Editorial. Dietary intake and blood levels of folate, a watersoluble

Transcription

1 Editorial Folate and Methylenetetrahydrofolate Reductase Polymorphisms: New Nutritional and Genetic Risk Factors for Pancreatic Cancer? Dietary intake and blood levels of folate, a watersoluble B vitamin and important co-factor in one carbon metabolism, 1 appear to be inversely associated with the risk of several malignancies including cancer of the colorectum, oropharynx, esophagus, stomach, lungs, breast, cervix and ovary, and neuroblastoma and leukemia. 2 The precise nature and magnitude of the relationship between folate status and the risk of these malignancies, however, have not been clearly established. 2 The mechanisms by which folate deficiency might enhance the development of these malignancies are in part related to the sole biochemical function known for folate, mediating the transfer of one carbon moieties. 1 As an essential co-factor for the de novo biosynthesis of purines and thymidylate (Figure 1), folate plays an important role in DNA synthesis, stability and integrity, and repair. 1 Folate is also an important cofactor in the remethylation of homocysteine to methionine, which is the precursor of S-adenosylmethionine (SAM), the primary methyl group donor for most biologic methylation reactions, including that of DNA (Figure 1). 1 In this role, folate might modulate DNA methylation, which is an important epigenetic determinant in gene expression (inverse relationship), in the maintenance of DNA integrity and stability, in chromosomal modifications, and in the development of mutations. 3 A growing body of evidence from in vitro, animal, and human studies indicates that folate deficiency is associated with DNA strand breaks, impaired DNA repair, uracil misincorporation, increased mutations, and aberrant DNA methylation, and that folate supplementation can correct some of these defects induced by folate deficiency. 4,5 The role of folate in the development of cancer has been further strengthened by the observations that genetic polymorphisms in the folate metabolic pathway modify the relationships and that the risk modification conferred by these polymorphisms is greatly influenced by folate status, alcohol (a well-known folate antagonist), cigarette smoking (which depletes systemic and intracellular folate), and the supply of other methyl group donors (eg, vitamins B 6 and B 12, and methionine) that are involved in one-carbon transfer reactions. 6,7 Among several genetic polymorphisms in the folate metabolic pathway identified and characterized thus far, single nucleotide polymorphisms (SNPs) in the 5,10- methylenetetrahydrofolate reductase (MTHFR) gene have garnered most attention. 6,7 Intracellular folate homeostasis depends on MTHFR, a critical enzyme in folate metabolism that catalyzes the irreversible conversion of 5,10-methylenetetrahydrofolate (5,10-methyleneTHF) to 5-methylTHF (Figure 1). The substrate 5,10-methyleneTHF, an intracellular coenzymatic form of folate, is the methyl donor for the nonreversible methylation, catalyzed by thymidylate synthase of deoxyuridine-5- monophosphate (dump) to deoxythymidine-5-monophosphate (dtmp), a precursor for DNA synthesis. The synthesis of dtmp results in the oxidation of 5,10-methyleneTHF to the inactive dihydrofolate, which can be converted back to THF by dihydrofolate reductase. 5,10- MethyleneTHF can also be oxidized to 10-formylTHF for de novo purine synthesis. Therefore, 5,10-methyleneTHF is critical to DNA synthesis and to maintaining deoxynucleotide pool. 5-MethylTHF, the product of MTHFR, is the predominant form of folate in blood and provides the methyl group for remethylation of homocysteine to methionine, thereby ensuring the provision of SAM necessary for biologic methylation reactions. A mutation (C T at nucleotide position 677) in the MTHFR gene, which results in an alanine to valine substitution, has been identified. 8,9 This mutation reduces MTHFR activity and increases thermolability of MTHFR, leading to lower levels of 5-methylTHF, an accumulation of 5,10-methyleneTHF, and increased plasma homocysteine (a sensitive inverse indicator of folate status) levels in individuals with a marginal folate status, changes in cellular composition of one carbon moieties, and genomic DNA hypomethylation in lymphocytes in conjunction with marginal folate status Recent biochemical and structural studies of Escherichia coli and human MTHFR have revealed that the MTHFR C677T polymorphism allows a faster dissociation of a critical stabilizing cofactor, flavine adenine dinucleotide (FAD) or a coenzyme form of riboflavin, from the mutant MTHFR compared with the wild-type MTHFR, resulting in thermolability and significantly reduced MTHFR activity. 15,16 The MTHFR C677T polymorphism is a common mutation, with an allele frequency of about 35% in the general North American population. 9,11,13 This polymorphism occurs frequently among white and Asian populations, with rates of 12% 15% for individuals who are homozygous (TT) for the mutation and up to 50% for individuals who are heterozygous (CT). 6,7 A second common polymorphism in the MTHFR gene (A C at nucleotide position 1298), which results in a glutamine to alanine substitution, has also been identified. 17,18 Similar to the C667T polymorphism, the A1298C polymorphism results in decreased MTHFR activity but does not result in a thermolabile protein. 17,18 Allele frequencies up to 33% have been previously reported for this polymorphism. 17,18 The CC prevalence in North American studies, which included mainly white subjects, was 7% 12%. 19 Unlike the C677T polymor- CLINICAL GASTROENTEROLOGY AND HEPATOLOGY 2005;3:

2 August 2005 EDITORIAL 739 Figure 1. Simplified scheme of folate metabolism. MTHFR catalyzes the irreversible conversion of 5,10-methyleneTHF to 5-methylTHF. B12, vitamin B12; DHFR, dihydrofolate reductase; CH 3, methyl group; CpG, cytosine-guanine dinucleotide sequence; SAH, S-adenosylhomocysteine. phism, the effect of the A1298C polymorphism on plasma homocysteine and folate levels is inconsistent, and its functional significance has not yet been well characterized. 18,20,21 However, there is preliminary evidence that under conditions of extreme folate depletion, this polymorphism might become clinically relevant. 22 The C677T and A1298C polymorphisms very rarely exist on the same allele, 17,18,23,24 and the combined MTHFR 677TT and 1298CC homozygous genotypes are exceedingly rare in the general population, 25 but 15% of a white population are heterozygous for both polymorphisms (ie, 677CT/1298AC). 17 These 677CT/1298AC individuals are clinically similar to individuals who are homozygous for the C677T polymorphism with respect to reduced MTHFR activity, higher plasma homocysteine, and reduced plasma folate levels. 17 Not surprisingly, very similar to the effects of folate deficiency, the MTHFR C677T polymorphism has been shown to modulate the risk of cancer. 6 Interestingly, however, the MTHFR C677T polymorphism modulates the risk of several malignancies in a site-specific manner. The C677T polymorphism appears to decrease the risk of colorectal cancer, hepatocellular carcinoma, acute lymphocytic leukemia in adults, certain infant/childhood leukemias, and lymphoma. 6,26 In contrast, the C677T polymorphism seems to increase the risk of cancer of the breast, endometrium, cervix, esophagus, stomach, and bladder. 6,26 This observation might be explained by different folate requirements and hence different susceptibility to folate deficiency and functional consequences among these tissues. 26 The majority of these studies have demonstrated that the cancer risk modification conferred by the C677T polymorphism is further modified by the status of folate and other methyl groups donors, alcohol, smoking, and other SNPs of the MTHFR and other genes involved in the folate metabolic pathway. 6,7,19,26,27 In contrast to the C677T polymorphism, the role of the MTHFR A1298C polymorphism in cancer development has been less extensively studied and is less convincing. However, some studies have found that the 1298CC genotype is associated with a decreased risk of leukemia and colorectal cancer with similar genenutrient/environment interactions observed with the C677T polymorphism. 19,27 Although it is still unclear how the MTHFR polymorphisms could modulate cancer risk depending on the supply of folate and other nutrients and factors that are involved in the folate metabolic pathway, a theoretically attractive hypothesis exists for colorectal cancer. 6,7,26 When the dietary methyl supply is high, individuals with the MTHFR C677T polymorphism might be at reduced risk of colorectal cancer because higher intracellular levels of 5,10-methyleneTHF might prevent imbalances of nucleotide pool during DNA synthesis, thereby ensuring DNA replication with a high fidelity, stable DNA integrity, and decreased mutagenesis (Figure 1). In contrast, when 5-methylTHF is depleted by alcohol consumption, cigarette smoking, or low folate intake, benefit of the MTHFR C677T polymorphism is offset. In this instance, abnormal DNA synthesis and deoxynucleotide pool imbalance that result from depleted intracellular 5,10-methyleneTHF might become the primary mechanisms of colorectal carcinogenesis (Figure 1). Furthermore, in this instance, DNA methylation might be affected because of reduced levels of 5-methylTHF resulting from insufficient supply from diet and reduced de novo methionine synthesis as a result of the MTHFR polymorphism (Figure 1). Recently, several epidemiologic studies have examined the relationship between folate status and the risk of pancreatic cancer. Adenocarcinoma of the pancreas is one of the deadliest and most catastrophic cancers. 28 Pancreatic cancer accounts for only 2% of all newly diagnosed cancers in the United States each year but for 5% of cancer mortality (the fifth most frequent cause of cancer death); only 20% of patients survive for more than 1 year. 28 Each year, approximately 30,000 Americans are diagnosed with pancreatic cancer, and about the same number of patients do not survive. 28 Most cases of pancreatic cancer are diagnosed in advanced, nonresectable stages because of the lack of effective means of early detection and prevention. 28 In addition to probable risk factors for pancreatic cancer including cigarette smoking, long-standing diabetes, chronic and hereditary pancreatitis, and family history of pancreatic cancer, 28 several nutritional factors have been observed to be positively (eg, alcohol, fat, meat, dairy products, energy, body mass index, carbohydrates, cholesterol, salt, and fried food) or inversely (eg, vegetables, fruits, fiber, an-

3 740 EDITORIAL CLINICAL GASTROENTEROLOGY AND HEPATOLOGY Vol. 3, No. 8 tioxidants, and physical activity) associated with the risk of pancreatic cancer. 28,29 Although for most dietary constituents, overall findings have been equivocal and tenuous at best, the portfolio of epidemiologic evidence suggests that dietary factors might account for 35% of pancreatic cancer. 28 For folate, several observations from preclinical studies suggest biologic plausibility for an association between folate status and the risk of pancreatic cancer. In rats, the pancreas contains a high concentration of folate derivatives including 5-methyl- THF and is highly susceptible to folate and other methyl group donor deficiency with biochemical, epigenetic, and clinical consequences including reduced methylation capacity, impaired exocrine pancreatic secretion, pancreatitis, abnormal acinar cell growth and differentiation, and potentiation of the carcinogenic effect of ethionine. 28,30 A case-control study from Australia showed that total intake of folic acid was inversely related to the risk of pancreatic cancer in a dose-dependent manner (P trend.002). 31 In this study, the risk of pancreatic cancer was reduced by 64% in individuals with the highest total intake of folic acid (odds ratio [OR], 0.36; 95% confidence interval [CI], ). 31 In a nested case-control study conducted within the Alpha-Tocopherol, Beta- Carotene Cancer Prevention Study cohort of male Finnish smokers, serum folate concentrations were inversely related to the risk of pancreatic cancer in a dose-dependent manner, with the highest serum levels having approximately half the risk of the lowest (OR, 0.45; 95% CI, ; P trend.009). 32 The similar inverse dose-dependent relationship was also observed for vitamin B A prospective analysis with the same cohort (n 27,101; 13 years of follow-up) also showed a significant dose-dependent inverse association between dietary folate intake and the risk of pancreatic cancer, with the highest dietary intake having half the risk of the lowest (hazards ratio, 0.52; 95% CI, ; P trend.05). 33 In this study, however, dietary intake of vitamins B 6 and B 12 and methionine was not associated with the risk of pancreatic cancer. 33 However, one case-control 34 and another prospective (the Nurses Health Study and the Health Professionals Follow-up Study; n 125,480; 14 years of follow-up) 35 studies conducted in the United States did not observe a significant association between dietary folate intake and the risk of pancreatic cancer. This discrepancy in the results concerning the effect of dietary folate intake on pancreatic cancer risk might be partially explained by differences in study design, cohorts, degree of dietary ascertainment of folate intake, food preparation methods and cooking, and ability to control potential confounders. In this issue of CLINICAL GASTROENTEROLOGY AND HEPA- TOLOGY, 2 studies have shown that the MTHFR C677T polymorphism might modulate the risk of pancreatic cancer. 36,37 In a case-control study from China involving 173 pancreatic cancer patients and 337 control subjects, both the MTHFR 677CT (OR, 2.60; 95% CI, ; P.0005) and 677TT (OR, 5.12; 95% CI, ; P.0001) genotypes significantly increased the risk of pancreatic cancer compared with the MTHFR CC (wildtype) genotype. 36 However, the MTHFR A1298C polymorphisms had no effect on the risk of pancreatic cancer. There was evidence for a more than additive joint effect of the C677T and A1298C polymorphisms on the risk of pancreatic cancer. The 677T-12989A haplotype was associated with a 2.3-fold increase in the risk of pancreatic cancer (OR, 2.29; 95% CI, ; P.0001). Furthermore, the MTHFR C677T polymorphism significantly interacted with smoking and alcohol to increase the risk of pancreatic cancer. Although this study was well designed and conducted, it is associated with usual inherent limitations of the retrospective analysis. One of the most limiting aspects of this study is the lack of information concerning dietary and supplemental intake and blood levels of folate and other methyl group donors. It has been well documented that the cancer risk modification conferred by the MTHFR C677T polymorphism is further modulated by the status of folate and methyl group donors. 6,7,19,26,27 For example, the protective effect of the MTHFR C677T polymorphism on colorectal cancer was evident only in individuals with adequate folate status. 6,7,19,26,27 In those with inadequate dietary intake or low blood levels of folate and methyl group donors, the protective effect conferred by the C677T polymorphism was largely abolished, and in fact, a significantly increased risk of colorectal cancer was observed in some studies. 6,7,19,26,27 The authors of this study, however, did provide information that a large proportion of Chinese adults have a low folate status in various regions of China including Beijing where the study was conducted. This implies that the observed increased risk of pancreatic cancer associated with the MTHFR C677T polymorphism was likely associated with inadequate folate status. Nevertheless, without this critical information concerning the status of folate and methyl group donors, the effect of the MTHFR C677T polymorphism on the risk of pancreatic cancer cannot be accurately ascertained. In contrast, the other case-control study from Johns Hopkins Hospital involving 303 patients with pancreatic cancer and 305 control subjects showed that neither the MTHFR C677T nor A1298C polymorphism significantly affected the risk of pancreatic cancer. 37 Furthermore, there was no significant interaction between these polymorphisms and smoking and diabetes. Similar to the other study, this study did not determine dietary intake and blood levels of folate and other methyl group donors and alcohol consumption that can modulate the effect of the MTHFR polymorphisms. Critical clinical information, including age, sex, ethnic makeup, and potential con-

4 August 2005 EDITORIAL 741 founders, was conspicuously lacking in this report. However, a separate study involving 82 patients with pancreatic (n 74) or biliary (n 8) cancer demonstrated that impaired MTHFR function rendered by the MTHFR C677T polymorphism or loss of heterozygosity (LOH) increased chromosomal deletions, especially at 20 folatesensitive fragile sites often observed in many cancers and at 12 tumor suppressor gene loci inactivated in pancreatic cancer. In yet another separate study involving 105 DNA samples from normal duodenum, 45 pancreaticobiliary cancer xenografts, and 68 cancer cell lines (including 24 pancreatic cancer cell lines), these investigators demonstrated that the MTHFR 677TT genotype and 677T with LOH were associated with a significantly lower degree of global DNA methylation compared with other genotypes (P.014). Therefore, this study showed that cancers with defective MTHFR genotypes are associated with DNA hypomethylation and more chromosome losses. Although the effect of the MTHFR C677T polymorphism on DNA methylation is consistent with the known biochemical function of MTHFR, its effect on chromosomal loss and genetic instability is counterintuitive. However, one possibility is that DNA hypomethylation resulting from the impaired MTHFR function might have caused genomic instability and chromosomal deletions. Because only few modifiable risk factors for pancreatic cancer exist, recent epidemiologic observations that folate status and the MTHFR polymorphisms might modulate the risk of pancreatic cancer underscore the need to further evaluate the role of folate and the MTHFR polymorphisms in the development and progression of this disease. More large scale studies (preferably international and cross-cultural prospective studies) with an accurate determination of dietary intake and blood levels of folate and other methyl group donors, alcohol, and smoking are required to clarify the role of folate and the MTHFR polymorphisms and their interactions in modifying pancreatic cancer risk. Future studies should also incorporate functionally relevant SNPs of other critical genes involved in the folate metabolic pathway to investigate potential nutrient-gene and gene-gene interactions in modifying pancreatic cancer risk. Studies with appropriate chemical and genetically predisposed animal models of pancreatic cancer are also needed to test the potential chemopreventive effect of folate supplementation on the development of pancreatic cancer. Furthermore, biologically plausible mechanisms need to be elucidated to fulfill Koch s criteria for causality. There are already several mechanistic studies investigating functional ramifications of altered DNA methylation, synthesis, and repair resulting from the MTHFR polymorphisms, overexpression, and inhibition in conjunction with folate status. 14,38,39 The puzzling site-specificity of cancer risk modification conferred by the MTHFR polymorphisms remains to be elucidated. If the MTHFR C677T polymorphism is indeed confirmed to increase the risk of pancreatic cancer in future studies, individuals with the MTHFR C677T polymorphism with inadequate folate intake or with significant alcohol and/or tobacco consumption could be potentially targeted for screening and early detection of pancreatic cancer and for folate supplementation to prevent the development of pancreatic cancer. However, folate supplementation is not without risks. Folate is generally regarded as safe and has long been presumed to be purely beneficial. 40 However, studies performed in animal models of colorectal cancer have shown that the dose and timing of folate intervention are critical in providing safe and effective chemoprevention; exceptionally high supplemental folate levels and folate intervention provided after microscopic neoplasia are established in the target tissue promote rather than suppress carcinogenesis. 41 Thus, the potential promoting effect of folate on the progression of preneoplastic or microscopic neoplastic lesions in the pancreas in individuals at high risk of developing pancreatic cancer is more than just a theoretical concern because the vast majority of the US and Canadian populations are exposed to high amounts of folic acid as a result of folic acid fortification and supplementation. 41 The expanding role of folate and SNPs in the folate metabolic pathway in cancer prevention has major public health implications. Although the jury is still out, the implications for folate and the MTHFR polymorphisms in pancreatic cancer prevention and risk modification remain provocative. YOUNG-IN KIM, MD*, *Departments of Medicine & Nutritional Sciences University of Toronto Division of Gastroenterology St Michael s Hospital Toronto, Ontario, Canada References 1. Shane B. Folate chemistry and metabolism. In: Bailey LB, ed. Folate in health and disease. New York: Marcel Dekker, 1995: Kim YI. Role of folate in colon cancer development and progression. J Nutr 2003;133:3731S 3739S. 3. Kim YI. Folate and DNA methylation: a mechanistic link between folate deficiency and colorectal cancer? Cancer Epidemiol Biomarkers Prev 2004;13: Kim YI. Folate and carcinogenesis: evidence, mechanisms, and implications. J Nutr Biochem 1999;10: Choi SW, Mason JB. Folate status: effects on pathways of colorectal carcinogenesis. J Nutr 2002;132:2413S 2418S. 6. Ueland PM, Hustad S, Schneede J, et al. Biological and clinical implications of the MTHFR C677T polymorphism. Trends Pharmacol Sci 2001;22: Bailey LB. Folate, methyl-related nutrients, alcohol, and the MTHFR 677C T polymorphism affect cancer risk: intake recommendations. J Nutr 2003;133:3748S 3753S. 8. Goyette P, Sumner JS, Milos R, et al. Human methylenetetrahydrofolate reductase: isolation of cdna, mapping and mutation identification. Nat Genet 1994;7:

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Genetic polymorphisms in methylenetetrahydrofolate reductase and thymidylate synthase and risk of pancreatic cancer. Clin Gastroenterol Hepatol 2005;3: Matsubayashi H, Skinner HG, Iacobuzio-Donahue C, et al. Pancreaticobiliary cancers with deficient methylenetetrahydrofolate reductase genotypes. Clin Gastroenterol Hepatol 2005;3: Sohn KJ, Croxford R, Yates Z, et al. Effect of the methylenetetrahydrofolate reductase C677T polymorphism on chemosensitivity of colon and breast cancer cells to 5-fluorouracil and methotrexate. J Natl Cancer Inst 2004;96: Stankova J, Shang J, Rozen R. Antisense inhibition of methylenetetrahydrofolate reductase reduces cancer cell survival in vitro and tumor growth in vivo. Clin Cancer Res 2005;11: Campbell NR. How safe are folic acid supplements? Arch Intern Med 1996;156: Kim YI. Will mandatory folic acid fortification prevent or promote cancer? Am J Clin Nutr 2004;80: PII: /S (05)