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)

A specific 'nested' reverse transcriptase/polymerase chain reaction (RT/PCR) procedure was used to characterize the expression patterns of PML-RAR-alpha chimeric mRNAs in 32 patients with acute promyelocytic leukemia (APL). The sensitivity of the technique was such that the fusion gene transcript could be detected from as little as 2.5 pg of total leukemic cell RNA against a background of 1 microgram of cellular RNA lacking the PML-RAR-alpha fusion gene transcript(s). In 19 cases the PML-RAR-alpha isoform referred to here as long was identified. A short isoform, which in comparison with the long form lacks three PML exons, was detected in 11 other cases. A third PML-RAR-alpha mRNA isoform, in which the most 3' PML exon present in the long-type isoform was truncated in its sequences lying immediately upstream of RAR-alpha B region, was found and characterized in a single patient. In one APL patient with a variant translocation t(11;17), the PCR product corresponding to PML-RAR-alpha chimeric mRNAs could not be amplified despite the presence of RAR-alpha gene rearrangement. Genomic and PCR analysis showed that the different PML-RAR-alpha isoforms found in APL patients arise as a result of distinct translocation breakpoints. In each case the exons encoding the B-F regions of RAR-alpha are expressed and are spliced downstream from variable PML gene exons. The 'nested' RT/PCR analysis of the PML-RAR-alpha fusion gene proved to be a rapid and sensitive tool for the diagnosis of the APL and for monitoring the residual APL chimeric mRNA expression during complete remission.
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PMID:Occurrence of distinct PML-RAR-alpha fusion gene isoforms in patients with acute promyelocytic leukemia detected by reverse transcriptase/polymerase chain reaction. 137 19

The t(15;17) translocation is specifically observed in patients with promyelocytic leukemia (AML3). The chromosomal rearrangement juxtaposes the retinoic acid receptor alpha (RAR alpha) and PML genes, resulting in PML/RAR alpha fusion transcripts. Our previous studies have shown that a polymerase chain reaction (PCR) amplification product could be obtained from the cDNA of the NB4 promyelocytic cell line from which the chimaeric PML/RAR alpha was cloned. We report here that in all 14 AML3 patients tested, reverse transcriptase-PCR (RT-PCR) allows the detection of three specific fusion products. In eight patients, one amplification product was detected corresponding to the previously described abnormal fusion. Five patients displayed a different amplified fragment corresponding to a different fusion point. One other patient always showed a third different-sized product. The different types of fusion transcripts amplified were correlated to the size of the abnormal RAR alpha transcripts detected in these patients by Northern analysis, but did not prove determinant for either the phenotypic features or the retinoic acid responsiveness in AML3 cells in this group of patients. The consistent identification by RT-PCR of the fusion of the PML and RAR alpha genes in AML3 patients suggest that this method will provide a useful tool for the diagnosis and detection of minimal residual disease in these patients.
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PMID:A PML/retinoic acid receptor alpha fusion transcript is constantly detected by RNA-based polymerase chain reaction in acute promyelocytic leukemia. 137 40

Variants of the t(15;17)(q22;q12-q21) chromosomal rearrangement associated with acute promyelocytic leukemia (APL) have been previously described and they frequently involve either chromosome 15 and/or 17. Previously we reported a rare variant t(11;17). We now describe two patients with myelodysplastic syndrome (MDS) that transformed to APL-like leukemia. Both had trisomy 11 at the diagnosis of APL-like leukemia. Following treatment for APL, patient 1 reverted to MDS and showed a normal karyotype. When leukemia recurred, his bone marrow karyotype was 47,XY,t(4;11), +11,der(22)t(1;22). Both patients were treated with all-trans retinoic acid (ATRA) for APL for 5 weeks, but failed to respond. The karyotype of patient 1 after ATRA treatment was 46,XY,t(4;11); the trisomy 11 had been lost and the bone marrow was replaced with immature myeloblasts without promyelocytes. In patient 2, the karyotype remained the same as at diagnosis, i.e., 47,X,-Y,dir ins(4;7),del(5), +6,del(7), +8, + 11,-18. Molecular analysis by reverse transcriptase PCR analysis showed the presence of wild type retinoic acid receptor alpha (RARA) and the absence of the PML-RARA chimeric gene associated with t(15;17). Additional analysis of PLZF, a new zinc finger gene associated with t(11;17), also showed the absence of this hybrid gene. These data support the concept that APL is a heterogeneous disorder and that variants with chromosome 11 rearrangement exist that do not respond to ATRA.
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PMID:Myelodysplastic syndrome transforming to acute promyelocytic-like leukemia with trisomy and rearrangement of chromosome 11. 751 69

The hierarchical level of stem cell involvement in acute promyelocytic leukemia (APL) characterized by the pathognomonic PML-RARA fusion gene is unknown. To determine if the cells of the primitive hematopoietic stem cell compartment are involved in the leukemic process, we have used molecular and cell sorting techniques in peripheral blood and bone marrow (BM) cells at diagnosis from three patients with APL and t(15; 17). In two of them, clonality analysis was also possible using the BstXI polymorphic site of the PGK gene. The PML-RARA fusion gene was readily identified by reverse transcriptase-polymerase chain reaction (RT-PCR) analysis of BM cells obtained at diagnosis in all three patients. These same samples were then used to sort CD34+ cells and their CD38+ and CD38- subsets by fluorescence-activated cell sorting. In both female patients, CD34+/CD38+ and CD34+/CD38- cell fractions were polyclonal using PCR, whereas a monoclonal pattern was identified at the BM sample obtained at diagnosis either by Southern blotting or by PCR. Because of the high sensitivity of the PCR analysis, the polyclonal pattern of these cell populations could mask the presence of a minor clone. To detect this clone, we preformed RT-PCR analysis for t(15; 17). In one female patient, the abnormal PML-RAR fusion gene was found only in the more mature CD34+/CD38+ cell fraction using a nested PCR approach, whereas the polyclonal CD34+/CD38- fraction was PML-RARA negative. These findings were confirmed in a third patient with APL in whom the PML-RARA transcripts were absent in the CD34+/CD38- cell fraction. To study the clonality at the level of clonogenic progenitors, we used in one patient PGK analysis by PCR of individual burst-forming units-erythroid and colony-forming units-granulocyte-macrophage obtained from the CD34+/CD38- and CD34+/CD38+ cell populations at diagnosis and from the BM sample obtained during remission. The two highly purified cell populations gave rise to morphologically normal colonies clonal for both the BstXI site containing (A) and the BstXI site lacking (B) PGK allelles, indicating their polyclonal content, a pattern that was also found in clonogenic progenitors obtained at remission. These findings strongly suggest that the primitive hematopoietic stem cells as defined by the CD34+/CD38- antigens are not involved by the neoplastic process in APL. These results may have important implications for autografting strategies of retinoic acid/chemotherapy-resistant or relapsed patients.
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PMID:Highly purified primitive hematopoietic stem cells are PML-RARA negative and generate nonclonal progenitors in acute promyelocytic leukemia. 753 93

The acute promyelocytic leukemia (APL)-specific t(15;17) chromosome abnormality is characterized at the molecular level by rearrangement of the PML and RAR alpha genes, resulting in fusion PML/RAR alpha mRNA and a chimeric protein. Besides its relevance in the pathogenesis of the disease, this hybrid gene represents a specific tumor marker that is rapidly detectable by reverse transcriptase-polymerase chain reaction (RT-PCR) in the RNA extracted from leukemic blasts. Several studies have highlighted the clinical relevance of PML/RAR alpha detection, which provides a specific diagnosis, prognostic information, and prediction of relapse when monitoring residual disease during the follow-up. In fact, this hybrid gene is detected in 100% of APLs. Rare cases of patients with a morphological diagnosis of FAB M3 AML who lack the specific PML/RAR alpha abnormality have been reported as being unresponsive to differentiation treatment. Finally, all the studies reported so far on PCR monitoring in APL have documented that the identification of small amounts of residual disease at remission strongly predicts impending relapse. Thus, RT-PCR of the hybrid PML/RAR alpha gene is currently performed prospectively as part of cooperative clinical trials aimed at better addressing post-remission treatment in APL.
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PMID:The PML/RAR alpha fusion gene in the diagnosis and monitoring of acute promyelocytic leukemia. 762 53

Of 113 acute promyelocytic leukemia cases documented to have diagnostic PML-RAR alpha hybrid mRNA, 10 cases (8.8%) had fusion sites in PML gene exon 6 (V-forms) rather than in the two common hybrid mRNA configurations resulting from breaksites in either PML gene intron 6 (L-forms) or intron 3 (S-forms). In 4 V-form cases, a common break/fusion site was discovered at PML gene nucleotide (nt) 1685, abutting a 3' cryptic splice donor sequence. The fusion site was proximal to the common site in 1 case and more distal in 5 cases. The open reading frame encoding a PML-RAR alpha gene was consistently preserved, either by an in-frame fusion site or by the insertion of 3 to 127 unidentified nts. In 2 V-form cases, hybridization analysis of the reverse transcriptase-polymerase chain reaction products with a PML-RAR alpha juction probe was required for discrimination from L-form cases. Two V-form subgroups were defined by in vitro sensitivity to all-trans retinoic acid (tRA)-induced differentiation: 4 of 4 cases tested with fusion sites at or 5' to nt 1685 (subgroup E6S) had reduced sensitivity (EC50 > or = 10(-7) mol/L), whereas 4 of 4 cases with fusion sites at or 3' to nt 1709 (subgroup E6L) had high sensitivity (EC50 < 10(-8) mol/L) indistinguishable from that of L-form and S-form cases. These results provide the first link between PML-RAR alpha configuration and tRA sensitivity in vitro and support the importance of subclassifying APL cases according to PML-RAR alpha transcript type.
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PMID:Characterization of acute promyelocytic leukemia cases with PML-RAR alpha break/fusion sites in PML exon 6: identification of a subgroup with decreased in vitro responsiveness to all-trans retinoic acid. 763 62

The WT 1 gene has been isolated as a tumor suppressor gene of Wilms' tumor. Using reverse transcriptase-polymerase chain reaction (RT-PCR), relative levels of the WT 1 gene expression was examined in 87 patients with acute leukemia, 25 with chronic myelogenous leukemia (CML), and 24 with non-Hodgkin's lymphoma (NHL). Significant levels of the WT 1 gene were expressed in all leukemia patients, and for CML the levels increased as the clinical phase progressed. No point mutations were found in the WT 1 gene when samples from 15 acute leukemia patients were subjected to PCR single-strand conformation polymorphism analysis. In striking contrast to acute leukemia, the levels of WT1 gene expression for NHL were significantly low or even undetectable. The levels of WT 1 gene expression inversely correlated with the prognosis of acute leukemia. The quantification of the WT 1 gene expression made it possible to detect minimal residual disease (MRD) in acute leukemia regardless of the presence of absence of tumor-specific DNA markers. Simultaneous monitoring of MRD by RT-PCR using primers for specific DNA markers in four patients (two AML-M3 with PML/RAR-alpha, one AML-M2 with AML1/ETO, and one CML with bcr/abl) detected MRD comparable to that obtained from quantitation of WT 1 gene expression. In a patient with acute promyelocytic leukemia, the limits of leukemic cell detection by RT-PCR using either WT 1 or PML/RAR-alpha gene primers were 10(-3)-10(-4) and 10(-4) for bone marrow, and 10(-5) and 10(-4) for peripheral blood, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)
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PMID:[WT 1 and leukemia]. 764 50

Acute promyelocytic leukaemia (APL) is characterized by t(15;17), which results in the formation of two chimaeric genes, PML-RAR alpha and RAR alpha-PML. PML-RAR alpha transcripts have been detected in all cases of APL whilst those of RAR alpha-PML have been detected in only about 67% of cases. We have used reverse transcriptase polymerase chain reaction (RT-PCR) to detect both fusion transcripts serially in 18 patients in remission of APL after chemotherapy and bone marrow transplantation. All patients were negative for PML-RAR alpha, whereas in six patients (remission 3-9 years) RAR alpha-PML was consistently detected. Only one patient at remission showed the 5' breakpoint RAR alpha-PML, with the rest showing the 3' breakpoint 144 bp RAR alpha-PML. The level of sensitivity for detecting RAR alpha-PML was some 10-fold higher than that for PML-RAR alpha. Serial negative tests for PML-RAR alpha have been correlated with durable remissions, suggesting possible eradication of residual leukaemia in APL. Our results, however, show persistence of t(15;17) cells expressing RAR alpha-PML fusion mRNA in patients in long-term remission of APL. They indicate that patients considered clinically 'cured' of APL still have molecular evidence of minimal residual disease and also provide further insight into the biology of acute myeloid leukaemia.
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PMID:Persistence of RAR alpha-PML fusion mRNA detected by reverse transcriptase polymerase chain reaction in patients in long-term remission of acute promyelocytic leukaemia. 764 2

Minimal residual disease (MRD) was prospectively monitored at the 10(-5) level by the reverse transcriptase-polymerase chain reaction (RT-PCR) of PML-retinoic acid receptor alpha (RARA) transcripts from 27 acute promyelocytic leukemia (APL) patients who achieved complete remission (CR) with all-trans retinoic acid and chemotherapy (previously untreated patients, 15; refractory to chemotherapy or relapsed, 12). The RNA quality from bone marrow cells was firstly assessed by gel electrophoresis to avoid false negativity because of the fragility of the APL cells and the PML-RARA transcripts. In 12 of 15 untreated patients, RT-PCR became negative during consolidation and intensification therapy 4-16 months after the initiation of therapy, whereas it remained positive in nine of 12 refractory patients. At the end of therapy, RT-PCR was negative in 14 patients and positive in 13 patients. The former patients remained in CR at median follow-up of 9 months after the end of therapy. In the latter, however, 10 patients relapsed at a median of 5 months after the end of therapy. These results suggest that the RT-PCR assay can evaluate the quality of CR in APL and predict subsequent relapse.
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PMID:Prognostic significance of the RT-PCR assay of PML-RARA transcripts in acute promyelocytic leukemia. The Leukemia Study Group of the Ministry of Health and Welfare (Kouseisho). 772 89

With the recent advances in molecular technology, diagnostic procedures of the diseases at a DNA level have been introduced in hematological fields. The diagnostic methods used are Southern blotting to detect gene rearrangements, Northern blotting to find gene expressions, RT-PCR (reverse transcriptase-polymerase chain reaction) to identify transcribed fusion messages, and PCR-SSCR (single strand conformation polymorphism) to detect mutated genes. Rearrangements within major Bcr (breakpoint cluster region) were observed in almost all cases in chronic myelogenous leukemia, and breakpoint were found within minor Bcr in Philadelphia-positive leukemia. The rearrangements within the second intron of the retinoic acid receptor-alpha and sixth intron (bcr 1), third intron (bcr 3) and sixth exon (bcr 2) of the PML gene were detected in all cases with acute promyelocytic leukemia. In malignant lymphoma, the rearrangements of immunoglobulin and T-cell receptor genes, and new genes such as Bcl-1, Bcl-2, Bcl-5, Tal-1, and Tal-2 were also reported and rearrangements of the Bcl-5 gene were found in this study using Bcl-5 specific probe which we have cloned. Point mutations and deletions of the genes involved in the coagulation and fibrinolysis system have been reported. One base insertion resulting in elongation of carboxy terminal region and one amino acid deletion in alpha 2-plasmin inhibitor gene were found in two cases of its deficiency. Further study revealed that mutated proteins were retained in the endoplasmic reticulum in the cells. With the development of the PCR method, identification of gene mutation is gradually carried out as a routine work.
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PMID:[Molecular study of hematological diseases]. 791 42


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