【作者】 门美超；

【导师】 冯永；

【作者基本信息】 中南大学 ， 耳鼻咽喉科学， 2011， 博士

【摘要】 全国性聋病分子流行病学研究数据显示,尽管遗传性耳聋具有广泛的遗传异质性,但大多数非综合征性遗传性耳聋仅由为数不多的几个基因突变所致,如GJB2基因、线粒体DNA基因、SLC26A4基因等,从而奠定了在中国开展耳聋基因诊断的理论基础,同时也催生出新的耳聋整体预防思路和方法。本课题致力于通过设计经济、高效、快速、高通量适合中国国情的耳聋基因诊断方法,为建立标准化、规范化的耳聋基因诊断、遗传咨询和产前诊断模式提供技术支持,使耳聋基因组研究所取得的伟大成果能够被广泛应用于中国临床遗传性耳聋病因学诊断和聋儿出生缺陷防控的实践,实现其“基础-临床”的顺利转化,从而推动国内临床耳聋基因诊断、遗传咨询和基于耳聋基因诊断的产前诊断工作的广泛开展,最终实现总体降低耳聋发生及出生缺陷的目标。本课题另外一个重要内容就是收集到湖南地区一个表现为X-连锁或母系遗传方式的遗传性耳聋大家系,通过对这一家系的临床特征及分子病因学研究,进一步探讨mtDNA突变同非综合征耳聋之间的关系,并为进行耳聋致病基因的功能研究打下了基础。本研究包括以下三个部分。第一部分运用PE-DHPLC技术快速检测中国人群非综合征型耳聋热点基因突变目的：建立针对中国人几种常见的遗传性耳聋基因型的基因诊断新方法。方法：PCR扩增的靶序列,经引物延伸,得到中国人非综合征性耳聋6个常见突变的特异性延伸片段（GJB2-235delC GJB2-299delAT、PDS IVS7-2A>G、PDS A2168G、mtDNA A1555G. mtDNA C1494T）,用全变性高效液相色谱分析延伸片段混合物,分离图谱可鉴定被检样本的基因型。结果：所选择的120例散发非综合征性耳聋样品的引物延伸变性高效液相色谱与经测序验证在结果吻合,前来遗传咨询的4个小家系的引物延伸变性高效液相色谱与测序结果亦相吻合,该法的准确率达100%。结论：该法是一种准确、高效的检测非综合征性耳聋基因突变的分析方法,可用于非综合征性耳聋基因及其他遗传疾病的诊断及遗传咨询。第二部分基因芯片技术在散发非综合征性耳聋基因诊断中的应用第一章基因芯片技术应用于耳聋基因检测的可行性研究目的：分析基因芯片技术应用于非综合征性耳聋基因检测的准确性,为建立新的基因检测方法提供依据。方法：通过对122例散发的非综合征性耳聋患者进行芯片及测序的双盲检测实验,验证GJB2₂35delC在这些散发样本中的分布情况结果：随机选取的122例样本的GJB2₂35delC的芯片检测与测序结果基本吻合,该法的准确率达89.9%。结论：该款芯片具有快速、高通量、高准确性、等特点,适合于大样本量遗传性耳聋的基因检测。第二章基因芯片检测技术在散发非综合征性耳聋患者中的应用目的：应用Goldengate耳聋基因芯片检测散发非综合征性耳聋患者,研究散发非综合征性耳聋的热点突变基因及耳聋基因位点的关联分析,为建立新的基因检测方法提供依据及模型。方法：对389例散发DNA样本（其中正常人DNA样本102例,散发非综合征性耳聋患者的DNA样本287例）进行芯片检测,应用关联分析研究耳聋及所选位点的相关性结果：6个位点WFS1_Leu829Pro、GJB3_Arg32Trp、OTOF IVS5+1、TMIE IVS2 2、PCDH15 3 BP DEL 5601 5603aac及SLC26A4_Gly497Ser与耳聋的关联分析为负相关；rs4809261、rs7421943、MYH14_Arg726Ser、TMPRSS3_PRO404LEU、rs878042、COCH_MET512THR、MYO3A_IVS7₂、TMC1_Tyr259Cys. GJB2_Arg165Trp、rs10515535、rs568619、rs7600176、rs3664、WHRN_IVS2+1、rs46791555及COL9A3_9BP_DEL₅41₅49ggtccccca与耳聋的关联分析为正相关。结论：基因芯片技术的建立及成功运用是对聋病基因检测手段的创新和突破,同时表明NSHL具有高度的遗传异质性。第三部分线粒体12SrRNA 1555A>G突变在一个中国耳聋大家系的特殊表现及分析目的线粒体突变是导致耳聋的重要原因,本文旨在探讨线粒体基因组1555A>G突变在一个中国耳聋大家系的特殊表现及分析。方法收集一遗传性耳聋大家系5代48人,提取样品DNA后,首先采用X-linkage连锁分析,未发现变异,再采用DNA直接测序法对家系成员进行线粒体全基因组、GJB2编码区及POU3F4基因进行序列分析。结果经过计算,连锁分析所有位点LOD值结果均小于1,不支持连锁；经mtDNA基因组序列分析发现12 SrRNA基因A1555G突变是此家系的唯一致病突变,同时患者均携带一新的变异14163C>T。结论线粒体A1555G突变是本家系耳聋的主要原因,环境因素及氨基甙类抗生素可能参与A1555G突变表型表达的调节,14163C>T为中国人群未报道新突变,可能对该家系A1555G突变所致耳聋的外显率及表型有影响。更多还原

【Abstract】 As the results of the national epidemiological for deafness show, hereditary hearing loss is extremely heterogeneous. Most of non-syndromic hereditary hearing losses, nevertheless, are found out to result from mutations of few genes, such as GJB2,mtDNA,SLC26A4,et al. This fact grounds the establishment of genetic testing and prenatal diagnosis for hearing loss in China. Our study is dedicated to found economic, high-effective, rapid and high-flux technologies for genetic testing of deafness, and standardize and normalize the contents and processes of genetic testing, consulting and prenatal diagnosis, to make the accomplishment of human genome project widely available in diagnosis for deafness and reduction of deaf baby, and to eventually promote genetic testing, consulting and prenatal diagnosis in China. Another role of our study is to investigate the clinical character and molecular genetic mechanism of a large family inherited as X-linked or maternally type of nonsyndromic deafness, which supply foundation for function study of genetic deafness genes. We carried out our research as the following three parts: Part one:Accurate and rapid diagnosis of common NSHL gene mutations by a multiplex primer extension and denaturing high-performance liquid chromatography techniqueObjectives:Nonsyndromic hearing loss （NSHL） is a common inherited disorder of the nervous system worldwide, and rapid and accurate genotyping methodologies for specific NSHL-causing gene mutations are needed for the diagnosis of the disease in different ethnic populations. Methods:In this study, we performed a novel multiplex primer extension （PE） reaction in combination with denaturing high-performance liquid chromatography （DHPLC） to simultaneously detect and genotype the 6 most common mutations in NSHL （GJB2-235delC, GJB2-299delAT, PDS-A2168G, PDS IVS7-2A>G, mtDNA-A1555G, and mtDNA-C1494T） in the Chinese population. This method involved the amplification of the target sequence, followed by a purification step, a multiplex PE reaction, and DHPLC analysis performed on the Transgenomic Wave DNA fragment analysis system under fully-denaturing conditions. Results:In a contrast analysis, this technique successfully and accurately genotyped 100%（120/120） of the samples previously detected by direct sequencing. For the four deafness genealogy for genetic consultion, this technique also successfully and accurately genotyped 100% of the members simutaniously detected by sequencing. Conclusion:The present study validated the combined PE/DHPLC approach as a simple, rapid, highly accurate, and cost-effective approach for genotyping common disease-causing mutations, and it strongly suggests that this technique may be successfully used in other genetic diseases.Part two:Application of DNA Chips in Clinical Genetic Testing for Sopratic genetic DeafnessChapter I:Feasibility Investigation of DNA Chips Applied in Clinical Genetic Testing for Sopratic genetic DeafnessObjectives:To investigate the feasibility of DNA Chips, and to found a new genetic detecting technology, we use the Illumina Goldengate gene chips in this part. Methods:In order to test the feasibility of DNA Chips, we detected the GJB2₂35delC mutation in 122 sporadic NSHL individuals through DNA Microarray and DNA sequencing at the same time. Results:The results get from the DNA Chips is the same as the traditional methods of sequencing. The detecting rate is 100%. Conclusion:Illumina GoldenGate DNA Chips appears to have some inherent advantages in genetic diagnosis of NSHL, such as low time consuming, high performance and accuracy, which make it fit to be used in clinic practice.Chapter II:Clinical Application of DNA Chips in Rapid Genetic Testing of Sopratic Non-Syndromic Hearing Loss Objectives:To detect the hot genes in sporadic NSHL patients and the association analysis between these genes and genetic deafness. Methods:389 sporadic patients were collected and detected by Illumina GoldenGate DNA Chips. Results:WFS1_Leu829Pro, GJB3_Arg32Trp, OTOF_IVS5+1, TMIE_IVS2₂, PCDH15_3BP_DEL₅601₅603aac and SLC26A4_Gly497Ser appeared negative correlation with NSHL; While rs480926、rs7421943、MYH14_Arg726Ser、TMPRSS3_PRO404LEU、rs878042、COCH MET512THR、MY03A IVS7 2、TMC1_Tyr259Cys、GJB2_Arg165Trp、rs10515535、rs568619. rs7600176、rs3664、WHRN（DFNB31）_IVS2+、rs4679155及COL9A3_9BP_DEL₅41₅49ggtccccca appeared positive correlation with NSHL Conclusion:The Illumina GoldenGate DNA Chips dependent new technique could be well applied for mutation detecting of deafness genes and confirmed the high genetic heterogeneity of genetic deafness.Part three:Special penetrance and analysis of hearing loss in a five-generation Chinese family with the mitochondrial 12S rRNA A1555G mutationObjectives:Mutations in mitochondrial DNA （mtDNA） have been found to be associated with sensorineural hearing loss. However, the variable clinical phenotype and incomplete penetrance of mtDNA 1555A>G-induced hearing loss complicate our understanding of this mutation Methods:Here, we performed clinical, genetic, and X-linkage analysis of a 5-generation Chinese family in which all the affected individuals were male. Results:Using X-linkage analysis and mtDNA sequencing, we detected an identical homoplasmic 1555A>G mutation in 9 individuals, and a previously unreported variant 14163C>T in mtDNA. Conclusions:Analysis of the complete mtDNA genome and x-linkage of this family revealed that the 1555A>G may lead to deafness. The 14163C>T is a never-reported variant and may synergism the development of the deafness.更多还原