Coronary-Heart-Disease-Associated Genetic Variant at the <i>COL4A1/COL4A2</i> Locus Affects <i>COL4A1/COL4A2</i> Expression, Vascular Cell Survival, Atherosclerotic Plaque Stability and Risk of Myocardial Infarction

Genome-wide association studies have revealed an association between coronary heart disease (CHD) and genetic variation on chromosome 13q34, with the lead single nucleotide polymorphism rs4773144 residing in the COL4A2 gene in this genomic region. We investigated the functional effects of this genetic variant. Analyses of primary cultures of vascular smooth muscle cells (SMCs) and endothelial cells (ECs) from different individuals showed a difference between rs4773144 genotypes in COL4A2 and COL4A1 expression levels, being lowest in the G/G genotype, intermediate in A/G and highest in A/A. Chromatin immunoprecipitation followed by allelic imbalance assays of primary cultures of SMCs and ECs that were of the A/G genotype revealed that the G allele had lower transcriptional activity than the A allele. Electrophoretic mobility shift assays and luciferase reporter gene assays showed that a short DNA sequence encompassing the rs4773144 site interacted with a nuclear protein, with lower efficiency for the G allele, and that the G allele sequence had lower activity in driving reporter gene expression. Analyses of cultured SMCs from different individuals demonstrated that cells of the G/G genotype had higher apoptosis rates. Immunohistochemical and histological examinations of ex vivo atherosclerotic coronary arteries from different individuals disclosed that atherosclerotic plaques with the G/G genotype had lower collagen IV abundance and thinner fibrous cap, a hallmark of unstable, rupture-prone plaques. A study of a cohort of patients with angiographically documented coronary artery disease showed that patients of the G/G genotype had higher rates of myocardial infarction, a phenotype often caused by plaque rupture. These results indicate that the CHD-related genetic variant at the COL4A2 locus affects COL4A2/COL4A1 expression, SMC survival, and atherosclerotic plaque stability, providing a mechanistic explanation for the association between the genetic variant and CHD risk.全基因组关联研究（Genome-wide association studies）已证实冠心病（coronary heart disease, CHD）与13号染色体q34区域的遗传变异存在关联，该区域关联最强的单核苷酸多态性rs4773144位于COL4A2基因内。本研究针对该遗传变异的功能效应展开了探究。对不同个体来源的血管平滑肌细胞（vascular smooth muscle cells, SMCs）与内皮细胞（endothelial cells, ECs）原代培养物的分析结果显示，rs4773144不同基因型的COL4A2与COL4A1表达水平存在显著差异：G/G基因型的表达水平最低，A/G基因型居中，A/A基因型的表达水平最高。针对携带A/G基因型的SMCs与ECs原代培养物开展的染色质免疫沉淀联合等位基因失衡分析表明，G等位基因的转录活性低于A等位基因。电泳迁移率变动分析（Electrophoretic mobility shift assays）与荧光素酶报告基因实验（luciferase reporter gene assays）结果显示，一段包含rs4773144位点的短DNA序列可与核蛋白结合，且G等位基因的结合效率更低；同时G等位基因序列驱动报告基因表达的活性也显著降低。对不同个体来源的培养SMCs的分析显示，携带G/G基因型的细胞凋亡率更高。对不同个体的离体动脉粥样硬化冠状动脉开展的免疫组织化学与组织学检查发现，携带G/G基因型的动脉粥样硬化斑块中IV型胶原蛋白（collagen IV）含量更低，纤维帽（fibrous cap）更薄——这正是不稳定、易破裂斑块的标志性特征。一项针对经血管造影确诊的冠状动脉疾病患者队列的研究显示，携带G/G基因型的患者心肌梗死（myocardial infarction）发生率更高，而心肌梗死通常由斑块破裂引发。上述结果表明，COL4A2基因位点处与冠心病相关的遗传变异可通过调控COL4A2/COL4A1的表达、SMCs存活以及动脉粥样硬化斑块稳定性，为该遗传变异与冠心病风险之间的关联提供了机制层面的解释。