Vascular Stiffness and Genetic Variation at the Endothelial Nitric Oxide Synthase Locus
The Framingham Heart Study
Arterial stiffness is a moderately heritable trait that is affected by alterations in the bioavailability of NO. Previous studies have found associations between variants in the gene for endothelial NO synthase (NOS3) and arterial properties. We previously identified a linkage peak for forward pressure wave amplitude in the immediate vicinity of NOS3. Therefore, we evaluated relations between arterial stiffness measures and common genetic variants at this locus. Eighteen single nucleotide polymorphisms capturing ≈90% of underlying common variation in NOS3 were genotyped in unrelated Framingham Heart Study participants (N=1157; 52.2% women; mean age: 62 years) with routinely ascertained tonometry data that provided 5 arterial phenotypes (forward and reflected pressure wave amplitude, central pulse pressure, carotid–femoral pulse wave velocity, and mean arterial pressure). In women but not men, the genotype for the common NOS3 missense mutation (Glu298Asp, rs1799983) was related to central pulse pressure (women: GG=53±0.9, GT=54±0.9, and TT=47±2.0 mm Hg, P=0.0047; men: GG=50±1.0, GT=49±0.9, and TT=47±1.8 mm Hg, P=0.30) and forward wave amplitude (women: GG=41±0.7, GT=42±0.7, and TT=38±1.6 mm Hg, P=0.029; men: GG=42±0.9, GT=41±0.8, and TT=39±1.5 mm Hg, P=0.47). The only other nominally significant sex-specific association in men but not women was between an intronic polymorphism (rs1800781) and reflected wave amplitude (women: AA=10.4±0.4, AG=11.1±0.6, and GG=8.9±2.2 mm Hg, P=0.50; men: AA=6.1±0.3, AG=7.3±0.5, and GG=11.3±2.3 mm Hg, P=0.014). After adjusting for multiple testing (18 polymorphisms and 5 phenotypes), these nominal associations were no longer significant. The present study was suggestive of modest relations between common genetic variants at the NOS3 locus and arterial stiffness.
Arterial stiffness increases with advancing age even in relatively healthy people who are at low risk for cardiovascular disease.1 The contribution of increased arterial stiffness to the pathogenesis of systolic hypertension, cardiovascular disease and damage, and dysfunction in the brain, kidneys, and other organs has stimulated interest in further defining factors that modulate arterial stiffness. We recently evaluated heritability and linkage for several key tonometry phenotypes, including forward pressure wave amplitude, reflected pressure wave amplitude, carotid–femoral pulse wave velocity (PWV), central pulse pressure, and true mean arterial pressure, and found moderate heritability (h2=0.21 to 0.48) and regions of significant (forward wave amplitude and reflected wave amplitude) or suggestive (carotid–femoral PWV, central pulse pressure, and true mean arterial pressure) linkage.2 Notably, we found a linkage peak for forward wave amplitude on chromosome 7 at 174 cM (logarithm of odds: 2.88; permuted P=0.017). The gene (NOS3) encoding endothelial cell NO synthase lies directly beneath this linkage peak.
Arterial tonometry has been routinely ascertained in the seventh examination cycle of the Framingham Heart Study (FHS) Offspring cohort. Common genetic variation and the linkage disequilibrium pattern at the NOS3 locus has been well characterized in FHS participants.3 Given the availability of these 2 data sets, the Framingham Offspring Study provided a unique opportunity to examine the association between genetic variation at the NOS3 locus and arterial stiffness.
The design of the FHS Offspring cohort has been described.4 Among the 3539 participants who attended the seventh examination (1998–2001), tonometry, which was initiated in early 1999, was attempted in 2640 participants; 2271 participants had complete, analyzable tonometry measurements of the brachial, radial, femoral, and carotid arteries. Separately, during the sixth examination (1995–1998), 1809 unrelated individuals provided blood samples for DNA extraction. FHS randomly selected these unrelated individuals for DNA collection without regard to any phenotype feature. In the present study, everyone who had tonometry measured was eligible provided they had DNA available. This resulted in 1157 participants who were eligible for genotype–tonometry association analyses. The Boston University Medical Center Institutional Review Board approved the study; participants gave written informed consent.
Noninvasive Tonometry Measures
Participants were studied in the supine position after several minutes of rest as described previously.1 Arterial tonometry, with simultaneous ECG, was obtained from the brachial, radial, femoral, and carotid arteries. Transit distances were assessed by body surface measurements from the suprasternal notch to each pulse recording site. Tonometry waveforms were analyzed as described previously.1 Carotid–femoral PWV was calculated from tonometry waveforms and body surface measurements. The central forward wave amplitude was defined as the difference between pressure at the waveform foot and pressure at the first systolic inflection point or peak of the carotid pressure waveform. Reflected wave pressure was defined as the difference between central systolic pressure and pressure at the forward wave peak.
Genotyping in the FHS
The NOS3 genotyping protocol and results have been described.3 We genotyped the previously reported common missense variant, Glu298Asp (rs1799983), 11 haplotype tagging single nucleotide polymorphisms (SNPs), and the previously studied T−786C promoter variant (rs2070744), as well as 5 additional SNPs that were redundant in Centre d’Etude du Polymorphisme Humain pedigrees (to help assess linkage disequilibrium block structure similarity between Centre d’Etude du Polymorphisme Humain and FHS samples). Thus, 18 NOS3 SNPs were genotyped in FHS (please see Table S1 available in a data supplement online at http://hyper.ahajournals.org). All of the SNPs were in Hardy–Weinberg equilibrium in the FHS sample (χ2 test P>0.05).3 As noted in a previous publication, these 18 genotyped SNPs capture ≈90% of common genetic variability at the NOS3 locus.3
Using a general model of inheritance we conducted multivariable linear regression analyses (SAS Proc GLM5) to test the null hypothesis that means for tonometry variables did not differ by SNP genotype. Because of previously reported sex differences in relations between vascular function and the NOS3 genotype,6,7 we report sex-specific analyses.
We adjusted for covariates associated previously with arterial function in our cohort, including the following: age, age2, sex (in sex-pooled models), heart rate, body mass index, total/high-density lipoprotein cholesterol ratio, triglycerides, fasting glucose, diabetes, prevalent cardiovascular disease, hormone replacement therapy, antihypertensive medication use, lipid-lowering medication, active smoking, smoking within 6 hours before the vascular test, and walk test before or after tonometry measurements. In analyses involving reflected pressure wave amplitude as the dependent variable, height and weight were included as covariates instead of body mass index. If we assume that the 17 covariates in our base model account for 25% of the variance in the arterial stiffness variable, then with our sample size of 1157 participants, we have 90% power to detect the addition of a genotype variable that explains as little as 0.7% of the variance in the stiffness variable.
To account for multiple statistical testing, we constructed null data sets through bootstrap resampling; genotypes and phenotypes were sampled randomly with replacement. For each of the 18 SNPs and 5 phenotypes (90 models), we ran the sex-pooled, multivariable-adjusted regression model 1000 times and evaluated the distribution of minimum P values. We obtained an empirical P value for the entire analysis by comparing the minimum nominal P value to the distribution of P values from the null data sets.
We examined 1157 (52.2% women) middle-aged-to-elderly FHS participants (Table 1) with the NOS3 genotype and tonometry measures. A comprehensive list of SNPs that were genotyped along with minor allele frequencies and genotyping call rates are provided in the online supplement (please see Table S1). In the sex-pooled sample, the genotype for the missense variant Glu298Asp (rs1799983) was associated with central pulse pressure, reflected wave amplitude, and mean arterial pressure (Table 2). Notably, only 1 other nominally significant association was found between SNP 16 (rs3918174) and mean arterial pressure (mean arterial pressure: AA= 90.9±0.4, AG=92.8±0.7, and GG=90.0±2.7 mm Hg; N: AA=812, AG=283, and GG=17; P=0.041).
In sex-specific analyses, Glu298Asp was related to central pulse pressure and forward and reflected pressure wave amplitude in women (Table 3). Women with the TT genotype had a central pulse pressure that was ≈6 to 7 mm Hg lower as compared with those with the GG or GT genotypes (Table 3). However, a test for a sex–genotype interaction was not significant for central pulse pressure (P=0.14), forward wave amplitude (P=0.25), or reflected wave amplitude (P=0.28). Similarly, tests for interactions between age and genotype were negative as well (P>0.05). The only other nominally significant association in sex-specific analyses was found between SNP 10 (rs1800781) and reflected wave amplitude in men (reflected wave: AA=6.1±0.3, AG=7.3±0.5, and GG=11.3±2.3 mm Hg; N: AA=375, AG=134, and GG=6; P=0.014) but not women (reflected wave: AA=10.4±0.4, AG=11.1±0.6, and GG=8.9±2.2 mm Hg; N: AA=430, AG=147, and GG=12; P=0.50). However, a test for an interaction between sex and genotype was not significant (P=0.38).
Accounting for Multiple Testing
We evaluated 18 SNPs and 5 phenotypes in sex-pooled models and found a minimum nominal P=0.011 (central pulse pressure; Table 2). With 1000 resampling procedure runs, this corresponds with an overall empirical P=0.53.
This study evaluated relations between common genetic variants in the NOS3 gene and several tonometry measures of arterial function and found that homozygosity for the common missense mutation (Glu298Asp) was associated with reduced central pulse pressure and forward wave amplitude, particularly in women. Women homozygous for the minor (T) allele had a central pulse pressure that was 6 to 7 mm Hg lower than women who were heterozygous or homozygous for the wild type (G) allele. These analyses in the unrelated Framingham Offspring subset were prompted by previous findings of linkage for forward wave amplitude in the immediate vicinity of NOS3 in analyses that were performed in the separate family based subset of Framingham Offspring study participants.2 Taken together, these 2 studies in different subgroups of an unselected, community-based sample provide support for a modest role of NOS3 in the modulation of large artery properties. However, it is important to note that in light of the large number of statistical comparisons that were required to comprehensively evaluate common genetic variability at the NOS3 locus, we are unable to exclude the possibility that the present findings represent a chance association.
Previous studies provide clear evidence for an important physiological role for NO in the modulation of large artery properties.8–11 Studies in the NOS3 knockout mouse have demonstrated an elevated pulse pressure.12 The foregoing studies suggest a biologically plausible role for NOS3 in the modulation of arterial properties and strengthen the prior probability of a true association between variants in the NOS3 gene and measures of arterial function. However, studies of the implications of the Glu298Asp mutation in humans are limited, and their conclusions are less concordant. A prior study in humans found no relation between the Glu298Asp mutation and carotid–femoral PWV, which is consistent with our findings.13 Another study in hypertensive individuals found a possible relation between the increase in pulse pressure with advancing age and the G298T polymorphism in women only.6 A recent study evaluated potential relations between the Glu298Asp polymorphism and carotid artery stiffness in a small sample of black and white young adults and found an association between presence of the T-allele (GT or TT) of the G894T polymorphism and lower carotid artery stiffness in blacks.14 This latter study parallels our finding that the T allele seems to offer a protective effect on measures of vascular function. Failure to see a significant effect in white individuals of European descent in the previous study may be attributable to grouping of GT and TT individuals in those analyses, which was done because of small sample size and may have obscured what appears to be an effect in TT homozygotes only in our study.
In contrast to our findings of a potentially beneficial effect of the T allele on measures of arterial function, early reports suggested that the T allele was associated with increased risk for hypertension.15 However, several large studies subsequently found no relation to hypertension and related phenotypes, including left ventricular mass and carotid intima–media thickness.16–18 Furthermore, data from Bogalusa suggested that the T allele was associated with lower long-term blood pressure in black women, which is consistent with our findings of a potentially protective effect of the T allele.19
Investigations of the effects of the Glu298Asp variant on in vitro endothelial cell NO synthase function have also yielded variable results. Early work suggested that the mutation increased susceptibility to intracellular cleavage20; however, more recent in vitro studies have challenged this assertion,21 and additional in vitro studies have not shown a functional difference between the 2 endothelial cell NO synthase alleles.22,23 Thus, although several studies, including ours, are supportive of a modest protective effect of the T allele on arterial function, the functional effects of the Glu298Asp mutation and its role in the pathogenesis of cardiovascular disease remain controversial.
Our analysis of relations between NOS3 variants and tonometry measures of arterial properties was prompted by our previous finding of a significant linkage peak for forward wave amplitude on chromosome 7 in the immediate vicinity of the NOS3 gene.2 Importantly, both forward wave amplitude and central pulse pressure, which is largely determined by forward wave amplitude, were associated with Glu298Asp in the present analysis, particularly in women (Table 3). However, it seems unlikely that the modest association between Glu298Asp fully accounts for the linkage peak found in the previous study, raising the possibility that variants in other genes in the vicinity of NOS3 may contribute to arterial properties and may account for the previously observed linkage peak. However, we could not evaluate this question directly, because the subsample genotyped in the present analysis consists of unrelated members of the Framingham offspring cohort, whereas our previous linkage analysis was performed in a separate subsample consisting of family members who have not been genotyped for the NOS3 SNPs.
There are several potential mechanisms for an effect of NOS3 mutations on arterial properties and forward wave amplitude. NO is a vasodilator and has well known short-term effects on muscular tone in large and especially in small arteries. We previously evaluated relations between brachial flow-mediated dilation and the NOS3 genotype in the present sample and found modest relations in general and no relation for the Glu298Asp polymorphism.3 However, long-term effects of NO on aortic structure and function may differ from the short-term effects in the brachial artery during reactive hyperemia. We speculate that long-term remodeling of aortic structure in response to variability in ambient flow may be impaired in a subset of individuals leading to increased forward wave amplitude and elevated pulse pressure. The present study suggests, however, that common genetic variation at the NOS3 locus accounts for only a modest proportion of the variance in tonometry phenotypes in this community-based sample.
Our study has several limitations. We have evaluated common variants in the NOS3 gene. There remains the possibility that multiple rare NOS3 mutations influence arterial stiffness measures. Our cohort was middle-aged to elderly and white, potentially limiting generalizability to individuals who are younger or other ethnicities/races. To reduce the effects of multiple testing, we evaluated only the general or codominant model of inheritance. As a result, we may have missed modest, nominally significant associations that were detectable using dominant or recessive models. However, the overall variance explained by such associations would necessarily be quite small to escape detection by the general model. We also did not evaluate haplotypes in the present analysis; thus, we may have missed associations attributable to mutations that are present on ≥1 haplotype but that are not sufficiently linked to any one of the specific SNPs that we evaluated. However, given the density of SNPs that we have evaluated and the comprehensive coverage that was achieved, it seems unlikely that we have missed an important association. Furthermore, the amount of statistical testing was already substantial (18 SNPs and 5 phenotypes, resulting in 90 models, which were then repeated in men and women separately). Thus, our comprehensive genotyping and phenotyping resulted in an empirical P value indicative of no association of the NOS3 locus with tonometry phenotypes if evaluated on a gene-wide scale. By addressing the false-positive rate through empirical methods, we may have increased our false-negative rate.24 As noted previously, rigid adherence to an empirical P<0.05 significance threshold across a study could be overly conservative and may obscure some true-positive associations.3,25 Study strengths distinguishing the present investigation include genotyping a comprehensive set of common variants in a large community-based cohort with routinely ascertained phenotypes and covariates, sex-specific analyses, and multiple statistical testing assessments.
Increased pulse pressure and large artery stiffness are important risk factors for several common diseases that contribute to considerable morbidity and mortality. Genetic factors appear to contribute substantially to individual differences in arterial properties. We demonstrated previously that a locus on chromosome 7 in the vicinity of the NOS3 gene contributes to variability in forward wave amplitude, which is the principal determinant of pulse pressure. The present study found that a common missense mutation in the NOS3 gene may account for a component of the variability in forward wave amplitude and pulse pressure at this locus; however, further studies are warranted to determine whether additional genes in this region contribute to arterial properties.
Sources of Funding
This work was supported by National Heart, Lung, and Blood Institute grant N01-HC-25195; CardioGenomics Programs for Genomic Applications (HL66582); grant HL60040; grant HL70100; grants HL71039, NO1-HV28178, and K24-HL-04334 (R.S.V.); and the Donald W. Reynolds Foundation.
G.F.M. is owner of Cardiovascular Engineering, Inc, a company that designs and manufactures devices that measure vascular stiffness. The company uses these devices in clinical trials that evaluate the effects of diseases and interventions on vascular stiffness. The remaining authors report no conflicts.
- Received November 27, 2006.
- Revision received December 15, 2006.
- Accepted March 8, 2007.
Mitchell GF, Parise H, Benjamin EJ, Larson MG, Keyes MJ, Vita JA, Vasan RS, Levy D. Changes in arterial stiffness and wave reflection with advancing age in healthy men and women: the Framingham Heart Study. Hypertension. 2004; 43: 1239–1245.
Mitchell GF, DeStefano AL, Larson MG, Benjamin EJ, Chen MH, Vasan RS, Vita JA, Levy D. Heritability and a genome-wide linkage scan for arterial stiffness, wave reflection, and mean arterial pressure: the Framingham Heart Study. Circulation. 2005; 112: 194–199.
Kathiresan S, Larson MG, Vasan RS, Guo CY, Vita JA, Mitchell GF, Keyes MJ, Newton-Cheh C, Musone SL, Lochner AL, Drake JA, Levy D, O’Donnell CJ, Hirschhorn JN, Benjamin EJ. Common genetic variation at the endothelial nitric oxide synthase locus and relations to brachial artery vasodilator function in the community. Circulation. 2005; 112: 1419–1427.
Kannel WB, Feinleib M, McNamara PM, Garrison RJ, Castelli WP. An investigation of coronary heart disease in families. The Framingham offspring study. Am J Epidemiol. 1979; 110: 281–290.
SAS Institute, Inc. SAS/STAT User’s Guide, Version 8.1. Cary, NC: SAS Institute, Inc; 2000.
Mourad JJ, Ducailar G, Rudnicki A, Lajemi M, Mimran A, Safar ME. Age-related increase of pulse pressure and gene polymorphisms in essential hypertension: a preliminary study. J Renin Angiotensin Aldosterone Syst. 2002; 3: 109–115.
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.
Fitch RM, Vergona R, Sullivan ME, Wang YX. Nitric oxide synthase inhibition increases aortic stiffness measured by pulse wave velocity in rats. Cardiovasc Res. 2001; 51: 351–358.
Kinlay S, Creager MA, Fukumoto M, Hikita H, Fang JC, Selwyn AP, Ganz P. Endothelium-derived nitric oxide regulates arterial elasticity in human arteries in vivo. Hypertension. 2001; 38: 1049–1053.
Ramsey MW, Goodfellow J, Jones CJ, Luddington LA, Lewis MJ, Henderson AH. Endothelial control of arterial distensibility is impaired in chronic heart failure. Circulation. 1995; 92: 3212–3219.
Wilkinson IB, Qasem A, McEniery CM, Webb DJ, Avolio AP, Cockcroft JR. Nitric oxide regulates local arterial distensibility in vivo. Circulation. 2002; 105: 213–217.
Chen W, Srinivasan SR, Bond MG, Tang R, Urbina EM, Li S, Boerwinkle E, Berenson GS. Nitric oxide synthase gene polymorphism (G894T) influences arterial stiffness in adults: the Bogalusa Heart Study. Am J Hypertens. 2004; 17: 553–559.
Miyamoto Y, Saito Y, Kajiyama N, Yoshimura M, Shimasaki Y, Nakayama M, Kamitani S, Harada M, Ishikawa M, Kuwahara K, Ogawa E, Hamanaka I, Takahashi N, Kaneshige T, Teraoka H, Akamizu T, Azuma N, Yoshimasa Y, Yoshimasa T, Itoh H, Masuda I, Yasue H, Nakao K. Endothelial nitric oxide synthase gene is positively associated with essential hypertension. Hypertension. 1998; 32: 3–8.
Karvonen J, Kauma H, Kervinen K, Rantala M, Ikaheimo M, Paivansalo M, Savolainen MJ, Kesaniemi YA. Endothelial nitric oxide synthase gene Glu298Asp polymorphism and blood pressure, left ventricular mass and carotid artery atherosclerosis in a population-based cohort. J Intern Med. 2002; 251: 102–110.
Kato N, Sugiyama T, Morita H, Nabika T, Kurihara H, Yamori Y, Yazaki Y. Lack of evidence for association between the endothelial nitric oxide synthase gene and hypertension. Hypertension. 1999; 33: 933–936.
Chen W, Srinivasan SR, Li S, Boerwinkle E, Berenson GS. Gender-specific influence of NO synthase gene on blood pressure since childhood: the Bogalusa Heart Study. Hypertension. 2004; 44: 668–673.
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 U S A. 2000; 97: 2832–2835.
Fairchild TA, Fulton D, Fontana JT, Gratton JP, McCabe TJ, Sessa WC. Acidic hydrolysis as a mechanism for the cleavage of the Glu(298)–>Asp variant of human endothelial nitric-oxide synthase. J Biol Chem. 2001; 276: 26674–26679.
Wacholder S, Chanock S, Garcia-Closas M, El Ghormli L, Rothman N. Assessing the probability that a positive report is false: an approach for molecular epidemiology studies. J Natl Cancer Inst. 2004; 96: 434–442.
Thomas DC, Clayton DG. Betting odds and genetic associations. J Natl Cancer Inst. 2004; 96: 421–423.