Based on a relatively large cohort, the present study analyzed the mutational spectrum of NSHI in the population of south China for the first time. It has been established that the common deafness mutations for the Chinese population are GJB2 c.235delC, c.299-300delAT, c.176-191del16, c.35delG; GJB3 c.538C > T; SLC26A4 c.IVS7-2A > G, c.2168A > G; and mitochondrial m.1555A > G and m.1494C > T [8, 10], whereas the major mutations in the south Chinese population consist of GJB2 c.235delC, c.109G > A, c.299-300delAT, c.176-191del16; SLC26A4 c.IVS7-2A > G and c.1229 T > C; and mitochondrial m.1555A > G. GJB2 exon sequencing is recommended for each individual because it is simple, cheap (25 RMB per sample), and can provide more information (18%) than gene chips . SLC26A4 exons sequencing is suggested for patients with EVA. An extended and systematic study of the complete mitochondrial DNA sequence may provide significant further information for assessment of patients with NSHI.
China consists of 56 ethnic countries that comprise a population of over 1.6 billion. Given the heterogeneity of genetic deafness, ethnic and regional specific factors are important. Although genetic analysis of patients from the northern , central , Xinjiang , Yunnan , and Tibet  areas of China have been reported, the mutational spectrum of NSHI in south China - consisting of the Guangdong province and the surrounding areas - remained unclear. A genetic study of the south China population may provide regional-specific mutational information to improve the efficacy of clinical genetic counseling. Therefore, the present results provide useful information for clinical application of mutant gene detection in patients with deafness.
GJB2 gene mutations are diverse in different ethnic regions (between 10–48.1%). The hereditary deafness website (http://hereditaryhearingloss.org) lists at least 111 GJB2 mutations. The c.35delG mutation is most prevalent in Europe and the United States, compared with c.235delC in East Asian populations and c.167delT in Jewish populations . We observed a GJB2 mutation distribution of approximately 20% that was responsible for NSHI in south China. The GJB2 mutation rate in south China was identical to that of central and northern China [4, 6–11]. The c.109G > A mutation was very common, although its pathogenicity remains controversial. A study indicated that the c.109G > A mutation had an allelic frequency of 6.7% (185/2744) in a Han patient group (excluding all cases with two clearly pathogenic mutations); this rate was significantly higher than the control population (2.8%, 17/602; P =0.0003) . These results support the conclusion that GJB2 c.109G > A should be reassigned to a pathogenic mutation. Our cell-based in vitro functional assays study also indicated that c.109G > A homozygote could impair connexin26 gap junctional coupling and should be assigned to pathogenic mutation (unpublished data). In addition, there is a lack of genetic information for the GJB2 mono-allelic mutation, which makes genetic counseling for these patients more difficult. The frequency of c.109G > A in control group is relatively high (6.1%) in our cohort. This may partially be attributed to the small sample sizes and/or selection bias; However, this frequency might reflect the prevalence of c.109G > A in this cohort and c.109G > A might be ascribed to founder effect in south Chinese. Further population-based and functional studies are recommended.
SLC26A4 mutations in Western countries and Asian populations show regional and ethnic diversity in their frequency and display mutational hot spots. For example, SLC26A4 mutations account for approximately 4% of NSHI in the Caucasian population . Evaluation of previous Chinese domestic studies indicates that the incidence of the SLC26A4 mutation may be between 12-14% in patients with NSHI [19, 20]. Our finding (13.1%) is consistent with these results, illuminating the feasibility of the SLC26A4 detection strategy inferred from north China [7, 9]. The c.IVS7-2A > G mutation is common in Eastern Asia. Several large cohort investigations have reported a high prevalence of c.IVS7-2A > G in the Chinese population (22.3-64.63%). In Koreans, the c.IVS7-2A > G mutation accounts for 20% of mutant SLC26A4 alleles and is the second most prevalent genetic variant . The c.IVS7-2A > G mutation has a low incidence of approximately 3% in Japanese individuals . The distribution of mutant SLC26A4 alleles revealed by our study suggests that mutation screening of four exons (exons 19, 10, 17 and 15) following c.IVS7-2A > G identification should be a priority for NSHI genetic testing in patients with EVA. Besides, previous studies [7, 10, 11, 14, 18] have revealed the close relationship between SLC26A4 mutations and EVA. The absence of SLC26A4 mutation in 37 patients without temporal CT in our cohort could preliminarily exclude the possibility of EVA in these patients.
Mitochondrial m.1555A > G mutation was detected at a lower rate in southern China compared with previous reports. The m.1555A > G mutation is observed in approximately 0.6-2.5% of Caucasian patients with NSHI , but more frequently in the Asian population, including China 2.9%, Japan 3% , and Indonesia 5.3% . The tRNAser(UCN) m.7445A > G and m.7444G > A mitochondrial mutations may be involved in clinical phenotype modifications. However, we failed to detect tRNAser(UCN) m.7445A > G and m.7444G > A mutations; therefore, they may be rare in the south China population.
Previous studies have found that digenic inheritance of NSHI is caused by mutations in the GJB2 and GJB3 genes . However, we observed that both GJB3 and GJB6 exhibited a low mutation rate [27, 28]. In the present study, 701 cases lacked the c.538C > T or c.547G > A mutations in GJB3. Additional screening of 200 subjects for mutations in the GJB3 and GJB6 coding regions did not identify any pathogenic mutations . Meanwhile, lack of WFS1 c.1901A > C, a rare low-frequency mutation , excludes the necessity of routine screening for this allele. Therefore, our preliminary results indicate that the GJB3, GJB6, and WFS1 c.1901A > C common gene mutations may be minor in NSHI and are not recommended as alleles for first-line screening.
Individualized genetic screening in south China may improve the NSHI diagnostic efficiency and positivity rate, to avoid unnecessary waste of economic and human resources. Therefore, larger epidemiological studies of the genetic etiology of NSHI are recommended due to the sample size limitations of the present study. New genetic technology, such as targeted exon sequencing, may shed further light on the genetic components of NSHI in patients that lack common gene mutations.