Genetic mutations of GJB2 and mitochondrial 12S rRNA in nonsyndromic hearing loss in Jiangsu Province of China

Hearing loss is caused by several environmental and genetic factors, and the proportion attributed to inherited causes is assumed at 50-60% [1]. About 70% of which are nonsyndromic hearing loss (NSHL) since hearing loss is the only symptom. Genetic hearing loss of nonsyndromic form can follow a pattern of autosomal dominant (DFNA), autosomal recessive (DFNB), or X-linked recessive, and mitochondrial inheritance [2].

To date, there are 134 genetic loci and 80 different genes that have been described for NSHL in human [3]. It is believed that alterations in several members of the connexin protein family and mtDNA 12S rRNA contribute to the development of the majority of genetic hearing losses, in which connexin 26 (GJB2) gene mutations are particularly an important cause of NSHL and mainly linked to pattern of autosomal recessive, and the single-nucleotide alteration mtDNA m.1555A > G has been identified as a major cause of aminoglycoside-induced NSHL [4‐8].

It is estimated that there are approximately 20 million babies born every year in China, of whom about 30,000 are expected to have congenital hearing loss [9]. Carrier frequencies of some mutational hot spots associated with NSHL from different areas and ethnic backgrounds have been reported [10‐15], but the molecular etiology of NSHL in the Chinese Jiangsu Province population has not been investigated systematically. To further extend the epidemiological data of common gene mutations in Chinese population, we screened the GJB2 and mitochondrial 12S rRNA genes to determine the etiology of hearing loss in Jiangsu Province.

Hearing loss is the most common neurosensory disorder in humans. Approximately half of the cases have a genetic etiology with extraordinary genetic heterogeneity. For many populations, the most common cause for NSHL is mutated Connexin 26, a gap junction protein encoded by the GJB2 gene, which is expressed in the cochlea and may play a role in K⁺ circulation between different partitions in the cochlea. GJB2 gene is composed of 2 exons separated by an intron. Its coding region is entirely contained in exon 2. The basal promoter activity resides in the first 128 nucleotides upstream of the transcription start point (TSP) and has two GC boxes, at position 281 and 293 from the TSP, which is important for transcription [17]. Previous reports have suggested that the mutation spectrum and prevalence of GJB2 vary significantly among different ethnic groups. GJB2 mutations with the c.35delG, c.167delT, c.235delC and c.427C > T alleles were responsible for a large proportion of NSHL in European, American, African and the Asian [18‐25]. In the Chinese populations, the 235delC is responsible for up to 21% of cases with NSHL [9].

Similar to other previous reports of China [10, 14, 26], our study showed that GJB2 mutations were an important cause of NSHL in Jiangsu deafness populations, and the most common GJB2 mutation detected in our study group was the c.235delC (102/658; 15.50% ). While the c.35delG mutation, which is prevalent in the Caucasians and rarely found in Chinese populations, was identified only in 2 patients. The four most prevalent mutations: c.235delC, c.299delAT, c.176del16, and c.35delG, account for 23.70% (156/658) of all mutant GJB2 alleles identified in Jiangsu Province. However, no mutations were found in GJB2 basal promoter region and exon 1 or splice sites, which suggested extremely low detection rate of GJB2 basal promoter region and exon 1 or splice sites mutation in this area.

One novel nucleotide alteration c.257C > G (T86R) in coding region of GJB2 gene were identified in two deaf siblings compound heterozygotes carrying c.605ins46 pathogenic mutation in the exon 2 of GJB2, this change has not been reported in Connexins and Deafness mutations database at http://​davinci.​crg.​es/​deafness/​. The heterozygotes were detected from their hearing parents, the father carried c.257C > G (T86R) mutation, while the mother with c.605ins46 mutation, the opposite alleles of the parents were both wild-type.

Although the majority of cases with NSHL are caused by nuclear gene mutations, it has become clear that mtDNA mutations can also cause deafness. In familial cases of ototoxicity, aminoglycoside hypersensitivity is often maternally transmitted, suggesting that the mutation in mtDNA is the molecular basis for this susceptibility. In the human mtDNA genome, m.1555A > G is the most common mutation in the gene. The 12S rRNA gene was proposed to be the primary targeting site for aminoglycosides. The identified nonsyndromic deafness-causing mtDNA mutations include m.1555A > G, m.1494C > T, m.1095 T > C and mutations at position 961 in the 12S rRNA gene [27‐32]. Currently, it is estimated that these mutations are present in about 3.10% of patients with NSHL, but it is expected that this number will increase as genetic testing becomes more readily available. In this study, 658 patients and all controls were screened for mtDNA 12S rRNA gene by PCR and DNA direct sequencing. We detected 30 variants in 12S rRNA gene. Among these, 39 patients carried the mtDNA 12S rRNA m.1555A > G mutation (5.93% ), which coincided with the previous report [15]. Four patients carried m.1494C > T mutation, 6 patients carried m.1095 T > C mutation, and 18 patients possess mutations at 961 position. A novel sequence variant, m.1222A > G in the 12S rRNA gene, was identified in one patient with profound hearing loss. Comparison of the variant frequencies in controls, assessment of nucleotide conservation among mammalian species, and structural analysis of the transcript were used to analyze the m.1222A > G mutation associated with hearing loss. To our knowledge, the homoplasmic m.1222A > G variant in 12S rRNA gene has not been reported to date. Conservation of the nucleotide among 22 primates and alteration of the predicted secondary structure of the 12S rRNA transcript suggest that the m.1222A > G variant might affect auditory function by changing the function of the 12S rRNA.

Similar to that of the GJB2 gene mutations, the association between mtDNA mutations and hearing loss has also a special ethnic and regional difference. In the NSHL cases of Jiangsu population, the incidence of the mtDNA mutation including m.1555A > G, m.1494C > T, m.1095 T > C and mutations at position 961 in the 12S rRNA gene appears to coincide with other populations as previous reports [15].

In summary, the results of our study indicate that the mutation spectrum and prevalence of GJB2 and mtDNA 12S rRNA genes in Jiangsu patients with hearing loss is similar to other areas of China. There are in total 31.46% of patients with NSHL carry deafness-causing mutations in GJB2 or mtDNA 12S rRNA gene. Mutation in GJB2 gene was the most common factor, mtDNA 12S rRNA also play an important part in the pathogenesis of hearing loss in Jiangsu Province areas. These results indicate the necessity of genetic screening for mutations of these genes in Jiangsu patients with NSHL.