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Methylenetetrahydrofolate reductase gene polymorphism
AJH
1998;11:1019 –1023
BRIEF COMMUNICATIONS
Methylenetetrahydrofolate Reductase Gene Polymorphism Relation to Blood Pressure and Cerebrovascular Disease Yukiko Nakata, Tomohiro Katsuya, Seiju Takami, Noriyuki Sato, Yuxiao Fu, Kazuhiko Ishikawa, Shin Takiuchi, Hiromi Rakugi, Tetsuro Miki, Jitsuo Higaki, and Toshio Ogihara
Hyperhomocysteinemia is reported to be associated with an increase in the incidence of ischemic heart disease and cerebrovascular disease. Genetic aberrations in methylenetetrahydrofolate reductase (MTHFR) may account for reduced enzyme activity and elevated plasma homocysteine level. A recent report revealed that a common mutation (677C to T; Ala to Val) in the MTHFR gene is associated with decreased specific MTHFR activity and with increased risk for coronary artery disease in the homozygous state (Val/Val). In the present study, we investigated whether the MTHFR gene is a genetic risk factor for cerebrovascular disease (CVD). To undertake a case-control study, we selected the patients with cerebral infarction (n 5 48) or cerebral hemorrhage (n 5 35) and examined the association between MTHFR gene polymorphism and CVD. The genotype distribution of the MTHFR gene was not significantly different between cases and controls. Because the possibility of matching the morbidity
H
of the effects of hypertension, the lack of association could not be excluded in the first study; however, we also examined whether the MTHFR mutation was associated with any clinical risk factor for CVD or with hypertension. It turned out that the subjects with the Val allele of the MTHFR gene had significantly lower blood pressure than the subjects with other genotypes in the general population (P 5 .02), and that the frequency of the Val/Val genotype in hypertensive subjects (n 5 173) was significantly lower than in control subjects (n 5 184) (P 5 .03). From these results, we conclude that the Val/Val homozygous state of the MTHFR gene increased the risk of thrombosis, but reduced the blood pressure, which resulted in the lack of increased risk for CVD. Am J Hypertens 1998;11:1019 –1023 © 1998 American Journal of Hypertension, Ltd. KEY WORDS:
Hyperhomocysteinemia, genetics, thrombosis, hypertension.
yperhomocysteinemia is known to be an independent risk factor for the development of coronary heart disease,1– 4 myocardial infarction,5 cerebrovascular dis6,7 ease (CVD), and venous thrombosis.8,9 The serum
homocysteine level is influenced by environmental as well as genetic factors. A high plasma concentration of homocysteine, derived from the demethylation of dietary methionine, may predispose to atherosclerosis by injuring the vascular endothelium, which results in
Received October 2, 1997. Accepted February 19, 1998. From the Department of Geriatric Medicine, Osaka University Medical School, Osaka, Japan. The contents of this paper were read at the twelfth meeting of the American Society of Hypertension in San Francisco, California, May 27–31, 1997.
Address correspondence and reprint requests to Toshio Ogihara, MD, PhD, Professor of Medicine, Department of Geriatric Medicine, Osaka University Medical School, 2-2 Yamada-oka, Suita, Osaka 565, Japan.
© 1998 by the American Journal of Hypertension, Ltd. Published by Elsevier Science, Inc.
0895-7061/98/$19.00 PII S0895-7061(98)00046-6
1020
NAKATA ET AL
endothelial dysfunction.10 For cardiovascular diseases, the enhanced risk associated with a 5-mol/L elevation of total plasma homocysteine was estimated to equal that of a 0.5-mmol/L increase in total cholesterol in a metaanalysis.11 A recent report revealed that a common mutation (677C to T; Ala to Val) in the methylenetetrahydrofolate reductase (MTHFR) gene is associated with decreased specific MTHFR activity and elevation of the homocysteine level in the Val/Val homozygous state.12 In this study, we investigated whether MTHFR gene polymorphism is a genetic risk factor for cerebrovascular disease. To clarify whether the MTHFR mutation itself is an independent risk factor for CVD or whether other confounding factors related to the MTHFR genotype affect the risk of CVD, we also examined the relations between MTHFR genotype polymorphism and classical risk factors for CVD. MATERIALS AND METHODS Subjects and Measurements In the present study, all subjects were Japanese and gave informed consent before participating in the research protocol, which was approved by each hospital’s ethics committee. Study 1: MTHFR Mutation and CVD Patients with documented CVD were identified from the clinical records from July 1992 to December 1995 at six hospitals in Osaka, Japan. Patients aged younger than 30 years and older than 80 years were excluded. In addition, subjects with risk factors for cardiogenic stroke, such as atrial fibrillation, valvular heart disease, endocarditis, acute myocardial infarction, dilated cardiomyopathy, and arteriosclerosis obliterans, were excluded. Final diagnosis of CVD was confirmed by brain computed tomography or brain magnetic resonance imaging. The criteria of the National Institute for Neurological Disorders and Stroke were applied in this study.13 Finally, we identified 83 patients with CVD, comprising cerebral infarction (n 5 48) and cerebral hemorrhage (n 5 35). Control subjects (n 5 105) without history of CVD were recruited from visitors to Sakuragaoka Hospital (Hyogo, Japan) who underwent general check-up from September 1991 to March 1993 (total number, 3920; 3103 men and 817 women), matching for age, gender, and morbidity of hypertension with patients. Study 2: MTHFR Mutation and Classical Risk Factors for CVD Because the morbidity of hypertension was matched between cases and controls, the effect of blood pressure on CVD risk could not be determined in Study 1. Therefore, the association between classical risk factors for CVD and MTHFR mutation was examined in the general population. The subjects (n 5 150) were recruited from the same population as in Study 1, excluding those receiving medication for hyperten-
AJH–AUGUST 1998 –VOL. 11, NO. 8, PART 1
sion, diabetes mellitus, or hyperlipidemia. Systolic and diastolic blood pressure in the sitting position, height, and body weight were obtained as physical findings. Fasting plasma glucose, total cholesterol, triglyceride, HDL cholesterol, and glycosylated hemoglobin (HbA1c) were measured by standard methods. Study 3: MTHFR Mutation and Essential Hypertension To examine whether there is a direct association between the MTHFR mutation and hypertension, patients with essential hypertension (n 5 173) and control subjects (n 5 184) were recruited from outpatients at Osaka University Medical School. All cases had a family history of hypertension in first-degree relatives and were diagnosed as having primary hypertension; those with secondary hypertension, diabetes mellitus, or apparent ischemic heart disease were excluded. The criteria for hypertension were defined as systolic blood pressure . 160 mm Hg, diastolic blood pressure . 95 mm Hg, or receiving antihypertensive therapy. Controls with no history of hypertension and without diabetes mellitus were recruited from the same population and were matched to patients for gender and age. Study 4: MTHFR Mutation and Plasma Homocysteine and Folate Levels To confirm the relationship between the MTHFR genotype and plasma homocysteine level in Japanese subjects, we measured homocysteine and folate levels using healthy middle-class volunteers in whom folate intake was adequate. Plasma homocysteine and folate levels were measured using high-performance liquid chromatography. Genotyping of MTHFR Gene Polymorphism DNA was extracted from 10 mL of whole blood using SepaGene (Sanko Junyaku Co., Tokyo, Japan). All polymerase chain reactions (PCR) to detect the MTHFR mutation were carried out with 100 ng of genomic DNA as a template, using a DNA Thermal Cycler PJ 2000 (Perkin Elmer Co., Norwalk, CT) and primers as previously reported.12 The sense and antisense primers were as follows; sense: 59TGA AGG AGA AGG TGT ATG AGG GA-39, antisense: 59-AGG ACG GTG CGG TGA GAG TG-39. Samples were amplified for 35 cycles consisting of denaturation at 94°C for 30 sec, annealing at 60°C for 60 sec, and extension at 72°C for 60 sec, followed by a final extension step at 72°C of 10 min. Because the nucleotide 677 mutation creates a restriction site for HinfI (Takara Shuzo Co. Ltd., Osaka, Japan), the genotype was determined by digestion with this enzyme, followed by 10% polyacrylamide gel electrophoresis and ethidium bromide staining. Statistical Analysis For all the statistical analyses, we used the computer software StatView 4.5J (Abacus Concepts Inc., Berkeley, CA). Alleles and genotype frequencies in cases and control subjects were com-
AJH–AUGUST 1998 –VOL. 11, NO. 8, PART 1
MTHFR GENE MUTATION AND RISK OF HYPERTENSION, CVD
1021
TABLE 1. DISTRIBUTION OF MTHFR GENOTYPES IN CVD SUBJECTS, HYPERTENSIVES, AND CONTROLS
Genotype CVD subjects Cerebral infarction
Cerebral hemorrhage Controls Hypertensives Controls
n
Ala/Ala (n [%])
Val/Ala (n [%])
Val/Val (n [%])
P
Genotype
n
83 48
31 (37.3) 19 (39.6)
41 (49.4) 23 (47.9)
11 (13.3) 6 (12.5)
NS* NS†
Hypertensives Controls
173 184
35 105 173 184
12 (34.3) 35 (33.3) 63 (36.4) 65 (35.3)
18 (51.4) 51 (48.6) 91 (52.6) 83 (45.1)
5 (14.3) 19 (18.1) 19 (11.0) 36 (19.6)
NS‡ .07
Ala/Ala, Val/Ala (n [%])
Val/Val (n [%])
154 (89.0) 19 (11.0) 148 (80.4) 36 (19.6) Odds ratio 5 1.97 (95% CI: 1.08–3.59)
P , .03
Hypertensives
CI: confidence interval. Significance of difference was examined by x2 analysis. * v controls; † v cerebral hemorrhage; ‡ v cerebral infarction.
pared by x2 test. The relationship between MTHFR genotypes and classical risk factors for CVD was assessed by one-way analysis of variance (ANOVA). P , .05 was considered significant.
hypertension in subjects with the Ala allele was 1.97 (95% CI: 1.08 –3.59), suggesting that the effect of the Val allele on hypertension acts in a recessive manner.
RESULTS Study 1: MTHFR Mutation and CVD The prevalence of hypertension was 70.1% in the CVD subjects and 65.8% in controls, supporting the possibility that the effect of hypertension was excluded in this study. The MTHFR genotype was clearly determined in all subjects. Genotype distribution was not significantly different from the Hardy-Weinberg equilibrium. The genotype distribution was not significantly different between the CVD and control, hemorrhage and control, infarction and control, or hemorrhage and infarction groups (Table 1). Furthermore, the MTHFR genotype was not associated with other classic risk factors for CVD (data not shown). Study 2: MTHFR Mutation and Classical Risk Factors for CVD Because we could not determine the genetic effect of MTHFR on blood pressure in Study 1, we examined whether MTHFR genotypes are associated with blood pressure in the general population. Both systolic and diastolic blood pressures were associated with the MTHFR genotype (F value 5 4.1, P , .02) (Figure 1). The subjects with the Val allele (Val/ Val and Val/Ala) had lower blood pressure than subjects with homozygous Ala alleles, for both systolic and diastolic blood pressures. However, no significant difference between heterozygous Val/Ala and homozygous Val/Val subjects was detected. Study 3: MTHFR Mutation and Essential Hypertension We also carried out a case-control study of hypertension. The frequency of the Val/Val genotype was significantly lower in hypertensives than in controls (P , .03) (Table 1). The estimated odds ratio for
FIGURE 1. Plasma homocysteine and folate levels in healthy subjects and association between MTHFR polymorphism and blood pressure. Association between MTHFR genotype and blood pressure was examined by ANOVA. Statistical significance differences between two groups was examined by Fisher’s PLSD test. (*P , .05, #P , .01). Ala/Ala, homozygous alanine allele; Val/ Ala, heterozygous valine and alanine alleles; Val/Val, homozygous alanine allele.
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NAKATA ET AL
Study 4: MTHFR Mutation and Plasma Homocysteine and Folate Levels To exclude the heterogeneity of diet, we measured the fasting plasma homocysteine and folic acid levels in normal middle-class volunteers. Both homocysteine and folate levels were associated with the MTHFR genotype. Subjects with the homozygous Val/Val genotype showed significantly higher homocysteine levels and lower folate levels than subjects with the homozygous Ala allele (Figure 1). DISCUSSION The main finding of the present study is that the Val allele of the MTHFR gene is not associated with cerebrovascular disease but is associated with lower blood pressure. Recent studies revealed that the Ala to Val mutation in the MTHFR gene is associated with increased plasma homocysteine levels and with thrombotic disease.1– 4 Plasma homocysteine levels were also higher in our study (Study 4) in subjects with the Val/Val genotype than in those with other genotypes (Figure 2), suggesting that the homozygous state associated with this mutation has an exaggerated hyperhomocysteinemic response and increased risk for thrombotic diseases, such as cerebrovascular disease. Recently, it was reported that homocysteine inhibits endothelial cell proliferation and stimulates vascular smooth muscle cell proliferation.14,15 Furthermore, there are some reports that homocysteine enhances endothelial cell-associated factor V activity16 and inhibits thrombomodulin surface expression,17 protein C activation,18 and tissue plasminogen activator binding.19 These results suggest that homocysteine may be involved in the atherosclerotic and thrombotic process by modulating vascular cell proliferation and promoting prothrombotic activities in the vascular wall. Thus, we hypothesized that the MTHFR mutation might increase the risk for CVD, and carried out an association study. However, we could not detect an association between the mutation of the MTHFR gene and CVD. In contrast, we found a unique association between the MTHFR gene and blood pressure in the general population. Subjects with the Val allele of the MTHFR gene showed lower blood pressure, both systolic and diastolic (Study 2). Furthermore, the frequency of the Val/Val genotype was significantly lower in hypertensive subjects than in control subjects (Study 3). A high homocysteine level in subjects with the homozygous Val/Val genotype was also confirmed in the Japanese population (Study 4). Summarizing the results of the studies,1– 4 the effects of the Val allele of the MTHFR gene were to increase the plasma homocysteine level, to increase the risk for thrombosis through hyperhomocysteinemia, and to decrease blood pressure. However, it did not increase
the CVD risk. Consequently, we speculate that the Val allele of the MTHFR gene increases the relative risk for thrombosis by increasing plasma homocysteine levels and decreasing blood pressure, which results in the lack of association between the MTHFR gene and CVD. Even though Wilken et al reported that the Val allele of the MTHFR gene was not a risk factor for coronary heart disease but that there was a weak association between the MTHFR gene and hypertension only in men,20 the discrepancy between their results and ours seems to be due to the strong effect of body mass index (BMI) on blood pressure or ethnic differences. In the present study, we observed no association between the MTHFR genotype and BMI, which was strongly associated with the MTHFR mutation in Wilken’s report. Though previous findings led us to believe that the Val allele favors an increase in blood pressure through stimulation of vascular smooth muscle cell proliferation, our study showed that the Val allele was associated with lower blood pressure. Considering other reports and our results, it seems that MTHFR gene polymorphisms have another role in the compensatory factor mechanism that attenuates the risk for CVD. We conclude that the Val allele of the MTHFR gene is not a genetic risk of CVD through its offset mechanism, predisposing individuals to thrombosis but lowering blood pressure. REFERENCES 1.
Clarke R, Daly L, Robinson K, et al: Hyperhomocysteinemia: an independent risk factor for vascular diseases. N Engl J Med 1991;324:1149 –1155.
2.
Gmurphy-Chutorian D, Alderman EL: The case that hyperhomocysteinemia is a risk for coronary artery disease. Am J Cardiol 1994;73:705–707.
3.
Arnrtson E, Refsum H, Bonaa KH, et al: Serum total homocysteine and coronary heart diseases. Int J Epidemiol 1995;24:704 –709.
4.
Robinson K, Mayer EL, Millert DP, et al: Hyperhomocysteinemia and low pyridoxalphosphate: common and independent reversible risk factors for coronary artery disease. Circulation 1995;92:2825–2830.
5.
Stampfer MJ, Malinow MR, Willett WC, et al: A prospective study of plasma homocysteine and risk of myocardial infarction in US physicians. JAMA 1992;24: 704 –709.
6.
Brattstrom L, Lindgren A, Israelsson B, et al: Hyperhomocysteinemia in stroke: prevalence, cause, and relationships to type of stroke and stroke risk factors. Eur J Clin Invest 1992;22:214 –221.
7.
Verhoef P, Hennekens CH, Mailnow MR, et al: A prospective study of plasma homocysteine and risk of ischemic stroke. Stroke 1994;25:1924 –1930.
8.
den Heijer M, Koster T, Blom HJ, et al: Hyperhomocysteinemia as a risk factor for deep-vein thrombosis. N Engl J Med 1996;334:759 –762.
9.
den Heijer M, Blom HJ, Gerrits WBJ, et al: Is hyperho-
AJH–AUGUST 1998 –VOL. 11, NO. 8, PART 1
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mocysteinemia a risk factor for recurrent venous thrombosis? Lancet 1995;345:882– 885. 10.
Boushey CJ, Beresford SA, Omenn GS, et al: A quantitative assessment of plasma homocysteine as a risk factor for vascular disease: probable benefits of increasing folic acid intakes. JAMA 1995;274:1049 –1057.
11.
Blom HJ, VandeMolen E: Pathobiochemical implications of hyperhomocysteinemia. Fibrinolysis 1991;8(suppl 2): 86– 87.
12.
Frosst P, Blom HJ, Milos R, et al: A candidate genetic risk factor for vascular disease: a common mutation in methylene tetrahydrofolate reductase. Nat Genet 1995; 10:111–113.
1023
15.
Tsai JC, Wang H, Perrella MA, et al: Induction of cyclin A gene expression by homocysteine in vascular endothelial cell activator. J Clin Invest 1986;97:146 –153.
16.
Rodger GM, Kante WH: Activation of endogenous factor V by a homocysteine-induced vascular endothelial cell activator. J Clin Invest 1986;97:1370 –1376.
17.
Lentz SR, Sadler JE: Inhibition of thrombomodulin surface expression and protein C activation by the thrombogenic agent homocysteine. J Clin Invest 1991;88:1906 – 1916.
18.
Rodger GM, Conn MT: Homocysteine, an atherogenic stimulus reduces protein C activation by arterial and venous endothelial cells. Blood 1990;75:895–901.
13.
Special report from the National Institute of Neurological Disorders and Stroke: Classification of cerebrovascular disease III. Stroke 1990;21:637– 676.
19.
Hajjar KA: Homocysteine-induced modulation of tissue plasminogen activator binding to its endothelial cell membrane receptor. J Clin Invest 1993;91:2873–2879.
14.
Tsai JC, Perrella MA, Yoshizumi M, et al: Promotion of vascular smooth muscle cell growth by homocysteine: a link to atherosclerosis. Proc Natl Acad Sci USA 1994; 91:6369 – 6373.
20.
Wilken DEL, Wang XL, Sim AS, et al: Distribution in healthy and coronary populations of the methylenetetrahydrofolate reductase (MTHFR) C677 T mutation. Arterioscler Thromb Vasc Biol 1996;16:878 – 882.
1998;11:1019 –1023
BRIEF COMMUNICATIONS
Methylenetetrahydrofolate Reductase Gene Polymorphism Relation to Blood Pressure and Cerebrovascular Disease Yukiko Nakata, Tomohiro Katsuya, Seiju Takami, Noriyuki Sato, Yuxiao Fu, Kazuhiko Ishikawa, Shin Takiuchi, Hiromi Rakugi, Tetsuro Miki, Jitsuo Higaki, and Toshio Ogihara
Hyperhomocysteinemia is reported to be associated with an increase in the incidence of ischemic heart disease and cerebrovascular disease. Genetic aberrations in methylenetetrahydrofolate reductase (MTHFR) may account for reduced enzyme activity and elevated plasma homocysteine level. A recent report revealed that a common mutation (677C to T; Ala to Val) in the MTHFR gene is associated with decreased specific MTHFR activity and with increased risk for coronary artery disease in the homozygous state (Val/Val). In the present study, we investigated whether the MTHFR gene is a genetic risk factor for cerebrovascular disease (CVD). To undertake a case-control study, we selected the patients with cerebral infarction (n 5 48) or cerebral hemorrhage (n 5 35) and examined the association between MTHFR gene polymorphism and CVD. The genotype distribution of the MTHFR gene was not significantly different between cases and controls. Because the possibility of matching the morbidity
H
of the effects of hypertension, the lack of association could not be excluded in the first study; however, we also examined whether the MTHFR mutation was associated with any clinical risk factor for CVD or with hypertension. It turned out that the subjects with the Val allele of the MTHFR gene had significantly lower blood pressure than the subjects with other genotypes in the general population (P 5 .02), and that the frequency of the Val/Val genotype in hypertensive subjects (n 5 173) was significantly lower than in control subjects (n 5 184) (P 5 .03). From these results, we conclude that the Val/Val homozygous state of the MTHFR gene increased the risk of thrombosis, but reduced the blood pressure, which resulted in the lack of increased risk for CVD. Am J Hypertens 1998;11:1019 –1023 © 1998 American Journal of Hypertension, Ltd. KEY WORDS:
Hyperhomocysteinemia, genetics, thrombosis, hypertension.
yperhomocysteinemia is known to be an independent risk factor for the development of coronary heart disease,1– 4 myocardial infarction,5 cerebrovascular dis6,7 ease (CVD), and venous thrombosis.8,9 The serum
homocysteine level is influenced by environmental as well as genetic factors. A high plasma concentration of homocysteine, derived from the demethylation of dietary methionine, may predispose to atherosclerosis by injuring the vascular endothelium, which results in
Received October 2, 1997. Accepted February 19, 1998. From the Department of Geriatric Medicine, Osaka University Medical School, Osaka, Japan. The contents of this paper were read at the twelfth meeting of the American Society of Hypertension in San Francisco, California, May 27–31, 1997.
Address correspondence and reprint requests to Toshio Ogihara, MD, PhD, Professor of Medicine, Department of Geriatric Medicine, Osaka University Medical School, 2-2 Yamada-oka, Suita, Osaka 565, Japan.
© 1998 by the American Journal of Hypertension, Ltd. Published by Elsevier Science, Inc.
0895-7061/98/$19.00 PII S0895-7061(98)00046-6
1020
NAKATA ET AL
endothelial dysfunction.10 For cardiovascular diseases, the enhanced risk associated with a 5-mol/L elevation of total plasma homocysteine was estimated to equal that of a 0.5-mmol/L increase in total cholesterol in a metaanalysis.11 A recent report revealed that a common mutation (677C to T; Ala to Val) in the methylenetetrahydrofolate reductase (MTHFR) gene is associated with decreased specific MTHFR activity and elevation of the homocysteine level in the Val/Val homozygous state.12 In this study, we investigated whether MTHFR gene polymorphism is a genetic risk factor for cerebrovascular disease. To clarify whether the MTHFR mutation itself is an independent risk factor for CVD or whether other confounding factors related to the MTHFR genotype affect the risk of CVD, we also examined the relations between MTHFR genotype polymorphism and classical risk factors for CVD. MATERIALS AND METHODS Subjects and Measurements In the present study, all subjects were Japanese and gave informed consent before participating in the research protocol, which was approved by each hospital’s ethics committee. Study 1: MTHFR Mutation and CVD Patients with documented CVD were identified from the clinical records from July 1992 to December 1995 at six hospitals in Osaka, Japan. Patients aged younger than 30 years and older than 80 years were excluded. In addition, subjects with risk factors for cardiogenic stroke, such as atrial fibrillation, valvular heart disease, endocarditis, acute myocardial infarction, dilated cardiomyopathy, and arteriosclerosis obliterans, were excluded. Final diagnosis of CVD was confirmed by brain computed tomography or brain magnetic resonance imaging. The criteria of the National Institute for Neurological Disorders and Stroke were applied in this study.13 Finally, we identified 83 patients with CVD, comprising cerebral infarction (n 5 48) and cerebral hemorrhage (n 5 35). Control subjects (n 5 105) without history of CVD were recruited from visitors to Sakuragaoka Hospital (Hyogo, Japan) who underwent general check-up from September 1991 to March 1993 (total number, 3920; 3103 men and 817 women), matching for age, gender, and morbidity of hypertension with patients. Study 2: MTHFR Mutation and Classical Risk Factors for CVD Because the morbidity of hypertension was matched between cases and controls, the effect of blood pressure on CVD risk could not be determined in Study 1. Therefore, the association between classical risk factors for CVD and MTHFR mutation was examined in the general population. The subjects (n 5 150) were recruited from the same population as in Study 1, excluding those receiving medication for hyperten-
AJH–AUGUST 1998 –VOL. 11, NO. 8, PART 1
sion, diabetes mellitus, or hyperlipidemia. Systolic and diastolic blood pressure in the sitting position, height, and body weight were obtained as physical findings. Fasting plasma glucose, total cholesterol, triglyceride, HDL cholesterol, and glycosylated hemoglobin (HbA1c) were measured by standard methods. Study 3: MTHFR Mutation and Essential Hypertension To examine whether there is a direct association between the MTHFR mutation and hypertension, patients with essential hypertension (n 5 173) and control subjects (n 5 184) were recruited from outpatients at Osaka University Medical School. All cases had a family history of hypertension in first-degree relatives and were diagnosed as having primary hypertension; those with secondary hypertension, diabetes mellitus, or apparent ischemic heart disease were excluded. The criteria for hypertension were defined as systolic blood pressure . 160 mm Hg, diastolic blood pressure . 95 mm Hg, or receiving antihypertensive therapy. Controls with no history of hypertension and without diabetes mellitus were recruited from the same population and were matched to patients for gender and age. Study 4: MTHFR Mutation and Plasma Homocysteine and Folate Levels To confirm the relationship between the MTHFR genotype and plasma homocysteine level in Japanese subjects, we measured homocysteine and folate levels using healthy middle-class volunteers in whom folate intake was adequate. Plasma homocysteine and folate levels were measured using high-performance liquid chromatography. Genotyping of MTHFR Gene Polymorphism DNA was extracted from 10 mL of whole blood using SepaGene (Sanko Junyaku Co., Tokyo, Japan). All polymerase chain reactions (PCR) to detect the MTHFR mutation were carried out with 100 ng of genomic DNA as a template, using a DNA Thermal Cycler PJ 2000 (Perkin Elmer Co., Norwalk, CT) and primers as previously reported.12 The sense and antisense primers were as follows; sense: 59TGA AGG AGA AGG TGT ATG AGG GA-39, antisense: 59-AGG ACG GTG CGG TGA GAG TG-39. Samples were amplified for 35 cycles consisting of denaturation at 94°C for 30 sec, annealing at 60°C for 60 sec, and extension at 72°C for 60 sec, followed by a final extension step at 72°C of 10 min. Because the nucleotide 677 mutation creates a restriction site for HinfI (Takara Shuzo Co. Ltd., Osaka, Japan), the genotype was determined by digestion with this enzyme, followed by 10% polyacrylamide gel electrophoresis and ethidium bromide staining. Statistical Analysis For all the statistical analyses, we used the computer software StatView 4.5J (Abacus Concepts Inc., Berkeley, CA). Alleles and genotype frequencies in cases and control subjects were com-
AJH–AUGUST 1998 –VOL. 11, NO. 8, PART 1
MTHFR GENE MUTATION AND RISK OF HYPERTENSION, CVD
1021
TABLE 1. DISTRIBUTION OF MTHFR GENOTYPES IN CVD SUBJECTS, HYPERTENSIVES, AND CONTROLS
Genotype CVD subjects Cerebral infarction
Cerebral hemorrhage Controls Hypertensives Controls
n
Ala/Ala (n [%])
Val/Ala (n [%])
Val/Val (n [%])
P
Genotype
n
83 48
31 (37.3) 19 (39.6)
41 (49.4) 23 (47.9)
11 (13.3) 6 (12.5)
NS* NS†
Hypertensives Controls
173 184
35 105 173 184
12 (34.3) 35 (33.3) 63 (36.4) 65 (35.3)
18 (51.4) 51 (48.6) 91 (52.6) 83 (45.1)
5 (14.3) 19 (18.1) 19 (11.0) 36 (19.6)
NS‡ .07
Ala/Ala, Val/Ala (n [%])
Val/Val (n [%])
154 (89.0) 19 (11.0) 148 (80.4) 36 (19.6) Odds ratio 5 1.97 (95% CI: 1.08–3.59)
P , .03
Hypertensives
CI: confidence interval. Significance of difference was examined by x2 analysis. * v controls; † v cerebral hemorrhage; ‡ v cerebral infarction.
pared by x2 test. The relationship between MTHFR genotypes and classical risk factors for CVD was assessed by one-way analysis of variance (ANOVA). P , .05 was considered significant.
hypertension in subjects with the Ala allele was 1.97 (95% CI: 1.08 –3.59), suggesting that the effect of the Val allele on hypertension acts in a recessive manner.
RESULTS Study 1: MTHFR Mutation and CVD The prevalence of hypertension was 70.1% in the CVD subjects and 65.8% in controls, supporting the possibility that the effect of hypertension was excluded in this study. The MTHFR genotype was clearly determined in all subjects. Genotype distribution was not significantly different from the Hardy-Weinberg equilibrium. The genotype distribution was not significantly different between the CVD and control, hemorrhage and control, infarction and control, or hemorrhage and infarction groups (Table 1). Furthermore, the MTHFR genotype was not associated with other classic risk factors for CVD (data not shown). Study 2: MTHFR Mutation and Classical Risk Factors for CVD Because we could not determine the genetic effect of MTHFR on blood pressure in Study 1, we examined whether MTHFR genotypes are associated with blood pressure in the general population. Both systolic and diastolic blood pressures were associated with the MTHFR genotype (F value 5 4.1, P , .02) (Figure 1). The subjects with the Val allele (Val/ Val and Val/Ala) had lower blood pressure than subjects with homozygous Ala alleles, for both systolic and diastolic blood pressures. However, no significant difference between heterozygous Val/Ala and homozygous Val/Val subjects was detected. Study 3: MTHFR Mutation and Essential Hypertension We also carried out a case-control study of hypertension. The frequency of the Val/Val genotype was significantly lower in hypertensives than in controls (P , .03) (Table 1). The estimated odds ratio for
FIGURE 1. Plasma homocysteine and folate levels in healthy subjects and association between MTHFR polymorphism and blood pressure. Association between MTHFR genotype and blood pressure was examined by ANOVA. Statistical significance differences between two groups was examined by Fisher’s PLSD test. (*P , .05, #P , .01). Ala/Ala, homozygous alanine allele; Val/ Ala, heterozygous valine and alanine alleles; Val/Val, homozygous alanine allele.
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Study 4: MTHFR Mutation and Plasma Homocysteine and Folate Levels To exclude the heterogeneity of diet, we measured the fasting plasma homocysteine and folic acid levels in normal middle-class volunteers. Both homocysteine and folate levels were associated with the MTHFR genotype. Subjects with the homozygous Val/Val genotype showed significantly higher homocysteine levels and lower folate levels than subjects with the homozygous Ala allele (Figure 1). DISCUSSION The main finding of the present study is that the Val allele of the MTHFR gene is not associated with cerebrovascular disease but is associated with lower blood pressure. Recent studies revealed that the Ala to Val mutation in the MTHFR gene is associated with increased plasma homocysteine levels and with thrombotic disease.1– 4 Plasma homocysteine levels were also higher in our study (Study 4) in subjects with the Val/Val genotype than in those with other genotypes (Figure 2), suggesting that the homozygous state associated with this mutation has an exaggerated hyperhomocysteinemic response and increased risk for thrombotic diseases, such as cerebrovascular disease. Recently, it was reported that homocysteine inhibits endothelial cell proliferation and stimulates vascular smooth muscle cell proliferation.14,15 Furthermore, there are some reports that homocysteine enhances endothelial cell-associated factor V activity16 and inhibits thrombomodulin surface expression,17 protein C activation,18 and tissue plasminogen activator binding.19 These results suggest that homocysteine may be involved in the atherosclerotic and thrombotic process by modulating vascular cell proliferation and promoting prothrombotic activities in the vascular wall. Thus, we hypothesized that the MTHFR mutation might increase the risk for CVD, and carried out an association study. However, we could not detect an association between the mutation of the MTHFR gene and CVD. In contrast, we found a unique association between the MTHFR gene and blood pressure in the general population. Subjects with the Val allele of the MTHFR gene showed lower blood pressure, both systolic and diastolic (Study 2). Furthermore, the frequency of the Val/Val genotype was significantly lower in hypertensive subjects than in control subjects (Study 3). A high homocysteine level in subjects with the homozygous Val/Val genotype was also confirmed in the Japanese population (Study 4). Summarizing the results of the studies,1– 4 the effects of the Val allele of the MTHFR gene were to increase the plasma homocysteine level, to increase the risk for thrombosis through hyperhomocysteinemia, and to decrease blood pressure. However, it did not increase
the CVD risk. Consequently, we speculate that the Val allele of the MTHFR gene increases the relative risk for thrombosis by increasing plasma homocysteine levels and decreasing blood pressure, which results in the lack of association between the MTHFR gene and CVD. Even though Wilken et al reported that the Val allele of the MTHFR gene was not a risk factor for coronary heart disease but that there was a weak association between the MTHFR gene and hypertension only in men,20 the discrepancy between their results and ours seems to be due to the strong effect of body mass index (BMI) on blood pressure or ethnic differences. In the present study, we observed no association between the MTHFR genotype and BMI, which was strongly associated with the MTHFR mutation in Wilken’s report. Though previous findings led us to believe that the Val allele favors an increase in blood pressure through stimulation of vascular smooth muscle cell proliferation, our study showed that the Val allele was associated with lower blood pressure. Considering other reports and our results, it seems that MTHFR gene polymorphisms have another role in the compensatory factor mechanism that attenuates the risk for CVD. We conclude that the Val allele of the MTHFR gene is not a genetic risk of CVD through its offset mechanism, predisposing individuals to thrombosis but lowering blood pressure. REFERENCES 1.
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