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Genetic frequencies related to severe or profound sensorineural hearing loss in Inner Mongolia Autonomous Region

Abstract The aim was to study the frequencies of common deafness-related mutations and their contribution to hearing loss in different regions of Inner Mongolia. A total of 738 deaf children were recruited from five different ethnic groups of Inner Mongolia, including Han Chinese (n=486), Mongolian (n=216), Manchurian (n=24), Hui (n=6) and Daur (n=6). Nine common mutations in four genes (GJB2, SLC26A4, GJB3 and mitochondrial MT-RNR1 gene) were detected by allele-specific PCR and universal array. At least one mutated allele was detected in 282 patients. Pathogenic mutations were detected in 168 patients: 114 were homozygotes and 54 were compound heterozygotes. The 114 patients were carriers of only one mutated allele. The frequency of GJB2 variants in Han Chinese (21.0%) was higher than that in Mongolians (16.7%), but not significantly different. On the other hand, the frequency of SLC26A4 variants in Han Chinese (14.8%) was lower than that in Mongolians (19.4%), but also not significantly different. The frequency of patients with pathogenic mutations was different in Ulanqab (21.4%), Xilingol (40.0%), Chifeng (40.0%), Hulunbeier (30.0%), Hohhot (26.3%), and in Baotou (0%). In conclusion, the frequency of mutated alleles in deafness-related genes did not differ between Han Chinese and Mongolians. However, differences in the distribution of common deafness-related mutations were found among the investigated areas of Inner Mongolia.


Introduction
Hearing loss is the most common sensory impairment that affects normal communication and life quality of the patients. About 70% of hearing loss is non-syndromic, and the remaining 30% is considered syndromic, accompanied by additional clinical abnormalities. Approximately 1 in 1,000 children is affected by prelingual hearing loss. The sensorineural hearing loss (SNHL) has been reported to occur in 2 of all newborns, and the ratio increases to 2.7 in 4 years-old children, and to 3.5 at puberty (Qing et al., 2015).
Various genetic and environmental causes are involved in the etiology of hearing loss (Hone andSmith, 2001, Mahboubi et al., 2012), which has increased in some places (Morton, 2002, Khabori and Patton, 2008, Sajjad et al., 2008. Genetic causes alone explain 50-70% of all cases (Petit et al., 2001, Smith et al., 2005. Genetic defects may cause susceptibility to environmental factors that contribute to late-onset deafness. Among a number of deafness-related genes (the Hereditary Hearing Loss Homepage, http://hereditaryhearingloss.org/), mutations in GJB2, SLC26A4, GJB3, and mitochondrial 12S rRNA gene (MT-RNR1) are found to be more frequent. Mutations in GJB2 and SLC26A4 have been reported to cause autosomal recessive nonsyndromic hearing loss, while the inheritance pattern of GJB3 mutations is not quite clear (Nishio and Usami, 2015). Mutations in these genes are estimated to explain more than 40% of non-syndromic hearing loss cases (Wu et al., 2011, Qing et al., 2015. Several common mutations have been identified in the four hearing loss-related genes. Ethnic differences exist in the genetic pathogenesis of deafness. In Caucasian, Asian, Ashkenazi Jew, and African populations, the most common mutations in the GJB2 gene are c.35delG, c.235delC, c.167delT, and c.427C > T, respectively. The second most common gene related to SNHL is SLC26A4. Mutations in this gene are considered major causes for enlarged vestibular aqueduct (EVA). Similar to GJB2, the frequency of SLC26A4 mutations also varies in different populations. The c.919-2A > G is a highly prevalent mutation in East Asian subjects but is rare in European lineages (Dai et al., 2009).
Microarray chip, a high-throughput and high-efficiency technology, has been widely used in gene mutation detection. Here in this study, we used a microarray chip developed by CapitalBio Corporation (Beijing, China) to detect deafness-related mutations. This microarray chip aims to detect frequent mutations that are related to hereditary hearing loss in Chinese population, as seen in large-scale epidemiological studies done across China (Wang et al., 2007, Dai et al., 2008, Chen et al., 2011. It enables simultaneous detection of nine common mutations in four genes of hereditary hearing loss, including c. 235delC, c.35delG, c.176del16, c.299- China is a country with multiple ethnicities. A recent research has shown that frequencies of mutations in MT-RNR1 (1555A > G or 1494C > T) in a group of multiethnic minorities was almost six times higher than that in the Han group of southwestern China. The authors studied mainly ethnic groups more prevalent in southwestern China, such as Miao, Tujia, Dong and Zhuang (Qing et al., 2015). Mongolians account for an important part of the Inner Mongolia population and make up for about 17.11% according to China's Sixth Population Census in 2010 (http://www.chinadaily.com.cn/china/2010census/). Han Chinese account for 79.54%, while the remaining minorities account for 3.36%. However, little is known about the frequency of mutations in the four deafness-related genes in Inner Mongolia, especially considering the two main ethnic groups (Han Chinese and Mongolian). In this study, we analyze nine common gene mutations (GJB2, SLC26A4, GJB3 and MT-RNR1) in 738 children with severe or profound hearing loss in Inner Mongolia. Ethnic and regional differences were also studied.

Subjects recruitment and clinical evaluation
From February 2011 to January 2013, 738 children with severe or profound hearing loss (456 male; 61.8%) were recruited for this study. All patients presented with congenital sensorineural deafness. Subjects from 12 main cities/leagues of Inner Mongolia Autonomous Region (Table 1) were selected in the Inner Mongolia People's Hospital, where they were treated with cochlear implants. The geographical areas from where subjects were recruited are shown in Figure 1. The 738 children with hearing impairment were all born in Inner Mongolia and belonged to five different ethnic groups, including Han Chinese (n=486), Mongolian (n=216), Manchurian (n=24), Hui (n=6) and Daur (n=6). The mean age of the participants was 4.24 ± 3.03 years, ranging from 12 months to 16 years. Clinical and general information was recorded including family history and past medical history. The study was performed under the approval of the Ethics Committee of Inner Mongolia People's Hospital. Signed informed consent was obtained from the legal guardians of the children.

Genetic analysis
Genetic analysis was conducted for all patients using genomic DNA from peripheral blood samples. The genetic screening included nine common mutations in four common non-syndromic hearing loss related genes, including GJB2 (NG_008358.1), SLC26A4 (NG_008489.1), GJB3 (NG_008309.1; NM_024009.2) and MT-RNR1 (NC_012920.1). The mutations were detected by allele-568 Liu et al. 1494C > T). The multiplex allele-specific PCR was performed as described previously (Qu et al., 2012) and the results of microarrays were scanned and analyzed by a LuxScan TM 10K/B Microarray Scanner (CapitalBio). The results were also validated by Sanger sequencing for wild and mutant types.

Statistical analysis
Data was analyzed using SPSS software (version 17.0). Statistical significance was evaluated using an c 2 test to compare the differences among the ethnic groups and cities/leagues. P < 0.05 was considered to be statistically significant.

Mutation frequencies of common deafness genes
At least one mutated allele was detected in 282 of the 738 deaf children, including 162 cases with GJB2 mutations, 114 cases with SLC26A4 mutations, and 6 with GJB3 mutations. Mutations of MT-RNR1 (m.1555A > G, m.1494C > T) were not detected in the sample. The frequencies of mutant alleles are shown in Table 2. Each allele was counted individually for compound heterozygotes. Thus, patients with two different mutated alleles were counted twice. Among all variants detected, the most frequent mutated alleles were GJB2 c.235delC (41.8%) and SLC26A4 c.919-2A > Ge (32.7%) ( Table 2).

Frequencies of mutations in Han Chinese and Mongolians
The frequency of mutated alleles in GJB2 in Han Chinese (21.0%, 102/486) was higher compared to that in Mongolians (16.7%, 36/216), but without significant difference (P=0.217). On the other hand, the frequency of mutated alleles in SLC26A4 was lower in Han Chinese (14.8%, SNHL in Inner Mongolia 569  (Table 3).

Mutations in different cities/leagues
As shown in Table 4, 168 patients showed pathogenic mutations, of which 114 had homozygous mutations and 54 were compound heterozygotes. The 114 patients were called "carriers" since they presented only one mutated allele. The mutation frequencies were higher in four cities/leagues including Ulanqab, Xilingol, Chifeng and Hulunbeier. In Hohhot, 30 patients (26.3%) were diagnosed as homozygotes or compound heterozygotes. However, in Baotou, a city located less than 200 kilometers away from Hohhot, no pathogenic mutation was detected. The frequencies were significantly different between the two main cities in Inner Mongolia, suggesting a difference in the geographical distribution of common deafnessrelated mutations.

Discussion
Mutations in various hearing loss-related genes have been studied worldwide, and several genes have been iden-tified as commonly related to hearing impairment, such as GJB2, SLC26A4, GJB3 and MT-RNR1 (mtDNA 12S rRNA gene). In the present study, mutation analysis was performed in 738 children with severe to profound SNHL in Inner Mongolia. Similar results have been reported by many studies. After screening for mutations of three prominent deafness-related genes (GJB2, SLC26A4 and mtDNA 12S rRNA) in 235 patients with hearing loss in the Yunnan province of China, 35.74% of patients showed genetic involvement, and 17.45%, 9.79%, and 8.51% of inherited hearing impairment were caused by GJB2, SLC26A4, and mtDNA 1555A > G mutations, respectively (Xin et al,. 2013). Mutations of the GJB2, SLC26A4, and mtDNA have also been screened in 1164 children with severe or profound SNHL in southwestern China. Approximately 28% (331/1164) of patients were found to carry mutations, of which 17.27%, 7.04%, and 4.12% were GJB2, SLC26A4, and mtDNA, respectively (Qing et al., 2015). GJB2 c.235delC and SLC26A4 c.919-2A > G have been widely known to be the most common mutations in Chinese population (Qu et al., 2012. Similarly, we also found that the frequencies of GJB2 c.235delC and SLC26A4 c.919-2A > G among all detected mutated alleles were the highest. The pathogenicity of GJB3 mutations has not been defined, and it is still unclear whether these mutations are related to autosomal dominant or recessive nonsyndromic hearing loss, especially in the case of mutation c.538C > T. The mutation in GJB3 (c.538C > T) has been first identified to be associated with autosomal dominant hearing impairment in two Chinese families (Xia et al., 1998). However, in a carrier screening for normal hearing female populations of childbearing age in South China, 9 subjects (in 7,263 women, 0.12%) were found carrying GJB3 570 Liu et al.  c.538C > T in a heterozygous state, suggesting that it might act as an autosomal recessive mutation . Moreover, the variant c.538C > T has been recategorized as "benign" lately, based on allele frequency (Shearer et al., 2014). Considering the uncertainty of the pathogenicity of this variant, patients with heterozygous GJB3 c.538C > T were counted as "carriers" in our study. Shearer et al. (2014) proposed a threshold of 0.0005 for considering a variant as pathogenic in an autosomal dominant fashion, in the general population. However, the allelic frequency was 0.004 (6/1476 alleles) in our study, suggesting a possible association with deafness. Further studies are needed to elucidate the pathogenicity of GJB3 variant c.538C > T. Previous reports have indicated that the prevalence of GJB2 mutations varies among different ethnic groups. The most common GJB2 mutation in Caucasians (c.35delG) has also been the most frequent mutation in patients of Uyghur ethnicity in Xinjiang province, where central and western Asian cultures converge on this northwestern border of China (Chen et al., 2011). The c.35delG has not been detected in our patients that were from five different ethnic groups in Inner Mongolia (Han, Mongolian, Manchurian, Hui and Daur). However, the c.235delC accounted for 41.8% of all GJB2 mutations detected in our study.
Inner Mongolia is located on the northern border of China, and is populated by a variety of ethnicities, but mainly by Han and Mongolian ethnicities. We found no significant difference in the frequency of GJB2 and SLC26A4 mutations between Han and Mongolian ethnicities. Similar results have been reported in southwestern China study, where no significant difference in frequencies of GJB2 and SLC26A4 mutations among China minorities and Han ethnicity populations were detected (Qing et al., 2015).
Although the frequencies of common deafnessrelated mutations are similar in many reports, local differences still exist. In our study, we found significant differences among different cities/leagues in Inner Mongolia. At least one mutated allele was detected in more than 50% of patients in four cities/leagues including Ulanqab, Xilingol, Chifeng and Hulunbeier. Interestingly, a significant difference in the frequency of deafness-related mutations was found between Hohhot and Baotou. This suggests a strong difference in mutation distribution. A possible explanation may be traced back to the beginning of the Ming and Qing dynasties, when a large population migration movement called "Zou Xikou" started (Yin and Bai, 2013). The migration from Shanxi province to Inner Mongolia lasted more than 300 years, and promoted the formation of Baotou. This migration might have contributed to the difference in frequencies of deafness-related mutations between Hohhot and Baotou. Further studies with more participants involved are needed to verify the results and elucidate possible causes.
The reported frequencies of common mutations in GJB2, SLC26A4, GJB3 and MT-RNR1 in children with SNHL in Inner Mongolia, present a conclusive molecular diagnosis of hearing loss. The low mutation frequency in patients from Baotou suggests that other mutations in other deafness-associated genes may explain hearing loss in this area.