Functional Assays to Screen Genomic Hits

描述（由申请人提供）：我们提出了一种综合实验系统，用于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病之一，美国每年仅因心源性猝死就导致超过 325,000 人死亡。这一重大公共卫生问题促使人们大力努力确定 传导系统疾病风险增加的遗传因素。大多数已识别的遗传变异本质上是非编码的，因此很难确定它们对基因功能和传导系统生物学的影响。除了这一困难之外，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。我们的建议直接解决了这些缺陷。利用 PI、ENCODE 项目的 PI 基因组学家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实验平台，从与 GWAS 传导系统表型相关的 LD 块中识别因果 SNP，并系统地测试这些 SNP 对心脏传导肌细胞和体内转基因小鼠中假定的转录增强子功能的影响。对于该项目的 R21 阶段，我们建议生成人类心脏的“迷你编码”，在全基因组范围内绘制人类心脏中假定的功能非编码序列的坐标。我们将把这些信息与包含与传导系统性状相关的非编码 SNP 的 50 个基因座中远距离染色质相互作用的 3D 图叠加。总之，这些数据将指出含有与传导系统特征相关的 SNP 的假定传导系统增强子的位置。在 R33 阶段，我们将利用多层平台从功能上探讨这些 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，我们开发了一种策略，从诱导多能干细胞（iPSC）中衍生出传导系统心肌细胞。这些细胞非常适合涉及传导系统生物学的功能实验。我们将测试这些细胞中含有疾病相关 SNP 的候选增强子，确定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技术进一步测试传导系统增强子的子集，以证明它们的体内调节特性。总之，我们提出的计划描述了一种逻辑的、逐步的方法来识别 LD 块内与心脏传导系统参数相关的因果 SNP，并开发了一个新颖的综合实验平台来从功能上确定这些与疾病相关的非编码 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.