RESUMEN
In acute myeloid leukaemia (AML), alterations in apoptotic pathways are crucial for treatment outcome, resulting either in refractoriness or in minimal residual disease (MRD). The apoptosis characteristics of MRD cells may differ from those at diagnosis and thereby determine the adequacy of further treatment. Such characteristics are largely unknown, since studies hereto are hampered by minimal cell availability. This study explores the applicability of the recently described RT-Multiplex Ligation-dependent Probe Amplification (RT-MLPA) for gene expression analysis of small amounts of RNA obtained from MRD cells. Reproducibility and dilution experiments showed that the relative expression of 37 apoptosis-related genes starting with only 1000 cells could be measured with 12% variation; for 100 cells, 31/37 genes could still be quantified, though expression variation increased. In practice 100-1000 leukemic cells can be obtained from bone marrow samples with clinically relevant MRD percentages of 0.01-0.1. Procedures often necessary to obtain AML blasts, that is, FACS-sorting, freeze-thawing or combinations are possible, provided that selected viable nonapoptotic cells are used. Concluding, RT-MLPA allows accurate gene expression profiling of MRD cells. This method will help to gain insight into the processes of MRD emergence and persistence in AML, which may ultimately guide new therapeutic strategies in AML.
Asunto(s)
Perfilación de la Expresión Génica , Leucemia Mieloide/genética , Neoplasia Residual/genética , Reacción en Cadena de la Polimerasa/métodos , Enfermedad Aguda , Humanos , Leucemia Mieloide/diagnóstico , Neoplasia Residual/diagnóstico , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Neoplásico/genética , ARN Neoplásico/metabolismo , Sensibilidad y EspecificidadRESUMEN
Hereditary non-polyposis colorectal cancer is an autosomal dominant condition due to germline mutations in DNA-mismatch-repair genes, in particular MLH1, MSH2 and MSH6. Here we describe the application of a novel technique for the detection of genomic deletions in MLH1 and MSH2. This method, called multiplex ligation-dependent probe amplification, is a quantitative multiplex PCR approach to determine the relative copy number of each MLH1 and MSH2 exon. Mutation screening of genes was performed in 126 colorectal cancer families selected on the basis of clinical criteria and in addition, for a subset of families, the presence of microsatellite instability (MSI-high) in tumours. Thirty-eight germline mutations were detected in 37 (29.4%) of these kindreds, 31 of which have a predicted pathogenic effect. Among families with MSI-high tumours 65.7% harboured germline gene defects. Genomic deletions accounted for 54.8% of the pathogenic mutations. A complete deletion of the MLH1 gene was detected in two families. The multiplex ligation-dependent probe amplification approach is a rapid method for the detection of genomic deletions in MLH1 and MSH2. In addition, it reveals alterations that might escape detection using conventional diagnostic techniques. Multiplex ligation-dependent probe amplification might be considered as an early step in the molecular diagnosis of hereditary non-polyposis colorectal cancer.
Asunto(s)
Neoplasias Colorrectales Hereditarias sin Poliposis/genética , Neoplasias Colorrectales/genética , Proteínas de Unión al ADN , Eliminación de Gen , Proteínas de Neoplasias/genética , Proteínas Proto-Oncogénicas/genética , Proteínas Adaptadoras Transductoras de Señales , Disparidad de Par Base/genética , Proteínas Portadoras , Estudios de Cohortes , Reparación del ADN/genética , Familia , Femenino , Mutación de Línea Germinal , Humanos , Masculino , Homólogo 1 de la Proteína MutL , Proteína 2 Homóloga a MutS , Proteínas Nucleares , Linaje , Reacción en Cadena de la PolimerasaRESUMEN
Several new loci were identified by a comprehensive analysis of loss of heterozygosity (LOH) using a subtraction library between matched normal and renal cell carcinoma (RCC) tissues. A total of 187 clones from the library, with a complexity of 1x10(4), were mapped, and 44 clusters of the mapped loci were subjected to LOH analysis using microsatellite markers. A total of 27 loci, which exhibited frequencies of LOH of at least 10% among 44 tumors, mostly clear-cell RCC, included several loci that were reported previously, such as, the von Hippel-Lindau gene, adenomatous polyposis coli, and interferon regulatory factor-1, as well as new loci, at 5q32-q34, 6q21-q22, 8p12, and others. These loci exhibited LOH among 11.8-93.8% of tumors, and most, if not all, were derived from the sites of hemizygous deletions. The minimum regions of LOH of chromosomes 5, 6, and 8 were 9.0, 10.3, and 0.775 Mb, respectively. The average distance between the cloned fragments on the chromosomes was 2.2 Mb in 187 clones, indicating that the minimum LOH size expected from this subtraction analysis was roughly 50 kb. Therefore, the strategy described here provides comprehensive analysis of LOH sites, which were mostly caused by hemizygous deletions.