广西遗传性耳聋高危人群致聋基因突变谱鉴定和功能研究

Hearing loss is among the top of the disabling diseases in China and more than half of hearing loss cases are in non-syndromic forms, i.e., hereditary. The mutation spectrum that causes hereditary hearing loss is characteristic of regional difference. Guangxi, a region where multiple ethic groups have lived together for thousands of years, is a remote place and isolated from the major Han people, and therefore is assumed to have its unique mutation spectrum. In a previous study involving 200 cases of hereditary hearing loss, we observed that only 10% were detectable by the current DNA microarray test that targets nine mutations sites of Chinese people. Taking advantage of DNA sequencing, we found more than 20 mutation types missed by the biochip test. These results strongly suggested that Guangxi indeed has a unique mutation spectrum of hereditary hearing loss. In this proposal, we aim to establish the Guangxi-specific mutation spectrum of 1000 cases with hereditary hearing loss and further establish an efficient screening strategy for hereditary hearing loss based on this mutation spectrum in Guangxi. We plan to do so by the combined use of DNA microarray, DNA sequencing, and exome-wide DNA capture and next generation sequencing: DNA microarray to pick up 9 common mutation sites in GJB2, SLC26A4, GJB3, and mitochondria 12S rRNA; sequencings to find new mutation sites in 101 genes that cause hereditary hearing loss. All the new mutation sites found will be further studied and confirmed by pedigree analysis, gene function analysis and gene mapping analysis. Meanwhile we plan to provide a novel gene mutation screening proposal based on multicolor melting curve analysis (MMCA) technology for Guangxi people. Fulfillment of the above goals will help to elucidate the molecular genetics mechanism of Guangxi-specific hereditary hearing loss and meanwhile offer the necessary technical expertise for prevention and control of hereditary hearing loss the Guangxi region.

我国耳聋高居各类残疾之首，其中至少一半与遗传因素有关，而遗传性耳聋基因突变谱存在区域性差异。广西具有数千年多民族聚居史，与广大汉族地区相对隔离。前期我们对广西200例耳聋患者的研究表明，现有检测9个突变的耳聋筛查芯片仅能检出其中的10%，利用DNA测序可发现20余种其它突变类型，提示该区具有独特的突变谱。本项目拟将分析人群扩大至1000例，利用基因芯片排查GJB2、SLC26A4、GJB3、线粒体 12SrRNA基因常见9个突变位点，再利用DNA测序、外显子捕获高通量测序技术（NGS）对101个已知耳聋相关基因及线粒体部分区域的全部外显子进行序列分析，结合核心家系的突变鉴定、功能分析以及新基因定位研究，绘制出广西耳聋基因的突变谱；并提供一种适合广西人群的基于多色探针熔解曲线分析技术的突变筛查方案，为阐明广西地区耳聋人群的分子遗传学特征提供理论依据，并为开展全区的耳聋防控工作奠定技术基础。

我国耳聋高居各类残疾之首，其中至少一半与遗传因素有关，而遗传性耳聋基因突变谱存在区域性差异。课题组收集了广西13个市22个机构共1428例耳聋患者（501个核心家系），建立了广西地区特色的遗传性耳聋资料库。利用遗传性耳聋基因芯片在1428例耳聋患者中，共检出携带遗传性耳聋相关基因突变的患者192例（13.45%）。国内首次建立了广西特色耳聋相关基因热点突变的多色探针熔解曲线分析新技术，并开发了遗传性耳聋基因检测试剂盒（荧光PCR熔解曲线法），可以检测出中国人群常见的4个基因的20种突变，利用该技术共检出遗传性耳聋相关基因突变的患者256例（17.93%），比用基因芯片技术提高了4.48%。广西耳聋患者GJB2、SLC26A4、12S rRNA的基因突变发生率（5.43%、11.27%和1.05%）均低于全国流行病学调查水平（21%、14.5%和4.4%）。汉族与壮族耳聋人群SLC26A4基因突变发生率高于GJB2基因，与全国流行病学调查数据不同。GJB2 基因c.235delC、SLC26A4 基因c.754 T>C、c.1229 C>T、c.919-2A>G为广西地区的四大热点突变。同时结合Sanger测序和高通量测序技术，遗传性耳聋基因突变诊断提高了20.35%，发现50多种基因新突变。课题组发明专利授权3件，发表论著10篇（其中SCI论文5篇），发表会议论文5篇，其中两篇论文获得第四届中国出生缺陷防控论坛优秀论文奖。培养博士研究生1人，在国家级继续教育培训班授课4次。举办6次培训班，共培养耳鼻喉医师、听力检测医师、护士、护师及基因检测人员达到2000余人次。本项目所建立新方法已在20多家县级以上医院、特殊教育学校及康复机构推广与应用，效果显著，提高了行业的技术水平，有助于实现国内地区大人群遗传性耳聋预防计划，提高人口质量。