Target Sequencing, Cell Experiments, and a Population Study Establish Endothelial Nitric Oxide Synthase ( eNOS ) Gene as Hypertension Susceptibility Gene

Abstract

Using GWAS in a case-control design, 7 we recently identified rs3918226 as a new hypertension susceptibility Abstract-A case-control study revealed association between hypertension and rs3918226 in the endothelial nitric oxide synthase (eNOS) gene promoter (minor/major allele, T/C allele). We aimed at substantiating these preliminary findings by target sequencing, cell experiments, and a population study. We sequenced the 140-kb genomic area encompassing the eNOS gene. In HeLa and HEK293T cells transfected with the eNOS promoter carrying either the T or the C allele, we quantified transcription by luciferase assay. In 2722 randomly recruited Europeans (53.0% women; mean age 40.1 years), we studied blood pressure change and incidence of hypertension in relation to rs3918226, using multivariable-adjusted models. Sequencing confirmed rs3918226, a binding site of E-twenty six transcription factors, as the single nucleotide polymorphism most closely associated with hypertension. In T compared with C transfected cells, eNOS promoter activity was from 20% to 40% (P<0.01) lower. In the population, systolic/diastolic blood pressure increased over 7.6 years (median) by 9.7/6.8 mm Hg in 28 TT homozygotes and by 3.8/1.9 mm Hg in 2694 C allele carriers (P≤0.0004). The blood pressure rise was 5.9 mm Hg systolic (confidence interval [CI], 0.6-11.1; P=0.028) and 4.8 mm Hg diastolic (CI, 1.5-8.2; P=0.0046) greater in TT homozygotes, with no differences between the CT and CC genotypes (P≥0.90). Among 2013 participants normotensive at baseline, 692 (34.4%) developed hypertension. The hazard ratio and attributable risk associated with TT homozygosity were 2.04 (CI, 1.24-3.37; P=0.0054) and 51.0%, respectively. In conclusion, rs3918226 in the eNOS promoter tags a hypertension susceptibility locus, TT homozygosity being associated with lesser transcription and higher risk of hypertension. Salvi et al Hypertension and eNOS 845locus. This locus lays in the promoter of the endothelial nitric oxide synthase (eNOS) gene, which encodes the enzyme that produces nitric oxide, a strong vasodilator with a key role in the regulation of systemic vascular resistance. GWAS usually points to genomic regions of interest in relation to a trait, but seldom directly identifies the causal or functional variant. In the present study, we aimed at consolidating the role of eNOS as a hypertension susceptibility gene by fine mapping the DNA sequence tagged by rs3918226, by studying the transcriptional functionality of the rs3918226 alleles in vitro, and by relating the change in BP over time to rs3918226 in a randomly recruited population sample. Methods Target SequencingFrom the HYPERGENES study, 7 we selected 44 hypertensive patients carrying ≥1 T allele and 48 healthy controls homozygous for the C allele. Analyses of the genetic data confirmed that all patients and controls were of continental Italian descent. We sequenced a 140-kb DNA region of chromosome, 7 which, in addition to eNOS, included KCNH2 mapping upstream and 6 genes mapping downstream: ATG...

The impact of genome‐wide association studies on the pathophysiology and therapy of cardiovascular disease

“…annot., bioinformatics annotation according to Braenne et al (2015) or others (Musunuru et al , 2010; Salvi et al , 2013); no data, no eQTL data or non‐coding variant; NA, not analysed.…”

“…Interestingly, the NOS3 gene has just recently been identified to be associated with CAD (Nikpay et al , 2015). Additionally, there is evidence that the particular variant (rs3918226), which is located in the promoter of the NOS3 gene, influences the expression of eNOS with a negative effect of the risk allele (Salvi et al , 2013). As briefly discussed above, the NOS3 gene has also been associated with blood pressure (Salvi et al , 2012, 2013).…”

Genetic and Molecular Aspects of Hypertension

“…Although majority of GWAS studied BP as a quantitative trait, only 2 studies analyzed hypertension as a dichotomous trait, 22,29 both of which resulted in identification of tractable signals in novel or established pathways of BP regulation. 22,[29][30][31][32] One variant (rs13333226) 22 is located in the promoter region of Uromodulin gene (UMOD), which is exclusively expressed in the kidney and possibly affects BP through a novel sodium homeostatic pathway, whereas second promoter SNP lies near the endothelial nitric oxide synthase (eNOS) gene, which is known to mediate vascular tone. 29,31 Among all the SNPs identified for quantitative BP, only 1 SNP (rs5068) has been reliably linked to a specific pathway (natriuretic peptide [NPPA/B]), whereas most of the remaining SNPs await robust elucidation of their causal genes and pathways (Table).…”

Association between endothelial NO synthase polymorphisms and arterial properties in the general population

“…In the Swedish population based INTERGENE study, rs3918226 was associated with arterial hypertension with the odds ratio of 1.32 [22]. In 2722 randomly recruited Europeans, systolic/ diastolic blood pressure increased over 7.6 years (median) by 9.7/ 6.8 mm Hg in 28 TT homozygotes and by 3.8/1.9 mm Hg in 2694 C allele carriers (P ≤ 0.0004) [19]. On the contrary, in an analysis of 18,436 Caucasian women who self-reported their blood pressure values, no connection with this SNP and blood pressure progression (OR, 95% confidence interval 1.00, 0.92-1.09) or incident hypertension (OR, 95% confidence interval 1.06, 0.99-1.14) was found [23].…”

“…A meta-analysis involving 21,714 subjects gave an independent confirmation that the T allele was associated with hypertension with an odds ratio (OR) of 1.34 [3]. Moreover, in T compared with C transfected cells, eNOS promoter activity was 20-40% lower [19]. Two studies investigated blood nitrite concentrations with respect to this SNP.…”