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

描述（由申请方提供）：血浆C-反应蛋白（CRP）水平是预测心血管疾病（CVD）未来风险的生物标志物。我们最近发现，CRP基因中几个常见的单核苷酸多态性（SNP）的等位基因与血浆CRP水平相关，并在功能上改变CRP启动子的调节。然而，CRP水平的大部分个体间差异仍然无法解释。常见的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SNP相比，存在对血浆CRP具有更大影响的罕见SNP并非不可能，并且这些罕见SNP可能解释血浆CRP中剩余差异的显著部分。本申请的目的是发现CRP基因中罕见的SNP，其与血浆CRP水平和动脉粥样硬化发病机制相关。 目的1：我们将通过对两组基因组进行重新测序来鉴定可能改变CRP水平的罕见SNP：A. CARDIA队列中CRP分布（以及年龄/性别匹配的对照）的高尾（N = 376）和低尾（N=376），该队列由4000多名年龄在38至50岁之间的个体组成，以及B。来自颈动脉粥样硬化疾病研究的严重狭窄病例（N=500）和对照组（N=500）。 目标二：我们将在功能上表征目的1中鉴定的pupirin功能性SNP，使用含有CRP-GFP融合基因的质粒的定点诱变以产生等位基因构建体，并对等位基因构建体的表达进行瞬时转染分析以评估对蛋白质和RNA水平的功能影响。

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.