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Duration of treatment of venous thromboembolism (VTE) and prevention of its recurrence represent significant problems of contemporary medicine, as the basic method of treatment - anticoagulation is frequently complicated by hemorrhage. Therefore, its duration is strictly defined and depends on existence of risk factors related to recurrence of thrombosis. Purpose of the conducted study was to establish the role of point mutations of prothrombin (PHG) - 20210G/A; Factor V Leiden (FVL) - 1691G/A and methylenetetrahydrofolate reductase (MTHFR) - 677C/T genes, i.e. inherited thrombophilia in the pathogenesis of proximal and distal lower extremity deep vein thrombosis in patients of the Georgian population, as in case of proximal thrombosis there is a higher risk of recurrent thrombosis. The above mutations were detected by PCR and single nucleotide primer extension reaction, followed by Enzyme Linked Immuno-Sorbent Assay (ELISA) in 61 patients with venous thromboembolism of various localizations, out of which: 49 patients were diagnosed with unprovoked proximal thromboembolism confirmed by objective studies and 12 patients were diagnosed with distal thromboembolism. The difference between the groups was evaluated by F (Fisher) precise criterion. According to statistical analysis of the results, incidence of FVL mutation in the group of patients with proximal thrombosis was significantly higher compared to patients with distal thrombosis 0.43 and 0.08 (p=0.0256), respectively. Similar tendencies were observed in case of carriage of prothrombin gene and MTHFR gene mutations, as their presence was higher in the group of patients having proximal thrombosis than in patients with distal thrombosis, however, this difference was not found to be statistically significant. It should be particularly mentioned that double or triple heterozygous or homozygous carriage of studied mutations with various options was confirmed in 15 of 61 patients and the above genotypes were observed only in the group of patients having proximal thrombosis. According to the results obtained from this investigation, it is possible to consider inherited thrombophilia, especially Leiden mutation and double or triple heterozygous and homozygous carriage of studied mutations in the Georgian population as an independent risk factor of development of proximal thrombosis, which on its part, represents one of the risk factors of development of recurrent thrombosis and conduct proper long-standing anticoagulation therapy and take secondary preventive measures in patients of similar genotype to reduce the risk of recurrent thrombosis.

Inherited thrombophilia means a predisposition of an individual to thrombosis caused by genetic disorders of homeostasis system. Purpose of the conducted study was to establish the role of point mutations of prothrombin (PGM) - 20210G/A; Factor V Leiden (FVL) - 1691G/A and methylenetetrahydrofolate reductase (MTHFR) - 677C/T genes, i.e. inherited thrombophilia in the pathogenesis of primary and recurrent venous thromboembolism in patients of the Georgian population. The above mentioned mutations were detected by PCR and single nucleotide primer extension reaction, followed by Enzyme Linked Immuno-Sorbent Assay (ELISA) in 93 patients with venous thromboembolism, out of which: 56 patients were diagnosed with unprovoked, primary thromboembolism confirmed by objective studies and 37 patients were diagnosed with recurrent thromboembolism. According to statistical analysis of the results, incidence of FVL mutation in the group of patients with recurrent thrombosis was significantly higher compared to patients with primary thrombosis - respectively 0.21 and 0.44 (p=0.0164<0.05). It should also be mentioned that homozygous carriage of FVL mutation was confirmed only with patients having recurrent thrombosis. Similar tendency was observed during study of prothrombin gene; however the difference was not statistically significant. Similar tendencies were not observed in case of homozygous carriage of MTHFR gene C677T mutation. Double and triple heterozygous/homozygous carriage of studied mutations (total of 20 cases) was observed in patients of both groups. Distribution of these genotypes in the recurrent thrombosis group was higher compared to patients with primary thrombosis - respectively 27% and 17.9%. Herewith, it should be mentioned that the patients with primary thrombosis were much younger than those with recurrent thrombosis and their age did not exceed 50 years. According to the results obtained by us, it is possible to consider Leiden mutation, especially its homozygous form and double/triple heterozygous/homozygous carriage of the studied mutations as an independent risk factor of development of recurrent thrombosis in the Georgian population and prolong anticoagulation therapy in patients of similar genotype as much as possible to prevent recurrent thrombosis and related complications.

Gene amplification, a key mechanism for oncogene activation and drug resistance in tumour cells, involves the generation and joining of DNA double-strand breaks. Amplified DNA can be carried either on intra-chromosomal arrays or on extra-chromosomal elements (double minutes). We previously showed that, in rodent cells deficient in DNA-PKcs, intra-chromosomal amplification is significantly enhanced. In the present work, we studied gene amplification in human HeLa cell lines in which the expression of the DNA-PKcs gene was constitutively inhibited by shRNAs. These cell lines showed an increased sensitivity to ionizing radiations, an enhanced frequency of chromosomal aberrations and an increased rate of occurrence of methotrexate resistant colonies compared to the control cell lines (6-18 times). The main mechanism of resistance to methotrexate was extra-chromosomal amplification of the dihydrofolate reductase gene. These results indicate that, in human cells, inhibition of DNA-PKcs gene expression favours gene amplification occurring via the production of double minutes. In addition, they show that cell lines constitutively expressing shRNAs are good model systems to study the role of specific functions in gene amplification.