High-throughput identification of causal variants underlying cardiac arrhythmia-related GWAS hits

项目摘要 心律失常是一个主要的临床问题，并可能导致心脏性猝死。基因组- 广泛关联研究（GWAS）已经确定了越来越多的与心脏病相关的序列变异， 心律失常和相关的心电图（ECG）特征，但这些GWAS命中的大多数属于非 编码区和它们的功能作用难以破译。我们假设大多数功能性的 与心律失常相关的非编码变体属于心脏顺式调节元件（克雷斯;即， 增强子/启动子），并通过破坏转录因子（TF）结合位点发挥其作用，从而 改变编码心脏蛋白，特别是离子通道及其调节因子的基因的表达水平。 确定潜在的心脏瓣膜相关GWAS命中的因果变异，并绘制瓣膜相关 克雷斯，我们建议实施一种称为CRE-seq（顺式调控元件测序分析）的技术。 在CRE-seq中，单个克雷斯与报告基因融合，每个基因含有独特的DNA条形码。所得 将由数千个构建体组成的CRE-报告基因文库导入活组织中， 通过使用RNA-seq计数条形码转录本来定量表达。CRE-seq有望大大加速 我们测量心脏疾病中顺式调节变体的影响的能力。为了实现这一目标，我们建议 两个具体目标。在目标1中，我们将使用CRE-seq在所有已知的GWAS中鉴定因果顺式调节变体， 与心律失常和相关性状相关的基因座。我们将测量顺式调节活性， 在小鼠体内心脏和人iPSC衍生的心肌细胞中， 腺相关病毒（AAV）介导的CRE-seq文库递送。然后，我们将评估 使用含有所有已知人类TF的蛋白质微阵列对TF结合的选择变体。最后，我们将 将我们的CRE-seq分析结果与心脏eQTL数据相关联。在目标2中，我们将建立一个模板 通过绘制人类心脏克雷斯的位置来解释罕见的心肌炎相关变异， 顺式调控逻辑我们将利用“捕获和克隆”策略进行CRE-seq文库构建， 允许分析长（即，~500 bp）在每个基因座上平铺报告基因。通过这种方式，我们将确定必要的TF 结合位点（TFBS），它们是罕见功能变体的可能靶点。接下来，我们将使用CRE-seq来分析 将所有可能的单核苷酸取代引入已鉴定的TFBS的效果。如目标1所示，我们将 在小鼠心脏和人iPSC衍生的心肌细胞中进行CRE-seq。综合来看，这两个 目的将使人类基因组中常见和罕见变异的功能解释成为可能， 从而便于评估患者的心脏病风险。

Project Summary Cardiac arrhythmias are a major clinical problem and can predispose to sudden cardiac death. Genome- wide association studies (GWAS) have identified a growing number of sequence variants associated with cardiac arrhythmias and related electrocardiogram (ECG) traits, but the majority of these GWAS hits fall within non- coding regions and their functional effects are difficult to decipher. We hypothesize that the majority of functional non-coding variants related to cardiac arrhythmias fall within cardiac cis-regulatory elements (CREs; i.e., enhancers/promoters), and exert their effects by disrupting transcription factor (TF) binding sites and thereby altering the expression level of genes encoding cardiac proteins, especially ion channels and their regulators. To identify causal variants underlying cardiac arrhythmia-related GWAS hits and to map arrhythmia-related CREs, we propose to implement a technique called CRE-seq (Cis-Regulatory Element analysis by sequencing). In CRE-seq, individual CREs are fused to reporter genes, each containing a unique DNA barcode. The resultant CRE-reporter library, consisting of thousands of constructs, is introduced into living tissue, and reporter gene expression is quantified by counting barcoded transcripts with RNA-seq. CRE-seq promises to greatly accelerate our ability to measure the effects of cis-regulatory variants in cardiac disease. To achieve this goal, we propose two Specific Aims. In Aim 1, we will use CRE-seq to identify causal cis-regulatory variants at all known GWAS loci associated with cardiac arrhythmias and related traits. We will measure the cis-regulatory activity of thousands of wild-type and variant CREs in mouse heart in vivo and in human iPSC-derived cardiomyocytes via adeno-associated virus (AAV)-mediated CRE-seq library delivery. We will then evaluate the functional effects of selected variants on TF binding using protein-microarrays containing all known human TFs. Lastly, we will correlate the results of our CRE-seq analyses with cardiac eQTL data. In Aim 2, we will establish a template for interpreting rare arrhythmia-related variants by mapping the location of human cardiac CREs and elucidating their cis-regulatory logic. We will utilize a 'capture and clone' strategy for CRE-seq library construction, which permits analysis of long (i.e., ~500 bp) tiled reporters at each locus. In this way, we will pinpoint essential TF binding sites (TFBSs) which are the likely targets of rare functional variants. Next, we will use CRE-seq to analyze the effects of introducing all possible single-nucleotide substitutions into identified TFBSs. As in Aim 1, we will perform CRE-seq in both mouse heart and human iPSC-derived cardiomyocytes. Taken together, these two Aims will enable functional interpretation of both common and rare variants in individual human genomes and thereby facilitate assessment of cardiac disease risk in patients.