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Previous issue date: 2010-06-30 / Objetivos: Descrever as alteracoes cromossomicas em policitemia vera (PV), trombocitemia essencial (TE) e mielofibrose primaria (MF). Verificar se a taxa de anormalidades cromossomicas e ampliada pela FISH nos casos com cariotipo normal e ausencia de metafases. Detectar a incidencia da mutacao JAK2 V617F em PV, TE e MF; de mutacoes no exon 12 do JAK2 em PV e de mutacoes MPL W515K/L em TE e MF. Correlacionar as alteracoes citogeneticas e moleculares encontradas com o grau de fibrose medular nos casos de MF; numero de leucocitos, plaquetas, hemoglobinas; e idade, ao diagnostico, em todos pacientes. Metodo: O estudo foi realizado em 20 casos de PV, 17 de TE e 21 de MF. O cariotipo por banda G foi realizado em amostras de medula ossea, semeadas em cultura de curta duracao (24h), sem mitogenos e processadas de forma habitual (Chauffaille, 2006). Parte da amostra (1mL) foi destinada a FISH, com sondas para as regioes: 20q12, 20q13.12, 13q14.3, 13qter (subtelomerica), 8p11.1-q11.1 (ƒ¿-satellite) e 9q12 (satellite III). A pesquisa das mutacoes JAK2V617F e MPL W515K/L foi realizada em DNA de sangue periferico, por PCR em tempo real, utilizando-se o kit JAK2 MutaScreenTM (Ipsogen). A pesquisa de mutacoes no exon 12 do JAK2 foi realizada por sequenciamento direto. Resultados: As alteracoes cromossomicas foram observadas em 11,8% das PV, 17,6% das MF e nenhuma das TE, nao havendo relacao entre dados clinicos avaliados e alteracoes cromossomicas. As anormalidades cromossomicas nao foram ampliadas pela FISH. JAK2 V617F foi observada em 90% das PV, 42,8% das MF e 47% das TE. Os pacientes com PV JAK2 V617F negativos apresentaram menores niveis de plaquetas em relacao aos PV V617F positivos (p<0,0001). MF V617F positivos apresentaram maiores graus de fibrose do que os V617F negativos (p=0,003). Nao foi detectada a presenca de mutacoes no exon 12 do JAK2 em pacientes com PV. MPL W515L foi observada em um caso de MF e em um de TE. Nao foi encontrada a mutacao MPL W515K nos pacientes com TE e MF. A paciente com TE MPL W515L positivo nao apresentou quadro clinico diferente dos demais pacientes com TE, enquanto que a paciente com MF MPL W515L positivo apresentou quadro clinico mais agressivo quando comparada aos demais pacientes com MF. O numero de alteracoes clonais nao mostrou diferenca quanto aos dados clinicos avaliados. Conclusoes: Os diferentes tipos de alteracoes clonais em neoplasias mieloproliferativas exaltam seus diferentes mecanismos fisiopatogenicos, auxiliando no diagnostico e compreensao da biologia destas doencas. Este estudo permitiu a caracterizacao citogenetica e molecular de PV, MF e TE. / Introcuction: Polycythemia vera (PV), essential thrombocythemia (ET) and myelofibrosis (MF) are clonal disorders of hematopoietic stem cell and clinical and biological aspects have in common that hinder their diagnosis, however, cytogenetic and molecular studies represent important tools to assist in this procedure. Therefore, the investigation of the presence of JAK2 V617F, mutations in exon 12 of JAK2, MPL W515K of MPLW515L and cytogenetic alterations per karyotype and FISH can provide a more detailed view for the diagnosis and prognosis of these diseases. In this study such cytogenetic and molecular changes were correlated with degree of fibrosis in cases of MF, the number of leukocytes, platelets, hemoglobin and age at diagnosis in PV, ET and MF Method: The karyotype by G-band was performed on samples of bone marrow, grown in culture for short duration (24h) without mitogens and processed as usual (Chauffaille, 2006). The sample (1 ml) was intended to FISH with probes for the regions: 20q12, 20q13.12, 13q14.3, 13qter, 8p11.1-q11.1 and 9q12. The investigation of JAK2 V617F and MPL W515K/L mutations was performed on DNA from peripheral blood by real time PCR, using the kit MutaScreenTM JAK2 (IPSOGEN). The search for mutations in exon 12 of JAK2 was performed by direct sequencing. Results: Chromosomal abnormalities were observed in 11.8% of PV, 17.6% of MF and none of the ET, no relation between clinical data and assessed chromosomal alterations. Chromosomal abnormalities were not amplified by FISH. JAK2 V617F was observed in 90% of PV, 42.8% of MF and 47% of ET. Patients with JAK2 V617F negative PV showed lower levels of platelets in relation to V617F positive PV (p <0.0001). MF V617F negative and MPL W515L positive showed higher degrees of fibrosis than V617F negative (p =0.003). Was not detected the presence of mutations in exon 12 of JAK2 in PV patients. MPL W515L was observed in a case of MF and a TE. No MPL W515K mutation was found in patients with ET and MF. The ET patient MPL W515L positive showed no clinical different from other patients with ET, whereas the patient with MF MPL W515L showed positive clinical more aggressive when compared to other patients with MF. The number of clonal abnormalities showed no difference in the clinical data evaluated. Conclusions: Different types of clonal abnormalities in myeloproliferative neoplasms exalt their different pathophysiological mechanism, aiding in the diagnosis and understanding of the biology of these diseases. This study allowed the cytogenetics and molecular characterization of PV, MF and ET. / TEDE / BV UNIFESP: Teses e dissertações
| Identifer | oai:union.ndltd.org:IBICT/oai:repositorio.unifesp.br:11600/9496 |
| Date | 30 June 2010 |
| Creators | Santos, Leonardo Caires dos [UNIFESP] |
| Contributors | Universidade Federal de São Paulo (UNIFESP), Chauffaille, Maria de Lourdes Lopes Ferrari [UNIFESP] |
| Publisher | Universidade Federal de São Paulo (UNIFESP) |
| Source Sets | IBICT Brazilian ETDs |
| Language | Portuguese |
| Detected Language | English |
| Type | info:eu-repo/semantics/publishedVersion, info:eu-repo/semantics/masterThesis |
| Format | 133 p. |
| Source | reponame:Repositório Institucional da UNIFESP, instname:Universidade Federal de São Paulo, instacron:UNIFESP |
| Rights | info:eu-repo/semantics/openAccess |
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