Single Nucleotide Polymorphism rs9277336 Controls the Nuclear Alpha Actinin 4‐Human Leukocyte Antigen‐DPA1 Axis and Pulmonary Endothelial Pathophenotypes in Pulmonary Arterial Hypertension

Background Pulmonary arterial hypertension (PAH) is a complex, fatal disease where disease severity has been associated with the single nucleotide polymorphism (SNP) rs2856830, located near the human leukocyte antigen DPA1 (HLA-DPA1) gene. We aimed to define the genetic architecture of functional variants associated with PAH disease severity by identifying allele-specific binding transcription factors and downstream targets that control endothelial pathophenotypes and PAH. Methods and Results Electrophoretic mobility shift assays of oligonucleotides containing SNP rs2856830 and 8 SNPs in linkage disequilibrium revealed functional SNPs via allele-imbalanced binding to human pulmonary arterial endothelial cell nuclear proteins. DNA pulldown proteomics identified SNP-binding proteins. SNP genotyping and clinical correlation analysis were performed in 84 patients with PAH at University of Pittsburgh Medical Center and in 679 patients with PAH in the <i>All of Us</i> database. SNP rs9277336 was identified as a functional SNP in linkage disequilibrium (<i>r</i>²>0.8) defined by rs2856830, and the minor allele was associated with decreased hospitalizations and improved cardiac output in patients with PAH, an index of disease severity. SNP pulldown proteomics showed allele-specific binding of nuclear ACTN4 (alpha actinin 4) protein to rs9277336 minor allele. Both ACTN4 and HLA-DPA1 were downregulated in pulmonary endothelium in human patients and rodent models of PAH. Via transcriptomic and phenotypic analyses, knockdown of HLA-DPA1 phenocopied knockdown of ACTN4, both similarly controlling cell structure pathways, immune pathways, and endothelial dysfunction. Conclusions We defined the pathogenic activity of functional SNP rs9277336, entailing the allele-specific binding of ACTN4 and controlling expression of the neighboring HLA-DPA1 gene. Through inflammatory or genetic means, downregulation of this ACTN4-HLA-DPA1 regulatory axis promotes endothelial pathophenotypes, providing a mechanistic explanation for the association between this SNP and PAH outcomes.