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Blood, Vol. 114, Issue 10, 2159-2167, September 3, 2009
Identification and molecular characterization of recurrent genomic deletions on 7p12 in the IKZF1 gene in a large cohort of BCR-ABL1–positive acute lymphoblastic leukemia patients: on behalf of Gruppo Italiano Malattie Ematologiche dell'Adulto Acute Leukemia Working Party (GIMEMA AL WP)
Blood Iacobucci et al.
114: 2159
Supplemental materials for: Iacobucci et al
Files in this Data Supplement:
- Table S6. IKZF1 genomic QPCR (PDF, 72.5 KB) -
Real-time genomic quantitative PCR (Q-PCR) of promoter and all IKZF1 exons was performed in order to confirm SNP results and to characterise the focal deletions. Genomic quantitative PCR of all 7 coding IKZF1 exons was performed for 8 BCR-ABL1–positive ALL cases and 2 germline samples to verify the extent of IKZF1 deletions, in 16 cases (13 BCR-ABL1–positive ALL samples and 3 germline samples) a subset of exons were tested to confirm the different focal IKZF1 deletions and in the remaining cases only qualitative PCR was performed to confirm the deletion and map the breakpoints. Genomic quantitative PCR results were expressed as IKZF1/RNAseP gqPCR ratios as described in Mullighan et al.7 IKZF1/RNAseP gqPCR ratios of less than 0.75 indicate heterozygous deletion and ratios of less than 0.3 indicate homozygous deletion. Samples labelled with “*” in the table had borderline results on qPCR, due to the their low blast cell percentage.
- Table S7. IKZF1 Δ4–7 breakpoints (PDF, 41.5 KB) -
Genomic amplification and sequencing of IKZF1 Δ4–7 deletions showed that the genomic breakpoints occurred in intron 3 and 7 and that the nucleotides which are inserted at the conjunction are patient specific. Heptamer RSSs were located immediately within the deleted segment or just before it. The heptamer RSSs are shown underlined and bold, the nucleotides matching the RSS exactly are shown in red and the AID consensus sequences are shown bold and in blue. NA= not available (IKZF1 sequencing for these cases was not performed or failed due to limited DNA).
- Table S8. IKZF1 Δ2–7 breakpoints (PDF, 29 KB) -
Amplification and sequencing of IKZF1 Δ4–7 deletions showed that the genomic breakpoints occurred in intron 1 and 7 and that the nucleotides which are inserted at the conjunction are patient specific. NA= not available (IKZF1 sequencing for these cases was not performed or failed due to limited DNA).
- Table S9. IKZF1 Δ4–7 and IKZF1 Δ2–7 breakpoints (PDF, 14.7 KB) -
Genomic breakpoints of the patient BCR-ABL1–#50 and BCR-ABL–#95 showing two different breakpoints on the two alleles.
- Table S10. IKZF1 Δ4–8 breakpoints (PDF, 12.6 KB)
- Table S11. IKZF1 deletions in other leukemias (PDF, 92.2 KB) -
Molecular and cytogenetic characteristics are reported in the Table. “Other” in the cases of acute leukemia myeloid (AML) indicates the presence of any of these chromosomal rearrangements: t(2;3)(p22–23)(q26–27), t(6;8)(q27;p12), t(2;13)(p21;q14), t(3;14)(p13;q32), t(1;3)(p36;q21), t(6;8)(q27;p12) and t(7;11)(p15;p15). Abbreviations: HES: Hypereosinophilic Syndrome; CML: chronic myeloid leukemia; CP: chronic phase; ly-BC: lymphoid blast crisis; my-BC: lymphoid blast crisis.
- Figure S1. Real-time quantitative PCR on cDNA samples demonstrated that genomic alteration in IKZF1 led to a significant down-modulation at the transcript level of the DNA binding isoforms (JPG, 94.7 KB)
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- Figure S2. Genomic PCR of IKZF1 Δ4–7 (JPG, 236 KB)
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Primers used were D1 and D2 (Table S2); the obtained PCR products were then directly sequenced to characterize the sequence flanking deletion breakpoints.
- Figure S3. Sequencing of RT-PCR products (A) and western blotting (B) confirmed the expression of the non–DNA-binding Ik6 isoforms in IKZF1 Δ4–7 deleted cases (JPG, 222 KB)
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Confocal images of leukemic cells from patients without IKZF1 Δ4–7 showed the characteristic multifocal nuclear localization pattern of Ikaros (e.g., bright green fluorescent rim surrounding the toto-labeled red nuclei) (C).
- Figure S4. Pherograms of sequencing of transcript generated following IKZF1 Δ2–7 breakpoints (JPG, 238 KB)
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- Figure S5. The sequence motifs, searched by fuzznuc, are listed (JPG, 309 KB)
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Motifs not shown in the figure have not been detected along the analyzed sequence.
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