新的家族性房间隔缺损致病基因的精细定位克隆及功能研究

Congenital atrial septal defect (CASD) is a common birth defect encountered in clinical practice, and is the leading etiology of birth defect-related deaths. Genetic defects are one of the important causes responsible for CASD. Therefore, based on the already obtained peripheral venous blood specimens from 56 families with CASD and 820 patients with idiopathic CASD, the present study aims to identify novel genes involved in the pathogenesis of CASD by direct PCR-sequencing of the candidate genes, genome-wide genotyping with Affymetrix gene chip of single nucleotide polymorphisms, or by sequencing of the positional candidate or newly cloned genes that are mapped by whole-genome scanning with microsatellite markers to genotype and make linkage analysis. The newly identified genes associated with CASD are cloned and transfected into the tool cells. The techniques of the reporter gene assays and electrophoretic mobility shift assays and others are used to explore the functional characteristics of the mutated genes. The sub-cellular distribution and trafficking of the mutated protein are investigated by laser scanning confocal fluorescence microscopy, thus revealing the mechanism of CASD ascribed to the identified culprit gene at cell level. The transgenic mouse model with myocardium-restricted overexpression of the mutated gene is generated by microinjection of the recombinant plasmid into the pronuclei of the fertilized oocytes and its cardiac phenotypes are examined by supersonic image and immunohistochemistry, hence demonstrating the mechanism of CASD at levels of animal organ and cardiac tissue. The findings not only contribute to reveal of novel mechanism implicated in CASD, but also help with the early diagnosis,prognostic appraisal, prenatal prophylaxis and the gene-specific therapy of CASD.

先天性房间隔缺损（CASD）是临床常见的出生缺陷和主要缺陷致死性疾病，遗传缺陷是其发生的重要原因。本研究拟在既得56个CASD家系和820例散发CASD患者基础上，运用候选基因策略直接PCR-测序筛查，应用Affymetrix基因芯片全基因组SNP分型或通过微卫星标记全基因组扫描、基因分型、连锁分析先进行基因定位，再测序筛查定位候选基因或新克隆的基因，以发现并克隆新的CASD致病基因。通过转染工具细胞，利用报告基因分析、电泳移动性分析等手段研究突变基因功能特性，应用激光共聚焦显微镜等研究突变体亚细胞定位分布，在细胞水平研究新CASD致病基因作用机制。通过受精卵显微注射生产心肌特异性表达CASD转基因小鼠模型,在整体器官及组织水平研究其致病作用机制。研制基于新突变的CASD基因诊断芯片。这不仅有助于揭示CASD发生的新机制，也有助于CASD患者早期诊断、预后评估、优生优育和基因特异治疗。

先天性房间隔缺损（CASD）是临床上常见的出生缺陷和主要的缺陷致死性疾病，遗传缺陷是其发生的重要原因。本研究在已经获得56个CASD家系和820例特发性CASD患者的基础上，运用候选基因策略直接PCR-测序筛查，应用Affymetrix基因芯片全基因组SNP分型或通过微卫星标记全基因组扫描、基因分型、连锁分析先进行基因定位，再测序筛查定位候选基因或新克隆的基因，以发现新的CASD致病基因。克隆新的CASD基因，转染工具细胞，利用报告基因分析、电泳移动性分析等手段研究突变基因的功能特性，应用激光共聚焦显微镜等研究突变体的亚细胞定位和分布，在细胞水平研究新的CASD致病基因的作用机制。通过受精卵显微注射生产心肌特异性表达CASD转基因小鼠模型,在整体器官及组织水平研究其致病作用及机制。.本研究发现了CASD相关基因的多个新突变如GATA4基因p.P36S、p.H190R、p.S262A和p.V399G等突变，分别发现于4例散发性ASD患者中。这些突变不存在于健康对照者，在人类SNP数据库中也没有报道。多物种GATA4序列比对显示这些被改变的氨基酸在进化上高度保守。对突变携带者的家系成员进行遗传分析表明在每个家系突变与ASD共分离。针对这些新突变的功能研究显示，突变蛋白的转录活性显著降低，通过“功能缺失性”效应影响心脏发育，导致CASD。这些研究结果不仅扩大了先天性心脏病相关基因的突变谱，也揭示了CASD新的分子机制，因而有助于CASD的早期诊断、预防咨询、危险分层、预后评估和基因特异性治疗。