基于家系的新一代测序探索冠心病分子遗传基础

Coronary artery disease (CAD) is the leading cause of death worldwide. Genetic factors contribute significantly to the development of CAD，however, more than 80% of susceptibility variants or genes for CAD remain to be identified (refered to as missing heritability). The long-term objective of this project is thus to identify novel genetic and molecular determinants for CAD. To achieve this goal, we have identified and clinically characterized 28 very large, multigenerational families, 511 core families with early onset CAD and more than 25,000 sporadic CAD patient. This has become a unique and highly valuable resource for discovering susceptibility genes and genomic variants that confer risk of CAD. We have completed a genome-wide linkage scan with 408 polymorphic markers that cover the entire human genome by every 10 cM in 28 families, and identified two highly significant CAD loci on chromosome 3q28 and 7p22.3, four significant loci and 11 loci with suggested linkage. The 3q28 and 9q22 CAD loci showed significant linkage in the 511 replication families, too. We propose to employ an innovative, integrated strategy that combines whole exome next generation sequencing and genome-wide linkage analysis in the 28 large families to identify genomic rare variants associated with CAD. Five affected family members and 1 unaffected subject from each of the 28 families will be subjected to whole exome sequencing, and novel rare genomic variants will be identified. Private variants will be characterized by simple co-segregation with disease in families to determine whether they are disease-causing mutations. Other rare variants will be analyzed for association with CAD in the 28 large families using family-based rare variant association studies . Positive associations will be validated in the familial replication population (511 families) and a population cohort with 25,000 CAD patients and 20,000 controls. We prioritize rare variants in the following succeeding order: (1) Rare variants under linkage peaks; (2) Rare variants at or near CAD loci identified by GWAS; (3) Rare variants outside of linkage peaks or GWAS loci. Functional and expression studies will be used to determine whether variants associated with CAD affect the function or expression of nearby genes. These studies should lead to the identification of new genomic variants that confer risk of CAD, which may explain a major portion of the phenomena of missing heritability that has puzzled the genetic field for many years. This study may lead to the identification of novel susceptibility genes and rare genomic variants associated with CAD, which could lead to more effective screening and early treatment of high-risk individuals. The study may identify unrecognized molecular pathways for the development of CAD, and could also suggest novel molecular targets for treatment, prevention, and drug development.

冠心病是致死率居首位疾病，遗传因素在其发生过程中起关键作用，但>80%相关基因有待发现。为识别新的冠心病基因，收集了28个大型多代冠心病家系（平均个体16人），511个早发冠心病核心家系（平均个体5人），以及25000例散发型冠心病病例样本，为发现冠心病基因、诠释其遗传基础与生物学通路提供了宝贵资源。已完成28个大型家系的全基因组连锁分析，发现了2新的冠心病高度连锁区域， 4显著连锁区域，以及11疑似连锁区域。本课题拟结合高通量测序技术和全基因组连锁分析，对28个家系进行全外显子测序，发现与家系中冠心病共分离突变，并利用基于家系的罕见变异关联分析方法，识别冠心病易感罕见变异，并在核心家系和散发验证群体中进行验证。对新发现冠心病相关基因和变异，在分子、细胞、个体水平进行功能分析。本课题有望识别新冠心病基因和生物学通路，诠释困惑遗传学领域的“遗传率缺失”现象，发现新的药物靶标。

本课题采用基于家系的连锁分析策略，结合新一代高通量全外显子测序技术，识别发掘与家族性冠心病相关的罕见变异，确认新的冠心病易感基因和位点，构建并完善影响冠心病的分子遗传网络，同时运用基因表达与功能研究等手段，甄别新的冠心病致病基因、易感基因及变异，深度诠释冠心病的分子遗传基础，为阐明冠心病的分子病理基础提供崭新的科学视角。. 经过本项目的实施，共发现5个新的冠心病基因，其中一个为新的致病基因（TM6SF2），4个为新的易感基因（C3AR1， C6，CAV1和TFPI）。我们采用eQTL分析、基因-基因互作分析、网络分析等，在冠心病家系以及散发病例中对冠心病易感基因、互作网络进行了研究，发现了多个冠心病易感位点，揭示了其调控网络。通过meta-GWAS和eQTL分析发现由易感基因MLH3驱动的冠心病易感基因的调控网路，通过遗传流行病学分析建立了冠心病基因APOA5，甘油三酯/高密度脂蛋白水平和冠心病发病风险的因果模型。我们对冠心病致病基因和易感基因进行了系统的功能研究，系统诠释了ADTRP基因调控内皮细胞功能的信号通路，如发现雄激素通过直接激活ADTRP转录，抑制动脉粥样硬化形成的关键过程。发现ADTRP通过TFPI影响斑马鱼血液系统发育，发现冠心病易感基因ADTRP与MIA3/TANGO1的基因间信号调控网络等，这些结果对于揭示冠心病分子遗传作用途径和机制具有重要意义。开发出新的冠心病、心衰研究领域无创治疗技术的发展，为通过简单的AGGF1蛋白质注射或者靶向递送预防、治疗心脏病奠定了基础。. 已在国际学术期刊发表研究论文35篇（均为通讯作者，其中第一标注14篇，第二标注12篇），申请专利2项（已授权1项）。所发表论文包括《Nature communication》、《Diabetes》、《Plos Genetics》、《Human molecular genetics》、《Journal of molecular and cellular cardiology》、《Journal of Biological Chemistry》、《Cardiovascular research》等国际权威期刊；培养博士后2名，博士研究生、硕士研究生共9名。