基于西北少数民族的聋病资源库构建及基因诊断策略研究

发布时间：2018-06-02 21:11

本文选题：耳聋 + 听力损失　；参考：《兰州大学》2014年博士论文

【摘要】：听力损失影响人的言语功能进而导致社会交流障碍,因其发病率高而成为医学界研究的热点。从1995年克隆第一个耳聋基因起,超过80个非综合征耳聋相关基因被发现,越来越多的聋病分子病因得以揭示。中国人口的多民族结构蕴含着丰富的遗传资源,特别是不同族源的少数民族人群更为珍贵。本研究旨在进行中国西北地区少数民族耳聋资源的收集,建立一个覆盖西北地区的包含主要少数民族的临床资源和遗传资源库。在此基础上,通过开展三种常见的耳聋相关基因的分子流行病学研究,并对一个耳聋家系进行候选基因突变研究,试图揭示这些基因在少数民族耳聋人群的流行状况,绘制各民族的聋病基因突变频谱,分析开展耳聋家系致病基因定位克隆的有效方法,探索针对少数民族的基因诊断策略。我们研究的主要内容由以下四部分组成：第一章：中国西北地区少数民族聋病资源库建设少数民族耳聋群体较为稀缺,遗传性耳聋资源有减少的趋势。为了保护少数民族的遗传性耳聋资源,我们研究建立西北地区少数民族的聋病资源库。研究中制定了资源库建设的规范流程,通过本地区各特教学校、各级残联及医院门诊等渠道收集耳聋散发病例及耳聋家系资源,进行问卷调查、物理检查、听力学检查并绘制家系图谱,签订知情同意书并抽取外周静脉血,提取基因组DNA,建立全套资料的数据化管理模式。我们共收集到少数民族1673例耳聋散发病例和146个遗传性耳聋家系,完成了临床和遗传资源的收集,为进一步开展遗传学研究积累了资源。我们认为需要进一步加强少数民族耳聋资源的保护,建立科学完善的资源库管理体系。第二章：西北少数民族非综合征感音神经性聋的GJB2基因突变筛查研究 GJB2基因是目前发现的先天性耳聋最常见的分子病因,GJB2基因编码缝隙连接蛋白Cx26,在内耳电解质交换和细胞间信息传递发挥重要作用,与人类听觉功能关系密切。我们在西北少数民族散发感音神经性聋患者群体中开展GJB2基因的突变研究,目的是分析该基因的流行状况,揭示少数民族的热点突变,探讨适合这些耳聋人群的基因诊断策略。研究中完成了共1330例非综合征感音神经性听力损失患者的GJB2基因的突变筛查,并且完成了同地区457例汉族耳聋患者的比较研究。结果在少数民族发现了11种突变形式,包括一种新的突变：c.257CG。少数民族共有202例患者发现突变或序列改变,纯合突变75例,杂合突变92例,复合杂合突变35例。这些基因型的突变频率为15.19%(202/1330),同地区汉族人群的突变频率为19.04%(87/457)。少数民族群体与汉族的突变频率之间没有显著性差异,比较各民族与汉族的GJB2基因突变频率差异,其中维吾尔族与汉族存在统计学差异,其他各民族之间无统计学差异。各民族GJB2基因突变基因型以框移突变为主,这类基因型决定了耳聋表型是以重度及极重度聋为多见。在少数民族人群中等位基因频率最高的四种突变是c.235delC、c.35delG、 c.109GA和c.299-300delAT,藏族的热点突变是c.235delC和c.109GA,东乡族的热点突变分别是c.299-300delAT和c.235delC,回族是c.235delC和c.109GA,哈萨克族是c.35delG,维吾尔族是c.235delC、c.35delG,汉族人群的热点突变是c.235delC、c.109GA和c.299-300delAT,各民族的GJB2基因突变频率与其种族背景相一致,其中哈萨克族和维吾尔族具有明显的高加索背景表现。研究中我们绘制了各民族完整的GJB2基因突变频谱,可以为针对各民族的个性化的基因诊断和大面积筛查提供方法学依据。另外考虑到基因的结构特点和突变位点的分散性,GJB2基因诊断模式以全编码序列的筛查为宜。第三章：SLC26A4基因及IntDNA1555AG突变在西北少数民族非综合征感音神经性听力损失患者的筛查研究前庭水管扩大是先天性耳聋最常见的内耳畸形,与这一疾病相关的SLC26A4基因与耳聋关系密切。线粒体DNAA1555G突变与氨基糖苷类抗生素导致耳聋有相关性,该突变检测有利于揭示药物性聋和环境因素的关系。我们对西北地区少数民族共1330例非综合征感音神经性聋患者,以PCR及直接测序检测SLC26A4基因第8、第19外显子的突变,运用PCR-RFLP筛查1ntDNA1555AG点突变。在共1330例少数民族患者中,筛查出SLC26A4基因c.919-2AG、c.2168AG、c.2162CT三种突变形式,检测到七种基因型,包括纯合26例,杂合40例,复合杂合突变5例,其中双等位基因的突变频率为2.33%。同期进行的本地区汉族457例聋人检测到6种基因型,包括纯合15例,杂合31例及复合杂合突变8例,双等位基因突变频率为5.03%。分析显示汉族同藏族、东乡族及维族的该基因突变频率有统计学差异,回族与东乡族和维族均有差异。研究证实c.919-2AG突变是汉族聋人的热点突变,这一突变在汉族与藏族、东乡族及维吾尔族均存在统计学差异,维族与回族及哈萨克族有差异,回族与东乡族存在差异。研究中检测出mtDNA1555AG突变的患者有28例,突变频率为2.11%,同地区457例汉族患者中突变者有32例,突变频率为7.00%,两者之间有显著性差异。在106例AAID患者中有9例检测到mtDNA1555AG突变,AAID患者中突变频率为8.49%。各少数民族耳聋患者频率比较显示汉族与回族、东乡族及维吾尔族之间的突变频率存在统计学差异。研究证实SLC26A4基因在各少数民族与汉族的突变频谱不同,热点突变各异,因此需要针对各少数民族研究其突变频谱,制定适合各民族的基因诊断策略和产前诊断方法。研究表明有必要在本地区少数民族耳聋人群中开展mtDNA1555AG突变的基因诊断,在散发耳聋患者、遗传性耳聋家系开展该突变检测,进行科学干预有助于延缓和减少药物性耳聋的发生。第四章常染色体显性遗传非综合征耳聋家系的候选基因分析遗传性疾病的基因定位克隆方法多样,需要根据遗传资源的特征选择合适的研究方法。本文旨在探索适宜于遗传性耳聋病因学研究的有效方法。针对一个遗传性耳聋大家系,我们系统进行临床资料和表型特点研究,并通过候选基因突变筛查方法,明确了该家系的致病基因。首先通过对家系中耳聋患者发病特点的分析,我们明确其遗传方式为常染色体显性遗传。总结本家系表型特点有：耳聋首先以高频听力损失为主,后期影响到全频听力,部分患者表现有前庭功能障碍的症状。通过对表型的详尽分析,研究中将候选基因局限在少数几个基因,进而对最符合表型特点的COCH基因全部外显子进行突变筛查,发现了该基因第8外显子c.485GA突变为致病突变,该突变与家系中遗传性状共分离。研究中发现该家系中听力损失外显率为100%,听力损失随年龄增长而逐渐加重,前庭功能障碍的外显率为22.22%,前庭障碍症状与听力损失的程度没有相关性。从听力学特点和前庭功能障碍特征判断,该家系与梅尼埃病的临床表现不符。我们认为遗传性耳聋家系的基因定位克隆,首选位置克隆和候选基因克隆法,通过连锁分析能够定位耳聋相关基因的位置,候选基因克隆法具有经济快捷的优势。收集详实的临床资料和听力学资料、充分分析家系临床特征对于选择适宜的研究方法是十分必要的。
[Abstract]:Hearing loss affects human speech function and leads to social communication disorders. Because of its high incidence, the first deafness gene has been cloned in 1995, and more than 80 non syndromic deafness related genes have been found. More and more deafness molecular causes have been revealed. The multi ethnic structure of Chinese population is rich in abundance. The rich genetic resources, especially the ethnic minority groups of different ethnic groups, are more precious. This study aims to collect the deafness resources of ethnic minorities in Northwest China and to establish a clinical resource and genetic resource bank covering the main ethnic minorities in Northwest China. On the basis of this, three common deafness related genes are carried out. The study of molecular epidemiology and the study of the candidate gene mutation of a deafness family, try to reveal the epidemic situation of these genes in the deaf people of ethnic minorities, draw the frequency spectrum of gene mutation of deaf disease in all ethnic groups, analyze the effective methods to carry out the gene location cloning of the deafness family, and explore the gene diagnosis strategy for the minority nationalities. The main contents of our research are composed of the following four parts: Chapter 1: the construction of the national minority deaf resource pool in Northwest China.
In order to protect the genetic deafness resources of ethnic minorities, we have studied and established the deafness Resource Bank of minority nationalities in Northwest China. In the study, we set up a standard process for the construction of the resource bank, through the local special education schools, all levels of the disabled and hospital outpatients, and so on. Channel collection of deafness sporadic cases and deafness family resources, questionnaire survey, physical examination, audiology examination and drawing of family map, sign informed consent and extraction of peripheral venous blood, extraction of genomic DNA, and establish a data management model of complete set of data. We collected 1673 cases of deafness and 146 heredity in Ethnic Minorities. The family of sexual deafness has completed the collection of clinical and genetic resources and accumulated resources for further genetic research. We think it is necessary to further strengthen the protection of the minority deafness resources and establish a scientific and perfect resource bank management system. The second chapter: the GJB2 gene mutation screening of the NNW non syndromic sensorineural hearing loss. Investigation and research
The GJB2 gene is the most common molecular cause of congenital deafness. The GJB2 gene encodes the gap junction protein Cx26, which plays an important role in the internal ear electrolyte exchange and intercellular information transmission, which is closely related to the human auditory function. We carry out the mutation of the GJB2 gene in the population of the sporadic sensorineural deafness of the minority nationalities in Northwest China. The purpose of this study was to analyze the epidemic situation of the gene, reveal the hot spot mutation of ethnic minorities and explore the genetic diagnosis strategy suitable for these deaf people. In this study, a total of 1330 cases of GJB2 gene mutation screening in a total of patients with sensorineural hearing loss were screened, and 457 cases of Han deafness in the same area were compared. Results 11 forms of mutation were found in ethnic minorities, including a new mutation: 202 cases of c.257CG. minorities found mutations or sequence changes, 75 homozygous mutations, 92 heterozygous mutations and 35 complex heterozygous mutations. The mutation frequency of these genotypes was 15.19% (202/ 1330), and the mutation frequency of the Han population in the same area was 19.04% (87). /457). There is no significant difference between the mutation frequency of the ethnic minority group and the Han nationality, compared with the GJB2 gene mutation frequency difference between the ethnic group and the Han nationality. There is a statistical difference between the Uygur and the Han nationality, and there is no statistical difference between the other ethnic groups. The mutation genotype of the GJB2 gene in each ethnic group is mainly based on the frame shift mutation, and this type of genotyping is determined. The deafness phenotype is more common with severe and extremely severe deafness. The four mutation of the highest allele frequency in minority groups is c.235delC, c.35delG, c.109GA and c.299-300delAT. The hot mutation of the Tibetan people is c.235delC and c.109GA. The hot mutations in the Dongxiang nationality are c.299-300delAT and c.235delC, and the Hui nationality is c.235delC and c.109GA, ha. The sak nationality is c.35delG, the Uygur nationality is c.235delC, c.35delG, the hot mutation of the Han population is c.235delC, c.109GA and c.299-300delAT. The mutation frequency of the GJB2 gene of each ethnic group is in accordance with its racial background. The Kazakh and Uygur ethnic groups have obvious Caucasus back scenery. In the study, we draw the complete GJB2 of the ethnic groups. The gene mutation spectrum can provide a methodological basis for individual genetic diagnosis and large area screening for various nationalities. In addition, considering the structural characteristics of the gene and the dispersity of the mutation sites, the GJB2 gene diagnosis model is suitable for the screening of the full coding sequence. The third chapter: the SLC26A4 gene and the IntDNA1555AG mutation in the northwest minority nationality. Screening study of non syndromic sensorineural hearing loss
The enlargement of the vestibular water pipe is the most common deafness of the congenital deafness. The SLC26A4 gene related to this disease is closely related to the deafness. The mitochondrial DNAA1555G mutation is associated with the deafness caused by aminoglycoside antibiotics. The mutation detection is beneficial to reveal the relationship between drug deafness and environmental factors. In 1330 patients with sensorineural hearing loss, SLC26A4 gene eighth, nineteenth exon mutations were detected by PCR and direct sequencing, and 1ntDNA1555AG point mutation was screened by PCR-RFLP. Among the 1330 ethnic minority patients, the SLC26A4 gene c.919-2AG, c.2168AG, c.2162CT were screened and seven genotypes were detected, including the homozygous 26. There were 40 heterozygosity and 5 complex heterozygosity mutations. The mutation frequency of the double allele was 6 genotypes in 457 cases of the Han nationality in the local Han nationality of 2.33%., including 15 homozygous, 31 heterozygosity and 8 heterozygosity mutations. The mutation frequency of the double allele was 5.03%. analysis of the Han nationality, the Dongxiang and the Uygur. There is a statistical difference between the frequency of the mutation and the difference between the Hui and the Dongxiang and the Uygur people. The study confirms that the c.919-2AG mutation is a hot mutation of the Han and the deaf people. This mutation is statistically different in the Han and Tibetan, Dongxiang and Uygur ethnic groups. There are differences between the Uygur and the Hui and Kazakh people, and there are differences in the Hui and Dongxiang ethnic groups. The study detected M There were 28 cases of tDNA1555AG mutation, the frequency of mutation was 2.11%, and there were 32 cases of mutation in 457 Han patients in the same area, and the mutation frequency was 7%. There was a significant difference between the two. 9 cases in 106 cases of AAID were detected mtDNA1555AG mutation, and the frequency of mutation in AAID patients was 8.49%. of the minority deafness patients compared with the Han and the Han people. The mutation frequency between the Hui nationality and the Uygur ethnic group is statistically different. The study confirms that the mutation frequency of the SLC26A4 gene is different in the ethnic minority and the Han nationality, and the mutation frequency is different. Therefore, it is necessary to study the mutation spectrum of the ethnic minorities and formulate the genetic diagnosis strategies and prenatal diagnosis methods suitable for each ethnic group. The study shows that the SLC26A4 gene is suitable for all ethnic minorities. It is necessary to carry out the genetic diagnosis of mtDNA1555AG mutation among the deafness people in the local minority, and to carry out the mutation detection in the patients with deafness and hereditary deafness, and to intervene scientifically can help delay and reduce the occurrence of drug-induced deafness.
Fourth chapter analysis of candidate genes in families with autosomal dominant hereditary nonsyndromic hearing loss
The gene location and cloning methods of genetic diseases are diverse and need to be selected according to the characteristics of genetic resources. This paper aims to explore an effective method suitable for the etiological study of hereditary deafness. Through the analysis of the characteristics of the deafness in the family, it is clear that the hereditary mode of the family is autosomal dominant. The phenotypic characteristics of the family are as follows: the hearing loss is mainly high frequency hearing loss, the latter affects the full frequency hearing, and some patients show the vestibular function barrier. After a detailed analysis of the phenotype, the candidate genes were limited to a few genes, and then all the exons of the COCH gene, the most conforming to the phenotypic characteristics, were screened, and the mutation of the eighth exon c.485GA was found to be a pathogenic mutation, and the mutation was separated from the genetic traits in the family. The hearing loss was 100%, the hearing loss increased with age, and the vestibular dysfunction was 22.22%. The vestibular disorder was not related to the degree of hearing loss. Judging from the characteristics of the hearing and the characteristics of the vestibule dysfunction, the family was not in accordance with the clinical manifestation of the disease. We thought hereditary ear was inherited. The gene location cloning of the deafness family, the preferred location clone and the candidate gene cloning method, can locate the position of the deafness related genes through linkage analysis. The candidate gene cloning method has an economical and quick advantage. The detailed clinical data and audiological data are collected. Necessary.
【学位授予单位】：兰州大学
【学位级别】：博士
【学位授予年份】：2014
【分类号】：R764.43

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