Angiotensinogen Polymorphisms and Elevated Blood Pressure in the General Population
The Copenhagen City Heart Study
In the present study, we tested the hypothesis that the Met235Thr and Thr174Met mutations were associated or not with elevated blood pressure. We genotyped 9100 women and men from the Danish general population, of whom 54% had elevated blood pressure. Of the 9100, 41% and 12% carried the Thr235 and Met174 mutations, respectively; the Met174 mutation always occurred on the same allele as the Thr235 mutation. On multifactorial logistic regression analysis, women homozygous for Thr235 versus noncarriers had an odds ratio for elevated blood pressure of 1.29 (95% CI 1.05 to 1.58), which increased to 1.50 (1.15 to 1.96) if they also were homozygous for Thr174 (noncarrier of Met174). Women homozygous for Thr235 also had an increased risk of isolated elevated systolic blood pressure (1.37; 1.02 to 1.84) and of mild blood pressure elevation (1.40; 1.10 to 1.77). We found no statistically significant association between elevated blood pressure and genotype in men or among genotype and systolic blood pressure, diastolic blood pressure, or pulse pressure in either gender. Homozygosity for both Thr235 and Thr174 was associated with a 10% increase in plasma angiotensinogen levels in both genders compared with homozygosity for Met235 and Thr174; however, systolic and diastolic blood pressures were positively correlated to plasma angiotensinogen levels in women only. In conclusion, in this large-scale study of the general population, double homozygosity for Thr235 and Thr174 in the angiotensinogen gene is associated with a 10% increase in angiotensinogen levels and is a risk factor for elevated blood pressure in women but not in men.
In 1992, Jeunemaitre et al1 reported an association between increased risk of elevated blood pressure and the Met235Thr and Thr174Met mutations in the angiotensinogen gene. This has been supported by some,2 3 but not all,4 5 6 7 8 later reports; however, all of these studies1 2 3 4 5 6 7 8 included relatively few individuals, increasing the risk of chance findings. Although 2 meta-analyses that included 5493 and 10 720 whites suggested a 15% to 32% increase in the risk of elevated blood pressure associated with Thr235,9 10 larger studies with more rigorous designs are clearly warranted to conclusively establish an association between hypertension and Thr235.
In the present study, we tested the hypothesis that the Met235Thr and Thr174Met mutations were associated or not with elevated blood pressure in the population at large. For this purpose, we examined 9100 individuals sampled from the adult Danish general population: The Copenhagen City Heart Study. This is the largest study to examine this hypothesis, and it is the only one to test the hypothesis in the general population separately in women and men.
The Copenhagen City Heart Study (third examination, 1991 to 1994) consists of 55% women and 45% men stratified into 10-year age groups from 20 to 80+ years, drawn randomly from the Copenhagen Central Population Register, with the aim of drawing a sample representative of the adult general population in Copenhagen.11 12 13 14 Less than 1% were nonwhite. For the present study, 9100 subjects (99%) were genotyped. All participants gave informed consent. The Danish Ethical Committee for Copenhagen and Frederiksberg approved this study (No. 100.2039/91). The procedures followed were in accordance with institutional guidelines.
Subjects reported on the use of medication, smoking status, physical activity, whether they had diabetes mellitus, and weekly alcohol consumption (g/wk). Women in addition reported on menopausal status and use of hormonal replacement therapy.
Elevated Blood Pressure
Elevated blood pressure was defined as systolic blood pressure of ≥140 mm Hg and/or diastolic blood pressure of ≥90 mm Hg,15 or treatment with antihypertensive medication. Isolated elevated systolic blood pressure was defined as systolic blood pressure of ≥140 mm Hg and diastolic blood pressure of ≤90 mm Hg.15 Subclassification of elevated blood pressure into mild (between 140/90 and 159/99 mm Hg), moderate (between 160/100 and 179/109 mm Hg), and severe (≥180/110 mm Hg) was as described previously.15 Pulse pressure was the difference between systolic and diastolic blood pressures. The phenotype elevated blood pressure was evenly distributed in the whole sample, and the laboratory technicians were blinded to the phenotype.
Blood pressure was measured by trained technicians using the London School of Hygiene sphygmomanometer on the left arm after 5 minutes’ rest with the subject in the sitting position. The fifth Korotkoff sound was used for diastolic pressure. The fall of the mercury column was set to 2 mm/s. The blood pressure cuff was 12×26 cm, but for subjects with an upper arm circumference of >46 cm, a cuff that measured 15×38 cm was used. Interobserver variation was tested and found to be statistically insignificant.
The Met235Thr and Thr174Met mutations in the angiotensinogen gene are caused by the substitution of tyrosine for cytosine at position 806 and of cytosine for tyrosine at position 623 of cDNA in exon 2 of the angiotensinogen gene.16 The presence of either or both mutations was determined in a single polymerase chain reaction (PCR); the following sense and antisense primers were used: 5′-AGTGACTATGGGGCGTGGTCCATGGGACC-3′ and 5′-GTTGAAAGCCAGGGTGCTGTCCACACTGACT-3′. The mismatch in the sense primer (underlined) creates a control site for the enzyme PshAI, whereas the mismatch in the antisense primer (underlined) creates a restriction site for PshAI when Thr235 is present. The PCR product was digested with NcoI (recognizing Thr174Met) and PshAI (recognizing Met235Thr) in the same reaction mixture, followed by separation on a 3% agarose gel. There were common bands of 23 and 8 bp due to the internal control sites for both enzymes and mutation-specific bands of 225 bp (174Met) and 186 or 411 bp (Thr235 with or without 174Met, respectively). This method thus enabled us to determine both genotypes and haplotypes.
With a 2-sided significance level of 0.05 and a power of 95%, with the aim to not overlook a mean difference in plasma angiotensinogen levels between genotypes of 208 nmol/L (269 ng/mL) as previously determined,1 we estimated that the number of subjects to be included was >47 for each genotype.17 Among the 6786 individuals homozygous for Thr174 in our study population, we randomly selected 300 men and women (40 to 67 years old), distributed equally between the 2 genders and among homozygotes, heterozygotes, and noncarriers of Thr235. We excluded individuals on medication or with conditions that potentially affect angiotensinogen levels (ie, those on medication that affect blood pressure, individuals on estrogen or other hormones, and individuals with suspected liver disease if they had aspartate aminotransferase levels of >3 times the upper normal limit or albumin levels of <340 μmol/L.
Angiotensinogen was first converted to angiotensin I by the addition of excess human renin in the presence of angiotensin I antibodies, followed by the measurement of angiotensin I levels by radioimmunoassay; the addition of antibodies prevents proteolytic degradation of angiotensin I. The intra-assay and interassay coefficients of variation were 2% and 5%, respectively.
Cholesterol was determined enzymatically (CHOD-PAP; Boehringer Mannheim). Body mass index (BMI) was calculated as weight divided by height squared (kg/m2).
Statistical analyses were performed for each gender separately with the Statistical Package for Social Sciences (SPSS) program; as in our other studies on polymorphisms, we a priori stratified for gender.11 14 A value of P<0.05 on a 2-sided test was considered significant. Correction for multiple comparisons was not performed in any of the analyses in the present study.
Logistic regression analysis that allowed for age (tertiles) alone or for age, BMI (tertiles), diabetes mellitus, smoking, plasma cholesterol (tertiles), alcohol consumption (tertiles), antihypertensive drug treatment, physical activity (4 levels), and menopausal status and hormonal replacement therapy in women explored the association between genotype and risk of elevated blood pressure; analysis on subgroups of mild, moderate, or severe elevation of blood pressure or isolated elevated systolic blood pressure excluded those on antihypertensive medication. Whether we allowed for age alone or the larger group of covariates, the results were similar; we have chosen to present the results after allowances for the larger group of covariates. Interaction was explored between genotype and gender, or the above mentioned risk factors on elevated blood pressure; logistic regression models included, besides age in tertiles, genotype, the risk factor in question, and an interaction term of the 2 latter factors.
ANOVA was used to test for differences in means of angiotensinogen levels, systolic blood pressure, diastolic blood pressure, and pulse pressure. Correlation between angiotensinogen levels and systolic blood pressure, diastolic blood pressure, pulse pressure, or BMI was examined with linear regression or ANCOVA.
The characteristics of participants from The Copenhagen City Heart Study are shown in Table 1⇓. Men and women are subdivided into those who were treated with medications known to affect blood pressure, either antihypertensive medication or medication for angina pectoris, heart failure, or cardiac arrhythmias, and those who were untreated. Of all participants, 54% had elevated blood pressure.
Genotype and Haplotype Frequencies
Relative frequencies of Met235Thr and Thr174Met in the angiotensinogen gene in 9100 individuals from the general population are shown in Table 2⇓. Frequencies of either substitution did not differ significantly from those predicted by the Hardy-Weinberg equilibrium (Met235Thr, P>0.70; Thr174Met, P>0.95) and were in accordance with frequencies observed in other studies of whites.1 4 5 6 8 18 The Met235Thr and Thr174Met mutations were in linkage disequilibrium (χ2=0.000): when present, the Met174 mutation always occurred on the same allele as the Thr235 mutation.
Relative haplotype frequencies were 0.59 for Met235/Thr174, 0.29 for Thr235/Thr174, and 0.12 for Thr235/Met174, respectively. The observed 6 possible genotype frequencies based on these 3 haplotypes were consistent with the expected frequencies according to the Hardy-Weinberg equilibrium (P>0.80).
Genotype and Elevated Blood Pressure
The odds ratio for elevated blood pressure in women homozygous for Thr235 versus noncarriers was 1.29 (95% CI 1.05 to 1.58) (Figure 1⇓, left top). The subgroup of these women who were also homozygous for the Thr174 (noncarrier of Met174) had an even higher odds ratio (1.50; 1.15 to 1.96) (Figure 1⇓, left bottom). In men, angiotensinogen genotype did not predict risk of elevated blood pressure (Figure 1⇓, right).
When individuals on antihypertensive medication were excluded, we also found an increased risk of elevated blood pressure in women homozygous for Thr235 versus noncarriers (1.32; 1.07 to 1.63), which increased in women also homozygous for Thr174 (1.51; 1.15 to 2.00). Furthermore, women homozygous for Thr235 versus noncarriers also had an increased risk of isolated elevated systolic blood pressure (1.37; 1.02 to 1.84), as well as mildly elevated blood pressure (1.40; 1.10 to 1.77); the latter increased in women who at the same time were homozygous for Thr174 (1.71; 1.26 to 2.32).
Apart from age and physical activity, which appeared to interact with the Thr174Met mutation on elevated blood pressure in men (P=0.03 and P=0.03, Table 3⇓, top), and cholesterol, which appeared to interact with the Met235Thr/Thr174Met genotype on elevated blood pressure in women (P=0.05, Table 3⇓, top), we found no other significant interactions. When the above mentioned potential interactions were explored with stratification by age, physical activity, and cholesterol, respectively, the observed irregular patterns were not biologically meaningful and thus suggested chance findings rather than plausible interactions.
Genotype and Antihypertensive Medication
Women who were double homozygous for Thr235 and Thr174 versus noncarriers had an increased risk of being on any antihypertensive medication (1.53; 1.12 to 2.09) but not of being on ≥2 versus none or 1 antihypertensive medication (1.17; 0.70 to 1.93). In men, there was no statistical evidence to suggest that genotype predicted treatment with any or ≥2 antihypertensive drugs.
Genotype and Blood Pressure
On ANOVA, diastolic blood pressure did not differ between genotype groups in either women or men (Figure 2⇓). Likewise, systolic blood pressure and pulse pressure did not differ between genotype groups (data not shown).
Genotype and Angiotensinogen Concentration
Among 300 randomly selected individuals, of whom all were homozygous for Thr174, plasma levels of angiotensinogen were significantly different by Met235Thr genotype in both women and men (ANOVA, P=0.01 and P=0.03; Figure 3⇓). Women who were homozygous for Met235Thr had higher levels than both heterozygotes and noncarriers (t test, P=0.03 and P=0.02). In men, homozygous Thr235 carriers had higher levels than noncarriers (P=0.02). Interestingly, women had higher mean angiotensinogen levels than men (P=0.01). The Met235Thr mutation accounted for 6% and 5% of the total variation in plasma angiotensinogen concentration in women and men, respectively (Table 4⇓).
Angiotensinogen Concentration and Blood Pressure
Systolic and diastolic blood pressures were positively correlated with plasma angiotensinogen levels in women; angiotensinogen accounted for 3% and 6% of the total variation in systolic and diastolic blood pressures when age was adjusted for (Table 4⇑). No significant correlation was found between angiotensinogen levels and pulse pressure in either gender or with systolic or diastolic blood pressure in men.
Angiotensinogen Concentration and BMI
BMI was positively correlated with plasma angiotensinogen levels and accounted for 3% of the total variation in women but not in men (Table 4⇑). Met235Thr genotype and BMI did not interact on plasma angiotensinogen levels.
New findings from this large-scale study of the general population include the following. (1) The risk of elevated blood pressure in women homozygous for Thr235 compared with noncarriers is elevated by 29% and increases to a 50% elevation in the subgroup of women who are also homozygous for Thr174 (noncarrier of Met174). (2) Double homozygosity for Thr235 and Thr174 versus noncarriers is associated with a 10% increase in the levels of plasma angiotensinogen in both genders; however, systolic and diastolic blood pressures were positively correlated to plasma angiotensinogen levels in women only.
Elevated Blood Pressure
Our results show that Thr235 homozygosity was associated with elevated blood pressure, isolated elevated systolic blood pressure, mildly elevated blood pressure, and use of antihypertensive medication in women, but not in men, and that these associations were strengthened when carriers homozygous for Thr235 in addition were homozygous for Thr174. In an attempt to question these associations, we measured plasma angiotensinogen levels: double homozygosity of Thr235 and Thr174 versus noncarriers was associated with 10% elevated plasma angiotensinogen levels in both genders. However, in accordance with the above mentioned gender-specific relationships. angiotensinogen levels were associated with blood pressure in women only. Nevertheless. although the data suggest that our observation of an association between Thr235 homozygosity and elevated blood pressure is a real observation, rather than a chance finding, it should be emphasized that we were not able to demonstrate an association between genotype and systolic blood pressure, diastolic blood pressure, or pulse pressure in either gender.
Previous studies never looked at the 6 combined genotypes of the Met235Thr/Thr174Met polymorphism separately but detected only either the 3 Met235Thr genotypes or the 3 Thr174Met genotypes.1 2 3 4 5 Therefore, the inconsistency of previous studies9 10 could be explained by the fact that individuals in these studies who were homozygous carriers of the Thr235 mutation represented a mixture of individuals with or without the Met174 mutation. In aggregate, however, previous studies support a 15% to 32% increase in the risk of elevated blood pressure associated with Thr235,9 10 similar to the 29% observed in the present study.
Only a few previous studies of the association between Thr235 and elevated blood pressure stratified for gender.5 19 Possibly due to the inclusion of only 170 and 408 female participants compared with our 5037 female participants, these studies were not able to find positive associations between the Thr235 mutation and elevated blood pressure in women. Nevertheless, gender-specific associations could be plausible, because an estrogen-related factor may mediate the angiotensinogen-associated genetic predisposition to elevated blood pressure.1 In support of this, the Thr235 mutation was significantly more frequent in women with preeclampsia and pregnancy-induced elevated blood pressure than in normotensive pregnant control subjects.20 Although our data seem to suggest that the risk of elevated blood pressure is increased in female Thr235 homozygotes, but not in male Thr235 homozygotes, it should be emphasized that genotype and gender did not interact statistically in the risk of elevated blood pressure.
In our sample, we previously examined the association between mutations in other candidate genes and elevated blood pressure: the ACE insertion/deletion polymorphism was not associated with variation in blood pressure,11 and heterozygosity for lipoprotein lipase deficiency likewise was not associated with blood pressure.12 However, subjects from the general population who were heterozygous for apoB Arg3500Gln had an odds ratio for hypertension of 10 (95% CI 2 to 51).13
Our demonstration of a 10% increase in plasma angiotensinogen levels in individuals who were double homozygous for Thr235 and Thr174 versus noncarriers agrees with previous studies showing a ≈20% increase in angiotensinogen levels in Thr235 homozygous hypertensive individuals versus noncarriers1 and a 13% increase in angiotensinogen levels in Thr235 homozygous normal children versus noncarriers.21 That the absolute mean angiotensinogen levels were higher in women than in men could be due to an estrogen-related factor.1 Positive correlations between plasma levels of angiotensinogen and blood pressure have been demonstrated previously.18 Our findings suggests that this correlation may be gender specific.
The renin-angiotensin system plays a crucial role in salt and water homeostasis and in the maintenance of vascular tone: the stimulation and inhibition of this system raises and lowers blood pressure, respectively. Angiotensinogen is converted by renin into angiotensin I, which is further converted into angiotensin II by ACE. Angiotensin II, which is the end product of the renin-angiotensin system, stimulates renal sodium reabsorption and vasoconstriction and thus raises blood pressure.
Our results imply that the Met235Thr mutation increases angiotensinogen levels in plasma, tending toward higher throughput in the renin-angiotensin system and thus higher levels of angiotensin II leading to higher blood pressure. Because we find that higher angiotensinogen levels are associated with higher blood pressure in women only, it also seems likely that the Thr235 mutation is associated with elevated blood pressure in women but not in men.
In our study, we measured blood pressure only once, and because blood pressure measurements vary considerably, this represents a clear limitation. Although misclassification bias of elevated blood pressure therefore is possible, this is mainly likely in individuals with diastolic and systolic blood pressure of ≈140/90 mm Hg, the diagnostic cutoff limits for elevated blood pressure,15 whereas individuals with very low or very high blood pressure or on antihypertensive medication are less likely to be misclassified. In contrast, when blood pressure is examined as a continuous covariate, misclassification is possible at all levels of blood pressure. This may therefore explain why genotype was associated with the dichotomized variable elevated blood pressure but not with blood pressure as a continuous covariate.
Because we genotyped 9100 individuals, we cannot exclude misclassification of a few individuals in our sample, but because genotype frequencies were in accordance with those predicted by the Hardy-Weinberg equilibrium and because genotyping as well as database entry was scrutinized by 2 different researchers, we believe that genotype misclassification at most is a minor problem in our study.
The possibility that our observation of a Thr235–elevated blood pressure association in women (but not in men) is a chance finding should also be considered. In favor of such an interpretation are the facts that (1) we did not document a similar association in men (or in the 2 genders combined), (2) genotype was not associated with systolic blood pressure, diastolic blood pressure, or pulse pressure in women, (3) the confidence intervals for the odds ratios in women were not far from overlapping 1.0, and (4) we previously used the same population to study other mutations on other end points,11 12 13 14 increasing the likelihood of chance findings. Arguing the interpretation of a chance finding are the following: (1) we observed associations between Thr235 and not only elevated blood pressure but also the use of antihypertensive medication; (2) the association among genotype, angiotensinogen levels, and blood pressure is consistent with a gender-specific association; (3) for other polymorphisms in consideration of other end points, we have on numerous occasions found gender-specific associations13 14 ; and (4) although we previously studied association with blood pressure for other mutations,11 12 13 this is our first study of mutations in which the primary end point is elevated blood pressure, which makes correction for multiple testing less relevant.
Finally, cross-sectional studies such as the present study may be confounded by hidden admixture or stratification and cryptic relatedness between cases that can inflate associations. Although we naturally cannot exclude such potential problems, we would like to point out that (1) we studied a very homogeneous sample drawn at random (after stratification on sex and age) to represent the adult Danish general population, (2) >99% of participants were white and of Danish descent, and (3) the response rate for this study was as high as 58%.11
Despite the limitations mentioned, our data suggest that in the population at large, double homozygosity for Thr235 and Thr174 in the angiotensinogen gene is associated with a 10% increase in angiotensinogen levels and is a risk factor for elevated blood pressure in women but not in men.
This study is supported by The Danish Heart Foundation, The Danish Medical Research Council, University of Copenhagen, The European Organization for the Control of Circulatory Diseases, The Beckett Fund, Manufacturer Frands Køhler Nielsen and Wife’s Grant, and King Christian the Xth Fund. We thank Marianne Lodahl for technical assistance and the participants of The Copenhagen City Heart Study for their willingness to participate.
- Received July 20, 2000.
- Revision received August 2, 2000.
- Accepted September 5, 2000.
Hingorani AD, Sharma P, Jia H, Hopper R, Brown MJ. Blood pressure and the M235T polymorphism of the angiotensinogen gene. Hypertension. 1996;28:907–911.
Kiema TR, Kauma H, Rantala AO, Lilja M, Reunanen A, Kesaniemi YA, Savolainen MJ. Variation at the angiotensin-converting enzyme gene and angiotensinogen gene loci in relation to blood pressure. Hypertension. 1996;28:1070–1075.
Kunz R, Kreutz R, Beige J, Distler A, Sharma AM. Association between the angiotensinogen 235T-variant and essential hypertension in whites: a systematic review and methodological appraisal. Hypertension. 1997;30:1331–1337.
Agerholm-Larsen B, Nordestgaard BG, Steffensen R, Sorensen TI, Jensen, Tybjaerg-Hansen A. ACE gene polymorphism: ischemic heart disease and longevity in 10 150 individuals: a case-referent and retrospective cohort study based on the Copenhagen City Heart Study. Circulation. 1997;95:2358–2367.
Nordestgaard BG, Abildgaard S, Wittrup HH, Steffensen R, Jensen G, Tybjaerg-Hansen A. Heterozygous lipoprotein lipase deficiency: frequency in the general population, effect on plasma lipid levels, and risk of ischemic heart disease. Circulation. 1997;96:1737–1744.
Agerholm-Larsen B, Nordestgaard BG, Steffensen R, Jensen G, Tybjaerg-Hansen A. Elevated HDL cholesterol is a risk factor for ischemic heart disease in white women when caused by a common mutation in cholesteryl ester transfer protein gene. Circulation. 2000;101:1907–1912.
Guidelines Subcommittee. World Health Organization-International Society of Hypertension Guidelines for the Management of Hypertension. J Hypertens. 1999;1999:17:151–183.
Fukamizu A, Takahashi S, Seo MS, Tada M, Tanimoto K, Uehara S, Murakami K. Structure and expression of the human angiotensinogen gene. J Biol Chem. 1990;265:7576–7582.
Armitage P, Berry G. The planning of statistical investigations. In: Statistical Methods in Medical Research, 3rd ed. Oxford, UK: Blackwell; 1994:175–206.
Schunkert H, Hense HW, Gimenez-Roqueplo AP, Stieber J, Keil U, Riegger GA, Jeunemaitre X. The angiotensinogen T235 variant and the use of antihypertensive drugs in a population-based cohort. Hypertension. 1997;29:628–633.
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.
Bloem LJ, Manatunga AK, Tewksbury DA, Pratt JH. The serum angiotensinogen concentration and variants of the angiotensinogen gene in white and black children. J Clin Invest. 1995;95:948–953.