Angiotensinogen Polymorphisms and Elevated Blood
Pressure in the General Population
The Copenhagen City Heart Study
Amar A. Sethi, Børge G. Nordestgaard, Birgit Agerholm-Larsen, Erik Frandsen,
Gorm Jensen, Anne Tybjærg-Hansen
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Abstract—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. (Hypertension. 2001;37:875-881.)
Key Words: angiotensinogen 䡲 blood pressure 䡲 genetics 䡲 hypertension, genetic 䡲 polymorphism
I
n 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 – 8 later
reports; however, all of these studies1– 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.
Methods
Subjects
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.11–14
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.
Received July 20, 2000; first decision August 2, 2000; revision accepted September 5, 2000.
From the Department of Clinical Biochemistry (A.A.S., B.G.N., B.A.-L.), Herlev University Hospital, Herlev, Denmark; The Copenhagen City Heart
Study (B.G.N., G.J., A.T.-H.), Bispebjerg University Hospital, Bispebjerg, Denmark; Department of Clinical Physiology and Nuclear Medicine (E.F.),
Glostrup University Hospital, Glostrup, Denmark; and Department of Clinical Biochemistry (A.T.H.), Rigshospitalet, Copenhagen University Hospital,
Copenhagen, Denmark.
Correspondence to Anne Tybjærg-Hansen, MD, DMSc, Department of Clinical Biochemistry KB3011, Rigshospitalet, Copenhagen University
Hospital, Blegdamsvej 9, DK-2100 Copenhagen Ø, Denmark. E-mail [email protected]
© 2001 American Heart Association, Inc.
Hypertension is available at http://www.hypertensionaha.org
875
876
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March 2001
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Elevated Blood Pressure
Statistical Analyses
Elevated blood pressure was defined as systolic blood pressure of
ⱖ140 mm Hg and/or diastolic blood pressure of ⱖ90 mm Hg,15 or
treatment with antihypertensive medication. Isolated elevated systolic blood pressure was defined as systolic blood pressure of
ⱖ140 mm Hg and diastolic blood pressure of ⱕ90 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 (ⱖ180/110 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.
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.
DNA Analyses
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.
Angiotensinogen Levels
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 ␮mol/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.
Other Analyses
Cholesterol was determined enzymatically (CHOD-PAP; Boehringer
Mannheim). Body mass index (BMI) was calculated as weight
divided by height squared (kg/m2).
Results
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 – 6,8,18 The
Met235Thr and Thr174Met mutations were in linkage disequilibrium (␹2⫽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 geno-
Sethi et al
TABLE 1.
Angiotensinogen Gene and Blood Pressure
877
Characteristics of Individuals Sampled From the General Population
Women
Treated*
(n⫽1179)
Age, y
Men
Untreated
(n⫽3858)
Treated*
(n⫽795)
Untreated
(n⫽3268)
68⫾0.3
56⫾0.3
68⫾0.4
54⫾0.3
Systolic blood
pressure, mm Hg
150⫾0.7
133⫾0.4
151⫾0.8
138⫾0.4
Diastolic blood
pressure, mm Hg
86⫾0.4
81⫾0.2
88⫾0.5
86⫾0.2
52⫾0.3
Pulse pressure, mm Hg
64⫾0.6
52⫾0.3
63⫾0.7
Body mass index, kg/m2
27⫾0.2
25⫾0.1
27⫾0.1
26⫾0.1
Cholesterol, mmol/L
6.7⫾0.04
6.2⫾0.02
6.2⫾0.04
5.9⫾0.02
Alcohol consumption, g/wk
57⫾3
74⫾2
150⫾5
168⫾3
Physically inactive, %
82
68
71
56
Elevated blood pressure, %
81
41
83
53
Mild
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䡠䡠䡠
25
䡠䡠䡠
33
Moderate
䡠䡠䡠
11
䡠䡠䡠
14
Severe
䡠䡠䡠
5
䡠䡠䡠
6
䡠䡠䡠
16
䡠䡠䡠
17
Isolated elevated systolic blood
pressure, %
Diabetes mellitus, %
Smokers, %
6
2
11
3
40
48
45
54
Values are given as mean⫾SEM or percent. Numbers may vary slightly according to availability of
data for individual covariates.
*Antihypertensive medication or medication for angina pectoris, heart failure, or cardiac
arrythmias.
type 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 ⱖ2 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 ⱖ2
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
TABLE 2. Relative Frequencies of Met235Thr and Thr174Met
in the Angiotensinogen Gene in 9100 Individuals Sampled From
the General Population
235 MM
235 MT
235 TT
174 TT
0.35
0.34
0.08
174 TM
0
0.15*
0.07
174 MM
0
0
0.01
*Mutations are always on the same allele.
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
878
Hypertension
March 2001
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Figure 2. Diastolic blood pressure by angiotensinogen genotype: mean levels and SEMs as a function of Met235Thr (left),
Thr174Met (middle), and Met235Thr/Thr174Met (right) genotypes by ANOVA in men and women. Individuals on medications
known to affect blood pressure (ie, antihypertensive medication
or medication for angina pectoris, heart failure, or cardiac arrhythmias; 1179 women and 795 men) were excluded from the
analyses. Results for systolic blood pressure and pulse pressure
were similar but not shown.
Figure 1. Risk of elevated blood pressure by angiotensinogen
genotype: odds ratios (95% CI) as a function of Met235Thr
(top), Thr174Met (middle), and Met235Thr/Thr174Met genotypes
(bottom) by multifactorial logistic regression analysis adjusted
for age, BMI, diabetes mellitus, smoking, cholesterol, alcohol
consumption, antihypertensive drug treatment, and physical
activity and in women in addition for menopausal status and
hormonal replacement therapy. When only age was allowed for,
the results were similar.
(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; angio-
tensinogen 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.
TABLE 3. P Values for Tests of Interaction on Elevated Blood Pressure Between
Genotype and Factors Known to Affect Blood Pressure
Women (n⫽4843)
Men (n⫽3968)
235
174
235/174
235
174
235/174
Gender
0.73
0.82
0.92
0.73
0.82
0.92
Age*
0.50
0.95
0.59
0.77
0.03
0.11
Body mass index*
0.24
0.21
0.36
0.17
0.12
0.35
Diabetes mellitus
0.60
0.96
0.69
0.12
0.91
0.39
Smoking
0.44
0.24
0.53
0.47
0.32
0.66
Cholesterol*
0.11
0.09
0.05
0.14
0.33
0.17
Alcohol consumption*
0.55
0.51
0.50
0.82
0.25
0.72
0.17
0.03
0.12
Physical activity
0.33
0.70
0.61
Menopausal status and HRT
0.95
0.31
0.83
䡠䡠䡠
䡠䡠䡠
䡠䡠䡠
235 indicates homozygosity, heterozygosity, or noncarrier status for 235T; 174, homozygosity,
heterozygosity, or noncarrier status for 174M; 235/174, the 6 possible genotype combinations; HRT,
hormonal replacement therapy.
P values are not corrected for multiple comparisons. Interaction between genotype and mentioned
risk factors on elevated blood pressure was explored with logistic regression models, including age
(tertiles), genotype, the risk factor in question, and an interaction term of the 2 latter factors. The
likelihood ratio test between the complete and reduced model determined statistical significance.
*Given in tertiles.
Sethi et al
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Figure 3. Plasma angiotensinogen levels adjusted for age by
Met235Thr genotypes in women and men (left and right); individuals on medications known to affect blood pressure or on
estrogen or other hormones and individuals with suspected liver
disease were excluded from these analyses. Results of ANOVA
(top) and post hoc t tests (bottom) are shown. Conversion factor
to nmol/L is 0.772.
Discussion
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
TABLE 4. Correlation Between Plasma Angiotensinogen Levels
and M235T Genotype, Blood Pressure, or Body Mass Index
Women (n⫽150)
r
r2
P
0.01
Systolic blood pressure
䡠䡠䡠
0.17
0.06
0.03
Diastolic blood pressure
0.25
Pulse pressure
Body mass index
Met235Thr
Men (n⫽150)
r
r2
P
0.03
0.04
䡠䡠䡠
0.11
0.05
0.01
0.16
0.06
0.002
0.11
0.01
0.17
0.01
0.00
0.88
0.07
0.01
0.39
0.17
0.03
0.05
0.00
0.00
0.87
Met235Thr indicates homozygosity, heterozygosity, or noncarrier status of
Thr235; all included individuals were homozygous for Thr174.
Angiotensinogen levels, blood pressure, and body mass index were age
adjusted. Systolic blood pressure and pulse pressure were transformed
logarithmically (log10) before statistical tests.
Angiotensinogen Gene and Blood Pressure
879
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 genderspecific 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.1–5 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
Angiotensinogen Levels
Our demonstration of a 10% increase in plasma angiotensinogen levels in individuals who were double homozygous
880
Hypertension
March 2001
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.
Mechanism
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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.
Study Limitations
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,11–14
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,11–13 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
Conclusions
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.
Acknowledgments
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.
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Amar A. Sethi, Børge G. Nordestgaard, Birgit Agerholm-Larsen, Erik Frandsen, Gorm Jensen
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Hypertension. 2001;37:875-881
doi: 10.1161/01.HYP.37.3.875
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