Translational Sciences

Myocardial Infarction–Associated SNP at 6p24 Interferes With MEF2 Binding and Associates With PHACTR1 Expression Levels in Human Coronary ArteriesSignificance

Mélissa Beaudoin, Rajat M. Gupta, Hong-Hee Won, Ken Sin Lo, Ron Do, Christopher A. Henderson, Claire Lavoie-St-Amour, Simon Langlois, Daniel Rivas, Stephanie Lehoux, Sekar Kathiresan, Jean-Claude Tardif, Kiran Musunuru, Guillaume Lettre
https://doi.org/10.1161/ATVBAHA.115.305534
Arteriosclerosis, Thrombosis, and Vascular Biology. 2015;35:1472-1479
Originally published April 2, 2015

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Abstract

Objective—Coronary artery disease (CAD), including myocardial infarction (MI), is the main cause of death in the world. Genome-wide association studies have identified dozens of single nucleotide polymorphisms (SNPs) associated with CAD/MI. One of the most robust CAD/MI genetic associations is with intronic SNPs in the gene PHACTR1 on chromosome 6p24. How these PHACTR1 SNPs influence CAD/MI risk, and whether PHACTR1 itself is the causal gene at the locus, is currently unknown.

Approach and Results—Using genetic fine-mapping and DNA resequencing experiments, we prioritized an intronic SNP (rs9349379) in PHACTR1 as causal variant. We showed that this variant is an expression quantitative trait locus for PHACTR1 expression in human coronary arteries. Experiments in endothelial cell extracts confirmed that alleles at rs9349379 are differentially bound by the transcription factors myocyte enhancer factor-2. We engineered a deletion of this myocyte enhancer factor-2–binding site using CRISPR/Cas9 genome-editing methodology. Heterozygous endothelial cells carrying this deletion express 35% less PHACTR1. Finally, we found no evidence that PHACTR1 expression levels are induced when stimulating human endothelial cells with vascular endothelial growth factor, tumor necrosis factor-α, or shear stress.

Conclusions—Our results establish a link between intronic SNPs in PHACTR1, myocyte enhancer factor-2 binding, and transcriptional functions at the locus, PHACTR1 expression levels in coronary arteries and CAD/MI risk. Because PHACTR1 SNPs are not associated with the traditional risk factors for CAD/MI (eg, blood lipids or pressure, diabetes mellitus), our results suggest that PHACTR1 may influence CAD/MI risk through as yet unknown mechanisms in the vascular endothelium.

Introduction

Coronary artery disease (CAD), including myocardial infarction (MI), remains the main cause of death and disability worldwide, despite a large number of efficient drugs to manage the traditional risk factors (eg, blood lipids, blood pressure, and heart rate).1 Recently, several new drugs have failed to show efficacy in large-scale clinical trials,2–4 emphasizing the need for new therapeutic targets. CAD/MI is heritable and the functional study of its genetic determinants could yield new biological pathways important for disease pathogenesis. Genome-wide association studies have already identified 45 single nucleotide polymorphisms (SNPs) robustly associated with CAD, including several markers that are not associated with the CAD epidemiological risk factors, such as hypertension, dyslipidemia, and type 2 diabetes mellitus.5 One of these CAD/MI loci is located on chromosome 6p24 and is defined by a group of SNPs in linkage disequilibrium within the third intron of the gene PHACTR1. In addition to the genetic association between PHACTR1 SNPs and MI6 or CAD,5,7–9 these 6p24 SNPs have also been robustly associated with coronary artery calcification,10 coronary artery stenosis,11 migraine,12 hemodynamic indexes,13 and cervical artery dissection.14

See accompanying editorial on page 1293

It is currently unknown if PHACTR1 is the causal gene at the locus, and if causal, what are the mechanisms underlying its effect on CAD/MI. The protein encoded by PHACTR1 was initially identified in a yeast 2-hybrid screen as a protein phosphatase 1 interactor.15 It is abundantly expressed in the nervous system, modulates protein phosphatase 1 activity in vitro and also interacts with actin.15 In human umbilical vein endothelial cells (HUVEC), it has been reported that PHACTR1 expression is induced on treatment with vascular endothelial growth factor (VEGF)16 and that PHACTR depletion induces apoptosis and decreases tube formation.17 In NIH3T3 fibroblasts, ectopically expressed PHACTR1 translocates to the nucleus after serum stimulation and this translocation depends on the competition of protein phosphatase 1 and G-actin for the PHACTR1 RPEL motifs and C-terminal domain.18 Disruption of these domains inhibits the cytoplasmic/nuclear localization of PHACTR1 and impairs actomyosin assembly.18 Recently, it was shown that PHACTR1 acts downstream of TGF-β to mediate actin reorganization and migration of breast cancer cells.19

The aim of our study was to determine if PHACTR1 is a good biological candidate for a role in CAD/MI. Here, we show that CAD/MI-associated SNPs at 6p24 correlate with PHACTR1 expression levels in coronary arteries. This result suggests that changes in PHACTR1 expression levels may modulate CAD/MI risk. We fine-mapped the association signal to an SNP in the third intron of PHACTR1, and showed that its alleles disrupt binding of the transcription factors myocyte enhancer factor (MEF) 2. Deleting this MEF2-binding site using CRISPR/Cas9 reduces PHACTR1 expression in endothelial cells. Our findings support a possible role for PHACTR1 in the vascular endothelium and provide a framework to further understand how PHACTR1 SNPs, which are not associated with the epidemiological risk factors of cardiovascular diseases, can influence CAD/MI risk.

Materials and Methods

Materials and methods are available in the online-only Data Supplement.

Results

SNPs at PHACTR1 Associate With MI in French Canadians

To characterize the genetic risk factors of CAD/MI in French Canadians, we genotyped SNPs previously associated with MI or CAD in 1176 MI cases and 1996 controls selected from the Montreal Heart Institute (MHI) Biobank (Table I in the online-only Data Supplement).5 Of the 45 SNPs that we genotyped successfully, 35 had an odds ratio consistent with the literature (binomial P=1.2×10−4) and 12 of these 35 SNPs were nominally significant (1-tailed P<0.05 and binomial P=1.5×10−6; Table II in the online-only Data Supplement). The strongest genetic association with MI risk that we observed was with rs12526453, an SNP located in the third intron of PHACTR1 (G allele odds ratio=0.79; P=8.4×10−4).6 To fine-map the genetic association between the PHACTR1 locus and MI risk in our population, we selected and genotyped 13 additional SNPs that cover common genetic variation at the locus based on genetic variation patterns in European-ancestry individuals from the 1000 Genomes Project (Table III in the online-only Data Supplement).20,21 We also imputed ungenotyped markers using reference haplotypes from the 1000 Genomes Project.21,22 After filtering on imputation quality, we analyzed association between MI and genotypes for 387 DNA sequence variants (Table IV in the online-only Data Supplement). The strongest association with MI was with a genotyped SNP, rs9349379, located ≈24 kilobases downstream of rs12526453 but still in intron 3 of PHACTR1 (G allele odds ratio=1.37; P=8.4×10−6; Figure 1; Table 1).23 When we conditioned on genotypes at rs9349379, no additional DNA markers were significantly associated with MI at the PHACTR1 locus (P>0.10).

View this table:
Table 1.

Association Results Between rs9349379 and MI in the MHI Biobank*

Figure 1.
Figure 1.

Association results between 387 genotyped or imputed DNA markers in PHACTR1 and myocardial infarction status in 3172 French Canadians from the Montreal Heart Institute Biobank. rs9349379 has the strongest association signal (P=8.4×10−6). We used the LocusZoom tool to plot association results.

We tested association between genotypes at PHACTR1 rs9349379 and several risk factors for MI in the MHI Biobank samples: hypertension and blood pressure, type 2 diabetes mellitus, low- and high-density lipoprotein–cholesterol levels, and smoking.24 After accounting for the number of phenotypes tested, none of the associations were significant (Table 1), consistent with results from large meta-analyses of genome-wide association results for these traits.25–27 This suggests that genetic variation at the PHACTR1 locus may influence MI risk through an unanticipated risk factor.

Coding Variants in PHACTR1 Are Not Associated With MI

We tried to link genetic variation at the PHACTR1 locus with a specific gene, focusing initially on the PHACTR1 gene itself. Identification of rare and functional DNA sequence variants by exon resequencing can be used to establish gene causality.28,29 As part of a parallel study, we resequenced all 14 exons of PHACTR1 in 500 early onset MI cases and 500 matched controls from the MHI Biobank (Table V in the online-only Data Supplement).30 We identified 4 novel rare exonic variants: 2 missense variants (ss836901033 [p.Ser190Pro] and ss836901061 [p.Glu196Lys]), as well as a 3′ splice site variant (exon 12, ss836901074) and a 3′ untranslated region variant (ss836901090; Table III in the online-only Data Supplement). We also queried data from the 1000 Genomes Project and found 3 additional rare nonsynonymous DNA sequence variants: 1 frameshift indel (rs36000655) and 2 missense variants (rs61746695 [p.Arg94Pro] and rs17602409 [p.Ile247Met]).

We genotyped 6 of these markers (the 3′ splice site variant ss836901074 failed assay design) in the MHI Biobank MI panel. The three 1000 Genomes Project variants were monomorphic in our DNA collection. After excluding the resequenced samples, the carrier frequencies for the remaining 3 PHACTR1 variants (ss836901033, ss836901061, and ss836901090) were 1.0% in MI cases and 0.7% in controls (0.3% and 0.1% in cases and controls, respectively, when restricting to missense variants only). The gene-based MI association results for the 3 coding variants or only the 2 missense variants were, P=0.38 and P=0.089 (In the Materials and Methods section of this article). We also genotyped the same 3 rare PHACTR1 coding variants in an additional subset of the MHI Biobank (870 MI cases and 1494 controls). The carrier frequencies were 1.4% in cases and 1.3% in controls for the 3 variants (P=0.95) and 0.2% in cases and 0.3% in controls for the 2 missense variants alone (P=0.51). Consistent with our results, rare coding variants in PHACTR1 were not associated with MI risk in a recent large-scale whole-exome sequencing project performed in 4703 MI cases and in 5090 controls (gene-based P>0.3; Table VI in the online-only Data Supplement).31 Thus, we cannot conclude that rare coding genetic variation implicates PHACTR1 in MI.

PHACTR1 rs9349379 is an eQTL in Human Coronary Arteries

Correlation between SNP genotypes and gene expression levels has also been used to identify causal genes involved in complex human diseases.32 PHACTR1 was originally identified as a gene highly expressed in the nervous system.15 We screened several human tissues and cell lines and detected PHACTR1 expression in the heart, aorta, and primary endothelial cells (Figure 2A).

Figure 2.
Figure 2.

PHACTR1 gene expression in human tissues. A, PHACTR1 expression levels were measured by quantitative polymerase chain reaction (experiment done in quadruplicates). Results were normalized on the housekeeping gene HPRT and calibrated on the expression in the heart (postinfarct). Error bars represent standard deviations. B, rs9349379 is an expression quantitative trait locus for PHACTR1 in human right coronary arteries (total n=25). The dashed line represents the best-fit regression line for PHACTR1 expression levels. HUVEC indicates human umbilical vein endothelial cells.

On the basis of these expression results and the reported association between PHACTR1 SNPs and CAD/MI, we obtained and extracted RNA from 25 human right coronary arteries to measure PHACTR1 transcript levels. These samples were from patients undergoing heart transplant at the MHI (Table VII in the online-only Data Supplement). We also genotyped PHACTR1 rs9349379 in the DNA of the same patients. We detected an association between genotypes at rs9349379 and PHACTR1 expression levels in this tissue (r2=0.21; P=0.018; Figure 2B). We also measured in the same coronary artery samples the expression of all coding genes located 1 megabase on either side of rs9349379 and found no association with genotypes at this marker (Figure I in the online-only Data Supplement). Thus, the association between genotypes and expression levels seems to be specific to PHACTR1 within this locus in human coronary arteries.

We genotyped and imputed PHACTR1 SNPs in the DNA extracted from the 25 right coronary artery donors, as described above (In the Materials and Methods section of this article; Table III in the online-only Data Supplement). When we conditioned on genotypes at rs9349379, no other markers were significantly associated with PHACTR1 expression levels, consistent with the MI genetic association results (Table VIII in the online-only Data Supplement). This suggests that rs9349379 is an expression quantitative trait locus (eQTL) for PHACTR1 in human right coronary arteries, providing an argument in favor of PHACTR1 being at least 1 of the genes at the 6p24 locus implicated in CAD/MI development. Because we could not identify an independent collection of human coronary arteries to replicate this eQTL effect, we turned to functional characterization to understand how genotypes at rs9349379 may modulate PHACTR1 expression levels.

rs9349379 Alleles Modulate MEF2 Binding at the PHACTR1 Locus

There are no genetic variants in the 1000 Genomes Project European populations in strong linkage disequilibrium with rs9349379 (no variants with r2≥0.8). We queried data from the ENCODE and Roadmap Epigenomics Projects to identify how genotypes at rs9349379 can influence PHACTR1 expression.33 There is no functional annotation in ENCODE for rs9349379. In the Roadmap Epigenomics data, chromatin state predictions based on histone tail modifications suggest that rs9349379 is located in a transcriptional enhancer in skeletal and stomach smooth muscle. However, we could not measure PHACTR1 expression in intestine smooth muscle, highlighting the importance to validate these bioinformatic predictions by direct biological experiments (Figure 2A). Although the Roadmap Epigenomics Project plans on testing several relevant human tissues for CAD/MI, notably the heart and aorta, these results are not yet publicly available.

Additional in silico searches revealed that rs9349379 lies within a predicted binding site for the transcription factors MEF2. MEF2 is a family of transcription factors encoded by 4 genes in humans (MEF2A-D) and is important for cellular differentiation and stress response.34 A previous report suggested that genetic variation in MEF2A might predispose to CAD risk,35 although this result is controversial.36,37 In the presence of the A allele at rs9349379, the DNA sequence matches perfectly the canonical MEF2-binding site except for a C-to-A change at the 5′ end of the motif (Figure 3). The G allele at rs9349379, associated with lower PHACTR1 expression in right coronary arteries, disrupts the MEF2-binding motif (Figure 3). To determine if rs9349379 is bound by MEF2, we performed electrophoretic mobility shift assays with HUVEC nuclear extracts. These experiments showed that the probe with the A allele at rs9349379, but not the G allele, is shifted by proteins in the nuclear extract (Figure 3). When we added an antibody that recognizes either MEF2A or MEF2C, we observed a supershift of probe A, indicating that MEF2 is one of the binding proteins (Figure 3). Furthermore, the interaction is specific as addition of excess unlabeled probe A, but not probe G, could efficiently disrupt the shift (Figure 3). Although we cannot completely rule out a role for MEF2C in the observed supershift, short interfering RNA–mediated knockdowns indicate that MEF2A is the most abundant MEF2 transcription factor in HUVEC that is recognized by the anti-MEF2 antibody (Figure II in the online-only Data Supplement). When we reduced the expression of MEF2A or MEF2C using short interfering RNA in HUVEC, we did not detect a reduction in PHACTR1 expression levels. However, the incomplete knockdowns of MEF2A and MEF2C (Figure II in the online-only Data Supplement), or other compensatory mechanisms, might explain the lack of effect on PHACTR1 expression.

Figure 3.
Figure 3.

PHACTR1-rs9349379 is differentially bound by myocyte enhancer factor (MEF) 2. Electrophoretic mobility shift assays with human umbilical vein endothelial cell nuclear extract. Probe C contains the canonical MEF2-binding site and acts as positive control. Probes A and G differ by their respective allele at rs9349379: the G allele disrupts the MEF2 consensus binding site. Only probe A shifts in the presence of nuclear extract (arrow, left), and the complex supershifts when an antibody against MEF2 is added (arrowhead, left). The binding between probe A and MEF2 is specific: excess unlabeled (cold) probe A, but not unlabeled probe G, competes and disrupts the interaction between labeled probe A and MEF2 (star, right).

MEF2-Binding Site at rs9349379 Controls PHACTR1 Expression In Vivo

To provide further in vivo evidence that the MEF2-binding site that overlaps with rs9349379 is important to modulate the expression of PHACTR1 in endothelial cells, we engineered its deletion using the CRISPR/Cas9 system (Figure 4A). Starting with human embryonic stem cells, we introduced a guide RNA that specifically targets rs9349379. After screening, we identified a clone that carries a heterozygous 34-base pairs deletion that removes rs9349379, as well as all but 1 base pairs of the MEF2-binding site (Figure 4A). We then differentiated wild-type and heterozygous human embryonic stem cells into endothelial cells using an established protocol (In the Materials and Methods section of this article).38,39 Endothelial cells that carry the 34-base pairs deletion at rs9349379 express 35% less PHACTR1 than cells homozygous for the high-expressing A allele at rs9349379 (Figure 4B). This represents a promising in vivo validation of the potential regulatory role of rs9349379 in the control of PHACTR1 expression in the vascular endothelium. This result is also consistent with our eQTL result.

Figure 4.
Figure 4.

A CRISPR/Cas9-induced deletion that encompasses rs9349379 and the myocyte enhancer factor (MEF) 2–binding site reduces PHACTR1 expression in endothelial cells. A, Schematic representation of the PHACTR1 locus. The star (bold, underline) corresponds to rs9349379 in intron 3 of PHACTR1 and the box highlights the DNA sequence targeted by the CRISPR guide RNA. We isolated a heterozygous clone that carries a 34-base pairs (bp) deletion (knockout [KO] allele) that removes rs9349379 (bold) and most of the MEF2-binding site (underline). B, Human endothelial cells that are heterozygous for the PHACTR1 34-bp deletion (HET) express 35% less PHACTR1 than wild-type (WT) cells (t test P=0.03). Data shown is mean±SD. The experiment was done in triplicate. SNP indicates single nucleotide polymorphism.

No Ectopic Induction of PHACTR1 Expression in HUVEC

In a previous report, VEGF treatment was shown to induce PHACTR1 expression in HUVEC.17 We attempted to reproduce this result using a similar protocol. First, we confirmed that our HUVEC can respond to VEGF by demonstrating the phosphorylation of the mitogen-activated protein kinase ERK (extracellular signal-regulated kinase) on VEGF treatment (Figure III in the online-only Data Supplement). However, we could not measure by quantitative polymerase chain reaction a significant difference in PHACTR1 expression levels using different VEGF concentrations and induction times (Figure 5A). We also tested the effect of tumor necrosis factor-α, an inflammatory molecule that can trigger endothelial dysfunction, and shear stress, a stimulus known to protect blood vessels from atherosclerosis, on PHACTR1 expression levels in HUVEC. In both the cases, we showed induction of the positive controls, NFKB1 for tumor necrosis factor-α and KLF2 for shear stress. However, we again did not measure a significant change in PHACTR1 expression levels (Figure 5B and 5C).

Figure 5.
Figure 5.

Different endothelial cell stimuli do not induce PHACTR1 expression levels as measured by quantitative polymerase chain reaction (PCR). A, Serum-starved human umbilical vein endothelial cells (HUVECs) were treated with vascular endothelial growth factor (VEGF; 20 ng/mL) for 5 minutes before RNA was extracted and PHACTR1 transcript levels measured. See Figure III in the online-only Data Supplement for the positive control of this experiment. B, HUVEC were treated with tumor necrosis factor (TNF)-α (10 ng/mL) for 16 hours. RNA was extracted and the levels of PHACTR1 and NFKB1 (positive control) were quantified by quantitative PCR. C, HUVEC were treated with no, low, or high shear stress. RNA was extracted 6 hours after the beginning of the experiments, and PHACTR1 and KLF2 (positive control) expression levels were measured by quantitative PCR. The same results were observed after 24 hours of shear stress. For all panels, data shown are mean±SDs. The VEGF and TNF-α experiments were done in triplicates; we had 4 replicates for the shear stress experiment. All comparisons are nonsignificant (t test P>0.05), except for NFKB1 without and with TNF-α treatment (P=7.8×10−5) and for KLF2 without and with high shear stress (P=0.0012).

Discussion

The genetic association between intronic SNPs in PHACTR1 and CAD/MI is robust, relatively strong for a genome-wide association studies finding (odds ratio for the risk allele at rs9349379 is 1.37 in the MHI Biobank) and pleiotropic (the same SNPs are also associated with coronary artery calcification and stenosis, migraine, hemodynamic indexes, and cervical artery dissection). It is different from many of the known CAD/MI-associated SNPs because it is not associated with the traditional risk factors, such as lipid levels, blood pressure, or diabetes mellitus. This observation is promising, in as much as understanding how this genetic variation influences CAD/MI risk may yield new insights into the biology of atherosclerosis and potentially, in the long-term, new therapeutic strategies.

To translate this genetic discovery, we first need to connect genetic variants with genes. Our own DNA resequencing project and a large whole-exome sequencing effort failed to identify coding variants in PHACTR1 that might directly implicate this gene in MI.30,31 Previous eQTL experiments to link CAD/MI-associated PHACTR1 SNPs with its expression levels have equally been unsuccessful, despite large sample sizes and a wide variety of tissues tested (leukocytes [including monocytes], liver, fat, skin, omentum, aortic media and adventitia, mammary artery, and lymphoblastoid cell lines).5,40 Reasoning that the transcriptional effect might be tissue-specific, we measured an association between genotypes at rs9349379 and PHACTR1 expression levels in human coronary arteries. This is an important result because it supports PHACTR1 as a potential causal gene at the locus and suggests that low PHACTR1 expression levels in coronary arteries increase CAD/MI risk. Although coronary artery is not a homogenous tissue, the lack of PHACTR1 expression in smooth muscle and of eQTL effect in leukocytes support the idea that the vascular endothelium may be the most relevant tissue to study how PHACTR1 functions influence CAD/MI.

A recent report showed that the G allele at PHACTR1-rs9349379 is associated with lower risk of cervical artery dissection.14 The same G allele is also associated with reduced migraine risk,12 a known risk factor for cervical artery dissection.41 These results are in sharp contrast with the reported association between the G allele at rs9349379 and increased risk of CAD or MI. This is not the first report of a genome-wide genetic association of opposite effects of the same SNP on 2 diseases.42 We will need to understand PHACTR1 biological activities to explain its opposite roles in cervical artery dissection and CAD/MI. Maybe PHACTR1 modulates a downstream pathway differently in carotid or cervical arteries than in coronary arteries? Or maybe it affects different biological pathways in these different artery tissues? Although to our knowledge it has not been tested, we would predict that rs9349379 would be an eQTL for PHACTR1 expression levels in human carotid or cervical arteries.

High-density genotyping and bioinformatic analyses pinpointed rs9349379 as a potential causal variant at the locus. Using in vitro assays, we showed that alleles at this intronic PHACTR1 SNP bind differentially members of the MEF2 transcription factor family. Furthermore, deletion of the MEF2-binding site that overlaps with rs9349379 reduced PHACTR1 expression in endothelial cells. A small exonic 21-base pairs deletion in MEF2A was originally described to cause familial CAD, although this result has not been widely replicated.35–37 It is important to emphasize that our results are not invalidated by the lack of consistent association between genetic variation at MEF2A and CAD. First, we do not know if MEF2A or MEF2C (or both) recognize in vivo the binding motif that we characterized at rs9349379. Second, we do not need variation in MEF2 expression levels or activities to explain the variation in PHACTR1 expression levels. Changes in PHACTR1 expression levels are controlled, at least in part, by genotypes at rs9349379. MEF2 transcription factors remain good candidates for a role in atherosclerosis as they are present in the endothelium and are involved in maintaining vascular integrity.34

A previous report suggested that PHACTR1 expression levels in HUVEC can be induced on VEGF treatment, but we were not able to reproduce this result, despite trying different VEGF concentrations and induction times.17 More generally, we explored if classic endothelial stimuli (VEGF, tumor necrosis factor-α, and shear stress) can induce PHACTR1 expression. Under our stimulation protocols, we failed to ectopically change PHACTR1 expression levels in HUVEC. These negative observations are informative in guiding future experiments to determine if other endothelial stresses, potentially through MEF2 activation, may act synergistically with genotypes at rs9349379 to influence CAD/MI risk. In conclusion, our results provide an initial model (CAD/MI-associated SNPs at 6p24 affects MEF2 binding and consequently PHACTR1 expression levels) and a candidate tissue (vascular endothelium) to further characterize how PHACTR1 influences atherosclerosis independently of the traditional CAD/MI risk factors.

Acknowledgments

We thank all participants and staff of the Montreal Heart Institute (MHI) Biobank. We acknowledge the technical support of the Beaulieu-Saucier MHI Pharmacogenomic Center for DNA sequencing and genotyping. We thank Chantal St-Cyr from the Réseau d’échange de tissus et d’échantillons biologiques for providing human coronary artery samples.

Sources of Funding

This work was funded by the Centre of Excellence in Personalized Medicine, the Fonds de Recherche Santé Québec, the Canadian Institute of Health Research (CIHR-MOP136979), and the Montreal Heart Institute Foundation (to G. Lettre), and the CIHR-MOP102489 (to S. Lehoux). S. Lehoux, J.-C. Tardif, and G. Lettre are Canada Research Chair holders. Sequencing of PHACTR1 was performed by the Myocardial Infarction Genetics Exome Sequencing Consortium with support from the National Heart, Lung, and Blood Institute and the National Human Genome Research Institute of the US National Institutes of Health (NIH). Grant support included RC2 HL-103010, RC2 HL-102923, RC2 HL-102924, RC2 HL-102925, RC2 HL-102926, and 5U54HG003067-11. S. Kathiresan is supported by a Research Scholar award from the Massachusetts General Hospital (MGH), the Howard Goodman Fellowship from MGH, the Donovan Family Foundation, R01HL107816, and a Transatlantic Networks of Excellence award from Fondation Leducq. R. Do is supported by a Banting Fellowship from the CIHR. K. Musunuru is supported by Grant R01-GM104464 from the US National Institutes of Health (NIH) and a Cardiovascular Program Pilot Grant from the Harvard Stem Cell Institute. R.M. Gupta is supported by the LaDue Fellowship at Harvard University and the Sarnoff Foundation Scholar Award. H.-H. Won is supported by a postdoctoral award from the American Heart Association (15POST23280019).

Disclosures

None.

Footnotes

  • The online-only Data Supplement is available with this article at http://atvb.ahajournals.org/lookup/suppl/doi:10.1161/ATVBAHA.115.305534/-/DC1.

  • Nonstandard Abbreviations and Acronyms
    CAD
    coronary artery disease
    HUVEC
    human umbilical vein endothelial cells
    MEF2
    myocyte enhancer factor-2
    MHI
    Montreal Heart Institute
    MI
    myocardial infarction
    NFKB1
    nuclear factor of kappa light polypeptide gene enhancer in B-cells 1
    PHACTR1
    phosphatase and actin regulator-1
    SNP
    single nucleotide polymorphism
    TGF-β
    transforming growth factor-β
    VEGF
    vascular endothelial growth factor

  • Received December 16, 2014.
  • Accepted March 18, 2015.

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Significance

Despite having efficient therapies and prevention strategies, coronary artery diseases remain one of the main causes of death in the World. Recent failed or futile clinical trials have highlighted the need to identify new therapeutic targets. Unbiased human genetic studies can provide such new entry points into disease pathophysiology. Genome-wide association studies for coronary artery disease risk have identified a robust signal for DNA polymorphisms within the PHACTR1 gene on chromosome 6. PHACTR1 single nucleotides polymorphisms are not associated with traditional atherosclerosis risk factors, such as blood lipids, hypertension, or diabetes mellitus. Our work establishes a link between these genetic variants and PHACTR1 expression levels in human coronary arteries through a potential effect on the binding of the transcription factors MEF2 at the locus. These results set the stage to explore how PHACTR1 functions in the vascular endothelium influence coronary artery disease risk.

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  • Myocardial Infarction–Associated SNP at 6p24 Interferes With MEF2 Binding and Associates With PHACTR1 Expression Levels in Human Coronary ArteriesSignificance
    Mélissa Beaudoin, Rajat M. Gupta, Hong-Hee Won, Ken Sin Lo, Ron Do, Christopher A. Henderson, Claire Lavoie-St-Amour, Simon Langlois, Daniel Rivas, Stephanie Lehoux, Sekar Kathiresan, Jean-Claude Tardif, Kiran Musunuru and Guillaume Lettre
    Arteriosclerosis, Thrombosis, and Vascular Biology. 2015;35:1472-1479, originally published April 2, 2015
    https://doi.org/10.1161/ATVBAHA.115.305534
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