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 . 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, 33–35]. 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, 33–35]. 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 . 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 . 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 .
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 . 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) . 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 .
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, 39–41], 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 . 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.