【作者】 张海军；

【导师】 单祥年；

【作者基本信息】 东南大学 ， 遗传学， 2006， 博士

【摘要】 在永久性保存遗传资源的基础上,对淮阴的一个A1555G突变相关耳聋大家系成员的线粒体基因组、可能的核修饰位点或基因进行研究,以期为阐明A1555G突变致聋分子机理和揭示母系遗传非综合征耳聋的本质等提供重要的分子生物学资料。一、在家系随访和临床医学检查的前提下,知情同意后,共收集到来自淮阴耳聋大家系的122例全血标本（包括85例母系成员、26例相关配偶对照和11例父系成员）,并采用EB病毒转化法成功构建了115株永生化细胞系,有效地保存了该大家系的遗传资源;二、共发现遗传性耳聋患者56例,且均为母系成员,表现出母系遗传的特征;耳聋患者个体间的听力损失程度和发病年龄差异较大,但均为双耳对称性、感音神经性高频听力损失,且多不伴有耳聋以外的其他病症,确认为非综合征型耳聋;根据构建的家系图谱分析,除线粒体DNA突变外,可能还有位于常染色体上的核基因以不规则显性遗传方式影响母系成员的临床表型;三、采用聚合酶链反应-限制片断长度多态性分析（PCR-restriction fragment length polymorphism, PCR-RFLP）和测序技术,检测了核心分支家系中26名母系成员的线粒体DNA上1555位点和7445位点的碱基变化,进而对该家系2名母系成员（一例听力正常,另一例具有严重耳聋症状）的线粒体全基因组和其他24名母系成员线粒体12S rRNA基因和tRNA^Ser（UCN）基因进行了全长测序。再次证明了A1555G突变是该家系成员致聋的分子生物学基础之一;与人类线粒体标准序列相比,发现临床表型完全不同两母系成员的线粒体基因组间无序列差异,均有31处序列变化,但只有A1555G和955-960insC为已知的可引起非综合征耳聋的突变,进一步研究发现该核心家系26例母系成员的线粒体基因组中除A1555G突变外,都同时存在有955-960 insC同质型突变,两突变共分离。另外,新发现一个线粒体DNA突变——7449insG,但该突变仅在2名母系成员中存在。推测955-960 insC突更多还原

【Abstract】 Following establishment of immortal lymphoblastoid cell lines for the Chinese extensive family with nonsyndromic deafness in Huaiyin, Jiangsu Province, the whole mitochondrial genome and two putative nuclear modifier genes were PCR amplified and directly sequenced, and the chromosomal region around marker D8S277, a promising modifier locus on 8p23.1, was fine-mapped, in order to identify whether other variants in the mitochondrial DNA, mutation(s) in Connexion 26 and MTO1, or D8S277, would contribute to the pathophysiological pathway in this Chinese deafness family associated A1555G mutation.I. Based on the clinical characterization, venous blood samples were obtained from 122 family members (including 85 matrilineal relatives, 11 patrilineal relatives and 26 spouse controls ) with informed consent, then 115 immortal lymphoblastoid cell lines were transformed using EB virus mediated by cyclosporin A.II. Clinical characterization showed that 56/85 maternal relatives, in this family, experienced hearing loss, whose severity and age-onset varied distinctly. However, their hearing loss shared some common features, being bilateral, sensorineural, and symmetric. And the pattern of inheritance in this family strongly indicates that the mitochondrial DNA mutation may be the main factor of deafness in this family, and that the nuclear modifier gene(s) may have involved in the development of deafness, which is in an irregular autosomal dominant model.III. PCR-RFLP (PCR-restriction fragment length polymorphism, PCR-RFLP) was used to screen both the nt1555 and the nt7445 of the mitochondrial DNA from 26 matrilineal members in the nuclear family; subsequently, the whole mitochondrial genomes from two matrilineal members (one with severe hearing loss, the other with normal hearing), 12S rRNA genes and tRNASer(UCN) from更多还原