In this large family-based case-control study of CRC risk we found that common variants in DHFR and MTR may be associated with a decrease in CRC risk in non-users of multivitamin supplements while there was no association in multivitamin users. No other significant associations were observed for common variants in these 15 FOCM-associated genes involved in nucleotide synthesis, methylation, vitamin B12 transport or polyamine synthesis. There was significant heterogeneity by multivitamin use for 4 tagSNPs in MAT2A and 1 tagSNP in MTRR although neither association was statistically significant.
Dihydrofolate reductase (DHFR) reduces dihydrofolate to tetrahydrofolate (THF). Failure of this reaction can trap folate moieties in an oxidized form that cannot be used for further one-carbon transfers. Inhibition of the DHFR reaction has been exploited for chemotherapy by the drug methotrexate (MTX) and increased DHFR activity is a common cause for resistance to MTX (29
). Reduction by DHFR is also involved in the utilization of folic acid, the fully oxidized folate form used for folate supplementation (30
Variation in DHFR seems to affect folate metabolism. Homozygosity for a 19bp deletion polymorphism in the 1st
intron of the DHFR
gene has been associated with increased DHFR mRNA (31
), a 14% decrease in plasma homocysteine (33
), increased red blood cell and plasma folate in women (34
), increased circulating folic acid in those with high folic acid intake (>500 µg/day) and decreased red blood cell folate in those consuming less than 250 µg/day (35
). A study that assessed the association between this polymorphism and CRC risk reported no association for either CpG island methylator positive (CIMP) or negative tumors (36
). Other promoter region (37
) or 3’UTR SNPs (38
) have been identified in DHFR but no SNPs in the coding region of the gene have been reported (33
The vitamin B12
-dependent enzyme methionine synthase (MTR) catalyses the transfer of a methyl group from 5-methyltetrahydrofolate (5-MTHF) to homocysteine to make methionine and tetrahydrofolate (THF). Interference with this process traps folates as 5-MTHF, which cannot be recycled for further one-carbon transfer reactions (39
). The de novo synthesis of methionine is the first step in the synthesis of the universal methyl donor S-adenosylmethionine (SAM) suggesting a possible mechanism by which genetic variation
in the MTR
gene may influence CRC risk. In this population one intronic tagSNP in MTR
(rs4659744) was associated with decreased CRC risk in non-multivitamin supplement users. The association between one non-synonymous SNP in the MTR
, D919G, rs1805087) and CRC risk has been studied by several groups (40
). The reported associations were inconsistent although a recent meta-analysis reported a small but significant decrease in CRC risk for the GG
genotype in European populations (47
). The D919G genotype (not linked to rs4659744) was not associated with risk in this population.
TagSNPs in two other genes, MTRR
were also modified significantly by multivitamin use, although the individual associations were not statistically significant in either multivitamin use group. MTRR reduces oxidized MTR and B12
after transfer of the methyl group from the B12
cofactor to homocysteine, while the MAT2A enzyme transfers the methyl group from methionine to SAM in an ATP-dependent reaction. The MTRR
SNPs rs2303080 and rs2287780 were significantly associated with CRC risk in a recent study of 24 non-synonymous SNPs in 13 folate pathway genes (42
) but were not associated with risk overall or in any subgroup in our study.
Martinez et al reported that the association between the MTHFR C677T TT
genotype and adenoma recurrence was limited to non-multivitamin supplement users with risk increased in that largely post-fortification population (48
). In the current study population, on the other hand, the MTHFR-677 TT
genotype was associated with a significantly decreased CRC risk in individuals not using a multivitamin supplement but an OR of approximately 1.0 in those using a multivitamin supplement, results that are similar to those reported here for the other folate pathway gene variants (21
Numerous other studies of the MTHFR C677T
polymorphisms and other FOCM-associated genes have assessed interactions between genotypes and indices of folate, alcohol, or other B-vitamins, although most of these studies were undertaken in populations not exposed to fortification of foods with folic acid (36
). All but 5 of these studies (36
) support the existence of such interactions. For the MTHFR C677T
genotype the majority of studies reported that the TT
genotype is more protective in those with a diet high in folate or other methyl-group nutrients or low in alcohol (42
) although other studies have reported no such trends or trends in the opposite direction (45
). Although none of these studies looked specifically at multivitamin supplement use, such supplement use is the major source of folates for those in the highest category (66
Clearly, we do not know for certain which nutrients or what combination of nutrients in multivitamins may modify the association between these FOCM-associated genetic variants and colorectal cancer risk. However, folic acid, B6 and B12 are likely to play the dominant roles. Additionally, in this study we used a tagSNP approach to assess genetic effects so it is not possible to predict how the variations tagged by these SNPs might interact with methyl-group nutrient status. It is notable, however, that over 80% of our study population was recruited after fortification of the North American food supply with folic acid, the synthetic folate used in fortified foods and supplements.
In our population, those not taking multivitamin supplements may have had folate levels more similar to those taking multivitamin supplements than to the nonmultivitamin supplement users in pre-fortification populations. The significant increase in plasma and red blood cell folate in both supplement users and non-users after fortification is well documented (67
). Thus, for genotype effects that are more relevant at higher folate levels we would expect to see associations in non-multivitamin supplement users in this population.
For the multivitamin supplement users the interaction between genotype and CRC risk may be more complex. First, genotype effects may become less important when mucosal folate levels are maximized, consistent with the OR’s close to 1.0 that we observed in multivitamin supplement users. Additionally, unmetabolized folic acid levels become measurable in the circulation at folic acid levels over 400 µg/d, due to saturation of the DHFR enzyme (30
). Such high folic acid intakes may be more prevalent in multivitamin users eating folic acid fortified foods (72
). The effect of this on cancer risk is unknown. One in vivo
) reported that increased unmetabolized folic acid was associated with a decrease in natural killer cell activity, suggesting a possible decrease in tumor surveillance. Additionally, sustained exposure to high levels of folic acid was associated with a significant decrease in folate uptake in human colon cancer cells in vitro
). Whether circulating folic acid levels among multivitamin users in fortified populations are high enough to have this effect in vivo
is not known but the possibility should be studied further as it suggests a paradoxical decrease in tissue folate stores among those with the highest folic acid intakes. Decreased folate uptake in those with higher folic acid intakes may nullify any protective effect of genotype. Alternatively, the lack of any effect of genotype on CRC risk in multivitamin users may be due to a higher progression risk in multivitamin users with pre-clinical lesions at the time fortification was instituted in 1998. Such an effect would be consistent with the increased neoplasia risk observed in recent studies of post folic acid fortified populations (14
), the dual effect of folic acid supplementation observed in animal studies (12
) and the co-incident rise in CRC incidence in the US and Canada around the time of fortification (19
). In this regard it may be of interest that the prevalence of multivitamin use was significantly higher in cases than controls in this study population (, p<0.01) suggesting increased CRC risk in supplement users. It is possible that this increase in CRC risk among supplement users ameliorated any protective effects attributable to genotype. Whether multivitamin use is a CRC risk factor in post-fortification populations requires further study.
The strengths of our study include the large number of subjects, the comprehensive approach to tagSNP selection and the use of a family-based design which limits the possibility of population stratification. This study has several limitations. In the main analysis we had limited statistical power for SNPs with a minor allele frequency (MAF) of 5% or less (60% power to detect an OR of 1.4). For SNPs with an MAF of 10% we had 80% power to detect an OR of 1.35. We did not have data on all potentially relevant FOCM pathway genes and may have missed some relevant tagSNPs. However, gene coverage ranged from 71% of all identified SNPs for CBS to 98% for SHMT1. The mean coverage was 89%. Although we did take multiple testing into account some possibility of a false positive result remains given the large number of parameters estimated. Similarly the stringent Bonferroni correction may have resulted in some false negatives in stratified analyses. Our study sample was selected to enrich the subject pool for those at higher CRC risk and recruited largely from populations with mandatory supplementation of foods with folic acid, potentially limiting the generalizability of our results.
In conclusion, in these data from a large population-based study we found significant associations between tagSNPs in DHFR and CRC risk in a study of 395 tagSNPs in 15 folate-pathway genes. Our data suggest that two linked tagSNPs in DHFR and one in MTR may mark genotypes that decrease CRC risk in non-multivitamin supplement users in this folate fortified population. Future studies of similarly fortified populations are required to replicate these results.