T-786C variation in the promoter sequence of human eNOS gene markedly influences its expression level.

Abstract

This study investigated the role of the T-786C polymorphism (SNP) in the 5'-flanking sequence of the endothelial nitric oxide synthase gene (eNOS) on its expression level in vascular endothelium with the ultimate goal of shedding more light on the mechanisms by which genetic variations of eNOS might affect the vascular level of nitric oxide (NO). Sequences in the 5'-flanking region of eNOS gene were PCR-amplified using genomic DNA templates isolated from blood samples collected from cardiovascular disease (CVD) patients. Two sequence-versions carrying the same SNP site were used; a short (345 bp) and an extended one (1,594 bp), numbered relative to the translational start site. All sequences were cloned into a promoter-less vector (pGL3-basic), which carries the firefly luciferase gene as a reporter. Genotyping of the T-786C polymorphism was performed using Sanger sequencing of the insert region. Luminescence levels were then recorded 24-48 h after transfecting human endothelial cell line (EA.hy926). Three genotypes were identified in the subject samples; TT, TC, or CC. The highest expression levels associated with the TT genotype, followed by the TC genotype, then the CC genotype. The extended sequence version produced higher expression levels compared to the shorter version. Our results provide evidence that the T allele at the T-786C SNP site of the eNOS gene results in increased expression of the enzyme, and consequently might provide a protective mechanism from CVD. The extended promoter sequence of eNOS resulted in higher expression of the gene, suggesting the presence of some essential binding sites for transcription enhancing proteins.