Physical and comparative mapping of candidate genes on the ... · Physical and comparative mapping...

Aus dem Institut für Tierzucht und Vererbungsforschung der

Tierärztlichen Hochschule Hannover

___________________________________________________________________

Physical and comparative mapping

of candidate genes on the horse genome

INAUGURAL-DISSERTATION

zur Erlangung des Grades einer

DOKTORIN DER VETERINÄRMEDIZIN

(Dr. med. vet.)

durch die Tierärztliche Hochschule Hannover

vorgelegt von

Dorothee Stübs geb. Müller

aus Heidelberg

Hannover 2006

Scientific supervisor: Univ.-Prof. Dr. Dr. habil. O. Distl

Examiner: Univ.-Prof. Dr. Dr. habil. O. Distl

Co-Examiner: Prof. Dr. Dr. habil. H. Niemann

Oral examination: 23.11.2006

This work was supported by grants from the German Research Council, DFG, Bonn,

Germany (DI 333/12-1).

To my beloved husband and our son

Parts of this work have been accepted for publication or have been published in the

following journals:

1. Animal Genetics

2. Cytogenetic and Genome Research

3. Archives for Animal Breeding

Table of contents

Chapter 1

Introduction 13

Chapter 2

Mapping the horse genome and its impact on equine genomics

for identification of genes for monogenic and complex traits 17

Abstract 19

Zusammenfassung 20

Introduction 20

Organization of the horse genome 21

Cytogenetic map 22

Synteny map/ Radiation hybrid map 22

Comparative map 23

Linkage map 25

Chromosomal abnormalities 26

Coat colours 26

Equine genetic diseases and the development of gene tests 31

Monogenic equine diseases 31

Genetically complex equine diseases 33

Conclusions and perspectives 36

References 37

Chapter 3

Assignment of the COMP gene to equine chromosome 21q12-q14

by FISH and confirmation by RH mapping 53

Source/description 55

Primer sequences 56

Chromosomal location 56

Radiation hybrid mapping/ PCR conditions 57

Comment 57

Acknowledgements 57

References 57

Figure 58

Chapter 4

Physical mapping of the PTHR1 gene to equine chromosome 16q21.2 59


Primer sequences 62


Radiation hybrid mapping/ PCR conditions 63

Comment 63

Acknowledgements 64

References 64

Figure 65

Chapter 5

Assignment of BGLAP, BMP2, CHST4, SLC1A3, SLC4A1, SLC9A5

And SLC20A1 to equine chromosomes by FISH and

confirmation by RH mapping 67


BAC library screening/ sequence analysis 71


Radiation hybrid mapping 72

Comment 73

Acknowledgements 73

References 73

Figures 74

Table 1 77

Table 2 80

Chapter 6

Physical mapping of the ATP2A2 gene to equine chromosome

8p14->p12 by FISH and confirmation by linkage and RH mapping 83

Rationale and significance 85

Materials and methods 85

BAC isolation and characterization of the equine ATP2A2 clone 85

PCR conditions and microsatellite analysis 87

Chromosome preparation 87

Fluorescence in situ hybridization 87

Radiation hybrid (RH) mapping 88

Linkage mapping 89

Results and discussion 89

Polymorphism 89


Acknowledgements 90

References 90

Figure 91

Table 92

Chapter 7

Assignment of the COL8A2 gene to equine chromosome 2p15-p16



Primer sequences 96

Chromosome location 96

Radiation hybrid mapping 97

Comment 97

Acknowledgements 98

References 98

Figure 98

Chapter 8

Assignment of the TYK2 gene to equine chromosome 7q12-q13


Background 101

Procedures 101

Results 103

Acknowledgements 103

References 104

Figure 104

Chapter 9

Assignment of CART1, COL9A3, GNRH2 and TCIRG1 to equine

chromosomes by FISH and confirmation by RH mapping 105

Background 107

Procedures 109

Results 110


References 112

Figures 115

Tables 117

Chapter 10

General discussion 119

Chapter 11

Summary 127

Chapter 12

Zusammenfassung 131

Appendix 139

List of publications 149


List of abbreviations

A adenine

BAC bacterial artificial colony

BLAST basic local alignment search tool

BLASTN basic local alignment search tool nucleotide

bp base pairs

C cytosine

cDNA complementary desoxyribonucleic acid

cen centromer

CHORI Children`s Hospital Oakland Research Institute

cM centiMorgan

cR centiRay

DAPI 4`,6`-diaminidino-2-phenylindole

DFG Deutsche Forschungsgemeinschaft (German Research Council)

DMSO dimethyl sulfoxide

DNA deoxyribonucleic acid

dNTPs deoxy nucleoside 5`triphosphates (N is A, C, G or T)

ECA Equus caballus autosome

EDM multiple epiphyseal dysplasia

EMBL European Molecular Biology Laboratory

F forward

FISH fluorescence in situ hybridisation

G guanine

GTG giemsa trypsin giemsa

HSA Homo sapiens autosome

IMAGE integrated molecular analysis of genomes and their expression

INRA Institut National de la Recherche Agronomique

ISCNH international system for chromosome nomenclature of the domestic horse

kb kilobase

LOD logarithm of the odds

Mb megabase

MED multiple epiphyseal dysplasia

mRNA messenger ribonucleic acid

MS microsatellite

NaH2PO4 Natriumdihydrogenphosphat

Na2HPO4 Dinatriumhydrogenphosphat

NCBI National Center for Biotechnology Information

no. number

NPL nonparametric linkage

OC osteochondrosis

OCD osteochondrosis dissecans

OMIM online mendelian inheritance in man

PCR polymerase chain reaction

POS position

QTL quantitative trait locus

R reverse

RET retention frequency

RH radiation hybrid

RZPD Resource Center/Primary Database, Berlin

SSC sus scrofa chromosome

STS sequence-tagged site

T thymine

TAMU Texas A & M University

TBE tris-borate-ethylenediamine tetraacetic acid

TE tris-ethylenediamine tetraacetic acid

Ta annealing temperature

UV ultraviolet

X-Gal 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside

Chapter 1

Introduction

Introduction

- 15 -

Introduction

The horse gene map was - compared with other farm animals - still in its infancy at

the beginning of this work, but it has quickly developed during the last few years.

Today, genome investigation in horses is aimed at the decoding of the whole horse

genome, as it has taken place for humans and several farm animals. Since the horse

has not only been used as a helpful worker during the last centuries, but has

developed into a dependable partner in equitation today, it is not surprising that the

bone and cartilage metabolism and affected diseases like osteochondrosis and

navicular disease play an interesting role in horse breeding in the meantime.

Osteochondrosis is a developmental orthopaedic disorder of growing animals. The

disease is accompanied by abnormal cartilage development in the joints of young

growing horses causing subchondral fractures, cysts, wear lines, chondromalacia or

cartilage flaps. It can also be manifested in the form of joint mice or free joint bodies

(chips) and is known as osteochondrosis dissecans (OCD). A number of articulations

can be affected, like the fetlock, hock, carpal, stifle, elbow, hip or vertebral joints. The

accumulation of the disease in several horse breeds and families (prevalence in

Warmblood and trotter horses between 10 and 79.5%) provides an indication of

involved hereditary factors, but responsible genes still have not been found until

today. Moreover, nutritional and endocrinological factors, skeletal growth rate and

biomechanical trauma seem to play an important role in Osteochondrosis; so the

disease appears to be multifactorial.

To contribute new results to the comparative human-equine gene map, altogether 17

candidate genes have been chosen. The objective of the present study is the

physical mapping of these candidate genes with the previously unknown localization

on the horse genome for the first time. Ten genes have been picked due to their

possible influence on osteochondrosis, five genes from different solute carrier

families, one gene as a candidate gene for pastern dermatitis. This work describes

the cytogenetical mapping of the candidate genes by FISH and the confirmation of

the localizations by radiation hybrid mapping and offers new findings, which are

partially surprising, in the end.

Introduction

- 16 -

Overview of chapter contents

The presented work consists of eleven chapters of which chapters 3-7 represent

already published articles.

Chapter 2 reviews the development and the actual situation of equine gene mapping

including physical gene maps, comparative gene maps, RH-maps, linkage maps and

describing the inheritance of coat colours and several hereditary diseases in horses.

Chapters 3-8 describe the physical mapping of 16 genes on the horse genome for

the first time.

In Chapter 8 and 9 the mapped genes that have not yet been published and the

mapping results are concluded and discussed.

Chapter 10 provides conclusions recapitulating chapters 3 to 9 and a general

discussion.

In Chapter 11 the whole work is summarized concisely, while Chapter 12 is a

detailed German summary.

All tables and figures belonging to the different chapters can be found at the end of

this work in the Appendix.

Chapter 2

Mapping the horse genome and its impact on equine

genomics for identification of genes for monogenic and

complex traits

D. Stübs and O. Distl

Institute for Animal Breeding and Genetics, University of Veterinary Medicine

Hannover, Foundation, Germany

Accepted for publication

In press: Archives for Animal Breeding

Mapping the horse genome and its impact on equine genomics for identification of genes for monogenic and complex traits

- 19 -

Mapping the horse genome and its impact on equine

genomics for identification of genes for monogenic and

complex traits

Abstract

Since the beginning of investigation in the horse genome in the early nineties, there

has been a great progress, especially during the last five years. At the beginning the

exploration of monogenic hereditary diseases was one of the main aims, and the

causal mutations of several diseases in the horse have been unravelled. The

inheritance of coat colours has been explored very detailed, and there exist gene

tests for different coat colours such as for chestnut, sabino and tobiano spotting,

silver dapple and cream dilution. Information about coat colours and inherited

diseases is very important for the breeders and helps avoiding the appearance of

lethal genetic factors or undesirable diseases. The most important achievements of

horse genome analysis were well-developed linkage, radiation hybrid and

cytogenetic genome maps including more than 2950 loci. These maps support

comparative analysis of equine hereditary diseases. The present known gene

mutations for five diseases in horses have human homologs. Studies on multifactorial

diseases such as osteochondrosis and navicular bone disease and on fertility and

temperament are underway. At the moment, the whole equine genome is sequenced

as it has been done for the human genome and also for other animal genomes.

Horse breeding will greatly benefit from identification of QTL for multifactorial traits

and gene mutations for congenital anomalies, diseases and performance traits.

Key words: Horse, genome, equine maps, coat colours, inherited diseases


- 20 -

Zusammenfassung

Titel der Arbeit: Kartierung des Pferdegenoms und Genomanalyse von

monogenen und komplexen Merkmalen beim Pferd

Seit den ersten Arbeiten zur Aufklärung von Genen beim Pferd in den 90er Jahren

wurden große Fortschritte in der Genomanalyse beim Pferd gemacht. Für fünf

monogen vererbte Krankheiten konnten die verantwortlichen Genmutationen

aufgeklärt werden. Die Vererbung der Fellfarben auf populationsgenetischer Ebene

ist beim Pferd weitgehend geklärt. Für einige Fellfarben, wie die Fuchsfarbe, die

Sabino- und Tobiano-Scheckung, den Silbergenort und Aufhellungsfaktor (Cream

Allel) am Albino Genort, konnten Gentests entwickelt werden. Diese Erkenntnisse

über die Farballele sind für die Pferdezucht wichtig, und Gentests für die Erkennung

von Anlageträgern helfen, das Auftreten von Erbfehlern und Krankheiten zu

vermeiden. Die bisher wichtigsten Fortschritte in der Genomanalyse waren die

Entwicklung von genetischen, zytogenetischen und Radiation Hybrid Karten, damit

Genorte für monogen und multifaktoriell vererbte Merkmale (QTL) kartiert und

anschließend vergleichend analysiert werden konnten. Bisher wurden mehr als 2950

Genloci auf diesen Karten kartiert. Arbeiten zu multifaktoriellen Krankheiten wie

Osteochondrose und Podotrochlose sowie zur Fruchtbarkeit sowie zu dem

Temperament sind auf dem Weg. Momentan wird das Pferdegenom komplett

sequenziert wie dies bereits für den Menschen und andere Haustiere durchgeführt

wurde. Die Pferdezucht wird davon für die Identifizierung von QTL und Aufklärung

von Genmutationen für angeborene Anomalien, Krankheiten und Leistungsmerkmale

erheblich profitieren.

Schlüsselwörter: Pferd, Genom, Genkarten, Fellfarben, erbliche Krankheiten

Introduction

The first animal genome sequence had been completed already when scientists

decoded the sequence of the nematode C. elegans (BETHESDA, 1998). The

exploration of mammalian genomes, especially in farm animals, proceeded very fast,

and there are several genomes of mammalian animals completed today (FADIEL et


- 21 -

al., 2005). Genome sequencing in other domestic animals, particularly in pets, forged

ahead in the last few years, too, because it was a matter of particular interest for

breeders. The chicken, dog, and cow genome have been wholly sequenced and

work has started in the pig and horse (BETHESDA, 2004).

Having the information on the complete genome sequence is the key on

understanding physiological and pathological processes and may revolutionize health

research. While scientific investigations had to be confined mainly to the analysis of

phenotypic data and pedigrees for familial diseases, today techniques include

fluorescence in situ hybridisation (JOHN et al., 1969), somatic cell hybrid panels and

radiation hybrid panels (STRACHAN and READ, 1996), comparative mapping

(O`BRIEN and GRAVES, 1991), sequence and mutation analysis, gene expression

using microarrays or quantitative real time techniques.

New information about mapped genes or markers is shortly posted on one of the

databases in the internet and is free available (HU et al., 2001).

The progress in investigation of the horse genome especially in the last ten years is

the result of concentrated and well-organized scientific work all over the world: 1995

was the year of the foundation of the International Equine Gene Mapping Workshop.

Since 1997 this foundation works together with the Horse Technical committee of the

National Research Sponsored Project-8 (NRSP-8) of the United States Department

of Agriculture National Animal Genome Research Program (USDA-NAGRP) with the

aim to decipher the horse genome as soon as possible.

Organization of the horse genome

The diploid genome of the domestic horse (Equus caballus) consists of 64

chromosomes, and 13 pairs of the autosomes are biarmed, while the other 18 pairs

of the autosomes are acrocentric. The X chromosome is large and metacentric, the Y

chromosome small and submetacentric. It is remarkable that the equus przewalski

genome consists of 66 chromosomes and that 12 pairs of its autosomes are

metacentric and 20 pairs are acrocentric. One pair of the horses` acrocentric

chromosomes (ECA5) contains two of the przewalski`s chromosomes (EPR23 and


- 22 -

EPR24), hence it is possible to mate przewalski and domestic horse and to produce

fertile offsprings (AHRENS and STRANZINGER, 2005).

Cytogenetic map

Physical identification of loci of interest can be achieved by fluorescence in situ

hybridisation (FISH). The first fluorescence in situ hybridisation in the horse was

reported by OAKENFULL et al. (1993) with the mapping of the alpha-globin gene

complex to horse chromosome (ECA) 13. BREEN et al. (1997) localized the first

equine markers when they mapped 18 microsatellites. RAUDSEPP et al. (1997)

cytogenetically mapped genes to the genome of the donkey. Lear et al. (2001),

RAUDSEPP et al. (2002), GODARD et al. (2000) used goat BAC clones to localize

44 coding sequences on the equine FISH map. LINDGREN et al. (2001) localized

another 13 genes by FISH and somatic cell hybrids. One of the first most extensive

FISH-map included 136 genes (MILENKOVIC et al. 2002), and to this cytogenetic

map further 165 genes were added by FISH and/or radiation hybrid (RH) mapping

(PERROCHEAU et al. 2005). The equine gene map now contains 713 genes, 441 on

the RH map, 511 on the cytogenetic and 238 on both maps.

Several of gene groups attracted special interest for mapping in different animals,

such as immunity-related genes: GUSTAFSON et al. (2003) constructed an ordered

BAC contig map of the equine major histocompatibility complex (MHC) and

TALLMADGE et al. (2003) mapped the ß2-microglobulin gene that is responsible for

the light chain of the MHC to ECA1q23-q25. MUSILOVA et al. (2005) mapped

another 19 horse immunity-related loci by FISH. NERGADZE et al. (2006) localized

the IL8 gene on ECA3q14. Others mapped genes related to the bone-, cartilage- and

collagene-metabolism. Genes eventually responsible for bone- or collagene-related

hereditary diseases in the horse were mapped by HILLYER et al. (2005), MÜLLER et

al. (2005), BÖNEKER et al. (2005) and DIERKS et al. (2006).

Synteny and radiation hybrid maps

Synteny mapping and radiation hybrid mapping in the horse work analogously to

those in humans, but the utilization of these techniques began more than 20 years


- 23 -

later than in humans. While there had been five synteny panels in the horse since

1992 (LEAR et al., 1992; WILLIAMS et al., 1993; BAILEY et al., 1995; RANEY et al.,

1998), the most important one was the UC Davis panel generated by SHIUE et al.

(1999). It included 450 markers and represents the origin for the developed horse-

human comparative maps (CAETANO et al., 1999).

A 3000-rad panel was developed by KIGUWA et al. (2000). Preliminary comparative

maps of horse chromosomes 1 to10 were created using this panel. A great progress

was the more extensive 5000-rad whole-genome-radiation hybrid panel that was

constructed using 92 horse x hamster hybrid cell lines and 730 equine markers (type

I and type II markers) (CHOWDHARY et al., 2003). This panel was one of the most

essential tools to construct high-resolution and comparative maps for ECA11

(CHOWDHARY et al. 2003), ECAX (RAUDSEPP et al., 2004), ECA17 (LEE et al.,

2004), ECA22 (GUSTAFSON-SEABURY et al. 2005), ECA4 (DIERKS et al., 2006),

equine homologs of HSA19 on ECA17, ECA10 and ECA21 (BRINKMEYER-

LANGFORD et al., 2005) and equine homologs of HSA2 on ECA6p, ECA15 and

ECA18 (WAGNER et al., 2006).

Comparative map

Comparative maps between horse and human and horse and mouse are the most

important ones. There also exist comparative maps between horse and other

mammalians e.g., between horse and donkey (RAUDSEPP et al., 1997, 1999, 2001;

RAUDSEPP and CHOWDHARY, 2001), horse and mountain zebra (RAUDSEPP et

al., 2002) and E. caballus and E. przewalski (MYKA et al., 2003). Comparative

chromosomal painting (Zoo-FISH) helps identifying conserved chromosomal

segments among species. Reciprocal painting of chromosomes and segments was

performed to compare the horse genome with that of the donkey (RAUDSEPP et al.,

1996; CHOWDHARY et al., 1998; YANG et al., 2004).

One approach to localize genes and to expand the comparative gene map between

horse and man is to pick genes or DNA-/RNA-sequences from genes physically

mapped and cloned in humans for screening filters of a genomic horse BAC library.

After verification of the selected gene sequence on the genomic equine BAC clone,


- 24 -

FISH is used for mapping this BAC clone on horse metaphase spreads. There are

three equine genomic BAC-libraries available: the equine BAC library of the

Chidren`s Hospital of Oakland Research Institute, CHORI-241 (GODARD et al.,

1998), one generated by the Texas A&M University (TAMU) and the other by the

Institut de la National Recherche Agronomique (INRA-LGBC library) (CHOWDHARY

et al., 2003).

CAETANO et al. (1999) published an extensive comparative map between horse and

man containing 68 equine type I loci. The most topical comparative map between

horse, donkey and human was generated by cross-species painting (YANG et al.,

2004). The human-donkey map was the first genome-wide comparative map

between these species generated by chromosome painting. This horse-human

comparative map agrees basically with the comparative map of CHOWDHARY et al.

(2003), except from varying results especially for ECA1. For several horse

chromosomes, detailed comparative maps have been developed. A detailed physical

map of the equine Y-chromosome was generated (RAUDSEPP et al., 2004) and

compared to the human and other mammalian species with the result that the equine

Y-chromosome shows the most homology with the Y-chromosome of the pig. A

comparative RH map for ECA17 was developed by LEE et al. (2004), and there

exists a detailed RH map of ECA22 comparing the equine loci to human, dog, cat,

bovids, elephant and bat, rodents, dolphins and other mammalians (GUSTAFSON-

SEABURY et al., 2005). This map covered estimated 64 Mb of physical length of

chromosome 22 (831 cR) and the 83 markers had an average distance of 10 cR. It

was remarkable, that the most common configuration for the ECA22 homologs

consisted for Hartmann’s zebra as expected, but also for carnivores, hippos,

primates, rabbits, squirrels and bats. The conserved synteny between the compared

animals can be taken as an indicator of the degree of putative ancestral relationship

between the different species. BRINKMEYER-LANGFORD et al. (2005) compared

equine homologs of HSA19 to other mammals using 89 loci for the 5000-rad-RH-

panel and four loci for FISH. The homologs to HSA19 were localized on ECA7, 21

and 10. This comparison demonstrated an overview of the evolution of putative

ancestral HSA19 chromosomal segments in more than 50 mammalian species.


- 25 -

PERROCHEAU et al. (2006) developed a medium density horse gene map using

323 genes evenly distributed over the human genome. They mapped 87 genes by

FISH and 186 genes by the equine 5000-rad RH panel and thereby detected

previously unknown homology between ECA27 and HSA8 as well as between

ECA12p and HSA11p.

Linkage map

Genetic and physical assignment of equine microsatellites began almost ten years

ago. BREEN et al. (1997) isolated 20 equine microsatellites from a genomic phage

library and mapped them by linkage mapping and FISH. GODARD et al. (1997)

assigned 36 new horse microsatellites, 11 of them from plasmid libraries and 25 from

a cosmid library.

The first equine linkage map included 140 markers (LINDGREN et al., 1998) and a

more extensive linkage map consisted of 161 loci in 29 linkage groups for 26

autosomes (GUERIN et al., 1999). The first map covering all autosomes and the sex-

chromosomes using 353 microsatellite-markers was generated by SWINBURNE et

al. (2000). The microsatellites were mapped to 42 linkage groups and covered 1780

cM of the horse genome.

A second generation linkage map was published by the International Equine Gene

Mapping Workshop (GUERIN et al. 2003). This linkage map was based on testing

503 half-sibling offspring from 13 sire families. The map included 344 markers in 34

linkage groups representing all 31 autosomes, but neither the X- nor the Y-

chromosome. The linkage groups covered 2262 cM with an average interval between

loci of 10.1 cM, ranging from 0 to 38.4. Another 61 new horse microsatellite loci were

assigned by SWINBURNE et al. (2003). TOZAKI et al. (2004) reported the

characterization of 341 newly isolated microsatellite markers of which 256 were

assigned to equine chromosomes.

PENEDO et al. (2005) added 359 microsatellites to the second generation linkage

map. Alltogether, this linkage map consisted of 766 markers assigned to the 30

autosomes and the X-chromosome spanning 3740 cM with an average marker

density of 6.3 cM. SWINBURNE et al. (2006) generated a linkage map including 742


- 26 -

markers in 32 linkage groups according to the 31 autosomes and the X-chromosome.

All linkage groups together span 2772 cM with an average interval of 3.7 cM between

the markers.

Chromosomal abnormalities

There exist several chromosomal abnormalities in the horse. Of great interest and

therefore well analyzed are those concerning the sex-chromosomes and influencing

the fertility of mares:

The most common abnormality in mares, X0 Gonadal Dysgenesis (X monosomy), is

characterised by the lack of one X-chromosome (63,X0) and was primarily described

by HUGHES et al. (1975). This defect was accompanied with infertility of the affected

mare. POWER (1986) reported another abnormality in horses, the XY Gonadal

Dysgenesis. The affected horses` phenotype is female, but their genotype is 64,XY

leading to infertility (BOWLING et al., 1987). There exist also infertile mares with the

genotype 65,XXX (CHANDLEY et al., 1975). KAKOI et al. (2005) published a

statistical evaluation of sex chromosome abnormalities and analyzed data of 17471

light-breed foals with the result that 0.15 % of the analyzed population showed an

XO-, 0.02% an XXY- (and/ or mosaics/chimaeras) and 0.01% an XXX-genotype.

Unlike the sex chromosomes, chromosomal abnormalities of the autosomes are

rarely encountered. Cases of trisomy are similar to human Downs and associated

with mental retardation failure to thrive (LEAR et al., 1999).

Coat colours

There exist several genetically characterized major genes that influence the coat

colour of a horse: Agouti-gene, Extension-gene, Cream dilution gene, Tobiano-gene,

White-gene, Greying hair-gene, Dun-gene, Roan-gene, Sabino-gene, Silver-gene,

and the Leopard Complex-gene. Knowledge about the inheritance of coat colours is

very important for the breeders, so it is comparatively well explorated and for several

genes exist gene tests, too. Chromosomal locations and mutations of genes

associated with equine coat colours are summarized in Table.


- 27 -

The White-gene and the Greying hair-gene can mask all the other coat colour genes,

so they are mentioned first.

White-gene (W-gene) and Sabino spotting

Horses with the dominant allele “W” lack pigment in skin and hair, so their coat colour

is white at birth, but eyes are dark or sometimes blue. The W-mutation was mapped

to a region of ECA3q22 where the equine KIT gene is located (MAU et al., 2004).

The homozygous “WW” is lethal at a very early stage of pregnancy as it is in mice,

too. Since the action of the dominant white allele seems not always to be fully

penetrant, some horses homozygously “WW” are born alive with the phenotype of

sabino spotting. All non-white horses are homozygous recessive “ww”. In contrast to

the greying gene, horses with the dominant “W” are not able to produce any pigment,

white horses with the “G”-gene instead can produce pigment but lose their pigment

with aging.

Sabino pattern is characterized by irregulary bordered white patches on the lower

limbs and face and often includes belly and interspersed white hairs on the

midsection. Sabino spotting is found in many light horse breeds such as Tennessee

walking horse, Missouri Foxtrotter, Shetland pony and American Paint Horse. A

single nucleotide exchange in the 3’-splice site of intron 16 of the equine KIT gene

causes a partial skipping of exon 17 and the Sabino spotting (BROOKS and BAILEY,

2005).

Furthermore, close association between KIT polymorphisms and roan coat colour

was detected in horses (MARKLUND et al., 1999).

Greying-gene (G-gene)

The dominant allele “G” is responsible for progressive greying in hairs. If a foal that

possesses the allele “G” is born coloured it will progressively turn white as an aged

animal. The Grey-locus was mapped to ECA25 by SWINBURNE et al. (2002) and by

HENNER et al. (2002). The mapping of the G-gene was of particular importance

because the Grey-gene and the appearance of melanomas in horses are associated:

grey horses get melanomas with a frequency of at least 80% in horses older than 10

years whereas melanomas are rare in non-grey horses. SWINBURNE et al. (2002)


- 28 -

localized the G-locus 13 cM proximal of the marker TKY316 and 17 cM distal of the

marker UCDEQ464 using a whole genome scan.

PIELBERG et al. (2005) refined the grey-locus to a region smaller than 6.9 Mb on

ECA25 using comparative mapping to mouse and human. In this refined region, no

obvious candidate genes for pigmentation disorders could be identified. Several

possible candidate genes responsible for different coat colours including agouti-

signaling-protein (ASIP), melanocortin-1-receptor (MC1R), tyrosinase-related protein

1 (TYRP1) and silver homolog (SILV, PMEL17) had been excluded before (RIEDER

et al., 2001; LOCKE et al., 2002).

Agouti-gene (A-gene)

The Agouti locus with the dominant “A” and recessive “a” allele is responsible for the

distribution pattern of black hair. The interaction with the extension-gene is

responsible if the horse is bay or black: A horse that is homozygous recessive “aa”

will be black if it also possesses a dominant “E” allele. If a horse possesses at least

one “A” allele together with the “E” allele, it will be bay. However, if a horse is

homozygous recessive “ee”, it will look sorrel or chestnut, irrespective if it carries an

“A” or “a”. This is important for the breeders, because it is possible that a sorrel or

chestnut horse can also produce blacks. RIEDER et al. (2001) characterized equine

MC1R (melanocortin-1-receptor) and ASIP (agouti signaling protein), and completed

a partial sequence of TYRP1. They detected an 11-bp deletion in exon 2 (ADEx2) of

ASIP which was completely associated with horse recessive black coat color in

horses of 9 different breeds. The frameshift initiated by ADEx2 was supposed to act

as a loss-of-function ASIP mutation.

Extension gene (E-gene)

The Extension locus controls the expression of black pigment in horses. The equine

melanocyte stimulating hormone receptor (MC1R) on ECA3 encodes the Extension

locus (MARKLUND et al., 1996). A missense mutation in MC1R was associated with

light colour, whereas the wildtype allele corresponded to dark pigmentation. A black

or bay horse possesses at least one “E” allele. A horse that is homozygous “ee” is

either chestnut or sorrel. For horse breeders where black colour is valued, it is


- 29 -

important, if a horse is homozygous “EE”, because that means that it will never have

a chestnut or sorrel foal.

Cream dilution gene (C-gene)

The dominant “C” allele causes a reduction in red pigment in hairs. A sorrel horse

inheriting one “C” allele is palomino (ee/Cc), a sorrel inheriting “C/C” is cremello

(ee/CC). A bay horse inheriting one “C” allele is buckskin (E-/Cc) and a horse

homozygous for “C” is a perlino (E-/CC). It is also possible that grey or dun horses

carry the “C” allel. The C-locus was localized to ECA21 and TYR (tyrosinase) could

be excluded as candidate for the cream dilution gene (Locke et al. 2001). MARIAT et

al. (2003) identified the causal mutation similarly to mice and humans in exon 2 of the

membrane associated transporter protein (MATP) gene mapping to ECA21p17. A

gene test based on this G to A transition could be developed to control if a horse

carries a dominant “C” allele.

Tobiano-gene (To-gene)

The “To” allele is dominantly inherited and is responsible for white spotting

characterized by distinct borders crossing the dorsal midline and including at least

one if not all four limbs. There was identified a MSPI polymorphism for the Tobiano-

locus in intron 13 of the equine homologue of the proto-oncogene c-kit (KIT) gene

(KM1 locus) which is strongly associated with the Tobiano gene but not causative

(BROOKS et al., 2002). If a horse does not possess the Tobiano-allele, the test result

will be designated KM0/KM0. If a horse carries one “To” allele, it will be tested

KM0/KM1, and if the test is KM1/KM1, the horse is probably a homozygous Tobiano.

However, solid-coloured horses can also possess the KM1 allele. This test is useful

for breeders valuing homozygous Tobianos, because they will always produce

spotted foals.

Silver-gene (Z-gene)

A mutation in the Silver (PMEL17) gene had recently been identified that produces in

black horses a chocolate coat colour with flaxen mane and tail and in bay horses

lightened coat colour in lower legs and flaxen mane and tail (BRUNBERG et al.,

2006). This mutation has no effect on chestnut-coloured horses. A missense

mutation in exon 11 changing arginine to cysteine (Arg618Cys) was likely to be


- 30 -

causative as complete association with the Silver phenotype was observed across

several horse breeds. A further silent mutation in intron 9 was also completely

associated with the Silver phenotype.

Appaloosa coat colour

TERRY et al. (2004) investigated the appaloosa coat colour which is characterized

by several different spotting patterns ranging from a few white specks on the rump to

an almost completely white animal. They excluded the KIT gene as possible

candidate gene causing Rw colour pattern in mice and also the candidate genes

microphthalmia-associated transcription factor (MITF) and mast cell growth factor

(MGF) (TERRY et al., 2001, 2002). They found that the autosomal incompletely

dominant gene LP (leopard complex) responsible for the appaloosa coat pattern in

horses mapped to ECA1q (TERRY et al., 2004).

Table

Molecular genetic characterization of coat colour loci in horse (Molekulargenetische

Charakterisierung von Farbgenorten beim Pferd)

Locus Gene ECA Mutation Reference

Agouti ASIP 22q15-q16 11 bp deletion RIEDER et al. (2001)

Extension MC1R 3p12 C>T missense

mutation

MARKLUND et al. (1996)

Cream MATP 21p17 G>A missense

mutation

MARIAT et al. (2003)

Sabino KIT 3q22 T>A mutation

(splice

mutation)

BROOKS and BAILEY

(2005)

Tobiano KIT 3q22 C>G missense

mutation

BROOKS et al. (2002)

Silver PMEL17 6q23 C>T missense

mutation

BRUNBERG et al. (2006)

ECA: Equus caballus autosome.


- 31 -

Equine genetic diseases and the development of gene tests

The most important information breeders take from gene investigation are probably

new results as to diseases or phenotypic features.

While inheritance of colours is usually comparatively simple with different alleles that

are either dominant or recessive responsible for all colours, investigation of

inheritance of diseases is often much more complicated, for example multifactorial

and influenced by lots of different genes. There are 188 diseases reported in the

horse that are discussed to be inherited. For 20 well-explorated diseases only a

single locus is responsible for the appearance.

This is probably one of the reasons for the fact that there are only a few gene tests

available for important diseases but for many of the possibly inherited diseases in the

horse the inheritance is still unknown.

Monogenic equine diseases

Lethal White Foal Syndrome (LWFS)

The Lethal White Foal Syndrome is observed in horse breeds with white coat

spotting patterns called overo, e.g., in Paints and Pintos. Affected foals are almost

completely white and die shortly after birth due to severe intestinal blockage resulting

from a lack of nerve cells in the distal portion of the large intestine (aganglionic

megacolon). The disease is similar to Hirschsprung disease in humans. As the

Hirschsprung disease is caused by a mutation in the endothelian B receptor gene

(EDNRB), METALLINOS et al. (1998) investigated the influence of the EDNRB in

horses with Lethal White Foal Syndrome and found an association between a

missense mutation in the EDNRB-gene and the appearance of LWFS like in humans

for the Hirschsprung disease. YANG et al. (1998) could show that a dinucleotide

TC>AG mutation was associated with LWFS when homozygous and with the overo

phenotype when heterozygous.

Herlitz junctional epidermolysis bullosa (epitheliogenesis imperfecta)

The pathological signs of epitheliogenesis imperfecta closely match a similar disease

in humans known as Herlitz junctional epidermolysis bullosa, which is caused by a

mutation in one of the genes (LAMA3, LAMB3 and LAMC2) coding for the subunits of


- 32 -

the laminin 5 protein. Herlitz junctional epidermolysis bullosa (HJEB) is a lethal

disease that causes blistering of the skin and mouth epithelia, and sloughing of

hooves in newborn foals, especially in American Saddlebred horses and Belgian

draft horses (JOHNSON et al., 1998). Pedigree studies demonstrated that the trait

followed a monogenic autosomal recessive inheritance pattern and a genetic

mapping study revealed that HJEB in American Saddlebred horses was linked to

microsatellites on ECA8q where LAMA3 is also located (LIETO and COTHRAN,

2003). SPIRITO et al. (2002) detected a cytosine insertion in exon 10 in the LAMC2

gene as being responsible for HJEB in Belgian draft horses and BAIRD et al. (2003)

developed a gene test that works with hair of the horses and is now available for

owners of registered horses. The same mutation was found to be associated with the

same condition in the French draft horse breeds, Trait Breton and Trait Comtois

(MILENKOVIC et al., 2003).

Hyperkalemic periodic paralysis

Hyperkalemic periodic paralysis (HYPP) is an inherited disease of the muscles that

causes attacks of paralysis that can lead to sudden death that especially affects

American Quarter horses. It is inherited as an autosomal codominant genetic defect

so that heterozygous horses are affected, too (ZEILMANN, 1993). HYPP is the result

of a missense mutation of the gene encoding the a-chain of the adult muscle sodium

channel. RUDOLPH et al. (1992) described the point mutation in transmembrane

domain IVS3 that is responsible for the occurrence of the disease. Even though the

attacks can be treated with azetazolamide (KOLLIAS-BAKER, 1999) and the disease

will not always lead to death, it is useful to test horses if they are carriers or not.

Severe Combined Immunodeficiency (SCID)

SCID is an inherited disease known in different species that always causes an early

death of affected animals because of the incapability to generate antigene-specific

immune responses; there exists a comparative study between the different

mechanisms that lead to SCID in humans, mice, horses and dogs (PERRYMAN,

2004). SCID is recessively inherited in Arabian horses. Horses affected with SCID

cannot react in a natural way of protection against diseases. They do not develop the

necessary active protection reactions because they lack intact lymphocytes. Due to


- 33 -

this reason, affected foals will not become older than six months. SHIN et al. (1997)

tested candidate genes from the mouse and could show that SCID is caused by a

frameshift mutation in the gene for DNA-dependent protein kinase catalytic subunit

(DNA-PK) mapping to horse chromosome 9. This mutation results in the lack of a full-

length kinase.

Glycogen Branching Enzyme Deficiency (GBED)

GBED is a glycogene storage disease similar to the human glycogen storage disease

type IV. The disease is lethal for the affected foals with a great variability of

symptoms ranging from abort or stillbirth until being euthanized due to weakness.

VALBERG et al. (2001) described the disease in American Quarter horses. Common

to GBED are the accumulation of unbranched polysaccharides in tissues and a

profound decrease of glycogen branching enzyme activity in cardiac and skeletal

muscle as well as in liver and peripheral blood cells of affected foals. The pedigree

analysis supported an autosomal recessive mode of inheritance.

WARD et al. (2003) mapped the GBE1 gene to ECA26q12-q13, and later on,

detected the C- to A-mutation at base 102 of the GBE1 gene causing the disease

(WARD et al., 2004). An autosomal recessive mode of inheritance was confirmed. It

is now possible to test horses whether they are carriers of the GBE1 allele.

Genetically complex equine diseases

Osteochondrosis (OC)

Osteochondrosis (OC) is a developmental orthopaedic disorder frequently observed

in young horses (VAN DE LEST et al., 1999). Signs of osteochondrosis are lesions of

the cartilage in the joints like subchondral fractures, subchondral cysts, wear lines,

chondromalacia, cartilage flaps and joint mice or free joint bodies (chips). Hereditary

factors play an important part in the pathogenesis of OC. An optimized microsatellite

marker set for complete genome scans in horses was developed including 155 highly

polymorphic markers equally distributed at a distance of about 20 cM. Data from 14

half-sib families of Hanoverian Warmblood horses were analysed using this marker

set to detect quantitative trait loci (QTL) with significant influence on the development

of OC (BÖNEKER et al., 2006; DIERKS and DISTL, 2006). QTL for osteochondrosis


- 34 -

in fetlock and hock joints were found on horse chromosomes 2, 3, 4, 5, 15, 16, 19,

and 21. In a population of South German coldblood horses a further whole genome

scan for OC in fetlock and hock joints was carried out (WITTWER et al., 2006;

WITTWER and DISTL, 2006). In total, 17 chromosome-wide significant QTL were

found on different equine chromosomes with influence on the development of OC in

fetlock and hock joints.

Navicular disease

Navicular disease (navicular syndrome or podotrochlosis) is a chronic and usually

progressive, degenerative alteration of the equine podotrochlea (RIJKENHUIZEN,

1989). Pathological alterations can primarily affect the navicular bone (os

sesamoideum distale), the navicular bursa (bursa podotrochlearis) or the distal end

of the deep digital flexor tendon. The disease may be part of the osteoarthritis

complex (SVALASTOGA and SMITH, 1983). A microsatellite marker set to be

applied in Hanoverian warmblood horses for a whole genome scan was prepared

and then a whole genome scan in 144 descendants of 17 Hanoverian warmblood

stallions was performed. The genotyped horses were randomly sampled from the

whole Hanoverian warmblood breeding district. QTL on different horse chromosomes

were identified such as on ECA2, 3, 4, and 10 (DIESTERBECK et al., 2006).

According to SVALASTOGA and SMITH (1983) increased bone marrow pressure

and lengthened contrast passage indicate similarities between osteoarthritis (OA) in

humans and navicular disease in horses. About 50 different positional candidate

genes have been reported for OA in humans. These candidate genes encode

different types of collagens, hormone receptors and interleukin receptors, growth

factors and metalloproteinases. About 13,966 equine cartilage expressed sequence

tags (ESTs) and further 23,171 ESTs from other cDNA libraries as well as BAC end

sequences or whole genome sequences can be used for identification of single

nucleotide polymorphisms in functional and positional candidate genes. However,

since navicular disease does not occur in humans, it is not clear whether genes for

OA are suitable candidates for navicular bone disease in horses.


- 35 -

Temperament

Temperament is considered important in horses as optimal performance is related

with a good temperament. The neurotransmitter-related dopamine D4 receptor

(DRD4) gene harbours many polymorphisms in the coding region and among them,

the variable number of tandem repeats polymorphism consisting of a 48-bp repeat

unit was shown to be associated with novelty-seeking in humans (BENJAMIN et al.,

1996; EBSTEIN et al., 1996). In horses, this part of DRD4 included repeats of an 18-

bp repeat unit (HASEGAWA et al., 2002). Two temperament scores, curiosity and

vigilance were associated with a single nucleotide polymorphism (A>G substitution)

of DRD4 but no difference in the number of repeats among the 136 two-year-old

thoroughbred horses was found (MOMOZAWA et al., 2005). Horses without the A

allele had higher curiosity and lower vigilance scores than those with the A allele.

Polymorphisms of the serotonin transporter (SLC6A4) gene were screened for

associations with an anxiety score in 67 Thoroughbred horses (MOMOZAWA et al.,

2006). However, no significant associations could be identified in this study.

Stallion fertility

Seminal plasma proteins that are produced by the epididymis and the accessory

glands play an important role in sperm maturation. Some of these proteins bind to

spermatozoa and act as binding partners in the female genital tract and others are

required to suppress adverse immune reactions directed against spermatozoa.

Among the seminal plasma proteins, cystein-rich secretory protein 3 (CRISP3) is

found in large amounts in the seminal plasma of stallions (SCHAMBONY et al.,

1998). Association studies for CRISP3 polymorphisms in 107 Hanoverian stallions

revealed a significant influence on fertility at artificial insemination of mares

(HAMANN et al., 2006). Stallions being heterozygous for an E208K mutation within

the CRISP3 gene had less insemination success than those without this mutation or

homozygous for this mutation.


- 36 -

Conclusions and perspectives

There has been great progress in the horse genome investigation especially during

the last ten years, but compared to the human genome or to animals like the mouse,

the horse gene map still shows many unexplored areas.

For the coat colours, the inheritance of the major colours is already resolved, but

for several possibly inherited diseases the mode of inheritance is still not detected

despite of intensive investigation in the last years, as for example for the appearance

of “parrot mouth”, “hyperelastosis cutis”, “laminitis”, “osteochondrosis” and many

more. It is always difficult if there are no suitable candidate genes from the human

because no equivalent diseases exist in human. Another problem is that many

diseases are not only characterized by a single mutation or a simple dominant or

recessive mode of inheritance but are multifactorial and many different genes or

environmental effects like feeding or movement influence the appearance of these

diseases.

The ongoing refinement of the comparative map between horse and human plays an

important role in genomic studies in the horse: Without the generation of tools like the

5000-rad-RH-panel by CHOWDHARY et al. (2003) it would not be possible to map

new loci as accurately as they can be localized today. Laborious methods like the

fluorescence in-situ hybridization (FISH) are increasingly eclipsed by methods that

are more timesaving and that lead to more new results in one experiment, like the

radiation hybrid panel mapping (RH).

Horse genome sequencing, funded by the National Human Genome Research

Institute, had started in February 2006 and the sequencing of the about 2.7 billion

DNA bases is nearly complete. A whole genome shotgun approach is being used for

sequencing a female thoroughbred named “Twilight”. A high-quality genome

sequence of Equus caballus will be elaborated together with an analysis of sequence

variation of 100,000 randomly chosen DNA segments from mares of seven additional

breeds. Breeds selected for this single nucleotide polymorphisms (SNPs) analysis

included thoroughbred, Quarterhorse, Arabian horse, American Standardbred pacer,

Akalteke, Andalusian and Icelandic horse. More than one million SNPs should be

identified. The horse genome project has emerged from an ongoing effort to highlight


- 37 -

the most evolutionarily conserved segments of the human genome by identifying

genetic similarities between several different mammalian genomes and human

genome. A high-quality horse genome sequence and the large catalogue of SNPs

will be the key to identify the genetic contributions to the aetiopathogenesis of

diseases, physiological and pathological processes and to the athletic performance

of horses in sports, agriculture and transportation.

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Chapter 3

Assignment of the COMP gene to equine chromosome

21q12-q14 by FISH and confirmation by RH mapping

D. Müller*, H. Kuiper*, S. Mömke*, C. Böneker*, C. Drögemüller*, B.P. Chowdhary†

and O. Distl*

*Institute for Animal Breeding and Genetics, University of Veterinary Medicine

Hannover, Germany. †Department of Veterinary Integrative Biosciences, College of

Veterinary Medicine and Biomedical Sciences, Texas A&M University, College

Station, TX 77843, USA

Accepted for publication 17 March 2005

Published in: Animal Genetics 36 (2005) 277-279

Assignment of the gene COMP gene to equine chromosome 21q12-q14 by FISH and confirmation by RH mapping

- 55 -

Assignment of the COMP gene to equine chromosome

21q12-q14 by FISH and confirmation by RH mapping

Source/description: The cartilage oligomeric matrix protein gene (COMP) maps to

HSA19p13.1 starting at 18,754,584 bp and ending at 18,763,114 bp and encodes a

524-kD protein that is expressed at high levels in the territorial matrix of

chondrocytes1. The human gene consists of 19 exons spanning 8531 bp and the

exons 1-3 are unique to COMP, whereas the exons 4-19 encode the EGF-like (type

II) repeats, calmodulin-like (type III) repeats (CLRs), and the C-terminal domain

correspond in sequence and intron location to the thrombospondin genes2. Mutations

in the COMP gene are responsible for both the autosomal dominantly inherited

pseudoachondroplasia (PSACH) and some types of multiple epiphyseal dysplasia

(MED), which are characterized by mild to severe short-limb dwarfism and early-

onset osteoarthritis in man2, 3, 4, 5. Further studies including COMP-null mice revealed

that the phenotype in PSACH and MED is caused not by the reduced amount of

COMP but by some other mechanism, such as folding defects or extracellular

assembly abnormalities due to dysfunctional mutated COMP 6.

The equine BAC library CHORI-241 was screened as per standard protocols

(http://bacpac.chori.org) with a heterologous 32P-labelled insert of a human COMP

cDNA clone (IMAGp998C026158) provided by the Resource Center/Primary

Database of the German Human Genome Project (http://www.rzpd.de/). A BAC clone

designated CH241-49F5 with an insert of approximately 160 kb was identified.

Following culture, BAC DNA was isolated using the Qiagen plasmid midi kit (Qiagen,

Hilden, Germany), and end-sequences of the BAC were obtained using the

ThermoSequenase kit (AmershamBiosciences, Freiburg, Germany) and a LI-COR

4200 automated sequencer (LI-COR, Inc., Lincoln, Nebraska, USA). The end

sequences are deposited in the EMBL nucleotide database (Accession nos.

AJ870433 and AJ870434). A BLASTN sequence comparison of the equine SP6 BAC

end sequence with the build 35.1 of the human genome sequence revealed a

significant match (BLAST E-value 2.6e-38) over 74 bp (93.2% identity) starting at

18,765,397 bp of HSA19p, approximately 11 kb downstream of human COMP.


- 56 -

Comparison of the T7 end sequence revealed a significant match (BLAST E-value

4.6e-72) over 143 bp (91.6 % identity) starting at 18,529,627 bp of HSA19p,

approximately 225 kb upstream of human COMP. The identity of the BAC clone with

respect to the presence of the COMP gene was further verified by obtaining primers

from an equine EST (Accession no. CX605670) corresponding to exons 14-18 of the

human gene, and using them for PCR amplification of the BAC DNA. Sequence of

the amplification product when compared with the human genome sequence (build

35.1) revealed a significant match (BLAST E-value 1e-36) over 124 bp (92 % identity)

starting at 18,756,781 within exon 16 of the human COMP gene.

Primer sequences: Primers for PCR amplification of the 214 bp BAC end sequence

(Accession no. AJ870434) and 166 bp internal BAC sequence corresponding to the

human COMP gene (Accession no. CX605670) were designed using the PRIMER3

software (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi).

T7 BAC end sequence:

F: 5’-CAC GTC TAG GCA CCT CTC CT-3’

R: 5’-GGC TTG CAT ATC CCC TCA C-3’

Internal BAC sequence:

F: 5’-GTT ACA CGG CCT TCA ATG G-3’

R: 5’-CCA CAT GAC CAC GTA GAA GC-3’

Chromosomal location: The equine BAC clone was labelled with digoxygenin using a

nick-translation mix (Roche Diagnostics, Mannheim, Germany). FISH on GTG-

banded horse chromosomes was performed using 500 ng of labelled DNA, and 20µg

sheared equine genomic DNA and 10 µg salmon sperm DNA as competitors. After

overnight hybridization, signal detection was performed using a Digoxygenin-FITC

Detection Kit (Qbiogene, Heidelberg, Germany). The chromosomes were

counterstained with DAPI (4’, 6’-diaminidino-2-phenylindole) and propidium iodide

and embedded in antifade. Previously photographed metaphase spreads were re-

examined for hybridization signals using a Zeiss Axioplan 2 microscope (Zeiss, Jena,

Germany) equipped for fluorescence. Identification of chromosomes followed the

international system for chromosome nomenclature of domestic horses (ISCNH


- 57 -

1997)7. The equine COMP gene was subsequently mapped to ECA21q12-q14

following examination of 35 metaphase spreads (Fig.1).

Radiation hybrid (RH) mapping/PCR conditions: The 5,000-rad TAMU equine

radiation hybrid panel8 was genotyped to map the T7 BAC end marker located about

227 kb upstream of COMP. PCR was carried out in a 20 µl volume containing 25 ng

of RH cell line DNA, 10 pmol of each primer and 0.85 U Taq polymerase (Qbiogene,

Heidelberg, Germany). The reaction conditions were as follows: denaturation at 94°C

for 5 min followed by 34 cycles of denaturation for 45 s at 94°C, annealing for 45 s at

62°C and extension for 5 min at 72°C. The PCR was completed with a final cooling at

4°C for 5 min. The amplified products were separated on a 1.5% agarose gel. After

scoring the signals, a two-point analysis9 was performed using RHMAPPER-1.22

(http://equine.cvm.tamu.edu/cgi-bin/ecarhmapper.cgi) against 861 equine markers

mapped previously on the first generation whole genome equine RH map. This

sequence tagged site (STS) marker showed a retention frequency of 13% and

showed close linkage to SG16 (11.99 cR; LOD >12.0) that is located on ECA21q13

by FISH10 and at 38 cR of ECA218 RH map. Thus the FISH and RH results

corroborate each other.

Comment: The physical assignment of the equine COMP gene on ECA21q12-q14 is

in agreement with comparative painting11 but does not agree with comparative

mapping of the current equine-human comparative map of ECA21 RH map, which

showed conserved synteny to HSA58,12.

Acknowledgements: This study was supported by grants of the German Research

Council, DFG, Bonn (DI 333/12-1).

References

1 Newton G. et al. (1994) Genomics 24, 435-9.

2 Briggs M.D. et al. (1995) Nature Genet 10, 330-6.

3 Briggs M.D. et al. (1998) Am J Hum Genet 62, 311-9.

4 Deere M. et al. (1998) Am J Med Genet 80, 510-3.

5 Mabuchi A. et al. (2003) Hum Genet 112, 84-90.

6 Svensson L. et al. (2002) Mol Cell Biol 22, 4366-71.


- 58 -

7 Bowling A.T. et al. (1997) Chromosome Res 5, 433-43.

8 Chowdhary B.P. et al. (2003) Genome Res 13, 742-51.

9 Slonim D. et al. (1997) J Comput Biol 4, 487-504.

10 Godard S. et al. (1997) Mamm Genome 8, 745-50.

11 Yang F. et al. (2004) Chromosome Res 12, 65-76.

12 Milenkovic D. et al. (2002) Mamm Genome 13, 524-34.

Figure 1 Chromosomal assignment of the equine BAC CH241-49F5 containing

COMP by FISH analysis. G-banded metaphase spread before (left) and after

hybridization (right). Double signals indicated by arrows are visible on both ECA21

chromosomes.

Chapter 4

Physical mapping of the PTHR1 gene to equine

chromosome 16q21.2

D. Müller*, H. Kuiper*, C. Böneker*, S. Mömke*, C. Drögemüller*, B.P. Chowdhary†

and O. Distl*


Hannover, Germany. †Department of Veterinary Integrative Biosciences, College of

Veterinary Medicine and Biomedical Sciences, Texas A&M University, College

Station, TX 77843, USA

Accepted for publication 22 March 2005


Physical mapping of the PTHR1 gene to equine chromosome 16q21.2

- 61 -

Physical mapping of the PTHR1 gene to equine

chromosome 16q21.2

Source/description: The parathyroid hormone receptor 1 (PTHR1) maps to

HSA3p22-p21.1 starting at 46,894,240 bp and encodes a receptor for both

parathyroid hormone and parathyroid hormone-related protein (PTHRP) that are

expressed in most tissues1. Three promoters, P1, P2, and P3, regulate the

expression of PTHR1 but the P3 promoter seems to be the most active in human

adult kidney and also active in human osteoblast-like cells2. The human gene

consists of 16 exons spanning 26054 bp3. The protein encoded by this gene is a

member of the G-protein coupled receptor family 24. The activity of this receptor is

mediated by G-proteins which activate adenylyl cyclase and also a

phosphatidylinositol-calcium second messenger system. Defects in this receptor are

known in human to be the cause of Jansen's metaphyseal chondrodysplasia (JMC)

and chondrodysplasia Blomstrand type (BOCD), as well as enchondromatosis.

Investigations in mouse models suggested that PTHRP mediates chondrocyte

differentiation during endochondral ossification.

The equine BAC library CHORI-241 was screened to isolate a genomic BAC clone

containing the PTHR1 gene. High density BAC colony filters were probed according

to the CHORI protocols (http://bacpac.chori.org) with a heterologous 32P-labelled

insert of a murine PTHR1 cDNA clone (IRAKp961D1425) provided by the Resource

Center/Primary Database of the German Human Genome Project

(http://www.rzpd.de/). An equine genomic BAC clone designated CH241-376I8 with

an insert of approximately 245 kb containing the PTHR1 gene was identified. BAC

DNA was prepared from the BAC clone using the Qiagen plasmid midi kit (Qiagen,

Hilden, Germany). BAC DNA sequences were obtained using the ThermoSequenase

kit (AmershamBiosciences, Freiburg, Germany) and a LI-COR 4200 automated

sequencer (LI-COR, Inc., Lincoln, Nebrasca, USA). The two CH241-376I8 end

sequences were deposited in the EMBL nucleotide database under accession

numbers AJ870493 and AJ870432, respectively. A BLASTN sequence comparison

of the equine SP6 BAC end sequence with the build 35.1 of the human genome


- 62 -

sequence revealed a significant match (BLAST E-value 5.8e-47) over 286 bp (identity

= 83.6%) starting at 47,110,626 bp of HSA3p22, which is approximately 190 kb

downstream of human PTHR1.

In order to verify the location of the PTHR1 gene on the equine CH241-376I8 BAC,

equine expressed sequence tagged (EST) sequences were identified by performing

a BLASTN search for est_others of mammalia by using the human mRNA sequence

of the PTHR1 gene. An equine EST (Accession no. CX605315) gave a significant hit

over 716 bp for exons 11-16 of the human PTHR1 gene (identity = 91 %, BLAST E-

value 0.0, Score 967). The equine EST sequence of exon 14 of the human PTHR1

gene was used to derive primers for PCR to obtain equine sequence of PTHR1 from

the CH241-376I8 BAC clone. A comparison of this amplification product with the

build 35.1 of the human genome sequence revealed a significant match (BLAST E-

value 2e-17) over 120 bp (identity = 88 %) starting at 46,919,031 within exon 14 of the

human PTHR1 gene.

Primer sequences: Primers for PCR amplification of a 212 bp fragment from the

CH241-376I8 T7 BAC end sequence (Accession no. AJ870432) and 120 bp internal

BAC sequence corresponding to the human PTHR1 gene (Accession no. CX605315)

were designed using the PRIMER3 software (http://frodo.wi.mit.edu/cgi-

bin/primer3/primer3_www.cgi).

T7 BAC end sequence:

F: 5’-GGT AGG GGA TGT TAT GGA GAG-3’

R: 5’-ATC ACC TGT CCT GGA AGA GC-3’

Internal BAC sequence corresponding to the human PTHR1 gene:

F: 5’-TCA AAT CCA CAC TGG TGC TC-3’

R: 5’-CGT AGT GCA TCT GGA CTT GC-3’

Chromosomal location: The equine BAC clone was labelled with digoxygenin by nick

translation using a nick-translation mix (Roche Diagnostics, Mannheim, Germany).

FISH on GTG-banded horse chromosomes was performed using 500 ng of

digoxygenin labelled BAC DNA, and 20 µg sheared total equine DNA and 20 µg

salmon sperm DNA as competitors. After hybridization overnight, signal detection

was performed using a Digoxygenin-FITC Detection Kit (Qbiogene, Heidelberg,


- 63 -

Germany). The chromosomes were counterstained with DAPI (4’,6’-diaminidino-2-

phenylindole) and propidium iodide and embedded in antifade. Previously

photographed metaphase spreads were re-examined for hybridization signals using a

Zeiss Axioplan 2 microscope (Zeiss, Jena, Germany) equipped for fluorescence.

Identification of chromosomes followed the international system for chromosome

nomenclature of domestic horses (ISCNH 1997)5.

The equine genomic BAC clone CH241-376I8 containing the PTHR1 gene was

located to ECA16q21.2 by examination of metaphase chromosomes of 35 cells

(Fig.1).

Radiation hybrid (RH) mapping/PCR conditions: To confirm the cytogenetic

assignment the 5,000-rad TAMU equine radiation hybrid panel6 was used to map the

T7 BAC end located about 192 kb downstream of PTHR1. PCR reaction using the

previously described primers for the T7 BAC end was carried out in a 20 µl reaction

containing 25 ng of RH cell line DNA, 10 pmol of each primer and 0.85 U Taq

polymerase (Qbiogene, Heidelberg, Germany). The reaction conditions started with a

denaturing step at 94°C for 5 min followed by 34 cycles using the following protocol:

denaturation for 45 s at 94°C, annealing for 45 s at 62°C and extension for 5 min at

72°C. The PCR was completed with a final cooling at 4°C for 5 min. PCR products

were separated on a 1.5% agarose gel. After scoring positive signals, a two-point

analysis7 was performed using RHMAPPER-1.22 (http://equine.cvm.tamu.edu/cgi-

bin/ecarhmapper.cgi) against 861 equine markers mapped previously on the first

generation whole genome equine 5,000-rad RH panel6. This sequence tagged site

(STS) marker showed a retention frequency of 17.4% and the RH mapping revealed

close linkage to PDCD6IP (programmed cell death 6-interacting gene, Accession no.

ECA001546) (26.79 cR; LOD >3.0). This gene had been previously mapped on

ECA16q21.3 by FISH and by RH mapping on ECA16 at 112.5 cR8. On the human

gene map, PDCD6IP is located at HSA3p22.3 starting at 33,815,086 bp and ending

at 33,883,502 bp (human genome map viewer build 35.1). Thus, the RH results

confirm the result obtained by FISH.

Comment: The physical assignment of the equine PTHR1 gene on ECA16q21.2

agrees with comparative mapping of the current equine-human comparative RH6 and


- 64 -

cytogenetic map8 at the chromosomal position of ECA16q21. These results confirm

the conserved synteny of ECA16 to a small segment of HSA3p represented by

cytogenetically mapped loci located on HSA3p21 (APEH, acylamino acidreleasing

enzyme), HSA3p21.3 (GPX1, glutathione peroxidase 1), HSA3p22 (TGFBR2,

transforming growth factor beta, type II), and HSA3p25 (HRH1, histamine receptor

H1)8.



References

1 Pausova Z. et al. (1994) Genomics 20, 20-6.

2 Manen D. et al. (2000) J Clin Endocr Metab 85, 3376-82.

3 McCuaig K.A. et al. (1994) Proc Nat Acad Sci 91, 5051-5.

4 Juppner H. et al. (1991) Science 254, 1024-6.

5 Bowling A.T. et al. (1997) Chromosome Res 5, 433-43.

6 Chowdhary B.P. et al. (2003) Genome Res 13, 742-51.




- 65 -

Figure 1 Chromosomal assignment of the equine BAC CH241-376I8 containing

PTHR1 by FISH analysis. G-banded metaphase spread before (left) and after

hybridization (right). Double signals indicated by arrows are visible on both ECA16

chromosomes.

Chapter 5

Assignment of BGLAP, BMP2, CHST4, SLC1A3, SLC4A1,

SLC9A5 and SLC20A1 to equine chromosomes by FISH

and confirmation by RH mapping

D. Müller*, H. Kuiper*, C. Böneker*, S. Mömke*, C. Drögemüller*, B.P. Chowdhary†

and O. Distl*


Hannover, Hannover, Germany. †Department of Veterinary Integrative Biosciences,

College of Veterinary Medicine and Biomedical Sciences, Texas A&M University,

College Station, TX 77843, USA

Accepted for publication 12 June 2005


Assignment of BGLAP, BMP2, CHST4, SLC1A3, SLC4A1, SLC9A5 and SLC20A1 to equine chromosomes by FISH and confirmation by RH mapping

- 69 -

Assignment of BGLAP, BMP2, CHST4, SLC1A3, SLC4A1,

SLC9A5, and SLC20A1 to equine chromosomes by FISH

and confirmation by RH mapping

Source/description: The human bone gamma-carboxyglutamate (gla) protein

(osteocalcin) gene (BGLAP) consists of four exons spanning 1,108 bp1. BGLAP

encodes a 100 amino acid protein that is expressed in osteoblasts but not in other

extracellular matrix producing cell types2. Polymorphisms of the osteocalcin gene are

associated with bone mineral density in postmenopausal women3. Through testing

for linkage and association simultaneously the osteocalcin gene was identified as a

quantitative trait locus (QTL) underlying hip bone mineral density variation4.

The bone morphogenetic protein 2 gene (BMP2) belongs to the transforming growth

factor-beta (TGFB) superfamily. The human gene consists of three exons spanning

10,563 bp5. BMP2 is regarded as a multifunctional cytokine involved in inducing the

formation and regeneration of cartilage and bone6, and it also participates in

organogenesis, cell differentiation, cell proliferation, and apoptosis. BMP2 has an

essential role in the regulation of the steps of chondrocyte and osteoblast

differentiation and it induces osteocalcin expression in osteoblasts7. The BMP2 gene

has been suggested as a candidate for fibrodysplasia (myositis) ossificans

progressiva in man, which is characterized by intermittently progressive ectopic

ossification and malformed big toes which are often monophalangic8.

The human carbohydrate (N-acetylglucosamine 6-O) sulfotransferase 4 (CHST4)

gene encodes a protein with 386 amino acids that is involved in in vitro enzymatic

synthesis of the disulfated disaccharide unit of corneal keratan sulfate9,10. The human

gene consists of two exons spanning 12,358 bp.

The solute carrier family 1 (glial high affinity glutamate transporter), member 3 or

excitatory amino acid transporter 1 gene (SLC1A3, EAAT1) belongs to the family of

high-affinity sodium-dependent transporter molecules that regulates neurotransmitter

concentrations at the excitatory glutamatergic synapses of the mammalian central

nervous system11,12. An important function of the SLC1A3 protein is the re-uptake of


- 70 -

secreted amino acid neurotransmitter, possibly maintaining extracellular amino acid

concentrations at nontoxic and nonepileptogenic levels11,12. The human SLC1A3

gene consists of 10 exons spanning 81,750 bp13 and encodes a 542-amino acid

glutamate transporter protein11,12. SLC1A3 appears to be related to the syndrome of

microcephaly and mental retardation in

association with an interstitial deletion of the distal chromosomal band of HSA5p1314.

Aberrant glutamate transporter expression is involved in Alzheimer's disease as a

mechanism of neurodegeneration as shown by expression patterns in a subset of

cortical pyramidal neurones in dementia cases with Alzheimer-type pathology15.

The solute carrier family 4, anion exchanger, member 1 (erythrocyte membrane

protein band 3, Diego blood group, band 3 of red cell membrane) gene (SLC4A1,

BND3) is the major glycoprotein of the erythrocyte membrane where it mediates

exchange of chloride and bicarbonate across the phospholipid bilayer and plays a

central role in carbon dioxide transport from tissues to lungs16,17. Many SLC4A1

mutations are known in man and these mutations can lead to two types of diseases;

destabilization of red cell membrane leading to hereditary spherocytosis and

defective kidney acid secretion leading to distal renal tubular acidosis18,19,20. Other

SLC4A1 mutations that do not give rise to disease result in novel blood group

antigens, which form the Diego blood group system. Southeast Asian ovalocytosis

(SAO, Melanesian ovalocytosis) results from the heterozygous presence of a deletion

in the SLC4A1 protein and is common in areas where Plasmodium falciparum

malaria is endemic21. The human SLC4A1 gene consists of 20 exons spanning

18,428 bp1.

The solute carrier family 9 (sodium/hydrogen exchanger), isoform 5 gene (SLC9A5,

NHE5) is the fifth member of plasma membrane proteins that mediate the exchange

of extracellular Na+ for intracellular H+1. Mammalian Na+/H+ exchangers (NHEs) are a

family of integral membrane proteins that play a central role in sodium, acid-base,

and cell volume homeostasis. Its function is dynamically regulated by

phosphatidylinositol 3'-kinase and by the state of F-actin assembly22. The human

SLC9A5 gene consists of 16 exons spanning 23,231 bp1 and encodes an 896-amino

acid plasma membrane protein22.


- 71 -

The solute carrier family 20 (phosphate transporter), member 1 gene (SLC20A1 or

GLVR1, gibbon ape leukaemia virus receptor 1) is a sodium-dependent phosphate

symporter which acts as a retrovirus receptor allowing infection of human and murine

cells for gibbon ape leukaemia virus23. The human SLC20A1 gene consists of 11

exons spanning 17,876 bp24 and encodes a 679-amino acid membrane protein

containing multiple transmembrane domains25.

BAC library screening/sequence analysis: The equine CHORI-241 BAC library was

screened for BAC clones containing the selected genes. High density BAC colony

filters were probed according to CHORI protocols (http://bacpac.chori.org) with

heterologous 32P-labelled inserts of the respective human or murine cDNA IMAGE

clones provided by the Resource Center/Primary Database of the German Human

Genome Project (http://www.rzpd.de/). BAC DNA was prepared from positive BAC

clones using the Qiagen plasmid midi kit (Qiagen, Hilden, Germany). BAC DNA end

sequences were obtained using the ThermoSequenase kit (AmershamBiosciences,

Freiburg, Germany) and a LI-COR 4200 automated sequencer (LI-COR Inc., Lincoln,

NE, USA). BAC subclone sequences were obtained for the BAC DNA containing the

SLCA1A3 gene because BLASTN sequence comparisons of SP6 and T7 termini of

this BAC clone gave no significant hits to human genome sequences (Build 35.1).

For this clone, BAC DNA was digested with EcoR I (New England Biolabs,

Schwalbach, Germany) and separated on 0.8% agarose gels in order to obtain

internal BAC DNA sequences. The resulting fragments were cloned into the

polylinker of pGEM-4Z (Promega, Mannheim, Germany) and then transformed into

XL1-Blue competent Escherichia coli. Montage Plasmid Miniprep kit (Millipore,

Molsheim, France) was used to isolate 16 subclones for sequencing. BLASTN

sequence comparisons of the equine BAC sequences were performed against Build

35.1 of the human genome sequence. In order to verify the selected genes on the

equine BAC clones, sequence primers were derived using human exonic sequences

of these genes. Amplification products of these internal BAC sequences were verified

by BLASTN sequence comparisons with the Build 35.1 of the human genome

sequence. Primers for PCR amplification from the BAC sequences were designed

using the PRIMER3 software (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi).


- 72 -

The results of the BLASTN sequence comparisons of the equine BAC end and

internal sequences with the Build 35.1 of the human genome sequences are shown

in Table 1.

Chromosomal location: The equine BAC clones were labelled with digoxygenin by

nick translation using a nick-translation mix (Roche Diagnostics, Mannheim,

Germany). FISH on GTG-banded horse chromosomes was performed using 500 ng

digoxygenin labelled BAC DNA, and 20 µg sheared total equine DNA and 10 µg

salmon sperm DNA as competitors. After hybridization overnight, signal detection

was performed using a digoxygenin-FITC detection kit (Qbiogene, Heidelberg,

Germany). The chromosomes were counterstained with DAPI (4’,6’-diaminidino-2-

phenylindole) and propidium iodide and embedded in antifade. Previously

photographed metaphase spreads were re-examined for hybridization signals using a

Zeiss Axioplan 2 microscope (Zeiss, Jena, Germany) equipped for fluorescence.

Identification of chromosomes followed the international system for chromosome

nomenclature of domestic horses (ISCNH 1997)26. The equine genomic BAC clones

containing the respective genes were located to equine chromosomes by

examination of at least 30 metaphase spreads (Fig. 1a-g).

Radiation hybrid mapping: The 5,000-rad TAMU equine radiation hybrid (RH) panel27

was genotyped to map the SP6 or T7 BAC end markers. PCR was carried out in a 20

µl reaction containing 25 ng RH cell line DNA, 15 pmol of each primer and 0.75 U

Taq polymerase (Qbiogene, Heidelberg, Germany). The reaction conditions started

with a denaturing step at 94°C for 5 min followed by 34 cycles using the following

protocol: denaturation for 45 s at 94°C, annealing for 45 s at 60°C and extension for

5 min at 72°C. The PCR was completed with a final cooling at 4 °C for 5 min. PCR

products were separated on a 1.5% agarose gel. After scoring positive signals, a

two-point analysis28 was performed using RHMAPPER-1.22

(http://equine.cvm.tamu.edu/cgi-bin/ecarhmapper.cgi) against 861 equine markers

mapped previously on the first generation whole genome equine RH map. The FISH

and RH mapping results and the conserved synteny to human chromosomes are

given in Table 2. The lod scores of all closest linked markers were > 3.0. The RH

results confirmed the results obtained by FISH.


- 73 -

Comment: The physical assignments of these seven equine genes agreed with the

current equine-human comparative RH map27,29.

Acknowledgement: This study was supported by grants of the German Research


References

1 International Human Genome Sequencing Consortium (2004) Nature 431, 931-45.

2 Kerner S. A. et al. (1989) Proc Natl Acad Sci USA 86, 4455-9.

3 Yamada Y. et al. (2003) J Clin Endocrinol Metab 88, 3372-8.

4 Deng H. W. et al. (2002) J Bone Miner Res 17, 678-86.

5 Deloukas P. et al. (2001) Nature 414, 865-71.

6 Majumdar M. K. et al. (2001) J Cell Physiol 189, 275-84.

7 Valcourt U. et al. (2002) J Biol Chem 277, 33545-58.

8 Connor J., Evans D.A.P. (1982) J Bone Joint Surg 64, 76-83.

9 Akama T.O. et al. (2002) J Biol Chem 277, 42505-13.

10 Martin J. et al. (2004) Nature 432, 988-94.

11 Arriza J. L. et al. (1994) J Neurosci 14, 5559-69.

12 Stoffel W. et al. (1996) FEBS Lett 386,189-93.

13 Schmutz J. et al. (2004) Nature 431, 268-74.

14 Keppen L. D. et al. (1992) Am J Med Genet 44, 356-60.

15 Scott H. L. et al. (2002) J Neurosci 22, RC206.

16 Palumbo A. P. et al. (1986) Am J Hum Genet 39, 307-16.

17 Langdon R. G., Holman V.P. (1988) Biochim Biophys Acta 945, 23-32.

18 Tanner M.J. (2002) Curr Opin Hematol 9,133-9.

19 Sritippayawan S. et al. (2004) Am J Kidney Dis 44, 64-70.

20 Shayakul C. et al. (2004) Nephrol Dial Transplant 19, 371-9.

21 Allen S. J. et al. (1999) Am J Trop Med Hyg 60, 1056-60.

22 Szaszi K. et al. (2002) J Biol Chem 277, 42623-32.

23 Kavanaugh M.P. et al. (1994) Proc Natl Acad Sci 91, 7071-5.

24 Palmer G. et al. (1999) Gene 226, 25-33.


- 74 -

25 O'Hara B. et al. (1990) Cell Growth Diff 1, 119-27.

26 Bowling A. T. et al. (1997) Chromosome Res 5, 433-43.

27 Chowdhary B. P. et al. (2003) Genome Res 13, 742-51.



Figure 1 Chromosomal assignment of the equine BACs containing a) BGLAP, b)

BMP2, c) CHST4, d) SLC1A3, e) SLC4A1, f) SLC9A5 and g) SLC20A5 by FISH

analysis. G-banded metaphase spread before (left) and after (right) hybridization.

Double signals indicated by arrows are visible on both equine chromosomes.

(a)

(b)


- 75 -

(c)

(d)

(e)


- 76 -

(f)

(g)


- 77 -

Table 1

Selected genes, their human location and cDNA clone identity, insert size of the

positive equine BAC clones, used BAC end sequence, Accession no., BLASTN

sequence comparisons of equine BAC sequences with human genome and their

locations on human genome (Build 35.1).

Human Human or murine BAC

location (bp)

cDNA clone identity insert size (kb)

Gene HSAa

(Build 35.1)

BAC end or internal (IN) sequence

Accession no.

SP6 AJ937344

T7 AJ937345

BGLAP

1q25-q31

153,025, 078 153,026, 185 IMAGp998K204334b

CHORI-241-256K13 195 IN AJ937346

T7 AJ871594

BMP2

20p12 6,697,207 6,707,769 IMAGp998G015200b

CHORI-241-159E8 230 IN CX593098

SP6 AJ937339

T7 AJ937340

CHST4

16q22.2

70,117, 560 70,129, 917 IMAGp998I158096b

CHORI-241-84A13 221 IN AJ937341

SCd AJ937342

SLC1A3

5p13

36,642, 446 36,724, 195 IRALp962P1337c

CHORI-241-406K6 200 IN AJ937343

T7 AJ937334

SLC4A1

17q21-q22

39,682, 566 39,700, 993 IMAGp998C13535c

CHORI-241-313L9 190 IN AJ937333


- 78 -

Table 1 continued

Human Human or murine BAC

location (bp)

cDNA clone identity insert size (kb)

Gene HSAa

(Build 35.1)


Accession no.

SP6 AJ937330 T7 AJ937331

SLC9A5

16q22.1

65,840, 365 65,863, 595 IMAGp998M093449c

CHORI-241-141P15 145 IN AJ937332

SP6 AJ937336

SLC20A1

2q11-q14

113,119, 758 113,137, 633 IRAKp961B1026b

CHORI-241-172C18 170 IN AJ937338

Table 1 continued

Identity Beginning of Distance to

Match to human

Gene BLAST E-Value

Length of match (bp) (%) sequence

alignment on HSA (bp)

selected gene (kb)

gene (Build 35.1)

1.4e-55 217 88% 153,247,625 222 exon 12 of MEF2D

41 92% 152,988,960 36 -

2.0e-5 58 87% 152,988,870 36 -

BGLAP

1.0e-4 46 89% 153,025,659 0 exon 3 of BGLAP

1.1e-134 385 85% 6,807,012 99 - BMP2

0.0 578 90% 6,706,890 0 exon 3 of BMP2


- 79 -

Table 1 continued

Identity Beginning of

Distance to

Match to human

Gene BLAST E-Value



selected gene (kb)

gene (Build 35.1)

2.0e-19 112 87% 69,910,086 208 -

4.0e-3 73 84% 70,131,053 1,1 -

4.0e-6 122 81% 70,131,184 1,1 -

5.0e-129 624 84% 70,128,532 0 exon 2 of CHST4

CHST4

4.0e-37 130 90% 70,128,362 0 exon 2 of CHST4

3.0e-11 236 86% 36,786,831 63 -

2.0e-61 408 83% 36,786,935 63 -

1.0e-111 443 84% 36,706,960 0

exon 4 and intron 4 of SLC1A3

SLC1A3

4.0e-13 110 80% 36,707,388 0 -

3.7e-41 120 87% 39,680,601 2 - SLC4A1

4.0e-53 288 82% 39,690,640 0 exon 10 of SLC4A1

4.0e-179 457 92% 65,911,858 48 exon 2 of FLJ40162

3.0e-15 57 96% 65,729,633 111 intron 8 of Lin10

3.0e-59 251 82% 65,862,389 0 exon 16 of SLC9A5

SLC9A5

3.0e-59 171 79% 65,862,807 0 exon 16 of SLC9A5


- 80 -

Table 1 continued

Identity Beginning of Distance to

Match to human

Gene BLAST E-Value



selected gene (kb)

gene (Build 35.1)

5.5e-61 190 91% 113,182,799 45 - SLC20A1

2.0e-88 252 91% 113,133,335 0 exon 8 of SLC20A1

a: HSA- homo sapiens autosome

b: human cDNA clone

c: murine cDNA clone

Table 2

Selected genes, equine BAC ends and PCR primer sequences used for radiation

hybrid (RH) mapping on the 5,000-rad TAMU equine RH panel27, equine genome

location by FISH and RH mapping, and conserved synteny to the human genome

(Build 35.1).

PCR Gene BAC enda Primer Sequences (5`- 3`)

(bp)

F: ATG GGT GGA TCT GGG TAC TG

BGLAP SP6 R: GGA GGA AGA CTG GCA CAG AT 175

F: TTT GGA CTG CTT AAA CCA TTG

BMP2 T7 R: CTT GCC CAA AGG ACC TCT C 226

F: CTG TTG TCT GCC ACT AAG AAT G

CHST4 SP6 R: TGT GTG CCA TTA GTG GAA TG 751

F: AAC AAG CCT TCAT CAT CAG C

SLC1A3 SCb R: ATT GGC TTT GTG TTT GCT TC 197

F: TTT TGC CTT AGT TCC GTG TC

SLC4A1 T7 R: ATA TGG TAC TTG GGC CTT GC 188


- 81 -

Table 2 continued

PCR Gene BAC enda Primer Sequences (5`- 3`)

(bp)

F: GCC AGA GGC TAT TCA TCG AC

SLC9A5 T7 R: AAG AGG GTC ACC TCA GCA AC 249

F: ACA ATA GCT GCC TGG CAT AG

SLC20A1 T7 R: TTC TGC CTG CAT TTA GCT TC 222

Table 2 continued

RH-mapping Location on horse genome

Conserved synteny to

Gene

Closest marker Distance (cR)f RET (%)g ECAd HSAe

BGLAP VHL66 51.42 20.70% 5p12-p13 1q21-q32

BMP2 HTG14 17.91 19.60% 22q14 20p12-p13

CHST4 AHT022 4.5 cR 21.70% 3p13 16q

SLC1A3 IL7R 0.0 19.60% 21q17 5p12-p14

SLC4A1 SG24 5.2 cR 26.10% 11p12.1-p12.3 17q21-q25

SLC9A5 CBFB 0.10 22.80% 3p13 16q12-q24

SLC20A1 IL1B 0.0 19.60% 15q13 2q13-q21

Chapter 6

Physical mapping of the ATP2A2 gene to equine

chromosome 8p14->p12 by FISH and confirmation by

linkage and RH mapping

D. Müller*, H. Kuiper*, C. Böneker*, C. Drögemüller, * J.E. Swinburne, M. Binns, B.P.

Chowdhary and O. Distl*


Hannover, Hannover, Germany. †Department of Veterinary Integrative Biosciences,

College of Veterinary Medicine and Biomedical Sciences, Texas A&M University,

College Station, TX 77843, USA

Accepted for publication 16 December 2005

Published in: Cytogenetic and Genome Research 114 (2006) 94

Physical mapping of the ATP2A2 gene to equine chromosome 8p12-p14 by FISH and confirmation by linkage and RH mapping

- 85 -

Physical mapping of the ATP2A2 gene to equine

chromosome 8p12-p14 by FISH and confirmation by

linkage and RH mapping

Rationale and significance

The ATPase, Ca++ transporting, cardiac muscle, slow twitch 2 gene (ATP2A2)

encodes one of the SERCA Ca(2+)-ATPases, which are intracellular pumps located

in the sarcoplasmic or endoplasmic reticula of muscle cells1,2. This enzyme catalyzes

the hydrolysis of ATP coupled with the translocation of calcium from the cytosol to

the sarcoplasmic reticulum lumen, and is involved in regulation of the

contraction/relaxation cycle1,2. ATP2A2 maps to HSA12q23-q24.13 at 109,182,152 to

109,251,615 and consists of 21 exons spanning 69464 bp4. Mutations in the ATP2A2

gene cause the autosomal dominantly inherited keratosis follicularis, also known as

Darier-White disease, characterized by loss of adhesion between epidermal cells and

abnormal keratinization5,6,7.

Further studies including a null mutation in one copy of the ATP2A2 gene in mice

revealed that serca2 protein levels were reduced by about a third, sarcoplasmic

reticulum calcium stores decreased and in isolated cardiomyocytes released. In

addition, these mice had reduced myocyte contractility8. Aged heterozygous mutant

(Atp2a2 +/-) mice developed squamous cell tumors, which led to the conclusion that

Atp2a2-haploinsufficiency predisposes murine keratinocytes to neoplasia, and that

perturbation of Ca(2+) homeostasis or signaling can be a primary initiating event in

cancer9.

Materials and methods

BAC isolation and characterization of the equine ATP2A2 clone

The equine BAC library CHORI-241 was screened for a BAC clone containing the

ATP2A2 gene. High density BAC colony filters were probed according to CHORI

protocols (http://bacpac.chori.org) with a heterologous 32P-labelled insert of a human

ATP2A2 cDNA clone (IMAGp998K2010072) from the Resource Center/Primary


- 86 -

Database of the German Human Genome Project (http://www.rzpd.de/). An equine

genomic BAC clone with an insert of approximately 220 kb containing the ATP2A2

gene was identified and designated CH241-167G5. BAC DNA was prepared from the

BAC clone using the Qiagen plasmid midi kit (Qiagen, Hilden, Germany). BAC DNA

end sequences were obtained using the ThermoSequenase kit

(AmershamBiosciences, Freiburg, Germany) and a LI-COR 4200 automated

sequencer (LI-COR Inc., Lincoln, NE, USA). The CH241-167G5 SP6 and T7 end

sequences were deposited in the EMBL nucleotide database under accession nos.

AJ937347 and AJ937348. A BLASTN sequence comparison of the equine SP6 BAC

end sequence with the Build 35.1 of the human genome sequence revealed four

significant matches (BLAST E-value of each match 1.4e-65). The first match was over

101 bp (identity = 89.1 %), which started at 109,369,448 bp of HSA12q24.11

approximately 118 kb downstream of human ATP2A2. The further BLASTN matches

over 63, 61, and 30 bp started at 109,369,792 bp, 109,369,269 bp and 109,369,371

bp of HSA12q24.11. Two of the three BLASTN matches were located within exon 1

of the HSU79274 (protein predicted by clone 23733) on HSA12q24.11.

The identity of the BAC clone with respect to the presence of the ATP2A2 gene was

further verified by obtaining DNA sequences from a shotgun plasmid library

generated from the BAC. For this, DNA from the clone CH241-167G5 was isolated

using the Qiagen Large Construct kit (Qiagen, Hilden, Germany). BAC DNA was

mechanically sheared to obtain fragments of approximately 2kb, and subsequently

used to construct a shotgun plasmid library. In total 96 plasmid subclones were

sequenced with the ThermoSequenase kit (AmershamBiosciences, Freiburg,

Germany) and a LICOR 4200 automated sequencer (LI-COR Inc., Lincoln, NE,

USA). Sequence data were analyzed with Sequencher 4.1.4 (GeneCodes, AnnArbor,

MI, USA). Sequences of the shotgun plasmid library (accession no. AJ937349) when

compared with the human genome sequence (Build 35.1) revealed a significant

match (BLAST E-value 1e-64) over 228 bp (87 % identity) starting at 109,223,486 with

exon 6 and parts of intron 5 and 6 of the human ATP2A2 gene. A (GT)25 dinucleotide

repeat was identified in the sequence was identified in the sequence, and flanking

PCR primers ATP2A2_MS5_F: 5`-TAA GAA GAA AGA GAG TGA TG-3`and


- 87 -

ATP2A2_MS5_R: 5`-GCT TTT CGT TTG GAA TAA ACC-3` were obtained for the

microsatellite (AJ937349) using the PRIMER3 software (http://frodo.wi.mit.edu/cgi-


PCR conditions and microsatellite analysis

The PCR amplification (12 µl final volume) was performed on a PTC 200TM thermal

cycler (MJ Research, Watertown, MA, USA) using 20 ng of genomic equine DNA, 1.2

µl 10 x PCR buffer, 0.24 µl DMSO, 0.2 µl dNTPs (5 mM each), 0.5 µl of each primer

(10 pmol/µl) and 0.5 Taq DNA polymerase (Qbiogene, Heidelberg, Germany). One

primer of the pair was endlabeled with fluorescent IRD 700 to enable flourescent

PCR fragment detection. Reaction started with a denaturing step at 94ºC for 4 min

followed by 35 cycles using the following protocol: denaturation for 30 s at 94ºC,

annealing for 30 s at 58ºC and extension for 45 s at 72ºC. The PCR was completed

with a final cooling at 4ºC for 20 min. Raw data were genotyped using Gene Profiler

3.55 software (Scanalytics Inc., Fairfax, USA). Marker characteristics were

determined by genotyping horses of three different breeds and the two three-

generation, full-sib reference families with 67 offspring created at the Animal Health

Trust, Newmarket, UK (NRF)13 using the above mentioned PCR primer pair. (Table

1).

Chromosome preparation

Equine metaphase spreads for FISH on GTG-banded chromosomes were prepared

using phytohemagglutinin stimulated blood lymphocytes from a normal horse. Cells

were harvested and slides prepared using standard cytogenetic techniques. Prior to

fluorescence in situ hybridization the chromosomes were GTG-banded and well-

banded metaphase chromosomes were photographed using a highly sensitive CCD

camera and IPLab 2.2.3 software (Scanalytics).

Fluorescence in situ hybridization

The equine BAC clone CH-241-167G5 containing the equine ATP2A2 gene was

labelled with digoxygenin by nick translation using a nick-translation mix (Roche


- 88 -

Diagnostics, Mannheim, Germany). FISH on GTG-banded horse chromosomes was

performed using 500 ng of digoxygenin labelled BAC DNA, and 20 µg sheared total

equine DNA and 10 µg salmon sperm DNA were used as competitors. After

hybridization overnight, signal detection was performed using a digoxygenin-FITC

detection kit (Qbiogene, Heidelberg, Germany). The chromosomes were

counterstained with DAPI (4’,6’-diaminidino-2-phenylindole) and propidium iodide and

embedded in antifade. Previously photographed metaphase spreads were re-

examined for hybridization signals using a Zeiss Axioplan 2 microscope (Zeiss, Jena,

Germany) equipped for fluorescence. Chromosomes were identified according to the

international system for chromosome nomenclature of domestic horses (ISCNH

1997)10. The equine genomic BAC clone CH241-167G5 containing the ATP2A2 gene

was mapped to ECA8p12-p14 following examination of 37 metaphase spreads (Fig.

1).

Probe name: CH241-167G5

Probe type: BAC clone from equine genomic BAC library CHORI-241

Insert size: 220 kb

Vector: pCMV-Sport6

Proof of authenticity: PCR and DNA sequencing

Gene reference for the human ATP2A2 gene: Sakunthabhai et al. (1999)

Radiation hybrid mapping

To confirm the cytogenetic assignment the 5,000-rad TAMU equine radiation hybrid

(RH) panel11 was genotyped to map the SP6 BAC end marker located about 118 kb

downstream of ATP2A2. PCR was carried out in a 20 µl reaction containing 25 ng

RH cell line DNA, 10 pmol of each primer and 0.85 U Taq polymerase (Qbiogene,

Heidelberg, Germany). The reaction conditions started with a denaturing step at 94°C

for 5 min followed by 34 cycles using the following protocol: denaturation for 45 s at

94°C, annealing for 45 s at 60°C and extension for 5 min at 72°C. The PCR was

completed with a final cooling at 4 °C for 5 min. PCR products were separated on a

1.5% agarose gel. After scoring positive signals, a two-point analysis12

(http://equine.cvm.tamu.edu/cgi-bin/ecarhmapper.cgi) was conducted to find


- 89 -

associations between ATP2A2 versus the markers of the first generation whole

genome equine RH map. This sequence tagged site (STS) marker showed a

retention frequency of 21.7 % and revealed close linkage to UM034 (22.06 cR; LOD

>3.0). The linked microsatellite marker had been previously mapped on ECA8 by RH

mapping11. Thus the FISH and RH results corroborate each other. The most closely

linked equine genes, SART3 (squamous cell carcinoma antigen recognised by T

cells 3) and FLJ11021 (similar to splicing factor, arginine/serine-rich 4), on ECA8

were also mapped on HSA12q24 and HSA12q24.3111.

Comment: The physical assignment of the equine ATP2A2 gene on ECA8p12-p14

agrees with the current equine-human comparative ECA8 RH map, which showed

conserved synteny to HSA12q2411.

Linkage mapping

The two full-sib reference families with 67 offspring (NRF)13 were genotyped to map

the microsatellite marker located within intron 5 of ATP2A2. PCR conditions, primer

pairs and fragment analysis were as described above. Linkage analysis was

performed using CRI-MAP program version 2.4 (Green et al. 1990). Maximum

likelyhood estimates of recombination fraction (theta) were calculated using the

TWO-POINT option with a lod-score threshold > 3 to determine linkage group and

chromosomal assignment versus the markers of the comprehensive horse linkage

map including 626 markers linearly ordered and 140 markers assigned to a

chromosomal region14.

Results and discussion

Chromosomal location

The equine genomic BAC clone CH241-167G5 containing the ATP2A2 gene was

mapped to ECA8p12-p14 following examination of 37 metaphases spreads (Fig.1).

Mapping data

Most precise location: ECA8p12-p14

Number of cells examined: 37


- 90 -

Number of cells with specific signals: 0(10), 1(4), 2(9), 3(7), 4(7) chromatids per cell

Location of background signals (site with > 2 signals): none observed

RH analysis of the sequence tagged site (STS) marker of the BAC clone CH241-

167G5 showed a retention frequency of 21.7%. Two-point linkage analysis revealed

close linkage to the microsatellite marker was previously mapped on ECA8 at 58.7

cM of the horse linkage map14 and by RH-mapping at 17.3 cR of ECARH08b11. The

microsatellite ATP2A2_MS5 showed recombination fractions of 0.06 with ECA

microsatellites LEX023 (Lod score = 23.7) at 71.0 cM and UCDEQ46 (Lod score =

17.8) at 58.7 cM. Thus the FISH, RH and genetic linkage results corroborate each

other. The most closely linked equine genes, SART3 (Squamous cell carcinoma

antigen recognised by T-cells 3) and FLJ11021 (similar to splicing factor,

arginine/serine-rich 4), on ECA8 were also mapped on HSA12q24 and

HSA12q24.3111.

The physical assignment of the equine ATP2A2 gene on ECA8p12-p14 agrees with

the current equine-human comparative ECA8 RH map, which showed conserved

synteny to HSA12q2411.

Acknowledgements

This study was supported by grants of the German Research Council (DFG), Bonn

(DI 333/12-1).

References

1 MacLennan D. H. et al. (1985) Nature 316, 696-700.

2 Ruiz-Perez V. L. et al. (1999) Hum Molec Gene. 8, 1621-30.

3 Otsu K. et al. (1993) Genomics 17, 507-9.

4 International Human Genome Sequencing Consortium (2004) Nature 431, 931-45.

5 Sakuntabhai A. et al. (1999) Nat Genet 21, 271-7.

6 Sakuntabhai A. et al. (1999) Hum Molec Genet 8, 1611-9.

7 Jacobsen N. J. O. et al. (1999) Hum Molec Genet 8, 1631-6.


- 91 -

8 Ji Y. et al. (2000) J Biol Chem 275, 38073-80.

9 Liu L. H. et al. (2001) J Biol Chem 276, 26737-40.




13 Swinburne et al. (2000) Genomics 66: 123-134

14 Penedo et al. (2005) Cytogenet. Genome Res. 111: 5-15

Figure 1 Chromosomal assignment of the equine BAC CH241-167G5 containing

ATP2A2 by FISH analysis. G-banded metaphase spread before (left) and after (right)

hybridization. Double signals indicated by arrows are visible on both ECA8

chromosomes.


- 92 -

Table 1

Characterization of the newly developed ATP2A2-associated microsatellite

ATP2A2_MS5

Breed/source Sample size No. of alleles

Allele size min-max

(bp)

Expected heterozygozity

PICa

South German coldblood 10 8 131-177 0.90 0.76 Saxon- Thuringa coldblood 6 5 131-177 0.83 0.68

Hannoverian warmblood 10 7 131-171 0.90 0.76

NRFb 67 8 135-171 0.87 0.72 aPolymorphism Information Content; Hardy-Weinberg equilibrium in each population

genotyped; bNRF:

Chapter 7

Assignment of the COL8A2 gene to equine chromosome

2p15-p16 by FISH and confirmation by RH mapping

C. Böneker*, D. Müller*, H. Kuiper*, C. Drögemüller*, B.P. Chowdhary+ and O. Distl*


Hannover, Hannover, Germany. +Department of Veterinary Integrative Biosciences,

College of Veterinary Medicine and Biomechanical Sciences, Texas A & M

University, College Station, TX 77843, USA.

Accepted for publication 1 May 2005


Assignment of the COL8A2 gene to equine chromosome 2p15-p16 by FISH and confirmation by RH-mapping

- 95 -

Assignment of the COL8A2 gene to equine chromosome

2p15-p16 by FISH and confirmation by RH mapping

Source/description: The human collagen, type VIII, alpha 2 (COL8A2) gene encodes

the alpha 2 chain of type VIII collagen, which is a major component of the Descemet

basement of corneal endothelial cells. The human gene encoding the alpha 2 chain

of type VIII collagen consists of two exons spanning about 5,005 bp. The COL8A2

gene has been assigned to HSA1p34.3-p32.3 at 36,229,939 bp to 36,234,943 bp1. A

region of 6 to 7 cM on human chromosome 1p34.3-p32, which includes the COL8A2

gene, was linked in a family with early-onset FECD (Fuchs endothelial corneal

dystrophy)2. Analysis of the coding sequence of COL8A2 defined a Gln 455 Lys

missense mutation in this family2. Analysis in other FECD patients demonstrated

further missense substitutions in familial and sporadic cases of FECD, as well as in a

single family with posterior polymorphous corneal dystrophy (PPCD)2. The corneal

endothelial dystrophies arise from dysfunction of the corneal epithelium leading to

corneal opacification2. The underlying pathogenesis of FECD and PPCD may be

related to a disturbance of the role of type-VIII collagen in its influence on the

terminal differentiation of the neural crest-derived corneal endothelial cell2, so that

COL8A2 is a candidate gene for corneal endothelial dystrophies.

The equine BAC CHORI-241 library was screened for a BAC clone containing the

COL8A2 gene. High-density BAC colony filters were probed according to CHORI

protocols (http://www.chori.org/bacpac/) with a heterologous 32P-labelled insert of a

human COL8A2 cDNA clone (IMAGp998A122908) from the Primary Database of the

Resource Center of the German Human Genome Project (http://www.rzpd.de/). An

equine genomic BAC clone with an insert of approximately 170 kb containing the

COL8A2 gene was identified and designated CH241-184G10. BAC DNA was

prepared from 100 mL overnight cultures using the Qiagen Midi plasmid kit according

to the modified protocol for BACs (Qiagen, Hilden, Germany). BAC ends were

sequenced using the ThermoSequenase kit (Amersham Biosciences, Freiburg,

Germany) and a LI-COR 4300 automated sequencer (LI-COR Inc., Lincoln, NE,

USA). The BAC end sequences were deposited in the EMBL nucleotide database


- 96 -

(accession nos. AJ891045 and AJ891046). BLASTN analysis of the CH241-184G10

SP6 BAC end against Build 35.1 of the human genome revealed a significant match

on HSA1p35-p34 located approximately 181 kb upstream of the human COL8A2

gene (identity = 88%, BLAST E-value 1e-41) in the human EIF2C1 gene starting at

36,048,900 bp in exon 14. Furthermore, a BLASTN sequence comparison of an

equine BAC sequence derived from an equine expressed sequence tag (EST,

accession no. CX605143) corresponding to human COL8A2 gave three significant

non-overlapping matches (BLAST E-value 1e-34) located in exon 2 of human COL8A2

gene starting at 36,231,100 bp (Build 35.1). The first match was over 107 bp (identity

= 84%), the second match over 111 bp (identity = 84%) and the third match over 165

bp (identity = 86%).

Primer sequences: Primers for PCR amplification of a 113 bp product from the

CH241-184G10 SP6 BAC end sequence (accession no. AJ891045) and a 590 bp

internal BAC sequence matching with exon 2 of human COL8A2 (accession no.

CX605143) were designed using the PRIMER3 software (http://frodo.wi.mit.edu/cgi-


SP6 BAC end sequence:

F: 5’-TGA GCC CCC AAG ACT ACA C-3’

R: 5’-TGA AGA TAC CAA GGG AGT GG-3’

Internal BAC sequence corresponding to the human COL8A2 gene:

F: 5'-CTG AGA AGA AAG GTG CCA AC-3'

R: 5'-CAG GAG CTG ATG AAT TTT CG-3'

Chromosome location: Equine metaphase spreads for fluorescence in situ

hybridisation (FISH) on GTG-banded chromosomes were prepared using pokeweed

mitogen stimulated blood lymphocytes. BAC clone CHORI241-184G10 was

digoxigenin labelled by nick translation using a nick translation mix (Roche

Diagnostics, Mannheim, Germany). FISH on the GTG-banded horse chromosomes

was performed using 750 ng digoxigenin-labelled BAC DNA, and 20 µg sheared total

equine DNA and 10 µg salmon sperm DNA as competitors. After hybridisation

overnight, signal detection was performed using a rhodamin detection kit (Qbiogene,

Heidelberg, Germany). The chromosomes were counterstained with DAPI (4’,6’-


- 97 -

diaminidino-2-phenylindole) and embedded in Vectashield Mounting Medium (Vector

Laboratories, Burlingame, USA). Metaphase chromosomes that had been previously

photographed with a CCD camera were re-examined after hybridisation with a Zeiss

Axioplan 2 microscope (Zeiss, Jena, Germany) equipped for fluorescence.

Chromosomes were identified according to the international system for chromosome

nomenclature of the domestic horse (ISCNH 1997)5. The equine genomic BAC clone

containing the COL8A2 gene was most precisely located to ECA2p15-p16 on

metaphase chromosomes of 30 cells (Fig.1).

Radiation hybrid mapping: To confirm the chromosomal location of the BAC clones,

the Texas A&M University equine 5,000-rad hybrid panel6

(http://equine.cvm.tamu.edu/cgi-bin/ecarhmapper.cgi) was used to map the

sequence tagged site (STS) marker of the SP6 BAC end located approximately 181

kb upstream of COL8A2. PCR reactions were performed in a total of 20 µl using

either 25 ng RH cell line DNA or 25 ng DNA from BAC clone CH241-184G10, 15

pmol of each primer and 0.75 U Taq polymerase (Qbiogene, Heidelberg, Germany).

Samples were denatured at 94 °C for 4 min followed by 35 cycles under the following

conditions: denaturation for 30 s at 94 °C, annealing for 60 s at 60 °C, and extension

for 40 s at 72 °C. The PCR was completed with a final cooling at 4 °C for 10 min.

PCR products were separated on a 1.5% agarose gel. The presence and absence of

a PCR product were scored and the results used for a two-point analysis with

RHMAPPER-1.227 to find markers co-segregating with COL8A2 on the equine

RH5,000 panel8. The retention frequency of the sequence tagged site (STS) marker of

the SP6 BAC end sequence was 14.1% and the two-point analysis revealed linkage

of CHORI241-184G10_SP6 at a distance of 22.06 cR to the RBBP4 gene. The

corresponding LOD score was 12. The RBBP4 gene had been previously located by

RH mapping at 0 cR on ECARH02b8 as well as on HSA1p35.1 at 31,785,917 bp to

32,815,347 bp (human genome map viewer build 35.1).

Comment: The RH and FISH mapping results of the equine COL8A2 gene on

ECA2p15-p16 agree with comparative mapping of the current equine-human

comparative RH and cytogenetic map8 of ECA2p, which shows conserved synteny to

HSA1p.


- 98 -



References

1 Muragaki Y. et al. (1991) J Biol Chem 266, 7721-7.

2 Biswas S. et al. (2001) Hum Mol Genet 10, 2415-23.

3 Perala M. et al. (1993) FEBS Lett 319, 177-80.

4 Warman M. L. et al. (1994) Genomics 23,158-62.


6 Chowdhary B. P. et al. (2002) Mamm Genome 13, 89-94.

7 Slonim D. et al. (1997) J Comp Biol 4, 487-504.


Figure 1 Chromosomal assignment of the equine BAC clone CH241-184G10

containing the COL8A2 gene by FISH analysis. (A) GTG-banded horse metaphase

spread. (B) Double signals visible on ECA2 are indicated by arrows.

Chapter 8

Assignment of the TYK2 gene to equine chromosome 7q12-

q13 by FISH and confirmation by RH mapping

Dorothee Stübs

Assignment of the TYK2 gene to equine chromosome 7q12-q13 by FISH and confirmation by RH mapping

- 101 -

Assignment of the TYK2 gene to equine chromosome 7q12-

q13 by FISH and confirmation by RH mapping

Background: The tyrosine kinase 2 gene (TYK2) is a member of the janus kinase

gene family and encodes an 1187 amino acid protein. All four members of the janus

kinase family JAK1, JAK2, JAK3, and TYK2 associate with various cytokine

receptors and mediate the signal transduction by tyrosine phosphorylation of

downstream targets (YAMOAKA et al., 2004). In addition to its signaling function in

humans, TYK2 is also necessary for the correct localization of the interferon receptor

1 (IFNAR1) in the cell membrane (RAGIMBEAU et al., 2003). Studies with Tyk2

deficient mice demonstrated that these mice are viable and show very specific

changes in several cytokine responses. Surprisingly, interferon a/ß signaling is only

impaired but not completely abolished in these mice (KARAGHIOSOFF et al., 2003).

Mutations in the murine Tyk2 gene are associated with increased susceptibility to

infectious and autoimmune diseases (SHAW et al., 2003). The human TYK2 gene

consists of 25 exons spanning 30003 bp on human chromosome 19p13.2 starting at

10,322,209 bp and ending at 10,352,211 bp (International Human Genome

Sequencing Consortium, 2004). The objective of this study was to determine the

chromosomal location of TYK2 in the horse by FISH and RH mapping.

Procedure:

BAC library screening/sequence analysis: The equine BAC library CHORI-241 was

screened to isolate a genomic BAC clone containing the TYK2 gene. High density

BAC colony filters were probed according to the CHORI protocols

(http://bacpac.chori.org) with a heterologous 32P-labelled insert of a human TYK2

cDNA clone (IRALp962L0830) provided by the Resource Center/Primary Database

of the German Human Genome Project (http://www.rzpd.de/). An equine genomic

BAC clone designated CH241-352C1 with an insert of approximately 100 kb

containing the TYK2 gene was identified. BAC DNA was prepared from the BAC

clone using the Qiagen plasmid midi kit (Qiagen, Hilden, Germany). BAC DNA


- 102 -


Freiburg, Germany) and a LI-COR 4200 automated sequencer (LI-COR, Inc.,

Lincoln, Nebr., USA). A BLASTN sequence comparison of the equine SP6 BAC end

sequence with the build 35.1 of the human genome sequence revealed a significant

match (BLAST E-value 6.0e-43) over 196 bp (identity = 87%) starting at 10,606,427

bp of HSA19p13.2, approximately 246 kb downstream of human TYK2.

Chromosomal location: The equine BAC clone was labelled with digoxygenin by nick

translation using a nick-translation mix (Roche Diagnostics, Mannheim, Germany).

FISH on GTG-banded horse chromosomes was performed using 500 ng of

digoxygenin labelled BAC DNA. 20 µg sheared total equine DNA and 10 µg salmon

sperm DNA were used as competitors in this experiment. After hybridization

overnight, signal detection was performed using a Digoxygenin-FITC Detection Kit

(Qbiogene, Heidelberg, Germany). The chromosomes were counterstained with

DAPI (4’,6’-diaminidino-2-phenylindole) and embedded in propidium iodide/antifade.

Metaphase chromosomes that had been previously photographed by using a highly

sensitive CCD camera were re-examined after hybridization with a Zeiss Axioplan 2

microscope (Zeiss, Jena, Germany) equipped for fluorescence. Identification of

chromosomes followed strictly the international system for chromosome

nomenclature of domestic horses (ISCNH 1997).

Primer sequences: Primers for PCR amplification of a 249 bp fragment were

designed from the CH241-352C1 SP6 BAC end sequence using the PRIMER3

software (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi).

F 5’-ATC ACT GTA GGG GGC AGA GA-3’

R 5’-CTG GTG TCT CTG TGC AGG AA-3’


assignment the 5,000 rad TAMU equine radiation hybrid panel (CHOWDHARY et al.,

2003) was used to map TYK2. PCR was carried out in a 20 µl reaction containing 25

ng of RH cell line DNA, 10 pmol of each primer and 0.85 U Taq polymerase

(Qbiogene, Heidelberg, Germany). The reaction conditions started with a denaturing

step at 94°C for 5 min followed by 34 cycles using the following protocol:

denaturation for 45 s at 94°C, annealing for 45 s at 60°C and extension for 5 min at


- 103 -

72°C. The PCR was completed with a final cooling at 4°C for 5 min. PCR products

were separated on a 1.5% agarose gel. After scoring positive signals, a two-point

analysis (http://equine.cvm.tamu.edu/cgi-bin/ecarhmapper.cgi) was conducted to find

associations between TYK2 versus the markers of the first generation whole genome

equine RH map.

Results:

The equine genomic BAC clone CH241-352C1 containing the TYK2 gene was

located to ECA 7q12-q13 by examination of metaphase chromosomes of 40 cells

(Fig. 1).

The sequence tagged site (STS) markers showed a retention frequency of 10.9%

and the RH mapping revealed close linkage to HTG33 (6.19 cR; LOD >3.0). The

linked microsatellite marker had been previously mapped on ECA 7 by radiation

hybrid mapping (CHOWDHARY et al., 2003). The RH result confirms the result

obtained by FISH.

The physical assignment of the equine TYK2 gene on ECA7q12-q13 did not agree

with the comparative mapping data of the current equine-human comparative map of

the centromeric region of ECA7p, which showed conserved synteny to HSA11

(CHOWDHARY et al., 2003; MILENKOVIC et al., 2002). Synteny detected here,

however, is in agreement with the actual comparative cytogenetic map

(PERROCHEAU et al., 2006).



References

CHOWDHARY, B.P. et al.: The first-generation whole-genome radiation hybrid map

in the horse identifies conserved segments in human and mouse genomes.

Genome Res., 13, (2003), 742-751


- 104 -

International Human Genome Sequencing Consortium:

Finishing the euchromatic sequence of the human genome. Nature 431 (2004),

931-945

KARAGHIOSOFF, M. et al.:

Central role for type 1 interferons and Tyk2 in lipopolysaccharide-induced

endotoxin shock. Nat. Immunol. 4 (2003), 471-477

MILENKOVIC, D. et al.:



RAGIMBEAU J. et al.:

The tyrosine kinase Tyk2 controls IFNAR1 cell surface expression. EMBO J. 22

(2003) 3, 537-547.

SHAW, M. H. et al.:

A natural mutation in the Tyk2 pseudokinase domain underlies altered

susceptibility of B10.Q/J mice to infection and autoimmunity. Proc. Natl. Acad.

Sci. USA 100 (2003), 11594-11599

YAMAOKA, K. et al.:

The Janus kinases (Jaks). Genome Biology 5 (2004) 253

Figure 1 Chromosomal assignment of the equine BAC containing TYK2 by FISH

analysis. G-banded metaphase spread before (left) and after (right) hybridization.

Double signals indicated by arrows are visible on both equine chromosomes.

Chapter 9

Assignment of CART1, COL9A3, GNRH2 and TCIRG1 to

equine chromosomes by FISH and confirmation by RH

mapping

Dorothee Stübs

Assignment of CART1, COL9A3, GNRH2 and TCIRG1 to equine chromosomes by FISH and confirmation by RH mapping

- 107 -

Assignment of CART1, COL9A3, GNRH2 and TCIRG1 to

equine chromosomes by FISH and confirmation by RH

mapping

Background:

Source/description: The cartilage paired-class homeoprotein 1 gene (CART1) maps

to HSA12q21.3-q22 starting at 84,176,353 bp and ending at 84,198,027 bp

(GORDON et al., 1996). It encodes a 326-amino acid homeoprotein containing a

paired-like domain that is expressed in the forebrain mesenchyme, branchial arches,

limb buds and cartilages during embryogenesis in rodents. It may also be involved in

development of the cervix (ZHAO et al., 1993). In humans the specific functions of

the CART1 gene are not yet known. The human gene consists of 4 exons spanning

21675 bp (GORDON et al., 1996). Mutations in the CART1 gene are associated in

mice with neural tube defects such as acrania and meroanencephaly. Cart1-

homozygous mutant mice are born alive with acrania and meroanencephaly but die

soon after birth. As the phenotype observed here resembles strikingly a

corresponding human syndrome caused by a neural tube closure defect, these mice

may provide a useful animal model for developing therapeutic protocols for neural

tube defects (ZHAO et al., 1996).

The collagen, type IX, alpha 3 (COL9A3) maps to HSA20q13.3 starting at 60,918,832

and ending at 60,942,955 (TILLER et al., 1998). It encodes alpha 3 type IX collagen

with 684 amino acids, one of the three alpha chains of type IX collagen, the major

collagen component of hyaline cartilage (BREWTON et al., 1995). The human gene

consists of 28 exons spanning 24124 bp (DELOUKAS et al., 2001). Type IX collagen

is a heterotrimeric molecule, which is usually found in tissues containing type II

collagen, a fibrillar collagen (MATSUI et al., 2003). Splice site mutations in the

COL9A3 gene are associated with multiple epiphyseal dysplasia (MED) (PAASSILTA

et al., 1999; BONNEMANN et al., 2000; LOHINIVA et al., 2000). The autosomal

dominantly inherited MED is characterized by knee pain, stiffness and difficulty

walking during childhood. Some patients develop hip arthrosis and require surgical


- 108 -

hip replacement after age 50 years. A splice acceptor mutation in intron 2 of the

COL9A3 gene causes autosomal dominant MED affecting predominantly the knee

joints and a mild proximal myopathy. An arg103-to-trp (trp3 allele) substitution in the

COL9A3 gene is supposed to increase the risk of lumbar disc disease about 3-fold

(PAASSILTA et al., 2001).

In addition to the hypothalamic decapeptide gonadotropin-releasing hormone 1

(GNRH1) regulating reproduction by serving as a signal from the hypothalamus to

pituitary gonadotropes, many vertebrate species express a second GNRH form,

GNRH2(WHITE et al., 1998). GNRH2 shares about 70% homology with the

mammalian hypothalamic neurohormone GNRH1, the primary regulator of

reproduction, but is encoded by a different gene. The gonadotropin-releasing

hormone 2 gene (GNRH2) maps to HSA20p13 starting at 2,972,268 and ending at

2,974,391 (International Human Genome Sequencing Consortium, 2004) and

encodes a 120 amino acid protein that is expressed in the midbrain (KASTEN et al.,

1996) and, in contrast to GNRH1, up to 30-fold higher levels outside the brain,

particularly in the kidney, bone marrow, and prostate (PAASSILTA et al., 2001). The

human gene consists of 4 exons spanning 2124 bp (DELOUKAS et al., 2001).

Exposure of normal or cancerous human or mouse T cells to GNRH2 triggered de

novo gene transcription and cell-surface expression of a 67-kD non-integrin laminin

receptor that is involved in cellular adhesion and migration and in tumor invasion and

metastasis. Furthermore, GNRH2 also induced adhesion to laminin and chemotaxis

toward SDF-1alpha, and augmented entry in vivo of metastatic T-lymphoma into the

spleen and bone marrow, which may be of clinical relevance (CHEN et al., 2002).

The T-cell immune response regulator 1 gene (TCIRG1) maps to HSA11q13.4-q13.5

starting at 67,563,059 bp and ending at 67,574,942 bp and consists of 20 exons

spanning 11884 bp. Through alternate splicing, this gene encodes the proteins

TIRC7 (essential in T-cell activation) and OC116 (osteoclast-specific subunit of the

vacuolar proton pump) with similarity to subunits of the vacuolar ATPase (V-

ATPase). V-ATPase is a multisubunit enzyme that mediates acidification of

eukaryotic intracellular organelles. V-ATPase is comprised of a cytosolic V1 domain

and a transmembrane V0 domain. TIRC7 contains 15 exons spanning 7.9 kb,


- 109 -

whereas OC116 contains 20 exons with the last 14 introns and exons being identical

to TIRC7. The encoded proteins seem to have different functions. In autosomal

recessive osteopetrosis patients TCIRG1 is mutated (SOBACCHI et al., 2001;

SCIMEKA et al., 2003). Inactivation of the Tcirg1 gene in mice caused osteoclast-rich

osteopetrosis (LI et al., 1999).

Procedure:

BAC library screening/sequence analysis: For isolating BAC clones containing the

selected genes, the equine CHORI-241 BAC library was screened. High density BAC

colony filters were probed according to the CHORI protocols (http://bacpac.chori.org)

with heterologous 32P-labelled inserts of the respective human cDNA IMAGE clones

provided by the Resource Center/Primary Database of the German Human Genome

Project (http://www.rzpd.de/). BAC DNA was prepared from the positive BAC clones

using the Qiagen plasmid midi kit (Qiagen, Hilden, Germany). BAC DNA end


Freiburg, Germany) and a LI-COR 4200 automated sequencer (LI-COR Inc., Lincoln,

Nebr., USA).

Chromosomal location: The equine BAC clones were labelled with digoxygenin by

nick translation using a nick-translation mix (Roche Diagnostics, Mannheim,

Germany). For TCIRG1 and TYK2, FISH on GTG-banded horse chromosomes was

performed using 500 ng digoxygenin labelled BAC DNA, and 20 µg sheared total

equine DNA and 10 µg salmon sperm DNA as competitors. For the remaining genes,

FISH was performed using 750 ng of digoxygenin labelled BAC DNA. 20 µg sheared

total equine DNA and 10 µg salmon sperm DNA were used as competitors in these

experiments. After hybridization overnight, signal detection was performed using a

digoxygenin-FITC detection kit (Qbiogene, Heidelberg, Germany). The chromosomes

were counterstained with DAPI (4’,6’-diaminidino-2-phenylindole) and propidium

iodide and embedded in antifade. Metaphase chromosomes that had been previously

photographed by using a highly sensitive CCD camera were re-examined after

hybridization with a Zeiss Axioplan 2 microscope (Zeiss, Jena, Germany) equipped


- 110 -

for fluorescence. Identification of chromosomes followed strictly the international

system for chromosome nomenclature of domestic horses (ISCNH 1997).

Primer sequences:

Primers for PCR amplification from the BAC sequences were designed using the

PRIMER3 software (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi). The

equine BAC sequences were then compared to the Build 35.1 of the human genome

sequences with BLASTN. The results are given in Table 1.


assignment the 5,000 rad TAMU equine radiation hybrid panel (CHOWDHARY et al.,

2003) was used to map the respective genes. PCR was carried out in a 20 µl

reaction containing 25 ng of RH cell line DNA, 10 pmol of each primer and 0.85 U

Taq polymerase (Qbiogene, Heidelberg, Germany). The reaction conditions started

with a denaturing step at 94°C for 5 min followed by 34 cycles using the following

protocol: denaturation for 45 s at 94°C, annealing for 45 s at 60°C and extension for

5 min at 72°C. The PCR was completed with a final cooling at 4°C for 5 min. PCR

products were separated on a 1.5% agarose gel. After scoring positive signals, a

two-point analysis (http://equine.cvm.tamu.edu/cgi-bin/ecarhmapper.cgi) (SLONIM et

al., 1997) was conducted to find associations between the selected genes and the

markers of the first generation whole genome equine RH map (CHOWDHARY et al.,

2003).

Results:

The equine genomic BAC clone CH241-482I10 containing the CART1 gene was

located to ECA28q13-q15 by examination of metaphase chromosomes of 30 cells

(Fig. 1a). The COL9A3 including equine genomic BAC clone CH241-311A7 was

located to ECA22q18-q19 by examination of metaphase chromosomes of 35 cells

(Fig. 1b), examination of metaphase chromosomes of 30 cells located the equine

genomic BAC clone CH241-33D15 containing the GNRH2 gene to ECA22q15 (Fig.

1c). The localisation of the equine genomic BAC clone CH241-526P8 containing the

TCIRG1 gene was confirmed to ECA12q14 by examination of metaphase

chromosomes of 35 cells (Fig. 1d).


- 111 -

Radiation hybrid (RH) mapping:

CART1: The retention frequency of the sequence tagged site (STS) markers was

16.3% and the RH mapping revealed close linkage to UM003 (2.12 cR; LOD >3.0).

The linked microsatellite had been previously mapped at 9.1 cM from the beginning

on ECA28 (GUERIN et al., 2003) and at 3.5 cR from HTG30 on ECARH28a

(CHOWDHARY et al., 2003).

COL9A3: STS-markers showed a retention frequency of 16.3% and the RH mapping

revealed close linkage to SG19 (2.53 cR; LOD >3.0) for COL9A3. The linked

microsatellite marker had been previously mapped on ECA22q19 by FISH (GODARD

et al., 1997), by linkage mapping at 54 cM of ECA22 (GUERIN et al., 2003), and by

radiation hybrid mapping at 285 cR from COR022 on ECARH22c (CHOWDHARY et

al., 2003).

GNRH2: The retention frequency shown by the STS-markers was 16.3% and the RH

mapping revealed close linkage to UMNE077 (21.20 cR; LOD >3.0). The linked

microsatellite marker had been previously mapped on ECA22 by radiation hybrid

mapping (CHOWDHARY et al., 2003).

TCIRG1: STS-markers showed a retention frequency of 28.3% and the RH mapping

revealed close linkage to ADRBK1 (adrenergic receptor beta kinase 1) (20.83 cR;

LOD >3.0) on ECA12. The STS-marker had been previously mapped on ECA12 at

164.8 cR by RH mapping (CHOWDHARY et al., 2003). The RH result confirmed the

result obtained by FISH. ADRBK1 is located on HSA 11cen-q13 starting at

66,790,663 bp and ending at 66,810,945 bp (human genome map viewer build 35.1).

In agreement with our human-equine comparative mapping results, the equine

CHRM1 (muscarin acetylcholine receptor I) gene was also cytogenetically located on

ECA12q14 (MILENKOVIC et al., 2002) and the human CHRM1 gene maps to

HSA11q13 starting at 62,432,728 bp and ending at 62,445,588 bp (human genome

map viewer build 35.1).

The physical assignment of the equine genes to the horse chromosomes agreed with

the comparative mapping data of the current equine-human comparative map

(PERROCHEAU et al., 2006).


- 112 -



References:

BREWTON, R.G. et al.:

Molecular cloning of the alpha 3 chain of human type IX collagen: Linkage of the

gene COL9A3 to chromosome 20q13.3. Genomics 30 (1995) 2, 329-336

BONNEMANN, C.G. et al.:

A mutation in the alpha 3 chain of type IX collagen causes autosomal dominant

multiple epiphyseal dysplasiawith mild myopathy. Proc. Nat. Acad. Sci. 97 (2000),

1212-1217

CHEN, A. et al.:

The neuropeptides GnRH-II and GnRH-1 are produced by human T-cells and

trigger laminin receptor gene expression, adhesion, chemotaxis and homing to

specific organs. Nat. Med. 8 (2002), 1421-1426

CHOWDHARY, B.P. et al.:

The first-generation whole-genome radiation hybrid map in the horse identifies

conserved segments in human and mouse genomes. Genome Res., 13, (2003),

742-751

DELOUKAS, P. et al.:

The DNA sequence and comparative analysis of human chromosome 20. Nature

414 (2001) 6866, 865-871

GODARD, S.:

Characterization, genetic and physical mapping analysis of 36 horse plasmid and

cosmid-derived microsatellites. Mamm. Genome, 8, (1997), 745-750

GORDON, D.F. et al.:

Human Cart-1: structural organization, chromosomal localization, and functional

analysis of a cartilage-specific homeodomain cDNA. DNA Cell Biol. 15 (1996) 7,

531-541


- 113 -

GUERIN, G. et al.:

The second generation of the International Equine Gene Mapping Workshop half-

sibling linkage map. Anim. Genet., 34, (2003), 161-168

International Human Genome Sequencing Consortium:

Finishing the euchromatic sequence of the human genome. Nature 431 (2004),

931-945

KASTEN, T.L. et al.:

Characterization of two new preproGnRH mRNAs in the tree shrew: first direct

evidence for mesencephalic GnRH gene expression in a placental mammal. Gen.

Comp. Endocr. 104 (1996), 7-19

KARAGHIOSOFF, M. et al.:

Central role for type 1 interferons and Tyk2 in lipopolysaccharide-induced

endotoxin shock. Nat. Immunol. 4 (2003), 471-477

LI, Y.-P. et al.:

Atp6i-deficient mice exhibit severe osteopetrosis due to lost of osteoclast-

mediated extracellular acidification. Nat. Genet. 23 (1999), 447-451

LOHINIVA, J. et al.:

Splicing mutations in the COL3 domain of collagen IX cause multiple epiphyseal

dysplasia. Am. J. Med. Genet. 90 (2000), 216-222.

MATSUI, Y. et al.:

Matrix-deposition of tryptophan-containing allelic variants of type 9 collagen in

developing human cartilage. Matrix Biol. 22 (2003), 123-129

MILENKOVIC, D. et al.:



PAASSILTA, P. et al.:

COL9A3: A third locus for multiple epiphyseal dysplasia. Am. J. Hum. Genet. 64

(1999), 1036-1044

PAASSILTA, P. et al.:

Identification of a novel common genetic risk factor for lumbar disk disease. J.

Am. Med. Ass. 285 (2001), 1843-1849


- 114 -

PERROCHEAU, M. et al.:

Construction of a medium-density horse gene map. Anim. Genet., 37, (2006),

145-155

SCIMECA, J.C. et al.:

Novel mutations in the TCIRG1 gene encoding the a 3 subunit of the vacuolar

proton pump in patients affected by infantile malignant osteopetrosis. Hum.

Mutat. 21 (2003), 151-157

SOBACCHI, C. et al.:

The mutational spectrum of human malignant autosomal recessive osteopetrosis.

Human Molecular Genetics 10 (2001) 17, 1767-1773

TILLER, G.E. et al.:

Physical and linkage mapping of the gene for the alpha 3 chain of type IX

collagen, COL9A3, to human chromosome 20q13.3. Cytogenet. Cell Genet. 81

(1998) 205-207

WHITE, R.B. et al.:

Second gene for gonadotropin-releasing hormone in humans. Proc. Natl. Acad.

Sci. USA 95 (1998) 1, 305-309

ZHAO, G.Q. et al.

Cartilage homeoprotein 1, a homeoprotein selectively expressed in chondrocytes.

Proc. Nat. Acad. Sci. USA 90 (1993) 8633-8637

ZHAO, Q. et al.:

Prenatal folic acid treatment suppresses acrania and meroanencephaly in mice

mutant for the Cart1 homeobox gene. Nat. Genet. 13 (1996) 275-283


- 115 -

Figure 1 Chromosomal assignment of the equine BACs containing a) CART1, b)

COL9A3, c) GNRH2, d) TCIRG1 by FISH analysis. G-banded metaphase spread

before (left) and after (right) hybridization. Double signals indicated by arrows are

visible on both equine chromosomes.

(a)

(b)


- 116 -

(c)

(d)


- 117 -

Table 1

Selected genes, their human location and cDNA clone identity, insert size of the

positive equine BAC clones, used BAC end sequence, Accession-no. of BAC end

sequence, BLASTN sequence comparisons of equine BAC sequences with human

genome.

Human Human or murine

location (bp)

cDNA clone identity

BAC Gene HSAa

(Build 35.1) insert size (kb)


CART1 12q21.3-q22

84,176,353 84,198,027 IMAGp998K149747b

CHORI-241-482I10 190 SP6

COL9A3 20q13.3 60,918,832, 60,942,955 IRALp962H0327b

CHORI-241-311A7 190 T7

GNRH2 20p13 2,972,268, 2,974,391 IMAGp998P018507

CHORI-241-33D15 180 SP6

TCIRG1 11q13.4-q13.5

67,563,059, 67,574,942 IMAGp998B1510243b

CHORI-241-526P8 180 T7

a: HSA: homo sapiens autosome

b: human cDNA clone



- 118 -

Table 1 continued

Identity Beginning of

Distance to

Gene BLAST E-Value

Length of match (bp)

(%) sequence alignment on HSA (bp)

selected gene (kb)

CART1 1.0e-30 292 82% 84,045,774 131

COL9A3 2.4e-52 198 88% 60,828,742 94

GNRH2 1.0e-74 155 88% 2,903,818 68

TCIRG1 2.0e-1 741 93% 67,694,235 140 a: HSA: homo sapiens autosome

b: human cDNA clone


Chapter 10

General discussion

General discussion

- 121 -

General discussion There has been a great progress in the development of comparative gene maps

between horse and man during the last years. However, while many studies

concentrated on the development of genetic linkage maps or comparative physical

maps using radiation hybrid mapping (Chowdhary et al. 2003), the determination of

cytogenetical anchorage of mapped genes was rare. Meanwhile, the comparative

equine-human gene map contains 713 genes, 441 on the RH map, 511 on the

cytogenetic and 238 on both maps (Perrocheau et al. 2006).

The most extensive comparative map developed from cytogenetically mapped genes

existing at the beginning of this work was published by Milenkovic et al. (2002) and

included 136 genes. The interval between the publication of this map and the next

extensive publication of a cytogenetic comparative map was almost three years. The

next extensive contribution to the cytogenetic map included the localization of 150

genes (Perrocheau et al. 2005). The actual comparative map of the horse is

developed by Perrrocheau et al. (2006) and added 130 newly cytogenetically

mapped BACs, so already 511 genes have been mapped cytogenetically today.

Between these milestones of cytogenetical mapping several genes have been

localized separately, especially immunity-related genes or genes concerning the

bone and cartilage metabolism and expanded the cytogenetic map of the horse

genome step by step as it is described in this work.

The contribution to the comparative horse genome map data resulting from this

thesis is the presentation of the mapping of 16 genes with previously unknown

localization. The genes have been mapped cytogenetically by FISH and the results

have been confirmed by radiation hybrid mapping afterwards. Linkage mapping had

been performed for ATP2A2_MS5.

At the beginning of this study, 17 genes have been chosen as candidate genes. Ten

genes have been chosen as candidate genes for osteochondrosis: BGLAP, BMP2,

CART1, COMP, GNRH2, POU1, PTHR1 and TYK2 have been selected due to their

locations in the vicinity of an identified QTL derived from QTL studies for OC in the

pig (Andersson-Eklund et al. 2000) as well as for their indicated role in the

development of cartilage growth. COL9A3 has been chosen because mutations in

General discussion

- 122 -

this gene cause autosomal dominantly inherited multiple epiphyseal dysplasia (MED)

in humans characterized by knee pain, stiffness and difficulty walking during

childhood - similar symptoms as in horses with OC (Paassilata et al. 1999). In

autosomal recessive osteopetrosis patients the picked candidate gene TCIRG1 is

mutated (Scimeca et al. 2003). Five genes from different solute carrier families

responsible for the production of several transporter or membrane molecules have

been localized in this work. Mutations in these genes are accompanied by different

disease patterns in humans. The human CHST4 gene encoding a protein that is

involved in enzymatic synthesis of the disulfated disaccharide unit of corneal keratan

sulfate (Akama et al. 2002) should also be mapped to the horse genome in this

study. The last gene, ATP2A2, has been chosen as a candidate gene for chronic

pastern dermatitis in coldblood horses. Mutations in this gene cause the autosomal

dominantly inherited condition keratosis follicularis in men.

All chosen candidate genes are of relevance for horse breeding, because they are

related to bone or cartilage metabolism or may contribute to inherited diseases.

Apart from two genes all localizations discovered for the candidate genes in this

thesis were in agreement with the former existing equine-human comparative maps

(Milenkovic et al. 2002, Chowdhary et al. 2003) and reflected the expectations arising

from those maps. One of the genes, SLC26A2, has only been localized by radiation

hybrid panel mapping because the localization by FISH and RH was already

published during this study by Brenig et al. (2004). Since there could not be found

positive signals on all filters of the equine CHORI-241 BAC library for the POU1F1

gene, no BAC clone was ordered and the gene could not be mapped in this study.

The COMP gene was expected to be localized on ECA10 or ECA7 according to the

existing comparative gene maps. The gene was definitely mapped to ECA21q13 by

FISH in this work. There had only been known synteny between ECA21q and HSA5

before, so the obtained result was in conflict with the existing comparative gene map.

The localization to ECA21q13 was confirmed by radiation hybrid mapping,

furthermore, so there has been shown previously unknown synteny between ECA21q

and HSA19p for the first time.

General discussion

- 123 -

For the TYK2 gene, FISH and RH-mapping results were also in conflict with the

existing comparative gene maps: TYK2 was mapped to ECA7q12-q13. There had

only been known synteny between ECA7q and HSA19q before, so synteny between

ECA7q and HSA19p has been detected in this work for the first time.

In the actual comparative cytogenetic map (Perrocheau et al. 2006), TYK2 was not

contained, but for another gene located in the centromeric region of ECA7q (NR1H2),

synteny between ECA7q11 and HSA19q had been shown. So the result detected in

this thesis was confirmed that there not only exists synteny between ECA7q and

HSA19q, but also between ECA7q and HSA19p.

Due to the possible influence of the mapped candidate genes for osteochondrosis,

former QTL studies for osteochondrosis identified important regions for ECA2 and

ECA4 and concentrated on those regions concerning the choice of candidate genes.

The mapped genes create a useful pool of genes as a basis for future studies

concerning the inheritance of diseases concerning the bone or cartilage metabolism

in horses.

As measured by the fact that the entire horse genome includes assumedly 20000 to

25000 genes, the contribution of 17 genes to the horse gene map seems not to be

great at first sight. But the systematic RH-mapping of genomic sequences as it is

done by some institutes at the moment was not the primary aim of this thesis.

Furthermore, it has to be considered that there can be faults if genes or genomic

sequences have only been RH-mapped.

This thesis represents a further step towards the construction of physical and

comparative maps of the horse genome, especially improving the physical anchorage

by FISH mapping, before whole genome sequencing is likely to be completed.

.

References

Akama T.O., Misra A.K., Hindsgaul O., Fukuda N.M. (2002). Enzymatic synthesis in

vitro of the disulfated disaccharide unit of corneal keratin sulfate. J. Biol. Chem

277: 42505-42513.

General discussion

- 124 -

Andersson-Eklund L., Uhlhorn H., Lundeheim N., Dalin G., Andersson L. (2000).

Mapping quantitative trait loci for principal components of bone measurements

and osteochondrosis scores in a wild boar x large wide intercross. Genet. Res.

75: 223-230.

Brenig B., Beck J., Hall A.J., Broad T.E., Chowdhary B.P., Piumi F. (2004).

Assignment of the equine solute carrier 26A2 gene (SLC26A2) to equine

chromosome 14q15? q21 (ECA14q15? q21) by in situ hybridization and

radiation hybrid panel mapping. Cytogenet. Genome Res. 107: 139.

Chowdhary B.P., Raudsepp T., Kata S.R., Goh G., Millon L.V., Allan V., Piumi F.,

Guerin G., Swinburne J., Binns M, Lear T.L., Mickelson J., Murray J., Antczak

D.F., Womack J.E., Skow L.C. (2003). The first-generation whole-genome

radiation hybrid map in the horse identifies conserved segments in human and

mouse genomes. Genome Res. 13: 742-751.

Milenkovic D., Oustry-Vaiman A., Lear T.L., Billault A., Mariat D., Piumi F., Schibler

L., Cribiu E., Guerin G. (2002). Cytogenetic localization of 136 genes in the

horse: Comparative mapping with the human genome. Mamm. Genome 13: 524-

34.

Paasilata P., Lohiniva J., Annunen S., Bonaventure J., Le Merrer M., Pai L., Ala

Kokko L. (1999). COL9A3: A third locus for multiple epiphyseal displasia. Am. J.

Hum. Genet. 64: 1036-1044.

Perrocheau M., Boutreux V., Chadi-Taourit S., Chowdhary B.P., Cribiu E.P., De Jong

P.J., Durkin K., Hasegawa T., Hirota K., Ianuzzi L., Incarnato D., Lear T.L.,

Raudsepp T., Perucatti A., Di Meo G.P., Zhu B., Guerin G. (2005). Cytogenetic

mapping of 150 genes on the horse genome. Plant and Animal Genome XIII

Conference, Januar 15-19, 2005, San Diego, CA.

Perrocheau M., Boutreux V., Chadi S., Mata X., Decaunes P., Raudsepp T., Durkin

K., Incarnato D., Iannuzzi L., Lear T.L., Hirota K., Hasegawa T., Zhu B., De Jong

P., Cribiu E.P., Chowdhary B.P., Guerin G. (2006). Construction of a medium-

density horse gene map. Anim. Genet. 37: 145-155.

General discussion

- 125 -

Scimeca J.C., Quincey D., Parrinello H., Romatet D., Grosgeorge J., Gaudray P.,

Philip N., Fischer A., Carle G.F. (2003). Novel mutations in the TCIRG1 gene

encoding the a3 subunit of the vacuolar proton pump in patients affected by

infantile malignant osteopetrosis. Hum. Mutat. 21: 151-157.

Chapter 11

Summary

Physical and comparative mapping of candidate genes on

the horse genome

Dorothee Stübs

Summary

- 129 -

Physical and comparative mapping of candidate genes on

the horse genome

The aim of this work was to create a contribution to the comparative physical gene

map, especially to the cytogenetic gene map of the horse by mapping candidate

genes using fluorescence in-situ hybridisation and radiation hybrid panel mapping.

First of all, 17 genes have been chosen as candidate genes. All chosen candidate

genes are of important relevance for the horse breeding, since they are all genes

possibly playing an important role concerning the inheritance of different diseases.

The genes BGLAP, BMP2, CART1, COMP, GNRH2, POU1F1, PTHR1, TCIRG1,

TYK2 and COL9A3 have been chosen as candidate genes for osteochondrosis. Five

genes from different solute carrier families, SLC1A3, SLC4A1, SLC9A5, SLC20A1

and SLC26A2, responsible for the production of several transporter or membrane

molecules whose alterations are causing different inherited diseases in humans,

have been localized in this work, too. Accessorily, the CHST4 gene encoding a

protein being involved in in vitro enzymatic synthesis of the disulfated disaccharide

unit of corneal sulphate and the ATP2A2 gene as candidate gene for pastern

dermatitis, should also be mapped to the horse genome in this study.

15 genes were mapped cytogenetically by using FISH on GTG-banded horse

chromosomes for the first time. One candidate gene, SLC26A2, has not been

mapped by FISH, because the localization of this gene was published in 2004 and

forestalled this study, when the gene was already RH-mapped but fluorescence in-

situ hybridisation had still not taken place. For the candidate gene POU1F1, no

orthologue equine BAC clone could be ordered, because there were no positive

signals on all eleven filters of the CHORI-241 BAC library, so the gene could not be

mapped.

To confirm the cytogenetic assignment, the genes were localized by using the 5,000

rad TAMU equine radiation hybrid panel. A two-point analysis

(http://equine.cvm.tamu.edu/cgi-bin/ecarhmapper.cgi) was conducted to find

associations between the candidate genes and the markers of the first generation

whole genome equine RH map. The results from RH-mapping confirmed the FISH-

Summary

- 130 -

results in all cases. The microsatellite ATP2A2_MS5 was also mapped to the equine

linkage map and corresponded to the results received by FISH and RH-mapping for

the gene ATP2A2.

For 14 genes, the results of the newly mapped genes reflected the expectations

arising from the known human-equine comparative map. In two cases (genes COMP

and TYK2), the results were in conflict with the former developed comparative map.

So far, conserved synteny had only been detected for ECA21q and HAS5 as well as

for ECA7q and HAS19q.

In this thesis, the gene COMP was mapped to ECA21q13 by FISH and by RH-

mapping, so conserved synteny between ECA21q and HAS19q has been detected

for the first time. For the candidate gene TYK2, the equine localization found by FISH

and RH-mapping was ECA7q12-q13, so conserved synteny between ECA7q and

HSA19p has been shown for the first time with this work, too.

Chapter 12

Erweiterte Zusammenfassung

Physikalische und vergleichende Kartierung von

Kandidatengenen auf dem Pferdegenom

Dorothee Stübs


- 133 -

Physikalische und vergleichende Kartierung von

Kandidatengenen im Pferdegenom

Einleitung

Vor dem Hintergrund der Erforschung erblicher und züchterisch relevanter

Krankheiten beim Pferd hat die Entwicklung der physikalischen Genkartierung beim

Pferd und die Entwicklung vergleichender Genkarten zwischen Mensch und Pferd in

den letzten Jahren große Fortschritte gemacht. Während die zytogenetische

Kartierung neuer Genorte mittels FISH zu Beginn der Genomforschung beim Pferd

im Vordergrund stand, expandiert die physikalische Kartierung mittels Radiation-

Hybrid-Kartierung dank der Entwicklung des 5000 rad-Panels in den letzten Jahren

stark. Die aktuelle vergleichende Genkarte umfasst 713 Gene, von denen 441 durch

Radiation-Hybrid (RH)-Kartierung und 511 Gene mittels Fluoreszenz-in-situ-

Hybridisierung (FISH) auf dem Pferdegenom lokalisiert wurden. 238 Gene wurden

sowohl auf der zytogenetischen als auch auf der RH-Karte lokalisiert.

Ziel dieser Arbeit ist es, einen Beitrag zur vergleichenden Genkarte zwischen

Mensch und Pferd zu leisten und gleichzeitig durch die Kartierung sowohl mittels

Fluoreszenz-in-situ-Hybridisierung als auch durch Radiation-Hybrid-Kartierung die

zytogenetische Verankerung in der vergleichenden Genkarte zu verbessern. 16

Kandidatengene sollen in dieser Arbeit zum ersten Mal auf den physikalischen

Genkarten beim Pferd lokalisiert werden.

Erweiterung der physikalischen Karten des Pferdes mittels Fluoreszenz-in-situ-

Hybridisierung (FISH) und Radiation-Hybrid (RH)-Kartierung

Auswahl der Kandidatengene

Insgesamt 17 Kandidatengene wurden für die physikalische Kartierung beim Pferd

ausgewählt. Einen Schwerpunkt bei der Kandidatengenauswahl bildeten zehn

Kandidatengene, die im Hinblick auf einen möglichen Zusammenhang mit

Osteochondrose ausgewählt wurden.


- 134 -

Bei der Osteochondrose handelt es sich um eine Störung der enchondralen

Ossifikation in den Gelenken junger Pferde, die sich durch Beeinträchtigungen im

Bewegungsablauf, Gelenkschwellung und Schmerzen äußern kann. Häufig betroffen

sind Fessel-, Sprung- und Kniegelenk, wobei auch Hüft-, Schulter- und

Wirbelgelenke betroffen sein können. Charakteristische Merkmale sind subchondrale

Knorpelfrakturen, subchondrale Knochenzysten, Knorpelusuren, Chondromalazie

und das Auftreten von Knorpelschuppen. Kommt es zum Vorhandensein freier

Knorpel- oder Knochenfragmente in einem Gelenk, wird die Krankheit als

Osteochondrosis dissecans bezeichnet.

Die Prävalenz von Osteochondrose beträgt bei verschiedenen

Warmblutpferderassen zwischen 10 und 79,5%. Für das Auftreten der Erkrankung

sind neben einer durch Heritabilitätsschätzungen belegten genetischen Komponente

auch die Ernährung, Skelettwachstumsraten, hormonelle Einflüsse sowie Traumata

von Bedeutung, sodass insgesamt von einem multifaktoriellen Geschehen

ausgegangen werden kann.

Die acht Kandidatengene BGLAP, BMP2, CART1, COMP, GNRH2, POU1, PTHR1

und TYK2 wurden auf Grund von QTL-Studien beim Schwein als möglicherweise am

Auftreten der Krankheit beteiligte Gene ausgewählt. Zusätzlich wurden das Gen

COL9A3, dessen Mutation beim Menschen für das Auftreten der autosomal dominant

vererbten Krankheit Multiple epiphsäre Dysplasie (MED) verantwortlich ist, und das

Gen TCIRG1, durch dessen Mutation es beim Menschen zum Krankheitsbild der

autosomal rezessiv vererbten Osteopetrose kommt, als Kandidatengene für OC

ausgewählt.

Fünf Kandidatengene gehören unterschiedlichen Solute Carrier-Familien an

(SLC1A3, SLC4A1, SLC9A5, SLC20A1 und SLC26A2) und codieren für diverse

Transport- und Membranproteine. Mutationen in diesen Genen gehen mit

unterschiedlichen Krankheitsbildern beim Menschen einher. So sind Mutationen des

SLC1A3-Gens, das für ein Transportprotein an glutamatergen Synapsen codiert,

beispielsweise mitbeteiligt am Auftreten von Alzheimer beim Menschen. Durch

Veränderungen des SLC4A1-Gens kann es beim Menschen zum Auftreten zweier

unterschiedlicher Krankheitsbilder kommen; außerdem sind Mutationen an diesem


- 135 -

Gen für die Sichelzellanämie in Malariaendemiegebieten verantwortlich. Das Protein,

für das das Gen SLC9A5 verantwortlich ist, gewährleistet den Na-/H-Austausch der

Zellmembranen. Das SLC20A1-Gen codiert für einen Natrium-abhängigen Phosphat-

Transporter, der bei der Infektion des Menschen mit dem Leukämievirus der

Gibbonaffen als Rezeptor für das Retrovirus dient.

Als weiteres Kandidatengen wurde das Gen CHST4 ausgewählt, das für ein Protein

codiert, das am enzymatischen Aufbau einer Untereinheit des Keratansulfats der

Cornea beteiligt ist.

Das Gen ATP2A2 wurde als Kandidatengen für Mauke ausgewählt. Veränderungen

des ATP2A2 Gens lösen beim Menschen follikuläre Keratose aus.

Material und Methoden

Isolierung equiner genomischer BAC-Klone

Vom RZPD (German Human Resource Center/Primary Database) wurden cDNA

IMAGE-Klone, die die Kandidatengene enthielten, bezogen. Diese wurden für die

Suche nach BAC-Klonen, die die orthologen equinen Sequenzen enthielten, mit 32P

markiert. Anschließend wurde eine radioaktive Hybridisierung mit den Filtern der

equinen genomischen CHORI-241-Genbank durchgeführt. Nach Auswertung wurden

für positive Signale die entsprechenden equinen genomischen BAC-Klone bestellt.

Für das Gen POU1F1 konnten auf keinem der Filter positive Signale identifiziert

werden, so dass für dieses Kandidatengen keine BAC-Klone bestellt werden konnten

und keine weiteren Studien erfolgten.

Die BAC-DNA wurde dann isoliert und die Länge der genomischen DNA-Fragmente

durch Pulsfeldgelelektrophorese ermittelt. Anschließend erfolgte die

Ansequenzierung der 3`- und 5`-Enden der BAC-Klone auf einem automatischen

Sequenziergerät (LI-COR 4200 oder 400). Zur Bestätigung der Richtigkeit der

Sequenzen wurden diese über das Programm BLASTN mit humanen

Datenbankeinträgen verglichen. Zu Beginn der Arbeit erfolgte für die equinen

genomischen BAC-Klone der Gene BMP2, COL9A3 und TCIRG1 zusätzlich eine

ECL-Hybridisierung zur Verifizierung.


- 136 -

Fluoreszenz-in-situ-Hybridisierung

Equine Blutlymphozyten wurden in einem Standard-Kulturmedium mit pokeweed-

Mitogen stimuliert und 72 Stunden im Brutschrank bei 38 Grad Celsius kultiviert. Die

Präparation der Metaphasechromosomen erfolgte nach zytogenetischen

Standardmethoden. Im Folgenden wurden eine GTG-Bänderung der Chromosomen

durchgeführt und gut gestreute Metaphasen fotografiert.

Die DNA der equinen BAC-Klone wurde über Nick-Translation mit Digoxygenin

markiert. Ihre Hybridisierung erfolgte über Nacht an den GTG-gebänderten equinen

Chromosomen. Als Kompetitor-DNA zum Binden repetitiver Sequenzen wurden

gescherte genomische equine DNA und Lachssperma-DNA verwendet. Mittels

Digoxigenin-FITC Detektionskit wurden die Signale der hybridisierten Proben

erfasst. Anschließend wurden die Chromosomen mit DAPI und Propidiumjodid

gegengefärbt und in Antifade eingebettet. Danach erfolgte die Überprüfung auf

Signale der vorher fotografierten Metaphasen und die Zuordnung zu den

Metaphasenchromosomen gemäß internationaler Nomenklatur für die

Pferdechromosomen (international system for chromosome nomenclature of

domestic horses, ISCNDH).

Radiation Hybrid (RH)-Kartierung

Für die Randsequenzen der equinen genomischen BAC-DNA wurden, wenn es

signifikante Übereinstimmungen mit den jeweiligen orthologen humanen Genen gab,

mit Hilfe der Primer3-Software Primersequenzen ermittelt. Für den orthologen BAC-

Klon des Gens SLC1A3 wurde für die Randsequenzen kein signifikanter Treffer zum

SLC1A3 Gen gefunden. Aus diesem Grund wurden eine Subklonierung des BAC-

Klons mit EcoR1 und die Ansequenzierung der Randsequenzen der isolierten

Subklon-DNA durchgeführt. Aus einer Subklon-Randsequenz mit signifikanter

Übereinstimmung mit dem SLC1A3 Gen konnten so Primer generiert werden.

Mittels PCR erfolgte dann die Typisierung des Primer-DNA-Fragments an den

Zelllinien des equinen 5,000 rad Texas A&M University RH Panels. Anschließend

wurde unter Verwendung von RHMAPPER -1.22 (http://equine.cvm.tamu.edu/cgi-

bin/ecarhmapper.cgi) die Auswertung durch Zwei-Punkt-Analyse mit den 861


- 137 -

Markern der equinen RH-Karte, die von Chowdhary et al. (2003) entwickelt worden

war, durchgeführt.

Ergebnisse

Insgesamt wurden von den 17 Kandidatengenen, deren Lokalisation auf dem

Pferdegenom vorher unbekannt war, 15 Gene mittels Fluoreszenz-in-situ-

Hybridisierung kartiert und ihre Lokalisation mittels Radiation Hybrid-Kartierung

bestätigt. Außerdem wurde die Lokalisation des im Gen ATP2A2 enthaltenen

Mikrosatelliten auf der Linkage-Karte des Pferdes ermittelt. Die Lokalisation des

Gens SLC26A6 wurde nur durch RH-Kartierung ermittelt, da noch vor Abschluss der

Kartierung mittels FISH die Kartierung des Gens von anderen Autoren veröffentlicht

wurde. Das Gen POU1F1 konnte nicht kartiert werden, da bei der radioaktiven

Hybridisierung auf allen elf Filtern der equinen genomischen CHORI-241 BAC-

Genbank keine Signale vorhanden waren. Die Ergebnisse der Kartierungen sind in

Tabelle 1 zusammengefasst.

Für 14 kartierte Gene wurden die Erwartungen, die aus den bekannten

vergleichenden Genkarten von Milenkovic et al. (2002) und Chowdhary et al. (2003)

resultierten, bestätigt. Die Lokalisationen der Gene COMP und TYK2 standen im

Widerspruch zu diesen vergleichenden Genkarten: Das COMP Gen wurde auf

ECA21q13 kartiert, das Gen TYK2 auf ECA7q12-q13. Mit diesen Ergebnissen

wurden erstmals Syntäniebeziehungen zwischen ECA21q und HSA19p sowie

zwischen ECA7q und HSA19p aufgezeigt.

Diskussion

Für insgesamt 14 kartierte Gene entsprach die Lokalisation auf dem Pferdegenom

den Erwartungen aus den zu diesem Zeitpunkt aktuellen vergleichenden Genkarten.

Das Gen COMP, für das laut vergleichenden Genkarten eine Lokalisierung auf ECA7

oder 10 erwartet worden wäre, wurde durch FISH auf ECA21q13 kartiert. Vorher

waren Synteniebeziehungen nur zwischen ECA21q und HSA5 gezeigt worden. Das

Ergebnis der RH-Kartierung bestätigte jedoch die Lokalisation. Auch für das Gen

TYK2 entsprach das Ergebnis der FISH-Kartierung nicht den gültigen vergleichenden


- 138 -

Genkarten: das Gen wurde auf ECA7q12-q13 kartiert. Bislang waren

Syntäniebeziehungen nur zwischen ECA7q und HSA19q bekannt gewesen. Auch

dieses Ergebnis wurde durch ein gleich lautendes Resultat der RH-Kartierung

untermauert.

In dieser Arbeit werden demzufolge erstmals neue Syntäniebeziehungen zwischen

ECA21q und HSA19q sowie zwischen ECA7q und HSA19p aufgezeigt. Das Gen

COMP ist in der aktuell gültigen vergleichenden Genkarte enthalten. Das Gen TYK2

ist in dieser vergleichenden Genkarte noch nicht enthalten, jedoch bestätigt die

Genkarte ebenfalls die festgestellte Syntäniebeziehung zwischen ECA7q und

HSA19p.

Insgesamt steht also der Beitrag neuer kartierter Gene für die physikalischen und

vergleichenden Genkarten beim Pferd als Ergebnis dieser Arbeit im Vordergrund.

Außerdem wurden in dieser Studie erstmals Syntäniebeziehungen zwischen ECA21q

und HSA19q sowie zwischen ECA7q und HSA19p festgestellt.

Chapter 13

Appendix

Appendix

- 141 -

Laboratory paraphernalia Equipment

Thermocycler

PTC-100TM ProgrammableThermal Controller (MJ Research, Watertown, USA)

PTC-100TM Peltier Thermal Cycler (MJ Research, Watertown, USA)

PTC-200TM 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 (Ammersham 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 1. OR (Heraeus, Osterode)

Speed Vac R Plus (Savant Instruments, Farmingdale, NY, USA)

Agarose gel electrophoresis and pulsed field gel electrophoresis

Electrophoresis chambers OWL Separation Systems, Portsmouth, NH, USA

Biometra Göttingen

BioRad, München

Generators 2301 Macrodrive 1 (LKB, Bromma, Sweden)

Power Pack 3,000 (BioRad, München)

Gel documentation system BioDocAnalyze 312 nm, Göttingen

Fluorescence-in-situ-hybridization

Zeiss Axioplan 2 microscope (Carl Zeiss, Jena)

SenSys Digital CCD Camera system, Kodak KAF 1400 (Fa. Photometrics, München)

Pipettes

MultipetteR plus (Eppendorf, Hamburg)

Appendix

- 142 -

PipetusR-akku (HirschmannR Laborgeräte GmbH & Co. KG, Eberstadt)

PipetmanR (P2, P10, P20, P100, P200, P1000) (Gilson Medical Electronics S.A.,

Villiers-le-bel, France)

Pipettor, Multi 12 Channel (0.1-10 µl) (MicronicR systems, Lelystad, The Netherlands)

ImpactR Pipettor 8-Channel (Matrix Technologies Corporation, Lowell, USA)

HAMILTON 8-channel syringe (Hamilton Company, Reno, NV, USA)

DistrimanR (Abimed, Langenfeld)

Others

Milli-QR biocel water purificationsystem (Millipore GmbH, Eschborn)

Incubator VT 5042 (Heraeus, Osterode)

UV-Illuminator 312 nm (Bachhofer, Reutlingen)

CentomatR R Desk-Shaker (B. Braun Melsungen AG, Melsungen)

Biophotometer (Eppendorf AG, Hamburg)

Kits

FISH

Dig-Nick Translation Mix (Roche Diagnostics, Mannheim, Germany)

Digoxigenin-FITC Detection Kit (Qbiogene, Heidelberg, Germany)

Rhodamin Detection Kit (Qbiogene, Heidelberg, Germany)

Isolation of DNA

HiSpeed Plasmid MIDI Kit (QIAGEN, Hilden)

Plasmid MIDI Kit (QIAGEN, Hilden)

Plasmid Mini Prep 96 Kit (Millipore GmbH, Eschborn)

Qiagen Large Construct Kit (QIAGEN, Hilden)

Radioactive Hybridization

RadPrime DNA Labelling System (Gibco-BRL, Eggenstein)

DNA purification

Montage PCR96 Cleanup Kit (Millipore GmbH, Eschborn)

Sequencing

ThermoSequenase Sequencing Kit (Amersham Biosciences, Freiburg, Germany)

Appendix

- 143 -

DYEnamic-ET-Terminator Cycle Sequencing Kit (Amersham Biosciences, Freiburg,

Germany)

Size standards

100 bp Ladder (New England Biolabs, Schwalbach Taunus)

1 kb Ladder (New England Biolabs, Schwalbach Taunus)

IRDyeTM 700 or 800 (LI-COR, Inc., Lincoln, NE, USA)

Reagents and buffers

APS solution (10%)

1g APS

10 ml H2O

Bromophenol blue solution

0.5 g bromophenol blue

10 ml 0.5 M EDTA solution

H2O ad 50 ml

dNTP solution

100 µl dATP [100mM]

100 µl dCTP [100mM]

100 µl dGTP [100mM]

100 µl dTTP [100mM]

1600 µl H2O the concentration of each dNTP in the ready-to-use solution is 5 mM

Gel solution

12.75 ml Urea/TBE solution (Roth, Karlsruhe)

2.25 ml Rotiphorese® Gel 40 (38% acrylamide and 2% bisacrylamide)

95 µl APS solution (10%)

9.5 µl TEMED

High stringency wash buffer II

1 mM Na2EDTA

40 mM NaHPO4, pH 7.2

7% SDS

Appendix

- 144 -

Hybridization solution II (Church buffer)

1% bovine serum albumine (BSA)

1 mM EDTA

0.5 M NaHPO4, pH 7.2

7% SDS

Loading buffer for agarose gels

EDTA, pH 8 100 mM

Ficoll 400 20% (w/v)

Bromophenol blue 0.25% (w/v)

Xylencyanol 0.25% (w/v)

Loading buffer for gel electrophoresis

2ml bromophenol blue solution

20 ml formamide

Low stringency wash buffer II

0.5% bovine serum albumine (BSA)

1 mM EDTA

40 mM NaHPO4, pH 7.2

5% SDS

TBE-buffer (1x)

100 ml TBE-buffer (10x)

900 ml H2O

TBE-buffer (10x)

108 g Tris [121.14 M]

55g boric acid [61.83 M]

7.44 g EDTA [372.24 M]

H2O ad 1000 ml

pH 8.0

Wash buffer for FISH

100 ml 1 mol Na2HPO4

100 ml 1 mol NaH2PO4

1 ml Tween 20

Appendix

- 145 -

Chemicals

a-[32P]-dCTP (50 µl, 3,000 Ci/mmol) (Hartmann Analytic GmbH, Braunschweig)

Agarose (Invitrogen, Paisley, UK)

Ampicillin (Serva, Heidelberg)

Boric acid = 99.8 %, p.a. (Carl Roth GmbH & Co, Karlsruhe)

Bromophenol blue (Merck KgaA, Darmstadt)

dATP, dCTP, dGTP, dTTP > 98 % (Carl Roth GmbH & Co, Karlsruhe)

Chloramphenicol (Serva, Heidelberg)

Colcemid (Biochrom AG, Berlin)

DAPI (Sigma-Aldrich Chemie GmbH, Taufkirchen)

Dinatriumhydrogenphosphat (Biochrom AG, Berlin)

DMSO = 99.5 %, p.a. (Carl Roth GmbH & Co, Karlsruhe)

dNTP-Mix (Qbiogene GmbH, Heidelberg)

EDTA = 99 %, p.a. (Carl Roth GmbH & Co, Karlsruhe)

Ethidium bromide (Carl Roth GmbH & Co, Karlsruhe)

Ethyl alcohol (AppliChem, Darmstadt)

FKS (Biochrom AG, Berlin)

Formamide = 99.5 %, p.a. (Carl Roth GmbH & Co, Karlsruhe)

LB (Luria Bertani) agar (Scharlau Microbiology, Barcelona, Spain)

LB (Luria Bertani) broth (Scharlau Microbiology, Barcelona, Spain)

Natriumdihydrogenphosphat (Biochrom AG, Berlin)

Paraffin (Merck KgaA, Darmstadt)

Penicillin/Streptomycin (Biochrom AG, Berlin)

Pokeweed mitogen (Biochrom AG, Berlin)

Propidiumjodid (Sigma-Aldrich Chemie GmbH, Taufkirchen)

Rotiphorese® Gel 40 (Carl Roth GmbH & Co, Karlsruhe)

RPMI 1640 (Biochrom AG, Berlin)

Trypsin (Biochrom AG, Berlin)

Tris PUFFERAN® = 99.9 % (Carl Roth GmbH & Co, Karlsruhe)

Twee20 (Carl Roth GmbH & Co, Karlsruhe)

Urea = 99.5 % (Carl Roth GmbH & Co, Karlsruhe)

Appendix

- 146 -

Water was taken from the water purification system Milli-Q®

X-Gal (AppliChem, Darmstadt)

Clones

All required clones have been ordered at the Resource Center/Primary Database

(RZPD, http://www.rzpd.de).

Enzymes

Taq-DNA-Polymerase 5 U/µl (Qbiogene GmbH, Heidelberg)

Incubation-Mix (10 x) T.Pol with MgCl2 [1.5 mM] (Qbiogene GmbH, Heidelberg)

The polymerase was always used in the presence of incubation Mix t.Pol 10 x buffer.

The enzymes SAC1, HIND III and ECO R1 (New England Biolabs, Schwalbach

Taunus) were used with the adequate 10 x enzyme buffer.

BAC-libraries and RH-panel

The gene-bank-screening was carried out on the CHORI-241 Equine BAC-Library

(http://bacpac.chori.org/equine241.htm). The RH-panel used was the Texas A & M

University equine Radiation Hybrid Panel (Chowdhary et al. 2002 and 2003).

Consumables

96 Well Multiply PCR plates, skirted (Sarstedt, Nümbrecht)

Centrifugal Tubes (Tierärztebedarf Lehneke, Schortens)

Combitips® plus (Eppendorf AG, Hamburg)

Microscope slide (Tierärztebedarf Lehneke, Schortens)

Pipette Tipps 0.1-10 µl, 5-200 µl (Carl Roth GmbH & Co, Karlsruhe)

Reaction tubes 1.5 and 2.0 ml (nerbe plus GmbH, Winsen/Luhe)

Reaction tubes 10 and 50 ml (Falcon) (Renner, Darmstadt)

Serological pipettes 2 and 10 ml (Biochrom AG, Berlin)

Thermo-fast 96 well plate, skirted (Abgene, Hamburg)

Tissue culture flasks (Biochrom AG, Berlin)

Appendix

- 147 -

X-ray films Kodak Biomax MS, developer and fixer (Eastman Kodak Company,

Rochester, NY, USA)

Software

BLASTN, trace archive http://www.ncbi.nlm.nih.gov

HORSEMAP database http://dga.jouy.inra.fr/cgibin/lgbc/loci_

IPLab 2.2.3 Scanalytics, Inc

Order of primers MWG Biotech-AG, Ebersberg

(https://ecom.mwgdna.com/register/index.tcl)

biomers.net GmbH, Ulm ([email protected])

Primer design http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi

Purchase of BACs http://bacpac.chori.org/

Repeat Masker http://www.repeatmasker.genome.washington.edu/

RHMAPPER-1.22 http://equine.cvm.tamu.edu/cgi-bin/ecarhmapper.cgi

Slonim et al. 1997

Sequencher 4.2 GeneCodes, Ann Arbor, MI, US

Chapter 14

List of publications

Publications

- 151 -

Journal articles

Müller D., Kuiper H., Mömke S., Böneker C., Drögemüller C., Chowdhary B.P. and

Distl O. (2005). Assignment of the COMP gene to equine chromosome 21q12-

q14 by FISH and confirmation by RH mapping. Animal Genetics 36: 277-279.

Müller D., Kuiper H., Böneker C., Mömke S., Drögemüller C., Chowdhary B.P. and

Distl O. (2005). Physical mapping of the PTHR1 gene to equine chromosome

16q21.2. Animal Genetics 36: 282-284.

Müller D., Kuiper H., Böneker C., Mömke S., Drögemüller C., Chowdhary B.P. and

Distl O. (2005). Assignment of BGLAP, BMP2, CHST4, SLC1A3, SLC4A1,

SLC9A5 and SLC20A1 to equine chromosomes by FISH and confirmation by

RH mapping. Animal Genetics 36: 457-460.

Müller D., Kuiper H., Böneker C., Drögemüller C., Swinburne J.E., Binns M.,

Chowdhary B.P. and Distl O. (2006). Physical mapping of the ATP2A2 gene to

equine chromosome 8p14->p12 by FISH and confirmation by linkage and RH

mapping. Cytogenetic and Genome Research 114: 94.

Stübs D., and Distl O. (2006). Mapping the horse genome and its impact on equine

genomics for identification of genes for monogenic and complex traits.

Accepted for publication in Archives for Animal Breeding.

Böneker C., Müller D., Kuiper H., Drögemüller C., Chowdhary B.P. and Distl

O.(2005). Assignment of the COL8A2 gene to equine chromosome 2p15-p16

by FISH and confirmation by RH mapping. Animal Genetics 36: 444-445.

Acknowledgements

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Acknowledgements

First of all, I want to thank Prof. Dr. Dr. habil. Ottmar Distl, the supervisor of my

doctoral thesis, for offering me an interesting work and for giving me the opportunity

to do the practical parts of this thesis before I finished my study of veterinary

sciences. I am very thankful for his support, his patience, his humor and his bavarian

dialect that always reminded me of my first semesters in Munic.

I also thank Prof. Dr. Cord Drögemüller and Prof. Dr. Tosso Leeb for their support

and advice concerning scientific questions.

I am most thankful to Dr. Heidi Kuiper for making me acquainted with the scientific,

especially with the cytogenetic working methods, for her scientific advice and her

invaluable support, the many answered questions, the constructive criticism in the

course of this work and for working and talking together in good athmosphere.

I wish to thank Flavia Martins-Wess, Simone Rak and Kathrin Löhring for helping me

at the beginning of this work, and I am also thankful to Flavia for being a friend and

the amusing aqua gym lessons we had.

I am very grateful to Dr. Stefanie Mömke, Dr. Anne Wöhlke, Dr. Claudia Dierks and

Dr. Andreas Spötter for their support and their open ear for my questions.

I wish to thank Heike Klippert-Hasberg, Stefan Neander and Diana Seinige for their

support and advice during the work in the laboratory.

My special thanks go to C. Mrusek and D. Böhm for their support in administrative

questions and for tolerating my dog in the office.

Furthermore, I want to thank Jörn Wrede for his help when my computer tried to

annoy me.

Acknowledgements

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I enjoyed working with so many friendly and ready to help persons at the Institute for

Animal Breeding and Genetics. Thanks to all of you - it was a great time!

I want to thank the clinic for horses for the cooperation concerning the horse blood

samples and I am grateful to all horses donating their blood to expand the horse

gene map.

Thanks to my brother Florian for helping me converting this file and for answering

many questions concerning problems with the computer.

My very special thanks go to my beloved husband Oli for supporting me and

especially for helping me attending to Finn during the last weeks writing this work in

Wolgast – I know I was intolerable sometimes… I am thankful to Finn for all the

minutes he gave me to write while he was sleeping. Last but not least I want to thank

Indie for the relaxing promenades and for always making me happy.

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