房颤相关 Connexin 基因突变的识别及功能分析

Since the applicant and colleagues identified the first 2 genes associated with atrial fibrillation, identification of the genes responsible for atrial fibrillation has been the hot research topic for the scholars from various countries. The cardiac gap junction channels assembled by connexins play important roles in the cardiac electrophysiology and the mutations of the connexin genes derived from atrial somatic cells may underlie atrial fibrillation. However, to date the associations of the germline connexin mutations with atrial fibrillation remain to be established. Recently, our research group found that germline connexin40 mutations may lead to atrial fibrillation by distinct molecular mechanisms, which suggests us to make a new hypothesis that germline connexin mutations are an important cause of atrial fibrillation. In order to substantiate the hypothesis, based on the original work this study will firstly aim to identify novel mutations of the connexin genes responsible for atrial fibrillation by sequencing all the 3 atrium-expressed connexin genes in patients with atrial fibrillation; secondly the identified connexin genes potentially implicated in the pathogenesis of atrial fibrillation will be cloned and functionally characterized by dual patch clamp; thirdly the subcellular distribution and trafficking of the mutated connexin proteins will be explored by laser scanning confocal fluorescence microscopy; finally the transgenic mouse model with myocardium-restricted overexpression of the mutated connexin gene will be generated by microinjection of the recombinant expression plasmid into the pronucleus of fertilized mouse oocytes and then be examined electrophysiolgically. Thereby the mechanism by which the identified culprit gene gives rise to atrial fibrillation will be demonstrated at both cell and animal levels. The findings will not only be of great original significance in revealing the molecular genetic basis and providing novel insight into the mechanism of atrial fibrillation, but will also likely make real contribution to the discovery of new drug target for the treatment of atrial fibrillation.

自从申请人及同事首先发现房颤相关基因以来，房颤致病基因的识别一直是各国学者研究的热点。心脏connexin（Cx）蛋白构成的缝隙连接通道在心脏电活动中具有重要作用，心房体细胞Cx基因突变可导致房颤。但生殖细胞Cx基因突变与房颤的关系目前仍待研究。最近本课题组发现生殖细胞源Cx40基因突变可通过不同的分子机制导致房颤，于是提出"生殖细胞源Cx基因突变是房颤的重要病因"这一新的假说。为此本研究拟在这些原创性工作的基础上，在房颤患者中测序筛查心房表达的全部3个Cx基因，以识别房颤相关Cx基因突变。克隆所识别的Cx基因，应用双膜片钳研究突变蛋白的功能特性，借助激光共聚焦显微镜分析突变蛋白的亚细胞分布，通过显微注射受精卵建立转基因小鼠模型，在细胞和整体水平研究该基因的作用机制。这不仅对于揭示房颤的分子遗传学基础，推动对房颤发生机制的认识具有重要原创意义，而且可能为发现新的药物治疗靶标做出现实贡献。

自从申请人及同事首先发现房颤相关基因以来，房颤致病基因的识别一直是各国学者研究的热点。心脏connexin（CX）蛋白构成的缝隙连接通道在心脏电活动中具有重要作用，心房体细胞CX基因突变可导致房颤。但生殖细胞CX基因突变与房颤的关系目前仍待研究。最近本课题组发现生殖细胞源CX40基因突变可通过不同的分子机制导致房颤，于是提出“生殖细胞源CX基因突变是房颤的重要病因”这一新的假说。为证实该假说，本研究拟在这些原创性工作的基础上，在房颤患者中测序筛查心房表达的全部3个CX基因，以识别房颤相关CX基因突变并揭示其致病机制。结果发现了多个房颤相关生殖细胞源CX40和CX45基因突变，功能研究表明这些突变均导致相应的CX蛋白所构成的缝隙连接通道的功能障碍。另外，运用直接候选基因策略还发现了多个房颤相关PITX2c、NKX2-5、NKX2-6和TBX5等转录因子基因突变，功能分析发现这些突变均导致相应蛋白的转录激活作用显著降低或完全丧失。这些研究结果不仅对于揭示房颤的分子遗传学基础，推动对房颤发生机制的认识具有重要原创意义，而且可能为发现新的药物治疗靶标做出现实贡献。.此外，还发现了转录因子基因GATA4、GATA5、GATA6、NKX2-5、PITX2、NKX2-6、HAND1、HAND2、TBX1、TBX5、TBX20和CASZ1功能缺失性突变可导致多种类型的先天性心脏病；转录因子基因GATA4、GATA5、GATA6、TBX5、TBX20、HAND1、NKX2-5和CASZ1功能缺失性突变可导致扩张性心肌病。这些研究结果不仅揭示先天性心脏病和扩张性心肌病的部分分子机制，也表明房颤、先天性心脏病和扩张性心肌病可能具有共同的心脏异常发育基础。