Colorectal Adenomas and the C677T MTHFR Polymorphism: Evidence for Gene-Environment Interaction?¹

Colorectal carcinogenesis is a multistage process during which global DNA methylation changes, hyperproliferation, adenoma formation and growth, specific somatic genetic changes, and malignant transformation are probably involved (1). Adenomatous polyps, which comprise about two-thirds of the polyps encountered in clinical settings, are considered precursors of colorectal cancer (2). There are a number of environmental factors that increase risk of colorectal neoplasia (3). Among the most consistent risk factors is a low intake of vegetables and perhaps fruit. A number of constituents of vegetables and fruit may contribute to this protective association, and one important constituent of vegetables and fruit is folate (4). In epidemiological observational studies, low-folate diets have been found to increase the risk of adenomatous polyps and colon cancer (5, 6, 7, 8, 9) and the recurrence of adenoma (10). Other dietary factors, including the vitamins B₁₂ and B₆, as well as methionine, play a role in folate metabolism (11, 12). There is very limited research on the risk of colon cancer or colorectal polyps associated with vitamins B₁₂ or B₆. Alcohol intake may be related to folate availability by affecting its intestinal absorption, metabolism, and renal excretion (13, 14, 15).

MTHFR³ is a key enzyme in folate metabolism (12). MTHFR plays a central role in the provision of methyl groups to the body by reducing 5,10-methylene-THF to 5-methyl-THF. 5-methyl-THF serves as a substrate for the remethylation of homocysteine to methionine, with subsequent production of SAM, the universal methyl donor in humans, required for DNA methylation. The methylation of homocysteine is catalyzed by the enzyme methionine synthase that requires the cofactor vitamin B₁₂. MTHFR is also linked to the production of dTMP via thymidylate synthase and to purine synthesis and, therefore, plays a role in the provision of nucleotides essential for DNA synthesis (12).

A defect in MTHFR could thus influence both DNA methylation and DNA synthesis; interactions with the nutritional cofactors vitamin B₁₂, vitamin B₆, and the substrate folate should be detectable. A common polymorphism in the MTHFR gene (C677T) has been identified; individuals with the variant MTHFR TT genotype have ≈30% of the in vitro enzyme activity seen in those with the CC wild-type (16). Heterozygotes (CT) show ≈65% of normal enzyme activity (16). The TT genotype is associated with higher plasma homocysteine levels and reduced plasma folate levels (17, 18, 19, 20, 21, 22, 23). Two previous studies have examined the relationship between the MTHFR genotype and colorectal cancer risk and observed significantly decreased risks with the variant TT genotype (24, 25).

We report here on the association between the MTHFR genotype and colorectal adenomatous polyps within a large clinic-based, case-control study. We investigated modifications in risk depending on nutrient intakes of folate, vitamin B₁₂, vitamin B₆, methionine, and other characteristics of the study population.

Subject recruitment for this case-control study has been described previously (26). Briefly, cases and controls were recruited through a large multiclinic private gastroenterology practice, DH, which conducts colonoscopies in 10 hospitals, and, at the time of this study, undertook ≈60% of all colonoscopies in metropolitan Minneapolis. All patients, ages 30–74 years, who were scheduled for colonoscopy at DH clinics between April 1991 and April 1994 were screened for eligibility (see below) and recruited prior to colonoscopy; recruitment at all 10 DH sites was initiated at the time of scheduling. The intent was to recruit subjects with both patient and recruiter blind to the final diagnosis. This study was approved by the internal review boards of the University of Minnesota and each DH endoscopy site. Written informed consent was obtained from each study participant.

Eligibility criteria for both cases and controls were: resident of Twin cities metropolitan area; ages 30–74 years; English speaking; no known genetic syndrome associated with predisposition to colonic neoplasia; no individual history of cancer (except nonmelanoma skin cancer); and no history of inflammatory bowel disease. In addition, cases were eligible if colonoscopy resulted in a first diagnosis of colon or rectal adenomatous polyps; controls had to be free of all polyps (hyperplastic or adenomatous) at colonoscopy. Indications for colonoscopy included bleeding, diagnostic/follow-up, family history, screening, and others (Table 1). These proportions differed somewhat by case-control status and age group but not by MTHFR genotype. Dietary intakes of nutrients in the MTHFR pathway did not differ by indication for colonoscopy, except for alcohol intake, which was significantly higher among those whose indication was bleeding compared with those with family history or screening as reported reasons.

The questionnaires were self-administered, and patients received all study material (including FFQs) before their clinic visit. At colonoscopy, the signed consent form and completed questionnaires were collected, and blood was drawn. The colonoscopy findings were recorded on standardized forms. Upon removal, polyps were examined histologically by the study pathologist using diagnostic criteria established for the National Polyp Study (27). Only participants with a complete colonoscopy reaching the cecum were eligible. The presence or absence of pathology was determined, and based on colonoscopy and pathology findings, participants were assigned to one of the following three groups: (a) adenomatous polyp group (defined as either adenomatous or mixed pathology, n = 575); (b) hyperplastic polyp-only group (n = 219); and (c) colonoscopy-negative group (controls, n = 708). Participants with polyps showing invasive carcinoma were excluded. The hyperplastic polyp group was treated as a separate group and is not considered further here. The participation rate for all colonoscoped patients was 68%.

Information on: (a) dietary intake; (b) physical activity; (c) smoking habits; (d) anthropometric measurements; (e) medical information; (f) demographic information; (g) reproductive history (women); and (h) family history of polyps and cancer, especially history of colon, breast, endometrial, or ovarian cancers, was collected. Study staff followed up by phone when data were incomplete. The dietary questionnaire was an adaptation of the Willett semiquantitative FFQ, which has been studied previously for validity and repeatability within the Nurses’ Health Study cohort (28), the Iowa Women’s Health Study cohort (29), and the Health Professionals Follow-up Study cohort (30). The FFQ included queries on the brand of breakfast cereal and the brand and frequency of multivitamin and individual vitamin supplement use. Correlation coefficients, on repeat administration, of this instrument in a similar study population were r = 0.62 for dietary folate, r = 0.67 for vitamin B₁₂ intake, and r = 0.99 for alcohol consumption (29). Giovannucci et al. (31) compared values derived from this questionnaire with RBC folate levels (an indicator of long-term folate status) and observed correlations of r = 0.55 for women and r = 0.56 for men.

At the clinic visit, venous blood was collected from each participant in two 20-ml ACD Vacutainer tubes. Buffy coats were isolated within 24 h of collection and frozen at −70°C until extraction and analysis. White cells, red cells, and plasma were separated according to a standardized protocol. White cells were stored, in appropriate cell culture medium, as multiple 0.5-ml aliquots, at −70°C for DNA extraction or preparation of cell lines. Red cells and plasma were also stored in multiple aliquots. Buffy coats were shipped in frequent batches to the University of Utah for extraction of DNA. DNA was extracted from buffy coats at the University of Utah using the Pure Gene DNA isolation kit (Gentra Systems, Inc., Minneapolis, MN). DNA was quantitated and examined for purity by UV absorption at 260 and 280 nm (ratio ≥ 1.8; Ref. 32). Extracted DNA was shipped to Seattle for genotyping analyses.

Determination of the C677T polymorphism was conducted at the Core Laboratory of the Public Health Sciences Division of the Fred Hutchinson Cancer Research Center. The MTHFR polymorphism at 677 bp was determined using the PCR/RFLP method described by Frosst et al. (16). PCR reactions were performed on a Deltacycler II thermal cycler (Ericomp, San Diego, CA) in 96-well plates. Primers (5′-TGA AGG AGA AGG TGT CTG CGG GA-3′) and (5′-AGG ACG GTG CGG TGA GAG TG-3′) were used to amplify a portion of the MTHFR sequence from 100 ng of genomic DNA in a 30-μl reaction containing 3 μl of 10× PCR buffer [100 mm Tris-HCl (pH 8.3) at 25°C, 500 mm KCl, 15 mm MgCl₂, and 0.01% (w/v) gelatin; Perkin-Elmer], 50 μg/ml BSA, 0.2 mm deoxynucleotide triphosphates, 0.2 μm each primer, and 1 unit of Taq DNA polymerase. The cycling conditions were: initial melting at 93°C for 5 min, then 30 amplification cycles of 93°C for 60 s, 58°C for 60 s, and 72°C for 60 s.

After amplification, the 198-bp MTHFR fragment was digested with HinfI in a 20-μl reaction containing 10 μl of PCR fragment, 2 μl of 10× buffer (H; Amersham Life Science; supplied by the manufacturer), and 4 units of HinfI at 37°C for 1 h. The digestion products were separated on a 3% NuSieve agarose gel (FHC Corp.), and the ethidium bromide-stained fragments were photographed on a UV transilluminator. Wild-type (CC) individuals were identified by only a 198-bp fragment, heterozygotes (CT) by both the 175/23 and 198-bp fragments, and homozygote variants (TT) by the 175-bp and the 23-bp (less visible) fragment. Blinded repeat genotyping of 20 DNA samples yielded a reproducibility of 100%. DNA quality or quantity was insufficient for MTHFR genotyping in 48 cases and 63 controls; thus, the final study population consisted of 527 cases and 645 controls.

Standard techniques for case-control studies were used. The measure of the association between MTHFR genotype and incidence of adenomatous polyps was the OR, which was estimated by logistic regression. ORs and 95% confidence intervals are presented. We evaluated the association between MTHFR genotype and adenomatous polyps first in the entire study population and subsequently separately in men and women. Further subsets of the population for analyses were based on age, polyp characteristics, and dietary intakes.

Effect modification was evaluated by stratification on the variable of interest, and ORs within each stratum were compared. Subsequently, the following potential confounding factors were evaluated: age, sex, race/ethnicity, hormone replacement therapy (ever/never), BMI, waist-to-hip ratio, pack-years of smoking, regular use of aspirin (at least one per week), regular use of nonsteroidal anti-inflammatory drugs (at least one per week), hours of physical activity, and the dietary intake variables kilocalories, dietary fiber, percentage kilocalories from fat, vitamin B₆, vitamin B₁₂, folate, and alcohol. After evaluation, a subset of these variables was maintained for multivariate adjustment: age, sex, BMI, percentage kilocalories from fat, dietary fiber intake (g), hormone replacement therapy (ever/never), and dietary intakes of folate, vitamin B₁₂, vitamin B₆, methionine, and alcohol. All adjustment variables were included in the model as continuous variables, with the exception of sex and hormone replacement therapy (ever/never). In general, the confounding effects of the variables listed were small; for consistency, the multivariate-adjusted estimates are reported throughout. Correlations between dietary intakes ranged from r = −0.05 (alcohol and vitamin B₆) to r = 0.47 (folate and vitamin B₁₂), and multivariate adjustment included all nutrients.

Statistical significance testing was conducted on several levels; differences in nutrient intakes and other population characteristics between cases and controls were evaluated by t tests and χ² tests. To assess the dose-response relationship between nutrient intakes or other variables and colorectal adenomas within each of the three genotypes, a test for trend was used. Effect modification of the relation between nutrients and other variables and risk of polyps by genotype was evaluated by testing for different slopes associated with nutrient intake across genotype. All tests of statistical significance were two-sided. SAS, version 6.11 (SAS Institute, Inc., Cary, NC), was used for all analyses.

As shown in Table 2, cases and controls in this study were similar with respect to the distribution of race/ethnicity, BMI, and regular use of aspirin. Cases were significantly older, more likely to be male, less likely (among women) to have ever used hormone replacement therapy, more likely to have never smoked, and less likely to have used nonsteroidal anti-inflammatory drugs. Cases were also less likely to report a family history of colon cancer; this somewhat paradoxical finding is almost certainly attributable to a greater likelihood of individuals with a family history seeking colonoscopies as a result of minor symptoms or for screening purposes (and thus obtaining insurance reimbursement). There was little difference in the distribution of MTHFR genotypes between cases and controls.

Table 3 shows some dietary intake variables in the study population that are of relevance to the MTHFR hypothesis. On the basis of the new 1998 recommended daily allowances for persons ages 51 and older issued by the Institute of Medicine (folate, 400 μg/day; vitamin B₁₂, 2.4 μg/day; and vitamin B₆, 1.5 mg/day for women, 1.7 mg/day for men), a substantial proportion of individuals appeared to have insufficient intakes of folate (33).

The variant MTHFR genotypes (CT and TT) were not significantly related to the risk of colorectal adenomas when compared with individuals with MTHFR CC genotype (Table 4). However, there appeared to be a weak trend toward an inverse association between the variant allele and colorectal adenoma risk (CC > CT > TT). This trend was apparent only among men, among those younger than age 60, those with two or more polyps, and those without a family history; however, confidence intervals for these subgroups are wide.

Associations between the MTHFR genotype and adenomatous polyps stratified by dietary intakes of several nutrients are shown in Table 5 and Fig. 1. Among those with high or medium intakes of folate, vitamins B₁₂ or B₆, or methionine, the TT genotype is associated with a slightly decreased risk. However, individuals with the MTHFR TT genotype and low intakes of folate, vitamin B₁₂, vitamin B₆ or methionine consistently comprise the stratum at highest risk. Within the TT genotype, increased risks associated with low nutrient intakes relative to high nutrient intakes range from 2-fold (methionine) to ≈3-fold (vitamins B₆ and B₁₂). To explore further the association with vitamin B₁₂ intake, we examined the major food sources of vitamin B₁₂, dairy products and meat. Only higher intakes of dairy products were associated with a decreased risk of adenomas, whereas meat intake did not follow that pattern.

Increasing alcohol intake (Table 5 and Fig. 2) was associated with increased risk only among individuals with the MTHFR CC genotype. In contrast, among those with the MTHFR TT genotype, the lowest risk was seen for those with moderate (<7 g/day) alcohol consumption.

The relations among dietary factors, MTHFR, and risk of adenomatous polyps were similar for men and women (data not shown); with the exception of vitamin B₁₂ intake in women (for which tertile cutpoints were different from these for men), individuals with low intakes and the MTHFR TT genotype were at highest risk compared with the other strata, and trends toward higher risk with low intakes were most pronounced within the MTHFR TT genotype.

There is evidence that nutrient absorption, in particular of vitamin B₁₂, is less efficient at older ages (34), which plausibly would exacerbate risk in those with low intakes. We therefore evaluated the association between MTHFR genotype and adenomatous polyps in a subset of the population, ages 60 and older. It appears that, with higher age, the increased risks associated with low nutrient intakes among those with the MTHFR TT genotype are much more pronounced. Fig. 3 demonstrates these trends, in particular for low vitamin B₁₂ (Fig. 3,b) and low vitamin B₆ (Fig. 3 c) intakes. ORs compared with the reference group of high intake/MTHFR CC genotype ranged between 2.3 (methionine) and 6.5 (vitamin B₁₂). However, confidence intervals for these estimates were wide.

In this case-control study, we observed a weak, statistically nonsignificant inverse association between the MTHFR TT genotype and risk of colorectal adenomas. More importantly, there was substantial modification of the risk associated with low nutrient intakes, depending on the MTHFR genotype; among individuals with the variant MTHFR TT genotype, those with low intakes of vitamin B₆ and B₁₂ were at substantially increased risk of colorectal adenomas compared with those with high intakes. Very consistently, individuals with the MTHFR TT genotype and low intakes of folate, vitamins B₁₂, B₆, or methionine were at highest risk compared with all other groups.

Previously, Ma et al. (24) and Chen et al. (25) reported a significantly decreased risk of colon cancer among men with the MTHFR TT genotype compared with the combined groups of MTHFR CC and CT genotypes in the Physician’s Health Study and Health Professionals Study. Those studies also observed that this inverse association was not seen in individuals with low dietary intakes of folate (or low plasma folate levels). Our study extends these findings toward a consistently increased risk of colorectal adenomas among those with the MTHFR TT genotype (= lowest MTHFR activity) with low nutrient intakes of either folate, vitamin B₁₂, B₆, or methionine and trends toward a decreased risk of colorectal adenomas among those with high nutrient intakes with the MTHFR TT genotype. The results are consistent with those of Ma et al. (24) and Chen et al. (25) in that among those with a TT genotype, a low-nutrient diet increases risk for both colorectal cancer and polyps. However, as far as we know, this is the first study to report a substantially increased risk for adenomas among those with MTHFR TT genotype and low-nutrient diets over those with the CC genotype (and low-nutrient diets), in particular among older individuals.

These results may be attributable to the metabolic position of the enzyme MTHFR, which is part of a complex metabolic entity involving both the generation of the universal methyl-group donor SAM and DNA synthesis via the creation of nucleotides (Fig. 4). There may be a balance between the provision of methyl groups and the supply of bases for DNA synthesis. Previously, James et al. (35) developed a model to explain the association between a methyl-group-deficient diet and nucleotide synthesis; furthermore, Ma et al. (24) discussed the inverse association of the MTHFR TT genotype with colon cancer risk based on the connection with nucleotide synthesis. From the data to date on the way in which the MTHFR genotype influences risk depending on nutrient status, we expand these models to explain these findings in terms of both reduced availability of methyl groups and reduced DNA synthesis capability. The key components of the model are: (a) the available pool of THF and 5,10-methylene-THF, the substrate for thymidylate synthase; and (b) the available pool of SAM, the universal methyl donor for methyltransferases, and an allosteric inhibitor of MTHFR.

Under conditions characterized by low nutrient intakes (Fig. 4), among those with the MTHFR TT genotype, both DNA methylation and DNA synthesis might be impaired, thus increasing the risk of colorectal adenomas. Among individuals with reduced MTHFR activity (genotype TT) and with low intakes of dietary folate and vitamin B₁₂, the activity of the enzyme methionine synthase would be expected to be low (due to lack of the cofactor vitamin B₁₂). This would result in reduced availability of SAM and elevated levels of homocysteine. With low SAM, minimal inhibition of MTHFR would occur; 5,10-methylene THF would continue to be converted to 5-methyl-THF, thus limiting the availability of 5,10-methylene-THF, the substrate for thymidylate synthesis. Low availability of vitamin B₆, the cofactor for serine-hydroxymethyltransferase, would further limit the availability of 5,10-methylene-THF. In addition to low SAM levels and a deficiency of methyl groups, decreased provision of dTMP and decreased synthesis of purines would probably result. Overall, one would expect in the presence of a combination of MTHFR TT genotype and low nutrient intakes both limited provision of methyl groups and DNA synthesis as a result of disturbances in nucleotide synthesis.

Conversely, in conditions characterized by high nutrient intakes (Fig. 4), among individuals with a MTHFR TT genotype, a slightly decreased risk may be observed. With high intakes of folate, vitamin B₁₂, and vitamin B₆, the recycling pathway of 5-methyl-THF to 5,10-methylene-THF would function at its full capacity (all cofactors would be available). Therefore, despite lower than normal MTHFR activity, persons with a TT genotype would have adequate quantities of SAM. Furthermore, the genetically determined reduction in activity of MTHFR, further reduced by inhibition via SAM, would result in an increased pool of 5,10-methylene-THF and provide a greater supply of this substrate for the use of thymidylate synthase and enhanced dTMP production. Similarly, adequate levels of THF for the formation of purines should be available under these circumstances. Overall, under the conditions of high nutrient intakes and the MTHFR TT genotype, adequate provision of SAM could be ensured, and enhanced production of pyrimidines and purines for DNA synthesis could result in a decreased risk of mutations in the colon.

DNA methylation is an essential mechanism of gene regulation, and disturbances may cause differential gene expression (36). In animal models, both folate and vitamin B₁₂ deficiencies can cause imbalances in DNA methylation (37, 38, 39, 40). Folate deficiency in rats has been shown to induce DNA strand breaks and hypomethylation within the p53 tumor suppressor gene (41, 42). Similarly, in rats both folate and vitamin B₁₂ deficiency have been shown to result in deoxynucleotide pool disturbances (35, 43, 44, 45), and decreases in the pyrimidines, dTMP and dTTP, as well as in the pools of dGTP and dATP were observed in methyl-deficient rats (35). Blount et al. (46) have shown that, in humans, folate deficiency can cause massive incorporation of uracil into DNA, resulting in chromosome breaks due to transient nicks. Both high DNA uracil levels and elevated micronuclei frequencies were reversible by folate administration (46). It has been proposed (47) that a chronic imbalance in deoxynucleotide triphosphate pools can contribute to carcinogenesis. The fidelity of DNA synthesis is critically dependent on the balance and availability of the deoxynucleotides (48). We hypothesize that among cells with a high replication rate, such as the colon epithelium, even transient imbalances in nucleotide synthesis may result in a greater mismatching of bases with subsequent potential for point mutations or chromosomal nicks.

Although reduction of plasma homocysteine with folate supplementation appears to be greater among individuals with the TT genotype (20, 49), there are no other data from humans directly or indirectly addressing the in vivo relation between the MTHFR genotype, nutrient intake, SAM production, methylation, and nucleotide synthesis. One supporting observation is that homocysteine levels are highest among those individuals with the TT genotype (17, 18, 19, 20, 21, 22, 23).

Vitamins B₁₂ and B₆ may play an important role in the association between MTHFR and colorectal adenomas; both vitamins are cofactors needed for the recycling of 5,10-methylene-THF. Data presented here as well as studies in other populations suggest that population folate intakes may be low, and that a substantial proportion of individuals may benefit from higher vitamin B₁₂ and possibly vitamin B₆ intakes (50, 51, 52). In a recent study, vitamin B₁₂ levels were inversely correlated with micronuclei formation, and supplementation with folate and vitamin B₁₂ reduced the occurrence of micronuclei (52). Although, more generally, the most substantial reduction in plasma homocysteine is observed with folic acid supplementation, supplements that contain vitamins B₁₂, B₆, and folate were, in several studies, more effective in lowering plasma homocysteine levels than folate alone (53, 54). These results and the data presented here suggest that adequate nutrition of the three B vitamins combined may provide the greatest benefits for colon neoplasia, even in the absence of obvious deficiency (33).

ORs for low nutrient intakes in conjunction with the MTHFR TT genotype were strikingly elevated among individuals ages 60 and older (Fig. 3). These estimates are, nonetheless, based on small numbers of individuals and need to be confirmed in other studies. For a variety of reasons, older individuals may have poorer absorption, particularly of vitamin B₁₂, even in the presence of adequate intakes (34, 55, 56, 57).

The findings on MTHFR genotype and alcohol consumption are unexpected. We found that only those with the MTHFR CC (wild-type) genotype were at increased risk associated with higher alcohol consumption, consistent with the general finding that alcohol is associated in this and other populations with an overall increased risk of adenomatous polyps. Among those with the TT genotype, alcohol was associated with decreased risk. The risk estimates were stable, and the interaction was statistically significant. Our results are in contrast to those of Ma et al. (24) and Chen et al. (25), who observed a higher risk for colon cancer in those with both MTHFR TT genotype and high alcohol consumption compared with those with low alcohol consumption and CC/CT genotypes combined. A similar tool for assessing alcohol intake was used in all studies. There are several explanations for this discrepancy: (a) effects of alcohol differ at early versus late stages of colon carcinogenesis; (b) chance; or (c) bias (it is unlikely, however, that reporting bias would differ by genotype). Some studies have shown that acetaldehyde (but not ethanol) can inhibit methyltransferases and methionine synthase in vitro and in vivo (58, 59). However, many of these experimental settings used alcohol in very high concentrations, which are unlikely to be comparable with the effects of moderate alcohol consumption.

The study population in this case-control study was not necessarily representative of the entire population, because only individuals who underwent colonoscopy were eligible. Individuals in the control group were more likely to have a positive family history as an indication for colonoscopy and probably underwent screening for these reasons. Those with diagnostic follow-up were underrepresented in the control group, which may have rendered the groups somewhat different. On the other hand, the major advantage of this clinic-based approach was that the presence of polyps was clearly established, and that the control group was free of any polyps. Studies that use a population-based control group will probably include a substantial proportion of individuals with undetected polyps, which may attenuate any study findings. Indications for colonoscopy were not related to MTHFR genotype or to intakes of the main nutrients; thus, there is unlikely to be a bias due to differences in indication for colonoscopy. Another strength of this case-control study was the relatively large study size, which allowed us to investigate gene-environment interactions.

The results from this case-control study indicate that associations between folate, vitamin B₁₂, vitamin B₆, and colorectal adenomas vary by MTHFR genotype. Previous inconsistencies in epidemiological studies with respect to the interaction between folate and other nutrients involved in this metabolic pathway (31, 60, 61) may be explained by this differential effect depending on genotype and perhaps on a complex interplay between specific nutrient availability and genotype.

We thank Dr. Arno Motulsky for valuable input to the study and Dale McLarren for assistance with the data management.