Comprehensive Characterization of a Novel Intronic Pseudo-Exon Inserted within an e14/a2 BCR-ABL Rearrangement in a Patient with Chronic Myeloid Leukemia

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Comprehensive Characterization of a Novel Intronic Pseudo-Exon Inserted within an e14/a2 BCR-ABL Rearrangement in a Patient with Chronic Myeloid Leukemia Nathalie Sorel, Caroline Mayeur-Rousse, Séverine Deverrière, Lydia Roy, Elisabeth Brottier-Mancini, François Guilhot, Ali G. Turhan, Jean Claude Chomel The Journal of Molecular Diagnostics Volume 12, Issue 4, Pages 520-524 (July 2010) DOI: 10.2353/jmoldx.2010.090218 Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 1 Detection of a novel intronic insertion within an e14/a2 BCR-ABL rearrangement. A: Using multiplex RT-PCR, an atypical BCR-ABL transcript was observed in the patient sample (p) as compared with e14/a2 (c1) and e1/a2 (c2) controls. Lane m: 100-bp ladder. B: Direct sequencing of the RT-PCR product revealed a 42-nucleotide insertion of ABL intron Ib within an e14/a2 BCR-ABL rearrangement. The resulting in-frame insertion of 15 amino acids was shown. C: A well-conserved donor splicing site was detected, at the genomic DNA level, in the 3′ end of the insertion. Uppercase and lowercase nucleotides corresponded respectively to pseudo-exonic and intronic sequences. The Journal of Molecular Diagnostics 2010 12, 520-524DOI: (10.2353/jmoldx.2010.090218) Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 2 Identification of the BCR-ABL breakpoint on genomic DNA. A: As we hypothesized that the BCR-ABL breakpoint occurred near the 5′ end of the inserted sequence, five primers located in BCR introns 13 and 14 and a reverse primer in ABL intron Ib (at the 3′ end of the insertion) were designed. B: Specific PCR amplifications were observed with two pairs of primers (d-z and e-z). The Journal of Molecular Diagnostics 2010 12, 520-524DOI: (10.2353/jmoldx.2010.090218) Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 3 Characterization of the consensus splice sites surrounding the 42-nucleotide insertion. Uppercase and lowercase nucleotides corresponded respectively to pseudo-exonic and intronic sequences. The BCR-ABL breakpoint occurred nine nucleotides upstream of the insertion. Well-conserved consensus splice sites were recognized in the sequence: donor site in ABL intron Ib, acceptor site at the BCR-ABL breakpoint, potential lariat branch point and polypyrimidine tract in BCR intron 14. The Journal of Molecular Diagnostics 2010 12, 520-524DOI: (10.2353/jmoldx.2010.090218) Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions

Figure 4 Consequences of an intronic insertion between BCR and ABL exons. A: The insertion conserves the reading frame between BCR and ABL exons. B: The inserted sequence disrupts the reading frame and leads to a premature stop codon. C: The insertion restores a reading frame with intact or truncated last BCR exon e8. The Journal of Molecular Diagnostics 2010 12, 520-524DOI: (10.2353/jmoldx.2010.090218) Copyright © 2010 American Society for Investigative Pathology and Association for Molecular Pathology Terms and Conditions