Obesity and the nitric oxide synthase 3 (NOS3) gene polymorphisms are associated with nitrite levels and hypertension. However, no study has tested the hypothesis that NOS3 tagSNPs rs3918226, rs3918188, rs743506 and rs7830 affect nitrite levels and are associated with hypertension in childhood obesity. We investigated the association of these NOS3 tagSNPs and the haplotypes formed by them with hypertension and with nitrite levels in children and adolescents with obesity and with obesity plus hypertension. We studied 355 subjects: 174 healthy (controls), 109 normotensive obese, and 72 obese children and adolescents with obesity plus hypertension. Genotypes were determined by Taqman allele discrimination assay and real-time PCR. We compared the distribution of NOS3 tagSNP genotypes, alleles and haplotypes in the three groups of subjects. Nitrite levels were determined by ozone-based chemiluminescence. Nitrite levels were affected by the rs3918226 polymorphism (P<0.05) but not by NOS3 haplotypes. There was no association between the tagSNPs studied and hypertension in children and adolescents. Our findings show that the NOS3 tagSNP rs3918226 is associated with NO production in children and adolescents, and suggest that this polymorphism may have an impact on cardiovascular health. Further studies are needed to better clarify the effects of this polymorphism on cardiovascular risk.
Childhood obesity is increasing in most countries and is an important risk factor for the development of disease and death in adulthood.1, 2, 3 Childhood obesity presents several clinical features, among them hypoadiponectinemia and reduced bioavailability of nitric oxide (NO).4, 5 These clinical features are responsible for, at least in part, the association between obesity and hypertension.4, 6
Adiponectin is an anti-inflammatory and antiatherogenic adipokine expressed by the adipose tissue in inverse proportion to adiposity in adults and children.5, 7 Moreover, adiponectin is also associated with hypertension due to its role in vascular homeostasis and NO synthesis.6, 8 Previous data have shown that NO bioavailability is positively affected by adiponectin concentrations in obese children and adolescents, suggesting that adiponectin may exert cardiovascular protective roles.9
NO is synthesized from L-arginine by at least three isoforms of NO synthases (NOSs), including the endothelial NOS (NOS3). Endogenous NO has important vasodilator properties, prevents leukocyte and platelet adhesion to vascular endothelium, and inhibits vascular smooth muscle cell migration and proliferation.10, 11 Conversely, reduced NO formation is associated with hypertension,12 and single-nucleotide polymorphisms (SNPs) of NOS3 gene were associated with hypertension, particularly combinations of three clinically relevant polymorphisms in the NOS3 gene: the SNPs rs2070744 in the promoter region, rs1799983 in exon 7, and a 27-bp variable number of tandem repeats in intron 4.13, 14, 15, 16, 17 More recently, other tagSNPs in the NOS3 gene, which represent the information of neighboring SNPs in linkage disequilibrium (rs3918188, rs743506 and rs7830), were associated with alterations in the vascular function and low levels of NO in adults.18, 19, 20 Importantly, one (rs3918226) of these tagSNPs was recently associated with hypertension in two large genome-wide association studies,21, 22 although it apparently does not affect NO formation in healthy subjects.23 However, the association of these NOS3 tagSNPs with hypertension and plasma NO levels in childhood obesity has not been studied so far. This interaction seems relevant because children and adolescents are exposed to environmental factors for shorter periods of time as compared to adult populations, and therefore the effects of NOS3 genotypes on the susceptibility to hypertension and on NO bioavailability could be more clearly detected in children and adolescents than in adults.
In this study we aimed at evaluating whether four NOS3 tagSNPs (rs3918226, rs3918188, rs743506 and rs7830) and the haplotypes formed by them are associated with hypertension, and with nitrite levels in children and adolescents with obesity, or with obesity and hypertension.
Materials and methods
This study was approved by the Institutional Review Board at the Federal University of Juiz de Fora, Brazil, for the inclusion of human subjects. Parents and children were informed of the nature and purpose of the study. Parents gave their written consent and children gave their verbal consent.
We studied 109 children and adolescents with obesity and 72 children and adolescents with obesity plus hypertension recruited from the Childhood Endocrinology Ambulatory of the IMEPEN Foundation at Juiz de Fora. The control group consisted of 174 healthy children and adolescents recruited from the local community.
All children underwent physical examination. Height was measured to the nearest 0.1 cm by using a wall-mounted stadiometer. The body weight was measured with a digital scale to the nearest 0.1 kg. Body mass index was calculated as the weight in kilograms divided by height in meters squared.
Obesity was defined as body mass index greater than the 95th percentile, matched according to age and sex.24 The waist circumference measurement was made at the midpoint between the bottom of the rib cage and above the top of the iliac crest. Systolic and diastolic blood pressures were measured at least three times and the presence of hypertension was defined as systolic and/or exceeding the 95th percentile.25
At the time of clinic attendance and after 12 hours fasting, venous blood samples were collected and genomic DNA was extracted from the cellular component of 1 ml of whole blood by a salting-out method and stored at −20 °C until analyzed.26
Lipid parameters (total cholesterol, triglycerides, high-density lipoprotein cholesterol), glucose and uric acid concentrations were determined in serum, with routine enzymatic methods using commercial kits (Labtest Diagnostic, SA, Lagoa Santa, Brazil). Low-density lipoprotein concentration was calculated according to the Friedewald formula.27 Insulin and adiponectin concentrations were measured in EDTA-plasma using an ELISA kit (Genese Diagnostics Products, Sao Paulo, Brazil).
Measurement of nitrite concentrations in whole-blood samples
To measure whole-blood nitrite concentrations in triplicate, venous blood samples were collected into tubes containing heparine and immediately mixed with a nitrite preservation solution in 5:1 dilution as previously described.28 Briefly, this solution contains 0.8 M ferricyanide and 1% NP-40. The samples were deproteinated with methanol (1:1) and centrifuged at 14 000 g for 5 min. Then, 200 μl supernatant was injected into the solution of acidified tri-iodide, purged with nitrogen in-line with a gas-phase chemiluminescence NO analyzer (Model 280, NO analyzer; Sievers, Boulder, CO, USA). Approximately 8 ml of triiodide solution (2.0 g of potassium iodide and 1.3 g of iodine dissolved in 40 ml of water with 140 ml of acetic acid) was placed in the purge vessel into which samples were injected. The triiodide solution reduced nitrites to NO gas, which was detected by the NO analyzer.29
Four tagging SNPs, rs3918188, rs3918226, rs743506 and rs7830, were selected from the Genome Variation Server of Seattle SNPs (http://gvs.gs.washington.edu/GVS/) using the parameters of r2⩾0.8 as threshold for clusters of linkage disequilibrium among polymorphisms and minor allele frequency⩾10% (23). Genotypes were determined by pre-designed Taqman Allele discrimination assays (rs3918188: C__29193459-10; rs3918226: primer forward: 5′-IndexTermAGCGTGCGTCACTGAATGA-3′, reverse 5′-IndexTermACACCCCCATGACTCAAGTG-3′ and probes 5′-IndexTermCAGGAAGCT[G/A]CCTTC-3′; rs743506: primer forward: 5′-IndexTermGCTGTCCCCTCCCTCTG-3′, reverse 5′-IndexTermGTGCCAGCTCCAGAGCAA-3′ and probes 5′-IndexTermCTCTCC[A/G]AGGCTCC-3′; rs7830: primer forward: 5′-IndexTermCCTAGATTGTGTGACTCCCTTCAG-3′, reverse 5′-IndexTermTGCATGACATTGAGAGCAAAGGT-3′ and probes 5′-IndexTermCTTTAGTC[A/C]CCAGCCTC-3′). PCR reactions and fluorescence measurements were performed in a Step One Plus real-time PCR equipment (Life Technologies, Carlsbad, CA, USA), and data were analyzed using the manufacturer’s software.
Data are reported as mean±s.d. The clinical characteristics of groups were compared by one-way ANOVA followed by Tukey’s post-hoc test. Categorical variables were compared between groups by χ2 tests. P<0.05 was considered statistically significant.
Haplotypes were inferred using the Bayesian statistics-based program PHASE version 2.1 (http://www.stat.washington.edu/stephens/software.html) to estimate the haplotype frequencies. The possible haplotypes including the alleles of four tagSNPs in the NOS3 gene studied (rs3918188, rs3918226, rs743506 and rs7830) were H1 (CCAC), H2 (CCAA), H3 (CCGC), H4 (CTAC), H5 (CTGC), H6 (ACAC), H7 (ACAA), H8 (ACGC) and H9 (ATAC).
Linear regression analysis and non-linear fitting routines were performed to assess univariate relations between variables (software JMP 5.0.1a; SAS Institute, Cary, NC, USA). In addition, a bivariate analysis was also used to assess for the potential confounding influence of each covariate on the relation between tagSNPs and haplotypes, and study group and nitrite levels. The variables of clinical importance, as identified by the bivariate approach, were then including in the final multiple linear regression models. In multiple linear regression analysis nitrite levels were considered as a dependent variable. Age, gender, group, acid uric levels, adiponectin levels and NOS3 genotypes or haplotypes were considered as independent variables. In multivariate logistic regression models the variable group was considered as a dependent variable. Age, gender, uric acid and adiponectin levels were considered as independent variables.
The clinical features of 355 children and adolescents evaluated are shown in Table 1. The obese group was slightly younger than the other groups (P<0.05). Compared to controls, the obese and hypertensive obese groups had higher body mass index, waist circumference, systolic blood pressure, diastolic blood pressure, low-density lipoprotein and triglycerides (all P<0.05). In contrast, high-density lipoprotein and adiponectin levels were lower in the obese and hypertensive obese groups compared with the control group (P<0.05). Interestingly, the nitrite level was lower only in the obese group (P<0.05).
The distribution of genotypes for the four polymorphisms studied here showed no deviation from Hardy–Weinberg equilibrium (all P>0.05). The frequencies of NOS3 genotypes and haplotypes in children and adolescents are shown in Tables 2 and 3, respectively.
The results were corrected for age, gender, uric acid and by adiponectin levels. When the allele and genotype frequencies for the control, obese and hypertensive obese groups were compared, no significant differences were found (P>0.05, Table 2). The haplotype analyses show that the haplotype 5-CTGC was associated with obesity (P=0.009, OR=37.68, CI 3.04–990) but not with hypertension (P>0.05; Table 3).
To assess the effects of genotypes and haplotypes of the NOS3 tagSNPs on nitrite plasma level, we performed a multiple linear regression analysis adjusted for gender, age, study group and plasma levels of uric acid and adiponectin (Tables 4 and 5, respectively). After adjustment for these selected variables, we found that nitrite concentrations were affected by the rs3918226 polymorphism (P<0.05, Table 4).
After adjustment for these selected variables, the haplotypes analyses showed that nitrite concentrations were not affected by NOS3 haplotypes (P>0.05, Table 5).
This is the first study to investigate whether NOS3 tagSNPs rs3918188, rs3918226, rs743506, and rs7830 and haplotypes affect blood pressure and nitrite levels in children and adolescents with obesity and with obesity plus hypertension, and whether these polymorphisms are associated with these clinical groups.
Although the main finding of the present study was that the CT genotype for rs3918226 is associated with higher nitrite level in children and adolescents after correction for selected variables, the other tagSNPs and the NOS3 haplotypes formed by all these tagSNPs are not associated with hypertension in children and adolescents after correction for multiple comparisons.
Nitrite levels were lower in obese groups, and although the latter is not a novelty, it is controversial in literature.4, 30, 31, 32 Our results are consistent with those of Gruber et al.,4 who showed reduced nitrite/nitrate levels (NOx) in juvenile obesity, and with Siervo and Bluck,32 who showed reduced NO synthesis in obesity and in metabolic syndrome.
Nitrite is considered a major storage pool for NO in the circulation, and it was suggested that two-thirds of basal plasma nitrite is derived from NOS3 activity.33 Pathological conditions such as hypertension, obesity and metabolic syndrome share a common feature, the endothelial dysfunction, which is characterized by reduced NO bioavailability.34, 35, 36 In obesity, important factors that could explain the reduced levels of nitrite are insulin resistance and increased oxidative stress.37 Importantly, eNOS uncoupling may severely impair NO production and also increase oxidative stress, which further reduces NO bioavailability.38, 39
In this study, both the obese group and the obese hypertensive group had significantly higher Homeostatic Model Assessment of Insulin Resistance (HOMA-IR) scores when compared to the control group, and this finding may indicate impaired insulin signaling in these groups. However, the obese hypertensive group showed nitrite levels that were not significantly lower than those found in the control group. It is generally accepted that obesity is associated with a compensatory increase in NO synthesis at the initial stage of obesity, which is followed by a decline,9 but further studies are needed to explore how cardiovascular risk factors such as hypertension affect NO bioavailability in children and adolescents.
Adiponectin has anti-inflammatory and antiatherogenic properties and is expressed by the adipose tissue in inverse proportion with adiposity;5, 7 previous data have shown that NO bioavailability is positively affected by adiponectin concentrations in obese children and adolescents.9 Interestingly, although adiponectin levels are lower in the hypertensive obese group compared with the obese group, nitrite concentrations are slightly higher in hypertensive obese compared with obese children. However, this suggestion does not take into consideration other factors that may affect both measurements, and is a not a valid conclusion derived from our multivariate logistic regression. The present results indicate that adiponectin levels are indeed reduced in obesity, and this is more important when obesity is associated with hypertension. These results are in agreement with other studies showing that adiponectin is inversely related with hypertension and obesity in children and adolescents.5, 6, 9
Adiponectin sensitizes the body to insulin and has an important role in the metabolism of glucose and lipids.5, 40, 41 It also stimulates NO formation by vascular endothelial cells after activating the phosphatidylinositide-3-kinase pathway, thus mimicking the effects of insulin on the synthesis of NO.40 For this reason, adiponectin was included in our linear regression model as an independent factor, so that we could correct for this important biochemical factor.
The effects of NOS3 polymorphisms on the susceptibility to hypertension and cardiovascular diseases have been widely studied.10, 11 Clinically relevant polymorphisms and their combinations within haplotypes affect both the susceptibility to hypertension and nitrite levels.13, 14, 15, 16, 17 However, tagSNPs provide further NOS3 genetic variation, which has been previously investigated.42, 43 In fact, haplotypes including the tagSNPs rs3918226 and rs743506 were associated with other conditions involving cardiovascular alterations.42, 43 Taken together, these findings suggest pathophysiological implications for these NOS3 tagSNPs.
We have previously shown that the NOS3 haplotype ‘C G A’ formed by tagSNPs rs3918188, rs743506 and rs7830 were associated with low circulating whole-blood nitrite levels in adults (23), but this polymorphism did not affect plasma nitrite levels in adults (20). Our findings reported here showed a possible effect of the tagSNP rs3918266 on nitrite levels, and suggest that this polymorphism may have an impact on cardiovascular health. Although we have not found an association with hypertension in childhood obesity in the present study, the rs3918226 in the NOS3 promoter is a susceptible locus to hypertension,21, 22 and it is associated with lesser eNOS transcription and higher risk of hypertension.25
It is noteworthy that the particular profile of the included patients allows the exclusion of all pharmacological interactions, as all patients were not taking any chronic anti-hypertensive treatment, and therefore our findings are not affected by drug effects.
Some limitations of the present study should be taken into account. It is possible that the low number of patients included here limited our conclusions in this matter. Another limitation is that subjects enrolled in our study were not on a particular diet before blood sampling, although they were on a 12-h fasting. Although it is possible that diet affects the circulating levels of NO metabolites, it has been shown that ∼70% of the circulating nitrite levels depend on constitutive nitric oxide synthase activity.33 Further studies are needed to better clarify the effects of this polymorphism on cardiovascular risk.
In conclusion, our findings suggest that NOS3 tagSNP rs3918226 affects nitrite levels and NO production in children and adolescents. However, the NOS3 tagSNPs rs3918188, rs3918226, rs743506, and rs7830 and haplotypes are not associated with hypertension in children and adolescents.
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This study was funded by Fundação de Amparo a Pesquisa do Estado de São Paulo (FAPESP-Brazil) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq-Brazil).
The authors declare no conflict of interest.
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de Miranda, J., Lacchini, R., Belo, V. et al. The effects of endothelial nitric oxide synthase tagSNPs on nitrite levels and risk of hypertension and obesity in children and adolescents. J Hum Hypertens 29, 109–114 (2015). https://doi.org/10.1038/jhh.2014.48
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