Methylenetetrahydrofolate reductase (MTHFR) is a key regulatory enzyme in the metabolism of folate, a nutrient that has been inversely related to colorectal cancer risk. The common C677T variant in the MTHFR gene results in a reduced activity of this enzyme, thereby increasing the availability of folate for the production of thymidylate and purine for DNA synthesis and repair. We investigated the association of the 677TT genotype with colorectal cancer in a case-control study of 822 cases and 2,021 controls nested within the Multiethnic Cohort Study. The Multiethnic Cohort Study is a large prospective study of men and women of Japanese, White, African American, Latino, and Native Hawaiian origin, residing in Hawaii and Los Angeles. After adjusting for covariates, we found an inverse association between colorectal cancer risk and the TT genotype, with odds ratios (OR; and 95% confidence intervals) for the CC, CT, and TT genotypes of 1.00, 1.01 (0.84-1.21), and 0.77 (0.58-1.03), respectively. This association was similar in both sexes, stronger at high levels of folate intake, and limited to light and nondrinkers (P for interaction with ethanol = 0.02). An analysis by subsite (rectum versus colon) and stage (regional/distant versus in situ/localized) showed that the inverse association with the TT genotype was limited to colon tumors, especially those diagnosed at an advanced stage. The OR for the TT versus CC genotype for early- and late-stage colon cancer was 0.88 (0.58-1.33) and 0.52 (0.32-0.85), respectively (P for difference in OR = 0.04). The frequency of the T allele was relatively low in African Americans (0.13) and Native Hawaiians (0.22), consistent with their greater likelihood of presenting at a late stage when diagnosed with colorectal cancer. This study corroborates previous findings of an inverse association of the MTHFR 677TT genotype with colorectal cancer, especially at high levels of folate and low levels of ethanol intake. It also suggests that this effect may be specific to advanced colon cancer.

Low intake and plasma levels of folate and use of alcohol have been associated with an increased risk of colorectal cancer (1-6). It has been proposed that this association may result from the disruption of nucleotide synthesis and/or intracellular methylation and that alcohol increases risk by acting as a folate antagonist (7). Deficiency in folic acid has been shown to lead to massive uracil incorporation into DNA and to double-strand chromosome breaks, a feature commonly seen in colorectal cancer (7, 8). Because folic acid is the primary methyl donor in cellular metabolism, folate deficiency also results in global hypomethylation of the genome, as well as simultaneous focal hypermethylation of CpG islands, which may both disrupt gene expression (9). Inherited sequence variants in folate metabolism–related genes may interact with low folate intake to modulate the effect of this nutrient.

The enzyme 5,10-methylenetetrahydrofolate reductase (MTHFR) plays a central role in folate metabolism, regulating the flow of folate between these two important pathways: production of thymidylate and purines for DNA synthesis and supply of methyl groups for the synthesis of methionine and DNA methylation (10). A common C677T substitution in the MTHFR gene, converting an alanine to a valine, results in a thermolabile enzyme with decreased activity. A second variant (A1298C) has also been shown to reduce MTHFR activity but to a lesser extent than C677T (11). The 677T variant has been associated with a lower risk of colorectal cancer, especially at high levels of folate intake (10, 12). Alcohol intake has been found to negate this effect (11). In contrast, most studies of colorectal adenomas have reported a direct association with the polymorphism at low levels of folate intake (12). Other B vitamins (e.g., vitamins B6 and B12) act as coenzymes in the same metabolic pathway and may also interact with the MTHFR polymorphism (10, 12).

We investigated the association of the MTHFR C677T polymorphism with colorectal cancer in a large case-control study nested in the Multiethnic Cohort Study. Our aims were to compare effects at various levels of folate and alcohol intakes in the Multiethnic Cohort population, which is notable for its wide range of dietary intake (13) and use the large sample size to examine effects by sex, anatomic subsite, and stage at diagnosis, which has not been done in previous studies.

The design and baseline characteristics of the Multiethnic Cohort have been described in detail elsewhere (13). In short, participants are Hawaii and Los Angeles residents who entered the cohort from 1993 to 1996 by completing a 26-page mail questionnaire about demographic factors, lifestyle (including diet and smoking), medical history, medication use, family history of common cancers, and, for women, reproductive history and hormone use. The cohort included 96,810 men and 118,441 women ages 45 to 75 years at cohort creation in 1993. Twenty-six percent were Japanese American, 23% White, 22% Latino, 16% African American, 7% Native Hawaiian, and 6% other ethnic/racial origin. Colorectal cancer cases were identified through the Rapid Reporting System of the Hawaii Tumor Registry and through quarterly linkage to the Los Angeles County Cancer Surveillance Program, two cancer registries that are members of the Surveillance, Epidemiology, and End Results program of the National Cancer Institute. This was complemented by annual linkages to the State of California's cancer registry. Incident colorectal cancer cases occurring since January 1995 and controls were contacted for donation of a blood sample. The median interval between diagnosis and blood draw was 14 months (interquartile range, 10-19) among cases. A sample of cohort participants was randomly selected to serve as controls at the onset of the nested case-control study. The selection was stratified by sex and race/ethnicity. Samples were collected at the subject's home, processed within 8 hours, and stored at −80°C. As of June 1999, the participation rate among cases was 74% and varied from 70% in African Americans to 81% in Latinos. The corresponding rate for controls was 66% and varied from 60% in African Americans to 71% in Whites.

The food frequency questionnaire asked about the frequency and amount of consumption for >140 food items during the last year. Photographs of foods, showing three different portion sizes, were used to facilitate quantification of intakes. Nutrients were computed by applying a food composition table to the daily grams of each food item and summing across items. The food composition data were primarily based on the U.S. Department of Agriculture's nutrient database (Handbook 8) and were supplemented with data from other research and commercial publications. A calibration study that compared diet reported on the questionnaire with three 24-hour recalls suggested that folate intake was adequately measured with correlation coefficients ranging across ethnic groups between 0.5 and 0.8 for women and 0.4 and 0.7 for men (14). Cohort members were asked about their usage during the last year of multivitamins/minerals and seven single vitamins. Supplemental folic acid intake was assessed from multivitamin usage. A composite nutrient content was assumed for multivitamins (15).

DNA was extracted from blood lymphocytes using a standard method (QIAamp DNA Blood Mini kit, Qiagen, Valencia, CA). Genotyping for the MTHFR C677T variant was carried out by PCR/RFLP as previously described (16). A subset of 826 female controls were also genotyped for the same variant in another study using the fluorogenic 5′-nuclease assay (TaqMan assay; ref. 17), resulting in perfect genotype concordance for 97% of the subjects.

The statistical analysis used unconditional logistic regression to compute odds ratios (OR) and 95% confidence intervals (95% CI) for exposures of interest (18). Fifty-one controls and 28 cases were excluded because of missing covariates, leaving 822 cases and 2,021 controls for the analysis. Genotypes were modeled as two dummy variables representing the three levels or as a gene dosage effect variable assigned a value of 1, 2, or 3 according to the number of variant alleles (zero, one, and two variant alleles, respectively). The final models were only adjusted for sex, age at blood draw, and ethnicity because further adjustment for other colorectal cancer risk factors (pack-years of cigarette smoking, body mass index, hours in vigorous activity, and dietary fiber and alcohol intakes) did not materially change the risk estimates (see Table 2). The likelihood ratio test was used to determine interaction among certain variables with respect to colorectal cancer. The test compares a main effects, no interaction model with a fully parameterized model containing all possible interaction terms for the variables of interest. Polytomous logistic regression was used to simultaneously estimate risk among controls and subgroups of cases, such as those diagnosed at a regional/distant stage versus in situ/localized stage and rectal versus colon cancer (19). A Wald test was used to statistically compare risk estimates across the case subgroups. Departure of the genotype distributions from the Hardy Weinberg equilibrium was tested with the χ2 test.

Table 1 compares characteristics of cases and controls. The ethnic distributions were different in cases and controls, with cases more likely to be of Japanese, and less likely to be of Hawaiian, ancestry. This indicates a need for adjustment for ethnicity and should not be interpreted as a difference in colorectal cancer risk, as the control distribution represents our sampling strategy of recruiting approximately equal numbers of controls for each ethnic group for the blood substudy rather than reflecting the population composition of the cohort. Cases more often had a family history of colorectal cancer among parents and siblings. On the average, cases were somewhat older, less physically active, and had a greater exposure to cigarette smoking. They also tended to consume less dietary fiber and folate, and to consume more ethanol, than controls.

Table 1.

Characterisitics of colorectal cancer cases and controls at baseline

Cases (n = 822)*Controls (n = 2,021)*
Japanese American (%) 33.7 22.0 
White (%) 19.0 20.3 
African American (%) 20.0 20.1 
Latino (%) 20.2 23.2 
Native Hawaiian (%) 7.2 14.3 
Male (%) 55.7 55.1 
Family history (%) 12.6 8.4 
Age at baseline (y) 63.5 ± 7.7 59.4 ± 8.7 
Age at blood draw (y) 67.2 ± 7.9 63.0 ± 8.7 
Body mass index (kg/m226.5 ± 5.0 26.4 ± 5.0 
Pack-years 12.6 ± 16.9 10.7 ± 14.7 
Vigorous activity (h/wk) 1.8 ± 2.5 2.1 ± 4.0 
Dietary fiber (g/d) 2.6 ± 1.8 3.0 ± 1.9 
Ethanol (g/d) 11.4 ± 31.4 9.6 ± 25.7 
Folate from foods (μg/d) 373 ± 226 398 ± 259 
Total folate (μg/d) 495 ± 317 548 ± 365 
Red meat (g/d) 61.5 ± 48.1 63.5 ± 53.0 
Cases (n = 822)*Controls (n = 2,021)*
Japanese American (%) 33.7 22.0 
White (%) 19.0 20.3 
African American (%) 20.0 20.1 
Latino (%) 20.2 23.2 
Native Hawaiian (%) 7.2 14.3 
Male (%) 55.7 55.1 
Family history (%) 12.6 8.4 
Age at baseline (y) 63.5 ± 7.7 59.4 ± 8.7 
Age at blood draw (y) 67.2 ± 7.9 63.0 ± 8.7 
Body mass index (kg/m226.5 ± 5.0 26.4 ± 5.0 
Pack-years 12.6 ± 16.9 10.7 ± 14.7 
Vigorous activity (h/wk) 1.8 ± 2.5 2.1 ± 4.0 
Dietary fiber (g/d) 2.6 ± 1.8 3.0 ± 1.9 
Ethanol (g/d) 11.4 ± 31.4 9.6 ± 25.7 
Folate from foods (μg/d) 373 ± 226 398 ± 259 
Total folate (μg/d) 495 ± 317 548 ± 365 
Red meat (g/d) 61.5 ± 48.1 63.5 ± 53.0 

NOTE: Data are mean ± SD except when specified as percentages.

*

Numbers of subjects were smaller for some risk factors due to missing data.

Family history of colorectal cancer in parents and siblings.

Folate from foods and supplements.

Based on the data for the controls, the frequency for the MTHFR 677T allele was 0.41 in Japanese Americans, 0.36 in Whites, 0.42 in Latinos, 0.13 in African Americans, and 0.22 in Native Hawaiians. These allele frequencies compare favorably with past reports (11). The genotype frequencies for the T allele among controls were consistent with the Hardy Weinberg equilibrium, except for Whites (Japanese American, P = 0.65; White, P = 0.01; Latino, P = 0.22; African American, P = 0.94, Native Hawaiian, P = 0.33).

After adjustment for ethnicity/race, sex and age at blood draw, the odds ratios for the MTHFR 677 CT and TT genotypes, compared with the CC genotype, were 1.01 (95% CI, 0.84-1.21) and 0.77 (95% CI, 0.58-1.03), with P for gene dosage effect = 0.18. These ORs were not modified after further adjustment for pack-years, body mass, vigorous physical activity, and dietary fiber and ethanol [1.0, 0.99 (95% CI, 0.81-1.20) and 0.78 (95% CI, 0.57-1.06); P for gene dosage effect, 0.19]. Consequently, this additional adjustment was not carried out in the rest of the analysis. The risk estimates for the CT and TT genotypes were similar in men [1.07 (95% CI, 0.53-1.15) and 0.78 (95% CI, 0.53-1.15); P for gene dosage, 0.46] and women [0.92 (95% CI, 0.49-1.22) and 0.75 (95% CI, 0.49-1.16); P for gene dosage, 0.21]. In other stratified analyses, the association with the TT genotype was statistically significant in Japanese Americans [OR for TT versus CC: 0.59 (95% CI, 0.36-0.95)], suggested for Whites [0.62 (95% CI, 0.34-1.15)] and not observed in Latinos [0.96 (95% CI, 0.56-1.15)]. There were only five cases each with the TT genotype for African Americans and Native Hawaiians and the risk estimates were unreliable in these groups.

Table 2 shows the joint effect of dietary folate and the TT genotype on colorectal cancer risk. The risk associated with the TT genotype was lowest among subjects with a high intake of folate. The OR for subjects with the TT genotype and total folate intake >median was significantly decreased (OR, 0.48; 95% CI, 0.33-0.72), compared with subjects with the CC or CT genotype and an intake ≤median. However, the test for interaction was not statistically significant. In contrast, in an analysis of the joint effect of ethanol and the TT genotype (Table 2), the inverse association with the TT genotype was only observed among subjects with ethanol intake ≤median (≤0.01 g/d; P for interaction, 0.02). The OR for the TT genotype among light and nondrinkers was 0.53 (95% CI, 0.34-0.82), whereas no corresponding decrease in risk with the TT genotype was observed among heavier drinkers. This pattern seemed to be similar in men and women and stronger for colon than rectal cancer (data not shown).

Table 2.

Joint effects of the MTHFR 677TT genotype and folate and alcohol intakes on colorectal cancer risk

GenotypeIntake*Food folate
Total folate
Ethanol
nOR§ (95% CI)nOR (95% CI)nOR (95% CI)
CC, CT ≤Median 363/854 1.00 396/829 1.00 317/748 1.00 
CC, CT >Median 350/883 0.91 (0.76-1.09) 317/908 0.66 (0.55-0.80) 396/989 1.05 (0.87-1.26) 
TT <Median 41/109 0.89 (0.60-1.32) 45/112 0.81 (0.56-1.19) 30/121 0.53 (0.34-0.82) 
TT >Median 42/139 0.62 (0.42-0.91) 38/136 0.48 (0.33-0.72) 53/127 1.06 (0.74-1.56) 
  P = 0.34  P = 0.69  P = 0.02  
GenotypeIntake*Food folate
Total folate
Ethanol
nOR§ (95% CI)nOR (95% CI)nOR (95% CI)
CC, CT ≤Median 363/854 1.00 396/829 1.00 317/748 1.00 
CC, CT >Median 350/883 0.91 (0.76-1.09) 317/908 0.66 (0.55-0.80) 396/989 1.05 (0.87-1.26) 
TT <Median 41/109 0.89 (0.60-1.32) 45/112 0.81 (0.56-1.19) 30/121 0.53 (0.34-0.82) 
TT >Median 42/139 0.62 (0.42-0.91) 38/136 0.48 (0.33-0.72) 53/127 1.06 (0.74-1.56) 
  P = 0.34  P = 0.69  P = 0.02  
*

The median intake was 322 μg/d for folate from foods, 450 μg/d for total folate, and 0.01 g/d for ethanol.

Folate from foods and supplements.

Number of cases/number of controls.

§

OR (95% CI) adjusted by logistic regression for age at blood draw, sex, and race/ethnicity.

P derived from a likelihood ratio test with 1 degree of freedom comparing a model with main effects for the MTHFR 677 genotype (CC and CT versus TT) and the dietary variable (≤median or >median) and a model with main effects and interaction terms.

To explore whether the type of categorization used for the dietary variables had any effect on the relationships noted above, we reran these interaction models successively using tertiles (Table 3) and quartiles (not shown) for the dietary variables. Although there was no gradient in risk for the TT genotype across tertiles of folate, the protective effect for the TT genotype was strongest in the group with the highest total intake for folate. We also explored the joint effects of the TT genotype with intakes of other B vitamins (Table 3). The protective effect of the TT genotype was limited to individuals in the highest two intake tertiles of riboflavin and vitamin B6. The effect of TT was also much stronger in nondrinkers than in the two intake groups of ethanol.

Table 3.

Combined effects of MTHFR C677T and B vitamin or alcohol intake on colorectal cancer risk

MTHFRNutrient intake*
T1
T2
T3
nOR (95% CI)nOR (95% CI)nOR (95% CI)P§
Folate from foods CC 1.00 119/282 0.90 (0.66-1.22) 124/366 0.70 (0.52-0.95)  
 CT 112/244 0.96 (0.70-1.31) 107/265 0.77 (0.56-1.06) 111/257 0.88 (0.64-1.21)  
 TT 25/68 0.74 (0.44-1.24) 32/90 0.70 (0.43-1.11) 26/90 0.56 (0.34-0.93) 0.55 
Folate from foods and supplements CC 141/320 1.00 123/317 0.84 (0.62-1.13) 119/334 0.69 (0.51-0.94)  
 CT 123/260 0.95 (0.70-1.30) 98/237 0.79 (0.57-1.09) 109/269 0.79 (0.57-1.08)  
 TT 26/73 0.74 (0.44-1.23) 35/79 0.90 (0.57-1.44) 22/96 0.39 (0.23-0.65) 0.22 
Vitamin B12 CC 118/259 1.00 131/327 0.86 (0.63-1.18) 134/385 0.82 (0.61-1.12)  
 CT 100/240 0.77 (0.55-1.08) 108/257 0.87 (0.63-1.21) 122/269 1.05 (0.76-1.45)  
 TT 26/58 0.80 (0.47-1.37) 23/91 0.51 (0.30-0.87) 34/99 0.77 (0.48-1.23) 0.16 
Vitamin B6 CC 147/302 1.00 97/285 0.63 (0.46-0.86) 139/384 0.68 (0.51-0.91)  
 CT 114/236 0.88 (0.64-1.20) 95/276 0.56 (0.41-0.78) 121/254 0.87 (0.64-1.20)  
 TT 25/62 0.70 (0.41-1.19) 30/93 0.54 (0.34-0.88) 28/93 0.53 (0.32-0.86) 0.34 
Riboflavin CC 134/291 1.00 115/297 0.80 (0.59-1.09) 134/383 0.76 (0.57-1.03)  
 CT 107/240 0.85 (0.62-1.18) 101/270 0.70 (0.50-0.97) 122/256 1.03 (0.75-1.41)  
 TT 29/57 0.96 (0.57-1.60) 27/94 0.52 (0.32-0.86) 27/97 0.59 (0.36-0.96) 0.12 
Ethanol CC 111/281 1.00 107/301 0.95 (0.68-1.32) 165/389 1.19 (0.88-1.62)  
 CT 81/202 0.97 (0.68-1.38) 109/237 1.08 (0.77-1.52) 140/327 1.14 (0.83-1.58)  
 TT 19/71 0.62 (0.35-1.10) 28/88 0.71 (0.43-1.18) 36/89 1.12 (0.70-1.81) 0.66 
MTHFRNutrient intake*
T1
T2
T3
nOR (95% CI)nOR (95% CI)nOR (95% CI)P§
Folate from foods CC 1.00 119/282 0.90 (0.66-1.22) 124/366 0.70 (0.52-0.95)  
 CT 112/244 0.96 (0.70-1.31) 107/265 0.77 (0.56-1.06) 111/257 0.88 (0.64-1.21)  
 TT 25/68 0.74 (0.44-1.24) 32/90 0.70 (0.43-1.11) 26/90 0.56 (0.34-0.93) 0.55 
Folate from foods and supplements CC 141/320 1.00 123/317 0.84 (0.62-1.13) 119/334 0.69 (0.51-0.94)  
 CT 123/260 0.95 (0.70-1.30) 98/237 0.79 (0.57-1.09) 109/269 0.79 (0.57-1.08)  
 TT 26/73 0.74 (0.44-1.23) 35/79 0.90 (0.57-1.44) 22/96 0.39 (0.23-0.65) 0.22 
Vitamin B12 CC 118/259 1.00 131/327 0.86 (0.63-1.18) 134/385 0.82 (0.61-1.12)  
 CT 100/240 0.77 (0.55-1.08) 108/257 0.87 (0.63-1.21) 122/269 1.05 (0.76-1.45)  
 TT 26/58 0.80 (0.47-1.37) 23/91 0.51 (0.30-0.87) 34/99 0.77 (0.48-1.23) 0.16 
Vitamin B6 CC 147/302 1.00 97/285 0.63 (0.46-0.86) 139/384 0.68 (0.51-0.91)  
 CT 114/236 0.88 (0.64-1.20) 95/276 0.56 (0.41-0.78) 121/254 0.87 (0.64-1.20)  
 TT 25/62 0.70 (0.41-1.19) 30/93 0.54 (0.34-0.88) 28/93 0.53 (0.32-0.86) 0.34 
Riboflavin CC 134/291 1.00 115/297 0.80 (0.59-1.09) 134/383 0.76 (0.57-1.03)  
 CT 107/240 0.85 (0.62-1.18) 101/270 0.70 (0.50-0.97) 122/256 1.03 (0.75-1.41)  
 TT 29/57 0.96 (0.57-1.60) 27/94 0.52 (0.32-0.86) 27/97 0.59 (0.36-0.96) 0.12 
Ethanol CC 111/281 1.00 107/301 0.95 (0.68-1.32) 165/389 1.19 (0.88-1.62)  
 CT 81/202 0.97 (0.68-1.38) 109/237 1.08 (0.77-1.52) 140/327 1.14 (0.83-1.58)  
 TT 19/71 0.62 (0.35-1.10) 28/88 0.71 (0.43-1.18) 36/89 1.12 (0.70-1.81) 0.66 
*

Number of cases/number of controls.

The intake tertile cut points were 253 and 412 μg/d for folate from foods, 322 and 590 μg/d for total folate, 2.86 and 4.99 μg/d for vitamin B12, 1.63 and 2.46 mg/d for vitamin B6, 1.38 and 2.13 mg/d for riboflavin, and 0 and 1.44 g/d for ethanol.

OR (95% CI) adjusted by logistic regression for age at blood draw, sex, and race/ethnicity.

§

P for interaction derived from a likelihood ratio test with 4 degrees of freedom comparing a model with main effects for the MTHFR 677 genotype (CC, CT, or TT) and the dietary variable (in tertiles) and a model with main effects and interaction terms.

Table 4 presents the odds ratios for the C677T polymorphism after stratification by anatomic subsite (colon and rectum) or stage at diagnosis (in situ/localized and regional/distant). The association with the TT genotype was not observed for rectal cancer and for early-stage colorectal cancer. In contrast, the TT genotype was associated with a 31% decreased risk of colon cancer (95% CI for OR, 0.49-0.96; P for gene dosage, 0.03) and a 37% decreased risk of advanced colorectal cancer (95% CI for OR, 0.42-0.94; P for gene dosage, 0.02). Similar odds ratios (all stages combined) were found for tumors of the left and right colon. The ORs (95% CI) for the CC, CT, and TT genotypes were 1.0, 1.0 (0.7-1.3) and 0.7 (0.5-1.1; P for gene dosage, 0.29) for the right colon and 1.0, 1.0 (0.8-1.3), and 0.6 (0.4-1.0; P for gene dosage, 0.12) for the left colon. Because colon cancer cases were more likely to be diagnosed at a late stage than rectal cancer cases, we ran a polytomous logistic regression comparing the risk associated with the genotypes in all four subsite/stage combinations (colon/early, colon/late, rectum/early and rectum/late; Table 5). This analysis showed that the association with the T variant allele was limited to colon cancer diagnosed at an advanced stage.

Table 4.

Colorectal cancer OR (95% CI) for MTHFR C677T by subsite or stage at diagnosis

CC
CT
TT
n*ORnOR (95% CI)nOR (95% CI)PP
Subsite         
    Colon 295/987 1.00 246/779 1.00 (0.81-1.23) 56/255 0.69 (0.49-0.96) 0.03 0.14 
    Rectum 99/987 1.00 90/779 0.99 (0.73-1.36) 31/255 1.01 (0.65-1.57) 0.97  
Stage at diagnosis         
    In situ/localized 202/987 1.00 200/779 1.15 (0.92-1.45) 53/255 0.95 (0.67-1.35) 0.65 0.12 
    Regional/distant 183/987 1.00 133/779 0.83 (0.65-1.08) 34/255 0.63 (0.42-0.94) 0.02  
CC
CT
TT
n*ORnOR (95% CI)nOR (95% CI)PP
Subsite         
    Colon 295/987 1.00 246/779 1.00 (0.81-1.23) 56/255 0.69 (0.49-0.96) 0.03 0.14 
    Rectum 99/987 1.00 90/779 0.99 (0.73-1.36) 31/255 1.01 (0.65-1.57) 0.97  
Stage at diagnosis         
    In situ/localized 202/987 1.00 200/779 1.15 (0.92-1.45) 53/255 0.95 (0.67-1.35) 0.65 0.12 
    Regional/distant 183/987 1.00 133/779 0.83 (0.65-1.08) 34/255 0.63 (0.42-0.94) 0.02  

NOTE: OR (95% CI) adjusted by logistic regression for age at blood draw, sex, and race/ethnicity.

*

Number of cases/number of controls. Seventy-nine cases were in situ, 376 localized, 350 regional/distant, and 17 unstaged.

P for gene dosage effect based on the on a variable assigned the value 1, 2, or 3, according to the subject's number of T alleles (0, 1, and 2, respectively).

P for difference in OR for the TT genotype between colon and rectal cases, and between regional/distant cases and in situ/localized cases as assessed by a Wald test under the polytomous logistic regression.

Table 5.

Colorectal cancer OR (95% CI) for MTHFR C677T by anatomic subsite and stage at diagnosis

StageCC
CT
TT
n*ORnOR (95% CI)nOR (95% CI)PP
Controls 987  779  255    
Colon cancer         
    In situ/localized 144 1.00 138 1.16 (0.89-1.52) 34 0.88 (0.58-1.33) 0.99 — 
    Regional/distant 145 1.00 104 0.84 (0.64-1.12) 22 0.52 (0.32-0.85) 0.01 0.04 
Rectal cancer         
    In situ/localized 58 1.00 61 1.12 (0.76-1.64) 19 1.01 (0.58-1.76) 0.80 0.82 
    Regional/distant 37 1.00 26 0.74 (0.44-1.26) 12 1.00 (0.50-2.00) 0.70 0.73 
StageCC
CT
TT
n*ORnOR (95% CI)nOR (95% CI)PP
Controls 987  779  255    
Colon cancer         
    In situ/localized 144 1.00 138 1.16 (0.89-1.52) 34 0.88 (0.58-1.33) 0.99 — 
    Regional/distant 145 1.00 104 0.84 (0.64-1.12) 22 0.52 (0.32-0.85) 0.01 0.04 
Rectal cancer         
    In situ/localized 58 1.00 61 1.12 (0.76-1.64) 19 1.01 (0.58-1.76) 0.80 0.82 
    Regional/distant 37 1.00 26 0.74 (0.44-1.26) 12 1.00 (0.50-2.00) 0.70 0.73 

NOTE: OR (95% CI) adjusted by polytomous logistic regression for age at blood draw, sex, and race/ethnicity.

*

Number of subjects. Seventy-nine cases were in situ, 376 localized, 350 regional/distant, and 17 unstaged.

P for gene dosage effect based on the on a variable assigned the value 1, 2, or 3 according to the subject's number of T alleles (0, 1, and 2, respectively).

P for pairwise difference in OR for the gene dosage variable for regional/distant colon cases, in situ/localized rectal cancer cases, and regional/distant rectal cancer cases compared with the in situ/localized colon cancer cases, as assessed by a Wald test under the polytomous logistic regression model.

In this large case-control study nested within a multiethnic cohort, we found an overall 23% decreased colorectal cancer risk for individuals with the MTHFR 677TT genotype. This association was similar in both sexes, stronger at high levels of folate intake, and limited to light and nondrinkers. The frequency of the T allele was found to differ markedly across ethnic/racial groups (lower in African Americans and Native Hawaiians and higher in Latinos and Japanese, compared with Whites). The power of the study was low to test for heterogeneity of effect across these populations. However, the analysis by subsite and stage showed that the association with the TT genotype was limited to advanced colon tumors.

Our results bring additional evidence for an inverse association between the MTHFR 677TT genotype and colorectal cancer. This association was first reported in two male Harvard cohorts (20, 21) and was reproduced in five of eight case-control studies to date (reviewed in ref. 11). Four of five past studies suggested interactions between folate and the TT genotype, with the inverse association being greatest among persons with the highest intake or plasma levels of folate (16, 20-23). Both studies that investigated the modifying effect of alcohol found a significant interaction between the TT genotype and alcohol, by which the inverse association with TT was only seen at low levels of ethanol intake (20, 21). Our results for the Multiethnic Cohort Study are remarkably consistent with those findings. Of note is that, in our population, the inverse association with alcohol intake was negated at a level of alcohol intake corresponding to one drink per week. This is in contrast to the two (predominantly White) male Harvard cohorts in which the effect was found to be absent over a level of approximately one drink per day (20, 21). Reasons for this discrepancy are not immediately apparent because similar methods of assessing alcohol intake were used. It is also unlikely to be due to a possible modifying effect of ALDH2*2 (24), an allele that is common in Japanese, because the exclusion of this group from our analysis did not substantially modify the results (P for interaction, 0.08).

Fewer studies compared results by anatomic subsite but similar main effect risk estimates were reported for the colon and rectum in the Physicians' Health Study (21). We are not aware of any past report by stage at diagnosis. Because our design was that of a case-control study, a certain percentage of cases died before we could contact them (24.9% for colon cancer cases; 23.5% for rectal cancer cases). As a result, the genotype frequencies could be misrepresented in our cases if the MTHFR C677T polymorphism were associated with survival after diagnosis. Few studies of this relationship have been published and the data have been inconsistent. In a study of 365 nonadjuvant-treated colorectal cancer patients, the TT genotype was associated with improved survival, but this association did not persist after adjustment for stage (25). Among 51 stage III colon cancer patients treated with 5-fluorouracil (5-FU), a common treatment for advanced colorectal cancer, presence of the 677T allele had little effect on survival (26). However, a study of 43 patients with metastatic colorectal cancer receiving 5-FU therapy showed that carriers of the 677T allele (n = 26) were more likely to respond to treatment than noncarriers (27). A similar finding was observed among 98 colorectal patients with nonresectable liver metastases treated with 5-FU (28). However, this better response to 5-FU for T-allele carriers did not translate into a survival advantage in this study (28). 5-FU sensitivity was also tested in 19 human cancer cell lines (head and neck, breast, and digestive tract) and found to be independent from the C677T polymorphism (29). However, human HCT116 colon cancer cells transfected with mutant 677T human MTHFR cDNA were recently found to have a decreased MTHFR activity, a changed intracellular folate distribution, an accelerated growth rate, an increased thymidylate synthase activity, and an increased sensitivity to 5-FU (30). Overall, however, the small sample sizes of the clinical studies and the high likelihood of publication bias preclude any robust conclusions (31).

Although we did not have information on the type of chemotherapy regimen received by cases in our study, we used the information collected by our Surveillance, Epidemiology, and End Results registries to evaluate the effect of the TT genotype on colorectal cancer risk among those who received chemotherapy as first course of treatment and those who did not. These analyses were run for all cases, limited to only advanced cases, and by colon and rectum subsite, using all controls in each model. A similar inverse association was observed independently of whether the patients received chemotherapy or not. For example, for patients with regional/distant colorectal cancer who received chemotherapy as part of their first course of treatment (n = 195), the OR for the CC, CT, and TT genotypes were 1.00, 0.95 (95% CI, 0.69-1.31) and 0.57 (95% CI, 0.33-0.99), respectively (P for gene dosage, 0.09). The corresponding ORs for those patients with regional/distant colorectal cancer who did not receive chemotherapy (n = 150) were 1.00, 0.65 (0.44-0.92), and 0.68 (0.39-1.20; P for gene dosage, 0.05). In addition, our overall risk estimates for the TT genotype were very similar to those found in studies where DNA was obtained before diagnosis (20, 21). Finally, the specificity of effect for advanced tumors was only observed for colon cancer and not rectal cancer and the proportion of patients missed due to deaths before contact was similar for both subsites (see above). Thus, we found no evidence that our stage-specific findings could be explained by survival bias.

Interestingly, the lower frequency of the T allele in African Americans and Native Hawaiians and the finding of a specificity of its protective effect against advanced colorectal cancer are consistent with the late-stage presentation and poorer survival observed for the disease in these ethnic groups (32-34). Similarly, the stronger effect for the TT genotype suggested for Japanese would be consistent with the early presentation and better survival of Japanese patients with colorectal cancer in Hawaii (32, 33).

Colon cancer is difficult to cure when the disease has spread outside the large intestine. Given the high frequency of the C allele and if its stronger association with advanced disease were to be confirmed, this allele may be responsible for a sizable portion of the morbidity and mortality from colorectal cancer, especially in populations with low total folate intake. Moreover, if the subsite specificity of the association can be replicated, it will be interesting to see whether the folic acid fortification of the diet initiated in the United States in 1998 will result in a greater decrease in rates, if any, for colon than rectal cancer.

In conclusion, these data corroborate previous findings of an inverse association of the MTHFR 677TT genotype with colorectal cancer, especially at high levels of folate and low levels of ethanol intake. They also suggest that this effect may be specific to advanced colon cancer.

Grant support: National Cancer Institute, U.S. Department of Health and Human Services, grants R01 CA 63464 and R01 CA54281.

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.

We thank Jana Koerte and Jennifer Yamamoto for assistance with data analysis, and Wendy Chang, Annette Jones, and Ann Seifried for their help with genotyping.

1
Freudenheim JL, Graham S, Marshall JR. Folate intake and carcinogenesis of the colon and rectum.
Int J Epidemiol
1991
;
20
:
368
–74.
2
Giovannucci E, Stampfer MJ, Colditz GA, et al. Folate, methionine and alcohol intake and risk of colorectal adenoma.
J Natl Cancer Inst
1993
;
85
:
875
–84.
3
Giovannucci E, Rimm EB, Ascherio A, Stampfer MJ, Colditz GA, Willett WC. Alcohol, low-methionine-low folate diets and risk of colon cancer in men.
J Natl Cancer Inst
1995
;
87
:
265
–73.
4
Giovannucci E, Stampfer MJ, Colditz GA, et al. Multivitamin use, folate and colon cancer in women in the Nurses' Health Study.
Ann Int Med
1998
;
129
:
517
–24.
5
Tseng M, Murray SC, Kupper LL, Sandler RS. Micronutrients and the risk of colorectal adenomas.
Am J Epidemiol
1996
;
144
:
1005
–14.
6
Bird C, Swendseid M, Witte J, et al. Red cell and plasma folate, folate consumption and risk of colorectal adenomatous polyps.
Cancer Epidemiol Biomarkers Prev
1995
;
144
:
1005
–14.
7
Ames BN. Micronutrient deficiencies. A major cause of DNA damage.
Ann N Y Acad Sci
1999
;
889
:
87
–106.
8
Vogelstein B, Fearon ER, Kern SE, et al. Allelotype of colorectal carcinomas.
Science
1989
;
244
:
207
–11.
9
Goelz SE, Vogelstein B, Hamilton SR, et al. Hypomethylation of DNA from benign and malignant human colon neoplasms.
Science
1985
;
228
:
187
–90.
10
Choi S-W, Mason JB. Folate status: effects on pathways of colorectal carcinogenesis.
J Nutr
2002
;
132
:
2413
–8S.
11
Weisberg I, Tran P, Christensen B, Sibani R, Rozen R. A second polymorphism in methylenetetrahydrofolate reductase (MTHFR) associated with decreased enzyme activity.
Mol Genet Metab
1998
;
64
:
169
–72.
12
Sharp L, Little J. Polymorphisms in genes involved in folate metabolism and colorectal neoplasia: a HuGE Review.
Am J Epidemiol
2004
;
159
:
423
–43.
13
Kolonel LN, Henderson BE, Hankin JH, et al. A multiethnic cohort in Hawaii and Los Angeles: baseline characteristics.
Am J Epidemiol
2000
;
151
:
346
–57.
14
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.
15
Murphy SP, Wilkens LR, Hankin JH, et al. Comparison of two instruments for quantifying intake of vitamin and mineral supplements: a brief questionnaire versus three 24-hour recalls.
Am J Epidemiol
2002
;
156
:
669
–75.
16
Le Marchand L, Donlon T, Hankin JH, Kolonel LN, Wilkens LR, Seifried A. B-vitamin intake, metabolic genes and colorectal cancer risk.
Cancer Causes Control
2002
;
13
:
239
–48.
17
Le Marchand L, Haiman CA, Wilkens LR, Kolonel LN, Henderson BE. MTHFR polymorphisms, diet, HRT and breast cancer risk: The Multiethnic Cohort Study.
Cancer Epidemiol Biomarkers Prev
2004
;
13
:
2071
–7.
18
Breslow NE, Day NE. Statistical methods in cancer research. Vol 1. The analysis of case-control studies. IARC scientific publication no. 32. Lyon (France): IARC; 1980.
19
Hosmer DW, Lemeshow S. Applied logistic regression. New York: John Wiley and Sons, Inc.; 1989.
20
Chen J, Giovannucci E, Kelsey K, et al. A methylenetetrahydrofolate reductase polymorphism and the risk of colorectal cancer.
Cancer Res
1996
;
56
:
4862
–4.
21
Ma J, Stampfer MJ, Giovannucci E, et al. Methylenetetrahydrofolate reductase polymorphism, dietary interactions and risk of colorectal cancer.
Cancer Res
1997
;
57
:
1098
–102.
22
Slattery ML, Potter JD, Samowitz W, Schaffer D, Leppert M. Methylenetetrahydrofolate reductase, diet, and risk of colon cancer.
Cancer Epidemiol Biomarkers Prev
1999
;
8
:
513
–8.
23
Keku T, William R, Worley K, et al. 5,10-Methylenetetrahydrofolate reductase codon 677 and 1298 polymorphisms and colon cancer in African Americans and whites.
Cancer Epidemiol Biomarkers Prev
2002
;
11
:
1611
–21.
24
Giovannucci E. Alcohol, one-carbon metabolism and colorectal cancer: recent insights from molecular studies.
J Nutr
2004
;
134
:
2475
–81S.
25
Shannon B, Gnanasampanthan S, Beilby J, Iacopetta B. A polymorphism in the methlenetetrahydrofolate reductase gene predisposes to colorectal cancers with microsatellite instability.
Gut
2002
;
50
:
520
–4.
26
Wisotzkey JD, Toman J, Bell T, Monk JS, Jones D. MTHFR (C677T) polymorphisms and stage III colon cancer: response to therapy.
Mol Diagn
1999
;
4
:
95
–9.
27
Cohen V, Panet-Raymont V, Sabbaghian N, Morin I, Batist G, Rozen R. Methylenetetrahydrofolate reductase polymorphism in advanced colorectal cancer: a novel genomic predictor of clinical response to fluoropyrimidine-based chemotherapy.
Clin Cancer Res
2003
;
9
:
1611
–5.
28
Etienne MC, Formento JL, Chazal M, et al. Methylenetetrahydrofolate reductase gene polymorphisms and response to fluorouracil-based treatment in advanced colorectal cancer patients.
Pharmacogenetics
2004
;
14
:
785
–92.
29
Etienne MC, Ilc K, Formento JL, et al. Thymidylate synthase and methylenetetrahydrofolate reductase gene polymorphisms: relationships with 5-fluorouracil sensitivity.
Br J Cancer
2004
;
90
:
526
–34.
30
Sohn KJ, Croxford R, Yates Z, Lucock M, Kim YI. Effect of methylenetetrahydrofolate reductase C677T polymorphism on chemosensitivity of colon and breast cancer cells to 5-fluorouracil and methotrexate.
J Natl Cancer Inst
2004
;
96
:
134
–44.
31
Ulrich CM, Robien K, McLeod HL. Cancer pharmacogenetics: polymorphisms, pathways and beyond.
Nat Rev Cancer
2003
;
3
:
912
–20.
32
Chen VW, Fenoglio-Preiser CM, Wu CX, et al. Aggressiveness of colon carcinoma in blacks and whites.
Cancer Epidemiol Biomarkers Prev
1997
;
6
:
1087
–93.
33
Wegner EL, Kolonel LN, Nomura AMY, Lee J. Racial and socioeconomic status differences in survival of colorectal cancer patients in Hawaii.
Cancer
1982
;
49
:
2208
–16.
34
Pagano IS, Morita SY, Dhakal S, Hundahl SA, Maskarinec G. Time dependent ethnic convergence in colorectal cancer survival in Hawaii.
BMC Cancer
2003
;
3
:
5
.