Folate, a B vitamin found primarily in fruits and vegetables, particularly in leafy greens, is believed to have a protective role in colorectal cancer (CRC) development. This view is supported by several prospective studies of folate intake^1–,⁶ as well as human intervention studies of folate supplementation on surrogate endpoints or provisional markers of CRC,^7–,⁹ and studies using animal models.^7,10

Although the evidence for a protective role for folate in CRC is convincing, epidemiological studies have focused overwhelmingly on dietary intake. In fact, to date only three prospective studies have assessed the effects of circulating folate levels on risk of CRC. The Physicians’ Health Study of men in the United States reported an increased risk of borderline statistical significance in subjects with plasma folate concentrations indicative of deficiency¹¹ whereas the Alpha-Tocopherol Beta-Carotene Study of male smokers in Finland found no association between serum folate concentrations and risk.¹² A statistically significant reduced CRC risk for the highest versus lowest quartile of serum folate was noted in the New York Women’s Health Study.¹³

The putative involvement of folate in CRC is based on its critical function as donor of one carbon groups for nucleotide synthesis and methylation reactions. The distribution of folate between these two pathways is determined in part by the activity of the methylenetetrahydrofolate reductase (MTHFR) enzyme. The substrate of the irreversible MTHFR reaction, 5,10-methylenetetrahydrofolate, promotes nucleotide synthesis. Especially important is the synthesis of thymidylate from uracil, which minimises uracil misincorporation in DNA and thus also double strand breaks, and benefits mismatch repair. The product of the MTHFR reaction, 5-methyltetrahydrofolate, provides one carbon groups for the methylation of the amino acid homocysteine to methionine, a reaction in which vitamin B12 acts as a coenzyme. Methionine is the precursor to S-adenosylmethionine, the universal one carbon donor for methylation reactions, including DNA methylation.

A common polymorphism in the MTHFR gene, 677C>T, results in a thermolabile enzyme with a reduction in activity of up to 70% in TT homozygotes.¹⁴ A second polymorphism, 1298A>C, has also been found to affect enzyme function, although to a lesser extent than 677C>T.^15,16 In CRC, evidence suggests a protective effect for the MTHFR 677C>T polymorphism,^11,17–,²² However, a few large studies have been less convincing.^23–,²⁶ Some reports suggest that the putative protective role may be limited to individuals with high folate status,^11,21,22 presumably because 5,10-methylenetetrahydrofolate levels are sufficient to overcome the reduced enzyme activity, resulting in adequate methylation and enhanced DNA synthesis and repair. This effect may be counteracted by low folate status, since in this case the mutation reduces an already low availability of one carbon groups for methylation reactions. Studies of the 1298A>C polymorphism have been less frequent and less consistent in their findings^{17,18,20,21,23,27,28} but inverse associations with CRC risk have been reported somewhat more frequently than positive associations.

Global DNA hypomethylation, associated with genome instability, is a common trait in tumours.²⁹ Hypermethylation of specific cytosine and guanine rich regions present in the promoters of approximately half of all human genes, called CpG islands, is also believed to be important in cancer development. CpG islands act as molecular switches, silencing gene expression when methylated. In the case of tumour suppressors, for example, promoter hypermethylation can thus aid tumorigenesis.²⁹ Although dietary methyl group supplementation has been reported to increase both genomic and promoter DNA methylation,^30,31 leading to gene silencing in the latter case, the extent to which folate status influences DNA methylation remains to be established.

The aim of this population based nested case referent study was to relate prediagnostic concentrations of plasma folate and homocysteine and the MTHFR 677C>T and 1298A>C polymorphisms to risk of developing CRC.

METHODS

RESULTS

A total of 260 CRC cases were identified and verified. Of these, 34 met one or more of the exclusion criteria, resulting in 226 CRC cases and 437 matched referents for inclusion in the study. DNA samples were unavailable for six cases and 22 referents. Baseline characteristics of the study subjects, as well as tumour site and stage, are presented in table 1⇓. Due to the all female MSP subcohort, women were somewhat overrepresented in the Northern Sweden Health and Disease Cohort and thus also in this study (60% of cases). Median age (25th–75th percentiles) of cases at recruitment to the cohort was 59.8 (50.3–60.2) years for the full study group and 63.4 (57.7–68.0) years for the MSP. Median follow up time between recruitment to the cohort and diagnosis of CRC was 4.2 (2.4–6.7) years for the full study group and 2.9 (1.4–4.2) years for the MSP. No statistically significant differences in median concentrations of plasma folate or homocysteine were observed between cases and referents. Plasma folate concentrations did not vary by fasting duration but homocysteine concentrations were significantly higher in subjects who had fasted less than four hours (p = 0.600 for folate, p<0.001 for homocysteine; Kruskal-Wallis test).

Plasma folate concentrations were significantly related to the risk of CRC in a bell-shaped manner (table 2⇓). Univariate ORs were, for the middle versus the lowest quintile, 2.26 (95% CI 1.29–3.95), and for the highest versus the lowest quintile, 1.55 (95% CI 0.85–2.85). Conditional logistic regression analysis was also performed using cut offs selected to explore the effects over a greater range of plasma folate concentrations. In this case, the OR for the highest versus the lowest category (⩾15.0 versus <5 nmol/l) was 1.10 (95% CI 0.52–2.32). Risk estimates were attenuated somewhat by adjustment for body mass index (BMI), current smoking, recreational and occupational physical activity levels, and alcohol consumption. In contrast with the findings for folate, plasma homocysteine concentrations were not significantly associated with CRC risk (table 2⇓).

Subgroup analyses for various baseline and clinical characteristics are presented in table 3⇓. In subjects with follow up times above the median of 4.2 years, plasma folate concentrations demonstrated a strong, positive, linear association with CRC risk (multivariate OR for highest versus lowest quintile 3.87 (95% CI 1.52–9.87); p trend = 0.007).

The MTHFR 677C>T and 1298A>C polymorphisms were in Hardy-Weinberg equilibrium in both referents and cases (p = 0.399 for 677C>T and p = 0.377 for 1298A>C for the full study group). Strong linkage disequilibrium between the two polymorphisms was also observed (D′ 1.0 (95% CI 0.96–1.0); r² 0.204; LOD 44.38). The estimated haplotype frequencies were 677C–1298A, 37.5%; 677C–1298C, 34.8%; 677T–1298A, 27.7%; and 677T–1298C, 0%. The 677C>T polymorphism, particularly TT homozygosity, was associated with lower plasma folate and higher homocysteine concentrations (Kruskal-Wallis test, p<0.001 for both). For 1298A>C, no statistically significant association with either plasma variable was observed.

The 677C>T polymorphism was inversely related to CRC risk (OR for TT versus CC 0.44 (95% CI 0.21–0.88); p trend = 0.048), a finding that was essentially unchanged in magnitude but attenuated in significance after adjustment for BMI, smoking, recreational and occupational physical activity, and alcohol consumption (OR 0.41 (95% CI 0.19–0.85); p trend = 0.062) (table 4⇓). In contrast with MTHFR 677C>T, a positive association with CRC risk was found for the 1298A>C mutation (OR for CC versus AA 1.52 (95% CI 0.90–2.57); p trend = 0.063), the p trend for which became statistically significant after adjustment for BMI, smoking, recreational and occupational physical activity, and alcohol consumption (p trend = 0.028) (table 4⇓). However, in a multivariate interaction analysis of genotype combinations, with 677CC-1298AA subjects as the reference group, ORs for the 1298A>C polymorphism were considerably lower (table 4⇓). When MTHFR 677C>T and 1298A>C were included in the same multivariate model, risk estimates were not materially affected, but none was statistically significant (data not shown).

A multivariate interaction analysis of CRC risk by combinations of MTHFR 677C>T genotype and plasma folate status is presented in fig 1⇓. Subjects were classified as having adequate or inadequate plasma folate status using a cut off of 6.8 nmol/l. For both folate categories, the CC and CT genotypes yielded very similar ORs and were therefore combined. Our observation of a reduced CRC risk in subjects homozygous for the T allele is shown here to be essentially independent of folate status.

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Figure 1

 Odds ratios (95% confidence interval) for colorectal cancer for combinations of plasma (P) folate status (cut off 6.8 nmol/l selected as a lower limit for adequate plasma folate status) and MTHFR 677 genotype (CC and CT yielded very similar odds ratios and were therefore combined), adjusted for body mass index, current smoking, recreational and occupational physical activity, and alcohol intake. Number of cases/referents: plasma folate ⩾6.8 nmol/l and MTHFR 677 CC or CT, 145/244; plasma folate ⩾6.8 nmol/l and MTHFR 677 TT, 4/18; plasma folate <6.8 nmol/l and MTHFR 677 CC or CT, 59/124; plasma folate <6.8 nmol/l and MTHFR 677 TT, 7/24.

DISCUSSION

In this prospective, population based study, we report a bell-shaped association between plasma folate concentrations and CRC risk. This observation is in contrast with considerable evidence supporting a protective effect for folate in CRC development.^1,7 Results also indicated a reduced CRC risk associated with the MTHFR 677C>T polymorphism that was present even in subjects with an inadequate folate status.

This is only the second prospective CRC study to incorporate data on plasma folate levels as well as both the MTHFR 677C>T and 1298A>C polymorphisms.²⁷ It is also the largest prospective study of circulating levels of folate and risk of CRC. Greater power would, however, have been desirable for the subgroup analyses. Although we were unable to control for a number of covariates, including other dietary intake variables, aspirin or non-steroidal anti-inflammatory drugs, and hereditary CRC, factors such as folate degradation during storage and seasonal fluctuations in folate status were accounted for by the matching process. Levels of red blood cell folate provide a more stable measure of folate status than do plasma concentrations but in large epidemiological studies a single measurement of plasma folate per subject appears to be adequate.³⁵

The main strength of this study was its prospective design, which minimised the problems of selection bias and reverse causation. Given the slow growth of CRC, undiagnosed tumours in the case group at baseline almost certainly existed. However, excluding subjects with follow up times less than one year did not materially affect risk estimates (data not shown).

Our results seem to suggest a reduced CRC risk for subjects with the lowest folate levels (table 2⇑). Although studies have provided some evidence that supraphysiological folate levels may enhance colorectal tumorigenesis,^36–,³⁸ a cancer preventative effect for folate deficiency seems less likely. However, the MTHFR 677C>T polymorphism, which limits the accessibility of folate for methylation reactions, has been reported to decrease the risk of C→T transitions in the p53 gene.³⁹ These mutations occur particularly often at methylated CpG island “hotspots” and are a common event in colorectal tumorigenesis. A decrease in the number and size of induced colorectal tumours in folate deficient rats has also been observed⁴⁰ but a review by Kim suggested that the quality of the test and control diets rather provides further evidence for a carcinogenic effect of supraphysiological folate intakes.⁷

Recently, the idea that folate might protect against CRC development but aid the progression of an established neoplasm has gained attention.^7,41,42 In the present study, however, a strong positive association between folate and CRC risk was apparent in subjects with follow up times over the median of 4.2 years, who would likely have had a lower prevalence of undiagnosed CRC at baseline (table 3⇑). Folate might be speculated to promote the transition from adenoma to carcinoma, a serious consideration given the high prevalence of adenoma in western populations, including Sweden.⁴³ However, this should also have resulted in a positive risk association in the subgroup with shorter follow up times. Of course, the observation may simply be a chance finding. It is none the less interesting and may warrant further study.

Plasma folate concentrations were considerably lower in the present study than in the two previous prospective reports of an inverse association between serum or plasma folate status and CRC risk, despite blood samples in those studies having been collected before the implementation of mandatory folic acid fortification in the USA.^11,13 For example, in the New York University Women’s Health Study,¹³ the lowest quartile cut off was comparable with our highest quintile cut off. Although our study included a greater proportion of fasting blood samples, we observed no variation in plasma folate concentrations by fasting duration. However, in northern Sweden, consumption of fruits and vegetables, major sources of folate in populations without fortification of foods, has been reported to be among the lowest in Europe.⁴⁴ Our study population may thus be better suited to investigating the effects of low plasma folate concentrations on CRC risk. The low folate status of the study group may also have precluded detection of a protective effect for high plasma folate concentrations. The observed bell-shaped, rather than linear, relationship between plasma folate concentrations and CRC risk supports this interpretation.

In the prospective Alpha-Tocopherol Beta-Carotene Study of male smokers in Finland,¹² both plasma folate concentrations and results were comparable with those of the present study. Thus an ethnic explanation for the discrepancy between our findings and the two previous American studies cannot be excluded.^11,13

In contrast with previous reports suggesting a possible positive association between homocysteine and CRC risk,^13,45 no clear relationship was observed in the present study. This may have been due to the relatively high homocysteine levels of the study subjects or possibly to confounding by folate, although including both plasma folate and homocysteine concentrations in the same multivariate model did not materially affect risk estimates (data not shown).

The MTHFR 677C>T polymorphisms were associated with a decreased CRC risk in this study, which is in line with several previous reports,^11,17–,²² However, as demonstrated in fig 1⇑, we found no evidence that this risk reduction might be eliminated or reversed when folate status is inadequate. This contradicts some findings,^11,21,22 including those of Ma et al, in which a similar figure was presented, but not other large dietary studies.^23,28 Although ours may be a chance finding due to the low number of TT homozygous subjects, it underscores the need for further investigation of the gene-nutrient interaction.

Our observation of an increased CRC risk in subjects with the MTHFR 1298A>C polymorphism contradicts a number of previous reports of a decreased, albeit seldom statistically significant, CRC risk associated with 1298CC homozygosity,^{17,18,21,23,27,28} but supports one large study.²⁰ Based on the interaction analysis in table 4⇑, it seems the association between MTHFR 1298A>C and CRC risk may have been largely due to the strong linkage disequilibrium with the MTHFR 677C>T polymorphism.

In light of accumulating evidence that dosage and timing may be critical to the effect of folate in carcinogenesis, concerns have been raised about the consequences of folic acid fortification on cancer incidence.⁴⁶ Supporting this are the results of the recent Aspirin-Folate Polyp Prevention Trial, in which folic acid supplementation was associated with a statistically significant 44% increase in the number of recurrent adenomas.³⁶ Our observations, which provide additional evidence for a detrimental component to the role of folate in colorectal tumorigenesis, may thus have implications in the ongoing debate in Europe concerning mandatory folic acid fortification of foods.

In conclusion, in this nested case referent study, plasma folate concentrations were associated with CRC risk in a bell-shaped manner. In subjects with longer follow up times, plasma folate concentrations were strongly positively related to CRC risk. The MTHFR 677C>T polymorphism was associated with a reduced risk of CRC that was independent of folate status. We speculate that inadequate folate status in the background population allowed us to investigate associations at the lower end of the biological folate spectrum. Without negating the possibility of a protective effect for high folate levels, our findings suggest that low folate status may reduce the risk of CRC.