The methylenetetrahydrofolate reductase gene is associated with increased cardiovascular risk in Japan, but not in other populations

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1 Atherosclerosis 153 (2000) The methylenetetrahydrofolate reductase gene is associated with increased cardiovascular risk in Japan, but not in other populations Sun Ha Jee a, *, Terri H. Beaty b, Il Suh c, Young-sup Yoon d, Lawrence J. Appel b a Department of Epidemiology and Disease Control, Yonsei Uni ersity Graduate School of Health Science and Management, CPO POB 8044, Seoul, South Korea b Department of Epidemiology, Johns Hopkins Uni ersity School of Hygiene and Public Health, Baltimore, MD, USA c Department of Pre enti e Medicine and Public Health, Yonsei Uni ersity College of Medicine, Seoul, South Korea d Di ision of Cardiology, Yonsei Cardio ascular Center, Yonsei Uni ersity College of Medicine, Seoul, South Korea Received 7 June 1999; received in revised form 13 December 1999; accepted 14 January 2000 Abstract The methylenetetrahydrofolate reductase (MTHFR) gene has been associated with increased risk for cardiovascular disease in some, but not all studies. Our data sources included a MEDLINE search of the literature published before December 1998, a bibliography review, and expert consultation. Of 23 studies initially identified, 18 (9855 persons) met the inclusion criteria. Information on sample size, study design, Hardy Weinberg equilibrium, method of genotype determination, plasma folate and homocysteine were abstracted by two reviewers using a standardized protocol. The overall odds ratio of the MTHFR gene on cardiovascular disease was estimated using the Mantel Haenzel method. From 12 studies with angiographically-confirmed coronary disease (CAD), the overall odds ratio (OR) for CAD among those with heterozygous (V/A) was 1.3 (95% CI, ), while it was 1.4 ( ) for the homozygous mutant (V/V) compared to those with homozygous normal (A/A). However, the overall odds ratio for CAD among those with the V/V genotype versus A/A genotype was not statistically significant (OR: 1.1; 95% CI: ) after excluding three Japanese studies. The corresponding OR for the three Japanese studies was 2.0 ( ). For six studies with myocardial infarction (MI), the overall OR of MI was 1.0 ( ) for those with the V/A genotype and 0.9 ( ) for those with the V/V genotype, respectively; none of these ORs for MI was statistically significant. The MTHFR gene is associated with increased risk for CAD in Japan, but not in other populations Elsevier Science Ireland Ltd. All rights reserved. Keywords: Genotype; MTHFR; Coronary disease; Myocardial infarction; Racial difference 1. Introduction * Corresponding author. Tel.: ; fax: address: jsunha@yumc.yonsei.ac.kr (S.H. Jee). Over the past decade, mild hyperhomocysteinemia has been recognized as a risk factor for occlusive arterial disease and thrombosis [1 5]. A meta-analysis of 27 studies investigating the relation between fasting total homocysteine (thcy) levels and coronary disease (CAD) yielded an odds ratio of 1.6 for men and 1.8 for women for every 5 mol/l increase in thcy plasma levels [6]. Homocysteine levels are influenced by environmental (folate, vitamin B6, and vitamin B12 intake) as well as genetic factors [2,3,7 9]. A common mutation of enzyme methylenetetrahydrofolate reductase (MTHFR) has been implicated in the development of hyperhomocysteinemia [10,11] and CAD. A missense mutation in the gene encoding MTHFR has recently been described as the molecular basis for this defect. MTHFR activity in the V/V genotype has been found to be reduced [12] and thcy significantly elevated [12,13] compared with the A/A and V/A genotypes. One [24], but not the other [40], meta-analysis has identified a positive association between the MTHFR gene and increased risk for CAD. Results of these /00/$ - see front matter 2000 Elsevier Science Ireland Ltd. All rights reserved. PII: S (00)

2 162 S.H. Jee et al. / Atherosclerosis 153 (2000) studies have been inconsistent, perhaps because of racial differences, small sample sizes or other design features. Pooling the results of case-control studies provided a means of exploring the basis for heterogeneity in study outcomes. The objective of this study was to quantitatively summarize the evidence for a relationship between the MTHFR gene, coronary disease and myocardial infarction. 2. Methods 2.1. Study selection Relevant studies in humans were identified by searching the MEDLINE database for the years 1988 through The search combined the following terms; methylenetetrahydrofolate reductase (MTHFR), homocysteine and cardiovascular disease. In addition, reference lists of identified studies and review articles were examined for other relevant studies. Finally, experts in the field were surveyed to identify additional studies. Twenty-three case-control studies [14 35] were identified. These articles were reviewed by two authors (Jee and Yoon) to determine whether they met predetermined criteria for inclusion in our subsequent analysis. Areas of disagreement or uncertainty were adjudicated by consensus. To be included, a study must have included the following criteria: [1] been conducted in humans; [2] assessed MTHFR genotype determination as an exposure using standardized laboratory methods; [12] and [3] reported the number of cases and controls with the different MTHFR genotypes. Studies [15,18 21,23 27] of CAD used angiographically confirmed occlusion as outcomes, while studies of myocardial infarction (MI) used WHO criteria for MI [16 18] which relies on symptoms, enzyme elevations or electrocardiographic changes, and clinical/past history [14,22,23] Data abstraction Information was abstracted on general characteristics (name of first author, year of publication, country of origin, sample size, mean age, gender of participants and body mass index), study design (case and control selection criteria), folate intake, plasma folate and thcy in the study population, method of MTHFR genotype determination including information on the polymerase chain reaction and enzyme used, and information on Hardy Weinberg equilibrium. If different outcomes were employed in the same report, they were analyzed as separate studies [18,23]. When cases of CAD or MI could not be separated, these studies were not included [27 34] Statistical analysis Alleic and genotypic frequencies were determined from observed genotype counts, and the expectations of the Hardy Weinberg equilibrium were evaluated by 2 analysis. Comparisons between genotypic frequencies were done using 2 analysis. A meta-analysis consists of tests for association and homogeneity. Homogeneity testing assesses the homogeneity of the different odds ratios determined in respective studies. The overall test for association then assesses the significance of the association between the C677T mutation and CAD or MI for all studies combined. To calculate the pooled effects of the MTHFR gene, each study was assigned a weight consisting of the reciprocal of variance of its odds-ratio estimate [36]. Estimations of the mean odds ratio of cardiovascular disease associated with those having the MTHFR gene and those with 95% confidence intervals were calculated using the Mantel Haenszel method [26]. 3. Results Table 1 shows the definition or criteria of cases and controls. The case-control studies were conducted between 1996 and 1998, and varied in size from 84 to 735 cases and from 73 to 1250 controls. The total number of participants was 9855 (4594 cases and 5261 controls). In 16 studies, healthy individuals were used as controls. In the remaining two studies, those subjects angiographically-confirmed as normal were used as controls. CAD patients with stenosis greater than 50% in at least one coronary were used as the CAD case group in eight studies [15] [18 21,23,25] and [27] whereas the four remaining studies [14,26,32,33] did not indicate which criteria of angiography was employed. In two studies for CAD and three studies for MI, the control group was matched for age and sex, whereas the remaining 13 studies did not indicate whether controls were matched for age and sex. All of the studies used a standardized laboratory method [12] to determine the MTHFR genotype. Three studies were conducted in Japan [20,25,33]; the others were conducted in Australia [15,19], the United States [18,23], Netherlands [21,24], Italy [26,32], and Germany [27]. Participant characteristics for the 18 case-control studies included in this meta-analysis are presented in Table 2. All of the studies were conducted in adults. Among cases, the mean age of participants was 60 (range of across studies). The corresponding mean age among controls was 56 (range of 53 65). Ten of the 18 studies included both men and women, while men were the sole participants in two studies. The remaining five studies did not specify the percentage of men.

3 S.H. Jee et al. / Atherosclerosis 153 (2000) Table 3 shows the genotypic and allelic distribution of the MTHFR gene and its Hardy Weinberg equilibrium. In all the studies included, the distributions of the genotypes were in the Hardy Weinberg equilibrium for both patients and controls. From 12 studies with angiographically-confirmed coronary disease (CAD), the frequency of the three genotypes among controls was A/A (homozygous normal), 43.6%; V/A (heterozygous), 45.2%; and V/V (homozygous mutant, 677 C T), 11.2%. These frequencies in cases were 42.1% for A/A, 43.9% for V/A, and 13.2% for V/V Coronary disease Twelve case-control studies examined the risk of CAD associated with those having the MTHFR gene. Genotype VV increased in the risk of CAD compared to the corresponding AA group in eight (66.7%) of the 12 studies. In just two of these studies, the relationship was statistically significant. Table 4 provides the overall odds ratios of CAD across these 12 case-control studies. Compared to the A/A genotype, the overall odds ratio for CAD was 1.3 (95% CI ) for the V/A genotype and 1.4 ( ) for the V/V genotype. However, after excluding the three Japanese studies [20,25,33] which had a strong association between MTHFR and CAD [20,25], the corresponding CAD odds ratios for the V/V orv/a genotypes were not statistically significant (1.1, ) and (1.1, ), respectively. For the three Japanese studies [20,25,33] the overall odds ratios were 1.6 ( ) for the VA genotype and 2.0 ( ) for the VV genotype. Table 1 Case and control characteristics of 18 case-control studies Authors (year) Cases Controls Number, outcome Selection criteria Number Selection criteria Izumi (1996) Schmitz (1996) 250 CAD 190 MI Angiographically demonstrated ischemic heart disease Clinical history and creatinine kinase rise Subjects without ischemic heart disease Random from residents, age and sex matched Wilcken (1996) 109 CAD 50% occlusion in at least in one coronary 225 Volunteers participating in a heart health education program Adams (1996) 310 MI WHO criteria 222 Adults visitors to patients with non-cardiovascular illness Ma (1996) 293 MI WHO criteria 290 Participants who were free of MI; matching of age, sex, and smoking Brugada a (1997a) 155 CAD 50% occlusion in at least in one coronary 155 Healthy Caucasian individuals, matched for age and sex Brugada (1997b) van Bockxmeer 79 MI 139 CAD WHO criteria 50% occlusion in at least in one coronary Healthy Caucasian individuals, matched for age and sex Residents of metropolitan Perth, with no (1997) history Morita (1997) 362 CAD 50% occlusion in at least in one coronary 778 Healthy volunteers from annual health examination Veroef (1997) 131 CAD 90% occlusion in one and 40 occlusion 100 Population-based controls in one additional coronary Schwartz (1997) 79 MI Cardiovascular health study 386 Residents of King, Pierce or Snohomish counties Anderson b 510 CAD 60% occlusion in at least in one coronary % occlusion in all major vessels (1997a) Anderson 200 MI History of MI 554 Patients selected on basis of being healthy (1997b) Kluijtmas (1997) 735 CAD Angiographically assessed CAD 1250 Healthy people Ou (1998) 510 CAD 60% occlusion in at least in one coronary 168 Patients with 10% luminal obstruction Girelli (1998) 278 CAD Angiographically documented multivessel 137 Angiographically documented normal CAD coronary arteries Reinhardt (1998) 180 CAD 50% occlusion in at least in one coronary 105 Healthy volunteers Abbate (1998) 84 CAD Angiographically documented CAD 106 Healthy subjects in the same age bracket a Brugada (1997): a, outcome was a CAD; b, outcome was a MI. b Anderson (1997): a, outcome was a CAD; b, outcome was a MI.

4 164 S.H. Jee et al. / Atherosclerosis 153 (2000) Table 2 Participants and study design characteristics of 18 case-control studies Author (year) Country Age (years) Percentage of males Plasma folate (nmol/l) Body mass index (kg/m 2 ) Mean Range Case Control Case Control Case Control Case Control Case Control Izumi (1996) Japan Schmitz (1996) USA Wilcken (1996) Australia c Adams (1996) UK Ma (1996) USA Brugada a (1997a) USA Brugada (1997b) USA van Bockxmeer (1997) Australia Morita (1997) Japan c Veroef (1997) Netherlands Schwartz (1997) USA Anderson (1997a) b USA Anderson (1997b) USA Kluijtmas (1997) Netherlands Ou (1998) Japan Girelli (1988) Italy Reinhardt (1998) Germany Abbate (1998) Italy Overall a Brugada (1997): a, outcome was a CAD; b, outcome was a MI. b Anderson (1997): a, outcome was a CAD; b, outcome was a MI. Note: means data was not available. c We assumed body mass index of case group was as same as that of control group.

5 S.H. Jee et al. / Atherosclerosis 153 (2000) Table 3 Genotypic and alleic distributions of the MTHFR polymorphism and Hardy Weinberg equilibrium of 18 case-control studies Authors (year) C677T(V) allelic frequency Genotype frequency (n) Hardy Weinberg equilibrium (%) Case Control Cases Controls Cases Cases VV VA AA VV VA AA 2 P-value 2 P-value Izumi (1996) Schmitz (1996) Wilcken (1996) Adams (1996) Ma (1996) Brugada a (1997a) Brugada (1997b) van Bockxmeer (1997) Morita (1997) Veroef (1997) Schwartz (1997) Anderson b (1997a) Anderson (1997b) Kluijtmas (1997) Ou (1998) Girelli (1988) Reinhardt (1998) Abbate (1998) Overall a Brugada (1997): a, outcome was a CAD; b, outcome was a MI. b Anderson (1997): a, outcome was a CAD; b, outcome was a MI. Note: means data was not available. Table 4 Odds ratios (OR) of coronary disease associated with MTHFR gene in 12 case-control studies No. of studies VV vs AA a VA vs AA a OR 95% CI P-value OR 95% CI P-value Overall Japanese studies [20,25,33] excluded Japanese studies [20,25,33] only a VV, homozygous for the mutant; VA, heterozygous; AA, homozygous normal Myocardial infarction Six studies examined the relationship between the risk of myocardial infarction (MI) and the MTHFR gene. Genotype VV increased the risk of MI compared to the corresponding AA group in four (66.7%) of six studies; while in none of these studies was the lower bound of the 95% confidence interval greater than one. Compared to those with the A/A genotype, the overall odds ratio for MI associated with the MTHFR gene was 1.0 ( ) among those with the V/A genotype and 0.9 ( ) among those with the V/V genotype (each P 0.05) Coronary disease or myocardial infarction Using data from all 18 studies, the occurrence of CAD or MI was assessed by the MTHFR genotype. Compared to the A/A genotype, the overall odds ratio for the combination of CAD and MI was 1.1 (95% CI, ) for the V/A genotype and 1.2 ( ) for the V/V genotype. After excluding the three Japanese studies, the corresponding odds ratios were 1.0 ( ) and 1.0 ( ). 4. Discussion Overall, compared with homozygous normal (A/A), the homozygous mutant (V/V) of the MTHFR gene was associated with about a 10% increased risk of the combination of CAD and MI, and a 40% increased risk of coronary disease, but not with myocardial infarction. However, the significant association of the

6 166 S.H. Jee et al. / Atherosclerosis 153 (2000) MTHFR gene on the combination of CAD and MI, and CAD disappeared after excluding three Japanese studies. The discrepancy between the results from the Japanese studies and other populations is unclear and may be due to differences in nutritional intake of co-factors required for the MTHFR pathway, such as vitamin B12 or folate, or to other ethnic differences (e.g. weight, BMI). Over the past decade, a consensus has emerged that elevated plasma homocysteine is an independent risk factor for coronary heart disease [37 39]. MTHFR, a folate-dependent enzyme, is essential for the remethylation of homocysteine. Frosst et al. [12] identified a single common mutation of the MTHFR gene that causes reduced activity and thermolability of the enzyme; while homozygosity for this mutant allele results in significantly-elevated plasma thcy levels. Therefore, in the Japanese studies, the finding of an increased risk of CAD associated with MTHFR is not surprising because MTHFR is strongly associated with an increased risk of plasma homocysteine. In particular, Morita [20] reported that 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. In our meta-analysis, the V allele has a codominant effect on coronary arterial risk among the Japanese studies. Previously, Kang et al. [10] reported that thermolabile MTHFR, which is thought to be correlated with the V/V genotype, can be an inherited risk factor for CAD. They documented a prevalence of thermolabile MTHFR of 17% in patients with CAD and of 5% in control subjects. Frosst et al. [12] documented that plasma homocysteine levels were significantly higher in patients with the V/V genotype than in patients with the A/A or V/A genotype. Although a difference in fasting homocysteine levels was small, previous studies have suggested that plasma homocysteine levels after dietary methionine loading are greatly affected by this mutation. These findings suggested that the V/V genotype of MTHFR, which can cause a predisposition to increased plasma homocysteine levels, may itself be an independent genetic risk factor for CAD. However, our meta analysis does not support the hypothesis that the MTHFR gene is an independent predictor of developing CAD. Still, the MTHFR gene was a strong predictor for CAD in Japanese individuals, who have a low fat diet and are generally lean. According to the present study, when BMI is high, the MTHFR gene has little effect on CAD (Fig. 1). Wilcken et al. [15] reported that the MTHFR mutation was associated with increased BMI, although MTHFR was still not associated with the number of diseased vessels. Therefore, it will be of great interest to determine the impact of this polymorphism in other lean populations, e.g. other East-Asian countries, and subsamples of other populations, e.g. populations with a lower intake of folate. In this study, unfortunately, we could not examine this hypothesis because only four studies reported the plasma intake level. According to the Frosst study [15], when folate consumption is high, this common genetic variant has little effect on thcy levels. It will be of great interest to determine the impact of this polymorphism in a general population sample with a lower intake of folate, as well as among younger individuals. Overall, we found no association between the MTHFR genotype and the risk of MI. However, because only six studies included this outcome, these analyses have relatively little power to test for an association between the risk of MI and MTHFR gene mutation. Schmitz (1996) [14] reported that only the survivors of MI entered their study, and this group may not be representative of all MI cases. However, in all the studies included, the distribution of genotypes were in Hardy Weinberg equilibrium for both patients and Fig. 1. Odds ratios of methylenetetrahydrofolate reductase gene on coronary disease by countries studied.

7 S.H. Jee et al. / Atherosclerosis 153 (2000) controls, which meant that there was no evidence of selected samples of cases and controls. Our results raise several important research questions. Specifically, what factors, genetic and environmental, account for the apparent association of MTHFR genotypes with CAD in Japan? In that country, the population tends to be leaner and to have lower levels of serum cholesterol than Western countries. Does leanness or lower cholesterol modify the risk of a relationship between MTHFR and CAD? Additional research, e.g. prospective observational studies in lean populations and those with a low intake of folate, is warranted. Acknowledgements This study was supported by a grant (No. HMP-98- M ) of the 1998 Good Health R&D Project, Ministry of Health and Welfare, Korea. References [1] Wilcken DEL, Wilcken B. The pathogenesis of coronary disease: a possible role for methionine metabolism. 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