基于深度突变扫描技术的遗传性耳聋GJB2基因错义变异功能研究

随着基因检测在临床上的广泛应用，新发现的临床意义不明变异(VUS)数量持续增长，成为阻碍临床诊断及干预的瓶颈。在我国，GJB2为遗传性耳聋最主要的致病基因之一，大量错义变异功能未知；其错义变异可导致非综合征型耳聋或综合征型耳聋，造成不同类型疾病的分子机制尚不明确。我们课题组综合运用合成生物学、CRISPR/Cas9基因编辑和高通量测序等手段建立了深度突变扫描技术，并成功应用于临床重要基因的高通量变异功能研究。在此基础上，本研究将构建包含GJB2基因所有可能错义变异(4294种)的饱和突变体文库，并通过诱导细胞凋亡、蛋白稳定性和半通道通透性三个实验系统性地评估GJB2基因错义变异对缝隙连接蛋白装配、运输以及通透性的影响。本项目将系统性阐明GJB2错义变异导致不同类型耳聋的分子机制，并大幅提升GJB2错义变异的解读能力，以期达到解决VUS解读困难的瓶颈问题，具有重要的科学意义及临床应用价值。

With the wide application of genetic tests in the etiological diagnosis of inherited disease, more variants of uncertain significance (VUS) have been found, hindering clinical diagnosis and intervention. In China, the most frequent cause of inherited deafness are pathogenic variants of GJB2 (Gap Junction Protein Beta 2) and a large number of missense variants are not functionally determined. In addition, GJB2 missense variants cause not only non-syndromic deafness but also syndromic deafness accompanied by skin diseases. The molecular mechanism of different phenotypes caused by different GJB2 missense variants is largely unknown. By using synthetic biology, CRISPR/Cas9 gene editing, and high-throughput sequencing technologies, we established deep mutational scanning (DMS) technology in our lab and successfully apply it to the massive functional variant classification of a clinically actionable gene. We will construct a saturated library of all possible 4294 GJB2 missense variants, design three functional assays to evaluate whether missense variants induce cell apoptosis, change protein stability and hemichannel permeability, and systematically study the effects of all GJB2 missense variants on the assembly, transportation, and permeability of gap junctions. This study has both scientific and clinical values, as it greatly improves the interpretation of GJB2 variants by solving the challenges of VUS interpretation, and reveal the molecular mechanism of different types of deafness caused by different GJB2 missense variants.