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

Circ Genom Precis Med. 2018;11:e002327. DOI: 10.1161/CIRCGEN.118.002327 October 2018 Xiao Wang, PhD Kiran Musunuru, MD, PhD, MPH 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 vascularspecific enhancer activity for PHACTR1.2 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.3 In 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 RESEARCH LETTER