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Acute Leukemias

Real-time PCR quantification of major Wilms’ tumor gene 1 (WT1) isoforms in acute myeloid leukemia, their characteristic expression patterns and possible functional consequences

Leukemia volume 26, pages 2086–2095 (2012)Cite this article

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Abstract

Wilms’ tumor gene 1 (WT1) functions including some contradictory effects may be explained by the presence and interactions of its isoforms, however, their evaluation has been so far complicated by several technical problems. We designed unique quantitative PCR systems for direct quantification of the major WT1 isoforms A[EX5−/KTS−], B[+/−], C[−/+] and D[+/+] and verified their sensitivity, specificity and reproducibility in extensive testing. With this method we evaluated WT1 total and isoform expression in 23 normal bone marrow (BM) samples, 73 childhood acute myeloid leukemia (AML), 20 childhood myelodysplastic syndrome (MDS), 9 childhood severe aplastic anemia (SAA), 30 adult AML and 29 adult MDS patients. WT1 isoform patterns showed differences among these samples and clustered them into groups representing the specific diagnoses (P<0.0001). Isoform profiles were independent of total WT1 expression and possess certain common features—overexpression of isoform D and EX5[+] variants. The KTS[+]/KTS[−] ratio was less variable than the EX5[+]/EX5[−] ratio and differed between children and adults (P<0.001); the EX5[+]/EX5[−] ratio varied between diagnoses (AML vs MDS, P<0.001). These findings bring new insights into WT1 isoform function and suggest that the ratio of WT1 isoforms, particularly EX5 variants, is probably crucial for the process of malignant transformation.

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Acknowledgements

We thank Ondrej Cinek from the Department of Pediatrics, Pavel Seeman from the Department of Pediatric Neurology, Zdenek Sedlacek from the Institute of Medical Biology and Genetics, 2nd Faculty of Medicine, Charles University in Prague, Olfert Landt from TIB-MOLBIOL, Berlin, Germany and Fiona Quinn from the Cancer Molecular Diagnostics Laboratory, St James’s Hospital, Dublin, for their invaluable advice and experienced opinions during method development and testing. We also appreciated the help of Katerina Muzikova, Ester Mejstrikova and Martina Sukova from the Department of Pediatric Hematology and Oncology, 2nd Faculty of Medicine, Charles University in Prague, and Jana Markova from the Institute of Hematology and Blood Transfusion, Prague, with the identification and initial processing of some samples. We thank the staff of the participating Czech Pediatric Hematology Working Group centers for cooperation. This study is supported by the grant IGA NS10488-3. KK was supported by the Charles University (GAUK 81709) solely and JT by the COST Program (OC 09051).

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Authors and Affiliations

  1. Department of Pediatric Hematology and Oncology, CLIP–Childhood Leukaemia Investigation Prague, 2nd Faculty of Medicine, Charles University, University Hospital Motol, Prague, Czech Republic

    K Kramarzova, J Stuchly, J Stary, J Trka & L Boublikova

  2. Department of Pediatric Hematology, Oncology and Hemostaseology, Goethe University, Hospital for children and adolescents II/III, Frankfurt am Main, Germany

    A Willasch

  3. Department of Pediatrics, University Hospital of Jena, Jena, Germany

    B Gruhn

  4. Institute of Hematology and Blood Transfusion, Prague, Czech Republic

    J Schwarz, J Cermak, K Machova-Polakova & O Fuchs

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  1. K Kramarzova
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Kramarzova, K., Stuchly, J., Willasch, A. et al. Real-time PCR quantification of major Wilms’ tumor gene 1 (WT1) isoforms in acute myeloid leukemia, their characteristic expression patterns and possible functional consequences. Leukemia 26, 2086–2095 (2012). https://doi.org/10.1038/leu.2012.76

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