Acta Oto-Laryngologica. 2014; 134: 924–929
ORIGINAL ARTICLE
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Analysis of common deafness gene mutations in deaf people from unique ethnic groups in Gansu Province, China
BAI-CHENG XU1*, PAN-PAN BIAN1*, XIAO-WEN LIU1, YI-MING ZHU1, XIAO-LONG YANG2, JIAN-LI MA3, XING-JIAN CHEN1, YAN-LI WANG1 & YU-FEN GUO1,4 1
Department of Otolaryngology-Head and Neck Surgery, Second Hospital of Lanzhou University, Lanzhou, 2Department of Otolaryngology-Head and Neck Surgery, People’s Hospital of Gansu Province, Lanzhou, 3Department of Otolaryngology-Head and Neck Surgery, General Hospital of Ningxia University, Yinchuan and 4Ministry of Health, Gansu Province, Lanzhou, China
Abstract Conclusions: The GJB2 gene mutation characteristic of Dongxiang was the interaction result of ethnic background and geographical environment, and Yugur exhibited the typical founder effect. The SLC26A4 gene mutation characteristic of Dongxiang was related to caucasian backgrounds and selection of purpose exons, i.e. ethnic background and the penetrance of ethnic specificity caused the low mtDNA1555A>G mutation frequency in Dongxiang. Objectives: To determine the prevalence of GJB2 and SLC26A4 genes and mtDNA1555A>G mutations and analyze the ethnic specificity in the non-syndromic sensorineural hearing loss (NSHL) of unique ethnic groups in Gansu Province. Methods: Peripheral blood samples were obtained from Dongxiang, Yugur, Bonan, and ethnic Han groups with moderately severe to profound NSHL in Gansu Province. Bidirectional sequencing (or enzyme digestion) was applied to identify the sequence variations. Results: The pathogenic allele frequency of the three gene mutations was different. The frequency of the GJB2 gene among the Dongxiang, Yugur, Bonan, and ethnic Han groups was 9.03%, 12.5%, 5.88%, and 12.17%, respectively. No difference was found between the ethnic groups. The frequencies of the SLC26A4 genes were 3.23%, 8.33%, 0%, and 9.81%, respectively. The mutation frequency of mtDNA1555A>G was 0%, 0%, 0%, and 6.03%, respectively. No difference was found between the ethnic groups, except for the Dongxiang and ethnic Han groups, both in SLC26A4 gene and mtDNA1555A>G.
Keywords: GJB2, SLC26A4, mtDNA1555A>G, Dongxiang, Bonan, Yugur, mutation
Introduction Deafness is a common human auditory disorder and an important factor in the quality of life within a population. Research has shown that the incidence of congenital deafness is 1/1000 to 1/500 [1]. For deaf people in China, the mutation frequencies of the GJB2 and SLC26A4 genes and mtDNA1555A>G were 20%, 8.95%, and 3.43%, respectively [2–4]. These values imply that the mutations of the GJB2 and SLC26A4 genes and mtDNA1555A>G cause 30% of congenital deafness cases.
GJB2 was found as the most common mutation gene in patients with non-syndromic sensorineural hearing loss (NSHL). GJB2 has specific mutation forms and frequencies in different races. The main mutation form is c.35delG in caucasians [5], c.167delT in Jews [6], c.427C>T in African negroids [7], and c.235delC in Worldwide mongoloids [3]. Another study of various ethnicities in China also suggested the ethnic specificity of GJB2 gene mutations. The pathogenic allele frequency of c.235delG was 23.7%, 18.3%, 13%, 10.1%, and 0.8% in Manchu, Mongolian, ethnic Han, Hui, and Tibetan
Correspondence: Professor Yu-Fen Guo, MD PhD, Department of Otolaryngology-Head and Neck Surgery, Second Hospital of Lanzhou University, No. 82, Cuiyingmen Street, Lanzhou City, 730030, Gansu Province, China. Tel: +86 09318942468. E-mail: [email protected] * These authors contributed equally to this work.
(Received 6 March 2014; accepted 12 May 2014) ISSN 0001-6489 print/ISSN 1651-2251 online 2014 Informa Healthcare DOI: 10.3109/00016489.2014.927588
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Deafness gene mutations in unique ethnic groups ethnicities, respectively. A statistical difference was found between races after statistical analysis [8]. Related research on the SLC26A4 gene also suggested racial specificity. The most common types of mutation are as follows: IVS7-2A>G and c.2168A>G among the mongoloids of mainland China [9], c.1246A>C and IVS8+1G>A among caucasians in the Nordic region [10], and c.2168A>G among Japanese and South Koreans [11,12]. Differences also existed among ethnic Chinese groups. The carrier rate of IVS7-2A>G was 13.88% in ethnic Hans, 0% in Tibetans, 0% in Uighurs, and 3.6% in southwestern ethnic minorities [13]. Mutations in mitochondrial DNA (mtDNA) are a common molecular cause of deafness after GJB2 and SLC26A4 mutations. According to the polymorphism of the nucleotide sequence, mtDNA is divided into types I and II [14]. Type II mtDNA is common among Asians (mainly mongoloid), which have higher mtDNA1555A>G mutation rates than caucasians [15]. Thus, mtDNA1555A>G also has racial specificity. Dongxiang, Yugur, and Bonan are unique ethnic groups that live in their own ethnic neighborhoods in Gansu Province, China. Conservative ethnic genetic information depends on different national religions, languages, and customs. These minority ethnic groups have different ethnic backgrounds. Dongxiang is the fusion of mongoloids and caucasians [16], Yugur has a clear caucasian racial background [17], and Bonan is mostly of mongoloid origin [18]. According to the molecular epidemiological study of these genes and mutation sites, research on mutation frequency, hot spot mutation, and ethnic specificity in unique ethnic groups will provide a basis for deafness prevention and genetic counseling. Material and methods
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acoustic immittance, and auditory-evoked brainstem response), and blood samples of the patients were collected by the Department of Otorhinolaryngology and Head and Neck Surgery in the Second Affiliated Hospital of Lanzhou University. General procedures for DNA isolation and sequencing Genomic DNA from peripheral blood leukocytes was obtained by the salting out method. All PCR amplified products for the target fragments of the three genes were purified with a Millipore (Shanghai, China) plate and were then sequenced with an ABI3730 (Shanghai, China). The sequence data were analyzed by aligning with reference sequences in NCBI (NC000013 for GJB2 gene, NT-007933 for SLC26A4 gene, AC-000021 for mtDNA1555A>G) via Sequence Scanner 1.0 (Ann Ardor, MI, USA) and DNAstar 7.0 software (Madison, WI, USA). Mutations or polymorphisms were identified according to reference sequences. GJB2 mutation screening The GJB2 gene has two exons, and the coding region is in exon2. We chose exon2 as the target. The primer pairs, PCR and AGE, were designed on the basis of previous reports by Guo et al. [3]. SLC24A4 mutation screening SLC26A4 gene has 21 exons in total. IVS7-2A>G and c.2168A>G were the main mutations in the study of the Chinese group. The former was located in the splice site of exon8, whereas the latter was located in exon19. For this reason, we selected exon8 and exon19 for the present study. The primer pairs, PCR and AGE, were designed on the basis of a previous report by Wang et al. [9].
Research subjects mtDNA1555A>G mutation screening In this study, 155 patients from the Dongxiang group (98 males and 57 females; average age of 28.27 ± 18.5 years), 17 patients from the Bonan group (11 males and 6 females; average age of 40.29 ± 14.91 years), and 12 patients from the Yugur group (8 males and 4 females; average age of 31.14 ± 21.64 years) had moderately severe, severe, or profound NSHL, respectively. We selected 464 patients from the ethnic Han group (262 males and 202 females; average age of 15 ± 3.58 years) as the control group. All patients were from different families whose hearing losses were not caused by auricle or middle ear diseases. The clinic dates, hearing test dates (including pure-tone audiometry,
PCR and Alw26I digestion analysis were designed on the basis of a previous report by Guo et al. [3]. The electrophoresis on the 2% agarose gel was run to examine the digested product. If the digested product showed the specific band for mtDNA1555A>G, the PCR product was verified by direct sequencing. Statistical analysis Mutation comparisons among ethnicities were performed by the chi-squared test via SPSS 19.0 software (Chicago, IL, USA). p values < 0.05 were considered statistically significant.
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Results
Table II. Mutation genotype statistical table for the SLC26A4 gene.
GJB2 mutations
Mutation genotype
Twelve subjects in the Dongxiang group (9 homozygous, 3 compound heterozygous), 1 subject in the Yugur group (compound heterozygous), 1 subject in the Bonan group (compound heterozygous), and 38 subjects in the ethnic Han group (homozygous) exhibited GJB2 gene mutations; the mutation frequencies were 7.74% (12/155), 8.33% (1/12), 5.88% (1/17), and 8.19% (38/464), respectively (Table I). No differences were found between the ethnic groups. The pathogenic allele frequency of the ethnic groups were 9.03% (28/310), 12.5% (3/24), 5.88% (2/34), and 12.17% (113/928), respectively (Table I). No differences were found between the ethnic groups. In the Dongxiang group, the pathogenic allele frequency of c.299_300delAT and c.235delC (3.87%, 12/310) were the hot spots and accounted for 42.9% (12/28) of the mutation alleles. The carrying rate of c.299_300delAT (4.51%, 7/155) was higher than that of c.235delC (3.87%, 6/155). The pathogenic allele frequency of c.35delG (8.33%, 2/24) was the highest in the Yugur group, accounting for 66.7% (2/3) of the mutation alleles. In the ethnic Han group, the pathogenic allele frequency of c.235delC (10.35%, 96/928) was the highest; the pathogenic allele frequency of c.299_300delAT was 0.43% (4/928). Both the pathogenic allele frequencies of c.299_300delAT in the Dongxiang group (p < 0.01, c2 = 18.942) and c.35delG in the Yugur group (p = 0.012) were significantly higher than for the Han group. The pathogenic allele frequency of
Table I. Mutation genotype statistical table for the GJB2 gene. Mutation genotype
Dongxiang Yugur Bonan Han
35delG/35delG
0
0
0
1
235delC/235delC
3
0
0
33
257C>G/257C>G
1
0
0
1
Dongxiang
Yugur
Bonan
Han
IVS7-2A>G/IVS7-2A>G
3
1
0
20
IVS7-2A>G/2162C>T
0
0
0
1
IVS7-2A>G/2168A>G
1
0
0
3
IVS7-2A>G/wt
0
0
0
38
2162C>T/wt
1
0
0
0
2168A>G/wt
1
0
0
5
c.235delC in the Dongxiang group was significantly lower than in the ethnic Han group (p < 0.01, c2 = 12.231). SLC26A4 mutations Four subjects in the Dongxiang group (3 homozygous, 1 compound heterozygous), 1 subject in the Yugur group (compound heterozygous), none of the Bonan group, and 24 subjects in the ethnic Han group (20 homozygous, 4 compound heterozygous) exhibited GJB2 gene mutations; their mutation frequencies were 2.58% (4/155), 8.33% (1/12), 0%, and 5.17% (24/464), respectively (Table II). No difference was found between the ethnic groups. The pathogenic allele frequencies of the ethnic groups were 3.23% (10/310), 8.33% (2/24), 0%, and 9.81% (91/928), respectively (Table II). No differences were found between the ethnic groups, except for the Dongxiang and ethnic Han groups. Two types of novel sequence variations, namely, c.918+48A>G and c.2090-51 insAAAC, were found in this study. A total of three types of mutations (IVS7-2A>G, c.2168A>G, and c.2162C>T) were detected. The pathogenic allele frequency of IVS7-2A>G was the highest among the Dongxiang, Yugur, and ethnic Han groups at 2.26% (7/310), 8.33% (2/24), and 8.84% (82/928), respectively (Table II). The pathogenic allele frequency of IVS7-2A>G in the Dongxiang group was lower than in the ethnic Han group (p < 0.01, c2 = 15.070). No differences were found among the Yugur and ethnic Han groups (p = 0.932, c2 = 0.007).
299-300delAT/299-300delAT
5
0
0
1
176-191del16/176-191del16
0
0
0
2
176-191del16/299-300delAT
0
0
1
0
mtDNA1555A>G
235delC/35delG
1
1
0
0
235delC/299-300delAT
2
0
0
0
35delG/wt
0
1
0
3
mtDNA1555A>G was not detected in the Dongxiang, Yugur, and Bonan groups. A total of 28 subjects in the ethnic Han group exhibited mtDNA1555A>G mutations with a mutation frequency of 6.03% (28/ 464). The mutation frequency of the Dongxiang group was lower than in the ethnic Han group (p = 0.002, c2 = 9.797). No statistically significant differences were found between the Yugur and ethnic
235delC/wt
3
0
0
30
176-191del16/wt
0
0
0
3
299-300delAT/wt
0
0
0
2
380G>A/wt
1
0
0
0
Deafness gene mutations in unique ethnic groups Han groups (p = 1.000) or between the Bonan and ethnic Han groups (p = 0.614).
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Discussion The unique ethnic groups of Dongxiang, Yugur, and Bonan in Gansu, China, have conservative genetic information. Furthermore, the characteristics of the GJB2 and SLC26A4 genes and mtDNA1555A>G mutations have obvious racial and ethnic specificity. Therefore, this study determined the prevalence and ethnic specificity of the GJB2 and SLC26A4 genes and the mtDNA1555A>G mutations among the unique ethnic groups. In the Dongxiang, Yugur, and Bonan groups, the pathogenic allele frequencies of the GJB2 gene were 9.03%, 12.5%, and 5.88%, respectively. No obvious differences were found between the unique ethnic groups and the ethnic Han group. Thus, GJB2 gene mutations are also an important cause of deafness in unique ethnic groups in Gansu Province, such as the ethnic Han group. The main forms of GJB2 gene mutations in the Dongxiang group were c.299-300delAT and c.235delC, similar to the ethnic Han group. The Dongxiang group was statistically higher in the pathogenic allele frequency of c.299-300delAT and lower in the pathogenic allele frequency of c.235delC than the ethnic Han group. By contrast, the carrying rate of c.299-300delAT was higher than that of c.235delC. Therefore, the most common mutation form of the Dongxiang group was c.299-300delAT, unlike in the ethnic Han group. The Dongxiang group traces its origins among the three theories of the Mongols, Hybrids, and Sarts. Among these theories, the Sarts are the most commonly accepted origin by the academe and the public. Sarts mainly originated from Turks, who belong to the yellow–white mixed race (mongoloid and caucasian) and fused with other minorities such as Mongolians [16]. c.235delC (mainly) and c.299-300delAT were the hot spots of mongoloids [3]. The caucasian hot spot is c.35delG [5]. Thus, the ethnic origin cannot fully explain why the proportion of c.299-300delAT was high in the Dongxiang group. In terms of mutation forms, the Dongxiang group was similar to the mongoloid group, thus showing that the mutation characteristics of the GJB2 gene in the Dongxiang group were close to the mongoloid group. This observation was in line with the ethnic background of the Dongxiang group. From genetics, the disease phenotype is related to gene mutation, gene interaction, and external factors (e.g. the environment). Therefore, we thought that the mutation characteristics of the GJB2 gene in the Dongxiang group were the result of different factors such as
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ethnic background, geographical environment difference, and ethnic migration. These factors need to be studied further. Current research suggests that the high incidence of c.35delG among caucasians was the expression of the founder effect. In this study, c.35delG accounted for 66.7% (2/3) of all pathogenic alleles and was the hot spot in the Yugur group. Considering the obvious caucasian ethnic background [17], this result could be considered an expression model of the founder effect wherein c.35delG was the hot spot of the GJB2 gene in the Yugur group. Given that only two pathogenic alleles (c.299300delAT and c.176-191del16) of the GJB2 gene were detected, no hot spot could be drawn in the Bonan group. This condition was influenced by the small population base, and more patients will be needed in future studies. In the Dongxiang group, the pathogenic allele frequency of the SLC26A4 gene was 3.23% (10/ 310) and the allele frequency of IVS7-2A>G was 2.26% (7/310); the latter was the hot spot and accounted for 70% (7/10) of all pathogenic alleles. From the statistical analysis, the Dongxiang group had lower pathogenic allele frequencies of SLC26A4 and IVS7-2A>G than the ethnic Han group. Previous studies confirmed that the pathogenic mutation detection rate of SLC26A4 in caucasians was obviously lower than in mongoloids among deaf people [19]. Considering that the Dongxiang group is a fusion of mongoloids and caucasians, we could easily determine that the lower pathogenic allele frequency of the Dongxiang than the ethnic Han group was a manifestation of racial specificity. The hot spot of the Dongxiang group was IVS7-2A>G, which is similar to the ethnic Han group. The hot spot of the caucasians (IVS8+1G>A) was not found. The mutation characteristics of the SLC26A4 gene in the Dongxiang were similar to mongoloids; this result was in line with the results for the GJB2 gene. However, the statistical analysis of the pathogenic allele frequency of SLC26A4 (exon8 and exon19) was different from GJB2. The Dongxiang group was smaller than the ethnic Han group; this result might be associated with the choice of purpose exons. The Dongxiang group has a caucasian background, but exon10 (wherein one of the hot spots of caucasians, c.1246A>G, was located) was not the purpose exon. Thus, further work on other exons, particularly exon10, is necessary. In this study, we found a new sequence change form (i.e. c.918+48A>G) in the Dongxiang group. This new sequence was a point mutation in the intron and was far from the exon. Therefore, this type of mutation was unlikely to have caused the disease.
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Only one homozygous mutation and hot spot, i.e. IVS7-2A>G, was detected in the Yugur group. The pathogenic and IVS7-2A>G allele frequencies were both similar to that of the ethnic Han group but were different from the ethnic specificity expression of the GJB2 gene in the Yugur group. The analysis of the GJB2 gene was validated considering the caucasian background of the Yugur group. The reasons for the abovementioned results were as follows. On one hand, bias easily appeared because the sample size was small. On the other hand, the problem of the purpose exons was similar to that of the Dongxiang group. Thus, we still need to enlarge the sample size and expand the exon number for the next step of our research. No pathogenic mutation was detected in the Bonan group; this result might be associated with the small sample size. We also found a new sequence change form in the Bonan group (i.e. c.2090-51insAAAC). Although this new sequence change form was an intron and is far from the exon, this sequence inserted four basic groups and largely affected the synthesis of the amino acids. This observation still needs to be verified in further studies. The caucasian background was the most important reason why the mtDNA1555A>G mutation frequency of the Dongxiang group was significantly lower than that of the ethnic Han group. The specificity of the mtDNA1555A>G deafness penetration could be another reason [20]. mtDNA1555A>G screening among normal-hearing people needs to be conducted to confirm this result. The mtDNA1555A>G mutation frequency of the Yugur and Bonan groups had no statistical differences from that of the ethnic Han group. The caucasian background of the Yugur group could explain the failure of detection. Given the probability of bias in the smaller sample sizes of the Yugur and Bonan groups, we need to enlarge the sample size and conduct thorough follow-up research. In conclusion, the GJB2 and SLC26A4 genes and the mtDNA1555A>G mutation in the unique ethnic groups had respective specificities. The ethnic background might be the main cause of this observation. The pathogenic allele frequency of the GJB2 gene in the unique ethnic groups was similar to that in the ethnic Han group. The mutation characteristic of the Dongxiang group was caused by the interaction of the ethnic background and geographical environment. The mutation characteristic of the Yugur group was the typical model for the founder effect. Moreover, the molecular epidemiological characteristics were uncertain. The hot spot of the SLC26A4 gene was IVS7-2A>G in both the Yugur and Bonan groups, similar to the ethnic Han group. The
pathogenic allele frequency of the Dongxiang group was lower than that of the ethnic Han group because of the caucasian background and the selection of purpose exons. The mutation characteristics of the Yugur group did not conform to the ethnic background; this result was associated with the small sample size and exon selection. A similar conclusion could not be made for the Bonan group. The ethnic background and penetration of ethnic specificity were the reasons why the mtDNA1555A>G mutation frequency was low in the Dongxiang group.
Acknowledgments This work was supported by grants from the National Natural Science Foundation of China (grant no. 81172765). Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
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[14] Horai S, Matsunaga E. Mitochondrial DNA polymorphism in Japanese. II. Analysis with restriction enzymes of four or five base pair recognition. Hum Genet 1986;72:105–17. [15] Hutchin T, Cortopassi G. Mitochondrial DNA haplotype predicts deafness risk. Am J Med Genet 1995;60:592. [16] Ma G, Zhao G. The population size and distribution of Dongxiang in China. Gansu Theory Research 2005;6:121–5. [17] Qian B. The transition and name of Sarig Uyghur. Social Sciences In Xinjiang 2004;6:98–104. [18] Ma Y, Wang L. The research description of Bonan. NW Ethno-National Studies 2004;1:203–7. [19] Albert S, Blons H, Jonard L, Feldmann D, Chauvin P, Loundon N, et al. SLC26A4 gene is frequently involved in nonsyndromic hearing impairment with enlarged vestibular aqueduct in Caucasian populations. Eur J Hum Genet 2006;14:773–9. [20] Fischel-Ghodsian N. Mitochondrial mutations and hearing loss: paradigm for mitochondrial genetics. Am J Hum Genet 1998;62:15–19.
ORIGINAL ARTICLE
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Analysis of common deafness gene mutations in deaf people from unique ethnic groups in Gansu Province, China
BAI-CHENG XU1*, PAN-PAN BIAN1*, XIAO-WEN LIU1, YI-MING ZHU1, XIAO-LONG YANG2, JIAN-LI MA3, XING-JIAN CHEN1, YAN-LI WANG1 & YU-FEN GUO1,4 1
Department of Otolaryngology-Head and Neck Surgery, Second Hospital of Lanzhou University, Lanzhou, 2Department of Otolaryngology-Head and Neck Surgery, People’s Hospital of Gansu Province, Lanzhou, 3Department of Otolaryngology-Head and Neck Surgery, General Hospital of Ningxia University, Yinchuan and 4Ministry of Health, Gansu Province, Lanzhou, China
Abstract Conclusions: The GJB2 gene mutation characteristic of Dongxiang was the interaction result of ethnic background and geographical environment, and Yugur exhibited the typical founder effect. The SLC26A4 gene mutation characteristic of Dongxiang was related to caucasian backgrounds and selection of purpose exons, i.e. ethnic background and the penetrance of ethnic specificity caused the low mtDNA1555A>G mutation frequency in Dongxiang. Objectives: To determine the prevalence of GJB2 and SLC26A4 genes and mtDNA1555A>G mutations and analyze the ethnic specificity in the non-syndromic sensorineural hearing loss (NSHL) of unique ethnic groups in Gansu Province. Methods: Peripheral blood samples were obtained from Dongxiang, Yugur, Bonan, and ethnic Han groups with moderately severe to profound NSHL in Gansu Province. Bidirectional sequencing (or enzyme digestion) was applied to identify the sequence variations. Results: The pathogenic allele frequency of the three gene mutations was different. The frequency of the GJB2 gene among the Dongxiang, Yugur, Bonan, and ethnic Han groups was 9.03%, 12.5%, 5.88%, and 12.17%, respectively. No difference was found between the ethnic groups. The frequencies of the SLC26A4 genes were 3.23%, 8.33%, 0%, and 9.81%, respectively. The mutation frequency of mtDNA1555A>G was 0%, 0%, 0%, and 6.03%, respectively. No difference was found between the ethnic groups, except for the Dongxiang and ethnic Han groups, both in SLC26A4 gene and mtDNA1555A>G.
Keywords: GJB2, SLC26A4, mtDNA1555A>G, Dongxiang, Bonan, Yugur, mutation
Introduction Deafness is a common human auditory disorder and an important factor in the quality of life within a population. Research has shown that the incidence of congenital deafness is 1/1000 to 1/500 [1]. For deaf people in China, the mutation frequencies of the GJB2 and SLC26A4 genes and mtDNA1555A>G were 20%, 8.95%, and 3.43%, respectively [2–4]. These values imply that the mutations of the GJB2 and SLC26A4 genes and mtDNA1555A>G cause 30% of congenital deafness cases.
GJB2 was found as the most common mutation gene in patients with non-syndromic sensorineural hearing loss (NSHL). GJB2 has specific mutation forms and frequencies in different races. The main mutation form is c.35delG in caucasians [5], c.167delT in Jews [6], c.427C>T in African negroids [7], and c.235delC in Worldwide mongoloids [3]. Another study of various ethnicities in China also suggested the ethnic specificity of GJB2 gene mutations. The pathogenic allele frequency of c.235delG was 23.7%, 18.3%, 13%, 10.1%, and 0.8% in Manchu, Mongolian, ethnic Han, Hui, and Tibetan
Correspondence: Professor Yu-Fen Guo, MD PhD, Department of Otolaryngology-Head and Neck Surgery, Second Hospital of Lanzhou University, No. 82, Cuiyingmen Street, Lanzhou City, 730030, Gansu Province, China. Tel: +86 09318942468. E-mail: [email protected] * These authors contributed equally to this work.
(Received 6 March 2014; accepted 12 May 2014) ISSN 0001-6489 print/ISSN 1651-2251 online 2014 Informa Healthcare DOI: 10.3109/00016489.2014.927588
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Deafness gene mutations in unique ethnic groups ethnicities, respectively. A statistical difference was found between races after statistical analysis [8]. Related research on the SLC26A4 gene also suggested racial specificity. The most common types of mutation are as follows: IVS7-2A>G and c.2168A>G among the mongoloids of mainland China [9], c.1246A>C and IVS8+1G>A among caucasians in the Nordic region [10], and c.2168A>G among Japanese and South Koreans [11,12]. Differences also existed among ethnic Chinese groups. The carrier rate of IVS7-2A>G was 13.88% in ethnic Hans, 0% in Tibetans, 0% in Uighurs, and 3.6% in southwestern ethnic minorities [13]. Mutations in mitochondrial DNA (mtDNA) are a common molecular cause of deafness after GJB2 and SLC26A4 mutations. According to the polymorphism of the nucleotide sequence, mtDNA is divided into types I and II [14]. Type II mtDNA is common among Asians (mainly mongoloid), which have higher mtDNA1555A>G mutation rates than caucasians [15]. Thus, mtDNA1555A>G also has racial specificity. Dongxiang, Yugur, and Bonan are unique ethnic groups that live in their own ethnic neighborhoods in Gansu Province, China. Conservative ethnic genetic information depends on different national religions, languages, and customs. These minority ethnic groups have different ethnic backgrounds. Dongxiang is the fusion of mongoloids and caucasians [16], Yugur has a clear caucasian racial background [17], and Bonan is mostly of mongoloid origin [18]. According to the molecular epidemiological study of these genes and mutation sites, research on mutation frequency, hot spot mutation, and ethnic specificity in unique ethnic groups will provide a basis for deafness prevention and genetic counseling. Material and methods
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acoustic immittance, and auditory-evoked brainstem response), and blood samples of the patients were collected by the Department of Otorhinolaryngology and Head and Neck Surgery in the Second Affiliated Hospital of Lanzhou University. General procedures for DNA isolation and sequencing Genomic DNA from peripheral blood leukocytes was obtained by the salting out method. All PCR amplified products for the target fragments of the three genes were purified with a Millipore (Shanghai, China) plate and were then sequenced with an ABI3730 (Shanghai, China). The sequence data were analyzed by aligning with reference sequences in NCBI (NC000013 for GJB2 gene, NT-007933 for SLC26A4 gene, AC-000021 for mtDNA1555A>G) via Sequence Scanner 1.0 (Ann Ardor, MI, USA) and DNAstar 7.0 software (Madison, WI, USA). Mutations or polymorphisms were identified according to reference sequences. GJB2 mutation screening The GJB2 gene has two exons, and the coding region is in exon2. We chose exon2 as the target. The primer pairs, PCR and AGE, were designed on the basis of previous reports by Guo et al. [3]. SLC24A4 mutation screening SLC26A4 gene has 21 exons in total. IVS7-2A>G and c.2168A>G were the main mutations in the study of the Chinese group. The former was located in the splice site of exon8, whereas the latter was located in exon19. For this reason, we selected exon8 and exon19 for the present study. The primer pairs, PCR and AGE, were designed on the basis of a previous report by Wang et al. [9].
Research subjects mtDNA1555A>G mutation screening In this study, 155 patients from the Dongxiang group (98 males and 57 females; average age of 28.27 ± 18.5 years), 17 patients from the Bonan group (11 males and 6 females; average age of 40.29 ± 14.91 years), and 12 patients from the Yugur group (8 males and 4 females; average age of 31.14 ± 21.64 years) had moderately severe, severe, or profound NSHL, respectively. We selected 464 patients from the ethnic Han group (262 males and 202 females; average age of 15 ± 3.58 years) as the control group. All patients were from different families whose hearing losses were not caused by auricle or middle ear diseases. The clinic dates, hearing test dates (including pure-tone audiometry,
PCR and Alw26I digestion analysis were designed on the basis of a previous report by Guo et al. [3]. The electrophoresis on the 2% agarose gel was run to examine the digested product. If the digested product showed the specific band for mtDNA1555A>G, the PCR product was verified by direct sequencing. Statistical analysis Mutation comparisons among ethnicities were performed by the chi-squared test via SPSS 19.0 software (Chicago, IL, USA). p values < 0.05 were considered statistically significant.
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Results
Table II. Mutation genotype statistical table for the SLC26A4 gene.
GJB2 mutations
Mutation genotype
Twelve subjects in the Dongxiang group (9 homozygous, 3 compound heterozygous), 1 subject in the Yugur group (compound heterozygous), 1 subject in the Bonan group (compound heterozygous), and 38 subjects in the ethnic Han group (homozygous) exhibited GJB2 gene mutations; the mutation frequencies were 7.74% (12/155), 8.33% (1/12), 5.88% (1/17), and 8.19% (38/464), respectively (Table I). No differences were found between the ethnic groups. The pathogenic allele frequency of the ethnic groups were 9.03% (28/310), 12.5% (3/24), 5.88% (2/34), and 12.17% (113/928), respectively (Table I). No differences were found between the ethnic groups. In the Dongxiang group, the pathogenic allele frequency of c.299_300delAT and c.235delC (3.87%, 12/310) were the hot spots and accounted for 42.9% (12/28) of the mutation alleles. The carrying rate of c.299_300delAT (4.51%, 7/155) was higher than that of c.235delC (3.87%, 6/155). The pathogenic allele frequency of c.35delG (8.33%, 2/24) was the highest in the Yugur group, accounting for 66.7% (2/3) of the mutation alleles. In the ethnic Han group, the pathogenic allele frequency of c.235delC (10.35%, 96/928) was the highest; the pathogenic allele frequency of c.299_300delAT was 0.43% (4/928). Both the pathogenic allele frequencies of c.299_300delAT in the Dongxiang group (p < 0.01, c2 = 18.942) and c.35delG in the Yugur group (p = 0.012) were significantly higher than for the Han group. The pathogenic allele frequency of
Table I. Mutation genotype statistical table for the GJB2 gene. Mutation genotype
Dongxiang Yugur Bonan Han
35delG/35delG
0
0
0
1
235delC/235delC
3
0
0
33
257C>G/257C>G
1
0
0
1
Dongxiang
Yugur
Bonan
Han
IVS7-2A>G/IVS7-2A>G
3
1
0
20
IVS7-2A>G/2162C>T
0
0
0
1
IVS7-2A>G/2168A>G
1
0
0
3
IVS7-2A>G/wt
0
0
0
38
2162C>T/wt
1
0
0
0
2168A>G/wt
1
0
0
5
c.235delC in the Dongxiang group was significantly lower than in the ethnic Han group (p < 0.01, c2 = 12.231). SLC26A4 mutations Four subjects in the Dongxiang group (3 homozygous, 1 compound heterozygous), 1 subject in the Yugur group (compound heterozygous), none of the Bonan group, and 24 subjects in the ethnic Han group (20 homozygous, 4 compound heterozygous) exhibited GJB2 gene mutations; their mutation frequencies were 2.58% (4/155), 8.33% (1/12), 0%, and 5.17% (24/464), respectively (Table II). No difference was found between the ethnic groups. The pathogenic allele frequencies of the ethnic groups were 3.23% (10/310), 8.33% (2/24), 0%, and 9.81% (91/928), respectively (Table II). No differences were found between the ethnic groups, except for the Dongxiang and ethnic Han groups. Two types of novel sequence variations, namely, c.918+48A>G and c.2090-51 insAAAC, were found in this study. A total of three types of mutations (IVS7-2A>G, c.2168A>G, and c.2162C>T) were detected. The pathogenic allele frequency of IVS7-2A>G was the highest among the Dongxiang, Yugur, and ethnic Han groups at 2.26% (7/310), 8.33% (2/24), and 8.84% (82/928), respectively (Table II). The pathogenic allele frequency of IVS7-2A>G in the Dongxiang group was lower than in the ethnic Han group (p < 0.01, c2 = 15.070). No differences were found among the Yugur and ethnic Han groups (p = 0.932, c2 = 0.007).
299-300delAT/299-300delAT
5
0
0
1
176-191del16/176-191del16
0
0
0
2
176-191del16/299-300delAT
0
0
1
0
mtDNA1555A>G
235delC/35delG
1
1
0
0
235delC/299-300delAT
2
0
0
0
35delG/wt
0
1
0
3
mtDNA1555A>G was not detected in the Dongxiang, Yugur, and Bonan groups. A total of 28 subjects in the ethnic Han group exhibited mtDNA1555A>G mutations with a mutation frequency of 6.03% (28/ 464). The mutation frequency of the Dongxiang group was lower than in the ethnic Han group (p = 0.002, c2 = 9.797). No statistically significant differences were found between the Yugur and ethnic
235delC/wt
3
0
0
30
176-191del16/wt
0
0
0
3
299-300delAT/wt
0
0
0
2
380G>A/wt
1
0
0
0
Deafness gene mutations in unique ethnic groups Han groups (p = 1.000) or between the Bonan and ethnic Han groups (p = 0.614).
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Discussion The unique ethnic groups of Dongxiang, Yugur, and Bonan in Gansu, China, have conservative genetic information. Furthermore, the characteristics of the GJB2 and SLC26A4 genes and mtDNA1555A>G mutations have obvious racial and ethnic specificity. Therefore, this study determined the prevalence and ethnic specificity of the GJB2 and SLC26A4 genes and the mtDNA1555A>G mutations among the unique ethnic groups. In the Dongxiang, Yugur, and Bonan groups, the pathogenic allele frequencies of the GJB2 gene were 9.03%, 12.5%, and 5.88%, respectively. No obvious differences were found between the unique ethnic groups and the ethnic Han group. Thus, GJB2 gene mutations are also an important cause of deafness in unique ethnic groups in Gansu Province, such as the ethnic Han group. The main forms of GJB2 gene mutations in the Dongxiang group were c.299-300delAT and c.235delC, similar to the ethnic Han group. The Dongxiang group was statistically higher in the pathogenic allele frequency of c.299-300delAT and lower in the pathogenic allele frequency of c.235delC than the ethnic Han group. By contrast, the carrying rate of c.299-300delAT was higher than that of c.235delC. Therefore, the most common mutation form of the Dongxiang group was c.299-300delAT, unlike in the ethnic Han group. The Dongxiang group traces its origins among the three theories of the Mongols, Hybrids, and Sarts. Among these theories, the Sarts are the most commonly accepted origin by the academe and the public. Sarts mainly originated from Turks, who belong to the yellow–white mixed race (mongoloid and caucasian) and fused with other minorities such as Mongolians [16]. c.235delC (mainly) and c.299-300delAT were the hot spots of mongoloids [3]. The caucasian hot spot is c.35delG [5]. Thus, the ethnic origin cannot fully explain why the proportion of c.299-300delAT was high in the Dongxiang group. In terms of mutation forms, the Dongxiang group was similar to the mongoloid group, thus showing that the mutation characteristics of the GJB2 gene in the Dongxiang group were close to the mongoloid group. This observation was in line with the ethnic background of the Dongxiang group. From genetics, the disease phenotype is related to gene mutation, gene interaction, and external factors (e.g. the environment). Therefore, we thought that the mutation characteristics of the GJB2 gene in the Dongxiang group were the result of different factors such as
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ethnic background, geographical environment difference, and ethnic migration. These factors need to be studied further. Current research suggests that the high incidence of c.35delG among caucasians was the expression of the founder effect. In this study, c.35delG accounted for 66.7% (2/3) of all pathogenic alleles and was the hot spot in the Yugur group. Considering the obvious caucasian ethnic background [17], this result could be considered an expression model of the founder effect wherein c.35delG was the hot spot of the GJB2 gene in the Yugur group. Given that only two pathogenic alleles (c.299300delAT and c.176-191del16) of the GJB2 gene were detected, no hot spot could be drawn in the Bonan group. This condition was influenced by the small population base, and more patients will be needed in future studies. In the Dongxiang group, the pathogenic allele frequency of the SLC26A4 gene was 3.23% (10/ 310) and the allele frequency of IVS7-2A>G was 2.26% (7/310); the latter was the hot spot and accounted for 70% (7/10) of all pathogenic alleles. From the statistical analysis, the Dongxiang group had lower pathogenic allele frequencies of SLC26A4 and IVS7-2A>G than the ethnic Han group. Previous studies confirmed that the pathogenic mutation detection rate of SLC26A4 in caucasians was obviously lower than in mongoloids among deaf people [19]. Considering that the Dongxiang group is a fusion of mongoloids and caucasians, we could easily determine that the lower pathogenic allele frequency of the Dongxiang than the ethnic Han group was a manifestation of racial specificity. The hot spot of the Dongxiang group was IVS7-2A>G, which is similar to the ethnic Han group. The hot spot of the caucasians (IVS8+1G>A) was not found. The mutation characteristics of the SLC26A4 gene in the Dongxiang were similar to mongoloids; this result was in line with the results for the GJB2 gene. However, the statistical analysis of the pathogenic allele frequency of SLC26A4 (exon8 and exon19) was different from GJB2. The Dongxiang group was smaller than the ethnic Han group; this result might be associated with the choice of purpose exons. The Dongxiang group has a caucasian background, but exon10 (wherein one of the hot spots of caucasians, c.1246A>G, was located) was not the purpose exon. Thus, further work on other exons, particularly exon10, is necessary. In this study, we found a new sequence change form (i.e. c.918+48A>G) in the Dongxiang group. This new sequence was a point mutation in the intron and was far from the exon. Therefore, this type of mutation was unlikely to have caused the disease.
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Only one homozygous mutation and hot spot, i.e. IVS7-2A>G, was detected in the Yugur group. The pathogenic and IVS7-2A>G allele frequencies were both similar to that of the ethnic Han group but were different from the ethnic specificity expression of the GJB2 gene in the Yugur group. The analysis of the GJB2 gene was validated considering the caucasian background of the Yugur group. The reasons for the abovementioned results were as follows. On one hand, bias easily appeared because the sample size was small. On the other hand, the problem of the purpose exons was similar to that of the Dongxiang group. Thus, we still need to enlarge the sample size and expand the exon number for the next step of our research. No pathogenic mutation was detected in the Bonan group; this result might be associated with the small sample size. We also found a new sequence change form in the Bonan group (i.e. c.2090-51insAAAC). Although this new sequence change form was an intron and is far from the exon, this sequence inserted four basic groups and largely affected the synthesis of the amino acids. This observation still needs to be verified in further studies. The caucasian background was the most important reason why the mtDNA1555A>G mutation frequency of the Dongxiang group was significantly lower than that of the ethnic Han group. The specificity of the mtDNA1555A>G deafness penetration could be another reason [20]. mtDNA1555A>G screening among normal-hearing people needs to be conducted to confirm this result. The mtDNA1555A>G mutation frequency of the Yugur and Bonan groups had no statistical differences from that of the ethnic Han group. The caucasian background of the Yugur group could explain the failure of detection. Given the probability of bias in the smaller sample sizes of the Yugur and Bonan groups, we need to enlarge the sample size and conduct thorough follow-up research. In conclusion, the GJB2 and SLC26A4 genes and the mtDNA1555A>G mutation in the unique ethnic groups had respective specificities. The ethnic background might be the main cause of this observation. The pathogenic allele frequency of the GJB2 gene in the unique ethnic groups was similar to that in the ethnic Han group. The mutation characteristic of the Dongxiang group was caused by the interaction of the ethnic background and geographical environment. The mutation characteristic of the Yugur group was the typical model for the founder effect. Moreover, the molecular epidemiological characteristics were uncertain. The hot spot of the SLC26A4 gene was IVS7-2A>G in both the Yugur and Bonan groups, similar to the ethnic Han group. The
pathogenic allele frequency of the Dongxiang group was lower than that of the ethnic Han group because of the caucasian background and the selection of purpose exons. The mutation characteristics of the Yugur group did not conform to the ethnic background; this result was associated with the small sample size and exon selection. A similar conclusion could not be made for the Bonan group. The ethnic background and penetration of ethnic specificity were the reasons why the mtDNA1555A>G mutation frequency was low in the Dongxiang group.
Acknowledgments This work was supported by grants from the National Natural Science Foundation of China (grant no. 81172765). Declaration of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper.
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