Hypermethylation of CpG islands in gene promoters is an important epigenetic alteration involved in carcinogenesis [¹]. In colorectal cancer (CRC), the CpG island methylator phenotype (CIMP) is characterized by frequent promoter CpG island hypermethylation [²]. However, little is known about potential determinants of this type of aberrant DNA methylation in CRC.

Folate and methionine are dietary methyl group donors that may be hypothesized to influence DNA methylation, whereas vitamins B2 and B6 potentially modulate the bioavailability of methyl groups [³, ⁴]. Low folate status or intake was suggested to decrease genomic methylation [⁵–⁸], while folate supplementation resulted in increased global DNA methylation in the colonic mucosa [⁹]. Adequate methyl donor intake possibly also prevents aberrant CpG island promoter hypermethylation. In this respect, a weak inverse association with gene promoter hypermethylation was suggested [¹⁰], although methyl donor intake and alcohol consumption, which may reduce the bioavailability of folate, were not associated with CIMP in CRC [¹¹]. Conversely, folate supplementation was suggested to increase promoter hypermethylation of multiple genes in colorectal mucosa [¹²], and circulating folate concentration was associated with increased gene promoter hypermethylation in colorectal tumors [¹³]. In addition, high vitamin B6 intake may be associated with increased MutL homologue 1 (MLH1) promoter methylation in CRC [¹⁴]. Apparently, the precise effect of methyl group bioavailability on gene promoter hypermethylation is still unclear and should be investigated further.

A potential effect of methyl donor intake on DNA methylation may be modified by polymorphisms in folate metabolizing enzymes. For example, the catalytic activity of the methylene tetrahydrofolate reductase (MTHFR) enzyme may be reduced in individuals carrying rare variants of the MTHFR c.665C > T (rs1801133) and c.1286A > C (rs1801131) polymorphisms [¹⁵, ¹⁶], which were also associated with the CIMP phenotype in colorectal cancer [¹⁷–¹⁹]. We previously observed inverse associations between methionine synthase (MTR) c.2756A > G (rs1805087) and CIMP, and between methionine synthase reductase (MTRR) c.66A > G (rs1801394) with MLH1 hypermethylation [²⁰]. Other enzymes involved in epigenetic regulation of gene expression are DNA methyltransferases (DNMTs) and histone methyltransferases (HMTs). However, whether an influence of methyl donor intake is modified by polymorphisms in such epigenetic regulators has not previously been studied in relation to CRC.

Here, we aimed to investigate associations between dietary folate, methionine, vitamins B2 and B6 with overall CRC, and risk of CRCs harboring CIMP, accounting for the occurrence of any, or combinations of rare variants of folate metabolizing enzymes MTHFR, MTR and MTRR, the DNA methyltransferase DNMT3B, and histone methyltransferases Euchromatin histone methyltransferase 1 (EHMT1), Euchromatin histone methyltransferase 2 (EHMT2) and PR domain zinc finger protein 2 (PRDM2) in the Netherlands Cohort Study on diet and cancer.

CRC risk was estimated over tertiles of folate intake, methionine, vitamins B2 and B6, among subjects homozygous for common alleles and among carriers of rare alleles. Folate or methionine intakes were not associated with CRC within either common homozygotes or within heterozygotes and rare homozygotes of any of the genotypes (Table 1). However, we observed a non-significant inverse association between vitamin B2 intake and CRC risk among subjects with the MTHFR c.665CC (rs1801133) common genotype (RR for the highest versus the lowest tertile of intake = 0.66, p_trend = 0.08, Table 2), and an inverse association among subjects with the common GG genotype of PRDM2 G > A (rs2235515, RR = 0.67, p_trend = 0.05). In addition, vitamin B2 was associated with reduced CRC risk in individuals carrying the variant allele of DNMT3B C > T (rs2424913, RR = 0.69, p_trend = 0.05). Conversely, subjects in the third tertile of vitamin B6 intake were at increased CRC risk when they carried the rare allele of DNMT3B C > T (rs406193, RR = 1.90, p_trend = 0.04), or the common allele of PRDM2 G > A (rs2235515, RR = 1.49, p_trend = 0.03). However, interactions between these dietary factors and genotypes were not statistically significant.

We also investigated the associations between methyl donor intake and CRC risk according to the number of rare alleles within each functional group (i.e. folate metabolizing enzymes, DNMT3B and histone methyltransferases). It appeared that methionine was inversely associated with CRC if subjects were homozygous to both of the common variants of the DNMT3B rs2424913 and rs406193 C > T SNPs (RR = 0.44, p_trend = 0.05, P_interaction = 0.07, Table 3). Moreover, relatively high vitamin B2 intake was associated with reduced CRC risk in subjects carrying less than one rare variant of folate metabolizing enzymes (RR = 0.30, p_trend = 0.005, P_interaction = 0.36, Table 4). No dietary associations were observed according to the number of rare alleles in the studied histone methyltransferases (Table 5).

With respect to CpG island promoter hypermethylation, we observed no overall associations between folate, methionine, vitamins B2 or B6 with CIMP (Table 6). Moreover, there were no clear associations between methyl donor intake and CIMP, MLH1 hypermethylation or MSI when accounting for genetic status of individuals (data not shown).

In the current prospective case-cohort study, we observed no clear associations between dietary folate and vitamin B6 with CRC risk when accounting for genetic variants of folate metabolizing enzymes, DNA methyltransferases, or histone methyltransferases. However, relatively high methionine intake may protect against CRC if enzymatic activity of DNMT3B is not affected by two C > T SNPs in its encoding gene. In addition, subjects with high vitamin B2 intake may be at reduced CRC risk in combination with optimal MTHFR activity in individuals homozygous for the common c.665CC (rs1801133) variant, with common PRDM2 GG (rs2235515) genotype and among those with the variant allele of DNMT3B C > T (rs2424913). We observed a strong inverse association between vitamin B2 intake and CRC risk among individuals carrying ≤ 1 rare allele in the combination of any of the folate metabolizing enzymes MTHFR, MTR, or MTRR. There were no associations with the CIMP phenotype overall, or within strata of the studied genotypes.

The MTHFR c.665C > T (rs1801133) polymorphism reduces binding of the MTHFR enzyme to its cofactor flavin adenine dinucleotide (FAD), a metabolite of vitamin B2, resulting in loss of enzymatic activity [¹⁵]. The potentially resulting reduced bioavailability of methyl groups may induce DNA hypomethylation in for example blood cells [³⁵, ³⁶] or CpG island promoter hypermethylation in CRC [¹⁹, ³⁷]. We observed an inverse association between vitamin B2 and CRC risk, predominantly among subjects homozygous for the MTHFR c.665CC (rs1801133) variant, suggesting that vitamin B2 may maximize the catalytic activity of MTHFR when binding to FAD is optimal. Similarly, it was recently observed that high vitamin B2 plasma concentrations, in combination with MTHFR c.665CC or CT genotypes, may reduce risk of CRA recurrence, whereas such an inverse association was not observed among individuals with the MTHFR c.665TT variant [³⁸].

The rare variant of another MTHFR polymorphism, MTHFR c.1286A > C (rs1801131), may also reduce enzymatic MTHFR activity [¹⁶], and was associated with CIMP in colorectal cancer [¹⁸], possibly in combination with low folate and methionine intakes and high alcohol consumption [¹⁷]. However, we previously observed that this polymorphism was neither associated with overall CRC or with the CIMP phenotype [³⁹], nor when methyl donor intake was accounted for in the current study. Possibly, the use of different panels to identify CIMP-high (a “classic” panel [¹⁷] or a new panel [¹⁸, ³⁹] which may be more robust [²]) may have contributed to this inconsistency. Moreover, different essays to measure DNA methylation were used, i.e. MSP [¹⁷, ³⁹] or a quantitative method [¹⁸]. However, in addition to this variety of approaches, it is also important to realize that the one-carbon metabolism is involved in both DNA synthesis as well as DNA methylation, both of which may have an effect on colorectal carcinogenesis [⁴⁰]. The relative contribution of each of these biological processes in carcinogenesis remains to be established and may not have been similar in the investigated study populations. Furthermore, global DNA hypomethylation and CIMP are possibly inversely associated in CRC [⁴¹], and methyl group donors may have an effect on both of these potentially distinct methylation-associated pathways in colorectal carcinogenesis. In this respect, low folate and high alcohol intakes were associated with LINE-1 hypomethylation as an indicator for global DNA hypomethylation [⁸], which is in agreement with in vivo experimental data [⁷].

We did not observe associations between methyl donor intake and the CIMP phenotype in CRC, either overall or after stratifying the analyses for the genetic variants of folate metabolizing enzymes or methyltransferases. Moreover, overall associations between methyl donor intake and CIMP in CRC were not observed in another population-based study [¹¹], while in the same cohort, a diet-gene association with CIMP in CRC was observed for only one out of thirteen one-carbon metabolism genes [¹⁷]. However, these studies, as well as our study, may have lacked adequate power to demonstrate such associations. The effect of methyl donor intake on gene promoter hypermethylation may indeed be weak though, and to demonstrate whether such an effect is modified by genetic variability of the methyl metabolism requires studies with large numbers of cases. Nonetheless, we did observe an inverse association between vitamin B2 and CRC risk in individuals carrying ≤1 variant allele out of the four studied SNPs of folate metabolizing enzymes, suggesting that the combination of common wild-type genotypes, which possibly results in higher bioavailability of methyl groups, protects against CRC in these people.

The findings of our study may indicate that relatively high methionine intake protects against CRC if enzymatic DNMT3B activity is not affected by two polymorphisms. DNMT3B activity, which may be increased by the DNMT3B C > T (rs2424913) polymorphism [⁴²], was associated with CIMP-high in CRC [⁴³], and with increased risk of various other types of cancer [⁴², ⁴⁴, ⁴⁵]. In addition, experimental research suggested that DNMT3B overexpression induced formation of tumors with promoter hypermethylation [⁴⁶]. DNMT3B C > T (rs2424913) was also associated with increased colorectal adenoma risk in individuals with low folate and methionine intakes [⁴⁷], suggesting a nutrient–gene interaction in colorectal carcinogenesis. In view of the function of the DNMT3B enzyme of incorporating methyl groups into DNA, an interaction between methionine intake, DNMT3B polymorphisms and CpG island hypermethylation may be expected, but we did not observe clear associations between methyl donor intake and CIMP, MLH1 hypermethylation or MSI when accounting for DNMT3B genotypes.

The potential protective effects of vitamin B2 or methionine may only be present among individuals with ≤1 polymorphism in folate metabolizing enzymes or among those with common wild-type genotypes of DNMT3B, respectively. This suggests that the occurrence of only one rare variant may be compensated for, but that the combination of several polymorphic genes may lead to disruption of a particular metabolic or regulatory function and to the abolishment of beneficial effects of nutrients. However, we should be careful in drawing definite conclusions because the sample size of our study may have been insufficient to conduct stratified analyses with adequate precision. Moreover, the P-values for interaction were not statistically significant, suggesting the absence of heterogeneity of diet associations with CRC across genotypes. In addition, we conducted several stratified analyses, and these multiple comparisons do not exclude the possibility of reporting chance findings. Nonetheless, although these observations are based on subgroup analyses, and thus have to be interpreted with some caution, this study may indicate that combining genotypes is important to reveal associations of dietary factors with cancer risk. Such an approach has not been followed in previous studies investigating associations between genetic factors and cancer risk, and we recommend that combinations of genotypes should be considered in addition to overall analyses in future studies.

Subgroup analyses in the present study indicated that vitamin B2 and methionine may protect against CRC among individuals who do not carry rare variants of folate metabolizing enzymes and a DNA methyltransferase. However, larger studies are needed to investigate a potential interaction between dietary methyl donor intake, genetic variation of folate metabolizing enzymes and epigenetic regulators, and methylation endpoints in CRC with more precision. Because multiple genes may collectively affect the folate metabolism, combining genotypes of related genes is a useful approach of investigating associations of dietary methyl donors and CRC.

The authors acknowledge Dr. M. Brink for the collection of the tissue samples. This study is funded by the Dutch Cancer Society (UM2004-3171 and UM99-1980).

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