迟发进展性遗传性聋致病基因SCD5的鉴定与功能分析

It is an important part of the pathogenesis study in hearing loss that the novel causing-gene of the late-set progressive hereditary hearing loss is cloned and studied. In the previous study, we have identified a mutation c.626G>C (p. W209S) in SCD5 gene in one large autosomal dominant late-set progressive hereditary hearing loss pedigree through SNP linkage analysis and the whole human exome sequencing and bioinformatics technique. The newly identified gene, SCD5, encodes stearoyl-CoA desaturase 5, which mainly expresses in human brains and is related with mouse Neuro2a cells' division and differentiation. But the potential role of mutated SCD5 in late-set progressive hereditary hearing loss remains unknown. In this study, we will investigate the expressing mRNA and protein product of SCD5 in cochlear and auditory nerves in different life stage, detect the altered function in Wnt signal transduction pathways that are known to modulate the processes of cell proliferation. Furthermore, a conditional cochlea SCD5 knockdown guinea pig model and an SCD5 c.626G>C mutation knockin guinea model will be made to study the hearing loss pathogenesis and SCD5 function. The lipo fraction and auditory physiology will be analized to see the difference among these animal models. Systemic phenotype, pathophysiology and biochemistry study will then be performed on the models. Thus it will be clarified that the pathogenesis of SCD5 gene mutations is responsible for late-set progressive hearing loss.

遗传性聋新基因的克隆与功能研究是耳聋发病机制研究的重要内容。本课题组前期工作在一大常显迟发进展性遗传性聋家系中发现SCD5基因新突变与表型共分离，蛋白结构预测为致病突变，SCD5表达蛋白硬脂酰辅酶A脱氢酶5是脂质代谢的关键酶，主要分布在脑组织，参与神经细胞的增殖分化和调控缝隙连接蛋白功能，提示SCD5突变可能导致迟发进展性遗传性聋。本项目在研究SCD5蛋白在内耳的表达分布和体外细胞模型证实该突变导致硬脂酰辅酶A脱氢酶5功能改变的基础上，拟构建SCD5基因表达选择性耳蜗敲低鼠模型和SCD5突变基因敲入鼠模型，检测SCD5在内耳和听觉神经通路的表达及突变鼠内耳细胞和听觉神经通路的形态学变化，听觉电生理改变，分析SCD5的相互作用分子及其功能，以及SCD5突变对内耳脂质代谢的影响，探索SCD5突变导致迟发进展性遗传性聋的分子机制，期望为阐明迟发进展性遗传性聋的发病机制提供思路。

SCD5基因和HOMER2基因突变均可引起常染色体显性遗传非综合征性耳聋，患者表现为迟发进展性聋。SCD5在豚鼠及人胎儿耳蜗螺旋神经节细胞和Corti器中表达，c.626G>C突变可抑制SCD5基因的促细胞增殖作用。本课题还研究了另一耳聋家系致病基因HOMER2的突变致病机制与听力表型的关系。通过构建HOMER2基因不同突变型表达载体，应用RT-PCR, 还原性、非还原性Western Blot，及免疫细胞荧光等技术，探讨HOMER2的突变对HOMER2多聚化功能的影响。应用CRISPR/Cas9基因编辑技术构建和人HOMER2基因c.840_841insC突变相对应的Homer2突变基因敲入模型小鼠。通过检测小鼠听力表型，及对小鼠耳蜗组织的研究，进一步在体内研究HOMER2基因在听觉通路中的作用及突变致聋的机制。实验研究发现HOMER2的插入突变导致蛋白截短，多聚化CC功能区及与CDC42结合功能区被破坏，蛋白表达量降低。截短的HOMER2蛋白的多聚化功能较野生型和已报道点突变型HOMER2蛋白显著降低。共聚焦显微镜观察三种HOMER2蛋白的亚细胞表达，发现野生型及已报道突变型HOMER2蛋白倾向于聚集成团分布，而该家系突变型HOMER2更倾向于弥漫性分布。与两家系的听力表型相符。HOMER蛋白多聚化是其发挥调控钙信号转导所必须的，蛋白多聚化功能的降低可能影响钙信号转导，进而影响耳蜗细胞功能导致耳聋。模型鼠相关实验尚在进行中，该部分实验的完成将会进一步揭示HOMER2基因在听觉中通路中的作用，及其突变导致耳聋的机制。本研究中的202家系是目前全球发现的第二个HOMER2基因突变导致的耳聋家系，该家系的发现充分佐证了HOMER2基因是人维持正常听力所必须的，丰富了耳聋基因谱。SCD5与HOMER2基因存在诸多共性，HOMER2相关研究的进行也有利于SCD5基因的致聋机制研究的推进。