Identifying Cis-Regulatory Variants, Genes, and Regulatory Networks Underlying QT Interval Variation

项目概要/摘要 心电图QT间期是心室复极的一个指标， 与心律失常和心源性猝死（SCD）风险相关的数量性状。遗传研究 在罕见的孟德尔长QT综合征和短QT综合征受试者中， ~20个编码电压门控离子通道、转运蛋白和调节心脏活动的相关蛋白的基因 电位时程（APD）或兴奋-收缩（EC）偶联。编码多态性在这些 影响群体复极变异性的基因非常少。相比之下，全基因组关联 在一般人群中进行的QT间期研究（GWAS）已经确定了常见的非编码变异， 三打基因座，共同解释~20%的加性方差。这些GWAS基因座中几乎有一半 已知调节心脏APD或EC偶联的基因，因此可能是致病基因。不过 实际致病基因的身份和与常见疾病相关的分子机制， 在这些基因座上的大部分非编码变体仍然未知。此外，大部分（~80%）的添加剂 差异仍未查明。利用已知基因的信息，在拟议的研究中，我们将解决 我们知识中的这些差距基于以下假设：a）影响活动的多种常见变体 几个顺式调节元件（克雷斯）的共同影响表达的GWAS信号的基础， 靶基因，和B）作为特定或相似疾病/性状基础的基因通常是功能相关的，且 功能相关的基因通常属于基因调控网络（GRNs），其成员是共调控的。 我们的目的是功能性地描述选择的QT间期GWAS基因座与可能的因果基因的先验证据 以及通过评估已知QT间期表达的扰动的影响来扩展基因发现 他们的GRN基因。我们的具体目标是：（1）在选定的QT间期确定功能性CRE变异体 GWAS基因座，通过评估所有与心脏开放染色质区域重叠的性状相关的常见变异， 在小鼠心肌细胞HL 1细胞中的高通量报告基因测定;以及将克雷斯及其变体与 基于成人表达数量性状基因座和染色质接触分析的推定靶基因 左心室组织和诱导多能干细胞衍生的心肌细胞（hiPSC-CM）;和（2）进行 新生大鼠中选定QT间期基因的基于CRISPR激活/干扰的表达扰动 心室肌细胞（NRVM），并评估转录组范围的影响，以确定候选的共调节基因 组;评估共调节基因组在亚阈值GWAS基因座中的富集;重叠共调节基因组 基因与QT GWAS基因座，以确定可能的因果基因;和评估干扰表达的影响， 在NRVM和hiPSC-CM中新鉴定的细胞电生理学基因。总的来说，拟议的研究 有望更好地了解QT间期变异的分子机制 和SCD风险，并作为复杂疾病和性状的GWAS基因座的功能表征的模型。

PROJECT SUMMARY/ABSTRACT Electrocardiographic QT interval, an index of ventricular repolarization, is a clinically relevant heritable quantitative trait associated with risk for cardiac arrhythmias and sudden cardiac death (SCD). Genetic studies in subjects with rare Mendelian long- and short-QT syndromes have identified rare, severe coding mutations in ~20 genes encoding voltage-gated ion channels, transporters and associated proteins that regulate cardiac action potential duration (APD) or excitation-contraction (EC) coupling. Prevalence of coding polymorphisms in these genes affecting population repolarization variability is extremely low. In contrast, genome-wide association studies (GWAS) of QT interval in the general population have identified common, noncoding variants at nearly three dozen loci, collectively explaining ~20% of the additive variance. Nearly half of these GWAS loci harbor genes known to regulate cardiac APD or EC coupling, thus serving as the likely causal genes. Nonetheless, the identities of the actual causal genes and molecular mechanisms underlying associations with the common and, largely noncoding variants at these loci, remain unknown. Moreover, a large fraction (~80%) of the additive variance remains unidentified. Leveraging information on known genes, in the proposed studies we will address these gaps in our knowledge based on the hypotheses that a) multiple common variants that impact the activities of several cis-regulatory elements (CREs) underlie a GWAS signal by collectively influencing the expression of a target gene, and b) genes underlying specific or similar diseases/traits are often functionally related, and that functionally related genes often belong to gene regulatory networks (GRNs), members of which are co-regulated. We aim to functionally characterize selected QT interval GWAS loci with a priori evidence for likely causal genes as well as extend gene discovery by assessing the impacts of perturbing the expression of known QT interval genes on their GRNs. Our specific aims are: (1) identification of functional CRE variants at selected QT interval GWAS loci by evaluating all trait-associated common variants overlapping cardiac open chromatin regions in high-throughput reporter assays in mouse cardiomyocyte HL1 cells; and to link CREs and their variants to putative target genes based on expression quantitative trait locus and chromatin contact analyses in human adult left ventricle tissue and induced pluripotent stem cells-derived cardiomyocytes (hiPSC-CMs); and (2) performing CRISPR activation/interference-based expression perturbations of selected QT interval genes in neonatal rat ventricular myocytes (NRVMs), and assessing transcriptome-wide effects to identify candidate co-regulated gene sets; evaluating the co-regulated gene sets for enrichment in sub-threshold GWAS loci; overlapping co-regulated genes with QT GWAS loci to identify likely causal genes; and assessing the impact of perturbing expression of newly identified genes on cellular electrophysiology in NRVMs and hiPSC-CMs. Together, the proposed studies are expected to lead to a better understanding of the molecular mechanisms underlying QT interval variation and SCD risk, and serve as a model for functional characterization of GWAS loci of complex diseases and traits.