Lack of Significance of the BRCA2 Promoter Methylation Status in Different Genotypes of the MTHFR a1298c Polymorphism in Ovarian Cancer Cases in Iran

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 (Permuth-Wey & Sellers, 2009; Swisher et al., 2009; Asadollahi et al., 2010). It is the ninth most common malignancy and the fifth most common cause of death from female cancers in the United States. In 2009, the American Cancer Society estimated that 21,550 women will be diagnosed with ovarian cancer. This disease often is sporadic (Holschneider & Berek, 2000; An et al., 2010). In ovarian cancer, at least 15 oncogenes, 16 candidate tumor suppressor genes and more than seven signaling pathways have been diagnosed. Aberrations in cell propagation, apoptosis and autophagy and alterations in cell sticking and motility all imply to disease progress and metastasis. Epigenetic events lead to changes in gene expression other than by means of DNA sequence alteration and include DNA methylation and histone modifications. From what is presently known, epigenetic have more and more become to be implied in the growth and progression of


Introduction
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 (Permuth-Wey & Sellers, 2009;Swisher et al., 2009;Asadollahi et al., 2010). It is the ninth most common malignancy and the fifth most common cause of death from female cancers in the United States. In 2009, the American Cancer Society estimated that 21,550 women will be diagnosed with ovarian cancer. This disease often is sporadic (Holschneider & Berek, 2000;An et al., 2010). In ovarian cancer, at least 15 oncogenes, 16 candidate tumor suppressor genes and more than seven signaling pathways have been diagnosed. Aberrations in cell propagation, apoptosis and autophagy and alterations in cell sticking and motility all imply to disease progress and metastasis. Epigenetic events lead to changes in gene expression other than by means of DNA sequence alteration and include DNA methylation and histone modifications. From what is presently known, epigenetic have more and more become to be implied in the growth and progression of ovarian cancer, and their gradual accumulation is related to advancing grade and stage of disease (Asadollahi et al., 2010).
DNA methylation at CpG sites in the regulatory region of a gene can alter gene expression, which has recently been considered an important characteristic of tumor growth and progression (Olek et al., 1996;Paige, 2003;Motoyama & Naka, 2004). DNA methylation refers to the covalent addition of a methyl group resulting from S-adenosyl-l-methionine to carbon 5 of the cytosine ring by DNA methyl transferases (Asadollahi et al., 2010). In the promoter of genes, aberrant methylation of CpG islands that often outcomes in atypical propagation or aberrant cell survival has been implicated as one of the main pathways involved in the progress of cancers, including ovarian cancer (Esteller et al., 2001;Widschwendter et al., 2001).
The importance of the role of unusual methylation in the development of cancer has become clear with the growing of genes that have been shown to be vulnerable to inactivation by promoter methylation (Costello et al., 2000;Kanai & Hirohashi, 2007). The BRCA2 protein is important in preserving genomic stability by interfere in repair of double-strand DNA breaks (Gudmundsdottir & Ashworth, 2006). Methylenetetrahydrofolate reductase (MTHFR) can catalyse the reduction of 5, 10-methylentetrahydrofolate to 5-methyltetrahydrofolate. This product remethylates homocysteine to methionine which is a system used in the methylation of DNA. A polymorphism which changes an A to C at position 1298 (a1298c) in MTHFR is a reasonably common variant of the gene. Neoplastic cells increase DNAmethyltransferase activity suggesting that methylation imbalance may contribute to tumor evolution (Lin et al., 2004;Jakubowska et al., 2007).
Because of the main role that MTHFR plays in the methylation status of genes, we designed assays to investigate the methylation status of the BRCA2 promoter and investigated any potential correlation with the MTHFRa1298c polymorphism in ovarian cancer samples with a view that it may provide a basis for a new biomarker for both the identification of patients at risk and early diagnosis of ovarian cancer. If a correlation could be established, a significant benefit would be the analysis of MTHFRa1298c polymorphism in tissue or blood, potentially leading to a non-invasive cancer susceptibility or cancer screening test. The ultimate goal of these studies would be to develop assays which could be used to reduce the incidence and mortality of ovarian cancer.

Subjects
This study was undertaken as part of the completion of a PhD thesis in anatomy. It was done in Department of Anatomy and Molecular Biology of Isfahan University of Medical Sciences from 2009 to 2011. In this study, 60 samples of ovarian cancer and 60 matched adjacent normal ovarian tissue samples with mullerian origin from the same patient were chosen in pathology wards of Isfahan hospitals. The methylation status of BRCA2 was studied using methylation specific PCR for both the cancer tissues and the matched controls. Polymorphisms of the MTHFR gene (a1298c) were evaluated using PCR-RFLP.

DNA extraction
DNA was extracted from 5-10 μm thick paraffinembedded tissue by adding lysis buffer (700µl of 0.1M NaOH, 1% SDS and 10 pellets of chelexe granules (Merk, Germany)) and incubated in boiling water for 20-40 minutes and subsequently centrifuged at 12,000 rpm for 10 minutes. The liquid phase was transferred to a new tube and an equal volume of phenol, chloroform-isoamyl alcohol (1:1) was added and centrifuged at 12,000 rpm for 10 minutes. The aqueous phase was transferred to a fresh tube and mixed with an equal volume of isopropanol-3 M sodium acetate (10:1) and centrifuged at 14,000 rpm for 10 minutes. The supernatant was discarded and 500 μl of 70% alcohol was added to the pellet. After mixing, the tubes were centrifuged at 14,000 rpm for 10 minutes and the aqueous supernatant was discarded. Tubes were incubated at room temperature until the pellets were dry and 50μl of DNase free water was added to dissolve the DNA pellet.

The assessment of the MTHFR a1298c polymorphism
The PCR-RFLP method was used to determine the MTHFR a1298c polymorphism. The a1298c nucleotide substitution creates a novel MboII restriction site. PCR was performed using specific primers (forward: CTT TGG GGA GCT GAA GGA CTA CTA C And reverse: CAC TTT GTG ACC ATT CCG GTT TG). The PCR mixture included 2.5-3 μl of template DNA, 10 mM tris-HCl (pH 8.3), 30 mM KCl, 1.5 mM MgCl 2 , 1 units of recombinant Taq DNA polymerase, 250 μM of each dNTP and 1 μl of each primer, made up to a total of 20 μl by adding distilled water. The DNA amplification was performed using the following program: 5-min at 94 °C, 35 cycle of (94 °C for 30 s, 60 °C for 30 s, 72 °C for 30 s) and 5 minutes at 72 °C. 10 µL of PCR product was mixed with 10U of HinfI for digestion at 37 °C overnight. For the analysis of the MTHFRa1298c polymorphisms, a CC genotype produced 84, 31, 30 and 18 bp fragments, AC genotypes produced 84, 56, 31, 30, 28 and 18 bp fragments while the AA genotypes produced five fragments of 56, 31, 30, 28 and 18 bp. Digestion products were visualized after electrophoresis on a 3% agarose gel stained with ethidium bromide (Lin et al., 2004;Jakubowska et al., 2007).

Modification of DNA
Cytosine nucleotides were changed to uracil by bisulfate treatment using the EZ DNA methylation kit (Zymo Research, USA) according to the manufacturer's guidelines. In brief, this procedure modifies unmethylated cytosines to uracil nucleotides but does not modify methylated cytosine nucleotides.

Methylation specific PCR
Bisulfate-modified DNA was amplified with PCR specific primers ( Table 1) that distinguish methylated (M) and unmethylated (U) DNA. Amplicon is 337 and 250 bp in length for unmethylated and methylated BRCA2, respectively. Contents of the PCR mixture included 2.5-3 μl of bisulphate treated template DNA, 10 mM tris-HCl (pH 8.3), 30 mM KCl, 1.5 mM MgCl2, 1 units of recombinant Taq DNA polymerase, 250 μM of each dNTP and 1 μl of each primer, made up to a total of 20 μl by adding distilled water. Thermocycling program is 94 °C for 5 min. followed by 35 cycles (94 °C for 30 seconds, 62 ºC for methylated primer and 56°C for unmethylated primer for 30 seconds, 72 °C for 30 second and 72 °C for 4 minutes. Fully unmethylated and methylated DNA after bisulfit conversion were used as unmethylated and  DOI:http://dx.doi.org/10.7314/APJCP.2012.13.5.1833  methylated controls. PCR products were analyzed on a 2% agarose gel ( Hilton et al., 2002;Varinderpal, 2004). The methylation status of the BRCA2 promoter in the different genotypes of MTHFR was assessed and the results are shown in Table 2 (The table shows methylation status of the BRCA2 promoter compared to the MTHFR polymorphism (AA, AC or CC). "Methylation" indicates those patients that had no detectable methylation in the BRCA2 promoter taken from normal tissue however; the tumor sample from the same patient was methylated. "Unmethylated" indicates a change in the promoter from methylated, in normal tissue, to unmethylate in the tumors sample.).
Finally, Statistical analyses, including chi-square tests, were performed using SPSS for Windows, version 16.0. All statistical tests were two sided, and P values less than .05 were considered statistically significant.

Results
Forty-seven of the 60 cases were unchanged in promoter methylation when comparing normal tissue to those taken from the same patients tumor, while 7 of them changed from unmethylated to methylated promoters and 6 cases changed from methylated to unmethylated in the promoter (Table 3) (Figure 1). 7 out of 60 cases (11.66%) were methylated and 53 out of these (88.33%) were unmethylated in promoter. In methylated cases, one out of seven cases had a CC genotype and the remaining 6 methylated cases had an AC genotype. The AA genotype was absent. In unmethylated cases, 34, 18, and one out of these had AC, AA, and CC genotypes, respectively (Table 2). There was no significant relationship between the methylation type of the BRCA2 promoter in different genotypes of MTHFRa1298c polymorphism in ovarian cancer; p=0.255.

Discussion
ADNA methylation is a distinctive and important process because it involves the covalent modification of a cell's genetic substance. Methylation as a mechanism for gene inactivation can occur in some gene promoter tumors. MTHFR plays a central role in the metabolism of folate and has been implicated in the etiology of cancer via its effects on DNA methylation (Lin et al., 2004;Jakubowska et al., 2007).
We studied the methylation status of the BRCA2 promoter in patients with different genotypes of the MTHFR gene (A or C nucleotides at position 1298) because of the importance that this gene may have in the development of ovarian carcinogenesis. We speculate that in our specimens, the methylation of the BRCA2 promoter can lead to gene silencing and that this may be responsible for the development of cancer in at least some of our patients. It should be mentioned that inactivation of BRCA2 may occur by mechanisms other than promoter methylation and this may also influence expression in our cohorts.
Methylation status of the gene promoter shows different results in numerous investigations (Hilton et al., 2002;Paige, 2003). One factor influencing the results could be caused by contamination of specimens with cells from nearby tissues during analysis and isolation of tumor samples. Unmethylated DNA from the normal cells might compromise the results of the methylation levels of the tumor tissue (Hilton et al., 2002). We arrange to determine if differences in the BRCA2 promoter methylation could be correlated to different genotypes of the MTHFR gene in cases (a1298c polymorphism). No significant correlation was observed between the different genotypes of MTHFR and the methylation type of the BRCA2 promoter. However, we observed the different genotypes of the MTHFR gene we found no correlation with changes in BRCA2 promoter methylation. Therefore, we concluded other factors affect the rate of promoter methylation of BRCA2 in the cohort studied here.
In conclusion, there was no relation between methylation types of the BRCA2 promoter in different genotype of MTHFRa1298c polymorphism in ovarian cancer.