先天性心脏病（CHD）是人类最常见的发育畸形和婴幼儿非感染性死亡的最常见病因，主要由遗传缺陷所致。本课题组在前期研究中通过对CHD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CHD基因座17q21.3，对该染色体区域的候选基因进行测序分析发现了CX45突变。基于CX45在心脏发育方面的关键作用及突变型CX45功能障碍，申请者提出“CX45可能是新的CHD致病基因”这一科学假说。为证实该假说，本研究拟继续在CHD患者中筛查CX45新突变；克隆CX45基因，构建其表达载体，借助激光共聚焦显微镜分析突变蛋白的亚细胞分布，分别应用双膜片钳技术和荧光染料转移技术研究突变缝隙连接通道的电生理特性和选择性通透性。这将不仅揭示CHD新的分子遗传学基础，还可能发现新的药物治疗靶标。

Congenital heart disease (CHD) represents the most common form of developmental abnormality in humans, and is the most common non-infectious cause of infant death. CHD is predominantly attributable to genetic defects. In recent studies, the present research group mapped a novel locus causally linked to CHD on human chromosome 17q21.3 by genome-wide scan with microsatellite markers, genotyping and linkage analysis. Sequence analysis of the candidate genes within this chromosomal region in the families with CHD led to identification of the CX45 mutation. Based on the crucial role of CX45 in the cardiac development and the dysfunctional effect of the CHD-associated CX45 mutation, the applicants made a scientific hypothesis that CX45 is likely to be a new gene underpinning CHD. In order to substantiate the hypothesis, the current study will continue to screen CX45 for novel mutations underlying CHD in the patients with CHD. The CX45 gene will be cloned and the plasmids expressing wild-type or mutant CX45 will be constructed. The subcellular distribution of the mutant CX45 protein will be analyzed by laser scanning confocal fluorescence microscopy. The electrophysiological characteristics and selective permeability of the mutant gap junction channels will be studied by using dual-patch clap and fluorescent dye transfer techniques, respectively. These studies will not only reveal novel molecular genetic basis of CHD, but will also discover potential new drug target for the treatment of CHD.

先天性心脏病（CHD）是人类最常见的发育畸形和婴幼儿非感染性死亡的最常见病因，主要由遗传缺陷所致。本课题组在前期研究中通过对CHD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CHD基因座17q21.3，对该染色体区域的候选基因进行测序分析发现了一个CX45基因错义突变即p.R184G突变。该突变仅存在于家系中的CHD患者，不存在于家系中的正常成员，也不存在于300名无血缘关系的健康对照者，多物种CX45 基因编码蛋白氨基酸序列比对显示突变位点氨基酸在进化上完全保守。计算机功能模拟表明该CX45基因突是致病突变。双膜片钳全细胞检测跨细胞缝隙连接通道电导发现，R184G纯合突变型CX45缝隙连接通道丧失了跨细胞电导功能，R184G杂合突变型CX45缝隙连接通道也丧失了跨细胞电导功能。应用激光共聚焦显微镜观察发现，R184G突变对CX45所构成的缝隙连接通道的选择性通透功能无显著性影响。全细胞双膜片钳研究发现，由野生型CX43和R184G 突变型CX45所构成的异质性缝隙连接通道的电导率显著降低，由野生型CX43和R184G 突变型CX45所构成的异质性缝隙连接通道的电压门控效应显著降低，由R184G 突变型CX45所构成的缝隙连接通道的电压门控效应完全丧失。另外，发现NR2F2、GATA4、MEF2C、MESP1和TBX1基因功能缺失性新突变可导致CHD。上述研究结果不仅揭示了CHD新的分子病因及其作用机制，也为CHD的防治提供了新的分子靶标，因而具有重要的科学意义和临床应用前景