Abstract P1017: Post-GWAS Functional Analysis Of CBLN2 Locus Reveals The Role Of CUX1 In The Inflammatory Pathogenesis Of Pulmonary Arterial Hypertension

Abstract

Background: Pulmonary arterial hypertension (PAH) is a deadly pulmonary vascular disease highly relevant to genetic susceptibility and acquired pathogenic triggers, especially inflammation. Chr18q22.3 near cerebellin 2 (CBLN2) gene is the first genetic locus identified by Genome-wide Association Study (GWAS) that is significantly associated with PAH risk in population of European ancestry. This locus includes a group of closely linked non-coding intergenic single nucleotide polymorphisms (SNPs) downstream of CBLN2. Although CBLN2 is upregulated in PAH and causes pulmonary vascular dysfunction, it remains unknown how these non-coding SNPs remotely affect CBLN2 and in turn alter the genetic susceptibility of PAH. Methods and Results: We applied Hi-C analysis, combined with electrophoretic mobility shift assay, to identify rs11151770 as a functional SNP (fSNP) characterized with allele-imbalanced nuclear protein binding and chromatin interaction with CBLN2 promoter, among 19 haplotype SNPs in a linkage disequilibrium (LD r 2 >0.8) in CBLN2 locus. As an intergenic non-coding SNP, rs11151770 is located 110 kbp downstream of CBLN2 promoter. Proteomic analysis revealed nuclear transcription factor CUX1 binds specifically to the risk allele A of rs11151770 but not to the non-risk allele C. Acting as the activator for the expression of target gene CBLN2, CUX1 knockdown prevented, but CUX1 overexpression induced, the pathophenotypes of human pulmonary arterial endothelial cells (PAECs) associated with PAH. CUX1-CBLN2 was upregulated by acquired inflammatory PAH trigger IL-1β and was increased in lung tissues extracted from PAH patients. SNP genotyping in PAH patients and non-PAH controls showed a 2.2-fold risk allele A enrichment only in patients with PAH associated with connective tissue disorders, supporting a pathogenic mechanism of an inflammation-sensitive pathway (CUX1-CBLN2) combined with a PAH susceptible genotype (risk allele A of rs11151770) for the manifestation of clinical PAH. Conclusion: Our post-GWAS functional genomics analysis revealed a CUX1-mediated inflammatory pathway and provided a mechanistic explanation for the GWAS-identified association between the genetic variants in CBLN2 locus and PAH pathogenesis.