遗传性中频耳聋新致病基因SLC44A4的认定及致聋机制研究

There are few reports about non-syndromic inherited mid-frequency deafness gene. We collected one non-syndromic mid-frequency deaf family which is autosomal dominant hereditary, including four generations of 37 people. After ruling out the common deafness genes, we preliminarily determined gene SLC44A4 (NM_025257) with Met156Val A > G point mutation cosegregating with the disease on depth sequencing of 3 affected and 1 normal individual of the family, which was verified by bioinformatics analysis and Sanger sequencing. After down-regulating slc44a4 in zebrafish, marked changes in the hearing related phenotypes could be observed, indicating SLC44A4 is probably a novel deafness gene in this family. On this basis, we intend to carry out the following researches: (1) to detect SLC44A4 expression in more patients with sensorineural hearing loss, and clear the mutation rate; (2) to observe the changes of auditory organs and function in zebrafish after down-regulating slc44a4 expression and CRISP/Cas9 fixed point mutation; (3)to detect choline intake and acetylcholine synthesis in 293T and SH–SY5Y cells after mutation and down-regulated expression of SLC44A4 , and clarify the molecular mechanism of deafness with SLC44A4 mutations.

遗传性非综合征型中频耳聋致病基因报道较少。本课题组收集了一个4代37人的非综合征型常染色体显性遗传性中频耳聋家系，在排除了常见的耳聋基因后，对其中3例患者与1例正常人进行深度测序，经生物信息学分析及Sanger法测序验证，初步确定SLC44A4基因（NM_025257）Met156Val A>G点突变与疾病共分离；下调斑马鱼的slc44a4基因，听觉相关表型发生明显改变，提示该基因极有可能是一个新的耳聋致病基因。在此基础上，本项目拟开展以下研究：1.在更多的感音神经性耳聋患者中检测SLC44A4基因，明确其突变率；2.应用模式生物斑马鱼，观察slc44a4下调及CRISP/Cas9定点突变后，听觉器官形态功能变化；3.在293T和SH-SY5Y两种细胞中，检测SLC44A4突变及表达下调后细胞胆碱摄入与乙酰胆碱合成的变化，阐明SLC44A4突变导致耳聋的分子机制。

本课题组就一个相对罕见的4代37人的非综合征型常染色体显性遗传性中频耳聋家系展开研究，初步确定SLC44A4基因（NM_025257）Met156Val A>G点突变与疾病共分离；下调斑马鱼的slc44a4基因，听觉相关表型发生明显改变，提示该基因极有可能是一个新的耳聋致病基因。在此基础上，本项目在1000耳聋散发病例中检测到SLC44A4一个新的Gly171Arg G511C致病位点变异，进一步证明其突变可引发耳聋；应用模式生物斑马鱼，观察到slc44a4下调显著影响听觉器官形态功能，应用人源SLC44A4 mRNA可有效挽回吗啡啉整体下调slc44a4后斑马鱼听觉表型异常，证实该基因与听觉功能密切相关；建立Slc44a4内耳及神经系统条件性敲除的小鼠模型进一步研究了SLC44A4与听觉功能的关系及突变致聋机制；构建稳定表达野生和突变型的神经母细胞瘤细胞SH-SY5Y，发现与野生型比较表达突变型SLC44A4的细胞胆碱摄入与乙酰胆碱合成水平明显低下，在细胞水平初步探明 SLC44A4 突变使支配外毛细胞的内侧橄榄耳蜗束传出系统重要神经递质Ach 合成分泌下降，致使外毛细胞功能异常及死亡，最终导致听力下降的致病机制。