【作者】 李庆忠；

【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 ， 耳鼻咽喉科学， 2005， 博士

【摘要】 过去的十年是遗传性耳聋研究飞速发展的不同寻常的十年。从1995年发现第一个非综合征型耳聋基因到现在,已经发现了39个非综合征型耳聋相关基因,其中包括编码肌球蛋白的细胞骨架蛋白、细胞外基质蛋白、通道和缝隙连接蛋白、转录因子基因,线粒体基因以及一些未知功能的基因等。这些研究成果使破解听觉基因,诠释听觉系统的分子机制越来越成为可能。随着人类基因组计划的顺利完成,越来越准确的基因组序列和功能信息的释放、遗传统计学方法的日臻完善、高密度遗传标记的发展和应用为人类耳聋基因定位克隆和分子流行病学研究提供了强有力的工具。本研究利用解放军总医院耳鼻咽喉研究所拥有的丰富的聋病遗传资源,在信息丰富的遗传家系中进行了具有创新意义的非综合征型遗传性耳聋大家系的基因定位克隆及散发病人中进行的耳聋相关基因的分子流行病学研究工作,得到了令人欣喜的研究结果,本研究包括如下两部分: 第一部分 遗传性耳聋致病基因的定位与克隆 在第一部分中主要对三个不同表型特征的中国耳聋大家系进行了定位与克隆研究。由此三个家系的研究组成了本部分的三个章节,分述如下: 第一章:X连锁先天性极重度耳聋POU3F4致病基因新突变的发现 本研究以一个5代相传、男性发病、CT扫描显示存在内耳畸形表现为先天性极重度耳聋家系(021家系)为研究对象。通过连锁分析的方法,将021家系的表型定位在X染色体上长臂Xq13.1-Xq23区域,遗传标记为.DXS983更多还原

【Abstract】 Over the past 10 years, remarkable progress has been made in the identification of new loci for non-syndromic hearing impairment (NSHI) and in the cloning of deafness genes. Since the first deafness gene was cloned in 1995, 39 genes for non-syndromic hearing loss have been identified. These genes encode a wide variety of protein classes: from myosins and other cytoskeletal proteins, channels and gap junction components, to transcription factors, extracellular matrix proteins and unknown proteins. These findings offer the promise to significantly expand our knowledge on the molecular mechanisms underlying the auditory and vestibular functions and on the pathophysiological mechanisms for hearing loss. With the completion of Human Genome Project, the release of massive genome sequence and function information, the advances in statistical genetics, and the use of the high-dense genetic markers provide powerful tool/information for genetic cloning and molecular epidemiology of human deafness.. This study used the plenty genetic resources established by the Otolaryngology Institute of PLA General Hospital, to hunt for the causative genes in three large Chinese pedigrees with non-syndromic hearing loss; and to investigate the molecular epidemiology of connexins in Chinese sporadic hearing-impaired cases. This thesis is divided into two main parts to present the results for the study.Part one: mapping and mutation screening of the causativeloci/genes in three hereditary hearing loss familiesThe purpose of this study was to map and to screen the genetic variants that cosegregated with the hereditary hearing loss in the three families.1. A causative gene was found in an X-linked congenital profound hearing loss familyThe clinic phenotype in family 021 was congenital profound hearing loss. The clinical investigations revealed the striking feature of the extremely high penetrance of deaf-mutism in the male members, but no penetrance in the female members. Linkage analysis was thus carried out under an X-link inheritance mode, with penetrance of 90%. The identified critical region spans a 29.53 cM region flanked by markers DXS983 and DXS1220. Maximal lod score of 3.21(theta=0.0) was observed at marker DXS990. Mutation screening of POU3F4 gene was conducted in family 021. A missense mutation (925T—C) was detected in all male patients, and in female carriers. The same mutation was not detected in other members of family 021 and in 110 unrelated controls with normal hearing. This mutation situated at POU homeodomain, was predicted to cause a conservative amino acid substitution (Ser309Pro). These data suggest that this mutation is responsible for the hearing loss in family 021.In short, a novel mutation of POU3F4 gene was identified to be the causative reason for the hearing loss in family 021. Further elucidation of the functional involvement of the mutation will help us to develop the method(s) for molecular diagnosis of the congenital hearing loss and to understand its role(s) in regulating the development of inner ear.2. AUNXl-a novel locus for auditory neuropathy was successfully mappedThe studied Family 015 was characterized with X-linked recessive non-syndromic auditory neuropathy. The disease locus for family 015 wasmapped into a chromosomal region defined by markers DXS1220 and DXS8084 via linkage analysis. Maximal lod score of 2.33 was observed at markers DXS1001, DXS1212, and DXS1211. The critical region spans a 28.09 cM chromosomal fragment. No known deaf genes or loci are localized within this region. Thus, AUNX1 (auditory neuropathy, X chromosome, locus 1) is a novel disease locus, and was approved by the International Committee of Human Genome Nomenclature.In short, a new locus (AUNX1) for non-syndromic auditory neuropathy was successfully mapped onto X chromosome using a large Chinese family.3. Mapping a disease locus for an autosomal-dominant inherited low-frequency sensorineural hearing loss (LFSNHL)Family 013 exhibited an autosomal dominant inheritance pattern of low frequency hearing loss. The disease locus was mapped into a chromosomal region flanked by markers D9S1677 to D9S1838 by linkage analysis. Maximal lod score of 4.57 was observed at marker D9S177. The region spans a 46.47 cM (28.54Mb) region. , containing DFNB31 and DFNB33 loci Because loci DFNB31 and DFNB33 follow a recessive mode(s) , the causative gene(s) underlying the low-frequency hearing loss in Family 013 might be in somewhere else.In short, we mapped low-frequency sensorineural hearing loss onto a chromosome 9 region via model-based linkage analysis of a well-characterized family. This region dose not overlaps any known loci for low-frequency hearing loss.Part two: molecular epidemiology of connexins was studied using Chinese sporadic hearing-impaired casesConnexin mutations were the major cause for autosomal recessive non-syndromic deafness (ARNSD). This study addressed the molecular更多还原