【作者】 王树辉；

【导师】 杨新明；

【作者基本信息】 中南大学 ， 耳鼻咽喉科学， 2008， 博士

【摘要】 目的：为明确湖南人群中非综合征性耳聋患者发病的遗传学基础,分析了患者GJB2基因突变的情况,并探索其发病的分子机制。方法：经过详细的病史询问和临床检查,收集了符合要求的、无亲缘关系的非综合征耳聋患者140例。PCR扩增患者GJB2基因的编码序列全长,纯化回收扩增产物,直接双向测序,用DNAStar软件分析测序结果,统计患者GJB2基因突变情况。选择研究中发现的突变,设计引物扩增突变体全长,将其构建到pEGFP表达载体,经菌液PCR检测、双酶切鉴定并测序验证之后,转染Hela细胞,行Western blot检测,荧光染色后在荧光显微镜下观察Cx26的表达情况,比较野生型与突变体之间亚细胞定位的差别；计数它们形成间隙连接通道的细胞数目,统计分析是否存在差异。结果：在140个无亲缘关系的耳聋患者中,共有56例检测到了Cx26基因突变,检出率达40%（56／140）；其中29例患者两个Cx26等位基因都发生突变,另27例患者只有一个Cx26等位基因突变,等位基因突变率为30.4%（85／280）。一共发现了10种不同的碱基变异,包括7种致病突变和3种多态。7种致病突变分别是无义突变c.139G>T；移码突变c.176₁91del 16和c.235delC;错义突变c.109G>A、c.344T>G、c.550C>T和c.571T>C。c.344T>G是一个新报道的错义突变。最常见的突变是235delC,共有27个患者检测到该突变,比例达19.3%（27／140）,其中纯合突变20例,复合杂合突变2例,杂合突变5例,占所有致病性等位基因的55.3%（47／85等位基因）。109G>A突变频率仅次于235delC,有25个患者检测到该突变,检出率达17.9%（25／140）,其中纯合突变7例,杂合突变18例,占所有致病性等位基因的37.6%（32／85等位基因）。选择在患者中发现的突变,构建了Cx26 wt及其突变体的真核表达载体：pEGFP-Cx26 wt, pEGFP-Cx26 V37I和pEGFP-Cx26F115C；转染Hela细胞后,成功表达Cx26 wt及其突变体的GFP融合蛋白；但是突变体在细胞定位和分布上与Cx26 wt无区别,形成通道的细胞数目也没有明显的差异。结论：Cx26是导致湖南人群NSHL的最常见的基因,其235delC突变是湖南人群NSHL最常见的突变；Cx26基因p.F115C和p.V37I两种突变体转染Hela细胞后,不影响其形成细胞间隙连接的能力,但其形成的间隙连接是否有正常功能还有待进一步研究。更多还原

【Abstract】 Objective:To identify genetic characteristics in patients of Hunan province with nonsydromic hearing loss (NSHL), determine the prevalence and spectrum of mutations in GJB2(Cx26) gene, explore the pathogenic molecular mechanism.Methods:140 sporadic cases of NSHL without genetic relationship were collected after enquiring history and clinical examinations. Molecular studies were performed by amplifing the coding region of Cx26 gene, purifing the PCR products, then bidirectional sequencing directly.Sequences were analysed by DNAStar software, determine the prevalence and spectrum of mutations in Cx26 gene. Cx26 gene mutants, which primers were designed to amplify,were selected, then constructed into pEGFP vector,transfected into Hela cells after identification by bacteria PCR, double restriction enzyme analysis and sequence. Western blot was performed. Expression of Cx26-EGFP protein were observed in fluorescence microscope after fluorescent staining,compare the differences of fluorescent localization between the wild type and the mutants.Account the mumber of cells that formed gap junction channels, statistic analysis were used to define whether there were differences.Results:Among the 140 NSHL patients without genetic relationship,56 patients were detected Cx26 gene mutation, the relevance ratio was 40%(56/140). Both of the two Cx26 alleles were mutated in 29 patients and one Cx26 allele in 27 patients, the rate of allele mutation was 30.4%(85/280).10 types of variations were detected, containing 7 types of pathogenic mutations and 3 types of polymorphisms. The 7 types of pathogenic mutations were nonsense mutation c.139G>T, frameshift mutation c.235delC and c.176-191del16, and missense mutation c.109G>A、c.344T>G、c.550C>T and c.571T>C. c.344T>G is a newly reported missense mutation. The c.235delC mutation which was detected in 27 patients was the most prevalent mutation, the relevance ratio was 19.3%(27/140), including homozygous mutation in 20 patients, heterozygous in 5 and compound heterozygous in 2, accounting for 55.3%(47/85)of the pathogenic alleles. The c.109G>A mutation which was detected in 25 patients was just next to c.235delC, the rate was 17.9%(25/140), including homozygous mutation in 7 patients and heterozygous in 18, accounting for 37.6%(32/85) of the pathogenic alleles. pEGFP-Cx26 wt, pEGFP-Cx26 V37I and pEGFP-Cx26 F115C eukaryotic expression vectors were constructed, after transfected into Hela cell,Cx26 wt and two mutants’Cx26 and GFP fusion protein were expressed successfully. The fluorescent localization and distribution assay of the two mutants showed no differences with the wildtype, as well as the numbers of cells that formed gap junction channels, indicating that they can not impair the conformation of the gap junctions. mutation is the most hot-spot in patients of Hunan province. The two mutants of Cx26,c.V371 and c.F115C,do not affect the conformation of gap junction after transfected into Hela cell,but whether the gap junction exist normal function need to do further research.更多还原