Genetic analysis of an Indian family with members affected withWaardenburg syndrome and Duchenne muscular dystrophy

Saketh Kapoor1 Parayil Sankaran Bindu2 Arun B Taly2 Sanjib Sinha2 Narayanappa Gayathri3

S Vasantha Rani4 Giriraj Ratan Chandak4 Arun Kumar1

1Department of Molecular Reproduction Development and Genetics Indian Institute of Science Bangalore India 2Departmentof Neurology National Institute of Mental Health and Neuro Sciences Bangalore India 3Department of Neuropathology NationalInstitute of Mental Health and Neuro Sciences Bangalore India 4Centre for Cellular and Molecular Biology Hyderabad India

Purpose Waardenburg syndrome (WS) is characterized by sensorineural hearing loss and pigmentation defects of theeye skin and hair It is caused by mutations in one of the following genes PAX3 (paired box 3) MITF (microphthalmia-associated transcription factor) EDNRB (endothelin receptor type B) EDN3 (endothelin 3) SNAI2 (snail homolog 2Drosophila) and SOX10 (SRY-box containing gene 10) Duchenne muscular dystrophy (DMD) is an X-linked recessivedisorder caused by mutations in the DMD gene The purpose of this study was to identify the genetic causes of WS andDMD in an Indian family with two patients one affected with WS and DMD and another one affected with only WSMethods Blood samples were collected from individuals for genomic DNA isolation To determine the linkage of thisfamily to the eight known WS loci microsatellite markers were selected from the candidate regions and used to genotypethe family Exon-specific intronic primers for EDN3 were used to amplify and sequence DNA samples from affectedindividuals to detect mutations A mutation in DMD was identified by multiplex PCR and multiplex ligation-dependentprobe amplification method using exon-specific probesResults Pedigree analysis suggested segregation of WS as an autosomal recessive trait in the family Haplotype analysissuggested linkage of the family to the WS4B (EDN3) locus DNA sequencing identified a novel missense mutation pT98Min EDN3 A deletion mutation was identified in DMDConclusions This study reports a novel missense mutation in EDN3 and a deletion mutation in DMD in the same Indianfamily The present study will be helpful in genetic diagnosis of this family and increases the mutation spectrum ofEDN3

Waardenburg syndrome (WS) is a hereditary auditory-pigmentary syndrome characterized by pigmentaryabnormalities of the hair including a white forelock andpremature graying pigmentary changes of the iris such asheterochromia irides and brilliant blue eyes lateraldisplacement of the medial canthi and lacrimal pointscongenital deafness and intestinal abnormalities [1] Theassociation of hearing loss pigmentary abnormalities andintestinal malformation results from an abnormalproliferation survival migration or differentiation of neuralcrest derived melanocytes of skin and inner ear glia andneurons of the peripheral and enteric nervous system [2] Theincidence of WS is around 1 in 42000 in the generalpopulation However it occurs in 5ndash6 of deaf individualsand is considered to be the most common autosomal-dominantform of syndromic deafness [3] It is clinically and geneticallyheterogeneous and is classified into four types (viz type I toIV) based on the presence of variable clinical characteristicsand additional symptoms WS type I is characterized by the

Correspondence to Professor Arun Kumar PhD Department ofMolecular Reproduction Development amp Genetics Indian Instituteof Science Bangalore 560 012 India Phone 91-80-2293 2998FAX 91-80-2360 0999 email karunmrdgiiscernetin

presence of dystopia canthorum (lateral displacement of innercanthi) WS type II is distinguished from type I by the absenceof dystopia canthorum WS type III patients have limbhypoplasia in addition to dystopia canthorum WS type IV hasan additional feature of Hirschsprung disease [2] Geneticanalysis has identified at least eight loci for WS WS1WS3on chromosome 2q361 WS2A on chromosome 3p141-p123 WS2B on chromosome 1p21-p133 WS2C onchromosome 8p23 WS2D on chromosome 8q1121 WS2EWS4C on chromosome 22q131 WS4A on chromosome13q223 and WS4B on chromosome 20q132-q133(OMIM) The genes for six loci are known WS1WS3-PAX3 (paired box 3) WS2A-MITF (micropthalmia-associated transcription factor) WS2D-SNAI2 (snail homolog2 Drosophila) WS2EWS4C-SOX10 (SRY-box containinggene 10) WS4A-EDNRB (endothelin receptor type B) andWS4B-EDN3 (endothelin 3) [4-12]

Duchenne muscular dystrophy (DMD) is the mostcommon X-linked recessive disease It is estimated to affect1 in 3500 newborn males worldwide [13] and caused bymutations in the DMD gene encoding a cytoskeletal proteindystrophin DMD is the largest human gene that spans gt2200kb of DNA and is composed of 79 exons [14] Deletionsaccount for approximately 65 of DMD mutations

Molecular Vision 2012 182022-2032 lthttpwwwmolvisorgmolvisv18a213gtReceived 9 June 2012 | Accepted 17 July 2012 | Published 20 July 2012

copy 2012 Molecular Vision

2022

duplications occur in approximately 6ndash10 of cases and theremaining 30ndash35 of mutations consist of small deletionsinsertions point mutations or splicing mutations most ofwhich introduce a premature stop codon [14-23]

Here we report on the genetic analysis of aconsanguineous family from the south Indian state ofKarnataka with members affected with two disordersWaardenburg syndrome and Duchenne muscular dystrophy

METHODSPatients We ascertained a consanguineous family (Figure 1)with one individual (IV-3) affected with WS and DMD andthe other one (IV-2) affected with only WS from a southIndian state of Karnataka in the Department of NeurologyNational Institute of Mental Health and Neuro SciencesBangalore Both affected individuals were examined byParayil Sankaran Bindu Arun B Taly and Sanjib Sinha Adetailed description of their clinical symptoms is given below

Individual IV-3mdashThe propositus a 9-year-old boy wasthe third child born to consanguineous parents after an

uneventful pregnancy and delivery His developmentalmilestones were normal except for speech delay A formalevaluation for the speech delay at the age of three yearsrevealed profound bilateral sensorineural hearing loss Sincethe age of six years he developed gradually progressivedifficulty in walking and getting up from sitting position Hehad blue iris faint white forelock confluent eyebrowsbilateral calf hypertrophy mild tendon Achilles contractureexaggerated lumbar lordosis and a waddling gait He hadsymmetric weakness of proximal muscles of all four limbs andtruncal muscles All stretch reflexes were sluggish except forthe ankle jerks Gowerrsquos sign was evident on getting up fromthe sitting position (Figure 2A) Ophthalmological evaluationshowed retina with visible choroidal vessels and depigmentediridis (Figure 2BG) Waardenburg index was 0572 thusruling out dystopia canthorum Laboratory evaluation showednormal hemogram and routine biochemical parametersSerum creatine phosphokinase was 3218 IUl (nlt170 IUl)Nerve conduction velocity studies that involved right medianulnar and common peroneal and sural nerve were normal

Figure 1 The haplotype analysis of thefamily with microsatellite markers fromthe EDN3 candidate region The diseasehaplotype 2ndash1 is shown by black barsNote the affected individuals (IV-2 andIV-3) are homozygous for the diseasehaplotype whereas both parents III- 1and III-2 are heterozygous for thedisease haplotype and are thereforecarriers for the mutation An arrowmarks the index case

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Figure 2 Clinical features of the affected individuals A Photograph of affected individual IV-3 Note the classical Gowerrsquos sign on tryingto get up from the sitting position B Fundus photograph of affected individual IV-3 Note the depigmented retina and underlying choroidvessels C-F Transverse sections of skeletal muscle C and D from an unrelated normal individual and E and F from affected individual IV-3Note normal polygonal myofibers with peripheral nuclei and uniform diameter in panel C (hematoxylin and eosin staining) and normal positiveimmunostaining of DMD protein along the sarcolemma in all the fibers in panel D Note rounding variation in diameter central nucleiregenerating fibers and fibrosis in panel E (hematoxylin and eosin staining) and total absence of DMD staining in all the fibers in panel FG Partial facial photograph of affected individual IV-3 showing blue iris H Partial facial photograph of affected individual IV-2 showingheterochromia of iris I Partial facial photograph of paternal grandmother II-2 showing heterochromia of iris

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Electromyography of right biceps and vastus lateralis revealeda myopathic pattern Audiometric evaluation showedprofound sensorineural hearing loss and absence of bilateralbrainstem auditory evoked responses Magnetic resonanceimaging of the brain showed normal myelination pattern Aleft quadriceps biopsy revealed marked variation in fiberdiameter with evidence of internal nuclei occasionalmyophagocytosis regenerating fiber and splitting suggestiveof muscular dystrophy On immunohistochemistry with ananti-dystrophin antibody (Novocastra Laboratories UK)there was absence of staining of dystrophin (Figure 2F) Theabove clinical symptoms suggested that this patient has bothWS and DMD

Individual IV-2mdashThe 11-year-old elder brother of theindex patient had a history of Hirschsprung disease thatrequired surgery at the age of 3 months He had irisdiscoloration at birth but did not report any hearing problemtill the age of 8 years On examination he had heterochromiaof iris (Figure 2H) and a normal neurologic examinationOphthalmological evaluation revealed hypopigmented irisand choroid with visualization of the choroidal vesselsWaardenburg index was 04059 thus ruling out dystopiacanthorum Audiometry revealed profound hearing loss onleft side with absence of auditory brainstem evoked responses

Laboratory evaluation showed normal creatine kinase levelsNerve conduction velocity studies and magnetic resonanceimaging of the brain were normal The above clinicalsymptoms suggested that he has only WS and it could be WStype IV (WS4)

A detailed clinical examination of their parents eldersister and paternal grandmother showed heterochromic irisonly in the grandmother II-2 (Figure 2I)Mutation analysis Following informed consent three-fivemilliliter of peripheral blood sample was collected in aVacutainer EDTATM tube (Beckton-Dickinson FranklinLakes NJ) from each individual for genomic DNA isolationusing a Wizardreg Genomic DNA Extraction Kit (PromegaMadison WI) This research followed the tenets of theDeclaration of Helsinki and the guidelines of the IndianCouncil of Medical Research New Delhi Although theclinical features in the affected individual IV-2 suggested thisfamily to be of WS type IV we still went ahead and selectedtwo microsatellite markers from each of the eight WS loci(Table 1) and used them to genotype the family according toKumar et al [24]

For mutational analysis the entire coding region of theEDN3 gene (GenBank NM_0001142) was amplified usingprimers that amplify all coding exons and their intron-exon

TABLE 1 MICROSATELLITE MARKERS FROM THE CANDIDATE REGIONS OF EIGHT KNOWN WS LOCI

Locus Chromosome location Gene MarkerWS1WS3 2q361 PAX3 D2S2197 D2S2300

WS2A 3p141-p123 MITF D3S1296 D3S1566WS2B 1p21-p133 D1S495 D1S248WS2C 8p23 D8S561 D8S1819WS2D 8q1121 SNAI2 D8S1716 D8S1745

WS2EWS4C 22q131 SOX10 D22S1045 D22S1156WS4A 13q223 EDNRB D13S1281 D13S160WS4B 20q132-q133 EDN3 D20S171 D20S173

Marker order was established using the sequence map from the UCSC Genome Bioinformatics site

TABLE 2 DETAILS OF PCR PRIMERS USED IN MUTATION ANALYSIS OF THE EDN3 GENE

Exon Primer Primer sequence (5prime to 3prime) Tm (degC) Amplicon size (bp)1 EN1bF Fgaaaagcccgagccacagccggc 64 379 EN1bR Rccgcgacgcacatcttctccgcg 2 EN2F Fcagacattttgcttgctccacc 62 528 EN2R Rcaggctctgggctaactgagc 3 EN3F Fggcggtggttctcgctccacac 56 372 EN3R Rcaggatgtgactgaactatccta 4 EN4F Ftggggaacgcactaatgtgctca 62 336 EN4R Ragaaacggtccaccaaaggcacc 5 EN5F Fttccagtctggtggtaggctcg 56 458 EN5R Rgtattgttaagtggggactctttg

Abbreviations F forward primer R reverse primer bp base pairs and Tm Annealing temperature Works with 5 DMSO

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T AB

LE 3

GEN

OTY

PES O

F IN

DIV

IDU

ALS

FOR M

AR

KER

S FR

OM

DIF

FER

ENT

WS

LOC

I

Gen

otyp

eL

ocus

Mar

ker

Indi

vidu

al II

I-1

Indi

vidu

al II

I-2

Indi

vidu

al IV

-1In

divi

dual

IV-2

Indi

vidu

al IV

-3W

S1W

S3D

2S21

971

21

21

11

12

2

D2S

2300

1 1

1 2

1 1

1 1

1 2

WS2

AD

3S12

962

31

22

31

31

2

D3S

1566

3 4

1 2

1 3

2 3

2 4

WS2

BD

1S49

51

32

41

22

31

4

D1S

248

1 2

2 2

2 2

1 2

2 2

WS2

CD

8S56

11

11

21

11

11

2

D8S

1819

1 1

2 3

1 2

1 2

1 3

WS2

DD

8S17

162

21

31

22

31

2

D8S

1745

1 2

1 2

1 2

1 1

2 2

WS2

EW

S4C

D22

S104

52

31

21

21

21

2

D22

S115

62

31

32

32

32

3W

S4A

D13

S128

12

21

21

21

21

2

D13

S160

1 2

2 2

1 2

1 2

1 2

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junctions (Table 2) Primers were designed using the genesequence from the UCSC Genome Bioinformatics siteSequences and PCR conditions of these primers are providedin Table 2 Mutations were identified by sequencing the PCRproducts from an affected individual from the family on anABIprism A370-automated sequencer (PE BiosystemsFoster City CA) Once a mutation was identified all members

of the family were examined for the presence of the mutationby sequencing

The mutation analysis of the DMD gene was performedusing multiplex PCR and multiplex ligation-dependent probeamplification (MLPA) technique and a kit from MRC-Holland Amsterdam the Netherlands according to themanufacturerrsquos recommendations The kit contains specificprobes for each of the 79 exons of the DMD gene on Xp212

Figure 3 Mutation analysis of the EDN3 gene in the family A Sequencing chromatograms of individuals from the family Note thehomozygous change CgtT in both affected individuals IV-2 and IV-3 (marked by arrows) The normal sibling IV-1 is homozygous for thewild-type allele whereas both parents (III-2 and III-3) and grandmother (II-2) are heterozygous for the change (see double peaks marked byarrows) B RFLP analysis to show segregation of the mutation Note affected individuals have only 528 bp fragment due to loss of the Eco72Isite the normal sibling has 345 and 183 bp fragments and both carrier parents have all the three fragments C Conservation of the threonine(T) residue in different orthologs The threonine residues are shown in bold letters The number refers to the position of amino acid residue

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and thus allows analysis of deletion andor duplication of oneor more of these exons Briefly 200 ng DNA was denaturedand hybridized overnight at 60 degC with the probe mixSamples were then treated with Ligase 65 for 15 min at 54 degCThe reactions were stopped by incubation at 98 degC for 5 minFinally PCR amplification was performed with the specificSALSA FAM PCR primers Amplification products were runon an ABI PRISM 3730 Genetic Analyzer (PE BiosystemsFoster City CA) and the obtained data were analyzed by usingthe Gene Marker 20 Software Deletions of a probersquosrecognition sequence on the X-chromosome will lead to acomplete absence of the corresponding probe amplificationproduct in males whereas female heterozygotes arerecognizable by a 35ndash50 reduction in relative peak area

RESULTS AND DISCUSSIONThe visual inspection of the family suggested segregation ofWS as an autosomal recessive trait (Figure 1) Haplotype

analysis using microsatellite markers from each of the eightknown WS loci suggested linkage of the family to the WS4Blocus on chromosome 20q132-q133 (Figure 1 and Table 1)indicating that WS is caused by a mutation in the EDN3 geneThe genotypes of individuals for microsatellite markers fromthe other seven WS loci are given in Table 3 To determinethe exact nature of the mutation the entire coding region ofEDN3 from the affected individual IV-2 was amplified using5 sets of PCR primers (Table 2) and sequenced The resultsrevealed a novel homozygous substitution mutation c293CgtT in exon 2 changing codon position 98 from threonineto methionine (pT98M Figure 3A) The affected sibling IV-3was also homozygous for the mutation both parents (III-1 andIII-2) and paternal grandmother (II-2) were heterozygous andthe normal sibling IV-1 was homozygous for the wild-typeallele (Figure 3A) The 528 bp wild-type exon 2 ampliconcontains a site for the restriction endonuclease Eco72I

Figure 4 Deletion analysis of the DMD gene A Results of multiplex PCR showing the deletion of exons 49 50 and 51 (marked by arrows)in affected individual IV-3 B MLPA analysis of the affected individual IV-3 showing the deletion of exons 49 50 and 51 (marked by arrows)Abbreviation N normal unrelated individual

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TAB

LE 4

MU

TATI

ON

S REP

OR

TED

SO FA

R IN

TH

E ED

N3

GEN

E

Sl

Mut

atio

nE

xon

Nat

ure

ofm

utat

ion

Stat

e of

zyg

osity

Eff

ect o

n pr

otei

nE

thni

c or

igin

of fa

mily

Ref

eren

ce

1c

163G

gtT (p

E55

X)

2N

onse

nse

Hom

ozyg

ous

Pred

icte

d to

resu

lt in

com

plet

e ab

senc

e of

act

ive

form

of p

eptid

eFr

ench

[26]

2c

262_

263d

elG

Cin

sT(p

Ala

88Se

rfsX

12)

2Fr

ames

hift

Hom

ozyg

ous

Pred

icte

d to

resu

lt in

com

plet

e ab

senc

e of

act

ive

form

of p

eptid

e1

Fren

ch amp

1B

osni

an[2

9]

3c

277C

gtG (p

R93

G)

2M

isse

nse

Hom

ozyg

ous

Pred

icte

d to

impa

ir fu

rin-m

edia

ted

prot

eoly

ticcl

eava

ge o

f pre

proe

ndot

helin

Egyp

tian

[30]

4c

286C

gtT (p

R96

C)

2M

isse

nse

Hom

ozyg

ous

Pred

icte

d to

impa

ir fu

rin-m

edia

ted

prot

eoly

ticcl

eava

ge o

f pre

proe

ndot

helin

Fren

ch[2

]

5c

293C

gtA (p

T98

K)

2M

isse

nse

Hom

ozyg

ous

Pred

icte

d to

resu

lt in

abs

ence

of a

ctiv

e fo

rm o

fpr

otei

nIn

dian

[2]

6c

293C

gtT (p

T98

M)

2M

isse

nse

Hom

ozyg

ous

Pred

icte

d to

resu

lt in

abs

ence

of a

ctiv

e fo

rm o

fpr

otei

nIn

dian

Pres

ent s

tudy

7c

335A

gtG (p

H11

2R)

2M

isse

nse

Hom

ozyg

ous

Cou

ld d

isru

pt th

e fo

rmat

ion

of d

isul

phid

e bo

ndSp

anis

h[2

9]8

c38

0AgtG

(pY

127C

)3

Mis

sens

eH

omoz

ygou

sC

ould

alte

r dis

ulph

ide

bond

form

atio

n in

the

endo

thel

in a

ndo

r the

ETl

ike

pept

ide

regi

ons

Indi

an[2

8]

9c

476G

gtT (p

C15

9F)

3M

isse

nse

Hom

ozyg

ous

Pres

umab

ly re

sults

in le

ss e

ffic

ient

cle

avag

e or

even

com

plet

e fa

ilure

of c

leav

age

of p

repr

o-en

doth

elin

3 Pa

kist

ani

[22

5]

10c

507C

gtA (p

C16

9X)

3N

onse

nse

Het

eroz

ygou

sPr

even

ts th

e di

sulp

hide

bon

d fo

rmat

ion

and

prob

ably

gen

erat

es a

n in

appr

opria

tely

cle

aved

in

activ

e pr

oend

othe

lin

Yug

osla

vian

[27]

11c

517T

gtC (p

C17

3R)

3M

isse

nse

Com

poun

dhe

tero

zygo

sity

with

pT9

8K

Prev

ents

the

disu

lphi

de b

ond

form

atio

nIn

dian

[2]

Molecular Vision 2012 182022-2032 lthttpwwwmolvisorgmolvisv18a213gt copy 2012 Molecular Vision

2029

cleaving it into two fragments of 345 bp and 183 bp Themutation abolishes this restriction site thus retaining theundigested 528 bp amplicon in affected indivuduals Wetherefore developed a restriction fragment lengthpolymorphism (RFLP) analysis to determine if the mutationwas present in 50 normal controls The analysis showedabsence of the mutation in 50 ethnically matched controlsfurther suggesting that the c293CgtT change is a mutation(data not shown) The analysis also showed segregation of themutation in the family (Figure 3B) In addition the pathogenicnature of the mutation also comes from the fact that threonineresidue at position 98 is conserved in different EDN3orthologs from human to zebra fish (Figure 3C) Accordingto the Mutation Taster the mutation is predicted to be diseasecausing with a p-value (probability) of 052 We then used twoother in silico methods PolyPhen-2 and SIFT to see the effectof this mutation on the protein function The effect of thismutation on EDN3 was predicted to be probably damaging byPolyPhen-2 with a score of 1 (score ranges from 0 to a positivenumber where 0 is neutral and a high positive number isdamaging) SIFT analysis predicted this mutation to bedamaging and intolerant with a score of zero (score rangesfrom 0 to 1 where 0 is damaging and 1 is neutral)

Multiplex PCR and MLPA analyses of the affectedindividual IV-3 showed that he has a deletion from exons 49ndash51 in the DMD gene (Figure 4) MLPA analysis showedabsence of the deletion in his mother (III-2) suggesting thatit is a de novo mutation (data not shown) His sister (IV-1)also did not have this mutation (data not shown) This deletioncauses a complete absence of the DMD protein in muscletissues (Figure 2F) of the affected individual and thus causingthe phenotype

A total of 10 mutations have been reported so far in theEDN3 gene in WS patients of different ethnic origins [2925-30] The novel mutation pT98M reported here along withtwo other mutations pH112R and pT98K affect one of the21 amino acids that constitute active peptide from amino acidpositions 97ndash117 in endothelin-3 [229] Further with themutation reported here the total number of mutations in theEDN3 gene is 11 (Table 4)

Endothelins (EDNs) are a family of three active peptidesEDN1 END2 and EDN3 that act as ligands and recognizedby two G-protein coupled heptahelical receptors known asendothelin receptors EDNRA and EDNRB EDN3preferentially binds the EDNRB [31] EDN3 is produced aspreproendothelin-3 encoded by the EDN3 gene Thebiologically active 21 amino acid long EDN3 is produced byproteolytic cleavage of preproendothelin-3 by endopeptidasesto yield proendothelin-3 which is finally cleaved byendothelin converting enzyme-1 to produce the mature activeEDN3 [3233] Studies have shown that EDN3EDNRBinteraction is required for the proper development of neuralcrest derived melanocytes and enteric neurons [34-36] Mouse

models for homozygous mutations in the EDNRB or EDN3genes show pigmentation anomaly aganglionic megacolonand cochlear disorder [3137] We suggest that the diseasephenotype in the present family could be due to the absenceof the active form of the protein as the mutation pT98M mightimpair the EDN3 activity Although both parents (III-1 andIII-2) and grandmother (II-2) are heterozygous for themutation (Figure 3) only the grandmother showedheterochromic iris (Figure 2I) However it is not completelyunexpected as heterozygous individuals for the EDN3mutations have been reported to show a few clinical featuresof WS [2529] The manifestation of a few of the WS clinicalsymptoms in heterozygous individuals could be due toenvironmental factors multigenic inheritance modifier genesor stochastic events on cell fate or cell differentiation in earlyembryogenesis [2527]

In summary we report an interesting Indian family withmembers affected with two different disorders WS and DMDThe WS is caused by a novel missense mutation whereasDMD resulted due to a deletion The present information willbe useful to provide rapid prenatal diagnosis and geneticcounseling for WS to the family and their relatives using aPCR-RFLP method developed during the study

ACKNOWLEDGMENTSWe thank the patients and their family members for theirinvolvement in the study We also thank Dr Robert Churchand two anonymous reviewers for their suggestions toimprove the manuscript

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2 Pingault V Dorothee E Florence D Goossens M Marlin SBondurand N Review and update of mutations causingWaardenburg syndrome Hum Mutat 2010 31391-406[PMID 20127975]

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7 Hoth CF Milunsky A Lipsky N Sheffer R Clarren SKBaldwin CT Mutations in the paired domain of the humanPAX3 gene cause Klein-Waardenburg syndrome (WS-III) as

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2030

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Articles are provided courtesy of Emory University and the Zhongshan Ophthalmic Center Sun Yat-sen University PR ChinaThe print version of this article was created on 17 July 2012 This reflects all typographical corrections and errata to the articlethrough that date Details of any changes may be found in the online version of the article

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