Author + information
- Received March 26, 1997
- Revision received July 15, 1997
- Accepted July 21, 1997
- Published online November 1, 1997.
- Jeffrey L Anderson, MD, FACCA,*,
- Gretchen J King, PhDA,
- Matthew J Thomson, BSA,
- Michael Todd, BSA,
- Tami L Bair, BSA,
- Joseph B Muhlestein, MD, FACCA and
- John.F Carlquist, PhDA
- ↵*Dr. Jeffrey L. Anderson, Division of Cardiology, LDS Hospital, 8th Avenue and C Street, Salt Lake City, Utah 84143.
Objectives. We sought to determine whether the C677T transition in the methylenetetrahydrofolate reductase (MTHFR) gene is associated with increased risk for coronary artery disease (CAD) or myocardial infarction (MI).
Background. Elevated plasma homocysteine has been identified as a risk factor for coronary atherosclerosis. Homocysteinemia may result from deficient MTHFR activity. A thermolabile form of MTHFR, associated with a C677T genetic transition, shows reduced activity and may be a risk factor for CAD.
Methods. Blood was withdrawn from patients undergoing coronary angiography, and DNA was extracted by a phenol-chloroform method. Genotyping was done by polymerase chain reaction (PCR) amplification of a 198-base pair segment of the MTHFR gene that brackets nucleotide 677. The amplicon was digested with the HinfI restriction enzyme. Products were visualized after electrophoresis in 1.5% agarose with ethidium bromide.
Results. Among 200 patients with a diagnosis of MI, the polymorphic allelic frequency was 33.3%, compared with 32.1% among 554 control subjects (p = 0.68); homozygosity was present in 11.5% of patients and 10.6% of control subjects (p = 0.74, odds ratio [OR] 1.09, 95% confidence interval [CI] 0.63 to 1.82). Among 510 patients with severe CAD (>60% stenosis), allelic frequency was 32.0%, compared with 34.8% for 168 subjects without CAD (<10% stenosis, p = 0.33); 11.2% of patients with CAD compared with 13.1% of control subjects were homozygous (p = 0.50, OR 0.83, 95% CI 0.5 to 1.40).
Conclusions. Patients with angiographic evidence of CAD or clinical MI do not show an increased frequency of the C677T transition in the MTHFR gene. Our findings do not support this polymorphism as a risk factor for CAD or MI in a predominantly white, well nourished population of unrestricted age.
Methylenetetrahydrofolate reductase (MTHFR) is an enzyme that reduces 5′,10′-methylenetetrahydrofolate to 5′-methylenetetrahydrofolate, the main circulating form of folate and the methyl donor for the remethylation of homocysteine to methionine. A thermolabile form of MTHFR has been identified [1, 2]and found to be caused by a missense mutation in its encoding gene, with the cytidine residue at nucleotide (nt) position 677 being replaced by thymidine (C677T), resulting in the substitution in the enzyme of a valine residue for alanine. The homozygous (+/+) genotype of this mutation has been found to specify a variant enzyme with reduced activity and to be associated with elevated total plasma homocysteine (tHCY) levels, particularly in the setting of low folate levels, as compared with the wild type (−/−) and heterozygous (+/−) genotypes [3–6].
It has been almost 30 years since hyperhomocysteinemia has been recognized to accelerate atherosclerosis [7, 8]. Moreover, in the past few years, moderate, clinically common elevations in tHCY levels have been associated with an increased risk of vascular disease [9–19], including coronary artery disease (CAD) [9, 10]and myocardial infarction (MI) [20, 21]. Elevations of tHCY might result from nutritional deficiencies (including folate, pyridoxal phosphate [B6] and methylcobalamin [B12] [5, 20–23]or genetically determined abnormalities of homocysteine metabolism (e.g., MTHFR), or a combination of these.
These considerations have raised the possibility that the relatively common C677T mutation in MTHFR might be an important genetic risk factor for vascular disease through its effects on homocysteine metabolism and tHCY [1–4, 24]. (A potential link of the MTHFR variant with congenital neural tube defects has also been proposed .) Recently, Kluijtmans et al. reported a threefold increase in the relative risk of premature vascular disease in homozygous individuals in a small case-control study. We therefore undertook a larger (n = 754), prospective study of the MTHFR polymorphism in a population defined by coronary angiography to test the hypothesis that the homozygous variant genotype is associated with an increased risk of MI and CAD.
1.1 Study Hypotheses
We postulated that the (+/+) mutant homozygote would be associated with an increased risk (odds ratio [OR] ≥1.5) for MI and CAD in patients studied by coronary angiography (recessive model). Second, we assessed whether the polymorphic C677T transition allele in the MTHFR gene would be associated with these diseases (dominant model).
The study sample consisted of patients undergoing coronary angiography because of either symptoms of suspected CAD or unrelated conditions requiring angiographic evaluation (e.g., valvular disease, cardiomyopathy). Patients were of unrestricted age and gender and gave written, informed consent for a blood draw at the time of angiography for use in confidential “deoxyribonucleic (DNA) bank” studies approved by the hospital’s Institutional Review Board. Subjects were residents of Utah, southwestern Idaho or southeastern Wyoming, a population that is ethnically primarily of Northern European (Anglo-Scandinavian) descent and genetically representative of U.S. Caucasians .
Key demographic characteristics were captured on computerized angiographic data forms, including age, gender and history of MI. Assessment of CAD was determined by a review of angiograms by the patient’s cardiologist and entered into the computer data base in a format modified after the CASS protocol [27, 28]. Patients were designated as having CAD if they had >60% stenosis of at least one coronary artery or its major branch and having no CAD if <10% stenosis was present in all major vessels. Patients with mild CAD (10% to 60% stenosis) were given an “indeterminate” CAD status. Final designations of MI and CAD status were made after considering arteriographic and ventriculographic results together with patient history, without knowledge of DNA genotype. Coronary angiograms were read without knowledge of genotypic results.
1.3 DNA Extraction
Blood (20 to 30 ml) was withdrawn and collected in EDTA at the time of angiography. The leukocyte buffy coat was recovered by centrifugation, washed in TNE buffer (10 mmol/liter Tris base, 10 mmol/liter sodium chloride, 1 mmol/liter EDTA), resuspended in 3 ml of a solution of sodium chloride (75 mmol/liter) and EDTA (24 mmol/liter) containing 250 μl of proteinase K (4 mg/ml) and 125 μl of 20% sodium dodecyl sulfate and incubated at 65°C overnight. The mixture then was combined with 0.5 vol equilibrated phenol and 0.5 vol chloroform:isoamyl alcohol (24:1), placed on a shaker for 1 h and then centrifuged at 1,000 gfor 10 min. The upper phase was removed, reextracted with 1 vol chloroform and centrifuged. The upper phase was collected and mixed with 0.1 vol 3 mol/liter sodium acetate (pH 5.2), shaken and combined with 2 vol cold isopropanol. Precipitated DNA was removed with a glass hook and resuspended in Tris/EDTA buffer (0.01 mol/liter Tris, 0.001 mol/liter EDTA, pH 8.0).
1.4 DNA Genotyping
Identification of the C to T transition at nt677 was done using the method of Frosst et al. . Previously published primers were used to amplify a 198-base pair (bp) segment containing nt677. The primers were (5′-3′)TGAAGGAGAAGGTGTCTGCGGGA and AGGACGGTGCGGTGAGAGTG. The amplification protocol consisted of an initial denaturation segment at 94°C for 5 min. After this, each cycle consisted of three segments (93°C for 50 s, 55°C for 50 s and 72°C for 30 s). The cycle was repeated 30 times, followed by an additional extension at 72°C for 5 min. The 198-bp product was treated with 5 U HinfI restriction endonuclease (3 to 4 h at 37°C), which recognizes a novel restriction site created by the C to T transition at nt677. The amplified product from the mutant gene was cleaved into 175-bp and 23-bp fragments by HinfI, which leaves the wild-type gene unaffected. After electrophoresis through 1.5% agarose gel, the digestion products were visualized by staining with 1 μg/ml of ethidium bromide. The gels were read without knowledge of the clinical and angiographic results by a single experienced reader. A representative gel is shown in Fig. 1.
A meta-analysis was performed as previously described , combining the present study with three recently published reports [30–32]examining the MTHFR C677T transition and risk of MI. Briefly, the meta-analysis consists of tests for association and homogeneity. Homogeneity testing assesses the reproducibility (homogeneity) of the different ORs determined in the respective studies. The overall test for association then assesses the significance of the association between the C677T mutation and MI for all studies combined.
1.6 Statistical Planning and Analysis
Power calculations indicated that to determine an OR of ≥1.5 for MI or CAD of the C677T transition polymorphism in the MTHFR gene with a power of 80% at an alpha level of 0.05 in a population with an incidence of the T allele of 35% [30, 31], a sample size of ∼375 subjects per group is required (GB-STAT for Windows). Accordingly, we assembled and studied a group of 754 subjects; 70% of the group had severe CAD and 28% had a history of MI.
Allelic and genotypic frequencies were determined from observed genotype counts, and the expectations of the Hardy-Weinberg equilibrium were evaluated by chi-square analysis. Comparisons between genotypic frequencies were done using chi-square analysis; ORs with 95% confidence intervals (CIs) were calculated as previously described .
2.1 Patient Groups
A total of 754 subjects were studied (average age 64 years, range 17 to 89); 200 had a history of MI and 510 had significant CAD. Key patient characteristics are summarized by disease subgroup in Table 1. As might be expected, those with CAD were older and more frequently men, smokers and diabetics. Those with MI were more frequently men and smokers. Blood pressure declined slightly after MI.
2.2 Genotypic and Allelic Frequencies
Genotypic and allelic frequencies for the study groups are shown in Table 2. The C677T (+) allelic frequency in control subjects without MI was 32.1%. Their genotypic frequencies were in agreement with those predicted by the Hardy-Weinberg equilibrium. These allelic and genotypic frequencies are also similar to those previously reported for U.S. physicians and in a Boston area study .
The C677T (+) allelic frequency in the control group without CAD was 34.8%. The genotypic frequencies in this control group were also in agreement with the frequencies predicted by the Hardy-Weinberg equilibrium.
2.3 Association Between MTHFR Polymorphism and MI
Among the 200 patients with a diagnosis of MI, the mutant C677T allelic frequency was 33.3%, compared with 32.1% among 554 control subjects (p = 0.68) (Table 2). The distribution of the genotypes was within the expectation of the Hardy-Weinberg equilibrium. Homozygosity for the (+/+) genotype among patients and control subjects was 11.5% and 10.6%, respectively (p = 0.74). The OR for MI was 1.09 (95% CI 0.63 to 1.82) for the (+/+) versus non(+/+) genotypes.
2.4 Association Between MTHFR Polymorphism and CAD
Among the 510 patients with severe CAD (>60% stenosis), the mutant C677T (+) allelic frequency was 32.0%, compared with 34.8% for 168 subjects without CAD (<10% stenosis) (p = 0.33); 11.2% of patients with CAD compared with 13.1% of control subjects were homozygous for the mutation (p = 0.50). The OR for CAD was 0.83 (95% CI 0.49 to 1.40) for the (+/+) versus non(+/+) genotypes. The distribution of the genotypes was within the expectation of the Hardy-Weinberg equilibrium.
2.5 Association of MTHFR Polymorphism With MI or CAD by Age and Other Prespecified Characteristics
The MTHFR polymorphism might affect risk primarily in a certain age group (i.e., younger patients). Thus, genotypic associations with disease were separately assessed by age (Table 3). For subjects <60 years of age, the OR for MI was 1.35 (95% CI 0.58 to 3.12) for the (+/+) versus non(+/+) genotypes, whereas it was 0.95 (95% CI 0.49 to 1.83) for those ≥60 years old. The OR of CAD was 1.40 (95% CI 0.56 to 3.50) for the (+/+) versus non(+/+) genotypes for subjects <60 years old, and it was 0.65 (95% CI 0.34 to 1.24) for those ≥60 years old. The possibility of associations between the MTHFR polymorphism and MI in other prespecified subgroups defined by baseline characteristics was also explored (Table 3), but no noteworthy trends were found.
3.1 Summary of Study Results
In our moderately large (n = 750), prospectively studied, angiographically defined sample group, we found no relation between the MTHFR C677T polymorphism and the risk of either MI (OR 1.09, p = 0.7) or angiographically defined CAD (OR 0.83, p = 0.5). Thus, in a generally well nourished, predominantly white, middle class population, this mutation does not appear to be a useful marker of increased cardiovascular risk. Although differences were not significant, a trend was noted for the (+/+) genotype with MI and CAD risk in younger patients that may deserve further exploration in larger studies with greater power, especially in patients with relative folate deficiency.
3.2 Comparison With Recent Published Reports
Our results do not confirm those of the small case-control study of Kluijtmans et al. , who reported a threefold increase in the relative risk of premature vascular disease in homozygous subjects, but are in keeping with three larger, more recent reports. In a Boston area health study report , MTHFR genotypic frequencies were similar for 190 patients with MI and 188 control subjects: 15.3% were homozygous for the variant polymorphism among patients compared with 14.4% among control subjects (relative risk 1.1, 95% CI 0.6 to 1.9). Stratification by folate intake did not alter the results. Plasma tHCY and folate levels were minimally higher and folate levels minimally lower among patients compared with control subjects, and levels did not differ significantly by genotype.
In a health study report of U.S. physicians , the frequency distribution of the MTHFR polymorphism was similar among patients and control subjects: the (+/+) genotype was present in 11% who developed MI (n = 293) and 13% who did not (n = 290) (relative risk 0.84, p = NS). In an exploratory analysis, the relative risk of MI in younger men (<60 years old) with low folate levels was 1.7 (95% CI 0.7 to 4.5) for the (+/+) genotype and 2.4 (95% CI 0.9 to 6.4) for the combined genotypes (+/−) and (+/+), each of which was compared with high folate and the wild type genotype (−/−) as reference. Neither comparison was significant. The study did confirm an association between the MTHFR polymorphism and tHCY levels, however, which averaged 12.6 ± 0.5 nmol/ml in (+/+) and 10.6 ± 0.3 in (−/−) subjects (p < 0.01). The difference was most marked among men with folate levels in the lowest quartile of the distribution of control subjects: 16.0 ± 1.1 versus 12.3 ± 0.6 nmol/ml, respectively (p < 0.001).
Van Bockxmeer et al. screened 555 whites with angiographic evidence of CAD and 143 community control subjects; 212 were categorized as to previous MI status. Homozygosity for the mutant C677T allele was found in 10.5% of control subjects, 10.6% of patients with CAD (<50 years old) and 9.1% of those with previous balloon angioplasty evaluated for restenosis. No relation was found between MI, CAD or restenosis and genotype. Plasma folate distributions were similar in control subjects and patients .
3.3 Overview of Studies
If this and the three other major prospective studies [30–32]are combined (Fig. 2), a relatively large experience with almost 2,000 patients and control subjects is obtained. The studies are consistent in showing no overall association between the MTHFR polymorphism and MI risk. The combined OR was 0.98 (95% CI 0.73 to 1.30). Except for the initial, small report that provoked further studies , these published observations are consistent in showing no evidence for an influence of the polymorphism on MI risk (or on angiographic CAD) in well nourished, mostly Caucasian populations.
3.4 Pathophysiologic Considerations
Why is there an absence of an effect of the polymorphism on MI risk when studies as a whole are consistent with an effect on tHCY levels, especially among those with lower folate intake? It appears likely that any effect of the genetic defect is indirect and acts through effects on tHCY levels. However, these effects may be of limited magnitude (mild or insignificant clinical impact), except in specific, vulnerable groups, such as young individuals with very low folate levels . Overall, among well nourished subjects, the genetic defect may be largely compensated for by adequate dietary folate intake. We did not have plasma samples available for folate and tHCY measurements, but in the other recent studies [30–32], they were measured in subsets of subjects, and the findings were consistent in showing little if any overall effect of the polymorphism on folate and tHCY levels. However, the results of these four moderately large studies in groups with favorable socioeconomic and nutritional status do not exclude the possibility of an effect in a younger sample group with substantially lower folate intake . Thus, additional studies in such groups should be encouraged.
In a prospectively studied, angiographically defined U.S. population of European descent, no relation of the MTHFR C677T mutation with the risk of either MI or angiographically defined CAD was found. This finding for MI is in contrast to molecular pathophysiologic considerations and an initial, small clinical study , but is consistent with three contemporary studies of moderate size [30–32]. We also observed an absence of an effect of genotype on angiographically defined CAD. These findings suggest that this mutation is not likely to be a useful marker of increased cardiovascular risk in this or similar groups. An effect of the polymorphism in younger subjects with substantially lower folate intake is still possible and should be explored in future studies.
☆ This study was supported in part by a grant from the Deseret Foundation, LDS Hospital, Salt Lake City, Utah.
- base pair
- coronary artery disease
- confidence interval
- deoxyribonucleic acid
- ethylenediaminetetraacetic acid
- total plasma homocysteine (level)
- myocardial infarction
- methylenetetrahydrofolate reductase
- odds ratio
- Received March 26, 1997.
- Revision received July 15, 1997.
- Accepted July 21, 1997.
- The American College of Cardiology
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