Published online 6 October 2008
Haematologica, Vol 93, Issue 12, 1814-1821 doi:10.3324/haematol.13260
Copyright © 2008 by Ferrata Storti Foundation

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Acute Lymphoblastic Leukemia

Identification of different Ikaros cDNA transcripts in Philadelphia-positive adult acute lymphoblastic leukemia by a high-throughput capillary electrophoresis sizing method

Ilaria Iacobucci¹, Annalisa Lonetti¹, Daniela Cilloni³, Francesca Messa³, Anna Ferrari¹, Roberta Zuntini², Simona Ferrari², Emanuela Ottaviani¹, Francesca Arruga³, Stefania Paolini¹, Cristina Papayannidis¹, Pier Paolo Piccaluga¹, Simona Soverini¹, Giuseppe Saglio³, Fabrizio Pane⁴, Anna Baruzzi⁵, Marco Vignetti⁶, Giorgio Berton⁵, Antonella Vitale⁶, Sabina Chiaretti⁶, Markus Müschen⁷, Robin Foà⁶, Michele Baccarani¹, Giovanni Martinelli¹

¹ Department of Hematology/Oncology "L. and A. Seràgnoli" S.Orsola Malpighi Hospital, University of Bologna, Bologna, Italy
² Medical Genetics Unit, S.Orsola-Malpighi University Hospital, Bologna, Italy
³ Department of Clinical and Biological Science, University of Turin at Orbassano, Turin, Italy
⁴ CEINGE Biotecnologie Avanzate and Department of Biochemistry and Medical Biotechnology, University of Naples Federico II, Naples, Italy
⁵ Department of Pathology, Section of General Pathology, University of Verona, Verona, Italy
⁶ "La Sapienza" University, Department of Cellular Biotechnologies and Hematology, Rome, Italy
⁷ Leukemia Research Program, Childrens’ Hospital Los Angeles, University of Southern California, CA, USA

Correspondence: Giovanni Martinelli, Molecular Biology Unit, Institute of Hematology and Medical Oncology "Seràgnoli", University of Bologna, Via Massarenti, 9, 40138 Bologna, Italy., E-mail:giovanni.martinelli2{at}unibo.it

Background: Ikaros is the prototypic member of a Kruppel-like zinc finger transcription factor subfamily that is required for normal hematopoietic cell differentiation and proliferation, particularly in the lymphoid lineages. Alternative splicing can generate multiple Ikaros isoforms that lack different numbers of exons and have different functions. Shorter isoforms, which lack the amino-terminal domain that mediates sequence-specific DNA binding, exert a dominant negative effect and inhibit the ability of longer heterodimer partners to bind DNA.

Design and Methods: In this study, we developed a high-throughput capillary electrophoresis sizing method to detect and quantify different Ikaros cDNA transcripts.

Results: We demonstrated that Philadelphia chromosome-positive acute lymphoblastic leukemia cells expressed high levels of the non-DNA-binding isoform Ik6 that was generated following IKZF1 genomic deletions (19/46 patients, 41%). Furthermore, a recurring 60 bp insertion immediately upstream of exon 5, at the exon 3/exon 5 junction, was frequently detected in the Ik2 and Ik4 isoforms. This insertion occurred either alone or together with an in-frame ten amino acid deletion that was due to a 30 bp loss at the end of exon 7. Both the alterations are due to the selection of alternative cryptic splice sites and have been suggested to cause impaired DNA-binding activity. Non-DNA-binding isoforms were localized in the cytoplasm whereas the DNA-binding isoforms were localized in the nucleus.

Conclusions: Our findings demonstrate that both aberrant splicing and genomic deletion leading to different non-DNA-binding Ikaros cDNA transcripts are common features of Philadelphia chromosome-positive acute lymphoblastic leukemia.

Key words: BCR-ABL1, acute lymphoblastic leukemia, Ikaros.