Variation at the Angiotensin-Converting Enzyme Gene and Angiotensinogen Gene Loci in Relation to Blood Pressure
To investigate whether the polymorphisms in the angiotensin-converting enzyme and angiotensinogen genes are associated with hypertension, we carried out a case-control study of 508 hypertensive and 523 control subjects randomly selected from the Social Insurance Institution register. The cohorts were well characterized and matched for age and sex. The insertion/deletion polymorphism of the angiotensin-converting enzyme gene and the methionine→threonine variant at position 235 of the angiotensinogen gene were determined by the polymerase chain reaction technique. The allele frequencies and genotype distributions of both polymorphisms were similar in hypertensive and control subjects. Systolic and diastolic pressures adjusted for age, body mass index, and alcohol consumption did not differ significantly between the different genotypes of the angiotensin-converting enzyme and angiotensinogen genes. The variation at the angiotensinogen and angiotensin-converting enzyme genes did not have any statistically significant synergistic effect on blood pressure levels. In conclusion, the polymorphisms in the renin-angiotensin cascade genes do not confer a significantly increased risk for the development of hypertension in this middle-aged, population-based cohort.
Essential hypertension is a complex disorder influenced by multiple genetic and environmental factors. The maintenance of BP is regulated largely by the complex RAS. Therefore, abnormalities of the RAS and its components may be associated with the predisposition to high BP. The RAS consists of ATG, renin enzyme, and ACE. ATG is primarily synthesized in the liver and released into the circulation, where it is cleaved by renin, generating inactive angiotensin I. ACE, which is present in nearly all mammalian tissues and body fluids,1 2 regulates BP by activating an inactive angiotensin I into a potent vasopressor, angiotensin II, and inactivating a potent vasodilator, bradykinin.3
Although the insertion/deletion (I/D) polymorphism of the ACE gene has been associated with plasma ACE concentrations,4 5 6 7 several studies have failed to show an association between hypertension and ACE polymorphism.5 6 8 9 The methionine→threonine variant at position 235 (M235T variant) of ATG has been considered as a marker for essential hypertension10 11 12 13 14 and an elevated plasma ATG concentration,10 13 although recently, Caulfield et al15 16 found a linkage and association in a family study but no association between the M235T variant and essential hypertension in a population sample of unrelated subjects. Although the polymorphisms of single genes seem to have no effect on BP, the interactions of the gene products within the same biochemical and physiological pathway (the RAS) may have synergistic effects on BP. Therefore, we investigated the association between the genetic variants of the RAS (ie, the ACE and ATG genes) and BP in a middle-aged, population-based cohort randomly selected from an ethnically homogenous population.
The Oulu Project Elucidating Risk of Atherosclerosis (OPERA) is a population-based, epidemiological study addressing the risk factors and disease end points of atherosclerotic cardiovascular diseases. The entire study cohort of OPERA forms the present study population. The hypertensive cohort (cases) consisted of 600 subjects (300 men and 300 women, aged 40 to 59 years at the time of selection) from the town of Oulu randomly selected by the Social Insurance Institution from the national register for reimbursement of hypertension medication. The Social Insurance Institution reimburses 75% (80% at the time of randomization) of the costs of drug treatment for moderate or severe hypertension. In the case of mild hypertension, the reimbursement is significantly lower, and these individuals are not listed as reimbursement recipients (ie, they are not included in the hypertensive cohort of the present study). To be eligible for the higher benefit, the hypertensive individual's diastolic BP level must have been above 105 mm Hg during a few months of follow-up, unless the individual already at presentation shows signs of left ventricular hypertrophy or other target-organ damage caused by hypertension. If the subject is young (men younger than 50 years, women younger than 40), has a family history of cardiovascular disease or sudden death at an early age, has diabetes or severe hyperlipidemia, or has systolic BP above 180 mm Hg, he or she is also eligible for the higher benefit when the diastolic BP level is above 95 mm Hg. In summary, the list of the reimbursement recipients consists of individuals with moderate or severe hypertension, individuals presenting with or developing target-organ damage, or individuals with a family history. For each year of birth (1931 to 1950), 15 hypertensive men and 15 hypertensive women were selected. For each hypertensive subject, an age- and sex-matched control subject was randomly selected from the national health register (including all inhabitants) excluding subjects with the right to reimbursement for hypertension medication.
The overall participation rate of the hypertensive subjects was 86.5% (261 men, 258 women) and of the control subjects was 87.7% (259 men, 267 women). Twenty-six men and 32 women among the control subjects were on chronic medication affecting BP because of the fact that BP-lowering drugs (in this case mainly β-blockers and diuretics) are commonly used also for other indications (eg, chest pain, ankle edema, essential tremor, palpitation, etc). Furthermore, in a smaller proportion (7 men and 14 women) of these 58 subjects, these drugs were used for the treatment of hypertension although the subjects were not listed as reimbursement recipients at the time of randomization because they had received the right to reimbursement after the randomization, or for any reason, had not requested the right to reimbursement.
Alcohol consumption was determined in an extensive interview concerning the amount of beer, wine, and strong alcoholic beverages consumed. In a questionnaire, all the participants were inquired in detail about their smoking habits, physical activity, use of medications, and medical history. Alcohol consumption was calculated as grams of absolute alcohol per week and smoking as the number of cigarettes smoked per day. Body mass index was calculated by dividing weight (kilograms) by the square of height (meters squared).
BP was measured with subjects in a sitting position from the right arm with an oscillometric device (Dinamap model 18465X, Critikon Ltd) after subjects had fasted overnight and rested 10 to 15 minutes. Three measurements at 1-minute intervals were made. The means of the last two measurements were used for analyses.
Genomic DNA was isolated from EDTA-containing blood with a salting-out method according to Miller et al.17 The I/D polymorphism of the ACE gene was detected by PCR. The primers were selected according to Rigat et al.18 Amplification reactions were performed in a final volume of 50 μL containing 67 mmol/L Tris-HCl (pH 8.8), 16 μmol/L (NH4)2SO4, 1.5 mmol/L MgCl2, 0.01% Tween 20, 50 pmol of both primers, 200 μmol/L of each dNTP, 325 ng genomic DNA, and 0.5 U Taq DNA polymerase (HyTest). DNA was amplified in a thermal cycler with 1 minute of denaturation at 94°C, 1 minute of annealing at 65°C, and 2 minutes of extension at 72°C for 30 cycles. In the last cycle, the extension step was carried out for 10 minutes. PCR products were separated on a 2% agarose gel containing ethidium bromide and visualized under UV lighting. The lengths of the fragments separated were 478 and 191 bp, corresponding to the I and D alleles, respectively. Since some previous reports19 20 have suggested that the D allele is preferentially amplified, the PCR reactions were reanalyzed in two different ways. The addition of dimethyl sulfoxide to the reaction mixture described above or the use of the insertion-specific primer20 confirmed the results of the original PCR reactions.
The detection of the M235T variant of the ATG gene was done with the PCR detection method. The primers were selected according to Russ et al.21 Amplification reactions were performed in a final volume of 25 μL as described above except that the initial denaturation was for 3 minutes at 90°C; 10 cycles of 94°C for 1 minute, 68°C for 1 minute, and 72°C for 1 minute; and 30 cycles of 90°C for 30 seconds, 68°C for 1 minute, and 72°C for 30 seconds followed by a final extension of 72°C for 10 minutes. Three microliters of unpurified PCR product was diluted to 10 μL in recommended restriction buffer containing 5 U of Tth 111 I enzyme (Pharmacia) and digested at 65°C under mineral oil for more than 3 hours. The digested PCR products were separated on a 3% agarose gel containing ethidium bromide and visualized under UV lighting. The lengths of the fragments separated were 165 bp, corresponding to the M235 allele (M), and 141 bp, corresponding to the digested T235 allele (T).21
Allele frequencies were estimated with the gene-counting method. A χ2 test was used for assessment of the fit of the observed allele frequencies to Hardy-Weinberg equilibrium and the difference in genotype distributions between the control and hypertensive groups. A previous study22 indicated that the frequency of the I allele among the hypertensive subjects would be approximately 56% and the expected prevalence in control subjects would not exceed 41%. With the expectation that a difference of this magnitude (odds ratio, 1.83) would strongly support the concept that the I allele is a significant genetic risk factor and with a two-sided α error of 0.05, the sample size of approximately 260 in each group would limit the β error to 0.07 (ie, the power of the study would be 93%). In addition, our control group had 80% power to show a difference of 9 mm Hg in systolic BP between subjects with the I/I genotype (n=95) and those with the D/D genotype (n=125) (α=0.05).
The continuous variables are expressed as mean±SD according to genotype (II, ID, DD and MM, MT, TT). Unpaired t test was used for comparison of the means of two groups. The differences between variables in the different genotype groups were compared by ANOVA. Because of skewed distribution, logarithmic transformation was used for alcohol consumption. A two-way ANOVA (3×3 factorial design) was used for the potential synergistic effect of the ACE and ATG genes on BP. Bonferroni's method was used for multiple comparisons between genotype classes. ANCOVA was used for adjustment of BP values for the effects of age, body mass index, and alcohol consumption. The adjustments were made separately for men and women. Multiple regression analysis was used for assessment of the quantitative effects of the covariates on BP levels. Covariates in the regression model were age, body mass index, alcohol consumption, ACE and ATG genotype, and the genotype interaction term (ACE×ATG). For the regression model, the genotype effect was assumed to be additive (with scores of 1, 2, and 3 assigned for genotypes II, ID, and DD, respectively), dominant (with scores of 0 for II and 1 for ID and DD combined), or recessive (with scores of 0 for II and ID combined and 1 for DD). A similar combination was used also for the ATG genotypes. The regression analysis was made separately for (control) men and women.
The Statistical Analysis System (SAS, version 6.08) was used in all statistical analyses.
Table 1⇓ shows the main characteristics of the control and hypertensive subjects by sex. DNA analysis was successful in 508 (ACE genotype was analyzed in 507) hypertensive and 523 control subjects. The prevalences of the ACE and ATG genotypes and allele frequencies in the subjects by sex are shown in Table 2⇓. No deviation from Hardy-Weinberg equilibrium was observed in any of the subgroups. Genotype prevalence and allele frequencies were calculated also in more severe categories of hypertension, ie, in subsets of the hypertensive subjects with (1) at least two ongoing antihypertensive drugs and (2) the onset of disease before age of 50. In women, the frequency of allele D of the ACE gene was higher both in all hypertensive subjects and in those with more severe disease than in control women. Exceptionally few control women had the DD genotype (23.8% versus 33.3% in control men). In men, no difference in allele frequency was seen. There were no differences in the allele distribution of the ATG gene.
We made further analyses to study the connection between BP and genotype among the control subjects. Subjects on BP-lowering drugs were excluded from the analyses. Table 3⇓ shows the main characteristics including systolic and diastolic BP values in control subjects according to sex and genotype. There was no significant association between BP level and genotype. Adjustment for the covariates did not change the findings (data not shown).
We also determined the effect of the ATG genotype on BP adjusted for the covariates in control subjects with different ACE genotypes. A significant synergistic effect of the ACE and ATG genes on BP was not observed, although in both men and women, BP tended to be highest in the IITT genotype (homozygous for both the ACE I and ATG T alleles) (Table 4⇓).
In multiple regression analysis with systolic BP as a dependent variable, neither the effect of the single genes nor the synergistic gene effect on the variance of BP was observed. Different assumptions about the type of the genotype effect (see “Statistical Analyses”) did not change the findings. The results of regression analysis are shown in Table 5⇓. This model was able to account for 9.4% (adjusted R2=.0943) of the total variance in systolic BP in men and 13.5% (adjusted R2=.1353) of the variance in women.
Our primary goal was to investigate whether the allele frequencies of the candidate genes differ between individuals with moderate or severe hypertension and control subjects. In addition, a secondary approach focused on the possible genetic effect on BP in the population-based control cohort. Since BP-lowering drugs are commonly used also for other indications and therefore might interfere with the measurement of the continuous variable to be analyzed, subjects receiving these drugs were excluded from the analysis of the secondary approach. Other environmental factors such as alcohol drinking and dietary habits were assumed to have more subtle effects on BP levels, and their role as confounders was handled by common statistical methods.
In the present research, we investigated two polymorphisms of the genes of the RAS in a well-characterized random population of hypertensive and control subjects matched for age and sex. Previous studies have suggested that allele I of the ACE gene is a marker for hypertension22 23 and that allele T235 of the ATG gene is associated with both hypertension10 11 12 13 14 and an increased ATG concentration,10 13 although opposite results have also been published.5 6 8 9 15 16 24 The present study did not show any association between allele I of ACE or the variant T235 of ATG and essential hypertension.
An individual's BP level is determined by genetic and environmental factors. In the present research investigating the role of genetic factors, we made a special attempt to exclude the possible bias caused by an uneven distribution of confounding factors between the genotypes. Therefore, we carefully controlled for the effects of environmental factors known to affect BP, such as alcohol intake, medication, and concomitant diseases. In the present study, all the subjects were middle-aged (from 40 to 61 years). Because of the high participation rate (more than 85% in all the groups), our results can be generalized to apply to the middle-aged population at large. Whether the associations of the studied genes with BP are different in subjects developing high BP at an early age remains to be studied.
The role of the RAS in BP control has been established.1 25 Since previous studies have shown that the I/D polymorphism in the ACE gene accounts for half of the variance of serum ACE levels and the D allele of the ACE gene is a marker for high levels of circulating ACE,4 5 6 7 it would be plausible to assume that individuals with a high BP have a high prevalence of the D allele. In the present study, the D allele was more common in hypertensive women than in control women, but the difference was entirely due to the exceptionally low prevalence (0.511) of the D allele among the control women compared with the male cohort and previous data.26 No significant difference in the prevalence of the D allele was observed between hypertensive and control men. Two previous studies have shown an association of ACE genotype with BP,22 23 whereas other studies have not.5 6 8 9 The latter findings are confirmed by the present study, which investigated the prevalence of the ACE gene polymorphism in 1031 unrelated subjects—the largest population-based cohort so far—randomly selected from the national health register. The lack of association may be related to the fact that although the D allele of ACE polymorphism is associated with high plasma ACE levels, it is not associated with a high concentration of angiotensin II in plasma.6
The M235T variant of the ATG gene was not consistently associated with high BP, a finding that is in accordance with recent studies.15 16 24 Jeunemaitre et al10 and Caulfield et al15 16 showed in linkage analyses a positive association between the ATG gene locus and hypertension. Furthermore, Jeunemaitre et al showed that this association was more pronounced in the more severe cases of hypertension and, moreover, observed a positive association between the M235T polymorphism and BP. Our results showing no association in more severe cases of disease are in disagreement with this10 and other11 12 14 studies that show a positive association between the M235T variant and BP or hypertension. The most probable explanation for differences may be the variability in the selection of hypertensive cases for the studies. As in all studies investigating associations between genetic polymorphisms and quantitative or qualitative traits, the genetic background and homogeneity of the study populations may influence the results. In other studies, hypertensive subjects have been selected by positive family history,10 15 16 from outpatient clinics for hypertension or cardiovascular diseases,11 12 14 or from a genetic isolate,27 whereas in our study, the hypertensive cohort represented a random sample of all middle-aged hypertensive individuals in the population. Although our results do not support the role of the ATG gene in hypertension, it should be noted that the gene effect in a general hypertensive population with a substantial heterogeneity of factors increasing BP—eg, obesity, salt intake, and alcohol consumption—would be difficult to observe. Since only 30% of the BP variability can be accounted for by heritable factors28 and the ATG gene polymorphism at best might account for 25% of the heritability factors,10 a sample size of several thousand would be necessary to document its effect in a general hypertensive population.
Interaction of gene products within biochemical and physiological pathways could contribute to the pathogenetic mechanism leading to elevated BP. Since the T235 allele of the ATG gene is a marker for an increased plasma ATG concentration10 13 and the D allele of the ACE gene is a marker for an elevated circulating ACE level,4 5 6 7 we hypothesized that the simultaneous presence of both of these markers in an individual might increase his or her risk for developing hypertension. We therefore determined the synergistic effect of the variation at the ATG and ACE genes on BP levels. In subjects with different ACE genotypes, the effect of the ATG genotype on BP adjusted for the covariates showed a trend only in the II genotype. Although this trend was consistently observed in both men and women as well as in both systolic and diastolic BPs, it was based on small counts in each cell and is likely to reflect chance observations.
In conclusion, we did not find any marked association between the two polymorphisms of the genes of the RAS and BP in a well-characterized random population of hypertensive and control subjects matched for age and sex.
Selected Abbreviations and Acronyms
|PCR||=||polymerase chain reaction|
This study was supported by grants from the Medical Council of the Academy of Finland and the Finnish Foundation for Cardiovascular Research. We gratefully acknowledge Helena Kalliokoski and Riitta Vanhanen for their excellent technical assistance.
Part of this work was presented at the 67th Scientific Sessions of the American Heart Association, Dallas, Tex, November 14-17, 1994, and published in abstract form (Circulation. 1994;90[suppl I]:I-130).
- Received December 27, 1995.
- Revision received February 21, 1996.
- Revision received July 3, 1996.
Ehlers MRW, Riordan JF. Angiotensin-converting enzyme: biochemistry and molecular biology. In: Laragh JH, Brenner BM, eds. Hypertension: Pathophysiology, Diagnosis and Management. New York, NY: Raven Press Publishers; 1990:1217-1231.
Lieberman J, Sastre A. Angiotensin-converting enzyme activity in postmortem human tissues. Lab Invest. 1983;359:641-644.
Erdös EG. Angiotensin I–converting enzyme and the changes in our concepts through the years. Hypertension. 1990;16:363-370.
Rigat B, Hubert C, Alhenc-Gelas F, Cambien F, Corvol P, Soubrier F. An insertion/deletion polymorphism in the angiotensin I-converting enzyme gene accounting for half the variance of serum enzyme levels. J Clin Invest. 1990;86:1343-1346.
Harrap SB, Davidson HR, Connor JM, Soubrier F, Corvol P, Fraser R, Foy CJW, Wat GCM. The angiotensin-converting enzyme gene and predisposition to high blood pressure. Hypertension. 1993;21:455-460.
Gardemann A, Weiß T, Schwartz O, Eberbach A, Katz N, Hehrlein FW, Tillmanns H, Waas W, Haberbosch W. Gene polymorphism but not catalytic activity of angiotensin I-converting enzyme is associated with coronary artery disease and myocardial infarction in low-risk patients. Circulation. 1995;92:2796-2799.
Hata A, Namikawa C, Sasaki M, Sato K, Nakamura T, Tamura K, Lalouel J-M. Angiotensinogen as a risk factor for essential hypertension in Japan. J Clin Invest. 1994;93:1285-1287.
Iwai N, Shimoike H, Ohmichi N, Kinoshita M. Angiotensinogen gene and blood pressure in the Japanese population. Hypertension. 1995;25(part 2):688-693.
Jeunemaitre X, Charru A, Chatellier G, Dumont C, Sassano P, Soubrier F, Ménard J, Corvol P. M235T variant of the human angiotensinogen gene in unselected hypertensive patients. J Hypertens. 1993;11(suppl 5):S80-S81.
Caulfield M, Lavender P, Newell-Price J, Farrall M, Kamdar S, Daniel H, Lawson M, De Freitas P, Fogarty P, Clark AJL. Linkage of the angiotensinogen gene locus to human essential hypertension in African Caribbeans. J Clin Invest. 1995;96:687-692.
Miller SA, Dykes DD, Polesky HF. A simple salting out procedure for extracting DNA from human nucleated cells. Nucleic Acids Res. 1988;16:1215.
Rigat B, Hubert C, Corvol P, Soubrier F. PCR detection of the insertion/deletion polymorphism of the human angiotensin converting enzyme gene (DCP1) (dipeptidyl carboxypeptidase 1). Nucleic Acids Res. 1992;20:1433.
Russ AP, Maerz W, Ruzicka V, Stein U, Groß W. Rapid detection of the hypertension-associated Met235→Thr allele of the human angiotensinogen gene. Hum Mol Genet. 1993;2:609-610.
Morris BJ, Zee RYL, Schrader AP. Different frequencies of angiotensin-converting enzyme genotypes in older hypertensive individuals. J Clin Invest. 1994;94:1085-1089.
Sealey JE, Laragh JH. The renin-angiotensin-aldosterone system for normal regulation of blood pressure and sodium and potassium homeostasis. In: Laragh JH, Brenner BM, eds. Hypertension: Pathophysiology, Diagnosis and Management. New York, NY: Raven Press Publishers; 1990:1287-1328.
Hegele RA, Brunt JH, Connelly PW. A polymorphism of the angiotensinogen gene associated with variation in blood pressure in a genetic isolate. Circulation. 1994;90:2207-2212.
Ward R. Familial aggregation and genetic epidemiology of blood pressure. In: Laragh JH, Brenner BM, eds. Hypertension: Pathophysiology, Diagnosis and Management. New York, NY: Raven Press Publishers; 1990:81-100.