The methylenetetrahydrofolate reductase gene polymorphism in Koreans with coronary artery disease

International Journal of Cardiology 78 (2001) 13–17 www.elsevier.com / locate / ijcard

The methylenetetrahydrofolate reductase gene polymorphism in Koreans with coronary artery disease a, b a a a Chul-Hyun Kim *, Kyu-Yoon Hwang , Tai-Myung Choi , Won-Yong Shin , Sae-Yong Hong a

Department of Internal Medicine, Soonchunhyang University Chunan Hospital, 23 -20 Bongmyung-Dong, Chunan, Chungnam, 330 -100, Republic of Korea b Department of Preventive Medicine, School of Medicine, Soonchunhyang University, Chungnam, Republic of Korea Received 22 March 2000; received in revised form 2 October 2000; accepted 18 October 2000

Abstract Hyperhomocysteinemia is a known risk factor of cardiovascular diseases. Methylenetetrahydrofolate reductase (MTHFR), involved in folate-dependant metabolism, is associated with homocysteine levels. We studied the associations among MTHFR genotypes, coronary artery disease (CAD), and homocysteine levels in 85 patients with CAD and 152 healthy subjects. The MTHFR genotypes and plasma homocysteine levels were determined. No significant difference in mutation of the MTHFR gene between two groups was observed (P.0.05). While the homozygous mutant genotype (V/ V) had the highest homocysteine levels compared to wild (A /A) and heterozygous mutant (A / V) genotypes, there were no significant differences in homocysteine levels among the MTHFR genotype groups. Homocysteine was significantly and inversely related to folate levels, the significant association in V/ V genotype (b coefficient521.954, P50.04). Our data suggested that MTHFR polymorphism was not associated with homocysteine levels, implying no association between gene polymorphism and CAD in Koreans.  2001 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Coronary artery disease; Homocysteine; Methylenetetrahydrofolate reductase; Polymorphism

1. Introduction Elevated plasma homocysteine has been identified as a risk factor for coronary atherosclerosis [1]. Hyperhomocysteinemia is caused by nutritional deficiencies or genetic defects in one of the enzymes involved in homocysteine metabolism. Methylenetetrahydrofolate reductase (MTHFR) catalyzes the reduction of 5,10 methylene tetrahydrofolate to 5 methyl tetrahydrofolate, the predominant circulatory

*Corresponding author. Tel.: 182-41-570-2125; fax: 182-41-5745762. E-mail address: [email protected] (C.-H. Kim).

form of folate and methyl donor for the remethylation of homocysteine to methionine. The role of the enzyme MTHFR in patients with coronary artery disease (CAD) has been assessed and found to be an abnormal thermolabile variant of this enzyme [2]. This MTHFR variant decreases specific MTHFR activity and results in elevated homocysteine concentrations in plasma [3]. It has been suggested that the variant gene could represent an important risk factor for vascular diseases [5,6]. However, recent studies found that the mutation of the MTHFR gene does not appear to be significantly associated with the risk for CAD [7]. There is considerable concern whether this mutation is important in the genesis of vascular disease [7–10].

0167-5273 / 01 / $ – see front matter  2001 Elsevier Science Ireland Ltd. All rights reserved. PII: S0167-5273( 00 )00431-9

14

C.-H. Kim et al. / International Journal of Cardiology 78 (2001) 13 – 17

Therefore, this study was designed to examine the prevalence of the MTHFR 677 mutation to assess the associations among MFHFR gene, CAD, and homocysteine levels in Koreans.

2. Materials and methods

2.1. Study subjects The study population was comprised of 152 healthy subjects and 85 patients with CAD. Healthy subjects were recruited through health examinations. Exclusions were made for individuals with any clinical diseases such as hypertension or ischemic heart disease. The patients diagnosed angiographically were enrolled at Soonchunhyang University Chunan Hospital (SUCH). Patients were considered as eligible subjects if a coronary angiogram revealed approximately 50% stenosis of at least one coronary artery. They were evaluated by historical ECG and enzyme documentation. The coronary angiogram was interpreted independently by two experienced cardiologists. All subjects did not take any vitamin supplements and showed normal renal function. This study was approved by the Institutional Board at SUCH. All subjects provided written informed consent, and participation was voluntary.

2.2. Biochemical assay

homocysteine, vitamin B 12 , and folate levels among genotype groups were assessed by ANOVA. The relationship between homocysteine and folate was assessed by correlation and simple linear regression analysis with stratification by the MTHFR gene. A value of P,0.05 was considered statistically significant. All statistical analysis was performed using a Stata program (Stata Release 5, College station, Texas).

3. Results

3.1. General characteristics Mean (S.D.) age of CAD patients was 61.6 (9.3) years old. Female patients accounted for 35.3% of CAD and were significantly older age (64.0 years old for females, 60.3 years old for males). Among 85 patients, heart attack signs were observed in 76.5%. Diabetes and hypertensive patients were 21.2% and 22.9%, respectively. 54.1% of the patients were current smokers.

3.2. Prevalence of the thermolabile MTHFR mutation The overall V allele frequency of the 677 C to T transition on the MTHFR gene did not show significant difference (33.5% for CAD patients, 31.6% for healthy subjects) (Fig. 1). No difference in genetic

After subjects fasted for 12 h, their blood was collected, centrifuged for plasma at 2000 g at 48C for 30 min, and stored at 2708C until assay. Total plasma homocysteine concentrations were determined by high performance liquid chromatography by using fluorescent detecting kits (HP1090 series II HPLC / FLD) [11]. Plasma folate and vitamin B 12 levels were measured using a radioassay method. Identification of the 677C to T transition in the MTHFR gene was performed as previous described [2,3].

2.3. Statistical analysis Data were presented as mean (S.D.) for continuous variables and frequency for categorical variables. Genotypic differences between two study groups were assessed using an x 2 test. Differences in

Fig. 1. Prevalence of methylenetetrahydrofolate (MTHFR) genetic polymorphism in coronary artery disease (CAD) patients and healthy subjects. Note on genotypes: A /A, homozygous wild type (alanine / alanine); A / V, heterozygous mutant (alanine / valine); V/ V, homozygous mutant (valine / valine).

C.-H. Kim et al. / International Journal of Cardiology 78 (2001) 13 – 17

15

Table 1 Homocysteine, folate, and vitamin B12 levels by methylene tetrahydrofolate (MTHFR) genetic polymorphism in coronary artery disease (CAD) patients MTHFR gene

Homocysteine (mmol / l) Mean (S.D.)

Folate (ng / ml) Mean (S.D.)

Vitamin B12 (pg / ml) Mean (S.D.)

Homozygous wild type (alanine / alanine) Heterozygous mutant (alanine / valine) Homozygous mutant (valine / valine) Total P-value*

12.2 (3.5) 12.6 (3.1) 13.5 (6.0) 12.6 (3.8) 0.588

4.8 (2.7) 4.0 (2.0) 4.6 (1.7) 4.4 (2.3) 0.342

802.5 (754.9) 651.4 (307.5) 766.5 (288.7) 723.7 (509.6) 0.445

*By one-way analysis of variance (ANOVA).

variation between two groups was observed (P5 0.862). Age and sex were not related to MTHFR genotypes in both groups (data not presented) [4].

3.3. Homocysteine, vitamin B12 , and folate levels The summary of homocysteine, folate, and vitamin B 12 concentrations is presented in Table 1. Overall mean homocysteine concentration in CAD patients was 12.6 mmol / l. Although the homozygous mutant genotype (V/ V) had the highest homocysteine concentration, no significant differences were observed for other genotypes (A /A and A / V) (P50.588). There were no significant differences for folate and vitamin B 12 among the MTHFR genotypic groups (P50.342 and 0.445, respectively).

3.4. Relation between homocysteine and folate Significant negative correlation between homocysteine and folate was found, but not with vitamin B 12 . The Pearson’s correlation coefficient (r) was 20.272 (P50.01) in CAD patients. In separate regression analysis, only homozygous mutant (V/ V) showed significant inverse relation (b coefficient521.954, P50.04, n514). Scatterplots and simple regression lines between homocysteine and folate are displayed in Fig. 2.

4. Discussion No associations between the MTHFR 677 polymorphism and coronary artery disease (CAD) were found in Koreans. Our result differed from Hirouki’s study, which reported that the V/ V genotype of MTHFR was significantly associated with CAD in

Japanese [6]. However, recent studies have reported the lack of association between the MTHFR mutation and CAD. Thus, the association is still controversial (Table 2). The distribution of MTHFR polymorphism was similar between CAD patients and control subjects [12]. Although it seems that the homozygous mutant (V/ V) is more common in Asians [6] than in Caucasians [12,13], generally the prevalence rates of gene variants were similar in CAD and the general population [7,8,]. Several studies have reported that elevated homocysteine concentrations were found in the homozygous mutant genotype (V/ V), especially combined with low folate [13–15]. Our results were consistent with other studies, but not statistical different among genotypes. Recently Chang et al. reported plasma homocysteine levels in healthy Koreans [16] showing that the homocysteine levels of 25th, 50th, 75th percentiles were 7.02 mmol / l, 9.61 mmol / l, and 12.4 mmol / l, respectively. In the current study, we observed more elevated plasma homocysteine concentrations in CAD patients than healthy Korean men (12.6 vs. 10.7 mmol / l). Consistent results of inverse relation between homocysteins and folate were observed in the current study as in previous reports [10,13,16]. The correlation coefficient was more significant and greater in the MTHFR homozygous mutant (V/ V) than in heterozygote (A / V) or homozyous wild (A /A) genotypes. This finding suggests homozygotes for this mutation have an exaggerated hyperhomocysteinic response to folate depletion. This study had some limitations. First, since the study was cross-sectional in design, we could not establish a temporal association. Second, healthy subjects were not confirmed by an angiographic finding as were CAD patients. Third, although age

C.-H. Kim et al. / International Journal of Cardiology 78 (2001) 13 – 17

16

Fig. 2. Scatterplots and regression lines between homocysteine and folate by MTHFR genetic polymorphism in CAD patients.

and sex were not significant confounding variables, other risk factors of CAD were not assessed and controlled. Finally, no association between MTHFR genotypes and CAD was likely due to the relatively

small number of study subjects and uncontrolled study design. In conclusion, despite the clear effect of the MTHFR polymorphism on increasing homocysteine

Table 2 Summary of methylenetetrahydrofolate (MTHFR) genetic polymorphism by selected studies Author (year)

Country

Subject

Ma et al. (1998)

USA

CAD Control

Verhoef et al. (1998)

USA

Kluijtmans et al. (1997)

No.

Genotype, n (%)

Finding

A /A

A/V

V/ V

293 290

136 (46.4) 137 (47.2)

124 (42.3) 116 (40.0)

33 (11.3) 39 (13.4)

Negative

CAD Control

500 500

230 (46.0) 228 (45.6)

209 (41.8) 200 (40.0)

61 (12.2) 72 (14.4)

Negative

Netherlands

CAD Control

735 1250

337 (45.9) 617 (49.4)

328 (44.6) 527 (42.2)

Morita et al. (1997)

Japan

CAD Control

362 778

117 (32.3) 338 (43.4)

188 (51.9) 361 (46.4)

57 (15.7) 79 (10.2)

Positive

¨ Tokgozoglu et al. (1999)

Turkey

CAD Control

151 91

69 (45.8) 47 (51.7)

71 (46.8) 39 (43.1)

11 (7.4) 5 (5.2)

Negative

70 (9.5) 106 (8.5)

Positive

C.-H. Kim et al. / International Journal of Cardiology 78 (2001) 13 – 17

levels, we did not observe associations among the MTHFR genotypes, the risk of CAD, and homocysteine concentration in Koreans. These results suggest that MTHFR gene is related to homocysteine metabolism but does not predict the risk of CAD as a susceptible genetic marker. Further controlled study is recommended to clarify the hypothesis.

References [1] Boushey CJ, Beresford SAA, Omenn GS, Motulsky AG. A quantitative assessment of plasma homocysteine as a risk factor for vascular disease: probable benefit of increasing folic acid intake. J Am Med Assoc 1995;274:1049–57. [2] Kang SS, Wong PWK, 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– 45. [3] Kang SS, Passen EL, Ruggie N, Wong PWK. Sora, H., Thermolabile defect of methylenetetrahydrofolate reductase in coronary artery disease. Circulation 1993;88:1463–9. [4] Frost P, Blom MJ, Lios R. A candidate genetic risk factor for vascular disease: a common mutation in methylenetetrahydrofolate reductase. Nat Genet 1995;10:111–3. [5] Kluijtmans LAJ, Kastelein JJP, Lindemans J et al. Thermolabile methylenetetrahydrofolate reductase in coronary artery disease. Circulation 1997;96:2573–7. [6] Morita H, Taguchi J, Kurihara H et al. Genetic polymorphism of 5,10 methylenetetrahydrofolate reductase (MTHFR) as a risk factor for coronary artery disease. Circulation 1997;95:2032–6. [7] Anderson JL, King GJ, Thomson MJ et al. A mutation in the methylenetetrahydrofolate reductase gene is not associated with increased risk for coronary artery disease or myocardial infarction. J Am Coll Cardiol 1997;30:1206–11.

17

[8] Bockxmeer FM, Mamotte CDS, Vasikaran SD, Tsyler RR. Methylenetetrahydrofolate reductase gene and coronary artery disease. Circulation 1997;95:21–3. [9] Verhoef P, Rimm EB, Hunter DJ, Chen J, Willett WC, Kelsey K, Stampfer MJ. A common mutation in the methylenetetrahydrofolate reductase gene and risk of coronary heart disease: result among U.S. men. J Am Coll Cardiol 1998;32:353–9. [10] Schwartz SM, Sisovick DS, Malinow MR et al. Myocardial infarction in young women in relation to plasma total homocysteine, folate, and a common variant in the methylenetetrahydrofolate reductase gene. Circulation 1997;96:412–7. [11] Ubbink JB, Vermaak WJH, Bissbort S. Rapid high performance liquid chromatographic assay for total homocysteine level in human serum. J Chromatogr 1991;565:441–6. [12] Ma J, Stampfer MJ, Henneken CH et al. Methylenetetrahydrofolate reductase polymorphism, plasma folate, homocysteine, and risk of myocardial infarction in US physicians. Circulation 1998;98:2510– 6. ¨ ¨ L, Wilcken DEL, Ohrvik [13] Brattstrom J, Brudin L. Common methylenetetrahydrofolate reductase gene mutation leads to hyperhomocysteinemia but not to vascular disease: the result of a metaanalysis. Circulation 1998;98:2520–6. ¨ ¨ [14] Tokgozoglu SL, Alikasifoglu M, Unsal I et al. Methylenetetrahydrofolate reductase genotype and the risk and extent of coronary artery disease in a population with low plasma folate. Heart 1999;81:518–22. [15] Deloughery TG, Evans A, Sadeghi A et al. Common mutation in methylenetetrahydrofolate reductase. Correlation with homocysteine metabolism and late onset vascular disease. Circulation 1996;94:3074–8. [16] Jang YS, Cho EU, Lee JH, Chung NS. Relationship between plasma homocysteine level and cardiovascular risk factors in healthy men. Kor Circ J 1999;29:135–45.