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）已经确定了与冠心病（CHD）显著相关的CXCL12位点的常见多态性。我们在多种族研究中重现了该基因座与冠心病风险的关联，并发现了一种新的变异，可增加冠心病风险，降低血浆CXCL12水平并改变CXCL12基因的表达。我们进一步假设，如果CXCL12与冠心病的发病有因果关系，那么其主要受体CXCR4的遗传变异也可能与冠心病有关。为了验证我们的假设，我们通过对63,000例冠心病患者和92,000例对照的常见变异遗传数据进行荟萃分析，并对50,000例冠心病患者和50,000例对照进行精细定位实验，研究了CXCR4位点的遗传变异。首先，我们能够在CXCR4位点确定一个与冠心病风险密切相关的常见变异（P = 4x10-7）。其次，精细定位研究的初步分析确定：(i) CXCR4启动子区域的低频变异与冠心病风险相关，在实验范围内显著水平（OR: 1.13； P值：7.1x10-6）； （ii） CXCR4基因中自然发生的罕见错义变异（I57L; OR: 2.16 P = 6x10-3）与冠心病风险增加相关。同样的分析还发现了CXCL12基因的低频错义变异（R125C; OR: 3.38; P = 0.01）与冠心病风险增加名义上相关。通过机制研究，我们能够进一步证明内皮特异性缺乏CXCR4导致小鼠动脉粥样硬化加速。基于这些发现，我们计划开展遗传学、生物标志物和机制研究，以探讨CXCL12/CXCR4通路在疾病风险中的潜在生物学机制和定向影响。这些证据将有助于优先考虑或取消已经将CXCL12视为冠心病潜在治疗靶点的现有治疗方案。特别是，我们将通过整合(i) 10,000例早发性心肌梗死病例（50岁以下的男性和女性）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。我们将在3项前瞻性研究和3项临床试验中，对12000名参与者（包括5500名冠心病患者和3000名高危冠心病患者）进行循环血浆CXCL12水平的测量。我们将整合血浆CXCL12水平、基因和冠心病风险的证据，以帮助评估血浆CXCL12作为“因果关系”标记的“方向性”和效用。我们将在CXCL12位点对人类和小鼠细胞中与冠心病风险以及小鼠动脉粥样硬化和心肌梗死相关的导联编码和非编码变异进行机制评估。我们还将对人类iPS细胞衍生的巨噬细胞和内皮细胞中的CXCR4遗传变异进行机制评估。我们将通过两种互补的方法获得基因型特异性的人类iPS细胞：(i)根据基因型召回参与者；（ii）通过TALENs进行靶向基因组编辑。我们将通过特异性的定量和功能分析，进一步开展CXCR4基因型表达和蛋白功能的研究。拟议研究的结果应该具有相当大的翻译意义。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，这些技能将有助于将研究结果转化为临床效益。

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.