基于大规模队列揭示血脂基因MCU和EEPD1在糖脂代谢调节中的作用及功能研究

Glycolipid metabolism disorder has been recognized as one pathogenesis mechanism for arteriosclerotic cardiovascular disease. By our genome-wide association study, we identified MCU and EEPD1 that reached chip-wide significance for lipid levels. The preliminary study suggests that MCU and EEPD1 modulate glycolipid metabolism disorder by targets and influence the progress of atherosclerosis. Toward achieving our overall objective, we will pursue the following specific aims:.Specific Aim 1: Using the strategy of exome sequencing in targeted regions, we will scan all exomes of MCU and EEPD1 genes to capture the rare variants associated with glucose and lipid levels. The potential pathogenic variation will be replicated in a prospective cohort. .Specific Aim 2: We will determine the RNA levels of MCU and EEPD1 in glycolipid metabolism disorder and coronary heart disease complicate with diabetes mellitus patients, and then evaluate the impact of variants on gene expression regulation, finally discover the role of MCU and EEPD1 for glycolipid metabolism disorder..Specific Aim 3: We will explore the potential novel mechanism of MCU, EEPD1 and their variants in modulating glucose and lipid metabolism in vitro. The role of MCU, EEPD1 and their variants in regulating glucose and lipid levels in mouse will be determined. We will create MCU gene-deficient mice and EEPD1-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 MCU and EEPD1 on animal phenotype and their roles in atherosclerosis will be investigated in vivo..This project will elucidate the roles and mechanisms of MCU and EEPD1 in glucose and lipid metabolism and atherosclerosis, and provide new strategies and novel drug targets for the prevention and control of major chronic vascular disease.

糖脂代谢紊乱是动脉粥样硬化性心血管病的病理学基础。申请人既往大规模基因组学研究发现MCU和EEPD1与血脂显著相关，进一步研究提示MCU和EEPD1可能参与糖脂代谢紊乱和动脉粥样硬化。本申请拟：1) 利用10万前瞻性队列人群筛选糖脂代谢紊乱个体对MCU和EEPD1进行靶向深度测序，鉴定少见致病突变；2) 在糖脂代谢紊乱个体中分析MCU、EEPD1基因的表达水平，确定突变体对基因表达的调控，揭示对糖脂代谢紊乱的影响；3) 在体外探索MCU、EEPD1基因及突变体调控糖脂代谢的新机制，在小鼠中明确MCU、EEPD1基因及突变体对血糖、血脂水平的作用，建立MCU、EEPD1基因分别与ApoE基因的双敲除小鼠，高脂饮食诱导AS，体内证明血脂关联基因在AS中的作用。本申请将阐明MCU、EEPD1基因在糖脂代谢紊乱作用及机制，为重大慢性血管性疾病的防治提供新的策略与潜在药物靶点。

糖脂代谢紊乱是冠心病等心血管疾病的病理学基础。申请人通过大规模基因组学研究发现MCU和EEPD1与血脂水平显著相关，但其功能和作用机制尚不清楚。本课题旨在研究血脂基因MCU和EEPD1在体内和体外对低密度脂蛋白胆固醇（LDL-C）、甘油三酯（TG）等水平的影响，揭示其调控血脂代谢紊乱的作用机制，并评估血脂基因变异在心血管疾病风险的预测价值，为心血管疾病的防治提供科学证据和潜在干预靶点。本课题主要开展了以下工作：.本研究从12万队列人群中挑选出1020例极端高和743例极端低LDL-C个体，开展全外显子组测序，鉴定出MCU和EEPD1基因的氨基酸编码变异，分析其与血脂水平的关系没有达到显著性，提示MCU和EEPD1基因可能通过非编码变异而不是编码变异发挥作用。.本课题分别建立了Mcu基因的全身敲除小鼠和Eepd1基因的肝脏特异性敲除小鼠，在动物水平证明Mcu和Eepd1基因均可通过PCSK9调控血浆LDL-C水平。在肝细胞HepG2中应用过表达和敲低策略，发现EEPD1可通过靶基因CYP1B1对PCSK9进行泛素化修饰，进而调控LDLR的蛋白表达水平。在细胞和动物水平证明miR-320b可直接靶向EEPD1基因调节巨噬细胞胆固醇外流，升高血浆LDL-C水平，进而加速动脉粥样硬化病变的发生。.同时本研究在4万人群队列中检测了500余个糖脂代谢和心血管疾病相关的遗传易感位点，评估其对心血管疾病发生的预测价值，建立了中国人群心血管疾病多基因遗传风险模型，揭示了血脂代谢紊乱的潜在关键干预靶点和通路，对心血管疾病的精准防治具有重要应用指导意义。进一步采用孟德尔随机化的方法，在跨种族人群中系统评价了不同种类降压药对糖脂代谢的影响，为高血压的个体化精准防治提供了科学证据。.本课题共发表科研论文6篇，其中5篇为SCI论文，授权专利4项，申请专利4项，并实现科研成果转化2000万元。