Methylenetetrahydrofolate Reductase Gene C677T Polymorphism and Its Association with Ovary Cancer

Cancer is a leading cause of death worldwide. It is estimated that the burden of cancer will increase up to 22.2 million new cases diagnosed annually worldwide by 2030, which represents an increase by 75% Compared with the statistics of 2008 [1,2]. Ovarian cancer is the most common cause of death from gynecological malignancies. In the early stages, women are generally asymptomatic or have non-specific symptoms, making early stage ovarian cancer difficult to diagnose [2-6]. It is the ninth most common malignancy and the fifth most common cause of death from female cancers in the United States [6].


Introduction
Cancer is a leading cause of death worldwide. It is estimated that the burden of cancer will increase up to 22.2 million new cases diagnosed annually worldwide by 2030, which represents an increase by 75% Compared with the statistics of 2008 [1,2]. Ovarian cancer is the most common cause of death from gynecological malignancies. In the early stages, women are generally asymptomatic or have non-specific symptoms, making early stage ovarian cancer difficult to diagnose [2][3][4][5][6]. It is the ninth most common malignancy and the fifth most common cause of death from female cancers in the United States [6].
The wide geographic variation in incidence rates points to the role of genetic and environmental factors in the pathogenesis of this cancer. Possible risk factors for ovarian cancer include family history, tobacco smoking, infertility, low parity, and hormone replacement therapy, while oral contraceptive use and fewer menstrual cycles are associated with decreased risk [7][8][9]. Deficiency of nutrients, such as vitamins and microelements, has also been associated with increased risk for ovarian cancer, whereas high fruit and vegetable intake may help prevent the disease [9,10]. The incidence rate varies geographic worldwide. The wide geographic variation at international levels of ovarian cancer in terms of incidence and mortality suggested the role of genetic and environmental factors in the pathogenesis of this cancer [11].
Folate has a key role in DNA synthesis and methylation, thus, adequate intake is essential to maintain DNA integrity, and its deficiency may increase the risk of mutation, and hence cancer, therefore many countries have introduced mandatory or voluntary fortification of grain products with folic acid. Low folate intake has been associated with an increased risk of several cancers, including breast, and endometrial cancer [8,[12][13][14]. Its influence on ovarian cancer risk is less clear [13,[15][16][17] and may depend on other unmeasured factors such as genetic variation in the folate metabolism pathway [18]. Folate availability for DNA synthesis and as a methyl donor for methylation depends not only on intake, but also on the activity of enzymes, including methylene tetrahydrofolate reductase (MTHFR), methionine synthase (MTR) and MTR reductase (MTRR) [18,19].
Methylenetetrahydrofolate reductase (MTHFR) is the enzyme responsible for the reduction of methylenetetrahydrofolate. Reduced MTHFR activity results in an increased requirement for folic acid to maintain normal homocysteine remethylation to methionine. In the absence of sufficient folic acid, intracellular homocysteine accumulates, methionine resynthesis is reduced and remethylation reactions are interrupted. Increased homocysteine and decreased methionine cause decreased SAM to S-adenosylhomocysteine (SAH) ratio, which takes part in methylation [20]. There are more than 40 polymorphisms reported in MTHFR gene and among them C677T variant is the most studied and clinically important. The C677T variant (rs 1801133; Ala 222 Val) has been associated with a decreased activity of MTHFR, and increased homocysteine level [21][22][23]. Mutant homozygous (TT) individuals have a decreased enzymatic activity ~70% and the heterozygote by 40%. A dysfunctional MTHFR leads to lower levels of SAM resulting into DNA hypomethylation. DNA hypomethylation increases the risk of many diseases and disorders like-neural tube

Publication bias
Publication bias was investigated by using funnel plots; viz. funnel plot of standard error by log odds ratio and funnel plot of precision (1/ standard error) by log odds ratio. Different statistical tests such as Begg and Mazumdar rank correlation [35] and Egger's regression intercept [36] were adopted to assess the publication bias. All p values are two tailed with a significance level at 0.05. All statistical analyses were undertaken using the freely available program MIX version 1.7 [37].

Eligible studies
Following these exclusions, 9 individual case-control studies with a total of 5,922 cases and 5,235 controls were included into this metaanalysis [9,11,18,19,[38][39][40][41][42]. One author18 collected case samples from three different centers (New England Case Control Study (NEC), Nurses' Health Study (NHS), and Mayo Clinic Ovarian Cancer Case Control Study (MAY)) and reported separately in article, so they were included as separate three studies. Hence, total numbers of 11 studies were included in the present meta-analysis. Three studies investigated Asian population [9,11,40] and other studies investigated Caucasian population [18,19,38,39,41,42].

Characteristics of included studies
In total 11 studies, total cases were 5,922 with CC (3032), CT (2308) and TT (582), and controls were 5,235 with CC (2511), CT (2176), and TT (548). In controls genotypes percentage of CC, CT and TT were 47.97%, 41.57% and 10.47% respectively. In total cases genotype percentage of CC, CT and TT was 51.2%, 39% and 9.8% respectively. Frequencies of CC and CT genotypes were highest in both cases and controls ( Table 1). Number of C and T alleles were also calculated and presented in Table 2. Five studies did not show any association  [18,19,38,39,42].

Meta-analysis:
The main analysis for investigating the association of the C677T allele T and the risk of developing ovary cancer relative to the allele C showed higher heterogeneity (P=0.0009, I 2 =65.2%) between the 11 studies; the fixed and random pooled OR did not significantly associated with ovary cancer (OR=1.04, 95% CI=0.99-1. 11

Statistical analysis
In allele contrast meta-analysis, sensitivity analysis performed by exclusion of the studies in which control population was not in Hardy Weinberg equilibrium, studies with small sample size (<100) and defects [24], cleft lip and palate [25], cardiovascular diseases [21], diabetes [26] and cancer [27] etc.
The different investigations reported the effect of MTHFR C677T polymorphism on ovarian cancer risk in different ethnicities may be due to differences in population background, study design, sample size, environmental factors, and chance variations. Further confirmation of such differential effects is therefore needed. Hence a meta-analysis was carried out to come to a conclusive result whether C677T polymorphism is a risk factor for ovary or not.

Selection of studies
Electronic searches were conducted using PubMed, Google Scholar, Elsevier and Springer link and all published manuscripts up to March, 2014 were considered in present meta-analysis. The following index terms were used for search 'MTHFR' 'Methylenetetrahydrofolate reductase', and 'C677T polymorphism', and 'Ovary cancer'. In addition, bibliographies of all articles and reviews were hand searched for additional suitable studies.

Data extraction
The following information was extracted from each eligible study: first author's name, journal name, year of publication, country name, number of cases and controls and genotyping method. Number of alleles or genotypes in both cases and controls were extracted or calculated from published data to recalculate ORs.

Inclusion and exclusion criteria
The inclusion criteria of studies were as follow: studies should: 1) Be original, 2) Used case control approach 3) Used PCR-RFLP method for MTHFR genotype analysis and 4) Published in a peer-reviewed journal. Studies were excluded if: 1) Their sample was not independent from that investigated in another study, 2) Incomplete raw data information and not providing complete information for number of allele and/or genotype calculation, 3) Studies based on pedigree data were excluded as they investigate linkage and not association.

Statistical analysis
The present meta-analysis examined the overall association of mutant T allele with the risk of ovary cancer relative to the C allele. The associations were indicated as odds ratios (ORs) with the corresponding 95% CI. A pooled OR was then estimated on the basis of the individual ORs. The OR was estimated either by using fixed effect [28] or random effect [29] models depending upon heterogeneity. When there is considerable heterogeneity between studies then the pooled OR is preferably estimated using the RE model [30]. Models were chosen based on the method described by Thakkinstian et al. [31], briefly calculating and comparing the ORs of T vs. C (allele contrast/additive), TT vs. CC (homozygote), CT vs. CC (co-dominant), TT+CT vs. CC (dominant) and TT vs. CT+CC (recessive) checking the heterogeneity and significance, then determining the best model.
The heterogeneity between studies was tested using the Q-statistic and heterogeneity between studies was quantified using the I 2 statistic [32,33]. If I 2 > 50% then random effect model was used (which gives wider confidence intervals) otherwise fixed effect model applied. Cumulative meta-analysis was also performed to observe the effect of subsequent addition of each study. Sensitivity analysis was performed to explore potential heterogeneity and verify the stability and robustness of the main results. Quality of each study was assessed using studies with high p values. Control population of one study [38] was not in HW equilibrium and heterogeneity did not decreased after exclusion of that study (p=0.007, I 2 =65.99%). Exclusion of one study with small sample size, less than 100 [41], also did not decreased heterogeneity (P heterogeneity =0.001, I 2 =65.49%). Similarly exclusion of five studies with very high p value [18,19,38,39] did not decrease heterogeneity (P heterogeneity =0.007, I 2 =65.99%) but increased odds ratio (OR=1.27, 95% CI=1.04-1.51 p=0.02) and showed significant association between C677T polymorphism and ovary cancer with allele contrast model (Figures 1 and 2).

Publication bias
Funnel plots, Begg's and Egger's test were performed to estimate the risk of publication bias. The shape of funnel plots in all contrast models showed obvious evidence of symmetry ( Figure 3). In addition, all the P values of Egger's test were more than 0.05, which provided statistical evidence for the symmetry of funnel plots in the meta-analysis (p=0.24 for T vs. C; p=0.95 for TT vs. CC; and p=0.29 for CT vs. CC; p=0.46 for TT+CT vs. CC; p=0.64 for TT vs. CT+CC) (

Discussion
Present meta-analysis of the association of the MTHFR C677T polymorphism with ovary cancer investigated 5,922 patients and 5,235 controls from 11 case-control studies. Overall meta-analysis did not detect significant genetic association between the MTHFR C677T polymorphism and ovarian cancer.
Folate, methionine, vitamin B6 and vitamin B12 may influence carcinogenesis due to their roles in the one-carbon metabolism pathway which is critical for DNA synthesis, methylation and repair. However, DNA synthesis is also integral to the process of tumor formation, and for many years anti-folates have been used to treat some cancers. Recent studies have suggested that rather than preventing cancer, high folate levels might promote progression of pre-neoplastic lesions to cancer (Table 5) [19], and that the introduction of mandatory folate fortification in Canada and the United States of America may have led to increases in colorectal cancer rates [19].
Epidemiologic studies have revealed that MTHFR polymorphisms are associated with an increased risk of esophageal cancer, gastric cancer, breast cancer, hepatocellular carcinoma, bladder cancer, and prostate cancer. Conversely, MTHFR polymorphisms have also been associated with a reduced risk of colon cancer, leukemia, and highly aggressive prostate cancer [43][44][45][46]. 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. This polymorphism might exert a protective effect, as observed for colorectal cancer [47], by increasing the levels of the MTHFR substrate, essential for DNA synthesis. The substrate of MTHFR enzyme, 5,10-methylenetetrahydrofolate, is involved in the conversion of deoxyuridylate monophosphate to deoxythymidylate monophosphate, and low levels of 5,10-methylenetetrahydrofolate would lead to an increased deoxyuridylate monophosphate/deoxythymidylate monophosphate ratio. In this situation, increased incorporation of uracil into DNA in place of thymine may follow, resulting in an increased chance of point mutations and DNA/chromosome breakage [48]. A less active form of MTHFR would lead, all other factors being equal, to an accumulation of 5,10-methylenetetrahydrofolate, thus a lower deoxyuridylate monophosphate/deoxythymidylate monophosphate ratio, and a presumably lower cancer risk [48].     Meta-analysis provides a standardized approach for exam ining the existing literature on a specific, possibly con troversial, issue to determine whether a conclusion can be reached regarding the effect of a polymorphism of low penetrant gene [49]. Several meta-analyses were published to assess the role of MTHFR polymorphism in cancer development like: breast cancer [50,51] lung cancer [52] colorectal cancer [53] pancreatic cancer [54] Esophageal cancer [55,56] and cervical cancer [57].
There were several limitations in present study: 1) Crude ORs was used in the pooled analysis without adjustment; 2) The robustness of every single study would be affected by the technique they used; 3) The relatively small sample sizes of some studies is included in the analysis; 4) Meta-analysis was restricted on only single polymorphism, other polymorphism of folate pathway genes should also be included in future meta-analysis and 5) Except one genetic polymorphism, other important factors such as age, ethnicity, and folate intake, and      smoking status were not considered. Present meta-analysis had several strength also like-(i) The publication bias was not detected in present meta-analysis, (ii) Pooled number of cases and controls from different studies significantly increased the statistical power of the analysis, (iii) Distribution of genotypes in controls except one study was in Hardy-Weinberg equilibrium.
In summary, the results of current meta-analysis indicated a lack of association between MTHFR C677T gene polymorphism and ovary cancer. The results of the present meta-analysis were based on relatively small numbers of studies, participants and higher between studies heterogeneity, hence must be interpreted with caution.