GJB2 mutation spectrum in 2063 Chinese patients with nonsyndromic hearing impairment

  • Pu Dai1Email author,

    Affiliated with

    • Fei Yu1,

      Affiliated with

      • Bing Han1,

        Affiliated with

        • Xuezhong Liu3,

          Affiliated with

          • Guojian Wang1,

            Affiliated with

            • Qi Li1,

              Affiliated with

              • Yongyi Yuan1,

                Affiliated with

                • Xin Liu1,

                  Affiliated with

                  • Deliang Huang1,

                    Affiliated with

                    • Dongyang Kang1,

                      Affiliated with

                      • Xin Zhang1,

                        Affiliated with

                        • Huijun Yuan1,

                          Affiliated with

                          • Kun Yao4,

                            Affiliated with

                            • Jinsheng Hao5,

                              Affiliated with

                              • Jia He6,

                                Affiliated with

                                • Yong He7,

                                  Affiliated with

                                  • Youqin Wang8,

                                    Affiliated with

                                    • Qing Ye8,

                                      Affiliated with

                                      • Youjun Yu9,

                                        Affiliated with

                                        • Hongyan Lin10,

                                          Affiliated with

                                          • Lijia Liu11,

                                            Affiliated with

                                            • Wei Deng12,

                                              Affiliated with

                                              • Xiuhui Zhu13,

                                                Affiliated with

                                                • Yiwen You14,

                                                  Affiliated with

                                                  • Jinghong Cui14,

                                                    Affiliated with

                                                    • Nongsheng Hou15,

                                                      Affiliated with

                                                      • Xuehai Xu16,

                                                        Affiliated with

                                                        • Jin Zhang17,

                                                          Affiliated with

                                                          • Liang Tang17,

                                                            Affiliated with

                                                            • Rendong Song18,

                                                              Affiliated with

                                                              • Yongjun Lin18,

                                                                Affiliated with

                                                                • Shuanzhu Sun19,

                                                                  Affiliated with

                                                                  • Ruining Zhang20,

                                                                    Affiliated with

                                                                    • Hao Wu21,

                                                                      Affiliated with

                                                                      • Yuebing Ma22,

                                                                        Affiliated with

                                                                        • Shanxiang Zhu23,

                                                                          Affiliated with

                                                                          • Bai-lin Wu24,

                                                                            Affiliated with

                                                                            • Dongyi Han1Email author and

                                                                              Affiliated with

                                                                              • Lee-Jun C Wong2Email author

                                                                                Affiliated with

                                                                                Journal of Translational Medicine20097:26

                                                                                DOI: 10.1186/1479-5876-7-26

                                                                                Received: 05 December 2008

                                                                                Accepted: 14 April 2009

                                                                                Published: 14 April 2009

                                                                                Abstract

                                                                                Background

                                                                                Mutations in GJB2 are the most common molecular defects responsible for autosomal recessive nonsyndromic hearing impairment (NSHI). The mutation spectra of this gene vary among different ethnic groups.

                                                                                Methods

                                                                                In order to understand the spectrum and frequency of GJB2 mutations in the Chinese population, the coding region of the GJB2 gene from 2063 unrelated patients with NSHI was PCR amplified and sequenced.

                                                                                Results

                                                                                A total of 23 pathogenic mutations were identified. Among them, five (p.W3X, c.99delT, c.155_c.158delTCTG, c.512_c.513insAACG, and p.Y152X) are novel. Three hundred and seven patients carry two confirmed pathogenic mutations, including 178 homozygotes and 129 compound heterozygotes. One hundred twenty five patients carry only one mutant allele. Thus, GJB2 mutations account for 17.9% of the mutant alleles in 2063 NSHI patients. Overall, 92.6% (684/739) of the pathogenic mutations are frame-shift truncation or nonsense mutations. The four prevalent mutations; c.235delC, c.299_c.300delAT, c.176_c.191del16, and c.35delG, account for 88.0% of all mutantalleles identified. The frequency of GJB2 mutations (alleles) varies from 4% to 30.4% among different regions of China. It also varies among different sub-ethnic groups.

                                                                                Conclusion

                                                                                In some regions of China, testing of the three most common mutations can identify at least one GJB2 mutant allele in all patients. In other regions such as Tibet, the three most common mutations account for only 16% the GJB2 mutant alleles. Thus, in this region, sequencing of GJB2 would be recommended. In addition, the etiology of more than 80% of the mutant alleles for NSHI in China remains to be identified. Analysis of other NSHI related genes will be necessary.

                                                                                Introduction

                                                                                Hearing impairment is the most common neurosensory disorder in humans. The reported incidence varies from 1 in 300 to 1 in 1000 children [14]. Approximately half of cases have a genetic etiology, including syndromic and non-syndromic forms, with extraordinary genetic heterogeneity. Non-syndromic deafness accounts for 60–70% of inherited hearing impairment. It involves more than 100 different genes with autosomal dominant (DFNA), autosomal recessive (DFNB), X-linked (DFN), and maternal inheritance [5], with autosomal recessive being the most common. For many populations, the most common cause for non-syndromic autosomal recessive hearing loss is mutated Connexin 26, a gap junction protein encoded by the GJB2 gene (MIM 121011) [613]. There are a few specific mutations in GJB2 gene that are associated with the autosomal dominant syndromic forms of deafness, and typically present with skin abnormalities including keratitis-ichthyosis [1416].

                                                                                Connexins are transmembrane proteins. Six monomers of connexin proteins associate to form a transmembrane hexameric gap junction hemi-channel called a connexon. Connexons embedded in the surfaces of adjacent cells associate to form an intercellular channel [17, 18]. In the inner ear, connexin 26 can be in association with other connexins to form heteromeric connexons. Gap junction channels can be homotypic or heterotypic. Connexin 26 gap junction channels recycle potassium ions as part of a mechanism of auditory signal transduction in inner ear [19].

                                                                                Mutations in three connexin (Cx) genes, GJB2 (Cx26), GJB6 (Cx30), and GJB3 (Cx31), have been identified and are known to cause hearing impairment [18, 19]. Sequence analysis of the GJB2 gene in subjects with autosomal recessive hearing impairment revealed that a high number of patients carried only one mutant allele. Some of these families showed clear evidence of linkage to the DFNB1 locus, which contains two genes, GJB2 and GJB6 [6, 20]. Further analysis demonstrated that some GJB2 heterozygotes also carried a truncating deletion of the GJB6 gene, encoding connexin 30, in trans [21, 22].

                                                                                To date, more than 150 mutations, polymorphisms, and unclassified variants have been described in the GJB2 gene http://​davinci.​crg.​es/​deafness. The mutation spectrum and prevalence of mutations vary significantly among different ethnic groups. Three mutations, c.35delG, c.167delT, and c.235delC, are found to be the most frequent mutations in Caucasian, Ashkenazi Jewish, and Asian populations, respectively [6, 7, 913, 20, 2326].

                                                                                In China, it is estimated that 30,000 babies are born with congenital hearing impairment every year [27]. The mutation spectrum of the GJB2 gene in Chinese patients with nonsyndromic hearing impairment (NSHI) has not been analyzed. Our recent study by screening for just the most common mutation, c.235delC, in 3004 Chinese NSHI patients revealed that 488 (16.3%) patients carried at least one c.235delC mutant allele, with 233 (7.8%) homozygotes and 255 (8.5%) heterozygotes [28], though the frequencies of homozygote and heterozygote of c.235delC varied from 0% to 14.7% and from 1.7% to 16.1% respectively in the populations examined in this study. Among different Chinese sub-ethnic groups the c.235delC allele frequency was the lowest (0.8%) in the Tibetan and the highest (31.0%) in Maan. These results highlight the need to sequence the entire GJB2 gene in order to more accurately establish the actual mutation frequency and mutation spectrum of GJB2 gene within various Chinese sub-populations. Our preliminary results reveal that other GJB2 mutations account for an additional 7.1% of NSHI patients from Qinghai, where only 7.1% patients carried at least one c.235delC mutation. Nevertheless, sequencing analysis of the entire coding region of the GJB2 gene in patients from Guangxi where the frequency of the c.235delC mutation is 3.4% reveals only one other mutation in 87 deaf patients. These results have two important implications: that the GJB2 gene needs to be sequenced in its entirety; and that mutations in genes responsible for NSHI other than GJB2 should be searched in patients who do not harbor two mutant alleles in the GJB2 gene. In this study, we report the results of sequencing the GJB2 gene in 2063 patients with NSHI from 23 different regions of China (Figure 1).
                                                                                http://static-content.springer.com/image/art%3A10.1186%2F1479-5876-7-26/MediaObjects/12967_2008_Article_337_Fig1_HTML.jpg
                                                                                Figure 1

                                                                                Geographic distribution and the proportion of patients carrying at least oneGJB2mutant allele in each region studied.

                                                                                Materials and methods

                                                                                Patients and DNA samples

                                                                                A total of 2063 unrelated NSHI students from 23 different regions of China were included in this sequencing study. The selection of samples was random regardless of the c.235delC genotype. The patients consisted of 1179 males and 884 females ranging in age from 2 to 30 years with an average age of 13.7 ± 4.5. The majority of patients were Han Chinese (1640), followed by Tibetan (122), minorities in the Southwest region (119), Hui (79), minorities in Xinjiang (62), Mongolian (21), Maan (18) and Korean (2). Ethnic subgroup designations were based on permanent residency documentation.

                                                                                This study was performed according to a protocol approved by the ethics committee of the Chinese PLA General Hospital. The subjects in this study were from deaf schools of each region and were recently described [28]. Only the unrelated patients with nonsyndromic hearing loss were included in this study. Parents were not included in this study. All patients showed moderate to profound bilateral sensorineural hearing impairment on audiograms and no pathient with mild hearing impairment was found in this cohort. In addition to the 2063 patients, 301 Han control individuals with normal hearing (either evaluated by pure tone audiometry or by self-assessment) from Beijing Capital (Northern) and Jiangsu Province (Eastern), two densely populated regions consisting of 98% Han Chinese, were also analyzed. DNA was extracted from peripheral blood leukocytes using a commercially available DNA extraction kit (Watson Biotechnologies Inc, Shanghai, China).

                                                                                Sequence analysis

                                                                                The coding exon (Exon2) and flanking intronic regions of GJB2 gene were PCR amplified with forward primer 5'TTGGTGTTTGCTCAGGAAGA 3' and reverse primer 5'GGCCTACAGGGGTTTCAAAT 3'. Among this study cohort, 851 patients from central China were also analyzed for mutations in Exon1 and flanking introns by PCR/sequencing. The PCR primers used are forward primer: 5'CTCATGGGGGCTCAAAGGAACTAGGAGATCGG3' and reverse primer 5'GGGGCTGGACCAACACACGTCCTTGGG3'. The PCR products were purified on Qia-quick spin columns (Qiagen, Valencia, CA) and sequenced using the BigDye Terminator Cycle Sequencing kit (version v3.1) and ABI 3130 automated DNA sequencers (Applied Biosystems, Foster City, CA, USA,) with Sequence Analysis Software (Sequencing Analysis version 3.7). DNA sequence variations were identified by comparison of subject DNA sequence to GJB2 reference sequences, Genebank Accession Number AY280971. Numbering of GJB2 begins with the nucleotide A of the ATG start codon in Exon2 as cDNA position number 1. The sequences were analyzed using Genetool Lite software and the GJB2 Genebank sequence. The presence of 309 kb deletion of GJB6 was analyzed by PCR method [21, 22]. A positive control of this deletion provided by Balin Wu (Department of Laboratory Medicine, Children's Hospital and Harvard Medical School, USA.) was used for the detection of deletion in GJB6 gene.

                                                                                Statistical analysis

                                                                                The statistical analysis was performed using SAS 9.1.3 software (SAS, Cary, North Carolina, USA).

                                                                                Results

                                                                                Mutations in GJB2 gene

                                                                                Sequencing of the coding region of the GJB2 gene revealed that at least 104 different genotypes were found in the 2063 patients (Table 1). Among them, 64 different genotypes harboring pathogenic mutations were found in 432 patients (Table 1). Three hundred and seven patients had two confirmed pathogenic mutations, including 178 homozygotes and 129 compound heterozygotes. One hundred twenty five patients carried one heterozygous pathogenic mutation without an identified second mutant allele. Thus, GJB2 mutant alleles account for 17.9% (739/4126) of the total alleles in 2063 NSHI patients. The most common genotype was homozygous c.235delC, followed by compound heterozygosity for c.235delC/c.299_300delAT, which accounted for 8.0% (164/2063) and 3.2% (66/2063) of NSHI patients respectively. The most common mutation c.235delC was in compound heterozygosity with 14 other different pathogenic mutations in 113 patients, and was present as a single heterozygous mutant allele in 68 patients. In addition, there were 23 different genotypes in patients carrying one allele of unclassified variants (Table 1). Twenty-three alterations were found, five (p.W3X, c.99delT, c.155_c.158delTCTG, p.Y152X, and c.512_c.513insAACG) of them were novel and pathogenic, and twelve (p,G21R, p,I30F, p.F31L, p.V37I, p.V63L, p.T123N, p.V153A, p.D159N, p.F191L, p.M195V, p.V198M, and p.I215N) are unclassified variants (Table 1 and Supplemental Table 1). The distribution of various genotypes in 23 regions (Figure 1) is detailed in Table 2 and Supplemental Table 2. The frequencies of the three most common GJB2 mutations in the 23 regions studied are listed in Table 2. The allele frequency of all mutations in the GJB2 gene in NSHI patients varied from 4.0% in Guangxi to 30.4% in Jiangsu (Table 2). Regions which appeared to have a higher frequency of the c.235delC mutation (Jiangsu, Inner Mongolia, Beijing, Hebei, Shanghai) also had a relatively high frequency of other GJB2 mutations (eg, the frequency of the c.235delC mutation in Jiangsu was as high as 20.6% and the frequencies of other mutations were also as high as 9.8%). Similarly, regions such as Shaanxi and Guangxi where the frequency of the c.235delC mutation is low (5.8 and 3.4% respectively), also had lower frequencies of other mutations (1.9 and 0.6% respectively). Patients from Tibet, Yunnan, Xinjiang, Heilongjiang, and Ningxia appear to have the most diverse mutation spectrum because uncommon mutations (except c.235delC, c.299_c.300delAT and c.176_c.191del16) comprise 84.2, 30.8, 26.1, 21.4, and 20.4%, respectively of overall GJB2 mutations in those regions.
                                                                                Table 1

                                                                                GJB2 genotypes of 2063 Chinese NSHI patients

                                                                                Allele 1

                                                                                Allele 2

                                                                                nucleotide change

                                                                                consequence or amino acid change

                                                                                category

                                                                                domain

                                                                                nucleotide change

                                                                                consequence or amino acid change

                                                                                Category

                                                                                domain

                                                                                Number of patientsd

                                                                                homozygous

                                                                                        

                                                                                c.35delG

                                                                                frame-shift

                                                                                pathogenic

                                                                                NT

                                                                                c.35delG

                                                                                frame-shift

                                                                                Pathogenic

                                                                                 

                                                                                2

                                                                                c.176_c.191del16

                                                                                fram shift

                                                                                pathogenic

                                                                                EC1

                                                                                c.176_c.191del16

                                                                                frame-shift

                                                                                Pathogenic

                                                                                EC1

                                                                                2

                                                                                c.235delC

                                                                                frame-shift

                                                                                pathogenic

                                                                                TM2

                                                                                c.235delC

                                                                                frame-shift

                                                                                Pathogenic

                                                                                TM2

                                                                                164

                                                                                c.299_c.300delAT

                                                                                frame-shift

                                                                                pathogenic

                                                                                CL

                                                                                c.299_c.300delAT

                                                                                frame-shift

                                                                                pathogenic

                                                                                CL

                                                                                8

                                                                                c.512_c.513insAACG

                                                                                frame-shift

                                                                                pathogenic

                                                                                EC2

                                                                                c.512_c.513insAACG

                                                                                frame-shift

                                                                                pathogenic

                                                                                EC2

                                                                                1

                                                                                c.605_c.606ins46

                                                                                frame-shift

                                                                                pathogenic

                                                                                TM4

                                                                                c.605_c.606ins46

                                                                                frame-shift

                                                                                pathogenic

                                                                                TM4

                                                                                1

                                                                                compound heterozygous

                                                                                c.9G>A, c.79G>A

                                                                                p.W3X, p.V27I

                                                                                pathogenic, polymophism

                                                                                NT, TM1

                                                                                c.427C>T

                                                                                p.R143W

                                                                                pathogenic

                                                                                TM3

                                                                                1

                                                                                c.35delG

                                                                                frame-shift

                                                                                pathogenic

                                                                                NT

                                                                                c.299_c.300delAT

                                                                                frame-shift

                                                                                pathogenic

                                                                                CL

                                                                                1

                                                                                c.35delG

                                                                                frame-shift

                                                                                pathogenic

                                                                                NT

                                                                                c.313_c.326del14

                                                                                frame-shift

                                                                                pathogenic

                                                                                CL

                                                                                1

                                                                                c.176_c.191del16

                                                                                frame-shift

                                                                                pathogenic

                                                                                EC1

                                                                                c.9G>A, c.79G>A

                                                                                p.W3X, p.V27I

                                                                                pathogenic, polymophism

                                                                                NT+TM1

                                                                                2

                                                                                c.176_c.191del16

                                                                                frame-shift

                                                                                pathogenic

                                                                                EC1

                                                                                c.299_c.300delAT

                                                                                frame-shift

                                                                                pathogenic

                                                                                CL

                                                                                4

                                                                                c.176_c.191del16

                                                                                frame-shift

                                                                                pathogenic

                                                                                EC1

                                                                                c.388_c.397del10

                                                                                frame-shift

                                                                                pathogenic

                                                                                 

                                                                                1

                                                                                c.235delC

                                                                                frame-shift

                                                                                pathogenic

                                                                                TM2

                                                                                c.9G>A, c.79G>A

                                                                                p.W3X, p.V27I

                                                                                pathogenic, polymophism

                                                                                NT+TM1

                                                                                2

                                                                                c.235delC

                                                                                frame-shift

                                                                                pathogenic

                                                                                TM2

                                                                                c.35delG

                                                                                frame-shift

                                                                                pathogenic

                                                                                NT

                                                                                1

                                                                                c.235delC

                                                                                frame-shift

                                                                                pathogenic

                                                                                TM2

                                                                                c.35insG

                                                                                frame-shift

                                                                                pathogenic

                                                                                NT

                                                                                2

                                                                                c.235delC

                                                                                frame-shift

                                                                                pathogenic

                                                                                TM2

                                                                                c.94C>T

                                                                                p.R32C

                                                                                pathogenic

                                                                                TM1

                                                                                1

                                                                                c.235delC

                                                                                frame-shift

                                                                                pathogenic

                                                                                TM2

                                                                                c.99delT

                                                                                frame-shift

                                                                                pathogenic

                                                                                TM1

                                                                                1

                                                                                c.235delC

                                                                                frame-shift

                                                                                pathogenic

                                                                                TM2

                                                                                c.139G>T

                                                                                p.E47X

                                                                                pathogenic

                                                                                EC1

                                                                                3

                                                                                c.235delC

                                                                                frame-shift

                                                                                pathogenic

                                                                                TM2

                                                                                c.155_c.158delTCTG

                                                                                frame-shift

                                                                                pathogenic

                                                                                EC1

                                                                                2

                                                                                c.235delC

                                                                                frame-shift

                                                                                pathogenic

                                                                                TM2

                                                                                c.176_191del16

                                                                                frame-shift

                                                                                pathogenic

                                                                                EC1

                                                                                18

                                                                                c.235delC

                                                                                frame-shift

                                                                                pathogenic

                                                                                TM2

                                                                                c.257C>G

                                                                                p.T86R

                                                                                pathogenic

                                                                                TM2

                                                                                6

                                                                                c.235delC

                                                                                frame-shift

                                                                                pathogenic

                                                                                TM2

                                                                                c.299_c.300delAT

                                                                                frame-shift

                                                                                pathogenic

                                                                                CL

                                                                                65

                                                                                c.235delC

                                                                                frame-shift

                                                                                pathogenic

                                                                                TM2

                                                                                c.299_c.300delAT, c.79G>A

                                                                                frame-shift, p.V27I

                                                                                frame-shift, polymorphism

                                                                                CL+TM1

                                                                                1

                                                                                c.235delC

                                                                                frame-shift

                                                                                pathogenic

                                                                                TM2

                                                                                c.313_c.326del14

                                                                                frame-shift

                                                                                pathogenic

                                                                                CL

                                                                                1

                                                                                c.235delC

                                                                                frame-shift

                                                                                pathogenic

                                                                                TM2

                                                                                c.427C>T

                                                                                p.R143W

                                                                                pathogenic

                                                                                TM3

                                                                                3

                                                                                c.235delC

                                                                                frame-shift

                                                                                pathogenic

                                                                                TM2

                                                                                c.512_c.513insAACG

                                                                                frame-shift

                                                                                pathogenic

                                                                                EC2

                                                                                6

                                                                                c.235delC

                                                                                frame-shift

                                                                                pathogenic

                                                                                TM2

                                                                                c.605_c.606ins46

                                                                                frame-shift

                                                                                pathogenic

                                                                                TM4

                                                                                1

                                                                                c.299_c.300delAT

                                                                                frame-shift

                                                                                pathogenic

                                                                                CL

                                                                                c.139G>A

                                                                                p.E47K

                                                                                pathogenic

                                                                                EC1

                                                                                1

                                                                                c.299_c.300delAT

                                                                                frame-shift

                                                                                pathogenic

                                                                                CL

                                                                                c.257C>G

                                                                                p.T86R

                                                                                pathogenic

                                                                                TM2

                                                                                1

                                                                                c.299_c.300delAT

                                                                                frame-shift

                                                                                pathogenic

                                                                                CL

                                                                                c.512_c.513insAACG

                                                                                frame-shift

                                                                                pathogenic

                                                                                EC2

                                                                                3

                                                                                c.456C>A

                                                                                p.Y152X

                                                                                pathogenic

                                                                                TM3, CL

                                                                                c.380G>A, c.79G>A, c.341A>G

                                                                                p.R127H, p.V27I, E114G

                                                                                pathogenic, polymophism

                                                                                TM1+CL

                                                                                1

                                                                                heterozygous (one mutant allele only)

                                                                                c.11G>A

                                                                                p.G4D

                                                                                pathogenic

                                                                                NT

                                                                                c.109G>A

                                                                                p.V37I

                                                                                see note

                                                                                TM1

                                                                                1

                                                                                c.11G>A

                                                                                p.G4D

                                                                                pathogenic

                                                                                NT

                                                                                Nv

                                                                                   

                                                                                2

                                                                                c.35delG

                                                                                frame-shift

                                                                                pathogenic

                                                                                NT

                                                                                c.79G>A, c.341A>G

                                                                                p.V27I p,E114G

                                                                                polymorphism

                                                                                TM1+CL

                                                                                1

                                                                                c.35delG

                                                                                frame-shift

                                                                                pathogenic

                                                                                NT

                                                                                Nv

                                                                                   

                                                                                4

                                                                                c.155_c.158delTCTG

                                                                                frame-shift

                                                                                pathogenic

                                                                                EC1

                                                                                c.341A>G, c.644T>A

                                                                                p.E114G, p.I215N

                                                                                polymorphism, unclassified

                                                                                CL+CT

                                                                                1

                                                                                c.176_c.191del16

                                                                                frame-shift

                                                                                pathogenic

                                                                                EC1

                                                                                Nv

                                                                                   

                                                                                2

                                                                                c.235delC

                                                                                frame-shift

                                                                                pathogenic

                                                                                TM2

                                                                                c.109G>A

                                                                                p.V37I

                                                                                see note

                                                                                TM1

                                                                                11

                                                                                c.235delC

                                                                                frame-shift

                                                                                pathogenic

                                                                                TM2

                                                                                c.79G>A

                                                                                p.V27I

                                                                                polymorphism

                                                                                TM1

                                                                                6

                                                                                c.235delC

                                                                                frame-shift

                                                                                pathogenic

                                                                                TM2

                                                                                c.79G>A, c.341A>G

                                                                                p.V27I, p.E114G

                                                                                polymorphism

                                                                                TM1+CL

                                                                                5

                                                                                c.235delC

                                                                                frame-shift

                                                                                pathogenic

                                                                                TM2

                                                                                c.341A>G

                                                                                p.E114G

                                                                                polymorphism

                                                                                CL

                                                                                2

                                                                                c.235delC

                                                                                frame-shift

                                                                                pathogenic

                                                                                TM2

                                                                                c.558G>A

                                                                                p.T186T

                                                                                polymorphism

                                                                                EC2

                                                                                1

                                                                                c.235delC

                                                                                frame-shift

                                                                                pathogenic

                                                                                TM2

                                                                                Nv

                                                                                   

                                                                                43

                                                                                c.253T>C

                                                                                p.S85P

                                                                                pathogenic

                                                                                TM2

                                                                                Nv

                                                                                   

                                                                                1

                                                                                c.299_c.300delAT

                                                                                frame-shift

                                                                                pathogenic

                                                                                CL

                                                                                c.109G>A

                                                                                p.V37I

                                                                                see note

                                                                                TM1

                                                                                1

                                                                                c.299_c.300delAT

                                                                                frame-shift

                                                                                pathogenic

                                                                                CL

                                                                                c.79G>A, c.341A>G

                                                                                p.V27I, p.E114G

                                                                                polymorphism

                                                                                TM1+CL

                                                                                1

                                                                                c.299_c.300delAT

                                                                                frame-shift

                                                                                pathogenic

                                                                                CL

                                                                                Nv

                                                                                   

                                                                                4

                                                                                c.380G>A, c.341A>G

                                                                                p.R127H, p.E114G

                                                                                pathogenic, polymophism

                                                                                CL+CL

                                                                                c.109G>A

                                                                                p.V37I

                                                                                see note

                                                                                TM1

                                                                                1

                                                                                c.380G>A

                                                                                p.R127H

                                                                                pathogenic

                                                                                CL

                                                                                c.109G>A

                                                                                p.V37I

                                                                                see note

                                                                                TM1

                                                                                1

                                                                                c.380G>A, c.109G>A

                                                                                p.R127H, p.V37I

                                                                                pathogenic, polymophism

                                                                                TM1+CL

                                                                                c.79G>A

                                                                                p.V27I

                                                                                polymorphism

                                                                                TM1

                                                                                1

                                                                                c.380G>A, c.79G>A

                                                                                p.R127H, p.V27I

                                                                                pathogenic, polymophism

                                                                                TM1+CL

                                                                                c.79G>A, c.341A>G

                                                                                p.V27I, p.E114G

                                                                                polymorphism

                                                                                TM1+CL

                                                                                1

                                                                                c.380G>A

                                                                                p.R127H

                                                                                pathogenic

                                                                                CL

                                                                                c.79G>A, c.341A>G

                                                                                p.V27I, p.E114G

                                                                                polymorphism

                                                                                TM1+CL

                                                                                9

                                                                                c.380G>A, c.147C>T

                                                                                p.R127H, p.A49A

                                                                                pathogenic, polymophism

                                                                                EC1+CL

                                                                                c.79G>A

                                                                                p.V27I

                                                                                polymorphism

                                                                                TM1

                                                                                1

                                                                                c.380G>A, c.608T>C

                                                                                p.R127H, p.I203T

                                                                                pathogenic, polymophism

                                                                                CL+TM4

                                                                                c.79G>A, c.341A>G

                                                                                p.V27I, p.E114G

                                                                                polymorphism

                                                                                TM1+CL

                                                                                1

                                                                                c.380G>A, c.608T>C

                                                                                p.R127H, p.I203T

                                                                                pathogenic, polymophism

                                                                                CL+TM4

                                                                                c.79G>A

                                                                                p.V27I

                                                                                polymorphism

                                                                                TM1

                                                                                1

                                                                                c.380G>A

                                                                                p.R127H

                                                                                pathogenic

                                                                                CL

                                                                                c.79G>A

                                                                                p.V27I

                                                                                polymorphism

                                                                                TM1

                                                                                4

                                                                                c.380G>A

                                                                                p.R127H

                                                                                pathogenic

                                                                                CL

                                                                                c.457G>A

                                                                                p.V153I

                                                                                polymorphism

                                                                                TM3

                                                                                1

                                                                                c.380G>A

                                                                                p.R127H

                                                                                pathogenic

                                                                                CL

                                                                                Nv

                                                                                   

                                                                                10

                                                                                c.416G>A

                                                                                p.S139N

                                                                                pathogenic

                                                                                CL

                                                                                c.79G>A, c.341A>G

                                                                                p.V27I, p.E114G

                                                                                polymorphism

                                                                                TM1+CL

                                                                                1

                                                                                c.416G>A

                                                                                p.S139N

                                                                                pathogenic

                                                                                CL

                                                                                Nv

                                                                                   

                                                                                1

                                                                                c.424_c.426del3

                                                                                p.del142F

                                                                                pathogenic

                                                                                TM3

                                                                                c.79G>A, c.341A>G, c.109G>A

                                                                                p.V27I, p.E114G, p.V37I

                                                                                polymorphisms, see note

                                                                                TM1+CL

                                                                                3

                                                                                c.424_c.426del3

                                                                                p.del142F

                                                                                pathogenic

                                                                                TM3

                                                                                c.79G>A, c.109G>A

                                                                                p.V27I, p.V37I

                                                                                polymorphisms, see note

                                                                                TM1

                                                                                1

                                                                                c.512_c.513insAACG

                                                                                frame-shift

                                                                                pathogenic

                                                                                EC2

                                                                                c.79G>A, c.368C>A

                                                                                p.V27I, p.T123N

                                                                                polymorphism, unclassified

                                                                                TM1+CL

                                                                                1

                                                                                c.512_c.513insAACG

                                                                                frame-shift

                                                                                pathogenic

                                                                                EC2

                                                                                Nv

                                                                                   

                                                                                1

                                                                                unclassified variant

                                                                                c.61G>C, c.79G>A

                                                                                p.G21R, p.V27I

                                                                                unclassified, polymorphism

                                                                                NT+TM1

                                                                                c.79G>A, c.341A>G

                                                                                p.V27I, p.E114G

                                                                                polymorphism

                                                                                TM1+CL

                                                                                1

                                                                                c.88A>T

                                                                                p.I30F

                                                                                unclassified

                                                                                TM1

                                                                                Nv

                                                                                   

                                                                                1

                                                                                c.93T>G

                                                                                p.F31L

                                                                                unclassified

                                                                                TM1

                                                                                c.79G>A, c.341A>G

                                                                                p.V27I, p.E114G

                                                                                polymorphism

                                                                                TM1+CL

                                                                                1

                                                                                c.187G>T

                                                                                p.V63L

                                                                                unclassified

                                                                                EC1

                                                                                Nv

                                                                                   

                                                                                2

                                                                                c.368C>A, c.79G>A

                                                                                p.T123N, p.V27I

                                                                                unclassified, polymorphism

                                                                                CL+TM1

                                                                                c.79G>A, c.341A>G

                                                                                p.V27I, p.E114G

                                                                                polymorphism

                                                                                TM1+CL

                                                                                1

                                                                                c.368C>A, c.79G>A

                                                                                p.T123N, p.V27I

                                                                                unclassified, polymorphism

                                                                                CL+TM1

                                                                                c.79G>A

                                                                                p.V27I

                                                                                polymorphism

                                                                                TM1

                                                                                3

                                                                                c.368C>A

                                                                                p.T123N

                                                                                unclassified

                                                                                CL

                                                                                c.79G>A

                                                                                p.V27I

                                                                                polymorphism

                                                                                TM1

                                                                                7

                                                                                c.368C>A, c.608T>C

                                                                                p.T123N, p.I203T

                                                                                unclassified, polymorphism

                                                                                CL+TM4

                                                                                c.79G>A

                                                                                p.V27I

                                                                                polymorphism

                                                                                TM1

                                                                                1

                                                                                c.458T>C

                                                                                p.V153A

                                                                                unclassified

                                                                                EC2

                                                                                c.608T>C

                                                                                p.I203T

                                                                                polymorphism

                                                                                TM4

                                                                                1

                                                                                c.571T>C, c.592G>A

                                                                                p.F191L, p.V198M

                                                                                unclassified

                                                                                TM4+TM4

                                                                                c.79G>A

                                                                                p.V27I

                                                                                polymorphism

                                                                                TM1

                                                                                1

                                                                                c.583A>G

                                                                                p.M195V

                                                                                unclassified

                                                                                TM4

                                                                                Nv

                                                                                   

                                                                                1

                                                                                c.583A>G

                                                                                p.M195V

                                                                                unclassified

                                                                                TM4

                                                                                c.79G>A, c.341A>G

                                                                                p.V27I, p.E114G

                                                                                polymorphism

                                                                                TM1+CL

                                                                                1

                                                                                c.592G>A, c.79G>A, c.341A>G

                                                                                p.V198M, p.V27I, p.E114G

                                                                                unclassified, polymorphism

                                                                                TM4+TM1+CL

                                                                                c.79G>A, c.341A>G

                                                                                p.V27I, p.E114G

                                                                                polymorphism

                                                                                TM1+CL

                                                                                1

                                                                                c.592G>A, c.79G>A

                                                                                p.V198M, p.V27I

                                                                                unclassified, polymorphism

                                                                                TM4+TM1

                                                                                c.79G>A, c.341A>G

                                                                                p.V27I, p.E114G

                                                                                polymorphism

                                                                                TM1+CL

                                                                                1

                                                                                c.592G>A

                                                                                p.V198M

                                                                                unclassified

                                                                                TM4

                                                                                c.79G>A, c.341A>G

                                                                                p.V27I, p.E114G

                                                                                polymorphism

                                                                                TM1+CL

                                                                                2

                                                                                c.475G>A

                                                                                p.D159N

                                                                                unclassified

                                                                                EC2

                                                                                Nv

                                                                                  

                                                                                TM1+CL

                                                                                1

                                                                                c644T>A, c.79G>A, c.341A>G

                                                                                p.I215N, p.V27I, p.E114G

                                                                                unclassified, polymorphism

                                                                                CT+TM1+CL

                                                                                c.79G>A, c.341A>G

                                                                                p.V27I, p.E114G

                                                                                polymorphism

                                                                                TM1+CL

                                                                                1

                                                                                c.644T>A

                                                                                p.I215N

                                                                                unclassified

                                                                                CT

                                                                                c.608T>C

                                                                                p.I203T

                                                                                polymorphism

                                                                                TM4

                                                                                1

                                                                                c.109G>A

                                                                                p.V37I

                                                                                see note

                                                                                TM1

                                                                                c.109G>A

                                                                                p.V37I

                                                                                see note

                                                                                TM1

                                                                                23

                                                                                c.109G>A

                                                                                p.V37I

                                                                                see note

                                                                                TM1

                                                                                c.79G>A, c.341A>G

                                                                                p.V27I, p.E114G

                                                                                polymorphism

                                                                                TM1+CL

                                                                                29

                                                                                c.109G>A

                                                                                p.V37I

                                                                                see note

                                                                                TM1

                                                                                c.79G>A

                                                                                p.V27I

                                                                                polymorphism

                                                                                TM1

                                                                                10

                                                                                c.109G>A

                                                                                p.V37I

                                                                                see note

                                                                                TM1

                                                                                c.608T>C

                                                                                p.I203T

                                                                                polymorphism

                                                                                TM4

                                                                                3

                                                                                c.109G>A

                                                                                p.V37I

                                                                                see note

                                                                                TM1

                                                                                Nv

                                                                                   

                                                                                91

                                                                                polymorphism

                                                                                c.79G>A, c.341A>G

                                                                                p.V27I, p.E114G

                                                                                polymorphism

                                                                                TM1+CL

                                                                                c.79G>A, c.341A>G

                                                                                p.V27I, p.E114G

                                                                                polymorphism

                                                                                TM1+CL

                                                                                90

                                                                                c.79G>A

                                                                                p.V27I

                                                                                polymorphism

                                                                                TM1

                                                                                c.79G>A

                                                                                p.V27I

                                                                                polymorphism

                                                                                TM1

                                                                                18

                                                                                c.79G>A, c.341A>G

                                                                                p.V27I, p.E114G

                                                                                polymorphism

                                                                                TM1+CL

                                                                                c.79G>A

                                                                                p.V27I

                                                                                polymorphism

                                                                                TM1

                                                                                42

                                                                                c.79G>A, c.341A>G

                                                                                p.V27I, p.E114G

                                                                                polymorphism

                                                                                TM1+CL

                                                                                c.341A>G

                                                                                p.E114G

                                                                                polymorphism

                                                                                 

                                                                                2

                                                                                c.79G>A, c.341A>G

                                                                                p.V27I, p.E114G

                                                                                polymorphism

                                                                                TM1+CL

                                                                                c.457G>A

                                                                                p.V153I

                                                                                polymorphism

                                                                                TM3

                                                                                1

                                                                                c.79G>A, c.341A>G

                                                                                p.V27I, p.E114G

                                                                                polymorphism

                                                                                TM1+CL

                                                                                c.608T>C

                                                                                p.I203T

                                                                                polymorphism

                                                                                TM4

                                                                                12

                                                                                c.79G>A, c.341A>G

                                                                                p.V27I, p.E114G

                                                                                polymorphism

                                                                                TM1+CL

                                                                                Nv

                                                                                   

                                                                                387

                                                                                c.79G>A

                                                                                p.V27I

                                                                                polymorphism

                                                                                TM1

                                                                                c.608T>C

                                                                                p.I203T

                                                                                polymorphism

                                                                                TM4

                                                                                5

                                                                                c.79G>A, c.608T>C

                                                                                p.V27I

                                                                                polymorphism

                                                                                TM1+TM4

                                                                                c.608T>C

                                                                                p.I203T

                                                                                polymorphism

                                                                                TM4

                                                                                1

                                                                                c.79G>A

                                                                                p.V27I

                                                                                polymorphism

                                                                                TM1

                                                                                Nv

                                                                                   

                                                                                202

                                                                                c.147C>T

                                                                                p.A49A

                                                                                polymorphism

                                                                                EC1

                                                                                Nv

                                                                                   

                                                                                1

                                                                                c.181A>G

                                                                                p.K61K

                                                                                polymorphism

                                                                                EC1

                                                                                Nv

                                                                                   

                                                                                1

                                                                                c.341A>G

                                                                                p.E114G

                                                                                polymorphism

                                                                                CL

                                                                                Nv

                                                                                   

                                                                                14

                                                                                c.438C>T

                                                                                p.F146F

                                                                                polymorphism

                                                                                TM3

                                                                                Nv

                                                                                   

                                                                                2

                                                                                c.608T>C

                                                                                p.I203T

                                                                                polymorphism

                                                                                TM4

                                                                                c.608T>C

                                                                                p.I203T

                                                                                polymorphism

                                                                                TM4

                                                                                3

                                                                                c.608T>C

                                                                                p.I203T

                                                                                polymorphism

                                                                                TM4

                                                                                Nv

                                                                                   

                                                                                28

                                                                                nv

                                                                                   

                                                                                Nv

                                                                                   

                                                                                638

                                                                                total

                                                                                       

                                                                                2063

                                                                                nv: no variant

                                                                                Note: p.V37I is controversy variant, see the discussion.

                                                                                Table 2

                                                                                Prevalence of GJB2 mutations in different areas of China

                                                                                  

                                                                                Number of NSHI

                                                                                c.235delC allele

                                                                                c.299_c.300delAT allele

                                                                                c.176_c.191del16 allele

                                                                                Uncommon mutant allele

                                                                                total number of mutant alleles(%)

                                                                                 

                                                                                total

                                                                                with two mutation

                                                                                1 allele with one mutaion

                                                                                number with 1 mutant allele (%)

                                                                                homo

                                                                                het

                                                                                total (%)a

                                                                                homo

                                                                                het

                                                                                total (%)a

                                                                                homo

                                                                                het

                                                                                total (%)a

                                                                                homo

                                                                                het

                                                                                total (%)a

                                                                                 

                                                                                Jiangsu

                                                                                102

                                                                                26

                                                                                10

                                                                                36 (35.3)

                                                                                12

                                                                                18

                                                                                42 (67.7)

                                                                                2

                                                                                7

                                                                                11 (17.7)

                                                                                1

                                                                                7

                                                                                9 (14.5)

                                                                                0

                                                                                0

                                                                                0

                                                                                30.4

                                                                                Nei Mongol

                                                                                115

                                                                                30

                                                                                5

                                                                                35 (30.4)

                                                                                14

                                                                                18

                                                                                46 (70.8)

                                                                                0

                                                                                11

                                                                                11 (16.9)

                                                                                0

                                                                                3

                                                                                3 (4.6)

                                                                                1

                                                                                3

                                                                                5 (7.7)

                                                                                28.3

                                                                                Beijing

                                                                                155

                                                                                37

                                                                                6

                                                                                43 (27.7)

                                                                                24

                                                                                13

                                                                                61 (76.3)

                                                                                0

                                                                                10

                                                                                10 (12.5)

                                                                                0

                                                                                0

                                                                                0

                                                                                0

                                                                                9

                                                                                9 (11.3)

                                                                                25.8

                                                                                Hebei

                                                                                64

                                                                                14

                                                                                3

                                                                                17 (26.6)

                                                                                7

                                                                                9

                                                                                23 (74.2)

                                                                                0

                                                                                3

                                                                                3 (9.7)

                                                                                0

                                                                                1

                                                                                1 (3.2)

                                                                                0

                                                                                4

                                                                                4 (12.9)

                                                                                24.2

                                                                                Shanghai

                                                                                31

                                                                                7

                                                                                1

                                                                                8 (25.8)

                                                                                3

                                                                                5

                                                                                11 (73.3)

                                                                                0

                                                                                2

                                                                                2 (13.3)

                                                                                0

                                                                                1

                                                                                1 (6.7)

                                                                                0

                                                                                1

                                                                                1 (6.7)

                                                                                24.2

                                                                                Heilongjiang

                                                                                36

                                                                                5

                                                                                4

                                                                                9 (25.0)

                                                                                1

                                                                                7

                                                                                9 (64.3)

                                                                                0

                                                                                2

                                                                                2 (14.3)

                                                                                0

                                                                                0

                                                                                0

                                                                                0

                                                                                3

                                                                                3 (21.4)

                                                                                19.4

                                                                                Guangdong

                                                                                77

                                                                                15

                                                                                4

                                                                                19 (24.7)

                                                                                10

                                                                                7

                                                                                27 (79.4)

                                                                                0

                                                                                4

                                                                                4 (11.8)

                                                                                0

                                                                                0

                                                                                0

                                                                                0

                                                                                3

                                                                                3 (8.8)

                                                                                22.1

                                                                                Sichuan

                                                                                109

                                                                                17

                                                                                8

                                                                                25 (22.9)

                                                                                10

                                                                                13

                                                                                33 (78.6)

                                                                                0

                                                                                3

                                                                                3 (7.1)

                                                                                0

                                                                                4

                                                                                4 (9.5)

                                                                                0

                                                                                2

                                                                                2 (4.8)

                                                                                19.3

                                                                                Shanxi

                                                                                57

                                                                                11

                                                                                2

                                                                                13 (22.8)

                                                                                4

                                                                                9

                                                                                17 (70.8)

                                                                                0

                                                                                5

                                                                                5 (20.8)

                                                                                0

                                                                                1

                                                                                1 (4.2)

                                                                                0

                                                                                1

                                                                                1 (4.2)

                                                                                21.1

                                                                                Gansu

                                                                                42

                                                                                7

                                                                                2

                                                                                9 (21.4)

                                                                                3

                                                                                5

                                                                                11 (68.8)

                                                                                0

                                                                                3

                                                                                3 (18.8)

                                                                                0

                                                                                0

                                                                                0

                                                                                0

                                                                                2

                                                                                2 (12.5)

                                                                                19

                                                                                Jilin

                                                                                57

                                                                                12

                                                                                0

                                                                                12 (21.1)

                                                                                7

                                                                                4

                                                                                18 (75.0)

                                                                                0

                                                                                5

                                                                                5 (21.0)

                                                                                0

                                                                                0

                                                                                0

                                                                                0

                                                                                1

                                                                                1 (4.0)

                                                                                21.1

                                                                                Fujian

                                                                                48

                                                                                6

                                                                                4

                                                                                10 (20.8)

                                                                                5

                                                                                4

                                                                                14 (87.5)

                                                                                0

                                                                                1

                                                                                1 (6.3)

                                                                                0

                                                                                0

                                                                                0

                                                                                0

                                                                                1

                                                                                1 (6.3)

                                                                                16.7

                                                                                Ningxia

                                                                                145

                                                                                20

                                                                                9

                                                                                29 (20.0)

                                                                                8

                                                                                14

                                                                                30 (61.2)

                                                                                1

                                                                                3

                                                                                5 (10.2)

                                                                                0

                                                                                4

                                                                                4 (8.2)

                                                                                0

                                                                                10

                                                                                10 (20.4)

                                                                                16.9

                                                                                Xinjiang

                                                                                136

                                                                                19

                                                                                8

                                                                                27 (19.9)

                                                                                9

                                                                                5

                                                                                23 (50.0)

                                                                                2

                                                                                4

                                                                                8 (17.4)

                                                                                0

                                                                                3

                                                                                3 (6.5)

                                                                                1

                                                                                10

                                                                                12 (26.1)

                                                                                16.9

                                                                                Hubei

                                                                                47

                                                                                7

                                                                                2

                                                                                9 (19.1)

                                                                                6

                                                                                2

                                                                                14 (87.5)

                                                                                0

                                                                                0

                                                                                0

                                                                                0

                                                                                0

                                                                                0

                                                                                0

                                                                                2

                                                                                2 (12.5)

                                                                                17

                                                                                Yunnan

                                                                                230

                                                                                23

                                                                                19

                                                                                42 (18.3)

                                                                                11

                                                                                14

                                                                                36 (55.4)

                                                                                1

                                                                                3

                                                                                5 (7.7)

                                                                                1

                                                                                2

                                                                                4 (6.2)

                                                                                1

                                                                                18

                                                                                20 (30.8)

                                                                                14.1

                                                                                Guiyang

                                                                                138

                                                                                23

                                                                                2

                                                                                25 (18.1)

                                                                                16

                                                                                9

                                                                                41 (85.4)

                                                                                0

                                                                                6

                                                                                6 (12.5)

                                                                                0

                                                                                0

                                                                                0

                                                                                0

                                                                                1

                                                                                1 (2.1)

                                                                                17.4

                                                                                Henan

                                                                                126

                                                                                16

                                                                                5

                                                                                21 (16.7)

                                                                                10

                                                                                8

                                                                                28 (75.7)

                                                                                0

                                                                                5

                                                                                5 (13.5)

                                                                                0

                                                                                0

                                                                                0

                                                                                0

                                                                                4

                                                                                4 (10.8)

                                                                                14.7

                                                                                Tibet

                                                                                118

                                                                                0

                                                                                19

                                                                                19 (16.1)

                                                                                0

                                                                                2

                                                                                2 (10.5)

                                                                                0

                                                                                1

                                                                                1 (5.3)

                                                                                0

                                                                                0

                                                                                0

                                                                                0

                                                                                16

                                                                                16 (84.2)

                                                                                8.1

                                                                                Qinghai

                                                                                56

                                                                                5

                                                                                3

                                                                                8 (14.3)

                                                                                1

                                                                                3

                                                                                5 (38.5)

                                                                                2

                                                                                2

                                                                                6 (46.2)

                                                                                0

                                                                                0

                                                                                0

                                                                                0

                                                                                2

                                                                                2 (15.4)

                                                                                11.6

                                                                                Anhui

                                                                                35

                                                                                3

                                                                                2

                                                                                5 (14.3)

                                                                                1

                                                                                4

                                                                                6 (75.0)

                                                                                0

                                                                                1

                                                                                1 (12.5)

                                                                                0

                                                                                1

                                                                                1 (12.5)

                                                                                0

                                                                                0

                                                                                0

                                                                                11.4

                                                                                Shaanxi

                                                                                52

                                                                                3

                                                                                2

                                                                                5 (9.6)

                                                                                1

                                                                                4

                                                                                6 (75.0)

                                                                                0

                                                                                1

                                                                                1 (12.5)

                                                                                0

                                                                                0

                                                                                0

                                                                                0

                                                                                1

                                                                                1 (12.5)

                                                                                7.7

                                                                                Guangxi

                                                                                87

                                                                                1

                                                                                5

                                                                                6 (6.9)

                                                                                1

                                                                                4

                                                                                6 (85.7)

                                                                                0

                                                                                0

                                                                                0

                                                                                0

                                                                                0

                                                                                0

                                                                                0

                                                                                1

                                                                                1 (14.3)

                                                                                4

                                                                                total

                                                                                2063

                                                                                307

                                                                                125

                                                                                432

                                                                                164

                                                                                181

                                                                                345

                                                                                8

                                                                                82

                                                                                90

                                                                                2

                                                                                27

                                                                                29

                                                                                3

                                                                                95

                                                                                98

                                                                                17.9

                                                                                homo: homozygous; het: hetrozygous; apercentage of total mutant alleles identified.

                                                                                Frame-shift and nonsense Pathogenic Mutations

                                                                                The c.235delC is the most prevalent mutation in the Chinese NSHI population with a total of 509 alleles (164 homozygous, 113 compound heterozygous with other pathogenic mutant alleles, and 68 one heterozygous allele only), followed by 98 c.299_c.300delAT mutant alleles (8 homozygotes, 76 compound heterozygotes, and 6 one allele heterozygotes), 31 c.176_c.191del16 mutant alleles (2 homozygous, 25 compound heterozygous and 2 with only one allele), and 12 c.35delG mutant alleles(2 homozygous, 3 compound heterozygous and 2 with only one allele) (Supplemental Table 1). The four prevalent mutations account for 88.0% (650/739) of all mutant alleles identified. Five novel mutations were identified in 20 patients; including two nonsense; p.W3X, p.Y152X, and 4 frame-shift truncation mutations; c.99delT, c.155–c.158 delTCTG, and c.512–c.513 insAACG. Among these, c.512–c.513insAACG occurs in 12 patients, including one homozygous from Yunnan. The novel truncation mutations account for only about 3.1% (23/739, Supplemental Table 1) of the overall GJB2 mutant alleles. The most prevalent Caucasian mutation, c.35delG, was found in 2 homozygous, 3 compound heterozygous, and 5 single allele heterozygous patients. Among the patients with c.35delG, 70% of patients (7/10) are Uigur from Xinjiang area. The c.35insG mutation was found in 2 patients (both are Hui people) compound heterozygous with the c.235delC mutation. Other reported frame-shift mutations; 1 c.388–c.397del10 and 3 c.605–c.606ins46, as well as nonsense mutations; 3 p.E47X, account for a small fraction (1.0%) of GJB2 mutant alleles. Overall, 92.6% (684/739) of the pathogenic mutations are frame-shift truncation or nonsense mutations, and they are predicted to cause loss of function of connexin 26. Only 6.9% (51/739) of the mutant alleles are reported missense mutations.

                                                                                Reported missense pathogenic mutations

                                                                                There are 8 reported missense pathogenic mutations and 1 in-frame deletion of 1 single amino acid, c.424_c.426del3 (p.del142F), which occurs in 4 heterozygous patients (Supplemental Table 1). The 8 missense mutations are p.G4D (3 heterozygous patients), p.R32C (one patient in compound heterozygosity with c.235delC), p.R143W (4 compound heterozygotes), p.T86R (all compound heterozygous, 6 with c.235delC and 1 with c.299_c.300delAT), p.R127H (one compound with p.Y152X, 31 single heterozygotes), p.S139N (2 single heterozygotes), p.E47K (one compound with c.299_c.300delAT), p.S85P (single heterozygote). All occur in an evolutionarily highly conserved region (Figure 2) [26, 29, 30].
                                                                                http://static-content.springer.com/image/art%3A10.1186%2F1479-5876-7-26/MediaObjects/12967_2008_Article_337_Fig2_HTML.jpg
                                                                                Figure 2

                                                                                Amino acid alignment of Connexin26 in different species.

                                                                                Unclassified Variants

                                                                                Twelve unclassified missense variants were identified. The p.G21R is most likely to be pathogenic based on its highly evolutionarily conserved nature and the dramatic effect of the amino acid substitutions on structure and ionic strength. The p.I215N variant is located in the conserved region of C-terminal ion channel domain. Replacing the hydrophobic amino acid isoleucine with a hydrophilic amino acid asparagine in this conserved region is expected to cause detrimental effect. This variant is also in compound heterozygous with a novel pathogenic mutation, c.155_c.158delTCTG. Thus, it is likely to be pathogenic.

                                                                                The missense variants, p.I30F, p.F31L, p.V63L, p.V153A, p.D159N, p.F191L, p.M195V, and p.V198M, do not involve drastic change in amino acid structure and polarity. They are all present as single heterozygous alleles without the presence of a second pathogenic mutant allele. Thus, their pathogenicity cannot be determined. Other changes of the same amino acids have been reported. For example, p.V63A has been reported as a novel variant, p.V153I and p.D159N were reported as a polymorphism [29]. The p.M195V and p.V198M, each occurs in two patients, without the second mutant allele. Each of the other variants occurs as heterozygous in one patient. None of these missense variants were detected in the control population.

                                                                                Uncharacterized Novel Silent Variants

                                                                                Several nucleotide substitutions do not result in amino acid change. These are p.A49A, p. K61K, p.F146F, and p.T186T (p.T186T is heterozygous with a single c.235delC). Although these nucleotide changes do not alter the encoded amino acids, we cannot exclude the possibility that they may activate an exonic splice enhancer and cause aberrant splicing. Alternatively, changes in triplet codon may affect the preference of codon usage or the stability of the mRNA, which in turn can affect the protein levels.

                                                                                Genotypes and Carrier Frequency in the Normal Control Population

                                                                                GJB2 is a small gene but harbors many mutations. Thus, the carrier frequency of GJB2 mutation in the Chinese population is not negligible. We sequenced the coding region of 301 normal control individuals of the Han ethnic group. Nine individuals were found to be heterozygous carriers of GJB2 pathogenic mutations; three had the c.235delC, three had the c.299_c.300delAT, and the c.512_c.513insAACG, c.35delG, and p.E47X mutation have been detected in single individuals (see Supplemental Table 3). Thus, the carrier frequency of GJB2 mutations in the control population is 3%.

                                                                                Frequencies of missense variants in patient and control populations

                                                                                The frequencies of common missense variants such as p.V37I, p.V27I, p.I203T, p.T123N, p.E114G in patients, control, and other Asian populations were compared (see Supplemental Table 4 and Table 5). The pathogenic role of p.V37I has been controversial [2426, 3033]. It was found that the p.V37I allele frequency was significantly higher in the Han patient group (excluding all cases with two clearly pathogenic mutations) than in the control group (6.7% and 2.8% respectively,. p = 0.0003), supporting a pathogenic role of p.V37I. The allele frequencies of p.V27I, p.E114G, p.I203T, and p.T123N were higher in the control group than in the Han patient group (excluding all cases with two clearly pathogenic mutations), arguing against their pathogenic role (see Supplemental Table 4 and Table 5).

                                                                                GJB2 mutation spectra among different sub-ethnic groups in China

                                                                                As indicated in Table 2, the frequency of GJB2 mutations varies from 4% in Guangxi to 30.4% in Jiangsu. These results suggest that the variation in mutation frequencies may be due to ethnic diversity in various regions. The total population of China is 1.3 billion and sub-populations of Han, Tibetan, Hui, Man, Mon, minorities in Xinjiang, and minorities in South-western China are 1137.4 million, 5.4 million, 9.8 million, 10.7 million, 5.8 million, 10.8 million, and 57.1 million, respectively (http://​www.​cnmuseum.​com/​intro/​renkou_​intro.​asp, http://​www.​xzqh.​org/​quhua/​index.​htm). We therefore analyzed the mutation frequencies in different sub-ethnic groups. As shown in Supplemental Table 6, Hui has the highest frequency of overall GJB2 mutations, followed by Han and minorities in Xinjiang with 20.3, 19.1, and 15.3% respectively. Tibetan and the minorities in the Southwest have lower mutation frequencies, 9.4 and 5.0% respectively, similar to the frequencies observed in corresponding regions. The majority of mutations found in this study were found in the Han patient group (1640 cases) only except c.35 insG that was in compound heterozygous with c.235delC found in two Hui patients. The common Caucasian mutation, c.35delG was mainly detected in the minorities of Xinjiang, and accounted for almost half of the GJB2 mutant alleles in minorities of Xinjiang (9 c.35delG/19 total mutant alleles). The finding of the c.35delG mutation in Xinjiang may be due in part to the close vicinity of Xinjiang to Russia and Eastern European countries, and possible admixture. The Maan sub-ethnic group also appears to have diverse GJB2 mutation spectrum because mutations other than c.235delC account for more than one third of the mutant alleles. The three most common mutations c.235delC, c.299_c.300delAT, and c.176_c.191del16 account for 100% of GJB2 mutations in 18 Mongolian individuals analyzed. However, the sample size is too small to be statistically significant.

                                                                                Discussion

                                                                                Previous reports have suggested that the prevalence of GJB2 mutations among different ethnic groups varies. In our patients, the most common Caucasian mutation, c.35delG was only found in 10 patients (seven of them were Uigur from Xinjiang). Instead, the c.235delC account for 68.9% of all GJB2 mutant alleles in our Chinese study population. These results support that the c.235delC mutation in connexin 26 gene is the most prevalent mutation in most Asian populations, including Han Chinese [11, 24, 30, 34]. The results from this study indicate that analysis of four common mutations, c.235delC, c.299_c.300delAT, c.176_c.191del16, and 35delG can detect 88.0% (650/739) of GJB2 mutations. In 13 regions of China, by analyzing these four mutations, we were able to identified at least one mutant allele in all studied patients with one or two GJB2 mutations (see Table 2 and Supplemental Table 2). In contrast, mutations in the GJB2 gene account for a variable proportion of the molecular etiology of NSHI in different regions and sub-ethnic groups in China. Our results have tremendous impact on the design of molecular diagnostic and carrier testing of NSHI families in China. For example, in addition to the three most common mutations of c.235delC, c.299_c.300delAT, c.176_c.191del16, for minorities in Xinjiang, testing of Caucasian c.35delG mutation should be included. In patients with Maan ethnic background, sequencing of the GJB2 coding region should be offered, since the analysis of three common mutations detects only 71% of GJB2 mutant alleles. In minorities from Southwest provinces, although the three most common mutations account for >90% of all GJB2 mutations, defects in GJB2 gene account for only a small fraction (5%, Supplemental Table 2 and Table 6) of mutant alleles in NSHI patients. Thus, in these groups, analysis of other NSHI related genes should be pursued.

                                                                                We recently reported that 7.8% of patients with autosomal recessive nonsyndromic hearing impairment in China were homozygous for the most common c.235delC mutation in GJB2 gene and 8.5% of them carried one mutant allele of the c.235delC mutation [28]. Sequencing of the coding region of the GJB2 gene reveals that 14.9% of the patients carry two pathogenic GJB2 mutation and 6.1% carry only one mutant allele. These results are comparable to other reported studies [7, 11, 13, 24, 29, 30, 3335]. The proportions of patients with GJB2 mutations carrying only one mutant allele vary among different regions, different sub-ethnic groups, and different countries [7, 11, 13, 24, 29, 30, 3335]. The observation that sequence analysis of GJB2 gene in subjects with autosomal recessive NSHI results in a high number of patients with only one GJB2 mutant allele has been puzzling [23]. Our unpublished data showed that no mutation were found in GJB2 Exon1 and its splicing sequence among 851 deaf individuals from Central China in this cohort which suggested extremely low detection rate of GJB2 Exon1 mutation among Chinese deaf population. For there is higher frequency of single heterozygous GJB2 mutation detected in the deaf population than in the normal population in this study, the further more extensive study of sequence change in GJB2 Exon1 or promoter area and 3'-UTR, fragment deletion neighboring GJB2 ORF region and digenic inheritance with other genes are already considered in this large Chinese deaf cohort for elucidating complex pathogenesis of GJB2 gene to hearing impairment. We already added a paragraph in discussion. Thus, a digenic hypothesis was proposed and mutations in two other connexin (Cx) genes, GJB6 for Cx30 and GJB3 for Cx31 were studied [21, 22, 36]. In families with clear evidence of linkage to the DFNB1 locus, which contains two genes, GJB2 and GJB6 [6, 20], a common 309 kb deletion, involving the coding region GJB6 gene upstream of GJB2 gene has been identified and found to account for up to 10% of DFNB1 alleles in Caucasians [22]. We analyzed the deletion in GJB6 gene in 372 patients from Inner Mongolia and central China, and deletions in GJB6 gene were not detected. Similar studies of GJB6 mutations in Taiwanese prelingual NSHI patients carrying one GJB2 mutant allele also did not detect any deleterious mutations in GJB6, consistent with our results [30].

                                                                                Although the spectrum of rare GJB2 mutations varies among sub-ethnic groups and in different regions of China, the same most common c.235delC mutation is shared. This observation is in agreement with the reports from the studies of other Asian NSHI patients [10, 11, 24, 30, 34]. However, instead of c.299_c.300delAT being the second most prevalent mutation, p.G45E accounts for 16% of the Japanese GJB2 mutations, while p.G4D accounts for 10.6% of Taiwanese GJB2 mutant alleles [10, 30]. The p.G45E mutation was not detected in our patients. The p.G4D mutation accounts for only 0.3% of GJB2 mutant alleles in Chinese NSHI patients and was recently reported in a US study [29, 30].

                                                                                Among the 23 pathogenic mutations, 14 cause truncated connexin 26 proteins due to nonsense or frame-shift mutations, 8 are missense mutations, and one is a deletion of one amino acid. These mutations occur along the coding region. The truncation mutations account for 92.6% of the mutant alleles. Amino acids sequence homology alignment reveals that all missense mutations and unclassified variants occur at an evolutionarily conserved amino acid (Figure 2).

                                                                                Three missense variants, p.V63L, p.V153A, and p.V198M, are located in extracelluar domain 1, 2, and transmembrane span 4, respectively, of connexin 26 protein. All these changes have not been reported in the Connexins and Deafness mutations database at http://​davinci.​crg.​es/​deafness. However, p.V63L has been found in 1 Taiwanese patient [30]. These three variants likely contribute to the pathogenesis of deafness, because (a) they were detected only in the patient group and not in 394 Japanese, 864 Taiwanese, 494 Korean and 301 Chinese (in this study) hearing normal subjects, and (b) they are evolutionarily conserved in xenopus, mouse, rat, sheep, orangutan, and human (Figure. 2). These variants were found in a heterozygous state in 4 unrelated patients who carried only one mutant allele.

                                                                                The pathogenicity of p.V37I is controversial. In a recent multicenter study, the p.V37I mutation was found to be associated with mild to moderate hearing impairment (median 25–40 dB) [37]. Our study revealed that p.V37I with an allele frequency of 6.7% (185/2744) in the Han patient group (excluding all cases with two clearly pathogenic mutations) is significantly higher compared with that (2.8%;17/602) found in the control population (p = 0.0003, see Supplemental Table 4 and Table 5), supporting Wu's opinion to reassignment of p. V37I from an allele variant to a pathogenic mutation [38].

                                                                                The p.T123N is an unclassified variant. It was counted as a mutation in Japanese group but a polymorphism in a Taiwanese study [10, 30]. We found a higher p.T123N allele frequency in the control group than in the patient group, suggesting that it may be neutral variant. However, its clinical implication is not clear at this time.

                                                                                The results of this study provide a great potential benefit for the clinical application of genetic testing for deafness. Based upon our preliminary data of molecular epidemiology of hearing impairment in China [28, 3941], Li has combined allele-specific PCR and universal array (ASPUA) methodologies for the detection of mutations causing hereditary hearing loss. It was employed for multiplex detection of 11 mutations in GJB2, GJB3, SLC26A4 and mitochondrial DNA causing hereditary hearing loss [42]. Although this simple screening chip only include probes and primers for the c.35delG, c.176_c.191del16, c.235delC, c.299_c.300delAT mutations of GJB2 gene, it can detect 88.0% (650/739) of GJB2 mutations among these 2063 deaf individuals, meanwhile, up to 88.9% (384/432) of 432 patients confirmed to carry at least one GJB2 mutation by sequencing in this study will be picked up by this fast screen method. The new methods for multiple mutation detection including ASPUA with capacity to test more gene loci have been under developed in our center, the data of this study will be crucial for the mutation selection in any new technology development for GJB2 gene testing in Chinese population.

                                                                                In summary, this study revealed a unique GJB2 mutation spectrum in Chinese patients with nonsyndromic hearing impairment. The c.235delC mutation is the most frequent mutation in Chinese patients. Testing of four common mutations, c.235delC, c.299_c.300delAT, c.176_c.191del16, and c.35delG can detect 88.0% of the GJB2 mutant alleles. However, in some regions or sub-ethnic groups, the GJB2 mutations only account for a small fraction of the NSHI mutant alleles. In these regions, analysis of NSHI related genes is necessary. The molecular defects of more than 80% of the mutant alleles for NSHI in China remain to be identified.

                                                                                Declarations

                                                                                Acknowledgements

                                                                                The authors would like to thank Dr. Dennis Johnson and Dr. Raye L. Alford for their valuable suggestions. This work was supported by the Chinese National Nature Science Foundation Research Grant 30728030,30872862, and Chinese Capital Medical Development Scientific Funding 2005-1032 to Dongyi Han.

                                                                                The authors also would like to thank the Fuyang School for the Deaf and Dumb (Anhui province), Beijing No.3 School for the Deaf (Beijing), Pinggu Special Education School (Beijing), Beijing Children's Hospital(Beijing), Fuzhou Special Education School (Fujian province), Lanzhou Convalescent Center for Deaf Children (Gansu province), Gansu Convalescent Ccenter for Deaf Children (Gansu province), Foshan School for the Deaf and Dumb (Guangdong province), Liuzhou School for the Bblind Deaf and Dumb (Guangxi province), Guiyang School for the Blind, Deaf and Dumb (Guizhou Province), Zhuozhou and Gaobeidian School for the Ddeaf and Dumb (Hebei province), Mudanjiang Special Education School (Heilongjiang province), Anyang Special Education School (Henan province), Wuhan Yimeng Convalescent Center for Deaf Children (Hubei province), Chifeng Special Education School (Inner Mongolia), Nantong School for the Deaf and Dumb (Jiangsu province), Haian School for the Deaf and Dumb (Jiangsu province), Haimen School for the Deaf and Dumb (Jiangsu province), Rugao School for the Deaf and Dumb (Jiangsu province), Tongzhou School for the Deaf and Dumb (Jiangsu province), Jilin Special Education School (Jilin province), Yinchuan School for the Blind, Deaf and Dumb (Ningxia Province), Xining Special Education School (Qinghai province), Changan School for the Deaf and Dumb (Shaanxi province), Affiliated Pediatric Medical Center of Shanghai Jiao Tong University (Shanghai), Yuncheng School for the Deaf and Dumb (Shanxi province), Yuncheng Disabled Person's Federation (Shanxi province), Yuncheng Convalescent Center for Deaf Children (Shanxi province), Chengdu Special Education School (Sichuan province), Urumchi School for the Deaf and Dumb (Xinjiang province), Korla School for the Deaf and Dumb (Xinjiang province), Kunming Huaxia Secondary School (Yunnan province), Kunming Convalescent Center for Deaf Children (Yunnan province), Lincang Special Education School (Yunnan province), Kunming Convalescent Center for Deaf Children (Yunnan province) and Lhasa Special Education School (Tibet municipality area) for their fundamental support and contributions to this work.

                                                                                Authors’ Affiliations

                                                                                (1)
                                                                                Department of Otolaryngology and Genetic Testing Center for Deafness, Chinese PLA General Hospital
                                                                                (2)
                                                                                Department of Molecular and Human Genetics, Baylor College of Medicine
                                                                                (3)
                                                                                Department of Otolaryngology, University of Miami
                                                                                (4)
                                                                                Department of Otolaryngology, Fuyang People's Hospital
                                                                                (5)
                                                                                Department of Otolaryngology, Beijing Children's Hospital
                                                                                (6)
                                                                                Department of Health Statistics, Second Military Medical University
                                                                                (7)
                                                                                Department of Otolaryngology, Fuzhou Second People's Hospital
                                                                                (8)
                                                                                Center of Hearing Rehabilitation, Guizhou People's Hospital
                                                                                (9)
                                                                                Department of Otolaryngology, Foshan First People's Hospital
                                                                                (10)
                                                                                Department of Otolaryngology, Anyang Stomatology Hospital
                                                                                (11)
                                                                                Department of Otolaryngology, Mudanjiang First People's Hospital
                                                                                (12)
                                                                                Department of Otolaryngology, PLA 161st Hospital
                                                                                (13)
                                                                                Department of Otolaryngology, Chifeng Second People's Hospital
                                                                                (14)
                                                                                Department of Otolaryngology, Affiliated Hospital of Nantong University
                                                                                (15)
                                                                                Department of Otolaryngology, Affiliated Hospital of Beihua University,Jilin
                                                                                (16)
                                                                                Department of Otolaryngology Head&neck Surgery, General Hospital of Lanzhou Area Command
                                                                                (17)
                                                                                Department of Otolaryngology, Urumchi People's Hospital
                                                                                (18)
                                                                                Department of Otolaryngology, Zhuozhou Second Central Hospital
                                                                                (19)
                                                                                Department of Otolaryngology, Datong Third People's Hospital
                                                                                (20)
                                                                                Department of Otolaryngology, Yuncheng Central Hospital
                                                                                (21)
                                                                                Department of Otolaryngology Head & Neck Surgery, Affiliated Xinhua Hospital of Shanghai Jiao Tong University
                                                                                (22)
                                                                                Department of Otolaryngology, General Hopital of Tibet Area Command
                                                                                (23)
                                                                                Institute of Geriatrics, Chinese PLA General Hospital
                                                                                (24)
                                                                                Division of Genetics and Metabolism, Children's Hospital Boston, Harvard Medical School

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                                                                                This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://​creativecommons.​org/​licenses/​by/​2.​0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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