【作者】 胡浩；

【导师】 邬玲仟； 梁德生； 潘乾；

【作者基本信息】 中南大学 ， 遗传学， 2008， 博士

【摘要】 耳聋是最常见的感觉障碍性疾病之一,新生儿重度听力障碍者约占1～3‰,其中约50%属于遗传性耳聋。根据是否伴有耳外组织的异常或病变可将其分为综合征性听力丧失(syndromic hearing loss,SHL)和非综合征性听力丧失(nonsyndromic hearing loss,NSHL),其中NSHL占70%以上。遗传性NSHL具有多种遗传方式,包括常染色体显性遗传(相关NSHL位点命名为DFNA)、常染色体隐性遗传(相关位点命名为DFNB)、X-连锁遗传(DFN)、Y-连锁遗传(DFNY)和母系遗传等。遗传性耳聋具有极强的遗传异质性,至2008年7月止,共定位耳聋相关染色体位点133个,克隆相关基因45个。其中,GJB2基因为最常见的耳聋相关基因,该基因的突变在高加索人群中与50%左右的先天性耳聋相关;其次为SLC26A4,占5%～10%,该基因突变导致的耳聋患者常伴有特征性的影像学表现,即前庭导水管扩张(Enlarged VestibularAqueduct,EVA);与线粒体基因突变相关的占1%左右。其余各型NSHL均仅占较小比例。结合相关领域研究的最新成果,建立了“遗传性非综合征型耳聋位点及相关基因数据库”和“遗传性非综合征型耳聋相关基因突变位点数据库”等遗传性NSHL相关数据库,并对“遗传性非综合征耳聋相关基因检测候选基因确定流程”进行了更新,使其更具操作性。从而使前期工作建立的“遗传性非综合征型神经性耳聋的基因诊断体系”更加完善。综合应用聚合酶链反应(Polymerase Chain Reaction,PCR)、变性高效液相色谱(Denatureing High Performance Liquid Chromatography,DHPLC)、限制性长度片段多态(Restricted Length FragmentPolymorphism,RFLP)和测序等技术,对1998年至2007年收集的407个耳聋家系进行GJB2基因编码区和线粒体12S基因和UCN基因突变热点位点筛查、对31个伴或不伴EVA表型的耳聋患者进行SLC26A4基因编码区的筛查,对阳性样品进行测序证实,为进一步明确SLC26A4基因相关耳聋患者的基因型与表型关系,对SLC26A4基因的调控序列以及调控基因FOXI1进行了检测。在407个耳聋家系中,共检测到24个家系先证者携带2条GJB2致病单体,15个家系先证者仅查及1条GJB2致病单体;27个家系先证者为线粒体A1555G同质型改变,其中两个家系先证者同时为G7444A同质型改变。31个伴或不伴EVA耳聋家系中,15个家系中的17名患者检测到2条SLC26A4致病单体,2名患者仅检测到1条致病单体;查及SLC26A4基因2种新突变:D87Y和F572L。SLC26A4基因调控序列及其调控基因的检测没有阳性发现。利用“遗传性非综合征型神经性耳聋的基因诊断体系”,选择合适的检测方法,针对性进行相应耳聋基因的检测,可高效、经济的完成耳聋患者的基因检测。本研究通过对各个基因突变谱的分析并结合文献报道,认为GJB2基因的235delC、线粒体DNA的A1555G和SLC26A4基因的IVS7-2A＞G系中国耳聋人群中的基因突变热点。更多还原

【Abstract】 Hearing loss is one of the most common sensory disorders. Profound hearing loss occurs in 1-2:1000 neonates and the cause is hereditary in about half. A distinction can be made between syndromic hearing loss (SHL), in which the hearing loss is companied by other specific abnormalities, and non-syndromic hearing loss (NSHL), in which there are no additional abnormalities (more than 70%). All types of hereditary mode can be observed in NSHL, including autosomal dominant (the associated NSHL gene loci are designated DFNA), autosomal recessive (DFNB), X-linked (DFN), Y-linked (DFNY) and maternal inheritance. NSHL is highly genetic heterogeneous. Up to July 2008, 133 NSHL gene loci have been mapped, and 45 genes have been identified. Of these GJB2 is the most common NSHL associated gene, which accounts for about 50% hereditary prelingual NSHL among Caucasian population. SLC26A4 comes next, accounts for 5%-10%, hearing loss with recessive mutations of SLC26A4 gene is usually associated with typical abnormalities of temporal bone, which is known as enlarged vestibular aqueduct (EVA). Mitochondrial DNA mutations account for about 1%. Other types of NSHL are very rare.By tracking the most updated research data of the field, two databases were set up, namely Hereditary NSHL Loci and Genes Database and Hereditary NSHL Gene Mutation Database. Meanwhile Flow Chart of NSHL Candidate Genes was also updated for easier manipulation. That significantly improved the previously established Genetic Diagnostic System for Hereditary Nonsyndromic Hearing Loss.Following polymerase chain reaction (PCR), coding sequence of GJB2 and mitochondrial genes of 12S and UCN were screened by denaturing high performance liquid chromatography (DHPLC) and restricted length fragment polymorphism (RFLP) with Alw26I and XbaI, respectively, among 407 hearing loss families collected from 1998 to 2007. Coding sequence of SLC26A4 was screened by DHPLC among 31 hearing loss families with or without EVA. All positive samples in screening were applied to direct sequencing. Mutation screening was also performed, by sequencing, to regulatory region of SLC26A4 and its regulatory gene F0XI1, in order to unveil the genotype-phenotype relationship.Among the 407 families, twenty-four probands were detected with two mutant alleles of GJB2; fifteen were with one mutant GJB2 allele. Twenty-seven probands had homogeneous mutation of A1555G of mtDNA, among which two also had homogeneous mutation of G7444A. Among those 31 families with or without EVA, seventeen patients from fifteen families were detected with two mutant alleles of SLC26A4; two were with one mutant SLC26A4 allele, none mutant alleles were detected in two EVA patients. None positive results got for the SLC26A4 regulatory components.Taking advantage of improved Genetic Diagnostic System for Hereditary Nonsyndromic Hearing Loss, with selected screening method, it could be efficient and economic for mutation screening of hereditary NSHL genes. According to published data and our own analysis, it could be concluded that 235delC of GJB2, A1555G of mtDNA, and IVS7-2A>G of SLC26A4 were hot spots of mutation among Chinese hearing loss population.更多还原