RESUMO
BACKGROUND: Myeloproliferative neoplasms (MPNs) include polycythemia vera (PV), essential thrombocythemia (ET), and primary myelofibrosis (PMF). Somatic mutations in exon 10 of the MPL (myeloproliferative leukemia virus oncogene) gene, mainly substitutions encoding W515 variants, have recently been described in a minority of patients with ET or PMF. We optimized analytically sensitive methods for detecting and genotyping MPL variants. METHODS: We used DNA previously isolated from circulating granulocytes of 60 patients with MPN that had previously been analyzed by high-resolution melting (HRM), direct sequencing, and the TaqMan allelic-discrimination assay. We developed conditions for enriching tumor mutant alleles with COLD-PCR (coamplification at lower denaturation temperature PCR) and coupled it with direct sequencing. Assays were designed for identifying MPL W515 substitutions with full COLD-PCR protocols. In parallel, we used innovative microarray substrates to develop assays for evaluating the mutant burden in granulocyte cells. RESULTS: Mutations that were present at very low levels in patients who had previously been scored as having an MPL variant by HRM and as wild type by direct sequencing were successfully identified in granulocyte DNA. Notably, the microarray approach displayed analytical sensitivities of 0.1% to 5% mutant allele, depending on the particular mutation. This analytical sensitivity is similar to that obtained with COLD-PCR. The assay requires no enrichment strategy and allows both the characterization of each variant allele and the evaluation of its proportion in every patient. CONCLUSIONS: These procedures, which are transferable to clinical diagnostic laboratories, can be used for detecting very low proportions of minority mutant alleles that cannot be identified by other, conventional methods.
Assuntos
Primers do DNA , Trombopoetina/genética , Éxons , Genótipo , Humanos , Mutação , Análise de Sequência com Séries de Oligonucleotídeos/métodos , Reação em Cadeia da Polimerase/métodos , Mielofibrose Primária/genética , Trombocitemia Essencial/genéticaRESUMO
Somatic mutations of MPL exon 10, mainly involving a W515 substitution, have been described in JAK2 (V617F)-negative patients with essential thrombocythemia and primary myelofibrosis. We used direct sequencing and high-resolution melt analysis to identify mutations of MPL exon 10 in 570 patients with myeloproliferative neoplasms, and allele specific PCR and deep sequencing to further characterize a subset of mutated patients. Somatic mutations were detected in 33 of 221 patients (15%) with JAK2 (V617F)-negative essential thrombocythemia or primary myelofibrosis. Only one patient with essential thrombocythemia carried both JAK2 (V617F) and MPL (W515L). High-resolution melt analysis identified abnormal patterns in all the MPL mutated cases, while direct sequencing did not detect the mutant MPL in one fifth of them. In 3 cases carrying double MPL mutations, deep sequencing analysis showed identical load and location in cis of the paired lesions, indicating their simultaneous occurrence on the same chromosome.
Assuntos
Éxons/genética , Sequenciamento de Nucleotídeos em Larga Escala , Mutação/genética , Transtornos Mieloproliferativos/genética , Receptores de Trombopoetina/genética , Adulto , Idoso , Idoso de 80 Anos ou mais , Sequência de Bases , Feminino , Humanos , Janus Quinase 2/genética , Masculino , Pessoa de Meia-Idade , Análise de Sequência de DNARESUMO
BACKGROUND: Essential thrombocythemia is a chronic myeloproliferative disorder; patients with this disorder have a propensity to develop thrombosis, myelofibrosis, and leukemia. DESIGN AND METHODS: We studied 605 patients with essential thrombocythemia (follow-up 4596 person-years) with the aim of defining prognostic factors for thrombosis, myelofibrosis, and leukemia during follow-up. RESULTS: Sixty-six patients (11%) developed thrombosis with a 10-year risk of 14%. Age >60 years (p<0.001) and a history of thrombosis (p=0.03) were independent risk factors for thrombosis. Progression to myelofibrosis occurred in 17 patients (2.8%) with a 10-year risk of 3.9%. Anemia at diagnosis of essential thrombocythemia was significantly correlated (p<0.001) with progression to myelofibrosis. Leukemia occurred in 14 patients (2.3%) at a median time of 11 years after the diagnosis of essential thrombocythemia; the risk was 2.6% at 10 years. Age >60 years (p=0.02) was significantly correlated with the development of leukemia. Cytotoxic treatment did not imply a higher risk of leukemia. At the time of the analysis, 64 of the 605 patients (10.6%) had died. The 10-year probability of survival was 88%, with a median survival of 22.3 years. Age >60 years (p<0.001) and history of thrombosis (p=0.001) were independent risk factors for survival. CONCLUSIONS: The findings from this study on a large series of patients treated according to current clinical practice provide reassurance that essential thrombocythemia is an indolent disorder and affected patients have a long survival. The main risk is thrombosis, while myelofibrosis and leukemia are rare and late complications.
Assuntos
Leucemia/fisiopatologia , Transtornos Mieloproliferativos/genética , Mielofibrose Primária/fisiopatologia , Trombocitemia Essencial/complicações , Trombose/fisiopatologia , Idoso , Progressão da Doença , Seguimentos , Humanos , Janus Quinase 2/genética , Leucemia/epidemiologia , Leucemia/etiologia , Pessoa de Meia-Idade , Mutação , Mielofibrose Primária/epidemiologia , Mielofibrose Primária/etiologia , Prognóstico , Fatores de Risco , Trombose/epidemiologia , Trombose/etiologia , Fatores de TempoRESUMO
BACKGROUND: A somatic gain-of-function mutation of the Janus kinase 2 (JAK2) gene has been identified in chronic myeloproliferative disorders, which appear to have a sporadic occurrence in most individuals. The authors studied the biologic significance of the JAK2 (V617F) mutation in familial myeloproliferative disorders. METHODS: Twenty pedigrees with familial chronic myeloproliferative disorders were identified through an investigation of family history in 264 patients with sporadic myeloproliferative disorders. A quantitative real-time polymerase chain reaction (qRT-PCR)-based allelic discrimination assay was employed for the detection of the V617F mutation in circulating granulocytes and T lymphocytes. An analysis of X-chromosome inactivation pattern was performed in female patients. RESULTS: Fourteen families had homogeneous phenotypes, and 6 families had mixed phenotypes. By using a qRT-PCR-based allelic discrimination assay, the JAK2 (V617F) mutation was detected in circulating granulocytes from 20 of 31 patients, but the mutation was not detected in T lymphocytes. Granulocyte mutant alleles ranged from 2.1% to 91.5% and, on average, increased with time. Discordant distribution of the JAK2 (V617F) mutation was observed in siblings with polycythemia vera. The proportion of granulocytes that carried the JAK2 (V617F) mutation was lower than the proportion of clonal granulocytes, as determined in an analysis of X-chromosome inactivation patterns in female patients. CONCLUSIONS: The current findings indicated that the JAK2 (V617F) mutation represents an acquired somatic mutation in patients with familial chronic myeloproliferative disorders and probably occurs as a secondary genetic event in the background of preexisting clonal hematopoiesis. Thus, a genetic predisposition to acquisition of JAK2 (V617F) is inherited in families with myeloproliferative disorders.
Assuntos
Cromossomos Humanos X/genética , Janus Quinase 2/genética , Mutação de Sentido Incorreto , Transtornos Mieloproliferativos/genética , Linhagem , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Criança , Progressão da Doença , Feminino , Testes Genéticos , Granulócitos/metabolismo , Humanos , Masculino , Pessoa de Meia-Idade , Inativação do Cromossomo X/genéticaRESUMO
Missense mutations in the ferroportin gene (SLC11A3) result in haemochromatosis type 4 [HFE4, Online Mendelian Inheritance in Man (OMIM) reference 606069] or ferroportin disease, an autosomal dominant disorder characterized by predominantly reticuloendothelial iron accumulation. To verify whether HFE4 is caused by defective iron recycling because of loss of functionality of ferroportin, we down-regulated SLC11A gene expression in human macrophages by using small interfering RNAs (siRNAs). Transfection experiments with ferroportin siRNAs resulted in a marked reduction (about two-thirds on average) in ferroportin mRNA levels as detected by quantitative real time polymerase chain reaction. When macrophages were grown in medium supplemented with iron, cells transfected with siRNAs displayed three- to eightfold increases in staining intensities following Perls reaction. These macrophages also showed significant increases in H-ferritin content. The observation that ferroportin mRNA down-regulation to levels compatible with haplo-insufficiency causes increased iron retention and H-ferritin synthesis in cultured macrophages has important implications. First, this indicates that ferroportin levels must be finely regulated in order to maintain cellular iron homeostasis, and that both copies of SLC11A3 must function efficiently to prevent iron accumulation. Second, this observation supports the hypothesis that reticuloendothelial iron overload in patients with ferroportin disease is caused by loss-of-function mutations in the SLC11A3 gene that mainly impair macrophage iron recycling.