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

EDN1 expression was 50-fold higher in ECs than in VSMCs.

“Though EDN1 expression was differentially regulated in both ECs and VSMCs, there was a 50-fold higher expression in ECs (Figure S3C).”

WT and Δ88 iPSCs differentiated into neural crest progenitor cells showed no differential gene expression.

“There was no differential gene expression between wild-type (WT) and Δ88 iPSCs differentiated to a non-vascular cell type, neural crest progenitor cells (Figure S3B).”

Human tissue epigenomic data showed an enhancer mark at rs9349379 only in aorta.

“Epigenomic data from human tissue revealed an enhancer signature at rs9349379 exclusively in aorta, suggesting a regulatory function for this SNP in the vasculature.”

• Also stated in: § A Vascular-Specific Regulatory Region at rs9349379 · plain

  “In these aortic samples, a strong enhancer signal overlies rs9349379 (Figure 2B), spanning 1 kb.”

In the UK Biobank PheWAS, rs9349379 showed no significant association with any other diseases.

“There was no significant association of rs9349379 with any other diseases in the UK Biobank PheWAS (Figure S1C).”

Gene set enrichment analysis showed strong enrichment for blood vessel development (FDR=1.64e-10), vasculature development (FDR=2.98e-10), and blood vessel morphogenesis (FDR=2.21e-9).

“Gene set enrichment analysis of the differentially expressed genes showed strong enrichment for blood vessel development (FDR = 1.64 × 10−10), vasculature development (FDR = 2.98 × 10−10), and blood vessel morphogenesis (FDR = 2.21 × 10−9).”

No H3K27Ac signal was detected at the 6p24 locus in the ENCODE phase 1 and 2 datasets.

“However, there was no detectable H3K27Ac signal at this locus in these datasets (Figure 2A).”

Five other genes within 1 Mb of rs9349379, including PHACTR1, showed no significant expression difference in either cell type.

“Expression of five other genes in the 1-Mb region surrounding rs9349379, including PHACTR1, did not differ significantly for either cell type.”

CRISPR-edited stem cell-derived endothelial cells show that rs9349379 controls EDN1 expression.

“CRISPR-edited stem cell-derived endothelial cells demonstrate rs9349379 regulates expression of endothelin 1 (EDN1), a gene located 600 kb upstream of PHACTR1.”

• Also stated in: § Targeted Deletion at the 6p24 Regulatory Element Increases EDN1 Expression in Vascular Cells · plain

  “In both iPSC-derived ECs and VSMCs, loss of the 88 bp flanking rs9349379 resulted in higher expression of the endothelin-1 (EDN1) gene compared with wild-type lines (Figure 3B).”

• AssumptionRests on an assumption: The genome-edited pluripotent stem cell-derived endothelial and smooth muscle cells faithfully model human vascular cell biology and the in vivo regulation of EDN1 by rs9349379.

The gene set enrichment results from Δ88 cells overlapped significantly with those from A/A versus G/G ESC-ECs.

“There was significant overlap in the gene set enrichment analysis represented by these differentially regulated genes and those from RNA-seq of the A/A and G/G ESC-ECs.”

The UK Biobank PheWAS reproduced prior associations for CAD and migraine headache (CAD p=1.16e-11; migraine p=0.000717).

“The UK Biobank PheWAS confirmed the previously published associations for CAD and migraine headache (Figure 1B; CAD p = 1.16 × 10−11; migraine p = 0.000717).”

After adjustment for rs9349379, no second independent CAD/MI signal remained at 6p24.

“This conditional analysis, after adjusting for rs9349379, showed no evidence of a second independent CAD/myocardial infarction (MI) association at this locus (Figure S1).”

EDN1, located over 600 kb away, was the only differentially regulated gene within 2 Mb of rs9349379.

“EDN1, a gene over 600 kb distal to rs9349379, was the only differentially regulated gene within 2 Mb of rs9349379 (Figure 4C).”

RNA-seq in six WT and six Δ88 cells identified 423 differentially regulated genes.

“RNA-seq was also conducted in six WT and six Δ88 cells, and, of the 173,259 analyzed transcripts, there were 423 differentially regulated genes (Figure S4F).”

In aortic samples, a strong enhancer signal spanning 1 kb overlapped rs9349379.

“In these aortic samples, a strong enhancer signal overlies rs9349379 (Figure 2B), spanning 1 kb.”

The PICS algorithm assigned rs9349379 a greater than 99% probability of being the causal SNP given the local haplotype structure.

“The Probabilistic Identification of Causal SNPs (PICS) algorithm (Farh et al., 2015) also identifies rs9349379 as the causal SNP, given the haplotype structure at the locus, with greater than 99% probability (Table S1).”

• cites Farh et al., 2015 sourceNot checked yet

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Differentially regulated genes included vascular wall mediators such as COL4A1, COL3A1, and FN1.

“The differentially regulated genes included several mediators of vascular wall integrity and structure, such as COL4A1, COL3A1, and FN1 (Figure S4E), which may be downstream effects of increased EDN1 expression in the G/G ESC-ECs (Amiri et al., 2004; Shi-Wen et al., 2001).”

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• cites Shi-Wen et al., 2001 sourceNot checked yet

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In iPSC-derived ECs and VSMCs, deleting the 88 bp around rs9349379 raised EDN1 expression relative to wild-type cells.

“In both iPSC-derived ECs and VSMCs, loss of the 88 bp flanking rs9349379 resulted in higher expression of the endothelin-1 (EDN1) gene compared with wild-type lines (Figure 3B).”

• AssumptionRests on an assumption: The genome-edited pluripotent stem cell-derived endothelial and smooth muscle cells faithfully model human vascular cell biology and the in vivo regulation of EDN1 by rs9349379.

All four aortic samples showed robust H3K27 acetylation at rs9349379.

“All four demonstrate robust H3K27 acetylation at rs9349379 (Figure S2).”

Genetic fine mapping singled out rs9349379 as the likely causal variant at 6p24.

“Through genetic fine mapping, we prioritized rs9349379, a common SNP in the third intron of the PHACTR1 gene, as the putative causal variant.”

• Also stated in: § Fine Mapping Prioritizes a Causal Variant at 6p24 · plain

  “The strongest association was for a single variant, rs9349379, with an association of 1.81 × 10−42 (odds ratio [OR] = 1.14), far exceeding the association signal for the next most highly associated SNP, rs78145402 (OR = 1.08, 4.69 × 10−17).”

Among 38,817 European-ancestry UK Biobank participants, the rs9349379 G allele was linked to lower arterial stiffness index (β=-0.077; 95% CI -0.034 to -0.119; p=0.0004).

“In 38,817 participants of European ancestry, the G allele at rs9349379 was associated with decreased arterial stiffness index, with β = −0.077 (95% confidence interval [CI]: −0.034, −0.119), p = 0.0004.”

IL-1α stimulation increased EDN1 expression in both WT and Δ88 ECs at mRNA (p=0.005) and protein (p=0.0004) levels.

“Stimulation with IL-1α increased EDN1 expression both in WT and D88 ECs at the mRNA (Figure 3D; p = 0.005) and protein levels (Figure 3E; p = 0.0004).”

A possible overlapping contact area was found intergenic to EDN1 and PHACTR1.

“There was a possible area of overlap intergenic to EDN1 and PHACTR1 (Figure 5A).”

The study demonstrates regulation of a gene located 600 kb from the causal variant.

“Here we demonstrate the regulation of a gene 600 kb from the causal variant.”

In this study, the causal variant's cis-regulatory effect was a 20% increase in EDN1 expression in genome-edited ECs homozygous for the G allele.

“In this study, the cis-regulatory effect of the causal variant is a 20% increase in EDN1 expression in the genome-edited ECs homozygous for the G allele.”

• AssumptionRests on an assumption: The genome-edited pluripotent stem cell-derived endothelial and smooth muscle cells faithfully model human vascular cell biology and the in vivo regulation of EDN1 by rs9349379.

This common contact site contains a series of strong enhancers, as shown by H3K27Ac signal.

“This common contact site also contains a series of strong enhancers, as demonstrated by H3K27Ac signal.”

• AssumptionRests on an assumption: The common contact site about 300 kb from both EDN1 and rs9349379 is the route by which the distal regulatory effect on EDN1 occurs.

In 99 healthy plasma samples, the rs9349379 G genotype was significantly associated with higher Big ET-1 levels under an additive model (p=0.00136).

“We found a significant association between G genotype at rs9349379 and higher Big ET-1 levels using an additive model of regression (Figure 6; p = 0.00136).”

4C-seq from the rs9349379 site showed a smaller contact region spanning chr6:12,690,000-13,350,000.

“4C-seq from the rs9349379 site demonstrated a smaller contact region that extended from chr6:12,690,000–13,350,000.”

The data point to a common contact site about 300 kb from both the EDN1 promoter and the rs9349379 regulatory element.

“Instead, the data point to a common contact site approximately 300 kb from both the EDN1 promoter and rs9349379 regulatory element.”

• AssumptionRests on an assumption: The common contact site about 300 kb from both EDN1 and rs9349379 is the route by which the distal regulatory effect on EDN1 occurs.

This region had high H3K27Ac signal in ECs, consistent with a super enhancer.

“Notably, this region featured high levels of H3K27Ac signal in ECs, consistent with a super enhancer (Figure 5B).”

Homozygous minor ESC-ECs produced more ET-1 protein in supernatants at 24 hr with or without IL-1α (unstimulated p=2.12e-5; IL-1α stimulated p=0.0006).

“The homozygous minor ESC-ECs also produced more ET-1 protein in the cell-free supernatants at 24 hr in the presence or absence of IL-1α stimulation (Figure 4E; unstimulated p = 2.12 × 10−5 and IL-1α stimulated p = 0.0006).”

The EDN1 promoter contacts multiple sites up to 500 kb away in both the 3′ and 5′ directions.

“The EDN1 promoter contacts multiple sites extending up to 500 kb in both the 3′ and 5′ directions.”

The common contact region contains four distinct H3K27Ac peaks.

“The common contact region contains four distinct peaks of H3K27Ac signal.”

The same chromatin structure appeared in 4C-seq from aortic ECs, iPSC-derived ECs, aortic VSMCs, and primary human monocytes.

“The same chromatin structure was seen in 4C-seq from aortic ECs, iPSC-derived ECs, aortic VSMCs, and primary human monocytes (Figure S5).”

Statistical evidence makes rs9349379 the probable causal SNP for the multiple vascular disease associations at 6p24.

“Thus, statistical evidence prioritizes rs9349379 as a likely causal SNP for the multiple vascular disease associations at this locus.”

Together, the genetic results point to a role for rs9349379 in human endothelial cell function.

“In aggregate, these genetic data suggest a role for rs9349379 in human endothelial cell function, and our subsequent experiments prioritized the use of endothelial cells to characterize underlying mechanisms.”

The aortic epigenomic data suggest that a gene regulatory function operates at this locus in vascular tissue.

“These data suggest a gene regulatory function exists at this locus in vascular tissue.”

The known vascular actions of EDN1 can account for the risk pattern seen across the five associated diseases.

“The known physiologic effects of EDN1 on the vasculature may explain the pattern of risk for the five associated diseases.”

• AssumptionRests on an assumption: The genome-edited pluripotent stem cell-derived endothelial and smooth muscle cells faithfully model human vascular cell biology and the in vivo regulation of EDN1 by rs9349379.
• AssumptionRests on an assumption: A roughly 20% change in EDN1 expression caused by rs9349379 is large enough to influence risk of the five vascular diseases.

Deleting the small regulatory region at rs9349379 increases EDN1 expression and ET-1 protein production in ECs and, less strongly, in VSMCs.

“These data demonstrate that deletion of a small region of apparent regulatory DNA at rs9349379 results in increased EDN1 expression and endothelin-1 (ET-1) protein production in ECs and to a lesser extent in VSMCs.”

• AssumptionRests on an assumption: The genome-edited pluripotent stem cell-derived endothelial and smooth muscle cells faithfully model human vascular cell biology and the in vivo regulation of EDN1 by rs9349379.

The PHACTR1 region at 6p24 is linked by genome-wide studies to five vascular conditions: high blood pressure, fibromuscular dysplasia, cervical artery dissection, migraine, and coronary heart disease.

“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, fibro-muscular dysplasia, and hypertension.”

• Also stated in: § Introduction · plain

  “Common DNA sequence variants at 6p24 have shown genome-wide significant association with early-onset myocardial infarction (Myocardial Infarction Genetics Consortium et al., 2009), CAD (CARDIoGRAMplusC4D Consortium et al., 2013; Nikpay et al., 2015), coronary artery calcification (O'Donnell et al., 2011), migraine headache (Anttila et al., 2013), cervical artery dissection (Debette et al., 2015),”

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• cites Anttila et al., 2013 sourceNot checked yet

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• cites Debette et al., 2015 sourceNot checked yet

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The distal cis-regulatory effect at 6q24 does not appear to occur through canonical enhancer-promoter contact.

“The mechanism of the distal cis-regulatory effect at 6q24 does not appear to be through canonical enhancer-promoter contact.”

• AssumptionRests on an assumption: The common contact site about 300 kb from both EDN1 and rs9349379 is the route by which the distal regulatory effect on EDN1 occurs.

The authors' data indicate that greater endothelial ET-1 production and subsequent ETA receptor binding promote atherosclerosis.

“Our data indicate that increased endothelial production of ET-1 and subsequent binding to the ETA receptor promotes atherosclerosis.”

• AssumptionRests on an assumption: The genome-edited pluripotent stem cell-derived endothelial and smooth muscle cells faithfully model human vascular cell biology and the in vivo regulation of EDN1 by rs9349379.
• AssumptionRests on an assumption: A roughly 20% change in EDN1 expression caused by rs9349379 is large enough to influence risk of the five vascular diseases.

The long-range chromatin loops suggest that rs9349379 regulates EDN1 without a traditional enhancer-promoter interaction.

“The significance of these long-range chromatin loops in the regulation of EDN1 merits further study, but they suggest that the regulatory effect of rs9349379 on EDN1 is not through traditional enhancer-promoter interactions.”

The link between the G allele and lower systolic blood pressure may be explained by ET-1 acting through the ETB receptor.

“Finally, the association of the G allele with lower systolic blood pressure (Surendran et al., 2016) may be explained by the action of ET-1 on the second endothelin receptor, ETB.”

• cites Surendran et al., 2016 sourceNot checked yet

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In summary, several lines of evidence indicate that a common DNA variant linked to five vascular diseases regulates EDN1 more than 600 kb away.

“In summary, we provide several lines of evidence that a common DNA variant associated with five vascular diseases regulates a gene, EDN1, located over 600 kb upstream in genomic distance.”

• AssumptionRests on an assumption: The genome-edited pluripotent stem cell-derived endothelial and smooth muscle cells faithfully model human vascular cell biology and the in vivo regulation of EDN1 by rs9349379.
• AssumptionRests on an assumption: A roughly 20% change in EDN1 expression caused by rs9349379 is large enough to influence risk of the five vascular diseases.