Identification of atrial fibrillation associated genes and functional non-coding variants

Abstract

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 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 co-localize 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.

Fig. 1

RNA-seq of human adult and fetal atria. a MA plot of left atrial and right atrial whole tissue expression (RPK). A total of 15,239 genes with RPKs > 1 are detected in both samples. b Volcano plot analysis of left versus right adult atrial expression. c Heatmap of left and right atria (n = 3) shows that these tissues cluster together. d Volcano plot of adult left atrial tissue versus PCM1-sorted left atrial nuclei (i.e., CM nuclei). e Heatmap of whole tissue (n = 3) and PCM1-enriched left atria (n = 2). f Volcano plot analysis of adult (n = 3) versus fetal (n = 2) atrial expression. g GO terms of top 500 expressed genes in fetal atria with rank and p-value. h GO terms of top 500 expressed genes in adult atria with rank and p-value. p-values were calculated using DAVID (based on Fischer-exact test) and Benjamini-Hochberg corrected. RPK reads per kilo base, PCM1 pericentriolar material 1, GO Gene Ontology

Fig. 2

Scoring and target genes. a Boxplot showing distribution of all genes included in this study. b Top 10 functional GO terms for all genes with score > 11 and p-value of enrichment. c Top 50 functional GO terms categorized. d Summary of potential AF target genes with a score > 11. Locus number, nearest gene, and target genes per locus are shown, with their scores. GO Gene ontology, AF atrial fibrillation

Fig. 3

Target genes and variant enhancer of SPATS2L locus. a SPATS2L locus showing TAD, variant region, (sub)threshold variants, CM PCHi-C, and RNA-seq. b Zoom of part of the variant region, showing PCHi-C and epigenetic datasets EMERGE and LA ATAC-seq. rs281766 is highlighted in yellow. c Information of variant rs281766, d TBX5 recognition motif and minor and major alleles of variant rs281766. e Table showing PCHi-C interaction with variant regions, expression per gene and final scores for the analyzed genes. TAD Topologically Associated Domain, CM cardiomyocyte, PCHi-C promoter capture Hi-C

Fig. 4

RNA-seq enrichment of AF target genes. Enrichment of expression of AF target genes (score > 11, −1 > L2FC > 1, padj < 0.05), comparing a enrichment of expression of LA and RA, b CM and whole tissue, c fetal and adult atria, and d zoom of fetal and adult atria. p-values are calculated with DESeq2 package (based on Wald-test) and corrected for multiple testing using false discovery rate method of Benjamini-Hochberg. Source data of all panels are provided as Source Data files. L2FC Log2-fold change, padj adjusted p-value, CM cardiomyocyte, LA left atria, RA right atria

Fig. 5

Variant region deletion shows distal regulatory element downstream of Gja1 in mouse. a Mouse region homologous to human AF associated GJA1 locus, showing gene, EMERGE and ATAC-seq tracks. Highlighted region is the 40 Kb deletion. b Browser view of human GJA1 locus, showing AF associated (subthreshold) SNPs in variant region (VR), TAD, genes, cardiac PCHi-C, ATAC-seq and EMERGE. Human homologous deleted region (highlighted). c Zoom of the human homologous deleted region (highlighted.) d Expression and PCHi-C values per gene in the TAD. e Corrected expression of genes as assessed by qPCR with Hprt and Eef1e1 as reference genes in atria and f brain. g Expression fold change of Gja1^RE1/RE1 (HOM) vs wild-type (WT) littermates as a negative control of P21 atria and h brain cortex. Source data of 5e–h are provided as Source Data files. Error bars represent SD, *p < 0.05, **p < 0.01, and ***p < 0.001 (two-tailed Student’s t-test). kb kilobase, SNP single nucleotide polymorphism, TAD topologically associated domain, PCHi-C promoter capture Hi-C, ATAC-seq assay for transposase-accessible chromatin sequencing, qPCR quantitative polymerase chain reaction, P21 postnatal day 21, SD standard deviation

Fig. 6

Identification of cardiac regulatory elements and functional AF-associated non-coding variants. a ATAC-seq motif enrichment, b Area under the receiver operating characteristic curves showing the improved prediction of the revised EMERGE, c Browser view of human heart enhancer prediction for brain, previous heart prediction and current heart prediction. hs1658 drives LacZ expression in the heart in the mouse embryo (Vista enhancer browser). d HOMER motif analysis on peak-calling ATAC-seq, EMERGE and overlapping peaks. e ATAC-seq and EMERGE peaks overlapping known AF GWAS variants, as well as variants that lie in promoters (red). f Allele-specific luciferase assay performed in iAM-1 cells. Error bars represent SD, *p < 0.05, **p < 0.01, and ***p < 0.001 (two-tailed Student’s t-test). Source data are provided as Source Data file. g SYNPO2L locus with GWAS variants, PCHi-C, ATAC-seq and EMERGE tracks. PCHi-C promoter capture Hi-C, ATAC-seq assay for transposase-accessible chromatin sequencing