Genetic Polymorphism of Methylenetetrahydrofolate Reductase and Myocardial Infarction
A Case-Control Study
Background Elevated total plasma homocyst(e)ine (tHcy; the composite of homocysteine-derived moieties in their oxidized and reduced forms) levels are a risk factor for coronary heart disease, stroke, and venous thrombosis. tHcy plasma levels are influenced by folate, vitamins B6 and B12, as well as by hereditary factors. A point mutation (C677T) in the gene encoding methylenetetrahydrofolate reductase, an enzyme involved in homocysteine remethylation, has been reported to render the enzyme thermolabile and less active and has been associated with elevated tHcy in homozygous carriers (+/+ genotype) as well as with increased risk of premature cardiovascular disease.
Methods and Results We investigated whether this mutation influences risk for myocardial infarction (MI) and plasma levels of tHcy and whether this effect may be modified by dietary folate intake in 190 MI cases and 188 control subjects from the Boston Area Health Study. Genotype frequencies were 37.8% for −/−, 47.8% for +/−, and 14.4% for +/+ in the control group and 50.0% for −/−, 34.7% for +/−, and 15.3% for +/+ in the case group. The relative risk for MI associated with the +/+ genotype (compared with +/− and −/−) was 1.1 (95% CI, 0.6 to 1.9; P=.8). Stratification by folate intake values above and below the median did not significantly alter these results. Plasma tHcy levels were 9.9±2.7 μmol/L in −/− individuals, 10.6±3.8 μmol/L in +/− individuals, and 9.1±2.3 μmol/L in +/+ individuals (Ptrend=NS; determined in 68 cases and 59 control subjects).
Conclusions Our data show that homozygosity for the C677T mutation in this largely white, middle-class US population is not associated with increased risk for MI, irrespective of folate intake. This suggests that this mutation does not represent a useful marker for increased cardiovascular risk in this and in similar populations.
Elevated total plasma homocyst(e)ine (tHcy) has been reported as an independent risk factor for coronary heart disease/myocardial infarction (CHD/MI),1 2 3 stroke,4 5 and deep venous thrombosis.6 A recent meta-analysis7 of 9 studies investigating the relation between fasting tHcy levels and CHD yielded an odds ratio of 1.6 for men and 1.8 for women for every 5 μmol/L increment of tHcy plasma levels.
Plasma tHcy levels are modulated by a complex interaction of environmental and genetic factors. Vitamins B6 (pyridoxal-phosphate) and B12 (methylcobalamin) and folate are essential coenzymes in homocysteine metabolism, and a correlation between low plasma levels of folate and elevated tHcy has been documented.7 Fasting levels of tHcy reflect mainly homocysteine remethylation, which is dependent on vitamin B12 and folate, whereas transsulfuration of homocysteine (dependent on pyridoxal phosphate) is thought to be reflected by levels of tHcy after an oral methionine load.
The enzyme methylenetetrahydrofolate reductase (MTHFR) reduces 5′,10′-methylenetetrahydrofolate to 5′-methyltetrahydrofolate, the main circulating form of folate, which is a cosubstrate in the remethylation of homocysteine to methionine. Complete deficiency of MTHFR, inherited as a rare recessive mendelian trait, results in excessive accumulation of tHcy and, among other manifestations, severe atherosclerotic and thromboembolic complications.
A less severe defect of this enzyme has previously been biochemically characterized and implicated in the development of hyperhomcyst(e)inemia8 9 and CHD.8 A missense mutation in the gene encoding MTHFR has recently been described10 as the molecular basis of this defect. This mutation (C677T), in which a cytidine residue at position 677 of the gene is replaced by thymidine, introduces a novel HinfI restriction site (+ allele). It results in the substitution of an alanine residue by valine, rendering the enzyme both thermolabile and less active. MTHFR activity in the +/+ genotype has been found reduced10 and tHcy significantly elevated10 11 compared with −/− and +/− genotypes, consistent with a loss-of-function, recessive phenotype. An effect modification of the mutation by folate plasma levels has been described, indicating that increased tHcy levels as a consequence of the mutation are present only if plasma folate levels are low.12
We recently reported that elevated fasting tHcy was a graded risk factor for risk of MI in a subgroup of the Boston Area Health Study (BAHS)13 and that intake and plasma levels of folate were inversely related to risk and tHcy levels. The impaired enzymatic activity of the mutant allele may represent an additional risk factor, as recently demonstrated in a small case-control study14 where +/+ individuals had a threefold increased relative risk of premature vascular disease. We therefore explored the relationship between the MTHFR mutation, plasma levels of tHcy, folate intake, and MI in a case-control study among 190 cases of MI and 188 age- and sex-matched control subjects from the BAHS.
The details of the BAHS population have been described previously.13 Briefly, eligible patients were men and women less than 76 years of age with a confirmed diagnosis of MI, each matched to a control subject of the same age (within 5 years) and sex without history of cardiovascular disease (CVD). Relevant information on tobacco use, dietary habits, physical activity, folate intake (dietary and from supplements; adjusted for total calorie intake), and past medical and family history was obtained from all study subjects. Weight, height, blood pressure, and body mass index were determined, and fasting venous blood samples were collected. Biochemical measurements were carried out according to previously described methods.13 Among a total of 340 cases with MI and 340 control subjects enrolled in the BAHS, DNA was available for genotyping in 190 cases and 188 control subjects, among them 147 matched case-control pairs; plasma levels of tHcy and folate were determined in an unselected subgroup of 68 cases and 59 control subjects only.
MTHFR Genotype Determination
A polymerase chain reaction–restriction fragment length polymorphism (PCR-RFLP) assay was carried out from whole blood with the use of GeneReleaser (Bioventures) according to the manufacturer's recommendations. Reagent concentrations in the 15 μL PCR reaction were 330 nmol/L each for sense (5′-CAA AGG CCA CCC CGA AGC-3′) and reverse (5′-AGG ACG GTG CGG TGA GAG TG-3′) primers, 166 μmol/L deoxynucleotide triphosphates, 2.5 mmol/L MgCl, and 0.15 units of Taq DNA-polymerase. Samples were amplified for 39 cycles consisting of denaturation at 94°C for 15 seconds, annealing at 58°C for 45 seconds, and extension at 72°C for 40 seconds, followed by a final extension step at 72°C of 5 minutes.
The resulting 246 base pair amplification product was incubated at 37°C for 6 hours with 2 units of the restriction endonuclease, HinfI (New England Biolabs), according to the manufacturer's recommendations, and restriction fragments were size-fractionated on 2% agarose gels. PCR results were scored blinded as to case-control status. Wherever there was any ambiguity, the PCR reaction, HinfI digestion, and scoring were repeated.
Alleles and genotype frequencies among cases and control subjects were counted and compared by χ2 test with Hardy-Weinberg predictions. Odds ratios with two-tailed P values and 95% CIs were calculated as a measure of the association of the MTHFR genotype with clinical outcome assuming a recessive model. Since there were no significant differences between matched and unmatched analyses, only unmatched data are presented. Analyses were carried out on raw data and after adjustment for a number of parameters known to contribute to the risk for cardiovascular disease by multiple logistic regression. To account for possible interactions of the mutation with folate intake, analyses were also performed after stratification by folate intake below and above the median.
Characteristics of case and control groups, with the expected significant differences in several recognized cardiovascular risk factors, are shown in Table 1⇓. No differences in any conventional risk parameter were found between MTHFR genotypes. Allele frequencies for wild-type (−) and mutant (+) alleles were 0.62 and 0.38 in control subjects, 0.67 and 0.33 in cases, and 0.65 and 0.35 in the total study population, respectively. Genotype frequencies were 37.8% for −/−, 47.8% for +/−, and 14.4 for +/+ in control subjects; 50.0% for −/−, 34.7% for +/−, and 15.3% for +/+ in cases; and 43.9% for −/−, 41.3% for +/−, and 14.8% for +/+ in both groups combined. Genotype frequencies did not deviate from the Hardy-Weinberg equilibrium in control subjects (χ2=0.014, P=.99), cases (χ2=4.37, P=.11), or the overall study group (χ2=0.94, P=.62).
Assuming a recessive model of inheritance (ie, +/+ versus +/− and −/− combined), +/+ individuals had a relative risk for MI of 1.1 (95% CI, 0.6 to 1.9; P=.8). Adjustment by multiple logistic regression analysis for cigarette and alcohol consumption, total calorie and saturated fat intake, body mass index, physical activity, past medical history, family history of heart disease, and plasma levels of LDL, HDL, and triglycerides did not materially alter these results (relative risk of MI after adjustment for these covariates was 1.1; 95% CI, 0.6 to 2.2; P=.8).
For subjects with folate intake values above and below the median, the relative risk was 1.3 (95% CI, 0.6 to 3.0; P=.5), and 0.9 (95% CI, 0.4 to 2.0; P=.8), respectively.
Plasma levels of tHcy, folate, and methionine, as well as folate intake values partitioned according to genotype and case-control status, are shown in Table 2⇓. While tHcy was minimally higher in cases compared with control subjects, and folate intake, plasma folate, and plasma methionine were slightly lower, none of these differences were statistically significant. Likewise, differences in these parameters between genotypes were not statistically significant.
In a sample of 190 cases with myocardial infarction and 188 healthy age- and sex-matched control subjects from the BAHS, we found no evidence for an association between the C677T polymorphism and risk for MI or for interactions between folate intake and effects of the mutation. Our results, derived from the largest study on the topic conducted so far, if confirmed by prospective studies, would suggest that this mutation is unlikely to represent an important risk factor for MI in white, middle-class US populations in which folate intake is adequate, such as the one studied. The fact that homozygosity for the mutation was not associated with elevated tHcy is not in concordance with previous studies10 11 14 and may be due to the relatively small number of subjects in whom tHcy levels were determined.
The reported detrimental effects of the C677T mutation on thermostability and enzymatic activity may depend on permissively low levels of folate. In subjects replete of folate and without other deficiencies of homocysteine-metabolizing enzymes, the mutation may thus be tolerated without consequences on biochemical or clinical phenotype, while in individuals with insufficient folate intake (and in those with concomitant other enzymatic deficits of homocysteine metabolism) the mutation may contribute to increased tHcy and heightened cardiovascular risk.
These possibilities, as well as potential differences between ethnic groups, cannot be excluded by the present study. A further limitation of this study is its retrospective character. Only survivors of MI entered this study, and this group may not be representative of all MI cases. The discrepant findings of our study and a previous report by Kluijtmans et al14 should not be seen as conflicting data, since the latter study included only 10 patients with MI as well as several cofactor-depleted subjects. While our study is significantly larger than previous investigations, it will be important to conduct additional, even larger studies that incorporate a broad range of folate intake values to fully assess the significance of this mutation.
This work was supported by Research Career Development Award K04-HL-03138-01 from the National Heart, Lung, and Blood Institute to Dr Lindpaintner and by a Scholarship Award of the “Studienstiftung des deutschen Volkes” to Dr Schmitz. We thank Martin van Denburgh for his help with statistical analyses.
- Received June 20, 1996.
- Revision received August 19, 1996.
- Accepted August 21, 1996.
- Copyright © 1996 by American Heart Association
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