Confirmation of Causal rs9349379- <i>PHACTR1</i> Expression Quantitative Trait Locus in Human-Induced Pluripotent Stem Cell Endothelial Cells

HomeCirculation: Genomic and Precision MedicineVol. 11, No. 10Confirmation of Causal rs9349379-PHACTR1 Expression Quantitative Trait Locus in Human-Induced Pluripotent Stem Cell Endothelial Cells Free AccessLetterPDF/EPUBAboutView PDFView EPUBSections ToolsAdd to favoritesDownload citationsTrack citationsPermissions ShareShare onFacebookTwitterLinked InMendeleyReddit Jump toFree AccessLetterPDF/EPUBConfirmation of Causal rs9349379-PHACTR1 Expression Quantitative Trait Locus in Human-Induced Pluripotent Stem Cell Endothelial Cells Xiao Wang, PhD and Kiran Musunuru, MD, PhD, MPH Xiao WangXiao Wang Division of Cardiology and Cardiovascular Institute, Department of Medicine, Perelman School of Medicine at the University of Pennsylvania, Philadelphia. and Kiran MusunuruKiran Musunuru Kiran Musunuru, MD, PhD, MPH, Division of Cardiology, Department of Medicine, Perelman School of Medicine at the University of Pennsylvania, 3400 Civic Center Blvd, Bldg 421, 11–104 SCTR, Philadelphia, PA 19104. Email E-mail Address: [email protected] Division of Cardiology and Cardiovascular Institute, Department of Medicine, Perelman School of Medicine at the University of Pennsylvania, Philadelphia. Originally published12 Oct 2018https://doi.org/10.1161/CIRCGEN.118.002327Circulation: Genomic and Precision Medicine. 2018;11:e002327Other version(s) of this articleYou are viewing the most recent version of this article. Previous versions: October 12, 2018: Previous Version of Record A chromosome 6p24 locus harboring the PHACTR1 gene has one of the strongest genome-wide association study signals for coronary artery disease1 and other vascular phenotypes. The variant in this locus with the strongest coronary artery disease association is rs9349379, located in an intron of PHACTR1.2 A previous report established an rs9349379-PHACTR1 expression quantitative trait locus (eQTL) in human coronary artery samples, with the European major allele (A) associated with higher PHACTR1 expression than the minor allele (G).2 Querying of the Genotype-Tissue Expression Project portal reveals strong rs9349379-PHACTR1 eQTLs with the same directionality in 3 human vascular tissues—tibial artery, coronary artery, and aorta (Figure [A]). The rs9349379 A allele conferred binding of myocyte enhancer factor-2 transcription factors in human umbilical vein endothelial cell nuclear extracts, binding that was disrupted by the G allele.2 Deletion of a 34-bp sequence around one of the rs9349379 A alleles in the HUES 9 human embryonic stem cell line (homozygous major) with CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat–associated 9), followed by differentiation into endothelial cells, resulted in lower PHACTR1 expression, consistent with but not proving that the A allele confers vascular-specific enhancer activity for PHACTR1.2Download figureDownload PowerPointFigure. PHACTR1 and EDN1 gene expression in vascular tissues and cells. A, Gene expression in 3 human vascular tissue types stratified by rs9349379 genotype. The Tukey box plots were generated by the Genotype-Tissue Expression Project portal (https://www.gtexportal.org/). B, Homozygous knock-in of the rs9349379 major allele (A) or minor allele (G) using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat–associated 9) and a targeting vector with puromycin resistance encoded within a scarless-excision piggyBac transposon. The protospacer is underlined, the protospacer-adjacent motif is bolded, and the position of rs9349379 is indicated in red. Sanger sequencing electropherograms of successfully targeted clones are shown. C, Gene expression in isogenic rs9349379 homozygous major (AA) and homozygous minor (GG) human-induced pluripotent stem cell (iPSC)–derived endothelial cells. Expression levels relative to HGPRT (reference gene) were quantified by the 2−ΔΔCt method. Data are displayed as Tukey box plots with all individual data points shown. P values were calculated with Mann-Whitney U tests.A subsequent report contradicted these findings.3 CRISPR/Cas9 deletion of an 88-bp sequence around both rs9349379 A alleles in HUES 9 cells (homozygous major) or both rs9349379 G alleles in DiPS 1016SevA (1016) induced pluripotent stem cells (homozygous minor), followed by differentiation into endothelial cells, resulted in unchanged PHACTR1 expression.3 Rather, there was higher expression of the EDN1 gene, 600 kb away from rs9349379, in the deleted cells of either background (AA or GG). Of note, these experiments do not permit attribution of the EDN1 expression change to rs9349379 allelic variation because the variant itself was not specifically altered. In a more directed experiment with CRISPR/Cas9 editing of the HUES 66 embryonic stem cell line (heterozygous) to generate isogenic rs9349379 homozygous major (AA) and homozygous minor (GG) lines, followed by differentiation into endothelial cells, there was lower EDN1 expression and unchanged PHACTR1 expression in AA cells.3 Notwithstanding that these newer data were inconsistent with the prior data, a model wherein altered EDN1 expression is the mechanism by which rs9349379 allelic variation modulates coronary artery disease risk and other vascular phenotypes was proposed.3In light of these contradictory studies, and mindful of past reports of the failure of human pluripotent stem cell–based models to replicate tissue eQTLs,4 we sought to replicate the experiment performed with isogenic rs9349379 homozygous major (AA) and homozygous minor (GG) lines, using a larger sample size (6 clones of each genotype instead of 3 clones each3). Starting with 1016 cells (homozygous minor, GG), we used CRISPR/Cas9 to introduce a piggyBac transposon harboring a puromycin selection cassette into a TTAA site near rs9349379 (Figure [B]) via homology-directed repair, using previously described techniques.5 In one targeting, the repair template had G at the site of rs9349379 (preserving the original genotype), whereas in a parallel targeting, the repair template had A (changing the genotype). In either targeting, several clones were identified with homozygous knock-in of the transposon and the rs9349379 allele. After each targeting, each set of clones with the same genotype was pooled and treated with piggyBac transposase as described previously,5 resulting in scarless removal of the puromycin selection cassettes. We expanded 6 clones of each genotype (GG or AA) and differentiated them in parallel into endothelial cells as described previously,2 with 7 biological replicates per clone.We used the same quantitative reverse transcriptase polymerase chain reaction reagents as the previous experiment3 to measure PHACTR1, EDN1, and HGPRT (reference gene) expression. Homozygous major (AA) endothelial cells had significantly higher PHACTR1 expression (22%; P=1×10−7) compared with homozygous minor (GG) endothelial cells, consistent with the A allele conferring enhancer activity for PHACTR1 and with previous rs9349379-PHACTR1 eQTL findings in vascular tissues (Figure [C]). In contrast, we observed no significant difference in EDN1 expression between alternative genotypes, with a trend toward higher expression in AA endothelial cells (rather than significantly lower expression, as observed in the previous experiment3).Thus, although our experiment did not replicate an endothelial rs9349379-EDN1 eQTL, it did confirm previously observed vascular rs9349379-PHACTR1 eQTLs and supported rs9349379 as the causal variant. Reasons for discrepancies between our experiment and the experiment we attempted to replicate could include the use of human pluripotent stem cell lines of different genetic backgrounds, differences in endothelial cell differentiation, differences in the genome-editing approach (the previous experiment's clones were each generated with 2 successive rounds of CRISPR/Cas9-mediated homology-directed repair), or simply the play of chance. We note that the Genotype-Tissue Expression Project portal shows no significant rs9349379-EDN1 eQTLs in 3 vascular tissues (Figure [A]). In light of our current results, we feel that it is premature to conclude that EDN1 is a causal gene responsible for the chromosome 6p24 coronary artery disease association, in preference to PHACTR1 being a causal gene. The data and cell lines that support the findings of this study are available from the corresponding author on reasonable request.Sources of FundingThis work was supported by an American Heart Association Postdoctoral Fellowship (Dr Wang) and National Institutes of Health grants R01-HL118744 and R01-GM104464 (Dr Musunuru).DisclosuresNone.FootnotesGuest Editor for this article was Christopher Semsarian, MBBS, PhD, MPH.https://www.ahajournals.org/journal/circgenKiran Musunuru, MD, PhD, MPH, Division of Cardiology, Department of Medicine, Perelman School of Medicine at the University of Pennsylvania, 3400 Civic Center Blvd, Bldg 421, 11–104 SCTR, Philadelphia, PA 19104. Email [email protected]comReferences1. Kathiresan S, et al; Myocardial Infarction Genetics Consortium. Genome-wide association of early-onset myocardial infarction with single nucleotide polymorphisms and copy number variants.Nat Genet. 2009; 41:334–341. doi: 10.1038/ng.327CrossrefMedlineGoogle Scholar2. Beaudoin M, et al. Myocardial infarction-associated SNP at 6p24 interferes with MEF2 binding and associates with PHACTR1 expression levels in human coronary arteries.Arterioscler Thromb Vasc Biol. 2015; 35:1472–1479. doi: 10.1161/ATVBAHA.115.305534LinkGoogle Scholar3. Gupta RM, et al. A genetic variant associated with five vascular diseases is a distal regulator of endothelin-1 gene expression.Cell. 2017; 170:522.e–533.e15. doi: 10.1016/j.cell.2017.06.049CrossrefGoogle Scholar4. Wang X, et al. Interrogation of the atherosclerosis-associated SORT1 (sortilin 1) locus with primary human hepatocytes, induced pluripotent stem cell-hepatocytes, and locus-humanized mice.Arterioscler Thromb Vasc Biol. 2018; 38:76–82. doi: 10.1161/ATVBAHA.117.310103LinkGoogle Scholar5. Pashos EE, et al. Large, diverse population cohorts of hiPSCs and derived hepatocyte-like cells reveal functional genetic variation at blood lipid-associated loci.Cell Stem Cell. 2017; 20:558.e–570.e10. doi: 10.1016/j.stem.2017.03.017CrossrefGoogle Scholar Previous Back to top Next FiguresReferencesRelatedDetailsCited By Wood A, Antonopoulos A, Chuaiphichai S, Kyriakou T, Diaz R, Al Hussaini A, Marsh A, Sian M, Meisuria M, McCann G, Rashbrook V, Drydale E, Draycott S, Polkinghorne M, Akoumianakis I, Antoniades C, Watkins H, Channon K, Adlam D and Douglas G (2022) PHACTR1 modulates vascular compliance but not endothelial function: a translational study , Cardiovascular Research, 10.1093/cvr/cvac092 Gupta R (2022) Causal Gene Confusion: The Complicated EDN1/PHACTR1 Locus for Coronary Artery Disease, Arteriosclerosis, Thrombosis, and Vascular Biology, 42:5, (610-612), Online publication date: 1-May-2022.Rubin S, Bougaran P, Martin S, Abelanet A, Delobel V, Pernot M, Jeanningros S, Bats M, Combe C, Dufourcq P, Debette S, Couffinhal T and Duplàa C (2022) PHACTR-1 (Phosphatase and Actin Regulator 1) Deficiency in Either Endothelial or Smooth Muscle Cells Does Not Predispose Mice to Nonatherosclerotic Arteriopathies in 3 Transgenic Mice, Arteriosclerosis, Thrombosis, and Vascular Biology, 42:5, (597-609), Online publication date: 1-May-2022. 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Musunuru K and Kathiresan S (2019) Genetics of Common, Complex Coronary Artery Disease, Cell, 10.1016/j.cell.2019.02.015, 177:1, (132-145), Online publication date: 1-Mar-2019. October 2018Vol 11, Issue 10 Advertisement Article InformationMetrics © 2018 American Heart Association, Inc.https://doi.org/10.1161/CIRCGEN.118.002327PMID: 30354304 Originally publishedOctober 12, 2018 Keywordsgene expressiongenomicsendothelial cellsgenome-wide association studystem cellsPDF download Advertisement SubjectsGene Expression and RegulationOmicsStem Cells

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