Dietary intake of folate, vitamin B₆, and vitamin B₁₂, genetic polymorphism of related enzymes, and risk of breast cancer: a case-control study in Brazilian women

Folate and other methyl-related B vitamins are essential nutrients which play important roles in DNA synthesis (genetics), repair, and methylation (epigenetics). These roles in turn indicate a potential association for these vitamins with the development of several types of cancer [1‐3]. For example, in one folate metabolism pathway, the enzyme 5,10-methylenetetrahydrofolate reductase (MTHFR) irreversibly catalyzes the conversion of 5,10-methylenetetrahydrofolate (5,10-methylene THF) to 5-methyltetrahydrofolate (5-methyl THF), the methyl donor in DNA methylation. In another, folate leads to purine synthesis in DNA repair [2‐4]. Activity of the MTHFR gene is attenuated by two common genetic polymorphisms, the MTHFR C677T and A1298C polymorphisms [2, 5]. Methionine synthase (MTR), which catalyzes the remethylation of homocysteine to methionine, has the common genetic polymorphism MTR A2756G [5, 6].

Several studies have suggested an inverse association between folate intake and breast cancer risk in all women [7‐13], pre- [7, 8], and postmenopausal women [7‐9, 12, 14, 15]. However, recent meta-analysis has shown no clear overall association between folate intake or folate levels in the blood and breast cancer risk [2, 16]. A number of studies have examined MTHFR and MTR polymorphisms [4‐6, 17‐25] and found that variant genotypes of MTHFR C677T are associated with an increased [4, 17, 22‐25] or decreased [5, 20] risk of breast cancer, while those of MTHFR A1298C are associated with a decreased risk [24] and those of MTR A2756G with a decreased risk [6]. Several studies have further reported on the interaction between these MTHFR genotypes and folate intake in affecting risk [4, 19]. In addition to the influence of genotype, DNA methylation and synthesis are also subject to the effects of other vitamins relevant to the one-carbon metabolism pathway, including vitamin B₆ (pyridoxine) as a cofactor of MTHFR and vitamin B₁₂ as a cofactor of MTR [4, 6, 19, 26]. Although a role for genotype in modifying the association between dietary intake and disease is biologically plausible [2, 4], research into the protective effects of increasing dietary intake of these B vitamins against breast cancer risk remains inconclusive [2, 27].

In our similar previous case-control study conducted in a Japanese population, we found no significant association between dietary folate intake, MTHFR and MTR genotype, and breast cancer risk [28]. Although the incidence of breast cancer is higher in Brazilian than Japanese women [29], the association between B vitamin intake in the diet, relevant genotypes, and breast cancer risk in Brazilian populations has not been investigated. Here, we conducted a hospital-based case-control study in São Paulo, Brazil, to investigate the role of MTHFR and MTR polymorphisms in modifying the association of folate and other B vitamin intake with breast cancer risk in Brazilian women.

Characteristics of case and control subjects at baseline are shown in Table 1. The breast cancer case group tended to include more smokers and have higher energy intake, but lower alcohol consumption, less moderate physical activity, fewer live births, and lower vitamin supplement use than the control group.

In this study, we found no overall association between folate, vitamin B_6, and vitamin B₁₂ intake and the risk of breast cancer. These findings are consistent with previous studies on folate [4, 14, 16, 20, 28, 33‐40], vitamin B₆ [4, 12, 28, 35, 39, 40], and vitamin B₁₂ [8, 15, 28, 39, 40].

Generally, folate is able to prevent the development of tumors before preneoplastic lesions have been established, but conversely increases tumorigenesis once lesions have been established [39, 41, 42]. Although increased folate intake may be beneficial in populations deficient in this nutrient, increased intake in women with already-sufficient levels of folate may provide no further benefit, or actually be harmful [34]. Folate intake in Brazilian women in the present study (inter-tertile range, 446.2–602.2 μg/day) was higher than that measured in a study of Japanese women (inter-tertile range, 386–501 μg/day) [28] and in a meta-analysis of Caucasian and Asian women (inter-tertile range, 77–255 to 147–359 μg/day and 132–304 to 206–473 μg/day) [2]. In this study, we observed an increased risk of breast cancer in premenopausal women with high folate intake. Folate levels may have been already sufficient in the lower intake group, and thus an increase in intake may on this basis have contributed to tumorigenesis.

Similarly, a study on plasma folate found that concentration was associated with an increase in breast cancer risk in premenopausal but not in postmenopausal women [39]. This study also indicated that the proliferation rate of breast epithelial cells may be higher in premenopausal than postmenopausal women, leaving less time for DNA repair in premenopausal women [39]. Further, a large population-based case-control study reported that high dietary vitamin B₆ intake was associated with an increased risk of breast cancer, but did not indicate the menopausal status of participants [8]. In contrast, another study found a decreased risk of breast cancer in postmenopausal women with high plasma levels of vitamin B₆ [39]. Our finding of a significant increase in breast cancer risk in postmenopausal women due to vitamin B₆ intake may thus be due to chance.

A Canadian study found that a high total folate intake, obtained mainly from nutritional supplements, significantly increased breast cancer risk by 32% (highest >853 μg/day versus lowest quintile ≤336 μg/day) in postmenopausal women [33]. The authors hypothesized that high folic acid concentrations may contribute to epigenetic changes in gene-regulatory mechanisms, which may result in gene silencing and enhanced cancer development, or may promote the growth of tumors expressing folate receptors [33, 41]. However, the mechanism of this effect remains unclear [41]. In our study, however, overall vitamin supplement users (defined as more than once-weekly use for at least one year) accounted for 11.8% of controls, and the distribution of supplement users was similar in the highest and lowest intake groups (data not shown). Because folate and other B vitamin intake in our population was thus obtained primarily from unfortified diets, the increased risk of breast cancer in premenopausal women with high folate intake was unlikely due to supplement use.

A recent meta-analysis indicated no overall significant association between the MTHFR C677T polymorphism and breast cancer risk in Caucasians and East Asians, or between the MTHFR A1298C polymorphism and risk in Caucasians [27]. Our study supports the lack of association found in this meta-analysis as well as in others studies in Caucasian and Asian women [1, 5, 43]. In contrast to another finding of the meta-analysis [27], however, which found an increased risk of breast cancer in premenopausal women with the MTHFR 677CT and TT genotype compared to those with the CC genotype, we did not observe any increase in risk in these groups. Further, contrary to the findings in a previous study conducted in Americans [1], we did not find an increased risk of breast cancer to be related to the genotype combination of MTHFR A1298C and C677T. This lack of concordance in findings may be due to the low proportion of the MTHFR 677T genotype in our study population or to compensation by the folate-replete status of patients for the effects of the variant alleles of the MTHFR A1298C and C677T genotypes [2]. Nevertheless, few studies have achieved conclusive results regarding whether functional polymorphisms in one-carbon metabolizing genes affect breast cancer risk associated with dietary intake of folate and other related B vitamins, the biological plausibility of this scenario notwithstanding [2].

The MTR gene contributes to alterations in the plasma levels of homocysteine and folate; specifically, subjects with the 2756AG and GG genotypes have lower levels of plasma homocysteine or higher levels of serum folate than those with the 2756AA genotype [44, 45]. When methionine levels are adequate, these variant polymorphisms with lower enzyme activity may also result in elevated homocysteine levels and DNA hypomethylation [46]. A Polish study reported the possible association between the hetero- and homozygote variants of the MTR A2756G and a decreased risk of breast cancer [6]. In the present study, however, we observed an increased risk of breast cancer in total subjects who had the MTR 2756GG genotype, a finding similar to that reported in a recent study conducted in Taiwan [46]. This observed increase may be due to the linkage between the MTR A2756G polymorphism and other genetic polymorphisms, or to the interaction between the MTR A2756G polymorphism and other genetic polymorphisms related to folate metabolism, such as that for methionine synthase reductase (MTRR G66A) [25, 45], cytosolic serine hydroxymethyltransferase (cSHMT C1420T) [21], or MTHFR C677T [47]. However, we observed no statistically significant associations between the combined genotypes of MTR A2756G and either MTHFR A1298C or C667T and breast cancer risk, nor could we test the joint effects of MTR with MTRR or cSHMT genes since MTRR and cSHMT genes were not available in the present study. Given the low frequency of the MTR 2756GG genotype in our study population, the significant finding of our present study may be the result of chance.

Previous authors have speculated that interactions between folate and MTHFR polymorphism may be elicited to show that (a) the lower activity of variant genotypes may increase the risk of breast cancer at low levels of dietary folate since less 5-methyl THF is made available for DNA methylation, and (b) variant genotypes might provide advantages over the wild genotype in folate-replete conditions since 5,10-methyl THF would be available for nucleotide synthesis [2, 4]. Supporting notion (a), we found an increased risk of breast cancer among women with the lowest folate intake who had the MTHFR 1298AC and CC genotype. Contrary to (a), however, we also saw a decrease in breast cancer risk among women with the lowest folate intake who had the MTHFR 677CT and TT genotype. Similar findings of decreased breast cancer risk in variant MTHFR C677T genotypes have been reported in previous studies in German [5], Japanese-American [20], and Chinese patients [10]. The reduced activity of the MTHFR C677T polymorphism was speculated to increase the pool of intracellular 5,10-methylene THF, which protects against potential defects in DNA synthesis, although circulating folate levels (5-methyl THF) tended to be lower [10]. Nevertheless, the inconsistencies in results between this and previous studies may be due to differences in ethnicity [10, 19], background dietary status [19, 37], or possibly chance. Given the above discussion regarding folate intake level, the increased risk of breast cancer observed here in patients with the MTHFR 1298AA and MTR 2756AA genotype in a higher tertile of folate intake may be due to the harmful effects brought about by excessive intake of folate. Notion (b) suggests that the lower activity of variant alleles of MTHFR A1298C provides advantages over the MTHFR 1298AA genotype, but with the compensation of sufficient folate intake, the lower enzyme activity was attenuated to result in the lack of significant association seen with the variant MTHFR A1298C genotypes in the higher category of folate intake. In addition, the observed trend to a decline in breast cancer risk with increased vitamin B₁₂ intake (P for trend = 0.073) and significant interaction between vitamin B₁₂ and MTHFR C677T polymorphism might be due to the function of vitamin B₁₂ as a cofactor in folate metabolisms [12, 19].

Several limitations of this study warrant mention. First, dietary intake of folate, vitamin B₆, and vitamin B₁₂ was assessed after breast cancer diagnosis and was therefore sensitive to recall bias. Case subjects may have been more motivated to recall diet than controls, and their recall may also have been inaccurate, depending on the belief of an association between diet and their disease. Second, although substantially high participation rates among both eligible case and control subjects minimized potential selection bias, the use of cancer-free patients as controls, whose dietary habits may have differed from the general population due to health consciousness or disease concerns, might have lead to selection bias. Third, as our subjects were recruited from eight hospitals in São Paulo, Brazil, they may not necessarily be representative of the general Brazilian population, and thus any extrapolation of results to the general population should be done cautiously. Fourth, our validation study found relatively low correlation coefficients between FFQ and DR-based nutrient intake estimates. Further, the validation study indicated that measurement errors in assessing nutrient intake by FFQ were unavoidable, although such random errors tend to result in null associations [48]. Fifth, although our quality control assessment of the genotyping showed high concordance rates between Genetic Lab. and our laboratory, we did not genotype the duplicate blinded samples of SNPs used in the present study and therefore cannot rule out the possibility of misclassification in genotyping status, although its effect on the results might be minimal. Sixth, stratified analyses were performed based on a relatively small number of cases, possibly limiting the interpretability of our results.

In this case-control study conducted in Brazilian women, we found no overall significant associations between dietary intake of folate, vitamin B₆, and vitamin B₁₂, MTHFR genotype, and breast cancer risk. However, we did identify an increased risk of breast cancer in total women with the MTR 2756GG genotype, increased risk associated with higher folate intake in premenopausal women, and gene-nutrient interactions. Further studies into these factors will help to elucidate the etiology of breast cancer.