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T Genotype and Folate Status Is a Determinant of Coronary Atherosclerosis Risk
* Department of Clinical and Experimental Medicine,
Institute of Biology and Genetics, and
Institute of Clinical Chemistry, University of Verona, Policlinico G.B. Rossi, 37134 Verona, Italy and
Vitamin Metabolism Laboratory, Jean Mayer U.S. Department of Agriculture Human Nutrition Research on Aging at Tufts University, Boston, MA 02111
2To whom correspondence should be addressed. E-mail: domenico.girelli{at}univr.it.
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ABSTRACT |
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 TOP ABSTRACT SUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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T polymorphism in the 5,10-methylenetetrahydrofolate reductase (MTHFR) gene interacts with folate status in determining elevated total plasma levels of homocysteine, a risk factor for coronary atherosclerotic disease (CAD). The present study had the following goals: 1) to define the 677 CT genotype-specific threshold values of both plasma and RBC folate, associated with hyperhomocysteinemia (>15 µmol/L); and 2) to determine the risk of CAD among subjects with levels of folate below the genotype-specific threshold considered at risk for hyperhomocysteinemia. We examined 655 subjects, with (433) or without (222) angiographically documented CAD. The MTHFR 677 CT genotype-specific threshold values of plasma folate corresponded to the 40th, 30th and 10th percentile in the TT, CT and CC genotype, respectively. A multivariate logistic regression analysis showed that the risk of CAD among subjects with plasma folate levels below the genotype-specific thresholds was 1.6 (95% CI, 1.04–2.46). Similar results were obtained when RBC folate was considered as a measure of folate status (odds ratio = 1.8, 95% CI, 1.03–3.15). A gene-nutrient interaction that defines a higher risk for CAD is determined by folate levels below specific thresholds, which differ depending on the MTHFR 677 CT genotype.
KEY WORDS: • MTHFR • folate • gene-nutrient interaction • coronary artery disease • homocysteine
There is growing interest in the importance of elevated plasma total homocysteine (tHcy) levels as a risk factor for coronary atherosclerotic disease (CAD) (1
–5
). The finding that most cases of mild hyperhomocysteinemia can be corrected with folic acid supplementation is also of interest (6
). To prevent the accumulation of homocysteine, adequate activity of 5,10-methylenetetrahydrofolate reductase (MTHFR, EC 1.5.1.20) must be maintained (7
). This enzyme catalyzes the reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, the main methyl donor for the remethylation of homocysteine to methionine (3
–5
). A point mutation, the 677 CT, in the MTHFR gene renders the enzyme thermolabile and less active (8
). Homozygosity (TT) for this mutation is common (10–18% in Caucasian populations) and is associated with mild hyperhomocysteinemia, particularly in subjects with low levels of plasma folate (9
). Extensive investigations on this polymorphism as a candidate genetic risk factor for CAD have given conflicting results [critically reviewed in (1
,10
,11
)]. Our previous data showed the existence of a gene-environment interaction between the MTHFR 677 CT and folate status not only in TT homozygous mutants but also in CT heterozygous subjects (12
). Such observations prompted us to study a larger sample of subjects with the following aims: 1) to define the 677 CT genotype-specific threshold values of both plasma and RBC folate associated with hyperhomocysteinemia considered clinically relevant for higher risk of CAD (>15 µmol/L); and 2) to determine the risk of CAD among subjects with levels of folate below the threshold who are considered at risk for hyperhomocysteinemia according to their specific genotype.
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SUBJECTS AND METHODS |
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TOPABSTRACTSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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The criteria for selection of the study population and laboratory methods were described in detail elsewhere (12
,13
). Briefly, between May 1996 and August 1998, we examined 655 unrelated patients referring to our Institute and/or to the Institute of Cardiovascular Surgery of the University of Verona. Of these 655 patients, 433 had angiographically proven CAD. The disease severity was evaluated by counting the number of major epicardial coronary arteries (left anterior descending, circumflex and right) affected with 
1 significant stenosis (50%). As a control group we considered 222 subjects with angiographically documented normal coronary arteries (in most cases, patients with valvular heart disease). To provide an objective definition of the atherosclerotic phenotype, subjects with nonsignificant coronary stenosis (<50%) were excluded. Controls were also required to have no history or evidence of atherosclerosis in other vascular areas. Both CAD patients and controls came from the same geographical area (Northern Italy) and had a similar socioeconomic background. At the time of blood sampling, a complete clinical history, which included specific reference to the use of any vitamin supplementation, was obtained by medical personnel by interviewing each study participant. Exclusion criteria included all of the conditions known to influence plasma tHcy levels such as the use of drugs interfering with tHcy metabolism, the presence of any major systemic acute illness in the previous 3 mo, serum creatinine levels 160 µmol/L and current or recent use of folate, vitamin B-12 or any multivitamin preparation. The study was approved by our institutional review boards and written informed consent was obtained from all of the patients.
Biochemical analysis.
Samples of venous blood were drawn from each subject after an overnight fast. For tHcy (the sum of homocysteine, homocystine and homocysteine-cysteine mixed disulfide, free and protein-bound), blood was collected into EDTA-containing vacuum tubes and kept on ice in the dark; plasma was separated within 90 min; plasma tHcy levels were determined by a HPLC method with fluorescent detection, according to Araki and Sako (14 ). Plasma folate was measured by an automated chemiluminescence method (Chiron Diagnostics, East Walpole, MA) (12 ).
For the RBC folate assay, RBC were isolated by centrifugation at 36,000 x g for 15 min within 1 h of collection, washed with 9 g/L of sodium chloride solution, and immediately stored at -70°C, until folate analysis. RBC folate was analyzed by affinity chromatography followed by reversed-phase chromatography with electrochemical detection, as previously described (15 ). An adequate sample for RBC folate analysis was available for 467 subjects.
Mutation analysis.
DNA was extracted from peripheral lymphocytes using the phenol/chloroform protocol, and the analysis of the 677 CT mutation in the MTHFR gene was performed by polymerase chain reaction and Hinf I digestion as described by Frosst et al. (8
).
Statistical analysis.
All calculations were performed with SPSS 10.0 statistical package (SPSS, Chicago, IL). Distributions of continuous variables in groups are expressed as means ± SD. Logarithmic transformation was performed on all skewed variables, including plasma tHcy, and plasma and RBC folate. Hence, geometric means with 95% CI are given for these variables. Quantitative data were assessed using Student’s t test or by analysis of covariance with Tukey’s post-hoc comparison of the means. Qualitative data were analyzed with the 
2 test. A value of P < 0.05 was considered significant. The correlation between plasma and RBC folate was evaluated using the Spearman coefficient.
Hyperhomocysteinemia was defined for plasma tHcy values 15 µmol/L, on the basis of a prevailing agreement in the literature (16
). Starting with the distributions of plasma and RBC folate levels in controls, we deduced for each MTHFR genotype the lowest level of folate required to keep mean homocysteine levels in a normal range (<15 µmol/L). On this basis, we defined the genotype-specific threshold values of either plasma or RBC folate. Subjects were then categorized into two groups: group A, with a "favorable" gene-environmental interaction (i.e., folate status higher than the genotype-specific threshold value), and group B, with an "unfavorable" interaction (i.e., folate status lower than the genotype-specific threshold value).
To assess the association of the different gene-nutrient interactions with coronary atherosclerosis, the odds ratio (OR) with 95% CI was first obtained by using univariate logistic regression analysis. Adjustments for all of the conventional risk factors for CAD were performed by multivariate logistic regression models.
|
RESULTS |
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TOPABSTRACTSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
|---|
T genotypes was similar in the two groups. The genotype-specific threshold levels of plasma folate (i.e., the lowest level still associated with plasma tHcy <15 µmol/L) corresponded to the 40th (11.1 nmol/L), 30th (10.2 nmol/L) and 10th (7.5 nmol/L) percentile among TT, CT, and CC subjects, respectively, of the control group. Subjects with folate levels below these threshold values had plasma tHcy levels of 25.6 (TT), 17.7 (CT) and 17.1 (CC) µmol/L, respectively (Table 2). These subjects were combined into a group considered to have an "unfavorable" gene-nutrient interaction (group A); the remaining subjects comprised a second group considered to have a "favorable" gene-nutrient status (group B). Plasma tHcy was 19.8 µmol/L (95% CI, 18.6–21.1) in group A vs. 14.3 µmol/L (95% CI, 13.8–14.7) in group B (P < 0.001). A greater proportion of the group with the "unfavorable" gene-environmental interaction were CAD vs. CAD-free subjects (33.9 vs. 24.3%; P = 0.011; OR = 1.6; 95% CI, 1.1–2.3) (Table 3).
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DISCUSSION |
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TOPABSTRACTSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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T polymorphism is not associated per se with a higher risk of CAD in this study population. This is not surprising, considering two peculiarities of this mutation. First, it is so common that a direct causal role in the pathogenesis of CAD, a potentially lethal disease, is unlikely. Second, the polymorphism impairs homocysteine metabolism only in presence of inadequate folate status (9
), which may result from consumption of a diet poor in green vegetables and fruits, the natural food sources of this vitamin. The presence of such a gene-nutrient interaction is consistent with the study performed by Guenther et al. (18 ) on a thermolabile MTHFR model expressed in E. coli in which they demonstrated that folate protects the MTHFR against the loss of its essential flavin cofactor.
The MTHFR-folate interaction reported in this study is graded, so that the TT homozygote mutants present a risk of hyperhomocysteinemia even with a marginal vitamin deficiency, whereas the CC wild type subjects develop hyperhomocysteinemia only if a severe folate deficiency occurs. To refine our data, we also considered RBC folate, which is a better indicator of long-term body folate stores (19 ). The genotype-specific threshold values were quite similar for RBC and plasma folate levels, as are the implications in terms of CAD risk.
Studies on the risk of CAD related to the 677 CT mutation (10
,11
) have offered a wide range of results, including a report of a protective effect of the TT genotype on endothelial function (20
). Several explanations may account for these discrepancies. The first is related to the different enrollment criteria used for cases and controls. Our clear-cut definition of the CAD phenotype likely reduced the chance of spurious results due to the inclusion of controls with substantial, though not yet clinically manifest CAD. Second, previous studies focused on the potential cardiovascular risk conferred by the TT genotype alone, without acknowledging the occurrence of hyperhomocysteinemia in CT and CC subjects with folate deficiency. Finally, the minority of studies providing information on folate status were conducted in populations heterogeneous in terms of exposure to grain products fortified with folic acid and/or use of multivitamin supplements, which are much more common in Canada/United States than in Europe. Indeed, a recent meta-analysis involving 11,162 cases and 12,758 controls suggested an increased risk for CAD associated with the MTHFR 677 TT genotype only among Europeans and not in North American populations (21
).
In this study, the effect of MTHFR-folate interaction on CAD risk was mild (OR = 1.6–1.8), as expected for a multifactorial disease. Our results in this study cannot resolve the issue of a potential causal relationship between MTHFR-folate interaction and CAD; other studies are required to address this specific topic. Caution is warranted in interpreting our data for a broad application, especially considering that our study design was based on the controversial assumption that plasma tHcy >15 µmol/L is unfavorable in terms of CAD risk (16
). Although virtually all of the retrospective and most of the prospective studies have demonstrated a positive association between plasma tHcy and atherosclerosis, definite proof that tHcy is a causal factor for CAD is still lacking (1
,22
). On the other hand, it has been proposed that folate deficiency may be the primary cause of the increased risk of CAD, with elevated tHcy acting as a marker of low folate status rather than a pathogenetic factor (22
). Indeed, our definition of adequate folate status as a genotype-specific composite variable shifts the focus from tHcy to low folate as the factor mediating the vascular lesions. Several epidemiologic studies, although not all, have suggested an inverse association between folate levels and the risk of CAD (23
–26
). Moreover, recent studies have demonstrated that folic acid supplementation can improve endothelial function, a surrogate end point for CAD risk, in hyperhomocysteinemic subjects (27
,28
). Schnyder et al. (29
) recently reported that vitamin supplementation effective in lowering tHcy reduced the rate of restenosis after coronary angioplasty. However, benefits of folates that are independent of the tHcy-lowering strategy have also been reported (22
). Although fortification of grain products was introduced by the U.S. FDA (30
) with the primary purpose of lowering the risk of neural-tube defects, an effect on reducing the rate of CAD may be detected as well. Nevertheless, within the debate over the role of widespread folic acid supplementation, the present study provides some evidence that is rather necessary to define an individual-specific folate requirement based on a given MTHFR genotype to modify the risk for CAD. This may also avoid the hazard of masking a concomitant vitamin B-12 deficiency potentially due to folic acid supplementation (30
). Our results point to a time of "nutritional ecogenetics," when a simple molecular test for the MTHFR 677 CT mutation may help formulate an individual’s optimal diet and/or vitamin supplementation requirement (30
) for reducing the risk of disease.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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3 Abbreviations used: CAD, coronary atherosclerotic disease; MTHFR, methylenetetrahydrofolate reductase; OR, odds ratio; tHcy, plasma levels of homocysteine.
Manuscript received 17 October 2002. Initial review completed 18 November 2002. Revision accepted 10 February 2003.
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LITERATURE CITED |
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TOPABSTRACTSUBJECTS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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