RESUMO
Interferon alpha (IFN-alpha) induces cytogenetic responses of variable degree in patients with CML. We sought to establish the relationship between BCR-ABL transcript numbers measured by competitive two-step reverse transcription polymerase chain reaction (RT-PCR) and cytogenetic status in CML patients treated with IFN-alpha. All 398 samples from 163 patients investigated by RT-PCR were positive for BCR-ABL transcripts. In order to standardize results for variability in RNA and cDNA quality, we quantified total ABL transcripts in each sample as internal control. The BCR-ABL/ABL ratios correlated with the cytogenetic results. Quantitative nested PCR allowed the detection of residual BCR-ABL transcripts in all complete cytogenetic responders on IFN-alpha. We conclude that competitive PCR with internal controls is a reliable method for monitoring patients on IFN-alpha and reduces the need for repeated marrow investigations.
Assuntos
Monitoramento de Medicamentos/métodos , Proteínas de Fusão bcr-abl/análise , Interferon-alfa/uso terapêutico , Leucemia Mielogênica Crônica BCR-ABL Positiva/terapia , Reação em Cadeia da Polimerase/métodos , Medula Óssea/patologia , DNA Complementar , Eritrócitos/patologia , Proteínas de Fusão bcr-abl/biossíntese , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/sangue , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/patologia , Proteínas Proto-Oncogênicas c-abl/análise , Reprodutibilidade dos Testes , Transcrição GênicaRESUMO
The gene (sigA) encoding the major sigma factor of the myxobacterium, Stigmatella aurantiaca, was cloned and sequenced. The deduced polypeptide contains 706 amino acids (aa) and has a deduced M(r) of 79,910. It exhibits four different aa sequence motifs which correlate with the conserved domains of the major sigma factors of Myxococcus xanthus (sigma 80), Escherichia coli (sigma 70) and Bacillus subtilis (sigma 43). The sigma factor (sigma A) was detected in crude lysates of vegetative cells and in cells of different developmental stages from S. aurantiaca with an antiserum to M. xanthus sigma 80 by Western blot analysis. The SigA polypeptide copurified with RNA polymerase from vegetative S. aurantiaca cells. The aa sequence of its N terminus matches a sequence located 25 codons downstream from the proposed start codon. The sigA gene was expressed in E. coli and the corresponding gene product cross-reacted with the SigA antiserum as a polypeptide of 100 kDa, which is identical in size to the sigma A detected in vegetative cells of S. aurantiaca.
Assuntos
Proteínas de Bactérias/genética , RNA Polimerases Dirigidas por DNA/genética , Genes Bacterianos/genética , Myxococcales/genética , Fator sigma/genética , Sequência de Aminoácidos , Proteínas de Bactérias/química , Sequência de Bases , Western Blotting , Clonagem Molecular , Reações Cruzadas , DNA Bacteriano/genética , Dados de Sequência Molecular , Myxococcales/crescimento & desenvolvimento , Proteínas Recombinantes de Fusão , Alinhamento de Sequência , Análise de Sequência de DNA , Homologia de Sequência de AminoácidosRESUMO
Interferon-alpha (IFN-alpha) induces cytogenetic responses of variable degree in patients with CML. We sought to establish the relationship between BCR-ABL transcript numbers measured by competitive two-step reverse transcription polymerase chain reaction (RT-PCR) and cytogenetic status in CML patients treated with IFN-alpha. All 305 samples from 133 patients investigated by RT-PCR were positive for BCR-ABL transcripts. In order to standardize results for variability in RNA and cDNA quality, we quantified total ABL transcripts in each sample as an internal control. The BCR-ABL : ABL ratios correlated well with the cytogenetic results. Quantitative nested PCR allowed the detection of residual BCR-ABL transcripts in all complete cytogenetic responders on IFN-alpha. We conclude that competitive PCR with internal controls is a reliable method for monitoring patients on IFN-alpha and reduces the need for repeated marrow investigations.
Assuntos
Antineoplásicos/uso terapêutico , Interferon-alfa/uso terapêutico , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/terapia , Reação em Cadeia da Polimerase/métodos , Citogenética , DNA de Neoplasias/sangue , DNA de Neoplasias/genética , Genes abl , Humanos , Prognóstico , RNA Neoplásico/sangue , RNA Neoplásico/genética , Fatores de TempoRESUMO
The degree of tumor load reduction after therapy, which is determined by the degrees of cytoreduction and cytogenetic response, is an important prognostic factor for patients with chronic myelogenous leukemia (CML). Conventional metaphase analysis is considered to be the "gold standard" for evaluating cytogenetic response. The frequency of cytogenetic analysis can be reduced considerably if patients are monitored by molecular methods, such as quantitative Southern blot, fluorescence in situ hybridization (FISH), quantitative western blot, or competitive reverse transcriptase polymerase chain reaction (RT-PCR). Molecular methods can be performed on peripheral blood specimens and are therefore less invasive than cytogenetic analyses of bone marrow metaphases. Furthermore these techniques are applicable to Ph-negative/BCR-AbL-positive cases. Results obtained by Southern blotting, western blotting, and FISH are readily quantifiable but their sensitivity is not generally superior to that of cytogenetic methods. RT-PCR is by far the most sensitive method. Quantitative RT-PCR analysis is the method of choice for monitoring patients after bone marrow transplantation (BMT). Using competitive PCR in patients after BMT, reappearance and/or rising levels of BCR-ABL transcripts can be detected prior to relapse. All complete cytogenetic responders to interferon-alpha are positive for BCR-ABL transcripts. The level of residual disease spans a range over found orders of magnitude. In both interferon-alpha-treated patients and patients after BMT a good correlation between BCR-ABL transcript numbers per microgram of RNA and cytogenetic results has been found. Variables in the competitive PCR assay may be controlled for by quantification of transcripts of the normal ABL gene as an internal standard. We suggest a stepwise strategy for diagnosis and follow-up of CML patients employing molecular methods.
Assuntos
Leucemia Mielogênica Crônica BCR-ABL Positiva/diagnóstico , Southern Blotting , Proteínas de Fusão bcr-abl/genética , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/terapia , Neoplasia Residual , Reação em Cadeia da PolimeraseRESUMO
Kabuki syndrome (OMIM 147920) is a rare disorder characterised by moderate intellectual disability, growth retardation, microcephaly and characteristic facial dysmorphic features which comprise long palpebral fissures, eversion of the lateral third of the eyelids and arched eyebrows with lateral sparseness. Mutations in MLL2 are the most frequent cause of this disorder. More than 100 MLL2 point mutations have been reported, but large intragenic deletions comprising one or more exons have not yet been identified. We report on a pair of monozygotic twin brothers in whom a deletion of 2 neighbouring exons was detected. The twins had the characteristic facial features of Kabuki syndrome, and they suffered from microcephaly, cleft lip and palate and congenital heart disease. Cleft lip and palate were left-sided in the first twin and right-sided in the second twin, i.e. they represented a mirror-image asymmetry. The intragenic deletion in these brothers broadens the spectrum of MLL2 mutations, and they provide a rare example of mirror-image asymmetry of congenital malformations in monozygotic twins.
Assuntos
Regulação Viral da Expressão Gênica , Genes , RNA Mensageiro/isolamento & purificação , Retroviridae/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa/métodos , Sequências Repetidas Terminais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Neoplasias da Mama/virologia , DNA Viral/genética , Feminino , Humanos , Mutagênese Insercional , Filogenia , Provírus/genética , RNA Mensageiro/genética , Transcrição Gênica , Células Tumorais CultivadasRESUMO
The DNA-dependent RNA polymerase (EC 2.7.7.6) of the myxobacterium Stigmatella aurantiaca has been purified. It shows three main polypeptide bands with apparent molecular weights of 146,000, 105,000, and 40,000 in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. beta and beta' subunits of the S. aurantiaca polymerase were shown to migrate in the 146,000-molecular-weight polypeptide band and the main sigma factor was shown to migrate in the 105,000-molecular-weight band by using heterologous antisera.
Assuntos
RNA Polimerases Dirigidas por DNA/isolamento & purificação , Myxococcales/genética , RNA Polimerases Dirigidas por DNA/química , RNA Polimerases Dirigidas por DNA/imunologia , Substâncias Macromoleculares , Peso Molecular , Especificidade da EspécieRESUMO
In the fruiting body-forming myxobacterium Stigmatella aurantiaca a 21,000-M(r) protein, SP21, is synthesized during fruiting, heat shock, and stress induced by oxygen limitation. The corresponding gene was isolated from a gene expression library in lambda gt11 with an antiserum to the purified protein. The DNA sequence of the gene reveals that SP21 is a member of the alpha-crystallin family of low-molecular-weight heat shock proteins.
Assuntos
Proteínas de Bactérias/biossíntese , Proteínas de Choque Térmico/biossíntese , Myxococcales/metabolismo , Sequência de Aminoácidos , Animais , Proteínas de Bactérias/genética , Sequência de Bases , Clonagem Molecular , Cristalinas/genética , Genes Bacterianos , Proteínas de Choque Térmico/genética , Temperatura Alta , Humanos , Dados de Sequência Molecular , Peso Molecular , Myxococcales/genética , Homologia de Sequência de AminoácidosRESUMO
Stigmatella aurantiaca is a gram-negative bacterium with a complex life cycle, including cellular aggregation resulting in the formation of a characteristic three-dimensional structure, the so-called fruiting body. During fruiting and upon chemical induction of sporulation, a major development-specific protein, SP21, is synthesized. SP21 was purified to homogeneity from the membranous fraction of chemically induced spores. Expression of SP21 was studied with an antiserum raised against the purified protein.
Assuntos
Proteínas de Bactérias/isolamento & purificação , Myxococcales/química , Sequência de Aminoácidos , Anticorpos Antibacterianos/imunologia , Proteínas de Bactérias/química , Proteínas de Bactérias/imunologia , Dados de Sequência Molecular , Peso Molecular , Esporos Bacterianos/químicaRESUMO
Within recent years, novel serological and molecular methods have been developed to improve the early diagnosis of invasive fungal infections especially in patients with malignant hematological diseases being at highest risk of developing these life-threatening infections. Early diagnosis is essential for adequate therapeutic management, which, however, often remains difficult since the diagnostic tools clinically used either lack specificity or sensitivity, or both at worst. The clinical value, the advantages and remaining problems of recently developed, more sensitive and specific serological assays and molecular methods are reviewed.
Assuntos
Aspergilose/diagnóstico , Candidíase/diagnóstico , Neoplasias Hematológicas/imunologia , Hospedeiro Imunocomprometido , Técnicas Microbiológicas , Infecções Oportunistas/diagnóstico , Aspergilose/microbiologia , Aspergillus/isolamento & purificação , Candida/isolamento & purificação , Candidíase/microbiologia , Humanos , Infecções Oportunistas/microbiologia , Reação em Cadeia da Polimerase/métodos , Sorologia/métodosRESUMO
The human mammary carcinoma cell line T47-D releases retrovirus-like particles of type B morphology in a steroid-dependent manner (I. Keydar, T. Ohno, R. Nayak, R. Sweet, F. Simoni, F. Weiss, S. Karby, R. Mesa-Tejada, and S. Spiegelman, Proc. Natl. Acad. Sci. USA 81:4188-4192, 1984). Furthermore, reverse transcriptase (RT) activity is found to be associated with particle preparations. Using a set of degenerate primers derived from a conserved region of retroviral pol genes, we repeatedly amplified three different retroviral sequences (MLN, FRD, and FTD) from purified T47-D particles in several RT-PCR experiments. Screening of a human genomic library and Southern blot analysis revealed that these sequences are of endogenous origin. ERV-MLN represents a multicopy family of human endogenous retroviral elements (HERVs) with two closely related copies and up to 20 more distantly related members. In contrast, ERV-FRD and ERV-FTD comprise only one copy and five to seven related elements per haploid human genome. DNA sequence analysis of the proviral pol region of ERV-MLN revealed an uninterrupted stretch of 241 amino acids that shows 65% identity with the RT of the type B-related HERV designated HERV-K10. ERV-FRD and ERV-FTD are defective type C-related HERVs. The pol gene of ERV-FRD displays a nucleotide homology of 54% to the gibbon ape leukemia virus, and the pol gene of ERV-FTD is about 67% homologous to members of the RTVL-I family of HERVs. Our results thus indicate that the retroviral particles released by the breast cancer cell line T47-D are probably generated by complementation of several endogenous proviruses and can package retroviral transcripts of different origins.
Assuntos
Betaretrovirus/classificação , Neoplasias da Mama/virologia , Gammaretrovirus/classificação , Retroviridae/classificação , Retroviridae/isolamento & purificação , Sequência de Aminoácidos , Sequência de Bases , Betaretrovirus/genética , Linhagem Celular , Primers do DNA , Gammaretrovirus/genética , Humanos , Dados de Sequência Molecular , Reação em Cadeia da Polimerase , RNA Mensageiro/biossíntese , RNA Mensageiro/isolamento & purificação , RNA Viral/isolamento & purificação , DNA Polimerase Dirigida por RNA/análise , Retroviridae/genética , Células Tumorais CultivadasRESUMO
We analysed 459 samples from 206 chronic myeloid leukaemia (CML) patients at diagnosis and during or after treatment with interferon-alpha (IFN-alpha) by quantitative Southern blot analysis for BCR rearrangement. In a minority (2%) of Ph-positive patients, no BCR rearrangement was detectable due to breakpoints outside the major breakpoint cluster region (M-bcr) or possibly due to M-bcr deletions. Results from 235 samples were compared with the proportion of Ph-positive metaphases found in contemporaneous bone marrow specimens analysed by conventional cytogenetics. The rank correlation between both methods was 0.82 (P < 0.001). The proportion of CML cells in samples determined by Southern blot analysis (BCR ratio) was significantly different between cytogenetically-defined minor, partial, and complete response groups (P < 0.001). Empirically-derived cut-off points in the BCR ratio were introduced in order to define molecular response groups for comparison to standard cytogenetic response groups: a BCR ratio of 0% was defined as complete molecular response and ratios of 1-24%, 25-50%, and > 50% were defined as partial, minor, and no molecular response, respectively. Using these cut-off points the concordance between both methods was 67% (P < 0.0001), a major cytogenetic response could be predicted or excluded in more than 90% of cases (P < 0.0001). Our findings demonstrated that quantitative Southern blot was as sensitive as cytogenetics and as peripheral blood samples are suitable for this technique it should be considered as the method of choice for routine monitoring IFN-alpha therapy in CML patients.
Assuntos
Interferon-alfa/uso terapêutico , Leucemia Mieloide de Fase Crônica/terapia , Proteínas Tirosina Quinases , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Southern Blotting , Criança , Feminino , Rearranjo Gênico , Humanos , Masculino , Pessoa de Meia-Idade , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-bcrRESUMO
The increasing incidence of aspergillosis, a life-threatening infection in immunocompromised patients, emphasizes the need to improve the currently limited diagnostic tools. We developed a two-step PCR assay that specifically amplifies a region of the 18S rRNA gene that is highly conserved in Aspergillus species. A number of primers with the least homology to equivalent human or Candida gene sequences were screened for the pairs that gave the highest sensitivity and specificity. No cross-reaction with the wide range of fungal and bacterial pathogens so far tested was observed. This assay allows direct and rapid detection of down to 10 fg of Aspergillus DNA corresponding to 1 to 5 CFU per ml of blood. A total of 315 blood and bronchoalveolar lavage samples from 140 subjects, including 93 patients at risk for invasive fungal disease, were screened. The result was a 100% correlation between positive histology, culture, or high-resolution computed tomography findings and PCR results. The test specificity was 89%. Our data point to the considerable potential clinical value of this simple, specific, rapid, and inexpensive PCR assay for improving the means of early diagnosis of systemic aspergillosis in high-risk patients.
Assuntos
Aspergilose/diagnóstico , Aspergillus/isolamento & purificação , Sangue/microbiologia , Líquido da Lavagem Broncoalveolar/microbiologia , Reação em Cadeia da Polimerase/métodos , Adulto , Idoso , Aspergilose/complicações , Aspergilose/microbiologia , Aspergillus/genética , Sequência de Bases , Primers do DNA , DNA Fúngico/análise , DNA Fúngico/genética , Genes de RNAr , Neoplasias Hematológicas/complicações , Humanos , Hospedeiro Imunocomprometido , Pessoa de Meia-Idade , Dados de Sequência Molecular , RNA Ribossômico 18S/genética , Sensibilidade e Especificidade , Especificidade da EspécieRESUMO
Bronchoalveolar lavage (BAL) samples from 67 patients who were at high risk for invasive aspergillosis were examined using a recently developed 2-step polymerase chain reaction (PCR) that detects =10 fg of Aspergillus DNA in blood and BAL samples in vitro. Thirteen of these patients had PCR and diagnostic results positive for Aspergillus infection. Four patients with possible invasive aspergillosis also had positive PCR results, and the remaining 50 had negative PCR results. In addition, 907 blood samples from 218 high-risk patients were screened. Thirty-three patients with positive PCR results had invasive aspergillosis; 148 patients had PCR and diagnostic results that were negative, and 34 patients with positive PCR results had nonconclusive clinical data. Both blood and BAL testing were performed for 45 patients. All 8 patients with proven invasive aspergillosis showed concordance of positive PCR results. Our data suggest that this PCR method has possible clinical value for confirming and improving the diagnosis of invasive aspergillosis in high-risk patients.
Assuntos
Aspergilose/diagnóstico , Aspergillus/isolamento & purificação , Líquido da Lavagem Broncoalveolar/microbiologia , DNA Fúngico/sangue , Hospedeiro Imunocomprometido , Reação em Cadeia da Polimerase/métodos , Adolescente , Adulto , Idoso , Idoso de 80 Anos ou mais , Aspergilose/microbiologia , Aspergillus/genética , Feminino , Humanos , Pneumopatias Fúngicas/diagnóstico , Pneumopatias Fúngicas/microbiologia , Masculino , Pessoa de Meia-IdadeRESUMO
We previously described that type B retrovirus-like particles released from the human mammary carcinoma cell line T47D are pseudotypes and package retroviral RNA of different origins (W. Seifarth, H. Skladny, F. Krieg-Schneider, A. Reichert, R. Hehlmann, and C. Leib-Mösch, J. Virol. 69:6408-6416, 1995). One preferentially packaged retroviral sequence, ERV-MLN, has now been used to isolate the corresponding full-length provirus from a human genomic library. The 9,315-bp proviral genome comprises a complete retroviral structure except for a 3' long terminal repeat (LTR) truncation. A lysine tRNA primer-binding site and phylogenetic analyses assign this human endogenous retroviral element, now called HERV-K-T47D, to the HML-4 subgroup of the HERV-K superfamily. The gag, prt, pol, and env genes exhibit 40 to 60% amino acid identity to HERV-K10. HERV-K-T47D is located on human chromosome 10, with five closely related elements on chromosomes 8, 9, 15, 16, and 19 and several hundred HERV-K-T47D-related solitary LTRs dispersed over the human genome. HERV-K-T47D-related sequences are detected in the genomes of higher primates and Old World monkeys but not in those of New World monkeys. High HERV-K-T47D transcription levels were observed in human placenta tissue, whereas transcription in T47D cells was strictly steroid dependent.
Assuntos
Mapeamento Cromossômico , Provírus/genética , Retroviridae/isolamento & purificação , Sequência de Aminoácidos , Animais , Sequência de Bases , Linhagem Celular , DNA Viral , Humanos , Dados de Sequência Molecular , Primatas , Sequências Repetitivas de Ácido Nucleico , Retroviridae/genética , Homologia de Sequência do Ácido Nucleico , Transcrição GênicaRESUMO
A substantial minority of patients with chronic myeloid leukaemia (CML) achieve a complete response to treatment with interferon-alpha (IFN-alpha), defined as the disappearance of Philadelphia chromosome positive metaphases or, for patients who are Philadelphia chromosome negative but BCR-ABL positive, the disappearance of the leukaemic clone as assayed by Southern blot. We have measured the levels of BCR-ABL transcripts in 20 such patients by quantitative PCR. Results were standardized for both quality and quantity of cDNA by quantification of ABL as an internal control. All 20 patients had evidence of residual disease; the median number of transcripts was 750/micrograms RNA (range 10-22,000) and the median BCR-ABL/ABL ratio was 0.17% (range 0.0008-3.6%). Our findings show that CML has not been eradicated in any patient and that the quantity of residual disease in complete responders may vary by as much as four orders of magnitude.
Assuntos
Proteínas de Fusão bcr-abl/biossíntese , Proteínas de Fusão bcr-abl/genética , Interferon-alfa/uso terapêutico , Leucemia Mielogênica Crônica BCR-ABL Positiva/genética , Adulto , Idoso , Southern Blotting , Feminino , Humanos , Leucemia Mielogênica Crônica BCR-ABL Positiva/terapia , Masculino , Pessoa de Meia-Idade , Reação em Cadeia da Polimerase , Indução de Remissão , Transcrição GênicaRESUMO
Interferon-alpha (IFN-alpha) induces cytogenetic responses of variable degree in patients with chronic myelogenous leukemia (CML). We sought to establish the relationship between BCR-ABL transcript numbers measured by competitive 2-step reverse transcription polymerase chain reaction (RT-PCR) and cytogenetic status in CML patients treated with IFN-alpha. A total of 250 peripheral blood and 55 bone marrow samples with 127 Philadelphia chromosome positive (Ph+) and 6 Ph-/BCR-ABL+ CML patients were investigated. Twenty-one patients were studied at diagnosis with IFN-alpha, 24 had a complete cytogenetic response, 21 a partial response, 12 a minor response, 26 no response, and 23 were unknown. Using nested RT-PCR, all 305 samples were positive for BCR-ABL transcripts. To standardize results for variability in RNA and cDNA quantity and quality, we quantified total ABL transcripts in each sample as internal control. The validity of ABL as internal control was shown by comparison with glucose-6-phosphate dehydrogenase transcript levels in 145 samples. The median BCR-ABL transcript numbers (and BCR-ABL/ABL ratios expressed as percentages) were 400/micrograms RNA (O.04%) in complete responders, 20,500/micrograms RNA (7.1%) in partial responders, 170,000/micrograms RNA (21.0%) in minor responders, and 430,000/micrograms RNA (58.7%) in nonresponders (P < .001). The cytogenetic results correlated with the BCR-ABL transcript numbers (r = .82; P < .001) and BCR-ABL/ABL ratios (r = .84; P < .001). Grouping the ratios BCR-ABL/ABL as less than 2%, 2% to 14% and greater than 14% to compare with cytogenetic complete response, partial response, and minor/nonresponse, the concordance between the two methods was 82% (chi2 P< .0001). We conclude that quantitative PCR with internal controls is as sensitive and reliable method for monitoring patients on IFN-alpha and reduces the need for repeated marrow investigations.
Assuntos
Proteínas de Fusão bcr-abl/genética , Leucemia Mielogênica Crônica BCR-ABL Positiva/diagnóstico , Adolescente , Adulto , Idoso , Sequência de Bases , Criança , Primers do DNA/química , Feminino , Humanos , Interferon-alfa/uso terapêutico , Cariotipagem , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Neoplasia Residual/diagnóstico , Reação em Cadeia da Polimerase/métodos , RNA Neoplásico/genéticaRESUMO
A novel variant of the chimeric BCR-ABL mRNA transcript was detected in a patient with Philadelphia chromosome-negative (Ph-) chronic myelogenous leukemia (CML) by multiplex reverse-transcription polymerase chain reaction (RT-PCR). Sequence analysis of the fusion region of the amplified cDNA fragment showed an in-frame joining of exon e6 of the BCR gene and exon a2 of the ABL gene, giving rise to an e6a2 BCR-ABL transcript. This finding was confirmed by Southern blot analysis using a specific probe corresponding to intron 6 of the BCR gene, whereas conventional Southern blot for rearrangement of the major breakpoint cluster region (M-bcr) was negative. Western blot studies detected a BCR-ABL protein slightly larger than p185 BCR-ABL. Metaphase fluorescence in situ hybridization showed an insertion of ABL material into the BCR region without reciprocal BCR translocation. The findings in this case show that atypical BCR-ABL transcripts are detectable even in Ph- CML patients without M-bcr-rearrangements. Multiplex PCR using primers that allow for amplification of all known BCR-ABL transcripts is an appropriate method to exclude these rare variants.