Molecular mechanisms linking the CXCL12 pathway to atherosclerosis

描述（由申请人提供）：全基因组关联研究（GWAS）已确定CXCL 12基因座的常见多态性与冠心病（CHD）显著相关。我们在多种族研究中再现了该位点与CHD风险的关联，并确定了一种新的变体，该变体增加CHD风险，降低血浆CXCL 12水平并改变CXCL 12基因的表达。我们进一步假设，如果CXCL 12与CHD的发病机制有因果关系，那么其主要受体CXCR 4的遗传变异也可能与CHD相关。为了验证我们的假设，我们通过对涉及约63，000例CHD病例和约92，000例对照的常见变异的遗传数据进行荟萃分析，并在约50，000例CHD病例和约50，000例对照中进行精细定位实验，研究了CXCR 4基因座的遗传变异。首先，我们能够确定CXCR 4基因座的一个常见变异，该变异与CHD风险密切相关（P = 4x 10 -7）。其次，对精细定位研究的初步分析确定了：（i）CXCR 4启动子区的一种低频变异，在实验范围内显著性水平（OR：1.13; P值：7.1x10-6）与CHD风险相关;（ii）CXCR 4基因中天然存在的一种罕见错义变异（I57 L; OR：2.16 P = 6x 10 -3）与CHD风险增加相关。同样的分析还发现了CXCL 12基因中的一个低频错义变异体（R125 C; OR：3.38; P = 0.01），名义上与CHD风险增加相关。通过机制研究，我们能够进一步证明内皮特异性CXCR 4缺陷导致小鼠动脉粥样硬化加速。利用这些发现，我们计划进行遗传、生物标志物和机制研究，以研究潜在的生物学机制和CXCL 12/CXCR 4通路对疾病风险的方向性影响。这些证据将有助于优先考虑或降低已经将CXCL 12视为CHD潜在治疗靶点的现有治疗方案的优先级。特别是，我们将通过整合（i）10，000例早发性MI病例（50岁的男性和女性）和10，000例对照的全外显子组测序研究来加强我们正在进行的精细定位实验。我们将进一步进行（ii）在三项前瞻性研究和三项临床试验中招募的12，000名参与者（包括5，500名发生CHD事件的参与者和3，000名高风险CHD组参与者）的循环血浆CXCL 12水平测量。我们将整合血浆CXCL 12水平、基因和CHD风险的证据，以帮助评估血浆CXCL 12作为“因果关系”标志物的“方向性”和实用性。我们将在人和鼠细胞中以及在小鼠动脉粥样硬化和心肌梗死中对CXCL 12位点与CHD风险相关的前导编码和非编码变体进行机械评估。我们还将从机制上评估人iPS细胞衍生的巨噬细胞和内皮细胞中的CXCR 4遗传变异。我们将通过两种互补方法获得基因型特异性人类iPS细胞：（i）基于基因型召回参与者和（ii）通过TALEN进行靶向基因组编辑。我们将进一步开展研究，通过特定的定量和功能分析，研究基因型CXCR 4的表达和蛋白质功能。从拟议的研究结果应该有相当大的翻译影响。这两个联合PI在血管生物标志物、流行病学、实验生物学和临床医学方面提供国际公认的跨学科专业知识，这些技能的结合将有助于将研究结果转化为临床获益。

DESCRIPTION (provided by applicant): Genome-wide association studies (GWAS) have identified common polymorphisms at the CXCL12 locus significantly associated with coronary heart disease (CHD). We have reproduced the association of this locus with CHD risk in multiethnic studies and have identified a novel variant that increases CHD risk, decreases plasma CXCL12 levels and alters expression of CXCL12 gene. We further hypothesized that if CXCL12 is causally involved in the pathogenesis of CHD than genetic variation at its primary receptor CXCR4 may also be associated with CHD. To test our hypothesis, we investigated genetic variation at the CXCR4 locus through meta-analyses of genetic data on common variants involving ~63,000 CHD cases and ~92,000 controls and fine-mapping experiments in ~50,000 CHD cases and ~50,000 controls. First, we were able to identify a common variant at the CXCR4 locus that is strongly associated with CHD risk (P = 4x10-7). Second, preliminary analyses of fine-mapping studies identified: (i) a low frequency variant in the promoter region of CXCR4 that associates with CHD risk at experiment-wide significance levels (OR: 1.13; P-value: 7.1x10-6) and (ii) a naturally occurring rare missense variant in the CXCR4 gene (I57L; OR: 2.16 P = 6x10-3) associated with increased CHD risk. The same analyses also identified a low-frequency missense variant in the CXCL12 gene (R125C; OR: 3.38; P = 0.01) nominally associated with increased CHD risk. Through mechanistic studies, we were able to further demonstrate that endothelial specific deficiency of CXCR4 leads to accelerated atherosclerosis in mice. Capitalizing on these findings, we plan to conduct genetic, biomarker, and mechanistic studies to investigate the underlying biological mechanisms and the directional impact of the CXCL12/CXCR4 pathway on disease risk. Such evidence will help prioritize or deprioritize existing therapeutic programs that are already considering CXCL12 as a potential therapeutic target in CHD. In particular, we will reinforce our ongoing fine-mapping experiments by integrating (i) whole-exome sequencing studies in 10,000 early-onset MI cases (men and women aged � 50 years) and 10,000 controls. We will further conduct (ii) measurements for circulating plasma CXCL12 levels in 12,000 participants (including 5,500 with incident CHD events and 3,000 participants in a high risk CHD group) enrolled in three prospective studies and three clinical trials. We will integrate evidence on plasma CXCL12 levels, genes and CHD risk to help assess "directionality" and utility of plasma CXCL12 as a marker of "causality". We will mechanistically evaluate at the CXCL12 locus the lead coding and non- coding variants associated with CHD risk in human and murine cells and on mouse atherosclerosis and myocardial infarction. We will also mechanistically evaluate CXCR4 genetic variation in human iPS cell derived macrophages and endothelial cells. We will obtain genotype specific human iPS cells through two complimentary approaches: (i) recall of participants based on genotype and (ii) targeted genome editing through TALENs. We will further conduct studies to investigate CXCR4 expression and protein function by genotype through specific quantitative and functional assays. Findings from the proposed study should have considerable translational implications. The two joint-PIs provide internationally recognized cross-disciplinary expertise in vascular biomarkers, epidemiology, experimental biology, and clinical medicine, a combination of skills that will help translate findings for clinical benefit.