Folate Pathway Gene MTHFR C677T Polymorphism and Risk of Lung Cancer in Asian Populations

Lung cancer has been the most common cancer in the world for several decades and there were an estimated 1.61 million new cases, representing 12.7% of all new cancers. It was also the most common cause of death from cancer, with 1.38 million deaths (18.2% of the total). The majority of the cases now occur in the developing countries (55%), a large increase since the estimates in 1980, when it was estimated that only 31% of lung cancer cases occurred in developing countries (Parkin et al., 2005; Ferlay et al., 2010). 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. Folate and methionine metabolism plays crucial roles in DNA synthesis and methylation (Sharp and Little, 2004). 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 (Duthie, 1999; Kim, 1999). Methylenetetrahydrofolate reductase (MTHFR) is the key enzyme in folate metabolism, and functional polymorphisms in the MTHFR gene have been seen with a highly variable prevalence in different ethnic populations and geographical areas (Sharp and Little, 2004). The human MTHFR gene contains 11 exons, located on chromosome 1p36.3 (Goyette et al., 1994, 1998), and encodes methylenetetrahydrofolate reductase


Introduction
Lung cancer has been the most common cancer in the world for several decades and there were an estimated 1.61 million new cases, representing 12.7% of all new cancers. It was also the most common cause of death from cancer, with 1.38 million deaths (18.2% of the total). The majority of the cases now occur in the developing countries (55%), a large increase since the estimates in 1980, when it was estimated that only 31% of lung cancer cases occurred in developing countries (Parkin et al., 2005;Ferlay et al., 2010). 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. Folate and methionine metabolism plays crucial roles in DNA synthesis and methylation (Sharp and Little, 2004). 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 (Duthie, 1999;Kim, 1999).
Methylenetetrahydrofolate reductase (MTHFR) is the key enzyme in folate metabolism, and functional polymorphisms in the MTHFR gene have been seen with a highly variable prevalence in different ethnic populations and geographical areas (Sharp and Little,

Vandana Rai
(MTHFR) a key enzyme in folate and homocysteine metabolism. MTHFR catalyzes the biologically irreversible reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, which provides the methyl group for the remethylation of homocysteine to methionine (Bailey and Gregory, 1999). The MTHFR enzyme is constituted by dimers in humans, each of which contains an N-terminal catalytic domain and a C-terminal regulatory domain. The monomer arrangement might be head to tail, with each catalytic domain flanked by a regulatory domain. Two common polymorphisms in the MTHFR gene have been described: C677T (A222V) in exon 4 and A1298C (E429A) in exon 7 (Frosst et al., 1995;Weisberg et al., 1998). The frequency of the MTHFR 677T allele varies in different ethnic and regional world populations for example, the allele frequency is 0.07 is Sub-Saharan Africans and 0.06 in Canadian Inuit, whereas in Asians the allele frequencies are 0.04-0.54 (Hegele et al., 1997;Pepe et al., 1998;Rai et al., 2010;. MTHFR functions in dimeric form and FAD works as a co-factor, but variant MTHFR (222V) dissociates into monomers and its enzymatic activity reduces. It is established by docking study that the mutant enzyme (222V) shows less affinity towards FAD than the wild enzyme (222A) (Yadav et al., 2011). The C677T polymorphism has been proven to affect the enzymatic activity and homocysteine level. Considering that methylation abnormalities appear to be important for the pathogenesis of many cancer types, many authors have examined the association between the genotype of the MTHFR C677T polymorphism with various cancers. Case-control studies investigating the association between the MTHFR C677T polymorphism and lung cancer have given controversial results. However, the results of these studies remain conflicting rather than conclusive. In consideration of the extensive role of MTHFR, a meta-analysis of all eligible case-control studies was carried out to estimate the overall lung cancer risk of these two polymorphisms and to quantify the potential between-study heterogeneity.

Search criteria
For electronic searches, the comprehensive search strategy was used to find eligible studies for present meta-analysis. Published studies were searched through PubMed, Google scholar, Elsevier and Springer Link databases, using following keywords "MTHFR", "methylenetetrahydrofolate reductase", and "Lung Cancer", in combination with "C677T", "mutation", "polymorphism". Furthermore, references of all relevant articles were retrieved to search for additional eligible studies.

Inclusion and exclusion criteria
The studies meeting the following criteria were included: (1) concerning the relationship between MTHFR C677T polymorphism and lung cancer; (2) case-control studies from Asian population; (3) providing complete data of cases and controls for calculating odd ratio (OR) with 95% confidence interval (CI); and (4) the distribution of the genotypes in control groups should be in Hardy-Weinberg equilibrium (HWE). The studies not reported the genotype/ allele numbers were excluded.

Data extraction
Following data were collected from included studies: first author name, year of publication, journal name, ethnicity, numbers of genotyped cases and controls. If studies contained overlapping cases and/or controls, the largest study was preferred.

Statistical analysis
For the control group of each study, the observed genotype frequencies of the MTHFR C677T polymorphism was assessed for Hardy-Weinberg equilibrium (HWE) using the x 2 test. The strength of association was accessed odds ratios (ORs) and 95% confidence intervals (CIs). The pooled ORs were performed for allele contrast (T vs C), codominant (CT vs CC), homozygote (TT vs CC), dominant (TT+CT vs CC), and recessive (TT vs CT+CC and CC) models. Heterogeneity assumption was evaluated by a chi-square based Q-test. A p-value of >0.05 for the Q-test indicated a lack of heterogeneity among the studies, the summary OR estimate was calculated by the fixed or random effects models (Mantel and Haenszel,1959;DerSimonian and Laird,1986). Heterogeneity was quantified with the I 2 metric (I 2 =(Q-df)/Q), which is independent of the number of studies in the meta-analysis. I 2 takes values of between 0 and 100%, with higher values denoting a greater degree of heterogeneity (Zintzaras and Hadjigeorgiou 2005;Zintzaras and Ioannidis, 2005;Zintzaras, 2007). Random effects modeling assume a genuine diversity in the results of various studies, and it incorporates a between-study variance into the calculations. Hence, when there is heterogeneity between studies then the pooled OR is preferably estimated using the RE model (Whitehead 2002).

Publication bias
The potential for publication bias was examined by a Begg's test (funnel plot method) and Egger's linear regression test (Begg and Mazumdar, 1994;Egger et al., 1997). p<0.05 was considered representative of statistical significance. All analyses were performed using the computer program MIX version 1.7 (Bax et al., 2006). A p value less than 0.05 was considered statistically significant, and all the p values were two sided.

Summary statistics
In all sixteen studies, total cases were 9,468 with CC  (3441), CT (4404) and TT (1623), and controls were 9,078 with CC (3,600), CT (4,115), and TT (1,363) genotypes. The number of cases varied from 59 to 3,939, with a mean of 676, and the numbers of controls varied from 78 to 2,803, with a mean of 648 (Table 2). In controls genotypes, percentage of CC, CT and TT were 39.66%, 45.33%, and 15.01% respectively. In total cases, genotype percentage of CC, CT, and TT was 36.34%, 46.15% and 17.14% respectively. Frequencies of CC and CT genotypes were highest in both cases and controls (Table 2). In cases and controls, the allele C was the most common. In seven studies OR is above one (Shen et al., 2005;Zhang et al., 2005;Hung et al., 2007;Yao et al., 2010;Cui et al., 2011;Kiyohara et al., 2011;Cheng et al., 2012).

Meta-analysis
An association was detected between the MTHFR C677T polymorphism and the susceptibility to lung cancer in Asian population in all the genetic models except co-dominant model using random effect model (for T  Figures 1-3).

Discussion
The results from the meta-analysis of 14 studies highlighted a higher risk of developing lung cancer for subjects carrying the MTHFR 677 TT genotype. Impaired MTHFR activity might influence cancer risk is determined by the level of S-adenosyl-L-methionine, the common donor of methyl that is necessary for maintenance of the methylation patterns in DNA. Changes in methylation modify DNA conformation and gene expression. A less active form of MTHFR leads to lower S-adenosyl-Lmethionine levels and consequently to hypomethylation; this phenomenon would be expected to increase the risk of some cancers (Stern et al., 2000). Similarly, low folate intake may modify cancer risk by inducing uracil misincorporation during DNA synthesis, leading to chromosomal damage, DNA strand breaks and impaired DNA repair, and DNA hypomethylation (Blount et al., 1997;Kim et al., 1997;Duthie, 1999;Kim, 1999).
MTHFR plays a central role in balancing DNA synthesis (which involves 5,10-methylentetrahydrofolate) and DNA methylation (which involves 5,10-methyltetrahydrofolate). Specifically, the 677T allele contributes to DNA hypomethylation, which in turn may lead to altered gene expression; at the same time, this polymorphism might exert a protective effect, as observed for colorectal cancer (Botto and Yang, 2000), by increasing the levels of the MTHFR substrate, essential for DNA synthesis.
There are few limitations in present meta-analysis likei) crude OR was used, ii) higher heterogeneity was observed, iii) controls were not uniform, iv) other genes of folate pathway was not considered and (iv) other risk factors among the subjects in the available studies, such as folate intake and smoking status etc were not considered. There is a need for larger and wider case-control studies to explore the role of other factors that are likely to cause Lung cancer. This is a meta-analysis with sufficient individual data to stratify results by ethnicity. This analysis supports conclusions that the T carriers genotype had increased risk of Lung cancer (TT vs CC: OR=1.25, 95%CI: 1.01-1.54, p=0.03) in the case of Asian population; this suggests the MTHFR C677T polymorphism may be associated with the risk of lung cancer. Future well designed large studies might be necessary to validate this association in different populations incorporated with micronutrient factors in the susceptibility of lung cancer.