WDR35和GALNT4基因在血管内皮细胞功能紊乱及动脉粥样硬化中的作用及机制研究

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.

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。

本课题组既往开展冠心病全基因组关联研究确定了4个新的冠心病易感区域，其中2p24.1区域WDR35基因、12q21.33区域GALNT4基因在冠心病病例和对照中表达差异显著，提示这些易感基因参与冠心病发生，但作用机制尚不清楚。本课题在分子细胞和动物水平试图揭示冠心病易感基因在内皮功能紊乱、血管稳态失衡和动脉粥样硬化发生中的作用机制。主要研究内容和结果如下：.在分子细胞水平，我们发现GALNT4基因可激活内皮细胞NF-κB信号通路，上调粘附分子ICAM-1、VCAM-1和MCP-1的表达，进而促进单核细胞粘附内皮细胞及内皮下迁移；与GALNT4基因同家族的GALNT3基因可调控p38/MAPK信号通路活性，促进HUVECs凋亡及MMP-2/14表达，从而导致内皮细胞损伤；WDR35基因可抑制内皮细胞增殖，促进凋亡、内皮细胞粘附因子表达，从而参与低氧介导的内皮细胞功能紊乱。.应用CRISPR/Cas9技术建立了内皮细胞特异性Wdr35fl/fl;Ts-Cre/+;ApoE-/-小鼠、Galnt4fl/fl;Ts-Cre/+;ApoE-/-小鼠、Galnt4fl/fl;Ts-Cre/+;LDLR-/-小鼠；进而应用高脂饮食诱导建立动脉粥样硬化模型，在动物水平明确冠心病易感基因在血管稳态失衡及动脉粥样硬化中的作用。.通过外显子芯片鉴别出了GALNT4、WDR35基因的编码变异位点，发现这些功能位点与冠心病和血脂水平的关联没有达到显著性，提示GALNT4和WDR35基因对疾病的作用主要是通过非编码位点而不是编码位点发挥作用。采用外显子组研究策略，在跨种族人群中开展冠心病和血脂异常基因组和外显子组研究，鉴定出50个基因区域的264个遗传变异与血脂水平显著相关，包括国际上首次报道的12个新的血脂易感基因和14个东亚人群种族特异性功能变异；并揭示了基因组少见功能变异对我国人群冠心病和血脂异常的影响。.本研究表明GALNT4和WDR35基因调节内皮细胞功能紊乱，与血管稳态失衡和动脉粥样硬化密切相关；大型跨种族人群冠心病和血脂外显子组研究鉴定的潜在功能位点，为解析冠心病遗传致病机制研究指明了潜在靶基因和功能变异。.本研究发表了10篇SCI论文，获得3项授权专利、1项教育部自然科学二等奖。