Functional Assays to Screen Genomic Hits

描述(由申请人提供)：我们提出了一个综合的实验系统来识别和功能表征与心脏传导特征和心律失常易感性相关的非编码遗传变异。传导系统疾病是最常见的心脏疾病之一，在美国，仅心脏性猝死每年就有超过325,000人死亡。这一重大的公共卫生问题促使人们加紧努力，以确定 导致传导系统疾病风险增加的遗传因素。大多数已发现的遗传变异本质上是非编码的，这使得确定它们对基因功能和传导系统生物学的影响变得困难。除了这一困难，普遍缺乏适当的实验平台来研究心脏系统生物学，如心脏传导系统培养的心肌细胞。我们的建议直接解决了这些不足之处。利用基因组学家、ENCODE项目中的PI和心脏传导系统病理学家的互补专业知识，我们建议创建一个多层次的实验平台，从与GWA型传导系统表型相关的LD块中识别原因SNPs，并系统地测试这些SNPs对其在心脏传导肌细胞和体内转基因小鼠中可能的转录增强功能的影响。对于这个项目的R21阶段，我们建议生成一个人类心脏的“迷你编码”，在全基因组范围内映射人类心脏中假定的功能非编码序列的坐标。我们将在包含与传导系统特征相关的非编码SNPs的50个基因座上，用远距离染色质相互作用的3D图覆盖这些信息。总之，这些数据将指向推测的传导系统增强子的位置，这些SNP含有与传导系统特征相关的SNP。在R33阶段，我们将利用多层平台从功能上询问这些SNP的影响。我们将首先测试R21组件中出现的候选增强剂。为此，我们开发了一种从诱导多能干细胞(IPSC)中衍生出传导系统心肌细胞的策略。这些细胞非常适合于涉及传导系统生物学的功能实验。我们将在这些细胞中测试含有疾病相关SNPs的候选增强子，建立它们的增强子属性以及等位基因特异性增强子效应。传导系统增强剂的一个子集将使用最先进的小鼠转基因技术进行进一步测试，以证明它们在体内的调节特性。总之，我们的计划描述了一种逻辑的、循序渐进的方法来识别与心脏传导系统参数相关的LD块中的原因SNPs，并开发了一个新的集成的实验平台来从功能上确定这些与疾病相关的非编码SNPs。

DESCRIPTION (provided by applicant): We propose an integrative experimental system to identify and functionally characterize noncoding genetic variants associated with cardiac conduction traits and susceptibility to arrhythmias. Conduction system diseases are among the most prevalent heart diseases, with sudden cardiac death alone responding for over 325,000 deaths in the US per year. This major public health issue has spurred intensive efforts to identify genetic factors underlying increased risk to conduction system diseases. Most genetic variants identified are noncoding in nature, making the determination of their impact on gene function and conduction system biology difficult to ascertain. Added to this difficulty, there is a generalized lack of proper experimental platforms to study cardiac system biology, such as cardiac conduction system myocytes in culture. Our proposal directly addresses these deficiencies. Capitalizing on the complementary expertise of the PIs, a genomicist that was a PI in the ENCODE project and a cardiac conduction system pathologist, we propose to create a multi-tiered experimental platform to identify causal SNPs from LD blocks associated with conduction system phenotypes from GWAS, and to systematically test for the impact of these SNPs on their putative transcriptional enhancer functions in cardiac conduction myocytes and in vivo, in transgenic mice. For the R21 phase of this project, we propose to generate a "mini-ENCODE" of the human heart, mapping genome-wide the coordinates of putative functional noncoding sequences in the human heart. We will overlay this information with a 3-D map of distant chromatin interactions in 50 loci containing noncoding SNPs associated with conduction system traits. Together, these data will point to the location of putative conduction system enhancers harboring SNPs associated with conduction system traits. In the R33 phase, we will utilize a multi-tiered platform to functionally interrogate the impact of these SNPs. We will initilly test candidate enhancers emerging from the R21 component. Toward that end, we developed a strategy to derive conduction system cardiomyocytes from induced pluripotent stem cells (iPSC). These cells are ideally suited for functional experiments involving conduction system biology. We will test candidate enhancers harboring disease-associated SNPs in these cells, establishing both their enhancer properties as well as allele-specific enhancer effects. A subset of conduction system enhancers will be further tested using state-of-the art mouse transgenics, to demonstrate their regulatory properties in vivo. Together, our proposed plan describes a logical, step-wise approach to identify causal SNPs within LD blocks associated with cardiac conduction system parameters and develops a novel and integrated experimental platform to functionally ascertain these disease-associated noncoding SNPs.