Yonago Acta medica 2001;44:79–83

A Molecular Variant of the Angiotensinogen Gene and Hypertension ina Case-Control Study in Japanese
Takuji Kishimoto, Akihiko Suyama, Yoneatsu Osaki, Tetsuya Miyamoto, Atsusi
Igarashi, Mikizoh Okamoto, Yoichi Kurosawa* and Soji Fukumoto†Department of Hygiene, *Department of Public Health and †Department of Parasitology,
Tottori University Faculty of Medicine, Yonago 683-0826 Japan
In order to examine the distribution of M235T (the substitution of threonine formethionine at position 235 codon) polymorphism genotypes of the angiotensinogen(AGT) gene and the relationship between M235T polymorphism of the AGT gene andhypertension, a descriptive study and a case-control study were performed among
Japanese workers. The subjects were 2042 workers at an occupational site in Shimane
Prefecture in Japan. The database was set up for the workers’ regular health examination in 1998. The M235T polymorphism of the AGT gene for each worker was definedby the mutant allele specific amplification (MASA) method. The rates of M235M (MM),
M235T (MT) and T235T (TT) genotypes were 3.9%, 30.7% and 65.5%, respectively.
The odds ratios of MT and TT against MM for hypertension by univariate analysis
were 0.77 (95% confidence interval (CI) 0.27–2.18) and 0.77 (95% CI 0.28–2.14), respectively. The odds ratios of MT and TT against MM for hypertension, adjusted for bodymass index, fasting blood sugar, drinking habits, cigarette smoking and exercise in alogistic regression model, were 0.90 (95% CI 0.29–2.74) and 0.87 (95% CI 0.30–2.58),
respectively. The data from this study suggests that there may be no relationship between
the M235T polymorphism of the AGT gene and hypertension. Further prospective
studies are needed to resolve this issue.
Key words: angiotensinogen; case-control study; genotype; hypertension; polymorphism

The angiotensinogen (AGT) gene variant,
M235T (mutation to the threonine of the methionine in amino acid codon 235), was first reported in the pathogenesis of essential hypertension by Jeunemaitre et al. (1992). Essential
hypertension is a multiple factorial disease thatis complicated by the interaction of a genetic
factor and an environmental factor (Soubrier etal., 1995). There have been many studies investigating the hypothesis that this polymorphism,
M235T, is one of the genetic factors of essential
hypertension (Hata et al., 1994; Tiila-Riikka etal., 1996; Borecki et al., 1997; Fu-Tien et al.,
1997; Frossard et al., 1998); however, despite
numerous studies the hypothesis remains controversial.
Most of the reports regarding the relationshipbetween M235T and hypertension implemented acase-control study using hospital-based datathat included a relatively small sample size.
Thus, we carried out a descriptive study and acase-control study using population-based datawith a large sample size in order to clarify the
involvement of M235T in hypertension.

Abbreviations: AGT, angiotensinogen; CI, confidence interval; DBP, diastolic blood pressure; MASA, mutant allele specific amplification; MM, genotype M235M; MT, genotype M235T; M235T, mutation to thethreonine of the methionine in amino acid codon 235; OD, odds ratio; PCR, polymerase chain reaction; SBP,
systolic blood pressure; TT, genotype T235T

79

T. Kishimoto et al.

the Korotkoff sound were used as SBP and DBP,
respectively. Measurement of the fasting blood
sugar value was carried out using an enzymatic
method (Hexokinase, G-6-PDH, Wako, Tokyo,
Japan). Drinking habits, cigarette smoking andexercise were assessed using a questionnaire.

Subjects and Methods

Subjects
The research was approved by the ethics committee of Tottori University Faculty of Medicine, and informed consent was obtained from2042 workers in Shimane Prefecture in Japan
who received their regular medical examinationin 1998. The case-control study comprised a
total of 402 individuals selected from the 2042
workers. The case and control individuals werematched by sex and age (difference within 2
years old). The following selection criteria for
hypertensives, borderline and normotensive
subjects were applied: (i) hypertensives, borderline and normotensive blood pressure as defined by systolic blood pressure (SBP) ≥ 160
mmHg and/or diastolic blood pressure (DBP) ≥95 mmHg, 140 mmHg ≤ SBP < 160 mmHg and/or 90 mmHg ≤ DBP < 95 mmHg, and SBP <140 mmHg and DBP < 90 mmHg, respectively;(ii) absence of secondary hypertension; and (iii)
absence of renal disease or renal insufficiency.

Identification of polymorphism

Genome DNA was prepared from white blood
cells with the use of a fully automatic nucleic
acid extractor (MFX-2000, Toyobo Co., Ltd.,
Osaka, Japan). The mutant allele specific amplification (MASA) method was used for the
analysis of the polymorphism (Takeda et al.,1993). Four primers were designed. The normal sense primer and antisense primer were 5'AAGACTGGCTGCTCCCTGAT-3' and 5'GCTGTCCACACTGGCTCCCG-3', respectively. The wild type primer and polymorphism
primer including the mutation part were 5'AAGACTGGCTGCTCCCTGAT-3' and 5'AAGACTGGCTGCTCCCTGAC-3', respectively. Polymerase chain reaction (PCR) was
performed in a TaKaRa PCR Thermal Cycler(Takara Co., Tokyo) with a 10 µL reaction
volume containing 10 mmol/L Tris-HCl (pHVarious measurements and survey of8.3), 50 mmol/L KCl, 25 µmol/L of each dNTP,
smoking history
1.25 µmol/L sense primer, 1 µmol/L antisense
Body mass index (BMI) was calculated by the primer, 2 µmol/L wild type primer, 2 µmol/L
following formula: (weight kg)/(height m)2. polymorphism primer, 0.1 U Ampli-Taq DNA
The blood pressure was measured in the right polymerase (Perkin Elmer, Foster City, CA) andupper arm with a standard sphygmomanometer 1.5 mmol/L MgCl2. The initial denaturation forin a sitting position. The 1st and 5th sound of 3 min at 95˚C was followed by 35 cycles ofdenaturation for 30 s at 95˚C, annealing for 30 s at 65˚C, and extension for
Table 1. Subjects related by angiotensinogen
60 s at 72˚C. PCR products werecodon 235 genotypes by age
electrophoresed in 6% polyacrylamide gels on a Model Cassette ElectroAge
Genotype
Total
phoresis Unit “DPC” (Daiichi Pure
MM
MT
TT
Chemical Co., Ltd., Tokyo) and DNA
10–19
1(100.0)
1 (100.0)
was visualized by ethidium bromide
20–29 13 ( 3.6) 124 ( 34.3) 224 (62.0) 361 (100.0)
staining.
30–39 18 ( 4.0) 134 ( 29.1) 304 (67.0) 454 (100.0)
40–49 21 ( 3.4) 197 ( 32.3) 391 (64.2) 609 (100.0)
50–59 21 ( 4.1) 143 ( 28.1) 345 (67.8) 509 (100.0)
60–69 4 ( 4.5) 22 ( 24.7)
63 (70.8)
89 (100.0)
70–79 2 (10.5)
7 ( 36.8)
10 (52.6)
19 (100.0)
Total 79 ( 3.9) 626 ( 30.7) 1337 (65.5) 2042 (100.0)
The percentage of individual genotypes by age is given inparentheses.

80Angiotensinogen gene and hypertension

Table 2. Subjects related by angiotensinogen codon 235 genotypes by blood pressure
SBP(mmHg)

DBP(mmHg)

Genotype
MT

MM

Total
TT

Normotensive < 140< 90
63 ( 79.7)
500 ( 79.9)
1046 ( 78.2)
1609 ( 78.8)
Borderline≥ 140, < 160≥ 90, < 95
7 ( 8.9)61 ( 9.7)
164 ( 12.3)
232 ( 11.4)
Hypertensive ≥ 160≥ 95
9 ( 11.4)
65 ( 10.4)
127 ( 9.5)201 ( 9.8)
Total
79 (100.0)
626 (100.0)
1337 (100.0)
2042 (100.0)
The percentage of individual normotensive, borderline and hypertensive is given in parentheses.
DBP, diastolic blood pressure; MM, M235M; MT, M235T; TT, T235T; SBP, systolic blood pressure.

genotype and age. The allele frequency of 235T
was 0.808. As shown in Table 2, there was no
significant difference between the normotensive, borderline and hypertensive subjects interms of the proportions of the genotypes. Table
3 shows the distribution of genotype, drinking
habits, cigarette smoking and exercise, and themean values of BMI and fasting blood sugar related to case and control. The mean values of
BMI and fasting blood sugar, and distributionof drinking habits in both case and control
groups were statistically different from eachother. In regard to the odds ratio (OD) for hypertension, the ODs of MT and TT against MM
by univariate analysis were 0.77 [95% confidence interval (CI) 0.27–2.18] and 0.77 (95%
CI 0.28–2.14) (Table 4). These ODs showedstatistically higher trends in BMI, fasting blood

Statistical analysis
SPSS software (version 8.0, SPSS Japan Inc.,Tokyo) was used for all statistical comparisons.
The χ2 statistic was calculated to test the distribution trend of each index by genotype. Relative risks were calculated by the logistic regression analysis method.

Results
The proportions of the genotype found in thisstudy for M235M (MM), M235T (MT) and
T235T (TT) were 3.9%, 30.7% and 65.5%, respectively (Table 1). There was no statisticallysignificant relationship between distribution of

Table 3. Distribution and mean value of various indices by case and control
ControlNormotensive
Genotype

Case
HypertensiveMM
7 ( 4)
9 ( 5)MT
66 ( 33)65 ( 32)TT
128 ( 64)
127 ( 63)
Total
201 (100)
201 (100)
BMI
Mean ± SD
23.2 ± 2.9
24.8 ± 3.3
Blood Sugar Mean ± SD (mg/dL)
102.6 ± 25.8
108.2 ± 22.6
Drinking
Never, sometimes
129 ( 64)
109 ( 54)
Everyday
72 ( 36)92 ( 46)
Total
201 (100)
201 (100)
Smoking
Never smoked or ex-smoker
141 ( 70)
129 ( 64)
Active smoker
60 ( 30)72 ( 37)
Total
201 (100)
201 (100)
Exercise
Many times
18 ( 9)14 ( 7)
Sometimes, never183 ( 91)
187 ( 93)
Total
201 (100)
201 (100)
The percentages are given in parentheses.
BMI, body mass index; MM, M235M; MT, M235T; TT, T235T.

81

Total
16 ( 4)131 ( 33)
255 ( 63)402 (100)24.0 ± 3.2
105.4 ± 24.4
238 ( 59)
164 ( 41)402 (100)
270 ( 67)132 ( 33)402 (100)
32 ( 8)370 ( 92)402 (100)

P value

0.877
0.000
0.021
0.043
0.19
0.447

T. Kishimoto et al.

Table 4. Hypertension risks estimates associated with angiotensinogen M235T gene
polymorphism and risk factors based on a case-control study
Univariate
Odds ratio
95% CI

Multivariate
Odds ratio
95% CI

Genotype (1) (MT versus MM)
0.766
0.269–2.179
0.895
0.293–2.735
Genotype (2) (TT versus MM)
0.772
0.279–2.135
0.873
0.295–2.583
BMI
1.191*
1.112–1.274
1.186*
1.107–1.271
Blood sugar
1.011*
1.001–1.021
1.006
0.997–1.015
Drinking
1.512*
1.012–2.260
1.450
0.941–2.234
Smoking
1.322
0.870–2.008
1.242
0.792–1.947
Exercise
1.337
0.631–2.831
1.289
0.589–2.825* P < 0.05.
BMI, body mass index; CI, confidence interval; MM, M235M; MT, M235T; TT, T235T.

in studies performed in Japan and in other countries (Jeunemaitre et al., 1992; Hata et al., 1994;
Soubrier et al., 1995; Tiila-Riikka et al., 1996;
Borecki et al., 1997; Fu-Tien et al., 1997; Frossard
et al., 1998). Kato et al. (1999) were the first to
cast doubt on this relationship. The different results for the relationship between genotypes andhypertension may be caused by the possibilityof bias in choosing controls, differences between races, regional differences in genetic background, differences in the age of subjects andmultifactors of the examined disease, because
most of the studies were hospital-based casecontrol studies in a small sample size (Singer etal., 1996).
To improve on the drawbacks of hospitalbased case-control studies in a small sample
size, we performed a population-based casecontrol study and obtained no correlations
between genotypes and hypertension. This
study was an improvement being performed ina population-based pattern, because a casecontrol study has many kinds of biases. It isvery difficult to choose a suitable control groupand it was considered necessary to evaluate therelationship by a cohort study which has few
information biases compared to a case-control
study.
In conclusion, there was no correlation between the M235T polymorphism of the AGT
gene and hypertension in a population-based
case-control study corrected for various factors
in Japanese subjects. We consider it necessary
to investigate further by using the method of acohort study.

sugar and drinking habits. When the multiple
risk factors of BMI, fasting blood sugar, drinking habits, cigarette smoking and exercise were
adjusted, the ODs of MT and TT against MM
were 0.90 (95% CI 0.29–2.74) and 0.87 (95%
CI 0.30–2.58), respectively, but these were notsignificant.

Discussion
In the population examined in this study, thefrequency of the wild type MM was the lowest,
while that of the mutant type TT was the highest. There were no significant differences in the
proportions of the genotypes among normotensive, borderline and hypertensive subjects.
The odds ratios of MT and TT against MM for
hypertension analyzed by correction using various factors were not significant, and there were
no correlations (0.90 and 0.87, respectively).
The allele frequency in the population was
higher than that of Caucasians (Jeunemaitre etal., 1992). This agreed with the results reported
in several studies on investigation of the allele
frequency in Japanese subjects (Iwai et al.,
1994; Kamitani et al., 1994; Morise et al., 1995;
Nishiuma et al., 1995; Sato et al., 1997). The
allele frequency was 0.808 in the present study,
and ranged from 0.604 to 0.860 in publishedstudies. This is probably because there are regional differences in the genetic background of
the native Japanese.
The relationship between this genetic polymorphism and hypertension has been suggested
82Angiotensinogen gene and hypertension

Acknowledgments: This research was supported inpart by a Grant-in-Aid (11470096) from the Ministryof Education, Japan.

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Received December 20, 2000; accepted January 10, 2001
Corresponding author: Prof. Takuji Kishimoto83