Methylenetetrahydrofolate reductase polymorphisms and interaction with smoking and alcohol consumption in lung cancer risk: a case-control study in a Japanese population

Lung cancer remains one of the major causes of mortality worldwide [1]. Although cigarette smoking is the primary risk factor for lung cancer, approximately one in 10 smokers develops lung cancer in their lifetime indicating an interindividual variation in susceptibility to tobacco smoke [2]. Other factors such as dietary factors may also play an important role in the etiology of lung cancer. Convincing evidence shows an inverse association between fruit and vegetable intake and lung cancer risk [3–5].

Genetic host factors have been implicated in some of the observed differences in susceptibility. To date, candidate susceptibility genes for lung cancer have been extensively studied, with most of the work focusing on mechanistically plausible polymorphisms in genes coding for enzymes involved in the activation, detoxification and repair of damage caused by tobacco smoke. In addition to metabolic polymorphisms, functional polymorphisms in folate metabolizing genes can also be good candidate susceptibility polymorphisms for lung cancer susceptibility. Folate, which is unsynthesizable by humans, is one of the major components of fruits and vegetables and may exert a beneficial impact on lung carcinogenesis [6]. Methylenetetrahydrofolate reductase (MTHFR), a key enzyme in folate metabolism, irreversibly catalyzes the conversion of 5,10-methylenetettrahydrofolate (5,10-methylene THF) to 5-methyltetrahydrofolate (5-methly THF). Two common functional MTHFR polymorphisms, C677T (rs1801133, A222V) and A1298C (rs1801131, E429A), have been the most studied. The TT genotype of the C677T polymorphism results in 30% enzyme activity in vitro compared with the CC genotype [7], whereas the CC genotype of the A1298C polymorphism has 60% enzyme activity of the AA genotype in vitro [8, 9]. Individuals with the genotypes involved in reduced enzyme activity had significantly increased homocysteine levels and decreased folate levels compared with individuals with their counterpart genotypes [10]. The importance of the MTHFR enzyme in cancer susceptibility arises from its involvement in two pathways of folate metabolism. 5,10-methylene THF is required for DNA synthesis and DNA repair, and 5-methyl THF is the methyl donor for regeneration of methionine from homocysteine for subsequent methylation reactions [11, 12]. Decrease in the activity of the MTHFR enzyme increases the pool of 5,10-methylene THF at the expense of the pool of 5-methyl THF (contributes to downstream methylation reactions by regeneration of methionine from homocysteine). Enhanced availability of 5,10-methylene THF in the DNA synthesis pathway reduces misincorporation of uracil into DNA, which might otherwise result in strand breaks during uracil excision repair, thus increasing the risk of chromosomal aberrations [11]. Therefore, it is probable that the decreased availability of 5-methyl THF for DNA methylation is the crucial mechanism behind the expected increased risk of lung cancer in subjects with the genotypes related to low MTHFR activity.

Lung cancer is a common disease that results from a complex interplay of genetic and environmental risk factors. It has been reported that there are lower circulating folate concentrations in smokers than in nonsmokers [13, 14] although a considerable portion of the effect of cigarette smoking on folate concentrations may be indirect (different intake of smokers and nonsmokers). Alcohol consumption has been shown to reduce folate bioavailability [15]. Smokers (established high risk population) or drinkers (suspected high risk population) with a genotype associated with decreased folate levels may be more susceptible to lung cancer than expected from the independent effects of the two (smoking/drinking and genetic) separate factors. As smoking and drinking may interact with the MTHFR genotypes to induce lung carcinogenesis, we conducted a case-control study of lung cancer in a Japanese population with special attention to the interaction between the MTHFR polymorphisms and either cigarette smoking or alcohol drinking.

The present study showed that the TT genotype of the MTHFR C677T polymorphism was significantly associated with an increased risk of lung cancer (OR = 2.27, 95% CI = 1.42 - 3.62, P < 0.01). Although the MTHFR A1298C polymorphism was not associated with lung cancer risk, there was a significant synergistic interaction between the A1298C polymorphism and alcohol consumption (P_interaciton = 0.049).

Among controls, the prevalences of the C allele of the C677T polymorphism and the A allele of the A1298C polymorphism were 64.1% and 79.1%, respectively (data not shown). According to the HapMap SNP database [21], the C allele frequency of the C677T polymorphism is most common among Yorubas (a West African ethnic group, 89.7%) and least common among Han Chinese (48.9%); Japanese (63.3%) and Caucasians (76.3%) have intermediate frequencies. The frequency of the C allele in our study was similar to the HapMap SNP database. Meanwhile, the A allele frequencies of the A1298C polymorphism among Caucasians, Han Chinese, Japanese and Yorubas were 64.2%, 80.0%, 82.2% and 89.2%, respectively, according to the HapMap SNP database [22]. The frequency of the A allele in our study was somewhat lower than the HapMap SNP database but similar to other Japanese populations [23–25] (79.0%, 78.4% and 78.9%).

As shown in Table 2, the TT genotype of the MTHFR C677T polymorphism was significantly associated with an increased risk of lung cancer (OR = 2.27, 95% CI = 1.42 - 3.62). Results in terms of the association between the C677T polymorphism and lung cancer yielded mixed, variously reporting an increased risk [26–31], a decreased risk [25, 32–35] or no association [36–39]. Two meta-analyses on the association between lung cancer and the MTHFR polymorphisms have been published in 2008 [40] and 2009 [41], respectively. The first meta-analysis [40] based on the published data from eight individual case-control studies [25–29, 32, 33, 37] reported that the summary OR for the TT genotype was 1.12 (95% CI = 0.97 -1.28) compared with the CC genotype. The second meta-analysis [41] reported that the summary OR for the TT genotype was 1.37 (95% CI = 1.02 - 1.84) when excluding studies conducted in the USA, where some common food items are regularly fortified with folate since 1998 [42]. Differences in ethnicity, dietary intake, exposure to environmental carcinogens and sample size may be responsible for the discrepancies in the results. It has been suggested that cancer risk associated with the MTHFR polymorphisms may be modulated by folate intake [43, 44]. Folate supplements (excessive) have been shown to increase folic acid levels, reduce homocysteine levels and then restore normal methionine levels, particularly in these individuals with the TT genotype of the MTHFR polymorphism [45, 46] although the mechanism remains unclear. Normal methionine levels may not induce aberrant DNA methylation [43]. When folate is excess, the C677T polymorphism may not affect lung cancer risk. Since Japanese drink several cups of green tea daily and consume substantial vegetables and fruits [47], folate insufficiency is rare. As the prevalence of folate supplement users is very low in Japan (0.1%) [48], excessive folate intake is also rare. When folate intake is sufficient, individuals with the TT genotype of the C677T polymorphism may have an increased risk of lung cancer, because decrease in MTHFR activity might lead to impairment of DNA methylation due to a reduction in the availability of 5-methyl THF.

The A1298C polymorphism was not associated with lung cancer risk in the present study. The A1298C polymorphism, which has been much less examined, was not associated with lung cancer risk in this study. Although the C677T and A1298C polymorphisms are in strong linkage disequilibrium, the different impacts of the two polymorphisms may be expected due to their location within the protein and subsequent effect on function. The meta-analysis [40] of seven studies [25–29, 33, 37] also showed no association between lung cancer and the A1298C polymorphism. Two [33, 38] of 10 studies [25–29, 31, 33, 34, 37, 38] showed a significant deleterious effect of the CC genotype of the A1298C polymorphism on lung cancer. The lower prevalence of the C allele of the A1298C polymorphism may make researchers less likely to detect a significant association. It is difficult to estimate accurately the combined effects of the C677T and A1298C polymorphisms on lung cancer risk in the current sample size. The TT genotype of the C677T polymorphism has a greater impact on enzyme function [7–9]. Moreover, several in vivo studies demonstrated an association between the TT genotype of the C677T polymorphism and increased total homocysteine plasma levels in healthy subjects while limited and inconsistent data on the role of the A1298C polymorphism as determinant of total homocysteine plasma levels are available [49, 50]. The C677T polymorphism may have confounded the results concerning the A1298C polymorphism. Further research on larger study populations is required before definitive conclusions can be drawn.

It is widely accepted that lung cancer development requires environmental factors acting on a genetically predisposed individual. Studying gene-environment interactions in relation to risk of lung cancer may be valuable because positive findings would clearly implicate the substrates with which the gene interacts as disease-causing exposures, clarify lung cancer etiology, and point to environmental modifications for disease prevention. We evaluated whether an interaction existed between cigarette smoking and the C677T or A1298C polymorphism (Tables 3 and 4). Interaction refers to the extent to which the joint effect of two risk factors on lung cancer differs from the independent effects of each of the factors. Two risk factors (MTHFR genotype and smoking/drinking) may act independently or interact thereby increasing or decreasing the effect of one another. A gene-environment interaction was suggested, with a combination of the TT genotype and smoking conferring significantly higher risk (OR = 6.16, 95% CI = 3.48 - 10.9), compared with at least one C allele and no history of smoking. Smoking (OR = 3.17) and the TT genotype of the C677T polymorphism (OR = 2.00) act independently (3.17*2.00 = 6.34 ≈ 6.16). There was no significant interaction between smoking and the C677T polymorphism with lung cancer. There was also no significant effect modification by the A1298C polymorphism in the association of smoking and lung cancer (P_interaction = 0.11). However, the risk estimates indicated that smoking and the "at-risk" genotype did not seem to be associated with increased risk among those already at increased risk due to one of the two factors (smoking/"at-risk" genotype). Eight studies examined the interaction between smoking and the C677T or A1298C polymorphism [25, 31–35, 38, 51]. Differences in the direction and the strength of the interaction were observed. The difference in circulating folate levels between different populations may partly account for the findings [29, 52]. Furthermore, the differences may indicate a difference in susceptibility among different populations to smoking-induced lung cancer.

We evaluated the interaction between the C677T or A1298C polymorphism and alcohol drinking (Tables 3 and 4). Drinking (OR = 1.76) and the TT genotype of the C677T polymorphism (OR = 2.00) act independently (1.76*2.00 = 3.52 ≈ 3.09). No interaction of drinking and the C677T polymorphism with lung cancer was detected while we found a significant interaction between drinking and the A1298C polymorphism (P_interaction = 0.049). Namely, excessive drinking (OR = 1.76) with the CC genotype (OR = 1.57) had an unexpectedly increased risk of lung cancer (1.76 *1.57 = 2.76 «7.23). As alcohol interferes with folate absorption and usage [53, 54] and serves as a methyl group antagonist [55], it is biologically plausible. Four studies examined the interaction between drinking and the C677T polymorphism [25, 29, 33, 36] or A1298C polymorphism [25, 33]. Statistically significant interaction between the A1298C polymorphism and alcohol consumption was observed in women (P_interaction = 0.021) but not in men (P_interaction = 0.569) [33]. Similarly, a significant interaction between drinking habits and the A1298C genotype was found (P_interaction = 0.025) but the CC genotype of the A1298C polymorphism was associated with a lower lung cancer risk (OR = 0.36, 95% CI = 0.12 - 1.04) [25]. Findings from gene-environment interaction analyses must be interpreted with caution due to reduced numbers of observations in the subgroups. Replication of findings is very important before any causal inference can be drawn. Testing replication in different populations is an important step.

Several limitations of this study warrant mention. Our study may have included a bias due to the self-reporting of smoking habits and alcohol consumption (misclassification bias). However, discrepancies between self-reported smoking habits and biochemical verification are minimal among the general population [56, 57]. Similarly, the validity of self-reports on alcohol consumption is generally high [58, 59]. Recall bias is one of well-recognized potential problems in case-control studies. Lung cancer patients may remember their exposure with a higher (lower) accuracy or completeness than healthy controls do (recall bias). To use hospital control is one way of minimizing the impact of recall bias because the controls would generally have the same incentive as the cases to remember events in the past [60]. Case-control studies tend to susceptible to selection bias, particularly in the control group. Controls should represent the source population from which the cases were drawn. As lung cancer patients are more likely to be more male sex and be less educated compared to controls [61], our results on sex ratio and educational status might not always suggest the existence of selection bias. When using hospital-based cases, it may not be possible to define the population which the cases were drawn. Hospital controls may be more appropriate because the study population can be defined as potential hospital users [60]. Generally, the reported participation rates were slightly higher in hospital-based case-control studies than in population-based case-control studies [62]. Participation rates of cases and controls were very high in this study. High participation rate may reduce the possibility of selection bias [62]. However, as the possibility of recall and selection biases could not be completely excluded in case-control studies, our findings should be interpreted with caution. Finally, we did not have data on dietary folate intake. Since smoking is associated with decreased circulating folate levels due to low folate intake [63, 64], the observed interaction between alcohol consumption and the A1298 C polymorphism may be distorted by residual confounding by smoking. Additional studies are warranted to replicate our and others' findings from case-control genetic association studies.

The TT genotype of the C677T polymorphism was significantly associated with an increased risk of lung cancer. Although the A1298C polymorphism was not associated with lung cancer, a significant interaction with drinking was observed. Smoking interacted with neither the C677T nor A1298C polymorphism in the development of lung cancer. Our results should be interpreted cautiously because we did not have data on dietary folate intake. Future studies involving larger control and case populations and better exposure histories will undoubtedly lead to a more thorough understanding of the role of MTHFR in lung cancer development.