Ανάλυση της έκφρασης του γονιδίου PRV1 στην αληθή πολυκυτταραιμία και τα θαλασσαιμικά σύνδρομα ως δείκτη ερυθροποίησης: μελέτη της πιθανής εμπλοκής του στους παθογενετικούς μηχανισμούς των ΧΜΥΣ

Abstract

Chronic Myeloproliferative Disorders CMPDs, which were first reported by Dameshek in 1951, are clonal hematopoietic stem cell disorders characterized by proliferation of 1 or more myeloid cell lineages in the bone marrow and increased numbers of mature or immature cells in peripheral blood. According to the Word Health Organization, CMPDs or Myeloproliferative Neoplasms MPNs, include Polycythemia Vera (PV), Essential Thrombocythemia (ET), Idiopathic Myelofibrosis (IMF), plus rarer subtypes such us chronic neutrophilic leukaemia (CNL), hypereosinophilic syndrome (HES) and chronic eosinophilic leukaemia (CEL). The identification in 2005 of the V617F mutation in the JAK2 gene in Chronic Myeloproliferative Disorders (CMPDs) or Myeloproliferative Neoplasms (MPNs ) may explain several characteristics of these diseases and provide a prognostic marker in clinical practice. The JAK2V617F mutation is present in 90% of Polycythemia Vera, PV, patients and in about a half of Essential Thrombocythemia, ET, and Idiopathic Myelofibrosis, IMF, patients respectively. The purpose of this study was the investigation of the V617F mutation of the JAK2 and the investigation of the over expression of PRV-1 gene in our patients. Also we try to understand the mechanism of the over expression of PRV-1 gene in the signalling pathway and the role in the pathogenesis of PV patients. To achieve this goal we used specific tyrosine kinase inhibitors, in specific cell lines (K562, HEL), such us Imatinib, Dasatinib, Sutent, Sorafenib and Erlotinib. Another purpose of this study was the investigation of the over expression of PRV-1 gene in patients, negative for the for the JAK2V617F mutation, and positive for the mutation in the exon 12 of the JAK2 gene. Also another purpose, was the investigation of the over expression of PRV-1 gene in patients, negative for the for the JAK2V617F mutation, and positive for the mutation in the thrombopoietin receptor c-MPL We took advantage of our patients and analyzed them for the identification of the V617F mutation and for the mutation load of the alleles carrying the V617F 205 mutation. The identification of the V617F mutation was detected by Amplification Refractory Mutation System-polymerase chain reaction (ARMs- PCR) assay performed in DNA samples extracted from mononuclear cells from the peripheral blood. This PCR assay allows detection of the mutation with sensitivity about 1-2% (Jones et al, 2005). The conclusions of our study: ?? Investigation of the JAK2V617F mutation and over expression of the PRV-1 gene in all our samples. Over expression of the PRV-1 in the positive homozygote samples more than heterozygote sample. (p : 0.0004). ?? Investigation of the mutation in the exon 12 of the JAK2 gene. Over expression of the PRV-1 gene in these 5 positive samples, similarity with the over expression observed in the positive PV samples for the JAK2V617F mutation. ?? Investigation of the mutation in the thrombopoietin receptor c-MPL (24 positive samples- 23 with ET and 1 with IMF). Also 20 patients were positive for the W515L and 4 for the W515K mutation. Over expression of the PRV-1 gene in these 24 positive samples, similarity with the over expression observed in the positive ET and IMF samples for the JAK2V617F mutation. ?? The purpose of this study was the investigation of the role of the HU in 7 PV patients and the role of this agent in the reduction of the over expression of the PRV-1 gene. We found that HU can not reduced the mutation load of the JAK2V617F mutation and it has none effect in the over expression of the PRV-1 gene. The conclusion, of the purpose of this study, has been carried out in 5 PV patients treated with the pegylated forms of the IFN-a-2a. The reduction of the mutation load started during the first year and it became undetectable after the 24 months of treatment a trend reported in (Kiladjian et al, 2008). Also we observed a reduction of the over expression of the PRV-1 gene similarity with the reduction of the JAK2V617 F mutation. In the case in which the reduction of the JAK2V617 F mutation 206 it became undetectable the level of the PRV-1 was normal. The level of these two markers remain lower after the last treatment for a few months ?? .Investigation of the mechanism of the over expression of PRV-1 gene in the signalling pathway. To achieve this goal we used specific tyrosine kinase inhibitors, in specific cell lines (K562, HEL), such us Imatinib, Dasatinib, Sutent, Sorafenib and Erlotinib. We found effect in the growth of the cell line K562 by the agent Imatinib, Dasatinib, Sutent, Sorafenib and not by the drug Erlotinib. We did not observed any effect at the over expression of the PRV-1 gene. Also we searched the effect of these drug in the cell line HEL. We found a positive effect in the growth of the cell line HEL and in the over expression of the PRV-1 gene by the drug Dasatinib, Sutent, Sorafenib.