The polymorphisms of genes involved in DNA methylation in patients with malignancies from West Ukraine

© 2016 I. Dmytruk et al.; Published by the Institute of Molecular Biology and Genetics, NAS of Ukraine on behalf of Biopolymers and Cell. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted reuse, distribution, and reproduction in any medium, provided the original work is properly cited


Introduction
Aberrant DNA methylation is a key epigenetic factor in tumorogenesis [1].The genomic methylation patterns depend of the one-carbon metabolizing enzymes, that involved in the provision of methyl groups, and DNA methyltransferases (DNMT) that transfer methyl groups from S-adenosylmethionine to DNA CpG dinucleotides [2,3].
A crucial role for the control on DNA methylation as well as DNA synthesis and repair mechanisms belongs to the provision of adequate pools of methyl groups through the functional activity of methylenetetrahydrofolate reductase (MTHFR), methionine synthase (MTR) and thymidylate synthase (TYMS) in one-carbon metabolism.MTHFR catalyzes the ir-reversible conversion of 5,10 -methylen-tetrahydrofolate (THF) to 5-methyl-THF.The 5,10-methylene-THF is used by TYMS in the methylation of dUMP to dTMP, which is the only de novo source of thymidine required for DNA synthesis and repair.The 5-methyl-THF is a methyl group donor for the remethylation of homocysteine to methionine catalysed by MTR [4].
There are a large number of molecular epidemiological studies about a role of the folate metabolic enzyme gene polymorphism in different kinds of malignancy: lung cancer [11], breast cancer [12][13][14][15], lymphoma [16,17].However, the association between polymorphism and cancer risk is still controversial.
It is well known that methylation of the promoter and exon regions is one of the major regulatory mechanisms of gene expression; thus, we hypothesized that a gene involved in the folate metabolism may be responsible for regulating the methylation status of other genes associated with cancer risk.Therefore, the aim of this study was to determine the occurrence of single-nucleotide polymorphisms in the genes encoding the folate metabolizing enzymes (MTHFR, MTR, TYMS) in a case-control study from West Ukraine for their potential role in the breast cancer risk and risk of leukemia.

Materials and Methods
Patients.For molecular genetic testing the DNA samples, that were obtained from the venous blood nuclear cells of the patients with cancer, were used.The patients were hospitalized in the Regional Specialized Children's Hospital, Lviv Regional Specialized Oncological Clinic and Lviv regional hospital.To form the patient groups a detailed medical anamnesis was obtained and clinical pictures of the oncological disease were estimated.The data were analyzed among 150 patients, who were separated into 2 groups in accordance with their diagnosis.The first research group contains 60 patients with leukemia and lymphoma (65 % -boys, 35 %girls) aged 2 to 16 years.The diagnosis of oncohematological pathology was established for the first time.The group includes the 17 patients with acute leukemia, 22 persons with Hodgkin's lymphoma and 21 patients with non-Hodgkin's lymphoma.90 patients with breast cancer compose the second research group: women with breast cancer (II -IV stages) aged 35 to 75 years with burdened family history of breast or ovarian cancer.The control group consist of 100 healthy patients without cancer pathology in anamnesis.All persons from the research and control groups were from West Ukraine.
All patients signed the informed consent for using their DNA samples for molecular-genetic testing.The experimental protocol was approved by the Bioethics Committee of SI "Institute of hereditary pathology NAMS of Ukraine" and complied with international standards.
Genotyping.The extraction and refinement of DNA from the leukocytes of the peripheral blood were conducted according to the salting-out protocol extraction [18].The amplification of DNA chain in vitro was performed using the method of polymerase chain reaction (PCR) [11,12].To detect the variable number tandem repeats (VNTR) for the TYMS gene, PCR product was separated on a 2 % agarose gel (Fig. 1).The samples showing the 2R/3R or 3R/3R genotypes were further analyzed for the 3RG>C polymorphism by the method of restriction fragment length polymorphism (RFLP) using HaeIII restriction enzyme (Fig. 2).
Genotyping of the MTHFR 677C>T and MTR 2756 A>G polymorphisms was performed by RFLP using the restriction enzymes HinfI and HaeIII respectively.The obtained restriction fragment was detected by 2 % agarose gels electrophoresis.
Statistics.The results were processed with the methods of variation statistics suitable for the biological studies and recommended for processing the molecular genetic research results: Chi-square analysis (χ2 tests), Hardy -Weinberg equilibrium, odds ratio (OR) with 95 % confidence interval (CI).

Results and Discussion
There are many data on the interrelation between polymorphisms of the gene involved in the methylation process and the cancer susceptibility but the results vary depending of the patient ethnicity.In this study we analyse functional polymorphisms in the genes encoding the one-carbon metabolism enzymes and compare it with other population study.We carried out the molecular genetic testing of the genotype and allele frequency of the MTHFR 677 C>T, MTR 2756 A>G and TYMS 2R/3R, TYMS 3R G>C polymorphisms among the patients with breast cancer, leukemia and of a control group.The established genotypes and alleles distribution of testing polymorphic loci in the research and control groups did not significantly differ from the theoretically expected frequency conformed to Hardy-Weinberg equilibrium (Table 1).We have determined and compared the alleles and genotypes frequency of the studied polymorphic loci in the control and case groups (Table 2).
The genotypes and allele distribution of the MTHFR 677 C>T polymorphic loci did not show significant difference between the case and control groups.(Table 2.).The frequency of low-expression 677 TT genotype was 8 % in the group of breast cancer and 7 % in the control group as much as in the group of patients with leukemia.The frequency of 677 T allele did not differ in the case groups compared to the control (34 %, 27 % vs. 31 %).OR calculation also did not show statistically significant result (Table 2).Our findings coincide with the results of German and Korean researches [19,20].The decreased susceptibility to acute lymphoblastic leukaemia was found in the West European populations [21].It was found that the 677C>T polymorphism is strongly associated with breast cancer in the East The polymorphisms of genes involved in DNA methylation in patients with malignancies from West Ukraine Asian populations [22].Therefore, our results seem to be more relevant to other population studies: the lack of association of the MTHFR polymorphisms with the breast cancer risk was observed in Indian women [23] and in the residents of Scotland [13], Finland [24] and Greece [25].
For better understanding of the clinical significance of the SNP and VNTR of TYMS, we analysed the frequency of comprehensive genotypes of 3R2R and 3R G>C polymorphisms in case and control groups.The allele and genotype frequency is summarized in Table 3.
The TYMS genotypes were each stratify into three groups because the statistical power was insufficient by analysis with more than three genotype groups.The TYMS functional groups were classified into the H-group (H/H and H/L) and L-group (L/L).The TYMS 3G allele was considered a high-expression allele and 2R or 3RC as low-expression alleles according to the in vitro functional analysis [26].Therefore, the TS genotypes of 2R/3G, 3C/3G, or 3G/3G were considered as high expression (H) type and 2R/2R, 2R/3C or 3C/3C -low expression (L) type.According to our results, there was no significant association between the genotype groups and cancer pathology (Table 2.).Thus, the frequency of H-genotype group amounts 15 % in the patients with leukemia, 14 % in the control group and the same in the patients with breast cancer.The following frequencies were detected in the L-genotype functional group: 85 %, 86 % and 86 % for the patients with leukemia, breast cancer and control group respectively.
Our results are very similar to those reported for the Spanish [27] and Mexican [28] women.The studies on other populations have found the significant associations between the TYMS gene TSER variations and breast cancer development [14].For the TYMS 2R3R polymorphisms was found a decreased susceptibility to acute lymfoblastic leukemia in the Western European [15] and Brazilian studies [30].Furthermore, the frequency of TS genotypes in the patients with leukemias United Kingdom [16,29] did not show any significant difference compared to the controls.
The MTR 2756 A>G genotypes analysis show a significantly lower frequency of MTR 2756 GG genotype in the group of patients with breast cancer (4 %) compared to the control group (8 %, P<0.05).
The distribution of the MTR 2756 AG genotypes and allele did not show significant difference in the control group and the group of patients with leukemia.The frequency of low enzyme activity 2756 GG genotype in the case group consists 5 % and in the control group 8 %.In the study on British population also was not found significant difference in the susceptibility to acute leukemia for the MTR 2756AG polymorphism [16].The association of this polymorphism has been reinforced in a meta-analysis, being the 2756 AA genotype reduced risk of developing childhood acute lymphoblastic leukemia [34].
In our research we also analyzed the distribution of different genotypes combination in group of patient with breast cancer, leukemia and control group.In fact, we compared combination of two and three genotypes of MTHFR 677C>T, MTR 2756 A>G, DNMT3B -149C>T, DNMT3B -579 G>T and TYMS 3R2R, 3R G>C polymorphisms (Table 4, Table 5).
Our findings show statistically significant difference in distribution of MTHFR 677677C>T / TYMS 3R2R, 3R G>C polymorphic loci genotypes combination in control group and group of women with breast cancer.The frequency of MTHFR 677CT/ TYMS LL genotypes was significant higher in control group (71 %) compared to group of patient with breast cancer (21 %), therefore, the frequency of MTHFR 677CT/ TYMS HL genotypes was significant higher in observed group with breast cancer (79 %) compared to control (29 %).The OR calculation show that carriers of MTHFR 677CT/ TYMS LL genotypes has reduced risk of breast cancer (OR = 0.11, CI 95 %: 0.04 -0.29), and the carriers of MTHFR 677CT/ TYMS HL genotypes, on the contrary, has increased breast cancer risk (OR = 9.04, CI 95 %: 3.50 -23.39).
The analysis of three genotype combination shown that carriers of MTHFR 677CC/ MTR 2756AG/ TYMS LL genotypes were over represented in cases (breast cancer group -67 %, group of leukemia -91 %) compared to controls (18 %).The presence of the MTHFR 677CC/ MTR 2756AG/ TYMS LL genotypes was associated with 9.33-fold risk of breast cancer and 46.6-fold risk of leukemia.In group of patient with leukemia was established higher frequency of MTHFR 677CT/ MTR 2756AG/ TYMS HH (0 %) and MTHFR 677CT/ MTR 2756AG/ TYMS LL (68 %) genotypes combination compared to control group (25 % and 75 %, respectively).These differences between the cases and controls were statistically significant (p ≤ 0.05).
In summary, we found that different genotypes combination of MTHFR, MTR and TYMS genes polymorphisms in the West Ukrainian population could mediate the risk of breast cancer and leukaemia via effects on pools of methyl groups needed for genes methylation.The reasons for contrary results obtained in our research and other studies remain ambiguous and might be attributed to the differences in ethnic backgrounds and the selection of the population studied, differences in sample sizes, and geneenvironment interactions.It is reasonable to repeat a comprehensive research in the whole country for clarifying a role of the genes involved in the DNA methylation process in cancer susceptibility.

Conclusion
Our findings show that West Ukrainian residents carrying at least one MTR 2756A allele have a significantly increased risk of breast cancer.Also, we find out, that susceptibility to breast cancer and leukaemia in West Ukrainians relate to the combination of few genotypes: MTHFR 677CC/ MTR 2756AG/ TYMS LL -increased the risk of breast cancer and leukaemia, 677CT/ TYMS HL -raise the risk of breast cancer and the presence of MTHFR 677CT/ TYMS LL genotypes acts as a protector factor for breast cancer development.

Funding
Expenses were partially covered by West Ukrainian Biomedical Research Centre (WUBMRC) grant.

Table 4 . Distribution of two genotypes combination of MTHFR 677C>T, MTR 2756 A>G, DNMT3B -149C>T, DNMT3B -579 G>T and TYMS 3R2R, 3R G>C polymorphisms among the patients with different types of cancer compared to the control group.
[14]08 -2.84) in the presence of at least one copy of the 2756 A allele.The association with breast cancer risk was found for the Brazilian[14], Indian