenburg syndrome The Waardenburg syndrome
(WS) manifests with sensorineural deafness and pigmentation defects in iris hair and
skin WS is classified into four types depending on the presence or absence of
additional symptoms which are caused by mutations in the five genes EDN3
EDNRB MITF PAX3 and SOX respectively
For another eight recently identified genes responsible for different forms of human
non-syndromic deafness including TMC1 TMIE USH1C MYH14 MYO3A PRES
WHRN and ESPN linkage and association analyses were performed using newly
developed SNPs
In the last years most projects have exploited canine traits for which either direct
candidate genes could be proposed and evaluated or for which large informative
pedigrees were available to enable linkage mapping to identify candidate regions A
major component of such research efforts comprised the cloning sequencing and
mapping of individual canine homologs of genes either proposed as candidate
genes or expected to be located in candidate regions This was necessary to identify
new informative polymorphisms (eg SNPs microsatellites) for high resolution
mapping of candidate regions and to examine each exon and exonintron boundary
for positional candidates Availability of the second version of the dog genome
assembly (build 21) of the NCBI database shortcuts this effort and increases the
investigators efficiency Now either additional candidate genes for canine congenital
sensorineural deafness can be found directly by its gene symbol in the 21 of the
General discussion
120
NCBIs genome annotation or if a candidate gene is not yet annotated a BLAST
(Basic Local Alignment Search Tool) search against the canine whole genome
shotgun (wgs) sequence resource can be used to obtain the sequence of the canine
genomic contigs containing the human homologous gene and thus intragenic
markers can be developed for subsequent linkage and association analyses
Over the past decade it has become increasingly clear how far structural and
functional homologies at the gene level extend across even distantly related species
Much is known about deafness-causing gene mutations in humans and mice
including the fact that the clinical and histopathological findings are often very similar
to those of deafness in Dalmatian dogs Thus genes responsible for non-syndromic
congenital hereditary deafness in humans seem to be appropriate candidate genes
for CCSD (Rak and Distl 2005) In this thesis we first concentrated on the candidate
gene approach combined with linkage analysis method using affected pedigree
members Once a significant linkage was found only the linked genes with the
required low error probability values were used for further molecular genetic analysis
The method of candidate gene approach using either gene-associated microsatellite
or alternatively SNP markers was applied for in total 32 candidate genes which
comprise nearly all of the identified mutated genes causing non-syndromic hereditary
hearing impairment in humans
Linkage and association analysis In principle linkage and association are totally different phenomena Linkage is a
relation between loci and association is a relation between alleles
Linkage means that a haplotype characterised by microsatellites or SNPs is
significantly more often present in family members with the phenotype under study
than expected by random assortment For construction of haplotypes sets of closely
linked genetic markers on the same chromosome are needed which tend to be
inherited together as they are not likely to be separated by recombination Linkage
creates associations within families but not among unrelated induviduals
Association is a statistical statement about the co-occurrence of alleles or
phenotypes Association analysis can be carried out as a method of genetic analysis
that compares the frequency of alleles between affected and unaffected individuals
across all families Thus for association family structures are not necessary A given
allele is considered to be associated with the disease if the presence of that allele
General discussion 121
explains a significant proportion of the phenotypic trait variation For association
studies the developing of a marker set consisting of SNPs rather than microsatellites
is needed
In this thesis a total of 32 candidate genes for canine congenital deafness were
analyzed which showed an appropriate clinical and histological disease pattern in
comparison to deafness in Dalmatian dogs Rak (2003) developed a set of 43
microsatellites for in total 24 candidate genes among them the CLDN14 gene on
canine chromosome (CFA) 31 and the MYH9 gene on CFA10 The GJA1 on CFA1
was also considered as a candidate gene for CCSD (Rak 2003) and therefore two
gene-associated microsatellites have been developed Recently it turned out that
GJA1 is not responsible for human sensorineural non-syndromic deafness but for a
human syndromic disorder that can be related with conductive deafness
By the use of a non-parametric linkage analysis using the existing set of 43
microsatellites associated to 24 candidate genes we found linkage to markers
associated to CLDN14 on CFA31 MYH9 on CFA10 and GJA1 on CFA1
For another another eight candidate genes it was possible to develop SNPs
Performing linkage analyses as well as association and haplotype studies it was
possible to exclude these eight candidate genes from being responsible for the
CCSD phenotype
Over the past ten years significant progress has been made in the identification of
deafness gene localisations Up to now approximately 120 loci have been reported
for both autosomal dominant and recessive forms of non-syndromic hereditary
deafness in humans and only for one third the responsible gene mutation could be
detected Thus it can be expected that additional potential human candidates for
CCSD in Dalmatian dogs will become available in future (Van Camp and Smith
2003)
The extreme heterogeneity of human deafness often hampered genetic studies
because many different genetic forms of hearing loss give rise to similar clinical
phenotypes and conversely mutations in the same gene can result in a variety of
clinical phenotypes In man genes that transport ions across membranes to
maintain appropriate solute concentration and pH as well as regulatory genes mainly
transcription factors and genes that play a part in structural integrity are essential
for the hearing process
General discussion
122
The results of this thesis indicate that the inheritance of hearing loss in Dalmatian
dogs is probably as heterogenic in origin as it is in humans Genetic heterogeneity
means that different mutations cause a similar phenotype the different mutations
can either be found at the same locus (allelic heterogeneity) or even at different loci
(non-allelic heterogeneity) As linkage was found for different candidate genes in
different families subsequently only the families indicating linkage were chosen for
further molecular analyses
GJA1 on CFA1 MYH9 on CFA10 and CLDN14 on CFA31 and their flanking regions
are further analyzed with a combined approach using microsatellite and SNP
markers
CFA1 By the use of GJA1-associated microsatellites one large French Dalmatian dog
family reached a Zmean value of 295 (plt0002) for GJA1_MS1 and a Zmean value
of 286 (plt0002) for GJA1_MS2 indicating linkage Subsequently a sequence
analysis of the GJA1 gene using the above mentioned French Dalmatian dog family
was performed None of the observed polymorphism did alter the predicted amino
acid sequence of GJA1 nor showed the identified haplotypes an association with the
CCSD phenotype Thus evaluation of the SNP markers debilitates the linkage to
CCSD for the French half-sib family It is unlikely that the GJA1 gene is involved in
the pathogenesis of CCSD in Dalmatian dogs To see whether significant test
statistics for other genomic regions on CFA1 and for more families can be shown a
non-parametric linkage analysis was performed with 27 microsatellite markers
covering CFA1 completely In total 176 animals were genotyped We could not find
linkage to any microsatellite in the analyzed families Furthermore it was revealed
that GJA1 is not a candidate gene for sensorineural non-syndromic deafness in
humans (WA Paznekas cited a personal communication from the senior author (W
E Nance) of the paper by Liu et al 2001) GJA1 is participating in a human
syndrome called oculodentodigital dysplasia (ODDD) that can be accompanied with
hearing impairment (Paznekas et al 2003) But the type of deafness in human
ODDD is conductive rather than sensorineural As deafness in dogs especially in
Dalmatians is almost exclusively caused by sensorineural non-syndromic forms also
known as cochleosaccular degeneration the GJA1 gene should not be considered
as a candidate gene for CCSD anymore
General discussion 123
CFA31 By the use of microsatellites as well as SNPs we found significant linkage with CCSD
for CLDN14-associated microsatellites in four full-sib and one half-sib Dalmatian dog
familiy with a Zmean value of 383 (plt000007) A mutation analysis was performed
for exon three as this is the only translated one in man None of the observed
polymorphisms did alter the predicted amino acid sequence However to clarify the
importance of the CLDN14 gene and its flanking regions in the CCSD phenotype
more SNPs have to be developed within the CLDN14 gene as well as in its flanking
regions with the aim to find significant linkage disequilibrium of SNP markers
CFA10 A significant co-segregation of markers alleles and the phenotypic expression of
deafness in a large sample of German Dalmatian dog families was determined for
one marker (Z-mean of 156 [p= 006] and a Lod score of 058 [p= 005]) associated
to the MYH9 gene Subsequently we evaluated whether MYH9 gene mutations are
responsible for CCSD in these Dalmatian dog families An initial priority in defining
gene structure is to obtain a full-length cDNA sequence and identify translational
initiation and termination sites and polyadenylation site(s) Exonintron structure can
then be determined by referencing the cDNA sequence against sequences of
cognate genomic DNA One popular method of obtaining full-length cDNA sequences
is the RACE (rapid amplification of cDNA ends) technique RACE-PCR is an anchor
PCR modification of RT-PCR The rationale is to amplify sequences between a single
previously characterised region in the mRNA (cDNA) and an anchor sequence that is
coupled to the 5 or the 3 end A primer is designed from the known internal
sequence and the second primer is selected from the relevant anchor sequence
To provide the genomic organization and the complete sequence of the canine
MYH9 gene the isolation of full length cDNAs was achieved with the help of a
modified rapid amplification of cDNA ends (RACE) protocol A mutation analysis was
performed to identify single nucleotide polymorphisms (SNPs) in this gene We
analyzed the association of the MYH9 haplotypes with the CCSD phenotype in three
families of Dalmatian dogs with frequent occurrence of CCSD and significant linkage
to gene-associated microsatellites Using the RT-PCR analyses for cDNA-genomic
sequence comparisons we detected that the canine MYH9 gene is bigger compared
to the human sequence due to the untranlated first exon in the 5rsquo-UTR
General discussion
124
The canine MYH9 gene consists of 41 exons with an untranslated exon 1 and
exonintron boundaries that conform perfectly to the GTAG rule
None of the observed polymorphisms did alter the predicted amino acid sequence of
MYH9 nor showed the identified haplotypes an association with the CCSD
phenotype
Thus these silent point mutations found in affected and unaffected Dalmatian dogs
do not seem to be responsible for the CCSD phenotype in these three families
To clarify if other regions on CFA10 are responsible for the CCSD phenotype we
added in a second step 27 microsatellite markers derived from the NCBI database to
cover CFA10 with a high density in the flanking region of the MYH9 gene A linkage
analysis was carried out for 23 Dalmatian dog families consisting of 176 animals that
were genotyped with the marker set of 27 microsatellite markers
We found significant linkage to microsatellites in the region spanning 36 Mb to 48 Mb
Consequently we screened this 12 Mb spanning region for SNPs Out of the 23
analyzed Dalmatian dog families five full-sib families were chosen to screen for
SNPs because of their obviously significant values at the above mentioned region
Twenty-six new SNP markers covering the region of 36 Mb to 48 Mb equally were
developed and added to the linkage analysis The significant Zmeans on CFA10 was
confirmed after adding the SNP markers Furthermore with the use of SNPs the
apparently linked region spanning 36 Mb to 48 Mb could be narrowed down to 5 Mb
spanning from 39 Mb to 44 Mb For this reason the use of SNPs in addition to
informative microsatellites appears highly recommendable
In further studies more SNPs have to be developed within the identified CCSD region
on CFA10 to localize the deafness causing gene or to find unambiguously associsted
SNP markers which could be used for a population-wide genetic test for CCSD
Chapter 10
Summary
Summary 127
Summary
Molecular genetic analysis of canine congenital sensorineural deafness in Dalmatian dogs
Katharina Mieskes (2006) The objective of the present study was to localize the gene or genomic region that is
involved in the development of canine congenital sensorineural deafness (CCSD) in
Dalmatian dogs
In man as in different dog breeds deafness is an often diagnosed disorder with the
Dalmatian dog showing the highest incidence Many genetic disorders in humans
and domestic dogs (Canis familiaris) demonstrate a high level of clinical and
molecular similarity
Altogether 39 genes have already been found causative for sensorineural non-
syndromic hearing impairment in humans Out of this 39 deafness causing genes a
total of 32 candidate genes were selected for canine congenital deafness which
showed an appropriate clinical and histological disease pattern in comparison to
deafness in Dalmatians dogs
On the one hand an existing set of 43 microsatllite markers for in total 24 candidate
genes were used for a non-parametric linkage analysis among them the claudin-14
(CLDN14) gene on canine chromosome (CFA) 31 and the myosin heavy polypeptide
9 (MYH9) gene on CFA10 The gap junction protein alpha 1 (GJA1) gene on CFA1
was also considered as a candidate gene for CCSD and thus GJA1-associated
microsatellites were part of the existing set Recently it turned out that GJA1 is not
responsible for human sensorineural non-syndromic deafness but for a human
syndromic disorder that can be related with conductive deafness In the last few
years more human deafness genes have been identified among them eight genes
that were considered as appropriate candidates for CCSD For these eight genes a
total of 21 SNPs were newly developed and used for non-parametric linkage and
association analyses
Summary
128
The used microsatellite and SNP markers derived either from a partial sequence
analysis of BAC clones each containing a different candidate gene or from
sequences deposited in the current dog genome assembly (Boxer genome assembly
21) of the NCBI GenBank We found significant linkage to markers associated to
CLDN14 on CFA31 MYH9 on CFA10 and GJA1 on CFA1 To substantiate the
linkage we searched for sequence variations within these three genes SNPs found
in intronic sequences of either gene were included in the linkage analyses Sequence
analysis neither revealed a causative mutation nor significant linkage disequilibrium
of SNP markers with CCSD Subsequently CFA1 10 and 31 were scanned
completely with microsatellite markers derived from the NCBI database with the
purpose to see if other regions on this three chromosomes harbour a gene that is
involved in the development of CCSD
The analyses of SNPs and more microsatellite markers on CFA1 revealed no
significant linkage to CCSD Thus it is unlikely that the canine chromosome 1 and
the GJA1 gene are responsible for the CCSD phenoptype As deafness in dogs
especially in Dalmatians is almost exclusively caused by sensorineural non-
syndromic forms the GJA1 gene should not be considered as a candidate gene for
CCSD anymore
On CFA10 we could exclude MYH9 for being causal for deafness but by adding
more microsatellites covering CFA10 completely we found significant linkage to the
CCSD phenotype in the region distal of MYH9 Hence new SNP-markers for fine
mapping the region spanning 36 to 48 Mb were developed by sequence analyses of
different Dalmatian dogs The search for SNPs was carried out on genomic
sequences of genes located in the significantly linked region The sequences of
these genomic sequences were derived from the NCBI GenBank The SNPs
confirmed the linkage and narrowed the region harbouring a causative CCSD gene
down to 5 Mb spanning from 39 to 44 Mb
After scanning CFA31 we could not exclude CLDN14 for being responsible for the
CCSD phenotype as microsatellite markers associated to CLDN14 indicated linkage
However to clarify the importance of CLDN14 in the CCSD phenotype more SNPs
have to be developed within the CLDN14 gene as well as in its flanking regions with
the aim to find linkage disequilibrium for SNP markers
Chapter 11
Erweiterte Zusammenfassung
Erweiterte Zusammenfassung 131
Erweiterte Zusammenfassung
Katharina Mieskes
Molekulargenetische Untersuchung der kongenitalen sensorineuralen Taubheit beim Dalmatiner
Einleitung Es gibt mehrere Moumlglichkeiten Taubheit zu klassifizieren Je nach den betroffenen
Strukturen des Gehoumlrorgans lassen sich konduktive und sensorineurale Formen
unterscheiden Erstere entstehen durch Stoumlrungen des Konduktionsmechanismus im
aumluszligeren Gehoumlrgang und im Mittelohr Ursachen fuumlr konduktive Houmlrstoumlrungen koumlnnen
zum Beispiel chronische Otitis externa oder media Frakturen von Mittelohranteilen
oder Tumoren sein Sehr viel oumlfter tritt die sensorineurale Taubheit auf bei der ein
Houmlrverlust als Folge von Transduktions- und Transmissionsstoumlrungen in der Cochlea
(Schnecke) sowie in peripheren und zentralen Anteilen des Nervus cochlearis auftritt
Neben der erblichen Form koumlnnen auch eine Otitis interna Tumoren oder
ototoxische Medikamente zur sensorineuralen Taubheit fuumlhren
Der Houmlrverlust kann bereits seit der Geburt bestehen (kongenital) oder er kann erst
im Laufe der weiteren Lebenszeit auftreten
Beim Menschen ist mehr als die Haumllfte der kongenitalen Taubheit erblich bedingt Die
erbliche kongenitale Taubheit kann als Teil einer multisystemischen Krankheit
(syndromisch) oder als Funktionsstoumlrung auftreten die auf das Ohr und das
vestibulaumlre System (nicht-syndromisch) begrenzt ist Da die nicht-syndromische
ererbte Taubheit fast ausschlieszliglich durch Cochleadefekte verursacht wird leiden die
Patienten unter sensorineuraler Taubheit Es wird geschaumltzt dass die nicht-
syndromische erbliche Taubheit 60-70 aller menschlichen Taubheitsfaumllle
verursacht Weiterhin sind 70-80 der Faumllle der nicht-syndromischen Taubheit auf
einen autosomal rezessiven Erbgang zuruumlck zu fuumlhren gefolgt von einem autosomal
dominanten Erbgang mit 10-20 der Faumllle und 1-2 der Faumllle sind X- gekoppelt Ein
noch geringerer Prozentsatz wird durch einen maternalen Erbgang verursacht
Von den ca 25000 bis 30000 menschlichen Genen ist nach Schaumltzungen bis zu 1
wichtig fuumlr den Houmlrprozess Annaumlhernd 120 Gene sind inzwischen identifiziert die fuumlr
Erweiterte Zusammenfassung
132
verschiedene Formen der menschlichen vererbten Taubheit verantwortlich sind
Darunter sind ungefaumlhr 80 Gene fuumlr die syndromische und 39 Gene fuumlr die nicht
syndromische vererbte Taubheit verantwortlich Insgesamt stellen die 120
identifizierten Gene vermutlich ein Drittel aller taubheitsverursachenden Gene dar
Die kongenitale sensorineurale Taubheit ist eine Erkrankung von der viele
Saumlugetierspezies betroffen sind Beim Hund werden uumlber 54 verschiedene Rassen
beschrieben bei welchen ein kongenitaler Houmlrverlust gehaumluft auftritt In zahlreichen
europaumlischen und amerikanischen Studien weisen hierbei die Dalmatiner mit 165-
30 die houmlchste Taubheitsinzidenz auf
Die histologischen Veraumlnderungen im Innenohr werden im Allgemeinen aumlhnlich wie
beim Menschen als cochleosacculaumlr beschrieben Die Degenerationen im Innenohr
schreiten bei betroffenen Tieren mit zunehmendem Alter fort und fuumlhren
normalerweise in einem Alter von drei bis vier Wochen spaumltestens aber in einem
Alter von etwa vier Monaten zu einem vollstaumlndigen ein- oder beidseitigen
Houmlrverlust
Eine objektive Diagnose der Taubheit besonders wenn sie nur einseitig ist stuumltzt
sich auf die brainstem auditory evoked response (BAER in Deutschland
audiometrischer Test) ein elektro-diagnostischer Test bei dem durch einen
bestimmten Reiz ausgeloumlste elektrische Aktivitaumlten (akustisch evozierte Potentiale) in
der Cochlea und den Nervenbahnen zur auditorischen Hirnrinde detektiert werden
Obwohl fuumlr den Dalmatiner die Erblichkeit der Erkrankung wiederholt nachgewiesen
werden konnte wurden fuumlr die verschiedenen untersuchten Dalmatinerpopulationen
auch sehr viele unterschiedliche Erbgaumlnge in Betracht gezogen Es ist bisher nicht
gelungen die genaue Anzahl der beteiligten Gene zu bestimmen Auch war es
bislang fuumlr keine der betroffenen Hunderassen moumlglich ein taubheitsverursachendes
Gen zu identifizieren
Bei deutschen Dalmatinern ergab eine Auswertung mittels komplexer
Segregationsanalysen dass ein Modell mit einem rezessiven Hauptgen und einer
polygenen Komponente das Auftreten der kongenitalen Taubheit in der untersuchten
Population am besten erklaumlrte Zusaumltzlich konnte ein Zusammenhang zu
Pigmentierungsgenorten nachgewiesen werden da Tiere mit blauer Augenfarbe
signifikant haumlufiger von Taubheit betroffen waren und gleichzeitig Tiere mit
Plattenzeichnung seltener kongenitalen Houmlrverlust zeigten
Erweiterte Zusammenfassung 133
Das Zuumlchten ein- oder beidseitig tauber Dalmatiner sowie Dalmatinern mit blauen
Augen ist in Deutschland schon laumlnger verboten und es wird von den
Zuchtverbaumlnden vorgeschrieben dass alle Dalmatinerwelpen in einem Alter von etwa
6 bis 8 Wochen audiometrisch untersucht werden Des Weiteren ist der Defekt
tierschutzrelevant nach sect 11b des Deutschen Tierschutzgesetzes da der artgemaumlszlige
Gebrauch eines Organs eingeschraumlnkt wird und eine Erblichkeit nachgewiesen
wurde Somit duumlrfen Tiere die Defektgentraumlger sind oder bei denen damit zu
rechnen ist nicht zur Zucht verwendet werden Waumlhrend einseitig taube Dalmatiner
als ganz normale Familienhunde gehalten werden koumlnnen sind beidseitig taube
Dalmatiner schwierig zu erziehen schreckhafter und daraus resultierend auch
oftmals aggressiv und bissig Aus diesen Gruumlnden werden fast alle Welpen mit
beidseitigem Houmlrverlust euthanasiert
Es hat sich gezeigt dass allein der Zuchtausschluss betroffener Tiere nicht genuumlgt
um die hohe Taubheitsinzidenz beim Dalmatiner deutlich zu reduzieren Aus diesen
Gruumlnden ist es von erheblicher Bedeutung die kongenitale sensorineurale Taubheit
beim Dalmatiner molekulargenetisch zu untersuchen um so ein
molekulargenetisches Testverfahren entwickeln zu koumlnnen welches auch die
Identifizierung von Anlagetraumlgern ermoumlglicht
Innerhalb des letzten Jahrzehnts wurden auch zwischen relativ unverwandten
Spezies die strukturellen und funktionellen Homologien auf genetischer Ebene
zunehmend verdeutlicht
Es wurde inzwischen eine groszlige Anzahl von taubheitsverursachenden
Genmutationen beim Menschen und der Maus aufgeklaumlrt Da auch die
histopathologischen Befunde beim Menschen und der Maus oftmals sehr aumlhnlich
denen beim Dalmatiner sind scheinen Gene die beim Menschen verantwortlich fuumlr
die congenitale sensorineurale Taubheit sind geeignete Kandidatengene fuumlr die
canine congenitale sensorineurale Taubheit zu sein
Das Ziel dieser Dissertation ist es die fuumlr die Taubheit kausalen Gene bzw Genorte
zu lokalisieren Um die kongenitale sensorineurale Taubheit beim Dalmatiner
moumlglichst effizient molekulargenetisch aufzuklaumlren wurden 32 Kandidatengene mit
genassoziierten Markern auf eine potenzielle Kopplung mit der caninen kongenitalen
sensorineuralen Taubheit untersucht
Die Genombereiche mit signifikanter Kopplung zur kongenitalen sensorineuralen
Taubheit wurden weitergehend molekulargenetisch analysiert
Erweiterte Zusammenfassung
134
Kopplungsanalyse fuumlr 24 Kandidatengene der caninen kongenitalen sensorineuralen Taubheit (CCSD) mit 43 genassoziierten Mikrosatelliten Markern
Material und Methoden
Fuumlr 24 Kandidatengene stand ein Mikrosatelliten Marker Set zur Verfuumlgung Das
Marker Set beinhaltete Mikrosatelliten fuumlr folgende Kandidatengene CDH23
CLDN14 COCH COL11A2 DFNA5 DIAPH1 EDN3 EDNRB EYA4 GJA1 GJB2
GJB6 MITF MYH9 MYO6 MYO7A MYO15A OTOF PAX3 POU4F3 SLC26A4
SOX10 TECTA und TMPRSS3 Als Familienmaterial fuumlr die Mikrosatellitenstudie
wurden 215 Dalmatiner verwendet Die Tiere verteilten sich auf 22 unverwandte
Vollgeschwisterfamilien und zwei paternale Halbgeschwistergruppen Die
genotypisierten Familien bestanden aus saumlmtlichen tauben Nachkommen (uni- oder
bilateral taub) ihren Eltern und einem bis vier nicht betroffenen Voll- bzw
Halbgeschwistern Mindestens zwei der Vollgeschwister einer Familie waren uni-
oder bilateral taub Der Phaumlnotyp der verwendeten Tiere wurde durch einen
audiometrischen Test bestimmt
Alle Mikrosatellitenmarker wurden uumlber PCR und Polyacrylamidgelelektrophorese
ausgewertet
Kopplungsanalyse
Die nicht-parametrische Kopplungsanalyse wurde unter Verwendung der Software
MERLIN (multipoint engine for rapid likelihood inference Version 0102)
durchgefuumlhrt und basierte auf dem identical-by-descent (IBD) Verfahren Dabei
wurden die Markerallele auf Kosegregation mit der phaumlnotypischen Auspraumlgung der
Erkrankung getestet Darauf folgend wurde die einer Normalverteilung folgende
Teststatistik fuumlr den Anteil von IBD-Markerallelen (Zmean) und ein daraus
abgeleiteter LOD-Score berechnet Als signifikant fuumlr die Kosegregation eines
Markerallels mit dem Phaumlnotyp der caninen kongenitalen sensorineuralen Taubheit
(CCSD) gelten Irrtumswahrscheinlichkeiten (p) von 005 oder kleiner
Die Daten wurden mit dem Softwarepaket SAS (Statistical Analysis System Version
913) bearbeitet um die Marker bezuumlglich Allelfrequenzen beobachteten und
erwarteten Heterozygotiegrad und PIC (Polymorphism information content)
charakterisieren zu koumlnnen
Erweiterte Zusammenfassung 135
Ergebnisse und Diskussion Das Mikrosatelliten Set bestand insgesamt aus 43 Markern mit einem bis drei
Markern pro Kandidatengen Zunaumlchst wurde fuumlr alle 24 Familien zusammen eine
Kopplungsanalyse durchgefuumlhrt Vermeintliche CCSD Loci wurden auf den caninen
Chromosomen (CFA) 1 10 12 20 und 31 lokalisiert Im Anschluss wurden die
Ergebnisse der Kopplungsanalyse auf Heterogenie zwischen den Familien uumlberpruumlft
Fuumlr einzelne Familien konnten houmlhere Teststatistiken und somit engere
Kopplungsbeziehungen als bei der Analyse aller Familien nachgewiesen werden
Eine signifikante Kopplung mit der kongenitalen sensorineuralen Taubheit wurde fuumlr
die Gene GJA1 auf CFA1 MYH9 auf CFA10 und CLDN14 auf CFA31 sowohl im
Gesamtmaterial wie in jeweils unterschiedlichen Gruppen von Dalmatinerfamilien
gefunden Es ist daher wahrscheinlich dass diese Gene oder Gene in ihrer naumlheren
Umgebung an der Entwicklung der Taubheit in den jeweiligen Familien involviert
sind Fuumlr die restlichen Kandidatengene lagen die Multipoint Teststatistiken fuumlr den
Anteil von IBD-Markerallelen (Zmean) und daraus abgeleiteten LOD-Scores bei
annaumlhernd Null und waren nicht signifikant
Die Ergebnisse dieser Teststatistiken zeigen dass der Erbgang der nicht-
syndromischen Taubheit bei Dalmatinern vermutlich ebenso heterogen ist wie er
sich beim Menschen darstellt
Dennoch stellt diese Studie den ersten Schritt dar um die CCSD verursachenden
Gene beim Dalmatiner zu identifizieren Die Gene GJA1 MYH9 und CLDN14 sowie
ihre naumlhere Umgebungen werden im weiteren Verlauf der Studie weitergehend
molekulargenetisch mit Hilfe von SNPs und Mikrosatelliten untersucht Aufgrund der
Heterogenitaumlt zwischen den Familien wurden fuumlr die weitere molekulargenetische
Analyse bestimmter Gene oder Genomregionen nur die Familien mit dem houmlchsten
Beitrag zu den Teststatistiken der Kopplungsanalyse (Zmean gt 1) ausgewaumlhlt
Erweiterte Zusammenfassung
136
Evaluierung von neu entwickelten SNP Markern assoziiert mit Kandidatengenen fuumlr die canine kongenitale sensorineurale Taubheit (CCSD) Material und Methoden Fuumlr acht Kandidatengene (TMC1 TMIE USH1C MYH14 MYO3A PRES WHRN
und ESPN) wurden intragenische Single Nucleotide Polymorphism (SNP) Marker neu
entwickelt
Als Familienmaterial fuumlr die Genotypisierung der SNPs wurde DNA von insgesamt 39
Dalmatinern verwendet die vier Vollgeschwisterfamilien angehoumlrten Mindestens
zwei der Vollgeschwister einer Familie waren uni- oder bilateral taub Der Phaumlnotyp
der verwendeten Tiere wurde durch einen audiometrischen Test bestimmt Die vier
Familien bestanden aus sechs bis zehn Vollgeschwistern und den dazugehoumlrenden
Elterntieren
Identifizierung von SNPs (Single Nucleotide Polymorphisms)
Die caninen Sequenzen der Kandidatengene wurden mit Hilfe der humanen mRNA
mittels BLAST (Basic Local Alignment Search Tool) Suche aus veroumlffentlichten
Sequenzen der NCBI Datenbank (Boxer genome assembly 21) entnommen Die
Exon Intron Grenzen wurden mit Hilfe des mRNA-to-genomic alignment Programms
Spidey bestimmt und Primer mit Hilfe des Primer3 Programms in die intronischen
Sequenzen gelegt Die SNPs wurden an den DNA-Sequenzen von Elterntieren der
betroffenen Dalmatinerfamilien identifiziert Die Amplifikate wurden mit Hilfe des
MegaBACE 1000 sequenziert und auf SNPs untersucht Bei Vorliegen eines oder
mehrerer SNPs wurde die entsprechende DNA-Sequenz der zugehoumlrigen
Nachkommen ebenfalls amplifiziert und sequenziert Die Auswertung erfolgte mit
Hilfe des Sequencher 42 Programms
Kopplungsanalyse
Die Genotypen der neuen SNP Marker wurden unter Verwendung des Programms
MERLIN ausgewertet Die Daten wurden mit dem Softwarepaket SAS (Statistical
Analysis System Version 913) bearbeitet um das Markerset bezuumlglich
Allelfrequenzen beobachteten Heterozygotiegrad und PIC (Polymorphism
information content) charakterisieren zu koumlnnen Des Weiteren wurde auf
Kopplungsungleichgewicht und die Assoziation der Haplotypen mit CCSD mittels
Erweiterte Zusammenfassung 137
des Programms CASECONTROL und HAPLOTYPE von SAS Genetics (Statistical
Analysis System Version 913 Cary NC USA) getestet
Ergebnisse und Diskussion Fuumlr acht Kandidatengene wurden mindestens zwei heterozygote SNPs entwickelt
Die nicht-parametrische Kopplungsanalyse zeigte keine signifikanten Teststatistiken
Offensichtlich war kein Haplotyp mit der caninen kongenitalen sensorineuralen
Taubheit (CCSD) in den untersuchten Familien assoziiert Da sowohl uni- und
bilateral taube Tiere als auch beidseitig houmlrende Tiere uumlber identische Haplotypen
verfuumlgen ist es unwahrscheinlich dass die Gene TMC1 TMIE USH1C MYH14
MYO3A PRES WHRN und ESPN an der Entstehung der CCSD beim Dalmatiner
beteiligt sind
Molekulargenetische Analyse des caninen myosin heavy polypeptide 9 non-muscle (MYH9) auf dem caninen Chromosom 10q232
Material und Methoden Klonierung und Sequenzierung von caniner MYH9 cDNA
Die canine MYH9 Gensequenz wurde mit Hilfe der BLAST Suche aus
veroumlffentlichten Sequenzen der NCBI Datenbank Die Isolierung der vollstaumlndigen
cDNA wurde durch ein Protokoll zur Amplifizierung von cDNA-Enden (rapid
amplification of cDNA ends [RACE]) erreicht Nach Klonierung und Sequenzierung
der RACE-Produkte wurden die Sequenzen mit Hilfe des Sequencher 42
Programms ausgewertet
Sequenzanalyse des caninen MYH9 Gens
Nach Fertigstellung des RACE Protokolls und Auswertung der Sequenzen wurden
die Exon Introngrenzen mit Hilfe des mRNA-to-genomic alignment Programms
Spidey lokalisiert Weiterhin wurden repetitive Elemente mit dem Programm
Repeatmaster 2 sowie der GC Gehalt mit der EBI toolbox CpG PlotCpGreport
ermittelt
Erweiterte Zusammenfassung
138
Mutatiosanalyse
Zur Identifizierung von Polymorphismen innerhalb der caninen MYH9 Sequenz
wurden die Exons mit ihren flankierenden intronischen Sequenzen mit Hilfe der PCR
amplifiziert und sequenziert Verwendet wurden hierfuumlr die DNA von insgesamt 16
Dalmatinern aus drei Familien Mindestens zwei der Vollgeschwister einer Familie
waren unilateral taub Der Phaumlnotyp der verwendeten Tiere wurde durch einen
audiometrischen Test bestimmt
PCR Primer wurden mit Hilfe des Primer3 Programms entwickelt PCR Reaktionen
fuumlr saumlmtliche Exons des MYH9 Gens mit ihren flankierenden Sequenzen wurden
durchgefuumlhrt die PCR Produkte sequenziert und die Sequenzen mit Hilfe des
Sequencher 42 Programms ausgewertet
Unter Verwendung der Software MERLIN (multipoint engine for rapid likelihood
inference Version 0910) wurde nach signifikanten nicht parametrischen LOD-
Scores fuumlr die Kosegregation von Markerallelen mit der phaumlnotypischen Auspraumlgung
der sensorineuralen Taubheit beim Dalmatiner gesucht
Ergebnisse und Diskussion
Analyse der genomischen Organisation und der cDNA des caninen MYH9 Gens
Eine vollstaumlndige cDNA Sequenz des caninen MYH9 Gens von 7484 bp konnte uumlber
die Reverse Transkription-PCR erhalten und bei der EMBL Nukleotid Datenbank
eingereicht werden Das canine MYH9 Gen besteht aus 41 Exons einschlieszliglich
einem untranslatierten ersten Exon und Exon Intron Grenzen die stets der GT AG
Regel folgen Der 5rsquo-UTR Bereich besteht aus 181 bp waumlhrend der 3rsquo-UTR aus 1432
bp besteht Weiterhin besitzt die canine MYH9 cDNA Sequenz einen offenen
Leserahmen von 5889 bp welcher ein Protein kodiert bestehend aus 1963
Aminosaumluren Die Groumlszlige der Exons variiert von 28 bis 1556 bp waumlhrend das
gesamte MYH9 Gens ungefaumlhr 90 kb umfasst Die Homologie der Sequenzen
zwischen dem humanen murinen und caninen MYH9 Gen ist limitiert auf die
kodierende Sequenz von Exon 2 bis 41
Mutations- und Haplotypenanalyse
Alle kodierenden Exons mit ihren flankierenden intronischen Sequenzen des MYH9
Gens konnten von den 16 Dalmatinern amplifiziert werden Die Sequenzen wurden
mit den entsprechenden Sequenzen vom Boxer aus der NCBI Genbank verglichen
Erweiterte Zusammenfassung 139
Die Suche nach Sequenzvariationen im MYH9 Gen lieszlig 22 SNPs erkennen wobei
die meisten Polymorphismen in den flankierenden Regionen der Exons gefunden
wurden Keiner der beobachteten Polymorphismen in den Exons aumlnderte die
Aminosaumluresequenz von MYH9 Auch zeigten die identifizierten Haplotypen keine
Assoziation mit dem CCSD Phaumlnotyp Multipoint Teststatistiken fuumlr den Anteil von
IBD-Markerallelen (Zmean) und ein daraus abgeleiteter LOD-Score lagen bei
annaumlhernd Null und waren nicht signifikant Zwei genassoziierte Mikrosatelliten
zeigten eine Kopplung diese kann jedoch mit Heterogenitaumlt zwischen den Familien
erklaumlrt werden
Fazit
Die Charakterisierung des Transkriptes und der genomischen Sequenz des caninen
MYH9 Gens zeigten einen konservierten Aufbau des Gens im Bezug auf das
humane orthologe Gen Aufgrund eines untranslatierten Exons im 5rsquo-UTR Bereich ist
das canine Gen groumlszliger als das humane Gen
Sowohl uni- oder bilateral taube und bilateral houmlrende Dalmatiner weisen identische
Haplotypen auf Daher sind in den untersuchten Dalmatinerfamilien die gefundenen
Polymorphismen offensichtlich nicht mit CCSD assoziiert Des Weiteren zeigt diese
Studie dass keine funktionellen Mutationen der vollstaumlndigen kodierenden Region
von MYH9 vorliegen Wir koumlnnen also abschlieszligend das MYH9 Gen als
Kandidatengen fuumlr die congenitale sensorineurale Taubheit beim Dalmatiner
ausschlieszligen
Erweiterte Zusammenfassung
140
Evaluierung von Mikrosatelliten und SNP Markern der caninen Chromosomen 1 10 und 31 auf Kopplung und Assoziation mit der caninen kongenitalen Taubheit Material und Methoden Pedigreematerial
Insgesamt wurden 176 Dalmatiner in die Untersuchung einbezogen Die Tiere
verteilten sich auf 23 unverwandte Vollgeschwisterfamilien und eine paternale
Halbgeschwistergruppe Die genotypisierten Familien bestanden aus saumlmtlichen
tauben Nachkommen (unilateral oder bilateral taub) ihren Eltern und einen bis vier
nicht betroffenen Voll- bzw Halbgeschwistern Mindestens zwei der Vollgeschwister
einer Familie waren unilateral oder bilateral taub Der Phaumlnotyp der verwendeten
Tiere wurde durch einen audiometrischen Test bestimmt
Alle 176 Dalmatiner wurden fuumlr die Mikrosatellitenstudie auf den caninen
Chromosomen 1 10 und 31 einbezogen Fuumlr die Feinkartierung mit Hilfe der SNPs
wurden dagegen nur noch die Dalmatinerfamilien mit einer eindeutigen Kopplung zu
der entsprechenden genomischen Region untersucht Fuumlr die Region auf CFA10
wurden dafuumlr 34 Tiere aus fuumlnf Vollgeschwisterfamilien ausgewaumlhlt und zur
Untersuchung des CLDN14 Gens auf CFA31 wurden 24 Tiere aus vier
Vollgeschwisterfamilien und 12 Tiere aus einer groszligen Halbgeschwisterfamilie
verwendet Fuumlr die Suche nach Polymorphismen im GJA1 Gen wurden 16 Tiere aus
einer groszligen Halbgeschwisterfamilie verwendet
Mikrosatellitenstudie
Fuumlr die Mikrosatellitenstudie von CFA1 10 und 31 wurden insgesamt 60 Marker
verwendet Mit jeweils 27 Mikrosatelliten wurde CFA1 und mit 27 Mikrosatelliten
wurde CFA10 vollstaumlndig abgedeckt Insgesamt 6 Marker wurden fuumlr CFA31
verwendet
Alle hier verwendeten Mikrosatelliten sind der NCBI Datenbank entnommen und
wurden uumlber PCR und Polyacrylamidgelelektrophorese ausgewertet
Identifizierung von SNPs (Single Nucleotide Polymorphisms)
Zur weiteren Abklaumlrung und zum Ausschluss falsch positiver Ergebnisse wurden
zusaumltzlich zu den Mikrosatelliten SNPs in den signifikanten Regionen auf CFA1 10
Erweiterte Zusammenfassung 141
und CFA 31 entwickelt und an den Familien die eine Kopplung zeigten
genotypisiert
Die caninen genomischen Sequenzen der untersuchten Gene wurden der NCBI
Datenbank (Boxer genome assembly 21) entnommen Exon Intron Grenzen wurden
bestimmt und Primer entwickelt Nach Durchfuumlhrung der PCR wurden die Amplifikate
mit Hilfe des MegaBACE 1000 sequenziert und auf SNPs untersucht Die
Auswertung erfolgte mit Hilfe des Sequencher 42 Programms
Die Genotypen der neuen SNP Marker wurden zu den Genotypen der fuumlr die
Kopplungsanalyse verwendeten Mikrosatelliten hinzugefuumlgt und gemeinsam unter
Verwendung des Programms MERLIN ausgewertet
Kopplungsanalyse
Unter Verwendung der Software MERLIN (multipoint engine for rapid likelihood
inference Version 0910) wurde nach signifikanten nicht parametrischen LOD-
Scores fuumlr die Kosegregation von Markerallelen mit der phaumlnotypischen Auspraumlgung
der sensorineuralen Taubheit beim Dalmatiner gesucht
Die Daten wurden mit dem Softwarepaket SAS (Statistical Analysis System Version
913) bearbeitet um die Marker bezuumlglich Allelfrequenzen beobachteten
Heterozygotiegrad und PIC (Polymorphism information content) charakterisieren zu
koumlnnen Des Weiteren wurde das Kopplungsungleichgewicht und die Assoziation der
Haplotypen mit CCSD mittles des Programms CASECONTROL und HAPLOTYPE
von SAS Genetics getestet
Ergebnisse und Diskussion CFA1
In einer groszligen Halbgeschwisterfamilie wurde eine signifikante Kopplung zu einem
GJA1 genassozierten Marker festgestellt Mit Hilfe der DNA von Tieren aus dieser
Halbgeschwisterfamilie wurde eine Sequenzanalyse des GJA1 Gens durchgefuumlhrt
Es wurden 3 SNPs im 3acute-untranslatierten Bereich gefunden Die identifizierten
Haplotypen zeigten keine Assoziation mit dem CCSD Phaumlnotyp Sowohl uni- oder
bilateral taube und bilateral houmlrende Dalmatiner wiesen identische Haplotypen auf
Daher sind in den untersuchten Dalmatinerfamilien die gefunden Polymorphismen
offensichtlich nicht mit CCSD assoziiert Auch wurde keine funktionelle Mutation in
der vollstaumlndigen kodierenden Region von GJA1 gefunden wurde Es ist daher
Erweiterte Zusammenfassung
142
unwahrscheinlich dass das GJA1 Gen an der Entwicklung der CCSD in der
untersuchten Halbgeschwisterfamilie beteiligt ist Die signifikante Kopplung der
GJA1-benachbarten Mikrosatelliten mit CCSD kann durch ein eng gekoppeltes Gen
hervorgerufen worden sein welches fuumlr die Entstehung der Taubheit in der
untersuchten Halbgeschwisterfamilie verantwortlich ist oder durch ein falsch-
positives Ergebnis der vorhergehend durchgefuumlhrten Mikrosatellitenstudie
Eine nicht-parametrische Kopplungsanalyse mit Hilfe von 27 Mikrosatelliten Marker
an 24 deutschen Dalmatinerfmilien wurde durchgefuumlhrt um abzuklaumlren ob andere
Genombereiche auf CFA1 signifikante Teststatistiken in mehr Familien zeigen Die
Kopplungsanalyse zeigte keine signifikanten Teststatistiken Es ist daher
unwahrscheinlich dass auf CFA1 Gene fuumlr die Entwicklung von CCSD in den
untersuchten Familien der aus Deutschland stammenden Dalmatiner verantwortlich
sind
Da kuumlrzlich festgestellt wurde dass das GJA1 Gen auch beim Menschen nicht fuumlr
eine Form der sensorineuralen nicht-syndromischen Taubheit verantwortlich ist
sondern an einer syndromischen Krankheit beteiligt ist die mit konduktiver Taubheit
einhergehen kann sollte das GJA1 Gen nicht laumlnger als ein Kandidatengen fuumlr
CCSD angesehen werden
CFA31
Auf CFA31 wurde durch eine nicht-parametrische Kopplungsanalyse von
Mikrosatelliten eine Kopplung eines CLDN14-benachbarten Markers mit der
sensorineuralen Taubheit beim Dalmatiner festgestellt Daraufhin wurden zusaumltzlich
zu den Mikrosatelliten acht SNPs entwickelt indem das einzige beim Menschen
translatierte Exon weite intronische Sequenzen sowie 5rsquo- und 3rsquo- untranslatierte
Bereiche des CLDN14 Gens einer Sequenzanalyse unterzogen wurden Nur zwei der
identifizierten SNPs waren polymorph in den untersuchten fuumlnf Dalmatinerfamilien
Sowohl uni- als auch bilateral taube und bilateral houmlrende Dalmatiner teilten
identische Haplotypen daher konnte keine Kosegregation mit tauben Dalmatinern
festgestellt werden
Da bislang keine funktionelle Mutation in der kodierenden Region von CLDN14
gefunden wurde ist es fuumlr eine eindeutige Aussage uumlber die Rolle des CLDN14
Gens im Entstehungsprozess der kongenitalen sensorineuralen Taubheit
Erweiterte Zusammenfassung 143
beim Dalmatiner notwendig noch weitere Marker im CLDN14 Gen sowie seiner
naumlheren Umgebung zu entwickeln und zu analysieren Da keine weitere Kopplung zu
einem Mikrosatelliten distal oder proximal des CLDN14 Gen festgestellt werden
konnte ist es wahrscheinlich dass entweder das CLDN14 Gen oder ein Gen in
seiner naumlheren Umgebung fuumlr die Entwicklung der sensorineuralen Taubheit in den
untersuchten Dalmatinerfamilien verantwortlich ist
CFA10
Auf CFA10 wurde durch eine nicht-parametrische Kopplungsanalyse von 27
Mikrosatelliten eine 12 Mb umfassende genomische Region mit einer signifikanten
Kopplung zur CCSD identifiziert Zur weiteren Feinkartierung wurden 26 neue SNPs
mit einem durchschnittlichen Abstand von 03 Mb fuumlr diesen Bereich entwickelt
Die SNPs waren groumlszligtenteils informativ im vorliegenden Familienmaterial und
bestaumltigten in einer folgenden Kopplungsanalyse die Lokalisation auf CFA10
deutlich Weiterhin konnte die gekoppelte Region auf einem 5 Mb umfassenden
Bereich von 39 bis 44 Mb eingegrenzt werden Es ist wahrscheinlich dass die
identifizierte CCSD Region ein Gen beinhaltet welches auf die Entstehung der
caninen kongenitalen Taubheit einwirkt
Es ist notwendig mehr SNPs zu entwickeln um das taubheitsverursachende Gen zu
lokalisieren oder einen eindeutig assoziierten SNP zu identifizieren
Chapter 12
References
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Chapter 13
Appendix
Appendix microsatellite marker III
HE
Te
79
5 7
98
76
9 5
12
71
3 8
33
70
2 4
35
72
7 7
00
54
6 4
77
48
8 3
08
44
8 5
40
67
4 8
05
76
2 6
54
70
8 7
73
78
1 6
87
65
4 6
26
51
9 5
89
76
4 1
60
PIC
d
72
2 8
41
67
0 4
85
67
1 7
85
57
2 4
07
61
4 6
29
35
8 3
30
40
0 2
90
35
0 4
60
70
0 7
90
76
2 6
21
63
8 7
26
77
1 6
26
60
1 6
13
38
7 5
52
80
0 1
40
Prim
er (r
ever
se)
5acute -gt
3acute
AA
CTG
TTTG
ATT
TTG
ATG
AG
GC
C
TTTG
ACC
TAC
ATA
TAA
AA
CA
AG
C
CA
CG
AA
GAA
AG
CC
ATG
GTT
T C
AA
CA
ATT
CC
TTTG
TTG
GTG
C
CTG
CC
AG
CTT
CC
TAC
AGC
T
GTG
GTC
ACA
AG
AC
TTTA
GC
C
CTG
CA
CC
AAA
GA
AA
CTC
C
GG
AC
CTA
TTC
TGA
AG
CC
TAA
C
TG
GC
CTT
TAA
TTTA
TCAT
GG
AG
A
TCA
GG
CC
CA
GA
TAG
TATG
C
TC
CTT
TGG
TTTT
TAG
CAG
GG
T
CTG
TGC
CC
AC
CTG
TGG
AG
C
AA
GA
GG
ATA
CC
GG
TGG
CA
G
CG
TGC
TTTG
TTA
TGG
CTT
GA
C
AG
CA
TGA
AG
GA
TCTC
TGA
CTA
A
CTA
AC
TCG
CC
AG
AC
TATT
C
CC
CTC
CAC
CA
ATC
TCTT
CA
C
GC
AA
AG
GTG
TATT
TAC
ATG
ATG
G
GG
CA
GC
CTT
ATT
ATT
CA
TGG
A
AC
AC
AG
GC
AC
AG
GA
GC
ATC
C
AG
CC
ATC
CA
GG
AA
ATC
G
CTG
AA
CTG
GTC
CC
TCAA
GC
A
GTC
ATG
CTG
ATT
TCTG
TGC
C
GA
CC
TATC
CA
TAC
ATG
CC
C
GAA
GG
AAG
GAA
GG
AAG
AAAA
GA
TTA
GTA
AA
GA
GA
CA
GC
TGA
TTG
CC
A
TCTA
CC
CTG
CA
ACC
CTG
TG
ATT
GA
GA
CC
CA
AG
ACTG
TTA
GTG
G
GTA
AC
AAC
CA
GAG
TGTG
TGTG
A
CG
TCG
AG
CTC
CTG
GC
AT
Prim
er (f
orw
ard)
5acute -
gt 3acute
GG
GTA
GA
TTTC
AA
CA
AAT
AG
TAC
TGG
T
AG
AA
AG
GC
CTG
GA
TGTC
G
AG
TAG
AA
GA
GG
CC
AC
GC
AA
A
TTT
TCC
AA
AA
TGA
AA
AC
CTG
C
AG
ATT
GTT
TATG
CA
GG
CA
TTG
A
GA
GTT
GAA
AG
GTT
GAA
AA
TGG
G
CTC
AG
AG
GC
AG
ATA
GA
AA
A
ATT
GA
TTC
ATG
ACC
CA
CTA
A
TTT
TGTC
AAG
CA
GC
CC
TATA
TC
TG
AA
ATG
GTA
CTT
CA
GC
ATC
G
AG
CTT
AG
CTT
AC
TGC
CC
TGG
T
CC
CC
GG
CC
CC
TGC
CC
GG
AG
T
AC
CAC
TGTC
ATT
TTTC
CA
TGC
C
CC
TTA
GG
AGG
AG
GC
AAG
AC
C
CA
AG
GG
GTA
TGTT
GTC
TATT
ACT
GG
GTG
GG
AA
ATG
TGA
CTC
TG
GG
GC
TGC
CC
ATT
TCTT
TAA
T T
TGC
CA
CAA
ATC
AC
TTAA
AG
G
GA
CTG
AG
TTC
TTTC
AG
CA
CA
GTG
A
AAAT
AAC
ACAG
CAT
CAG
G
GA
ATC
CC
CA
ATG
TAC
ATG
GC
A
AC
AG
GC
GG
ATT
CTG
TAG
C
AC
TGG
CC
AAA
GA
GTA
CA
AG
G
CC
TTG
AC
TCA
GC
AGC
CC
TAC
A
AA
GA
TTG
TCTT
GA
CAC
GC
TG
GG
GTC
CTG
GG
ATC
AA
GC
C
AC
TGA
CTG
ATG
TCC
TGTG
CG
A
AA
AA
GTG
TAG
AG
CTT
TCTT
CA
AA
T
AA
ATT
CA
ATA
AG
CC
ATG
GA
GG
A
CC
TCC
AAG
ATG
GC
TCTT
GA
ATc
(degC
) 60
58
58
56
58
60
54
56
60
60
58
60
60
60
60
60
60
60
60
52
56
58
54
62
60
60
62
62
60
60
Pro
duct
si
ze (b
p)
380-
400
337-
409
267-
304
225-
235
312-
336
350-
394
155-
161
126-
156
510-
570
436-
468
189-
191
72-8
0 13
0-13
6 34
0-35
0 14
5-15
3 98
-102
22
0-24
0 21
2-24
0 34
7-40
3 32
5-34
1 25
1-27
9 32
5-36
1 19
7-24
5 30
9-40
9 34
1-37
5 21
5-23
1 30
8-31
6 16
2-17
4 46
8-50
2 18
2-19
0
Alle
les
6 14
8 4 7 11
3 6 8 7 2 2 3 4 2 3 5 8 12
7 8 7 14
10
9 7 3 3 15
2
Mbb
54
1
49
19
8 2
18
23
5 2
47
26
0 3
02
35
3 3
98
58
8 6
26
63
9 7
61
77
5 7
78
78
6 7
98
85
4 1
060
1
090
1
100
1
100
1
110
1
130
1
150
1
170
3
5
12
4 1
63
CFA
a
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10
10
10
Tabl
e 1
Cha
ract
eris
tics
of m
icro
sate
llite
mar
kers
sel
ecte
d fo
r CFA
1 1
0 an
d 31
com
pris
ing
in to
tal 6
0 m
arke
rs
(Con
tinue
d on
nex
t pag
e) T
he m
icro
sate
llite
mar
kers
wer
e al
l der
ived
from
the
NC
BI d
atab
ase
(http
w
ebn
cbin
lmn
ihg
ov)
Mar
ker
FH33
25
FH36
03
FH23
13
RE
N18
9I14
FH
3219
FH
3413
R
EN
136G
19
RE
N13
5K06
FH
3922
FH
3300
C
014
24
AH
T107
C
012
51
RE
N17
2C02
D
0150
5 R
EN
04H
08
RE
N15
9F24
FH
3894
FH
2309
R
EN
06N
11
FH23
26
FH33
22
FH35
05
FH25
98
FH26
34
FH22
94
RE
N22
9P15
FH
2537
FH
4081
C
107
81
Appendix microsatellite marker
IV
H
ETe
64
0 6
80
63
0 6
51
54
6 7
06
81
0 6
14
30
2 7
42
34
4 7
40
63
0 8
67
82
2 6
70
75
0 6
80
60
0 4
70
31
0 5
70
80
1 5
80
83
1 5
10
64
0 6
90
19
0 8
22
PIC
d
53
0 6
90
52
2 5
89
80
0 6
70
75
9 5
24
24
7 7
09
26
9 6
90
50
0 7
98
75
5 5
50
69
0 5
30
56
0 3
40
26
0 5
00
80
1 5
30
74
0 3
70
64
0 6
70
25
0 7
21
Prim
er (r
ever
se)
5acute -gt
3acute
AG
GC
CA
GC
AC
CA
TAA
CTC
AG
C
CTC
TCC
TCC
TTC
CC
CTT
CTC
A
GTG
CTG
TCC
ATT
GC
AA
GTC
T
GC
TCC
CC
TGTG
TTC
TGAG
G
CTG
GTG
ACTC
AGA
GAA
GTC
C
GG
AG
TTG
ATA
GTA
AA
TCTT
TGC
GG
T
GC
CC
TATT
CA
TTC
AA
CTG
G
AC
AC
TCTG
GTG
CA
AG
CG
AC
TC
TTC
TAC
TGA
AG
CA
TCC
GC
CT
GA
TTTT
CTC
TCTG
TCC
AC
TT
AA
CA
GC
ATT
TCA
GAC
AGA
GG
G
GG
TTTT
GG
CTG
TGA
GG
ATA
G
AG
TTC
CAG
ATC
GAG
TCC
CA
C
CA
CAT
GAT
TTC
ACTT
GTA
TATG
G
CC
TGA
GG
GTT
TTTA
CAG
TATG
G
TG
AA
ATC
TGG
GA
TTC
TGA
TAC
C
CC
AA
GC
TTTC
AC
CTA
ATC
AC
C
GA
TATT
TTTC
TCTC
CC
AC
CC
C
AC
ATT
TCTA
GG
CAG
TGG
CA
C
TGTC
CC
ATC
TAA
AG
GA
TAG
G
AG
TAA
TGC
TGC
CC
ACG
GA
AC
G
GC
TGG
AA
CC
TTC
CC
CTT
TAG
A
GC
TCTT
ACA
CG
CA
TTG
AG
G
AA
AC
GG
AG
TTC
CA
TCTC
TGG
C
AC
AG
TGA
GG
AG
GG
GTG
G
TG
TAA
CC
CTC
CTG
TGA
TATG
A
TGTG
AA
CC
CC
GC
CC
AATA
G
AA
GTG
AC
TGA
GTA
CC
TGA
AA
TCG
G
CC
TCTG
CC
TCTG
CC
TCTG
T G
AG
CC
CTG
TTC
TCA
GG
TTG
Prim
er (f
orw
ard)
5acute -
gt 3acute
AG
AC
CC
CC
CC
ACTA
CC
CC
AC
GC
GTT
AGC
TGC
CA
TCA
TCTT
G
CTG
GTT
GG
AG
CA
ATA
CC
AA
G
CC
ATT
CA
TGTT
GTT
GC
AG
ATG
T
AA
GG
AG
GG
CA
CTT
GAT
GG
C
TTA
TGG
CA
TTTG
GC
CTG
TC
AA
GG
AG
CAT
CTT
CC
AG
AAC
C
CG
CTC
ATG
CA
AG
TCA
TCA
CA
T C
TGG
CTT
AAA
CC
AC
TGG
TCA
C
CTT
CC
ATC
CC
GTT
GTG
TGT
ATT
CC
CC
AGC
GAT
ACC
A C
TTG
AG
TGG
CTT
GA
GTG
GC
TAC
T
TCTT
GC
TTC
TTG
AA
GTA
AG
CC
T
TGC
CC
GTC
CTA
TAC
TCC
TG
GA
AG
GTG
GTT
ATT
GTC
CTT
GG
G
AA
CC
ACA
GA
AG
AG
CTG
GA
A
GC
ATT
TGA
TGA
AA
TAA
AG
GG
A
GA
ATG
AA
AA
CG
GA
GC
AG
CA
G
CC
TTC
AAC
AC
CC
ATA
GC
TC
TC
AG
CA
ACTA
TAC
ATT
TAA
GA
GC
A
ATG
GA
CA
AATG
AA
CA
AA
AG
T
GTG
AC
TTTC
TTA
TCC
GC
CC
C
CC
AG
AA
AC
TCA
ACTG
ATG
C
TG
GA
CG
CTA
AG
CC
TGAC
TTT
CC
ATA
AG
ATA
CTC
AG
AA
AC
ATG
CA
C
AC
CAT
AA
ATG
GA
TGG
ATA
G
CA
GTG
AG
CA
AA
GC
AA
ATG
AA
C
CC
ATT
AG
CA
AA
TGA
CTG
GG
A
AA
GA
ATG
GG
AA
AA
CTG
ATA
A
AA
GG
TAG
TCC
CA
CG
ATC
CTC
ATc
(degC
) 60
60
60
60
60
60
60
60
60
60
60
60
60
62
60
60
60
60
60
60
60
60
60
60
60
60
60
60
60
60
Pro
duct
si
ze (b
p)
100-
110
112-
124
194-
206
210-
230
309-
341
350-
362
276-
300
96-1
30
130-
138
120-
138
200-
204
278-
296
157-
165
178-
254
358-
368
327-
337
380-
404
160-
170
320-
330
165-
169
230-
232
300-
310
276-
380
255-
263
258-
278
106-
120
164-
180
568-
608
223-
239
172-
186
Alle
les
3 5 3 5 8 4 7 4 2 5 3 7 5 9 6 4 7 3 4 2 2 3 11
5 6 2 4 7 4 8
Mbb
24
9 3
23
34
9 3
67
38
9 3
94
40
1 4
10
45
8 4
64
48
0 4
88
51
5 5
64
56
8 5
97
62
7 6
33
63
7 6
53
67
3 6
85
69
1 7
08
0
66
9
7
28
1 2
96
37
5
CFA
a
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
31
31
31
31
31
31
Tabl
e 1
(con
tinue
d)
Mar
ker
RE
N06
H21
R
EN
68B
08
FH30
55
C10
769
FH
3302
R
EN
181G
20
FH34
03
G02
512
RE
N15
0B12
A
HT1
01
RE
N73
F08
ZUB
EC
A1
C10
16
FH24
22
FH34
48
FH26
65
DTR
105
C
106
02
RE
N16
1L12
C
0410
7 R
EN
91H
07
RE
N15
4O19
FH
3381
R
EN
154G
10
FH21
89
RV
C11
R
EN
43H
24
FH22
39
RE
N11
0K04
FH
2712
a Can
ine
chro
mos
ome
b Pos
ition
of m
arke
r (M
b) c A
nnea
ling
tem
pera
ture
d P
olym
orph
ism
info
rmat
ion
cont
ent (
)
e obse
rved
Het
eroz
ygos
ity
Appendix itemisation of alleles V
Table 2 Itemisation of alleles to bases for SNPs in the pedigrees
Code no 1 2 3 4
Base Adenine Cytosine Guanine Thymine
Appendix laboratory paraphernalia
VI
Laboratory paraphernalia Equipment Thermocycler
PTC-100trade Programmable Thermal Controller (MJ Research Watertown USA)
PTC-100trade Peltier Thermal Cycler (MJ Research Watertown USA)
PTC-200trade Peltier Thermal Cycler (MJ Research Watertown USA)
Automated sequencers
LI-COR Gene Read IR 4200 DNA Analyzer (LI-COR Inc Lincoln NE USA)
LI-COR Gene Read IR 4300 DNA Analyzer (LI-COR Inc Lincoln NE USA)
MegaBACE 1000 (Amersham Biosciences Freiburg)
Centrifuges
Sigma centrifuge 4-15 (Sigma Laborzentrifugen GmbH Osterode)
Desk-centrifuge 5415D (Eppendorf Hamburg)
Biofuge stratos (Heraeus Osterode)
Centrifuge Centrikon H 401 (Kontron Gosheim)
Megafuge 1OR (Heraeus Osterode)
Speed Vacreg Plus (Savant Instruments Farmingdale NY USA)
Agarose gel electrophoresis and pulsed field gel electrophoresis
Electrophoresis chambers OWL Separation Systems Portsmouth NH USA
Biometra Goumlttingen
BioRad Muumlnchen
Generators 2301 Macrodrive 1 (LKB Bromma Sweden)
Power Pac 3000 (BioRad Muumlnchen)
Gel documentation system BioDocAnalyze 312 nm Goumlttingen
Appendix laboratory paraphernalia VII
Others
Milli-Qreg biocel water purification system (Millipore GmbH Eschborn)
Incubator VT 5042 (Heraeus Osterode)
UV-Illuminator 312 nm (Bachhofer Reutlingen)
Centomatreg R Desk-Shaker (B Braun Melsungen AG Melsungen)
Biophotometer (Eppendorf AG Hamburg)
Kits
DNA purification
Montage PCR96 Cleanup Kit (Millipore GmbH Eschborn)
Cloning
Invitrogen TA cloningreg kit (Invitrogen Karlsruhe Germany)
Isolation of DNA
QIAamp 96 DNA Blood Kit (QIAGEN Hilden)
Plasmid Mini Prep 96 Kit (Millipore GmbH Eschborn)
Sequencing
ThermoSequenase Sequencing Kit (Amersham Biosciences Freiburg)
DYEnamic-ET-Terminator Cycle Sequencing Kit (Amersham Biosciences Freiburg
Germany)
RACE
FirstChoiceTM RNA ligase-mediated (RLM)-RACE kit (Ambion Europe Huntingdon
UK)
Appendix laboratory paraphernalia
VIII
RNA Total RNA (Biocat Heidelberg Germany)
Size standards
100 bp Ladder (New England Biolabs Schwalbach Taunus)
1 kb Ladder (New England Biolabs Schwalbach Taunus)
IRDyetrade 700 or 800 (LI-COR Inc Lincoln NE USA)
Reagents and buffers APS solution (10 )
1 g APS
10 ml H2O
Bromophenol blue solution
05 g bromophenol blue
10 ml 05 M EDTA solution
H2O ad 50 ml
dNTP solution
100 microl dATP [100 mM]
100 microl dCTP [100 mM]
100 microl dGTP [100 mM]
100 microl dTTP [100 mM]
1600 microl H2O
the concentration of each dNTP in the ready-to-use solution is 5 mM
Gel solution
1275 ml UreaTBE solution (Roth Karlsruhe)
225 ml Rotiphoresereg Gel 40 (38 acrylamide and 2 bisacrylamide)
95 microl APS solution (10 )
95 microl TEMED
Appendix laboratory paraphernalia IX
Loading buffer for agarose gels
EDTA pH 8 100 mM
Ficoll 400 20 (wv)
Bromophenol blue 025 (wv)
Xylencyanol 025 (wv)
Loading buffer for gel electrophoresis
2 ml bromophenol blue solution
20 ml formamide
TBE-buffer (1x)
100 ml TBE-buffer (10x)
900 ml H2O
TBE-buffer (10x)
108 g Tris [12114 M]
55 g boric acid [6183 M]
744 g EDTA [37224 M]
H2O ad 1000 ml
pH 80
UreaTBE solution (6 )
425 g urea [6006 M]
250 ml H2O
100 ml TBE-buffer (10x)
solubilise in a water bath at 65degC
H2O ad 850 ml
Appendix laboratory paraphernalia
X
Chemicals Agarose (Invitrogen Paisley UK)
Ammonium persulfate (APS) ge 98 (Sigma-Aldrich Chemie GmbH Steinheim)
Ampicillin (Serva Heidelberg)
Boric acid ge 998 pa (Carl Roth GmbH amp Co Karlsruhe)
Bromophenol blue (Merck KgaA Darmstadt)
dATP dCTP dGTP dTTP gt 98 (Carl Roth GmbH amp Co Karlsruhe)
Chloramphenicol (Serva Heidelberg)
DMSO ge 995 pa (Carl Roth GmbH amp Co Karlsruhe)
dNTP-Mix (Qbiogene GmbH Heidelberg)
EDTA ge 99 pa (Carl Roth GmbH amp Co Karlsruhe)
Ethidium bromide (Carl Roth GmbH amp Co Karlsruhe)
Ethyl alcohol (AppliChem Darmstadt)
Formamide ge 995 pa (Carl Roth GmbH amp Co Karlsruhe)
LB (Luria Bertani) agar (Scharlau Microbiology Barcelona Spain)
Paraffin (Merck KgaA Darmstadt)
RotiphoreseregGel40 (Carl Roth GmbH amp Co Karlsruhe)
SephadexTM G-50 Superfine (Amersham Biosciences Freiburg)
TEMED 99 pa (Carl Roth GmbH amp Co Karlsruhe)
Tris PUFFERANreg ge 999 pa (Carl Roth GmbH amp Co Karlsruhe)
Urea ge 995 pa (Carl Roth GmbH amp Co Karlsruhe)
Water was taken from the water purification system Milli-Qreg
X-Gal (AppliChem Darmstadt)
Enzymes Taq-DNA-Polymerase 5 Umicrol (Qbiogene GmbH Heidelberg)
Taq-DNA-Polymerase 5 U microl (Qiagen Hilden Germany)
Incubation Mix (10x) TPol with MgCl2 [15 mM] (Qbiogene GmbH Heidelberg)
The polymerase was always used in the presence of incubation Mix TPol 10x buffer
The encyme ECO RI (New England Biolabs Schwalbach Taunus) were used with
the adequate 10x encyme buffer
Appendix laboratory paraphernalia XI
Consumables
96 Well Multiply PCR plates skirted (Sarstedt Nuumlmbrecht)
Combitipsreg plus (Eppendorf AG Hamburg)
Pipette tips 01 ndash 10 microl 01 ndash 10 microl 5 ndash 200 microl (Carl Roth GmbH amp Co Karlsruhe)
Reaction tubes 15 and 20 ml (nerbe plus GmbH WinsenLuhe)
Reaction tubes 10 und 50 ml (Falcon) (Renner Darmstadt)
Thermo-fast 96 well plate skirted (ABgene Hamburg)
Software BLAST trace archive httpwwwncbinlmnihgov
httpwwwensemblorg
EBI toolbox httpwwwebiacukToolssequencehtml
MERLIN 0102 package httpwwwsphumicheducsgabecasisMerlin
Order of primers MWG Biotech-AG Ebersberg (httpsecom
mwgdnacomregisterindextcl)
biomersnet GmbH Ulm (orderbiomersnet)
PED50 Dr H Plendl et al (2005) Institute for Human Genetics
Kiel
Primer design httpfrodowimiteducgi-binprimer3primer3_ wwwcgi
Repeat Masker httpwwwrepeatmaskergenome washingtonedu
Sequencher 42 GeneCodes Ann Arbor MI USA
Spidey httpwwwncbinlmnihgovIEBResearch
OstellSpideyindexhtml
SUN Ultra Enterprise 450 Sun microsystems
XIII
List of publications Journal articles 1 MIESKES K WOumlHLKE A DROumlGEMUumlLLER C DISTL O 2006 Molecular
characterization of the canine myosin heavy polypeptide 9 non-muscle (MYH9)
gene on dog chromosome 10q232 Submitted for publication in Gene
2 MIESKES K DISTL O 2006 Evaluation of 17 single nucleotide
polymorphisms (SNPs) in six candidate genes for hereditary non-syndromic deafness
in Dalmatian dogs Submitted for publication in Journal of Heredity
3 MIESKES K DISTL O 2006 Evaluation of the TMC1 and TMIE genes as
candidates for hereditary non-syndromic deafness in Dalmatian dogs Submitted for
publication in Animal Genetics
XV
Acknowledgements
First of all I wish to thank Prof Dr Dr habil Ottmar Distl the supervisor of my
doctoral thesis for his academic guidance and support of this work
I am very thankful to Heike Klippert-Hasberg and Stefan Neander for technical
expertise and assistance
I am very grateful to Joumlrn Wrede for his support during the statistical analyses and his
help with computer problems
I wish to express my appreciation to the Gesellschaft zur Foumlrderung Kynologischer
Forschung (GKF) eV Germany for funding this work with a grant
I am appreciative to all Dalmatian breeders and owners for providing me blood
samples and the results of the BAER tests
My special thanks go to all colleagues and friends of the Institute for Animal Breeding
and Genetics of the University of Veterinary Medicine Hannover for their support
humour and the friendly atmosphere in the laboratory
Last but not least I wish to thank my family for their support during the work on this
thesis
Parts of this work have been submitted for publication in the following journals
1 Gene
2 Journal of Heredity
3 Animal Genetics
Contents
1 Introduction 1
2 A comparative overview of the molecular genetics of non-syndromic deafness in dogs and humans 5
Abstract 7
The structure of the ear 7
Deafness in man 8
Deafness in dogs 9
The canine genome project 11
3 Linkage analysis of gene-associated microsatellite markers with congenital sensorineural deafness in Dalmatian dogs 23
Abstract 25
Introduction 25
Material and methods 26
Results and discussion 28
4 Evaluation of eight candidate genes for canine congenital sensorineural deafness in Dalmatian dogs 41
Abstract 43
Introduction 43
Material and methods 44
Results and discussion 46
5 Molecular characterization of the canine myosin heavy polypeptide 9 non-muscle (MYH9) gene on dog chromosome 10q232 63
Abstract 65
Introduction 65
Materials and Methods 67
Results and Discussion 69
6 Identification of a 5 Mb region on canine chromosome 10 harbouring a causative gene responsible for congenital sensorineural deafness in German Dalmatian dogs 81
Abstract 83
Introduction 83
Material and methods 84
Results 86
Discussion 87
7 Analysis of canine chromosome 1 and the Gap junction protein alpha 1 (GJA1) gene in Dalmatian dogs segregating for congenital sensorineural deafness 97
Abstract 99
Introduction 99
Material and methods 100
Results and discussion 102
8 Analysis of canine chromosome 31 and the claudin-14 (CLDN14) gene in Dalmatian dogs segregating for congenital sensorineural deafness 107
Abstract 109
Introduction 109
Material and methods 110
Results and discussion 112
9 General Discussion 117
The candidate gene approach 119
Linkage and association analysis 120
CFA1 122
CFA31 123
CFA10 123
10 Summary 125
11 Erweiterte Zusammenfassung 129
12 References 145
13 Appendix I 14 List of publications XIII
Abbreviations
List of abbreviations
A adenine
Acc No accession number
ACTG1 actin gamma 1
AEP akustisch evozierte Potentiale (acoustically evoked potentials)
APS ammonium persulphate
AT annealing temperature
BAC bacterial artificial chromosome
BAER brain stem auditory evoked response
BLAST Basic Local Alignment Search Tool
bp base pair
C cytosine
CCSD canine congenital sensorineural deafness
CDH23 cadherin related 23
cDNA copy desoxyribonucleic acid
CFA chromosome of Canis familiaris
CLDN14 claudin-14
cM centiMorgan
COCH coagulation factor C homolog cochlin
COL11A2 collagen type XI alpha 2
CRYM crystallin mu
CSD cochleosaccular degeneration
CX connexin
DFN x-linked deafness locus
DFNA autosomal dominant deafness locus
DFNA5 deafness autosomal dominant 5
DFNB autosomal recessive deafness locus
DIAPH1 diaphanous homolog 1 (Drosophila)
DMSO dimethyl sulfoxide
Abbreviations
DNA deoxyribonucleic acid
dNTPs deoxy nucleoside 5rsquotriphosphates (N is ACG or T)
EDN3 endothelin 3
EDNRB endothelin receptor type B
EDTA ethylenediamine tetraaceticacid
EMBL European Molecular Biology Laboratory
ESPN espin
EST expressed sequence tag
EYA4 eyes absent homolog 4 (Drosophila)
F forward
FISH fluorescence in situ hybridisation
G guanine
GJA1 gap junction protein alpha 1 43kD (connexin 43)
GJB2 gap junction protein beta 2 26k (connexin 26)
GJB3 gap junction protein beta 3 31kDa (connexin 31)
GJB6 gap junction protein beta 6 (connexin 30)
GKF Gesellschaft zur Foumlrderung Kynologischer Forschung (Society for the
Advancement of Cynological Research)
HET observed heterozygocity
HE expected heterozygosity value
HSA chromosome of Homo sapiens
IBD identical by descent
IRD infrared dye
KCNQ4 potassium voltage-gated channel KQT-like subfamily member 4
Kb kilobase
LD linkage disequilibrium
LINE long interspersed nuclear element
LOD logarithm of the odds
M molar
Mb megabase
Merlin multipoint engine for rapid likelihood inference
MITF microphthalmia-associated transcription factor
Abbreviations
MS microsatellite
MTRNR1 mitochondrially encoded 12S RNA
MTTS1 mitochondrially encoded tRNA serine 1 (UCN)
MYH9 myosin heavy polypeptide 9 non-muscle
MYH14 myosin heavy polypeptide 14
MYO1A myosin IA
MYO3A myosin IIIA
MYO6 myosin VI
MYO7A myosin VIIA
MYO15A myosin XVA
NCBI National Center for Biotechnology Information
NMMHC-A nonmuscle myosin heavy chain-A
ODDD oculodentodigital dysplasia
OMIM Online Mendelian Inheritance in Man
OTOA Otoancorin
OTOF otoferlin
P error probability
PAX3 paired box gene 3 (Waardenburg syndrome 1)
PCDH15 Protocadherin-15
PCR polymerase chain reaction
PIC polymorphism information content
POU3F4 POU domain class 3 transcription factor 4
POU4F3 POU domain class 4 transcription factor 3
PRES solute carrier family 26 member 5 (prestin)
QTL quantitative trait locus
R reverse
RACE rapid amplification of cDNA ends
RH radiation-hybrid
RLM RNA ligase-mediated
RNA ribonucleic acid
RT-PCR reverse transcription polymerase chain reaction
SAS Statistical Analysis System
Abbreviations
SH1 Src homology 1
SINE short interspersed nuclear element
SLC26A4 solute carrier family 26 member 4 (pendrin)
SLC26A5 solute carrier family 26 member 5 (prestin)
SNP single nucleotide polymorphism
SOX10 SRY (sex determining region Y)-box 10
STRC stereocilin
STS sequence-tagged site
T thymine
TBE tris-borate-ethylenediamine tetraacetic acid
TECTA tectorin alpha
TEMED NNNrsquoNrsquo-tetramethylenediamine
TFCP2L3 transcription factor CP2-like 3
TJ tight juncions
TMC1 transmembrane channel-like gene 1
TMIE transmembran inner ear gene
TMPRSS3 transmembrane protease serine 3
U unit
USH1C Usher syndrome 1C
UTR untranslated region
WFS1 Wolfram syndrome 1 (wolframin)
wgs whole genome shotgun
WHRN whirlin
WS Waardenburg syndrome
Chapter 1
Introduction
Introduction 3
Introduction
Canine congenital sensorineural deafness (CCSD) has often been reported in the
literature and occurs in more than 54 different breeds of dogs with the Dalmatian dog
showing the highest incidence The inheritance and segregation of a major gene in
CCSD has been demonstrated in different Dalmatian dog populations But although
several studies have demonstrated the mode of inheritance in Dalmatian dogs no
universally accepted mode of inheritance for the other dog breeds affected by CCSD
has yet been identified
Breeding with blue-eyed Dalmatian dogs and unilaterally or bilaterally deaf dams and
sires is forbidden by paragraph 11b of the German animal welfare laws and thus the
hearing status of all Dalmatian dogs has to be tested as a puppy at about six to eight
weeks old using the brain stem auditory evoked response (BAER) test As deaf dogs
are very difficult to raise and often become aggressive and snappish from fear most
puppies suffering from bilateral hearing loss are euthanized However it has been
shown in recent years that auditory testing does not seem to be an effective way of
clearly reducing the high incidence of deafness in this breed Thus prevention of
CCSD cannot be achieved alone by exclusion of affected animals from breeding
Consequently a molecular genetic approach toward unravelling the responsible
genes in carriers is urgently needed
Many genetic disorders in humans and domestic dogs (Canis familiaris) demonstrate
a high level of clinical and molecular similarity Therefore the mutated genes in
human hereditary deafness seemed to be appropriate candidates for canine
congenital sensorineural deafness
The objective of the present study is to localize the gene that is involved in the
development of CCSD in Dalmatian dogs In order to achieve this goal successively
32 canidate genes were evaluated by means of linkage analyses using microsatellite
markers and single nucleotide polymorphisms (SNPs) This candidate gene
approach using gene-associated markers for linkage studies in families segregating
for deafness turned out to be little effective Therefore the canine chromosomes
(CFA) 1 CFA10 and CFA31 were scanned entirely with microsatellite markers
Additionally single nucleotide polymorphisms (SNPs) were developed for fine
mapping the identified CCSD regions
Introduction 4
Overview of chapter contents Chapter 2 reviews the identified 39 mutated genes causing non-syndromic hereditary
hearing impairment in humans Parallels and differences in canine and human
deafness are shown including the clinical signs inheritance patterns and
histopathology We located the humane deafness genes in the canine genome and
discussed the advantages of comparative genomics and different molecular genetic
approaches
In Chapter 3 an existing set of 43 microsatellite markers for in total 24 candidate
genes were used for a non-parametric linkage analysis with congenital sensorineural
deafness (CCSD) in Dalmatian dog families segregating for deafness
In Chapter 4 newly developed SNP markers associated with in total eight candidate
genes were evaluated for CCSD in Dalmatian dogs
In Chapter 5 the molecular characterization of the canine myosin heavy polypeptide
9 non-muscle (MYH9) gene on dog chromosome 10q232 is described
Chapter 6 7 and 8 present linkage analyses performed in Dalmatian dog families
segregating for congenital sensorineural deafness using microsatellite markers on
canine chromosome (CFA) 1 CFA10 and CFA31 and the results of fine mapping
regions linked with the CCSD phenotype using newly developed SNPs
Chapter 9 provides a general discussion and conclusions referring to Chapters 1-8
Chapter 10 is a concise English summary of this thesis while Chapter 11 is an
expanded detailed German summary which takes into consideration the overall
research context
Chapter 2
A comparative overview of the molecular genetics of non-syndromic deafness
in dogs and humans
Non-syndromic deafness in dogs and humans 7
A comparative overview of the molecular genetics of non-syndromic deafness in dogs and humans
Abstract
In man as in different dog breeds deafness is an often diagnosed disorder with the
Dalmatian dog showing the highest incidence Deafness in Dalmatian dogs is clearly
heritable and the presence of a recessive major gene affecting the disorder was
shown in several Dalmatian dog populations
This Chapter provides an overview of the identified 39 mutated genes causing
human non-syndromic hereditary hearing impairment as well as of the five genes
responsible for Waardenburg syndrome in humans We point out their cytogenetic
and genomic localisations in man and dog and compare the genomic and mRNA
sequences of these genes between man and dog Moreover an overview is given on
deafness genes-associated markers identified in Dalmatian dogs and on candidate
genes characterized in dogs
The structure of the ear The inner ear consists of the semicircular canals the vestibule and the cochlea
whereas the vestibule and the semicircular canals are concerned with vestibular
function (balance) and the cocnlea is concerned with hearing Reissneracutes membrane
and the basilar membrane divide the cochlea longitudinally into three scalae the
scala vestibule the scala media and the scala tympani The process of transduction
occurs in the structures within scala media sitting on the basilar membrane and
comprising the organ of Corti Cutting the cochlea tube cross sectionally the scala
media is more or less triangular formed by Reissneracutes membrane basilar
membrane and a structure called the stria vascularis The fluid that fills scala
tympani and scala vestibule is called perilymph the fluid that fills scala media is
called endolymph The organ of Corti rests on the basilar membrane within scala
media The cochlea contains an array of highly specialized cells arranged in a highly
Non-syndromic deafness in dogs and humans 8
specialized manner Two types of cells in the organ of Corti are support cells and
hair cells The hair cells are the receptor cells that trancsduce sound
When a sound wave brings physical displacement of the membranes separating the
perilymph from the endolymph they cause the organ of Corti to move and the hair
cells on it are scraped along the bottom of the tectorial membrane The tectorial
membrane is firmly anchored to the bone Relative movement of the organ of Corti
and its hair cells with respect to the tectorial membrane is the source of the
deformation of the hair cells microvilli The hair cells are so constructed that any
deformation of their microvilli will cause a change in the overall membrane potential
of the cell This signal is detected by the fibers from the cells in the spiral ganglion
These fibers are neural elements and they carry their own depolarization wave into
the auditory region of the brain
Deafness in man There are various ways to categorise deafness The two main types of deafness are
classified based on which portions of the auditory system are affected conductive
hearing loss occurs when when sound is not conducted efficiently through the outer
andor middle part of the ear Much more common is the sensorineural hearing loss
Sensorineural hearing loss occurs when there is damage to the inner ear (cochlea)
or to the nerve pathways from the inner ear (retrocochlear) to the brain Most cases
of sensorineural hearing loss are due to cochlear defects (Petit et al 2001)
Hearing loss can be present at birth (congenital) or become evident later in life
(acquired) Congenital deafness similarly may or may not be genetic In fact more
than half of congenital hearing loss is inherited Alternatively congenital deafness
may be due to a condition or infection to which the mother was exposed during
pregnancy Furthermore congenital hereditary deafness may occur as part of a
multisystem disease (syndromic) or as a disorder restricted to the ear and vestibular
system (non-syndromic) As non-syndromic hereditary hearing impairment is almost
exclusively caused by cochlear defects affected patients suffer from sensorineural
hearing loss In Table 1 and 2 the genes underlying human hereditary non-
syndromic deafness as a result of cochlear defects in consequence of primary
defects in hair cells non-sensory cells and the tectorial membrane or unknown cell
type are shown Non-syndromic deafness is estimated to cause 60-70 of cases of
congenital hereditary deafness in man (Morton 1991) In 70-80 of all cases this
Non-syndromic deafness in dogs and humans 9
non-syndromic form of deafness shows an autosomal recessive inheritance followed
by an autosomal dominant inheritance in 10-20 of all cases and 1-2 of all cases
are X-linked A maternally inherited form may also occur (Van Camp and Smith
2003)
Non-syndromic forms of hereditary deafness are classified by their mode of
inheritance DFN DFNA and DFNB refer to deafness forms inherited on the X
chromosome-linked autosomal dominant and autosomal recessive modes of
transmission respectively
Human hereditary isolated hearing loss is genetically heterogeneous (Petit et al
2001) Up to 1 of the human genes are estimated to be necessary for hearing
(Friedmann and Griffith 2003) Today approximately 120 genes for human
hereditary deafness have been identified approximately 80 for syndromic and 39 for
non-syndromic hereditary deafness which is suspected to be one-third of the total
(Nance 2003) In Table 1 the identified 39 mutated genes causing non-syndromic
hereditary hearing impairment in humans are shown Out of the 39 genes 15 genes
cause autosomal recessive and 15 genes cause autosomal dominant forms six
genes are involved in both recessive and dominant forms one gene causes X-linked
and two a maternally inherited form (The Hereditary Hearing Loss Hompage
httpwebhostuaacbehhh)
Furthermore several hundred forms of syndromes with hearing loss have been
documented in humans (Van Camp and Smith 2003) One is the human
Waardenburg syndrome (WS) which manifests itself with sensorineural deafness
and pigmentation defects in the iris hair and skin The WS is classified into four
types depending on the presence or absence of additional symptoms which are
caused by mutations in the five genes EDN3 EDNRB MITF PAX3 and SOX10
respectively These genes are shown in Table 3 They are known to be expressed in
the neural crest (EDN3 EDNRB PAX3 SOX10 ) or directly in the melanocytes
(MITF) and are inter alia involved in migration differentiation or survival of
melanocytes respectively (Bondurand et al 2000)
Deafness in dogs Congenital sensorineural deafness (CSD) has been reported in a variety of mammal
species other than humans ranging from mice to dogs guinea pigs and mink
Canine congenital deafness has often been reported in the literature and occurs in
Non-syndromic deafness in dogs and humans 10
more than 54 different breeds of dogs according to Strain (1996 and 2004) The
breeds with the highest incidence include Dalmatian dogs Bull Terrier English
Cocker Spaniel English Setter Australian Cattle Dog Australian Shepherd West-
Highland-White-Terrier Dobermann and Dogo Argentino The incidence of canine
congenital deafness is highest in Dalmatian dogs of which 165 to 30 exhibit
unilateral or bilateral hearing loss (Famula et al 1996 Wood and Lakhani 1997
Muhle et al 2002 Juraschko et al 2003a Rak and Distl 2005) The inheritance
and segregation of a major gene in canine congenital sensorineural deafness
(CCSD) has been demonstrated in different Dalmatian dog populations (Famula et
al 2000 Muhle et al 2002 Juraschko et al 2003b) But although several studies
have demonstrated the mode of inheritance in Dalmatian dogs no universally
accepted mode of inheritance for the other dog breeds affected by CCSD has yet
been identified
Congenital sensorineural hearing impairment can be recognised in dogs at four to
eight weeks of age (Strain 1996) while histological studies of deaf Dalmatian dogs
have shown that the degeneration of the inner ear structures begins as early as one
day after birth and is histologically clearly evident by four weeks of age (Johnsson et
al 1973) In 70 of the cases with human hereditary deafness the histological
pattern is known as cochleo-saccular degeneration (CSD) (Lalwani et al 1997)
commonly known as Scheibe dysplasia with preservation of the pars superior of the
membranous labyrinth and an unremarkable bony labyrinth As in man also in many
affected dog breeds the histological pattern of congenital sensorineural deafness is
known as cochleo-saccular degeneration
Breeding with blue-eyed Dalmatian dogs and unilaterally or bilaterally deaf dams and
sires is forbidden by paragraph 11b of the German animal welfare laws and thus the
hearing status of all Dalmatian dogs has to be tested as a puppy at about six to eight
weeks old using the brainstem auditory evoked response (BAER) test that detects
electrical activity in the cochlea and auditory pathways in the brain Although the
BAER test is a reliable method for identifying unilaterally and bilaterally deaf dogs it
does not seem to be an effective way of clearly reducing the incidence of deafness in
affected breeds particularly in a recessive mode of inheritance so that hearing dogs
can still be genetic carriers Furthermore deaf dogs are very difficult to raise and
often become aggressive and snappish from fear consequently most puppies
Non-syndromic deafness in dogs and humans 11
suffering from bilateral hearing loss are euthanized Thus prevention of CCSD
cannot be achieved alone by exclusion of affected animals from breeding and
consequently a molecular genetic approach toward unravelling the responsible
genes in carriers is urgently needed
Over the past decade it has become increasingly clear how far structural and
functional homologies at the gene level extend across even distantly related species
Much is known about deafness-causing gene mutations in humans and mice
including the fact that the clinical and histopathological findings are often very similar
to those of deafness in Dalmatian dogs Thus despite the genetical heterogeneity of
human non-syndromic deafness the genes that are responsible for non-syndromic
congenital hereditary deafness in humans (Table 1) seemed to be appropriate
candidate genes for CCSD especially in Dalmatian dogs (Rak and Distl 2005) The
genes that are mutated in the human WS (Table 2) were selected as candidates
because the WS phenotype where the deafness is associated with pigmentation
defects seems to be similar to the phenotype of most affected dog breeds (Strain
and Tedford 1996) Both Juraschko et al (2003a) and Strain et al (1992) have
demonstrated that patched Dalmatians are less likely to be deaf than unpatched
animals and blue-eyed Dalmatians are more likely to be affected from hearing
impairment than brown-eyed animals
In an attempt to achieve the aim of molecular genetic diagnosis of CCSD carriers
Rak et al (2003) and co-workers (Droumlgemuumlller et al 2002 Kuiper et al 2002 Rak
et al 2002a 2002b 2003) already mapped 24 potential candidate genes for
sensorineural deafness in dogs by fluorescence in situ hybridization and a radiation
hybrid panel to 16 different canine chromosomes
The canine genome project In December 2005 an international research team led by scientists at the Broad
Institute of MIT and Harvard achieved the completion of a high-quality genome
sequence of the domestic dog together with a catalog of 25 million specific genetic
differences across several dog breeds (Lindblad-Toh et al 2005) The authors found
that humans share more of their ancestral DNA with dogs than with mice confirming
the utility of dog genetics for understanding human disease Furthermore the
physiology disease presentation and clinical response of dogs often mimic human
Non-syndromic deafness in dogs and humans 12
diseases closely As indicated above hearing impairment seemed to be no
exception
In the last years most projects have exploited canine traits for which either direct
candidate genes could be proposed and evaluated or for which large informative
pedigrees were available to enable linkage mapping to identify candidate regions A
major component of such research efforts comprised the cloning sequencing and
mapping of individual canine homologs of genes either proposed as candidate
genes or expected to be located in candidate regions This was necessary to
identify new informative polymorphisms (eg single nucleotide polymorphisms
(SNPs) microsatellites) for high resolution mapping of candidate regions and to
examine each exon and exonintron boundary for positional candidates Availability
of the second version of the dog genome assembly (build 21) of the NCBI database
shortcut this effort and increase the investigators efficency
The current RH map with 3200 markers provides a good estimate of the order and
physical spacing (ie in base pairs) of markers along canine chromosomes (Guyon
et al 2003) and was recently complemented by the construction of a 4249-marker
integrated canine genome RH map that consists of 900 genes 1589 microsatellites
and 1760 BAC end markers (Breen et al 2004) all included and available in the
NCBI database The second version 1 of the NCBIs genome annotation consists of
large contigs covering all canine chromosomes given with their located markers and
genes The great majority of genes are derived by automated computational analysis
using the gene prediction method GNOMON
With this help either additional candidate genes for canine CSD can be found directly
by its gene symbol in the 21 of the NCBIs genome annotation or if a candidate gene
is yet not annotated a BLAST (Basic Local Alignment Search Tool) search versus
the canine whole genome shotgun (wgs) sequence resource can be used to obtain
the sequence of the canine genomic contigs containing the human homologous
gene The localisation of all 39 known human non-syndromic hereditary deafness
genes in the canine genome with the corresponding accession numbers of the contig
and if available the accession number of the genomic sequence and mRNA of the
canine gene are shown in Tables 4 and 5 Furthermore the identity of canine and
human or mouse mRNA is shown in Table 5 The average identity of canine and
human mRNA is with 088 percent higher than the average identity of canine and
mouse mRNA with 084 percent Canine sequences that correspond to the human
Non-syndromic deafness in dogs and humans 13
candidate gene can now be used to find microsatellite or SNP markers associated to
the respective canine gene These markers can be used for linkage and haplotype
studies in dog families segregating for deafness
Table 7 shows the microsatellite and SNP markers developed for in total 32
candidate genes for CCSD
The candidate genes for which a set of in total 43 microsatellite marker were
designed by Rak (2003) included the following 24 genes CDH23 CLDN14 COCH
COL11A2 DFNA5 DIAPH1 EDN3 EDNRB EYA4 GJA1 GJB2 GJB6 MITF
MYH9 MYO6 MYO7A MYO15A OTOF PAX3 POU4F3 SLC26A4 SOX10
TECTA and TMPRSS3 This existing set of 43 microsatellite markers for 24
candidate genes were used for linkage and haplotype studies in Dalmatian dog
families segregating for deafness (Chapter 3) These 24 genes are known to be
involved either in human non-syndromic deafness or in the human Waardenburg
syndrome For another eight candidate genes including TMC1 TMIE USH1C
MYH14 MYO3A PRES WHRN and ESPN SNP markers were newly developed
(Chapter 4) and subsequently used for linkage and association analyses in
Dalmatian dog families segregating for deafness These genes are also involved in
human non-syndromic deafness
Non-syndromic deafness in dogs and humans 14
Tabele 1 Genes responsible for non-syndromic congenital hereditary deafness in
humans
Inheritance Gene Gene product Type of molecule Locus namea
ACTG1 γ-Actin cytoskeletal protein DFNA20 DFNA26
COCH Cochlin extracellular matrix component DFNA9
COL11A2 Collagen XI (α2-chain) extracellular matrix component DFNA13
CRYM micro-Cristallin thyroid hormone-binding protein DFNAb DFNA5 Unidentified Unidentified DFNA5 DIAPH1 Diaphanous-1 cytoskeleton regulatory protein DFNA1 EYA4 EYA4 transcriptional coactivator DFNA10 GJB3 Connexin-31 gap junction protein DFNA2 KCNQ4 KCNQ4 K+ channel subunit DFNA2 MYH14 Myosin IIC motor protein DFNA4 MYH9 Myosin IIA motor protein DFNA17 MYO1A Myosin IA motor protein DFNA48 POU4F3 POU4F3 transcription factor DFNA15 TFCP2L3 TFCP2L3 transcription factor DFNA28
Autosomal dominant
WFS1 Wolframin endoplasmic-reticulum membrane protein DFNA6 DFNA14
CDH23 Cadherin-23 cell-adhesion protein DFNB12 CLDN14 Claudin-14 tight-junction protein DFNB29 ESPN Espin actin-bundling protein DFNB36 DFNAb MYO15 Myosin XV motor protein DFNB3 MYO3A Myosin IIIA motor protein DFNB30 OTOA Otoancorin cell-surface protein DFNB22 OTOF Otoferlin putative vesicle traffic protein DFNB9 PCDH15 Protocadherin-15 cell-adhesion protein DFNB23 SLC26A4 Pendrin IminusndashClminus transporter DFNB4 SLC26A5 Prestin anion transporter DFNB61 STRC Stereocilin DFNB16
TMIE TMIE transmembrane domain- containing protein DFNB6
TMPRSS3 TMPRSS3 transmembrane serine protease DFNB8 DFNB10 USH1C Harmonin PDZ domain-containing protein DFNB18
Autosomal recessive
WHRN Whirlin PDZ domain-containing protein DFNB31 GJB2 Connexin-26 gap junction protein DFNB1 DFNA3 GJB6 Connexin-30 gap junction protein DFNB1 DFNA3 MYO6 Myosin VI motor protein DFNA22DFNB37MYO7A Myosin VIIA motor protein DFNB2DFNA11
TECTA α-Tectorin extracellular matrix component DFNA8 DFNA12DFNB21
Autosomal dominant and autosomal recessive
TMC1 TMC1 transmembrane channel-like protein
DFNB7 DFNB11DFNA36
X-linked POU3F4 POU3F4 transcription factor DFN3
MTRNR1 Mitochondrial 12S rRNA not defined
nomenclature Mitochondrial
MTTS1 Mitochondrial 12S rRNA not defined
nomenclature a Autosomal recessive loci are designated DFNB autosomal dominant loci DFNA
Non-syndromic deafness in dogs and humans 15
Table 2 Genes underlying hereditary non-syndromic deafness as a result of primary
defects in hair cells non-sensory cells and the tectorial membrane or unknown cell
type
Primary defect
Gene
Hair cells MYO7A MYO15 MYO6 MYO3A MYO1A ACTG1 USH1C
WHRN CDH23 PCDH15 TMIE STRC SLC26A5 ESPN
KCNQ4 TMC1 OTOF POU4F3
Non-sensory cells
GJB2 GJB6 GJB3 SLC26A4 CRYM OTOA CLDN14
COCH TMPRSS3 MYH9 MYH14 EYA4 POU3F4
Tectorial membrane
COL11A2 TECTA
Unknown
DIAPH1 DFNA5 WFS1 TFCP2L3 MTRNR1 MTTS1
Table 3 Genes involved in the human Waardenburg syndrome
Inheritance Gene Gene product Type of molecule Type
EDN3 endothelin 3 vasoconstricted peptide WS type IV4
EDNRBendothelin
receptor type B receptor protein WS type IV4
MITF
microphthalmia-
associated
transcription
factor
transcription factor WS type II2
PAX3 paired box 3 DNA-binding protein WS type I1and III 2
SOX10 SRY-box 10 transcription factor WS type IV4 1Type I Dystopia canthorum 2Type II No dystopia canthorum 3Klein-Waardenburg syndrome (type III) Type I and upper limb abnormalities 4Waardenburg-Shah syndrome (type IV) Type II and Hirschsprung disease
(autosomal recessive inheritance)
Non-syndromic deafness in dogs and humans 16
Leng
th o
f co
ntig
(bp)
7799
0652
3821
0901
7077
2986
7251
5492
5989
7527
4533
7677
6093
8239
9498
326
6961
0260
1859
5814
6421
1953
Scor
e of
co
ntig
614
938
792
567
300
715
323
535
1208
1634
923
E-va
lue
of
cont
ig
4 E
-172
0 0
2 E
-158
1 e-
78
0
2 e-
85
7 E
-149
0 0 0
Acc
No
of
WG
S co
ntig
unkn
own
NW
_876
311
NW
_876
295
NW
_876
327
NW
_876
254
NW
_876
321
NW
_876
292
NW
_876
258
NW
_876
315
NW
_876
269
NW
_876
278
NW
_876
259
Mb
from
to
unkn
own
254
12
578
337
93
379
132
11
323
563
56
5
272
12
723
393
33
943
411
64
123
632
86
331
292
82
955
209
32
094
101
81
019
Gen
e lo
catio
n on
C
FA
unkn
own
4 31
8 12
6 2 14
5 1 25
15
Can
ine
gene
al
iase
s
none
none
LOC
4877
51
LOC
4906
40
LOC
4817
34
LOC
4798
18
none
LOC
6112
23
LOC
4896
31
EYA4
GJB
2
LOC
4824
86
Acc
No
hu
man
m
RN
A
NM
_001
614
NM
_022
124
NM
_144
492
NM
_004
086
NM
_080
680
NM
_001
888
NM
_005
219
NM
_004
403
NM
_031
475
NM
_172
105
NM
_004
004
NM
_024
009
Gen
e lo
catio
n on
H
SA
17
10
21
14
6 16
5 7 1 6 13
1
Tabl
e 4
Loca
lisat
ion
of h
uman
non
-syn
drom
ic h
ered
itary
dea
fnes
s ge
nes
in th
e ca
nine
gen
ome
the
cani
ne
gene
loc
alis
atio
n in
meg
abas
es (
Mb)
th
e ac
cess
ion
num
ber
(Acc
No
) of
the
who
le g
enom
e sh
otgu
n (W
GS
) con
tig c
onta
inin
g th
e hu
man
hom
olog
ous
gene
and
the
corr
espo
ndin
g E
-val
ue s
core
and
leng
th o
f th
e co
ntig
Hum
an
deaf
ness
ge
ne
AC
TG1
CD
H23
CLD
N14
CO
CH
CO
L11A
2
CR
YM
DIA
PH1
DFN
A5
ESPN
EYA
4
GJB
2
GJB
3
der
ived
from
the
NC
BIs
can
ine
geno
me
anno
tatio
n ve
rsio
n 2
1
Non-syndromic deafness in dogs and humans 17
Leng
th o
f co
ntig
6421
1953
5300
4996
5294
2087
2607
3285
1654
5469
1249
9463
7251
5492
5102
4781
5989
7527
5159
1990
1284
7264
7521
5785
Scor
e of
co
ntig
525
521
973
2256
348
567
1236
404
337
383
604
1715
E-va
lue
of
cont
ig
3 e-
146
1 e-
144
0 0
9 e-
93
2 e-
158
0
3 e-
109
3 e-
89
7 e-
103
2 e-
169
0
Acc
No
of
WG
S co
ntig
unkn
own
NW
_876
259
NW
_876
270
NW
_876
251
NW
_876
313
NW
_876
250
NW
_876
290
NW
_876
254
NW
_876
273
NW
_876
321
NW
_876
263
NW
_876
283
NW
_879
563
Mb
from
to
unkn
own
521
52
3
109
24
109
35
311
33
119
443
64
441
415
41
7
103
41
056
404
14
050
245
42
460
261
32
619
235
02
359
371
43
769
674
86
748
Gen
e lo
catio
n on
C
FA
25
15
1 10
5 10
2 12
21
6 17
26
X
Can
ine
gene
al
iase
s
none
LOC
4824
51
none
LOC
4812
80
LOC
4795
22
LOC
4744
10
LOC
4871
06
LOC
4818
84
LOC
4851
74
LOC
6086
55
LOC
6079
61
none
LOC
4919
88
Acc
No
hu
man
m
RN
A
NM
_006
783
NM
_004
700
NM
_024
729
NM
_002
473
NM
_016
239
NM
_005
379
NM
_017
433
XM
_376
516
NM
_000
260
NM
_144
672
NM
_194
248
NM
_033
056
NM
_000
307
Gen
e lo
catio
n on
H
SA
13
1 19
22
17
12
10
6 11
16
2 10
X
Tabl
e 4
(con
tinue
d)
Hum
an
deaf
ness
ge
ne
GJB
6
KC
NQ
4
MYH
14
MYH
9
MYO
15
MYO
1A
MYO
3A
MYO
6
MYO
7A
OTO
A
OTO
F
PCD
H15
POU
3F4
der
ived
from
the
NC
BIs
can
ine
geno
me
anno
tatio
n ve
rsio
n 2
1
Non-syndromic deafness in dogs and humans 18
Leng
th o
f co
ntig
4533
7677
2532
0482
2532
0482
4020
6070
3002
9677
2968
9717
5300
4996
3309
7591
3821
0901
5102
4781
1104
8438
6535
5756
Scor
e of
co
ntig
1404
283
283
1683
883
529
354
216
198
354
2238
689
E-va
lue
of
cont
ig
0
6 e-
73
2 E
-73
0 0
5 e-
147
2 e-
94
4 e-
53
9 e-
48
2 E
-94
0 0
Acc
No
of
WG
S co
ntig
NW
_876
292
NW
_876
265
NW
_876
265
NW
_876
294
NW
_876
312
NW
_876
255
NW
_876
270
NW
_876
272
NW
_876
295
NW
_876
273
NW
_876
256
NW
_876
253
Mb
from
to
436
14
361
158
61
592
197
91
982
134
31
345
158
81
595
622
63
7
880
88
822
450
54
505
390
33
905
432
44
328
414
94
151
716
47
172
Gen
e lo
catio
n on
C
FA
2 18
18
30
5 13
1 20
31
21
13
11
Can
ine
gene
al
iase
s
LOC
4872
00
LOC
4832
63
LOC
4832
74
LOC
4782
78
LOC
4893
57
LOC
4819
85
LOC
4841
68
LOC
6093
50
LO
C61
0987
LOC
6108
50
LOC
4821
13
LOC
6125
88
Acc
No
hu
man
m
RN
A
NM
_002
700
NM
_000
441
NM
_206
883
NM
_153
700
NM
_005
422
NM
_024
915
NM
_138
691
NM
_147
196
NM
_024
022
NM
_153
676
NM
_006
005
NM
_015
404
Gen
e lo
catio
n on
H
SA
5 7 7 15
11
8 9 3 21
11
4 9
Tabl
e 4
(con
tinue
d)
Hum
an
deaf
ness
ge
ne
POU
4F3
SLC
26A
4
SLC
26A
5
STR
C
TEC
TA
TFC
P2L3
TMC
1
TMIE
TMPR
SS3
USH
1C
WFS
1
WH
RN
der
ived
from
the
NC
BIs
can
ine
geno
me
anno
tatio
n ve
rsio
n 2
1
Non-syndromic deafness in dogs and humans 19
Leng
th o
f co
ntig
(bp)
4776
3139
5561
1003
2518
2130
3091
5115
5294
2087
Scor
e of
co
ntig
262
721
2927
967
1179
E-va
lue
of
cont
ig
8e-6
7
0 0 0 0
Acc
No
of
WG
S co
ntig
NW
_876
277
NW
_876
274
NW
_876
271
NW
_876
304
NW
_876
251
Mb
from
to
470
14
703
343
63
438
248
52
488
313
43
144
297
52
976
Gen
e lo
catio
n on
C
FA
24
22
20
37
10
Can
ine
gene
al
iase
s
ED
N3
ED
NR
B
MIT
F
PAX3
LOC
4812
58
Acc
No
hu
man
m
RN
A
NM
_207
032
NM
_000
115
NM
_198
159
NM
_181
457
NM
_006
941
Gen
e lo
catio
n on
H
SA
20
13
3 2 22
Tabl
e 5
Loca
lisat
ion
of g
enes
invo
lved
in th
e hu
man
Waa
rden
burg
syn
drom
e (W
S)
in th
e ca
nine
gen
ome
th
e ca
nine
gen
e lo
calis
atio
n in
meg
abas
es (
Mb)
th
e ac
cess
ion
num
ber
(Acc
No
) of
the
who
le g
enom
e sh
otgu
n (W
GS
) con
tig c
onta
inin
g th
e hu
man
hom
olog
ous
gene
and
the
corr
espo
ndin
g E
-val
ue s
core
and
le
ngth
of t
he c
ontig
Hum
an
deaf
ness
ge
ne
EDN
3
EDN
RB
MIT
F
PAX3
SOX1
0
der
ived
from
the
NC
BIs
can
ine
geno
me
anno
tatio
n ve
rsio
n 2
1
Non-syndromic deafness in dogs and humans 20
Table 6 Canine candidate genes for CCSD with their accession number (AccNo) of
the genomic sequence and mRNA and if available the percent identity of canine and
human or mouse mRNA Canine candidate gene
Acc No canine genomic sequence
Acc No canine mRNA (predicted)
Canine mRNA (bp)
Identity () of canine and human mRNA
Identity () of canine and mouse mRNA
ACTG1 none1 none unknown unknown unknown CDH23 none1 none unknown 8800 8690 CLDN14 NC_006613 XM_544876 714 8880 8460 COCH NC_006590 XM_547762 3050 8770 8170
COL11A2 NC_006594 XM_857285 5202 8970 8740 CRYM NC_006588 XM_845361 1279 8720 7890
DIAPH1 none1 none unknown 8630 unknown DFNA5 NC_006596 XM_848863 1512 8320 7680 ESPN NC_006587 XM_546751 2565 8930 8270 EYA4 NC_006583 XM_541108 3969 9240 8120 GJB2 NC_006607 XM_543177 2208 7860 6720 GJB3 NC_006597 XM_539603 1305 8550 8090 GJB6 none none unknown unknown unknown
KCNQ4 NC_006597 XM_539568 1836 9350 8760 MYH14 none1 none unknown 8770 8220 MYH9 NC_006592 XM_538401 6201 9130 8940
MYO15 NC_006587 XM_536660 10128 8500 7960 MYO1A NC_006592 XM_531642 3388 8600 8350 MYO3A NC_006584 XM_544234 5589 8510 8910 MYO6 NC_006594 XM_862465 3983 9250 8610
MYO7A NC_006603 XM_542292 6519 9190 8880 OTOA NC_006588 XM_845746 3558 8740 7990 OTOF NC_006599 XM_844665 5994 9020 8780
PCDH15 none1 none unknown 8210 7390 POU3F4 NC_006621 XM_549108 1086 9190 9060 POU4F3 NC_006584 XM_544328 1017 9380 9160 SLC26A4 NC_006600 XM_540382 2382 7970 8240 SLC26A5 NC_006600 XM_540393 2235 9250 8740
STRC NC_006612 XM_535452 5301 8980 8520 TECTA NC_006587 XM_546475 7136 9110 8510
TFCP2L3 NC_006595 XM_539106 2127 9030 8760 TMC1 NC_006583 XM_541284 2580 8860 8740 TMIE NC_006602 XM_846596 396 9110 8910
TMPRSS3 NC_006613 XM_848589 1542 8500 8340 USH1C NC_006603 XM_860072 1730 9010 8670 WFS1 NC_006595 XM_539234 2667 8510 8420 WHRN NC_006593 XM_850321 2817 8440 8110 EDN3 NC_006606 NM_001002942 1976 747 716
EDNRB NC_006604 NM_001010943 1329 889 827 MITF NC_006602 XM_850501 1590 934 862 PAX3 NC_006619 XM_545664 1474 869 861
SOX10 NC_006592 XM_538379 1987 926 900 derived from the NCBIs canine genome annotation version 21 1 the definite region in the whole genome sequence (WGS) contig was used to get the percent identity of mRNAs
Non-syndromic deafness in dogs and humans 21
Table 7 Microsatellite markers and single nucleotide polymorphisms (SNPs)
of canine candidate genes for canine congenital sensorineural deafness in
Dalmatian dogs
Canine candidate gene
Number of gene-associated
microsatellites
Number of gene-associated
SNPs
CDH23 2 0 CLDN14 3 8 COCH 2 0
COL11A2 2 0 DIAPH1 2 0 DFNA5 2 0 ESPN 0 5 EYA4 2 0 GJB2 3 0 GJB3 1 0 GJB6 1 0
MYH14 0 2 MYH9 2 22
MYO15 2 0 MYO3A 0 3 MYO6 1 0
MYO7A 3 0 OTOF 1 0 PAX3 1 0
POU4F3 1 0 SLC26A4 1 0 SLC26A5 0 2 TECTA 2 0 TMC1 1 1 TMIE 1 3
TMPRSS3 2 0 USH1C 0 2 WHRN 0 3
Chapter 3
Linkage analysis of gene-associated microsatellite markers with
congenital sensorineural deafness in Dalmatian dogs
Linkage analysis of gene-associated microsatellites 25
Linkage analysis of gene-associated microsatellite markers with congenital sensorineural deafness in Dalmatian dogs
Abstract Deafness is a disorder often diagnosed in different dog breeds In this Chapter an
existing set of 43 microsatellite markers associated with in total 24 candidate genes
for canine congenital sensorineural deafness (CCSD) were used for linkage and
haplotype analyses in a large Dalmatian dog population with frequent occurrence of
CCSD We found significant linkage for the genes GJA1 MYH9 and CLDN14 As
linkage was found for different candidate genes in different families the results of
these test statistics indicate that the inheritance of non-syndromic deafness in
Dalmatian dogs is heterogenic in origin
Introduction
Canine congenital sensorineural deafness (CCSD) has been reported to occur in
more than 54 different breeds of dogs (Strain 1996) As in man also in dog breeds
the most commonly observed histological pattern of degenerative inner ear changes
is known as the cochleo-saccular or Scheibe type of end organ degeneration
The incidence of this congenital anomaly is highest in Dalmatian dogs of which 165-
30 exhibit unilateral or bilateral hearing loss (Famula et al 1996 Wood and
Lakhani 1997 Muhle et al 2002 Juraschko et al 2003a Rak and Distl 2005) The
inheritance and segregation of a major gene in CCSD has been demonstrated in
different Dalmatian dog populations (Famula et al 2000 Muhle et al 2002 Juraschko et al 2003b) Moreover deafness in Dalmatian dogs seems to be
pigment-associated (Greibrokk 1994 Holliday et al 1992 Juraschko et al 2003a
2003b Mair 1976 Strain et al 1992 Strain 1996)
No gene mutation has yet been identified that is responsible for CCSD in Dalmatian
dogs or in one of the various other dog breeds that suffer from inherited hearing
impairment Since mutations in various genes have already been found to be the
cause of sensorineural hearing impairment in humans or mice 24 of these genes
Linkage analysis of gene-associated microsatellites
26
were considered as candidates for CCSD in Dalmatian dogs (Rak et al 2005)
Details of the 24 candidate genes are given in Table 1
Rak et al (2003) and co-workers (Droumlgemuumlller et al 2002 Kuiper et al 2002 Rak et
al 2002a 2002b 2003) mapped this 24 potential candidate genes for sensorineural
deafness in dogs by fluorescence in situ hybridization and a radiation hybrid panel
Subsequently Rak (2003) developed altogether 43 new highly polymorphic DNA
markers for the 24 candidate genes including CDH23 CLDN14 COCH COL11A2
DFNA5 DIAPH1 EDN3 EDNRB EYA4 GJA1 GJB2 GJB6 MITF MYH9 MYO6
MYO7A MYO15A OTOF PAX3 POU4F3 SLC26A4 SOX10 TECTA and
TMPRSS3 (Table 2)
Among the 24 candidate genes seven genes cause autosomal dominant non-
syndromic forms of deafness seven cause autosomal recessive forms and five
genes cause both recessive and dominant forms of non-syndromic deafness in
different human families segregating for either forms
The functions of these 19 deafness-causing genes are diverse and include gap
junctions and tight junctions (GJA1 GJB2 GJB6 CLDN14) ion channels (SLC26A4)
and ion channel activators (TMPRSS3) Included are also unconventional myosins
(MYO6 MYO7A MYH9 MYO15A) transcription factors (POU4F3 EYA4) as well as
extracellular matrix components (COCH COL11A2 TECTA) a cytoskeleton
regulatory protein (DIAPH1) a cell-adhesion protein (CDH13) and genes with
unknown or only suspected functions (DFNA5 OTOF) The 24 candidates also
include five genes which are mutated in the human Waardenburg syndrome (WS)
The WS is classified into four types depending on the presence or absence of
additional symptoms which are caused by mutations in the five genes EDN3
EDNRB MITF PAX3 and SOX10 respectively The objective of the current study
was to use this set of markers developed by Rak (2003) for a non-parametric linkage
analysis with CCSD in a German and French Dalmatian dog population
Material and methods Pedigree material
For the linkage analysis we used DNA from altogether 215 animals belonging to a
total of 24 Dalmatian dog families The families included 22 full-sib families and one
large paternal half-sib family of German Dalmatian dogs as well as 46 animals of a
Linkage analysis of gene-associated microsatellites 27
large paternal half-sib family of French Dalmatian dogs All families were segregating
for CCSD The genotyped families included all affected dogs (unilaterally and
bilaterally deaf) their parents if available and one to four unaffected animals At least
two of the full sibs of each family were unilaterally deaf
In total these 24 families included 402 individuals with an average family size of 168
ranging from 5 to 116 animals and covering two to four generations The hearing
status of 344 dogs was examined by veterinarians using the BAER (brain stem
auditory evoked response) test and the other animals included in the pedigree being
not BAER tested were used to construct relationships among CSD affected dogs
The prevalence of CSD in this pedigree was 285
Microsatellite marker analysis An existing marker set consisting of 43 microsatellite markers (Table 2) was used for
linkage analysis This set included 36 markers developed by Rak (2003) and 7
markers of the RH map available at httpwww-recomgenuniv-rennes1frdoggyhtml
For most of the 24 candidate genes two markers were available for two of the
candidates three markers were available but for seven candidate genes the set
contains only one marker The marker set is composed of 33 perfect repeats two
imperfect six compound-perfect and two compound-imperfect repeats
The majority (674) of the 43 markers in the set was represented by dinucleotide
repeats 209 were tetranucleotide repeats 47 trinucleotide repeats and 23
pentanucleotide repeats In addition one marker (23) was a compound di-
tetranucleotide and another one (23) was a compound tetra-pentanucleotide
repeat The average number of alleles was 35 with a minimum of 2 and a maximum
of 8 different alleles per marker
PCR reactions for microsatellites were carried out in 12 microl reaction mixtures
containing 2 microl genomic DNA 12 microl 10x PCR buffer (Qbiogene Heidelberg
Germany) 024 microl dimethyl sulfoxide (DMSO) 02 microl dNTPs (100 microM) 01 microl Taq
Polymerase (5Umicrol) (Qbiogene) 06 microl (10 microM) 5rsquo-IRD700 or IRD800 labelled forward
primer 06 microl (10 microM) unlabelled reverse primer The PCR reactions were carried out
in MJ Research thermocyclers with the following program 4 min at 94 degC followed
by 32 cycles of 30 sec at 94degC 30 sec at maximum annealing temperature and a
final extension of 45 sec at 72degC PCR-products were diluted with formamide loading
buffer in ratios from 110 to 140 and then size-fractionated by gel electrophoresis on
Linkage analysis of gene-associated microsatellites
28
automated LI-COR 42004300 sequencers (LI-COR inc Lincoln NE USA) using
denaturing 4 and 6 polyacrylamide gels (RotiphoreseregGel 40 Roth Karlsruhe
Germany)
To localize the 24 candidate genes and their associated microsatellites exactly the
canine candidate gene sequences were derived from sequences deposited in the
current dog genome assembly (Boxer genome assembly 21) of the NCBI GenBank
by BLAST (Basic Local Alignment Search Tool) search
(httpwwwncbinlmnihgovBLAST) using the human reference mRNA sequence
(Table 3)
Linkage analysis
Multipoint linkage and haplotype analyses were performed using the MERLIN
software version 0102 (multipoint engine for rapid likelihood inference Center for
Statistical Genetics University of Michigan MI USA Abecasis et al 2002) The test
statistics Z-mean and Lod score were used to test for the proportion of alleles shared
by affected individuals identical by descent (IBD) for the considered marker loci
Linkage analyses were performed regarding the marker set consisting of 43 gene-
associated microsatellite markers Linkage analysis was at first carried out for all 24
families conjoined After this the families were scanned separately
The data of the genotypes was additionally analyzed using SASGenetics (Statistical
Analysis System Version 913 SAS Institute Inc Cary NC USA 2005) to specify
the number of alleles of each marker the allele frequency the observed (HET) and
expected (HE) heterozygosity and the polymorphism information content (PIC)
(Table 4 and 5)
Results and discussion
Test statistics for all families conjoined are given in Table 6 Significant CCSD loci
were located on different chromosomes The loci were located on canine
chromosome (CFA) 1 10 12 20 and 31 Linkage analyses per family indicated even
higher test statistics for subgroups of families (Table 7) Scanning only families with
Zmeans gt1 test statistics for linkage increased for the genes GJA1 on CFA1 MYH9
on CFA10 and CLDN14 on CFA31 with congenital sensorineural deafness in different
Dalmatian dog families (Table 7) Therefore it is probable that these genes or genes
Linkage analysis of gene-associated microsatellites 29
in their flanking regions are involved in the development of the disease in the
respective familes The results of this test statistics indicate that the inheritance of
non-syndromic hearing loss in Dalmatian dogs is probably as heterogenic in origin as
it is in humans Genetic heterogeneity means that different mutations cause the same
phenotype or disease the different mutations can either be found at the same locus
(allelic heterogeneity) or even at different loci (non-allelic heterogeneity)
The extreme heterogeneity of human deafness often hampered genetic studies
because many different genetic forms of hearing loss give rise to similar clinical
phenotypes and conversely mutations in the same gene can result in a variety of
clinical phenotypes In man genes that transport ions across membranes to maintain
appropriate solute concentration and pH as well as regulatory genes mainly
transcription factors and genes that play a part in structural integrity are essential for
the hearing process
However this study was a first step in identifying genes responsible for CCSD in
Dalmatian dogs The genes GJA1 MYH9 and CLDN14 and their flanking regions will
be further analyzed with a combined approach using microsatellites and single
nucleotide polymorphisms (SNPs) (Chapter 6-9) As linkage was found for different
candidate genes in different families subsequently only the families indicating
linkage will be chosen for further molecular analyses of the respective gene
To confirm the result of this study the density of the intragenic markers has to be
increased The current RH map with 3200 markers provides a good estimation of the
order and physical spacing (ie in base pairs) of markers along canine
chromosomes (Guyon et al 2003) and was recently complemented by the
construction of a 4249-marker integrated canine genome RH map which consists of
900 genes 1589 microsatellites and 1760 BAC end markers (Breen et al 2004) all
included and available in the NCBI database (httpwebncbinlmnihgov)
Thus microsatellites derived from the NCBI database could be used to confirm the
linkage Alternatively BLAST searches versus the canine whole genome shotgun
(wgs) sequence resource were perfomed to localize the genes exactly and to obtain
the sequence of the canine genomic contigs containing the human homologous gene
The results of the BLAST searches of the 24 candidate genes against the Boxer
genome assembly 21 are shown in Table 3 The genomic sequence of the
respective candidate gene can now be used to search for intragenic SNPs as these
polymorphisms are the most abundant and useful markers for fine mapping
Linkage analysis of gene-associated microsatellites
30
Development of SNPs requires sequencing of DNA for the respective genomic
regions of the parents with the aim to identify heterozygous base pair exchanges
After a heterozygous base pair is found the whole family can be genotyped for this
informative SNP marker These polymorphisms can than be used for linkage
analyses as well as association studies
Fine mapping using SNP markers for all genes indicating linkage with CCSD
identified by this study should enable us to detect mutations responsible for CCSD in
parts of the Dalmatian dog population
Linkage analysis of gene-associated microsatellites 31
Table 1 Details of the 24 selected human candidate genes
Symbol Gene name Locus name1 Most important reference
CDH23 cadherin related 23 DFNB12 Bork et al 2001
CLDN14 claudin 14 DFNB29 Wilcox et al 2001
COCH coagulation factor C homolog cochlin DFNA9 Robertson et al 1998
COL11A2 collagen type XI alpha 2 DFNA13 McGuirt et al 1999
DFNA5 deafness autosomal dominant 5 DFNA5 Van Laer et al 1998
DIAPH1 diaphanous homolog 1 (Drosophila) DFNA1 Lynch et al 1997
EDN3 endothelin 3 WS type IV Edery et al 1996
EDNRB endothelin receptor type B WS type IV Attie et al 1995
EYA4 eyes absent homolog 4 (Drosophila) DFNA10 Wayne et al 2001
GJA1 gap junction protein alpha 1 43kD (connexin 43)
2 Liu et al 2001
GJB2 gap junction protein beta 2 26k (connexin 26) DFNA3 DFNB1 Kelsell et al 1997
GJB6 gap junction protein beta 6 (connexin 30) DFNA3 DFNB1 Grifa et al 1999
Del Castillo et al 2002
MITF microphthalmia-associated transcription factor WS type II Tassabehji et al 1994
MYH9 myosin heavy polypeptide 9 non-muscle DFNA17 Lalwani et al 2000
MYO6 myosin VI DFNA22 DFNB37 Melchionda et al 2001 Ahmed et al 2003
MYO7A myosin VIIA DFNA11 DFNB2 Liu et al 1997a Liu et al 1997b Weil et al 1997
MYO15A myosin XVA DFNB3 Wang et al 1998
OTOF otoferlin DFNB9 Yasunaga et al 1999
PAX3 paired box gene 3 (Waardenburg syndrome 1) WS type I WS type III Hoth et al 1993 Tassabehji
et al 1992
POU4F3 POU domain class 4 transcription factor 3 DFNA15 Vahava et al 1998
SLC26A4 solute carrier family 26 member 4 DFNB4 Li et al 1998
SOX10 SRY (sex determining region Y)-box 10 WS type IV Pingault et al 1998
TECTA tectorin alpha DFNB21 DFNA8DFNA12
Mustapha et al 1999 Verhoeven et al 1998
TMPRSS3 transmembrane protease serine 3 DFNB8DFNB10 Scott et al 2001
1 Autosomal recessive loci are designated DFNB autosomal dominant loci DFNA 2 To our knowledge no deafness locus has been determined for this autosomal recessive gene
Linkage analysis of gene-associated microsatellites
32
HET
()
638
338
366
544
616
632
456
656
433
614
458
586
529
PIC
()
752
548
336
484
775
70
63
9
74
6
61
6
83
7
62
762
621
No
of
alle
les
6 4 4 4 9 8 6 6 6 13 5 11 5
PCR
pr
oduc
t (b
p)
175
109
123
156
146
179
259
151
241
219
186
214
191
AT
(degC
)
62 60 56 60 60 62 58 60 62 58 58 60 58
Prim
er s
eque
nces
(5rsquorarr
3rsquo)
F A
ATC
AG
GA
GG
GG
TGAG
TGTG
R
CC
CC
CAG
CTC
ATA
CA
TTC
TC
F C
CTG
TAC
TGA
ATG
CTT
GA
GG
R
CA
TCTC
TAG
AA
GA
AG
CC
TCC
F
TC
AC
ATA
GC
ATT
ATA
TATG
GA
C
R T
TGA
GAT
GG
CTC
TTAC
TGA
G
F T
CG
ATG
ATG
CTT
TCTG
GTT
G
R A
GG
CTG
TGA
AA
TGG
ATG
GA
G
F G
AG
AA
GC
AC
CA
GG
CA
TAG
G
R T
GG
TTTA
GC
AA
GG
CTG
TTC
C
F T
GA
CA
TAC
GG
AG
GAC
CA
AG
AG
R C
CC
CTC
CC
TTG
CTC
TATC
TC
F T
GC
CC
CTC
AG
AG
ATA
ATC
AC
R
CTT
CA
ATTA
TAC
AC
ACA
GG
TAC
F
TG
AA
TATG
GG
GC
TGAG
GA
AG
R
TTC
TCC
CTC
TGC
CTG
TGTC
F
GG
TTTA
GC
AC
TGC
CTT
CA
GC
R
CA
TTA
AG
CA
TCTG
GC
ATG
TGG
F
GA
AA
AC
TCA
GA
TTA
GC
CTG
G
R A
TCTT
GA
GA
GC
AA
AGG
TTG
TG
F T
GG
TTA
GG
GC
ATG
ATTC
CA
G
R C
ATG
TATA
AA
GA
GTA
ATG
CC
AG
F
CG
GG
AG
AGG
GTT
TGAC
TAC
R
CTC
CG
TATT
GC
TCA
TCTT
TCC
F
AG
CTT
CC
CTT
CTC
TGA
GA
C
R G
AG
AA
TAG
AG
TTTG
TGC
TCA
G
Rep
eat
(TTT
A)1
5 (A
CC
)9(A
TC)3
(A
TTT)
7(G
TTT)
5 (C
A)2
1 (G
A)2
0 (C
TTT)
~20
(GA
)13
(AC
)20(
AG)9
(C
T)16
TT
(CT)
5 (A
AA
AT)
~25
(CT)
11(G
T)4(
CT)
2 (A
TTT(
T))2
3 (A
C)2
2
Mar
ker n
ame
CD
H23
_MS1
C
DH
23_M
S2_F
2 C
LDN
14_M
S1
CLD
N14
_MS
2 C
LDN
14_M
S3
CO
CH
_MS1
C
OC
H_M
S2
CO
L11A
2_M
S1
CO
L11A
2_M
S3
DFN
A5_
MS
1 D
FNA
5 _M
S2
DIA
PH
1 _M
S1
DIA
PH
1_M
S2
Mar
ker o
rigin
RP
CI8
1-99
C20
R
PC
I81-
99C
20
RP
CI8
1-97
L17
RP
CI8
1-97
L17
RP
CI8
1-97
L17
RP
CI8
1-32
1I4
RP
CI8
1-32
1I4
RP
CI8
1-24
M6
RP
CI8
1-24
M6
RP
CI8
1-66
C12
R
PC
I81-
66C
12
RP
CI8
1-36
2I5
RP
CI8
1-36
2I5
Tabl
e 2
Can
dida
te g
ene-
asso
ciat
ed m
arke
rs f
or c
anin
e de
afne
ss d
evel
oped
by
Rak
(20
03)
For
each
gen
e th
e m
icro
sate
llite-
base
d m
arke
r th
e m
arke
r or
igin
rep
eat t
ype
PC
R p
rimer
s w
ith o
ptim
ised
con
ditio
ns a
nd th
e nu
mbe
r of
alle
les
pol
ymor
phis
m
info
rmat
ion
cont
ent (
PIC
) and
obs
erve
d he
tero
zygo
sity
(HE
T) fo
r all
dogs
gen
otyp
ed a
re s
how
n
Can
dida
te
gene
CD
H23
C
LDN
14
CO
CH
C
OL1
1A2
DFN
A5
DIA
PH
1
Linkage analysis of gene-associated microsatellites 33
HET
()
815
345
695
578
562
508
583
441
75
63
7
33
414
30
76
7
44
8
PIC
()
853
679
798
774
652
63
72
3
59
5
83
2
77
9
50
5
54
1
40
4
88
9
59
1
No
of
alle
les
14 6 7 6 9 5 9 6 13 13 4 6 4 14 6
PCR
pr
oduc
t (b
p)
147
131
258
227
134
190
148
195
218
260
181
164 96
227 94
AT
(degC
)
60 58 58 58 60 60 58 62 58 58 62 56 58 58 62
Prim
er s
eque
nces
(5rsquorarr
3rsquo)
F G
CTA
GG
AA
AA
ATC
CG
CA
ATG
R
GAC
CC
CC
TAG
GAC
ACC
AAC
F
GA
GA
ATT
GG
GC
ATG
GG
CAG
A
R T
GA
CTT
TATC
AC
TGG
TCTT
TG
F T
TATG
CAG
CC
CA
TGAC
AA
TC
R C
AA
GG
GA
AC
TCA
AAG
GC
TTG
F
TG
GA
CC
AG
GTC
AGTT
TGTG
R
TC
TGC
CTG
TGTC
TCTG
CC
F
ATG
GC
ATG
AA
GA
GG
ATA
CC
G
R A
GG
AC
AGG
TGAC
GG
CTC
TAC
F
GC
TAG
TAC
TCG
ATT
GTG
GTC
R
TC
ATG
GG
TTG
TGA
GA
TCC
AG
F T
TAA
TTTG
CTC
GTC
TTC
CTG
R
TG
TAA
GC
TCC
ACG
GA
TCA
CC
F
CTC
TCTT
GG
TCTC
CC
TCTG
C
R G
GG
AG
TAG
GG
GTG
GAG
TAG
G
F G
GTG
TTTC
CTT
TCC
TTTT
CT
R G
GTG
TTC
TCTC
CC
TTTC
TCT
F C
TCTA
TGA
AA
GG
TGA
TTG
CC
R
CA
GC
CAT
AC
AA
ATG
AGA
ATT
G
F C
TAC
AG
TGA
ATC
AG
CA
CA
GAC
R
CA
GC
CTT
GA
CTG
TTTC
TTTG
G
F T
GA
TTG
AC
TCTA
CTT
TAC
AC
A
R T
ATA
TTA
GG
CG
GTT
TTC
TTC
T F
AC
CC
AG
GTG
GC
CTG
ATTG
R
GC
AC
GC
AC
GTT
CTC
TCTT
TC
F G
AA
TGC
CC
TTC
ACC
TTG
AA
A
R G
GA
AA
AG
GA
GA
GA
TGA
TGC
C
F T
CTT
CC
TTG
GA
AA
GG
GA
AC
TC
R T
GC
CC
TAA
CA
CTT
GG
AA
TGG
Rep
eat
(TA
GA
)12
(GA
)25
(GT)
10(A
T)13
(A
G)2
1 (G
T)16
(A
G)1
2 (C
A)1
2 (G
A)8
(C
TTT)
~20
(GA
AA
)~25
(G
T)12
(C
A)1
5 (A
G)1
1 (G
AA
A)~
43
(AC
)13
Mar
ker n
ame
ED
N3_
MS
1 E
DN
RB
_M
S1
EY
A4_
MS
1 E
YA
4_M
S2
GJA
1_M
S1
GJA
1_M
S2
GJB
2_M
S1
GJB
2_M
S2
GJB
6_M
S2
GJB
2+6_
MS1
M
ITF
_MS
2 M
ITF
_MS
3 M
YH
9_M
S2
MY
H9_
MS
3 M
YO
6_M
S2
Mar
ker o
rigin
RP
CI8
1-36
6E14
Z
emke
et a
l (1
999)
R
PC
I81-
301N
19
RP
CI8
1-30
1N19
R
PC
I81-
370A
16
RP
CI8
1-37
0A16
R
PC
I81-
133O
22
RP
CI8
1-13
3O22
R
PC
I81-
343C
15
FH
2324
R
PC
I81-
119P
24
RE
N10
0J13
R
PC
I81-
374A
12
FH
2293
R
PC
I81-
156P
14
Tabl
e 2
(con
tinue
d)
Can
dida
te g
ene
ED
N3
ED
NR
B
EY
A4
GJA
1 G
JB2
GJB
6 M
ITF
MY
H9
MY
O6
Linkage analysis of gene-associated microsatellites
34
HET
()
359
366
431
566
51
50
5
28
3
35
8
79
9
70
4
69
2
23
9
68 71
698
PIC
()
496
623
627
765
635
756
502
575
756
76
70
1
44
2
75
4
84
5
80
6
No
of
alle
les
5 4 6 13 7 8 5 3 10 11 8 8 8 10 11
PCR
pr
oduc
t (b
p)
293
166
100
137
174
197
164
201
160
297
267
140
276
228
260
AT
(degC
)
56 60 56 62 62 62 60 60 58 58 56 58 58 62 60
Prim
er s
eque
nces
(5rsquorarr
3rsquo)
F T
GG
TTA
AA
AC
ATT
AA
AC
TTA
TAG
R
TA
GTA
TATA
GA
GA
TGC
AA
TGG
F
CA
TTG
GG
TGC
TTTC
CTG
TTC
R
TG
GA
GC
TGC
AG
GTA
TAG
CC
F
CC
AG
GC
ATT
CG
AG
GG
TG
R C
AG
AA
CTT
GA
GG
AAC
CA
TAG
F
CC
ATG
AAC
TTTG
TGG
AA
CTG
C
R A
AA
GG
GTT
GC
TGTG
GA
GA
TG
F A
GG
CA
GG
TTC
ATC
TGTG
TCC
R
TC
CC
AG
AC
CC
AG
CTA
CA
TTC
F
CA
GC
CAA
CTG
TATT
CTC
CTT
G
R A
TCTT
GA
GC
CC
TGC
ATT
AG
G
F G
AA
GC
GA
GG
AG
AG
ACA
GTC
C
R A
AG
GA
AG
CC
TCC
TGAC
AA
CC
F
CA
GG
GTC
AG
GC
TCTA
TGC
TC
R T
CC
TATC
ATC
CG
GC
TTTG
AC
F
TC
TGG
ATTG
TGG
TCA
CA
AC
C
R A
CTG
GAC
AC
TTC
TTTT
CA
GAC
G
F A
AA
GTG
GC
TGG
TTC
GG
AA
G
R A
GC
AG
CA
GC
ATA
CA
TTC
CTC
F
AA
GTA
GA
TCC
TATT
ATC
GTG
G
R A
GTT
TCA
GTG
TCTG
TTA
AA
TAG
F
CC
GG
ATT
TCTG
AG
GAG
GC
R
CA
TGC
TCTT
CA
CC
AGA
AC
C
F T
CA
GC
ATG
GA
TTTT
GTA
AA
ATC
R
GG
AC
TGC
GTG
GA
CAT
CTG
F
AC
AC
GG
TTC
TCG
CTG
ATG
TG
R T
GA
AG
GG
GA
TTG
AAC
AG
AG
G
F T
TCA
TTC
CG
AG
GTT
CTA
AC
TG
R C
AC
CA
TCTC
GTA
GC
CTT
TATC
Rep
eat
(TC
)13
(AC
)12
(GT)
12
(AC
)18
(GT)
12
(TA
)3(T
G)9
(TA
)2(C
A)2
C
(TA
AA
)5
(AA
T)18
(C
T)13
(T
G)1
3 (C
T)17
(T
AA
A)1
4 (G
T)3
CC
(GT)
19
(ATT
T)12
(T
C)8
(CA)
5(C
G)2
(CA)
9 (G
T)16
Mar
ker n
ame
MY
O7A
_M
S1
MY
O7A
_M
S2
MY
O7A
_M
S3
MY
O15
A _
MS
1 M
YO
15A
_M
S2
OTO
F _M
S1
PA
X3_
MS
1 P
AX
3_M
S2
PO
U4F
3_M
S4
SLC
26A
4_M
S2
SO
X10
_MS
2 T
EC
TA _
MS
1 T
EC
TA _
MS
2 T
MP
RS
S3_
MS
1 T
MP
RS
S3_
MS
2
Mar
ker o
rigin
RP
CI8
1-19
3deg2
RP
CI8
1-19
3deg2
AH
TH29
8 R
PC
I81-
362deg
13
RP
CI8
1-36
2deg13
R
PC
I81-
198L
15
RP
CI8
1-25
7H23
R
PC
I81-
257H
23
G2C
024
66
RP
CI8
1-47
P17
RP
CI8
1-50
5H2
RP
CI8
1-59
C2
RP
CI8
1-59
C2
RP
CI8
1-12
5P17
A
HTH
246
Tabl
e 2
(con
tinue
d)
Can
dida
te
gene
MY
O7A
M
YO
15A
O
TOF
PA
X3
PO
U4F
3 S
LC26
A4
SO
X10
TE
CTA
TM
PR
SS
3
Linkage analysis of gene-associated microsatellites 35
Table 3 The gene-associated microsatellites (MS) for 24 candidate genes of canine
congenital sensorineural deafness were localized using BLAST searches against the
Boxer genome assembly 21 The accession numbers (AccNo) of the whole
genome shotgun (WGS) contigs containing the genes as well as their associated
microsatellites are given
Microsatellite Acc No of WGS contig CFA Location of
Gene (Mb) Location of
MS (bp) Location of
MS in relation to gene
CDH23_MS1 NW_876311 4 2541025780 25510 intragenic
CDH23_MS2 NW_876311 4 2541025780 25630 intragenic
CLDN14_MS1 NW_876295 31 3379533796 33790 proximal
CLDN14_MS2 NW_876295 31 3379533796 33950 distal
CLDN14_MS3 NW_876295 31 3379533796 33790 proximal
COCH_MS1 NW_876327 8 1321513232 13225 intragenic
COCH_MS2 NW_876327 8 1321513232 13290 distal
COL11A2_MS1 NW_876254 12 56315659 5608 proximal
COL11A2_MS3 NW_876254 12 56315659 5578 proximal
DFNA5_MS1 NW_876258 14 4116941237 41135 proximal
DFNA5_MS2 NW_876258 14 4116941237 41250 distal
DIAPH1_MS1 NW_876292 2 3933039430 39382 intragenic
DIAPH1_MS2 NW_876292 2 3933039430 39552 distal
EDN3_MS1 NW_876277 24 4701347032 47057 distal
EDNRB_MS1 Zemke et al (1999) 22 3436334383 34354 proximal
EYA4_MS1 NW_876269 1 2928029550 29531 intragenic
EYA4_MS2 NW_876269 1 2928029550 29500 intragenic
GJA1_MS1 NW_876269 1 6399463996 64150 distal
GJA1_MS2 NW_876269 1 6399463996 64160 distal
GJB2_MS1 NW_8762781 25 2093620942 20938 intragenic
GJB2_MS2 NW_8762781 25 2093620942 20863 proximal
GJB2+6_MS1 FH2324 25 2093620942 17543 proximal
GJB3_MS2 NW_876259 15 1018310194 4530 proximal
GJB6_MS2 NW_876278 25 2090420906 20953 distal
Linkage analysis of gene-associated microsatellites
36
Table 3 (continued)
Microsatellite Acc No of WGS contig CFA Location of
Gene (Mb) Location of
MS (bp) Location of
MS in relation to gene
MITF_MS2 NW_876271 20 2485324884 24844 proximal
MITF_MS3 REN100J13 20 2485324884 25668 distal
MYH9_MS2 NW_876251 10 3113531193 31244 distal
MYH9_MS3 FH2293 10 3113531193 31696 distal
MYO15A_MS1 NW_876313 5 4436944419 44330 proximal
MYO15A_MS2 NW_876313 5 4436944419 44409 intragenic
MYO6_MS2 NW_876254 12 4041740504 40534 distal MYO7A_MS1 NW_876273 21 2454324609 24465 proximal
MYO7A_MS2 NW_876273 21 2454324609 24566 intragenic
MYO7A_MS3 AHTH298 21 2454324609 24594 distal
OTOF_MS1 NW_876263 17 2350223595 23463 proximal
PAX3_MS1 NW_876304 37 3134831445 31426 intragenic
PAX3_MS2 NW_876304 37 3134831445 31481 distal
POU4F3_MS4 G2C02466 2 4361043612 - -
SLC26A4_MS2 NW_876265 18 1586715927 15960 distal
SOX10_MS2 NW_876251 10 2975129762 29740 proximal
TECTA_MS1 NW_876312 5 1588515954 15910 intragenic
TECTA_MS2 NW_876312 5 1588515954 15930 intragenic
TMPRSS3_MS1 NW_876295 31 3903539054 38942 proximal
TMPRSS3_MS2 AHTH246 31 3903539054 38508 proximal
Linkage analysis of gene-associated microsatellites 37
Table 4 Number of alleles observed (HET) and expected (HE) heterozygosity and
polymorphism information content (PIC) for the developed marker-set
Feature Mean SD Min Max
No of alleles 75 31 3 14
hO () 703 122 370 898
hE () 532 151 239 815
PIC () 667 130 336 889
Table 5 Number of alleles per microsatellite locus and their PIC () values of the
developed marker-set
No of alleles per
microsatellite
Number of marker
loci PIC ()
3 1 575
4 6 483
5 5 574
6 11 652
7 2 716
8 5 671
9 3 717
10 2 800
11 3 776
13 4 803
14 2 871
Linkage analysis of gene-associated microsatellites
38
Table 6 Significant test statistics for linkage analysis carried out for all 24 genotyped
families conjoined Zmeans and LOD scores are given with their respective error
probabilities for the gene-associated markers of the candidate genes CLDN14
COL11A2 GJA1 MITF MYH9 and SOX10
Marker Location on canine chromosome (CFA)
Zmean pZmean LOD score pLOD
CLDN14_MS1 31q15 134 009 086 002
CLDN14_MS2 31q15 168 005 105 001
CLDN14_MS3 31q15 108 014 049 007
COL11A2_MS1 12q11-q12 166 005 085 002
COL11A2_MS3 12q11-q12 167 005 078 003
GJA1_MS1 1q24-q25 151 007 118 001
GJA1_MS2 1q24-q25 151 007 118 001
MITF_MS2 20q13 101 02 080 003
MITF_MS3 20q13 121 011 104 001
MYH9_MS2 10q232 080 02 018 02
MYH9_MS3 10q232 175 004 097 002
SOX10_MS2 10q21-q23 146 007 110 001
Linkage analysis of gene-associated microsatellites 39
Table 7 Significant test statistics for linkage analyses carried out each family
separately Zmeans and LOD scores are given with their respective error probabilities
for the gene-associated markers of the candidate genes CLDN14 MYH9 and GJA1
Gene-associated
marker
Number of families with significant linkage to
CCSD
Number of corresponding
family members
Zmean pZmean LOD-score pLOD
CLDN14_MS1 51 40 278 0003 112 0011
CLDN14_MS2 383 000007 170 0003
CLDN14_MS3 281 0002 113 0011
MYH9_MS2 32 21 081 02 023 02
MYH9_MS3
(=FH2293) 156 006 058 005
GJA1_MS1 13 46 295 0002 052 006
GJA1_MS2 286 0002 051 006 1 families with significant linkage include four German full-sib families and one
German half-sib family 2 families with significant linkage include three German full-sib families 3 families with significant linkage include one French half-sib family
Chapter 4
Evaluation of eight candidate genes for canine congenital sensorineural deafness
in Dalmatian dogs
Evaluation of eight candidate genes for CCSD 43
Evaluation of eight candidate genes for canine congenital sensorineural deafness in Dalmatian dogs Abstract
In this study we have been focusing on genomic loci that encode various enzymes
and transporters involved in the hearing process in humans We developed intragenic
markers for the canine genes ESPN MYH14 MYO3A PRES TMC1 TMIE USH1C
and WHRN on canine chromosomes (CFA) 1 2 5 11 18 20 and 21 which have
been shown to be responsible for human hereditary deafness and to employ these
newly developed markers for non-parametric linkage analyses with canine congenital
sensorineural deafness (CCSD) We screened DNA from altogether six Dalmatian
dogs which represent the parents of four families for single nucleotide polymorphisms
(SNPs) in the eight candidate genes by means of direct sequencing combined with a
polymerase chain reaction method for amplifying genomic DNA We characterized 20
SNPs and one insertion-deletion polymorphism For the TMC1 and TMIE genes we
additionally genotyped one microsatellite marker each The families used for
subsequent genotyping of the markers included 39 members from four full-sib
families with frequent occurrence of CCSD We concluded that mutations in ESPN
MYH14 MYO3A PRES TMC1 TMIE USH1C and WHRN do not play a role in
CCSD of the Dalmatian dog population investigated here
Introduction Over the past ten years significant progress has been made in the identification of
genes causing different forms of human deafness Currently 39 of the genes
responsible for non-syndromic hearing impairment have been identified in different
human populations (The Hereditary Hearing Loss Hompage
httpwebhostuaacbehhh Van Camp and Smith 2003)
Since non-syndromic hereditary hearing impairment is almost exclusively caused by
cochlear defects affected patients suffer from sensorineural hearing loss
Evaluation of eight candidate genes for CCSD
44
The most common histopathologic finding in cases of profound congenital deafness
in humans is the cochleosaccular degeneration (CSD) It is estimated to occur in
approximately 70 of cases in man and also in dog breeds the histological pattern is
known as cochleosaccular degeneration
Many genetic disorders in humans and domestic dogs (Canis familiaris) demonstrate
a high level of clinical and molecular similarity Therefore genes responsible for
human hereditary deafness seem to be appropriate candidate genes for CCSD
especially in Dalmatian dogs
In this report we provide 21 single-nucleotide polymorphisms (SNPs) and two
microsatellite markers in altogether eight selected human candidate genes (Table 1)
This eight candidate genes were only recently identified for being responsible for
different form of human non-syndromic deafness In order to evaluate whether any of
this candidate genes is responsible for congenital sensorineural deafness in
Dalmatian dogs we analyzed the association of the ESPN MYH14 MYO3A PRES
TMC1 TMIE USH1C and WHRN haplotypes with the CCSD phenotype in four
families of Dalmatian dogs with frequent occurrence of CCSD
Material and methods Pedigree structure and sampling
For the linkage analysis we used blood samples from 39 Dalmatian dogs They
belong to four full-sib families segregating for CCSD At least two of the full sibs of
each family were unilaterally deaf The phenotype of the affected animals had been
confirmed by brainstem auditory evoked response (BAER) that detects electrical
activity in the cochlea and auditory pathways in the brain
The families consisted of eight to 12 individuals In two families a blood sample of the
sire and dam respectively was not available Screening for SNPs was performed by
comparative sequencing of genomic DNA from the parents of the families used for
linkage analyses
SNP and microsatellite marker identification for genotyping
The canine ESPN MYH14 MYO3A PRES TMC1 TMIE USH1C and WHRN gene
sequences was derived from sequences deposited in the current dog genome
assembly (Boxer genome assembly 21) of the NCBI GenBank (Table 2) by BLAST
Evaluation of eight candidate genes for CCSD 45
(Basic Local Alignment Search Tool) search (httpwwwncbinlmnihgovBLAST)
using the human ESPN MYH14 MYO3A PRES TMC1 TMIE USH1C and WHRN
reference mRNA sequence
We compared the canine genomic DNA sequence from the eight candidate genes to
canine cDNA fragments in the canine EST database using the BLASTN program
For the localization of the exonintron boundaries canine or alternatively human
mRNA sequences were used for the mRNA-to-genomic alignment program Spidey
(httpwwwncbinlmnihgovIEBResearchOstellSpideyindexhtml) The human
and canine mRNA sequences which were used to determine the exon organization of
the candidate genes are given in Table 2
For each of the eight candidate genes we designed intragenic primer pairs to amplifly
intronic sequences yielding products with a length of 560 to 670 bp PCR primers
were developed with the Primer3 program (httpfrodowimiteducgi-
binprimer3primer3_wwwcgi) The PCR reactions were performed in a total of 50 microl
containing 125 microM dNTPs 25 pmol of each primer the reaction buffer supplied by
the manufacturer (Qbiogene Heidelberg Germany) and 1 U Taq polymerase After
a 4 min initial denaturation at 95degC 35 cycles of 30 sec at 94degC 45 sec at 58degC and
80 sec at 72degC were performed in a MJ Research thermocycler (Biozym Hessisch
Oldendorf Germany) The obtained PCR products were directly sequenced with the
DYEnamic ET Terminator kit (AmershamBiosciences Freiburg Germany) and a
MegaBACE 1000 capillary sequencer (AmershamBiosciences) using the PCR
primers as sequencing primers Sequence data were analyzed with Sequencher 42
(GeneCodes Ann Arbor MI)
In the first step sequence analyses were performed for PCR products of the parents
of four full-sib families If a heterozygous SNP was found for one or both parents all
progeny of the respective families were analyzed for that SNP Additionally to the
SNPs we used two microsatellite markers for linkage analyses We identified one
intragenic microsatelllite within the TMC1 gene As we could develop only one SNP
for the TMIE gene we additionally genotyped one microsatellite marker derived from
the NCBI database (httpwebncbinlmnihgov) located very closely to the TMIE
gene (Table 3)
Evaluation of eight candidate genes for CCSD
46
SNP marker analysis
A total of 21 SNPs were identified within ESPN MYH14 MYO3A PRES TMC1
TMIE USH1C and WHRN (Table 3) and used for linkage analysis Of all SNPs six
out of the observed 21 SNPs were polymorphic in all four examined families Out of
the 21 SNPs eleven SNPs were present in family 1 18 SNPs were heterozygous for
one or both parents in family 2 and 15 SNPs could be used for linkage analysis in
family 3 and 4 respectively (Table 4)
The most frequent form of SNPs with a frequency of 238 was the CT transition
motif The scarcest one was the CG transversion motif with a frequency of 48
respectively
Linkage and haplotype analysis Multipoint linkage and haplotype analysis were performed using MERLIN version
0102 (multipoint engine for rapid likelihood inference Center for Statistical Genetics
University of Michigan MI USA Abecasis et al 2002) The test statistics Z-mean
and Lod score were used to test for the proportion of alleles shared by affected
individuals identical by descent (IBD) for the considered marker loci The data of the
genotypes was additionally computed using the software package SAS Genetics
(Statistical Analysis System Version 913 SAS Institute Inc Cary NC USA 2005)
to specify the observed heterozygocity values (HET) and the polymorphism
information content (PIC)
Furthermore linkage disequilibrium (LD) and association of haplotypes with CCSD
was tested using the procedures CASECONTROL and HAPLOTYPE of
SASGenetics (Statistical Analysis System version 913 Cary NC USA)
Results and discussion The non-parametric multipoint linkage analysis in four full-sib families did not show
significant test statistics The highest Z-mean value was 046 the highest LOD Score
was 008 while the error probabilities ranged from 03 to 08 (Table 5) The maximum
achievable Z-mean was 448 and the corresponding value for the LOD score was
160 Marker-trait association tests for haplotypes of the candidate gene markers
were not significant Obviously no haplotype was associated with CCSD in these
Dalmatian dog families as demonstrated (Figure 1 2 3 4 5 6 7 and 8) The
Evaluation of eight candidate genes for CCSD 47
paternal and maternal haplotypes as far as they could be estimated were distributed
among the affected progeny closely to the expected proportion of 50 and thus no
excess of a certain haplotype could be observed in the affected dogs
Due to the fact that both animals with unilateral or bilaterally hearing loss and
bilateral hearing animals shared identical haplotypes it is unlikely that the ESPN
MYH14 MYO3A PRES TMC1 TMIE USH1C and WHRN genes are involved in the
pathogenesis of CCSD in Dalmatian dogs However the ESPN MYH14 MYO3A
PRES TMC1 TMIE USH1C and WHRN gene-associated marker may serve for
further linkage studies in other Dalmatian dog populations and dog breeds other than
Dalmatians
Evaluation of eight candidate genes for CCSD
48
Table 1 Details of the six selected human candidate genes
Gene symbol
Gene name Locus name1 Most important reference
ESPN espin DFNB36 Naz et al (2004)
MYH14 myosin heavy polypeptide 14 DFNA4 Donaudy et al (2004)
MYO3A myosin IIIA DFNB30 Walsh et al (2002)
PRES solute carrier family 26
member 5 (prestin) DFNB61
Zheng et al (2000) Liu et
al (2003)
TMC1 transmembrane channel-like
gene 1
DFNB7
DFNA36 Kurima et al (2002)
TMIE transmembran inner ear DFNB6 Naz et al 2002
USH1C Usher syndrome 1C DFNB18 Verpy et al (2000) Ouyang
et al (2002)
WHRN whirlin DFNB31 Mburu et al (2003)
1 Autosomal recessive loci are designated DFNB autosomal dominant loci DFNA
Evaluation of eight candidate genes for CCSD 49
Table 2 Selected human candidate genes with their location on HSA and CFA and
corresponding accession numbers
Gene symbol Gene
location on HSA1
Acc No 3 human mRNA
Gene location
on CFA2
Acc No 3 canine
genomic sequence
Acc No 3 canine mRNA
ESPN 1 NM_031475 5 NC_006587 XM_546751
MYH14 19 NM_024729 1 NW_876270 -
MYO3A 10 NM_017433 2 NC_006584 XM_544234
PRES 7 NM_206883 18 NC_006600 XM_540393
TMC1 9 NM_138691 1 NC_006583 XM_541284
TMIE 3 NM_147196 20 NC_006602 XM_846596
USH1C 11 NM_153676 21 NC_006603 XM_860072
WHRN 9 NM_015404 11 NC_006593 XM_850321
1 Homo sapiens autosome 2 Canis familiaris autosome 3 Accession number
Evaluation of eight candidate genes for CCSD
50
Table 3 The 21 newly developed intragenic SNPs and two microsatellite markers for
Dalmatian dogs in the candidate genes ESPN MYH14 MYO3A PRES TMC1
TMIE USH1C and WHRN with their corresponding primers the SNP motif the
product size and the annealing temperature the observed heterozygosity (HET) and
polymorphism information content (PIC)
SNP Primer F1 (5acute -gt 3acute) SNP motif primer R2 (5acute -gt 3acute)
Bp3 AT4 PIC ()
HET()
ESPN_SNP1
ACCAGCACCCTCTCCAACTA
AGGAATTCACAA (CT)CACACATACA
ACTCAAGCTCAGGGTGTGGT
565 60 9 10
ESPN_SNP2
ACCAGCACCCTCTCCAACTA
ATGGCTGGCGCT (AG)GAGGCTGCCC
ACTCAAGCTCAGGGTGTGGT
565 60 27 41
ESPN_SNP3
ACCAGCACCCTCTCCAACTA
ACACTCTTCCCA (CT)GGCTGGCGCT
ACTCAAGCTCAGGGTGTGGT
565 60 23 32
ESPN_SNP4
ACCAGCACCCTCTCCAACTA
TGGGAAGAGGGA (AG)GGGGGAGCAT
ACTCAAGCTCAGGGTGTGGT
565 60 23 32
ESPN_SNP5
ACCAGCACCCTCTCCAACTA
GAGTGGGCCAGG (CT)TGGGAAGAGG
ACTCAAGCTCAGGGTGTGGT
565 60 28 42
MYH14_SNP1
CTCTCCCCAACTCAGTTCCA
ACGTGTATTCGG (GT)CGCTTTTATT
GTGATAGGGACGAGCAGCAT
670 60 35 42
MYH14_SNP2
CATGGGACCGTTCCTACACT
AGCCTCGTTTAA (CT)CTAAAAGGAA
GCTCAATAGGCACGACATCA
640 60 34 39
Evaluation of eight candidate genes for CCSD 51
Table 3 (continued)
SNP Primer F1 (5acute -gt 3acute) SNP motif primer R2 (5acute -gt 3acute)
Bp3 AT4 PIC ()
HET()
MYO3A_SNP1
AATGCTTGAGTTTGGGATGC
GGCAGTCCCATG (GT)CCCTTATAAG
ACCTAATTGCCCAGATGCAG
650 60 37 77
MYO3A_SNP2
AATGCTTGAGTTTGGGATGC
GTGGAGAGCCAC (CG)TTGGGAGAGG
ACCTAATTGCCCAGATGCAG
650 60 37 70
MYO3A_SNP3
AACCTCCTGGCGTAGTATTCC
CATTACCTATTT (AT)GATCCTTATA
TTTTCCACTTCAGGCACACA
650 60 25 36
PRES_SNP1
CCCTTACCCCATACCATTCC
GATAGACTTCCT (AG)CCCTCAGACT
TTCAGGACAGCATCATCTGC
560 60 37 64
PRES_SNP2
CCCTTACCCCATACCATTCC
TGATGTCTGCTG (AT)TAACCCATTC
TTCAGGACAGCATCATCTGC
560 60 37 66
TMC1_SNP1
GCAACCTCTCGGTTTATCCA
CGTGAAGTGCCC (AT)TTGATGGAAA
AAGCTGGGGAAGTGGATATGT
610 60 57 37
TMC1_SNP2
GCAACCTCTCGGTTTATCCA
GGAGACATTACC (AG)TGAAGTGCCC
AAGCTGGGGAAGTGGATATGT
610 60 47 29
TMC1_SNP3
GGAAGCAAGACTGAGGTTGG
AGGCTTTTTAAA (AG)CTGTTCTGGG
CTGCTGCATTTGCCTGTAAG
650 60 48 30
TMIE_SNP1
AGAACACCACCGTCTCCTTG
CAAGGCGACGCC (AT)GTGCTGTCCT
GCCTCTGGTCAGAAGAGGTG
625 60 59 36
Evaluation of eight candidate genes for CCSD
52
Table 3 (continued)
SNP Primer F1 (5acute -gt 3acute) SNP motif primer R2 (5acute -gt 3acute)
Bp3 AT4 PIC()
HET()
USH1C_SNP2
CTCCCGGTCTGTCAGGAAC
GGCCTGGGGGGA (AC)AAGCGGACGG
ATGGCATCGACTTCTCCAAC
560 60 37 35
USH1C_SNP4
CTCCCGGTCTGTCAGGAAC
GGTCTCAGACCG (AC)GGCAGGGAGA
ATGGCATCGACTTCTCCAAC
560 60 37 37
WHRN_SNP1
TTCACCTCCAGGATCTGGTC
CCTGAGCCCGAG (CT)CCACGCTGCT
GGCTACTTTTCTTCCCCCTTT
600 60 25 37
WHRN_SNP2
TTCACCTCCAGGATCTGGTC
GGTCACGGGGGC (CT)CCGGGAGGTT
GGCTACTTTTCTTCCCCCTTT
600 59 24 33
WHRN_SNP3
TTCACCTCCAGGATCTGGTC
GTCCGAGTCCCG (AG)CCCCAGCCTG
GGCTACTTTTCTTCCCCCTTT
600 60 34 55
Microsatellite marker
Primers (forward reverse ) 5acute -gt 3acute Bp AT PIC()
HET()
TMC1_MS1
GCCCCCAGCTAAAAAGAGAA
TTCTCTTCCTCCCTCCTGTTC
220-220 60 76 57
FH2158 ATGGCCACATCACCCTAGTC
CTCTCTCTGCATCTCTCATGAA
274-302 58 57 66
Microsatllite marker derived from the NCBI database (httpwebncbinlmnihgov) 1 Forward 2 Reverse 3 Product size (basepairs) 4 Annealing temperature (degC)
Evaluation of eight candidate genes for CCSD 53
Table 4 The 21 newly developed intragenic SNPs for Dalmatian dogs in the
candidate genes ESPN MYH14 MYO3A PRES TMC1 TMIE USH1C and WHRN
with their nucleotide polymorphism allele and genotype frequencies
Gene symbol Fam1 Nucleotide polymorphism
Allele frequencies
Genotype frequencies2
ESPN_SNP1 4 CgtT 083017 840
ESPN_SNP2 2 3 4 AgtG 074026 15160
ESPN_SNP3 2 3 TgtC 068032 07120
ESPN_SNP4 2 3 GgtA 068032 07120
ESPN_SNP5 2 3 4 CgtT 074026 15160
MYH14_SNP1 2 3 4 GgtT 058041 51610
MYH14_SNP2 2 3 4 CgtT 058041 51610
MYO3A_SNP1 1 2 GgtT 062038 5111
MYO3A_SNP2 1 2 CgtG 062038 5111
MYO3A_SNP3 1 2 4 TgtA 076024 15140
PRES_SNP1 1 2 3 4 AgtG 058042 10254
PRES_SNP2 1 2 3 4 TgtA 058042 10254
TMC1_SNP1 1 2 3 4 AgtT 056044 11226
TMC1_SNP2 1 2 3 4 AgtG 076024 20190
TMC1_SNP3 1 2 3 4 AgtG 074026 19200
TMIE_SNP1 1 2 3 4 AgtT 058042 12216
USH1C_SNP2 1 3 4 AgtC 053047 9147
USH1C_SNP4 1 3 4 AgtC 053047 9147
WHRN_SNP1 2 CgtT 075025 360
WHRN_SNP2 2 CgtT 075025 360
WHRN_SNP3 2 3 4 InDel3 069031 13171 1 Number of families which were informative for the respective SNPs 2 Frequence of genotypes (first number number of animals homozygous for allele 1
second number heterozygous third number homozygous for allele 2) 3 Insertiondeletion of one base-pair (adenine)
Evaluation of eight candidate genes for CCSD
54
Table 5 Linkage analyses for families 1 2 3 and 4 for the 21 SNP and two
microsatellite markers within the eight candidate genes regarding Zmean LOD score
and error probabilities (p-values)
Gene symbol Marker Zmean pz-value1 LOD score pL-value2
ESPN ESPN_SNP1 014 04 002 04
ESPN_SNP2 014 04 002 04
ESPN_SNP3 014 04 002 04
ESPN_SNP4 014 04 002 04
ESPN_SNP5 014 04 002 04
MYH14 MYH14_SNP1 -089 08 -019 08
MYH14_SNP1 -089 08 -019 08
MYO3A MYO3A_SNP1 -049 07 -011 08
MYO3A_SNP2 -049 07 -011 08
MYO3A_SNP3 -049 07 -011 08
PRES PRES_SNP1 -094 08 -019 08
PRES_SNP2 -094 08 -019 08
TMC1 TMC1_SNP1 -034 06 -008 07
TMC1_SNP2 -034 06 -008 07
TMC1_SNP3 -034 06 -008 07
TMC1_MS1 -035 06 -008 07
TMIE TMIE_SNP1 013 04 003 03
FH2158 -056 07 -013 08
USH1C USH1C_SNP2 018 04 04 03
USH1C_SNP4 018 04 04 03
WHRN WHRN_SNP1 046 03 008 03
WHRN_SNP2 046 03 008 03
WHRN_SNP3 046 03 008 03 1 Error probability regarding Zmean 2 Error probability regarding LOD score
Microsatellite marker
Evaluation of eight candidate genes for CCSD 55
Figu
re 1
SN
P-h
aplo
type
s of
the
ES
PN
gen
e in
all
anal
yzed
Dal
mat
ian
dog
fam
ilies
The
hapl
otyp
es b
elon
ging
to F
amily
2 3
and
4 re
gard
ing
the
gene
ES
PN
sho
w n
o as
soci
atio
n w
ith th
e C
CS
D p
heno
type
In
the
thre
e fa
milie
s th
e ha
plot
ype
21
43
2 is
the
mos
t com
mon
one
bei
ng p
rese
nt in
72
o
f all
anim
als
It is
follo
wed
by
the
hapl
otyp
e 2
32
14
with
18
a
nd 4
34
34
with
6
Nor
mal
hea
ring
anim
als
as
wel
l as
thei
r de
af s
iblin
gs s
how
all
thes
e th
ree
hapl
otyp
es T
here
is n
o re
com
bina
tion
of th
e ha
plot
ypes
of t
he E
SP
N g
ene
in th
e th
ree
fam
ilies
Evaluation of eight candidate genes for CCSD 56
Figu
re 2
SN
P-h
aplo
type
s of
the
MY
H14
gen
e in
all
anal
yzed
Dal
mat
ian
dog
fam
ilies
Evaluation of eight candidate genes for CCSD 57
Figu
re 3
SN
P-h
aplo
type
s of
the
MY
O3A
gen
e in
all
anal
yzed
Dal
mat
ian
dog
fam
ilies
Evaluation of eight candidate genes for CCSD 58
Figu
re 4
SN
P-h
aplo
type
s of
the
PR
ES
gen
e in
all
anal
yzed
Dal
mat
ian
dog
fam
ilies
Evaluation of eight candidate genes for CCSD 59
Figu
re 5
SN
P- a
nd m
icro
sate
llite-
hapl
otyp
es o
f the
TM
C1
gene
in a
ll an
alyz
ed D
alm
atia
n
Evaluation of eight candidate genes for CCSD 60
Figu
re 6
SN
P- a
nd m
icro
sate
llite-
hapl
otyp
es o
f the
TM
IE g
ene
in a
ll an
alyz
ed D
alm
atia
n do
g fa
milie
s
Evaluation of eight candidate genes for CCSD 61
Figu
re 7
SN
P-h
aplo
type
s of
the
US
H1C
gen
e in
all
anal
yzed
Dal
mat
ian
dog
fam
ilies
Evaluation of eight candidate genes for CCSD 62
Figu
re 8
SN
P-h
aplo
type
s of
the
WH
RN
gen
e in
all
anal
yzed
Dal
mat
ian
dog
fam
ilies
Chapter 5
Molecular characterization of the canine myosin heavy polypeptide 9
non-muscle (MYH9) gene on dog chromosome 10q232
Canine MYH9 gene 65
Molecular characterization of the canine myosin heavy polypeptide 9 non-muscle (MYH9) gene on dog chromosome 10q232 Abstract A missense mutation of the Myosin heavy polypeptide 9 (MYH9) gene which
encodes the nonmuscle myosin heavy chain-A caused non-syndromic sensorineural
deafness in a human family which was characterized by cochleosaccular
degeneration In the present study we evaluated whether MYH9 gene mutations are
responsible for canine congenital sensorineural deafness (CCSD) in Dalmatian dogs
As described in Chapter 3 two MYH9 gene-associated microsatellites were
genotyped in 25 Dalmatian dog families segregating for CCSD We could find
significant linkage with CCSD for the microsatellite marker MYH9_MS3 (=FH2293)
We used data deposited in the NCBI to assemble the canine MYH9 gene DNA
sequence Characterization of the canine MYH9 gene revealed that the canine gene
consists of 41 exons spanning approximately 90 kb
We detected 22 single nucleotide polymorphisms (SNPs) in a mutation scan of
altogether 16 Dalmatian dogs from three families which showed significant linkage
between the deafness phenotype and the MYH9 gene-associated microsatellite
None of the SNPs affects the amino acid sequence of MYH9 We concluded that the
exons of the canine MYH9 gene do not play a role in Dalmatian CCSD Availability of
the microsatellite marker SNPs and DNA sequence reported in this study enhance
evaluation of the MYH9 gene as a CCSD candidate gene in other Dalmatian dog
populations and other dog breeds affected by CCSD
Introduction Myosin is a functional protein associated with cellular movement cell division muscle
contraction and other functions Members of the myosin super-family are
distinguished from the myosin heavy chains that play crucial roles in cellular
processes The human MYH9 gene consists of 40 exons and spans 67959 bp
Canine MYH9 gene
66
Toothaker et al (1991) mapped a nonmuscle myosin heavy chain gene to human
chromosome (HSA) 22q123-q131 Simons et al (1991) likewise mapped the gene
to HSA22q112 which they designated nonmuscle myosin heavy chain-A (NMMHC-
A)
The similarities between the autosomal dominant giant-platelet disorders May-
Hegglin anomaly Fechtner syndrome and Sebastian syndrome and refinement of the
disease loci for May-Hegglin anomaly and Fechtner syndrome to an overlapping
region of 480 kb on human chromosome 22 suggested that all these three disorders
may be allelic Among the identified candidate genes was the gene encoding
nonmuscle myosin heavy-chain A (MYH9) The May-HegglinFechtner Syndrome
Consortium (2000) demonstrated that mutations in MYH9 result in one of the three
disorders mentioned above The same Consortium also speculated that mutations in
MYH9 may also play a role in another autosomal dominant disorder a form of
nonsyndromic deafness characterized by progressive hearing impairment and
cochleosaccular degeneration This autosomal dominant form of human
nonsyndromic hereditary deafness (DFNA17) was described by Lalwani et al (1999)
They studied a five-generation American family previously reported by Lalwani et al
(1997) with deafness caused by cochleosaccular degeneration (CSD) CSD is the
most common histopathologic finding in cases of profound congenital deafness and
is estimated to occur in approximately 70 of cases in man
DFNA17 maps to the same region as MYH9 Because of the importance of myosins
in hearing Lalwani et al (2000) tested MYH9 as a candidate gene for DFNA17 and
demonstrated a missense mutation in the MYH9 gene in affected members of a
kindred with DFNA17 They found a G-to-A transition at nucleotide 2114 in the MYH9
gene This missense mutation changed codon 705 from an invariant arginine to a
histidine within a highly conserved Src homology 1 (SH1) linker region Previous
studies had shown that modification of amino acid residues within the SH1 helix
causes dysfunction of the ATPase activity of the motor domain in myosin
Many genetic disorders in humans and domestic dogs (Canis familiaris) demonstrate
a high level of clinical and molecular similarity Rak et al (2003) mapped 20 potential
candidate genes for sensorineural deafness in dogs by fluorescence in situ
hybridization and a radiation hybrid panel among them the MYH9 gene that was
assigned to canine chromosome (CFA) 10q232 Congenital sensorineural deafness
has been reported for approximately 54 different breeds of dogs (Strain 1996)
Canine MYH9 gene 67
The incidence of this congenital anomaly is highest in Dalmatian dogs of which 165-
30 exhibit unilateral or bilateral hearing loss (Famula et al 1996 Wood and
Lakhani 1997 Muhle et al 2002 Juraschko et al 2003a Rak and Distl 2005) The
inheritance and segregation of a major gene in CCSD has been demonstrated in
different Dalmatian dog populations (Famula et al 2000 Muhle et al 2002
Juraschko et al 2003b)
In dog breeds the histological pattern is known as cochleosaccular degeneration
commonly known as Scheibe dysplasia as it is described in approximately 70 of
cases with human hereditary deafness (Strain 1996) Therefore the MYH9 gene
seems to be a perfect candidate gene for CCSD especially in Dalmatian dogs
In this report we provide the genomic organization and the complete sequence of the
canine MYH9 gene A mutation analysis was performed to identify single nucleotide
polymorphisms (SNPs) in this gene In order to evaluate whether the MYH9 gene is
responsible for congenital sensorineural deafness in Dalmatian dogs we analyzed
the association of the MYH9 haplotypes with the CCSD phenotype in three families of
Dalmatian dogs with frequent occurrence of CCSD and significant linkage to the
gene-associated microsatellite MYH9_MS3 (Chapter 3)
Materials and Methods
Cloning and sequencing of canine MYH9 cDNA
The canine MYH9 gene sequence was derived from sequences deposited in the
current dog genome assembly (Boxer genome assembly 21) of the NCBI GenBank
(Genbank Acc No NW_1398701) by BLAST (Basic Local Alignment Search Tool)
search (httpwwwncbinlmnihgovBLAST) using the human MYH9 reference
mRNA sequence (Genbank Acc No NM_002473)
The distance between MYH9 and the microsatellite MYH9_MS2 is about 181 kb
whereas the microsatellite MYH9_MS3 (=FH2293) places about 511 kb from MYH9
The isolation of full length cDNAs was achieved by a modified rapid amplification of
cDNA ends (RACE) protocol Total RNA from dog lung of a normal female Beagle
(Biocat Heidelberg Germany) was used for the overlapping 5rsquo- and 3rsquo-RACE
products with the FirstChoiceTM RNA ligase-mediated (RLM)-RACE kit (Ambion
Europe Huntingdon UK) according to the protocols of the manufacturer Reverse
transcription polymerase chain reaction (RT-PCR) using two pairs of nested
Canine MYH9 gene
68
gene-specific and supplied 5rsquo-adaptor-specific primers were used to amplify the
complete 5rsquo-end of the investigated cDNA Similarly the 3rsquo-end were amplified using
two pairs of nested gene-specific primers in combination with supplied 3rsquo-adaptor-
specific primers Additionally RT-PCR using six pairs of gene-specific primers were
used to amplify the complete sequence of the investigated cDNAs
RACE products were cloned into pCRR21 vector using the Invitrogen TA cloning kit
(Invitrogen Karlsruhe Germany) Several clones for each cDNA were sequenced
with the ThermoSequenase kit (AmershamBiosciences Freiburg Germany) and a
LI-COR 4300 DNA Sequencer (LICOR inc Bad Homburg Germany) Sequence
data were analyzed with Sequencher 42 (GeneCodes Ann Arbor MI)
Sequence analysis of canine MYH9 gene For the localization of the exonintron boundaries after cloning and sequencing of full
length canine cDNAs (as described above) the mRNA-to-genomic alignment program
Spidey (httpwwwncbinlmnihgovIEBResearchOstellSpideyindexhtml) was
used Repetitive elements were analyzed with Repeatmasker 2
(httprepeatmaskergenomewashingtonedu) The GC content was calculated with
the EBI toolbox CpG PlotCpGreport (httpwwwebiacukToolssequencehtml)
Mutation analysis To identify polymorphisms within the canine MYH9 sequence exons with flanking
regions were PCR amplified and sequenced from 16 Dalmatian dogs which
represent three families consisting of three to six full sibs and at the best of both
parents At least two of the full sibs of each family were unilaterally deaf The
phenotype of the affected animals had been confirmed by brainstem auditory evoked
response (BAER) that detects electrical activity in the cochlea and auditory pathways
in the brain PCR primers and conditions for the amplification of MYH9 exons with
flanking sequences and microsatellite flanking primers for PCR reactions are given in
Table 1 PCR primers were developed with the Primer3 program
(httpfrodowimiteducgi-binprimer3primer3_wwwcgi) The PCR reactions for
exons and their flanking sequences were performed in a total of 50 microl containing 125
microM dNTPs 25 pmol of each primer the reaction buffer supplied by the manufacturer
(Qiagen Hilden Germany) and 1 U Taq polymerase After a 4 min initial
denaturation at 95 degC 35 cycles of 30 sec at 94 degC 45 sec at 58 degC and 80 sec
Canine MYH9 gene 69
at 72 degC were performed in a MJ Research thermocycler (Biozym Hessisch
Oldendorf Germany) The obtained PCR products were directly sequenced with the
DYEnamic ET Terminator kit (AmershamBiosciences Freiburg Germany) and a
MegaBACE 1000 capillary sequencer (AmershamBiosciences) using the PCR
primers as sequencing primers Sequence data were analyzed with Sequencher 42
(GeneCodes Ann Arbor MI)
Multipoint linkage and haplotype analysis were performed using MERLIN version
0102 (multipoint engine for rapid likelihood inference Center for Statistical Genetics
University of Michigan MI USA Abecasis et al 2002) The test statistics Z-mean
and Lod score were used to test for the proportion of alleles shared by affected
individuals identical by descent (IBD) for the considered marker loci
Linkage means that a haplotype characterized by microsatellites or SNPs is
significantly more often present in family members with the phenotype under study
than expected by random assortment For construction of haplotypes sets of closely
linked genetic markers on the same chromosome are needed which tend to be
inherited together as they are not likely to be separated by recombination
Association analysis can be carried out as a method of genetic analysis that
compares the frequency of alleles between affected and unaffected individuals
across all families A given allele is considered to be associated with the disease if
the presence of that allele explains a significant proportion of the phenotypic trait
variation
Results and Discussion
Analysis of the genomic organization and cDNA of the canine MYH9 gene
A full-length cDNA sequence of 7484 bp of the canine MYH9 gene was obtained by
using the RACE protocol The obtained RT-PCR products were sequenced and the
generated sequence data were submitted to the EMBL nucleotide database (Acc No
AM086385)
Using the RT-PCR analyses for cDNA-genomic sequence comparisons we detected
that the canine MYH9 gene consists of 41 exons with an untranslated exon 1 and
exonintron boundaries that conform perfectly to the GTAG rule (Table 2)
The 5rsquo-UTR consists of 181 bp while the 3rsquo-UTR consists of 1432 bp Assuming that
the homologous ATG start codon as in man is used the canine MYH9 cDNA
Canine MYH9 gene
70
contains an open reading frame of 5889 bp encoding a protein of 1963 amino acids
A canonical polyadenylation signal AAUAAA is located approximately 14 kb
downstream of the stop codon
The exon sizes range from 28 to 1556 bp the introns between these exons span
between 86 and 13493 bp the total size of the canine MYH9 gene is approximately
90 kb However the sequence homology between the human murine and canine
MYH9 gene is limited to the coding sequence of exon 2 to 41 The coding sequence
of canine MYH9 displays 926 and 899 similarities to the human and murine
MYH9 gene respectively In the untranslated regions the sequence similarity
between dog and human and mouse is rather low The canine MYH9 protein shows
982 and 968 identities to the orthologous human and murine protein
respectively (Fig 2)
The repeat content in the 90 kb region of the canine MYH9 gene is 1458 Most of
the repetitive elements belong to the SINE family (892) followed by the fraction of
the LINEs (306) other repetitive elements constitute 165 respectively The
entire canine MYH9 gene has an overall GC-content of 54 The canine MYH9 gene
contains 12 CpG islands (Gardiner-Garden and Frommer 1987) distributed over the
entire gene whereas by far the longest one with a length of 789 bp can be found in
the region of the second exon or in the first translated exon respectively (GC content
of 50 over 200 bp)
Mutation and haplotype analysis
All coding exons with flanking intronic regions of MYH9 could be amplified from the
examined 16 dogs and the sequences were compared to the Boxer genome
assembly 21 of the NCBI Gen Bank (Genbank Acc No NW_1398701)
The search for sequence variations within the MYH9 gene revealed a total of 22
SNPs shown in Table 3 Most of the polymorphisms were found in the flanking
regions of exons only 3 were within exons Only five out of the observed 22 SNPs
were polymorphic in all three examined families
None of the observed polymorphism did alter the predicted amino acid sequence of
MYH9 nor the identified hapltypes showed an association with the CCSD phenotype
Multipoint test statistics for MYH9 alleles identical by descent were close to zero (Z-
mean 035 to -035 Lod score 013 to -007) and not significant (p= 04 to 07)
However the microsatellite MYH9_MS3 (=FH2293) showed a Z-mean of 156
Canine MYH9 gene 71
(p= 006) and a Lod score of 058 (p= 005) indicating linkage The reason for this
result was heterogeneity among these three families For family 1 and 2 the test
statistics indicated linkage (Z-mean 191 with p= 003 Lod score 058 with p= 005)
whereas for familiy 3 the Z-mean and Lod score was -050 (p= 07) and -008 (p=
07)
Thus these silent point mutations found in affected and unaffected Dalmatian dogs
seem not responsible for the CCSD phenotype in these three families
Conclusions
The characterization of the transcript and genomic sequences of canine MYH9 gene
revealed a conserved organization with respect to the human orthologs In general
the gene size in dog is bigger compared to the human sequence due to the
untranlated first exon in the 5rsquo-UTR Several single nucleotide polymorphisms in the
canine MYH9 gene were identified However because of the fact that both animals
with unilateral hearing loss and bilateral hearing animals shared identical
haplotypes these polymorphisms are obviously not associated with CCSD in these
Dalmatian dog families Furthermore the present study revealed no functional
mutations of the complete coding region of MYH9 We can exclude the MYH9 gene
as a candidate gene for congenital sensorineunal deafness in Dalmatian dogs
However the MYH9 gene sequence SNPs and microsatellite markers reported in
this study enhance evaluation of the MYH9 gene in other Dalmatian dog populations
and dog breeds other than Dalmatians
Canine MYH9 gene
72
Table 1 PCR primers for the amplification of all MYH9 exons
Exon Primer Sequence (5rsquo ndash 3rsquo) Product
size (bp)
Annealing temperature
(degC) exon 1 MYH9_Ex1_F GCC TGG TTT GAC AAG TTT TG MYH9_Ex1_R TTC CTC AAG TCC CCT ACT CC 560 58 exon 2 MYH9_Ex2_F AGG ACC CTC ACC CAA CAC MYH9_Ex2_R GCA CAG AGT CTG GAC AAG AAT G 621 58 exon 3 MYH9_Ex3_F CAC CCC TCA ATA CTC AGT GC MYH9_Ex3_R GAG AAG ACT CCT GCC TCT CC 588 58 exon 4 MYH9_Ex4_F GGC AAT GTG TTT GAT TGT ACT G MYH9_Ex4_R TTT CCA AAG CAA CTC AGA AAG 542 58 exon 5 -6 MYH9_Ex56_F ACA GAG CAG GCG TGA CAG MYH9_Ex56_R GAG GAC AAG AGC TGG AAG C 724 58 exon 7 -8 MYH9_Ex78_F AAG CGG AAA GTT GCC TTC MYH9_Ex78_R ATG GCA CAA TGA CCA GTC TC 699 58 exon 9 MYH9_Ex9_F TGG GCA CAG TTT TTG TCT G MYH9_Ex9_R CAC ACA ATC AAG GGC TTT TC 590 58 exon 10 MYH9_Ex10_F GGA GCC AGT TGT CCT AGT AGT G MYH9_Ex10_R GCC AGG TGC CTA TCA CTG 540 58 exon 11 MYH9_Ex11_F TGT TTC GAA GCG TTT TTA CC MYH9_Ex11_R GGT CCC ATT GTT TCT AGC AC 551 58 exon 12 MYH9_Ex12_F TTT CCC AAG CAG CTA GAA AG MYH9_Ex12_R ACT GGG TGG TCT TGA GAC TG 586 58 exon 13 MYH9_Ex13_F CTG CCT TTG GTG ACA AGT G MYH9_Ex13_R GCA AGC CCT AAG TAC AAT GG 548 58 exon 14 MYH9_Ex14_F ATA CCA GGT CAA GGG GTG TC MYH9_Ex14_R TCA CCT GGT TCT AGG TGG AG 524 58 exon 15 MYH9_Ex15_F CGT TTG GAG TTG AT CCT AGC MYH9_Ex15_R AGC CAA GTT TCC TGA CTG TG 566 58 exon 16 MYH9_Ex16_F AGA TAG GCT GCA GTG GTA GC MYH9_Ex16_R CAG TAG CAA ACA GGG CTA GG 568 58 exon 17 MYH9_Ex17_F AAC GGT CCA ATG TGA GTT TC MYH9_Ex17_R ACC AGC TCA CAA CAG TAC CC 559 58 exon 18 MYH9_Ex18_F TGT AGG CTG TGA TTG CAG AG MYH9_Ex18_R CCC TCT CTC AAA CAA ATT GC 586 58 exon 19 MYH9_Ex19_F CCT TGG TGA AGG AAA GTG G MYH9_Ex19_R GCT GTG TGC AGG TTC TGA C 657 58 exon 20 MYH9_Ex20_F AGC AGC ATA GCA CTG AAT CC MYH9_Ex20_R TTC CCA TCT CTC ATG TTC TTG 579 58
Canine MYH9 gene 73
Table 1 (continued)
Exon Primer Sequence (5rsquo ndash 3rsquo) Product
size (bp)
Annealing temperature
(degC) exons 21 MYH9_Ex21_F CTC CTG AGT GTT GGG TTA GC MYH9_Ex21_R CTC TAA CAG GCA GCC CAA G 555 58 exon 22 MYH9_Ex22_F GGA GGA AAC GCA GAA ATC C MYH9_Ex22_R ACG GAC ACC CAG GAA AAC 660 58 exon 23 MYH9_Ex23_F CAG GGG TGC TAG TGA TCT TTC MYH9_Ex23_R AGA GCT GGG CTC AGA CCT AC 630 58 exon 24 MYH9_Ex24_F TCA GGG ATG TGC TTT CTT TC MYH9_Ex24_R CCC CAA GCA CAT CTG TAG TC 563 58 exon 24 MYH9_Ex24_F TCA GGG ATG TGC TTT CTT TC MYH9_Ex24_R CCC CAA GCA CAT CTG TAG TC 563 58 exon 25 -26 MYH9_Ex2526F GGC TGC TCT TCT CTG AAG C MYH9_Ex2526R GGA GTC AGG CTT CCA GTA AAG 851 58 exon 27 -28 MYH9_Ex2728F AGC ACA TTC TGG ATC TGG AG MYH9_Ex2728R GCG ACA GCT GAA TTC CTG 752 58 exon 29 MYH9_Ex29_F CAG CTT CAG GAC ACA CAG G MYH9_Ex29_R CTC TGG GTC TCC CCT GTC 601 58 exon 30 MYH9_Ex30_F GAG GCT TGG TGT ATG TCT CG MYH9_Ex30_R TGG TTC CAT AGA AAC GAT CTG 592 58 exon 31 - 32 MYH9_Ex3132F CAT TCC CCA AGG AGT TTT G MYH9_Ex3132R AAC ACA GCC CAG GGA AAG 770 58 exon 33 MYH9_Ex33_F CCT GGA ACA AGG CTG AGA AC MYH9_Ex33_R TCT CAG AGC AGC TTT GCT TC 600 58 exon 34 MYH9_Ex34_F GGT GTG CTT GGT AAA TCC TG MYH9_Ex34_R CAC AAA GCC TCA TTT CAC G 611 58 exon 34 - 36 MYH9_Ex3536F AGG TCA CGG TCT GGG TTC T MYH9_Ex3536R CAA GGG AGG CTG TTG CAG 401 60 exon 37 MYH9_Ex37_F CTC CCT TGG TTT CCC TCT CT MYH9_Ex37_R CAG GTC AGG AGG GGA CAT 488 60 exon 38 -39 MYH9_Ex3839F AGG CCT AAG GAC AGC TCA TC MYH9_Ex3839R CCA GGG CAC AGA TAG AAC C 545 58 exon 40 MYH9_Ex40_F1 GTC CCT CTA AAG ATG GCT TTG MYH9_Ex40_R1 TGG GGA GGA ACG ACT CTC 844 58 MYH9_Ex40_F2 TGG GTG ATT GGG GAA GAG MYH9_Ex40_R2 CCA GAA GAT GCA CAA AGT AGG 981 58
Canine MYH9 gene
74
Table 2 Exonintron boundaries of the canine MYH9 gene
3rsquo-Splice site Exon 5rsquo-Splice site
Intron phase
Intron size
-20 (exon 1 162 bp) GTTCAGgtgagcgagccccgc
gt30000 bp
-19 +333 gctgtagGTCCTGGCTGTGA (exon 2 352 bp) CGTAAgtggggcctggggcc
0
4922 bp
+334 +490 tctttagACCTATTCAGGCC (exon 3 157 bp) GCAAGgtgatgcctcggcctt
1
13493 bp
+491 +518 gtttcagACCGAGAGGACCA (exon 4 28 bp) TGCACgtaagtgtttctgcc
2
803 bp
+519 +612 tcggcagGGGTGAGTCTGGA (exon 5 94 bp) ACCAGgtgagtgcgggcctt
0
4077 bp
+613 +705 cttgtagGGTGAGTTGGAGA (exon 6 93 bp) GATTTgtgagtagcggccag
0
427 bp
+706 +769 tttttagGGCAAATTCATTC (exon 7 64 bp) GACTTgtatccttggttgat
1
738 bp
+770 +868 tgcaaagATCTTCTGGAGAA (exon 8 99 bp) GAAGAgtgagtagtgagccc
1
343 bp
+869 +1012 ctttcagCTGATCTCCTGTT (exon 9 144 bp) AATGGgtacagagctggccc
1
749 bp
+1013 +1108 cccgaagGCTTGTTGCGAGT (exon 10 96 bp) CACAGgtaacgcctgtggtcc
1
1041 bp
+1109 +1227 atcgcagCTGCCCAGAAAGT (exon 11 119 bp) AACAGgtaggctctctggcc
0
1877 bp
+1228 +1380 accccagGCTGACTTTGCCA (exon 12 153 bp) TTGATgtgagctcctctcct
0
922 bp
+1381 +1554 cctgcagCTGAACTCCTTCG (exon 13 174 bp) AGCCAgtaagagccagacct
0
1801 bp
+1555 +1728 ttcccagGCAGGGCCCCCTG (exon 14 174 bp) GCAAGgtgaggcaagcacgt
0
2049 bp
+1729 +1843 cttgcagGTGGACTACAAAG (exon 15 115 bp) GGATGgtacgtgccccctgg
1
1877 bp
+1844 +2037 tttgcagTGGACCGCATCAT (exon 16 194 bp) AGAAGgtgcgggtgctgggc
0
343 bp
+2038 +2159 tctccagGCTGGCAAACTGG (exon 17 122 bp) CAGAGgtgagcggggcctgt
1
835 bp
+2160 +2229 ccctcagATATGAGATCTTG (exon 18 70 bp) TCATGgtgagtagagctgct
2
1418 bp
+2230 +2390 cgtctagATCAAAGCCCTGG (exon 19 161 bp) AGGAAgtgagttcctaaagg
1
851 bp
+2391 +2499 tccccagGGCCTTTGCCAAG (exon 20 109 bp) CCAAGgtgggttctggaagg
2
1428 bp
+2500 +2631 cttctagGTCAAGCCACTGC (exon 21 132 bp) CCCAGgtaggtgtcagcagg
2
398 bp
+2632 +2838 ttggcagCTCATGGCAGAGA (exon 22 207 bp) TCCAAgtgagccccttccct
2
488 bp
Canine MYH9 gene 75
Table 2 (continued)
3rsquo-Splice site Exon 5rsquo-Splice site
Intron phase
Intron size
+2839 +2976 atggtagGAACTGGAGGAGC (exon 23 138 bp) CCAAGgtgaggcccgtggtg
2
971 bp
+2977 +3100 cccccagGAGAAGAAGCTTC (exon 24 124 bp) GGAAGgtaagagggtaacag
0
1603 bp
+3101 +3272 tcctcagAGCGCCTCCGAAG (exon 25 172 bp) GCCAGgtgagggcctcgctt
1
719 bp
+3273 +3485 ggcagagTGGAGGAGGAAGC (exon 26 213 bp) CTCAGgtgtgccatgcggcc
1
270 bp
+3486 +3630 tctccagGTCCAAACGCGAA (exon 27 145 bp) AACGGgtgcgtgggctcccc
2
480 bp
+3631 +3837 ccaccagGTGAAAGCCAACT (exon 28 207 bp) TGCAGgtgaggcctccatgg
2
232 bp
+3838 +3941 gccacagGTTGAGCTGGACA (exon 29 104 bp) ACACAgtaaaggtggcagcg
1
204 bp
+3942 +4094 ggtgtagGAGCTGCTCCAGG (exon 30 153 bp) CCCAGgttcttatcggcatg
1
1083 bp
+4095 +4343 tgcccagGTGACGGACATGA (exon 31 249 bp) ACCAGgtgtgggccccaggc
1
1298 bp
+4344 +4556 tccccagCTACTGGCCGAGG (exon 32 213 bp) AGAGCgtgagtaccatgtca
1
150 bp
+4557 +4769 gtggcagGTCCACGAGCTGG (exon 33 213 bp) GACAGgtgtggaggccctcc
1
303 bp
+4770 +4931 gccccagGTGCGGGAGATGG (exon 34 162 bp) TGCAGgtgaggtcggctggg
1
1173 bp
+4932 +5060 accccagGCCCAGATGAAGG (exon 35 129 bp) AGGAGgtgggccgggcctca
1
941 bp
+5061 +5149 atggcagGAACTGGCAGCTG (exon 36 89 bp) AAGGGgtgagccgtggcgtg
0
92 bp
+5150 +5273 cacccagAGCTCTGGCATTG (exon 37 124 bp) TGCAGgtgggcggggtcggg
2
224 bp
+5274 +5482 cccacagATCGACCAGATCA (exon 38 209 bp) ACCAAgtaggtgttcctgcc
1
563 bp
+5483 +5591 ctcgcagGGAGCGCCAGGCA (exon 39 109 bp) ACCAGgtgtgtgtgcacacc
2
86 bp
+5592 +5763 tctgcagGCCGACAAGGCAT (exon 40 172 bp) CTCAGgtgagccctgcctcc
0
739 bp
+5764 +7295 tgcaaagATCTTCTGGAGAA (exon 41 1532 bp) CGTCACAATAAAGAGAACCA
Exon sequences are shown in uppercase letters and intron sequences in lowercase
letters Untranslated regions are shown in italics The conserved GTAG exonintron
junctions are shown in boldface type For the last exon the polyadenylation signal is
shown underlined instead of an exonintron junction Position +1 corresponds to the
adenine of the translation initiation codon ATG
Canine MYH9 gene
76
16
TT
TC
G
A
TC
A
A C
A
CT
T
C
GC
G
G
AG
C
C
AG
G
A
CT
C
T
GA
A
G
CC
C
T
CC
T
C
96
96
229
23
7
15
TT
TC
G
A
TC
A
A C
A
CT
T
C
GC
G
G
AG
C
C
AG
G
A
CT
C
T
GA
A
G
CC
C
T
CC
T
C
96
96
229
22
9
14
deaf
T
T T
C
GA
T
C
AA
CA
C
T
TC
G
C
GG
A
G
CC
A
G
GA
C
T
CT
G
A
AG
C
C
CT
C
C
TC
96
96
22
9
229
13
deaf
T
T T
C
GA
T
C
AA
CA
C
T
TC
G
C
GG
A
G
CC
A
G
GA
C
T
CT
G
A
AG
C
C
CT
C
C
TC
96
96
22
9
229
12
deaf
T
T C
C
AA
CC
A
A A
A T
T C
C
CC
G
G
GG
C
C
GG
A
A C
C
CC
A
A G
G
CC
T
T C
C
CC
96
10
0 24
1
229
11
deaf
T
T C
C
AA
CC
A
A A
A T
T C
C
CC
G
G
GG
C
C
GG
A
A C
C
CC
A
A G
G
CC
T
T C
C
CC
96
10
0 24
1
229
10
TT
TC
G
A
TC
A
A C
A
CT
T
C
GC
G
G
AG
C
C
AG
G
A
CT
C
T
GA
A
G
CC
C
T
CC
T
C
96
96
237
22
9
Fam
ily 3
9 T
T C
C
AA
CC
A
A A
A T
T C
C
CC
G
G
GG
C
C
- - CC
C
C
AA
GG
C
C
TT
CC
C
C
96
100
229
24
1
8 T
T C
T
AG
C
T
AA
AA
TT
CC
C
C
GG
G
G
CT
G
G
AA
CC
C
C
AA
GG
C
C
TT
CC
C
C
96
96
225
25
3
7 de
af
TC
C
C
AG
C
T
AT
A
A T
T C
C
CC
G
A
GG
C
T
GG
A
G
CC
C
C
AA
GG
C
T
TC
C
T
CC
96
89
22
1
237
6 de
af
TC
C
C
AG
C
T
AT
A
A T
T C
C
CC
G
A
GG
C
T
GG
A
G
CC
C
C
AA
GG
C
T
TC
C
T
CC
96
89
22
1
237 Fa
mily
2
5 T
C
CC
A
G
CT
A
T
AA
TT
CC
C
C
GA
G
G
CT
G
G
AG
C
C
CC
A
A G
G
CT
T
C
CT
C
C
96
89
221
23
7
4 de
af
TT
CC
A
A C
C
AA
AA
TT
CC
C
C
GG
G
G
CC
G
G
AG
C
C
CC
A
A G
G
CC
T
T C
C
CC
96
96
23
7
257
3 de
af
TC
C
C
AG
C
T
AT
A
A T
T C
C
CC
G
A
GG
C
T
GG
G
A
CC
C
C
AA
GG
C
T
TC
C
T
CC
96
96
23
7
257
2 T
C
CC
A
G
CT
A
T
AA
TT
CC
C
C
GA
G
G
CT
G
G
AA
CC
C
C
AA
GG
C
T
TC
C
T
CC
96
96
23
7
257 Fa
mily
1
1 C
T
CC
G
A
TC
T
A A
A T
T C
C
CC
A
G
GG
T
C
GG
A
G
CC
C
C
AA
GG
T
C
CT
T
C
CC
96
96
25
7
257
Ref
eren
ce
se
quen
ce
NW
_139
870
T C
A T A A T C
C
G
G
C
G
A C
C
A G
C
T C
C
posi
tion1
212
9
1 4
4 2
39
125
1
32
316
3
78
42
218
1
8 1
75
24
13
8
207
2
63
287
9
22
40
59
64
Pos
ition
with
in M
YH
9 In
tron
34
Exo
n 4
Intro
n 14
15
Intro
n 15
16
Intro
n 16
17
Intro
n 18
19
Intro
n 20
21
Intro
n 20
21
Intro
n 25
26
Intro
n 25
26
Intro
n 26
27
Intro
n 27
28
Exo
n 29
In
tron
293
0 In
tron
303
1 E
xon
32
Intro
n 32
33
Intro
n 32
33
Intro
n 34
35
Intro
n 38
39
Intro
n 38
39
Intro
n 38
39
Mic
rosa
tellit
e M
YH
9_M
S2
Mic
rosa
tellit
e M
YH
9_M
S3
(=FH
2293
)
1 N
umbe
ring
refe
rs to
the
posi
tion
of th
e po
lym
orph
ic n
ucle
otid
e w
ithin
the
give
n ex
on
or in
tron
Tabl
e 3
Hap
loty
pes
of th
e ca
nine
MY
H9
gene
in16
Dal
mat
ian
dogs
Canine MYH9 gene 77
Figure 1 Family structure of the analyzed Dalmatian dog families Hearing status was tested by brain auditory evoked response (BAER)
Canine MYH9 gene
78
Figure 2 Alignment of the canine MYH9 protein with orthologous human and murine
MYH9 protein sequences The sequences where derived from GenBank entries with
the accessions NP_002464 (human MYH9) and NP_071855 (mouse MYH9)
Identical residues are indicated by asterisk beneath the alignment while dots and
colons represent similar or very similar amino acids respectively
dog MAQQAADKYLYVDKNFINNPLAQADWAAKKLVWVPSDKSGFEPASLKEEVGEEAIVELVE 60 human MAQQAADKYLYVDKNFINNPLAQADWAAKKLVWVPSDKSGFEPASLKEEVGEEAIVELVE 60 mouse MAQQAADKYLYVDKNFINNPLAQADWAAKKLVWVPSSKNGFEPASLKEEVGEEAIVELVE 60 dog NGKKVKVNKDDIQKMNPPKFSKVEDMAELTCLNEASVLHNLKERYYSGLIYTYSGLFCVV 120 human NGKKVKVNKDDIQKMNPPKFSKVEDMAELTCLNEASVLHNLKERYYSGLIYTYSGLFCVV 120 mouse NGKKVKVNKDDIQKMNPPKFSKVEDMAELTCLNEASVLHNLKERYYSGLIYTYSGLFCVV 120 dog INPYKNLPIYSEEIVEMYKGKKRHEMPPHIYAITDTAYRSMMQDREDQSILCTGESGAGK 180 human INPYKNLPIYSEEIVEMYKGKKRHEMPPHIYAITDTAYRSMMQDREDQSILCTGESGAGK 180 mouse INPYKNLPIYSEEIVEMYKGKKRHEMPPHIYAITDTAYRSMMQDREDQSILCTGESGAGK 180 dog TENTKKVIQYLAHVASSHKSKKDQGELERQLLQANPILEAFGNAKTVKNDNSSRFGKFIR 240 human TENTKKVIQYLAYVASSHKSKKDQGELERQLLQANPILEAFGNAKTVKNDNSSRFGKFIR 240 mouse TENTKKVIQYLAHVASSHKSKKDQGELERQLLQANPILEAFGNAKTVKNDNSSRFGKFIR 240 dog INFDVNGYIVGANIETYLLEKSRAIRQAKEERTFHIFYYLLSGAGEHLKTDLLLEPYNKY 300 human INFDVNGYIVGANIETYLLEKSRAIRQAKEERTFHIFYYLLSGAGEHLKTDLLLEPYNKY 300 mouse INFDVNGYIVGANIETYLLEKSRAIRQAKEERTFHIFYYLLSGAGEHLKTDLLLEPYNKY 300 dog RFLSNGHVTIPGQQDKDMFQETMEAMRIMGIPEEEQMGLLRVISGVLQLGNIVFKKERNT 360 human RFLSNGHVTIPGQQDKDMFQETMEAMRIMGIPEEEQMGLLRVISGVLQLGNIVFKKERNT 360 mouse RFLSNGHVTIPGQQDKDMFQETMEAMRIMGIPEDEQMGLLRVISGVLQLGNIAFKKERNT 360 dog DQASMPDNTGNASAQKVSHLLGINVTDFTRGILTPRIKVGRDYVQKAQTKEQADFAIEAL 420 human DQASMPDNT---AAQKVSHLLGINVTDFTRGILTPRIKVGRDYVQKAQTKEQADFAIEAL 417 mouse DQASMPDNT---AAQKVSHLLGINVTDFTRGILTPRIKVGRDYVQKAQTKEQADFAIEAL 417 dog AKATYERMFRWLVLRINKALDKTKRQGASFIGILDIAGFEIFDLNSFEQLCINYTNEKLQ 480 human AKATYERMFRWLVLRINKALDKTKRQGASFIGILDIAGFEIFDLNSFEQLCINYTNEKLQ 477 mouse AKATYERMFRWLVLRINKALDKTKRQGASFIGILDIAGFEIFDLNSFEQLCINYTNEKLQ 477 dog QLFNHTMFILEQEEYQREGIEWNFIDFGLDLQPCIDLIEKPAGPPGILALLDEECWFPKA 540 human QLFNHTMFILEQEEYQREGIEWNFIDFGLDLQPCIDLIEKPAGPPGILALLDEECWFPKA 537 mouse QLFNHTMFILEQEEYQREGIEWNFIDFGLDLQPCIDLIEKPAGPPGILALLDEECWFPKA 537 dog TDKSFVEKVVQEQGTHPKFQKPKQLKDKADFCIIHYAGKVDYKADEWLMKNMDPLNDNIA 600 human TDKSFVEKVMQEQGTHPKFQKPKQLKDKADFCIIHYAGKVDYKADEWLMKNMDPLNDNIA 597 mouse TDKSFVEKVVQEQGTHPKFQKPKQLKDKADFCIIHYAGKVDYKADEWLMKNMDPLNDNIA 597
Canine MYH9 gene 79
dog TLLHQSSDKFVSELWKDVDRIIGLDQVAGMSETALPGAFKTRKGMFRTVGQLYKEQLAKL 660 human TLLHQSSDKFVSELWKDVDRIIGLDQVAGMSETALPGAFKTRKGMFRTVGQLYKEQLAKL 657 mouse TLLHQSSDKFVSELWKDVDRIIGLDQVAGMSETALPGAFKTRKGMFRTVGQLYKEQLAKL 657 dog MATLRNTNPNFVRCIIPNHEKKAGKLDPHLVLDQLRCNGVLEGIRICRQGFPNRVVFQEF 720 human MATLRNTNPNFVRCIIPNHEKKAGKLDPHLVLDQLRCNGVLEGIRICRQGFPNRVVFQEF 717 mouse MATLRNTNPNFVRCIIPNHEKKAGKLDPHLVLDQLRCNGVLEGIRICRQGFPNRVVFQEF 717 dog RQRYEILTPNSIPKGFMDGKQACVLMIKALELDSNLYRIGQSKVFFRAGVLAHLEEERDL 780 human RQRYEILTPNSIPKGFMDGKQACVLMIKALELDSNLYRIGQSKVFFRAGVLAHLEEERDL 777 mouse RQRYEILTPNSIPKGFMDGKQACVLMIKALELDSNLYRIGQSKVFFRAGVLAHLEEERDL 777 dog KITDVIIGFQACCRGYLARKAFAKRQQQLTAMKVLQRNCAAYLKLRNWQWWRLFTKVKPL 840 human KITDVIIGFQACCRGYLARKAFAKRQQQLTAMKVLQRNCAAYLKLRNWQWWRLFTKVKPL 837 mouse KITDVIIGFQACCRGYLARKAFAKRQQQLTAMKVLQRNCAAYLRLRNWQWWRLFTKVKPL 837 dog LQVSRQEEEMMAKEEELVKVREKQLAAENRLTEMETLQSQLMAEKLQLQEQLQAETELCA 900 human LQVSRQEEEMMAKEEELVKVREKQLAAENRLTEMETLQSQLMAEKLQLQEQLQAETELCA 897 mouse LNSIRHEDELLAKEAELTKVREKHLAAENRLTEMETMQSQLMAEKLQLQEQLQAETELCA 897 dog EAEELRARLTAKKQELEEICHDLEARVEEEEERCQHLQAEKKKMQQNIQELEEQLEEEES 960 human EAEELRARLTAKKQELEEICHDLEARVEEEEERCQHLQAEKKKMQQNIQELEEQLEEEES 957 mouse EAEELRARLTAKKQELEEICHDLEARVEEEEERCQYLQAEKKKMQQNIQELEEQLEEEES 957 dog ARQKLQLEKVTTEAKLKKLEEDQIIMEDQNCKLAKEKKLLEDRIAEFTTNLMEEEEKSKS 1020 human ARQKLQLEKVTTEAKLKKLEEEQIILEDQNCKLAKEKKLLEDRIAEFTTNLTEEEEKSKS 1017 mouse ARQKLQLEKVTTEAKLKKLEEDQIIMEDQNCKLAKEKKLLEDRVAEFTTNLMEEEEKSKS 1017 dog LAKLKNKHEAMITDLEERLRREEKQRQELEKTRRKLEGDSTDLNDQIAELQAQIAELKMQ 1080 human LAKLKNKHEAMITDLEERLRREEKQRQELEKTRRKLEGDSTDLSDQIAELQAQIAELKMQ 1077 mouse LAKLKNKHEAMITDLEERLRREEKQRQELEKTRRKLEGDSTDLSDQIAELQAQIAELKMQ 1077 dog LAKKEEELQAALARVEEEATQKNMALKKIRELESQISELQEDLESERASRNKAEKQKRDL 1140 human LAKKEEELQAALARVEEEAAQKNMALKKIRELESQISELQEDLESERASRNKAEKQKRDL 1137 mouse LAKKEEELQAALARVEEEAAQKNMALKKIRELETQISELQEDLESERASRNKAEKQKRDL 1137 dog GEELEALKTELEDTLDSTAAQQELRSKREQEVNILKKTLEEEARTHEAQIQEMRQKHSQA 1200 human GEELEALKTELEDTLDSTAAQQELRSKREQEVNILKKTLEEEAKTHEAQIQEMRQKHSQA 1197 mouse GEELEALKTELEDTLDSTAAQQELRSKREQEVSILKKTLEDEAKTHEAQIQEMRQKHSQA 1197 dog VEELAEQLEQTKRVKANLEKAKQTLENERGELANEVKVLQQGKGDSEHKRKKAEAQLQEL 1260 human VEELAEQLEQTKRVKANLEKAKQTLENERGELANEVKVLLQGKGDSEHKRKKVEAQLQEL 1257 mouse VEELADQLEQTKRVKATLEKAKQTLENERGELANEVKALLQGKGDSEHKRKKVEAQLQEL 1257 dog QVKFTEGERVRTELADKVTKLQVELDNVMGLLTQSDSKSSKLTKDFSALESQLQDTQELL 1320 human QVKFNEGERVRTELADKVTKLQVELDNVTGLLSQSDSKSSKLTKDFSALESQLQDTQELL 1317 mouse QVKFSEGERVRTELADKVTKLQVELDSVTGLLSQSDSKSSKLTKDFSALESQLQDTQELL 1317
Canine MYH9 gene
80
dog QEENRQKLSLSTKLKQMEDEKNSFKEQLEEEEEAKRNLEKQIATLHAQVTDMKKKMEDGV 1380 human QEENRQKLSLSTKLKQVEDEKNSFREQLEEEEEAKHNLEKQIATLHAQVADMKKKMEDSV 1377 mouse QEENRQKLSLSTKLKQMEDEKNSFREQLEEEEEAKRNLEKQIATLHAQVTDMKKKMEDGV 1377 dog GCLETAEEAKRKLQKDLEGLGQRYEEKVAAYDKLEKTKTRLQQELDDLLVDLDHQRQTAS 1440 human GCLETAEEVKRKLQKDLEGLSQRHEEKVAAYDKLEKTKTRLQQELDDLLVDLDHQRQSAC 1437 mouse GCLETAEEAKRRLQKDLEGLSQRLEEKVAAYDKLEKTKTRLQQELDDLLVDLDHQRQSVS 1437 dog NLEKKQKKFDQLLAEEKTISAKYAEERDRAEAEAREKETKALSLARALEEAMEQKAELER 1500 human NLEKKQKKFDQLLAEEKTISAKYAEERDRAEAEAREKETKALSLARALEEAMEQKAELER 1497 mouse NLEKKQKKFDQLLAEEKTISAKYAEERDRAEAEAREKETKALSLARALEEAMEQKAELER 1497 dog LNKQFRTEMEDLMSSKDDVGKSVHELEKSKRALEQQVEEMKTQLEELEDELQATEDAKLR 1560 human LNKQFRTEMEDLMSSKDDVGKSVHELEKSKRALEQQVEEMKTQLEELEDELQATEDAKLR 1557 mouse LNKQFRTEMEDLMSSKDDVGKSVHELEKSKRALEQQVEEMKTQLEELEDELQATEDAKLR 1557 dog LEVNLQAMKAQFERDLQGRDEQSEEKKKQLVRQVREMEAELEDEKKQRSMAVAARKKLEM 1620 human LEVNLQAMKAQFERDLQGRDEQSEEKKKQLVRQVREMEAELEDERKQRSMAVAARKKLEM 1617 mouse LEVNLQAMKAQFERDLQGRDEQSEEKKKQLVRQVREMEAELEDERKQRSMAMAARKKLEM 1617 dog DLKDLEAHIDSANKNRDEAIKQLRKLQAQMKDCVRELDDTRASREEILAQAKENEKKMKS 1680 human DLKDLEAHIDSANKNRDEAIKQLRKLQAQMKDCMRELDDTRASREEILAQAKENEKKLKS 1677 mouse DLKDLEAHIDTANKNREEAIKQLRKLQAQMKDCMRELDDTRASREEILAQAKENEKKLKS 1677 dog MEAEMIQLQEELAAAERAKRQAQQERDELADEIANSSGKGALALEEKRRLEARIAQLEEE 1740 human MEAEMIQLQEELAAAERAKRQAQQERDELADEIANSSGKGALALEEKRRLEARIAQLEEE 1737 mouse MEAEMIQLQEELAAAERAKRQAQQERDELADEIANSSGKGALALEEKRRLEARIALLEEE 1737 dog LEEEQGNTELVNDRLKKANLQIDQINTDLNLERSHAQKNENARQQLERQNKELKVKLQEM 1800 human LEEEQGNTELINDRLKKANLQIDQINTDLNLERSHAQKNENARQQLERQNKELKVKLQEM 1797 mouse LEEEQGNTELINDRLKKANLQIDQINTDLNLERSHAQKNENARQQLERQNKELKAKLQEM 1797 dog EGTVKSKYKASITALEAKIAQLEEQLDNETKERQAACKQVRRAEKKLKDVLLQVDDERRN 1860 human EGTVKSKYKASITALEAKIAQLEEQLDNETKERQAACKQVRRTEKKLKDVLLQVDDERRN 1857 mouse ESAVKSKYKASIAALEAKIAQLEEQLDNETKERQAASKQVRRTEKKLKDVLLQVEDERRN 1857 dog AEQFKDQADKASTRLKQLKRQLEEAEEEAQRANASRRKLQRELEDATETADAMNREVSSL 1920 human AEQYKDQADKASTRLKQLKRQLEEAEEEAQRANASRRKLQRELEDATETADAMNREVSSL 1917 mouse AEQFKDQADKASTRLKQLKRQLEEAEEEAQRANASRRKLQRELEDATETADAMNREVSSL 1917 dog KNKLRRGDLPFVVPRRVARKGAGDCSDEEVDGKADGAEAKAAE 1963 human KNKLRRGDLPFVVPRRMARKGAGDGSDEEVDGKADGAEAKPAE 1960 mouse KNKLRRGDLPFVVTRRIVRKGTGDCSDEEVDGKADGADAKAAE 1960
Chapter 6
Identification of a 5 Mb region on canine chromosome 10
harbouring a causative gene responsible for congenital sensorineural deafness
in German Dalmatian dogs
Fine mapping of CFA10 83
Identification of a 5 Mb region on canine chromosome 10 harbouring a causative gene responsible for congenital sensorineural deafness in German Dalmatian dogs
Abstract
In the present study we evaluated whether the canine chromosome (CFA) 10
harbours a gene responsible for canine congenital sensorineural deafness (CCSD) in
Dalmatian dogs Altogether 27 microsatellite markers covering the whole CFA10
were genotyped in 176 Dalmatian dogs with frequent occurrence of CCSD
Significant linkage between the deafness phenotype and microsatellites located in a
region of 36 Mb to 48 Mb on CFA10 was found In order to refine the location of the
causative canine congenital sensorineural deafness (CCSD) gene we used data
deposited in the NCBI to search for single nucleotide polymorphisms (SNPs) in the
intronic sequences of the canine genes located on CFA10 in this region We
characterized 26 SNPs and used them for non-parametric linkage and association
analyses with CCSD of altogether 34 Dalmatian dogs from five full-sib families We
could find significant linkage to SNPs located in the region spanning 39 Mb to 44 Mb
and significant haplotype-trait association for SNPs in this region These results
enforce further evaluation of this 5 Mb region with the aim to detect the gene
responsible for CCSD in Dalmatian dogs
Introduction
Of the identified genes responsible for different forms of sensorineural non-syndromic
deafness in humans Rak et al (2003) and co-workers (Droumlgemuumlller et al 2002
Kuiper et al 2002 Rak et al 2002a 2002b 2003) considered 24 genes as
candidates for sensorineural deafness in dogs among them the MYH9 gene on
CFA10 Subsequently Rak (2003) developed a set of microsatellite markers for the
respective 24 candidate genes
As described in chapter 3 we could show significant linkage of CCSD with the MYH9
associated microsaellite MYH9_MS3 (=FH2293) in different German Dalmatian dog
Fine mapping of CFA10
84
families However as described in Chapter 5 we already excluded MYH9 for being
responsible for the CCSD phenotype in German Dalmatian dog families segregating
for CCSD by comparative sequencing of genomic sequences from deaf and normal
hearing Dalmatian dogs and additionally by sequencing of full length canine cDNA
The reason for the MYH9_MS3 (=FH2293) indicating linkage may be caused by a
closely linked gene involved in CCSD Thus the objective of the present study was to
perform a scan of canine chromosome 10 using microsatellite markers and single
nucleotide polymorphisms (SNPs) in order to evaluate whether CFA10 harbours a
gene responsible for the deafness phenotype in Dalmatian dogs Additionally we
analyzed the association of the CCSD phenotype with a large number of newly
developed SNPs located in the genomic deafness region on CFA10
Material and methods Pedigree material
For the linkage analysis of the microsatellite marker on CFA10 we used DNA from
altogether 176 animals belonging to 22 full-sib families and one large paternal half-
sib family of German Dalmatian dogs All families were segregating for CCSD The
genotyped families included all the affected dogs (unilaterally and bilaterally deaf)
their parents if available and one to four unaffected full-sibs At least two of the full
sibs of each family were unilaterally deaf The phenotype of the affected animals had
been confirmed by brainstem auditory evoked response (BAER) that detects
electrical activity in the cochlea and auditory pathways in the brain
Screening for SNPs was performed by comparative sequencing of DNA from parents
of five families with significant linkage of microsatellites located in the region
spanning 36 Mb to 48 Mb to CCSD For the linkage analysis concerning the SNPs we
then used blood samples from 34 Dalmatian dogs consisting of the progeny and their
parents of the abovementioned five full-sib families of Dalmatian dogs with frequent
occurrence of CCSD The families consisted of five to nine individuals and their
parents
Microsatellite marker analysis
We used 27 microsatellite marker derived from the NCBI database
(httpwebncbinlmnihgov) to cover CFA10 completely (Appendix Table 1)
Fine mapping of CFA10 85
PCR reactions for microsatellites were carried out in 12 microl reaction mixtures
containing 2 microl genomic DNA 12 microl 10x PCR buffer (Qbiogene Heidelberg
Germany) 024 microl dimethyl sulfoxide (DMSO) 02 microl dNTPs (100 microM) 01 microl Taq
Polymerase (5Umicrol) (Qbiogene) 06 microl (10 microM) 5rsquo-IRD700 or IRD800 labelled forward
primer 06 microl (10 microM) unlabelled reverse primer The PCR reactions were carried out
in MJ Research thermocyclers with the following program 4 min at 94 degC followed
by 32 cycles of 30 sec at 94degC 30 sec at maximum annealing temperature and a
final extension of 45 sec at 72degC PCR-products were diluted with formamide loading
buffer in ratios from 110 to 140 and then size-fractionated by gel electrophoresis on
automated LI-COR 42004300 sequencers (LI-COR inc Lincoln NE USA) using
denaturing 4 and 6 polyacrylamide gels (RotiphoreseregGel 40 Roth Karlsruhe
Germany)
Development of single nucleotide polymorphisms (SNPs)
We developed SNPs for the region between 36 Mb to 48 Mb on CFA10 previously
proven to be linked to the CCSD phenotype The SNPs for this region were derived
from one 5294 Mb large whole genome shotgun sequence (NW_876251) deposited
in the current dog genome assembly (Boxer genome assembly 21) of the NCBI
GenBank The canine genomic sequences and mRNA of the genes that were used
for the analysis were also derived from sequences deposited in the current dog
genome assembly (Boxer genome assembly 21) of the NCBI GenBank (Table 1)
In total 72 primer pairs were designed most of them located intragenic in intronic
sequences or alternatively in flanking regions of the 5rsquo- or 3rsquondashends of the respective
gene yielding products with a length of around 600 bp PCR primers were developed
with the Primer3 program (httpfrodowimiteducgi-binprimer3primer3_wwwcgi)
The PCR reactions were performed in a total of 50 microl containing 125 microM dNTPs 25
pmol of each primer the reaction buffer supplied by the manufacturer (Qiagen
Hilden Germany) and 1 U Taq polymerase After a 4 min initial denaturation at
95degC 35 cycles of 30 sec at 94degC 45 sec at 58degC and 80 sec at 72degC were
performed in a MJ Research thermocycler (Biozym Hessisch Oldendorf Germany)
The obtained PCR products were directly sequenced with the DYEnamic ET
Terminator kit (AmershamBiosciences Freiburg Germany) and a MegaBACE 1000
capillary sequencer (AmershamBiosciences) using the PCR primers as sequencing
primers Sequence data were analyzed with Sequencher 42
Fine mapping of CFA10
86
(GeneCodes Ann Arbor MI USA) If a heterozygous SNP was found for one or both
parents all progeny of the respective families were analyzed for that SNP
We developed altogether 26 SNPs with 20 SNPs located intragenic in intronic
sequences and 6 SNPs located very closely at the 5rsquo- or 3rsquo-ends respectively with
one to four SNPs per gene (Table 3)
The newly identified 26 SNPs are shown in Tabele 2 Only SNPs are listed and
chosen for linkage analyses that were heterozygous for one or both parents of at
least two of the five families Of all SNPs only one was heterozygous in all families
(Table 3) The most frequent form of SNPs with a frequency of 423 was the AG
transition motif The scarcest one with a frequency of 385 was the CG and the
AC transversion motif respectively
Linkage analysis
Multipoint non-parametric linkage and haplotype analysis were performed using the
MERLIN software version 0102 (multipoint engine for rapid likelihood inference
Center for Statistical Genetics University of Michigan MI USA Abecasis et al
2002) The test statistics Z-mean and Lod score were used to test for the proportion
of alleles shared by affected individuals identical by descent (IBD) for the considered
marker loci
In a first step linkage analysis was performed regarding the 27 marker covering the
whole CFA10 In a second step a linkage analysis was performed including the newly
added SNPs spanning the region 36 Mb to 48 Mb on CFA10
The observed heterozygosity (HET) and the polymorphism information content (PIC)
were calculated using the software package SASGenetics (Statistical Analysis
System Version 913 SAS Institute Inc Cary NC USA 2005)
Linkage disequilibrium (LD) and association of haplotypes with CCSD were tested
using the procedures CASECONTROL and HAPLOTYPE of SASGenetics
(Statistical Analysis System version 913 Cary NC USA)
Results
A linkage analysis was at first carried out for the 176 animals that were analyzed with
27 microsatellite markers covering the whole CFA10
Fine mapping of CFA10 87
The microsatellite FH3302 positioned at 3887 Mb showed the highest Zmean with a
value of 234 (p= 001) and a Lod score of 121 (p= 0009) at 3887 Mb A Zmean
value of 217 (p= 0015) and a Lod score of 12 (p= 0009) was reached for the
microsatellite REN181G20 positioned at 3936 Mb indicating linkage but all Zmean
values and error probabilities of eight microsatellite markers in the interval from 3666
Mb up to 488 Mb were almost as high Consequently we screened the region
spanning from 36 Mb to 48 Mb for SNPs Out of the 23 analyzed Dalmatian dog
families five full-sib families were chosen to screen for SNPs because of their
significant contribution to the test statistics
Twenty-six new SNP markers covering the region of 36 Mb to 48 Mb equally were
developed and added to the linkage analysis These SNPs had PIC values ranging
from 013 to 037
Figure 1 shows the Zmeans and LOD scores for all 27 microsatellite markers on
CFA10 harbouring a genomic deafness region Figure 2 shows the corresponding p-
values Adding the SNPs in the interval spanning 36 Mb to 48 Mb we could narrow
the significant region down to 5 Mb spanning 39 Mb to 44 Mb (Table 4 Figures 3 and
4)
Haplotype-trait association test statistics for the SNPs 16-23 located in the interval
from 39 Mb to 44 Mb were significant However the marker-trait association test
failed the 5 threshold of the error probability (p= 007) but was lowest for all
possible haplotype-trait combinations The χ2ndashtests of the procedure
CASECONTROL were not significant indicating that a SNP for the causative
deafness gene was not yet found
Discussion
We used 27 microsatellite marker and 26 newly developed SNP markers with the
intention to create a dense map for linkage analysis of CFA10 especially the region
spanning 36 Mb to 48 Mb proven to be linked to the CCSD phenotype All SNP
markers were chosen due to their heterozygosity in one or both parents of at least
two families
The significant Zmeans on CFA10 reported for the chromosome scan using only
microsatellites was confirmed by adding the SNP markers Furthermore with the use
of SNPs the apparently linked region from 36 Mb to 48 Mb could be narrowed down
Fine mapping of CFA10
88
to 5 Mb spanning 39 Mb to 44 Mb For this reason the use of SNPs in addition to
informative microsatellites appears highly recommendable
The identified CCSD region spanning 5 Mb might be participating in the development
of CCSD in the analyzed Dalmatian dog families
However genes that are known to be involved in the hearing process or known to be
expressed in the inner ear are not annotated at the syntenic human region on homo
sapiens autosome (HSA) 2 spanning from 100 Mb to 108 Mb Therefore a well-
defined candidate gene for canine hereditary deafness in the abovementioned 5 Mb
does not exist This means that for all genes in this region informative SNPs have to
be developed and tested for linkage disequilibrium and association with CCSD
Despite the remarkable progress that has been made in the past few years in
identifying deafness genes in man and mouse it is suspected that only one-third of all
deafness causing genes have been identified so far Therefore more SNPs have to
be developed within the identified region on CFA10 to localize the deafness causing
gene or to find unambiguously associated SNP markers which could be used for a
population-wide genetic test for CCSD
Fine mapping of CFA10 89
Table 1 Canine genes where gene-associated SNPs could be developed with their
exact location on CFA10 and their corresponding accession numbers (Acc No)
Gene symbol Gene description Position in
Mb
Acc No canine genomic sequence
Acc No canine mRNA
LOC474535 similar to hypothetical protein MGC40397 36493650 NC_006592 XM_531764
LOC474536 similar to KM-102-derived reductase-like factor
37023714 NC_006592 XM_531765
LOC610953 similar to NHP2-like protein 1 37293729 NC_006592 XM_848546
LOC481302 similar to C2orf26 protein 37363737 NC_006592 XM_848552
LOC610991 hypothetical protein LOC610991 38103820 NC_006592 XM_848591
LOC611007 similar to eukaryotic translation elongation factor 1 alpha 2
38353835 NC_006592 XM_848614
LOC474541 similar to GRIP coiled-coil protein GCC185 isoform a
38503857 NC_006592 XM_531770
LOC474542 similar to Sulfotransferase 1C2 38613862 NC_006592 XM_531771
LOC481308 similar to Keratin type I cytoskeletal 18 38653865 NC_006592 XM_538429
LOC474543 similar to Sulfotransferase K1 38683870 NC_006592 XM_857994
LOC609217 similar to family with sequence similarity 32 member A like
39453945 NC_006592 XM_858065
LOC611115 similar to D-PCa-2 protein isoform c 39843984 NC_006592 XM_848756
LOC481325 similar to ubiquitin-conjugating enzyme E2C
42564272 NC_006592 XM_538446
LOC481330 similar to Interleukin-1 receptor type II precursor
44084413 NC_006592 XM_538451
LOC611493 hypothetical protein LOC611493 44384440 NC_006592 XM_863243
LOC481337 similar to DNA repair protein REV1
46264637 NC_006592 XM_538458
LOC611728 similar to 3-hydroxyanthranilate 34-dioxygenase
48494852 NC_006592 XM_849433
Predicted gene derived from the dog genome assembly (build 21) that used gene
prediction method GNOMON supported by EST evidence
Fine mapping of CFA10
90
Table 2 The 26 newly developed SNPs for Dalmatian dogs located in the region
spanning 36 Mb to 48 Mb on CFA10 with their corresponding primers the SNP motif
the product size and the annealing temperature
Gene
description SNP
Location
(intron or
5rsquo3rsquo-end)
Primer F1 (5acute -gt 3acute)
SNP motif
Primer R2 (5acute -gt 3acute)
Bp3 AT4
LOC474535
SNP_1
intron
ACCCAAGCCTAACTGCAGAA
ACCCCAGTCT(CG)GCCAGAGCTGTT
590 60
SNP_2 intron TGTGTGCATA(AG)TGTGCTGAGCCT
TCATCTGTTAAAACAGGGGTGAT
LOC474536
SNP_3
intron
CCAGTTAATGATTGTTTCGTTGA
AAGCTGCTTT(AC)CACCCCCATCAG
TCATTCCTGCTGTTGTGCTC
590 60
LOC610953
SNP_4
intron
CTGTCTTGGGGACTGTTTGC
AAGGCAGACG(CT)AATGACTGAGGC
600 60
SNP_5 intron GACTTGAAGC(CT)ACCACAGATACT
GCCATCACGATGAACTCAGA
LOC481302
SNP_6
3rsquo-end
AATTGAGGCCGAAGTCCAAT
CTTTTCCCCA(GT)GCCACCCCTCTG
GAGCACTATTTACGATACAAACAGGA
610 60
LOC610991
SNP_7
intron
CATGCATGATGCCCAGAGTA
CCCAAAGCAC(AG)CTGTGATTTAAT
AGGGCTTCCTGGGAAAAGT
600 60
LOC611007
SNP_8
intron
CAGACCAACAGTGACCCAGA
TAGGCATACC(GT)TCAGTCCTAAAG
GCCTGTTGTGGGCAGAGTAT
480 60
LOC474541
SNP_9
intron
ACTGAGCCAAAGGTGGATTG
AGAGAATAGC(AG)CTGTGTTTTACA
ACCTGCACATCGGGATTTAG
575 60
Fine mapping of CFA10 91
Table 2 (continued)
Gene-
symbol SNP
Location
(intron or
5rsquo3rsquo-end)
Primer F1 (5acute -gt 3acute)
SNP motif
Primer R2 (5acute -gt 3acute)
Bp3 AT4
LOC474542
SNP_10
intron
CTTCCCCAGGAGAGAGTGAC
AATATGATCA(CT)ATTTAAAGAAAT
CTTTTGTCAACATCCCCTTCA
560 60
LOC481308
SNP_11
3rsquo-end
ACCCATTGTCTCTCCAGCAC
CCACATAACT(GT)AGCATCCCTAGC
600 60
TGATGATGTAAGTTGGCCTCA
LOC474543
SNP_12
intron
TTGAAGTTGTGTGAGTAAATGAAAGA
CAATATATAA(AG)CATTTGCTACAA 600 60
SNP_13 intron TTGAGATGAA(AT)ATAATAGAGCTG
SNP_14 intron CGATTTTCCA(AG)GACATGGAGATG
SNP_15 intron ACTGAACCAA(AG)TGTGCAAAGCAT
GGAAACCATGCAGTCTTTGG
LOC609217
SNP_16
5rsquo-end
TGGCCTCATTTTCCAGTATG
GAAGGAGTTA(CT)ACAGTGAAGATA
580 60
SNP_17 5rsquo-end AAGTGATCAA(AG)CCAGTGCATTCT
GGCAATTACCCTGAGTGGTG
LOC611115
SNP_18
3rsquo-end
GGGCTGTCTTAGAGGTGCTG
TGTGGTCTCA(CT)ACACTTCCTGAG
590 60
SNP_19 3rsquo-end ATTGCAAGTG(GT)CCTGTGATGGCC
CTTCTTTGGGCAGGAAAGTG
LOC481325
SNP_20
intron
AAAATGATTGATCGCAAAAGAAA
AATTACTGTA(CT)AACAGTATCAGA
600 60
SNP_21 intron TAACAGTATC(AG)GAATAAAAGTTT
TTCTGTGATTGCACTGACCG
Fine mapping of CFA10
92
Table 2 (continued)
Gene-
symbol SNP
Location
(intron
or 5rsquo3rsquo-
end)
Primer F1 (5acute -gt 3acute)
SNP motif
Primer R2 (5acute -gt 3acute)
Bp3 AT4
LOC481330
SNP_22
intron
GAAAGGCCTGGGTTCAAAA
GGCAGGGAGG(AG)TCACCATCGTTC
AATTTCCCCAAATGCCTCAC
575 60
LOC611493
SNP_23
intron
GCATGAAGGAGCCCTATGTC
CCAAGAGTCC(AT)GCCCAACACCCT
GGAGGGATGGCATTCTATGA
590 60
LOC481337
SNP_24
intron
GGCTGAGGAGATTGTGTTTCA
GCTGATATTT(AG)GCCTTCTGAGAT
620 60
SNP_25 intron AAAGCCAGAC(AG)CTTTGCCATGGT
CAGCTCCCTGTAATGGGAAA
LOC611728
SNP_26
intron
TCCTACTCCCATCACTTCCAA
CCACACTGGG(GT)CCTGGGATGAGG
CACAGCTCCATGTAGGTCCA
620 60
hypothetical gene found in the 2 version 1 of the NCBIs genome annotation 1 Forward 2 Reverse 3 Product size (basepairs) 4 Annealing temperature (degC)
Fine mapping of CFA10 93
Table 3 The 26 newly developed intragenic SNPs for Dalmatian dogs with their
nucleotide polymorphism allele and genotype frequencies observed heterozygosity
(HET) and polymorphism information content (PIC)
SNP Fam1 Nucleotide
polymorphism
Allele
frequencies
Genotype
frequencies2 PIC HET
SNP_1 125 CgtG 042054 3135 036 055
SNP_2 124 AgtG 064039 91013 037 044
SNP_3 2345 AgtC 041049 2156 033 047
SNP_4 345 CgtT 065035 6140 035 067
SNP_5 34 CgtT 065035 490 028 042
SNP_6 345 CgtT 066034 6130 035 039
SNP_7 1345 AgtG 054046 6174 037 053
SNP_8 145 GgtT 075025 01010 029 045
SNP_9 1345 AgtG 052054 6145 037 047
SNP_10 1234 CgtT 057043 7173 037 068
SNP_11 14 GgtT 065035 5120 035 062
SNP_12 15 AgtG 028072 097 030 044
SNP_13 45 AgtT 077023 870 017 021
SNP_14 5 AgtG 036064 052 013 015
SNP_15 134 AgtG 034066 0157 034 059
SNP_16 145 CgtT 030070 3812 033 036
SNP_17 245 AgtG 058042 6113 030 032
SNP_18 123 CgtT 037063 1126 027 035
SNP_19 123 GgtT 045055 3115 030 032
SNP_20 2345 CgtT 032068 1129 029 039
SNP_21 1235 AgtG 063037 883 037 052
SNP_22 1234 AgtG 068032 10102 030 034
SNP_23 12345 AgtT 030070 21313 033 046
SNP_24 124 AgtG 047053 667 033 019
SNP_25 234 AgtG 053047 3132 035 053
SNP_26 234 GgtT 060040 3120 026 0391 Number of families which were informative for the respective SNPs 2 Genotypes are given as number of animals [homozygous for allele 1heterozygous
homozygous for allele 2]
Fine mapping of CFA10
94
Table 4 Linkage analysis for canine chromosome 10 regarding the region spanning
39 Mb to 44 Mb with the significant Zmean LOD score and error probabilities (p-
values)
Marker Location (Mb) Zmean pz-value1 LOD-score pL-value2
SNP_16 39453 262 0004 123 0009
SNP_17 39455 262 0004 123 0009
SNP_18 39840 261 0004 123 0009
SNP_19 39843 261 0004 123 0009
SNP_20 4260 255 0005 118 0010
SNP_21 4270 255 0005 117 0010
SNP_22 4405 317 00008 131 0007
SNP_23 4439 337 00004 134 0007 1 Error probability regarding Zmean 2 Error probability regarding LOD score
Fine mapping of CFA10 95
Figure 1 Zmeans and LOD scores for 27 microsatellite markers on CFA10
harbouring a congenital sensorineural deafness region (number of families 23
number of genotyped dogs 176)
Figure 2 P-values for Zmeans and LOD scores of 27 microsatellite markers on
CFA10 harbouring a congenital sensorineural deafness region (number of families
23 number of genotyped dogs 176)
Fine mapping of CFA10
96
Figure 3 Zmeans and LOD scores of 53 markers on CFA10 harbouring a congenital
sensorineural deafness region (number of families 5 number of genotyped dogs 34)
Figure 4 P-values for Zmeans and LOD scores of 53 markers on CFA10 for the
region between 30 to 60 Mb harbouring a congenital sensorineural deafness region
(number of families 5 number of genotyped dogs 34)
CCSD region (SNP 16-23)
Chapter 7
Analysis of canine chromosome 1 and the Gap junction protein alpha 1 (GJA1) gene
in Dalmatian dogs segregating for congenital sensorineural deafness
CFA1 and the GJA1 gene 99
Analysis of canine chromosome 1 and the Gap junction protein alpha 1 (GJA1) gene in Dalmatian dogs segregating for congenital sensorineural deafness Abstract Two microsatellite markers associated to the GJA1 (alternatively connexin 43) gene
showed significant linkage with canine congenital deafness (CCSD) in a large French
half-sib family (Chapter 3) PCR products of this half-sib family were used to perform
a sequence analysis in order to evaluate whether GJA1 is responsible for CCSD As
the linkage could not be confirmed we used altogether 27 microsatellite markers for
a scan of canine chromosome (CFA) 1 with 23 Dalmatian dog families segregating
for CCSD A non-parametric linkage analysis was performed to see whether
significant test statistics for other genomic regions on CFA1 and for more families can
be shown As a result we could not find linkage to any microsatellite in the analyzed
families
Introduction Rak (2003) considered in total 24 genes as candidates for sensorineural deafness in
dogs among them the Gap junction protein alpha 1 (GJA1) or connexin 43 gene on
canine chromosome (CFA) 1 A marker set of altogether 43 microsatellites were
developed by Rak (2003) among them two microsatellite marker associated to the
GJA1 gene
GJA1 or connexin 43 is a member of the connexin gene family and a component of
gap junctions Mutations in 4 members of the connexin gene family have been shown
to underlie distinct genetic forms of deafness including GJB2 (connexin [CX] 26)
GJB3 (CX31) GJB6 (CX30) and GJB1 (CX32)
Liu et al (2001) reported mutations in GJA1 in association with sensorineural
recessive deafness in man However these mutations have recently been shown to
involve the pseudogene of GJA1 on human chromosome (HSA) 5 and not the
CFA1 and the GJA1 gene
100
GJA1 gene on HSA 6 (WA Paznekas cited a personal communication from the
senior author (W E Nance) of the paper by Liu et al 2001)
In previously performed studies (Chapter 3) one large French Dalmatian dog family
with frequent occurrence of CCSD showed linkage to microsatellites associated to
the GJA1 gene
In this report we performed a mutation analysis of the GJA1 gene sequence to
identify polymorphisms In order to evaluate whether the GJA1 gene is responsible
for congenital sensorineural deafness in Dalmatian dogs we analyzed the
association of the GJA1 haplotypes with the CCSD phenotype Furthermore we
employed 27 microsatellite markers covering the entire CFA1 and used them for a
non-parametric linkage analysis with CCSD in a Dalmatian dog population of 176
animals with frequent occurrence of CCSD
Material and methods Pedigree material
For the linkage analysis of the microsatellite marker we used DNA from altogether
176 animals belonging to 22 full-sib families and one large paternal half-sib family of
German Dalmatian dogs All families were segregating for CCSD The genotyped
families included all affected dogs (unilaterally and bilaterally deaf) their parents if
available and one to four unaffected animals At least two of the full sibs of each
family were unilaterally or bilaterally deaf The phenotype of the affected animals had
been confirmed by brainstem auditory evoked response (BAER) that detects
electrical activity in the cochlea and auditory pathways in the brain
Mutation analysis and screening for SNPs was performed by comparative
sequencing of DNA from 16 animals consisting of the parents and their progenies of
one large half-sib family of French Dalmatian dogs which showed significant linkage
to GJA1-associated microsatellites (Chapter 3)
Sequencing of canine genomic DNA and mutation analysis
The canine GJA1 gene sequence was derived from sequences deposited in the
current dog genome assembly (Boxer genome assembly 21) of the NCBI GenBank
(Genbank Acc No NC_006583) We compared the canine genomic DNA sequence
to canine cDNA fragments in the canine EST database using the
CFA1 and the GJA1 gene 101
BLASTN (Basic Local Alignment Search Tool Nucleotide) program Three canine
ESTs (Acc No DN880776 BU744925 and DN369900) were used to confirm the
assembly of the GJA1 gene
To identify polymorphisms within the canine GJA1 sequence the gene consisting of
one 1251 bp spanning exon were PCR amplified and sequenced from 16 French
Dalmatian dogs Additionally large sequences from the 3acute-end were amplified Primer
pairs were designed yielding products with a length of around 600 bp PCR primers
were developed with the Primer3 program (httpfrodowimiteducgi-
binprimer3primer3_wwwcgi) The PCR reactions were performed in a total of 50 microl
containing 125 microM dNTPs 25 pmol of each primer the reaction buffer supplied by
the manufacturer (Qbiogene Heidelberg Germany) and 1 U Taq polymerase After
a 4 min initial denaturation at 95degC 35 cycles of 30 sec at 94degC 45 sec at 58degC and
80 sec at 72degC were performed in a MJ Research thermocycler (Biozym Hessisch
Oldendorf Germany)
The obtained PCR products were directly sequenced with the DYEnamic ET
Terminator kit (AmershamBiosciences Freiburg Germany) and a MegaBACE 1000
capillary sequencer (AmershamBiosciences) using the PCR primers as sequencing
primers Sequence data were analyzed with Sequencher 42
(GeneCodes Ann Arbor MI USA)
Microsatellite marker analysis
We used 27 microsatellite marker derived from the NCBI database
(httpwebncbinlmnihgov) to cover CFA1 completely (Appendix Table 1)
PCR reactions for microsatellites were carried out in 12 microl reaction mixtures
containing 2 microl genomic DNA 12 microl 10x PCR buffer (Qbiogene) 024 microl dimethyl
sulfoxide (DMSO) 02 microl dNTPs (100 microM) 01 microl Taq Polymerase (5Umicrol)
(Qbiogene) 06 microl (10 microM) 5rsquo-IRD700 or IRD800 labelled forward primer 06 microl (10
microM) unlabelled reverse primer The PCR reactions were carried out in MJ Research
thermocyclers with the following program 4 min at 94 degC followed by 32 cycles of 30
sec at 94degC 30 sec at maximum annealing temperature and a final extension of 45
sec at 72degC PCR-products were diluted with formamide loading buffer in ratios from
110 to 140 and then size-fractionated by gel electrophoresis on automated LI-COR
42004300 sequencers (LI-COR inc Lincoln NE USA) using denaturing 4 and 6
polyacrylamide gels (RotiphoreseregGel 40 Roth Karlsruhe Germany)
CFA1 and the GJA1 gene
102
Linkage and haplotype analysis
Multipoint non-parametric linkage and haplotype analyses were performed using the
MERLIN software version 0102 (multipoint engine for rapid likelihood inference
Center for Statistical Genetics University of Michigan MI USA Abecasis et al
2002) The test statistics Z-mean and Lod score were used to test for the proportion
of alleles shared by affected individuals identical by descent (IBD) for the considered
marker loci
A non-parametric linkage analysis was performed with 27 microsatellite markers in 23
Dalmatian dog families consisting of 176 Dalmatian dogs The results were added to
the linkage analysis performed in Chapter 3
Results and discussion
In previously performed studies (Chapter 3) one French Dalmatian dog family
reached a Zmean value of 295 (plt0002) for GJA1_MS1 and a Zmean value of 286
(plt0002) for GJA1_MS2 indicating linkage Both markers were associated with the
GJA1 gene
By sequence analysis we revealed a total of 3 SNPs found in sequences of the 3acute-
UTR shown in Table 1 None of the polymorphisms were polymorphic in all parents of
the examined French half-sib family Neither of the observed polymorphism did alter
the predicted amino acid sequence of GJA1 nor showed the identified haplotypes an
association with the CCSD phenotype (Figure 1)
There is no recombination of the haplotypes of the GJA1 gene in this family The
paternal and maternal haplotypes as far as they could be estimated were distributed
among the affected progeny closely to the expected proportion of 50 and therefore
no excess of a certain haplotype could be observed in the affected dogs
Thus evaluation of the SNP markers debilitates the linkage to CCSD for the French
half-sib family Because of the fact that both animals with unilateral hearing loss and
bilateral hearing animals shared identical haplotypes these polymorphisms are
obviously not associated with CCSD in these Dalmatian dog families Furthermore
the present study revealed no functional mutations of the complete coding region of
GJA1 It is therefore unlikely that the GJA1 gene is involved in the pathogenesis of
CCSD in the analyzed French Dalmatian dogs The reason for GJA1_MS1 and
GJA1_MS2 indicating linkage may be caused by a closely linked gene involved in
CFA1 and the GJA1 gene 103
CCSD among the half-sib family of French Dalmatian dogs or by a false positive
result of the microsatellite study performed in Chapter 3
To clarify whether significant test statistics for other genomic regions on CFA1 and
for more families can be shown we used 27 microsatellite markers derived from the
NCBI database (httpwebncbinlmnihgov) to cover CFA1 completely A linkage
analysis was carried out after genotyping 176 German Dalmatian dogs with a set of
27 microsatellite markers The results of this linkage analysis were added to the
results of the test statistics for the microsatellites GJA1_MS1 and GJA1_MS2
(Chapter 3)
As a result we could not find linkage to any microsatellite in the analyzed families
(Figure 2 and 3) It is unlikely that the canine chromosome 1 harbours genomic
regions that are involved in the development of CCSD in the analyzed Dalmatian dog
families
With hindsight it was revealed that GJA1 is not responsible for sensorineural non-
syndromic deafness in humans as Liu et al (2001) has published GJA1 is
participating in a human syndrome called oculodentodigital dysplasia (ODDD) that
can be accompanied with hearing impairment (Paznekas et al 2003) But the type of
deafness in human ODDD differs from the typical hearing loss associated with other
connexin mutations because it is conductive rather than sensorineural
As deafness in dogs especially in Dalmatians is almost exclusively caused by
sensorineural non-syndromic forms also known as cochleosaccular degeneration
the GJA1 gene should not be considered as a candidate gene for CCSD anymore
CFA1 and the GJA1 gene
104
Table 1 Three newly developed intragenic SNPs and two microsatellite markers for
Dalmatian dogs in the candidate gene GJA1 with their corresponding primers the
SNP motif the product size and the annealing temperature
SNP
Primer F (5acute -gt 3acute)
SNP motif
Primer R (5acute -gt 3acute)
Product
size (bp)
Annealing
temperatur
GJA1_SNP1+2
CACCTTAGGCGTTCATTTTG
CCGGGGAG(AG)AAAA(AG)AAAAATACTT
TGGCTTGATTCCCTGACTC
650 58
GJA1_SNP3
TCTGAAATGTAATCATGGATGC
CAGAACTTGTAT(AT)CTGTTAAGAG
AATCACACAGGATATAGAGGCTATC
600 58
Microsatellite
marker Primers (forward reverse ) 5acute -gt 3acute
Product
size (bp)
Annealing
temperatur
GJA1_MS1
ATGGCATGAAGAGGATACCG
AGGACAGGTGACGGCTCTAC
134 60
GJA1_MS2
GCTAGTACTCGATTGTGGTC
TCATGGGTTGTGAGATCCAG
190 60
CFA1 and the GJA1 gene 105
Figu
re 1
Hap
loty
pes
of th
e G
JA1
gene
in th
e an
alyz
ed D
alm
atia
n do
g fa
mily
CFA1 and the GJA1 gene
106
Figure 2 Zmeans and LOD scores for 29 microsatellite markers on CFA1 (number of
families 23 number of genotyped dogs 176)
Figure 3 P-values for Zmeans and LOD scores of 29 microsatellite markers on CFA1
(number of families 23 number of genotyped dogs 176)
Chapter 8
Analysis of canine chromosome 31 and the claudin-14 (CLDN14) gene
in Dalmatian dogs segregating for congenital sensorineural deafness
CFA31 and the CLDN14 gene 109
Analysis of canine chromosome 31 and the claudin-14 (CLDN14) gene in Dalmatian dogs segregating for congenital sensorineural deafness Abstract A previously accomplished linkage analysis revealed significant linkage of
microsatellite markers associated with the CLDN14 gene on canine chromosome
(CFA) 31 with the phenotype of canine sensorineural deafness (Chapter 3) The
objective of the present study was to perform a sequence analysis in order to find
single nucleotide polymorphisms (SNPs) for the CLDN14 gene and additionally to
use a set of six microsatellite markers evenly distributed on CFA31 for non-
parametric linkage analysis with the aim to verify the significant test statistics shown
in Chapter 3
Introduction
Of the genes responsible for different forms of sensorineural non-syndromic deafness
in humans Rak (2003) considered 24 genes as candidates for sensorineural
deafness in dogs among them the CLDN14 gene on canine chromosome (CFA) 31 CLDN14 encodes a member of the claudin family which comprises the major
components of tight juncions (TJ) The human CLDN14 gene consists of one
tranlatete exon and six exons in the 5rsquountranlated region Five splice isoformes are
identified so far (Wilcox et al 2001 Wattenhofer et al 2005)
For the compartmentalization of perilymph and endolymph in the inner ear the
leakage of solutes through a paracellular pathway must be prevented by tight
junctions TJ are an intercellular junction found at the most apical region of polarised
epithelial and endothelial cells at which adjacent plasma membranes are joined
tightly together separating apical membranes and basolateral menbranes They are
specialised membrane domains containing branching strands of integral proteins and
create a primary barrier preventing paracellular transport of solutes and restricting
lateral diffusion of membrane lipids and proteins (Schneeberger and Lynch 2004)
CFA31 and the CLDN14 gene
110
The important role of the CLDN14 gene in the TJ of the inner ear was demonstrated
by Wilcox et al (2001) when he identified mutations in the CLDN14 gene responsible
for a hereditary human deafness in families segregating for congenital recessive
deafness (DFNB29)
In previously performed studies one large half-sib family as well as several full-sib
families of German Dalmatian dog indicated linkage to CLDN14 gene-associated
markers (Chapter 3)
A sequence analysis was performed to identify single nucleotide polymorphisms
(SNPs) either intergenic located or alternatively in the flanking 5rsquo- and 3rsquo-Regions
Furthermore we employed microsatellite markers covering CFA31 and used them for
a non-parametric linkage analysis with CCSD in a German Dalmatian dog population
of 176 animals with frequent occurrence of CCSD
Material and methods Pedigree material
For the linkage analysis of the microsatellite marker we used DNA from altogether
176 animals belonging to 22 full-sib families and one large paternal half-sib family of
German Dalmatian dogs All families were segregating for CCSD The genotyped
families included all the affected dogs (unilaterally and bilaterally deaf) their parents
if available and one to four unaffected animals At least two of the full sibs of each
family were unilaterally or bilaterally deaf The phenotype of the affected animals had
been confirmed by brainstem auditory evoked response (BAER) that detects
electrical activity in the cochlea and auditory pathways in the brain
Mutation analysis and screening for SNPs was performed by comparative
sequencing of DNA from 36 animals consisting of the parents and their progenies of
one half-sib family and four full-sib families of Dalmatian dogs which showed
significant linkage to a CLDN14-associated microsatellite (Chapter 3)
Sequencing of canine genomic DNA and development of single nucleotide
polymorphisms (SNPs)
The canine CLDN14 gene sequence was derived from sequences deposited in the
current dog genome assembly (Boxer genome assembly 21) of the NCBI GenBank
(Genbank Acc No NW_876295) by BLAST (Basic Local Alignment Search Tool)
CFA31 and the CLDN14 gene 111
search (httpwwwncbinlmnihgovBLAST) using the human CLDN14 reference
mRNA sequence (Genbank Acc No NM_144492)
We compared the canine genomic DNA sequence to canine cDNA fragments in the
canine EST database using the BLASTN program As no ESTs could be found
human mRNA sequences were used for the localization of the exonintron
boundaries using the mRNA-to-genomic alignment program Spidey
(httpwwwncbinlmnihgovIEBResearchOstellSpideyindexhtml)
We screened exon three for mutations as this exon is the only translated one in man
Additionally we screened large intronic sequences and sequences located in the 5rsquo-
and 3rsquo-UTR of the CLDN14 gene for Polymorphisms For this purpose primer pairs
were designed yielding products with a length of around 600 bp
PCR primers were developed with the Primer3 program (httpfrodowimiteducgi-
binprimer3primer3_wwwcgi) The PCR reactions were performed in a total of 50 microl
containing 125 microM dNTPs 25 pmol of each primer the reaction buffer supplied by
the manufacturer (Qbiogene Heidelberg Germany) and 1 U Taq polymerase After
a 4 min initial denaturation at 95degC 35 cycles of 30 sec at 94degC 45 sec at 58degC and
80 sec at 72degC were performed in a MJ Research thermocycler (Biozym Hessisch
Oldendorf Germany)
The obtained PCR products were directly sequenced with the DYEnamic ET
Terminator kit (AmershamBiosciences Freiburg Germany) and a MegaBACE 1000
capillary sequencer (AmershamBiosciences) using the PCR primers as sequencing
primers Sequence data were analyzed with Sequencher 42 (GeneCodes Ann
Arbor MI USA) The eight newly developed SNPs are shown in Table 1
Microsatellite marker analysis
In total six microsatellite marker were derived from the NCBI database
(httpwebncbinlmnihgov) to cover CFA31 completely (Appendix Table 1)
PCR reactions for microsatellites were carried out in 12 microl reaction mixtures
containing 2 microl genomic DNA 12 microl 10x PCR buffer (Qbiogene) 024 microl dimethyl
sulfoxide (DMSO) 02 microl dNTPs (100 microM) 01 microl Taq Polymerase (5Umicrol)
(Qbiogene) 06 microl (10 microM) 5rsquo-IRD700 or IRD800 labelled forward primer 06 microl (10
microM) unlabelled reverse primer The PCR reactions were carried out in MJ Research
thermocyclers with the following program 4 min at 94 degC followed by 32 cycles of 30
sec at 94degC 30 sec at maximum annealing temperature and a final extension of
CFA31 and the CLDN14 gene
112
45 sec at 72degC PCR-products were diluted with formamide loading buffer in ratios
from 110 to 140 and then size-fractionated by gel electrophoresis on automated LI-
COR 42004300 sequencers (LI-COR inc Lincoln NE USA) using denaturing 4
and 6 polyacrylamide gels (RotiphoreseregGel 40 Roth Karlsruhe Germany)
Linkage and haplotype analysis
Multipoint non-parametric linkage and haplotype analysis were performed using
MERLIN version 0102 (multipoint engine for rapid likelihood inference Center for
Statistical Genetics University of Michigan MI USA Abecasis et al 2002) The test
statistics Z-mean and Lod score were used to test for the proportion of alleles shared
by affected individuals identical by descent (IBD) for the considered marker loci A
linkage analysis was performed with 6 microsatellite markers on 176 Dalmatian dogs
The results were added to the linkage analysis performed in Chapter 3
Results and discussion CLDN14-associted microsatellites were significantly linked with CCSD in a scan of
candidate genes using 24 families with 215 Dalmatian dogs (Chapter 3) Selection of
four full-sib families and one half-sib family with the highest tests statistics lead to a
Zmean value of 383 (plt000007) for the CLDN14 gene-associated marker
CLDN14_MS2
To substantiate the linkage to the CLDN14 gene we searched for sequence
variations within the CLDN14 gene in four full-sib families and one half-sib family with
the highest contribution to the test statistics shown in Chapter 3 Most of the identified
polymorphisms were found in intronic sequences none were within exon three None
of the observed polymorphism did alter the predicted amino acid sequence of exon
three We revealed a total of eight SNPs (Table 1) but only two (SNP_4 and SNP_5)
out of the identified eight SNPs were polymorphic in the examined families Both
unilaterally and bilaterally deaf animals as well as normal hearing animals showed
identical haplotypes for these two polymorphic SNPs and thus no co-segregation with
the deaf Dalmatian dogs could be revealed (Figure 1 and 2) In this study we did not
identify any causal mutations for CCSD in the analyzed Dalmatian dogs Despite this
results a mutation outside of the translated genomic regions analyzed here may exist
that would affect CLDN14 expression
CFA31 and the CLDN14 gene 113
To clarify if other regions on CFA31 are responsible for the CCSD phenotype
additionally six microsatellite markers covering CFA31 were tested in 23 Dalmatian
dog families consisting of 176 individuals A non-parametric linkage analysis was
performed regarding these 23 families The results of this linkage analysis were
added to the results of the test statistics for the CLDN14 gene-associated
microsatellites (Chapter 3)
We could only find significant linkage with CCSD for CLDN14-associted
microsatellites in the abovementioned five families The two microsatellites located
most closely were proximal and distal 4 Mb off the CLDN14 gene REN110K04 with a
Zmean value of -009 (plt05) and FH2712 with a Zmean value of 022 (plt03) not
indicating linkage
It can not be excluded that CLDN14 or genes in its flanking regions are involved in
the development of CCSD in the analyzed Dalmatian dog families Other genes than
the CLDN14 that are known to be involved in the hearing process or known to be
expressed in the inner ear are not annotated at the syntenic human region of homo
sapiens autosome (HSA) 21q223 Therefore despite the CLDN14 gene no other
well-defined candidate gene for canine hereditary deafness exists in the linked
region Despite the remarkable progress that has been made in the past few years in
identifying deafness genes in man and mouse it is suspected that only one-third of all
deafness causing genes have been identified so far and thus it seems possible that
other genes in the flanking region of the CLDN14 gene are involved in the
development of the disease Anyway to clarify the importance of CLDN14 in the
CCSD phenotype more SNPs have to be developed within the CLDN14 gene as well
as in its flanking regions with the aim to find significant linkage disequilibrium of SNP
markers with CCSD
CFA31 and the CLDN14 gene
114
Table 1 The 8 newly developed SNPs for Dalmatian dogs in the CLDN14 gene on
CFA31 with their corresponding primers the SNP motif the product size and the
annealing temperature
SNP
Location
(intron or
5rsquo3rsquo-UTR)
Primer F1 (5acute -gt 3acute)
SNP motif
Primer R2 (5acute -gt 3acute)
Bp3 AT4
CLDN14_SNP1
intron
GACCATATGTTTGTGGCC
CTTCCAGGGAAA(AT)TGTCGTAGCC
CGTCAGGATGTTGGTGCC
580 60
CLDN14_SNP2
inton
GACCATATGTTTGTGGCC
GAAATTGTCGTA(AG)CCCGGCCGCT
CGTCAGGATGTTGGTGCC
580 60
CLDN14_SNP3
3rsquo-UTR
CTGCCTTCAAGGACAACC
CCAGAGGAATAA(CT)ATGATCGTGA
GATGAGTATCAGCCCAGC
550 60
CLDN14_SNP4
3rsquo-UTR
CTGCCTTCAAGGACAACC
ACCACCGCACAC(CT)GTCACAGCTT
GATGAGTATCAGCCCAGC
550 60
CLDN14_SNP5 3rsquo-UTR
CATGCCTTTGTCCCAAACTT
GAGACCCTCTGG(CT)TCCTTTTGGC
GTACCTGTTGCCTGGGTTGT
610 60
CLDN14_SNP6 5rsquo-UTR
CCTTCATCCTTTCTGGTTGA
GCTCACAGTCCC(AC)ATGGGGACAT
GGGGAGCATAATGTGGTCAT
585 60
CLDN14_SNP7 5rsquo-UTR
TGAACTGGTCCCAAGGAAAG
GCACGACCGAGC(CT)CTGGCTTTAC
GGGATGAGAGGGAGGTTTTT
580 60
CLDN14_SNP8 3rsquo-UTR
AATGCCTATCCCTTCTTTGGA
CACGTTACTGTG(AG)ACCTCTCTAC
GCAGGCTTTCCTCAAGTGTC
680 60
1 Forward 2 Reverse 3 Product size (basepairs) 4 Annealing temperature (degC)
CFA31 and the CLDN14 gene 115
Figure 1 Haplotypes of the CLDN14 gene-associated markers SNP_4 and SNP_5 in
the analyzed four Dalmatian dog full-sib families
CFA31 and the CLDN14 gene
116
Figu
re 2
Hap
loty
pes
of th
e C
LDN
14 g
ene-
asso
ciat
ed m
arke
rs S
NP
_4 a
nd S
NP
_5 in
the
anal
yzed
Dal
mat
ian
dog
half-
sib
fam
ily
Chapter 9
General discussion
General discussion 119
General discussion
The candidate gene approach In the first step of this thesis candidate genes for canine congenital sensorineural
deafness (CCSD) in Dalmatian dogs were analyzed by means of microsatellite
markers or alternatively by single nucleotide polymorphisms (SNPs)
The candidate genes for which a set of in total 43 microsatellites was available
included the following 24 genes CDH23 CLDN14 COCH COL11A2 DFNA5
DIAPH1 EDN3 EDNRB EYA4 GJA1 GJB2 GJB6 MITF MYH9 MYO6 MYO7A
MYO15A OTOF PAX3 POU4F3 SLC26A4 SOX10 TECTA and TMPRSS3
(Rak 2003) These genes are known to be involved either in human non-syndromic
deafness or in the human Waardenburg syndrome The Waardenburg syndrome
(WS) manifests with sensorineural deafness and pigmentation defects in iris hair and
skin WS is classified into four types depending on the presence or absence of
additional symptoms which are caused by mutations in the five genes EDN3
EDNRB MITF PAX3 and SOX respectively
For another eight recently identified genes responsible for different forms of human
non-syndromic deafness including TMC1 TMIE USH1C MYH14 MYO3A PRES
WHRN and ESPN linkage and association analyses were performed using newly
developed SNPs
In the last years most projects have exploited canine traits for which either direct
candidate genes could be proposed and evaluated or for which large informative
pedigrees were available to enable linkage mapping to identify candidate regions A
major component of such research efforts comprised the cloning sequencing and
mapping of individual canine homologs of genes either proposed as candidate
genes or expected to be located in candidate regions This was necessary to identify
new informative polymorphisms (eg SNPs microsatellites) for high resolution
mapping of candidate regions and to examine each exon and exonintron boundary
for positional candidates Availability of the second version of the dog genome
assembly (build 21) of the NCBI database shortcuts this effort and increases the
investigators efficiency Now either additional candidate genes for canine congenital
sensorineural deafness can be found directly by its gene symbol in the 21 of the
General discussion
120
NCBIs genome annotation or if a candidate gene is not yet annotated a BLAST
(Basic Local Alignment Search Tool) search against the canine whole genome
shotgun (wgs) sequence resource can be used to obtain the sequence of the canine
genomic contigs containing the human homologous gene and thus intragenic
markers can be developed for subsequent linkage and association analyses
Over the past decade it has become increasingly clear how far structural and
functional homologies at the gene level extend across even distantly related species
Much is known about deafness-causing gene mutations in humans and mice
including the fact that the clinical and histopathological findings are often very similar
to those of deafness in Dalmatian dogs Thus genes responsible for non-syndromic
congenital hereditary deafness in humans seem to be appropriate candidate genes
for CCSD (Rak and Distl 2005) In this thesis we first concentrated on the candidate
gene approach combined with linkage analysis method using affected pedigree
members Once a significant linkage was found only the linked genes with the
required low error probability values were used for further molecular genetic analysis
The method of candidate gene approach using either gene-associated microsatellite
or alternatively SNP markers was applied for in total 32 candidate genes which
comprise nearly all of the identified mutated genes causing non-syndromic hereditary
hearing impairment in humans
Linkage and association analysis In principle linkage and association are totally different phenomena Linkage is a
relation between loci and association is a relation between alleles
Linkage means that a haplotype characterised by microsatellites or SNPs is
significantly more often present in family members with the phenotype under study
than expected by random assortment For construction of haplotypes sets of closely
linked genetic markers on the same chromosome are needed which tend to be
inherited together as they are not likely to be separated by recombination Linkage
creates associations within families but not among unrelated induviduals
Association is a statistical statement about the co-occurrence of alleles or
phenotypes Association analysis can be carried out as a method of genetic analysis
that compares the frequency of alleles between affected and unaffected individuals
across all families Thus for association family structures are not necessary A given
allele is considered to be associated with the disease if the presence of that allele
General discussion 121
explains a significant proportion of the phenotypic trait variation For association
studies the developing of a marker set consisting of SNPs rather than microsatellites
is needed
In this thesis a total of 32 candidate genes for canine congenital deafness were
analyzed which showed an appropriate clinical and histological disease pattern in
comparison to deafness in Dalmatian dogs Rak (2003) developed a set of 43
microsatellites for in total 24 candidate genes among them the CLDN14 gene on
canine chromosome (CFA) 31 and the MYH9 gene on CFA10 The GJA1 on CFA1
was also considered as a candidate gene for CCSD (Rak 2003) and therefore two
gene-associated microsatellites have been developed Recently it turned out that
GJA1 is not responsible for human sensorineural non-syndromic deafness but for a
human syndromic disorder that can be related with conductive deafness
By the use of a non-parametric linkage analysis using the existing set of 43
microsatellites associated to 24 candidate genes we found linkage to markers
associated to CLDN14 on CFA31 MYH9 on CFA10 and GJA1 on CFA1
For another another eight candidate genes it was possible to develop SNPs
Performing linkage analyses as well as association and haplotype studies it was
possible to exclude these eight candidate genes from being responsible for the
CCSD phenotype
Over the past ten years significant progress has been made in the identification of
deafness gene localisations Up to now approximately 120 loci have been reported
for both autosomal dominant and recessive forms of non-syndromic hereditary
deafness in humans and only for one third the responsible gene mutation could be
detected Thus it can be expected that additional potential human candidates for
CCSD in Dalmatian dogs will become available in future (Van Camp and Smith
2003)
The extreme heterogeneity of human deafness often hampered genetic studies
because many different genetic forms of hearing loss give rise to similar clinical
phenotypes and conversely mutations in the same gene can result in a variety of
clinical phenotypes In man genes that transport ions across membranes to
maintain appropriate solute concentration and pH as well as regulatory genes mainly
transcription factors and genes that play a part in structural integrity are essential
for the hearing process
General discussion
122
The results of this thesis indicate that the inheritance of hearing loss in Dalmatian
dogs is probably as heterogenic in origin as it is in humans Genetic heterogeneity
means that different mutations cause a similar phenotype the different mutations
can either be found at the same locus (allelic heterogeneity) or even at different loci
(non-allelic heterogeneity) As linkage was found for different candidate genes in
different families subsequently only the families indicating linkage were chosen for
further molecular analyses
GJA1 on CFA1 MYH9 on CFA10 and CLDN14 on CFA31 and their flanking regions
are further analyzed with a combined approach using microsatellite and SNP
markers
CFA1 By the use of GJA1-associated microsatellites one large French Dalmatian dog
family reached a Zmean value of 295 (plt0002) for GJA1_MS1 and a Zmean value
of 286 (plt0002) for GJA1_MS2 indicating linkage Subsequently a sequence
analysis of the GJA1 gene using the above mentioned French Dalmatian dog family
was performed None of the observed polymorphism did alter the predicted amino
acid sequence of GJA1 nor showed the identified haplotypes an association with the
CCSD phenotype Thus evaluation of the SNP markers debilitates the linkage to
CCSD for the French half-sib family It is unlikely that the GJA1 gene is involved in
the pathogenesis of CCSD in Dalmatian dogs To see whether significant test
statistics for other genomic regions on CFA1 and for more families can be shown a
non-parametric linkage analysis was performed with 27 microsatellite markers
covering CFA1 completely In total 176 animals were genotyped We could not find
linkage to any microsatellite in the analyzed families Furthermore it was revealed
that GJA1 is not a candidate gene for sensorineural non-syndromic deafness in
humans (WA Paznekas cited a personal communication from the senior author (W
E Nance) of the paper by Liu et al 2001) GJA1 is participating in a human
syndrome called oculodentodigital dysplasia (ODDD) that can be accompanied with
hearing impairment (Paznekas et al 2003) But the type of deafness in human
ODDD is conductive rather than sensorineural As deafness in dogs especially in
Dalmatians is almost exclusively caused by sensorineural non-syndromic forms also
known as cochleosaccular degeneration the GJA1 gene should not be considered
as a candidate gene for CCSD anymore
General discussion 123
CFA31 By the use of microsatellites as well as SNPs we found significant linkage with CCSD
for CLDN14-associated microsatellites in four full-sib and one half-sib Dalmatian dog
familiy with a Zmean value of 383 (plt000007) A mutation analysis was performed
for exon three as this is the only translated one in man None of the observed
polymorphisms did alter the predicted amino acid sequence However to clarify the
importance of the CLDN14 gene and its flanking regions in the CCSD phenotype
more SNPs have to be developed within the CLDN14 gene as well as in its flanking
regions with the aim to find significant linkage disequilibrium of SNP markers
CFA10 A significant co-segregation of markers alleles and the phenotypic expression of
deafness in a large sample of German Dalmatian dog families was determined for
one marker (Z-mean of 156 [p= 006] and a Lod score of 058 [p= 005]) associated
to the MYH9 gene Subsequently we evaluated whether MYH9 gene mutations are
responsible for CCSD in these Dalmatian dog families An initial priority in defining
gene structure is to obtain a full-length cDNA sequence and identify translational
initiation and termination sites and polyadenylation site(s) Exonintron structure can
then be determined by referencing the cDNA sequence against sequences of
cognate genomic DNA One popular method of obtaining full-length cDNA sequences
is the RACE (rapid amplification of cDNA ends) technique RACE-PCR is an anchor
PCR modification of RT-PCR The rationale is to amplify sequences between a single
previously characterised region in the mRNA (cDNA) and an anchor sequence that is
coupled to the 5 or the 3 end A primer is designed from the known internal
sequence and the second primer is selected from the relevant anchor sequence
To provide the genomic organization and the complete sequence of the canine
MYH9 gene the isolation of full length cDNAs was achieved with the help of a
modified rapid amplification of cDNA ends (RACE) protocol A mutation analysis was
performed to identify single nucleotide polymorphisms (SNPs) in this gene We
analyzed the association of the MYH9 haplotypes with the CCSD phenotype in three
families of Dalmatian dogs with frequent occurrence of CCSD and significant linkage
to gene-associated microsatellites Using the RT-PCR analyses for cDNA-genomic
sequence comparisons we detected that the canine MYH9 gene is bigger compared
to the human sequence due to the untranlated first exon in the 5rsquo-UTR
General discussion
124
The canine MYH9 gene consists of 41 exons with an untranslated exon 1 and
exonintron boundaries that conform perfectly to the GTAG rule
None of the observed polymorphisms did alter the predicted amino acid sequence of
MYH9 nor showed the identified haplotypes an association with the CCSD
phenotype
Thus these silent point mutations found in affected and unaffected Dalmatian dogs
do not seem to be responsible for the CCSD phenotype in these three families
To clarify if other regions on CFA10 are responsible for the CCSD phenotype we
added in a second step 27 microsatellite markers derived from the NCBI database to
cover CFA10 with a high density in the flanking region of the MYH9 gene A linkage
analysis was carried out for 23 Dalmatian dog families consisting of 176 animals that
were genotyped with the marker set of 27 microsatellite markers
We found significant linkage to microsatellites in the region spanning 36 Mb to 48 Mb
Consequently we screened this 12 Mb spanning region for SNPs Out of the 23
analyzed Dalmatian dog families five full-sib families were chosen to screen for
SNPs because of their obviously significant values at the above mentioned region
Twenty-six new SNP markers covering the region of 36 Mb to 48 Mb equally were
developed and added to the linkage analysis The significant Zmeans on CFA10 was
confirmed after adding the SNP markers Furthermore with the use of SNPs the
apparently linked region spanning 36 Mb to 48 Mb could be narrowed down to 5 Mb
spanning from 39 Mb to 44 Mb For this reason the use of SNPs in addition to
informative microsatellites appears highly recommendable
In further studies more SNPs have to be developed within the identified CCSD region
on CFA10 to localize the deafness causing gene or to find unambiguously associsted
SNP markers which could be used for a population-wide genetic test for CCSD
Chapter 10
Summary
Summary 127
Summary
Molecular genetic analysis of canine congenital sensorineural deafness in Dalmatian dogs
Katharina Mieskes (2006) The objective of the present study was to localize the gene or genomic region that is
involved in the development of canine congenital sensorineural deafness (CCSD) in
Dalmatian dogs
In man as in different dog breeds deafness is an often diagnosed disorder with the
Dalmatian dog showing the highest incidence Many genetic disorders in humans
and domestic dogs (Canis familiaris) demonstrate a high level of clinical and
molecular similarity
Altogether 39 genes have already been found causative for sensorineural non-
syndromic hearing impairment in humans Out of this 39 deafness causing genes a
total of 32 candidate genes were selected for canine congenital deafness which
showed an appropriate clinical and histological disease pattern in comparison to
deafness in Dalmatians dogs
On the one hand an existing set of 43 microsatllite markers for in total 24 candidate
genes were used for a non-parametric linkage analysis among them the claudin-14
(CLDN14) gene on canine chromosome (CFA) 31 and the myosin heavy polypeptide
9 (MYH9) gene on CFA10 The gap junction protein alpha 1 (GJA1) gene on CFA1
was also considered as a candidate gene for CCSD and thus GJA1-associated
microsatellites were part of the existing set Recently it turned out that GJA1 is not
responsible for human sensorineural non-syndromic deafness but for a human
syndromic disorder that can be related with conductive deafness In the last few
years more human deafness genes have been identified among them eight genes
that were considered as appropriate candidates for CCSD For these eight genes a
total of 21 SNPs were newly developed and used for non-parametric linkage and
association analyses
Summary
128
The used microsatellite and SNP markers derived either from a partial sequence
analysis of BAC clones each containing a different candidate gene or from
sequences deposited in the current dog genome assembly (Boxer genome assembly
21) of the NCBI GenBank We found significant linkage to markers associated to
CLDN14 on CFA31 MYH9 on CFA10 and GJA1 on CFA1 To substantiate the
linkage we searched for sequence variations within these three genes SNPs found
in intronic sequences of either gene were included in the linkage analyses Sequence
analysis neither revealed a causative mutation nor significant linkage disequilibrium
of SNP markers with CCSD Subsequently CFA1 10 and 31 were scanned
completely with microsatellite markers derived from the NCBI database with the
purpose to see if other regions on this three chromosomes harbour a gene that is
involved in the development of CCSD
The analyses of SNPs and more microsatellite markers on CFA1 revealed no
significant linkage to CCSD Thus it is unlikely that the canine chromosome 1 and
the GJA1 gene are responsible for the CCSD phenoptype As deafness in dogs
especially in Dalmatians is almost exclusively caused by sensorineural non-
syndromic forms the GJA1 gene should not be considered as a candidate gene for
CCSD anymore
On CFA10 we could exclude MYH9 for being causal for deafness but by adding
more microsatellites covering CFA10 completely we found significant linkage to the
CCSD phenotype in the region distal of MYH9 Hence new SNP-markers for fine
mapping the region spanning 36 to 48 Mb were developed by sequence analyses of
different Dalmatian dogs The search for SNPs was carried out on genomic
sequences of genes located in the significantly linked region The sequences of
these genomic sequences were derived from the NCBI GenBank The SNPs
confirmed the linkage and narrowed the region harbouring a causative CCSD gene
down to 5 Mb spanning from 39 to 44 Mb
After scanning CFA31 we could not exclude CLDN14 for being responsible for the
CCSD phenotype as microsatellite markers associated to CLDN14 indicated linkage
However to clarify the importance of CLDN14 in the CCSD phenotype more SNPs
have to be developed within the CLDN14 gene as well as in its flanking regions with
the aim to find linkage disequilibrium for SNP markers
Chapter 11
Erweiterte Zusammenfassung
Erweiterte Zusammenfassung 131
Erweiterte Zusammenfassung
Katharina Mieskes
Molekulargenetische Untersuchung der kongenitalen sensorineuralen Taubheit beim Dalmatiner
Einleitung Es gibt mehrere Moumlglichkeiten Taubheit zu klassifizieren Je nach den betroffenen
Strukturen des Gehoumlrorgans lassen sich konduktive und sensorineurale Formen
unterscheiden Erstere entstehen durch Stoumlrungen des Konduktionsmechanismus im
aumluszligeren Gehoumlrgang und im Mittelohr Ursachen fuumlr konduktive Houmlrstoumlrungen koumlnnen
zum Beispiel chronische Otitis externa oder media Frakturen von Mittelohranteilen
oder Tumoren sein Sehr viel oumlfter tritt die sensorineurale Taubheit auf bei der ein
Houmlrverlust als Folge von Transduktions- und Transmissionsstoumlrungen in der Cochlea
(Schnecke) sowie in peripheren und zentralen Anteilen des Nervus cochlearis auftritt
Neben der erblichen Form koumlnnen auch eine Otitis interna Tumoren oder
ototoxische Medikamente zur sensorineuralen Taubheit fuumlhren
Der Houmlrverlust kann bereits seit der Geburt bestehen (kongenital) oder er kann erst
im Laufe der weiteren Lebenszeit auftreten
Beim Menschen ist mehr als die Haumllfte der kongenitalen Taubheit erblich bedingt Die
erbliche kongenitale Taubheit kann als Teil einer multisystemischen Krankheit
(syndromisch) oder als Funktionsstoumlrung auftreten die auf das Ohr und das
vestibulaumlre System (nicht-syndromisch) begrenzt ist Da die nicht-syndromische
ererbte Taubheit fast ausschlieszliglich durch Cochleadefekte verursacht wird leiden die
Patienten unter sensorineuraler Taubheit Es wird geschaumltzt dass die nicht-
syndromische erbliche Taubheit 60-70 aller menschlichen Taubheitsfaumllle
verursacht Weiterhin sind 70-80 der Faumllle der nicht-syndromischen Taubheit auf
einen autosomal rezessiven Erbgang zuruumlck zu fuumlhren gefolgt von einem autosomal
dominanten Erbgang mit 10-20 der Faumllle und 1-2 der Faumllle sind X- gekoppelt Ein
noch geringerer Prozentsatz wird durch einen maternalen Erbgang verursacht
Von den ca 25000 bis 30000 menschlichen Genen ist nach Schaumltzungen bis zu 1
wichtig fuumlr den Houmlrprozess Annaumlhernd 120 Gene sind inzwischen identifiziert die fuumlr
Erweiterte Zusammenfassung
132
verschiedene Formen der menschlichen vererbten Taubheit verantwortlich sind
Darunter sind ungefaumlhr 80 Gene fuumlr die syndromische und 39 Gene fuumlr die nicht
syndromische vererbte Taubheit verantwortlich Insgesamt stellen die 120
identifizierten Gene vermutlich ein Drittel aller taubheitsverursachenden Gene dar
Die kongenitale sensorineurale Taubheit ist eine Erkrankung von der viele
Saumlugetierspezies betroffen sind Beim Hund werden uumlber 54 verschiedene Rassen
beschrieben bei welchen ein kongenitaler Houmlrverlust gehaumluft auftritt In zahlreichen
europaumlischen und amerikanischen Studien weisen hierbei die Dalmatiner mit 165-
30 die houmlchste Taubheitsinzidenz auf
Die histologischen Veraumlnderungen im Innenohr werden im Allgemeinen aumlhnlich wie
beim Menschen als cochleosacculaumlr beschrieben Die Degenerationen im Innenohr
schreiten bei betroffenen Tieren mit zunehmendem Alter fort und fuumlhren
normalerweise in einem Alter von drei bis vier Wochen spaumltestens aber in einem
Alter von etwa vier Monaten zu einem vollstaumlndigen ein- oder beidseitigen
Houmlrverlust
Eine objektive Diagnose der Taubheit besonders wenn sie nur einseitig ist stuumltzt
sich auf die brainstem auditory evoked response (BAER in Deutschland
audiometrischer Test) ein elektro-diagnostischer Test bei dem durch einen
bestimmten Reiz ausgeloumlste elektrische Aktivitaumlten (akustisch evozierte Potentiale) in
der Cochlea und den Nervenbahnen zur auditorischen Hirnrinde detektiert werden
Obwohl fuumlr den Dalmatiner die Erblichkeit der Erkrankung wiederholt nachgewiesen
werden konnte wurden fuumlr die verschiedenen untersuchten Dalmatinerpopulationen
auch sehr viele unterschiedliche Erbgaumlnge in Betracht gezogen Es ist bisher nicht
gelungen die genaue Anzahl der beteiligten Gene zu bestimmen Auch war es
bislang fuumlr keine der betroffenen Hunderassen moumlglich ein taubheitsverursachendes
Gen zu identifizieren
Bei deutschen Dalmatinern ergab eine Auswertung mittels komplexer
Segregationsanalysen dass ein Modell mit einem rezessiven Hauptgen und einer
polygenen Komponente das Auftreten der kongenitalen Taubheit in der untersuchten
Population am besten erklaumlrte Zusaumltzlich konnte ein Zusammenhang zu
Pigmentierungsgenorten nachgewiesen werden da Tiere mit blauer Augenfarbe
signifikant haumlufiger von Taubheit betroffen waren und gleichzeitig Tiere mit
Plattenzeichnung seltener kongenitalen Houmlrverlust zeigten
Erweiterte Zusammenfassung 133
Das Zuumlchten ein- oder beidseitig tauber Dalmatiner sowie Dalmatinern mit blauen
Augen ist in Deutschland schon laumlnger verboten und es wird von den
Zuchtverbaumlnden vorgeschrieben dass alle Dalmatinerwelpen in einem Alter von etwa
6 bis 8 Wochen audiometrisch untersucht werden Des Weiteren ist der Defekt
tierschutzrelevant nach sect 11b des Deutschen Tierschutzgesetzes da der artgemaumlszlige
Gebrauch eines Organs eingeschraumlnkt wird und eine Erblichkeit nachgewiesen
wurde Somit duumlrfen Tiere die Defektgentraumlger sind oder bei denen damit zu
rechnen ist nicht zur Zucht verwendet werden Waumlhrend einseitig taube Dalmatiner
als ganz normale Familienhunde gehalten werden koumlnnen sind beidseitig taube
Dalmatiner schwierig zu erziehen schreckhafter und daraus resultierend auch
oftmals aggressiv und bissig Aus diesen Gruumlnden werden fast alle Welpen mit
beidseitigem Houmlrverlust euthanasiert
Es hat sich gezeigt dass allein der Zuchtausschluss betroffener Tiere nicht genuumlgt
um die hohe Taubheitsinzidenz beim Dalmatiner deutlich zu reduzieren Aus diesen
Gruumlnden ist es von erheblicher Bedeutung die kongenitale sensorineurale Taubheit
beim Dalmatiner molekulargenetisch zu untersuchen um so ein
molekulargenetisches Testverfahren entwickeln zu koumlnnen welches auch die
Identifizierung von Anlagetraumlgern ermoumlglicht
Innerhalb des letzten Jahrzehnts wurden auch zwischen relativ unverwandten
Spezies die strukturellen und funktionellen Homologien auf genetischer Ebene
zunehmend verdeutlicht
Es wurde inzwischen eine groszlige Anzahl von taubheitsverursachenden
Genmutationen beim Menschen und der Maus aufgeklaumlrt Da auch die
histopathologischen Befunde beim Menschen und der Maus oftmals sehr aumlhnlich
denen beim Dalmatiner sind scheinen Gene die beim Menschen verantwortlich fuumlr
die congenitale sensorineurale Taubheit sind geeignete Kandidatengene fuumlr die
canine congenitale sensorineurale Taubheit zu sein
Das Ziel dieser Dissertation ist es die fuumlr die Taubheit kausalen Gene bzw Genorte
zu lokalisieren Um die kongenitale sensorineurale Taubheit beim Dalmatiner
moumlglichst effizient molekulargenetisch aufzuklaumlren wurden 32 Kandidatengene mit
genassoziierten Markern auf eine potenzielle Kopplung mit der caninen kongenitalen
sensorineuralen Taubheit untersucht
Die Genombereiche mit signifikanter Kopplung zur kongenitalen sensorineuralen
Taubheit wurden weitergehend molekulargenetisch analysiert
Erweiterte Zusammenfassung
134
Kopplungsanalyse fuumlr 24 Kandidatengene der caninen kongenitalen sensorineuralen Taubheit (CCSD) mit 43 genassoziierten Mikrosatelliten Markern
Material und Methoden
Fuumlr 24 Kandidatengene stand ein Mikrosatelliten Marker Set zur Verfuumlgung Das
Marker Set beinhaltete Mikrosatelliten fuumlr folgende Kandidatengene CDH23
CLDN14 COCH COL11A2 DFNA5 DIAPH1 EDN3 EDNRB EYA4 GJA1 GJB2
GJB6 MITF MYH9 MYO6 MYO7A MYO15A OTOF PAX3 POU4F3 SLC26A4
SOX10 TECTA und TMPRSS3 Als Familienmaterial fuumlr die Mikrosatellitenstudie
wurden 215 Dalmatiner verwendet Die Tiere verteilten sich auf 22 unverwandte
Vollgeschwisterfamilien und zwei paternale Halbgeschwistergruppen Die
genotypisierten Familien bestanden aus saumlmtlichen tauben Nachkommen (uni- oder
bilateral taub) ihren Eltern und einem bis vier nicht betroffenen Voll- bzw
Halbgeschwistern Mindestens zwei der Vollgeschwister einer Familie waren uni-
oder bilateral taub Der Phaumlnotyp der verwendeten Tiere wurde durch einen
audiometrischen Test bestimmt
Alle Mikrosatellitenmarker wurden uumlber PCR und Polyacrylamidgelelektrophorese
ausgewertet
Kopplungsanalyse
Die nicht-parametrische Kopplungsanalyse wurde unter Verwendung der Software
MERLIN (multipoint engine for rapid likelihood inference Version 0102)
durchgefuumlhrt und basierte auf dem identical-by-descent (IBD) Verfahren Dabei
wurden die Markerallele auf Kosegregation mit der phaumlnotypischen Auspraumlgung der
Erkrankung getestet Darauf folgend wurde die einer Normalverteilung folgende
Teststatistik fuumlr den Anteil von IBD-Markerallelen (Zmean) und ein daraus
abgeleiteter LOD-Score berechnet Als signifikant fuumlr die Kosegregation eines
Markerallels mit dem Phaumlnotyp der caninen kongenitalen sensorineuralen Taubheit
(CCSD) gelten Irrtumswahrscheinlichkeiten (p) von 005 oder kleiner
Die Daten wurden mit dem Softwarepaket SAS (Statistical Analysis System Version
913) bearbeitet um die Marker bezuumlglich Allelfrequenzen beobachteten und
erwarteten Heterozygotiegrad und PIC (Polymorphism information content)
charakterisieren zu koumlnnen
Erweiterte Zusammenfassung 135
Ergebnisse und Diskussion Das Mikrosatelliten Set bestand insgesamt aus 43 Markern mit einem bis drei
Markern pro Kandidatengen Zunaumlchst wurde fuumlr alle 24 Familien zusammen eine
Kopplungsanalyse durchgefuumlhrt Vermeintliche CCSD Loci wurden auf den caninen
Chromosomen (CFA) 1 10 12 20 und 31 lokalisiert Im Anschluss wurden die
Ergebnisse der Kopplungsanalyse auf Heterogenie zwischen den Familien uumlberpruumlft
Fuumlr einzelne Familien konnten houmlhere Teststatistiken und somit engere
Kopplungsbeziehungen als bei der Analyse aller Familien nachgewiesen werden
Eine signifikante Kopplung mit der kongenitalen sensorineuralen Taubheit wurde fuumlr
die Gene GJA1 auf CFA1 MYH9 auf CFA10 und CLDN14 auf CFA31 sowohl im
Gesamtmaterial wie in jeweils unterschiedlichen Gruppen von Dalmatinerfamilien
gefunden Es ist daher wahrscheinlich dass diese Gene oder Gene in ihrer naumlheren
Umgebung an der Entwicklung der Taubheit in den jeweiligen Familien involviert
sind Fuumlr die restlichen Kandidatengene lagen die Multipoint Teststatistiken fuumlr den
Anteil von IBD-Markerallelen (Zmean) und daraus abgeleiteten LOD-Scores bei
annaumlhernd Null und waren nicht signifikant
Die Ergebnisse dieser Teststatistiken zeigen dass der Erbgang der nicht-
syndromischen Taubheit bei Dalmatinern vermutlich ebenso heterogen ist wie er
sich beim Menschen darstellt
Dennoch stellt diese Studie den ersten Schritt dar um die CCSD verursachenden
Gene beim Dalmatiner zu identifizieren Die Gene GJA1 MYH9 und CLDN14 sowie
ihre naumlhere Umgebungen werden im weiteren Verlauf der Studie weitergehend
molekulargenetisch mit Hilfe von SNPs und Mikrosatelliten untersucht Aufgrund der
Heterogenitaumlt zwischen den Familien wurden fuumlr die weitere molekulargenetische
Analyse bestimmter Gene oder Genomregionen nur die Familien mit dem houmlchsten
Beitrag zu den Teststatistiken der Kopplungsanalyse (Zmean gt 1) ausgewaumlhlt
Erweiterte Zusammenfassung
136
Evaluierung von neu entwickelten SNP Markern assoziiert mit Kandidatengenen fuumlr die canine kongenitale sensorineurale Taubheit (CCSD) Material und Methoden Fuumlr acht Kandidatengene (TMC1 TMIE USH1C MYH14 MYO3A PRES WHRN
und ESPN) wurden intragenische Single Nucleotide Polymorphism (SNP) Marker neu
entwickelt
Als Familienmaterial fuumlr die Genotypisierung der SNPs wurde DNA von insgesamt 39
Dalmatinern verwendet die vier Vollgeschwisterfamilien angehoumlrten Mindestens
zwei der Vollgeschwister einer Familie waren uni- oder bilateral taub Der Phaumlnotyp
der verwendeten Tiere wurde durch einen audiometrischen Test bestimmt Die vier
Familien bestanden aus sechs bis zehn Vollgeschwistern und den dazugehoumlrenden
Elterntieren
Identifizierung von SNPs (Single Nucleotide Polymorphisms)
Die caninen Sequenzen der Kandidatengene wurden mit Hilfe der humanen mRNA
mittels BLAST (Basic Local Alignment Search Tool) Suche aus veroumlffentlichten
Sequenzen der NCBI Datenbank (Boxer genome assembly 21) entnommen Die
Exon Intron Grenzen wurden mit Hilfe des mRNA-to-genomic alignment Programms
Spidey bestimmt und Primer mit Hilfe des Primer3 Programms in die intronischen
Sequenzen gelegt Die SNPs wurden an den DNA-Sequenzen von Elterntieren der
betroffenen Dalmatinerfamilien identifiziert Die Amplifikate wurden mit Hilfe des
MegaBACE 1000 sequenziert und auf SNPs untersucht Bei Vorliegen eines oder
mehrerer SNPs wurde die entsprechende DNA-Sequenz der zugehoumlrigen
Nachkommen ebenfalls amplifiziert und sequenziert Die Auswertung erfolgte mit
Hilfe des Sequencher 42 Programms
Kopplungsanalyse
Die Genotypen der neuen SNP Marker wurden unter Verwendung des Programms
MERLIN ausgewertet Die Daten wurden mit dem Softwarepaket SAS (Statistical
Analysis System Version 913) bearbeitet um das Markerset bezuumlglich
Allelfrequenzen beobachteten Heterozygotiegrad und PIC (Polymorphism
information content) charakterisieren zu koumlnnen Des Weiteren wurde auf
Kopplungsungleichgewicht und die Assoziation der Haplotypen mit CCSD mittels
Erweiterte Zusammenfassung 137
des Programms CASECONTROL und HAPLOTYPE von SAS Genetics (Statistical
Analysis System Version 913 Cary NC USA) getestet
Ergebnisse und Diskussion Fuumlr acht Kandidatengene wurden mindestens zwei heterozygote SNPs entwickelt
Die nicht-parametrische Kopplungsanalyse zeigte keine signifikanten Teststatistiken
Offensichtlich war kein Haplotyp mit der caninen kongenitalen sensorineuralen
Taubheit (CCSD) in den untersuchten Familien assoziiert Da sowohl uni- und
bilateral taube Tiere als auch beidseitig houmlrende Tiere uumlber identische Haplotypen
verfuumlgen ist es unwahrscheinlich dass die Gene TMC1 TMIE USH1C MYH14
MYO3A PRES WHRN und ESPN an der Entstehung der CCSD beim Dalmatiner
beteiligt sind
Molekulargenetische Analyse des caninen myosin heavy polypeptide 9 non-muscle (MYH9) auf dem caninen Chromosom 10q232
Material und Methoden Klonierung und Sequenzierung von caniner MYH9 cDNA
Die canine MYH9 Gensequenz wurde mit Hilfe der BLAST Suche aus
veroumlffentlichten Sequenzen der NCBI Datenbank Die Isolierung der vollstaumlndigen
cDNA wurde durch ein Protokoll zur Amplifizierung von cDNA-Enden (rapid
amplification of cDNA ends [RACE]) erreicht Nach Klonierung und Sequenzierung
der RACE-Produkte wurden die Sequenzen mit Hilfe des Sequencher 42
Programms ausgewertet
Sequenzanalyse des caninen MYH9 Gens
Nach Fertigstellung des RACE Protokolls und Auswertung der Sequenzen wurden
die Exon Introngrenzen mit Hilfe des mRNA-to-genomic alignment Programms
Spidey lokalisiert Weiterhin wurden repetitive Elemente mit dem Programm
Repeatmaster 2 sowie der GC Gehalt mit der EBI toolbox CpG PlotCpGreport
ermittelt
Erweiterte Zusammenfassung
138
Mutatiosanalyse
Zur Identifizierung von Polymorphismen innerhalb der caninen MYH9 Sequenz
wurden die Exons mit ihren flankierenden intronischen Sequenzen mit Hilfe der PCR
amplifiziert und sequenziert Verwendet wurden hierfuumlr die DNA von insgesamt 16
Dalmatinern aus drei Familien Mindestens zwei der Vollgeschwister einer Familie
waren unilateral taub Der Phaumlnotyp der verwendeten Tiere wurde durch einen
audiometrischen Test bestimmt
PCR Primer wurden mit Hilfe des Primer3 Programms entwickelt PCR Reaktionen
fuumlr saumlmtliche Exons des MYH9 Gens mit ihren flankierenden Sequenzen wurden
durchgefuumlhrt die PCR Produkte sequenziert und die Sequenzen mit Hilfe des
Sequencher 42 Programms ausgewertet
Unter Verwendung der Software MERLIN (multipoint engine for rapid likelihood
inference Version 0910) wurde nach signifikanten nicht parametrischen LOD-
Scores fuumlr die Kosegregation von Markerallelen mit der phaumlnotypischen Auspraumlgung
der sensorineuralen Taubheit beim Dalmatiner gesucht
Ergebnisse und Diskussion
Analyse der genomischen Organisation und der cDNA des caninen MYH9 Gens
Eine vollstaumlndige cDNA Sequenz des caninen MYH9 Gens von 7484 bp konnte uumlber
die Reverse Transkription-PCR erhalten und bei der EMBL Nukleotid Datenbank
eingereicht werden Das canine MYH9 Gen besteht aus 41 Exons einschlieszliglich
einem untranslatierten ersten Exon und Exon Intron Grenzen die stets der GT AG
Regel folgen Der 5rsquo-UTR Bereich besteht aus 181 bp waumlhrend der 3rsquo-UTR aus 1432
bp besteht Weiterhin besitzt die canine MYH9 cDNA Sequenz einen offenen
Leserahmen von 5889 bp welcher ein Protein kodiert bestehend aus 1963
Aminosaumluren Die Groumlszlige der Exons variiert von 28 bis 1556 bp waumlhrend das
gesamte MYH9 Gens ungefaumlhr 90 kb umfasst Die Homologie der Sequenzen
zwischen dem humanen murinen und caninen MYH9 Gen ist limitiert auf die
kodierende Sequenz von Exon 2 bis 41
Mutations- und Haplotypenanalyse
Alle kodierenden Exons mit ihren flankierenden intronischen Sequenzen des MYH9
Gens konnten von den 16 Dalmatinern amplifiziert werden Die Sequenzen wurden
mit den entsprechenden Sequenzen vom Boxer aus der NCBI Genbank verglichen
Erweiterte Zusammenfassung 139
Die Suche nach Sequenzvariationen im MYH9 Gen lieszlig 22 SNPs erkennen wobei
die meisten Polymorphismen in den flankierenden Regionen der Exons gefunden
wurden Keiner der beobachteten Polymorphismen in den Exons aumlnderte die
Aminosaumluresequenz von MYH9 Auch zeigten die identifizierten Haplotypen keine
Assoziation mit dem CCSD Phaumlnotyp Multipoint Teststatistiken fuumlr den Anteil von
IBD-Markerallelen (Zmean) und ein daraus abgeleiteter LOD-Score lagen bei
annaumlhernd Null und waren nicht signifikant Zwei genassoziierte Mikrosatelliten
zeigten eine Kopplung diese kann jedoch mit Heterogenitaumlt zwischen den Familien
erklaumlrt werden
Fazit
Die Charakterisierung des Transkriptes und der genomischen Sequenz des caninen
MYH9 Gens zeigten einen konservierten Aufbau des Gens im Bezug auf das
humane orthologe Gen Aufgrund eines untranslatierten Exons im 5rsquo-UTR Bereich ist
das canine Gen groumlszliger als das humane Gen
Sowohl uni- oder bilateral taube und bilateral houmlrende Dalmatiner weisen identische
Haplotypen auf Daher sind in den untersuchten Dalmatinerfamilien die gefundenen
Polymorphismen offensichtlich nicht mit CCSD assoziiert Des Weiteren zeigt diese
Studie dass keine funktionellen Mutationen der vollstaumlndigen kodierenden Region
von MYH9 vorliegen Wir koumlnnen also abschlieszligend das MYH9 Gen als
Kandidatengen fuumlr die congenitale sensorineurale Taubheit beim Dalmatiner
ausschlieszligen
Erweiterte Zusammenfassung
140
Evaluierung von Mikrosatelliten und SNP Markern der caninen Chromosomen 1 10 und 31 auf Kopplung und Assoziation mit der caninen kongenitalen Taubheit Material und Methoden Pedigreematerial
Insgesamt wurden 176 Dalmatiner in die Untersuchung einbezogen Die Tiere
verteilten sich auf 23 unverwandte Vollgeschwisterfamilien und eine paternale
Halbgeschwistergruppe Die genotypisierten Familien bestanden aus saumlmtlichen
tauben Nachkommen (unilateral oder bilateral taub) ihren Eltern und einen bis vier
nicht betroffenen Voll- bzw Halbgeschwistern Mindestens zwei der Vollgeschwister
einer Familie waren unilateral oder bilateral taub Der Phaumlnotyp der verwendeten
Tiere wurde durch einen audiometrischen Test bestimmt
Alle 176 Dalmatiner wurden fuumlr die Mikrosatellitenstudie auf den caninen
Chromosomen 1 10 und 31 einbezogen Fuumlr die Feinkartierung mit Hilfe der SNPs
wurden dagegen nur noch die Dalmatinerfamilien mit einer eindeutigen Kopplung zu
der entsprechenden genomischen Region untersucht Fuumlr die Region auf CFA10
wurden dafuumlr 34 Tiere aus fuumlnf Vollgeschwisterfamilien ausgewaumlhlt und zur
Untersuchung des CLDN14 Gens auf CFA31 wurden 24 Tiere aus vier
Vollgeschwisterfamilien und 12 Tiere aus einer groszligen Halbgeschwisterfamilie
verwendet Fuumlr die Suche nach Polymorphismen im GJA1 Gen wurden 16 Tiere aus
einer groszligen Halbgeschwisterfamilie verwendet
Mikrosatellitenstudie
Fuumlr die Mikrosatellitenstudie von CFA1 10 und 31 wurden insgesamt 60 Marker
verwendet Mit jeweils 27 Mikrosatelliten wurde CFA1 und mit 27 Mikrosatelliten
wurde CFA10 vollstaumlndig abgedeckt Insgesamt 6 Marker wurden fuumlr CFA31
verwendet
Alle hier verwendeten Mikrosatelliten sind der NCBI Datenbank entnommen und
wurden uumlber PCR und Polyacrylamidgelelektrophorese ausgewertet
Identifizierung von SNPs (Single Nucleotide Polymorphisms)
Zur weiteren Abklaumlrung und zum Ausschluss falsch positiver Ergebnisse wurden
zusaumltzlich zu den Mikrosatelliten SNPs in den signifikanten Regionen auf CFA1 10
Erweiterte Zusammenfassung 141
und CFA 31 entwickelt und an den Familien die eine Kopplung zeigten
genotypisiert
Die caninen genomischen Sequenzen der untersuchten Gene wurden der NCBI
Datenbank (Boxer genome assembly 21) entnommen Exon Intron Grenzen wurden
bestimmt und Primer entwickelt Nach Durchfuumlhrung der PCR wurden die Amplifikate
mit Hilfe des MegaBACE 1000 sequenziert und auf SNPs untersucht Die
Auswertung erfolgte mit Hilfe des Sequencher 42 Programms
Die Genotypen der neuen SNP Marker wurden zu den Genotypen der fuumlr die
Kopplungsanalyse verwendeten Mikrosatelliten hinzugefuumlgt und gemeinsam unter
Verwendung des Programms MERLIN ausgewertet
Kopplungsanalyse
Unter Verwendung der Software MERLIN (multipoint engine for rapid likelihood
inference Version 0910) wurde nach signifikanten nicht parametrischen LOD-
Scores fuumlr die Kosegregation von Markerallelen mit der phaumlnotypischen Auspraumlgung
der sensorineuralen Taubheit beim Dalmatiner gesucht
Die Daten wurden mit dem Softwarepaket SAS (Statistical Analysis System Version
913) bearbeitet um die Marker bezuumlglich Allelfrequenzen beobachteten
Heterozygotiegrad und PIC (Polymorphism information content) charakterisieren zu
koumlnnen Des Weiteren wurde das Kopplungsungleichgewicht und die Assoziation der
Haplotypen mit CCSD mittles des Programms CASECONTROL und HAPLOTYPE
von SAS Genetics getestet
Ergebnisse und Diskussion CFA1
In einer groszligen Halbgeschwisterfamilie wurde eine signifikante Kopplung zu einem
GJA1 genassozierten Marker festgestellt Mit Hilfe der DNA von Tieren aus dieser
Halbgeschwisterfamilie wurde eine Sequenzanalyse des GJA1 Gens durchgefuumlhrt
Es wurden 3 SNPs im 3acute-untranslatierten Bereich gefunden Die identifizierten
Haplotypen zeigten keine Assoziation mit dem CCSD Phaumlnotyp Sowohl uni- oder
bilateral taube und bilateral houmlrende Dalmatiner wiesen identische Haplotypen auf
Daher sind in den untersuchten Dalmatinerfamilien die gefunden Polymorphismen
offensichtlich nicht mit CCSD assoziiert Auch wurde keine funktionelle Mutation in
der vollstaumlndigen kodierenden Region von GJA1 gefunden wurde Es ist daher
Erweiterte Zusammenfassung
142
unwahrscheinlich dass das GJA1 Gen an der Entwicklung der CCSD in der
untersuchten Halbgeschwisterfamilie beteiligt ist Die signifikante Kopplung der
GJA1-benachbarten Mikrosatelliten mit CCSD kann durch ein eng gekoppeltes Gen
hervorgerufen worden sein welches fuumlr die Entstehung der Taubheit in der
untersuchten Halbgeschwisterfamilie verantwortlich ist oder durch ein falsch-
positives Ergebnis der vorhergehend durchgefuumlhrten Mikrosatellitenstudie
Eine nicht-parametrische Kopplungsanalyse mit Hilfe von 27 Mikrosatelliten Marker
an 24 deutschen Dalmatinerfmilien wurde durchgefuumlhrt um abzuklaumlren ob andere
Genombereiche auf CFA1 signifikante Teststatistiken in mehr Familien zeigen Die
Kopplungsanalyse zeigte keine signifikanten Teststatistiken Es ist daher
unwahrscheinlich dass auf CFA1 Gene fuumlr die Entwicklung von CCSD in den
untersuchten Familien der aus Deutschland stammenden Dalmatiner verantwortlich
sind
Da kuumlrzlich festgestellt wurde dass das GJA1 Gen auch beim Menschen nicht fuumlr
eine Form der sensorineuralen nicht-syndromischen Taubheit verantwortlich ist
sondern an einer syndromischen Krankheit beteiligt ist die mit konduktiver Taubheit
einhergehen kann sollte das GJA1 Gen nicht laumlnger als ein Kandidatengen fuumlr
CCSD angesehen werden
CFA31
Auf CFA31 wurde durch eine nicht-parametrische Kopplungsanalyse von
Mikrosatelliten eine Kopplung eines CLDN14-benachbarten Markers mit der
sensorineuralen Taubheit beim Dalmatiner festgestellt Daraufhin wurden zusaumltzlich
zu den Mikrosatelliten acht SNPs entwickelt indem das einzige beim Menschen
translatierte Exon weite intronische Sequenzen sowie 5rsquo- und 3rsquo- untranslatierte
Bereiche des CLDN14 Gens einer Sequenzanalyse unterzogen wurden Nur zwei der
identifizierten SNPs waren polymorph in den untersuchten fuumlnf Dalmatinerfamilien
Sowohl uni- als auch bilateral taube und bilateral houmlrende Dalmatiner teilten
identische Haplotypen daher konnte keine Kosegregation mit tauben Dalmatinern
festgestellt werden
Da bislang keine funktionelle Mutation in der kodierenden Region von CLDN14
gefunden wurde ist es fuumlr eine eindeutige Aussage uumlber die Rolle des CLDN14
Gens im Entstehungsprozess der kongenitalen sensorineuralen Taubheit
Erweiterte Zusammenfassung 143
beim Dalmatiner notwendig noch weitere Marker im CLDN14 Gen sowie seiner
naumlheren Umgebung zu entwickeln und zu analysieren Da keine weitere Kopplung zu
einem Mikrosatelliten distal oder proximal des CLDN14 Gen festgestellt werden
konnte ist es wahrscheinlich dass entweder das CLDN14 Gen oder ein Gen in
seiner naumlheren Umgebung fuumlr die Entwicklung der sensorineuralen Taubheit in den
untersuchten Dalmatinerfamilien verantwortlich ist
CFA10
Auf CFA10 wurde durch eine nicht-parametrische Kopplungsanalyse von 27
Mikrosatelliten eine 12 Mb umfassende genomische Region mit einer signifikanten
Kopplung zur CCSD identifiziert Zur weiteren Feinkartierung wurden 26 neue SNPs
mit einem durchschnittlichen Abstand von 03 Mb fuumlr diesen Bereich entwickelt
Die SNPs waren groumlszligtenteils informativ im vorliegenden Familienmaterial und
bestaumltigten in einer folgenden Kopplungsanalyse die Lokalisation auf CFA10
deutlich Weiterhin konnte die gekoppelte Region auf einem 5 Mb umfassenden
Bereich von 39 bis 44 Mb eingegrenzt werden Es ist wahrscheinlich dass die
identifizierte CCSD Region ein Gen beinhaltet welches auf die Entstehung der
caninen kongenitalen Taubheit einwirkt
Es ist notwendig mehr SNPs zu entwickeln um das taubheitsverursachende Gen zu
lokalisieren oder einen eindeutig assoziierten SNP zu identifizieren
Chapter 12
References
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MELDRIM J MELNIKOV A MENEUS L MIHALEV A MIHOVA T MILLER K
MITTELMAN R MLENGA V MULRAIN L MUNSON G NAVIDI A
NAYLOR J NGUYEN T NGUYEN N NGUYEN C NGUYEN T NICOL R
NORBU N NORBU C NOVOD N NYIMA T OLANDT P ONEILL B
ONEILL K OSMAN S OYONO L PATTI C PERRIN D PHUNKHANG P
PIERRE F PRIEST M RACHUPKA A RAGHURAMAN S RAMEAU R
RAY V RAYMOND C REGE F RISE C ROGERS J ROGOV P SAHALIE J
SETTIPALLI S SHARPE T SHEA T SHEEHAN M SHERPA N SHI J
SHIH D SLOAN J SMITH C SPARROW T STALKER J STANGE-
THOMANN N STAVROPOULOS S STONE C STONE S SYKES S
TCHUINGA P TENZING P TESFAYE S THOULUTSANG D
THOULUTSANG Y TOPHAM K TOPPING I TSAMLA T VASSILIEV H
VENKATARAMAN V VO A WANGCHUK T WANGDI T WEIAND M
WILKINSON J WILSON A YADAV S YANG S YANG X YOUNG G YU Q
ZAINOUN J ZEMBEK L ZIMMER A LANDER ES (2005) Genome sequence
comparative analysis and haplotype structure of the domestic dog Nature
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GANGAROSSA S CARIDI G BORDO D LO NIGRO C GHIGGERI GM
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Chapter 13
Appendix
Appendix microsatellite marker III
HE
Te
79
5 7
98
76
9 5
12
71
3 8
33
70
2 4
35
72
7 7
00
54
6 4
77
48
8 3
08
44
8 5
40
67
4 8
05
76
2 6
54
70
8 7
73
78
1 6
87
65
4 6
26
51
9 5
89
76
4 1
60
PIC
d
72
2 8
41
67
0 4
85
67
1 7
85
57
2 4
07
61
4 6
29
35
8 3
30
40
0 2
90
35
0 4
60
70
0 7
90
76
2 6
21
63
8 7
26
77
1 6
26
60
1 6
13
38
7 5
52
80
0 1
40
Prim
er (r
ever
se)
5acute -gt
3acute
AA
CTG
TTTG
ATT
TTG
ATG
AG
GC
C
TTTG
ACC
TAC
ATA
TAA
AA
CA
AG
C
CA
CG
AA
GAA
AG
CC
ATG
GTT
T C
AA
CA
ATT
CC
TTTG
TTG
GTG
C
CTG
CC
AG
CTT
CC
TAC
AGC
T
GTG
GTC
ACA
AG
AC
TTTA
GC
C
CTG
CA
CC
AAA
GA
AA
CTC
C
GG
AC
CTA
TTC
TGA
AG
CC
TAA
C
TG
GC
CTT
TAA
TTTA
TCAT
GG
AG
A
TCA
GG
CC
CA
GA
TAG
TATG
C
TC
CTT
TGG
TTTT
TAG
CAG
GG
T
CTG
TGC
CC
AC
CTG
TGG
AG
C
AA
GA
GG
ATA
CC
GG
TGG
CA
G
CG
TGC
TTTG
TTA
TGG
CTT
GA
C
AG
CA
TGA
AG
GA
TCTC
TGA
CTA
A
CTA
AC
TCG
CC
AG
AC
TATT
C
CC
CTC
CAC
CA
ATC
TCTT
CA
C
GC
AA
AG
GTG
TATT
TAC
ATG
ATG
G
GG
CA
GC
CTT
ATT
ATT
CA
TGG
A
AC
AC
AG
GC
AC
AG
GA
GC
ATC
C
AG
CC
ATC
CA
GG
AA
ATC
G
CTG
AA
CTG
GTC
CC
TCAA
GC
A
GTC
ATG
CTG
ATT
TCTG
TGC
C
GA
CC
TATC
CA
TAC
ATG
CC
C
GAA
GG
AAG
GAA
GG
AAG
AAAA
GA
TTA
GTA
AA
GA
GA
CA
GC
TGA
TTG
CC
A
TCTA
CC
CTG
CA
ACC
CTG
TG
ATT
GA
GA
CC
CA
AG
ACTG
TTA
GTG
G
GTA
AC
AAC
CA
GAG
TGTG
TGTG
A
CG
TCG
AG
CTC
CTG
GC
AT
Prim
er (f
orw
ard)
5acute -
gt 3acute
GG
GTA
GA
TTTC
AA
CA
AAT
AG
TAC
TGG
T
AG
AA
AG
GC
CTG
GA
TGTC
G
AG
TAG
AA
GA
GG
CC
AC
GC
AA
A
TTT
TCC
AA
AA
TGA
AA
AC
CTG
C
AG
ATT
GTT
TATG
CA
GG
CA
TTG
A
GA
GTT
GAA
AG
GTT
GAA
AA
TGG
G
CTC
AG
AG
GC
AG
ATA
GA
AA
A
ATT
GA
TTC
ATG
ACC
CA
CTA
A
TTT
TGTC
AAG
CA
GC
CC
TATA
TC
TG
AA
ATG
GTA
CTT
CA
GC
ATC
G
AG
CTT
AG
CTT
AC
TGC
CC
TGG
T
CC
CC
GG
CC
CC
TGC
CC
GG
AG
T
AC
CAC
TGTC
ATT
TTTC
CA
TGC
C
CC
TTA
GG
AGG
AG
GC
AAG
AC
C
CA
AG
GG
GTA
TGTT
GTC
TATT
ACT
GG
GTG
GG
AA
ATG
TGA
CTC
TG
GG
GC
TGC
CC
ATT
TCTT
TAA
T T
TGC
CA
CAA
ATC
AC
TTAA
AG
G
GA
CTG
AG
TTC
TTTC
AG
CA
CA
GTG
A
AAAT
AAC
ACAG
CAT
CAG
G
GA
ATC
CC
CA
ATG
TAC
ATG
GC
A
AC
AG
GC
GG
ATT
CTG
TAG
C
AC
TGG
CC
AAA
GA
GTA
CA
AG
G
CC
TTG
AC
TCA
GC
AGC
CC
TAC
A
AA
GA
TTG
TCTT
GA
CAC
GC
TG
GG
GTC
CTG
GG
ATC
AA
GC
C
AC
TGA
CTG
ATG
TCC
TGTG
CG
A
AA
AA
GTG
TAG
AG
CTT
TCTT
CA
AA
T
AA
ATT
CA
ATA
AG
CC
ATG
GA
GG
A
CC
TCC
AAG
ATG
GC
TCTT
GA
ATc
(degC
) 60
58
58
56
58
60
54
56
60
60
58
60
60
60
60
60
60
60
60
52
56
58
54
62
60
60
62
62
60
60
Pro
duct
si
ze (b
p)
380-
400
337-
409
267-
304
225-
235
312-
336
350-
394
155-
161
126-
156
510-
570
436-
468
189-
191
72-8
0 13
0-13
6 34
0-35
0 14
5-15
3 98
-102
22
0-24
0 21
2-24
0 34
7-40
3 32
5-34
1 25
1-27
9 32
5-36
1 19
7-24
5 30
9-40
9 34
1-37
5 21
5-23
1 30
8-31
6 16
2-17
4 46
8-50
2 18
2-19
0
Alle
les
6 14
8 4 7 11
3 6 8 7 2 2 3 4 2 3 5 8 12
7 8 7 14
10
9 7 3 3 15
2
Mbb
54
1
49
19
8 2
18
23
5 2
47
26
0 3
02
35
3 3
98
58
8 6
26
63
9 7
61
77
5 7
78
78
6 7
98
85
4 1
060
1
090
1
100
1
100
1
110
1
130
1
150
1
170
3
5
12
4 1
63
CFA
a
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10
10
10
Tabl
e 1
Cha
ract
eris
tics
of m
icro
sate
llite
mar
kers
sel
ecte
d fo
r CFA
1 1
0 an
d 31
com
pris
ing
in to
tal 6
0 m
arke
rs
(Con
tinue
d on
nex
t pag
e) T
he m
icro
sate
llite
mar
kers
wer
e al
l der
ived
from
the
NC
BI d
atab
ase
(http
w
ebn
cbin
lmn
ihg
ov)
Mar
ker
FH33
25
FH36
03
FH23
13
RE
N18
9I14
FH
3219
FH
3413
R
EN
136G
19
RE
N13
5K06
FH
3922
FH
3300
C
014
24
AH
T107
C
012
51
RE
N17
2C02
D
0150
5 R
EN
04H
08
RE
N15
9F24
FH
3894
FH
2309
R
EN
06N
11
FH23
26
FH33
22
FH35
05
FH25
98
FH26
34
FH22
94
RE
N22
9P15
FH
2537
FH
4081
C
107
81
Appendix microsatellite marker
IV
H
ETe
64
0 6
80
63
0 6
51
54
6 7
06
81
0 6
14
30
2 7
42
34
4 7
40
63
0 8
67
82
2 6
70
75
0 6
80
60
0 4
70
31
0 5
70
80
1 5
80
83
1 5
10
64
0 6
90
19
0 8
22
PIC
d
53
0 6
90
52
2 5
89
80
0 6
70
75
9 5
24
24
7 7
09
26
9 6
90
50
0 7
98
75
5 5
50
69
0 5
30
56
0 3
40
26
0 5
00
80
1 5
30
74
0 3
70
64
0 6
70
25
0 7
21
Prim
er (r
ever
se)
5acute -gt
3acute
AG
GC
CA
GC
AC
CA
TAA
CTC
AG
C
CTC
TCC
TCC
TTC
CC
CTT
CTC
A
GTG
CTG
TCC
ATT
GC
AA
GTC
T
GC
TCC
CC
TGTG
TTC
TGAG
G
CTG
GTG
ACTC
AGA
GAA
GTC
C
GG
AG
TTG
ATA
GTA
AA
TCTT
TGC
GG
T
GC
CC
TATT
CA
TTC
AA
CTG
G
AC
AC
TCTG
GTG
CA
AG
CG
AC
TC
TTC
TAC
TGA
AG
CA
TCC
GC
CT
GA
TTTT
CTC
TCTG
TCC
AC
TT
AA
CA
GC
ATT
TCA
GAC
AGA
GG
G
GG
TTTT
GG
CTG
TGA
GG
ATA
G
AG
TTC
CAG
ATC
GAG
TCC
CA
C
CA
CAT
GAT
TTC
ACTT
GTA
TATG
G
CC
TGA
GG
GTT
TTTA
CAG
TATG
G
TG
AA
ATC
TGG
GA
TTC
TGA
TAC
C
CC
AA
GC
TTTC
AC
CTA
ATC
AC
C
GA
TATT
TTTC
TCTC
CC
AC
CC
C
AC
ATT
TCTA
GG
CAG
TGG
CA
C
TGTC
CC
ATC
TAA
AG
GA
TAG
G
AG
TAA
TGC
TGC
CC
ACG
GA
AC
G
GC
TGG
AA
CC
TTC
CC
CTT
TAG
A
GC
TCTT
ACA
CG
CA
TTG
AG
G
AA
AC
GG
AG
TTC
CA
TCTC
TGG
C
AC
AG
TGA
GG
AG
GG
GTG
G
TG
TAA
CC
CTC
CTG
TGA
TATG
A
TGTG
AA
CC
CC
GC
CC
AATA
G
AA
GTG
AC
TGA
GTA
CC
TGA
AA
TCG
G
CC
TCTG
CC
TCTG
CC
TCTG
T G
AG
CC
CTG
TTC
TCA
GG
TTG
Prim
er (f
orw
ard)
5acute -
gt 3acute
AG
AC
CC
CC
CC
ACTA
CC
CC
AC
GC
GTT
AGC
TGC
CA
TCA
TCTT
G
CTG
GTT
GG
AG
CA
ATA
CC
AA
G
CC
ATT
CA
TGTT
GTT
GC
AG
ATG
T
AA
GG
AG
GG
CA
CTT
GAT
GG
C
TTA
TGG
CA
TTTG
GC
CTG
TC
AA
GG
AG
CAT
CTT
CC
AG
AAC
C
CG
CTC
ATG
CA
AG
TCA
TCA
CA
T C
TGG
CTT
AAA
CC
AC
TGG
TCA
C
CTT
CC
ATC
CC
GTT
GTG
TGT
ATT
CC
CC
AGC
GAT
ACC
A C
TTG
AG
TGG
CTT
GA
GTG
GC
TAC
T
TCTT
GC
TTC
TTG
AA
GTA
AG
CC
T
TGC
CC
GTC
CTA
TAC
TCC
TG
GA
AG
GTG
GTT
ATT
GTC
CTT
GG
G
AA
CC
ACA
GA
AG
AG
CTG
GA
A
GC
ATT
TGA
TGA
AA
TAA
AG
GG
A
GA
ATG
AA
AA
CG
GA
GC
AG
CA
G
CC
TTC
AAC
AC
CC
ATA
GC
TC
TC
AG
CA
ACTA
TAC
ATT
TAA
GA
GC
A
ATG
GA
CA
AATG
AA
CA
AA
AG
T
GTG
AC
TTTC
TTA
TCC
GC
CC
C
CC
AG
AA
AC
TCA
ACTG
ATG
C
TG
GA
CG
CTA
AG
CC
TGAC
TTT
CC
ATA
AG
ATA
CTC
AG
AA
AC
ATG
CA
C
AC
CAT
AA
ATG
GA
TGG
ATA
G
CA
GTG
AG
CA
AA
GC
AA
ATG
AA
C
CC
ATT
AG
CA
AA
TGA
CTG
GG
A
AA
GA
ATG
GG
AA
AA
CTG
ATA
A
AA
GG
TAG
TCC
CA
CG
ATC
CTC
ATc
(degC
) 60
60
60
60
60
60
60
60
60
60
60
60
60
62
60
60
60
60
60
60
60
60
60
60
60
60
60
60
60
60
Pro
duct
si
ze (b
p)
100-
110
112-
124
194-
206
210-
230
309-
341
350-
362
276-
300
96-1
30
130-
138
120-
138
200-
204
278-
296
157-
165
178-
254
358-
368
327-
337
380-
404
160-
170
320-
330
165-
169
230-
232
300-
310
276-
380
255-
263
258-
278
106-
120
164-
180
568-
608
223-
239
172-
186
Alle
les
3 5 3 5 8 4 7 4 2 5 3 7 5 9 6 4 7 3 4 2 2 3 11
5 6 2 4 7 4 8
Mbb
24
9 3
23
34
9 3
67
38
9 3
94
40
1 4
10
45
8 4
64
48
0 4
88
51
5 5
64
56
8 5
97
62
7 6
33
63
7 6
53
67
3 6
85
69
1 7
08
0
66
9
7
28
1 2
96
37
5
CFA
a
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
31
31
31
31
31
31
Tabl
e 1
(con
tinue
d)
Mar
ker
RE
N06
H21
R
EN
68B
08
FH30
55
C10
769
FH
3302
R
EN
181G
20
FH34
03
G02
512
RE
N15
0B12
A
HT1
01
RE
N73
F08
ZUB
EC
A1
C10
16
FH24
22
FH34
48
FH26
65
DTR
105
C
106
02
RE
N16
1L12
C
0410
7 R
EN
91H
07
RE
N15
4O19
FH
3381
R
EN
154G
10
FH21
89
RV
C11
R
EN
43H
24
FH22
39
RE
N11
0K04
FH
2712
a Can
ine
chro
mos
ome
b Pos
ition
of m
arke
r (M
b) c A
nnea
ling
tem
pera
ture
d P
olym
orph
ism
info
rmat
ion
cont
ent (
)
e obse
rved
Het
eroz
ygos
ity
Appendix itemisation of alleles V
Table 2 Itemisation of alleles to bases for SNPs in the pedigrees
Code no 1 2 3 4
Base Adenine Cytosine Guanine Thymine
Appendix laboratory paraphernalia
VI
Laboratory paraphernalia Equipment Thermocycler
PTC-100trade Programmable Thermal Controller (MJ Research Watertown USA)
PTC-100trade Peltier Thermal Cycler (MJ Research Watertown USA)
PTC-200trade Peltier Thermal Cycler (MJ Research Watertown USA)
Automated sequencers
LI-COR Gene Read IR 4200 DNA Analyzer (LI-COR Inc Lincoln NE USA)
LI-COR Gene Read IR 4300 DNA Analyzer (LI-COR Inc Lincoln NE USA)
MegaBACE 1000 (Amersham Biosciences Freiburg)
Centrifuges
Sigma centrifuge 4-15 (Sigma Laborzentrifugen GmbH Osterode)
Desk-centrifuge 5415D (Eppendorf Hamburg)
Biofuge stratos (Heraeus Osterode)
Centrifuge Centrikon H 401 (Kontron Gosheim)
Megafuge 1OR (Heraeus Osterode)
Speed Vacreg Plus (Savant Instruments Farmingdale NY USA)
Agarose gel electrophoresis and pulsed field gel electrophoresis
Electrophoresis chambers OWL Separation Systems Portsmouth NH USA
Biometra Goumlttingen
BioRad Muumlnchen
Generators 2301 Macrodrive 1 (LKB Bromma Sweden)
Power Pac 3000 (BioRad Muumlnchen)
Gel documentation system BioDocAnalyze 312 nm Goumlttingen
Appendix laboratory paraphernalia VII
Others
Milli-Qreg biocel water purification system (Millipore GmbH Eschborn)
Incubator VT 5042 (Heraeus Osterode)
UV-Illuminator 312 nm (Bachhofer Reutlingen)
Centomatreg R Desk-Shaker (B Braun Melsungen AG Melsungen)
Biophotometer (Eppendorf AG Hamburg)
Kits
DNA purification
Montage PCR96 Cleanup Kit (Millipore GmbH Eschborn)
Cloning
Invitrogen TA cloningreg kit (Invitrogen Karlsruhe Germany)
Isolation of DNA
QIAamp 96 DNA Blood Kit (QIAGEN Hilden)
Plasmid Mini Prep 96 Kit (Millipore GmbH Eschborn)
Sequencing
ThermoSequenase Sequencing Kit (Amersham Biosciences Freiburg)
DYEnamic-ET-Terminator Cycle Sequencing Kit (Amersham Biosciences Freiburg
Germany)
RACE
FirstChoiceTM RNA ligase-mediated (RLM)-RACE kit (Ambion Europe Huntingdon
UK)
Appendix laboratory paraphernalia
VIII
RNA Total RNA (Biocat Heidelberg Germany)
Size standards
100 bp Ladder (New England Biolabs Schwalbach Taunus)
1 kb Ladder (New England Biolabs Schwalbach Taunus)
IRDyetrade 700 or 800 (LI-COR Inc Lincoln NE USA)
Reagents and buffers APS solution (10 )
1 g APS
10 ml H2O
Bromophenol blue solution
05 g bromophenol blue
10 ml 05 M EDTA solution
H2O ad 50 ml
dNTP solution
100 microl dATP [100 mM]
100 microl dCTP [100 mM]
100 microl dGTP [100 mM]
100 microl dTTP [100 mM]
1600 microl H2O
the concentration of each dNTP in the ready-to-use solution is 5 mM
Gel solution
1275 ml UreaTBE solution (Roth Karlsruhe)
225 ml Rotiphoresereg Gel 40 (38 acrylamide and 2 bisacrylamide)
95 microl APS solution (10 )
95 microl TEMED
Appendix laboratory paraphernalia IX
Loading buffer for agarose gels
EDTA pH 8 100 mM
Ficoll 400 20 (wv)
Bromophenol blue 025 (wv)
Xylencyanol 025 (wv)
Loading buffer for gel electrophoresis
2 ml bromophenol blue solution
20 ml formamide
TBE-buffer (1x)
100 ml TBE-buffer (10x)
900 ml H2O
TBE-buffer (10x)
108 g Tris [12114 M]
55 g boric acid [6183 M]
744 g EDTA [37224 M]
H2O ad 1000 ml
pH 80
UreaTBE solution (6 )
425 g urea [6006 M]
250 ml H2O
100 ml TBE-buffer (10x)
solubilise in a water bath at 65degC
H2O ad 850 ml
Appendix laboratory paraphernalia
X
Chemicals Agarose (Invitrogen Paisley UK)
Ammonium persulfate (APS) ge 98 (Sigma-Aldrich Chemie GmbH Steinheim)
Ampicillin (Serva Heidelberg)
Boric acid ge 998 pa (Carl Roth GmbH amp Co Karlsruhe)
Bromophenol blue (Merck KgaA Darmstadt)
dATP dCTP dGTP dTTP gt 98 (Carl Roth GmbH amp Co Karlsruhe)
Chloramphenicol (Serva Heidelberg)
DMSO ge 995 pa (Carl Roth GmbH amp Co Karlsruhe)
dNTP-Mix (Qbiogene GmbH Heidelberg)
EDTA ge 99 pa (Carl Roth GmbH amp Co Karlsruhe)
Ethidium bromide (Carl Roth GmbH amp Co Karlsruhe)
Ethyl alcohol (AppliChem Darmstadt)
Formamide ge 995 pa (Carl Roth GmbH amp Co Karlsruhe)
LB (Luria Bertani) agar (Scharlau Microbiology Barcelona Spain)
Paraffin (Merck KgaA Darmstadt)
RotiphoreseregGel40 (Carl Roth GmbH amp Co Karlsruhe)
SephadexTM G-50 Superfine (Amersham Biosciences Freiburg)
TEMED 99 pa (Carl Roth GmbH amp Co Karlsruhe)
Tris PUFFERANreg ge 999 pa (Carl Roth GmbH amp Co Karlsruhe)
Urea ge 995 pa (Carl Roth GmbH amp Co Karlsruhe)
Water was taken from the water purification system Milli-Qreg
X-Gal (AppliChem Darmstadt)
Enzymes Taq-DNA-Polymerase 5 Umicrol (Qbiogene GmbH Heidelberg)
Taq-DNA-Polymerase 5 U microl (Qiagen Hilden Germany)
Incubation Mix (10x) TPol with MgCl2 [15 mM] (Qbiogene GmbH Heidelberg)
The polymerase was always used in the presence of incubation Mix TPol 10x buffer
The encyme ECO RI (New England Biolabs Schwalbach Taunus) were used with
the adequate 10x encyme buffer
Appendix laboratory paraphernalia XI
Consumables
96 Well Multiply PCR plates skirted (Sarstedt Nuumlmbrecht)
Combitipsreg plus (Eppendorf AG Hamburg)
Pipette tips 01 ndash 10 microl 01 ndash 10 microl 5 ndash 200 microl (Carl Roth GmbH amp Co Karlsruhe)
Reaction tubes 15 and 20 ml (nerbe plus GmbH WinsenLuhe)
Reaction tubes 10 und 50 ml (Falcon) (Renner Darmstadt)
Thermo-fast 96 well plate skirted (ABgene Hamburg)
Software BLAST trace archive httpwwwncbinlmnihgov
httpwwwensemblorg
EBI toolbox httpwwwebiacukToolssequencehtml
MERLIN 0102 package httpwwwsphumicheducsgabecasisMerlin
Order of primers MWG Biotech-AG Ebersberg (httpsecom
mwgdnacomregisterindextcl)
biomersnet GmbH Ulm (orderbiomersnet)
PED50 Dr H Plendl et al (2005) Institute for Human Genetics
Kiel
Primer design httpfrodowimiteducgi-binprimer3primer3_ wwwcgi
Repeat Masker httpwwwrepeatmaskergenome washingtonedu
Sequencher 42 GeneCodes Ann Arbor MI USA
Spidey httpwwwncbinlmnihgovIEBResearch
OstellSpideyindexhtml
SUN Ultra Enterprise 450 Sun microsystems
XIII
List of publications Journal articles 1 MIESKES K WOumlHLKE A DROumlGEMUumlLLER C DISTL O 2006 Molecular
characterization of the canine myosin heavy polypeptide 9 non-muscle (MYH9)
gene on dog chromosome 10q232 Submitted for publication in Gene
2 MIESKES K DISTL O 2006 Evaluation of 17 single nucleotide
polymorphisms (SNPs) in six candidate genes for hereditary non-syndromic deafness
in Dalmatian dogs Submitted for publication in Journal of Heredity
3 MIESKES K DISTL O 2006 Evaluation of the TMC1 and TMIE genes as
candidates for hereditary non-syndromic deafness in Dalmatian dogs Submitted for
publication in Animal Genetics
XV
Acknowledgements
First of all I wish to thank Prof Dr Dr habil Ottmar Distl the supervisor of my
doctoral thesis for his academic guidance and support of this work
I am very thankful to Heike Klippert-Hasberg and Stefan Neander for technical
expertise and assistance
I am very grateful to Joumlrn Wrede for his support during the statistical analyses and his
help with computer problems
I wish to express my appreciation to the Gesellschaft zur Foumlrderung Kynologischer
Forschung (GKF) eV Germany for funding this work with a grant
I am appreciative to all Dalmatian breeders and owners for providing me blood
samples and the results of the BAER tests
My special thanks go to all colleagues and friends of the Institute for Animal Breeding
and Genetics of the University of Veterinary Medicine Hannover for their support
humour and the friendly atmosphere in the laboratory
Last but not least I wish to thank my family for their support during the work on this
thesis
Contents
1 Introduction 1
2 A comparative overview of the molecular genetics of non-syndromic deafness in dogs and humans 5
Abstract 7
The structure of the ear 7
Deafness in man 8
Deafness in dogs 9
The canine genome project 11
3 Linkage analysis of gene-associated microsatellite markers with congenital sensorineural deafness in Dalmatian dogs 23
Abstract 25
Introduction 25
Material and methods 26
Results and discussion 28
4 Evaluation of eight candidate genes for canine congenital sensorineural deafness in Dalmatian dogs 41
Abstract 43
Introduction 43
Material and methods 44
Results and discussion 46
5 Molecular characterization of the canine myosin heavy polypeptide 9 non-muscle (MYH9) gene on dog chromosome 10q232 63
Abstract 65
Introduction 65
Materials and Methods 67
Results and Discussion 69
6 Identification of a 5 Mb region on canine chromosome 10 harbouring a causative gene responsible for congenital sensorineural deafness in German Dalmatian dogs 81
Abstract 83
Introduction 83
Material and methods 84
Results 86
Discussion 87
7 Analysis of canine chromosome 1 and the Gap junction protein alpha 1 (GJA1) gene in Dalmatian dogs segregating for congenital sensorineural deafness 97
Abstract 99
Introduction 99
Material and methods 100
Results and discussion 102
8 Analysis of canine chromosome 31 and the claudin-14 (CLDN14) gene in Dalmatian dogs segregating for congenital sensorineural deafness 107
Abstract 109
Introduction 109
Material and methods 110
Results and discussion 112
9 General Discussion 117
The candidate gene approach 119
Linkage and association analysis 120
CFA1 122
CFA31 123
CFA10 123
10 Summary 125
11 Erweiterte Zusammenfassung 129
12 References 145
13 Appendix I 14 List of publications XIII
Abbreviations
List of abbreviations
A adenine
Acc No accession number
ACTG1 actin gamma 1
AEP akustisch evozierte Potentiale (acoustically evoked potentials)
APS ammonium persulphate
AT annealing temperature
BAC bacterial artificial chromosome
BAER brain stem auditory evoked response
BLAST Basic Local Alignment Search Tool
bp base pair
C cytosine
CCSD canine congenital sensorineural deafness
CDH23 cadherin related 23
cDNA copy desoxyribonucleic acid
CFA chromosome of Canis familiaris
CLDN14 claudin-14
cM centiMorgan
COCH coagulation factor C homolog cochlin
COL11A2 collagen type XI alpha 2
CRYM crystallin mu
CSD cochleosaccular degeneration
CX connexin
DFN x-linked deafness locus
DFNA autosomal dominant deafness locus
DFNA5 deafness autosomal dominant 5
DFNB autosomal recessive deafness locus
DIAPH1 diaphanous homolog 1 (Drosophila)
DMSO dimethyl sulfoxide
Abbreviations
DNA deoxyribonucleic acid
dNTPs deoxy nucleoside 5rsquotriphosphates (N is ACG or T)
EDN3 endothelin 3
EDNRB endothelin receptor type B
EDTA ethylenediamine tetraaceticacid
EMBL European Molecular Biology Laboratory
ESPN espin
EST expressed sequence tag
EYA4 eyes absent homolog 4 (Drosophila)
F forward
FISH fluorescence in situ hybridisation
G guanine
GJA1 gap junction protein alpha 1 43kD (connexin 43)
GJB2 gap junction protein beta 2 26k (connexin 26)
GJB3 gap junction protein beta 3 31kDa (connexin 31)
GJB6 gap junction protein beta 6 (connexin 30)
GKF Gesellschaft zur Foumlrderung Kynologischer Forschung (Society for the
Advancement of Cynological Research)
HET observed heterozygocity
HE expected heterozygosity value
HSA chromosome of Homo sapiens
IBD identical by descent
IRD infrared dye
KCNQ4 potassium voltage-gated channel KQT-like subfamily member 4
Kb kilobase
LD linkage disequilibrium
LINE long interspersed nuclear element
LOD logarithm of the odds
M molar
Mb megabase
Merlin multipoint engine for rapid likelihood inference
MITF microphthalmia-associated transcription factor
Abbreviations
MS microsatellite
MTRNR1 mitochondrially encoded 12S RNA
MTTS1 mitochondrially encoded tRNA serine 1 (UCN)
MYH9 myosin heavy polypeptide 9 non-muscle
MYH14 myosin heavy polypeptide 14
MYO1A myosin IA
MYO3A myosin IIIA
MYO6 myosin VI
MYO7A myosin VIIA
MYO15A myosin XVA
NCBI National Center for Biotechnology Information
NMMHC-A nonmuscle myosin heavy chain-A
ODDD oculodentodigital dysplasia
OMIM Online Mendelian Inheritance in Man
OTOA Otoancorin
OTOF otoferlin
P error probability
PAX3 paired box gene 3 (Waardenburg syndrome 1)
PCDH15 Protocadherin-15
PCR polymerase chain reaction
PIC polymorphism information content
POU3F4 POU domain class 3 transcription factor 4
POU4F3 POU domain class 4 transcription factor 3
PRES solute carrier family 26 member 5 (prestin)
QTL quantitative trait locus
R reverse
RACE rapid amplification of cDNA ends
RH radiation-hybrid
RLM RNA ligase-mediated
RNA ribonucleic acid
RT-PCR reverse transcription polymerase chain reaction
SAS Statistical Analysis System
Abbreviations
SH1 Src homology 1
SINE short interspersed nuclear element
SLC26A4 solute carrier family 26 member 4 (pendrin)
SLC26A5 solute carrier family 26 member 5 (prestin)
SNP single nucleotide polymorphism
SOX10 SRY (sex determining region Y)-box 10
STRC stereocilin
STS sequence-tagged site
T thymine
TBE tris-borate-ethylenediamine tetraacetic acid
TECTA tectorin alpha
TEMED NNNrsquoNrsquo-tetramethylenediamine
TFCP2L3 transcription factor CP2-like 3
TJ tight juncions
TMC1 transmembrane channel-like gene 1
TMIE transmembran inner ear gene
TMPRSS3 transmembrane protease serine 3
U unit
USH1C Usher syndrome 1C
UTR untranslated region
WFS1 Wolfram syndrome 1 (wolframin)
wgs whole genome shotgun
WHRN whirlin
WS Waardenburg syndrome
Chapter 1
Introduction
Introduction 3
Introduction
Canine congenital sensorineural deafness (CCSD) has often been reported in the
literature and occurs in more than 54 different breeds of dogs with the Dalmatian dog
showing the highest incidence The inheritance and segregation of a major gene in
CCSD has been demonstrated in different Dalmatian dog populations But although
several studies have demonstrated the mode of inheritance in Dalmatian dogs no
universally accepted mode of inheritance for the other dog breeds affected by CCSD
has yet been identified
Breeding with blue-eyed Dalmatian dogs and unilaterally or bilaterally deaf dams and
sires is forbidden by paragraph 11b of the German animal welfare laws and thus the
hearing status of all Dalmatian dogs has to be tested as a puppy at about six to eight
weeks old using the brain stem auditory evoked response (BAER) test As deaf dogs
are very difficult to raise and often become aggressive and snappish from fear most
puppies suffering from bilateral hearing loss are euthanized However it has been
shown in recent years that auditory testing does not seem to be an effective way of
clearly reducing the high incidence of deafness in this breed Thus prevention of
CCSD cannot be achieved alone by exclusion of affected animals from breeding
Consequently a molecular genetic approach toward unravelling the responsible
genes in carriers is urgently needed
Many genetic disorders in humans and domestic dogs (Canis familiaris) demonstrate
a high level of clinical and molecular similarity Therefore the mutated genes in
human hereditary deafness seemed to be appropriate candidates for canine
congenital sensorineural deafness
The objective of the present study is to localize the gene that is involved in the
development of CCSD in Dalmatian dogs In order to achieve this goal successively
32 canidate genes were evaluated by means of linkage analyses using microsatellite
markers and single nucleotide polymorphisms (SNPs) This candidate gene
approach using gene-associated markers for linkage studies in families segregating
for deafness turned out to be little effective Therefore the canine chromosomes
(CFA) 1 CFA10 and CFA31 were scanned entirely with microsatellite markers
Additionally single nucleotide polymorphisms (SNPs) were developed for fine
mapping the identified CCSD regions
Introduction 4
Overview of chapter contents Chapter 2 reviews the identified 39 mutated genes causing non-syndromic hereditary
hearing impairment in humans Parallels and differences in canine and human
deafness are shown including the clinical signs inheritance patterns and
histopathology We located the humane deafness genes in the canine genome and
discussed the advantages of comparative genomics and different molecular genetic
approaches
In Chapter 3 an existing set of 43 microsatellite markers for in total 24 candidate
genes were used for a non-parametric linkage analysis with congenital sensorineural
deafness (CCSD) in Dalmatian dog families segregating for deafness
In Chapter 4 newly developed SNP markers associated with in total eight candidate
genes were evaluated for CCSD in Dalmatian dogs
In Chapter 5 the molecular characterization of the canine myosin heavy polypeptide
9 non-muscle (MYH9) gene on dog chromosome 10q232 is described
Chapter 6 7 and 8 present linkage analyses performed in Dalmatian dog families
segregating for congenital sensorineural deafness using microsatellite markers on
canine chromosome (CFA) 1 CFA10 and CFA31 and the results of fine mapping
regions linked with the CCSD phenotype using newly developed SNPs
Chapter 9 provides a general discussion and conclusions referring to Chapters 1-8
Chapter 10 is a concise English summary of this thesis while Chapter 11 is an
expanded detailed German summary which takes into consideration the overall
research context
Chapter 2
A comparative overview of the molecular genetics of non-syndromic deafness
in dogs and humans
Non-syndromic deafness in dogs and humans 7
A comparative overview of the molecular genetics of non-syndromic deafness in dogs and humans
Abstract
In man as in different dog breeds deafness is an often diagnosed disorder with the
Dalmatian dog showing the highest incidence Deafness in Dalmatian dogs is clearly
heritable and the presence of a recessive major gene affecting the disorder was
shown in several Dalmatian dog populations
This Chapter provides an overview of the identified 39 mutated genes causing
human non-syndromic hereditary hearing impairment as well as of the five genes
responsible for Waardenburg syndrome in humans We point out their cytogenetic
and genomic localisations in man and dog and compare the genomic and mRNA
sequences of these genes between man and dog Moreover an overview is given on
deafness genes-associated markers identified in Dalmatian dogs and on candidate
genes characterized in dogs
The structure of the ear The inner ear consists of the semicircular canals the vestibule and the cochlea
whereas the vestibule and the semicircular canals are concerned with vestibular
function (balance) and the cocnlea is concerned with hearing Reissneracutes membrane
and the basilar membrane divide the cochlea longitudinally into three scalae the
scala vestibule the scala media and the scala tympani The process of transduction
occurs in the structures within scala media sitting on the basilar membrane and
comprising the organ of Corti Cutting the cochlea tube cross sectionally the scala
media is more or less triangular formed by Reissneracutes membrane basilar
membrane and a structure called the stria vascularis The fluid that fills scala
tympani and scala vestibule is called perilymph the fluid that fills scala media is
called endolymph The organ of Corti rests on the basilar membrane within scala
media The cochlea contains an array of highly specialized cells arranged in a highly
Non-syndromic deafness in dogs and humans 8
specialized manner Two types of cells in the organ of Corti are support cells and
hair cells The hair cells are the receptor cells that trancsduce sound
When a sound wave brings physical displacement of the membranes separating the
perilymph from the endolymph they cause the organ of Corti to move and the hair
cells on it are scraped along the bottom of the tectorial membrane The tectorial
membrane is firmly anchored to the bone Relative movement of the organ of Corti
and its hair cells with respect to the tectorial membrane is the source of the
deformation of the hair cells microvilli The hair cells are so constructed that any
deformation of their microvilli will cause a change in the overall membrane potential
of the cell This signal is detected by the fibers from the cells in the spiral ganglion
These fibers are neural elements and they carry their own depolarization wave into
the auditory region of the brain
Deafness in man There are various ways to categorise deafness The two main types of deafness are
classified based on which portions of the auditory system are affected conductive
hearing loss occurs when when sound is not conducted efficiently through the outer
andor middle part of the ear Much more common is the sensorineural hearing loss
Sensorineural hearing loss occurs when there is damage to the inner ear (cochlea)
or to the nerve pathways from the inner ear (retrocochlear) to the brain Most cases
of sensorineural hearing loss are due to cochlear defects (Petit et al 2001)
Hearing loss can be present at birth (congenital) or become evident later in life
(acquired) Congenital deafness similarly may or may not be genetic In fact more
than half of congenital hearing loss is inherited Alternatively congenital deafness
may be due to a condition or infection to which the mother was exposed during
pregnancy Furthermore congenital hereditary deafness may occur as part of a
multisystem disease (syndromic) or as a disorder restricted to the ear and vestibular
system (non-syndromic) As non-syndromic hereditary hearing impairment is almost
exclusively caused by cochlear defects affected patients suffer from sensorineural
hearing loss In Table 1 and 2 the genes underlying human hereditary non-
syndromic deafness as a result of cochlear defects in consequence of primary
defects in hair cells non-sensory cells and the tectorial membrane or unknown cell
type are shown Non-syndromic deafness is estimated to cause 60-70 of cases of
congenital hereditary deafness in man (Morton 1991) In 70-80 of all cases this
Non-syndromic deafness in dogs and humans 9
non-syndromic form of deafness shows an autosomal recessive inheritance followed
by an autosomal dominant inheritance in 10-20 of all cases and 1-2 of all cases
are X-linked A maternally inherited form may also occur (Van Camp and Smith
2003)
Non-syndromic forms of hereditary deafness are classified by their mode of
inheritance DFN DFNA and DFNB refer to deafness forms inherited on the X
chromosome-linked autosomal dominant and autosomal recessive modes of
transmission respectively
Human hereditary isolated hearing loss is genetically heterogeneous (Petit et al
2001) Up to 1 of the human genes are estimated to be necessary for hearing
(Friedmann and Griffith 2003) Today approximately 120 genes for human
hereditary deafness have been identified approximately 80 for syndromic and 39 for
non-syndromic hereditary deafness which is suspected to be one-third of the total
(Nance 2003) In Table 1 the identified 39 mutated genes causing non-syndromic
hereditary hearing impairment in humans are shown Out of the 39 genes 15 genes
cause autosomal recessive and 15 genes cause autosomal dominant forms six
genes are involved in both recessive and dominant forms one gene causes X-linked
and two a maternally inherited form (The Hereditary Hearing Loss Hompage
httpwebhostuaacbehhh)
Furthermore several hundred forms of syndromes with hearing loss have been
documented in humans (Van Camp and Smith 2003) One is the human
Waardenburg syndrome (WS) which manifests itself with sensorineural deafness
and pigmentation defects in the iris hair and skin The WS is classified into four
types depending on the presence or absence of additional symptoms which are
caused by mutations in the five genes EDN3 EDNRB MITF PAX3 and SOX10
respectively These genes are shown in Table 3 They are known to be expressed in
the neural crest (EDN3 EDNRB PAX3 SOX10 ) or directly in the melanocytes
(MITF) and are inter alia involved in migration differentiation or survival of
melanocytes respectively (Bondurand et al 2000)
Deafness in dogs Congenital sensorineural deafness (CSD) has been reported in a variety of mammal
species other than humans ranging from mice to dogs guinea pigs and mink
Canine congenital deafness has often been reported in the literature and occurs in
Non-syndromic deafness in dogs and humans 10
more than 54 different breeds of dogs according to Strain (1996 and 2004) The
breeds with the highest incidence include Dalmatian dogs Bull Terrier English
Cocker Spaniel English Setter Australian Cattle Dog Australian Shepherd West-
Highland-White-Terrier Dobermann and Dogo Argentino The incidence of canine
congenital deafness is highest in Dalmatian dogs of which 165 to 30 exhibit
unilateral or bilateral hearing loss (Famula et al 1996 Wood and Lakhani 1997
Muhle et al 2002 Juraschko et al 2003a Rak and Distl 2005) The inheritance
and segregation of a major gene in canine congenital sensorineural deafness
(CCSD) has been demonstrated in different Dalmatian dog populations (Famula et
al 2000 Muhle et al 2002 Juraschko et al 2003b) But although several studies
have demonstrated the mode of inheritance in Dalmatian dogs no universally
accepted mode of inheritance for the other dog breeds affected by CCSD has yet
been identified
Congenital sensorineural hearing impairment can be recognised in dogs at four to
eight weeks of age (Strain 1996) while histological studies of deaf Dalmatian dogs
have shown that the degeneration of the inner ear structures begins as early as one
day after birth and is histologically clearly evident by four weeks of age (Johnsson et
al 1973) In 70 of the cases with human hereditary deafness the histological
pattern is known as cochleo-saccular degeneration (CSD) (Lalwani et al 1997)
commonly known as Scheibe dysplasia with preservation of the pars superior of the
membranous labyrinth and an unremarkable bony labyrinth As in man also in many
affected dog breeds the histological pattern of congenital sensorineural deafness is
known as cochleo-saccular degeneration
Breeding with blue-eyed Dalmatian dogs and unilaterally or bilaterally deaf dams and
sires is forbidden by paragraph 11b of the German animal welfare laws and thus the
hearing status of all Dalmatian dogs has to be tested as a puppy at about six to eight
weeks old using the brainstem auditory evoked response (BAER) test that detects
electrical activity in the cochlea and auditory pathways in the brain Although the
BAER test is a reliable method for identifying unilaterally and bilaterally deaf dogs it
does not seem to be an effective way of clearly reducing the incidence of deafness in
affected breeds particularly in a recessive mode of inheritance so that hearing dogs
can still be genetic carriers Furthermore deaf dogs are very difficult to raise and
often become aggressive and snappish from fear consequently most puppies
Non-syndromic deafness in dogs and humans 11
suffering from bilateral hearing loss are euthanized Thus prevention of CCSD
cannot be achieved alone by exclusion of affected animals from breeding and
consequently a molecular genetic approach toward unravelling the responsible
genes in carriers is urgently needed
Over the past decade it has become increasingly clear how far structural and
functional homologies at the gene level extend across even distantly related species
Much is known about deafness-causing gene mutations in humans and mice
including the fact that the clinical and histopathological findings are often very similar
to those of deafness in Dalmatian dogs Thus despite the genetical heterogeneity of
human non-syndromic deafness the genes that are responsible for non-syndromic
congenital hereditary deafness in humans (Table 1) seemed to be appropriate
candidate genes for CCSD especially in Dalmatian dogs (Rak and Distl 2005) The
genes that are mutated in the human WS (Table 2) were selected as candidates
because the WS phenotype where the deafness is associated with pigmentation
defects seems to be similar to the phenotype of most affected dog breeds (Strain
and Tedford 1996) Both Juraschko et al (2003a) and Strain et al (1992) have
demonstrated that patched Dalmatians are less likely to be deaf than unpatched
animals and blue-eyed Dalmatians are more likely to be affected from hearing
impairment than brown-eyed animals
In an attempt to achieve the aim of molecular genetic diagnosis of CCSD carriers
Rak et al (2003) and co-workers (Droumlgemuumlller et al 2002 Kuiper et al 2002 Rak
et al 2002a 2002b 2003) already mapped 24 potential candidate genes for
sensorineural deafness in dogs by fluorescence in situ hybridization and a radiation
hybrid panel to 16 different canine chromosomes
The canine genome project In December 2005 an international research team led by scientists at the Broad
Institute of MIT and Harvard achieved the completion of a high-quality genome
sequence of the domestic dog together with a catalog of 25 million specific genetic
differences across several dog breeds (Lindblad-Toh et al 2005) The authors found
that humans share more of their ancestral DNA with dogs than with mice confirming
the utility of dog genetics for understanding human disease Furthermore the
physiology disease presentation and clinical response of dogs often mimic human
Non-syndromic deafness in dogs and humans 12
diseases closely As indicated above hearing impairment seemed to be no
exception
In the last years most projects have exploited canine traits for which either direct
candidate genes could be proposed and evaluated or for which large informative
pedigrees were available to enable linkage mapping to identify candidate regions A
major component of such research efforts comprised the cloning sequencing and
mapping of individual canine homologs of genes either proposed as candidate
genes or expected to be located in candidate regions This was necessary to
identify new informative polymorphisms (eg single nucleotide polymorphisms
(SNPs) microsatellites) for high resolution mapping of candidate regions and to
examine each exon and exonintron boundary for positional candidates Availability
of the second version of the dog genome assembly (build 21) of the NCBI database
shortcut this effort and increase the investigators efficency
The current RH map with 3200 markers provides a good estimate of the order and
physical spacing (ie in base pairs) of markers along canine chromosomes (Guyon
et al 2003) and was recently complemented by the construction of a 4249-marker
integrated canine genome RH map that consists of 900 genes 1589 microsatellites
and 1760 BAC end markers (Breen et al 2004) all included and available in the
NCBI database The second version 1 of the NCBIs genome annotation consists of
large contigs covering all canine chromosomes given with their located markers and
genes The great majority of genes are derived by automated computational analysis
using the gene prediction method GNOMON
With this help either additional candidate genes for canine CSD can be found directly
by its gene symbol in the 21 of the NCBIs genome annotation or if a candidate gene
is yet not annotated a BLAST (Basic Local Alignment Search Tool) search versus
the canine whole genome shotgun (wgs) sequence resource can be used to obtain
the sequence of the canine genomic contigs containing the human homologous
gene The localisation of all 39 known human non-syndromic hereditary deafness
genes in the canine genome with the corresponding accession numbers of the contig
and if available the accession number of the genomic sequence and mRNA of the
canine gene are shown in Tables 4 and 5 Furthermore the identity of canine and
human or mouse mRNA is shown in Table 5 The average identity of canine and
human mRNA is with 088 percent higher than the average identity of canine and
mouse mRNA with 084 percent Canine sequences that correspond to the human
Non-syndromic deafness in dogs and humans 13
candidate gene can now be used to find microsatellite or SNP markers associated to
the respective canine gene These markers can be used for linkage and haplotype
studies in dog families segregating for deafness
Table 7 shows the microsatellite and SNP markers developed for in total 32
candidate genes for CCSD
The candidate genes for which a set of in total 43 microsatellite marker were
designed by Rak (2003) included the following 24 genes CDH23 CLDN14 COCH
COL11A2 DFNA5 DIAPH1 EDN3 EDNRB EYA4 GJA1 GJB2 GJB6 MITF
MYH9 MYO6 MYO7A MYO15A OTOF PAX3 POU4F3 SLC26A4 SOX10
TECTA and TMPRSS3 This existing set of 43 microsatellite markers for 24
candidate genes were used for linkage and haplotype studies in Dalmatian dog
families segregating for deafness (Chapter 3) These 24 genes are known to be
involved either in human non-syndromic deafness or in the human Waardenburg
syndrome For another eight candidate genes including TMC1 TMIE USH1C
MYH14 MYO3A PRES WHRN and ESPN SNP markers were newly developed
(Chapter 4) and subsequently used for linkage and association analyses in
Dalmatian dog families segregating for deafness These genes are also involved in
human non-syndromic deafness
Non-syndromic deafness in dogs and humans 14
Tabele 1 Genes responsible for non-syndromic congenital hereditary deafness in
humans
Inheritance Gene Gene product Type of molecule Locus namea
ACTG1 γ-Actin cytoskeletal protein DFNA20 DFNA26
COCH Cochlin extracellular matrix component DFNA9
COL11A2 Collagen XI (α2-chain) extracellular matrix component DFNA13
CRYM micro-Cristallin thyroid hormone-binding protein DFNAb DFNA5 Unidentified Unidentified DFNA5 DIAPH1 Diaphanous-1 cytoskeleton regulatory protein DFNA1 EYA4 EYA4 transcriptional coactivator DFNA10 GJB3 Connexin-31 gap junction protein DFNA2 KCNQ4 KCNQ4 K+ channel subunit DFNA2 MYH14 Myosin IIC motor protein DFNA4 MYH9 Myosin IIA motor protein DFNA17 MYO1A Myosin IA motor protein DFNA48 POU4F3 POU4F3 transcription factor DFNA15 TFCP2L3 TFCP2L3 transcription factor DFNA28
Autosomal dominant
WFS1 Wolframin endoplasmic-reticulum membrane protein DFNA6 DFNA14
CDH23 Cadherin-23 cell-adhesion protein DFNB12 CLDN14 Claudin-14 tight-junction protein DFNB29 ESPN Espin actin-bundling protein DFNB36 DFNAb MYO15 Myosin XV motor protein DFNB3 MYO3A Myosin IIIA motor protein DFNB30 OTOA Otoancorin cell-surface protein DFNB22 OTOF Otoferlin putative vesicle traffic protein DFNB9 PCDH15 Protocadherin-15 cell-adhesion protein DFNB23 SLC26A4 Pendrin IminusndashClminus transporter DFNB4 SLC26A5 Prestin anion transporter DFNB61 STRC Stereocilin DFNB16
TMIE TMIE transmembrane domain- containing protein DFNB6
TMPRSS3 TMPRSS3 transmembrane serine protease DFNB8 DFNB10 USH1C Harmonin PDZ domain-containing protein DFNB18
Autosomal recessive
WHRN Whirlin PDZ domain-containing protein DFNB31 GJB2 Connexin-26 gap junction protein DFNB1 DFNA3 GJB6 Connexin-30 gap junction protein DFNB1 DFNA3 MYO6 Myosin VI motor protein DFNA22DFNB37MYO7A Myosin VIIA motor protein DFNB2DFNA11
TECTA α-Tectorin extracellular matrix component DFNA8 DFNA12DFNB21
Autosomal dominant and autosomal recessive
TMC1 TMC1 transmembrane channel-like protein
DFNB7 DFNB11DFNA36
X-linked POU3F4 POU3F4 transcription factor DFN3
MTRNR1 Mitochondrial 12S rRNA not defined
nomenclature Mitochondrial
MTTS1 Mitochondrial 12S rRNA not defined
nomenclature a Autosomal recessive loci are designated DFNB autosomal dominant loci DFNA
Non-syndromic deafness in dogs and humans 15
Table 2 Genes underlying hereditary non-syndromic deafness as a result of primary
defects in hair cells non-sensory cells and the tectorial membrane or unknown cell
type
Primary defect
Gene
Hair cells MYO7A MYO15 MYO6 MYO3A MYO1A ACTG1 USH1C
WHRN CDH23 PCDH15 TMIE STRC SLC26A5 ESPN
KCNQ4 TMC1 OTOF POU4F3
Non-sensory cells
GJB2 GJB6 GJB3 SLC26A4 CRYM OTOA CLDN14
COCH TMPRSS3 MYH9 MYH14 EYA4 POU3F4
Tectorial membrane
COL11A2 TECTA
Unknown
DIAPH1 DFNA5 WFS1 TFCP2L3 MTRNR1 MTTS1
Table 3 Genes involved in the human Waardenburg syndrome
Inheritance Gene Gene product Type of molecule Type
EDN3 endothelin 3 vasoconstricted peptide WS type IV4
EDNRBendothelin
receptor type B receptor protein WS type IV4
MITF
microphthalmia-
associated
transcription
factor
transcription factor WS type II2
PAX3 paired box 3 DNA-binding protein WS type I1and III 2
SOX10 SRY-box 10 transcription factor WS type IV4 1Type I Dystopia canthorum 2Type II No dystopia canthorum 3Klein-Waardenburg syndrome (type III) Type I and upper limb abnormalities 4Waardenburg-Shah syndrome (type IV) Type II and Hirschsprung disease
(autosomal recessive inheritance)
Non-syndromic deafness in dogs and humans 16
Leng
th o
f co
ntig
(bp)
7799
0652
3821
0901
7077
2986
7251
5492
5989
7527
4533
7677
6093
8239
9498
326
6961
0260
1859
5814
6421
1953
Scor
e of
co
ntig
614
938
792
567
300
715
323
535
1208
1634
923
E-va
lue
of
cont
ig
4 E
-172
0 0
2 E
-158
1 e-
78
0
2 e-
85
7 E
-149
0 0 0
Acc
No
of
WG
S co
ntig
unkn
own
NW
_876
311
NW
_876
295
NW
_876
327
NW
_876
254
NW
_876
321
NW
_876
292
NW
_876
258
NW
_876
315
NW
_876
269
NW
_876
278
NW
_876
259
Mb
from
to
unkn
own
254
12
578
337
93
379
132
11
323
563
56
5
272
12
723
393
33
943
411
64
123
632
86
331
292
82
955
209
32
094
101
81
019
Gen
e lo
catio
n on
C
FA
unkn
own
4 31
8 12
6 2 14
5 1 25
15
Can
ine
gene
al
iase
s
none
none
LOC
4877
51
LOC
4906
40
LOC
4817
34
LOC
4798
18
none
LOC
6112
23
LOC
4896
31
EYA4
GJB
2
LOC
4824
86
Acc
No
hu
man
m
RN
A
NM
_001
614
NM
_022
124
NM
_144
492
NM
_004
086
NM
_080
680
NM
_001
888
NM
_005
219
NM
_004
403
NM
_031
475
NM
_172
105
NM
_004
004
NM
_024
009
Gen
e lo
catio
n on
H
SA
17
10
21
14
6 16
5 7 1 6 13
1
Tabl
e 4
Loca
lisat
ion
of h
uman
non
-syn
drom
ic h
ered
itary
dea
fnes
s ge
nes
in th
e ca
nine
gen
ome
the
cani
ne
gene
loc
alis
atio
n in
meg
abas
es (
Mb)
th
e ac
cess
ion
num
ber
(Acc
No
) of
the
who
le g
enom
e sh
otgu
n (W
GS
) con
tig c
onta
inin
g th
e hu
man
hom
olog
ous
gene
and
the
corr
espo
ndin
g E
-val
ue s
core
and
leng
th o
f th
e co
ntig
Hum
an
deaf
ness
ge
ne
AC
TG1
CD
H23
CLD
N14
CO
CH
CO
L11A
2
CR
YM
DIA
PH1
DFN
A5
ESPN
EYA
4
GJB
2
GJB
3
der
ived
from
the
NC
BIs
can
ine
geno
me
anno
tatio
n ve
rsio
n 2
1
Non-syndromic deafness in dogs and humans 17
Leng
th o
f co
ntig
6421
1953
5300
4996
5294
2087
2607
3285
1654
5469
1249
9463
7251
5492
5102
4781
5989
7527
5159
1990
1284
7264
7521
5785
Scor
e of
co
ntig
525
521
973
2256
348
567
1236
404
337
383
604
1715
E-va
lue
of
cont
ig
3 e-
146
1 e-
144
0 0
9 e-
93
2 e-
158
0
3 e-
109
3 e-
89
7 e-
103
2 e-
169
0
Acc
No
of
WG
S co
ntig
unkn
own
NW
_876
259
NW
_876
270
NW
_876
251
NW
_876
313
NW
_876
250
NW
_876
290
NW
_876
254
NW
_876
273
NW
_876
321
NW
_876
263
NW
_876
283
NW
_879
563
Mb
from
to
unkn
own
521
52
3
109
24
109
35
311
33
119
443
64
441
415
41
7
103
41
056
404
14
050
245
42
460
261
32
619
235
02
359
371
43
769
674
86
748
Gen
e lo
catio
n on
C
FA
25
15
1 10
5 10
2 12
21
6 17
26
X
Can
ine
gene
al
iase
s
none
LOC
4824
51
none
LOC
4812
80
LOC
4795
22
LOC
4744
10
LOC
4871
06
LOC
4818
84
LOC
4851
74
LOC
6086
55
LOC
6079
61
none
LOC
4919
88
Acc
No
hu
man
m
RN
A
NM
_006
783
NM
_004
700
NM
_024
729
NM
_002
473
NM
_016
239
NM
_005
379
NM
_017
433
XM
_376
516
NM
_000
260
NM
_144
672
NM
_194
248
NM
_033
056
NM
_000
307
Gen
e lo
catio
n on
H
SA
13
1 19
22
17
12
10
6 11
16
2 10
X
Tabl
e 4
(con
tinue
d)
Hum
an
deaf
ness
ge
ne
GJB
6
KC
NQ
4
MYH
14
MYH
9
MYO
15
MYO
1A
MYO
3A
MYO
6
MYO
7A
OTO
A
OTO
F
PCD
H15
POU
3F4
der
ived
from
the
NC
BIs
can
ine
geno
me
anno
tatio
n ve
rsio
n 2
1
Non-syndromic deafness in dogs and humans 18
Leng
th o
f co
ntig
4533
7677
2532
0482
2532
0482
4020
6070
3002
9677
2968
9717
5300
4996
3309
7591
3821
0901
5102
4781
1104
8438
6535
5756
Scor
e of
co
ntig
1404
283
283
1683
883
529
354
216
198
354
2238
689
E-va
lue
of
cont
ig
0
6 e-
73
2 E
-73
0 0
5 e-
147
2 e-
94
4 e-
53
9 e-
48
2 E
-94
0 0
Acc
No
of
WG
S co
ntig
NW
_876
292
NW
_876
265
NW
_876
265
NW
_876
294
NW
_876
312
NW
_876
255
NW
_876
270
NW
_876
272
NW
_876
295
NW
_876
273
NW
_876
256
NW
_876
253
Mb
from
to
436
14
361
158
61
592
197
91
982
134
31
345
158
81
595
622
63
7
880
88
822
450
54
505
390
33
905
432
44
328
414
94
151
716
47
172
Gen
e lo
catio
n on
C
FA
2 18
18
30
5 13
1 20
31
21
13
11
Can
ine
gene
al
iase
s
LOC
4872
00
LOC
4832
63
LOC
4832
74
LOC
4782
78
LOC
4893
57
LOC
4819
85
LOC
4841
68
LOC
6093
50
LO
C61
0987
LOC
6108
50
LOC
4821
13
LOC
6125
88
Acc
No
hu
man
m
RN
A
NM
_002
700
NM
_000
441
NM
_206
883
NM
_153
700
NM
_005
422
NM
_024
915
NM
_138
691
NM
_147
196
NM
_024
022
NM
_153
676
NM
_006
005
NM
_015
404
Gen
e lo
catio
n on
H
SA
5 7 7 15
11
8 9 3 21
11
4 9
Tabl
e 4
(con
tinue
d)
Hum
an
deaf
ness
ge
ne
POU
4F3
SLC
26A
4
SLC
26A
5
STR
C
TEC
TA
TFC
P2L3
TMC
1
TMIE
TMPR
SS3
USH
1C
WFS
1
WH
RN
der
ived
from
the
NC
BIs
can
ine
geno
me
anno
tatio
n ve
rsio
n 2
1
Non-syndromic deafness in dogs and humans 19
Leng
th o
f co
ntig
(bp)
4776
3139
5561
1003
2518
2130
3091
5115
5294
2087
Scor
e of
co
ntig
262
721
2927
967
1179
E-va
lue
of
cont
ig
8e-6
7
0 0 0 0
Acc
No
of
WG
S co
ntig
NW
_876
277
NW
_876
274
NW
_876
271
NW
_876
304
NW
_876
251
Mb
from
to
470
14
703
343
63
438
248
52
488
313
43
144
297
52
976
Gen
e lo
catio
n on
C
FA
24
22
20
37
10
Can
ine
gene
al
iase
s
ED
N3
ED
NR
B
MIT
F
PAX3
LOC
4812
58
Acc
No
hu
man
m
RN
A
NM
_207
032
NM
_000
115
NM
_198
159
NM
_181
457
NM
_006
941
Gen
e lo
catio
n on
H
SA
20
13
3 2 22
Tabl
e 5
Loca
lisat
ion
of g
enes
invo
lved
in th
e hu
man
Waa
rden
burg
syn
drom
e (W
S)
in th
e ca
nine
gen
ome
th
e ca
nine
gen
e lo
calis
atio
n in
meg
abas
es (
Mb)
th
e ac
cess
ion
num
ber
(Acc
No
) of
the
who
le g
enom
e sh
otgu
n (W
GS
) con
tig c
onta
inin
g th
e hu
man
hom
olog
ous
gene
and
the
corr
espo
ndin
g E
-val
ue s
core
and
le
ngth
of t
he c
ontig
Hum
an
deaf
ness
ge
ne
EDN
3
EDN
RB
MIT
F
PAX3
SOX1
0
der
ived
from
the
NC
BIs
can
ine
geno
me
anno
tatio
n ve
rsio
n 2
1
Non-syndromic deafness in dogs and humans 20
Table 6 Canine candidate genes for CCSD with their accession number (AccNo) of
the genomic sequence and mRNA and if available the percent identity of canine and
human or mouse mRNA Canine candidate gene
Acc No canine genomic sequence
Acc No canine mRNA (predicted)
Canine mRNA (bp)
Identity () of canine and human mRNA
Identity () of canine and mouse mRNA
ACTG1 none1 none unknown unknown unknown CDH23 none1 none unknown 8800 8690 CLDN14 NC_006613 XM_544876 714 8880 8460 COCH NC_006590 XM_547762 3050 8770 8170
COL11A2 NC_006594 XM_857285 5202 8970 8740 CRYM NC_006588 XM_845361 1279 8720 7890
DIAPH1 none1 none unknown 8630 unknown DFNA5 NC_006596 XM_848863 1512 8320 7680 ESPN NC_006587 XM_546751 2565 8930 8270 EYA4 NC_006583 XM_541108 3969 9240 8120 GJB2 NC_006607 XM_543177 2208 7860 6720 GJB3 NC_006597 XM_539603 1305 8550 8090 GJB6 none none unknown unknown unknown
KCNQ4 NC_006597 XM_539568 1836 9350 8760 MYH14 none1 none unknown 8770 8220 MYH9 NC_006592 XM_538401 6201 9130 8940
MYO15 NC_006587 XM_536660 10128 8500 7960 MYO1A NC_006592 XM_531642 3388 8600 8350 MYO3A NC_006584 XM_544234 5589 8510 8910 MYO6 NC_006594 XM_862465 3983 9250 8610
MYO7A NC_006603 XM_542292 6519 9190 8880 OTOA NC_006588 XM_845746 3558 8740 7990 OTOF NC_006599 XM_844665 5994 9020 8780
PCDH15 none1 none unknown 8210 7390 POU3F4 NC_006621 XM_549108 1086 9190 9060 POU4F3 NC_006584 XM_544328 1017 9380 9160 SLC26A4 NC_006600 XM_540382 2382 7970 8240 SLC26A5 NC_006600 XM_540393 2235 9250 8740
STRC NC_006612 XM_535452 5301 8980 8520 TECTA NC_006587 XM_546475 7136 9110 8510
TFCP2L3 NC_006595 XM_539106 2127 9030 8760 TMC1 NC_006583 XM_541284 2580 8860 8740 TMIE NC_006602 XM_846596 396 9110 8910
TMPRSS3 NC_006613 XM_848589 1542 8500 8340 USH1C NC_006603 XM_860072 1730 9010 8670 WFS1 NC_006595 XM_539234 2667 8510 8420 WHRN NC_006593 XM_850321 2817 8440 8110 EDN3 NC_006606 NM_001002942 1976 747 716
EDNRB NC_006604 NM_001010943 1329 889 827 MITF NC_006602 XM_850501 1590 934 862 PAX3 NC_006619 XM_545664 1474 869 861
SOX10 NC_006592 XM_538379 1987 926 900 derived from the NCBIs canine genome annotation version 21 1 the definite region in the whole genome sequence (WGS) contig was used to get the percent identity of mRNAs
Non-syndromic deafness in dogs and humans 21
Table 7 Microsatellite markers and single nucleotide polymorphisms (SNPs)
of canine candidate genes for canine congenital sensorineural deafness in
Dalmatian dogs
Canine candidate gene
Number of gene-associated
microsatellites
Number of gene-associated
SNPs
CDH23 2 0 CLDN14 3 8 COCH 2 0
COL11A2 2 0 DIAPH1 2 0 DFNA5 2 0 ESPN 0 5 EYA4 2 0 GJB2 3 0 GJB3 1 0 GJB6 1 0
MYH14 0 2 MYH9 2 22
MYO15 2 0 MYO3A 0 3 MYO6 1 0
MYO7A 3 0 OTOF 1 0 PAX3 1 0
POU4F3 1 0 SLC26A4 1 0 SLC26A5 0 2 TECTA 2 0 TMC1 1 1 TMIE 1 3
TMPRSS3 2 0 USH1C 0 2 WHRN 0 3
Chapter 3
Linkage analysis of gene-associated microsatellite markers with
congenital sensorineural deafness in Dalmatian dogs
Linkage analysis of gene-associated microsatellites 25
Linkage analysis of gene-associated microsatellite markers with congenital sensorineural deafness in Dalmatian dogs
Abstract Deafness is a disorder often diagnosed in different dog breeds In this Chapter an
existing set of 43 microsatellite markers associated with in total 24 candidate genes
for canine congenital sensorineural deafness (CCSD) were used for linkage and
haplotype analyses in a large Dalmatian dog population with frequent occurrence of
CCSD We found significant linkage for the genes GJA1 MYH9 and CLDN14 As
linkage was found for different candidate genes in different families the results of
these test statistics indicate that the inheritance of non-syndromic deafness in
Dalmatian dogs is heterogenic in origin
Introduction
Canine congenital sensorineural deafness (CCSD) has been reported to occur in
more than 54 different breeds of dogs (Strain 1996) As in man also in dog breeds
the most commonly observed histological pattern of degenerative inner ear changes
is known as the cochleo-saccular or Scheibe type of end organ degeneration
The incidence of this congenital anomaly is highest in Dalmatian dogs of which 165-
30 exhibit unilateral or bilateral hearing loss (Famula et al 1996 Wood and
Lakhani 1997 Muhle et al 2002 Juraschko et al 2003a Rak and Distl 2005) The
inheritance and segregation of a major gene in CCSD has been demonstrated in
different Dalmatian dog populations (Famula et al 2000 Muhle et al 2002 Juraschko et al 2003b) Moreover deafness in Dalmatian dogs seems to be
pigment-associated (Greibrokk 1994 Holliday et al 1992 Juraschko et al 2003a
2003b Mair 1976 Strain et al 1992 Strain 1996)
No gene mutation has yet been identified that is responsible for CCSD in Dalmatian
dogs or in one of the various other dog breeds that suffer from inherited hearing
impairment Since mutations in various genes have already been found to be the
cause of sensorineural hearing impairment in humans or mice 24 of these genes
Linkage analysis of gene-associated microsatellites
26
were considered as candidates for CCSD in Dalmatian dogs (Rak et al 2005)
Details of the 24 candidate genes are given in Table 1
Rak et al (2003) and co-workers (Droumlgemuumlller et al 2002 Kuiper et al 2002 Rak et
al 2002a 2002b 2003) mapped this 24 potential candidate genes for sensorineural
deafness in dogs by fluorescence in situ hybridization and a radiation hybrid panel
Subsequently Rak (2003) developed altogether 43 new highly polymorphic DNA
markers for the 24 candidate genes including CDH23 CLDN14 COCH COL11A2
DFNA5 DIAPH1 EDN3 EDNRB EYA4 GJA1 GJB2 GJB6 MITF MYH9 MYO6
MYO7A MYO15A OTOF PAX3 POU4F3 SLC26A4 SOX10 TECTA and
TMPRSS3 (Table 2)
Among the 24 candidate genes seven genes cause autosomal dominant non-
syndromic forms of deafness seven cause autosomal recessive forms and five
genes cause both recessive and dominant forms of non-syndromic deafness in
different human families segregating for either forms
The functions of these 19 deafness-causing genes are diverse and include gap
junctions and tight junctions (GJA1 GJB2 GJB6 CLDN14) ion channels (SLC26A4)
and ion channel activators (TMPRSS3) Included are also unconventional myosins
(MYO6 MYO7A MYH9 MYO15A) transcription factors (POU4F3 EYA4) as well as
extracellular matrix components (COCH COL11A2 TECTA) a cytoskeleton
regulatory protein (DIAPH1) a cell-adhesion protein (CDH13) and genes with
unknown or only suspected functions (DFNA5 OTOF) The 24 candidates also
include five genes which are mutated in the human Waardenburg syndrome (WS)
The WS is classified into four types depending on the presence or absence of
additional symptoms which are caused by mutations in the five genes EDN3
EDNRB MITF PAX3 and SOX10 respectively The objective of the current study
was to use this set of markers developed by Rak (2003) for a non-parametric linkage
analysis with CCSD in a German and French Dalmatian dog population
Material and methods Pedigree material
For the linkage analysis we used DNA from altogether 215 animals belonging to a
total of 24 Dalmatian dog families The families included 22 full-sib families and one
large paternal half-sib family of German Dalmatian dogs as well as 46 animals of a
Linkage analysis of gene-associated microsatellites 27
large paternal half-sib family of French Dalmatian dogs All families were segregating
for CCSD The genotyped families included all affected dogs (unilaterally and
bilaterally deaf) their parents if available and one to four unaffected animals At least
two of the full sibs of each family were unilaterally deaf
In total these 24 families included 402 individuals with an average family size of 168
ranging from 5 to 116 animals and covering two to four generations The hearing
status of 344 dogs was examined by veterinarians using the BAER (brain stem
auditory evoked response) test and the other animals included in the pedigree being
not BAER tested were used to construct relationships among CSD affected dogs
The prevalence of CSD in this pedigree was 285
Microsatellite marker analysis An existing marker set consisting of 43 microsatellite markers (Table 2) was used for
linkage analysis This set included 36 markers developed by Rak (2003) and 7
markers of the RH map available at httpwww-recomgenuniv-rennes1frdoggyhtml
For most of the 24 candidate genes two markers were available for two of the
candidates three markers were available but for seven candidate genes the set
contains only one marker The marker set is composed of 33 perfect repeats two
imperfect six compound-perfect and two compound-imperfect repeats
The majority (674) of the 43 markers in the set was represented by dinucleotide
repeats 209 were tetranucleotide repeats 47 trinucleotide repeats and 23
pentanucleotide repeats In addition one marker (23) was a compound di-
tetranucleotide and another one (23) was a compound tetra-pentanucleotide
repeat The average number of alleles was 35 with a minimum of 2 and a maximum
of 8 different alleles per marker
PCR reactions for microsatellites were carried out in 12 microl reaction mixtures
containing 2 microl genomic DNA 12 microl 10x PCR buffer (Qbiogene Heidelberg
Germany) 024 microl dimethyl sulfoxide (DMSO) 02 microl dNTPs (100 microM) 01 microl Taq
Polymerase (5Umicrol) (Qbiogene) 06 microl (10 microM) 5rsquo-IRD700 or IRD800 labelled forward
primer 06 microl (10 microM) unlabelled reverse primer The PCR reactions were carried out
in MJ Research thermocyclers with the following program 4 min at 94 degC followed
by 32 cycles of 30 sec at 94degC 30 sec at maximum annealing temperature and a
final extension of 45 sec at 72degC PCR-products were diluted with formamide loading
buffer in ratios from 110 to 140 and then size-fractionated by gel electrophoresis on
Linkage analysis of gene-associated microsatellites
28
automated LI-COR 42004300 sequencers (LI-COR inc Lincoln NE USA) using
denaturing 4 and 6 polyacrylamide gels (RotiphoreseregGel 40 Roth Karlsruhe
Germany)
To localize the 24 candidate genes and their associated microsatellites exactly the
canine candidate gene sequences were derived from sequences deposited in the
current dog genome assembly (Boxer genome assembly 21) of the NCBI GenBank
by BLAST (Basic Local Alignment Search Tool) search
(httpwwwncbinlmnihgovBLAST) using the human reference mRNA sequence
(Table 3)
Linkage analysis
Multipoint linkage and haplotype analyses were performed using the MERLIN
software version 0102 (multipoint engine for rapid likelihood inference Center for
Statistical Genetics University of Michigan MI USA Abecasis et al 2002) The test
statistics Z-mean and Lod score were used to test for the proportion of alleles shared
by affected individuals identical by descent (IBD) for the considered marker loci
Linkage analyses were performed regarding the marker set consisting of 43 gene-
associated microsatellite markers Linkage analysis was at first carried out for all 24
families conjoined After this the families were scanned separately
The data of the genotypes was additionally analyzed using SASGenetics (Statistical
Analysis System Version 913 SAS Institute Inc Cary NC USA 2005) to specify
the number of alleles of each marker the allele frequency the observed (HET) and
expected (HE) heterozygosity and the polymorphism information content (PIC)
(Table 4 and 5)
Results and discussion
Test statistics for all families conjoined are given in Table 6 Significant CCSD loci
were located on different chromosomes The loci were located on canine
chromosome (CFA) 1 10 12 20 and 31 Linkage analyses per family indicated even
higher test statistics for subgroups of families (Table 7) Scanning only families with
Zmeans gt1 test statistics for linkage increased for the genes GJA1 on CFA1 MYH9
on CFA10 and CLDN14 on CFA31 with congenital sensorineural deafness in different
Dalmatian dog families (Table 7) Therefore it is probable that these genes or genes
Linkage analysis of gene-associated microsatellites 29
in their flanking regions are involved in the development of the disease in the
respective familes The results of this test statistics indicate that the inheritance of
non-syndromic hearing loss in Dalmatian dogs is probably as heterogenic in origin as
it is in humans Genetic heterogeneity means that different mutations cause the same
phenotype or disease the different mutations can either be found at the same locus
(allelic heterogeneity) or even at different loci (non-allelic heterogeneity)
The extreme heterogeneity of human deafness often hampered genetic studies
because many different genetic forms of hearing loss give rise to similar clinical
phenotypes and conversely mutations in the same gene can result in a variety of
clinical phenotypes In man genes that transport ions across membranes to maintain
appropriate solute concentration and pH as well as regulatory genes mainly
transcription factors and genes that play a part in structural integrity are essential for
the hearing process
However this study was a first step in identifying genes responsible for CCSD in
Dalmatian dogs The genes GJA1 MYH9 and CLDN14 and their flanking regions will
be further analyzed with a combined approach using microsatellites and single
nucleotide polymorphisms (SNPs) (Chapter 6-9) As linkage was found for different
candidate genes in different families subsequently only the families indicating
linkage will be chosen for further molecular analyses of the respective gene
To confirm the result of this study the density of the intragenic markers has to be
increased The current RH map with 3200 markers provides a good estimation of the
order and physical spacing (ie in base pairs) of markers along canine
chromosomes (Guyon et al 2003) and was recently complemented by the
construction of a 4249-marker integrated canine genome RH map which consists of
900 genes 1589 microsatellites and 1760 BAC end markers (Breen et al 2004) all
included and available in the NCBI database (httpwebncbinlmnihgov)
Thus microsatellites derived from the NCBI database could be used to confirm the
linkage Alternatively BLAST searches versus the canine whole genome shotgun
(wgs) sequence resource were perfomed to localize the genes exactly and to obtain
the sequence of the canine genomic contigs containing the human homologous gene
The results of the BLAST searches of the 24 candidate genes against the Boxer
genome assembly 21 are shown in Table 3 The genomic sequence of the
respective candidate gene can now be used to search for intragenic SNPs as these
polymorphisms are the most abundant and useful markers for fine mapping
Linkage analysis of gene-associated microsatellites
30
Development of SNPs requires sequencing of DNA for the respective genomic
regions of the parents with the aim to identify heterozygous base pair exchanges
After a heterozygous base pair is found the whole family can be genotyped for this
informative SNP marker These polymorphisms can than be used for linkage
analyses as well as association studies
Fine mapping using SNP markers for all genes indicating linkage with CCSD
identified by this study should enable us to detect mutations responsible for CCSD in
parts of the Dalmatian dog population
Linkage analysis of gene-associated microsatellites 31
Table 1 Details of the 24 selected human candidate genes
Symbol Gene name Locus name1 Most important reference
CDH23 cadherin related 23 DFNB12 Bork et al 2001
CLDN14 claudin 14 DFNB29 Wilcox et al 2001
COCH coagulation factor C homolog cochlin DFNA9 Robertson et al 1998
COL11A2 collagen type XI alpha 2 DFNA13 McGuirt et al 1999
DFNA5 deafness autosomal dominant 5 DFNA5 Van Laer et al 1998
DIAPH1 diaphanous homolog 1 (Drosophila) DFNA1 Lynch et al 1997
EDN3 endothelin 3 WS type IV Edery et al 1996
EDNRB endothelin receptor type B WS type IV Attie et al 1995
EYA4 eyes absent homolog 4 (Drosophila) DFNA10 Wayne et al 2001
GJA1 gap junction protein alpha 1 43kD (connexin 43)
2 Liu et al 2001
GJB2 gap junction protein beta 2 26k (connexin 26) DFNA3 DFNB1 Kelsell et al 1997
GJB6 gap junction protein beta 6 (connexin 30) DFNA3 DFNB1 Grifa et al 1999
Del Castillo et al 2002
MITF microphthalmia-associated transcription factor WS type II Tassabehji et al 1994
MYH9 myosin heavy polypeptide 9 non-muscle DFNA17 Lalwani et al 2000
MYO6 myosin VI DFNA22 DFNB37 Melchionda et al 2001 Ahmed et al 2003
MYO7A myosin VIIA DFNA11 DFNB2 Liu et al 1997a Liu et al 1997b Weil et al 1997
MYO15A myosin XVA DFNB3 Wang et al 1998
OTOF otoferlin DFNB9 Yasunaga et al 1999
PAX3 paired box gene 3 (Waardenburg syndrome 1) WS type I WS type III Hoth et al 1993 Tassabehji
et al 1992
POU4F3 POU domain class 4 transcription factor 3 DFNA15 Vahava et al 1998
SLC26A4 solute carrier family 26 member 4 DFNB4 Li et al 1998
SOX10 SRY (sex determining region Y)-box 10 WS type IV Pingault et al 1998
TECTA tectorin alpha DFNB21 DFNA8DFNA12
Mustapha et al 1999 Verhoeven et al 1998
TMPRSS3 transmembrane protease serine 3 DFNB8DFNB10 Scott et al 2001
1 Autosomal recessive loci are designated DFNB autosomal dominant loci DFNA 2 To our knowledge no deafness locus has been determined for this autosomal recessive gene
Linkage analysis of gene-associated microsatellites
32
HET
()
638
338
366
544
616
632
456
656
433
614
458
586
529
PIC
()
752
548
336
484
775
70
63
9
74
6
61
6
83
7
62
762
621
No
of
alle
les
6 4 4 4 9 8 6 6 6 13 5 11 5
PCR
pr
oduc
t (b
p)
175
109
123
156
146
179
259
151
241
219
186
214
191
AT
(degC
)
62 60 56 60 60 62 58 60 62 58 58 60 58
Prim
er s
eque
nces
(5rsquorarr
3rsquo)
F A
ATC
AG
GA
GG
GG
TGAG
TGTG
R
CC
CC
CAG
CTC
ATA
CA
TTC
TC
F C
CTG
TAC
TGA
ATG
CTT
GA
GG
R
CA
TCTC
TAG
AA
GA
AG
CC
TCC
F
TC
AC
ATA
GC
ATT
ATA
TATG
GA
C
R T
TGA
GAT
GG
CTC
TTAC
TGA
G
F T
CG
ATG
ATG
CTT
TCTG
GTT
G
R A
GG
CTG
TGA
AA
TGG
ATG
GA
G
F G
AG
AA
GC
AC
CA
GG
CA
TAG
G
R T
GG
TTTA
GC
AA
GG
CTG
TTC
C
F T
GA
CA
TAC
GG
AG
GAC
CA
AG
AG
R C
CC
CTC
CC
TTG
CTC
TATC
TC
F T
GC
CC
CTC
AG
AG
ATA
ATC
AC
R
CTT
CA
ATTA
TAC
AC
ACA
GG
TAC
F
TG
AA
TATG
GG
GC
TGAG
GA
AG
R
TTC
TCC
CTC
TGC
CTG
TGTC
F
GG
TTTA
GC
AC
TGC
CTT
CA
GC
R
CA
TTA
AG
CA
TCTG
GC
ATG
TGG
F
GA
AA
AC
TCA
GA
TTA
GC
CTG
G
R A
TCTT
GA
GA
GC
AA
AGG
TTG
TG
F T
GG
TTA
GG
GC
ATG
ATTC
CA
G
R C
ATG
TATA
AA
GA
GTA
ATG
CC
AG
F
CG
GG
AG
AGG
GTT
TGAC
TAC
R
CTC
CG
TATT
GC
TCA
TCTT
TCC
F
AG
CTT
CC
CTT
CTC
TGA
GA
C
R G
AG
AA
TAG
AG
TTTG
TGC
TCA
G
Rep
eat
(TTT
A)1
5 (A
CC
)9(A
TC)3
(A
TTT)
7(G
TTT)
5 (C
A)2
1 (G
A)2
0 (C
TTT)
~20
(GA
)13
(AC
)20(
AG)9
(C
T)16
TT
(CT)
5 (A
AA
AT)
~25
(CT)
11(G
T)4(
CT)
2 (A
TTT(
T))2
3 (A
C)2
2
Mar
ker n
ame
CD
H23
_MS1
C
DH
23_M
S2_F
2 C
LDN
14_M
S1
CLD
N14
_MS
2 C
LDN
14_M
S3
CO
CH
_MS1
C
OC
H_M
S2
CO
L11A
2_M
S1
CO
L11A
2_M
S3
DFN
A5_
MS
1 D
FNA
5 _M
S2
DIA
PH
1 _M
S1
DIA
PH
1_M
S2
Mar
ker o
rigin
RP
CI8
1-99
C20
R
PC
I81-
99C
20
RP
CI8
1-97
L17
RP
CI8
1-97
L17
RP
CI8
1-97
L17
RP
CI8
1-32
1I4
RP
CI8
1-32
1I4
RP
CI8
1-24
M6
RP
CI8
1-24
M6
RP
CI8
1-66
C12
R
PC
I81-
66C
12
RP
CI8
1-36
2I5
RP
CI8
1-36
2I5
Tabl
e 2
Can
dida
te g
ene-
asso
ciat
ed m
arke
rs f
or c
anin
e de
afne
ss d
evel
oped
by
Rak
(20
03)
For
each
gen
e th
e m
icro
sate
llite-
base
d m
arke
r th
e m
arke
r or
igin
rep
eat t
ype
PC
R p
rimer
s w
ith o
ptim
ised
con
ditio
ns a
nd th
e nu
mbe
r of
alle
les
pol
ymor
phis
m
info
rmat
ion
cont
ent (
PIC
) and
obs
erve
d he
tero
zygo
sity
(HE
T) fo
r all
dogs
gen
otyp
ed a
re s
how
n
Can
dida
te
gene
CD
H23
C
LDN
14
CO
CH
C
OL1
1A2
DFN
A5
DIA
PH
1
Linkage analysis of gene-associated microsatellites 33
HET
()
815
345
695
578
562
508
583
441
75
63
7
33
414
30
76
7
44
8
PIC
()
853
679
798
774
652
63
72
3
59
5
83
2
77
9
50
5
54
1
40
4
88
9
59
1
No
of
alle
les
14 6 7 6 9 5 9 6 13 13 4 6 4 14 6
PCR
pr
oduc
t (b
p)
147
131
258
227
134
190
148
195
218
260
181
164 96
227 94
AT
(degC
)
60 58 58 58 60 60 58 62 58 58 62 56 58 58 62
Prim
er s
eque
nces
(5rsquorarr
3rsquo)
F G
CTA
GG
AA
AA
ATC
CG
CA
ATG
R
GAC
CC
CC
TAG
GAC
ACC
AAC
F
GA
GA
ATT
GG
GC
ATG
GG
CAG
A
R T
GA
CTT
TATC
AC
TGG
TCTT
TG
F T
TATG
CAG
CC
CA
TGAC
AA
TC
R C
AA
GG
GA
AC
TCA
AAG
GC
TTG
F
TG
GA
CC
AG
GTC
AGTT
TGTG
R
TC
TGC
CTG
TGTC
TCTG
CC
F
ATG
GC
ATG
AA
GA
GG
ATA
CC
G
R A
GG
AC
AGG
TGAC
GG
CTC
TAC
F
GC
TAG
TAC
TCG
ATT
GTG
GTC
R
TC
ATG
GG
TTG
TGA
GA
TCC
AG
F T
TAA
TTTG
CTC
GTC
TTC
CTG
R
TG
TAA
GC
TCC
ACG
GA
TCA
CC
F
CTC
TCTT
GG
TCTC
CC
TCTG
C
R G
GG
AG
TAG
GG
GTG
GAG
TAG
G
F G
GTG
TTTC
CTT
TCC
TTTT
CT
R G
GTG
TTC
TCTC
CC
TTTC
TCT
F C
TCTA
TGA
AA
GG
TGA
TTG
CC
R
CA
GC
CAT
AC
AA
ATG
AGA
ATT
G
F C
TAC
AG
TGA
ATC
AG
CA
CA
GAC
R
CA
GC
CTT
GA
CTG
TTTC
TTTG
G
F T
GA
TTG
AC
TCTA
CTT
TAC
AC
A
R T
ATA
TTA
GG
CG
GTT
TTC
TTC
T F
AC
CC
AG
GTG
GC
CTG
ATTG
R
GC
AC
GC
AC
GTT
CTC
TCTT
TC
F G
AA
TGC
CC
TTC
ACC
TTG
AA
A
R G
GA
AA
AG
GA
GA
GA
TGA
TGC
C
F T
CTT
CC
TTG
GA
AA
GG
GA
AC
TC
R T
GC
CC
TAA
CA
CTT
GG
AA
TGG
Rep
eat
(TA
GA
)12
(GA
)25
(GT)
10(A
T)13
(A
G)2
1 (G
T)16
(A
G)1
2 (C
A)1
2 (G
A)8
(C
TTT)
~20
(GA
AA
)~25
(G
T)12
(C
A)1
5 (A
G)1
1 (G
AA
A)~
43
(AC
)13
Mar
ker n
ame
ED
N3_
MS
1 E
DN
RB
_M
S1
EY
A4_
MS
1 E
YA
4_M
S2
GJA
1_M
S1
GJA
1_M
S2
GJB
2_M
S1
GJB
2_M
S2
GJB
6_M
S2
GJB
2+6_
MS1
M
ITF
_MS
2 M
ITF
_MS
3 M
YH
9_M
S2
MY
H9_
MS
3 M
YO
6_M
S2
Mar
ker o
rigin
RP
CI8
1-36
6E14
Z
emke
et a
l (1
999)
R
PC
I81-
301N
19
RP
CI8
1-30
1N19
R
PC
I81-
370A
16
RP
CI8
1-37
0A16
R
PC
I81-
133O
22
RP
CI8
1-13
3O22
R
PC
I81-
343C
15
FH
2324
R
PC
I81-
119P
24
RE
N10
0J13
R
PC
I81-
374A
12
FH
2293
R
PC
I81-
156P
14
Tabl
e 2
(con
tinue
d)
Can
dida
te g
ene
ED
N3
ED
NR
B
EY
A4
GJA
1 G
JB2
GJB
6 M
ITF
MY
H9
MY
O6
Linkage analysis of gene-associated microsatellites
34
HET
()
359
366
431
566
51
50
5
28
3
35
8
79
9
70
4
69
2
23
9
68 71
698
PIC
()
496
623
627
765
635
756
502
575
756
76
70
1
44
2
75
4
84
5
80
6
No
of
alle
les
5 4 6 13 7 8 5 3 10 11 8 8 8 10 11
PCR
pr
oduc
t (b
p)
293
166
100
137
174
197
164
201
160
297
267
140
276
228
260
AT
(degC
)
56 60 56 62 62 62 60 60 58 58 56 58 58 62 60
Prim
er s
eque
nces
(5rsquorarr
3rsquo)
F T
GG
TTA
AA
AC
ATT
AA
AC
TTA
TAG
R
TA
GTA
TATA
GA
GA
TGC
AA
TGG
F
CA
TTG
GG
TGC
TTTC
CTG
TTC
R
TG
GA
GC
TGC
AG
GTA
TAG
CC
F
CC
AG
GC
ATT
CG
AG
GG
TG
R C
AG
AA
CTT
GA
GG
AAC
CA
TAG
F
CC
ATG
AAC
TTTG
TGG
AA
CTG
C
R A
AA
GG
GTT
GC
TGTG
GA
GA
TG
F A
GG
CA
GG
TTC
ATC
TGTG
TCC
R
TC
CC
AG
AC
CC
AG
CTA
CA
TTC
F
CA
GC
CAA
CTG
TATT
CTC
CTT
G
R A
TCTT
GA
GC
CC
TGC
ATT
AG
G
F G
AA
GC
GA
GG
AG
AG
ACA
GTC
C
R A
AG
GA
AG
CC
TCC
TGAC
AA
CC
F
CA
GG
GTC
AG
GC
TCTA
TGC
TC
R T
CC
TATC
ATC
CG
GC
TTTG
AC
F
TC
TGG
ATTG
TGG
TCA
CA
AC
C
R A
CTG
GAC
AC
TTC
TTTT
CA
GAC
G
F A
AA
GTG
GC
TGG
TTC
GG
AA
G
R A
GC
AG
CA
GC
ATA
CA
TTC
CTC
F
AA
GTA
GA
TCC
TATT
ATC
GTG
G
R A
GTT
TCA
GTG
TCTG
TTA
AA
TAG
F
CC
GG
ATT
TCTG
AG
GAG
GC
R
CA
TGC
TCTT
CA
CC
AGA
AC
C
F T
CA
GC
ATG
GA
TTTT
GTA
AA
ATC
R
GG
AC
TGC
GTG
GA
CAT
CTG
F
AC
AC
GG
TTC
TCG
CTG
ATG
TG
R T
GA
AG
GG
GA
TTG
AAC
AG
AG
G
F T
TCA
TTC
CG
AG
GTT
CTA
AC
TG
R C
AC
CA
TCTC
GTA
GC
CTT
TATC
Rep
eat
(TC
)13
(AC
)12
(GT)
12
(AC
)18
(GT)
12
(TA
)3(T
G)9
(TA
)2(C
A)2
C
(TA
AA
)5
(AA
T)18
(C
T)13
(T
G)1
3 (C
T)17
(T
AA
A)1
4 (G
T)3
CC
(GT)
19
(ATT
T)12
(T
C)8
(CA)
5(C
G)2
(CA)
9 (G
T)16
Mar
ker n
ame
MY
O7A
_M
S1
MY
O7A
_M
S2
MY
O7A
_M
S3
MY
O15
A _
MS
1 M
YO
15A
_M
S2
OTO
F _M
S1
PA
X3_
MS
1 P
AX
3_M
S2
PO
U4F
3_M
S4
SLC
26A
4_M
S2
SO
X10
_MS
2 T
EC
TA _
MS
1 T
EC
TA _
MS
2 T
MP
RS
S3_
MS
1 T
MP
RS
S3_
MS
2
Mar
ker o
rigin
RP
CI8
1-19
3deg2
RP
CI8
1-19
3deg2
AH
TH29
8 R
PC
I81-
362deg
13
RP
CI8
1-36
2deg13
R
PC
I81-
198L
15
RP
CI8
1-25
7H23
R
PC
I81-
257H
23
G2C
024
66
RP
CI8
1-47
P17
RP
CI8
1-50
5H2
RP
CI8
1-59
C2
RP
CI8
1-59
C2
RP
CI8
1-12
5P17
A
HTH
246
Tabl
e 2
(con
tinue
d)
Can
dida
te
gene
MY
O7A
M
YO
15A
O
TOF
PA
X3
PO
U4F
3 S
LC26
A4
SO
X10
TE
CTA
TM
PR
SS
3
Linkage analysis of gene-associated microsatellites 35
Table 3 The gene-associated microsatellites (MS) for 24 candidate genes of canine
congenital sensorineural deafness were localized using BLAST searches against the
Boxer genome assembly 21 The accession numbers (AccNo) of the whole
genome shotgun (WGS) contigs containing the genes as well as their associated
microsatellites are given
Microsatellite Acc No of WGS contig CFA Location of
Gene (Mb) Location of
MS (bp) Location of
MS in relation to gene
CDH23_MS1 NW_876311 4 2541025780 25510 intragenic
CDH23_MS2 NW_876311 4 2541025780 25630 intragenic
CLDN14_MS1 NW_876295 31 3379533796 33790 proximal
CLDN14_MS2 NW_876295 31 3379533796 33950 distal
CLDN14_MS3 NW_876295 31 3379533796 33790 proximal
COCH_MS1 NW_876327 8 1321513232 13225 intragenic
COCH_MS2 NW_876327 8 1321513232 13290 distal
COL11A2_MS1 NW_876254 12 56315659 5608 proximal
COL11A2_MS3 NW_876254 12 56315659 5578 proximal
DFNA5_MS1 NW_876258 14 4116941237 41135 proximal
DFNA5_MS2 NW_876258 14 4116941237 41250 distal
DIAPH1_MS1 NW_876292 2 3933039430 39382 intragenic
DIAPH1_MS2 NW_876292 2 3933039430 39552 distal
EDN3_MS1 NW_876277 24 4701347032 47057 distal
EDNRB_MS1 Zemke et al (1999) 22 3436334383 34354 proximal
EYA4_MS1 NW_876269 1 2928029550 29531 intragenic
EYA4_MS2 NW_876269 1 2928029550 29500 intragenic
GJA1_MS1 NW_876269 1 6399463996 64150 distal
GJA1_MS2 NW_876269 1 6399463996 64160 distal
GJB2_MS1 NW_8762781 25 2093620942 20938 intragenic
GJB2_MS2 NW_8762781 25 2093620942 20863 proximal
GJB2+6_MS1 FH2324 25 2093620942 17543 proximal
GJB3_MS2 NW_876259 15 1018310194 4530 proximal
GJB6_MS2 NW_876278 25 2090420906 20953 distal
Linkage analysis of gene-associated microsatellites
36
Table 3 (continued)
Microsatellite Acc No of WGS contig CFA Location of
Gene (Mb) Location of
MS (bp) Location of
MS in relation to gene
MITF_MS2 NW_876271 20 2485324884 24844 proximal
MITF_MS3 REN100J13 20 2485324884 25668 distal
MYH9_MS2 NW_876251 10 3113531193 31244 distal
MYH9_MS3 FH2293 10 3113531193 31696 distal
MYO15A_MS1 NW_876313 5 4436944419 44330 proximal
MYO15A_MS2 NW_876313 5 4436944419 44409 intragenic
MYO6_MS2 NW_876254 12 4041740504 40534 distal MYO7A_MS1 NW_876273 21 2454324609 24465 proximal
MYO7A_MS2 NW_876273 21 2454324609 24566 intragenic
MYO7A_MS3 AHTH298 21 2454324609 24594 distal
OTOF_MS1 NW_876263 17 2350223595 23463 proximal
PAX3_MS1 NW_876304 37 3134831445 31426 intragenic
PAX3_MS2 NW_876304 37 3134831445 31481 distal
POU4F3_MS4 G2C02466 2 4361043612 - -
SLC26A4_MS2 NW_876265 18 1586715927 15960 distal
SOX10_MS2 NW_876251 10 2975129762 29740 proximal
TECTA_MS1 NW_876312 5 1588515954 15910 intragenic
TECTA_MS2 NW_876312 5 1588515954 15930 intragenic
TMPRSS3_MS1 NW_876295 31 3903539054 38942 proximal
TMPRSS3_MS2 AHTH246 31 3903539054 38508 proximal
Linkage analysis of gene-associated microsatellites 37
Table 4 Number of alleles observed (HET) and expected (HE) heterozygosity and
polymorphism information content (PIC) for the developed marker-set
Feature Mean SD Min Max
No of alleles 75 31 3 14
hO () 703 122 370 898
hE () 532 151 239 815
PIC () 667 130 336 889
Table 5 Number of alleles per microsatellite locus and their PIC () values of the
developed marker-set
No of alleles per
microsatellite
Number of marker
loci PIC ()
3 1 575
4 6 483
5 5 574
6 11 652
7 2 716
8 5 671
9 3 717
10 2 800
11 3 776
13 4 803
14 2 871
Linkage analysis of gene-associated microsatellites
38
Table 6 Significant test statistics for linkage analysis carried out for all 24 genotyped
families conjoined Zmeans and LOD scores are given with their respective error
probabilities for the gene-associated markers of the candidate genes CLDN14
COL11A2 GJA1 MITF MYH9 and SOX10
Marker Location on canine chromosome (CFA)
Zmean pZmean LOD score pLOD
CLDN14_MS1 31q15 134 009 086 002
CLDN14_MS2 31q15 168 005 105 001
CLDN14_MS3 31q15 108 014 049 007
COL11A2_MS1 12q11-q12 166 005 085 002
COL11A2_MS3 12q11-q12 167 005 078 003
GJA1_MS1 1q24-q25 151 007 118 001
GJA1_MS2 1q24-q25 151 007 118 001
MITF_MS2 20q13 101 02 080 003
MITF_MS3 20q13 121 011 104 001
MYH9_MS2 10q232 080 02 018 02
MYH9_MS3 10q232 175 004 097 002
SOX10_MS2 10q21-q23 146 007 110 001
Linkage analysis of gene-associated microsatellites 39
Table 7 Significant test statistics for linkage analyses carried out each family
separately Zmeans and LOD scores are given with their respective error probabilities
for the gene-associated markers of the candidate genes CLDN14 MYH9 and GJA1
Gene-associated
marker
Number of families with significant linkage to
CCSD
Number of corresponding
family members
Zmean pZmean LOD-score pLOD
CLDN14_MS1 51 40 278 0003 112 0011
CLDN14_MS2 383 000007 170 0003
CLDN14_MS3 281 0002 113 0011
MYH9_MS2 32 21 081 02 023 02
MYH9_MS3
(=FH2293) 156 006 058 005
GJA1_MS1 13 46 295 0002 052 006
GJA1_MS2 286 0002 051 006 1 families with significant linkage include four German full-sib families and one
German half-sib family 2 families with significant linkage include three German full-sib families 3 families with significant linkage include one French half-sib family
Chapter 4
Evaluation of eight candidate genes for canine congenital sensorineural deafness
in Dalmatian dogs
Evaluation of eight candidate genes for CCSD 43
Evaluation of eight candidate genes for canine congenital sensorineural deafness in Dalmatian dogs Abstract
In this study we have been focusing on genomic loci that encode various enzymes
and transporters involved in the hearing process in humans We developed intragenic
markers for the canine genes ESPN MYH14 MYO3A PRES TMC1 TMIE USH1C
and WHRN on canine chromosomes (CFA) 1 2 5 11 18 20 and 21 which have
been shown to be responsible for human hereditary deafness and to employ these
newly developed markers for non-parametric linkage analyses with canine congenital
sensorineural deafness (CCSD) We screened DNA from altogether six Dalmatian
dogs which represent the parents of four families for single nucleotide polymorphisms
(SNPs) in the eight candidate genes by means of direct sequencing combined with a
polymerase chain reaction method for amplifying genomic DNA We characterized 20
SNPs and one insertion-deletion polymorphism For the TMC1 and TMIE genes we
additionally genotyped one microsatellite marker each The families used for
subsequent genotyping of the markers included 39 members from four full-sib
families with frequent occurrence of CCSD We concluded that mutations in ESPN
MYH14 MYO3A PRES TMC1 TMIE USH1C and WHRN do not play a role in
CCSD of the Dalmatian dog population investigated here
Introduction Over the past ten years significant progress has been made in the identification of
genes causing different forms of human deafness Currently 39 of the genes
responsible for non-syndromic hearing impairment have been identified in different
human populations (The Hereditary Hearing Loss Hompage
httpwebhostuaacbehhh Van Camp and Smith 2003)
Since non-syndromic hereditary hearing impairment is almost exclusively caused by
cochlear defects affected patients suffer from sensorineural hearing loss
Evaluation of eight candidate genes for CCSD
44
The most common histopathologic finding in cases of profound congenital deafness
in humans is the cochleosaccular degeneration (CSD) It is estimated to occur in
approximately 70 of cases in man and also in dog breeds the histological pattern is
known as cochleosaccular degeneration
Many genetic disorders in humans and domestic dogs (Canis familiaris) demonstrate
a high level of clinical and molecular similarity Therefore genes responsible for
human hereditary deafness seem to be appropriate candidate genes for CCSD
especially in Dalmatian dogs
In this report we provide 21 single-nucleotide polymorphisms (SNPs) and two
microsatellite markers in altogether eight selected human candidate genes (Table 1)
This eight candidate genes were only recently identified for being responsible for
different form of human non-syndromic deafness In order to evaluate whether any of
this candidate genes is responsible for congenital sensorineural deafness in
Dalmatian dogs we analyzed the association of the ESPN MYH14 MYO3A PRES
TMC1 TMIE USH1C and WHRN haplotypes with the CCSD phenotype in four
families of Dalmatian dogs with frequent occurrence of CCSD
Material and methods Pedigree structure and sampling
For the linkage analysis we used blood samples from 39 Dalmatian dogs They
belong to four full-sib families segregating for CCSD At least two of the full sibs of
each family were unilaterally deaf The phenotype of the affected animals had been
confirmed by brainstem auditory evoked response (BAER) that detects electrical
activity in the cochlea and auditory pathways in the brain
The families consisted of eight to 12 individuals In two families a blood sample of the
sire and dam respectively was not available Screening for SNPs was performed by
comparative sequencing of genomic DNA from the parents of the families used for
linkage analyses
SNP and microsatellite marker identification for genotyping
The canine ESPN MYH14 MYO3A PRES TMC1 TMIE USH1C and WHRN gene
sequences was derived from sequences deposited in the current dog genome
assembly (Boxer genome assembly 21) of the NCBI GenBank (Table 2) by BLAST
Evaluation of eight candidate genes for CCSD 45
(Basic Local Alignment Search Tool) search (httpwwwncbinlmnihgovBLAST)
using the human ESPN MYH14 MYO3A PRES TMC1 TMIE USH1C and WHRN
reference mRNA sequence
We compared the canine genomic DNA sequence from the eight candidate genes to
canine cDNA fragments in the canine EST database using the BLASTN program
For the localization of the exonintron boundaries canine or alternatively human
mRNA sequences were used for the mRNA-to-genomic alignment program Spidey
(httpwwwncbinlmnihgovIEBResearchOstellSpideyindexhtml) The human
and canine mRNA sequences which were used to determine the exon organization of
the candidate genes are given in Table 2
For each of the eight candidate genes we designed intragenic primer pairs to amplifly
intronic sequences yielding products with a length of 560 to 670 bp PCR primers
were developed with the Primer3 program (httpfrodowimiteducgi-
binprimer3primer3_wwwcgi) The PCR reactions were performed in a total of 50 microl
containing 125 microM dNTPs 25 pmol of each primer the reaction buffer supplied by
the manufacturer (Qbiogene Heidelberg Germany) and 1 U Taq polymerase After
a 4 min initial denaturation at 95degC 35 cycles of 30 sec at 94degC 45 sec at 58degC and
80 sec at 72degC were performed in a MJ Research thermocycler (Biozym Hessisch
Oldendorf Germany) The obtained PCR products were directly sequenced with the
DYEnamic ET Terminator kit (AmershamBiosciences Freiburg Germany) and a
MegaBACE 1000 capillary sequencer (AmershamBiosciences) using the PCR
primers as sequencing primers Sequence data were analyzed with Sequencher 42
(GeneCodes Ann Arbor MI)
In the first step sequence analyses were performed for PCR products of the parents
of four full-sib families If a heterozygous SNP was found for one or both parents all
progeny of the respective families were analyzed for that SNP Additionally to the
SNPs we used two microsatellite markers for linkage analyses We identified one
intragenic microsatelllite within the TMC1 gene As we could develop only one SNP
for the TMIE gene we additionally genotyped one microsatellite marker derived from
the NCBI database (httpwebncbinlmnihgov) located very closely to the TMIE
gene (Table 3)
Evaluation of eight candidate genes for CCSD
46
SNP marker analysis
A total of 21 SNPs were identified within ESPN MYH14 MYO3A PRES TMC1
TMIE USH1C and WHRN (Table 3) and used for linkage analysis Of all SNPs six
out of the observed 21 SNPs were polymorphic in all four examined families Out of
the 21 SNPs eleven SNPs were present in family 1 18 SNPs were heterozygous for
one or both parents in family 2 and 15 SNPs could be used for linkage analysis in
family 3 and 4 respectively (Table 4)
The most frequent form of SNPs with a frequency of 238 was the CT transition
motif The scarcest one was the CG transversion motif with a frequency of 48
respectively
Linkage and haplotype analysis Multipoint linkage and haplotype analysis were performed using MERLIN version
0102 (multipoint engine for rapid likelihood inference Center for Statistical Genetics
University of Michigan MI USA Abecasis et al 2002) The test statistics Z-mean
and Lod score were used to test for the proportion of alleles shared by affected
individuals identical by descent (IBD) for the considered marker loci The data of the
genotypes was additionally computed using the software package SAS Genetics
(Statistical Analysis System Version 913 SAS Institute Inc Cary NC USA 2005)
to specify the observed heterozygocity values (HET) and the polymorphism
information content (PIC)
Furthermore linkage disequilibrium (LD) and association of haplotypes with CCSD
was tested using the procedures CASECONTROL and HAPLOTYPE of
SASGenetics (Statistical Analysis System version 913 Cary NC USA)
Results and discussion The non-parametric multipoint linkage analysis in four full-sib families did not show
significant test statistics The highest Z-mean value was 046 the highest LOD Score
was 008 while the error probabilities ranged from 03 to 08 (Table 5) The maximum
achievable Z-mean was 448 and the corresponding value for the LOD score was
160 Marker-trait association tests for haplotypes of the candidate gene markers
were not significant Obviously no haplotype was associated with CCSD in these
Dalmatian dog families as demonstrated (Figure 1 2 3 4 5 6 7 and 8) The
Evaluation of eight candidate genes for CCSD 47
paternal and maternal haplotypes as far as they could be estimated were distributed
among the affected progeny closely to the expected proportion of 50 and thus no
excess of a certain haplotype could be observed in the affected dogs
Due to the fact that both animals with unilateral or bilaterally hearing loss and
bilateral hearing animals shared identical haplotypes it is unlikely that the ESPN
MYH14 MYO3A PRES TMC1 TMIE USH1C and WHRN genes are involved in the
pathogenesis of CCSD in Dalmatian dogs However the ESPN MYH14 MYO3A
PRES TMC1 TMIE USH1C and WHRN gene-associated marker may serve for
further linkage studies in other Dalmatian dog populations and dog breeds other than
Dalmatians
Evaluation of eight candidate genes for CCSD
48
Table 1 Details of the six selected human candidate genes
Gene symbol
Gene name Locus name1 Most important reference
ESPN espin DFNB36 Naz et al (2004)
MYH14 myosin heavy polypeptide 14 DFNA4 Donaudy et al (2004)
MYO3A myosin IIIA DFNB30 Walsh et al (2002)
PRES solute carrier family 26
member 5 (prestin) DFNB61
Zheng et al (2000) Liu et
al (2003)
TMC1 transmembrane channel-like
gene 1
DFNB7
DFNA36 Kurima et al (2002)
TMIE transmembran inner ear DFNB6 Naz et al 2002
USH1C Usher syndrome 1C DFNB18 Verpy et al (2000) Ouyang
et al (2002)
WHRN whirlin DFNB31 Mburu et al (2003)
1 Autosomal recessive loci are designated DFNB autosomal dominant loci DFNA
Evaluation of eight candidate genes for CCSD 49
Table 2 Selected human candidate genes with their location on HSA and CFA and
corresponding accession numbers
Gene symbol Gene
location on HSA1
Acc No 3 human mRNA
Gene location
on CFA2
Acc No 3 canine
genomic sequence
Acc No 3 canine mRNA
ESPN 1 NM_031475 5 NC_006587 XM_546751
MYH14 19 NM_024729 1 NW_876270 -
MYO3A 10 NM_017433 2 NC_006584 XM_544234
PRES 7 NM_206883 18 NC_006600 XM_540393
TMC1 9 NM_138691 1 NC_006583 XM_541284
TMIE 3 NM_147196 20 NC_006602 XM_846596
USH1C 11 NM_153676 21 NC_006603 XM_860072
WHRN 9 NM_015404 11 NC_006593 XM_850321
1 Homo sapiens autosome 2 Canis familiaris autosome 3 Accession number
Evaluation of eight candidate genes for CCSD
50
Table 3 The 21 newly developed intragenic SNPs and two microsatellite markers for
Dalmatian dogs in the candidate genes ESPN MYH14 MYO3A PRES TMC1
TMIE USH1C and WHRN with their corresponding primers the SNP motif the
product size and the annealing temperature the observed heterozygosity (HET) and
polymorphism information content (PIC)
SNP Primer F1 (5acute -gt 3acute) SNP motif primer R2 (5acute -gt 3acute)
Bp3 AT4 PIC ()
HET()
ESPN_SNP1
ACCAGCACCCTCTCCAACTA
AGGAATTCACAA (CT)CACACATACA
ACTCAAGCTCAGGGTGTGGT
565 60 9 10
ESPN_SNP2
ACCAGCACCCTCTCCAACTA
ATGGCTGGCGCT (AG)GAGGCTGCCC
ACTCAAGCTCAGGGTGTGGT
565 60 27 41
ESPN_SNP3
ACCAGCACCCTCTCCAACTA
ACACTCTTCCCA (CT)GGCTGGCGCT
ACTCAAGCTCAGGGTGTGGT
565 60 23 32
ESPN_SNP4
ACCAGCACCCTCTCCAACTA
TGGGAAGAGGGA (AG)GGGGGAGCAT
ACTCAAGCTCAGGGTGTGGT
565 60 23 32
ESPN_SNP5
ACCAGCACCCTCTCCAACTA
GAGTGGGCCAGG (CT)TGGGAAGAGG
ACTCAAGCTCAGGGTGTGGT
565 60 28 42
MYH14_SNP1
CTCTCCCCAACTCAGTTCCA
ACGTGTATTCGG (GT)CGCTTTTATT
GTGATAGGGACGAGCAGCAT
670 60 35 42
MYH14_SNP2
CATGGGACCGTTCCTACACT
AGCCTCGTTTAA (CT)CTAAAAGGAA
GCTCAATAGGCACGACATCA
640 60 34 39
Evaluation of eight candidate genes for CCSD 51
Table 3 (continued)
SNP Primer F1 (5acute -gt 3acute) SNP motif primer R2 (5acute -gt 3acute)
Bp3 AT4 PIC ()
HET()
MYO3A_SNP1
AATGCTTGAGTTTGGGATGC
GGCAGTCCCATG (GT)CCCTTATAAG
ACCTAATTGCCCAGATGCAG
650 60 37 77
MYO3A_SNP2
AATGCTTGAGTTTGGGATGC
GTGGAGAGCCAC (CG)TTGGGAGAGG
ACCTAATTGCCCAGATGCAG
650 60 37 70
MYO3A_SNP3
AACCTCCTGGCGTAGTATTCC
CATTACCTATTT (AT)GATCCTTATA
TTTTCCACTTCAGGCACACA
650 60 25 36
PRES_SNP1
CCCTTACCCCATACCATTCC
GATAGACTTCCT (AG)CCCTCAGACT
TTCAGGACAGCATCATCTGC
560 60 37 64
PRES_SNP2
CCCTTACCCCATACCATTCC
TGATGTCTGCTG (AT)TAACCCATTC
TTCAGGACAGCATCATCTGC
560 60 37 66
TMC1_SNP1
GCAACCTCTCGGTTTATCCA
CGTGAAGTGCCC (AT)TTGATGGAAA
AAGCTGGGGAAGTGGATATGT
610 60 57 37
TMC1_SNP2
GCAACCTCTCGGTTTATCCA
GGAGACATTACC (AG)TGAAGTGCCC
AAGCTGGGGAAGTGGATATGT
610 60 47 29
TMC1_SNP3
GGAAGCAAGACTGAGGTTGG
AGGCTTTTTAAA (AG)CTGTTCTGGG
CTGCTGCATTTGCCTGTAAG
650 60 48 30
TMIE_SNP1
AGAACACCACCGTCTCCTTG
CAAGGCGACGCC (AT)GTGCTGTCCT
GCCTCTGGTCAGAAGAGGTG
625 60 59 36
Evaluation of eight candidate genes for CCSD
52
Table 3 (continued)
SNP Primer F1 (5acute -gt 3acute) SNP motif primer R2 (5acute -gt 3acute)
Bp3 AT4 PIC()
HET()
USH1C_SNP2
CTCCCGGTCTGTCAGGAAC
GGCCTGGGGGGA (AC)AAGCGGACGG
ATGGCATCGACTTCTCCAAC
560 60 37 35
USH1C_SNP4
CTCCCGGTCTGTCAGGAAC
GGTCTCAGACCG (AC)GGCAGGGAGA
ATGGCATCGACTTCTCCAAC
560 60 37 37
WHRN_SNP1
TTCACCTCCAGGATCTGGTC
CCTGAGCCCGAG (CT)CCACGCTGCT
GGCTACTTTTCTTCCCCCTTT
600 60 25 37
WHRN_SNP2
TTCACCTCCAGGATCTGGTC
GGTCACGGGGGC (CT)CCGGGAGGTT
GGCTACTTTTCTTCCCCCTTT
600 59 24 33
WHRN_SNP3
TTCACCTCCAGGATCTGGTC
GTCCGAGTCCCG (AG)CCCCAGCCTG
GGCTACTTTTCTTCCCCCTTT
600 60 34 55
Microsatellite marker
Primers (forward reverse ) 5acute -gt 3acute Bp AT PIC()
HET()
TMC1_MS1
GCCCCCAGCTAAAAAGAGAA
TTCTCTTCCTCCCTCCTGTTC
220-220 60 76 57
FH2158 ATGGCCACATCACCCTAGTC
CTCTCTCTGCATCTCTCATGAA
274-302 58 57 66
Microsatllite marker derived from the NCBI database (httpwebncbinlmnihgov) 1 Forward 2 Reverse 3 Product size (basepairs) 4 Annealing temperature (degC)
Evaluation of eight candidate genes for CCSD 53
Table 4 The 21 newly developed intragenic SNPs for Dalmatian dogs in the
candidate genes ESPN MYH14 MYO3A PRES TMC1 TMIE USH1C and WHRN
with their nucleotide polymorphism allele and genotype frequencies
Gene symbol Fam1 Nucleotide polymorphism
Allele frequencies
Genotype frequencies2
ESPN_SNP1 4 CgtT 083017 840
ESPN_SNP2 2 3 4 AgtG 074026 15160
ESPN_SNP3 2 3 TgtC 068032 07120
ESPN_SNP4 2 3 GgtA 068032 07120
ESPN_SNP5 2 3 4 CgtT 074026 15160
MYH14_SNP1 2 3 4 GgtT 058041 51610
MYH14_SNP2 2 3 4 CgtT 058041 51610
MYO3A_SNP1 1 2 GgtT 062038 5111
MYO3A_SNP2 1 2 CgtG 062038 5111
MYO3A_SNP3 1 2 4 TgtA 076024 15140
PRES_SNP1 1 2 3 4 AgtG 058042 10254
PRES_SNP2 1 2 3 4 TgtA 058042 10254
TMC1_SNP1 1 2 3 4 AgtT 056044 11226
TMC1_SNP2 1 2 3 4 AgtG 076024 20190
TMC1_SNP3 1 2 3 4 AgtG 074026 19200
TMIE_SNP1 1 2 3 4 AgtT 058042 12216
USH1C_SNP2 1 3 4 AgtC 053047 9147
USH1C_SNP4 1 3 4 AgtC 053047 9147
WHRN_SNP1 2 CgtT 075025 360
WHRN_SNP2 2 CgtT 075025 360
WHRN_SNP3 2 3 4 InDel3 069031 13171 1 Number of families which were informative for the respective SNPs 2 Frequence of genotypes (first number number of animals homozygous for allele 1
second number heterozygous third number homozygous for allele 2) 3 Insertiondeletion of one base-pair (adenine)
Evaluation of eight candidate genes for CCSD
54
Table 5 Linkage analyses for families 1 2 3 and 4 for the 21 SNP and two
microsatellite markers within the eight candidate genes regarding Zmean LOD score
and error probabilities (p-values)
Gene symbol Marker Zmean pz-value1 LOD score pL-value2
ESPN ESPN_SNP1 014 04 002 04
ESPN_SNP2 014 04 002 04
ESPN_SNP3 014 04 002 04
ESPN_SNP4 014 04 002 04
ESPN_SNP5 014 04 002 04
MYH14 MYH14_SNP1 -089 08 -019 08
MYH14_SNP1 -089 08 -019 08
MYO3A MYO3A_SNP1 -049 07 -011 08
MYO3A_SNP2 -049 07 -011 08
MYO3A_SNP3 -049 07 -011 08
PRES PRES_SNP1 -094 08 -019 08
PRES_SNP2 -094 08 -019 08
TMC1 TMC1_SNP1 -034 06 -008 07
TMC1_SNP2 -034 06 -008 07
TMC1_SNP3 -034 06 -008 07
TMC1_MS1 -035 06 -008 07
TMIE TMIE_SNP1 013 04 003 03
FH2158 -056 07 -013 08
USH1C USH1C_SNP2 018 04 04 03
USH1C_SNP4 018 04 04 03
WHRN WHRN_SNP1 046 03 008 03
WHRN_SNP2 046 03 008 03
WHRN_SNP3 046 03 008 03 1 Error probability regarding Zmean 2 Error probability regarding LOD score
Microsatellite marker
Evaluation of eight candidate genes for CCSD 55
Figu
re 1
SN
P-h
aplo
type
s of
the
ES
PN
gen
e in
all
anal
yzed
Dal
mat
ian
dog
fam
ilies
The
hapl
otyp
es b
elon
ging
to F
amily
2 3
and
4 re
gard
ing
the
gene
ES
PN
sho
w n
o as
soci
atio
n w
ith th
e C
CS
D p
heno
type
In
the
thre
e fa
milie
s th
e ha
plot
ype
21
43
2 is
the
mos
t com
mon
one
bei
ng p
rese
nt in
72
o
f all
anim
als
It is
follo
wed
by
the
hapl
otyp
e 2
32
14
with
18
a
nd 4
34
34
with
6
Nor
mal
hea
ring
anim
als
as
wel
l as
thei
r de
af s
iblin
gs s
how
all
thes
e th
ree
hapl
otyp
es T
here
is n
o re
com
bina
tion
of th
e ha
plot
ypes
of t
he E
SP
N g
ene
in th
e th
ree
fam
ilies
Evaluation of eight candidate genes for CCSD 56
Figu
re 2
SN
P-h
aplo
type
s of
the
MY
H14
gen
e in
all
anal
yzed
Dal
mat
ian
dog
fam
ilies
Evaluation of eight candidate genes for CCSD 57
Figu
re 3
SN
P-h
aplo
type
s of
the
MY
O3A
gen
e in
all
anal
yzed
Dal
mat
ian
dog
fam
ilies
Evaluation of eight candidate genes for CCSD 58
Figu
re 4
SN
P-h
aplo
type
s of
the
PR
ES
gen
e in
all
anal
yzed
Dal
mat
ian
dog
fam
ilies
Evaluation of eight candidate genes for CCSD 59
Figu
re 5
SN
P- a
nd m
icro
sate
llite-
hapl
otyp
es o
f the
TM
C1
gene
in a
ll an
alyz
ed D
alm
atia
n
Evaluation of eight candidate genes for CCSD 60
Figu
re 6
SN
P- a
nd m
icro
sate
llite-
hapl
otyp
es o
f the
TM
IE g
ene
in a
ll an
alyz
ed D
alm
atia
n do
g fa
milie
s
Evaluation of eight candidate genes for CCSD 61
Figu
re 7
SN
P-h
aplo
type
s of
the
US
H1C
gen
e in
all
anal
yzed
Dal
mat
ian
dog
fam
ilies
Evaluation of eight candidate genes for CCSD 62
Figu
re 8
SN
P-h
aplo
type
s of
the
WH
RN
gen
e in
all
anal
yzed
Dal
mat
ian
dog
fam
ilies
Chapter 5
Molecular characterization of the canine myosin heavy polypeptide 9
non-muscle (MYH9) gene on dog chromosome 10q232
Canine MYH9 gene 65
Molecular characterization of the canine myosin heavy polypeptide 9 non-muscle (MYH9) gene on dog chromosome 10q232 Abstract A missense mutation of the Myosin heavy polypeptide 9 (MYH9) gene which
encodes the nonmuscle myosin heavy chain-A caused non-syndromic sensorineural
deafness in a human family which was characterized by cochleosaccular
degeneration In the present study we evaluated whether MYH9 gene mutations are
responsible for canine congenital sensorineural deafness (CCSD) in Dalmatian dogs
As described in Chapter 3 two MYH9 gene-associated microsatellites were
genotyped in 25 Dalmatian dog families segregating for CCSD We could find
significant linkage with CCSD for the microsatellite marker MYH9_MS3 (=FH2293)
We used data deposited in the NCBI to assemble the canine MYH9 gene DNA
sequence Characterization of the canine MYH9 gene revealed that the canine gene
consists of 41 exons spanning approximately 90 kb
We detected 22 single nucleotide polymorphisms (SNPs) in a mutation scan of
altogether 16 Dalmatian dogs from three families which showed significant linkage
between the deafness phenotype and the MYH9 gene-associated microsatellite
None of the SNPs affects the amino acid sequence of MYH9 We concluded that the
exons of the canine MYH9 gene do not play a role in Dalmatian CCSD Availability of
the microsatellite marker SNPs and DNA sequence reported in this study enhance
evaluation of the MYH9 gene as a CCSD candidate gene in other Dalmatian dog
populations and other dog breeds affected by CCSD
Introduction Myosin is a functional protein associated with cellular movement cell division muscle
contraction and other functions Members of the myosin super-family are
distinguished from the myosin heavy chains that play crucial roles in cellular
processes The human MYH9 gene consists of 40 exons and spans 67959 bp
Canine MYH9 gene
66
Toothaker et al (1991) mapped a nonmuscle myosin heavy chain gene to human
chromosome (HSA) 22q123-q131 Simons et al (1991) likewise mapped the gene
to HSA22q112 which they designated nonmuscle myosin heavy chain-A (NMMHC-
A)
The similarities between the autosomal dominant giant-platelet disorders May-
Hegglin anomaly Fechtner syndrome and Sebastian syndrome and refinement of the
disease loci for May-Hegglin anomaly and Fechtner syndrome to an overlapping
region of 480 kb on human chromosome 22 suggested that all these three disorders
may be allelic Among the identified candidate genes was the gene encoding
nonmuscle myosin heavy-chain A (MYH9) The May-HegglinFechtner Syndrome
Consortium (2000) demonstrated that mutations in MYH9 result in one of the three
disorders mentioned above The same Consortium also speculated that mutations in
MYH9 may also play a role in another autosomal dominant disorder a form of
nonsyndromic deafness characterized by progressive hearing impairment and
cochleosaccular degeneration This autosomal dominant form of human
nonsyndromic hereditary deafness (DFNA17) was described by Lalwani et al (1999)
They studied a five-generation American family previously reported by Lalwani et al
(1997) with deafness caused by cochleosaccular degeneration (CSD) CSD is the
most common histopathologic finding in cases of profound congenital deafness and
is estimated to occur in approximately 70 of cases in man
DFNA17 maps to the same region as MYH9 Because of the importance of myosins
in hearing Lalwani et al (2000) tested MYH9 as a candidate gene for DFNA17 and
demonstrated a missense mutation in the MYH9 gene in affected members of a
kindred with DFNA17 They found a G-to-A transition at nucleotide 2114 in the MYH9
gene This missense mutation changed codon 705 from an invariant arginine to a
histidine within a highly conserved Src homology 1 (SH1) linker region Previous
studies had shown that modification of amino acid residues within the SH1 helix
causes dysfunction of the ATPase activity of the motor domain in myosin
Many genetic disorders in humans and domestic dogs (Canis familiaris) demonstrate
a high level of clinical and molecular similarity Rak et al (2003) mapped 20 potential
candidate genes for sensorineural deafness in dogs by fluorescence in situ
hybridization and a radiation hybrid panel among them the MYH9 gene that was
assigned to canine chromosome (CFA) 10q232 Congenital sensorineural deafness
has been reported for approximately 54 different breeds of dogs (Strain 1996)
Canine MYH9 gene 67
The incidence of this congenital anomaly is highest in Dalmatian dogs of which 165-
30 exhibit unilateral or bilateral hearing loss (Famula et al 1996 Wood and
Lakhani 1997 Muhle et al 2002 Juraschko et al 2003a Rak and Distl 2005) The
inheritance and segregation of a major gene in CCSD has been demonstrated in
different Dalmatian dog populations (Famula et al 2000 Muhle et al 2002
Juraschko et al 2003b)
In dog breeds the histological pattern is known as cochleosaccular degeneration
commonly known as Scheibe dysplasia as it is described in approximately 70 of
cases with human hereditary deafness (Strain 1996) Therefore the MYH9 gene
seems to be a perfect candidate gene for CCSD especially in Dalmatian dogs
In this report we provide the genomic organization and the complete sequence of the
canine MYH9 gene A mutation analysis was performed to identify single nucleotide
polymorphisms (SNPs) in this gene In order to evaluate whether the MYH9 gene is
responsible for congenital sensorineural deafness in Dalmatian dogs we analyzed
the association of the MYH9 haplotypes with the CCSD phenotype in three families of
Dalmatian dogs with frequent occurrence of CCSD and significant linkage to the
gene-associated microsatellite MYH9_MS3 (Chapter 3)
Materials and Methods
Cloning and sequencing of canine MYH9 cDNA
The canine MYH9 gene sequence was derived from sequences deposited in the
current dog genome assembly (Boxer genome assembly 21) of the NCBI GenBank
(Genbank Acc No NW_1398701) by BLAST (Basic Local Alignment Search Tool)
search (httpwwwncbinlmnihgovBLAST) using the human MYH9 reference
mRNA sequence (Genbank Acc No NM_002473)
The distance between MYH9 and the microsatellite MYH9_MS2 is about 181 kb
whereas the microsatellite MYH9_MS3 (=FH2293) places about 511 kb from MYH9
The isolation of full length cDNAs was achieved by a modified rapid amplification of
cDNA ends (RACE) protocol Total RNA from dog lung of a normal female Beagle
(Biocat Heidelberg Germany) was used for the overlapping 5rsquo- and 3rsquo-RACE
products with the FirstChoiceTM RNA ligase-mediated (RLM)-RACE kit (Ambion
Europe Huntingdon UK) according to the protocols of the manufacturer Reverse
transcription polymerase chain reaction (RT-PCR) using two pairs of nested
Canine MYH9 gene
68
gene-specific and supplied 5rsquo-adaptor-specific primers were used to amplify the
complete 5rsquo-end of the investigated cDNA Similarly the 3rsquo-end were amplified using
two pairs of nested gene-specific primers in combination with supplied 3rsquo-adaptor-
specific primers Additionally RT-PCR using six pairs of gene-specific primers were
used to amplify the complete sequence of the investigated cDNAs
RACE products were cloned into pCRR21 vector using the Invitrogen TA cloning kit
(Invitrogen Karlsruhe Germany) Several clones for each cDNA were sequenced
with the ThermoSequenase kit (AmershamBiosciences Freiburg Germany) and a
LI-COR 4300 DNA Sequencer (LICOR inc Bad Homburg Germany) Sequence
data were analyzed with Sequencher 42 (GeneCodes Ann Arbor MI)
Sequence analysis of canine MYH9 gene For the localization of the exonintron boundaries after cloning and sequencing of full
length canine cDNAs (as described above) the mRNA-to-genomic alignment program
Spidey (httpwwwncbinlmnihgovIEBResearchOstellSpideyindexhtml) was
used Repetitive elements were analyzed with Repeatmasker 2
(httprepeatmaskergenomewashingtonedu) The GC content was calculated with
the EBI toolbox CpG PlotCpGreport (httpwwwebiacukToolssequencehtml)
Mutation analysis To identify polymorphisms within the canine MYH9 sequence exons with flanking
regions were PCR amplified and sequenced from 16 Dalmatian dogs which
represent three families consisting of three to six full sibs and at the best of both
parents At least two of the full sibs of each family were unilaterally deaf The
phenotype of the affected animals had been confirmed by brainstem auditory evoked
response (BAER) that detects electrical activity in the cochlea and auditory pathways
in the brain PCR primers and conditions for the amplification of MYH9 exons with
flanking sequences and microsatellite flanking primers for PCR reactions are given in
Table 1 PCR primers were developed with the Primer3 program
(httpfrodowimiteducgi-binprimer3primer3_wwwcgi) The PCR reactions for
exons and their flanking sequences were performed in a total of 50 microl containing 125
microM dNTPs 25 pmol of each primer the reaction buffer supplied by the manufacturer
(Qiagen Hilden Germany) and 1 U Taq polymerase After a 4 min initial
denaturation at 95 degC 35 cycles of 30 sec at 94 degC 45 sec at 58 degC and 80 sec
Canine MYH9 gene 69
at 72 degC were performed in a MJ Research thermocycler (Biozym Hessisch
Oldendorf Germany) The obtained PCR products were directly sequenced with the
DYEnamic ET Terminator kit (AmershamBiosciences Freiburg Germany) and a
MegaBACE 1000 capillary sequencer (AmershamBiosciences) using the PCR
primers as sequencing primers Sequence data were analyzed with Sequencher 42
(GeneCodes Ann Arbor MI)
Multipoint linkage and haplotype analysis were performed using MERLIN version
0102 (multipoint engine for rapid likelihood inference Center for Statistical Genetics
University of Michigan MI USA Abecasis et al 2002) The test statistics Z-mean
and Lod score were used to test for the proportion of alleles shared by affected
individuals identical by descent (IBD) for the considered marker loci
Linkage means that a haplotype characterized by microsatellites or SNPs is
significantly more often present in family members with the phenotype under study
than expected by random assortment For construction of haplotypes sets of closely
linked genetic markers on the same chromosome are needed which tend to be
inherited together as they are not likely to be separated by recombination
Association analysis can be carried out as a method of genetic analysis that
compares the frequency of alleles between affected and unaffected individuals
across all families A given allele is considered to be associated with the disease if
the presence of that allele explains a significant proportion of the phenotypic trait
variation
Results and Discussion
Analysis of the genomic organization and cDNA of the canine MYH9 gene
A full-length cDNA sequence of 7484 bp of the canine MYH9 gene was obtained by
using the RACE protocol The obtained RT-PCR products were sequenced and the
generated sequence data were submitted to the EMBL nucleotide database (Acc No
AM086385)
Using the RT-PCR analyses for cDNA-genomic sequence comparisons we detected
that the canine MYH9 gene consists of 41 exons with an untranslated exon 1 and
exonintron boundaries that conform perfectly to the GTAG rule (Table 2)
The 5rsquo-UTR consists of 181 bp while the 3rsquo-UTR consists of 1432 bp Assuming that
the homologous ATG start codon as in man is used the canine MYH9 cDNA
Canine MYH9 gene
70
contains an open reading frame of 5889 bp encoding a protein of 1963 amino acids
A canonical polyadenylation signal AAUAAA is located approximately 14 kb
downstream of the stop codon
The exon sizes range from 28 to 1556 bp the introns between these exons span
between 86 and 13493 bp the total size of the canine MYH9 gene is approximately
90 kb However the sequence homology between the human murine and canine
MYH9 gene is limited to the coding sequence of exon 2 to 41 The coding sequence
of canine MYH9 displays 926 and 899 similarities to the human and murine
MYH9 gene respectively In the untranslated regions the sequence similarity
between dog and human and mouse is rather low The canine MYH9 protein shows
982 and 968 identities to the orthologous human and murine protein
respectively (Fig 2)
The repeat content in the 90 kb region of the canine MYH9 gene is 1458 Most of
the repetitive elements belong to the SINE family (892) followed by the fraction of
the LINEs (306) other repetitive elements constitute 165 respectively The
entire canine MYH9 gene has an overall GC-content of 54 The canine MYH9 gene
contains 12 CpG islands (Gardiner-Garden and Frommer 1987) distributed over the
entire gene whereas by far the longest one with a length of 789 bp can be found in
the region of the second exon or in the first translated exon respectively (GC content
of 50 over 200 bp)
Mutation and haplotype analysis
All coding exons with flanking intronic regions of MYH9 could be amplified from the
examined 16 dogs and the sequences were compared to the Boxer genome
assembly 21 of the NCBI Gen Bank (Genbank Acc No NW_1398701)
The search for sequence variations within the MYH9 gene revealed a total of 22
SNPs shown in Table 3 Most of the polymorphisms were found in the flanking
regions of exons only 3 were within exons Only five out of the observed 22 SNPs
were polymorphic in all three examined families
None of the observed polymorphism did alter the predicted amino acid sequence of
MYH9 nor the identified hapltypes showed an association with the CCSD phenotype
Multipoint test statistics for MYH9 alleles identical by descent were close to zero (Z-
mean 035 to -035 Lod score 013 to -007) and not significant (p= 04 to 07)
However the microsatellite MYH9_MS3 (=FH2293) showed a Z-mean of 156
Canine MYH9 gene 71
(p= 006) and a Lod score of 058 (p= 005) indicating linkage The reason for this
result was heterogeneity among these three families For family 1 and 2 the test
statistics indicated linkage (Z-mean 191 with p= 003 Lod score 058 with p= 005)
whereas for familiy 3 the Z-mean and Lod score was -050 (p= 07) and -008 (p=
07)
Thus these silent point mutations found in affected and unaffected Dalmatian dogs
seem not responsible for the CCSD phenotype in these three families
Conclusions
The characterization of the transcript and genomic sequences of canine MYH9 gene
revealed a conserved organization with respect to the human orthologs In general
the gene size in dog is bigger compared to the human sequence due to the
untranlated first exon in the 5rsquo-UTR Several single nucleotide polymorphisms in the
canine MYH9 gene were identified However because of the fact that both animals
with unilateral hearing loss and bilateral hearing animals shared identical
haplotypes these polymorphisms are obviously not associated with CCSD in these
Dalmatian dog families Furthermore the present study revealed no functional
mutations of the complete coding region of MYH9 We can exclude the MYH9 gene
as a candidate gene for congenital sensorineunal deafness in Dalmatian dogs
However the MYH9 gene sequence SNPs and microsatellite markers reported in
this study enhance evaluation of the MYH9 gene in other Dalmatian dog populations
and dog breeds other than Dalmatians
Canine MYH9 gene
72
Table 1 PCR primers for the amplification of all MYH9 exons
Exon Primer Sequence (5rsquo ndash 3rsquo) Product
size (bp)
Annealing temperature
(degC) exon 1 MYH9_Ex1_F GCC TGG TTT GAC AAG TTT TG MYH9_Ex1_R TTC CTC AAG TCC CCT ACT CC 560 58 exon 2 MYH9_Ex2_F AGG ACC CTC ACC CAA CAC MYH9_Ex2_R GCA CAG AGT CTG GAC AAG AAT G 621 58 exon 3 MYH9_Ex3_F CAC CCC TCA ATA CTC AGT GC MYH9_Ex3_R GAG AAG ACT CCT GCC TCT CC 588 58 exon 4 MYH9_Ex4_F GGC AAT GTG TTT GAT TGT ACT G MYH9_Ex4_R TTT CCA AAG CAA CTC AGA AAG 542 58 exon 5 -6 MYH9_Ex56_F ACA GAG CAG GCG TGA CAG MYH9_Ex56_R GAG GAC AAG AGC TGG AAG C 724 58 exon 7 -8 MYH9_Ex78_F AAG CGG AAA GTT GCC TTC MYH9_Ex78_R ATG GCA CAA TGA CCA GTC TC 699 58 exon 9 MYH9_Ex9_F TGG GCA CAG TTT TTG TCT G MYH9_Ex9_R CAC ACA ATC AAG GGC TTT TC 590 58 exon 10 MYH9_Ex10_F GGA GCC AGT TGT CCT AGT AGT G MYH9_Ex10_R GCC AGG TGC CTA TCA CTG 540 58 exon 11 MYH9_Ex11_F TGT TTC GAA GCG TTT TTA CC MYH9_Ex11_R GGT CCC ATT GTT TCT AGC AC 551 58 exon 12 MYH9_Ex12_F TTT CCC AAG CAG CTA GAA AG MYH9_Ex12_R ACT GGG TGG TCT TGA GAC TG 586 58 exon 13 MYH9_Ex13_F CTG CCT TTG GTG ACA AGT G MYH9_Ex13_R GCA AGC CCT AAG TAC AAT GG 548 58 exon 14 MYH9_Ex14_F ATA CCA GGT CAA GGG GTG TC MYH9_Ex14_R TCA CCT GGT TCT AGG TGG AG 524 58 exon 15 MYH9_Ex15_F CGT TTG GAG TTG AT CCT AGC MYH9_Ex15_R AGC CAA GTT TCC TGA CTG TG 566 58 exon 16 MYH9_Ex16_F AGA TAG GCT GCA GTG GTA GC MYH9_Ex16_R CAG TAG CAA ACA GGG CTA GG 568 58 exon 17 MYH9_Ex17_F AAC GGT CCA ATG TGA GTT TC MYH9_Ex17_R ACC AGC TCA CAA CAG TAC CC 559 58 exon 18 MYH9_Ex18_F TGT AGG CTG TGA TTG CAG AG MYH9_Ex18_R CCC TCT CTC AAA CAA ATT GC 586 58 exon 19 MYH9_Ex19_F CCT TGG TGA AGG AAA GTG G MYH9_Ex19_R GCT GTG TGC AGG TTC TGA C 657 58 exon 20 MYH9_Ex20_F AGC AGC ATA GCA CTG AAT CC MYH9_Ex20_R TTC CCA TCT CTC ATG TTC TTG 579 58
Canine MYH9 gene 73
Table 1 (continued)
Exon Primer Sequence (5rsquo ndash 3rsquo) Product
size (bp)
Annealing temperature
(degC) exons 21 MYH9_Ex21_F CTC CTG AGT GTT GGG TTA GC MYH9_Ex21_R CTC TAA CAG GCA GCC CAA G 555 58 exon 22 MYH9_Ex22_F GGA GGA AAC GCA GAA ATC C MYH9_Ex22_R ACG GAC ACC CAG GAA AAC 660 58 exon 23 MYH9_Ex23_F CAG GGG TGC TAG TGA TCT TTC MYH9_Ex23_R AGA GCT GGG CTC AGA CCT AC 630 58 exon 24 MYH9_Ex24_F TCA GGG ATG TGC TTT CTT TC MYH9_Ex24_R CCC CAA GCA CAT CTG TAG TC 563 58 exon 24 MYH9_Ex24_F TCA GGG ATG TGC TTT CTT TC MYH9_Ex24_R CCC CAA GCA CAT CTG TAG TC 563 58 exon 25 -26 MYH9_Ex2526F GGC TGC TCT TCT CTG AAG C MYH9_Ex2526R GGA GTC AGG CTT CCA GTA AAG 851 58 exon 27 -28 MYH9_Ex2728F AGC ACA TTC TGG ATC TGG AG MYH9_Ex2728R GCG ACA GCT GAA TTC CTG 752 58 exon 29 MYH9_Ex29_F CAG CTT CAG GAC ACA CAG G MYH9_Ex29_R CTC TGG GTC TCC CCT GTC 601 58 exon 30 MYH9_Ex30_F GAG GCT TGG TGT ATG TCT CG MYH9_Ex30_R TGG TTC CAT AGA AAC GAT CTG 592 58 exon 31 - 32 MYH9_Ex3132F CAT TCC CCA AGG AGT TTT G MYH9_Ex3132R AAC ACA GCC CAG GGA AAG 770 58 exon 33 MYH9_Ex33_F CCT GGA ACA AGG CTG AGA AC MYH9_Ex33_R TCT CAG AGC AGC TTT GCT TC 600 58 exon 34 MYH9_Ex34_F GGT GTG CTT GGT AAA TCC TG MYH9_Ex34_R CAC AAA GCC TCA TTT CAC G 611 58 exon 34 - 36 MYH9_Ex3536F AGG TCA CGG TCT GGG TTC T MYH9_Ex3536R CAA GGG AGG CTG TTG CAG 401 60 exon 37 MYH9_Ex37_F CTC CCT TGG TTT CCC TCT CT MYH9_Ex37_R CAG GTC AGG AGG GGA CAT 488 60 exon 38 -39 MYH9_Ex3839F AGG CCT AAG GAC AGC TCA TC MYH9_Ex3839R CCA GGG CAC AGA TAG AAC C 545 58 exon 40 MYH9_Ex40_F1 GTC CCT CTA AAG ATG GCT TTG MYH9_Ex40_R1 TGG GGA GGA ACG ACT CTC 844 58 MYH9_Ex40_F2 TGG GTG ATT GGG GAA GAG MYH9_Ex40_R2 CCA GAA GAT GCA CAA AGT AGG 981 58
Canine MYH9 gene
74
Table 2 Exonintron boundaries of the canine MYH9 gene
3rsquo-Splice site Exon 5rsquo-Splice site
Intron phase
Intron size
-20 (exon 1 162 bp) GTTCAGgtgagcgagccccgc
gt30000 bp
-19 +333 gctgtagGTCCTGGCTGTGA (exon 2 352 bp) CGTAAgtggggcctggggcc
0
4922 bp
+334 +490 tctttagACCTATTCAGGCC (exon 3 157 bp) GCAAGgtgatgcctcggcctt
1
13493 bp
+491 +518 gtttcagACCGAGAGGACCA (exon 4 28 bp) TGCACgtaagtgtttctgcc
2
803 bp
+519 +612 tcggcagGGGTGAGTCTGGA (exon 5 94 bp) ACCAGgtgagtgcgggcctt
0
4077 bp
+613 +705 cttgtagGGTGAGTTGGAGA (exon 6 93 bp) GATTTgtgagtagcggccag
0
427 bp
+706 +769 tttttagGGCAAATTCATTC (exon 7 64 bp) GACTTgtatccttggttgat
1
738 bp
+770 +868 tgcaaagATCTTCTGGAGAA (exon 8 99 bp) GAAGAgtgagtagtgagccc
1
343 bp
+869 +1012 ctttcagCTGATCTCCTGTT (exon 9 144 bp) AATGGgtacagagctggccc
1
749 bp
+1013 +1108 cccgaagGCTTGTTGCGAGT (exon 10 96 bp) CACAGgtaacgcctgtggtcc
1
1041 bp
+1109 +1227 atcgcagCTGCCCAGAAAGT (exon 11 119 bp) AACAGgtaggctctctggcc
0
1877 bp
+1228 +1380 accccagGCTGACTTTGCCA (exon 12 153 bp) TTGATgtgagctcctctcct
0
922 bp
+1381 +1554 cctgcagCTGAACTCCTTCG (exon 13 174 bp) AGCCAgtaagagccagacct
0
1801 bp
+1555 +1728 ttcccagGCAGGGCCCCCTG (exon 14 174 bp) GCAAGgtgaggcaagcacgt
0
2049 bp
+1729 +1843 cttgcagGTGGACTACAAAG (exon 15 115 bp) GGATGgtacgtgccccctgg
1
1877 bp
+1844 +2037 tttgcagTGGACCGCATCAT (exon 16 194 bp) AGAAGgtgcgggtgctgggc
0
343 bp
+2038 +2159 tctccagGCTGGCAAACTGG (exon 17 122 bp) CAGAGgtgagcggggcctgt
1
835 bp
+2160 +2229 ccctcagATATGAGATCTTG (exon 18 70 bp) TCATGgtgagtagagctgct
2
1418 bp
+2230 +2390 cgtctagATCAAAGCCCTGG (exon 19 161 bp) AGGAAgtgagttcctaaagg
1
851 bp
+2391 +2499 tccccagGGCCTTTGCCAAG (exon 20 109 bp) CCAAGgtgggttctggaagg
2
1428 bp
+2500 +2631 cttctagGTCAAGCCACTGC (exon 21 132 bp) CCCAGgtaggtgtcagcagg
2
398 bp
+2632 +2838 ttggcagCTCATGGCAGAGA (exon 22 207 bp) TCCAAgtgagccccttccct
2
488 bp
Canine MYH9 gene 75
Table 2 (continued)
3rsquo-Splice site Exon 5rsquo-Splice site
Intron phase
Intron size
+2839 +2976 atggtagGAACTGGAGGAGC (exon 23 138 bp) CCAAGgtgaggcccgtggtg
2
971 bp
+2977 +3100 cccccagGAGAAGAAGCTTC (exon 24 124 bp) GGAAGgtaagagggtaacag
0
1603 bp
+3101 +3272 tcctcagAGCGCCTCCGAAG (exon 25 172 bp) GCCAGgtgagggcctcgctt
1
719 bp
+3273 +3485 ggcagagTGGAGGAGGAAGC (exon 26 213 bp) CTCAGgtgtgccatgcggcc
1
270 bp
+3486 +3630 tctccagGTCCAAACGCGAA (exon 27 145 bp) AACGGgtgcgtgggctcccc
2
480 bp
+3631 +3837 ccaccagGTGAAAGCCAACT (exon 28 207 bp) TGCAGgtgaggcctccatgg
2
232 bp
+3838 +3941 gccacagGTTGAGCTGGACA (exon 29 104 bp) ACACAgtaaaggtggcagcg
1
204 bp
+3942 +4094 ggtgtagGAGCTGCTCCAGG (exon 30 153 bp) CCCAGgttcttatcggcatg
1
1083 bp
+4095 +4343 tgcccagGTGACGGACATGA (exon 31 249 bp) ACCAGgtgtgggccccaggc
1
1298 bp
+4344 +4556 tccccagCTACTGGCCGAGG (exon 32 213 bp) AGAGCgtgagtaccatgtca
1
150 bp
+4557 +4769 gtggcagGTCCACGAGCTGG (exon 33 213 bp) GACAGgtgtggaggccctcc
1
303 bp
+4770 +4931 gccccagGTGCGGGAGATGG (exon 34 162 bp) TGCAGgtgaggtcggctggg
1
1173 bp
+4932 +5060 accccagGCCCAGATGAAGG (exon 35 129 bp) AGGAGgtgggccgggcctca
1
941 bp
+5061 +5149 atggcagGAACTGGCAGCTG (exon 36 89 bp) AAGGGgtgagccgtggcgtg
0
92 bp
+5150 +5273 cacccagAGCTCTGGCATTG (exon 37 124 bp) TGCAGgtgggcggggtcggg
2
224 bp
+5274 +5482 cccacagATCGACCAGATCA (exon 38 209 bp) ACCAAgtaggtgttcctgcc
1
563 bp
+5483 +5591 ctcgcagGGAGCGCCAGGCA (exon 39 109 bp) ACCAGgtgtgtgtgcacacc
2
86 bp
+5592 +5763 tctgcagGCCGACAAGGCAT (exon 40 172 bp) CTCAGgtgagccctgcctcc
0
739 bp
+5764 +7295 tgcaaagATCTTCTGGAGAA (exon 41 1532 bp) CGTCACAATAAAGAGAACCA
Exon sequences are shown in uppercase letters and intron sequences in lowercase
letters Untranslated regions are shown in italics The conserved GTAG exonintron
junctions are shown in boldface type For the last exon the polyadenylation signal is
shown underlined instead of an exonintron junction Position +1 corresponds to the
adenine of the translation initiation codon ATG
Canine MYH9 gene
76
16
TT
TC
G
A
TC
A
A C
A
CT
T
C
GC
G
G
AG
C
C
AG
G
A
CT
C
T
GA
A
G
CC
C
T
CC
T
C
96
96
229
23
7
15
TT
TC
G
A
TC
A
A C
A
CT
T
C
GC
G
G
AG
C
C
AG
G
A
CT
C
T
GA
A
G
CC
C
T
CC
T
C
96
96
229
22
9
14
deaf
T
T T
C
GA
T
C
AA
CA
C
T
TC
G
C
GG
A
G
CC
A
G
GA
C
T
CT
G
A
AG
C
C
CT
C
C
TC
96
96
22
9
229
13
deaf
T
T T
C
GA
T
C
AA
CA
C
T
TC
G
C
GG
A
G
CC
A
G
GA
C
T
CT
G
A
AG
C
C
CT
C
C
TC
96
96
22
9
229
12
deaf
T
T C
C
AA
CC
A
A A
A T
T C
C
CC
G
G
GG
C
C
GG
A
A C
C
CC
A
A G
G
CC
T
T C
C
CC
96
10
0 24
1
229
11
deaf
T
T C
C
AA
CC
A
A A
A T
T C
C
CC
G
G
GG
C
C
GG
A
A C
C
CC
A
A G
G
CC
T
T C
C
CC
96
10
0 24
1
229
10
TT
TC
G
A
TC
A
A C
A
CT
T
C
GC
G
G
AG
C
C
AG
G
A
CT
C
T
GA
A
G
CC
C
T
CC
T
C
96
96
237
22
9
Fam
ily 3
9 T
T C
C
AA
CC
A
A A
A T
T C
C
CC
G
G
GG
C
C
- - CC
C
C
AA
GG
C
C
TT
CC
C
C
96
100
229
24
1
8 T
T C
T
AG
C
T
AA
AA
TT
CC
C
C
GG
G
G
CT
G
G
AA
CC
C
C
AA
GG
C
C
TT
CC
C
C
96
96
225
25
3
7 de
af
TC
C
C
AG
C
T
AT
A
A T
T C
C
CC
G
A
GG
C
T
GG
A
G
CC
C
C
AA
GG
C
T
TC
C
T
CC
96
89
22
1
237
6 de
af
TC
C
C
AG
C
T
AT
A
A T
T C
C
CC
G
A
GG
C
T
GG
A
G
CC
C
C
AA
GG
C
T
TC
C
T
CC
96
89
22
1
237 Fa
mily
2
5 T
C
CC
A
G
CT
A
T
AA
TT
CC
C
C
GA
G
G
CT
G
G
AG
C
C
CC
A
A G
G
CT
T
C
CT
C
C
96
89
221
23
7
4 de
af
TT
CC
A
A C
C
AA
AA
TT
CC
C
C
GG
G
G
CC
G
G
AG
C
C
CC
A
A G
G
CC
T
T C
C
CC
96
96
23
7
257
3 de
af
TC
C
C
AG
C
T
AT
A
A T
T C
C
CC
G
A
GG
C
T
GG
G
A
CC
C
C
AA
GG
C
T
TC
C
T
CC
96
96
23
7
257
2 T
C
CC
A
G
CT
A
T
AA
TT
CC
C
C
GA
G
G
CT
G
G
AA
CC
C
C
AA
GG
C
T
TC
C
T
CC
96
96
23
7
257 Fa
mily
1
1 C
T
CC
G
A
TC
T
A A
A T
T C
C
CC
A
G
GG
T
C
GG
A
G
CC
C
C
AA
GG
T
C
CT
T
C
CC
96
96
25
7
257
Ref
eren
ce
se
quen
ce
NW
_139
870
T C
A T A A T C
C
G
G
C
G
A C
C
A G
C
T C
C
posi
tion1
212
9
1 4
4 2
39
125
1
32
316
3
78
42
218
1
8 1
75
24
13
8
207
2
63
287
9
22
40
59
64
Pos
ition
with
in M
YH
9 In
tron
34
Exo
n 4
Intro
n 14
15
Intro
n 15
16
Intro
n 16
17
Intro
n 18
19
Intro
n 20
21
Intro
n 20
21
Intro
n 25
26
Intro
n 25
26
Intro
n 26
27
Intro
n 27
28
Exo
n 29
In
tron
293
0 In
tron
303
1 E
xon
32
Intro
n 32
33
Intro
n 32
33
Intro
n 34
35
Intro
n 38
39
Intro
n 38
39
Intro
n 38
39
Mic
rosa
tellit
e M
YH
9_M
S2
Mic
rosa
tellit
e M
YH
9_M
S3
(=FH
2293
)
1 N
umbe
ring
refe
rs to
the
posi
tion
of th
e po
lym
orph
ic n
ucle
otid
e w
ithin
the
give
n ex
on
or in
tron
Tabl
e 3
Hap
loty
pes
of th
e ca
nine
MY
H9
gene
in16
Dal
mat
ian
dogs
Canine MYH9 gene 77
Figure 1 Family structure of the analyzed Dalmatian dog families Hearing status was tested by brain auditory evoked response (BAER)
Canine MYH9 gene
78
Figure 2 Alignment of the canine MYH9 protein with orthologous human and murine
MYH9 protein sequences The sequences where derived from GenBank entries with
the accessions NP_002464 (human MYH9) and NP_071855 (mouse MYH9)
Identical residues are indicated by asterisk beneath the alignment while dots and
colons represent similar or very similar amino acids respectively
dog MAQQAADKYLYVDKNFINNPLAQADWAAKKLVWVPSDKSGFEPASLKEEVGEEAIVELVE 60 human MAQQAADKYLYVDKNFINNPLAQADWAAKKLVWVPSDKSGFEPASLKEEVGEEAIVELVE 60 mouse MAQQAADKYLYVDKNFINNPLAQADWAAKKLVWVPSSKNGFEPASLKEEVGEEAIVELVE 60 dog NGKKVKVNKDDIQKMNPPKFSKVEDMAELTCLNEASVLHNLKERYYSGLIYTYSGLFCVV 120 human NGKKVKVNKDDIQKMNPPKFSKVEDMAELTCLNEASVLHNLKERYYSGLIYTYSGLFCVV 120 mouse NGKKVKVNKDDIQKMNPPKFSKVEDMAELTCLNEASVLHNLKERYYSGLIYTYSGLFCVV 120 dog INPYKNLPIYSEEIVEMYKGKKRHEMPPHIYAITDTAYRSMMQDREDQSILCTGESGAGK 180 human INPYKNLPIYSEEIVEMYKGKKRHEMPPHIYAITDTAYRSMMQDREDQSILCTGESGAGK 180 mouse INPYKNLPIYSEEIVEMYKGKKRHEMPPHIYAITDTAYRSMMQDREDQSILCTGESGAGK 180 dog TENTKKVIQYLAHVASSHKSKKDQGELERQLLQANPILEAFGNAKTVKNDNSSRFGKFIR 240 human TENTKKVIQYLAYVASSHKSKKDQGELERQLLQANPILEAFGNAKTVKNDNSSRFGKFIR 240 mouse TENTKKVIQYLAHVASSHKSKKDQGELERQLLQANPILEAFGNAKTVKNDNSSRFGKFIR 240 dog INFDVNGYIVGANIETYLLEKSRAIRQAKEERTFHIFYYLLSGAGEHLKTDLLLEPYNKY 300 human INFDVNGYIVGANIETYLLEKSRAIRQAKEERTFHIFYYLLSGAGEHLKTDLLLEPYNKY 300 mouse INFDVNGYIVGANIETYLLEKSRAIRQAKEERTFHIFYYLLSGAGEHLKTDLLLEPYNKY 300 dog RFLSNGHVTIPGQQDKDMFQETMEAMRIMGIPEEEQMGLLRVISGVLQLGNIVFKKERNT 360 human RFLSNGHVTIPGQQDKDMFQETMEAMRIMGIPEEEQMGLLRVISGVLQLGNIVFKKERNT 360 mouse RFLSNGHVTIPGQQDKDMFQETMEAMRIMGIPEDEQMGLLRVISGVLQLGNIAFKKERNT 360 dog DQASMPDNTGNASAQKVSHLLGINVTDFTRGILTPRIKVGRDYVQKAQTKEQADFAIEAL 420 human DQASMPDNT---AAQKVSHLLGINVTDFTRGILTPRIKVGRDYVQKAQTKEQADFAIEAL 417 mouse DQASMPDNT---AAQKVSHLLGINVTDFTRGILTPRIKVGRDYVQKAQTKEQADFAIEAL 417 dog AKATYERMFRWLVLRINKALDKTKRQGASFIGILDIAGFEIFDLNSFEQLCINYTNEKLQ 480 human AKATYERMFRWLVLRINKALDKTKRQGASFIGILDIAGFEIFDLNSFEQLCINYTNEKLQ 477 mouse AKATYERMFRWLVLRINKALDKTKRQGASFIGILDIAGFEIFDLNSFEQLCINYTNEKLQ 477 dog QLFNHTMFILEQEEYQREGIEWNFIDFGLDLQPCIDLIEKPAGPPGILALLDEECWFPKA 540 human QLFNHTMFILEQEEYQREGIEWNFIDFGLDLQPCIDLIEKPAGPPGILALLDEECWFPKA 537 mouse QLFNHTMFILEQEEYQREGIEWNFIDFGLDLQPCIDLIEKPAGPPGILALLDEECWFPKA 537 dog TDKSFVEKVVQEQGTHPKFQKPKQLKDKADFCIIHYAGKVDYKADEWLMKNMDPLNDNIA 600 human TDKSFVEKVMQEQGTHPKFQKPKQLKDKADFCIIHYAGKVDYKADEWLMKNMDPLNDNIA 597 mouse TDKSFVEKVVQEQGTHPKFQKPKQLKDKADFCIIHYAGKVDYKADEWLMKNMDPLNDNIA 597
Canine MYH9 gene 79
dog TLLHQSSDKFVSELWKDVDRIIGLDQVAGMSETALPGAFKTRKGMFRTVGQLYKEQLAKL 660 human TLLHQSSDKFVSELWKDVDRIIGLDQVAGMSETALPGAFKTRKGMFRTVGQLYKEQLAKL 657 mouse TLLHQSSDKFVSELWKDVDRIIGLDQVAGMSETALPGAFKTRKGMFRTVGQLYKEQLAKL 657 dog MATLRNTNPNFVRCIIPNHEKKAGKLDPHLVLDQLRCNGVLEGIRICRQGFPNRVVFQEF 720 human MATLRNTNPNFVRCIIPNHEKKAGKLDPHLVLDQLRCNGVLEGIRICRQGFPNRVVFQEF 717 mouse MATLRNTNPNFVRCIIPNHEKKAGKLDPHLVLDQLRCNGVLEGIRICRQGFPNRVVFQEF 717 dog RQRYEILTPNSIPKGFMDGKQACVLMIKALELDSNLYRIGQSKVFFRAGVLAHLEEERDL 780 human RQRYEILTPNSIPKGFMDGKQACVLMIKALELDSNLYRIGQSKVFFRAGVLAHLEEERDL 777 mouse RQRYEILTPNSIPKGFMDGKQACVLMIKALELDSNLYRIGQSKVFFRAGVLAHLEEERDL 777 dog KITDVIIGFQACCRGYLARKAFAKRQQQLTAMKVLQRNCAAYLKLRNWQWWRLFTKVKPL 840 human KITDVIIGFQACCRGYLARKAFAKRQQQLTAMKVLQRNCAAYLKLRNWQWWRLFTKVKPL 837 mouse KITDVIIGFQACCRGYLARKAFAKRQQQLTAMKVLQRNCAAYLRLRNWQWWRLFTKVKPL 837 dog LQVSRQEEEMMAKEEELVKVREKQLAAENRLTEMETLQSQLMAEKLQLQEQLQAETELCA 900 human LQVSRQEEEMMAKEEELVKVREKQLAAENRLTEMETLQSQLMAEKLQLQEQLQAETELCA 897 mouse LNSIRHEDELLAKEAELTKVREKHLAAENRLTEMETMQSQLMAEKLQLQEQLQAETELCA 897 dog EAEELRARLTAKKQELEEICHDLEARVEEEEERCQHLQAEKKKMQQNIQELEEQLEEEES 960 human EAEELRARLTAKKQELEEICHDLEARVEEEEERCQHLQAEKKKMQQNIQELEEQLEEEES 957 mouse EAEELRARLTAKKQELEEICHDLEARVEEEEERCQYLQAEKKKMQQNIQELEEQLEEEES 957 dog ARQKLQLEKVTTEAKLKKLEEDQIIMEDQNCKLAKEKKLLEDRIAEFTTNLMEEEEKSKS 1020 human ARQKLQLEKVTTEAKLKKLEEEQIILEDQNCKLAKEKKLLEDRIAEFTTNLTEEEEKSKS 1017 mouse ARQKLQLEKVTTEAKLKKLEEDQIIMEDQNCKLAKEKKLLEDRVAEFTTNLMEEEEKSKS 1017 dog LAKLKNKHEAMITDLEERLRREEKQRQELEKTRRKLEGDSTDLNDQIAELQAQIAELKMQ 1080 human LAKLKNKHEAMITDLEERLRREEKQRQELEKTRRKLEGDSTDLSDQIAELQAQIAELKMQ 1077 mouse LAKLKNKHEAMITDLEERLRREEKQRQELEKTRRKLEGDSTDLSDQIAELQAQIAELKMQ 1077 dog LAKKEEELQAALARVEEEATQKNMALKKIRELESQISELQEDLESERASRNKAEKQKRDL 1140 human LAKKEEELQAALARVEEEAAQKNMALKKIRELESQISELQEDLESERASRNKAEKQKRDL 1137 mouse LAKKEEELQAALARVEEEAAQKNMALKKIRELETQISELQEDLESERASRNKAEKQKRDL 1137 dog GEELEALKTELEDTLDSTAAQQELRSKREQEVNILKKTLEEEARTHEAQIQEMRQKHSQA 1200 human GEELEALKTELEDTLDSTAAQQELRSKREQEVNILKKTLEEEAKTHEAQIQEMRQKHSQA 1197 mouse GEELEALKTELEDTLDSTAAQQELRSKREQEVSILKKTLEDEAKTHEAQIQEMRQKHSQA 1197 dog VEELAEQLEQTKRVKANLEKAKQTLENERGELANEVKVLQQGKGDSEHKRKKAEAQLQEL 1260 human VEELAEQLEQTKRVKANLEKAKQTLENERGELANEVKVLLQGKGDSEHKRKKVEAQLQEL 1257 mouse VEELADQLEQTKRVKATLEKAKQTLENERGELANEVKALLQGKGDSEHKRKKVEAQLQEL 1257 dog QVKFTEGERVRTELADKVTKLQVELDNVMGLLTQSDSKSSKLTKDFSALESQLQDTQELL 1320 human QVKFNEGERVRTELADKVTKLQVELDNVTGLLSQSDSKSSKLTKDFSALESQLQDTQELL 1317 mouse QVKFSEGERVRTELADKVTKLQVELDSVTGLLSQSDSKSSKLTKDFSALESQLQDTQELL 1317
Canine MYH9 gene
80
dog QEENRQKLSLSTKLKQMEDEKNSFKEQLEEEEEAKRNLEKQIATLHAQVTDMKKKMEDGV 1380 human QEENRQKLSLSTKLKQVEDEKNSFREQLEEEEEAKHNLEKQIATLHAQVADMKKKMEDSV 1377 mouse QEENRQKLSLSTKLKQMEDEKNSFREQLEEEEEAKRNLEKQIATLHAQVTDMKKKMEDGV 1377 dog GCLETAEEAKRKLQKDLEGLGQRYEEKVAAYDKLEKTKTRLQQELDDLLVDLDHQRQTAS 1440 human GCLETAEEVKRKLQKDLEGLSQRHEEKVAAYDKLEKTKTRLQQELDDLLVDLDHQRQSAC 1437 mouse GCLETAEEAKRRLQKDLEGLSQRLEEKVAAYDKLEKTKTRLQQELDDLLVDLDHQRQSVS 1437 dog NLEKKQKKFDQLLAEEKTISAKYAEERDRAEAEAREKETKALSLARALEEAMEQKAELER 1500 human NLEKKQKKFDQLLAEEKTISAKYAEERDRAEAEAREKETKALSLARALEEAMEQKAELER 1497 mouse NLEKKQKKFDQLLAEEKTISAKYAEERDRAEAEAREKETKALSLARALEEAMEQKAELER 1497 dog LNKQFRTEMEDLMSSKDDVGKSVHELEKSKRALEQQVEEMKTQLEELEDELQATEDAKLR 1560 human LNKQFRTEMEDLMSSKDDVGKSVHELEKSKRALEQQVEEMKTQLEELEDELQATEDAKLR 1557 mouse LNKQFRTEMEDLMSSKDDVGKSVHELEKSKRALEQQVEEMKTQLEELEDELQATEDAKLR 1557 dog LEVNLQAMKAQFERDLQGRDEQSEEKKKQLVRQVREMEAELEDEKKQRSMAVAARKKLEM 1620 human LEVNLQAMKAQFERDLQGRDEQSEEKKKQLVRQVREMEAELEDERKQRSMAVAARKKLEM 1617 mouse LEVNLQAMKAQFERDLQGRDEQSEEKKKQLVRQVREMEAELEDERKQRSMAMAARKKLEM 1617 dog DLKDLEAHIDSANKNRDEAIKQLRKLQAQMKDCVRELDDTRASREEILAQAKENEKKMKS 1680 human DLKDLEAHIDSANKNRDEAIKQLRKLQAQMKDCMRELDDTRASREEILAQAKENEKKLKS 1677 mouse DLKDLEAHIDTANKNREEAIKQLRKLQAQMKDCMRELDDTRASREEILAQAKENEKKLKS 1677 dog MEAEMIQLQEELAAAERAKRQAQQERDELADEIANSSGKGALALEEKRRLEARIAQLEEE 1740 human MEAEMIQLQEELAAAERAKRQAQQERDELADEIANSSGKGALALEEKRRLEARIAQLEEE 1737 mouse MEAEMIQLQEELAAAERAKRQAQQERDELADEIANSSGKGALALEEKRRLEARIALLEEE 1737 dog LEEEQGNTELVNDRLKKANLQIDQINTDLNLERSHAQKNENARQQLERQNKELKVKLQEM 1800 human LEEEQGNTELINDRLKKANLQIDQINTDLNLERSHAQKNENARQQLERQNKELKVKLQEM 1797 mouse LEEEQGNTELINDRLKKANLQIDQINTDLNLERSHAQKNENARQQLERQNKELKAKLQEM 1797 dog EGTVKSKYKASITALEAKIAQLEEQLDNETKERQAACKQVRRAEKKLKDVLLQVDDERRN 1860 human EGTVKSKYKASITALEAKIAQLEEQLDNETKERQAACKQVRRTEKKLKDVLLQVDDERRN 1857 mouse ESAVKSKYKASIAALEAKIAQLEEQLDNETKERQAASKQVRRTEKKLKDVLLQVEDERRN 1857 dog AEQFKDQADKASTRLKQLKRQLEEAEEEAQRANASRRKLQRELEDATETADAMNREVSSL 1920 human AEQYKDQADKASTRLKQLKRQLEEAEEEAQRANASRRKLQRELEDATETADAMNREVSSL 1917 mouse AEQFKDQADKASTRLKQLKRQLEEAEEEAQRANASRRKLQRELEDATETADAMNREVSSL 1917 dog KNKLRRGDLPFVVPRRVARKGAGDCSDEEVDGKADGAEAKAAE 1963 human KNKLRRGDLPFVVPRRMARKGAGDGSDEEVDGKADGAEAKPAE 1960 mouse KNKLRRGDLPFVVTRRIVRKGTGDCSDEEVDGKADGADAKAAE 1960
Chapter 6
Identification of a 5 Mb region on canine chromosome 10
harbouring a causative gene responsible for congenital sensorineural deafness
in German Dalmatian dogs
Fine mapping of CFA10 83
Identification of a 5 Mb region on canine chromosome 10 harbouring a causative gene responsible for congenital sensorineural deafness in German Dalmatian dogs
Abstract
In the present study we evaluated whether the canine chromosome (CFA) 10
harbours a gene responsible for canine congenital sensorineural deafness (CCSD) in
Dalmatian dogs Altogether 27 microsatellite markers covering the whole CFA10
were genotyped in 176 Dalmatian dogs with frequent occurrence of CCSD
Significant linkage between the deafness phenotype and microsatellites located in a
region of 36 Mb to 48 Mb on CFA10 was found In order to refine the location of the
causative canine congenital sensorineural deafness (CCSD) gene we used data
deposited in the NCBI to search for single nucleotide polymorphisms (SNPs) in the
intronic sequences of the canine genes located on CFA10 in this region We
characterized 26 SNPs and used them for non-parametric linkage and association
analyses with CCSD of altogether 34 Dalmatian dogs from five full-sib families We
could find significant linkage to SNPs located in the region spanning 39 Mb to 44 Mb
and significant haplotype-trait association for SNPs in this region These results
enforce further evaluation of this 5 Mb region with the aim to detect the gene
responsible for CCSD in Dalmatian dogs
Introduction
Of the identified genes responsible for different forms of sensorineural non-syndromic
deafness in humans Rak et al (2003) and co-workers (Droumlgemuumlller et al 2002
Kuiper et al 2002 Rak et al 2002a 2002b 2003) considered 24 genes as
candidates for sensorineural deafness in dogs among them the MYH9 gene on
CFA10 Subsequently Rak (2003) developed a set of microsatellite markers for the
respective 24 candidate genes
As described in chapter 3 we could show significant linkage of CCSD with the MYH9
associated microsaellite MYH9_MS3 (=FH2293) in different German Dalmatian dog
Fine mapping of CFA10
84
families However as described in Chapter 5 we already excluded MYH9 for being
responsible for the CCSD phenotype in German Dalmatian dog families segregating
for CCSD by comparative sequencing of genomic sequences from deaf and normal
hearing Dalmatian dogs and additionally by sequencing of full length canine cDNA
The reason for the MYH9_MS3 (=FH2293) indicating linkage may be caused by a
closely linked gene involved in CCSD Thus the objective of the present study was to
perform a scan of canine chromosome 10 using microsatellite markers and single
nucleotide polymorphisms (SNPs) in order to evaluate whether CFA10 harbours a
gene responsible for the deafness phenotype in Dalmatian dogs Additionally we
analyzed the association of the CCSD phenotype with a large number of newly
developed SNPs located in the genomic deafness region on CFA10
Material and methods Pedigree material
For the linkage analysis of the microsatellite marker on CFA10 we used DNA from
altogether 176 animals belonging to 22 full-sib families and one large paternal half-
sib family of German Dalmatian dogs All families were segregating for CCSD The
genotyped families included all the affected dogs (unilaterally and bilaterally deaf)
their parents if available and one to four unaffected full-sibs At least two of the full
sibs of each family were unilaterally deaf The phenotype of the affected animals had
been confirmed by brainstem auditory evoked response (BAER) that detects
electrical activity in the cochlea and auditory pathways in the brain
Screening for SNPs was performed by comparative sequencing of DNA from parents
of five families with significant linkage of microsatellites located in the region
spanning 36 Mb to 48 Mb to CCSD For the linkage analysis concerning the SNPs we
then used blood samples from 34 Dalmatian dogs consisting of the progeny and their
parents of the abovementioned five full-sib families of Dalmatian dogs with frequent
occurrence of CCSD The families consisted of five to nine individuals and their
parents
Microsatellite marker analysis
We used 27 microsatellite marker derived from the NCBI database
(httpwebncbinlmnihgov) to cover CFA10 completely (Appendix Table 1)
Fine mapping of CFA10 85
PCR reactions for microsatellites were carried out in 12 microl reaction mixtures
containing 2 microl genomic DNA 12 microl 10x PCR buffer (Qbiogene Heidelberg
Germany) 024 microl dimethyl sulfoxide (DMSO) 02 microl dNTPs (100 microM) 01 microl Taq
Polymerase (5Umicrol) (Qbiogene) 06 microl (10 microM) 5rsquo-IRD700 or IRD800 labelled forward
primer 06 microl (10 microM) unlabelled reverse primer The PCR reactions were carried out
in MJ Research thermocyclers with the following program 4 min at 94 degC followed
by 32 cycles of 30 sec at 94degC 30 sec at maximum annealing temperature and a
final extension of 45 sec at 72degC PCR-products were diluted with formamide loading
buffer in ratios from 110 to 140 and then size-fractionated by gel electrophoresis on
automated LI-COR 42004300 sequencers (LI-COR inc Lincoln NE USA) using
denaturing 4 and 6 polyacrylamide gels (RotiphoreseregGel 40 Roth Karlsruhe
Germany)
Development of single nucleotide polymorphisms (SNPs)
We developed SNPs for the region between 36 Mb to 48 Mb on CFA10 previously
proven to be linked to the CCSD phenotype The SNPs for this region were derived
from one 5294 Mb large whole genome shotgun sequence (NW_876251) deposited
in the current dog genome assembly (Boxer genome assembly 21) of the NCBI
GenBank The canine genomic sequences and mRNA of the genes that were used
for the analysis were also derived from sequences deposited in the current dog
genome assembly (Boxer genome assembly 21) of the NCBI GenBank (Table 1)
In total 72 primer pairs were designed most of them located intragenic in intronic
sequences or alternatively in flanking regions of the 5rsquo- or 3rsquondashends of the respective
gene yielding products with a length of around 600 bp PCR primers were developed
with the Primer3 program (httpfrodowimiteducgi-binprimer3primer3_wwwcgi)
The PCR reactions were performed in a total of 50 microl containing 125 microM dNTPs 25
pmol of each primer the reaction buffer supplied by the manufacturer (Qiagen
Hilden Germany) and 1 U Taq polymerase After a 4 min initial denaturation at
95degC 35 cycles of 30 sec at 94degC 45 sec at 58degC and 80 sec at 72degC were
performed in a MJ Research thermocycler (Biozym Hessisch Oldendorf Germany)
The obtained PCR products were directly sequenced with the DYEnamic ET
Terminator kit (AmershamBiosciences Freiburg Germany) and a MegaBACE 1000
capillary sequencer (AmershamBiosciences) using the PCR primers as sequencing
primers Sequence data were analyzed with Sequencher 42
Fine mapping of CFA10
86
(GeneCodes Ann Arbor MI USA) If a heterozygous SNP was found for one or both
parents all progeny of the respective families were analyzed for that SNP
We developed altogether 26 SNPs with 20 SNPs located intragenic in intronic
sequences and 6 SNPs located very closely at the 5rsquo- or 3rsquo-ends respectively with
one to four SNPs per gene (Table 3)
The newly identified 26 SNPs are shown in Tabele 2 Only SNPs are listed and
chosen for linkage analyses that were heterozygous for one or both parents of at
least two of the five families Of all SNPs only one was heterozygous in all families
(Table 3) The most frequent form of SNPs with a frequency of 423 was the AG
transition motif The scarcest one with a frequency of 385 was the CG and the
AC transversion motif respectively
Linkage analysis
Multipoint non-parametric linkage and haplotype analysis were performed using the
MERLIN software version 0102 (multipoint engine for rapid likelihood inference
Center for Statistical Genetics University of Michigan MI USA Abecasis et al
2002) The test statistics Z-mean and Lod score were used to test for the proportion
of alleles shared by affected individuals identical by descent (IBD) for the considered
marker loci
In a first step linkage analysis was performed regarding the 27 marker covering the
whole CFA10 In a second step a linkage analysis was performed including the newly
added SNPs spanning the region 36 Mb to 48 Mb on CFA10
The observed heterozygosity (HET) and the polymorphism information content (PIC)
were calculated using the software package SASGenetics (Statistical Analysis
System Version 913 SAS Institute Inc Cary NC USA 2005)
Linkage disequilibrium (LD) and association of haplotypes with CCSD were tested
using the procedures CASECONTROL and HAPLOTYPE of SASGenetics
(Statistical Analysis System version 913 Cary NC USA)
Results
A linkage analysis was at first carried out for the 176 animals that were analyzed with
27 microsatellite markers covering the whole CFA10
Fine mapping of CFA10 87
The microsatellite FH3302 positioned at 3887 Mb showed the highest Zmean with a
value of 234 (p= 001) and a Lod score of 121 (p= 0009) at 3887 Mb A Zmean
value of 217 (p= 0015) and a Lod score of 12 (p= 0009) was reached for the
microsatellite REN181G20 positioned at 3936 Mb indicating linkage but all Zmean
values and error probabilities of eight microsatellite markers in the interval from 3666
Mb up to 488 Mb were almost as high Consequently we screened the region
spanning from 36 Mb to 48 Mb for SNPs Out of the 23 analyzed Dalmatian dog
families five full-sib families were chosen to screen for SNPs because of their
significant contribution to the test statistics
Twenty-six new SNP markers covering the region of 36 Mb to 48 Mb equally were
developed and added to the linkage analysis These SNPs had PIC values ranging
from 013 to 037
Figure 1 shows the Zmeans and LOD scores for all 27 microsatellite markers on
CFA10 harbouring a genomic deafness region Figure 2 shows the corresponding p-
values Adding the SNPs in the interval spanning 36 Mb to 48 Mb we could narrow
the significant region down to 5 Mb spanning 39 Mb to 44 Mb (Table 4 Figures 3 and
4)
Haplotype-trait association test statistics for the SNPs 16-23 located in the interval
from 39 Mb to 44 Mb were significant However the marker-trait association test
failed the 5 threshold of the error probability (p= 007) but was lowest for all
possible haplotype-trait combinations The χ2ndashtests of the procedure
CASECONTROL were not significant indicating that a SNP for the causative
deafness gene was not yet found
Discussion
We used 27 microsatellite marker and 26 newly developed SNP markers with the
intention to create a dense map for linkage analysis of CFA10 especially the region
spanning 36 Mb to 48 Mb proven to be linked to the CCSD phenotype All SNP
markers were chosen due to their heterozygosity in one or both parents of at least
two families
The significant Zmeans on CFA10 reported for the chromosome scan using only
microsatellites was confirmed by adding the SNP markers Furthermore with the use
of SNPs the apparently linked region from 36 Mb to 48 Mb could be narrowed down
Fine mapping of CFA10
88
to 5 Mb spanning 39 Mb to 44 Mb For this reason the use of SNPs in addition to
informative microsatellites appears highly recommendable
The identified CCSD region spanning 5 Mb might be participating in the development
of CCSD in the analyzed Dalmatian dog families
However genes that are known to be involved in the hearing process or known to be
expressed in the inner ear are not annotated at the syntenic human region on homo
sapiens autosome (HSA) 2 spanning from 100 Mb to 108 Mb Therefore a well-
defined candidate gene for canine hereditary deafness in the abovementioned 5 Mb
does not exist This means that for all genes in this region informative SNPs have to
be developed and tested for linkage disequilibrium and association with CCSD
Despite the remarkable progress that has been made in the past few years in
identifying deafness genes in man and mouse it is suspected that only one-third of all
deafness causing genes have been identified so far Therefore more SNPs have to
be developed within the identified region on CFA10 to localize the deafness causing
gene or to find unambiguously associated SNP markers which could be used for a
population-wide genetic test for CCSD
Fine mapping of CFA10 89
Table 1 Canine genes where gene-associated SNPs could be developed with their
exact location on CFA10 and their corresponding accession numbers (Acc No)
Gene symbol Gene description Position in
Mb
Acc No canine genomic sequence
Acc No canine mRNA
LOC474535 similar to hypothetical protein MGC40397 36493650 NC_006592 XM_531764
LOC474536 similar to KM-102-derived reductase-like factor
37023714 NC_006592 XM_531765
LOC610953 similar to NHP2-like protein 1 37293729 NC_006592 XM_848546
LOC481302 similar to C2orf26 protein 37363737 NC_006592 XM_848552
LOC610991 hypothetical protein LOC610991 38103820 NC_006592 XM_848591
LOC611007 similar to eukaryotic translation elongation factor 1 alpha 2
38353835 NC_006592 XM_848614
LOC474541 similar to GRIP coiled-coil protein GCC185 isoform a
38503857 NC_006592 XM_531770
LOC474542 similar to Sulfotransferase 1C2 38613862 NC_006592 XM_531771
LOC481308 similar to Keratin type I cytoskeletal 18 38653865 NC_006592 XM_538429
LOC474543 similar to Sulfotransferase K1 38683870 NC_006592 XM_857994
LOC609217 similar to family with sequence similarity 32 member A like
39453945 NC_006592 XM_858065
LOC611115 similar to D-PCa-2 protein isoform c 39843984 NC_006592 XM_848756
LOC481325 similar to ubiquitin-conjugating enzyme E2C
42564272 NC_006592 XM_538446
LOC481330 similar to Interleukin-1 receptor type II precursor
44084413 NC_006592 XM_538451
LOC611493 hypothetical protein LOC611493 44384440 NC_006592 XM_863243
LOC481337 similar to DNA repair protein REV1
46264637 NC_006592 XM_538458
LOC611728 similar to 3-hydroxyanthranilate 34-dioxygenase
48494852 NC_006592 XM_849433
Predicted gene derived from the dog genome assembly (build 21) that used gene
prediction method GNOMON supported by EST evidence
Fine mapping of CFA10
90
Table 2 The 26 newly developed SNPs for Dalmatian dogs located in the region
spanning 36 Mb to 48 Mb on CFA10 with their corresponding primers the SNP motif
the product size and the annealing temperature
Gene
description SNP
Location
(intron or
5rsquo3rsquo-end)
Primer F1 (5acute -gt 3acute)
SNP motif
Primer R2 (5acute -gt 3acute)
Bp3 AT4
LOC474535
SNP_1
intron
ACCCAAGCCTAACTGCAGAA
ACCCCAGTCT(CG)GCCAGAGCTGTT
590 60
SNP_2 intron TGTGTGCATA(AG)TGTGCTGAGCCT
TCATCTGTTAAAACAGGGGTGAT
LOC474536
SNP_3
intron
CCAGTTAATGATTGTTTCGTTGA
AAGCTGCTTT(AC)CACCCCCATCAG
TCATTCCTGCTGTTGTGCTC
590 60
LOC610953
SNP_4
intron
CTGTCTTGGGGACTGTTTGC
AAGGCAGACG(CT)AATGACTGAGGC
600 60
SNP_5 intron GACTTGAAGC(CT)ACCACAGATACT
GCCATCACGATGAACTCAGA
LOC481302
SNP_6
3rsquo-end
AATTGAGGCCGAAGTCCAAT
CTTTTCCCCA(GT)GCCACCCCTCTG
GAGCACTATTTACGATACAAACAGGA
610 60
LOC610991
SNP_7
intron
CATGCATGATGCCCAGAGTA
CCCAAAGCAC(AG)CTGTGATTTAAT
AGGGCTTCCTGGGAAAAGT
600 60
LOC611007
SNP_8
intron
CAGACCAACAGTGACCCAGA
TAGGCATACC(GT)TCAGTCCTAAAG
GCCTGTTGTGGGCAGAGTAT
480 60
LOC474541
SNP_9
intron
ACTGAGCCAAAGGTGGATTG
AGAGAATAGC(AG)CTGTGTTTTACA
ACCTGCACATCGGGATTTAG
575 60
Fine mapping of CFA10 91
Table 2 (continued)
Gene-
symbol SNP
Location
(intron or
5rsquo3rsquo-end)
Primer F1 (5acute -gt 3acute)
SNP motif
Primer R2 (5acute -gt 3acute)
Bp3 AT4
LOC474542
SNP_10
intron
CTTCCCCAGGAGAGAGTGAC
AATATGATCA(CT)ATTTAAAGAAAT
CTTTTGTCAACATCCCCTTCA
560 60
LOC481308
SNP_11
3rsquo-end
ACCCATTGTCTCTCCAGCAC
CCACATAACT(GT)AGCATCCCTAGC
600 60
TGATGATGTAAGTTGGCCTCA
LOC474543
SNP_12
intron
TTGAAGTTGTGTGAGTAAATGAAAGA
CAATATATAA(AG)CATTTGCTACAA 600 60
SNP_13 intron TTGAGATGAA(AT)ATAATAGAGCTG
SNP_14 intron CGATTTTCCA(AG)GACATGGAGATG
SNP_15 intron ACTGAACCAA(AG)TGTGCAAAGCAT
GGAAACCATGCAGTCTTTGG
LOC609217
SNP_16
5rsquo-end
TGGCCTCATTTTCCAGTATG
GAAGGAGTTA(CT)ACAGTGAAGATA
580 60
SNP_17 5rsquo-end AAGTGATCAA(AG)CCAGTGCATTCT
GGCAATTACCCTGAGTGGTG
LOC611115
SNP_18
3rsquo-end
GGGCTGTCTTAGAGGTGCTG
TGTGGTCTCA(CT)ACACTTCCTGAG
590 60
SNP_19 3rsquo-end ATTGCAAGTG(GT)CCTGTGATGGCC
CTTCTTTGGGCAGGAAAGTG
LOC481325
SNP_20
intron
AAAATGATTGATCGCAAAAGAAA
AATTACTGTA(CT)AACAGTATCAGA
600 60
SNP_21 intron TAACAGTATC(AG)GAATAAAAGTTT
TTCTGTGATTGCACTGACCG
Fine mapping of CFA10
92
Table 2 (continued)
Gene-
symbol SNP
Location
(intron
or 5rsquo3rsquo-
end)
Primer F1 (5acute -gt 3acute)
SNP motif
Primer R2 (5acute -gt 3acute)
Bp3 AT4
LOC481330
SNP_22
intron
GAAAGGCCTGGGTTCAAAA
GGCAGGGAGG(AG)TCACCATCGTTC
AATTTCCCCAAATGCCTCAC
575 60
LOC611493
SNP_23
intron
GCATGAAGGAGCCCTATGTC
CCAAGAGTCC(AT)GCCCAACACCCT
GGAGGGATGGCATTCTATGA
590 60
LOC481337
SNP_24
intron
GGCTGAGGAGATTGTGTTTCA
GCTGATATTT(AG)GCCTTCTGAGAT
620 60
SNP_25 intron AAAGCCAGAC(AG)CTTTGCCATGGT
CAGCTCCCTGTAATGGGAAA
LOC611728
SNP_26
intron
TCCTACTCCCATCACTTCCAA
CCACACTGGG(GT)CCTGGGATGAGG
CACAGCTCCATGTAGGTCCA
620 60
hypothetical gene found in the 2 version 1 of the NCBIs genome annotation 1 Forward 2 Reverse 3 Product size (basepairs) 4 Annealing temperature (degC)
Fine mapping of CFA10 93
Table 3 The 26 newly developed intragenic SNPs for Dalmatian dogs with their
nucleotide polymorphism allele and genotype frequencies observed heterozygosity
(HET) and polymorphism information content (PIC)
SNP Fam1 Nucleotide
polymorphism
Allele
frequencies
Genotype
frequencies2 PIC HET
SNP_1 125 CgtG 042054 3135 036 055
SNP_2 124 AgtG 064039 91013 037 044
SNP_3 2345 AgtC 041049 2156 033 047
SNP_4 345 CgtT 065035 6140 035 067
SNP_5 34 CgtT 065035 490 028 042
SNP_6 345 CgtT 066034 6130 035 039
SNP_7 1345 AgtG 054046 6174 037 053
SNP_8 145 GgtT 075025 01010 029 045
SNP_9 1345 AgtG 052054 6145 037 047
SNP_10 1234 CgtT 057043 7173 037 068
SNP_11 14 GgtT 065035 5120 035 062
SNP_12 15 AgtG 028072 097 030 044
SNP_13 45 AgtT 077023 870 017 021
SNP_14 5 AgtG 036064 052 013 015
SNP_15 134 AgtG 034066 0157 034 059
SNP_16 145 CgtT 030070 3812 033 036
SNP_17 245 AgtG 058042 6113 030 032
SNP_18 123 CgtT 037063 1126 027 035
SNP_19 123 GgtT 045055 3115 030 032
SNP_20 2345 CgtT 032068 1129 029 039
SNP_21 1235 AgtG 063037 883 037 052
SNP_22 1234 AgtG 068032 10102 030 034
SNP_23 12345 AgtT 030070 21313 033 046
SNP_24 124 AgtG 047053 667 033 019
SNP_25 234 AgtG 053047 3132 035 053
SNP_26 234 GgtT 060040 3120 026 0391 Number of families which were informative for the respective SNPs 2 Genotypes are given as number of animals [homozygous for allele 1heterozygous
homozygous for allele 2]
Fine mapping of CFA10
94
Table 4 Linkage analysis for canine chromosome 10 regarding the region spanning
39 Mb to 44 Mb with the significant Zmean LOD score and error probabilities (p-
values)
Marker Location (Mb) Zmean pz-value1 LOD-score pL-value2
SNP_16 39453 262 0004 123 0009
SNP_17 39455 262 0004 123 0009
SNP_18 39840 261 0004 123 0009
SNP_19 39843 261 0004 123 0009
SNP_20 4260 255 0005 118 0010
SNP_21 4270 255 0005 117 0010
SNP_22 4405 317 00008 131 0007
SNP_23 4439 337 00004 134 0007 1 Error probability regarding Zmean 2 Error probability regarding LOD score
Fine mapping of CFA10 95
Figure 1 Zmeans and LOD scores for 27 microsatellite markers on CFA10
harbouring a congenital sensorineural deafness region (number of families 23
number of genotyped dogs 176)
Figure 2 P-values for Zmeans and LOD scores of 27 microsatellite markers on
CFA10 harbouring a congenital sensorineural deafness region (number of families
23 number of genotyped dogs 176)
Fine mapping of CFA10
96
Figure 3 Zmeans and LOD scores of 53 markers on CFA10 harbouring a congenital
sensorineural deafness region (number of families 5 number of genotyped dogs 34)
Figure 4 P-values for Zmeans and LOD scores of 53 markers on CFA10 for the
region between 30 to 60 Mb harbouring a congenital sensorineural deafness region
(number of families 5 number of genotyped dogs 34)
CCSD region (SNP 16-23)
Chapter 7
Analysis of canine chromosome 1 and the Gap junction protein alpha 1 (GJA1) gene
in Dalmatian dogs segregating for congenital sensorineural deafness
CFA1 and the GJA1 gene 99
Analysis of canine chromosome 1 and the Gap junction protein alpha 1 (GJA1) gene in Dalmatian dogs segregating for congenital sensorineural deafness Abstract Two microsatellite markers associated to the GJA1 (alternatively connexin 43) gene
showed significant linkage with canine congenital deafness (CCSD) in a large French
half-sib family (Chapter 3) PCR products of this half-sib family were used to perform
a sequence analysis in order to evaluate whether GJA1 is responsible for CCSD As
the linkage could not be confirmed we used altogether 27 microsatellite markers for
a scan of canine chromosome (CFA) 1 with 23 Dalmatian dog families segregating
for CCSD A non-parametric linkage analysis was performed to see whether
significant test statistics for other genomic regions on CFA1 and for more families can
be shown As a result we could not find linkage to any microsatellite in the analyzed
families
Introduction Rak (2003) considered in total 24 genes as candidates for sensorineural deafness in
dogs among them the Gap junction protein alpha 1 (GJA1) or connexin 43 gene on
canine chromosome (CFA) 1 A marker set of altogether 43 microsatellites were
developed by Rak (2003) among them two microsatellite marker associated to the
GJA1 gene
GJA1 or connexin 43 is a member of the connexin gene family and a component of
gap junctions Mutations in 4 members of the connexin gene family have been shown
to underlie distinct genetic forms of deafness including GJB2 (connexin [CX] 26)
GJB3 (CX31) GJB6 (CX30) and GJB1 (CX32)
Liu et al (2001) reported mutations in GJA1 in association with sensorineural
recessive deafness in man However these mutations have recently been shown to
involve the pseudogene of GJA1 on human chromosome (HSA) 5 and not the
CFA1 and the GJA1 gene
100
GJA1 gene on HSA 6 (WA Paznekas cited a personal communication from the
senior author (W E Nance) of the paper by Liu et al 2001)
In previously performed studies (Chapter 3) one large French Dalmatian dog family
with frequent occurrence of CCSD showed linkage to microsatellites associated to
the GJA1 gene
In this report we performed a mutation analysis of the GJA1 gene sequence to
identify polymorphisms In order to evaluate whether the GJA1 gene is responsible
for congenital sensorineural deafness in Dalmatian dogs we analyzed the
association of the GJA1 haplotypes with the CCSD phenotype Furthermore we
employed 27 microsatellite markers covering the entire CFA1 and used them for a
non-parametric linkage analysis with CCSD in a Dalmatian dog population of 176
animals with frequent occurrence of CCSD
Material and methods Pedigree material
For the linkage analysis of the microsatellite marker we used DNA from altogether
176 animals belonging to 22 full-sib families and one large paternal half-sib family of
German Dalmatian dogs All families were segregating for CCSD The genotyped
families included all affected dogs (unilaterally and bilaterally deaf) their parents if
available and one to four unaffected animals At least two of the full sibs of each
family were unilaterally or bilaterally deaf The phenotype of the affected animals had
been confirmed by brainstem auditory evoked response (BAER) that detects
electrical activity in the cochlea and auditory pathways in the brain
Mutation analysis and screening for SNPs was performed by comparative
sequencing of DNA from 16 animals consisting of the parents and their progenies of
one large half-sib family of French Dalmatian dogs which showed significant linkage
to GJA1-associated microsatellites (Chapter 3)
Sequencing of canine genomic DNA and mutation analysis
The canine GJA1 gene sequence was derived from sequences deposited in the
current dog genome assembly (Boxer genome assembly 21) of the NCBI GenBank
(Genbank Acc No NC_006583) We compared the canine genomic DNA sequence
to canine cDNA fragments in the canine EST database using the
CFA1 and the GJA1 gene 101
BLASTN (Basic Local Alignment Search Tool Nucleotide) program Three canine
ESTs (Acc No DN880776 BU744925 and DN369900) were used to confirm the
assembly of the GJA1 gene
To identify polymorphisms within the canine GJA1 sequence the gene consisting of
one 1251 bp spanning exon were PCR amplified and sequenced from 16 French
Dalmatian dogs Additionally large sequences from the 3acute-end were amplified Primer
pairs were designed yielding products with a length of around 600 bp PCR primers
were developed with the Primer3 program (httpfrodowimiteducgi-
binprimer3primer3_wwwcgi) The PCR reactions were performed in a total of 50 microl
containing 125 microM dNTPs 25 pmol of each primer the reaction buffer supplied by
the manufacturer (Qbiogene Heidelberg Germany) and 1 U Taq polymerase After
a 4 min initial denaturation at 95degC 35 cycles of 30 sec at 94degC 45 sec at 58degC and
80 sec at 72degC were performed in a MJ Research thermocycler (Biozym Hessisch
Oldendorf Germany)
The obtained PCR products were directly sequenced with the DYEnamic ET
Terminator kit (AmershamBiosciences Freiburg Germany) and a MegaBACE 1000
capillary sequencer (AmershamBiosciences) using the PCR primers as sequencing
primers Sequence data were analyzed with Sequencher 42
(GeneCodes Ann Arbor MI USA)
Microsatellite marker analysis
We used 27 microsatellite marker derived from the NCBI database
(httpwebncbinlmnihgov) to cover CFA1 completely (Appendix Table 1)
PCR reactions for microsatellites were carried out in 12 microl reaction mixtures
containing 2 microl genomic DNA 12 microl 10x PCR buffer (Qbiogene) 024 microl dimethyl
sulfoxide (DMSO) 02 microl dNTPs (100 microM) 01 microl Taq Polymerase (5Umicrol)
(Qbiogene) 06 microl (10 microM) 5rsquo-IRD700 or IRD800 labelled forward primer 06 microl (10
microM) unlabelled reverse primer The PCR reactions were carried out in MJ Research
thermocyclers with the following program 4 min at 94 degC followed by 32 cycles of 30
sec at 94degC 30 sec at maximum annealing temperature and a final extension of 45
sec at 72degC PCR-products were diluted with formamide loading buffer in ratios from
110 to 140 and then size-fractionated by gel electrophoresis on automated LI-COR
42004300 sequencers (LI-COR inc Lincoln NE USA) using denaturing 4 and 6
polyacrylamide gels (RotiphoreseregGel 40 Roth Karlsruhe Germany)
CFA1 and the GJA1 gene
102
Linkage and haplotype analysis
Multipoint non-parametric linkage and haplotype analyses were performed using the
MERLIN software version 0102 (multipoint engine for rapid likelihood inference
Center for Statistical Genetics University of Michigan MI USA Abecasis et al
2002) The test statistics Z-mean and Lod score were used to test for the proportion
of alleles shared by affected individuals identical by descent (IBD) for the considered
marker loci
A non-parametric linkage analysis was performed with 27 microsatellite markers in 23
Dalmatian dog families consisting of 176 Dalmatian dogs The results were added to
the linkage analysis performed in Chapter 3
Results and discussion
In previously performed studies (Chapter 3) one French Dalmatian dog family
reached a Zmean value of 295 (plt0002) for GJA1_MS1 and a Zmean value of 286
(plt0002) for GJA1_MS2 indicating linkage Both markers were associated with the
GJA1 gene
By sequence analysis we revealed a total of 3 SNPs found in sequences of the 3acute-
UTR shown in Table 1 None of the polymorphisms were polymorphic in all parents of
the examined French half-sib family Neither of the observed polymorphism did alter
the predicted amino acid sequence of GJA1 nor showed the identified haplotypes an
association with the CCSD phenotype (Figure 1)
There is no recombination of the haplotypes of the GJA1 gene in this family The
paternal and maternal haplotypes as far as they could be estimated were distributed
among the affected progeny closely to the expected proportion of 50 and therefore
no excess of a certain haplotype could be observed in the affected dogs
Thus evaluation of the SNP markers debilitates the linkage to CCSD for the French
half-sib family Because of the fact that both animals with unilateral hearing loss and
bilateral hearing animals shared identical haplotypes these polymorphisms are
obviously not associated with CCSD in these Dalmatian dog families Furthermore
the present study revealed no functional mutations of the complete coding region of
GJA1 It is therefore unlikely that the GJA1 gene is involved in the pathogenesis of
CCSD in the analyzed French Dalmatian dogs The reason for GJA1_MS1 and
GJA1_MS2 indicating linkage may be caused by a closely linked gene involved in
CFA1 and the GJA1 gene 103
CCSD among the half-sib family of French Dalmatian dogs or by a false positive
result of the microsatellite study performed in Chapter 3
To clarify whether significant test statistics for other genomic regions on CFA1 and
for more families can be shown we used 27 microsatellite markers derived from the
NCBI database (httpwebncbinlmnihgov) to cover CFA1 completely A linkage
analysis was carried out after genotyping 176 German Dalmatian dogs with a set of
27 microsatellite markers The results of this linkage analysis were added to the
results of the test statistics for the microsatellites GJA1_MS1 and GJA1_MS2
(Chapter 3)
As a result we could not find linkage to any microsatellite in the analyzed families
(Figure 2 and 3) It is unlikely that the canine chromosome 1 harbours genomic
regions that are involved in the development of CCSD in the analyzed Dalmatian dog
families
With hindsight it was revealed that GJA1 is not responsible for sensorineural non-
syndromic deafness in humans as Liu et al (2001) has published GJA1 is
participating in a human syndrome called oculodentodigital dysplasia (ODDD) that
can be accompanied with hearing impairment (Paznekas et al 2003) But the type of
deafness in human ODDD differs from the typical hearing loss associated with other
connexin mutations because it is conductive rather than sensorineural
As deafness in dogs especially in Dalmatians is almost exclusively caused by
sensorineural non-syndromic forms also known as cochleosaccular degeneration
the GJA1 gene should not be considered as a candidate gene for CCSD anymore
CFA1 and the GJA1 gene
104
Table 1 Three newly developed intragenic SNPs and two microsatellite markers for
Dalmatian dogs in the candidate gene GJA1 with their corresponding primers the
SNP motif the product size and the annealing temperature
SNP
Primer F (5acute -gt 3acute)
SNP motif
Primer R (5acute -gt 3acute)
Product
size (bp)
Annealing
temperatur
GJA1_SNP1+2
CACCTTAGGCGTTCATTTTG
CCGGGGAG(AG)AAAA(AG)AAAAATACTT
TGGCTTGATTCCCTGACTC
650 58
GJA1_SNP3
TCTGAAATGTAATCATGGATGC
CAGAACTTGTAT(AT)CTGTTAAGAG
AATCACACAGGATATAGAGGCTATC
600 58
Microsatellite
marker Primers (forward reverse ) 5acute -gt 3acute
Product
size (bp)
Annealing
temperatur
GJA1_MS1
ATGGCATGAAGAGGATACCG
AGGACAGGTGACGGCTCTAC
134 60
GJA1_MS2
GCTAGTACTCGATTGTGGTC
TCATGGGTTGTGAGATCCAG
190 60
CFA1 and the GJA1 gene 105
Figu
re 1
Hap
loty
pes
of th
e G
JA1
gene
in th
e an
alyz
ed D
alm
atia
n do
g fa
mily
CFA1 and the GJA1 gene
106
Figure 2 Zmeans and LOD scores for 29 microsatellite markers on CFA1 (number of
families 23 number of genotyped dogs 176)
Figure 3 P-values for Zmeans and LOD scores of 29 microsatellite markers on CFA1
(number of families 23 number of genotyped dogs 176)
Chapter 8
Analysis of canine chromosome 31 and the claudin-14 (CLDN14) gene
in Dalmatian dogs segregating for congenital sensorineural deafness
CFA31 and the CLDN14 gene 109
Analysis of canine chromosome 31 and the claudin-14 (CLDN14) gene in Dalmatian dogs segregating for congenital sensorineural deafness Abstract A previously accomplished linkage analysis revealed significant linkage of
microsatellite markers associated with the CLDN14 gene on canine chromosome
(CFA) 31 with the phenotype of canine sensorineural deafness (Chapter 3) The
objective of the present study was to perform a sequence analysis in order to find
single nucleotide polymorphisms (SNPs) for the CLDN14 gene and additionally to
use a set of six microsatellite markers evenly distributed on CFA31 for non-
parametric linkage analysis with the aim to verify the significant test statistics shown
in Chapter 3
Introduction
Of the genes responsible for different forms of sensorineural non-syndromic deafness
in humans Rak (2003) considered 24 genes as candidates for sensorineural
deafness in dogs among them the CLDN14 gene on canine chromosome (CFA) 31 CLDN14 encodes a member of the claudin family which comprises the major
components of tight juncions (TJ) The human CLDN14 gene consists of one
tranlatete exon and six exons in the 5rsquountranlated region Five splice isoformes are
identified so far (Wilcox et al 2001 Wattenhofer et al 2005)
For the compartmentalization of perilymph and endolymph in the inner ear the
leakage of solutes through a paracellular pathway must be prevented by tight
junctions TJ are an intercellular junction found at the most apical region of polarised
epithelial and endothelial cells at which adjacent plasma membranes are joined
tightly together separating apical membranes and basolateral menbranes They are
specialised membrane domains containing branching strands of integral proteins and
create a primary barrier preventing paracellular transport of solutes and restricting
lateral diffusion of membrane lipids and proteins (Schneeberger and Lynch 2004)
CFA31 and the CLDN14 gene
110
The important role of the CLDN14 gene in the TJ of the inner ear was demonstrated
by Wilcox et al (2001) when he identified mutations in the CLDN14 gene responsible
for a hereditary human deafness in families segregating for congenital recessive
deafness (DFNB29)
In previously performed studies one large half-sib family as well as several full-sib
families of German Dalmatian dog indicated linkage to CLDN14 gene-associated
markers (Chapter 3)
A sequence analysis was performed to identify single nucleotide polymorphisms
(SNPs) either intergenic located or alternatively in the flanking 5rsquo- and 3rsquo-Regions
Furthermore we employed microsatellite markers covering CFA31 and used them for
a non-parametric linkage analysis with CCSD in a German Dalmatian dog population
of 176 animals with frequent occurrence of CCSD
Material and methods Pedigree material
For the linkage analysis of the microsatellite marker we used DNA from altogether
176 animals belonging to 22 full-sib families and one large paternal half-sib family of
German Dalmatian dogs All families were segregating for CCSD The genotyped
families included all the affected dogs (unilaterally and bilaterally deaf) their parents
if available and one to four unaffected animals At least two of the full sibs of each
family were unilaterally or bilaterally deaf The phenotype of the affected animals had
been confirmed by brainstem auditory evoked response (BAER) that detects
electrical activity in the cochlea and auditory pathways in the brain
Mutation analysis and screening for SNPs was performed by comparative
sequencing of DNA from 36 animals consisting of the parents and their progenies of
one half-sib family and four full-sib families of Dalmatian dogs which showed
significant linkage to a CLDN14-associated microsatellite (Chapter 3)
Sequencing of canine genomic DNA and development of single nucleotide
polymorphisms (SNPs)
The canine CLDN14 gene sequence was derived from sequences deposited in the
current dog genome assembly (Boxer genome assembly 21) of the NCBI GenBank
(Genbank Acc No NW_876295) by BLAST (Basic Local Alignment Search Tool)
CFA31 and the CLDN14 gene 111
search (httpwwwncbinlmnihgovBLAST) using the human CLDN14 reference
mRNA sequence (Genbank Acc No NM_144492)
We compared the canine genomic DNA sequence to canine cDNA fragments in the
canine EST database using the BLASTN program As no ESTs could be found
human mRNA sequences were used for the localization of the exonintron
boundaries using the mRNA-to-genomic alignment program Spidey
(httpwwwncbinlmnihgovIEBResearchOstellSpideyindexhtml)
We screened exon three for mutations as this exon is the only translated one in man
Additionally we screened large intronic sequences and sequences located in the 5rsquo-
and 3rsquo-UTR of the CLDN14 gene for Polymorphisms For this purpose primer pairs
were designed yielding products with a length of around 600 bp
PCR primers were developed with the Primer3 program (httpfrodowimiteducgi-
binprimer3primer3_wwwcgi) The PCR reactions were performed in a total of 50 microl
containing 125 microM dNTPs 25 pmol of each primer the reaction buffer supplied by
the manufacturer (Qbiogene Heidelberg Germany) and 1 U Taq polymerase After
a 4 min initial denaturation at 95degC 35 cycles of 30 sec at 94degC 45 sec at 58degC and
80 sec at 72degC were performed in a MJ Research thermocycler (Biozym Hessisch
Oldendorf Germany)
The obtained PCR products were directly sequenced with the DYEnamic ET
Terminator kit (AmershamBiosciences Freiburg Germany) and a MegaBACE 1000
capillary sequencer (AmershamBiosciences) using the PCR primers as sequencing
primers Sequence data were analyzed with Sequencher 42 (GeneCodes Ann
Arbor MI USA) The eight newly developed SNPs are shown in Table 1
Microsatellite marker analysis
In total six microsatellite marker were derived from the NCBI database
(httpwebncbinlmnihgov) to cover CFA31 completely (Appendix Table 1)
PCR reactions for microsatellites were carried out in 12 microl reaction mixtures
containing 2 microl genomic DNA 12 microl 10x PCR buffer (Qbiogene) 024 microl dimethyl
sulfoxide (DMSO) 02 microl dNTPs (100 microM) 01 microl Taq Polymerase (5Umicrol)
(Qbiogene) 06 microl (10 microM) 5rsquo-IRD700 or IRD800 labelled forward primer 06 microl (10
microM) unlabelled reverse primer The PCR reactions were carried out in MJ Research
thermocyclers with the following program 4 min at 94 degC followed by 32 cycles of 30
sec at 94degC 30 sec at maximum annealing temperature and a final extension of
CFA31 and the CLDN14 gene
112
45 sec at 72degC PCR-products were diluted with formamide loading buffer in ratios
from 110 to 140 and then size-fractionated by gel electrophoresis on automated LI-
COR 42004300 sequencers (LI-COR inc Lincoln NE USA) using denaturing 4
and 6 polyacrylamide gels (RotiphoreseregGel 40 Roth Karlsruhe Germany)
Linkage and haplotype analysis
Multipoint non-parametric linkage and haplotype analysis were performed using
MERLIN version 0102 (multipoint engine for rapid likelihood inference Center for
Statistical Genetics University of Michigan MI USA Abecasis et al 2002) The test
statistics Z-mean and Lod score were used to test for the proportion of alleles shared
by affected individuals identical by descent (IBD) for the considered marker loci A
linkage analysis was performed with 6 microsatellite markers on 176 Dalmatian dogs
The results were added to the linkage analysis performed in Chapter 3
Results and discussion CLDN14-associted microsatellites were significantly linked with CCSD in a scan of
candidate genes using 24 families with 215 Dalmatian dogs (Chapter 3) Selection of
four full-sib families and one half-sib family with the highest tests statistics lead to a
Zmean value of 383 (plt000007) for the CLDN14 gene-associated marker
CLDN14_MS2
To substantiate the linkage to the CLDN14 gene we searched for sequence
variations within the CLDN14 gene in four full-sib families and one half-sib family with
the highest contribution to the test statistics shown in Chapter 3 Most of the identified
polymorphisms were found in intronic sequences none were within exon three None
of the observed polymorphism did alter the predicted amino acid sequence of exon
three We revealed a total of eight SNPs (Table 1) but only two (SNP_4 and SNP_5)
out of the identified eight SNPs were polymorphic in the examined families Both
unilaterally and bilaterally deaf animals as well as normal hearing animals showed
identical haplotypes for these two polymorphic SNPs and thus no co-segregation with
the deaf Dalmatian dogs could be revealed (Figure 1 and 2) In this study we did not
identify any causal mutations for CCSD in the analyzed Dalmatian dogs Despite this
results a mutation outside of the translated genomic regions analyzed here may exist
that would affect CLDN14 expression
CFA31 and the CLDN14 gene 113
To clarify if other regions on CFA31 are responsible for the CCSD phenotype
additionally six microsatellite markers covering CFA31 were tested in 23 Dalmatian
dog families consisting of 176 individuals A non-parametric linkage analysis was
performed regarding these 23 families The results of this linkage analysis were
added to the results of the test statistics for the CLDN14 gene-associated
microsatellites (Chapter 3)
We could only find significant linkage with CCSD for CLDN14-associted
microsatellites in the abovementioned five families The two microsatellites located
most closely were proximal and distal 4 Mb off the CLDN14 gene REN110K04 with a
Zmean value of -009 (plt05) and FH2712 with a Zmean value of 022 (plt03) not
indicating linkage
It can not be excluded that CLDN14 or genes in its flanking regions are involved in
the development of CCSD in the analyzed Dalmatian dog families Other genes than
the CLDN14 that are known to be involved in the hearing process or known to be
expressed in the inner ear are not annotated at the syntenic human region of homo
sapiens autosome (HSA) 21q223 Therefore despite the CLDN14 gene no other
well-defined candidate gene for canine hereditary deafness exists in the linked
region Despite the remarkable progress that has been made in the past few years in
identifying deafness genes in man and mouse it is suspected that only one-third of all
deafness causing genes have been identified so far and thus it seems possible that
other genes in the flanking region of the CLDN14 gene are involved in the
development of the disease Anyway to clarify the importance of CLDN14 in the
CCSD phenotype more SNPs have to be developed within the CLDN14 gene as well
as in its flanking regions with the aim to find significant linkage disequilibrium of SNP
markers with CCSD
CFA31 and the CLDN14 gene
114
Table 1 The 8 newly developed SNPs for Dalmatian dogs in the CLDN14 gene on
CFA31 with their corresponding primers the SNP motif the product size and the
annealing temperature
SNP
Location
(intron or
5rsquo3rsquo-UTR)
Primer F1 (5acute -gt 3acute)
SNP motif
Primer R2 (5acute -gt 3acute)
Bp3 AT4
CLDN14_SNP1
intron
GACCATATGTTTGTGGCC
CTTCCAGGGAAA(AT)TGTCGTAGCC
CGTCAGGATGTTGGTGCC
580 60
CLDN14_SNP2
inton
GACCATATGTTTGTGGCC
GAAATTGTCGTA(AG)CCCGGCCGCT
CGTCAGGATGTTGGTGCC
580 60
CLDN14_SNP3
3rsquo-UTR
CTGCCTTCAAGGACAACC
CCAGAGGAATAA(CT)ATGATCGTGA
GATGAGTATCAGCCCAGC
550 60
CLDN14_SNP4
3rsquo-UTR
CTGCCTTCAAGGACAACC
ACCACCGCACAC(CT)GTCACAGCTT
GATGAGTATCAGCCCAGC
550 60
CLDN14_SNP5 3rsquo-UTR
CATGCCTTTGTCCCAAACTT
GAGACCCTCTGG(CT)TCCTTTTGGC
GTACCTGTTGCCTGGGTTGT
610 60
CLDN14_SNP6 5rsquo-UTR
CCTTCATCCTTTCTGGTTGA
GCTCACAGTCCC(AC)ATGGGGACAT
GGGGAGCATAATGTGGTCAT
585 60
CLDN14_SNP7 5rsquo-UTR
TGAACTGGTCCCAAGGAAAG
GCACGACCGAGC(CT)CTGGCTTTAC
GGGATGAGAGGGAGGTTTTT
580 60
CLDN14_SNP8 3rsquo-UTR
AATGCCTATCCCTTCTTTGGA
CACGTTACTGTG(AG)ACCTCTCTAC
GCAGGCTTTCCTCAAGTGTC
680 60
1 Forward 2 Reverse 3 Product size (basepairs) 4 Annealing temperature (degC)
CFA31 and the CLDN14 gene 115
Figure 1 Haplotypes of the CLDN14 gene-associated markers SNP_4 and SNP_5 in
the analyzed four Dalmatian dog full-sib families
CFA31 and the CLDN14 gene
116
Figu
re 2
Hap
loty
pes
of th
e C
LDN
14 g
ene-
asso
ciat
ed m
arke
rs S
NP
_4 a
nd S
NP
_5 in
the
anal
yzed
Dal
mat
ian
dog
half-
sib
fam
ily
Chapter 9
General discussion
General discussion 119
General discussion
The candidate gene approach In the first step of this thesis candidate genes for canine congenital sensorineural
deafness (CCSD) in Dalmatian dogs were analyzed by means of microsatellite
markers or alternatively by single nucleotide polymorphisms (SNPs)
The candidate genes for which a set of in total 43 microsatellites was available
included the following 24 genes CDH23 CLDN14 COCH COL11A2 DFNA5
DIAPH1 EDN3 EDNRB EYA4 GJA1 GJB2 GJB6 MITF MYH9 MYO6 MYO7A
MYO15A OTOF PAX3 POU4F3 SLC26A4 SOX10 TECTA and TMPRSS3
(Rak 2003) These genes are known to be involved either in human non-syndromic
deafness or in the human Waardenburg syndrome The Waardenburg syndrome
(WS) manifests with sensorineural deafness and pigmentation defects in iris hair and
skin WS is classified into four types depending on the presence or absence of
additional symptoms which are caused by mutations in the five genes EDN3
EDNRB MITF PAX3 and SOX respectively
For another eight recently identified genes responsible for different forms of human
non-syndromic deafness including TMC1 TMIE USH1C MYH14 MYO3A PRES
WHRN and ESPN linkage and association analyses were performed using newly
developed SNPs
In the last years most projects have exploited canine traits for which either direct
candidate genes could be proposed and evaluated or for which large informative
pedigrees were available to enable linkage mapping to identify candidate regions A
major component of such research efforts comprised the cloning sequencing and
mapping of individual canine homologs of genes either proposed as candidate
genes or expected to be located in candidate regions This was necessary to identify
new informative polymorphisms (eg SNPs microsatellites) for high resolution
mapping of candidate regions and to examine each exon and exonintron boundary
for positional candidates Availability of the second version of the dog genome
assembly (build 21) of the NCBI database shortcuts this effort and increases the
investigators efficiency Now either additional candidate genes for canine congenital
sensorineural deafness can be found directly by its gene symbol in the 21 of the
General discussion
120
NCBIs genome annotation or if a candidate gene is not yet annotated a BLAST
(Basic Local Alignment Search Tool) search against the canine whole genome
shotgun (wgs) sequence resource can be used to obtain the sequence of the canine
genomic contigs containing the human homologous gene and thus intragenic
markers can be developed for subsequent linkage and association analyses
Over the past decade it has become increasingly clear how far structural and
functional homologies at the gene level extend across even distantly related species
Much is known about deafness-causing gene mutations in humans and mice
including the fact that the clinical and histopathological findings are often very similar
to those of deafness in Dalmatian dogs Thus genes responsible for non-syndromic
congenital hereditary deafness in humans seem to be appropriate candidate genes
for CCSD (Rak and Distl 2005) In this thesis we first concentrated on the candidate
gene approach combined with linkage analysis method using affected pedigree
members Once a significant linkage was found only the linked genes with the
required low error probability values were used for further molecular genetic analysis
The method of candidate gene approach using either gene-associated microsatellite
or alternatively SNP markers was applied for in total 32 candidate genes which
comprise nearly all of the identified mutated genes causing non-syndromic hereditary
hearing impairment in humans
Linkage and association analysis In principle linkage and association are totally different phenomena Linkage is a
relation between loci and association is a relation between alleles
Linkage means that a haplotype characterised by microsatellites or SNPs is
significantly more often present in family members with the phenotype under study
than expected by random assortment For construction of haplotypes sets of closely
linked genetic markers on the same chromosome are needed which tend to be
inherited together as they are not likely to be separated by recombination Linkage
creates associations within families but not among unrelated induviduals
Association is a statistical statement about the co-occurrence of alleles or
phenotypes Association analysis can be carried out as a method of genetic analysis
that compares the frequency of alleles between affected and unaffected individuals
across all families Thus for association family structures are not necessary A given
allele is considered to be associated with the disease if the presence of that allele
General discussion 121
explains a significant proportion of the phenotypic trait variation For association
studies the developing of a marker set consisting of SNPs rather than microsatellites
is needed
In this thesis a total of 32 candidate genes for canine congenital deafness were
analyzed which showed an appropriate clinical and histological disease pattern in
comparison to deafness in Dalmatian dogs Rak (2003) developed a set of 43
microsatellites for in total 24 candidate genes among them the CLDN14 gene on
canine chromosome (CFA) 31 and the MYH9 gene on CFA10 The GJA1 on CFA1
was also considered as a candidate gene for CCSD (Rak 2003) and therefore two
gene-associated microsatellites have been developed Recently it turned out that
GJA1 is not responsible for human sensorineural non-syndromic deafness but for a
human syndromic disorder that can be related with conductive deafness
By the use of a non-parametric linkage analysis using the existing set of 43
microsatellites associated to 24 candidate genes we found linkage to markers
associated to CLDN14 on CFA31 MYH9 on CFA10 and GJA1 on CFA1
For another another eight candidate genes it was possible to develop SNPs
Performing linkage analyses as well as association and haplotype studies it was
possible to exclude these eight candidate genes from being responsible for the
CCSD phenotype
Over the past ten years significant progress has been made in the identification of
deafness gene localisations Up to now approximately 120 loci have been reported
for both autosomal dominant and recessive forms of non-syndromic hereditary
deafness in humans and only for one third the responsible gene mutation could be
detected Thus it can be expected that additional potential human candidates for
CCSD in Dalmatian dogs will become available in future (Van Camp and Smith
2003)
The extreme heterogeneity of human deafness often hampered genetic studies
because many different genetic forms of hearing loss give rise to similar clinical
phenotypes and conversely mutations in the same gene can result in a variety of
clinical phenotypes In man genes that transport ions across membranes to
maintain appropriate solute concentration and pH as well as regulatory genes mainly
transcription factors and genes that play a part in structural integrity are essential
for the hearing process
General discussion
122
The results of this thesis indicate that the inheritance of hearing loss in Dalmatian
dogs is probably as heterogenic in origin as it is in humans Genetic heterogeneity
means that different mutations cause a similar phenotype the different mutations
can either be found at the same locus (allelic heterogeneity) or even at different loci
(non-allelic heterogeneity) As linkage was found for different candidate genes in
different families subsequently only the families indicating linkage were chosen for
further molecular analyses
GJA1 on CFA1 MYH9 on CFA10 and CLDN14 on CFA31 and their flanking regions
are further analyzed with a combined approach using microsatellite and SNP
markers
CFA1 By the use of GJA1-associated microsatellites one large French Dalmatian dog
family reached a Zmean value of 295 (plt0002) for GJA1_MS1 and a Zmean value
of 286 (plt0002) for GJA1_MS2 indicating linkage Subsequently a sequence
analysis of the GJA1 gene using the above mentioned French Dalmatian dog family
was performed None of the observed polymorphism did alter the predicted amino
acid sequence of GJA1 nor showed the identified haplotypes an association with the
CCSD phenotype Thus evaluation of the SNP markers debilitates the linkage to
CCSD for the French half-sib family It is unlikely that the GJA1 gene is involved in
the pathogenesis of CCSD in Dalmatian dogs To see whether significant test
statistics for other genomic regions on CFA1 and for more families can be shown a
non-parametric linkage analysis was performed with 27 microsatellite markers
covering CFA1 completely In total 176 animals were genotyped We could not find
linkage to any microsatellite in the analyzed families Furthermore it was revealed
that GJA1 is not a candidate gene for sensorineural non-syndromic deafness in
humans (WA Paznekas cited a personal communication from the senior author (W
E Nance) of the paper by Liu et al 2001) GJA1 is participating in a human
syndrome called oculodentodigital dysplasia (ODDD) that can be accompanied with
hearing impairment (Paznekas et al 2003) But the type of deafness in human
ODDD is conductive rather than sensorineural As deafness in dogs especially in
Dalmatians is almost exclusively caused by sensorineural non-syndromic forms also
known as cochleosaccular degeneration the GJA1 gene should not be considered
as a candidate gene for CCSD anymore
General discussion 123
CFA31 By the use of microsatellites as well as SNPs we found significant linkage with CCSD
for CLDN14-associated microsatellites in four full-sib and one half-sib Dalmatian dog
familiy with a Zmean value of 383 (plt000007) A mutation analysis was performed
for exon three as this is the only translated one in man None of the observed
polymorphisms did alter the predicted amino acid sequence However to clarify the
importance of the CLDN14 gene and its flanking regions in the CCSD phenotype
more SNPs have to be developed within the CLDN14 gene as well as in its flanking
regions with the aim to find significant linkage disequilibrium of SNP markers
CFA10 A significant co-segregation of markers alleles and the phenotypic expression of
deafness in a large sample of German Dalmatian dog families was determined for
one marker (Z-mean of 156 [p= 006] and a Lod score of 058 [p= 005]) associated
to the MYH9 gene Subsequently we evaluated whether MYH9 gene mutations are
responsible for CCSD in these Dalmatian dog families An initial priority in defining
gene structure is to obtain a full-length cDNA sequence and identify translational
initiation and termination sites and polyadenylation site(s) Exonintron structure can
then be determined by referencing the cDNA sequence against sequences of
cognate genomic DNA One popular method of obtaining full-length cDNA sequences
is the RACE (rapid amplification of cDNA ends) technique RACE-PCR is an anchor
PCR modification of RT-PCR The rationale is to amplify sequences between a single
previously characterised region in the mRNA (cDNA) and an anchor sequence that is
coupled to the 5 or the 3 end A primer is designed from the known internal
sequence and the second primer is selected from the relevant anchor sequence
To provide the genomic organization and the complete sequence of the canine
MYH9 gene the isolation of full length cDNAs was achieved with the help of a
modified rapid amplification of cDNA ends (RACE) protocol A mutation analysis was
performed to identify single nucleotide polymorphisms (SNPs) in this gene We
analyzed the association of the MYH9 haplotypes with the CCSD phenotype in three
families of Dalmatian dogs with frequent occurrence of CCSD and significant linkage
to gene-associated microsatellites Using the RT-PCR analyses for cDNA-genomic
sequence comparisons we detected that the canine MYH9 gene is bigger compared
to the human sequence due to the untranlated first exon in the 5rsquo-UTR
General discussion
124
The canine MYH9 gene consists of 41 exons with an untranslated exon 1 and
exonintron boundaries that conform perfectly to the GTAG rule
None of the observed polymorphisms did alter the predicted amino acid sequence of
MYH9 nor showed the identified haplotypes an association with the CCSD
phenotype
Thus these silent point mutations found in affected and unaffected Dalmatian dogs
do not seem to be responsible for the CCSD phenotype in these three families
To clarify if other regions on CFA10 are responsible for the CCSD phenotype we
added in a second step 27 microsatellite markers derived from the NCBI database to
cover CFA10 with a high density in the flanking region of the MYH9 gene A linkage
analysis was carried out for 23 Dalmatian dog families consisting of 176 animals that
were genotyped with the marker set of 27 microsatellite markers
We found significant linkage to microsatellites in the region spanning 36 Mb to 48 Mb
Consequently we screened this 12 Mb spanning region for SNPs Out of the 23
analyzed Dalmatian dog families five full-sib families were chosen to screen for
SNPs because of their obviously significant values at the above mentioned region
Twenty-six new SNP markers covering the region of 36 Mb to 48 Mb equally were
developed and added to the linkage analysis The significant Zmeans on CFA10 was
confirmed after adding the SNP markers Furthermore with the use of SNPs the
apparently linked region spanning 36 Mb to 48 Mb could be narrowed down to 5 Mb
spanning from 39 Mb to 44 Mb For this reason the use of SNPs in addition to
informative microsatellites appears highly recommendable
In further studies more SNPs have to be developed within the identified CCSD region
on CFA10 to localize the deafness causing gene or to find unambiguously associsted
SNP markers which could be used for a population-wide genetic test for CCSD
Chapter 10
Summary
Summary 127
Summary
Molecular genetic analysis of canine congenital sensorineural deafness in Dalmatian dogs
Katharina Mieskes (2006) The objective of the present study was to localize the gene or genomic region that is
involved in the development of canine congenital sensorineural deafness (CCSD) in
Dalmatian dogs
In man as in different dog breeds deafness is an often diagnosed disorder with the
Dalmatian dog showing the highest incidence Many genetic disorders in humans
and domestic dogs (Canis familiaris) demonstrate a high level of clinical and
molecular similarity
Altogether 39 genes have already been found causative for sensorineural non-
syndromic hearing impairment in humans Out of this 39 deafness causing genes a
total of 32 candidate genes were selected for canine congenital deafness which
showed an appropriate clinical and histological disease pattern in comparison to
deafness in Dalmatians dogs
On the one hand an existing set of 43 microsatllite markers for in total 24 candidate
genes were used for a non-parametric linkage analysis among them the claudin-14
(CLDN14) gene on canine chromosome (CFA) 31 and the myosin heavy polypeptide
9 (MYH9) gene on CFA10 The gap junction protein alpha 1 (GJA1) gene on CFA1
was also considered as a candidate gene for CCSD and thus GJA1-associated
microsatellites were part of the existing set Recently it turned out that GJA1 is not
responsible for human sensorineural non-syndromic deafness but for a human
syndromic disorder that can be related with conductive deafness In the last few
years more human deafness genes have been identified among them eight genes
that were considered as appropriate candidates for CCSD For these eight genes a
total of 21 SNPs were newly developed and used for non-parametric linkage and
association analyses
Summary
128
The used microsatellite and SNP markers derived either from a partial sequence
analysis of BAC clones each containing a different candidate gene or from
sequences deposited in the current dog genome assembly (Boxer genome assembly
21) of the NCBI GenBank We found significant linkage to markers associated to
CLDN14 on CFA31 MYH9 on CFA10 and GJA1 on CFA1 To substantiate the
linkage we searched for sequence variations within these three genes SNPs found
in intronic sequences of either gene were included in the linkage analyses Sequence
analysis neither revealed a causative mutation nor significant linkage disequilibrium
of SNP markers with CCSD Subsequently CFA1 10 and 31 were scanned
completely with microsatellite markers derived from the NCBI database with the
purpose to see if other regions on this three chromosomes harbour a gene that is
involved in the development of CCSD
The analyses of SNPs and more microsatellite markers on CFA1 revealed no
significant linkage to CCSD Thus it is unlikely that the canine chromosome 1 and
the GJA1 gene are responsible for the CCSD phenoptype As deafness in dogs
especially in Dalmatians is almost exclusively caused by sensorineural non-
syndromic forms the GJA1 gene should not be considered as a candidate gene for
CCSD anymore
On CFA10 we could exclude MYH9 for being causal for deafness but by adding
more microsatellites covering CFA10 completely we found significant linkage to the
CCSD phenotype in the region distal of MYH9 Hence new SNP-markers for fine
mapping the region spanning 36 to 48 Mb were developed by sequence analyses of
different Dalmatian dogs The search for SNPs was carried out on genomic
sequences of genes located in the significantly linked region The sequences of
these genomic sequences were derived from the NCBI GenBank The SNPs
confirmed the linkage and narrowed the region harbouring a causative CCSD gene
down to 5 Mb spanning from 39 to 44 Mb
After scanning CFA31 we could not exclude CLDN14 for being responsible for the
CCSD phenotype as microsatellite markers associated to CLDN14 indicated linkage
However to clarify the importance of CLDN14 in the CCSD phenotype more SNPs
have to be developed within the CLDN14 gene as well as in its flanking regions with
the aim to find linkage disequilibrium for SNP markers
Chapter 11
Erweiterte Zusammenfassung
Erweiterte Zusammenfassung 131
Erweiterte Zusammenfassung
Katharina Mieskes
Molekulargenetische Untersuchung der kongenitalen sensorineuralen Taubheit beim Dalmatiner
Einleitung Es gibt mehrere Moumlglichkeiten Taubheit zu klassifizieren Je nach den betroffenen
Strukturen des Gehoumlrorgans lassen sich konduktive und sensorineurale Formen
unterscheiden Erstere entstehen durch Stoumlrungen des Konduktionsmechanismus im
aumluszligeren Gehoumlrgang und im Mittelohr Ursachen fuumlr konduktive Houmlrstoumlrungen koumlnnen
zum Beispiel chronische Otitis externa oder media Frakturen von Mittelohranteilen
oder Tumoren sein Sehr viel oumlfter tritt die sensorineurale Taubheit auf bei der ein
Houmlrverlust als Folge von Transduktions- und Transmissionsstoumlrungen in der Cochlea
(Schnecke) sowie in peripheren und zentralen Anteilen des Nervus cochlearis auftritt
Neben der erblichen Form koumlnnen auch eine Otitis interna Tumoren oder
ototoxische Medikamente zur sensorineuralen Taubheit fuumlhren
Der Houmlrverlust kann bereits seit der Geburt bestehen (kongenital) oder er kann erst
im Laufe der weiteren Lebenszeit auftreten
Beim Menschen ist mehr als die Haumllfte der kongenitalen Taubheit erblich bedingt Die
erbliche kongenitale Taubheit kann als Teil einer multisystemischen Krankheit
(syndromisch) oder als Funktionsstoumlrung auftreten die auf das Ohr und das
vestibulaumlre System (nicht-syndromisch) begrenzt ist Da die nicht-syndromische
ererbte Taubheit fast ausschlieszliglich durch Cochleadefekte verursacht wird leiden die
Patienten unter sensorineuraler Taubheit Es wird geschaumltzt dass die nicht-
syndromische erbliche Taubheit 60-70 aller menschlichen Taubheitsfaumllle
verursacht Weiterhin sind 70-80 der Faumllle der nicht-syndromischen Taubheit auf
einen autosomal rezessiven Erbgang zuruumlck zu fuumlhren gefolgt von einem autosomal
dominanten Erbgang mit 10-20 der Faumllle und 1-2 der Faumllle sind X- gekoppelt Ein
noch geringerer Prozentsatz wird durch einen maternalen Erbgang verursacht
Von den ca 25000 bis 30000 menschlichen Genen ist nach Schaumltzungen bis zu 1
wichtig fuumlr den Houmlrprozess Annaumlhernd 120 Gene sind inzwischen identifiziert die fuumlr
Erweiterte Zusammenfassung
132
verschiedene Formen der menschlichen vererbten Taubheit verantwortlich sind
Darunter sind ungefaumlhr 80 Gene fuumlr die syndromische und 39 Gene fuumlr die nicht
syndromische vererbte Taubheit verantwortlich Insgesamt stellen die 120
identifizierten Gene vermutlich ein Drittel aller taubheitsverursachenden Gene dar
Die kongenitale sensorineurale Taubheit ist eine Erkrankung von der viele
Saumlugetierspezies betroffen sind Beim Hund werden uumlber 54 verschiedene Rassen
beschrieben bei welchen ein kongenitaler Houmlrverlust gehaumluft auftritt In zahlreichen
europaumlischen und amerikanischen Studien weisen hierbei die Dalmatiner mit 165-
30 die houmlchste Taubheitsinzidenz auf
Die histologischen Veraumlnderungen im Innenohr werden im Allgemeinen aumlhnlich wie
beim Menschen als cochleosacculaumlr beschrieben Die Degenerationen im Innenohr
schreiten bei betroffenen Tieren mit zunehmendem Alter fort und fuumlhren
normalerweise in einem Alter von drei bis vier Wochen spaumltestens aber in einem
Alter von etwa vier Monaten zu einem vollstaumlndigen ein- oder beidseitigen
Houmlrverlust
Eine objektive Diagnose der Taubheit besonders wenn sie nur einseitig ist stuumltzt
sich auf die brainstem auditory evoked response (BAER in Deutschland
audiometrischer Test) ein elektro-diagnostischer Test bei dem durch einen
bestimmten Reiz ausgeloumlste elektrische Aktivitaumlten (akustisch evozierte Potentiale) in
der Cochlea und den Nervenbahnen zur auditorischen Hirnrinde detektiert werden
Obwohl fuumlr den Dalmatiner die Erblichkeit der Erkrankung wiederholt nachgewiesen
werden konnte wurden fuumlr die verschiedenen untersuchten Dalmatinerpopulationen
auch sehr viele unterschiedliche Erbgaumlnge in Betracht gezogen Es ist bisher nicht
gelungen die genaue Anzahl der beteiligten Gene zu bestimmen Auch war es
bislang fuumlr keine der betroffenen Hunderassen moumlglich ein taubheitsverursachendes
Gen zu identifizieren
Bei deutschen Dalmatinern ergab eine Auswertung mittels komplexer
Segregationsanalysen dass ein Modell mit einem rezessiven Hauptgen und einer
polygenen Komponente das Auftreten der kongenitalen Taubheit in der untersuchten
Population am besten erklaumlrte Zusaumltzlich konnte ein Zusammenhang zu
Pigmentierungsgenorten nachgewiesen werden da Tiere mit blauer Augenfarbe
signifikant haumlufiger von Taubheit betroffen waren und gleichzeitig Tiere mit
Plattenzeichnung seltener kongenitalen Houmlrverlust zeigten
Erweiterte Zusammenfassung 133
Das Zuumlchten ein- oder beidseitig tauber Dalmatiner sowie Dalmatinern mit blauen
Augen ist in Deutschland schon laumlnger verboten und es wird von den
Zuchtverbaumlnden vorgeschrieben dass alle Dalmatinerwelpen in einem Alter von etwa
6 bis 8 Wochen audiometrisch untersucht werden Des Weiteren ist der Defekt
tierschutzrelevant nach sect 11b des Deutschen Tierschutzgesetzes da der artgemaumlszlige
Gebrauch eines Organs eingeschraumlnkt wird und eine Erblichkeit nachgewiesen
wurde Somit duumlrfen Tiere die Defektgentraumlger sind oder bei denen damit zu
rechnen ist nicht zur Zucht verwendet werden Waumlhrend einseitig taube Dalmatiner
als ganz normale Familienhunde gehalten werden koumlnnen sind beidseitig taube
Dalmatiner schwierig zu erziehen schreckhafter und daraus resultierend auch
oftmals aggressiv und bissig Aus diesen Gruumlnden werden fast alle Welpen mit
beidseitigem Houmlrverlust euthanasiert
Es hat sich gezeigt dass allein der Zuchtausschluss betroffener Tiere nicht genuumlgt
um die hohe Taubheitsinzidenz beim Dalmatiner deutlich zu reduzieren Aus diesen
Gruumlnden ist es von erheblicher Bedeutung die kongenitale sensorineurale Taubheit
beim Dalmatiner molekulargenetisch zu untersuchen um so ein
molekulargenetisches Testverfahren entwickeln zu koumlnnen welches auch die
Identifizierung von Anlagetraumlgern ermoumlglicht
Innerhalb des letzten Jahrzehnts wurden auch zwischen relativ unverwandten
Spezies die strukturellen und funktionellen Homologien auf genetischer Ebene
zunehmend verdeutlicht
Es wurde inzwischen eine groszlige Anzahl von taubheitsverursachenden
Genmutationen beim Menschen und der Maus aufgeklaumlrt Da auch die
histopathologischen Befunde beim Menschen und der Maus oftmals sehr aumlhnlich
denen beim Dalmatiner sind scheinen Gene die beim Menschen verantwortlich fuumlr
die congenitale sensorineurale Taubheit sind geeignete Kandidatengene fuumlr die
canine congenitale sensorineurale Taubheit zu sein
Das Ziel dieser Dissertation ist es die fuumlr die Taubheit kausalen Gene bzw Genorte
zu lokalisieren Um die kongenitale sensorineurale Taubheit beim Dalmatiner
moumlglichst effizient molekulargenetisch aufzuklaumlren wurden 32 Kandidatengene mit
genassoziierten Markern auf eine potenzielle Kopplung mit der caninen kongenitalen
sensorineuralen Taubheit untersucht
Die Genombereiche mit signifikanter Kopplung zur kongenitalen sensorineuralen
Taubheit wurden weitergehend molekulargenetisch analysiert
Erweiterte Zusammenfassung
134
Kopplungsanalyse fuumlr 24 Kandidatengene der caninen kongenitalen sensorineuralen Taubheit (CCSD) mit 43 genassoziierten Mikrosatelliten Markern
Material und Methoden
Fuumlr 24 Kandidatengene stand ein Mikrosatelliten Marker Set zur Verfuumlgung Das
Marker Set beinhaltete Mikrosatelliten fuumlr folgende Kandidatengene CDH23
CLDN14 COCH COL11A2 DFNA5 DIAPH1 EDN3 EDNRB EYA4 GJA1 GJB2
GJB6 MITF MYH9 MYO6 MYO7A MYO15A OTOF PAX3 POU4F3 SLC26A4
SOX10 TECTA und TMPRSS3 Als Familienmaterial fuumlr die Mikrosatellitenstudie
wurden 215 Dalmatiner verwendet Die Tiere verteilten sich auf 22 unverwandte
Vollgeschwisterfamilien und zwei paternale Halbgeschwistergruppen Die
genotypisierten Familien bestanden aus saumlmtlichen tauben Nachkommen (uni- oder
bilateral taub) ihren Eltern und einem bis vier nicht betroffenen Voll- bzw
Halbgeschwistern Mindestens zwei der Vollgeschwister einer Familie waren uni-
oder bilateral taub Der Phaumlnotyp der verwendeten Tiere wurde durch einen
audiometrischen Test bestimmt
Alle Mikrosatellitenmarker wurden uumlber PCR und Polyacrylamidgelelektrophorese
ausgewertet
Kopplungsanalyse
Die nicht-parametrische Kopplungsanalyse wurde unter Verwendung der Software
MERLIN (multipoint engine for rapid likelihood inference Version 0102)
durchgefuumlhrt und basierte auf dem identical-by-descent (IBD) Verfahren Dabei
wurden die Markerallele auf Kosegregation mit der phaumlnotypischen Auspraumlgung der
Erkrankung getestet Darauf folgend wurde die einer Normalverteilung folgende
Teststatistik fuumlr den Anteil von IBD-Markerallelen (Zmean) und ein daraus
abgeleiteter LOD-Score berechnet Als signifikant fuumlr die Kosegregation eines
Markerallels mit dem Phaumlnotyp der caninen kongenitalen sensorineuralen Taubheit
(CCSD) gelten Irrtumswahrscheinlichkeiten (p) von 005 oder kleiner
Die Daten wurden mit dem Softwarepaket SAS (Statistical Analysis System Version
913) bearbeitet um die Marker bezuumlglich Allelfrequenzen beobachteten und
erwarteten Heterozygotiegrad und PIC (Polymorphism information content)
charakterisieren zu koumlnnen
Erweiterte Zusammenfassung 135
Ergebnisse und Diskussion Das Mikrosatelliten Set bestand insgesamt aus 43 Markern mit einem bis drei
Markern pro Kandidatengen Zunaumlchst wurde fuumlr alle 24 Familien zusammen eine
Kopplungsanalyse durchgefuumlhrt Vermeintliche CCSD Loci wurden auf den caninen
Chromosomen (CFA) 1 10 12 20 und 31 lokalisiert Im Anschluss wurden die
Ergebnisse der Kopplungsanalyse auf Heterogenie zwischen den Familien uumlberpruumlft
Fuumlr einzelne Familien konnten houmlhere Teststatistiken und somit engere
Kopplungsbeziehungen als bei der Analyse aller Familien nachgewiesen werden
Eine signifikante Kopplung mit der kongenitalen sensorineuralen Taubheit wurde fuumlr
die Gene GJA1 auf CFA1 MYH9 auf CFA10 und CLDN14 auf CFA31 sowohl im
Gesamtmaterial wie in jeweils unterschiedlichen Gruppen von Dalmatinerfamilien
gefunden Es ist daher wahrscheinlich dass diese Gene oder Gene in ihrer naumlheren
Umgebung an der Entwicklung der Taubheit in den jeweiligen Familien involviert
sind Fuumlr die restlichen Kandidatengene lagen die Multipoint Teststatistiken fuumlr den
Anteil von IBD-Markerallelen (Zmean) und daraus abgeleiteten LOD-Scores bei
annaumlhernd Null und waren nicht signifikant
Die Ergebnisse dieser Teststatistiken zeigen dass der Erbgang der nicht-
syndromischen Taubheit bei Dalmatinern vermutlich ebenso heterogen ist wie er
sich beim Menschen darstellt
Dennoch stellt diese Studie den ersten Schritt dar um die CCSD verursachenden
Gene beim Dalmatiner zu identifizieren Die Gene GJA1 MYH9 und CLDN14 sowie
ihre naumlhere Umgebungen werden im weiteren Verlauf der Studie weitergehend
molekulargenetisch mit Hilfe von SNPs und Mikrosatelliten untersucht Aufgrund der
Heterogenitaumlt zwischen den Familien wurden fuumlr die weitere molekulargenetische
Analyse bestimmter Gene oder Genomregionen nur die Familien mit dem houmlchsten
Beitrag zu den Teststatistiken der Kopplungsanalyse (Zmean gt 1) ausgewaumlhlt
Erweiterte Zusammenfassung
136
Evaluierung von neu entwickelten SNP Markern assoziiert mit Kandidatengenen fuumlr die canine kongenitale sensorineurale Taubheit (CCSD) Material und Methoden Fuumlr acht Kandidatengene (TMC1 TMIE USH1C MYH14 MYO3A PRES WHRN
und ESPN) wurden intragenische Single Nucleotide Polymorphism (SNP) Marker neu
entwickelt
Als Familienmaterial fuumlr die Genotypisierung der SNPs wurde DNA von insgesamt 39
Dalmatinern verwendet die vier Vollgeschwisterfamilien angehoumlrten Mindestens
zwei der Vollgeschwister einer Familie waren uni- oder bilateral taub Der Phaumlnotyp
der verwendeten Tiere wurde durch einen audiometrischen Test bestimmt Die vier
Familien bestanden aus sechs bis zehn Vollgeschwistern und den dazugehoumlrenden
Elterntieren
Identifizierung von SNPs (Single Nucleotide Polymorphisms)
Die caninen Sequenzen der Kandidatengene wurden mit Hilfe der humanen mRNA
mittels BLAST (Basic Local Alignment Search Tool) Suche aus veroumlffentlichten
Sequenzen der NCBI Datenbank (Boxer genome assembly 21) entnommen Die
Exon Intron Grenzen wurden mit Hilfe des mRNA-to-genomic alignment Programms
Spidey bestimmt und Primer mit Hilfe des Primer3 Programms in die intronischen
Sequenzen gelegt Die SNPs wurden an den DNA-Sequenzen von Elterntieren der
betroffenen Dalmatinerfamilien identifiziert Die Amplifikate wurden mit Hilfe des
MegaBACE 1000 sequenziert und auf SNPs untersucht Bei Vorliegen eines oder
mehrerer SNPs wurde die entsprechende DNA-Sequenz der zugehoumlrigen
Nachkommen ebenfalls amplifiziert und sequenziert Die Auswertung erfolgte mit
Hilfe des Sequencher 42 Programms
Kopplungsanalyse
Die Genotypen der neuen SNP Marker wurden unter Verwendung des Programms
MERLIN ausgewertet Die Daten wurden mit dem Softwarepaket SAS (Statistical
Analysis System Version 913) bearbeitet um das Markerset bezuumlglich
Allelfrequenzen beobachteten Heterozygotiegrad und PIC (Polymorphism
information content) charakterisieren zu koumlnnen Des Weiteren wurde auf
Kopplungsungleichgewicht und die Assoziation der Haplotypen mit CCSD mittels
Erweiterte Zusammenfassung 137
des Programms CASECONTROL und HAPLOTYPE von SAS Genetics (Statistical
Analysis System Version 913 Cary NC USA) getestet
Ergebnisse und Diskussion Fuumlr acht Kandidatengene wurden mindestens zwei heterozygote SNPs entwickelt
Die nicht-parametrische Kopplungsanalyse zeigte keine signifikanten Teststatistiken
Offensichtlich war kein Haplotyp mit der caninen kongenitalen sensorineuralen
Taubheit (CCSD) in den untersuchten Familien assoziiert Da sowohl uni- und
bilateral taube Tiere als auch beidseitig houmlrende Tiere uumlber identische Haplotypen
verfuumlgen ist es unwahrscheinlich dass die Gene TMC1 TMIE USH1C MYH14
MYO3A PRES WHRN und ESPN an der Entstehung der CCSD beim Dalmatiner
beteiligt sind
Molekulargenetische Analyse des caninen myosin heavy polypeptide 9 non-muscle (MYH9) auf dem caninen Chromosom 10q232
Material und Methoden Klonierung und Sequenzierung von caniner MYH9 cDNA
Die canine MYH9 Gensequenz wurde mit Hilfe der BLAST Suche aus
veroumlffentlichten Sequenzen der NCBI Datenbank Die Isolierung der vollstaumlndigen
cDNA wurde durch ein Protokoll zur Amplifizierung von cDNA-Enden (rapid
amplification of cDNA ends [RACE]) erreicht Nach Klonierung und Sequenzierung
der RACE-Produkte wurden die Sequenzen mit Hilfe des Sequencher 42
Programms ausgewertet
Sequenzanalyse des caninen MYH9 Gens
Nach Fertigstellung des RACE Protokolls und Auswertung der Sequenzen wurden
die Exon Introngrenzen mit Hilfe des mRNA-to-genomic alignment Programms
Spidey lokalisiert Weiterhin wurden repetitive Elemente mit dem Programm
Repeatmaster 2 sowie der GC Gehalt mit der EBI toolbox CpG PlotCpGreport
ermittelt
Erweiterte Zusammenfassung
138
Mutatiosanalyse
Zur Identifizierung von Polymorphismen innerhalb der caninen MYH9 Sequenz
wurden die Exons mit ihren flankierenden intronischen Sequenzen mit Hilfe der PCR
amplifiziert und sequenziert Verwendet wurden hierfuumlr die DNA von insgesamt 16
Dalmatinern aus drei Familien Mindestens zwei der Vollgeschwister einer Familie
waren unilateral taub Der Phaumlnotyp der verwendeten Tiere wurde durch einen
audiometrischen Test bestimmt
PCR Primer wurden mit Hilfe des Primer3 Programms entwickelt PCR Reaktionen
fuumlr saumlmtliche Exons des MYH9 Gens mit ihren flankierenden Sequenzen wurden
durchgefuumlhrt die PCR Produkte sequenziert und die Sequenzen mit Hilfe des
Sequencher 42 Programms ausgewertet
Unter Verwendung der Software MERLIN (multipoint engine for rapid likelihood
inference Version 0910) wurde nach signifikanten nicht parametrischen LOD-
Scores fuumlr die Kosegregation von Markerallelen mit der phaumlnotypischen Auspraumlgung
der sensorineuralen Taubheit beim Dalmatiner gesucht
Ergebnisse und Diskussion
Analyse der genomischen Organisation und der cDNA des caninen MYH9 Gens
Eine vollstaumlndige cDNA Sequenz des caninen MYH9 Gens von 7484 bp konnte uumlber
die Reverse Transkription-PCR erhalten und bei der EMBL Nukleotid Datenbank
eingereicht werden Das canine MYH9 Gen besteht aus 41 Exons einschlieszliglich
einem untranslatierten ersten Exon und Exon Intron Grenzen die stets der GT AG
Regel folgen Der 5rsquo-UTR Bereich besteht aus 181 bp waumlhrend der 3rsquo-UTR aus 1432
bp besteht Weiterhin besitzt die canine MYH9 cDNA Sequenz einen offenen
Leserahmen von 5889 bp welcher ein Protein kodiert bestehend aus 1963
Aminosaumluren Die Groumlszlige der Exons variiert von 28 bis 1556 bp waumlhrend das
gesamte MYH9 Gens ungefaumlhr 90 kb umfasst Die Homologie der Sequenzen
zwischen dem humanen murinen und caninen MYH9 Gen ist limitiert auf die
kodierende Sequenz von Exon 2 bis 41
Mutations- und Haplotypenanalyse
Alle kodierenden Exons mit ihren flankierenden intronischen Sequenzen des MYH9
Gens konnten von den 16 Dalmatinern amplifiziert werden Die Sequenzen wurden
mit den entsprechenden Sequenzen vom Boxer aus der NCBI Genbank verglichen
Erweiterte Zusammenfassung 139
Die Suche nach Sequenzvariationen im MYH9 Gen lieszlig 22 SNPs erkennen wobei
die meisten Polymorphismen in den flankierenden Regionen der Exons gefunden
wurden Keiner der beobachteten Polymorphismen in den Exons aumlnderte die
Aminosaumluresequenz von MYH9 Auch zeigten die identifizierten Haplotypen keine
Assoziation mit dem CCSD Phaumlnotyp Multipoint Teststatistiken fuumlr den Anteil von
IBD-Markerallelen (Zmean) und ein daraus abgeleiteter LOD-Score lagen bei
annaumlhernd Null und waren nicht signifikant Zwei genassoziierte Mikrosatelliten
zeigten eine Kopplung diese kann jedoch mit Heterogenitaumlt zwischen den Familien
erklaumlrt werden
Fazit
Die Charakterisierung des Transkriptes und der genomischen Sequenz des caninen
MYH9 Gens zeigten einen konservierten Aufbau des Gens im Bezug auf das
humane orthologe Gen Aufgrund eines untranslatierten Exons im 5rsquo-UTR Bereich ist
das canine Gen groumlszliger als das humane Gen
Sowohl uni- oder bilateral taube und bilateral houmlrende Dalmatiner weisen identische
Haplotypen auf Daher sind in den untersuchten Dalmatinerfamilien die gefundenen
Polymorphismen offensichtlich nicht mit CCSD assoziiert Des Weiteren zeigt diese
Studie dass keine funktionellen Mutationen der vollstaumlndigen kodierenden Region
von MYH9 vorliegen Wir koumlnnen also abschlieszligend das MYH9 Gen als
Kandidatengen fuumlr die congenitale sensorineurale Taubheit beim Dalmatiner
ausschlieszligen
Erweiterte Zusammenfassung
140
Evaluierung von Mikrosatelliten und SNP Markern der caninen Chromosomen 1 10 und 31 auf Kopplung und Assoziation mit der caninen kongenitalen Taubheit Material und Methoden Pedigreematerial
Insgesamt wurden 176 Dalmatiner in die Untersuchung einbezogen Die Tiere
verteilten sich auf 23 unverwandte Vollgeschwisterfamilien und eine paternale
Halbgeschwistergruppe Die genotypisierten Familien bestanden aus saumlmtlichen
tauben Nachkommen (unilateral oder bilateral taub) ihren Eltern und einen bis vier
nicht betroffenen Voll- bzw Halbgeschwistern Mindestens zwei der Vollgeschwister
einer Familie waren unilateral oder bilateral taub Der Phaumlnotyp der verwendeten
Tiere wurde durch einen audiometrischen Test bestimmt
Alle 176 Dalmatiner wurden fuumlr die Mikrosatellitenstudie auf den caninen
Chromosomen 1 10 und 31 einbezogen Fuumlr die Feinkartierung mit Hilfe der SNPs
wurden dagegen nur noch die Dalmatinerfamilien mit einer eindeutigen Kopplung zu
der entsprechenden genomischen Region untersucht Fuumlr die Region auf CFA10
wurden dafuumlr 34 Tiere aus fuumlnf Vollgeschwisterfamilien ausgewaumlhlt und zur
Untersuchung des CLDN14 Gens auf CFA31 wurden 24 Tiere aus vier
Vollgeschwisterfamilien und 12 Tiere aus einer groszligen Halbgeschwisterfamilie
verwendet Fuumlr die Suche nach Polymorphismen im GJA1 Gen wurden 16 Tiere aus
einer groszligen Halbgeschwisterfamilie verwendet
Mikrosatellitenstudie
Fuumlr die Mikrosatellitenstudie von CFA1 10 und 31 wurden insgesamt 60 Marker
verwendet Mit jeweils 27 Mikrosatelliten wurde CFA1 und mit 27 Mikrosatelliten
wurde CFA10 vollstaumlndig abgedeckt Insgesamt 6 Marker wurden fuumlr CFA31
verwendet
Alle hier verwendeten Mikrosatelliten sind der NCBI Datenbank entnommen und
wurden uumlber PCR und Polyacrylamidgelelektrophorese ausgewertet
Identifizierung von SNPs (Single Nucleotide Polymorphisms)
Zur weiteren Abklaumlrung und zum Ausschluss falsch positiver Ergebnisse wurden
zusaumltzlich zu den Mikrosatelliten SNPs in den signifikanten Regionen auf CFA1 10
Erweiterte Zusammenfassung 141
und CFA 31 entwickelt und an den Familien die eine Kopplung zeigten
genotypisiert
Die caninen genomischen Sequenzen der untersuchten Gene wurden der NCBI
Datenbank (Boxer genome assembly 21) entnommen Exon Intron Grenzen wurden
bestimmt und Primer entwickelt Nach Durchfuumlhrung der PCR wurden die Amplifikate
mit Hilfe des MegaBACE 1000 sequenziert und auf SNPs untersucht Die
Auswertung erfolgte mit Hilfe des Sequencher 42 Programms
Die Genotypen der neuen SNP Marker wurden zu den Genotypen der fuumlr die
Kopplungsanalyse verwendeten Mikrosatelliten hinzugefuumlgt und gemeinsam unter
Verwendung des Programms MERLIN ausgewertet
Kopplungsanalyse
Unter Verwendung der Software MERLIN (multipoint engine for rapid likelihood
inference Version 0910) wurde nach signifikanten nicht parametrischen LOD-
Scores fuumlr die Kosegregation von Markerallelen mit der phaumlnotypischen Auspraumlgung
der sensorineuralen Taubheit beim Dalmatiner gesucht
Die Daten wurden mit dem Softwarepaket SAS (Statistical Analysis System Version
913) bearbeitet um die Marker bezuumlglich Allelfrequenzen beobachteten
Heterozygotiegrad und PIC (Polymorphism information content) charakterisieren zu
koumlnnen Des Weiteren wurde das Kopplungsungleichgewicht und die Assoziation der
Haplotypen mit CCSD mittles des Programms CASECONTROL und HAPLOTYPE
von SAS Genetics getestet
Ergebnisse und Diskussion CFA1
In einer groszligen Halbgeschwisterfamilie wurde eine signifikante Kopplung zu einem
GJA1 genassozierten Marker festgestellt Mit Hilfe der DNA von Tieren aus dieser
Halbgeschwisterfamilie wurde eine Sequenzanalyse des GJA1 Gens durchgefuumlhrt
Es wurden 3 SNPs im 3acute-untranslatierten Bereich gefunden Die identifizierten
Haplotypen zeigten keine Assoziation mit dem CCSD Phaumlnotyp Sowohl uni- oder
bilateral taube und bilateral houmlrende Dalmatiner wiesen identische Haplotypen auf
Daher sind in den untersuchten Dalmatinerfamilien die gefunden Polymorphismen
offensichtlich nicht mit CCSD assoziiert Auch wurde keine funktionelle Mutation in
der vollstaumlndigen kodierenden Region von GJA1 gefunden wurde Es ist daher
Erweiterte Zusammenfassung
142
unwahrscheinlich dass das GJA1 Gen an der Entwicklung der CCSD in der
untersuchten Halbgeschwisterfamilie beteiligt ist Die signifikante Kopplung der
GJA1-benachbarten Mikrosatelliten mit CCSD kann durch ein eng gekoppeltes Gen
hervorgerufen worden sein welches fuumlr die Entstehung der Taubheit in der
untersuchten Halbgeschwisterfamilie verantwortlich ist oder durch ein falsch-
positives Ergebnis der vorhergehend durchgefuumlhrten Mikrosatellitenstudie
Eine nicht-parametrische Kopplungsanalyse mit Hilfe von 27 Mikrosatelliten Marker
an 24 deutschen Dalmatinerfmilien wurde durchgefuumlhrt um abzuklaumlren ob andere
Genombereiche auf CFA1 signifikante Teststatistiken in mehr Familien zeigen Die
Kopplungsanalyse zeigte keine signifikanten Teststatistiken Es ist daher
unwahrscheinlich dass auf CFA1 Gene fuumlr die Entwicklung von CCSD in den
untersuchten Familien der aus Deutschland stammenden Dalmatiner verantwortlich
sind
Da kuumlrzlich festgestellt wurde dass das GJA1 Gen auch beim Menschen nicht fuumlr
eine Form der sensorineuralen nicht-syndromischen Taubheit verantwortlich ist
sondern an einer syndromischen Krankheit beteiligt ist die mit konduktiver Taubheit
einhergehen kann sollte das GJA1 Gen nicht laumlnger als ein Kandidatengen fuumlr
CCSD angesehen werden
CFA31
Auf CFA31 wurde durch eine nicht-parametrische Kopplungsanalyse von
Mikrosatelliten eine Kopplung eines CLDN14-benachbarten Markers mit der
sensorineuralen Taubheit beim Dalmatiner festgestellt Daraufhin wurden zusaumltzlich
zu den Mikrosatelliten acht SNPs entwickelt indem das einzige beim Menschen
translatierte Exon weite intronische Sequenzen sowie 5rsquo- und 3rsquo- untranslatierte
Bereiche des CLDN14 Gens einer Sequenzanalyse unterzogen wurden Nur zwei der
identifizierten SNPs waren polymorph in den untersuchten fuumlnf Dalmatinerfamilien
Sowohl uni- als auch bilateral taube und bilateral houmlrende Dalmatiner teilten
identische Haplotypen daher konnte keine Kosegregation mit tauben Dalmatinern
festgestellt werden
Da bislang keine funktionelle Mutation in der kodierenden Region von CLDN14
gefunden wurde ist es fuumlr eine eindeutige Aussage uumlber die Rolle des CLDN14
Gens im Entstehungsprozess der kongenitalen sensorineuralen Taubheit
Erweiterte Zusammenfassung 143
beim Dalmatiner notwendig noch weitere Marker im CLDN14 Gen sowie seiner
naumlheren Umgebung zu entwickeln und zu analysieren Da keine weitere Kopplung zu
einem Mikrosatelliten distal oder proximal des CLDN14 Gen festgestellt werden
konnte ist es wahrscheinlich dass entweder das CLDN14 Gen oder ein Gen in
seiner naumlheren Umgebung fuumlr die Entwicklung der sensorineuralen Taubheit in den
untersuchten Dalmatinerfamilien verantwortlich ist
CFA10
Auf CFA10 wurde durch eine nicht-parametrische Kopplungsanalyse von 27
Mikrosatelliten eine 12 Mb umfassende genomische Region mit einer signifikanten
Kopplung zur CCSD identifiziert Zur weiteren Feinkartierung wurden 26 neue SNPs
mit einem durchschnittlichen Abstand von 03 Mb fuumlr diesen Bereich entwickelt
Die SNPs waren groumlszligtenteils informativ im vorliegenden Familienmaterial und
bestaumltigten in einer folgenden Kopplungsanalyse die Lokalisation auf CFA10
deutlich Weiterhin konnte die gekoppelte Region auf einem 5 Mb umfassenden
Bereich von 39 bis 44 Mb eingegrenzt werden Es ist wahrscheinlich dass die
identifizierte CCSD Region ein Gen beinhaltet welches auf die Entstehung der
caninen kongenitalen Taubheit einwirkt
Es ist notwendig mehr SNPs zu entwickeln um das taubheitsverursachende Gen zu
lokalisieren oder einen eindeutig assoziierten SNP zu identifizieren
Chapter 12
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ROBERTSON NG LU L HELLER S MERCHANT SN EAVEY RD MCKENNA M
NADOL JB JR MIYAMOTO RT LINTHICUM FH JR LUBIANCA NETO JF
HUDSPETH AJ SEIDMAN CE MORTON CC SEIDMAN JG (1998)
Mutations in a novel cochlear gene cause DFNA9 a human nonsyndromic
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SCHNEEBERGER EE LYNCH RD (2004) The tight junction a multifunctional
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GUIPPONI M WANG J KAWASAKI K ASAKAWA S MINOSHIMA S
YOUNUS F MEHDI SQ RADHAKRISHNA U PAPASAVVAS MP
GEHRIG C ROSSIER C KOROSTISHEVSKY M GAL A SHIMIZU N
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SIMONS M WANG M MCBRIDE OW KAWAMOTO S YAMAKAWA K GDULA D
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539
STRAIN GM KEARNEY MT GIGNAC IJ LEVESQUE DC NELSON HJ
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TASSABEHJI M READ AP NEWTON VE HARRIS R BALLING R GRUSS P
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VAHAVA O MORELL R LYNCH ED WEISS S KAGAN ME AHITUV N
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VAN LAER L HUIZING EH VERSTREKEN M VAN ZUIJLEN D WAUTERS JG
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VERPY E LEIBOVICI M ZWAENEPOEL I LIU XZ GAL A SALEM N
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WANG A LIANG Y FRIDELL RA PROBST FJ WILCOX ER TOUCHMAN JW
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WATTENHOFER M REYMOND A FALCIOLA V CHAROLLAIS A CAILLE D
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Chapter 13
Appendix
Appendix microsatellite marker III
HE
Te
79
5 7
98
76
9 5
12
71
3 8
33
70
2 4
35
72
7 7
00
54
6 4
77
48
8 3
08
44
8 5
40
67
4 8
05
76
2 6
54
70
8 7
73
78
1 6
87
65
4 6
26
51
9 5
89
76
4 1
60
PIC
d
72
2 8
41
67
0 4
85
67
1 7
85
57
2 4
07
61
4 6
29
35
8 3
30
40
0 2
90
35
0 4
60
70
0 7
90
76
2 6
21
63
8 7
26
77
1 6
26
60
1 6
13
38
7 5
52
80
0 1
40
Prim
er (r
ever
se)
5acute -gt
3acute
AA
CTG
TTTG
ATT
TTG
ATG
AG
GC
C
TTTG
ACC
TAC
ATA
TAA
AA
CA
AG
C
CA
CG
AA
GAA
AG
CC
ATG
GTT
T C
AA
CA
ATT
CC
TTTG
TTG
GTG
C
CTG
CC
AG
CTT
CC
TAC
AGC
T
GTG
GTC
ACA
AG
AC
TTTA
GC
C
CTG
CA
CC
AAA
GA
AA
CTC
C
GG
AC
CTA
TTC
TGA
AG
CC
TAA
C
TG
GC
CTT
TAA
TTTA
TCAT
GG
AG
A
TCA
GG
CC
CA
GA
TAG
TATG
C
TC
CTT
TGG
TTTT
TAG
CAG
GG
T
CTG
TGC
CC
AC
CTG
TGG
AG
C
AA
GA
GG
ATA
CC
GG
TGG
CA
G
CG
TGC
TTTG
TTA
TGG
CTT
GA
C
AG
CA
TGA
AG
GA
TCTC
TGA
CTA
A
CTA
AC
TCG
CC
AG
AC
TATT
C
CC
CTC
CAC
CA
ATC
TCTT
CA
C
GC
AA
AG
GTG
TATT
TAC
ATG
ATG
G
GG
CA
GC
CTT
ATT
ATT
CA
TGG
A
AC
AC
AG
GC
AC
AG
GA
GC
ATC
C
AG
CC
ATC
CA
GG
AA
ATC
G
CTG
AA
CTG
GTC
CC
TCAA
GC
A
GTC
ATG
CTG
ATT
TCTG
TGC
C
GA
CC
TATC
CA
TAC
ATG
CC
C
GAA
GG
AAG
GAA
GG
AAG
AAAA
GA
TTA
GTA
AA
GA
GA
CA
GC
TGA
TTG
CC
A
TCTA
CC
CTG
CA
ACC
CTG
TG
ATT
GA
GA
CC
CA
AG
ACTG
TTA
GTG
G
GTA
AC
AAC
CA
GAG
TGTG
TGTG
A
CG
TCG
AG
CTC
CTG
GC
AT
Prim
er (f
orw
ard)
5acute -
gt 3acute
GG
GTA
GA
TTTC
AA
CA
AAT
AG
TAC
TGG
T
AG
AA
AG
GC
CTG
GA
TGTC
G
AG
TAG
AA
GA
GG
CC
AC
GC
AA
A
TTT
TCC
AA
AA
TGA
AA
AC
CTG
C
AG
ATT
GTT
TATG
CA
GG
CA
TTG
A
GA
GTT
GAA
AG
GTT
GAA
AA
TGG
G
CTC
AG
AG
GC
AG
ATA
GA
AA
A
ATT
GA
TTC
ATG
ACC
CA
CTA
A
TTT
TGTC
AAG
CA
GC
CC
TATA
TC
TG
AA
ATG
GTA
CTT
CA
GC
ATC
G
AG
CTT
AG
CTT
AC
TGC
CC
TGG
T
CC
CC
GG
CC
CC
TGC
CC
GG
AG
T
AC
CAC
TGTC
ATT
TTTC
CA
TGC
C
CC
TTA
GG
AGG
AG
GC
AAG
AC
C
CA
AG
GG
GTA
TGTT
GTC
TATT
ACT
GG
GTG
GG
AA
ATG
TGA
CTC
TG
GG
GC
TGC
CC
ATT
TCTT
TAA
T T
TGC
CA
CAA
ATC
AC
TTAA
AG
G
GA
CTG
AG
TTC
TTTC
AG
CA
CA
GTG
A
AAAT
AAC
ACAG
CAT
CAG
G
GA
ATC
CC
CA
ATG
TAC
ATG
GC
A
AC
AG
GC
GG
ATT
CTG
TAG
C
AC
TGG
CC
AAA
GA
GTA
CA
AG
G
CC
TTG
AC
TCA
GC
AGC
CC
TAC
A
AA
GA
TTG
TCTT
GA
CAC
GC
TG
GG
GTC
CTG
GG
ATC
AA
GC
C
AC
TGA
CTG
ATG
TCC
TGTG
CG
A
AA
AA
GTG
TAG
AG
CTT
TCTT
CA
AA
T
AA
ATT
CA
ATA
AG
CC
ATG
GA
GG
A
CC
TCC
AAG
ATG
GC
TCTT
GA
ATc
(degC
) 60
58
58
56
58
60
54
56
60
60
58
60
60
60
60
60
60
60
60
52
56
58
54
62
60
60
62
62
60
60
Pro
duct
si
ze (b
p)
380-
400
337-
409
267-
304
225-
235
312-
336
350-
394
155-
161
126-
156
510-
570
436-
468
189-
191
72-8
0 13
0-13
6 34
0-35
0 14
5-15
3 98
-102
22
0-24
0 21
2-24
0 34
7-40
3 32
5-34
1 25
1-27
9 32
5-36
1 19
7-24
5 30
9-40
9 34
1-37
5 21
5-23
1 30
8-31
6 16
2-17
4 46
8-50
2 18
2-19
0
Alle
les
6 14
8 4 7 11
3 6 8 7 2 2 3 4 2 3 5 8 12
7 8 7 14
10
9 7 3 3 15
2
Mbb
54
1
49
19
8 2
18
23
5 2
47
26
0 3
02
35
3 3
98
58
8 6
26
63
9 7
61
77
5 7
78
78
6 7
98
85
4 1
060
1
090
1
100
1
100
1
110
1
130
1
150
1
170
3
5
12
4 1
63
CFA
a
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10
10
10
Tabl
e 1
Cha
ract
eris
tics
of m
icro
sate
llite
mar
kers
sel
ecte
d fo
r CFA
1 1
0 an
d 31
com
pris
ing
in to
tal 6
0 m
arke
rs
(Con
tinue
d on
nex
t pag
e) T
he m
icro
sate
llite
mar
kers
wer
e al
l der
ived
from
the
NC
BI d
atab
ase
(http
w
ebn
cbin
lmn
ihg
ov)
Mar
ker
FH33
25
FH36
03
FH23
13
RE
N18
9I14
FH
3219
FH
3413
R
EN
136G
19
RE
N13
5K06
FH
3922
FH
3300
C
014
24
AH
T107
C
012
51
RE
N17
2C02
D
0150
5 R
EN
04H
08
RE
N15
9F24
FH
3894
FH
2309
R
EN
06N
11
FH23
26
FH33
22
FH35
05
FH25
98
FH26
34
FH22
94
RE
N22
9P15
FH
2537
FH
4081
C
107
81
Appendix microsatellite marker
IV
H
ETe
64
0 6
80
63
0 6
51
54
6 7
06
81
0 6
14
30
2 7
42
34
4 7
40
63
0 8
67
82
2 6
70
75
0 6
80
60
0 4
70
31
0 5
70
80
1 5
80
83
1 5
10
64
0 6
90
19
0 8
22
PIC
d
53
0 6
90
52
2 5
89
80
0 6
70
75
9 5
24
24
7 7
09
26
9 6
90
50
0 7
98
75
5 5
50
69
0 5
30
56
0 3
40
26
0 5
00
80
1 5
30
74
0 3
70
64
0 6
70
25
0 7
21
Prim
er (r
ever
se)
5acute -gt
3acute
AG
GC
CA
GC
AC
CA
TAA
CTC
AG
C
CTC
TCC
TCC
TTC
CC
CTT
CTC
A
GTG
CTG
TCC
ATT
GC
AA
GTC
T
GC
TCC
CC
TGTG
TTC
TGAG
G
CTG
GTG
ACTC
AGA
GAA
GTC
C
GG
AG
TTG
ATA
GTA
AA
TCTT
TGC
GG
T
GC
CC
TATT
CA
TTC
AA
CTG
G
AC
AC
TCTG
GTG
CA
AG
CG
AC
TC
TTC
TAC
TGA
AG
CA
TCC
GC
CT
GA
TTTT
CTC
TCTG
TCC
AC
TT
AA
CA
GC
ATT
TCA
GAC
AGA
GG
G
GG
TTTT
GG
CTG
TGA
GG
ATA
G
AG
TTC
CAG
ATC
GAG
TCC
CA
C
CA
CAT
GAT
TTC
ACTT
GTA
TATG
G
CC
TGA
GG
GTT
TTTA
CAG
TATG
G
TG
AA
ATC
TGG
GA
TTC
TGA
TAC
C
CC
AA
GC
TTTC
AC
CTA
ATC
AC
C
GA
TATT
TTTC
TCTC
CC
AC
CC
C
AC
ATT
TCTA
GG
CAG
TGG
CA
C
TGTC
CC
ATC
TAA
AG
GA
TAG
G
AG
TAA
TGC
TGC
CC
ACG
GA
AC
G
GC
TGG
AA
CC
TTC
CC
CTT
TAG
A
GC
TCTT
ACA
CG
CA
TTG
AG
G
AA
AC
GG
AG
TTC
CA
TCTC
TGG
C
AC
AG
TGA
GG
AG
GG
GTG
G
TG
TAA
CC
CTC
CTG
TGA
TATG
A
TGTG
AA
CC
CC
GC
CC
AATA
G
AA
GTG
AC
TGA
GTA
CC
TGA
AA
TCG
G
CC
TCTG
CC
TCTG
CC
TCTG
T G
AG
CC
CTG
TTC
TCA
GG
TTG
Prim
er (f
orw
ard)
5acute -
gt 3acute
AG
AC
CC
CC
CC
ACTA
CC
CC
AC
GC
GTT
AGC
TGC
CA
TCA
TCTT
G
CTG
GTT
GG
AG
CA
ATA
CC
AA
G
CC
ATT
CA
TGTT
GTT
GC
AG
ATG
T
AA
GG
AG
GG
CA
CTT
GAT
GG
C
TTA
TGG
CA
TTTG
GC
CTG
TC
AA
GG
AG
CAT
CTT
CC
AG
AAC
C
CG
CTC
ATG
CA
AG
TCA
TCA
CA
T C
TGG
CTT
AAA
CC
AC
TGG
TCA
C
CTT
CC
ATC
CC
GTT
GTG
TGT
ATT
CC
CC
AGC
GAT
ACC
A C
TTG
AG
TGG
CTT
GA
GTG
GC
TAC
T
TCTT
GC
TTC
TTG
AA
GTA
AG
CC
T
TGC
CC
GTC
CTA
TAC
TCC
TG
GA
AG
GTG
GTT
ATT
GTC
CTT
GG
G
AA
CC
ACA
GA
AG
AG
CTG
GA
A
GC
ATT
TGA
TGA
AA
TAA
AG
GG
A
GA
ATG
AA
AA
CG
GA
GC
AG
CA
G
CC
TTC
AAC
AC
CC
ATA
GC
TC
TC
AG
CA
ACTA
TAC
ATT
TAA
GA
GC
A
ATG
GA
CA
AATG
AA
CA
AA
AG
T
GTG
AC
TTTC
TTA
TCC
GC
CC
C
CC
AG
AA
AC
TCA
ACTG
ATG
C
TG
GA
CG
CTA
AG
CC
TGAC
TTT
CC
ATA
AG
ATA
CTC
AG
AA
AC
ATG
CA
C
AC
CAT
AA
ATG
GA
TGG
ATA
G
CA
GTG
AG
CA
AA
GC
AA
ATG
AA
C
CC
ATT
AG
CA
AA
TGA
CTG
GG
A
AA
GA
ATG
GG
AA
AA
CTG
ATA
A
AA
GG
TAG
TCC
CA
CG
ATC
CTC
ATc
(degC
) 60
60
60
60
60
60
60
60
60
60
60
60
60
62
60
60
60
60
60
60
60
60
60
60
60
60
60
60
60
60
Pro
duct
si
ze (b
p)
100-
110
112-
124
194-
206
210-
230
309-
341
350-
362
276-
300
96-1
30
130-
138
120-
138
200-
204
278-
296
157-
165
178-
254
358-
368
327-
337
380-
404
160-
170
320-
330
165-
169
230-
232
300-
310
276-
380
255-
263
258-
278
106-
120
164-
180
568-
608
223-
239
172-
186
Alle
les
3 5 3 5 8 4 7 4 2 5 3 7 5 9 6 4 7 3 4 2 2 3 11
5 6 2 4 7 4 8
Mbb
24
9 3
23
34
9 3
67
38
9 3
94
40
1 4
10
45
8 4
64
48
0 4
88
51
5 5
64
56
8 5
97
62
7 6
33
63
7 6
53
67
3 6
85
69
1 7
08
0
66
9
7
28
1 2
96
37
5
CFA
a
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
31
31
31
31
31
31
Tabl
e 1
(con
tinue
d)
Mar
ker
RE
N06
H21
R
EN
68B
08
FH30
55
C10
769
FH
3302
R
EN
181G
20
FH34
03
G02
512
RE
N15
0B12
A
HT1
01
RE
N73
F08
ZUB
EC
A1
C10
16
FH24
22
FH34
48
FH26
65
DTR
105
C
106
02
RE
N16
1L12
C
0410
7 R
EN
91H
07
RE
N15
4O19
FH
3381
R
EN
154G
10
FH21
89
RV
C11
R
EN
43H
24
FH22
39
RE
N11
0K04
FH
2712
a Can
ine
chro
mos
ome
b Pos
ition
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Appendix itemisation of alleles V
Table 2 Itemisation of alleles to bases for SNPs in the pedigrees
Code no 1 2 3 4
Base Adenine Cytosine Guanine Thymine
Appendix laboratory paraphernalia
VI
Laboratory paraphernalia Equipment Thermocycler
PTC-100trade Programmable Thermal Controller (MJ Research Watertown USA)
PTC-100trade Peltier Thermal Cycler (MJ Research Watertown USA)
PTC-200trade Peltier Thermal Cycler (MJ Research Watertown USA)
Automated sequencers
LI-COR Gene Read IR 4200 DNA Analyzer (LI-COR Inc Lincoln NE USA)
LI-COR Gene Read IR 4300 DNA Analyzer (LI-COR Inc Lincoln NE USA)
MegaBACE 1000 (Amersham Biosciences Freiburg)
Centrifuges
Sigma centrifuge 4-15 (Sigma Laborzentrifugen GmbH Osterode)
Desk-centrifuge 5415D (Eppendorf Hamburg)
Biofuge stratos (Heraeus Osterode)
Centrifuge Centrikon H 401 (Kontron Gosheim)
Megafuge 1OR (Heraeus Osterode)
Speed Vacreg Plus (Savant Instruments Farmingdale NY USA)
Agarose gel electrophoresis and pulsed field gel electrophoresis
Electrophoresis chambers OWL Separation Systems Portsmouth NH USA
Biometra Goumlttingen
BioRad Muumlnchen
Generators 2301 Macrodrive 1 (LKB Bromma Sweden)
Power Pac 3000 (BioRad Muumlnchen)
Gel documentation system BioDocAnalyze 312 nm Goumlttingen
Appendix laboratory paraphernalia VII
Others
Milli-Qreg biocel water purification system (Millipore GmbH Eschborn)
Incubator VT 5042 (Heraeus Osterode)
UV-Illuminator 312 nm (Bachhofer Reutlingen)
Centomatreg R Desk-Shaker (B Braun Melsungen AG Melsungen)
Biophotometer (Eppendorf AG Hamburg)
Kits
DNA purification
Montage PCR96 Cleanup Kit (Millipore GmbH Eschborn)
Cloning
Invitrogen TA cloningreg kit (Invitrogen Karlsruhe Germany)
Isolation of DNA
QIAamp 96 DNA Blood Kit (QIAGEN Hilden)
Plasmid Mini Prep 96 Kit (Millipore GmbH Eschborn)
Sequencing
ThermoSequenase Sequencing Kit (Amersham Biosciences Freiburg)
DYEnamic-ET-Terminator Cycle Sequencing Kit (Amersham Biosciences Freiburg
Germany)
RACE
FirstChoiceTM RNA ligase-mediated (RLM)-RACE kit (Ambion Europe Huntingdon
UK)
Appendix laboratory paraphernalia
VIII
RNA Total RNA (Biocat Heidelberg Germany)
Size standards
100 bp Ladder (New England Biolabs Schwalbach Taunus)
1 kb Ladder (New England Biolabs Schwalbach Taunus)
IRDyetrade 700 or 800 (LI-COR Inc Lincoln NE USA)
Reagents and buffers APS solution (10 )
1 g APS
10 ml H2O
Bromophenol blue solution
05 g bromophenol blue
10 ml 05 M EDTA solution
H2O ad 50 ml
dNTP solution
100 microl dATP [100 mM]
100 microl dCTP [100 mM]
100 microl dGTP [100 mM]
100 microl dTTP [100 mM]
1600 microl H2O
the concentration of each dNTP in the ready-to-use solution is 5 mM
Gel solution
1275 ml UreaTBE solution (Roth Karlsruhe)
225 ml Rotiphoresereg Gel 40 (38 acrylamide and 2 bisacrylamide)
95 microl APS solution (10 )
95 microl TEMED
Appendix laboratory paraphernalia IX
Loading buffer for agarose gels
EDTA pH 8 100 mM
Ficoll 400 20 (wv)
Bromophenol blue 025 (wv)
Xylencyanol 025 (wv)
Loading buffer for gel electrophoresis
2 ml bromophenol blue solution
20 ml formamide
TBE-buffer (1x)
100 ml TBE-buffer (10x)
900 ml H2O
TBE-buffer (10x)
108 g Tris [12114 M]
55 g boric acid [6183 M]
744 g EDTA [37224 M]
H2O ad 1000 ml
pH 80
UreaTBE solution (6 )
425 g urea [6006 M]
250 ml H2O
100 ml TBE-buffer (10x)
solubilise in a water bath at 65degC
H2O ad 850 ml
Appendix laboratory paraphernalia
X
Chemicals Agarose (Invitrogen Paisley UK)
Ammonium persulfate (APS) ge 98 (Sigma-Aldrich Chemie GmbH Steinheim)
Ampicillin (Serva Heidelberg)
Boric acid ge 998 pa (Carl Roth GmbH amp Co Karlsruhe)
Bromophenol blue (Merck KgaA Darmstadt)
dATP dCTP dGTP dTTP gt 98 (Carl Roth GmbH amp Co Karlsruhe)
Chloramphenicol (Serva Heidelberg)
DMSO ge 995 pa (Carl Roth GmbH amp Co Karlsruhe)
dNTP-Mix (Qbiogene GmbH Heidelberg)
EDTA ge 99 pa (Carl Roth GmbH amp Co Karlsruhe)
Ethidium bromide (Carl Roth GmbH amp Co Karlsruhe)
Ethyl alcohol (AppliChem Darmstadt)
Formamide ge 995 pa (Carl Roth GmbH amp Co Karlsruhe)
LB (Luria Bertani) agar (Scharlau Microbiology Barcelona Spain)
Paraffin (Merck KgaA Darmstadt)
RotiphoreseregGel40 (Carl Roth GmbH amp Co Karlsruhe)
SephadexTM G-50 Superfine (Amersham Biosciences Freiburg)
TEMED 99 pa (Carl Roth GmbH amp Co Karlsruhe)
Tris PUFFERANreg ge 999 pa (Carl Roth GmbH amp Co Karlsruhe)
Urea ge 995 pa (Carl Roth GmbH amp Co Karlsruhe)
Water was taken from the water purification system Milli-Qreg
X-Gal (AppliChem Darmstadt)
Enzymes Taq-DNA-Polymerase 5 Umicrol (Qbiogene GmbH Heidelberg)
Taq-DNA-Polymerase 5 U microl (Qiagen Hilden Germany)
Incubation Mix (10x) TPol with MgCl2 [15 mM] (Qbiogene GmbH Heidelberg)
The polymerase was always used in the presence of incubation Mix TPol 10x buffer
The encyme ECO RI (New England Biolabs Schwalbach Taunus) were used with
the adequate 10x encyme buffer
Appendix laboratory paraphernalia XI
Consumables
96 Well Multiply PCR plates skirted (Sarstedt Nuumlmbrecht)
Combitipsreg plus (Eppendorf AG Hamburg)
Pipette tips 01 ndash 10 microl 01 ndash 10 microl 5 ndash 200 microl (Carl Roth GmbH amp Co Karlsruhe)
Reaction tubes 15 and 20 ml (nerbe plus GmbH WinsenLuhe)
Reaction tubes 10 und 50 ml (Falcon) (Renner Darmstadt)
Thermo-fast 96 well plate skirted (ABgene Hamburg)
Software BLAST trace archive httpwwwncbinlmnihgov
httpwwwensemblorg
EBI toolbox httpwwwebiacukToolssequencehtml
MERLIN 0102 package httpwwwsphumicheducsgabecasisMerlin
Order of primers MWG Biotech-AG Ebersberg (httpsecom
mwgdnacomregisterindextcl)
biomersnet GmbH Ulm (orderbiomersnet)
PED50 Dr H Plendl et al (2005) Institute for Human Genetics
Kiel
Primer design httpfrodowimiteducgi-binprimer3primer3_ wwwcgi
Repeat Masker httpwwwrepeatmaskergenome washingtonedu
Sequencher 42 GeneCodes Ann Arbor MI USA
Spidey httpwwwncbinlmnihgovIEBResearch
OstellSpideyindexhtml
SUN Ultra Enterprise 450 Sun microsystems
XIII
List of publications Journal articles 1 MIESKES K WOumlHLKE A DROumlGEMUumlLLER C DISTL O 2006 Molecular
characterization of the canine myosin heavy polypeptide 9 non-muscle (MYH9)
gene on dog chromosome 10q232 Submitted for publication in Gene
2 MIESKES K DISTL O 2006 Evaluation of 17 single nucleotide
polymorphisms (SNPs) in six candidate genes for hereditary non-syndromic deafness
in Dalmatian dogs Submitted for publication in Journal of Heredity
3 MIESKES K DISTL O 2006 Evaluation of the TMC1 and TMIE genes as
candidates for hereditary non-syndromic deafness in Dalmatian dogs Submitted for
publication in Animal Genetics
XV
Acknowledgements
First of all I wish to thank Prof Dr Dr habil Ottmar Distl the supervisor of my
doctoral thesis for his academic guidance and support of this work
I am very thankful to Heike Klippert-Hasberg and Stefan Neander for technical
expertise and assistance
I am very grateful to Joumlrn Wrede for his support during the statistical analyses and his
help with computer problems
I wish to express my appreciation to the Gesellschaft zur Foumlrderung Kynologischer
Forschung (GKF) eV Germany for funding this work with a grant
I am appreciative to all Dalmatian breeders and owners for providing me blood
samples and the results of the BAER tests
My special thanks go to all colleagues and friends of the Institute for Animal Breeding
and Genetics of the University of Veterinary Medicine Hannover for their support
humour and the friendly atmosphere in the laboratory
Last but not least I wish to thank my family for their support during the work on this
thesis
6 Identification of a 5 Mb region on canine chromosome 10 harbouring a causative gene responsible for congenital sensorineural deafness in German Dalmatian dogs 81
Abstract 83
Introduction 83
Material and methods 84
Results 86
Discussion 87
7 Analysis of canine chromosome 1 and the Gap junction protein alpha 1 (GJA1) gene in Dalmatian dogs segregating for congenital sensorineural deafness 97
Abstract 99
Introduction 99
Material and methods 100
Results and discussion 102
8 Analysis of canine chromosome 31 and the claudin-14 (CLDN14) gene in Dalmatian dogs segregating for congenital sensorineural deafness 107
Abstract 109
Introduction 109
Material and methods 110
Results and discussion 112
9 General Discussion 117
The candidate gene approach 119
Linkage and association analysis 120
CFA1 122
CFA31 123
CFA10 123
10 Summary 125
11 Erweiterte Zusammenfassung 129
12 References 145
13 Appendix I 14 List of publications XIII
Abbreviations
List of abbreviations
A adenine
Acc No accession number
ACTG1 actin gamma 1
AEP akustisch evozierte Potentiale (acoustically evoked potentials)
APS ammonium persulphate
AT annealing temperature
BAC bacterial artificial chromosome
BAER brain stem auditory evoked response
BLAST Basic Local Alignment Search Tool
bp base pair
C cytosine
CCSD canine congenital sensorineural deafness
CDH23 cadherin related 23
cDNA copy desoxyribonucleic acid
CFA chromosome of Canis familiaris
CLDN14 claudin-14
cM centiMorgan
COCH coagulation factor C homolog cochlin
COL11A2 collagen type XI alpha 2
CRYM crystallin mu
CSD cochleosaccular degeneration
CX connexin
DFN x-linked deafness locus
DFNA autosomal dominant deafness locus
DFNA5 deafness autosomal dominant 5
DFNB autosomal recessive deafness locus
DIAPH1 diaphanous homolog 1 (Drosophila)
DMSO dimethyl sulfoxide
Abbreviations
DNA deoxyribonucleic acid
dNTPs deoxy nucleoside 5rsquotriphosphates (N is ACG or T)
EDN3 endothelin 3
EDNRB endothelin receptor type B
EDTA ethylenediamine tetraaceticacid
EMBL European Molecular Biology Laboratory
ESPN espin
EST expressed sequence tag
EYA4 eyes absent homolog 4 (Drosophila)
F forward
FISH fluorescence in situ hybridisation
G guanine
GJA1 gap junction protein alpha 1 43kD (connexin 43)
GJB2 gap junction protein beta 2 26k (connexin 26)
GJB3 gap junction protein beta 3 31kDa (connexin 31)
GJB6 gap junction protein beta 6 (connexin 30)
GKF Gesellschaft zur Foumlrderung Kynologischer Forschung (Society for the
Advancement of Cynological Research)
HET observed heterozygocity
HE expected heterozygosity value
HSA chromosome of Homo sapiens
IBD identical by descent
IRD infrared dye
KCNQ4 potassium voltage-gated channel KQT-like subfamily member 4
Kb kilobase
LD linkage disequilibrium
LINE long interspersed nuclear element
LOD logarithm of the odds
M molar
Mb megabase
Merlin multipoint engine for rapid likelihood inference
MITF microphthalmia-associated transcription factor
Abbreviations
MS microsatellite
MTRNR1 mitochondrially encoded 12S RNA
MTTS1 mitochondrially encoded tRNA serine 1 (UCN)
MYH9 myosin heavy polypeptide 9 non-muscle
MYH14 myosin heavy polypeptide 14
MYO1A myosin IA
MYO3A myosin IIIA
MYO6 myosin VI
MYO7A myosin VIIA
MYO15A myosin XVA
NCBI National Center for Biotechnology Information
NMMHC-A nonmuscle myosin heavy chain-A
ODDD oculodentodigital dysplasia
OMIM Online Mendelian Inheritance in Man
OTOA Otoancorin
OTOF otoferlin
P error probability
PAX3 paired box gene 3 (Waardenburg syndrome 1)
PCDH15 Protocadherin-15
PCR polymerase chain reaction
PIC polymorphism information content
POU3F4 POU domain class 3 transcription factor 4
POU4F3 POU domain class 4 transcription factor 3
PRES solute carrier family 26 member 5 (prestin)
QTL quantitative trait locus
R reverse
RACE rapid amplification of cDNA ends
RH radiation-hybrid
RLM RNA ligase-mediated
RNA ribonucleic acid
RT-PCR reverse transcription polymerase chain reaction
SAS Statistical Analysis System
Abbreviations
SH1 Src homology 1
SINE short interspersed nuclear element
SLC26A4 solute carrier family 26 member 4 (pendrin)
SLC26A5 solute carrier family 26 member 5 (prestin)
SNP single nucleotide polymorphism
SOX10 SRY (sex determining region Y)-box 10
STRC stereocilin
STS sequence-tagged site
T thymine
TBE tris-borate-ethylenediamine tetraacetic acid
TECTA tectorin alpha
TEMED NNNrsquoNrsquo-tetramethylenediamine
TFCP2L3 transcription factor CP2-like 3
TJ tight juncions
TMC1 transmembrane channel-like gene 1
TMIE transmembran inner ear gene
TMPRSS3 transmembrane protease serine 3
U unit
USH1C Usher syndrome 1C
UTR untranslated region
WFS1 Wolfram syndrome 1 (wolframin)
wgs whole genome shotgun
WHRN whirlin
WS Waardenburg syndrome
Chapter 1
Introduction
Introduction 3
Introduction
Canine congenital sensorineural deafness (CCSD) has often been reported in the
literature and occurs in more than 54 different breeds of dogs with the Dalmatian dog
showing the highest incidence The inheritance and segregation of a major gene in
CCSD has been demonstrated in different Dalmatian dog populations But although
several studies have demonstrated the mode of inheritance in Dalmatian dogs no
universally accepted mode of inheritance for the other dog breeds affected by CCSD
has yet been identified
Breeding with blue-eyed Dalmatian dogs and unilaterally or bilaterally deaf dams and
sires is forbidden by paragraph 11b of the German animal welfare laws and thus the
hearing status of all Dalmatian dogs has to be tested as a puppy at about six to eight
weeks old using the brain stem auditory evoked response (BAER) test As deaf dogs
are very difficult to raise and often become aggressive and snappish from fear most
puppies suffering from bilateral hearing loss are euthanized However it has been
shown in recent years that auditory testing does not seem to be an effective way of
clearly reducing the high incidence of deafness in this breed Thus prevention of
CCSD cannot be achieved alone by exclusion of affected animals from breeding
Consequently a molecular genetic approach toward unravelling the responsible
genes in carriers is urgently needed
Many genetic disorders in humans and domestic dogs (Canis familiaris) demonstrate
a high level of clinical and molecular similarity Therefore the mutated genes in
human hereditary deafness seemed to be appropriate candidates for canine
congenital sensorineural deafness
The objective of the present study is to localize the gene that is involved in the
development of CCSD in Dalmatian dogs In order to achieve this goal successively
32 canidate genes were evaluated by means of linkage analyses using microsatellite
markers and single nucleotide polymorphisms (SNPs) This candidate gene
approach using gene-associated markers for linkage studies in families segregating
for deafness turned out to be little effective Therefore the canine chromosomes
(CFA) 1 CFA10 and CFA31 were scanned entirely with microsatellite markers
Additionally single nucleotide polymorphisms (SNPs) were developed for fine
mapping the identified CCSD regions
Introduction 4
Overview of chapter contents Chapter 2 reviews the identified 39 mutated genes causing non-syndromic hereditary
hearing impairment in humans Parallels and differences in canine and human
deafness are shown including the clinical signs inheritance patterns and
histopathology We located the humane deafness genes in the canine genome and
discussed the advantages of comparative genomics and different molecular genetic
approaches
In Chapter 3 an existing set of 43 microsatellite markers for in total 24 candidate
genes were used for a non-parametric linkage analysis with congenital sensorineural
deafness (CCSD) in Dalmatian dog families segregating for deafness
In Chapter 4 newly developed SNP markers associated with in total eight candidate
genes were evaluated for CCSD in Dalmatian dogs
In Chapter 5 the molecular characterization of the canine myosin heavy polypeptide
9 non-muscle (MYH9) gene on dog chromosome 10q232 is described
Chapter 6 7 and 8 present linkage analyses performed in Dalmatian dog families
segregating for congenital sensorineural deafness using microsatellite markers on
canine chromosome (CFA) 1 CFA10 and CFA31 and the results of fine mapping
regions linked with the CCSD phenotype using newly developed SNPs
Chapter 9 provides a general discussion and conclusions referring to Chapters 1-8
Chapter 10 is a concise English summary of this thesis while Chapter 11 is an
expanded detailed German summary which takes into consideration the overall
research context
Chapter 2
A comparative overview of the molecular genetics of non-syndromic deafness
in dogs and humans
Non-syndromic deafness in dogs and humans 7
A comparative overview of the molecular genetics of non-syndromic deafness in dogs and humans
Abstract
In man as in different dog breeds deafness is an often diagnosed disorder with the
Dalmatian dog showing the highest incidence Deafness in Dalmatian dogs is clearly
heritable and the presence of a recessive major gene affecting the disorder was
shown in several Dalmatian dog populations
This Chapter provides an overview of the identified 39 mutated genes causing
human non-syndromic hereditary hearing impairment as well as of the five genes
responsible for Waardenburg syndrome in humans We point out their cytogenetic
and genomic localisations in man and dog and compare the genomic and mRNA
sequences of these genes between man and dog Moreover an overview is given on
deafness genes-associated markers identified in Dalmatian dogs and on candidate
genes characterized in dogs
The structure of the ear The inner ear consists of the semicircular canals the vestibule and the cochlea
whereas the vestibule and the semicircular canals are concerned with vestibular
function (balance) and the cocnlea is concerned with hearing Reissneracutes membrane
and the basilar membrane divide the cochlea longitudinally into three scalae the
scala vestibule the scala media and the scala tympani The process of transduction
occurs in the structures within scala media sitting on the basilar membrane and
comprising the organ of Corti Cutting the cochlea tube cross sectionally the scala
media is more or less triangular formed by Reissneracutes membrane basilar
membrane and a structure called the stria vascularis The fluid that fills scala
tympani and scala vestibule is called perilymph the fluid that fills scala media is
called endolymph The organ of Corti rests on the basilar membrane within scala
media The cochlea contains an array of highly specialized cells arranged in a highly
Non-syndromic deafness in dogs and humans 8
specialized manner Two types of cells in the organ of Corti are support cells and
hair cells The hair cells are the receptor cells that trancsduce sound
When a sound wave brings physical displacement of the membranes separating the
perilymph from the endolymph they cause the organ of Corti to move and the hair
cells on it are scraped along the bottom of the tectorial membrane The tectorial
membrane is firmly anchored to the bone Relative movement of the organ of Corti
and its hair cells with respect to the tectorial membrane is the source of the
deformation of the hair cells microvilli The hair cells are so constructed that any
deformation of their microvilli will cause a change in the overall membrane potential
of the cell This signal is detected by the fibers from the cells in the spiral ganglion
These fibers are neural elements and they carry their own depolarization wave into
the auditory region of the brain
Deafness in man There are various ways to categorise deafness The two main types of deafness are
classified based on which portions of the auditory system are affected conductive
hearing loss occurs when when sound is not conducted efficiently through the outer
andor middle part of the ear Much more common is the sensorineural hearing loss
Sensorineural hearing loss occurs when there is damage to the inner ear (cochlea)
or to the nerve pathways from the inner ear (retrocochlear) to the brain Most cases
of sensorineural hearing loss are due to cochlear defects (Petit et al 2001)
Hearing loss can be present at birth (congenital) or become evident later in life
(acquired) Congenital deafness similarly may or may not be genetic In fact more
than half of congenital hearing loss is inherited Alternatively congenital deafness
may be due to a condition or infection to which the mother was exposed during
pregnancy Furthermore congenital hereditary deafness may occur as part of a
multisystem disease (syndromic) or as a disorder restricted to the ear and vestibular
system (non-syndromic) As non-syndromic hereditary hearing impairment is almost
exclusively caused by cochlear defects affected patients suffer from sensorineural
hearing loss In Table 1 and 2 the genes underlying human hereditary non-
syndromic deafness as a result of cochlear defects in consequence of primary
defects in hair cells non-sensory cells and the tectorial membrane or unknown cell
type are shown Non-syndromic deafness is estimated to cause 60-70 of cases of
congenital hereditary deafness in man (Morton 1991) In 70-80 of all cases this
Non-syndromic deafness in dogs and humans 9
non-syndromic form of deafness shows an autosomal recessive inheritance followed
by an autosomal dominant inheritance in 10-20 of all cases and 1-2 of all cases
are X-linked A maternally inherited form may also occur (Van Camp and Smith
2003)
Non-syndromic forms of hereditary deafness are classified by their mode of
inheritance DFN DFNA and DFNB refer to deafness forms inherited on the X
chromosome-linked autosomal dominant and autosomal recessive modes of
transmission respectively
Human hereditary isolated hearing loss is genetically heterogeneous (Petit et al
2001) Up to 1 of the human genes are estimated to be necessary for hearing
(Friedmann and Griffith 2003) Today approximately 120 genes for human
hereditary deafness have been identified approximately 80 for syndromic and 39 for
non-syndromic hereditary deafness which is suspected to be one-third of the total
(Nance 2003) In Table 1 the identified 39 mutated genes causing non-syndromic
hereditary hearing impairment in humans are shown Out of the 39 genes 15 genes
cause autosomal recessive and 15 genes cause autosomal dominant forms six
genes are involved in both recessive and dominant forms one gene causes X-linked
and two a maternally inherited form (The Hereditary Hearing Loss Hompage
httpwebhostuaacbehhh)
Furthermore several hundred forms of syndromes with hearing loss have been
documented in humans (Van Camp and Smith 2003) One is the human
Waardenburg syndrome (WS) which manifests itself with sensorineural deafness
and pigmentation defects in the iris hair and skin The WS is classified into four
types depending on the presence or absence of additional symptoms which are
caused by mutations in the five genes EDN3 EDNRB MITF PAX3 and SOX10
respectively These genes are shown in Table 3 They are known to be expressed in
the neural crest (EDN3 EDNRB PAX3 SOX10 ) or directly in the melanocytes
(MITF) and are inter alia involved in migration differentiation or survival of
melanocytes respectively (Bondurand et al 2000)
Deafness in dogs Congenital sensorineural deafness (CSD) has been reported in a variety of mammal
species other than humans ranging from mice to dogs guinea pigs and mink
Canine congenital deafness has often been reported in the literature and occurs in
Non-syndromic deafness in dogs and humans 10
more than 54 different breeds of dogs according to Strain (1996 and 2004) The
breeds with the highest incidence include Dalmatian dogs Bull Terrier English
Cocker Spaniel English Setter Australian Cattle Dog Australian Shepherd West-
Highland-White-Terrier Dobermann and Dogo Argentino The incidence of canine
congenital deafness is highest in Dalmatian dogs of which 165 to 30 exhibit
unilateral or bilateral hearing loss (Famula et al 1996 Wood and Lakhani 1997
Muhle et al 2002 Juraschko et al 2003a Rak and Distl 2005) The inheritance
and segregation of a major gene in canine congenital sensorineural deafness
(CCSD) has been demonstrated in different Dalmatian dog populations (Famula et
al 2000 Muhle et al 2002 Juraschko et al 2003b) But although several studies
have demonstrated the mode of inheritance in Dalmatian dogs no universally
accepted mode of inheritance for the other dog breeds affected by CCSD has yet
been identified
Congenital sensorineural hearing impairment can be recognised in dogs at four to
eight weeks of age (Strain 1996) while histological studies of deaf Dalmatian dogs
have shown that the degeneration of the inner ear structures begins as early as one
day after birth and is histologically clearly evident by four weeks of age (Johnsson et
al 1973) In 70 of the cases with human hereditary deafness the histological
pattern is known as cochleo-saccular degeneration (CSD) (Lalwani et al 1997)
commonly known as Scheibe dysplasia with preservation of the pars superior of the
membranous labyrinth and an unremarkable bony labyrinth As in man also in many
affected dog breeds the histological pattern of congenital sensorineural deafness is
known as cochleo-saccular degeneration
Breeding with blue-eyed Dalmatian dogs and unilaterally or bilaterally deaf dams and
sires is forbidden by paragraph 11b of the German animal welfare laws and thus the
hearing status of all Dalmatian dogs has to be tested as a puppy at about six to eight
weeks old using the brainstem auditory evoked response (BAER) test that detects
electrical activity in the cochlea and auditory pathways in the brain Although the
BAER test is a reliable method for identifying unilaterally and bilaterally deaf dogs it
does not seem to be an effective way of clearly reducing the incidence of deafness in
affected breeds particularly in a recessive mode of inheritance so that hearing dogs
can still be genetic carriers Furthermore deaf dogs are very difficult to raise and
often become aggressive and snappish from fear consequently most puppies
Non-syndromic deafness in dogs and humans 11
suffering from bilateral hearing loss are euthanized Thus prevention of CCSD
cannot be achieved alone by exclusion of affected animals from breeding and
consequently a molecular genetic approach toward unravelling the responsible
genes in carriers is urgently needed
Over the past decade it has become increasingly clear how far structural and
functional homologies at the gene level extend across even distantly related species
Much is known about deafness-causing gene mutations in humans and mice
including the fact that the clinical and histopathological findings are often very similar
to those of deafness in Dalmatian dogs Thus despite the genetical heterogeneity of
human non-syndromic deafness the genes that are responsible for non-syndromic
congenital hereditary deafness in humans (Table 1) seemed to be appropriate
candidate genes for CCSD especially in Dalmatian dogs (Rak and Distl 2005) The
genes that are mutated in the human WS (Table 2) were selected as candidates
because the WS phenotype where the deafness is associated with pigmentation
defects seems to be similar to the phenotype of most affected dog breeds (Strain
and Tedford 1996) Both Juraschko et al (2003a) and Strain et al (1992) have
demonstrated that patched Dalmatians are less likely to be deaf than unpatched
animals and blue-eyed Dalmatians are more likely to be affected from hearing
impairment than brown-eyed animals
In an attempt to achieve the aim of molecular genetic diagnosis of CCSD carriers
Rak et al (2003) and co-workers (Droumlgemuumlller et al 2002 Kuiper et al 2002 Rak
et al 2002a 2002b 2003) already mapped 24 potential candidate genes for
sensorineural deafness in dogs by fluorescence in situ hybridization and a radiation
hybrid panel to 16 different canine chromosomes
The canine genome project In December 2005 an international research team led by scientists at the Broad
Institute of MIT and Harvard achieved the completion of a high-quality genome
sequence of the domestic dog together with a catalog of 25 million specific genetic
differences across several dog breeds (Lindblad-Toh et al 2005) The authors found
that humans share more of their ancestral DNA with dogs than with mice confirming
the utility of dog genetics for understanding human disease Furthermore the
physiology disease presentation and clinical response of dogs often mimic human
Non-syndromic deafness in dogs and humans 12
diseases closely As indicated above hearing impairment seemed to be no
exception
In the last years most projects have exploited canine traits for which either direct
candidate genes could be proposed and evaluated or for which large informative
pedigrees were available to enable linkage mapping to identify candidate regions A
major component of such research efforts comprised the cloning sequencing and
mapping of individual canine homologs of genes either proposed as candidate
genes or expected to be located in candidate regions This was necessary to
identify new informative polymorphisms (eg single nucleotide polymorphisms
(SNPs) microsatellites) for high resolution mapping of candidate regions and to
examine each exon and exonintron boundary for positional candidates Availability
of the second version of the dog genome assembly (build 21) of the NCBI database
shortcut this effort and increase the investigators efficency
The current RH map with 3200 markers provides a good estimate of the order and
physical spacing (ie in base pairs) of markers along canine chromosomes (Guyon
et al 2003) and was recently complemented by the construction of a 4249-marker
integrated canine genome RH map that consists of 900 genes 1589 microsatellites
and 1760 BAC end markers (Breen et al 2004) all included and available in the
NCBI database The second version 1 of the NCBIs genome annotation consists of
large contigs covering all canine chromosomes given with their located markers and
genes The great majority of genes are derived by automated computational analysis
using the gene prediction method GNOMON
With this help either additional candidate genes for canine CSD can be found directly
by its gene symbol in the 21 of the NCBIs genome annotation or if a candidate gene
is yet not annotated a BLAST (Basic Local Alignment Search Tool) search versus
the canine whole genome shotgun (wgs) sequence resource can be used to obtain
the sequence of the canine genomic contigs containing the human homologous
gene The localisation of all 39 known human non-syndromic hereditary deafness
genes in the canine genome with the corresponding accession numbers of the contig
and if available the accession number of the genomic sequence and mRNA of the
canine gene are shown in Tables 4 and 5 Furthermore the identity of canine and
human or mouse mRNA is shown in Table 5 The average identity of canine and
human mRNA is with 088 percent higher than the average identity of canine and
mouse mRNA with 084 percent Canine sequences that correspond to the human
Non-syndromic deafness in dogs and humans 13
candidate gene can now be used to find microsatellite or SNP markers associated to
the respective canine gene These markers can be used for linkage and haplotype
studies in dog families segregating for deafness
Table 7 shows the microsatellite and SNP markers developed for in total 32
candidate genes for CCSD
The candidate genes for which a set of in total 43 microsatellite marker were
designed by Rak (2003) included the following 24 genes CDH23 CLDN14 COCH
COL11A2 DFNA5 DIAPH1 EDN3 EDNRB EYA4 GJA1 GJB2 GJB6 MITF
MYH9 MYO6 MYO7A MYO15A OTOF PAX3 POU4F3 SLC26A4 SOX10
TECTA and TMPRSS3 This existing set of 43 microsatellite markers for 24
candidate genes were used for linkage and haplotype studies in Dalmatian dog
families segregating for deafness (Chapter 3) These 24 genes are known to be
involved either in human non-syndromic deafness or in the human Waardenburg
syndrome For another eight candidate genes including TMC1 TMIE USH1C
MYH14 MYO3A PRES WHRN and ESPN SNP markers were newly developed
(Chapter 4) and subsequently used for linkage and association analyses in
Dalmatian dog families segregating for deafness These genes are also involved in
human non-syndromic deafness
Non-syndromic deafness in dogs and humans 14
Tabele 1 Genes responsible for non-syndromic congenital hereditary deafness in
humans
Inheritance Gene Gene product Type of molecule Locus namea
ACTG1 γ-Actin cytoskeletal protein DFNA20 DFNA26
COCH Cochlin extracellular matrix component DFNA9
COL11A2 Collagen XI (α2-chain) extracellular matrix component DFNA13
CRYM micro-Cristallin thyroid hormone-binding protein DFNAb DFNA5 Unidentified Unidentified DFNA5 DIAPH1 Diaphanous-1 cytoskeleton regulatory protein DFNA1 EYA4 EYA4 transcriptional coactivator DFNA10 GJB3 Connexin-31 gap junction protein DFNA2 KCNQ4 KCNQ4 K+ channel subunit DFNA2 MYH14 Myosin IIC motor protein DFNA4 MYH9 Myosin IIA motor protein DFNA17 MYO1A Myosin IA motor protein DFNA48 POU4F3 POU4F3 transcription factor DFNA15 TFCP2L3 TFCP2L3 transcription factor DFNA28
Autosomal dominant
WFS1 Wolframin endoplasmic-reticulum membrane protein DFNA6 DFNA14
CDH23 Cadherin-23 cell-adhesion protein DFNB12 CLDN14 Claudin-14 tight-junction protein DFNB29 ESPN Espin actin-bundling protein DFNB36 DFNAb MYO15 Myosin XV motor protein DFNB3 MYO3A Myosin IIIA motor protein DFNB30 OTOA Otoancorin cell-surface protein DFNB22 OTOF Otoferlin putative vesicle traffic protein DFNB9 PCDH15 Protocadherin-15 cell-adhesion protein DFNB23 SLC26A4 Pendrin IminusndashClminus transporter DFNB4 SLC26A5 Prestin anion transporter DFNB61 STRC Stereocilin DFNB16
TMIE TMIE transmembrane domain- containing protein DFNB6
TMPRSS3 TMPRSS3 transmembrane serine protease DFNB8 DFNB10 USH1C Harmonin PDZ domain-containing protein DFNB18
Autosomal recessive
WHRN Whirlin PDZ domain-containing protein DFNB31 GJB2 Connexin-26 gap junction protein DFNB1 DFNA3 GJB6 Connexin-30 gap junction protein DFNB1 DFNA3 MYO6 Myosin VI motor protein DFNA22DFNB37MYO7A Myosin VIIA motor protein DFNB2DFNA11
TECTA α-Tectorin extracellular matrix component DFNA8 DFNA12DFNB21
Autosomal dominant and autosomal recessive
TMC1 TMC1 transmembrane channel-like protein
DFNB7 DFNB11DFNA36
X-linked POU3F4 POU3F4 transcription factor DFN3
MTRNR1 Mitochondrial 12S rRNA not defined
nomenclature Mitochondrial
MTTS1 Mitochondrial 12S rRNA not defined
nomenclature a Autosomal recessive loci are designated DFNB autosomal dominant loci DFNA
Non-syndromic deafness in dogs and humans 15
Table 2 Genes underlying hereditary non-syndromic deafness as a result of primary
defects in hair cells non-sensory cells and the tectorial membrane or unknown cell
type
Primary defect
Gene
Hair cells MYO7A MYO15 MYO6 MYO3A MYO1A ACTG1 USH1C
WHRN CDH23 PCDH15 TMIE STRC SLC26A5 ESPN
KCNQ4 TMC1 OTOF POU4F3
Non-sensory cells
GJB2 GJB6 GJB3 SLC26A4 CRYM OTOA CLDN14
COCH TMPRSS3 MYH9 MYH14 EYA4 POU3F4
Tectorial membrane
COL11A2 TECTA
Unknown
DIAPH1 DFNA5 WFS1 TFCP2L3 MTRNR1 MTTS1
Table 3 Genes involved in the human Waardenburg syndrome
Inheritance Gene Gene product Type of molecule Type
EDN3 endothelin 3 vasoconstricted peptide WS type IV4
EDNRBendothelin
receptor type B receptor protein WS type IV4
MITF
microphthalmia-
associated
transcription
factor
transcription factor WS type II2
PAX3 paired box 3 DNA-binding protein WS type I1and III 2
SOX10 SRY-box 10 transcription factor WS type IV4 1Type I Dystopia canthorum 2Type II No dystopia canthorum 3Klein-Waardenburg syndrome (type III) Type I and upper limb abnormalities 4Waardenburg-Shah syndrome (type IV) Type II and Hirschsprung disease
(autosomal recessive inheritance)
Non-syndromic deafness in dogs and humans 16
Leng
th o
f co
ntig
(bp)
7799
0652
3821
0901
7077
2986
7251
5492
5989
7527
4533
7677
6093
8239
9498
326
6961
0260
1859
5814
6421
1953
Scor
e of
co
ntig
614
938
792
567
300
715
323
535
1208
1634
923
E-va
lue
of
cont
ig
4 E
-172
0 0
2 E
-158
1 e-
78
0
2 e-
85
7 E
-149
0 0 0
Acc
No
of
WG
S co
ntig
unkn
own
NW
_876
311
NW
_876
295
NW
_876
327
NW
_876
254
NW
_876
321
NW
_876
292
NW
_876
258
NW
_876
315
NW
_876
269
NW
_876
278
NW
_876
259
Mb
from
to
unkn
own
254
12
578
337
93
379
132
11
323
563
56
5
272
12
723
393
33
943
411
64
123
632
86
331
292
82
955
209
32
094
101
81
019
Gen
e lo
catio
n on
C
FA
unkn
own
4 31
8 12
6 2 14
5 1 25
15
Can
ine
gene
al
iase
s
none
none
LOC
4877
51
LOC
4906
40
LOC
4817
34
LOC
4798
18
none
LOC
6112
23
LOC
4896
31
EYA4
GJB
2
LOC
4824
86
Acc
No
hu
man
m
RN
A
NM
_001
614
NM
_022
124
NM
_144
492
NM
_004
086
NM
_080
680
NM
_001
888
NM
_005
219
NM
_004
403
NM
_031
475
NM
_172
105
NM
_004
004
NM
_024
009
Gen
e lo
catio
n on
H
SA
17
10
21
14
6 16
5 7 1 6 13
1
Tabl
e 4
Loca
lisat
ion
of h
uman
non
-syn
drom
ic h
ered
itary
dea
fnes
s ge
nes
in th
e ca
nine
gen
ome
the
cani
ne
gene
loc
alis
atio
n in
meg
abas
es (
Mb)
th
e ac
cess
ion
num
ber
(Acc
No
) of
the
who
le g
enom
e sh
otgu
n (W
GS
) con
tig c
onta
inin
g th
e hu
man
hom
olog
ous
gene
and
the
corr
espo
ndin
g E
-val
ue s
core
and
leng
th o
f th
e co
ntig
Hum
an
deaf
ness
ge
ne
AC
TG1
CD
H23
CLD
N14
CO
CH
CO
L11A
2
CR
YM
DIA
PH1
DFN
A5
ESPN
EYA
4
GJB
2
GJB
3
der
ived
from
the
NC
BIs
can
ine
geno
me
anno
tatio
n ve
rsio
n 2
1
Non-syndromic deafness in dogs and humans 17
Leng
th o
f co
ntig
6421
1953
5300
4996
5294
2087
2607
3285
1654
5469
1249
9463
7251
5492
5102
4781
5989
7527
5159
1990
1284
7264
7521
5785
Scor
e of
co
ntig
525
521
973
2256
348
567
1236
404
337
383
604
1715
E-va
lue
of
cont
ig
3 e-
146
1 e-
144
0 0
9 e-
93
2 e-
158
0
3 e-
109
3 e-
89
7 e-
103
2 e-
169
0
Acc
No
of
WG
S co
ntig
unkn
own
NW
_876
259
NW
_876
270
NW
_876
251
NW
_876
313
NW
_876
250
NW
_876
290
NW
_876
254
NW
_876
273
NW
_876
321
NW
_876
263
NW
_876
283
NW
_879
563
Mb
from
to
unkn
own
521
52
3
109
24
109
35
311
33
119
443
64
441
415
41
7
103
41
056
404
14
050
245
42
460
261
32
619
235
02
359
371
43
769
674
86
748
Gen
e lo
catio
n on
C
FA
25
15
1 10
5 10
2 12
21
6 17
26
X
Can
ine
gene
al
iase
s
none
LOC
4824
51
none
LOC
4812
80
LOC
4795
22
LOC
4744
10
LOC
4871
06
LOC
4818
84
LOC
4851
74
LOC
6086
55
LOC
6079
61
none
LOC
4919
88
Acc
No
hu
man
m
RN
A
NM
_006
783
NM
_004
700
NM
_024
729
NM
_002
473
NM
_016
239
NM
_005
379
NM
_017
433
XM
_376
516
NM
_000
260
NM
_144
672
NM
_194
248
NM
_033
056
NM
_000
307
Gen
e lo
catio
n on
H
SA
13
1 19
22
17
12
10
6 11
16
2 10
X
Tabl
e 4
(con
tinue
d)
Hum
an
deaf
ness
ge
ne
GJB
6
KC
NQ
4
MYH
14
MYH
9
MYO
15
MYO
1A
MYO
3A
MYO
6
MYO
7A
OTO
A
OTO
F
PCD
H15
POU
3F4
der
ived
from
the
NC
BIs
can
ine
geno
me
anno
tatio
n ve
rsio
n 2
1
Non-syndromic deafness in dogs and humans 18
Leng
th o
f co
ntig
4533
7677
2532
0482
2532
0482
4020
6070
3002
9677
2968
9717
5300
4996
3309
7591
3821
0901
5102
4781
1104
8438
6535
5756
Scor
e of
co
ntig
1404
283
283
1683
883
529
354
216
198
354
2238
689
E-va
lue
of
cont
ig
0
6 e-
73
2 E
-73
0 0
5 e-
147
2 e-
94
4 e-
53
9 e-
48
2 E
-94
0 0
Acc
No
of
WG
S co
ntig
NW
_876
292
NW
_876
265
NW
_876
265
NW
_876
294
NW
_876
312
NW
_876
255
NW
_876
270
NW
_876
272
NW
_876
295
NW
_876
273
NW
_876
256
NW
_876
253
Mb
from
to
436
14
361
158
61
592
197
91
982
134
31
345
158
81
595
622
63
7
880
88
822
450
54
505
390
33
905
432
44
328
414
94
151
716
47
172
Gen
e lo
catio
n on
C
FA
2 18
18
30
5 13
1 20
31
21
13
11
Can
ine
gene
al
iase
s
LOC
4872
00
LOC
4832
63
LOC
4832
74
LOC
4782
78
LOC
4893
57
LOC
4819
85
LOC
4841
68
LOC
6093
50
LO
C61
0987
LOC
6108
50
LOC
4821
13
LOC
6125
88
Acc
No
hu
man
m
RN
A
NM
_002
700
NM
_000
441
NM
_206
883
NM
_153
700
NM
_005
422
NM
_024
915
NM
_138
691
NM
_147
196
NM
_024
022
NM
_153
676
NM
_006
005
NM
_015
404
Gen
e lo
catio
n on
H
SA
5 7 7 15
11
8 9 3 21
11
4 9
Tabl
e 4
(con
tinue
d)
Hum
an
deaf
ness
ge
ne
POU
4F3
SLC
26A
4
SLC
26A
5
STR
C
TEC
TA
TFC
P2L3
TMC
1
TMIE
TMPR
SS3
USH
1C
WFS
1
WH
RN
der
ived
from
the
NC
BIs
can
ine
geno
me
anno
tatio
n ve
rsio
n 2
1
Non-syndromic deafness in dogs and humans 19
Leng
th o
f co
ntig
(bp)
4776
3139
5561
1003
2518
2130
3091
5115
5294
2087
Scor
e of
co
ntig
262
721
2927
967
1179
E-va
lue
of
cont
ig
8e-6
7
0 0 0 0
Acc
No
of
WG
S co
ntig
NW
_876
277
NW
_876
274
NW
_876
271
NW
_876
304
NW
_876
251
Mb
from
to
470
14
703
343
63
438
248
52
488
313
43
144
297
52
976
Gen
e lo
catio
n on
C
FA
24
22
20
37
10
Can
ine
gene
al
iase
s
ED
N3
ED
NR
B
MIT
F
PAX3
LOC
4812
58
Acc
No
hu
man
m
RN
A
NM
_207
032
NM
_000
115
NM
_198
159
NM
_181
457
NM
_006
941
Gen
e lo
catio
n on
H
SA
20
13
3 2 22
Tabl
e 5
Loca
lisat
ion
of g
enes
invo
lved
in th
e hu
man
Waa
rden
burg
syn
drom
e (W
S)
in th
e ca
nine
gen
ome
th
e ca
nine
gen
e lo
calis
atio
n in
meg
abas
es (
Mb)
th
e ac
cess
ion
num
ber
(Acc
No
) of
the
who
le g
enom
e sh
otgu
n (W
GS
) con
tig c
onta
inin
g th
e hu
man
hom
olog
ous
gene
and
the
corr
espo
ndin
g E
-val
ue s
core
and
le
ngth
of t
he c
ontig
Hum
an
deaf
ness
ge
ne
EDN
3
EDN
RB
MIT
F
PAX3
SOX1
0
der
ived
from
the
NC
BIs
can
ine
geno
me
anno
tatio
n ve
rsio
n 2
1
Non-syndromic deafness in dogs and humans 20
Table 6 Canine candidate genes for CCSD with their accession number (AccNo) of
the genomic sequence and mRNA and if available the percent identity of canine and
human or mouse mRNA Canine candidate gene
Acc No canine genomic sequence
Acc No canine mRNA (predicted)
Canine mRNA (bp)
Identity () of canine and human mRNA
Identity () of canine and mouse mRNA
ACTG1 none1 none unknown unknown unknown CDH23 none1 none unknown 8800 8690 CLDN14 NC_006613 XM_544876 714 8880 8460 COCH NC_006590 XM_547762 3050 8770 8170
COL11A2 NC_006594 XM_857285 5202 8970 8740 CRYM NC_006588 XM_845361 1279 8720 7890
DIAPH1 none1 none unknown 8630 unknown DFNA5 NC_006596 XM_848863 1512 8320 7680 ESPN NC_006587 XM_546751 2565 8930 8270 EYA4 NC_006583 XM_541108 3969 9240 8120 GJB2 NC_006607 XM_543177 2208 7860 6720 GJB3 NC_006597 XM_539603 1305 8550 8090 GJB6 none none unknown unknown unknown
KCNQ4 NC_006597 XM_539568 1836 9350 8760 MYH14 none1 none unknown 8770 8220 MYH9 NC_006592 XM_538401 6201 9130 8940
MYO15 NC_006587 XM_536660 10128 8500 7960 MYO1A NC_006592 XM_531642 3388 8600 8350 MYO3A NC_006584 XM_544234 5589 8510 8910 MYO6 NC_006594 XM_862465 3983 9250 8610
MYO7A NC_006603 XM_542292 6519 9190 8880 OTOA NC_006588 XM_845746 3558 8740 7990 OTOF NC_006599 XM_844665 5994 9020 8780
PCDH15 none1 none unknown 8210 7390 POU3F4 NC_006621 XM_549108 1086 9190 9060 POU4F3 NC_006584 XM_544328 1017 9380 9160 SLC26A4 NC_006600 XM_540382 2382 7970 8240 SLC26A5 NC_006600 XM_540393 2235 9250 8740
STRC NC_006612 XM_535452 5301 8980 8520 TECTA NC_006587 XM_546475 7136 9110 8510
TFCP2L3 NC_006595 XM_539106 2127 9030 8760 TMC1 NC_006583 XM_541284 2580 8860 8740 TMIE NC_006602 XM_846596 396 9110 8910
TMPRSS3 NC_006613 XM_848589 1542 8500 8340 USH1C NC_006603 XM_860072 1730 9010 8670 WFS1 NC_006595 XM_539234 2667 8510 8420 WHRN NC_006593 XM_850321 2817 8440 8110 EDN3 NC_006606 NM_001002942 1976 747 716
EDNRB NC_006604 NM_001010943 1329 889 827 MITF NC_006602 XM_850501 1590 934 862 PAX3 NC_006619 XM_545664 1474 869 861
SOX10 NC_006592 XM_538379 1987 926 900 derived from the NCBIs canine genome annotation version 21 1 the definite region in the whole genome sequence (WGS) contig was used to get the percent identity of mRNAs
Non-syndromic deafness in dogs and humans 21
Table 7 Microsatellite markers and single nucleotide polymorphisms (SNPs)
of canine candidate genes for canine congenital sensorineural deafness in
Dalmatian dogs
Canine candidate gene
Number of gene-associated
microsatellites
Number of gene-associated
SNPs
CDH23 2 0 CLDN14 3 8 COCH 2 0
COL11A2 2 0 DIAPH1 2 0 DFNA5 2 0 ESPN 0 5 EYA4 2 0 GJB2 3 0 GJB3 1 0 GJB6 1 0
MYH14 0 2 MYH9 2 22
MYO15 2 0 MYO3A 0 3 MYO6 1 0
MYO7A 3 0 OTOF 1 0 PAX3 1 0
POU4F3 1 0 SLC26A4 1 0 SLC26A5 0 2 TECTA 2 0 TMC1 1 1 TMIE 1 3
TMPRSS3 2 0 USH1C 0 2 WHRN 0 3
Chapter 3
Linkage analysis of gene-associated microsatellite markers with
congenital sensorineural deafness in Dalmatian dogs
Linkage analysis of gene-associated microsatellites 25
Linkage analysis of gene-associated microsatellite markers with congenital sensorineural deafness in Dalmatian dogs
Abstract Deafness is a disorder often diagnosed in different dog breeds In this Chapter an
existing set of 43 microsatellite markers associated with in total 24 candidate genes
for canine congenital sensorineural deafness (CCSD) were used for linkage and
haplotype analyses in a large Dalmatian dog population with frequent occurrence of
CCSD We found significant linkage for the genes GJA1 MYH9 and CLDN14 As
linkage was found for different candidate genes in different families the results of
these test statistics indicate that the inheritance of non-syndromic deafness in
Dalmatian dogs is heterogenic in origin
Introduction
Canine congenital sensorineural deafness (CCSD) has been reported to occur in
more than 54 different breeds of dogs (Strain 1996) As in man also in dog breeds
the most commonly observed histological pattern of degenerative inner ear changes
is known as the cochleo-saccular or Scheibe type of end organ degeneration
The incidence of this congenital anomaly is highest in Dalmatian dogs of which 165-
30 exhibit unilateral or bilateral hearing loss (Famula et al 1996 Wood and
Lakhani 1997 Muhle et al 2002 Juraschko et al 2003a Rak and Distl 2005) The
inheritance and segregation of a major gene in CCSD has been demonstrated in
different Dalmatian dog populations (Famula et al 2000 Muhle et al 2002 Juraschko et al 2003b) Moreover deafness in Dalmatian dogs seems to be
pigment-associated (Greibrokk 1994 Holliday et al 1992 Juraschko et al 2003a
2003b Mair 1976 Strain et al 1992 Strain 1996)
No gene mutation has yet been identified that is responsible for CCSD in Dalmatian
dogs or in one of the various other dog breeds that suffer from inherited hearing
impairment Since mutations in various genes have already been found to be the
cause of sensorineural hearing impairment in humans or mice 24 of these genes
Linkage analysis of gene-associated microsatellites
26
were considered as candidates for CCSD in Dalmatian dogs (Rak et al 2005)
Details of the 24 candidate genes are given in Table 1
Rak et al (2003) and co-workers (Droumlgemuumlller et al 2002 Kuiper et al 2002 Rak et
al 2002a 2002b 2003) mapped this 24 potential candidate genes for sensorineural
deafness in dogs by fluorescence in situ hybridization and a radiation hybrid panel
Subsequently Rak (2003) developed altogether 43 new highly polymorphic DNA
markers for the 24 candidate genes including CDH23 CLDN14 COCH COL11A2
DFNA5 DIAPH1 EDN3 EDNRB EYA4 GJA1 GJB2 GJB6 MITF MYH9 MYO6
MYO7A MYO15A OTOF PAX3 POU4F3 SLC26A4 SOX10 TECTA and
TMPRSS3 (Table 2)
Among the 24 candidate genes seven genes cause autosomal dominant non-
syndromic forms of deafness seven cause autosomal recessive forms and five
genes cause both recessive and dominant forms of non-syndromic deafness in
different human families segregating for either forms
The functions of these 19 deafness-causing genes are diverse and include gap
junctions and tight junctions (GJA1 GJB2 GJB6 CLDN14) ion channels (SLC26A4)
and ion channel activators (TMPRSS3) Included are also unconventional myosins
(MYO6 MYO7A MYH9 MYO15A) transcription factors (POU4F3 EYA4) as well as
extracellular matrix components (COCH COL11A2 TECTA) a cytoskeleton
regulatory protein (DIAPH1) a cell-adhesion protein (CDH13) and genes with
unknown or only suspected functions (DFNA5 OTOF) The 24 candidates also
include five genes which are mutated in the human Waardenburg syndrome (WS)
The WS is classified into four types depending on the presence or absence of
additional symptoms which are caused by mutations in the five genes EDN3
EDNRB MITF PAX3 and SOX10 respectively The objective of the current study
was to use this set of markers developed by Rak (2003) for a non-parametric linkage
analysis with CCSD in a German and French Dalmatian dog population
Material and methods Pedigree material
For the linkage analysis we used DNA from altogether 215 animals belonging to a
total of 24 Dalmatian dog families The families included 22 full-sib families and one
large paternal half-sib family of German Dalmatian dogs as well as 46 animals of a
Linkage analysis of gene-associated microsatellites 27
large paternal half-sib family of French Dalmatian dogs All families were segregating
for CCSD The genotyped families included all affected dogs (unilaterally and
bilaterally deaf) their parents if available and one to four unaffected animals At least
two of the full sibs of each family were unilaterally deaf
In total these 24 families included 402 individuals with an average family size of 168
ranging from 5 to 116 animals and covering two to four generations The hearing
status of 344 dogs was examined by veterinarians using the BAER (brain stem
auditory evoked response) test and the other animals included in the pedigree being
not BAER tested were used to construct relationships among CSD affected dogs
The prevalence of CSD in this pedigree was 285
Microsatellite marker analysis An existing marker set consisting of 43 microsatellite markers (Table 2) was used for
linkage analysis This set included 36 markers developed by Rak (2003) and 7
markers of the RH map available at httpwww-recomgenuniv-rennes1frdoggyhtml
For most of the 24 candidate genes two markers were available for two of the
candidates three markers were available but for seven candidate genes the set
contains only one marker The marker set is composed of 33 perfect repeats two
imperfect six compound-perfect and two compound-imperfect repeats
The majority (674) of the 43 markers in the set was represented by dinucleotide
repeats 209 were tetranucleotide repeats 47 trinucleotide repeats and 23
pentanucleotide repeats In addition one marker (23) was a compound di-
tetranucleotide and another one (23) was a compound tetra-pentanucleotide
repeat The average number of alleles was 35 with a minimum of 2 and a maximum
of 8 different alleles per marker
PCR reactions for microsatellites were carried out in 12 microl reaction mixtures
containing 2 microl genomic DNA 12 microl 10x PCR buffer (Qbiogene Heidelberg
Germany) 024 microl dimethyl sulfoxide (DMSO) 02 microl dNTPs (100 microM) 01 microl Taq
Polymerase (5Umicrol) (Qbiogene) 06 microl (10 microM) 5rsquo-IRD700 or IRD800 labelled forward
primer 06 microl (10 microM) unlabelled reverse primer The PCR reactions were carried out
in MJ Research thermocyclers with the following program 4 min at 94 degC followed
by 32 cycles of 30 sec at 94degC 30 sec at maximum annealing temperature and a
final extension of 45 sec at 72degC PCR-products were diluted with formamide loading
buffer in ratios from 110 to 140 and then size-fractionated by gel electrophoresis on
Linkage analysis of gene-associated microsatellites
28
automated LI-COR 42004300 sequencers (LI-COR inc Lincoln NE USA) using
denaturing 4 and 6 polyacrylamide gels (RotiphoreseregGel 40 Roth Karlsruhe
Germany)
To localize the 24 candidate genes and their associated microsatellites exactly the
canine candidate gene sequences were derived from sequences deposited in the
current dog genome assembly (Boxer genome assembly 21) of the NCBI GenBank
by BLAST (Basic Local Alignment Search Tool) search
(httpwwwncbinlmnihgovBLAST) using the human reference mRNA sequence
(Table 3)
Linkage analysis
Multipoint linkage and haplotype analyses were performed using the MERLIN
software version 0102 (multipoint engine for rapid likelihood inference Center for
Statistical Genetics University of Michigan MI USA Abecasis et al 2002) The test
statistics Z-mean and Lod score were used to test for the proportion of alleles shared
by affected individuals identical by descent (IBD) for the considered marker loci
Linkage analyses were performed regarding the marker set consisting of 43 gene-
associated microsatellite markers Linkage analysis was at first carried out for all 24
families conjoined After this the families were scanned separately
The data of the genotypes was additionally analyzed using SASGenetics (Statistical
Analysis System Version 913 SAS Institute Inc Cary NC USA 2005) to specify
the number of alleles of each marker the allele frequency the observed (HET) and
expected (HE) heterozygosity and the polymorphism information content (PIC)
(Table 4 and 5)
Results and discussion
Test statistics for all families conjoined are given in Table 6 Significant CCSD loci
were located on different chromosomes The loci were located on canine
chromosome (CFA) 1 10 12 20 and 31 Linkage analyses per family indicated even
higher test statistics for subgroups of families (Table 7) Scanning only families with
Zmeans gt1 test statistics for linkage increased for the genes GJA1 on CFA1 MYH9
on CFA10 and CLDN14 on CFA31 with congenital sensorineural deafness in different
Dalmatian dog families (Table 7) Therefore it is probable that these genes or genes
Linkage analysis of gene-associated microsatellites 29
in their flanking regions are involved in the development of the disease in the
respective familes The results of this test statistics indicate that the inheritance of
non-syndromic hearing loss in Dalmatian dogs is probably as heterogenic in origin as
it is in humans Genetic heterogeneity means that different mutations cause the same
phenotype or disease the different mutations can either be found at the same locus
(allelic heterogeneity) or even at different loci (non-allelic heterogeneity)
The extreme heterogeneity of human deafness often hampered genetic studies
because many different genetic forms of hearing loss give rise to similar clinical
phenotypes and conversely mutations in the same gene can result in a variety of
clinical phenotypes In man genes that transport ions across membranes to maintain
appropriate solute concentration and pH as well as regulatory genes mainly
transcription factors and genes that play a part in structural integrity are essential for
the hearing process
However this study was a first step in identifying genes responsible for CCSD in
Dalmatian dogs The genes GJA1 MYH9 and CLDN14 and their flanking regions will
be further analyzed with a combined approach using microsatellites and single
nucleotide polymorphisms (SNPs) (Chapter 6-9) As linkage was found for different
candidate genes in different families subsequently only the families indicating
linkage will be chosen for further molecular analyses of the respective gene
To confirm the result of this study the density of the intragenic markers has to be
increased The current RH map with 3200 markers provides a good estimation of the
order and physical spacing (ie in base pairs) of markers along canine
chromosomes (Guyon et al 2003) and was recently complemented by the
construction of a 4249-marker integrated canine genome RH map which consists of
900 genes 1589 microsatellites and 1760 BAC end markers (Breen et al 2004) all
included and available in the NCBI database (httpwebncbinlmnihgov)
Thus microsatellites derived from the NCBI database could be used to confirm the
linkage Alternatively BLAST searches versus the canine whole genome shotgun
(wgs) sequence resource were perfomed to localize the genes exactly and to obtain
the sequence of the canine genomic contigs containing the human homologous gene
The results of the BLAST searches of the 24 candidate genes against the Boxer
genome assembly 21 are shown in Table 3 The genomic sequence of the
respective candidate gene can now be used to search for intragenic SNPs as these
polymorphisms are the most abundant and useful markers for fine mapping
Linkage analysis of gene-associated microsatellites
30
Development of SNPs requires sequencing of DNA for the respective genomic
regions of the parents with the aim to identify heterozygous base pair exchanges
After a heterozygous base pair is found the whole family can be genotyped for this
informative SNP marker These polymorphisms can than be used for linkage
analyses as well as association studies
Fine mapping using SNP markers for all genes indicating linkage with CCSD
identified by this study should enable us to detect mutations responsible for CCSD in
parts of the Dalmatian dog population
Linkage analysis of gene-associated microsatellites 31
Table 1 Details of the 24 selected human candidate genes
Symbol Gene name Locus name1 Most important reference
CDH23 cadherin related 23 DFNB12 Bork et al 2001
CLDN14 claudin 14 DFNB29 Wilcox et al 2001
COCH coagulation factor C homolog cochlin DFNA9 Robertson et al 1998
COL11A2 collagen type XI alpha 2 DFNA13 McGuirt et al 1999
DFNA5 deafness autosomal dominant 5 DFNA5 Van Laer et al 1998
DIAPH1 diaphanous homolog 1 (Drosophila) DFNA1 Lynch et al 1997
EDN3 endothelin 3 WS type IV Edery et al 1996
EDNRB endothelin receptor type B WS type IV Attie et al 1995
EYA4 eyes absent homolog 4 (Drosophila) DFNA10 Wayne et al 2001
GJA1 gap junction protein alpha 1 43kD (connexin 43)
2 Liu et al 2001
GJB2 gap junction protein beta 2 26k (connexin 26) DFNA3 DFNB1 Kelsell et al 1997
GJB6 gap junction protein beta 6 (connexin 30) DFNA3 DFNB1 Grifa et al 1999
Del Castillo et al 2002
MITF microphthalmia-associated transcription factor WS type II Tassabehji et al 1994
MYH9 myosin heavy polypeptide 9 non-muscle DFNA17 Lalwani et al 2000
MYO6 myosin VI DFNA22 DFNB37 Melchionda et al 2001 Ahmed et al 2003
MYO7A myosin VIIA DFNA11 DFNB2 Liu et al 1997a Liu et al 1997b Weil et al 1997
MYO15A myosin XVA DFNB3 Wang et al 1998
OTOF otoferlin DFNB9 Yasunaga et al 1999
PAX3 paired box gene 3 (Waardenburg syndrome 1) WS type I WS type III Hoth et al 1993 Tassabehji
et al 1992
POU4F3 POU domain class 4 transcription factor 3 DFNA15 Vahava et al 1998
SLC26A4 solute carrier family 26 member 4 DFNB4 Li et al 1998
SOX10 SRY (sex determining region Y)-box 10 WS type IV Pingault et al 1998
TECTA tectorin alpha DFNB21 DFNA8DFNA12
Mustapha et al 1999 Verhoeven et al 1998
TMPRSS3 transmembrane protease serine 3 DFNB8DFNB10 Scott et al 2001
1 Autosomal recessive loci are designated DFNB autosomal dominant loci DFNA 2 To our knowledge no deafness locus has been determined for this autosomal recessive gene
Linkage analysis of gene-associated microsatellites
32
HET
()
638
338
366
544
616
632
456
656
433
614
458
586
529
PIC
()
752
548
336
484
775
70
63
9
74
6
61
6
83
7
62
762
621
No
of
alle
les
6 4 4 4 9 8 6 6 6 13 5 11 5
PCR
pr
oduc
t (b
p)
175
109
123
156
146
179
259
151
241
219
186
214
191
AT
(degC
)
62 60 56 60 60 62 58 60 62 58 58 60 58
Prim
er s
eque
nces
(5rsquorarr
3rsquo)
F A
ATC
AG
GA
GG
GG
TGAG
TGTG
R
CC
CC
CAG
CTC
ATA
CA
TTC
TC
F C
CTG
TAC
TGA
ATG
CTT
GA
GG
R
CA
TCTC
TAG
AA
GA
AG
CC
TCC
F
TC
AC
ATA
GC
ATT
ATA
TATG
GA
C
R T
TGA
GAT
GG
CTC
TTAC
TGA
G
F T
CG
ATG
ATG
CTT
TCTG
GTT
G
R A
GG
CTG
TGA
AA
TGG
ATG
GA
G
F G
AG
AA
GC
AC
CA
GG
CA
TAG
G
R T
GG
TTTA
GC
AA
GG
CTG
TTC
C
F T
GA
CA
TAC
GG
AG
GAC
CA
AG
AG
R C
CC
CTC
CC
TTG
CTC
TATC
TC
F T
GC
CC
CTC
AG
AG
ATA
ATC
AC
R
CTT
CA
ATTA
TAC
AC
ACA
GG
TAC
F
TG
AA
TATG
GG
GC
TGAG
GA
AG
R
TTC
TCC
CTC
TGC
CTG
TGTC
F
GG
TTTA
GC
AC
TGC
CTT
CA
GC
R
CA
TTA
AG
CA
TCTG
GC
ATG
TGG
F
GA
AA
AC
TCA
GA
TTA
GC
CTG
G
R A
TCTT
GA
GA
GC
AA
AGG
TTG
TG
F T
GG
TTA
GG
GC
ATG
ATTC
CA
G
R C
ATG
TATA
AA
GA
GTA
ATG
CC
AG
F
CG
GG
AG
AGG
GTT
TGAC
TAC
R
CTC
CG
TATT
GC
TCA
TCTT
TCC
F
AG
CTT
CC
CTT
CTC
TGA
GA
C
R G
AG
AA
TAG
AG
TTTG
TGC
TCA
G
Rep
eat
(TTT
A)1
5 (A
CC
)9(A
TC)3
(A
TTT)
7(G
TTT)
5 (C
A)2
1 (G
A)2
0 (C
TTT)
~20
(GA
)13
(AC
)20(
AG)9
(C
T)16
TT
(CT)
5 (A
AA
AT)
~25
(CT)
11(G
T)4(
CT)
2 (A
TTT(
T))2
3 (A
C)2
2
Mar
ker n
ame
CD
H23
_MS1
C
DH
23_M
S2_F
2 C
LDN
14_M
S1
CLD
N14
_MS
2 C
LDN
14_M
S3
CO
CH
_MS1
C
OC
H_M
S2
CO
L11A
2_M
S1
CO
L11A
2_M
S3
DFN
A5_
MS
1 D
FNA
5 _M
S2
DIA
PH
1 _M
S1
DIA
PH
1_M
S2
Mar
ker o
rigin
RP
CI8
1-99
C20
R
PC
I81-
99C
20
RP
CI8
1-97
L17
RP
CI8
1-97
L17
RP
CI8
1-97
L17
RP
CI8
1-32
1I4
RP
CI8
1-32
1I4
RP
CI8
1-24
M6
RP
CI8
1-24
M6
RP
CI8
1-66
C12
R
PC
I81-
66C
12
RP
CI8
1-36
2I5
RP
CI8
1-36
2I5
Tabl
e 2
Can
dida
te g
ene-
asso
ciat
ed m
arke
rs f
or c
anin
e de
afne
ss d
evel
oped
by
Rak
(20
03)
For
each
gen
e th
e m
icro
sate
llite-
base
d m
arke
r th
e m
arke
r or
igin
rep
eat t
ype
PC
R p
rimer
s w
ith o
ptim
ised
con
ditio
ns a
nd th
e nu
mbe
r of
alle
les
pol
ymor
phis
m
info
rmat
ion
cont
ent (
PIC
) and
obs
erve
d he
tero
zygo
sity
(HE
T) fo
r all
dogs
gen
otyp
ed a
re s
how
n
Can
dida
te
gene
CD
H23
C
LDN
14
CO
CH
C
OL1
1A2
DFN
A5
DIA
PH
1
Linkage analysis of gene-associated microsatellites 33
HET
()
815
345
695
578
562
508
583
441
75
63
7
33
414
30
76
7
44
8
PIC
()
853
679
798
774
652
63
72
3
59
5
83
2
77
9
50
5
54
1
40
4
88
9
59
1
No
of
alle
les
14 6 7 6 9 5 9 6 13 13 4 6 4 14 6
PCR
pr
oduc
t (b
p)
147
131
258
227
134
190
148
195
218
260
181
164 96
227 94
AT
(degC
)
60 58 58 58 60 60 58 62 58 58 62 56 58 58 62
Prim
er s
eque
nces
(5rsquorarr
3rsquo)
F G
CTA
GG
AA
AA
ATC
CG
CA
ATG
R
GAC
CC
CC
TAG
GAC
ACC
AAC
F
GA
GA
ATT
GG
GC
ATG
GG
CAG
A
R T
GA
CTT
TATC
AC
TGG
TCTT
TG
F T
TATG
CAG
CC
CA
TGAC
AA
TC
R C
AA
GG
GA
AC
TCA
AAG
GC
TTG
F
TG
GA
CC
AG
GTC
AGTT
TGTG
R
TC
TGC
CTG
TGTC
TCTG
CC
F
ATG
GC
ATG
AA
GA
GG
ATA
CC
G
R A
GG
AC
AGG
TGAC
GG
CTC
TAC
F
GC
TAG
TAC
TCG
ATT
GTG
GTC
R
TC
ATG
GG
TTG
TGA
GA
TCC
AG
F T
TAA
TTTG
CTC
GTC
TTC
CTG
R
TG
TAA
GC
TCC
ACG
GA
TCA
CC
F
CTC
TCTT
GG
TCTC
CC
TCTG
C
R G
GG
AG
TAG
GG
GTG
GAG
TAG
G
F G
GTG
TTTC
CTT
TCC
TTTT
CT
R G
GTG
TTC
TCTC
CC
TTTC
TCT
F C
TCTA
TGA
AA
GG
TGA
TTG
CC
R
CA
GC
CAT
AC
AA
ATG
AGA
ATT
G
F C
TAC
AG
TGA
ATC
AG
CA
CA
GAC
R
CA
GC
CTT
GA
CTG
TTTC
TTTG
G
F T
GA
TTG
AC
TCTA
CTT
TAC
AC
A
R T
ATA
TTA
GG
CG
GTT
TTC
TTC
T F
AC
CC
AG
GTG
GC
CTG
ATTG
R
GC
AC
GC
AC
GTT
CTC
TCTT
TC
F G
AA
TGC
CC
TTC
ACC
TTG
AA
A
R G
GA
AA
AG
GA
GA
GA
TGA
TGC
C
F T
CTT
CC
TTG
GA
AA
GG
GA
AC
TC
R T
GC
CC
TAA
CA
CTT
GG
AA
TGG
Rep
eat
(TA
GA
)12
(GA
)25
(GT)
10(A
T)13
(A
G)2
1 (G
T)16
(A
G)1
2 (C
A)1
2 (G
A)8
(C
TTT)
~20
(GA
AA
)~25
(G
T)12
(C
A)1
5 (A
G)1
1 (G
AA
A)~
43
(AC
)13
Mar
ker n
ame
ED
N3_
MS
1 E
DN
RB
_M
S1
EY
A4_
MS
1 E
YA
4_M
S2
GJA
1_M
S1
GJA
1_M
S2
GJB
2_M
S1
GJB
2_M
S2
GJB
6_M
S2
GJB
2+6_
MS1
M
ITF
_MS
2 M
ITF
_MS
3 M
YH
9_M
S2
MY
H9_
MS
3 M
YO
6_M
S2
Mar
ker o
rigin
RP
CI8
1-36
6E14
Z
emke
et a
l (1
999)
R
PC
I81-
301N
19
RP
CI8
1-30
1N19
R
PC
I81-
370A
16
RP
CI8
1-37
0A16
R
PC
I81-
133O
22
RP
CI8
1-13
3O22
R
PC
I81-
343C
15
FH
2324
R
PC
I81-
119P
24
RE
N10
0J13
R
PC
I81-
374A
12
FH
2293
R
PC
I81-
156P
14
Tabl
e 2
(con
tinue
d)
Can
dida
te g
ene
ED
N3
ED
NR
B
EY
A4
GJA
1 G
JB2
GJB
6 M
ITF
MY
H9
MY
O6
Linkage analysis of gene-associated microsatellites
34
HET
()
359
366
431
566
51
50
5
28
3
35
8
79
9
70
4
69
2
23
9
68 71
698
PIC
()
496
623
627
765
635
756
502
575
756
76
70
1
44
2
75
4
84
5
80
6
No
of
alle
les
5 4 6 13 7 8 5 3 10 11 8 8 8 10 11
PCR
pr
oduc
t (b
p)
293
166
100
137
174
197
164
201
160
297
267
140
276
228
260
AT
(degC
)
56 60 56 62 62 62 60 60 58 58 56 58 58 62 60
Prim
er s
eque
nces
(5rsquorarr
3rsquo)
F T
GG
TTA
AA
AC
ATT
AA
AC
TTA
TAG
R
TA
GTA
TATA
GA
GA
TGC
AA
TGG
F
CA
TTG
GG
TGC
TTTC
CTG
TTC
R
TG
GA
GC
TGC
AG
GTA
TAG
CC
F
CC
AG
GC
ATT
CG
AG
GG
TG
R C
AG
AA
CTT
GA
GG
AAC
CA
TAG
F
CC
ATG
AAC
TTTG
TGG
AA
CTG
C
R A
AA
GG
GTT
GC
TGTG
GA
GA
TG
F A
GG
CA
GG
TTC
ATC
TGTG
TCC
R
TC
CC
AG
AC
CC
AG
CTA
CA
TTC
F
CA
GC
CAA
CTG
TATT
CTC
CTT
G
R A
TCTT
GA
GC
CC
TGC
ATT
AG
G
F G
AA
GC
GA
GG
AG
AG
ACA
GTC
C
R A
AG
GA
AG
CC
TCC
TGAC
AA
CC
F
CA
GG
GTC
AG
GC
TCTA
TGC
TC
R T
CC
TATC
ATC
CG
GC
TTTG
AC
F
TC
TGG
ATTG
TGG
TCA
CA
AC
C
R A
CTG
GAC
AC
TTC
TTTT
CA
GAC
G
F A
AA
GTG
GC
TGG
TTC
GG
AA
G
R A
GC
AG
CA
GC
ATA
CA
TTC
CTC
F
AA
GTA
GA
TCC
TATT
ATC
GTG
G
R A
GTT
TCA
GTG
TCTG
TTA
AA
TAG
F
CC
GG
ATT
TCTG
AG
GAG
GC
R
CA
TGC
TCTT
CA
CC
AGA
AC
C
F T
CA
GC
ATG
GA
TTTT
GTA
AA
ATC
R
GG
AC
TGC
GTG
GA
CAT
CTG
F
AC
AC
GG
TTC
TCG
CTG
ATG
TG
R T
GA
AG
GG
GA
TTG
AAC
AG
AG
G
F T
TCA
TTC
CG
AG
GTT
CTA
AC
TG
R C
AC
CA
TCTC
GTA
GC
CTT
TATC
Rep
eat
(TC
)13
(AC
)12
(GT)
12
(AC
)18
(GT)
12
(TA
)3(T
G)9
(TA
)2(C
A)2
C
(TA
AA
)5
(AA
T)18
(C
T)13
(T
G)1
3 (C
T)17
(T
AA
A)1
4 (G
T)3
CC
(GT)
19
(ATT
T)12
(T
C)8
(CA)
5(C
G)2
(CA)
9 (G
T)16
Mar
ker n
ame
MY
O7A
_M
S1
MY
O7A
_M
S2
MY
O7A
_M
S3
MY
O15
A _
MS
1 M
YO
15A
_M
S2
OTO
F _M
S1
PA
X3_
MS
1 P
AX
3_M
S2
PO
U4F
3_M
S4
SLC
26A
4_M
S2
SO
X10
_MS
2 T
EC
TA _
MS
1 T
EC
TA _
MS
2 T
MP
RS
S3_
MS
1 T
MP
RS
S3_
MS
2
Mar
ker o
rigin
RP
CI8
1-19
3deg2
RP
CI8
1-19
3deg2
AH
TH29
8 R
PC
I81-
362deg
13
RP
CI8
1-36
2deg13
R
PC
I81-
198L
15
RP
CI8
1-25
7H23
R
PC
I81-
257H
23
G2C
024
66
RP
CI8
1-47
P17
RP
CI8
1-50
5H2
RP
CI8
1-59
C2
RP
CI8
1-59
C2
RP
CI8
1-12
5P17
A
HTH
246
Tabl
e 2
(con
tinue
d)
Can
dida
te
gene
MY
O7A
M
YO
15A
O
TOF
PA
X3
PO
U4F
3 S
LC26
A4
SO
X10
TE
CTA
TM
PR
SS
3
Linkage analysis of gene-associated microsatellites 35
Table 3 The gene-associated microsatellites (MS) for 24 candidate genes of canine
congenital sensorineural deafness were localized using BLAST searches against the
Boxer genome assembly 21 The accession numbers (AccNo) of the whole
genome shotgun (WGS) contigs containing the genes as well as their associated
microsatellites are given
Microsatellite Acc No of WGS contig CFA Location of
Gene (Mb) Location of
MS (bp) Location of
MS in relation to gene
CDH23_MS1 NW_876311 4 2541025780 25510 intragenic
CDH23_MS2 NW_876311 4 2541025780 25630 intragenic
CLDN14_MS1 NW_876295 31 3379533796 33790 proximal
CLDN14_MS2 NW_876295 31 3379533796 33950 distal
CLDN14_MS3 NW_876295 31 3379533796 33790 proximal
COCH_MS1 NW_876327 8 1321513232 13225 intragenic
COCH_MS2 NW_876327 8 1321513232 13290 distal
COL11A2_MS1 NW_876254 12 56315659 5608 proximal
COL11A2_MS3 NW_876254 12 56315659 5578 proximal
DFNA5_MS1 NW_876258 14 4116941237 41135 proximal
DFNA5_MS2 NW_876258 14 4116941237 41250 distal
DIAPH1_MS1 NW_876292 2 3933039430 39382 intragenic
DIAPH1_MS2 NW_876292 2 3933039430 39552 distal
EDN3_MS1 NW_876277 24 4701347032 47057 distal
EDNRB_MS1 Zemke et al (1999) 22 3436334383 34354 proximal
EYA4_MS1 NW_876269 1 2928029550 29531 intragenic
EYA4_MS2 NW_876269 1 2928029550 29500 intragenic
GJA1_MS1 NW_876269 1 6399463996 64150 distal
GJA1_MS2 NW_876269 1 6399463996 64160 distal
GJB2_MS1 NW_8762781 25 2093620942 20938 intragenic
GJB2_MS2 NW_8762781 25 2093620942 20863 proximal
GJB2+6_MS1 FH2324 25 2093620942 17543 proximal
GJB3_MS2 NW_876259 15 1018310194 4530 proximal
GJB6_MS2 NW_876278 25 2090420906 20953 distal
Linkage analysis of gene-associated microsatellites
36
Table 3 (continued)
Microsatellite Acc No of WGS contig CFA Location of
Gene (Mb) Location of
MS (bp) Location of
MS in relation to gene
MITF_MS2 NW_876271 20 2485324884 24844 proximal
MITF_MS3 REN100J13 20 2485324884 25668 distal
MYH9_MS2 NW_876251 10 3113531193 31244 distal
MYH9_MS3 FH2293 10 3113531193 31696 distal
MYO15A_MS1 NW_876313 5 4436944419 44330 proximal
MYO15A_MS2 NW_876313 5 4436944419 44409 intragenic
MYO6_MS2 NW_876254 12 4041740504 40534 distal MYO7A_MS1 NW_876273 21 2454324609 24465 proximal
MYO7A_MS2 NW_876273 21 2454324609 24566 intragenic
MYO7A_MS3 AHTH298 21 2454324609 24594 distal
OTOF_MS1 NW_876263 17 2350223595 23463 proximal
PAX3_MS1 NW_876304 37 3134831445 31426 intragenic
PAX3_MS2 NW_876304 37 3134831445 31481 distal
POU4F3_MS4 G2C02466 2 4361043612 - -
SLC26A4_MS2 NW_876265 18 1586715927 15960 distal
SOX10_MS2 NW_876251 10 2975129762 29740 proximal
TECTA_MS1 NW_876312 5 1588515954 15910 intragenic
TECTA_MS2 NW_876312 5 1588515954 15930 intragenic
TMPRSS3_MS1 NW_876295 31 3903539054 38942 proximal
TMPRSS3_MS2 AHTH246 31 3903539054 38508 proximal
Linkage analysis of gene-associated microsatellites 37
Table 4 Number of alleles observed (HET) and expected (HE) heterozygosity and
polymorphism information content (PIC) for the developed marker-set
Feature Mean SD Min Max
No of alleles 75 31 3 14
hO () 703 122 370 898
hE () 532 151 239 815
PIC () 667 130 336 889
Table 5 Number of alleles per microsatellite locus and their PIC () values of the
developed marker-set
No of alleles per
microsatellite
Number of marker
loci PIC ()
3 1 575
4 6 483
5 5 574
6 11 652
7 2 716
8 5 671
9 3 717
10 2 800
11 3 776
13 4 803
14 2 871
Linkage analysis of gene-associated microsatellites
38
Table 6 Significant test statistics for linkage analysis carried out for all 24 genotyped
families conjoined Zmeans and LOD scores are given with their respective error
probabilities for the gene-associated markers of the candidate genes CLDN14
COL11A2 GJA1 MITF MYH9 and SOX10
Marker Location on canine chromosome (CFA)
Zmean pZmean LOD score pLOD
CLDN14_MS1 31q15 134 009 086 002
CLDN14_MS2 31q15 168 005 105 001
CLDN14_MS3 31q15 108 014 049 007
COL11A2_MS1 12q11-q12 166 005 085 002
COL11A2_MS3 12q11-q12 167 005 078 003
GJA1_MS1 1q24-q25 151 007 118 001
GJA1_MS2 1q24-q25 151 007 118 001
MITF_MS2 20q13 101 02 080 003
MITF_MS3 20q13 121 011 104 001
MYH9_MS2 10q232 080 02 018 02
MYH9_MS3 10q232 175 004 097 002
SOX10_MS2 10q21-q23 146 007 110 001
Linkage analysis of gene-associated microsatellites 39
Table 7 Significant test statistics for linkage analyses carried out each family
separately Zmeans and LOD scores are given with their respective error probabilities
for the gene-associated markers of the candidate genes CLDN14 MYH9 and GJA1
Gene-associated
marker
Number of families with significant linkage to
CCSD
Number of corresponding
family members
Zmean pZmean LOD-score pLOD
CLDN14_MS1 51 40 278 0003 112 0011
CLDN14_MS2 383 000007 170 0003
CLDN14_MS3 281 0002 113 0011
MYH9_MS2 32 21 081 02 023 02
MYH9_MS3
(=FH2293) 156 006 058 005
GJA1_MS1 13 46 295 0002 052 006
GJA1_MS2 286 0002 051 006 1 families with significant linkage include four German full-sib families and one
German half-sib family 2 families with significant linkage include three German full-sib families 3 families with significant linkage include one French half-sib family
Chapter 4
Evaluation of eight candidate genes for canine congenital sensorineural deafness
in Dalmatian dogs
Evaluation of eight candidate genes for CCSD 43
Evaluation of eight candidate genes for canine congenital sensorineural deafness in Dalmatian dogs Abstract
In this study we have been focusing on genomic loci that encode various enzymes
and transporters involved in the hearing process in humans We developed intragenic
markers for the canine genes ESPN MYH14 MYO3A PRES TMC1 TMIE USH1C
and WHRN on canine chromosomes (CFA) 1 2 5 11 18 20 and 21 which have
been shown to be responsible for human hereditary deafness and to employ these
newly developed markers for non-parametric linkage analyses with canine congenital
sensorineural deafness (CCSD) We screened DNA from altogether six Dalmatian
dogs which represent the parents of four families for single nucleotide polymorphisms
(SNPs) in the eight candidate genes by means of direct sequencing combined with a
polymerase chain reaction method for amplifying genomic DNA We characterized 20
SNPs and one insertion-deletion polymorphism For the TMC1 and TMIE genes we
additionally genotyped one microsatellite marker each The families used for
subsequent genotyping of the markers included 39 members from four full-sib
families with frequent occurrence of CCSD We concluded that mutations in ESPN
MYH14 MYO3A PRES TMC1 TMIE USH1C and WHRN do not play a role in
CCSD of the Dalmatian dog population investigated here
Introduction Over the past ten years significant progress has been made in the identification of
genes causing different forms of human deafness Currently 39 of the genes
responsible for non-syndromic hearing impairment have been identified in different
human populations (The Hereditary Hearing Loss Hompage
httpwebhostuaacbehhh Van Camp and Smith 2003)
Since non-syndromic hereditary hearing impairment is almost exclusively caused by
cochlear defects affected patients suffer from sensorineural hearing loss
Evaluation of eight candidate genes for CCSD
44
The most common histopathologic finding in cases of profound congenital deafness
in humans is the cochleosaccular degeneration (CSD) It is estimated to occur in
approximately 70 of cases in man and also in dog breeds the histological pattern is
known as cochleosaccular degeneration
Many genetic disorders in humans and domestic dogs (Canis familiaris) demonstrate
a high level of clinical and molecular similarity Therefore genes responsible for
human hereditary deafness seem to be appropriate candidate genes for CCSD
especially in Dalmatian dogs
In this report we provide 21 single-nucleotide polymorphisms (SNPs) and two
microsatellite markers in altogether eight selected human candidate genes (Table 1)
This eight candidate genes were only recently identified for being responsible for
different form of human non-syndromic deafness In order to evaluate whether any of
this candidate genes is responsible for congenital sensorineural deafness in
Dalmatian dogs we analyzed the association of the ESPN MYH14 MYO3A PRES
TMC1 TMIE USH1C and WHRN haplotypes with the CCSD phenotype in four
families of Dalmatian dogs with frequent occurrence of CCSD
Material and methods Pedigree structure and sampling
For the linkage analysis we used blood samples from 39 Dalmatian dogs They
belong to four full-sib families segregating for CCSD At least two of the full sibs of
each family were unilaterally deaf The phenotype of the affected animals had been
confirmed by brainstem auditory evoked response (BAER) that detects electrical
activity in the cochlea and auditory pathways in the brain
The families consisted of eight to 12 individuals In two families a blood sample of the
sire and dam respectively was not available Screening for SNPs was performed by
comparative sequencing of genomic DNA from the parents of the families used for
linkage analyses
SNP and microsatellite marker identification for genotyping
The canine ESPN MYH14 MYO3A PRES TMC1 TMIE USH1C and WHRN gene
sequences was derived from sequences deposited in the current dog genome
assembly (Boxer genome assembly 21) of the NCBI GenBank (Table 2) by BLAST
Evaluation of eight candidate genes for CCSD 45
(Basic Local Alignment Search Tool) search (httpwwwncbinlmnihgovBLAST)
using the human ESPN MYH14 MYO3A PRES TMC1 TMIE USH1C and WHRN
reference mRNA sequence
We compared the canine genomic DNA sequence from the eight candidate genes to
canine cDNA fragments in the canine EST database using the BLASTN program
For the localization of the exonintron boundaries canine or alternatively human
mRNA sequences were used for the mRNA-to-genomic alignment program Spidey
(httpwwwncbinlmnihgovIEBResearchOstellSpideyindexhtml) The human
and canine mRNA sequences which were used to determine the exon organization of
the candidate genes are given in Table 2
For each of the eight candidate genes we designed intragenic primer pairs to amplifly
intronic sequences yielding products with a length of 560 to 670 bp PCR primers
were developed with the Primer3 program (httpfrodowimiteducgi-
binprimer3primer3_wwwcgi) The PCR reactions were performed in a total of 50 microl
containing 125 microM dNTPs 25 pmol of each primer the reaction buffer supplied by
the manufacturer (Qbiogene Heidelberg Germany) and 1 U Taq polymerase After
a 4 min initial denaturation at 95degC 35 cycles of 30 sec at 94degC 45 sec at 58degC and
80 sec at 72degC were performed in a MJ Research thermocycler (Biozym Hessisch
Oldendorf Germany) The obtained PCR products were directly sequenced with the
DYEnamic ET Terminator kit (AmershamBiosciences Freiburg Germany) and a
MegaBACE 1000 capillary sequencer (AmershamBiosciences) using the PCR
primers as sequencing primers Sequence data were analyzed with Sequencher 42
(GeneCodes Ann Arbor MI)
In the first step sequence analyses were performed for PCR products of the parents
of four full-sib families If a heterozygous SNP was found for one or both parents all
progeny of the respective families were analyzed for that SNP Additionally to the
SNPs we used two microsatellite markers for linkage analyses We identified one
intragenic microsatelllite within the TMC1 gene As we could develop only one SNP
for the TMIE gene we additionally genotyped one microsatellite marker derived from
the NCBI database (httpwebncbinlmnihgov) located very closely to the TMIE
gene (Table 3)
Evaluation of eight candidate genes for CCSD
46
SNP marker analysis
A total of 21 SNPs were identified within ESPN MYH14 MYO3A PRES TMC1
TMIE USH1C and WHRN (Table 3) and used for linkage analysis Of all SNPs six
out of the observed 21 SNPs were polymorphic in all four examined families Out of
the 21 SNPs eleven SNPs were present in family 1 18 SNPs were heterozygous for
one or both parents in family 2 and 15 SNPs could be used for linkage analysis in
family 3 and 4 respectively (Table 4)
The most frequent form of SNPs with a frequency of 238 was the CT transition
motif The scarcest one was the CG transversion motif with a frequency of 48
respectively
Linkage and haplotype analysis Multipoint linkage and haplotype analysis were performed using MERLIN version
0102 (multipoint engine for rapid likelihood inference Center for Statistical Genetics
University of Michigan MI USA Abecasis et al 2002) The test statistics Z-mean
and Lod score were used to test for the proportion of alleles shared by affected
individuals identical by descent (IBD) for the considered marker loci The data of the
genotypes was additionally computed using the software package SAS Genetics
(Statistical Analysis System Version 913 SAS Institute Inc Cary NC USA 2005)
to specify the observed heterozygocity values (HET) and the polymorphism
information content (PIC)
Furthermore linkage disequilibrium (LD) and association of haplotypes with CCSD
was tested using the procedures CASECONTROL and HAPLOTYPE of
SASGenetics (Statistical Analysis System version 913 Cary NC USA)
Results and discussion The non-parametric multipoint linkage analysis in four full-sib families did not show
significant test statistics The highest Z-mean value was 046 the highest LOD Score
was 008 while the error probabilities ranged from 03 to 08 (Table 5) The maximum
achievable Z-mean was 448 and the corresponding value for the LOD score was
160 Marker-trait association tests for haplotypes of the candidate gene markers
were not significant Obviously no haplotype was associated with CCSD in these
Dalmatian dog families as demonstrated (Figure 1 2 3 4 5 6 7 and 8) The
Evaluation of eight candidate genes for CCSD 47
paternal and maternal haplotypes as far as they could be estimated were distributed
among the affected progeny closely to the expected proportion of 50 and thus no
excess of a certain haplotype could be observed in the affected dogs
Due to the fact that both animals with unilateral or bilaterally hearing loss and
bilateral hearing animals shared identical haplotypes it is unlikely that the ESPN
MYH14 MYO3A PRES TMC1 TMIE USH1C and WHRN genes are involved in the
pathogenesis of CCSD in Dalmatian dogs However the ESPN MYH14 MYO3A
PRES TMC1 TMIE USH1C and WHRN gene-associated marker may serve for
further linkage studies in other Dalmatian dog populations and dog breeds other than
Dalmatians
Evaluation of eight candidate genes for CCSD
48
Table 1 Details of the six selected human candidate genes
Gene symbol
Gene name Locus name1 Most important reference
ESPN espin DFNB36 Naz et al (2004)
MYH14 myosin heavy polypeptide 14 DFNA4 Donaudy et al (2004)
MYO3A myosin IIIA DFNB30 Walsh et al (2002)
PRES solute carrier family 26
member 5 (prestin) DFNB61
Zheng et al (2000) Liu et
al (2003)
TMC1 transmembrane channel-like
gene 1
DFNB7
DFNA36 Kurima et al (2002)
TMIE transmembran inner ear DFNB6 Naz et al 2002
USH1C Usher syndrome 1C DFNB18 Verpy et al (2000) Ouyang
et al (2002)
WHRN whirlin DFNB31 Mburu et al (2003)
1 Autosomal recessive loci are designated DFNB autosomal dominant loci DFNA
Evaluation of eight candidate genes for CCSD 49
Table 2 Selected human candidate genes with their location on HSA and CFA and
corresponding accession numbers
Gene symbol Gene
location on HSA1
Acc No 3 human mRNA
Gene location
on CFA2
Acc No 3 canine
genomic sequence
Acc No 3 canine mRNA
ESPN 1 NM_031475 5 NC_006587 XM_546751
MYH14 19 NM_024729 1 NW_876270 -
MYO3A 10 NM_017433 2 NC_006584 XM_544234
PRES 7 NM_206883 18 NC_006600 XM_540393
TMC1 9 NM_138691 1 NC_006583 XM_541284
TMIE 3 NM_147196 20 NC_006602 XM_846596
USH1C 11 NM_153676 21 NC_006603 XM_860072
WHRN 9 NM_015404 11 NC_006593 XM_850321
1 Homo sapiens autosome 2 Canis familiaris autosome 3 Accession number
Evaluation of eight candidate genes for CCSD
50
Table 3 The 21 newly developed intragenic SNPs and two microsatellite markers for
Dalmatian dogs in the candidate genes ESPN MYH14 MYO3A PRES TMC1
TMIE USH1C and WHRN with their corresponding primers the SNP motif the
product size and the annealing temperature the observed heterozygosity (HET) and
polymorphism information content (PIC)
SNP Primer F1 (5acute -gt 3acute) SNP motif primer R2 (5acute -gt 3acute)
Bp3 AT4 PIC ()
HET()
ESPN_SNP1
ACCAGCACCCTCTCCAACTA
AGGAATTCACAA (CT)CACACATACA
ACTCAAGCTCAGGGTGTGGT
565 60 9 10
ESPN_SNP2
ACCAGCACCCTCTCCAACTA
ATGGCTGGCGCT (AG)GAGGCTGCCC
ACTCAAGCTCAGGGTGTGGT
565 60 27 41
ESPN_SNP3
ACCAGCACCCTCTCCAACTA
ACACTCTTCCCA (CT)GGCTGGCGCT
ACTCAAGCTCAGGGTGTGGT
565 60 23 32
ESPN_SNP4
ACCAGCACCCTCTCCAACTA
TGGGAAGAGGGA (AG)GGGGGAGCAT
ACTCAAGCTCAGGGTGTGGT
565 60 23 32
ESPN_SNP5
ACCAGCACCCTCTCCAACTA
GAGTGGGCCAGG (CT)TGGGAAGAGG
ACTCAAGCTCAGGGTGTGGT
565 60 28 42
MYH14_SNP1
CTCTCCCCAACTCAGTTCCA
ACGTGTATTCGG (GT)CGCTTTTATT
GTGATAGGGACGAGCAGCAT
670 60 35 42
MYH14_SNP2
CATGGGACCGTTCCTACACT
AGCCTCGTTTAA (CT)CTAAAAGGAA
GCTCAATAGGCACGACATCA
640 60 34 39
Evaluation of eight candidate genes for CCSD 51
Table 3 (continued)
SNP Primer F1 (5acute -gt 3acute) SNP motif primer R2 (5acute -gt 3acute)
Bp3 AT4 PIC ()
HET()
MYO3A_SNP1
AATGCTTGAGTTTGGGATGC
GGCAGTCCCATG (GT)CCCTTATAAG
ACCTAATTGCCCAGATGCAG
650 60 37 77
MYO3A_SNP2
AATGCTTGAGTTTGGGATGC
GTGGAGAGCCAC (CG)TTGGGAGAGG
ACCTAATTGCCCAGATGCAG
650 60 37 70
MYO3A_SNP3
AACCTCCTGGCGTAGTATTCC
CATTACCTATTT (AT)GATCCTTATA
TTTTCCACTTCAGGCACACA
650 60 25 36
PRES_SNP1
CCCTTACCCCATACCATTCC
GATAGACTTCCT (AG)CCCTCAGACT
TTCAGGACAGCATCATCTGC
560 60 37 64
PRES_SNP2
CCCTTACCCCATACCATTCC
TGATGTCTGCTG (AT)TAACCCATTC
TTCAGGACAGCATCATCTGC
560 60 37 66
TMC1_SNP1
GCAACCTCTCGGTTTATCCA
CGTGAAGTGCCC (AT)TTGATGGAAA
AAGCTGGGGAAGTGGATATGT
610 60 57 37
TMC1_SNP2
GCAACCTCTCGGTTTATCCA
GGAGACATTACC (AG)TGAAGTGCCC
AAGCTGGGGAAGTGGATATGT
610 60 47 29
TMC1_SNP3
GGAAGCAAGACTGAGGTTGG
AGGCTTTTTAAA (AG)CTGTTCTGGG
CTGCTGCATTTGCCTGTAAG
650 60 48 30
TMIE_SNP1
AGAACACCACCGTCTCCTTG
CAAGGCGACGCC (AT)GTGCTGTCCT
GCCTCTGGTCAGAAGAGGTG
625 60 59 36
Evaluation of eight candidate genes for CCSD
52
Table 3 (continued)
SNP Primer F1 (5acute -gt 3acute) SNP motif primer R2 (5acute -gt 3acute)
Bp3 AT4 PIC()
HET()
USH1C_SNP2
CTCCCGGTCTGTCAGGAAC
GGCCTGGGGGGA (AC)AAGCGGACGG
ATGGCATCGACTTCTCCAAC
560 60 37 35
USH1C_SNP4
CTCCCGGTCTGTCAGGAAC
GGTCTCAGACCG (AC)GGCAGGGAGA
ATGGCATCGACTTCTCCAAC
560 60 37 37
WHRN_SNP1
TTCACCTCCAGGATCTGGTC
CCTGAGCCCGAG (CT)CCACGCTGCT
GGCTACTTTTCTTCCCCCTTT
600 60 25 37
WHRN_SNP2
TTCACCTCCAGGATCTGGTC
GGTCACGGGGGC (CT)CCGGGAGGTT
GGCTACTTTTCTTCCCCCTTT
600 59 24 33
WHRN_SNP3
TTCACCTCCAGGATCTGGTC
GTCCGAGTCCCG (AG)CCCCAGCCTG
GGCTACTTTTCTTCCCCCTTT
600 60 34 55
Microsatellite marker
Primers (forward reverse ) 5acute -gt 3acute Bp AT PIC()
HET()
TMC1_MS1
GCCCCCAGCTAAAAAGAGAA
TTCTCTTCCTCCCTCCTGTTC
220-220 60 76 57
FH2158 ATGGCCACATCACCCTAGTC
CTCTCTCTGCATCTCTCATGAA
274-302 58 57 66
Microsatllite marker derived from the NCBI database (httpwebncbinlmnihgov) 1 Forward 2 Reverse 3 Product size (basepairs) 4 Annealing temperature (degC)
Evaluation of eight candidate genes for CCSD 53
Table 4 The 21 newly developed intragenic SNPs for Dalmatian dogs in the
candidate genes ESPN MYH14 MYO3A PRES TMC1 TMIE USH1C and WHRN
with their nucleotide polymorphism allele and genotype frequencies
Gene symbol Fam1 Nucleotide polymorphism
Allele frequencies
Genotype frequencies2
ESPN_SNP1 4 CgtT 083017 840
ESPN_SNP2 2 3 4 AgtG 074026 15160
ESPN_SNP3 2 3 TgtC 068032 07120
ESPN_SNP4 2 3 GgtA 068032 07120
ESPN_SNP5 2 3 4 CgtT 074026 15160
MYH14_SNP1 2 3 4 GgtT 058041 51610
MYH14_SNP2 2 3 4 CgtT 058041 51610
MYO3A_SNP1 1 2 GgtT 062038 5111
MYO3A_SNP2 1 2 CgtG 062038 5111
MYO3A_SNP3 1 2 4 TgtA 076024 15140
PRES_SNP1 1 2 3 4 AgtG 058042 10254
PRES_SNP2 1 2 3 4 TgtA 058042 10254
TMC1_SNP1 1 2 3 4 AgtT 056044 11226
TMC1_SNP2 1 2 3 4 AgtG 076024 20190
TMC1_SNP3 1 2 3 4 AgtG 074026 19200
TMIE_SNP1 1 2 3 4 AgtT 058042 12216
USH1C_SNP2 1 3 4 AgtC 053047 9147
USH1C_SNP4 1 3 4 AgtC 053047 9147
WHRN_SNP1 2 CgtT 075025 360
WHRN_SNP2 2 CgtT 075025 360
WHRN_SNP3 2 3 4 InDel3 069031 13171 1 Number of families which were informative for the respective SNPs 2 Frequence of genotypes (first number number of animals homozygous for allele 1
second number heterozygous third number homozygous for allele 2) 3 Insertiondeletion of one base-pair (adenine)
Evaluation of eight candidate genes for CCSD
54
Table 5 Linkage analyses for families 1 2 3 and 4 for the 21 SNP and two
microsatellite markers within the eight candidate genes regarding Zmean LOD score
and error probabilities (p-values)
Gene symbol Marker Zmean pz-value1 LOD score pL-value2
ESPN ESPN_SNP1 014 04 002 04
ESPN_SNP2 014 04 002 04
ESPN_SNP3 014 04 002 04
ESPN_SNP4 014 04 002 04
ESPN_SNP5 014 04 002 04
MYH14 MYH14_SNP1 -089 08 -019 08
MYH14_SNP1 -089 08 -019 08
MYO3A MYO3A_SNP1 -049 07 -011 08
MYO3A_SNP2 -049 07 -011 08
MYO3A_SNP3 -049 07 -011 08
PRES PRES_SNP1 -094 08 -019 08
PRES_SNP2 -094 08 -019 08
TMC1 TMC1_SNP1 -034 06 -008 07
TMC1_SNP2 -034 06 -008 07
TMC1_SNP3 -034 06 -008 07
TMC1_MS1 -035 06 -008 07
TMIE TMIE_SNP1 013 04 003 03
FH2158 -056 07 -013 08
USH1C USH1C_SNP2 018 04 04 03
USH1C_SNP4 018 04 04 03
WHRN WHRN_SNP1 046 03 008 03
WHRN_SNP2 046 03 008 03
WHRN_SNP3 046 03 008 03 1 Error probability regarding Zmean 2 Error probability regarding LOD score
Microsatellite marker
Evaluation of eight candidate genes for CCSD 55
Figu
re 1
SN
P-h
aplo
type
s of
the
ES
PN
gen
e in
all
anal
yzed
Dal
mat
ian
dog
fam
ilies
The
hapl
otyp
es b
elon
ging
to F
amily
2 3
and
4 re
gard
ing
the
gene
ES
PN
sho
w n
o as
soci
atio
n w
ith th
e C
CS
D p
heno
type
In
the
thre
e fa
milie
s th
e ha
plot
ype
21
43
2 is
the
mos
t com
mon
one
bei
ng p
rese
nt in
72
o
f all
anim
als
It is
follo
wed
by
the
hapl
otyp
e 2
32
14
with
18
a
nd 4
34
34
with
6
Nor
mal
hea
ring
anim
als
as
wel
l as
thei
r de
af s
iblin
gs s
how
all
thes
e th
ree
hapl
otyp
es T
here
is n
o re
com
bina
tion
of th
e ha
plot
ypes
of t
he E
SP
N g
ene
in th
e th
ree
fam
ilies
Evaluation of eight candidate genes for CCSD 56
Figu
re 2
SN
P-h
aplo
type
s of
the
MY
H14
gen
e in
all
anal
yzed
Dal
mat
ian
dog
fam
ilies
Evaluation of eight candidate genes for CCSD 57
Figu
re 3
SN
P-h
aplo
type
s of
the
MY
O3A
gen
e in
all
anal
yzed
Dal
mat
ian
dog
fam
ilies
Evaluation of eight candidate genes for CCSD 58
Figu
re 4
SN
P-h
aplo
type
s of
the
PR
ES
gen
e in
all
anal
yzed
Dal
mat
ian
dog
fam
ilies
Evaluation of eight candidate genes for CCSD 59
Figu
re 5
SN
P- a
nd m
icro
sate
llite-
hapl
otyp
es o
f the
TM
C1
gene
in a
ll an
alyz
ed D
alm
atia
n
Evaluation of eight candidate genes for CCSD 60
Figu
re 6
SN
P- a
nd m
icro
sate
llite-
hapl
otyp
es o
f the
TM
IE g
ene
in a
ll an
alyz
ed D
alm
atia
n do
g fa
milie
s
Evaluation of eight candidate genes for CCSD 61
Figu
re 7
SN
P-h
aplo
type
s of
the
US
H1C
gen
e in
all
anal
yzed
Dal
mat
ian
dog
fam
ilies
Evaluation of eight candidate genes for CCSD 62
Figu
re 8
SN
P-h
aplo
type
s of
the
WH
RN
gen
e in
all
anal
yzed
Dal
mat
ian
dog
fam
ilies
Chapter 5
Molecular characterization of the canine myosin heavy polypeptide 9
non-muscle (MYH9) gene on dog chromosome 10q232
Canine MYH9 gene 65
Molecular characterization of the canine myosin heavy polypeptide 9 non-muscle (MYH9) gene on dog chromosome 10q232 Abstract A missense mutation of the Myosin heavy polypeptide 9 (MYH9) gene which
encodes the nonmuscle myosin heavy chain-A caused non-syndromic sensorineural
deafness in a human family which was characterized by cochleosaccular
degeneration In the present study we evaluated whether MYH9 gene mutations are
responsible for canine congenital sensorineural deafness (CCSD) in Dalmatian dogs
As described in Chapter 3 two MYH9 gene-associated microsatellites were
genotyped in 25 Dalmatian dog families segregating for CCSD We could find
significant linkage with CCSD for the microsatellite marker MYH9_MS3 (=FH2293)
We used data deposited in the NCBI to assemble the canine MYH9 gene DNA
sequence Characterization of the canine MYH9 gene revealed that the canine gene
consists of 41 exons spanning approximately 90 kb
We detected 22 single nucleotide polymorphisms (SNPs) in a mutation scan of
altogether 16 Dalmatian dogs from three families which showed significant linkage
between the deafness phenotype and the MYH9 gene-associated microsatellite
None of the SNPs affects the amino acid sequence of MYH9 We concluded that the
exons of the canine MYH9 gene do not play a role in Dalmatian CCSD Availability of
the microsatellite marker SNPs and DNA sequence reported in this study enhance
evaluation of the MYH9 gene as a CCSD candidate gene in other Dalmatian dog
populations and other dog breeds affected by CCSD
Introduction Myosin is a functional protein associated with cellular movement cell division muscle
contraction and other functions Members of the myosin super-family are
distinguished from the myosin heavy chains that play crucial roles in cellular
processes The human MYH9 gene consists of 40 exons and spans 67959 bp
Canine MYH9 gene
66
Toothaker et al (1991) mapped a nonmuscle myosin heavy chain gene to human
chromosome (HSA) 22q123-q131 Simons et al (1991) likewise mapped the gene
to HSA22q112 which they designated nonmuscle myosin heavy chain-A (NMMHC-
A)
The similarities between the autosomal dominant giant-platelet disorders May-
Hegglin anomaly Fechtner syndrome and Sebastian syndrome and refinement of the
disease loci for May-Hegglin anomaly and Fechtner syndrome to an overlapping
region of 480 kb on human chromosome 22 suggested that all these three disorders
may be allelic Among the identified candidate genes was the gene encoding
nonmuscle myosin heavy-chain A (MYH9) The May-HegglinFechtner Syndrome
Consortium (2000) demonstrated that mutations in MYH9 result in one of the three
disorders mentioned above The same Consortium also speculated that mutations in
MYH9 may also play a role in another autosomal dominant disorder a form of
nonsyndromic deafness characterized by progressive hearing impairment and
cochleosaccular degeneration This autosomal dominant form of human
nonsyndromic hereditary deafness (DFNA17) was described by Lalwani et al (1999)
They studied a five-generation American family previously reported by Lalwani et al
(1997) with deafness caused by cochleosaccular degeneration (CSD) CSD is the
most common histopathologic finding in cases of profound congenital deafness and
is estimated to occur in approximately 70 of cases in man
DFNA17 maps to the same region as MYH9 Because of the importance of myosins
in hearing Lalwani et al (2000) tested MYH9 as a candidate gene for DFNA17 and
demonstrated a missense mutation in the MYH9 gene in affected members of a
kindred with DFNA17 They found a G-to-A transition at nucleotide 2114 in the MYH9
gene This missense mutation changed codon 705 from an invariant arginine to a
histidine within a highly conserved Src homology 1 (SH1) linker region Previous
studies had shown that modification of amino acid residues within the SH1 helix
causes dysfunction of the ATPase activity of the motor domain in myosin
Many genetic disorders in humans and domestic dogs (Canis familiaris) demonstrate
a high level of clinical and molecular similarity Rak et al (2003) mapped 20 potential
candidate genes for sensorineural deafness in dogs by fluorescence in situ
hybridization and a radiation hybrid panel among them the MYH9 gene that was
assigned to canine chromosome (CFA) 10q232 Congenital sensorineural deafness
has been reported for approximately 54 different breeds of dogs (Strain 1996)
Canine MYH9 gene 67
The incidence of this congenital anomaly is highest in Dalmatian dogs of which 165-
30 exhibit unilateral or bilateral hearing loss (Famula et al 1996 Wood and
Lakhani 1997 Muhle et al 2002 Juraschko et al 2003a Rak and Distl 2005) The
inheritance and segregation of a major gene in CCSD has been demonstrated in
different Dalmatian dog populations (Famula et al 2000 Muhle et al 2002
Juraschko et al 2003b)
In dog breeds the histological pattern is known as cochleosaccular degeneration
commonly known as Scheibe dysplasia as it is described in approximately 70 of
cases with human hereditary deafness (Strain 1996) Therefore the MYH9 gene
seems to be a perfect candidate gene for CCSD especially in Dalmatian dogs
In this report we provide the genomic organization and the complete sequence of the
canine MYH9 gene A mutation analysis was performed to identify single nucleotide
polymorphisms (SNPs) in this gene In order to evaluate whether the MYH9 gene is
responsible for congenital sensorineural deafness in Dalmatian dogs we analyzed
the association of the MYH9 haplotypes with the CCSD phenotype in three families of
Dalmatian dogs with frequent occurrence of CCSD and significant linkage to the
gene-associated microsatellite MYH9_MS3 (Chapter 3)
Materials and Methods
Cloning and sequencing of canine MYH9 cDNA
The canine MYH9 gene sequence was derived from sequences deposited in the
current dog genome assembly (Boxer genome assembly 21) of the NCBI GenBank
(Genbank Acc No NW_1398701) by BLAST (Basic Local Alignment Search Tool)
search (httpwwwncbinlmnihgovBLAST) using the human MYH9 reference
mRNA sequence (Genbank Acc No NM_002473)
The distance between MYH9 and the microsatellite MYH9_MS2 is about 181 kb
whereas the microsatellite MYH9_MS3 (=FH2293) places about 511 kb from MYH9
The isolation of full length cDNAs was achieved by a modified rapid amplification of
cDNA ends (RACE) protocol Total RNA from dog lung of a normal female Beagle
(Biocat Heidelberg Germany) was used for the overlapping 5rsquo- and 3rsquo-RACE
products with the FirstChoiceTM RNA ligase-mediated (RLM)-RACE kit (Ambion
Europe Huntingdon UK) according to the protocols of the manufacturer Reverse
transcription polymerase chain reaction (RT-PCR) using two pairs of nested
Canine MYH9 gene
68
gene-specific and supplied 5rsquo-adaptor-specific primers were used to amplify the
complete 5rsquo-end of the investigated cDNA Similarly the 3rsquo-end were amplified using
two pairs of nested gene-specific primers in combination with supplied 3rsquo-adaptor-
specific primers Additionally RT-PCR using six pairs of gene-specific primers were
used to amplify the complete sequence of the investigated cDNAs
RACE products were cloned into pCRR21 vector using the Invitrogen TA cloning kit
(Invitrogen Karlsruhe Germany) Several clones for each cDNA were sequenced
with the ThermoSequenase kit (AmershamBiosciences Freiburg Germany) and a
LI-COR 4300 DNA Sequencer (LICOR inc Bad Homburg Germany) Sequence
data were analyzed with Sequencher 42 (GeneCodes Ann Arbor MI)
Sequence analysis of canine MYH9 gene For the localization of the exonintron boundaries after cloning and sequencing of full
length canine cDNAs (as described above) the mRNA-to-genomic alignment program
Spidey (httpwwwncbinlmnihgovIEBResearchOstellSpideyindexhtml) was
used Repetitive elements were analyzed with Repeatmasker 2
(httprepeatmaskergenomewashingtonedu) The GC content was calculated with
the EBI toolbox CpG PlotCpGreport (httpwwwebiacukToolssequencehtml)
Mutation analysis To identify polymorphisms within the canine MYH9 sequence exons with flanking
regions were PCR amplified and sequenced from 16 Dalmatian dogs which
represent three families consisting of three to six full sibs and at the best of both
parents At least two of the full sibs of each family were unilaterally deaf The
phenotype of the affected animals had been confirmed by brainstem auditory evoked
response (BAER) that detects electrical activity in the cochlea and auditory pathways
in the brain PCR primers and conditions for the amplification of MYH9 exons with
flanking sequences and microsatellite flanking primers for PCR reactions are given in
Table 1 PCR primers were developed with the Primer3 program
(httpfrodowimiteducgi-binprimer3primer3_wwwcgi) The PCR reactions for
exons and their flanking sequences were performed in a total of 50 microl containing 125
microM dNTPs 25 pmol of each primer the reaction buffer supplied by the manufacturer
(Qiagen Hilden Germany) and 1 U Taq polymerase After a 4 min initial
denaturation at 95 degC 35 cycles of 30 sec at 94 degC 45 sec at 58 degC and 80 sec
Canine MYH9 gene 69
at 72 degC were performed in a MJ Research thermocycler (Biozym Hessisch
Oldendorf Germany) The obtained PCR products were directly sequenced with the
DYEnamic ET Terminator kit (AmershamBiosciences Freiburg Germany) and a
MegaBACE 1000 capillary sequencer (AmershamBiosciences) using the PCR
primers as sequencing primers Sequence data were analyzed with Sequencher 42
(GeneCodes Ann Arbor MI)
Multipoint linkage and haplotype analysis were performed using MERLIN version
0102 (multipoint engine for rapid likelihood inference Center for Statistical Genetics
University of Michigan MI USA Abecasis et al 2002) The test statistics Z-mean
and Lod score were used to test for the proportion of alleles shared by affected
individuals identical by descent (IBD) for the considered marker loci
Linkage means that a haplotype characterized by microsatellites or SNPs is
significantly more often present in family members with the phenotype under study
than expected by random assortment For construction of haplotypes sets of closely
linked genetic markers on the same chromosome are needed which tend to be
inherited together as they are not likely to be separated by recombination
Association analysis can be carried out as a method of genetic analysis that
compares the frequency of alleles between affected and unaffected individuals
across all families A given allele is considered to be associated with the disease if
the presence of that allele explains a significant proportion of the phenotypic trait
variation
Results and Discussion
Analysis of the genomic organization and cDNA of the canine MYH9 gene
A full-length cDNA sequence of 7484 bp of the canine MYH9 gene was obtained by
using the RACE protocol The obtained RT-PCR products were sequenced and the
generated sequence data were submitted to the EMBL nucleotide database (Acc No
AM086385)
Using the RT-PCR analyses for cDNA-genomic sequence comparisons we detected
that the canine MYH9 gene consists of 41 exons with an untranslated exon 1 and
exonintron boundaries that conform perfectly to the GTAG rule (Table 2)
The 5rsquo-UTR consists of 181 bp while the 3rsquo-UTR consists of 1432 bp Assuming that
the homologous ATG start codon as in man is used the canine MYH9 cDNA
Canine MYH9 gene
70
contains an open reading frame of 5889 bp encoding a protein of 1963 amino acids
A canonical polyadenylation signal AAUAAA is located approximately 14 kb
downstream of the stop codon
The exon sizes range from 28 to 1556 bp the introns between these exons span
between 86 and 13493 bp the total size of the canine MYH9 gene is approximately
90 kb However the sequence homology between the human murine and canine
MYH9 gene is limited to the coding sequence of exon 2 to 41 The coding sequence
of canine MYH9 displays 926 and 899 similarities to the human and murine
MYH9 gene respectively In the untranslated regions the sequence similarity
between dog and human and mouse is rather low The canine MYH9 protein shows
982 and 968 identities to the orthologous human and murine protein
respectively (Fig 2)
The repeat content in the 90 kb region of the canine MYH9 gene is 1458 Most of
the repetitive elements belong to the SINE family (892) followed by the fraction of
the LINEs (306) other repetitive elements constitute 165 respectively The
entire canine MYH9 gene has an overall GC-content of 54 The canine MYH9 gene
contains 12 CpG islands (Gardiner-Garden and Frommer 1987) distributed over the
entire gene whereas by far the longest one with a length of 789 bp can be found in
the region of the second exon or in the first translated exon respectively (GC content
of 50 over 200 bp)
Mutation and haplotype analysis
All coding exons with flanking intronic regions of MYH9 could be amplified from the
examined 16 dogs and the sequences were compared to the Boxer genome
assembly 21 of the NCBI Gen Bank (Genbank Acc No NW_1398701)
The search for sequence variations within the MYH9 gene revealed a total of 22
SNPs shown in Table 3 Most of the polymorphisms were found in the flanking
regions of exons only 3 were within exons Only five out of the observed 22 SNPs
were polymorphic in all three examined families
None of the observed polymorphism did alter the predicted amino acid sequence of
MYH9 nor the identified hapltypes showed an association with the CCSD phenotype
Multipoint test statistics for MYH9 alleles identical by descent were close to zero (Z-
mean 035 to -035 Lod score 013 to -007) and not significant (p= 04 to 07)
However the microsatellite MYH9_MS3 (=FH2293) showed a Z-mean of 156
Canine MYH9 gene 71
(p= 006) and a Lod score of 058 (p= 005) indicating linkage The reason for this
result was heterogeneity among these three families For family 1 and 2 the test
statistics indicated linkage (Z-mean 191 with p= 003 Lod score 058 with p= 005)
whereas for familiy 3 the Z-mean and Lod score was -050 (p= 07) and -008 (p=
07)
Thus these silent point mutations found in affected and unaffected Dalmatian dogs
seem not responsible for the CCSD phenotype in these three families
Conclusions
The characterization of the transcript and genomic sequences of canine MYH9 gene
revealed a conserved organization with respect to the human orthologs In general
the gene size in dog is bigger compared to the human sequence due to the
untranlated first exon in the 5rsquo-UTR Several single nucleotide polymorphisms in the
canine MYH9 gene were identified However because of the fact that both animals
with unilateral hearing loss and bilateral hearing animals shared identical
haplotypes these polymorphisms are obviously not associated with CCSD in these
Dalmatian dog families Furthermore the present study revealed no functional
mutations of the complete coding region of MYH9 We can exclude the MYH9 gene
as a candidate gene for congenital sensorineunal deafness in Dalmatian dogs
However the MYH9 gene sequence SNPs and microsatellite markers reported in
this study enhance evaluation of the MYH9 gene in other Dalmatian dog populations
and dog breeds other than Dalmatians
Canine MYH9 gene
72
Table 1 PCR primers for the amplification of all MYH9 exons
Exon Primer Sequence (5rsquo ndash 3rsquo) Product
size (bp)
Annealing temperature
(degC) exon 1 MYH9_Ex1_F GCC TGG TTT GAC AAG TTT TG MYH9_Ex1_R TTC CTC AAG TCC CCT ACT CC 560 58 exon 2 MYH9_Ex2_F AGG ACC CTC ACC CAA CAC MYH9_Ex2_R GCA CAG AGT CTG GAC AAG AAT G 621 58 exon 3 MYH9_Ex3_F CAC CCC TCA ATA CTC AGT GC MYH9_Ex3_R GAG AAG ACT CCT GCC TCT CC 588 58 exon 4 MYH9_Ex4_F GGC AAT GTG TTT GAT TGT ACT G MYH9_Ex4_R TTT CCA AAG CAA CTC AGA AAG 542 58 exon 5 -6 MYH9_Ex56_F ACA GAG CAG GCG TGA CAG MYH9_Ex56_R GAG GAC AAG AGC TGG AAG C 724 58 exon 7 -8 MYH9_Ex78_F AAG CGG AAA GTT GCC TTC MYH9_Ex78_R ATG GCA CAA TGA CCA GTC TC 699 58 exon 9 MYH9_Ex9_F TGG GCA CAG TTT TTG TCT G MYH9_Ex9_R CAC ACA ATC AAG GGC TTT TC 590 58 exon 10 MYH9_Ex10_F GGA GCC AGT TGT CCT AGT AGT G MYH9_Ex10_R GCC AGG TGC CTA TCA CTG 540 58 exon 11 MYH9_Ex11_F TGT TTC GAA GCG TTT TTA CC MYH9_Ex11_R GGT CCC ATT GTT TCT AGC AC 551 58 exon 12 MYH9_Ex12_F TTT CCC AAG CAG CTA GAA AG MYH9_Ex12_R ACT GGG TGG TCT TGA GAC TG 586 58 exon 13 MYH9_Ex13_F CTG CCT TTG GTG ACA AGT G MYH9_Ex13_R GCA AGC CCT AAG TAC AAT GG 548 58 exon 14 MYH9_Ex14_F ATA CCA GGT CAA GGG GTG TC MYH9_Ex14_R TCA CCT GGT TCT AGG TGG AG 524 58 exon 15 MYH9_Ex15_F CGT TTG GAG TTG AT CCT AGC MYH9_Ex15_R AGC CAA GTT TCC TGA CTG TG 566 58 exon 16 MYH9_Ex16_F AGA TAG GCT GCA GTG GTA GC MYH9_Ex16_R CAG TAG CAA ACA GGG CTA GG 568 58 exon 17 MYH9_Ex17_F AAC GGT CCA ATG TGA GTT TC MYH9_Ex17_R ACC AGC TCA CAA CAG TAC CC 559 58 exon 18 MYH9_Ex18_F TGT AGG CTG TGA TTG CAG AG MYH9_Ex18_R CCC TCT CTC AAA CAA ATT GC 586 58 exon 19 MYH9_Ex19_F CCT TGG TGA AGG AAA GTG G MYH9_Ex19_R GCT GTG TGC AGG TTC TGA C 657 58 exon 20 MYH9_Ex20_F AGC AGC ATA GCA CTG AAT CC MYH9_Ex20_R TTC CCA TCT CTC ATG TTC TTG 579 58
Canine MYH9 gene 73
Table 1 (continued)
Exon Primer Sequence (5rsquo ndash 3rsquo) Product
size (bp)
Annealing temperature
(degC) exons 21 MYH9_Ex21_F CTC CTG AGT GTT GGG TTA GC MYH9_Ex21_R CTC TAA CAG GCA GCC CAA G 555 58 exon 22 MYH9_Ex22_F GGA GGA AAC GCA GAA ATC C MYH9_Ex22_R ACG GAC ACC CAG GAA AAC 660 58 exon 23 MYH9_Ex23_F CAG GGG TGC TAG TGA TCT TTC MYH9_Ex23_R AGA GCT GGG CTC AGA CCT AC 630 58 exon 24 MYH9_Ex24_F TCA GGG ATG TGC TTT CTT TC MYH9_Ex24_R CCC CAA GCA CAT CTG TAG TC 563 58 exon 24 MYH9_Ex24_F TCA GGG ATG TGC TTT CTT TC MYH9_Ex24_R CCC CAA GCA CAT CTG TAG TC 563 58 exon 25 -26 MYH9_Ex2526F GGC TGC TCT TCT CTG AAG C MYH9_Ex2526R GGA GTC AGG CTT CCA GTA AAG 851 58 exon 27 -28 MYH9_Ex2728F AGC ACA TTC TGG ATC TGG AG MYH9_Ex2728R GCG ACA GCT GAA TTC CTG 752 58 exon 29 MYH9_Ex29_F CAG CTT CAG GAC ACA CAG G MYH9_Ex29_R CTC TGG GTC TCC CCT GTC 601 58 exon 30 MYH9_Ex30_F GAG GCT TGG TGT ATG TCT CG MYH9_Ex30_R TGG TTC CAT AGA AAC GAT CTG 592 58 exon 31 - 32 MYH9_Ex3132F CAT TCC CCA AGG AGT TTT G MYH9_Ex3132R AAC ACA GCC CAG GGA AAG 770 58 exon 33 MYH9_Ex33_F CCT GGA ACA AGG CTG AGA AC MYH9_Ex33_R TCT CAG AGC AGC TTT GCT TC 600 58 exon 34 MYH9_Ex34_F GGT GTG CTT GGT AAA TCC TG MYH9_Ex34_R CAC AAA GCC TCA TTT CAC G 611 58 exon 34 - 36 MYH9_Ex3536F AGG TCA CGG TCT GGG TTC T MYH9_Ex3536R CAA GGG AGG CTG TTG CAG 401 60 exon 37 MYH9_Ex37_F CTC CCT TGG TTT CCC TCT CT MYH9_Ex37_R CAG GTC AGG AGG GGA CAT 488 60 exon 38 -39 MYH9_Ex3839F AGG CCT AAG GAC AGC TCA TC MYH9_Ex3839R CCA GGG CAC AGA TAG AAC C 545 58 exon 40 MYH9_Ex40_F1 GTC CCT CTA AAG ATG GCT TTG MYH9_Ex40_R1 TGG GGA GGA ACG ACT CTC 844 58 MYH9_Ex40_F2 TGG GTG ATT GGG GAA GAG MYH9_Ex40_R2 CCA GAA GAT GCA CAA AGT AGG 981 58
Canine MYH9 gene
74
Table 2 Exonintron boundaries of the canine MYH9 gene
3rsquo-Splice site Exon 5rsquo-Splice site
Intron phase
Intron size
-20 (exon 1 162 bp) GTTCAGgtgagcgagccccgc
gt30000 bp
-19 +333 gctgtagGTCCTGGCTGTGA (exon 2 352 bp) CGTAAgtggggcctggggcc
0
4922 bp
+334 +490 tctttagACCTATTCAGGCC (exon 3 157 bp) GCAAGgtgatgcctcggcctt
1
13493 bp
+491 +518 gtttcagACCGAGAGGACCA (exon 4 28 bp) TGCACgtaagtgtttctgcc
2
803 bp
+519 +612 tcggcagGGGTGAGTCTGGA (exon 5 94 bp) ACCAGgtgagtgcgggcctt
0
4077 bp
+613 +705 cttgtagGGTGAGTTGGAGA (exon 6 93 bp) GATTTgtgagtagcggccag
0
427 bp
+706 +769 tttttagGGCAAATTCATTC (exon 7 64 bp) GACTTgtatccttggttgat
1
738 bp
+770 +868 tgcaaagATCTTCTGGAGAA (exon 8 99 bp) GAAGAgtgagtagtgagccc
1
343 bp
+869 +1012 ctttcagCTGATCTCCTGTT (exon 9 144 bp) AATGGgtacagagctggccc
1
749 bp
+1013 +1108 cccgaagGCTTGTTGCGAGT (exon 10 96 bp) CACAGgtaacgcctgtggtcc
1
1041 bp
+1109 +1227 atcgcagCTGCCCAGAAAGT (exon 11 119 bp) AACAGgtaggctctctggcc
0
1877 bp
+1228 +1380 accccagGCTGACTTTGCCA (exon 12 153 bp) TTGATgtgagctcctctcct
0
922 bp
+1381 +1554 cctgcagCTGAACTCCTTCG (exon 13 174 bp) AGCCAgtaagagccagacct
0
1801 bp
+1555 +1728 ttcccagGCAGGGCCCCCTG (exon 14 174 bp) GCAAGgtgaggcaagcacgt
0
2049 bp
+1729 +1843 cttgcagGTGGACTACAAAG (exon 15 115 bp) GGATGgtacgtgccccctgg
1
1877 bp
+1844 +2037 tttgcagTGGACCGCATCAT (exon 16 194 bp) AGAAGgtgcgggtgctgggc
0
343 bp
+2038 +2159 tctccagGCTGGCAAACTGG (exon 17 122 bp) CAGAGgtgagcggggcctgt
1
835 bp
+2160 +2229 ccctcagATATGAGATCTTG (exon 18 70 bp) TCATGgtgagtagagctgct
2
1418 bp
+2230 +2390 cgtctagATCAAAGCCCTGG (exon 19 161 bp) AGGAAgtgagttcctaaagg
1
851 bp
+2391 +2499 tccccagGGCCTTTGCCAAG (exon 20 109 bp) CCAAGgtgggttctggaagg
2
1428 bp
+2500 +2631 cttctagGTCAAGCCACTGC (exon 21 132 bp) CCCAGgtaggtgtcagcagg
2
398 bp
+2632 +2838 ttggcagCTCATGGCAGAGA (exon 22 207 bp) TCCAAgtgagccccttccct
2
488 bp
Canine MYH9 gene 75
Table 2 (continued)
3rsquo-Splice site Exon 5rsquo-Splice site
Intron phase
Intron size
+2839 +2976 atggtagGAACTGGAGGAGC (exon 23 138 bp) CCAAGgtgaggcccgtggtg
2
971 bp
+2977 +3100 cccccagGAGAAGAAGCTTC (exon 24 124 bp) GGAAGgtaagagggtaacag
0
1603 bp
+3101 +3272 tcctcagAGCGCCTCCGAAG (exon 25 172 bp) GCCAGgtgagggcctcgctt
1
719 bp
+3273 +3485 ggcagagTGGAGGAGGAAGC (exon 26 213 bp) CTCAGgtgtgccatgcggcc
1
270 bp
+3486 +3630 tctccagGTCCAAACGCGAA (exon 27 145 bp) AACGGgtgcgtgggctcccc
2
480 bp
+3631 +3837 ccaccagGTGAAAGCCAACT (exon 28 207 bp) TGCAGgtgaggcctccatgg
2
232 bp
+3838 +3941 gccacagGTTGAGCTGGACA (exon 29 104 bp) ACACAgtaaaggtggcagcg
1
204 bp
+3942 +4094 ggtgtagGAGCTGCTCCAGG (exon 30 153 bp) CCCAGgttcttatcggcatg
1
1083 bp
+4095 +4343 tgcccagGTGACGGACATGA (exon 31 249 bp) ACCAGgtgtgggccccaggc
1
1298 bp
+4344 +4556 tccccagCTACTGGCCGAGG (exon 32 213 bp) AGAGCgtgagtaccatgtca
1
150 bp
+4557 +4769 gtggcagGTCCACGAGCTGG (exon 33 213 bp) GACAGgtgtggaggccctcc
1
303 bp
+4770 +4931 gccccagGTGCGGGAGATGG (exon 34 162 bp) TGCAGgtgaggtcggctggg
1
1173 bp
+4932 +5060 accccagGCCCAGATGAAGG (exon 35 129 bp) AGGAGgtgggccgggcctca
1
941 bp
+5061 +5149 atggcagGAACTGGCAGCTG (exon 36 89 bp) AAGGGgtgagccgtggcgtg
0
92 bp
+5150 +5273 cacccagAGCTCTGGCATTG (exon 37 124 bp) TGCAGgtgggcggggtcggg
2
224 bp
+5274 +5482 cccacagATCGACCAGATCA (exon 38 209 bp) ACCAAgtaggtgttcctgcc
1
563 bp
+5483 +5591 ctcgcagGGAGCGCCAGGCA (exon 39 109 bp) ACCAGgtgtgtgtgcacacc
2
86 bp
+5592 +5763 tctgcagGCCGACAAGGCAT (exon 40 172 bp) CTCAGgtgagccctgcctcc
0
739 bp
+5764 +7295 tgcaaagATCTTCTGGAGAA (exon 41 1532 bp) CGTCACAATAAAGAGAACCA
Exon sequences are shown in uppercase letters and intron sequences in lowercase
letters Untranslated regions are shown in italics The conserved GTAG exonintron
junctions are shown in boldface type For the last exon the polyadenylation signal is
shown underlined instead of an exonintron junction Position +1 corresponds to the
adenine of the translation initiation codon ATG
Canine MYH9 gene
76
16
TT
TC
G
A
TC
A
A C
A
CT
T
C
GC
G
G
AG
C
C
AG
G
A
CT
C
T
GA
A
G
CC
C
T
CC
T
C
96
96
229
23
7
15
TT
TC
G
A
TC
A
A C
A
CT
T
C
GC
G
G
AG
C
C
AG
G
A
CT
C
T
GA
A
G
CC
C
T
CC
T
C
96
96
229
22
9
14
deaf
T
T T
C
GA
T
C
AA
CA
C
T
TC
G
C
GG
A
G
CC
A
G
GA
C
T
CT
G
A
AG
C
C
CT
C
C
TC
96
96
22
9
229
13
deaf
T
T T
C
GA
T
C
AA
CA
C
T
TC
G
C
GG
A
G
CC
A
G
GA
C
T
CT
G
A
AG
C
C
CT
C
C
TC
96
96
22
9
229
12
deaf
T
T C
C
AA
CC
A
A A
A T
T C
C
CC
G
G
GG
C
C
GG
A
A C
C
CC
A
A G
G
CC
T
T C
C
CC
96
10
0 24
1
229
11
deaf
T
T C
C
AA
CC
A
A A
A T
T C
C
CC
G
G
GG
C
C
GG
A
A C
C
CC
A
A G
G
CC
T
T C
C
CC
96
10
0 24
1
229
10
TT
TC
G
A
TC
A
A C
A
CT
T
C
GC
G
G
AG
C
C
AG
G
A
CT
C
T
GA
A
G
CC
C
T
CC
T
C
96
96
237
22
9
Fam
ily 3
9 T
T C
C
AA
CC
A
A A
A T
T C
C
CC
G
G
GG
C
C
- - CC
C
C
AA
GG
C
C
TT
CC
C
C
96
100
229
24
1
8 T
T C
T
AG
C
T
AA
AA
TT
CC
C
C
GG
G
G
CT
G
G
AA
CC
C
C
AA
GG
C
C
TT
CC
C
C
96
96
225
25
3
7 de
af
TC
C
C
AG
C
T
AT
A
A T
T C
C
CC
G
A
GG
C
T
GG
A
G
CC
C
C
AA
GG
C
T
TC
C
T
CC
96
89
22
1
237
6 de
af
TC
C
C
AG
C
T
AT
A
A T
T C
C
CC
G
A
GG
C
T
GG
A
G
CC
C
C
AA
GG
C
T
TC
C
T
CC
96
89
22
1
237 Fa
mily
2
5 T
C
CC
A
G
CT
A
T
AA
TT
CC
C
C
GA
G
G
CT
G
G
AG
C
C
CC
A
A G
G
CT
T
C
CT
C
C
96
89
221
23
7
4 de
af
TT
CC
A
A C
C
AA
AA
TT
CC
C
C
GG
G
G
CC
G
G
AG
C
C
CC
A
A G
G
CC
T
T C
C
CC
96
96
23
7
257
3 de
af
TC
C
C
AG
C
T
AT
A
A T
T C
C
CC
G
A
GG
C
T
GG
G
A
CC
C
C
AA
GG
C
T
TC
C
T
CC
96
96
23
7
257
2 T
C
CC
A
G
CT
A
T
AA
TT
CC
C
C
GA
G
G
CT
G
G
AA
CC
C
C
AA
GG
C
T
TC
C
T
CC
96
96
23
7
257 Fa
mily
1
1 C
T
CC
G
A
TC
T
A A
A T
T C
C
CC
A
G
GG
T
C
GG
A
G
CC
C
C
AA
GG
T
C
CT
T
C
CC
96
96
25
7
257
Ref
eren
ce
se
quen
ce
NW
_139
870
T C
A T A A T C
C
G
G
C
G
A C
C
A G
C
T C
C
posi
tion1
212
9
1 4
4 2
39
125
1
32
316
3
78
42
218
1
8 1
75
24
13
8
207
2
63
287
9
22
40
59
64
Pos
ition
with
in M
YH
9 In
tron
34
Exo
n 4
Intro
n 14
15
Intro
n 15
16
Intro
n 16
17
Intro
n 18
19
Intro
n 20
21
Intro
n 20
21
Intro
n 25
26
Intro
n 25
26
Intro
n 26
27
Intro
n 27
28
Exo
n 29
In
tron
293
0 In
tron
303
1 E
xon
32
Intro
n 32
33
Intro
n 32
33
Intro
n 34
35
Intro
n 38
39
Intro
n 38
39
Intro
n 38
39
Mic
rosa
tellit
e M
YH
9_M
S2
Mic
rosa
tellit
e M
YH
9_M
S3
(=FH
2293
)
1 N
umbe
ring
refe
rs to
the
posi
tion
of th
e po
lym
orph
ic n
ucle
otid
e w
ithin
the
give
n ex
on
or in
tron
Tabl
e 3
Hap
loty
pes
of th
e ca
nine
MY
H9
gene
in16
Dal
mat
ian
dogs
Canine MYH9 gene 77
Figure 1 Family structure of the analyzed Dalmatian dog families Hearing status was tested by brain auditory evoked response (BAER)
Canine MYH9 gene
78
Figure 2 Alignment of the canine MYH9 protein with orthologous human and murine
MYH9 protein sequences The sequences where derived from GenBank entries with
the accessions NP_002464 (human MYH9) and NP_071855 (mouse MYH9)
Identical residues are indicated by asterisk beneath the alignment while dots and
colons represent similar or very similar amino acids respectively
dog MAQQAADKYLYVDKNFINNPLAQADWAAKKLVWVPSDKSGFEPASLKEEVGEEAIVELVE 60 human MAQQAADKYLYVDKNFINNPLAQADWAAKKLVWVPSDKSGFEPASLKEEVGEEAIVELVE 60 mouse MAQQAADKYLYVDKNFINNPLAQADWAAKKLVWVPSSKNGFEPASLKEEVGEEAIVELVE 60 dog NGKKVKVNKDDIQKMNPPKFSKVEDMAELTCLNEASVLHNLKERYYSGLIYTYSGLFCVV 120 human NGKKVKVNKDDIQKMNPPKFSKVEDMAELTCLNEASVLHNLKERYYSGLIYTYSGLFCVV 120 mouse NGKKVKVNKDDIQKMNPPKFSKVEDMAELTCLNEASVLHNLKERYYSGLIYTYSGLFCVV 120 dog INPYKNLPIYSEEIVEMYKGKKRHEMPPHIYAITDTAYRSMMQDREDQSILCTGESGAGK 180 human INPYKNLPIYSEEIVEMYKGKKRHEMPPHIYAITDTAYRSMMQDREDQSILCTGESGAGK 180 mouse INPYKNLPIYSEEIVEMYKGKKRHEMPPHIYAITDTAYRSMMQDREDQSILCTGESGAGK 180 dog TENTKKVIQYLAHVASSHKSKKDQGELERQLLQANPILEAFGNAKTVKNDNSSRFGKFIR 240 human TENTKKVIQYLAYVASSHKSKKDQGELERQLLQANPILEAFGNAKTVKNDNSSRFGKFIR 240 mouse TENTKKVIQYLAHVASSHKSKKDQGELERQLLQANPILEAFGNAKTVKNDNSSRFGKFIR 240 dog INFDVNGYIVGANIETYLLEKSRAIRQAKEERTFHIFYYLLSGAGEHLKTDLLLEPYNKY 300 human INFDVNGYIVGANIETYLLEKSRAIRQAKEERTFHIFYYLLSGAGEHLKTDLLLEPYNKY 300 mouse INFDVNGYIVGANIETYLLEKSRAIRQAKEERTFHIFYYLLSGAGEHLKTDLLLEPYNKY 300 dog RFLSNGHVTIPGQQDKDMFQETMEAMRIMGIPEEEQMGLLRVISGVLQLGNIVFKKERNT 360 human RFLSNGHVTIPGQQDKDMFQETMEAMRIMGIPEEEQMGLLRVISGVLQLGNIVFKKERNT 360 mouse RFLSNGHVTIPGQQDKDMFQETMEAMRIMGIPEDEQMGLLRVISGVLQLGNIAFKKERNT 360 dog DQASMPDNTGNASAQKVSHLLGINVTDFTRGILTPRIKVGRDYVQKAQTKEQADFAIEAL 420 human DQASMPDNT---AAQKVSHLLGINVTDFTRGILTPRIKVGRDYVQKAQTKEQADFAIEAL 417 mouse DQASMPDNT---AAQKVSHLLGINVTDFTRGILTPRIKVGRDYVQKAQTKEQADFAIEAL 417 dog AKATYERMFRWLVLRINKALDKTKRQGASFIGILDIAGFEIFDLNSFEQLCINYTNEKLQ 480 human AKATYERMFRWLVLRINKALDKTKRQGASFIGILDIAGFEIFDLNSFEQLCINYTNEKLQ 477 mouse AKATYERMFRWLVLRINKALDKTKRQGASFIGILDIAGFEIFDLNSFEQLCINYTNEKLQ 477 dog QLFNHTMFILEQEEYQREGIEWNFIDFGLDLQPCIDLIEKPAGPPGILALLDEECWFPKA 540 human QLFNHTMFILEQEEYQREGIEWNFIDFGLDLQPCIDLIEKPAGPPGILALLDEECWFPKA 537 mouse QLFNHTMFILEQEEYQREGIEWNFIDFGLDLQPCIDLIEKPAGPPGILALLDEECWFPKA 537 dog TDKSFVEKVVQEQGTHPKFQKPKQLKDKADFCIIHYAGKVDYKADEWLMKNMDPLNDNIA 600 human TDKSFVEKVMQEQGTHPKFQKPKQLKDKADFCIIHYAGKVDYKADEWLMKNMDPLNDNIA 597 mouse TDKSFVEKVVQEQGTHPKFQKPKQLKDKADFCIIHYAGKVDYKADEWLMKNMDPLNDNIA 597
Canine MYH9 gene 79
dog TLLHQSSDKFVSELWKDVDRIIGLDQVAGMSETALPGAFKTRKGMFRTVGQLYKEQLAKL 660 human TLLHQSSDKFVSELWKDVDRIIGLDQVAGMSETALPGAFKTRKGMFRTVGQLYKEQLAKL 657 mouse TLLHQSSDKFVSELWKDVDRIIGLDQVAGMSETALPGAFKTRKGMFRTVGQLYKEQLAKL 657 dog MATLRNTNPNFVRCIIPNHEKKAGKLDPHLVLDQLRCNGVLEGIRICRQGFPNRVVFQEF 720 human MATLRNTNPNFVRCIIPNHEKKAGKLDPHLVLDQLRCNGVLEGIRICRQGFPNRVVFQEF 717 mouse MATLRNTNPNFVRCIIPNHEKKAGKLDPHLVLDQLRCNGVLEGIRICRQGFPNRVVFQEF 717 dog RQRYEILTPNSIPKGFMDGKQACVLMIKALELDSNLYRIGQSKVFFRAGVLAHLEEERDL 780 human RQRYEILTPNSIPKGFMDGKQACVLMIKALELDSNLYRIGQSKVFFRAGVLAHLEEERDL 777 mouse RQRYEILTPNSIPKGFMDGKQACVLMIKALELDSNLYRIGQSKVFFRAGVLAHLEEERDL 777 dog KITDVIIGFQACCRGYLARKAFAKRQQQLTAMKVLQRNCAAYLKLRNWQWWRLFTKVKPL 840 human KITDVIIGFQACCRGYLARKAFAKRQQQLTAMKVLQRNCAAYLKLRNWQWWRLFTKVKPL 837 mouse KITDVIIGFQACCRGYLARKAFAKRQQQLTAMKVLQRNCAAYLRLRNWQWWRLFTKVKPL 837 dog LQVSRQEEEMMAKEEELVKVREKQLAAENRLTEMETLQSQLMAEKLQLQEQLQAETELCA 900 human LQVSRQEEEMMAKEEELVKVREKQLAAENRLTEMETLQSQLMAEKLQLQEQLQAETELCA 897 mouse LNSIRHEDELLAKEAELTKVREKHLAAENRLTEMETMQSQLMAEKLQLQEQLQAETELCA 897 dog EAEELRARLTAKKQELEEICHDLEARVEEEEERCQHLQAEKKKMQQNIQELEEQLEEEES 960 human EAEELRARLTAKKQELEEICHDLEARVEEEEERCQHLQAEKKKMQQNIQELEEQLEEEES 957 mouse EAEELRARLTAKKQELEEICHDLEARVEEEEERCQYLQAEKKKMQQNIQELEEQLEEEES 957 dog ARQKLQLEKVTTEAKLKKLEEDQIIMEDQNCKLAKEKKLLEDRIAEFTTNLMEEEEKSKS 1020 human ARQKLQLEKVTTEAKLKKLEEEQIILEDQNCKLAKEKKLLEDRIAEFTTNLTEEEEKSKS 1017 mouse ARQKLQLEKVTTEAKLKKLEEDQIIMEDQNCKLAKEKKLLEDRVAEFTTNLMEEEEKSKS 1017 dog LAKLKNKHEAMITDLEERLRREEKQRQELEKTRRKLEGDSTDLNDQIAELQAQIAELKMQ 1080 human LAKLKNKHEAMITDLEERLRREEKQRQELEKTRRKLEGDSTDLSDQIAELQAQIAELKMQ 1077 mouse LAKLKNKHEAMITDLEERLRREEKQRQELEKTRRKLEGDSTDLSDQIAELQAQIAELKMQ 1077 dog LAKKEEELQAALARVEEEATQKNMALKKIRELESQISELQEDLESERASRNKAEKQKRDL 1140 human LAKKEEELQAALARVEEEAAQKNMALKKIRELESQISELQEDLESERASRNKAEKQKRDL 1137 mouse LAKKEEELQAALARVEEEAAQKNMALKKIRELETQISELQEDLESERASRNKAEKQKRDL 1137 dog GEELEALKTELEDTLDSTAAQQELRSKREQEVNILKKTLEEEARTHEAQIQEMRQKHSQA 1200 human GEELEALKTELEDTLDSTAAQQELRSKREQEVNILKKTLEEEAKTHEAQIQEMRQKHSQA 1197 mouse GEELEALKTELEDTLDSTAAQQELRSKREQEVSILKKTLEDEAKTHEAQIQEMRQKHSQA 1197 dog VEELAEQLEQTKRVKANLEKAKQTLENERGELANEVKVLQQGKGDSEHKRKKAEAQLQEL 1260 human VEELAEQLEQTKRVKANLEKAKQTLENERGELANEVKVLLQGKGDSEHKRKKVEAQLQEL 1257 mouse VEELADQLEQTKRVKATLEKAKQTLENERGELANEVKALLQGKGDSEHKRKKVEAQLQEL 1257 dog QVKFTEGERVRTELADKVTKLQVELDNVMGLLTQSDSKSSKLTKDFSALESQLQDTQELL 1320 human QVKFNEGERVRTELADKVTKLQVELDNVTGLLSQSDSKSSKLTKDFSALESQLQDTQELL 1317 mouse QVKFSEGERVRTELADKVTKLQVELDSVTGLLSQSDSKSSKLTKDFSALESQLQDTQELL 1317
Canine MYH9 gene
80
dog QEENRQKLSLSTKLKQMEDEKNSFKEQLEEEEEAKRNLEKQIATLHAQVTDMKKKMEDGV 1380 human QEENRQKLSLSTKLKQVEDEKNSFREQLEEEEEAKHNLEKQIATLHAQVADMKKKMEDSV 1377 mouse QEENRQKLSLSTKLKQMEDEKNSFREQLEEEEEAKRNLEKQIATLHAQVTDMKKKMEDGV 1377 dog GCLETAEEAKRKLQKDLEGLGQRYEEKVAAYDKLEKTKTRLQQELDDLLVDLDHQRQTAS 1440 human GCLETAEEVKRKLQKDLEGLSQRHEEKVAAYDKLEKTKTRLQQELDDLLVDLDHQRQSAC 1437 mouse GCLETAEEAKRRLQKDLEGLSQRLEEKVAAYDKLEKTKTRLQQELDDLLVDLDHQRQSVS 1437 dog NLEKKQKKFDQLLAEEKTISAKYAEERDRAEAEAREKETKALSLARALEEAMEQKAELER 1500 human NLEKKQKKFDQLLAEEKTISAKYAEERDRAEAEAREKETKALSLARALEEAMEQKAELER 1497 mouse NLEKKQKKFDQLLAEEKTISAKYAEERDRAEAEAREKETKALSLARALEEAMEQKAELER 1497 dog LNKQFRTEMEDLMSSKDDVGKSVHELEKSKRALEQQVEEMKTQLEELEDELQATEDAKLR 1560 human LNKQFRTEMEDLMSSKDDVGKSVHELEKSKRALEQQVEEMKTQLEELEDELQATEDAKLR 1557 mouse LNKQFRTEMEDLMSSKDDVGKSVHELEKSKRALEQQVEEMKTQLEELEDELQATEDAKLR 1557 dog LEVNLQAMKAQFERDLQGRDEQSEEKKKQLVRQVREMEAELEDEKKQRSMAVAARKKLEM 1620 human LEVNLQAMKAQFERDLQGRDEQSEEKKKQLVRQVREMEAELEDERKQRSMAVAARKKLEM 1617 mouse LEVNLQAMKAQFERDLQGRDEQSEEKKKQLVRQVREMEAELEDERKQRSMAMAARKKLEM 1617 dog DLKDLEAHIDSANKNRDEAIKQLRKLQAQMKDCVRELDDTRASREEILAQAKENEKKMKS 1680 human DLKDLEAHIDSANKNRDEAIKQLRKLQAQMKDCMRELDDTRASREEILAQAKENEKKLKS 1677 mouse DLKDLEAHIDTANKNREEAIKQLRKLQAQMKDCMRELDDTRASREEILAQAKENEKKLKS 1677 dog MEAEMIQLQEELAAAERAKRQAQQERDELADEIANSSGKGALALEEKRRLEARIAQLEEE 1740 human MEAEMIQLQEELAAAERAKRQAQQERDELADEIANSSGKGALALEEKRRLEARIAQLEEE 1737 mouse MEAEMIQLQEELAAAERAKRQAQQERDELADEIANSSGKGALALEEKRRLEARIALLEEE 1737 dog LEEEQGNTELVNDRLKKANLQIDQINTDLNLERSHAQKNENARQQLERQNKELKVKLQEM 1800 human LEEEQGNTELINDRLKKANLQIDQINTDLNLERSHAQKNENARQQLERQNKELKVKLQEM 1797 mouse LEEEQGNTELINDRLKKANLQIDQINTDLNLERSHAQKNENARQQLERQNKELKAKLQEM 1797 dog EGTVKSKYKASITALEAKIAQLEEQLDNETKERQAACKQVRRAEKKLKDVLLQVDDERRN 1860 human EGTVKSKYKASITALEAKIAQLEEQLDNETKERQAACKQVRRTEKKLKDVLLQVDDERRN 1857 mouse ESAVKSKYKASIAALEAKIAQLEEQLDNETKERQAASKQVRRTEKKLKDVLLQVEDERRN 1857 dog AEQFKDQADKASTRLKQLKRQLEEAEEEAQRANASRRKLQRELEDATETADAMNREVSSL 1920 human AEQYKDQADKASTRLKQLKRQLEEAEEEAQRANASRRKLQRELEDATETADAMNREVSSL 1917 mouse AEQFKDQADKASTRLKQLKRQLEEAEEEAQRANASRRKLQRELEDATETADAMNREVSSL 1917 dog KNKLRRGDLPFVVPRRVARKGAGDCSDEEVDGKADGAEAKAAE 1963 human KNKLRRGDLPFVVPRRMARKGAGDGSDEEVDGKADGAEAKPAE 1960 mouse KNKLRRGDLPFVVTRRIVRKGTGDCSDEEVDGKADGADAKAAE 1960
Chapter 6
Identification of a 5 Mb region on canine chromosome 10
harbouring a causative gene responsible for congenital sensorineural deafness
in German Dalmatian dogs
Fine mapping of CFA10 83
Identification of a 5 Mb region on canine chromosome 10 harbouring a causative gene responsible for congenital sensorineural deafness in German Dalmatian dogs
Abstract
In the present study we evaluated whether the canine chromosome (CFA) 10
harbours a gene responsible for canine congenital sensorineural deafness (CCSD) in
Dalmatian dogs Altogether 27 microsatellite markers covering the whole CFA10
were genotyped in 176 Dalmatian dogs with frequent occurrence of CCSD
Significant linkage between the deafness phenotype and microsatellites located in a
region of 36 Mb to 48 Mb on CFA10 was found In order to refine the location of the
causative canine congenital sensorineural deafness (CCSD) gene we used data
deposited in the NCBI to search for single nucleotide polymorphisms (SNPs) in the
intronic sequences of the canine genes located on CFA10 in this region We
characterized 26 SNPs and used them for non-parametric linkage and association
analyses with CCSD of altogether 34 Dalmatian dogs from five full-sib families We
could find significant linkage to SNPs located in the region spanning 39 Mb to 44 Mb
and significant haplotype-trait association for SNPs in this region These results
enforce further evaluation of this 5 Mb region with the aim to detect the gene
responsible for CCSD in Dalmatian dogs
Introduction
Of the identified genes responsible for different forms of sensorineural non-syndromic
deafness in humans Rak et al (2003) and co-workers (Droumlgemuumlller et al 2002
Kuiper et al 2002 Rak et al 2002a 2002b 2003) considered 24 genes as
candidates for sensorineural deafness in dogs among them the MYH9 gene on
CFA10 Subsequently Rak (2003) developed a set of microsatellite markers for the
respective 24 candidate genes
As described in chapter 3 we could show significant linkage of CCSD with the MYH9
associated microsaellite MYH9_MS3 (=FH2293) in different German Dalmatian dog
Fine mapping of CFA10
84
families However as described in Chapter 5 we already excluded MYH9 for being
responsible for the CCSD phenotype in German Dalmatian dog families segregating
for CCSD by comparative sequencing of genomic sequences from deaf and normal
hearing Dalmatian dogs and additionally by sequencing of full length canine cDNA
The reason for the MYH9_MS3 (=FH2293) indicating linkage may be caused by a
closely linked gene involved in CCSD Thus the objective of the present study was to
perform a scan of canine chromosome 10 using microsatellite markers and single
nucleotide polymorphisms (SNPs) in order to evaluate whether CFA10 harbours a
gene responsible for the deafness phenotype in Dalmatian dogs Additionally we
analyzed the association of the CCSD phenotype with a large number of newly
developed SNPs located in the genomic deafness region on CFA10
Material and methods Pedigree material
For the linkage analysis of the microsatellite marker on CFA10 we used DNA from
altogether 176 animals belonging to 22 full-sib families and one large paternal half-
sib family of German Dalmatian dogs All families were segregating for CCSD The
genotyped families included all the affected dogs (unilaterally and bilaterally deaf)
their parents if available and one to four unaffected full-sibs At least two of the full
sibs of each family were unilaterally deaf The phenotype of the affected animals had
been confirmed by brainstem auditory evoked response (BAER) that detects
electrical activity in the cochlea and auditory pathways in the brain
Screening for SNPs was performed by comparative sequencing of DNA from parents
of five families with significant linkage of microsatellites located in the region
spanning 36 Mb to 48 Mb to CCSD For the linkage analysis concerning the SNPs we
then used blood samples from 34 Dalmatian dogs consisting of the progeny and their
parents of the abovementioned five full-sib families of Dalmatian dogs with frequent
occurrence of CCSD The families consisted of five to nine individuals and their
parents
Microsatellite marker analysis
We used 27 microsatellite marker derived from the NCBI database
(httpwebncbinlmnihgov) to cover CFA10 completely (Appendix Table 1)
Fine mapping of CFA10 85
PCR reactions for microsatellites were carried out in 12 microl reaction mixtures
containing 2 microl genomic DNA 12 microl 10x PCR buffer (Qbiogene Heidelberg
Germany) 024 microl dimethyl sulfoxide (DMSO) 02 microl dNTPs (100 microM) 01 microl Taq
Polymerase (5Umicrol) (Qbiogene) 06 microl (10 microM) 5rsquo-IRD700 or IRD800 labelled forward
primer 06 microl (10 microM) unlabelled reverse primer The PCR reactions were carried out
in MJ Research thermocyclers with the following program 4 min at 94 degC followed
by 32 cycles of 30 sec at 94degC 30 sec at maximum annealing temperature and a
final extension of 45 sec at 72degC PCR-products were diluted with formamide loading
buffer in ratios from 110 to 140 and then size-fractionated by gel electrophoresis on
automated LI-COR 42004300 sequencers (LI-COR inc Lincoln NE USA) using
denaturing 4 and 6 polyacrylamide gels (RotiphoreseregGel 40 Roth Karlsruhe
Germany)
Development of single nucleotide polymorphisms (SNPs)
We developed SNPs for the region between 36 Mb to 48 Mb on CFA10 previously
proven to be linked to the CCSD phenotype The SNPs for this region were derived
from one 5294 Mb large whole genome shotgun sequence (NW_876251) deposited
in the current dog genome assembly (Boxer genome assembly 21) of the NCBI
GenBank The canine genomic sequences and mRNA of the genes that were used
for the analysis were also derived from sequences deposited in the current dog
genome assembly (Boxer genome assembly 21) of the NCBI GenBank (Table 1)
In total 72 primer pairs were designed most of them located intragenic in intronic
sequences or alternatively in flanking regions of the 5rsquo- or 3rsquondashends of the respective
gene yielding products with a length of around 600 bp PCR primers were developed
with the Primer3 program (httpfrodowimiteducgi-binprimer3primer3_wwwcgi)
The PCR reactions were performed in a total of 50 microl containing 125 microM dNTPs 25
pmol of each primer the reaction buffer supplied by the manufacturer (Qiagen
Hilden Germany) and 1 U Taq polymerase After a 4 min initial denaturation at
95degC 35 cycles of 30 sec at 94degC 45 sec at 58degC and 80 sec at 72degC were
performed in a MJ Research thermocycler (Biozym Hessisch Oldendorf Germany)
The obtained PCR products were directly sequenced with the DYEnamic ET
Terminator kit (AmershamBiosciences Freiburg Germany) and a MegaBACE 1000
capillary sequencer (AmershamBiosciences) using the PCR primers as sequencing
primers Sequence data were analyzed with Sequencher 42
Fine mapping of CFA10
86
(GeneCodes Ann Arbor MI USA) If a heterozygous SNP was found for one or both
parents all progeny of the respective families were analyzed for that SNP
We developed altogether 26 SNPs with 20 SNPs located intragenic in intronic
sequences and 6 SNPs located very closely at the 5rsquo- or 3rsquo-ends respectively with
one to four SNPs per gene (Table 3)
The newly identified 26 SNPs are shown in Tabele 2 Only SNPs are listed and
chosen for linkage analyses that were heterozygous for one or both parents of at
least two of the five families Of all SNPs only one was heterozygous in all families
(Table 3) The most frequent form of SNPs with a frequency of 423 was the AG
transition motif The scarcest one with a frequency of 385 was the CG and the
AC transversion motif respectively
Linkage analysis
Multipoint non-parametric linkage and haplotype analysis were performed using the
MERLIN software version 0102 (multipoint engine for rapid likelihood inference
Center for Statistical Genetics University of Michigan MI USA Abecasis et al
2002) The test statistics Z-mean and Lod score were used to test for the proportion
of alleles shared by affected individuals identical by descent (IBD) for the considered
marker loci
In a first step linkage analysis was performed regarding the 27 marker covering the
whole CFA10 In a second step a linkage analysis was performed including the newly
added SNPs spanning the region 36 Mb to 48 Mb on CFA10
The observed heterozygosity (HET) and the polymorphism information content (PIC)
were calculated using the software package SASGenetics (Statistical Analysis
System Version 913 SAS Institute Inc Cary NC USA 2005)
Linkage disequilibrium (LD) and association of haplotypes with CCSD were tested
using the procedures CASECONTROL and HAPLOTYPE of SASGenetics
(Statistical Analysis System version 913 Cary NC USA)
Results
A linkage analysis was at first carried out for the 176 animals that were analyzed with
27 microsatellite markers covering the whole CFA10
Fine mapping of CFA10 87
The microsatellite FH3302 positioned at 3887 Mb showed the highest Zmean with a
value of 234 (p= 001) and a Lod score of 121 (p= 0009) at 3887 Mb A Zmean
value of 217 (p= 0015) and a Lod score of 12 (p= 0009) was reached for the
microsatellite REN181G20 positioned at 3936 Mb indicating linkage but all Zmean
values and error probabilities of eight microsatellite markers in the interval from 3666
Mb up to 488 Mb were almost as high Consequently we screened the region
spanning from 36 Mb to 48 Mb for SNPs Out of the 23 analyzed Dalmatian dog
families five full-sib families were chosen to screen for SNPs because of their
significant contribution to the test statistics
Twenty-six new SNP markers covering the region of 36 Mb to 48 Mb equally were
developed and added to the linkage analysis These SNPs had PIC values ranging
from 013 to 037
Figure 1 shows the Zmeans and LOD scores for all 27 microsatellite markers on
CFA10 harbouring a genomic deafness region Figure 2 shows the corresponding p-
values Adding the SNPs in the interval spanning 36 Mb to 48 Mb we could narrow
the significant region down to 5 Mb spanning 39 Mb to 44 Mb (Table 4 Figures 3 and
4)
Haplotype-trait association test statistics for the SNPs 16-23 located in the interval
from 39 Mb to 44 Mb were significant However the marker-trait association test
failed the 5 threshold of the error probability (p= 007) but was lowest for all
possible haplotype-trait combinations The χ2ndashtests of the procedure
CASECONTROL were not significant indicating that a SNP for the causative
deafness gene was not yet found
Discussion
We used 27 microsatellite marker and 26 newly developed SNP markers with the
intention to create a dense map for linkage analysis of CFA10 especially the region
spanning 36 Mb to 48 Mb proven to be linked to the CCSD phenotype All SNP
markers were chosen due to their heterozygosity in one or both parents of at least
two families
The significant Zmeans on CFA10 reported for the chromosome scan using only
microsatellites was confirmed by adding the SNP markers Furthermore with the use
of SNPs the apparently linked region from 36 Mb to 48 Mb could be narrowed down
Fine mapping of CFA10
88
to 5 Mb spanning 39 Mb to 44 Mb For this reason the use of SNPs in addition to
informative microsatellites appears highly recommendable
The identified CCSD region spanning 5 Mb might be participating in the development
of CCSD in the analyzed Dalmatian dog families
However genes that are known to be involved in the hearing process or known to be
expressed in the inner ear are not annotated at the syntenic human region on homo
sapiens autosome (HSA) 2 spanning from 100 Mb to 108 Mb Therefore a well-
defined candidate gene for canine hereditary deafness in the abovementioned 5 Mb
does not exist This means that for all genes in this region informative SNPs have to
be developed and tested for linkage disequilibrium and association with CCSD
Despite the remarkable progress that has been made in the past few years in
identifying deafness genes in man and mouse it is suspected that only one-third of all
deafness causing genes have been identified so far Therefore more SNPs have to
be developed within the identified region on CFA10 to localize the deafness causing
gene or to find unambiguously associated SNP markers which could be used for a
population-wide genetic test for CCSD
Fine mapping of CFA10 89
Table 1 Canine genes where gene-associated SNPs could be developed with their
exact location on CFA10 and their corresponding accession numbers (Acc No)
Gene symbol Gene description Position in
Mb
Acc No canine genomic sequence
Acc No canine mRNA
LOC474535 similar to hypothetical protein MGC40397 36493650 NC_006592 XM_531764
LOC474536 similar to KM-102-derived reductase-like factor
37023714 NC_006592 XM_531765
LOC610953 similar to NHP2-like protein 1 37293729 NC_006592 XM_848546
LOC481302 similar to C2orf26 protein 37363737 NC_006592 XM_848552
LOC610991 hypothetical protein LOC610991 38103820 NC_006592 XM_848591
LOC611007 similar to eukaryotic translation elongation factor 1 alpha 2
38353835 NC_006592 XM_848614
LOC474541 similar to GRIP coiled-coil protein GCC185 isoform a
38503857 NC_006592 XM_531770
LOC474542 similar to Sulfotransferase 1C2 38613862 NC_006592 XM_531771
LOC481308 similar to Keratin type I cytoskeletal 18 38653865 NC_006592 XM_538429
LOC474543 similar to Sulfotransferase K1 38683870 NC_006592 XM_857994
LOC609217 similar to family with sequence similarity 32 member A like
39453945 NC_006592 XM_858065
LOC611115 similar to D-PCa-2 protein isoform c 39843984 NC_006592 XM_848756
LOC481325 similar to ubiquitin-conjugating enzyme E2C
42564272 NC_006592 XM_538446
LOC481330 similar to Interleukin-1 receptor type II precursor
44084413 NC_006592 XM_538451
LOC611493 hypothetical protein LOC611493 44384440 NC_006592 XM_863243
LOC481337 similar to DNA repair protein REV1
46264637 NC_006592 XM_538458
LOC611728 similar to 3-hydroxyanthranilate 34-dioxygenase
48494852 NC_006592 XM_849433
Predicted gene derived from the dog genome assembly (build 21) that used gene
prediction method GNOMON supported by EST evidence
Fine mapping of CFA10
90
Table 2 The 26 newly developed SNPs for Dalmatian dogs located in the region
spanning 36 Mb to 48 Mb on CFA10 with their corresponding primers the SNP motif
the product size and the annealing temperature
Gene
description SNP
Location
(intron or
5rsquo3rsquo-end)
Primer F1 (5acute -gt 3acute)
SNP motif
Primer R2 (5acute -gt 3acute)
Bp3 AT4
LOC474535
SNP_1
intron
ACCCAAGCCTAACTGCAGAA
ACCCCAGTCT(CG)GCCAGAGCTGTT
590 60
SNP_2 intron TGTGTGCATA(AG)TGTGCTGAGCCT
TCATCTGTTAAAACAGGGGTGAT
LOC474536
SNP_3
intron
CCAGTTAATGATTGTTTCGTTGA
AAGCTGCTTT(AC)CACCCCCATCAG
TCATTCCTGCTGTTGTGCTC
590 60
LOC610953
SNP_4
intron
CTGTCTTGGGGACTGTTTGC
AAGGCAGACG(CT)AATGACTGAGGC
600 60
SNP_5 intron GACTTGAAGC(CT)ACCACAGATACT
GCCATCACGATGAACTCAGA
LOC481302
SNP_6
3rsquo-end
AATTGAGGCCGAAGTCCAAT
CTTTTCCCCA(GT)GCCACCCCTCTG
GAGCACTATTTACGATACAAACAGGA
610 60
LOC610991
SNP_7
intron
CATGCATGATGCCCAGAGTA
CCCAAAGCAC(AG)CTGTGATTTAAT
AGGGCTTCCTGGGAAAAGT
600 60
LOC611007
SNP_8
intron
CAGACCAACAGTGACCCAGA
TAGGCATACC(GT)TCAGTCCTAAAG
GCCTGTTGTGGGCAGAGTAT
480 60
LOC474541
SNP_9
intron
ACTGAGCCAAAGGTGGATTG
AGAGAATAGC(AG)CTGTGTTTTACA
ACCTGCACATCGGGATTTAG
575 60
Fine mapping of CFA10 91
Table 2 (continued)
Gene-
symbol SNP
Location
(intron or
5rsquo3rsquo-end)
Primer F1 (5acute -gt 3acute)
SNP motif
Primer R2 (5acute -gt 3acute)
Bp3 AT4
LOC474542
SNP_10
intron
CTTCCCCAGGAGAGAGTGAC
AATATGATCA(CT)ATTTAAAGAAAT
CTTTTGTCAACATCCCCTTCA
560 60
LOC481308
SNP_11
3rsquo-end
ACCCATTGTCTCTCCAGCAC
CCACATAACT(GT)AGCATCCCTAGC
600 60
TGATGATGTAAGTTGGCCTCA
LOC474543
SNP_12
intron
TTGAAGTTGTGTGAGTAAATGAAAGA
CAATATATAA(AG)CATTTGCTACAA 600 60
SNP_13 intron TTGAGATGAA(AT)ATAATAGAGCTG
SNP_14 intron CGATTTTCCA(AG)GACATGGAGATG
SNP_15 intron ACTGAACCAA(AG)TGTGCAAAGCAT
GGAAACCATGCAGTCTTTGG
LOC609217
SNP_16
5rsquo-end
TGGCCTCATTTTCCAGTATG
GAAGGAGTTA(CT)ACAGTGAAGATA
580 60
SNP_17 5rsquo-end AAGTGATCAA(AG)CCAGTGCATTCT
GGCAATTACCCTGAGTGGTG
LOC611115
SNP_18
3rsquo-end
GGGCTGTCTTAGAGGTGCTG
TGTGGTCTCA(CT)ACACTTCCTGAG
590 60
SNP_19 3rsquo-end ATTGCAAGTG(GT)CCTGTGATGGCC
CTTCTTTGGGCAGGAAAGTG
LOC481325
SNP_20
intron
AAAATGATTGATCGCAAAAGAAA
AATTACTGTA(CT)AACAGTATCAGA
600 60
SNP_21 intron TAACAGTATC(AG)GAATAAAAGTTT
TTCTGTGATTGCACTGACCG
Fine mapping of CFA10
92
Table 2 (continued)
Gene-
symbol SNP
Location
(intron
or 5rsquo3rsquo-
end)
Primer F1 (5acute -gt 3acute)
SNP motif
Primer R2 (5acute -gt 3acute)
Bp3 AT4
LOC481330
SNP_22
intron
GAAAGGCCTGGGTTCAAAA
GGCAGGGAGG(AG)TCACCATCGTTC
AATTTCCCCAAATGCCTCAC
575 60
LOC611493
SNP_23
intron
GCATGAAGGAGCCCTATGTC
CCAAGAGTCC(AT)GCCCAACACCCT
GGAGGGATGGCATTCTATGA
590 60
LOC481337
SNP_24
intron
GGCTGAGGAGATTGTGTTTCA
GCTGATATTT(AG)GCCTTCTGAGAT
620 60
SNP_25 intron AAAGCCAGAC(AG)CTTTGCCATGGT
CAGCTCCCTGTAATGGGAAA
LOC611728
SNP_26
intron
TCCTACTCCCATCACTTCCAA
CCACACTGGG(GT)CCTGGGATGAGG
CACAGCTCCATGTAGGTCCA
620 60
hypothetical gene found in the 2 version 1 of the NCBIs genome annotation 1 Forward 2 Reverse 3 Product size (basepairs) 4 Annealing temperature (degC)
Fine mapping of CFA10 93
Table 3 The 26 newly developed intragenic SNPs for Dalmatian dogs with their
nucleotide polymorphism allele and genotype frequencies observed heterozygosity
(HET) and polymorphism information content (PIC)
SNP Fam1 Nucleotide
polymorphism
Allele
frequencies
Genotype
frequencies2 PIC HET
SNP_1 125 CgtG 042054 3135 036 055
SNP_2 124 AgtG 064039 91013 037 044
SNP_3 2345 AgtC 041049 2156 033 047
SNP_4 345 CgtT 065035 6140 035 067
SNP_5 34 CgtT 065035 490 028 042
SNP_6 345 CgtT 066034 6130 035 039
SNP_7 1345 AgtG 054046 6174 037 053
SNP_8 145 GgtT 075025 01010 029 045
SNP_9 1345 AgtG 052054 6145 037 047
SNP_10 1234 CgtT 057043 7173 037 068
SNP_11 14 GgtT 065035 5120 035 062
SNP_12 15 AgtG 028072 097 030 044
SNP_13 45 AgtT 077023 870 017 021
SNP_14 5 AgtG 036064 052 013 015
SNP_15 134 AgtG 034066 0157 034 059
SNP_16 145 CgtT 030070 3812 033 036
SNP_17 245 AgtG 058042 6113 030 032
SNP_18 123 CgtT 037063 1126 027 035
SNP_19 123 GgtT 045055 3115 030 032
SNP_20 2345 CgtT 032068 1129 029 039
SNP_21 1235 AgtG 063037 883 037 052
SNP_22 1234 AgtG 068032 10102 030 034
SNP_23 12345 AgtT 030070 21313 033 046
SNP_24 124 AgtG 047053 667 033 019
SNP_25 234 AgtG 053047 3132 035 053
SNP_26 234 GgtT 060040 3120 026 0391 Number of families which were informative for the respective SNPs 2 Genotypes are given as number of animals [homozygous for allele 1heterozygous
homozygous for allele 2]
Fine mapping of CFA10
94
Table 4 Linkage analysis for canine chromosome 10 regarding the region spanning
39 Mb to 44 Mb with the significant Zmean LOD score and error probabilities (p-
values)
Marker Location (Mb) Zmean pz-value1 LOD-score pL-value2
SNP_16 39453 262 0004 123 0009
SNP_17 39455 262 0004 123 0009
SNP_18 39840 261 0004 123 0009
SNP_19 39843 261 0004 123 0009
SNP_20 4260 255 0005 118 0010
SNP_21 4270 255 0005 117 0010
SNP_22 4405 317 00008 131 0007
SNP_23 4439 337 00004 134 0007 1 Error probability regarding Zmean 2 Error probability regarding LOD score
Fine mapping of CFA10 95
Figure 1 Zmeans and LOD scores for 27 microsatellite markers on CFA10
harbouring a congenital sensorineural deafness region (number of families 23
number of genotyped dogs 176)
Figure 2 P-values for Zmeans and LOD scores of 27 microsatellite markers on
CFA10 harbouring a congenital sensorineural deafness region (number of families
23 number of genotyped dogs 176)
Fine mapping of CFA10
96
Figure 3 Zmeans and LOD scores of 53 markers on CFA10 harbouring a congenital
sensorineural deafness region (number of families 5 number of genotyped dogs 34)
Figure 4 P-values for Zmeans and LOD scores of 53 markers on CFA10 for the
region between 30 to 60 Mb harbouring a congenital sensorineural deafness region
(number of families 5 number of genotyped dogs 34)
CCSD region (SNP 16-23)
Chapter 7
Analysis of canine chromosome 1 and the Gap junction protein alpha 1 (GJA1) gene
in Dalmatian dogs segregating for congenital sensorineural deafness
CFA1 and the GJA1 gene 99
Analysis of canine chromosome 1 and the Gap junction protein alpha 1 (GJA1) gene in Dalmatian dogs segregating for congenital sensorineural deafness Abstract Two microsatellite markers associated to the GJA1 (alternatively connexin 43) gene
showed significant linkage with canine congenital deafness (CCSD) in a large French
half-sib family (Chapter 3) PCR products of this half-sib family were used to perform
a sequence analysis in order to evaluate whether GJA1 is responsible for CCSD As
the linkage could not be confirmed we used altogether 27 microsatellite markers for
a scan of canine chromosome (CFA) 1 with 23 Dalmatian dog families segregating
for CCSD A non-parametric linkage analysis was performed to see whether
significant test statistics for other genomic regions on CFA1 and for more families can
be shown As a result we could not find linkage to any microsatellite in the analyzed
families
Introduction Rak (2003) considered in total 24 genes as candidates for sensorineural deafness in
dogs among them the Gap junction protein alpha 1 (GJA1) or connexin 43 gene on
canine chromosome (CFA) 1 A marker set of altogether 43 microsatellites were
developed by Rak (2003) among them two microsatellite marker associated to the
GJA1 gene
GJA1 or connexin 43 is a member of the connexin gene family and a component of
gap junctions Mutations in 4 members of the connexin gene family have been shown
to underlie distinct genetic forms of deafness including GJB2 (connexin [CX] 26)
GJB3 (CX31) GJB6 (CX30) and GJB1 (CX32)
Liu et al (2001) reported mutations in GJA1 in association with sensorineural
recessive deafness in man However these mutations have recently been shown to
involve the pseudogene of GJA1 on human chromosome (HSA) 5 and not the
CFA1 and the GJA1 gene
100
GJA1 gene on HSA 6 (WA Paznekas cited a personal communication from the
senior author (W E Nance) of the paper by Liu et al 2001)
In previously performed studies (Chapter 3) one large French Dalmatian dog family
with frequent occurrence of CCSD showed linkage to microsatellites associated to
the GJA1 gene
In this report we performed a mutation analysis of the GJA1 gene sequence to
identify polymorphisms In order to evaluate whether the GJA1 gene is responsible
for congenital sensorineural deafness in Dalmatian dogs we analyzed the
association of the GJA1 haplotypes with the CCSD phenotype Furthermore we
employed 27 microsatellite markers covering the entire CFA1 and used them for a
non-parametric linkage analysis with CCSD in a Dalmatian dog population of 176
animals with frequent occurrence of CCSD
Material and methods Pedigree material
For the linkage analysis of the microsatellite marker we used DNA from altogether
176 animals belonging to 22 full-sib families and one large paternal half-sib family of
German Dalmatian dogs All families were segregating for CCSD The genotyped
families included all affected dogs (unilaterally and bilaterally deaf) their parents if
available and one to four unaffected animals At least two of the full sibs of each
family were unilaterally or bilaterally deaf The phenotype of the affected animals had
been confirmed by brainstem auditory evoked response (BAER) that detects
electrical activity in the cochlea and auditory pathways in the brain
Mutation analysis and screening for SNPs was performed by comparative
sequencing of DNA from 16 animals consisting of the parents and their progenies of
one large half-sib family of French Dalmatian dogs which showed significant linkage
to GJA1-associated microsatellites (Chapter 3)
Sequencing of canine genomic DNA and mutation analysis
The canine GJA1 gene sequence was derived from sequences deposited in the
current dog genome assembly (Boxer genome assembly 21) of the NCBI GenBank
(Genbank Acc No NC_006583) We compared the canine genomic DNA sequence
to canine cDNA fragments in the canine EST database using the
CFA1 and the GJA1 gene 101
BLASTN (Basic Local Alignment Search Tool Nucleotide) program Three canine
ESTs (Acc No DN880776 BU744925 and DN369900) were used to confirm the
assembly of the GJA1 gene
To identify polymorphisms within the canine GJA1 sequence the gene consisting of
one 1251 bp spanning exon were PCR amplified and sequenced from 16 French
Dalmatian dogs Additionally large sequences from the 3acute-end were amplified Primer
pairs were designed yielding products with a length of around 600 bp PCR primers
were developed with the Primer3 program (httpfrodowimiteducgi-
binprimer3primer3_wwwcgi) The PCR reactions were performed in a total of 50 microl
containing 125 microM dNTPs 25 pmol of each primer the reaction buffer supplied by
the manufacturer (Qbiogene Heidelberg Germany) and 1 U Taq polymerase After
a 4 min initial denaturation at 95degC 35 cycles of 30 sec at 94degC 45 sec at 58degC and
80 sec at 72degC were performed in a MJ Research thermocycler (Biozym Hessisch
Oldendorf Germany)
The obtained PCR products were directly sequenced with the DYEnamic ET
Terminator kit (AmershamBiosciences Freiburg Germany) and a MegaBACE 1000
capillary sequencer (AmershamBiosciences) using the PCR primers as sequencing
primers Sequence data were analyzed with Sequencher 42
(GeneCodes Ann Arbor MI USA)
Microsatellite marker analysis
We used 27 microsatellite marker derived from the NCBI database
(httpwebncbinlmnihgov) to cover CFA1 completely (Appendix Table 1)
PCR reactions for microsatellites were carried out in 12 microl reaction mixtures
containing 2 microl genomic DNA 12 microl 10x PCR buffer (Qbiogene) 024 microl dimethyl
sulfoxide (DMSO) 02 microl dNTPs (100 microM) 01 microl Taq Polymerase (5Umicrol)
(Qbiogene) 06 microl (10 microM) 5rsquo-IRD700 or IRD800 labelled forward primer 06 microl (10
microM) unlabelled reverse primer The PCR reactions were carried out in MJ Research
thermocyclers with the following program 4 min at 94 degC followed by 32 cycles of 30
sec at 94degC 30 sec at maximum annealing temperature and a final extension of 45
sec at 72degC PCR-products were diluted with formamide loading buffer in ratios from
110 to 140 and then size-fractionated by gel electrophoresis on automated LI-COR
42004300 sequencers (LI-COR inc Lincoln NE USA) using denaturing 4 and 6
polyacrylamide gels (RotiphoreseregGel 40 Roth Karlsruhe Germany)
CFA1 and the GJA1 gene
102
Linkage and haplotype analysis
Multipoint non-parametric linkage and haplotype analyses were performed using the
MERLIN software version 0102 (multipoint engine for rapid likelihood inference
Center for Statistical Genetics University of Michigan MI USA Abecasis et al
2002) The test statistics Z-mean and Lod score were used to test for the proportion
of alleles shared by affected individuals identical by descent (IBD) for the considered
marker loci
A non-parametric linkage analysis was performed with 27 microsatellite markers in 23
Dalmatian dog families consisting of 176 Dalmatian dogs The results were added to
the linkage analysis performed in Chapter 3
Results and discussion
In previously performed studies (Chapter 3) one French Dalmatian dog family
reached a Zmean value of 295 (plt0002) for GJA1_MS1 and a Zmean value of 286
(plt0002) for GJA1_MS2 indicating linkage Both markers were associated with the
GJA1 gene
By sequence analysis we revealed a total of 3 SNPs found in sequences of the 3acute-
UTR shown in Table 1 None of the polymorphisms were polymorphic in all parents of
the examined French half-sib family Neither of the observed polymorphism did alter
the predicted amino acid sequence of GJA1 nor showed the identified haplotypes an
association with the CCSD phenotype (Figure 1)
There is no recombination of the haplotypes of the GJA1 gene in this family The
paternal and maternal haplotypes as far as they could be estimated were distributed
among the affected progeny closely to the expected proportion of 50 and therefore
no excess of a certain haplotype could be observed in the affected dogs
Thus evaluation of the SNP markers debilitates the linkage to CCSD for the French
half-sib family Because of the fact that both animals with unilateral hearing loss and
bilateral hearing animals shared identical haplotypes these polymorphisms are
obviously not associated with CCSD in these Dalmatian dog families Furthermore
the present study revealed no functional mutations of the complete coding region of
GJA1 It is therefore unlikely that the GJA1 gene is involved in the pathogenesis of
CCSD in the analyzed French Dalmatian dogs The reason for GJA1_MS1 and
GJA1_MS2 indicating linkage may be caused by a closely linked gene involved in
CFA1 and the GJA1 gene 103
CCSD among the half-sib family of French Dalmatian dogs or by a false positive
result of the microsatellite study performed in Chapter 3
To clarify whether significant test statistics for other genomic regions on CFA1 and
for more families can be shown we used 27 microsatellite markers derived from the
NCBI database (httpwebncbinlmnihgov) to cover CFA1 completely A linkage
analysis was carried out after genotyping 176 German Dalmatian dogs with a set of
27 microsatellite markers The results of this linkage analysis were added to the
results of the test statistics for the microsatellites GJA1_MS1 and GJA1_MS2
(Chapter 3)
As a result we could not find linkage to any microsatellite in the analyzed families
(Figure 2 and 3) It is unlikely that the canine chromosome 1 harbours genomic
regions that are involved in the development of CCSD in the analyzed Dalmatian dog
families
With hindsight it was revealed that GJA1 is not responsible for sensorineural non-
syndromic deafness in humans as Liu et al (2001) has published GJA1 is
participating in a human syndrome called oculodentodigital dysplasia (ODDD) that
can be accompanied with hearing impairment (Paznekas et al 2003) But the type of
deafness in human ODDD differs from the typical hearing loss associated with other
connexin mutations because it is conductive rather than sensorineural
As deafness in dogs especially in Dalmatians is almost exclusively caused by
sensorineural non-syndromic forms also known as cochleosaccular degeneration
the GJA1 gene should not be considered as a candidate gene for CCSD anymore
CFA1 and the GJA1 gene
104
Table 1 Three newly developed intragenic SNPs and two microsatellite markers for
Dalmatian dogs in the candidate gene GJA1 with their corresponding primers the
SNP motif the product size and the annealing temperature
SNP
Primer F (5acute -gt 3acute)
SNP motif
Primer R (5acute -gt 3acute)
Product
size (bp)
Annealing
temperatur
GJA1_SNP1+2
CACCTTAGGCGTTCATTTTG
CCGGGGAG(AG)AAAA(AG)AAAAATACTT
TGGCTTGATTCCCTGACTC
650 58
GJA1_SNP3
TCTGAAATGTAATCATGGATGC
CAGAACTTGTAT(AT)CTGTTAAGAG
AATCACACAGGATATAGAGGCTATC
600 58
Microsatellite
marker Primers (forward reverse ) 5acute -gt 3acute
Product
size (bp)
Annealing
temperatur
GJA1_MS1
ATGGCATGAAGAGGATACCG
AGGACAGGTGACGGCTCTAC
134 60
GJA1_MS2
GCTAGTACTCGATTGTGGTC
TCATGGGTTGTGAGATCCAG
190 60
CFA1 and the GJA1 gene 105
Figu
re 1
Hap
loty
pes
of th
e G
JA1
gene
in th
e an
alyz
ed D
alm
atia
n do
g fa
mily
CFA1 and the GJA1 gene
106
Figure 2 Zmeans and LOD scores for 29 microsatellite markers on CFA1 (number of
families 23 number of genotyped dogs 176)
Figure 3 P-values for Zmeans and LOD scores of 29 microsatellite markers on CFA1
(number of families 23 number of genotyped dogs 176)
Chapter 8
Analysis of canine chromosome 31 and the claudin-14 (CLDN14) gene
in Dalmatian dogs segregating for congenital sensorineural deafness
CFA31 and the CLDN14 gene 109
Analysis of canine chromosome 31 and the claudin-14 (CLDN14) gene in Dalmatian dogs segregating for congenital sensorineural deafness Abstract A previously accomplished linkage analysis revealed significant linkage of
microsatellite markers associated with the CLDN14 gene on canine chromosome
(CFA) 31 with the phenotype of canine sensorineural deafness (Chapter 3) The
objective of the present study was to perform a sequence analysis in order to find
single nucleotide polymorphisms (SNPs) for the CLDN14 gene and additionally to
use a set of six microsatellite markers evenly distributed on CFA31 for non-
parametric linkage analysis with the aim to verify the significant test statistics shown
in Chapter 3
Introduction
Of the genes responsible for different forms of sensorineural non-syndromic deafness
in humans Rak (2003) considered 24 genes as candidates for sensorineural
deafness in dogs among them the CLDN14 gene on canine chromosome (CFA) 31 CLDN14 encodes a member of the claudin family which comprises the major
components of tight juncions (TJ) The human CLDN14 gene consists of one
tranlatete exon and six exons in the 5rsquountranlated region Five splice isoformes are
identified so far (Wilcox et al 2001 Wattenhofer et al 2005)
For the compartmentalization of perilymph and endolymph in the inner ear the
leakage of solutes through a paracellular pathway must be prevented by tight
junctions TJ are an intercellular junction found at the most apical region of polarised
epithelial and endothelial cells at which adjacent plasma membranes are joined
tightly together separating apical membranes and basolateral menbranes They are
specialised membrane domains containing branching strands of integral proteins and
create a primary barrier preventing paracellular transport of solutes and restricting
lateral diffusion of membrane lipids and proteins (Schneeberger and Lynch 2004)
CFA31 and the CLDN14 gene
110
The important role of the CLDN14 gene in the TJ of the inner ear was demonstrated
by Wilcox et al (2001) when he identified mutations in the CLDN14 gene responsible
for a hereditary human deafness in families segregating for congenital recessive
deafness (DFNB29)
In previously performed studies one large half-sib family as well as several full-sib
families of German Dalmatian dog indicated linkage to CLDN14 gene-associated
markers (Chapter 3)
A sequence analysis was performed to identify single nucleotide polymorphisms
(SNPs) either intergenic located or alternatively in the flanking 5rsquo- and 3rsquo-Regions
Furthermore we employed microsatellite markers covering CFA31 and used them for
a non-parametric linkage analysis with CCSD in a German Dalmatian dog population
of 176 animals with frequent occurrence of CCSD
Material and methods Pedigree material
For the linkage analysis of the microsatellite marker we used DNA from altogether
176 animals belonging to 22 full-sib families and one large paternal half-sib family of
German Dalmatian dogs All families were segregating for CCSD The genotyped
families included all the affected dogs (unilaterally and bilaterally deaf) their parents
if available and one to four unaffected animals At least two of the full sibs of each
family were unilaterally or bilaterally deaf The phenotype of the affected animals had
been confirmed by brainstem auditory evoked response (BAER) that detects
electrical activity in the cochlea and auditory pathways in the brain
Mutation analysis and screening for SNPs was performed by comparative
sequencing of DNA from 36 animals consisting of the parents and their progenies of
one half-sib family and four full-sib families of Dalmatian dogs which showed
significant linkage to a CLDN14-associated microsatellite (Chapter 3)
Sequencing of canine genomic DNA and development of single nucleotide
polymorphisms (SNPs)
The canine CLDN14 gene sequence was derived from sequences deposited in the
current dog genome assembly (Boxer genome assembly 21) of the NCBI GenBank
(Genbank Acc No NW_876295) by BLAST (Basic Local Alignment Search Tool)
CFA31 and the CLDN14 gene 111
search (httpwwwncbinlmnihgovBLAST) using the human CLDN14 reference
mRNA sequence (Genbank Acc No NM_144492)
We compared the canine genomic DNA sequence to canine cDNA fragments in the
canine EST database using the BLASTN program As no ESTs could be found
human mRNA sequences were used for the localization of the exonintron
boundaries using the mRNA-to-genomic alignment program Spidey
(httpwwwncbinlmnihgovIEBResearchOstellSpideyindexhtml)
We screened exon three for mutations as this exon is the only translated one in man
Additionally we screened large intronic sequences and sequences located in the 5rsquo-
and 3rsquo-UTR of the CLDN14 gene for Polymorphisms For this purpose primer pairs
were designed yielding products with a length of around 600 bp
PCR primers were developed with the Primer3 program (httpfrodowimiteducgi-
binprimer3primer3_wwwcgi) The PCR reactions were performed in a total of 50 microl
containing 125 microM dNTPs 25 pmol of each primer the reaction buffer supplied by
the manufacturer (Qbiogene Heidelberg Germany) and 1 U Taq polymerase After
a 4 min initial denaturation at 95degC 35 cycles of 30 sec at 94degC 45 sec at 58degC and
80 sec at 72degC were performed in a MJ Research thermocycler (Biozym Hessisch
Oldendorf Germany)
The obtained PCR products were directly sequenced with the DYEnamic ET
Terminator kit (AmershamBiosciences Freiburg Germany) and a MegaBACE 1000
capillary sequencer (AmershamBiosciences) using the PCR primers as sequencing
primers Sequence data were analyzed with Sequencher 42 (GeneCodes Ann
Arbor MI USA) The eight newly developed SNPs are shown in Table 1
Microsatellite marker analysis
In total six microsatellite marker were derived from the NCBI database
(httpwebncbinlmnihgov) to cover CFA31 completely (Appendix Table 1)
PCR reactions for microsatellites were carried out in 12 microl reaction mixtures
containing 2 microl genomic DNA 12 microl 10x PCR buffer (Qbiogene) 024 microl dimethyl
sulfoxide (DMSO) 02 microl dNTPs (100 microM) 01 microl Taq Polymerase (5Umicrol)
(Qbiogene) 06 microl (10 microM) 5rsquo-IRD700 or IRD800 labelled forward primer 06 microl (10
microM) unlabelled reverse primer The PCR reactions were carried out in MJ Research
thermocyclers with the following program 4 min at 94 degC followed by 32 cycles of 30
sec at 94degC 30 sec at maximum annealing temperature and a final extension of
CFA31 and the CLDN14 gene
112
45 sec at 72degC PCR-products were diluted with formamide loading buffer in ratios
from 110 to 140 and then size-fractionated by gel electrophoresis on automated LI-
COR 42004300 sequencers (LI-COR inc Lincoln NE USA) using denaturing 4
and 6 polyacrylamide gels (RotiphoreseregGel 40 Roth Karlsruhe Germany)
Linkage and haplotype analysis
Multipoint non-parametric linkage and haplotype analysis were performed using
MERLIN version 0102 (multipoint engine for rapid likelihood inference Center for
Statistical Genetics University of Michigan MI USA Abecasis et al 2002) The test
statistics Z-mean and Lod score were used to test for the proportion of alleles shared
by affected individuals identical by descent (IBD) for the considered marker loci A
linkage analysis was performed with 6 microsatellite markers on 176 Dalmatian dogs
The results were added to the linkage analysis performed in Chapter 3
Results and discussion CLDN14-associted microsatellites were significantly linked with CCSD in a scan of
candidate genes using 24 families with 215 Dalmatian dogs (Chapter 3) Selection of
four full-sib families and one half-sib family with the highest tests statistics lead to a
Zmean value of 383 (plt000007) for the CLDN14 gene-associated marker
CLDN14_MS2
To substantiate the linkage to the CLDN14 gene we searched for sequence
variations within the CLDN14 gene in four full-sib families and one half-sib family with
the highest contribution to the test statistics shown in Chapter 3 Most of the identified
polymorphisms were found in intronic sequences none were within exon three None
of the observed polymorphism did alter the predicted amino acid sequence of exon
three We revealed a total of eight SNPs (Table 1) but only two (SNP_4 and SNP_5)
out of the identified eight SNPs were polymorphic in the examined families Both
unilaterally and bilaterally deaf animals as well as normal hearing animals showed
identical haplotypes for these two polymorphic SNPs and thus no co-segregation with
the deaf Dalmatian dogs could be revealed (Figure 1 and 2) In this study we did not
identify any causal mutations for CCSD in the analyzed Dalmatian dogs Despite this
results a mutation outside of the translated genomic regions analyzed here may exist
that would affect CLDN14 expression
CFA31 and the CLDN14 gene 113
To clarify if other regions on CFA31 are responsible for the CCSD phenotype
additionally six microsatellite markers covering CFA31 were tested in 23 Dalmatian
dog families consisting of 176 individuals A non-parametric linkage analysis was
performed regarding these 23 families The results of this linkage analysis were
added to the results of the test statistics for the CLDN14 gene-associated
microsatellites (Chapter 3)
We could only find significant linkage with CCSD for CLDN14-associted
microsatellites in the abovementioned five families The two microsatellites located
most closely were proximal and distal 4 Mb off the CLDN14 gene REN110K04 with a
Zmean value of -009 (plt05) and FH2712 with a Zmean value of 022 (plt03) not
indicating linkage
It can not be excluded that CLDN14 or genes in its flanking regions are involved in
the development of CCSD in the analyzed Dalmatian dog families Other genes than
the CLDN14 that are known to be involved in the hearing process or known to be
expressed in the inner ear are not annotated at the syntenic human region of homo
sapiens autosome (HSA) 21q223 Therefore despite the CLDN14 gene no other
well-defined candidate gene for canine hereditary deafness exists in the linked
region Despite the remarkable progress that has been made in the past few years in
identifying deafness genes in man and mouse it is suspected that only one-third of all
deafness causing genes have been identified so far and thus it seems possible that
other genes in the flanking region of the CLDN14 gene are involved in the
development of the disease Anyway to clarify the importance of CLDN14 in the
CCSD phenotype more SNPs have to be developed within the CLDN14 gene as well
as in its flanking regions with the aim to find significant linkage disequilibrium of SNP
markers with CCSD
CFA31 and the CLDN14 gene
114
Table 1 The 8 newly developed SNPs for Dalmatian dogs in the CLDN14 gene on
CFA31 with their corresponding primers the SNP motif the product size and the
annealing temperature
SNP
Location
(intron or
5rsquo3rsquo-UTR)
Primer F1 (5acute -gt 3acute)
SNP motif
Primer R2 (5acute -gt 3acute)
Bp3 AT4
CLDN14_SNP1
intron
GACCATATGTTTGTGGCC
CTTCCAGGGAAA(AT)TGTCGTAGCC
CGTCAGGATGTTGGTGCC
580 60
CLDN14_SNP2
inton
GACCATATGTTTGTGGCC
GAAATTGTCGTA(AG)CCCGGCCGCT
CGTCAGGATGTTGGTGCC
580 60
CLDN14_SNP3
3rsquo-UTR
CTGCCTTCAAGGACAACC
CCAGAGGAATAA(CT)ATGATCGTGA
GATGAGTATCAGCCCAGC
550 60
CLDN14_SNP4
3rsquo-UTR
CTGCCTTCAAGGACAACC
ACCACCGCACAC(CT)GTCACAGCTT
GATGAGTATCAGCCCAGC
550 60
CLDN14_SNP5 3rsquo-UTR
CATGCCTTTGTCCCAAACTT
GAGACCCTCTGG(CT)TCCTTTTGGC
GTACCTGTTGCCTGGGTTGT
610 60
CLDN14_SNP6 5rsquo-UTR
CCTTCATCCTTTCTGGTTGA
GCTCACAGTCCC(AC)ATGGGGACAT
GGGGAGCATAATGTGGTCAT
585 60
CLDN14_SNP7 5rsquo-UTR
TGAACTGGTCCCAAGGAAAG
GCACGACCGAGC(CT)CTGGCTTTAC
GGGATGAGAGGGAGGTTTTT
580 60
CLDN14_SNP8 3rsquo-UTR
AATGCCTATCCCTTCTTTGGA
CACGTTACTGTG(AG)ACCTCTCTAC
GCAGGCTTTCCTCAAGTGTC
680 60
1 Forward 2 Reverse 3 Product size (basepairs) 4 Annealing temperature (degC)
CFA31 and the CLDN14 gene 115
Figure 1 Haplotypes of the CLDN14 gene-associated markers SNP_4 and SNP_5 in
the analyzed four Dalmatian dog full-sib families
CFA31 and the CLDN14 gene
116
Figu
re 2
Hap
loty
pes
of th
e C
LDN
14 g
ene-
asso
ciat
ed m
arke
rs S
NP
_4 a
nd S
NP
_5 in
the
anal
yzed
Dal
mat
ian
dog
half-
sib
fam
ily
Chapter 9
General discussion
General discussion 119
General discussion
The candidate gene approach In the first step of this thesis candidate genes for canine congenital sensorineural
deafness (CCSD) in Dalmatian dogs were analyzed by means of microsatellite
markers or alternatively by single nucleotide polymorphisms (SNPs)
The candidate genes for which a set of in total 43 microsatellites was available
included the following 24 genes CDH23 CLDN14 COCH COL11A2 DFNA5
DIAPH1 EDN3 EDNRB EYA4 GJA1 GJB2 GJB6 MITF MYH9 MYO6 MYO7A
MYO15A OTOF PAX3 POU4F3 SLC26A4 SOX10 TECTA and TMPRSS3
(Rak 2003) These genes are known to be involved either in human non-syndromic
deafness or in the human Waardenburg syndrome The Waardenburg syndrome
(WS) manifests with sensorineural deafness and pigmentation defects in iris hair and
skin WS is classified into four types depending on the presence or absence of
additional symptoms which are caused by mutations in the five genes EDN3
EDNRB MITF PAX3 and SOX respectively
For another eight recently identified genes responsible for different forms of human
non-syndromic deafness including TMC1 TMIE USH1C MYH14 MYO3A PRES
WHRN and ESPN linkage and association analyses were performed using newly
developed SNPs
In the last years most projects have exploited canine traits for which either direct
candidate genes could be proposed and evaluated or for which large informative
pedigrees were available to enable linkage mapping to identify candidate regions A
major component of such research efforts comprised the cloning sequencing and
mapping of individual canine homologs of genes either proposed as candidate
genes or expected to be located in candidate regions This was necessary to identify
new informative polymorphisms (eg SNPs microsatellites) for high resolution
mapping of candidate regions and to examine each exon and exonintron boundary
for positional candidates Availability of the second version of the dog genome
assembly (build 21) of the NCBI database shortcuts this effort and increases the
investigators efficiency Now either additional candidate genes for canine congenital
sensorineural deafness can be found directly by its gene symbol in the 21 of the
General discussion
120
NCBIs genome annotation or if a candidate gene is not yet annotated a BLAST
(Basic Local Alignment Search Tool) search against the canine whole genome
shotgun (wgs) sequence resource can be used to obtain the sequence of the canine
genomic contigs containing the human homologous gene and thus intragenic
markers can be developed for subsequent linkage and association analyses
Over the past decade it has become increasingly clear how far structural and
functional homologies at the gene level extend across even distantly related species
Much is known about deafness-causing gene mutations in humans and mice
including the fact that the clinical and histopathological findings are often very similar
to those of deafness in Dalmatian dogs Thus genes responsible for non-syndromic
congenital hereditary deafness in humans seem to be appropriate candidate genes
for CCSD (Rak and Distl 2005) In this thesis we first concentrated on the candidate
gene approach combined with linkage analysis method using affected pedigree
members Once a significant linkage was found only the linked genes with the
required low error probability values were used for further molecular genetic analysis
The method of candidate gene approach using either gene-associated microsatellite
or alternatively SNP markers was applied for in total 32 candidate genes which
comprise nearly all of the identified mutated genes causing non-syndromic hereditary
hearing impairment in humans
Linkage and association analysis In principle linkage and association are totally different phenomena Linkage is a
relation between loci and association is a relation between alleles
Linkage means that a haplotype characterised by microsatellites or SNPs is
significantly more often present in family members with the phenotype under study
than expected by random assortment For construction of haplotypes sets of closely
linked genetic markers on the same chromosome are needed which tend to be
inherited together as they are not likely to be separated by recombination Linkage
creates associations within families but not among unrelated induviduals
Association is a statistical statement about the co-occurrence of alleles or
phenotypes Association analysis can be carried out as a method of genetic analysis
that compares the frequency of alleles between affected and unaffected individuals
across all families Thus for association family structures are not necessary A given
allele is considered to be associated with the disease if the presence of that allele
General discussion 121
explains a significant proportion of the phenotypic trait variation For association
studies the developing of a marker set consisting of SNPs rather than microsatellites
is needed
In this thesis a total of 32 candidate genes for canine congenital deafness were
analyzed which showed an appropriate clinical and histological disease pattern in
comparison to deafness in Dalmatian dogs Rak (2003) developed a set of 43
microsatellites for in total 24 candidate genes among them the CLDN14 gene on
canine chromosome (CFA) 31 and the MYH9 gene on CFA10 The GJA1 on CFA1
was also considered as a candidate gene for CCSD (Rak 2003) and therefore two
gene-associated microsatellites have been developed Recently it turned out that
GJA1 is not responsible for human sensorineural non-syndromic deafness but for a
human syndromic disorder that can be related with conductive deafness
By the use of a non-parametric linkage analysis using the existing set of 43
microsatellites associated to 24 candidate genes we found linkage to markers
associated to CLDN14 on CFA31 MYH9 on CFA10 and GJA1 on CFA1
For another another eight candidate genes it was possible to develop SNPs
Performing linkage analyses as well as association and haplotype studies it was
possible to exclude these eight candidate genes from being responsible for the
CCSD phenotype
Over the past ten years significant progress has been made in the identification of
deafness gene localisations Up to now approximately 120 loci have been reported
for both autosomal dominant and recessive forms of non-syndromic hereditary
deafness in humans and only for one third the responsible gene mutation could be
detected Thus it can be expected that additional potential human candidates for
CCSD in Dalmatian dogs will become available in future (Van Camp and Smith
2003)
The extreme heterogeneity of human deafness often hampered genetic studies
because many different genetic forms of hearing loss give rise to similar clinical
phenotypes and conversely mutations in the same gene can result in a variety of
clinical phenotypes In man genes that transport ions across membranes to
maintain appropriate solute concentration and pH as well as regulatory genes mainly
transcription factors and genes that play a part in structural integrity are essential
for the hearing process
General discussion
122
The results of this thesis indicate that the inheritance of hearing loss in Dalmatian
dogs is probably as heterogenic in origin as it is in humans Genetic heterogeneity
means that different mutations cause a similar phenotype the different mutations
can either be found at the same locus (allelic heterogeneity) or even at different loci
(non-allelic heterogeneity) As linkage was found for different candidate genes in
different families subsequently only the families indicating linkage were chosen for
further molecular analyses
GJA1 on CFA1 MYH9 on CFA10 and CLDN14 on CFA31 and their flanking regions
are further analyzed with a combined approach using microsatellite and SNP
markers
CFA1 By the use of GJA1-associated microsatellites one large French Dalmatian dog
family reached a Zmean value of 295 (plt0002) for GJA1_MS1 and a Zmean value
of 286 (plt0002) for GJA1_MS2 indicating linkage Subsequently a sequence
analysis of the GJA1 gene using the above mentioned French Dalmatian dog family
was performed None of the observed polymorphism did alter the predicted amino
acid sequence of GJA1 nor showed the identified haplotypes an association with the
CCSD phenotype Thus evaluation of the SNP markers debilitates the linkage to
CCSD for the French half-sib family It is unlikely that the GJA1 gene is involved in
the pathogenesis of CCSD in Dalmatian dogs To see whether significant test
statistics for other genomic regions on CFA1 and for more families can be shown a
non-parametric linkage analysis was performed with 27 microsatellite markers
covering CFA1 completely In total 176 animals were genotyped We could not find
linkage to any microsatellite in the analyzed families Furthermore it was revealed
that GJA1 is not a candidate gene for sensorineural non-syndromic deafness in
humans (WA Paznekas cited a personal communication from the senior author (W
E Nance) of the paper by Liu et al 2001) GJA1 is participating in a human
syndrome called oculodentodigital dysplasia (ODDD) that can be accompanied with
hearing impairment (Paznekas et al 2003) But the type of deafness in human
ODDD is conductive rather than sensorineural As deafness in dogs especially in
Dalmatians is almost exclusively caused by sensorineural non-syndromic forms also
known as cochleosaccular degeneration the GJA1 gene should not be considered
as a candidate gene for CCSD anymore
General discussion 123
CFA31 By the use of microsatellites as well as SNPs we found significant linkage with CCSD
for CLDN14-associated microsatellites in four full-sib and one half-sib Dalmatian dog
familiy with a Zmean value of 383 (plt000007) A mutation analysis was performed
for exon three as this is the only translated one in man None of the observed
polymorphisms did alter the predicted amino acid sequence However to clarify the
importance of the CLDN14 gene and its flanking regions in the CCSD phenotype
more SNPs have to be developed within the CLDN14 gene as well as in its flanking
regions with the aim to find significant linkage disequilibrium of SNP markers
CFA10 A significant co-segregation of markers alleles and the phenotypic expression of
deafness in a large sample of German Dalmatian dog families was determined for
one marker (Z-mean of 156 [p= 006] and a Lod score of 058 [p= 005]) associated
to the MYH9 gene Subsequently we evaluated whether MYH9 gene mutations are
responsible for CCSD in these Dalmatian dog families An initial priority in defining
gene structure is to obtain a full-length cDNA sequence and identify translational
initiation and termination sites and polyadenylation site(s) Exonintron structure can
then be determined by referencing the cDNA sequence against sequences of
cognate genomic DNA One popular method of obtaining full-length cDNA sequences
is the RACE (rapid amplification of cDNA ends) technique RACE-PCR is an anchor
PCR modification of RT-PCR The rationale is to amplify sequences between a single
previously characterised region in the mRNA (cDNA) and an anchor sequence that is
coupled to the 5 or the 3 end A primer is designed from the known internal
sequence and the second primer is selected from the relevant anchor sequence
To provide the genomic organization and the complete sequence of the canine
MYH9 gene the isolation of full length cDNAs was achieved with the help of a
modified rapid amplification of cDNA ends (RACE) protocol A mutation analysis was
performed to identify single nucleotide polymorphisms (SNPs) in this gene We
analyzed the association of the MYH9 haplotypes with the CCSD phenotype in three
families of Dalmatian dogs with frequent occurrence of CCSD and significant linkage
to gene-associated microsatellites Using the RT-PCR analyses for cDNA-genomic
sequence comparisons we detected that the canine MYH9 gene is bigger compared
to the human sequence due to the untranlated first exon in the 5rsquo-UTR
General discussion
124
The canine MYH9 gene consists of 41 exons with an untranslated exon 1 and
exonintron boundaries that conform perfectly to the GTAG rule
None of the observed polymorphisms did alter the predicted amino acid sequence of
MYH9 nor showed the identified haplotypes an association with the CCSD
phenotype
Thus these silent point mutations found in affected and unaffected Dalmatian dogs
do not seem to be responsible for the CCSD phenotype in these three families
To clarify if other regions on CFA10 are responsible for the CCSD phenotype we
added in a second step 27 microsatellite markers derived from the NCBI database to
cover CFA10 with a high density in the flanking region of the MYH9 gene A linkage
analysis was carried out for 23 Dalmatian dog families consisting of 176 animals that
were genotyped with the marker set of 27 microsatellite markers
We found significant linkage to microsatellites in the region spanning 36 Mb to 48 Mb
Consequently we screened this 12 Mb spanning region for SNPs Out of the 23
analyzed Dalmatian dog families five full-sib families were chosen to screen for
SNPs because of their obviously significant values at the above mentioned region
Twenty-six new SNP markers covering the region of 36 Mb to 48 Mb equally were
developed and added to the linkage analysis The significant Zmeans on CFA10 was
confirmed after adding the SNP markers Furthermore with the use of SNPs the
apparently linked region spanning 36 Mb to 48 Mb could be narrowed down to 5 Mb
spanning from 39 Mb to 44 Mb For this reason the use of SNPs in addition to
informative microsatellites appears highly recommendable
In further studies more SNPs have to be developed within the identified CCSD region
on CFA10 to localize the deafness causing gene or to find unambiguously associsted
SNP markers which could be used for a population-wide genetic test for CCSD
Chapter 10
Summary
Summary 127
Summary
Molecular genetic analysis of canine congenital sensorineural deafness in Dalmatian dogs
Katharina Mieskes (2006) The objective of the present study was to localize the gene or genomic region that is
involved in the development of canine congenital sensorineural deafness (CCSD) in
Dalmatian dogs
In man as in different dog breeds deafness is an often diagnosed disorder with the
Dalmatian dog showing the highest incidence Many genetic disorders in humans
and domestic dogs (Canis familiaris) demonstrate a high level of clinical and
molecular similarity
Altogether 39 genes have already been found causative for sensorineural non-
syndromic hearing impairment in humans Out of this 39 deafness causing genes a
total of 32 candidate genes were selected for canine congenital deafness which
showed an appropriate clinical and histological disease pattern in comparison to
deafness in Dalmatians dogs
On the one hand an existing set of 43 microsatllite markers for in total 24 candidate
genes were used for a non-parametric linkage analysis among them the claudin-14
(CLDN14) gene on canine chromosome (CFA) 31 and the myosin heavy polypeptide
9 (MYH9) gene on CFA10 The gap junction protein alpha 1 (GJA1) gene on CFA1
was also considered as a candidate gene for CCSD and thus GJA1-associated
microsatellites were part of the existing set Recently it turned out that GJA1 is not
responsible for human sensorineural non-syndromic deafness but for a human
syndromic disorder that can be related with conductive deafness In the last few
years more human deafness genes have been identified among them eight genes
that were considered as appropriate candidates for CCSD For these eight genes a
total of 21 SNPs were newly developed and used for non-parametric linkage and
association analyses
Summary
128
The used microsatellite and SNP markers derived either from a partial sequence
analysis of BAC clones each containing a different candidate gene or from
sequences deposited in the current dog genome assembly (Boxer genome assembly
21) of the NCBI GenBank We found significant linkage to markers associated to
CLDN14 on CFA31 MYH9 on CFA10 and GJA1 on CFA1 To substantiate the
linkage we searched for sequence variations within these three genes SNPs found
in intronic sequences of either gene were included in the linkage analyses Sequence
analysis neither revealed a causative mutation nor significant linkage disequilibrium
of SNP markers with CCSD Subsequently CFA1 10 and 31 were scanned
completely with microsatellite markers derived from the NCBI database with the
purpose to see if other regions on this three chromosomes harbour a gene that is
involved in the development of CCSD
The analyses of SNPs and more microsatellite markers on CFA1 revealed no
significant linkage to CCSD Thus it is unlikely that the canine chromosome 1 and
the GJA1 gene are responsible for the CCSD phenoptype As deafness in dogs
especially in Dalmatians is almost exclusively caused by sensorineural non-
syndromic forms the GJA1 gene should not be considered as a candidate gene for
CCSD anymore
On CFA10 we could exclude MYH9 for being causal for deafness but by adding
more microsatellites covering CFA10 completely we found significant linkage to the
CCSD phenotype in the region distal of MYH9 Hence new SNP-markers for fine
mapping the region spanning 36 to 48 Mb were developed by sequence analyses of
different Dalmatian dogs The search for SNPs was carried out on genomic
sequences of genes located in the significantly linked region The sequences of
these genomic sequences were derived from the NCBI GenBank The SNPs
confirmed the linkage and narrowed the region harbouring a causative CCSD gene
down to 5 Mb spanning from 39 to 44 Mb
After scanning CFA31 we could not exclude CLDN14 for being responsible for the
CCSD phenotype as microsatellite markers associated to CLDN14 indicated linkage
However to clarify the importance of CLDN14 in the CCSD phenotype more SNPs
have to be developed within the CLDN14 gene as well as in its flanking regions with
the aim to find linkage disequilibrium for SNP markers
Chapter 11
Erweiterte Zusammenfassung
Erweiterte Zusammenfassung 131
Erweiterte Zusammenfassung
Katharina Mieskes
Molekulargenetische Untersuchung der kongenitalen sensorineuralen Taubheit beim Dalmatiner
Einleitung Es gibt mehrere Moumlglichkeiten Taubheit zu klassifizieren Je nach den betroffenen
Strukturen des Gehoumlrorgans lassen sich konduktive und sensorineurale Formen
unterscheiden Erstere entstehen durch Stoumlrungen des Konduktionsmechanismus im
aumluszligeren Gehoumlrgang und im Mittelohr Ursachen fuumlr konduktive Houmlrstoumlrungen koumlnnen
zum Beispiel chronische Otitis externa oder media Frakturen von Mittelohranteilen
oder Tumoren sein Sehr viel oumlfter tritt die sensorineurale Taubheit auf bei der ein
Houmlrverlust als Folge von Transduktions- und Transmissionsstoumlrungen in der Cochlea
(Schnecke) sowie in peripheren und zentralen Anteilen des Nervus cochlearis auftritt
Neben der erblichen Form koumlnnen auch eine Otitis interna Tumoren oder
ototoxische Medikamente zur sensorineuralen Taubheit fuumlhren
Der Houmlrverlust kann bereits seit der Geburt bestehen (kongenital) oder er kann erst
im Laufe der weiteren Lebenszeit auftreten
Beim Menschen ist mehr als die Haumllfte der kongenitalen Taubheit erblich bedingt Die
erbliche kongenitale Taubheit kann als Teil einer multisystemischen Krankheit
(syndromisch) oder als Funktionsstoumlrung auftreten die auf das Ohr und das
vestibulaumlre System (nicht-syndromisch) begrenzt ist Da die nicht-syndromische
ererbte Taubheit fast ausschlieszliglich durch Cochleadefekte verursacht wird leiden die
Patienten unter sensorineuraler Taubheit Es wird geschaumltzt dass die nicht-
syndromische erbliche Taubheit 60-70 aller menschlichen Taubheitsfaumllle
verursacht Weiterhin sind 70-80 der Faumllle der nicht-syndromischen Taubheit auf
einen autosomal rezessiven Erbgang zuruumlck zu fuumlhren gefolgt von einem autosomal
dominanten Erbgang mit 10-20 der Faumllle und 1-2 der Faumllle sind X- gekoppelt Ein
noch geringerer Prozentsatz wird durch einen maternalen Erbgang verursacht
Von den ca 25000 bis 30000 menschlichen Genen ist nach Schaumltzungen bis zu 1
wichtig fuumlr den Houmlrprozess Annaumlhernd 120 Gene sind inzwischen identifiziert die fuumlr
Erweiterte Zusammenfassung
132
verschiedene Formen der menschlichen vererbten Taubheit verantwortlich sind
Darunter sind ungefaumlhr 80 Gene fuumlr die syndromische und 39 Gene fuumlr die nicht
syndromische vererbte Taubheit verantwortlich Insgesamt stellen die 120
identifizierten Gene vermutlich ein Drittel aller taubheitsverursachenden Gene dar
Die kongenitale sensorineurale Taubheit ist eine Erkrankung von der viele
Saumlugetierspezies betroffen sind Beim Hund werden uumlber 54 verschiedene Rassen
beschrieben bei welchen ein kongenitaler Houmlrverlust gehaumluft auftritt In zahlreichen
europaumlischen und amerikanischen Studien weisen hierbei die Dalmatiner mit 165-
30 die houmlchste Taubheitsinzidenz auf
Die histologischen Veraumlnderungen im Innenohr werden im Allgemeinen aumlhnlich wie
beim Menschen als cochleosacculaumlr beschrieben Die Degenerationen im Innenohr
schreiten bei betroffenen Tieren mit zunehmendem Alter fort und fuumlhren
normalerweise in einem Alter von drei bis vier Wochen spaumltestens aber in einem
Alter von etwa vier Monaten zu einem vollstaumlndigen ein- oder beidseitigen
Houmlrverlust
Eine objektive Diagnose der Taubheit besonders wenn sie nur einseitig ist stuumltzt
sich auf die brainstem auditory evoked response (BAER in Deutschland
audiometrischer Test) ein elektro-diagnostischer Test bei dem durch einen
bestimmten Reiz ausgeloumlste elektrische Aktivitaumlten (akustisch evozierte Potentiale) in
der Cochlea und den Nervenbahnen zur auditorischen Hirnrinde detektiert werden
Obwohl fuumlr den Dalmatiner die Erblichkeit der Erkrankung wiederholt nachgewiesen
werden konnte wurden fuumlr die verschiedenen untersuchten Dalmatinerpopulationen
auch sehr viele unterschiedliche Erbgaumlnge in Betracht gezogen Es ist bisher nicht
gelungen die genaue Anzahl der beteiligten Gene zu bestimmen Auch war es
bislang fuumlr keine der betroffenen Hunderassen moumlglich ein taubheitsverursachendes
Gen zu identifizieren
Bei deutschen Dalmatinern ergab eine Auswertung mittels komplexer
Segregationsanalysen dass ein Modell mit einem rezessiven Hauptgen und einer
polygenen Komponente das Auftreten der kongenitalen Taubheit in der untersuchten
Population am besten erklaumlrte Zusaumltzlich konnte ein Zusammenhang zu
Pigmentierungsgenorten nachgewiesen werden da Tiere mit blauer Augenfarbe
signifikant haumlufiger von Taubheit betroffen waren und gleichzeitig Tiere mit
Plattenzeichnung seltener kongenitalen Houmlrverlust zeigten
Erweiterte Zusammenfassung 133
Das Zuumlchten ein- oder beidseitig tauber Dalmatiner sowie Dalmatinern mit blauen
Augen ist in Deutschland schon laumlnger verboten und es wird von den
Zuchtverbaumlnden vorgeschrieben dass alle Dalmatinerwelpen in einem Alter von etwa
6 bis 8 Wochen audiometrisch untersucht werden Des Weiteren ist der Defekt
tierschutzrelevant nach sect 11b des Deutschen Tierschutzgesetzes da der artgemaumlszlige
Gebrauch eines Organs eingeschraumlnkt wird und eine Erblichkeit nachgewiesen
wurde Somit duumlrfen Tiere die Defektgentraumlger sind oder bei denen damit zu
rechnen ist nicht zur Zucht verwendet werden Waumlhrend einseitig taube Dalmatiner
als ganz normale Familienhunde gehalten werden koumlnnen sind beidseitig taube
Dalmatiner schwierig zu erziehen schreckhafter und daraus resultierend auch
oftmals aggressiv und bissig Aus diesen Gruumlnden werden fast alle Welpen mit
beidseitigem Houmlrverlust euthanasiert
Es hat sich gezeigt dass allein der Zuchtausschluss betroffener Tiere nicht genuumlgt
um die hohe Taubheitsinzidenz beim Dalmatiner deutlich zu reduzieren Aus diesen
Gruumlnden ist es von erheblicher Bedeutung die kongenitale sensorineurale Taubheit
beim Dalmatiner molekulargenetisch zu untersuchen um so ein
molekulargenetisches Testverfahren entwickeln zu koumlnnen welches auch die
Identifizierung von Anlagetraumlgern ermoumlglicht
Innerhalb des letzten Jahrzehnts wurden auch zwischen relativ unverwandten
Spezies die strukturellen und funktionellen Homologien auf genetischer Ebene
zunehmend verdeutlicht
Es wurde inzwischen eine groszlige Anzahl von taubheitsverursachenden
Genmutationen beim Menschen und der Maus aufgeklaumlrt Da auch die
histopathologischen Befunde beim Menschen und der Maus oftmals sehr aumlhnlich
denen beim Dalmatiner sind scheinen Gene die beim Menschen verantwortlich fuumlr
die congenitale sensorineurale Taubheit sind geeignete Kandidatengene fuumlr die
canine congenitale sensorineurale Taubheit zu sein
Das Ziel dieser Dissertation ist es die fuumlr die Taubheit kausalen Gene bzw Genorte
zu lokalisieren Um die kongenitale sensorineurale Taubheit beim Dalmatiner
moumlglichst effizient molekulargenetisch aufzuklaumlren wurden 32 Kandidatengene mit
genassoziierten Markern auf eine potenzielle Kopplung mit der caninen kongenitalen
sensorineuralen Taubheit untersucht
Die Genombereiche mit signifikanter Kopplung zur kongenitalen sensorineuralen
Taubheit wurden weitergehend molekulargenetisch analysiert
Erweiterte Zusammenfassung
134
Kopplungsanalyse fuumlr 24 Kandidatengene der caninen kongenitalen sensorineuralen Taubheit (CCSD) mit 43 genassoziierten Mikrosatelliten Markern
Material und Methoden
Fuumlr 24 Kandidatengene stand ein Mikrosatelliten Marker Set zur Verfuumlgung Das
Marker Set beinhaltete Mikrosatelliten fuumlr folgende Kandidatengene CDH23
CLDN14 COCH COL11A2 DFNA5 DIAPH1 EDN3 EDNRB EYA4 GJA1 GJB2
GJB6 MITF MYH9 MYO6 MYO7A MYO15A OTOF PAX3 POU4F3 SLC26A4
SOX10 TECTA und TMPRSS3 Als Familienmaterial fuumlr die Mikrosatellitenstudie
wurden 215 Dalmatiner verwendet Die Tiere verteilten sich auf 22 unverwandte
Vollgeschwisterfamilien und zwei paternale Halbgeschwistergruppen Die
genotypisierten Familien bestanden aus saumlmtlichen tauben Nachkommen (uni- oder
bilateral taub) ihren Eltern und einem bis vier nicht betroffenen Voll- bzw
Halbgeschwistern Mindestens zwei der Vollgeschwister einer Familie waren uni-
oder bilateral taub Der Phaumlnotyp der verwendeten Tiere wurde durch einen
audiometrischen Test bestimmt
Alle Mikrosatellitenmarker wurden uumlber PCR und Polyacrylamidgelelektrophorese
ausgewertet
Kopplungsanalyse
Die nicht-parametrische Kopplungsanalyse wurde unter Verwendung der Software
MERLIN (multipoint engine for rapid likelihood inference Version 0102)
durchgefuumlhrt und basierte auf dem identical-by-descent (IBD) Verfahren Dabei
wurden die Markerallele auf Kosegregation mit der phaumlnotypischen Auspraumlgung der
Erkrankung getestet Darauf folgend wurde die einer Normalverteilung folgende
Teststatistik fuumlr den Anteil von IBD-Markerallelen (Zmean) und ein daraus
abgeleiteter LOD-Score berechnet Als signifikant fuumlr die Kosegregation eines
Markerallels mit dem Phaumlnotyp der caninen kongenitalen sensorineuralen Taubheit
(CCSD) gelten Irrtumswahrscheinlichkeiten (p) von 005 oder kleiner
Die Daten wurden mit dem Softwarepaket SAS (Statistical Analysis System Version
913) bearbeitet um die Marker bezuumlglich Allelfrequenzen beobachteten und
erwarteten Heterozygotiegrad und PIC (Polymorphism information content)
charakterisieren zu koumlnnen
Erweiterte Zusammenfassung 135
Ergebnisse und Diskussion Das Mikrosatelliten Set bestand insgesamt aus 43 Markern mit einem bis drei
Markern pro Kandidatengen Zunaumlchst wurde fuumlr alle 24 Familien zusammen eine
Kopplungsanalyse durchgefuumlhrt Vermeintliche CCSD Loci wurden auf den caninen
Chromosomen (CFA) 1 10 12 20 und 31 lokalisiert Im Anschluss wurden die
Ergebnisse der Kopplungsanalyse auf Heterogenie zwischen den Familien uumlberpruumlft
Fuumlr einzelne Familien konnten houmlhere Teststatistiken und somit engere
Kopplungsbeziehungen als bei der Analyse aller Familien nachgewiesen werden
Eine signifikante Kopplung mit der kongenitalen sensorineuralen Taubheit wurde fuumlr
die Gene GJA1 auf CFA1 MYH9 auf CFA10 und CLDN14 auf CFA31 sowohl im
Gesamtmaterial wie in jeweils unterschiedlichen Gruppen von Dalmatinerfamilien
gefunden Es ist daher wahrscheinlich dass diese Gene oder Gene in ihrer naumlheren
Umgebung an der Entwicklung der Taubheit in den jeweiligen Familien involviert
sind Fuumlr die restlichen Kandidatengene lagen die Multipoint Teststatistiken fuumlr den
Anteil von IBD-Markerallelen (Zmean) und daraus abgeleiteten LOD-Scores bei
annaumlhernd Null und waren nicht signifikant
Die Ergebnisse dieser Teststatistiken zeigen dass der Erbgang der nicht-
syndromischen Taubheit bei Dalmatinern vermutlich ebenso heterogen ist wie er
sich beim Menschen darstellt
Dennoch stellt diese Studie den ersten Schritt dar um die CCSD verursachenden
Gene beim Dalmatiner zu identifizieren Die Gene GJA1 MYH9 und CLDN14 sowie
ihre naumlhere Umgebungen werden im weiteren Verlauf der Studie weitergehend
molekulargenetisch mit Hilfe von SNPs und Mikrosatelliten untersucht Aufgrund der
Heterogenitaumlt zwischen den Familien wurden fuumlr die weitere molekulargenetische
Analyse bestimmter Gene oder Genomregionen nur die Familien mit dem houmlchsten
Beitrag zu den Teststatistiken der Kopplungsanalyse (Zmean gt 1) ausgewaumlhlt
Erweiterte Zusammenfassung
136
Evaluierung von neu entwickelten SNP Markern assoziiert mit Kandidatengenen fuumlr die canine kongenitale sensorineurale Taubheit (CCSD) Material und Methoden Fuumlr acht Kandidatengene (TMC1 TMIE USH1C MYH14 MYO3A PRES WHRN
und ESPN) wurden intragenische Single Nucleotide Polymorphism (SNP) Marker neu
entwickelt
Als Familienmaterial fuumlr die Genotypisierung der SNPs wurde DNA von insgesamt 39
Dalmatinern verwendet die vier Vollgeschwisterfamilien angehoumlrten Mindestens
zwei der Vollgeschwister einer Familie waren uni- oder bilateral taub Der Phaumlnotyp
der verwendeten Tiere wurde durch einen audiometrischen Test bestimmt Die vier
Familien bestanden aus sechs bis zehn Vollgeschwistern und den dazugehoumlrenden
Elterntieren
Identifizierung von SNPs (Single Nucleotide Polymorphisms)
Die caninen Sequenzen der Kandidatengene wurden mit Hilfe der humanen mRNA
mittels BLAST (Basic Local Alignment Search Tool) Suche aus veroumlffentlichten
Sequenzen der NCBI Datenbank (Boxer genome assembly 21) entnommen Die
Exon Intron Grenzen wurden mit Hilfe des mRNA-to-genomic alignment Programms
Spidey bestimmt und Primer mit Hilfe des Primer3 Programms in die intronischen
Sequenzen gelegt Die SNPs wurden an den DNA-Sequenzen von Elterntieren der
betroffenen Dalmatinerfamilien identifiziert Die Amplifikate wurden mit Hilfe des
MegaBACE 1000 sequenziert und auf SNPs untersucht Bei Vorliegen eines oder
mehrerer SNPs wurde die entsprechende DNA-Sequenz der zugehoumlrigen
Nachkommen ebenfalls amplifiziert und sequenziert Die Auswertung erfolgte mit
Hilfe des Sequencher 42 Programms
Kopplungsanalyse
Die Genotypen der neuen SNP Marker wurden unter Verwendung des Programms
MERLIN ausgewertet Die Daten wurden mit dem Softwarepaket SAS (Statistical
Analysis System Version 913) bearbeitet um das Markerset bezuumlglich
Allelfrequenzen beobachteten Heterozygotiegrad und PIC (Polymorphism
information content) charakterisieren zu koumlnnen Des Weiteren wurde auf
Kopplungsungleichgewicht und die Assoziation der Haplotypen mit CCSD mittels
Erweiterte Zusammenfassung 137
des Programms CASECONTROL und HAPLOTYPE von SAS Genetics (Statistical
Analysis System Version 913 Cary NC USA) getestet
Ergebnisse und Diskussion Fuumlr acht Kandidatengene wurden mindestens zwei heterozygote SNPs entwickelt
Die nicht-parametrische Kopplungsanalyse zeigte keine signifikanten Teststatistiken
Offensichtlich war kein Haplotyp mit der caninen kongenitalen sensorineuralen
Taubheit (CCSD) in den untersuchten Familien assoziiert Da sowohl uni- und
bilateral taube Tiere als auch beidseitig houmlrende Tiere uumlber identische Haplotypen
verfuumlgen ist es unwahrscheinlich dass die Gene TMC1 TMIE USH1C MYH14
MYO3A PRES WHRN und ESPN an der Entstehung der CCSD beim Dalmatiner
beteiligt sind
Molekulargenetische Analyse des caninen myosin heavy polypeptide 9 non-muscle (MYH9) auf dem caninen Chromosom 10q232
Material und Methoden Klonierung und Sequenzierung von caniner MYH9 cDNA
Die canine MYH9 Gensequenz wurde mit Hilfe der BLAST Suche aus
veroumlffentlichten Sequenzen der NCBI Datenbank Die Isolierung der vollstaumlndigen
cDNA wurde durch ein Protokoll zur Amplifizierung von cDNA-Enden (rapid
amplification of cDNA ends [RACE]) erreicht Nach Klonierung und Sequenzierung
der RACE-Produkte wurden die Sequenzen mit Hilfe des Sequencher 42
Programms ausgewertet
Sequenzanalyse des caninen MYH9 Gens
Nach Fertigstellung des RACE Protokolls und Auswertung der Sequenzen wurden
die Exon Introngrenzen mit Hilfe des mRNA-to-genomic alignment Programms
Spidey lokalisiert Weiterhin wurden repetitive Elemente mit dem Programm
Repeatmaster 2 sowie der GC Gehalt mit der EBI toolbox CpG PlotCpGreport
ermittelt
Erweiterte Zusammenfassung
138
Mutatiosanalyse
Zur Identifizierung von Polymorphismen innerhalb der caninen MYH9 Sequenz
wurden die Exons mit ihren flankierenden intronischen Sequenzen mit Hilfe der PCR
amplifiziert und sequenziert Verwendet wurden hierfuumlr die DNA von insgesamt 16
Dalmatinern aus drei Familien Mindestens zwei der Vollgeschwister einer Familie
waren unilateral taub Der Phaumlnotyp der verwendeten Tiere wurde durch einen
audiometrischen Test bestimmt
PCR Primer wurden mit Hilfe des Primer3 Programms entwickelt PCR Reaktionen
fuumlr saumlmtliche Exons des MYH9 Gens mit ihren flankierenden Sequenzen wurden
durchgefuumlhrt die PCR Produkte sequenziert und die Sequenzen mit Hilfe des
Sequencher 42 Programms ausgewertet
Unter Verwendung der Software MERLIN (multipoint engine for rapid likelihood
inference Version 0910) wurde nach signifikanten nicht parametrischen LOD-
Scores fuumlr die Kosegregation von Markerallelen mit der phaumlnotypischen Auspraumlgung
der sensorineuralen Taubheit beim Dalmatiner gesucht
Ergebnisse und Diskussion
Analyse der genomischen Organisation und der cDNA des caninen MYH9 Gens
Eine vollstaumlndige cDNA Sequenz des caninen MYH9 Gens von 7484 bp konnte uumlber
die Reverse Transkription-PCR erhalten und bei der EMBL Nukleotid Datenbank
eingereicht werden Das canine MYH9 Gen besteht aus 41 Exons einschlieszliglich
einem untranslatierten ersten Exon und Exon Intron Grenzen die stets der GT AG
Regel folgen Der 5rsquo-UTR Bereich besteht aus 181 bp waumlhrend der 3rsquo-UTR aus 1432
bp besteht Weiterhin besitzt die canine MYH9 cDNA Sequenz einen offenen
Leserahmen von 5889 bp welcher ein Protein kodiert bestehend aus 1963
Aminosaumluren Die Groumlszlige der Exons variiert von 28 bis 1556 bp waumlhrend das
gesamte MYH9 Gens ungefaumlhr 90 kb umfasst Die Homologie der Sequenzen
zwischen dem humanen murinen und caninen MYH9 Gen ist limitiert auf die
kodierende Sequenz von Exon 2 bis 41
Mutations- und Haplotypenanalyse
Alle kodierenden Exons mit ihren flankierenden intronischen Sequenzen des MYH9
Gens konnten von den 16 Dalmatinern amplifiziert werden Die Sequenzen wurden
mit den entsprechenden Sequenzen vom Boxer aus der NCBI Genbank verglichen
Erweiterte Zusammenfassung 139
Die Suche nach Sequenzvariationen im MYH9 Gen lieszlig 22 SNPs erkennen wobei
die meisten Polymorphismen in den flankierenden Regionen der Exons gefunden
wurden Keiner der beobachteten Polymorphismen in den Exons aumlnderte die
Aminosaumluresequenz von MYH9 Auch zeigten die identifizierten Haplotypen keine
Assoziation mit dem CCSD Phaumlnotyp Multipoint Teststatistiken fuumlr den Anteil von
IBD-Markerallelen (Zmean) und ein daraus abgeleiteter LOD-Score lagen bei
annaumlhernd Null und waren nicht signifikant Zwei genassoziierte Mikrosatelliten
zeigten eine Kopplung diese kann jedoch mit Heterogenitaumlt zwischen den Familien
erklaumlrt werden
Fazit
Die Charakterisierung des Transkriptes und der genomischen Sequenz des caninen
MYH9 Gens zeigten einen konservierten Aufbau des Gens im Bezug auf das
humane orthologe Gen Aufgrund eines untranslatierten Exons im 5rsquo-UTR Bereich ist
das canine Gen groumlszliger als das humane Gen
Sowohl uni- oder bilateral taube und bilateral houmlrende Dalmatiner weisen identische
Haplotypen auf Daher sind in den untersuchten Dalmatinerfamilien die gefundenen
Polymorphismen offensichtlich nicht mit CCSD assoziiert Des Weiteren zeigt diese
Studie dass keine funktionellen Mutationen der vollstaumlndigen kodierenden Region
von MYH9 vorliegen Wir koumlnnen also abschlieszligend das MYH9 Gen als
Kandidatengen fuumlr die congenitale sensorineurale Taubheit beim Dalmatiner
ausschlieszligen
Erweiterte Zusammenfassung
140
Evaluierung von Mikrosatelliten und SNP Markern der caninen Chromosomen 1 10 und 31 auf Kopplung und Assoziation mit der caninen kongenitalen Taubheit Material und Methoden Pedigreematerial
Insgesamt wurden 176 Dalmatiner in die Untersuchung einbezogen Die Tiere
verteilten sich auf 23 unverwandte Vollgeschwisterfamilien und eine paternale
Halbgeschwistergruppe Die genotypisierten Familien bestanden aus saumlmtlichen
tauben Nachkommen (unilateral oder bilateral taub) ihren Eltern und einen bis vier
nicht betroffenen Voll- bzw Halbgeschwistern Mindestens zwei der Vollgeschwister
einer Familie waren unilateral oder bilateral taub Der Phaumlnotyp der verwendeten
Tiere wurde durch einen audiometrischen Test bestimmt
Alle 176 Dalmatiner wurden fuumlr die Mikrosatellitenstudie auf den caninen
Chromosomen 1 10 und 31 einbezogen Fuumlr die Feinkartierung mit Hilfe der SNPs
wurden dagegen nur noch die Dalmatinerfamilien mit einer eindeutigen Kopplung zu
der entsprechenden genomischen Region untersucht Fuumlr die Region auf CFA10
wurden dafuumlr 34 Tiere aus fuumlnf Vollgeschwisterfamilien ausgewaumlhlt und zur
Untersuchung des CLDN14 Gens auf CFA31 wurden 24 Tiere aus vier
Vollgeschwisterfamilien und 12 Tiere aus einer groszligen Halbgeschwisterfamilie
verwendet Fuumlr die Suche nach Polymorphismen im GJA1 Gen wurden 16 Tiere aus
einer groszligen Halbgeschwisterfamilie verwendet
Mikrosatellitenstudie
Fuumlr die Mikrosatellitenstudie von CFA1 10 und 31 wurden insgesamt 60 Marker
verwendet Mit jeweils 27 Mikrosatelliten wurde CFA1 und mit 27 Mikrosatelliten
wurde CFA10 vollstaumlndig abgedeckt Insgesamt 6 Marker wurden fuumlr CFA31
verwendet
Alle hier verwendeten Mikrosatelliten sind der NCBI Datenbank entnommen und
wurden uumlber PCR und Polyacrylamidgelelektrophorese ausgewertet
Identifizierung von SNPs (Single Nucleotide Polymorphisms)
Zur weiteren Abklaumlrung und zum Ausschluss falsch positiver Ergebnisse wurden
zusaumltzlich zu den Mikrosatelliten SNPs in den signifikanten Regionen auf CFA1 10
Erweiterte Zusammenfassung 141
und CFA 31 entwickelt und an den Familien die eine Kopplung zeigten
genotypisiert
Die caninen genomischen Sequenzen der untersuchten Gene wurden der NCBI
Datenbank (Boxer genome assembly 21) entnommen Exon Intron Grenzen wurden
bestimmt und Primer entwickelt Nach Durchfuumlhrung der PCR wurden die Amplifikate
mit Hilfe des MegaBACE 1000 sequenziert und auf SNPs untersucht Die
Auswertung erfolgte mit Hilfe des Sequencher 42 Programms
Die Genotypen der neuen SNP Marker wurden zu den Genotypen der fuumlr die
Kopplungsanalyse verwendeten Mikrosatelliten hinzugefuumlgt und gemeinsam unter
Verwendung des Programms MERLIN ausgewertet
Kopplungsanalyse
Unter Verwendung der Software MERLIN (multipoint engine for rapid likelihood
inference Version 0910) wurde nach signifikanten nicht parametrischen LOD-
Scores fuumlr die Kosegregation von Markerallelen mit der phaumlnotypischen Auspraumlgung
der sensorineuralen Taubheit beim Dalmatiner gesucht
Die Daten wurden mit dem Softwarepaket SAS (Statistical Analysis System Version
913) bearbeitet um die Marker bezuumlglich Allelfrequenzen beobachteten
Heterozygotiegrad und PIC (Polymorphism information content) charakterisieren zu
koumlnnen Des Weiteren wurde das Kopplungsungleichgewicht und die Assoziation der
Haplotypen mit CCSD mittles des Programms CASECONTROL und HAPLOTYPE
von SAS Genetics getestet
Ergebnisse und Diskussion CFA1
In einer groszligen Halbgeschwisterfamilie wurde eine signifikante Kopplung zu einem
GJA1 genassozierten Marker festgestellt Mit Hilfe der DNA von Tieren aus dieser
Halbgeschwisterfamilie wurde eine Sequenzanalyse des GJA1 Gens durchgefuumlhrt
Es wurden 3 SNPs im 3acute-untranslatierten Bereich gefunden Die identifizierten
Haplotypen zeigten keine Assoziation mit dem CCSD Phaumlnotyp Sowohl uni- oder
bilateral taube und bilateral houmlrende Dalmatiner wiesen identische Haplotypen auf
Daher sind in den untersuchten Dalmatinerfamilien die gefunden Polymorphismen
offensichtlich nicht mit CCSD assoziiert Auch wurde keine funktionelle Mutation in
der vollstaumlndigen kodierenden Region von GJA1 gefunden wurde Es ist daher
Erweiterte Zusammenfassung
142
unwahrscheinlich dass das GJA1 Gen an der Entwicklung der CCSD in der
untersuchten Halbgeschwisterfamilie beteiligt ist Die signifikante Kopplung der
GJA1-benachbarten Mikrosatelliten mit CCSD kann durch ein eng gekoppeltes Gen
hervorgerufen worden sein welches fuumlr die Entstehung der Taubheit in der
untersuchten Halbgeschwisterfamilie verantwortlich ist oder durch ein falsch-
positives Ergebnis der vorhergehend durchgefuumlhrten Mikrosatellitenstudie
Eine nicht-parametrische Kopplungsanalyse mit Hilfe von 27 Mikrosatelliten Marker
an 24 deutschen Dalmatinerfmilien wurde durchgefuumlhrt um abzuklaumlren ob andere
Genombereiche auf CFA1 signifikante Teststatistiken in mehr Familien zeigen Die
Kopplungsanalyse zeigte keine signifikanten Teststatistiken Es ist daher
unwahrscheinlich dass auf CFA1 Gene fuumlr die Entwicklung von CCSD in den
untersuchten Familien der aus Deutschland stammenden Dalmatiner verantwortlich
sind
Da kuumlrzlich festgestellt wurde dass das GJA1 Gen auch beim Menschen nicht fuumlr
eine Form der sensorineuralen nicht-syndromischen Taubheit verantwortlich ist
sondern an einer syndromischen Krankheit beteiligt ist die mit konduktiver Taubheit
einhergehen kann sollte das GJA1 Gen nicht laumlnger als ein Kandidatengen fuumlr
CCSD angesehen werden
CFA31
Auf CFA31 wurde durch eine nicht-parametrische Kopplungsanalyse von
Mikrosatelliten eine Kopplung eines CLDN14-benachbarten Markers mit der
sensorineuralen Taubheit beim Dalmatiner festgestellt Daraufhin wurden zusaumltzlich
zu den Mikrosatelliten acht SNPs entwickelt indem das einzige beim Menschen
translatierte Exon weite intronische Sequenzen sowie 5rsquo- und 3rsquo- untranslatierte
Bereiche des CLDN14 Gens einer Sequenzanalyse unterzogen wurden Nur zwei der
identifizierten SNPs waren polymorph in den untersuchten fuumlnf Dalmatinerfamilien
Sowohl uni- als auch bilateral taube und bilateral houmlrende Dalmatiner teilten
identische Haplotypen daher konnte keine Kosegregation mit tauben Dalmatinern
festgestellt werden
Da bislang keine funktionelle Mutation in der kodierenden Region von CLDN14
gefunden wurde ist es fuumlr eine eindeutige Aussage uumlber die Rolle des CLDN14
Gens im Entstehungsprozess der kongenitalen sensorineuralen Taubheit
Erweiterte Zusammenfassung 143
beim Dalmatiner notwendig noch weitere Marker im CLDN14 Gen sowie seiner
naumlheren Umgebung zu entwickeln und zu analysieren Da keine weitere Kopplung zu
einem Mikrosatelliten distal oder proximal des CLDN14 Gen festgestellt werden
konnte ist es wahrscheinlich dass entweder das CLDN14 Gen oder ein Gen in
seiner naumlheren Umgebung fuumlr die Entwicklung der sensorineuralen Taubheit in den
untersuchten Dalmatinerfamilien verantwortlich ist
CFA10
Auf CFA10 wurde durch eine nicht-parametrische Kopplungsanalyse von 27
Mikrosatelliten eine 12 Mb umfassende genomische Region mit einer signifikanten
Kopplung zur CCSD identifiziert Zur weiteren Feinkartierung wurden 26 neue SNPs
mit einem durchschnittlichen Abstand von 03 Mb fuumlr diesen Bereich entwickelt
Die SNPs waren groumlszligtenteils informativ im vorliegenden Familienmaterial und
bestaumltigten in einer folgenden Kopplungsanalyse die Lokalisation auf CFA10
deutlich Weiterhin konnte die gekoppelte Region auf einem 5 Mb umfassenden
Bereich von 39 bis 44 Mb eingegrenzt werden Es ist wahrscheinlich dass die
identifizierte CCSD Region ein Gen beinhaltet welches auf die Entstehung der
caninen kongenitalen Taubheit einwirkt
Es ist notwendig mehr SNPs zu entwickeln um das taubheitsverursachende Gen zu
lokalisieren oder einen eindeutig assoziierten SNP zu identifizieren
Chapter 12
References
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Chapter 13
Appendix
Appendix microsatellite marker III
HE
Te
79
5 7
98
76
9 5
12
71
3 8
33
70
2 4
35
72
7 7
00
54
6 4
77
48
8 3
08
44
8 5
40
67
4 8
05
76
2 6
54
70
8 7
73
78
1 6
87
65
4 6
26
51
9 5
89
76
4 1
60
PIC
d
72
2 8
41
67
0 4
85
67
1 7
85
57
2 4
07
61
4 6
29
35
8 3
30
40
0 2
90
35
0 4
60
70
0 7
90
76
2 6
21
63
8 7
26
77
1 6
26
60
1 6
13
38
7 5
52
80
0 1
40
Prim
er (r
ever
se)
5acute -gt
3acute
AA
CTG
TTTG
ATT
TTG
ATG
AG
GC
C
TTTG
ACC
TAC
ATA
TAA
AA
CA
AG
C
CA
CG
AA
GAA
AG
CC
ATG
GTT
T C
AA
CA
ATT
CC
TTTG
TTG
GTG
C
CTG
CC
AG
CTT
CC
TAC
AGC
T
GTG
GTC
ACA
AG
AC
TTTA
GC
C
CTG
CA
CC
AAA
GA
AA
CTC
C
GG
AC
CTA
TTC
TGA
AG
CC
TAA
C
TG
GC
CTT
TAA
TTTA
TCAT
GG
AG
A
TCA
GG
CC
CA
GA
TAG
TATG
C
TC
CTT
TGG
TTTT
TAG
CAG
GG
T
CTG
TGC
CC
AC
CTG
TGG
AG
C
AA
GA
GG
ATA
CC
GG
TGG
CA
G
CG
TGC
TTTG
TTA
TGG
CTT
GA
C
AG
CA
TGA
AG
GA
TCTC
TGA
CTA
A
CTA
AC
TCG
CC
AG
AC
TATT
C
CC
CTC
CAC
CA
ATC
TCTT
CA
C
GC
AA
AG
GTG
TATT
TAC
ATG
ATG
G
GG
CA
GC
CTT
ATT
ATT
CA
TGG
A
AC
AC
AG
GC
AC
AG
GA
GC
ATC
C
AG
CC
ATC
CA
GG
AA
ATC
G
CTG
AA
CTG
GTC
CC
TCAA
GC
A
GTC
ATG
CTG
ATT
TCTG
TGC
C
GA
CC
TATC
CA
TAC
ATG
CC
C
GAA
GG
AAG
GAA
GG
AAG
AAAA
GA
TTA
GTA
AA
GA
GA
CA
GC
TGA
TTG
CC
A
TCTA
CC
CTG
CA
ACC
CTG
TG
ATT
GA
GA
CC
CA
AG
ACTG
TTA
GTG
G
GTA
AC
AAC
CA
GAG
TGTG
TGTG
A
CG
TCG
AG
CTC
CTG
GC
AT
Prim
er (f
orw
ard)
5acute -
gt 3acute
GG
GTA
GA
TTTC
AA
CA
AAT
AG
TAC
TGG
T
AG
AA
AG
GC
CTG
GA
TGTC
G
AG
TAG
AA
GA
GG
CC
AC
GC
AA
A
TTT
TCC
AA
AA
TGA
AA
AC
CTG
C
AG
ATT
GTT
TATG
CA
GG
CA
TTG
A
GA
GTT
GAA
AG
GTT
GAA
AA
TGG
G
CTC
AG
AG
GC
AG
ATA
GA
AA
A
ATT
GA
TTC
ATG
ACC
CA
CTA
A
TTT
TGTC
AAG
CA
GC
CC
TATA
TC
TG
AA
ATG
GTA
CTT
CA
GC
ATC
G
AG
CTT
AG
CTT
AC
TGC
CC
TGG
T
CC
CC
GG
CC
CC
TGC
CC
GG
AG
T
AC
CAC
TGTC
ATT
TTTC
CA
TGC
C
CC
TTA
GG
AGG
AG
GC
AAG
AC
C
CA
AG
GG
GTA
TGTT
GTC
TATT
ACT
GG
GTG
GG
AA
ATG
TGA
CTC
TG
GG
GC
TGC
CC
ATT
TCTT
TAA
T T
TGC
CA
CAA
ATC
AC
TTAA
AG
G
GA
CTG
AG
TTC
TTTC
AG
CA
CA
GTG
A
AAAT
AAC
ACAG
CAT
CAG
G
GA
ATC
CC
CA
ATG
TAC
ATG
GC
A
AC
AG
GC
GG
ATT
CTG
TAG
C
AC
TGG
CC
AAA
GA
GTA
CA
AG
G
CC
TTG
AC
TCA
GC
AGC
CC
TAC
A
AA
GA
TTG
TCTT
GA
CAC
GC
TG
GG
GTC
CTG
GG
ATC
AA
GC
C
AC
TGA
CTG
ATG
TCC
TGTG
CG
A
AA
AA
GTG
TAG
AG
CTT
TCTT
CA
AA
T
AA
ATT
CA
ATA
AG
CC
ATG
GA
GG
A
CC
TCC
AAG
ATG
GC
TCTT
GA
ATc
(degC
) 60
58
58
56
58
60
54
56
60
60
58
60
60
60
60
60
60
60
60
52
56
58
54
62
60
60
62
62
60
60
Pro
duct
si
ze (b
p)
380-
400
337-
409
267-
304
225-
235
312-
336
350-
394
155-
161
126-
156
510-
570
436-
468
189-
191
72-8
0 13
0-13
6 34
0-35
0 14
5-15
3 98
-102
22
0-24
0 21
2-24
0 34
7-40
3 32
5-34
1 25
1-27
9 32
5-36
1 19
7-24
5 30
9-40
9 34
1-37
5 21
5-23
1 30
8-31
6 16
2-17
4 46
8-50
2 18
2-19
0
Alle
les
6 14
8 4 7 11
3 6 8 7 2 2 3 4 2 3 5 8 12
7 8 7 14
10
9 7 3 3 15
2
Mbb
54
1
49
19
8 2
18
23
5 2
47
26
0 3
02
35
3 3
98
58
8 6
26
63
9 7
61
77
5 7
78
78
6 7
98
85
4 1
060
1
090
1
100
1
100
1
110
1
130
1
150
1
170
3
5
12
4 1
63
CFA
a
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10
10
10
Tabl
e 1
Cha
ract
eris
tics
of m
icro
sate
llite
mar
kers
sel
ecte
d fo
r CFA
1 1
0 an
d 31
com
pris
ing
in to
tal 6
0 m
arke
rs
(Con
tinue
d on
nex
t pag
e) T
he m
icro
sate
llite
mar
kers
wer
e al
l der
ived
from
the
NC
BI d
atab
ase
(http
w
ebn
cbin
lmn
ihg
ov)
Mar
ker
FH33
25
FH36
03
FH23
13
RE
N18
9I14
FH
3219
FH
3413
R
EN
136G
19
RE
N13
5K06
FH
3922
FH
3300
C
014
24
AH
T107
C
012
51
RE
N17
2C02
D
0150
5 R
EN
04H
08
RE
N15
9F24
FH
3894
FH
2309
R
EN
06N
11
FH23
26
FH33
22
FH35
05
FH25
98
FH26
34
FH22
94
RE
N22
9P15
FH
2537
FH
4081
C
107
81
Appendix microsatellite marker
IV
H
ETe
64
0 6
80
63
0 6
51
54
6 7
06
81
0 6
14
30
2 7
42
34
4 7
40
63
0 8
67
82
2 6
70
75
0 6
80
60
0 4
70
31
0 5
70
80
1 5
80
83
1 5
10
64
0 6
90
19
0 8
22
PIC
d
53
0 6
90
52
2 5
89
80
0 6
70
75
9 5
24
24
7 7
09
26
9 6
90
50
0 7
98
75
5 5
50
69
0 5
30
56
0 3
40
26
0 5
00
80
1 5
30
74
0 3
70
64
0 6
70
25
0 7
21
Prim
er (r
ever
se)
5acute -gt
3acute
AG
GC
CA
GC
AC
CA
TAA
CTC
AG
C
CTC
TCC
TCC
TTC
CC
CTT
CTC
A
GTG
CTG
TCC
ATT
GC
AA
GTC
T
GC
TCC
CC
TGTG
TTC
TGAG
G
CTG
GTG
ACTC
AGA
GAA
GTC
C
GG
AG
TTG
ATA
GTA
AA
TCTT
TGC
GG
T
GC
CC
TATT
CA
TTC
AA
CTG
G
AC
AC
TCTG
GTG
CA
AG
CG
AC
TC
TTC
TAC
TGA
AG
CA
TCC
GC
CT
GA
TTTT
CTC
TCTG
TCC
AC
TT
AA
CA
GC
ATT
TCA
GAC
AGA
GG
G
GG
TTTT
GG
CTG
TGA
GG
ATA
G
AG
TTC
CAG
ATC
GAG
TCC
CA
C
CA
CAT
GAT
TTC
ACTT
GTA
TATG
G
CC
TGA
GG
GTT
TTTA
CAG
TATG
G
TG
AA
ATC
TGG
GA
TTC
TGA
TAC
C
CC
AA
GC
TTTC
AC
CTA
ATC
AC
C
GA
TATT
TTTC
TCTC
CC
AC
CC
C
AC
ATT
TCTA
GG
CAG
TGG
CA
C
TGTC
CC
ATC
TAA
AG
GA
TAG
G
AG
TAA
TGC
TGC
CC
ACG
GA
AC
G
GC
TGG
AA
CC
TTC
CC
CTT
TAG
A
GC
TCTT
ACA
CG
CA
TTG
AG
G
AA
AC
GG
AG
TTC
CA
TCTC
TGG
C
AC
AG
TGA
GG
AG
GG
GTG
G
TG
TAA
CC
CTC
CTG
TGA
TATG
A
TGTG
AA
CC
CC
GC
CC
AATA
G
AA
GTG
AC
TGA
GTA
CC
TGA
AA
TCG
G
CC
TCTG
CC
TCTG
CC
TCTG
T G
AG
CC
CTG
TTC
TCA
GG
TTG
Prim
er (f
orw
ard)
5acute -
gt 3acute
AG
AC
CC
CC
CC
ACTA
CC
CC
AC
GC
GTT
AGC
TGC
CA
TCA
TCTT
G
CTG
GTT
GG
AG
CA
ATA
CC
AA
G
CC
ATT
CA
TGTT
GTT
GC
AG
ATG
T
AA
GG
AG
GG
CA
CTT
GAT
GG
C
TTA
TGG
CA
TTTG
GC
CTG
TC
AA
GG
AG
CAT
CTT
CC
AG
AAC
C
CG
CTC
ATG
CA
AG
TCA
TCA
CA
T C
TGG
CTT
AAA
CC
AC
TGG
TCA
C
CTT
CC
ATC
CC
GTT
GTG
TGT
ATT
CC
CC
AGC
GAT
ACC
A C
TTG
AG
TGG
CTT
GA
GTG
GC
TAC
T
TCTT
GC
TTC
TTG
AA
GTA
AG
CC
T
TGC
CC
GTC
CTA
TAC
TCC
TG
GA
AG
GTG
GTT
ATT
GTC
CTT
GG
G
AA
CC
ACA
GA
AG
AG
CTG
GA
A
GC
ATT
TGA
TGA
AA
TAA
AG
GG
A
GA
ATG
AA
AA
CG
GA
GC
AG
CA
G
CC
TTC
AAC
AC
CC
ATA
GC
TC
TC
AG
CA
ACTA
TAC
ATT
TAA
GA
GC
A
ATG
GA
CA
AATG
AA
CA
AA
AG
T
GTG
AC
TTTC
TTA
TCC
GC
CC
C
CC
AG
AA
AC
TCA
ACTG
ATG
C
TG
GA
CG
CTA
AG
CC
TGAC
TTT
CC
ATA
AG
ATA
CTC
AG
AA
AC
ATG
CA
C
AC
CAT
AA
ATG
GA
TGG
ATA
G
CA
GTG
AG
CA
AA
GC
AA
ATG
AA
C
CC
ATT
AG
CA
AA
TGA
CTG
GG
A
AA
GA
ATG
GG
AA
AA
CTG
ATA
A
AA
GG
TAG
TCC
CA
CG
ATC
CTC
ATc
(degC
) 60
60
60
60
60
60
60
60
60
60
60
60
60
62
60
60
60
60
60
60
60
60
60
60
60
60
60
60
60
60
Pro
duct
si
ze (b
p)
100-
110
112-
124
194-
206
210-
230
309-
341
350-
362
276-
300
96-1
30
130-
138
120-
138
200-
204
278-
296
157-
165
178-
254
358-
368
327-
337
380-
404
160-
170
320-
330
165-
169
230-
232
300-
310
276-
380
255-
263
258-
278
106-
120
164-
180
568-
608
223-
239
172-
186
Alle
les
3 5 3 5 8 4 7 4 2 5 3 7 5 9 6 4 7 3 4 2 2 3 11
5 6 2 4 7 4 8
Mbb
24
9 3
23
34
9 3
67
38
9 3
94
40
1 4
10
45
8 4
64
48
0 4
88
51
5 5
64
56
8 5
97
62
7 6
33
63
7 6
53
67
3 6
85
69
1 7
08
0
66
9
7
28
1 2
96
37
5
CFA
a
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
31
31
31
31
31
31
Tabl
e 1
(con
tinue
d)
Mar
ker
RE
N06
H21
R
EN
68B
08
FH30
55
C10
769
FH
3302
R
EN
181G
20
FH34
03
G02
512
RE
N15
0B12
A
HT1
01
RE
N73
F08
ZUB
EC
A1
C10
16
FH24
22
FH34
48
FH26
65
DTR
105
C
106
02
RE
N16
1L12
C
0410
7 R
EN
91H
07
RE
N15
4O19
FH
3381
R
EN
154G
10
FH21
89
RV
C11
R
EN
43H
24
FH22
39
RE
N11
0K04
FH
2712
a Can
ine
chro
mos
ome
b Pos
ition
of m
arke
r (M
b) c A
nnea
ling
tem
pera
ture
d P
olym
orph
ism
info
rmat
ion
cont
ent (
)
e obse
rved
Het
eroz
ygos
ity
Appendix itemisation of alleles V
Table 2 Itemisation of alleles to bases for SNPs in the pedigrees
Code no 1 2 3 4
Base Adenine Cytosine Guanine Thymine
Appendix laboratory paraphernalia
VI
Laboratory paraphernalia Equipment Thermocycler
PTC-100trade Programmable Thermal Controller (MJ Research Watertown USA)
PTC-100trade Peltier Thermal Cycler (MJ Research Watertown USA)
PTC-200trade Peltier Thermal Cycler (MJ Research Watertown USA)
Automated sequencers
LI-COR Gene Read IR 4200 DNA Analyzer (LI-COR Inc Lincoln NE USA)
LI-COR Gene Read IR 4300 DNA Analyzer (LI-COR Inc Lincoln NE USA)
MegaBACE 1000 (Amersham Biosciences Freiburg)
Centrifuges
Sigma centrifuge 4-15 (Sigma Laborzentrifugen GmbH Osterode)
Desk-centrifuge 5415D (Eppendorf Hamburg)
Biofuge stratos (Heraeus Osterode)
Centrifuge Centrikon H 401 (Kontron Gosheim)
Megafuge 1OR (Heraeus Osterode)
Speed Vacreg Plus (Savant Instruments Farmingdale NY USA)
Agarose gel electrophoresis and pulsed field gel electrophoresis
Electrophoresis chambers OWL Separation Systems Portsmouth NH USA
Biometra Goumlttingen
BioRad Muumlnchen
Generators 2301 Macrodrive 1 (LKB Bromma Sweden)
Power Pac 3000 (BioRad Muumlnchen)
Gel documentation system BioDocAnalyze 312 nm Goumlttingen
Appendix laboratory paraphernalia VII
Others
Milli-Qreg biocel water purification system (Millipore GmbH Eschborn)
Incubator VT 5042 (Heraeus Osterode)
UV-Illuminator 312 nm (Bachhofer Reutlingen)
Centomatreg R Desk-Shaker (B Braun Melsungen AG Melsungen)
Biophotometer (Eppendorf AG Hamburg)
Kits
DNA purification
Montage PCR96 Cleanup Kit (Millipore GmbH Eschborn)
Cloning
Invitrogen TA cloningreg kit (Invitrogen Karlsruhe Germany)
Isolation of DNA
QIAamp 96 DNA Blood Kit (QIAGEN Hilden)
Plasmid Mini Prep 96 Kit (Millipore GmbH Eschborn)
Sequencing
ThermoSequenase Sequencing Kit (Amersham Biosciences Freiburg)
DYEnamic-ET-Terminator Cycle Sequencing Kit (Amersham Biosciences Freiburg
Germany)
RACE
FirstChoiceTM RNA ligase-mediated (RLM)-RACE kit (Ambion Europe Huntingdon
UK)
Appendix laboratory paraphernalia
VIII
RNA Total RNA (Biocat Heidelberg Germany)
Size standards
100 bp Ladder (New England Biolabs Schwalbach Taunus)
1 kb Ladder (New England Biolabs Schwalbach Taunus)
IRDyetrade 700 or 800 (LI-COR Inc Lincoln NE USA)
Reagents and buffers APS solution (10 )
1 g APS
10 ml H2O
Bromophenol blue solution
05 g bromophenol blue
10 ml 05 M EDTA solution
H2O ad 50 ml
dNTP solution
100 microl dATP [100 mM]
100 microl dCTP [100 mM]
100 microl dGTP [100 mM]
100 microl dTTP [100 mM]
1600 microl H2O
the concentration of each dNTP in the ready-to-use solution is 5 mM
Gel solution
1275 ml UreaTBE solution (Roth Karlsruhe)
225 ml Rotiphoresereg Gel 40 (38 acrylamide and 2 bisacrylamide)
95 microl APS solution (10 )
95 microl TEMED
Appendix laboratory paraphernalia IX
Loading buffer for agarose gels
EDTA pH 8 100 mM
Ficoll 400 20 (wv)
Bromophenol blue 025 (wv)
Xylencyanol 025 (wv)
Loading buffer for gel electrophoresis
2 ml bromophenol blue solution
20 ml formamide
TBE-buffer (1x)
100 ml TBE-buffer (10x)
900 ml H2O
TBE-buffer (10x)
108 g Tris [12114 M]
55 g boric acid [6183 M]
744 g EDTA [37224 M]
H2O ad 1000 ml
pH 80
UreaTBE solution (6 )
425 g urea [6006 M]
250 ml H2O
100 ml TBE-buffer (10x)
solubilise in a water bath at 65degC
H2O ad 850 ml
Appendix laboratory paraphernalia
X
Chemicals Agarose (Invitrogen Paisley UK)
Ammonium persulfate (APS) ge 98 (Sigma-Aldrich Chemie GmbH Steinheim)
Ampicillin (Serva Heidelberg)
Boric acid ge 998 pa (Carl Roth GmbH amp Co Karlsruhe)
Bromophenol blue (Merck KgaA Darmstadt)
dATP dCTP dGTP dTTP gt 98 (Carl Roth GmbH amp Co Karlsruhe)
Chloramphenicol (Serva Heidelberg)
DMSO ge 995 pa (Carl Roth GmbH amp Co Karlsruhe)
dNTP-Mix (Qbiogene GmbH Heidelberg)
EDTA ge 99 pa (Carl Roth GmbH amp Co Karlsruhe)
Ethidium bromide (Carl Roth GmbH amp Co Karlsruhe)
Ethyl alcohol (AppliChem Darmstadt)
Formamide ge 995 pa (Carl Roth GmbH amp Co Karlsruhe)
LB (Luria Bertani) agar (Scharlau Microbiology Barcelona Spain)
Paraffin (Merck KgaA Darmstadt)
RotiphoreseregGel40 (Carl Roth GmbH amp Co Karlsruhe)
SephadexTM G-50 Superfine (Amersham Biosciences Freiburg)
TEMED 99 pa (Carl Roth GmbH amp Co Karlsruhe)
Tris PUFFERANreg ge 999 pa (Carl Roth GmbH amp Co Karlsruhe)
Urea ge 995 pa (Carl Roth GmbH amp Co Karlsruhe)
Water was taken from the water purification system Milli-Qreg
X-Gal (AppliChem Darmstadt)
Enzymes Taq-DNA-Polymerase 5 Umicrol (Qbiogene GmbH Heidelberg)
Taq-DNA-Polymerase 5 U microl (Qiagen Hilden Germany)
Incubation Mix (10x) TPol with MgCl2 [15 mM] (Qbiogene GmbH Heidelberg)
The polymerase was always used in the presence of incubation Mix TPol 10x buffer
The encyme ECO RI (New England Biolabs Schwalbach Taunus) were used with
the adequate 10x encyme buffer
Appendix laboratory paraphernalia XI
Consumables
96 Well Multiply PCR plates skirted (Sarstedt Nuumlmbrecht)
Combitipsreg plus (Eppendorf AG Hamburg)
Pipette tips 01 ndash 10 microl 01 ndash 10 microl 5 ndash 200 microl (Carl Roth GmbH amp Co Karlsruhe)
Reaction tubes 15 and 20 ml (nerbe plus GmbH WinsenLuhe)
Reaction tubes 10 und 50 ml (Falcon) (Renner Darmstadt)
Thermo-fast 96 well plate skirted (ABgene Hamburg)
Software BLAST trace archive httpwwwncbinlmnihgov
httpwwwensemblorg
EBI toolbox httpwwwebiacukToolssequencehtml
MERLIN 0102 package httpwwwsphumicheducsgabecasisMerlin
Order of primers MWG Biotech-AG Ebersberg (httpsecom
mwgdnacomregisterindextcl)
biomersnet GmbH Ulm (orderbiomersnet)
PED50 Dr H Plendl et al (2005) Institute for Human Genetics
Kiel
Primer design httpfrodowimiteducgi-binprimer3primer3_ wwwcgi
Repeat Masker httpwwwrepeatmaskergenome washingtonedu
Sequencher 42 GeneCodes Ann Arbor MI USA
Spidey httpwwwncbinlmnihgovIEBResearch
OstellSpideyindexhtml
SUN Ultra Enterprise 450 Sun microsystems
XIII
List of publications Journal articles 1 MIESKES K WOumlHLKE A DROumlGEMUumlLLER C DISTL O 2006 Molecular
characterization of the canine myosin heavy polypeptide 9 non-muscle (MYH9)
gene on dog chromosome 10q232 Submitted for publication in Gene
2 MIESKES K DISTL O 2006 Evaluation of 17 single nucleotide
polymorphisms (SNPs) in six candidate genes for hereditary non-syndromic deafness
in Dalmatian dogs Submitted for publication in Journal of Heredity
3 MIESKES K DISTL O 2006 Evaluation of the TMC1 and TMIE genes as
candidates for hereditary non-syndromic deafness in Dalmatian dogs Submitted for
publication in Animal Genetics
XV
Acknowledgements
First of all I wish to thank Prof Dr Dr habil Ottmar Distl the supervisor of my
doctoral thesis for his academic guidance and support of this work
I am very thankful to Heike Klippert-Hasberg and Stefan Neander for technical
expertise and assistance
I am very grateful to Joumlrn Wrede for his support during the statistical analyses and his
help with computer problems
I wish to express my appreciation to the Gesellschaft zur Foumlrderung Kynologischer
Forschung (GKF) eV Germany for funding this work with a grant
I am appreciative to all Dalmatian breeders and owners for providing me blood
samples and the results of the BAER tests
My special thanks go to all colleagues and friends of the Institute for Animal Breeding
and Genetics of the University of Veterinary Medicine Hannover for their support
humour and the friendly atmosphere in the laboratory
Last but not least I wish to thank my family for their support during the work on this
thesis
Abbreviations
List of abbreviations
A adenine
Acc No accession number
ACTG1 actin gamma 1
AEP akustisch evozierte Potentiale (acoustically evoked potentials)
APS ammonium persulphate
AT annealing temperature
BAC bacterial artificial chromosome
BAER brain stem auditory evoked response
BLAST Basic Local Alignment Search Tool
bp base pair
C cytosine
CCSD canine congenital sensorineural deafness
CDH23 cadherin related 23
cDNA copy desoxyribonucleic acid
CFA chromosome of Canis familiaris
CLDN14 claudin-14
cM centiMorgan
COCH coagulation factor C homolog cochlin
COL11A2 collagen type XI alpha 2
CRYM crystallin mu
CSD cochleosaccular degeneration
CX connexin
DFN x-linked deafness locus
DFNA autosomal dominant deafness locus
DFNA5 deafness autosomal dominant 5
DFNB autosomal recessive deafness locus
DIAPH1 diaphanous homolog 1 (Drosophila)
DMSO dimethyl sulfoxide
Abbreviations
DNA deoxyribonucleic acid
dNTPs deoxy nucleoside 5rsquotriphosphates (N is ACG or T)
EDN3 endothelin 3
EDNRB endothelin receptor type B
EDTA ethylenediamine tetraaceticacid
EMBL European Molecular Biology Laboratory
ESPN espin
EST expressed sequence tag
EYA4 eyes absent homolog 4 (Drosophila)
F forward
FISH fluorescence in situ hybridisation
G guanine
GJA1 gap junction protein alpha 1 43kD (connexin 43)
GJB2 gap junction protein beta 2 26k (connexin 26)
GJB3 gap junction protein beta 3 31kDa (connexin 31)
GJB6 gap junction protein beta 6 (connexin 30)
GKF Gesellschaft zur Foumlrderung Kynologischer Forschung (Society for the
Advancement of Cynological Research)
HET observed heterozygocity
HE expected heterozygosity value
HSA chromosome of Homo sapiens
IBD identical by descent
IRD infrared dye
KCNQ4 potassium voltage-gated channel KQT-like subfamily member 4
Kb kilobase
LD linkage disequilibrium
LINE long interspersed nuclear element
LOD logarithm of the odds
M molar
Mb megabase
Merlin multipoint engine for rapid likelihood inference
MITF microphthalmia-associated transcription factor
Abbreviations
MS microsatellite
MTRNR1 mitochondrially encoded 12S RNA
MTTS1 mitochondrially encoded tRNA serine 1 (UCN)
MYH9 myosin heavy polypeptide 9 non-muscle
MYH14 myosin heavy polypeptide 14
MYO1A myosin IA
MYO3A myosin IIIA
MYO6 myosin VI
MYO7A myosin VIIA
MYO15A myosin XVA
NCBI National Center for Biotechnology Information
NMMHC-A nonmuscle myosin heavy chain-A
ODDD oculodentodigital dysplasia
OMIM Online Mendelian Inheritance in Man
OTOA Otoancorin
OTOF otoferlin
P error probability
PAX3 paired box gene 3 (Waardenburg syndrome 1)
PCDH15 Protocadherin-15
PCR polymerase chain reaction
PIC polymorphism information content
POU3F4 POU domain class 3 transcription factor 4
POU4F3 POU domain class 4 transcription factor 3
PRES solute carrier family 26 member 5 (prestin)
QTL quantitative trait locus
R reverse
RACE rapid amplification of cDNA ends
RH radiation-hybrid
RLM RNA ligase-mediated
RNA ribonucleic acid
RT-PCR reverse transcription polymerase chain reaction
SAS Statistical Analysis System
Abbreviations
SH1 Src homology 1
SINE short interspersed nuclear element
SLC26A4 solute carrier family 26 member 4 (pendrin)
SLC26A5 solute carrier family 26 member 5 (prestin)
SNP single nucleotide polymorphism
SOX10 SRY (sex determining region Y)-box 10
STRC stereocilin
STS sequence-tagged site
T thymine
TBE tris-borate-ethylenediamine tetraacetic acid
TECTA tectorin alpha
TEMED NNNrsquoNrsquo-tetramethylenediamine
TFCP2L3 transcription factor CP2-like 3
TJ tight juncions
TMC1 transmembrane channel-like gene 1
TMIE transmembran inner ear gene
TMPRSS3 transmembrane protease serine 3
U unit
USH1C Usher syndrome 1C
UTR untranslated region
WFS1 Wolfram syndrome 1 (wolframin)
wgs whole genome shotgun
WHRN whirlin
WS Waardenburg syndrome
Chapter 1
Introduction
Introduction 3
Introduction
Canine congenital sensorineural deafness (CCSD) has often been reported in the
literature and occurs in more than 54 different breeds of dogs with the Dalmatian dog
showing the highest incidence The inheritance and segregation of a major gene in
CCSD has been demonstrated in different Dalmatian dog populations But although
several studies have demonstrated the mode of inheritance in Dalmatian dogs no
universally accepted mode of inheritance for the other dog breeds affected by CCSD
has yet been identified
Breeding with blue-eyed Dalmatian dogs and unilaterally or bilaterally deaf dams and
sires is forbidden by paragraph 11b of the German animal welfare laws and thus the
hearing status of all Dalmatian dogs has to be tested as a puppy at about six to eight
weeks old using the brain stem auditory evoked response (BAER) test As deaf dogs
are very difficult to raise and often become aggressive and snappish from fear most
puppies suffering from bilateral hearing loss are euthanized However it has been
shown in recent years that auditory testing does not seem to be an effective way of
clearly reducing the high incidence of deafness in this breed Thus prevention of
CCSD cannot be achieved alone by exclusion of affected animals from breeding
Consequently a molecular genetic approach toward unravelling the responsible
genes in carriers is urgently needed
Many genetic disorders in humans and domestic dogs (Canis familiaris) demonstrate
a high level of clinical and molecular similarity Therefore the mutated genes in
human hereditary deafness seemed to be appropriate candidates for canine
congenital sensorineural deafness
The objective of the present study is to localize the gene that is involved in the
development of CCSD in Dalmatian dogs In order to achieve this goal successively
32 canidate genes were evaluated by means of linkage analyses using microsatellite
markers and single nucleotide polymorphisms (SNPs) This candidate gene
approach using gene-associated markers for linkage studies in families segregating
for deafness turned out to be little effective Therefore the canine chromosomes
(CFA) 1 CFA10 and CFA31 were scanned entirely with microsatellite markers
Additionally single nucleotide polymorphisms (SNPs) were developed for fine
mapping the identified CCSD regions
Introduction 4
Overview of chapter contents Chapter 2 reviews the identified 39 mutated genes causing non-syndromic hereditary
hearing impairment in humans Parallels and differences in canine and human
deafness are shown including the clinical signs inheritance patterns and
histopathology We located the humane deafness genes in the canine genome and
discussed the advantages of comparative genomics and different molecular genetic
approaches
In Chapter 3 an existing set of 43 microsatellite markers for in total 24 candidate
genes were used for a non-parametric linkage analysis with congenital sensorineural
deafness (CCSD) in Dalmatian dog families segregating for deafness
In Chapter 4 newly developed SNP markers associated with in total eight candidate
genes were evaluated for CCSD in Dalmatian dogs
In Chapter 5 the molecular characterization of the canine myosin heavy polypeptide
9 non-muscle (MYH9) gene on dog chromosome 10q232 is described
Chapter 6 7 and 8 present linkage analyses performed in Dalmatian dog families
segregating for congenital sensorineural deafness using microsatellite markers on
canine chromosome (CFA) 1 CFA10 and CFA31 and the results of fine mapping
regions linked with the CCSD phenotype using newly developed SNPs
Chapter 9 provides a general discussion and conclusions referring to Chapters 1-8
Chapter 10 is a concise English summary of this thesis while Chapter 11 is an
expanded detailed German summary which takes into consideration the overall
research context
Chapter 2
A comparative overview of the molecular genetics of non-syndromic deafness
in dogs and humans
Non-syndromic deafness in dogs and humans 7
A comparative overview of the molecular genetics of non-syndromic deafness in dogs and humans
Abstract
In man as in different dog breeds deafness is an often diagnosed disorder with the
Dalmatian dog showing the highest incidence Deafness in Dalmatian dogs is clearly
heritable and the presence of a recessive major gene affecting the disorder was
shown in several Dalmatian dog populations
This Chapter provides an overview of the identified 39 mutated genes causing
human non-syndromic hereditary hearing impairment as well as of the five genes
responsible for Waardenburg syndrome in humans We point out their cytogenetic
and genomic localisations in man and dog and compare the genomic and mRNA
sequences of these genes between man and dog Moreover an overview is given on
deafness genes-associated markers identified in Dalmatian dogs and on candidate
genes characterized in dogs
The structure of the ear The inner ear consists of the semicircular canals the vestibule and the cochlea
whereas the vestibule and the semicircular canals are concerned with vestibular
function (balance) and the cocnlea is concerned with hearing Reissneracutes membrane
and the basilar membrane divide the cochlea longitudinally into three scalae the
scala vestibule the scala media and the scala tympani The process of transduction
occurs in the structures within scala media sitting on the basilar membrane and
comprising the organ of Corti Cutting the cochlea tube cross sectionally the scala
media is more or less triangular formed by Reissneracutes membrane basilar
membrane and a structure called the stria vascularis The fluid that fills scala
tympani and scala vestibule is called perilymph the fluid that fills scala media is
called endolymph The organ of Corti rests on the basilar membrane within scala
media The cochlea contains an array of highly specialized cells arranged in a highly
Non-syndromic deafness in dogs and humans 8
specialized manner Two types of cells in the organ of Corti are support cells and
hair cells The hair cells are the receptor cells that trancsduce sound
When a sound wave brings physical displacement of the membranes separating the
perilymph from the endolymph they cause the organ of Corti to move and the hair
cells on it are scraped along the bottom of the tectorial membrane The tectorial
membrane is firmly anchored to the bone Relative movement of the organ of Corti
and its hair cells with respect to the tectorial membrane is the source of the
deformation of the hair cells microvilli The hair cells are so constructed that any
deformation of their microvilli will cause a change in the overall membrane potential
of the cell This signal is detected by the fibers from the cells in the spiral ganglion
These fibers are neural elements and they carry their own depolarization wave into
the auditory region of the brain
Deafness in man There are various ways to categorise deafness The two main types of deafness are
classified based on which portions of the auditory system are affected conductive
hearing loss occurs when when sound is not conducted efficiently through the outer
andor middle part of the ear Much more common is the sensorineural hearing loss
Sensorineural hearing loss occurs when there is damage to the inner ear (cochlea)
or to the nerve pathways from the inner ear (retrocochlear) to the brain Most cases
of sensorineural hearing loss are due to cochlear defects (Petit et al 2001)
Hearing loss can be present at birth (congenital) or become evident later in life
(acquired) Congenital deafness similarly may or may not be genetic In fact more
than half of congenital hearing loss is inherited Alternatively congenital deafness
may be due to a condition or infection to which the mother was exposed during
pregnancy Furthermore congenital hereditary deafness may occur as part of a
multisystem disease (syndromic) or as a disorder restricted to the ear and vestibular
system (non-syndromic) As non-syndromic hereditary hearing impairment is almost
exclusively caused by cochlear defects affected patients suffer from sensorineural
hearing loss In Table 1 and 2 the genes underlying human hereditary non-
syndromic deafness as a result of cochlear defects in consequence of primary
defects in hair cells non-sensory cells and the tectorial membrane or unknown cell
type are shown Non-syndromic deafness is estimated to cause 60-70 of cases of
congenital hereditary deafness in man (Morton 1991) In 70-80 of all cases this
Non-syndromic deafness in dogs and humans 9
non-syndromic form of deafness shows an autosomal recessive inheritance followed
by an autosomal dominant inheritance in 10-20 of all cases and 1-2 of all cases
are X-linked A maternally inherited form may also occur (Van Camp and Smith
2003)
Non-syndromic forms of hereditary deafness are classified by their mode of
inheritance DFN DFNA and DFNB refer to deafness forms inherited on the X
chromosome-linked autosomal dominant and autosomal recessive modes of
transmission respectively
Human hereditary isolated hearing loss is genetically heterogeneous (Petit et al
2001) Up to 1 of the human genes are estimated to be necessary for hearing
(Friedmann and Griffith 2003) Today approximately 120 genes for human
hereditary deafness have been identified approximately 80 for syndromic and 39 for
non-syndromic hereditary deafness which is suspected to be one-third of the total
(Nance 2003) In Table 1 the identified 39 mutated genes causing non-syndromic
hereditary hearing impairment in humans are shown Out of the 39 genes 15 genes
cause autosomal recessive and 15 genes cause autosomal dominant forms six
genes are involved in both recessive and dominant forms one gene causes X-linked
and two a maternally inherited form (The Hereditary Hearing Loss Hompage
httpwebhostuaacbehhh)
Furthermore several hundred forms of syndromes with hearing loss have been
documented in humans (Van Camp and Smith 2003) One is the human
Waardenburg syndrome (WS) which manifests itself with sensorineural deafness
and pigmentation defects in the iris hair and skin The WS is classified into four
types depending on the presence or absence of additional symptoms which are
caused by mutations in the five genes EDN3 EDNRB MITF PAX3 and SOX10
respectively These genes are shown in Table 3 They are known to be expressed in
the neural crest (EDN3 EDNRB PAX3 SOX10 ) or directly in the melanocytes
(MITF) and are inter alia involved in migration differentiation or survival of
melanocytes respectively (Bondurand et al 2000)
Deafness in dogs Congenital sensorineural deafness (CSD) has been reported in a variety of mammal
species other than humans ranging from mice to dogs guinea pigs and mink
Canine congenital deafness has often been reported in the literature and occurs in
Non-syndromic deafness in dogs and humans 10
more than 54 different breeds of dogs according to Strain (1996 and 2004) The
breeds with the highest incidence include Dalmatian dogs Bull Terrier English
Cocker Spaniel English Setter Australian Cattle Dog Australian Shepherd West-
Highland-White-Terrier Dobermann and Dogo Argentino The incidence of canine
congenital deafness is highest in Dalmatian dogs of which 165 to 30 exhibit
unilateral or bilateral hearing loss (Famula et al 1996 Wood and Lakhani 1997
Muhle et al 2002 Juraschko et al 2003a Rak and Distl 2005) The inheritance
and segregation of a major gene in canine congenital sensorineural deafness
(CCSD) has been demonstrated in different Dalmatian dog populations (Famula et
al 2000 Muhle et al 2002 Juraschko et al 2003b) But although several studies
have demonstrated the mode of inheritance in Dalmatian dogs no universally
accepted mode of inheritance for the other dog breeds affected by CCSD has yet
been identified
Congenital sensorineural hearing impairment can be recognised in dogs at four to
eight weeks of age (Strain 1996) while histological studies of deaf Dalmatian dogs
have shown that the degeneration of the inner ear structures begins as early as one
day after birth and is histologically clearly evident by four weeks of age (Johnsson et
al 1973) In 70 of the cases with human hereditary deafness the histological
pattern is known as cochleo-saccular degeneration (CSD) (Lalwani et al 1997)
commonly known as Scheibe dysplasia with preservation of the pars superior of the
membranous labyrinth and an unremarkable bony labyrinth As in man also in many
affected dog breeds the histological pattern of congenital sensorineural deafness is
known as cochleo-saccular degeneration
Breeding with blue-eyed Dalmatian dogs and unilaterally or bilaterally deaf dams and
sires is forbidden by paragraph 11b of the German animal welfare laws and thus the
hearing status of all Dalmatian dogs has to be tested as a puppy at about six to eight
weeks old using the brainstem auditory evoked response (BAER) test that detects
electrical activity in the cochlea and auditory pathways in the brain Although the
BAER test is a reliable method for identifying unilaterally and bilaterally deaf dogs it
does not seem to be an effective way of clearly reducing the incidence of deafness in
affected breeds particularly in a recessive mode of inheritance so that hearing dogs
can still be genetic carriers Furthermore deaf dogs are very difficult to raise and
often become aggressive and snappish from fear consequently most puppies
Non-syndromic deafness in dogs and humans 11
suffering from bilateral hearing loss are euthanized Thus prevention of CCSD
cannot be achieved alone by exclusion of affected animals from breeding and
consequently a molecular genetic approach toward unravelling the responsible
genes in carriers is urgently needed
Over the past decade it has become increasingly clear how far structural and
functional homologies at the gene level extend across even distantly related species
Much is known about deafness-causing gene mutations in humans and mice
including the fact that the clinical and histopathological findings are often very similar
to those of deafness in Dalmatian dogs Thus despite the genetical heterogeneity of
human non-syndromic deafness the genes that are responsible for non-syndromic
congenital hereditary deafness in humans (Table 1) seemed to be appropriate
candidate genes for CCSD especially in Dalmatian dogs (Rak and Distl 2005) The
genes that are mutated in the human WS (Table 2) were selected as candidates
because the WS phenotype where the deafness is associated with pigmentation
defects seems to be similar to the phenotype of most affected dog breeds (Strain
and Tedford 1996) Both Juraschko et al (2003a) and Strain et al (1992) have
demonstrated that patched Dalmatians are less likely to be deaf than unpatched
animals and blue-eyed Dalmatians are more likely to be affected from hearing
impairment than brown-eyed animals
In an attempt to achieve the aim of molecular genetic diagnosis of CCSD carriers
Rak et al (2003) and co-workers (Droumlgemuumlller et al 2002 Kuiper et al 2002 Rak
et al 2002a 2002b 2003) already mapped 24 potential candidate genes for
sensorineural deafness in dogs by fluorescence in situ hybridization and a radiation
hybrid panel to 16 different canine chromosomes
The canine genome project In December 2005 an international research team led by scientists at the Broad
Institute of MIT and Harvard achieved the completion of a high-quality genome
sequence of the domestic dog together with a catalog of 25 million specific genetic
differences across several dog breeds (Lindblad-Toh et al 2005) The authors found
that humans share more of their ancestral DNA with dogs than with mice confirming
the utility of dog genetics for understanding human disease Furthermore the
physiology disease presentation and clinical response of dogs often mimic human
Non-syndromic deafness in dogs and humans 12
diseases closely As indicated above hearing impairment seemed to be no
exception
In the last years most projects have exploited canine traits for which either direct
candidate genes could be proposed and evaluated or for which large informative
pedigrees were available to enable linkage mapping to identify candidate regions A
major component of such research efforts comprised the cloning sequencing and
mapping of individual canine homologs of genes either proposed as candidate
genes or expected to be located in candidate regions This was necessary to
identify new informative polymorphisms (eg single nucleotide polymorphisms
(SNPs) microsatellites) for high resolution mapping of candidate regions and to
examine each exon and exonintron boundary for positional candidates Availability
of the second version of the dog genome assembly (build 21) of the NCBI database
shortcut this effort and increase the investigators efficency
The current RH map with 3200 markers provides a good estimate of the order and
physical spacing (ie in base pairs) of markers along canine chromosomes (Guyon
et al 2003) and was recently complemented by the construction of a 4249-marker
integrated canine genome RH map that consists of 900 genes 1589 microsatellites
and 1760 BAC end markers (Breen et al 2004) all included and available in the
NCBI database The second version 1 of the NCBIs genome annotation consists of
large contigs covering all canine chromosomes given with their located markers and
genes The great majority of genes are derived by automated computational analysis
using the gene prediction method GNOMON
With this help either additional candidate genes for canine CSD can be found directly
by its gene symbol in the 21 of the NCBIs genome annotation or if a candidate gene
is yet not annotated a BLAST (Basic Local Alignment Search Tool) search versus
the canine whole genome shotgun (wgs) sequence resource can be used to obtain
the sequence of the canine genomic contigs containing the human homologous
gene The localisation of all 39 known human non-syndromic hereditary deafness
genes in the canine genome with the corresponding accession numbers of the contig
and if available the accession number of the genomic sequence and mRNA of the
canine gene are shown in Tables 4 and 5 Furthermore the identity of canine and
human or mouse mRNA is shown in Table 5 The average identity of canine and
human mRNA is with 088 percent higher than the average identity of canine and
mouse mRNA with 084 percent Canine sequences that correspond to the human
Non-syndromic deafness in dogs and humans 13
candidate gene can now be used to find microsatellite or SNP markers associated to
the respective canine gene These markers can be used for linkage and haplotype
studies in dog families segregating for deafness
Table 7 shows the microsatellite and SNP markers developed for in total 32
candidate genes for CCSD
The candidate genes for which a set of in total 43 microsatellite marker were
designed by Rak (2003) included the following 24 genes CDH23 CLDN14 COCH
COL11A2 DFNA5 DIAPH1 EDN3 EDNRB EYA4 GJA1 GJB2 GJB6 MITF
MYH9 MYO6 MYO7A MYO15A OTOF PAX3 POU4F3 SLC26A4 SOX10
TECTA and TMPRSS3 This existing set of 43 microsatellite markers for 24
candidate genes were used for linkage and haplotype studies in Dalmatian dog
families segregating for deafness (Chapter 3) These 24 genes are known to be
involved either in human non-syndromic deafness or in the human Waardenburg
syndrome For another eight candidate genes including TMC1 TMIE USH1C
MYH14 MYO3A PRES WHRN and ESPN SNP markers were newly developed
(Chapter 4) and subsequently used for linkage and association analyses in
Dalmatian dog families segregating for deafness These genes are also involved in
human non-syndromic deafness
Non-syndromic deafness in dogs and humans 14
Tabele 1 Genes responsible for non-syndromic congenital hereditary deafness in
humans
Inheritance Gene Gene product Type of molecule Locus namea
ACTG1 γ-Actin cytoskeletal protein DFNA20 DFNA26
COCH Cochlin extracellular matrix component DFNA9
COL11A2 Collagen XI (α2-chain) extracellular matrix component DFNA13
CRYM micro-Cristallin thyroid hormone-binding protein DFNAb DFNA5 Unidentified Unidentified DFNA5 DIAPH1 Diaphanous-1 cytoskeleton regulatory protein DFNA1 EYA4 EYA4 transcriptional coactivator DFNA10 GJB3 Connexin-31 gap junction protein DFNA2 KCNQ4 KCNQ4 K+ channel subunit DFNA2 MYH14 Myosin IIC motor protein DFNA4 MYH9 Myosin IIA motor protein DFNA17 MYO1A Myosin IA motor protein DFNA48 POU4F3 POU4F3 transcription factor DFNA15 TFCP2L3 TFCP2L3 transcription factor DFNA28
Autosomal dominant
WFS1 Wolframin endoplasmic-reticulum membrane protein DFNA6 DFNA14
CDH23 Cadherin-23 cell-adhesion protein DFNB12 CLDN14 Claudin-14 tight-junction protein DFNB29 ESPN Espin actin-bundling protein DFNB36 DFNAb MYO15 Myosin XV motor protein DFNB3 MYO3A Myosin IIIA motor protein DFNB30 OTOA Otoancorin cell-surface protein DFNB22 OTOF Otoferlin putative vesicle traffic protein DFNB9 PCDH15 Protocadherin-15 cell-adhesion protein DFNB23 SLC26A4 Pendrin IminusndashClminus transporter DFNB4 SLC26A5 Prestin anion transporter DFNB61 STRC Stereocilin DFNB16
TMIE TMIE transmembrane domain- containing protein DFNB6
TMPRSS3 TMPRSS3 transmembrane serine protease DFNB8 DFNB10 USH1C Harmonin PDZ domain-containing protein DFNB18
Autosomal recessive
WHRN Whirlin PDZ domain-containing protein DFNB31 GJB2 Connexin-26 gap junction protein DFNB1 DFNA3 GJB6 Connexin-30 gap junction protein DFNB1 DFNA3 MYO6 Myosin VI motor protein DFNA22DFNB37MYO7A Myosin VIIA motor protein DFNB2DFNA11
TECTA α-Tectorin extracellular matrix component DFNA8 DFNA12DFNB21
Autosomal dominant and autosomal recessive
TMC1 TMC1 transmembrane channel-like protein
DFNB7 DFNB11DFNA36
X-linked POU3F4 POU3F4 transcription factor DFN3
MTRNR1 Mitochondrial 12S rRNA not defined
nomenclature Mitochondrial
MTTS1 Mitochondrial 12S rRNA not defined
nomenclature a Autosomal recessive loci are designated DFNB autosomal dominant loci DFNA
Non-syndromic deafness in dogs and humans 15
Table 2 Genes underlying hereditary non-syndromic deafness as a result of primary
defects in hair cells non-sensory cells and the tectorial membrane or unknown cell
type
Primary defect
Gene
Hair cells MYO7A MYO15 MYO6 MYO3A MYO1A ACTG1 USH1C
WHRN CDH23 PCDH15 TMIE STRC SLC26A5 ESPN
KCNQ4 TMC1 OTOF POU4F3
Non-sensory cells
GJB2 GJB6 GJB3 SLC26A4 CRYM OTOA CLDN14
COCH TMPRSS3 MYH9 MYH14 EYA4 POU3F4
Tectorial membrane
COL11A2 TECTA
Unknown
DIAPH1 DFNA5 WFS1 TFCP2L3 MTRNR1 MTTS1
Table 3 Genes involved in the human Waardenburg syndrome
Inheritance Gene Gene product Type of molecule Type
EDN3 endothelin 3 vasoconstricted peptide WS type IV4
EDNRBendothelin
receptor type B receptor protein WS type IV4
MITF
microphthalmia-
associated
transcription
factor
transcription factor WS type II2
PAX3 paired box 3 DNA-binding protein WS type I1and III 2
SOX10 SRY-box 10 transcription factor WS type IV4 1Type I Dystopia canthorum 2Type II No dystopia canthorum 3Klein-Waardenburg syndrome (type III) Type I and upper limb abnormalities 4Waardenburg-Shah syndrome (type IV) Type II and Hirschsprung disease
(autosomal recessive inheritance)
Non-syndromic deafness in dogs and humans 16
Leng
th o
f co
ntig
(bp)
7799
0652
3821
0901
7077
2986
7251
5492
5989
7527
4533
7677
6093
8239
9498
326
6961
0260
1859
5814
6421
1953
Scor
e of
co
ntig
614
938
792
567
300
715
323
535
1208
1634
923
E-va
lue
of
cont
ig
4 E
-172
0 0
2 E
-158
1 e-
78
0
2 e-
85
7 E
-149
0 0 0
Acc
No
of
WG
S co
ntig
unkn
own
NW
_876
311
NW
_876
295
NW
_876
327
NW
_876
254
NW
_876
321
NW
_876
292
NW
_876
258
NW
_876
315
NW
_876
269
NW
_876
278
NW
_876
259
Mb
from
to
unkn
own
254
12
578
337
93
379
132
11
323
563
56
5
272
12
723
393
33
943
411
64
123
632
86
331
292
82
955
209
32
094
101
81
019
Gen
e lo
catio
n on
C
FA
unkn
own
4 31
8 12
6 2 14
5 1 25
15
Can
ine
gene
al
iase
s
none
none
LOC
4877
51
LOC
4906
40
LOC
4817
34
LOC
4798
18
none
LOC
6112
23
LOC
4896
31
EYA4
GJB
2
LOC
4824
86
Acc
No
hu
man
m
RN
A
NM
_001
614
NM
_022
124
NM
_144
492
NM
_004
086
NM
_080
680
NM
_001
888
NM
_005
219
NM
_004
403
NM
_031
475
NM
_172
105
NM
_004
004
NM
_024
009
Gen
e lo
catio
n on
H
SA
17
10
21
14
6 16
5 7 1 6 13
1
Tabl
e 4
Loca
lisat
ion
of h
uman
non
-syn
drom
ic h
ered
itary
dea
fnes
s ge
nes
in th
e ca
nine
gen
ome
the
cani
ne
gene
loc
alis
atio
n in
meg
abas
es (
Mb)
th
e ac
cess
ion
num
ber
(Acc
No
) of
the
who
le g
enom
e sh
otgu
n (W
GS
) con
tig c
onta
inin
g th
e hu
man
hom
olog
ous
gene
and
the
corr
espo
ndin
g E
-val
ue s
core
and
leng
th o
f th
e co
ntig
Hum
an
deaf
ness
ge
ne
AC
TG1
CD
H23
CLD
N14
CO
CH
CO
L11A
2
CR
YM
DIA
PH1
DFN
A5
ESPN
EYA
4
GJB
2
GJB
3
der
ived
from
the
NC
BIs
can
ine
geno
me
anno
tatio
n ve
rsio
n 2
1
Non-syndromic deafness in dogs and humans 17
Leng
th o
f co
ntig
6421
1953
5300
4996
5294
2087
2607
3285
1654
5469
1249
9463
7251
5492
5102
4781
5989
7527
5159
1990
1284
7264
7521
5785
Scor
e of
co
ntig
525
521
973
2256
348
567
1236
404
337
383
604
1715
E-va
lue
of
cont
ig
3 e-
146
1 e-
144
0 0
9 e-
93
2 e-
158
0
3 e-
109
3 e-
89
7 e-
103
2 e-
169
0
Acc
No
of
WG
S co
ntig
unkn
own
NW
_876
259
NW
_876
270
NW
_876
251
NW
_876
313
NW
_876
250
NW
_876
290
NW
_876
254
NW
_876
273
NW
_876
321
NW
_876
263
NW
_876
283
NW
_879
563
Mb
from
to
unkn
own
521
52
3
109
24
109
35
311
33
119
443
64
441
415
41
7
103
41
056
404
14
050
245
42
460
261
32
619
235
02
359
371
43
769
674
86
748
Gen
e lo
catio
n on
C
FA
25
15
1 10
5 10
2 12
21
6 17
26
X
Can
ine
gene
al
iase
s
none
LOC
4824
51
none
LOC
4812
80
LOC
4795
22
LOC
4744
10
LOC
4871
06
LOC
4818
84
LOC
4851
74
LOC
6086
55
LOC
6079
61
none
LOC
4919
88
Acc
No
hu
man
m
RN
A
NM
_006
783
NM
_004
700
NM
_024
729
NM
_002
473
NM
_016
239
NM
_005
379
NM
_017
433
XM
_376
516
NM
_000
260
NM
_144
672
NM
_194
248
NM
_033
056
NM
_000
307
Gen
e lo
catio
n on
H
SA
13
1 19
22
17
12
10
6 11
16
2 10
X
Tabl
e 4
(con
tinue
d)
Hum
an
deaf
ness
ge
ne
GJB
6
KC
NQ
4
MYH
14
MYH
9
MYO
15
MYO
1A
MYO
3A
MYO
6
MYO
7A
OTO
A
OTO
F
PCD
H15
POU
3F4
der
ived
from
the
NC
BIs
can
ine
geno
me
anno
tatio
n ve
rsio
n 2
1
Non-syndromic deafness in dogs and humans 18
Leng
th o
f co
ntig
4533
7677
2532
0482
2532
0482
4020
6070
3002
9677
2968
9717
5300
4996
3309
7591
3821
0901
5102
4781
1104
8438
6535
5756
Scor
e of
co
ntig
1404
283
283
1683
883
529
354
216
198
354
2238
689
E-va
lue
of
cont
ig
0
6 e-
73
2 E
-73
0 0
5 e-
147
2 e-
94
4 e-
53
9 e-
48
2 E
-94
0 0
Acc
No
of
WG
S co
ntig
NW
_876
292
NW
_876
265
NW
_876
265
NW
_876
294
NW
_876
312
NW
_876
255
NW
_876
270
NW
_876
272
NW
_876
295
NW
_876
273
NW
_876
256
NW
_876
253
Mb
from
to
436
14
361
158
61
592
197
91
982
134
31
345
158
81
595
622
63
7
880
88
822
450
54
505
390
33
905
432
44
328
414
94
151
716
47
172
Gen
e lo
catio
n on
C
FA
2 18
18
30
5 13
1 20
31
21
13
11
Can
ine
gene
al
iase
s
LOC
4872
00
LOC
4832
63
LOC
4832
74
LOC
4782
78
LOC
4893
57
LOC
4819
85
LOC
4841
68
LOC
6093
50
LO
C61
0987
LOC
6108
50
LOC
4821
13
LOC
6125
88
Acc
No
hu
man
m
RN
A
NM
_002
700
NM
_000
441
NM
_206
883
NM
_153
700
NM
_005
422
NM
_024
915
NM
_138
691
NM
_147
196
NM
_024
022
NM
_153
676
NM
_006
005
NM
_015
404
Gen
e lo
catio
n on
H
SA
5 7 7 15
11
8 9 3 21
11
4 9
Tabl
e 4
(con
tinue
d)
Hum
an
deaf
ness
ge
ne
POU
4F3
SLC
26A
4
SLC
26A
5
STR
C
TEC
TA
TFC
P2L3
TMC
1
TMIE
TMPR
SS3
USH
1C
WFS
1
WH
RN
der
ived
from
the
NC
BIs
can
ine
geno
me
anno
tatio
n ve
rsio
n 2
1
Non-syndromic deafness in dogs and humans 19
Leng
th o
f co
ntig
(bp)
4776
3139
5561
1003
2518
2130
3091
5115
5294
2087
Scor
e of
co
ntig
262
721
2927
967
1179
E-va
lue
of
cont
ig
8e-6
7
0 0 0 0
Acc
No
of
WG
S co
ntig
NW
_876
277
NW
_876
274
NW
_876
271
NW
_876
304
NW
_876
251
Mb
from
to
470
14
703
343
63
438
248
52
488
313
43
144
297
52
976
Gen
e lo
catio
n on
C
FA
24
22
20
37
10
Can
ine
gene
al
iase
s
ED
N3
ED
NR
B
MIT
F
PAX3
LOC
4812
58
Acc
No
hu
man
m
RN
A
NM
_207
032
NM
_000
115
NM
_198
159
NM
_181
457
NM
_006
941
Gen
e lo
catio
n on
H
SA
20
13
3 2 22
Tabl
e 5
Loca
lisat
ion
of g
enes
invo
lved
in th
e hu
man
Waa
rden
burg
syn
drom
e (W
S)
in th
e ca
nine
gen
ome
th
e ca
nine
gen
e lo
calis
atio
n in
meg
abas
es (
Mb)
th
e ac
cess
ion
num
ber
(Acc
No
) of
the
who
le g
enom
e sh
otgu
n (W
GS
) con
tig c
onta
inin
g th
e hu
man
hom
olog
ous
gene
and
the
corr
espo
ndin
g E
-val
ue s
core
and
le
ngth
of t
he c
ontig
Hum
an
deaf
ness
ge
ne
EDN
3
EDN
RB
MIT
F
PAX3
SOX1
0
der
ived
from
the
NC
BIs
can
ine
geno
me
anno
tatio
n ve
rsio
n 2
1
Non-syndromic deafness in dogs and humans 20
Table 6 Canine candidate genes for CCSD with their accession number (AccNo) of
the genomic sequence and mRNA and if available the percent identity of canine and
human or mouse mRNA Canine candidate gene
Acc No canine genomic sequence
Acc No canine mRNA (predicted)
Canine mRNA (bp)
Identity () of canine and human mRNA
Identity () of canine and mouse mRNA
ACTG1 none1 none unknown unknown unknown CDH23 none1 none unknown 8800 8690 CLDN14 NC_006613 XM_544876 714 8880 8460 COCH NC_006590 XM_547762 3050 8770 8170
COL11A2 NC_006594 XM_857285 5202 8970 8740 CRYM NC_006588 XM_845361 1279 8720 7890
DIAPH1 none1 none unknown 8630 unknown DFNA5 NC_006596 XM_848863 1512 8320 7680 ESPN NC_006587 XM_546751 2565 8930 8270 EYA4 NC_006583 XM_541108 3969 9240 8120 GJB2 NC_006607 XM_543177 2208 7860 6720 GJB3 NC_006597 XM_539603 1305 8550 8090 GJB6 none none unknown unknown unknown
KCNQ4 NC_006597 XM_539568 1836 9350 8760 MYH14 none1 none unknown 8770 8220 MYH9 NC_006592 XM_538401 6201 9130 8940
MYO15 NC_006587 XM_536660 10128 8500 7960 MYO1A NC_006592 XM_531642 3388 8600 8350 MYO3A NC_006584 XM_544234 5589 8510 8910 MYO6 NC_006594 XM_862465 3983 9250 8610
MYO7A NC_006603 XM_542292 6519 9190 8880 OTOA NC_006588 XM_845746 3558 8740 7990 OTOF NC_006599 XM_844665 5994 9020 8780
PCDH15 none1 none unknown 8210 7390 POU3F4 NC_006621 XM_549108 1086 9190 9060 POU4F3 NC_006584 XM_544328 1017 9380 9160 SLC26A4 NC_006600 XM_540382 2382 7970 8240 SLC26A5 NC_006600 XM_540393 2235 9250 8740
STRC NC_006612 XM_535452 5301 8980 8520 TECTA NC_006587 XM_546475 7136 9110 8510
TFCP2L3 NC_006595 XM_539106 2127 9030 8760 TMC1 NC_006583 XM_541284 2580 8860 8740 TMIE NC_006602 XM_846596 396 9110 8910
TMPRSS3 NC_006613 XM_848589 1542 8500 8340 USH1C NC_006603 XM_860072 1730 9010 8670 WFS1 NC_006595 XM_539234 2667 8510 8420 WHRN NC_006593 XM_850321 2817 8440 8110 EDN3 NC_006606 NM_001002942 1976 747 716
EDNRB NC_006604 NM_001010943 1329 889 827 MITF NC_006602 XM_850501 1590 934 862 PAX3 NC_006619 XM_545664 1474 869 861
SOX10 NC_006592 XM_538379 1987 926 900 derived from the NCBIs canine genome annotation version 21 1 the definite region in the whole genome sequence (WGS) contig was used to get the percent identity of mRNAs
Non-syndromic deafness in dogs and humans 21
Table 7 Microsatellite markers and single nucleotide polymorphisms (SNPs)
of canine candidate genes for canine congenital sensorineural deafness in
Dalmatian dogs
Canine candidate gene
Number of gene-associated
microsatellites
Number of gene-associated
SNPs
CDH23 2 0 CLDN14 3 8 COCH 2 0
COL11A2 2 0 DIAPH1 2 0 DFNA5 2 0 ESPN 0 5 EYA4 2 0 GJB2 3 0 GJB3 1 0 GJB6 1 0
MYH14 0 2 MYH9 2 22
MYO15 2 0 MYO3A 0 3 MYO6 1 0
MYO7A 3 0 OTOF 1 0 PAX3 1 0
POU4F3 1 0 SLC26A4 1 0 SLC26A5 0 2 TECTA 2 0 TMC1 1 1 TMIE 1 3
TMPRSS3 2 0 USH1C 0 2 WHRN 0 3
Chapter 3
Linkage analysis of gene-associated microsatellite markers with
congenital sensorineural deafness in Dalmatian dogs
Linkage analysis of gene-associated microsatellites 25
Linkage analysis of gene-associated microsatellite markers with congenital sensorineural deafness in Dalmatian dogs
Abstract Deafness is a disorder often diagnosed in different dog breeds In this Chapter an
existing set of 43 microsatellite markers associated with in total 24 candidate genes
for canine congenital sensorineural deafness (CCSD) were used for linkage and
haplotype analyses in a large Dalmatian dog population with frequent occurrence of
CCSD We found significant linkage for the genes GJA1 MYH9 and CLDN14 As
linkage was found for different candidate genes in different families the results of
these test statistics indicate that the inheritance of non-syndromic deafness in
Dalmatian dogs is heterogenic in origin
Introduction
Canine congenital sensorineural deafness (CCSD) has been reported to occur in
more than 54 different breeds of dogs (Strain 1996) As in man also in dog breeds
the most commonly observed histological pattern of degenerative inner ear changes
is known as the cochleo-saccular or Scheibe type of end organ degeneration
The incidence of this congenital anomaly is highest in Dalmatian dogs of which 165-
30 exhibit unilateral or bilateral hearing loss (Famula et al 1996 Wood and
Lakhani 1997 Muhle et al 2002 Juraschko et al 2003a Rak and Distl 2005) The
inheritance and segregation of a major gene in CCSD has been demonstrated in
different Dalmatian dog populations (Famula et al 2000 Muhle et al 2002 Juraschko et al 2003b) Moreover deafness in Dalmatian dogs seems to be
pigment-associated (Greibrokk 1994 Holliday et al 1992 Juraschko et al 2003a
2003b Mair 1976 Strain et al 1992 Strain 1996)
No gene mutation has yet been identified that is responsible for CCSD in Dalmatian
dogs or in one of the various other dog breeds that suffer from inherited hearing
impairment Since mutations in various genes have already been found to be the
cause of sensorineural hearing impairment in humans or mice 24 of these genes
Linkage analysis of gene-associated microsatellites
26
were considered as candidates for CCSD in Dalmatian dogs (Rak et al 2005)
Details of the 24 candidate genes are given in Table 1
Rak et al (2003) and co-workers (Droumlgemuumlller et al 2002 Kuiper et al 2002 Rak et
al 2002a 2002b 2003) mapped this 24 potential candidate genes for sensorineural
deafness in dogs by fluorescence in situ hybridization and a radiation hybrid panel
Subsequently Rak (2003) developed altogether 43 new highly polymorphic DNA
markers for the 24 candidate genes including CDH23 CLDN14 COCH COL11A2
DFNA5 DIAPH1 EDN3 EDNRB EYA4 GJA1 GJB2 GJB6 MITF MYH9 MYO6
MYO7A MYO15A OTOF PAX3 POU4F3 SLC26A4 SOX10 TECTA and
TMPRSS3 (Table 2)
Among the 24 candidate genes seven genes cause autosomal dominant non-
syndromic forms of deafness seven cause autosomal recessive forms and five
genes cause both recessive and dominant forms of non-syndromic deafness in
different human families segregating for either forms
The functions of these 19 deafness-causing genes are diverse and include gap
junctions and tight junctions (GJA1 GJB2 GJB6 CLDN14) ion channels (SLC26A4)
and ion channel activators (TMPRSS3) Included are also unconventional myosins
(MYO6 MYO7A MYH9 MYO15A) transcription factors (POU4F3 EYA4) as well as
extracellular matrix components (COCH COL11A2 TECTA) a cytoskeleton
regulatory protein (DIAPH1) a cell-adhesion protein (CDH13) and genes with
unknown or only suspected functions (DFNA5 OTOF) The 24 candidates also
include five genes which are mutated in the human Waardenburg syndrome (WS)
The WS is classified into four types depending on the presence or absence of
additional symptoms which are caused by mutations in the five genes EDN3
EDNRB MITF PAX3 and SOX10 respectively The objective of the current study
was to use this set of markers developed by Rak (2003) for a non-parametric linkage
analysis with CCSD in a German and French Dalmatian dog population
Material and methods Pedigree material
For the linkage analysis we used DNA from altogether 215 animals belonging to a
total of 24 Dalmatian dog families The families included 22 full-sib families and one
large paternal half-sib family of German Dalmatian dogs as well as 46 animals of a
Linkage analysis of gene-associated microsatellites 27
large paternal half-sib family of French Dalmatian dogs All families were segregating
for CCSD The genotyped families included all affected dogs (unilaterally and
bilaterally deaf) their parents if available and one to four unaffected animals At least
two of the full sibs of each family were unilaterally deaf
In total these 24 families included 402 individuals with an average family size of 168
ranging from 5 to 116 animals and covering two to four generations The hearing
status of 344 dogs was examined by veterinarians using the BAER (brain stem
auditory evoked response) test and the other animals included in the pedigree being
not BAER tested were used to construct relationships among CSD affected dogs
The prevalence of CSD in this pedigree was 285
Microsatellite marker analysis An existing marker set consisting of 43 microsatellite markers (Table 2) was used for
linkage analysis This set included 36 markers developed by Rak (2003) and 7
markers of the RH map available at httpwww-recomgenuniv-rennes1frdoggyhtml
For most of the 24 candidate genes two markers were available for two of the
candidates three markers were available but for seven candidate genes the set
contains only one marker The marker set is composed of 33 perfect repeats two
imperfect six compound-perfect and two compound-imperfect repeats
The majority (674) of the 43 markers in the set was represented by dinucleotide
repeats 209 were tetranucleotide repeats 47 trinucleotide repeats and 23
pentanucleotide repeats In addition one marker (23) was a compound di-
tetranucleotide and another one (23) was a compound tetra-pentanucleotide
repeat The average number of alleles was 35 with a minimum of 2 and a maximum
of 8 different alleles per marker
PCR reactions for microsatellites were carried out in 12 microl reaction mixtures
containing 2 microl genomic DNA 12 microl 10x PCR buffer (Qbiogene Heidelberg
Germany) 024 microl dimethyl sulfoxide (DMSO) 02 microl dNTPs (100 microM) 01 microl Taq
Polymerase (5Umicrol) (Qbiogene) 06 microl (10 microM) 5rsquo-IRD700 or IRD800 labelled forward
primer 06 microl (10 microM) unlabelled reverse primer The PCR reactions were carried out
in MJ Research thermocyclers with the following program 4 min at 94 degC followed
by 32 cycles of 30 sec at 94degC 30 sec at maximum annealing temperature and a
final extension of 45 sec at 72degC PCR-products were diluted with formamide loading
buffer in ratios from 110 to 140 and then size-fractionated by gel electrophoresis on
Linkage analysis of gene-associated microsatellites
28
automated LI-COR 42004300 sequencers (LI-COR inc Lincoln NE USA) using
denaturing 4 and 6 polyacrylamide gels (RotiphoreseregGel 40 Roth Karlsruhe
Germany)
To localize the 24 candidate genes and their associated microsatellites exactly the
canine candidate gene sequences were derived from sequences deposited in the
current dog genome assembly (Boxer genome assembly 21) of the NCBI GenBank
by BLAST (Basic Local Alignment Search Tool) search
(httpwwwncbinlmnihgovBLAST) using the human reference mRNA sequence
(Table 3)
Linkage analysis
Multipoint linkage and haplotype analyses were performed using the MERLIN
software version 0102 (multipoint engine for rapid likelihood inference Center for
Statistical Genetics University of Michigan MI USA Abecasis et al 2002) The test
statistics Z-mean and Lod score were used to test for the proportion of alleles shared
by affected individuals identical by descent (IBD) for the considered marker loci
Linkage analyses were performed regarding the marker set consisting of 43 gene-
associated microsatellite markers Linkage analysis was at first carried out for all 24
families conjoined After this the families were scanned separately
The data of the genotypes was additionally analyzed using SASGenetics (Statistical
Analysis System Version 913 SAS Institute Inc Cary NC USA 2005) to specify
the number of alleles of each marker the allele frequency the observed (HET) and
expected (HE) heterozygosity and the polymorphism information content (PIC)
(Table 4 and 5)
Results and discussion
Test statistics for all families conjoined are given in Table 6 Significant CCSD loci
were located on different chromosomes The loci were located on canine
chromosome (CFA) 1 10 12 20 and 31 Linkage analyses per family indicated even
higher test statistics for subgroups of families (Table 7) Scanning only families with
Zmeans gt1 test statistics for linkage increased for the genes GJA1 on CFA1 MYH9
on CFA10 and CLDN14 on CFA31 with congenital sensorineural deafness in different
Dalmatian dog families (Table 7) Therefore it is probable that these genes or genes
Linkage analysis of gene-associated microsatellites 29
in their flanking regions are involved in the development of the disease in the
respective familes The results of this test statistics indicate that the inheritance of
non-syndromic hearing loss in Dalmatian dogs is probably as heterogenic in origin as
it is in humans Genetic heterogeneity means that different mutations cause the same
phenotype or disease the different mutations can either be found at the same locus
(allelic heterogeneity) or even at different loci (non-allelic heterogeneity)
The extreme heterogeneity of human deafness often hampered genetic studies
because many different genetic forms of hearing loss give rise to similar clinical
phenotypes and conversely mutations in the same gene can result in a variety of
clinical phenotypes In man genes that transport ions across membranes to maintain
appropriate solute concentration and pH as well as regulatory genes mainly
transcription factors and genes that play a part in structural integrity are essential for
the hearing process
However this study was a first step in identifying genes responsible for CCSD in
Dalmatian dogs The genes GJA1 MYH9 and CLDN14 and their flanking regions will
be further analyzed with a combined approach using microsatellites and single
nucleotide polymorphisms (SNPs) (Chapter 6-9) As linkage was found for different
candidate genes in different families subsequently only the families indicating
linkage will be chosen for further molecular analyses of the respective gene
To confirm the result of this study the density of the intragenic markers has to be
increased The current RH map with 3200 markers provides a good estimation of the
order and physical spacing (ie in base pairs) of markers along canine
chromosomes (Guyon et al 2003) and was recently complemented by the
construction of a 4249-marker integrated canine genome RH map which consists of
900 genes 1589 microsatellites and 1760 BAC end markers (Breen et al 2004) all
included and available in the NCBI database (httpwebncbinlmnihgov)
Thus microsatellites derived from the NCBI database could be used to confirm the
linkage Alternatively BLAST searches versus the canine whole genome shotgun
(wgs) sequence resource were perfomed to localize the genes exactly and to obtain
the sequence of the canine genomic contigs containing the human homologous gene
The results of the BLAST searches of the 24 candidate genes against the Boxer
genome assembly 21 are shown in Table 3 The genomic sequence of the
respective candidate gene can now be used to search for intragenic SNPs as these
polymorphisms are the most abundant and useful markers for fine mapping
Linkage analysis of gene-associated microsatellites
30
Development of SNPs requires sequencing of DNA for the respective genomic
regions of the parents with the aim to identify heterozygous base pair exchanges
After a heterozygous base pair is found the whole family can be genotyped for this
informative SNP marker These polymorphisms can than be used for linkage
analyses as well as association studies
Fine mapping using SNP markers for all genes indicating linkage with CCSD
identified by this study should enable us to detect mutations responsible for CCSD in
parts of the Dalmatian dog population
Linkage analysis of gene-associated microsatellites 31
Table 1 Details of the 24 selected human candidate genes
Symbol Gene name Locus name1 Most important reference
CDH23 cadherin related 23 DFNB12 Bork et al 2001
CLDN14 claudin 14 DFNB29 Wilcox et al 2001
COCH coagulation factor C homolog cochlin DFNA9 Robertson et al 1998
COL11A2 collagen type XI alpha 2 DFNA13 McGuirt et al 1999
DFNA5 deafness autosomal dominant 5 DFNA5 Van Laer et al 1998
DIAPH1 diaphanous homolog 1 (Drosophila) DFNA1 Lynch et al 1997
EDN3 endothelin 3 WS type IV Edery et al 1996
EDNRB endothelin receptor type B WS type IV Attie et al 1995
EYA4 eyes absent homolog 4 (Drosophila) DFNA10 Wayne et al 2001
GJA1 gap junction protein alpha 1 43kD (connexin 43)
2 Liu et al 2001
GJB2 gap junction protein beta 2 26k (connexin 26) DFNA3 DFNB1 Kelsell et al 1997
GJB6 gap junction protein beta 6 (connexin 30) DFNA3 DFNB1 Grifa et al 1999
Del Castillo et al 2002
MITF microphthalmia-associated transcription factor WS type II Tassabehji et al 1994
MYH9 myosin heavy polypeptide 9 non-muscle DFNA17 Lalwani et al 2000
MYO6 myosin VI DFNA22 DFNB37 Melchionda et al 2001 Ahmed et al 2003
MYO7A myosin VIIA DFNA11 DFNB2 Liu et al 1997a Liu et al 1997b Weil et al 1997
MYO15A myosin XVA DFNB3 Wang et al 1998
OTOF otoferlin DFNB9 Yasunaga et al 1999
PAX3 paired box gene 3 (Waardenburg syndrome 1) WS type I WS type III Hoth et al 1993 Tassabehji
et al 1992
POU4F3 POU domain class 4 transcription factor 3 DFNA15 Vahava et al 1998
SLC26A4 solute carrier family 26 member 4 DFNB4 Li et al 1998
SOX10 SRY (sex determining region Y)-box 10 WS type IV Pingault et al 1998
TECTA tectorin alpha DFNB21 DFNA8DFNA12
Mustapha et al 1999 Verhoeven et al 1998
TMPRSS3 transmembrane protease serine 3 DFNB8DFNB10 Scott et al 2001
1 Autosomal recessive loci are designated DFNB autosomal dominant loci DFNA 2 To our knowledge no deafness locus has been determined for this autosomal recessive gene
Linkage analysis of gene-associated microsatellites
32
HET
()
638
338
366
544
616
632
456
656
433
614
458
586
529
PIC
()
752
548
336
484
775
70
63
9
74
6
61
6
83
7
62
762
621
No
of
alle
les
6 4 4 4 9 8 6 6 6 13 5 11 5
PCR
pr
oduc
t (b
p)
175
109
123
156
146
179
259
151
241
219
186
214
191
AT
(degC
)
62 60 56 60 60 62 58 60 62 58 58 60 58
Prim
er s
eque
nces
(5rsquorarr
3rsquo)
F A
ATC
AG
GA
GG
GG
TGAG
TGTG
R
CC
CC
CAG
CTC
ATA
CA
TTC
TC
F C
CTG
TAC
TGA
ATG
CTT
GA
GG
R
CA
TCTC
TAG
AA
GA
AG
CC
TCC
F
TC
AC
ATA
GC
ATT
ATA
TATG
GA
C
R T
TGA
GAT
GG
CTC
TTAC
TGA
G
F T
CG
ATG
ATG
CTT
TCTG
GTT
G
R A
GG
CTG
TGA
AA
TGG
ATG
GA
G
F G
AG
AA
GC
AC
CA
GG
CA
TAG
G
R T
GG
TTTA
GC
AA
GG
CTG
TTC
C
F T
GA
CA
TAC
GG
AG
GAC
CA
AG
AG
R C
CC
CTC
CC
TTG
CTC
TATC
TC
F T
GC
CC
CTC
AG
AG
ATA
ATC
AC
R
CTT
CA
ATTA
TAC
AC
ACA
GG
TAC
F
TG
AA
TATG
GG
GC
TGAG
GA
AG
R
TTC
TCC
CTC
TGC
CTG
TGTC
F
GG
TTTA
GC
AC
TGC
CTT
CA
GC
R
CA
TTA
AG
CA
TCTG
GC
ATG
TGG
F
GA
AA
AC
TCA
GA
TTA
GC
CTG
G
R A
TCTT
GA
GA
GC
AA
AGG
TTG
TG
F T
GG
TTA
GG
GC
ATG
ATTC
CA
G
R C
ATG
TATA
AA
GA
GTA
ATG
CC
AG
F
CG
GG
AG
AGG
GTT
TGAC
TAC
R
CTC
CG
TATT
GC
TCA
TCTT
TCC
F
AG
CTT
CC
CTT
CTC
TGA
GA
C
R G
AG
AA
TAG
AG
TTTG
TGC
TCA
G
Rep
eat
(TTT
A)1
5 (A
CC
)9(A
TC)3
(A
TTT)
7(G
TTT)
5 (C
A)2
1 (G
A)2
0 (C
TTT)
~20
(GA
)13
(AC
)20(
AG)9
(C
T)16
TT
(CT)
5 (A
AA
AT)
~25
(CT)
11(G
T)4(
CT)
2 (A
TTT(
T))2
3 (A
C)2
2
Mar
ker n
ame
CD
H23
_MS1
C
DH
23_M
S2_F
2 C
LDN
14_M
S1
CLD
N14
_MS
2 C
LDN
14_M
S3
CO
CH
_MS1
C
OC
H_M
S2
CO
L11A
2_M
S1
CO
L11A
2_M
S3
DFN
A5_
MS
1 D
FNA
5 _M
S2
DIA
PH
1 _M
S1
DIA
PH
1_M
S2
Mar
ker o
rigin
RP
CI8
1-99
C20
R
PC
I81-
99C
20
RP
CI8
1-97
L17
RP
CI8
1-97
L17
RP
CI8
1-97
L17
RP
CI8
1-32
1I4
RP
CI8
1-32
1I4
RP
CI8
1-24
M6
RP
CI8
1-24
M6
RP
CI8
1-66
C12
R
PC
I81-
66C
12
RP
CI8
1-36
2I5
RP
CI8
1-36
2I5
Tabl
e 2
Can
dida
te g
ene-
asso
ciat
ed m
arke
rs f
or c
anin
e de
afne
ss d
evel
oped
by
Rak
(20
03)
For
each
gen
e th
e m
icro
sate
llite-
base
d m
arke
r th
e m
arke
r or
igin
rep
eat t
ype
PC
R p
rimer
s w
ith o
ptim
ised
con
ditio
ns a
nd th
e nu
mbe
r of
alle
les
pol
ymor
phis
m
info
rmat
ion
cont
ent (
PIC
) and
obs
erve
d he
tero
zygo
sity
(HE
T) fo
r all
dogs
gen
otyp
ed a
re s
how
n
Can
dida
te
gene
CD
H23
C
LDN
14
CO
CH
C
OL1
1A2
DFN
A5
DIA
PH
1
Linkage analysis of gene-associated microsatellites 33
HET
()
815
345
695
578
562
508
583
441
75
63
7
33
414
30
76
7
44
8
PIC
()
853
679
798
774
652
63
72
3
59
5
83
2
77
9
50
5
54
1
40
4
88
9
59
1
No
of
alle
les
14 6 7 6 9 5 9 6 13 13 4 6 4 14 6
PCR
pr
oduc
t (b
p)
147
131
258
227
134
190
148
195
218
260
181
164 96
227 94
AT
(degC
)
60 58 58 58 60 60 58 62 58 58 62 56 58 58 62
Prim
er s
eque
nces
(5rsquorarr
3rsquo)
F G
CTA
GG
AA
AA
ATC
CG
CA
ATG
R
GAC
CC
CC
TAG
GAC
ACC
AAC
F
GA
GA
ATT
GG
GC
ATG
GG
CAG
A
R T
GA
CTT
TATC
AC
TGG
TCTT
TG
F T
TATG
CAG
CC
CA
TGAC
AA
TC
R C
AA
GG
GA
AC
TCA
AAG
GC
TTG
F
TG
GA
CC
AG
GTC
AGTT
TGTG
R
TC
TGC
CTG
TGTC
TCTG
CC
F
ATG
GC
ATG
AA
GA
GG
ATA
CC
G
R A
GG
AC
AGG
TGAC
GG
CTC
TAC
F
GC
TAG
TAC
TCG
ATT
GTG
GTC
R
TC
ATG
GG
TTG
TGA
GA
TCC
AG
F T
TAA
TTTG
CTC
GTC
TTC
CTG
R
TG
TAA
GC
TCC
ACG
GA
TCA
CC
F
CTC
TCTT
GG
TCTC
CC
TCTG
C
R G
GG
AG
TAG
GG
GTG
GAG
TAG
G
F G
GTG
TTTC
CTT
TCC
TTTT
CT
R G
GTG
TTC
TCTC
CC
TTTC
TCT
F C
TCTA
TGA
AA
GG
TGA
TTG
CC
R
CA
GC
CAT
AC
AA
ATG
AGA
ATT
G
F C
TAC
AG
TGA
ATC
AG
CA
CA
GAC
R
CA
GC
CTT
GA
CTG
TTTC
TTTG
G
F T
GA
TTG
AC
TCTA
CTT
TAC
AC
A
R T
ATA
TTA
GG
CG
GTT
TTC
TTC
T F
AC
CC
AG
GTG
GC
CTG
ATTG
R
GC
AC
GC
AC
GTT
CTC
TCTT
TC
F G
AA
TGC
CC
TTC
ACC
TTG
AA
A
R G
GA
AA
AG
GA
GA
GA
TGA
TGC
C
F T
CTT
CC
TTG
GA
AA
GG
GA
AC
TC
R T
GC
CC
TAA
CA
CTT
GG
AA
TGG
Rep
eat
(TA
GA
)12
(GA
)25
(GT)
10(A
T)13
(A
G)2
1 (G
T)16
(A
G)1
2 (C
A)1
2 (G
A)8
(C
TTT)
~20
(GA
AA
)~25
(G
T)12
(C
A)1
5 (A
G)1
1 (G
AA
A)~
43
(AC
)13
Mar
ker n
ame
ED
N3_
MS
1 E
DN
RB
_M
S1
EY
A4_
MS
1 E
YA
4_M
S2
GJA
1_M
S1
GJA
1_M
S2
GJB
2_M
S1
GJB
2_M
S2
GJB
6_M
S2
GJB
2+6_
MS1
M
ITF
_MS
2 M
ITF
_MS
3 M
YH
9_M
S2
MY
H9_
MS
3 M
YO
6_M
S2
Mar
ker o
rigin
RP
CI8
1-36
6E14
Z
emke
et a
l (1
999)
R
PC
I81-
301N
19
RP
CI8
1-30
1N19
R
PC
I81-
370A
16
RP
CI8
1-37
0A16
R
PC
I81-
133O
22
RP
CI8
1-13
3O22
R
PC
I81-
343C
15
FH
2324
R
PC
I81-
119P
24
RE
N10
0J13
R
PC
I81-
374A
12
FH
2293
R
PC
I81-
156P
14
Tabl
e 2
(con
tinue
d)
Can
dida
te g
ene
ED
N3
ED
NR
B
EY
A4
GJA
1 G
JB2
GJB
6 M
ITF
MY
H9
MY
O6
Linkage analysis of gene-associated microsatellites
34
HET
()
359
366
431
566
51
50
5
28
3
35
8
79
9
70
4
69
2
23
9
68 71
698
PIC
()
496
623
627
765
635
756
502
575
756
76
70
1
44
2
75
4
84
5
80
6
No
of
alle
les
5 4 6 13 7 8 5 3 10 11 8 8 8 10 11
PCR
pr
oduc
t (b
p)
293
166
100
137
174
197
164
201
160
297
267
140
276
228
260
AT
(degC
)
56 60 56 62 62 62 60 60 58 58 56 58 58 62 60
Prim
er s
eque
nces
(5rsquorarr
3rsquo)
F T
GG
TTA
AA
AC
ATT
AA
AC
TTA
TAG
R
TA
GTA
TATA
GA
GA
TGC
AA
TGG
F
CA
TTG
GG
TGC
TTTC
CTG
TTC
R
TG
GA
GC
TGC
AG
GTA
TAG
CC
F
CC
AG
GC
ATT
CG
AG
GG
TG
R C
AG
AA
CTT
GA
GG
AAC
CA
TAG
F
CC
ATG
AAC
TTTG
TGG
AA
CTG
C
R A
AA
GG
GTT
GC
TGTG
GA
GA
TG
F A
GG
CA
GG
TTC
ATC
TGTG
TCC
R
TC
CC
AG
AC
CC
AG
CTA
CA
TTC
F
CA
GC
CAA
CTG
TATT
CTC
CTT
G
R A
TCTT
GA
GC
CC
TGC
ATT
AG
G
F G
AA
GC
GA
GG
AG
AG
ACA
GTC
C
R A
AG
GA
AG
CC
TCC
TGAC
AA
CC
F
CA
GG
GTC
AG
GC
TCTA
TGC
TC
R T
CC
TATC
ATC
CG
GC
TTTG
AC
F
TC
TGG
ATTG
TGG
TCA
CA
AC
C
R A
CTG
GAC
AC
TTC
TTTT
CA
GAC
G
F A
AA
GTG
GC
TGG
TTC
GG
AA
G
R A
GC
AG
CA
GC
ATA
CA
TTC
CTC
F
AA
GTA
GA
TCC
TATT
ATC
GTG
G
R A
GTT
TCA
GTG
TCTG
TTA
AA
TAG
F
CC
GG
ATT
TCTG
AG
GAG
GC
R
CA
TGC
TCTT
CA
CC
AGA
AC
C
F T
CA
GC
ATG
GA
TTTT
GTA
AA
ATC
R
GG
AC
TGC
GTG
GA
CAT
CTG
F
AC
AC
GG
TTC
TCG
CTG
ATG
TG
R T
GA
AG
GG
GA
TTG
AAC
AG
AG
G
F T
TCA
TTC
CG
AG
GTT
CTA
AC
TG
R C
AC
CA
TCTC
GTA
GC
CTT
TATC
Rep
eat
(TC
)13
(AC
)12
(GT)
12
(AC
)18
(GT)
12
(TA
)3(T
G)9
(TA
)2(C
A)2
C
(TA
AA
)5
(AA
T)18
(C
T)13
(T
G)1
3 (C
T)17
(T
AA
A)1
4 (G
T)3
CC
(GT)
19
(ATT
T)12
(T
C)8
(CA)
5(C
G)2
(CA)
9 (G
T)16
Mar
ker n
ame
MY
O7A
_M
S1
MY
O7A
_M
S2
MY
O7A
_M
S3
MY
O15
A _
MS
1 M
YO
15A
_M
S2
OTO
F _M
S1
PA
X3_
MS
1 P
AX
3_M
S2
PO
U4F
3_M
S4
SLC
26A
4_M
S2
SO
X10
_MS
2 T
EC
TA _
MS
1 T
EC
TA _
MS
2 T
MP
RS
S3_
MS
1 T
MP
RS
S3_
MS
2
Mar
ker o
rigin
RP
CI8
1-19
3deg2
RP
CI8
1-19
3deg2
AH
TH29
8 R
PC
I81-
362deg
13
RP
CI8
1-36
2deg13
R
PC
I81-
198L
15
RP
CI8
1-25
7H23
R
PC
I81-
257H
23
G2C
024
66
RP
CI8
1-47
P17
RP
CI8
1-50
5H2
RP
CI8
1-59
C2
RP
CI8
1-59
C2
RP
CI8
1-12
5P17
A
HTH
246
Tabl
e 2
(con
tinue
d)
Can
dida
te
gene
MY
O7A
M
YO
15A
O
TOF
PA
X3
PO
U4F
3 S
LC26
A4
SO
X10
TE
CTA
TM
PR
SS
3
Linkage analysis of gene-associated microsatellites 35
Table 3 The gene-associated microsatellites (MS) for 24 candidate genes of canine
congenital sensorineural deafness were localized using BLAST searches against the
Boxer genome assembly 21 The accession numbers (AccNo) of the whole
genome shotgun (WGS) contigs containing the genes as well as their associated
microsatellites are given
Microsatellite Acc No of WGS contig CFA Location of
Gene (Mb) Location of
MS (bp) Location of
MS in relation to gene
CDH23_MS1 NW_876311 4 2541025780 25510 intragenic
CDH23_MS2 NW_876311 4 2541025780 25630 intragenic
CLDN14_MS1 NW_876295 31 3379533796 33790 proximal
CLDN14_MS2 NW_876295 31 3379533796 33950 distal
CLDN14_MS3 NW_876295 31 3379533796 33790 proximal
COCH_MS1 NW_876327 8 1321513232 13225 intragenic
COCH_MS2 NW_876327 8 1321513232 13290 distal
COL11A2_MS1 NW_876254 12 56315659 5608 proximal
COL11A2_MS3 NW_876254 12 56315659 5578 proximal
DFNA5_MS1 NW_876258 14 4116941237 41135 proximal
DFNA5_MS2 NW_876258 14 4116941237 41250 distal
DIAPH1_MS1 NW_876292 2 3933039430 39382 intragenic
DIAPH1_MS2 NW_876292 2 3933039430 39552 distal
EDN3_MS1 NW_876277 24 4701347032 47057 distal
EDNRB_MS1 Zemke et al (1999) 22 3436334383 34354 proximal
EYA4_MS1 NW_876269 1 2928029550 29531 intragenic
EYA4_MS2 NW_876269 1 2928029550 29500 intragenic
GJA1_MS1 NW_876269 1 6399463996 64150 distal
GJA1_MS2 NW_876269 1 6399463996 64160 distal
GJB2_MS1 NW_8762781 25 2093620942 20938 intragenic
GJB2_MS2 NW_8762781 25 2093620942 20863 proximal
GJB2+6_MS1 FH2324 25 2093620942 17543 proximal
GJB3_MS2 NW_876259 15 1018310194 4530 proximal
GJB6_MS2 NW_876278 25 2090420906 20953 distal
Linkage analysis of gene-associated microsatellites
36
Table 3 (continued)
Microsatellite Acc No of WGS contig CFA Location of
Gene (Mb) Location of
MS (bp) Location of
MS in relation to gene
MITF_MS2 NW_876271 20 2485324884 24844 proximal
MITF_MS3 REN100J13 20 2485324884 25668 distal
MYH9_MS2 NW_876251 10 3113531193 31244 distal
MYH9_MS3 FH2293 10 3113531193 31696 distal
MYO15A_MS1 NW_876313 5 4436944419 44330 proximal
MYO15A_MS2 NW_876313 5 4436944419 44409 intragenic
MYO6_MS2 NW_876254 12 4041740504 40534 distal MYO7A_MS1 NW_876273 21 2454324609 24465 proximal
MYO7A_MS2 NW_876273 21 2454324609 24566 intragenic
MYO7A_MS3 AHTH298 21 2454324609 24594 distal
OTOF_MS1 NW_876263 17 2350223595 23463 proximal
PAX3_MS1 NW_876304 37 3134831445 31426 intragenic
PAX3_MS2 NW_876304 37 3134831445 31481 distal
POU4F3_MS4 G2C02466 2 4361043612 - -
SLC26A4_MS2 NW_876265 18 1586715927 15960 distal
SOX10_MS2 NW_876251 10 2975129762 29740 proximal
TECTA_MS1 NW_876312 5 1588515954 15910 intragenic
TECTA_MS2 NW_876312 5 1588515954 15930 intragenic
TMPRSS3_MS1 NW_876295 31 3903539054 38942 proximal
TMPRSS3_MS2 AHTH246 31 3903539054 38508 proximal
Linkage analysis of gene-associated microsatellites 37
Table 4 Number of alleles observed (HET) and expected (HE) heterozygosity and
polymorphism information content (PIC) for the developed marker-set
Feature Mean SD Min Max
No of alleles 75 31 3 14
hO () 703 122 370 898
hE () 532 151 239 815
PIC () 667 130 336 889
Table 5 Number of alleles per microsatellite locus and their PIC () values of the
developed marker-set
No of alleles per
microsatellite
Number of marker
loci PIC ()
3 1 575
4 6 483
5 5 574
6 11 652
7 2 716
8 5 671
9 3 717
10 2 800
11 3 776
13 4 803
14 2 871
Linkage analysis of gene-associated microsatellites
38
Table 6 Significant test statistics for linkage analysis carried out for all 24 genotyped
families conjoined Zmeans and LOD scores are given with their respective error
probabilities for the gene-associated markers of the candidate genes CLDN14
COL11A2 GJA1 MITF MYH9 and SOX10
Marker Location on canine chromosome (CFA)
Zmean pZmean LOD score pLOD
CLDN14_MS1 31q15 134 009 086 002
CLDN14_MS2 31q15 168 005 105 001
CLDN14_MS3 31q15 108 014 049 007
COL11A2_MS1 12q11-q12 166 005 085 002
COL11A2_MS3 12q11-q12 167 005 078 003
GJA1_MS1 1q24-q25 151 007 118 001
GJA1_MS2 1q24-q25 151 007 118 001
MITF_MS2 20q13 101 02 080 003
MITF_MS3 20q13 121 011 104 001
MYH9_MS2 10q232 080 02 018 02
MYH9_MS3 10q232 175 004 097 002
SOX10_MS2 10q21-q23 146 007 110 001
Linkage analysis of gene-associated microsatellites 39
Table 7 Significant test statistics for linkage analyses carried out each family
separately Zmeans and LOD scores are given with their respective error probabilities
for the gene-associated markers of the candidate genes CLDN14 MYH9 and GJA1
Gene-associated
marker
Number of families with significant linkage to
CCSD
Number of corresponding
family members
Zmean pZmean LOD-score pLOD
CLDN14_MS1 51 40 278 0003 112 0011
CLDN14_MS2 383 000007 170 0003
CLDN14_MS3 281 0002 113 0011
MYH9_MS2 32 21 081 02 023 02
MYH9_MS3
(=FH2293) 156 006 058 005
GJA1_MS1 13 46 295 0002 052 006
GJA1_MS2 286 0002 051 006 1 families with significant linkage include four German full-sib families and one
German half-sib family 2 families with significant linkage include three German full-sib families 3 families with significant linkage include one French half-sib family
Chapter 4
Evaluation of eight candidate genes for canine congenital sensorineural deafness
in Dalmatian dogs
Evaluation of eight candidate genes for CCSD 43
Evaluation of eight candidate genes for canine congenital sensorineural deafness in Dalmatian dogs Abstract
In this study we have been focusing on genomic loci that encode various enzymes
and transporters involved in the hearing process in humans We developed intragenic
markers for the canine genes ESPN MYH14 MYO3A PRES TMC1 TMIE USH1C
and WHRN on canine chromosomes (CFA) 1 2 5 11 18 20 and 21 which have
been shown to be responsible for human hereditary deafness and to employ these
newly developed markers for non-parametric linkage analyses with canine congenital
sensorineural deafness (CCSD) We screened DNA from altogether six Dalmatian
dogs which represent the parents of four families for single nucleotide polymorphisms
(SNPs) in the eight candidate genes by means of direct sequencing combined with a
polymerase chain reaction method for amplifying genomic DNA We characterized 20
SNPs and one insertion-deletion polymorphism For the TMC1 and TMIE genes we
additionally genotyped one microsatellite marker each The families used for
subsequent genotyping of the markers included 39 members from four full-sib
families with frequent occurrence of CCSD We concluded that mutations in ESPN
MYH14 MYO3A PRES TMC1 TMIE USH1C and WHRN do not play a role in
CCSD of the Dalmatian dog population investigated here
Introduction Over the past ten years significant progress has been made in the identification of
genes causing different forms of human deafness Currently 39 of the genes
responsible for non-syndromic hearing impairment have been identified in different
human populations (The Hereditary Hearing Loss Hompage
httpwebhostuaacbehhh Van Camp and Smith 2003)
Since non-syndromic hereditary hearing impairment is almost exclusively caused by
cochlear defects affected patients suffer from sensorineural hearing loss
Evaluation of eight candidate genes for CCSD
44
The most common histopathologic finding in cases of profound congenital deafness
in humans is the cochleosaccular degeneration (CSD) It is estimated to occur in
approximately 70 of cases in man and also in dog breeds the histological pattern is
known as cochleosaccular degeneration
Many genetic disorders in humans and domestic dogs (Canis familiaris) demonstrate
a high level of clinical and molecular similarity Therefore genes responsible for
human hereditary deafness seem to be appropriate candidate genes for CCSD
especially in Dalmatian dogs
In this report we provide 21 single-nucleotide polymorphisms (SNPs) and two
microsatellite markers in altogether eight selected human candidate genes (Table 1)
This eight candidate genes were only recently identified for being responsible for
different form of human non-syndromic deafness In order to evaluate whether any of
this candidate genes is responsible for congenital sensorineural deafness in
Dalmatian dogs we analyzed the association of the ESPN MYH14 MYO3A PRES
TMC1 TMIE USH1C and WHRN haplotypes with the CCSD phenotype in four
families of Dalmatian dogs with frequent occurrence of CCSD
Material and methods Pedigree structure and sampling
For the linkage analysis we used blood samples from 39 Dalmatian dogs They
belong to four full-sib families segregating for CCSD At least two of the full sibs of
each family were unilaterally deaf The phenotype of the affected animals had been
confirmed by brainstem auditory evoked response (BAER) that detects electrical
activity in the cochlea and auditory pathways in the brain
The families consisted of eight to 12 individuals In two families a blood sample of the
sire and dam respectively was not available Screening for SNPs was performed by
comparative sequencing of genomic DNA from the parents of the families used for
linkage analyses
SNP and microsatellite marker identification for genotyping
The canine ESPN MYH14 MYO3A PRES TMC1 TMIE USH1C and WHRN gene
sequences was derived from sequences deposited in the current dog genome
assembly (Boxer genome assembly 21) of the NCBI GenBank (Table 2) by BLAST
Evaluation of eight candidate genes for CCSD 45
(Basic Local Alignment Search Tool) search (httpwwwncbinlmnihgovBLAST)
using the human ESPN MYH14 MYO3A PRES TMC1 TMIE USH1C and WHRN
reference mRNA sequence
We compared the canine genomic DNA sequence from the eight candidate genes to
canine cDNA fragments in the canine EST database using the BLASTN program
For the localization of the exonintron boundaries canine or alternatively human
mRNA sequences were used for the mRNA-to-genomic alignment program Spidey
(httpwwwncbinlmnihgovIEBResearchOstellSpideyindexhtml) The human
and canine mRNA sequences which were used to determine the exon organization of
the candidate genes are given in Table 2
For each of the eight candidate genes we designed intragenic primer pairs to amplifly
intronic sequences yielding products with a length of 560 to 670 bp PCR primers
were developed with the Primer3 program (httpfrodowimiteducgi-
binprimer3primer3_wwwcgi) The PCR reactions were performed in a total of 50 microl
containing 125 microM dNTPs 25 pmol of each primer the reaction buffer supplied by
the manufacturer (Qbiogene Heidelberg Germany) and 1 U Taq polymerase After
a 4 min initial denaturation at 95degC 35 cycles of 30 sec at 94degC 45 sec at 58degC and
80 sec at 72degC were performed in a MJ Research thermocycler (Biozym Hessisch
Oldendorf Germany) The obtained PCR products were directly sequenced with the
DYEnamic ET Terminator kit (AmershamBiosciences Freiburg Germany) and a
MegaBACE 1000 capillary sequencer (AmershamBiosciences) using the PCR
primers as sequencing primers Sequence data were analyzed with Sequencher 42
(GeneCodes Ann Arbor MI)
In the first step sequence analyses were performed for PCR products of the parents
of four full-sib families If a heterozygous SNP was found for one or both parents all
progeny of the respective families were analyzed for that SNP Additionally to the
SNPs we used two microsatellite markers for linkage analyses We identified one
intragenic microsatelllite within the TMC1 gene As we could develop only one SNP
for the TMIE gene we additionally genotyped one microsatellite marker derived from
the NCBI database (httpwebncbinlmnihgov) located very closely to the TMIE
gene (Table 3)
Evaluation of eight candidate genes for CCSD
46
SNP marker analysis
A total of 21 SNPs were identified within ESPN MYH14 MYO3A PRES TMC1
TMIE USH1C and WHRN (Table 3) and used for linkage analysis Of all SNPs six
out of the observed 21 SNPs were polymorphic in all four examined families Out of
the 21 SNPs eleven SNPs were present in family 1 18 SNPs were heterozygous for
one or both parents in family 2 and 15 SNPs could be used for linkage analysis in
family 3 and 4 respectively (Table 4)
The most frequent form of SNPs with a frequency of 238 was the CT transition
motif The scarcest one was the CG transversion motif with a frequency of 48
respectively
Linkage and haplotype analysis Multipoint linkage and haplotype analysis were performed using MERLIN version
0102 (multipoint engine for rapid likelihood inference Center for Statistical Genetics
University of Michigan MI USA Abecasis et al 2002) The test statistics Z-mean
and Lod score were used to test for the proportion of alleles shared by affected
individuals identical by descent (IBD) for the considered marker loci The data of the
genotypes was additionally computed using the software package SAS Genetics
(Statistical Analysis System Version 913 SAS Institute Inc Cary NC USA 2005)
to specify the observed heterozygocity values (HET) and the polymorphism
information content (PIC)
Furthermore linkage disequilibrium (LD) and association of haplotypes with CCSD
was tested using the procedures CASECONTROL and HAPLOTYPE of
SASGenetics (Statistical Analysis System version 913 Cary NC USA)
Results and discussion The non-parametric multipoint linkage analysis in four full-sib families did not show
significant test statistics The highest Z-mean value was 046 the highest LOD Score
was 008 while the error probabilities ranged from 03 to 08 (Table 5) The maximum
achievable Z-mean was 448 and the corresponding value for the LOD score was
160 Marker-trait association tests for haplotypes of the candidate gene markers
were not significant Obviously no haplotype was associated with CCSD in these
Dalmatian dog families as demonstrated (Figure 1 2 3 4 5 6 7 and 8) The
Evaluation of eight candidate genes for CCSD 47
paternal and maternal haplotypes as far as they could be estimated were distributed
among the affected progeny closely to the expected proportion of 50 and thus no
excess of a certain haplotype could be observed in the affected dogs
Due to the fact that both animals with unilateral or bilaterally hearing loss and
bilateral hearing animals shared identical haplotypes it is unlikely that the ESPN
MYH14 MYO3A PRES TMC1 TMIE USH1C and WHRN genes are involved in the
pathogenesis of CCSD in Dalmatian dogs However the ESPN MYH14 MYO3A
PRES TMC1 TMIE USH1C and WHRN gene-associated marker may serve for
further linkage studies in other Dalmatian dog populations and dog breeds other than
Dalmatians
Evaluation of eight candidate genes for CCSD
48
Table 1 Details of the six selected human candidate genes
Gene symbol
Gene name Locus name1 Most important reference
ESPN espin DFNB36 Naz et al (2004)
MYH14 myosin heavy polypeptide 14 DFNA4 Donaudy et al (2004)
MYO3A myosin IIIA DFNB30 Walsh et al (2002)
PRES solute carrier family 26
member 5 (prestin) DFNB61
Zheng et al (2000) Liu et
al (2003)
TMC1 transmembrane channel-like
gene 1
DFNB7
DFNA36 Kurima et al (2002)
TMIE transmembran inner ear DFNB6 Naz et al 2002
USH1C Usher syndrome 1C DFNB18 Verpy et al (2000) Ouyang
et al (2002)
WHRN whirlin DFNB31 Mburu et al (2003)
1 Autosomal recessive loci are designated DFNB autosomal dominant loci DFNA
Evaluation of eight candidate genes for CCSD 49
Table 2 Selected human candidate genes with their location on HSA and CFA and
corresponding accession numbers
Gene symbol Gene
location on HSA1
Acc No 3 human mRNA
Gene location
on CFA2
Acc No 3 canine
genomic sequence
Acc No 3 canine mRNA
ESPN 1 NM_031475 5 NC_006587 XM_546751
MYH14 19 NM_024729 1 NW_876270 -
MYO3A 10 NM_017433 2 NC_006584 XM_544234
PRES 7 NM_206883 18 NC_006600 XM_540393
TMC1 9 NM_138691 1 NC_006583 XM_541284
TMIE 3 NM_147196 20 NC_006602 XM_846596
USH1C 11 NM_153676 21 NC_006603 XM_860072
WHRN 9 NM_015404 11 NC_006593 XM_850321
1 Homo sapiens autosome 2 Canis familiaris autosome 3 Accession number
Evaluation of eight candidate genes for CCSD
50
Table 3 The 21 newly developed intragenic SNPs and two microsatellite markers for
Dalmatian dogs in the candidate genes ESPN MYH14 MYO3A PRES TMC1
TMIE USH1C and WHRN with their corresponding primers the SNP motif the
product size and the annealing temperature the observed heterozygosity (HET) and
polymorphism information content (PIC)
SNP Primer F1 (5acute -gt 3acute) SNP motif primer R2 (5acute -gt 3acute)
Bp3 AT4 PIC ()
HET()
ESPN_SNP1
ACCAGCACCCTCTCCAACTA
AGGAATTCACAA (CT)CACACATACA
ACTCAAGCTCAGGGTGTGGT
565 60 9 10
ESPN_SNP2
ACCAGCACCCTCTCCAACTA
ATGGCTGGCGCT (AG)GAGGCTGCCC
ACTCAAGCTCAGGGTGTGGT
565 60 27 41
ESPN_SNP3
ACCAGCACCCTCTCCAACTA
ACACTCTTCCCA (CT)GGCTGGCGCT
ACTCAAGCTCAGGGTGTGGT
565 60 23 32
ESPN_SNP4
ACCAGCACCCTCTCCAACTA
TGGGAAGAGGGA (AG)GGGGGAGCAT
ACTCAAGCTCAGGGTGTGGT
565 60 23 32
ESPN_SNP5
ACCAGCACCCTCTCCAACTA
GAGTGGGCCAGG (CT)TGGGAAGAGG
ACTCAAGCTCAGGGTGTGGT
565 60 28 42
MYH14_SNP1
CTCTCCCCAACTCAGTTCCA
ACGTGTATTCGG (GT)CGCTTTTATT
GTGATAGGGACGAGCAGCAT
670 60 35 42
MYH14_SNP2
CATGGGACCGTTCCTACACT
AGCCTCGTTTAA (CT)CTAAAAGGAA
GCTCAATAGGCACGACATCA
640 60 34 39
Evaluation of eight candidate genes for CCSD 51
Table 3 (continued)
SNP Primer F1 (5acute -gt 3acute) SNP motif primer R2 (5acute -gt 3acute)
Bp3 AT4 PIC ()
HET()
MYO3A_SNP1
AATGCTTGAGTTTGGGATGC
GGCAGTCCCATG (GT)CCCTTATAAG
ACCTAATTGCCCAGATGCAG
650 60 37 77
MYO3A_SNP2
AATGCTTGAGTTTGGGATGC
GTGGAGAGCCAC (CG)TTGGGAGAGG
ACCTAATTGCCCAGATGCAG
650 60 37 70
MYO3A_SNP3
AACCTCCTGGCGTAGTATTCC
CATTACCTATTT (AT)GATCCTTATA
TTTTCCACTTCAGGCACACA
650 60 25 36
PRES_SNP1
CCCTTACCCCATACCATTCC
GATAGACTTCCT (AG)CCCTCAGACT
TTCAGGACAGCATCATCTGC
560 60 37 64
PRES_SNP2
CCCTTACCCCATACCATTCC
TGATGTCTGCTG (AT)TAACCCATTC
TTCAGGACAGCATCATCTGC
560 60 37 66
TMC1_SNP1
GCAACCTCTCGGTTTATCCA
CGTGAAGTGCCC (AT)TTGATGGAAA
AAGCTGGGGAAGTGGATATGT
610 60 57 37
TMC1_SNP2
GCAACCTCTCGGTTTATCCA
GGAGACATTACC (AG)TGAAGTGCCC
AAGCTGGGGAAGTGGATATGT
610 60 47 29
TMC1_SNP3
GGAAGCAAGACTGAGGTTGG
AGGCTTTTTAAA (AG)CTGTTCTGGG
CTGCTGCATTTGCCTGTAAG
650 60 48 30
TMIE_SNP1
AGAACACCACCGTCTCCTTG
CAAGGCGACGCC (AT)GTGCTGTCCT
GCCTCTGGTCAGAAGAGGTG
625 60 59 36
Evaluation of eight candidate genes for CCSD
52
Table 3 (continued)
SNP Primer F1 (5acute -gt 3acute) SNP motif primer R2 (5acute -gt 3acute)
Bp3 AT4 PIC()
HET()
USH1C_SNP2
CTCCCGGTCTGTCAGGAAC
GGCCTGGGGGGA (AC)AAGCGGACGG
ATGGCATCGACTTCTCCAAC
560 60 37 35
USH1C_SNP4
CTCCCGGTCTGTCAGGAAC
GGTCTCAGACCG (AC)GGCAGGGAGA
ATGGCATCGACTTCTCCAAC
560 60 37 37
WHRN_SNP1
TTCACCTCCAGGATCTGGTC
CCTGAGCCCGAG (CT)CCACGCTGCT
GGCTACTTTTCTTCCCCCTTT
600 60 25 37
WHRN_SNP2
TTCACCTCCAGGATCTGGTC
GGTCACGGGGGC (CT)CCGGGAGGTT
GGCTACTTTTCTTCCCCCTTT
600 59 24 33
WHRN_SNP3
TTCACCTCCAGGATCTGGTC
GTCCGAGTCCCG (AG)CCCCAGCCTG
GGCTACTTTTCTTCCCCCTTT
600 60 34 55
Microsatellite marker
Primers (forward reverse ) 5acute -gt 3acute Bp AT PIC()
HET()
TMC1_MS1
GCCCCCAGCTAAAAAGAGAA
TTCTCTTCCTCCCTCCTGTTC
220-220 60 76 57
FH2158 ATGGCCACATCACCCTAGTC
CTCTCTCTGCATCTCTCATGAA
274-302 58 57 66
Microsatllite marker derived from the NCBI database (httpwebncbinlmnihgov) 1 Forward 2 Reverse 3 Product size (basepairs) 4 Annealing temperature (degC)
Evaluation of eight candidate genes for CCSD 53
Table 4 The 21 newly developed intragenic SNPs for Dalmatian dogs in the
candidate genes ESPN MYH14 MYO3A PRES TMC1 TMIE USH1C and WHRN
with their nucleotide polymorphism allele and genotype frequencies
Gene symbol Fam1 Nucleotide polymorphism
Allele frequencies
Genotype frequencies2
ESPN_SNP1 4 CgtT 083017 840
ESPN_SNP2 2 3 4 AgtG 074026 15160
ESPN_SNP3 2 3 TgtC 068032 07120
ESPN_SNP4 2 3 GgtA 068032 07120
ESPN_SNP5 2 3 4 CgtT 074026 15160
MYH14_SNP1 2 3 4 GgtT 058041 51610
MYH14_SNP2 2 3 4 CgtT 058041 51610
MYO3A_SNP1 1 2 GgtT 062038 5111
MYO3A_SNP2 1 2 CgtG 062038 5111
MYO3A_SNP3 1 2 4 TgtA 076024 15140
PRES_SNP1 1 2 3 4 AgtG 058042 10254
PRES_SNP2 1 2 3 4 TgtA 058042 10254
TMC1_SNP1 1 2 3 4 AgtT 056044 11226
TMC1_SNP2 1 2 3 4 AgtG 076024 20190
TMC1_SNP3 1 2 3 4 AgtG 074026 19200
TMIE_SNP1 1 2 3 4 AgtT 058042 12216
USH1C_SNP2 1 3 4 AgtC 053047 9147
USH1C_SNP4 1 3 4 AgtC 053047 9147
WHRN_SNP1 2 CgtT 075025 360
WHRN_SNP2 2 CgtT 075025 360
WHRN_SNP3 2 3 4 InDel3 069031 13171 1 Number of families which were informative for the respective SNPs 2 Frequence of genotypes (first number number of animals homozygous for allele 1
second number heterozygous third number homozygous for allele 2) 3 Insertiondeletion of one base-pair (adenine)
Evaluation of eight candidate genes for CCSD
54
Table 5 Linkage analyses for families 1 2 3 and 4 for the 21 SNP and two
microsatellite markers within the eight candidate genes regarding Zmean LOD score
and error probabilities (p-values)
Gene symbol Marker Zmean pz-value1 LOD score pL-value2
ESPN ESPN_SNP1 014 04 002 04
ESPN_SNP2 014 04 002 04
ESPN_SNP3 014 04 002 04
ESPN_SNP4 014 04 002 04
ESPN_SNP5 014 04 002 04
MYH14 MYH14_SNP1 -089 08 -019 08
MYH14_SNP1 -089 08 -019 08
MYO3A MYO3A_SNP1 -049 07 -011 08
MYO3A_SNP2 -049 07 -011 08
MYO3A_SNP3 -049 07 -011 08
PRES PRES_SNP1 -094 08 -019 08
PRES_SNP2 -094 08 -019 08
TMC1 TMC1_SNP1 -034 06 -008 07
TMC1_SNP2 -034 06 -008 07
TMC1_SNP3 -034 06 -008 07
TMC1_MS1 -035 06 -008 07
TMIE TMIE_SNP1 013 04 003 03
FH2158 -056 07 -013 08
USH1C USH1C_SNP2 018 04 04 03
USH1C_SNP4 018 04 04 03
WHRN WHRN_SNP1 046 03 008 03
WHRN_SNP2 046 03 008 03
WHRN_SNP3 046 03 008 03 1 Error probability regarding Zmean 2 Error probability regarding LOD score
Microsatellite marker
Evaluation of eight candidate genes for CCSD 55
Figu
re 1
SN
P-h
aplo
type
s of
the
ES
PN
gen
e in
all
anal
yzed
Dal
mat
ian
dog
fam
ilies
The
hapl
otyp
es b
elon
ging
to F
amily
2 3
and
4 re
gard
ing
the
gene
ES
PN
sho
w n
o as
soci
atio
n w
ith th
e C
CS
D p
heno
type
In
the
thre
e fa
milie
s th
e ha
plot
ype
21
43
2 is
the
mos
t com
mon
one
bei
ng p
rese
nt in
72
o
f all
anim
als
It is
follo
wed
by
the
hapl
otyp
e 2
32
14
with
18
a
nd 4
34
34
with
6
Nor
mal
hea
ring
anim
als
as
wel
l as
thei
r de
af s
iblin
gs s
how
all
thes
e th
ree
hapl
otyp
es T
here
is n
o re
com
bina
tion
of th
e ha
plot
ypes
of t
he E
SP
N g
ene
in th
e th
ree
fam
ilies
Evaluation of eight candidate genes for CCSD 56
Figu
re 2
SN
P-h
aplo
type
s of
the
MY
H14
gen
e in
all
anal
yzed
Dal
mat
ian
dog
fam
ilies
Evaluation of eight candidate genes for CCSD 57
Figu
re 3
SN
P-h
aplo
type
s of
the
MY
O3A
gen
e in
all
anal
yzed
Dal
mat
ian
dog
fam
ilies
Evaluation of eight candidate genes for CCSD 58
Figu
re 4
SN
P-h
aplo
type
s of
the
PR
ES
gen
e in
all
anal
yzed
Dal
mat
ian
dog
fam
ilies
Evaluation of eight candidate genes for CCSD 59
Figu
re 5
SN
P- a
nd m
icro
sate
llite-
hapl
otyp
es o
f the
TM
C1
gene
in a
ll an
alyz
ed D
alm
atia
n
Evaluation of eight candidate genes for CCSD 60
Figu
re 6
SN
P- a
nd m
icro
sate
llite-
hapl
otyp
es o
f the
TM
IE g
ene
in a
ll an
alyz
ed D
alm
atia
n do
g fa
milie
s
Evaluation of eight candidate genes for CCSD 61
Figu
re 7
SN
P-h
aplo
type
s of
the
US
H1C
gen
e in
all
anal
yzed
Dal
mat
ian
dog
fam
ilies
Evaluation of eight candidate genes for CCSD 62
Figu
re 8
SN
P-h
aplo
type
s of
the
WH
RN
gen
e in
all
anal
yzed
Dal
mat
ian
dog
fam
ilies
Chapter 5
Molecular characterization of the canine myosin heavy polypeptide 9
non-muscle (MYH9) gene on dog chromosome 10q232
Canine MYH9 gene 65
Molecular characterization of the canine myosin heavy polypeptide 9 non-muscle (MYH9) gene on dog chromosome 10q232 Abstract A missense mutation of the Myosin heavy polypeptide 9 (MYH9) gene which
encodes the nonmuscle myosin heavy chain-A caused non-syndromic sensorineural
deafness in a human family which was characterized by cochleosaccular
degeneration In the present study we evaluated whether MYH9 gene mutations are
responsible for canine congenital sensorineural deafness (CCSD) in Dalmatian dogs
As described in Chapter 3 two MYH9 gene-associated microsatellites were
genotyped in 25 Dalmatian dog families segregating for CCSD We could find
significant linkage with CCSD for the microsatellite marker MYH9_MS3 (=FH2293)
We used data deposited in the NCBI to assemble the canine MYH9 gene DNA
sequence Characterization of the canine MYH9 gene revealed that the canine gene
consists of 41 exons spanning approximately 90 kb
We detected 22 single nucleotide polymorphisms (SNPs) in a mutation scan of
altogether 16 Dalmatian dogs from three families which showed significant linkage
between the deafness phenotype and the MYH9 gene-associated microsatellite
None of the SNPs affects the amino acid sequence of MYH9 We concluded that the
exons of the canine MYH9 gene do not play a role in Dalmatian CCSD Availability of
the microsatellite marker SNPs and DNA sequence reported in this study enhance
evaluation of the MYH9 gene as a CCSD candidate gene in other Dalmatian dog
populations and other dog breeds affected by CCSD
Introduction Myosin is a functional protein associated with cellular movement cell division muscle
contraction and other functions Members of the myosin super-family are
distinguished from the myosin heavy chains that play crucial roles in cellular
processes The human MYH9 gene consists of 40 exons and spans 67959 bp
Canine MYH9 gene
66
Toothaker et al (1991) mapped a nonmuscle myosin heavy chain gene to human
chromosome (HSA) 22q123-q131 Simons et al (1991) likewise mapped the gene
to HSA22q112 which they designated nonmuscle myosin heavy chain-A (NMMHC-
A)
The similarities between the autosomal dominant giant-platelet disorders May-
Hegglin anomaly Fechtner syndrome and Sebastian syndrome and refinement of the
disease loci for May-Hegglin anomaly and Fechtner syndrome to an overlapping
region of 480 kb on human chromosome 22 suggested that all these three disorders
may be allelic Among the identified candidate genes was the gene encoding
nonmuscle myosin heavy-chain A (MYH9) The May-HegglinFechtner Syndrome
Consortium (2000) demonstrated that mutations in MYH9 result in one of the three
disorders mentioned above The same Consortium also speculated that mutations in
MYH9 may also play a role in another autosomal dominant disorder a form of
nonsyndromic deafness characterized by progressive hearing impairment and
cochleosaccular degeneration This autosomal dominant form of human
nonsyndromic hereditary deafness (DFNA17) was described by Lalwani et al (1999)
They studied a five-generation American family previously reported by Lalwani et al
(1997) with deafness caused by cochleosaccular degeneration (CSD) CSD is the
most common histopathologic finding in cases of profound congenital deafness and
is estimated to occur in approximately 70 of cases in man
DFNA17 maps to the same region as MYH9 Because of the importance of myosins
in hearing Lalwani et al (2000) tested MYH9 as a candidate gene for DFNA17 and
demonstrated a missense mutation in the MYH9 gene in affected members of a
kindred with DFNA17 They found a G-to-A transition at nucleotide 2114 in the MYH9
gene This missense mutation changed codon 705 from an invariant arginine to a
histidine within a highly conserved Src homology 1 (SH1) linker region Previous
studies had shown that modification of amino acid residues within the SH1 helix
causes dysfunction of the ATPase activity of the motor domain in myosin
Many genetic disorders in humans and domestic dogs (Canis familiaris) demonstrate
a high level of clinical and molecular similarity Rak et al (2003) mapped 20 potential
candidate genes for sensorineural deafness in dogs by fluorescence in situ
hybridization and a radiation hybrid panel among them the MYH9 gene that was
assigned to canine chromosome (CFA) 10q232 Congenital sensorineural deafness
has been reported for approximately 54 different breeds of dogs (Strain 1996)
Canine MYH9 gene 67
The incidence of this congenital anomaly is highest in Dalmatian dogs of which 165-
30 exhibit unilateral or bilateral hearing loss (Famula et al 1996 Wood and
Lakhani 1997 Muhle et al 2002 Juraschko et al 2003a Rak and Distl 2005) The
inheritance and segregation of a major gene in CCSD has been demonstrated in
different Dalmatian dog populations (Famula et al 2000 Muhle et al 2002
Juraschko et al 2003b)
In dog breeds the histological pattern is known as cochleosaccular degeneration
commonly known as Scheibe dysplasia as it is described in approximately 70 of
cases with human hereditary deafness (Strain 1996) Therefore the MYH9 gene
seems to be a perfect candidate gene for CCSD especially in Dalmatian dogs
In this report we provide the genomic organization and the complete sequence of the
canine MYH9 gene A mutation analysis was performed to identify single nucleotide
polymorphisms (SNPs) in this gene In order to evaluate whether the MYH9 gene is
responsible for congenital sensorineural deafness in Dalmatian dogs we analyzed
the association of the MYH9 haplotypes with the CCSD phenotype in three families of
Dalmatian dogs with frequent occurrence of CCSD and significant linkage to the
gene-associated microsatellite MYH9_MS3 (Chapter 3)
Materials and Methods
Cloning and sequencing of canine MYH9 cDNA
The canine MYH9 gene sequence was derived from sequences deposited in the
current dog genome assembly (Boxer genome assembly 21) of the NCBI GenBank
(Genbank Acc No NW_1398701) by BLAST (Basic Local Alignment Search Tool)
search (httpwwwncbinlmnihgovBLAST) using the human MYH9 reference
mRNA sequence (Genbank Acc No NM_002473)
The distance between MYH9 and the microsatellite MYH9_MS2 is about 181 kb
whereas the microsatellite MYH9_MS3 (=FH2293) places about 511 kb from MYH9
The isolation of full length cDNAs was achieved by a modified rapid amplification of
cDNA ends (RACE) protocol Total RNA from dog lung of a normal female Beagle
(Biocat Heidelberg Germany) was used for the overlapping 5rsquo- and 3rsquo-RACE
products with the FirstChoiceTM RNA ligase-mediated (RLM)-RACE kit (Ambion
Europe Huntingdon UK) according to the protocols of the manufacturer Reverse
transcription polymerase chain reaction (RT-PCR) using two pairs of nested
Canine MYH9 gene
68
gene-specific and supplied 5rsquo-adaptor-specific primers were used to amplify the
complete 5rsquo-end of the investigated cDNA Similarly the 3rsquo-end were amplified using
two pairs of nested gene-specific primers in combination with supplied 3rsquo-adaptor-
specific primers Additionally RT-PCR using six pairs of gene-specific primers were
used to amplify the complete sequence of the investigated cDNAs
RACE products were cloned into pCRR21 vector using the Invitrogen TA cloning kit
(Invitrogen Karlsruhe Germany) Several clones for each cDNA were sequenced
with the ThermoSequenase kit (AmershamBiosciences Freiburg Germany) and a
LI-COR 4300 DNA Sequencer (LICOR inc Bad Homburg Germany) Sequence
data were analyzed with Sequencher 42 (GeneCodes Ann Arbor MI)
Sequence analysis of canine MYH9 gene For the localization of the exonintron boundaries after cloning and sequencing of full
length canine cDNAs (as described above) the mRNA-to-genomic alignment program
Spidey (httpwwwncbinlmnihgovIEBResearchOstellSpideyindexhtml) was
used Repetitive elements were analyzed with Repeatmasker 2
(httprepeatmaskergenomewashingtonedu) The GC content was calculated with
the EBI toolbox CpG PlotCpGreport (httpwwwebiacukToolssequencehtml)
Mutation analysis To identify polymorphisms within the canine MYH9 sequence exons with flanking
regions were PCR amplified and sequenced from 16 Dalmatian dogs which
represent three families consisting of three to six full sibs and at the best of both
parents At least two of the full sibs of each family were unilaterally deaf The
phenotype of the affected animals had been confirmed by brainstem auditory evoked
response (BAER) that detects electrical activity in the cochlea and auditory pathways
in the brain PCR primers and conditions for the amplification of MYH9 exons with
flanking sequences and microsatellite flanking primers for PCR reactions are given in
Table 1 PCR primers were developed with the Primer3 program
(httpfrodowimiteducgi-binprimer3primer3_wwwcgi) The PCR reactions for
exons and their flanking sequences were performed in a total of 50 microl containing 125
microM dNTPs 25 pmol of each primer the reaction buffer supplied by the manufacturer
(Qiagen Hilden Germany) and 1 U Taq polymerase After a 4 min initial
denaturation at 95 degC 35 cycles of 30 sec at 94 degC 45 sec at 58 degC and 80 sec
Canine MYH9 gene 69
at 72 degC were performed in a MJ Research thermocycler (Biozym Hessisch
Oldendorf Germany) The obtained PCR products were directly sequenced with the
DYEnamic ET Terminator kit (AmershamBiosciences Freiburg Germany) and a
MegaBACE 1000 capillary sequencer (AmershamBiosciences) using the PCR
primers as sequencing primers Sequence data were analyzed with Sequencher 42
(GeneCodes Ann Arbor MI)
Multipoint linkage and haplotype analysis were performed using MERLIN version
0102 (multipoint engine for rapid likelihood inference Center for Statistical Genetics
University of Michigan MI USA Abecasis et al 2002) The test statistics Z-mean
and Lod score were used to test for the proportion of alleles shared by affected
individuals identical by descent (IBD) for the considered marker loci
Linkage means that a haplotype characterized by microsatellites or SNPs is
significantly more often present in family members with the phenotype under study
than expected by random assortment For construction of haplotypes sets of closely
linked genetic markers on the same chromosome are needed which tend to be
inherited together as they are not likely to be separated by recombination
Association analysis can be carried out as a method of genetic analysis that
compares the frequency of alleles between affected and unaffected individuals
across all families A given allele is considered to be associated with the disease if
the presence of that allele explains a significant proportion of the phenotypic trait
variation
Results and Discussion
Analysis of the genomic organization and cDNA of the canine MYH9 gene
A full-length cDNA sequence of 7484 bp of the canine MYH9 gene was obtained by
using the RACE protocol The obtained RT-PCR products were sequenced and the
generated sequence data were submitted to the EMBL nucleotide database (Acc No
AM086385)
Using the RT-PCR analyses for cDNA-genomic sequence comparisons we detected
that the canine MYH9 gene consists of 41 exons with an untranslated exon 1 and
exonintron boundaries that conform perfectly to the GTAG rule (Table 2)
The 5rsquo-UTR consists of 181 bp while the 3rsquo-UTR consists of 1432 bp Assuming that
the homologous ATG start codon as in man is used the canine MYH9 cDNA
Canine MYH9 gene
70
contains an open reading frame of 5889 bp encoding a protein of 1963 amino acids
A canonical polyadenylation signal AAUAAA is located approximately 14 kb
downstream of the stop codon
The exon sizes range from 28 to 1556 bp the introns between these exons span
between 86 and 13493 bp the total size of the canine MYH9 gene is approximately
90 kb However the sequence homology between the human murine and canine
MYH9 gene is limited to the coding sequence of exon 2 to 41 The coding sequence
of canine MYH9 displays 926 and 899 similarities to the human and murine
MYH9 gene respectively In the untranslated regions the sequence similarity
between dog and human and mouse is rather low The canine MYH9 protein shows
982 and 968 identities to the orthologous human and murine protein
respectively (Fig 2)
The repeat content in the 90 kb region of the canine MYH9 gene is 1458 Most of
the repetitive elements belong to the SINE family (892) followed by the fraction of
the LINEs (306) other repetitive elements constitute 165 respectively The
entire canine MYH9 gene has an overall GC-content of 54 The canine MYH9 gene
contains 12 CpG islands (Gardiner-Garden and Frommer 1987) distributed over the
entire gene whereas by far the longest one with a length of 789 bp can be found in
the region of the second exon or in the first translated exon respectively (GC content
of 50 over 200 bp)
Mutation and haplotype analysis
All coding exons with flanking intronic regions of MYH9 could be amplified from the
examined 16 dogs and the sequences were compared to the Boxer genome
assembly 21 of the NCBI Gen Bank (Genbank Acc No NW_1398701)
The search for sequence variations within the MYH9 gene revealed a total of 22
SNPs shown in Table 3 Most of the polymorphisms were found in the flanking
regions of exons only 3 were within exons Only five out of the observed 22 SNPs
were polymorphic in all three examined families
None of the observed polymorphism did alter the predicted amino acid sequence of
MYH9 nor the identified hapltypes showed an association with the CCSD phenotype
Multipoint test statistics for MYH9 alleles identical by descent were close to zero (Z-
mean 035 to -035 Lod score 013 to -007) and not significant (p= 04 to 07)
However the microsatellite MYH9_MS3 (=FH2293) showed a Z-mean of 156
Canine MYH9 gene 71
(p= 006) and a Lod score of 058 (p= 005) indicating linkage The reason for this
result was heterogeneity among these three families For family 1 and 2 the test
statistics indicated linkage (Z-mean 191 with p= 003 Lod score 058 with p= 005)
whereas for familiy 3 the Z-mean and Lod score was -050 (p= 07) and -008 (p=
07)
Thus these silent point mutations found in affected and unaffected Dalmatian dogs
seem not responsible for the CCSD phenotype in these three families
Conclusions
The characterization of the transcript and genomic sequences of canine MYH9 gene
revealed a conserved organization with respect to the human orthologs In general
the gene size in dog is bigger compared to the human sequence due to the
untranlated first exon in the 5rsquo-UTR Several single nucleotide polymorphisms in the
canine MYH9 gene were identified However because of the fact that both animals
with unilateral hearing loss and bilateral hearing animals shared identical
haplotypes these polymorphisms are obviously not associated with CCSD in these
Dalmatian dog families Furthermore the present study revealed no functional
mutations of the complete coding region of MYH9 We can exclude the MYH9 gene
as a candidate gene for congenital sensorineunal deafness in Dalmatian dogs
However the MYH9 gene sequence SNPs and microsatellite markers reported in
this study enhance evaluation of the MYH9 gene in other Dalmatian dog populations
and dog breeds other than Dalmatians
Canine MYH9 gene
72
Table 1 PCR primers for the amplification of all MYH9 exons
Exon Primer Sequence (5rsquo ndash 3rsquo) Product
size (bp)
Annealing temperature
(degC) exon 1 MYH9_Ex1_F GCC TGG TTT GAC AAG TTT TG MYH9_Ex1_R TTC CTC AAG TCC CCT ACT CC 560 58 exon 2 MYH9_Ex2_F AGG ACC CTC ACC CAA CAC MYH9_Ex2_R GCA CAG AGT CTG GAC AAG AAT G 621 58 exon 3 MYH9_Ex3_F CAC CCC TCA ATA CTC AGT GC MYH9_Ex3_R GAG AAG ACT CCT GCC TCT CC 588 58 exon 4 MYH9_Ex4_F GGC AAT GTG TTT GAT TGT ACT G MYH9_Ex4_R TTT CCA AAG CAA CTC AGA AAG 542 58 exon 5 -6 MYH9_Ex56_F ACA GAG CAG GCG TGA CAG MYH9_Ex56_R GAG GAC AAG AGC TGG AAG C 724 58 exon 7 -8 MYH9_Ex78_F AAG CGG AAA GTT GCC TTC MYH9_Ex78_R ATG GCA CAA TGA CCA GTC TC 699 58 exon 9 MYH9_Ex9_F TGG GCA CAG TTT TTG TCT G MYH9_Ex9_R CAC ACA ATC AAG GGC TTT TC 590 58 exon 10 MYH9_Ex10_F GGA GCC AGT TGT CCT AGT AGT G MYH9_Ex10_R GCC AGG TGC CTA TCA CTG 540 58 exon 11 MYH9_Ex11_F TGT TTC GAA GCG TTT TTA CC MYH9_Ex11_R GGT CCC ATT GTT TCT AGC AC 551 58 exon 12 MYH9_Ex12_F TTT CCC AAG CAG CTA GAA AG MYH9_Ex12_R ACT GGG TGG TCT TGA GAC TG 586 58 exon 13 MYH9_Ex13_F CTG CCT TTG GTG ACA AGT G MYH9_Ex13_R GCA AGC CCT AAG TAC AAT GG 548 58 exon 14 MYH9_Ex14_F ATA CCA GGT CAA GGG GTG TC MYH9_Ex14_R TCA CCT GGT TCT AGG TGG AG 524 58 exon 15 MYH9_Ex15_F CGT TTG GAG TTG AT CCT AGC MYH9_Ex15_R AGC CAA GTT TCC TGA CTG TG 566 58 exon 16 MYH9_Ex16_F AGA TAG GCT GCA GTG GTA GC MYH9_Ex16_R CAG TAG CAA ACA GGG CTA GG 568 58 exon 17 MYH9_Ex17_F AAC GGT CCA ATG TGA GTT TC MYH9_Ex17_R ACC AGC TCA CAA CAG TAC CC 559 58 exon 18 MYH9_Ex18_F TGT AGG CTG TGA TTG CAG AG MYH9_Ex18_R CCC TCT CTC AAA CAA ATT GC 586 58 exon 19 MYH9_Ex19_F CCT TGG TGA AGG AAA GTG G MYH9_Ex19_R GCT GTG TGC AGG TTC TGA C 657 58 exon 20 MYH9_Ex20_F AGC AGC ATA GCA CTG AAT CC MYH9_Ex20_R TTC CCA TCT CTC ATG TTC TTG 579 58
Canine MYH9 gene 73
Table 1 (continued)
Exon Primer Sequence (5rsquo ndash 3rsquo) Product
size (bp)
Annealing temperature
(degC) exons 21 MYH9_Ex21_F CTC CTG AGT GTT GGG TTA GC MYH9_Ex21_R CTC TAA CAG GCA GCC CAA G 555 58 exon 22 MYH9_Ex22_F GGA GGA AAC GCA GAA ATC C MYH9_Ex22_R ACG GAC ACC CAG GAA AAC 660 58 exon 23 MYH9_Ex23_F CAG GGG TGC TAG TGA TCT TTC MYH9_Ex23_R AGA GCT GGG CTC AGA CCT AC 630 58 exon 24 MYH9_Ex24_F TCA GGG ATG TGC TTT CTT TC MYH9_Ex24_R CCC CAA GCA CAT CTG TAG TC 563 58 exon 24 MYH9_Ex24_F TCA GGG ATG TGC TTT CTT TC MYH9_Ex24_R CCC CAA GCA CAT CTG TAG TC 563 58 exon 25 -26 MYH9_Ex2526F GGC TGC TCT TCT CTG AAG C MYH9_Ex2526R GGA GTC AGG CTT CCA GTA AAG 851 58 exon 27 -28 MYH9_Ex2728F AGC ACA TTC TGG ATC TGG AG MYH9_Ex2728R GCG ACA GCT GAA TTC CTG 752 58 exon 29 MYH9_Ex29_F CAG CTT CAG GAC ACA CAG G MYH9_Ex29_R CTC TGG GTC TCC CCT GTC 601 58 exon 30 MYH9_Ex30_F GAG GCT TGG TGT ATG TCT CG MYH9_Ex30_R TGG TTC CAT AGA AAC GAT CTG 592 58 exon 31 - 32 MYH9_Ex3132F CAT TCC CCA AGG AGT TTT G MYH9_Ex3132R AAC ACA GCC CAG GGA AAG 770 58 exon 33 MYH9_Ex33_F CCT GGA ACA AGG CTG AGA AC MYH9_Ex33_R TCT CAG AGC AGC TTT GCT TC 600 58 exon 34 MYH9_Ex34_F GGT GTG CTT GGT AAA TCC TG MYH9_Ex34_R CAC AAA GCC TCA TTT CAC G 611 58 exon 34 - 36 MYH9_Ex3536F AGG TCA CGG TCT GGG TTC T MYH9_Ex3536R CAA GGG AGG CTG TTG CAG 401 60 exon 37 MYH9_Ex37_F CTC CCT TGG TTT CCC TCT CT MYH9_Ex37_R CAG GTC AGG AGG GGA CAT 488 60 exon 38 -39 MYH9_Ex3839F AGG CCT AAG GAC AGC TCA TC MYH9_Ex3839R CCA GGG CAC AGA TAG AAC C 545 58 exon 40 MYH9_Ex40_F1 GTC CCT CTA AAG ATG GCT TTG MYH9_Ex40_R1 TGG GGA GGA ACG ACT CTC 844 58 MYH9_Ex40_F2 TGG GTG ATT GGG GAA GAG MYH9_Ex40_R2 CCA GAA GAT GCA CAA AGT AGG 981 58
Canine MYH9 gene
74
Table 2 Exonintron boundaries of the canine MYH9 gene
3rsquo-Splice site Exon 5rsquo-Splice site
Intron phase
Intron size
-20 (exon 1 162 bp) GTTCAGgtgagcgagccccgc
gt30000 bp
-19 +333 gctgtagGTCCTGGCTGTGA (exon 2 352 bp) CGTAAgtggggcctggggcc
0
4922 bp
+334 +490 tctttagACCTATTCAGGCC (exon 3 157 bp) GCAAGgtgatgcctcggcctt
1
13493 bp
+491 +518 gtttcagACCGAGAGGACCA (exon 4 28 bp) TGCACgtaagtgtttctgcc
2
803 bp
+519 +612 tcggcagGGGTGAGTCTGGA (exon 5 94 bp) ACCAGgtgagtgcgggcctt
0
4077 bp
+613 +705 cttgtagGGTGAGTTGGAGA (exon 6 93 bp) GATTTgtgagtagcggccag
0
427 bp
+706 +769 tttttagGGCAAATTCATTC (exon 7 64 bp) GACTTgtatccttggttgat
1
738 bp
+770 +868 tgcaaagATCTTCTGGAGAA (exon 8 99 bp) GAAGAgtgagtagtgagccc
1
343 bp
+869 +1012 ctttcagCTGATCTCCTGTT (exon 9 144 bp) AATGGgtacagagctggccc
1
749 bp
+1013 +1108 cccgaagGCTTGTTGCGAGT (exon 10 96 bp) CACAGgtaacgcctgtggtcc
1
1041 bp
+1109 +1227 atcgcagCTGCCCAGAAAGT (exon 11 119 bp) AACAGgtaggctctctggcc
0
1877 bp
+1228 +1380 accccagGCTGACTTTGCCA (exon 12 153 bp) TTGATgtgagctcctctcct
0
922 bp
+1381 +1554 cctgcagCTGAACTCCTTCG (exon 13 174 bp) AGCCAgtaagagccagacct
0
1801 bp
+1555 +1728 ttcccagGCAGGGCCCCCTG (exon 14 174 bp) GCAAGgtgaggcaagcacgt
0
2049 bp
+1729 +1843 cttgcagGTGGACTACAAAG (exon 15 115 bp) GGATGgtacgtgccccctgg
1
1877 bp
+1844 +2037 tttgcagTGGACCGCATCAT (exon 16 194 bp) AGAAGgtgcgggtgctgggc
0
343 bp
+2038 +2159 tctccagGCTGGCAAACTGG (exon 17 122 bp) CAGAGgtgagcggggcctgt
1
835 bp
+2160 +2229 ccctcagATATGAGATCTTG (exon 18 70 bp) TCATGgtgagtagagctgct
2
1418 bp
+2230 +2390 cgtctagATCAAAGCCCTGG (exon 19 161 bp) AGGAAgtgagttcctaaagg
1
851 bp
+2391 +2499 tccccagGGCCTTTGCCAAG (exon 20 109 bp) CCAAGgtgggttctggaagg
2
1428 bp
+2500 +2631 cttctagGTCAAGCCACTGC (exon 21 132 bp) CCCAGgtaggtgtcagcagg
2
398 bp
+2632 +2838 ttggcagCTCATGGCAGAGA (exon 22 207 bp) TCCAAgtgagccccttccct
2
488 bp
Canine MYH9 gene 75
Table 2 (continued)
3rsquo-Splice site Exon 5rsquo-Splice site
Intron phase
Intron size
+2839 +2976 atggtagGAACTGGAGGAGC (exon 23 138 bp) CCAAGgtgaggcccgtggtg
2
971 bp
+2977 +3100 cccccagGAGAAGAAGCTTC (exon 24 124 bp) GGAAGgtaagagggtaacag
0
1603 bp
+3101 +3272 tcctcagAGCGCCTCCGAAG (exon 25 172 bp) GCCAGgtgagggcctcgctt
1
719 bp
+3273 +3485 ggcagagTGGAGGAGGAAGC (exon 26 213 bp) CTCAGgtgtgccatgcggcc
1
270 bp
+3486 +3630 tctccagGTCCAAACGCGAA (exon 27 145 bp) AACGGgtgcgtgggctcccc
2
480 bp
+3631 +3837 ccaccagGTGAAAGCCAACT (exon 28 207 bp) TGCAGgtgaggcctccatgg
2
232 bp
+3838 +3941 gccacagGTTGAGCTGGACA (exon 29 104 bp) ACACAgtaaaggtggcagcg
1
204 bp
+3942 +4094 ggtgtagGAGCTGCTCCAGG (exon 30 153 bp) CCCAGgttcttatcggcatg
1
1083 bp
+4095 +4343 tgcccagGTGACGGACATGA (exon 31 249 bp) ACCAGgtgtgggccccaggc
1
1298 bp
+4344 +4556 tccccagCTACTGGCCGAGG (exon 32 213 bp) AGAGCgtgagtaccatgtca
1
150 bp
+4557 +4769 gtggcagGTCCACGAGCTGG (exon 33 213 bp) GACAGgtgtggaggccctcc
1
303 bp
+4770 +4931 gccccagGTGCGGGAGATGG (exon 34 162 bp) TGCAGgtgaggtcggctggg
1
1173 bp
+4932 +5060 accccagGCCCAGATGAAGG (exon 35 129 bp) AGGAGgtgggccgggcctca
1
941 bp
+5061 +5149 atggcagGAACTGGCAGCTG (exon 36 89 bp) AAGGGgtgagccgtggcgtg
0
92 bp
+5150 +5273 cacccagAGCTCTGGCATTG (exon 37 124 bp) TGCAGgtgggcggggtcggg
2
224 bp
+5274 +5482 cccacagATCGACCAGATCA (exon 38 209 bp) ACCAAgtaggtgttcctgcc
1
563 bp
+5483 +5591 ctcgcagGGAGCGCCAGGCA (exon 39 109 bp) ACCAGgtgtgtgtgcacacc
2
86 bp
+5592 +5763 tctgcagGCCGACAAGGCAT (exon 40 172 bp) CTCAGgtgagccctgcctcc
0
739 bp
+5764 +7295 tgcaaagATCTTCTGGAGAA (exon 41 1532 bp) CGTCACAATAAAGAGAACCA
Exon sequences are shown in uppercase letters and intron sequences in lowercase
letters Untranslated regions are shown in italics The conserved GTAG exonintron
junctions are shown in boldface type For the last exon the polyadenylation signal is
shown underlined instead of an exonintron junction Position +1 corresponds to the
adenine of the translation initiation codon ATG
Canine MYH9 gene
76
16
TT
TC
G
A
TC
A
A C
A
CT
T
C
GC
G
G
AG
C
C
AG
G
A
CT
C
T
GA
A
G
CC
C
T
CC
T
C
96
96
229
23
7
15
TT
TC
G
A
TC
A
A C
A
CT
T
C
GC
G
G
AG
C
C
AG
G
A
CT
C
T
GA
A
G
CC
C
T
CC
T
C
96
96
229
22
9
14
deaf
T
T T
C
GA
T
C
AA
CA
C
T
TC
G
C
GG
A
G
CC
A
G
GA
C
T
CT
G
A
AG
C
C
CT
C
C
TC
96
96
22
9
229
13
deaf
T
T T
C
GA
T
C
AA
CA
C
T
TC
G
C
GG
A
G
CC
A
G
GA
C
T
CT
G
A
AG
C
C
CT
C
C
TC
96
96
22
9
229
12
deaf
T
T C
C
AA
CC
A
A A
A T
T C
C
CC
G
G
GG
C
C
GG
A
A C
C
CC
A
A G
G
CC
T
T C
C
CC
96
10
0 24
1
229
11
deaf
T
T C
C
AA
CC
A
A A
A T
T C
C
CC
G
G
GG
C
C
GG
A
A C
C
CC
A
A G
G
CC
T
T C
C
CC
96
10
0 24
1
229
10
TT
TC
G
A
TC
A
A C
A
CT
T
C
GC
G
G
AG
C
C
AG
G
A
CT
C
T
GA
A
G
CC
C
T
CC
T
C
96
96
237
22
9
Fam
ily 3
9 T
T C
C
AA
CC
A
A A
A T
T C
C
CC
G
G
GG
C
C
- - CC
C
C
AA
GG
C
C
TT
CC
C
C
96
100
229
24
1
8 T
T C
T
AG
C
T
AA
AA
TT
CC
C
C
GG
G
G
CT
G
G
AA
CC
C
C
AA
GG
C
C
TT
CC
C
C
96
96
225
25
3
7 de
af
TC
C
C
AG
C
T
AT
A
A T
T C
C
CC
G
A
GG
C
T
GG
A
G
CC
C
C
AA
GG
C
T
TC
C
T
CC
96
89
22
1
237
6 de
af
TC
C
C
AG
C
T
AT
A
A T
T C
C
CC
G
A
GG
C
T
GG
A
G
CC
C
C
AA
GG
C
T
TC
C
T
CC
96
89
22
1
237 Fa
mily
2
5 T
C
CC
A
G
CT
A
T
AA
TT
CC
C
C
GA
G
G
CT
G
G
AG
C
C
CC
A
A G
G
CT
T
C
CT
C
C
96
89
221
23
7
4 de
af
TT
CC
A
A C
C
AA
AA
TT
CC
C
C
GG
G
G
CC
G
G
AG
C
C
CC
A
A G
G
CC
T
T C
C
CC
96
96
23
7
257
3 de
af
TC
C
C
AG
C
T
AT
A
A T
T C
C
CC
G
A
GG
C
T
GG
G
A
CC
C
C
AA
GG
C
T
TC
C
T
CC
96
96
23
7
257
2 T
C
CC
A
G
CT
A
T
AA
TT
CC
C
C
GA
G
G
CT
G
G
AA
CC
C
C
AA
GG
C
T
TC
C
T
CC
96
96
23
7
257 Fa
mily
1
1 C
T
CC
G
A
TC
T
A A
A T
T C
C
CC
A
G
GG
T
C
GG
A
G
CC
C
C
AA
GG
T
C
CT
T
C
CC
96
96
25
7
257
Ref
eren
ce
se
quen
ce
NW
_139
870
T C
A T A A T C
C
G
G
C
G
A C
C
A G
C
T C
C
posi
tion1
212
9
1 4
4 2
39
125
1
32
316
3
78
42
218
1
8 1
75
24
13
8
207
2
63
287
9
22
40
59
64
Pos
ition
with
in M
YH
9 In
tron
34
Exo
n 4
Intro
n 14
15
Intro
n 15
16
Intro
n 16
17
Intro
n 18
19
Intro
n 20
21
Intro
n 20
21
Intro
n 25
26
Intro
n 25
26
Intro
n 26
27
Intro
n 27
28
Exo
n 29
In
tron
293
0 In
tron
303
1 E
xon
32
Intro
n 32
33
Intro
n 32
33
Intro
n 34
35
Intro
n 38
39
Intro
n 38
39
Intro
n 38
39
Mic
rosa
tellit
e M
YH
9_M
S2
Mic
rosa
tellit
e M
YH
9_M
S3
(=FH
2293
)
1 N
umbe
ring
refe
rs to
the
posi
tion
of th
e po
lym
orph
ic n
ucle
otid
e w
ithin
the
give
n ex
on
or in
tron
Tabl
e 3
Hap
loty
pes
of th
e ca
nine
MY
H9
gene
in16
Dal
mat
ian
dogs
Canine MYH9 gene 77
Figure 1 Family structure of the analyzed Dalmatian dog families Hearing status was tested by brain auditory evoked response (BAER)
Canine MYH9 gene
78
Figure 2 Alignment of the canine MYH9 protein with orthologous human and murine
MYH9 protein sequences The sequences where derived from GenBank entries with
the accessions NP_002464 (human MYH9) and NP_071855 (mouse MYH9)
Identical residues are indicated by asterisk beneath the alignment while dots and
colons represent similar or very similar amino acids respectively
dog MAQQAADKYLYVDKNFINNPLAQADWAAKKLVWVPSDKSGFEPASLKEEVGEEAIVELVE 60 human MAQQAADKYLYVDKNFINNPLAQADWAAKKLVWVPSDKSGFEPASLKEEVGEEAIVELVE 60 mouse MAQQAADKYLYVDKNFINNPLAQADWAAKKLVWVPSSKNGFEPASLKEEVGEEAIVELVE 60 dog NGKKVKVNKDDIQKMNPPKFSKVEDMAELTCLNEASVLHNLKERYYSGLIYTYSGLFCVV 120 human NGKKVKVNKDDIQKMNPPKFSKVEDMAELTCLNEASVLHNLKERYYSGLIYTYSGLFCVV 120 mouse NGKKVKVNKDDIQKMNPPKFSKVEDMAELTCLNEASVLHNLKERYYSGLIYTYSGLFCVV 120 dog INPYKNLPIYSEEIVEMYKGKKRHEMPPHIYAITDTAYRSMMQDREDQSILCTGESGAGK 180 human INPYKNLPIYSEEIVEMYKGKKRHEMPPHIYAITDTAYRSMMQDREDQSILCTGESGAGK 180 mouse INPYKNLPIYSEEIVEMYKGKKRHEMPPHIYAITDTAYRSMMQDREDQSILCTGESGAGK 180 dog TENTKKVIQYLAHVASSHKSKKDQGELERQLLQANPILEAFGNAKTVKNDNSSRFGKFIR 240 human TENTKKVIQYLAYVASSHKSKKDQGELERQLLQANPILEAFGNAKTVKNDNSSRFGKFIR 240 mouse TENTKKVIQYLAHVASSHKSKKDQGELERQLLQANPILEAFGNAKTVKNDNSSRFGKFIR 240 dog INFDVNGYIVGANIETYLLEKSRAIRQAKEERTFHIFYYLLSGAGEHLKTDLLLEPYNKY 300 human INFDVNGYIVGANIETYLLEKSRAIRQAKEERTFHIFYYLLSGAGEHLKTDLLLEPYNKY 300 mouse INFDVNGYIVGANIETYLLEKSRAIRQAKEERTFHIFYYLLSGAGEHLKTDLLLEPYNKY 300 dog RFLSNGHVTIPGQQDKDMFQETMEAMRIMGIPEEEQMGLLRVISGVLQLGNIVFKKERNT 360 human RFLSNGHVTIPGQQDKDMFQETMEAMRIMGIPEEEQMGLLRVISGVLQLGNIVFKKERNT 360 mouse RFLSNGHVTIPGQQDKDMFQETMEAMRIMGIPEDEQMGLLRVISGVLQLGNIAFKKERNT 360 dog DQASMPDNTGNASAQKVSHLLGINVTDFTRGILTPRIKVGRDYVQKAQTKEQADFAIEAL 420 human DQASMPDNT---AAQKVSHLLGINVTDFTRGILTPRIKVGRDYVQKAQTKEQADFAIEAL 417 mouse DQASMPDNT---AAQKVSHLLGINVTDFTRGILTPRIKVGRDYVQKAQTKEQADFAIEAL 417 dog AKATYERMFRWLVLRINKALDKTKRQGASFIGILDIAGFEIFDLNSFEQLCINYTNEKLQ 480 human AKATYERMFRWLVLRINKALDKTKRQGASFIGILDIAGFEIFDLNSFEQLCINYTNEKLQ 477 mouse AKATYERMFRWLVLRINKALDKTKRQGASFIGILDIAGFEIFDLNSFEQLCINYTNEKLQ 477 dog QLFNHTMFILEQEEYQREGIEWNFIDFGLDLQPCIDLIEKPAGPPGILALLDEECWFPKA 540 human QLFNHTMFILEQEEYQREGIEWNFIDFGLDLQPCIDLIEKPAGPPGILALLDEECWFPKA 537 mouse QLFNHTMFILEQEEYQREGIEWNFIDFGLDLQPCIDLIEKPAGPPGILALLDEECWFPKA 537 dog TDKSFVEKVVQEQGTHPKFQKPKQLKDKADFCIIHYAGKVDYKADEWLMKNMDPLNDNIA 600 human TDKSFVEKVMQEQGTHPKFQKPKQLKDKADFCIIHYAGKVDYKADEWLMKNMDPLNDNIA 597 mouse TDKSFVEKVVQEQGTHPKFQKPKQLKDKADFCIIHYAGKVDYKADEWLMKNMDPLNDNIA 597
Canine MYH9 gene 79
dog TLLHQSSDKFVSELWKDVDRIIGLDQVAGMSETALPGAFKTRKGMFRTVGQLYKEQLAKL 660 human TLLHQSSDKFVSELWKDVDRIIGLDQVAGMSETALPGAFKTRKGMFRTVGQLYKEQLAKL 657 mouse TLLHQSSDKFVSELWKDVDRIIGLDQVAGMSETALPGAFKTRKGMFRTVGQLYKEQLAKL 657 dog MATLRNTNPNFVRCIIPNHEKKAGKLDPHLVLDQLRCNGVLEGIRICRQGFPNRVVFQEF 720 human MATLRNTNPNFVRCIIPNHEKKAGKLDPHLVLDQLRCNGVLEGIRICRQGFPNRVVFQEF 717 mouse MATLRNTNPNFVRCIIPNHEKKAGKLDPHLVLDQLRCNGVLEGIRICRQGFPNRVVFQEF 717 dog RQRYEILTPNSIPKGFMDGKQACVLMIKALELDSNLYRIGQSKVFFRAGVLAHLEEERDL 780 human RQRYEILTPNSIPKGFMDGKQACVLMIKALELDSNLYRIGQSKVFFRAGVLAHLEEERDL 777 mouse RQRYEILTPNSIPKGFMDGKQACVLMIKALELDSNLYRIGQSKVFFRAGVLAHLEEERDL 777 dog KITDVIIGFQACCRGYLARKAFAKRQQQLTAMKVLQRNCAAYLKLRNWQWWRLFTKVKPL 840 human KITDVIIGFQACCRGYLARKAFAKRQQQLTAMKVLQRNCAAYLKLRNWQWWRLFTKVKPL 837 mouse KITDVIIGFQACCRGYLARKAFAKRQQQLTAMKVLQRNCAAYLRLRNWQWWRLFTKVKPL 837 dog LQVSRQEEEMMAKEEELVKVREKQLAAENRLTEMETLQSQLMAEKLQLQEQLQAETELCA 900 human LQVSRQEEEMMAKEEELVKVREKQLAAENRLTEMETLQSQLMAEKLQLQEQLQAETELCA 897 mouse LNSIRHEDELLAKEAELTKVREKHLAAENRLTEMETMQSQLMAEKLQLQEQLQAETELCA 897 dog EAEELRARLTAKKQELEEICHDLEARVEEEEERCQHLQAEKKKMQQNIQELEEQLEEEES 960 human EAEELRARLTAKKQELEEICHDLEARVEEEEERCQHLQAEKKKMQQNIQELEEQLEEEES 957 mouse EAEELRARLTAKKQELEEICHDLEARVEEEEERCQYLQAEKKKMQQNIQELEEQLEEEES 957 dog ARQKLQLEKVTTEAKLKKLEEDQIIMEDQNCKLAKEKKLLEDRIAEFTTNLMEEEEKSKS 1020 human ARQKLQLEKVTTEAKLKKLEEEQIILEDQNCKLAKEKKLLEDRIAEFTTNLTEEEEKSKS 1017 mouse ARQKLQLEKVTTEAKLKKLEEDQIIMEDQNCKLAKEKKLLEDRVAEFTTNLMEEEEKSKS 1017 dog LAKLKNKHEAMITDLEERLRREEKQRQELEKTRRKLEGDSTDLNDQIAELQAQIAELKMQ 1080 human LAKLKNKHEAMITDLEERLRREEKQRQELEKTRRKLEGDSTDLSDQIAELQAQIAELKMQ 1077 mouse LAKLKNKHEAMITDLEERLRREEKQRQELEKTRRKLEGDSTDLSDQIAELQAQIAELKMQ 1077 dog LAKKEEELQAALARVEEEATQKNMALKKIRELESQISELQEDLESERASRNKAEKQKRDL 1140 human LAKKEEELQAALARVEEEAAQKNMALKKIRELESQISELQEDLESERASRNKAEKQKRDL 1137 mouse LAKKEEELQAALARVEEEAAQKNMALKKIRELETQISELQEDLESERASRNKAEKQKRDL 1137 dog GEELEALKTELEDTLDSTAAQQELRSKREQEVNILKKTLEEEARTHEAQIQEMRQKHSQA 1200 human GEELEALKTELEDTLDSTAAQQELRSKREQEVNILKKTLEEEAKTHEAQIQEMRQKHSQA 1197 mouse GEELEALKTELEDTLDSTAAQQELRSKREQEVSILKKTLEDEAKTHEAQIQEMRQKHSQA 1197 dog VEELAEQLEQTKRVKANLEKAKQTLENERGELANEVKVLQQGKGDSEHKRKKAEAQLQEL 1260 human VEELAEQLEQTKRVKANLEKAKQTLENERGELANEVKVLLQGKGDSEHKRKKVEAQLQEL 1257 mouse VEELADQLEQTKRVKATLEKAKQTLENERGELANEVKALLQGKGDSEHKRKKVEAQLQEL 1257 dog QVKFTEGERVRTELADKVTKLQVELDNVMGLLTQSDSKSSKLTKDFSALESQLQDTQELL 1320 human QVKFNEGERVRTELADKVTKLQVELDNVTGLLSQSDSKSSKLTKDFSALESQLQDTQELL 1317 mouse QVKFSEGERVRTELADKVTKLQVELDSVTGLLSQSDSKSSKLTKDFSALESQLQDTQELL 1317
Canine MYH9 gene
80
dog QEENRQKLSLSTKLKQMEDEKNSFKEQLEEEEEAKRNLEKQIATLHAQVTDMKKKMEDGV 1380 human QEENRQKLSLSTKLKQVEDEKNSFREQLEEEEEAKHNLEKQIATLHAQVADMKKKMEDSV 1377 mouse QEENRQKLSLSTKLKQMEDEKNSFREQLEEEEEAKRNLEKQIATLHAQVTDMKKKMEDGV 1377 dog GCLETAEEAKRKLQKDLEGLGQRYEEKVAAYDKLEKTKTRLQQELDDLLVDLDHQRQTAS 1440 human GCLETAEEVKRKLQKDLEGLSQRHEEKVAAYDKLEKTKTRLQQELDDLLVDLDHQRQSAC 1437 mouse GCLETAEEAKRRLQKDLEGLSQRLEEKVAAYDKLEKTKTRLQQELDDLLVDLDHQRQSVS 1437 dog NLEKKQKKFDQLLAEEKTISAKYAEERDRAEAEAREKETKALSLARALEEAMEQKAELER 1500 human NLEKKQKKFDQLLAEEKTISAKYAEERDRAEAEAREKETKALSLARALEEAMEQKAELER 1497 mouse NLEKKQKKFDQLLAEEKTISAKYAEERDRAEAEAREKETKALSLARALEEAMEQKAELER 1497 dog LNKQFRTEMEDLMSSKDDVGKSVHELEKSKRALEQQVEEMKTQLEELEDELQATEDAKLR 1560 human LNKQFRTEMEDLMSSKDDVGKSVHELEKSKRALEQQVEEMKTQLEELEDELQATEDAKLR 1557 mouse LNKQFRTEMEDLMSSKDDVGKSVHELEKSKRALEQQVEEMKTQLEELEDELQATEDAKLR 1557 dog LEVNLQAMKAQFERDLQGRDEQSEEKKKQLVRQVREMEAELEDEKKQRSMAVAARKKLEM 1620 human LEVNLQAMKAQFERDLQGRDEQSEEKKKQLVRQVREMEAELEDERKQRSMAVAARKKLEM 1617 mouse LEVNLQAMKAQFERDLQGRDEQSEEKKKQLVRQVREMEAELEDERKQRSMAMAARKKLEM 1617 dog DLKDLEAHIDSANKNRDEAIKQLRKLQAQMKDCVRELDDTRASREEILAQAKENEKKMKS 1680 human DLKDLEAHIDSANKNRDEAIKQLRKLQAQMKDCMRELDDTRASREEILAQAKENEKKLKS 1677 mouse DLKDLEAHIDTANKNREEAIKQLRKLQAQMKDCMRELDDTRASREEILAQAKENEKKLKS 1677 dog MEAEMIQLQEELAAAERAKRQAQQERDELADEIANSSGKGALALEEKRRLEARIAQLEEE 1740 human MEAEMIQLQEELAAAERAKRQAQQERDELADEIANSSGKGALALEEKRRLEARIAQLEEE 1737 mouse MEAEMIQLQEELAAAERAKRQAQQERDELADEIANSSGKGALALEEKRRLEARIALLEEE 1737 dog LEEEQGNTELVNDRLKKANLQIDQINTDLNLERSHAQKNENARQQLERQNKELKVKLQEM 1800 human LEEEQGNTELINDRLKKANLQIDQINTDLNLERSHAQKNENARQQLERQNKELKVKLQEM 1797 mouse LEEEQGNTELINDRLKKANLQIDQINTDLNLERSHAQKNENARQQLERQNKELKAKLQEM 1797 dog EGTVKSKYKASITALEAKIAQLEEQLDNETKERQAACKQVRRAEKKLKDVLLQVDDERRN 1860 human EGTVKSKYKASITALEAKIAQLEEQLDNETKERQAACKQVRRTEKKLKDVLLQVDDERRN 1857 mouse ESAVKSKYKASIAALEAKIAQLEEQLDNETKERQAASKQVRRTEKKLKDVLLQVEDERRN 1857 dog AEQFKDQADKASTRLKQLKRQLEEAEEEAQRANASRRKLQRELEDATETADAMNREVSSL 1920 human AEQYKDQADKASTRLKQLKRQLEEAEEEAQRANASRRKLQRELEDATETADAMNREVSSL 1917 mouse AEQFKDQADKASTRLKQLKRQLEEAEEEAQRANASRRKLQRELEDATETADAMNREVSSL 1917 dog KNKLRRGDLPFVVPRRVARKGAGDCSDEEVDGKADGAEAKAAE 1963 human KNKLRRGDLPFVVPRRMARKGAGDGSDEEVDGKADGAEAKPAE 1960 mouse KNKLRRGDLPFVVTRRIVRKGTGDCSDEEVDGKADGADAKAAE 1960
Chapter 6
Identification of a 5 Mb region on canine chromosome 10
harbouring a causative gene responsible for congenital sensorineural deafness
in German Dalmatian dogs
Fine mapping of CFA10 83
Identification of a 5 Mb region on canine chromosome 10 harbouring a causative gene responsible for congenital sensorineural deafness in German Dalmatian dogs
Abstract
In the present study we evaluated whether the canine chromosome (CFA) 10
harbours a gene responsible for canine congenital sensorineural deafness (CCSD) in
Dalmatian dogs Altogether 27 microsatellite markers covering the whole CFA10
were genotyped in 176 Dalmatian dogs with frequent occurrence of CCSD
Significant linkage between the deafness phenotype and microsatellites located in a
region of 36 Mb to 48 Mb on CFA10 was found In order to refine the location of the
causative canine congenital sensorineural deafness (CCSD) gene we used data
deposited in the NCBI to search for single nucleotide polymorphisms (SNPs) in the
intronic sequences of the canine genes located on CFA10 in this region We
characterized 26 SNPs and used them for non-parametric linkage and association
analyses with CCSD of altogether 34 Dalmatian dogs from five full-sib families We
could find significant linkage to SNPs located in the region spanning 39 Mb to 44 Mb
and significant haplotype-trait association for SNPs in this region These results
enforce further evaluation of this 5 Mb region with the aim to detect the gene
responsible for CCSD in Dalmatian dogs
Introduction
Of the identified genes responsible for different forms of sensorineural non-syndromic
deafness in humans Rak et al (2003) and co-workers (Droumlgemuumlller et al 2002
Kuiper et al 2002 Rak et al 2002a 2002b 2003) considered 24 genes as
candidates for sensorineural deafness in dogs among them the MYH9 gene on
CFA10 Subsequently Rak (2003) developed a set of microsatellite markers for the
respective 24 candidate genes
As described in chapter 3 we could show significant linkage of CCSD with the MYH9
associated microsaellite MYH9_MS3 (=FH2293) in different German Dalmatian dog
Fine mapping of CFA10
84
families However as described in Chapter 5 we already excluded MYH9 for being
responsible for the CCSD phenotype in German Dalmatian dog families segregating
for CCSD by comparative sequencing of genomic sequences from deaf and normal
hearing Dalmatian dogs and additionally by sequencing of full length canine cDNA
The reason for the MYH9_MS3 (=FH2293) indicating linkage may be caused by a
closely linked gene involved in CCSD Thus the objective of the present study was to
perform a scan of canine chromosome 10 using microsatellite markers and single
nucleotide polymorphisms (SNPs) in order to evaluate whether CFA10 harbours a
gene responsible for the deafness phenotype in Dalmatian dogs Additionally we
analyzed the association of the CCSD phenotype with a large number of newly
developed SNPs located in the genomic deafness region on CFA10
Material and methods Pedigree material
For the linkage analysis of the microsatellite marker on CFA10 we used DNA from
altogether 176 animals belonging to 22 full-sib families and one large paternal half-
sib family of German Dalmatian dogs All families were segregating for CCSD The
genotyped families included all the affected dogs (unilaterally and bilaterally deaf)
their parents if available and one to four unaffected full-sibs At least two of the full
sibs of each family were unilaterally deaf The phenotype of the affected animals had
been confirmed by brainstem auditory evoked response (BAER) that detects
electrical activity in the cochlea and auditory pathways in the brain
Screening for SNPs was performed by comparative sequencing of DNA from parents
of five families with significant linkage of microsatellites located in the region
spanning 36 Mb to 48 Mb to CCSD For the linkage analysis concerning the SNPs we
then used blood samples from 34 Dalmatian dogs consisting of the progeny and their
parents of the abovementioned five full-sib families of Dalmatian dogs with frequent
occurrence of CCSD The families consisted of five to nine individuals and their
parents
Microsatellite marker analysis
We used 27 microsatellite marker derived from the NCBI database
(httpwebncbinlmnihgov) to cover CFA10 completely (Appendix Table 1)
Fine mapping of CFA10 85
PCR reactions for microsatellites were carried out in 12 microl reaction mixtures
containing 2 microl genomic DNA 12 microl 10x PCR buffer (Qbiogene Heidelberg
Germany) 024 microl dimethyl sulfoxide (DMSO) 02 microl dNTPs (100 microM) 01 microl Taq
Polymerase (5Umicrol) (Qbiogene) 06 microl (10 microM) 5rsquo-IRD700 or IRD800 labelled forward
primer 06 microl (10 microM) unlabelled reverse primer The PCR reactions were carried out
in MJ Research thermocyclers with the following program 4 min at 94 degC followed
by 32 cycles of 30 sec at 94degC 30 sec at maximum annealing temperature and a
final extension of 45 sec at 72degC PCR-products were diluted with formamide loading
buffer in ratios from 110 to 140 and then size-fractionated by gel electrophoresis on
automated LI-COR 42004300 sequencers (LI-COR inc Lincoln NE USA) using
denaturing 4 and 6 polyacrylamide gels (RotiphoreseregGel 40 Roth Karlsruhe
Germany)
Development of single nucleotide polymorphisms (SNPs)
We developed SNPs for the region between 36 Mb to 48 Mb on CFA10 previously
proven to be linked to the CCSD phenotype The SNPs for this region were derived
from one 5294 Mb large whole genome shotgun sequence (NW_876251) deposited
in the current dog genome assembly (Boxer genome assembly 21) of the NCBI
GenBank The canine genomic sequences and mRNA of the genes that were used
for the analysis were also derived from sequences deposited in the current dog
genome assembly (Boxer genome assembly 21) of the NCBI GenBank (Table 1)
In total 72 primer pairs were designed most of them located intragenic in intronic
sequences or alternatively in flanking regions of the 5rsquo- or 3rsquondashends of the respective
gene yielding products with a length of around 600 bp PCR primers were developed
with the Primer3 program (httpfrodowimiteducgi-binprimer3primer3_wwwcgi)
The PCR reactions were performed in a total of 50 microl containing 125 microM dNTPs 25
pmol of each primer the reaction buffer supplied by the manufacturer (Qiagen
Hilden Germany) and 1 U Taq polymerase After a 4 min initial denaturation at
95degC 35 cycles of 30 sec at 94degC 45 sec at 58degC and 80 sec at 72degC were
performed in a MJ Research thermocycler (Biozym Hessisch Oldendorf Germany)
The obtained PCR products were directly sequenced with the DYEnamic ET
Terminator kit (AmershamBiosciences Freiburg Germany) and a MegaBACE 1000
capillary sequencer (AmershamBiosciences) using the PCR primers as sequencing
primers Sequence data were analyzed with Sequencher 42
Fine mapping of CFA10
86
(GeneCodes Ann Arbor MI USA) If a heterozygous SNP was found for one or both
parents all progeny of the respective families were analyzed for that SNP
We developed altogether 26 SNPs with 20 SNPs located intragenic in intronic
sequences and 6 SNPs located very closely at the 5rsquo- or 3rsquo-ends respectively with
one to four SNPs per gene (Table 3)
The newly identified 26 SNPs are shown in Tabele 2 Only SNPs are listed and
chosen for linkage analyses that were heterozygous for one or both parents of at
least two of the five families Of all SNPs only one was heterozygous in all families
(Table 3) The most frequent form of SNPs with a frequency of 423 was the AG
transition motif The scarcest one with a frequency of 385 was the CG and the
AC transversion motif respectively
Linkage analysis
Multipoint non-parametric linkage and haplotype analysis were performed using the
MERLIN software version 0102 (multipoint engine for rapid likelihood inference
Center for Statistical Genetics University of Michigan MI USA Abecasis et al
2002) The test statistics Z-mean and Lod score were used to test for the proportion
of alleles shared by affected individuals identical by descent (IBD) for the considered
marker loci
In a first step linkage analysis was performed regarding the 27 marker covering the
whole CFA10 In a second step a linkage analysis was performed including the newly
added SNPs spanning the region 36 Mb to 48 Mb on CFA10
The observed heterozygosity (HET) and the polymorphism information content (PIC)
were calculated using the software package SASGenetics (Statistical Analysis
System Version 913 SAS Institute Inc Cary NC USA 2005)
Linkage disequilibrium (LD) and association of haplotypes with CCSD were tested
using the procedures CASECONTROL and HAPLOTYPE of SASGenetics
(Statistical Analysis System version 913 Cary NC USA)
Results
A linkage analysis was at first carried out for the 176 animals that were analyzed with
27 microsatellite markers covering the whole CFA10
Fine mapping of CFA10 87
The microsatellite FH3302 positioned at 3887 Mb showed the highest Zmean with a
value of 234 (p= 001) and a Lod score of 121 (p= 0009) at 3887 Mb A Zmean
value of 217 (p= 0015) and a Lod score of 12 (p= 0009) was reached for the
microsatellite REN181G20 positioned at 3936 Mb indicating linkage but all Zmean
values and error probabilities of eight microsatellite markers in the interval from 3666
Mb up to 488 Mb were almost as high Consequently we screened the region
spanning from 36 Mb to 48 Mb for SNPs Out of the 23 analyzed Dalmatian dog
families five full-sib families were chosen to screen for SNPs because of their
significant contribution to the test statistics
Twenty-six new SNP markers covering the region of 36 Mb to 48 Mb equally were
developed and added to the linkage analysis These SNPs had PIC values ranging
from 013 to 037
Figure 1 shows the Zmeans and LOD scores for all 27 microsatellite markers on
CFA10 harbouring a genomic deafness region Figure 2 shows the corresponding p-
values Adding the SNPs in the interval spanning 36 Mb to 48 Mb we could narrow
the significant region down to 5 Mb spanning 39 Mb to 44 Mb (Table 4 Figures 3 and
4)
Haplotype-trait association test statistics for the SNPs 16-23 located in the interval
from 39 Mb to 44 Mb were significant However the marker-trait association test
failed the 5 threshold of the error probability (p= 007) but was lowest for all
possible haplotype-trait combinations The χ2ndashtests of the procedure
CASECONTROL were not significant indicating that a SNP for the causative
deafness gene was not yet found
Discussion
We used 27 microsatellite marker and 26 newly developed SNP markers with the
intention to create a dense map for linkage analysis of CFA10 especially the region
spanning 36 Mb to 48 Mb proven to be linked to the CCSD phenotype All SNP
markers were chosen due to their heterozygosity in one or both parents of at least
two families
The significant Zmeans on CFA10 reported for the chromosome scan using only
microsatellites was confirmed by adding the SNP markers Furthermore with the use
of SNPs the apparently linked region from 36 Mb to 48 Mb could be narrowed down
Fine mapping of CFA10
88
to 5 Mb spanning 39 Mb to 44 Mb For this reason the use of SNPs in addition to
informative microsatellites appears highly recommendable
The identified CCSD region spanning 5 Mb might be participating in the development
of CCSD in the analyzed Dalmatian dog families
However genes that are known to be involved in the hearing process or known to be
expressed in the inner ear are not annotated at the syntenic human region on homo
sapiens autosome (HSA) 2 spanning from 100 Mb to 108 Mb Therefore a well-
defined candidate gene for canine hereditary deafness in the abovementioned 5 Mb
does not exist This means that for all genes in this region informative SNPs have to
be developed and tested for linkage disequilibrium and association with CCSD
Despite the remarkable progress that has been made in the past few years in
identifying deafness genes in man and mouse it is suspected that only one-third of all
deafness causing genes have been identified so far Therefore more SNPs have to
be developed within the identified region on CFA10 to localize the deafness causing
gene or to find unambiguously associated SNP markers which could be used for a
population-wide genetic test for CCSD
Fine mapping of CFA10 89
Table 1 Canine genes where gene-associated SNPs could be developed with their
exact location on CFA10 and their corresponding accession numbers (Acc No)
Gene symbol Gene description Position in
Mb
Acc No canine genomic sequence
Acc No canine mRNA
LOC474535 similar to hypothetical protein MGC40397 36493650 NC_006592 XM_531764
LOC474536 similar to KM-102-derived reductase-like factor
37023714 NC_006592 XM_531765
LOC610953 similar to NHP2-like protein 1 37293729 NC_006592 XM_848546
LOC481302 similar to C2orf26 protein 37363737 NC_006592 XM_848552
LOC610991 hypothetical protein LOC610991 38103820 NC_006592 XM_848591
LOC611007 similar to eukaryotic translation elongation factor 1 alpha 2
38353835 NC_006592 XM_848614
LOC474541 similar to GRIP coiled-coil protein GCC185 isoform a
38503857 NC_006592 XM_531770
LOC474542 similar to Sulfotransferase 1C2 38613862 NC_006592 XM_531771
LOC481308 similar to Keratin type I cytoskeletal 18 38653865 NC_006592 XM_538429
LOC474543 similar to Sulfotransferase K1 38683870 NC_006592 XM_857994
LOC609217 similar to family with sequence similarity 32 member A like
39453945 NC_006592 XM_858065
LOC611115 similar to D-PCa-2 protein isoform c 39843984 NC_006592 XM_848756
LOC481325 similar to ubiquitin-conjugating enzyme E2C
42564272 NC_006592 XM_538446
LOC481330 similar to Interleukin-1 receptor type II precursor
44084413 NC_006592 XM_538451
LOC611493 hypothetical protein LOC611493 44384440 NC_006592 XM_863243
LOC481337 similar to DNA repair protein REV1
46264637 NC_006592 XM_538458
LOC611728 similar to 3-hydroxyanthranilate 34-dioxygenase
48494852 NC_006592 XM_849433
Predicted gene derived from the dog genome assembly (build 21) that used gene
prediction method GNOMON supported by EST evidence
Fine mapping of CFA10
90
Table 2 The 26 newly developed SNPs for Dalmatian dogs located in the region
spanning 36 Mb to 48 Mb on CFA10 with their corresponding primers the SNP motif
the product size and the annealing temperature
Gene
description SNP
Location
(intron or
5rsquo3rsquo-end)
Primer F1 (5acute -gt 3acute)
SNP motif
Primer R2 (5acute -gt 3acute)
Bp3 AT4
LOC474535
SNP_1
intron
ACCCAAGCCTAACTGCAGAA
ACCCCAGTCT(CG)GCCAGAGCTGTT
590 60
SNP_2 intron TGTGTGCATA(AG)TGTGCTGAGCCT
TCATCTGTTAAAACAGGGGTGAT
LOC474536
SNP_3
intron
CCAGTTAATGATTGTTTCGTTGA
AAGCTGCTTT(AC)CACCCCCATCAG
TCATTCCTGCTGTTGTGCTC
590 60
LOC610953
SNP_4
intron
CTGTCTTGGGGACTGTTTGC
AAGGCAGACG(CT)AATGACTGAGGC
600 60
SNP_5 intron GACTTGAAGC(CT)ACCACAGATACT
GCCATCACGATGAACTCAGA
LOC481302
SNP_6
3rsquo-end
AATTGAGGCCGAAGTCCAAT
CTTTTCCCCA(GT)GCCACCCCTCTG
GAGCACTATTTACGATACAAACAGGA
610 60
LOC610991
SNP_7
intron
CATGCATGATGCCCAGAGTA
CCCAAAGCAC(AG)CTGTGATTTAAT
AGGGCTTCCTGGGAAAAGT
600 60
LOC611007
SNP_8
intron
CAGACCAACAGTGACCCAGA
TAGGCATACC(GT)TCAGTCCTAAAG
GCCTGTTGTGGGCAGAGTAT
480 60
LOC474541
SNP_9
intron
ACTGAGCCAAAGGTGGATTG
AGAGAATAGC(AG)CTGTGTTTTACA
ACCTGCACATCGGGATTTAG
575 60
Fine mapping of CFA10 91
Table 2 (continued)
Gene-
symbol SNP
Location
(intron or
5rsquo3rsquo-end)
Primer F1 (5acute -gt 3acute)
SNP motif
Primer R2 (5acute -gt 3acute)
Bp3 AT4
LOC474542
SNP_10
intron
CTTCCCCAGGAGAGAGTGAC
AATATGATCA(CT)ATTTAAAGAAAT
CTTTTGTCAACATCCCCTTCA
560 60
LOC481308
SNP_11
3rsquo-end
ACCCATTGTCTCTCCAGCAC
CCACATAACT(GT)AGCATCCCTAGC
600 60
TGATGATGTAAGTTGGCCTCA
LOC474543
SNP_12
intron
TTGAAGTTGTGTGAGTAAATGAAAGA
CAATATATAA(AG)CATTTGCTACAA 600 60
SNP_13 intron TTGAGATGAA(AT)ATAATAGAGCTG
SNP_14 intron CGATTTTCCA(AG)GACATGGAGATG
SNP_15 intron ACTGAACCAA(AG)TGTGCAAAGCAT
GGAAACCATGCAGTCTTTGG
LOC609217
SNP_16
5rsquo-end
TGGCCTCATTTTCCAGTATG
GAAGGAGTTA(CT)ACAGTGAAGATA
580 60
SNP_17 5rsquo-end AAGTGATCAA(AG)CCAGTGCATTCT
GGCAATTACCCTGAGTGGTG
LOC611115
SNP_18
3rsquo-end
GGGCTGTCTTAGAGGTGCTG
TGTGGTCTCA(CT)ACACTTCCTGAG
590 60
SNP_19 3rsquo-end ATTGCAAGTG(GT)CCTGTGATGGCC
CTTCTTTGGGCAGGAAAGTG
LOC481325
SNP_20
intron
AAAATGATTGATCGCAAAAGAAA
AATTACTGTA(CT)AACAGTATCAGA
600 60
SNP_21 intron TAACAGTATC(AG)GAATAAAAGTTT
TTCTGTGATTGCACTGACCG
Fine mapping of CFA10
92
Table 2 (continued)
Gene-
symbol SNP
Location
(intron
or 5rsquo3rsquo-
end)
Primer F1 (5acute -gt 3acute)
SNP motif
Primer R2 (5acute -gt 3acute)
Bp3 AT4
LOC481330
SNP_22
intron
GAAAGGCCTGGGTTCAAAA
GGCAGGGAGG(AG)TCACCATCGTTC
AATTTCCCCAAATGCCTCAC
575 60
LOC611493
SNP_23
intron
GCATGAAGGAGCCCTATGTC
CCAAGAGTCC(AT)GCCCAACACCCT
GGAGGGATGGCATTCTATGA
590 60
LOC481337
SNP_24
intron
GGCTGAGGAGATTGTGTTTCA
GCTGATATTT(AG)GCCTTCTGAGAT
620 60
SNP_25 intron AAAGCCAGAC(AG)CTTTGCCATGGT
CAGCTCCCTGTAATGGGAAA
LOC611728
SNP_26
intron
TCCTACTCCCATCACTTCCAA
CCACACTGGG(GT)CCTGGGATGAGG
CACAGCTCCATGTAGGTCCA
620 60
hypothetical gene found in the 2 version 1 of the NCBIs genome annotation 1 Forward 2 Reverse 3 Product size (basepairs) 4 Annealing temperature (degC)
Fine mapping of CFA10 93
Table 3 The 26 newly developed intragenic SNPs for Dalmatian dogs with their
nucleotide polymorphism allele and genotype frequencies observed heterozygosity
(HET) and polymorphism information content (PIC)
SNP Fam1 Nucleotide
polymorphism
Allele
frequencies
Genotype
frequencies2 PIC HET
SNP_1 125 CgtG 042054 3135 036 055
SNP_2 124 AgtG 064039 91013 037 044
SNP_3 2345 AgtC 041049 2156 033 047
SNP_4 345 CgtT 065035 6140 035 067
SNP_5 34 CgtT 065035 490 028 042
SNP_6 345 CgtT 066034 6130 035 039
SNP_7 1345 AgtG 054046 6174 037 053
SNP_8 145 GgtT 075025 01010 029 045
SNP_9 1345 AgtG 052054 6145 037 047
SNP_10 1234 CgtT 057043 7173 037 068
SNP_11 14 GgtT 065035 5120 035 062
SNP_12 15 AgtG 028072 097 030 044
SNP_13 45 AgtT 077023 870 017 021
SNP_14 5 AgtG 036064 052 013 015
SNP_15 134 AgtG 034066 0157 034 059
SNP_16 145 CgtT 030070 3812 033 036
SNP_17 245 AgtG 058042 6113 030 032
SNP_18 123 CgtT 037063 1126 027 035
SNP_19 123 GgtT 045055 3115 030 032
SNP_20 2345 CgtT 032068 1129 029 039
SNP_21 1235 AgtG 063037 883 037 052
SNP_22 1234 AgtG 068032 10102 030 034
SNP_23 12345 AgtT 030070 21313 033 046
SNP_24 124 AgtG 047053 667 033 019
SNP_25 234 AgtG 053047 3132 035 053
SNP_26 234 GgtT 060040 3120 026 0391 Number of families which were informative for the respective SNPs 2 Genotypes are given as number of animals [homozygous for allele 1heterozygous
homozygous for allele 2]
Fine mapping of CFA10
94
Table 4 Linkage analysis for canine chromosome 10 regarding the region spanning
39 Mb to 44 Mb with the significant Zmean LOD score and error probabilities (p-
values)
Marker Location (Mb) Zmean pz-value1 LOD-score pL-value2
SNP_16 39453 262 0004 123 0009
SNP_17 39455 262 0004 123 0009
SNP_18 39840 261 0004 123 0009
SNP_19 39843 261 0004 123 0009
SNP_20 4260 255 0005 118 0010
SNP_21 4270 255 0005 117 0010
SNP_22 4405 317 00008 131 0007
SNP_23 4439 337 00004 134 0007 1 Error probability regarding Zmean 2 Error probability regarding LOD score
Fine mapping of CFA10 95
Figure 1 Zmeans and LOD scores for 27 microsatellite markers on CFA10
harbouring a congenital sensorineural deafness region (number of families 23
number of genotyped dogs 176)
Figure 2 P-values for Zmeans and LOD scores of 27 microsatellite markers on
CFA10 harbouring a congenital sensorineural deafness region (number of families
23 number of genotyped dogs 176)
Fine mapping of CFA10
96
Figure 3 Zmeans and LOD scores of 53 markers on CFA10 harbouring a congenital
sensorineural deafness region (number of families 5 number of genotyped dogs 34)
Figure 4 P-values for Zmeans and LOD scores of 53 markers on CFA10 for the
region between 30 to 60 Mb harbouring a congenital sensorineural deafness region
(number of families 5 number of genotyped dogs 34)
CCSD region (SNP 16-23)
Chapter 7
Analysis of canine chromosome 1 and the Gap junction protein alpha 1 (GJA1) gene
in Dalmatian dogs segregating for congenital sensorineural deafness
CFA1 and the GJA1 gene 99
Analysis of canine chromosome 1 and the Gap junction protein alpha 1 (GJA1) gene in Dalmatian dogs segregating for congenital sensorineural deafness Abstract Two microsatellite markers associated to the GJA1 (alternatively connexin 43) gene
showed significant linkage with canine congenital deafness (CCSD) in a large French
half-sib family (Chapter 3) PCR products of this half-sib family were used to perform
a sequence analysis in order to evaluate whether GJA1 is responsible for CCSD As
the linkage could not be confirmed we used altogether 27 microsatellite markers for
a scan of canine chromosome (CFA) 1 with 23 Dalmatian dog families segregating
for CCSD A non-parametric linkage analysis was performed to see whether
significant test statistics for other genomic regions on CFA1 and for more families can
be shown As a result we could not find linkage to any microsatellite in the analyzed
families
Introduction Rak (2003) considered in total 24 genes as candidates for sensorineural deafness in
dogs among them the Gap junction protein alpha 1 (GJA1) or connexin 43 gene on
canine chromosome (CFA) 1 A marker set of altogether 43 microsatellites were
developed by Rak (2003) among them two microsatellite marker associated to the
GJA1 gene
GJA1 or connexin 43 is a member of the connexin gene family and a component of
gap junctions Mutations in 4 members of the connexin gene family have been shown
to underlie distinct genetic forms of deafness including GJB2 (connexin [CX] 26)
GJB3 (CX31) GJB6 (CX30) and GJB1 (CX32)
Liu et al (2001) reported mutations in GJA1 in association with sensorineural
recessive deafness in man However these mutations have recently been shown to
involve the pseudogene of GJA1 on human chromosome (HSA) 5 and not the
CFA1 and the GJA1 gene
100
GJA1 gene on HSA 6 (WA Paznekas cited a personal communication from the
senior author (W E Nance) of the paper by Liu et al 2001)
In previously performed studies (Chapter 3) one large French Dalmatian dog family
with frequent occurrence of CCSD showed linkage to microsatellites associated to
the GJA1 gene
In this report we performed a mutation analysis of the GJA1 gene sequence to
identify polymorphisms In order to evaluate whether the GJA1 gene is responsible
for congenital sensorineural deafness in Dalmatian dogs we analyzed the
association of the GJA1 haplotypes with the CCSD phenotype Furthermore we
employed 27 microsatellite markers covering the entire CFA1 and used them for a
non-parametric linkage analysis with CCSD in a Dalmatian dog population of 176
animals with frequent occurrence of CCSD
Material and methods Pedigree material
For the linkage analysis of the microsatellite marker we used DNA from altogether
176 animals belonging to 22 full-sib families and one large paternal half-sib family of
German Dalmatian dogs All families were segregating for CCSD The genotyped
families included all affected dogs (unilaterally and bilaterally deaf) their parents if
available and one to four unaffected animals At least two of the full sibs of each
family were unilaterally or bilaterally deaf The phenotype of the affected animals had
been confirmed by brainstem auditory evoked response (BAER) that detects
electrical activity in the cochlea and auditory pathways in the brain
Mutation analysis and screening for SNPs was performed by comparative
sequencing of DNA from 16 animals consisting of the parents and their progenies of
one large half-sib family of French Dalmatian dogs which showed significant linkage
to GJA1-associated microsatellites (Chapter 3)
Sequencing of canine genomic DNA and mutation analysis
The canine GJA1 gene sequence was derived from sequences deposited in the
current dog genome assembly (Boxer genome assembly 21) of the NCBI GenBank
(Genbank Acc No NC_006583) We compared the canine genomic DNA sequence
to canine cDNA fragments in the canine EST database using the
CFA1 and the GJA1 gene 101
BLASTN (Basic Local Alignment Search Tool Nucleotide) program Three canine
ESTs (Acc No DN880776 BU744925 and DN369900) were used to confirm the
assembly of the GJA1 gene
To identify polymorphisms within the canine GJA1 sequence the gene consisting of
one 1251 bp spanning exon were PCR amplified and sequenced from 16 French
Dalmatian dogs Additionally large sequences from the 3acute-end were amplified Primer
pairs were designed yielding products with a length of around 600 bp PCR primers
were developed with the Primer3 program (httpfrodowimiteducgi-
binprimer3primer3_wwwcgi) The PCR reactions were performed in a total of 50 microl
containing 125 microM dNTPs 25 pmol of each primer the reaction buffer supplied by
the manufacturer (Qbiogene Heidelberg Germany) and 1 U Taq polymerase After
a 4 min initial denaturation at 95degC 35 cycles of 30 sec at 94degC 45 sec at 58degC and
80 sec at 72degC were performed in a MJ Research thermocycler (Biozym Hessisch
Oldendorf Germany)
The obtained PCR products were directly sequenced with the DYEnamic ET
Terminator kit (AmershamBiosciences Freiburg Germany) and a MegaBACE 1000
capillary sequencer (AmershamBiosciences) using the PCR primers as sequencing
primers Sequence data were analyzed with Sequencher 42
(GeneCodes Ann Arbor MI USA)
Microsatellite marker analysis
We used 27 microsatellite marker derived from the NCBI database
(httpwebncbinlmnihgov) to cover CFA1 completely (Appendix Table 1)
PCR reactions for microsatellites were carried out in 12 microl reaction mixtures
containing 2 microl genomic DNA 12 microl 10x PCR buffer (Qbiogene) 024 microl dimethyl
sulfoxide (DMSO) 02 microl dNTPs (100 microM) 01 microl Taq Polymerase (5Umicrol)
(Qbiogene) 06 microl (10 microM) 5rsquo-IRD700 or IRD800 labelled forward primer 06 microl (10
microM) unlabelled reverse primer The PCR reactions were carried out in MJ Research
thermocyclers with the following program 4 min at 94 degC followed by 32 cycles of 30
sec at 94degC 30 sec at maximum annealing temperature and a final extension of 45
sec at 72degC PCR-products were diluted with formamide loading buffer in ratios from
110 to 140 and then size-fractionated by gel electrophoresis on automated LI-COR
42004300 sequencers (LI-COR inc Lincoln NE USA) using denaturing 4 and 6
polyacrylamide gels (RotiphoreseregGel 40 Roth Karlsruhe Germany)
CFA1 and the GJA1 gene
102
Linkage and haplotype analysis
Multipoint non-parametric linkage and haplotype analyses were performed using the
MERLIN software version 0102 (multipoint engine for rapid likelihood inference
Center for Statistical Genetics University of Michigan MI USA Abecasis et al
2002) The test statistics Z-mean and Lod score were used to test for the proportion
of alleles shared by affected individuals identical by descent (IBD) for the considered
marker loci
A non-parametric linkage analysis was performed with 27 microsatellite markers in 23
Dalmatian dog families consisting of 176 Dalmatian dogs The results were added to
the linkage analysis performed in Chapter 3
Results and discussion
In previously performed studies (Chapter 3) one French Dalmatian dog family
reached a Zmean value of 295 (plt0002) for GJA1_MS1 and a Zmean value of 286
(plt0002) for GJA1_MS2 indicating linkage Both markers were associated with the
GJA1 gene
By sequence analysis we revealed a total of 3 SNPs found in sequences of the 3acute-
UTR shown in Table 1 None of the polymorphisms were polymorphic in all parents of
the examined French half-sib family Neither of the observed polymorphism did alter
the predicted amino acid sequence of GJA1 nor showed the identified haplotypes an
association with the CCSD phenotype (Figure 1)
There is no recombination of the haplotypes of the GJA1 gene in this family The
paternal and maternal haplotypes as far as they could be estimated were distributed
among the affected progeny closely to the expected proportion of 50 and therefore
no excess of a certain haplotype could be observed in the affected dogs
Thus evaluation of the SNP markers debilitates the linkage to CCSD for the French
half-sib family Because of the fact that both animals with unilateral hearing loss and
bilateral hearing animals shared identical haplotypes these polymorphisms are
obviously not associated with CCSD in these Dalmatian dog families Furthermore
the present study revealed no functional mutations of the complete coding region of
GJA1 It is therefore unlikely that the GJA1 gene is involved in the pathogenesis of
CCSD in the analyzed French Dalmatian dogs The reason for GJA1_MS1 and
GJA1_MS2 indicating linkage may be caused by a closely linked gene involved in
CFA1 and the GJA1 gene 103
CCSD among the half-sib family of French Dalmatian dogs or by a false positive
result of the microsatellite study performed in Chapter 3
To clarify whether significant test statistics for other genomic regions on CFA1 and
for more families can be shown we used 27 microsatellite markers derived from the
NCBI database (httpwebncbinlmnihgov) to cover CFA1 completely A linkage
analysis was carried out after genotyping 176 German Dalmatian dogs with a set of
27 microsatellite markers The results of this linkage analysis were added to the
results of the test statistics for the microsatellites GJA1_MS1 and GJA1_MS2
(Chapter 3)
As a result we could not find linkage to any microsatellite in the analyzed families
(Figure 2 and 3) It is unlikely that the canine chromosome 1 harbours genomic
regions that are involved in the development of CCSD in the analyzed Dalmatian dog
families
With hindsight it was revealed that GJA1 is not responsible for sensorineural non-
syndromic deafness in humans as Liu et al (2001) has published GJA1 is
participating in a human syndrome called oculodentodigital dysplasia (ODDD) that
can be accompanied with hearing impairment (Paznekas et al 2003) But the type of
deafness in human ODDD differs from the typical hearing loss associated with other
connexin mutations because it is conductive rather than sensorineural
As deafness in dogs especially in Dalmatians is almost exclusively caused by
sensorineural non-syndromic forms also known as cochleosaccular degeneration
the GJA1 gene should not be considered as a candidate gene for CCSD anymore
CFA1 and the GJA1 gene
104
Table 1 Three newly developed intragenic SNPs and two microsatellite markers for
Dalmatian dogs in the candidate gene GJA1 with their corresponding primers the
SNP motif the product size and the annealing temperature
SNP
Primer F (5acute -gt 3acute)
SNP motif
Primer R (5acute -gt 3acute)
Product
size (bp)
Annealing
temperatur
GJA1_SNP1+2
CACCTTAGGCGTTCATTTTG
CCGGGGAG(AG)AAAA(AG)AAAAATACTT
TGGCTTGATTCCCTGACTC
650 58
GJA1_SNP3
TCTGAAATGTAATCATGGATGC
CAGAACTTGTAT(AT)CTGTTAAGAG
AATCACACAGGATATAGAGGCTATC
600 58
Microsatellite
marker Primers (forward reverse ) 5acute -gt 3acute
Product
size (bp)
Annealing
temperatur
GJA1_MS1
ATGGCATGAAGAGGATACCG
AGGACAGGTGACGGCTCTAC
134 60
GJA1_MS2
GCTAGTACTCGATTGTGGTC
TCATGGGTTGTGAGATCCAG
190 60
CFA1 and the GJA1 gene 105
Figu
re 1
Hap
loty
pes
of th
e G
JA1
gene
in th
e an
alyz
ed D
alm
atia
n do
g fa
mily
CFA1 and the GJA1 gene
106
Figure 2 Zmeans and LOD scores for 29 microsatellite markers on CFA1 (number of
families 23 number of genotyped dogs 176)
Figure 3 P-values for Zmeans and LOD scores of 29 microsatellite markers on CFA1
(number of families 23 number of genotyped dogs 176)
Chapter 8
Analysis of canine chromosome 31 and the claudin-14 (CLDN14) gene
in Dalmatian dogs segregating for congenital sensorineural deafness
CFA31 and the CLDN14 gene 109
Analysis of canine chromosome 31 and the claudin-14 (CLDN14) gene in Dalmatian dogs segregating for congenital sensorineural deafness Abstract A previously accomplished linkage analysis revealed significant linkage of
microsatellite markers associated with the CLDN14 gene on canine chromosome
(CFA) 31 with the phenotype of canine sensorineural deafness (Chapter 3) The
objective of the present study was to perform a sequence analysis in order to find
single nucleotide polymorphisms (SNPs) for the CLDN14 gene and additionally to
use a set of six microsatellite markers evenly distributed on CFA31 for non-
parametric linkage analysis with the aim to verify the significant test statistics shown
in Chapter 3
Introduction
Of the genes responsible for different forms of sensorineural non-syndromic deafness
in humans Rak (2003) considered 24 genes as candidates for sensorineural
deafness in dogs among them the CLDN14 gene on canine chromosome (CFA) 31 CLDN14 encodes a member of the claudin family which comprises the major
components of tight juncions (TJ) The human CLDN14 gene consists of one
tranlatete exon and six exons in the 5rsquountranlated region Five splice isoformes are
identified so far (Wilcox et al 2001 Wattenhofer et al 2005)
For the compartmentalization of perilymph and endolymph in the inner ear the
leakage of solutes through a paracellular pathway must be prevented by tight
junctions TJ are an intercellular junction found at the most apical region of polarised
epithelial and endothelial cells at which adjacent plasma membranes are joined
tightly together separating apical membranes and basolateral menbranes They are
specialised membrane domains containing branching strands of integral proteins and
create a primary barrier preventing paracellular transport of solutes and restricting
lateral diffusion of membrane lipids and proteins (Schneeberger and Lynch 2004)
CFA31 and the CLDN14 gene
110
The important role of the CLDN14 gene in the TJ of the inner ear was demonstrated
by Wilcox et al (2001) when he identified mutations in the CLDN14 gene responsible
for a hereditary human deafness in families segregating for congenital recessive
deafness (DFNB29)
In previously performed studies one large half-sib family as well as several full-sib
families of German Dalmatian dog indicated linkage to CLDN14 gene-associated
markers (Chapter 3)
A sequence analysis was performed to identify single nucleotide polymorphisms
(SNPs) either intergenic located or alternatively in the flanking 5rsquo- and 3rsquo-Regions
Furthermore we employed microsatellite markers covering CFA31 and used them for
a non-parametric linkage analysis with CCSD in a German Dalmatian dog population
of 176 animals with frequent occurrence of CCSD
Material and methods Pedigree material
For the linkage analysis of the microsatellite marker we used DNA from altogether
176 animals belonging to 22 full-sib families and one large paternal half-sib family of
German Dalmatian dogs All families were segregating for CCSD The genotyped
families included all the affected dogs (unilaterally and bilaterally deaf) their parents
if available and one to four unaffected animals At least two of the full sibs of each
family were unilaterally or bilaterally deaf The phenotype of the affected animals had
been confirmed by brainstem auditory evoked response (BAER) that detects
electrical activity in the cochlea and auditory pathways in the brain
Mutation analysis and screening for SNPs was performed by comparative
sequencing of DNA from 36 animals consisting of the parents and their progenies of
one half-sib family and four full-sib families of Dalmatian dogs which showed
significant linkage to a CLDN14-associated microsatellite (Chapter 3)
Sequencing of canine genomic DNA and development of single nucleotide
polymorphisms (SNPs)
The canine CLDN14 gene sequence was derived from sequences deposited in the
current dog genome assembly (Boxer genome assembly 21) of the NCBI GenBank
(Genbank Acc No NW_876295) by BLAST (Basic Local Alignment Search Tool)
CFA31 and the CLDN14 gene 111
search (httpwwwncbinlmnihgovBLAST) using the human CLDN14 reference
mRNA sequence (Genbank Acc No NM_144492)
We compared the canine genomic DNA sequence to canine cDNA fragments in the
canine EST database using the BLASTN program As no ESTs could be found
human mRNA sequences were used for the localization of the exonintron
boundaries using the mRNA-to-genomic alignment program Spidey
(httpwwwncbinlmnihgovIEBResearchOstellSpideyindexhtml)
We screened exon three for mutations as this exon is the only translated one in man
Additionally we screened large intronic sequences and sequences located in the 5rsquo-
and 3rsquo-UTR of the CLDN14 gene for Polymorphisms For this purpose primer pairs
were designed yielding products with a length of around 600 bp
PCR primers were developed with the Primer3 program (httpfrodowimiteducgi-
binprimer3primer3_wwwcgi) The PCR reactions were performed in a total of 50 microl
containing 125 microM dNTPs 25 pmol of each primer the reaction buffer supplied by
the manufacturer (Qbiogene Heidelberg Germany) and 1 U Taq polymerase After
a 4 min initial denaturation at 95degC 35 cycles of 30 sec at 94degC 45 sec at 58degC and
80 sec at 72degC were performed in a MJ Research thermocycler (Biozym Hessisch
Oldendorf Germany)
The obtained PCR products were directly sequenced with the DYEnamic ET
Terminator kit (AmershamBiosciences Freiburg Germany) and a MegaBACE 1000
capillary sequencer (AmershamBiosciences) using the PCR primers as sequencing
primers Sequence data were analyzed with Sequencher 42 (GeneCodes Ann
Arbor MI USA) The eight newly developed SNPs are shown in Table 1
Microsatellite marker analysis
In total six microsatellite marker were derived from the NCBI database
(httpwebncbinlmnihgov) to cover CFA31 completely (Appendix Table 1)
PCR reactions for microsatellites were carried out in 12 microl reaction mixtures
containing 2 microl genomic DNA 12 microl 10x PCR buffer (Qbiogene) 024 microl dimethyl
sulfoxide (DMSO) 02 microl dNTPs (100 microM) 01 microl Taq Polymerase (5Umicrol)
(Qbiogene) 06 microl (10 microM) 5rsquo-IRD700 or IRD800 labelled forward primer 06 microl (10
microM) unlabelled reverse primer The PCR reactions were carried out in MJ Research
thermocyclers with the following program 4 min at 94 degC followed by 32 cycles of 30
sec at 94degC 30 sec at maximum annealing temperature and a final extension of
CFA31 and the CLDN14 gene
112
45 sec at 72degC PCR-products were diluted with formamide loading buffer in ratios
from 110 to 140 and then size-fractionated by gel electrophoresis on automated LI-
COR 42004300 sequencers (LI-COR inc Lincoln NE USA) using denaturing 4
and 6 polyacrylamide gels (RotiphoreseregGel 40 Roth Karlsruhe Germany)
Linkage and haplotype analysis
Multipoint non-parametric linkage and haplotype analysis were performed using
MERLIN version 0102 (multipoint engine for rapid likelihood inference Center for
Statistical Genetics University of Michigan MI USA Abecasis et al 2002) The test
statistics Z-mean and Lod score were used to test for the proportion of alleles shared
by affected individuals identical by descent (IBD) for the considered marker loci A
linkage analysis was performed with 6 microsatellite markers on 176 Dalmatian dogs
The results were added to the linkage analysis performed in Chapter 3
Results and discussion CLDN14-associted microsatellites were significantly linked with CCSD in a scan of
candidate genes using 24 families with 215 Dalmatian dogs (Chapter 3) Selection of
four full-sib families and one half-sib family with the highest tests statistics lead to a
Zmean value of 383 (plt000007) for the CLDN14 gene-associated marker
CLDN14_MS2
To substantiate the linkage to the CLDN14 gene we searched for sequence
variations within the CLDN14 gene in four full-sib families and one half-sib family with
the highest contribution to the test statistics shown in Chapter 3 Most of the identified
polymorphisms were found in intronic sequences none were within exon three None
of the observed polymorphism did alter the predicted amino acid sequence of exon
three We revealed a total of eight SNPs (Table 1) but only two (SNP_4 and SNP_5)
out of the identified eight SNPs were polymorphic in the examined families Both
unilaterally and bilaterally deaf animals as well as normal hearing animals showed
identical haplotypes for these two polymorphic SNPs and thus no co-segregation with
the deaf Dalmatian dogs could be revealed (Figure 1 and 2) In this study we did not
identify any causal mutations for CCSD in the analyzed Dalmatian dogs Despite this
results a mutation outside of the translated genomic regions analyzed here may exist
that would affect CLDN14 expression
CFA31 and the CLDN14 gene 113
To clarify if other regions on CFA31 are responsible for the CCSD phenotype
additionally six microsatellite markers covering CFA31 were tested in 23 Dalmatian
dog families consisting of 176 individuals A non-parametric linkage analysis was
performed regarding these 23 families The results of this linkage analysis were
added to the results of the test statistics for the CLDN14 gene-associated
microsatellites (Chapter 3)
We could only find significant linkage with CCSD for CLDN14-associted
microsatellites in the abovementioned five families The two microsatellites located
most closely were proximal and distal 4 Mb off the CLDN14 gene REN110K04 with a
Zmean value of -009 (plt05) and FH2712 with a Zmean value of 022 (plt03) not
indicating linkage
It can not be excluded that CLDN14 or genes in its flanking regions are involved in
the development of CCSD in the analyzed Dalmatian dog families Other genes than
the CLDN14 that are known to be involved in the hearing process or known to be
expressed in the inner ear are not annotated at the syntenic human region of homo
sapiens autosome (HSA) 21q223 Therefore despite the CLDN14 gene no other
well-defined candidate gene for canine hereditary deafness exists in the linked
region Despite the remarkable progress that has been made in the past few years in
identifying deafness genes in man and mouse it is suspected that only one-third of all
deafness causing genes have been identified so far and thus it seems possible that
other genes in the flanking region of the CLDN14 gene are involved in the
development of the disease Anyway to clarify the importance of CLDN14 in the
CCSD phenotype more SNPs have to be developed within the CLDN14 gene as well
as in its flanking regions with the aim to find significant linkage disequilibrium of SNP
markers with CCSD
CFA31 and the CLDN14 gene
114
Table 1 The 8 newly developed SNPs for Dalmatian dogs in the CLDN14 gene on
CFA31 with their corresponding primers the SNP motif the product size and the
annealing temperature
SNP
Location
(intron or
5rsquo3rsquo-UTR)
Primer F1 (5acute -gt 3acute)
SNP motif
Primer R2 (5acute -gt 3acute)
Bp3 AT4
CLDN14_SNP1
intron
GACCATATGTTTGTGGCC
CTTCCAGGGAAA(AT)TGTCGTAGCC
CGTCAGGATGTTGGTGCC
580 60
CLDN14_SNP2
inton
GACCATATGTTTGTGGCC
GAAATTGTCGTA(AG)CCCGGCCGCT
CGTCAGGATGTTGGTGCC
580 60
CLDN14_SNP3
3rsquo-UTR
CTGCCTTCAAGGACAACC
CCAGAGGAATAA(CT)ATGATCGTGA
GATGAGTATCAGCCCAGC
550 60
CLDN14_SNP4
3rsquo-UTR
CTGCCTTCAAGGACAACC
ACCACCGCACAC(CT)GTCACAGCTT
GATGAGTATCAGCCCAGC
550 60
CLDN14_SNP5 3rsquo-UTR
CATGCCTTTGTCCCAAACTT
GAGACCCTCTGG(CT)TCCTTTTGGC
GTACCTGTTGCCTGGGTTGT
610 60
CLDN14_SNP6 5rsquo-UTR
CCTTCATCCTTTCTGGTTGA
GCTCACAGTCCC(AC)ATGGGGACAT
GGGGAGCATAATGTGGTCAT
585 60
CLDN14_SNP7 5rsquo-UTR
TGAACTGGTCCCAAGGAAAG
GCACGACCGAGC(CT)CTGGCTTTAC
GGGATGAGAGGGAGGTTTTT
580 60
CLDN14_SNP8 3rsquo-UTR
AATGCCTATCCCTTCTTTGGA
CACGTTACTGTG(AG)ACCTCTCTAC
GCAGGCTTTCCTCAAGTGTC
680 60
1 Forward 2 Reverse 3 Product size (basepairs) 4 Annealing temperature (degC)
CFA31 and the CLDN14 gene 115
Figure 1 Haplotypes of the CLDN14 gene-associated markers SNP_4 and SNP_5 in
the analyzed four Dalmatian dog full-sib families
CFA31 and the CLDN14 gene
116
Figu
re 2
Hap
loty
pes
of th
e C
LDN
14 g
ene-
asso
ciat
ed m
arke
rs S
NP
_4 a
nd S
NP
_5 in
the
anal
yzed
Dal
mat
ian
dog
half-
sib
fam
ily
Chapter 9
General discussion
General discussion 119
General discussion
The candidate gene approach In the first step of this thesis candidate genes for canine congenital sensorineural
deafness (CCSD) in Dalmatian dogs were analyzed by means of microsatellite
markers or alternatively by single nucleotide polymorphisms (SNPs)
The candidate genes for which a set of in total 43 microsatellites was available
included the following 24 genes CDH23 CLDN14 COCH COL11A2 DFNA5
DIAPH1 EDN3 EDNRB EYA4 GJA1 GJB2 GJB6 MITF MYH9 MYO6 MYO7A
MYO15A OTOF PAX3 POU4F3 SLC26A4 SOX10 TECTA and TMPRSS3
(Rak 2003) These genes are known to be involved either in human non-syndromic
deafness or in the human Waardenburg syndrome The Waardenburg syndrome
(WS) manifests with sensorineural deafness and pigmentation defects in iris hair and
skin WS is classified into four types depending on the presence or absence of
additional symptoms which are caused by mutations in the five genes EDN3
EDNRB MITF PAX3 and SOX respectively
For another eight recently identified genes responsible for different forms of human
non-syndromic deafness including TMC1 TMIE USH1C MYH14 MYO3A PRES
WHRN and ESPN linkage and association analyses were performed using newly
developed SNPs
In the last years most projects have exploited canine traits for which either direct
candidate genes could be proposed and evaluated or for which large informative
pedigrees were available to enable linkage mapping to identify candidate regions A
major component of such research efforts comprised the cloning sequencing and
mapping of individual canine homologs of genes either proposed as candidate
genes or expected to be located in candidate regions This was necessary to identify
new informative polymorphisms (eg SNPs microsatellites) for high resolution
mapping of candidate regions and to examine each exon and exonintron boundary
for positional candidates Availability of the second version of the dog genome
assembly (build 21) of the NCBI database shortcuts this effort and increases the
investigators efficiency Now either additional candidate genes for canine congenital
sensorineural deafness can be found directly by its gene symbol in the 21 of the
General discussion
120
NCBIs genome annotation or if a candidate gene is not yet annotated a BLAST
(Basic Local Alignment Search Tool) search against the canine whole genome
shotgun (wgs) sequence resource can be used to obtain the sequence of the canine
genomic contigs containing the human homologous gene and thus intragenic
markers can be developed for subsequent linkage and association analyses
Over the past decade it has become increasingly clear how far structural and
functional homologies at the gene level extend across even distantly related species
Much is known about deafness-causing gene mutations in humans and mice
including the fact that the clinical and histopathological findings are often very similar
to those of deafness in Dalmatian dogs Thus genes responsible for non-syndromic
congenital hereditary deafness in humans seem to be appropriate candidate genes
for CCSD (Rak and Distl 2005) In this thesis we first concentrated on the candidate
gene approach combined with linkage analysis method using affected pedigree
members Once a significant linkage was found only the linked genes with the
required low error probability values were used for further molecular genetic analysis
The method of candidate gene approach using either gene-associated microsatellite
or alternatively SNP markers was applied for in total 32 candidate genes which
comprise nearly all of the identified mutated genes causing non-syndromic hereditary
hearing impairment in humans
Linkage and association analysis In principle linkage and association are totally different phenomena Linkage is a
relation between loci and association is a relation between alleles
Linkage means that a haplotype characterised by microsatellites or SNPs is
significantly more often present in family members with the phenotype under study
than expected by random assortment For construction of haplotypes sets of closely
linked genetic markers on the same chromosome are needed which tend to be
inherited together as they are not likely to be separated by recombination Linkage
creates associations within families but not among unrelated induviduals
Association is a statistical statement about the co-occurrence of alleles or
phenotypes Association analysis can be carried out as a method of genetic analysis
that compares the frequency of alleles between affected and unaffected individuals
across all families Thus for association family structures are not necessary A given
allele is considered to be associated with the disease if the presence of that allele
General discussion 121
explains a significant proportion of the phenotypic trait variation For association
studies the developing of a marker set consisting of SNPs rather than microsatellites
is needed
In this thesis a total of 32 candidate genes for canine congenital deafness were
analyzed which showed an appropriate clinical and histological disease pattern in
comparison to deafness in Dalmatian dogs Rak (2003) developed a set of 43
microsatellites for in total 24 candidate genes among them the CLDN14 gene on
canine chromosome (CFA) 31 and the MYH9 gene on CFA10 The GJA1 on CFA1
was also considered as a candidate gene for CCSD (Rak 2003) and therefore two
gene-associated microsatellites have been developed Recently it turned out that
GJA1 is not responsible for human sensorineural non-syndromic deafness but for a
human syndromic disorder that can be related with conductive deafness
By the use of a non-parametric linkage analysis using the existing set of 43
microsatellites associated to 24 candidate genes we found linkage to markers
associated to CLDN14 on CFA31 MYH9 on CFA10 and GJA1 on CFA1
For another another eight candidate genes it was possible to develop SNPs
Performing linkage analyses as well as association and haplotype studies it was
possible to exclude these eight candidate genes from being responsible for the
CCSD phenotype
Over the past ten years significant progress has been made in the identification of
deafness gene localisations Up to now approximately 120 loci have been reported
for both autosomal dominant and recessive forms of non-syndromic hereditary
deafness in humans and only for one third the responsible gene mutation could be
detected Thus it can be expected that additional potential human candidates for
CCSD in Dalmatian dogs will become available in future (Van Camp and Smith
2003)
The extreme heterogeneity of human deafness often hampered genetic studies
because many different genetic forms of hearing loss give rise to similar clinical
phenotypes and conversely mutations in the same gene can result in a variety of
clinical phenotypes In man genes that transport ions across membranes to
maintain appropriate solute concentration and pH as well as regulatory genes mainly
transcription factors and genes that play a part in structural integrity are essential
for the hearing process
General discussion
122
The results of this thesis indicate that the inheritance of hearing loss in Dalmatian
dogs is probably as heterogenic in origin as it is in humans Genetic heterogeneity
means that different mutations cause a similar phenotype the different mutations
can either be found at the same locus (allelic heterogeneity) or even at different loci
(non-allelic heterogeneity) As linkage was found for different candidate genes in
different families subsequently only the families indicating linkage were chosen for
further molecular analyses
GJA1 on CFA1 MYH9 on CFA10 and CLDN14 on CFA31 and their flanking regions
are further analyzed with a combined approach using microsatellite and SNP
markers
CFA1 By the use of GJA1-associated microsatellites one large French Dalmatian dog
family reached a Zmean value of 295 (plt0002) for GJA1_MS1 and a Zmean value
of 286 (plt0002) for GJA1_MS2 indicating linkage Subsequently a sequence
analysis of the GJA1 gene using the above mentioned French Dalmatian dog family
was performed None of the observed polymorphism did alter the predicted amino
acid sequence of GJA1 nor showed the identified haplotypes an association with the
CCSD phenotype Thus evaluation of the SNP markers debilitates the linkage to
CCSD for the French half-sib family It is unlikely that the GJA1 gene is involved in
the pathogenesis of CCSD in Dalmatian dogs To see whether significant test
statistics for other genomic regions on CFA1 and for more families can be shown a
non-parametric linkage analysis was performed with 27 microsatellite markers
covering CFA1 completely In total 176 animals were genotyped We could not find
linkage to any microsatellite in the analyzed families Furthermore it was revealed
that GJA1 is not a candidate gene for sensorineural non-syndromic deafness in
humans (WA Paznekas cited a personal communication from the senior author (W
E Nance) of the paper by Liu et al 2001) GJA1 is participating in a human
syndrome called oculodentodigital dysplasia (ODDD) that can be accompanied with
hearing impairment (Paznekas et al 2003) But the type of deafness in human
ODDD is conductive rather than sensorineural As deafness in dogs especially in
Dalmatians is almost exclusively caused by sensorineural non-syndromic forms also
known as cochleosaccular degeneration the GJA1 gene should not be considered
as a candidate gene for CCSD anymore
General discussion 123
CFA31 By the use of microsatellites as well as SNPs we found significant linkage with CCSD
for CLDN14-associated microsatellites in four full-sib and one half-sib Dalmatian dog
familiy with a Zmean value of 383 (plt000007) A mutation analysis was performed
for exon three as this is the only translated one in man None of the observed
polymorphisms did alter the predicted amino acid sequence However to clarify the
importance of the CLDN14 gene and its flanking regions in the CCSD phenotype
more SNPs have to be developed within the CLDN14 gene as well as in its flanking
regions with the aim to find significant linkage disequilibrium of SNP markers
CFA10 A significant co-segregation of markers alleles and the phenotypic expression of
deafness in a large sample of German Dalmatian dog families was determined for
one marker (Z-mean of 156 [p= 006] and a Lod score of 058 [p= 005]) associated
to the MYH9 gene Subsequently we evaluated whether MYH9 gene mutations are
responsible for CCSD in these Dalmatian dog families An initial priority in defining
gene structure is to obtain a full-length cDNA sequence and identify translational
initiation and termination sites and polyadenylation site(s) Exonintron structure can
then be determined by referencing the cDNA sequence against sequences of
cognate genomic DNA One popular method of obtaining full-length cDNA sequences
is the RACE (rapid amplification of cDNA ends) technique RACE-PCR is an anchor
PCR modification of RT-PCR The rationale is to amplify sequences between a single
previously characterised region in the mRNA (cDNA) and an anchor sequence that is
coupled to the 5 or the 3 end A primer is designed from the known internal
sequence and the second primer is selected from the relevant anchor sequence
To provide the genomic organization and the complete sequence of the canine
MYH9 gene the isolation of full length cDNAs was achieved with the help of a
modified rapid amplification of cDNA ends (RACE) protocol A mutation analysis was
performed to identify single nucleotide polymorphisms (SNPs) in this gene We
analyzed the association of the MYH9 haplotypes with the CCSD phenotype in three
families of Dalmatian dogs with frequent occurrence of CCSD and significant linkage
to gene-associated microsatellites Using the RT-PCR analyses for cDNA-genomic
sequence comparisons we detected that the canine MYH9 gene is bigger compared
to the human sequence due to the untranlated first exon in the 5rsquo-UTR
General discussion
124
The canine MYH9 gene consists of 41 exons with an untranslated exon 1 and
exonintron boundaries that conform perfectly to the GTAG rule
None of the observed polymorphisms did alter the predicted amino acid sequence of
MYH9 nor showed the identified haplotypes an association with the CCSD
phenotype
Thus these silent point mutations found in affected and unaffected Dalmatian dogs
do not seem to be responsible for the CCSD phenotype in these three families
To clarify if other regions on CFA10 are responsible for the CCSD phenotype we
added in a second step 27 microsatellite markers derived from the NCBI database to
cover CFA10 with a high density in the flanking region of the MYH9 gene A linkage
analysis was carried out for 23 Dalmatian dog families consisting of 176 animals that
were genotyped with the marker set of 27 microsatellite markers
We found significant linkage to microsatellites in the region spanning 36 Mb to 48 Mb
Consequently we screened this 12 Mb spanning region for SNPs Out of the 23
analyzed Dalmatian dog families five full-sib families were chosen to screen for
SNPs because of their obviously significant values at the above mentioned region
Twenty-six new SNP markers covering the region of 36 Mb to 48 Mb equally were
developed and added to the linkage analysis The significant Zmeans on CFA10 was
confirmed after adding the SNP markers Furthermore with the use of SNPs the
apparently linked region spanning 36 Mb to 48 Mb could be narrowed down to 5 Mb
spanning from 39 Mb to 44 Mb For this reason the use of SNPs in addition to
informative microsatellites appears highly recommendable
In further studies more SNPs have to be developed within the identified CCSD region
on CFA10 to localize the deafness causing gene or to find unambiguously associsted
SNP markers which could be used for a population-wide genetic test for CCSD
Chapter 10
Summary
Summary 127
Summary
Molecular genetic analysis of canine congenital sensorineural deafness in Dalmatian dogs
Katharina Mieskes (2006) The objective of the present study was to localize the gene or genomic region that is
involved in the development of canine congenital sensorineural deafness (CCSD) in
Dalmatian dogs
In man as in different dog breeds deafness is an often diagnosed disorder with the
Dalmatian dog showing the highest incidence Many genetic disorders in humans
and domestic dogs (Canis familiaris) demonstrate a high level of clinical and
molecular similarity
Altogether 39 genes have already been found causative for sensorineural non-
syndromic hearing impairment in humans Out of this 39 deafness causing genes a
total of 32 candidate genes were selected for canine congenital deafness which
showed an appropriate clinical and histological disease pattern in comparison to
deafness in Dalmatians dogs
On the one hand an existing set of 43 microsatllite markers for in total 24 candidate
genes were used for a non-parametric linkage analysis among them the claudin-14
(CLDN14) gene on canine chromosome (CFA) 31 and the myosin heavy polypeptide
9 (MYH9) gene on CFA10 The gap junction protein alpha 1 (GJA1) gene on CFA1
was also considered as a candidate gene for CCSD and thus GJA1-associated
microsatellites were part of the existing set Recently it turned out that GJA1 is not
responsible for human sensorineural non-syndromic deafness but for a human
syndromic disorder that can be related with conductive deafness In the last few
years more human deafness genes have been identified among them eight genes
that were considered as appropriate candidates for CCSD For these eight genes a
total of 21 SNPs were newly developed and used for non-parametric linkage and
association analyses
Summary
128
The used microsatellite and SNP markers derived either from a partial sequence
analysis of BAC clones each containing a different candidate gene or from
sequences deposited in the current dog genome assembly (Boxer genome assembly
21) of the NCBI GenBank We found significant linkage to markers associated to
CLDN14 on CFA31 MYH9 on CFA10 and GJA1 on CFA1 To substantiate the
linkage we searched for sequence variations within these three genes SNPs found
in intronic sequences of either gene were included in the linkage analyses Sequence
analysis neither revealed a causative mutation nor significant linkage disequilibrium
of SNP markers with CCSD Subsequently CFA1 10 and 31 were scanned
completely with microsatellite markers derived from the NCBI database with the
purpose to see if other regions on this three chromosomes harbour a gene that is
involved in the development of CCSD
The analyses of SNPs and more microsatellite markers on CFA1 revealed no
significant linkage to CCSD Thus it is unlikely that the canine chromosome 1 and
the GJA1 gene are responsible for the CCSD phenoptype As deafness in dogs
especially in Dalmatians is almost exclusively caused by sensorineural non-
syndromic forms the GJA1 gene should not be considered as a candidate gene for
CCSD anymore
On CFA10 we could exclude MYH9 for being causal for deafness but by adding
more microsatellites covering CFA10 completely we found significant linkage to the
CCSD phenotype in the region distal of MYH9 Hence new SNP-markers for fine
mapping the region spanning 36 to 48 Mb were developed by sequence analyses of
different Dalmatian dogs The search for SNPs was carried out on genomic
sequences of genes located in the significantly linked region The sequences of
these genomic sequences were derived from the NCBI GenBank The SNPs
confirmed the linkage and narrowed the region harbouring a causative CCSD gene
down to 5 Mb spanning from 39 to 44 Mb
After scanning CFA31 we could not exclude CLDN14 for being responsible for the
CCSD phenotype as microsatellite markers associated to CLDN14 indicated linkage
However to clarify the importance of CLDN14 in the CCSD phenotype more SNPs
have to be developed within the CLDN14 gene as well as in its flanking regions with
the aim to find linkage disequilibrium for SNP markers
Chapter 11
Erweiterte Zusammenfassung
Erweiterte Zusammenfassung 131
Erweiterte Zusammenfassung
Katharina Mieskes
Molekulargenetische Untersuchung der kongenitalen sensorineuralen Taubheit beim Dalmatiner
Einleitung Es gibt mehrere Moumlglichkeiten Taubheit zu klassifizieren Je nach den betroffenen
Strukturen des Gehoumlrorgans lassen sich konduktive und sensorineurale Formen
unterscheiden Erstere entstehen durch Stoumlrungen des Konduktionsmechanismus im
aumluszligeren Gehoumlrgang und im Mittelohr Ursachen fuumlr konduktive Houmlrstoumlrungen koumlnnen
zum Beispiel chronische Otitis externa oder media Frakturen von Mittelohranteilen
oder Tumoren sein Sehr viel oumlfter tritt die sensorineurale Taubheit auf bei der ein
Houmlrverlust als Folge von Transduktions- und Transmissionsstoumlrungen in der Cochlea
(Schnecke) sowie in peripheren und zentralen Anteilen des Nervus cochlearis auftritt
Neben der erblichen Form koumlnnen auch eine Otitis interna Tumoren oder
ototoxische Medikamente zur sensorineuralen Taubheit fuumlhren
Der Houmlrverlust kann bereits seit der Geburt bestehen (kongenital) oder er kann erst
im Laufe der weiteren Lebenszeit auftreten
Beim Menschen ist mehr als die Haumllfte der kongenitalen Taubheit erblich bedingt Die
erbliche kongenitale Taubheit kann als Teil einer multisystemischen Krankheit
(syndromisch) oder als Funktionsstoumlrung auftreten die auf das Ohr und das
vestibulaumlre System (nicht-syndromisch) begrenzt ist Da die nicht-syndromische
ererbte Taubheit fast ausschlieszliglich durch Cochleadefekte verursacht wird leiden die
Patienten unter sensorineuraler Taubheit Es wird geschaumltzt dass die nicht-
syndromische erbliche Taubheit 60-70 aller menschlichen Taubheitsfaumllle
verursacht Weiterhin sind 70-80 der Faumllle der nicht-syndromischen Taubheit auf
einen autosomal rezessiven Erbgang zuruumlck zu fuumlhren gefolgt von einem autosomal
dominanten Erbgang mit 10-20 der Faumllle und 1-2 der Faumllle sind X- gekoppelt Ein
noch geringerer Prozentsatz wird durch einen maternalen Erbgang verursacht
Von den ca 25000 bis 30000 menschlichen Genen ist nach Schaumltzungen bis zu 1
wichtig fuumlr den Houmlrprozess Annaumlhernd 120 Gene sind inzwischen identifiziert die fuumlr
Erweiterte Zusammenfassung
132
verschiedene Formen der menschlichen vererbten Taubheit verantwortlich sind
Darunter sind ungefaumlhr 80 Gene fuumlr die syndromische und 39 Gene fuumlr die nicht
syndromische vererbte Taubheit verantwortlich Insgesamt stellen die 120
identifizierten Gene vermutlich ein Drittel aller taubheitsverursachenden Gene dar
Die kongenitale sensorineurale Taubheit ist eine Erkrankung von der viele
Saumlugetierspezies betroffen sind Beim Hund werden uumlber 54 verschiedene Rassen
beschrieben bei welchen ein kongenitaler Houmlrverlust gehaumluft auftritt In zahlreichen
europaumlischen und amerikanischen Studien weisen hierbei die Dalmatiner mit 165-
30 die houmlchste Taubheitsinzidenz auf
Die histologischen Veraumlnderungen im Innenohr werden im Allgemeinen aumlhnlich wie
beim Menschen als cochleosacculaumlr beschrieben Die Degenerationen im Innenohr
schreiten bei betroffenen Tieren mit zunehmendem Alter fort und fuumlhren
normalerweise in einem Alter von drei bis vier Wochen spaumltestens aber in einem
Alter von etwa vier Monaten zu einem vollstaumlndigen ein- oder beidseitigen
Houmlrverlust
Eine objektive Diagnose der Taubheit besonders wenn sie nur einseitig ist stuumltzt
sich auf die brainstem auditory evoked response (BAER in Deutschland
audiometrischer Test) ein elektro-diagnostischer Test bei dem durch einen
bestimmten Reiz ausgeloumlste elektrische Aktivitaumlten (akustisch evozierte Potentiale) in
der Cochlea und den Nervenbahnen zur auditorischen Hirnrinde detektiert werden
Obwohl fuumlr den Dalmatiner die Erblichkeit der Erkrankung wiederholt nachgewiesen
werden konnte wurden fuumlr die verschiedenen untersuchten Dalmatinerpopulationen
auch sehr viele unterschiedliche Erbgaumlnge in Betracht gezogen Es ist bisher nicht
gelungen die genaue Anzahl der beteiligten Gene zu bestimmen Auch war es
bislang fuumlr keine der betroffenen Hunderassen moumlglich ein taubheitsverursachendes
Gen zu identifizieren
Bei deutschen Dalmatinern ergab eine Auswertung mittels komplexer
Segregationsanalysen dass ein Modell mit einem rezessiven Hauptgen und einer
polygenen Komponente das Auftreten der kongenitalen Taubheit in der untersuchten
Population am besten erklaumlrte Zusaumltzlich konnte ein Zusammenhang zu
Pigmentierungsgenorten nachgewiesen werden da Tiere mit blauer Augenfarbe
signifikant haumlufiger von Taubheit betroffen waren und gleichzeitig Tiere mit
Plattenzeichnung seltener kongenitalen Houmlrverlust zeigten
Erweiterte Zusammenfassung 133
Das Zuumlchten ein- oder beidseitig tauber Dalmatiner sowie Dalmatinern mit blauen
Augen ist in Deutschland schon laumlnger verboten und es wird von den
Zuchtverbaumlnden vorgeschrieben dass alle Dalmatinerwelpen in einem Alter von etwa
6 bis 8 Wochen audiometrisch untersucht werden Des Weiteren ist der Defekt
tierschutzrelevant nach sect 11b des Deutschen Tierschutzgesetzes da der artgemaumlszlige
Gebrauch eines Organs eingeschraumlnkt wird und eine Erblichkeit nachgewiesen
wurde Somit duumlrfen Tiere die Defektgentraumlger sind oder bei denen damit zu
rechnen ist nicht zur Zucht verwendet werden Waumlhrend einseitig taube Dalmatiner
als ganz normale Familienhunde gehalten werden koumlnnen sind beidseitig taube
Dalmatiner schwierig zu erziehen schreckhafter und daraus resultierend auch
oftmals aggressiv und bissig Aus diesen Gruumlnden werden fast alle Welpen mit
beidseitigem Houmlrverlust euthanasiert
Es hat sich gezeigt dass allein der Zuchtausschluss betroffener Tiere nicht genuumlgt
um die hohe Taubheitsinzidenz beim Dalmatiner deutlich zu reduzieren Aus diesen
Gruumlnden ist es von erheblicher Bedeutung die kongenitale sensorineurale Taubheit
beim Dalmatiner molekulargenetisch zu untersuchen um so ein
molekulargenetisches Testverfahren entwickeln zu koumlnnen welches auch die
Identifizierung von Anlagetraumlgern ermoumlglicht
Innerhalb des letzten Jahrzehnts wurden auch zwischen relativ unverwandten
Spezies die strukturellen und funktionellen Homologien auf genetischer Ebene
zunehmend verdeutlicht
Es wurde inzwischen eine groszlige Anzahl von taubheitsverursachenden
Genmutationen beim Menschen und der Maus aufgeklaumlrt Da auch die
histopathologischen Befunde beim Menschen und der Maus oftmals sehr aumlhnlich
denen beim Dalmatiner sind scheinen Gene die beim Menschen verantwortlich fuumlr
die congenitale sensorineurale Taubheit sind geeignete Kandidatengene fuumlr die
canine congenitale sensorineurale Taubheit zu sein
Das Ziel dieser Dissertation ist es die fuumlr die Taubheit kausalen Gene bzw Genorte
zu lokalisieren Um die kongenitale sensorineurale Taubheit beim Dalmatiner
moumlglichst effizient molekulargenetisch aufzuklaumlren wurden 32 Kandidatengene mit
genassoziierten Markern auf eine potenzielle Kopplung mit der caninen kongenitalen
sensorineuralen Taubheit untersucht
Die Genombereiche mit signifikanter Kopplung zur kongenitalen sensorineuralen
Taubheit wurden weitergehend molekulargenetisch analysiert
Erweiterte Zusammenfassung
134
Kopplungsanalyse fuumlr 24 Kandidatengene der caninen kongenitalen sensorineuralen Taubheit (CCSD) mit 43 genassoziierten Mikrosatelliten Markern
Material und Methoden
Fuumlr 24 Kandidatengene stand ein Mikrosatelliten Marker Set zur Verfuumlgung Das
Marker Set beinhaltete Mikrosatelliten fuumlr folgende Kandidatengene CDH23
CLDN14 COCH COL11A2 DFNA5 DIAPH1 EDN3 EDNRB EYA4 GJA1 GJB2
GJB6 MITF MYH9 MYO6 MYO7A MYO15A OTOF PAX3 POU4F3 SLC26A4
SOX10 TECTA und TMPRSS3 Als Familienmaterial fuumlr die Mikrosatellitenstudie
wurden 215 Dalmatiner verwendet Die Tiere verteilten sich auf 22 unverwandte
Vollgeschwisterfamilien und zwei paternale Halbgeschwistergruppen Die
genotypisierten Familien bestanden aus saumlmtlichen tauben Nachkommen (uni- oder
bilateral taub) ihren Eltern und einem bis vier nicht betroffenen Voll- bzw
Halbgeschwistern Mindestens zwei der Vollgeschwister einer Familie waren uni-
oder bilateral taub Der Phaumlnotyp der verwendeten Tiere wurde durch einen
audiometrischen Test bestimmt
Alle Mikrosatellitenmarker wurden uumlber PCR und Polyacrylamidgelelektrophorese
ausgewertet
Kopplungsanalyse
Die nicht-parametrische Kopplungsanalyse wurde unter Verwendung der Software
MERLIN (multipoint engine for rapid likelihood inference Version 0102)
durchgefuumlhrt und basierte auf dem identical-by-descent (IBD) Verfahren Dabei
wurden die Markerallele auf Kosegregation mit der phaumlnotypischen Auspraumlgung der
Erkrankung getestet Darauf folgend wurde die einer Normalverteilung folgende
Teststatistik fuumlr den Anteil von IBD-Markerallelen (Zmean) und ein daraus
abgeleiteter LOD-Score berechnet Als signifikant fuumlr die Kosegregation eines
Markerallels mit dem Phaumlnotyp der caninen kongenitalen sensorineuralen Taubheit
(CCSD) gelten Irrtumswahrscheinlichkeiten (p) von 005 oder kleiner
Die Daten wurden mit dem Softwarepaket SAS (Statistical Analysis System Version
913) bearbeitet um die Marker bezuumlglich Allelfrequenzen beobachteten und
erwarteten Heterozygotiegrad und PIC (Polymorphism information content)
charakterisieren zu koumlnnen
Erweiterte Zusammenfassung 135
Ergebnisse und Diskussion Das Mikrosatelliten Set bestand insgesamt aus 43 Markern mit einem bis drei
Markern pro Kandidatengen Zunaumlchst wurde fuumlr alle 24 Familien zusammen eine
Kopplungsanalyse durchgefuumlhrt Vermeintliche CCSD Loci wurden auf den caninen
Chromosomen (CFA) 1 10 12 20 und 31 lokalisiert Im Anschluss wurden die
Ergebnisse der Kopplungsanalyse auf Heterogenie zwischen den Familien uumlberpruumlft
Fuumlr einzelne Familien konnten houmlhere Teststatistiken und somit engere
Kopplungsbeziehungen als bei der Analyse aller Familien nachgewiesen werden
Eine signifikante Kopplung mit der kongenitalen sensorineuralen Taubheit wurde fuumlr
die Gene GJA1 auf CFA1 MYH9 auf CFA10 und CLDN14 auf CFA31 sowohl im
Gesamtmaterial wie in jeweils unterschiedlichen Gruppen von Dalmatinerfamilien
gefunden Es ist daher wahrscheinlich dass diese Gene oder Gene in ihrer naumlheren
Umgebung an der Entwicklung der Taubheit in den jeweiligen Familien involviert
sind Fuumlr die restlichen Kandidatengene lagen die Multipoint Teststatistiken fuumlr den
Anteil von IBD-Markerallelen (Zmean) und daraus abgeleiteten LOD-Scores bei
annaumlhernd Null und waren nicht signifikant
Die Ergebnisse dieser Teststatistiken zeigen dass der Erbgang der nicht-
syndromischen Taubheit bei Dalmatinern vermutlich ebenso heterogen ist wie er
sich beim Menschen darstellt
Dennoch stellt diese Studie den ersten Schritt dar um die CCSD verursachenden
Gene beim Dalmatiner zu identifizieren Die Gene GJA1 MYH9 und CLDN14 sowie
ihre naumlhere Umgebungen werden im weiteren Verlauf der Studie weitergehend
molekulargenetisch mit Hilfe von SNPs und Mikrosatelliten untersucht Aufgrund der
Heterogenitaumlt zwischen den Familien wurden fuumlr die weitere molekulargenetische
Analyse bestimmter Gene oder Genomregionen nur die Familien mit dem houmlchsten
Beitrag zu den Teststatistiken der Kopplungsanalyse (Zmean gt 1) ausgewaumlhlt
Erweiterte Zusammenfassung
136
Evaluierung von neu entwickelten SNP Markern assoziiert mit Kandidatengenen fuumlr die canine kongenitale sensorineurale Taubheit (CCSD) Material und Methoden Fuumlr acht Kandidatengene (TMC1 TMIE USH1C MYH14 MYO3A PRES WHRN
und ESPN) wurden intragenische Single Nucleotide Polymorphism (SNP) Marker neu
entwickelt
Als Familienmaterial fuumlr die Genotypisierung der SNPs wurde DNA von insgesamt 39
Dalmatinern verwendet die vier Vollgeschwisterfamilien angehoumlrten Mindestens
zwei der Vollgeschwister einer Familie waren uni- oder bilateral taub Der Phaumlnotyp
der verwendeten Tiere wurde durch einen audiometrischen Test bestimmt Die vier
Familien bestanden aus sechs bis zehn Vollgeschwistern und den dazugehoumlrenden
Elterntieren
Identifizierung von SNPs (Single Nucleotide Polymorphisms)
Die caninen Sequenzen der Kandidatengene wurden mit Hilfe der humanen mRNA
mittels BLAST (Basic Local Alignment Search Tool) Suche aus veroumlffentlichten
Sequenzen der NCBI Datenbank (Boxer genome assembly 21) entnommen Die
Exon Intron Grenzen wurden mit Hilfe des mRNA-to-genomic alignment Programms
Spidey bestimmt und Primer mit Hilfe des Primer3 Programms in die intronischen
Sequenzen gelegt Die SNPs wurden an den DNA-Sequenzen von Elterntieren der
betroffenen Dalmatinerfamilien identifiziert Die Amplifikate wurden mit Hilfe des
MegaBACE 1000 sequenziert und auf SNPs untersucht Bei Vorliegen eines oder
mehrerer SNPs wurde die entsprechende DNA-Sequenz der zugehoumlrigen
Nachkommen ebenfalls amplifiziert und sequenziert Die Auswertung erfolgte mit
Hilfe des Sequencher 42 Programms
Kopplungsanalyse
Die Genotypen der neuen SNP Marker wurden unter Verwendung des Programms
MERLIN ausgewertet Die Daten wurden mit dem Softwarepaket SAS (Statistical
Analysis System Version 913) bearbeitet um das Markerset bezuumlglich
Allelfrequenzen beobachteten Heterozygotiegrad und PIC (Polymorphism
information content) charakterisieren zu koumlnnen Des Weiteren wurde auf
Kopplungsungleichgewicht und die Assoziation der Haplotypen mit CCSD mittels
Erweiterte Zusammenfassung 137
des Programms CASECONTROL und HAPLOTYPE von SAS Genetics (Statistical
Analysis System Version 913 Cary NC USA) getestet
Ergebnisse und Diskussion Fuumlr acht Kandidatengene wurden mindestens zwei heterozygote SNPs entwickelt
Die nicht-parametrische Kopplungsanalyse zeigte keine signifikanten Teststatistiken
Offensichtlich war kein Haplotyp mit der caninen kongenitalen sensorineuralen
Taubheit (CCSD) in den untersuchten Familien assoziiert Da sowohl uni- und
bilateral taube Tiere als auch beidseitig houmlrende Tiere uumlber identische Haplotypen
verfuumlgen ist es unwahrscheinlich dass die Gene TMC1 TMIE USH1C MYH14
MYO3A PRES WHRN und ESPN an der Entstehung der CCSD beim Dalmatiner
beteiligt sind
Molekulargenetische Analyse des caninen myosin heavy polypeptide 9 non-muscle (MYH9) auf dem caninen Chromosom 10q232
Material und Methoden Klonierung und Sequenzierung von caniner MYH9 cDNA
Die canine MYH9 Gensequenz wurde mit Hilfe der BLAST Suche aus
veroumlffentlichten Sequenzen der NCBI Datenbank Die Isolierung der vollstaumlndigen
cDNA wurde durch ein Protokoll zur Amplifizierung von cDNA-Enden (rapid
amplification of cDNA ends [RACE]) erreicht Nach Klonierung und Sequenzierung
der RACE-Produkte wurden die Sequenzen mit Hilfe des Sequencher 42
Programms ausgewertet
Sequenzanalyse des caninen MYH9 Gens
Nach Fertigstellung des RACE Protokolls und Auswertung der Sequenzen wurden
die Exon Introngrenzen mit Hilfe des mRNA-to-genomic alignment Programms
Spidey lokalisiert Weiterhin wurden repetitive Elemente mit dem Programm
Repeatmaster 2 sowie der GC Gehalt mit der EBI toolbox CpG PlotCpGreport
ermittelt
Erweiterte Zusammenfassung
138
Mutatiosanalyse
Zur Identifizierung von Polymorphismen innerhalb der caninen MYH9 Sequenz
wurden die Exons mit ihren flankierenden intronischen Sequenzen mit Hilfe der PCR
amplifiziert und sequenziert Verwendet wurden hierfuumlr die DNA von insgesamt 16
Dalmatinern aus drei Familien Mindestens zwei der Vollgeschwister einer Familie
waren unilateral taub Der Phaumlnotyp der verwendeten Tiere wurde durch einen
audiometrischen Test bestimmt
PCR Primer wurden mit Hilfe des Primer3 Programms entwickelt PCR Reaktionen
fuumlr saumlmtliche Exons des MYH9 Gens mit ihren flankierenden Sequenzen wurden
durchgefuumlhrt die PCR Produkte sequenziert und die Sequenzen mit Hilfe des
Sequencher 42 Programms ausgewertet
Unter Verwendung der Software MERLIN (multipoint engine for rapid likelihood
inference Version 0910) wurde nach signifikanten nicht parametrischen LOD-
Scores fuumlr die Kosegregation von Markerallelen mit der phaumlnotypischen Auspraumlgung
der sensorineuralen Taubheit beim Dalmatiner gesucht
Ergebnisse und Diskussion
Analyse der genomischen Organisation und der cDNA des caninen MYH9 Gens
Eine vollstaumlndige cDNA Sequenz des caninen MYH9 Gens von 7484 bp konnte uumlber
die Reverse Transkription-PCR erhalten und bei der EMBL Nukleotid Datenbank
eingereicht werden Das canine MYH9 Gen besteht aus 41 Exons einschlieszliglich
einem untranslatierten ersten Exon und Exon Intron Grenzen die stets der GT AG
Regel folgen Der 5rsquo-UTR Bereich besteht aus 181 bp waumlhrend der 3rsquo-UTR aus 1432
bp besteht Weiterhin besitzt die canine MYH9 cDNA Sequenz einen offenen
Leserahmen von 5889 bp welcher ein Protein kodiert bestehend aus 1963
Aminosaumluren Die Groumlszlige der Exons variiert von 28 bis 1556 bp waumlhrend das
gesamte MYH9 Gens ungefaumlhr 90 kb umfasst Die Homologie der Sequenzen
zwischen dem humanen murinen und caninen MYH9 Gen ist limitiert auf die
kodierende Sequenz von Exon 2 bis 41
Mutations- und Haplotypenanalyse
Alle kodierenden Exons mit ihren flankierenden intronischen Sequenzen des MYH9
Gens konnten von den 16 Dalmatinern amplifiziert werden Die Sequenzen wurden
mit den entsprechenden Sequenzen vom Boxer aus der NCBI Genbank verglichen
Erweiterte Zusammenfassung 139
Die Suche nach Sequenzvariationen im MYH9 Gen lieszlig 22 SNPs erkennen wobei
die meisten Polymorphismen in den flankierenden Regionen der Exons gefunden
wurden Keiner der beobachteten Polymorphismen in den Exons aumlnderte die
Aminosaumluresequenz von MYH9 Auch zeigten die identifizierten Haplotypen keine
Assoziation mit dem CCSD Phaumlnotyp Multipoint Teststatistiken fuumlr den Anteil von
IBD-Markerallelen (Zmean) und ein daraus abgeleiteter LOD-Score lagen bei
annaumlhernd Null und waren nicht signifikant Zwei genassoziierte Mikrosatelliten
zeigten eine Kopplung diese kann jedoch mit Heterogenitaumlt zwischen den Familien
erklaumlrt werden
Fazit
Die Charakterisierung des Transkriptes und der genomischen Sequenz des caninen
MYH9 Gens zeigten einen konservierten Aufbau des Gens im Bezug auf das
humane orthologe Gen Aufgrund eines untranslatierten Exons im 5rsquo-UTR Bereich ist
das canine Gen groumlszliger als das humane Gen
Sowohl uni- oder bilateral taube und bilateral houmlrende Dalmatiner weisen identische
Haplotypen auf Daher sind in den untersuchten Dalmatinerfamilien die gefundenen
Polymorphismen offensichtlich nicht mit CCSD assoziiert Des Weiteren zeigt diese
Studie dass keine funktionellen Mutationen der vollstaumlndigen kodierenden Region
von MYH9 vorliegen Wir koumlnnen also abschlieszligend das MYH9 Gen als
Kandidatengen fuumlr die congenitale sensorineurale Taubheit beim Dalmatiner
ausschlieszligen
Erweiterte Zusammenfassung
140
Evaluierung von Mikrosatelliten und SNP Markern der caninen Chromosomen 1 10 und 31 auf Kopplung und Assoziation mit der caninen kongenitalen Taubheit Material und Methoden Pedigreematerial
Insgesamt wurden 176 Dalmatiner in die Untersuchung einbezogen Die Tiere
verteilten sich auf 23 unverwandte Vollgeschwisterfamilien und eine paternale
Halbgeschwistergruppe Die genotypisierten Familien bestanden aus saumlmtlichen
tauben Nachkommen (unilateral oder bilateral taub) ihren Eltern und einen bis vier
nicht betroffenen Voll- bzw Halbgeschwistern Mindestens zwei der Vollgeschwister
einer Familie waren unilateral oder bilateral taub Der Phaumlnotyp der verwendeten
Tiere wurde durch einen audiometrischen Test bestimmt
Alle 176 Dalmatiner wurden fuumlr die Mikrosatellitenstudie auf den caninen
Chromosomen 1 10 und 31 einbezogen Fuumlr die Feinkartierung mit Hilfe der SNPs
wurden dagegen nur noch die Dalmatinerfamilien mit einer eindeutigen Kopplung zu
der entsprechenden genomischen Region untersucht Fuumlr die Region auf CFA10
wurden dafuumlr 34 Tiere aus fuumlnf Vollgeschwisterfamilien ausgewaumlhlt und zur
Untersuchung des CLDN14 Gens auf CFA31 wurden 24 Tiere aus vier
Vollgeschwisterfamilien und 12 Tiere aus einer groszligen Halbgeschwisterfamilie
verwendet Fuumlr die Suche nach Polymorphismen im GJA1 Gen wurden 16 Tiere aus
einer groszligen Halbgeschwisterfamilie verwendet
Mikrosatellitenstudie
Fuumlr die Mikrosatellitenstudie von CFA1 10 und 31 wurden insgesamt 60 Marker
verwendet Mit jeweils 27 Mikrosatelliten wurde CFA1 und mit 27 Mikrosatelliten
wurde CFA10 vollstaumlndig abgedeckt Insgesamt 6 Marker wurden fuumlr CFA31
verwendet
Alle hier verwendeten Mikrosatelliten sind der NCBI Datenbank entnommen und
wurden uumlber PCR und Polyacrylamidgelelektrophorese ausgewertet
Identifizierung von SNPs (Single Nucleotide Polymorphisms)
Zur weiteren Abklaumlrung und zum Ausschluss falsch positiver Ergebnisse wurden
zusaumltzlich zu den Mikrosatelliten SNPs in den signifikanten Regionen auf CFA1 10
Erweiterte Zusammenfassung 141
und CFA 31 entwickelt und an den Familien die eine Kopplung zeigten
genotypisiert
Die caninen genomischen Sequenzen der untersuchten Gene wurden der NCBI
Datenbank (Boxer genome assembly 21) entnommen Exon Intron Grenzen wurden
bestimmt und Primer entwickelt Nach Durchfuumlhrung der PCR wurden die Amplifikate
mit Hilfe des MegaBACE 1000 sequenziert und auf SNPs untersucht Die
Auswertung erfolgte mit Hilfe des Sequencher 42 Programms
Die Genotypen der neuen SNP Marker wurden zu den Genotypen der fuumlr die
Kopplungsanalyse verwendeten Mikrosatelliten hinzugefuumlgt und gemeinsam unter
Verwendung des Programms MERLIN ausgewertet
Kopplungsanalyse
Unter Verwendung der Software MERLIN (multipoint engine for rapid likelihood
inference Version 0910) wurde nach signifikanten nicht parametrischen LOD-
Scores fuumlr die Kosegregation von Markerallelen mit der phaumlnotypischen Auspraumlgung
der sensorineuralen Taubheit beim Dalmatiner gesucht
Die Daten wurden mit dem Softwarepaket SAS (Statistical Analysis System Version
913) bearbeitet um die Marker bezuumlglich Allelfrequenzen beobachteten
Heterozygotiegrad und PIC (Polymorphism information content) charakterisieren zu
koumlnnen Des Weiteren wurde das Kopplungsungleichgewicht und die Assoziation der
Haplotypen mit CCSD mittles des Programms CASECONTROL und HAPLOTYPE
von SAS Genetics getestet
Ergebnisse und Diskussion CFA1
In einer groszligen Halbgeschwisterfamilie wurde eine signifikante Kopplung zu einem
GJA1 genassozierten Marker festgestellt Mit Hilfe der DNA von Tieren aus dieser
Halbgeschwisterfamilie wurde eine Sequenzanalyse des GJA1 Gens durchgefuumlhrt
Es wurden 3 SNPs im 3acute-untranslatierten Bereich gefunden Die identifizierten
Haplotypen zeigten keine Assoziation mit dem CCSD Phaumlnotyp Sowohl uni- oder
bilateral taube und bilateral houmlrende Dalmatiner wiesen identische Haplotypen auf
Daher sind in den untersuchten Dalmatinerfamilien die gefunden Polymorphismen
offensichtlich nicht mit CCSD assoziiert Auch wurde keine funktionelle Mutation in
der vollstaumlndigen kodierenden Region von GJA1 gefunden wurde Es ist daher
Erweiterte Zusammenfassung
142
unwahrscheinlich dass das GJA1 Gen an der Entwicklung der CCSD in der
untersuchten Halbgeschwisterfamilie beteiligt ist Die signifikante Kopplung der
GJA1-benachbarten Mikrosatelliten mit CCSD kann durch ein eng gekoppeltes Gen
hervorgerufen worden sein welches fuumlr die Entstehung der Taubheit in der
untersuchten Halbgeschwisterfamilie verantwortlich ist oder durch ein falsch-
positives Ergebnis der vorhergehend durchgefuumlhrten Mikrosatellitenstudie
Eine nicht-parametrische Kopplungsanalyse mit Hilfe von 27 Mikrosatelliten Marker
an 24 deutschen Dalmatinerfmilien wurde durchgefuumlhrt um abzuklaumlren ob andere
Genombereiche auf CFA1 signifikante Teststatistiken in mehr Familien zeigen Die
Kopplungsanalyse zeigte keine signifikanten Teststatistiken Es ist daher
unwahrscheinlich dass auf CFA1 Gene fuumlr die Entwicklung von CCSD in den
untersuchten Familien der aus Deutschland stammenden Dalmatiner verantwortlich
sind
Da kuumlrzlich festgestellt wurde dass das GJA1 Gen auch beim Menschen nicht fuumlr
eine Form der sensorineuralen nicht-syndromischen Taubheit verantwortlich ist
sondern an einer syndromischen Krankheit beteiligt ist die mit konduktiver Taubheit
einhergehen kann sollte das GJA1 Gen nicht laumlnger als ein Kandidatengen fuumlr
CCSD angesehen werden
CFA31
Auf CFA31 wurde durch eine nicht-parametrische Kopplungsanalyse von
Mikrosatelliten eine Kopplung eines CLDN14-benachbarten Markers mit der
sensorineuralen Taubheit beim Dalmatiner festgestellt Daraufhin wurden zusaumltzlich
zu den Mikrosatelliten acht SNPs entwickelt indem das einzige beim Menschen
translatierte Exon weite intronische Sequenzen sowie 5rsquo- und 3rsquo- untranslatierte
Bereiche des CLDN14 Gens einer Sequenzanalyse unterzogen wurden Nur zwei der
identifizierten SNPs waren polymorph in den untersuchten fuumlnf Dalmatinerfamilien
Sowohl uni- als auch bilateral taube und bilateral houmlrende Dalmatiner teilten
identische Haplotypen daher konnte keine Kosegregation mit tauben Dalmatinern
festgestellt werden
Da bislang keine funktionelle Mutation in der kodierenden Region von CLDN14
gefunden wurde ist es fuumlr eine eindeutige Aussage uumlber die Rolle des CLDN14
Gens im Entstehungsprozess der kongenitalen sensorineuralen Taubheit
Erweiterte Zusammenfassung 143
beim Dalmatiner notwendig noch weitere Marker im CLDN14 Gen sowie seiner
naumlheren Umgebung zu entwickeln und zu analysieren Da keine weitere Kopplung zu
einem Mikrosatelliten distal oder proximal des CLDN14 Gen festgestellt werden
konnte ist es wahrscheinlich dass entweder das CLDN14 Gen oder ein Gen in
seiner naumlheren Umgebung fuumlr die Entwicklung der sensorineuralen Taubheit in den
untersuchten Dalmatinerfamilien verantwortlich ist
CFA10
Auf CFA10 wurde durch eine nicht-parametrische Kopplungsanalyse von 27
Mikrosatelliten eine 12 Mb umfassende genomische Region mit einer signifikanten
Kopplung zur CCSD identifiziert Zur weiteren Feinkartierung wurden 26 neue SNPs
mit einem durchschnittlichen Abstand von 03 Mb fuumlr diesen Bereich entwickelt
Die SNPs waren groumlszligtenteils informativ im vorliegenden Familienmaterial und
bestaumltigten in einer folgenden Kopplungsanalyse die Lokalisation auf CFA10
deutlich Weiterhin konnte die gekoppelte Region auf einem 5 Mb umfassenden
Bereich von 39 bis 44 Mb eingegrenzt werden Es ist wahrscheinlich dass die
identifizierte CCSD Region ein Gen beinhaltet welches auf die Entstehung der
caninen kongenitalen Taubheit einwirkt
Es ist notwendig mehr SNPs zu entwickeln um das taubheitsverursachende Gen zu
lokalisieren oder einen eindeutig assoziierten SNP zu identifizieren
Chapter 12
References
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MELDRIM J MELNIKOV A MENEUS L MIHALEV A MIHOVA T MILLER K
MITTELMAN R MLENGA V MULRAIN L MUNSON G NAVIDI A
NAYLOR J NGUYEN T NGUYEN N NGUYEN C NGUYEN T NICOL R
NORBU N NORBU C NOVOD N NYIMA T OLANDT P ONEILL B
ONEILL K OSMAN S OYONO L PATTI C PERRIN D PHUNKHANG P
PIERRE F PRIEST M RACHUPKA A RAGHURAMAN S RAMEAU R
RAY V RAYMOND C REGE F RISE C ROGERS J ROGOV P SAHALIE J
SETTIPALLI S SHARPE T SHEA T SHEEHAN M SHERPA N SHI J
SHIH D SLOAN J SMITH C SPARROW T STALKER J STANGE-
THOMANN N STAVROPOULOS S STONE C STONE S SYKES S
TCHUINGA P TENZING P TESFAYE S THOULUTSANG D
THOULUTSANG Y TOPHAM K TOPPING I TSAMLA T VASSILIEV H
VENKATARAMAN V VO A WANGCHUK T WANGDI T WEIAND M
WILKINSON J WILSON A YADAV S YANG S YANG X YOUNG G YU Q
ZAINOUN J ZEMBEK L ZIMMER A LANDER ES (2005) Genome sequence
comparative analysis and haplotype structure of the domestic dog Nature
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GANGAROSSA S CARIDI G BORDO D LO NIGRO C GHIGGERI GM
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Chapter 13
Appendix
Appendix microsatellite marker III
HE
Te
79
5 7
98
76
9 5
12
71
3 8
33
70
2 4
35
72
7 7
00
54
6 4
77
48
8 3
08
44
8 5
40
67
4 8
05
76
2 6
54
70
8 7
73
78
1 6
87
65
4 6
26
51
9 5
89
76
4 1
60
PIC
d
72
2 8
41
67
0 4
85
67
1 7
85
57
2 4
07
61
4 6
29
35
8 3
30
40
0 2
90
35
0 4
60
70
0 7
90
76
2 6
21
63
8 7
26
77
1 6
26
60
1 6
13
38
7 5
52
80
0 1
40
Prim
er (r
ever
se)
5acute -gt
3acute
AA
CTG
TTTG
ATT
TTG
ATG
AG
GC
C
TTTG
ACC
TAC
ATA
TAA
AA
CA
AG
C
CA
CG
AA
GAA
AG
CC
ATG
GTT
T C
AA
CA
ATT
CC
TTTG
TTG
GTG
C
CTG
CC
AG
CTT
CC
TAC
AGC
T
GTG
GTC
ACA
AG
AC
TTTA
GC
C
CTG
CA
CC
AAA
GA
AA
CTC
C
GG
AC
CTA
TTC
TGA
AG
CC
TAA
C
TG
GC
CTT
TAA
TTTA
TCAT
GG
AG
A
TCA
GG
CC
CA
GA
TAG
TATG
C
TC
CTT
TGG
TTTT
TAG
CAG
GG
T
CTG
TGC
CC
AC
CTG
TGG
AG
C
AA
GA
GG
ATA
CC
GG
TGG
CA
G
CG
TGC
TTTG
TTA
TGG
CTT
GA
C
AG
CA
TGA
AG
GA
TCTC
TGA
CTA
A
CTA
AC
TCG
CC
AG
AC
TATT
C
CC
CTC
CAC
CA
ATC
TCTT
CA
C
GC
AA
AG
GTG
TATT
TAC
ATG
ATG
G
GG
CA
GC
CTT
ATT
ATT
CA
TGG
A
AC
AC
AG
GC
AC
AG
GA
GC
ATC
C
AG
CC
ATC
CA
GG
AA
ATC
G
CTG
AA
CTG
GTC
CC
TCAA
GC
A
GTC
ATG
CTG
ATT
TCTG
TGC
C
GA
CC
TATC
CA
TAC
ATG
CC
C
GAA
GG
AAG
GAA
GG
AAG
AAAA
GA
TTA
GTA
AA
GA
GA
CA
GC
TGA
TTG
CC
A
TCTA
CC
CTG
CA
ACC
CTG
TG
ATT
GA
GA
CC
CA
AG
ACTG
TTA
GTG
G
GTA
AC
AAC
CA
GAG
TGTG
TGTG
A
CG
TCG
AG
CTC
CTG
GC
AT
Prim
er (f
orw
ard)
5acute -
gt 3acute
GG
GTA
GA
TTTC
AA
CA
AAT
AG
TAC
TGG
T
AG
AA
AG
GC
CTG
GA
TGTC
G
AG
TAG
AA
GA
GG
CC
AC
GC
AA
A
TTT
TCC
AA
AA
TGA
AA
AC
CTG
C
AG
ATT
GTT
TATG
CA
GG
CA
TTG
A
GA
GTT
GAA
AG
GTT
GAA
AA
TGG
G
CTC
AG
AG
GC
AG
ATA
GA
AA
A
ATT
GA
TTC
ATG
ACC
CA
CTA
A
TTT
TGTC
AAG
CA
GC
CC
TATA
TC
TG
AA
ATG
GTA
CTT
CA
GC
ATC
G
AG
CTT
AG
CTT
AC
TGC
CC
TGG
T
CC
CC
GG
CC
CC
TGC
CC
GG
AG
T
AC
CAC
TGTC
ATT
TTTC
CA
TGC
C
CC
TTA
GG
AGG
AG
GC
AAG
AC
C
CA
AG
GG
GTA
TGTT
GTC
TATT
ACT
GG
GTG
GG
AA
ATG
TGA
CTC
TG
GG
GC
TGC
CC
ATT
TCTT
TAA
T T
TGC
CA
CAA
ATC
AC
TTAA
AG
G
GA
CTG
AG
TTC
TTTC
AG
CA
CA
GTG
A
AAAT
AAC
ACAG
CAT
CAG
G
GA
ATC
CC
CA
ATG
TAC
ATG
GC
A
AC
AG
GC
GG
ATT
CTG
TAG
C
AC
TGG
CC
AAA
GA
GTA
CA
AG
G
CC
TTG
AC
TCA
GC
AGC
CC
TAC
A
AA
GA
TTG
TCTT
GA
CAC
GC
TG
GG
GTC
CTG
GG
ATC
AA
GC
C
AC
TGA
CTG
ATG
TCC
TGTG
CG
A
AA
AA
GTG
TAG
AG
CTT
TCTT
CA
AA
T
AA
ATT
CA
ATA
AG
CC
ATG
GA
GG
A
CC
TCC
AAG
ATG
GC
TCTT
GA
ATc
(degC
) 60
58
58
56
58
60
54
56
60
60
58
60
60
60
60
60
60
60
60
52
56
58
54
62
60
60
62
62
60
60
Pro
duct
si
ze (b
p)
380-
400
337-
409
267-
304
225-
235
312-
336
350-
394
155-
161
126-
156
510-
570
436-
468
189-
191
72-8
0 13
0-13
6 34
0-35
0 14
5-15
3 98
-102
22
0-24
0 21
2-24
0 34
7-40
3 32
5-34
1 25
1-27
9 32
5-36
1 19
7-24
5 30
9-40
9 34
1-37
5 21
5-23
1 30
8-31
6 16
2-17
4 46
8-50
2 18
2-19
0
Alle
les
6 14
8 4 7 11
3 6 8 7 2 2 3 4 2 3 5 8 12
7 8 7 14
10
9 7 3 3 15
2
Mbb
54
1
49
19
8 2
18
23
5 2
47
26
0 3
02
35
3 3
98
58
8 6
26
63
9 7
61
77
5 7
78
78
6 7
98
85
4 1
060
1
090
1
100
1
100
1
110
1
130
1
150
1
170
3
5
12
4 1
63
CFA
a
1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 10
10
10
Tabl
e 1
Cha
ract
eris
tics
of m
icro
sate
llite
mar
kers
sel
ecte
d fo
r CFA
1 1
0 an
d 31
com
pris
ing
in to
tal 6
0 m
arke
rs
(Con
tinue
d on
nex
t pag
e) T
he m
icro
sate
llite
mar
kers
wer
e al
l der
ived
from
the
NC
BI d
atab
ase
(http
w
ebn
cbin
lmn
ihg
ov)
Mar
ker
FH33
25
FH36
03
FH23
13
RE
N18
9I14
FH
3219
FH
3413
R
EN
136G
19
RE
N13
5K06
FH
3922
FH
3300
C
014
24
AH
T107
C
012
51
RE
N17
2C02
D
0150
5 R
EN
04H
08
RE
N15
9F24
FH
3894
FH
2309
R
EN
06N
11
FH23
26
FH33
22
FH35
05
FH25
98
FH26
34
FH22
94
RE
N22
9P15
FH
2537
FH
4081
C
107
81
Appendix microsatellite marker
IV
H
ETe
64
0 6
80
63
0 6
51
54
6 7
06
81
0 6
14
30
2 7
42
34
4 7
40
63
0 8
67
82
2 6
70
75
0 6
80
60
0 4
70
31
0 5
70
80
1 5
80
83
1 5
10
64
0 6
90
19
0 8
22
PIC
d
53
0 6
90
52
2 5
89
80
0 6
70
75
9 5
24
24
7 7
09
26
9 6
90
50
0 7
98
75
5 5
50
69
0 5
30
56
0 3
40
26
0 5
00
80
1 5
30
74
0 3
70
64
0 6
70
25
0 7
21
Prim
er (r
ever
se)
5acute -gt
3acute
AG
GC
CA
GC
AC
CA
TAA
CTC
AG
C
CTC
TCC
TCC
TTC
CC
CTT
CTC
A
GTG
CTG
TCC
ATT
GC
AA
GTC
T
GC
TCC
CC
TGTG
TTC
TGAG
G
CTG
GTG
ACTC
AGA
GAA
GTC
C
GG
AG
TTG
ATA
GTA
AA
TCTT
TGC
GG
T
GC
CC
TATT
CA
TTC
AA
CTG
G
AC
AC
TCTG
GTG
CA
AG
CG
AC
TC
TTC
TAC
TGA
AG
CA
TCC
GC
CT
GA
TTTT
CTC
TCTG
TCC
AC
TT
AA
CA
GC
ATT
TCA
GAC
AGA
GG
G
GG
TTTT
GG
CTG
TGA
GG
ATA
G
AG
TTC
CAG
ATC
GAG
TCC
CA
C
CA
CAT
GAT
TTC
ACTT
GTA
TATG
G
CC
TGA
GG
GTT
TTTA
CAG
TATG
G
TG
AA
ATC
TGG
GA
TTC
TGA
TAC
C
CC
AA
GC
TTTC
AC
CTA
ATC
AC
C
GA
TATT
TTTC
TCTC
CC
AC
CC
C
AC
ATT
TCTA
GG
CAG
TGG
CA
C
TGTC
CC
ATC
TAA
AG
GA
TAG
G
AG
TAA
TGC
TGC
CC
ACG
GA
AC
G
GC
TGG
AA
CC
TTC
CC
CTT
TAG
A
GC
TCTT
ACA
CG
CA
TTG
AG
G
AA
AC
GG
AG
TTC
CA
TCTC
TGG
C
AC
AG
TGA
GG
AG
GG
GTG
G
TG
TAA
CC
CTC
CTG
TGA
TATG
A
TGTG
AA
CC
CC
GC
CC
AATA
G
AA
GTG
AC
TGA
GTA
CC
TGA
AA
TCG
G
CC
TCTG
CC
TCTG
CC
TCTG
T G
AG
CC
CTG
TTC
TCA
GG
TTG
Prim
er (f
orw
ard)
5acute -
gt 3acute
AG
AC
CC
CC
CC
ACTA
CC
CC
AC
GC
GTT
AGC
TGC
CA
TCA
TCTT
G
CTG
GTT
GG
AG
CA
ATA
CC
AA
G
CC
ATT
CA
TGTT
GTT
GC
AG
ATG
T
AA
GG
AG
GG
CA
CTT
GAT
GG
C
TTA
TGG
CA
TTTG
GC
CTG
TC
AA
GG
AG
CAT
CTT
CC
AG
AAC
C
CG
CTC
ATG
CA
AG
TCA
TCA
CA
T C
TGG
CTT
AAA
CC
AC
TGG
TCA
C
CTT
CC
ATC
CC
GTT
GTG
TGT
ATT
CC
CC
AGC
GAT
ACC
A C
TTG
AG
TGG
CTT
GA
GTG
GC
TAC
T
TCTT
GC
TTC
TTG
AA
GTA
AG
CC
T
TGC
CC
GTC
CTA
TAC
TCC
TG
GA
AG
GTG
GTT
ATT
GTC
CTT
GG
G
AA
CC
ACA
GA
AG
AG
CTG
GA
A
GC
ATT
TGA
TGA
AA
TAA
AG
GG
A
GA
ATG
AA
AA
CG
GA
GC
AG
CA
G
CC
TTC
AAC
AC
CC
ATA
GC
TC
TC
AG
CA
ACTA
TAC
ATT
TAA
GA
GC
A
ATG
GA
CA
AATG
AA
CA
AA
AG
T
GTG
AC
TTTC
TTA
TCC
GC
CC
C
CC
AG
AA
AC
TCA
ACTG
ATG
C
TG
GA
CG
CTA
AG
CC
TGAC
TTT
CC
ATA
AG
ATA
CTC
AG
AA
AC
ATG
CA
C
AC
CAT
AA
ATG
GA
TGG
ATA
G
CA
GTG
AG
CA
AA
GC
AA
ATG
AA
C
CC
ATT
AG
CA
AA
TGA
CTG
GG
A
AA
GA
ATG
GG
AA
AA
CTG
ATA
A
AA
GG
TAG
TCC
CA
CG
ATC
CTC
ATc
(degC
) 60
60
60
60
60
60
60
60
60
60
60
60
60
62
60
60
60
60
60
60
60
60
60
60
60
60
60
60
60
60
Pro
duct
si
ze (b
p)
100-
110
112-
124
194-
206
210-
230
309-
341
350-
362
276-
300
96-1
30
130-
138
120-
138
200-
204
278-
296
157-
165
178-
254
358-
368
327-
337
380-
404
160-
170
320-
330
165-
169
230-
232
300-
310
276-
380
255-
263
258-
278
106-
120
164-
180
568-
608
223-
239
172-
186
Alle
les
3 5 3 5 8 4 7 4 2 5 3 7 5 9 6 4 7 3 4 2 2 3 11
5 6 2 4 7 4 8
Mbb
24
9 3
23
34
9 3
67
38
9 3
94
40
1 4
10
45
8 4
64
48
0 4
88
51
5 5
64
56
8 5
97
62
7 6
33
63
7 6
53
67
3 6
85
69
1 7
08
0
66
9
7
28
1 2
96
37
5
CFA
a
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
10
31
31
31
31
31
31
Tabl
e 1
(con
tinue
d)
Mar
ker
RE
N06
H21
R
EN
68B
08
FH30
55
C10
769
FH
3302
R
EN
181G
20
FH34
03
G02
512
RE
N15
0B12
A
HT1
01
RE
N73
F08
ZUB
EC
A1
C10
16
FH24
22
FH34
48
FH26
65
DTR
105
C
106
02
RE
N16
1L12
C
0410
7 R
EN
91H
07
RE
N15
4O19
FH
3381
R
EN
154G
10
FH21
89
RV
C11
R
EN
43H
24
FH22
39
RE
N11
0K04
FH
2712
a Can
ine
chro
mos
ome
b Pos
ition
of m
arke
r (M
b) c A
nnea
ling
tem
pera
ture
d P
olym
orph
ism
info
rmat
ion
cont
ent (
)
e obse
rved
Het
eroz
ygos
ity
Appendix itemisation of alleles V
Table 2 Itemisation of alleles to bases for SNPs in the pedigrees
Code no 1 2 3 4
Base Adenine Cytosine Guanine Thymine
Appendix laboratory paraphernalia
VI
Laboratory paraphernalia Equipment Thermocycler
PTC-100trade Programmable Thermal Controller (MJ Research Watertown USA)
PTC-100trade Peltier Thermal Cycler (MJ Research Watertown USA)
PTC-200trade Peltier Thermal Cycler (MJ Research Watertown USA)
Automated sequencers
LI-COR Gene Read IR 4200 DNA Analyzer (LI-COR Inc Lincoln NE USA)
LI-COR Gene Read IR 4300 DNA Analyzer (LI-COR Inc Lincoln NE USA)
MegaBACE 1000 (Amersham Biosciences Freiburg)
Centrifuges
Sigma centrifuge 4-15 (Sigma Laborzentrifugen GmbH Osterode)
Desk-centrifuge 5415D (Eppendorf Hamburg)
Biofuge stratos (Heraeus Osterode)
Centrifuge Centrikon H 401 (Kontron Gosheim)
Megafuge 1OR (Heraeus Osterode)
Speed Vacreg Plus (Savant Instruments Farmingdale NY USA)
Agarose gel electrophoresis and pulsed field gel electrophoresis
Electrophoresis chambers OWL Separation Systems Portsmouth NH USA
Biometra Goumlttingen
BioRad Muumlnchen
Generators 2301 Macrodrive 1 (LKB Bromma Sweden)
Power Pac 3000 (BioRad Muumlnchen)
Gel documentation system BioDocAnalyze 312 nm Goumlttingen
Appendix laboratory paraphernalia VII
Others
Milli-Qreg biocel water purification system (Millipore GmbH Eschborn)
Incubator VT 5042 (Heraeus Osterode)
UV-Illuminator 312 nm (Bachhofer Reutlingen)
Centomatreg R Desk-Shaker (B Braun Melsungen AG Melsungen)
Biophotometer (Eppendorf AG Hamburg)
Kits
DNA purification
Montage PCR96 Cleanup Kit (Millipore GmbH Eschborn)
Cloning
Invitrogen TA cloningreg kit (Invitrogen Karlsruhe Germany)
Isolation of DNA
QIAamp 96 DNA Blood Kit (QIAGEN Hilden)
Plasmid Mini Prep 96 Kit (Millipore GmbH Eschborn)
Sequencing
ThermoSequenase Sequencing Kit (Amersham Biosciences Freiburg)
DYEnamic-ET-Terminator Cycle Sequencing Kit (Amersham Biosciences Freiburg
Germany)
RACE
FirstChoiceTM RNA ligase-mediated (RLM)-RACE kit (Ambion Europe Huntingdon
UK)
Appendix laboratory paraphernalia
VIII
RNA Total RNA (Biocat Heidelberg Germany)
Size standards
100 bp Ladder (New England Biolabs Schwalbach Taunus)
1 kb Ladder (New England Biolabs Schwalbach Taunus)
IRDyetrade 700 or 800 (LI-COR Inc Lincoln NE USA)
Reagents and buffers APS solution (10 )
1 g APS
10 ml H2O
Bromophenol blue solution
05 g bromophenol blue
10 ml 05 M EDTA solution
H2O ad 50 ml
dNTP solution
100 microl dATP [100 mM]
100 microl dCTP [100 mM]
100 microl dGTP [100 mM]
100 microl dTTP [100 mM]
1600 microl H2O
the concentration of each dNTP in the ready-to-use solution is 5 mM
Gel solution
1275 ml UreaTBE solution (Roth Karlsruhe)
225 ml Rotiphoresereg Gel 40 (38 acrylamide and 2 bisacrylamide)
95 microl APS solution (10 )
95 microl TEMED
Appendix laboratory paraphernalia IX
Loading buffer for agarose gels
EDTA pH 8 100 mM
Ficoll 400 20 (wv)
Bromophenol blue 025 (wv)
Xylencyanol 025 (wv)
Loading buffer for gel electrophoresis
2 ml bromophenol blue solution
20 ml formamide
TBE-buffer (1x)
100 ml TBE-buffer (10x)
900 ml H2O
TBE-buffer (10x)
108 g Tris [12114 M]
55 g boric acid [6183 M]
744 g EDTA [37224 M]
H2O ad 1000 ml
pH 80
UreaTBE solution (6 )
425 g urea [6006 M]
250 ml H2O
100 ml TBE-buffer (10x)
solubilise in a water bath at 65degC
H2O ad 850 ml
Appendix laboratory paraphernalia
X
Chemicals Agarose (Invitrogen Paisley UK)
Ammonium persulfate (APS) ge 98 (Sigma-Aldrich Chemie GmbH Steinheim)
Ampicillin (Serva Heidelberg)
Boric acid ge 998 pa (Carl Roth GmbH amp Co Karlsruhe)
Bromophenol blue (Merck KgaA Darmstadt)
dATP dCTP dGTP dTTP gt 98 (Carl Roth GmbH amp Co Karlsruhe)
Chloramphenicol (Serva Heidelberg)
DMSO ge 995 pa (Carl Roth GmbH amp Co Karlsruhe)
dNTP-Mix (Qbiogene GmbH Heidelberg)
EDTA ge 99 pa (Carl Roth GmbH amp Co Karlsruhe)
Ethidium bromide (Carl Roth GmbH amp Co Karlsruhe)
Ethyl alcohol (AppliChem Darmstadt)
Formamide ge 995 pa (Carl Roth GmbH amp Co Karlsruhe)
LB (Luria Bertani) agar (Scharlau Microbiology Barcelona Spain)
Paraffin (Merck KgaA Darmstadt)
RotiphoreseregGel40 (Carl Roth GmbH amp Co Karlsruhe)
SephadexTM G-50 Superfine (Amersham Biosciences Freiburg)
TEMED 99 pa (Carl Roth GmbH amp Co Karlsruhe)
Tris PUFFERANreg ge 999 pa (Carl Roth GmbH amp Co Karlsruhe)
Urea ge 995 pa (Carl Roth GmbH amp Co Karlsruhe)
Water was taken from the water purification system Milli-Qreg
X-Gal (AppliChem Darmstadt)
Enzymes Taq-DNA-Polymerase 5 Umicrol (Qbiogene GmbH Heidelberg)
Taq-DNA-Polymerase 5 U microl (Qiagen Hilden Germany)
Incubation Mix (10x) TPol with MgCl2 [15 mM] (Qbiogene GmbH Heidelberg)
The polymerase was always used in the presence of incubation Mix TPol 10x buffer
The encyme ECO RI (New England Biolabs Schwalbach Taunus) were used with
the adequate 10x encyme buffer
Appendix laboratory paraphernalia XI
Consumables
96 Well Multiply PCR plates skirted (Sarstedt Nuumlmbrecht)
Combitipsreg plus (Eppendorf AG Hamburg)
Pipette tips 01 ndash 10 microl 01 ndash 10 microl 5 ndash 200 microl (Carl Roth GmbH amp Co Karlsruhe)
Reaction tubes 15 and 20 ml (nerbe plus GmbH WinsenLuhe)
Reaction tubes 10 und 50 ml (Falcon) (Renner Darmstadt)
Thermo-fast 96 well plate skirted (ABgene Hamburg)
Software BLAST trace archive httpwwwncbinlmnihgov
httpwwwensemblorg
EBI toolbox httpwwwebiacukToolssequencehtml
MERLIN 0102 package httpwwwsphumicheducsgabecasisMerlin
Order of primers MWG Biotech-AG Ebersberg (httpsecom
mwgdnacomregisterindextcl)
biomersnet GmbH Ulm (orderbiomersnet)
PED50 Dr H Plendl et al (2005) Institute for Human Genetics
Kiel
Primer design httpfrodowimiteducgi-binprimer3primer3_ wwwcgi
Repeat Masker httpwwwrepeatmaskergenome washingtonedu
Sequencher 42 GeneCodes Ann Arbor MI USA
Spidey httpwwwncbinlmnihgovIEBResearch
OstellSpideyindexhtml
SUN Ultra Enterprise 450 Sun microsystems
XIII
List of publications Journal articles 1 MIESKES K WOumlHLKE A DROumlGEMUumlLLER C DISTL O 2006 Molecular
characterization of the canine myosin heavy polypeptide 9 non-muscle (MYH9)
gene on dog chromosome 10q232 Submitted for publication in Gene
2 MIESKES K DISTL O 2006 Evaluation of 17 single nucleotide
polymorphisms (SNPs) in six candidate genes for hereditary non-syndromic deafness
in Dalmatian dogs Submitted for publication in Journal of Heredity
3 MIESKES K DISTL O 2006 Evaluation of the TMC1 and TMIE genes as
candidates for hereditary non-syndromic deafness in Dalmatian dogs Submitted for
publication in Animal Genetics
XV
Acknowledgements
First of all I wish to thank Prof Dr Dr habil Ottmar Distl the supervisor of my
doctoral thesis for his academic guidance and support of this work
I am very thankful to Heike Klippert-Hasberg and Stefan Neander for technical
expertise and assistance
I am very grateful to Joumlrn Wrede for his support during the statistical analyses and his
help with computer problems
I wish to express my appreciation to the Gesellschaft zur Foumlrderung Kynologischer
Forschung (GKF) eV Germany for funding this work with a grant
I am appreciative to all Dalmatian breeders and owners for providing me blood
samples and the results of the BAER tests
My special thanks go to all colleagues and friends of the Institute for Animal Breeding
and Genetics of the University of Veterinary Medicine Hannover for their support
humour and the friendly atmosphere in the laboratory
Last but not least I wish to thank my family for their support during the work on this
thesis