Introduction

Lung Cancer (LC) has become the most common type of cancer worldwide with the increase in smoking habit [1]. It is responsible for 12.8% of the cancer cases and 17.8% of cancer related deaths worldwide [2]. Just in 2001, LC caused more than one million deaths [1, 3]. Although smoking is the primary risk factor for lung cancer, LC develops in less than 20% of people who smoke throughout their life [4]. This shows that genetic susceptibility and nutritional habits of an individual increase the risk of LC [5, 6]. The risk of lung cancer development is increased by active and passive smoking, carcinogens in polluted air, carcinogen activation by various occupational exposures as well as by individual genetic factors [7]. The genes coding some enzymes like polycyclic aromatic hydrocarbons and aromatic amines, which act in the detoxification and metabolic activation of pulmonary carcinogens in humans, are known to be polymorphic [8].

Numerous epidemiological studies have pointed out that low dietary folate intake is an important factor in development of cancer including lung, breast, colorectum, bladder and pancreas [9–13]. Folate is involved in DNA methylation, synthesis, and repair [14]. In all of these events, folate serves as one-carbon donor and its metabolic product provides methyl group for both synthesis of methionine and DNA methylation [15]. Folate deficiency may cause uracil misincorporation and subsequent DNA instability, retarded DNA repair capacity for oxidative or alkylating damage, and global and proto-oncogenic DNA hypomethylation [14].

MTHFR is an FAD-dependent enzyme, which catalyzes the irreversible conversion of 5,10 methylenetetrahydrofolate to 5-methyltetrahydrofolate. Five-Methyltetrahydrofolate in turn serves as a methyl donor in the remethylation of homocysteine (Hcy) to methionine. The enzyme, therefore, resides at an important metabolic branch point directing the folate pool towards Hcy remethylation and DNA methylation at the expense of DNA and RNA biosynthesis [16]. There are two well-described polymorphisms in the MTHFR gene: A1298C and C677T. The C677T polymorphism occurs in exon 4 and results in alanine to valine substitution at codon 222 [17]. It has been shown that individuals who have homozygous mutation at this codon lose MTHFR enzyme activity approximately by 70% [18]. A1298C polymorphism resides in exon 7 of MTHFR gene and results in glutamate-to-alanine substitution at codon 429 [19].

In this study, the relation between MTHFR Gene Polymorphism and LC risk and the frequency of this polymorphism were investigated in the population of Central Anatolia.

Study population

The study was approved by the Ethics Committee of Medical faculty of Cumhuriyet University, Sivas, Turkey, and written informed consent was obtained from all individuals or their legal guardians. This study involved 64 patients whose definitive diagnosis of LC was made in our thoracic diseases clinic between 2007 and 2009. The patients were genotyped and were compared histologically in terms of small-cell carcinoma (SCLC) and non-small cell carcinoma (NSCLC). A control group consisting of 61 people were formed. The age, gender, smoking habit, body mass index (BMI), alcohol consumption of the patient and control groups was recorded. The patients were divided into 3 groups according to their smoking habits as: none smokers (NS), who never smoked throughout their life, ex-smokers (ES) who smoked before but stopped smoking for at least the last one year, active smokers (AS) who started smoking before and continued smoking within the last one year. Active smokers (AS) and ex-smokers (ES) were included in the ever smoking group. Smoking period was calculated as pack-years of smoking. The patients were then allocated into two groups in terms of alcohol consumption habits: “ever drinkers” who used alcohol at least once a week for one year or more up until now and “never drinkers” as the rest of the patients.

Mutation analysis

Total genomic DNA was extracted from the peripheral blood samples using DNA isolation kit according to manufacturer’s instruction (Invitek Invisorb Spin Blood Kit, Germany). Isolated DNA samples were screened for two MTHFR gene polymorphism (C677T and A1298C) using reverse hybridization strip assay (Vienna Lab, CVD Strip Assay, GMBH, Austria). Relevant gene sequences were simultaneously in vitro amplified and biotin labeled in a single (multiplex) amplification reaction. PCR conditions were as follows: pre-PCR, 2 min at 95°C; denaturation, 15 s at 95°C; annealing 30 s at 56°C; extension 30 s at 72°C, and final extension 3 min at 72°C. The amplification products were selectively hybridized to a test strip, which contains allele-specific (wild type and mutant) oligonucleotide probes immobilized as an array of parallel lines. Bound biotinylated sequences were detected using streptavidin-alkaline phosphatise and color substrates. Hybridization was performed in an automated incubator (Auto-LIPA, İnnogenetics).

Statistical analysis

Statistical analysis was performed using SPSS 14.0 program (SPSS Inc., Chicago, IL, USA). Student’s t test was performed for BMI, pack-year of smoking, and mean age. χ2 test was used to evaluate alcohol consumption, gender differences, allele and genotype frequency. The odds ratios (OR) for relation between genotypes and the risk in the formation of LC were calculated at 95% confidence interval, and P < 0.05 was considered statistically significant.

Results

The patient group consisted of 54 male (84%) and 10 female (16%) subjects. The mean age of the patients was 60.8 ± 9.0 years (Range: 32–77 years). The control group consisted of 52 male (85%) and 9 female (15%) subjects. The mean age of the control group subjects was 58.7 ± 8.7 years (Range: 28–75 years). No statistically significant differences were found between the age, gender, and alcohol intake of the patient and control groups. Pack-year of smoking of the patients (32.83 ± 13.5 year) was higher than that of the control group (24.3 ± 11.5 year) (P < 0.001). Body mass index (BMI) of the patients (23.1 ± 5.5) was lower than BMI of the control group (P < 0.001). The characteristics of the patients and the control group are shown in Table 1.

Table 1 The characteristics of cancer patients and the control group
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Distribution of MTHFR variant allele and genotypes in the patient and control groups is presented in Table 2. While MTHFR 677T and 677C allele frequencies were 0.33 and 0.67 in cancer patients respectively, they were 0.29 and 0.71 in the control group. Similarly, while MTHFR 1298C and 1298A frequencies were 0.33 and 0.67 in cancer patients, they were 0.31 and 0.69 in the control group respectively. There were no differences between the patient and control groups for these variant alleles (For C677T, P = 0.445 and for A1298C, P = 0.841). When 677TT genotype of MTHFR was compared to its 677CC genotype, a 2.4 times higher risk of LC was determined with 677TT genotype (Table 2). Similarly, when MTHFR homozygote mutant MTHFR 1298CC genotype was compared to wild type 1298AA genotype, there was a 1.5 times higher risk of LC (Table 2). When genotype frequencies were evaluated for genders and smoking status, 677TT genotype of MTHFR presented 3 times higher risk of LC in smokers, whereas the risk was 1.3 times in 1298CC genotype (Table 3). Similarly, while 677TT genotype MTHFR showed 3 times higher risk of LC in men, the risk was 1.6 times higher in 1298CC genotype (Table 3).

Table 2 The distribution of the MTHFR C677T and ve A1298C genotype and frequency of the allele in the patient and control groups
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Table 3 The association of AC CA and MTHRF polymorphism with gender and smoking status
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When genotype frequencies of NSCLC and SCLC histological types were evaluated, it was determined that MTHFR 677CT and 677TT genotypes were 2.1 and 3 times higher respectively in the NSCLC group compared to the SCLC group (Table 4). The frequencies of the 1298AC and 1298CC genotypes of MTHFR were 2.6 and 2.4 times higher in NSCLC group compared to the SCLC group (Table 4).

Table 4 The association between histological type and MTHRF gene polymorphism
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Discussion

To the best of our knowledge, this study, which evaluated the relation between MTHFR gene polymorphism and risk of LC development in 64 patients and 61 control subjects from Sivas, the Central Anatolia Region of Turkey, is the first of its kind in Turkey.

Enzyme activity has been shown to decrease in people with 677TT genotype [18]. MTHFR gene presents wide-spread polymorphism in two different regions. From the exon 4, 222nd codon belonging to the gene region, normally alanin aminoacid is synthesized, but during this process, missence mutation that develops in the form of leads to amionoacid synthesis rather than valin alanin synthesis. This leads to a reduction in the enzyme activity. Reduction in the MTHFR enzymatic activity may increase the risk of cancer development as a result of the change in the DNA methylation associated with reduced level of 5-methyltetrahydrofolate. In conformity with this hypothesis, in people with 677TT genotype, higher risk of breast and esophagus cancer has been reported [20–22]. Few studies have evaluated the relationship between MTHFR C677T polymorphism and LC risk. Siemianowicz et al. [23], in their case-control study on 146 patients and 44 controls, determined a significant relationship between 677TT genotype and LC risk. However, in the study by Shen et al. [24] on 550 patients and 554 controls, no significant relationship was determined between C677T polymorphism and LC risk. In another study with 59 patients and 232 controls, no relationship was found between 677TT genotype and LC risk (OR, 0.71; 95% CI, 0.39–1.30) [25]. In our study, in subjects with 677TT genotype, the risk of LC was 2.4 times higher than the risk in the control subjects. However, this increase was not statistically significant.

MTHFR A1298C polymorphism leads to the reduction of enzyme activity by replacement of glutamic acid with alanine in the 7th exon 429th codon [19]. In two earlier studies [23, 24], no significant relationship was found between 1289T and TT polymorphism and LC risk. Qiuling et al. [2] found no statistically significant relationships although MTHFR 1298C and CC genotypes had a higher risk of LC. In our study, MTHFR1298CC genotype presented lower rate of risk for LC than MTHFR 677TT genotype, but a higher rate of risk than MTHFR 1298AA genotype. The difference, however, was not statistically significant.

The degree of smoking-associated DNA damage in the tumor and adjacent tissues of females has been reported to be higher than that of both smoking and non-smoking males [28–30]. In one study, it was suggested that women tended to have less capacity for DNA repair than men [12]. These findings suggest that intake of food that enhance the capacity for DNA repair and synthesis may prove more beneficial for women than men. Qiuling et al. [26], in their case-controlled study, evaluated the relationship between gender and MTHFR polymorphism and found that 1289CC genotype showed a higher risk for LC (OR, 2.09; 95% CI, 1.32–3.29), whereas 677TT genotype had a lower risk for LC (OR, 0.60; 95% CI, 0.40–0.92). These rates were not statistically significant in males (for 1289CC: OR, 0.95; 95% CI, 0.62–1.45; for 677TT: OR, 1.09: 95% CI, 0.72–1.65). In the same study, there was a higher rate of 1298CC in smoking females (OR, 2.25; CI, 1.19–4.23), while it was lower in smoking males (OR;0.44: CI, 0.20–0.95). Suzuki et al. [27] determined no significant relationships between 1298CC and 677TT genotypes and LC risk in smoking individuals. In our study, although the rates of 1298CC and 677TT genotypes were higher in the male LC cases, the differences were not statistically significant (For 1298CC: OR, 3.05:CI, 0.52–10.68 and for 677CC: OR, 1.64: CI, 0.32–9.14). The rates of these two genotypes in females were the same, and the differences were not statistically significant (for both genotypes: OR, 0.95: CI, 0.03–35.77). However, this may have been due to the low number of female cases included in the study. In our study, the rates of 1298CC and 677TT genotypes were higher in smoking individuals and LC cases. However, the difference was not statistically significant.

Literature presents few studies that have evaluated the relationship between NSCLC and SCLC and MTHFR gene polymorphism. Siemianowicz et al. [23] determined a significant relationship between 677TT genotype and risk of NSCLC development. In another study [27], it was determined that those with MTHFR 677T and 1298C alleles had lower risk of squamous/small cell carcinoma (P = 0.029). In our study, the rates of 677T and 677TT alleles were 2.13 and 3 times higher in the NSCLC cases than in the SCLC cases. However, the differences were not statistically significant. Similarly, although the rates of 1298C and 1298CC genotypes were 2.6 and 2.4 times higher in the NSCLC group, the differences were not statistically significant.

The values of the present study for some parameters were either higher of different from those of the control group, but the differences were not statistically significant. This may have been associated with the limited number of patients. Another drawback of our study was the lack of reliable information provided by the patients on their nutritional habits and lack of sufficient diet analysis for folate intake. Studies with larger series are needed to confirm our findings for our country.

The distribution of alleles in 61 individuals with no cancer were as follows MTHFR 677CC genotype: 48%, CT genotype: 48%, and TT genotype: 5%. These rates have been reported to be as follows in different regions of the world: for Canada, 43, +6, and 11% respectively in a control group of 260 individuals [31]; for Texas: 44, 46, and 11% respectively in a control group of 1141 individuals [26]; for California: 45, 43, and 12% respectively in a control group of 1964 individuals [32]; and for Japan: 37, 46, and 17% respectively in a control group of 1030 individuals [27].

According to our results, the rates of TT genotypes were less than half those of the other reported series, while the rates for the other genotypes were similar to those reported in the literature. In our study, the distribution of MTHFR 1298AA, AC, and CC genotypes were 46, 48, and 75% respectively. In the study from Texas [26], these rates were found to be 49, 44, and 8%; from California (33), 47, 42, and 11%; from Japan [27], 63, 31, and 4%; from Italy [33], 49, 43, and 8%. The data collected in this study from the city of Sivas vicinity in the Mid-Anatolia region of Turkey were compatible with the literature.

In conclusion, the rates of 677TT and 1298CC alleles were higher in those with LC, and the rates for smokers were 3.05 and 1.29 times; for males, 3.05 and 1.64 times, and for those with NSCLC, 3.0 and 2.44 times higher. The distribution of the alleles was similar to those reported for different parts of the world. However, further studies with larger series of Turkish cases are needed to confirm our results.