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Essential hypertension results from the interaction of several genetic and environmental factors. Identification of genetic factors that modulate blood pressure (BP) response to interventions can lead to improved strategies for prevention and control. The purpose of this study was to identify genes that modulate BP response to dietary interventions.
We used data and samples collected in two randomized feeding studies to determine the extent to which genetic architecture is associated with the effect on BP of sodium intake and the Dietary approaches to Stop Hypertension (DASH) dietary pattern. Participants in both trials were adults with above-optimal BP or unmedicated stage 1 hypertension. Genomic DNa was typed for several candidate genes.
The effect of sodium intake on BP differed by genotype at the angiotensinogen, β2-adrenergic receptor, and kallikrein loci. The effect of DASH dietary pattern on BP differed by genotype at the β2-adrenergic receptor locus.
These findings have implications for understanding the mechanism(s) through which diet affects BP, the heterogeneity of these effects, and the extent to which dietary interventions can modulate genetic predisposition.
Essential hypertension results from the interaction of several genetic and environmental factors. Genes that regulate the renin–angiotensin–aldosterone system, adrenergic activity, and renal sodium excretion have been implicated in normal and abnormal blood pressure (BP) regulation.1–7 In addition, genes regulating these systems have been implicated in BP response to dietary intervention.8–10 For example, we have previously reported that salt sensitivity may be linked to genotype at the β2-adrenergic receptor,9 and that BP response to the Dietary Approaches to Stop Hypertension (DASH) dietary pattern varies based on angiotensinogen genotype.10
Thus, there is evidence that genetic predisposition modulates the BP response to diet, and therefore modifiable behavior (i.e., diet) can mitigate or enhance the effects of genetic predisposition. Increasing our understanding of this relationship can lead to physiologically targeted treatment and increased understanding of pathophysiology. We used data and samples collected in two randomized feeding studies11,12 to identify genes that modulate BP response to dietary intervention.
We report data from the DASH and DASH-Sodium trials. The design and results of both studies,13,14 as well as a description of the dietary patterns used in these studies,15 have been presented elsewhere and are briefly described below. Both protocols as well as this analysis were approved by the institutional review board at each study site. All participants included in this analysis provided written informed consent for genetic analyses related to BP.
DASH was a multicenter, randomized–controlled feeding study testing the effects of dietary patterns, independent of sodium intake or weight loss, on BP.11 Following a 3-week run-in on the reference (Typical American) diet, nondiabetic adults with high-normal BP (JNC-6 criteria16) or unmedicated stage 1 hypertension were randomly assigned to eat one of three dietary patterns for 8 weeks. Sodium content was held constant at ~150 mmol/day for a 2,100 kcal energy intake (corresponding to typical US consumption), and weight was kept stable by adjusting energy intake as needed. All meals, snacks, and beverages were provided to study participants, and at least one meal per day was consumed at the study site on weekdays. The primary outcome measure was the average of multiple BP measurements at baseline and at the end of the 8-week feeding intervention.
Two of the three dietary patterns evaluated in DASH (a Typical American diet and the DASH diet) are the focus of this article. The Typical American diet was based on typical intake in the most recent National Health and Nutrition Evaluation Survey (NHANES).17 By contrast, the DASH diet emphasized fruits, vegetables, and low-fat dairy foods (Table 1). Nutrient composition was confirmed by chemical analysis.
The DASH-Sodium Trial was a multi-center, randomized feeding trial that compared the effects on BP of three levels of sodium intake in two dietary patterns. These dietary patterns corresponded to the Typical American and DASH diets from the DASH Trial.12 The three sodium levels were defined as “higher” (target of 150 mmol/day with 2,100 kcal energy intake), “intermediate” (100 mmol/day), and “lower” (50 mmol/day). The actual sodium content of the diet was proportionate to the total energy intake of the individual participant, and average 24-h excretion of sodium for the study population as a whole was 142, 107, and 65 mmol/day for the higher, intermediate, and lower sodium levels, respectively.
Adults with entry criteria similar to DASH initially consumed the Typical American diet at the higher sodium level for a 2-week run-in period, after which they were randomly assigned to either continue this diet or to start consuming the DASH diet. Within each of these two parallel arms, participants consumed the three sodium levels for 30 days each, in a randomly determined order, according to a traditional crossover design layout. Weight was kept stable and the primary outcome measure was BP measured at baseline and at the end of each 30-day intervention feeding period.
In both DASH and DASH-Sodium, dietary adherence was assessed via daily diaries, observation of on-site meal consumption, and measurement of 24-h urinary excretion of micronutrients.
Of the 459 DASH and 412 DASH-Sodium participants who were randomized, we excluded the 154 DASH participants who were not assigned to either the Typical American or DASH dietary patterns and an additional 27 individuals (from either trial) who did not self-identify as either white/Caucasian or black/African American. This resulted in a sample of 690 individuals who were potentially eligible for this analysis. We further excluded 59 individuals (8.6%) without an adequate buffy coat sample for extraction of DNA, leaving a total 631 individuals. Of these 631 participants, 17 had participated in both DASH and DASH-Sodium. We chose to exclude them from the DASH participants and retain them in the DASH-Sodium participants, leaving a total of 614 individuals (218 from DASH and 396 from DASH-Sodium) for the current analysis
Each BP measurement consisted of the average of two seated measurements obtained in the right arm using a random-zero sphygmomanometer. All BP measurers were trained and certified using a well-established protocol14,18 and were blinded to treatment assignment. Baseline BP was defined as the average of at least four daily measurements obtained during screening and run-in, and as the average of at least four daily measurements obtained during the final week of each intervention feeding period.13,18 Fewer than 3% of BP outcomes (35/1,695) were missing and replaced by either baseline BP or earlier intervention BPs.
Genomic DNA was extracted from buffy coat samples using standard procedures.
We selected candidate genes that were known to regulate BP, sodium homeostasis, or vascular tone, including genes encoding angiotensinogen, angiotensin receptor, renin, β2-adrenergic receptor, kallikrein, adducin, and aldosterone synthase. From these genes, we identified single-nucleotide polymorphisms (SNPs) or structural variation either from existing data in the literature or from public SNP data bases. All variants in Table 2 were genotyped, but only eight were included in the association analysis (Table 2); the other SNPs were excluded because of insufficient sample size (n < 5 for at least one genotype) or excessive missing data (>30% individuals missing genotype).
All genotyping was performed at the Harvard Partners Genotyping Facility (Cambridge, MA) by laboratory technicians who were blinded to the participants’ identity, demographic characteristics, diet assignment, and study outcomes. SNP genotyping was performed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry using the Sequenom mass spectrometry system (Sequenom, San Diego, CA). Deletion and insertion were determined by using Taqman SNP allelic discrimination on the ABI 7700/7900 (Applied Biosystems, Foster City, CA).
Using the exact tests in Genetic Data Analysis,19 we initially tested for violations of Hardy–Weinberg equilibrium and deviations from linkage disequilibrium. We also examined gene frequencies by self-reported race (black vs. white).
For analyses of the primary outcomes (i.e., effects on BP of sodium intake and the DASH dietary pattern), we fit linear models that included baseline and end-of-intervention BP measurements as continuous variables, with adjustment for clinical site, study cohort, and, for DASH-Sodium data, for carryover effects. In order to be able to incorporate data from the DASH and DASH-Sodium trials into a single analysis, we treated the DASH trial as an incomplete replication of the DASH-Sodium trial in which participants consumed the higher sodium level only and data for the remaining sodium levels were missing by design. DASH participants, therefore, contribute to the analysis of DASH diet effects, but not sodium effects, whereas DASH-Sodium participants contribute to both analyses. Analyses were performed using the PROC-MIXED procedure in SAS, version 8.2, and an exchangeable covariance matrix was assumed for the repeated measurements for each person.
The basic model fully saturated the eight cells of the design matrix (two diet arms by three sodium levels plus baseline), with different parameterizations used to test specific hypotheses of interest. In order to assess effects within subgroups defined by various genotypes, we used gene–environment interactions to fit a fully saturated design matrix within each genetic subgroup, but assumed constant site, cohort, and carryover effects, and a constant covariance matrix, across subgroups. Evidence of gene–environment interactions (i.e., effect modification) was assessed by examining the significance of gene–diet and gene–sodium interactions in these models using a linear term for genotype. We included covariate terms for race, gender, and age in all models to adjust for their potentially confounding effects, and in addition performed additional analyses stratified by race.
Due to the nature of the design, sodium effects are effectively evaluated as within person contrasts within each diet arm, whereas the DASH effect is inherently a between-subject contrast in which separate effects were estimated for each sodium level while adjusting for baseline differences in BP between participants in the two diet arms. Because previous analyses showed that the effects of the DASH diet and reduced sodium intake were subadditive,12 for this article we chose to focus on the effect of the DASH vs. Typical American diets in the context of the higher sodium level and the effect of the higher vs. lower sodium levels in the context of the Typical American dietary pattern. This was done to maximize our power to detect effects. Consequently, our estimates of the DASH diet effect use data from all 614 subjects, whereas our estimates of the higher vs. lower sodium effect are based on the 204 DASH-Sodium participants who consumed the Typical American diet.
We considered our primary analysis to be exploratory and report results without adjustment for multiple comparisons. However, in a secondary analysis, we used the methods of Benjamini and Hochberg in order to adjust for multiple comparisons in our tests of whether the effects on BP of either the DASH diet or sodium intake vary by genotype.20 Specifically, for each of these two broad questions (combining across SNPs and BP outcomes), we separately adjusted our levels of statistical significance to assure that the false discovery rate (i.e., the probability that a test declared to be significant really is not significant) to be 5%. We applied this adjustment to all trend tests to determine whether BP effects vary by genotype, with separate adjustments for analyses in the total population, in whites, and in blacks.
The sample size was fixed at the number of individuals from the parent studies (614 for assessment of DASH effects and 204 for assessment of sodium effects). Power for testing both main effects on BP and differences across genotypes was affected by genotype frequency. For testing main effects of the DASH diet or reduced sodium for participants of a given genotype, power exceeded 80% for detecting a change of 2 mm Hg if the genotypic frequency was at least 60%, and power exceeded 90% for detecting a change of 3 mm Hg if the genotypic frequency was at least 35%. For detecting differences within treatment group across genotypes, power exceeded 80% to detect a difference of 5 mm Hg if the more common homozygous state occurred in 49% of individuals and the less common homozygous genotype in 13%, as is the case, for example, for the angiotensinogen marker AGT_M235T. Larger differences in genotype proportions were associated with greater power to detect the same difference in BP response.
Of the 614 individuals included in these analyses, 59% were black, 55% were women, mean age was 46.8 years, and 36% were hypertensive. Mean BP at entry was 133.4/85.3 mm Hg (Table 3). These characteristics were similar for the DASH and Typical American diet groups.
Dietary adherence was excellent.11,12 Based on mean 24-h urinary excretion data, sodium intake was comparable for the two dietary patterns in DASH, and in DASH-Sodium showed the anticipated separation across the lower, intermediate, and higher levels. Urinary potassium excretion reflected the increased fruit and vegetable content of the DASH diet relative to the Typical American diet, and, in DASH-Sodium, showed good stability across sodium levels. Body weight remained within 2% of baseline values.
As previously reported, reducing sodium intake from the higher to the lower level (in the context of the Typical American diet) was associated with a 6.7/3.5 mm Hg reduction in BP (P < 0.001).12 The DASH diet (in the context of higher sodium intake) reduced BP by 5.5/3.0 mm Hg in the DASH study11 and by 5.9/2.9 mm Hg in the DASH-Sodium study (P < 0.001 in both studies).12
We found no violation of Hardy–Weinberg equilibrium assumptions. Genotype frequencies for each marker, overall and by race, are provided in Table 4. Certain markers showed striking race differences in genotype frequencies. Consequently, tests for association were performed both overall (adjusted for race) and stratified by race.
In the context of the Typical American diet, the effect of the lower sodium intake (relative to the higher sodium intake) on systolic BP (SBP) was evident across all genotypes studied. Estimated SBP reductions ranged from ~3 to 9 mm Hg, and in almost all genotypes the change was statistically significant.
The effect of sodium intake on SBP differed by genotype (P value for trend < 0.05) for angiotensinogen, β2-adrenergic receptor, and kallikrein SNPs. Figure 1a displays the relationship between the M235T marker of the angiotensinogen gene and SBP change. SBP was reduced by 8.2 mm Hg (95% confidence interval (CI) –9.7 to –6.6) in individuals with the GG genotype compared to 4.3 mm Hg (95% CI –6.9 to –1.6) in those with the AA genotype, with a significant trend across GG, GA, and AA genotypes (P = 0.01). Figure 1a further suggests that this association differed by race, but the trend did not reach statistical significance in either racial group. However, the data suggest that the lack of significance at least in whites may have been related to insufficient power. We also tested the angiotensinogen marker AGT_C_20A. Because the GG genotype was only present in 3% of individuals (see Table 4), a trend test across genotypes was not meaningful for this marker. However, the BP response to reduced sodium intake was greater in individuals with the GT genotype (mean –8.3 mm Hg, 95% CI –10.5 to –6.2) compared to the TT genotype (mean –6.63 mm Hg, 95% CI –7.9 to –5.4).
Figure 1b shows the relationship between genotype at the C79G marker of the β2-adrenergic receptor. SBP was reduced by 2.8 mm Hg (95% CI –6.2 to 0.5) in individuals with the GG genotype and by 8.7 mm Hg (95% CI –10.2 to –7.3) in those homozygous for the C allele in the overall sample. The effect of increasing numbers of C alleles was highly significant (P value for trend = 0.00002). A similar pattern was seen with the A46G polymorphism of the β2-drenergic receptor (ADR) gene, with a P value for trend of 0.006 in the overall sample (Figure 1c). Trends appeared similar in black and white subgroups, but only achieved statistical significance for the C79G marker in blacks.
Figure 1d demonstrates the significant association between genotype of the kallikrein marker and sodium effect, and this relationship was also significant in blacks.
There was no significant relationship between sodium effect on SBP and genotype at the markers for angiotensin-II receptor (AGTR_A166C), aldosterone synthase (CYP11B2_C_344T), and α-adducin (ADD1_G460W).
Similar to the sodium effect, the DASH diet (compared to the Typical American diet) was associated with a statistically significant decrease in SBP in almost all genotypes, with estimated reductions in SBP ranging from 3.9 to 9.8 mm Hg. However, this effect varied significantly only for the β2-drenergic receptor gene (trend P value = 0.05 for the C79G gene marker). Figure 2a suggests that this relationship was mainly due to a trend across genotypes in blacks. Figure 2b suggests a similar relationship for the ADBR2_A46G marker, but the trend was not statistically significant overall or in either race group.
Although the prespecified primary outcome was SBP, we also performed trend tests for associations between genotype and both mean arterial pressure and diastolic BP. The association between sodium effect and genotype was consistently significant for SBP, mean arterial pressure and diastolic BP for AGT_M235T, ADRB_A46G and ADRB_C79G genotypes, but was significant only for SBP for KLK genotype. The association between DASH effect on SBP and ADRB genotype was not seen for mean arterial pressure or diastolic BP, but there was a significant association between AGT_M235T and DASH effect on mean arterial pressure and diastolic BP that was not significant for SBP.
Secondary analysis, employing the false discovery rate adjustment for multiple comparisons,20 confirmed an association of genotype at the angiotensinogen, β2-drenergic receptor, and kallikrein loci with SBP response to reduced sodium intake. Other associations were not statistically significant after this adjustment.
In well-characterized adults with pre- or Stage 1 hypertension, studied during controlled dietary intake, we found that genotype at two angiotensinogen SNPs, two β2-adrenergic receptor SNPs, and the Q121E SNP of the kallikrein gene were associated with the extent to which BP fell with reduced sodium intake. We found that the ADBR2_C79G SNP was significantly associated with the extent to which BP fell with DASH dietary pattern, with a qualitatively similar association for the ADBR2_A46G SNP. The difference in effect between favorable and unfavorable genotypes was as much as a two- to threefold difference in the extent of BP-lowering. In both dietary interventions, there was a trend for racial differences in these relationships. Although many of these associations were not statistically significant in a secondary analysis with a fairly conservative adjusted for multiple comparisons, such an adjustment may have led to insufficient power. Given the exploratory nature of this study, statistically significant associations in the unadjusted analysis warrant further consideration.
The particular genes implicated in this study are important in normal and abnormal regulation of BP, so it is interesting that they may also be involved in BP response to environmental (i.e., dietary) factors. For example, several previous studies have demonstrated associations between BP phenotypes and polymorphisms of the angiotensinogen gene. This gene has been associated with elevated levels of circulating angiotensinogen1–3 with the presence of essential hypertension,2–5 with family history of hypertension,8 and possibly with hypertension in pregnancy.6 Thus, genotype at the angiotensinogen locus appears to confer susceptibility to hypertension. In the Trials of Hypertension Prevention, AGT genotype was associated with the impact of reduced sodium intake on the incidence of hypertension in whites.1 Our data extend these observations to identify two polymorphisms that are associated with BP response to reduced sodium intake.
The role of AGT genotype in modulating response to DASH is less clear. In this study, there is no significant association between genotype at two AGT SNPs and BP response to DASH. However, in a previous analysis of DASH participants only, the BP response to the DASH diet trended greater in the AA genotype of the G-6A AGT SNP and least in the GG genotype.10 The lack of consistency between the two analyses raise the possibility that the association may be spurious or complex, but other data suggest that DASH lowers BP through effects on the renin–angiotensin–aldosterone system,21 raising the likelihood that genetic variation influences the effect of DASH on renin– angiotensin–aldosterone system, perhaps at other loci. Future research is needed to clarify this relationship.
The β2-adrenergic receptor (β2-ADR) gene is also highly implicated in normal and abnormal BP regulation. We and others have previously reported a significant association between β2-ADR genotype and the presence of hypertension in unrelated individuals, families, and twins.7,22–24 Further, in 109 African-American sib-pairs we found preliminary evidence of linkage of β2-ADR gene and salt sensitivity.9 In this study, the observed association between two β2-ADR SNPs and BP response to reduced sodium intake strongly suggests that this locus modulates dietary sodium sensitivity. These findings are consistent with those of Pojoga et al. who reported greater salt sensitivity associated with the A allele of A46G and the C allele of C79G.25 In addition, this study confirms the previously reported association between β2-ADR genotype and response to DASH dietary pattern.10
The role of the kallikrein gene in BP regulation is less well-established. Renal kallikrein acts on the substrate kininogen to form lysyl-bradykinin (kallidin), a potent vasodilator that also promotes sodium excretion. Kallikrein excretion is decreased with hypertension26–29 and increases with higher potassium intake.27,30 Thus, the relationship between BP and dietary sodium/potassium ratio may be mediated through effects on the kallikrein system. Our data suggest that the BP response to sodium intake, but not (potassium-rich) DASH is associated with genotype at the kallikrein locus, but the significance of this relationship is unclear.
Although we did not explore the relationship between genotype and biochemical response to diet in this analysis, it is known that both sodium reduction and DASH dietary pattern increase plasma renin activity and serum aldosterone,31 and that genotype can influence the magnitude of this effect.25 Further research is needed to determine the mechanism(s) through which genotype (and presumably gene product) influences BP response to diet.
It is common to consider the extent to which genotype modulates BP response to a particular intervention. This is a useful approach for understanding mechanisms and identifying targets of drug therapy. However, it is also useful to consider the extent to which dietary intervention can mitigate or enhance a genetic predisposition, with the goal of targeting an intervention to those genetically predisposed to respond to it. For example, current guidelines recommend that all individuals with prehypertension or hypertension eat the DASH dietary pattern, reduce sodium intake, and lose weight if overweight or obese.16 Changing behavior is difficult, and it may be that changing several behaviors is more difficult than focusing on a single behavior change. Identifying which interventions are likely to have the greatest effect on BP for a given individual may lead to a specific lifestyle prescription that is more likely to be successfully adopted. In this context, this study lays groundwork for targeting behavioral interventions to genotype, a strategy that may become more feasible and attractive as genetic screening becomes more commonplace.
Our study is consistent with data from other clinical conditions. For example, a genotype of the transcription factor 7-like 2 gene confers excess risk of type 2 diabetes mellitus, but the difference in risk between deleterious genotype and favorable genotype is eliminated by a lifestyle intervention.32 Similarly, a genetic variant of the 5-lipoxygenase gene promoter is associated with atherosclerosis, as indicated by carotid artery intimal–medial thickness, but the expression of excess risk is modulated by dietary intake of both arachidonic and linoleic acid.33 Thus, investigation of gene–environment interactions, when the environment refers to dietary intake, may prove useful in developing individualized, targeted behavioral interventions.
The strengths of this study derive mainly from the study design. Previous studies of gene–diet association generally depend on inferences about dietary intake from food records, 24-h excretion data or response to dietary supplement or infusion of saline. Most data are restricted to the effect of sodium rather than other nutrients or overall dietary pattern, and the populations studied are small and not racially diverse.34 In contrast, our sample size was large, the population diverse and well-characterized, BP response was measured using precise, standardized, unbiased methods, and the interventions were provided in the context of controlled feeding studies. Nutrient intake was controlled and quantified, and confounders were mitigated (e.g., body weight remained stable). A potential limitation of this study is that we focused on several candidate genes rather than screening for all possible polymorphisms associated with BP response to diet. However, although genome-wide association studies can detect genes not previously suspected of having a role in the disease under study, they are highly prone to false positive results.35 Our candidate gene approach increases confidence in the validity of the results. In addition, as so much is known about systems involved in BP regulation, a focus on genes regulating these systems could lead more efficiently to deeper understanding. Nonetheless, to better understand the universe of genes associated with BP response to DASH and sodium reduction, future research should include genome-wide association studies. In addition, our results should be verified in independent populations. Although we focus here on BP effects, the possibility that the same genetic polymorphisms might also affect the relationship between BP and clinical outcomes should also be investigated.
In summary, our study demonstrates a relationship between angiotensinogen, β2-adrenergic receptor and kallikrein geno-type and the effect of sodium intake and the DASH dietary pattern on BP. These findings have implications for understanding the mechanism(s) through which diet affects BP, the heterogeneity of these effects, and the extent to which dietary interventions can modulate genetic predisposition.
This study was supported by National Institutes of Health grants R01HL57114, R01 HL77234, and K24 DK63214.
Disclosure: The authors declared no conflict of interest.