We previously reported (J. Chen et al., Cancer Res., 56:4862–4864, 1996; J. Ma et al., Cancer Res., 57: 1098–1102, 1997) that a 5,10-methylenetetrahydrofolate reductase (MTHFR) polymorphism (677C→T, alaval) was associated with lower risk of colorectal cancer. In this study, we examined the relationship of a polymorphism (2756A→G, aspgly) in the gene (MTR) for methionine synthase, another important enzyme in the same folate/methionine/homocyst(e)ine metabolic pathway, with risk of colorectal cancer among 356 cases and 476 cancer-free controls. The frequency of the homozygous variant genotype (gly/gly) was slightly lower among cases (3%) than controls (5%). The odds ratio for the gly/gly genotype was 0.59 [95% confidence interval (CI), 0.27–1.27] compared with those with the homozygous wild type (asp/asp). There were no significant differences in plasma levels of folate, vitamin B12, and homocyst(e)ine (tHcy) among the MTR genotypes, in contrast to the MTHFR polymorphism. However, similar to the interaction observed for the MTHFR polymorphism among men who consumed less than 1 alcoholic drink/day, those with the gly/gly genotype had a lower risk of colorectal cancer with an odds ratio of 0.27 (95% CI, 0.09–0.81) compared with those with the asp/asp genotype. The possible association of the MTR polymorphism with lower risk of colorectal cancer especially among those with low alcohol consumption, in the same direction as for the MTHFR polymorphism, is intriguing. However, our study had limited statistical power because of the low frequency of the MTR variant genotype, which is reflected in the wide CIs. Hence, these findings need to be confirmed in larger populations.

Methionine synthase, a vitamin B12-dependent enzyme, plays an important role in folate metabolism (1). It catalyzes the transfer of a methyl group from 5-methyltetrahydrofolate to homocysteine, producing methionine and tetrahydrofolate. Methionine synthase is critical for maintaining adequate intracellular methionine, an essential amino acid and the precursor of SAM.4 SAM is a crucial methyl group donor involved in over 100 methylation reactions including DNA methylation. Hypomethylation of the promoter regions of proto-oncogenes (2) or hypermethylation of these regions of tumor suppressor genes (3) may cause selective growth and transformation of cells. De novo methylation of CpG islands within the promoter regions of growth-regulatory genes, which may inactivate their transcription, is frequently observed in colonic tumors (4). Methionine synthase is also essential for maintaining adequate intracellular folate pools and ensuring that homocysteine concentrations do not reach toxic levels. Severe deficiency of vitamin B12 or of methionine synthase causes hypomethioninemia, hyperhomocysteinemia, and homocystinuria (5). It may also result in an accumulation of 5-methyltetrahydrofolate and depletion of intracellular folate derivatives including 5,10-methylenetetrahydrofolate required for thymidylate biosynthesis, the basis of the well-documented “methyl folate trap” leading to deoxynucleotide pool imbalances and megaloblastic anemia (1, 6, 7). This leads to the accumulation of deoxyuridylate in DNA, and removal of this abnormal base may damage DNA, perhaps leading to strand breaks commonly seen in colorectal cancers (7, 8). Pernicious anemia, caused by vitamin B12 malabsorption, has been associated with an elevated risk of cancer of the esophagus, stomach, and colon (9, 10). Recently, a polymorphism in the methionine synthase gene (MTR; 2756A→G), resulting in the substitution of aspartic acid (D919) by glycine (G), was identified in patients with methionine synthase deficiency and was found to be polymorphic among healthy controls (11).

We previously reported, from the same group of participants (12, 13), that a polymorphism (677C→T) that encodes a thermolabile MTHFR and causes a decrease in plasma 5-methyltetrahydrofolate was associated with a lower risk of colorectal cancer. This protective effect could result from increased availability of intracellular 5,10-methylenetetrahydrofolate and consequent reductions of uracil incorporation into DNA or from decreased SAM levels leading to DNA hypomethylation. Low folate intake or high alcohol consumption (which interferes with folate metabolism) seemed to negate some of the protective effect (12, 13). In the present study nested in two large cohorts, we examined the association of the MTR polymorphism with the risk of colorectal cancer and whether the association differs by plasma levels of folate, vitamin B12, tHcy, or alcohol intake. We hypothesize that, if the variant genotype (gly/gly) of this MTR polymorphism is associated with a decreased activity of methionine synthase, men with the gly/gly genotype would have lower cellular methionine and folate derivatives, elevated tHcy levels, and an increased risk of colorectal cancer. Alternatively, lower methionine and SAM may lead to DNA hypomethylation, which would modify the cancer risk. For comparison, we also present some results for the MTHFR polymorphism.

Study Population.

The PHS is a randomized, double-blind, placebo-controlled 2 × 2 factorial trial of low-dose aspirin and β-carotene among 22,071 predominantly Caucasian-American male physicians, ages 40–84 years. Blood samples were collected at baseline, in 1982, from 14,916 (68%) of the randomized physicians. Alcohol consumption (drinks of beer, wine, or liquor) was ascertained from the baseline questionnaire. The men were subsequently followed for incident cancer through biannual mailed questionnaires. After 13 years of follow-up, 212 cases of colorectal cancer were identified and confirmed by medical records. Three hundred forty-six men who were free from diagnosed cancer at the time of case ascertainment were selected as controls and were matched on age (1 year) and smoking status (never, past, current).

The HPFS is a prospective study of 51,529 predominantly Caucasian-American male health professionals, ages 40–75, enrolled in 1986. Alcohol consumption (drinks of beer, wine, or liquor) was ascertained from a semiquantitative food frequency questionnaire at baseline. Blood samples were collected between 1993 and 1994 from 18,025 participants, among whom 144 had been diagnosed with colorectal cancer between 1986 and 1994; 130 cancer-free men were selected as controls. These cancer cases were also confirmed by medical records. The participants were predominantly Caucasians (over 95% in both the PHS and the HPFS).

MTR Genotype and Other Laboratory Assays.

DNA from cases and controls from both studies was extracted and MTR genotype was analyzed in Dr. Rozen’s laboratory; investigators and laboratory personnel were blinded to case-control status. The presence of the mutation was determined by PCR of genomic DNA, followed by HaeIII restriction digestion, as previously described (11). Because plasma levels of folate, vitamin B12, and tHcy may be altered by change of diet or treatment after the diagnosis of cancer, these biomarkers were measured only for the PHS participants. Samples from cases and their matched controls were assayed in the same batch to minimize interassay variability, and aliquots from a pool of quality control plasma were inserted randomly. Laboratory personel were unable to distinguish among case, control, and quality control samples. They were also unaware of the genotype status. Plasma levels of folate and vitamin B12 were determined using a radioassay kit (Ciba-Corning, Walpole, MA) in the laboratory of the Jean Mayer United States Department of Agriculture Human Nutrition Research Center on Aging at Tufts University, Boston, MA. Total tHcy levels were determined in the same laboratory as described previously(14). The average intra-assay coefficient of variation for folate, vitamin B12, and tHcy were 6.8%, 3.0%, and 2.9%, respectively.

Statistical Analyses.

We examined the age-adjusted OR and 95% CI for the association of the MTR genotype with the risk of developing colorectal cancer in the PHS and HPFS separately and combined. We conducted unconditional logistic regression analysis because of the combined study population. Using conditional logistic analysis yielded similar results when the matched case-control subgroup was examined. Prospective data on blood nutrients levels were availible only for the PHS colorectal cancer cases and controls. The age-adjusted geometric mean of plasma folate (because of the skewed distribution), mean vitamin B12, and tHcy concentration within strata of the MTR and MTHFR genotypes by case-control status were calculated by analysis of covariance. In the PHS, we assessed the age-adjusted ORs for the joint effect of the MTR and MTHFR genotypes and status of plasma folate, vitamin B12, and tHcy (categorized into two groups based on control tertile distribution, lower one-third versus upper two-thirds for folate and vitamin B12, lower two-thirds versus upper one-third for tHcy) using an indicator variable for each category in logistic regression models. Because alcohol consumption was measured prospectively in both the PHS and the HPFS, we assessed the joint effect of the MTR genotype and alcohol consumption (<1 drink/day, and ≥1 drink/day) within the PHS as well as the PHS and the HPFS combined. We compared the log likelihood statistics of the main effect model with the joint effect model to assess interaction. Because of small numbers in some of the stratified analyses, we used an exact statistic method (LogXact; Ref. 15) and obtained virtually identical results; we, therefore, present all of the results from unconditional logistic regression analyses. All of the Ps are two-sided, and all of the analyses were done using SAS (16).

The overall frequency of the homozygous variant gly/gly genotype was 5% among controls and 3% among cases (Table 1). The gly allele frequencies were 0.19 among controls and 0.17 among cases, not materially different from the 0.15 reported by Leclerc et al.(11) and the 0.16 reported by van der Put et al.(17). The genotype and allele frequencies were similar in the two cohorts. Overall, there was a nonsignificant inverse association of the gly/gly genotype with risk of colorectal cancer (OR, 0.59; 95% CI, 0.27–1.27) compared with the asp/asp genotype (Table 1).

Plasma folate, tHcy, and B12 were measured only among the PHS participants. There were no apparent associations between the MTR homozygous variant gly/gly genotype and plasma levels of folate and tHcy (Table 2). Similar to our previous observation using the microbiological method for folate measurement (13), the MTHFR val/val genotype was significantly associated with lower plasma folate levels determined by the radioassay method, among both cases and controls (Table 2). The observation that the MTHFR homozygous variant genotype val/val was significantly associated with lower plasma folate levels is also consistent with findings from a separate case-control study of myocardial infarction nested in the PHS (18). In the present study, we also measured plasma vitamin B12 and tHcy levels and observed significantly higher tHcy levels among cases with the MTHFR val/val genotype (Table 2). Although men with the MTR homozygous variant gly/gly genotype had higher levels of vitamin B12 and men with the MTHFR val/val genotype had lower levels, none of these differences were statistically significant.

We examined the association of the MTR genotype and the risk of colorectal cancer according to plasma levels of folate, vitamin B12, and tHcy in comparison with the MTHFR genotype. Because of the low prevalence of the gly/gly genotype, we categorized plasma folate, vitamin B12, and tHcy into two groups based on the distribution among controls. For the MTHFR genotype, as we previously reported (13), there was a significant 70% decrease (OR, 0.29; 95% CI, 0.12–0.73) in risk among men with the val/val genotype and with plasma folate levels in the upper two tertiles of the control distribution compared with those with the ala/ala or val/ala genotype in the same folate category (Table 3). In the present analysis, we found that men with the homozygous val/val genotype and plasma tHcy levels in the lower two tertiles also had a significant 75% decrease (OR, 0.25; 95% CI, 0.08–0.74) in risk (Table 3). For the MTR genotype, we observed a nonsignificant 50% decrease (OR, 0.51; 95% CI, 0.14–1.90) in risk among men with the gly/gly genotype and with plasma folate levels in the upper two tertiles compared with those with the gly/asp or asp/asp genotype in the same folate category (Table 3). However, there was no apparent interaction between the MTR genotype and plasma tHcy (Table 3) on the risk of colorectal cancer. No apparent interactions were observed between vitamin B12 and both genotypes (data not shown). Adjustment for body mass index, alcohol intake, multivitamin intake, and aspirin assignment did not change the results.

Alcohol consumption was assessed at baseline in both the PHS (1982) and the HPFS (1986). We examined the alcohol-genotype interaction within the PHS cohort as well as combined with the HPFS participants. Similar to our previous observation of the interaction between the MTHFR genotype and alcohol intake on risk (12, 13), we found a significant interaction between the MTR genotype and alcohol intake (Pinteraction < 0.01) in the PHS. Among men with alcohol intake less than 1 drink/day, those with the gly/gly genotype had an OR of 0.10 (95% CI, 0.01–0.81), those with the gly/asp genotype had an OR of 0.70 (95% CI, 0.44–1.12) compared with those with the asp/asp genotype. Compared with the same reference group, among men with alcohol intake one drink/day or more, those with the gly/gly genotype had an OR of 3.79 (95% CI, 0.71–20.22), those with the gly/asp genotype had an OR of 1.14 (95% CI, 0.61–2.13). The combined PHS and HPFS data showed a similar association (Pinteraction = 0.04; Table 4). Excluding men with MTHFR val/val genotype (n = 50) or further adjusting for body mass index, multivitamin intake, and aspirin assignment yielded similar results.

Although the 2756A→G polymorphism of MTR was first identified among patients with a deficiency of methionine synthase and among normal controls, the biological impact of this polymorphism is unknown (11). This A→G substitution at bp 2756 causes a substitution of glycine for aspartic acid (D919G). D919 corresponds to Q893 in the cobalamin-dependent Escherichia coli methionine synthase. In this highly homologous bacterial enzyme, this residue is at the penultimate position in a long helix that leads out of the cobalamin domain to the SAM-binding domain (19). It has been postulated that the glycine residue, a strong helix breaker compared with aspartic acid, could affect the secondary structure of the protein and, therefore, have functional consequences, perhaps leading to altered levels of vitamin B12, folate, or tHcy (17). However, our observation that the MTR polymorphism was not associated with plasma levels of folate, vitamin B12, or tHcy suggests that this aspartic acid-to-glycine change may not significantly deteriorate methionine synthase activity. In a recent Dutch study of patients and mothers of children with neural tube defects, patients with arterial disease, and population-based controls, the MTR polymorphism was also not associated with plasma tHcy levels (17). One alternative explanation is that homocysteine may be remethylated to methionine through an alternative pathway by betaine-homocysteine methyltransferase (Fig. 1;20). Although the main physiological role of that enzyme is to catabolize excess betaine (21), it also participates in regulating tissue levels of methionine and in removing excess homocysteine during stress (22). Under normal conditions, the activity of this enzyme increases substantially as a result of (a) inadequate methionine intake (23); (b) inactivation of methionine synthase by nitrous oxide (24); (c) impaired methionine synthase due to ethanol administration (25); or possibly (d) being in the presence of the variant MTR genotype.

Overall, we found that the MTR gly/gly genotype was associated with a nonsignificant 40% decrease in risk of colorectal cancer, in the same direction as we previous reported from the same participants for the MTHFR polymorphism (12, 13). The decreased risk was consistent among the PHS and the HPFS participants analyzed separately, but the CIs were wide, reflecting the small number of participants carrying the gly/gly genotype. Decreased activity of MTR or MTHFR would be expected to lower SAM levels leading to decreased DNA methylation. DNA hypomethylation has been suggested to suppress tumor growth (26, 27). Alternatively, because the activities of MTHFR and methionine synthase were significantly increased in tumor-bearing animals because of increased methylation reactions for tumor growth (28, 29, 30, 31, 32), these polymorphisms may inhibit the excessive use of methionine by tumor cells. This inhibition probably acts on a late stage of tumorigenesis because neither the MTHFR nor the MTR polymorphisms are associated with risk of colorectal adenoma, the immediate precursor of colorectal cancer (33).

Because plasma folate levels reflect both genetic and dietary variation, and ethanol can interfere with folate and methyl group metabolism as well as methionine synthase activity, moderate enzymatic changes due to genetic polymorphisms may behave differently according to the intake of alcohol (12, 13). The interactions between the MTR genotype and alcohol intake are consistent with what we observed for the MTHFR genotype. The increased risk for colorectal cancer conferred by high alcohol intake may overcome the protective effect of these polymorphisms (12, 13). Also, the influence of alcohol on cancer risk may differ among individuals with different genetic susceptibility. Among 10 cases and 21 controls who had the MTR variant genotype gly/gly in our analysis, men reporting one or more drinks/day had about a 10-fold higher risk than those who drank less. Although we observed similar results within the two cohorts as well as in the combined analysis, our study has limited statistical power for both main effect and stratified analyses because of the low prevalence of the variant MTR genotype, which is reflected in the wide CIs. The possible association of this MTR genotype with the risk of colorectal cancer, especially among individuals with low alcohol intake, merits further study in larger populations.

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

Supported by research Grants CA 42182 and CA 40360 from NIH and the Medical Research Council of Canada.

                        
4

The abbreviations used are: SAM, S-adenosylmethionine; MTHFR, 5,10-methylenetetrahydrofolate reductase; tHcy, homocyst(e)ine; OR, odds ratio; CI, confidence interval; PHS, Physician’s Health Study; HPFS, Health Professional Follow-up Study.

Fig. 1.

Role of methionine synthase (MS) and MTHFR in folate/homocyst(e)ine/methionine metabolism.

Fig. 1.

Role of methionine synthase (MS) and MTHFR in folate/homocyst(e)ine/methionine metabolism.

Close modal
Table 1

Frequency of MTR genotypes and age-adjusted risk of colorectal cancer by MTR genotype among participants of the Physicians’ Health Study (PHS) and the Health Professional Follow-up Study (HPFS)

MTR genotypeCasesControlsOR95% CI
N(%)N(%)
PHS       
 asp/asp 145 (68) 235 (68) 1.00 ref.a 
 asp/gly 61 (29) 95 (27) 1.04 0.71–1.53 
 gly/gly (3) 16 (5) 0.63 0.24–1.64 
 Total 212  346    
HPFS       
 asp/asp 103 (72) 82 (63) 1.00 ref. 
 asp/gly 37 (26) 42 (32) 0.71 0.42–1.20 
 gly/gly (3) (5) 0.53 0.14–1.93 
 Total 144  130    
PHS and HPFS       
 asp/asp 248 (70) 317 (66) 1.00 ref. 
 asp/gly 98 (28) 137 (29) 0.92 0.67–1.25 
 gly/gly 10 (3) 22 (5) 0.59 0.27–1.27 
 Total 356  476    
MTR genotypeCasesControlsOR95% CI
N(%)N(%)
PHS       
 asp/asp 145 (68) 235 (68) 1.00 ref.a 
 asp/gly 61 (29) 95 (27) 1.04 0.71–1.53 
 gly/gly (3) 16 (5) 0.63 0.24–1.64 
 Total 212  346    
HPFS       
 asp/asp 103 (72) 82 (63) 1.00 ref. 
 asp/gly 37 (26) 42 (32) 0.71 0.42–1.20 
 gly/gly (3) (5) 0.53 0.14–1.93 
 Total 144  130    
PHS and HPFS       
 asp/asp 248 (70) 317 (66) 1.00 ref. 
 asp/gly 98 (28) 137 (29) 0.92 0.67–1.25 
 gly/gly 10 (3) 22 (5) 0.59 0.27–1.27 
 Total 356  476    
a

ref., reference.

Table 2

Age-adjusted mean of folate (geometric mean), B12, and homocyst(e)ine by case/control status and genotypes of MTR and MTHFR among the PHS participants

CaseControls
NMeanNMean
MTR genotype     
 Folate     
  asp/asp 126 4.13 218 4.29 
  gly/asp 53 4.17 87 3.80 
  gly/gly 4.60 14 3.88 
B12     
  asp/asp 126 452 218 477 
  gly/asp 53 494 87 476 
  gly/gly 542 14 506 
 Homocyst(e)ine     
  asp/asp 115 12.9 200 12.3 
  gly/asp 49 11.6 79 11.9 
  gly/gly 12.8 11 11.4 
MTHFR genotype     
 Folate     
  ala/ala 81 4.54 138 4.39 
  val/ala 81 3.87 116 4.07 
  val/val 14 3.12a 46 3.50a 
B12     
  ala/ala 81 471 138 503 
  val/ala 81 462 116 468 
  val/val 14 431 46 440 
 Homocyst(e)ine     
  ala/ala 79 12.0 135 12.2 
  val/ala 78 12.3 111 11.8 
  val/val 13 17.6b 43 13.1 
CaseControls
NMeanNMean
MTR genotype     
 Folate     
  asp/asp 126 4.13 218 4.29 
  gly/asp 53 4.17 87 3.80 
  gly/gly 4.60 14 3.88 
B12     
  asp/asp 126 452 218 477 
  gly/asp 53 494 87 476 
  gly/gly 542 14 506 
 Homocyst(e)ine     
  asp/asp 115 12.9 200 12.3 
  gly/asp 49 11.6 79 11.9 
  gly/gly 12.8 11 11.4 
MTHFR genotype     
 Folate     
  ala/ala 81 4.54 138 4.39 
  val/ala 81 3.87 116 4.07 
  val/val 14 3.12a 46 3.50a 
B12     
  ala/ala 81 471 138 503 
  val/ala 81 462 116 468 
  val/val 14 431 46 440 
 Homocyst(e)ine     
  ala/ala 79 12.0 135 12.2 
  val/ala 78 12.3 111 11.8 
  val/val 13 17.6b 43 13.1 
a

P ≤ 0.05.

b

P ≤ 0.01 for val/valgenotype versus ala/ala genotype.

Table 3

Age-adjusted risk of colorectal cancer according to MTR and MTHFR genotypes and levels of folate and homocyst(e)ine among the PHS participants

Folate (ng/ml)Homocyst(e)ine (mg/dl)
Tertile 1Tertile 2 and 3Tertile 1 and 2Tertile 3
MTR asp/asp or gly/asp     
 Number 51/102 128/203 100/186 64/93 
 OR (95% CI) 0.84 (0.56–1.27) 1.00 (ref) 1.00 (ref) 1.21 (0.81–1.82) 
MTR gly/gly     
 Number 3/4 3/10 4/7 2/4 
 OR (95% CI) 1.25 (0.27–5.69) 0.51 (0.14–1.90) 1.10 (0.31–3.90) 1.05 (0.19–5.85) 
 Pinteraction = 0.22  Pinteraction = 0.37  
     
MTHFR ala/ala or val/ala     
 Number 45/83 117/171 100/167 57/79 
 OR (95% CI) 0.88 (0.56–1.36) 1.00 (ref) 1.00 (ref) 1.16 (0.76–1.78) 
MTHFR val/val     
 Number 8/18 6/28 4/25 9/18 
 OR (95% CI) 0.57 (0.24–1.38) 0.29 (0.12–0.73) 0.25 (0.08–0.74) 0.69 (0.29–1.62) 
 Pinteraction = 0.16  Pinteraction = 0.24  
Folate (ng/ml)Homocyst(e)ine (mg/dl)
Tertile 1Tertile 2 and 3Tertile 1 and 2Tertile 3
MTR asp/asp or gly/asp     
 Number 51/102 128/203 100/186 64/93 
 OR (95% CI) 0.84 (0.56–1.27) 1.00 (ref) 1.00 (ref) 1.21 (0.81–1.82) 
MTR gly/gly     
 Number 3/4 3/10 4/7 2/4 
 OR (95% CI) 1.25 (0.27–5.69) 0.51 (0.14–1.90) 1.10 (0.31–3.90) 1.05 (0.19–5.85) 
 Pinteraction = 0.22  Pinteraction = 0.37  
     
MTHFR ala/ala or val/ala     
 Number 45/83 117/171 100/167 57/79 
 OR (95% CI) 0.88 (0.56–1.36) 1.00 (ref) 1.00 (ref) 1.16 (0.76–1.78) 
MTHFR val/val     
 Number 8/18 6/28 4/25 9/18 
 OR (95% CI) 0.57 (0.24–1.38) 0.29 (0.12–0.73) 0.25 (0.08–0.74) 0.69 (0.29–1.62) 
 Pinteraction = 0.16  Pinteraction = 0.24  
Table 4

Age-adjusted risk of colorectal cancer according to MTR genotype and alcohol intake among United States physicians and health professionals in the PHS and HPFS

MTR genotypeAlcohol intake
<1 drink/day≥1 drink/day
asp/asp   
 Number 162/210 86/104 
 OR (95% CI) 1.00 (ref.) 1.04 (0.73–1.49) 
gly/asp   
 Number 57/86 41/50 
 OR (95% CI) 0.88 (0.59–1.30) 1.03 (0.64–1.63) 
gly/gly   
 Number 4/19 6/3 
 OR (95% CI) 0.27 (0.09–0.81) 2.64 (0.65–10.82) 
 Ptrend = 0.04 Ptrend = 0.52 
 Pinteraction = 0.04  
MTR genotypeAlcohol intake
<1 drink/day≥1 drink/day
asp/asp   
 Number 162/210 86/104 
 OR (95% CI) 1.00 (ref.) 1.04 (0.73–1.49) 
gly/asp   
 Number 57/86 41/50 
 OR (95% CI) 0.88 (0.59–1.30) 1.03 (0.64–1.63) 
gly/gly   
 Number 4/19 6/3 
 OR (95% CI) 0.27 (0.09–0.81) 2.64 (0.65–10.82) 
 Ptrend = 0.04 Ptrend = 0.52 
 Pinteraction = 0.04  

We thank the participants of the Physicians’ Health Study for their cooperation and participation. The authors are also indebted to Kathryn Starzyk, Rachel Adams, Xiaoyang Liu and Stefanie Parker for their expert and unfailing assistance. We are grateful to Dr. Klaus Lindpaintner who prepared the DNA, and to Nelly Sabbaghian and Marie Nadeau for technical assistance.

1
Banerjee R. V., Matthews R. G. Cobalamin-dependent methionine synthase.
FASEB J.
,
4
:
1450
-1459,  
1990
.
2
Fearon E. R., Vogelstein B. A genetic model for colorectal tumorigenesis.
Cell
,
61
:
759
-767,  
1990
.
3
Greenblatt M. S., Bennett W. P., Hollstein M., Harris C. C. Mutations in the p53 tumor suppressor gene: clues to cancer etiology and molecular pathogenesis.
Cancer Res.
,
54
:
4855
-4878,  
1994
.
4
Issa J-P., Ottaviano Y. L., Celano P., Hamilton S. R., Davidson N. E., Baylin S. B. Methylation of the oestrogen receptor CpG island links aging and neoplasia in human colon.
Nat. Genet.
,
7
:
536
-540,  
1994
.
5
Watkins D., Rosenblatt D. S. Functional methionine synthase deficiency (cb1E and cb1G): clinical and biochemical heterogeneity.
Am. J. Med. Genet.
,
34
:
427
-434,  
1989
.
6
Fowler B., Whitehouse C., Wenzel F., Wraith J. E. Methionine and serine formation in control and mutant cultured fibroblasts: evidence for methyl trapping and characterization of remethylation defects.
Pediatr. Res.
,
41
:
145
-151,  
1997
.
7
Blount B. C., Ames B. N. DNA damage in folate deficiency.
Baillieres Clin. Haematol.
,
8
:
461
-478,  
1995
.
8
Vogelstein B., Fearon E. R., Kern S. E., Hamilton S. R., Preisinger A. C., Nakamura Y., White R. Allelotype of colorectal carcinomas.
Science (Washington DC)
,
244
:
207
-211,  
1989
.
9
Talley N. J., Chute C. G., Larson D. E., Epstein R., Lydick E. G., Melton L. J. Risk for colorectal adenocarcinoma in pernicious anemia: a population-based cohort study.
Ann. Intern. Med.
,
111
:
738
-742,  
1989
.
10
Hsing A. W., Hansson L. E., McLaughlin J. K., Myren O., Blot W. J., Ekbom A., Fraumeni J. F., Jr. Pernicious anemia and subsequent cancer. A population-based cohort study.
Cancer (Phila.)
,
71
:
745
-750,  
1993
.
11
Leclerc D., Campeau E., Goyette P., Adjalla C. E., Christensen B., Ross M., Eydoux P., Rosenblatt D. S., Rozen R., Gravel R. A. Human methionine synthase: cDNA cloning and identification of mutations in patients of the cblG complementation group of folate/cobalamin disorders.
Hum. Mol. Genet.
,
5
:
1967
-1974,  
1996
.
12
Chen J., Giovannucci E., Kelsey K., Rimm E. B., Stampfer M. J., Colditz G. A., Spiegelman D., Willett W. C., Hunter D. J. A methylenetetrahydrofolate reductase polymorphism and the risk of colorectal cancer.
Cancer Res.
,
56
:
4862
-4864,  
1996
.
13
Ma J., Stampfer M., Giovannucci E., Artigas C., Hunter D., Fuchs C., Willett W., Selhub J., Hennekens C., Rozen R. Methylenetetrahydrofolate reductase polymorphism, dietary interactions, and risk of colorectal cancer.
Cancer Res.
,
57
:
1098
-1102,  
1997
.
14
Tucker K. L., Mahnken B., Wilson P. W. F., Jacques P., Selhub J. Folic acid fortification of the food supply: potential benefits and risks for the elderly population.
J. Am. Med. Assoc.
,
276
:
1879
-1885,  
1996
.
15
Mehta C., Patel N. LogXact Users Manual Cytel Software Corporation Cambridge, MA  
1993
.
16
SAS Institute. I. SAS/STATR User’s Guide Version 6. Cary, NC: SAS Institute Inc., 1989.
17
van der Put N. M. J., van der Molen E. F., Kluijtmans L. A. J., Heil S. G., Trijbels J. M. F., Eskes T. K. A. B., Van Oppenraajj-Emmerzaal D., Banerjee R., Blom H. J. Sequence analysis of the coding region of human methionine synthase relevance to hyperhomocysteinaemia in neural-tube defects and vascular disease.
QJM
,
90
:
511
-517,  
1997
.
18
Ma J., Stampfer M. J., Hennekens C. H., Frosst P., Selhub J., Horsford J., Malinow M. R., Willett W. C., Rozen R. Methylenetetrahydrofolate reductase polymorphism, plasma folate, homocysteine, and risk of myocardial infarction in U. S. physicians.
Circulation
,
94
:
2410
-2416,  
1996
.
19
Luschinsky Drennan C., Huang S., Drummond J. T., Matthews R. G., Ludwig M. L. How a protein binds B12: a 30 A X-ray structure of B12-binding domains of methionine synthase.
Science (Washington DC)
,
266
:
1669
-1674,  
1994
.
20
Green R., Jacobsen D. W. Clinical implications of hyperhomocysteinemia Marcel Dekker, Inc. New York  
1992
.
21
Finkelstein J. D., Martin J. J., Harris B. J., Kyle W. E. Regulation of hepatic betaine-homocysteine methyltransferase by dietary betaine.
J. Nutr.
,
113
:
519
-521,  
1983
.
22
Finkelstein J. D., Martin J. J., Harris B. J., Kyle W. E. Regulation of the betaine content of rat liver.
Arch. Biochem. Biophys.
,
218
:
169
-173,  
1982
.
23
Finkelstein J. D., Harris B. J., Martin J. J., Kyle W. E. Regulation of hepatic betaine-homocysteine methyltransferase by dietary methionine.
Biochem. Biophys. Res. Commun.
,
108
:
344
-348,  
1982
.
24
Lumb M., Sharrer N., Deacon R., Jennings P., Purkiss P., Perry J., Chanarin I. Effects of nitrous oxide-induced inactivation of cobalamin on methionine and S-adenosylmethionine metabolism in the rat.
Biochim. Biophys. Acta
,
756
:
354
-359,  
1983
.
25
Barak A. J., Beckenhauer H. C., Tuma D. J., Donahue T. M. J. Adaptive increase in betaine: homocysteine methyltransferase activity maintains hepatic S-adenosylmethionine levels in ethanol-treated rats.
IRCS (Int. Res. Commun. Syst.) Med Sci
,
12
:
866
-877,  
1984
.
26
Laird P., Jackson-Grusby L., Fazeli A., Dickinson S., Jung W., Li E., Weinberg R., Jaenisch R. Suppression of intestinal neoplasia by DNA hypomethylation.
Cell
,
81
:
197
-205,  
1995
.
27
Bender C. M., Pao M. M., Jones P. A. Inhibition of DNA methylation by 5-aza-2′-deoxycytidine suppresses the growth of human tumor cell lines.
Cancer Res.
,
58
:
95
-101,  
1998
.
28
Crossman M. R., Finkelstein J. D., Kyle W. E., Morris H. P. The enzymology of methionine metabolism in rat hepatomas.
Cancer Res.
,
34
:
794
-800,  
1974
.
29
Grzelakowska-Sztabert B., Chnurzynska M. M. W., Sikora E. Age- and tumor-related changes in methionine biosynthesis in mice.
Cancer Lett.
,
32
:
207
-217,  
1986
.
30
Hoffman R. M. Altered methionine metabolism, DNA methylation and oncogene expression in carcinogenesis.
Biochim. Biophys. Acta
,
738
:
49
-87,  
1984
.
31
Stern P. H., Hoffman R. M. Elevated overall rates of transmethylation in cell lines from diverse human tumors.
In Vitro (Rockville)
,
20
:
663
-670,  
1984
.
32
Breillout F., Antoine E., Poupon M. F. Methionine dependency of malignant tumors: a possible approach for therapy.
J. Natl. Cancer Inst.
,
82
:
1628
-1632,  
1990
.
33
Chen J., Giovannucci E., Hankinson S. E., Willett W. C., Spiegelman D., Kelsey K. T., Hunter D. J. A prospective study of methylenetetrahydrofolate reductase and methionine synthase gene polymorphism and risk of colorectal adenoma.
Carcinogenesis (Lond.)
,
19
:
2129
-2132,  
1998
.