AWARD NUMBER: DAMD17-02-1-0606 TITLE: Folate and Breast Cancer: Role of Intake, Blood Levels, and Metabolic Gene Polymorphisms

Folate plays an important role in DNA methylation, synthesis, and repair; intake has been associated with breast cancer. The folate-metabolizing enzyme, methylenetetrahydrofolate reductase (MTHFR) is polymorphic at nucleotides 677 (C3T) and 1298 (A3C), resulting in allozymes with decreased activity. We evaluated these two common polymorphisms and their effects on the folate intake and breast cancer risk association in a population-based case-control study of 1144 breast cancer cases and 1236 controls using a PCRRFLP-based assay. All subjects completed in-person interviews, which included a food frequency questionnaire. Unconditional logistic regression models were used to calculate odds ratios and their 95% confidence intervals, after adjusting for potential confounding factors. Cases and controls were similar in the distribution of MTHFR polymorphisms at codons 677 (41.4% cases and 41.8% controls carried the T allele) and 1298 (17.6% cases and 17.5% controls carried the C allele). An inverse association of breast cancer risk with folate intake was observed in all genotype groups, particularly among subjects with the 677TT genotype. Compared with those with the 677CC genotype and high folate, the adjusted odds ratios (95% confidence intervals) associated with low folate intake were 1.94 (1.15–3.26), 2.17 (1.34–3.51), and 2.51 (1.37–4.60) for subjects who had CC, CT, and TT genotypes (p for interaction, 0.05). No modifying effect of A1298C genotypes on the association of folate intake with breast cancer risk was observed. Results of this study suggest that the MTHFR C677T polymorphisms may modify the association between dietary folate intake and breast cancer risk. Introduction Folate is involved in DNA methylation, synthesis, and repair. Low intake of folate may increase risk for several cancers, including breast cancer (1, 2). The enzyme methylenetetrahydrofolate reductase (MTHFR) irreversibly catalyzes 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, the donor for the remethylation of homocysteine to methionine, the precursor for the universal methyl donor, S-adenosylmethionine (3, 4). Folate that is not converted through this pathway can be used for purine synthesis or the conversion of uracil to thymine, which is used for DNA synthesis and repair (5). Two common polymorphisms in the MTHFR gene have been characterized (6, 7). The 677C3 T polymorphism codes for an alanine to valine substitution in the N-terminal catalytic domain and results in an allozyme with 65% and 30% of the wild-type homozygote activity for heterozygotes and homozygotes of the variant allele, respectively (6, 8). The A3 C polymorphism at nucleotide 1298 codes for an alanine to glutamine substitution in the C-terminal regulatory domain (7). Individuals homozygous for the 1298C allele have approximately the same enzyme activity as those heterozygous for the 677T allele (7, 8). The C677T polymorphism has been examined in relation to several cancers (2, 9). In most studies of colorectal neoplasms, the MTHFR 677TT genotype has been associated with an overall reduction in risk, reduced risk among those with higher intakes of folate (10–13), or increased risk among those with lower folate intakes (13–15). MTHFR has not been as well studied in relation to breast cancer risk. Only three small studies have evaluated the association between MTHFR genotype and breast cancer (16–18). The results from these studies have been inconsistent. Only one study assessed both the C677T and A1298C polymorphisms and their possible joint effect with folate intake (18). However, in that study, only 60 cases were included. We reported recently that folate intake was inversely associated with breast cancer risk in a large population-based, case-control study among Chinese women in Shanghai (19). In an extension of these results, we investigated whether this association may be modified by MTHFR genotypes. Materials and Methods The Shanghai Breast Cancer Study is a population-based, casecontrol study conducted in urban Shanghai, China during 1996–1998. This study was approved by the committees for the use of human subjects in all collaborating institutions. Detailed study methods have been published previously (20). Subjects. All incident breast cancer cases newly diagnosed during the study period and meeting the eligibility criteria were identified through a rapid case-ascertainment system suppleReceived 8/8/03; revised 9/15/03; accepted 9/26/03. Grant support: This research was supported by United States Public Health Service Grant RO1CA64277 from the National Cancer Institute. Dr. Shrubsole is supported, in part, by DAMD17-02-1-0606 from the United States Army Medical Research and Materiel Command. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Requests for reprints: Wei Zheng, Center for Health Services Research, Vanderbilt University Medical Center, Nashville, TN 37232-8300. Phone: (615) 9360682; Fax: (615) 936-1269 E-mail: wei.zheng@vanderbilt.edu. 190 Vol. 13, 190–196, February 2004 Cancer Epidemiology, Biomarkers & Prevention