Functional Assays to Screen Genomic Hits

描述（由申请人提供）：我们提出了一个综合实验系统，以识别和功能表征与心脏传导性状和心律失常易感性相关的非编码遗传变异。传导系统疾病是最普遍的心脏病之一，仅心脏性猝死一项就导致美国每年超过325，000人死亡。这一重大的公共卫生问题促使人们加紧努力， 遗传因素导致传导系统疾病的风险增加。大多数已确定的遗传变异在本质上是非编码的，使得它们对基因功能和传导系统生物学的影响难以确定。除了这个困难之外，还普遍缺乏适当的实验平台来研究心脏系统生物学，例如培养的心脏传导系统肌细胞。我们的建议直接针对这些不足之处。利用PI的互补专业知识，基因组学家是ENCODE项目中的PI和心脏传导系统病理学家，我们建议创建一个多层实验平台，以识别与GWAS传导系统表型相关的LD块的因果SNP，并系统地测试这些SNP对心脏传导肌细胞和体内假定的转录增强子功能的影响，在转基因小鼠中。对于该项目的R21阶段，我们建议生成人类心脏的“迷你编码”，在人类心脏中绘制全基因组推定功能非编码序列的坐标。我们将覆盖这一信息与3D地图的遥远的染色质相互作用在50个位点含有非编码单核苷酸多态性与传导系统性状。总之，这些数据将指向假定的传导系统增强子的位置，这些增强子包含与传导系统性状相关的SNP。在R33阶段，我们将利用一个多层次的平台来功能性地询问这些SNP的影响。我们将首先测试从R21组分中出现的候选增强子。为此，我们开发了一种从诱导多能干细胞（iPSC）中获得传导系统心肌细胞的策略。这些细胞非常适合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携带疾病相关SNP的候选增强子，建立它们的增强子特性以及等位基因特异性增强子效应。将使用最先进的小鼠转基因进一步测试传导系统增强子的子集，以证明其体内调节特性。总之，我们提出的计划描述了一种逻辑，逐步的方法来确定与心脏传导系统参数相关的LD块内的因果SNP，并开发了一种新的综合实验平台，以在功能上确定这些疾病相关的非编码SNP。

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.