心肌袖发育关键基因PITX2c突变导致房颤的机制研究

Atrial fibrillation is the most common type of cardiac arrhythmia encountered in clinical practice and is associated with substantially increased morbidity and mortality. Genetic defects are one of the important causes responsible for atrial fibrillation. In previous studies, the current research group mapped a locus susceptible to atrial fibrillation to chromosomal 4q25, and identified multiple gain-of-function mutations by analysis of the PITX2c gene, which is located on chromosomal 4q25 and key to the development of myocardial sleeves, in patients with atrial fibrillation. Considering the important role of PITX2c in the cardiovascular development, the applicant makes a new hypothesis that PITX2c gain-of-function mutations are an important cause underlying atrial fibrillation. In order to substantiate the hypothesis, based on the established genetic resource bank for atrial fibrillation, this study is aimed to identify novel PITX2c mutations associated with atrial fibrillation. The identified PITX2c mutants will be cloned and their appropriate expressing plasmids will be constructed. The recombinant plasmids will be transfected into tool cells. The transcriptional activity of the mutated proteins will be explored by using a luciferase reporter assay system for promoter activity, then the binding ability of the mutated proteins to the target promoter will be investigated by electrophoretic mobility shift assay, and the sub-cellular distribution of the mutated proteins will be observed and analyzed under laser scanning confocal microscopy, thus revealing the mechanism at cell level. The transgenic mouse model with myocardium-specific expression of the mutant PITX2c gene will be generated by microinjection of mutant PITX2c-expressing plasmid into the pronuclei of fertilized oocytes and will be studied systemically, so unraveling the mechanism at animal level. These findings in theory will provide novel insight into the mechanism involved in the pathogenesis of atrial fibrillation, and in practice will potentially provide new molecular target for the effective treatment of atrial fibrillation.

房颤是临床上最常见的心律失常，可显著增加患者的病残率和病死率，遗传缺陷是其重要原因之一。本课题组在前期研究中将一房颤易感基因座定位于染色体4q25区域，在房颤患者中对该区域心肌袖发育关键基因PITX2c进行分析发现多个功能获得性突变。鉴于PITX2c在心血管发育方面的重要作用，申请者提出"PITX2c功能获得性突变是特发性房颤的重要病因"这一新的假说。为了证实该假说，本研究拟基于已经建立的房颤遗传资源库识别房颤相关新的PITX2c基因突变，克隆所识别的突变型PITX2c基因，构建其表达载体，转染工具细胞，应用启动子活性分析、电泳移动性分析和蛋白质示踪等技术研究突变蛋白的转录活性、结合力和亚细胞定位，在细胞水平揭示该基因的致病机制；通过显微注射受精卵建立转基因动物模型，在整体水平研究该基因的作用机制。该项目的成功实施，将在理论上揭示房颤发生的新机制，在实践上为房颤的有效防治提供新的分子靶标。

房颤是临床上最常见的心律失常，可显著增加患者的病残率和病死率，遗传缺陷是其重要原因之一。本课题组在前期研究中将一房颤易感基因座定位于染色体4q25 区域，在房颤患者中对该区域心肌袖发育关键基因PITX2c进行分析发现多个功能获得性突变。鉴于PITX2c在心血管发育方面的重要作用，我们在已经建立的房颤遗传资源库中识别了房颤相关新的PITX2c基因突变c.619A>G，即p.M207V突变。多物种PITX2c蛋白之氨基酸序列比对分析显示，突变位点的蛋氨酸在物种进化上完全保守，提示该氨基酸具有重要的功能作用。构建了野生型PITX2c基因的真核表达载体，发现M207V突变型PITX2c的转录激活作用较野生型的显著增强，显著增加CX40、CX43、SCN1B、KCNQ1、KCNH2和SCN5A的表达，增加了房颤的易感性。