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. Author manuscript; available in PMC: 2012 Dec 3.
Published in final edited form as: Toxicol Lett. 2008 Sep 16;184(2):73–80. doi: 10.1016/j.toxlet.2008.09.003

Review and pooled analysis of studies on MTHFR C677T polymorphism and esophageal cancer

SM Langevin 1,2, D Lin 3, K Matsuo 4, CM Gao 5, T Takezaki 6, RZ Stolzenberg-Solomon 7, M Vasavi 8, Q Hasan 8,9, E Taioli 1,2,*
PMCID: PMC3512563  NIHMSID: NIHMS418994  PMID: 18840514

Abstract

Esophageal cancer has been associated with tobacco and alcohol consumption, gastric reflux, exposure to nitrosamines from food or other environmental sources, and diets lacking folate. Susceptibility to esophageal cancer may be modified by functional polymorphisms in genes along the folate metabolic pathway, such as methylenetetrahydrofolate reductase (MTHFR). The C677T polymorphism is the most common functional variant, leading to a reduction in enzyme activity. We report a pooled analysis of 5 studies on the association of MTHFR C677T polymorphism and esophageal cancer, including 725 cases and 1531 controls. A significant association between the MTHFR 677 TT genotype and esophageal cancer was observed (OR = 2.63, 95% CI: 1.75–3.94), although there was significant heterogeneity between studies. A sensitivity analysis excluded one study; the association between TT genotype and esophageal cancer was still present, although of reduced magnitude (OR = 1.57, 95% CI: 0.96–2.56). A significant interaction between smoking and TT genotype on esophageal cancer risk was observed, while no interaction was observed between alcohol consumption and genotype.

Keywords: Methylenetetrahydrofolate reductase, Esophagus, Genetic epidemiology, case control studies

GENE AND GENE FUNCTION

The 5,10-methylenetetrahydrofolate reductase (MTHFR) gene contains a 2.0 kb coding region with 11 exons, and is located on chromosome 1p36.31,2. The gene product of MTHFR is a 77-kd protein of the same name (EC 1.5.1.20). Methylenetetrahydrofolate reductase is a key enzyme in folate metabolism that catalyzes the conversion of intracellular 5,10-methylenetetrahydrofolate (5,10-methylene THF) to 5-methyltetrahydrofolate (5-methyl THF), the predominant circulatory form of folate and primary methyl donor for the remethylation of homocysteine to methionine2–5 (figure 1). Methionine is a substrate for s-adenosylmethionine (SAM), which is the principal biological methyl group donor for the DNA methylation process5.

Figure 1.

Figure 1

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Simplified folate metabolic pathway showing the effects of alcohol and tobacco

MTHFR, FOLATE METABOLISM, AND CANCER RISK

The involvement of MTHFR in the folate metabolic pathway may affect cancer risk according to two primary mechanisms. Increased activity level of MTHFR enzyme facilitates the conversion of 5,10-methylene THF to 5-methyl THF. Low cytosolic levels of 5,10-methylene THF reduce the synthesis of deoxythymidylate monophosphate, and increase the deoxyuridylate monophosphate / deoxythymidylate monophosphate (dUMP/dTMP) ratio, thus inducing uracil misincorporation into DNA. This may result in point mutations and/or chromosomal breaks6. The second mechanism involves a decrease in available 5-methyl THF, which causes a decline in the conversion of homocysteine to methionine. This diminishes the availability of SAM, resulting in DNA hypomethylation, a process associated with carcinogenesis7,8. Folate deficiency leads to a reduction of both intra- and extracellular folate, and therefore is believed to increase cancer risk through both mechanisms described above9.

GENE VARIANT AND FREQUENCY

Approximately 60 polymorphisms, as well as 41 rare but deleterious mutations have been described in the MTHFR gene10. The most common functional variant and most studied to date is the thermolabile MTHFR C677T polymorphism (rs1801133). The C677T variant is a C to T transition in exon 4 at nucleotide 677, resulting in the conversion of alanine to valine at position 222 of the MTHFR amino acid sequence4,5. Heterozygotes (CT) and homozygotes (TT) for the C677T variant have respectively 65% and 30% of the MTHFR enzyme activity observed in homozygous wild type subjects (CC)11. As a result, TT homozygotes have been associated with lower serum folate levels and higher homocysteine levels than their wild type homozygous counterparts12–14.

The frequency of MTHFR C677T varies by ethnicity. The T allele frequency ranges from 0.25–0.44 in European Caucasians, 0.18–0.42 in Asians, and 0.12–0.24 in African Americans15. The prevalence of the TT genotype ranges from 8–18% in Caucasians, 11–16% in Asians, 21–25% in Latinos, and 0–6% for those of African descent16.

DISEASE

It is estimated that there were 15,650 new cases of esophageal cancer in the US in 2007, with 13,940 attributable deaths17, and a 16% 5-year survival rate. Esophageal cancer is the eighth most common cancer in the world, with approximately 462,000 cases and 386,000 deaths worldwide in 2002, making it the 6th most common cause of cancer--related deaths18.

There is substantial geographic variation in the occurrence of esophageal cancer, with nearly 20-fold difference in male incidence between areas at high-risk such as Eastern Asia and low-risk Western Africa19. Other regions with relatively high incidence and mortality include Eastern Africa, Southern Africa, South Central Asia, Northern Europe, and South America (males). Age-adjusted incidence rates in less-developed geographic regions are approximately double compared with those observed in more-developed regions, and age-adjusted mortality rates are 5 to 6-fold higher19. (Figure 2) The estimated age-adjusted 5-year survival rate in developed countries is 15% for males and 8% for females, while it is 17% and 16% respectively in developing countries18. This cancer occurs more frequently in men than women18. Globally, most esophageal cancers are squamous, and originate in the middle to lower third of the esophagus. However, there has been a recent increase in esophageal adenocarcinoma in Western countries, accounting for about 27% of esophageal cancers in men and 16% in women20. Esophageal cancer is associated with exposure to tobacco and alcohol consumption, gastric reflux or other chronic trauma, exposure to nitrosamines via food or environment, and a diet low in folate18,21.

Figure 2.

Figure 2

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Worldwide age-standardized mortality rates for esophageal cancer by gender64

GENE-ENVIRONMENT AND GENE-GENE INTERACTIONS

An interaction between folate status and the MTHFR C677T polymorphism has been described. Subjects who are homozygous for the MTHFR C677T variant and have a low folate status have been reported to have relative DNA hypomethylation compared with wild-type subjects, while no such effect is observed in subjects with high folate status22. Nutrients such as B6, B12, riboflavin, and methionine are involved in the folate metabolic pathway (figure 1). Flavin adenine dinucleotide (FAD), a phosphorylated form of riboflavin, acts as a cofactor for MTHFR. Increased levels of riboflavin significantly decrease serum homocysteine in subjects carrying the MTHFR 677 TT genotype but not in subjects with other MTHFR 677 genotypes23,24.

Tobacco smoke has been shown to transform folate and B12 co-enzymes into biologically inactive compounds25,26. Alcohol acts as a folate antagonist, resulting in malabsorption, increased folate excretion, or abnormalities in folate metabolism through the inhibition of methionine synthase (MTR)27,28. These effects are illustrated in figure 1. An interaction between MTHFR C677T and alcohol consumption has been reported in gastric and colon cancers; alcohol consumption has been associated with increased risk of gastric cancer in subjects with the MTHFR 677 TT genotype29,30; in heavy drinkers, TT genotype has been associated with higher risk of colon cancer compared with the wild type (CC), whereas TT genotype appears to have a protective effect in non-heavy drinkers31,32. However, the epidemiological data on MTHFR C677T and esophageal cancer do not seem to follow the prediction derived from experimental studies or from epidemiological findings from studies conducted on other gastrointestinal cancers. Yang33 reported a significant inverse trend for esophageal cancer across MTHFR 677 genotypes in heavy drinkers (CC > CT > TT; p = 0.024), while this was not observed in non-heavy drinkers (p = 0.46). Stolzenberg-Solomon29 reported no interaction between MTHFR C677T and esophageal cancer and alcohol use during the previous 12 months. Yang33 found no significant interaction between the MTHFR C677T polymorphism and smoking on esophageal cancer risk.

Few gene-gene interactions have been reported in the literature regarding MTHFR C677T and esophageal cancer. The interaction between the MTHFR C677T and A1298C polymorphisms was evaluated in a study on incident and prevalent esophageal cancer cases34, and reported a joint effect of 677 CT and 1298 CC genotypes on esophageal cancer risk, although no significant interaction was found. In a recent case-control study, Wang35 reported an interaction between MTHFR C677T and SHMT1 C1420T polymorphisms on esophageal squamous cell carcinoma; an increased cancer risk in individuals with wild-type SHMT and variant MTHFR TT genotypes was found.

OBJECTIVE

There have been mixed reports in the literature regarding the association of MTHFR C677T polymorphism with esophageal cancer29,33–39 (table 1). A recent meta--analysis of 7 publications on the association of MTHFR C677T and esophageal cancer3 observed significant heterogeneity among the studies, and therefore no summary estimate was reported. We conducted a pooled analysis to assess the overall effect of MTHFR C677T polymorphism on esophageal cancer risk and to test for possible gene-environment interactions with smoking and alcohol consumption.

Table 1.

Summary of published studies on the association between MTHFR C677T and esophageal cancer

Study Cases Controls Country Ethnicity Control
Source		 Crude ORCT
(95% CI)		 Crude ORTT
(95% CI)		 Crude ORCT+TT
(95% CI)		 Adj ORCT
(95% CI)		 Adj ORTT
(95% CI)		 Adj ORCT+TT
(95% CI)
Song34 * 240 360 China Asian Population 2.98 (1.87–4.75) 6.52 (3.89–10.92) 3.92 (2.51–6.11) 3.14 (1.94–5.08)a 6.18 (3.32–11.51)a Not Reported
Stolzenberg-Solomon29 * 129 398 China Asian Population 0.78 (0.45–1.37) 1.09 (0.61–1.96) 0.90 (0.53–1.52) 0.86 (0.48–1.54)b 1.24 (0.68–2.26)b Not Reported
Gao36 * 138 222 China Asian Population Not Reported Not Reported 1.25 (0.81–1.93) Not Reported Not Reported Not Reported
Zhang (Caucasian)39 241 256 Germany Caucasian Population 1.15 (0.79–1.68) 1.04 (0.59–1.82) 1.12 (0.78–1.61) Not Reported Not Reported 1.03 (0.46–2.33)a,e
Kureshi37 34 54 Pakistan Caucasian Population 0.97 (0.39–2.41) ---- 0.79 (0.33–1.93) Not Reported Not Reported Not Reported
Wang LD38 275 315 China Asian Population 1.07 (0.69–1.65) 1.76 (1.13–2.75) 1.35 (0.90–2.01) 0.96 (0.61–1.50)c 1.58 (0.99–2.50)c Not Reported
Yang33 * 165 493 Japan Asian Hospital 1.07 (0.73–1.56) 0.74 (0.42–1.30) 0.98 (0.68–1.41) 0.97 (0.63–1.49)d 0.66 (0.35–1.25)d Not Reported
Wang Y35, Zhang (Asian) 39^ 584 540 China Asian Population 1.83 (1.30–2.58) 2.16 (1.53–3.06) 1.98 (1.44–2.72) 1.81 (1.28–2.54)c 2.13 (1.50–3.02)c Not Reported
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Kureshi 2004 had 0 esophageal cancer cases with MTHFR 677 TT genotypes

a

Odds Ratio (OR) adjusted for age, gender, and smoking status

b

Reports Relative Risk (RR) adjusted for age, gender, smoking status, BMI, and alcohol status

c

Odds Ratio (OR) adjusted for age and gender

d

Odds Ratio (OR) adjusted for smoking status, alcohol status, and folate status

e

This study reported OR for CC using CT+TT as baseline for comparison, therefore the inverse of the OR was calculated

*

Included in pooled analysis. Pooled analysis also includes 1 unpublished case-control data set

^

Overlaps Asian subjects in Zhang39 study

DATA COLLECTION

The pooled-analysis on the association between MTHFR C677T and esophageal cancer was conducted using data available in the GSEC database40. Briefly, GSEC is an international collaborative initiative aimed at collection of published and unpublished data sets on metabolic polymorphisms and cancer risk.

A MEDLINE literature search for case-control studies published between 1966 and July 1, 2007 on the association of MTHFR C677T polymorphism and esophageal cancer was conducted using the search term: (mthfr OR methylenetetrahydrofolate reductase) AND (esophagus OR esophageal) AND (cancer OR carcinoma), yielding 24 results. Of those, 14 were excluded; two publications because they were a review41 and a meta-analysis3; six because they reported the effect of MTHFR C677T polymorphism on treatment outcomes42–47; two because they evaluated the association of MTHFR C677T polymorphism and gastric cancer48,49; one because it reported on MTHFR A1298C polymorphism50; and one because it was an update on the status of the HERPACC-II project51 and did not report case-control data. Two studies evaluated MTHFR promoter methylation, but did not report case-control MTHFR 677 genotype data52,53. After exclusions, 8 studies with unique case-control data sets were identified29,33–39. Authors of all relevant publications were contacted and their data was requested for inclusion in the GSEC database. Data sets were required to have case-control data for MTHFR C677T and esophageal cancer with MTHFR 677 genotypes in Hardy-Weinberg equilibrium to be included in the analysis54. We received data from 4 of the 8 case--control studies29,33,34,36 and from 1 unpublished case-control data set (Vasavi et al), for a total of 725 cases and 1,621 controls. A description of the pooled data set is provided in table 2.

Table 2.

Description of the data available in the GSEC pooled data set

Cases (n) Controls (n)
Age p = 0.56
    Mean (years) 57.3 (725) 57.6 (1531)
Gender p = 0.05
    Male 68.0% (493) 72.0% (1103)
    Female 32.0% (232) 28.0% (428)
Race p < 0.01
    Asian 92.8% (673) 96.7% (1481)
    Caucasian 7.2% (52) 3.3% (50)
Smoking Status p < 0.01
    Never-Smoker 38.2% (276) 45.2% (688)
    Ever-Smoker 61.8% (446) 54.8% (835)
Alcohol Use^ p < 0.001
    Never-Drinker 30.9% (149) 41.1% (479)
    Ever-Drinker 69.2% (334) 58.9% (687)
Histology*
    Squamous Cell 98.1% (579) -----
    Adenocarcinoma 1.9% (11) -----
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^

Alcohol data was unavailable for one of the five studies (Song et al34)

*

Histology data was unavailable for one of the five studies (Gao et al36)

STATISTICAL ANALYSIS

Adjusted odds ratios and 95% confidence intervals for MTHFR C677T were estimated using unconditional logistic regression, including study, age, gender, and smoking status as covariates in the model. Interactions were assessed between MTHFR 677 genotype and smoking status, and between MTHFR 677 genotype and alcohol use. For all included studies, smoking and alcohol status was derived from direct interview or self administered questionnaire data. No validation of smoking status by cotinine was available. Age and gender data was 100% complete; smoking data was complete in 99.5% of the subjects (11 missing); and alcohol data was available for 73.1% of subjects (607 missing, study by Song et al34). Heterogeneity was tested using a Q-statistic55 with p-values < 0.05, calculated using crude ORs from the individual studies. Egger’s56 and Begg’s57 tests were performed to evaluate publication bias among the pooled studies. Funnel plots were generated for a visual representation of potential publication bias (not shown). When heterogeneity between studies was observed, sensitivity analysis was performed by restricting the pooled analysis to those studies without significant heterogeneity, testing the robustness of the results.

Hardy-Weinberg equilibrium was verified on all pooled studies58 with exact methods using REAP v. 4.0 (University of Maine, Orono, ME). All other analyses were performed using Intercooled Stata 10 (Stata Corp, College Station, TX).

RESULTS

The results of the pooled analysis are summarized in table 3. There was a significant overall association between MTHFR 677 TT genotype (OR = 2.63, 95% CI: 1.75 – 3.94), but not MTHFR 677 CT genotype (OR = 1.22, 95% CI: 0.84–1.77) and esophageal cancer, with a significant trend across genotypes (CC < CT < TT; p < 0.01). However, significant heterogeneity was observed (CT: p < 0.01; TT: p < 0.01). No evidence of publication bias was found for the CT genotype (p = 0.57) nor for the TT genotype (p = 0.15).

Table 3.

Association between MTHFR 677 genotype and esophageal cancer in the GSEC pooled analysis

Genotype Cases Controls Adj OR (95% CI) Trend
Overall* p < 0.01
    CC 209 486 1.00 (Ref)
    CT 328 731 1.22 (0.84–1.77)
    TT 188 314 2.63 (1.75–3.94)
Never-Smokers # p < 0.01
    CC 77 221 1.00 (Ref)
    CT 106 325 1.23 (0.83–1.81)
    TT 93 142 2.57 (1.68–3.93)
Ever-Smokers # p = 0.16
    CC 130 264 1.00 (Ref)
    CT 221 400 1.25 (0.94–1.65)
    TT 95 171 1.26 (0.89–1.78)
Never-Drinkers* p = 0.65
    CC 58 142 1.00 (Ref)
    CT 51 216 0.81 (0.47–1.38)
    TT 40 121 1.10 (0.61–1.98)
Ever-Drinkers* p = 0.37
    CC 122 219 1.00 (Ref)
    CT 158 339 0.92 (0.67–1.25)
    TT 54 129 0.83 (0.55–1.25)
Squamous Cell Carcinoma* P < 0.01
    CC 157 486 1.00 (Ref)
    CT 260 731 1.36 (1.05–1.76)
    TT 162 314 2.00 (1.49–2.69)
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*

ORs are adjusted for study, age, race, gender, and smoking habit.

#

ORs are adjusted for study, age, race, and gender

The sensitivity analysis indicated that removal of one study34 from the analysis reduced the evidence of heterogeneity (CT: p = 0.47; TT: p = 0.60). The direction of the overall association of TT genotype with esophageal cancer was similar, although the magnitude of the association decreased (OR = 1.57, 95% CI: 0.96–2.56). No association was observed for CT genotype and esophageal cancer (OR = 0.88, 95% CI: 0.57–1.36). After performing sensitivity analysis there was still no evidence of publication bias for TT genotype (p = 0.96), but there was evidence of publication bias for CT genotype (p = 0.05).

The analysis was then restricted to squamous cell carcinoma (n = 579); similar results were observed for the TT genotype (OR = 2.00, 95% CI: 1.49–2.69), and the CT genotype (OR = 1.36, 95% CI: 1.05–1.76). There were insufficient cases to analyze the association of MTHFR C677T and esophageal adenocarcinoma.

A significant positive association between the MTHFR 677 TT genotype and esophageal cancer was observed in never smokers (OR = 2.57, 95% CI: 1.68–3.93), but not for the CT genotype (OR = 1.23, 95% CI: 0.83–1.81), with a positive trend across genotypes (CC < CT < TT; p < 0.01). Among ever-smokers, there was no association between the genotype and esophageal cancer (TT: OR = 1.26, 95% CI: 0.89–1.78; CT: OR = 1.25, 95% CI: 0.94–1.65; trend: p = 0.16). A significant interaction was identified between smoking and the MTHFR 677 genotype in both the full pooled analysis and in the analysis restricted to the homogeneous studies.

We further evaluated the effect of cumulative smoking dose, measured in average packs of cigarettes smoked per year (pack-years), on esophageal cancer risk according to the MTHFR 677 genotype (table 4). There was no effect of the genotype on the risk of the esophageal cancer associated with smoking dose.

Table 4.

Association between MTHFR 677 genotype and esophageal cancer by smoking dose in the GSEC pooled analysis

Genotype Cases Controls Adj OR (95% CI)
1st Tertile (≤ 20 pack-years)
    CC 19 63 1.00 (Ref)
    CT 61 99 2.39 (1.21–4.72)
    TT 33 72 1.37 (0.65–2.90)
2nd Tertile (21–39 pack-years)
    CC 33 71 1.00 (Ref)
    CT 63 114 1.19 (0.70–2.01)
    TT 23 40 1.24 (0.63–2.45)
3rd Tertile (≥ 40 pack-years)
    CC 30 84 1.00 (Ref)
    CT 54 122 1.27 (0.74–2.16)
    TT 22 33 1.81 (0.91–3.58)
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No significant interaction was found between alcohol consumption and the MTHFR 677 genotype. No significant three-way interaction between alcohol, smoking, and MTHFR 677 genotype was observed.

DISCUSSION

This pooled analysis, containing 725 cases and 1,531 controls from 5 studies, suggests an overall association between MTHFR 677 TT genotype and esophageal cancer, although with some heterogeneity between studies, and a non significant association between CT genotype and esophageal cancer. A recent meta-analysis by Larsson (2006)3 also examined the association between MTHFR C677T and esophageal cancer, but did not report a summary estimate due to significant heterogeneity between published studies. Meta-analyses and/or pooled-analyses examining the role of MTHFR C677T and other cancers have reported significant positive associations with gastric cancer, multiple myeloma, and breast cancer (postmenopausal women)15,59–61; while significant inverse associations have been reported for leukemia (adults) and colorectal cancer60. However, a recent meta-analysis reported no significant association for MTHFR C677T and lung cancer62.

Additionally, we report a significant negative interaction between smoking and MTHFR 677 genotype. The direction of the interaction is contrary to the expected effect, given that tobacco smoke has been reported to impair folate status through biological activation of folate and B12. Since low folate status has been shown to interact with MTHFR C677T 22, it would be expected that TT genotype would be associated with increased risk compared with CC genotype in smokers. We did not observe an interaction between MTHFR 677 genotype and alcohol. In a previous study, Yang et al33 observed no interaction between smoking and MTHFR C677T polymorphism on esophageal cancer risk, but reported an interaction between alcohol consumption and genotype, observing an inverse trend across genotypes (CC > CT > TT) in heavy drinkers. As with tobacco smoke, alcohol has been shown to act as a folate antagonist (figure 1). It is possible that individual folate levels may play a role in the interaction between alcohol, smoking of the MTHFR C677T polymorphism; unfortunately we did not have sufficient individual folate data for use as a covariate in the pooled analysis.

The validity of the present pooled analysis may be limited because the available data only includes Asian and a small proportion of Caucasian subjects. However, this is representative of the published literature, which contains 8 studies; 6 of which report on Asian populations29,33–36,38, 1 on Caucasians37, and 1 on both Asian and Caucasian subjects39. Currently, there is no literature evaluating the association between the MTHFR C677T polymorphism and esophageal cancer in races other than Asian and Caucasian, such as Latinos, Native Americans, Aboriginals, or people of African decent.

A strength of our analysis, and pooled analyses in general, is that the aggregation of data from multiple studies may afford better statistical power necessary to detect an association if one exists. The pooled analysis has advantages over meta-analysis63, allowing for adjustment for potential confounders, testing of interactions, and stratification. Thus, we were able to adjust for age, gender, race and smoking status; test for interactions between alcohol consumption, smoking, and MTHFR C677T; and stratify our results by smoking habit, which would not be possible with meta-analysis given the aggregate nature of the data available in the published studies.

In conclusion, we report an association of MTHFR 677 TT genotype and esophageal cancer, and an interaction between smoking and genotype. However, the folate metabolic pathway is very complex, and is mediated by a variety of gene products, folate, and nutrient cofactors. Future research on MTHFR C677T and esophageal cancer should be further targeted at the interactive effects of dietary and environmental factors, and gene-gene interactions. Large-scale studies are required to whether an interaction exists between MTHFR C677T and folate levels on esophageal cancer, and if this modifies the risk of esophageal cancer associated with both smoking and alcohol.

ACKNOWLEDGEMENTS

Environmental Cancer Risk, Nutrition and Individual Susceptibility (ECNIS); the authors thank Barbara M. Stadterman, GSEC administrator, for her support (National Institutes of Health grant P30CA047904-19S1; NIH/NCI SPORE in Head and Neck Cancer 5P50CA097190-02).

Footnotes

POTENTIAL CONFLICTS OF INTEREST

None declared

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