Abstract
Mutations in GJB2, the gene encoding connexin-26 at the DFNB1 locus on 13q12, are found in as many as 50% of subjects with autosomal recessive, nonsyndromic prelingual hearing impairment. However, genetic diagnosis is complicated by the fact that 10%–50% of affected subjects with GJB2 mutations carry only one mutant allele. Recently, a deletion truncating the GJB6 gene (encoding connexin-30), near GJB2 on 13q12, was shown to be the accompanying mutation in ∼50% of these deaf GJB2 heterozygotes in a cohort of Spanish patients, thus becoming second only to 35delG at GJB2 as the most frequent mutation causing prelingual hearing impairment in Spain. Here, we present data from a multicenter study in nine countries that shows that the deletion is present in most of the screened populations, with higher frequencies in France, Spain, and Israel, where the percentages of unexplained GJB2 heterozygotes fell to 16.0%–20.9% after screening for the del(GJB6-D13S1830) mutation. Our results also suggest that additional mutations remain to be identified, either in DFNB1 or in other unlinked genes involved in epistatic interactions with GJB2. Analysis of haplotypes associated with the deletion revealed a founder effect in Ashkenazi Jews and also suggested a common founder for countries in Western Europe. These results have important implications for the diagnosis and counseling of families with DFNB1 deafness.
Hearing impairment is the most common sensory disorder. In developed countries, >60% of the cases are due to genetic causes (Petit et al. 2001). Nonsyndromic forms, in which the hearing deficit is the only clinical sign, are highly heterogeneous, with >80 loci already reported and 30 genes identified so far (Hereditary Hearing Loss Homepage). Hearing impairment that manifests before speech acquisition (i.e., with prelingual onset) is mainly inherited in an autosomal recessive pattern, with 31 different loci and 16 currently identified genes (Hereditary Hearing Loss Homepage).
The DFNB1 locus for nonsyndromic, autosomal recessive, prelingual hearing impairment (MIM 220290) was mapped to the 13q12 region (Guilford et al. 1994). This locus contains the GJB2 gene (MIM 121011), encoding connexin-26 (Cx26), a transmembrane protein subunit of intercellular gap junctions (Kelsell et al. 1997). Six monomers of connexin bind together to form a hexamer (connexon), which, in turn, docks with another connexon on the surface of an adjacent cell to form an intercellular gap-junction channel (Goodenough et al. 1996; Kumar and Gilula 1996). In the cochlea, there are two networks of gap junctions, the epithelial cell system and the connective tissue cell system, which are thought to be involved in the recycling of potassium back into the cochlear endolymph, where it plays an essential role in sound transduction (Kikuchi et al. 2000). In fact, targeted inactivation of the GJB2 gene in the inner-ear epithelial network led to hearing impairment in mice, demonstrating the key role of this network in cochlear function and cell survival (Cohen-Salmon et al. 2002). Several different connexins have been shown to participate in these gap junction systems. To date, mutations in the genes encoding three of these connexins (GJB2 for Cx26, GJB6 for Cx30 [MIM 604418], and GJB3 for Cx31 [MIM 603324]) are known to result in hearing impairment (Kelsell et al. 1997; Xia et al. 1998; Grifa et al. 1999). Among them, GJB2 stands out, because mutations in this gene account for as many as 50% of all cases of prelingual hearing impairment in many populations (Rabionet et al. 2000). More than 80 different mutations in GJB2 have been described in subjects with hearing impairment (Connexin-Deafness Homepage). Molecular testing for GJB2 mutations has rapidly become the standard of care for the diagnosis and counseling of patients with nonsyndromic hearing impairment of unknown cause.
Mutation screening of GJB2 in subjects with autosomal recessive hearing impairment has, however, revealed an unexpected problem—namely, a high number of patients carrying only one mutant allele. Exhaustive screening of the coding region (fully contained in exon 2), exon 1, and splice sites did not reveal any mutation in the second allele. These cases accounted for 10%–50% of all deaf subjects with at least one GJB2 mutation. These findings could be attributed to intrinsic limitations in the techniques used for mutation screening, to the high frequency of carriers for some GJB2 mutations, or to the existence of mutations in other noncoding parts of the gene that have not yet been identified. However, it was also suspected that other mutations might exist, in the DFNB1 locus but not in the GJB2 gene, that could provide an explanation for the high proportion of heterozygous affected subjects. Recently, this hypothesis received experimental support from the finding of a novel class of mutations in the DFNB1 locus, which were deletions not affecting GJB2 but truncating the neighboring GJB6 gene, which encodes Cx30. These deletions were found accompanying in trans the only GJB2 mutant allele in heterozygous affected subjects (double heterozygosity) (Lerer et al. 2001; del Castillo et al. 2002; Pallares-Ruiz et al. 2002) and were also found to be the cause of deafness in three unrelated affected subjects who were homozygous for the deletion (del Castillo et al. 2002; Pallares-Ruiz et al. 2002). In one study, the deletion breakpoint junction was isolated and sequenced, revealing the loss of a DNA segment of ∼342 kb, with one breakpoint inside the GJB6 coding region (del Castillo et al. 2002). This deletion, named “del(GJB6-D13S1830),” was the accompanying mutation in ∼50% of the deaf GJB2 heterozygotes (del Castillo et al. 2002). It remained to be determined whether the deletions detected by other groups (Lerer et al. 2001; Pallares-Ruiz et al. 2002) were also del(GJB6-D13S1830).
These findings may indicate a digenic pattern of inheritance of hearing impairment from mutations involving GJB2 and GJB6. This hypothesis is supported by several facts: (i) both Cx26 and Cx30 are expressed in the same inner-ear structures (Lautermann et al. 1998, 1999), (ii) connexons composed of Cx26 can bind connexons composed of Cx30 to form heterotypic gap-junction channels (Dahl et al. 1996), (iii) a mutation in GJB6 was reported in a case of autosomal dominant hearing impairment (Grifa et al. 1999), and (iv) Cx30-deficient mice exhibit a severe constitutive hearing impairment and lack an endocochlear potential (Teubner et al. 2003). However, the fact that point mutations in GJB6 have not yet been found in cases of autosomal recessive hearing impairment argues against this hypothesis. An alternative explanation is that the deletion may eliminate an upstream regulatory element for GJB2 that is essential for the normal expression of this gene in the inner ear. So far, such an element has not been found.
Given the association previously found between GJB2 monoallelic mutations and the del(GJB6-D13S1830) mutation in several studies (Lerer et al. 2001; del Castillo et al. 2002; Pallares-Ruiz et al. 2002), we investigated the contribution of this deletion to hearing impairment in nine countries. A genetic analysis of five microsatellite markers flanking the deletion breakpoints was also conducted, to determine the haplotypes associated with the del(GJB6-D13S1830) mutation and to explore its evolutionary origins. The implications of our results for molecular diagnosis of DFNB1 mutations are presented.
The study was performed on probands with nonsyndromic prelingual hearing impairment from nine countries (table 1). In Spain, Israel, and the United States, two independent studies were performed. After getting written informed consent, blood samples were obtained, and DNA was extracted by standard procedures. Testing for the del(GJB6-D13S1830) mutation was performed using the previously reported primers GJB6-1R (forward) and BKR-1 (reverse) (del Castillo et al. 2002), as well as a modification of the method to positively detect a wild-type product by adding another reverse primer that is located in the deleted segment of GJB6 (R2, 5′-TCATCGGGGGTGTCAACAAACA-3′). When these three primers were used together, two different PCR products were obtained—GJB6-1R→R2 (681 bp) and GJB6-1R→BKR-1 (460 bp)—allowing for discrimination between wild-type subjects (681-bp product), homozygotes for the deletion (460-bp product), and heterozygotes (both products) in a single test. In all subjects carrying the deletion, the PCR product that contains the breakpoint junction of the deletion was sequenced, to confirm that the breakpoint junction was identical in all cases. Thus, we confirmed that all the deletions reported by Lerer et al. (2001) were del(GJB6-D13S1830). In an independent study, it was also shown that the deletions reported by Pallares-Ruiz et al. (2002) were del(GJB6-D13S1830) (A. F. Roux, personal communication).
Table 1.
Results from the Screenings for the del(GJB6-D13S1830) Mutation
Country/Laboratory | No. of (del[GJB6-D13S1830]+GJB2) Double Heterozygotes/No. of GJB2 Heterozygotes | No. of del(GJB6-D13S1830) Homozygotes/Total Screened |
Spain/Madrida | 29/68 (42.6%)b | 1/425 (.2%) + 2 heterozygotesc |
Spain/Barcelona | 5/35 (14.3%) | 1/236 (.4%) |
Italy | 0/31 (.0%) | 0/238 (.0%) + 1 heterozygotec |
France | 23/60 (38.3%) | 0/208 (.0%) + 1 heterozygotec |
Belgium | 2/19 (10.5%) | 0/151 (.0%) |
United Kingdom | 6/19 (31.6%) | Not performed |
Israel/Tel Aviv | 7/20 (35.0%) | 1/191 (.5%) |
Israel/Jerusalem | 5/7 (71.4%) | Not performed |
United States/Virginia | 14/88 (15.9%) | 1/486 (.2%) + 4 heterozygotesc |
United States/Iowa | 7/95 (7.4%) | Not performed |
Brazil | 2/9 (22.2%) | Not performed |
Australia | 2/29 (6.9%) | Not performed |
This work expands the results of a previous study (del Castillo et al. 2002).
By performing haplotype analysis for genetic markers from 13q12, linkage to DFNB1 was excluded in 11 cases. After correction, the figures are 29/57 (50.9%).
Number of del(GJB6-D13S1830) heterozygotes with no mutation in GJB2.
Two different screenings were performed, the first on deaf subjects carrying only one GJB2 mutant allele, which had been found during routine testing for GJB2 mutations (tables 1 and 2); and the second on subjects with nonsyndromic prelingual hearing impairment, carrying no mutation in GJB2 (table 1). The frequency of the del(GJB6-D13S1830) allele among DFNB1 alleles is shown in table 3.
Table 2.
Impact of Screening for del(GJB6-D13S1830) on the Elucidation of Cases with Only One Mutant GJB2 Allele
No. of Monoallelic Subjects/No. of Monoallelic +No. of Biallelic Subjectsb |
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Country/Laboratory | GJB2 Testinga | Before Screeningfor the Deletion | After Screeningfor the Deletion |
Spain/Madrid | Ex2-CR, Ex1, SpS | 68/244 (27.9%) | 39/244 (16.0%)c |
Spain/Barcelona | Ex2-CR, Ex1, SpS | 35/68 (51.5%) | 30/68 (44.1%) |
Italy | Ex2-CR | 31/278 (11.2%) | 31/278 (11.2%) |
France | Ex2-CR, Ex1, SpS | 60/177 (33.9%) | 37/177 (20.9%) |
Belgium | Ex2-CR | 19/86 (22.1%) | 17/86 (19.8%) |
United Kingdom | Ex2-CR | 19/64 (29.7%) | 13/64 (20.3%) |
Israel/Tel Aviv | Ex2-CR, Ex1, SpS | 20/75 (26.7%) | 13/75 (17.3%) |
Israel/Jerusalem | Ex2-CR, Ex1, SpS | 7/44 (15.9%) | 2/44 (4.5%) |
United States/Virginia | Ex2-CR, Ex1, SpS | 88/251 (35.1%) | 74/251 (29.5%) |
United States/Iowa | Ex2-CR, Ex1, SpS | 95/305 (31.1%) | 88/305 (28.9%) |
Brazil | Ex2-CR, Ex1, SpS | 9/21 (42.9%) | 7/21 (33.3%) |
Australia | Ex2-CR, Ex1, SpS | 29/102 (28.4%) | 27/102 (26.5%) |
Ex2-CR = exon 2 coding region; Ex1 = entire exon 1; SpS = splice sites.
“Monoallelic” refers to subjects carrying only one mutant GJB2 allele; “biallelic” refers to subjects carrying two mutant GJB2 alleles.
After excluding cases not linked to DFNB1 according to haplotype analysis, results were 28/244 (11.5%).
Table 3.
Frequency of the del(GJB6-D13S1830) Allele among the DFNB1 Alleles
Country/Laboratory | No. of del(GJB6-D13S1830) Alleles/Total No. of DFNB1 allelesa |
Spain/Madrid | 31/408 (7.6%) |
Spain/Barcelona | 7/72 (9.7%) |
Italy | 0/494 (0%) |
France | 23/280 (8.2%) |
Belgium | 2/138 (1.4%) |
United Kingdom | 6/102 (5.9%) |
Israel/Tel Aviv | 9/126 (7.1%) |
Israel/Jerusalem | 5/84 (6.0%) |
USA/Virginia | 16/356 (4.5%) |
USA/Iowa | 7/434 (1.6%) |
Brazil | 2/28 (7.1%) |
Australia | 2/150 (1.3%) |
Only cases in which both DFNB1 alleles were identified contribute to this total.
The del(GJB6-D13S1830) allele is most frequent in Spain, France, the United Kingdom, Israel, and Brazil, accounting for 5.9%–9.7% of all the DFNB1 alleles (table 3). Its frequency is lower in Belgium and Australia (1.3%–1.4%), and it has not been found among Italian GJB2 unelucidated heterozygotes. In the United States, two different screenings yielded different results: a moderate frequency (4.5%, Virginia cohort) or a low frequency similar to those of Belgium and Australia (1.6%, Iowa cohort). A likely explanation for the higher frequency observed in the Virginia cohort is that it contains a higher proportion of individuals of Spanish descent than does the Iowa cohort, but the contribution of other factors cannot be excluded (for instance, differences in the proportion of probands who were the offspring of intermarriages among deaf people).
Before the screenings reported here, subjects with only one mutant GJB2 allele accounted for 11.2%–51.5% of those with (one or two) mutations in GJB2 in our cohorts of cases. The lowest percentage was observed in Italy (11.2%), a result that is consistent with the absence of the deletion in that screening. In screenings performed on large cohorts (>50 subjects) in other countries, data were, in general, rather homogeneous (22.1%–35.1%). Differences between the two screenings in Spain are probably due to regional bias in the Barcelona cohort (40% of cases are from the same Spanish region, Cantabria). It is noteworthy that screenings not including exon 1 and splice sites show percentages that are similar to those that do include them, a result that suggests a low frequency for mutations in these noncoding parts of the gene.
As expected, the observed frequencies for the del(GJB6-D13S1830) allele correlate with the percentage of cases with only one mutant GJB2 allele that were elucidated by the finding of the deletion (table 1). The highest figures correspond to France, Spain, Israel, and the United Kingdom (31.6%–71.4%). Differences between the two Israeli studies, both performed on Ashkenazi Jews, are likely to be due to the small size of the cohorts that were analyzed. On the other end, Australia, the United States, and Belgium show much lower percentages of elucidated cases (6.9%–15.9%). A slightly higher percentage was found in an independent study performed in the United States (Stevenson et al. 2003), with the deletion accounting for 20% of GJB2 deaf heterozygotes. After screening for the deletion, cases that remain not elucidated fell to 16.0%–20.9% in France, Spain, and Israel (results from larger cohorts) and are now closer to data from Italy (11.2%) (table 2). Moreover, in the screening performed by the Madrid team in Spain, genotyping and haplotype analysis for genetic markers close to GJB2 allowed us to exclude linkage to DFNB1 in 11 of 39 unelucidated cases, with the percentage of unelucidated cases then decreasing to 11.5% (table 2). In contrast, these figures remain high in all the other countries (19.8%–33.3% in Belgium, the United Kingdom, Australia, the United States, and Brazil). It is remarkable that Spanish subjects carrying a single mutation in GJB2 but without linkage to DFNB1 (coincidental carriers) account for at least 4.5% (11 of 244; data from the Madrid laboratory) of cases with GJB2 mutations. It must be taken into account that linkage to DFNB1 cannot be tested in simplex (sporadic) cases, impeding the detection of additional coincidental carriers, and so this figure undoubtedly is higher. However, even if we assume a higher percentage of coincidental carriers, it clearly emerges that other DFNB1 mutations remain to be identified in most countries.
The frequency of the del(GJB6-D13S1830) mutation in all populations is not high enough to result in a large number of homozygous subjects. They represent <0.5% of all cases of prelingual hearing impairment without mutations in GJB2 (table 1; none were born from consanguineous parents). It is noteworthy that four screenings detected del(GJB6-D13S1830) heterozygotes without accompanying DFNB1 mutation (table 1), which are to be added to the cases lacking elucidation.
We investigated the evolutionary origins of the deletion by studying haplotypes associated with this mutation. Five microsatellite markers closely flanking the deletion breakpoints were selected for this study. Their relative order and physical distances were as follows: D13S1835–117 kb–(TG)n–42 kb–D13S141–68 kb–(GAAA)n–4 kb–deletion proximal breakpoint–309 kb–deletion distal breakpoint–233 kb–D13S1831. Note that the deletion size, according to the latest sequencing data, is 309 kb (National Center for Biotechnology Information database, Homo sapiens genome view, build 33, contig NT_009799). Conditions for the PCR amplification of these markers have been reported elsewhere (Hudson et al. 1992; Kibar et al. 1999; Lerer et al. 2001). Cases suitable for haplotype analysis were genotyped for these markers, but we report here only those cases in which the haplotype associated with the deletion could be determined unambiguously. These included 52 nonrelated chromosomes: 28 from Spain (including 1 of Russian origin), 9 from Israel, 5 from France, 3 from the United Kingdom, 3 from the United States, 2 from Brazil, and 2 from Australia (tables 4 and 5). Allele sizes were determined by DNA sequencing of a control sample, which was shared by all the laboratories in this multicenter study and was used as a standard in genotyping assays. To allow other laboratories to compare their data with those reported in this work, we provide allele sizes for individual 134702, available from CEPH (Dib et al. 1996) (table 4).
Table 4.
Haplotypes Associated with the del(GJB6-D13S1830) Mutation
Allele in Haplotype |
|||||||||||||||||
Markera | Heterozygosityb(%) | A1 | A2 | A3 | A4 | B1 | B2 | B3 | B4 | B5 | B6 | C1 | D1 | E1 | E2 | F1 | Genotypefor CEPHIndividual134702 |
D13S1835 | 78 | 134 | 134 | 134 | 136 | 132 | 132 | 134 | 136 | 136 | 138 | 136 | 134 | 138 | 165 | 138 | 134/136 |
(TG)n | 65 | 204 | 204 | 204 | 204 | 208 | 208 | 208 | 208 | 208 | 208 | 210 | 204 | 208 | 208 | 208 | 206/208 |
D13S141 | 55 | 124 | 124 | 124 | 124 | 124 | 124 | 124 | 124 | 124 | 124 | 124 | 126 | 126 | 126 | 124 | 126/126 |
(GAAA)n | 79 | 209 | 209 | 209 | 209 | 209 | 209 | 209 | 209 | 209 | 209 | 209 | 209 | 209 | 209 | 205 | 209/216 |
D13S1831 | 84 | 96 | 103 | 105 | 105 | 87 | 105 | 105 | 87 | 105 | 105 | 105 | 105 | 105 | 105 | 101 | 99/101 |
Relative order and physical distances are as follows: D13S1835–117 kb–(TG)n–42 kb–D13S141–68 kb–(GAAA)n–4 kb–deletion proximal breakpoint–309 kb–deletion distal breakpoint–233 kb–D13S1831.
Calculated from 100 Spanish control chromosomes.
Table 5.
Distribution of Haplotypes Associated with the Deletion in Different Populations
Haplotype | N | Distribution |
Group A: | ||
A1 | 1 | Spain (Russian origin) |
A2 | 1 | Australia |
A3 | 21 | 11 Spain (10 Madrid, 1 Barcelona) 8 Israel (6 Tel Aviv, 2 Jerusalem), 2 France |
A4 | 1 |
Israel (Jerusalem) |
Total | 24 | |
Group B: | ||
B1 | 7 | Spain (Madrid) |
B2 | 2 | Spain (Madrid) |
B3 | 1 | Spain (Madrid) |
B4 | 1 | Spain (Madrid) |
B5 | 3 | 2 Spain (Madrid), 1 United Kingdom |
B6 | 9 |
2 Spain (Madrid), 2 France, 1 United Kingdom, 1 Australia 1 United States (Virginia), 2 Brazil (Portuguese origin) |
Total | 23 | |
Group C (C1) | 1 | France |
Group D (D1) | 1 | United States (Virginia) |
Group E: | ||
E1 | 1 | United Kingdom |
E2 | 1 |
Spain (Madrid) |
Total | 2 | |
Group F (F1) | 1 |
United States (Virginia) |
Total | 52 |
In 51 of 52 chromosomes carrying the deletion, we observed association with allele 209 from marker (GAAA)n, which is at a distance of only 4 kb from the deletion proximal breakpoint (frequency of this allele: 0.415 in Spain and 0.364 in Ashkenazi Jews). When considering a core haplotype constituted by markers (TG)n, D13S141, and (GAAA)n, we could define six different haplogroups, A–F (table 4). Finally, an expanded haplotype with all the five markers revealed 15 variants associated with the deletion (table 4). When examining the geographic distribution of haplotypes (table 5), it is remarkable that all nine Israeli chromosomes belong to group A (eight A3 and one A4; these two haplotypes differ only in D13S1835). Spanish haplotypes are mainly concentrated in groups A and B (11 A3, 15 B, and 1 E2), and the two Brazilian chromosomes are B6. Most chromosomes from other countries (France, the United Kingdom, and Australia) also belong to groups A and B. It is noteworthy that two of three chromosomes from the United States belong to groups D and F.
Our results show a clear founder effect for the del(GJB6-D13S1830) mutation in Ashkenazi Jews in Israel. Although the size of the sample should be increased to reach firmer conclusions in most of the analyzed populations, our data also suggest a common founder for the deletion in some countries in western Europe, since it is very scarce in Italy and has a low frequency in Belgium, whereas it is quite frequent in Spain and France, in Brazilian subjects of Portuguese origin, and—although to a lesser extent—in the United Kingdom (in all of these cases, the deletion is mainly associated with haplogroups A and B). The fact that the Ashkenazi affected subjects share a common haplotype along 464 kb contrasts with the diversity of haplotypes found in populations from western Europe, suggesting an older origin for the deletion in these countries. The existence of haplogroup F also suggests that the deletion could have other independent origins.
Inherited hearing impairment shows almost unparalleled genetic heterogeneity, not only in terms of numbers of genes and mutations, but also in prevalence of specific mutations among different populations. Therefore, data from genetic epidemiological studies are essential for the design of molecular diagnostic protocols well suited for each population. Our study provides some conclusions that should serve to improve the molecular diagnosis of DFNB1 cases. Given the simplicity of the test for the detection of the del(GJB6-D13S1830) mutation, it should be performed for all subjects with prelingual hearing impairment, at least in populations having higher frequencies. Our work also highlights the importance of performing haplotype analysis, when possible, in subjects with only one mutant GJB2 allele, to identify carriers whose hearing impairment might be due to mutations in a different gene or to hypothetical epistatic interactions between GJB2 mutations and other unlinked gene(s). This step would also help to concentrate research on the elucidation of cases consistent with linkage to DFNB1 by investigating other parts of GJB2 (promoter, 3′ UTR) and by searching for hypothetical point mutations in GJB6 or other DNA rearrangements in the DFNB1 locus.
Acknowledgments
We thank the patients and their relatives, for their kind cooperation in this study, and Federación Española de Asociaciones de Padres y Amigos de los Sordos, for their enthusiastic support of this research. F.J.d.C. and M.V. were recipients of fellowships from the Comunidad de Madrid. A.A. was a recipient of a fellowship from Fondo de Investigaciones Sanitarias. This work was supported by European Community grant QLG2-CT-1999-00988, Comisión Asesora Interministerial de Ciencia y Tecnología of Spanish Ministerio de Ciencia y Tecnología grant SAF99-0025 (to F.M.), Spanish Fondo de Investigaciones Sanitarias grants FIS 00/0244 and FIS PI020807 (to I.d.C.), a grant from the Flemish Fund for Scientific Research (to G.V.C.), a grant from the Israel Ministry of Science, Culture and Sport (to K.B.A.), a grant from The Garnett Passe and Rodney Williams Memorial Foundation (to H.H.M.D.), National Institutes of Health grant RO1-DC02842 (to R.J.H.S.), and a Consiglio Nazionale della Ricerche–Genomica Funzionale grant (to P.G.).
Electronic-Database Information
The accession number and URLs for data presented herein are as follows:
- Connexin-Deafness Homepage: http://www.crg.es/deafness/
- Hereditary Hearing Loss Homepage, http://www.uia.ac.be/dnalab/hhh/
- National Center for Biotechnology Information, http://www.ncbi.nlm.nih.gov/ (for contig NT_009799)
- Online Mendelian Inheritance in Man (OMIM), http://www.ncbi.nlm.nih.gov/Omim/ (for DFNB1, GJB2, GJB6, and GJB3)
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