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

项目摘要/摘要 心电图QT间期是一项反映心室复极的指标，是一种临床相关的遗传性指标。 与心律失常和心脏性猝死(SCD)风险相关的数量性状。遗传学研究 在罕见的孟德尔长QT间期综合征和短QT间期综合征的受试者中，发现了罕见的、严重的编码突变 ~20个基因编码电压门控离子通道、转运体和相关蛋白，调节心脏活动 电位时程(Apd)或兴奋-收缩(EC)耦合。这些基因中编码多态的流行程度 影响种群复极化变异性的基因极低。相比之下，全基因组关联 对普通人群QT间期的研究发现，普通人群中常见的非编码变异体 36个基因座，总共解释了~20%的加性方差。其中近一半的GWA微基因座 已知的基因调节心脏动作电位或内皮细胞偶联，因此可能是致病基因。尽管如此， 实际致病基因的身份和潜在的与共同的和， 这些基因座上的大部分非编码变异体仍然未知。此外，很大一部分(~80%)的添加剂 差异仍未确定。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量 几个顺式调控元件(CRE)共同影响一个GWAS信号的表达 目标基因，以及b)特定或类似疾病/性状的潜在基因通常是功能相关的，并且 功能相关的基因通常属于基因调控网络(GRN)，其中的成员是共同调控的。 我们的目标是利用可能的致病基因的先验证据对选定的QT间期Gwas基因座进行功能特征分析 以及通过评估干扰已知QT间期表达的影响来扩大基因发现 他们的GRN上的基因。我们的具体目标是：(1)识别特定QT间期的功能性Cre变异体 通过评估所有与心脏开放染色质区域重叠的性状相关常见变异 在小鼠心肌细胞HL1细胞中的高通量报告分析；并将Cres及其变异体与 基于成人基因表达、数量性状基因座和染色质接触分析的可能靶基因 左心室组织和诱导多能干细胞来源的心肌细胞(hiPSC-CMS)；以及(2) 新生大鼠部分QT间期基因的CRISPR激活/干扰表达扰动 心室肌细胞(NRVMs)，并评估转录组的影响以确定候选的共同调节基因 SET；评估共调控的基因组在阈值以下的GWA基因座的富集性；重叠的共调控 用QT Gwas基因座识别可能的致病基因；并评估干扰表达的影响 NRVMS和HiPSC-CMS中新发现的细胞电生理学基因。总之，拟议的研究 有望更好地理解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GWA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.