Abstract 755: A Functional Single-Nucleotide Polymorphism (−254 C to G) in the TRPC6 Gene is Associated with Idiopathic Pulmonary Arterial Hypertension
Background & Hypothesis: Sustained contraction and excessive proliferation of pulmonary artery smooth muscle cells (PASMC), mediated by an increase in cytosolic Ca2+, play a critical role in the development of idiopathic pulmonary arterial hypertension (IPAH). Recently, we demonstrated that upregulation of TRPC6 channel expression contributes to the excessive PASMC proliferation in IPAH patients. This study aimed at testing whether single nucleotide polymorphisms (SNPs) of TRPC6 gene affects TRPC6 expression in IPAH-PASMC.
Methods & Results: Genomic DNA were isolated from blood samples of normal subjects (n=72) and patients with IPAH (n=126) and chronic thromboembolic pulmonary hypertension (CTEPH; n=53). Three biallelic SNPs, −361 (A/T), −254 (C/G) and −218 (C/T) were identified in the 2,000-bp sequence upstream of the transcriptional start site of TRPC6. The allele frequency of the −361 and −218 SNPs were not different among the 3 groups, the −254 (C/G) SNP had a significantly higher allele frequency in IPAH patients (P<0.05) than in normal subjects and CTEPH patients. Genotype data showed that 25% IPAH patients carried the −254G allele (compared with ~12% normal subjects and CTEPH patients). Among them, 6.3% IPAH patients carried homozygous −254G/G, while no one in normal subjects carried −254G/G. Interestingly, the −254 (C to G) nucleotide variation in TRPC6 generates an additional binding sequence for transcription factor, NF-κB. Electrophoretic mobility shift assays confirmed that −254G site had NF-κB DNA-binding activity. DNA fragments of TRPC6 gene (−1622 to +110 bp) containing −254C or −254G were cloned and fused to the promoterless LacZ reporter vector, respectively. Activation of NF-κB via TNF-α only stimulated promoter activity of −254G reporter vector, but not the −254C vector. Moreover, specific inhibition of NF-κB by overexpression IκBα superrepressor significantly decreased TRPC6 protein expression in PASMC from IPAH patients who carry the −254G allele.
Conclusions: These results suggest that −254 (C to G) SNP of TRPC6 gene predisposes individuals to high-risk of IPAH by modifying NF-κB-mediated TRPC6 expression, and may play a mechanistic role in the vascular remodeling intrinsic to the pathogenesis of IPAH.