模拟人DFNA64型耳聋Smac/DIABLO小鼠模型的建立及致聋机制研究

Hearing Loss is a common disease causes human disability. The identification of the genes underlying hereditary hearing loss and to decipher its mechanism will be important to prevent the recurrence of hearing loss. This research field has always had originally innovative achievements. By linkage analysis and candidate genes screening, the applicant firstly identified a heterozygous Smac/DIABLO mutation in a six-generation Chinese kindred characterized of dominant progressive non-syndromic hearing loss, designated as DFNA64. With several cell models, our group used a functional study approach to demonstrate that the Smac/DIABLOS71L mutant triggers significant degradation of both wild type and mutant Smac/DIABLO, rendering host mitochondria susceptible for calcium-induced membrane potential loss. These results were published on Am.J.Hum.Genet., 2011 (IF=11.68, the applicant was the first author). However, it remains to be determined which degradation pathway is activated by SmacS71L. In this proposal, we are going to establish a mouse model that expresses a mutant allele SmacS71L in SmacWT background to study the accumulative effects and progressive hearing loss. This study will evaluate the possibility and the security of utilizing P7C3 like mitochondria protective compound for potential therapeutic benefit to protect hair cell mitochondrial membrane potential and attenuate the progression of hearing loss in DFNA64 individuals. This would represent a new direction for possible intervention of progressive hearing loss among the general deaf population.

耳聋是严重高发的致残性疾病。新致聋基因的发现及其致聋机制的诠释在耳聋防治研究领域一直占据着重要的源头创新地位。在前期工作中，本课题申请人将一个中国耳聋巨大家系定位于DFNA64新位点，并在国际上首次报道了DFNA64型耳聋的致病基因Smac/DIABLO，发现SmacS71L 突变体可引起细胞内野生型和突变型Smac大量降解，引发线粒体膜电位慢性损伤，最终导致内耳毛细胞死亡，初步研究结果发表于Am. J. Hum. Genet，2011（影响因子11.68，申请人为第一作者）。本申请课题拟建立模拟导致人类DFNA64型耳聋的SmacS71L 突变的knock-in小鼠模型，深入研究Smac/DIABLO的致聋机制，评估线粒体膜电位保护性药物对DFNA64型耳聋症状前预防作用的有效性和安全性，为遗传性耳聋的诊断治疗与药物设计提供理论依据，为迟发性耳聋群体的药物干预研究开辟新的方向。

SMAC/DIABLO是本课题组在一个常染色体显性遗传非综合征型耳聋家系中定位的新的耳聋基因，并确定了该家系的致病突变（c.377C>T，p.S126L）。SMAC/DIABLO 是线粒体内膜上非常重要的细胞凋亡促进蛋白，前期研究提示，p.S126L 突变体相对正常对照对于外界刺激更加敏感，可引发线粒体膜电位慢性损伤。我们成功构建了Diablo基因的knock-out小鼠以及模拟人相同突变的knock-in小鼠模型。对突变型及野生型小鼠进行听觉脑干反应和耳蜗石蜡切片的HE染色，在突变型和野生型小鼠间均未发现具有显著差异。然而，在已知的34个DFNA基因中，有17个基因构建了相关的动物模型，仅有6个基因有听力表型的异常。因此，我们的工作显示，动物模型并不能很好的复制人的相关表型变化，需要探索新的研究方案对SMAC/DIABLO的致病机制进行研究。

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