遗传性耳聋新致病基因THOC1的听觉功能及致聋机制研究

In a dominant family segregated with late-onset, progressive, non-syndromic hearing impairment, linkage analysis revealed a 1.4Mb candidate region on chromosome 18p11.3 that contains THOC1. Whole-exome sequencing identified a heterozygous p.L183V variant of THOC1 as the only candidate mutation segregating with the hearing loss within the family. Antibody immunostaining analysis showed THOC1 was highly expressed in mouse hair cells. Consistently, we observed that thoc1 was specifically expressed in the zebrafish developing neuromasts, particularly enriched in the hair cells. In the thoc1 mutants and morphants, the hair cell number in each neuromast and in the whole embryo is dramatically decreased. In addition, both human THOC1 mRNA and zebrafish thoc1 mRNA significantly rescued the hair cell defects caused by loss of Thoc1. While the mutated THOC1 (p.L183V) mRNAs showed remarkable less capacity to restore the hair cell defects. Furthermore we demonstrated that thoc1 deficiency induced hair cell apoptosis. In this study, knock-in mouse will be generated for functional studies of the causative gene THOC1 of nonsyndromic deafness. In addition, several type of technologies, including: biochemical analysis, molecular biological analysis, electrophysiological analysis, live imaging, etc., will be employed to elucidate the complex mechanism of the novel deafness THOC1caused by identified gene mutations. The studies in this project will increase our knowledge of the molecular bases of nonsyndromic hereditary hearing loss, and provide new opportunities for the diagnosis and treatment of this disease in the hearing loss population.

本课题组收集一个常染色体显性非综合征遗传性耳聋大家系SH，已将该家系致聋基因定位在常染色体18p11.3区域（最大LOD值4.93），候选区域位于rs4797697与rs11080868之间，约1.4Mb大小,该区域不包含已知耳聋基因；全基因组外显子测序联合连锁定位，发现了一个耳聋新致病基因及其突变位点:THOC1（NM_005131),c.C547G,p.L183V; 后续经直接测序证实该突变位点在SH家系中共分离；前期功能实验已证实THOC1蛋白表达于小鼠内耳的外毛细胞和内毛细胞；同时发现THOC1在斑马鱼神经丘的毛细胞中特异性的表达。接着我们建立了THOC1敲除与敲低的斑马鱼模型，发现THOC1缺失可以导致斑马鱼毛细胞的个数显著减少，促进斑马鱼神经丘中毛细胞异常的凋亡。我们拟构建THOC1p.L183V基因敲入小鼠模型，对THOC1及其突变进行深入的听觉功能研究，探索其具体发病机制。

本项目通过遗传性耳聋大家系的收集，利用连锁分析联合全基因组外显子测序鉴定了致聋基因Thoc1基因及突变位点，后续的Thoc1内耳功能研究通过构建动物模型（斑马鱼与条件性敲除小鼠模型）结合听觉生理学、内耳病理学及分子生物学等手段，对新耳聋致病基因Thoc1开展深入的功能研究，在分子水平上探讨了Thoc1基因相关的听觉功能和相关聋病发病机制，为遗传性耳聋的诊断、预防和治疗的个体化精准化奠定了坚实的理论基础。本研究预期目标分阶段按期实现，完成情况良好，利用CRISPR/Cas9系统构建了Thoc1基因突变的斑马鱼品系，首先发现Thoc1缺失导致斑马鱼毛细胞数量的显著减少，接着发现Thoc1基因突变斑马鱼毛细胞发生了凋亡，且与P53信号通路有关，发现Thoc1突变可激活P53信号通路导致毛细胞凋亡。在上述研究工作的基础上，进一步成功构建了条件性敲除的Thoc1基因flox小鼠，发现在P49-P63时 Thoc1 CKO小鼠ABR听力阈值逐渐升高，且差异逐渐增大，上述构建的Thoc1基因内耳条件性敲除小鼠的听力表型及其功能研究发现：Thoc1基因并不影响小鼠耳蜗毛细胞的发育，但影响成年小鼠耳蜗毛细胞的存活，Thoc1 基因功能缺陷可在小鼠中造成迟发性耳聋。由此模拟了人类耳聋大家系迟发性、随着年龄的增大进行性加重的听力表型。本研究首次发现了一个新的与毛细胞凋亡相关的关键基因Thoc1，研究证实Thoc1基因缺陷致聋机制本质上属于内耳毛细胞凋亡一类病变，而凋亡又与老年性耳聋密切相关，可以为老年性聋的防治提供了新的切入点，以上原创性的研究工作将为聋病的分子诊断、预防、新药物开发和基因治疗奠定坚实的理论基础，为尽早实现聋病的个体化精准防治寻找突破口，最终造福广大耳聋患者。共发表资金标注的 SCI 论文 3 篇。培养四名硕士研究生，其中一名硕士已顺利毕业。