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Carcinogenesis Advance Access originally published online on April 29, 2007
Carcinogenesis 2007 28(8):1718-1725; doi:10.1093/carcin/bgm104
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© The Author 2007. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Impact of one-carbon metabolism-related gene polymorphisms on risk of lung cancer in Japan: a case–control study

Takeshi Suzuki1,2, Keitaro Matsuo1,6,*, Akio Hiraki1, Toshiko Saito1, Shigeki Sato2, Yasushi Yatabe3, Tetsuya Mitsudomi4, Toyoaki Hida5, Ryuzo Ueda2 and Kazuo Tajima1,6

1 Division of Epidemiology and Prevention, Aichi Cancer Center Research Institute, 1-1 Kanokoden, Chikusa-ku, Nagoya 464-8681, Japan
2 Department of Internal Medicine and Molecular Science, Nagoya City University Graduate School of Medical Science, 1 Kawasumi, Mizuho-cho, Mizuho-ku, Nagoya 467-8601, Japan
3 Department of Pathology and Molecular Diagnostics, Aichi Cancer Center Hospital, 1-1 Kanokoden, Chikusa-ku, Nagoya 464-8681, Japan
4 Department of Thoracic Surgery, Aichi Cancer Center Hospital, 1-1 Kanokoden, Chikusa-ku, Nagoya 464-8681, Japan
5 Department of Thoracic Oncology, Aichi Cancer Center Hospital, 1-1 Kanokoden, Chikusa-ku, Nagoya 464-8681, Japan
6 Department of Epidemiology, Nagoya University Graduate School of Medicine, 65 Tsuruma-cho, Showa-ku, Nagoya 466-8550, Japan

* To whom correspondence should be addressed. Tel: +81 52 762 6111; Fax: +81 52 763 5233; Email: kmatsuo{at}aichi-cc.jp


   Abstract
Top
 Abstract
Introduction
 Materials and methods
 Results
 Discussion
 References
 
There is substantial evidence that the decreased risk of lung cancer with high intake of vegetables and fruits is linked to folate as a specific nutrient. Functional polymorphisms in genes encoding one-carbon metabolism enzymes, methylenetetrahydrofolate reductase (MTHFR C677T and A1298C), methionine synthase (MTR A2756G), methionine synthase reductase (MTRR A66G) and thymidylate synthase, influence folate metabolism and thus might be suspected of impacting on lung cancer risk. We therefore conducted a case–control study with 515 lung cancer cases newly and histologically diagnosed and 1030 age- and sex-matched non-cancer controls to clarify associations with these five polymorphisms according to lung cancer subtype. Gene–environment interactions with smoking and drinking habit and folate consumption were also evaluated by logistic regression analysis. None of the polymorphisms showed any significant impact on lung cancer overall risk by genotype alone, but on histology-based analysis increase in MTHFR 677T and 1298C alleles was associated with reduced risk of squamous/small cell carcinoma (P = 0.029), especially among heavy smokers (P = 0.035), whereas the MTHFR 677TT genotype was linked to decreased risk for these subtypes among heavy drinkers (odds ratio = 0.17, 95% confidence interval: 0.03–0.98). In addition, we found interactions between the MTRR A66G polymorphism and smoking (P = 0.015) and the MTHFR A1298C polymorphism and alcohol consumption (P = 0.025) for risk of lung cancer overall. In conclusion, the results suggest that MTHFR polymorphisms contribute to risk of squamous/small cell carcinomas of the lung, along with possible interactions among folate metabolism-related polymorphisms and smoking/drinking habits. Further evaluation is warranted.

Abbreviations: CI, confidence interval; FFQ, food frequency questionnaire; 5,10-methylene THF, 5,10-methylenetetrahydrofolate; MTHFR, methylenetetrahydrofolate reductase; MTR, methionine synthase; MTRR, methionine synthase reductase; OR, odds ratio; PCR, polymerase chain reaction; 2R, two repeat; TS, thymidylate synthase; VNTR, variable number of tandem repeat


   Introduction
Top
 Abstract
 Introduction
Materials and methods
 Results
 Discussion
 References
 
Lung cancer, with its four major histological types (adenocarcinoma, squamous cell carcinoma, large cell carcinoma and small cell carcinoma), currently claims >55 000 lives annually in Japan and has become the leading cause of cancer death (1). Despite rapid advances in treatment over recent decades, the prognosis has not greatly improved. Therefore, efforts toward primary prevention in addition to early detection have come under the spotlight.

Many epidemiological studies have provided evidence that high consumption of vegetables and fruits is associated with a reduced risk of lung cancer (2–4). Folate is one of the constituents found in vegetables and fruits, and dietary folate may be one of the micronutrients that provide protection against lung carcinogenesis (5–7).

Biological functions of folate within so-called ‘one-carbon metabolism’ are to facilitate de novo deoxynucleoside triphosphate synthesis and to provide methyl groups required for intracellular methylation reactions. Folate deficiency is thought to increase the risk of cancer through impaired DNA repair synthesis and disruption of DNA methylation that may lead to proto-oncogene activation (8–10).

Methylenetetrahydrofolate reductase (MTHFR), methionine synthase (MTR), methionine synthase reductase (MTRR) and thymidylate synthase (TS) play important and interrelated roles in folate metabolism (Figure 1). The MTHFR reduces 5,10-methylenetetrahydrofolate (5,10-methylene THF) to 5-methyl THF, the primary circulating form of folate (11). The TS catalyzes the conversion of deoxyuridine monophosphate to deoxythymidine monophosphate using 5,10-methylene THF (12). The MTHFR product, 5-methyl THF, is the methyl group donor for the remethylation of homocysteine to methionine catalyzed by MTR (13). MTR activity is maintained by MTRR (14). Polymorphisms in the genes for MTHFR C677T and A1298C, MTR A2756G, MTRR A66G and TS 28 bp variable number of tandem repeat (VNTR) in the promoter region are known to have functional relevance (15). Thus, they might play roles in the etiology of lung cancer in combination with environmental factors such as folate consumption. Since information for this area of lung cancer is limited (16–22), we conducted the present case–control study, taking tobacco smoking, alcohol drinking and intake of folate into consideration.


Figure 1
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  		Fig. 1. Overview of folate metabolism. Enzymes with polymorphisms investigated in this study are boxed. THF, tetrahydrofolate; DHF, dihydrofolate; dUMP, deoxyuridine monophosphate; dTMP, deoxythymidine monophosphate; SAM, S-adenosylmethionine and SAH, S-adenosylhomocysteine.

 

   Materials and methods
Top
 Abstract
 Introduction
 Materials and methods
Results
 Discussion
 References
 
Subjects
The cases were 515 patients who were newly and histologically diagnosed as having lung cancer and not having any earlier history of cancer. Controls (n = 1030) were randomly selected and matched by age (±3 years) and sex to cases with a 1:2 case–control ratio from among the 2395 cancer-free individuals. All the subjects were recruited in the framework of the Hospital-based Epidemiologic Research Program at Aichi Cancer Center, as described elsewhere (23,24). In brief, information on lifestyle factors was collected using a self-administered questionnaire, checked by a trained interviewer, from all first-visit out-patients at Aichi Cancer Center Hospital aged 18–79 who were enrolled in Hospital-based Epidemiologic Research Program at Aichi Cancer Center between January 2001 and November 2005. Out-patients were also asked to provide blood samples. Each patient was asked about his or her lifestyle when healthy or before the current symptoms developed. Approximately 95% of eligible subjects complete the questionnaire and 60% provide blood samples. The data were loaded into a Hospital-based Epidemiologic Research Program at Aichi Cancer Center database and routinely linked with the hospital-based cancer registry system to update the data on cancer incidence. All participants gave written informed consent and the study was approved by Institutional Ethical Committee of Aichi Cancer Center.

Genotyping of MTHFR, MTR, MTRR and TS
DNA from each subject was extracted from the buffy coat fraction using BioRobot EZ1 and an EZ1 DNA Blood 350 ml Kit (Qiagen, Tokyo, Japan). The genotyping method was described in our previous reports with the polymerase chain reaction (PCR) TaqMan method using the GeneAmp PCR System 9700 or the 7500 Fast Real-Time PCR system (Applied Biosystems, Foster City, CA). Briefly, for the MTHFR C677T (dbSNP ID: rs677) and A1298C (rs1801131), as well as MTR A2756G (rs1805087) and MTRR A66G (rs1801394) polymorphisms, extracted DNA was amplified with validated probes (assay IDs: C__11975651_10, C__850486_20, C__12005959_10 and C__3068176_10, respectively; Applied Biosystems). The TS VNTR polymorphism was defined by PCR using 5'-CGTGGCTCCTGCGTTTCC-3' and 5'-GAGCCGGCCACAGGCAT-3' primers. In our laboratory, quality of genotyping is routinely assessed statistically using the Hardy–Weinberg test. When allelic distributions for controls depart from the Hardy–Weinberg frequency, genotyping is assessed using another method.

Intake assessment for folate and other nutrients
The consumption of folate and other nutrients was determined using a food frequency questionnaire (FFQ), described in detail elsewhere (25,26). Briefly, the FFQ consisted of 47 single food items with frequencies in the eight categories. We estimated the average daily intake of nutrients by multiplying the food intake (in grams) or serving size by the nutrient content per 100 g of food as listed in standard tables of food composition. Consumption of folate and other vitamins from supplements was not considered in total consumption because the questionnaire for multivitamins was not quantitative. Energy-adjusted intake of nutrients was calculated by the residual method (27). The FFQ was validated by referring to a 3-day weighed dietary record as a standard, which showed reproducibility and validity to be acceptable (28). The deattenuated correlation coefficients for energy-adjusted intakes of folate were 0.36 in men and 0.38 in women.

Consumption of tobacco and alcohol
Cumulative smoking dose was evaluated as pack-years, the product of the number of packs consumed per day and years of smoking. Smoking habit was entered for four categories of never, former and current smokers of <40 and ≥40 pack-years. Former smokers were defined as those who quit smoking at least 1 year before the survey. Consumption of each type of beverage (Japanese sake, beer, shochu, whiskey and wine) was determined by the average number of drinks per day, which was then converted into a Japanese sake (rice wine) equivalent. One drink equates to one ‘go’ (180 ml) of Japanese sake, which contains 23 g of ethanol, equivalent to one large bottle (633 ml) of beer, two shots (60 ml) of whiskey and two and a half glasses of wine (200 ml). One drink of ‘shochu’ (distilled spirit), which contains 25% ethanol, was rated as 108 ml. Total amount of alcohol consumption was estimated as the summarized amount of pure alcohol consumption (gram per drink) of Japanese sake, beer, shochu, whiskey and wine among current regular drinkers. Drinking habit was entered for four categories of never, former, current moderate and heavy drinkers. Heavy drinkers were defined as those currently drinking alcoholic beverages 5 days or more per week in a daily amount of 46 g (two Japanese drinks) or more, whereas moderate drinkers were defined as those currently consuming less frequently than 5 days/week, in lower amounts, or both. Former drinkers were defined as those who quit drinking at least 1 year before the survey. Former or current smokers and drinkers were categorized as ‘smokers’ and ‘drinkers’, respectively.

Statistical analysis
To assess the strength of the associations between polymorphic genes involved in folate metabolism and risk of lung cancer, odds ratio (ORs) with 95% confidence intervals (CIs) were estimated using age- and sex-matched conditional logistic models adjusted for potential confounders. For stratified and interaction analysis by smoking and drinking habit and folate intake, an unconditional logistic regression model was used because the matching was not retained after stratification by smoking and drinking habit and folate intake. Folate and other nutrient intakes were categorized into three groups as: first, second and third tertiles of dietary intake among controls. Potential confounders considered in the multivariate analyses were age, sex, smoking habit (never smokers, former smokers, current smokers of <40 or ≥40 pack-years), drinking habit (never drinkers, former drinkers, moderate drinkers or heavy drinkers), body mass index (<18.5, 18.5–24.9 or ≥25.0), total energy intake (as a continuous variable), dietary carotene intake (µg/day, tertiles), dietary vitamin C intake (mg/day, tertiles), dietary vitamin E intake (mg/day, tertiles), dietary folate intake (µg/day, tertiles), multivitamin use (at least once per week for 1 year or longer: yes or no) and referral pattern (patient’s discretion, family recommendation, referral from other clinics, secondary screening after primary screening or others). Missing values for each covariate were treated as an additional category in the variable and were included in the logistic model.

For the histology-based analysis, we combined squamous cell carcinoma and small cell carcinoma, because tumors of these subtypes were small in number and both are consistently more related with smoking as compared with adenocarcinomas. Considering potential effects of two polymorphisms (MTHFR C677T and MTHFR A1298C) on lung risk, we evaluated associations with their combined genotypes. Trend of genotype was assessed by score test applying score for each genotype (0, homozygous for reference allele or combined reference genotypes; 1, heterozygote or one reference genotype and 2, homozygous non-reference allele or non-reference genotype).

Gene–environment interactions between smoking and drinking habit and folate intake and genotypes in each polymorphism were evaluated under the multiplicative assumption. Products of scores for genotype (0, homozygous; 1, heterozygote and 2, homozygous or 0, referent alleles and 1, non-referent alleles) and smoking habit (0, non-smoker and 1, smoker), drinking habit (0, non-drinker and 1, drinker), folate intake (0, tertile 1 and 1, tertile 2 + 3) or combined smoking–drinking habit (0, non-smoker and non-drinker; 1, smoker and non-drinker or drinker and non-smoker and 2, smoker/drinker) were included as interaction terms. Differences in categorized demographic variables between the cases and controls were tested by the Chi-squared test. Mean values for age and total energy intake were compared for cases and controls by the Student’s t-test. Accordance with the Hardy–Weinberg equilibrium was checked for controls using the Chi-squared test and the exact P-value was used to assess any discrepancies between genotypes and allele frequencies. A P-value <0.05 was considered statistically significant. All analyses were performed using STATA version 9 (Stata Corp., College Station, TX).


   Results
Top
 Abstract
 Introduction
 Materials and methods
 Results
Discussion
 References
 
Data from 515 lung cancer cases, comprising 316 (61.4%) adenocarcinomas, 91 (17.7%) squamous cell carcinomas, 55 (10.7%) small cell carcinomas, 40 (7.8%) large cell carcinomas and 13 (2.5%) others, and 1030 controls were available for analysis. Table I shows the distribution of cases and controls by background characteristics. Age and sex were appropriately matched. Smoking habits differed to a large extent between cases and controls. The proportion of 40 pack-years or more current smokers in cases was significantly higher than controls. Heavy drinkers in the cases were significantly higher than for the controls. Among cases, the proportion of lower body mass index was higher, consistent with previous study (29). Total energy intake did not differ between cases and controls. Significant lower intake of dietary carotene was found among the cases. For other nutrients lower proportions of the highest intake group among the cases also were found, including for folate, but these were not statistically significant. With regard to referral pattern, referral from other clinics was frequent, whereas patient discretion and secondary screening after primary screening were less common among the case group than the control group.


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  		Table I. Characteristics of cases and controls

 
Table II shows genotype distributions for MTHFR, MTR, MTRR and TS and their ORs and 95% CIs for lung cancer risk according to histological subtypes. The genotype frequencies for all the polymorphisms were in accordance with the Hardy–Weinberg law in controls: MTHFR C677T (P = 0.17), MTHFR A1298C (P = 0.51), MTR A2756G (P = 0.17), MTRR A66G (P = 0.85) and TS VNTR (P = 0.51). On analysis of lung cancer overall, a slightly reduced risk was observed with the MTHFR 677TT genotype, but without statistical significance. The genotype frequencies for TS VNTR were quite varied; however, two repeat (2R) and three repeat alleles were dominant. The 2R/2R genotype showed decreased risk of lung cancer as compared with the non-2R homozygous, although again this was not significant. On subanalysis according to histological subtypes, the combination of MTHFR C677T and A1298C polymorphisms showed a significant decreased risk of squamous/small cell carcinoma among individuals with two or more MTHFR 677T and/or 1298C alleles (OR = 0.34, 95% CI: 0.13–0.92, trend P = 0.029), compared with those with MTHFR 677CC and 1298AA genotypes. In contrast, none of the polymorphisms showed any significant impact on adenocarcinoma risk.


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  		Table II. MTHFR, MTR, MTRR and TS genotype distributions, and ORs for lung cancer according to histology

 
To further evaluate the impact of MTHFR polymorphisms with regard to squamous/small cell carcinoma, we conducted stratified analysis by smoking and drinking habit (Table III). Among heavy drinkers, the MTHFR 677TT genotype conferred a significant decreased risk (OR = 0.17, 95% CI: 0.03–0.98, trend P = 0.041). A significant decreased risk among 40 pack-years or more current smokers was observed as number of MTHFR 677T or 1298C alleles increased (trend P = 0.035). No clear association was found for lung cancers overall or for adenocarcinomas in the stratified analysis (data not shown).


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  		Table III. Stratification analysis by smoking and drinking habit for the MTHFR polymorphisms in squamous/small cell carcinoma

 
Table IV shows data for the combinations of gene and environmental factors with reference to lung cancer overall risk. The interaction with smoking was significant for the MTRR A66G genotype (P = 0.015). Among non-smokers, risk was reduced with increase in the number of MTRR G alleles, whereas a trend for increased risk was observed among smokers. A significant interaction between drinking habits and the MTHFR A1298C genotype was found (P = 0.025). These two interactions were especially noteworthy for adenocarcinomas when histology-based analyses were conducted (data not shown). We were not able to analyze the smoking interaction for squamous/small cell due to insufficient number of non-smokers in this category. No obvious interaction was found between folate intake and the polymorphisms.


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  		Table IV. Interaction between MTHFR, MTR, MTRR and TS polymorphisms and smoking and drinking habit and folate intake for lung cancer risk

 
Considering the possible effects of both tobacco smoking and alcohol drinking on folate, we further examined the impact of four-way combinations of these two factors, folate intake and the polymorphisms on lung cancer risk (Table V). The MTRR A66G genotype showed a significant interaction among the subjects with tertiles 2 or 3 of folate intake (P = 0.023). The risk with the MTRR 66GG was consistently decreased among non-smoker/non-drinker subjects with adequate folate intake (OR = 0.20, 95% CI: 0.04–0.91).


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  		Table V. Impact of combination of smoking and drinking habit by folate intake and the polymorphisms on lung cancer risk

 

   Discussion
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
References
 
The present study showed a significant impact of MTHFR C677T and MTHFR A1298C in combination for risk of the most smoking related subtypes of lung cancer, squamous and small cell carcinomas. Moreover, this effect was prominent among heavy smokers. The MTHFR 677TT genotype was inversely associated with squamous/small cell carcinoma risk among heavy drinkers. In combination analysis of smoking, drinking and folate consumption, several potential gene–environment interactions were suggested, between (i) the MTRR A66G polymorphism and smoking and (ii) the MTHFR A1298C polymorphism and alcohol consumption.

High dietary intake of folate has been found to decrease the risk of lung cancer in several epidemiological studies (5–7). Although our result for folate did not reach statistically significance, the observed trend was accordant with other studies. Two small-sized clinical trials found folate and vitamin B12 supplementation to reverse atypia among patients with bronchial squamous metaplasia, a precursor of squamous cell carcinoma of the lung (30,31). One might therefore hypothesize a protective effect of folate on lung cancer, but there are also epidemiological studies providing no support for this concept (32–35). Considering the fact that functional polymorphisms in folate-related genes may contribute to alteration of folate metabolism (15), it is biologically plausible to hypothesize that the polymorphisms or the gene–environment interactions rather than the folate intake alone have the impact on lung cancer risk.

Hitherto, only a few studies have investigated associations between one-carbon metabolism-related gene polymorphisms and lung cancer risk. The MTHFR 677TT genotype has been reported to decrease risk of lung cancer in female Caucasians (20), but the results were inconsistent in other case–control studies (17,19). The MTHFR 1298CC and MTRR 66AG or GG genotypes were associated with significantly increased risk (20,21), whereas MTR and TS enhancer region polymorphisms in the Caucasians studies demonstrated no link (21,22). Our results of overall analysis added evidence for a null association in this controversial issue. However, of note in this study was the fact that MTHFR 677T and/or MTHFR 1298C alleles were associated with reduced risk of squamous/small cell carcinomas, especially among heavy smokers and drinkers. It has been shown that subjects with the MTHFR 677TT and MTHFR 1298CC genotypes have a reduction in enzyme activity compared with the wild-type homozygous, 677CC and 1298AA genotypes (36–38). This would lead to high 5,10-methylene THF concentrations, which may provide more one-carbon groups for thymidylate synthesis, thereby enhancing DNA synthesis and repair ability. Thus, it is biologically reasonable that individuals harboring the MTHFR 677T and MTHFR 1298C alleles among heavy smokers and drinkers have lower risk of squamous/small cell carcinoma development, given that carcinogenesis is strongly related with the accumulation of DNA damage. To our knowledge, this is the first indication of protective effects of combinations of MTHFR polymorphisms for this histologic subtype. These data provide support for the hypothesis of links between one-carbon metabolism and tobacco and alcohol influence on squamous/small cell carcinoma carcinogenesis. Regarding other body sites, our previous study on esophagus cancers, which are almost all squamous cell carcinomas in Japan, demonstrated that the MTHFR 677TT had the protective effects among heavy drinkers, consistent with the present study (39).

One difficulty exists in distinguishing effects of smoking and drinking on lung cancer risk. In the present study, of 33 heavy drinkers in squamous/small cell carcinoma cases, 24 (72.7%) cases were heavy smokers, so we may not conclude an independent protective effect of MTHFR 677TT genotypes among heavy drinkers, although adjustment for smoking habits was performed. On the other hand, all cases with squamous/small cell carcinomas were smokers except one and 60% (85/142) in this subtype were heavy smokers (40 pack-years or more). Alcohol drinking as well as tobacco smoking is considered to induce DNA damage and resultant modification of nucleotides (40,41). In addition, high intake of alcohol can lead to folate depletion (42). Therefore, it is within expectation that the MTHFR 677T allele, associated with high 5,10-methylene THF concentrations, may have the potential to protect against squamous/small cell carcinomas in tobacco consumers drinking large amounts of alcohol.

It was previously reported that lung cancer risk is higher with the MTRR 66AG/GG genotypes than the MTRR 66AA genotype among former smokers, but this did not extend to never and current smokers (21). Here, interaction between this gene and smoking habit was observed. Furthermore, the MTRR GG genotype exhibited a protective effect in low-risk subjects (non-smokers/non-drinkers with adequate folate intake). Several cytogenetic biomarker studies suggested that some polymorphisms involved in metabolic activation/deactivation or in DNA repair have been expected to be of special importance in modulating tobacco and alcohol carcinogen effects (43). A recent study reported a positive association with the modulating effect of the MTRR polymorphism on micronucleus frequency in peripheral blood lymphocytes, one of the cytogenetic markers (44), which is probably to increase by smoking (45) and drinking (46). The higher micronucleus frequency recorded in MTRR 66GG genotype with respect to AG or AA genotype is suggestive of a role of this polymorphism in modulation of chromosome stability, so that the findings may be consistent with our results. Further studies on the underlying mechanisms of the MTRR polymorphism thus appear warranted.

We found an interaction between the drinking habit and MTHFR A1298C polymorphisms for lung cancer risk, with decreased risk among non-drinkers. A Caucasian study showed that the MTHFR 1298CC genotype elevated risk among both drinkers and non-drinkers but only in women (20). The MTHFR A1298C is associated with decreased enzymatic activity (37,38) and would be expected to exert a similar effect to MTHFR C677T, with mutant alleles more protective among drinkers (27,39). There is no clear biological explanation for our results, and we cannot rule out the possibility that our observations for MTHFR A1298C were due to chance. Replication in a future study is needed.

Several potential limitations of the present study warrant consideration. First, internal validity of this hospital-based study is a potential threat to causal inference. We used non-cancer patients at our hospital as controls, given the likelihood that our cases arose within this population base, but individuals selected randomly from our control population were earlier shown to be similar to the general population in terms of the exposure of interest (47). Equivalence in the genotype distribution for the MTHFR C677T polymorphism between our controls and the general population has also been reported (48). To account for variation between cases and controls, we adjusted for referral pattern to our hospital. Second, as with other case–control studies, this study may suffer from recall bias. Although the questionnaires were completed before the diagnosis in our hospital, in some cases, patients referred from other institutions might have known the diagnosis. Third, we used a self-administered questionnaire to evaluate the nutrients intake, including folate. Data obtained from FFQ may not reflect intake as accurately as those from other methods, such as biological markers. We could not find any association with intake of vitamin C and E or folate for lung cancer risk, contrasting with our previous demonstration using the same population of protective effects of vegetables and fruits (4). The estimation of consumption by FFQ may be one possible explanation for this apparent anomaly. However, the reproducibility and validity of the FFQ were acceptable (28). We could not consider consumption of folate from supplements in total consumption, but the proportion of user with folate supplement is very low in Japan (0.1%) (49). Lastly, the limited number of cases, especially in subanalysis, is another factor and replication of our findings in a larger study is warranted.

In conclusion, we observed significant associations between MTHFR C677T and combined MTHFR C677T/A1298C polymorphisms and squamous/small cell carcinoma risk among heavy smokers and drinkers. Moreover, interactions between MTRR polymorphisms and smoking as well as the MTHFR A1298C polymorphism and alcohol consumption were also suggested. Our results thus support the hypothesis that folate metabolism-related gene polymorphisms may play a role in the genesis of lung cancer in combination with environmental factors. Replication in large epidemiological studies as well as studies of the mechanisms of the metabolisms is to be recommended.


   Acknowledgments
 
Authors are grateful to the staff of the Division of Epidemiology and Prevention at Aichi Cancer Center Research Institute for their assistance. The study was supported by a Grant-in-Aid for Scientific Research from the Ministry of Education, Science, Sports, Culture and Technology of Japan and by a Grant-in-Aid for the Third Term Comprehensive 10-Year Strategy for Cancer Control from the Ministry of Health, Labour and Welfare of Japan.

Conflict of Interest statement: None declared.


   References
Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 

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Received February 19, 2007; revised April 4, 2007; accepted April 21, 2007.


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