职业性噪声听力损失敏感人群全外显子SNP特征分析及遗传易感标志的研究

发布时间：2018-05-19 22:10

本文选题：外显子测序 + 单核苷酸多态性　；参考：《东南大学》2016年硕士论文

【摘要】：噪声性听力损失(Noise-induced Hearing Loss, NIHL)是长期接触有害噪声引起的进行性感音神经性听力损伤。NIHL是由环境因素和遗传因素交互作用发生的。以往研究关注较长时间噪声暴露的遗传易感性,但我们先期的研究发现,新入职的青年工人,排除其他听力损失的原因,在职业噪声暴露三年内存在听力损失的风险,为职业性噪声暴露的敏感人群。本研究主要探讨该NIHL敏感人群的遗传易感性。首先采用全外显子组测序法对5例短期内接触噪声发生NIHL的青年工人进行外显子测序,对比SNP(亚洲人群频率筛选)数据库,筛选候选的易感SNP位点；进而对候选SNP位点与青年工人较短时间发生NIHL的相关性进行验证,并进一步探讨候选SNP位点与一般噪声暴露人群NIHL的相关性；最后通过构建NIHL动物模型,检测候选SNP的基因在噪声暴露后mRNA的相对表达水平,以确定靶基因是否在转录水平上对噪声暴露进行应答,在机制上验证易感性关联的可能性。1.噪声敏感人群的全外显子组测序及候选SNP筛选对5例噪声暴露三年内就发生NEHL的新入职青年工人进行全外显子组测序,测序过程包括DNA提取、DNA质检、文库构建、外显子区域捕获、高通量测序、生物信息学分析。通过比对SNP(亚洲人群频率筛选)数据库,根据SNP位点位于外显子区域、突变为非同义突变、4个以上测序样本都发生突变,而正常汉族人群中等位基因频率突小于0.2等标准纳入目的靶基因。一共筛选出27个SNP位点,其中5个测序样本都突变的SNP位点有6个,包括TTLL4 rs3731877、STK36rs1344642、BSPH1 rs60213124. HGC6.3 rs76543658、COL28A1 rs6952195、COL28A1 rs55745506。其中TTLL4基因编码的蛋白是一种催化微管蛋白络氨酸残基磷酸化的蛋白,在细胞的生长、繁殖、分化过程中具有重要的作用；STK36基因编码的蛋白属于丝氨酸/苏氨酸激酶家族；STK36基因的缺陷可能导致运动纤毛功能性缺失：BSPH1基因编码粘附相关蛋白,参与机体能量代谢；基因库中对于HGC6.3基因尚无明确定义：COL28A1基因编码的蛋白质属于胶原蛋白家族,是一种生物性高分子化合物,在人体细胞中扮演结合组织的角色。2.候选SNP与噪声性听力损失遗传易感性的研究2.1外显子测序目的靶基因位点多态性与NIHL的相关性分析本研究着重对TTLL4 rs3731877、STK36 rs 1344642、BSPH1 rs60213124等三个SNP位点进行验证。采用1：1配对病例对照方法,首先以19对接噪工龄小于3年的发生NIHL的工人和对照为研究对象,应用聚合酶链反应和直接测序法,分析候选SNP与NIHL易感性的相关关系；进而以177对发生NIHL的工人和对照为研究对象,对目的靶基因的多态性与NIHL易感性进行分析,并对靶基因多态性与NIHL的相关性进行了年龄、工龄分层分析。研究结果显示在短期内接触噪声发生NIHL的青年工人中,BSPH1rs60213124位点的G、A等位基因频率分布在组间存在统计学差异(x2=6.33,P0.05)：显性模型(AA/AG VS.GG)分析结果显示携带AA/AG基因型的个体患NIHL的危险度是携带GG基因型个体的7.65倍(OR=7.65,95%CI=1.37-42.71).未发现BSPH1 rs60213124、TTLL4 rs3731877、STK36 re1344642位点多态性与其他工龄和年龄组发生NIHL的相关性。研究结果提示,BSPH1 rs60213124位点可能是短期内接触噪声青年工人发生NIHL的易感基因位点,但结果需要加大样本量进一步研究。2.2GJB2、NCL、CDH23、PCDH15基因多态性与NIHL的相关性分析本研究旨在探讨GJB2基因rs2274083、rs2284084、rs72474224、NCL基因rs7598759、CDH23基因rs 10999947、PCDH15基因rs4935502、re108252693等位点的多态性与NIHL的相关性。研究结果表明,GJB2基因rs2274084的基因型分布在两组组间存在统计学差异(x2=8.51,P=0.014),等位基因分布在两组间也存在统计学差异(x2=8.06,P=0.005),携带CC基因型的个体患NIHL的危险度是携带TT基因型个体的2.17倍(OR=2.17,95%CI=1.04-4.53).对该位点进行工龄分层分析,在小于5年工龄组和大于15年工龄组,该位点基因型分布和等位基因频率分布在病例组和对照组之间均存在统计学差异,并且大于15年工龄组的携带CC基因型个体罹患NIHL的危险度要高于小于5年工龄组携带CC基因型的个体。CDH23rs10999947位点基因型分布在病例组和对照组之间存在统计学差异(X2=8.82,P=0.01),携带GG基因型的个体患NIHL的危险度是AA基因型个体的4.03倍(OR=4.03,95%CI=1.44-11.3)。该位点隐形模型分析结果显示,携带GG/AG基因型的个体患NIHL的危险度是AA基因型个体的4.14倍(OR=4.14,95%CI=1.51-11.34)。对该SNP位点进行人群年龄与工龄分层分析,结果显示在年龄35-45岁段,该位点多态性与NIHL存在统计学相关(x2=6.3,P=0.04)。研究结果提示,GJB2基因rs2274084和CDH23基因rS10999947位点可能是NIHL的易感基因位点。3. NIHL动物模型的构建及耳蜗靶基因mRNA表达的检测采用白噪声暴露小鼠的方式构建NIHL的动物模型,对小鼠耳蜗内靶基因在噪声暴露后mRNA相对表达进行检测,以判断目的靶基因是否在转录水平上在参与了NIHL的发生过程。ABR检测结果显示,噪声暴露后两组小鼠听阈值组间存在统计学差异(P0.05),并且随着电测听频率的升高,听阈差值存在增大的趋势。基底膜铺片可见噪声组小鼠耳蜗毛细胞出现缺损、倒伏严重。应用荧光定量PCR对目的靶基因的表达进行检测,发现GJB2基因、CDH23基因、PCDH 15基因mRNA的相对表达量组间差异存在统计学差异(P0.05)。而对于其他靶基因,本研究未发现mRNA表达的差异。结果提示本研究构建NIHL动物模型成功,GJB2基因、CDH23基因、PCDH15基因可能参与了NIHL的发生过程。4.总结经外显子测序和比对SNP(亚洲人群频率筛选)数据库,初步获得6个可能的青年工人短期内噪声暴露发生NIHL的候选SNP位点,经以接噪工龄小于3年发生NIHL的工人和对照为研究对象,分析候选SNP与NIHL易感性的相关关系,发现BSPH1rs60213124位点可能是短期内接触噪声青年工人发生NIHL的易感基因位点。对GJB2、NCL、CDH23、PCDH15基因多态性与NIHL的相关性分析发现GJB2基因rs2274084和CDH23基因rs10999947位点与NIHL存在着相关性,GJB2基因rs2274084位点的CC基因型和CDH23基因rs10999947位点的GG基因型可能是噪声性听力损失的易感基因型。采用白噪声暴露小鼠的方式成功构建NIHL的动物模型,对靶基因mRNA的表达检测,发现GJB2基因、CDH23基因、PCDH 15基因mRNA的相对表达量在噪声暴露组和对照组之间存在差异,GJB2基因、CDH23基因、PCDH15基因可能在转录水平上参与了NIHL的发生过程。
[Abstract]:Noise induced hearing loss (Noise-induced Hearing Loss, NIHL) is a long-term exposure to noxious noise caused by sexy sound neurogenic hearing impairment..NIHL is caused by the interaction of environmental factors and genetic factors. Previous studies focused on the genetic susceptibility to prolonged exposure to noise, but our previous study found that new recruits were recruited. Workers, excluding other hearing loss, have the risk of hearing loss within three years of occupational noise exposure. This is a sensitive group of occupational noise exposure. This study mainly discusses the genetic susceptibility to the NIHL sensitive population. First, 5 young workers with short term exposure to noise are revealed by the exon sequencing method. Subsequencing and comparing the SNP (Asian population frequency screening) database to screen candidate susceptible SNP loci, and then verify the correlation between the candidate SNP loci and the young workers in the short time of NIHL, and further explore the correlation between the candidate SNP loci and the NIHL in the general noise exposed population. Finally, the NIHL animal model is constructed and the detection weather is detected. The relative expression level of the SNP gene after noise exposure was selected to determine whether the target gene responds to the noise exposure at the transcriptional level, and the possibility of the susceptibility association is verified by the mechanism. The exon sequencing and candidate SNP screening of the.1. noise sensitive population in 5 cases of NEHL in the three year of noise exposure are the new recruits. The whole exon group was sequenced, and the sequencing process included DNA extraction, DNA quality inspection, library construction, exon capture, high throughput sequencing, bioinformatics analysis. By comparing the SNP (Asian population frequency screening) database, the SNP loci in exons were mutated into non synonymous mutations, and more than 4 sequencing samples were mutated, and positive The target gene of secondary gene frequency was less than 0.2 in the Han population. 27 SNP loci were screened out, of which 5 of the sequencing samples had 6 SNP loci, including TTLL4 rs3731877, STK36rs1344642, BSPH1 rs60213124. HGC6.3 rs76543658, COL28A1 rs6952195, and COL28A1 genes encoded eggs White is a protein that catalyzes the phosphorylation of the microtubulin complexate residue, which plays an important role in the growth, reproduction and differentiation of the cells; the protein encoded by the STK36 gene belongs to the serine / threonine kinase family; the defects of the STK36 gene may lead to the functional deletion of the sports cilium: the BSPH1 gene encodes the adhesion related protein and participates in the protein gene The energy metabolism of the body; there is no clear definition of HGC6.3 gene in the gene pool: the protein encoded by the COL28A1 gene belongs to the collagen family, is a biological polymer compound, plays the role of the binding tissue in human cells,.2. candidate SNP and the susceptibility of noise induced hearing loss, 2.1 exon sequencing target Analysis of the correlation between polymorphism of gene locus and NIHL, this study focuses on three SNP loci, such as TTLL4 rs3731877, STK36 RS 1344642, BSPH1 rs60213124 and other SNP loci. In order to analyze the correlation between candidate SNP and NIHL susceptibility, the polymorphism of target gene and NIHL susceptibility were analyzed with 177 pairs of NIHL workers and controls, and the correlation between target gene polymorphism and NIHL was analyzed. Among young workers who were born with NIHL, the frequency distribution of G and A alleles at the BSPH1rs60213124 locus was statistically different (x2=6.33, P0.05). The dominant model (AA/AG VS.GG) analysis showed that the risk of NIHL with AA/AG genotype was 7.65 times as large as that of the GG genotype (OR=7.65,95%CI=1.37-42.71). 13124, TTLL4 rs3731877, STK36 re1344642 locus polymorphism is associated with the occurrence of NIHL in other working age groups and age groups. The results suggest that BSPH1 rs60213124 loci may be a susceptible gene locus for NIHL in young workers exposed to noise in the short term, but the results need to be increased to further study.2.2GJB2, NCL, CDH23, PCDH15 gene polymorphism. The correlation analysis between sex and NIHL was aimed at exploring the correlation between the polymorphism of the GJB2 gene rs2274083, rs2284084, rs72474224, NCL gene rs7598759, CDH23 gene RS 10999947, PCDH15 gene rs4935502, re108252693 and other loci. X2=8.51, P=0.014), the distribution of alleles in the two groups was also statistically different (x2=8.06, P=0.005), and the risk of NIHL with the CC genotype was 2.17 times that of the TT genotype (OR=2.17,95%CI=1.04-4.53). The distribution and allele frequency distribution were statistically different between the case group and the control group, and the risk degree of NIHL in the CC genotype individuals with the 15 year old group was higher than that of the individuals carrying the CC genotype in the group less than 5 years old. The genotype distribution of the.CDH23rs10999947 loci of the individuals carrying the CC genotype was statistically significant between the case group and the control group. X2=8.82 (P=0.01), the risk degree of NIHL in individuals carrying GG genotype was 4.03 times (OR=4.03,95%CI=1.44-11.3) of AA genotype individuals. The stealth model analysis showed that the risk of NIHL in individuals carrying the GG/AG genotype was 4.14 times (OR=4.14,95%CI=1.51-11.34) for AA genotype individuals. Age and age stratification analysis showed that the polymorphism of the site was statistically related to NIHL (x2=6.3, P=0.04) at age 35-45 years. The results suggest that the rS10999947 locus of the GJB2 gene rs2274084 and CDH23 gene may be the construction of the.3. NIHL animal model of the susceptible gene locus of NIHL and the detection of the mRNA expression of the cochlear target gene. The animal model of NIHL was constructed by noise exposed mice, and the relative expression of mRNA in the mouse cochlea target gene was detected after noise exposure to determine whether the target gene was involved in the transcription of NIHL at the transcriptional level and.ABR detection results showed that there was a statistical difference between the two groups of hearing threshold groups after the noise exposure (P0.05). And with the increase of the frequency of electrical audiometry, the difference of hearing threshold increased. The cochlear hair cells in the group of basal membrane were visible and the lodging was serious. The expression of target gene was detected by fluorescence quantitative PCR. The difference of the relative expression of the relative expression of the GJB2 gene, CDH23 gene and PCDH 15 gene mRNA was found to be statistically significant. P0.05. But for other target genes, there is no difference in the expression of mRNA in this study. The results suggest that the NIHL animal model is successful in this study. The GJB2 gene, CDH23 gene, and PCDH15 gene may be involved in the occurrence of NIHL,.4. summarizes the exon sequencing and compares SNP (Asian population frequency screening) database, initially obtaining 6 possible In the short term, young workers exposed to the candidate SNP loci of NIHL, the workers and controls who were less than 3 years older than 3 years, analyzed the correlation between the candidate SNP and the NIHL susceptibility, and found that the BSPH1rs60213124 locus may be the susceptible gene locus of NIHL in young workers exposed to noise in the short term. For GJB2, NCL, CDH23. The correlation analysis between PCDH15 gene polymorphism and NIHL found that there was a correlation between the rs10999947 locus of the GJB2 gene rs2274084 and CDH23 gene and NIHL. The CC genotypes of the rs2274084 locus of the GJB2 gene and the CDH23 gene rs10999947 loci may be the susceptible genotypes of noise induced hearing loss. The animal model of NIHL was constructed and the expression of the target gene mRNA was detected. The relative expression of the GJB2, CDH23 and PCDH 15 gene mRNA was found to be different between the noise exposed group and the control group. The GJB2, CDH23, and PCDH15 genes may be involved in the occurrence of NIHL at the transcription level.
【学位授予单位】：东南大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R135.8

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