The enzyme 5,10-methylenetetrahydrofolate reductase (MTHFR) catalyses the reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, which is the carbon donor for de novo methionine synthesis and DNA methylation. Approximately 10% of Caucasian populations are homozygous for a common C→T substitution at nucleotide 677 of the MTHFR gene. This mutation converts an alanine to a valine at codon 225 and is associated with reduced enzyme activity and increased thermolability [1]. The potential influence of MTHFR activity on DNA methylation and on the availability of uridylates and thymidylates for DNA synthesis and repair makes MTHFR an attractive candidate for a cancer predisposing gene [2, 3].

Deficiencies in thymidylate have been shown to increase the rate of misincorporation of uridylate into DNA and may in turn lead to an increased rate of DNA strand breaks and other chromosomal damage [4]. Consequently, reduced MTHFR activity, which leads to increased levels of cytosolic 5,10-methylenetetrahydrofolate available for thymidylate synthesis, may protect cells from DNA damage induced by uridylate misincorporation. Consistent with this model, several independent studies have shown that the mutant (valine) allele of MTHFR may confer a twofold reduction in risk of colon cancer [2, 5] and a fourfold reduction in risk of acute lymphocytic leukemia [6]. Mutant MTHFR has also been associated with a 2.8-fold increase in risk of endometrial cancer [7] and a 2.9-fold increase in risk of cervical intraepithelial neoplasia [8]. In the latter situation the increased cancer risk associated with mutant MTHFR might be explained by impairment of DNA methylation due to a reduction in the availability of 5-methyltetrahydrofolate. This is a plausible model because DNA methylation plays a critical role in regulation of gene expression and maintenance of genomic stability [9, 10] and aberrations in normal methylation patterns have been associated with the development of cancer [10, 11]. More recently, Stern et al.[12] demonstrated that individuals who are homozygous for the mutant C677T MTHFR polymorphism show a higher degree of genomic DNA hypomethylation than do homozygotes for the wild-type MTHFR.

The role of folate in breast cancer has been investigated in several dietary studies and most have shown folate consumption to be inversely related to breast cancer risk [13–16]. By contrast, few studies have investigated the influence of the MTHFR C677T polymorphism on breast cancer risk. In one study the frequency of the low MTHFR activity valine allele was reported to occur significantly more frequently in Jewish women diagnosed with bilateral breast cancer or combined breast and ovarian cancer [17]. Another study of unselected breast cancer cases among a Scottish population did not detect any association with the C677T polymorphism and breast cancer risk [18], although the study involved only 62 cases and 66 controls and lacked statistical power.

Given the plausible role of variations in MTHFR activity in breast cancer susceptibility we undertook an analysis of the C677T polymorphism in a case-control study of 335 women with breast cancer and 233 controls.

The frequency of the valine allele among the breast cancer cohort was 0.34, which was higher than that of the controls (0.30), but this was not statistically significant (P = 0.30). The combined number of valine/valine homozygotes and alanine/valine heterozygotes was significantly higher among the breast cancer cases as a whole than among the controls (OR 1.43, 95% CI 1.02–2.00). The significant excess of valine genotypes was only observed among the breast cancer cases diagnosed under the age of 40 years (OR 1.64, 95% CI 1.12–2.41). Interestingly, the valine heterozygotes had a significantly increased OR (OR 1.7, 95% CI 1.2–2.6) while the homozygotes did not (OR 1.4, 95% CI 0.7–2.7). This may be due to the effect of the small number of valine homozygotes.

The potential influence of MTHFR activity on DNA methylation and on the availability of uridylates and thymidylates for DNA synthesis and repair makes MTHFR an attractive candidate cancer-predisposing gene. Reduced MTHFR activity leads to increased levels of cytosolic 5,10-methylenetetrahydrofolate, which may protect cells from DNA damage induced by uridylate misincorporation. Indeed, several independent studies have shown that the mutant valine allele of MTHFR may reduce the risk of colon cancer [2, 5, 20] and acute lymphocytic leukemia [6]. Conversely, mutant MTHFR has also been associated with an increase in risk of endometrial cancer [7] and cervical intraepithelial neoplasia [8]. An increased cancer risk conferred by the valine allele may be explained by impairment of DNA methylation due to a reduction in the availability of 5-methyltetrahydrofolate.

Only two previous studies have examined the relationship between the MTHFR C677T polymorphism and breast cancer risk. Gershoni-Baruch et al.[17] reported that the 677T allele occurred significantly more frequently in Jewish women diagnosed with bilateral breast cancer or combined breast and ovarian cancer. Sharp et al. [18] did not observe any significant difference in the genotype distributions within a case-control study of unselected breast cancers from a Scottish population. Comparison of the number of cases that were compound homozygotes of the low-activity 677TT genotype and/or another low-activity MTHFR variant (1298 CC) revealed a significant reduction in risk (OR 0.26), suggesting that low MTHFR activity may protect against breast cancer. While this association is intriguing, the reliability of the conclusion is tempered by the fact that the study included only 62 cases and 66 controls.

Given the plausible role of variations in MTHFR activity in breast cancer susceptibility and the conflicting conclusions of previous studies we undertook an analysis of the C677T polymorphism in a more substantial number of cancers and controls. Our study revealed a significant excess of valine genotypes among the early onset breast cancers (OR 1.43). In contrast to the study by Gershoni-Baruch et al. [17], we did not observe a significant increase in the valine allele frequency among the women diagnosed with bilateral breast cancer or with a family history of breast cancer (although it was higher than in the controls). The data presented by Sharp et al. [18] suggest that low MTHFR activity is associated with a decrease rather than an increase in breast cancer risk. The reliability of their conclusions, however, is limited by the lack of statistical power. The data from our study of 335 cases suggests that, in fact, low MTHFR activity increases the risk of breast cancer. We recognise that the conflicting conclusions may be attributable to the different characteristics of the breast cancer populations studied. Our study included both familial and early onset breast cancers while those of Sharp et al. [18] were unselected cases and those of Gershoni-Baruch et al.[17] were unselected and familial cases occurring among women of Jewish ancestry.

In conclusion, we suggest that low MTHFR activity is associated with an increased risk of early onset breast cancer. It is possible, however, that chance or confounding factors such as ethnicity and the influence of known risk factors such as oral contraceptive use may have generated a false positive result. Consequently it will be important to replicate our findings in larger population-based case-control studies. In addition, it will be important for future studies to incorporate detailed dietary information, because deficiencies in MTHFR activity may be compensated for by increased folate intake and therefore may confound interpretation of the genetic data.