Background: The MTHFR C677T TT genotype is associated with a 15% to 18% reduction in colorectal cancer risk, but it is not clear if other variants of the gene are associated with colorectal cancer risk.

Methods: We used a tagSNP approach to comprehensively evaluate associations between variation in the MTHFR gene and colorectal cancer risk using a large family-based case-control study of 1,750 population-based and 245 clinic-based families from the Colon Cancer Family Registry. We assessed 22 TagSNPs, selected based on pairwise r2 >95%, using the Haploview Tagger and genotyped the TagSNPs on the Illumina GoldenGate or Sequenom platforms. The association between single nucleotide polymorphisms and colorectal cancer was assessed using log-additive, codominant, and recessive models.

Results: From studying the population-based families, the C677T (rs1801133) and A1298C (rs1801131) polymorphisms were associated with a decreased colorectal cancer risk overall [odds ratio (OR), 0.81; 95% confidence interval (95% CI), 0.63-1.04; and OR, 0.82; 95% CI, 0.64-1.07, respectively]. The 677 TT genotype was associated with a decreased risk of microsatellite-stable/microsatellite-low tumors (OR, 0.69; 95% CI, 0.49-0.97) and an increased risk of microsatellite-high tumors (OR, 2.22; 95% CI, 0.91-5.43; Pinteraction = 0.01), as well as an increased risk of proximal cancers and a decreased risk of distal and rectal cancers (Pinteraction = 0.02). No other single nucleotide polymorphism was associated with risk overall or within subgroups.

Conclusion: The 677 TT and 1298 CC genotypes may each be associated with a decrease in colorectal cancer risk. We observed little evidence of additional genetic variability in the MTHFR gene relevant to colorectal cancer risk. Cancer Epidemiol Biomarkers Prev; 19(1); 89–100

5,10-Methylenetetrahydrofolate reductase (MTHFR) is a key enzyme in folate-associated one-carbon metabolism. The MTHFR enzyme is a flavin adenine dinucleotide–dependent enzyme that irreversibly reduces 5,10-methyltetrahdrofolate to 5-methyltetrahydrofolate, acting at the junction of two critical uses for folate-associated one-carbon groups, nucleotide synthesis and synthesizing the universal methyl donor S-adenosylmethionine. The MTHFR gene is polymorphic and two common nonsynonymous single nucleotide polymorphisms (SNP), C677T (A222V; rs1801133) and A1298C (E429A; rs1801131), have been extensively studied for associations with colorectal cancer. Both genotypes have been associated with decreased enzyme function in vitro, with reductions of ∼60% for the 677 TT genotype (1, 2) and 30% for the 1298 CC genotype (3, 4). Four recent meta-analyses of these MTHFR genotypes and colorectal cancer risk overall, using data from 22 studies, reported a modest but statistically significant 15% to 18% decrease in risk for the 677 TT genotype (5-8). A similar inverse association was also reported for the 1298 CC genotype on colorectal cancer risk (5, 7). Many studies reported that the decreased colorectal cancer risk associated with the 677 TT genotype was observed mainly in those with higher folate availability (9-14). However, all of these studies were conducted in populations not exposed to supplementation of the food supply with folic acid.

More recently, studies have assessed associations between an increased number of polymorphic loci in the MTHFR gene and other health outcomes using tagging SNP or haplotype-based approaches for SNP selection (15, 16). Liu et al. (15) reported statistically significant associations between two SNPs and lung cancer risk, whereas in another study, three MTHFR SNPs were significantly associated with lean body mass after correction for multiple testing (16). These data suggest that there may be additional functionally significant variants in the MTHFR gene, but whether such variants are important determinants of colorectal cancer risk is unknown.

In the present study, we used a tagSNP approach to comprehensively evaluate associations between variation in the MTHFR gene, including the two known functional polymorphisms, and colorectal cancer risk in a large family-based case-control study based on the Colon Cancer Family Registry (C-CFR). We also assessed whether associations between SNPs and colorectal cancer risk were modified by dietary or total folate intake, folate supplement, or multivitamin use and assessed potential heterogeneity of the MTHFR SNP to colorectal cancer associations by selected tumor characteristics.

Data for this study were obtained through the C-CFR, a National Cancer Institute (NCI)–funded registry of colorectal cancer cases, unaffected family members, and population-based controls, which uses comprehensive and standardized methods for data collection and genotyping. Detailed information about the C-CFR can be found in a recent report by Newcomb et al. (17) and at the CFR Web site.12

Case and Control Ascertainment

The C-CFR is an international collaborative study initiated in 1997 with the goal of creating a resource for the study of the genetic epidemiology of colorectal cancer. Subject recruitment at the different C-CFR sites is described in detail by Newcomb et al. (17) and is described only briefly here. Participants were recruited from six centers including centers in the University of Southern California Consortium (Arizona, Cleveland Clinic, Colorado, Dartmouth, Minnesota, North Carolina, and University of Southern California), Hawaii (Honolulu), Fred Hutchinson Cancer Research Center (Seattle, WA), Mayo Clinic (Rochester, MN), Cancer Care Ontario (Toronto, Canada), and University of Melbourne (Victoria, Australia) using population-based and clinic-based ascertainment strategies. All centers except Fred Hutchinson Cancer Research Center oversampled cases with multiple first-degree relatives reporting colorectal cancer or colorectal cancer cases diagnosed under age 50 y to target families with excess colorectal cancer risk. First-degree and some second-degree relatives with colorectal cancer were also recruited from families with multiple colorectal cancer cases. For all centers, unaffected siblings or, when necessary, second-degree relatives were recruited as controls. The clinic-based sample represents multiple-case families at high risk of Hereditary Non-Polyposis Colon Cancer or other familial colorectal cancer phenotypes.

We used a case/unaffected sibling control design with data from both population-based and clinic-based families in the main effect analyses. There were too few clinic-based case/control pairs for stratified analyses so all stratified analyses used the population-based families only. Cases were probands and siblings diagnosed with colorectal cancer and controls were siblings without colorectal cancer at the time of ascertainment. All cases were interviewed within 5 y of diagnosis (73% within 2 y). We excluded monozygous twins. In addition, we also genotyped a random set of unrelated population-based controls (n = 265) from one of the C-CFR sites (Fred Hutchinson Cancer Research Center). All subjects signed an informed consent before providing data to the C-CFR.

SNP Selection

TagSNPs were selected using Haploview Tagger (18) using the following criteria: minor allele frequency (MAF) of >5%, pairwise r2 of >0.95, and distance from closest SNP of >60 bp on the Illumina platform. The linkage disequilibrium blocks were determined using data from HapMap data release no. 16c.1, June 2005, on National Center for Biotechnology Information B34 assembly, dbSNP b124. For each gene, we extended the 5′- and 3′-untranslated regions (UTR) to include the 5′-UTR and 3′-UTR most SNP within the linkage disequilibrium (LD) block (∼10 kb upstream and 5 kb downstream). In regions of no or low LD, SNPs with an MAF of >5% at a density of ∼1 per kb were selected from either HapMap or dbSNP. Finally, nonsynonymous SNPs and expert-curated SNPs regardless of MAF were included. In this analysis, we report results for 22 tagSNPs and 3 candidate nonsynonymous SNPs in one gene central to regulation of the folate pathway, MTHFR.

SNP Genotyping

SNPs were genotyped on the Illumina GoldenGate platform (19). We implemented a series of quality control checks based on the Illumina metrics, and SNPs were excluded from analysis based on the following criteria: GenTrain score of <0.4, 10%GC score of <0.25, AB T Dev of >0.1239, call rate of <0.95, more than two P-P-C errors, or less than three discordance with HapMap. Interplate and intraplate replicates were included, and SNPs were excluded from the analysis if there were more than two errors on the replicate genotypes. In addition, genotype data from 30 CEPH trios (Coriell Cell Repository, Camden, NJ) were used to confirm reliability and reproducibility of the genotyping. SNPs were excluded from the analysis if more than three discordant genotypes were discovered in comparison with the genotypes from the International HapMap Project (20). We performed additional genotyping using Sequenom's iPLEX Gold for two SNPs (rs17376328 and rs2050265) that were not successfully genotyped on the Illumina platform. PCR and extension primers for these two SNPs were designed using the MassARRAY Assay Design 3.0 software (Sequenom, Inc.) and are available upon request. PCR amplification and single base extension reactions were done according to the manufacturer's instructions. Extension product sizes were determined by mass spectrometry using Sequenom's Compact matrix-assisted laser desorption/ionization–time-of-flight mass spectrometer. The resulting mass spectra were converted to genotype data using SpectroTYPER-RT software.

Two SNPs were excluded because they failed genotyping (rs17375901 and rs3753582—one nonsynonymous SNP (rs2274976, R593Q) was excluded due to missing genotypes for over 900 individuals and one SNP (rs7533315) was excluded because it was significantly out of Hardy-Weinberg equilibrium at a significance level of <0.001 (P = 4.74E-08). This analysis reports on the remaining 22 SNPs.

Microsatellite Instability Testing

All available tumors from the C-CFR's Jeremy Jass Memorial Pathology Bank were assayed for instability at the following 10 microsatellites: BAT25, BAT26, BAT40, BAT34C4, D5S346, D17S250, D18S55, D10S197, ACTC, and MYCL as described previously (17). Only subjects with clear results for at least four markers were included. Microsatellite Instability data were available for 1,200 (66.4%) of cases. Instability at >30% of the tested loci was defined as microsatellite instability high (MSI-H); instability at >10% of loci but <30% of loci was defined as microsatellite instability low (MSI-L); and those with instability at 0 loci were categorized as microsatellite stable (MSS). Due to the small numbers and the lack of evidence for a separate effect in these data, the MSI-L cases were combined with the MSS cases in the analysis.

Folate Supplement Use, Multivitamin Use, and Colon Subsite

A standard risk factor questionnaire was administered to all participants in both populations at the time of recruitment and was available for 1,782 cases and 2,815 controls (∼98% of the study population for this analysis). The questionnaire collected information on demographic factors; personal and family cancer histories, including personal history of colorectal polyps, colorectal, and other cancers; and other risk factors including selected medication use, use of a folate supplement and multivitamin at least twice weekly for more than a month in the 2 y before recruitment; tobacco and alcohol use history; physical activity and selected dietary preferences (e.g., cooking preference for red meat); and reproductive history (in women). Alcohol use was queried for three time periods (20s and 30s, 40s, and since turning 50). In this analysis, we summed across all three periods to get an estimate of lifetime use. Weekly alcohol intake was calculated as the sum of drinks per week from beer, wine, and liquor.

Dietary Folate Intake

Estimated dietary folate intake during the same 2-year time period, available for 585 cases and 837 controls (approximately one-third of the study population), was estimated from a validated food frequency questionnaire developed at the University of Hawaii (21) and available only for subjects recruited in North America, excluding those from the Mayo clinic and Fred Hutchinson Cancer Research Center (which did not administer a food frequency questionnaire). All food frequency data were collected after 1998, when supplementation of the food supply with 140 μg folic acid per 100 g of cereal products became mandatory. Dietary folate intake was estimated using a food composition table that included that supplementation in one analysis and also, in a separate analysis, where we ignored this supplementation. For the postsupplementation analysis, dietary and total folate were estimated using dietary folate equivalents to allow for the different bioavailability of folic acid.

Tumor Subsite

Tumor subsite was obtained from the pathology report and was available for 1,734 (96.0%) of cases. Right colon was defined as occurring in the cecum through the splenic flexure; left colon included the descending colon through the sigmoid colon; and rectal tumors included the rectosigmoid junction and the rectum.

Statistical Analysis

MAF was estimated from the genotype data from unrelated population-based controls. Pairwise linkage disequilibrium between SNPs was estimated using the square of the correlation coefficient (R2) between markers. In the analysis of main effects, the population- and clinic-based data were analyzed separately. We used multivariable conditional logistic regression with sibship as the matching factor to estimate main effects and stratum-specific odds ratios (OR). We controlled for age and sex in all analyses. Additional control for folic acid supplement use (yes/no), multivitamin use (yes/no), and alcohol intake (none, 1-7 drinks/wk and >7 drinks/wk) did not change the results. In addition, we present only the age- and sex-adjusted models here. Except for the C677T and A1298C polymorphisms, for which there are data supporting a recessive model, we assumed a log-additive model to assess genotype/colorectal cancer associations. Because prior data suggest specific effects for C677T and A1298C, P values for these two SNPs were not corrected for multiple testing. For the log-additive model, P values for the remaining SNPs were adjusted for multiple testing, taking into account correlated tagSNPs, using a modified test of Conneely and Boehnke (22), which is valid only for one–degree of freedom (df) tests. As a secondary analysis, we assessed all the genotype effects in a codominant model, comparing heterozygotes and homozygotes for the minor allele to those homozygous for the major allele, using dummy variables to obtain the OR and 95% confidence interval (95% CI) for each genotype and a 2-df likelihood ratio to test to estimate P values for each comparison. Third, we assessed the possibility for recessive effects for all SNPs, comparing those homozygous for the minor allele to those with one or no minor alleles. The results using the recessive models were the same as those using the codominant models, and only the codominant model results are included here. For multiple df likelihood ratio tests, the Bonferroni method was used to reset the significance level to 0.00227.

All analyses within the exposure strata were specified in advance based on indications for potential effect modification in the literature. Stratum-specific ORs were estimated among population-based families to evaluate heterogeneity by MSI (MSS/MSI-L and MSI-H), tumor subsite (right, left, rectum/rectosigmoid junction), regular use of a folate or multivitamin supplement (yes or no), and dietary folate intake (dichotomized at the median of the control population). Because not all centers recruited subjects from populations with folic acid fortification of the food supply (i.e., Australia and New Zealand), we assessed potential heterogeneity by center. Finally, we considered whether inclusion of cases recruited >2 y after diagnosis resulted in biased estimates by comparing SNP OR estimates for cases diagnosed under and over 2 y after diagnosis. There was no heterogeneity for either variable. For all stratified analyses, we included interaction terms in the regression models to get the interaction P values and used a 2-df (MSI status) or 3-df (tumor subsite) likelihood ratio test to assess heterogeneity. All statistical analyses were conducted using the R programming language and SAS v9.1.

Table 1 describes the MTHFR SNPs included in this study. Characteristics of the study population are presented in Table 2. There were 1,806 population-based cases with 2,879 sibling controls. For the clinic-based population, there were 269 cases with 475 sibling controls. Both study populations were mainly Caucasian: 87.5% of cases and 87.3% of controls in the population-based sample and 97.3% of cases and 97.5% of controls in the clinic-based sample reported their race/ethnic group as white. In the population-based sample, 17% were from Ontario, Canada; 64% were from the four U.S. sites; and 19% were from Australia or New Zealand. For the clinic-based population, none of the participants were from Canada; 14% were from the USC consortium in the United States; 41% were from Australia or New Zealand; 45% were from the Mayo Clinic in the United States; and no cases or controls were from the Hawaii or Seattle sites. Sixty-five percent of population-based cases reported no family history of colorectal cancer; 30% reported at least one first-degree relative with a family colorectal cancer history; and family history data were missing for 4.6%. In the clinic-based cases, 37.5% reported no first-degree relative with colorectal cancer; 26% reported at least one first-degree relative with colorectal cancer; and family history was missing for 36.4%.

Table 1.

Primary SNP data for the 22 SNPs

SNPLocation in gene/protein changePositionBase change in assayMAF* population-based families
rs11121832 Intron 11782707 A/G 0.25 
rs12121543 Intron 11777258 A/G 0.24 
rs12404124 Intron 11796456 C/T 0.41 
rs13306556 Intron 11774697 A/G 0.10 
rs1476413 Intron 11774887 A/G 0.27 
rs17037390 Intron 11783430 A/G 0.16 
rs17037396 Intron 11784634 C/T 0.11 
rs17037425 Intron 11792970 C/T 0.14 
rs17421462 Flanking 3′-UTR 11779434 A/G 0.08 
rs17421511 Intron 11780375 A/C 0.16 
rs1801131 E428A 11777063 A/G 0.31 
rs1801133 A221V 11778956 C/T 0.32 
rs1994798 Intron 11777342 A/G 0.41 
rs3737964 Intron 11789631 C/T 0.25 
rs3737965 Intron 11789038 C/T 0.06 
rs4846048 Intron 11768839 C/T 0.29 
rs4846049 Intron 11772952 C/T 0.32 
rs4846054 Intron 11791817 G/T 0.40 
rs6541003 3′-UTR 11778454 G/T 0.40 
rs9651118 Intron 11784801 A/G 0.23 
rs17376328 5′ UTR 11799249 G/A 0.05 
rs2050265 5′UTR 11802286 T/C 0.16 
SNPLocation in gene/protein changePositionBase change in assayMAF* population-based families
rs11121832 Intron 11782707 A/G 0.25 
rs12121543 Intron 11777258 A/G 0.24 
rs12404124 Intron 11796456 C/T 0.41 
rs13306556 Intron 11774697 A/G 0.10 
rs1476413 Intron 11774887 A/G 0.27 
rs17037390 Intron 11783430 A/G 0.16 
rs17037396 Intron 11784634 C/T 0.11 
rs17037425 Intron 11792970 C/T 0.14 
rs17421462 Flanking 3′-UTR 11779434 A/G 0.08 
rs17421511 Intron 11780375 A/C 0.16 
rs1801131 E428A 11777063 A/G 0.31 
rs1801133 A221V 11778956 C/T 0.32 
rs1994798 Intron 11777342 A/G 0.41 
rs3737964 Intron 11789631 C/T 0.25 
rs3737965 Intron 11789038 C/T 0.06 
rs4846048 Intron 11768839 C/T 0.29 
rs4846049 Intron 11772952 C/T 0.32 
rs4846054 Intron 11791817 G/T 0.40 
rs6541003 3′-UTR 11778454 G/T 0.40 
rs9651118 Intron 11784801 A/G 0.23 
rs17376328 5′ UTR 11799249 G/A 0.05 
rs2050265 5′UTR 11802286 T/C 0.16 

*MAF was estimated from genotype data from unrelated population-based controls.

Table 2.

Selected characteristics of the study population

Population-based familiesClinic-based families
Cases (n = 1,806)Sibling controls (n = 2,879)P*Cases (n = 269)Sibling controls (n = 475)P
Person characteristic 
    Mean age ± SD 53.5 ± 10.9 54.0 ± 11.8 <0.01 49.1 ± 11.4 51.4 ± 11.8 <0.01 
    Sex, no. (%) 
        Male 927 (51.3) 1278 (44.4) <0.01 133 (49.4) 204 (42.9) 0.19 
        Female 879 (48.7) 1601 (55.6)  136 (50.6) 271 (57.1)  
    Race, no. (%) 
        Non-Hispanic white 1580 (87.5) 2512 (87.3) 1.00 262 (97.4) 463 (97.5) 1.00 
        Black 32 (1.8) 42 (1.5)  1 (0.4) 2 (0.4)  
        Asian 69 (3.8) 113 (3.9)  0 (0) 0 (0)  
        Other 104 (5.8) 189 (6.6)  5 (1.9) 9 (1.9)  
        Unknown/missing 21 (1.2) 23 (0.8)  1 (0.4) 1 (0.2)  
    Center, no. (%) 
        Ontario, Canada 308 (17.1) 515 (17.9) — 0 (0) 0 (0) 1.00 
        USC Consortium, U.S. 384 (21.3) 519 (18.0)  38 (14.1) 48 (10.1)  
        Melbourne, Australia 344 (19.0) 611 (21.2)  110 (40.9) 213 (44.8)  
        Hawaii, U.S. 63 (3.5) 103 (3.6)  0 (0) 0 (0)  
        Mayo Foundation, U.S. 282 (15.6) 526 (18.3)  121 (45.0) 214 (45.1)  
        Seattle, U.S. 425 (23.5) 605 (21.0)  0 (0) 0 (0)  
Family history of colorectal cancer, no. (%) 
No first -degree relative 1177 (65.2)   101 (37.5)   
At least first-degree relative 546 (30.2) — — 70 (26.0) — — 
Unknown/Missing 83 (4.6)   98 (36.4)   
    BMI (kg/m2) 
        15-18 (underweight) 22 (1.2) 25 (0.9) <0.01 6 (2.2) 12 (2.5) 0.82 
        18-25 (normal) 629 (34.8) 1155 (40.1)  97 (36.1) 174 (36.6)  
        25-30 (overweight) 670 (37.1) 1036 (36.0)  100 (37.2) 174 (36.6)  
        30+ (obese) 422 (23.4) 594 (20.6)  53 (19.7) 92 (19.4)  
        Unknown/missing 63 (3.5) 69 (2.4)  13 (4.8) 23 (4.8)  
    Alcohol use (drinks/wk) 
        None 467 (25.9) 829 (28.8) 0.08 76 (28.3) 132 (27.8) 0.43 
        1-7 (moderate) 857 (47.5) 1353 (47.0)  124 (46.1) 226 (47.2)  
        8+ (heavy) 229 (12.7) 362 (12.6)  39 (14.5) 61 (12.8)  
        Unknown/missing 253 (14.0) 335 (11.6)  30 (11.2) 56 (11.8)  
    Smoking 
        Never 781 (43.2) 1309 (45.5) 0.24 138 (51.3) 226 (47.6) <0.01 
        Former 632 (35.0) 1001 (34.8)  58 (21.6) 153 (32.2)  
        Current 343 (19.0) 509 (17.7)  66 (24.5) 87 (18.3)  
        Unknown/missing 50 (2.8) 60 (2.1)  7 (2.6) 9 (1.9)  
    Folate supplements§ 
        No 1586 (87.8) 2557 (88.8) 0.14 233 (86.6) 411 (91.3) 0.25 
        Yes 196 (10.9) 274 (9.5)  31 (11.5) 39 (8.7)  
        Unknown/missing 24 (1.3) 48 (1.7)  5 (1.9) 11 (2.3)  
Multivitamins§ 
        No 820 (45.4) 1497 (52.0) <0.01 138 (51.3) 267 (56.2) 0.27 
        Yes 971 (53.8) 1346 (46.8)  129 (48.0) 200 (42.1)  
    Calcium supplements§ 
        No 1335 (73.9) 2063 (71.7) 0.03 208 (77.3) 345 (76.7) 0.33 
        Yes 459 (25.4) 785 (27.3)  56 (20.8) 105 (23.3)  
Unknown/missing 12 (0.7) 31 (1.1)  5 (1.9) 2 (0.4)  
    Dietary folate (μg; mean ± SD) 327.4 ± 118.7 334.1 ± 126.8 0.32 349.6 ± 154.9 346.0 ± 145.0 0.95 
    Total folate DFE (mean ± SD) 477 ± 265.6 525.4 ± 439.7 <0.01 606.5 ± 463.1 549.6 ± 322.2 0.29 
    Dietary B12 (mean ± SD) 3.0 ± 1.2 2.9 ± 1.3 0.67 3.1 ± 1.4 3.0 ± 1.4 0.36 
    Total B12 (mean ± SD) 6.2 ± 6.4 7.4 ± 11.8 <0.01 10.0 ± 17.6 8.8 ± 10.0 0.41 
    Dietary B6 (mean ± SD) 1.1 ± 0.4 1.1 ± 0.4 0.26 1.1 ± 0.5 1.1 ± 0.4 0.64 
    Total B6 (mean ± SD) 1.9 ± 2.0 2.3 ± 3.8 <0.01 3.0 ± 6.0 2.6 ± 3.3 0.48 
Tumor characteristics 
    Site, no. (%) 
        Right colon 598 (33.1) —  85 (31.6) —  
        Left colon 525 (29.1)   44 (16.4)   
        Rectum 593 (32.8)   77 (28.6)   
        Unknown/missing 90 (5.0)   63 (23.4)   
    MSI, no. (%) 
        MSS 855 (47.3) —  61 (22.7) —  
        MSI-L 151 (8.4)   14 (5.2)   
        MSI-H 179 (9.9)   55 (20.4)   
        Unknown/missing 621 (34.4)   139 (51.7)   
Population-based familiesClinic-based families
Cases (n = 1,806)Sibling controls (n = 2,879)P*Cases (n = 269)Sibling controls (n = 475)P
Person characteristic 
    Mean age ± SD 53.5 ± 10.9 54.0 ± 11.8 <0.01 49.1 ± 11.4 51.4 ± 11.8 <0.01 
    Sex, no. (%) 
        Male 927 (51.3) 1278 (44.4) <0.01 133 (49.4) 204 (42.9) 0.19 
        Female 879 (48.7) 1601 (55.6)  136 (50.6) 271 (57.1)  
    Race, no. (%) 
        Non-Hispanic white 1580 (87.5) 2512 (87.3) 1.00 262 (97.4) 463 (97.5) 1.00 
        Black 32 (1.8) 42 (1.5)  1 (0.4) 2 (0.4)  
        Asian 69 (3.8) 113 (3.9)  0 (0) 0 (0)  
        Other 104 (5.8) 189 (6.6)  5 (1.9) 9 (1.9)  
        Unknown/missing 21 (1.2) 23 (0.8)  1 (0.4) 1 (0.2)  
    Center, no. (%) 
        Ontario, Canada 308 (17.1) 515 (17.9) — 0 (0) 0 (0) 1.00 
        USC Consortium, U.S. 384 (21.3) 519 (18.0)  38 (14.1) 48 (10.1)  
        Melbourne, Australia 344 (19.0) 611 (21.2)  110 (40.9) 213 (44.8)  
        Hawaii, U.S. 63 (3.5) 103 (3.6)  0 (0) 0 (0)  
        Mayo Foundation, U.S. 282 (15.6) 526 (18.3)  121 (45.0) 214 (45.1)  
        Seattle, U.S. 425 (23.5) 605 (21.0)  0 (0) 0 (0)  
Family history of colorectal cancer, no. (%) 
No first -degree relative 1177 (65.2)   101 (37.5)   
At least first-degree relative 546 (30.2) — — 70 (26.0) — — 
Unknown/Missing 83 (4.6)   98 (36.4)   
    BMI (kg/m2) 
        15-18 (underweight) 22 (1.2) 25 (0.9) <0.01 6 (2.2) 12 (2.5) 0.82 
        18-25 (normal) 629 (34.8) 1155 (40.1)  97 (36.1) 174 (36.6)  
        25-30 (overweight) 670 (37.1) 1036 (36.0)  100 (37.2) 174 (36.6)  
        30+ (obese) 422 (23.4) 594 (20.6)  53 (19.7) 92 (19.4)  
        Unknown/missing 63 (3.5) 69 (2.4)  13 (4.8) 23 (4.8)  
    Alcohol use (drinks/wk) 
        None 467 (25.9) 829 (28.8) 0.08 76 (28.3) 132 (27.8) 0.43 
        1-7 (moderate) 857 (47.5) 1353 (47.0)  124 (46.1) 226 (47.2)  
        8+ (heavy) 229 (12.7) 362 (12.6)  39 (14.5) 61 (12.8)  
        Unknown/missing 253 (14.0) 335 (11.6)  30 (11.2) 56 (11.8)  
    Smoking 
        Never 781 (43.2) 1309 (45.5) 0.24 138 (51.3) 226 (47.6) <0.01 
        Former 632 (35.0) 1001 (34.8)  58 (21.6) 153 (32.2)  
        Current 343 (19.0) 509 (17.7)  66 (24.5) 87 (18.3)  
        Unknown/missing 50 (2.8) 60 (2.1)  7 (2.6) 9 (1.9)  
    Folate supplements§ 
        No 1586 (87.8) 2557 (88.8) 0.14 233 (86.6) 411 (91.3) 0.25 
        Yes 196 (10.9) 274 (9.5)  31 (11.5) 39 (8.7)  
        Unknown/missing 24 (1.3) 48 (1.7)  5 (1.9) 11 (2.3)  
Multivitamins§ 
        No 820 (45.4) 1497 (52.0) <0.01 138 (51.3) 267 (56.2) 0.27 
        Yes 971 (53.8) 1346 (46.8)  129 (48.0) 200 (42.1)  
    Calcium supplements§ 
        No 1335 (73.9) 2063 (71.7) 0.03 208 (77.3) 345 (76.7) 0.33 
        Yes 459 (25.4) 785 (27.3)  56 (20.8) 105 (23.3)  
Unknown/missing 12 (0.7) 31 (1.1)  5 (1.9) 2 (0.4)  
    Dietary folate (μg; mean ± SD) 327.4 ± 118.7 334.1 ± 126.8 0.32 349.6 ± 154.9 346.0 ± 145.0 0.95 
    Total folate DFE (mean ± SD) 477 ± 265.6 525.4 ± 439.7 <0.01 606.5 ± 463.1 549.6 ± 322.2 0.29 
    Dietary B12 (mean ± SD) 3.0 ± 1.2 2.9 ± 1.3 0.67 3.1 ± 1.4 3.0 ± 1.4 0.36 
    Total B12 (mean ± SD) 6.2 ± 6.4 7.4 ± 11.8 <0.01 10.0 ± 17.6 8.8 ± 10.0 0.41 
    Dietary B6 (mean ± SD) 1.1 ± 0.4 1.1 ± 0.4 0.26 1.1 ± 0.5 1.1 ± 0.4 0.64 
    Total B6 (mean ± SD) 1.9 ± 2.0 2.3 ± 3.8 <0.01 3.0 ± 6.0 2.6 ± 3.3 0.48 
Tumor characteristics 
    Site, no. (%) 
        Right colon 598 (33.1) —  85 (31.6) —  
        Left colon 525 (29.1)   44 (16.4)   
        Rectum 593 (32.8)   77 (28.6)   
        Unknown/missing 90 (5.0)   63 (23.4)   
    MSI, no. (%) 
        MSS 855 (47.3) —  61 (22.7) —  
        MSI-L 151 (8.4)   14 (5.2)   
        MSI-H 179 (9.9)   55 (20.4)   
        Unknown/missing 621 (34.4)   139 (51.7)   

Abbreviations: DFE, dietary folate equivalent; BMI, body mass index.

*P values using a 1-df likelihood test from a conditional logistic regression model.

Includes individuals who self-identified themselves as Hispanic, Native, Hawaiian/Pacific Islander, and mixed race.

Self-reported weight and height (2 y before the questionnaire completion date) used to calculate body mass index.

§Ever use of supplements regularly during the 2 y before recruitment (at least twice per week for more than a month).

Calorie-adjusted calculated from the food frequency questionnaire using postfortification food composition tables (n cases = 585; n controls = 837).

Approximately 47% of subjects with risk factor data in each population reported drinking a moderate amount of alcohol (1-7 drinks per week) and ∼10% of each study population reported taking a folic acid supplement. In the population-based families, 54% of cases and 47% of controls reported taking a multivitamin regularly in the 2 years before enrollment in this study (P < 0.01). Population-based cases were significantly younger (P < 0.01), more likely to be male (P < 0.01), and had a significantly higher body mass index (P < 0.01). Among the subjects with food frequency questionnaire data (32.4% of cases and 29.1% of controls), cases were less likely to take calcium supplements (P = 0.04), had lower total folate intake (P = 0.02), and lower total B12 intake (0.019). In the clinic-based families, cases were significantly younger (P < 0.01), more likely to be smokers (P < 0.01), and more likely to be very active (P < 0.01). Cases in the clinic-based population were more likely to have tumors with MSI-H (20.4% and 9.9% in the clinic-based and population-based samples, respectively) and less likely to have MSS tumors (22.7% and 47.3%, respectively).

We evaluated 22 SNPs in MTHFR with MAFs ranging from 0.05 to 0.41 (Table 1). The results of the main effect analyses are presented in Table 3. We observed a borderline statistically significant decrease in colorectal cancer risk (OR, 0.81; 95% CI, 0.63-1.04; P = 0.10) for the 677 TT genotype (rs1801133) relative to the CC and CT genotypes in the population-based series. There was a nonstatistically significant inverse association for the 677 TT relative to the CT and TT genotypes in the clinic-based population (OR, 0.59; 95% CI, 0.31-1.12; P = 0.11). The 1298 CC genotype (rs1801131) relative to the AC and AA genotypes was also associated with a borderline statistically significant decrease in colorectal cancer risk among the population-based families (OR, 0.82; 95% CI, 0.64-1.07; P = 0.14) but not among the clinic-based families (OR, 1.02; 95% CI, 0.53-1.98; P = 0.95). Under the assumption of a log-additive model, no other SNP was statistically significantly associated with colorectal cancer risk in either study population after the correction for multiple testing (Table 3). When we assessed associations assuming a codominant model, homozygosity for the minor allele was nominally associated with colorectal cancer risk for five SNPs in the population-based cases (rs12404124, rs1994798, rs4846048, rs4846049, and rs6541003), but no P value was significant at the Bonferroni-adjusted P value (Table 3). In the clinic-based cases, no SNPs were associated with risk in the codominant models. The results for the recessive models were essentially the same as those for the codominant models.

Table 3.

Single SNP analysis: colorectal cancer risk by SNP, analysis model, and study population

Population-based sample*
SNPLog-additive modelCorrected PCodominant model: heterozygotesPCodominant model: homozygotesP2-df LRT P
OR (95% CI)OR (95% CI)OR (95A% CI)
rs11121832 0.95 (0.82-1.10) 0.93 0.96 (0.81-1.14) 0.63 0.89 (0.64-1.25) 0.51 0.79 
rs12121543 0.94 (0.81-1.08) 0.94 0.89 (0.75-1.05) 0.17 1.00 (0.71-1.41) 1.00 0.34 
rs12404124 0.90 (0.79-1.02) 0.54 0.96 (0.81-1.14) 0.62 0.77 (0.60-1.00) 0.05 0.13 
rs13306556 1.01 (0.82-1.23) 0.99 0.98 (0.79-1.22) 0.86 1.22 (0.63-2.39) 0.56 0.83 
rs1476413 0.95 (0.82-1.09) 0.95 0.93 (0.78-1.10) 0.38 0.93 (0.67-1.30) 0.68 0.68 
rs17037390 0.86 (0.73-1.02) 0.56 0.86 (0.72-1.04) 0.13 0.75 (0.45-1.25) 0.26 0.26 
rs17037396 1.00 (0.83-1.22) 0.98 0.94 (0.76-1.17) 0.59 1.34 (0.71-2.53) 0.37 0.50 
rs17037425 0.90 (0.75-1.07) 0.80 0.91 (0.75-1.10) 0.34 0.74 (0.42-1.28) 0.27 0.46 
rs17421462 1.01 (0.81-1.26) 1.00 0.94 (0.74-1.18) 0.59 1.97 (0.83-4.67) 0.12 0.24 
rs17421511 0.93 (0.79-1.10) 0.95 0.93 (0.77-1.12) 0.46 0.88 (0.54-1.45) 0.63 0.74 
rs1801131§ 0.82 (0.64-1.07) 0.14 0.90 (0.76-1.07) 0.23 0.76 (0.56-1.02) 0.07 0.19 
rs1801133§ 0.81 (0.63-1.04) 0.10 1.10 (0.93-1.31) 0.26 0.88 (0.66-1.17) 0.36 0.14 
rs1994798 0.89 (0.78-1.00) 0.44 0.95 (0.80-1.14) 0.59 0.76 (0.59-0.98) 0.03 0.08 
rs3737964 0.95 (0.83-1.09) 0.93 0.96 (0.81-1.13) 0.61 0.89 (0.64-1.24) 0.50 0.78 
rs3737965 0.90 (0.68-1.17) 0.95 0.90 (0.68-1.19) 0.45 0.77 (0.22-2.70) 0.69 0.73 
rs4846048 0.87 (0.76-1.00) 0.42 0.93 (0.78-1.10) 0.37 0.71 (0.52-0.96) 0.03 0.09 
rs4846049 0.87 (0.76-0.99) 0.37 0.89 (0.75-1.05) 0.16 0.74 (0.56-0.99) 0.04 0.13 
rs4846054 0.91 (0.80-1.03) 0.64 0.97 (0.82-1.14) 0.75 0.79 (0.61-1.03) 0.08 0.17 
rs6541003 0.89 (0.79-1.01) 0.52 0.96 (0.81-1.14) 0.62 0.77 (0.59-0.99) 0.05 0.12 
rs9651118 1.15 (1.00-1.33) 0.45 1.26 (1.06-1.50) 0.01 1.09 (0.76-1.55) 0.65 0.03 
rs17376328 1.04 (0.78-1.38) 1.00 1.06 (0.80-1.42) 0.67 0.43 (0.05-3.43) 0.43 0.67 
rs2050265 0.87 (0.73-1.03) 0.58 0.88 (0.73-1.07) 0.21 0.68 (0.41-1.15) 0.15 0.26 
 
Clinic-based families 
rs11121832 1.09 (0.77-1.54) 1.00 1.28 (0.81-2.02) 0.30 0.91 (0.41-2.03) 0.83 0.83 
rs12121543 1.12 (0.81-1.56) 0.99 1.05 (0.70-1.59) 0.81 1.41 (0.64-3.10) 0.39 0.71 
rs12404124 1.00 (0.74-1.36) 1.00 0.94 (0.61-1.46) 0.79 1.02 (0.55-1.89) 0.95 0.93 
rs13306556 1.12 (0.68-1.83) 0.93 1.12 (0.68-1.87) 0.65 1.14 (0.11-11.8) 0.91 0.91 
rs1476413 1.03 (0.74-1.44) 1.00 1.02 (0.68-1.52) 0.93 1.09 (0.50-2.38) 0.84 0.98 
rs17037390 0.90 (0.59-1.38) 1.00 0.90 (0.56-1.43) 0.65 0.83 (0.23-3.07) 0.78 0.89 
rs17037396 1.07 (0.66-1.75) 1.00 1.11 (0.67-1.84) 0.69 0.73 (0.06-8.58) 0.81 0.89 
rs17037425 0.99 (0.64-1.53) 1.00 1.01 (0.63-1.61) 0.97 0.79 (0.14-4.59) 0.79 0.95 
rs17421462 1.32 (0.78-2.24) 0.87 1.16 (0.67-2.00) 0.59 9.91(1.25-78.4) 0.03 0.12 
rs17421511 0.98 (0.64-1.50) 1.00 1.19 (0.77-1.83) 0.43 0.27 (0.05-1.48) 0.13 0.09 
rs1801131* 1.02 (0.53-1.98) 0.95 0.98 (0.65-1.47) 0.92 1.01 (0.48-2.10) 0.99 0.98 
rs1801133* 0.59 (0.31-1.12) 0.10 1.21 (0.82-1.80) 0.33 0.69 (0.33-1.44) 0.32 0.16 
rs1994798 1.08 (0.78-1.49) 1.00 1.04 (0.67-1.61) 0.86 1.19 (0.62-2.27) 0.60 0.85 
rs3737964 1.06 (0.76-1.47) 1.00 1.23 (0.78-1.93) 0.37 0.90 (0.43-1.87) 0.78 0.49 
rs3737965 2.04 (1.07-3.88) 0.43 2.04 (1.07-3.88) 0.03 NA NA 0.17 
rs4846048 1.02 (0.72-1.44) 1.00 1.27 (0.81-1.98) 0.30 0.85 (0.42-1.74) 0.66 0.33 
rs4846049 0.96 (0.69-1.34) 1.00 0.98 (0.65-1.48) 0.93 0.91 (0.44-1.88) 0.79 0.96 
rs4846054 1.07 (0.78-1.45) 1.00 1.03 (0.67-1.60) 0.88 1.15 (0.62-2.15) 0.66 0.89 
rs6541003 1.06 (0.76-1.46) 1.00 0.93 (0.60-1.43) 0.73 1.17 (0.61-2.25) 0.64 0.69 
rs9651118 0.95 (0.68-1.33) 1.00 1.08 (0.70-1.68) 0.73 0.71 (0.35-1.48) 0.36 0.64 
rs17376328 0.64 (0.32-1.32) 0.81 0.64 (0.32-1.32) 0.23 NA NA 0.46 
rs2050265 0.92 (0.60-1.40) 1.00 0.92 (0.57-1.46) 0.71 0.85 (0.23-3.12) 0.80 0.93 
Population-based sample*
SNPLog-additive modelCorrected PCodominant model: heterozygotesPCodominant model: homozygotesP2-df LRT P
OR (95% CI)OR (95% CI)OR (95A% CI)
rs11121832 0.95 (0.82-1.10) 0.93 0.96 (0.81-1.14) 0.63 0.89 (0.64-1.25) 0.51 0.79 
rs12121543 0.94 (0.81-1.08) 0.94 0.89 (0.75-1.05) 0.17 1.00 (0.71-1.41) 1.00 0.34 
rs12404124 0.90 (0.79-1.02) 0.54 0.96 (0.81-1.14) 0.62 0.77 (0.60-1.00) 0.05 0.13 
rs13306556 1.01 (0.82-1.23) 0.99 0.98 (0.79-1.22) 0.86 1.22 (0.63-2.39) 0.56 0.83 
rs1476413 0.95 (0.82-1.09) 0.95 0.93 (0.78-1.10) 0.38 0.93 (0.67-1.30) 0.68 0.68 
rs17037390 0.86 (0.73-1.02) 0.56 0.86 (0.72-1.04) 0.13 0.75 (0.45-1.25) 0.26 0.26 
rs17037396 1.00 (0.83-1.22) 0.98 0.94 (0.76-1.17) 0.59 1.34 (0.71-2.53) 0.37 0.50 
rs17037425 0.90 (0.75-1.07) 0.80 0.91 (0.75-1.10) 0.34 0.74 (0.42-1.28) 0.27 0.46 
rs17421462 1.01 (0.81-1.26) 1.00 0.94 (0.74-1.18) 0.59 1.97 (0.83-4.67) 0.12 0.24 
rs17421511 0.93 (0.79-1.10) 0.95 0.93 (0.77-1.12) 0.46 0.88 (0.54-1.45) 0.63 0.74 
rs1801131§ 0.82 (0.64-1.07) 0.14 0.90 (0.76-1.07) 0.23 0.76 (0.56-1.02) 0.07 0.19 
rs1801133§ 0.81 (0.63-1.04) 0.10 1.10 (0.93-1.31) 0.26 0.88 (0.66-1.17) 0.36 0.14 
rs1994798 0.89 (0.78-1.00) 0.44 0.95 (0.80-1.14) 0.59 0.76 (0.59-0.98) 0.03 0.08 
rs3737964 0.95 (0.83-1.09) 0.93 0.96 (0.81-1.13) 0.61 0.89 (0.64-1.24) 0.50 0.78 
rs3737965 0.90 (0.68-1.17) 0.95 0.90 (0.68-1.19) 0.45 0.77 (0.22-2.70) 0.69 0.73 
rs4846048 0.87 (0.76-1.00) 0.42 0.93 (0.78-1.10) 0.37 0.71 (0.52-0.96) 0.03 0.09 
rs4846049 0.87 (0.76-0.99) 0.37 0.89 (0.75-1.05) 0.16 0.74 (0.56-0.99) 0.04 0.13 
rs4846054 0.91 (0.80-1.03) 0.64 0.97 (0.82-1.14) 0.75 0.79 (0.61-1.03) 0.08 0.17 
rs6541003 0.89 (0.79-1.01) 0.52 0.96 (0.81-1.14) 0.62 0.77 (0.59-0.99) 0.05 0.12 
rs9651118 1.15 (1.00-1.33) 0.45 1.26 (1.06-1.50) 0.01 1.09 (0.76-1.55) 0.65 0.03 
rs17376328 1.04 (0.78-1.38) 1.00 1.06 (0.80-1.42) 0.67 0.43 (0.05-3.43) 0.43 0.67 
rs2050265 0.87 (0.73-1.03) 0.58 0.88 (0.73-1.07) 0.21 0.68 (0.41-1.15) 0.15 0.26 
 
Clinic-based families 
rs11121832 1.09 (0.77-1.54) 1.00 1.28 (0.81-2.02) 0.30 0.91 (0.41-2.03) 0.83 0.83 
rs12121543 1.12 (0.81-1.56) 0.99 1.05 (0.70-1.59) 0.81 1.41 (0.64-3.10) 0.39 0.71 
rs12404124 1.00 (0.74-1.36) 1.00 0.94 (0.61-1.46) 0.79 1.02 (0.55-1.89) 0.95 0.93 
rs13306556 1.12 (0.68-1.83) 0.93 1.12 (0.68-1.87) 0.65 1.14 (0.11-11.8) 0.91 0.91 
rs1476413 1.03 (0.74-1.44) 1.00 1.02 (0.68-1.52) 0.93 1.09 (0.50-2.38) 0.84 0.98 
rs17037390 0.90 (0.59-1.38) 1.00 0.90 (0.56-1.43) 0.65 0.83 (0.23-3.07) 0.78 0.89 
rs17037396 1.07 (0.66-1.75) 1.00 1.11 (0.67-1.84) 0.69 0.73 (0.06-8.58) 0.81 0.89 
rs17037425 0.99 (0.64-1.53) 1.00 1.01 (0.63-1.61) 0.97 0.79 (0.14-4.59) 0.79 0.95 
rs17421462 1.32 (0.78-2.24) 0.87 1.16 (0.67-2.00) 0.59 9.91(1.25-78.4) 0.03 0.12 
rs17421511 0.98 (0.64-1.50) 1.00 1.19 (0.77-1.83) 0.43 0.27 (0.05-1.48) 0.13 0.09 
rs1801131* 1.02 (0.53-1.98) 0.95 0.98 (0.65-1.47) 0.92 1.01 (0.48-2.10) 0.99 0.98 
rs1801133* 0.59 (0.31-1.12) 0.10 1.21 (0.82-1.80) 0.33 0.69 (0.33-1.44) 0.32 0.16 
rs1994798 1.08 (0.78-1.49) 1.00 1.04 (0.67-1.61) 0.86 1.19 (0.62-2.27) 0.60 0.85 
rs3737964 1.06 (0.76-1.47) 1.00 1.23 (0.78-1.93) 0.37 0.90 (0.43-1.87) 0.78 0.49 
rs3737965 2.04 (1.07-3.88) 0.43 2.04 (1.07-3.88) 0.03 NA NA 0.17 
rs4846048 1.02 (0.72-1.44) 1.00 1.27 (0.81-1.98) 0.30 0.85 (0.42-1.74) 0.66 0.33 
rs4846049 0.96 (0.69-1.34) 1.00 0.98 (0.65-1.48) 0.93 0.91 (0.44-1.88) 0.79 0.96 
rs4846054 1.07 (0.78-1.45) 1.00 1.03 (0.67-1.60) 0.88 1.15 (0.62-2.15) 0.66 0.89 
rs6541003 1.06 (0.76-1.46) 1.00 0.93 (0.60-1.43) 0.73 1.17 (0.61-2.25) 0.64 0.69 
rs9651118 0.95 (0.68-1.33) 1.00 1.08 (0.70-1.68) 0.73 0.71 (0.35-1.48) 0.36 0.64 
rs17376328 0.64 (0.32-1.32) 0.81 0.64 (0.32-1.32) 0.23 NA NA 0.46 
rs2050265 0.92 (0.60-1.40) 1.00 0.92 (0.57-1.46) 0.71 0.85 (0.23-3.12) 0.80 0.93 

Abbreviations: LRT, likelihood ratio test; NA, not applicable.

*Based on a minimum of 1,702 cases and 27,26 controls from 1,647 discordant sibships.

ORs estimated assuming a log-additive model and controlling for age and sex.

ORs estimated assuming a codominant model and controlling for age and sex.

§ORs estimate assuming a recessive genotype effect and controlling for age and sex.

P value not corrected for multiple testing.

OR could not be estimated because there were 0 subjects in the case or control group or the number was too small for a valid estimate.

Among those with food frequency questionnaire data, we found no evidence of heterogeneity for MTHFR genotypes when stratified by dietary or total (food plus supplement) folate intake using either prefortification or postfortification estimates (data not shown). Folate and multivitamin supplement use (yes/no) in the 2 years before diagnosis or recruitment was available for the 98% of the study population with completed risk factor questionnaire data. The 677 TT genotype was associated with a decrease in colorectal cancer risk only in nonusers of a folic acid supplement (OR, 0.78; 95% CI, 0.60-1.01; P = 0.06; and OR, 1.16; 95% CI, 0.65-2.08; P = 0.40 in folic acid supplement nonusers and users, respectively), although the group of folic acid supplement users was small and neither the interaction term (P = 0.55) nor the P for heterogeneity (P = 0.08) were statistically significant. The results were similar when we stratified on multivitamin use (OR, 0.68; 95% CI, 0.49-0.95; P = 0.02; and OR, 0.97; 95% CI, 0.72-1.32; P = 0.87 in multivitamin nonusers and users, respectively). Again, neither the P for the interaction (P = 0.48) nor heterogeneity (P = 0.07) were statistically significant. The same pattern was evident for the 1298 CC genotype. There were no other interactions with folate or multivitamin supplement use for any SNP.

We also assessed heterogeneity of the MTHFR effect by tumor microsatellite instability among the population-based families (Table 4). The 677 TT genotype was associated with decreased risk of MSS/MSI-L colorectal cancer (OR, 0.69; 95% CI, (0.50-0.97; P = 0.03) and increased risk of an MSI-H tumor (OR, 2.22; 95% CI, 0.91-5.43; P = 0.08; Pinteraction = 0.02; Pheterogeneity = 0.01). The 1298 CC genotype was associated with a nonstatistically significant decreased risk of MSS/MSI-L (OR, 0.77; 95% CI, 0.54-1.08; P = 0.13) and MSI-H (OR, 0.51; 95% CI, 0.24-1.08; P = 0.08) tumors. There was no heterogeneity by MSI status for the other SNPs.

Table 4.

Colorectal cancer risk by MTHFR genotype and MSI status

SNPMSS + MSI-L*PMSI-HPPinteraction
rs11121832 1.04 (0.86-1.27)§ 0.66 1.05 (0.66-1.69) 0.23 0.97 
rs12121543 0.94 (0.78-1.14) 0.54 0.97 (0.59-1.60) 0.83 0.91 
rs12404124 0.96 (0.81-1.13) 0.61 0.90 (0.59-1.37) 0.61 0.77 
rs13306556 1.03 (0.80-1.33) 0.83 0.93 (0.47-1.87) 0.85 0.79 
rs1476413 0.97 (0.81-1.17) 0.75 1.05 (0.65-1.71) 0.83 0.74 
rs17037390 0.88 (0.70-1.09) 0.25 0.76 (0.44-1.33) 0.34 0.64 
rs17037396 0.97 (0.76-1.25) 0.84 1.11 (0.60-2.07) 0.74 0.69 
rs17037425 0.95 (0.76-1.19) 0.65 0.82 (0.47-1.42) 0.48 0.63 
rs17421462 1.25 (0.92-1.70) 0.16 1.00 (0.53-1.87) 0.99 0.56 
rs17421511 0.97 (0.77-1.20) 0.75 1.08 (0.63-1.85) 0.78 0.71 
rs1801131 0.77 (0.54-1.08) 0.13 0.51 (0.24-1.08) 0.08 0.36 
rs1801133 0.69 (0.50-0.97) 0.03 2.22 (0.91-5.43) 0.08 0.01 
rs1994798 0.94 (0.80-1.11) 0.50 0.85 (0.55-1.31) 0.47 0.65 
rs3737964 1.01 (0.83-1.22) 0.92 1.08 (0.68-1.72) 0.75 0.80 
rs3737965 0.98 (0.70-1.39) 0.92 0.81 (0.33-2.00) 0.66 0.69 
rs4846048 0.94 (0.78-1.13) 0.52 0.92 (0.59-1.43) 0.71 0.93 
rs4846049 0.91 (0.76-1.08) 0.27 0.85 (0.54-1.35) 0.49 0.79 
rs4846054 0.99 (0.84-1.17) 0.88 0.91 (0.59-1.40) 0.68 0.74 
rs6541003 0.98 (0.83-1.16) 0.86 0.87 (0.57-1.34) 0.54 0.61 
rs9651118 1.07 (0.88-1.30) 0.48 0.68 (0.41-1.12) 0.13 0.12 
rs17376328 0.86 (0.59-1.25) 0.43 1.41 (0.58-3.45) 0.45 0.32 
rs2050265 0.87 (0.69-1.08) 0.21 0.90 (0.51-1.58) 0.71 0.91 
SNPMSS + MSI-L*PMSI-HPPinteraction
rs11121832 1.04 (0.86-1.27)§ 0.66 1.05 (0.66-1.69) 0.23 0.97 
rs12121543 0.94 (0.78-1.14) 0.54 0.97 (0.59-1.60) 0.83 0.91 
rs12404124 0.96 (0.81-1.13) 0.61 0.90 (0.59-1.37) 0.61 0.77 
rs13306556 1.03 (0.80-1.33) 0.83 0.93 (0.47-1.87) 0.85 0.79 
rs1476413 0.97 (0.81-1.17) 0.75 1.05 (0.65-1.71) 0.83 0.74 
rs17037390 0.88 (0.70-1.09) 0.25 0.76 (0.44-1.33) 0.34 0.64 
rs17037396 0.97 (0.76-1.25) 0.84 1.11 (0.60-2.07) 0.74 0.69 
rs17037425 0.95 (0.76-1.19) 0.65 0.82 (0.47-1.42) 0.48 0.63 
rs17421462 1.25 (0.92-1.70) 0.16 1.00 (0.53-1.87) 0.99 0.56 
rs17421511 0.97 (0.77-1.20) 0.75 1.08 (0.63-1.85) 0.78 0.71 
rs1801131 0.77 (0.54-1.08) 0.13 0.51 (0.24-1.08) 0.08 0.36 
rs1801133 0.69 (0.50-0.97) 0.03 2.22 (0.91-5.43) 0.08 0.01 
rs1994798 0.94 (0.80-1.11) 0.50 0.85 (0.55-1.31) 0.47 0.65 
rs3737964 1.01 (0.83-1.22) 0.92 1.08 (0.68-1.72) 0.75 0.80 
rs3737965 0.98 (0.70-1.39) 0.92 0.81 (0.33-2.00) 0.66 0.69 
rs4846048 0.94 (0.78-1.13) 0.52 0.92 (0.59-1.43) 0.71 0.93 
rs4846049 0.91 (0.76-1.08) 0.27 0.85 (0.54-1.35) 0.49 0.79 
rs4846054 0.99 (0.84-1.17) 0.88 0.91 (0.59-1.40) 0.68 0.74 
rs6541003 0.98 (0.83-1.16) 0.86 0.87 (0.57-1.34) 0.54 0.61 
rs9651118 1.07 (0.88-1.30) 0.48 0.68 (0.41-1.12) 0.13 0.12 
rs17376328 0.86 (0.59-1.25) 0.43 1.41 (0.58-3.45) 0.45 0.32 
rs2050265 0.87 (0.69-1.08) 0.21 0.90 (0.51-1.58) 0.71 0.91 

*Based on a minimum of 958 cases and 1,532 controls from 931 discordant sibships.

Based on a minimum of 175 cases and 255 controls from 166 discordant sibships.

P value for the multiplicative interaction term [MSI status * genotype (coded as 0, 1, or 2 for the presence of the minor allele)].

§Except as noted, all ORs were estimated assuming a log-additive model and controlling for age and sex.

ORs estimated from a recessive model and controlling for age and sex.

The overall test for interaction between the three subsites for the 677 TT genotype was statistically significant (Pinteraction = 0.01; 3 df likelihood ratio P value for heterogeneity = 0.02). The MTHFR 677 TT genotype was associated with an increase in the risk of proximal tumors (OR, 1.40; 95% CI, 0.88-2.23; P = 0.16) and a decrease in the risk for distal (OR, 0.69; 95% CI, 0.43-1.10; P = 0.12) and rectal (OR, 0.62; 95% CI, 0.41-0.93; P = 0.02) tumors. There was no evidence for heterogeneity in risk by tumor subsite for the MTHFR 1298 CC genotype (OR, 0.70; 95% CI, 0.45-1.07 for proximal colon; OR, 1.22; 95% CI, 0.74-1.99 for distal colon; and OR, 0.69; 95% CI, 0.44-1.10 for rectal tumors; Pinteraction = 0.95). No other SNP was differentially associated with tumor subsite.

We also conducted a stratified analysis to examine possible modification by age (<65/>65 years) or sex. There was no modification by age or sex for any SNP (data not shown).

In the current study, we assessed a comprehensive set of SNPs that characterized the genetic variation of the MTHFR gene, including 10-kb 5′ of the transcription start site and 5-kb into the 3′-UTR. To our knowledge, this is the most comprehensive analysis of genetic variation in the MTHFR gene in relation to colorectal cancer risk completed to date. Our data from both the population- and clinic-based series are consistent with inverse associations for the 677 TT (rs1801133; A222V) and the 1298 CC genotypes (rs1801131; E429A). Stratification by folate and multivitamin supplement use suggested that 677 TT and 1298 CC genotypes may be associated with decreased colorectal cancer risk only among nonusers of folate or multivitamin supplements, but neither interaction was statistically significant. Our data also support a positive association between the 677 TT genotype and the MSI-H phenotype and tumors of the proximal colon (wherein most MSI-H tumors are located); conversely, the results suggested an inverse association between this SNP and MSS/MSI-L tumors and tumors in the distal colon and rectum. No other SNPs were significantly associated with risk overall or in any subgroup.

Our data are consistent with four recent meta-analyses that suggested 15% to 18% reductions in colorectal cancer risk for the 677 TT genotype (5-8). The A1298C polymorphism has been also well studied. Although the functional effects of the C allele are unclear (2), two meta-analyses (5, 7) suggest that homozygosity for the enzyme with a C at nucleotide 1298 is associated with an approximately 10% to 15% decrease in colorectal cancer risk, consistent with the estimate in the current study. Several studies published after the meta-analyses were completed have reported increased risk associated with the 677 TT genotype (23-25), whereas others reported no association (26-28) or a decreased risk for those with the variant genotype (29). Chang et al. (9) reported a significant increase in risk for the 677 TT genotype in those with low folate intake. For the 1298 CC genotype, two studies reported an increase in risk (27, 28), one reported a decreased risk (29), and one reported no association with risk (9). Additionally, one recent study of a Hereditary Non-Polyposis Colon Cancer cohort reported an increase in the age of colorectal cancer onset among those with the 1298 CC or joint 677T/1298C genotypes, suggesting a protective effect of these variant alleles in Hereditary Non-Polyposis Colon Cancer kindreds (30). Variability in folate availability in these different source populations may explain the diverse findings, suggesting that future meta-analyses should account for such differences.

Data from observational studies suggest that the phenotype of the MTHFR valine protein (677 TT genotype) depends significantly on folate availability (31-34). A recent in vitro study in HCT116 colon carcinoma cells reported that the valine protein (TT genotype) was associated with increased genomic DNA methylation if folate is adequate but shows a significant decrease in genomic methylation when folate is deficient (35), supporting the impression that folate availability is a modifier of genotype effect. This is consistent with the biochemical changes in the valine-containing enzyme, which show that the enzyme is stabilized by the addition of 5-methyltetrahydrofolate to the culture medium (2). Therefore, we considered whether folate availability might modify associations between other SNPs and colorectal cancer risk as well. Although there was no heterogeneity by dietary or total folate consumption in the subgroup of participants with food frequency data, in the data from the whole study population, homozygosity for the MTHFR 677 T allele was significantly associated with decreased risk only in nonmultivitamin users, a result that was suggested for nonusers of folate supplements as well. These results conflict with most previous reports of nonfolate-supplemented populations, which suggest that the 677 TT genotype is protective mainly for those with higher folate availability (9-14), although other studies have not observed this difference (29, 36-38). However, in the current study, neither the interactions nor the tests for heterogeneity were statistically significant. Therefore, this finding may be due to chance. On the other hand, one difference between our study population and those of the earlier studies is the likely higher folic acid levels to which most of our study population had been exposed during the 2 years preceding their recruitment, due to fortification of the food supply. It is likely that nonsupplement users in the current population have a greater folate intake than the subjects in the prefortification study populations (39) with levels more similar to those with higher folate intakes in previous studies. Subjects taking supplements in the current population showed little association with colorectal cancer risk, as one would expect if there were a stabilization of the enzyme in the presence of high-folate availability. Whether postsupplement folate levels are relevant to the lower risk we observed in 677 TT homozygotes who were not taking folate or multivitamin supplements is unclear and must be assessed in future studies of populations with similar levels of fortification.

In stratified analyses, we found that the 677 TT genotype was associated with a decreased risk of MSS tumors and tumors in the distal colon or rectum while being associated with an increased risk for MSI-H tumors and tumors in proximal colon. The literature on the relationship between the 677 TT genotype and MSI status is mixed. Most studies have reported an increased risk for MSI-H tumors in those homozygous for the 677 TT genotype (9, 40-42), but some studies have reported no difference between MSI subgroups (43, 44) or a decrease in the risk of MSI-H tumors in those homozygous for the 677 T allele (45, 46). Our data are also consistent with those of a recent study of Australian colorectal cancer patients, which reported a significant increase in the risk of proximal and decreased risk of distal colorectal cancers in those with the 677 TT genotype (47), adding to the general consensus that those with the 677 TT genotype may have an increased risk for tumors with the MSI-H phenotype and for tumors of the proximal colon. These two findings are likely to reflect the same association because data strongly suggest that the majority of MSI-H tumors develop in proximal colon and rarely in distal colon or rectum (47, 48). We did not observe any modification by MSI status for the 1298 CC genotype, a result that is consistent with those of other reports (41, 45, 46). It is unclear why the association between the 1298 genotype and MSI status should be different from that of the 677 genotype.

This study has several strengths. In addition to the large sample size, we had a comprehensive approach to identifying genetic variation in the MTHFR gene, a family-based design which minimized the probability of population stratification, included detailed risk factor information on all our subjects, and had the ability to assess possible heterogeneity of genotype effects by folate nutrition, multivitamin use, MSI status, and tumor subsite. The ability to assess genotype associations in two separate samples is another strength.

Weaknesses of this study include the possibility that we have missed an important source of genetic variability because we used public databases to define SNPs; these databases are also incomplete. Additionally, although the case-unaffected sibling design is more powerful for assessing gene-environment interactions, it is less powerful for detecting main effects (49). Thus, our study may have been underpowered in the main effect analyses. In addition, we did not have the dietary data from all the participants and not all cases provided tumor tissue for MSI analysis, limiting the statistical power for analyses involving diet and MSI status; however, it is unlikely that the availability of dietary or MSI data are associated with genotype, so this should not have resulted in any bias in the observed ORs. As in any case-control study, dietary intake information was assessed after the diagnosis in cases, so the information may be affected by recall bias. We were unable to genotype the rare R593Q (G1793A, rs2274976) polymorphism, so we could not assess whether this SNP was associated with risk overall or in any subgroup. The functional consequences of the R593Q polymorphism are not completely clear but some studies have suggested associations with various outcomes (50-54). Several studies have shown the A allele for this SNP to be in cis with the 1298 C allele (53, 55, 56). Finally, many of the protocols used in the C-CFR were designed to oversample cases with a greater risk for a family history of colorectal cancer, which may decrease the generalizability of our findings. Additionally, in the population-based sample, ∼90% of our study population was Caucasian and from the folate-supplemented populations in the United States or Canada, whereas this percentage was ∼100% in the clinic-based subjects. Only 19% of the population-based sample was not of North American origin and none of the subjects in either population came from Latin America, South America, or Western Europe, all countries with significantly lower folate availability. To the extent that MTHFR genotype modifies risk differently in different countries, perhaps associated with differences in folate intake and the prevalence of smoking and alcohol use, our results may not generalize to all relevant populations.

In this study, using a tagSNP approach, we did not find strong evidence for additional functional genetic variation in the MTHFR gene for colorectal cancer risk. Our data suggest that the well-known C677T and A1298C variants are the most relevant MTHFR variants for colorectal cancer risk and that there may be heterogeneity in the risk associated with the C677T TT variant by MSI status, tumor subsite and, possibly, by folate or multivitamin supplement use in folic acid–supplemented populations.

D. Conti is an advisor to Pfizer, Inc. P. Limburg is an advisor to Genomic Health, Inc.

We thank Margreet Luchtenborg, Maj Earle, Barbara Saltzman, Kathy Kennedy, Darin Taverna, Chris Edlund, Matt Westlake, Paul Mosquin, Darshana Daftary, Douglas Snazel, Allyson Templeton, Terry Teitsch, Helen Chen, Maggie Angelakos, Paul Mosquin for their support in data collection and management and all the individuals who participated in the C-CFR.

Grant Support: NCI, NIH under RFA # CA-95-011 and through cooperative agreements with the Australasian Colorectal Cancer Family Registry (U01 CA097735), the USC Familial Colorectal Neoplasia Collaborative Group (U01 CA074799), the Mayo Clinic Cooperative Family Registry for Colon Cancer Studies (U01 CA074800), the Ontario Registry for Studies of Familial Colorectal Cancer (U01 CA074783), the Seattle Colorectal Cancer Family Registry (U01 CA074794), and the University of Hawaii Colorectal Cancer Family Registry (U01 CA074806) as well as NCI T32 CA009142 (J.N. Poynter), NCI R01 CA112237 (R.W. Haile), and NCI PO1 CA41108 CA-23074 and CA 956060 (M.E. Martinez). P.T. Campbell and J.C. Figueiredo were supported in part by NCI of Canada post-PhD Fellowships (#18735 and #17602).

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.

1
Frosst
P
,
Blom
HJ
,
Milos
R
, et al
. 
A candidate genetic risk factor for vascular disease: a common mutation in methylenetetrahydrofolate reductase
.
Nat Genet
1995
;
10
:
111
3
.
2
Yamada
K
,
Chen
Z
,
Rozen
R
, et al
. 
Effects of common polymorphisms on the properties of recombinant human methylenetetrahydrofolate reductase
.
Proc Natl Acad Sci U S A
2001
;
98
:
14853
8
.
3
van der Put
NM
,
Gabreels
F
,
Stevens
EM
, et al
. 
A second common mutation in the methylenetetrahydrofolate reductase gene: an additional risk factor for neural-tube defects?
Am J Hum Genet
1998
;
62
:
1044
51
.
4
Weisberg
IS
,
Jacques
PF
,
Selhub
J
, et al
. 
The 1298A->C polymorphism in methylenetetrahydrofolate reductase (MTHFR): in vitro expression and association with homocysteine
.
Atherosclerosis
2001
;
156
:
409
15
.
5
Huang
Y
,
Han
S
,
Li
Y
, et al
. 
Different roles of MTHFR C677T and A1298C polymorphisms in colorectal adenoma and colorectal cancer: a meta-analysis
.
J Hum Genet
2007
;
52
:
73
85
.
6
Hubner
RA
,
Houlston
RS
. 
MTHFR C677T and colorectal cancer risk: a meta-analysis of 25 populations
.
Int J Cancer
2007
;
120
:
1027
35
.
7
Kono
S
,
Chen
K
. 
Genetic polymorphisms of methylenetetrahydrofolate reductase and colorectal cancer and adenoma
.
Cancer Sci
2005
;
96
:
535
42
.
8
Little
J
,
Sharp
L
,
Duthie
S
, et al
. 
Colon cancer and genetic variation in folate metabolism: the clinical bottom line
.
J Nutr
2003
;
133
:
3758
66S
.
9
Chang
SC
,
Lin
PC
,
Lin
JK
, et al
. 
Role of MTHFR polymorphisms and folate levels in different phenotypes of sporadic colorectal cancers
.
Int J Colorectal Dis
2007
;
22
:
483
9
.
10
Chen
J
,
Giovannucci
E
,
Kelsey
K
, et al
. 
A methylenetetrahydrofolate reductase polymorphism and the risk of colorectal cancer
.
Cancer Res
1996
;
56
:
4862
4
.
11
Le Marchand
L
,
Wilkens
LR
,
Kolonel
LN
, et al
. 
The MTHFR C677T polymorphism and colorectal cancer: the multiethnic cohort study
.
Cancer Epidemiol Biomarkers Prev
2005
;
14
:
1198
203
.
12
Ma
J
,
Stampfer
MJ
,
Giovannucci
E
, et al
. 
Methylenetetrahydrofolate reductase polymorphism, dietary interactions, and risk of colorectal cancer
.
Cancer Res
1997
;
57
:
1098
102
.
13
Slattery
ML
,
Potter
JD
,
Samowitz
W
, et al
. 
Methylenetetrahydrofolate reductase, diet, and risk of colon cancer
.
Cancer Epidemiol Biomarkers Prev
1999
;
8
:
513
8
.
14
Yin
G
,
Kono
S
,
Toyomura
K
, et al
. 
Methylenetetrahydrofolate reductase C677T and A1298C polymorphisms and colorectal cancer: the Fukuoka Colorectal Cancer Study
.
Cancer Sci
2004
;
95
:
908
13
.
15
Liu
H
,
Jin
G
,
Wang
H
, et al
. 
Association of polymorphisms in one-carbon metabolizing genes and lung cancer risk: a case-control study in Chinese population
.
Lung Cancer
2008
;
61
:
21
9
.
16
Liu
X
,
Zhao
LJ
,
Liu
YJ
, et al
. 
The MTHFR gene polymorphism is associated with lean body mass but not fat body mass
.
Hum Genet
2008
;
123
:
189
96
.
17
Newcomb
PA
,
Baron
J
,
Cotterchio
M
, et al
. 
Colon Cancer Family Registry: an international resource for studies of the genetic epidemiology of colon cancer
.
Cancer Epidemiol Biomarkers Prev
2007
;
16
:
2331
43
.
18
Barrett
JC
,
Fry
B
,
Maller
J
, et al
. 
Haploview: analysis and visualization of LD and haplotype maps
.
Bioinformatics
2005
;
21
:
263
5
.
19
Shen
R
,
Fan
JB
,
Campbell
D
, et al
. 
High-throughput SNP genotyping on universal bead arrays
.
Mutat Res
2005
;
573
:
70
82
.
20
The International HapMap project
. 
A haplotype map of the human genome
.
Nature
2005
;
437
:
1299
320
.
21
Stram
DO
,
Hankin
JH
,
Wilkens
LR
, et al
. 
Calibration of the dietary questionnaire for a multiethnic cohort in Hawaii and Los Angeles
.
Am J Epidemiol
2000
;
151
:
358
70
.
22
Conneely
KN
,
Boehnke
M
. 
So many correlated tests, so little time! Rapid adjustment of P values for multiple correlated tests
.
Am J Hum Genet
2007
;
81
:
1158
68
.
23
Cao
HX
,
Gao
CM
,
Takezaki
T
, et al
. 
Genetic polymorphisms of methylenetetrahydrofolate reductase and susceptibility to colorectal cancer
.
Asian Pac J Cancer Prev
2008
;
9
:
203
8
.
24
Gallegos-Arreola
MP
,
Garcia-Ortiz
JE
,
Figuera
LE
, et al
. 
Association of the 677C ->T polymorphism in the MTHFR gene with colorectal cancer in Mexican patients
.
Cancer Genomics Proteomics
2009
;
6
:
183
8
.
25
Guerreiro
CS
,
Carmona
B
,
Goncalves
S
, et al
. 
Risk of colorectal cancer associated with the C677T polymorphism in 5,10-methylenetetrahydrofolate reductase in Portuguese patients depends on the intake of methyl-donor nutrients
.
Am J Clin Nutr
2008
;
88
:
1413
8
.
26
Derwinger
K
,
Wettergren
Y
,
Odin
E
, et al
. 
A study of the MTHFR gene polymorphism C677T in colorectal cancer
.
Clin Colorectal Cancer
2009
;
8
:
43
8
.
27
Kury
S
,
Buecher
B
,
Robiou-du-Pont
S
, et al
. 
Low-penetrance alleles predisposing to sporadic colorectal cancers: a French case-controlled genetic association study
.
BMC Cancer
2008
;
8
:
326
.
28
Lightfoot
TJ
,
Barrett
JH
,
Bishop
T
, et al
. 
Methylene tetrahydrofolate reductase genotype modifies the chemopreventive effect of folate in colorectal adenoma, but not colorectal cancer
.
Cancer Epidemiol Biomarkers Prev
2008
;
17
:
2421
30
.
29
Sharp
L
,
Little
J
,
Brockton
NT
, et al
. 
Polymorphisms in the methylenetetrahydrofolate reductase (MTHFR) gene, intakes of folate and related B vitamins and colorectal cancer: a case-control study in a population with relatively low folate intake
.
Br J Nutr
2008
;
99
:
379
89
.
30
Reeves
SG
,
Meldrum
C
,
Groombridge
C
, et al
. 
MTHFR 677 C>T and 1298 A>C polymorphisms and the age of onset of colorectal cancer in hereditary nonpolyposis colorectal cancer
.
Eur J Hum Genet
2009
;
17
:
629
35
.
31
Friso
S
,
Choi
SW
,
Girelli
D
, et al
. 
A common mutation in the 5,10-methylenetetrahydrofolate reductase gene affects genomic DNA methylation through an interaction with folate status
.
Proc Natl Acad Sci U S A
2002
;
99
:
5606
11
.
32
Jacques
PF
,
Bostom
AG
,
Williams
RR
, et al
. 
Relation between folate status, a common mutation in methylenetetrahydrofolate reductase, and plasma homocysteine concentrations
.
Circulation
1996
;
93
:
7
9
.
33
Malinow
MR
,
Nieto
FJ
,
Kruger
WD
, et al
. 
The effects of folic acid supplementation on plasma total homocysteine are modulated by multivitamin use and methylenetetrahydrofolate reductase genotypes
.
Arterioscler Thromb Vasc Biol
1997
;
17
:
1157
62
.
34
Nelen
WL
,
Blom
HJ
,
Thomas
CM
, et al
. 
Methylenetetrahydrofolate reductase polymorphism affects the change in homocysteine and folate concentrations resulting from low dose folic acid supplementation in women with unexplained recurrent miscarriages
.
J Nutr
1998
;
128
:
1336
41
.
35
Sohn
KJ
,
Jang
H
,
Campan
M
, et al
. 
The methylenetetrahydrofolate reductase C677T mutation induces cell-specific changes in genomic DNA methylation and uracil misincorporation: a possible molecular basis for the site-specific cancer risk modification
.
Int J Cancer
2009
;
124
:
1999
2005
.
36
Jiang
Q
,
Chen
K
,
Ma
X
, et al
. 
Diets, polymorphisms of methylenetetrahydrofolate reductase, and the susceptibility of colon cancer and rectal cancer
.
Cancer Detect Prev
2005
;
29
:
146
54
.
37
Matsuo
K
,
Hamajima
N
,
Hirai
T
, et al
. 
Methionine synthase reductase gene A66G polymorphism is associated with risk of colorectal cancer
.
Asian Pac J Cancer Prev
2002
;
3
:
353
9
.
38
Matsuo
K
,
Ito
H
,
Wakai
K
, et al
. 
One-carbon metabolism related gene polymorphisms interact with alcohol drinking to influence the risk of colorectal cancer in Japan
.
Carcinogenesis
2005
;
26
:
2164
71
.
39
Dietrich
M
,
Brown
CJ
,
Block
G
. 
The effect of folate fortification of cereal-grain products on blood folate status, dietary folate intake, and dietary folate sources among adult non-supplement users in the United States
.
J Am Coll Nutr
2005
;
24
:
266
74
.
40
Clarizia
AD
,
Bastos-Rodrigues
L
,
Pena
HB
, et al
. 
Relationship of the methylenetetrahydrofolate reductase C677T polymorphism with microsatellite instability and promoter hypermethylation in sporadic colorectal cancer
.
Genet Mol Res
2006
;
5
:
315
22
.
41
Hubner
RA
,
Lubbe
S
,
Chandler
I
, et al
. 
MTHFR C677T has differential influence on risk of MSI and MSS colorectal cancer
.
Hum Mol Genet
2007
;
16
:
1072
7
.
42
Shannon
B
,
Gnanasampanthan
S
,
Beilby
J
, et al
. 
A polymorphism in the methylenetetrahydrofolate reductase gene predisposes to colorectal cancers with microsatellite instability
.
Gut
2002
;
50
:
520
4
.
43
Plaschke
J
,
Schwanebeck
U
,
Pistorius
S
, et al
. 
Methylenetetrahydrofolate reductase polymorphisms and risk of sporadic and hereditary colorectal cancer with or without microsatellite instability
.
Cancer Lett
2003
;
191
:
179
85
.
44
Ulrich
CM
,
Curtin
K
,
Potter
JD
, et al
. 
Polymorphisms in the reduced folate carrier, thymidylate synthase, or methionine synthase and risk of colon cancer
.
Cancer Epidemiol Biomarkers Prev
2005
;
14
:
2509
16
.
45
Eaton
AM
,
Sandler
R
,
Carethers
JM
, et al
. 
5,10-Methylenetetrahydrofolate reductase 677 and 1298 polymorphisms, folate intake, and microsatellite instability in colon cancer
.
Cancer Epidemiol Biomarkers Prev
2005
;
14
:
2023
9
.
46
Toffoli
G
,
Gafa
R
,
Russo
A
, et al
. 
Methylenetetrahydrofolate reductase 677 C->T polymorphism and risk of proximal colon cancer in north Italy
.
Clin Cancer Res
2003
;
9
:
743
8
.
47
Iacopetta
B
,
Heyworth
J
,
Girschik
J
, et al
. 
The MTHFR C677T and ΔDNMT3B C-149T polymorphisms confer different risks for right- and left-sided colorectal cancer
.
Int J Cancer
2009
;
125
:
84
90
.
48
Iacopetta
B
. 
Are there two sides to colorectal cancer?
Int J Cancer
2002
;
101
:
403
8
.
49
Witte
JS
,
Gauderman
WJ
,
Thomas
DC
. 
Asymptotic bias and efficiency in case-control studies of candidate genes and gene-environment interactions: basic family designs
.
Am J Epidemiol
1999
;
149
:
693
705
.
50
Huemer
M
,
Vonblon
K
,
Fodinger
M
, et al
. 
Total homocysteine, folate, and cobalamin, and their relation to genetic polymorphisms, lifestyle and body mass index in healthy children and adolescents
.
Pediatr Res
2006
;
60
:
764
9
.
51
Melo
SS
,
Persuhn
DC
,
Meirelles
MS
, et al
. 
G1793A polymorphisms in the methylene-tetrahydrofolate gene: effect of folic acid on homocysteine levels
.
Mol Nutr Food Res
2006
;
50
:
769
74
.
52
Winkelmayer
WC
,
Huber
A
,
Wagner
OF
, et al
. 
Associations between MTHFR 1793G>A and plasma total homocysteine, folate, and vitamin B in kidney transplant recipients
.
Kidney Int
2005
;
67
:
1980
5
.
53
Winkelmayer
WC
,
Sunder-Plassmann
G
,
Huber
A
, et al
. 
Patterns of co-occurrence of three single nucleotide polymorphisms of the 5,10-methylenetetrahydrofolate reductase gene in kidney transplant recipients
.
Eur J Clin Invest
2004
;
34
:
613
8
.
54
Kebert
CB
,
Eichner
JE
,
Moore
WE
, et al
. 
Relationship of the 1793G-A and 677C-T polymorphisms of the 5,10-methylenetetrahydrofolate reductase gene to coronary artery disease
.
Dis Markers
2006
;
22
:
293
301
.
55
Rady
PL
,
Szucs
S
,
Grady
J
, et al
. 
Genetic polymorphisms of methylenetetrahydrofolate reductase (MTHFR) and methionine synthase reductase (MTRR) in ethnic populations in Texas; a report of a novel MTHFR polymorphic site, G1793A
.
Am J Med Genet
2002
;
107
:
162
8
.
56
Wakutani
Y
,
Kowa
H
,
Kusumi
M
, et al
. 
A haplotype of the methylenetetrahydrofolate reductase gene is protective against late-onset Alzheimer's disease
.
Neurobiol Aging
2004
;
25
:
291
4
.