Abstract

Background:

The W allele of the G460W polymorphism in the adducin-1 gene has been occasionally associated with increased blood pressure (BP). The aim of this study was to test whether the G460W variant is associated with BP levels and BP progression rate and whether G460W associations with BP are affected by sex, body mass index (BMI), or age.

Methods:

The G460W polymorphism was genotyped in the population-based Malmö Diet and Cancer–cardiovascular arm (MDC-CVA; n = 6103), of whom 53% had also been examined 11 ± 4.4 years earlier in the Malmö Preventive Project (MPP).

Results:

Among subjects without antihypertensive treatment (AHT) in the MDC-CVA (n = 5009), there was no difference between carriers (38%) and noncarriers (62%) of the W allele in systolic BP (139.2 ± 18.2 v 139.2 ± 18.5 mm Hg; P = .99) or diastolic BP (85.9 ± 9.1 v 86.1 ± 9.2 mm Hg; P = .49). In subjects free from AHT in the MPP and MDC (n = 2637) there was no difference between carriers (38%) and noncarriers (62%) in progression of systolic BP (2.0 ± 2.5 v 2.0 ± 2.7 mm Hg/year; P = .45) or diastolic BP (0.59 ± 1.6 v 0.56 ± 1.5 mm Hg/year; P = .66) from MPP to MDC. At MDC-CVA BP was influenced by interaction between the G460W and BMI (P = .02 for systolic BP and P = .002 for diastolic BP) and by interaction between G460W and sex (P = .03 for systolic BP and P = .02 for diastolic BP), a result further confirmed by stratified analysis showing that female carriers of the W allele belonging to the upper tertile of BMI had increased systolic BP (146.1 ± 18.6 v 141.2 ± 18.6 mm Hg; P < .001), diastolic BP (88.7 ± 8.7 v 86.1 ± 8.7 mm Hg; P < .001), and prevalence of hypertension (72.5% v 61.8 %; P = .001).

Conclusions:

Our data suggest that the G460W polymorphism influences BP when BMI and sex are taken into account. Am J Hypertens 2007;20: 981–989 © 2007 American Journal of Hypertension, Ltd.

High blood pressure (BP) even within the normal range is responsible for millions of deaths and disabilities throughout the world.1 Blood pressure is a complex trait, and genetic and environmental factors have a complex interrelation to determine individual BP values.2

Adducin is a heterodymeric cytoskeletal protein highly conserved through phylogenesis. Adducin promotes the organization of the spectrin–actin lattice by favoring its binding and controlling the rate of actin polymerization as an end-capping actin protein.3,4

Adducin is formed by three subunits encoded by three genes (ADD-1, ADD-2, and ADD-3). These proteins and the genes encoding them have received attention, as adducin polymorphisms have been shown to affect BP both in rats and humans through an enhanced constitutive sodium reabsorption.5–9 In humans two polymorphisms of the ADD-1 gene lead to amino acid substitution: G460W and S586C6. Rat ADD-1 tyrosine at amino acid position 316 and human tryptophane at amino acid position 460 bind with higher affinity and activate the Na-K-ATPase more strongly than the respective normal protein.10

In addition, expression of the hypertensive rat 316Y or human 460W variant of adducin into normal renal epithelial cells recreates the hypertensive phenotype with higher Na-K-ATPase activity, μ2-subunit hyperphosphorylation, and impaired Na-K-ATPase endocytosis.11

A recent review reported that several linkage studies, exploring DNA markers very close to the ADD-1 locus, and 18 of 20 association studies taking into account variables reflecting body sodium or the renin-angiotensin system, have shown positive results regarding the effect of the G460W variant of ADD-1 on BP and hypertension. Furthermore, 12 of 16 studies found that the ADD-1 polymorphism is associated with stroke, coronary artery disease, or renal and vascular dysfunction.12

Nevertheless, there have been several studies in different populations, where no effect of the 460W allele on either BP or hypertension has been found,12–15 and in one study, the G460 allele was associated with hypertension.16

Blood pressure is a complex phenotype, and the effect of single gene variants is likely to be quite small. It is likely that many single gene variants can be detected only when other factors, genetic or environmental, allow them to be expressed.7,8,12,17,18

The aim of the present study was to test the possible association of the G460W polymorphism with cross-sectional BP levels or BP change over time and whether possible G460W associations with BP are affected by sex, body mass index (BMI), or age.

Methods

The population studied was taken from the Malmö Diet and Cancer (MDC) study cohort.19 Blood pressure, along with other cardiovascular risk factors, was measured in a random sample of the MDC, referred to as the MDC–cardiovascular arm (MDC-CVA) (n = 6103). Successfully extracted genomic DNA in MDC-CVA, which was required for inclusion in the present study, was available on 6055 subjects, but 50 people were excluded from the analysis because of unsuccessful genotyping (discussed later). The study of BP as a continuous variable in MDC-CVA was restricted to subjects free from antihypertensive medication (n = 5009), whereas the patients on antihypertensive medication (n = 996) were included when the dichotomized phenotype of “hypertension” and “normotension” was studied. Of the untreated subjects in MDC-CVA we were able to study the BP change over time in 2637 subjects who previously had been investigated in another cohort study from Malmö—the Malmö Preventive Project (MPP)—and were free from antihypertensive medication also at MPP, which was performed approximately 10 years before MDC-CVA.20

Subjects followed from MPP to MDC-CVA, and who were free from antihypertensive treatment at both examinations (n = 2637), had a mean “follow-up time” of 11.2 ± 4.4 years (range, 1.0 to 19.3 years).

All study participants had given written informed consent. The Ethics Committee of the Medical Faculty of Lund University approved the study. The procedures were in accordance with the institutional guidelines.

Clinical characteristics of all subjects are shown in Table 1.

Table 1

Anthropometric and metabolic features of the subjects investigated in the Malmö Diet and Cancer–cardiovascular arm (MDC-CVA) and the Malmö Preventive Project (MPP)

Variables MPP to MDC-CVA without AHT (N = 2637) MDC-CVA without AHT (N = 5009) MDC-CVA with AHT (N = 996) 
Sex, male (%) 54.9 41.8 44.7 
Age (y) 47.0 ± 5.7 57.1 ± 5.9 59.3 ± 5.6 
BMI (kg/m224.1 ± 3 25.5 ± 3.8 27.5 ± 4.5 
SBP (mm Hg) 122.4 ± 13.2 139.2 ± 18.3 152.3 ± 19.4 
DBP (mm Hg) 81.9 ± 8.4 86.0 ± 9.1 92.0 ± 9.6 
ΔSBP (mm Hg/y) 2.0 ± 2.7   
ΔDBP (mm Hg/y) 0.58 ± 1.54   
ΔSBP (%/y) 1.7 ± 2.3   
ΔDBP (%/y) 0.77 ± 2.1   
Variables MPP to MDC-CVA without AHT (N = 2637) MDC-CVA without AHT (N = 5009) MDC-CVA with AHT (N = 996) 
Sex, male (%) 54.9 41.8 44.7 
Age (y) 47.0 ± 5.7 57.1 ± 5.9 59.3 ± 5.6 
BMI (kg/m224.1 ± 3 25.5 ± 3.8 27.5 ± 4.5 
SBP (mm Hg) 122.4 ± 13.2 139.2 ± 18.3 152.3 ± 19.4 
DBP (mm Hg) 81.9 ± 8.4 86.0 ± 9.1 92.0 ± 9.6 
ΔSBP (mm Hg/y) 2.0 ± 2.7   
ΔDBP (mm Hg/y) 0.58 ± 1.54   
ΔSBP (%/y) 1.7 ± 2.3   
ΔDBP (%/y) 0.77 ± 2.1   

AHT = antihypertensive treatment; BMI = body mass index; DBP = diastolic blood pressure; SBP = systolic blood pressure.

Data are presented either as mean ± standard deviation or percentage.

Phenotyping

Blood pressure was measured twice by specially trained nurses in the right brachial artery in the supine position after 5 min of rest using a mercury sphygmomanometer, and the average of the two values was taken as the BP. Korotkoff sounds corresponding to phase I were used to define the systolic BP and phase V, the diastolic BP.

Blood pressure change over time from MPP to MDC-CVA was expressed as mm Hg increase of BP per year: BP at MDC-CVA – BP at MPP/follow-up time (in years). To adjust for BP values at baseline, the BP change over time was also expressed as percent increase in BP per year: BP at MDC-CVA – BP at MPP/follow-up time (in years) / BP at MPP × 100. Hypertension was defined as being on antihypertensive treatment or having systolic or diastolic BP ≥140/90 mm Hg according to current diagnostic criteria and normotension as having systolic and diastolic BP <140/90 mm Hg.21 Body mass index was calculated as the ratio of the weight in kilograms to the square of the height in meters.

Genetic Tests

DNA was extracted from frozen granulocyte or buffy coat samples using QIAamp-96 spin blood kits (QIAGEN, VWR, Stockholm, Sweden) at the DNA extraction facility supported by SWEGENE. The G460W SNP in the ADD-1 gene (dbSNP accession number, rs4961) was genotyped. Genotyping was performed using the ABI 7900 (Applied Biosystems, Foster City, CA) using—forward primer: 5′-GAGAAGACAAGATGGCTGAACTCT-3′, reverse primer: 5′-GTCTTCGACTTGGGACTGCTT-3′—synthesized by Applied Biosystems. The TaqMan MGB probes were custom synthesized by Applied Biosystems—Wild-type (A): G460 probe: 5′-VIC-CATTCTGCCCTTCCTC-3′, Mutant W460 probe: 5′-FAM-ATTCTGCCATTCCTC-3′.

The polymerase chain reactions (PCR) were run in the TaqMan Universal Master mix (Applied Biosystems), according to manufacturer recommendations. The TaqMan assay plates were transferred to a Prism 7900HT instrument (Applied Biosystems) in which the fluorescence intensity in each well of the plate was read. Fluorescence data files from each plate were analyzed using automated software (SDS 2.1; Applied Biosystems).22

Statistics

All data were analyzed with SPSS statistical software (version 12.0.1; SPSS Inc., Chicago, IL). Frequency differences were analyzed by χ2 test. Continuous variables are presented as mean ± standard deviation (SD). Significance of differences in continuous variables was tested by t-test. Multiple linear regression and multiple logistic regression analysis were used in the multivariate models, with BP and hypertension status, respectively, as dependent variables and genotype, age, sex, and BMI as independent variables. Multiple linear regression analysis was used also to test for interaction of genotype and either age, sex, and BMI regarding effect on BP. All tests were two-sided, and throughout P < .05 was considered statistically significant.

Results

In the total population (n = 6055), the genotyping success rate was 99.2% (n = 6005) including patients on antihypertensive medication. Of the successfully genotyped subjects, 3772 (62.8%) were homozygotes for the wild-type G allele, 1983 were heterozygotes (33.0%), and 250 (4.2%) were homozygotes for the variant W allele. This finding is in line with previous reports in white subjects.12

Genotype distributions, in all groups of subjects studied, were in accordance with Hardy-Weinberg equilibrium (data not shown).

Blood pressure at MDC-CVA and BP change over time from MPP to MDC-CVA were similar in all genotype groups (Fig. 1). In subjects who were normotensive at MPP, there was no difference in the proportion of subjects who converted from normotension to hypertension from MPP to MDC-CVA between carriers of 460W (37.9%) and carriers of the wild-type genotype (38.0%) (P = .98). Adjustments for age, sex, and BMI, which were independently related to both systolic and diastolic BP, did not change any of these results (data not shown).

Systolic (SBP) and diastolic (DBP) blood-pressure values at MDC-CVA (a, b) and systolic (Delta SBP) and diastolic (Delta DBP) blood pressure change from MPP to MDC-CVA (c, d) according to genotypes and after stratification for sex (n = 5009). Data are presented as mean (bars) and SEM (error bars). G460G = white bars; 460W = black bars. MDC-CVA = Malmö Diet and Cancer–cardiovascular arm; MPP = Malmö Preventive Project; n.s. = not significant.

At MDC-CVA, BP was influenced by significant interaction between the G460W and BMI as well as by interaction between G460W and sex (Tables 2 and 3), indicating that the 460W variant, together with female sex, and 460W variant, together with increasing BMI, is associated with higher systolic and diastolic BP.

Table 3

Linear regression analysis of diastolic blood pressure at MDC-CVA (n = 5002*)

Variables Regression coefficient Standard error Standardized coefficient 95% CI of regression coefficient P 
Intercept 70.16 1.65  67.16/74.42 <.001 
Sex −3.22 0.32 −0.17 −3.84/−2.60 <.001 
Age (y) 0.14 0.02 0.09 0.10/0.18 <.001 
BMI (kg/m20.50 0.04 0.21 0.42/0.58 <.001 
ADD-1_G460W −18.5 5.11 −0.98 −28.49/−8.46 <.001 
ADD-1_G460W × SEX§ 8.09 2.91 0.73 2.38/13.8 .006 
ADD-1_G460W × BMI§ 0.66 0.19 0.90 0.27/1.04 .001 
ADD-1_G460W × BMI§ × SEX§ −0.27 0.11 −0.62 −0.48/−0.049 .016 
Model     <.001 
Variables Regression coefficient Standard error Standardized coefficient 95% CI of regression coefficient P 
Intercept 70.16 1.65  67.16/74.42 <.001 
Sex −3.22 0.32 −0.17 −3.84/−2.60 <.001 
Age (y) 0.14 0.02 0.09 0.10/0.18 <.001 
BMI (kg/m20.50 0.04 0.21 0.42/0.58 <.001 
ADD-1_G460W −18.5 5.11 −0.98 −28.49/−8.46 <.001 
ADD-1_G460W × SEX§ 8.09 2.91 0.73 2.38/13.8 .006 
ADD-1_G460W × BMI§ 0.66 0.19 0.90 0.27/1.04 .001 
ADD-1_G460W × BMI§ × SEX§ −0.27 0.11 −0.62 −0.48/−0.049 .016 
Model     <.001 

Abbreviations as in Tables 1 and 2.

The interaction term ADD-1_G460W × AGE was discarded from the regression model because it was not significant.

*

Seven subjects were not included in the analysis due to missed BMI;

Male sex is coded as 1 and female sex as 2;

For the ADD-1_G460W polymorphism “wild-type” subjects carrying two G alleles are coded as 0 and W carriers as 1;

§

The statistical variables used for the interaction (ADD-1_G460W × SEX, ADD-1_G460W × BMI, ADD-1_G460W × BMI × SEX) have been computed by multiplying the ADD-1_G460W genotype, respectively, with sex, BMI and sex multiplied for BMI.

Table 2

Linear regression analysis of systolic blood pressure at MDC-CVA (n = 5002*)

Variables Regression coefficient Standard error Standardized coefficient 95% CI of regression coefficient P 
Intercept 70.62 3.21  64.32/76.92 <.001 
Sex −3.33 0.61 −0.09 −4.53/−2.13 <.001 
Age (y) 0.93 0.04 0.30 0.85/1.07 <.001 
BMI (kg/m20.82 0.08 0.17 0.66/0.97 <.001 
ADD-1_G460W −11.95 5.91 −0.31 −19.61/−4.29 .002 
ADD-1_G460W × SEX§ 2.27 1.01 0.10 0.30/4.25 .024 
ADD-1_G460W × BMI§ 0.33 0.13 0.23 0.07/0.59 .013 
Model     <.001 
Variables Regression coefficient Standard error Standardized coefficient 95% CI of regression coefficient P 
Intercept 70.62 3.21  64.32/76.92 <.001 
Sex −3.33 0.61 −0.09 −4.53/−2.13 <.001 
Age (y) 0.93 0.04 0.30 0.85/1.07 <.001 
BMI (kg/m20.82 0.08 0.17 0.66/0.97 <.001 
ADD-1_G460W −11.95 5.91 −0.31 −19.61/−4.29 .002 
ADD-1_G460W × SEX§ 2.27 1.01 0.10 0.30/4.25 .024 
ADD-1_G460W × BMI§ 0.33 0.13 0.23 0.07/0.59 .013 
Model     <.001 

ADD1 = adducin 1; CI = confidence interval; other abbreviations as in Table 1.

The interaction terms ADD-1_G460W × AGE and ADD-1_G460W × BMI × SEX were discarded from the regression model because they are not significant.

*

Seven subjects were not included in the analysis due to missed BMI;

Male sex is coded as 1 and female sex as 2;

For the ADD-1_G460W polymorphism “wild-type” subjects carrying two G alleles are coded as 0 and W carriers as 1;

§

The statistical variables used for the interaction (ADD-1_G460W × SEX, ADD-1_G460W × BMI, ADD-1_G460W × BMI × SEX) have been computed by multiplying the ADD-1_G460W genotype, respectively, with sex, BMI, and sex multiplied for BMI.

A stratified analysis of the data confirmed that subjects in the upper tertile of BMI (BMI >26.65 kg/m2) expressing the W allele had higher systolic and diastolic BP compared with noncarriers (145.4 ± 18 v 143.4 ± 18.2; P = .03 for systolic BP and 90.0 ± 9.0 v 88.3 ± 9.1, P < .001 for diastolic BP; Fig. 2). Further stratification for sex showed that the association was present only in women in the upper tertile of BMI (146.1 ± 18.6 v 141.2 ± 18.6, P < .001 for systolic BP and 88.7 ± 8.7 v 86.1 ± 8.7, P < .001 for diastolic BP; Fig. 2).

Systolic (SBP) and diastolic (DBP) blood-pressure values at Malmö Diet and Cancer–cardiovascular arm according to genotypes in women (a, c; n = 2912) and men (b, d; n = 2090) after stratification for body mass index (BMI) (tertiles). Data are presented as mean (bars) and SEM (error bars). G460G = white bars; 460W = black bars. Seven subjects were not included in the analysis because of missing value of BMI. Other abbreviations as in Fig. 1.

In the analysis of the dichotomous variable of hypertension/normotension at MDC-CVA, thereby allowing inclusion also of subjects on antihypertensive medication who were excluded in the analyses of the continuous phenotype of BP, there was no difference in the prevalence of the W allele in hypertensive patients compared with normotensive subjects (37.1% v 37.4%, P = .78). Similar to the continuous variables of systolic and diastolic BP, logistic regression revealed that hypertension prevalence was influenced by a significant interaction between the G460W and BMI, as well as by interaction between G460W and Sex (Table 4), indicating that the 460W variant, together with female sex, and 460W variant, together with increasing BMI, is associated with a higher prevalence of hypertension.

Table 4

Logistic regression analysis of hypertension prevalence at MDC-CVA (n = 5002*)

Variables Regression coefficient Standard error OR 95% CI P 
Intercept −6.78 0.38 0.001  <.001 
Sex −0.46 0.07 0.63 0.55/0.73 <.001 
Age (y) 0.08 0.005 1.09 1.08/1.10 <.001 
BMI (kg/m20.11 0.01 1.12 1.10/1.14 <.001 
ADD-1_G460W −4.16 1.34 0.016 0.001/0.22 .002 
ADD-1_G460W × SEX§ 1.89 0.76 6.6 1.50/29.1 .013 
ADD-1_G460W × BMI§ 0.14 0.052 1.16 1.043/1.28 .006 
ADD-1_G460W × BMI§ × SEX§ −0.062 0.030 0.94 0.89/0.99 .036 
Model     <.001 
Variables Regression coefficient Standard error OR 95% CI P 
Intercept −6.78 0.38 0.001  <.001 
Sex −0.46 0.07 0.63 0.55/0.73 <.001 
Age (y) 0.08 0.005 1.09 1.08/1.10 <.001 
BMI (kg/m20.11 0.01 1.12 1.10/1.14 <.001 
ADD-1_G460W −4.16 1.34 0.016 0.001/0.22 .002 
ADD-1_G460W × SEX§ 1.89 0.76 6.6 1.50/29.1 .013 
ADD-1_G460W × BMI§ 0.14 0.052 1.16 1.043/1.28 .006 
ADD-1_G460W × BMI§ × SEX§ −0.062 0.030 0.94 0.89/0.99 .036 
Model     <.001 

OR = odds ratio; other abbreviations as in Tables 1 and 2.

The interaction term ADD-1_G460W × AGE was discarded from the regression model because it was not significant.

*

Seven subjects were not included in the analysis due to missed BMI;

Male sex is coded as 1 and female sex as 2;

For the ADD-1_G460W polymorphism “wild-type” subjects carrying two G alleles are coded as 0 and W carriers as 1;

§

The statistical variables used for the interaction (ADD-1_G460W × SEX, ADD-1_G460W × BMI, ADD-1_G460W × BMI × SEX) have been computed by multiplying the ADD-1_G460W genotype, respectively, with sex, BMI, and sex multiplied for BMI.

Stratified analysis showed a significantly higher prevalence of the W allele in hypertensive overweight patients compared with normotensive subjects (Table 5). Again, the significant association was present only in women (Table 5).

Table 5

Distribution of normotensive and hypertensive subjects at MDC-CVA according to genotype and after stratification for sex and BMI (tertiles) (n = 5998)

 All (N = 5998* Male subjects (N = 2540)  Female subjects (N = 3465)  
BMI (tertiles) G460G (N = 1277) W carriers (N = 719) P G460G (N = 600) W carriers (N = 349) P G460G (N = 677) W carriers (N = 370) P 
BMI ≥27.00 (N = 1996)          
 Hyp 969 (62.5) 581 (37.5) .012 495 (63.2) 288 (36.8) .99 474 (61.8) 293 (38.2) .001 
 Nor 308 (69.1) 138 (30.9)  105 (63.3) 61 (36.7)  203 (72.5) 77 (27.5)  
 G460G (N = 1258) W carriers (N = 795)  G460G (N = 620) W carriers (N = 346)  G460G (N = 638) W carriers (N = 398)  
BMI ≥23.91 <27.00 (N = 2002)          
 Hyp 795 (63.8) 451 (36.2) .25 407 (65.6) 213 (34.4) .21 388 (62.0) 238 (38.0) .75 
 Nor 463 (61.2) 293 (38.8)  213 (61.6) 133 (38.4)  250 (61.0) 160 (39.0)  
 G460G (N = 1235) W carriers (N = 765)  G460G (N = 383) W carriers (N = 237)  G460G (N = 852) W carriers (N = 528)  
BMI <23.91 (N = 2000)          
 Hyp 644 (62.8) 382 (37.2) .36 223 (64.3) 124 (35.7) .16 421 (62.0) 258 (38.0) .87 
 Nor 591 (60.7) 383 (39.3)  160 (58.6) 113 (42.4)  431 (61.5) 270 (38.5)  
 All (N = 5998* Male subjects (N = 2540)  Female subjects (N = 3465)  
BMI (tertiles) G460G (N = 1277) W carriers (N = 719) P G460G (N = 600) W carriers (N = 349) P G460G (N = 677) W carriers (N = 370) P 
BMI ≥27.00 (N = 1996)          
 Hyp 969 (62.5) 581 (37.5) .012 495 (63.2) 288 (36.8) .99 474 (61.8) 293 (38.2) .001 
 Nor 308 (69.1) 138 (30.9)  105 (63.3) 61 (36.7)  203 (72.5) 77 (27.5)  
 G460G (N = 1258) W carriers (N = 795)  G460G (N = 620) W carriers (N = 346)  G460G (N = 638) W carriers (N = 398)  
BMI ≥23.91 <27.00 (N = 2002)          
 Hyp 795 (63.8) 451 (36.2) .25 407 (65.6) 213 (34.4) .21 388 (62.0) 238 (38.0) .75 
 Nor 463 (61.2) 293 (38.8)  213 (61.6) 133 (38.4)  250 (61.0) 160 (39.0)  
 G460G (N = 1235) W carriers (N = 765)  G460G (N = 383) W carriers (N = 237)  G460G (N = 852) W carriers (N = 528)  
BMI <23.91 (N = 2000)          
 Hyp 644 (62.8) 382 (37.2) .36 223 (64.3) 124 (35.7) .16 421 (62.0) 258 (38.0) .87 
 Nor 591 (60.7) 383 (39.3)  160 (58.6) 113 (42.4)  431 (61.5) 270 (38.5)  

Hyp = hypertensive subjects; Nor = normotensive subjects; other abbreviations as in Tables 1 and 2.

Data are presented as absolute number of subjects (percentage of subjects).

*

Seven subjects were not included in the analysis because of missing value of BMI.

Discussion

We found that there is no overall association between the G460W polymorphism and BP in a large Swedish population-based sample. However, there was significant interaction between the G460W polymorphism and both sex and BMI regarding BP and hypertension prevalence.

The kidney plays a major role in BP homeostasis and hypertension development, and functional variants of genes that interfere with sodium reabsorption are attractive candidate genes. Bianchi et al12 focused a lot of attention on ADD-1, a cytoskeletal protein that has been shown to augment the activity of the Na-K pump. The Na-K pump is an ATPase that drives the reabsorption of sodium in the kidney, but it is present and active in several other tissues, including vascular smooth muscle cells (VSMC). An enhanced activity of the Na-K pump in the SMC could putatively lead to enhanced vasodilatation with diminished peripheral resistance and lower BP. Thus, it is possible that the effect of the functional G460W polymorphism, enhancing the Na-K pump activity in the kidney with consequent augmented sodium handling, under different conditions, could be blurred or even counteracted from the effect elicited in other tissues such as VSMC. Thus, most studies controlling for body sodium or renin-angiotensin-aldosterone system (RAAS) activity have found positive associations between the W allele and higher BP.12 Also, in our sample we could not find a simple association of the G460W polymorphism with BP, but when the interaction of this genetic variant with sex and BMI was considered, a positive association resulted, evident both for systolic and diastolic BP, suggesting that the effect of the polymorphism is unmasked when these two well-established BP covariates are taken into account. A stratified analysis of the data confirmed that subjects with BMI in the upper tertile and expressing the W allele have higher systolic BP, diastolic BP, and hypertension prevalence with respect to wild-type carriers. Interestingly, the effect of the W allele was particularly evident in women.

It is possible that lack of or even reversed associations between G460W and BP, when significant cofactors are not taken into account, are due to the single gene effect of G460W being diluted by the polygenic background of BP, assuming that the G460W primarily affects the sodium-sensitive component of BP. In addition, one could speculate that the importance of the renal versus the VSMC effects of the G460W is likely to be relatively greater in subjects with endothelial dysfunction, as the latter could be expected to blunt the vasodilatory effects of the W allele in SMC but not the enhanced sodium transport induced by W allele in renal tubular cells.

Endothelial dysfunction, as well as salt sensitivity, have been associated with both increasing age and obesity.23–27 Regarding sex, it has to be underlined that in our sample women are mostly postmenopausal (Table 1), which has been found associated with both salt sensitivity28 and endothelial dysfunction.29

Previous studies have shown that homozygosity for the W allele is significantly associated with reduced renal plasma flow and glomerular filtration rate as compared with the wild-type variant30 and that acute changes in body sodium may differently affect BP in humans as a function of the α-adducin genotype.8

All of these effects could be difficult to detect at the general population level, where other counterbalancing effects may be active. Interaction and stratified analysis can help to unravel the biological complexity of genetic variants that act in concert with other factors. The importance of interaction analyses in the interpretation of single gene effects in complex traits such as BP is further emphasized because the G460W polymorphism was negatively associated with BP when the interaction terms of genotype/sex and genotype/BMI were simultaneously taken into account. Genetic and environmental heterogeneity of BP homeostasis and essential hypertension development have made it difficult to repeatedly link genetic variants to BP and hypertension in humans. Among genes that exert their roles in sodium handling, ADD-1 is one of the most attractive candidates, but at the general population level its effect has not invariably been confirmed in previous studies. Nevertheless, only minor effects are expected from a single nucleotide polymorphism (SNP) and these effects could be blurred from complex gene–gene and gene–environment interactions. Studies of adequate size are needed to analyze whether the effects of these genetic variants could be exacerbated by other factors such as obesity or sex. Thus, major efforts are needed in the future to specifically define and characterize more homogeneous groups of people where specific genetic variants could have a primary role in determining BP levels.

In conclusion, we found that the G460W polymorphism does not play a major role either in BP or hypertension development in the general population when possible interactions are disregarded. Positive interaction of G460W with sex and BMI and the result of the stratified analysis suggest that this polymorphism could be of primary importance for BP homeostasis and hypertension prevalence in obese women, probably in addition to a salt-sensitive background. Further studies of adequate size are needed to confirm our findings and to explore the hypothesis that salt sensitivity could be the factor subtending the effect of the G460W polymorphism of the ADD-1 gene on BP at the general population level.

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Author notes

*
This study was supported by grants from the Swedish Medical Research Council, the Swedish Heart and Lung Foundation, the Medical Faculty of Lund University, Malmö University Hospital, the Albert Påhlsson Research Foundation, the Crafoord Foundation, the Ernhold Lundströms Research Foundation, the Region Skane, Hulda and Conrad Mossfelt Foundation, King Gustaf V and Queen Victoria Foundation, and the Lennart Hansson Memorial Fund. The authors acknowledge the Knut and Alice Wallenberg Foundation for its economic support of the SWEGENE DNA extraction facility.