Post-GWAS Analyses Define Pathogenic Mechanisms of Functional SNPs Relevant for Pulmonary Arterial Hypertension
Year: 2021
Type: conference-abstract
Abstract: Rationale: Pulmonary arterial hypertension (PAH) is a rare but morbid disease and highly relevant to genetic susceptibility. Genome-wide Association Studies (GWAS) have identified 12 single nucleotide polymorphisms (SNPs) on 9 PAH-associated genetic loci. However, the mechanism of how these SNPs contribute to PAH pathogenesis remains unknown. In general, GWAS is unable to identify the causative functional SNPs (fSNP) in linkage disequilibrium (LD) that carries many other nonrandomly disease-associated SNPs. Even where a fSNP is identified, it is equally difficult to define the mechanisms by which a fSNP regulates the risk gene expression. Methods: We have developed a novel sequential methodology for rapid identification and characterize diseaseassociated fSNPs. First, we applied Reel-seq (Regulatory element-sequencing), an EMSA-based high-throughput technique, to identify fSNPs in a synthetic DNA library containing 500 SNPs associated with 9 PAH-loci in LDs revealed by GWAS. Second, we applied SDCP-MS (SNP-specific DNA competition pulldown-mass spectrometry) to identify proteins that specifically bind to fSNPs, and then validated these SNP-bound proteins using AIDP-Wb (allele-imbalanced DNA pulldown-Western blot) by showing allele-imbalanced binding of these proteins to their fSNPs. The regulation of PAH risk gene expression by these fSNP-binding proteins were determined in human PAECs and confirmed in PAH patients. Results: From 500 PAH-associated SNPs, 28 fSNPs were identified and subsequently confirmed by EMSA. By using SDCP-MS, cut-like homeobox1 (CUX1) was found to specifically bind to fSNP rs11151770A, the risk allele associated with a 1.97 folds increase in PAH susceptibility in GWAS. This fSNP located 54Kb downstream of Cerebellin 2 Precursor (CBLN2) gene. The specific binding of CUX1 to rs11151770A allele was confirmed by AIDP-Wb and luciferase reporter assays. CUX1 knockdown in human PAECs downregulated CBLN2 expression. Conversely, lentiviral-mediated CUX1 overexpression upregulated CBLN2. CUX1 overexpression in PAECs decreased angiogenic potential and proliferation but increased apoptosis, which were reversed by concomitant knockdown of CBLN2. Correspondingly, in human PAECs exposed to PAH triggers hypoxia or IL-1, and in lung tissues isolated from PAH patients, CUX1 expression was increased, with a simultaneous increase of CBLN2 expression. Conclusion: By using a functional genomic approach, we demonstrate that CUX1 regulates the expression of target gene CBLN2 via binding to the PAHassociated fSNP rs11151770. Alteration of CBLN2 expression, in turn, controls crucial endothelial function relevant to PAH. These results identify a novel role of CUX1 in PAH, clarifying a decade-long mystery surrounding the pathogenic function of this PAH-associated locus and providing a potential target for PAH intervention.
Institution: University of Pittsburgh
Language: English
Subfield: Pulmonary and Respiratory Medicine
Field: Medicine
Domain: Health Sciences
Open Access status: gold
OpenAlex ID: W4247454161