Investigation of mitochondrial DNA polymorphisms in patients with hematological malignancy

1Diskapi Yildirim Beyazit Training and Research Hospital, Department of Medical Genetics, Ankara / TURKEY 2Ataturk University, Faculty of Medicine, Department of Medical Genetics, Erzurum / TURKEY 3Ataturk University, Faculty of Medicine Department of Internal Medicine, Division of Hematology, Erzurum / TURKEY 4 Medical Park Antalya Hospital Complex, Department of Internal Medicine, Division of Hematology, Antalya / TURKEY Hematolojik maligniteli hastalarda mitokondriyal DNA polimorfizmlerinin araştırılması mitochondrial DNA polymorphisms Turkish Journal of Clinics and Laboratory


Introduction
Mitochondria are cytoplasmic organelles that play a role in the regulation of many important physiological processes in the cell. It plays a role in the production of ATP required for the cell, modulation of the oxidation-reduction process called redox, production of reactive oxygen species (ROS), control of cytosolic calcium levels and Fe / S biogenesis, intrinsic pathway of apoptosis and the formation of some cytosolic precursors [1,2]. The mitochondrial genome, which accounts for about 0.5-1% of the total DNA in the cell, contains 37 genes. In these genes, one single nucleotide polymorphism (SNP) is observed for every 13 base pair, these SNPs may cause an increased risk of various types of cancer according to the studies [3]. The mechanism underlying the relationship between specific mtDNA SNPs and malignancy susceptibility is still unclear [4]. It has been reported in the literature that sequence variants of mitochondrial NADH dehydrogenase, Cytochrome b, Cytochrome C Oxidase I, 12S rRNA, Cytochrome C Oxidase II and tRNA genes can affect the OXPHOS process, cause an increase in ROS production and eventually initiate carcinogenesis [5][6][7][8].
In this study, we aimed to examine 13 SNPs of these six genes from the mtDNAs of patients diagnosed with hematological malignancy using the Polymerase Chain Reaction-Restriction Fragment Length Polymorphism (PCR-RFLP) method and to investigate the possible importance of these SNPs in cancer etiopathogenesis.

Data of Patients
This study was conducted in Medical Genetics Department of Ataturk University between September 2011 and December 2011 in order to determine the relationship between mtDNA polymorphisms and disease etiology in patients with hematological malignancy. According to the World Health Organization diagnostic criteria [9,10], 80 patients between the ages of 18-82 who were diagnosed with acute myeloid/ lymphoid and chronic myeloid/lymphoid leukemia and 80 healthy individuals between the ages of 18-87 were included in this study. Information on the demographic and clinical characteristics of the patients was obtained from the patients and their medical records.  to the manufacturer's DNA-mini kit (Qiagen, Turkey) protocol. Isolated mtDNA samples were stored at -20°C. DNA quality was measured and standardized by spectrophotometry.

PCR amplification of mtDNA fragment
The SNP regions examined in this study are located in genes that play important roles in the OXHPOS pathway and are classified as "benign and likely benign" according to recent guidelines [11] (Tab.1).
In this research, one 0.5 ml PCR tube was labeled for each individual. 50 µl of the mixture was prepared for PCR. 5 µl DNA solution, 10 µl sterile double distilled water, 14 µl PCR master mix and 1.5 µl primer mix (Metabion, Germany) were used for the area concerned. While preparing the primary mixture; 5 µl of each forward and reverse primer of the relevant region was taken and diluted with 90 µl distilled water (Tab.2).    In this study, XP Thermal Cycler (Bioer, China) device was used. PCR was performed with 35 cycles of denaturation: at 95 ° C for 1 min; annealing: at 58 ° C for 1 min; elongation: at 72 ° C for 1 min". From obtained products, 4 µl of each was taken and mixed with 0.4 µl DNA dye (Metabion, Germany) and it was run at 120 V current for 20 minutes using 1.8% agarose gel in an electrophoresis system (Wealtec, USA). At the gel-imaging system (Syngene, USA) the band lengths were compared with marker DNA (Thermo Fermentas, Turkey) under UV light. It was decided that the PCR process was successful for the samples with the desired band lengths.

Restriction enzymes application
It was used the NCBI database for determine the sequence and location of the gene regions where the mtDNA SNPs to be investigated are located (https://www.ncbi.nlm.nih. gov/nuccore). NEBcutter V2.0 program was selected for determination of suitable enzyme and products to be used for cutting the obtained sequence (http://nc2.neb.com/ NEBcutter2/?name). For each sample 0.5 µl SNP region determination enzyme (Thermo Fermentas, Turkey) and 8 µl of PCR product were mixed. In the condition of 9 µl of distilled water and 3 µl of buffer (Thermo Fermentas, Turkey) were added to this mixture. The resulting mixture was incubated at 37 ° C for 1 night (overnight   The Cytochrome C Oxidase II region 8252 (C>G) polymorphism was observed as homoplasmic in 4 (5%) of the patients and heteroplasmic in 11 (13.75%). In the control group, 3 people (3.75%) were found to be homoplasmic and 10 (12.5%) were heteroplasmic in terms of this polymorphism. No significant difference was found by the statistical comparison of both groups (p = 0.895) (Fig.1).
In terms of 13704 (C>T) polymorphism on ND5 region; only 1 (1.25%) person in the control group and 12 (15%) people in the patient group were found to be homoplasmic. Homoplasmy is statistically significant in the patient group compared to the healthy controls group (p=0.001). This polymorphism showed no significant difference between patient subgroups and gender (Fig.2).

Discussion
Most of the human mitochondrial genome contains genes that encode as opposed to the nuclear genome, and the evolutionary rate of the mitochondrial genome is 5-10 times greater than the nuclear genome. Therefore, it is thought that mitochondrial genome polymorphisms may contribute to functional differences between individuals in terms of bioenergetic efficiency, metabolic rate, oxygen consumption and ROS production compared to the nuclear genome [12].
It is shown that various polymorphisms could be effective in the carcinogenesis process by increasing ROS production. Increased ROS levels may cause activation of an oncogenic pathway, which is a risk factor in cancer development, as well as changes in the efficacy of an apoptotic reaction that has a protective effect in the last stages of cancer development [13,14]. Many parameters related to nuclear DNA, which is known to be important in the diagnosis and progression of the disease in hematological malignancies, have been defined [15]. In addition, in this disease group, various mutations and polymorphisms associated with the disease have been reported in mitochondrial DNA. In studies investigating the relationship between mtDNA polymorphisms and cancer; it has been argued that these variants may be important for determining DNA damage and individual susceptibility to carcinogens and can be considered as a molecular marker in various types of cancer [16]. These variations have shown that not only the risk of developing cancer, but also the occurrence of cancer-related symptoms, cancer treatment, and disease outcomes [2,17].
The importance of mtDNA variants in hematological malignancies was first described by Clayton et al. in 1967, with a study conducted on leukemia patients. They argued that mtDNA has an important role in the etiology and treatment of acute and chronic leukemias [18]. In subsequent studies, Gatterman et al. suggested that mtDNA variants may contribute to the development of leukemia by increasing clonal expression in myelodysplastic syndrome (MDS) patients [19]. Wulfert et al. found a high proportion of mtDNA variants in patients with MDS and myeloproliferative disease. These variants concentrated in the control region of tRNA and rRNA, it has been detected less in the coding genes regions [20]. Similarly, in the studies of Yao et al. on leukemia patients, mtDNA variations were mostly observed in the control region [21]. In other studies; it has been claimed that the variations detected in D-Loop, the control region of mtDNA, contribute to the carcinogenesis process and are significantly associated with disease progression [22][23][24]. Cerezo et al. argued that various variants detected in mtDNAs of CLL patients may cause mtDNA instability and that these variants may contribute to the tumoral process even if they cannot be shown as the primary cause of CLL [25]. Carew et al. also detected many polymorphisms and mutations in the mtDNAs of CLL patients. In these patients after chemotherapy, there was an increase in mutation density. A positive correlation between superoxide anion production and mtDNA mutations has been observed [26]. Grist et al. in AML and ALL patients [27], Shin et al. in myelodysplasia patients [28], and Monnat et al. in leukemia and lymphoma cell lines [29] found various mutations and SNPs of mtDNA in their studies. Linnartz et al. identified various variants in genes encoding rRNA, tRNA and polypeptide in mtDNAs of AML patients developing secondary MDS and claimed that these variants may have a role in malignancy development [30]. He et al. identified many mtDNA variants in leukemia patients and specifically claimed that a variant in the cytochrome b region could be a clonal marker [31].
In this study, 13 SNPs in six gene of encoding mtDNA in 80 patients with hematological malignancies were examined by PCR-RFLP method. PCR-RFLP method was preferred because it is a reliable, sensitive and inexpensive method that does not require any funding for us. The limitations of this study were the heterogeneity of subgroup diagnoses of hematologic malignancy patients and the relatively small size of our case series, and the inability to rule out environmental and / or individual factors that could cause cancer susceptibility. Although there are mtDNA studies in hematological malignancy in the literature, the selected SNPs of our study were not studied and there is no information about their importance in hematological malignancies. In our study, evaluated SNPs are m.15315C>T, m.740G>A, m.680T>C, m.7319T>C, m.7444G>A, m.7660G>A, m.7975A>G, m.8310T>C, m. 8014A>G, m.8113C>A, m.8152G>A, and all of them were observed normal for the patient and control groups. Although individuals with heteroplasmic and homoplasmic genotype were found in the patient and control groups in the investigation of Cytochrome C Oxidase II-8252 (C>G) polymorphism, it showed no statistically significant (p=0.895).
One of the regions of the oxidative phosphorylation system (OXPHOS) that contains common defects is the last component of the electron transport chain of cytochrome c oxidase and provides the transfer of electrons from cytochrome c to oxygen. Larger-scale studies are necessary to explain the possible role of SNPs in this region as well as other coding gene regions, due to its indisputable importance in OXPHOS in cancer investigation. In our study, ND-5 13704 (C>T) polymorphism of the Complex I region was found to be significantly positive in patients diagnosed with hematological malignancy compared to healthy controls (p=0.001). In literature genomic changes of Complex I have been reported in many cancer patients It has been suggested that SNPs in this region cause oxidative damage by causing an increase in ROS production and may facilitate the development of neoplastic transformation and metastasis [32,33]. The polymorphisms of this gene was studied for the first time by Canter et al. and an increased risk of breast cancer was observed in polymorphic women [32]. This region SNPs have also shown an increased risk of prostate and esophageal cancer [33]. La Biche et al. claimed that the expression of the ND5 Complex I subunit in metastatic lymphoma cells was increased compared to nonmetastatic cells and that increased ND5 expression may play a role in lymphoma metastasis [34,35]. Studies on these Complex I respiratory defects have shown an increase in the levels of bcl-2, an anti-apoptotic protein, and a decrease in Bax, a pro-apoptotic protein, in cells [36,37]. Allegra et al. suggested that Complex I plays an important role in the metabolic processing of carcinogens and alterations in the function of this complex may contribute to the accumulation of mutagens [33]. Vanniarajan et al. claimed that the ND5 gene region is the hot spot region of the mitochondrial genome [38].

Conclusion
In our study, the presence of this polymorphism in the ND5 region, known as a hot spot in the literature, was found to be statistically significantly higher in patients compared to healthy ones. As a result, the 13704 (C>T) polymorphism in the ND5 region may play a role in the carcinogenesis process and contribute to the formation of hematological malignancy.