a Division of Cardiology, Okazaki City Hospital, Okazaki, Japan
b Department of Cardiology, Nagoya University Graduate School of Medicine, Nagoya, Japan
c Department of Clinical Pathophysiology, Nagoya University Graduate School of Medicine, Nagoya, Japan
d Department of Human Functional Genomics, Life Science Research Centre, Mie University, 1515 Kamihama, Tsu, Mie 514-8507, Japan
e Division of Cardiology, Kosei Hospital, Anjo, Japan
* Corresponding author. Tel.: +81-59-231-5387; fax: +81-59-231-5388
E-mail address: yamada{at}gene.mie-u.ac.jp
Received 28 October 2003; revised 16 February 2004; accepted 19 February 2004 See page 900 for the editorial comment on this article1
Abstract
Aims The aim of the study was to identify genes that confer susceptibility to coronary artery spasm and clarify the interaction between genetic and environmental factors in this condition.
Methods and results The study population comprised 2188 Japanese individuals, including 593 subjects with coronary artery spasm (453 men, 140 women) and 1595 controls (762 men, 833 women). The genotypes for 35 polymorphisms of 29 candidate genes were determined with an allele-specific DNA primerprobe assay. Multivariable logistic regression analysis adjusted for age, body mass index, and the prevalence of smoking, hypertension, diabetes mellitus, hypercholesterolaemia, and hyperuricaemia revealed a significant association with coronary artery spasm of one polymorphism (242CT in the NADH/NADPH oxidase p22 phox gene) in men and two polymorphisms (1171/5A
6A in the stromelysin-1 gene and 634C
G in the interleukin-6 gene) in women. A stepwise forward selection procedure revealed that smoking was the most important risk factor for coronary artery spasm and that the effects of these polymorphisms on this condition were statistically independent of smoking.
Conclusion The NADH/NADPH oxidase p22 phox gene is a susceptibility locus for coronary artery spasm in men, and the stromelysin-1 and interleukin-6 genes are susceptibility loci in women.
Key Words: Coronary artery spasm Vasospastic angina Myocardial infarction Genetics Polymorphism Coronary risk factor
Introduction
Coronary artery spasm plays an important role in the aetiology of vasospastic angina and acute coronary syndrome.1 Although calcium-channel blockers, such as diltiazem and nitrates, are effective in its treatment, the molecular mechanisms that underlie this condition remain unclear. We previously showed that smoking is a major risk factor for vasospastic angina without significant coronary artery narrowing.2 The prevalence of coronary artery spasm was also shown to be higher among Japanese individuals than in Italian Caucasians.3 This racial difference could not be explained by conventional risk factors because the Japanese subjects had lower serum concentrations of cholesterol and triglycerides and a similar prevalence of hypertension and smoking compared with the Caucasians.3 These observations suggest that genetic factors and geneenvironment interactions may contribute to the pathophysiology of coronary artery spasm. Genetic epidemiological studies have suggested that several genetic variants, including those of the genes for endothelial nitric oxide synthase,4,5 angiotensin-converting enzyme,6 angiotensin II receptor type 1,7 and paraoxonase,8 increase the risk for coronary artery spasm, but the genes that contribute to genetic susceptibility to this condition remain to be identified definitively.
We made a large-scale association study for 35 polymorphisms of 29 candidate genes and coronary artery spasm. The purpose of this study was to identify genes that confer susceptibility to coronary artery spasm and to clarify the geneenvironment interactions that underlie the condition. Such information may facilitate assessment of the genetic risk for this condition and contribute to the primary prevention of vasospastic angina and acute coronary syndrome.
Methods
Study population
The study population comprised 2188 unrelated Japanese individuals (1215 men, 973 women) who either visited outpatient clinics of or were admitted to one of the participating hospitals (Appendix A) between July 1994 and December 2001. The 593 subjects with coronary artery spasm (453 men, 140 women) all underwent coronary angiography and left ventriculography. All subjects with coronary artery spasm had angina pectoris at rest. Among these subjects, 68 individuals (54 men, 14 women) who had a previous myocardial infarction were also included in our previous study.9 Coronary artery spasm associated with ST-segment elevation occurring spontaneously or induced by intracoronary injection of acetylcholine was documented angiographically for each patient.
The 1595 control subjects (762 men, 833 women) were recruited from individuals who attended outpatient clinics of the cardiology departments of the participating hospitals for a medical check-up. These subjects exhibited normal electrocardiograms at rest and no signs of myocardial ischaemia in exercise stress testing. They had no history of myocardial infarction, angina pectoris, or coronary artery spasm. These subjects either had or did not have conventional risk factors for coronary artery disease, including regular cigarette smoking (10 cigarettes daily), obesity (body mass index
26 kg/m2), hypertension (systolic blood pressure
140 mmHg or diastolic blood pressure
90 mmHg, or both), diabetes mellitus (fasting blood glucose
6.93 mmol/l or haemoglobin A1c
6.5%, or both), hypercholesterolaemia (serum total cholesterol
5.72 mmol/l), and hyperuricaemia (serum uric acid
0.46 mmol/l for men or
0.33 mmol/l for women). The study protocol complies with the declaration of Helsinki and was approved by the Committees on the Ethics of Human Research of Nagoya University Graduate School of Medicine, Okazaki City Hospital, Kosei Hospital, and Nagoya Daini Red Cross Hospital. Written informed consent was obtained from each participant.
Cardiac catheterisation
All medications were discontinued at least 48 h before coronary angiography. Coronary artery spasm was induced as described previously,4,5,10 with minor modifications. In brief, after baseline angiography of the left and right coronary arteries, two doses (50 and 100 µg) of acetylcholine were injected sequentially into the left coronary artery; angiography was performed 1 min after each injection. Acetylcholine (50 µg) was then injected into the right coronary artery and angiography was performed again. Coronary artery spasm was defined as either total or 90% occlusion of the epicardial coronary arteries associated with chest pain and ischaemic ST-segment elevation. Finally, after intracoronary injection of 1 mg of isosorbide dinitrate, left and right coronary arteriograms were obtained in multiple projections. In all patients, luminal narrowing of coronary arteries was <25%, which was a post hoc finding. In subjects with a previous myocardial infarction, coronary artery spasm was provoked in the stable chronic phase.
Selection of candidate gene polymorphisms
With the use of public databases, we selected 29 candidate genes that have been characterised and are believed to be associated with coronary artery spasm on the basis of a comprehensive overview of vascular biology, platelet and leukocyte biology, the coagulation and fibrinolysis cascades, as well as lipid and glucose metabolism and other metabolic factors. We further selected 35 polymorphisms of these genes most located in the promoter region, exons, or splice donor or acceptor sites in introns that might be expected to affect the expression or function of the encoded protein (Table 1).
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Statistical analysis
We performed statistical analyses for men and women separately. Quantitative clinical data were compared between subjects with coronary artery spasm and controls using the unpaired Student's test. Qualitative data were compared by the
test. Allele frequencies were estimated by the gene counting method, and the
test was used to identify significant departures from HardyWeinberg equilibrium. We performed a multivariable logistic regression analysis to adjust for risk factors, with coronary artery spasm as a dependent variable and independent variables including age, body mass index, smoking status (0=nonsmoker, 1=smoker), metabolic variables (0=no history of hypertension, diabetes mellitus, hypercholesterolaemia, or hyperuricaemia; 1=positive history), and genotype of each polymorphism. There were three genotypes for each polymorphism: wild-type homozygote, heterozygote, and variant homozygote. A dominant genetic model was defined as a model comprising two groups: wild-type homozygotes and the combined group of heterozygotes and variant homozygotes. A recessive model was also defined as a model of two groups: variant homozygotes and the combined group of wild-type homozygotes and heterozygotes. An additive model was defined as a model consisting of the three genotype groups. Each genotype was assessed according to dominant, recessive, and additive genetic models, and the
value (Wald test), odds ratio, and 95% confidence interval (profile likelihood ratio method) were calculated (JMP version 5; SAS Institute, Cary, NC, USA). We also performed a stepwise forward selection procedure to examine the effects of genotypes and other characteristics on coronary artery spasm. In this analysis, the dependent variable was coronary artery spasm and independent variables included age, body mass index, smoking status (0=nonsmoker, 1=smoker), metabolic variables (0=no history of hypertension, diabetes mellitus, hypercholesterolaemia, or hyperuricaemia; 1=positive history), and genotype of p22-PHOX (dominant or additive model) for men or of MMP3 (dominant or additive model) and IL6 (recessive model) for women. The significance levels for inclusion in and exclusion from the model were 25% and 10%, respectively. Given the multiple comparisons of genotypes with coronary artery spasm, we adopted a strict criterion of statistical significance to avoid type I error; a
value of
0.005 was considered statistically significant for association. For other clinical background data, a
value of
0.05 was considered statistically significant. Statistical significance was examined by two-sided tests.
Results
The characteristics of all 2188 study participants (1215 men, 973 women) are shown in Table 2. For men, the prevalence of smoking and hypercholesterolaemia was significantly greater and age was significantly younger in subjects with coronary artery spasm than in controls. For women, the prevalence of smoking, hypertension, and hyperuricaemia was significantly greater and age was significantly younger in subjects with coronary artery spasm than in controls.
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Finally, we performed a stepwise forward selection procedure to examine the effects of genotypes for p22-PHOX, MMP3, and IL6 and other characteristics on coronary artery spasm (Table 5). For men, smoking, age, p22-PHOX genotype, and hypercholesterolaemia, in descending order of statistical significance, affected the prevalence of coronary artery spasm. For women, smoking, MMP3 genotype, hypertension, IL6 genotype, and age significantly influenced the prevalence of coronary artery spasm.
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The major causes of coronary artery spasm may include endothelial dysfunction10,11 and an enhanced vasoconstriction response of vascular smooth muscle cells12 that result in disruption of the control of vasomotor tone. In addition, vasoactive substances released from activated platelets11 and underlying atherosclerotic changes13 may contribute to this condition. The genes now shown to be associated with coronary artery spasm may play roles in oxidative stress in the vessel wall (p22-PHOX), vascular matrix metabolism (MMP3), and vascular inflammation (IL6). Among the nine polymorphisms related to coronary artery spasm in the present study, only the 786TC polymorphism of the endothelial nitric oxide synthase gene (NOS3) has previously been associated with this condition.4,5 The
allele of this polymorphism in the
flanking region of NOS3 was shown to be a risk factor for coronary artery spasm.4 Functional analysis using a luciferase reporter gene assay demonstrated that the promoter activity of the 786C allele was reduced relative to that of the 786T allele.
Because interactions between genetic and environmental factors may be important in the aetiology of coronary artery spasm, we examined the effects of genotypes and conventional risk factors for coronary artery disease on the prevalence of this condition. Our results indicate that smoking is the most important environmental factor for coronary artery spasm in both men and women. This observation is consistent with the results of our previous study2 and is supported by the fact that smoking impairs endothelium-dependent dilation of coronary arteries in humans.14,15 Smoke extracts contain oxygen free radicals that inactivate nitric oxide and directly damage endothelial cells.15 We thus confirmed that smoking cessation is important in the prevention of coronary artery spasm. A stepwise forward selection procedure revealed that, in addition to smoking, age, p22-PHOX genotype, and hypercholesterolaemia significantly influenced the prevalence of coronary artery spasm in men, and that MMP3 genotype, hypertension, IL6 genotype, and age significantly affected the prevalence of this condition in women, indicating that the effects of these factors were statistically independent of smoking.
NADH/NADPH oxidase is present in vascular endothelial and smooth muscle cells and appears to be a major source of vascular superoxide in human atherosclerosis.16 The p22 phox subunit of NADH/NADPH oxidase is required for oxidative activity in smooth muscle cells and is expressed in human coronary arteries.17 The 242CT (His72Tyr) polymorphism of p22-PHOX was previously shown to be associated with coronary artery disease, with the T allele protecting against this condition.18 The T allele of this polymorphism was also associated with both reduced NADH/NADPH oxidase activity and reduced superoxide production in human blood vessels.19 Given that oxidative stress is implicated as an important factor in coronary artery spasm,8 the 242C
T polymorphism of p22-PHOX might be expected to play a role in predisposition to this condition. Our results now show that this polymorphism was indeed significantly associated with coronary artery spasm in men, with the T allele representing a protective factor, consistent with the results of previous studies.18,19
Stromelysin-1 catalyses the degradation of many of the constituents of the extracellular matrix found in atherosclerotic plaques.20 The 6A allele of the 1171/5A6A polymorphism of MMP3 was previously associated with an increased rate of progression of coronary atherosclerosis in a male population in England.21 The 6A/6A genotype was also associated with restenosis after conventional balloon angioplasty in a French population22 and with an increased intima-media thickness of the carotid artery in Finnish men23 and in men and women in New York.24 Consistent with these previous observations,2124 we have now shown that the 6A allele of the 1171/5A
6A polymorphism of MMP3 is a risk factor for coronary artery spasm in women. The distribution of MMP3 genotypes in control women was not in HardyWeinberg equilibrium, however, and differed significantly from the distribution in a Japanese population of a previous study25 (5A allele, 32% and 18%; 6A allele, 68% and 82% in our study and the previous study, respectively;
,
test). The deviation from HardyWeinberg equilibrium may be attributable to a selection bias of control subjects, given that they were recruited from individuals seen in outpatient clinics rather than from the general population.
Interleukin-6 plays a key role in driving the acute inflammatory response and in regulating the production of acute-phase proteins such as C-reactive protein.26 Interleukin-6 was shown to be associated with markers of endothelial dysfunction such as chemokine and adhesion molecule release.27 Interleukin-6 may contribute to the inflammatory response by activating endothelial cells28 and stimulating the synthesis of fibrinogen.29 It might thus be important in the pathogenesis of inflammation associated with coronary artery disease. Furthermore, unstable angina has been associated with activation of circulating neutrophils and with increased serum concentrations of C-reactive protein and interleukin-6.30 These various observations thus suggest that interleukin-6 may play an important role in the pathogenesis of coronary artery spasm. Our results now show that the 634CG polymorphism of IL6 was significantly associated with the prevalence of coronary artery spasm in women, with the GG genotype representing a risk factor for this condition.
In general, since the total risk of coronary artery disease in women lags behind that of men by 10 years, the mechanism underlying the risk for this condition in women may differ from that of men at each age. A role for genetic factors in the greater risk of mortality from coronary artery disease in women aged 65 years than in men of the same age was demonstrated in a study of Swedish twins,31 suggesting that such factors may play a more important role in the development of myocardial infarction in women of this age group than in men of the same age. Furthermore, the prevalence of smoking, which is the most important risk factor for coronary artery spasm,2 was much greater in men than in women in the present study. We therefore hypothesised that polymorphisms associated with coronary artery spasm might differ between men and women and performed association studies for men and women separately. Indeed, the polymorphisms significantly associated with coronary artery spasm did differ between men and women. The gender difference in the association of polymorphisms with coronary artery spasm might be attributable, at least in part, to the difference in serum oestrogen concentration between men and women. Oestrogen exerts various favourable effects on vasomotor function, including stimulation of the production of nitric oxide and prostaglandin I2, as well as inhibition of the release of endothelin-1 by vascular endothelial cells.32 In addition, given that the 35 polymorphisms examined in the present study likely represent only a small proportion of those potentially associated with coronary artery spasm, it remains possible that further investigations will uncover polymorphisms associated with this condition in both men and women.
The present study has several limitations: (1) Although we selected as controls individuals with no history of myocardial infarction, angina pectoris, or coronary artery spasm, with normal electrocardiograms at rest and no signs of myocardial ischaemia during exercise stress testing, without performing coronary angiography or provocation testing for coronary artery spasm we could not exclude the possibility that some of these subjects were affected by coronary artery spasm. (2) Given the important racial differences in the prevalence of coronary artery spasm between Caucasians and Japanese,3,33 our findings can be applied only to Asian populations. (3) Although smooth muscle hyperreactivity is an important cause of coronary artery spasm, polymorphisms in genes potentially associated with smooth muscle function were not included in the present analysis. (4) The alleles of p22-PHOX and MMP3 associated with coronary artery spasm were also present in most controls as well as in subjects with coronary artery spasm, indicating a simultaneous contribution of other genetic or environmental factors to coronary artery spasm. (5) Given the multiple comparisons of genotypes with coronary artery spasm in the present study, it is not possible to completely exclude potential statistical errors such as false positives, although we adopted a strict criterion of statistical significance (P<0.005) for association. (6) It is also possible that one or more of the polymorphisms associated with coronary artery spasm in our study are in linkage disequilibrium with polymorphisms of other nearby genes that are actually responsible for the development of this condition.
Despite these limitations, our present results indicate that p22-PHOX is a susceptibility locus for coronary artery spasm in Japanese men, and that MMP3 and IL6 constitute such loci in Japanese women. Genotyping of these polymorphisms may prove informative for assessment of the genetic risk for coronary artery spasm, and may thereby contribute to the primary prevention of vasospastic angina and acute coronary syndrome.
The following physicians and institutions participated in this study: Y. Suzuki, H. Kataoka, Y. Morimoto, T. Tajika, T. Tanaka, and H. Ishihara (Okazaki City Hospital); H. Horibe, M. Yanase, K. Takemoto, and S. Shimizu (Kosei Hospital); T. Okada and H. Hirayama (Nagoya Daini Red Cross Hospital); H. Izawa, A. Yamada, and T. Kato (Nagoya University Hospital).
See Table 6.
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This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science, and Technology of Japan (M.Y. and Y.Y.) and by a grant from the Mitsui Life Social Welfare Foundation (M.Y.).
Footnotes
1 doi:10.1016/j.ehj.2004.04.013
References
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