Assessing the Impact of Rare Polymorphism at CRP on CRP Levels & Atherosclerosis

描述(由申请人提供)：血浆C-反应蛋白(CRP)水平是预测未来心血管疾病(CVD)风险的生物标志物。我们最近发现，CRP基因中几个常见的单核苷酸多态(SNPs)等位基因与血浆CRP水平相关，并在功能上改变了CRP启动子的调节。然而，C反应蛋白水平的大部分个体间差异仍未得到解释。普通的SNPs往往比罕见的SNPs更古老，因此经历了更长期的自然选择。因此，与已知的、常见的SNP相比，罕见的SNPs对血浆CRP的影响并不是不可能的，这些罕见的SNPs可能解释了血浆CRP剩余变异的很大一部分。这项应用的目的是发现CRP基因中罕见的SNP，这些SNP与血浆CRP水平和动脉粥样硬化的发病机制具有功能相关性。 目的1：我们将通过对两个小组进行重新测序来识别可能改变CRP水平的罕见SNPs：A.CARDIA队列中CRP分布的高(N=376)和低(N=376)尾(以及年龄/性别匹配的对照)，超过4000人，年龄在38到50岁之间，以及B.颈动脉粥样硬化性疾病研究中的严重狭窄病例(N=500)和对照组(N=500)。 目的2：我们将通过对含有CRP-GFP融合基因的质粒进行定点突变来产生等位基因结构，并对等位基因结构的表达进行瞬时转染分析，以评估功能对蛋白质和RNA水平的影响，从而从功能上表征目标1中确定的功能SNPs。

DESCRIPTION (provided by applicant): Plasma C-Reactive Protein (CRP) level is a biomarker that predicts future risk of cardiovascular disease (CVD). We recently demonstrated that alleles at several common single nucleotide polymorphisms (SNPs) in the CRP gene correlate with plasma CRP levels, and functionally alter the regulation of the CRP promoter. However, the majority of the inter-individual variance in CRP levels remains unexplained. Common SNPs tend to be older than rare SNPs, and therefore have been exposed to longer term natural selection. Thus, it is not unlikely that rare SNPs exist with larger effects on plasma CRP than the known, common SNPs, and these rare SNPs might explain a significant fraction of the remaining variance in plasma CRP. The goal of this application is to discover rare SNPs in the CRP gene, which are of functional relevance in relation to plasma CRP levels and atherosclerotic pathogenesis. Aim 1: We will identify rare SNPs likely to alter CRP levels by resequencing two panels: A. the high (N=376) and low (N=376) tails of the CRP distribution (and age/gender matched controls) in the CARDIA cohort, a cohort of more than 4000 individuals, ages 38 to 50, and B. Severely stenosed cases (N=500) and controls (N=500) from a study of carotid atherosclerotic disease. Aim 2: We will functionally characterize putatively functional SNPs identified in Aim 1, using site-directed mutagenesis of a plasmid containing a CRP-GFP fusion gene to generate allelic constructs, and transient transfection analysis of expression from the allelic constructs to assess functional impacts on protein and RNA levels.