In Vivo Analysis of a Noncoding Susceptibility Region for Coronary Artery Disease

描述(由申请人提供)：大型流行病学研究表明，遗传因素对冠状动脉粥样硬化的发展有重要作用，冠状动脉粥样硬化是西方国家发病率和死亡率的主要原因。通过全基因组关联研究(GWASs)，我们和其他人已经确定了9p21上58kb的非编码区与冠状动脉疾病(CAD)之间的强烈联系。由于该区间不包含任何编码蛋白质的基因，因此该基因座上的序列变异影响CAD的机制尚不清楚。为了提供一个动物模型来研究这个与CAD相关的非编码区，我们从小鼠基因组中删除了这个同源区间(chr4？70kb)，并表明它对邻近缺失的两个基因(CDKN2a和CDKN2b)的心脏表达有显著影响。因此，这项应用的中心目标是使用这个和其他基因工程小鼠来揭示这个非编码区间中的序列变异导致CAD易感性的机制。其具体目的是：(1)鉴定在chr4？70kb小鼠中异常表达的基因/途径；(2)通过转基因小鼠增强子实验鉴定和鉴定缺失区间内的转录增强子；(3)测试我们在连锁不平衡区间中已确定的增强子中与CAD相关的序列变异对体内基因表达的影响；(4)确定chr4？70kb缺失对小鼠动脉粥样硬化易感性和细胞增殖的影响。这些研究将探索人类9p21连锁不平衡区域如何导致CAD易感性的机制，并作为将非编码区发生的疾病相关DNA序列变异与临床相关表型联系起来的范例。与公共卫生相关：全基因组关联研究(GWAS)揭示了一些导致人类疾病的基因组区间，但缺乏蛋白质编码基因。值得注意的是，由于人类染色体9p21非编码区的序列变异，许多人患冠状动脉疾病的风险大幅增加，但致病机制仍然不明。因此，我们建议利用基因工程小鼠模型在受控实验室环境中研究这些非编码序列变化如何导致人类心脏病。

DESCRIPTION (provided by applicant): Large epidemiological studies have indicated that genetic factors contribute significantly to the development of coronary atherosclerosis, a major cause of morbidity and mortality in Western countries. Through genome-wide association studies (GWASs), we and others have identified a strong link between a 58kb noncoding region on 9p21 and coronary artery disease (CAD). The mechanism by which sequence variation at this locus influences CAD is not known since the interval does not contain any protein-coding genes. To provide an animal model to study this CAD-associated noncoding region, we have deleted the orthologous interval (chr4?70kb) from the mouse genome and show that it significantly impacts on cardiac expression of two genes (Cdkn2a and Cdkn2b) adjacent to the deletion. Accordingly, the central goal of this application is to use this and other genetically engineered mice to uncover the mechanism by which sequence variation in this noncoding interval confers susceptibility to CAD. The specific aims are to (1) identify genes/pathways abnormally expressed in the chr4?70kb mice, (2) to identify and characterize transcriptional enhancers within the deleted interval through a transgenic mouse enhancer assay, (3) to test CAD-associated sequence variants in the enhancers that we have identified in the linkage disequilibrium interval for their impact on gene expression in vivo, and (4) to determine the effect of the chr4?70kb deletion on murine atherosclerosis susceptibility and cellular proliferation. These studies will explore the mechanism of how the human 9p21 linkage disequilibrium region causes CAD susceptibility and serve as a paradigm for relating disease-associated DNA sequence variants occurring in noncoding regions to clinically relevant phenotypes. PUBLIC HEALTH RELEVANCE: Genome-wide association studies (GWASs) have revealed a number of genomic intervals that contribute to human disease but are devoid of protein-coding genes. Remarkably, many individuals have a substantially increased risk for developing coronary artery disease due to sequence variations in a noncoding region of human chromosome 9p21, yet the disease- causing mechanism remains cryptic. Accordingly, we propose to utilize genetically engineered mouse models to study in a controlled laboratory setting how these noncoding sequence changes lead to heart disease in humans.