RESUMO
INTRODUCTION: Diagnosing BCR-ABL negative myeloproliferative neoplasms (MPN) may be challenging due to overlapping features and lack of robust discriminatory parameters, especially between essential thrombocythemia (ET) and prefibrotic myelofibrosis (MF). Circulating immature hematopoietic cells are variably present in polycythemia vera (PV), ET, and MF. The C-type lectin hMICL is aberrantly expressed on hematopoietic stem cells in the majority of acute myeloid leukemia patients. However, the hMICL expression in MPN, having varying propensity of leukemic transformation, is unsettled. We hypothesized that enumeration of immature cells by flow cytometry (FCM) could be a discriminatory tool in MPN diagnostics. METHODS: By FCM, we quantified circulating stem cells with aberrant hMICL expression in 39 MPN patients, 10 age-matched controls, and in leukapheresis products from 10 patients with lymphoproliferative neoplasms. The utility of the FCM assay for discriminating MPN entities was evaluated by applying ROC curve analysis. RESULTS: While hMICL was absent in control samples, MF patients had significantly more hMICL+ stem cells (median 15.2%) than PV and ET (0.0%, P = .001 and 0.0%, P = .002, respectively). By ROC curve analysis, the presence of hMICL+ stem cells (>0 cells) in peripheral blood reliably discriminates MF from ET and PV with a sensitivity of 80% and a specificity of 97%. CONCLUSION: Enumeration of circulating hMICL+ stem cells by FCM can discriminate between MPN phenotypes and holds potential for monitoring disease evolution.
Assuntos
Lectinas Tipo C/análise , Células Neoplásicas Circulantes/metabolismo , Mielofibrose Primária/diagnóstico , Receptores Mitogênicos/análise , Células-Tronco/patologia , Adulto , Idoso , Estudos de Casos e Controles , Contagem de Células , Diagnóstico Diferencial , Citometria de Fluxo , Humanos , Pessoa de Meia-Idade , Células Neoplásicas Circulantes/patologia , Policitemia Vera/diagnóstico , Trombocitemia Essencial/diagnósticoRESUMO
Silencing of the cyclin-dependent kinase inhibitor gene p15INK4B by cytosine methylation of the promoter region has been associated with some types of hematological malignancy. To study in detail the patterns of p15INK4B methylation in patients with acute myeloid leukemia, we adopted a novel approach based on PCR amplification of bisulfite-treated DNA followed by resolution of differentially methylated sequences by denaturing gradient gel electrophoresis. This method visually displays the degree and heterogeneity of DNA methylation and enables the isolation and characterization of distinct clonotypic epigenotypes. A surprisingly high degree of intra- and interindividual heterogeneity of p15INK4B methylation was observed in the 65 acute myeloid leukemia patients examined. Methylation was detected in 46 (71%) of the patients and was observed more frequently in the French-American-British subtypes M1/M2 than in M4/M5 (P < 0.025). Examination of the same panel of samples using a highly sensitive methylation-specific PCR method showed methylated p15INK4B alleles in 61 (94%) of the samples. We present evidence that the higher frequency of p15INK4B methylation determined by methylation-specific PCR may, at least in part, be due to the presence of a small fraction of p15INK4B-methylated lymphocytes in normal blood.
Assuntos
Proteínas de Ciclo Celular , Metilação de DNA , Leucemia Mieloide Aguda/genética , Fatores de Transcrição/genética , Proteínas Supressoras de Tumor , Alelos , Sequência de Bases , Inibidor de Quinase Dependente de Ciclina p15 , Humanos , Leucócitos Mononucleares/metabolismo , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , Regiões Promotoras GenéticasRESUMO
Serial quantification of BCR-ABL1 mRNA is an important therapeutic indicator in chronic myeloid leukaemia, but there is a substantial variation in results reported by different laboratories. To improve comparability, an internationally accepted plasmid certified reference material (CRM) was developed according to ISO Guide 34:2009. Fragments of BCR-ABL1 (e14a2 mRNA fusion), BCR and GUSB transcripts were amplified and cloned into pUC18 to yield plasmid pIRMM0099. Six different linearised plasmid solutions were produced with the following copy number concentrations, assigned by digital PCR, and expanded uncertainties: 1.08±0.13 × 10(6), 1.08±0.11 × 10(5), 1.03±0.10 × 10(4), 1.02±0.09 × 10(3), 1.04±0.10 × 10(2) and 10.0±1.5 copies/µl. The certification of the material for the number of specific DNA fragments per plasmid, copy number concentration of the plasmid solutions and the assessment of inter-unit heterogeneity and stability were performed according to ISO Guide 35:2006. Two suitability studies performed by 63 BCR-ABL1 testing laboratories demonstrated that this set of 6 plasmid CRMs can help to standardise a number of measured transcripts of e14a2 BCR-ABL1 and three control genes (ABL1, BCR and GUSB). The set of six plasmid CRMs is distributed worldwide by the Institute for Reference Materials and Measurements (Belgium) and its authorised distributors (https://ec.europa.eu/jrc/en/reference-materials/catalogue/; CRM code ERM-AD623a-f).
Assuntos
Proteínas de Fusão bcr-abl/genética , Proteínas de Fusão bcr-abl/metabolismo , Plasmídeos/genética , Reação em Cadeia da Polimerase em Tempo Real/normas , Calibragem , Clonagem Molecular , DNA , Proteínas de Escherichia coli/genética , Dosagem de Genes , Humanos , Proteínas de Membrana Transportadoras/genética , Proteínas Proto-Oncogênicas c-bcr/genética , RNA Mensageiro/metabolismo , Padrões de ReferênciaAssuntos
Metilação de DNA , DNA de Neoplasias/metabolismo , Regulação Leucêmica da Expressão Gênica , Leucemia Linfocítica Crônica de Células B/metabolismo , Linfoma de Célula do Manto/metabolismo , Proteínas de Neoplasias/biossíntese , Proteínas do Tecido Nervoso/biossíntese , Receptores Imunológicos/biossíntese , Feminino , Humanos , Leucemia Linfocítica Crônica de Células B/patologia , Linfoma de Célula do Manto/patologia , Masculino , Proteínas RoundaboutRESUMO
BACKGROUND: Previous studies suggest a deleterious effect of cigarette smoking on semen quality, but their results have not been consistent. We studied the association between current smoking and semen characteristics and hormonal levels in a large group of healthy men. METHODS: From 1987 to 2004, seven separate occupational or environmental semen quality studies were co-ordinated by our department. A total of 2562 men participated, each providing semen and blood sample and answering a questionnaire about lifestyle and factors related to health. Appropriate semen and smoking data were available for 2542 men. RESULTS: Adjusting for study, age and other covariates, we observed an inverse dose-response relation between smoking and semen volume, total sperm count and percentage motile sperm. Heavy smokers had a 19% lower sperm concentration than non-smokers. We found a positive dose-response relationship between smoking and testosterone, LH and the LH/free testosterone ratios. CONCLUSION: Current smoking in adult life moderately impairs the semen quality. It is well known that semen quality is associated to fecundity. Therefore, it would be sensible to advise men to abstain from smoking to avoid decreased fecundity.
Assuntos
Sêmen/citologia , Sêmen/fisiologia , Fumar/efeitos adversos , Adulto , Azoospermia/etiologia , Estudos Transversais , Humanos , Masculino , Razão de Chances , Oligospermia/etiologia , Fatores de Risco , Contagem de EspermatozoidesRESUMO
BACKGROUND: Most PCR assays for detection of 5-methylcytosine in genomic DNA entail a two-step procedure, comprising initial PCR amplification and subsequent product analysis in separate operations that usually require manual transfer. These methods generally provide information about methylation of only a few CpG dinucleotides within the target sequence. METHODS: An in-tube methylation assay is described that integrates amplification of bisulfite-treated DNA and melting analysis by using a thermal cycler coupled to a fluorometer (LightCycler). DNA melting curves were acquired by measuring the fluorescence of a double-stranded DNA-binding dye (SYBR Green I) during a linear temperature transition. RESULTS: Analysis of a region comprising 11 CpG sites at the SNRPN promoter CpG island showed that the melting temperature (T(m)) differed by approximately 3 degrees C between unmethylated and fully methylated alleles. This assay could easily distinguish patients with Prader-Willi syndrome or Angelman syndrome from individuals without these conditions. Melting curve analysis also allowed resolution of methylation "mosaicism" at the p15(Ink4b) promoter in bone marrow samples from patients with acute myeloid leukemia (AML). AML samples representing pools of heterogeneously methylated p15(Ink4b) alleles showed broadened melting peaks with overall T(m)s between those of the unmethylated and fully methylated alleles. CONCLUSIONS: Integration of PCR and fluorescence melting analysis may be useful for simple and cost-effective detection of aberrant methylation patterns.
Assuntos
Proteínas de Ciclo Celular , Inibidor p16 de Quinase Dependente de Ciclina , Impressões Digitais de DNA/métodos , Proteínas Supressoras de Tumor , Doença Aguda , Síndrome de Angelman/genética , Autoantígenos/genética , Medula Óssea/metabolismo , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Inibidor de Quinase Dependente de Ciclina p15 , Metilação de DNA , Fluorometria , Humanos , Indicadores e Reagentes , Leucemia Mieloide/genética , Reação em Cadeia da Polimerase/métodos , Síndrome de Prader-Willi/genética , Ribonucleoproteínas Nucleares Pequenas/genética , Sulfitos , Proteínas Centrais de snRNPRESUMO
The candidate tumour suppressor gene MMAC1/PTEN located at chromosome 10q23.3 has been reported to be frequently mutated in a number of solid tumours. Less is known about its status in leukaemia. In the present study we first analysed 13 leukaemia cell lines for mutations and homozygous deletions in MMAC1/PTEN using PCR and denaturing gradient gel electrophoresis (DGGE). We identified an intragenic deletion including MMAC1/PTEN exons 2-5 in an acute myelocytic leukaemia cell line, HL-60 blast, and an insertion of four nucleotides in exon 5 in an acute monocytic leukaemia cell line, U937. Analysis of 59 patients with acute myeloid leukaemia (AML), 26 patients with myelodysplastic syndromes (MDS) and 10 patients with chronic myeloid leukaemia (CML) only revealed a polymorphic base substitution in codon 44 in one AML patient, suggesting that mutations in the MMAC1/PTEN gene are infrequent genetic aberrations in myeloid leukaemia.
Assuntos
Genes Supressores de Tumor/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mieloide/genética , Síndromes Mielodisplásicas/genética , Monoéster Fosfórico Hidrolases/genética , Proteínas Supressoras de Tumor , Doença Aguda , Sequência de Bases , Análise Mutacional de DNA , DNA de Neoplasias/genética , Eletroforese em Gel de Ágar , Éxons/genética , Deleção de Genes , Células HL-60 , Humanos , Mutação , PTEN Fosfo-Hidrolase , Reação em Cadeia da Polimerase , Células Tumorais Cultivadas , Células U937RESUMO
Denaturing gradient gel electrophoresis (DGGE) in combination with PCR and 'GC-clamping' has proven highly efficient as a method for detection of DNA sequence differences. Due to strand dissociation phenomena, however, its use has been limited to the analysis of sequences with a relatively low content of GC pairs. This paper describes how treatment of template DNA with sodium bisulphite drastically lowers the melting temperature of very GC-rich sequences and renders them amenable to DGGE analysis. We demonstrate the use of bisulphite DGGE for rapid and efficient detection of mutations in the p16(INK4/CDKN2) tumour suppressor gene.