doi: 10.1161/01.HYP.0000013703.07316.7F
(Hypertension. 2002;39:919.)
© 2002 American Heart Association, Inc.
Scientific Contributions |
The T-786
C Mutation in Endothelial Nitric Oxide Synthase Is Associated With Hypertension
From the University of Calgary Departments of Medicine and Medical Genetics (M.E.H., H.G.P., S.V., P.J.B., S.E., C.J., T.J.A.), Calgary, Alberta; Department of Medicine (E.L., F.C.), McGill University, Montreal, Quebec.
Correspondence to Todd J. Anderson, MD, FRCPC, Associate Professor of Medicine, Division of Cardiology, Foothills Hospital, 1403-29th St NW, Calgary, AB CANADA, T2N-2T9. E-mail todd.anderson{at}calgaryhealthregion.ca
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Abstract |
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Although the pathogenic mechanisms involved in predisposing individuals to hypertension are not well defined, evidence is accumulating that suggests a strong genetic transmission. Animal studies and some clinical investigations have revealed that aberrant NO production may be an important contributing factor. Indeed, a missense mutation in the endothelial NO gene caused by a Glu298Asp alteration has been strongly associated with essential hypertension, coronary artery spasm, and myocardial infarction. Recently, another point mutation caused by a T-786
C transition in the 5'-flanking region of the endothelial NO synthase gene has been identified and, like the Glu298Asp mutation, is associated with coronary artery spasm. The present study was conducted to determine the effect of the T-786C point mutation on hypertension. We investigated the interaction between the endothelial NO synthase T-786C polymorphism and blood pressure in a large (n=705) clinically healthy population. Allele frequencies for the T and C alleles were 62% and 38%, translating into 39%, 46% and 15% of the population having the T/T, T/C, and C/C genotypes, respectively, for the T-786C point mutation. Subjects with the C/C genotype had significantly higher systolic blood pressures and were 2.16(95% confidence interval, 1.3 to 3.7) more likely to be hypertensive. Therefore, the -786 C/C genotype in NO synthase is a significant contributing factor for increasing the risk of essential hypertension.
Key Words: nitric oxide • hypertension, essential • mutation • blood pressure • polymorphism • nitric oxide synthase
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Introduction |
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The pathogenesis of hypertension is multifactorial and involves both genetic and environmental mechanisms. Accumulating evidence from clinical and animal studies suggests that an alteration in NO metabolism may be a contributing factor in the pathogenesis of hypertension.1,2
The potent vasodilator properties of NO in the resistance arteries, coupled with the genetic basis of hypertension, suggests that mutations affecting the endothelial NO (eNOS) gene, and consequently impairing NO release, might contribute to increased vascular resistance and in turn an elevation in systemic blood pressure (BP). Recent investigations have demonstrated that mutations affecting the eNOS gene may lead to impaired NO release. The G-to-T conversion at nucleotide 894 in the eNOS gene, which introduces an aspartic acid in place of a glutamic acid residue (Glu298Asp), is associated with an increased risk for myocardial infarction,3 hypertension,4,5 and coronary artery spasm.6 Another variant in the eNOS 5'-flanking region gene has been identified. The variant is a result of a thymidine being replaced by a cytosine at nucleotide -786 (T-786C).7 Like the Glu298Asp mutation, one manifestation of the T-786C mutation is increased risk for the risk of coronary spasm.7 In a Japanese population, subjects with the T-786C homozygous genotype were at 3 times the risk for coronary spasm compared with those with the C/T or the T/T genotypes.7 The role of the T-786C eNOS mutation toward the pathogenesis of hypertension has not been well studied. The objectives of the present study were to identify the prevalence of the eNOS T-786C mutation and to investigate the effects of the genotypes on BP in a healthy cohort without overt vascular disease. We report a significant association between the C allele and essential hypertension in this population.
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Methods |
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Subjects
The study population reported herein is a subgroup from a larger longitudinal clinical trial designed to assess the predictive value of brachial artery flow-mediated vasodilation to predict cardiovascular end points (Firemen And Their Endothelium [FATE] study). The cohort consisted of active and retired middle-aged men from the Calgary and Red Deer Fire departments. Patients were excluded if they had a previous history of documented coronary artery disease, peripheral vascular disease, or cerebrovascular disease. BP was measured on 2 occasions, in a quiet room after 15 minutes in a supine position, by use of a recently calibrated sphygmomanometer. Patients were considered to be hypertensive if their systolic or diastolic BP was >140 mm Hg or >95 mm Hg, respectively; were reported to have hypertension; or were on antihypertensive therapy. Genotyping and BP were measured in 705 subjects who were enrolled and agreed to participate in the study. Informed consent was obtained from all subjects enrolled in the study. The study was approved by the University of Calgary ethics committee.
Genetic Analysis
Genomic DNA was isolated from circulating lymphocytes. The eNOS T-786C mutation was analyzed by polymerase chain reaction (PCR) using the following primers: 5'ATG CTC CCA GGG CAT CA-3' and 5'GTC CTT GAG TCT GAC ATT AGG G-3'.7 The reaction was performed in 50-µL tubes containing 5 µL of 10xTaq buffer, 2.5 µL of a 10 µmol/L solution of each primer, 5.0 µL of 2.0 mmol/L dNTP mix, 1.0 µL of 50 mmol/L MgCl2, 28.5 µL of dH20, 5 U of Taq polymerase, and 50 ng of sample DNA. The reaction was run under the following conditions: 94°C for 7 minutes and then cycled 30 times at 94°C for 30 seconds, 57°C for 30 seconds, and 72°C for 30 seconds, followed by 72°C for 7 minutes; 7.5 µL of the PCR product was digested with 10 U of NgoM IV 0.5 µL of 10x react 9 (GIBCO) and 1.5 µL of dH20. The C allele introduces a cut site for NgoM IV, allowing differentiation from the T genotype when the PCR products are separated on a 4% Nusieve agarose gel.
Statistical Analysis
Data variables are described as mean±SD. Comparisons between the hypertensive and normotensive individuals were assessed using a t test, with unequal variances when appropriate. One-way ANOVA followed by Scheffe’s post hoc analysis was used to compare BP variables stratified by the eNOS T-786C genotypes. Logistic regression was used to compare discrete data. Multiple regressions and multiple logistic stepwise regression were used to assess interactions between age triglycerides and body mass index and the eNOS genotypes. Data analyses were performed using the statistical program Stata.
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Results |
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A total of 705 patients had both their BP and eNOS T-786
C genotypes identified. Mean age, weight, cholesterol, serum triglycerides, systolic BP, diastolic BP, and mean arterial BP are summarized in Table 1, subdivided into hypertensive, normotensive, and all individuals. The hypertensive individuals were significantly older and had an elevated body mass and higher serum triglycerides compared with levels of the normotensive patients. The groups were similar in terms of cholesterol values.
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Genetic Analysis
The distribution of the genotypes for the T-786C is shown in Table 2. Allele frequencies for the T and C alleles were 62% and 38%, translating into 39%, 46%, and 15% of the population having the T/T, T/C, and C/C genotypes, respectively, for the T-786C point mutation. Mean arterial and diastolic BP were not statistical different among the various genotypes. Mean systolic BP was significantly elevated in patients with the C/C genotype compared with those with the T/T genotype for the eNOS T-786C point mutation (P<0.05) (Table 2). Likewise, 22.6% of subjects with the C/C were hypertensive compared with 12.4% and 14.8% of the subjects with the T/T and T/C genotypes, respectively.
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Logistic regression revealed that the C/C genotype was significantly more prevalent in the hypertensive group compared with those with the T/T and T/C genotype, only after adjusting for age triglycerides and body mass index (Odds ratio, 1.4; P=0.09 unadjusted; P=0.02 adjusted) (Table 3). Subjects with the C/C genotype were 2.16 (P<0.01) times more likely to be hypertensive compared with only those with T/T genotypes, and the difference remained evident after adjusting for age triglycerides and body mass index (P=0.02).
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Discussion |
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Here we report that the eNOS T-786
C mutation in the 5'-flanking region of eNOS is significantly associated with hypertension in a healthy adult male population. Mean systolic BP was 4.4 mm Hg (P<0.05) higher in the homozygous C/C subjects compared with subjects with the T/T genotype. Furthermore, there were significantly more individuals with the C/C genotype and hypertension compared with either the T/T and T/C groups combined or only those with the T/T genotype. NO is produced from the amino acid arginine in response to a number of physiological stimuli.8 Mechanical stress and hormonal signals simulate eNOS, promoting the release of NO and subsequently inducing vascular smooth muscle cell relaxation.9 Animal models with impaired NO bioavailability invariably develop hypertension, highlighting the role of eNOS in BP homeostasis.2 Moreover, the use of NOS inhibitors to generate models of hypertension attests to the role of NOS in maintenance of BP.10
Hypertensive patients have impaired NO-mediated dilation of the small resistance vessels, and it is thought that this directly leads to an elevation in BP.11,12 Clinical investigations have revealed that hypertensive patients have reduced vasodilatation induced by acetylcholine with preserved endothelium-independent relaxation to sodium nitroprusside.11,13 Similarly, a separate study demonstrated that patients with essential hypertension have lower plasma and urinary nitrate levels compared with those of normotensive individuals.1 Collectively these studies suggest that eNOS plays a prominent role not only in the maintenance of systemic BP but also in the pathogenesis of hypertension.
There is a strong genetic contribution to hypertension, in which persons <50 years of age with hypertension are 4 times more likely to have a first-degree relative with essential hypertension.14 It has been estimated that 25% to 60% of hypertension relates directly to inherited traits. Although impaired endothelial function is not a universal finding in hypertensive patients,15 Taddei et al16 determined that endothelial dysfunction antedates the development of hypertension; offspring of parents with hypertension have diminished endothelium-dependent vasodilatation in the prehypertensive state. This is further supported by studies that have demonstrated that eNOS knockout mice have significantly elevated BPs.2
Nakayama et al,7 using a luciferase reporter construct, demonstrated that the C/C genotype was associated with a 52% and 62% decrease in promoter activity under normoxic and hypoxic conditions, respectively, compared with the wild-type promoter sequence. The investigators also found a strong association between the C allele and coronary spasm, suggesting that the decreased expression of eNOS has important clinical implications.7 Another in vivo study reported that the -786 C allele decreased eNOS mRNA levels and serum nitrite and nitrate.17 Our data extends these results and suggests that the C allele in eNOS may be an important contributor to essential hypertension, given that patients with the C/C genotype have a significantly elevated systolic BP (Table 2) and are over-represented in the hypertensive group (Table 3). However, a similar study by Kajiyama et al18 concluded that the eNOS -786 C allele was not associated with essential hypertension. The apparent discrepancies between the studies may be explained by the much higher frequency of the C allele in our white population, allowing a larger number of homozygous patients to be analyzed. This discrepancy in allelic frequencies between ethnic groups requires further investigation.
It has previously been demonstrated that smoking further amplifies the risk for coronary artery spasm in the subjects with the C/C genotype.19 We were unable to investigate this interaction because of an insufficient number of smokers in our cohort. Other genetic and environmental interactions with the eNOS T-786C point mutation remain largely unknown. For example, the high prevalence of the -786 C allele may allow for significant interactions in cis or trans with the Glu298Asp genotypes, further potentiating a deficiency in NO. It is possible that a combination of the 2 Glu298Asp and the T-786C polymorphisms could have an amplifying effect, increasing the risk for hypertension above that of a single alteration in the eNOS gene.
Limitations
Our cohort is relatively large, exclusively male, and more physically active compared with the general population. Hence, the results from this study must be interpolated to other population with some degree of caution. Conversely, we could be underestimating the role of the eNOS -786 allele, given that only 15% of our cohort presented with essential hypertension.
In conclusion the T-786C transition in the 5'-flanking region of the endothelial NO gene may be a significant contributing factor to essential hypertension. These results further support the central role of endothelial-derived NO in the pathogenesis of essential hypertension.
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Acknowledgments |
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We acknowledge Annette Robertson, RN, Heather Conradson, BN, Kathy Hildebrand, BN, Debbie Houston, Marinda Fung, and Patti Robinson for their expert nursing and technical support. The FATE study is supported by an unrestricted grant from Pfizer, Canada, and the Canadian Institutes of Health Research. This study was also funded by the Alberta Heart and Stroke Foundation. Dr Verma is a MRC Fellow, and Dr Anderson is a Scholar of the Alberta Heritage Foundation for Medical Research.
Received August 23, 2001; first decision January 4, 2002; accepted February 8, 2002.
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References |
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