American Journal of Medical Genetics 137A:255–258 (2005)

High Prevalence of the W24X Mutation in the GeneEncoding Connexin-26 (GJB2) in Spanish Romani (Gypsies)With Autosomal Recessive Non-Syndromic Hearing LossAraceli Alvarez,1 Ignacio del Castillo,1 Manuela Villamar,1 Luis A. Aguirre,1 Anna Gonzalez-Neira,2

Alicia Lopez-Nevot,3 Miguel A. Moreno-Pelayo,1 and Felipe Moreno1*1Unidad de Genetica Molecular, Hospital Ramon y Cajal, Madrid, Spain2Unitat de Biologia Evolutiva, Facultat de Ciencies de la Salut i de la Vida, Universitat Pompeu Fabra, Barcelona, Spain3Servicio de ORL, Hospital Virgen de las Nieves, Granada, Spain

Molecular testing for mutations in the geneencoding connexin-26 (GJB2) at the DFNB1 locushas become the standard of care for geneticdiagnosis and counseling of autosomal recessivenon-syndromic hearing impairment (ARNSHI).The spectrum of mutations in GJB2 varies con-siderably among the populations, different allelespredominating in different ethnic groups. A cohortof 34 families of Spanish Romani (gypsies) withARNSHI was screened for mutations in GJB2. Wefound that DFNB1 deafness accounts for 50% of allARNSHI in Spanish gypsies. The predominatingallele is W24X (79% of the DFNB1 alleles), and35delG is the second most common allele (17%).An allele-specific PCR test was developed for thedetection of the W24X mutation. By using thistest, carrier frequencies were determined in twosample groups of gypsies from different Spanishregions (Andalusia and Catalonia), being 4% and0%, respectively. Haplotype analysis for micro-satellite markers closely flanking the GJB2 generevealed five different haplotypes associatedwith the W24X mutation, all sharing the sameallele from marker D13S141, suggesting that afounder effect for this mutation is responsible forits high prevalence among Spanish gypsies.� 2005 Wiley-Liss, Inc.

KEY WORDS: hearing impairment;DFNB1;GJB2;connexin-26; genetic epidemio-logy; gypsies

INTRODUCTION

Hearing impairment is a highly prevalent sensory disorderresulting from a variety of causes. In developed countries,

genetic causes are thought to account for more than 60% ofcases [Petit et al., 2001]. Non-syndromic hearing impairment(NSHI) includes a large number of conditions in which thehearing deficit is the only clinical sign. In amajority of cases it isinherited in an autosomal recessive pattern, with 38 differentloci and 20 genes known to date [Van Camp and Smith, 2005].

This almost unparalleled genetic heterogeneity complicatesthe molecular diagnosis of NSHI. Furthermore, the epidemio-logical data that have been collected to date indicate thatthere is also a great diversity in the prevalence of each geneticsubtype of NSHI in different populations. For example, muta-tions in GJB2, the gene encoding the gap-junction proteinconnexin-26, in the DFNB1 locus on 13q12, account for 0%–50% of all cases of autosomal recessive NSHI in differentcountries [Kenneson et al., 2002]. Moreover, GJB2 mutationspectra are also diverse. In most of the studies, a single allelepredominates, namely 35delG in Caucasians (up to 85% of allDFNB1 alleles in unrelated biallelic subjects) [Kenneson et al.,2002]; 167delT in Ashkenazi Jews (76%–81%) [Morell et al.,1998; Lerer et al., 2000; Sobe et al., 1999, 2000]; 235delC inJapanese (34%–75%) [Abe et al., 2000; Fuse et al., 1999; Kudoet al., 2000; Ohtsuka et al., 2003]; Chinese (58%–96%) [Liuet al., 2002a,b; Wang et al., 2002; Hwa et al., 2003]; andKoreans (56%) [Park et al., 2000]; R143W in Ghana (93%)[Brobby et al., 1998; Hamelmann et al., 2001]; and W24X inIndia (83%–96%) [Maheshwari etal., 2003;RamShankaretal.,2003]. A few other alleles have a moderate frequency, andmany are private mutations, having been reported in only oneor a few pedigrees. Identifying the most frequent mutationsleading to NSHI in a given population must contribute todevelop molecular diagnostic protocols well suited for thatpopulation,witha fewspecific tests capable of detectingmost ofmutant alleles. Here, we report data on the frequency andmutation spectrum of DFNB1 deafness in a cohort of SpanishGypsies with autosomal recessive NSHI.

MATERIALS AND METHODS

We enrolled 34 unrelated families of gypsies with autosomalrecessive NSHI, collected from all over Spain. Six of themincluded only one affected subject (simplex cases), and 28 hadat least two affected subjects (multiplex cases). In all these34 families, the mode of inheritance was compatible withan autosomal recessive pattern. Clinical examination of theaffected subjects excluded syndromic features, as well asputative environmental causes of their hearing impairment.Written informed consent was obtained from all the subjectsthatwereenrolled in this study.DNAextractionwasperformedfrom peripheral blood samples by standard procedures.

Detection of the 35delGmutation inGJB2was carried out aspreviously reported [Alvarez et al., 2003]. The screening forothermutationswas performed byDNAheteroduplex analysisof four overlapping PCR products spanning the GJB2 coding

Grant sponsor: European Community; Grant number: QLG2-CT-1999-00988; Grant sponsor: CAICYT of Spanish Ministerio deCiencia y Tecnologıa; Grant number: SAF2002-03966; Grantsponsor: Spanish ResearchNetwork on the Genetic andMolecularBases of Hearing Disorders; Grant number: FIS G03/203; Grantsponsor: Programa Ramon y Cajal; Grant sponsor: Spanish Fondode Investigaciones Sanitarias; Grant number: FIS PI020807.

*Correspondence to: Felipe Moreno, Unidad de GeneticaMolecular, Hospital Ramon y Cajal, Carretera de Colmenar, Km 9,28034 Madrid, Spain. E-mail: fmoreno.hrc@salud.madrid.org

Received 16 February 2005; Accepted 31 May 2005

DOI 10.1002/ajmg.a.30884

� 2005 Wiley-Liss, Inc.

region contained in exon 2, on Mutation Detection Enhance-ment gels (MDE, FMC Bioproducts, Rockland, ME, USA).Primers were as follows: Fragment 1, forward primer: 50-CAAACCGCCCAGAGTAGAAG-30; reverse: 50-GTGATCGTA-GCACACGTTCTTG-30. Fragment 2, forward: 50-CCAGGCTG-CAAGAACGTGTG-30; reverse: 50-TCGAAGATGACCCGGAA-GAA-30. Fragment 3, forward: 50-TCGAGGAGATCAAAACCC-AGAAG-30; reverse: 50-GCAAATTCCAGACACTGCAATCA-30.Fragment 4, forward: 50-GCCTTGTCCCAACACTGTGGACT-30; reverse: 50-TGAGCACGGGTTGCCTCATC-30. Heterodu-plex analysis was carried out on one patient and one parentfrom each family. In each positive case, the mutation wasidentified by DNA sequencing in an ABI Prism 310 GeneticAnalyzer. Detection of the del(GJB6-D13S1830) mutation wasperformed as previously reported [del Castillo et al., 2003].

Primers for allele-specific PCR for the detection of theW24Xmutationwereas follows:W24X-A3MUT, 50-CACCAGCATTG-GAAAGAACTA-30, and W24X-B, 50-GGGAGATGGGGAAG-TAGTGAT-30, for amplification of a 167-bp DNA segment onlyfrom W24X carriers; and those for fragment 4 (see above) foramplification of a 230-bp DNA segment of the GJB2 codingregion, which is used as a control to check the efficiency of thePCR. Multiplex PCR amplification with all four primers wasperformed in a Perkin-Elmer GeneAmp PCR System 9600,using the following program: one cycle of denaturation at 948Cfor 2 min; 30 cycles of denaturation at 948C for 40 sec, andannealing at 608C for 40 sec; and a final extension step of 728Cfor 7min. The reactionwas performed in afinal volume of 15 ml,at a final concentration of 1.5 mMMgCl2, using Fast Start TaqDNA polymerase (Roche, Basel, Switzerland). PCR productswere resolved by agarose gel electrophoresis (1.5% agarosegels).

Conditions for PCR amplification of microsatellite markersD13S141 [Hudson et al., 1992], D13S175 [Dib et al., 1996],(TG)n and (GAAA)n [Lerer et al., 2001] have been reportedpreviously.

RESULTS

We screened a cohort of 34 unrelated Spanish Romani(gypsy) families with autosomal recessive NSHI for mutationsin the GJB2 gene. In 18 cases (15 familial, 3 simplex), wedetectedpathogenicmutations inGJB2 (Tables I and II).Threeof these 18 cases carried only one mutant GJB2 allele. Since a309-kb deletion, del(GJB6-D13S1830), truncating the geneencoding connexin-30 (GJB6) has been shown to be the accom-

panying mutation in up to 50% of these unresolved hetero-zygotes from different populations [del Castillo et al., 2003],we screened those three cases for this deletion. It was foundin trans with the mutant GJB2 allele in two cases. In theremaining family, haplotype analysis allowed us to excludelinkage to DFNB1, indicating that the affected subject withtheW24Xmutationwas just a coincidental carrier. In addition,we found the non-pathogenic R127H allele in seven unrelatedaffected subjectswhodidnot carryany otherDFNB1mutation.Altogether, our data indicate that DFNB1 deafness accountsfor 50% of all autosomal recessive NSHI among Spanishgypsies.

Given the high prevalence of W24X among the RomaniDFNB1 alleles (Table II), we investigated the frequency of thismutation in two different sample groups of Spanish gypsies,one from Granada (Andalusia) and the other from Barcelona(Catalonia). An allele-specific PCR assay was developed andused to perform the screening (see Materials and Methods).We found the W24X mutation in 3 of 76 subjects (4.0%) inGranada’s sample group, but in none of 79 subjects in thesample group from Barcelona.

We investigated the evolutionary origins of W24X by study-ing haplotypes associated with this mutation. Four micro-satellitemarkers closely flanking theGJB2 genewere selectedfor this study. Their relative order and physical distanceswereas follows: (TG)n—42 kb—D13S141—37 kb—GJB2—29 kb—(GAAA)n—56 kb—D13S175. Cases suitable for haplotypeanalysis were genotyped for these markers, but here we onlyreport data on 20 chromosomes from unrelated cases in whichthe haplotype associated with the mutation could be deter-minedunambiguously (Table III).Allele sizeswere determinedby DNA sequencing of a control sample, which was used as astandard in genotyping assays. To allow other laboratories tocompare their datawith those reported in thiswork,weprovide

TABLE I. DFNB1 Genotypes Containing Pathogenic Mutations Found in Spanish Gypsies WithAutosomal Recessive NSHI

Genotype No. cases Type Ethnic origin

W24X/W24X 7 All familial(2 consanguineous)

Romani

35delG/W24X 6 2 familial, 2 simplex Romani1 simplex 50% Romani,

50% non-Romani1 familial 50% Romani,

50% unknownW24X/W77R 1 Familial 50% Romani,

50% non-Romani35delG/W24Xa; W24X/W24X 1 Familial RomaniW24X/del(GJB6-D13S1830) 2 1 familial Romani

1 familial 50% Romani,50% unknown

W24X/wild-typeb 1 Familial RomaniTotal 18 15 familial, 3 simplex

aBoth genotypes found in the same pedigree.bNot linked to DFNB1, according to haplotype analysis.

TABLE II. Occurrence of Pathogenic DFNB1 Alleles of RomaniOrigin in Spanish Gypsies

Allele Numbera Frequency (%)

W24X 23 79.335delG 5 17.2del(GJB6-D13S1830) 1 3.5Total 29 100.0

aOnly unrelated alleles of Romani origin in biallelic DFNB1 subjects wereincluded.

256 Alvarez et al.

allele sizes for individual 134702 (DNA sample available fromthe CEPH) [Dib et al., 1996] (Table III). We found five differenthaplotypes associated with W24X, all of them sharing allele124 from marker D13S141. Allele 208 from marker (TG)nwas found in 18 of 20 (90%) chromosomes carrying W24X(haplotypes A, B, and C) (Table III).

DISCUSSION

Epidemiological data on the prevalence and mutationspectrumof eachgenetic subtype ofNSHI in a givenpopulationare essential to improve the efficiency and the cost ofmoleculardiagnosis of these heterogeneous conditions. Here we reportdata on Spanish gypsies, a population of more than 500,000subjects distributed all over the country [Kalaydjieva et al.,2001]. In this sample, the DFNB1 subtype accounts for 50% ofall autosomal recessive NSHI, whereas in the rest of theSpanish population it is 30%–37% [Rabionet et al., 2000; ourunpublished data]. The spectra of mutations in the DFNB1locus are also clearly different. The predominating allele in theSpanish gypsy subpopulation, W24X, accounts for 79% of theDFNB1 alleles, whereas the most frequent allele in the non-Romani Spanish population, 35delG (55%–66% of the DFNB1alleles) [Rabionet et al., 2000; our unpublished data], onlyranks second in gypsies (17%). The del(GJB6-D13S1830)mutation, ranking second in the non-Romani Spanish popula-tion, is also present in gypsies, but with a low frequency.Conversely, W24X was found in the non-Romani Spanishpopulation in only 3 of 253 (1.2%) unrelated DFNB1 bialleliccases [unpublished data], and in one of these cases the ethnicorigin of the affected subject could not be investigated. Ourdata suggest that W24X would be a Romani founder mutationand that 35delG and del(GJB6-D13S1830) would have beenimported from non-Romani populations. It is remarkable thatthere are no unresolved GJB2 heterozygotes in our cohort ofRomani families after screening for del(GJB6-D13S1830). Ofnote, all the Romani DFNB1 genotypes found in this studycarry at least one W24X mutant allele, what should be takeninto account when performing molecular diagnosis of DFNB1hearing impairment in this ethnic minority. Here we havedescribed an allele-specific PCR test, of diagnostic value for thedetection of this mutation.

The W24X (c.71G>A) mutation was first described in aPakistani family [Kelsell et al., 1997], and later on it was alsofound in several Asian families [Scott et al., 1998; Kudo et al.,2001; Rickard et al., 2001; Najmabadi et al., 2002]. Recent dataindicate that it is the predominating DFNB1 allele in India[Maheshwari et al., 2003; RamShankar et al., 2003]. Duringthepreparation of this report,we learned thatW24Xwas foundto be the most common DFNB1 allele among Slovak gypsieswith autosomal recessive NSHI [Minarik et al., 2003]. Ourfindings in Spanish gypsies are confirmatory of the ethnic

origin of this mutation. In the Slovak and the Spanish cohorts,the second most common allele is 35delG. Furthermore, thenon-pathogenic R127H allele is frequent in both cohorts.Sample numbers are still small, however, to allow comparisonof other less common mutant alleles in the two populations.It is remarkable that the W24X carrier frequency in differentRomani subgroups of samples is highly variable, ranging from0.0% to 26.1% in four Slovak subgroups [Minarik et al., 2003],and being 0.0% to 4.0% in two Spanish subgroups. This situa-tion is most likely a consequence of the social structure of theRomani people, which must be considered a conglomerate ofgenetically isolated founder populations, with a high degree ofendogamy [Kalaydjieva et al., 2001].

The Romani people traces their origins to the Indiansubcontinent, from where they moved in successive migra-tions, arriving in Europe in the 11th century. By the 15thcentury, they were present in most of European countries[Kalaydjieva et al., 2001]. The finding of a high prevalence oftheW24Xmutation inSlovakandSpanish gypsies and in Indiatraces the origins of the mutation to the Indian subcontinentand suggests that this GJB2 mutant allele may be the com-monest in other European Romani populations. In fact, W24Xhas been reported in Czech gypsies [Seeman et al., 2004], and,without any indication of ethnicity, in Turkey [Uyguner et al.,2003], Hungary [Toth et al., 2004], Austria [Frei et al., 2002],Greece [Pampanos et al., 2002], and France [Roux et al., 2004],countries which have significant Romani minorities. In thiswork, we have found five different haplotypes associated withthe W24X mutation, all of them sharing the same allele frommarker D13S141, and 90% sharing also the same allele frommarker (TG)n, suggesting that a founder effect for thismutation is responsible for its high prevalence among Spanishgypsies. In fact, a founder effect for W24X has been postulatedalso in the Indian population [RamShankar et al., 2003].Comparison with haplotypes of other W24X carriers fromEurope and Asia, will help to understand better the evolu-tionary origins of this mutation.

ACKNOWLEDGMENTS

We thank the patients and the clinicians who participatedin this study, and FIAPAS for their enthusiastic support ofthis research. A.A., M.V., and L.A. were recipients of fellow-ships fromFondode InvestigacionesSanitarias,ComunidaddeMadrid, and Organizacion Nacional de Ciegos Espanoles,respectively. This work was supported by grants from theEuropean Community (QLG2-CT-1999-00988), CAICYT ofSpanish Ministerio de Ciencia y Tecnologıa (SAF2002-03966,to F.M.), Spanish Research Network on the Genetic andMolecular Bases of Hearing Disorders (FIS G03/203, to F.M.),Programa Ramon y Cajal (to I.d.C.), and Spanish Fondo deInvestigaciones Sanitarias (FIS PI020807, to I.d.C.).

TABLE III. Haplotypes of Romani Origin Associated With the W24X Mutation in the GJB2 Gene

Markera

Haplotypes Genotype forCEPH individual

134702

Allele frequency in controls

A B C D E Barcelona (N¼94) Granada (N¼ 140)

(TG)n 208 208 208 212 204 206/208 ND NDD13S141 124 124 124 124 124 126/126 124: 68% 124: 68%(GAAA)n 240 240 244 240 244 209/216 240: 27%; 244: 5% 240: 17%; 244: 5%D13S175 103 105 103 103 103 103/105 ND NDNumber of haplotypes(N¼ 20)

7 2 9 1 1

ND, not determined.aRelative order and physical distances are as follows: (TG)n—42 kb—D13S141—37 kb—GJB2—29 kb—(GAAA)n—56 kb—D13S175.

DFNB1 Mutations in Spanish Gypsies 257

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