冠心病及心肌梗死是人类主要死因之一。申请人既往完成的中国人群冠心病GWAS 研究鉴定了4个新的易感区域。前期研究发现其中的WDR35、GALNT4基因mRNA在冠心病病例、对照样本中表达差异显著，提示WDR35、GALNT4基因是冠心病易感基因，但其在冠心病发生中的作用机制不明确。本申请拟进行以下研究：1）采用基因过表达和沉默策略，构建腺病毒介导的易感基因表达载体，在分子、细胞水平研究其在血管内皮细胞功能紊乱中的作用及机制；2）应用冠心病核心家系样本，对WDR35、GALNT4基因外显子进行深度测序，鉴定少见致病突变，并进行功能研究；3）建立WDR35、GALNT4基因分别与ApoE基因的双敲除小鼠，高脂饮食诱导动脉粥样硬化，从体内证明易感基因在血管内皮细胞功能紊乱及动脉粥样硬化中的作用。本申请将阐明冠心病易感区域在疾病发生中的作用及机制，为重大慢性血管性疾病的防治提供新的策略与药物靶点。

Dysfunction of blood vessel is the initiating agent for atherosclerosis. Coronary artery disease (CAD), the leading cause of human mortality and disability worldwide, has been shown to be highly heritable. By our genome-wide association study (GWAS) in Chinese Han population, we identified 4 novel loci (2p24.1, 4q32.1, 6p21.32, 12q21.33) for CAD reaching genome-wide significance. Pinpointing the genes and mechanism underlying the identified genetic loci has been challenging because most common variations have only subtle functional consequences. We have found that the expression of WDR35 and GALNT4 genes located at susceptibility regions 2p24.1 and 12q21.33, respectively, was of significant difference between CAD patients and controls, suggesting that these genes might be involved in the pathogenesis of CAD, but detailed roles and mechanisms are still unclear. The overall objective of present project is to elucidate the precise molecular mechanisms linking genetic variations to the vascular physiology in vitro and in vivo. Toward achieving our overall objective, we will pursue the following specific aims:.Specific Aim 1: We will construct expression vectors mediated by adenovirus of susceptibility genes to determine the effects of overexpression or knockdown of these genes on the NO production, apoptosis or proliferation of vascular endothelial cells, the adhesion of leukocyte to vascular endothelial cells or platelets, and finally define their roles in the physiological or pathological processes of endothelial cells. .Specific Aim 2: Using the strategy of exome sequencing in targeted regions, we will scan all exomes of WDR35 and GALNT4 genes to capture the rare variants associated with CAD in 212 members. The potential pathogenic variation will be replicated in a total of 2000 cases and controls. We will determine the presence or absence of rare genetic variation in these regions involving in the pathogenesis of CAD..Specific Aim 3: We will create endothelium-specific WDR35 gene-deficient mice and GALNT4-deficient mice, and then mate the knockout mice with ApoE-/- mice to generate double knockout mice. Double knockout mice will be fed with the Western-type diet to establish the animal model of atherosclerosis, and the effects of susceptibility genes on animal phenotype and their roles in vascular homeostasis will be investigated in vivo. .This project will elucidate the roles and mechanisms of these CAD susceptibility gene and pathogenic mutations, and provide new strategies and novel drug targets for the prevention and control of major chronic vascular disease.