Abstract
BACKGROUND
Polymorphisms in the endothelial nitric oxide synthase (NOS3) gene increase susceptibility to hypertension and cardiovascular disease. We examined genetic and pharmacogenetic associations between NOS3 polymorphisms, blood pressure (BP) control, and cardiovascular events in elderly, hypertensive coronary artery disease (CAD) patients.
METHODS
Patients with CAD were randomly assigned to either verapamil SR– or atenolol-based antihypertensive treatment and followed for cardiovascular events. Cases (all-cause death, nonfatal myocardial infarction (MI), or nonfatal stroke) and an age-, sex-, race/ethnicity-matched control population were genotyped for the -786T>C and Glu298>Asp polymorphisms in NOS3. On-treatment BP and BP control were compared across genotype groups. Logistic regression was performed to estimate odds ratios (ORs) for the -786T>C and Glu298>Asp polymorphisms in the combined population and in randomized treatment groups.
RESULTS
Genotype data were available for 256 cases and 769 controls. Among controls, mean on-treatment BP differed according to -786T>C genotype (T/T 137/78 mm Hg, T/C 133/76 mm Hg, C/C 133/75 mm Hg; P = 0.0007 for systolic, P = 0.09 for diastolic) which corresponded to differing rates of BP control (T/T 63%, T/C 72%, C/C 88%; P = 0.002). Neither polymorphisms was associated with case status, with or without regard to assigned treatment.
CONCLUSIONS
The -786T>C, but not the Glu298>Asp variant of NOS3, may correlate with BP but do not appear to be associated with incident cardiovascular events in patients with established cardiovascular disease. The antihypertensive treatment approach did not appear to alter the genetic contribution to either BP control or cardiovascular events.
Nitric oxide (NO) is a critical mediator of vascular tone that also has antiplatelet, antiproliferative, antimitogenic, and anti-inflammatory properties. Dysregulation of NO homeostasis is central to many cardiovascular conditions.1 Therefore, gene polymorphisms that affect the expression or activity of endothelial NO synthase (eNOS) may be important in cardiovascular outcomes and/or response to treatment. Attention has focused on two single-nucleotide polymorphisms (SNPs) in the gene encoding eNOS, NOS3, based on their frequency in the population and functional consequences. A common SNP in the promoter region of NOS3, -786T>C, reduces gene expression.2–4 Another nonsynonymous SNP at codon 298 (Glu298>Asp; 894G>T) increases susceptibility to proteolytic cleavage,5 reducing NO bioavailability.6–8 These NOS3 SNPs have been implicated in hypertension and coronary artery disease (CAD).9,10
In the current investigation, we examined whether common NOS3 SNPs influence the success of antihypertensive therapy and are associated with incident cardiovascular events among CAD patients in the INternational VErapamil SR/trandolapril STudy (INVEST). Because antihypertensive drugs differ in their effects on NO homeostasis,11 it may be possible to optimize future antihypertensive therapy based on patient genotype. Accordingly, we hypothesized that NOS3 SNPs were associated with blood pressure (BP) control and risk of cardiovascular events in CAD patients based on assigned antihypertensive treatment.
METHODS
INVEST design and methods
INVEST was a prospective, randomized trial of β-blocker- vs. calcium antagonist–based antihypertensive therapy in 22,576 CAD patients with hypertension. 12 Hypertensive patients over 50 years old with CAD were randomly assigned to either an atenolol- or verapamil SR–based antihypertensive strategies. Medications were titrated to achieve BP goals of Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure VI based on the average of two seated BP measurements, as previously described.12 Patients were seen every 6 weeks for the first 6 months, and then every 6 months thereafter until 2 years after the last patient was enrolled. In patients assigned to atenolol, hydrochlorothiazide was added, followed by trandolapril. In patients assigned to verapamil SR, trandolapril was added, followed by hydrochlorothiazide. Dose adjustments were made before adding drugs. Trandolapril was recommended for all patients with diabetes, heart failure, or renal insufficiency. The primary outcome was a composite of the first occurrence of all-cause mortality, nonfatal myocardial infarction (MI), and nonfatal stroke. In the overall INVEST population, the treatment strategies were equivalent in terms of the incidence of the primary outcome and BP control.12
INVEST-GENES case–control study design
Informed consent for genetic studies was obtained and DNA samples were collected from 5,979 INVEST participants residing in the United States. Within this cohort, 258 patients experienced the primary outcome after a median (interquartile range) of 2.8 (2.3–3.1) years of follow-up and all of these cases were included in this analysis (99 deaths, 81 nonfatal MIs, and 86 nonfatal strokes; patients may have had more than one event). All events were adjudicated by a committee blinded to treatment strategy. Patients not experiencing a primary outcome event during follow-up were randomly selected as controls at a ratio of 3:1 (n = 774) with frequency-matching to cases by age, sex, and race/ethnicity.
DNA collection and genotyping
Genomic DNA was isolated from mouthwash. Pyrosequencing (Biotage, Uppsala, Sweden) was used to genotype NOS3 -786T>C (rs2070744), and allelic discrimination (7900HT TaqMan; Applied Biosystems, Foster City, CA) was used to genotype Glu298>Asp (rs1799983). Genotype accuracy was verified by blind duplicate regenotyping of ~5% of the samples and demonstrated to be 97 and 100% concordant for the -786T>C and Glu298>Asp polymorphisms. Seven samples could not be genotyped. To address the potential issue of population stratification, 87 ancestry informative markers were genotyped using either allele-specific PCR with universal energy transfer labeled primers or competitive allele-specific PCR at Prevention Genetics (Marshfield, WI).
Statistics
Statistical analyses were performed using SAS version 9.1 (SAS Institute, Cary, NC). Hardy–Weinberg equilibrium was tested for each racial/ethnic group using χ2-analysis. Linkage disequilibrium (r2) between the SNPs in each racial/ethnic group was estimated using Haploview.13 Baseline characteristics were compared by case–control status and genotype using χ2-tests for categorical data, and analysis of variance or a nonparametric equivalent for continuous data.
Genotype associations with mean on-treatment BP, BP at last follow-up, and BP control rates were evaluated using analysis of covariance or logistic regression, adjusting for age, sex, race/ethnicity, African and Native American ancestry (estimated by maximum likelihood from the 87 ancestry informative markers), body mass index, history of renal insufficiency, diabetes, case status, and baseline systolic and diastolic BP. Genotype was treated categorically (2 degrees of freedom). Mean on-treatment BP was defined as the average BP from 6 months after the start of treatment (i.e., end of protocol defined titration phase) to end of follow-up. BP control was defined as <140/<90 or <130/<80 mm Hg in patients with diabetes or renal insufficiency. Based on the potential relationship between BP and case status, BP analyses were also carried out for the case and control groups separately to minimize confounding.
Logistic regression was performed to estimate odds ratios (ORs) and 95% confidence intervals (95% CIs) for case status, tested as a composite of the outcomes and then each individually. Genotype was treated categorically using the common allele as the reference (i.e., -786C and Asp298 are the risk alleles). Additive, dominant, and recessive models were also tested. Testing was performed for the overall population and within the randomized treatment groups. Logistic regression models adjusted for age, sex, race/ethnicity, proportion of West African and Native American ancestry and assigned treatment. Other potential confounders were selected using the stepwise procedure (P < 0.1 for entry, P < 0.05 for retention): history of heart failure, MI, diabetes, stroke or transient ischemic attack, renal insufficiency, dyslipidemia, left ventricular hypertrophy, peripheral vascular disease, stable angina, unstable angina, arrhythmia, cancer, ever-smoking, body mass index, and baseline systolic and diastolic BPs.
The significance threshold for associations with BP and incident cardiovascular events was set at P < 0.05 for tests of global genotype association in the overall population, because this study would represent replication of previous analyses, and P < 0.025 for testing within the treatment arms. The case–control association study had >80% power to detect an OR of 1.40 using an additive model for minor allele frequencies >0.25.
As an exploratory analysis to identify pharmacogenetic interactions and other modifiers of genotype effects, stratified analyses were performed for the cardiovascular events according to added trandolapril and hydrochlorothiazide use, race/ethnicity, sex, age >70 years, smoking (current and ever), and histories of MI, stroke or transient ischemic attack, and diabetes. The rationale for this analysis is that genotype associations may be detected only in patients who have not had a history of MI, for instance, as both cases and noncases had a history of MI, and that this is also the phenotype of interest. Stratified analysis of death, nonfatal MI, and nonfatal stroke was performed only after significant associations with the combined group. The significance threshold for stratified risk estimates was set at P < 0.0024 to account for 21 association tests in the specified subgroups.
RESULTS
Baseline characteristics
Baseline characteristics for the 1,025 patients (256 cases, 769 controls) who were successfully genotyped at either locus appear in Table 1. Both loci were in Hardy–Weinberg equilibrium. The minor allele frequencies for -786T>C in whites, Hispanics, and blacks were 0.37, 0.31, and 0.18, respectively. The minor allele frequencies for Glu298>Asp in whites, Hispanics, and blacks were 0.33, 0.28, and 0.18, respectively. Low linkage disequilibrium was present between the two SNPs (white r2 0.13, Hispanic r2 0.13, black r2 0.03). Demographic and clinical characteristics did not differ significantly by genotype except as follows: diabetes was less prevalent in patients with the -786C allele (T/T 36% vs. T/C 28% vs. C/C 27%; P = 0.03), and left ventricular hypertrophy was more prevalent in patients with the Asp298 allele (Glu/Glu 12% vs. Glu/Asp 15% vs. Asp/Asp 21%; P = 0.008). The median (interquartile range) time to first event or censoring was 1.9 years (1.0–2.5) and 2.9 years (2.5–3.3) for cases and controls, respectively, but follow-up did not differ by genotype or treatment arm.
Table 1.
Baseline characteristics
| Cases (n = 256) | Controls (n = 769) | |
|---|---|---|
| Atenolol strategy | 120 (46.9) | 399 (51.9) |
| Demographic | ||
| Age, mean (s.d.) | 71.5 (9.9) | 70.2 (9.2) |
| Age >70, n (%) | 146 (57.3) | 414 (54.1) |
| Female, n (%) | 131 (51.2) | 392 (51.0) |
| Race/ethnicity, n (%) | ||
| White | 157 (61.3) | 471 (61.3) |
| Hispanic | 63 (24.6) | 197 (25.6) |
| Black | 36 (14.1) | 101 (13.1) |
| BMI, mean (s.d.), kg/m2* | 27.5 (4.8) | 29.0 (5.6) |
| Medical history, n (%) | ||
| History of MI* | 95 (37.1) | 228 (29.7) |
| Heart failure (class I–III)* | 28 (10.9) | 29 (3.8) |
| Stable angina | 151 (59.0) | 481 (62.6) |
| Unstable angina | 38 (14.8) | 97 (12.6) |
| Dyslipidemia | 179 (69.9) | 519 (67.5) |
| LVH | 44 (17.2) | 134 (17.4) |
| Arrhythmia | 25 (9.8) | 74 (9.6) |
| Stroke or TIA | 36 (14.1) | 71 (9.2) |
| PVD* | 43 (16.8) | 88 (11.4) |
| Renal insufficiency* | 14 (5.5) | 18 (2.3) |
| Diabetes* | 102 (39.8) | 223 (29.0) |
| Obese* | 60 (23.4) | 298 (38.8) |
| Cancer | 20 (7.8) | 46 (6.0) |
| Ever-smoker | 132 (51.6) | 354 (46.0) |
| BP | ||
| Systolic BP*, mean (s.d.) | 151 (19) | 148 (19) |
| Diastolic BP, mean (s.d.) | 84 (11) | 83 (11) |
| Heart rate, mean (s.d.) | 75 (8) | 75 (10) |
| BP controlled, n (%) | 48 (18.8) | 187 (24.3) |
BMI, body mass index; BP, blood pressure; LVH, left ventricular hypertrophy; MI, myocardial infarction; PVD, peripheral vascular disease; TIA, transient ischemic attack.
P < 0.05 for cases vs. controls.
BP
Significant differences were noted across -786T>C genotype groups in the average on-treatment systolic BP (T/T 137/78 mm Hg, T/C 135/77 mm Hg, C/C 134/75 mm Hg; P = 0.002 for systolic, P = 0.02 for diastolic) and systolic BP at the last follow-up (T/T 135/77 mm Hg, T/C 133/76 mm Hg, C/C 129/74 mm Hg; P = 0.002 for systolic, P = 0.01 for diastolic). Accordingly, BP control rates at last follow-up were greater in patients with increasing number of -786C alleles (T/T 64%, T/C 71%, C/C 77%; P = 0.004). When evaluated by case– control status, -786T>C genotype associations with BP were noted only in controls (data not shown). Add-on drug use did not differ by -786T>C genotype in a consistent manner (Table 2). The Glu298>Asp polymorphism was not significantly associated with any of the BP response phenotypes in the overall population (data not shown). In analysis by atenolol/verapamil SR use, the BP differences for the -786T>C SNP were significant only in patients receiving verapamil SR, although the results in atenolol-treated patients followed the same trend (Table 2). The interaction P values between NOS3 -786T>C and treatment were significant for mean on-treatment systolic BP (P = 0.03), but not for diastolic BP (P = 0.15), final systolic BP (P = 0.30) or diastolic BP (P = 0.81), or rate of BP control (P = 0.54).
Table 2.
NOS3 -786T>C genotype associations with blood pressure
| Atenolol strategy | Verapamil SR strategy | |||||||
|---|---|---|---|---|---|---|---|---|
| -786T/T (n = 236) | -786T/C (n = 223) | -786C/C (n = 56) | Pa | -786T/T (n = 216) | -786T/C (n = 225) | -786C/C (n = 60) | Pa | |
| On-treatment, mean (s.d.) | ||||||||
| Systolic BP | 136 (11) | 136 (13) | 134 (14) | 0.55 | 137 (12) | 134 (10) | 133 (9) | <0.0001 |
| Diastolic BP | 77 (7) | 77 (6) | 75 (7) | 0.62 | 78 (8) | 76 (7) | 75 (6) | 0.007 |
| Final visit, mean (s.d.) | ||||||||
| Systolic BP | 134 (17) | 133 (18) | 130 (14) | 0.28 | 136 (18) | 132 (15) | 128 (14) | 0.0004 |
| Diastolic BP | 77 (9) | 76 (9) | 73 (9) | 0.04 | 77 (11) | 75 (9) | 74 (9) | 0.12 |
| BP controlled at final visit, n (%)b | 161 (64) | 162 (68) | 46 (75) | 0.16 | 142 (63) | 169 (73) | 50 (80) | 0.004 |
| Antihypertensive drug use, n (%) | ||||||||
| HCTZ | 32 (15) | 40 (19) | 8 (15) | 11 (5) | 18 (9) | 3 (5) | ||
| Trandolapril | 36 (16) | 31 (15) | 7 (13) | 0.71 | 60 (30) | 57 (30) | 15 (26) | 0.55 |
| HCTZ + trandolapril | 155 (70) | 141 (67) | 40 (73) | 132 (65) | 120 (62) | 40 (69) | ||
BP, blood pressure; HCTZ, hydrochlorothiazide.
P values based on analysis of covariance for mean BPs or logistic regression for BP control and adjusted for age, sex, race/ethnicity, African and Native American ancestry, body mass index, history of renal insufficiency, diabetes, case status, and baseline systolic and diastolic BP.
BP control defined as <140/<90 or <130/<80 with a history of diabetes or renal disease.
Cardiovascular events
In the overall population, the -786T>C and Glu298>Asp SNPs were not associated with case status when death, MI and stroke were combined or evaluated individually (Table 3). NOS3 genotype influences on adverse cardiovascular events did not differ according to antihypertensive treatment strategy (-786T>C × treatment strategy interaction P value = 0.18; Glu298>Asp × treatment strategy interaction P value = 0.33). However, a trend toward increased risk was seen for the -786T>C polymorphism and composite case status in patients assigned to atenolol treatment (Table 3). The CIs for the strata were overlapped. Similar trends were noted for -786C allele in the atenolol-treated patients using additive (P = 0.05) and dominant (P = 0.047) coding.
Table 3.
NOS3 -786T>C and Glu298>Asp associations with cardiovascular events
| Adjusted odds ratio (95% confidence interval) | |||
|---|---|---|---|
| -786T>C | T/C vs. T/T | C/C vs. T/T | P* |
| All cases | |||
| Overall population | 1.19 (0.86–1.64) | 1.40 (0.85–2.30) | 0.35 |
| Atenolol | 1.58 (0.99–2.54) | 1.73 (0.82–3.68) | 0.12 |
| Verapamil SR | 0.89 (0.57–1.41) | 1.08 (0.55–2.14) | 0.82 |
| Death | |||
| Overall population | 1.32 (0.81–2.14) | 1.71 (0.83–3.54) | 0.28 |
| Atenolol | 1.94 (0.92–4.10) | 2.11 (0.67–6.61) | 0.19 |
| Verapamil SR | 0.88 (0.45–1.71) | 1.31 (0.51–3.39) | 0.71 |
| Nonfatal MI | |||
| Overall population | 0.82 (0.49–1.38) | 1.13 (0.51–2.51) | 0.65 |
| Atenolol | 1.13 (0.55–2.32) | 1.24 (0.41–3.74) | 0.91 |
| Verapamil SR | 0.48 (0.22–1.05) | 0.58 (0.18–1.88) | 0.17 |
| Nonfatal stroke | |||
| Overall population | 1.44 (0.87–2.39) | 1.32 (0.58–2.99) | 0.36 |
| Atenolol | 1.65 (0.78–3.49) | 1.63 (0.47–5.63) | 0.40 |
| Verapamil SR | 1.32 (0.64–2.72) | 1.17 (0.38–3.65) | 0.76 |
| Glu298>Asp | Glu/Asp vs. Glu/Glu | Asp/Asp vs. Glu/Glu | |
| All cases | |||
| Overall population | 1.25 (0.91–1.71) | 1.29 (0.74–2.26) | 0.34 |
| Atenolol | 1.32 (0.83–2.11) | 1.86 (0.81–4.30) | 0.26 |
| Verapamil SR | 1.22 (0.78–1.90) | 0.99 (0.46–2.12) | 0.66 |
| Death | |||
| Overall population | 1.28 (0.79–2.07) | 1.73 (0.81–3.71) | 0.31 |
| Atenolol | 1.11 (0.53–2.32) | 1.98 (0.58–6.74) | 0.55 |
| Verapamil SR | 1.31 (0.68–2.53) | 1.46 (0.55–3.86) | 0.64 |
| Nonfatal MI | |||
| Overall population | 1.52 (0.91–2.52) | 0.73 (0.24–2.21) | 0.17 |
| Atenolol | 1.51 (0.74–3.06) | 1.46 (0.37–5.72) | 0.51 |
| Verapamil SR | 1.36 (0.65–2.84) | 0.24 (0.03–1.95) | 0.23 |
| Nonfatal stroke | |||
| Overall population | 1.14 (0.69–1.88) | 1.28 (0.55–2.99) | 0.80 |
| Atenolol | 1.44 (0.69–2.96) | 1.55 (0.38–6.27) | 0.58 |
| Verapamil SR | 0.79 (0.38–1.64) | 1.13 (0.38–3.36) | 0.75 |
MI, myocardial infarction.
P values for Wald global test.
In the exploratory subgroup analyses, an association between Glu298>Asp and case status was present only in men but again the association was only nominally significant (additive OR 1.52, 95% CI 1.11–2.09; P = 0.01). Additional nominally significant associations were noted in patients >70 years for the -786T>C SNP, and among ever-smokers and nondiabetics for Glu298>Asp (data not shown). However, the associations did not remain significant in any of the strata after adjustment for multiple comparisons (P > 0.0024 in all strata). As such, genotype associations with death, nonfatal MI, or nonfatal stroke were not evaluated. Of note, no interaction was present between either NOS3 genotype and race/ethnicity or smoking status, factors that have contributed to the heterogeneity of NOS3 and other genetic associations in previous studies (data not shown).14,15
DISCUSSION
NOS3 is a biologically attractive candidate gene for cardiovascular risk given role of NO in vascular function. The current case–control study focused on cardiovascular events in hypertensive CAD patients. The data from INVEST used in this analysis suggest that common NOS3 polymorphisms are associated with the level of BP control (-786T>C) in hypertensive CAD patients receiving an aggressive multi-drug antihypertensive regimen, but are not associated with cardiovascular events.
Angiotensin-converting enzyme inhibitors, dihydropyridine calcium antagonists, and vasodilating β-blockers work in part by altering NO production.11 However, the impact of variability in eNOS function on the BP-lowering response to these and other antihypertensive drugs remains largely unexplored.16,17 In our investigation, we did not identify any association between the NOS3 SNPs and baseline BP, but found that patients with NOS3 polymorphisms had relatively lower BP over the course of treatment and improved rates of BP control. Although the association was significant only for those assigned the verapamil SR strategy, similar trends were observed in the atenolol strategy. Thus, with aggressive multi-drug regimens as required in INVEST, regardless of the type of drug used, patients with the -786T>C polymorphism may be more responsive to therapy. The exact mechanism of the more prominent association observed in verapamil-treated patients is unknown. However, because of distinct mechanisms of action, it is plausible that differential pharmacological modulation of NO balance by calcium antagonists and β-blockers may explain the differences in the strength of the NOS3 genetic association with treatment BP.18 Interestingly, our findings support prior evidence that atenolol does not affect endothelial function or alter NO bioavailability.19,20 The 4–7 mm Hg difference across genotype groups in the verapamil SR arm represents a clinically significant difference; achieving sustained decreases in systolic BP of 12 mm Hg prevents 1 death for every 11 patients treated.21 NOS3 polymorphisms have not been consistently associated with BP phenotypes9,10,22 although our data suggest that NOS3 genotype might contribute to response heterogeneity with some antihypertensive drugs, and pharmacogenetic studies should be pursued to more fully characterize this mechanistically unclear, but biologically plausible gene–drug relationship.
Although the influence of NOS3 SNPs on BP remains somewhat inconclusive, meta-analyses suggest a more consistent role for NOS3 SNPs in CAD.9 However, we did not identify any association between NOS3 polymorphisms and three risk phenotypes for death, MI, or stroke. Aberrant NO metabolism arising from genetic polymorphisms in NOS3 may precede development of overt cardiovascular disease. Thus, given that all INVEST-GENES patients had CAD and associated vascular dysfunction, it is likely that enrichment with such patients diminished our ability to detect genotypic differences in cardiovascular risk. However, even when excluding patients with more severe manifestations of vascular disease such as history of MI or stroke, we still did not observe any associations. Others have similarly reported no association with incident cardiovascular events in CAD patients23 and many of the studies demonstrating significant associations compared cardiovascular disease cases to control populations without known cardiovascular disease.9 Our results do not discount the fact that NOS3 SNPs may be associated with atherosclerotic cardiovascular disease, although our data suggest that NOS3 SNPs may not be important prognostic markers for acute myocardial or cerebrovascular phenotypes in elderly hypertensive patients with established CAD. Furthermore, the findings of our study may reflect the limitations of genetic testing for complex disease risk, in that the test may be less informative in patients who are aggressively treated or have relatively advanced disease.
Although the events in INVEST were adjudicated and the antihypertensive therapies followed a rigid protocol, several limitations remain. First, as previously noted, the association with cardiovascular risk may not have been detected due to the fact that all of the controls had CAD, biasing our assessments toward the null. Second, population stratification could be a concern given that INVEST-GENES enrolled a very large proportion of admixed Hispanic patients, although we did control for ancestry with a large panel of ancestry informative markers. Third, the composite case group is heterogeneous. However, we use it as a screening analysis given our relatively low power to examine associations with the individual endpoints in the context of number of comparisons incurred in analyzing them all. Last, we noted a qualitative interaction between genotype and antihypertensive treatment, but our investigation may have been underpowered to detect gene–environment inter actions. Thus, we are not able to draw definitive conclusions from the exploratory stratified analyses. Additionally, the NOS3 polymorphisms only appear to increase risk by ~20%.9 The point estimates we obtained were consistent with this relatively small increase in risk, and this effect is smaller than we were powered to detect, as reflected by the width of the CIs. However, of the 22 studies of -786T>C and 42 studies of Glu298>Asp cited in prior meta-analysis,9 only four were larger than INVEST-GENES and adequately powered to detect ORs of 1.3 or smaller. It is notable that none of the adequately powered studies revealed a significant association.9
In conclusion, the standard for defining genetic associations, for candidate gene and genome-wide studies alike, is now predicated on replication in independent populations or biological plausibility based on functional studies. Nonvalidating reports of neutral associations are equally important.24 In meta-analysis, NOS3 SNPs do appear to associate with cardiovascular disease, although publication bias has been suggested and NOS3 SNP associations in the literature are heterogeneous. This may be driven by the complexity and variety of phenotypes under investigation, population differences, and differences in case ascertainment or control populations. In our investigation, NOS3 SNPs were significantly associated with the extent of BP control in aggressively treated hypertensive CAD patients, but were not significant predictors of death, nonfatal MI, or nonfatal stroke, despite observed differences in the treated BP levels. Additionally, the genetic associations, or lack thereof, did not appear to be influenced by the specific antihypertensive treatment or clinical factors, such as smoking. Although genetic variation in NOS3 may promote the development of cardiovascular disease and impact BP responses to antihypertensive therapy, it does not appear to contribute substantially to clinical events such as death, MI, and stroke in patients with established cardiovascular disease.
Acknowledgments
This project was funded by National Institutes of Health (Hl074730 and Hl69758 to J.A.J. and C.J.P.), RR17568 to the university of Florida College of Pharmacy, a grant from Abbott Pharmaceuticals (to J.A.J. and C.J.P.), a university of Florida Opportunity Fund grant, (to J.A.J. and C.J.P.) and the American Heart Association Postdoctoral Fellowship (0625619B to M.A.P.). We thank Greg Welder and jennifer Adicks for laboratory assistance.
Footnotes
Disclosure: J.A.J., C.J.P., and R.M.C. received grant funding from Abbott laboratories. C.J.P. has been a consultant for Abbott laboratories. R.M.C. and C.J.P. hold US patent 5,991,731 related to INVEST.
References
- 1.Forstermann U, Munzel T. Endothelial nitric oxide synthase in vascular disease: from marvel to menace. Circulation. 2006;113:1708–1714. doi: 10.1161/CIRCULATIONAHA.105.602532. [DOI] [PubMed] [Google Scholar]
- 2.Iwai C, Akita H, Kanazawa K, Shiga N, Terashima M, Matsuda Y, Takai E, Miyamoto Y, Shimizu M, Kajiya T, Hayashi T, Yokoyama M. Arg389Gly polymorphism of the human beta1-adrenergic receptor in patients with nonfatal acute myocardial infarction. Am Heart J. 2003;146:106–109. doi: 10.1016/S0002-8703(03)00110-8. [DOI] [PubMed] [Google Scholar]
- 3.Nakayama M, Yasue H, Yoshimura M, Shimasaki Y, Kugiyama K, Ogawa H, Motoyama T, Saito Y, Ogawa Y, Miyamoto Y, Nakao K. T-786-->C mutation in the 5′-flanking region of the endothelial nitric oxide synthase gene is associated with coronary spasm. Circulation. 1999;99:2864–2870. doi: 10.1161/01.cir.99.22.2864. [DOI] [PubMed] [Google Scholar]
- 4.Cattaruzza M, Guzik TJ, Slodowski W, Pelvan A, Becker J, Halle M, Buchwald AB, Channon KM, Hecker M. Shear stress insensitivity of endothelial nitric oxide synthase expression as a genetic risk factor for coronary heart disease. Circ Res. 2004;95:841–847. doi: 10.1161/01.RES.0000145359.47708.2f. [DOI] [PubMed] [Google Scholar]
- 5.Tesauro M, Thompson WC, Rogliani P, Qi L, Chaudhary PP, Moss J. Intracellular processing of endothelial nitric oxide synthase isoforms associated with differences in severity of cardiopulmonary diseases: cleavage of proteins with aspartate vs. glutamate at position 298. Proc Natl Acad Sci USA. 2000;97:2832–2835. doi: 10.1073/pnas.97.6.2832. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Persu A, Stoenoiu MS, Messiaen T, Davila S, Robino C, El-Khattabi O, Mourad M, Horie S, Feron O, Balligand JL, Wattiez R, Pirson Y, Chauveau D, Lens XM, Devuyst O. Modifier effect of ENOS in autosomal dominant polycystic kidney disease. Hum Mol Genet. 2002;11:229–241. doi: 10.1093/hmg/11.3.229. [DOI] [PubMed] [Google Scholar]
- 7.Savvidou MD, Vallance PJ, Nicolaides KH, Hingorani AD. Endothelial nitric oxide synthase gene polymorphism and maternal vascular adaptation to pregnancy. Hypertension. 2001;38:1289–1293. doi: 10.1161/hy1201.097305. [DOI] [PubMed] [Google Scholar]
- 8.Leeson CP, Hingorani AD, Mullen MJ, Jeerooburkhan N, Kattenhorn M, Cole TJ, Muller DP, Lucas A, Humphries SE, Deanfield JE. Glu298Asp endothelial nitric oxide synthase gene polymorphism interacts with environmental and dietary factors to influence endothelial function. Circ Res. 2002;90:1153–1158. doi: 10.1161/01.res.0000020562.07492.d4. [DOI] [PubMed] [Google Scholar]
- 9.Casas JP, Cavalleri GL, Bautista LE, Smeeth L, Humphries SE, Hingorani AD. Endothelial nitric oxide synthase gene polymorphisms and cardiovascular disease: a HuGE review. Am J Epidemiol. 2006;164:921–935. doi: 10.1093/aje/kwj302. [DOI] [PubMed] [Google Scholar]
- 10.Zintzaras E, Kitsios G, Stefanidis I. Endothelial NO synthase gene polymorphisms and hypertension: a meta-analysis. Hypertension. 2006;48:700–710. doi: 10.1161/01.HYP.0000238124.91161.02. [DOI] [PubMed] [Google Scholar]
- 11.Ignarro LJ, Napoli C, Loscalzo J. Nitric oxide donors and cardiovascular agents modulating the bioactivity of nitric oxide: an overview. Circ Res. 2002;90:21–28. doi: 10.1161/hh0102.102330. [DOI] [PubMed] [Google Scholar]
- 12.Pepine CJ, Handberg EM, Cooper-DeHoff RM, Marks RG, Kowey P, Messerli FH, Mancia G, Cangiano JL, Garcia-Barreto D, Keltai M, Erdine S, Bristol HA, Kolb HR, Bakris GL, Cohen JD, Parmley WW. A calcium antagonist vs a non-calcium antagonist hypertension treatment strategy for patients with coronary artery disease. The International Verapamil-Trandolapril Study (INVEST): a randomized controlled trial. JAMA. 2003;290:2805–2816. doi: 10.1001/jama.290.21.2805. [DOI] [PubMed] [Google Scholar]
- 13.Barrett JC, Fry B, Maller J, Daly MJ. Haploview: analysis and visualization of LD and haplotype maps. Bioinformatics. 2005;21:263–265. doi: 10.1093/bioinformatics/bth457. [DOI] [PubMed] [Google Scholar]
- 14.Lee CR, North KE, Bray MS, Avery CL, Mosher MJ, Couper DJ, Coresh J, Folsom AR, Boerwinkle E, Heiss G, Zeldin DC. NOS3 polymorphisms, cigarette smoking, and cardiovascular disease risk: the Atherosclerosis Risk in Communities study. Pharmacogenet Genomics. 2006;16:891–899. doi: 10.1097/01.fpc.0000236324.96056.16. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 15.Johnson JA. Ethnic differences in cardiovascular drug response: potential contribution of pharmacogenetics. Circulation. 2008;118:1383–1393. doi: 10.1161/CIRCULATIONAHA.107.704023. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Turner ST, Chapman AB, Schwartz GL, Boerwinkle E. Effects of endothelial nitric oxide synthase, alpha-adducin, and other candidate gene polymorphisms on blood pressure response to hydrochlorothiazide. Am J Hypertens. 2003;16:834–839. doi: 10.1016/s0895-7061(03)01011-2. [DOI] [PubMed] [Google Scholar]
- 17.Arnett DK, Claas SA, Glasser SP. Pharmacogenetics of antihypertensive treatment. Vascul Pharmacol. 2006;44:107–118. doi: 10.1016/j.vph.2005.09.010. [DOI] [PubMed] [Google Scholar]
- 18.Rossi GP, Taddei S, Virdis A, Cavallin M, Ghiadoni L, Favilla S, Versari D, Sudano I, Pessina AC, Salvetti A. The T-786C and Glu298Asp polymorphisms of the endothelial nitric oxide gene affect the forearm blood flow responses of Caucasian hypertensive patients. J Am Coll Cardiol. 2003;41:938–945. doi: 10.1016/s0735-1097(02)03011-5. [DOI] [PubMed] [Google Scholar]
- 19.Pasini AF, Garbin U, Stranieri C, Boccioletti V, Mozzini C, Manfro S, Pasini A, Cominacini M, Cominacini L. Nebivolol treatment reduces serum levels of asymmetric dimethylarginine and improves endothelial dysfunction in essential hypertensive patients. Am J Hypertens. 2008;21:1251–1257. doi: 10.1038/ajh.2008.260. [DOI] [PubMed] [Google Scholar]
- 20.Fratta Pasini A, Garbin U, Nava MC, Stranieri C, Davoli A, Sawamura T, Lo Cascio V, Cominacini L. Nebivolol decreases oxidative stress in essential hypertensive patients and increases nitric oxide by reducing its oxidative inactivation. J Hypertens. 2005;23:589–596. doi: 10.1097/01.hjh.0000160216.86597.ff. [DOI] [PubMed] [Google Scholar]
- 21.Chobanian AV, Bakris GL, Black HR, Cushman WC, Green LA, Izzo JL, Jr, Jones DW, Materson BJ, Oparil S, Wright JT, Jr, Roccella EJ. The National High Blood Pressure Education Program Coordinating C. Seventh report of the Joint National Committee on Prevention, Detection, Evaluation, and Treatment of High Blood Pressure. Hypertension. 2003;42:1206–1252. doi: 10.1161/01.HYP.0000107251.49515.c2. [DOI] [PubMed] [Google Scholar]
- 22.Pereira TV, Rudnicki M, Cheung BM, Baum L, Yamada Y, Oliveira PS, Pereira AC, Krieger JE. Three endothelial nitric oxide (NOS3) gene polymorphisms in hypertensive and normotensive individuals: meta-analysis of 53 studies reveals evidence of publication bias. J Hypertens. 2007;25:1763–1774. doi: 10.1097/HJH.0b013e3281de740d. [DOI] [PubMed] [Google Scholar]
- 23.Agema WR, de Maat MP, Zwinderman AH, Kastelein JJ, Rabelink TJ, van Boven AJ, Feskens EJ, Boer JM, van der Wall EE, Jukema JW. An integrated evaluation of endothelial constitutive nitric oxide synthase polymorphisms and coronary artery disease in men. Clin Sci. 2004;107:255–261. doi: 10.1042/CS20030360. [DOI] [PubMed] [Google Scholar]
- 24.Morgan TM, Krumholz HM, Lifton RP, Spertus JA. Nonvalidation of reported genetic risk factors for acute coronary syndrome in a large-scale replication study. JAMA. 2007;297:1551–1561. doi: 10.1001/jama.297.14.1551. [DOI] [PubMed] [Google Scholar]