This Article Abstract Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Morita, H. Articles by Yazaki, Y. Search for Related Content PubMed PubMed Citation Articles by Morita, H. Articles by Yazaki, Y.

(Circulation. 1997;95:2032-2036.)
© 1997 American Heart Association, Inc.

Articles

Genetic Polymorphism of 5,10-Methylenetetrahydrofolate Reductase (MTHFR) as a Risk Factor for Coronary Artery Disease

Hiroyuki Morita, MD; Jun-ichi Taguchi, MD; Hiroki Kurihara, MD; Masao Kitaoka, MD; Hideaki Kaneda, MD; Yukiko Kurihara, MD; Koji Maemura, MD; Tohru Minamino, MD; Minoru Ohno, MD; Kazuhide Yamaoki, MD; Ken Ogasawara, MD; Tadanori Aizawa, MD; Shin Suzuki, MD; Yoshio Yazaki, MD

the Third Department of Internal Medicine (H.M., H. Kurihara, Y.K., K.M., T.S., T.M., K.Y., Y.Y.) and the First Department of Internal Medicine (J.T., M.O.), Faculty of Medicine, University of Tokyo; Sakakibara Heart Institute (M.K.); Cardiovascular Institute Hospital, Tokyo (H. Kaneda, K.O., T.A.); and Chiba-nishi Hospital, Chiba (S.S.), Japan.

Correspondence to Hiroki Kurihara, MD, The Third Department of Internal Medicine, Faculty of Medicine, University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113, Japan. E-mail kuri-tky{at}umin.u-tokyo.ac.jp

	Abstract

Top Abstract Introduction Methods Results Discussion References

 
Background Epidemiological studies have identified hyperhomocyst(e)inemia as an independent risk factor for coronary artery disease (CAD). Recently, the alanine/valine (A/V) polymorphism of the 5,10-methylenetetrahydrofolate reductase (MTHFR) gene, one of the key enzymes catalyzing remethylation of homocysteine, has been reported. The VV genotype correlates with increased plasma homocyst(e)ine levels as a result of the reduced activity and increased thermolability of this enzyme. In this study, we examined the distribution of the MTHFR genotypes in Japanese men and the association between the VV genotype and CAD.

Methods and Results The diagnoses of CAD of all the studied patients were confirmed by coronary angiography. The MTHFR genotype was analyzed by PCR followed by HinfI digestion. In 778 healthy male subjects, the frequency of the V allele was 0.33, comparable to that in a French Canadian population. In 362 patients with CAD, the VV genotype was significantly more frequent than in control subjects (16% versus 10%, P=.0067). The association of the VV genotype with CAD was further increased in patients with 99% stenotic lesions (18%, P=.0010), whereas no significant association with the VV genotype was observed in patients without a 99% stenosis. When the genotype frequency was compared among patients with different numbers of stenotic coronary arteries, the frequency of the VV genotype was significantly higher in patients with triple-vessel disease (26%) than in patients with single- or double-vessel disease (15% and 14%, respectively).

Conclusions The VV genotype of MTHFR was also common in the Japanese population and was significantly associated with CAD. The frequency of this genotype in particular was correlated with the severity of disease. The VV genotype associated with a predisposition to increased plasma homocyst(e)ine levels may represent a genetic risk factor for CAD.

Key Words: genetics • myocardial infarction • risk factors • coronary disease

	Introduction

Top Abstract Introduction Methods Results Discussion References

 
Homocysteine is a sulfur-containing amino acid generated as an intermediate product in methionine metabolism. Three key enzymes essential for normal homocysteine metabolism have been identified: cystathionine ß-synthase, methyltetrahydrofolate homocysteine methyltransferase, and MTHFR. Recently, hyperhomocyst(e)inemia has emerged as an independent risk factor for atherosclerosis.^{1 2} Homocystinuria, a rare autosomal recessive disease due to cystathionine ß-synthase deficiency, is characterized by premature atherosclerosis and thromboembolism as well as ocular, skeletal, and neurogenic complications.³ ,⁴ Heterozygosity for cystathionine ß-synthase deficiency and other causes, such as vitamins B₆, B₁₂, or folate deficiency, can lead to milder homocyst(e)inemia, which has also been shown to be associated with atherosclerosis and thrombotic diseases, including CAD.^{5 6 7 8 9 10} A recent study indicated that only a mild increase in plasma homocyst(e)ine levels was associated with a high incidence of acute myocardial infarction.¹¹

Abnormalities of MTHFR, the enzyme catalyzing remethylation of homocysteine, can also cause hyperhomocyst(e)inemia. The clinical features of severe MTHFR deficiency are reminiscent of those of cystathionine ß-synthase deficiency. A variant of MTHFR with reduced activity and increased thermolability has been reported to be associated with the development of CAD.¹² Recently, Frosst et al¹³ demonstrated that a common point mutation causing Ala-to-Val substitution correlates with the characteristics of thermolabile MTHFR. The plasma homocyst(e)ine levels in individuals homozygous for this mutation were significantly higher than those of the other individuals.¹³ These findings suggest that this mutation may be a risk factor for CAD. In the present study, we analyzed the genotype of MTHFR for this mutation in a Japanese male population. Then we compared the frequency of the genotypes between patients with CAD and normal subjects to identify this mutation of the MTHFR gene as an important coronary risk factor.

	Methods

Top Abstract Introduction Methods Results Discussion References

 
Subjects
The study population comprised 778 healthy subjects and 362 patients with CAD. All the subjects were men of Japanese origin. Healthy volunteers (26 to 86 years old; mean, 48±10 years) were recruited at the annual health examination at a city clinic. Patients with CAD (>50% narrowing in at least one major coronary artery) were consecutively enrolled at the time of diagnostic cardiac catheterization. The mean age of all the patients was 62±9 years. Coronary angiograms were interpreted by two or more independent, experienced cardiologists. At study entry, 202 patients had clinical evidence of acute or previous MI, 20 had crescendo-type unstable angina, and 140 had stable angina. Informed consent was obtained from every subject after a full explanation of the study, which was approved by the Ethics Committee of the University of Tokyo.

By means of coronary angiography, the patient population was divided into patients with 99% stenotic lesion (group A, n=218; mean age, 61±9 years) and patients without 99% lesion (group B, n=144; mean age, 62=9 years). The percentage of patients with clinical evidence of MI was 86% in group A and 10% in group B. Each subpopulation was further divided into three subgroups on the basis of the number of stenotic major coronary arteries.

Genetic Analysis
Venous blood samples were collected in tubes containing Na₂EDTA and applied to genomic DNA extracting columns (QIAamp blood kit, Qiagen) according to the manufacturer's protocol. PCR was performed on the genomic DNA samples with a GeneAmp PCR kit (Perkin-Elmer Cetus) and primers as previously reported.¹³ The sense and antisense primers were 5'-TGAAGGAGAAGGTGTCTGCGGGA-3' and 5'-AGGACGGTGCGGTGAGAGTG-3', respectively. Thirty-five cycles (95°C for 60 seconds, 62°C for 90 seconds, 72°C for 60 seconds) were used to amplify 198-bp products. The amplified fragments were cut with HinfI, which can recognize the C-to-T substitution in the fragments. Because this one nucleotide substitution corresponds to a conversion of Ala-to-Val residue in the MTHFR encoding region, the two different alleles were designated A (Ala) and V (Val). The 198-bp fragment derived from the A allele is not digested by HinfI, whereas the fragment of the same length from the V allele is digested by HinfI into 175- and 23-bp fragments. The HinfI-treated PCR fragments were electrophoresed in 9.6% polyacrylamide gels and stained with ethidium bromide.

Measurement of Plasma Homocyst(e)ine Levels
In 198 of the enrolled patients with CAD, we measured plasma homocyst(e)ine levels at the time of coronary angiography. Fasting venous blood was drawn on the morning of catheterization, and plasma homocyst(e)ine levels were determined as total homocysteine by high performance liquid chromatography with fluorescence detection as previously described.¹⁴

Statistical Analysis
Quantitative data were analyzed with univariate analysis with the Mann-Whitney rank-sum test and expressed as mean±SD. Qualitative data were analyzed with a ² test. The relationship between the MTHFR genotype and CAD was assessed by a linear trend test. A value of P<<.05 was considered significant.

	Results

Top Abstract Introduction Methods Results Discussion References

 
Distribution of MTHFR Genotypes in Healthy Japanese Men
First, we analyzed the distribution of the MTHFR genotypes in a Japanese population. As shown in the previous report by Frosst et al,¹³ the three MTHFR genotypes for Ala-to-Val mutation (AA, AV, and VV) were diagnosed by digestion of the 198-bp PCR products by HinfI. In the screening of 778 healthy Japanese men, the allele frequency of the V mutation was 0.33. The distribution of the three genotypes was as follows: AA genotype, 43.4%; AV genotype, 46.4%; and VV genotype, 10.2%. This genotype distribution was compatible with the Hardy-Weinberg equilibrium.

To rule out the age dependence of the MTHFR genotype distribution, we divided the subjects into two subgroups according to age and compared the genotype distribution between the subgroups. As shown in Table 1, the distribution of the MTHFR genotype was almost identical between the two subgroups and compatible with Hardy-Weinberg equilibrium in each group, suggesting that there is no apparent selection for a specific MTHFR genotype, at least around 40 years of age. In the following study, all the 778 normal subjects served as the control group.

View this table: [in this window] [in a new window]   Table 1.

Association of VV Genotype of MTHFR With CAD
Next, we examined the distribution of the MTHFR genotypes in Japanese male patients with CAD. Of the 362 patients, the AA genotype was found in 32.3%, the AV genotype in 51.9%, and the VV genotype in 15.7% (Table 1). The allele frequency of the V mutation was significantly higher in the CAD group than in the control group (0.42 versus 0.33, P=.0001). The association was stronger in homozygous than in heterozygous patients (odds ratios of 2.08 and 1.50, respectively; P<<.0001), suggesting a codominant effect of the V allele on coronary risk. The VV genotype was significantly more frequent in patients with CAD than in healthy subjects (P=.0067). The odds ratio of the VV genotype for CAD was 1.65. Clinical characteristics for the three genotype groups of the 362 patients are shown in Table 2. There was no difference among the genotypes for any of the variables examined.

View this table: [in this window] [in a new window]   Table 2.

The association of the VV genotype with CAD was further studied in terms of the severity of disease (Table 3). In group A (patients with 99% stenotic lesions), the association between the VV genotype and CAD was highly significant (P=.0010), with the odds ratio compared with the normal subjects of 2.0. In group B (patients without 99% lesions), in contrast, no significant association between the VV genotype and CAD was observed. In each group, the correlation between the VV genotype and CAD and the number of stenotic major coronary arteries was tested. In group A, the frequencies of the VV genotype were 15%, 14%, and 26% for patients with single-, double-, and triple-vessel disease, respectively. The frequency of the VV genotype in patients with triple-vessel disease was significantly higher than that in patients with single- or double-vessel disease (P=.031) and higher than that in normal subjects (P<.0001). In group B, no association was detected between the VV genotype and the number of stenotic coronary arteries.

View this table: [in this window] [in a new window]   Table 3.

Correlation Between MTHFR Genotype and Plasma Homocyst(e)ine Levels
Plasma homocyst(e)ine levels were determined in 198 of 362 patients with CAD. Plasma homocyst(e)ine levels were significantly higher in patients with the VV genotype than in patients with the AA or AV genotype (16.4±6.2 µmol/L, n=29, versus 14.5±3.6 µmol/L, n=169; P=.021).

	Discussion

Top Abstract Introduction Methods Results Discussion References

 
The major findings of the present study are that the mutation of MTHFR reported by Frosst et al¹³ is also common in the Japanese population and that this mutation is significantly associated with CAD. In particular, the frequency of this mutation was correlated with the severity of stenotic lesions (the presence of 99% stenosis) and the number of stenotic coronary arteries, suggesting that this mutation is closely associated with the severity of CAD and the occurrence of MI.

The allele frequency of the mutation (designated the V allele) in the Japanese (Mongoloid) population was 0.33, which is comparable to that in the French Canadian (Caucasian) population reported by Frosst et al (Table 1).¹³ The distribution was compatible with Hardy-Weinberg equilibrium. These results suggest that this mutation is highly prevalent beyond ethnic groups and can be regarded as a balanced polymorphism that escaped natural selection through the long history of humanity. Such a polymorphism does not usually cause a serious lethal disorder as a single factor. This idea is supported by the present finding that the distribution of the MTHFR genotype is not grossly affected by age. Rather, it is likely that such a polymorphism can contribute to the pathogenesis of prevalent multifactorial diseases, such as atherosclerosis, in cooperation with other factors, such as environmental ones.

The present study first demonstrated the association between the genotype homozygous for this mutation (the VV genotype) and CAD. The trend test on genotypes suggested that the V allele has a codominant effect on the coronary risk. In addition, the VV genotype and the severity of CAD were significantly correlated. Because no difference in conventional coronary risk factors was detected among the genotypes, the association of the VV genotype with CAD seems to be independent of other risk factors. Previously, Kang et al¹² reported that thermolabile MTHFR, which is thought to be correlated with the VV genotype, can be an inherited risk factor for CAD. They demonstrated that a prevalence of thermolabile MTHFR was found in 17% of patients with CAD and 5% of control subjects. These numbers are comparable to the frequency of the VV genotype in our present study. In addition, plasma homocyst(e)ine levels were significantly higher in patients with the VV genotype than patients with the AA or AV genotype. Although the difference in plasma homocyst(e)ine levels is small, previous studies suggest that plasma homocyst(e)ine levels after dietary methionine loading are greatly affected by this mutation.¹³ These findings strongly suggest that the VV genotype of MTHFR, which can cause a predisposition to increased plasma homocyst(e)ine levels, is an independent genetic risk factor for CAD.

In addition to epidemiological studies that revealed the association between hyperhomocyst(e)inemia and vascular diseases, experimental studies have suggested that high plasma homocyst(e)ine levels can cause atherogenic and thrombotic states. Harker et al⁵ demonstrated that infusion of homocysteine into baboons resulted in patchy desquamation of vascular endothelium in an acute phase and neointimal formation composed of proliferating smooth muscle cells in a chronic phase, which was prevented by antiplatelet agents. Although the precise mechanism for the atherogenic effects of homocysteine has not been elucidated, various in vitro studies have proposed possible targets of homocysteine. Homocysteine has a direct cytotoxic effect on cultured endothelial cells, which is prevented by catalase.^{15 16 17} Recently, Tsai et al^{18 19} reported that homocysteine inhibits endothelial cell proliferation, whereas homocysteine induces cyclin D1 and cyclin A expression and stimulates vascular smooth muscle cell proliferation. Furthermore, homocysteine enhances endothelial cell–associated factor V activity²⁰ and inhibits thrombomodulin surface expression,²¹ protein C activation,²² tissue-type plasminogen activator binding,²³ and anticoagulant heparan sulfate expression²⁴ in endothelial cells. Homocysteine is also shown to increase thromboxane A₂ formation in platelets, which may be coupled with platelet activation.²⁵ Thus, homocysteine may contribute to the atherosclerotic and thrombotic process by modulating vascular cell proliferation and promoting prothrombotic activities in the vascular wall. These effects of homocysteine may explain the close correlation between the VV genotype and the presence of CAD.

Recently, several studies have shown that plasma concentrations of folate and vitamins B₆ and B₁₂ are negatively correlated with plasma homocyst(e)ine levels,^{7 26 27 28} and increased intake of these vitamins can reduce plasma homocyst(e)ine levels in patients with CAD.^{29 30 31} At present, the beneficial effect of lowering plasma homocyst(e)ine by vitamins on cardiovascular risk has not yet been established. The identification of the VV genotype of MTHFR as a genetic coronary risk factor may contribute to the understanding of the pathological role of homocysteine and the effectiveness of vitamin supplementation for the prevention of CAD.

After we submitted the present article for publication, a report by Wilcken et al³² on the distribution of the MTHFR genotype in patients with CAD was published. They demonstrated that the MTHFR genotype is not associated with CAD in an Australian population. The discrepancy between their results and ours may be due to ethnic differences. Inclusion of both sexes in the study by Wilcken et al may be another explanation, but they stated that the distribution of the genotypes between male and female patients was the same. Further studies are needed to assess which populations are susceptible to this novel coronary risk factor.

	Selected Abbreviations and Acronyms

 

CAD = coronary artery disease MI = myocardial infarction MTHFR = methylenetetrahydrofolate reductase PCR = polymerase chain reaction

	Acknowledgments

 
This work was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, and Culture, Japan; the Sankyo Foundation; Kowa Life Science Foundation; Japan Cardiovascular Research Foundation; Kanae Foundation of Research for New Medicine; TMFC; and the Ryoichi Naito Foundation for Medical Research. We wish to thank Midori Hayashi for her excellent technical assistance and Dr Isamu Ono (Nikon Clinic) for recruitment of healthy volunteers. We also wish to thank Dr Takashi Kadowaki (University of Tokyo) for helpful discussion.

	Footnotes

 
The first two authors contributed equally to this work.

Received August 16, 1996; revision received November 19, 1996; accepted November 25, 1996.

	References

Top Abstract Introduction Methods Results Discussion References

 

1. McCully KS. Homocysteine and vascular disease. Nat Med. 1996;2:386-389.[Medline] [Order article via Infotrieve]
   
2. Mayer EL, Jacobsen DW, Robinson K. Homocysteine and coronary atherosclerosis. J Am Coll Cardiol. 1996;27:517-527.[Abstract]
   
3. Boers GH, Smals AG, Trijbels FJ, Fowler B, Bakkeren JA, Schoonderwaldt HC, Kleijer WJ, Kloppenborg PW. Heterozygosity for homocystinuria in premature peripheral and cerebral occlusive arterial disease. N Engl J Med. 1985;313:709-715.[Abstract]
   
4. Mudd SH, Skovby F, Levy HL, Pettigrew KD, Wilcken B, Pyeritz RE, Andria G, Boers GH, Bromberg IL, Cerone R, Fowler B, Grobe H, Schmidt H, Schweitzer L. The natural history of homocystinuria due to cystathionine beta-synthase deficiency. Am J Hum Genet. 1985;37:1-31.[Medline] [Order article via Infotrieve]
   
5. Harker LA, Ross R, Slichter SJ, Scott CR. Homocystine-induced arteriosclerosis: the role of endothelial cell injury and platelet response in its genesis. J Clin Invest. 1976;58:731-741.
   
6. Malinow MR, Kang SS, Taylor LM, Wong PWK, Coull B, Inahara T, Mukerjee D, Sexton G, Upson B. Prevalence of hyperhomocyst(e)inemia in patients with peripheral arterial occlusive disease. Circulation. 1989;79:1180-1188.[Abstract/Free Full Text]
   
7. Clarke R, Daly L, Robinson K, Naughten E, Cahalane S, Fowler B, Graham I. Hyperhomocysteinemia: an independent risk factor for vascular disease. N Engl J Med. 1991;324:1149-1155.[Abstract]
   
8. Selhub J, Jacques PF, Bostom AG, D'Agostino RB, Wilson PW, Belanger AJ, O'Leary DH, Wolf PA, Schaefer EJ, Rosenberg IH. Association between plasma homocysteine concentrations and extracranial carotid-artery stenosis. N Engl J Med. 1995;332:286-291.[Abstract/Free Full Text]
   
9. den Heijer M, Koster T, Blom HJ, Bos GMJ, Briet E, Reitsma PH, Vandenbroucke JP, Rosendaal FR. Hyperhomocysteinemia as a risk factor for deep-vein thrombosis. N Engl J Med. 1996;334:759-762.[Abstract/Free Full Text]
   
10. Mandel H, Brenner B, Berant M, Rosenberg N, Lanir N, Jakobs C, Fowler B, Seligsohn U. Coexistence of hereditary homocystinuria and factor V Leiden: effect on thrombosis. N Engl J Med. 1996;334:763-768.[Abstract/Free Full Text]
    
11. Stampfer MJ, Malinow MR, Willett WC, Newcomer LM, Upson B, Ullmann D, Tishler PV, Hennekens CH. A prospective study of plasma homocyst(e)ine and risk of myocardial infarction in US physicians. JAMA. 1992;268:877-881.[Abstract]
    
12. Kang SS, Wong PW, Susmano A, Sora J, Norusis M, Ruggie N. Thermolabile methylenetetrahydrofolate reductase: an inherited risk factor for coronary artery disease. Am J Hum Genet. 1991;48:536-545.[Medline] [Order article via Infotrieve]
    
13. Frosst P, Blom HJ, Milos R, Goyette P, Sheppard CA, Matthews RG, Boers GJ, den Heijer M, Kluijtmans LA, van den Heuvel LP, Rozen R. A candidate genetic risk factor for vascular disease: a common mutation in methylenetetrahydrofolate reductase. Nat Genet. 1995;10:111-113.[Medline] [Order article via Infotrieve]
    
14. Araki A, Sako Y. Determination of free and total homocysteine in human plasma by high-performance liquid chromatography with fluorescence detection. J Chromatogr. 1987;422:43-52.[Medline] [Order article via Infotrieve]
    
15. Wall RT, Harlan JM, Harker LA, Striker GE. Homocysteine-induced endothelial cell injury in vitro: a model for the study of vascular injury. Thromb Res. 1980;18:113-121.[Medline] [Order article via Infotrieve]
    
16. Starkebaum G, Harlan JM. Endothelial cell injury due to copper-catalyzed hydrogen peroxide generation from homocysteine. J Clin Invest. 1986;77:1370-1376.
    
17. Dudman NP, Hicks C, Lynch JF, Wilcken DE, Wang J. Homocysteine thiolactone disposal by human arterial endothelial cells and serum in vitro. Arterioscler Thromb. 1991;11:663-670.[Abstract/Free Full Text]
    
18. Tsai J-C, Perrella MA, Yoshizumi M, Hsieh CM, Haber E, Schlegel R, Lee ME. Promotion of vascular smooth muscle cell growth by homocysteine: a link to atherosclerosis. Proc Natl Acad Sci U S A. 1994;91:6369-6373.[Abstract/Free Full Text]
    
19. Tsai J-C, Wang H, Perrella MA, Yoshizumi M, Sibinga ES, Tan LC, Haber E, Chang TH-T, Schlegel R, Lee ME. Induction of cyclin A gene expression by homocysteine in vascular smooth muscle cells. J Clin Invest. 1996;97:146-153.[Medline] [Order article via Infotrieve]
    
20. Rodgers GM, Kane WH. Activation of endogenous factor V by a homocysteine-induced vascular endothelial cell activator. J Clin Invest. 1986;77:1909-1916.
    
21. Lentz SR, Sadler JE. Inhibition of thrombomodulin surface expression and protein C activation by the thrombogenic agent homocysteine. J Clin Invest. 1991;88:1906-1914.
    
22. Rodgers GM, Conn MT. Homocysteine, an atherogenic stimulus, reduces protein C activation by arterial and venous endothelial cells. Blood. 1990;75:895-901.[Abstract/Free Full Text]
    
23. Hajjar KA. Homocysteine-induced modulation of tissue plasminogen activator binding to its endothelial cell membrane receptor. J Clin Invest. 1993;91:2873-2879.
    
24. Nishinaga M, Ozawa T, Shimada K. Homocysteine, a thrombogenic agent, suppresses anticoagulant heparan sulfate expression in cultured porcine aortic endothelial cells. J Clin Invest. 1993;92:1381-1386.
    
25. Di Minno G, Davi G, Margaglione M, Cirillo F, Grandone E, Ciabattoni G, Catalano I, Strisciuglio P, Andria G, Patrono C, Mancini M. Abnormally high thromboxane biosynthesis in homozygous homocystinuria: evidence for platelet involvement and probucol-sensitive mechanism. J Clin Invest. 1993;92:1400-1406.
    
26. Israelsson B, Brattstrom LE, Hultberg BL. Homocysteine and myocardial infarction. Atherosclerosis. 1988;71:227-233.[Medline] [Order article via Infotrieve]
    
27. Wu LL, Wu J, Hunt SC, James BC, Vincent GM, Williams RR, Hopkins PN. Plasma homocyst(e)ine as a risk factor for early familial coronary artery disease. Clin Chem. 1994;40:552-561.[Abstract/Free Full Text]
    
28. Pancharuniti N, Lewis CA, Sauberlich HE, Perkins LL, Go RC, Alvarez JO, Macaluso M, Acton RT, Copeland RB, Cousins AL, Gore TB, Cornwell PE, Roseman JM. Plasma homocyst(e)ine, folate, and vitamin B12 concentrations and risk for early-onset coronary artery disease. Am J Clin Nutr. 1994;59:940-948.[Abstract/Free Full Text]
    
29. Dudman NP, Wilcken DE, Wang J, Lynch JF, Macey D, Lundberg P. Disordered methionine/homocysteine metabolism in premature vascular disease: its occurrence, cofactor therapy, and enzymology. Arterioscler Thromb. 1993;13:1253-1260.[Abstract/Free Full Text]
    
30. Landgren F, Israelsson B, Lindgren A, Hultberg B, Andersson A, Brattstrom L. Plasma homocysteine in acute myocardial infarction: homocysteine-lowering effect of folic acid. J Intern Med. 1995;237:381-388.[Medline] [Order article via Infotrieve]
    
31. Naurath HJ, Joosten E, Riezler R, Stabler SP, Allen RH, Lindenbaum J. Effects of vitamin B12, folate, and vitamin B6 supplements in elderly people with normal serum vitamin concentrations. Lancet. 1995;346:85-89.[Medline] [Order article via Infotrieve]
    
32. Wilcken DEL, Wang XL, Sim AS, McCredie RM. Distribution in healthy and coronary populations of the methylenetetrahydrofolate reductase (MTHFR) C677T mutation. Arterioscler Thromb Vasc Biol. 1996;16:878-882.[Abstract/Free Full Text]
    

This article has been cited by other articles:

				A. L. Moens, C. J. Vrints, M. J. Claeys, J.-P. Timmermans, H. C. Champion, and D. A. Kass Mechanisms and potential therapeutic targets for folic acid in cardiovascular disease Am J Physiol Heart Circ Physiol, May 1, 2008; 294(5): H1971 - H1977. [Abstract] [Full Text] [PDF]

				B. Smolkova, M. Dusinska, K. Raslova, M. Barancokova, A. Kazimirova, A. Horska, V. Spustova, and A. Collins Folate levels determine effect of antioxidant supplementation on micronuclei in subjects with cardiovascular risk Mutagenesis, November 1, 2004; 19(6): 469 - 476. [Abstract] [Full Text] [PDF]

				J J McCarthy, A Parker, R Salem, D J Moliterno, Q Wang, E F Plow, S Rao, G Shen, W J Rogers, L K Newby, et al. Large scale association analysis for identification of genes underlying premature coronary heart disease: cumulative perspective from analysis of 111 candidate genes J. Med. Genet., May 1, 2004; 41(5): 334 - 341. [Abstract] [Full Text] [PDF]

				L. D. Spotila, P. F. Jacques, P. B. Berger, K. V. Ballman, R. C. Ellison, and R. Rozen Age Dependence of the Influence of Methylenetetrahydrofolate Reductase Genotype on Plasma Homocysteine Level Am. J. Epidemiol., November 1, 2003; 158(9): 871 - 877. [Abstract] [Full Text] [PDF]

				R. Meleady, P. M Ueland, H. Blom, A. S Whitehead, H. Refsum, L. E Daly, S. E. Vollset, C. Donohue, B. Giesendorf, I. M Graham, et al. Thermolabile methylenetetrahydrofolate reductase, homocysteine, and cardiovascular disease risk: the European Concerted Action Project Am. J. Clinical Nutrition, January 1, 2003; 77(1): 63 - 70. [Abstract] [Full Text] [PDF]

				M. Klerk, P. Verhoef, R. Clarke, H. J. Blom, F. J. Kok, E. G. Schouten, and and the MTHFR Studies Collaboration Group MTHFR 677C->T Polymorphism and Risk of Coronary Heart Disease: A Meta-analysis JAMA, October 23, 2002; 288(16): 2023 - 2031. [Abstract] [Full Text] [PDF]

				B. N Ames, I. Elson-Schwab, and E. A Silver High-dose vitamin therapy stimulates variant enzymes with decreased coenzyme binding affinity (increased Km): relevance to genetic disease and polymorphisms Am. J. Clinical Nutrition, April 1, 2002; 75(4): 616 - 658. [Abstract] [Full Text] [PDF]

				M.C. Verhaar, E. Stroes, and T.J. Rabelink Folates and Cardiovascular Disease Arterioscler. Thromb. Vasc. Biol., January 1, 2002; 22(1): 6 - 13. [Abstract] [Full Text] [PDF]

				K. Matsuo, R. Suzuki, N. Hamajima, M. Ogura, Y. Kagami, H. Taji, E. Kondoh, S. Maeda, S. Asakura, S. Kaba, et al. Association between polymorphisms of folate- and methionine-metabolizing enzymes and susceptibility to malignant lymphoma Blood, May 15, 2001; 97(10): 3205 - 3209. [Abstract] [Full Text] [PDF]

				H. R. Thompson, G. M. Jones, and M. R. Narkewicz Ontogeny of hepatic enzymes involved in serine- and folate-dependent one-carbon metabolism in rabbits Am J Physiol Gastrointest Liver Physiol, May 1, 2001; 280(5): G873 - G878. [Abstract] [Full Text] [PDF]

				T. Kosokabe, K. Okumura, T. Sone, J. Kondo, H. Tsuboi, H. Mukawa, T. Tomida, T. Suzuki, H. Kamiya, H. Matsui, et al. Relation of a Common Methylenetetrahydrofolate Reductase Mutation and Plasma Homocysteine With Intimal Hyperplasia After Coronary Stenting Circulation, April 24, 2001; 103(16): 2048 - 2054. [Abstract] [Full Text] [PDF]

				Z. Chen, A. C. Karaplis, S. L. Ackerman, I. P. Pogribny, S. Melnyk, S. Lussier-Cacan, M. F. Chen, A. Pai, S. W.M. John, R. S. Smith, et al. Mice deficient in methylenetetrahydrofolate reductase exhibit hyperhomocysteinemia and decreased methylation capacity, with neuropathology and aortic lipid deposition Hum. Mol. Genet., March 1, 2001; 10(5): 433 - 443. [Abstract] [Full Text] [PDF]

				L.A.J Kluijtmans and A.S Whitehead Methylenetetrahydrofolate reductase genotypes and predisposition to atherothrombotic disease. Evidence that all three MTHFR C677T genotypes confer different levels of risk Eur. Heart J., February 2, 2001; 22(4): 294 - 299. [Abstract] [PDF]

				S.-M. Saw, J.-M. Yuan, C.-N. Ong, K. Arakawa, H.-P. Lee, G. A Coetzee, and M. C Yu Genetic, dietary, and other lifestyle determinants of plasma homocysteine concentrations in middle-aged and older Chinese men and women in Singapore Am. J. Clinical Nutrition, February 1, 2001; 73(2): 232 - 239. [Abstract] [Full Text] [PDF]

				H. Morita, H. Kurihara, S. Yoshida, Y. Saito, T. Shindo, Y. Oh-hashi, Y. Kurihara, Y. Yazaki, and R. Nagai Diet-Induced Hyperhomocysteinemia Exacerbates Neointima Formation in Rat Carotid Arteries After Balloon Injury Circulation, January 2, 2001; 103(1): 133 - 139. [Abstract] [Full Text] [PDF]

				E. E Delvin, R. Rozen, A. Merouani, J. Genest Jr, and M. Lambert Influence of methylenetetrahydrofolate reductase genotype, age, vitamin B-12, and folate status on plasma homocysteine in children Am. J. Clinical Nutrition, December 1, 2000; 72(6): 1469 - 1473. [Abstract] [Full Text] [PDF]

				T C F Sykes, C Fegan, and D Mosquera Thrombophilia, polymorphisms, and vascular disease Mol. Pathol., December 1, 2000; 53(6): 300 - 306. [Abstract] [Full Text]

				J. C. Chambers, H. Ireland, E. Thompson, P. Reilly, O. A. Obeid, H. Refsum, P. Ueland, D. A. Lane, and J. S. Kooner Methylenetetrahydrofolate Reductase 677 C->T Mutation and Coronary Heart Disease Risk in UK Indian Asians Arterioscler. Thromb. Vasc. Biol., November 1, 2000; 20(11): 2448 - 2452. [Abstract] [Full Text] [PDF]

				P. M Ueland, H. Refsum, S. A. Beresford, and S. E. Vollset The controversy over homocysteine and cardiovascular risk Am. J. Clinical Nutrition, August 1, 2000; 72(2): 324 - 332. [Abstract] [Full Text] [PDF]

				G. L. Booth, E. E.L. Wang, and with the Canadian Task Force on Preventive Health Preventive health care, 2000 update: screening and management of hyperhomocysteinemia for the prevention of coronary artery disease events Can. Med. Assoc. J., July 1, 2000; 163(1): 21 - 29. [Abstract] [Full Text] [PDF]

				S. Barbaux, L. A.J. Kluijtmans, and A. S. Whitehead Accurate and Rapid ""Multiplex Heteroduplexing"" Method for Genotyping Key Enzymes Involved in Folate/Homocysteine Metabolism Clin. Chem., July 1, 2000; 46(7): 907 - 912. [Abstract] [Full Text] [PDF]

				H. KIMURA, F. GEJYO, S. SUZUKI, and R. MIYAZAKI The C677T Methylenetetrahydrofolate Reductase Gene Mutation in Hemodialysis Patients J. Am. Soc. Nephrol., May 1, 2000; 11(5): 885 - 893. [Abstract] [Full Text]

				A. Mager, P. Tiqva, D. L. Brattstrom, L. Brudin, P. D. E.L. Wilcken, and J. Ohrvik Methylenetetrahydrofolate Reductase Gene and Coronary Artery Disease Response Circulation, April 25, 2000; 101 (16): e172 - e173. [Full Text] [PDF]

				R. P. Murphy, C. Donoghue, R. J. Nallen, M. D'Mello, C. Regan, A. S. Whitehead, and D. J. Fitzgerald Prospective Evaluation of the Risk Conferred by Factor V Leiden and Thermolabile Methylenetetrahydrofolate Reductase Polymorphisms in Pregnancy Arterioscler. Thromb. Vasc. Biol., January 1, 2000; 20(1): 266 - 270. [Abstract] [Full Text] [PDF]

				A. Mager, S. Lalezari, T. Shohat, Y. Birnbaum, Y. Adler, N. Magal, and M. Shohat Methylenetetrahydrofolate Reductase Genotypes and Early-Onset Coronary Artery Disease Circulation, December 14, 1999; 100(24): 2406 - 2410. [Abstract] [Full Text] [PDF]

				I. Bova, J. Chapman, C. Sylantiev, A. D. Korczyn, and N. M. Bornstein The A677V Methylenetetrahydrofolate Reductase Gene Polymorphism and Carotid Atherosclerosis Stroke, October 1, 1999; 30(10): 2180 - 2182. [Abstract] [Full Text] [PDF]

				B. M. McQuillan, J. P. Beilby, M. Nidorf, P. L. Thompson, and J. Hung Hyperhomocysteinemia but Not the C677T Mutation of Methylenetetrahydrofolate Reductase Is an Independent Risk Determinant of Carotid Wall Thickening : The Perth Carotid Ultrasound Disease Assessment Study (CUDAS) Circulation, May 11, 1999; 99(18): 2383 - 2388. [Abstract] [Full Text] [PDF]

				S L Tokgözoglu, M Alikasifoglu, I Ünsal, E Atalar, K Aytemir, N Özer, K Övünç, O Usal, S Kes, and E Tunçbilek Methylene tetrahydrofolate reductase genotype and the risk and extent of coronary artery disease in a population with low plasma folate Heart, May 1, 1999; 81(5): 518 - 522. [Abstract] [Full Text]

				J. D. Spence, M. R. Malinow, P. A. Barnett, A. J. Marian, D. Freeman, and R. A. Hegele Plasma Homocyst(e)ine Concentration, But Not MTHFR Genotype, Is Associated With Variation in Carotid Plaque Area Stroke, May 1, 1999; 30(5): 969 - 973. [Abstract] [Full Text] [PDF]

				A. Gardemann, H. Weidemann, M. Philipp, N. Katz, H. Tillmanns, F. W. Hehrlein, and W. Haberbosch The TT genotype of the methylenetetrahydrofolate reductase C677T gene polymorphism is associated with the extent of coronary atherosclerosis in patients at high risk for coronary artery disease Eur. Heart J., April 2, 1999; 20(8): 584 - 592. [Abstract] [PDF]

A. Inbal, D. Freimark, B. Modan, A. Chetrit, S. Matetzky, N. Rosenberg, R. Dardik, Z. Baron, and U. Seligsohn Synergistic Effects of Prothrombotic Polymorphisms and Atherogenic Factors on the Risk of Myocardial Infarction in Young Males Blood, April 1, 1999; 93(7): 2186 - 2190. [Abstract] [Full Text]