Published online before print May 29, 2003, doi: 10.1161/01.STR.0000075769.09092.82
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(Stroke. 2003;34:1628.)
© 2003 American Heart Association, Inc.
Original Contributions |
Association of Methylenetetrahydrofolate Reductase Gene Polymorphism With Carotid Atherosclerosis Depending on Smoking Status in a Japanese General Population
From the Department of Preventive Cardiology, National Cardiovascular Center (N.I., Y.K., T.M., S.B., J.O., H.T.), and Department of Geriatric Medicine, Osaka University Graduate School of Medicine (N.I., T.K., T.A., T.O.),Suita, Osaka, Japan.
Correspondence to Tomohiro Katsuya, MD, PhD, Department of Geriatric Medicine, Osaka University Graduate School of Medicine, 2-2 No. B6, Yamada-Oka, Suita, Osaka 565-0871, Japan. E-mail katsuya{at}geriat.med.osaka-u.ac.jp
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Abstract |
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Background and Purpose— The association of the methylenetetrahydrofolate reductase gene (MTHFR) with carotid atherosclerosis remains inconsistent. This may be due to small sample size and inappropriate analysis. We investigated the association of C677T/MTHFR with blood pressure and carotid atherosclerosis in a Japanese general population.
Methods— Subjects (30 to 89 years of age; 1693 women, 1554 men) who gave informed consent were randomly selected from a general population in Suita, Japan. MTHFR genotypes were determined by TaqMan polymerase chain reaction. Carotid atherosclerosis was evaluated by high-resolution ultrasonography with atherosclerotic indexes of intimal-medial thickness (IMT), maximum IMT in the common carotid artery (CCA), plaque score, and stenosis (>50%).
Results— Age-adjusted diastolic blood pressure was significantly higher in women with the TT genotype than in those with the CC genotype. In a recessive model (CC+CT versus TT), all adjusted odds ratios for hypertension and >50% stenosis in women were 1.42 and 3.42 (95% confidence intervals, 1.01 to 1.99 and 1.23 to 9.53), respectively. In women, maximum IMT in CCA for smokers with the TT genotype was significantly higher than for smokers with the CC genotype and nonsmokers with the TT genotype (P<0.05).
Conclusions— Our study suggests that the MTHFR TT genotype is a risk factor for hypertension and carotid stenosis in women. Significant interactions between C677T/MTHFR and smoking on maximum IMT in CCA were observed in women but not in men. Smoking cessation for subjects with the TT genotype is important in the prevention of cerebrovascular disease.
Key Words: amine oxidoreductases • blood pressure • carotid arteries • Japan • risk factors
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Introduction |
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Hyperhomocysteinemia is associated with increased risk of atherosclerotic vascular disease.1 The association of plasma total homocysteine concentration with atherosclerosis has been the subject of a number of clinical studies that have consistently linked moderate hyperhomocysteinemia with peripheral vascular disease, cerebrovascular disease, and coronary heart disease.2–5
Plasma total homocysteine levels are regulated mainly by 5,10-methylenetetrahydrofolate reductase, which is involved in the folate-dependent remethylation of homocysteine to methionine. Frosst et al6 suggested that the C677T polymorphism in the methylenetetrahydrofolate reductase gene (MTHFR) is a candidate risk factor for vascular disease. The metabolic changes associated with C677T/MTHFR are postulated to modify the predisposition to diseases associated with folate deficiency.7 Particular emphasis has been given to the role of C677T/MTHFR in cardiovascular8 and cerebrovascular disease9 and venous thrombosis.10
On the other hand, technical improvements in carotid ultrasonography have revealed new risk factors for stroke in its wide use. Some studies have demonstrated a close correlation between carotid ultrasound measurement, usually of carotid intimal-medial wall thickness (IMT), and the severity of extracranial carotid atherosclerosis.11,12 Plasma total homocysteine levels have also been associated with more advanced carotid atherosclerosis in elderly subjects.3,13 However, there have been controversies among their results. Most studies have failed to show an association between C677T/MTHFR and atherosclerotic disease.14,15 These inconsistencies may be due to small sample size, combined-sex analysis, and lack of consideration of lifestyle. In this study, we examined the effect of C677T/MTHFR on carotid atherosclerosis and blood pressure (BP) in a large genetic epidemiological study, the Suita Study.
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Materials and Methods |
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Subject Population
The Suita Study was based on a random sample of 14 200 Japanese residents of Suita, a city located in the second-largest urban area in Japan, Osaka.16 These 14 200 residents between 30 and 89 years of age were arbitrarily selected from the municipality population registry, stratified by sex and 10-year age groups. We sent these residents letters to ask if they were willing to participate in this study from 1989 with a cohort base; by February 1007, 51.7% of the subjects (n=7347) had paid an initial visit to the National Cardiovascular Center (NCVC). The participants have visited NCVC every 2 years since then for regular health checkups. In addition to routine blood examinations that included total serum cholesterol, high-density lipoprotein (HDL) cholesterol, triglycerides, glucose, glycosylated hemoglobin A1C (HbA1C), systolic BP (SBP), and diastolic BP (DBP), DNA was extracted from an extra 5 mL blood withdrawn from those who underwent general examinations at NCVC between May 1996 and February 1998. Ninety percent of the subjects who visited NCVC during this period gave informed consent for genetic analysis of 13 genes including MTHFR and storage of a DNA sample and were enrolled in the present study. The study protocol of genetic analysis was approved by the ethics committee of Osaka University. Three physicians performed the carotid ultrasonic examinations. Finally, the subjects in the present study included 1693 women and 1553 men 30 to 89 years of age who attended regular health checkups and subsequently underwent ultrasonic examinations and genetic analysis.
Measurements
The subjects’ BPs were measured after at least 10 minutes of rest in the sitting position. The mean value of 2 measurements of SBP or DBP obtained by a physician using a mercury sphygmomanometer (recorded >3 minutes apart) was used for the analysis. Hypertension was defined as a mean SBP of 
160 mm Hg, a mean DBP of 95 mm Hg, or current use of antihypertensive medication.
The subjects were classified as current smokers or drinkers if they smoked or drank. Hypercholesterolemia was defined as serum total cholesterol levels 220 mg/dL or current use of antihyperlipidemic medication. Diabetes was defined as fasting plasma glucose levels 7.0 mmol/L (126 mg/dL) or nonfasting glucose levels 11.1 mmol/L (200 mg/dL), HbA1C 6.5%, or current use of antidiabetic medication. Body mass index (BMI) was calculated as weight (in kilograms) divided by height (in meters) squared.
Blood samples drawn from the subjects after 12 hours of fasting were collected in EDTA-containing tubes. Total cholesterol and HDL cholesterol levels were measured with an autoanalyzer (Toshiba TBA-80) in accordance with the Lipid Standardization Program of the US Centers for Disease Control and Prevention through the Osaka Medical Center for Health Science and Promotion, Japan.17 Among 3247 subjects, 1541 (820 women, 721 men) underwent measurement of fasting total plasma homocysteine levels by high-performance liquid chromatography.18
Carotid Ultrasound Measurements
Details of the carotid ultrasonic examination methods have been previously published.16 We used a high-resolution B-mode ultrasonic machine with a 7.5-MHz transducer yielding an axial resolution of 0.1 mm. The regions from 30 mm proximal to the beginning of the dilation of the bifurcation bulb to 15 mm distal to the flow divider of both common carotid arteries (CCAs) were scanned. All measurements were made at the time of scanning with the electronic caliper and were recorded on photocopies. IMT was measured on a longitudinal scan of the CCAs at a point 10 mm proximal to the beginning of the dilation of each carotid artery bulb. IMT was defined as the mean of the IMT of the proximal and distal walls at the point of measurement. Maximum IMT in the CCA and maximum IMT were defined as the maximum IMT in the scanned CCA area and the maximum IMT in the entire scanned area, respectively. We defined a plaque, a focal IMT thickening, as an area where IMT 1.1 mm and calculated plaque score by totaling the maximum thickness of all the plaques in the scanned area. Finally, we defined stenosis as a condition in which a plaque occupied more than half of the lumen circumference of an artery on a cross-sectional scan. We performed color-flow Doppler examination to confirm the presence of stenosis.
MTHFR Genotype Determination With TaqMan Polymerase Chain Reaction Method
Genomic DNA was extracted from peripheral blood lymphocytes by standard procedures with a QIAamp DNA Blood Kit (Qiangen Inc). To deal with a large number of samples, we introduced the TaqMan polymerase chain reaction (PCR) method (Applied Biosystems). In the current investigation, we prepared 2 probes: C allele–specific probe, 5' Tet-TCT GCG GGA GcC GAT TTC ATC ATC-Tamra-3', and T allele–specific probe, 5'-Fam-TCT GCG GGA GtC GAT TTC ATC ATC-Tamra-3'. Primer design for PCR of the flanking region of C677T/MTHFR was as follows: forward, 5'-GGC TGA CCT GAA GCA CTT GAA-3'; reverse, 5'-GCG GAA GAA TGT GTC ATC CT-3'. PCR was carried out with a thermal cycler (GeneAmp, PCR System 9700, Applied Biosystems). PCR was performed according to the following conditions: initial denaturation at 95°C for 10 minutes, followed by 40 cycles of 95°C for 15 seconds and 60°C for 60 seconds. The fluorescence level of PCR products was measured with an ABI PRISM 7200 and 7900 Sequence Detector (Applied Biosystems), resulting in clear identification of the 3 genotypes of C677T/MTHFR.
Statistical Analysis
The number of subjects was restricted to 3247 who had complete data, including C677T/MTHFR and carotid ultrasonographic measurements. Analysis of variance was used to compare mean values between groups, and if overall significance was demonstrated, the intergroup difference was assessed by means of a general linear model. Frequencies were compared by 
2 analysis.
Associations of C677T/MTHFR with BP were investigated by sex through logistic regression analysis considering potential confounding risk variables, including age, BMI, present illness (hyperlipidemia and diabetes mellitus), lifestyle (smoking and drinking), and antihypertensive medication. The genotype effect was examined according to a dominant (TT+CT versus CC) and a recessive (TT versus CT+CC) model. For multivariate risk predictors, the adjusted odds ratios (ORs) were given with the 95% confidence intervals (CIs). The relationships in men and women between C677T/MTHFR and hypertensive risk were expressed in terms of ORs adjusted for possible confounding effects. The association of C677T/MTHFR with carotid atherosclerotic index was also investigated by sex through logistic regression analysis considering potential confounding risk variables. Partial correlation coefficients between plasma total homocysteine and carotid atherosclerotic indexes by sex and C677T/MTHFR were determined. In addition, gene and environmental interactions were calculated with the following logistic regression model: logit p=ß0+ßgxg+ßexe+ßgexgxe, where xg and xe are genetic and environmental data, respectively; ß0 is an intercept term; ßg is the main effect due to genes; and ße is the main effect of the environment. The coefficient ßge of the product xgxe estimates the gene and environmental interaction on the logit scale.19 All analyses were performed with SAS statistical software (release 6.12, SAS Institute Inc).
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Results |
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Basic Characteristics of Subjects in the Suita Study
As shown in Table 1, age, SBP, DBP, BMI, total cholesterol, HDL cholesterol, IMT, maximum IMT in CCA, plaque score, CCA stenosis (
50%), percentage of current smokers, percentage of current drinkers, prevalence of hypertension, prevalence of diabetes mellitus, and total plasma homocysteine levels were significantly higher in men than in women. Only the frequency of hyperlipidemia was significantly higher in women than in men.
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C677T/MTHFR, Hypertension, and Plasma Homocysteine Levels
The frequencies of C677T/MTHFR in women were 37.5% for CC, 47.2% for CT, and 15.3% for TT genotypes, whereas those in men were 36.2% for CC, 47.8% for CT, and 16.0% for TT genotypes. There was no significant difference in allele frequencies between age groups (2=1.07, df=2, P=0.59). The genotype distribution of C677T/MTHFR was not significantly deviated from Hardy-Weinberg’s expectation in men or women. In women, SBP and DBP increased according to the number of T677 alleles of MTHFR, but the association was not statistically significant. Only DBP in TT women was significantly higher in those with the C677 allele after age adjustment. In the recessive model (CT+CC versus TT), however, C677T/MTHFR was significantly associated with the prevalence of hypertension, and the all adjusted OR for hypertension was 1.42 (95% CI, 1.01 to 1.99) in women (Table 2).
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Figure 1 shows plasma total homocysteine levels according to genotype of C677T/MTHFR in men and women. Mean plasma total homocysteine levels in subjects with the TT genotype was significantly higher than that in subjects with the CC or CT genotype.
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P<0.0001 vs CT subjects.
Carotid Atherosclerotic Index and C677T/MTHFR
Carotid atherosclerotic indexes (IMT, maximum IMT in CCA, maximum IMT, and plaque score) were evaluated in men and women separately, according to C677T/MTHFR genotype (Table 3). In women with the CT genotype, age-adjusted IMT, maximum IMT in CCA, and all adjusted maximum IMT in CCA were significantly thicker than in those with the CC genotype. However, there was no difference between subjects with the TT and CC genotypes in any atherosclerotic indexes.
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In contrast, C677T/MTHFR gave a significantly increased risk for stenosis (>50%) of CCA in women. In a recessive model (CC+CT versus TT), the all adjusted OR for stenosis (>50%) was 3.42 (95% CI, 1.23 to 9.53) in women and 1.41 (95% CI, 0.76 to 2.63) in men.
Partial correlation coefficients between plasma total homocysteine levels and carotid atherosclerotic index by C677T/MTHFR genotype are shown in Table 4. Positive relationships were found between plasma total homocysteine levels and IMT in men with the CC genotype and maximum IMT in CCA for men. These associations were stronger in men than in women.
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Interaction Between C677T/MTHFR and Lifestyle on Carotid Atherosclerotic Index According to Sex
Figure 2 shows the association of IMT and maximum IMT in CCA with C677T/MTHFR according to smoking and drinking status. In women with the CC or CT genotype, IMT in smokers was significantly higher than in nonsmokers. In women with the TT genotype, maximum IMT in CCA in smokers and drinkers was significantly higher than that in nonsmokers and nondrinkers, respectively (Figure 2–A2, P<0.05 for interaction; Figure 2–B2). In men with the CC or TT genotype, IMT and maximum IMT in CCA were significantly higher in smokers than in nonsmokers (Figure 2–A1).
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Discussion |
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The present study showed that the TT genotype of C677T/MTHFR was significantly associated with the prevalence of hypertension (OR, 1.15) and carotid stenosis (<50%) in women but not in men. In addition, the specific genotype of C677T/MTHFR affected maximum IMT in CCA in the interaction with smoking in women. These results show an association of C677T/MTHFR with BP and carotid atherosclerosis on the basis of gene and environmental interaction, which has not been previously reported.
Although previous studies showed that subjects with the TT genotype of C677T/MTHFR are associated with an increased risk of cardiovascular disease via an increase in plasma homocysteine levels,2,6,20 the conclusion is still controversial.7,14,15,21,22 The inconsistencies may be attributed to small sample size, combined-sex analysis, and no inclusion of lifestyle factors such as smoking and drinking. One should be aware that detecting gene and environmental interactions could require a substantially larger sample size than the sample size necessary for detecting genetic or environmental effects alone.23 Thus, we examined the effect of C677T/MTHFR in a large general population with various phenotypes that included plasma homocysteine levels, atheromatous indexes, smoking and drinking status, and relevant basic characteristics.
It can be questioned why the TT genotype of C677T/MTHFR is not unequivocally associated with increased cardiovascular risk,5 based on the argument that the gene is a strong predictor of hyperhomocysteinemia in general populations.6,24,25 It could be attributed to the close relationship between plasma homocysteine levels and folate metabolism. Several reports revealed that plasma total homocysteine levels become elevated only in folate-deficient subjects with the TT genotype7,25,26 and that the slope of regression lines relating total homocysteine to folate increases in the order of CC, CT, and TT genotypes.15,24 In other words, if folate intake is sufficient, subjects with the TT genotype would not have increased risk of cardiovascular disease via hyperhomocysteinemia.
Under stratification by sex, we observed that the TT genotype was independently associated with DBP and carotid stenosis in women and showed a greater disadvantage in female smokers and drinkers. Even though homocysteine would injure the endothelium of small arteries at an early stage27 and endothelial dysfunction plays a critical role in the early events of atherosclerosis,28 we currently have no definitive answer to explain the results. However, it seems to be an important finding that most of the positive results in the present study were obtained only in women. As supporting data of our results, a female-specific significant association with the TT genotype was also reported in the predisposition to ischemic stroke29 and asymptomatic carotid atherosclerosis.30 Motti et al31 reported that sex differentiation is independently associated with homocysteine. Plasma homocysteine levels are significantly higher in healthy men than in women, which is consistent with our results (Table 1). In addition, homocysteine levels are reported to be lower in premenopausal women than in men and postmenopausal women. Furthermore, a recent report suggested that total homocysteine levels were significantly correlated with fat-free mass and testosterone and inversely with estradiol. The sex difference with regard to total homocysteine levels was explained primarily by differences in fat-free mass but also by estradiol concentration. Those results might be a feasible explanation for the lack of association in men.32 However, there was no association between C677T/MTHFR and carotid atherosclerosis in premenopausal and postmenopausal women (data not shown). This result suggests that estrogen might have a protective effect against homocysteinemia but not atherosclerosis via C677T/MTHFR. Indeed, previous reports did not find such a specific advantage in the relationship between C677T/MTHFR and coronary artery disease in young woman in a small Caucasian population.33,34
Disadvantages of our study design were that only half of the subjects had their total plasma homocysteine levels analyzed. This is not a serious limitation, however, because the association between C677T/MTHFR and plasma homocysteine levels has already been demonstrated in several large studies.7,26 Another disadvantage was that we had no data on the physical activity and nutrition of the subjects, but these data were also supported by previous studies. The dietary intake of folate, vitamin B6, and B12 is inversely (negatively) correlated with plasma homocysteine35,36; physical activity is also inversely associated with plasma homocysteine.37 There is a need for additional prospective studies with data on relevant confounders that have sufficient power to examine the association between homocysteine concentration and stroke risk, whether linear or threshold, and to study interactions between homocysteine, other dietary markers, and established stroke risk factors such as smoking and hypertension. Similarly, the evidence linking hyperhomocysteinemia with hypertension is limited and inconsistent. Ultimately, the case for a causal role of elevated homocysteine levels in vascular disease, including hypertension and stroke, will depend on data from randomly controlled trials of homocysteine-lowering interventions.
In summary, the present study shows that the homozygous T677 allele of C677T/MTHFR is a risk factor for hypertension and carotid stenosis in women. In addition, smoking increased IMT in CCA in women with the TT genotype. In the near future, physicians might use the genotypic data of C677T/MTHFR to modify their patients’ lifestyles to prevent cardiovascular disease.
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Acknowledgments |
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This study was supported by a grant-in-aid (H14-027) from the Japanese Ministry of Health, Labor, and Welfare; grants-in-aid for scientific research (12557063, 13770349, 13204050, 13670709) from the Ministry of Education, Science, Sports and Culture of Japan; and research grants from the Takeda Medical Foundation, Salt Science Research Foundation, Osaka Medical Research Foundation for Incurable Diseases, Yokoyama Foundation for Clinical Pharmacology and Therapeutics, Kanae Foundation for Life and Socio-Medical Science, Osakagas Group Welfare Foundation, Osaka Kidney Foundation, Japan, Preventive Arteriosclerosis Research Association, and Kurozumi Medical Foundation. We would like to express our deepest gratitude to the following people for their continuous support of our population survey in this area: Dr Otosaburo Hishikawa (president), Dr Katsuyuki Kawanishi (committee member in chief for the city health checkup service), and other members of Suita City Medical Association, and Shigeru Kobayashi, director of the City Health Center. We would also like to express our greatest thanks to the members of our attendants’ society (Satsuki-Junyu-kai) for their cooperation and assistance in our survey on risk factors and preventive activity on cardiovascular diseases. We are also deeply grateful to Dr Soichiro Kitamura, president of the National Cardiovascular Center, for consideration of our research.
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Footnotes |
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This article encompasses the doctoral dissertation of Nozomu Inamoto.
Received October 16, 2002; revision received December 25, 2002; accepted January 23, 2003.
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