Circulation research, 06 April 2020 Identification of Functional Variant Enhancers Associated with Atrial Fibrillation. van Ouwerkerk AF, Bosada F, Liu J, Zhang J, van Duijvenboden K, Chaffin M, Tucker N, Pijnappels DA, Ellinor PT, Barnett P, de Vries AA, Christoffels VM

Rationale

Genome-wide association studies (GWAS) have identified a large number of common variants (single nucleotide polymorphisms) associated with atrial fibrillation (AF). These variants are located mainly in non-coding 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 as well as 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 GWAS 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 (STARR-seq) 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 STARR-seq 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 a RE containing non-coding variants in the Hcn4 locus of the mouse genome. Mice heterozygous for the RE deletion showed bradycardia, sinus node dysfunction and selective loss of Hcn4 expression. Conclusions

We have identified REs at multiple genetic loci for AF and found that loss of an RE at the HCN4 locus 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 GWAS loci.

Circ Res. 2020 Apr