【作者】 刘穹；

【导师】 韩东一； 王秋菊；

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

【摘要】 致聋基因的定位与克隆一直是全球遗传学和耳鼻喉科学学者共同致力研究的焦点。从1988年,第一个非综合征型耳聋基因位点被确定,截止至2008年5月,非综合征型遗传性耳聋的研究取得了巨大的成就,共定位了144个基因座位,克隆了49个基因。从1995年,第一个非综合征型耳聋基因被克隆,到2006年,耳聋基因的定位与克隆一直保持高速增长,平均每年定位8.3个位点,克隆4个基因。在这其中也包含了中国学者大量深入、细致的工作。早在1998年,夏家辉院士就克隆了GJB3基因——DFNA2基因座位的责任基因之一。到目前,中国学者共报告了13个基因座位(13/144,占9%),其中7个与国外报道的耳聋基因座位重合,6个是新的耳聋基因座位(6/144,4.2%)。遗传性耳聋基因的定位与克隆研究前景非常可观,有近2/3的基因座还没有找到责任基因,还有更多的新的耳聋基因等待着人们去发现。然而机遇与挑战并存,从2007年开始,遗传性耳聋的研究工作遭遇瓶颈,耳聋基因定位与克隆的速度明显减缓,成功定位和克隆的耳聋基因的数量逐年减少,2007-2008两年间只发现了4个耳聋基因新座位,克隆了2个新耳聋基因。为探索打破瓶颈,推动遗传性耳聋的分子机制研究进展,本研究立足于中国耳聋人群,采用位置候选基因克隆策略,进行了非综合征型遗传性耳聋大家系的基因定位克隆和候选基因筛查研究工作,成功定位了2个耳聋家系,发现了一个新耳聋基因座位——DFNA61,并利用一种新方法为发现新的耳聋基因进行了尝试,具体内容包括如下三部分。第一部分DFNA61型中高频听力损失家系(0703271家系)新基因定位研究本研究对一个罕见的中国遗传性中高频听力损失耳聋家系(0703271家系)进行基因定位克隆研究。该家系耳聋表型表现为一种常染色体显性遗传、迟发型、渐进性的、为中频为主的听力损失。家系共有3代,21名成员,10名耳聋患者。通过全基因组基因定位扫描连锁分析,在17号染色体上D17S1852微卫星标记处取得了最大LOD值3.45,定位区段位于D17S804至D17S969之间6.74cM的区域内。在此定位区段内尚无听力损失位点的报道,因此这是一个新的基因座位,我们将其命名为DFNA61。DFNA61基因座内共有14个编码蛋白基因,本研究下一步将对其展开筛查,希望早日找到DFNA61的致病基因。第二部分遗传性迟发型听力损失(W727家系)致病基因的精细定位克隆研究几乎所有的DFNA位点都具有迟发型听力损失的表型,而且遗传性迟发型听力损失对于研究听力的“提前衰老”,进而为老年性耳聋提供研究思路意义重大,因此,本课题组一直致力于遗传性迟发型听力损失的研究。本研究对2007年初步定位在9号染色体上(LOD=2.06 D9S157)的中国迟发型听力损失(W727)家系进行了进一步的精细基因定位与克隆研究。在W727家系原有基础上新增加了15名家系成员:包括6名耳聋患者和9名听力正常者,结果在1号染色体D1S2797处,取得LOD值=3.75的结果,将W727家系定位在1号染色体的D1S255和D1S2890之间18.9cM的区域内。该位点与DFNA2有部分重合,首选该DFNA2责任基因GJB3基因作为侯选基因进行突变检测,未发现致病突变。另一个DFNA2责任基因——KCNQ4基因的筛查正在进行,如果发现致病突变,则可以确定W727家系的致病基因;如果KCNQ4不是W727家系的致病基因,则可以推测该基因座位存在一个新的耳聋基因,继续研究,力争发现一个新的耳聋基因。第三部分人类遗传疾病基因模块化方法预测和验证聋病相关基因耳聋基因的定位只是破译耳聋基因研究的第一步,在已经定位的基因座中寻找到致病目标基因是一项更为艰难的工作。人类遗传疾病基因预测的模块化方法是近些年来出现的一种建立于生物信息学基础之上、利用基因网络手段对遗传疾病致病基因进行预测的新方法。为了给遗传性耳聋的致病基因克隆提供新的思路和方法,也对这种新方法的效能进行验证,本研究对2005年已经定位在9号染色体D9S165-D9S1874之间约4.12cM的区域内的常染色体显性遗传性耳聋大家系—686家系,采用了人类遗传疾病基因预测的模块化方法中的CIPher模型和Endeavour在线工具两种方法进行了致病基因的预测。研究结果发现共有6个基因:TLN1(细胞骨架蛋白基因),STOML2(溴化丙胺太林相关蛋白基因),AQP3(水通道蛋白基因),DNAI1(动力蛋白基因),C9ORF24(假基因),CCIN(精子细胞内基本蛋白基因),GALT(1-磷酸半乳糖尿苷酸转移酶基因)入选。首先选择AQP3基因在25名家系成员(包括8名耳聋患者)中进行了筛查。在筛查,我们发现了两个多态性改变:390C>T/390C>T和394G>A/WT。其中后者可引起氨基酸改变(ASP132ASN),进而可能使蛋白的空间构象发生变化,对其功能造成一定影响。家系中发生394G>A/WT突变的4名成员可能都是患病状态,在目前可以确定的家系正常人中尚未发现这种突变。本研究探索了一种新型的研究思路来思考和发现新的耳聋致病基因,具有一定的指导意义。更多还原

【Abstract】 The mapping and cloning of deafness genes is a hot focus that geneticists and otolaryngologists.The great progress of the research on hereditary non-syndromic deafness has been made since the first non-syndromic deafness locus was mapped in 1988.Until May 2008,144 loci are located,from which and 49 genes are cloned.The mapping and cloning on of deafness genes grew rapidly since the first gene of non-syndromic deafness was cloned in 1995.On average, 8.3 loci and 4 genes were identified every year,from 1995 to 2006.Chinese scholars achieved great success during the period.In 1999,Academician Xia Jia-hui et al from Chinese Academy of Science cloned the underling gene GJB3 for DFNA2,which was the only gene cloned by Chinese scientist.Up to now, Chinese scientist have discovered 13 loci(13/144,9%),7 of which were the same as the previous identified,and the other 6 were novel(6/144,4.2%).The future for positional cloning of deafness gene is bright.The underling genes in nearly 2/3 of the identified loci remain to be cloned,in addition to continuously identified genes/loci.However,those opportunities parallel with challises.The year of 2007 was a bottle-neck in the history of genetic studies of hearing loss.The speed for mapping and cloning of deafness genes was dramatically reduced,indicated by less successful attempts of positional cloning annually discovered.Only 4 loci and 2 genes were discovered during 2007 and 2008.In this study,we worked on the research of three non-syndromic hereditary deafness pedigrees by the strategy of positional cloning and screening the candidate genes based on the deaf people in China.We succeeded in locating 2 loci in two pedigrees and identifying a novel deafness locus named DFNA61 and attempted to predict causative genes in a deafness family mapped before.The research included three parts. Part one:mapping of the novel locus DFNA61 for late-onset mid to high frequency autosomal dominant hereditary hearing lossIn this study,mapping and cloning of the underlying gene for a scarce frait of hereditary deafness in a Chinese family suffering from middle and high-frequency hearing loss was performed.The phenotypic characteristics included autosomal dominant inheritance,late-onset occurence,progressivity and the hearing loss mainly in middle frequencies hearing.The family included 21 members in 3 generations,of which 10 members were deaf.We got the maximum LOD value of 3.45 on chromosome 17 with peak microsatellite marker D17S1852 by using genome-wide genesan and linkage analysis,and the linked region, defined by markers D17S804 and D17S969,was 6.74 cM long.No known hearing loss locus/gene was reported previously in this region,and thus,this locus was named as DFNA61.It contains 14 protein-coding genes,and further effort will be directed to identification of the causative genes in the locus.Part two:fine mapping of the linkage for hereditary late-onset hearing loss(W727 family)Almost all the DFNA loci have been linked to late-onset hearing loss phenotypes.Studies of hereditary late-onset hearing loss might give insights for early aging in hearing or age-related hearing loss.Consequently,our group have worked in this area for many years.In this study,we fine-mapped the locus for a Chinese late-onset hearing loss family named W727,which was previously mapped on chromosome 9(LOD=2.06,D9S157) in 2007.This family was extended by adding 15 new members including 6 deaf and 9 normal subjects.We got the maximum LOD value of 3.75 on Maker D1S2797 on chromosome 1,the interval of 18.9cM distance was defined by markers D1S255 and D1S2890.This interval overlapped with DFNA2 overlaps in part,we screened the mutations of GJB3 gene,one of responsible genes for DFNA2,but no mutation was found.In the next step,we will screen the other gene(KCNQ4).If no causative mutataion was found either,the underlying gene for this family might be novel. Part three:Network-based global inference and identification of human deafness genes by using modularization methodMapping deafness gene was just the first step in genetic research,it followed by difficult task finding the causative gene in the locus.Network-based global inference was a newly development bioinfornatics method to predict causative genes via the gene network.In order to verify the potential of this method for prioritizing the genes to be cloned,we applied this method tp predict the putative for causative gene for autonomic dominant deafness in family 686,which had been mapped on to a Chromosome 9 interval of 4.12 cM,flanked by markers D9S165 and D9S1874.Two different modular methods,CIPher model and Endeavour online tools were implemented,leading to identification of 7 genes were chosen,including TLN1,STOML,AQP3,DNAI1,C9ORF24,CCIN, and GALT.We first selected AQP3 gene to screen 25 family members including 8 deafness patients.Two polymorphisms were found:390C＞T/390C＞T and 394G＞A/WT.The latter can change the 132nd amino acid,from Asp to Asn, possibly leading to changes both in functionalities and spatial structure of the resulted protein.Reasonably,the 4 members with this functional polymorphism night be affected,soon or later.However,none of the healthy members in this family had this mutation.This study demonstrate that in-silico bioinformatics methods might provide effective alternative or pfiofitizing the hearing loss gene to be loned.更多还原