Rationale: Genome-wide association studies have identified a large number of common variants (single-nucleotide polymorphisms) associated with atrial fibrillation (AF). These variants are located... Show moreRationale: Genome-wide association studies have identified a large number of common variants (single-nucleotide polymorphisms) associated with atrial fibrillation (AF). These variants are located mainly in noncoding regions of the genome and likely include variants that modulate the function of transcriptional regulatory elements (REs) such as enhancers. However, the actual REs modulated by variants and the target genes of such REs remain to be identified. Thus, the biological mechanisms by which genetic variation promotes AF has thus far remained largely unexplored. Objective: To identify REs in genome-wide association study loci that are influenced by AF-associated variants. Methods and Results: We screened 2.45 Mbp of human genomic DNA containing 12 strongly AF-associated loci for RE activity using self-transcribing active regulatory region sequencing and a recently generated monoclonal line of conditionally immortalized rat atrial myocytes. We identified 444 potential REs, 55 of which contain AF-associated variants (P<10(-8)). Subsequently, using an adaptation of the self-transcribing active regulatory region sequencing approach, we identified 24 variant REs with allele-specific regulatory activity. By mining available chromatin conformation data, the possible target genes of these REs were mapped. To define the physiological function and target genes of such REs, we deleted the orthologue of an RE containing noncoding variants in theHcn4(potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channel 4) locus of the mouse genome. Mice heterozygous for the RE deletion showed bradycardia, sinus node dysfunction, and selective loss ofHcn4expression. Conclusions: We have identified REs at multiple genetic loci for AF and found that loss of an RE at theHCN4locus results in sinus node dysfunction and reduced gene expression. Our approach can be broadly applied to facilitate the identification of human disease-relevant REs and target genes at cardiovascular genome-wide association studies loci. Show less
Ouwerkerk, A.F. van; Bosada, F.M.; Duijvenboden, K. van; Hill, M.C.; Montefiori, L.E.; Scholman, K.T.; ... ; Christoffels, V.M. 2019
Disease-associated genetic variants that lie in non-coding regions found by genome-wide association studies are thought to alter the functionality of transcription regulatory elements and target... Show moreDisease-associated genetic variants that lie in non-coding regions found by genome-wide association studies are thought to alter the functionality of transcription regulatory elements and target gene expression. To uncover causal genetic variants, variant regulatory elements and their target genes, here we cross-reference human transcriptomic, epigenomic and chromatin conformation datasets. Of 104 genetic variant regions associated with atrial fibrillation candidate target genes are prioritized. We optimize EMERGE enhancer prediction and use accessible chromatin profiles of human atrial cardiomyocytes to more accurately predict cardiac regulatory elements and identify hundreds of sub-threshold variants that colocalize with regulatory elements. Removal of mouse homologues of atrial fibrillation-associated regions in vivo uncovers a distal regulatory region involved in Gja1 (Cx43) expression. Our analyses provide a shortlist of genes likely affected by atrial fibrillation-associated variants and provide variant regulatory elements in each region that link genetic variation and target gene regulation, helping to focus future investigations. Show less
