| Noise-induced hearing loss (NIHL) is a slowly, progressively sensorineural hearing loss that is often caused by daily exposure to detrimental levels of noise. NIHL is a complex disease that results from the interaction between environmental and genetic factors. Researches usually paid special attention to the hereditary susceptibility of a long period of noise exposure. However, our previous study found that young workers who exposed to noise less than 3 years had developed NIHL when excluded other factors of hearing loss, which might be the susceptible population to occupational noise-exposure. In this study, the whole exome sequencing was used to detecte of DNA samples from 5 young workers who developed NIHL in a short period of noise exposure and the candidate susceptible SNPs were screened by comparing the sequenc ing results with the database of Asian population SNP. Therelation between the candidate susceptible SNPs and the NIHL of the susceptible population was then verified. The correlation between the susceptibility SNPs and NIHL of noise-exposure workers was further analyzed by paired case-control study. Finally, a mice model of NIHL was built and the mRNA expression of the target genes in mice cochela after noise exposure was detected to comfirm the response of the target genes to noise exposure.1. The whole exome sequencing of noise susceptible population and the screening of candidate SNPsThe whole exome sequencing was detected in DNA samples from 5 young workers who developed NIHL in a short period of noise exposure. The procedure of exon sequencing included DNA extraction? DNA quality testing library construction exoncapture?high through sequencing and bioinformatics analysis. Comparing with the database of Asian population SNP, The target genes were screened based on following screening criteria:the location of variation was in exon area, the mutation was non-synonymous, five samples were all mutated, the mutation rate was less than 0.2 in normal Asian population. The results showed that 27 candidate SNPs were selected in the study, including 6 SNPs of 100% mutation genotype in 5 samples: TTLL4 rs3731877?STK36rs 1344642?BSPH1 rs60213124?HGC6.3rs76543658, COL28A1 rs6952195, COL28A1 rs55745506. The protein encoded by TTLL4 gene is tubulin tyrosine ligase like 4 and it plays an important role in the growth and differentiation of cells. The protein encoded by STK36 gene belongs to the family of serine/threonine kinase, the defect of STK36 gene may results in the functionality defect of motile cilia. The protein encoded by BSPH1 gene is binder protein homolog 1, it may participate in the metabolism of energy. There was no clear clue of HGC6.3 in the database. The protein encoded by COL28A1 gene belongs to the family of collagen and it plays an important role of combining organization.2. The study of the correlation of candidate SNPs and NIHL susceptibility2.1 The correlation between the polymorphism of candidate SNPs from whole-exome sequencing and NIHL susceptibilityThree candidate SNPs including TTLL4 rs3731877, STK36 rsl344642, BSPH1 rs60213124 were selected and 1:1 matched case-control design was used in this study. The correlation of the candidate SNPs screened from exon sequencing and N1HL susceptibility in the group of 19 young workers who developed NIHL in a short period of noise exposure and its paired controls were detected. Then the correlation of the candidate SNPs and NIHL susceptibility in 354 normal noise exposure workers, including 177 cases and 177 controls were analyzed. We also did the stratification analysis between target genes and NIHL susceptibility. The results showed the allele frequency of BSPHI gene rs60213124 locus among the cases and controls were statistically significantly different (?2=6.33, P<0.05) in the group of young workers who exposed by noise in a short period. The individual carrying AA/AG genotype had a 7.65 fold risk of NIHL comparing with individual with GG genotype. The association betweenNIHL susceptibility and BSPH1 rs60213124?TTLL4 rs3731877?STK36 rs 1344642 in other age groups and seniority groups were not found. It suggested the locus of BSPH1 gene rs60213124 might be the NIHL susceptibility gene locus of young workers who developed NIHL in a short period of noise exposue, but the results need to be verified in further study with a larger sample size.2.2 The correlation of the polymorphism of GJB2, NCL, CDH23, PCDH15 and NIHL susceptibilityThe correlation between NIHL susceptibility and rs2274083, rs2284084, rs72474224, rs7598759, rs 10999947, rs493550 and rs 108252693 was analyzed in this study. The results showed the genotype frequencies of GJB2 gene rs2274084 locus among case and control were significantly different (x2=8.51. P=0.014) and the allele frequencies of this locus among case and control were also significantly different (?2=8.06, P=0.005),Therisk of NIHL in individuals with CC genotype was 2.17 fold of individuals with AA genotype (OR=2.17,95%CI=1.04-4.53). The results of Stratified analysis showed that there were significant differences in the genotype and allele distribution between cases and controls in the groups of noise exposure time (< 5 years and> 15 years). Individuals with CC genotype over 15 years noise exposure had a higher risk of NIHL comparing with groups in noise exposure time (<5 years). Besides, there was correlationship between CDH23 rs 10999947 and NIHL susceptibility (x2=8.82, P=0.01), the risk of NIHL in individuals with GG genotype was 4.03 fold of individuals carrying AA genotype(OR=4.03,95%CI=1.44-11.3). The result of the invisibility model showed that the risk of NIHL in individuals with GG/AG genotype was 4.14 fold of individuals with AA genotype (OR=4.14,95%CI=1.51-11.34). The result of Stratified analysis showed that there was statistical correlation between the SNP polymorphism and NIHL (x2=6.3, P=0.04) in the age of 35 to 45 years old. The study indicated that the locus of CDH23 rs10999947 and GJB2 gene rs2274084 might be the NIHL susceptibility gene locus.3. The construction of NIHL mice model and the detection of target gene mRNA expression in mice cochelaThe mice were exposed to white noise to build the model of NIHL, and then the mRNA expression of target genes in mice cochlea was detected in order to make sure if the target genes participate in the process of NIHL in the transcriptional level. The results of ABR detection showed that the threshold of ABR among case and control was significantly different (P<0.05), the D-value of threshold had a trend of increasing along with the rising of frequency of brainstem electric response audiometry. The result of Basement membrane surface preparation showed that hair cells were defected, fell hardly in case group on contrary to the control group. The results indicated that the mice model of NIHL was successfully constructed. The mRNA expression of target genes in mice cochlea was then detected by real-time quantitative PCR, The results showed the mRNA relative expression of GJB2 gene, CDH23 gene and PCDH15 gene among case and control were statistically significantly different between cases and controls. This indicated GJB2 gene, CDH23 gene and PCDH15 gene had participated in the process of NIHL in the transcriptional level.4. ConclusionsSix NIHL candidate SNPs of 100% mutation genotype in 5 samples were selected via whole exome sequencing by comparing with Asian population SNP database. The locus of BSPHl gene rs60213124 might be the NIHL susceptibility gene locus of young workers who developed NIHL in a short period of noise exposue through the paired case-control study.The study of correlation between NIHL and GJB2, NCL, CDH23?PCDH15 gene polymorphism showed that there was a significant correlation between NIHL susceptibility and the locus of GJB2 rs2274084 and CDH23 rs10999947. It indicated that the locus of CDH23 rs10999947 and GJB2 gene rs2274084 might be the NIHL susceptibility gene locus.The mice model of NIHL was successfully constructed. The study of mRNA relative expression of candidate genes indicated that GJB2, CDH23 and PCDH15 gene had participated in the process of NIHL in the transcriptional level. |
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