RESUMO
To determine efficiently the clonality of B-cell lymphoproliferative disorders, we modified an immunoglobulin heavy-chain (IGH) gene rearrangement polymerase chain reaction (PCR) assay that requires only a single primer germline variable (VH) and joining (JH) pair and does not involve nested priming, blot hybridization, radioactivity, or sequencing of the amplified PCR product. This simple PCR technique enabled detection of IGH gene rearrangements in as little as 10 pg (one cell equivalent) of DNA or when the clonal-to-polyclonal B-cell ratio was experimentally set at 1:1000. We detected IGH gene rearrangements in 83.5% (71 of 85) of clonal B-cell processes, a sensitivity approaching that of more cumbersome multiple primer and nested primer assays. Moreover, this technique is equally effective with fixed tissues, either B5 or formalin, and can be performed on minute samples, histologic sections, fine-needle aspirates, or cerebrospinal fluids. When compared with conventional Southern blot analysis using a genomic JH probe, the PCR assay demonstrated IGH gene rearrangements in 82% (37 of 45) of B-cell processes positive by Southern blot. No false-positive results were observed in 29 negative control tissues. We now use IGH gene PCR routinely in our laboratory for the detection of clonal B-cells in virtually any tissue sample as an aid in early diagnosis, staging, and monitoring, and the Southern blot procedure is reserved for only a minority of diagnostic cases. for only a minority of diagnostic cases.
Assuntos
Southern Blotting/métodos , Rearranjo Gênico de Cadeia Pesada de Linfócito B/genética , Reação em Cadeia da Polimerase/métodos , Linfócitos B/imunologia , Sequência de Bases , DNA de Neoplasias/genética , Humanos , Transtornos Linfoproliferativos/genética , Transtornos Linfoproliferativos/imunologia , Dados de Sequência Molecular , Sensibilidade e EspecificidadeRESUMO
Acute promyelocytic leukemia (APML) almost always involves a chromosomal translocation t(15:17) that results in the fusion of the retinoic acid receptor alpha (RAR alpha) gene with a transcription factor gene called PML. Several cases of APML with t(11;17) have recently been described, involving fusion of the RAR alpha gene with a new zinc finger gene named PLZF. We report here a second non-classical translocation, t(5;17), with a rearranged RAR alpha gene in a child with APML. Based on restriction endonuclease analysis, the rearrangement of RAR alpha occurred within the second intron, the common breakpoint site for t(15;17). The leukemic cells in the bone marrow aspirate were a mixture of hypergranular and hypogranular bilobed promyelocytes. Although less than 1% abnormal promyelocytes were identified after induction therapy, cytogenetics revealed persistent t(5;17). Therefore, the child was treated with all-trans-retinoic acid (ATRA). There was no disease progression, and one marrow was interpreted as remission, with confirmation by cytogenetics which failed to reveal the translocation. However, disease reoccurred shortly after completion of ATRA. This poor response to ATRA may be an additional characteristic associated with non-classical translocations in APML. The identification of a second variant translocation involving the RAR alpha gene in APML suggests yet another RAR alpha rearrangement related to neoplastic myelopoiesis.
Assuntos
Cromossomos Humanos Par 17 , Leucemia Promielocítica Aguda/genética , Receptores do Ácido Retinoico/genética , Translocação Genética , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Medula Óssea/patologia , Pré-Escolar , Cromossomos Humanos Par 11 , Cromossomos Humanos Par 15 , Citarabina/administração & dosagem , Daunorrubicina/administração & dosagem , Etoposídeo/uso terapêutico , Feminino , Humanos , Cariotipagem , Leucemia Promielocítica Aguda/tratamento farmacológico , Leucemia Promielocítica Aguda/patologia , Receptor alfa de Ácido Retinoico , Tioguanina/administração & dosagemRESUMO
Standard electrophoretic methods for the diagnosis of hemoglobinopathies are confounded in individuals chronically transfused. We present the accurate diagnosis of sickle cell disease in two such transfused patients by the application of polymerase chain reaction technology to analyze patient's hemoglobin beta-chain genes directly.
Assuntos
Anemia Falciforme/diagnóstico , Reação Transfusional , Anemia Falciforme/sangue , Anemia Falciforme/genética , Sequência de Bases , Criança , Pré-Escolar , Fatores de Confusão Epidemiológicos , DNA/análise , Feminino , Genoma Humano , Hemoglobinas/genética , Humanos , Transplante de Rim , Masculino , Ácido Metilmalônico/sangue , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Fatores de TempoRESUMO
Mutants of Drosophila melanogaster that are sensitive to chemical mutagens were analyzed for sensitivity to X-rays and for the capacity to repair single-strand DNA breaks induced by X-rays. Analysis of X-ray sensitivity demonstrated that 74% of the mutants assayed display some X-ray sensitivity, with 75% of the sensitive lines being extremely sensitive. Repair of single-strand breaks was assayed after both high and low doses of irradiation in order to permit detection of repair over a wide range of damage. The results of this investigation fail to show a correlation between X-ray sensitivity and this particular repair process. Repair of single-strand breaks is therefore mediated by repair processes unrelated to those that are disrupted in the current mutant collection.
