GJB2 and GJB6 Mutations Genotypic and Phenotypic Correlations in a Large Cohort of Hearing-Impaired Patients

ORIGINAL ARTICLE GJB2 and GJB6 Mutations Genotypic and Phenotypic Correlations in a Large Cohort of Hearing-Impaired Patients

Sandrine Marlin, MD, PhD; Delphine Feldmann, MD, PhD; Hélène Blons, MD, PhD; Natalie Loundon, MD; Isabelle Rouillon, MD; Sébastien Albert, MD; Pierre Chauvin, PhD; Eréa-Noël Garabédian, MD; Rémy Couderc, MD, PhD; Sylvie Odent, MD; Alain Joannard, MD; Sébastien Schmerber, MD; Bruno Delobel, MD; Jacques Leman, MD; Hubert Journel, MD; Hélène Catros, MD; Cédric Lemarechal, MD; Hélène Dollfus, MD, PhD; Marie-Madeleine Eliot, MD; Jean-Louis Delaunoy, MD; Albert David, MD; Catherine Calais, MD; Valérie Drouin-Garraud, MD; Marie-Françoise Obstoy, MD; Cyril Goizet, MD; Françoise Duriez, MD; Florence Fellmann, MD; Jocelyne Hélias, MD; Jacqueline Vigneron, MD; Bettina Montaut, MD; Dominique Matin-Coignard, MD; Laurence Faivre, MD; Clarisse Baumann, MD; Patricia Lewin, MD; Christine Petit, PhD; Françoise Denoyelle, MD, PhD

Objectives: To analyze the clinical features of hearing all mutated alleles and was observed in 25 of 93 com- impairment and to search for correlations with the geno- pound heterozygous patients. Three novel GJB2 muta- type in patients with DFNB1. tions, 355del9, V95M, and 573delCA, were identified. Hearing impairment was frequently less severe in com- Design: Case series. pound heterozygotes 35delG/L90P and 35delG/N206S than in 35delG homozygotes. Moderate or mild hearing Setting: Collaborative study in referral centers, insti- impairment was more frequent in patients with 1 or 2 tutional practice. noninactivating mutations than in patients with 2 inactivating mutations. Of 93 patients, hearing loss was stable Patients: A total of 256 hearing-impaired patients se- in 73, progressive in 21, and fluctuant in 2. Progressive lected on the basis of the presence of biallelic mutations hearing loss was more frequent in patients with 1 or 2 in GJB2 or the association of 1 GJB2 mutation with the GJB6 deletion (GJB6-D13S1830)del. noninactivating mutations than in those with 2 inactivating mutations. In 49 families, hearing loss was com- Main Outcome Measures: The prevalence of GJB2 mu- pared between siblings with similar genotypes, and vari- tations and the GJB6 deletion and audiometric pheno- ability in terms of severity was found in 18 families (37%). types related to the most frequent genotypes. Conclusion: Genotype may affect deafness severity, but Results: Twenty-nine different GJB2 mutations were environmental and other genetic factors may also modu- identified. Allelic frequency of 35delG was 69%, and the late the severity and evolution of GJB2-GJB6 deafness. other common mutations, 313del14, E47X, Q57X, and L90P, accounted for 2.6% to 2.9% of the variants. Con- cerning GJB6,(GJB6-D13S1830)del accounted for 5% of Arch Otolaryngol Head Neck Surg. 2005;131:481-487

EARING LOSS IS THE MOST has been recognized to account for a large frequent sensory deficit. proportion of cases of nonsyndromic au- One child in 1000 is born tosomal recessive deafness (DFNB1). Fur- with severe or profound thermore, the gene that encodes con- deafness, and another 1 in nexin 26, GJB2, has been identified as 1000H becomes deaf before adulthood. Re- causative in DFNB1.1 The GJB2 gene is also cent work in human genetics indicates that involved in an autosomal dominant form 60% to 80% of cases of congenital deaf- of deafness, DFNA3, and in syndromes ness are of genetic origin in developed such as Vohwinkel or keratitis-ichtyosis- countries1 and that nonsyndromic senso- deafness syndrome. The GJB6 gene, which rineural hearing loss (NSSNHL) is mainly encodes connexin 30, maps close to GJB2. autosomal recessive. Since 1994, more than The GJB6 gene was first described as caus- 80 loci for NSSNHL have been identified, ative in a rare dominant form of deaf- 1 Author Affiliations are listed at and 35 different genes have been cloned. ness, DFNA3, and its implication in the end of this article. Despite extensive genetic heterogeneity, NSSNHL was ascertained through the Financial Disclosure: None. a single locus on chromosome band 13q12 identification of a large deletion that in-

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©2005 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/27/2021 cludes the GJB6 5Ј-end noncoding region and most of high-resolution temporal bone computed tomography the coding region. This genetic alteration was found in shows no inner ear malformation. Understanding the un- patients with hearing impairment who carry a GJB2 mu- derlying causes of the variability in DFNB1 deafness is tation in trans and at a homozygous state in some fami- of major importance in terms of genetic counseling. One lies.2-4 In those patients, it is still not clear whether the of these causes may be the GJB2-GJB6 genotype. There- deafness is due to a digenic pattern of inheritance or fore, the first step is to study genotype-phenotype cor- whether the deletion removes common GJB6 and GJB2 relations, and a large series of patients is necessary to vali- regulatory elements.5 date these correlations. Only 2 studies have been More than 70 different GJB2 mutations have been re- conducted to date, 1 in a small cohort of 31 patients with ported in the connexin-deafness database6 for recessive biallelic mutations26 and the second in a series of 277 pa- deafness (DFNB1), and 6 have been associated with a tients of various geographic origins.27 These previous stud- dominant form of inheritance (DFNA3). Three variants ies have shown, first, that homozygotes for truncating are of major importance in terms of frequency. In white mutations are more likely to express more severe hear- populations, GJB2 35delG was found to be the most fre- ing loss than other genotypes and, second, that some rare quent mutation in hearing-impaired children.7-10 The 2 genotypes could induce a less severe phenotype com- other mutations, 167delT and 235delC, are the most compared with 35delG homozygotes. mon pathogenic alleles in Ashkenazi Jews and Asians, re- In this article, we report on the genotypes and phe- spectively,11-13 2 common deletions with carrier frequen- notypes of 256 hearing-impaired patients with biallelic cies of 4% and 1%, respectively.12,13 The pathogenic role GJB2 or GJB6 alterations analyzed and documented be- of GJB2 mutations has been clearly established for many tween January 1, 1995, and March 1, 2004, following ho- variants but remains controversial for missense muta- mogenous guidelines through a prospective research col- tions such as M34T, V37I, and R127H. The M34T varia- laborative study conducted in France. tion was first described as a dominant mutation,14 but the description of normal-hearing carriers abolished this hy- METHODS pothesis.15 Furthermore, the description of normal- hearing individuals with compound heterozygotes car- CLINICAL EVALUATION rying an M34T allele and a GJB2 mutated allele in trans raised the possibility that M34T was a nonpathogenic vari- 16-18 Clinical data and samples were obtained from a prospective col- ant in vivo. Furthermore, the M34T allele has been lection compiled between January 1, 1995, and March 1, 2004, observed in the general population with a high fre- through a broad national program on deafness. A standard- quency of 1.2% to 1.5%.15,19 This high allele frequency ized database was developed using a software program (File- was confirmed in a large study20 of the general popula- Maker Pro 5; FileMaker Inc, Santa Clara, Calif). Clinical fea- tion of the South of France (81 of 7032 chromosomes) tures included age at onset; hearing thresholds; audiometric and in a study18 of normal-hearing individuals (4 of 232 configuration; evolution within 2-, 5-, and 10-year periods; bal- chromosomes). Because M34T allele frequency is not sig- ance symptoms and tests; pedigree; inner ear computed tomo- nificantly higher in patients with hearing loss than in the graphic scans; and genetic assessment. All data were reviewed general population, this suggests that M34T is a com- by a clinical geneticist (S.M.) who specializes in the treatment 18-20 of patients with hearing impairments. A subgroup of 256 pa- mon polymorphism. However, in vitro functional stud- tients with biallelic alterations in GJB2 or GJB6 as described in ies provide evidence that M34T could have an effect on the introduction was extracted from the database for the fol- 1 deafness. Thus, some researchers supported the hypoth- lowing study. Hearing loss was sporadic in 158 families and esis that the phenotypic consequences of the M34T al- familial (2 affected siblings in the same family) in 49 families. lele depend on the GJB2 mutated allele in trans or on the After medical questioning to determine age at deafness onset genetic background of the patients.15,21 Similarly, V37I and to exclude environmental causes, individuals with hear- and R127H mutations have also been described in pa- ing impairments underwent an otoscopic examination of the tients with nonsyndromic sensorineural hearing loss, and ear and a nose, throat, and general examination, with routine in vitro studies have suggested that these variations could assessment for signs suggestive of syndromic deafness per- act as recessive mutations.1 However, there is no signifi- formed by a clinical geneticist. Participants also underwent an ophthalmologic evaluation, including funduscopy and inves- cant increase in V37I and R127H allele frequency in deaf 15,20 tigation for hematuria and proteinuria. An electrocardiogram populations compared with general populations. Fur- was recorded. High-resolution temporal bone computed to- thermore, a high V37I allele frequency was recently ob- mography was performed. Hearing-impaired children and their served in the general population of Taiwan (8.5%; 35 of parents underwent pure-tone audiometry with a diagnostic au- 410 chromosomes).22 Also, a normal-hearing individual diometer in a soundproof room, with recording of pure-tone with 35delG/R127H was previously described.17 These air and bone conduction thresholds. Air conduction pure- observations suggest that M34T, V37I, and R127H could tone average (ACPTA) thresholds in the conversational fre- have no pathogenic effects. quencies (0.5, 1, 2, and 4 kHz) were calculated for each deaf ear and were used to grade the severity of deafness. Four lev- Nonsyndromic autosomal recessive deafness (DFNB1) Ͻ Յ has been reported previously.10,23-25 It is characterized by els were defined: mild (20 dB ACPTA 39 dB), moderate (40 dBϽACPTAՅ69 dB), severe (70 dBϽACPTAՅ89 dB), and pro- a prelingual onset. The severity of the deafness varies from found (Ն90 dB). The severity of deafness was defined by the mild to profound and may even vary among siblings. Hear- degree of hearing loss of the better ear. In accordance with the ing loss is generally stable but is occasionally progres- European Working Group on Genetics of Hearing Impair- sive or fluctuant. Audiometric curves are either flat or ment criteria, hearing loss was considered to be progressive when sloping, and hearing loss affects all frequencies. Finally, the patient lost more than 15 dB in the ACPTA thresholds in

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©2005 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/27/2021 the conversational frequencies as determined by comparing the results of 2 reliable audiometric tests performed at least 10 years Table 1. Frequency of Each GJB2 Mutation and of the GJB6 apart, and we added to this criterion a more than 8-dB loss in Deletion in 207 Unrelated Patients* tests performed 5 years apart. We considered deafness to be fluctuant when the hearing level had risen by more than 10 dB be- Mutation Alleles, No. tween 2 successive audiograms. c.35delG 287 For intrafamilial comparisons, we considered that an intra- (GJB6-D13S1830) del 21 familial variation was present when 1 sibling had severe or pro- c.313_326del (313del14) 12 found deafness and the other had moderate or mild deafness c.139G→T (E47X) 12 with a minimum 15-dB difference between ACPTA thresh- c.169C→T (Q57X) 11 olds. Blood samples on EDTA and informed consent were ob- c.269T←C (L90P) 11 tained from the patients and their parents for subsequent ge- c.617A→G (N206S) 6 netic analysis. The study was approved by the Committee for IVS1 ϩ 1G→A6 the Protection of Individuals in Biochemical Research, as re- c.167delT 6 quired by French legislation. c.358_360del (E120del)† 6 c.71G→A (W24X) 4 c. 283G→A (V95M) 4 c.229T←C (W77R) 4 MUTATION ANALYSIS c.1A→G (M1V) 3 c.551G→C (R184P) 3 c.31_68del (31del38) 2 Genomic DNA was isolated from whole blood using various ex- c.95G→A (R32H) 2 traction methods. Analysis of GJB2 was performed on some of c.633T→A (C211X) 2 23 the samples using direct sequencing and on some using dena- c.35insG 1 turing high-performance liquid chromatography followed by se- c.94C→T (R32C) 1 quencing. The GJB2 coding exon was amplified in 2 fragments. c.119C→A (A40E) 1 The following primer pairs were used: CX26ex2S: 5ЈGCA TTC c.235delC (235delC) 1 GTC TTT TCC AGA GCA and CX26DG2AS: 5ЈGAG CCT TCG c.246C→G (I82M) 1 ATG CGG ACC TT for the first fragment, CX26 2A: 5ЈGAG CCT c.269insT 1 TCG ATG CGG ACC TT and CX26ex2AS: TCA TCC CTC TCA c.333_334del (333-334delAA) 1 TGC TGT CT for the second fragment. Fragments were ampli- c.355_364del (355del9) 1 fied using AmpliTaq Gold (Applied Biosystems, Courtaboeuf, c.416G→A (S139N) 1 France). The first fragment was amplified using the following c.465T→A (Y155X) 1 program: 95°C for 8 minutes, 35 cycles at 95°C for 30 seconds, c.452T→G (M151R) 1 60°C for 30 seconds, and 72°C for 1 minute. The second frag- c.575_576del (575delCA)† 1 ment was amplified using touchdown polymerase chain reac- Total 414 tion: 95°C for 8 minutes, 10 cycles at 95°C for 25 seconds, 64°C (–1°C) for 25 seconds, 72°C for 30 seconds, 25 cycles at 95°C *The mutation nomenclature follows international nomenclature recommendations: c. designates the mutated nucleotide numbering with the for 25 seconds, 54°C for 25 seconds, and 72°C for 30 seconds. complementary DNA reference sequence. The mutation name at the protein To detect homozygous mutations, all amplification prod- level or the usual name found in the connexin-deafness database is given in ucts were mixed and denatured for 5 minutes at 95°C with nor- parentheses. mal control polymerase chain reaction to allow the generation †When the mutation occurs in a repeated sequence, the highest 3Ј of heteroduplex. Both mixes were run on a Wave 2100A ap- position was adopted. paratus (Transgenomic; Courtaboeuf) at 63°C. Additional analyses were performed at 64°C to analyze from codon 71 to codon 121 and at 57°C to analyze from codon 200 to codon 227 for STATISTICAL ANALYSIS the first and the second fragments, respectively. Denaturing high- performance liquid chromatography variant profiles were se- All statistical comparisons between groups were performed us- quenced using a Big Dye Terminator V.2 kit, following the manu- ing the Mantel-Haenszel ␹2 test, or the Fisher exact test when facturer’s instructions, on an ABI310 (Applied Biosystems) numbers were small. For genotype-phenotype correlation analy- application. The GJB2 exon 1 was only sequenced when a mono- sis, patients were classified into 2 groups: those with severe or allelic mutation was found in exon 2. Exon 1 was amplified us- profound defects and those with mild or moderate defects. This ing the Expand Long Template polymerase chain reaction sys- distribution is medically justified in terms of implications of tem (Roche, Meylan, France) with forward and reverse primers the hearing impairment on patient care. CX26 E1F 5ЈTCC GTA ACT TTC CCA GTC TCC GAG GGA AGA GG and CX26 ER1R 5ЈCCC AAG GAC GTG TGT TGG TCC AGC CCC. Sequencing was run as already described. Con- RESULTS cerning GJB6, specific polymerase chain reaction assay using the method described by del Castillo et al3 was performed to GJB2-GJB6 MUTATION SPECTRUM detect the presence of the (GJB6-D13S1830)del deletion when no mutation was identified or when a monoallelic mutation was A total of 29 different mutations of GJB2 and the GJB6 identified in GJB2. This alteration was then ascertained by se- deletion (GJB6-D13S1830)del were identified in 207 quencing. Mutation segregation was confirmed by molecular unrelated patients (Table 1). As expected, the 35delG analysis of other members of the family to study the linkage between mutations and disease. mutation was the most frequent, accounting for 69% of Patients with GJB2 variations considered to be polymor- the alleles. Six other mutations demonstrated an allele phisms or having an unclear pathogenic nature, that is, G4D, frequency higher than 2%: the GJB6 deletion (5%), V27I, M34T, V37I, F83L, R127H, E114G, V153I, c.-15C→T, 313del14 (2.9%), E47X (2.9%), L90P, and Q57X c.-34C→T, and c.558G→C, were excluded from the study. (2.6%).

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Patients With Hearing Loss, No. P Genotype Profound Severe Moderate Mild Total Value* 35delG/35delG 82 25 22 2 131 Reference (GJB6-D13S1830)del/35delG 17 2 2 0 21 .53 L90P/35delG 2 0 6 2 10 Ͻ.001 Q57X/35delG 5 5 0 0 10 .36 313del14/35delG 7 2 0 0 9 .36 E120del/35delG 1 1 0 2 4 .13 167delT/35delG 3 1 0 0 4 Ͼ.99 N206S/35delG 0 0 0 3 3 .005 Other genotypes 29 16 18 1 64 .03 Total 146 52 48 10 256

*Two-tailed Fisher exact test after grouping profound and severe deafness vs moderate and mild hearing loss.

GJB2 mutations. Twenty-five patients were identified as Table 3. Hearing Loss in 7 Patients Homozygous (GJB6-D13S1830)del and GJB2 compound heterozy- for a Rare GJB2 Mutation gotes. Not included in the 256 patients, we identified 16 patients as compound heterozygotes with M34T and a Mutation Type of Hearing Loss Patients, No. GJB2 mutation, 2 as homozygous for M34T, 5 as com- N206S/N206S Profound 1 pound heterozygous for V37I and a GJB2 mutation, and Mild 1 4 as homozygous for V37I, with 1 patient homozygous W24X/W24X Profound 1 Q57X/Q57X Profound 1 for R127H. 313del14/313del14 Severe 1 The hearing defect was profound in 146 patients, se- Moderate 1 vere in 52, moderate in 48, and mild in 10. To evaluate E120del/E120del Moderate 1 the impact on the deafness phenotype of a non-35delG mutation, we compared the phenotype of 35delG compound heterozygous with 35delG homozygous patients (Table 2). No difference in deafness severity was ob- Three new mutations were identified: 355del9 was observed with the GJB6 deletion or the GJB2 mutations served in 2 siblings associated in trans with 35delG, Q57X, 313del14, E120del, and 167delT. In contrast, the M151R was present in a sporadic case associated in trans L90P/35delG and N206S/35delG compound heterozy- with V95M, and 573delCA was identified in a sporadic gotes were statistically associated with a less severe phe- case associated in trans with 35delG. These mutations notype than 35delG homozygotes. were not observed in 100 chromosomes of normal- The hearing loss of 7 patients homozygous for a rare hearing individuals. The 355del9 mutation leads to the mutation is reported in Table 3. Mutations were clas- deletion of 3 amino acids, glutamic acids 119 and 120, sified according to their possible effect on the connexin and isoleucine 121, located in the second intracytoplas- 26 protein. Frameshifts, nonsense mutations, deletions, mic domain. Because it does not disrupt the open read- and insertions causing a disruption of the open reading ing frame, 335del9 probably does not modify the rest of frame were considered to be inactivating mutations. Mis- the protein. The M151R (c.452T→G) is located in the sense mutations, deletions, or insertions of 3 or a mul- third transmembrane segment of the protein. Methio- tiple of 3 nucleotides were considered to be noninacti- nine 151 is evolutionarily conserved in all the species stud- vating mutations. Moderate or mild hearing loss was more ied: Homo sapiens, Mus musculus, Cavia porcellus, Crice- frequent in patients with 2 noninactivating mutations or tus griseus, Macaca mulatta, and Pongo pygmaeus. The with 1 inactivating and 1 noninactivating mutation (20 573delCA mutation induces the creation of a stop codon of 38 patients) compared with patients with 2 inactivat- at position 195 and modifies the amino acids between ing mutations (38 of 218 patients; P=.001) (Figure 1). 191 and 195. We compared hearing loss between 2 siblings bear- ing the same genotype in 49 families. In 18 families (37%), DEAFNESS PHENOTYPE IN PATIENTS WITH the severity of the defect differed between siblings, that DFNB1 AND GENOTYPE-PHENOTYPE is, profound or severe in one sibling and moderate or mild CORRELATIONS in the other. Intrafamilial variability was present in 13 (35%) of 37 families with 2 inactivating mutations and In this study, genotypes and phenotypes of 256 patients in 5 (42%) of 12 families with 1 or 2 noninactivating mu- were analyzed. Genotyping of the 256 patients revealed tations, and this difference was not statistically signifi- that 131 were 35delG homozygotes and 72 were com- cant (P=.74). pound heterozygous for 35delG and a rare GJB2 muta- The evolution of the hearing defect was studied us- tion. Seven patients were homozygous for a rare GJB2 mu- ing tonal audiometric tests in 96 patients. The hearing tation and 21 were compound heterozygous for 2 rare level was stable in 73 patients (76%), progressive in 21

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©2005 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/27/2021 (22%), and fluctuant in 2 (2%). There was no statisti- 100 cally significant difference between the ages of patients with stable hearing loss and those with progressive hear- 80 ing loss: 20 years (range, 7-50 years) vs 19.3 years (range, 6-56 years), by Mann-Whitney-Wilcoxon test. Deafness 60 Mild evolution was found to significantly depend on geno- Moderate Severe Figure 2 40 type (P=.01) ( ). Profound Hearing Loss, % 20 COMMENT 0 I/I NI/I, NI/NI To date, the most frequent form of NSSNHL is DFNB1, Mutation due to mutations in GJB2 and to GJB6 deletion. DFNB1 Figure 1. Hearing loss in 3 classes of GJB2-GJB6 genotypes. Inactivating (I) represents up to 40% of NSSNHL cases in some popula- mutations were frameshift and nonsense mutations, insertions, and tions.28 In France, it has recently been shown that DFNB1 deletions, with a disrupted open reading frame. Noninactivating (NI) accounts for 32% of NSSNHL.29 In the present study, we mutations were missense mutations, deletions, or insertions of 3 or a multiple of 3 nucleotides. A significant difference (P=.001) was observed report molecular and phenotypic analyses of 256 pa- between the 2 genotype groups using the 2-tailed Fisher exact test. A tients with DFNB1, the largest cohort of patients with reduced contingency table combines in one group profound and severe DFNB1 reported in any single country using standard- deafness and in another group moderate and mild hearing loss. The ized data collection. Thirty different GJB2-GJB6 muta- reference groups were those with two I mutations. tions are reported; 35delG is the most frequent mutation observed, with an allele frequency of 69% (287 of 100 414). The 35delG mutation is the most frequent in white patients with DFNB1, but the allelic frequencies re- 80 ported vary by country: 88% in Italy, 55% in Spain, and 58.3% in the South of France, which is a lower figure than 60 Fluctuant in the present study.20,30,31 The second most frequent al- Progressive lele is the large deletion involving GJB6,(GJB6-D13S1830) 40 Stable del, which accounts for 5% of the alleles. The (GJB6- Hearing Loss, % 20 D13S1830)del allele frequency is high in Spain (7.6%- 9.7%), Israel (6%-7.1%), and the United Kingdom (5.9%) 0 but low in Italy and Belgium (1.4%).32,33 The highest I/I I/NI, NI/NI (GJB6-D13S1830)del frequency was observed in the South Mutation of France, where it accounts for 15% (9 of 60) of the mu- 20 Figure 2. Evolution of the hearing defect in GJB2-GJB6 genotype groups. tated alleles. Analysis of haplotypes associated with the Inactivating (I) mutations were frameshift and nonsense mutations, deletion suggests a common founder for Western Euro- insertions, and deletions, with a disrupted open reading frame. pean countries,32 possibly originating in the South of Noninactivating (NI) mutations were insertions and deletions without a disrupted open reading frame and missense mutations. The hearing loss France or in Spain, where the frequency is the highest. evolution was different in patients with two I mutations and in those with two Four other mutations were recurrent, with an allele fre- NI mutations or two I and one NI mutation: P=.01, 2-tailed Fisher exact test. quency greater than 2%: 310del14, E47X, L90P, and The reference groups were those with two I mutations. Q57X. Interestingly, 310del14 and L90P have frequently been found in Eastern European popula- ally depends on the less severe mutation.42 The distribu- tions.26,34-38 In our French cohort, 310del14 and L90P rep- tion of patients with profound and severe or moderate resented 3% and 2.5% of the alleles, respectively, possibly and mild hearing loss did not differ from that of the 35delG owing to Eastern migrations. In Ashkenazi Jews and Pal- homozygote group for the following mutations: GJB6 de- estinians, the most common GJB2 mutation is the 167delT letion, Q57X, 313del14, E102del, and 167delT. Con- mutation.11,39,40 In our French cohort, this variant rep- versely, L90P and N206S were associated with a higher resented only 6 of the 414 alleles. The 235delC Asiatic proportion of moderate or mild hearing loss. L90P has mutation12,13 was present with a 167delT deletion in trans already been described as being associated with nonsyn- in 1 family of Chinese origins. We did not identify the dromic sensorineural hearing loss43 in many studies. Be- African R143W GJB2 mutation41 in any of our patients. cause L90P is not observed in the general popula- In addition to the prevalence of GJB2-GJB6 mutations, tion,19,20 this mutation is considered to be deleterious. This we described 3 new mutations most likely to have patho- study did not include patients who carry mutations with genic consequences: 355del9, M151R, and 573delCA. controversial effects, such as M34T, V37I, and R127H, This study reports clinical findings for 256 patients which are frequent and mostly associated with a mild phe- with DFNB1. Profound or severe hearing loss was pres- notype. Nevertheless, we observed a significant milder ent in 198 patients (77%) and in 107 (82%) of 131 pa- hearing loss in patients with at least 1 noninactivating tients homozygous for 35delG. To study the effect of rare mutation. This could be explained by a residual activity mutations on the phenotype, we compared the pheno- of some connexin 26 mutants. type of patients carrying a rare mutation associated with In addition, we showed that the severity of the hear- 35delG with the phenotype of 35delG homozygous pa- ing defect can differ between siblings. Indeed, an intra- tients. Indeed, the phenotype of recessive disease usu- familial deafness variation was demonstrated in 36% of

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©2005 American Medical Association. All rights reserved. Downloaded From: https://jamanetwork.com/ on 09/27/2021 the families carrying various genotypes. This is in agree- tique, Hôpital Robert-Debré, Paris (Dr Baumann); Labo- ment with previous study results,23 and our study found ratoire Pasteur Cerba, Cergy-Pontoise (Dr Lewin); and an intrafamilial hearing loss variation even in cases of mild Unité de Génétique des Déficits Sensoriels, INSERM U587, mutations. This observation demonstrates that the DFNB1 Institut Pasteur, Paris (Dr Petit), France. phenotype is determined not only by the GJB2-GJB6 geno- Correspondence: Françoise Denoyelle, MD, PhD, and San- type but also by environmental factors and genetic back- drine Marlin, MD, PhD, Service d’ORL et de Chirurgie Cer- ground. vico-faciale, Unité de Génétique Médicale, Hôpital d’Enfants Finally, we observed that DFNB1 hearing loss is mostly Armand-Trousseau, AP-HP, 26 Avenue du Dr Arnold Net- stable (76%) but can be progressive and, in rare cases, ter, 75012 Paris, France ([email protected] and fluctuant. Although Cryns et al27 found no evidence of [email protected]). progressive hearing loss, this pattern has been observed Previous Presentation: This study was presented as a in patients with DFNB1.23,26 Also, we observed that pro- poster at the 54th Meeting of the American Society of Hu- gressive hearing loss was more frequent in patients with man Genetics; October 26-30, 2004; Toronto, Ontario. mild genotypes. It would be useful to examine the effect Funding/Support: This work was supported by the Fon- of environmental factors, such as infections or medica- dation pour la Recherche Medicale, the Association tions, on hearing loss increase in patients with DFNB1. Française Contre Les Myopathies, the Institut National This is the first homogenous study of GJB2 genotype- de la Sante et de la Recherche Medicale (INSERM), and phenotype correlations. It reviews the spectrum and dis- the Association “S’entendre,” Paris, France. tribution of mutations in France, confirms previous study Acknowledgment: We thank Catherine Magnier, DETAB, results, and broadens the spectrum of mild mutation. In- Corinne Chauve, DETAB, Isabelle Sargis, BTS; Christina deed, genotype is linked to phenotype for specific mu- Das Neves, DETAB, and France Laroze, DETAB, for their tations, but the role of genetic background and environ- excellent technical assistance. mental factors cannot be discounted, as shown by intrafamilial variability. Many factors remain to be studied to help with the genetic counseling and treatment of REFERENCES patients with NSSNHL. 1. Petit C, Levilliers J, Marlin S, Hardelin JP. Hereditary hearing loss. In: Scriver C, Submitted for Publication: February 8, 2005; accepted Beaudet A, Sly W, Valle D, Childs B, Kinzler K, Vogelstein B, eds. The Metabolic and Molecular Bases of Inherited Disease. 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