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

高通量识别心律失常相关 GWAS 命中的因果变异

基本信息

批准号：
10191029
负责人：
JOSEPH CORBO
金额：
$ 72.45万
依托单位：
WASHINGTON UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-07-01 至 2024-04-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10191029
关键词：
Address Affinity Arrhythmia Bar Codes Binding Binding Sites Biological Assay Cardiac Cardiac Myocytes Chromatin Clinical Complement DNA Data Dependovirus Diagnosis Disease Electrocardiogram Enhancers Etiology Evaluation Future Gene Expression Genes Genomic Segment Genotype-Tissue Expression Project Goals Heart Heart Diseases Human Human Activities Human Genome Individual Ion Channel Knowledge Lead Libraries Linkage Disequilibrium Location Logic Maps Measures Mediating Mus Mutation Nucleotides Paired Comparison Patients Play Process Protein Microchips Proteins Regulatory Element Reporter Reporter Genes Resolution Role Techniques Technology Tissues Transcript Untranslated RNA Variant base causal variant disorder risk falls genetic disorder diagnosis genome wide association study genome-wide heart disease risk human data improved in vivo individual patient induced pluripotent stem cell interest new technology novel promoter rare variant risk stratification sudden cardiac death trait transcription factor transcriptome sequencing

项目摘要

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.

项目总结