房颤易感基因HCN2的定位克隆与功能研究

Atrial fibrillation is the most common form of sustained cardiac arrhythmia, accounting for substantially increased morbidity and mortality. Genetic defects are one of the major causes responsible for atrial fibrillation. In previous studies,the present research group mapped a novel locus susceptible to atrial fibrillation on human chromosome 19p13.3 by genome-wide scan with microsatellite markers,genotyping and linkage analysis. Sequence analysis of the positional candidate genes in this chromosomal region in the families with atrial fibrillation revealed mutations in the HCN2 gene. Based on the important role of HCN2 in the cardiac electrophysiology,the applicants proposed a scientific hypothesis that HCN2 is likely to be a novel gene predisposing to atrial fibrillation. In order to substantiate the hypothesis, based on the original work mentioned above the current study will firstly aim to identify novel HCN2 mutations associated with atrial fibrillation by sequencing the HCN2 gene in patients with atrial fibrillation; secondly the identified HCN2 gene potentially implicated in the pathogenesis of atrial fibrillation will be cloned and functionally characterized by patch clamp; thirdly the subcellular distribution and trafficking of the mutated HCN2 protein will be explored by laser scanning confocal fluorescence microscopy; finally the transgenic mouse model with myocardium-restricted overexpression of the mutant HCN2 gene will be generated by microinjection of the recombinant expression plasmid into the pronucleus of fertilized mouse oocytes and then be investigated electrophysiolgically. Thereby the mechanism by which the identified culprit gene gives rise to atrial fibrillation will be unveiled at both cell and animal levels. The findings will not only be of important 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.

房颤是最常见的持续性心律失常，可显著增加患者的病残率和病死率，遗传缺陷是其重要病因之一。本课题组在前期研究中通过对房颤家系进行微卫星标记全基因组扫描、基因分型和连锁分析定位了1个新的房颤易感基因座19p13.3，在房颤家系对该染色体区域的候选基因基因进行测序分析发现了HCN2基因突变。基于HCN2在心脏电生理方面的重要作用，申请者提出"HCN2可能是新的房颤易感基因"这一科学假说。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HCN2以识别新的房颤相关HCN2基因突变；克隆所识别的HCN2基因，应用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，推进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。

房颤是最常见的持续性心律失常，可显著增加患者的病残率和病死率，遗传缺陷是其重要病因之一。本课题组在前期研究中通过对房颤家系进行微卫星标记全基因组扫描、基因分型和连锁分析定位了1个新的房颤易感基因座19p13.3，在房颤家系对该染色体区域的候选基因基因进行测序分析发现了HCN2基因突变。基于HCN2在心脏电生理方面的重要作用，申请者提出“HCN2可能是新的房颤易感基因”这一科学假说。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HCN2以识别新的房颤相关HCN2基因突变并揭示其致病机制。结果发现了多个房颤相关HCN2基因突变，功能研究表明这些突变均导致相应的HCN2蛋白所构成的钾离子通道的功能障碍。另外，运用直接候选基因策略还发现了多个房颤相关PITX2c、SHOX2和TBX5等转录因子基因突变，功能分析发现房颤相关PITX2c突变导致相应蛋白的转录激活作用显著增强，而房颤相关SHOX2和TBX5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，而且有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ISL1、GATA6、MEF2C、NR2F2、GATA4、TBX1、NKX2-5、TBX20、HAND1、MESP1、HAND2、PITX2和CASZ1功能缺失性突变可导致多种类型的先天性心血管疾病；转录因子基因ISL1、HAND2、ZBTB17、MEF2C、CASZ1、NKX2-5、HAND1、TBX5和TBX20功能缺失性突变可导致扩张性心肌病。这些研究结果不仅仅揭示了先天性心脏病和扩张性心肌病的新的分子遗传学机制，也表明一部分房颤、先天性心脏病和扩张性心肌病可能具有相同的心脏异常发育基础。