Association between MTHFR C677T polymorphism and folate, vitamin B12, homocysteine, and DNA fragmentation in patients with ovarian cancer Over kanserli hastalarda MTHFR C677T polimorfizmi ile folat, vitamin B12, homosistein düzeyleri ve DNA kırıkları arasındaki ilişki

Objective: Methylenetetrahydrofolate reductase (MTHFR) is an important enzyme that regulates the metabolism of methionine and folate. MTHFR C677T polymorphism was reported to be associated with breast and ovarian cancer. The aim of this study was to evaluate the association between the MTHFR C677T (rs1801133) polymorphism and homocysteine, vitamin B12, and folate levels, and DNA fragmentation in patients with ovarian cancer and healthy controls. Materials and methods : This case-control study was con-ducted in Istanbul University Cerrahpasa Medical Faculty. We studied 50 ovarian cancer patients and 54 healthy controls. The MTHFR C677T polymorphism was determined by PCR followed by restriction fragment length polymorphism (RFLP) and agarose gel electrophoresis. DNA fragmentation was assessed by the comet assay. Homocysteine levels were measured by ELISA, whereas vitamin B 12 and folate levels were measured by chemiluminescence methods. Results : We found no correlation between the MTHFR C677T polymorphism and ovarian cancer. No significant difference was found in homocysteine, folate, and vitamin B 12 levels between patient and control groups. Increased DNA fragmentation was detected in patients with ovarian cancer. Conclusion : Our findings suggest that MTHFR C677T polymorphism, as well as homocysteine, folic acid, and vitamin B12 levels, are not associated with an increased risk for ovarian cancer.


Introduction
Ovarian cancer is the most prevalent gynecological cancer, and is the most common type of epithelial cancer. Two-thirds of the cases are diagnosed in a late stage, as they remain asymptomatic until metastasis occurs. Moreover, ovarian cancer has the highest overall mortality rate among all fatal tumors in women [1][2][3].
In this scenario, folate and methionine play a significant role in DNA synthesis, repair, and methylation, and methylenetetrahydrofolate reductase (MTHFR) is a key enzyme in the regulation of both folate and methionine metabolism. MTHFR catalyzes the conversion of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, which ultimately donates the methyl group to homocysteine for further DNA methylation. The conversion to 5-methyltetrahydrofolate is, therefore, essential for DNA methylation. Decreased MTHFR activity leads to decreased availability of methyl groups for DNA methylation, and may thereby affect the expression of tumor suppressor genes as well as overall DNA stability [4][5][6]. Additionally, in vitro studies in human lymphocytes have shown that low folate serum levels are associated with misincorporation of dUMP into DNA and leading to double-strand breakage [7,8]. It has also been argued that low folate levels, along with gene polymorphism-associated hyperhomocysteinemia, might be associated with neural tube defects [9].
A common MTHFR variant C677T (rs1801133), resulting in an alanine to valine amino acid substitution, is associated with reduced MTHFR enzymatic activity. C677T MTHFR gene polymorphism was also reported as a risk factor for breast, ovarian, and colorectal cancer [10][11][12]. Previous studies have shown that this polymorphism was also increased in Polish BRCA1 mutation carriers [11], and that 677T homozygosity was associated with bilateral mammary cancer and with both breast and ovarian carcinoma among Jewish BRCA1/2 mutation carriers [12].
Moreover, deficiency of methionine formation from homocysteine, due to reduced MTHFR activity, results in reduced methionine levels and excess of homocysteine with toxic effect for the organism [13].
The degree of the genetic damage in ovarian cancer patients was assessed by single-cell gel electrophoresis, also known as the comet assay [14,15]. This technique was used to evidence the effects of the oxidative damage induced by ionizing ultraviolet radiation on lymphocytes. In the current study, we investigated the MTHFR gene C677T polymorphism in ovarian cancer patients and compared them to healthy controls. We also analyzed homocysteine, folate, and vitamin B12 levels, which play crucial roles in MTHFR metabolism. Furthermore, DNA damage in patients with different stages of ovarian cancer was evidenced by the comet assay, in order to unveil the mechanisms responsible for its pathogenesis.

Materials and methods
The study cohort included 50 ovarian cancer patients (age 52.88 ± 12.83 years old) from the Department of Obstetrics and Gynecology of the Haseki Education and Research Hospital, and 54 healthy subjects (age 50.75 ± 9.64 years old), without cancer or cardiovascular disease history, who underwent a regular check-up from Cerrahpasa Medical Faculty Hospital. All patients and controls provided informed consent before being enrolled in the study. This study was approved by the Ethics Committee of the Cerrahpasa Medical Faculty (#28113). All patients and controls were from the Istanbul district, with middle-income brackets. Patients and controls were not doing regular exercise. All patients who underwent blood sample collection were not subjected to chemotherapy or anticancer therapy.
Genomic DNA was isolated from peripheral blood using a commercial kit (Roche Diagnostics GmbH, Mannheim, Germany) according to the manufacturer's instructions. MTHFR C677T (rs1801133) polymorphism was determined by PCR followed by restriction fragment length polymorphism (RFLP), as previously described [16]. PCR products digested by HinfI resulted in one 198-bp fragment in wild-type homozygotes (CC), two 175 and 23 bp fragments in mutated homozygotes (TT), and three 198, 175, and 23 bp fragments in heterozygotes (CT).
Homocysteine levels were determined by ELISA [The Axis ® Homocysteine Enzyme Immunoassay (EIA), Scotland] and vitamin B12, and folate levels were determined by chemiluminescence method (Access Immunoassay Systems, Beckman Coulter, USA). DNA fragmentation in patients with ovarian cancer was assessed by the comet assay, as previously reported [14]. For the comet assay, 5 mL of peripheral blood were taken and placed into a heparinized tube. Lymphocytes were isolated by the Ficoll-Histopaque method. Briefly, the basic steps of the comet assay included the preparation of microscopic slides layered with cells embedded in agarose gel, lysis of the cells to release the DNA, electrophoresis, neutralization of the alkali, drying, DNA staining by ethidium bromide (20 mg/mL), and scoring [14]. Cells were assessed visually and scores from 0 (undamaged) to 4 (maximally damaged) were assigned according to the tail intensity (size and shape). Statistical comparison of DNA damage between patient and control groups was performed using the method reported by Feng et al. [17]. All experiments were performed by fluorescent microscopy (Olympus).
Statistical analyses (mean, standard deviation, and percentages) were performed using SPSS 21 software. One-way analysis of variance (ANOVA) was used to compare folate, homocysteine, vitamin B12, and comet assay results between the genotypic sub-groups of cases and controls. The Chi-square (χ 2 ) test was used to compare the association between the genotypes and alleles in cases and controls, and to test for deviation of genotype distribution from the Hardy-Weinberg equilibrium (HWE). A p-value < 0.05 was considered statistically significant.
Homocysteine, folate, vitamin B12 levels, and comet assay scores for ovarian cancer patients and healthy controls are shown in Table 2. We found no significant differences regarding homocysteine (p = 0.6), folic acid (p = 0.92), and vitamin B12 plasma levels between ovarian cancer patients and controls (p = 0.14). We compared the genotypes of patients and controls according to homocysteine, folate, vitamin B12 levels and comet assay scores. The comet assay scores of CC and CT genotypes showed a significant difference between patients and controls ( Table 3). The comet assay scores also showed a significant increase in DNA fragmentation among ovarian cancer patients compared to controls (p < 0.01) ( Figure 2).
Homocysteine, folic acid, vitamin B12 levels, and comet assay scores in association with the tumor grades of ovarian cancer in our cohort of patients are shown in Table 4. Comet assay scores were significantly higher for grade 3 than for grade 1 and 2 tumors (p = 0.0007) ( Table 4).

Discussion
Ovarian cancer is the fourth leading cause of cancerrelated deaths worldwide, following lung, breast, and colon cancer [1]. Several studies proved that the risk of having ovarian cancer at any stage of a woman's life is 1.42% [18]. The disease is usually rare before the age of 40, increasing steeply and showing a peak after 65 years of age [19]. Sazci et al. studied the allelic frequencies of C677T and A1298C polymorphisms in the MTHFR gene in 1684 randomized individuals from the Turkish population [20]. They reported the frequencies of C677T genotypes as 42.9% CT, 47.4% CC, and 9.6% TT. In our cohort, we found the CT genotype frequency to be 55.8%, CC genotype 35.6%, and TT genotype 8.6%, in accordance to the previously mentioned findings.
Previous studies on the effects of MTHFR C677T polymorphism showed an increased breast cancer risk in premenopausal women associated with the TT genotype [21]. In Korean population, the MTHFR C677T polymorphism has been reported as a risk factor for cervical cancer [22]. Additional studies indicate that MTHFR C677T polymorphism frequencies are higher in BRCA1 mutation carriers, with higher rates of TT homozygotes in women with bilateral breast and ovarian cancer [11]. Another study demonstrated that 677T homozygosity is associated with bilateral mammary and joint breast and ovarian carcinoma among Jewish BRCA1/2 carriers [12]. In a review by Pu et al. an association between the MTHFR C677T polymorphism and susceptibility of ovarian cancer in Asian population suggests that the TT genotype may act as a risk factor for ovarian cancer among Asians but not among Caucasians [23]. Moreover, a meta-analysis including 3379 ovarian cancer cases and 4078 controls evidenced that MTHFR C677T polymorphism is not associated with ovarian cancer in Caucasians, whereas the T variant may contribute to increase the risk in East Asians [24]. Finally, it was suggested that the MTHFR T allele increases the risk for ovarian carcinoma development in three times [25]. In our study, there were no significant differences in MTHFR C667T genotype frequencies between the ovarian cancer patients and the control group. Our findings were consistent with the studies of Pu et al. and Ding et al. since our patient and control groups were composed of Turkish Caucasians [23,24]. Wu et al. reported an association between folic acid deficiency and MTHFR C677T polymorphism in human peripheral blood lymphocyte cytotoxicity [26]. DNA methylation defects may result in carcinogenesis by causing genomic instability and mutations [27]. Chen et al. found that DNA hypomethylation leads to an increase in mutation rates [28]. The present study was unable to confirm the role of the TT genotype in the development of ovarian cancer, given its small sample size (50 ovarian cancer patients and 54 controls) as we only selected patients who were not receiving chemotherapeutic medication before enrollment. The homocysteine levels were not significantly different from the control group between the different C677T genotypes. Our findings regarding homocysteine levels could be explained by an improved quality of life and an increased folic acid intake.
Some studies speculate that changes in folic acid metabolism might contribute to the oncogenic development of both ovarian and colorectal cancer [10,29]. Given its essential role in DNA synthesis, repair, and methylation, it has been hypothesized that dietary folate consumption may influence cancer risk [30]. A decrease in folic acid levels may increase DNA instability. However, in our study, no significant differences were observed in the folic acid levels between ovarian cancer patients and controls. Breast cancer has been also reported as a manifestation of abnormal genetic and epigenetic changes. A disruption in folate metabolism might affect genetic and epigenetic changes influencing gene expression, through DNA methylation, and genome integrity, through DNA synthesis and repair [27]. The present study showed decreased folic acid levels in ovarian cancer patients carrying the TT genotype compared to controls, however, this difference could not reach statistical significance given the limited sample size. In our study, the level of genetic damage in cancer patients was assessed by the comet assay. Lymphocytes circulate through different organs, including the lung, before they return to peripheral blood. They can circulate for years or even decades, accumulating DNA mutations produced by the exposure to different mutagenic agents. The comet assay has previously been used to measure DNA damage by irradiating the peripheral lymphocytes from bladder cancer patients and showing a higher degree of DNA damage in patients compared to controls [31]. Baltaci et al. used the comet assay to show the degree of genetic damage in ovarian cancer patients evidencing higher DNA fragmentation in correlation with increasing histopathological grades of cancer [15]. In our study, we found that DNA fragmentation in the ovarian cancer group was significantly higher than that in the control group, in accordance with previous findings [15]. Furthermore, the comparison of the histopathological grades among ovarian cancer patients, by the comet assay, showed that DNA fragmentation was higher in patients with grade 3 cancer compared to those with grade 1 or 2. Therefore, we suggest that DNA fragmentation increases with the level of malignancy of the tumor.
In conclusion, our results disprove the correlation between the MTHFR C677T polymorphism and ovarian cancer. Furthermore, no significant differences were found in homocysteine, folic acid, and vitamin B12 levels between ovarian cancer patients and controls. We showed that patients with the TT genotype exhibit low folate levels. In addition, increased DNA fragmentation was detected in the ovarian cancer patient group. DNA fragmentation degree was the highest in grade 3 ovarian cancer suggesting an association between DNA fragmentation and carcinogenesis progression. Larger patient cohorts will be necessary to confirm the association between homocysteine, folic acid, vitamin B12, and the MTHFR C677T polymorphism in ovarian cancer.