Research ArticleTargeted Next Generation Sequencing Identifies NovelMutations in RP1 as a Relatively Common Cause of AutosomalRecessive Rod-Cone Dystrophy

Said El Shamieh123 Elise Boulanger-Scemama123 Marie-Elise Lancelot123

Aline Antonio123 Vanessa Deacutemontant123 Christel Condroyer123 Meacutelanie Letexier4

Jean-Paul Saraiva4 Saddek Mohand-Sad1235 Joseacute-Alain Sahel1235678

Isabelle Audo12358 and Christina Zeitz123

1 INSERM U968 75012 Paris France2Sorbonne Universites UPMC University Paris 06 UMR S 968 Institut de la Vision 75012 Paris France3CNRS UMR 7210 75012 Paris France4IntegraGen SA Genopole Campus 1 Building G8 91030 Evry France5CentreHospitalier National drsquoOphtalmologie des Quinze-Vingts DHUViewMaintain INSERM-DHOSCIC 1423 75012 Paris France6Fondation Ophtalmologique Adolphe de Rothschild 75019 Paris France7Academie des Sciences-Institut de France 75006 Paris France8University College London Institute of Ophthalmology 11-43 Bath Street London EC1V 9EL UK

Correspondence should be addressed to Isabelle Audo isabelleaudoinsermfr and Christina Zeitz christinazeitzinsermfr

Received 27 June 2014 Accepted 10 July 2014

Academic Editor Calvin Yu-Chian Chen

Copyright copy 2015 Said El Shamieh et alThis is an open access article distributed under theCreative CommonsAttribution Licensewhich permits unrestricted use distribution and reproduction in any medium provided the original work is properly cited

We report ophthalmic and genetic findings in families with autosomal recessive rod-cone dystrophy (arRCD) and RP1mutationsDetailed ophthalmic examination was performed in 242 sporadic and arRCD subjects Genomic DNA was investigated using ourcustomized next generation sequencing panel targeting up to 123 genes implicated in inherited retinal disorders Stringent filteringcoupled with Sanger sequencing and followed by cosegregation analysis was performed to confirm biallelism and the implicationof the most likely disease causing variants Sequencing identified 9 RP1 mutations in 7 index cases Eight of the mutations werenovel and all cosegregated with severe arRCD phenotype found associated with additional macular changes Among the identifiedmutations 4 belong to a region previously associated with arRCD and 5 others in a region previously associated with adRCDOur prevalence studies showed that RP1 mutations account for up to 25 of arRCD These results point out for the necessityof sequencing RP1 when genetically investigating sporadic and arRCD It further highlights the interest of unbiased sequencingtechnique which allows investigating the implication of the same gene in different modes of inheritance Finally it reports thatdifferent regions of RP1 can also lead to arRCD

1 Introduction

Rod-cone dystrophy (RCD) also known as retinitis pigmen-tosa is a heterogeneous group of inherited disorders affectingprimary rod photoreceptors in the majority of cases withsecondary cone degeneration [1 2] Population-based studiesshowed that 1 in 4000 individuals is affected around the

world [1] Patients diagnosed with RCD initially complainof night blindness due to rod dysfunction followed by pro-gressive visual field constriction abnormal color vision andeventually loss of central vision due to cone photoreceptorinvolvement [1]

RCD is inherited as a Mendelian trait in most cases [3]On the basis of its mode of inheritance and prevalence

Hindawi Publishing CorporationBioMed Research InternationalVolume 2015 Article ID 485624 11 pageshttpdxdoiorg1011552015485624

2 BioMed Research International

RCD can be divided into 3 groups autosomal dominant(ad) (30ndash40) autosomal recessive (ar) (50ndash60) and X-linked (xl) (5ndash15) [3] To date mutations in at least 53genes were reported to cause nonsyndromic RCD (till 25June 2014 httpssphutheduretnet) Prevalence studiesrevealed rhodopsin (RHO) retinitis pigmentosa GTPase reg-ulator (RPGR) and usherin (USH2A) as being the mostfrequently mutated genes in adRCD [4 5] xlRCD [4] andarRCD respectively [6] Of note is that many other geneswith lower prevalence are also implicated in the geneticetiology of RCD [7 8] Mutations in RP1 were first shownto cause adRCD [9ndash11] however since 2005 articles haveshed light on its implication in arRCD etiology [12ndash20] RP1mutations were shown to account for asymp55 and asymp1 ofadRCD and arRCD cases respectively [8ndash20] InterestinglyAvila-Fernandez et al [12] reported that a founder nonsensemutation in the Spanish population pSer542lowast is responsiblefor 45 of arRCD cases suggesting that RP1 mutations aremore prevalent in arRCD than previously thought [12]

Retinitis pigmentosa 1 (RP1) is a photoreceptor-specificgene encoding a protein regulated by oxygen [10] RP1 proteinis required for correct orientation and higher-order stackingof outer segment disks [21] and was shown to be part of thephotoreceptor axoneme [22] RP1 localizes to the connectingcilia of photoreceptors and may assist in maintenance ofciliary structure or transport down the photoreceptor [22]Like many retinal degeneration genes the mechanism bywhich mutations in RP1 lead to photoreceptor cell death isstill unclear

We developed an unbiased and time-efficient retinal genenext generation sequencing array (NGS) which was furtherrevised and improved to target more than 120 genes impli-cated in inherited retinal diseases (IRDs) (list available uponrequest) [23] Using this NGS panel we screened a total of242 subjects with sporadic and recessive RCD in order todetect disease causingmutations and to report the prevalenceof pathogenic mutations in RP1 causing arRCD

2 Methods

21 Ethics Statement and Clinical Diagnosis of Rod-ConeDystrophy The study protocol adhered to the tenets of theDeclaration of Helsinki and was approved by the localEthics Committee (CPP Ile de France V) Informed writtenconsent was obtained from each study participant Indexpatients underwent full ophthalmic examination as previ-ously described [23]

22 Targeted Next Generation Sequencing A cohort of 242subjects affected with sporadic and arRCD was investigatedin the present study Prior to NGS screening moleculargenetic analysis with microarray (Asper Ophthalmics TartuEstonia) followed by direct Sanger sequencing of EYS andC2orf71 (major and minor genes implicated in RCD newlydiscovered at the beginning of our study) was performedin 201 index subjects (82) [2 24] As RPGR exon ORF15(MIM 312610) is not targeted by existing NGS panels weexcluded mutations in this ldquohot spotrdquo by Sanger sequencing

Although our NGS panel was selected from the SureSelectHuman All Exon Kits Version 4 (Agilent Massy France)this design was improved after analyzing the first 83 subjectswith sporadic and arRCD More precisely a total of asymp300Kbregions were added in order to cover all the previouslynontargeted regions Thus whereas the first design coveredthe exons and the flanking intronic regions of 120 genesimplicated in IRDs the second covered 123 genes in totalThe eArray web-based probe design tool was used for thispurpose (httpsearraychemagilentcomearray) All probeswere designed and synthesized by Agilent Technologies(Santa Clara CA USA) Sequence capture enrichment andelution were performed according to Agilentrsquos instructionsThe complete details were described elsewhere [23]

23 Assembly and Variant Calling Sequence reads werealigned to the reference human genome (UCSC hg19)using CASAVA17 software (Illumina) and the ELANDv2alignment algorithm Sequence variation annotation wasperformed using the IntegraGen in-house pipeline whichconsisted of gene annotation (RefSeq) detection of knownsingle nucleotide polymorphisms (dbSNP 135) followed bymutation characterization (missense intronic synonymousnonsense splice site and insertionsdeletions)

24 Quality Control and Coverage Assessment The firstNGS retinal panel harbored 120 IRDs genes encompassing321240 kb length per sample However after improvementthe same panel contained asymp600Kb and covered 123 IRDgenes The depth of coverage was calculated by counting thenumber of sequenced bases mapping to the target regionsMean depth of coverage was calculated per base pair for allsamples however only the results of subjects having RP1mutations were shown

25 Discrete Filtering of Annotated Variants In order to iden-tify disease causing mutations among nonpathogenic singlenucleotide polymorphisms we used a filtering approachagainst a set of polymorphisms that are available in the publicdatabases dbSNP 137 1000 Genomes Project [25] HapMap[26] and Exome Variant Server [27] with removal of variantswith a minor allele frequency (MAF) ge 0005 in case ofpresumed autosomal recessive mode of inheritance

26 Pathogenicity Assessment We stratified candidate muta-tions based on their functional class by giving a priorityto frameshifts stop codons and disruptions of canonicalsplice sites variants [28] For missense changes amino acidconservation across 46 different species was studied using theUCSC Genome Browser [29] If no amino acid change wasfound then the residue was considered as ldquohighly conservedrdquoIf a different change was seen in less than four species and notin the primates then it was considered as ldquomoderately con-servedrdquo and if a change was present in 5ndash7 it was consideredas ldquomarginally conservedrdquo otherwise the amino acid residuewas considered as ldquonot conservedrdquo Pathogenic predictionwas performed using two software programs Polyphen2 [30]and SIFT [31] based on specieshomologue conservation

BioMed Research International 3

putative structural domains and 3D structures (if available)Analysis of potential splice site variant consequences whenrelevant was done using human splicing finder [32]

27 Known Genotype-Phenotype Correlations The searchfor previous genotype-phenotype associations was done bysearching numerous literature databases including OnlineMendelian Inheritance in Man (httpomimorg) HumanGene Mutation Database [33] Leiden Open Variation Data-base [34] and RetNet (httpssphutheduretnet)

28 Validation of the Identified Variants and CosegregationAnalyses Sanger sequencing was performed to validate dis-ease causing mutations in RP1 The respective primer infor-mation can be communicated on request In addition bloodsamples were collected from additional family memberswhen possible and cosegregation analyses on extracted DNAwere performed as previously described [35 36]

3 Results

31 Clinical Data Clinical data are summarized in Table 1Among identified patients 5 were females 2 were maleand ages at time of examination ranged from 25 to 42 Allpatients were diagnosed before age 20 mostly based on nightblindness from early childhood and secondary central visionloss They all showed severe RCD with constricted visualfields no detectable responses on full field electroretinogramand both peripheral involvement and macular involvement(Figure 1 presents fundus pictures of patient II1 (CIC01245)in family F752 as an example) Comparing visual acuity andvisual fields for these arRCD patients with those of adRCDcases published by Audo and coworkers [8] we noticed amore severe phenotype in recessive cases However morecases with RP1mutations would be needed to draw statisticalconclusion

32 Sequencing Statistics In index patients the overallsequencing coverage of the target regions was ge88 for a 25Xdepth of coverage in each of the chromosomes (Figure 2(a))resulting a mean sequencing depth of 299 times per baseMean sequencing results per base in each target chromosomegene regions were shown in Figure 2(b) It is of importanceto mention that lt1 of target regions were not covered at allThese were fragments of 120 bp belonging in 66 of the casesonly to a fraction of an exonThe remaining uncovered targetscorresponded each to an entire exon in genes such as CHMPDZD7 RP9 CC2D2A IMPDH1 CNGA1 and EYS

33 Detection of Disease Causing Mutations in RP1 GeneAfter data filtering the total number of putative diseasecausing variants was reduced by 993Thus in total filteringenriched the percentage of putative disease causingmutationsfrom 07 (253339 variants) to 333 (925 variants) in the 7subjects presented here (Table 2) These subjects exhibit RP1mutations in the last exon 4 that are predicted to lead to apremature stop codon We found 9 pathogenic mutations inRP1 among which one (pSer542lowast in CIC00445) was already

reported by Avila-Fernandez et al [12] as a founder nonsensemutation in the Spanish population responsible for 45of arRCD Although F303 is from French origin we cannotexclude the possibility of a founder effect of pSer542lowast in ourcohort

Patient family F303 II1 (CIC00445) was found to carrycompound heterozygous variants a nonsense mutationc1625CgtG leading to a predicted premature stop (pSer542lowast)and a deletion c4587 4590delTAAG leading to a frameshiftand a premature termination codon (pSer1529Argfslowast9)(Table 2 Figure 3) Patient family F752 II1 (CIC01245)was also found to carry compound heterozygous variantsa 1 bp duplication c2025dupA leading to pSer676Ilefslowast22and a 1 bp deletion c2377delA leading to pArg793Glufslowast55(Table 2)

Patients from family F335 III1 (CIC00491) family F674III6 (CIC01106) family F782 II5 (CIC01300) family F1941III1 (CIC04130) and family F3110 III5 (CIC05941) werefound to carry homozygous deletions c4089 4092delAAGAleading to pArg1364Valfslowast8 c1205delG leading topGly402Alafslowast7 c1719 1723delCTCAA leading topSer574Cysfslowast7 c1329delG leading to pLys443Asnfslowast12and c2391 2392delAA leading to pAsp799lowast resp) (Table 2and Figure 3) It is important to note that consanguinity wasreported in families F335 F674 F782 and F1941

AllRP1mutations detected byNGSwere further validatedby Sanger sequencing All variants cosegregatedwith the phe-notype in available family members Based on the previousfindings the measured prevalence of RP1-associated arRCDin this cohort is asymp25

4 Discussion

The current study further demonstrates the usefulness ofNGS as a comprehensive genetic diagnostic tool for IRDswith further impact on patients counseling and participationfor potential therapeutic trials Our study applied to a largecohort of sporadic and autosomal recessive cases of RCDidentifies 8 novel mutations in a gene not classically screenedin arRCD by other methods such as Sanger sequencing ormicroarray analysis outlining the interest of this massiveparallel sequencing method Consequently a prevalence ofRP1 mutation in 25 of sporadic or arRCD cases in theEuropean population is herein reported

RP1 is a 15 kb single copy gene clustering the small armof the chromosome 8 (8q121) It encodes a 2506 aminoacid protein having a molecular weight of 241 kDa contain-ing a Drosophila melanogaster bifocal (BIF) (amino acid486ndash635) and two doublecortin domains Whereas the BIFdomain helps to maintain the photoreceptor morphogenesisdoublecortin domains bind microtubules and regulate theirpolymerization [22] Along with RP1L1 (Retinitis Pigmentosa1-like 1 another retinal-specific protein) RP1 plays essentialand synergistic roles in outer segment morphogenesis of rodphotoreceptors [22]

To date at least 50 mutations in RP1 were identified inRCD [8 12ndash20] the majority of which are located in its

4 BioMed Research International

Table1Clinicaldataof

the7

indexpatie

ntsw

ithRP

1recessiv

emutations

Patie

ntAge

attim

eof

testing

Age

ofon

set

Sex

Family

histo

ry

Symptom

sat

timeo

fdiagno

sis

BCVA

ODO

SWith

refractio

n

Color

visio

n(15desaturated

Hue)

Bino

cular

kinetic

visualfield

(III4e

stim

ulus)

FFand

mfERG

Fund

usexam

ination

FAF

Sd-O

CT

F303II1

(CIC00

445)

426

FNoother

affectedFM

fro

mFrance

Night

blindn

ess

Handmotion

inbo

theyes

Impo

ssibledu

eto

lowvisio

n

Redu

cedto

perip

heral

islands

ofperceptio

n

Both

undetectable

Paleop

ticdisc

narrow

edbloo

dvessels

macular

atroph

icchangesand

optic

nerve

drusen

Hypoautofl

uorescence

inthem

acular

region

Thinning

ofou

terretina

inthe

macular

region

F335III1

(CIC00

491)

363

M

Twoother

brothers

affected

parentsfi

rst

cousins

Night

blindn

essa

ndrapid

decreased

visio

n

LPin

both

eyes

Impo

ssibledu

eto

lowvisio

n

Impo

ssible

duetolow

visio

n

Both

undetectable

Widespread

RPEchanges

andretin

alatroph

yin

both

thep

eriphery

andthem

acular

area

Widespreadlossof

FAF

Widespread

thinning

ofou

terretina

F674III6

(CIC01106)

2519

F

Parentsfi

rst

cousinsfrom

Turkeyone

femalea

ndmalec

ousin

saffectedalso

from

acon

-sang

uineou

sun

ion

Night

blindn

essa

nddecreased

visio

n

HMminus3(minus1)

0∘ 2016

0minus3

(minus050)0∘

Dyschromatop

siawith

nospecific

axis

Redu

cedto

5central

degrees

Both

undetectable

Well-c

olored

optic

disc

and

nonarrow

ingof

retin

alvessels

RP

Echangesin

thep

eriphery

andmacular

atroph

icchanges

Hypoautofl

uorescence

inthem

acular

region

andou

tside

the

vascular

arcades

Thinning

ofou

terretina

inthe

macular

region

F752II1

(CIC01245)

31Ea

rlyteens

FTw

osisters

affected

Night

blindn

ess

2063plano

(minus3)

180∘

2050plano

(minus17

5)180∘

Deutandefecton

both

eyes

Redu

cedto

10∘

times20∘

Both

undetectable

Paleop

ticdisc

headnarrowed

retin

alvessels

andRP

Echangesinthe

perip

hery

with

somem

acular

atroph

icchanges

Hypoautofl

uorescence

inthem

acular

region

andou

tside

the

vascular

arcades

Thinning

ofou

terretina

inthe

macular

region

F782

II5

(CIC01300)

279

MParentsfrom

Algeriafirst

cousins

Night

blindn

essa

nddecreased

visio

n

2050minus92

5(minus250)15∘

2050

minus9(minus17

5)100∘

mdash

Redu

cedto

the10

central

degrees

Both

undetectable

Mild

optic

disc

pallo

ratroph

icmacular

changesand

perip

heral

pigm

ent

depo

sits

Hypoautofl

uorescence

inthem

acular

region

Thinning

ofou

terretina

inthe

macular

BioMed Research International 5

Table1Con

tinued

Patie

ntAge

attim

eof

testing

Age

ofon

set

Sex

Family

histo

ry

Symptom

sat

timeo

fdiagno

sis

BCVA

ODO

SWith

refractio

n

Color

visio

n(15desaturated

Hue)

Bino

cular

kinetic

visualfield

(III4e

stim

ulus)

FFand

mfERG

Fund

usexam

ination

FAF

Sd-O

CT

F1941III1

(CIC04

130)

30child

hood

FParentsfrom

Algeriafirst

cousins

Night

blindn

ess

2010

0minus425

(minus12

5)150∘

2080minus425

(minus12

5)150∘

Normalatthe

saturatedtest

Redu

cedto

the10

central

degrees

Both

undetectable

Well-c

olored

optic

disc

but

narrow

edretin

alvessels

RPE

changesinthe

perip

hery

and

macular

atroph

icchanges

Hypoautofl

uorescence

inthem

acular

region

andou

tside

the

vascular

arcades

Thinning

ofou

terretina

inthe

macular

region

F3110

III5

(CIC05941)

275

F

One

cousin

onmother

sidem

ayhave

RCD

Night

blindn

essa

nddecreased

visio

n

2012

5+2

(minus2)

95∘

2012

5+175

(minus2)

70∘

c

Dyschromatop

siawith

nospecific

axis

Redu

cedto

the10

central

degrees

Both

undetectable

Paleop

ticdisc

narrow

edretin

alvessels

and

RPE

changesinthe

perip

hery

with

somem

acular

atroph

icchanges

Hypoautofl

uorescence

inthem

acular

region

andou

tside

the

vascular

arcades

Thinning

ofou

terretina

inthe

macular

region

FfemaleFM

fam

ilymem

berMm

aleBC

VAbestcorrected

visualacuityO

Doculadextra

(right

eye)O

Soculas

inistra

(lefteye)FFandmfERG

full-fi

eldandmultifocalER

GFAFfund

usautoflu

orescence

Sd-O

CTspectrald

omainop

ticalcoherencetom

ograph

yRP

Eretin

alpigm

entepitheliumLP

light

perceptio

nHMhandmotion

6 BioMed Research International

Table2Listof

mutations

detected

bynext

generatio

nsequ

encing

after

applying

relevant

filters

Patie

ntGene

Exon

Allelestate

Nucleotidee

xchange

Proteineffect

rsID

Con

servation

Polyoh

en2SIFT

Pathogenicity

Note

F303

II1(C

IC00

445)

NPH

P412

HTZ

AgtG

pSer481As

nno

NC

BT

Neutral

RP1

4HTZ

c162

5CgtG

pSer542lowast

mdashmdash

mdashmdash

Disease

causing

RM

[12]

RP1

4HTZ

c458

745

90delTAAG

pSer1529A

rgfslowast

9mdash

mdashmdash

mdashDisease

causing

NM

F335

III1

(CIC00

491)

PROM1

4HTZ

TgtC

pIle

178V

almdash

NC

BT

Neutral

GPR9

829

HTZ

GgtA

pArg2128Gln

rs149390094

NC

BT

Neutral

RP1

4HMZ

c408

940

92delAAG

ApArg1364

Valfslowast

8mdash

mdashmdash

mdashDisease

causing

NM

F674

III6

(CIC01106)

USH

2A39

HTZ

TgtG

pSer2450A

rgNo

HC

PD

DProb

ably

diseasec

ausin

gRP

14

HMZ

c120

5delG

pGly40

2Alafslowast

7mdash

mdashmdash

mdashDisease

causing

NM

F752

II1(C

IC01245)

USH

1C17

HTZ

GgtA

pArg477Trp

TMPES

P11

17532053

HC

PD

DProb

ablydisease

causing

PDE6

B10

HTZ

TgtC

pThr432Ile

mdashHC

BT

Uncertain

pathogenicity

RP1

4HTZ

c202

5dup

ApSer676Ile

fslowast

22mdash

mdashmdash

mdashDisease

causing

NM

RP1

4HTZ

c2377d

elA

pArg793G

lufslowast

55mdash

mdashmdash

mdashDisease

causing

NM

F782II5

(CIC01300)

RP1

4HMZ

c17191723delCTC

AA

pSer574Cy

sfslowast7

mdashmdash

mdashmdash

Disease

causing

NM

TULP

15

HMZ

c395

418dup

pAs

p124

Glu131del

rs63749128

mdashmdash

mdashNeutral

F1941III1

(CIC04130)

PCDH15

33HTZ

CgtT

pArg1889His

rs145851144

NC

BT

Neutral

C2orf71

1HTZ

CgtA

pArg656Ser

rs201980758

NC

BT

Neutral

CACN

A2D4

Exon

37-

Intro

n37

HTZ

CgtT

mdashrs80092457

NC

mdashmdash

Neutral

RP1

4HMZ

c1329d

elG

pLys443

Asnfslowast

12mdash

mdashmdash

mdashDisease

causing

NM

F3110

III5

(CIC05941)

EYS

6HTZ

CgtT

pSer326As

nrs112822256

NC

BT

Neutral

MER

TK8

HTZ

CgtG

pArg421Trp

rs138908058

NC

BD

Neutral

PRPF

621

HTZ

AgtG

pVal915M

etrs139778757

MC

PD

DUncertain

pathogenicity

TULP

114

HTZ

GgtA

pAla496Th

rrs141980901

MC

BD

Neutral

EYS

26HTZ

GgtA

pLys1365G

lurs16895519

NC

BD

Neutral

MER

TK18

HTZ

GgtC

pGlu823G

lnrs55924349

MC

BD

Neutral

RP1

4HMZ

c239

12392

delAA

pAsp79

9lowastmdash

mdashmdash

mdashDisease

causing

NM

Prob

ablydiseasec

ausin

gmutations

areh

ighlighted

inbo

ld

Bbenign

HMZ

homozygou

sHTZ

heterozygou

sM

Cmarginally

conservedNC

notcon

servedN

Mn

ovelmutation

RMrecurrent

mutation

TtoleratedPD

possib

lydamaging

BioMed Research International 7

(a)

(b) (c)

(d)

(e) (f)

Figure 1 Ophthalmic features of family F752 II1 (CIC01245) fundus color photographs ((a) and (d) for right and left eye resp)autofluorescence ((b) and (e) for right and left eye resp) and spectral domain optical coherence tomography horizontal macula scans ((c)and (f) for right and left eye resp) showing severe rod-cone dystrophy signs with macular involvement

last exon (exon 4) and shown to be transmitted in an auto-somal dominant mode of inheritance Most of RP1 diseasecausing variants represent nonsense mutations deletions orinsertions In mammalian genes nonsense mutations lead tounstable mRNAs that are degraded by nonsense-mediated

decay (NMD)However exceptionsmight arise when prema-ture stop codons occur in the last exon [37]These variants arethought to abolish RP1 function by resulting in a truncatedprotein lacking important functional domains although stillable to interact with some of its protein partner(s) [21] The

8 BioMed Research International

0 2 4 6 8 10 12 14 16 18 20 22

87888990919293949596979899100

Chromosome

Cov

erag

e at d

epth

25

X (

)

F303 CIC00445F335 CIC00491F674 CIC01106F752 CIC01245

F782 CIC01300

F3110 CIC05941F1941 CIC04130

(a)

0 2 4 6 8 10 12 14 16 18 20 22

Chromosome

100

150

200

250

300

350

400

Mea

n de

pth

per b

ase

F303 CIC00445F335 CIC00491F674 CIC01106 F3110 CIC05941F752 CIC01245

F782 CIC01300F1941 CIC04130

(b)

Figure 2 Sequencing statistics in index patients (a)The overall sequencing coverage of the target regions at 25X depth of coverage is shownin each of the chromosomes No values were indicated for chromosomes 13 18 21 and 22 as they were not targeted The term chromosome23 was used to designate the X chromosome F1941 III1 (CIC04130) and F3110 III5 (CIC05941) showed the lowest coverage results (b) Theaverage mean depth per base pair is shown for each of the chromosomes Most targets showed coverage around 300 times

1 1 1

1 2 3 4 5 6

2 3 4

4

5 632

2

1

1

1 2 3

4

5

11

2 21

2 2

3

2

3

6 7 8

4

41 5

(I)

(II)

(III)

(a) CIC00445 II1 F303 (b) CIC00491 III1 F335

(f) CIC04130 III1 F1941 (g) CIC05941 III5 F3110

1 2 3 4

1

1

1

2

2

3

3

4

4

5

5

6 7 8 9 10 65

43

21

1

1

2 3 4 5

432

2

1 2

2 3 4 5 61

1

(I)

(II)

(III)

(IV)

M1 c1625CgtGpSer542lowastM2 c4587 4590delTAAG pSer1529Argfslowast9

[M1][M2]

M3 c4089 4092delAAGApArg1364Valfslowast8

[M3][M3]

M4 c1205delG pGly402Alafslowast7

[M4][M4]

M5 c2025dupA pSer676Ilefslowast22M6 c2377delA pArg793Glufslowast55

[M5][M6]

M7 c1719 1723delCTCAA pSer574Cysfslowast7 M8 c1329delG pLys443Asnfslowast12

[M8][M8]

[M7][M7]

[=][=]

M9 c2391 2392delAA pAsp799lowast

[M9][M9]

[M9][=]

(c) CIC01106 III6 F674 (d) CIC01245 II1 F752

(e) CIC01300 II5 F782

Figure 3 Pedigrees of seven families with RP1 mutations underlying autosomal recessive rod-cone dystrophy Affected and unaffectedindividuals are represented by shapes filled with black and white colors respectively Men and women are indicated by squares and circlesrespectively Index subjects are marked by Consanguinity is marked by a double horizontal line

BioMed Research International 9

4

adRCD

arRCD

Class I

Class II (hot spot)

Class III Class III Class IV

321 BIF

pA

sp202

Glu

pThr373

Ile

pA

rg1519

Glu

fsX2

pA

rg1652

Leu

pCy

s2033

Tyr

pA

la669

Thr

pT8

65

Leu8

66

delIn

s

pA

sp984

Gly

pSe

r152

9Arg

fslowast9

pA

rg15

19G

lufslowast

2

pPr

o164

8Ser

fslowast2

pA

sn17

51Le

ufslowast4

pA

sn94

9Lys

fslowast32

pSe

r542lowast

pSe

r574

Cysfs

lowast7

pSe

r676

Ilefslowast

22

pA

rg79

3Glu

fslowast55

pA

sp79

9lowast

pM

et50

0Ser

fslowast33

pPr

o658

Hisf

slowast4

pLy

s673

Arg

fslowast9

pLy

s675

Asn

fslowast7

pA

rg67

7lowast

pA

rg67

7Asp

fslowast5

pG

ln67

9lowastp

Tyr6

85lowast

pG

ln68

6lowastp

Gln

689lowast

pG

lu70

0lowastp

Gly

706V

alfslowast7

pLy

s722

Glu

fslowast16

pG

ly72

3lowastp

Gly

724G

lufslowast

9

pile

725A

rgfslowast

6

pIle

725Th

rfslowast13

pIle

725A

spfslowast

4

pG

lu72

9Lys

fslowast9

pThr7

36Ile

fslowast4

pCy

s744

lowast

pSe

r747

Valfs

lowast16

pA

sn74

8Ile

fslowast15

pA

rg75

9lowastp

Leu7

62

Asn763

pL

eu762

Tyrfs

lowast17

pA

sn76

3Leu

fslowast11

pA

sn76

9Arg

fslowast6

pLy

s778

lowast

pSe

r779

lowast

pSe

r862

lowast

pIle

864L

ysfslowast

11

pTy

r105

3Thrfs

lowast4

pSe

r2A

rgfslowast16

pH

is31A

rgfslowast

9

pVa

l157

Trpf

slowast16

pA

la22

1Gly

fslowast43

pPr

o229

Gln

fslowast35

pA

rg33

8lowast

pA

rg39

6lowastp

Gly

402A

lafslowast7

pLy

s443

Asn

fslowast12

pG

lu48

8lowast

pTr

p113

1lowast

pG

ly11

40ly

sfslowast4

pA

sn11

43Ile

fslowast25

pG

lu12

27M

etfslowast

29

pA

rg13

64Va

lfslowast8

pLy

s151

8lowast

pA

rg87

2Thrfs

lowast2

pSe

r911

lowast

Figure 4 Schematic presentation of RP1 disease causing mutations Disease causing mutations were represented based on the classificationby Chen and coworkers [13] Mutations responsible for recessive arRCD were shown in the upper half whereas mutations causing adRCDwere shown in the lower half pGly402Alafslowast7 pLys443Asnfslowast12 pArg1364Valfslowast8 and pSer1529Argfslowast9 belong to class III AlthoughpSer574Cysfslowast7 pSer676Ilefslowast22 pArg793Glufslowast55 and pAsp799lowast are class II mutations these variants do not cause adRCD but arRCDinstead Amino acid modifications shown in red and blue represent novel frameshift or nonsense mutations and the recurrent pSer542lowastmutation respectively Protein localization of pSer542lowast was highlighted in blue as it marked a recurrent mutation adRCD autosomaldominant rod-cone dystrophy arRCD autosomal recessive rod-cone dystrophy BIF drosophila melanogaster bifocal

latter observation is supported by finding that RP1 mutantmRNA is expressed in a human cell line carrying a homozy-gous pArg677lowast mutation [21]

Based on Chen et al [13] RP1 truncating mutations lead-ing to arRCD or adRCD can be divided into four distinctgroups Class I is composed of truncating mutations locatedin exons 2 and 3These variants are sensitive toNMDand thusare considered as true loss-of-function alleles (Figure 4) [13]Class II involves truncating mutations that are located in aspot between codons 500 and 1053 in exon 4 [13] the so calledldquoRP1 hot spotrdquo The ldquohot spotrdquo variants tend to be insensitiveto NMD process and thus result in a protein with a potentialdominant negative effect leading to adRCD (Figure 4) [13]Class III includes truncating mutations insensible to NMDlocated between codons 264 and 499 and between codons1054 to 1751 in exon 4 These truncating proteins result in aloss of function leading to arRCD (Figure 4) [13] Finally classIV includes protein-truncating mutations near the 31015840 endof the fourth exon (Figure 4) [13] Most likely the resultingproteins display only aminor loss of their C-terminal portionpreserving the majority of functional domains and keeping aresidual activity According to the classification of Chen et al[13] pGly402Alafslowast7 pLys443Asnfslowast12 pArg1364Valfslowast8and pSer1529Argfslowast9 belong to class III (Figure 4)

The predicted physiopathology for pSer542lowastpSer574Cysfslowast7 pSer676Ilefslowast22 pArg793Glufslowast55 andpAsp799lowast is more complex According to Chenrsquos classi-fication these frameshift deletions and nonsense mutationsshould belong to class II previously only associated withadRCD However herein they are causing presumablyarRCD (Figure 4) To further confirm these findings clinicaland genetic testing of the reported unaffected parents shouldbe done

Based on the previous findings we speculate that the clas-sification by Chen and coworkers does not hold true for allmutations Supporting this statement Avila-Fernandez et al[12] reported the same nonsense mutation (pSer542lowast) foundin (F303 II1 (CIC00445)) and located at the 51015840 end of theldquohot spotrdquo to cause arRP in the Spanish population [12]These observations are of interest as they point out for animplication of hot spot region for adRCD-RP1mutation alsoin case of arRCD Future studies will need to clarify why someclass II mutations lead to adRCD and others to arRCD

Patients with arRCD and RP1 mutations show a moresevere disease than adRCD-RP1 mutant patients with mac-ular atrophy in all our cases This was first outlined byLafont et al [17] When patients are presenting with latesevere disease the diagnostic distinction between RCD with

10 BioMed Research International

initial rod involvement and cone-rod dystrophy (CRD) withinitial cone involvement is difficult Of note is that one ofthe patients (CIC01300) in the present study was initiallyclassified as possibly having severe CRD and his diagnosiswas actually revisited after NGS resultsThis also outlines theinterest of unbiased massive parallel sequencing methods fora more precise clinical diagnostic in case of end stage diseaseThis point will most likely become evenmore critical with theperspective of therapeutic trials

41 Strength and Limitations We estimate that 1 of ourtarget regions were not covered Partially uncovered exonsare a real common issue when capturing the DNA sequencesusing commercially available probes this bias might implya loss of some candidate variants However we found thatrate of 1 is very reasonable when compared with otherNGS panels In addition in order to exclude the possibilityof finding other candidate variants we have sequenced bySanger method the majority of these regions Five of ourpatients carried homozygous RP1 mutations For four of thesubjects carrying homozygous variants namely CIC00491F335 CIC01106 F674 CIC01300 F782 and CIC04130 F1941co-segregation analysis needs to be done to confirm auto-somal recessive inheritance but we do not have access toparentrsquos DNA CIC05941 was the only one not to reportclear consanguinity in the family and we cannot exclude thepossibility of a large deletion on the second allele of RP1gene Again DNA of the father not available for us wouldbe helpful to prove autosomal recessive inheritance and thehomozygous state of the mutation

In conclusion we have reported 9 mutations in RP1 ofwhich 8 were novel causing arRCD [8 12ndash20] Interestingly aprevalence ofasymp25points out for the necessity of sequencingRP1 in sporadic and recessive cases of RCD Further func-tional studies are needed to understand the impact of RP1structure on its function at the molecular level such a stepwould strengthen our knowledge in the physiology of retinalphotoreceptors

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Isabelle Audo and Christina Zeitz contributed equally to thiswork

Acknowledgments

The authors express their sincere gratitude to the familieswho participated in this study and to the clinical staff fortheir help in clinical data and DNA collection This workwas supported by Foundation Voir et Entendre (CZ) PrixDalloz for ldquola recherche en ophtalmologierdquo (CZ) FoundationFighting Blindness (FFB) [CD-CL-0808-0466-CHNO] (IA)and FFB center (FFB grantC-GE-0912-0601-INSERM02)

Prix de la Fondation de lrsquoŒil (IA) Ville de Paris and RegionIle de France and the French State program ldquoInvestissementsdrsquoAvenirrdquo managed by the Agence Nationale de la Recherche[LIFESENSES ANR-10-LABX-65]

References

[1] D T Hartong E L Berson and T P Dryja ldquoRetinitis pigmen-tosardquoThe Lancet vol 368 no 9549 pp 1795ndash1809 2006

[2] I Audo M Lancelot S Mohand-Saıd et al ldquoNovel C2orf71mutations account for approximately sim1 of cases in a largeFrench arRP cohortrdquoHumanMutation vol 32 no 4 pp E2091ndashE2103 2011

[3] A Anasagasti C Irigoyen O Barandika A Lopez de Munainand J Ruiz-Ederra ldquoCurrent mutation discovery approaches inRetinitis PigmentosardquoVision Research vol 75 pp 117ndash129 2012

[4] L S Sullivan S J Bowne D G Birch et al ldquoPrevalence ofdisease-causingmutations in families with autosomal dominantretinitis pigmentosa a screen of known genes in 200 familiesrdquoInvestigative Ophthalmology andVisual Science vol 47 no 7 pp3052ndash3064 2006

[5] B J Seyedahmadi C Rivolta J A Keene E L Berson andT P Dryja ldquoComprehensive screening of the USH2A gene inUsher syndrome type II and non-syndromic recessive retinitispigmentosardquo Experimental Eye Research vol 79 no 2 pp 167ndash173 2004

[6] A Avila-Fernandez D Cantalapiedra E Aller et al ldquoMutationanalysis of 272 Spanish families affected by autosomal recessiveretinitis pigmentosa using a genotyping microarrayrdquoMolecularVision vol 16 pp 2550ndash2558 2010

[7] S El Shamieh M Neuille A Terray et al ldquoWhole-exomesequencing identifies KIZ as a ciliary gene associated withautosomal-recessive rod-cone dystrophyrdquo American Journal ofHuman Genetics vol 94 no 4 pp 625ndash633 2014

[8] I Audo S Mohand-Saıd C M Dhaenens et al ldquoRP1 andautosomal dominant rod-cone dystrophy novel mutations areview of published variants and genotype-phenotype correla-tionrdquo Human Mutation vol 33 no 1 pp 73ndash80 2012

[9] X Guillonneau N I Piriev M Danciger et al ldquoA nonsensemutation in a novel gene is associated with retinitis pigmentosain a family linked to the RP1 locusrdquoHumanMolecular Geneticsvol 8 no 8 pp 1541ndash1546 1999

[10] E A Pierce T Quinn T Meehan T L McGee E L Bersonand T P Dryja ldquoMutations in a gene encoding a new oxygen-regulated photoreceptor protein cause dominant retinitis pig-mentosardquo Nature Genetics vol 22 no 3 pp 248ndash254 1999

[11] L S Sullivan J R Heckenlively S J Bowne et al ldquoMutations ina novel retina-specific gene cause autosomal dominant retinitispigmentosardquo Nature Genetics vol 22 no 3 pp 255ndash259 1999

[12] A Avila-Fernandez M Corton K M Nishiguchi et al ldquoIden-tification of an RP1 prevalent founder mutation and relatedphenotype in Spanish patients with early-onset autosomalrecessive retinitisrdquo Ophthalmology vol 119 no 12 pp 2616ndash2621 2012

[13] L J Chen T Y Y Lai P O S Tam et al ldquoCompound heter-ozygosity of two novel truncation mutations in RP1 causingautosomal recessive retinitis pigmentosardquo InvestigativeOphthal-mology and Visual Science vol 51 no 4 pp 2236ndash2242 2010

[14] S Khaliq A Abid M Ismail et al ldquoNovel association of RP1gene mutations with autosomal recessive retinitis pigmentosardquoJournal of Medical Genetics vol 42 no 5 pp 436ndash438 2005

BioMed Research International 11

[15] A M Siemiatkowska G D N Astuti K Arimadyo et alldquoIdentification of a novel nonsense mutation in RP1 that causesautosomal recessive retinitis pigmentosa in an IndonesianfamilyrdquoMolecular Vision vol 18 pp 2411ndash2419 2012

[16] B Bocquet N A Marzouka M Hebrard et al ldquoHomozygositymapping in autosomal recessive retinitis pigmentosa familiesdetects novel mutationsrdquo Molecular Vision vol 19 pp 2487ndash2500 2013

[17] E M Lafont G Manes G Senechal et al ldquoPatients with reti-nitis pigmentosa due to RP1 mutations show greater severity inrecessive than in dominant casesrdquo Journal of Clinical amp Experi-mental Ophthalmology vol 2 article 194 2011

[18] M Al-Rashed L Abu Safieh H Alkuraya et al ldquoRP1 and reti-nitis pigmentosa report of novel mutations and insight intomutational mechanismrdquo British Journal of Ophthalmology vol96 no 7 pp 1018ndash1022 2012

[19] S A Riazuddin A Shahzadi C Zeitz et al ldquoA mutation inSLC24A1 implicated in autosomal-recessive congenital station-ary night blindnessrdquoThe American Journal of Human Geneticsvol 87 no 4 pp 523ndash531 2010

[20] H P Singh S Jalali R Narayanan and C Kannabiran ldquoGe-netic analysis of indian families with autosomal recessiveretinitis pigmentosa by homozygosity screeningrdquo InvestigativeOphthalmology and Visual Science vol 50 no 9 pp 4065ndash40712009

[21] Q Liu A Lyubarsky J H Skalet E N Pugh Jr and E A PierceldquoRP1 is required for the correct stacking of outer segment discsrdquoInvestigative Ophthalmology ampVisual Science vol 44 no 10 pp4171ndash4183 2003

[22] Q Liu J Zuo and E A Pierce ldquoThe retinitis pigmentosa 1protein is a photoreceptor microtubule-associated proteinrdquoJournal of Neuroscience vol 24 no 29 pp 6427ndash6436 2004

[23] I Audo K M Bujakowska T Leveillard et al ldquoDevelop-ment and application of a next-generation-sequencing (NGS)approach to detect known and novel gene defects underlyingretinal diseasesrdquo Orphanet Journal of Rare Diseases vol 7 no 1article 8 2012

[24] I Audo J A Sahel S Mohand-Saıd et al ldquoEYS is a major genefor rod-cone dystrophies in Francerdquo Human Mutation vol 31no 5 pp E1406ndashE1435 2010

[25] T G P Consortium ldquoA map of human genome variation frompopulation-scale sequencingrdquo Nature vol 467 no 7319 pp1061ndash1073 2010

[26] DMAltshuler RAGibbs L Peltonen et al ldquoIntegrating com-mon and rare genetic variation in diverse human populationsrdquoNature vol 467 no 7311 pp 52ndash58 2010

[27] J A Tennessen AW Bigham T D OrsquoConnor et al ldquoEvolutionand functional impact of rare coding variation from deepsequencing of human exomesrdquo Science vol 337 no 6090 pp64ndash69 2012

[28] M J Bamshad S BNgAW Bighamet al ldquoExome sequencingas a tool for Mendelian disease gene discoveryrdquo Nature ReviewsGenetics vol 12 no 11 pp 745ndash755 2011

[29] W J Kent C W Sugnet T S Furey et al ldquoThe human genomebrowser at UCSCrdquo Genome Research vol 12 no 6 pp 996ndash1006 2002

[30] I A Adzhubei S Schmidt L Peshkin et al ldquoA method andserver for predicting damaging missense mutationsrdquo NatureMethods vol 7 no 4 pp 248ndash249 2010

[31] P Kumar S Henikoff and P C Ng ldquoPredicting the effects ofcoding non-synonymous variants on protein function using the

SIFT algorithmrdquo Nature Protocols vol 4 no 7 pp 1073ndash10822009

[32] F O Desmet D Hamroun M Lalande G Collod-Beroud MClaustres and C Beroud ldquoHuman splicing finder an onlinebioinformatics tool to predict splicing signalsrdquo Nucleic AcidsResearch vol 37 no 9 article no e67 2009

[33] P D Stenson E V Ball M Mort A D Phillips K Shaw andD N Cooper ldquoThe Human Gene Mutation Database (HGMD)and its exploitation in the fields of personalized genomics andmolecular evolutionrdquo in Current Protocols in Bioinformaticschapter 1ndash13 2012

[34] I F A C Fokkema J T Den Dunnen and P E M TaschnerldquoLOVD easy creation of a locus-specific sequence variationdatabase using an ldquoLSDB-in-a-Boxrdquo approachrdquo Human Muta-tion vol 26 no 2 pp 63ndash68 2005

[35] S A Miller D D Dykes and H F Polesky ldquoA simple saltingout procedure for extracting DNA from human nucleated cellsrdquoNucleic Acids Research vol 16 no 3 p 1215 1988

[36] C Zeitz B Kloeckener-Gruissem U Forster et al ldquoMutationsin CABP4 the gene encoding the Ca2+-binding protein 4cause autosomal recessive night blindnessrdquoAmerican Journal ofHuman Genetics vol 79 no 4 pp 657ndash667 2006

[37] S A Riazuddin F Zulfiqar Q Zhang et al ldquoAutosomal reces-sive retinitis pigmentosa is associated with mutations in RP1 inthree consanguineous Pakistani familiesrdquo Investigative Ophthal-mology and Visual Science vol 46 no 7 pp 2264ndash2270 2005

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

2 BioMed Research International

RCD can be divided into 3 groups autosomal dominant(ad) (30ndash40) autosomal recessive (ar) (50ndash60) and X-linked (xl) (5ndash15) [3] To date mutations in at least 53genes were reported to cause nonsyndromic RCD (till 25June 2014 httpssphutheduretnet) Prevalence studiesrevealed rhodopsin (RHO) retinitis pigmentosa GTPase reg-ulator (RPGR) and usherin (USH2A) as being the mostfrequently mutated genes in adRCD [4 5] xlRCD [4] andarRCD respectively [6] Of note is that many other geneswith lower prevalence are also implicated in the geneticetiology of RCD [7 8] Mutations in RP1 were first shownto cause adRCD [9ndash11] however since 2005 articles haveshed light on its implication in arRCD etiology [12ndash20] RP1mutations were shown to account for asymp55 and asymp1 ofadRCD and arRCD cases respectively [8ndash20] InterestinglyAvila-Fernandez et al [12] reported that a founder nonsensemutation in the Spanish population pSer542lowast is responsiblefor 45 of arRCD cases suggesting that RP1 mutations aremore prevalent in arRCD than previously thought [12]

Retinitis pigmentosa 1 (RP1) is a photoreceptor-specificgene encoding a protein regulated by oxygen [10] RP1 proteinis required for correct orientation and higher-order stackingof outer segment disks [21] and was shown to be part of thephotoreceptor axoneme [22] RP1 localizes to the connectingcilia of photoreceptors and may assist in maintenance ofciliary structure or transport down the photoreceptor [22]Like many retinal degeneration genes the mechanism bywhich mutations in RP1 lead to photoreceptor cell death isstill unclear

We developed an unbiased and time-efficient retinal genenext generation sequencing array (NGS) which was furtherrevised and improved to target more than 120 genes impli-cated in inherited retinal diseases (IRDs) (list available uponrequest) [23] Using this NGS panel we screened a total of242 subjects with sporadic and recessive RCD in order todetect disease causingmutations and to report the prevalenceof pathogenic mutations in RP1 causing arRCD

2 Methods

21 Ethics Statement and Clinical Diagnosis of Rod-ConeDystrophy The study protocol adhered to the tenets of theDeclaration of Helsinki and was approved by the localEthics Committee (CPP Ile de France V) Informed writtenconsent was obtained from each study participant Indexpatients underwent full ophthalmic examination as previ-ously described [23]

22 Targeted Next Generation Sequencing A cohort of 242subjects affected with sporadic and arRCD was investigatedin the present study Prior to NGS screening moleculargenetic analysis with microarray (Asper Ophthalmics TartuEstonia) followed by direct Sanger sequencing of EYS andC2orf71 (major and minor genes implicated in RCD newlydiscovered at the beginning of our study) was performedin 201 index subjects (82) [2 24] As RPGR exon ORF15(MIM 312610) is not targeted by existing NGS panels weexcluded mutations in this ldquohot spotrdquo by Sanger sequencing

Although our NGS panel was selected from the SureSelectHuman All Exon Kits Version 4 (Agilent Massy France)this design was improved after analyzing the first 83 subjectswith sporadic and arRCD More precisely a total of asymp300Kbregions were added in order to cover all the previouslynontargeted regions Thus whereas the first design coveredthe exons and the flanking intronic regions of 120 genesimplicated in IRDs the second covered 123 genes in totalThe eArray web-based probe design tool was used for thispurpose (httpsearraychemagilentcomearray) All probeswere designed and synthesized by Agilent Technologies(Santa Clara CA USA) Sequence capture enrichment andelution were performed according to Agilentrsquos instructionsThe complete details were described elsewhere [23]

23 Assembly and Variant Calling Sequence reads werealigned to the reference human genome (UCSC hg19)using CASAVA17 software (Illumina) and the ELANDv2alignment algorithm Sequence variation annotation wasperformed using the IntegraGen in-house pipeline whichconsisted of gene annotation (RefSeq) detection of knownsingle nucleotide polymorphisms (dbSNP 135) followed bymutation characterization (missense intronic synonymousnonsense splice site and insertionsdeletions)

24 Quality Control and Coverage Assessment The firstNGS retinal panel harbored 120 IRDs genes encompassing321240 kb length per sample However after improvementthe same panel contained asymp600Kb and covered 123 IRDgenes The depth of coverage was calculated by counting thenumber of sequenced bases mapping to the target regionsMean depth of coverage was calculated per base pair for allsamples however only the results of subjects having RP1mutations were shown

25 Discrete Filtering of Annotated Variants In order to iden-tify disease causing mutations among nonpathogenic singlenucleotide polymorphisms we used a filtering approachagainst a set of polymorphisms that are available in the publicdatabases dbSNP 137 1000 Genomes Project [25] HapMap[26] and Exome Variant Server [27] with removal of variantswith a minor allele frequency (MAF) ge 0005 in case ofpresumed autosomal recessive mode of inheritance

26 Pathogenicity Assessment We stratified candidate muta-tions based on their functional class by giving a priorityto frameshifts stop codons and disruptions of canonicalsplice sites variants [28] For missense changes amino acidconservation across 46 different species was studied using theUCSC Genome Browser [29] If no amino acid change wasfound then the residue was considered as ldquohighly conservedrdquoIf a different change was seen in less than four species and notin the primates then it was considered as ldquomoderately con-servedrdquo and if a change was present in 5ndash7 it was consideredas ldquomarginally conservedrdquo otherwise the amino acid residuewas considered as ldquonot conservedrdquo Pathogenic predictionwas performed using two software programs Polyphen2 [30]and SIFT [31] based on specieshomologue conservation

BioMed Research International 3

putative structural domains and 3D structures (if available)Analysis of potential splice site variant consequences whenrelevant was done using human splicing finder [32]

27 Known Genotype-Phenotype Correlations The searchfor previous genotype-phenotype associations was done bysearching numerous literature databases including OnlineMendelian Inheritance in Man (httpomimorg) HumanGene Mutation Database [33] Leiden Open Variation Data-base [34] and RetNet (httpssphutheduretnet)

28 Validation of the Identified Variants and CosegregationAnalyses Sanger sequencing was performed to validate dis-ease causing mutations in RP1 The respective primer infor-mation can be communicated on request In addition bloodsamples were collected from additional family memberswhen possible and cosegregation analyses on extracted DNAwere performed as previously described [35 36]

3 Results

31 Clinical Data Clinical data are summarized in Table 1Among identified patients 5 were females 2 were maleand ages at time of examination ranged from 25 to 42 Allpatients were diagnosed before age 20 mostly based on nightblindness from early childhood and secondary central visionloss They all showed severe RCD with constricted visualfields no detectable responses on full field electroretinogramand both peripheral involvement and macular involvement(Figure 1 presents fundus pictures of patient II1 (CIC01245)in family F752 as an example) Comparing visual acuity andvisual fields for these arRCD patients with those of adRCDcases published by Audo and coworkers [8] we noticed amore severe phenotype in recessive cases However morecases with RP1mutations would be needed to draw statisticalconclusion

32 Sequencing Statistics In index patients the overallsequencing coverage of the target regions was ge88 for a 25Xdepth of coverage in each of the chromosomes (Figure 2(a))resulting a mean sequencing depth of 299 times per baseMean sequencing results per base in each target chromosomegene regions were shown in Figure 2(b) It is of importanceto mention that lt1 of target regions were not covered at allThese were fragments of 120 bp belonging in 66 of the casesonly to a fraction of an exonThe remaining uncovered targetscorresponded each to an entire exon in genes such as CHMPDZD7 RP9 CC2D2A IMPDH1 CNGA1 and EYS

33 Detection of Disease Causing Mutations in RP1 GeneAfter data filtering the total number of putative diseasecausing variants was reduced by 993Thus in total filteringenriched the percentage of putative disease causingmutationsfrom 07 (253339 variants) to 333 (925 variants) in the 7subjects presented here (Table 2) These subjects exhibit RP1mutations in the last exon 4 that are predicted to lead to apremature stop codon We found 9 pathogenic mutations inRP1 among which one (pSer542lowast in CIC00445) was already

reported by Avila-Fernandez et al [12] as a founder nonsensemutation in the Spanish population responsible for 45of arRCD Although F303 is from French origin we cannotexclude the possibility of a founder effect of pSer542lowast in ourcohort

Patient family F303 II1 (CIC00445) was found to carrycompound heterozygous variants a nonsense mutationc1625CgtG leading to a predicted premature stop (pSer542lowast)and a deletion c4587 4590delTAAG leading to a frameshiftand a premature termination codon (pSer1529Argfslowast9)(Table 2 Figure 3) Patient family F752 II1 (CIC01245)was also found to carry compound heterozygous variantsa 1 bp duplication c2025dupA leading to pSer676Ilefslowast22and a 1 bp deletion c2377delA leading to pArg793Glufslowast55(Table 2)

Patients from family F335 III1 (CIC00491) family F674III6 (CIC01106) family F782 II5 (CIC01300) family F1941III1 (CIC04130) and family F3110 III5 (CIC05941) werefound to carry homozygous deletions c4089 4092delAAGAleading to pArg1364Valfslowast8 c1205delG leading topGly402Alafslowast7 c1719 1723delCTCAA leading topSer574Cysfslowast7 c1329delG leading to pLys443Asnfslowast12and c2391 2392delAA leading to pAsp799lowast resp) (Table 2and Figure 3) It is important to note that consanguinity wasreported in families F335 F674 F782 and F1941

AllRP1mutations detected byNGSwere further validatedby Sanger sequencing All variants cosegregatedwith the phe-notype in available family members Based on the previousfindings the measured prevalence of RP1-associated arRCDin this cohort is asymp25

4 Discussion

The current study further demonstrates the usefulness ofNGS as a comprehensive genetic diagnostic tool for IRDswith further impact on patients counseling and participationfor potential therapeutic trials Our study applied to a largecohort of sporadic and autosomal recessive cases of RCDidentifies 8 novel mutations in a gene not classically screenedin arRCD by other methods such as Sanger sequencing ormicroarray analysis outlining the interest of this massiveparallel sequencing method Consequently a prevalence ofRP1 mutation in 25 of sporadic or arRCD cases in theEuropean population is herein reported

RP1 is a 15 kb single copy gene clustering the small armof the chromosome 8 (8q121) It encodes a 2506 aminoacid protein having a molecular weight of 241 kDa contain-ing a Drosophila melanogaster bifocal (BIF) (amino acid486ndash635) and two doublecortin domains Whereas the BIFdomain helps to maintain the photoreceptor morphogenesisdoublecortin domains bind microtubules and regulate theirpolymerization [22] Along with RP1L1 (Retinitis Pigmentosa1-like 1 another retinal-specific protein) RP1 plays essentialand synergistic roles in outer segment morphogenesis of rodphotoreceptors [22]

To date at least 50 mutations in RP1 were identified inRCD [8 12ndash20] the majority of which are located in its

4 BioMed Research International

Table1Clinicaldataof

the7

indexpatie

ntsw

ithRP

1recessiv

emutations

Patie

ntAge

attim

eof

testing

Age

ofon

set

Sex

Family

histo

ry

Symptom

sat

timeo

fdiagno

sis

BCVA

ODO

SWith

refractio

n

Color

visio

n(15desaturated

Hue)

Bino

cular

kinetic

visualfield

(III4e

stim

ulus)

FFand

mfERG

Fund

usexam

ination

FAF

Sd-O

CT

F303II1

(CIC00

445)

426

FNoother

affectedFM

fro

mFrance

Night

blindn

ess

Handmotion

inbo

theyes

Impo

ssibledu

eto

lowvisio

n

Redu

cedto

perip

heral

islands

ofperceptio

n

Both

undetectable

Paleop

ticdisc

narrow

edbloo

dvessels

macular

atroph

icchangesand

optic

nerve

drusen

Hypoautofl

uorescence

inthem

acular

region

Thinning

ofou

terretina

inthe

macular

region

F335III1

(CIC00

491)

363

M

Twoother

brothers

affected

parentsfi

rst

cousins

Night

blindn

essa

ndrapid

decreased

visio

n

LPin

both

eyes

Impo

ssibledu

eto

lowvisio

n

Impo

ssible

duetolow

visio

n

Both

undetectable

Widespread

RPEchanges

andretin

alatroph

yin

both

thep

eriphery

andthem

acular

area

Widespreadlossof

FAF

Widespread

thinning

ofou

terretina

F674III6

(CIC01106)

2519

F

Parentsfi

rst

cousinsfrom

Turkeyone

femalea

ndmalec

ousin

saffectedalso

from

acon

-sang

uineou

sun

ion

Night

blindn

essa

nddecreased

visio

n

HMminus3(minus1)

0∘ 2016

0minus3

(minus050)0∘

Dyschromatop

siawith

nospecific

axis

Redu

cedto

5central

degrees

Both

undetectable

Well-c

olored

optic

disc

and

nonarrow

ingof

retin

alvessels

RP

Echangesin

thep

eriphery

andmacular

atroph

icchanges

Hypoautofl

uorescence

inthem

acular

region

andou

tside

the

vascular

arcades

Thinning

ofou

terretina

inthe

macular

region

F752II1

(CIC01245)

31Ea

rlyteens

FTw

osisters

affected

Night

blindn

ess

2063plano

(minus3)

180∘

2050plano

(minus17

5)180∘

Deutandefecton

both

eyes

Redu

cedto

10∘

times20∘

Both

undetectable

Paleop

ticdisc

headnarrowed

retin

alvessels

andRP

Echangesinthe

perip

hery

with

somem

acular

atroph

icchanges

Hypoautofl

uorescence

inthem

acular

region

andou

tside

the

vascular

arcades

Thinning

ofou

terretina

inthe

macular

region

F782

II5

(CIC01300)

279

MParentsfrom

Algeriafirst

cousins

Night

blindn

essa

nddecreased

visio

n

2050minus92

5(minus250)15∘

2050

minus9(minus17

5)100∘

mdash

Redu

cedto

the10

central

degrees

Both

undetectable

Mild

optic

disc

pallo

ratroph

icmacular

changesand

perip

heral

pigm

ent

depo

sits

Hypoautofl

uorescence

inthem

acular

region

Thinning

ofou

terretina

inthe

macular

BioMed Research International 5

Table1Con

tinued

Patie

ntAge

attim

eof

testing

Age

ofon

set

Sex

Family

histo

ry

Symptom

sat

timeo

fdiagno

sis

BCVA

ODO

SWith

refractio

n

Color

visio

n(15desaturated

Hue)

Bino

cular

kinetic

visualfield

(III4e

stim

ulus)

FFand

mfERG

Fund

usexam

ination

FAF

Sd-O

CT

F1941III1

(CIC04

130)

30child

hood

FParentsfrom

Algeriafirst

cousins

Night

blindn

ess

2010

0minus425

(minus12

5)150∘

2080minus425

(minus12

5)150∘

Normalatthe

saturatedtest

Redu

cedto

the10

central

degrees

Both

undetectable

Well-c

olored

optic

disc

but

narrow

edretin

alvessels

RPE

changesinthe

perip

hery

and

macular

atroph

icchanges

Hypoautofl

uorescence

inthem

acular

region

andou

tside

the

vascular

arcades

Thinning

ofou

terretina

inthe

macular

region

F3110

III5

(CIC05941)

275

F

One

cousin

onmother

sidem

ayhave

RCD

Night

blindn

essa

nddecreased

visio

n

2012

5+2

(minus2)

95∘

2012

5+175

(minus2)

70∘

c

Dyschromatop

siawith

nospecific

axis

Redu

cedto

the10

central

degrees

Both

undetectable

Paleop

ticdisc

narrow

edretin

alvessels

and

RPE

changesinthe

perip

hery

with

somem

acular

atroph

icchanges

Hypoautofl

uorescence

inthem

acular

region

andou

tside

the

vascular

arcades

Thinning

ofou

terretina

inthe

macular

region

FfemaleFM

fam

ilymem

berMm

aleBC

VAbestcorrected

visualacuityO

Doculadextra

(right

eye)O

Soculas

inistra

(lefteye)FFandmfERG

full-fi

eldandmultifocalER

GFAFfund

usautoflu

orescence

Sd-O

CTspectrald

omainop

ticalcoherencetom

ograph

yRP

Eretin

alpigm

entepitheliumLP

light

perceptio

nHMhandmotion

6 BioMed Research International

Table2Listof

mutations

detected

bynext

generatio

nsequ

encing

after

applying

relevant

filters

Patie

ntGene

Exon

Allelestate

Nucleotidee

xchange

Proteineffect

rsID

Con

servation

Polyoh

en2SIFT

Pathogenicity

Note

F303

II1(C

IC00

445)

NPH

P412

HTZ

AgtG

pSer481As

nno

NC

BT

Neutral

RP1

4HTZ

c162

5CgtG

pSer542lowast

mdashmdash

mdashmdash

Disease

causing

RM

[12]

RP1

4HTZ

c458

745

90delTAAG

pSer1529A

rgfslowast

9mdash

mdashmdash

mdashDisease

causing

NM

F335

III1

(CIC00

491)

PROM1

4HTZ

TgtC

pIle

178V

almdash

NC

BT

Neutral

GPR9

829

HTZ

GgtA

pArg2128Gln

rs149390094

NC

BT

Neutral

RP1

4HMZ

c408

940

92delAAG

ApArg1364

Valfslowast

8mdash

mdashmdash

mdashDisease

causing

NM

F674

III6

(CIC01106)

USH

2A39

HTZ

TgtG

pSer2450A

rgNo

HC

PD

DProb

ably

diseasec

ausin

gRP

14

HMZ

c120

5delG

pGly40

2Alafslowast

7mdash

mdashmdash

mdashDisease

causing

NM

F752

II1(C

IC01245)

USH

1C17

HTZ

GgtA

pArg477Trp

TMPES

P11

17532053

HC

PD

DProb

ablydisease

causing

PDE6

B10

HTZ

TgtC

pThr432Ile

mdashHC

BT

Uncertain

pathogenicity

RP1

4HTZ

c202

5dup

ApSer676Ile

fslowast

22mdash

mdashmdash

mdashDisease

causing

NM

RP1

4HTZ

c2377d

elA

pArg793G

lufslowast

55mdash

mdashmdash

mdashDisease

causing

NM

F782II5

(CIC01300)

RP1

4HMZ

c17191723delCTC

AA

pSer574Cy

sfslowast7

mdashmdash

mdashmdash

Disease

causing

NM

TULP

15

HMZ

c395

418dup

pAs

p124

Glu131del

rs63749128

mdashmdash

mdashNeutral

F1941III1

(CIC04130)

PCDH15

33HTZ

CgtT

pArg1889His

rs145851144

NC

BT

Neutral

C2orf71

1HTZ

CgtA

pArg656Ser

rs201980758

NC

BT

Neutral

CACN

A2D4

Exon

37-

Intro

n37

HTZ

CgtT

mdashrs80092457

NC

mdashmdash

Neutral

RP1

4HMZ

c1329d

elG

pLys443

Asnfslowast

12mdash

mdashmdash

mdashDisease

causing

NM

F3110

III5

(CIC05941)

EYS

6HTZ

CgtT

pSer326As

nrs112822256

NC

BT

Neutral

MER

TK8

HTZ

CgtG

pArg421Trp

rs138908058

NC

BD

Neutral

PRPF

621

HTZ

AgtG

pVal915M

etrs139778757

MC

PD

DUncertain

pathogenicity

TULP

114

HTZ

GgtA

pAla496Th

rrs141980901

MC

BD

Neutral

EYS

26HTZ

GgtA

pLys1365G

lurs16895519

NC

BD

Neutral

MER

TK18

HTZ

GgtC

pGlu823G

lnrs55924349

MC

BD

Neutral

RP1

4HMZ

c239

12392

delAA

pAsp79

9lowastmdash

mdashmdash

mdashDisease

causing

NM

Prob

ablydiseasec

ausin

gmutations

areh

ighlighted

inbo

ld

Bbenign

HMZ

homozygou

sHTZ

heterozygou

sM

Cmarginally

conservedNC

notcon

servedN

Mn

ovelmutation

RMrecurrent

mutation

TtoleratedPD

possib

lydamaging

BioMed Research International 7

(a)

(b) (c)

(d)

(e) (f)

Figure 1 Ophthalmic features of family F752 II1 (CIC01245) fundus color photographs ((a) and (d) for right and left eye resp)autofluorescence ((b) and (e) for right and left eye resp) and spectral domain optical coherence tomography horizontal macula scans ((c)and (f) for right and left eye resp) showing severe rod-cone dystrophy signs with macular involvement

last exon (exon 4) and shown to be transmitted in an auto-somal dominant mode of inheritance Most of RP1 diseasecausing variants represent nonsense mutations deletions orinsertions In mammalian genes nonsense mutations lead tounstable mRNAs that are degraded by nonsense-mediated

decay (NMD)However exceptionsmight arise when prema-ture stop codons occur in the last exon [37]These variants arethought to abolish RP1 function by resulting in a truncatedprotein lacking important functional domains although stillable to interact with some of its protein partner(s) [21] The

8 BioMed Research International

0 2 4 6 8 10 12 14 16 18 20 22

87888990919293949596979899100

Chromosome

Cov

erag

e at d

epth

25

X (

)

F303 CIC00445F335 CIC00491F674 CIC01106F752 CIC01245

F782 CIC01300

F3110 CIC05941F1941 CIC04130

(a)

0 2 4 6 8 10 12 14 16 18 20 22

Chromosome

100

150

200

250

300

350

400

Mea

n de

pth

per b

ase

F303 CIC00445F335 CIC00491F674 CIC01106 F3110 CIC05941F752 CIC01245

F782 CIC01300F1941 CIC04130

(b)

Figure 2 Sequencing statistics in index patients (a)The overall sequencing coverage of the target regions at 25X depth of coverage is shownin each of the chromosomes No values were indicated for chromosomes 13 18 21 and 22 as they were not targeted The term chromosome23 was used to designate the X chromosome F1941 III1 (CIC04130) and F3110 III5 (CIC05941) showed the lowest coverage results (b) Theaverage mean depth per base pair is shown for each of the chromosomes Most targets showed coverage around 300 times

1 1 1

1 2 3 4 5 6

2 3 4

4

5 632

2

1

1

1 2 3

4

5

11

2 21

2 2

3

2

3

6 7 8

4

41 5

(I)

(II)

(III)

(a) CIC00445 II1 F303 (b) CIC00491 III1 F335

(f) CIC04130 III1 F1941 (g) CIC05941 III5 F3110

1 2 3 4

1

1

1

2

2

3

3

4

4

5

5

6 7 8 9 10 65

43

21

1

1

2 3 4 5

432

2

1 2

2 3 4 5 61

1

(I)

(II)

(III)

(IV)

M1 c1625CgtGpSer542lowastM2 c4587 4590delTAAG pSer1529Argfslowast9

[M1][M2]

M3 c4089 4092delAAGApArg1364Valfslowast8

[M3][M3]

M4 c1205delG pGly402Alafslowast7

[M4][M4]

M5 c2025dupA pSer676Ilefslowast22M6 c2377delA pArg793Glufslowast55

[M5][M6]

M7 c1719 1723delCTCAA pSer574Cysfslowast7 M8 c1329delG pLys443Asnfslowast12

[M8][M8]

[M7][M7]

[=][=]

M9 c2391 2392delAA pAsp799lowast

[M9][M9]

[M9][=]

(c) CIC01106 III6 F674 (d) CIC01245 II1 F752

(e) CIC01300 II5 F782

Figure 3 Pedigrees of seven families with RP1 mutations underlying autosomal recessive rod-cone dystrophy Affected and unaffectedindividuals are represented by shapes filled with black and white colors respectively Men and women are indicated by squares and circlesrespectively Index subjects are marked by Consanguinity is marked by a double horizontal line

BioMed Research International 9

4

adRCD

arRCD

Class I

Class II (hot spot)

Class III Class III Class IV

321 BIF

pA

sp202

Glu

pThr373

Ile

pA

rg1519

Glu

fsX2

pA

rg1652

Leu

pCy

s2033

Tyr

pA

la669

Thr

pT8

65

Leu8

66

delIn

s

pA

sp984

Gly

pSe

r152

9Arg

fslowast9

pA

rg15

19G

lufslowast

2

pPr

o164

8Ser

fslowast2

pA

sn17

51Le

ufslowast4

pA

sn94

9Lys

fslowast32

pSe

r542lowast

pSe

r574

Cysfs

lowast7

pSe

r676

Ilefslowast

22

pA

rg79

3Glu

fslowast55

pA

sp79

9lowast

pM

et50

0Ser

fslowast33

pPr

o658

Hisf

slowast4

pLy

s673

Arg

fslowast9

pLy

s675

Asn

fslowast7

pA

rg67

7lowast

pA

rg67

7Asp

fslowast5

pG

ln67

9lowastp

Tyr6

85lowast

pG

ln68

6lowastp

Gln

689lowast

pG

lu70

0lowastp

Gly

706V

alfslowast7

pLy

s722

Glu

fslowast16

pG

ly72

3lowastp

Gly

724G

lufslowast

9

pile

725A

rgfslowast

6

pIle

725Th

rfslowast13

pIle

725A

spfslowast

4

pG

lu72

9Lys

fslowast9

pThr7

36Ile

fslowast4

pCy

s744

lowast

pSe

r747

Valfs

lowast16

pA

sn74

8Ile

fslowast15

pA

rg75

9lowastp

Leu7

62

Asn763

pL

eu762

Tyrfs

lowast17

pA

sn76

3Leu

fslowast11

pA

sn76

9Arg

fslowast6

pLy

s778

lowast

pSe

r779

lowast

pSe

r862

lowast

pIle

864L

ysfslowast

11

pTy

r105

3Thrfs

lowast4

pSe

r2A

rgfslowast16

pH

is31A

rgfslowast

9

pVa

l157

Trpf

slowast16

pA

la22

1Gly

fslowast43

pPr

o229

Gln

fslowast35

pA

rg33

8lowast

pA

rg39

6lowastp

Gly

402A

lafslowast7

pLy

s443

Asn

fslowast12

pG

lu48

8lowast

pTr

p113

1lowast

pG

ly11

40ly

sfslowast4

pA

sn11

43Ile

fslowast25

pG

lu12

27M

etfslowast

29

pA

rg13

64Va

lfslowast8

pLy

s151

8lowast

pA

rg87

2Thrfs

lowast2

pSe

r911

lowast

Figure 4 Schematic presentation of RP1 disease causing mutations Disease causing mutations were represented based on the classificationby Chen and coworkers [13] Mutations responsible for recessive arRCD were shown in the upper half whereas mutations causing adRCDwere shown in the lower half pGly402Alafslowast7 pLys443Asnfslowast12 pArg1364Valfslowast8 and pSer1529Argfslowast9 belong to class III AlthoughpSer574Cysfslowast7 pSer676Ilefslowast22 pArg793Glufslowast55 and pAsp799lowast are class II mutations these variants do not cause adRCD but arRCDinstead Amino acid modifications shown in red and blue represent novel frameshift or nonsense mutations and the recurrent pSer542lowastmutation respectively Protein localization of pSer542lowast was highlighted in blue as it marked a recurrent mutation adRCD autosomaldominant rod-cone dystrophy arRCD autosomal recessive rod-cone dystrophy BIF drosophila melanogaster bifocal

latter observation is supported by finding that RP1 mutantmRNA is expressed in a human cell line carrying a homozy-gous pArg677lowast mutation [21]

Based on Chen et al [13] RP1 truncating mutations lead-ing to arRCD or adRCD can be divided into four distinctgroups Class I is composed of truncating mutations locatedin exons 2 and 3These variants are sensitive toNMDand thusare considered as true loss-of-function alleles (Figure 4) [13]Class II involves truncating mutations that are located in aspot between codons 500 and 1053 in exon 4 [13] the so calledldquoRP1 hot spotrdquo The ldquohot spotrdquo variants tend to be insensitiveto NMD process and thus result in a protein with a potentialdominant negative effect leading to adRCD (Figure 4) [13]Class III includes truncating mutations insensible to NMDlocated between codons 264 and 499 and between codons1054 to 1751 in exon 4 These truncating proteins result in aloss of function leading to arRCD (Figure 4) [13] Finally classIV includes protein-truncating mutations near the 31015840 endof the fourth exon (Figure 4) [13] Most likely the resultingproteins display only aminor loss of their C-terminal portionpreserving the majority of functional domains and keeping aresidual activity According to the classification of Chen et al[13] pGly402Alafslowast7 pLys443Asnfslowast12 pArg1364Valfslowast8and pSer1529Argfslowast9 belong to class III (Figure 4)

The predicted physiopathology for pSer542lowastpSer574Cysfslowast7 pSer676Ilefslowast22 pArg793Glufslowast55 andpAsp799lowast is more complex According to Chenrsquos classi-fication these frameshift deletions and nonsense mutationsshould belong to class II previously only associated withadRCD However herein they are causing presumablyarRCD (Figure 4) To further confirm these findings clinicaland genetic testing of the reported unaffected parents shouldbe done

Based on the previous findings we speculate that the clas-sification by Chen and coworkers does not hold true for allmutations Supporting this statement Avila-Fernandez et al[12] reported the same nonsense mutation (pSer542lowast) foundin (F303 II1 (CIC00445)) and located at the 51015840 end of theldquohot spotrdquo to cause arRP in the Spanish population [12]These observations are of interest as they point out for animplication of hot spot region for adRCD-RP1mutation alsoin case of arRCD Future studies will need to clarify why someclass II mutations lead to adRCD and others to arRCD

Patients with arRCD and RP1 mutations show a moresevere disease than adRCD-RP1 mutant patients with mac-ular atrophy in all our cases This was first outlined byLafont et al [17] When patients are presenting with latesevere disease the diagnostic distinction between RCD with

10 BioMed Research International

initial rod involvement and cone-rod dystrophy (CRD) withinitial cone involvement is difficult Of note is that one ofthe patients (CIC01300) in the present study was initiallyclassified as possibly having severe CRD and his diagnosiswas actually revisited after NGS resultsThis also outlines theinterest of unbiased massive parallel sequencing methods fora more precise clinical diagnostic in case of end stage diseaseThis point will most likely become evenmore critical with theperspective of therapeutic trials

41 Strength and Limitations We estimate that 1 of ourtarget regions were not covered Partially uncovered exonsare a real common issue when capturing the DNA sequencesusing commercially available probes this bias might implya loss of some candidate variants However we found thatrate of 1 is very reasonable when compared with otherNGS panels In addition in order to exclude the possibilityof finding other candidate variants we have sequenced bySanger method the majority of these regions Five of ourpatients carried homozygous RP1 mutations For four of thesubjects carrying homozygous variants namely CIC00491F335 CIC01106 F674 CIC01300 F782 and CIC04130 F1941co-segregation analysis needs to be done to confirm auto-somal recessive inheritance but we do not have access toparentrsquos DNA CIC05941 was the only one not to reportclear consanguinity in the family and we cannot exclude thepossibility of a large deletion on the second allele of RP1gene Again DNA of the father not available for us wouldbe helpful to prove autosomal recessive inheritance and thehomozygous state of the mutation

In conclusion we have reported 9 mutations in RP1 ofwhich 8 were novel causing arRCD [8 12ndash20] Interestingly aprevalence ofasymp25points out for the necessity of sequencingRP1 in sporadic and recessive cases of RCD Further func-tional studies are needed to understand the impact of RP1structure on its function at the molecular level such a stepwould strengthen our knowledge in the physiology of retinalphotoreceptors

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Isabelle Audo and Christina Zeitz contributed equally to thiswork

Acknowledgments

The authors express their sincere gratitude to the familieswho participated in this study and to the clinical staff fortheir help in clinical data and DNA collection This workwas supported by Foundation Voir et Entendre (CZ) PrixDalloz for ldquola recherche en ophtalmologierdquo (CZ) FoundationFighting Blindness (FFB) [CD-CL-0808-0466-CHNO] (IA)and FFB center (FFB grantC-GE-0912-0601-INSERM02)

Prix de la Fondation de lrsquoŒil (IA) Ville de Paris and RegionIle de France and the French State program ldquoInvestissementsdrsquoAvenirrdquo managed by the Agence Nationale de la Recherche[LIFESENSES ANR-10-LABX-65]

References

[1] D T Hartong E L Berson and T P Dryja ldquoRetinitis pigmen-tosardquoThe Lancet vol 368 no 9549 pp 1795ndash1809 2006

[2] I Audo M Lancelot S Mohand-Saıd et al ldquoNovel C2orf71mutations account for approximately sim1 of cases in a largeFrench arRP cohortrdquoHumanMutation vol 32 no 4 pp E2091ndashE2103 2011

[3] A Anasagasti C Irigoyen O Barandika A Lopez de Munainand J Ruiz-Ederra ldquoCurrent mutation discovery approaches inRetinitis PigmentosardquoVision Research vol 75 pp 117ndash129 2012

[4] L S Sullivan S J Bowne D G Birch et al ldquoPrevalence ofdisease-causingmutations in families with autosomal dominantretinitis pigmentosa a screen of known genes in 200 familiesrdquoInvestigative Ophthalmology andVisual Science vol 47 no 7 pp3052ndash3064 2006

[5] B J Seyedahmadi C Rivolta J A Keene E L Berson andT P Dryja ldquoComprehensive screening of the USH2A gene inUsher syndrome type II and non-syndromic recessive retinitispigmentosardquo Experimental Eye Research vol 79 no 2 pp 167ndash173 2004

[6] A Avila-Fernandez D Cantalapiedra E Aller et al ldquoMutationanalysis of 272 Spanish families affected by autosomal recessiveretinitis pigmentosa using a genotyping microarrayrdquoMolecularVision vol 16 pp 2550ndash2558 2010

[7] S El Shamieh M Neuille A Terray et al ldquoWhole-exomesequencing identifies KIZ as a ciliary gene associated withautosomal-recessive rod-cone dystrophyrdquo American Journal ofHuman Genetics vol 94 no 4 pp 625ndash633 2014

[8] I Audo S Mohand-Saıd C M Dhaenens et al ldquoRP1 andautosomal dominant rod-cone dystrophy novel mutations areview of published variants and genotype-phenotype correla-tionrdquo Human Mutation vol 33 no 1 pp 73ndash80 2012

[9] X Guillonneau N I Piriev M Danciger et al ldquoA nonsensemutation in a novel gene is associated with retinitis pigmentosain a family linked to the RP1 locusrdquoHumanMolecular Geneticsvol 8 no 8 pp 1541ndash1546 1999

[10] E A Pierce T Quinn T Meehan T L McGee E L Bersonand T P Dryja ldquoMutations in a gene encoding a new oxygen-regulated photoreceptor protein cause dominant retinitis pig-mentosardquo Nature Genetics vol 22 no 3 pp 248ndash254 1999

[11] L S Sullivan J R Heckenlively S J Bowne et al ldquoMutations ina novel retina-specific gene cause autosomal dominant retinitispigmentosardquo Nature Genetics vol 22 no 3 pp 255ndash259 1999

[12] A Avila-Fernandez M Corton K M Nishiguchi et al ldquoIden-tification of an RP1 prevalent founder mutation and relatedphenotype in Spanish patients with early-onset autosomalrecessive retinitisrdquo Ophthalmology vol 119 no 12 pp 2616ndash2621 2012

[13] L J Chen T Y Y Lai P O S Tam et al ldquoCompound heter-ozygosity of two novel truncation mutations in RP1 causingautosomal recessive retinitis pigmentosardquo InvestigativeOphthal-mology and Visual Science vol 51 no 4 pp 2236ndash2242 2010

[14] S Khaliq A Abid M Ismail et al ldquoNovel association of RP1gene mutations with autosomal recessive retinitis pigmentosardquoJournal of Medical Genetics vol 42 no 5 pp 436ndash438 2005

BioMed Research International 11

[15] A M Siemiatkowska G D N Astuti K Arimadyo et alldquoIdentification of a novel nonsense mutation in RP1 that causesautosomal recessive retinitis pigmentosa in an IndonesianfamilyrdquoMolecular Vision vol 18 pp 2411ndash2419 2012

[16] B Bocquet N A Marzouka M Hebrard et al ldquoHomozygositymapping in autosomal recessive retinitis pigmentosa familiesdetects novel mutationsrdquo Molecular Vision vol 19 pp 2487ndash2500 2013

[17] E M Lafont G Manes G Senechal et al ldquoPatients with reti-nitis pigmentosa due to RP1 mutations show greater severity inrecessive than in dominant casesrdquo Journal of Clinical amp Experi-mental Ophthalmology vol 2 article 194 2011

[18] M Al-Rashed L Abu Safieh H Alkuraya et al ldquoRP1 and reti-nitis pigmentosa report of novel mutations and insight intomutational mechanismrdquo British Journal of Ophthalmology vol96 no 7 pp 1018ndash1022 2012

[19] S A Riazuddin A Shahzadi C Zeitz et al ldquoA mutation inSLC24A1 implicated in autosomal-recessive congenital station-ary night blindnessrdquoThe American Journal of Human Geneticsvol 87 no 4 pp 523ndash531 2010

[20] H P Singh S Jalali R Narayanan and C Kannabiran ldquoGe-netic analysis of indian families with autosomal recessiveretinitis pigmentosa by homozygosity screeningrdquo InvestigativeOphthalmology and Visual Science vol 50 no 9 pp 4065ndash40712009

[21] Q Liu A Lyubarsky J H Skalet E N Pugh Jr and E A PierceldquoRP1 is required for the correct stacking of outer segment discsrdquoInvestigative Ophthalmology ampVisual Science vol 44 no 10 pp4171ndash4183 2003

[22] Q Liu J Zuo and E A Pierce ldquoThe retinitis pigmentosa 1protein is a photoreceptor microtubule-associated proteinrdquoJournal of Neuroscience vol 24 no 29 pp 6427ndash6436 2004

[23] I Audo K M Bujakowska T Leveillard et al ldquoDevelop-ment and application of a next-generation-sequencing (NGS)approach to detect known and novel gene defects underlyingretinal diseasesrdquo Orphanet Journal of Rare Diseases vol 7 no 1article 8 2012

[24] I Audo J A Sahel S Mohand-Saıd et al ldquoEYS is a major genefor rod-cone dystrophies in Francerdquo Human Mutation vol 31no 5 pp E1406ndashE1435 2010

[25] T G P Consortium ldquoA map of human genome variation frompopulation-scale sequencingrdquo Nature vol 467 no 7319 pp1061ndash1073 2010

[26] DMAltshuler RAGibbs L Peltonen et al ldquoIntegrating com-mon and rare genetic variation in diverse human populationsrdquoNature vol 467 no 7311 pp 52ndash58 2010

[27] J A Tennessen AW Bigham T D OrsquoConnor et al ldquoEvolutionand functional impact of rare coding variation from deepsequencing of human exomesrdquo Science vol 337 no 6090 pp64ndash69 2012

[28] M J Bamshad S BNgAW Bighamet al ldquoExome sequencingas a tool for Mendelian disease gene discoveryrdquo Nature ReviewsGenetics vol 12 no 11 pp 745ndash755 2011

[29] W J Kent C W Sugnet T S Furey et al ldquoThe human genomebrowser at UCSCrdquo Genome Research vol 12 no 6 pp 996ndash1006 2002

[30] I A Adzhubei S Schmidt L Peshkin et al ldquoA method andserver for predicting damaging missense mutationsrdquo NatureMethods vol 7 no 4 pp 248ndash249 2010

[31] P Kumar S Henikoff and P C Ng ldquoPredicting the effects ofcoding non-synonymous variants on protein function using the

SIFT algorithmrdquo Nature Protocols vol 4 no 7 pp 1073ndash10822009

[32] F O Desmet D Hamroun M Lalande G Collod-Beroud MClaustres and C Beroud ldquoHuman splicing finder an onlinebioinformatics tool to predict splicing signalsrdquo Nucleic AcidsResearch vol 37 no 9 article no e67 2009

[33] P D Stenson E V Ball M Mort A D Phillips K Shaw andD N Cooper ldquoThe Human Gene Mutation Database (HGMD)and its exploitation in the fields of personalized genomics andmolecular evolutionrdquo in Current Protocols in Bioinformaticschapter 1ndash13 2012

[34] I F A C Fokkema J T Den Dunnen and P E M TaschnerldquoLOVD easy creation of a locus-specific sequence variationdatabase using an ldquoLSDB-in-a-Boxrdquo approachrdquo Human Muta-tion vol 26 no 2 pp 63ndash68 2005

[35] S A Miller D D Dykes and H F Polesky ldquoA simple saltingout procedure for extracting DNA from human nucleated cellsrdquoNucleic Acids Research vol 16 no 3 p 1215 1988

[36] C Zeitz B Kloeckener-Gruissem U Forster et al ldquoMutationsin CABP4 the gene encoding the Ca2+-binding protein 4cause autosomal recessive night blindnessrdquoAmerican Journal ofHuman Genetics vol 79 no 4 pp 657ndash667 2006

[37] S A Riazuddin F Zulfiqar Q Zhang et al ldquoAutosomal reces-sive retinitis pigmentosa is associated with mutations in RP1 inthree consanguineous Pakistani familiesrdquo Investigative Ophthal-mology and Visual Science vol 46 no 7 pp 2264ndash2270 2005

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

BioMed Research International 3

putative structural domains and 3D structures (if available)Analysis of potential splice site variant consequences whenrelevant was done using human splicing finder [32]

27 Known Genotype-Phenotype Correlations The searchfor previous genotype-phenotype associations was done bysearching numerous literature databases including OnlineMendelian Inheritance in Man (httpomimorg) HumanGene Mutation Database [33] Leiden Open Variation Data-base [34] and RetNet (httpssphutheduretnet)

28 Validation of the Identified Variants and CosegregationAnalyses Sanger sequencing was performed to validate dis-ease causing mutations in RP1 The respective primer infor-mation can be communicated on request In addition bloodsamples were collected from additional family memberswhen possible and cosegregation analyses on extracted DNAwere performed as previously described [35 36]

3 Results

31 Clinical Data Clinical data are summarized in Table 1Among identified patients 5 were females 2 were maleand ages at time of examination ranged from 25 to 42 Allpatients were diagnosed before age 20 mostly based on nightblindness from early childhood and secondary central visionloss They all showed severe RCD with constricted visualfields no detectable responses on full field electroretinogramand both peripheral involvement and macular involvement(Figure 1 presents fundus pictures of patient II1 (CIC01245)in family F752 as an example) Comparing visual acuity andvisual fields for these arRCD patients with those of adRCDcases published by Audo and coworkers [8] we noticed amore severe phenotype in recessive cases However morecases with RP1mutations would be needed to draw statisticalconclusion

32 Sequencing Statistics In index patients the overallsequencing coverage of the target regions was ge88 for a 25Xdepth of coverage in each of the chromosomes (Figure 2(a))resulting a mean sequencing depth of 299 times per baseMean sequencing results per base in each target chromosomegene regions were shown in Figure 2(b) It is of importanceto mention that lt1 of target regions were not covered at allThese were fragments of 120 bp belonging in 66 of the casesonly to a fraction of an exonThe remaining uncovered targetscorresponded each to an entire exon in genes such as CHMPDZD7 RP9 CC2D2A IMPDH1 CNGA1 and EYS

33 Detection of Disease Causing Mutations in RP1 GeneAfter data filtering the total number of putative diseasecausing variants was reduced by 993Thus in total filteringenriched the percentage of putative disease causingmutationsfrom 07 (253339 variants) to 333 (925 variants) in the 7subjects presented here (Table 2) These subjects exhibit RP1mutations in the last exon 4 that are predicted to lead to apremature stop codon We found 9 pathogenic mutations inRP1 among which one (pSer542lowast in CIC00445) was already

reported by Avila-Fernandez et al [12] as a founder nonsensemutation in the Spanish population responsible for 45of arRCD Although F303 is from French origin we cannotexclude the possibility of a founder effect of pSer542lowast in ourcohort

Patient family F303 II1 (CIC00445) was found to carrycompound heterozygous variants a nonsense mutationc1625CgtG leading to a predicted premature stop (pSer542lowast)and a deletion c4587 4590delTAAG leading to a frameshiftand a premature termination codon (pSer1529Argfslowast9)(Table 2 Figure 3) Patient family F752 II1 (CIC01245)was also found to carry compound heterozygous variantsa 1 bp duplication c2025dupA leading to pSer676Ilefslowast22and a 1 bp deletion c2377delA leading to pArg793Glufslowast55(Table 2)

Patients from family F335 III1 (CIC00491) family F674III6 (CIC01106) family F782 II5 (CIC01300) family F1941III1 (CIC04130) and family F3110 III5 (CIC05941) werefound to carry homozygous deletions c4089 4092delAAGAleading to pArg1364Valfslowast8 c1205delG leading topGly402Alafslowast7 c1719 1723delCTCAA leading topSer574Cysfslowast7 c1329delG leading to pLys443Asnfslowast12and c2391 2392delAA leading to pAsp799lowast resp) (Table 2and Figure 3) It is important to note that consanguinity wasreported in families F335 F674 F782 and F1941

AllRP1mutations detected byNGSwere further validatedby Sanger sequencing All variants cosegregatedwith the phe-notype in available family members Based on the previousfindings the measured prevalence of RP1-associated arRCDin this cohort is asymp25

4 Discussion

The current study further demonstrates the usefulness ofNGS as a comprehensive genetic diagnostic tool for IRDswith further impact on patients counseling and participationfor potential therapeutic trials Our study applied to a largecohort of sporadic and autosomal recessive cases of RCDidentifies 8 novel mutations in a gene not classically screenedin arRCD by other methods such as Sanger sequencing ormicroarray analysis outlining the interest of this massiveparallel sequencing method Consequently a prevalence ofRP1 mutation in 25 of sporadic or arRCD cases in theEuropean population is herein reported

RP1 is a 15 kb single copy gene clustering the small armof the chromosome 8 (8q121) It encodes a 2506 aminoacid protein having a molecular weight of 241 kDa contain-ing a Drosophila melanogaster bifocal (BIF) (amino acid486ndash635) and two doublecortin domains Whereas the BIFdomain helps to maintain the photoreceptor morphogenesisdoublecortin domains bind microtubules and regulate theirpolymerization [22] Along with RP1L1 (Retinitis Pigmentosa1-like 1 another retinal-specific protein) RP1 plays essentialand synergistic roles in outer segment morphogenesis of rodphotoreceptors [22]

To date at least 50 mutations in RP1 were identified inRCD [8 12ndash20] the majority of which are located in its

4 BioMed Research International

Table1Clinicaldataof

the7

indexpatie

ntsw

ithRP

1recessiv

emutations

Patie

ntAge

attim

eof

testing

Age

ofon

set

Sex

Family

histo

ry

Symptom

sat

timeo

fdiagno

sis

BCVA

ODO

SWith

refractio

n

Color

visio

n(15desaturated

Hue)

Bino

cular

kinetic

visualfield

(III4e

stim

ulus)

FFand

mfERG

Fund

usexam

ination

FAF

Sd-O

CT

F303II1

(CIC00

445)

426

FNoother

affectedFM

fro

mFrance

Night

blindn

ess

Handmotion

inbo

theyes

Impo

ssibledu

eto

lowvisio

n

Redu

cedto

perip

heral

islands

ofperceptio

n

Both

undetectable

Paleop

ticdisc

narrow

edbloo

dvessels

macular

atroph

icchangesand

optic

nerve

drusen

Hypoautofl

uorescence

inthem

acular

region

Thinning

ofou

terretina

inthe

macular

region

F335III1

(CIC00

491)

363

M

Twoother

brothers

affected

parentsfi

rst

cousins

Night

blindn

essa

ndrapid

decreased

visio

n

LPin

both

eyes

Impo

ssibledu

eto

lowvisio

n

Impo

ssible

duetolow

visio

n

Both

undetectable

Widespread

RPEchanges

andretin

alatroph

yin

both

thep

eriphery

andthem

acular

area

Widespreadlossof

FAF

Widespread

thinning

ofou

terretina

F674III6

(CIC01106)

2519

F

Parentsfi

rst

cousinsfrom

Turkeyone

femalea

ndmalec

ousin

saffectedalso

from

acon

-sang

uineou

sun

ion

Night

blindn

essa

nddecreased

visio

n

HMminus3(minus1)

0∘ 2016

0minus3

(minus050)0∘

Dyschromatop

siawith

nospecific

axis

Redu

cedto

5central

degrees

Both

undetectable

Well-c

olored

optic

disc

and

nonarrow

ingof

retin

alvessels

RP

Echangesin

thep

eriphery

andmacular

atroph

icchanges

Hypoautofl

uorescence

inthem

acular

region

andou

tside

the

vascular

arcades

Thinning

ofou

terretina

inthe

macular

region

F752II1

(CIC01245)

31Ea

rlyteens

FTw

osisters

affected

Night

blindn

ess

2063plano

(minus3)

180∘

2050plano

(minus17

5)180∘

Deutandefecton

both

eyes

Redu

cedto

10∘

times20∘

Both

undetectable

Paleop

ticdisc

headnarrowed

retin

alvessels

andRP

Echangesinthe

perip

hery

with

somem

acular

atroph

icchanges

Hypoautofl

uorescence

inthem

acular

region

andou

tside

the

vascular

arcades

Thinning

ofou

terretina

inthe

macular

region

F782

II5

(CIC01300)

279

MParentsfrom

Algeriafirst

cousins

Night

blindn

essa

nddecreased

visio

n

2050minus92

5(minus250)15∘

2050

minus9(minus17

5)100∘

mdash

Redu

cedto

the10

central

degrees

Both

undetectable

Mild

optic

disc

pallo

ratroph

icmacular

changesand

perip

heral

pigm

ent

depo

sits

Hypoautofl

uorescence

inthem

acular

region

Thinning

ofou

terretina

inthe

macular

BioMed Research International 5

Table1Con

tinued

Patie

ntAge

attim

eof

testing

Age

ofon

set

Sex

Family

histo

ry

Symptom

sat

timeo

fdiagno

sis

BCVA

ODO

SWith

refractio

n

Color

visio

n(15desaturated

Hue)

Bino

cular

kinetic

visualfield

(III4e

stim

ulus)

FFand

mfERG

Fund

usexam

ination

FAF

Sd-O

CT

F1941III1

(CIC04

130)

30child

hood

FParentsfrom

Algeriafirst

cousins

Night

blindn

ess

2010

0minus425

(minus12

5)150∘

2080minus425

(minus12

5)150∘

Normalatthe

saturatedtest

Redu

cedto

the10

central

degrees

Both

undetectable

Well-c

olored

optic

disc

but

narrow

edretin

alvessels

RPE

changesinthe

perip

hery

and

macular

atroph

icchanges

Hypoautofl

uorescence

inthem

acular

region

andou

tside

the

vascular

arcades

Thinning

ofou

terretina

inthe

macular

region

F3110

III5

(CIC05941)

275

F

One

cousin

onmother

sidem

ayhave

RCD

Night

blindn

essa

nddecreased

visio

n

2012

5+2

(minus2)

95∘

2012

5+175

(minus2)

70∘

c

Dyschromatop

siawith

nospecific

axis

Redu

cedto

the10

central

degrees

Both

undetectable

Paleop

ticdisc

narrow

edretin

alvessels

and

RPE

changesinthe

perip

hery

with

somem

acular

atroph

icchanges

Hypoautofl

uorescence

inthem

acular

region

andou

tside

the

vascular

arcades

Thinning

ofou

terretina

inthe

macular

region

FfemaleFM

fam

ilymem

berMm

aleBC

VAbestcorrected

visualacuityO

Doculadextra

(right

eye)O

Soculas

inistra

(lefteye)FFandmfERG

full-fi

eldandmultifocalER

GFAFfund

usautoflu

orescence

Sd-O

CTspectrald

omainop

ticalcoherencetom

ograph

yRP

Eretin

alpigm

entepitheliumLP

light

perceptio

nHMhandmotion

6 BioMed Research International

Table2Listof

mutations

detected

bynext

generatio

nsequ

encing

after

applying

relevant

filters

Patie

ntGene

Exon

Allelestate

Nucleotidee

xchange

Proteineffect

rsID

Con

servation

Polyoh

en2SIFT

Pathogenicity

Note

F303

II1(C

IC00

445)

NPH

P412

HTZ

AgtG

pSer481As

nno

NC

BT

Neutral

RP1

4HTZ

c162

5CgtG

pSer542lowast

mdashmdash

mdashmdash

Disease

causing

RM

[12]

RP1

4HTZ

c458

745

90delTAAG

pSer1529A

rgfslowast

9mdash

mdashmdash

mdashDisease

causing

NM

F335

III1

(CIC00

491)

PROM1

4HTZ

TgtC

pIle

178V

almdash

NC

BT

Neutral

GPR9

829

HTZ

GgtA

pArg2128Gln

rs149390094

NC

BT

Neutral

RP1

4HMZ

c408

940

92delAAG

ApArg1364

Valfslowast

8mdash

mdashmdash

mdashDisease

causing

NM

F674

III6

(CIC01106)

USH

2A39

HTZ

TgtG

pSer2450A

rgNo

HC

PD

DProb

ably

diseasec

ausin

gRP

14

HMZ

c120

5delG

pGly40

2Alafslowast

7mdash

mdashmdash

mdashDisease

causing

NM

F752

II1(C

IC01245)

USH

1C17

HTZ

GgtA

pArg477Trp

TMPES

P11

17532053

HC

PD

DProb

ablydisease

causing

PDE6

B10

HTZ

TgtC

pThr432Ile

mdashHC

BT

Uncertain

pathogenicity

RP1

4HTZ

c202

5dup

ApSer676Ile

fslowast

22mdash

mdashmdash

mdashDisease

causing

NM

RP1

4HTZ

c2377d

elA

pArg793G

lufslowast

55mdash

mdashmdash

mdashDisease

causing

NM

F782II5

(CIC01300)

RP1

4HMZ

c17191723delCTC

AA

pSer574Cy

sfslowast7

mdashmdash

mdashmdash

Disease

causing

NM

TULP

15

HMZ

c395

418dup

pAs

p124

Glu131del

rs63749128

mdashmdash

mdashNeutral

F1941III1

(CIC04130)

PCDH15

33HTZ

CgtT

pArg1889His

rs145851144

NC

BT

Neutral

C2orf71

1HTZ

CgtA

pArg656Ser

rs201980758

NC

BT

Neutral

CACN

A2D4

Exon

37-

Intro

n37

HTZ

CgtT

mdashrs80092457

NC

mdashmdash

Neutral

RP1

4HMZ

c1329d

elG

pLys443

Asnfslowast

12mdash

mdashmdash

mdashDisease

causing

NM

F3110

III5

(CIC05941)

EYS

6HTZ

CgtT

pSer326As

nrs112822256

NC

BT

Neutral

MER

TK8

HTZ

CgtG

pArg421Trp

rs138908058

NC

BD

Neutral

PRPF

621

HTZ

AgtG

pVal915M

etrs139778757

MC

PD

DUncertain

pathogenicity

TULP

114

HTZ

GgtA

pAla496Th

rrs141980901

MC

BD

Neutral

EYS

26HTZ

GgtA

pLys1365G

lurs16895519

NC

BD

Neutral

MER

TK18

HTZ

GgtC

pGlu823G

lnrs55924349

MC

BD

Neutral

RP1

4HMZ

c239

12392

delAA

pAsp79

9lowastmdash

mdashmdash

mdashDisease

causing

NM

Prob

ablydiseasec

ausin

gmutations

areh

ighlighted

inbo

ld

Bbenign

HMZ

homozygou

sHTZ

heterozygou

sM

Cmarginally

conservedNC

notcon

servedN

Mn

ovelmutation

RMrecurrent

mutation

TtoleratedPD

possib

lydamaging

BioMed Research International 7

(a)

(b) (c)

(d)

(e) (f)

Figure 1 Ophthalmic features of family F752 II1 (CIC01245) fundus color photographs ((a) and (d) for right and left eye resp)autofluorescence ((b) and (e) for right and left eye resp) and spectral domain optical coherence tomography horizontal macula scans ((c)and (f) for right and left eye resp) showing severe rod-cone dystrophy signs with macular involvement

last exon (exon 4) and shown to be transmitted in an auto-somal dominant mode of inheritance Most of RP1 diseasecausing variants represent nonsense mutations deletions orinsertions In mammalian genes nonsense mutations lead tounstable mRNAs that are degraded by nonsense-mediated

decay (NMD)However exceptionsmight arise when prema-ture stop codons occur in the last exon [37]These variants arethought to abolish RP1 function by resulting in a truncatedprotein lacking important functional domains although stillable to interact with some of its protein partner(s) [21] The

8 BioMed Research International

0 2 4 6 8 10 12 14 16 18 20 22

87888990919293949596979899100

Chromosome

Cov

erag

e at d

epth

25

X (

)

F303 CIC00445F335 CIC00491F674 CIC01106F752 CIC01245

F782 CIC01300

F3110 CIC05941F1941 CIC04130

(a)

0 2 4 6 8 10 12 14 16 18 20 22

Chromosome

100

150

200

250

300

350

400

Mea

n de

pth

per b

ase

F303 CIC00445F335 CIC00491F674 CIC01106 F3110 CIC05941F752 CIC01245

F782 CIC01300F1941 CIC04130

(b)

Figure 2 Sequencing statistics in index patients (a)The overall sequencing coverage of the target regions at 25X depth of coverage is shownin each of the chromosomes No values were indicated for chromosomes 13 18 21 and 22 as they were not targeted The term chromosome23 was used to designate the X chromosome F1941 III1 (CIC04130) and F3110 III5 (CIC05941) showed the lowest coverage results (b) Theaverage mean depth per base pair is shown for each of the chromosomes Most targets showed coverage around 300 times

1 1 1

1 2 3 4 5 6

2 3 4

4

5 632

2

1

1

1 2 3

4

5

11

2 21

2 2

3

2

3

6 7 8

4

41 5

(I)

(II)

(III)

(a) CIC00445 II1 F303 (b) CIC00491 III1 F335

(f) CIC04130 III1 F1941 (g) CIC05941 III5 F3110

1 2 3 4

1

1

1

2

2

3

3

4

4

5

5

6 7 8 9 10 65

43

21

1

1

2 3 4 5

432

2

1 2

2 3 4 5 61

1

(I)

(II)

(III)

(IV)

M1 c1625CgtGpSer542lowastM2 c4587 4590delTAAG pSer1529Argfslowast9

[M1][M2]

M3 c4089 4092delAAGApArg1364Valfslowast8

[M3][M3]

M4 c1205delG pGly402Alafslowast7

[M4][M4]

M5 c2025dupA pSer676Ilefslowast22M6 c2377delA pArg793Glufslowast55

[M5][M6]

M7 c1719 1723delCTCAA pSer574Cysfslowast7 M8 c1329delG pLys443Asnfslowast12

[M8][M8]

[M7][M7]

[=][=]

M9 c2391 2392delAA pAsp799lowast

[M9][M9]

[M9][=]

(c) CIC01106 III6 F674 (d) CIC01245 II1 F752

(e) CIC01300 II5 F782

Figure 3 Pedigrees of seven families with RP1 mutations underlying autosomal recessive rod-cone dystrophy Affected and unaffectedindividuals are represented by shapes filled with black and white colors respectively Men and women are indicated by squares and circlesrespectively Index subjects are marked by Consanguinity is marked by a double horizontal line

BioMed Research International 9

4

adRCD

arRCD

Class I

Class II (hot spot)

Class III Class III Class IV

321 BIF

pA

sp202

Glu

pThr373

Ile

pA

rg1519

Glu

fsX2

pA

rg1652

Leu

pCy

s2033

Tyr

pA

la669

Thr

pT8

65

Leu8

66

delIn

s

pA

sp984

Gly

pSe

r152

9Arg

fslowast9

pA

rg15

19G

lufslowast

2

pPr

o164

8Ser

fslowast2

pA

sn17

51Le

ufslowast4

pA

sn94

9Lys

fslowast32

pSe

r542lowast

pSe

r574

Cysfs

lowast7

pSe

r676

Ilefslowast

22

pA

rg79

3Glu

fslowast55

pA

sp79

9lowast

pM

et50

0Ser

fslowast33

pPr

o658

Hisf

slowast4

pLy

s673

Arg

fslowast9

pLy

s675

Asn

fslowast7

pA

rg67

7lowast

pA

rg67

7Asp

fslowast5

pG

ln67

9lowastp

Tyr6

85lowast

pG

ln68

6lowastp

Gln

689lowast

pG

lu70

0lowastp

Gly

706V

alfslowast7

pLy

s722

Glu

fslowast16

pG

ly72

3lowastp

Gly

724G

lufslowast

9

pile

725A

rgfslowast

6

pIle

725Th

rfslowast13

pIle

725A

spfslowast

4

pG

lu72

9Lys

fslowast9

pThr7

36Ile

fslowast4

pCy

s744

lowast

pSe

r747

Valfs

lowast16

pA

sn74

8Ile

fslowast15

pA

rg75

9lowastp

Leu7

62

Asn763

pL

eu762

Tyrfs

lowast17

pA

sn76

3Leu

fslowast11

pA

sn76

9Arg

fslowast6

pLy

s778

lowast

pSe

r779

lowast

pSe

r862

lowast

pIle

864L

ysfslowast

11

pTy

r105

3Thrfs

lowast4

pSe

r2A

rgfslowast16

pH

is31A

rgfslowast

9

pVa

l157

Trpf

slowast16

pA

la22

1Gly

fslowast43

pPr

o229

Gln

fslowast35

pA

rg33

8lowast

pA

rg39

6lowastp

Gly

402A

lafslowast7

pLy

s443

Asn

fslowast12

pG

lu48

8lowast

pTr

p113

1lowast

pG

ly11

40ly

sfslowast4

pA

sn11

43Ile

fslowast25

pG

lu12

27M

etfslowast

29

pA

rg13

64Va

lfslowast8

pLy

s151

8lowast

pA

rg87

2Thrfs

lowast2

pSe

r911

lowast

Figure 4 Schematic presentation of RP1 disease causing mutations Disease causing mutations were represented based on the classificationby Chen and coworkers [13] Mutations responsible for recessive arRCD were shown in the upper half whereas mutations causing adRCDwere shown in the lower half pGly402Alafslowast7 pLys443Asnfslowast12 pArg1364Valfslowast8 and pSer1529Argfslowast9 belong to class III AlthoughpSer574Cysfslowast7 pSer676Ilefslowast22 pArg793Glufslowast55 and pAsp799lowast are class II mutations these variants do not cause adRCD but arRCDinstead Amino acid modifications shown in red and blue represent novel frameshift or nonsense mutations and the recurrent pSer542lowastmutation respectively Protein localization of pSer542lowast was highlighted in blue as it marked a recurrent mutation adRCD autosomaldominant rod-cone dystrophy arRCD autosomal recessive rod-cone dystrophy BIF drosophila melanogaster bifocal

latter observation is supported by finding that RP1 mutantmRNA is expressed in a human cell line carrying a homozy-gous pArg677lowast mutation [21]

Based on Chen et al [13] RP1 truncating mutations lead-ing to arRCD or adRCD can be divided into four distinctgroups Class I is composed of truncating mutations locatedin exons 2 and 3These variants are sensitive toNMDand thusare considered as true loss-of-function alleles (Figure 4) [13]Class II involves truncating mutations that are located in aspot between codons 500 and 1053 in exon 4 [13] the so calledldquoRP1 hot spotrdquo The ldquohot spotrdquo variants tend to be insensitiveto NMD process and thus result in a protein with a potentialdominant negative effect leading to adRCD (Figure 4) [13]Class III includes truncating mutations insensible to NMDlocated between codons 264 and 499 and between codons1054 to 1751 in exon 4 These truncating proteins result in aloss of function leading to arRCD (Figure 4) [13] Finally classIV includes protein-truncating mutations near the 31015840 endof the fourth exon (Figure 4) [13] Most likely the resultingproteins display only aminor loss of their C-terminal portionpreserving the majority of functional domains and keeping aresidual activity According to the classification of Chen et al[13] pGly402Alafslowast7 pLys443Asnfslowast12 pArg1364Valfslowast8and pSer1529Argfslowast9 belong to class III (Figure 4)

The predicted physiopathology for pSer542lowastpSer574Cysfslowast7 pSer676Ilefslowast22 pArg793Glufslowast55 andpAsp799lowast is more complex According to Chenrsquos classi-fication these frameshift deletions and nonsense mutationsshould belong to class II previously only associated withadRCD However herein they are causing presumablyarRCD (Figure 4) To further confirm these findings clinicaland genetic testing of the reported unaffected parents shouldbe done

Based on the previous findings we speculate that the clas-sification by Chen and coworkers does not hold true for allmutations Supporting this statement Avila-Fernandez et al[12] reported the same nonsense mutation (pSer542lowast) foundin (F303 II1 (CIC00445)) and located at the 51015840 end of theldquohot spotrdquo to cause arRP in the Spanish population [12]These observations are of interest as they point out for animplication of hot spot region for adRCD-RP1mutation alsoin case of arRCD Future studies will need to clarify why someclass II mutations lead to adRCD and others to arRCD

Patients with arRCD and RP1 mutations show a moresevere disease than adRCD-RP1 mutant patients with mac-ular atrophy in all our cases This was first outlined byLafont et al [17] When patients are presenting with latesevere disease the diagnostic distinction between RCD with

10 BioMed Research International

initial rod involvement and cone-rod dystrophy (CRD) withinitial cone involvement is difficult Of note is that one ofthe patients (CIC01300) in the present study was initiallyclassified as possibly having severe CRD and his diagnosiswas actually revisited after NGS resultsThis also outlines theinterest of unbiased massive parallel sequencing methods fora more precise clinical diagnostic in case of end stage diseaseThis point will most likely become evenmore critical with theperspective of therapeutic trials

41 Strength and Limitations We estimate that 1 of ourtarget regions were not covered Partially uncovered exonsare a real common issue when capturing the DNA sequencesusing commercially available probes this bias might implya loss of some candidate variants However we found thatrate of 1 is very reasonable when compared with otherNGS panels In addition in order to exclude the possibilityof finding other candidate variants we have sequenced bySanger method the majority of these regions Five of ourpatients carried homozygous RP1 mutations For four of thesubjects carrying homozygous variants namely CIC00491F335 CIC01106 F674 CIC01300 F782 and CIC04130 F1941co-segregation analysis needs to be done to confirm auto-somal recessive inheritance but we do not have access toparentrsquos DNA CIC05941 was the only one not to reportclear consanguinity in the family and we cannot exclude thepossibility of a large deletion on the second allele of RP1gene Again DNA of the father not available for us wouldbe helpful to prove autosomal recessive inheritance and thehomozygous state of the mutation

In conclusion we have reported 9 mutations in RP1 ofwhich 8 were novel causing arRCD [8 12ndash20] Interestingly aprevalence ofasymp25points out for the necessity of sequencingRP1 in sporadic and recessive cases of RCD Further func-tional studies are needed to understand the impact of RP1structure on its function at the molecular level such a stepwould strengthen our knowledge in the physiology of retinalphotoreceptors

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Isabelle Audo and Christina Zeitz contributed equally to thiswork

Acknowledgments

The authors express their sincere gratitude to the familieswho participated in this study and to the clinical staff fortheir help in clinical data and DNA collection This workwas supported by Foundation Voir et Entendre (CZ) PrixDalloz for ldquola recherche en ophtalmologierdquo (CZ) FoundationFighting Blindness (FFB) [CD-CL-0808-0466-CHNO] (IA)and FFB center (FFB grantC-GE-0912-0601-INSERM02)

Prix de la Fondation de lrsquoŒil (IA) Ville de Paris and RegionIle de France and the French State program ldquoInvestissementsdrsquoAvenirrdquo managed by the Agence Nationale de la Recherche[LIFESENSES ANR-10-LABX-65]

References

[1] D T Hartong E L Berson and T P Dryja ldquoRetinitis pigmen-tosardquoThe Lancet vol 368 no 9549 pp 1795ndash1809 2006

[2] I Audo M Lancelot S Mohand-Saıd et al ldquoNovel C2orf71mutations account for approximately sim1 of cases in a largeFrench arRP cohortrdquoHumanMutation vol 32 no 4 pp E2091ndashE2103 2011

[3] A Anasagasti C Irigoyen O Barandika A Lopez de Munainand J Ruiz-Ederra ldquoCurrent mutation discovery approaches inRetinitis PigmentosardquoVision Research vol 75 pp 117ndash129 2012

[4] L S Sullivan S J Bowne D G Birch et al ldquoPrevalence ofdisease-causingmutations in families with autosomal dominantretinitis pigmentosa a screen of known genes in 200 familiesrdquoInvestigative Ophthalmology andVisual Science vol 47 no 7 pp3052ndash3064 2006

[5] B J Seyedahmadi C Rivolta J A Keene E L Berson andT P Dryja ldquoComprehensive screening of the USH2A gene inUsher syndrome type II and non-syndromic recessive retinitispigmentosardquo Experimental Eye Research vol 79 no 2 pp 167ndash173 2004

[6] A Avila-Fernandez D Cantalapiedra E Aller et al ldquoMutationanalysis of 272 Spanish families affected by autosomal recessiveretinitis pigmentosa using a genotyping microarrayrdquoMolecularVision vol 16 pp 2550ndash2558 2010

[7] S El Shamieh M Neuille A Terray et al ldquoWhole-exomesequencing identifies KIZ as a ciliary gene associated withautosomal-recessive rod-cone dystrophyrdquo American Journal ofHuman Genetics vol 94 no 4 pp 625ndash633 2014

[8] I Audo S Mohand-Saıd C M Dhaenens et al ldquoRP1 andautosomal dominant rod-cone dystrophy novel mutations areview of published variants and genotype-phenotype correla-tionrdquo Human Mutation vol 33 no 1 pp 73ndash80 2012

[9] X Guillonneau N I Piriev M Danciger et al ldquoA nonsensemutation in a novel gene is associated with retinitis pigmentosain a family linked to the RP1 locusrdquoHumanMolecular Geneticsvol 8 no 8 pp 1541ndash1546 1999

[10] E A Pierce T Quinn T Meehan T L McGee E L Bersonand T P Dryja ldquoMutations in a gene encoding a new oxygen-regulated photoreceptor protein cause dominant retinitis pig-mentosardquo Nature Genetics vol 22 no 3 pp 248ndash254 1999

[11] L S Sullivan J R Heckenlively S J Bowne et al ldquoMutations ina novel retina-specific gene cause autosomal dominant retinitispigmentosardquo Nature Genetics vol 22 no 3 pp 255ndash259 1999

[12] A Avila-Fernandez M Corton K M Nishiguchi et al ldquoIden-tification of an RP1 prevalent founder mutation and relatedphenotype in Spanish patients with early-onset autosomalrecessive retinitisrdquo Ophthalmology vol 119 no 12 pp 2616ndash2621 2012

[13] L J Chen T Y Y Lai P O S Tam et al ldquoCompound heter-ozygosity of two novel truncation mutations in RP1 causingautosomal recessive retinitis pigmentosardquo InvestigativeOphthal-mology and Visual Science vol 51 no 4 pp 2236ndash2242 2010

[14] S Khaliq A Abid M Ismail et al ldquoNovel association of RP1gene mutations with autosomal recessive retinitis pigmentosardquoJournal of Medical Genetics vol 42 no 5 pp 436ndash438 2005

BioMed Research International 11

[15] A M Siemiatkowska G D N Astuti K Arimadyo et alldquoIdentification of a novel nonsense mutation in RP1 that causesautosomal recessive retinitis pigmentosa in an IndonesianfamilyrdquoMolecular Vision vol 18 pp 2411ndash2419 2012

[16] B Bocquet N A Marzouka M Hebrard et al ldquoHomozygositymapping in autosomal recessive retinitis pigmentosa familiesdetects novel mutationsrdquo Molecular Vision vol 19 pp 2487ndash2500 2013

[17] E M Lafont G Manes G Senechal et al ldquoPatients with reti-nitis pigmentosa due to RP1 mutations show greater severity inrecessive than in dominant casesrdquo Journal of Clinical amp Experi-mental Ophthalmology vol 2 article 194 2011

[18] M Al-Rashed L Abu Safieh H Alkuraya et al ldquoRP1 and reti-nitis pigmentosa report of novel mutations and insight intomutational mechanismrdquo British Journal of Ophthalmology vol96 no 7 pp 1018ndash1022 2012

[19] S A Riazuddin A Shahzadi C Zeitz et al ldquoA mutation inSLC24A1 implicated in autosomal-recessive congenital station-ary night blindnessrdquoThe American Journal of Human Geneticsvol 87 no 4 pp 523ndash531 2010

[20] H P Singh S Jalali R Narayanan and C Kannabiran ldquoGe-netic analysis of indian families with autosomal recessiveretinitis pigmentosa by homozygosity screeningrdquo InvestigativeOphthalmology and Visual Science vol 50 no 9 pp 4065ndash40712009

[21] Q Liu A Lyubarsky J H Skalet E N Pugh Jr and E A PierceldquoRP1 is required for the correct stacking of outer segment discsrdquoInvestigative Ophthalmology ampVisual Science vol 44 no 10 pp4171ndash4183 2003

[22] Q Liu J Zuo and E A Pierce ldquoThe retinitis pigmentosa 1protein is a photoreceptor microtubule-associated proteinrdquoJournal of Neuroscience vol 24 no 29 pp 6427ndash6436 2004

[23] I Audo K M Bujakowska T Leveillard et al ldquoDevelop-ment and application of a next-generation-sequencing (NGS)approach to detect known and novel gene defects underlyingretinal diseasesrdquo Orphanet Journal of Rare Diseases vol 7 no 1article 8 2012

[24] I Audo J A Sahel S Mohand-Saıd et al ldquoEYS is a major genefor rod-cone dystrophies in Francerdquo Human Mutation vol 31no 5 pp E1406ndashE1435 2010

[25] T G P Consortium ldquoA map of human genome variation frompopulation-scale sequencingrdquo Nature vol 467 no 7319 pp1061ndash1073 2010

[26] DMAltshuler RAGibbs L Peltonen et al ldquoIntegrating com-mon and rare genetic variation in diverse human populationsrdquoNature vol 467 no 7311 pp 52ndash58 2010

[27] J A Tennessen AW Bigham T D OrsquoConnor et al ldquoEvolutionand functional impact of rare coding variation from deepsequencing of human exomesrdquo Science vol 337 no 6090 pp64ndash69 2012

[28] M J Bamshad S BNgAW Bighamet al ldquoExome sequencingas a tool for Mendelian disease gene discoveryrdquo Nature ReviewsGenetics vol 12 no 11 pp 745ndash755 2011

[29] W J Kent C W Sugnet T S Furey et al ldquoThe human genomebrowser at UCSCrdquo Genome Research vol 12 no 6 pp 996ndash1006 2002

[30] I A Adzhubei S Schmidt L Peshkin et al ldquoA method andserver for predicting damaging missense mutationsrdquo NatureMethods vol 7 no 4 pp 248ndash249 2010

[31] P Kumar S Henikoff and P C Ng ldquoPredicting the effects ofcoding non-synonymous variants on protein function using the

SIFT algorithmrdquo Nature Protocols vol 4 no 7 pp 1073ndash10822009

[32] F O Desmet D Hamroun M Lalande G Collod-Beroud MClaustres and C Beroud ldquoHuman splicing finder an onlinebioinformatics tool to predict splicing signalsrdquo Nucleic AcidsResearch vol 37 no 9 article no e67 2009

[33] P D Stenson E V Ball M Mort A D Phillips K Shaw andD N Cooper ldquoThe Human Gene Mutation Database (HGMD)and its exploitation in the fields of personalized genomics andmolecular evolutionrdquo in Current Protocols in Bioinformaticschapter 1ndash13 2012

[34] I F A C Fokkema J T Den Dunnen and P E M TaschnerldquoLOVD easy creation of a locus-specific sequence variationdatabase using an ldquoLSDB-in-a-Boxrdquo approachrdquo Human Muta-tion vol 26 no 2 pp 63ndash68 2005

[35] S A Miller D D Dykes and H F Polesky ldquoA simple saltingout procedure for extracting DNA from human nucleated cellsrdquoNucleic Acids Research vol 16 no 3 p 1215 1988

[36] C Zeitz B Kloeckener-Gruissem U Forster et al ldquoMutationsin CABP4 the gene encoding the Ca2+-binding protein 4cause autosomal recessive night blindnessrdquoAmerican Journal ofHuman Genetics vol 79 no 4 pp 657ndash667 2006

[37] S A Riazuddin F Zulfiqar Q Zhang et al ldquoAutosomal reces-sive retinitis pigmentosa is associated with mutations in RP1 inthree consanguineous Pakistani familiesrdquo Investigative Ophthal-mology and Visual Science vol 46 no 7 pp 2264ndash2270 2005

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

4 BioMed Research International

Table1Clinicaldataof

the7

indexpatie

ntsw

ithRP

1recessiv

emutations

Patie

ntAge

attim

eof

testing

Age

ofon

set

Sex

Family

histo

ry

Symptom

sat

timeo

fdiagno

sis

BCVA

ODO

SWith

refractio

n

Color

visio

n(15desaturated

Hue)

Bino

cular

kinetic

visualfield

(III4e

stim

ulus)

FFand

mfERG

Fund

usexam

ination

FAF

Sd-O

CT

F303II1

(CIC00

445)

426

FNoother

affectedFM

fro

mFrance

Night

blindn

ess

Handmotion

inbo

theyes

Impo

ssibledu

eto

lowvisio

n

Redu

cedto

perip

heral

islands

ofperceptio

n

Both

undetectable

Paleop

ticdisc

narrow

edbloo

dvessels

macular

atroph

icchangesand

optic

nerve

drusen

Hypoautofl

uorescence

inthem

acular

region

Thinning

ofou

terretina

inthe

macular

region

F335III1

(CIC00

491)

363

M

Twoother

brothers

affected

parentsfi

rst

cousins

Night

blindn

essa

ndrapid

decreased

visio

n

LPin

both

eyes

Impo

ssibledu

eto

lowvisio

n

Impo

ssible

duetolow

visio

n

Both

undetectable

Widespread

RPEchanges

andretin

alatroph

yin

both

thep

eriphery

andthem

acular

area

Widespreadlossof

FAF

Widespread

thinning

ofou

terretina

F674III6

(CIC01106)

2519

F

Parentsfi

rst

cousinsfrom

Turkeyone

femalea

ndmalec

ousin

saffectedalso

from

acon

-sang

uineou

sun

ion

Night

blindn

essa

nddecreased

visio

n

HMminus3(minus1)

0∘ 2016

0minus3

(minus050)0∘

Dyschromatop

siawith

nospecific

axis

Redu

cedto

5central

degrees

Both

undetectable

Well-c

olored

optic

disc

and

nonarrow

ingof

retin

alvessels

RP

Echangesin

thep

eriphery

andmacular

atroph

icchanges

Hypoautofl

uorescence

inthem

acular

region

andou

tside

the

vascular

arcades

Thinning

ofou

terretina

inthe

macular

region

F752II1

(CIC01245)

31Ea

rlyteens

FTw

osisters

affected

Night

blindn

ess

2063plano

(minus3)

180∘

2050plano

(minus17

5)180∘

Deutandefecton

both

eyes

Redu

cedto

10∘

times20∘

Both

undetectable

Paleop

ticdisc

headnarrowed

retin

alvessels

andRP

Echangesinthe

perip

hery

with

somem

acular

atroph

icchanges

Hypoautofl

uorescence

inthem

acular

region

andou

tside

the

vascular

arcades

Thinning

ofou

terretina

inthe

macular

region

F782

II5

(CIC01300)

279

MParentsfrom

Algeriafirst

cousins

Night

blindn

essa

nddecreased

visio

n

2050minus92

5(minus250)15∘

2050

minus9(minus17

5)100∘

mdash

Redu

cedto

the10

central

degrees

Both

undetectable

Mild

optic

disc

pallo

ratroph

icmacular

changesand

perip

heral

pigm

ent

depo

sits

Hypoautofl

uorescence

inthem

acular

region

Thinning

ofou

terretina

inthe

macular

BioMed Research International 5

Table1Con

tinued

Patie

ntAge

attim

eof

testing

Age

ofon

set

Sex

Family

histo

ry

Symptom

sat

timeo

fdiagno

sis

BCVA

ODO

SWith

refractio

n

Color

visio

n(15desaturated

Hue)

Bino

cular

kinetic

visualfield

(III4e

stim

ulus)

FFand

mfERG

Fund

usexam

ination

FAF

Sd-O

CT

F1941III1

(CIC04

130)

30child

hood

FParentsfrom

Algeriafirst

cousins

Night

blindn

ess

2010

0minus425

(minus12

5)150∘

2080minus425

(minus12

5)150∘

Normalatthe

saturatedtest

Redu

cedto

the10

central

degrees

Both

undetectable

Well-c

olored

optic

disc

but

narrow

edretin

alvessels

RPE

changesinthe

perip

hery

and

macular

atroph

icchanges

Hypoautofl

uorescence

inthem

acular

region

andou

tside

the

vascular

arcades

Thinning

ofou

terretina

inthe

macular

region

F3110

III5

(CIC05941)

275

F

One

cousin

onmother

sidem

ayhave

RCD

Night

blindn

essa

nddecreased

visio

n

2012

5+2

(minus2)

95∘

2012

5+175

(minus2)

70∘

c

Dyschromatop

siawith

nospecific

axis

Redu

cedto

the10

central

degrees

Both

undetectable

Paleop

ticdisc

narrow

edretin

alvessels

and

RPE

changesinthe

perip

hery

with

somem

acular

atroph

icchanges

Hypoautofl

uorescence

inthem

acular

region

andou

tside

the

vascular

arcades

Thinning

ofou

terretina

inthe

macular

region

FfemaleFM

fam

ilymem

berMm

aleBC

VAbestcorrected

visualacuityO

Doculadextra

(right

eye)O

Soculas

inistra

(lefteye)FFandmfERG

full-fi

eldandmultifocalER

GFAFfund

usautoflu

orescence

Sd-O

CTspectrald

omainop

ticalcoherencetom

ograph

yRP

Eretin

alpigm

entepitheliumLP

light

perceptio

nHMhandmotion

6 BioMed Research International

Table2Listof

mutations

detected

bynext

generatio

nsequ

encing

after

applying

relevant

filters

Patie

ntGene

Exon

Allelestate

Nucleotidee

xchange

Proteineffect

rsID

Con

servation

Polyoh

en2SIFT

Pathogenicity

Note

F303

II1(C

IC00

445)

NPH

P412

HTZ

AgtG

pSer481As

nno

NC

BT

Neutral

RP1

4HTZ

c162

5CgtG

pSer542lowast

mdashmdash

mdashmdash

Disease

causing

RM

[12]

RP1

4HTZ

c458

745

90delTAAG

pSer1529A

rgfslowast

9mdash

mdashmdash

mdashDisease

causing

NM

F335

III1

(CIC00

491)

PROM1

4HTZ

TgtC

pIle

178V

almdash

NC

BT

Neutral

GPR9

829

HTZ

GgtA

pArg2128Gln

rs149390094

NC

BT

Neutral

RP1

4HMZ

c408

940

92delAAG

ApArg1364

Valfslowast

8mdash

mdashmdash

mdashDisease

causing

NM

F674

III6

(CIC01106)

USH

2A39

HTZ

TgtG

pSer2450A

rgNo

HC

PD

DProb

ably

diseasec

ausin

gRP

14

HMZ

c120

5delG

pGly40

2Alafslowast

7mdash

mdashmdash

mdashDisease

causing

NM

F752

II1(C

IC01245)

USH

1C17

HTZ

GgtA

pArg477Trp

TMPES

P11

17532053

HC

PD

DProb

ablydisease

causing

PDE6

B10

HTZ

TgtC

pThr432Ile

mdashHC

BT

Uncertain

pathogenicity

RP1

4HTZ

c202

5dup

ApSer676Ile

fslowast

22mdash

mdashmdash

mdashDisease

causing

NM

RP1

4HTZ

c2377d

elA

pArg793G

lufslowast

55mdash

mdashmdash

mdashDisease

causing

NM

F782II5

(CIC01300)

RP1

4HMZ

c17191723delCTC

AA

pSer574Cy

sfslowast7

mdashmdash

mdashmdash

Disease

causing

NM

TULP

15

HMZ

c395

418dup

pAs

p124

Glu131del

rs63749128

mdashmdash

mdashNeutral

F1941III1

(CIC04130)

PCDH15

33HTZ

CgtT

pArg1889His

rs145851144

NC

BT

Neutral

C2orf71

1HTZ

CgtA

pArg656Ser

rs201980758

NC

BT

Neutral

CACN

A2D4

Exon

37-

Intro

n37

HTZ

CgtT

mdashrs80092457

NC

mdashmdash

Neutral

RP1

4HMZ

c1329d

elG

pLys443

Asnfslowast

12mdash

mdashmdash

mdashDisease

causing

NM

F3110

III5

(CIC05941)

EYS

6HTZ

CgtT

pSer326As

nrs112822256

NC

BT

Neutral

MER

TK8

HTZ

CgtG

pArg421Trp

rs138908058

NC

BD

Neutral

PRPF

621

HTZ

AgtG

pVal915M

etrs139778757

MC

PD

DUncertain

pathogenicity

TULP

114

HTZ

GgtA

pAla496Th

rrs141980901

MC

BD

Neutral

EYS

26HTZ

GgtA

pLys1365G

lurs16895519

NC

BD

Neutral

MER

TK18

HTZ

GgtC

pGlu823G

lnrs55924349

MC

BD

Neutral

RP1

4HMZ

c239

12392

delAA

pAsp79

9lowastmdash

mdashmdash

mdashDisease

causing

NM

Prob

ablydiseasec

ausin

gmutations

areh

ighlighted

inbo

ld

Bbenign

HMZ

homozygou

sHTZ

heterozygou

sM

Cmarginally

conservedNC

notcon

servedN

Mn

ovelmutation

RMrecurrent

mutation

TtoleratedPD

possib

lydamaging

BioMed Research International 7

(a)

(b) (c)

(d)

(e) (f)

Figure 1 Ophthalmic features of family F752 II1 (CIC01245) fundus color photographs ((a) and (d) for right and left eye resp)autofluorescence ((b) and (e) for right and left eye resp) and spectral domain optical coherence tomography horizontal macula scans ((c)and (f) for right and left eye resp) showing severe rod-cone dystrophy signs with macular involvement

last exon (exon 4) and shown to be transmitted in an auto-somal dominant mode of inheritance Most of RP1 diseasecausing variants represent nonsense mutations deletions orinsertions In mammalian genes nonsense mutations lead tounstable mRNAs that are degraded by nonsense-mediated

decay (NMD)However exceptionsmight arise when prema-ture stop codons occur in the last exon [37]These variants arethought to abolish RP1 function by resulting in a truncatedprotein lacking important functional domains although stillable to interact with some of its protein partner(s) [21] The

8 BioMed Research International

0 2 4 6 8 10 12 14 16 18 20 22

87888990919293949596979899100

Chromosome

Cov

erag

e at d

epth

25

X (

)

F303 CIC00445F335 CIC00491F674 CIC01106F752 CIC01245

F782 CIC01300

F3110 CIC05941F1941 CIC04130

(a)

0 2 4 6 8 10 12 14 16 18 20 22

Chromosome

100

150

200

250

300

350

400

Mea

n de

pth

per b

ase

F303 CIC00445F335 CIC00491F674 CIC01106 F3110 CIC05941F752 CIC01245

F782 CIC01300F1941 CIC04130

(b)

Figure 2 Sequencing statistics in index patients (a)The overall sequencing coverage of the target regions at 25X depth of coverage is shownin each of the chromosomes No values were indicated for chromosomes 13 18 21 and 22 as they were not targeted The term chromosome23 was used to designate the X chromosome F1941 III1 (CIC04130) and F3110 III5 (CIC05941) showed the lowest coverage results (b) Theaverage mean depth per base pair is shown for each of the chromosomes Most targets showed coverage around 300 times

1 1 1

1 2 3 4 5 6

2 3 4

4

5 632

2

1

1

1 2 3

4

5

11

2 21

2 2

3

2

3

6 7 8

4

41 5

(I)

(II)

(III)

(a) CIC00445 II1 F303 (b) CIC00491 III1 F335

(f) CIC04130 III1 F1941 (g) CIC05941 III5 F3110

1 2 3 4

1

1

1

2

2

3

3

4

4

5

5

6 7 8 9 10 65

43

21

1

1

2 3 4 5

432

2

1 2

2 3 4 5 61

1

(I)

(II)

(III)

(IV)

M1 c1625CgtGpSer542lowastM2 c4587 4590delTAAG pSer1529Argfslowast9

[M1][M2]

M3 c4089 4092delAAGApArg1364Valfslowast8

[M3][M3]

M4 c1205delG pGly402Alafslowast7

[M4][M4]

M5 c2025dupA pSer676Ilefslowast22M6 c2377delA pArg793Glufslowast55

[M5][M6]

M7 c1719 1723delCTCAA pSer574Cysfslowast7 M8 c1329delG pLys443Asnfslowast12

[M8][M8]

[M7][M7]

[=][=]

M9 c2391 2392delAA pAsp799lowast

[M9][M9]

[M9][=]

(c) CIC01106 III6 F674 (d) CIC01245 II1 F752

(e) CIC01300 II5 F782

Figure 3 Pedigrees of seven families with RP1 mutations underlying autosomal recessive rod-cone dystrophy Affected and unaffectedindividuals are represented by shapes filled with black and white colors respectively Men and women are indicated by squares and circlesrespectively Index subjects are marked by Consanguinity is marked by a double horizontal line

BioMed Research International 9

4

adRCD

arRCD

Class I

Class II (hot spot)

Class III Class III Class IV

321 BIF

pA

sp202

Glu

pThr373

Ile

pA

rg1519

Glu

fsX2

pA

rg1652

Leu

pCy

s2033

Tyr

pA

la669

Thr

pT8

65

Leu8

66

delIn

s

pA

sp984

Gly

pSe

r152

9Arg

fslowast9

pA

rg15

19G

lufslowast

2

pPr

o164

8Ser

fslowast2

pA

sn17

51Le

ufslowast4

pA

sn94

9Lys

fslowast32

pSe

r542lowast

pSe

r574

Cysfs

lowast7

pSe

r676

Ilefslowast

22

pA

rg79

3Glu

fslowast55

pA

sp79

9lowast

pM

et50

0Ser

fslowast33

pPr

o658

Hisf

slowast4

pLy

s673

Arg

fslowast9

pLy

s675

Asn

fslowast7

pA

rg67

7lowast

pA

rg67

7Asp

fslowast5

pG

ln67

9lowastp

Tyr6

85lowast

pG

ln68

6lowastp

Gln

689lowast

pG

lu70

0lowastp

Gly

706V

alfslowast7

pLy

s722

Glu

fslowast16

pG

ly72

3lowastp

Gly

724G

lufslowast

9

pile

725A

rgfslowast

6

pIle

725Th

rfslowast13

pIle

725A

spfslowast

4

pG

lu72

9Lys

fslowast9

pThr7

36Ile

fslowast4

pCy

s744

lowast

pSe

r747

Valfs

lowast16

pA

sn74

8Ile

fslowast15

pA

rg75

9lowastp

Leu7

62

Asn763

pL

eu762

Tyrfs

lowast17

pA

sn76

3Leu

fslowast11

pA

sn76

9Arg

fslowast6

pLy

s778

lowast

pSe

r779

lowast

pSe

r862

lowast

pIle

864L

ysfslowast

11

pTy

r105

3Thrfs

lowast4

pSe

r2A

rgfslowast16

pH

is31A

rgfslowast

9

pVa

l157

Trpf

slowast16

pA

la22

1Gly

fslowast43

pPr

o229

Gln

fslowast35

pA

rg33

8lowast

pA

rg39

6lowastp

Gly

402A

lafslowast7

pLy

s443

Asn

fslowast12

pG

lu48

8lowast

pTr

p113

1lowast

pG

ly11

40ly

sfslowast4

pA

sn11

43Ile

fslowast25

pG

lu12

27M

etfslowast

29

pA

rg13

64Va

lfslowast8

pLy

s151

8lowast

pA

rg87

2Thrfs

lowast2

pSe

r911

lowast

Figure 4 Schematic presentation of RP1 disease causing mutations Disease causing mutations were represented based on the classificationby Chen and coworkers [13] Mutations responsible for recessive arRCD were shown in the upper half whereas mutations causing adRCDwere shown in the lower half pGly402Alafslowast7 pLys443Asnfslowast12 pArg1364Valfslowast8 and pSer1529Argfslowast9 belong to class III AlthoughpSer574Cysfslowast7 pSer676Ilefslowast22 pArg793Glufslowast55 and pAsp799lowast are class II mutations these variants do not cause adRCD but arRCDinstead Amino acid modifications shown in red and blue represent novel frameshift or nonsense mutations and the recurrent pSer542lowastmutation respectively Protein localization of pSer542lowast was highlighted in blue as it marked a recurrent mutation adRCD autosomaldominant rod-cone dystrophy arRCD autosomal recessive rod-cone dystrophy BIF drosophila melanogaster bifocal

latter observation is supported by finding that RP1 mutantmRNA is expressed in a human cell line carrying a homozy-gous pArg677lowast mutation [21]

Based on Chen et al [13] RP1 truncating mutations lead-ing to arRCD or adRCD can be divided into four distinctgroups Class I is composed of truncating mutations locatedin exons 2 and 3These variants are sensitive toNMDand thusare considered as true loss-of-function alleles (Figure 4) [13]Class II involves truncating mutations that are located in aspot between codons 500 and 1053 in exon 4 [13] the so calledldquoRP1 hot spotrdquo The ldquohot spotrdquo variants tend to be insensitiveto NMD process and thus result in a protein with a potentialdominant negative effect leading to adRCD (Figure 4) [13]Class III includes truncating mutations insensible to NMDlocated between codons 264 and 499 and between codons1054 to 1751 in exon 4 These truncating proteins result in aloss of function leading to arRCD (Figure 4) [13] Finally classIV includes protein-truncating mutations near the 31015840 endof the fourth exon (Figure 4) [13] Most likely the resultingproteins display only aminor loss of their C-terminal portionpreserving the majority of functional domains and keeping aresidual activity According to the classification of Chen et al[13] pGly402Alafslowast7 pLys443Asnfslowast12 pArg1364Valfslowast8and pSer1529Argfslowast9 belong to class III (Figure 4)

The predicted physiopathology for pSer542lowastpSer574Cysfslowast7 pSer676Ilefslowast22 pArg793Glufslowast55 andpAsp799lowast is more complex According to Chenrsquos classi-fication these frameshift deletions and nonsense mutationsshould belong to class II previously only associated withadRCD However herein they are causing presumablyarRCD (Figure 4) To further confirm these findings clinicaland genetic testing of the reported unaffected parents shouldbe done

Based on the previous findings we speculate that the clas-sification by Chen and coworkers does not hold true for allmutations Supporting this statement Avila-Fernandez et al[12] reported the same nonsense mutation (pSer542lowast) foundin (F303 II1 (CIC00445)) and located at the 51015840 end of theldquohot spotrdquo to cause arRP in the Spanish population [12]These observations are of interest as they point out for animplication of hot spot region for adRCD-RP1mutation alsoin case of arRCD Future studies will need to clarify why someclass II mutations lead to adRCD and others to arRCD

Patients with arRCD and RP1 mutations show a moresevere disease than adRCD-RP1 mutant patients with mac-ular atrophy in all our cases This was first outlined byLafont et al [17] When patients are presenting with latesevere disease the diagnostic distinction between RCD with

10 BioMed Research International

initial rod involvement and cone-rod dystrophy (CRD) withinitial cone involvement is difficult Of note is that one ofthe patients (CIC01300) in the present study was initiallyclassified as possibly having severe CRD and his diagnosiswas actually revisited after NGS resultsThis also outlines theinterest of unbiased massive parallel sequencing methods fora more precise clinical diagnostic in case of end stage diseaseThis point will most likely become evenmore critical with theperspective of therapeutic trials

41 Strength and Limitations We estimate that 1 of ourtarget regions were not covered Partially uncovered exonsare a real common issue when capturing the DNA sequencesusing commercially available probes this bias might implya loss of some candidate variants However we found thatrate of 1 is very reasonable when compared with otherNGS panels In addition in order to exclude the possibilityof finding other candidate variants we have sequenced bySanger method the majority of these regions Five of ourpatients carried homozygous RP1 mutations For four of thesubjects carrying homozygous variants namely CIC00491F335 CIC01106 F674 CIC01300 F782 and CIC04130 F1941co-segregation analysis needs to be done to confirm auto-somal recessive inheritance but we do not have access toparentrsquos DNA CIC05941 was the only one not to reportclear consanguinity in the family and we cannot exclude thepossibility of a large deletion on the second allele of RP1gene Again DNA of the father not available for us wouldbe helpful to prove autosomal recessive inheritance and thehomozygous state of the mutation

In conclusion we have reported 9 mutations in RP1 ofwhich 8 were novel causing arRCD [8 12ndash20] Interestingly aprevalence ofasymp25points out for the necessity of sequencingRP1 in sporadic and recessive cases of RCD Further func-tional studies are needed to understand the impact of RP1structure on its function at the molecular level such a stepwould strengthen our knowledge in the physiology of retinalphotoreceptors

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Isabelle Audo and Christina Zeitz contributed equally to thiswork

Acknowledgments

The authors express their sincere gratitude to the familieswho participated in this study and to the clinical staff fortheir help in clinical data and DNA collection This workwas supported by Foundation Voir et Entendre (CZ) PrixDalloz for ldquola recherche en ophtalmologierdquo (CZ) FoundationFighting Blindness (FFB) [CD-CL-0808-0466-CHNO] (IA)and FFB center (FFB grantC-GE-0912-0601-INSERM02)

Prix de la Fondation de lrsquoŒil (IA) Ville de Paris and RegionIle de France and the French State program ldquoInvestissementsdrsquoAvenirrdquo managed by the Agence Nationale de la Recherche[LIFESENSES ANR-10-LABX-65]

References

[1] D T Hartong E L Berson and T P Dryja ldquoRetinitis pigmen-tosardquoThe Lancet vol 368 no 9549 pp 1795ndash1809 2006

[2] I Audo M Lancelot S Mohand-Saıd et al ldquoNovel C2orf71mutations account for approximately sim1 of cases in a largeFrench arRP cohortrdquoHumanMutation vol 32 no 4 pp E2091ndashE2103 2011

[3] A Anasagasti C Irigoyen O Barandika A Lopez de Munainand J Ruiz-Ederra ldquoCurrent mutation discovery approaches inRetinitis PigmentosardquoVision Research vol 75 pp 117ndash129 2012

[4] L S Sullivan S J Bowne D G Birch et al ldquoPrevalence ofdisease-causingmutations in families with autosomal dominantretinitis pigmentosa a screen of known genes in 200 familiesrdquoInvestigative Ophthalmology andVisual Science vol 47 no 7 pp3052ndash3064 2006

[5] B J Seyedahmadi C Rivolta J A Keene E L Berson andT P Dryja ldquoComprehensive screening of the USH2A gene inUsher syndrome type II and non-syndromic recessive retinitispigmentosardquo Experimental Eye Research vol 79 no 2 pp 167ndash173 2004

[6] A Avila-Fernandez D Cantalapiedra E Aller et al ldquoMutationanalysis of 272 Spanish families affected by autosomal recessiveretinitis pigmentosa using a genotyping microarrayrdquoMolecularVision vol 16 pp 2550ndash2558 2010

[7] S El Shamieh M Neuille A Terray et al ldquoWhole-exomesequencing identifies KIZ as a ciliary gene associated withautosomal-recessive rod-cone dystrophyrdquo American Journal ofHuman Genetics vol 94 no 4 pp 625ndash633 2014

[8] I Audo S Mohand-Saıd C M Dhaenens et al ldquoRP1 andautosomal dominant rod-cone dystrophy novel mutations areview of published variants and genotype-phenotype correla-tionrdquo Human Mutation vol 33 no 1 pp 73ndash80 2012

[9] X Guillonneau N I Piriev M Danciger et al ldquoA nonsensemutation in a novel gene is associated with retinitis pigmentosain a family linked to the RP1 locusrdquoHumanMolecular Geneticsvol 8 no 8 pp 1541ndash1546 1999

[10] E A Pierce T Quinn T Meehan T L McGee E L Bersonand T P Dryja ldquoMutations in a gene encoding a new oxygen-regulated photoreceptor protein cause dominant retinitis pig-mentosardquo Nature Genetics vol 22 no 3 pp 248ndash254 1999

[11] L S Sullivan J R Heckenlively S J Bowne et al ldquoMutations ina novel retina-specific gene cause autosomal dominant retinitispigmentosardquo Nature Genetics vol 22 no 3 pp 255ndash259 1999

[12] A Avila-Fernandez M Corton K M Nishiguchi et al ldquoIden-tification of an RP1 prevalent founder mutation and relatedphenotype in Spanish patients with early-onset autosomalrecessive retinitisrdquo Ophthalmology vol 119 no 12 pp 2616ndash2621 2012

[13] L J Chen T Y Y Lai P O S Tam et al ldquoCompound heter-ozygosity of two novel truncation mutations in RP1 causingautosomal recessive retinitis pigmentosardquo InvestigativeOphthal-mology and Visual Science vol 51 no 4 pp 2236ndash2242 2010

[14] S Khaliq A Abid M Ismail et al ldquoNovel association of RP1gene mutations with autosomal recessive retinitis pigmentosardquoJournal of Medical Genetics vol 42 no 5 pp 436ndash438 2005

BioMed Research International 11

[15] A M Siemiatkowska G D N Astuti K Arimadyo et alldquoIdentification of a novel nonsense mutation in RP1 that causesautosomal recessive retinitis pigmentosa in an IndonesianfamilyrdquoMolecular Vision vol 18 pp 2411ndash2419 2012

[16] B Bocquet N A Marzouka M Hebrard et al ldquoHomozygositymapping in autosomal recessive retinitis pigmentosa familiesdetects novel mutationsrdquo Molecular Vision vol 19 pp 2487ndash2500 2013

[17] E M Lafont G Manes G Senechal et al ldquoPatients with reti-nitis pigmentosa due to RP1 mutations show greater severity inrecessive than in dominant casesrdquo Journal of Clinical amp Experi-mental Ophthalmology vol 2 article 194 2011

[18] M Al-Rashed L Abu Safieh H Alkuraya et al ldquoRP1 and reti-nitis pigmentosa report of novel mutations and insight intomutational mechanismrdquo British Journal of Ophthalmology vol96 no 7 pp 1018ndash1022 2012

[19] S A Riazuddin A Shahzadi C Zeitz et al ldquoA mutation inSLC24A1 implicated in autosomal-recessive congenital station-ary night blindnessrdquoThe American Journal of Human Geneticsvol 87 no 4 pp 523ndash531 2010

[20] H P Singh S Jalali R Narayanan and C Kannabiran ldquoGe-netic analysis of indian families with autosomal recessiveretinitis pigmentosa by homozygosity screeningrdquo InvestigativeOphthalmology and Visual Science vol 50 no 9 pp 4065ndash40712009

[21] Q Liu A Lyubarsky J H Skalet E N Pugh Jr and E A PierceldquoRP1 is required for the correct stacking of outer segment discsrdquoInvestigative Ophthalmology ampVisual Science vol 44 no 10 pp4171ndash4183 2003

[22] Q Liu J Zuo and E A Pierce ldquoThe retinitis pigmentosa 1protein is a photoreceptor microtubule-associated proteinrdquoJournal of Neuroscience vol 24 no 29 pp 6427ndash6436 2004

[23] I Audo K M Bujakowska T Leveillard et al ldquoDevelop-ment and application of a next-generation-sequencing (NGS)approach to detect known and novel gene defects underlyingretinal diseasesrdquo Orphanet Journal of Rare Diseases vol 7 no 1article 8 2012

[24] I Audo J A Sahel S Mohand-Saıd et al ldquoEYS is a major genefor rod-cone dystrophies in Francerdquo Human Mutation vol 31no 5 pp E1406ndashE1435 2010

[25] T G P Consortium ldquoA map of human genome variation frompopulation-scale sequencingrdquo Nature vol 467 no 7319 pp1061ndash1073 2010

[26] DMAltshuler RAGibbs L Peltonen et al ldquoIntegrating com-mon and rare genetic variation in diverse human populationsrdquoNature vol 467 no 7311 pp 52ndash58 2010

[27] J A Tennessen AW Bigham T D OrsquoConnor et al ldquoEvolutionand functional impact of rare coding variation from deepsequencing of human exomesrdquo Science vol 337 no 6090 pp64ndash69 2012

[28] M J Bamshad S BNgAW Bighamet al ldquoExome sequencingas a tool for Mendelian disease gene discoveryrdquo Nature ReviewsGenetics vol 12 no 11 pp 745ndash755 2011

[29] W J Kent C W Sugnet T S Furey et al ldquoThe human genomebrowser at UCSCrdquo Genome Research vol 12 no 6 pp 996ndash1006 2002

[30] I A Adzhubei S Schmidt L Peshkin et al ldquoA method andserver for predicting damaging missense mutationsrdquo NatureMethods vol 7 no 4 pp 248ndash249 2010

[31] P Kumar S Henikoff and P C Ng ldquoPredicting the effects ofcoding non-synonymous variants on protein function using the

SIFT algorithmrdquo Nature Protocols vol 4 no 7 pp 1073ndash10822009

[32] F O Desmet D Hamroun M Lalande G Collod-Beroud MClaustres and C Beroud ldquoHuman splicing finder an onlinebioinformatics tool to predict splicing signalsrdquo Nucleic AcidsResearch vol 37 no 9 article no e67 2009

[33] P D Stenson E V Ball M Mort A D Phillips K Shaw andD N Cooper ldquoThe Human Gene Mutation Database (HGMD)and its exploitation in the fields of personalized genomics andmolecular evolutionrdquo in Current Protocols in Bioinformaticschapter 1ndash13 2012

[34] I F A C Fokkema J T Den Dunnen and P E M TaschnerldquoLOVD easy creation of a locus-specific sequence variationdatabase using an ldquoLSDB-in-a-Boxrdquo approachrdquo Human Muta-tion vol 26 no 2 pp 63ndash68 2005

[35] S A Miller D D Dykes and H F Polesky ldquoA simple saltingout procedure for extracting DNA from human nucleated cellsrdquoNucleic Acids Research vol 16 no 3 p 1215 1988

[36] C Zeitz B Kloeckener-Gruissem U Forster et al ldquoMutationsin CABP4 the gene encoding the Ca2+-binding protein 4cause autosomal recessive night blindnessrdquoAmerican Journal ofHuman Genetics vol 79 no 4 pp 657ndash667 2006

[37] S A Riazuddin F Zulfiqar Q Zhang et al ldquoAutosomal reces-sive retinitis pigmentosa is associated with mutations in RP1 inthree consanguineous Pakistani familiesrdquo Investigative Ophthal-mology and Visual Science vol 46 no 7 pp 2264ndash2270 2005

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

BioMed Research International 5

Table1Con

tinued

Patie

ntAge

attim

eof

testing

Age

ofon

set

Sex

Family

histo

ry

Symptom

sat

timeo

fdiagno

sis

BCVA

ODO

SWith

refractio

n

Color

visio

n(15desaturated

Hue)

Bino

cular

kinetic

visualfield

(III4e

stim

ulus)

FFand

mfERG

Fund

usexam

ination

FAF

Sd-O

CT

F1941III1

(CIC04

130)

30child

hood

FParentsfrom

Algeriafirst

cousins

Night

blindn

ess

2010

0minus425

(minus12

5)150∘

2080minus425

(minus12

5)150∘

Normalatthe

saturatedtest

Redu

cedto

the10

central

degrees

Both

undetectable

Well-c

olored

optic

disc

but

narrow

edretin

alvessels

RPE

changesinthe

perip

hery

and

macular

atroph

icchanges

Hypoautofl

uorescence

inthem

acular

region

andou

tside

the

vascular

arcades

Thinning

ofou

terretina

inthe

macular

region

F3110

III5

(CIC05941)

275

F

One

cousin

onmother

sidem

ayhave

RCD

Night

blindn

essa

nddecreased

visio

n

2012

5+2

(minus2)

95∘

2012

5+175

(minus2)

70∘

c

Dyschromatop

siawith

nospecific

axis

Redu

cedto

the10

central

degrees

Both

undetectable

Paleop

ticdisc

narrow

edretin

alvessels

and

RPE

changesinthe

perip

hery

with

somem

acular

atroph

icchanges

Hypoautofl

uorescence

inthem

acular

region

andou

tside

the

vascular

arcades

Thinning

ofou

terretina

inthe

macular

region

FfemaleFM

fam

ilymem

berMm

aleBC

VAbestcorrected

visualacuityO

Doculadextra

(right

eye)O

Soculas

inistra

(lefteye)FFandmfERG

full-fi

eldandmultifocalER

GFAFfund

usautoflu

orescence

Sd-O

CTspectrald

omainop

ticalcoherencetom

ograph

yRP

Eretin

alpigm

entepitheliumLP

light

perceptio

nHMhandmotion

6 BioMed Research International

Table2Listof

mutations

detected

bynext

generatio

nsequ

encing

after

applying

relevant

filters

Patie

ntGene

Exon

Allelestate

Nucleotidee

xchange

Proteineffect

rsID

Con

servation

Polyoh

en2SIFT

Pathogenicity

Note

F303

II1(C

IC00

445)

NPH

P412

HTZ

AgtG

pSer481As

nno

NC

BT

Neutral

RP1

4HTZ

c162

5CgtG

pSer542lowast

mdashmdash

mdashmdash

Disease

causing

RM

[12]

RP1

4HTZ

c458

745

90delTAAG

pSer1529A

rgfslowast

9mdash

mdashmdash

mdashDisease

causing

NM

F335

III1

(CIC00

491)

PROM1

4HTZ

TgtC

pIle

178V

almdash

NC

BT

Neutral

GPR9

829

HTZ

GgtA

pArg2128Gln

rs149390094

NC

BT

Neutral

RP1

4HMZ

c408

940

92delAAG

ApArg1364

Valfslowast

8mdash

mdashmdash

mdashDisease

causing

NM

F674

III6

(CIC01106)

USH

2A39

HTZ

TgtG

pSer2450A

rgNo

HC

PD

DProb

ably

diseasec

ausin

gRP

14

HMZ

c120

5delG

pGly40

2Alafslowast

7mdash

mdashmdash

mdashDisease

causing

NM

F752

II1(C

IC01245)

USH

1C17

HTZ

GgtA

pArg477Trp

TMPES

P11

17532053

HC

PD

DProb

ablydisease

causing

PDE6

B10

HTZ

TgtC

pThr432Ile

mdashHC

BT

Uncertain

pathogenicity

RP1

4HTZ

c202

5dup

ApSer676Ile

fslowast

22mdash

mdashmdash

mdashDisease

causing

NM

RP1

4HTZ

c2377d

elA

pArg793G

lufslowast

55mdash

mdashmdash

mdashDisease

causing

NM

F782II5

(CIC01300)

RP1

4HMZ

c17191723delCTC

AA

pSer574Cy

sfslowast7

mdashmdash

mdashmdash

Disease

causing

NM

TULP

15

HMZ

c395

418dup

pAs

p124

Glu131del

rs63749128

mdashmdash

mdashNeutral

F1941III1

(CIC04130)

PCDH15

33HTZ

CgtT

pArg1889His

rs145851144

NC

BT

Neutral

C2orf71

1HTZ

CgtA

pArg656Ser

rs201980758

NC

BT

Neutral

CACN

A2D4

Exon

37-

Intro

n37

HTZ

CgtT

mdashrs80092457

NC

mdashmdash

Neutral

RP1

4HMZ

c1329d

elG

pLys443

Asnfslowast

12mdash

mdashmdash

mdashDisease

causing

NM

F3110

III5

(CIC05941)

EYS

6HTZ

CgtT

pSer326As

nrs112822256

NC

BT

Neutral

MER

TK8

HTZ

CgtG

pArg421Trp

rs138908058

NC

BD

Neutral

PRPF

621

HTZ

AgtG

pVal915M

etrs139778757

MC

PD

DUncertain

pathogenicity

TULP

114

HTZ

GgtA

pAla496Th

rrs141980901

MC

BD

Neutral

EYS

26HTZ

GgtA

pLys1365G

lurs16895519

NC

BD

Neutral

MER

TK18

HTZ

GgtC

pGlu823G

lnrs55924349

MC

BD

Neutral

RP1

4HMZ

c239

12392

delAA

pAsp79

9lowastmdash

mdashmdash

mdashDisease

causing

NM

Prob

ablydiseasec

ausin

gmutations

areh

ighlighted

inbo

ld

Bbenign

HMZ

homozygou

sHTZ

heterozygou

sM

Cmarginally

conservedNC

notcon

servedN

Mn

ovelmutation

RMrecurrent

mutation

TtoleratedPD

possib

lydamaging

BioMed Research International 7

(a)

(b) (c)

(d)

(e) (f)

Figure 1 Ophthalmic features of family F752 II1 (CIC01245) fundus color photographs ((a) and (d) for right and left eye resp)autofluorescence ((b) and (e) for right and left eye resp) and spectral domain optical coherence tomography horizontal macula scans ((c)and (f) for right and left eye resp) showing severe rod-cone dystrophy signs with macular involvement

last exon (exon 4) and shown to be transmitted in an auto-somal dominant mode of inheritance Most of RP1 diseasecausing variants represent nonsense mutations deletions orinsertions In mammalian genes nonsense mutations lead tounstable mRNAs that are degraded by nonsense-mediated

decay (NMD)However exceptionsmight arise when prema-ture stop codons occur in the last exon [37]These variants arethought to abolish RP1 function by resulting in a truncatedprotein lacking important functional domains although stillable to interact with some of its protein partner(s) [21] The

8 BioMed Research International

0 2 4 6 8 10 12 14 16 18 20 22

87888990919293949596979899100

Chromosome

Cov

erag

e at d

epth

25

X (

)

F303 CIC00445F335 CIC00491F674 CIC01106F752 CIC01245

F782 CIC01300

F3110 CIC05941F1941 CIC04130

(a)

0 2 4 6 8 10 12 14 16 18 20 22

Chromosome

100

150

200

250

300

350

400

Mea

n de

pth

per b

ase

F303 CIC00445F335 CIC00491F674 CIC01106 F3110 CIC05941F752 CIC01245

F782 CIC01300F1941 CIC04130

(b)

Figure 2 Sequencing statistics in index patients (a)The overall sequencing coverage of the target regions at 25X depth of coverage is shownin each of the chromosomes No values were indicated for chromosomes 13 18 21 and 22 as they were not targeted The term chromosome23 was used to designate the X chromosome F1941 III1 (CIC04130) and F3110 III5 (CIC05941) showed the lowest coverage results (b) Theaverage mean depth per base pair is shown for each of the chromosomes Most targets showed coverage around 300 times

1 1 1

1 2 3 4 5 6

2 3 4

4

5 632

2

1

1

1 2 3

4

5

11

2 21

2 2

3

2

3

6 7 8

4

41 5

(I)

(II)

(III)

(a) CIC00445 II1 F303 (b) CIC00491 III1 F335

(f) CIC04130 III1 F1941 (g) CIC05941 III5 F3110

1 2 3 4

1

1

1

2

2

3

3

4

4

5

5

6 7 8 9 10 65

43

21

1

1

2 3 4 5

432

2

1 2

2 3 4 5 61

1

(I)

(II)

(III)

(IV)

M1 c1625CgtGpSer542lowastM2 c4587 4590delTAAG pSer1529Argfslowast9

[M1][M2]

M3 c4089 4092delAAGApArg1364Valfslowast8

[M3][M3]

M4 c1205delG pGly402Alafslowast7

[M4][M4]

M5 c2025dupA pSer676Ilefslowast22M6 c2377delA pArg793Glufslowast55

[M5][M6]

M7 c1719 1723delCTCAA pSer574Cysfslowast7 M8 c1329delG pLys443Asnfslowast12

[M8][M8]

[M7][M7]

[=][=]

M9 c2391 2392delAA pAsp799lowast

[M9][M9]

[M9][=]

(c) CIC01106 III6 F674 (d) CIC01245 II1 F752

(e) CIC01300 II5 F782

Figure 3 Pedigrees of seven families with RP1 mutations underlying autosomal recessive rod-cone dystrophy Affected and unaffectedindividuals are represented by shapes filled with black and white colors respectively Men and women are indicated by squares and circlesrespectively Index subjects are marked by Consanguinity is marked by a double horizontal line

BioMed Research International 9

4

adRCD

arRCD

Class I

Class II (hot spot)

Class III Class III Class IV

321 BIF

pA

sp202

Glu

pThr373

Ile

pA

rg1519

Glu

fsX2

pA

rg1652

Leu

pCy

s2033

Tyr

pA

la669

Thr

pT8

65

Leu8

66

delIn

s

pA

sp984

Gly

pSe

r152

9Arg

fslowast9

pA

rg15

19G

lufslowast

2

pPr

o164

8Ser

fslowast2

pA

sn17

51Le

ufslowast4

pA

sn94

9Lys

fslowast32

pSe

r542lowast

pSe

r574

Cysfs

lowast7

pSe

r676

Ilefslowast

22

pA

rg79

3Glu

fslowast55

pA

sp79

9lowast

pM

et50

0Ser

fslowast33

pPr

o658

Hisf

slowast4

pLy

s673

Arg

fslowast9

pLy

s675

Asn

fslowast7

pA

rg67

7lowast

pA

rg67

7Asp

fslowast5

pG

ln67

9lowastp

Tyr6

85lowast

pG

ln68

6lowastp

Gln

689lowast

pG

lu70

0lowastp

Gly

706V

alfslowast7

pLy

s722

Glu

fslowast16

pG

ly72

3lowastp

Gly

724G

lufslowast

9

pile

725A

rgfslowast

6

pIle

725Th

rfslowast13

pIle

725A

spfslowast

4

pG

lu72

9Lys

fslowast9

pThr7

36Ile

fslowast4

pCy

s744

lowast

pSe

r747

Valfs

lowast16

pA

sn74

8Ile

fslowast15

pA

rg75

9lowastp

Leu7

62

Asn763

pL

eu762

Tyrfs

lowast17

pA

sn76

3Leu

fslowast11

pA

sn76

9Arg

fslowast6

pLy

s778

lowast

pSe

r779

lowast

pSe

r862

lowast

pIle

864L

ysfslowast

11

pTy

r105

3Thrfs

lowast4

pSe

r2A

rgfslowast16

pH

is31A

rgfslowast

9

pVa

l157

Trpf

slowast16

pA

la22

1Gly

fslowast43

pPr

o229

Gln

fslowast35

pA

rg33

8lowast

pA

rg39

6lowastp

Gly

402A

lafslowast7

pLy

s443

Asn

fslowast12

pG

lu48

8lowast

pTr

p113

1lowast

pG

ly11

40ly

sfslowast4

pA

sn11

43Ile

fslowast25

pG

lu12

27M

etfslowast

29

pA

rg13

64Va

lfslowast8

pLy

s151

8lowast

pA

rg87

2Thrfs

lowast2

pSe

r911

lowast

Figure 4 Schematic presentation of RP1 disease causing mutations Disease causing mutations were represented based on the classificationby Chen and coworkers [13] Mutations responsible for recessive arRCD were shown in the upper half whereas mutations causing adRCDwere shown in the lower half pGly402Alafslowast7 pLys443Asnfslowast12 pArg1364Valfslowast8 and pSer1529Argfslowast9 belong to class III AlthoughpSer574Cysfslowast7 pSer676Ilefslowast22 pArg793Glufslowast55 and pAsp799lowast are class II mutations these variants do not cause adRCD but arRCDinstead Amino acid modifications shown in red and blue represent novel frameshift or nonsense mutations and the recurrent pSer542lowastmutation respectively Protein localization of pSer542lowast was highlighted in blue as it marked a recurrent mutation adRCD autosomaldominant rod-cone dystrophy arRCD autosomal recessive rod-cone dystrophy BIF drosophila melanogaster bifocal

latter observation is supported by finding that RP1 mutantmRNA is expressed in a human cell line carrying a homozy-gous pArg677lowast mutation [21]

Based on Chen et al [13] RP1 truncating mutations lead-ing to arRCD or adRCD can be divided into four distinctgroups Class I is composed of truncating mutations locatedin exons 2 and 3These variants are sensitive toNMDand thusare considered as true loss-of-function alleles (Figure 4) [13]Class II involves truncating mutations that are located in aspot between codons 500 and 1053 in exon 4 [13] the so calledldquoRP1 hot spotrdquo The ldquohot spotrdquo variants tend to be insensitiveto NMD process and thus result in a protein with a potentialdominant negative effect leading to adRCD (Figure 4) [13]Class III includes truncating mutations insensible to NMDlocated between codons 264 and 499 and between codons1054 to 1751 in exon 4 These truncating proteins result in aloss of function leading to arRCD (Figure 4) [13] Finally classIV includes protein-truncating mutations near the 31015840 endof the fourth exon (Figure 4) [13] Most likely the resultingproteins display only aminor loss of their C-terminal portionpreserving the majority of functional domains and keeping aresidual activity According to the classification of Chen et al[13] pGly402Alafslowast7 pLys443Asnfslowast12 pArg1364Valfslowast8and pSer1529Argfslowast9 belong to class III (Figure 4)

The predicted physiopathology for pSer542lowastpSer574Cysfslowast7 pSer676Ilefslowast22 pArg793Glufslowast55 andpAsp799lowast is more complex According to Chenrsquos classi-fication these frameshift deletions and nonsense mutationsshould belong to class II previously only associated withadRCD However herein they are causing presumablyarRCD (Figure 4) To further confirm these findings clinicaland genetic testing of the reported unaffected parents shouldbe done

Based on the previous findings we speculate that the clas-sification by Chen and coworkers does not hold true for allmutations Supporting this statement Avila-Fernandez et al[12] reported the same nonsense mutation (pSer542lowast) foundin (F303 II1 (CIC00445)) and located at the 51015840 end of theldquohot spotrdquo to cause arRP in the Spanish population [12]These observations are of interest as they point out for animplication of hot spot region for adRCD-RP1mutation alsoin case of arRCD Future studies will need to clarify why someclass II mutations lead to adRCD and others to arRCD

Patients with arRCD and RP1 mutations show a moresevere disease than adRCD-RP1 mutant patients with mac-ular atrophy in all our cases This was first outlined byLafont et al [17] When patients are presenting with latesevere disease the diagnostic distinction between RCD with

10 BioMed Research International

initial rod involvement and cone-rod dystrophy (CRD) withinitial cone involvement is difficult Of note is that one ofthe patients (CIC01300) in the present study was initiallyclassified as possibly having severe CRD and his diagnosiswas actually revisited after NGS resultsThis also outlines theinterest of unbiased massive parallel sequencing methods fora more precise clinical diagnostic in case of end stage diseaseThis point will most likely become evenmore critical with theperspective of therapeutic trials

41 Strength and Limitations We estimate that 1 of ourtarget regions were not covered Partially uncovered exonsare a real common issue when capturing the DNA sequencesusing commercially available probes this bias might implya loss of some candidate variants However we found thatrate of 1 is very reasonable when compared with otherNGS panels In addition in order to exclude the possibilityof finding other candidate variants we have sequenced bySanger method the majority of these regions Five of ourpatients carried homozygous RP1 mutations For four of thesubjects carrying homozygous variants namely CIC00491F335 CIC01106 F674 CIC01300 F782 and CIC04130 F1941co-segregation analysis needs to be done to confirm auto-somal recessive inheritance but we do not have access toparentrsquos DNA CIC05941 was the only one not to reportclear consanguinity in the family and we cannot exclude thepossibility of a large deletion on the second allele of RP1gene Again DNA of the father not available for us wouldbe helpful to prove autosomal recessive inheritance and thehomozygous state of the mutation

In conclusion we have reported 9 mutations in RP1 ofwhich 8 were novel causing arRCD [8 12ndash20] Interestingly aprevalence ofasymp25points out for the necessity of sequencingRP1 in sporadic and recessive cases of RCD Further func-tional studies are needed to understand the impact of RP1structure on its function at the molecular level such a stepwould strengthen our knowledge in the physiology of retinalphotoreceptors

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Isabelle Audo and Christina Zeitz contributed equally to thiswork

Acknowledgments

The authors express their sincere gratitude to the familieswho participated in this study and to the clinical staff fortheir help in clinical data and DNA collection This workwas supported by Foundation Voir et Entendre (CZ) PrixDalloz for ldquola recherche en ophtalmologierdquo (CZ) FoundationFighting Blindness (FFB) [CD-CL-0808-0466-CHNO] (IA)and FFB center (FFB grantC-GE-0912-0601-INSERM02)

Prix de la Fondation de lrsquoŒil (IA) Ville de Paris and RegionIle de France and the French State program ldquoInvestissementsdrsquoAvenirrdquo managed by the Agence Nationale de la Recherche[LIFESENSES ANR-10-LABX-65]

References

[1] D T Hartong E L Berson and T P Dryja ldquoRetinitis pigmen-tosardquoThe Lancet vol 368 no 9549 pp 1795ndash1809 2006

[2] I Audo M Lancelot S Mohand-Saıd et al ldquoNovel C2orf71mutations account for approximately sim1 of cases in a largeFrench arRP cohortrdquoHumanMutation vol 32 no 4 pp E2091ndashE2103 2011

[3] A Anasagasti C Irigoyen O Barandika A Lopez de Munainand J Ruiz-Ederra ldquoCurrent mutation discovery approaches inRetinitis PigmentosardquoVision Research vol 75 pp 117ndash129 2012

[4] L S Sullivan S J Bowne D G Birch et al ldquoPrevalence ofdisease-causingmutations in families with autosomal dominantretinitis pigmentosa a screen of known genes in 200 familiesrdquoInvestigative Ophthalmology andVisual Science vol 47 no 7 pp3052ndash3064 2006

[5] B J Seyedahmadi C Rivolta J A Keene E L Berson andT P Dryja ldquoComprehensive screening of the USH2A gene inUsher syndrome type II and non-syndromic recessive retinitispigmentosardquo Experimental Eye Research vol 79 no 2 pp 167ndash173 2004

[6] A Avila-Fernandez D Cantalapiedra E Aller et al ldquoMutationanalysis of 272 Spanish families affected by autosomal recessiveretinitis pigmentosa using a genotyping microarrayrdquoMolecularVision vol 16 pp 2550ndash2558 2010

[7] S El Shamieh M Neuille A Terray et al ldquoWhole-exomesequencing identifies KIZ as a ciliary gene associated withautosomal-recessive rod-cone dystrophyrdquo American Journal ofHuman Genetics vol 94 no 4 pp 625ndash633 2014

[8] I Audo S Mohand-Saıd C M Dhaenens et al ldquoRP1 andautosomal dominant rod-cone dystrophy novel mutations areview of published variants and genotype-phenotype correla-tionrdquo Human Mutation vol 33 no 1 pp 73ndash80 2012

[9] X Guillonneau N I Piriev M Danciger et al ldquoA nonsensemutation in a novel gene is associated with retinitis pigmentosain a family linked to the RP1 locusrdquoHumanMolecular Geneticsvol 8 no 8 pp 1541ndash1546 1999

[10] E A Pierce T Quinn T Meehan T L McGee E L Bersonand T P Dryja ldquoMutations in a gene encoding a new oxygen-regulated photoreceptor protein cause dominant retinitis pig-mentosardquo Nature Genetics vol 22 no 3 pp 248ndash254 1999

[11] L S Sullivan J R Heckenlively S J Bowne et al ldquoMutations ina novel retina-specific gene cause autosomal dominant retinitispigmentosardquo Nature Genetics vol 22 no 3 pp 255ndash259 1999

[12] A Avila-Fernandez M Corton K M Nishiguchi et al ldquoIden-tification of an RP1 prevalent founder mutation and relatedphenotype in Spanish patients with early-onset autosomalrecessive retinitisrdquo Ophthalmology vol 119 no 12 pp 2616ndash2621 2012

[13] L J Chen T Y Y Lai P O S Tam et al ldquoCompound heter-ozygosity of two novel truncation mutations in RP1 causingautosomal recessive retinitis pigmentosardquo InvestigativeOphthal-mology and Visual Science vol 51 no 4 pp 2236ndash2242 2010

[14] S Khaliq A Abid M Ismail et al ldquoNovel association of RP1gene mutations with autosomal recessive retinitis pigmentosardquoJournal of Medical Genetics vol 42 no 5 pp 436ndash438 2005

BioMed Research International 11

[15] A M Siemiatkowska G D N Astuti K Arimadyo et alldquoIdentification of a novel nonsense mutation in RP1 that causesautosomal recessive retinitis pigmentosa in an IndonesianfamilyrdquoMolecular Vision vol 18 pp 2411ndash2419 2012

[16] B Bocquet N A Marzouka M Hebrard et al ldquoHomozygositymapping in autosomal recessive retinitis pigmentosa familiesdetects novel mutationsrdquo Molecular Vision vol 19 pp 2487ndash2500 2013

[17] E M Lafont G Manes G Senechal et al ldquoPatients with reti-nitis pigmentosa due to RP1 mutations show greater severity inrecessive than in dominant casesrdquo Journal of Clinical amp Experi-mental Ophthalmology vol 2 article 194 2011

[18] M Al-Rashed L Abu Safieh H Alkuraya et al ldquoRP1 and reti-nitis pigmentosa report of novel mutations and insight intomutational mechanismrdquo British Journal of Ophthalmology vol96 no 7 pp 1018ndash1022 2012

[19] S A Riazuddin A Shahzadi C Zeitz et al ldquoA mutation inSLC24A1 implicated in autosomal-recessive congenital station-ary night blindnessrdquoThe American Journal of Human Geneticsvol 87 no 4 pp 523ndash531 2010

[20] H P Singh S Jalali R Narayanan and C Kannabiran ldquoGe-netic analysis of indian families with autosomal recessiveretinitis pigmentosa by homozygosity screeningrdquo InvestigativeOphthalmology and Visual Science vol 50 no 9 pp 4065ndash40712009

[21] Q Liu A Lyubarsky J H Skalet E N Pugh Jr and E A PierceldquoRP1 is required for the correct stacking of outer segment discsrdquoInvestigative Ophthalmology ampVisual Science vol 44 no 10 pp4171ndash4183 2003

[22] Q Liu J Zuo and E A Pierce ldquoThe retinitis pigmentosa 1protein is a photoreceptor microtubule-associated proteinrdquoJournal of Neuroscience vol 24 no 29 pp 6427ndash6436 2004

[23] I Audo K M Bujakowska T Leveillard et al ldquoDevelop-ment and application of a next-generation-sequencing (NGS)approach to detect known and novel gene defects underlyingretinal diseasesrdquo Orphanet Journal of Rare Diseases vol 7 no 1article 8 2012

[24] I Audo J A Sahel S Mohand-Saıd et al ldquoEYS is a major genefor rod-cone dystrophies in Francerdquo Human Mutation vol 31no 5 pp E1406ndashE1435 2010

[25] T G P Consortium ldquoA map of human genome variation frompopulation-scale sequencingrdquo Nature vol 467 no 7319 pp1061ndash1073 2010

[26] DMAltshuler RAGibbs L Peltonen et al ldquoIntegrating com-mon and rare genetic variation in diverse human populationsrdquoNature vol 467 no 7311 pp 52ndash58 2010

[27] J A Tennessen AW Bigham T D OrsquoConnor et al ldquoEvolutionand functional impact of rare coding variation from deepsequencing of human exomesrdquo Science vol 337 no 6090 pp64ndash69 2012

[28] M J Bamshad S BNgAW Bighamet al ldquoExome sequencingas a tool for Mendelian disease gene discoveryrdquo Nature ReviewsGenetics vol 12 no 11 pp 745ndash755 2011

[29] W J Kent C W Sugnet T S Furey et al ldquoThe human genomebrowser at UCSCrdquo Genome Research vol 12 no 6 pp 996ndash1006 2002

[30] I A Adzhubei S Schmidt L Peshkin et al ldquoA method andserver for predicting damaging missense mutationsrdquo NatureMethods vol 7 no 4 pp 248ndash249 2010

[31] P Kumar S Henikoff and P C Ng ldquoPredicting the effects ofcoding non-synonymous variants on protein function using the

SIFT algorithmrdquo Nature Protocols vol 4 no 7 pp 1073ndash10822009

[32] F O Desmet D Hamroun M Lalande G Collod-Beroud MClaustres and C Beroud ldquoHuman splicing finder an onlinebioinformatics tool to predict splicing signalsrdquo Nucleic AcidsResearch vol 37 no 9 article no e67 2009

[33] P D Stenson E V Ball M Mort A D Phillips K Shaw andD N Cooper ldquoThe Human Gene Mutation Database (HGMD)and its exploitation in the fields of personalized genomics andmolecular evolutionrdquo in Current Protocols in Bioinformaticschapter 1ndash13 2012

[34] I F A C Fokkema J T Den Dunnen and P E M TaschnerldquoLOVD easy creation of a locus-specific sequence variationdatabase using an ldquoLSDB-in-a-Boxrdquo approachrdquo Human Muta-tion vol 26 no 2 pp 63ndash68 2005

[35] S A Miller D D Dykes and H F Polesky ldquoA simple saltingout procedure for extracting DNA from human nucleated cellsrdquoNucleic Acids Research vol 16 no 3 p 1215 1988

[36] C Zeitz B Kloeckener-Gruissem U Forster et al ldquoMutationsin CABP4 the gene encoding the Ca2+-binding protein 4cause autosomal recessive night blindnessrdquoAmerican Journal ofHuman Genetics vol 79 no 4 pp 657ndash667 2006

[37] S A Riazuddin F Zulfiqar Q Zhang et al ldquoAutosomal reces-sive retinitis pigmentosa is associated with mutations in RP1 inthree consanguineous Pakistani familiesrdquo Investigative Ophthal-mology and Visual Science vol 46 no 7 pp 2264ndash2270 2005

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

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The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

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Signal TransductionJournal of

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ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Nucleic AcidsJournal of

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Enzyme Research

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International Journal of

Microbiology

6 BioMed Research International

Table2Listof

mutations

detected

bynext

generatio

nsequ

encing

after

applying

relevant

filters

Patie

ntGene

Exon

Allelestate

Nucleotidee

xchange

Proteineffect

rsID

Con

servation

Polyoh

en2SIFT

Pathogenicity

Note

F303

II1(C

IC00

445)

NPH

P412

HTZ

AgtG

pSer481As

nno

NC

BT

Neutral

RP1

4HTZ

c162

5CgtG

pSer542lowast

mdashmdash

mdashmdash

Disease

causing

RM

[12]

RP1

4HTZ

c458

745

90delTAAG

pSer1529A

rgfslowast

9mdash

mdashmdash

mdashDisease

causing

NM

F335

III1

(CIC00

491)

PROM1

4HTZ

TgtC

pIle

178V

almdash

NC

BT

Neutral

GPR9

829

HTZ

GgtA

pArg2128Gln

rs149390094

NC

BT

Neutral

RP1

4HMZ

c408

940

92delAAG

ApArg1364

Valfslowast

8mdash

mdashmdash

mdashDisease

causing

NM

F674

III6

(CIC01106)

USH

2A39

HTZ

TgtG

pSer2450A

rgNo

HC

PD

DProb

ably

diseasec

ausin

gRP

14

HMZ

c120

5delG

pGly40

2Alafslowast

7mdash

mdashmdash

mdashDisease

causing

NM

F752

II1(C

IC01245)

USH

1C17

HTZ

GgtA

pArg477Trp

TMPES

P11

17532053

HC

PD

DProb

ablydisease

causing

PDE6

B10

HTZ

TgtC

pThr432Ile

mdashHC

BT

Uncertain

pathogenicity

RP1

4HTZ

c202

5dup

ApSer676Ile

fslowast

22mdash

mdashmdash

mdashDisease

causing

NM

RP1

4HTZ

c2377d

elA

pArg793G

lufslowast

55mdash

mdashmdash

mdashDisease

causing

NM

F782II5

(CIC01300)

RP1

4HMZ

c17191723delCTC

AA

pSer574Cy

sfslowast7

mdashmdash

mdashmdash

Disease

causing

NM

TULP

15

HMZ

c395

418dup

pAs

p124

Glu131del

rs63749128

mdashmdash

mdashNeutral

F1941III1

(CIC04130)

PCDH15

33HTZ

CgtT

pArg1889His

rs145851144

NC

BT

Neutral

C2orf71

1HTZ

CgtA

pArg656Ser

rs201980758

NC

BT

Neutral

CACN

A2D4

Exon

37-

Intro

n37

HTZ

CgtT

mdashrs80092457

NC

mdashmdash

Neutral

RP1

4HMZ

c1329d

elG

pLys443

Asnfslowast

12mdash

mdashmdash

mdashDisease

causing

NM

F3110

III5

(CIC05941)

EYS

6HTZ

CgtT

pSer326As

nrs112822256

NC

BT

Neutral

MER

TK8

HTZ

CgtG

pArg421Trp

rs138908058

NC

BD

Neutral

PRPF

621

HTZ

AgtG

pVal915M

etrs139778757

MC

PD

DUncertain

pathogenicity

TULP

114

HTZ

GgtA

pAla496Th

rrs141980901

MC

BD

Neutral

EYS

26HTZ

GgtA

pLys1365G

lurs16895519

NC

BD

Neutral

MER

TK18

HTZ

GgtC

pGlu823G

lnrs55924349

MC

BD

Neutral

RP1

4HMZ

c239

12392

delAA

pAsp79

9lowastmdash

mdashmdash

mdashDisease

causing

NM

Prob

ablydiseasec

ausin

gmutations

areh

ighlighted

inbo

ld

Bbenign

HMZ

homozygou

sHTZ

heterozygou

sM

Cmarginally

conservedNC

notcon

servedN

Mn

ovelmutation

RMrecurrent

mutation

TtoleratedPD

possib

lydamaging

BioMed Research International 7

(a)

(b) (c)

(d)

(e) (f)

Figure 1 Ophthalmic features of family F752 II1 (CIC01245) fundus color photographs ((a) and (d) for right and left eye resp)autofluorescence ((b) and (e) for right and left eye resp) and spectral domain optical coherence tomography horizontal macula scans ((c)and (f) for right and left eye resp) showing severe rod-cone dystrophy signs with macular involvement

last exon (exon 4) and shown to be transmitted in an auto-somal dominant mode of inheritance Most of RP1 diseasecausing variants represent nonsense mutations deletions orinsertions In mammalian genes nonsense mutations lead tounstable mRNAs that are degraded by nonsense-mediated

decay (NMD)However exceptionsmight arise when prema-ture stop codons occur in the last exon [37]These variants arethought to abolish RP1 function by resulting in a truncatedprotein lacking important functional domains although stillable to interact with some of its protein partner(s) [21] The

8 BioMed Research International

0 2 4 6 8 10 12 14 16 18 20 22

87888990919293949596979899100

Chromosome

Cov

erag

e at d

epth

25

X (

)

F303 CIC00445F335 CIC00491F674 CIC01106F752 CIC01245

F782 CIC01300

F3110 CIC05941F1941 CIC04130

(a)

0 2 4 6 8 10 12 14 16 18 20 22

Chromosome

100

150

200

250

300

350

400

Mea

n de

pth

per b

ase

F303 CIC00445F335 CIC00491F674 CIC01106 F3110 CIC05941F752 CIC01245

F782 CIC01300F1941 CIC04130

(b)

Figure 2 Sequencing statistics in index patients (a)The overall sequencing coverage of the target regions at 25X depth of coverage is shownin each of the chromosomes No values were indicated for chromosomes 13 18 21 and 22 as they were not targeted The term chromosome23 was used to designate the X chromosome F1941 III1 (CIC04130) and F3110 III5 (CIC05941) showed the lowest coverage results (b) Theaverage mean depth per base pair is shown for each of the chromosomes Most targets showed coverage around 300 times

1 1 1

1 2 3 4 5 6

2 3 4

4

5 632

2

1

1

1 2 3

4

5

11

2 21

2 2

3

2

3

6 7 8

4

41 5

(I)

(II)

(III)

(a) CIC00445 II1 F303 (b) CIC00491 III1 F335

(f) CIC04130 III1 F1941 (g) CIC05941 III5 F3110

1 2 3 4

1

1

1

2

2

3

3

4

4

5

5

6 7 8 9 10 65

43

21

1

1

2 3 4 5

432

2

1 2

2 3 4 5 61

1

(I)

(II)

(III)

(IV)

M1 c1625CgtGpSer542lowastM2 c4587 4590delTAAG pSer1529Argfslowast9

[M1][M2]

M3 c4089 4092delAAGApArg1364Valfslowast8

[M3][M3]

M4 c1205delG pGly402Alafslowast7

[M4][M4]

M5 c2025dupA pSer676Ilefslowast22M6 c2377delA pArg793Glufslowast55

[M5][M6]

M7 c1719 1723delCTCAA pSer574Cysfslowast7 M8 c1329delG pLys443Asnfslowast12

[M8][M8]

[M7][M7]

[=][=]

M9 c2391 2392delAA pAsp799lowast

[M9][M9]

[M9][=]

(c) CIC01106 III6 F674 (d) CIC01245 II1 F752

(e) CIC01300 II5 F782

Figure 3 Pedigrees of seven families with RP1 mutations underlying autosomal recessive rod-cone dystrophy Affected and unaffectedindividuals are represented by shapes filled with black and white colors respectively Men and women are indicated by squares and circlesrespectively Index subjects are marked by Consanguinity is marked by a double horizontal line

BioMed Research International 9

4

adRCD

arRCD

Class I

Class II (hot spot)

Class III Class III Class IV

321 BIF

pA

sp202

Glu

pThr373

Ile

pA

rg1519

Glu

fsX2

pA

rg1652

Leu

pCy

s2033

Tyr

pA

la669

Thr

pT8

65

Leu8

66

delIn

s

pA

sp984

Gly

pSe

r152

9Arg

fslowast9

pA

rg15

19G

lufslowast

2

pPr

o164

8Ser

fslowast2

pA

sn17

51Le

ufslowast4

pA

sn94

9Lys

fslowast32

pSe

r542lowast

pSe

r574

Cysfs

lowast7

pSe

r676

Ilefslowast

22

pA

rg79

3Glu

fslowast55

pA

sp79

9lowast

pM

et50

0Ser

fslowast33

pPr

o658

Hisf

slowast4

pLy

s673

Arg

fslowast9

pLy

s675

Asn

fslowast7

pA

rg67

7lowast

pA

rg67

7Asp

fslowast5

pG

ln67

9lowastp

Tyr6

85lowast

pG

ln68

6lowastp

Gln

689lowast

pG

lu70

0lowastp

Gly

706V

alfslowast7

pLy

s722

Glu

fslowast16

pG

ly72

3lowastp

Gly

724G

lufslowast

9

pile

725A

rgfslowast

6

pIle

725Th

rfslowast13

pIle

725A

spfslowast

4

pG

lu72

9Lys

fslowast9

pThr7

36Ile

fslowast4

pCy

s744

lowast

pSe

r747

Valfs

lowast16

pA

sn74

8Ile

fslowast15

pA

rg75

9lowastp

Leu7

62

Asn763

pL

eu762

Tyrfs

lowast17

pA

sn76

3Leu

fslowast11

pA

sn76

9Arg

fslowast6

pLy

s778

lowast

pSe

r779

lowast

pSe

r862

lowast

pIle

864L

ysfslowast

11

pTy

r105

3Thrfs

lowast4

pSe

r2A

rgfslowast16

pH

is31A

rgfslowast

9

pVa

l157

Trpf

slowast16

pA

la22

1Gly

fslowast43

pPr

o229

Gln

fslowast35

pA

rg33

8lowast

pA

rg39

6lowastp

Gly

402A

lafslowast7

pLy

s443

Asn

fslowast12

pG

lu48

8lowast

pTr

p113

1lowast

pG

ly11

40ly

sfslowast4

pA

sn11

43Ile

fslowast25

pG

lu12

27M

etfslowast

29

pA

rg13

64Va

lfslowast8

pLy

s151

8lowast

pA

rg87

2Thrfs

lowast2

pSe

r911

lowast

Figure 4 Schematic presentation of RP1 disease causing mutations Disease causing mutations were represented based on the classificationby Chen and coworkers [13] Mutations responsible for recessive arRCD were shown in the upper half whereas mutations causing adRCDwere shown in the lower half pGly402Alafslowast7 pLys443Asnfslowast12 pArg1364Valfslowast8 and pSer1529Argfslowast9 belong to class III AlthoughpSer574Cysfslowast7 pSer676Ilefslowast22 pArg793Glufslowast55 and pAsp799lowast are class II mutations these variants do not cause adRCD but arRCDinstead Amino acid modifications shown in red and blue represent novel frameshift or nonsense mutations and the recurrent pSer542lowastmutation respectively Protein localization of pSer542lowast was highlighted in blue as it marked a recurrent mutation adRCD autosomaldominant rod-cone dystrophy arRCD autosomal recessive rod-cone dystrophy BIF drosophila melanogaster bifocal

latter observation is supported by finding that RP1 mutantmRNA is expressed in a human cell line carrying a homozy-gous pArg677lowast mutation [21]

Based on Chen et al [13] RP1 truncating mutations lead-ing to arRCD or adRCD can be divided into four distinctgroups Class I is composed of truncating mutations locatedin exons 2 and 3These variants are sensitive toNMDand thusare considered as true loss-of-function alleles (Figure 4) [13]Class II involves truncating mutations that are located in aspot between codons 500 and 1053 in exon 4 [13] the so calledldquoRP1 hot spotrdquo The ldquohot spotrdquo variants tend to be insensitiveto NMD process and thus result in a protein with a potentialdominant negative effect leading to adRCD (Figure 4) [13]Class III includes truncating mutations insensible to NMDlocated between codons 264 and 499 and between codons1054 to 1751 in exon 4 These truncating proteins result in aloss of function leading to arRCD (Figure 4) [13] Finally classIV includes protein-truncating mutations near the 31015840 endof the fourth exon (Figure 4) [13] Most likely the resultingproteins display only aminor loss of their C-terminal portionpreserving the majority of functional domains and keeping aresidual activity According to the classification of Chen et al[13] pGly402Alafslowast7 pLys443Asnfslowast12 pArg1364Valfslowast8and pSer1529Argfslowast9 belong to class III (Figure 4)

The predicted physiopathology for pSer542lowastpSer574Cysfslowast7 pSer676Ilefslowast22 pArg793Glufslowast55 andpAsp799lowast is more complex According to Chenrsquos classi-fication these frameshift deletions and nonsense mutationsshould belong to class II previously only associated withadRCD However herein they are causing presumablyarRCD (Figure 4) To further confirm these findings clinicaland genetic testing of the reported unaffected parents shouldbe done

Based on the previous findings we speculate that the clas-sification by Chen and coworkers does not hold true for allmutations Supporting this statement Avila-Fernandez et al[12] reported the same nonsense mutation (pSer542lowast) foundin (F303 II1 (CIC00445)) and located at the 51015840 end of theldquohot spotrdquo to cause arRP in the Spanish population [12]These observations are of interest as they point out for animplication of hot spot region for adRCD-RP1mutation alsoin case of arRCD Future studies will need to clarify why someclass II mutations lead to adRCD and others to arRCD

Patients with arRCD and RP1 mutations show a moresevere disease than adRCD-RP1 mutant patients with mac-ular atrophy in all our cases This was first outlined byLafont et al [17] When patients are presenting with latesevere disease the diagnostic distinction between RCD with

10 BioMed Research International

initial rod involvement and cone-rod dystrophy (CRD) withinitial cone involvement is difficult Of note is that one ofthe patients (CIC01300) in the present study was initiallyclassified as possibly having severe CRD and his diagnosiswas actually revisited after NGS resultsThis also outlines theinterest of unbiased massive parallel sequencing methods fora more precise clinical diagnostic in case of end stage diseaseThis point will most likely become evenmore critical with theperspective of therapeutic trials

41 Strength and Limitations We estimate that 1 of ourtarget regions were not covered Partially uncovered exonsare a real common issue when capturing the DNA sequencesusing commercially available probes this bias might implya loss of some candidate variants However we found thatrate of 1 is very reasonable when compared with otherNGS panels In addition in order to exclude the possibilityof finding other candidate variants we have sequenced bySanger method the majority of these regions Five of ourpatients carried homozygous RP1 mutations For four of thesubjects carrying homozygous variants namely CIC00491F335 CIC01106 F674 CIC01300 F782 and CIC04130 F1941co-segregation analysis needs to be done to confirm auto-somal recessive inheritance but we do not have access toparentrsquos DNA CIC05941 was the only one not to reportclear consanguinity in the family and we cannot exclude thepossibility of a large deletion on the second allele of RP1gene Again DNA of the father not available for us wouldbe helpful to prove autosomal recessive inheritance and thehomozygous state of the mutation

In conclusion we have reported 9 mutations in RP1 ofwhich 8 were novel causing arRCD [8 12ndash20] Interestingly aprevalence ofasymp25points out for the necessity of sequencingRP1 in sporadic and recessive cases of RCD Further func-tional studies are needed to understand the impact of RP1structure on its function at the molecular level such a stepwould strengthen our knowledge in the physiology of retinalphotoreceptors

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Isabelle Audo and Christina Zeitz contributed equally to thiswork

Acknowledgments

The authors express their sincere gratitude to the familieswho participated in this study and to the clinical staff fortheir help in clinical data and DNA collection This workwas supported by Foundation Voir et Entendre (CZ) PrixDalloz for ldquola recherche en ophtalmologierdquo (CZ) FoundationFighting Blindness (FFB) [CD-CL-0808-0466-CHNO] (IA)and FFB center (FFB grantC-GE-0912-0601-INSERM02)

Prix de la Fondation de lrsquoŒil (IA) Ville de Paris and RegionIle de France and the French State program ldquoInvestissementsdrsquoAvenirrdquo managed by the Agence Nationale de la Recherche[LIFESENSES ANR-10-LABX-65]

References

[1] D T Hartong E L Berson and T P Dryja ldquoRetinitis pigmen-tosardquoThe Lancet vol 368 no 9549 pp 1795ndash1809 2006

[2] I Audo M Lancelot S Mohand-Saıd et al ldquoNovel C2orf71mutations account for approximately sim1 of cases in a largeFrench arRP cohortrdquoHumanMutation vol 32 no 4 pp E2091ndashE2103 2011

[3] A Anasagasti C Irigoyen O Barandika A Lopez de Munainand J Ruiz-Ederra ldquoCurrent mutation discovery approaches inRetinitis PigmentosardquoVision Research vol 75 pp 117ndash129 2012

[4] L S Sullivan S J Bowne D G Birch et al ldquoPrevalence ofdisease-causingmutations in families with autosomal dominantretinitis pigmentosa a screen of known genes in 200 familiesrdquoInvestigative Ophthalmology andVisual Science vol 47 no 7 pp3052ndash3064 2006

[5] B J Seyedahmadi C Rivolta J A Keene E L Berson andT P Dryja ldquoComprehensive screening of the USH2A gene inUsher syndrome type II and non-syndromic recessive retinitispigmentosardquo Experimental Eye Research vol 79 no 2 pp 167ndash173 2004

[6] A Avila-Fernandez D Cantalapiedra E Aller et al ldquoMutationanalysis of 272 Spanish families affected by autosomal recessiveretinitis pigmentosa using a genotyping microarrayrdquoMolecularVision vol 16 pp 2550ndash2558 2010

[7] S El Shamieh M Neuille A Terray et al ldquoWhole-exomesequencing identifies KIZ as a ciliary gene associated withautosomal-recessive rod-cone dystrophyrdquo American Journal ofHuman Genetics vol 94 no 4 pp 625ndash633 2014

[8] I Audo S Mohand-Saıd C M Dhaenens et al ldquoRP1 andautosomal dominant rod-cone dystrophy novel mutations areview of published variants and genotype-phenotype correla-tionrdquo Human Mutation vol 33 no 1 pp 73ndash80 2012

[9] X Guillonneau N I Piriev M Danciger et al ldquoA nonsensemutation in a novel gene is associated with retinitis pigmentosain a family linked to the RP1 locusrdquoHumanMolecular Geneticsvol 8 no 8 pp 1541ndash1546 1999

[10] E A Pierce T Quinn T Meehan T L McGee E L Bersonand T P Dryja ldquoMutations in a gene encoding a new oxygen-regulated photoreceptor protein cause dominant retinitis pig-mentosardquo Nature Genetics vol 22 no 3 pp 248ndash254 1999

[11] L S Sullivan J R Heckenlively S J Bowne et al ldquoMutations ina novel retina-specific gene cause autosomal dominant retinitispigmentosardquo Nature Genetics vol 22 no 3 pp 255ndash259 1999

[12] A Avila-Fernandez M Corton K M Nishiguchi et al ldquoIden-tification of an RP1 prevalent founder mutation and relatedphenotype in Spanish patients with early-onset autosomalrecessive retinitisrdquo Ophthalmology vol 119 no 12 pp 2616ndash2621 2012

[13] L J Chen T Y Y Lai P O S Tam et al ldquoCompound heter-ozygosity of two novel truncation mutations in RP1 causingautosomal recessive retinitis pigmentosardquo InvestigativeOphthal-mology and Visual Science vol 51 no 4 pp 2236ndash2242 2010

[14] S Khaliq A Abid M Ismail et al ldquoNovel association of RP1gene mutations with autosomal recessive retinitis pigmentosardquoJournal of Medical Genetics vol 42 no 5 pp 436ndash438 2005

BioMed Research International 11

[15] A M Siemiatkowska G D N Astuti K Arimadyo et alldquoIdentification of a novel nonsense mutation in RP1 that causesautosomal recessive retinitis pigmentosa in an IndonesianfamilyrdquoMolecular Vision vol 18 pp 2411ndash2419 2012

[16] B Bocquet N A Marzouka M Hebrard et al ldquoHomozygositymapping in autosomal recessive retinitis pigmentosa familiesdetects novel mutationsrdquo Molecular Vision vol 19 pp 2487ndash2500 2013

[17] E M Lafont G Manes G Senechal et al ldquoPatients with reti-nitis pigmentosa due to RP1 mutations show greater severity inrecessive than in dominant casesrdquo Journal of Clinical amp Experi-mental Ophthalmology vol 2 article 194 2011

[18] M Al-Rashed L Abu Safieh H Alkuraya et al ldquoRP1 and reti-nitis pigmentosa report of novel mutations and insight intomutational mechanismrdquo British Journal of Ophthalmology vol96 no 7 pp 1018ndash1022 2012

[19] S A Riazuddin A Shahzadi C Zeitz et al ldquoA mutation inSLC24A1 implicated in autosomal-recessive congenital station-ary night blindnessrdquoThe American Journal of Human Geneticsvol 87 no 4 pp 523ndash531 2010

[20] H P Singh S Jalali R Narayanan and C Kannabiran ldquoGe-netic analysis of indian families with autosomal recessiveretinitis pigmentosa by homozygosity screeningrdquo InvestigativeOphthalmology and Visual Science vol 50 no 9 pp 4065ndash40712009

[21] Q Liu A Lyubarsky J H Skalet E N Pugh Jr and E A PierceldquoRP1 is required for the correct stacking of outer segment discsrdquoInvestigative Ophthalmology ampVisual Science vol 44 no 10 pp4171ndash4183 2003

[22] Q Liu J Zuo and E A Pierce ldquoThe retinitis pigmentosa 1protein is a photoreceptor microtubule-associated proteinrdquoJournal of Neuroscience vol 24 no 29 pp 6427ndash6436 2004

[23] I Audo K M Bujakowska T Leveillard et al ldquoDevelop-ment and application of a next-generation-sequencing (NGS)approach to detect known and novel gene defects underlyingretinal diseasesrdquo Orphanet Journal of Rare Diseases vol 7 no 1article 8 2012

[24] I Audo J A Sahel S Mohand-Saıd et al ldquoEYS is a major genefor rod-cone dystrophies in Francerdquo Human Mutation vol 31no 5 pp E1406ndashE1435 2010

[25] T G P Consortium ldquoA map of human genome variation frompopulation-scale sequencingrdquo Nature vol 467 no 7319 pp1061ndash1073 2010

[26] DMAltshuler RAGibbs L Peltonen et al ldquoIntegrating com-mon and rare genetic variation in diverse human populationsrdquoNature vol 467 no 7311 pp 52ndash58 2010

[27] J A Tennessen AW Bigham T D OrsquoConnor et al ldquoEvolutionand functional impact of rare coding variation from deepsequencing of human exomesrdquo Science vol 337 no 6090 pp64ndash69 2012

[28] M J Bamshad S BNgAW Bighamet al ldquoExome sequencingas a tool for Mendelian disease gene discoveryrdquo Nature ReviewsGenetics vol 12 no 11 pp 745ndash755 2011

[29] W J Kent C W Sugnet T S Furey et al ldquoThe human genomebrowser at UCSCrdquo Genome Research vol 12 no 6 pp 996ndash1006 2002

[30] I A Adzhubei S Schmidt L Peshkin et al ldquoA method andserver for predicting damaging missense mutationsrdquo NatureMethods vol 7 no 4 pp 248ndash249 2010

[31] P Kumar S Henikoff and P C Ng ldquoPredicting the effects ofcoding non-synonymous variants on protein function using the

SIFT algorithmrdquo Nature Protocols vol 4 no 7 pp 1073ndash10822009

[32] F O Desmet D Hamroun M Lalande G Collod-Beroud MClaustres and C Beroud ldquoHuman splicing finder an onlinebioinformatics tool to predict splicing signalsrdquo Nucleic AcidsResearch vol 37 no 9 article no e67 2009

[33] P D Stenson E V Ball M Mort A D Phillips K Shaw andD N Cooper ldquoThe Human Gene Mutation Database (HGMD)and its exploitation in the fields of personalized genomics andmolecular evolutionrdquo in Current Protocols in Bioinformaticschapter 1ndash13 2012

[34] I F A C Fokkema J T Den Dunnen and P E M TaschnerldquoLOVD easy creation of a locus-specific sequence variationdatabase using an ldquoLSDB-in-a-Boxrdquo approachrdquo Human Muta-tion vol 26 no 2 pp 63ndash68 2005

[35] S A Miller D D Dykes and H F Polesky ldquoA simple saltingout procedure for extracting DNA from human nucleated cellsrdquoNucleic Acids Research vol 16 no 3 p 1215 1988

[36] C Zeitz B Kloeckener-Gruissem U Forster et al ldquoMutationsin CABP4 the gene encoding the Ca2+-binding protein 4cause autosomal recessive night blindnessrdquoAmerican Journal ofHuman Genetics vol 79 no 4 pp 657ndash667 2006

[37] S A Riazuddin F Zulfiqar Q Zhang et al ldquoAutosomal reces-sive retinitis pigmentosa is associated with mutations in RP1 inthree consanguineous Pakistani familiesrdquo Investigative Ophthal-mology and Visual Science vol 46 no 7 pp 2264ndash2270 2005

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

BioMed Research International 7

(a)

(b) (c)

(d)

(e) (f)

Figure 1 Ophthalmic features of family F752 II1 (CIC01245) fundus color photographs ((a) and (d) for right and left eye resp)autofluorescence ((b) and (e) for right and left eye resp) and spectral domain optical coherence tomography horizontal macula scans ((c)and (f) for right and left eye resp) showing severe rod-cone dystrophy signs with macular involvement

last exon (exon 4) and shown to be transmitted in an auto-somal dominant mode of inheritance Most of RP1 diseasecausing variants represent nonsense mutations deletions orinsertions In mammalian genes nonsense mutations lead tounstable mRNAs that are degraded by nonsense-mediated

decay (NMD)However exceptionsmight arise when prema-ture stop codons occur in the last exon [37]These variants arethought to abolish RP1 function by resulting in a truncatedprotein lacking important functional domains although stillable to interact with some of its protein partner(s) [21] The

8 BioMed Research International

0 2 4 6 8 10 12 14 16 18 20 22

87888990919293949596979899100

Chromosome

Cov

erag

e at d

epth

25

X (

)

F303 CIC00445F335 CIC00491F674 CIC01106F752 CIC01245

F782 CIC01300

F3110 CIC05941F1941 CIC04130

(a)

0 2 4 6 8 10 12 14 16 18 20 22

Chromosome

100

150

200

250

300

350

400

Mea

n de

pth

per b

ase

F303 CIC00445F335 CIC00491F674 CIC01106 F3110 CIC05941F752 CIC01245

F782 CIC01300F1941 CIC04130

(b)

Figure 2 Sequencing statistics in index patients (a)The overall sequencing coverage of the target regions at 25X depth of coverage is shownin each of the chromosomes No values were indicated for chromosomes 13 18 21 and 22 as they were not targeted The term chromosome23 was used to designate the X chromosome F1941 III1 (CIC04130) and F3110 III5 (CIC05941) showed the lowest coverage results (b) Theaverage mean depth per base pair is shown for each of the chromosomes Most targets showed coverage around 300 times

1 1 1

1 2 3 4 5 6

2 3 4

4

5 632

2

1

1

1 2 3

4

5

11

2 21

2 2

3

2

3

6 7 8

4

41 5

(I)

(II)

(III)

(a) CIC00445 II1 F303 (b) CIC00491 III1 F335

(f) CIC04130 III1 F1941 (g) CIC05941 III5 F3110

1 2 3 4

1

1

1

2

2

3

3

4

4

5

5

6 7 8 9 10 65

43

21

1

1

2 3 4 5

432

2

1 2

2 3 4 5 61

1

(I)

(II)

(III)

(IV)

M1 c1625CgtGpSer542lowastM2 c4587 4590delTAAG pSer1529Argfslowast9

[M1][M2]

M3 c4089 4092delAAGApArg1364Valfslowast8

[M3][M3]

M4 c1205delG pGly402Alafslowast7

[M4][M4]

M5 c2025dupA pSer676Ilefslowast22M6 c2377delA pArg793Glufslowast55

[M5][M6]

M7 c1719 1723delCTCAA pSer574Cysfslowast7 M8 c1329delG pLys443Asnfslowast12

[M8][M8]

[M7][M7]

[=][=]

M9 c2391 2392delAA pAsp799lowast

[M9][M9]

[M9][=]

(c) CIC01106 III6 F674 (d) CIC01245 II1 F752

(e) CIC01300 II5 F782

Figure 3 Pedigrees of seven families with RP1 mutations underlying autosomal recessive rod-cone dystrophy Affected and unaffectedindividuals are represented by shapes filled with black and white colors respectively Men and women are indicated by squares and circlesrespectively Index subjects are marked by Consanguinity is marked by a double horizontal line

BioMed Research International 9

4

adRCD

arRCD

Class I

Class II (hot spot)

Class III Class III Class IV

321 BIF

pA

sp202

Glu

pThr373

Ile

pA

rg1519

Glu

fsX2

pA

rg1652

Leu

pCy

s2033

Tyr

pA

la669

Thr

pT8

65

Leu8

66

delIn

s

pA

sp984

Gly

pSe

r152

9Arg

fslowast9

pA

rg15

19G

lufslowast

2

pPr

o164

8Ser

fslowast2

pA

sn17

51Le

ufslowast4

pA

sn94

9Lys

fslowast32

pSe

r542lowast

pSe

r574

Cysfs

lowast7

pSe

r676

Ilefslowast

22

pA

rg79

3Glu

fslowast55

pA

sp79

9lowast

pM

et50

0Ser

fslowast33

pPr

o658

Hisf

slowast4

pLy

s673

Arg

fslowast9

pLy

s675

Asn

fslowast7

pA

rg67

7lowast

pA

rg67

7Asp

fslowast5

pG

ln67

9lowastp

Tyr6

85lowast

pG

ln68

6lowastp

Gln

689lowast

pG

lu70

0lowastp

Gly

706V

alfslowast7

pLy

s722

Glu

fslowast16

pG

ly72

3lowastp

Gly

724G

lufslowast

9

pile

725A

rgfslowast

6

pIle

725Th

rfslowast13

pIle

725A

spfslowast

4

pG

lu72

9Lys

fslowast9

pThr7

36Ile

fslowast4

pCy

s744

lowast

pSe

r747

Valfs

lowast16

pA

sn74

8Ile

fslowast15

pA

rg75

9lowastp

Leu7

62

Asn763

pL

eu762

Tyrfs

lowast17

pA

sn76

3Leu

fslowast11

pA

sn76

9Arg

fslowast6

pLy

s778

lowast

pSe

r779

lowast

pSe

r862

lowast

pIle

864L

ysfslowast

11

pTy

r105

3Thrfs

lowast4

pSe

r2A

rgfslowast16

pH

is31A

rgfslowast

9

pVa

l157

Trpf

slowast16

pA

la22

1Gly

fslowast43

pPr

o229

Gln

fslowast35

pA

rg33

8lowast

pA

rg39

6lowastp

Gly

402A

lafslowast7

pLy

s443

Asn

fslowast12

pG

lu48

8lowast

pTr

p113

1lowast

pG

ly11

40ly

sfslowast4

pA

sn11

43Ile

fslowast25

pG

lu12

27M

etfslowast

29

pA

rg13

64Va

lfslowast8

pLy

s151

8lowast

pA

rg87

2Thrfs

lowast2

pSe

r911

lowast

Figure 4 Schematic presentation of RP1 disease causing mutations Disease causing mutations were represented based on the classificationby Chen and coworkers [13] Mutations responsible for recessive arRCD were shown in the upper half whereas mutations causing adRCDwere shown in the lower half pGly402Alafslowast7 pLys443Asnfslowast12 pArg1364Valfslowast8 and pSer1529Argfslowast9 belong to class III AlthoughpSer574Cysfslowast7 pSer676Ilefslowast22 pArg793Glufslowast55 and pAsp799lowast are class II mutations these variants do not cause adRCD but arRCDinstead Amino acid modifications shown in red and blue represent novel frameshift or nonsense mutations and the recurrent pSer542lowastmutation respectively Protein localization of pSer542lowast was highlighted in blue as it marked a recurrent mutation adRCD autosomaldominant rod-cone dystrophy arRCD autosomal recessive rod-cone dystrophy BIF drosophila melanogaster bifocal

latter observation is supported by finding that RP1 mutantmRNA is expressed in a human cell line carrying a homozy-gous pArg677lowast mutation [21]

Based on Chen et al [13] RP1 truncating mutations lead-ing to arRCD or adRCD can be divided into four distinctgroups Class I is composed of truncating mutations locatedin exons 2 and 3These variants are sensitive toNMDand thusare considered as true loss-of-function alleles (Figure 4) [13]Class II involves truncating mutations that are located in aspot between codons 500 and 1053 in exon 4 [13] the so calledldquoRP1 hot spotrdquo The ldquohot spotrdquo variants tend to be insensitiveto NMD process and thus result in a protein with a potentialdominant negative effect leading to adRCD (Figure 4) [13]Class III includes truncating mutations insensible to NMDlocated between codons 264 and 499 and between codons1054 to 1751 in exon 4 These truncating proteins result in aloss of function leading to arRCD (Figure 4) [13] Finally classIV includes protein-truncating mutations near the 31015840 endof the fourth exon (Figure 4) [13] Most likely the resultingproteins display only aminor loss of their C-terminal portionpreserving the majority of functional domains and keeping aresidual activity According to the classification of Chen et al[13] pGly402Alafslowast7 pLys443Asnfslowast12 pArg1364Valfslowast8and pSer1529Argfslowast9 belong to class III (Figure 4)

The predicted physiopathology for pSer542lowastpSer574Cysfslowast7 pSer676Ilefslowast22 pArg793Glufslowast55 andpAsp799lowast is more complex According to Chenrsquos classi-fication these frameshift deletions and nonsense mutationsshould belong to class II previously only associated withadRCD However herein they are causing presumablyarRCD (Figure 4) To further confirm these findings clinicaland genetic testing of the reported unaffected parents shouldbe done

Based on the previous findings we speculate that the clas-sification by Chen and coworkers does not hold true for allmutations Supporting this statement Avila-Fernandez et al[12] reported the same nonsense mutation (pSer542lowast) foundin (F303 II1 (CIC00445)) and located at the 51015840 end of theldquohot spotrdquo to cause arRP in the Spanish population [12]These observations are of interest as they point out for animplication of hot spot region for adRCD-RP1mutation alsoin case of arRCD Future studies will need to clarify why someclass II mutations lead to adRCD and others to arRCD

Patients with arRCD and RP1 mutations show a moresevere disease than adRCD-RP1 mutant patients with mac-ular atrophy in all our cases This was first outlined byLafont et al [17] When patients are presenting with latesevere disease the diagnostic distinction between RCD with

10 BioMed Research International

initial rod involvement and cone-rod dystrophy (CRD) withinitial cone involvement is difficult Of note is that one ofthe patients (CIC01300) in the present study was initiallyclassified as possibly having severe CRD and his diagnosiswas actually revisited after NGS resultsThis also outlines theinterest of unbiased massive parallel sequencing methods fora more precise clinical diagnostic in case of end stage diseaseThis point will most likely become evenmore critical with theperspective of therapeutic trials

41 Strength and Limitations We estimate that 1 of ourtarget regions were not covered Partially uncovered exonsare a real common issue when capturing the DNA sequencesusing commercially available probes this bias might implya loss of some candidate variants However we found thatrate of 1 is very reasonable when compared with otherNGS panels In addition in order to exclude the possibilityof finding other candidate variants we have sequenced bySanger method the majority of these regions Five of ourpatients carried homozygous RP1 mutations For four of thesubjects carrying homozygous variants namely CIC00491F335 CIC01106 F674 CIC01300 F782 and CIC04130 F1941co-segregation analysis needs to be done to confirm auto-somal recessive inheritance but we do not have access toparentrsquos DNA CIC05941 was the only one not to reportclear consanguinity in the family and we cannot exclude thepossibility of a large deletion on the second allele of RP1gene Again DNA of the father not available for us wouldbe helpful to prove autosomal recessive inheritance and thehomozygous state of the mutation

In conclusion we have reported 9 mutations in RP1 ofwhich 8 were novel causing arRCD [8 12ndash20] Interestingly aprevalence ofasymp25points out for the necessity of sequencingRP1 in sporadic and recessive cases of RCD Further func-tional studies are needed to understand the impact of RP1structure on its function at the molecular level such a stepwould strengthen our knowledge in the physiology of retinalphotoreceptors

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Isabelle Audo and Christina Zeitz contributed equally to thiswork

Acknowledgments

The authors express their sincere gratitude to the familieswho participated in this study and to the clinical staff fortheir help in clinical data and DNA collection This workwas supported by Foundation Voir et Entendre (CZ) PrixDalloz for ldquola recherche en ophtalmologierdquo (CZ) FoundationFighting Blindness (FFB) [CD-CL-0808-0466-CHNO] (IA)and FFB center (FFB grantC-GE-0912-0601-INSERM02)

Prix de la Fondation de lrsquoŒil (IA) Ville de Paris and RegionIle de France and the French State program ldquoInvestissementsdrsquoAvenirrdquo managed by the Agence Nationale de la Recherche[LIFESENSES ANR-10-LABX-65]

References

[1] D T Hartong E L Berson and T P Dryja ldquoRetinitis pigmen-tosardquoThe Lancet vol 368 no 9549 pp 1795ndash1809 2006

[2] I Audo M Lancelot S Mohand-Saıd et al ldquoNovel C2orf71mutations account for approximately sim1 of cases in a largeFrench arRP cohortrdquoHumanMutation vol 32 no 4 pp E2091ndashE2103 2011

[3] A Anasagasti C Irigoyen O Barandika A Lopez de Munainand J Ruiz-Ederra ldquoCurrent mutation discovery approaches inRetinitis PigmentosardquoVision Research vol 75 pp 117ndash129 2012

[4] L S Sullivan S J Bowne D G Birch et al ldquoPrevalence ofdisease-causingmutations in families with autosomal dominantretinitis pigmentosa a screen of known genes in 200 familiesrdquoInvestigative Ophthalmology andVisual Science vol 47 no 7 pp3052ndash3064 2006

[5] B J Seyedahmadi C Rivolta J A Keene E L Berson andT P Dryja ldquoComprehensive screening of the USH2A gene inUsher syndrome type II and non-syndromic recessive retinitispigmentosardquo Experimental Eye Research vol 79 no 2 pp 167ndash173 2004

[6] A Avila-Fernandez D Cantalapiedra E Aller et al ldquoMutationanalysis of 272 Spanish families affected by autosomal recessiveretinitis pigmentosa using a genotyping microarrayrdquoMolecularVision vol 16 pp 2550ndash2558 2010

[7] S El Shamieh M Neuille A Terray et al ldquoWhole-exomesequencing identifies KIZ as a ciliary gene associated withautosomal-recessive rod-cone dystrophyrdquo American Journal ofHuman Genetics vol 94 no 4 pp 625ndash633 2014

[8] I Audo S Mohand-Saıd C M Dhaenens et al ldquoRP1 andautosomal dominant rod-cone dystrophy novel mutations areview of published variants and genotype-phenotype correla-tionrdquo Human Mutation vol 33 no 1 pp 73ndash80 2012

[9] X Guillonneau N I Piriev M Danciger et al ldquoA nonsensemutation in a novel gene is associated with retinitis pigmentosain a family linked to the RP1 locusrdquoHumanMolecular Geneticsvol 8 no 8 pp 1541ndash1546 1999

[10] E A Pierce T Quinn T Meehan T L McGee E L Bersonand T P Dryja ldquoMutations in a gene encoding a new oxygen-regulated photoreceptor protein cause dominant retinitis pig-mentosardquo Nature Genetics vol 22 no 3 pp 248ndash254 1999

[11] L S Sullivan J R Heckenlively S J Bowne et al ldquoMutations ina novel retina-specific gene cause autosomal dominant retinitispigmentosardquo Nature Genetics vol 22 no 3 pp 255ndash259 1999

[12] A Avila-Fernandez M Corton K M Nishiguchi et al ldquoIden-tification of an RP1 prevalent founder mutation and relatedphenotype in Spanish patients with early-onset autosomalrecessive retinitisrdquo Ophthalmology vol 119 no 12 pp 2616ndash2621 2012

[13] L J Chen T Y Y Lai P O S Tam et al ldquoCompound heter-ozygosity of two novel truncation mutations in RP1 causingautosomal recessive retinitis pigmentosardquo InvestigativeOphthal-mology and Visual Science vol 51 no 4 pp 2236ndash2242 2010

[14] S Khaliq A Abid M Ismail et al ldquoNovel association of RP1gene mutations with autosomal recessive retinitis pigmentosardquoJournal of Medical Genetics vol 42 no 5 pp 436ndash438 2005

BioMed Research International 11

[15] A M Siemiatkowska G D N Astuti K Arimadyo et alldquoIdentification of a novel nonsense mutation in RP1 that causesautosomal recessive retinitis pigmentosa in an IndonesianfamilyrdquoMolecular Vision vol 18 pp 2411ndash2419 2012

[16] B Bocquet N A Marzouka M Hebrard et al ldquoHomozygositymapping in autosomal recessive retinitis pigmentosa familiesdetects novel mutationsrdquo Molecular Vision vol 19 pp 2487ndash2500 2013

[17] E M Lafont G Manes G Senechal et al ldquoPatients with reti-nitis pigmentosa due to RP1 mutations show greater severity inrecessive than in dominant casesrdquo Journal of Clinical amp Experi-mental Ophthalmology vol 2 article 194 2011

[18] M Al-Rashed L Abu Safieh H Alkuraya et al ldquoRP1 and reti-nitis pigmentosa report of novel mutations and insight intomutational mechanismrdquo British Journal of Ophthalmology vol96 no 7 pp 1018ndash1022 2012

[19] S A Riazuddin A Shahzadi C Zeitz et al ldquoA mutation inSLC24A1 implicated in autosomal-recessive congenital station-ary night blindnessrdquoThe American Journal of Human Geneticsvol 87 no 4 pp 523ndash531 2010

[20] H P Singh S Jalali R Narayanan and C Kannabiran ldquoGe-netic analysis of indian families with autosomal recessiveretinitis pigmentosa by homozygosity screeningrdquo InvestigativeOphthalmology and Visual Science vol 50 no 9 pp 4065ndash40712009

[21] Q Liu A Lyubarsky J H Skalet E N Pugh Jr and E A PierceldquoRP1 is required for the correct stacking of outer segment discsrdquoInvestigative Ophthalmology ampVisual Science vol 44 no 10 pp4171ndash4183 2003

[22] Q Liu J Zuo and E A Pierce ldquoThe retinitis pigmentosa 1protein is a photoreceptor microtubule-associated proteinrdquoJournal of Neuroscience vol 24 no 29 pp 6427ndash6436 2004

[23] I Audo K M Bujakowska T Leveillard et al ldquoDevelop-ment and application of a next-generation-sequencing (NGS)approach to detect known and novel gene defects underlyingretinal diseasesrdquo Orphanet Journal of Rare Diseases vol 7 no 1article 8 2012

[24] I Audo J A Sahel S Mohand-Saıd et al ldquoEYS is a major genefor rod-cone dystrophies in Francerdquo Human Mutation vol 31no 5 pp E1406ndashE1435 2010

[25] T G P Consortium ldquoA map of human genome variation frompopulation-scale sequencingrdquo Nature vol 467 no 7319 pp1061ndash1073 2010

[26] DMAltshuler RAGibbs L Peltonen et al ldquoIntegrating com-mon and rare genetic variation in diverse human populationsrdquoNature vol 467 no 7311 pp 52ndash58 2010

[27] J A Tennessen AW Bigham T D OrsquoConnor et al ldquoEvolutionand functional impact of rare coding variation from deepsequencing of human exomesrdquo Science vol 337 no 6090 pp64ndash69 2012

[28] M J Bamshad S BNgAW Bighamet al ldquoExome sequencingas a tool for Mendelian disease gene discoveryrdquo Nature ReviewsGenetics vol 12 no 11 pp 745ndash755 2011

[29] W J Kent C W Sugnet T S Furey et al ldquoThe human genomebrowser at UCSCrdquo Genome Research vol 12 no 6 pp 996ndash1006 2002

[30] I A Adzhubei S Schmidt L Peshkin et al ldquoA method andserver for predicting damaging missense mutationsrdquo NatureMethods vol 7 no 4 pp 248ndash249 2010

[31] P Kumar S Henikoff and P C Ng ldquoPredicting the effects ofcoding non-synonymous variants on protein function using the

SIFT algorithmrdquo Nature Protocols vol 4 no 7 pp 1073ndash10822009

[32] F O Desmet D Hamroun M Lalande G Collod-Beroud MClaustres and C Beroud ldquoHuman splicing finder an onlinebioinformatics tool to predict splicing signalsrdquo Nucleic AcidsResearch vol 37 no 9 article no e67 2009

[33] P D Stenson E V Ball M Mort A D Phillips K Shaw andD N Cooper ldquoThe Human Gene Mutation Database (HGMD)and its exploitation in the fields of personalized genomics andmolecular evolutionrdquo in Current Protocols in Bioinformaticschapter 1ndash13 2012

[34] I F A C Fokkema J T Den Dunnen and P E M TaschnerldquoLOVD easy creation of a locus-specific sequence variationdatabase using an ldquoLSDB-in-a-Boxrdquo approachrdquo Human Muta-tion vol 26 no 2 pp 63ndash68 2005

[35] S A Miller D D Dykes and H F Polesky ldquoA simple saltingout procedure for extracting DNA from human nucleated cellsrdquoNucleic Acids Research vol 16 no 3 p 1215 1988

[36] C Zeitz B Kloeckener-Gruissem U Forster et al ldquoMutationsin CABP4 the gene encoding the Ca2+-binding protein 4cause autosomal recessive night blindnessrdquoAmerican Journal ofHuman Genetics vol 79 no 4 pp 657ndash667 2006

[37] S A Riazuddin F Zulfiqar Q Zhang et al ldquoAutosomal reces-sive retinitis pigmentosa is associated with mutations in RP1 inthree consanguineous Pakistani familiesrdquo Investigative Ophthal-mology and Visual Science vol 46 no 7 pp 2264ndash2270 2005

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

8 BioMed Research International

0 2 4 6 8 10 12 14 16 18 20 22

87888990919293949596979899100

Chromosome

Cov

erag

e at d

epth

25

X (

)

F303 CIC00445F335 CIC00491F674 CIC01106F752 CIC01245

F782 CIC01300

F3110 CIC05941F1941 CIC04130

(a)

0 2 4 6 8 10 12 14 16 18 20 22

Chromosome

100

150

200

250

300

350

400

Mea

n de

pth

per b

ase

F303 CIC00445F335 CIC00491F674 CIC01106 F3110 CIC05941F752 CIC01245

F782 CIC01300F1941 CIC04130

(b)

Figure 2 Sequencing statistics in index patients (a)The overall sequencing coverage of the target regions at 25X depth of coverage is shownin each of the chromosomes No values were indicated for chromosomes 13 18 21 and 22 as they were not targeted The term chromosome23 was used to designate the X chromosome F1941 III1 (CIC04130) and F3110 III5 (CIC05941) showed the lowest coverage results (b) Theaverage mean depth per base pair is shown for each of the chromosomes Most targets showed coverage around 300 times

1 1 1

1 2 3 4 5 6

2 3 4

4

5 632

2

1

1

1 2 3

4

5

11

2 21

2 2

3

2

3

6 7 8

4

41 5

(I)

(II)

(III)

(a) CIC00445 II1 F303 (b) CIC00491 III1 F335

(f) CIC04130 III1 F1941 (g) CIC05941 III5 F3110

1 2 3 4

1

1

1

2

2

3

3

4

4

5

5

6 7 8 9 10 65

43

21

1

1

2 3 4 5

432

2

1 2

2 3 4 5 61

1

(I)

(II)

(III)

(IV)

M1 c1625CgtGpSer542lowastM2 c4587 4590delTAAG pSer1529Argfslowast9

[M1][M2]

M3 c4089 4092delAAGApArg1364Valfslowast8

[M3][M3]

M4 c1205delG pGly402Alafslowast7

[M4][M4]

M5 c2025dupA pSer676Ilefslowast22M6 c2377delA pArg793Glufslowast55

[M5][M6]

M7 c1719 1723delCTCAA pSer574Cysfslowast7 M8 c1329delG pLys443Asnfslowast12

[M8][M8]

[M7][M7]

[=][=]

M9 c2391 2392delAA pAsp799lowast

[M9][M9]

[M9][=]

(c) CIC01106 III6 F674 (d) CIC01245 II1 F752

(e) CIC01300 II5 F782

Figure 3 Pedigrees of seven families with RP1 mutations underlying autosomal recessive rod-cone dystrophy Affected and unaffectedindividuals are represented by shapes filled with black and white colors respectively Men and women are indicated by squares and circlesrespectively Index subjects are marked by Consanguinity is marked by a double horizontal line

BioMed Research International 9

4

adRCD

arRCD

Class I

Class II (hot spot)

Class III Class III Class IV

321 BIF

pA

sp202

Glu

pThr373

Ile

pA

rg1519

Glu

fsX2

pA

rg1652

Leu

pCy

s2033

Tyr

pA

la669

Thr

pT8

65

Leu8

66

delIn

s

pA

sp984

Gly

pSe

r152

9Arg

fslowast9

pA

rg15

19G

lufslowast

2

pPr

o164

8Ser

fslowast2

pA

sn17

51Le

ufslowast4

pA

sn94

9Lys

fslowast32

pSe

r542lowast

pSe

r574

Cysfs

lowast7

pSe

r676

Ilefslowast

22

pA

rg79

3Glu

fslowast55

pA

sp79

9lowast

pM

et50

0Ser

fslowast33

pPr

o658

Hisf

slowast4

pLy

s673

Arg

fslowast9

pLy

s675

Asn

fslowast7

pA

rg67

7lowast

pA

rg67

7Asp

fslowast5

pG

ln67

9lowastp

Tyr6

85lowast

pG

ln68

6lowastp

Gln

689lowast

pG

lu70

0lowastp

Gly

706V

alfslowast7

pLy

s722

Glu

fslowast16

pG

ly72

3lowastp

Gly

724G

lufslowast

9

pile

725A

rgfslowast

6

pIle

725Th

rfslowast13

pIle

725A

spfslowast

4

pG

lu72

9Lys

fslowast9

pThr7

36Ile

fslowast4

pCy

s744

lowast

pSe

r747

Valfs

lowast16

pA

sn74

8Ile

fslowast15

pA

rg75

9lowastp

Leu7

62

Asn763

pL

eu762

Tyrfs

lowast17

pA

sn76

3Leu

fslowast11

pA

sn76

9Arg

fslowast6

pLy

s778

lowast

pSe

r779

lowast

pSe

r862

lowast

pIle

864L

ysfslowast

11

pTy

r105

3Thrfs

lowast4

pSe

r2A

rgfslowast16

pH

is31A

rgfslowast

9

pVa

l157

Trpf

slowast16

pA

la22

1Gly

fslowast43

pPr

o229

Gln

fslowast35

pA

rg33

8lowast

pA

rg39

6lowastp

Gly

402A

lafslowast7

pLy

s443

Asn

fslowast12

pG

lu48

8lowast

pTr

p113

1lowast

pG

ly11

40ly

sfslowast4

pA

sn11

43Ile

fslowast25

pG

lu12

27M

etfslowast

29

pA

rg13

64Va

lfslowast8

pLy

s151

8lowast

pA

rg87

2Thrfs

lowast2

pSe

r911

lowast

Figure 4 Schematic presentation of RP1 disease causing mutations Disease causing mutations were represented based on the classificationby Chen and coworkers [13] Mutations responsible for recessive arRCD were shown in the upper half whereas mutations causing adRCDwere shown in the lower half pGly402Alafslowast7 pLys443Asnfslowast12 pArg1364Valfslowast8 and pSer1529Argfslowast9 belong to class III AlthoughpSer574Cysfslowast7 pSer676Ilefslowast22 pArg793Glufslowast55 and pAsp799lowast are class II mutations these variants do not cause adRCD but arRCDinstead Amino acid modifications shown in red and blue represent novel frameshift or nonsense mutations and the recurrent pSer542lowastmutation respectively Protein localization of pSer542lowast was highlighted in blue as it marked a recurrent mutation adRCD autosomaldominant rod-cone dystrophy arRCD autosomal recessive rod-cone dystrophy BIF drosophila melanogaster bifocal

latter observation is supported by finding that RP1 mutantmRNA is expressed in a human cell line carrying a homozy-gous pArg677lowast mutation [21]

Based on Chen et al [13] RP1 truncating mutations lead-ing to arRCD or adRCD can be divided into four distinctgroups Class I is composed of truncating mutations locatedin exons 2 and 3These variants are sensitive toNMDand thusare considered as true loss-of-function alleles (Figure 4) [13]Class II involves truncating mutations that are located in aspot between codons 500 and 1053 in exon 4 [13] the so calledldquoRP1 hot spotrdquo The ldquohot spotrdquo variants tend to be insensitiveto NMD process and thus result in a protein with a potentialdominant negative effect leading to adRCD (Figure 4) [13]Class III includes truncating mutations insensible to NMDlocated between codons 264 and 499 and between codons1054 to 1751 in exon 4 These truncating proteins result in aloss of function leading to arRCD (Figure 4) [13] Finally classIV includes protein-truncating mutations near the 31015840 endof the fourth exon (Figure 4) [13] Most likely the resultingproteins display only aminor loss of their C-terminal portionpreserving the majority of functional domains and keeping aresidual activity According to the classification of Chen et al[13] pGly402Alafslowast7 pLys443Asnfslowast12 pArg1364Valfslowast8and pSer1529Argfslowast9 belong to class III (Figure 4)

The predicted physiopathology for pSer542lowastpSer574Cysfslowast7 pSer676Ilefslowast22 pArg793Glufslowast55 andpAsp799lowast is more complex According to Chenrsquos classi-fication these frameshift deletions and nonsense mutationsshould belong to class II previously only associated withadRCD However herein they are causing presumablyarRCD (Figure 4) To further confirm these findings clinicaland genetic testing of the reported unaffected parents shouldbe done

Based on the previous findings we speculate that the clas-sification by Chen and coworkers does not hold true for allmutations Supporting this statement Avila-Fernandez et al[12] reported the same nonsense mutation (pSer542lowast) foundin (F303 II1 (CIC00445)) and located at the 51015840 end of theldquohot spotrdquo to cause arRP in the Spanish population [12]These observations are of interest as they point out for animplication of hot spot region for adRCD-RP1mutation alsoin case of arRCD Future studies will need to clarify why someclass II mutations lead to adRCD and others to arRCD

Patients with arRCD and RP1 mutations show a moresevere disease than adRCD-RP1 mutant patients with mac-ular atrophy in all our cases This was first outlined byLafont et al [17] When patients are presenting with latesevere disease the diagnostic distinction between RCD with

10 BioMed Research International

initial rod involvement and cone-rod dystrophy (CRD) withinitial cone involvement is difficult Of note is that one ofthe patients (CIC01300) in the present study was initiallyclassified as possibly having severe CRD and his diagnosiswas actually revisited after NGS resultsThis also outlines theinterest of unbiased massive parallel sequencing methods fora more precise clinical diagnostic in case of end stage diseaseThis point will most likely become evenmore critical with theperspective of therapeutic trials

41 Strength and Limitations We estimate that 1 of ourtarget regions were not covered Partially uncovered exonsare a real common issue when capturing the DNA sequencesusing commercially available probes this bias might implya loss of some candidate variants However we found thatrate of 1 is very reasonable when compared with otherNGS panels In addition in order to exclude the possibilityof finding other candidate variants we have sequenced bySanger method the majority of these regions Five of ourpatients carried homozygous RP1 mutations For four of thesubjects carrying homozygous variants namely CIC00491F335 CIC01106 F674 CIC01300 F782 and CIC04130 F1941co-segregation analysis needs to be done to confirm auto-somal recessive inheritance but we do not have access toparentrsquos DNA CIC05941 was the only one not to reportclear consanguinity in the family and we cannot exclude thepossibility of a large deletion on the second allele of RP1gene Again DNA of the father not available for us wouldbe helpful to prove autosomal recessive inheritance and thehomozygous state of the mutation

In conclusion we have reported 9 mutations in RP1 ofwhich 8 were novel causing arRCD [8 12ndash20] Interestingly aprevalence ofasymp25points out for the necessity of sequencingRP1 in sporadic and recessive cases of RCD Further func-tional studies are needed to understand the impact of RP1structure on its function at the molecular level such a stepwould strengthen our knowledge in the physiology of retinalphotoreceptors

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Isabelle Audo and Christina Zeitz contributed equally to thiswork

Acknowledgments

The authors express their sincere gratitude to the familieswho participated in this study and to the clinical staff fortheir help in clinical data and DNA collection This workwas supported by Foundation Voir et Entendre (CZ) PrixDalloz for ldquola recherche en ophtalmologierdquo (CZ) FoundationFighting Blindness (FFB) [CD-CL-0808-0466-CHNO] (IA)and FFB center (FFB grantC-GE-0912-0601-INSERM02)

Prix de la Fondation de lrsquoŒil (IA) Ville de Paris and RegionIle de France and the French State program ldquoInvestissementsdrsquoAvenirrdquo managed by the Agence Nationale de la Recherche[LIFESENSES ANR-10-LABX-65]

References

[1] D T Hartong E L Berson and T P Dryja ldquoRetinitis pigmen-tosardquoThe Lancet vol 368 no 9549 pp 1795ndash1809 2006

[2] I Audo M Lancelot S Mohand-Saıd et al ldquoNovel C2orf71mutations account for approximately sim1 of cases in a largeFrench arRP cohortrdquoHumanMutation vol 32 no 4 pp E2091ndashE2103 2011

[3] A Anasagasti C Irigoyen O Barandika A Lopez de Munainand J Ruiz-Ederra ldquoCurrent mutation discovery approaches inRetinitis PigmentosardquoVision Research vol 75 pp 117ndash129 2012

[4] L S Sullivan S J Bowne D G Birch et al ldquoPrevalence ofdisease-causingmutations in families with autosomal dominantretinitis pigmentosa a screen of known genes in 200 familiesrdquoInvestigative Ophthalmology andVisual Science vol 47 no 7 pp3052ndash3064 2006

[5] B J Seyedahmadi C Rivolta J A Keene E L Berson andT P Dryja ldquoComprehensive screening of the USH2A gene inUsher syndrome type II and non-syndromic recessive retinitispigmentosardquo Experimental Eye Research vol 79 no 2 pp 167ndash173 2004

[6] A Avila-Fernandez D Cantalapiedra E Aller et al ldquoMutationanalysis of 272 Spanish families affected by autosomal recessiveretinitis pigmentosa using a genotyping microarrayrdquoMolecularVision vol 16 pp 2550ndash2558 2010

[7] S El Shamieh M Neuille A Terray et al ldquoWhole-exomesequencing identifies KIZ as a ciliary gene associated withautosomal-recessive rod-cone dystrophyrdquo American Journal ofHuman Genetics vol 94 no 4 pp 625ndash633 2014

[8] I Audo S Mohand-Saıd C M Dhaenens et al ldquoRP1 andautosomal dominant rod-cone dystrophy novel mutations areview of published variants and genotype-phenotype correla-tionrdquo Human Mutation vol 33 no 1 pp 73ndash80 2012

[9] X Guillonneau N I Piriev M Danciger et al ldquoA nonsensemutation in a novel gene is associated with retinitis pigmentosain a family linked to the RP1 locusrdquoHumanMolecular Geneticsvol 8 no 8 pp 1541ndash1546 1999

[10] E A Pierce T Quinn T Meehan T L McGee E L Bersonand T P Dryja ldquoMutations in a gene encoding a new oxygen-regulated photoreceptor protein cause dominant retinitis pig-mentosardquo Nature Genetics vol 22 no 3 pp 248ndash254 1999

[11] L S Sullivan J R Heckenlively S J Bowne et al ldquoMutations ina novel retina-specific gene cause autosomal dominant retinitispigmentosardquo Nature Genetics vol 22 no 3 pp 255ndash259 1999

[12] A Avila-Fernandez M Corton K M Nishiguchi et al ldquoIden-tification of an RP1 prevalent founder mutation and relatedphenotype in Spanish patients with early-onset autosomalrecessive retinitisrdquo Ophthalmology vol 119 no 12 pp 2616ndash2621 2012

[13] L J Chen T Y Y Lai P O S Tam et al ldquoCompound heter-ozygosity of two novel truncation mutations in RP1 causingautosomal recessive retinitis pigmentosardquo InvestigativeOphthal-mology and Visual Science vol 51 no 4 pp 2236ndash2242 2010

[14] S Khaliq A Abid M Ismail et al ldquoNovel association of RP1gene mutations with autosomal recessive retinitis pigmentosardquoJournal of Medical Genetics vol 42 no 5 pp 436ndash438 2005

BioMed Research International 11

[15] A M Siemiatkowska G D N Astuti K Arimadyo et alldquoIdentification of a novel nonsense mutation in RP1 that causesautosomal recessive retinitis pigmentosa in an IndonesianfamilyrdquoMolecular Vision vol 18 pp 2411ndash2419 2012

[16] B Bocquet N A Marzouka M Hebrard et al ldquoHomozygositymapping in autosomal recessive retinitis pigmentosa familiesdetects novel mutationsrdquo Molecular Vision vol 19 pp 2487ndash2500 2013

[17] E M Lafont G Manes G Senechal et al ldquoPatients with reti-nitis pigmentosa due to RP1 mutations show greater severity inrecessive than in dominant casesrdquo Journal of Clinical amp Experi-mental Ophthalmology vol 2 article 194 2011

[18] M Al-Rashed L Abu Safieh H Alkuraya et al ldquoRP1 and reti-nitis pigmentosa report of novel mutations and insight intomutational mechanismrdquo British Journal of Ophthalmology vol96 no 7 pp 1018ndash1022 2012

[19] S A Riazuddin A Shahzadi C Zeitz et al ldquoA mutation inSLC24A1 implicated in autosomal-recessive congenital station-ary night blindnessrdquoThe American Journal of Human Geneticsvol 87 no 4 pp 523ndash531 2010

[20] H P Singh S Jalali R Narayanan and C Kannabiran ldquoGe-netic analysis of indian families with autosomal recessiveretinitis pigmentosa by homozygosity screeningrdquo InvestigativeOphthalmology and Visual Science vol 50 no 9 pp 4065ndash40712009

[21] Q Liu A Lyubarsky J H Skalet E N Pugh Jr and E A PierceldquoRP1 is required for the correct stacking of outer segment discsrdquoInvestigative Ophthalmology ampVisual Science vol 44 no 10 pp4171ndash4183 2003

[22] Q Liu J Zuo and E A Pierce ldquoThe retinitis pigmentosa 1protein is a photoreceptor microtubule-associated proteinrdquoJournal of Neuroscience vol 24 no 29 pp 6427ndash6436 2004

[23] I Audo K M Bujakowska T Leveillard et al ldquoDevelop-ment and application of a next-generation-sequencing (NGS)approach to detect known and novel gene defects underlyingretinal diseasesrdquo Orphanet Journal of Rare Diseases vol 7 no 1article 8 2012

[24] I Audo J A Sahel S Mohand-Saıd et al ldquoEYS is a major genefor rod-cone dystrophies in Francerdquo Human Mutation vol 31no 5 pp E1406ndashE1435 2010

[25] T G P Consortium ldquoA map of human genome variation frompopulation-scale sequencingrdquo Nature vol 467 no 7319 pp1061ndash1073 2010

[26] DMAltshuler RAGibbs L Peltonen et al ldquoIntegrating com-mon and rare genetic variation in diverse human populationsrdquoNature vol 467 no 7311 pp 52ndash58 2010

[27] J A Tennessen AW Bigham T D OrsquoConnor et al ldquoEvolutionand functional impact of rare coding variation from deepsequencing of human exomesrdquo Science vol 337 no 6090 pp64ndash69 2012

[28] M J Bamshad S BNgAW Bighamet al ldquoExome sequencingas a tool for Mendelian disease gene discoveryrdquo Nature ReviewsGenetics vol 12 no 11 pp 745ndash755 2011

[29] W J Kent C W Sugnet T S Furey et al ldquoThe human genomebrowser at UCSCrdquo Genome Research vol 12 no 6 pp 996ndash1006 2002

[30] I A Adzhubei S Schmidt L Peshkin et al ldquoA method andserver for predicting damaging missense mutationsrdquo NatureMethods vol 7 no 4 pp 248ndash249 2010

[31] P Kumar S Henikoff and P C Ng ldquoPredicting the effects ofcoding non-synonymous variants on protein function using the

SIFT algorithmrdquo Nature Protocols vol 4 no 7 pp 1073ndash10822009

[32] F O Desmet D Hamroun M Lalande G Collod-Beroud MClaustres and C Beroud ldquoHuman splicing finder an onlinebioinformatics tool to predict splicing signalsrdquo Nucleic AcidsResearch vol 37 no 9 article no e67 2009

[33] P D Stenson E V Ball M Mort A D Phillips K Shaw andD N Cooper ldquoThe Human Gene Mutation Database (HGMD)and its exploitation in the fields of personalized genomics andmolecular evolutionrdquo in Current Protocols in Bioinformaticschapter 1ndash13 2012

[34] I F A C Fokkema J T Den Dunnen and P E M TaschnerldquoLOVD easy creation of a locus-specific sequence variationdatabase using an ldquoLSDB-in-a-Boxrdquo approachrdquo Human Muta-tion vol 26 no 2 pp 63ndash68 2005

[35] S A Miller D D Dykes and H F Polesky ldquoA simple saltingout procedure for extracting DNA from human nucleated cellsrdquoNucleic Acids Research vol 16 no 3 p 1215 1988

[36] C Zeitz B Kloeckener-Gruissem U Forster et al ldquoMutationsin CABP4 the gene encoding the Ca2+-binding protein 4cause autosomal recessive night blindnessrdquoAmerican Journal ofHuman Genetics vol 79 no 4 pp 657ndash667 2006

[37] S A Riazuddin F Zulfiqar Q Zhang et al ldquoAutosomal reces-sive retinitis pigmentosa is associated with mutations in RP1 inthree consanguineous Pakistani familiesrdquo Investigative Ophthal-mology and Visual Science vol 46 no 7 pp 2264ndash2270 2005

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

BioMed Research International 9

4

adRCD

arRCD

Class I

Class II (hot spot)

Class III Class III Class IV

321 BIF

pA

sp202

Glu

pThr373

Ile

pA

rg1519

Glu

fsX2

pA

rg1652

Leu

pCy

s2033

Tyr

pA

la669

Thr

pT8

65

Leu8

66

delIn

s

pA

sp984

Gly

pSe

r152

9Arg

fslowast9

pA

rg15

19G

lufslowast

2

pPr

o164

8Ser

fslowast2

pA

sn17

51Le

ufslowast4

pA

sn94

9Lys

fslowast32

pSe

r542lowast

pSe

r574

Cysfs

lowast7

pSe

r676

Ilefslowast

22

pA

rg79

3Glu

fslowast55

pA

sp79

9lowast

pM

et50

0Ser

fslowast33

pPr

o658

Hisf

slowast4

pLy

s673

Arg

fslowast9

pLy

s675

Asn

fslowast7

pA

rg67

7lowast

pA

rg67

7Asp

fslowast5

pG

ln67

9lowastp

Tyr6

85lowast

pG

ln68

6lowastp

Gln

689lowast

pG

lu70

0lowastp

Gly

706V

alfslowast7

pLy

s722

Glu

fslowast16

pG

ly72

3lowastp

Gly

724G

lufslowast

9

pile

725A

rgfslowast

6

pIle

725Th

rfslowast13

pIle

725A

spfslowast

4

pG

lu72

9Lys

fslowast9

pThr7

36Ile

fslowast4

pCy

s744

lowast

pSe

r747

Valfs

lowast16

pA

sn74

8Ile

fslowast15

pA

rg75

9lowastp

Leu7

62

Asn763

pL

eu762

Tyrfs

lowast17

pA

sn76

3Leu

fslowast11

pA

sn76

9Arg

fslowast6

pLy

s778

lowast

pSe

r779

lowast

pSe

r862

lowast

pIle

864L

ysfslowast

11

pTy

r105

3Thrfs

lowast4

pSe

r2A

rgfslowast16

pH

is31A

rgfslowast

9

pVa

l157

Trpf

slowast16

pA

la22

1Gly

fslowast43

pPr

o229

Gln

fslowast35

pA

rg33

8lowast

pA

rg39

6lowastp

Gly

402A

lafslowast7

pLy

s443

Asn

fslowast12

pG

lu48

8lowast

pTr

p113

1lowast

pG

ly11

40ly

sfslowast4

pA

sn11

43Ile

fslowast25

pG

lu12

27M

etfslowast

29

pA

rg13

64Va

lfslowast8

pLy

s151

8lowast

pA

rg87

2Thrfs

lowast2

pSe

r911

lowast

Figure 4 Schematic presentation of RP1 disease causing mutations Disease causing mutations were represented based on the classificationby Chen and coworkers [13] Mutations responsible for recessive arRCD were shown in the upper half whereas mutations causing adRCDwere shown in the lower half pGly402Alafslowast7 pLys443Asnfslowast12 pArg1364Valfslowast8 and pSer1529Argfslowast9 belong to class III AlthoughpSer574Cysfslowast7 pSer676Ilefslowast22 pArg793Glufslowast55 and pAsp799lowast are class II mutations these variants do not cause adRCD but arRCDinstead Amino acid modifications shown in red and blue represent novel frameshift or nonsense mutations and the recurrent pSer542lowastmutation respectively Protein localization of pSer542lowast was highlighted in blue as it marked a recurrent mutation adRCD autosomaldominant rod-cone dystrophy arRCD autosomal recessive rod-cone dystrophy BIF drosophila melanogaster bifocal

latter observation is supported by finding that RP1 mutantmRNA is expressed in a human cell line carrying a homozy-gous pArg677lowast mutation [21]

Based on Chen et al [13] RP1 truncating mutations lead-ing to arRCD or adRCD can be divided into four distinctgroups Class I is composed of truncating mutations locatedin exons 2 and 3These variants are sensitive toNMDand thusare considered as true loss-of-function alleles (Figure 4) [13]Class II involves truncating mutations that are located in aspot between codons 500 and 1053 in exon 4 [13] the so calledldquoRP1 hot spotrdquo The ldquohot spotrdquo variants tend to be insensitiveto NMD process and thus result in a protein with a potentialdominant negative effect leading to adRCD (Figure 4) [13]Class III includes truncating mutations insensible to NMDlocated between codons 264 and 499 and between codons1054 to 1751 in exon 4 These truncating proteins result in aloss of function leading to arRCD (Figure 4) [13] Finally classIV includes protein-truncating mutations near the 31015840 endof the fourth exon (Figure 4) [13] Most likely the resultingproteins display only aminor loss of their C-terminal portionpreserving the majority of functional domains and keeping aresidual activity According to the classification of Chen et al[13] pGly402Alafslowast7 pLys443Asnfslowast12 pArg1364Valfslowast8and pSer1529Argfslowast9 belong to class III (Figure 4)

The predicted physiopathology for pSer542lowastpSer574Cysfslowast7 pSer676Ilefslowast22 pArg793Glufslowast55 andpAsp799lowast is more complex According to Chenrsquos classi-fication these frameshift deletions and nonsense mutationsshould belong to class II previously only associated withadRCD However herein they are causing presumablyarRCD (Figure 4) To further confirm these findings clinicaland genetic testing of the reported unaffected parents shouldbe done

Based on the previous findings we speculate that the clas-sification by Chen and coworkers does not hold true for allmutations Supporting this statement Avila-Fernandez et al[12] reported the same nonsense mutation (pSer542lowast) foundin (F303 II1 (CIC00445)) and located at the 51015840 end of theldquohot spotrdquo to cause arRP in the Spanish population [12]These observations are of interest as they point out for animplication of hot spot region for adRCD-RP1mutation alsoin case of arRCD Future studies will need to clarify why someclass II mutations lead to adRCD and others to arRCD

Patients with arRCD and RP1 mutations show a moresevere disease than adRCD-RP1 mutant patients with mac-ular atrophy in all our cases This was first outlined byLafont et al [17] When patients are presenting with latesevere disease the diagnostic distinction between RCD with

10 BioMed Research International

initial rod involvement and cone-rod dystrophy (CRD) withinitial cone involvement is difficult Of note is that one ofthe patients (CIC01300) in the present study was initiallyclassified as possibly having severe CRD and his diagnosiswas actually revisited after NGS resultsThis also outlines theinterest of unbiased massive parallel sequencing methods fora more precise clinical diagnostic in case of end stage diseaseThis point will most likely become evenmore critical with theperspective of therapeutic trials

41 Strength and Limitations We estimate that 1 of ourtarget regions were not covered Partially uncovered exonsare a real common issue when capturing the DNA sequencesusing commercially available probes this bias might implya loss of some candidate variants However we found thatrate of 1 is very reasonable when compared with otherNGS panels In addition in order to exclude the possibilityof finding other candidate variants we have sequenced bySanger method the majority of these regions Five of ourpatients carried homozygous RP1 mutations For four of thesubjects carrying homozygous variants namely CIC00491F335 CIC01106 F674 CIC01300 F782 and CIC04130 F1941co-segregation analysis needs to be done to confirm auto-somal recessive inheritance but we do not have access toparentrsquos DNA CIC05941 was the only one not to reportclear consanguinity in the family and we cannot exclude thepossibility of a large deletion on the second allele of RP1gene Again DNA of the father not available for us wouldbe helpful to prove autosomal recessive inheritance and thehomozygous state of the mutation

In conclusion we have reported 9 mutations in RP1 ofwhich 8 were novel causing arRCD [8 12ndash20] Interestingly aprevalence ofasymp25points out for the necessity of sequencingRP1 in sporadic and recessive cases of RCD Further func-tional studies are needed to understand the impact of RP1structure on its function at the molecular level such a stepwould strengthen our knowledge in the physiology of retinalphotoreceptors

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Isabelle Audo and Christina Zeitz contributed equally to thiswork

Acknowledgments

The authors express their sincere gratitude to the familieswho participated in this study and to the clinical staff fortheir help in clinical data and DNA collection This workwas supported by Foundation Voir et Entendre (CZ) PrixDalloz for ldquola recherche en ophtalmologierdquo (CZ) FoundationFighting Blindness (FFB) [CD-CL-0808-0466-CHNO] (IA)and FFB center (FFB grantC-GE-0912-0601-INSERM02)

Prix de la Fondation de lrsquoŒil (IA) Ville de Paris and RegionIle de France and the French State program ldquoInvestissementsdrsquoAvenirrdquo managed by the Agence Nationale de la Recherche[LIFESENSES ANR-10-LABX-65]

References

[1] D T Hartong E L Berson and T P Dryja ldquoRetinitis pigmen-tosardquoThe Lancet vol 368 no 9549 pp 1795ndash1809 2006

[2] I Audo M Lancelot S Mohand-Saıd et al ldquoNovel C2orf71mutations account for approximately sim1 of cases in a largeFrench arRP cohortrdquoHumanMutation vol 32 no 4 pp E2091ndashE2103 2011

[3] A Anasagasti C Irigoyen O Barandika A Lopez de Munainand J Ruiz-Ederra ldquoCurrent mutation discovery approaches inRetinitis PigmentosardquoVision Research vol 75 pp 117ndash129 2012

[4] L S Sullivan S J Bowne D G Birch et al ldquoPrevalence ofdisease-causingmutations in families with autosomal dominantretinitis pigmentosa a screen of known genes in 200 familiesrdquoInvestigative Ophthalmology andVisual Science vol 47 no 7 pp3052ndash3064 2006

[5] B J Seyedahmadi C Rivolta J A Keene E L Berson andT P Dryja ldquoComprehensive screening of the USH2A gene inUsher syndrome type II and non-syndromic recessive retinitispigmentosardquo Experimental Eye Research vol 79 no 2 pp 167ndash173 2004

[6] A Avila-Fernandez D Cantalapiedra E Aller et al ldquoMutationanalysis of 272 Spanish families affected by autosomal recessiveretinitis pigmentosa using a genotyping microarrayrdquoMolecularVision vol 16 pp 2550ndash2558 2010

[7] S El Shamieh M Neuille A Terray et al ldquoWhole-exomesequencing identifies KIZ as a ciliary gene associated withautosomal-recessive rod-cone dystrophyrdquo American Journal ofHuman Genetics vol 94 no 4 pp 625ndash633 2014

[8] I Audo S Mohand-Saıd C M Dhaenens et al ldquoRP1 andautosomal dominant rod-cone dystrophy novel mutations areview of published variants and genotype-phenotype correla-tionrdquo Human Mutation vol 33 no 1 pp 73ndash80 2012

[9] X Guillonneau N I Piriev M Danciger et al ldquoA nonsensemutation in a novel gene is associated with retinitis pigmentosain a family linked to the RP1 locusrdquoHumanMolecular Geneticsvol 8 no 8 pp 1541ndash1546 1999

[10] E A Pierce T Quinn T Meehan T L McGee E L Bersonand T P Dryja ldquoMutations in a gene encoding a new oxygen-regulated photoreceptor protein cause dominant retinitis pig-mentosardquo Nature Genetics vol 22 no 3 pp 248ndash254 1999

[11] L S Sullivan J R Heckenlively S J Bowne et al ldquoMutations ina novel retina-specific gene cause autosomal dominant retinitispigmentosardquo Nature Genetics vol 22 no 3 pp 255ndash259 1999

[12] A Avila-Fernandez M Corton K M Nishiguchi et al ldquoIden-tification of an RP1 prevalent founder mutation and relatedphenotype in Spanish patients with early-onset autosomalrecessive retinitisrdquo Ophthalmology vol 119 no 12 pp 2616ndash2621 2012

[13] L J Chen T Y Y Lai P O S Tam et al ldquoCompound heter-ozygosity of two novel truncation mutations in RP1 causingautosomal recessive retinitis pigmentosardquo InvestigativeOphthal-mology and Visual Science vol 51 no 4 pp 2236ndash2242 2010

[14] S Khaliq A Abid M Ismail et al ldquoNovel association of RP1gene mutations with autosomal recessive retinitis pigmentosardquoJournal of Medical Genetics vol 42 no 5 pp 436ndash438 2005

BioMed Research International 11

[15] A M Siemiatkowska G D N Astuti K Arimadyo et alldquoIdentification of a novel nonsense mutation in RP1 that causesautosomal recessive retinitis pigmentosa in an IndonesianfamilyrdquoMolecular Vision vol 18 pp 2411ndash2419 2012

[16] B Bocquet N A Marzouka M Hebrard et al ldquoHomozygositymapping in autosomal recessive retinitis pigmentosa familiesdetects novel mutationsrdquo Molecular Vision vol 19 pp 2487ndash2500 2013

[17] E M Lafont G Manes G Senechal et al ldquoPatients with reti-nitis pigmentosa due to RP1 mutations show greater severity inrecessive than in dominant casesrdquo Journal of Clinical amp Experi-mental Ophthalmology vol 2 article 194 2011

[18] M Al-Rashed L Abu Safieh H Alkuraya et al ldquoRP1 and reti-nitis pigmentosa report of novel mutations and insight intomutational mechanismrdquo British Journal of Ophthalmology vol96 no 7 pp 1018ndash1022 2012

[19] S A Riazuddin A Shahzadi C Zeitz et al ldquoA mutation inSLC24A1 implicated in autosomal-recessive congenital station-ary night blindnessrdquoThe American Journal of Human Geneticsvol 87 no 4 pp 523ndash531 2010

[20] H P Singh S Jalali R Narayanan and C Kannabiran ldquoGe-netic analysis of indian families with autosomal recessiveretinitis pigmentosa by homozygosity screeningrdquo InvestigativeOphthalmology and Visual Science vol 50 no 9 pp 4065ndash40712009

[21] Q Liu A Lyubarsky J H Skalet E N Pugh Jr and E A PierceldquoRP1 is required for the correct stacking of outer segment discsrdquoInvestigative Ophthalmology ampVisual Science vol 44 no 10 pp4171ndash4183 2003

[22] Q Liu J Zuo and E A Pierce ldquoThe retinitis pigmentosa 1protein is a photoreceptor microtubule-associated proteinrdquoJournal of Neuroscience vol 24 no 29 pp 6427ndash6436 2004

[23] I Audo K M Bujakowska T Leveillard et al ldquoDevelop-ment and application of a next-generation-sequencing (NGS)approach to detect known and novel gene defects underlyingretinal diseasesrdquo Orphanet Journal of Rare Diseases vol 7 no 1article 8 2012

[24] I Audo J A Sahel S Mohand-Saıd et al ldquoEYS is a major genefor rod-cone dystrophies in Francerdquo Human Mutation vol 31no 5 pp E1406ndashE1435 2010

[25] T G P Consortium ldquoA map of human genome variation frompopulation-scale sequencingrdquo Nature vol 467 no 7319 pp1061ndash1073 2010

[26] DMAltshuler RAGibbs L Peltonen et al ldquoIntegrating com-mon and rare genetic variation in diverse human populationsrdquoNature vol 467 no 7311 pp 52ndash58 2010

[27] J A Tennessen AW Bigham T D OrsquoConnor et al ldquoEvolutionand functional impact of rare coding variation from deepsequencing of human exomesrdquo Science vol 337 no 6090 pp64ndash69 2012

[28] M J Bamshad S BNgAW Bighamet al ldquoExome sequencingas a tool for Mendelian disease gene discoveryrdquo Nature ReviewsGenetics vol 12 no 11 pp 745ndash755 2011

[29] W J Kent C W Sugnet T S Furey et al ldquoThe human genomebrowser at UCSCrdquo Genome Research vol 12 no 6 pp 996ndash1006 2002

[30] I A Adzhubei S Schmidt L Peshkin et al ldquoA method andserver for predicting damaging missense mutationsrdquo NatureMethods vol 7 no 4 pp 248ndash249 2010

[31] P Kumar S Henikoff and P C Ng ldquoPredicting the effects ofcoding non-synonymous variants on protein function using the

SIFT algorithmrdquo Nature Protocols vol 4 no 7 pp 1073ndash10822009

[32] F O Desmet D Hamroun M Lalande G Collod-Beroud MClaustres and C Beroud ldquoHuman splicing finder an onlinebioinformatics tool to predict splicing signalsrdquo Nucleic AcidsResearch vol 37 no 9 article no e67 2009

[33] P D Stenson E V Ball M Mort A D Phillips K Shaw andD N Cooper ldquoThe Human Gene Mutation Database (HGMD)and its exploitation in the fields of personalized genomics andmolecular evolutionrdquo in Current Protocols in Bioinformaticschapter 1ndash13 2012

[34] I F A C Fokkema J T Den Dunnen and P E M TaschnerldquoLOVD easy creation of a locus-specific sequence variationdatabase using an ldquoLSDB-in-a-Boxrdquo approachrdquo Human Muta-tion vol 26 no 2 pp 63ndash68 2005

[35] S A Miller D D Dykes and H F Polesky ldquoA simple saltingout procedure for extracting DNA from human nucleated cellsrdquoNucleic Acids Research vol 16 no 3 p 1215 1988

[36] C Zeitz B Kloeckener-Gruissem U Forster et al ldquoMutationsin CABP4 the gene encoding the Ca2+-binding protein 4cause autosomal recessive night blindnessrdquoAmerican Journal ofHuman Genetics vol 79 no 4 pp 657ndash667 2006

[37] S A Riazuddin F Zulfiqar Q Zhang et al ldquoAutosomal reces-sive retinitis pigmentosa is associated with mutations in RP1 inthree consanguineous Pakistani familiesrdquo Investigative Ophthal-mology and Visual Science vol 46 no 7 pp 2264ndash2270 2005

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

10 BioMed Research International

initial rod involvement and cone-rod dystrophy (CRD) withinitial cone involvement is difficult Of note is that one ofthe patients (CIC01300) in the present study was initiallyclassified as possibly having severe CRD and his diagnosiswas actually revisited after NGS resultsThis also outlines theinterest of unbiased massive parallel sequencing methods fora more precise clinical diagnostic in case of end stage diseaseThis point will most likely become evenmore critical with theperspective of therapeutic trials

41 Strength and Limitations We estimate that 1 of ourtarget regions were not covered Partially uncovered exonsare a real common issue when capturing the DNA sequencesusing commercially available probes this bias might implya loss of some candidate variants However we found thatrate of 1 is very reasonable when compared with otherNGS panels In addition in order to exclude the possibilityof finding other candidate variants we have sequenced bySanger method the majority of these regions Five of ourpatients carried homozygous RP1 mutations For four of thesubjects carrying homozygous variants namely CIC00491F335 CIC01106 F674 CIC01300 F782 and CIC04130 F1941co-segregation analysis needs to be done to confirm auto-somal recessive inheritance but we do not have access toparentrsquos DNA CIC05941 was the only one not to reportclear consanguinity in the family and we cannot exclude thepossibility of a large deletion on the second allele of RP1gene Again DNA of the father not available for us wouldbe helpful to prove autosomal recessive inheritance and thehomozygous state of the mutation

In conclusion we have reported 9 mutations in RP1 ofwhich 8 were novel causing arRCD [8 12ndash20] Interestingly aprevalence ofasymp25points out for the necessity of sequencingRP1 in sporadic and recessive cases of RCD Further func-tional studies are needed to understand the impact of RP1structure on its function at the molecular level such a stepwould strengthen our knowledge in the physiology of retinalphotoreceptors

Conflict of Interests

The authors declare that there is no conflict of interestsregarding the publication of this paper

Authorsrsquo Contribution

Isabelle Audo and Christina Zeitz contributed equally to thiswork

Acknowledgments

The authors express their sincere gratitude to the familieswho participated in this study and to the clinical staff fortheir help in clinical data and DNA collection This workwas supported by Foundation Voir et Entendre (CZ) PrixDalloz for ldquola recherche en ophtalmologierdquo (CZ) FoundationFighting Blindness (FFB) [CD-CL-0808-0466-CHNO] (IA)and FFB center (FFB grantC-GE-0912-0601-INSERM02)

Prix de la Fondation de lrsquoŒil (IA) Ville de Paris and RegionIle de France and the French State program ldquoInvestissementsdrsquoAvenirrdquo managed by the Agence Nationale de la Recherche[LIFESENSES ANR-10-LABX-65]

References

[1] D T Hartong E L Berson and T P Dryja ldquoRetinitis pigmen-tosardquoThe Lancet vol 368 no 9549 pp 1795ndash1809 2006

[2] I Audo M Lancelot S Mohand-Saıd et al ldquoNovel C2orf71mutations account for approximately sim1 of cases in a largeFrench arRP cohortrdquoHumanMutation vol 32 no 4 pp E2091ndashE2103 2011

[3] A Anasagasti C Irigoyen O Barandika A Lopez de Munainand J Ruiz-Ederra ldquoCurrent mutation discovery approaches inRetinitis PigmentosardquoVision Research vol 75 pp 117ndash129 2012

[4] L S Sullivan S J Bowne D G Birch et al ldquoPrevalence ofdisease-causingmutations in families with autosomal dominantretinitis pigmentosa a screen of known genes in 200 familiesrdquoInvestigative Ophthalmology andVisual Science vol 47 no 7 pp3052ndash3064 2006

[5] B J Seyedahmadi C Rivolta J A Keene E L Berson andT P Dryja ldquoComprehensive screening of the USH2A gene inUsher syndrome type II and non-syndromic recessive retinitispigmentosardquo Experimental Eye Research vol 79 no 2 pp 167ndash173 2004

[6] A Avila-Fernandez D Cantalapiedra E Aller et al ldquoMutationanalysis of 272 Spanish families affected by autosomal recessiveretinitis pigmentosa using a genotyping microarrayrdquoMolecularVision vol 16 pp 2550ndash2558 2010

[7] S El Shamieh M Neuille A Terray et al ldquoWhole-exomesequencing identifies KIZ as a ciliary gene associated withautosomal-recessive rod-cone dystrophyrdquo American Journal ofHuman Genetics vol 94 no 4 pp 625ndash633 2014

[8] I Audo S Mohand-Saıd C M Dhaenens et al ldquoRP1 andautosomal dominant rod-cone dystrophy novel mutations areview of published variants and genotype-phenotype correla-tionrdquo Human Mutation vol 33 no 1 pp 73ndash80 2012

[9] X Guillonneau N I Piriev M Danciger et al ldquoA nonsensemutation in a novel gene is associated with retinitis pigmentosain a family linked to the RP1 locusrdquoHumanMolecular Geneticsvol 8 no 8 pp 1541ndash1546 1999

[10] E A Pierce T Quinn T Meehan T L McGee E L Bersonand T P Dryja ldquoMutations in a gene encoding a new oxygen-regulated photoreceptor protein cause dominant retinitis pig-mentosardquo Nature Genetics vol 22 no 3 pp 248ndash254 1999

[11] L S Sullivan J R Heckenlively S J Bowne et al ldquoMutations ina novel retina-specific gene cause autosomal dominant retinitispigmentosardquo Nature Genetics vol 22 no 3 pp 255ndash259 1999

[12] A Avila-Fernandez M Corton K M Nishiguchi et al ldquoIden-tification of an RP1 prevalent founder mutation and relatedphenotype in Spanish patients with early-onset autosomalrecessive retinitisrdquo Ophthalmology vol 119 no 12 pp 2616ndash2621 2012

[13] L J Chen T Y Y Lai P O S Tam et al ldquoCompound heter-ozygosity of two novel truncation mutations in RP1 causingautosomal recessive retinitis pigmentosardquo InvestigativeOphthal-mology and Visual Science vol 51 no 4 pp 2236ndash2242 2010

[14] S Khaliq A Abid M Ismail et al ldquoNovel association of RP1gene mutations with autosomal recessive retinitis pigmentosardquoJournal of Medical Genetics vol 42 no 5 pp 436ndash438 2005

BioMed Research International 11

[15] A M Siemiatkowska G D N Astuti K Arimadyo et alldquoIdentification of a novel nonsense mutation in RP1 that causesautosomal recessive retinitis pigmentosa in an IndonesianfamilyrdquoMolecular Vision vol 18 pp 2411ndash2419 2012

[16] B Bocquet N A Marzouka M Hebrard et al ldquoHomozygositymapping in autosomal recessive retinitis pigmentosa familiesdetects novel mutationsrdquo Molecular Vision vol 19 pp 2487ndash2500 2013

[17] E M Lafont G Manes G Senechal et al ldquoPatients with reti-nitis pigmentosa due to RP1 mutations show greater severity inrecessive than in dominant casesrdquo Journal of Clinical amp Experi-mental Ophthalmology vol 2 article 194 2011

[18] M Al-Rashed L Abu Safieh H Alkuraya et al ldquoRP1 and reti-nitis pigmentosa report of novel mutations and insight intomutational mechanismrdquo British Journal of Ophthalmology vol96 no 7 pp 1018ndash1022 2012

[19] S A Riazuddin A Shahzadi C Zeitz et al ldquoA mutation inSLC24A1 implicated in autosomal-recessive congenital station-ary night blindnessrdquoThe American Journal of Human Geneticsvol 87 no 4 pp 523ndash531 2010

[20] H P Singh S Jalali R Narayanan and C Kannabiran ldquoGe-netic analysis of indian families with autosomal recessiveretinitis pigmentosa by homozygosity screeningrdquo InvestigativeOphthalmology and Visual Science vol 50 no 9 pp 4065ndash40712009

[21] Q Liu A Lyubarsky J H Skalet E N Pugh Jr and E A PierceldquoRP1 is required for the correct stacking of outer segment discsrdquoInvestigative Ophthalmology ampVisual Science vol 44 no 10 pp4171ndash4183 2003

[22] Q Liu J Zuo and E A Pierce ldquoThe retinitis pigmentosa 1protein is a photoreceptor microtubule-associated proteinrdquoJournal of Neuroscience vol 24 no 29 pp 6427ndash6436 2004

[23] I Audo K M Bujakowska T Leveillard et al ldquoDevelop-ment and application of a next-generation-sequencing (NGS)approach to detect known and novel gene defects underlyingretinal diseasesrdquo Orphanet Journal of Rare Diseases vol 7 no 1article 8 2012

[24] I Audo J A Sahel S Mohand-Saıd et al ldquoEYS is a major genefor rod-cone dystrophies in Francerdquo Human Mutation vol 31no 5 pp E1406ndashE1435 2010

[25] T G P Consortium ldquoA map of human genome variation frompopulation-scale sequencingrdquo Nature vol 467 no 7319 pp1061ndash1073 2010

[26] DMAltshuler RAGibbs L Peltonen et al ldquoIntegrating com-mon and rare genetic variation in diverse human populationsrdquoNature vol 467 no 7311 pp 52ndash58 2010

[27] J A Tennessen AW Bigham T D OrsquoConnor et al ldquoEvolutionand functional impact of rare coding variation from deepsequencing of human exomesrdquo Science vol 337 no 6090 pp64ndash69 2012

[28] M J Bamshad S BNgAW Bighamet al ldquoExome sequencingas a tool for Mendelian disease gene discoveryrdquo Nature ReviewsGenetics vol 12 no 11 pp 745ndash755 2011

[29] W J Kent C W Sugnet T S Furey et al ldquoThe human genomebrowser at UCSCrdquo Genome Research vol 12 no 6 pp 996ndash1006 2002

[30] I A Adzhubei S Schmidt L Peshkin et al ldquoA method andserver for predicting damaging missense mutationsrdquo NatureMethods vol 7 no 4 pp 248ndash249 2010

[31] P Kumar S Henikoff and P C Ng ldquoPredicting the effects ofcoding non-synonymous variants on protein function using the

SIFT algorithmrdquo Nature Protocols vol 4 no 7 pp 1073ndash10822009

[32] F O Desmet D Hamroun M Lalande G Collod-Beroud MClaustres and C Beroud ldquoHuman splicing finder an onlinebioinformatics tool to predict splicing signalsrdquo Nucleic AcidsResearch vol 37 no 9 article no e67 2009

[33] P D Stenson E V Ball M Mort A D Phillips K Shaw andD N Cooper ldquoThe Human Gene Mutation Database (HGMD)and its exploitation in the fields of personalized genomics andmolecular evolutionrdquo in Current Protocols in Bioinformaticschapter 1ndash13 2012

[34] I F A C Fokkema J T Den Dunnen and P E M TaschnerldquoLOVD easy creation of a locus-specific sequence variationdatabase using an ldquoLSDB-in-a-Boxrdquo approachrdquo Human Muta-tion vol 26 no 2 pp 63ndash68 2005

[35] S A Miller D D Dykes and H F Polesky ldquoA simple saltingout procedure for extracting DNA from human nucleated cellsrdquoNucleic Acids Research vol 16 no 3 p 1215 1988

[36] C Zeitz B Kloeckener-Gruissem U Forster et al ldquoMutationsin CABP4 the gene encoding the Ca2+-binding protein 4cause autosomal recessive night blindnessrdquoAmerican Journal ofHuman Genetics vol 79 no 4 pp 657ndash667 2006

[37] S A Riazuddin F Zulfiqar Q Zhang et al ldquoAutosomal reces-sive retinitis pigmentosa is associated with mutations in RP1 inthree consanguineous Pakistani familiesrdquo Investigative Ophthal-mology and Visual Science vol 46 no 7 pp 2264ndash2270 2005

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

BioMed Research International 11

[15] A M Siemiatkowska G D N Astuti K Arimadyo et alldquoIdentification of a novel nonsense mutation in RP1 that causesautosomal recessive retinitis pigmentosa in an IndonesianfamilyrdquoMolecular Vision vol 18 pp 2411ndash2419 2012

[16] B Bocquet N A Marzouka M Hebrard et al ldquoHomozygositymapping in autosomal recessive retinitis pigmentosa familiesdetects novel mutationsrdquo Molecular Vision vol 19 pp 2487ndash2500 2013

[17] E M Lafont G Manes G Senechal et al ldquoPatients with reti-nitis pigmentosa due to RP1 mutations show greater severity inrecessive than in dominant casesrdquo Journal of Clinical amp Experi-mental Ophthalmology vol 2 article 194 2011

[18] M Al-Rashed L Abu Safieh H Alkuraya et al ldquoRP1 and reti-nitis pigmentosa report of novel mutations and insight intomutational mechanismrdquo British Journal of Ophthalmology vol96 no 7 pp 1018ndash1022 2012

[19] S A Riazuddin A Shahzadi C Zeitz et al ldquoA mutation inSLC24A1 implicated in autosomal-recessive congenital station-ary night blindnessrdquoThe American Journal of Human Geneticsvol 87 no 4 pp 523ndash531 2010

[20] H P Singh S Jalali R Narayanan and C Kannabiran ldquoGe-netic analysis of indian families with autosomal recessiveretinitis pigmentosa by homozygosity screeningrdquo InvestigativeOphthalmology and Visual Science vol 50 no 9 pp 4065ndash40712009

[21] Q Liu A Lyubarsky J H Skalet E N Pugh Jr and E A PierceldquoRP1 is required for the correct stacking of outer segment discsrdquoInvestigative Ophthalmology ampVisual Science vol 44 no 10 pp4171ndash4183 2003

[22] Q Liu J Zuo and E A Pierce ldquoThe retinitis pigmentosa 1protein is a photoreceptor microtubule-associated proteinrdquoJournal of Neuroscience vol 24 no 29 pp 6427ndash6436 2004

[23] I Audo K M Bujakowska T Leveillard et al ldquoDevelop-ment and application of a next-generation-sequencing (NGS)approach to detect known and novel gene defects underlyingretinal diseasesrdquo Orphanet Journal of Rare Diseases vol 7 no 1article 8 2012

[24] I Audo J A Sahel S Mohand-Saıd et al ldquoEYS is a major genefor rod-cone dystrophies in Francerdquo Human Mutation vol 31no 5 pp E1406ndashE1435 2010

[25] T G P Consortium ldquoA map of human genome variation frompopulation-scale sequencingrdquo Nature vol 467 no 7319 pp1061ndash1073 2010

[26] DMAltshuler RAGibbs L Peltonen et al ldquoIntegrating com-mon and rare genetic variation in diverse human populationsrdquoNature vol 467 no 7311 pp 52ndash58 2010

[27] J A Tennessen AW Bigham T D OrsquoConnor et al ldquoEvolutionand functional impact of rare coding variation from deepsequencing of human exomesrdquo Science vol 337 no 6090 pp64ndash69 2012

[28] M J Bamshad S BNgAW Bighamet al ldquoExome sequencingas a tool for Mendelian disease gene discoveryrdquo Nature ReviewsGenetics vol 12 no 11 pp 745ndash755 2011

[29] W J Kent C W Sugnet T S Furey et al ldquoThe human genomebrowser at UCSCrdquo Genome Research vol 12 no 6 pp 996ndash1006 2002

[30] I A Adzhubei S Schmidt L Peshkin et al ldquoA method andserver for predicting damaging missense mutationsrdquo NatureMethods vol 7 no 4 pp 248ndash249 2010

[31] P Kumar S Henikoff and P C Ng ldquoPredicting the effects ofcoding non-synonymous variants on protein function using the

SIFT algorithmrdquo Nature Protocols vol 4 no 7 pp 1073ndash10822009

[32] F O Desmet D Hamroun M Lalande G Collod-Beroud MClaustres and C Beroud ldquoHuman splicing finder an onlinebioinformatics tool to predict splicing signalsrdquo Nucleic AcidsResearch vol 37 no 9 article no e67 2009

[33] P D Stenson E V Ball M Mort A D Phillips K Shaw andD N Cooper ldquoThe Human Gene Mutation Database (HGMD)and its exploitation in the fields of personalized genomics andmolecular evolutionrdquo in Current Protocols in Bioinformaticschapter 1ndash13 2012

[34] I F A C Fokkema J T Den Dunnen and P E M TaschnerldquoLOVD easy creation of a locus-specific sequence variationdatabase using an ldquoLSDB-in-a-Boxrdquo approachrdquo Human Muta-tion vol 26 no 2 pp 63ndash68 2005

[35] S A Miller D D Dykes and H F Polesky ldquoA simple saltingout procedure for extracting DNA from human nucleated cellsrdquoNucleic Acids Research vol 16 no 3 p 1215 1988

[36] C Zeitz B Kloeckener-Gruissem U Forster et al ldquoMutationsin CABP4 the gene encoding the Ca2+-binding protein 4cause autosomal recessive night blindnessrdquoAmerican Journal ofHuman Genetics vol 79 no 4 pp 657ndash667 2006

[37] S A Riazuddin F Zulfiqar Q Zhang et al ldquoAutosomal reces-sive retinitis pigmentosa is associated with mutations in RP1 inthree consanguineous Pakistani familiesrdquo Investigative Ophthal-mology and Visual Science vol 46 no 7 pp 2264ndash2270 2005

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology