From genotype to phenotype in a GWAS locus: the role of REST in atherosclerosis

项目摘要 K08临床科学家导师研究职业发展奖是一项为期五年的计划，旨在 促进Marios Arvantis博士(PI)发展成为血管遗传学的独立医生兼研究员。 动脉粥样硬化性心血管疾病(ASCVD)是一种主要的公共卫生负担，占 美国每年有60万人死亡。ASCVD是高度遗传性的全基因组关联研究 已经发现了许多候选基因组基因座，它们会增加人群中患病的风险，因此 提供了一扇通往新疗法的窗口。然而，大多数ASCVD的基因组风险基因座仍未被发现 它们是如何导致疾病风险的。 PI之前发表的工作主要集中在基因组风险基因座的机制解释上 心血管疾病，包括开发一种新的贝叶斯方法，称为CAFEH，以确定优先顺序 基因组座位上的靶组织和基因。我们对ASCVD遗传基础的初步分析 揭示了内皮细胞在ASCVD遗传性方面的丰富，我们已经使用这些方法来优先考虑 4号染色体基因座被预测通过改变RE-1沉默的表达而影响ASCVD风险 内皮细胞中的转录因子(REST)基因。K08项目将通过以下方式探索监管机制 哪些休息基因和基因影响动脉粥样硬化的发生发展。AIM 1将采用CRISPR-CAS9 编辑人类干细胞，然后将其分化为内皮细胞，以确定原因变体 以及介导4Q12冠脉疾病基因座关联的上游转录因子。 Aim 2将定义受内皮细胞静息影响的远端基因和通路，并研究它们的细胞 结果，首先评估REST在内皮细胞向间充质转化中的作用。AIM 3将使用 他莫昔芬诱导的血管内皮细胞特异性REST基因敲除小鼠模型的体内效应 内皮细胞与动脉粥样硬化。 该项目的成功得到了多学科指导团队的支持，包括 一位血管生物学家(Charles Lowenstein博士)，一位计算生物学家(Alexis Battle博士)，以及一位功能性 遗传学专家(安德鲁·麦卡利安博士)与血管生物学专家咨询委员会一起， 细胞分化与动脉粥样硬化(哈里·迪茨博士、权楚兰博士和托马斯·奎特莫斯博士)。这 获奖期将帮助PI提高他们的基因组学技能，获得新的湿实验室技能并生成初步数据 成功争取R01资金，以便将基因洞察转化为新的机制 ASCVD。

Project Summary This K08 mentored clinical scientist research career development award is a five-year program designed to facilitate Dr. Marios Arvanitis’ (PI) development into an independent physician-investigator in vascular genetics. Atherosclerotic cardiovascular disease (ASCVD) is a major public health burden that accounts for over 600,000 deaths in the United States each year. ASCVD is highly heritable and genome-wide association studies have discovered many candidate genomic loci that increase the risk of the disease in the population, thereby providing a window to novel therapies. However, most genomic risk loci for ASCVD remain unexplored in terms of how they lead to disease risk. Previously published work by the PI has focused on the mechanistic interpretation of genomic risk loci for cardiovascular disease, including the development of a novel Bayesian method, called CAFEH, to prioritize the target tissue and genes in genomic loci. Our preliminary analyses of the genetic underpinnings of ASCVD reveal that endothelial cells are enriched for ASCVD heritability, and we have used those methods to prioritize a chromosome 4 locus that is predicted to affect ASCVD risk via altering the expression of the RE-1 silencing transcription factor (REST) gene in endothelial cells. This K08 project will explore the regulatory mechanisms via which the REST locus and gene influence the development of atherosclerosis. Aim 1 will employ CRISPR-Cas9 editing in human stem cells which will then be differentiated into endothelial cells to identify the causal variants and the upstream transcription factors that mediate the association in the 4q12 coronary disease GWAS locus. Aim 2 will define distal genes and pathways affected by REST in the endothelium and investigate their cellular consequences, starting with evaluating the role of REST in endothelial to mesenchymal transition. Aim 3 will use a tamoxifen inducible endothelial specific Rest knock-out mouse model to evaluate the in vivo effects of Rest in the endothelium and atherosclerosis. The success of this project is guaranteed by the support of a multidisciplinary mentoring team including a vascular biologist (Dr. Charles Lowenstein), a computational biologist (Dr. Alexis Battle), and a functional genetics expert (Dr. Andrew McCallion), along with an advisory committee of experts in vascular biology, stem cell differentiation and atherosclerosis (Dr. Harry Dietz, Dr. Chulan Kwon and Dr. Thomas Quertermous). This award period will help the PI boost their genomics skills, acquire new wet lab skills and generate preliminary data to successfully compete for R01 funding in order to translate the genetic insights into novel mechanisms for ASCVD.