遗传性耳聋新的致病基因ABCC1的鉴定及致病机制研究

Combining whole-exome sequencing and linkage analysis, we have identified a novel heterozygous mutation (c.1769 A>G) in ABCC1 gene from a Chinese pedigree, which showed autosomal dominant non-syndromic hearing loss. We confirmed other mutations of this gene in other hearing loss families and sporadic hearing loss patients. It suggests that ABCC1 may be a novel pathogenic gene in Chinese deafness population. With current knowledge of ABCC1 and our preliminary studies, it implied that abnormalities in the transport of cochlear stria vascularis may be related to deafness caused by mutation of ABCC1 gene. This project intends: ① expanding the mutation detection of this gene in Chinese deafness population, to understand the mutation frequency of ABCC1 in Chinese deafness population and hot spot mutations; ② constructing ABCC1 wild-type and mutant over-expression plasmids and transfecting them to cells, to study in-vitro cytological changes; ③ constructing Abcc1 c.1769 A> G knock-in (KI) mice by CRISP/Cas9 technology, to identify the different phenotypes between wild-type mice and KI mice. Taking together, this project will help us to understand the ABCC1 induced deafness mechanism through human, cells, animal these three levels, to clarify pathological changes of ABCC1, and lays the foundation for the genetic diagnosis and treatment of hereditary hearing loss in future.

对收集的一个常染色体显性非综合征性耳聋家系，运用全外显子二代测序结合连锁定位分析，发现ABCC1基因一个杂合突变c.1769A>G与该家系耳聋患者共分离，进一步在其它耳聋家系和散发患者中也发现了该基因的突变，提示ABCC1可能是中国耳聋人群中一个新的致病基因。根据该基因研究背景和我们的前期研究推测：耳蜗血管纹转运功能异常可能与该基因突变致聋有关。本项目拟：①扩大该基因在中国耳聋人群中的检测，了解ABCC1在中国耳聋人群的发生频率和热点突变；②构建ABCC1野生型和突变型表达质粒转染细胞，研究体外细胞学功能改变；③用CRISP/Cas9技术构建该突变的基因敲入(KI)小鼠模型，鉴定野生型和KI小鼠耳聋表型差异，从组织病理、血管纹通透性及转运功能等方面探讨ABCC1致聋机制；从而在人体、细胞、动物三个层面明确该基因的致聋性，为下一步开展遗传性耳聋基因诊断及矫正治疗打下基础。

本课题以一个湖南常染色体显性遗传性耳聋大家系为研究切入点，从耳聋基因的筛查鉴定、细胞及动物实验三个方面分别进行耳聋基因致病性及致病机制的研究。课题组首先运用梯级遗传分析法，寻找并鉴定出耳聋新致病基因ABCC1，随后扩大了ABCC1基因在217例语后聋患者及200个听力正常人ABCC1全部编码区和剪接位点区的变异筛查；构建了ABCC1野生型和突变型c.1796A>G点突变的表达质粒，并比较了野生型和突变体的蛋白表达量、稳定性、亚细胞定位、转运功能；随后构建了Abcc1基因点突变和基因敲除两种小鼠，完成了不同年龄鼠的听力学表型和耳蜗形态学的鉴定工作，及在庆大霉素耳毒性中的作用。结果发现：ABCC1是耳聋新致病基因，突变后的mRNA表达水平下降、蛋白的转运能力下降，变异后的蛋白不仅表达在细胞膜上，在细胞质中也出现了表达；成功构建Abcc1点突变KI小鼠及基因敲除KO小鼠，两种小鼠在SPF级饲养环境下，都没有显示出明显的听力学及形态学改变，KO小鼠直至老年，外毛细胞才出现较严重的缺失，说明ABCC1基因可能与年龄相关的听力下降相关。同时Abcc1 KO鼠对于庆大霉素的耳毒性更易感，这与外排障碍、耳蜗内药物残留增加有关。从人体、细胞、动物三个层面，研究耳聋基因ABCC1致聋机制，为遗传性耳聋的基因诊断及治疗打下基础。

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