【作者】 朱玉华；

【导师】 翟所强； 袁慧军； 戴朴；

【作者基本信息】 中国人民解放军军医进修学院 ， 耳鼻咽喉科学， 2009， 博士

【摘要】 耳聋是导致言语交流障碍的常见疾病,由环境因素和遗传因素共同致病。由遗传因素引起的耳聋不仅包括单基因遗传性耳聋(如:非综合征型耳聋),多基因遗传性耳聋(如:老年性耳聋),还包括线粒体相关性耳聋(如:药物性耳聋)等。以上各方面耳聋遗传病学的研究为聋病的防治奠定了坚实的基础。随着人类基因组计划(Human genome project,HGP)的深入和完成,国际人类基因组单体型图计划(简称HapMap计划)的实施和不断完善,在遗传学高速发展的后基因组时代,遗传性耳聋的研究面临着前所未有的机遇和挑战。现如今,在现代分子遗传学研究技术平台的发展中应运而生的第三代遗传学标记单核苷酸多态(Single nucleotide polymorphism,SNP)在众多领域中发挥着重要作用,将其应用于遗传性耳聋致病基因及易感基因的定位、克隆研究也将成为遗传性耳聋研究的流行趋势和关注热点。在本研究中,我们应用单核苷酸多态作为遗传标记,在一个常染色体显性非综合征型耳聋家系中成功定位及筛查出一个全新的耳聋相关基因;探讨了常见耳聋基因GJB2、12S rRNA和GSTT1/GSTM1基因多态性与老年性耳聋遗传易感性的关系;以及追溯了线粒体12SrRNA C1494T突变的起源。一、非综合征型遗传性听力损失家系致病基因的定位、筛查及鉴定研究本研究选用单核苷酸多态(SNP)及微卫星标记(STR)作为遗传标记,利用连锁分析的方法,分步对一个常染色体显性遗传的非综合征型耳聋大家系(HN-W078)进行了耳聋致病基因的定位、筛查和鉴定工作。HN-W078家系为一个5代相传的耳聋大家系,耳聋患者的听力学表型为迟发性、渐进性、以高频听力下降为主的感音神经性耳聋。首先,选取SNP作为遗传标记,利用Affymetrix 5.0 SNP基因芯片进行全基因组扫描及连锁分析,将HN-W078家系的致病基因初步定位于12号染色体12q24.31-12q24.32区域(最大LOD值=5.656);之后,选取初步定位区域内及附近的16个微卫星标记进行精细定位及单倍体型分析,将该致病基因定位于微卫星标记D12S86和D12S1612之间的区域(最大LOD值=5.35)。通过定位区域候选基因直接测序筛查的方法,我们在DFNA60基因的EXON4外显子中发现了与此家系疾病表型共分离的突变位点c.377C＞T(p.S126L)。对整个家系和500个听力正常人进行该位点的酶切检测,均证明只有家系中的耳聋患者携带此突变。通过免疫组织化学方法显示,DFNA60基因编码的蛋白在耳蜗中多处表达,包括Corti’s器,血管纹,螺旋韧带等。此结果为阐明DFNA60基因突变引起的迟发性非综合征型耳聋的致病机制奠定了基础。(注:此位点及致病基因为全新发现,尚未发表,均暂用DFNA60表示)。二、老年性耳聋遗传易感基因的筛查老年性耳聋,亦指年龄相关性耳聋,是指随着年龄的增加逐渐出现的听力下降。随着现代生活环境的影响,衰老的过程逐渐提前,听觉器官也不例外。本研究通过普查的方法共收集到662名不同听力水平的中老年人研究对象(40岁至90岁);根据听力下降水平共分为四组;首先选取GJB2基因、12s rRNA基因及GSTT1/GSTM1基因作为老年性耳聋的遗传易感候选基因,通过直接测序和多重PCR的方法,研究了GJB2、12S rRNA和GSTT1/GSTM1基因与老年性耳聋遗传易感性的关系,初步探讨了不同听力水平中老年人的老年性耳聋遗传易感性差异的遗传学基础。关联性分析结果显示,GJB2基因的109G＞A、608T＞C杂合突变和12S rRNA A827G同质性突变均可增加老年性耳聋的遗传易感性,但没有检测到GSTT1/GSTM1基因的多态与老年性耳聋遗传易感性的关联性。本研究不仅为大规模、多中心、多学科联合研究老年性耳聋遗传易感基因收集了宝贵的老年性耳聋研究对象资源,同时提供了常见耳聋基因与老年性耳聋遗传易感性关联性分析的基础。三、线粒体12S rRNA C1494T突变的遗传学及起源性分析线粒体12S rRNA基因是一个与耳聋相关的热点基因,其中位于高度保守区域的12S rRNAA1555G和C1494T突变与药物性耳聋和非综合征型耳聋密切相关。迄今为止,一共报道了8个12S rRNA C1494T突变的药物性耳聋和非综合征型耳聋中国家系,这些家系内及家系间母系亲属的发病年龄、外显率以及听力下降程度均存在一定的差异,这说明12S rRNA C1494T突变不是影响耳聋表型的唯一因素,药物、线粒体单体型、核修饰基因以及线粒体第二突变均可能影响耳聋的外显率。本研究对基于聋病分子流行病调查发现的13个12S rRNA C1494T突变家系进行了线粒体基因组全序列测定以及全面的遗传学特征和单体型分析。通过线粒体基因组全序列分析,排除了线粒体DNA第二突变位点以及mt DNA变异体对12S rRNA C1494T突变耳聋家系外显率的影响;通过13个家系先证者的线粒体单体型分析发现,来自中国7个不同省份的13个12S rRNA C1494T突变耳聋家系分别属于A,B,D,D4,D4b2,F1,M,M7c,N9a1,和H2b单体型,明确了12S rRNA C1494T突变在进化过程中具有多起源性。对以上结果的综合分析表明,氨基糖甙类药物是目前唯一明确的12S rRNA C1494T突变外显率的影响因素,12S rRNAC1494T突变携带者避免接触氨基糖甙类药物是预防药物性耳聋最有效的方法。此研究为在正常人群中大规模筛查12S rRNA C1494T突变提供了确实的遗传学理论基础。更多还原

【Abstract】 Hearing impairment is the most common perception disorder in the world. It is caused by both environmental factors and genetic factors.The hearing loss attributable to genetic factors include not only single gene genetic deafness(such as non-syndromic hearing loss),but also multi-gene genetic deafness(such as age-related heating loss),as well as mitochondrion-related deafness(such as aminoglycoside-induced hearing loss).The research of over all aspects of genetic hearing loss has provided a solid foundation for prevention and treatment of hearing loss.With the gradual completion of the Human Genome Project(Human genome project,HGP),implementation and constant improvement of the International Human Genome HapMap Project(HapMap Project),in the post-genomic era with the rapid development of the genetics,the research of the genetic hearing loss is faced with unprecedented opportunities and challenges.In recent years,the third generation of genetic markers,single nucleotide polymorphisms(SNPs),which came into being because of demand with the development of modern molecular genetics technology platform,were applied in many fields and played an important role in many aspects.It will be the new concerns and fashion trends to apply the SNP in the mapping and cloning pathogenic gene or susceptibility gene of genetic heating loss.In our research,by taking single nucleotide polymorphisms as the genetic marker,we successfully mapped and identified a novel heating loss locus and gene in an autosomal dominant non-syndromic deafness family,completed the screening of common deafness gene GJB2,12Sr RNA and GSTT1/GSTM1 for the susceptibility of presbyacusis,and traced the origin of mitochondrial 12SrRNA C 1494T mutation.Part 1:Mapping,screening and identification of pathogenic gene in one autosomal-dominant nonsyndromic hearing loss familyIn this study,by taking both SNP and STR as the genetic markers,and making use of the linkage analysis,we successfully mapped,screened and identified the pathogenic gene in one autosomal-dominant nonsyndromic hearing loss family step by step.Family HN-W078 was a large Chinese family with hearing loss transmitted through 5 generations.The clinical evaluation of audiology revealed that the hearing loss was late-onset and gradual sensorineural hearing loss which first developed in high frequencies.First of all,we mapped the locus on chromosome 12q24.31-q24.32 region(two-point lod-score of 5.656) with application of the Affymetrix 5.0 SNP Genechip,and then by refined mapping and analysis of haplotype,we mapped the same locus to the region between D12S86 and D 12S 1612(two-point lod-score of 5.35) with application of 16 microsatellite markers.By direct sequencing of candidate genes in mapping region,we found a missense mutation c.377C＞T(p.S126L) in EXON4 of DFNA60 gene,which was totally segregated with the disease phenotype.We confirmed that only the patients in HN-W078 carried this mutation by failing in detecting this mutation in 500 normal subjects.With the method of immunohistochemistry,we found out that the DFNA60 widely expressed in the cochlear,including the organ of Corti,stria vascularis,and spiral ligament,and so on.It had laid the foundation for pathogenic mechanism study of deafness caused by DFNA60 gene mutation.(The locus and gene is novel and has not been published anywhere,it is represented by DFNA60 temporarily).Part 2:Genetic screening for susceptibility genes of presbyacusisPresbyacusis,also refers to age-related hearing loss,is gradual hearing loss with age.With the environmental impact of modern life,the process of aging is being in advance,no exception of the hearing organ.In our study,662 elder people with different age(40-90years old) and different hearing threshold levels through census were collected.According to audiological data,all elder were divided into four groups.Through the direct sequencing and multi-polymerase chain reaction,we had investigated the relationship of GJB2,12S rRNA and GSTT1/GSTM1 genes and susceptibility of presbyacusis and initially explored the hereditary agent of susceptibility for presbyacusis.The findings indicated that 109G＞A and 608T＞C heterozygotic mutations of GJB2 gene,and homoplasmy A827G mutation of mitochondrial 12S rRNA may increase susceptibility of presbyacusis.No relationship was deteced between GSTT1/GSTM1 gene and presbyacusis.In summary,this study not only collected the large scale of subjects with presbyacusis for large-scale,multi-center,multi-disciplinary joint research of presbyacusis’ genetic susceptibility,but also provided foundation of analysis for relationship of common deafness genes and genetic susceptibility of presbyacusis.Part 3:Genetics and origin analysis of mitochondrial 12S rRNA C1494T mutationMitochondrial 12S rRNA has been shown to be the hot spot for mutations associated with both aminoglycoside-induced and nonsyndromic hearing loss.The 12S rRNA A1555G and C1494T mutations in the highly conserved A-site of the 12SrRNA have been associated with both aminoglycoside-induced and nonsyndromic hearing loss in many families worldwid.Previous investigations revealed the highly variable penetrance and expressivity of hearing loss in 8 Han Chinese families carrying the C1494T mutation.Matrilineal relatives within and among families carrying the C1494T mutation exhibited a wide range of penetrance,severity,and age-of-onset in hearing loss.These results indicated that the mtDNA mutation 12S rRNA C1494T itself is insufficient to produce the clinical phenotype.Therefore,other modifiers including aminoglycosides,nuclear modifier genes,second mutation,and mitochondrial haplotypes may modulate the phenotypic manifestations of the C1494T mutation.In this study,we reported the clinical,genetic,and molecular characteristics of 13 Han Chinese families with aminoglycoside-induced and nonsyndromic bilateral hearing loss collected by screening of nationwide molecular epidemic.Through the whole mitochondrial genome DNA sequence analysis,we precluded the possibility that the second mutations might significantly modify the manifestation of the C1494T mutation. Through analysis of haplotypes of the probands,13 families come from 7 different provience were classified into 10 haplogroups by the distinct sets of mtDNA polymorphisms belonged to different haplogroups,including haplogroups A,B,D, D4,D4b2,F1,M,M7c,N9al,and H2b.This result suggested that the C1494T mutation occurred sporadically and might be multi-origin mutation through evolution of the mtDNA in China,and these mtDNA haplogroup-specific variants may not play an important role in the phenotypic expression of the C1494T mutation in these Chinese families with different penetrance of hearing loss. Therefore,aminoglycoside-antibiotics is solo well-established factor to contribute to the deafness expression of the C 1494T mutation.So prevention by avoiding the administration of aminoglycoside in individuals carrying C1494T mutation is the most effective way to protect their vulnerable hearing,and a systematic and extended mutational screening of the 12S rRNA C1494T mutation not only for hearing loss patients but also for normal Chinese subjects is necessary before the use of aminoglycosides.更多还原