EPISTATIC REGULATORY MECHANISMS OF CORONARY HEART DISEASE RISK

项目概述这项为期五年的计划的目的是为申请者提供一个综合性和个性化的培训计划，使其能够过渡到心血管科学领域的一个独立的学术职位。长期目标是通过研究全基因组关联上游的信号机制来发现心血管疾病的新疗法。申请者已经在心血管药理学、生理学和信号转导方面有很强的背景，采用了体外和体内的实验方法。这一职业发展计划(K99阶段)将提供人类遗传学和基因组分析方面的额外培训，采用创新的下一代测序技术，以及复杂的基因靶向方法，以研究与冠心病相关的因果变异的调节功能。申请者还将在斯坦福大学接受大量的非正式和指导性培训，如基因组和统计分析等专业领域，以及专业发展技能，这对申请者获得自主权和开始富有成效的职业生涯至关重要。 一名独立调查员。在医学博士Thomas Querterous博士和召集的咨询委员会(Assimes博士、曹博士、Pritchard博士和格林利夫博士)的专家指导下，申请者将获得必要的指导和资源，以实现这些目标并在R00阶段有效地过渡到独立。这项建议的研究主题通过确定常见基因变异和可遗传冠心病之间缺失的机制联系，填补了该领域的一个重大知识空白。患动脉粥样硬化性冠心病(CHD)的风险高达一半是由基因决定的。全基因组关联研究现在已经确定了多个独立的遗传区域，这些区域加在一起只占冠心病估计遗传率(~60%)的一小部分(~10%)。预测了常见易感区域之间的基因-基因(上位性)和基因-环境相互作用，以解释这种“隐藏的”遗传性。然而，基因变异改变疾病风险的因果机制在很大程度上仍不清楚。破译这些调控网络预计将揭示对疾病风险和治疗发展战略的新见解。下一代测序方法现在加速了我们对非编码变异如何改变疾病相关基因表达的理解。这个项目的目标是研究导致风险的分子和遗传相互作用，这两个基因是血管发育转录因子TCF21和血小板衍生生长因子D(PDGFD)。这两个基因都与多个种族/民族的心脏病风险有关，独立于传统的危险因素发挥作用，是心脏发育和损伤过程中平滑肌细胞反应的关键调节因子。我们先前发现了一种TCF21关联的调节机制，涉及冠状动脉平滑肌细胞中TCF21基因表达的转录和转录后调节。最近，我们系统地定位了TCF21转录因子蛋白来直接结合和反式激活PDGFD基因，并观察到TCF21和PDGFD在人类动脉粥样硬化病变中的表达呈正相关。一致地，PDGF介导的信号在TCF21结合位点和CHD危险基因总体上是丰富的。基于这些发现，我们假设TCF21的调控可能在SMC对血管壁损伤的表型反应中起关键作用，并且TCF21和PDGFD基因座之间的分子和遗传相互作用增强了这些基因的表达，导致冠状动脉粥样硬化风险增加。这一假说将通过追求以下具体目标进行实证检验：1)剖析TCF21和PDGFD基因座调控变异的原因机制。2)研究TCF21和PDGFD基因座之间的分子相互作用。为了实现这些目标，我们将把人类冠状动脉原发病变组织的高通量基因组学与RNA引导的人类冠状动脉SMC和小鼠胚胎体内的基因组靶向相结合。最终，这项工作将为靶向与不同人群中“隐藏的”CHD风险相关的关键途径提供新的线索，并使下一代疗法的开发成为可能。

DESCRIPTION (provided by applicant): PROJECT SUMMARY The purpose of this five-year proposal is to provide an integrative and personalized training program for the applicant to transition into an independent academic position in the cardiovascular sciences. The long-term goal is to discover new treatments for cardiovascular disease through investigating the signaling mechanisms upstream of genome-wide associations. The applicant already has a strong background in cardiovascular pharmacology, physiology, and signal transduction by employing both in vitro and in vivo experimental approaches. This career development plan (K99 phase) will provide additional training in human genetics and genomic analyses employing innovative next-generation sequencing technologies, as well as sophisticated gene-targeting approaches to investigate the regulatory function of causal variants associated with coronary heart disease. The applicant will also receive a wealth of informal and didactic training at Stanford University i specialized areas such as genomic and statistical analyses, and professional development skills, which will be critical for the applicant to gain autonomy and launch a productive career as an independent investigator. Under the expert mentorship of Dr. Thomas Quertermous, MD and the assembled advisory committee (Dr. Assimes, Dr. Tsao, Dr. Pritchard, and Dr. Greenleaf) the applicant will receive the necessary guidance and resources to accomplish these goals and efficiently transition to independence during the R00 phase. The research topic of this proposal fulfills a significant knowledge gap in the field by identifying the missing mechanistic links between common genetic variants and heritable coronary heart disease. As much as half the risk of developing atherosclerotic coronary heart disease (CHD) is genetically predetermined. Genome-wide association studies have now identified multiple independent genetic regions which together account for a fraction (~10%) of the estimated heritability for coronary heart disease (~60%). Gene-gene (epistatic) and gene-environment interactions between common susceptibility regions are predicted to explain this "hidden" heritability. However, the causal mechanisms by which genetic variation alters disease risk remain largely unknown. Deciphering these regulatory networks is predicted to reveal new insights into disease risk and therapeutic development strategies. Next-generation sequencing approaches have now accelerated our understanding of how non-coding variation alters disease-related gene expression. The goal of this project is to investigate the molecular and genetic interactions responsible for driving risk t two genes, the vascular development transcription factor, TCF21, and the platelet-derived growth factor D (PDGFD). Both of these genes have been associated with heart disease risk in multiple racial/ethnic groups, act independently of traditional risk factors, and are key regulator of smooth muscle cell responses during cardiac development and injury. We previously identified a regulatory mechanism for the TCF21 association involving both transcriptional and post-transcriptional regulation of TCF21 gene expression in coronary artery smooth muscle cells. Recently, we systematically mapped TCF21 transcription factor protein to directly bind and trans-activate the PDGFD locus, and observed a positive correlation of TCF21 and PDGFD expression in human atherosclerotic lesions. Consistently, PDGF-mediated signaling was enriched at TCF21 binding sites and CHD risk loci overall. Based on these findings, we hypothesize that regulation of TCF21 may be pivotal to the SMC phenotypic response to injury in the vessel wall, and that molecular and genetic interactions between the TCF21 and PDGFD loci potentiates expression of these genes, leading to increased coronary atherosclerosis risk. This hypothesis will be empirically tested by pursuing the following specific aims: 1) Dissect the causal mechanisms of regulatory variants at TCF21 and PDGFD loci. 2) Investigate the molecular interactions between the TCF21 and PDGFD loci. To achieve these aims, we will integrate high-throughput genomics in primary diseased human coronary artery tissue with RNA-guided genome targeting in human coronary artery SMC and in mouse embryos in vivo. Ultimately, this work will shed new light on targeting critical pathways associated with "hidden" CHD risk in various human populations, and enable the development of the next generation of therapeutics.