A Functional 12T-insertion polymorphism in the <i>ATP1A1</i> promoter confers decreased susceptibility to hypertension in a male Sardinian population

Identification of susceptibility genes for essential hypertension in humans has been a challenge due to its multifactorial pathogenesis complicated by gene-gene and gene-environment interactions, developmental programing and sex specific differences. These concurrent features make identification of causal hypertension susceptibility genes with a single approach difficult, thus requiring multiple lines of evidence involving genetic, biochemical and biological experimentation to establish causal functional mutations. Here we report experimental evidence encompassing genetic, biochemical and in vivo modeling that altogether support ATP1A1 as a hypertension susceptibility gene in males in Sardinia, Italy. ATP1A1 encodes the α1Na,K-ATPase isoform, the sole sodium pump in vascular endothelial and renal tubular epithelial cells. DNA-sequencing detected a 12-nucleotide long thymidine (12T) insertion(ins)/deletion(del) polymorphism within a poly-T sequence (38T vs 26T) in the ATP1A1 5’-regulatory region associated with hypertension in a male Sardinian population. The 12T-insertion allele confers decreased susceptibility to hypertension (P = 0.035; OR = 0.50 [0.28–0.93]) accounting for 12.1 mmHg decrease in systolic BP (P = 0.02) and 6.6 mmHg in diastolic BP (P = 0.046). The ATP1A1 promoter containing the 12T-insertion exhibited decreased transcriptional activity in in vitro reporter-assay systems, indicating decreased α1Na,K-ATPase expression with the 12T-insertion, compared with the 12T-deletion ATP1A1 promoter. To test the effects of decreased α1Na,K-ATPase expression on blood pressure, we measured blood pressure by radiotelemetry in three month-old, highly inbred heterozygous knockout ATP1A1+/− male mice with resultant 58% reduction in ATP1A1 protein levels. Male ATP1A1+/− mice showed significantly lower blood pressure (P &#60; 0.03) than age-matched male wild-type littermate controls. Concordantly, lower ATP1A1 expression is expected to lower Na-reabsorption in the kidney thereby decreasing sodium-associated risk for hypertension and sodium-induced endothelial stiffness and dysfunction. Altogether, data support ATP1A1 as a hypertension susceptibility gene in a male Sardinian population, and mandate further investigation of its involvement in hypertension in the general population

A functional 12T-insertion polymorphism in the ATP1A1 promoter confers decreased susceptibility to hypertension in a male Sardinian population.

Identification of susceptibility genes for essential hypertension in humans has been a challenge due to its multifactorial pathogenesis complicated by gene-gene and gene-environment interactions, developmental programing and sex specific differences. These concurrent features make identification of causal hypertension susceptibility genes with a single approach difficult, thus requiring multiple lines of evidence involving genetic, biochemical and biological experimentation to establish causal functional mutations. Here we report experimental evidence encompassing genetic, biochemical and in vivo modeling that altogether support ATP1A1 as a hypertension susceptibility gene in males in Sardinia, Italy. ATP1A1 encodes the α1Na,K-ATPase isoform, the sole sodium pump in vascular endothelial and renal tubular epithelial cells. DNA-sequencing detected a 12-nucleotide long thymidine (12T) insertion(ins)/deletion(del) polymorphism within a poly-T sequence (38T vs 26T) in the ATP1A1 5'-regulatory region associated with hypertension in a male Sardinian population. The 12T-insertion allele confers decreased susceptibility to hypertension (P = 0.035; OR = 0.50 [0.28-0.93]) accounting for 12.1 mmHg decrease in systolic BP (P = 0.02) and 6.6 mmHg in diastolic BP (P = 0.046). The ATP1A1 promoter containing the 12T-insertion exhibited decreased transcriptional activity in in vitro reporter-assay systems, indicating decreased α1Na,K-ATPase expression with the 12T-insertion, compared with the 12T-deletion ATP1A1 promoter. To test the effects of decreased α1Na,K-ATPase expression on blood pressure, we measured blood pressure by radiotelemetry in three month-old, highly inbred heterozygous knockout ATP1A1+/- male mice with resultant 58% reduction in ATP1A1 protein levels. Male ATP1A1+/- mice showed significantly lower blood pressure (P < 0.03) than age-matched male wild-type littermate controls. Concordantly, lower ATP1A1 expression is expected to lower Na-reabsorption in the kidney thereby decreasing sodium-associated risk for hypertension and sodium-induced endothelial stiffness and dysfunction. Altogether, data support ATP1A1 as a hypertension susceptibility gene in a male Sardinian population, and mandate further investigation of its involvement in hypertension in the general population

Analysis of <i>ATP1A1</i> (12T-ins/del) variants based on blood pressure as a quantitative trait.

<p>Blood pressures were adjusted for age, body mass index and case/control status.</p><p>^a Number of individuals</p><p>^b Systolic blood pressure in mmHg</p><p>^c Diastolic blood pressure in mmHg</p><p>s.e.m., standard error of the mean</p><p>Δ SBP, difference in systolic blood pressure</p><p>Δ DBP, difference in diastolic blood pressure</p><p><i>P</i>, Mann-Whitney Rank Sum Test <i>P</i> values.</p><p>Analysis of <i>ATP1A1</i> (12T-ins/del) variants based on blood pressure as a quantitative trait.</p

Identification of the 12T-ins/del polymorphism in the <i>ATP1A1</i> promoter region and transcriptional activity of <i>ATP1A1</i> promoter variants.

<p>(A) Nucleotide sequence spanning the poly-T sequence involved in the 12T-ins/del polymorphism from the two <i>ATP1A1</i> (p12T ins, p12T del) reporter gene constructs utilized in the transcriptional assays. On right detection of 12T-insertion and 12T-deletion alleles by PCR-amplification followed by denaturing polyacrylamide gel (6%) electrophoresis used for genotyping of the Sardinian cohort. (B) Illustration of the <i>ATP1A1</i> promoter region. Non-coding exon is presented as open box and exon encoding the NH₂-terminal region is presented as black box. Sequence and location of the <i>ATP1A1</i> 12T-ins/del polymorphism is shown. The positions of TATAAA box, INR (initiator) and DPE (downstream promoter element) core promoter elements within <i>ATP1A1</i> promoter are shown. Core promoter elements were identified based on 100% homology with corresponding consensus sequences [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116724#pone.0116724.ref039" target="_blank">39</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0116724#pone.0116724.ref040" target="_blank">40</a>]. (C) Schematic of two <i>ATP1A1</i> (p12T ins, p12T del) reporter gene constructs. (D) Relative transcriptional activity of 12T-insertion (p12T ins) and 12T-deletion (p12T del) gene constructs in Cos1, HEK293 and MDA-MB-468 cells. *, <i>P</i> < 0.05; **, <i>P</i> < 0.01 (two-tailed student <i>t</i>-test).</p

Analysis of ATP1A1 protein expression, blood pressure, heart rate and activity in heterozygous <i>ATP1A1^+/−</i> and wild-type male mice.

<p>(A) Western blot analysis of <i>ATP1A1^+/−</i> and wild type mouse whole kidney extracts (30 μg) reacted with anti-mouse ATP1A1 and anti-mouse βActin polypeptides. (B) Densitometry analysis of samples shown in (A) detecting 58% decrease ATP1A1 levels in <i>ATP1A1^+/−</i> kidneys. (C) Mean systolic blood pressure ± sem (SBP; mmHg). (D) Mean heart rate ± sem (beats/min; BPM). (E) Mean activity ± sem (Counts/min) in <i>ATP1A1^+/−</i> (solid bars, n = 4) and wild-type (open bars, n = 5) male mice. * <i>P</i> < 0.03, ** <i>P</i> = 0.015 (two-tailed student <i>t</i>-test).</p

Characteristics of the study population.

<p>Characteristics of the study population.</p

Physical activity attenuates but does not eliminate coronary heart disease risk amongst adults with risk factors: EPIC-CVD case-cohort study

This study aimed to evaluate the association between physical activity and the incidence of coronary heart disease (CHD) in individuals with and without CHD risk factors. EPIC-CVD is a case-cohort study of 29 333 participants that included 13 582 incident CHD cases and a randomly selected sub-cohort nested within the European Prospective Investigation into Cancer and Nutrition (EPIC) cohort. Self-reported physical activity was summarized using the Cambridge physical activity index (inactive, moderately inactive, moderately active, and active). Participants were categorized into sub-groups based on the presence or the absence of the following risk factors: obesity (body mass index ≥30 kg/m2 ), hypercholesterolaemia (total cholesterol ≥6.2 mmol/L), history of diabetes, hypertension (self-reported or ≥140/90 mmHg), and current smoking. Prentice-weighted Cox regression was used to assess the association between physical activity and incident CHD events (non-fatal and fatal). Compared to inactive participants without the respective CHD risk factor (referent), excess CHD risk was highest in physically inactive and lowest in moderately active participants with CHD risk factors. Corresponding excess CHD risk estimates amongst those with obesity were 47% [95% confidence interval (CI) 32–64%] and 21% (95%CI 2–44%), with hypercholesterolaemia were 80% (95%CI 55–108%) and 48% (95%CI 22–81%), with hypertension were 80% (95%CI 65– 96%) and 49% (95%CI 28–74%), with diabetes were 142% (95%CI 63–260%), and 100% (95%CI 32–204%), and amongst smokers were 152% (95%CI 122–186%) and 109% (95%CI 74–150%). In people with CHD risk factors, moderate physical activity, equivalent to 40 mins of walking per day, attenuates but does not completely offset CHD ris