应用小鼠模型研究一新耳聋基因Tmem132e在遗传性耳聋发生中的作用机制

Hearing loss (HL) is the most common birth defect in developed countries. At least 50% of persons with HL results from genetic factor.Inherited HL are almost always monogenic. 70% of inherited HL are nonsyndromic, with HL being the only disorder. NSHL is inherited mainly(80%) in an autosomal recessive nonsyndromic hearing loss(ARNSHL). In our previous studies, a consanguineous family with ARNSHL from Shandong province was investigated by our research team and located to 17q11.2-12(DFNB99)by performing homozygosity mapping and linkage analysis. Furthermore, by using Exome sequencing, one missense mutation(G1259A) in TMEM132E gene was detected. This mutation cosegregated with HL in this family and was not present in controls. The mainly expression of TMEM132E in hair cell of the cochlea is observed in mouse by using immunofluorescene. In addition, knockdown of Tmem132e in zebrafish resulted in impairments in mechanotransduction of hair cells. Co-injection of human wild-type TMEM132E mRNA,rather than the mutant TMEM132E mRNA, could rescue the phenotypes. These results strongly suggest that mutation in TMEM132E is responsible for DFNB99. As the function of TMEM132E remains obscure, it is optimal to elucidate its role in the auditory system in the mouse models which have provided an invaluable tool for studying advanced hearing mechanisms in a way that could not have been achieved only by human studies. We will create conditional knockout mouse of Tmem132e gene by cross-mating Tmem132e flox/flox mouse with Foxg1 Cre/+ mouse in which Cre mainly express in the iner ear. The hearing in the knockout mouse will be evaluatd with measuring auditory brainstem response and endocochlear potential.Vestibular defect will be assessed by swimming and reaching response. Inner ear development and defects will be followed by using HE staining of cochlear frozen section, cochlear whole mount immunohistochemistry, scanning and transmission electron microscopy , as well as by paintfill assay. 4-Di-2-ASP uptake of whole cochleae and the whole cell patch clamp recording will be used to examine mechanosensory function in hair cell.The results obtained above will be confirmed by performing rescue test with viral expression vectors encoding human wild type or mutant TMEM132E. At last, it will be helpful to elucidate the role of Tmem132e gene in the inner ear and the pathologic mechanisms of deafness caused by TMEM132E gene mutation .

耳聋是导致语言交流障碍的最常见疾病，遗传因素导致的耳聋超过50%。分离鉴定新的耳聋基因，阐明耳聋发生的分子机制，将有助于我们制定防聋措施和治聋方案。本课题组在前期研究中发现一个新的、功能未知的基因Tmem132e突变导致DFNB99,且基因功能与毛细胞结构和功能相关。本项目拟通过构建Tmem132e基因内耳条件敲除小鼠，采用听觉脑干诱发电位测定、耳蜗内电位检测、基底膜铺片免疫组化、扫描电镜、透射电镜和膜片钳全细胞记录等方法，分析Tmem132e基因敲除对内耳尤其是毛细胞形态结构和功能的影响，并通过病毒介导的拯救实验加以验证，从而阐明Tmem132e基因在内耳中的作用和致聋机制，同时初探基因治疗的有效性。

耳聋是导致语言交流障碍的最常见疾病。分离鉴定新的耳聋基因，阐明耳聋发生的分子机制，将有助于我们制定防聋措施和治聋方案。本课题组在前期研究中发现一个新的、功能未知的基因 Tmem132e 突变导致 DFNB99,且基因功能与毛细胞结构和功能相关。. 本项目通过与专业的动物模型制作实验室合作得到 Tmem132e flox/+ 小鼠，雌雄交配得到Tmem132e flox/flox 小鼠,然后分别与Atoh1-Cre、Pax2-Cre和 Sox2-Cre小鼠交配，得到毛细胞敲除的Atoh1-Cre+/-；Tmem132eflox／flox小鼠、整个内耳敲除的Pax2-Cre+/-；Tmem132eflox／flox 小鼠和全身敲除的Sox2-Cre+/-；Tmem132eΔ／ Δ小鼠。对以上3种小鼠听性脑干反应(ABR)检测发现仅有Sox2-Cre+/-；Tmem132eΔ／ Δ小鼠听力严重受损，其他两种小鼠模型无听力改变，证实TMEM132E基因是耳聋基因，但其机制可能不完全限制于内耳耳蜗、螺旋神经节，可能也与听力产生其它环节有关。同时采用基底膜铺片结合共聚焦显微镜分析蛋白在毛细胞中亚定位情况，认为该蛋白不在毛细胞纤毛束表达而是细胞质内。进一步利用斑马鱼模型研究Tmem132e的功能。采用膜片钳技术结合配置浸入式镜头的显微镜，对5dpf的MO和MC斑马鱼进行微音电位的测定，发现MO斑马鱼的微音电位显著降低；而对斑马鱼耳囊显微注射荧光物质，发现耳囊中毛细胞丧失对该物质的摄取，从而提示耳囊中Tmem132e基因对毛细胞的机械-电传导功能有影响，可能是致聋机制之一。对TMEM132E基因在耳聋机制中的研究将有助于我们对听力机制的了解，为耳聋的预防治疗提供理论基础。.