Abstract 12700: Post-GWAS Functional SNP Analysis to Define Genetic and Acquired Pathogenesis of Pulmonary Arterial Hypertension

Abstract

Introduction: Pulmonary arterial hypertension (PAH) is an enigmatic and morbid disease where insights are emerging regarding genetic susceptibility. Genome-wide Association Studies (GWAS) have identified genomic SNPs associated with increased PAH risk. It has been challenging to define the mechanisms underlying the contribution of non-coding PAH-associated functional SNPs (fSNPs) to the pathogenesis of this disease. Methods: We developed a post-GWAS functional genomics strategy to define the causative fSNPs and the associated biological mechanisms. It includes Reel-seq (Regulatory element-sequencing), an EMSA-based technique to identify fSNPs in a synthetic SNP library; SDCP-MS (SNP-specific DNA competition pulldown-mass spectrometry) to identify proteins that specifically bind to fSNPs; and AIDP-Wb (allele-imbalanced DNA pulldown-Western blot) to show allele-imbalanced binding of these proteins to fSNPs. The regulation of risk gene expression by fSNP-binding proteins and their pathogenicity were determined in human pulmonary arterial endothelial cells (PAECs) and confirmed in PAH animal models and patients. Results: An intergenic SNP rs4738801 was identified as a fSNP by Reel-seq. This non-coding fSNP resides in a remote upstream enhancer region of SOX17, an endothelial effector increasingly associated with PAH pathogenesis. Using SDCP-MS and AIDP-Wb, we found that transcription factor FUBP1 binds to rs4738801 risk allele C with lower affinity than non-risk allele G, resulting in a decrease in SOX17 expression. FUBP1 and target gene SOX17 controlled PAH-associated pathophenotypes in PAECs, including proliferation, apoptosis, and angiogenesis. Downregulated by major acquired PAH trigger hypoxia, FUBP1 and SOX17 were decreased in lungs and pulmonary ECs isolated from PAH patients and mouse models. A 3.8-fold enrichment of risk allele C was found in patients with PAH induced by hypoxia, but not in PAH associated with connective tissue disease or other etiology. CONCLUSION: Our functional genomics findings identify a novel role of FUBP1 in the functional regulation of SOX17 locus and elucidate a pathogenic mechanism that combines the acquired PAH-triggering factors and altered genetic susceptibility.