神经嵴异常相关综合征型耳聋的遗传因素分析及其致病机制研究

Over 400 types of syndromic hearing loss (SHL) has been reported now. However, it is difficult to study the genetic basis and pathogenesis of SHL systematically, because of the strong clinical and genetic heterogeneity of SHL.The incidence of most SHL was low. Waardenburg syndrome (WS), Congenital microtia syndrome, and Large vestibular aquduct syndrome (LVAS) are three common SHL in clinical. The dysfunction of neural crest cells caused by the gene interaction network extending from SOX10 and PAX3 not only could lead to WS , but also involved in multiple craniofacial syndrome including microtia, which indicated that there was a commonality in the pathogenesis of WS and microtia syndrome. Interestingly, our previous studies also confirmed the interaction of PAX3, SOX10 and MITF in the pathogenesis of WS. The hypothesis of this study is that the neural crest gene interaction network might play an important role in the SHL phenotypes including pigment abnormalities, microtia and expanded vestibular aqueduct. Based on the hypothesis, we will collect pedigrees and Sporadic cases related to the phenotypes mentioned above,and Exome-sequencing will be performed to gene mapping and cloning of the common SHL in this study. And then, we will try to find out the potential common pathway and pathogenic mechanisms of the neural crest abnormal gene interaction network involved in the syndromic deafness phenotypes mentioned above at the molecular and cellular(induced pluripotent stem cells (iPSCs) technology) levels, respectively. Our study will enrich the molecular genetic background of SHL, provide new ideas for studies of SHL, and lay the research foundation for further exploration of the new intervention strategies of SHL.

综合征型耳聋（SHL）现已报道400多种，其极强的临床和遗传异质性使得系统地研究其遗传基础及发病机制十分困难。SHL绝大部分少见，临床常见的有Waardenburg综合征（WS）、先天性小耳畸形综合征、前庭导水管扩大综合征等。以SOX10与PAX3为中心的基因作用网络引起的神经嵴细胞功能异常与WS、小耳畸形及前庭导水管扩大等表型相关。本课题组的前期研究也证实该基因网络参与WS的发病机制。本研究拟以神经嵴异常相关基因网络可能参与上述SHL表型的发病机制作为研究假设，收集上述表型的家系及病例，使用全外显子测序定位克隆SHL新基因，从分子和细胞水平（诱导多潜能干细胞（iPSCs）技术）系统的研究导致SHL的共同通路和致病机制。本研究将丰富SHL的分子遗传背景，为SHL的系统研究提供新思路并为进一步的干预治疗奠定研究基础

本课题以 SOX10与 PAX3为中心的与神经嵴细胞的迁移和分化有关的基因作用网络为理论指导依据，寻找并克隆综合征型耳聋新致病基因或者新发突变，解释PAX3、SOX10 和新发致病基因如何通过影响神经嵴细胞的发育和迁移并最终致聋的基本分子机制。本课题在研究过程中继续通过门诊、住院患者及其他途径扩大收集国人神经嵴异常相关综合征型耳聋患者家系及散发病例，并利用直接测序、高通量二代测序及拷贝数变异检测等多种方法对国人该类综合征型耳聋患者进行有针对性的突变筛查和相关体外功能研究。本研究团队创造性的采用基于Illumina® Miseq测序平台的高通量二代测序技术，分别针对Waardenburg综合征的6 个致病基因（MITF、SOX10、PAX3、EDN3、EDNRB、SNAI2）和前庭导水管扩大（EVA）的3个致病基因（SLC26A4、FOXI1及KCNJ10）建立了目标区域的高通量测序体系及CNVplexTM高通量拷贝数检测体系，并研发出WS 与EVA两个综合征型耳聋基因诊断试剂盒。我们以色素型综合征型耳聋为主要研究对象，通过一系列体外细胞实验，从分子和细胞水平探索研究神经嵴异常相关综合征型耳聋的共同通路和致病机制，通过该类耳聋相关的基因MITF分别与IRF4和LEF1如何协同调控TYR基因进行了相关体外实验研究：MITF与IRF4、LEF-1协同激活TYR基因表达的相关功能实验。对直接测序法发现WS和EVA新发突变并进行了相关功能试验验证；随后建立了携带有 WS 和EVA患者特异性遗传背景信息的iPSCs。本课题通过研究新鉴定的 SHL 致病基因突变与神经嵴细胞迁移分化有关的致病性，研究致病基因或者基因突变致病的分子机制；同时也为今后的干预治疗奠定前期研究基础。