DIAPH1基因拷贝数变异导致遗传性耳聋的分子机制研究

Hereditary hearing loss is one of the most common disorders in Otolaryngology. According to previous study, 130 genes had been reported relating to hereditary hearing loss. The mutations of the human genome can mainly be divided into 2 parts: point mutations and structural variations. However, Copy Number Variations (CNVs) account for most of the structural variations and are with much more higher frequency than point mutations. Prior studies mainly focused on point mutations whereas Copy Number Variations were almost overlooked. In this study, the DIAPH1 knocked-out C57BL/6J mouse would be used to build up different types of CNVs models with different kinds of virus vectors, the effects from CNVs on the supporting cells, hair cells, stria vascularis and lateral wall cells in the inner ear would also be detected by the study of morphology; the study on electrophysiology would be used to detect the effect from CNVs on cell membrane potential. The treatment effect would be observed on the CNVs mouse model after the adenoviral vectors with different CNVs are ejected in. The molecular mechanisms will be explored at the different levels of the gene, protein, cell, tissue, organ and whole body by both in vitro and in vivo methods. The results will provide the theory basis for the diagnosis before the human embryo implantation, antenatal diagnosis and the genetic counseling. This study will potentially contribute to reducing birth defects caused by CNVs and also provide the theoretical basis for gene therapy of CNV-associated deafness in future.

遗传性耳聋是耳鼻喉科最常见的疾病之一。现已报道有130多种基因与遗传性耳聋相关。但以往研究主要集中在基因点突变，而结构性变异对耳聋的影响在国内外均罕有报道。在前期研究中本课题组已利用二代测序研究了130个耳聋基因在中国散发耳聋患者中拷贝数变异分布，发现DIAPH1基因拷贝数变异频率最高。本项目拟基于DIAPH1基因敲除C57BL/6J小鼠，利用病毒载体表达技术构建具有不同拷贝数DIAPH1基因小鼠模型，通过形态学方法，研究该基因拷贝数变异对小鼠耳蜗支持细胞、毛细胞等结构的影响；利用电生理学实验，研究DIAPH1基因拷贝数变异对小鼠耳蜗细胞膜电位的影响；利用过表达系统，评估不同拷贝数腺病毒载体对疾病模型的治疗效果。本项目拟从基因、蛋白、细胞及动物水平阐述DIAPH1基因拷贝数变异的致聋机制，为拷贝数变异相关的胚胎植入前诊断、产前诊断及遗传咨询提供依据，为遗传性耳聋的治疗奠定理论基础。

背景:.遗传性耳聋是耳鼻喉科最常见的疾病之一，明确新的耳聋基因及新的突变位点功能对防聋治聋至关重要。 .主要研究内容、重要结果及关键数据:.第一个家系中我们同时发现GJB2基因新的点突变（chr13:20763656 T>G）及USH2A基因第六个外显子发生的拷贝数变异(chr1: 215820813-215821122)。.第二个家系发现了一对双胞胎线粒体大片段缺失的突变，对两名KSS（Kearns Sayre Syndrome）患者的mtDNA进行了测序分析，发现其线粒体DNA中存在大片段缺失（A large 6, 600bp mtDNA deletion, ranging from position ChM 8, 702 to 15, 302）。.第三个家系是常染色体显性遗传耳聋家系，经过全外显子测序分析，我们最终确定PLAT、LCE1E、MCM4、PHGDH、ICOSLG、NBPF10、NBPF10、KRTAP5-1、MUC6、MUC5AC、PRAMEF25、MUC3A、CNTNAP3B为可疑致病基因。以上基因均为未报道过的新基因，还需进一步缩小致病范围。.第四个家系为非综合征型常染色体显性致病的耳聋家系，现已排除已知基因致病郑使用全外显子测序方法进行检测。.对中国散发感音神经性耳聋患者常见耳聋基因分析的研究结果与以往的报道的GJB2、SLC26A4及线粒体 12SrRNA的发病率不同，根据发现致病突变的概率进行排序，分别为 GJB2、DSPP、SLC26A4、COCH、ESRRB、MYO6、SOX2、TMPRSS3 及 WFS1。 .利用CRISPR/Cas9技术进行diaph1基因大片段敲除小鼠模型的构建。.科学意义：.已知致病基因分子遗传学筛查提示这四个家系致病基因为非已知致病基因，可能为新基因突变或多基因协同致病。.散发病例的研究为耳聋基因检测突变分布特征及耳聋基因的筛查提供了新的科学的参考依据。.DIAPH1基因拷贝数变异的致聋机制的研究，为拷贝数变异相关的胚胎植入前诊断、产前诊断及遗传咨询提供依据，为遗传性耳聋的治疗奠定理论基础。