Spectrum and Frequency of the GJB2 Gene Pathogenic Variants in a Large Cohort of Patients with Hearing Impairment Living in a Subarctic Region of Russia (the Sakha Republic)

Pathogenic variants in the GJB2 gene, encoding connexin 26, are known to be a major cause of hearing impairment (HI). More than 300 allelic variants have been identified in the GJB2 gene. Spectrum and allelic frequencies of the GJB2 gene vary significantly among different ethnic groups worldwide. Until now, the spectrum and frequency of the pathogenic variants in exon 1, exon 2 and the flanking intronic regions of the GJB2 gene have not been described thoroughly in the Sakha Republic (Yakutia), which is located in a subarctic region in Russia. The complete sequencing of the non-coding and coding regions of the GJB2 gene was performed in 393 patients with HI (Yakuts—296, Russians—51, mixed and other ethnicities—46) and in 187 normal hearing individuals of Yakut (n = 107) and Russian (n = 80) populations. In the total sample (n = 580), we revealed 12 allelic variants of the GJB2 gene, 8 of which were recessive pathogenic variants. Ten genotypes with biallelic recessive pathogenic variants in the GJB2 gene (in a homozygous or a compound heterozygous state) were found in 192 out of 393 patients (48.85%). We found that the most frequent GJB2 pathogenic variant in the Yakut patients was c.-23+1G>A (51.82%) and that the second most frequent was c.109G>A (2.37%), followed by c.35delG (1.64%). Pathogenic variants с.35delG (22.34%), c.-23+1G>A (5.31%), and c.313_326del14 (2.12%) were found to be the most frequent among the Russian patients. The carrier frequencies of the c.-23+1G>A and с.109G>A pathogenic variants in the Yakut control group were 10.20% and 2.80%, respectively. The carrier frequencies of с.35delG and c.101T>C were identical (2.5%) in the Russian control group. We found that the contribution of the GJB2 gene pathogenic variants in HI in the population of the Sakha Republic (48.85%) was the highest among all of the previously studied regions of Asia. We suggest that extensive accumulation of the c.-23+1G>A pathogenic variant in the indigenous Yakut population (92.20% of all mutant chromosomes in patients) and an extremely high (10.20%) carrier frequency in the control group may indicate a possible selective advantage for the c.-23+1G>A carriers living in subarctic climate.

in Russia. The complete sequencing of the non-coding and coding regions of the GJB2 gene was performed in 393 patients with HI (Yakuts-296, Russians-51, mixed and other ethnicities-46) and in 187 normal hearing individuals of Yakut (n = 107) and Russian (n = 80) populations. In the total sample (n = 580), we revealed 12 allelic variants of the GJB2 gene, 8 of which were recessive pathogenic variants. Ten genotypes with biallelic recessive pathogenic variants in the GJB2 gene (in a homozygous or a compound heterozygous state) were found in 192 out of 393 patients (48.85%). We found that the most frequent GJB2 pathogenic variant in the Yakut patients was c.-23+1G>A (51.82%) and that the second most frequent was c.109G>A (2.37%), followed by c.35delG (1.64%). Pathogenic variants с.35delG (22.34%), c.-23+1G>A (5.31%), and c.313_326del14 (2.12%) were found to be the most frequent among the Russian patients. The carrier frequencies of the c.-23 +1G>A and с.109G>A pathogenic variants in the Yakut control group were 10.20% and 2.80%, respectively. The carrier frequencies of с.35delG and c.101T>C were identical (2.5%) in the Russian control group. We found that the contribution of the GJB2 gene pathogenic variants in HI in the population of the Sakha Republic (48.85%) was the highest among all of the previously studied regions of Asia. We suggest that extensive accumulation of the c.-23+1G>A pathogenic variant in the indigenous Yakut population (92.20% of all mutant chromosomes in patients) and an extremely high (10.20%) carrier frequency in the control group may indicate a possible selective advantage for the c.-23+1G>A carriers living in subarctic climate.
Preliminary mutational analysis of the coding region (exon 2) of the GJB2 gene in patients with HI from the Sakha Republic (Yakutia) located in subarctic region of Russia (Northeast Asia) revealed the presence of the GJB2 pathogenic variants in 50.1% of patients of Caucasian origin (Russians, Ukrainians, and Ingushes) and only in 7.2% of the Yakut patients (indigenous population of the Sakha Republic) [83]. Subsequent mutational analysis of the non-coding region of the GJB2 gene revealed a large cohort of Yakut patients with HI who were homozygous for the splice site pathogenic variant c.-23+1G>A (70 unrelated patients in total) [84]. Nevertheless, until now, the spectrum and frequency of all pathogenic variants in exon 1, exon 2 and the flanking intronic regions of the GJB2 gene in the Sakha Republic have not been described thoroughly.
In this study, we present updated data on the spectrum and frequency of the GJB2 gene sequence variants (exon 1, exon 2 and flanking intronic regions) in the extended cohort of patients with HI (n = 393) of different ethnicities and in normal hearing individuals (n = 187) living in the Sakha Republic.

Materials and Methods Patients
Data on individuals with HI were obtained from the Republican Hospital # 1 of the National Medical Centre (Yakutsk, Russian Federation) and the Republican special residential schools for the deaf and hard-of-hearing children (Yakutsk, Russian Federation). The genomic DNA samples of 393 patients with HI from 360 unrelated families were collected from 2005 to 2010. The majority of patients were Yakuts (75.3%; n = 296), Russian patients (12.9%; n = 51), and patients of mixed and other ethnicities (11.7%; n = 46) ( Table 1). Audiograms of patients demonstrated variability in bilateral sensorineural HI (from mild to profound). In most cases, the hearing thresholds were determined by pure-tone audiometry, using a clinical tonal audiometer GSI 60 (Grason-Stadler, Madison, WI, USA) in a soundproof room according to the current clinical standards. Air-conduction thresholds were obtained at 0.125, 0.25, 0.5, 1, 2, 4, and 8 kHz. Severity of hearing loss was defined as mild (25-40 dB), moderate (41-70 dB), severe (71-90 dB) or profound (above 90 dB).

Control group
The control group was represented by 187 unrelated normal hearing individuals of Yakut (n = 107) and Russian (n = 80) ethnicities living in different districts of the Sakha Republic (Table 1). Blood samples were collected after written informed consent. The carrier frequency of the major GJB2 pathogenic variants c.-23+1G>A and c.35delG in Yakuts was calculated by a compilation of corresponding data from previous studies [85,86]. Differences in the c.-23+1G>A pathogenic variant frequencies between the study groups (95% credible interval) were computed with the 'Sampling' software kindly provided by V. Macaulay and adapted by M. Metspalu (Estonian Biocentre, Tartu, Estonia).
Sequence analysis of the GJB2 gene DNA was extracted from the blood leukocyte fraction using the phenol-chloroform method. Amplification of non-coding (exon 1), coding (exon 2) and flanking intronic regions of the GJB2 gene was conducted with PCR on a MJ Mini (Bio-Rad) thermocycler using primers 5'-CCGGGAAGCTCTGAGGAC-3' and 5'-GCAACCGCTCTGGGTCTC-3' for amplification of exon 1 [55] and 5'-TCGGCCCCAGTGGTACAG-3' and 5'-CTGGGCAATGCGTTAAACTGG -3' for amplification of exon 2 [32,58,59]. The PCR products were subjected to direct sequencing using the same primers on ABI PRISM 3130XL (Applied Biosystems, USA) Genomics Core Facility, Institute of Chemical Biology and Fundamental Medicine, Siberian Branch of the Russian Academy of Sciences, Novosibirsk, Russia). DNA sequences variations were identified through comparison with the GJB2 gene reference sequences М86849.2 and U43932.1 (GenBank).

GJB2 genotypes in patients
Twenty one different GJB2 genotypes were identified in all patients with HI (n = 393). Among them, ten pathogenic (biallelic recessive pathogenic variants in a homozygous or compound heterozygous state) GJB2 genotypes were found in 192 patients (48.85%). Four common pathogenic GJB2 genotypes were presented with a frequency >1%: с. Spectrum and contribution of the GJB2 gene pathogenic variants in HI in two different ethnic groups of patients We found different contributions of the GJB2 gene pathogenic variants in HI among two major different ethnic groups of patients (Yakuts and Russians). The presence of biallelic recessive GJB2 pathogenic variants were detected in 157 out of 296 Yakut patients with HI (53.04%) ( Table 2). Pathogenic variant c.-23+1G>A was the most frequent (93.63% of all mutant chromosomes) among the three recessive GJB2 pathogenic variants detected in Yakut patients ( Fig  2). The HI in 17 out of 51 Russian patients (33.33%) was caused by the presence of biallelic recessive GJB2 pathogenic variants. Particularly, c.35delG was the most frequent (61.76% of all mutant chromosomes) among the five pathogenic variants found in the Russian patients (c.-23+1G>A, c.35delG, с.313_326del14, c.333_334delAA, and с.167delT) (Fig 2). Three pathogenic GJB2 genotypes accounted for HI in 39.13% of patients of the other ethnicities (Fig 2).

Distribution of HI caused by the presence of biallelic GJB2 recessive pathogenic variants in the Sakha Republic
We analyzed the distribution of the GJB2 genotypes with biallelic recessive pathogenic variants in the Sakha Republic (S1 Table and Fig 3). The average rate of HI caused by the biallelic GJB2  pathogenic variants was 2.00±0.14 per 10,000, with the highest prevalence in the Nyurbinskiy (9.50±1.94) and Churapchinskiy (7.84±1.96) districts of the Sakha Republic.

Discussion
In this study, we present updated data on the spectrum and frequency of the GJB2 gene allelic variants (exon 1, exon 2 and flanking intronic region) in a large cohort of patients with HI (n = 393) and in normal hearing individuals (n = 187) living in the Sakha Republic (in total, n = 580). The majority of patients with HI were Yakuts (indigenous population of the Sakha Republic) and Russians, with the minority being from other ethnic groups (Ukrainians, Evenks, Evens, and Tatars) and individuals of mixed ethnicity. In total, in the studied samples (n = 580), we revealed 12 allelic variants of the GJB2 gene, eight of which were recessive pathogenic variants. All identified sequence variants were found in the coding region (exon 2) of GJB2, except for the splice site pathogenic variant c.-23 +1G>А, which is located in the non-coding region (intron 1) of GJB2 (Fig 1). The c.-23 +1G>A (42.28%), с.35delG (5.92%), and c.109G>A (1.92%) pathogenic variants were found to be common (>1%) in the total patient samples.
Data on the territorial distribution of HI caused by genetic factors are of great importance for the clinical evaluation of deaf people and for estimating recurrence risks for their families. In this study, we estimated the distribution of the GJB2 genotypes with biallelic recessive pathogenic variants in the Sakha Republic (average rate was found to be 2.00±0.14 per 10,000) (S1 Table). The highest prevalence of HI, caused by biallelic GJB2 recessive pathogenic variants, was registered in the Nyurbinskiy (9.50±1.94) and Churapchinskiy (7.84±1.96) districts of the Sakha Republic (Fig 3). These findings are comparable with our recent data on the extensive accumulation of the c.-23+1G>A splice site pathogenic variant in the GJB2 gene as a result of the founder effect [84]. Reconstruction of 140 haplotypes with c.-23+1G>A demonstrated that the most recombined haplotypes (more ancient) were found in the same districts (Nyurbinskiy and Churapchinskiy) of the Sakha Republic [84]. The age of c.-23+1G>A in the Yakut population was estimated at approximately 800 years [84]. A more ancient age of the common GJB2 pathogenic variants was shown for c.35delG in the Caucasian populations (approximately Prevalence of congenital HI caused by biallelic GJB2 pathogenic variants in the Sakha Republic. Note: The territory of the Sakha Republic is shown in blue (bottom map). HI rates were calculated per 10,000 people, and appropriate data are presented only for the districts and cities of the Sakha Republic with population more than 10,000. Detailed data are presented in S1 Table. doi:10.1371/journal.pone.0156300.g003 GJB2-Pathogenic Variants in Patients with Hearing Impairment in Subarctic Part of Russia 10000 years) [88], c.235delC in East Asian populations (approximately 11500 years) [89], and p.Trp24 Ã in India (approximately 8800 years) [43].
In total, from all of the samples, we identified 10 pathogenic genotypes (with biallelic recessive GJB2 pathogenic variants) in 192 out of 393 studied patients (48.85%). Thus, 48.85% of HI in patients in the Sakha Republic could be caused by the GJB2 pathogenic variants. Previous reports suggested that the contribution of the GJB2 pathogenic variants in HI in Asian populations was lower than in Europe and the US. The low contribution of the GJB2 pathogenic variants in HI was demonstrated in Mongolia (4.5%) [45], Japan (7.52%) [46], Thailand (8.4%) [47] and Korea (8.2%) [42]. A higher contribution of the GJB2 pathogenic variants in HI was found in China (14.9%) [35], Iran (16.1%) [50] and India (21.1%) [39]. Therefore, our results indicate that the contribution of the GJB2 pathogenic variants to HI (48.85%) in the Sakha Republic located in subarctic part of Russia was the highest among all studied Asian regions.
We estimated that the total carrier frequency of the GJB2 pathogenic variants in the Yakuts was 0.143 (Table 4). Based on this data, the expected rate of patients with HI (homozygous or compound heterozygous for the GJB2 gene pathogenic variants) should be approximately 0.005 in the Yakut population (466,492 in total) or approximately 50 per 10,000 people, which is substantially higher than what we found (Fig 3). This bias could be explained by two reasons: first, such theoretical calculations cannot be applied to a relatively small isolated and subdivided Yakut population, and second, there is a possible underestimation of hearing-impaired people due to the known phenotypic variability of HI (from profound to mild) caused by pathogenic variants in GJB2 gene. In total, 85% of patients demonstrated severe to profound HI, while 14% displayed moderate HI, and 1% displayed mild HI [84].
In contrast, an extremely high prevalence of the c.-23+1G>A pathogenic variant in the indigenous people living in the subarctic region of Russia (up to 13.3% in some sub-populations of the Yakuts) is comparable with the carrier frequency of the HbS allele associated with sickle cell anemia in Africa (a frequency of 10% and higher of the HbS allele was registered only in certain areas of sub-Saharan Africa) [86,90]. The worldwide carriers' frequency for different GJB2 pathogenic variants is very high [3,85], suggesting a common selective advantage for heterozygous GJB2 variants on a global scale. The GJB2 heterozygote advantage might consist of increased resistance to gastrointestinal infections due to the epithelial barrier thickening, as suggested in previous studies [91][92][93][94][95][96]. We suggest a similar mechanism of heterozygous advantage for the c.-23+1G>A carriers, although further comprehensive studies are needed to elucidate the special features related to the subarctic climate of the Sakha Republic.

Conclusions
We found that the contribution of the GJB2 gene pathogenic variants to HI in the population of the Sakha Republic (48.85%) was the highest among all of the regions of Asia studied previously. We suggest that extensive accumulation of the c.-23+1G>A pathogenic variant in the indigenous Yakut population (92.20% of all mutant chromosomes in patients and an extremely high (10.20%) carrier frequency in the control group) may indicate the possible selective advantage of the c.-23+1G>A carriers living in the subarctic climate.
Supporting Information S1