A Genetic Variant Associated with Five Vascular Diseases Is a Distal Regulator of Endothelin-1 Gene Expression

Abstract

Genome-wide association studies (GWASs) implicate the PHACTR1 locus (6p24) in risk for five vascular diseases, including coronary artery disease, migraine headache, cervical artery dissection, fibromuscular dysplasia, and hypertension. Through genetic fine mapping, we prioritized rs9349379, a common SNP in the third intron of the PHACTR1 gene, as the putative causal variant. Epigenomic data from human tissue revealed an enhancer signature at rs9349379 exclusively in aorta, suggesting a regulatory function for this SNP in the vasculature. CRISPR-edited stem cell-derived endothelial cells demonstrate rs9349379 regulates expression of endothelin 1 (EDN1), a gene located 600 kb upstream of PHACTR1. The known physiologic effects of EDN1 on the vasculature may explain the pattern of risk for the five associated diseases. Overall, these data illustrate the integration of genetic, phenotypic, and epigenetic analysis to identify the biologic mechanism by which a common, non-coding variant can distally regulate a gene and contribute to the pathogenesis of multiple vascular diseases.

Keywords: SNP; cardiovascular diseases; coronary artery disease; endothelial cells; endothelin-1; epigenomics; genetic enhancer elements; genome-wide association study; hypertension; migraine disorders.

Figure 1. Lead SNP in 6p24 Locus, rs9349379, Is Associated with Multiple Vascular Diseases

(A) Locus Zoom Plot of 6p24 associations for CAD/MI. Each variant identified in 1000 Genomes Project sequencing is represented by a dot, with color representing linkage disequilibrium (LD) with the lead SNP. A single variant, rs9349379, demonstrates the most significant association with CAD/MI (p = 1.81 × 10⁻⁴¹). No variants in the locus are in significant LD with rs9349379 (r² >0.5). (B) Multiple disease associations of rs9349379 minor allele (G) with vascular disorders. The minor allele (G) is associated with increased risk of CAD and coronary calcification. The minor allele is alternatively associated with reduced risk of cervical dissection, migraine headache, fibromuscular dysplasia, and hypertension. CAD and migraine headache associations are confirmed in UK Biobank data.

Figure 2. H3K27 Acetylation Identifies a Vascular-Specific Regulatory Element at rs9349379

(A) Chromatin immunoprecipitation sequencing (ChIP-seq) for H3K27Ac signal at the 6p24 locus shows no evidence of an enhancer or promoter element within 100 kb of rs9349379 in non-vascular tissues. (B) Arterial tissue from the Aorta shows a strong H3K27Ac peak at rs9349379, with no signal at the locus for H3K4me3, H3K36me3, H3K27me3, or H3K9me3. All data are plotted as rpm/bp for ChIP-seq conducted in one representative tissue sample of each type.

Figure 3. Targeted Deletion at 6p24 Causal SNP Increases EDN1 Expression in Stem Cell-Derived Vascular Cells

(A) Generation of cell lines with 88-bp deletion at rs9349379, with two sgRNAs flanking the rs9349379 regulatory region. (B) iPSC-derived ECs and VSMCs have increased EDN1 expression after deletion of the rs9349379 regulatory region (Δ88) with flanking CRISPR/Cas9 nucleases. Other genes in the 6p24 locus are minimally expressed and are not significantly altered (*p = 0.028 and **p = 0.01). (C) ESC-derived ECs have increased EDN1 expression with Δ88 deletion (*p = 0.0004). (D) EDN1 expression increases in both WT and Δ88 iPSC-ECs in response to IL-1α stimulation (50 ng/mL) (*p = 0.03 and **p = 0.005). (E) Increased Big ET-1 protein level before and after IL-1α stimulation in Δ88 iPSC-derived ECs (*p = 0.003 and **p = 0.0004). All data represent mean ± SEM of three independently differentiated clones in each group.

Figure 4. Allelic Series at rs9349379 Demonstrates cis-Regulatory Effect on EDN1 Expression

(A) Generation of an allelic series of stem cell lines at rs9349379. A heterozygous stem cell line (Hues66) was edited to first insert PAM sites, and then it was edited in a second round to generate three homozygous clones for both the major (A) and minor (G) alleles. (B) Differential gene expression analysis of RNA-seq from AA versus GG ESC-derived ECs shows 273 differentially expressed transcripts (FDR < 0.05), with clustering by genotype at rs9349379. (C) RNA-seq expression for genes in the 6p24 locus demonstrates significantly increased expression of EDN1 in G/G ESC-derived ECs compared with A/A isogenic control. Other locus genes show minimal expression in transcripts per million reads (tpm). (D) The qPCR in separately differentiated ESC-ECs validates the expression difference for EDN1 (*p = 7.59 × 10⁻⁷) and TBC1D7 (**p = 1.75 × 10⁻⁵). (E) Increased Big ET-1 expression before and after IL-1α stimulation in homozygous minor (G/G) ESC-derived ECs (*p = 2.12 × 10⁻⁵ and **p = 0.0006). (F) Homozygous minor ESC-derived VSMCs demonstrate higher expression of EDN1 (*p = 0.012), while no other 6p24 locus genes show differential expression. All data represent mean ± SEM of three independently differentiated clones of each genotype.

Figure 5. Very Low Contact between EDN1 Promoter and rs9349379 by 3D Chromatin Structure

(A) 4C-seq in human coronary artery ECs demonstrates 3D chromatin loops from the EDN1 promoter viewpoint (red) and rs9349379 SNP viewpoint (blue). The EDN1 promoter contacts multiple sites extending up to 500 kb in both the 3′ and 5′ directions. 4C-seq from the rs9349379 viewpoint demonstrates a smaller contact region that extends from chr6:12,690,000 to 13,350,000. There is a common contact area intergenic to EDN1 and PHACTR1 (*). (B) Super enhancer at chr6:12,490,000–12,590,000 seen in H3K27Ac ChIP-seq in human umbilical vein endothelial cell (HUVEC), iPSC-derived EC, and embryonic-derived EC. The super enhancer is within the same transcriptionally associated domain as EDN1, and it also includes the common contact sites of the EDN1 promoter and rs9349379 regulatory element. HUVEC, iPSC-EC-B, and embryonic-EC were generated from publicly available data. (C) Demonstration of strong enhancer activity for each of the four discrete regions that comprise the super enhancer. The 2-kb segments were tested in a luciferase expression assay and compared with empty vector (EV); each region had a 2- to 12-fold induction of luciferase signal.

Figure 6. Minor Allele at rs9349379 Increases Plasma Levels of Big ET-1 in Healthy Subjects

Association between circulating levels of ET-1 precursor protein and genotype at rs9349379 in human plasma samples. Each copy of G allele results in higher Big ET-1 expression (n=33 for each rs9349379 genotype; p = 0.00136, additive model of regression).

Figure 7. The Relationship between rs9349379 Genotype, Endothelin-1, and Risk of Five Vascular Diseases

(A) The cis-regulatory effect of the G allele at rs9349379 is associated with higher EDN1 expression and higher ET-1 secreted protein. The G allele at rs9349379 is associated with increased risk of CAD/MI and lower risk of migraine headache, cervical artery dissection, fibromuscular dysplasia, and hypertension. (B) Proposed model for ET-1 function and risk of five vascular diseases. Activation of ET_A receptor results in vasoconstriction, VSMC proliferation, ECM production, and fibrosis. These downstream effects of ET-1 result in increased risk of CAD/MI and decreased risk of migraine headache, cervical artery dissection, and fibromuscular dysplasia. ET_B receptors are predominantly in large arteries and the renal collecting system. Higher ET-1 levels may result in hypotension via ET_B-induced nitric oxide and prostacyclin production, resultant vasodilation, diuresis, and natriuresis.