Endothelial Nitric Oxide Synthase Gene Polymorphism and

Acute Myocardial Infarction

Kiyoshi Hibi, Tomoaki Ishigami, Kouichi Tamura, Shunsaku Mizushima, Nobuo Nyui, Takayuki Fujita,
Hisao Ochiai, Masami Kosuge, Yasujirou Watanabe, Yuzuru Yoshii, Minoru Kihara, Kazuo Kimura,
Masao Ishii, Satoshi Umemura

Abstract—Recently a point mutation of guanine to thymine at nucleotide position 1917 in the endothelial nitric oxide
synthase (eNOS) gene has been reported to be associated with coronary artery spasm. In addition, a significant
association of the 4a/b polymorphism in intron 4 of the eNOS gene with coronary artery disease has been reported.
However, the implications of these polymorphisms with respect to acute myocardial infarction (AMI) remain to be
established. We conducted a case-control study of 226 patients with AMI and 357 healthy gender- and age-matched
control subjects. In the former group, coronary angiograms were evaluated according to angiographic criteria based on
the number of diseased vessels ($75%) and the number of stenotic lesions ($50%). Homozygosity for the Glu-Asp298
polymorphism existed in 5 of 226 patients with AMI (2.2%) but not in any of the 357 control subjects (P5.0085).
However, when we evaluated the coronary angiograms of 226 case patients, there was no difference in the number of
diseased vessels or the number of stenotic lesions between the patients with this homozygote and those without it. By
contrast, there was no evidence of a significant increase in the risk of AMI or the severity of coronary atherosclerosis
among individuals with the a/a genotype of the eNOS4a/b polymorphism. Our results imply that patients who are
homozygous for the Glu-Asp298 polymorphism may be genetically predisposed to AMI; however, this mutation
apparently is not related to the severity of coronary atherosclerosis. Further studies are needed to confirm our results and
characterize the molecular mechanisms by which eNOS is involved in susceptibility to AMI. (Hypertension.
1998;32:521-526.)
Key Words: endothelium-derived relaxing factor n genes n myocardial infarction n atherosclerosis n angiography

S ince the identification of nitric oxide (NO) as an impor-

tant endothelium-derived relaxing factor, there has been
an explosion of new information on the physiological and
pathophysiological roles of NO. NO is synthesized from the
amino acid L-arginine by a family of enzymes, referred to as
NO synthase (NOS). Three distinct isoforms of NOS have
been identified to date.1 The inducible NOS is expressed in
vessel walls and macrophages by certain cytokines and
endotoxin lipopolysaccharides in pathological conditions.2
The constitutive neuronal NOS is expressed in the central and
peripheral nervous systems as well as in macula densa of
kidney. It plays important roles in physiological3 and patho-
physiological4 conditions. The constitutive endothelial NO
synthase (eNOS) is expressed in the endothelium, where it
produces NO from L-arginine. NO diffuses from the endo-
thelium to the vascular smooth muscle cells, where it in-
creases the concentration of cGMP by stimulating soluble
guanylate cyclase, leading to vascular relaxation.1
Several studies suggest that the basal release of NO by the
endothelium contributes to basal vascular tone5,6 and regu-
lates blood flow and blood pressure. Recent reports have
suggested a possible role of NO in the pathogenesis of
coronary spasm.7 NO also inhibits the proliferation of smooth
muscle cells.8 Furthermore, NO protects against platelet
aggregation in vitro9 and in vivo10 and inhibits platelet
adhesion to vascular endothelium.11 In addition, NO inhibits
leukocyte adhesion to endothelium.12 All of these processes
are important events during atherogenesis. Dysfunction of
this important mechanism may promote atherogenesis by
exposing the arterial wall to the direct vasoconstrictor effects
of factors that mediate vasospasm and increase the risk of
thrombosis, leading to acute myocardial infarction (AMI).
Among the reported polymorphisms of the eNOS gene, a
significant association of the 4a/b polymorphism in intron 4
of the eNOS gene with coronary artery disease (CAD) has
been reported.13 In addition, recent preliminary data indicate
that the Glu-Asp298 polymorphism in exon 7 of the eNOS
gene is associated with coronary spasm,14 although the
implications of these polymorphisms with respect to AMI
remain to be established.

Received February 20, 1998; first decision March 12, 1998; revision accepted April 13, 1998.
From the Second Department of Internal Medicine (K.H., S.U., T.I., K.T., N.N., T.F., H.O., M.K., Y.W., M.K., K.K., M.I.) and the Department of
Public Health (S.M.), Yokohama City University School of Medicine; and the Department of Cardiology, Saiseikai Yokohama City Nanbu Hospital
(Y.Y.), Yokohama, Kanagawa, Japan.
Correspondence to Satoshi Umemura, MD, Second Department of Internal Medicine, Yokohama City University School of Medicine, 3–9, Fukuura,
Kanazawa-Ku, Yokohama 236, Japan. E-mail hibikiyo@yellow.med.yokohama-cu.ac.jp
© 1998 American Heart Association, Inc.

521
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522 eNOS Gene and Myocardial Infarction

Methods
Study Population
The Glu-Asp298 genotype of the eNOS gene was determined in 226
patients at first presentation of AMI and in 482 control subjects. We
also determined the eNOS4a/b polymorphism in this population.
Adult Japanese patients who satisfied the World Health Organization
criteria for myocardial infarction15 were eligible for the study (185
men, mean age 61.5 years; 41 women, mean age 71.3 years). All
enrolled patients had angiographically documented coronary artery
narrowing, exceeding 75% luminal diameter, and underwent percu-
taneous transluminal coronary angioplasty, thrombolytic therapy, or
both.
A total of 482 control subjects (285 men, mean age 60.0 years; 197
women, mean age 60.5 years) who came to the Health Checkup Center
of Nanasawa Rehabilitation Hospital for their regular checkup were
enrolled in the study. They were genetically unrelated and were living in
Kanagawa, Japan. The control individuals had no symptoms of CAD
and had normal ECG results. All patients and control subjects gave their
informed consent to participate in this study. Because the gender
distributions were different between the patient and control groups
(P,0.0001) and the female control subjects were younger than female
patients (P,0.0001), we recruited 357 control subjects who were
matched to the case patients for gender and age for case-control
comparisons (Tables 1 through 4).

Genotyping A, Representative polyacrylamide gel stained with ethidium bro-

DNA was extracted from peripheral leukocytes. For detection of
Glu-Asp298 polymorphism of the eNOS gene, we used primer pairs
to amplify a part of the eNOS gene containing exon 7 by polymerase
chain reaction (PCR). Primer pairs for PCR were as follows: sense
59-TCC CTG AGG AGG GCA TGA GGC T-39 and antisense
59-TGA GGG TCA CAC AGG TTC CT-39. Samples were amplified
for 30 cycles, consisting of denaturation at 94°C for 1 minute,
annealing at 61°C for 1 minute, and extension at 72°C for 1 minute.
The resulting 457-bp amplification product was incubated at 37°C
for at least 20 hours with 8 U of the restriction enzyme BanII (New
England Biolabs Inc). The amplified fragments were digested by
BanII into smaller fragments (137 and 320 bp). In the case of a G to
T substitution at position 1917 of the eNOS gene, a BanII restriction
site is lost. The restricted fragments were separated on 8% polyacryl-
amide gels with ethidium bromide staining (Figure, A). The PCR
isoform typing results were checked in 10% of the samples by the
direct sequencing of amplified DNA. All fragments that were not
cleaved were also confirmed by direct sequencing (Figure, B) to
avoid mistyping. In brief, after removal of dNTP and primers by
columns, each sample was subjected to cycle sequencing using a dye
terminator cycle-sequencing kit (Perkin-Elmer) according to the
supplier’s instructions. Electrophoresis was performed with the use
of a DNA sequencer (model 310, version 2.1.1, Perkin-Elmer).
The eNOS4a/b gene polymorphism was detected by the method of
Wang et al13 with minor modifications. Briefly, the DNA samples
were subjected to amplification by PCR using primer pairs that flank
the region of the 27-bp direct repeat in intron 4 of the eNOS gene.
Primer pairs for PCR were as follows: sense 59-AGG CCC TAT
GGT AGT GCC TTT-39 and antisense 59-TCT CTT AGT GCT GTG
GTC AC-39. The amplified fragments were separated on 4% poly-
acrylamide gels with ethidium bromide staining.
Clinical and Laboratory Measurements
All participants completed a standard questionnaire on personal
medical history, family history, and smoking habits. The subject was
considered to be a current daily smoker if she or he had regularly
smoked at least 5 cigarettes per day for at least the previous 3 months
or had stopped smoking for ,1 year. Subjects who had stopped
smoking for at least 1 year were classified as ex-smokers. Thus,
smokers were classified as nonsmokers, current smokers, or ex-
smokers.16 Hypertensive patients with AMI were defined as those in
whom hypertension had been diagnosed previously or who had a
history of antihypertensive therapy. Hypertension in control subjects
was arbitrarily defined as systolic pressure $140 mm Hg and/or
mide and photographed under UV transillumination. PCR prod-
ucts were amplified from human genomic DNA and digested by
restriction enzyme BanII. Homozygotes with a G to T substitu-
tion at nucleotide position 1917 (T/T) showed a single band at
457 bp. Homozygotes with G at this position (G/G) showed 2
bands at 320 bp and 137 bp. Heterozygotes for this mutation
(G/T) showed 3 bands at 457 bp, 320 bp, and 137 bp. B, eNOS
exon 7 was amplified from genomic DNA of healthy control sub-
jects (left) and patients with AMI (right) and sequenced directly,
revealing a homozygous G to T transition at nucleotide position
1917.
diastolic pressure $90 mm Hg.17 Diabetes mellitus was defined as a
prior diagnosis of the disease, a history of antidiabetic medication, or
a plasma fasting glucose level of $7.8 mmol/L on 2 or more
occasions. Serum total cholesterol (Chol), triglyceride, HDL choles-
terol (HDL-C), and glucose levels in blood specimens obtained in the
morning after a 12-hour fast were measured by an automated
enzymatic assay. Blood specimens were taken from the patients with
AMI at least 1 week after infarction.
Angiographic Criteria
The severity of CAD was evaluated from coronary angiograms on
the basis of the number of diseased vessels with stenosis of $75%
and the number of lesions with stenosis $50% as reported
previously.18
Statistical Analysis
All statistical analyses were conducted with use of the SPSS
statistical package, version 6.1. Data are expressed as mean6SEM.
The frequencies of the alleles and genotypes were compared between
patient and control groups by the x2 test when appropriate. The
distributions of genotype frequencies in control subjects, patients, or
the overall study group were compared by the x2 test with the values
predicted by the Hardy-Weinberg equilibrium model. Given a
recessive model of inheritance, Fisher’s exact test was used to
compare the distribution of 2 genotype subgroups by construction of
232 contingency tables19 when the smallest of the 4 expected
numbers was ,5. The distributions of gender (male or female),
smoking, presence of hypertension, and diabetes mellitus among the
3 genotypes of patients were analyzed by construction of 332
contingency tables and x2 analysis. One-way ANOVA was used to
analyze the relations between genotypes and the general character-
istics or severity seen on coronary angiograms in the patient group.

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 Hibi et al September 1998 523

TABLE 1. Characteristics of Patients and Control Subjects TABLE 3. eNOS4a/b Genotypes and Frequency of Alleles
Patients Controls Genotypes Control AMI
Variable (n5226) (n5357) P and Alleles (n5357) (n5226)
Age, y 63.360.7 62.860.7 0.666 Genotypes
M/F 185/41 285/72 0.547 b/b 284 (79.6%) 174 (77.0%)
BMI, kg/m2 23.860.2 23.660.2 0.479 b/a 68 (19.0%) 48 (21.2%)
Smokers, non/ex-/current 42/19/165 161/75/121 ,0.0001* a/a 5 (1.4%) 4 (1.8%)
Chol/HDL-C 5.2760.15 4.3860.13 ,0.0001* Recessive model*
Uric acid, mmol/L 383.9617.8 344.566.5 0.058 b/b1b/a 352 (98.6%) 222 (98.2%)
Hypertension, with/without 131/95 160/197 0.002* aa 5 (1.4%) 4 (1.8%)
Diabetes mellitus, with/without 73/153 54/302 ,0.0001* Alleles†
*P,0.05. Student’s t test or x2 (232 or 233 contingency table) analysis 4b 0.891 0.876
was used to assess various phenotypic values of frequency. 4a 0.109 0.124
*P50.74; †x251.09, P50.30.
Because the number of stenoses was not distributed normally,
statistical tests were performed on square root–transformed stenoses. subjects from 3 different families and confirmed that the
Glu-Asp298 polymorphism of the eNOS gene is inherited in
Results
a simple mendelian fashion (data not shown). Representative
Clinical Variables in Patients With AMI and genotyping results for subjects with each genotype are shown
Control Subjects in panel A of the Figure. The genotype and allele frequency
Table 1 compares clinical characteristics between the patients of the polymorphism in patients and control subjects are
with AMI and the control subjects. As we recruited control shown in Table 2. Genotype frequencies did not deviate from
subjects who were matched to case patients for gender and the Hardy-Weinberg equilibrium in control subjects, patients,
age, these variables were not different between patients with or the overall study group. The allele frequencies of these
AMI and control subjects. As expected, in a study of eNOS genes were similar in these groups. However, when we
myocardial infarction, patients with AMI had higher values of assumed a recessive model of inheritance (ie, T/T versus G/T
Chol/HDL-C and higher prevalence of hypertension and and G/G combined), the frequency of T/T homozygotes in
diabetes mellitus. There was a higher frequency of current patients with AMI was significantly higher than that in
smokers among patients with AMI, whereas the frequency of healthy control subjects (5 of 226 patients and none of 357
past smokers was lower than in the control group. The controls, respectively; P50.0085).
frequency of nonsmokers was significantly lower among
patients with AMI than control subjects ( x 2 547.0, 4a/b Polymorphism of eNOS Gene
P,0.0001). By contrast, the genotype distribution and allele frequency of
4a/b polymorphism were similar between patients with AMI
Distributions of Genotype and Allele Frequencies and healthy control subjects (Table 3). When we assumed a
in eNOS Gene Variants in Patients With AMI and recessive model of inheritance (ie, a/a versus b/a and b/b
Control Subjects combined), the frequency of a/a homozygotes was not differ-
Glu-Asp298 Polymorphism of eNOS Gene ent between patients with AMI and control subjects (4 of 226
A total of 226 patients with AMI and 357 healthy Japanese patients and 5 of 399 controls; P50.74).
subjects were enrolled in the study. We also examined 18
Genotype Frequencies of Glu-Asp298
Polymorphism in Prespecified Study Subgroups
TABLE 2. Glu-Asp298 Genotypes and Frequency of Alleles
We analyzed the data by stratifying patients with AMI and
Genotypes Control AMI control subjects according to age, gender, and other estab-
and Alleles (n5357) (n5226) lished risk factors for CAD (Table 4). In the subgroups of age
Genotypes .60 years, male, body mass index (BMI) #25 kg/m2, without
GG 295 (82.6%) 189 (83.6%) hypertension, and without diabetes mellitus, the frequencies
GT 62 (17.4%) 32 (14.2%) of T/T homozygotes in patients with AMI were still signifi-
TT 0 (0.0%) 5 (2.2%) cantly higher than those in control subjects.
Recessive model*
Relationship Between Demographic Characteristics
GG1GT 357 (100%) 221 (97.8%)
and Severity of CAD and Genotypes in Patients
TT 0 (0.0%) 5 (2.2%) With AMI
Alleles†
Glu-Asp298 Polymorphism of eNOS Gene
G (Glu) 0.913 0.907 When clinical and laboratory values were compared among
T (Asp) 0.087 0.093 genotypes in the patients with AMI, no significant difference
*P50.0085; †x 50.22, P50.64.
2
was noted (Table 5). When the severity of CAD determined

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524 eNOS Gene and Myocardial Infarction

TABLE 4. Genotype Frequencies of Glu-Asp298 Polymorphism diseased vessels or the number of stenotic lesions (vessel: b/b
in Prespecified Study Subgroups 1.460.1, b/a 1.360.1, a/a 1.060.0, P50.29; stenoses: b/b
Genotype (GG1GT/TT) 2.060.1, b/a 1.860.1, a/a 1.760.3, P50.33).

Subgroups Patients Controls P Discussion

Age, y We performed a case-control study of the endothelial NOS
#60 (n5218) 92/1 125/0 0.427 locus and found a significant association between homozy-
.60 (n5365) 129/4 232/0 0.017* gotes for Glu-Asp298 polymorphism and the occurrence of
Gender
AMI. Lack of an increased risk of AMI in the eNOS GT
heterozygotes suggests that the risk of AMI posed by the
Male (n5470) 181/4 285/0 0.024*
eNOS T allele is not dominantly expressed and that the
Female (n5113) 40/1 72/0 0.362
increased risk is confined to eNOS TT homozygotes. To our
BMI, kg/m2 knowledge, this is the first study to implicate Glu-Asp298
#25 (n5391) 147/4 240/0 0.022* polymorphism of the eNOS gene as a genetic risk factor for
.25 (n5192) 74/1 117/0 0.391 AMI. Although Bonnardeaux et al20 reported no association
Smoking between the eNOS gene and essential hypertension, the
Current or ex-smoker 180/4 196/0 0.054 results of our study and a recent investigation by Wang et al13
Nonsmoker 41/1 161/0 0.207 suggest that the eNOS gene is related to CAD.
Chol/HDL-C Although patients with AMI had higher values of Chol/
HDL-C and higher prevalence of hypertension, diabetes
,4.5 (n5304) 78/1 255/0 0.260
mellitus, and smoking status (both current and ex-smokers)
$4.5 (n5279) 143/4 132/0 0.125
than age- and gender-matched control subjects (Table 1), no
Hypertension evidence of increased frequency of eNOS TT homozygotes
With (n5291) 129/2 160/0 0.202 was found in high-risk subgroups in analyses stratified by
Without (n5292) 92/3 197/0 0.034* well-established risk factors for CAD such as BMI, smoking
No diabetes mellitus (n5455) 148/5 302/0 0.004* status, Chol/HDL-C, hypertension, and diabetes mellitus
*P,0.05. (Table 4). Thus, it is not likely that our findings were due to
selection bias.
In addition to established risk factors, genetic risk factors
by coronary angiogram was compared among genotypes, may have important roles in the pathogenesis of coronary
there were no differences in the number of diseased vessels or atherosclerosis. Identification of these genetic risk factors is
the number of stenotic lesions (Table 5). expected to enhance our understanding of the molecular basis
for atherosclerosis. Case-control studies can detect weak
4a/b Polymorphism of eNOS Gene susceptibility genes in polygenic diseases such as CAD.
Similarly, there were no differences among genotypes in Using this approach, we and others have identified gene
clinical and laboratory values (data not shown). There also polymorphisms, including the M235T variant of angioten-
were no differences among genotypes in the number of sinogen,21 the DD genotype of the angiotensin-converting

TABLE 5. General Characteristics and Severity of Coronary Atherosclerosis of

Patients in Each Glu-Asp298 Genotype Subgroup
Genotype

GG GT TT
Variable (n5189) (n532) (n55) P
Age, y 63.460.8 62.461.9 64.666.0 0.87
Gender, M/F 32/157 8/24 1/4 0.54
BMI, kg/m2 23.860.3 23.860.5 22.761.6 0.76
Nonsmoker/current and ex-smoker 51/138 11/21 1/4 0.58
Hypertension, with/without 106/83 23/9 2/3 0.18
Diabetes mellitus, with/without 63/126 10/22 0/5 0.29
Total cholesterol, mmol/L 4.9460.07 5.1560.19 4.5460.31 0.33
Triglyceride, mmol/L 1.2360.05 1.3560.12 1.3860.25 0.55
HDL-C, mmol/L 1.0360.02 1.1560.42 1.0160.23 0.18
Vessels 1.460.1 1.560.1 1.260.2 0.39
Stenosis 1.960.1 2.160.1 1.860.3 0.43
Values are presented as mean6SEM. Vessels indicates number of diseased vessels; Stenosis,
square root of number of stenoses.

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enzyme gene,22 and the E4 allele of the apolipoprotein E
gene,23 as genetic risk factors for coronary atherosclerosis. In
the present study, we report for the first time that a Glu-
Asp298 polymorphism of the eNOS gene is also a genetic
risk factor for AMI. However, since the TT genotype existed
in only 5 of 226 patients (2.2%) with AMI, this genotype can
explain only a small part of genetic susceptibility to AMI.
Studies indicate that AMI results from 2 main processes:
coronary atherosclerosis and the formation of a platelet
aggregate at the site of a ruptured coronary atherosclerotic
plaque. Coronary artery spasm may also play a part in the
pathogenesis of AMI and sudden death. Our study provides
no information about mechanisms by which Glu-Asp298
polymorphism of the eNOS gene predisposes patients to
AMI. However, a recent study showed that NG-nitro-L-
arginine methyl ester–induced chronic inhibition of NO
production accelerated neointima formation and impaired
endothelial function in hypercholesterolemic rabbits.24 An-
other group reported that chronic administration of
L-arginine, the precursor of NO, improved endothelium-
dependent vasorelaxation in a similar animal model,25 indi-
cating that continuous release of NO by endothelium inhibits
the progression of atherosclerosis. NO plays important roles
in inhibiting platelet activation and adhesion to the endothe-
lium10,11 as well as monocyte adhesion. In addition, a recent
report suggests that there is a deficiency of both basal and
stimulated NO activity in patients with coronary spastic
angina, indicating pivotal roles of NO in the pathogenesis of
coronary artery spasm,7 which is now considered an early
stage of coronary atherosclerosis.26 Although the mechanism
by which Glu-Asp298 polymorphism confers susceptibility to
AMI is not clear, this polymorphism is probably not athero-
genic, since the severity of coronary atherosclerosis, as
assessed on the basis of coronary angiograms, was similar
among the different genotypes. This is consistent with the
recent report by Quyyumi et al,27 who showed no correlation
between the angiographic severity of coronary atherosclerosis
and the magnitude of depression in basal NO activity.
A recent report by Wang et al13 showed an association
between the eNOS4a/a genotype and CAD. They also found
that the eNOS4a/a genotype was associated with increased
severity of coronary atherosclerosis, which is smoking de-
pendent. In the present study, the genotype distribution of
4a/b polymorphism was similar in patients with AMI and
healthy control subjects, and the severity of coronary athero-
sclerosis did not differ according to genotype in the patients
with AMI, even when we analyzed the data by stratifying
patients according to smoking status (nonsmokers, light
current or ex-smokers, medium current or ex-smokers, or
heavy current or ex-smokers; data not shown). One explana-
tion of the discrepancy between our study and the previous
one may be patient selection. Wang et al studied white
patients with CAD who were referred to their hospital,
whereas our patients were Japanese survivors of AMI who
underwent coronary angiography at our hospital. Alterna-
tively, the small sample size of patients with AMI may also
explain the differences in the results of these studies.
Our study has several limitations. The first is the lack of
functional studies. Whether the Glu-Asp298 polymorphism
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Hibi et al September 1998 525
functionally underlies a mechanism leading to AMI should be
determined. Second, the T allele of the Glu-Asp298 poly-
morphism and the 4a allele of the eNOS4a/b polymorphism
both have an estimated frequency of only 0.1, and the
association between Glu-Asp298 polymorphism and AMI is
based on only 5 individuals who were homozygous for this
polymorphism. Furthermore, this association is only valid
under the presumption of a recessive gene effect. Thus, a
larger sample should be examined to confirm the relation
between these polymorphisms and AMI. Third, because our
results are limited to the subgroup of survivors of AMI but
not to the entire group of patients with CAD, these observa-
tions need further confirmation using prospective study de-
sign and in other subgroups of patients with CAD.
In summary, we have identified a new genetic risk factor for
AMI. Our results imply that homozygosity for the Glu-Asp298
polymorphism of the eNOS gene may be involved in predispo-
sition to AMI. However, this polymorphism can explain only a
small part of genetic susceptibility to AMI. Further studies are
needed to characterize the molecular mechanisms by which
eNOS is involved in susceptibility to AMI.
Acknowledgments
We thank Mayumi Iwaki for her technical assistance. We also thank
Dr Hiroshi Umeda, Department of Urology, Dokkyou University
School of Medicine, Tochigi, Japan, for his help.
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Endothelial Nitric Oxide Synthase Gene Polymorphism and Acute Myocardial Infarction
Kiyoshi Hibi, Tomoaki Ishigami, Kouichi Tamura, Shunsaku Mizushima, Nobuo Nyui,
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Kihara, Kazuo Kimura, Masao Ishii and Satoshi Umemura

Hypertension. 1998;32:521-526
doi: 10.1161/01.HYP.32.3.521
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