【作者】 马建鹂；

【导师】 郭玉芬；

【作者基本信息】 兰州大学 ， 耳鼻咽喉科学， 2013， 硕士

【摘要】 耳聋是导致言语交流障碍最主要的原因,严重影响人口质量,病因包括遗传因素和环境因素,其中遗传因素占60-80%,在遗传因素中70%为非综合征型感音神经性耳聋（nonsyndromic sensorineural hearing loss,NSHL）目前发现与耳聋相关的基因超过140个,其中线粒体DNA12Sr RNA1555A>G （m.1555>G）, GJB2和SLC26A4突变是最常见的NSHL致病基因,国内外多年的研究证实上述三种基因突变所致的NSHL在不同种族、不同地域有很大的差异,包括不同的突变形式和突变频率。回族在中国是比较特殊的一个民族,历史上,回族是阿拉伯人的后裔,后经不断的迁徙、融合和演变,与许多民族之间相互融合,形成了一个特殊的群体。中国回族人口总数逾千万,其中一半以上聚集于中国西北地区,呈“大分散,小聚集”的分布特点,目前国内尚无关于回族聋病基因详细的研究报道,本研究收集来自中国西北地区的420例回族NSHL患者作为研究对象,探究上述三种基因的热点突变和突变频率,分析其特变特征与种族起源的相关性,为该地区回族耳聋人群的分子病因学诊断和遗传咨询提供分子流行病学基础,为政府制定因地制宜的防聋治聋策略提供依据。本研究通过对来自中国西北地区（包括甘肃、宁夏、青海、新疆和陕西）的420例回族非综合征型感音神经性耳聋（nonsyndromic sensorineural hearing loss,NSHL）患者进行药物相关性耳聋基因m.1555A>G、GJB2及SLC26A4的突变特征分析,探究三种常见聋病基因在该地区回族耳聋人群的热点突变和突变频率。m.1555A>G突变是氨基糖苷类抗生素致聋（aminoglycoside antibiotic-induced deafness, AAID）的分子学病因,因此对患者的临床病因进行分析,结果显示,420例回族NSHL患者中285例为先天性耳聋,占所有耳聋患者的67.86%（285/420）,其中有耳聋家族遗传病史患者40例；108例为药物性耳聋,占所有患者的25.71%（108/420）,其中氨基糖苷类抗生素（aminoglycoside antibiotic, AmAn）所致耳聋占所有药物性耳聋的84.26%（91/108）；其他病因引起的耳聋患者27例。由此可见,先天性耳聋（包括遗传性聋）和药物性耳聋是该地区回族耳聋发病的主要原因。基因检查结果发现11例患者为m.1555A>G均质性突变,突变携带频率为2.62%（11/420）。在11例m.1555A>G基因突变患者中,6例有明确的AmAn用药史,占病因明确患者的54.55%,一例为可疑药物性聋患者。因此,与AmAn致聋相关的m.1555A>G基因筛查对该地区回族人群防聋意义重大。通过对420例回族NSHL患者进行常见致聋基因GJB2基因的编码区和SLC26A4基因的第8、19外显子的检测,绘制中国西北地区回族NSHL患者GJB2基因突变图谱,明确两种基因在中国西北地区回族NSHL耳聋患者中的突变频率及主要的突变方式,分析其与种族起源间的关系。盐析法提取基因组DNA后运用多聚酶链反应（polymerase chain reaction, PCR）技术扩增GJB2基因的编码区和SLC26A4基因第8、19外显子,然后对上述PCR产物进行测序（上海美吉生物医药科技有限公司—北京测序部）。将生物公司反馈的测序结果利用Sequence Scanner v1.0和SeqMan与来自NCBI的标准序列（mtDNA:NC012920.1;GJB2: NG₀08358.1; SLC26A4:N T₀07933.15）进行对比。结果发现420耳聋患者中41例为GJB2基因突变所致,包括纯合和复合杂合,占所有患者的9.76%（41/420）,是最常见的致聋病因。其中c.235delC等位基因突变频率为6.90%（58/840）,占所有致病等位基因的51.33%（58/113）,是GJB2最常见的突变方式；20例为SLC26A4双等位基因改变,占所有患者的20/420（4.76%）,其中c.919-2A>G等位基因突变频率为5.0%（42/840）,占所有等位基因碱基改变的68.85%（42/61）,是SLC26A4的热点突变形式。通过上述三种常见聋病基因的检测,为该地区回族耳聋人群的基因诊断、遗传咨询及治疗提供分子流行病学依据。更多还原

【Abstract】 Deafness, seriously affects the quality of population, is the most significant cause of linguistic barrier, the causes of which including inherit(60-80%) and environmental factors. Nonsyndromic sensorineural hearing loss(NSHL) accounts for70%in inherit factors.To date, there are more than140genes related hearing loss, mitochondria DNA12Sr RNA1555A>G, GJB2and SLC26A4gene are the most common deafness-related-genes of NSHL. Lots of studies proved that the mutation frequency and mutation pattern are disparate in different areas and races. Hui, the descendant of the Arabs, was a special nationality in China after constantly evolution, migration and fusion with other nationalities in history. Total of Hui ethnic group population is more than ten million in China, more than half of which live in Northwest China. So far, there was no detailed research report about Hui ethnic group, in this study we collected420Hui NSHL patients to discuss the hot-spot mutation, frequency and the relation-ship betweem characteristic of genes mutation and origin of the hui nationality,the results of which will provides the epidemiology basis on molecular etiology and genetic counseling for this population, at the same time, provides scientifice evidence for the government in order to draw up strategy in the work of prevention and treatment for deafness in different races and areas.The aim of this study is to analysis the characteristic of m.1555A>G, GJB2and SLC26A4genes mutation in Hui patients with non-syndromic hearing loss in Northwest China(Gansu, Ningxia, Qinghai, Xinjiang and Shaanxi), probing into the frequency and hotspot mutation types of three frequent deafness-correlative genes.Because m.1555A>G mutation is the molecular etiology of aminoglycoside antibiotic-induced deafness(AAID), we analysis the clinical etiology of all patients, the results showed that285(67.86%) patients are congenital deafness, in which40(9.52%)patients had a family history;108(108/420) had the history of prescribing drugs,in addition,91(84.26%) of these objects had a history of using aminoglycoside antibiotic(AmAn) Moreover,27patients were caused by other etiologies. Thus, it can be seen that congenital deafness(including Hereditary hearing loss) and hearing loss induced by drugs are the most common factors. Genetic testing showed that11(2.62%) cases were caused by m.1555A>G homozygous mutation in420patients with NSHL,6of whom had a specific history of AmAn(6/11=54.55%), and one case was doubtful. Therefore, there has a great significance of m.1555A>G gene screening in order to prevent the deafness occurring in Hui population in Northwest China.At the same time,we analysis the characteristic of GJB2and SLC26A4genes mutation in420Hui ethnic group patients with NSHL and draw the map of GJB2gene mutation investigate the mutation frequency and forms of two genes. Amplified the target gene by polymerase chain reaction (PCR) after extracting genomic DNA from whole blood, then direct sequencing was used to the coding region of GJB2gene,exon8and19of SLC26A4gene. Results showed that41(9.76%) cases,including homozygote and compound heterozygote,were caused by GJB2gene mutation, which was the most frequent deafness-related gene.The allel frequency of c.235delC accounts for6.90%,as well as the most frequent(51.33%) mutational pattern in GJB2gene.20patients(4.76%) were found carring two allel mutations in SLC26A4gene.The allel frequency of c.919-2A>G is5.0%,accounts for a total of68.85%in all base alterations of SLC26A4gene,is the major mutant form of SLC26A4gene.Through this study we can provide the molecular epidemiology basis for Hui ethnic group patients with NSHL from Northwest China in genetic diagnosis, genetic counseling and therapy by associated testing of the involved three genes.更多还原