Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:2.7.7.49 (reverse transcriptase)
31,746 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The resolving power of RT-PCR is limited by the efficiency of RNA-to-cDNA conversion. Methods to determine this efficiency, using a real-time PCR assay for quantifying AML1-MTG 8 [t(8;21)] fusion gene transcripts, are described. The efficiency is shown to be directly proportional to RNA template levels. The Moloney murine leukemia virus (MMLV) reverse transcriptase enzyme's conversion efficiency was calculated to be approximately 20%. The efficiency was even lower (6%) when target templates were rare (single molecules) in the RT reactions. Levels of nonspecific or background RNA present in the RT reaction reduced the reverse transcriptase's conversion efficiency. This background effect was particularly pronounced when the specific template was present in rare amounts.
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PMID:Low efficiency of the Moloney murine leukemia virus reverse transcriptase during reverse transcription of rare t(8;21) fusion gene transcripts. 1196 98

Studies on monozygotic twins with concordant leukemia and retrospective scrutiny of neonatal blood spots of patients with leukemia indicate that chromosomal translocations characteristic of pediatric leukemia often arise prenatally, probably as initiating events. The modest concordance rate for leukemia in identical twins ( approximately 5%), protracted latency, and transgenic modeling all suggest that additional postnatal exposure and/or genetic events are required for clinically overt leukemia development. This notion leads to the prediction that chromosome translocations, functional fusion genes, and preleukemic clones should be present in the blood of healthy newborns at a rate that is significantly greater than the cumulative risk of the corresponding leukemia. Using parallel reverse transcriptase-PCR and real-time PCR (Taqman) screening, we find that the common leukemia fusion genes, TEL-AML1 or AML1-ETO, are present in cord bloods at a frequency that is 100-fold greater than the risk of the corresponding leukemia. Single-cell analysis by cell enrichment and immunophenotype/fluorescence in situ hybridization multicolor staining confirmed the presence of translocations in restricted cell types corresponding to the B lymphoid or myeloid lineage of the leukemias that normally harbor these fusion genes. The frequency of positive cells (10(-4) to 10(-3)) indicates substantial clonal expansion of a progenitor population. These data have significant implications for the pathogenesis, natural history, and etiology of childhood leukemia.
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PMID:Chromosome translocations and covert leukemic clones are generated during normal fetal development. 1204 36

One important question in stem cell biology of childhood acute lymphoblastic leukemia (ALL) is whether immature CD34+CD19- cells are part of the leukemic cell clone. CD34+CD19- cells from the bone marrow of 9 children with TEL/AML1-positive ALL were purified by flow sorting and subjected to reverse transcriptase-polymerase chain reaction (RT-PCR), fluorescence in situ hybridization, and methylcellulose cultures. In 3 of 8 patients analyzed by RT-PCR, no TEL/AML1-positive cells could be detected in the CD34+CD19- cell fraction. Altogether, the percentage of TEL/AML1-positive cells was low: 1.6% (n = 8; SD 2.2%) by nested real-time RT-PCR and 2.5% (n = 5; SD 2.6%) by fluorescence in situ hybridization. This correlated with the percentage of contaminating CD19+ leukemic cells in the CD34+CD19- cell fraction in 6 control sorts (mean 4.6%, SD 3.6%), indicating that the low levels of leukemic cells detected in the CD34+CD19- cell fraction could be attributed to sorter errors. Methylcellulose cultures in 3 patients provided further evidence that CD34+CD19- cells represent a candidate normal cell population. The clonogenicity of the CD34+CD19- cell fraction was similar to normal progenitors, including growth of primitive granulocyte, erythroid, macrophage, megakaryocyte colony-forming units. Each of 92 colonies from cultures with CD34+CD19- cells tested negative for TEL/AML1. In conclusion, our data support the hypothesis that the leukemia in TEL/AML1-positive childhood ALL originates in a CD19+ lymphoid progenitor. This has many therapeutic implications, eg, for purging of autologous stem cell products, flow cytometric monitoring of minimal residual disease, and targeting immunotherapy against the leukemic cell clone.
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PMID:Immature CD34+CD19- progenitor/stem cells in TEL/AML1-positive acute lymphoblastic leukemia are genetically and functionally normal. 1209 59

A 63-year-old male was diagnosed as chronic myelomonocytic leukemia with normal karyotype in September 1998. He developed acute myelogenous leukemia (AML-M4Eo) in September 2001. The cytogenetic analysis disclosed double t(3;21) at a ratio of 1/20, and the reverse transcriptase-polymerase chain reaction showed AML1/EVI-1 mRNA. He could not achieve complete remission after two courses of induction chemotherapy, and his leukemia cells carrying double t(3;21) were relatively increased. He died of interstitial pneumonia in December 2001. This is the first leukemia case with double t(3;21) and this chromosomal abnormality might play a role in leukemia cell proliferation by generating two AML1/EVI-1 genes.
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PMID:[Double t (3; 21) in acute myelomonocytic leukemia transformed from chronic myelomonocytic leukemia]. 1241 94

We report a case of acute myeloid leukemia (AML), M2 subtype according to the French-American-British (FAB) classification, with extramedullary myeloblastoma of the uterus and a masked type of variant translocation of t(8;21)(q22;q22). A 45-year-old Japanese woman presented with metrorrhagia, and AML (M2) with uterine invasion was diagnosed. The patient received an allogeneic peripheral blood stem cell transplantation after remission, and her pelvis was irradiated locally. Cytogenetic study at first showed t(8;17)(q22;p13) by G-banding. Spectral karyotyping (SKY) analysis modified this interpretation to a 3-way translocation involving chromosomes 8,17, and 21 and identified a masked type of variant t(8;21)(q22;q22) translocation. Results of fluorescence in situ hybridization using the AML1/ETO probe, and of detection of the AML1/ETO fusion transcript by reverse transcriptase-polymerase chain reaction were consistent with the karyotyping result. SKY analysis is useful to compensate for the limitations of cytogenetic studies.
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PMID:Acute myeloid leukemia (FAB-M2) with a masked type of t(8;21) translocation revealed by spectral karyotyping. 1246 97

A 43-year-old man was diagnosed with acute myelocytic leukemia with cellular maturation (AML-M2, according to the French-American-British classification criteria). A cytogenetic study with a G-banding method initially reported the karyotype as 45,X,-Y; however, dual-color, dual-fusion fluorescence in situ hybridization (FISH) with probes for the AML1 and the ETO genes showed an unusual pattern of signals, presenting one fusion signal on chromosome 21. Molecular study by reverse transcriptase polymerase chain reaction revealed the presence of a typical AML1/ETO chimeric gene. FISH with whole-chromosome painting probes targeting chromosomes 8 and 21 revealed insertion of part of 8 chromosome into the long arm of chromosome 21. We concluded that complicated translocations involving chromosomes 8 and 21 in this patient resulted in the development of the chimeric gene, AML1/ETO, on the long arm of chromosome 21. This aberrant location of AML1/ETO gene and the final karyotype of 45,X,-Y,ins(21;8)(q22;q22q22) could not be determined without molecular analysis. This abnormality is considered a masked t(8;21).
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PMID:Insertion (21;8)(q22;q22q22): a masked t(8;21) in a patient with acute myelocytic leukemia. 1462 63

A quantitative nested reverse transcriptase polymerase chain reaction (QN-RT-PCR) method was developed using a plasmid cDNA containing the AML1/ETO (MTG8) fusion transcript from Kasumi-1 cells, an acute-myelogenous leukemia cell line with the t(8;21) translocation. In this method, the plasmid was detectable at a concentration of 10(-17) m. The fusion transcript in a mixture of 10(7) Rice94 (Burkitt lymphoma cell line) cells containing two Kasumi-1 cells was detectable at 10(-17) m. In a previously published real-time PCR method, the plasmid containing the fusion transcript was detectable at 10(-16) m or higher, and 20 or more Kasumi-1 cells were detectable in 10(7) Rice94 cells. Thus, this QN-RT-PCR method is more sensitive than the real-time PCR. When the same samples were examined by real-time PCR and our QN-RT-PCR method, in one patient in clinical remission after chemotherapy and allogeneic-bone marrow transplantation (BMT), the transcript was detected by QN-RT-PCR 60 days prior to hematological relapse, in contrast to 10 days before hematological relapse by real-time PCR. The transcript level was below 10(-17) m (undetectable) with this QN-RT-PCR in patients in clinical remission after chemotherapy and BMT, while it was 10(-15)-10(-16) m in patients in clinical remission after chemotherapy alone. The quantitative difference of the transcript level in minimal residual disease (MRD) between these two different types of clinical remission was estimated to be at least 10(2)-fold. This QN-RT-PCR method is useful for predicting hematological relapse and for quantitatively estimating MRD in different types of clinical remission.
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PMID:Quantitative nested reverse transcriptase PCR vs. real-time PCR for measuring AML1/ETO (MTG8) transcripts. 1505 4

The most common gene fusion (up to 25%) in childhood acute lymphoblastic leukemia (ALL) is that between ETV6 and RUNX1 (previously TEL and AML1, respectively; we here use the old nomenclature, for ease of reference to the literature). We determined the incidence of TEL/AML1 translocation with reverse transcriptase polymerase chain reaction (RT-PCR) and flow-cytometric immunophenotyping of newly diagnosed pediatric acute lymphoblastic leukemia patients in Thailand. The TEL/AML1 fusion genes were cloned into plasmids and sequenced. The variant found was confirmed with restriction fragment length polymorphism (RFLP) using SphI restriction endonuclease. Of 35 ALL patients, we found an incidence of 8.6% of TEL/AML1 translocation in ALL patients (12% of B-lineage ALL), which is lower than that reported in caucasians but is similar to that reported in Japanese and Koreans. All the translocation-positive patients had B-lineage common ALL, expressing CD10+. Interestingly, the two TEL/AML1 subclones were CD20 negative, and one subclone expressed a myelocytic marker (CD33+). Two TEL/AML1 subclones from bone marrow of ALL patients were isolated and sequenced. One was a wild type and the other was a variant having A --> G substitution at nucleotide 73 from the 5' end. The substitution nucleotide was located in the AML1 region. The clinical relevance of the variant is to be investigated.
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PMID:Cloning and sequencing of ETV6/RUNX1 (TEL/AML1) variant in acute lymphoblastic leukemia. 1510 90

Association of long-term clinical remission and molecular disease-eradication is well established in acute myeloid leukemia (AML) patients with t(15;17) and inv(16). In patients with t(8;21) positive AML no consensus exists over the disappearance of the AML1/ETO fusion transcript during the course of disease and most studies reported reverse transcriptase polymerase chain reaction (RT-PCR) positivity as a common finding after consolidation chemotherapy, autologous and allogeneic stem cell transplantation (alloSCT). In our single center study, we performed RT-PCR monitoring in 14 patients with t(8;21) in CR1 (n = 13) and/or CR2 (n = 4). The median number of bone marrow (BM) and/or peripheral blood (PB) samples per patient was 18 (range, 2-43). In 5 out of 6 cases relapse occurred after persistence of minimal residual disease (MRD) in CR for 4-14 months. The sixth patient relapsed despite molecular remission (MR) in BM and PB for 3 months, molecular relapse preceded hematological relapse for 7 months. Eleven patients with a median follow-up of 7.8 (range, 1.5-15.4) years are in persistent CR and MR after consolidation chemotherapy (n = 7). mainly with repetitive cycles of high-dose Ara-C, autologous (n = 1) or myeloablative allogeneic (n = 3) stem cell transplantation. Molecular remission was attained immediately after alloSCT, but after 6-26 months in CR in patients with consolidation chemotherapy. In 7 patients, MRD was only studied in long-term remission. In conclusion, long-term CR was associated with persistent molecular disease-eradication. In our patients, molecular remission was a prerequisite but not a guarantee for long-term disease-free survival. Hematological relapse never occurred without prior molecular relapse. Due to the slow kinetics of AML1/ETO after consolidation chemotherapy the value of qualitative RT-PCR to predict early relapse is limited. In this situation quantitative RT-PCR might help to define individual relapse risk and to improve as well as facilitate clinical decision-making.
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PMID:Molecular disease eradication is a prerequisite for long-term remission in patients with t(8;21) positive acute myeloid leukemia: a single center study. 1529 57

Germ-line heterozygous mutations in the hematopoietic transcription factor AML1 (RUNX1) have been identified in patients with familial platelet disorder with predisposition to acute myelogenous leukemia (FPD/AML), which is characterized by thrombocytopenia, abnormal platelet function, and propensity to myeloid malignancies. We identified a novel mutation in the AML1 gene in an FPD/AML pedigree characterized by a single nucleotide deletion that generates a frameshift and premature chain termination (Pro218fs-Ter225). Both wild-type and mutant transcripts were expressed in affected individuals by allele-specific reverse transcriptase-polymerase chain reaction (RT-PCR). Thrombopoietin (TPO) binds to the Mpl receptor and is the major regulator of megakaryopoiesis. To explore the mechanisms underlying thrombocytopenia, we studied the TPO/Mpl pathway in this newly identified pedigree. TPO levels were mildly to moderately elevated. On flow cytometry and immunoblotting, Mpl receptor expression was decreased and TPO-induced signaling was impaired. While no mutations were identified in the MPL gene by sequence analysis, low MPL mRNA levels were found, suggesting decreased gene expression. Of particular interest, several AML1-binding motifs are present in the MPL promoter, suggesting MPL is an AML1 target. In conclusion, we identified a C-terminal AML1 mutation that leads to a decrease in Mpl receptor expression, providing a potential explanation for thrombocytopenia in this FPD/AML pedigree.
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PMID:Low Mpl receptor expression in a pedigree with familial platelet disorder with predisposition to acute myelogenous leukemia and a novel AML1 mutation. 1574 Dec 16


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