PMC3567982_01
Cytogenetic and fluorescence in situ hybridization studies of the AML patient. The bone marrow sample was cultured for 24 h following standard procedures. Chromosome banding analysis of the bone marrow was performed after Leichmann's staining and the karyotype was described according to ISCN. A) Bone marrow karyotype obtained by GTL banding technique exhibiting a t(3;11)(p11;p15) as the sole chromosome abnormality, suggesting the involvement of the NUP98 gene located at 11p15. Derivative chromosomes 3 and 11 are marked with arrows. BAC clones for NUP98 and POU1F1 were selected using the UCSC Human Genome Browser and obtained from the BACPAC Resource Center. B) Dual color, dual fusion FISH analysis performed on metaphases using BAC probes for NUP98 (CTD-3082 N2, RP11-161I4) labeled in green and for POU1F1 (CTD-2372C6) labeled in red, showed the presence of fusion signals on der(3) and der(11) and isolated red and green signals on normal chromosomes 3 and 11, respectively.
1476-4598-12-5-1.jpg
PMC3567982_01_1476-4598-12-5-1.jpg
F1
The bone marrow karyotype revealed a t(3;11)(p11;p15) as the sole cytogenetic abnormality in all 30 metaphases analyzed (Figure 1A), suggesting the involvement of the NUP98 gene located in 11p15. FISH analysis revealed the presence of fusion signals on the der (3) and on the der (11) chromosomes, strongly suggesting the presence of a rearrangement involving NUP98 and POU1F1 (Figure 1B). We found an internal tandem mutation of the FLT3 gene, as shown by the presence of an amplification product of approximately 350 bp (Additional file 2: Figure S1).
Characterization of the NUP98-POU1F1 fusion. A) RT-PCR analysis with one antisense primer on POU1F1 exon 5 and three sense primers on NUP98 exons 9, 10, and 11 (lanes 3, 5, and 7) showed the presence of PCR fragments of 362, 242, and 101 bp, respectively, suggestive of a fusion between NUP98 exon 11 and POU1F1 exon 5. Additional RT-PCR analysis with several NUP98 and POU1F1 primer combinations gave additional support to this hypothesis since no amplification was observed (lanes 2, 4, 6, 8, and 9). Lane 10: RNA integrity check (370 bp B2M gene fragment). Lanes 1 and 11: 100 bp molecular marker. B) Sequence analysis followed by a BLAST search confirmed that NUP98 exon 11 was fused in-frame with nucleotide 730 of the POU1F1 transcript (arrow). C) Schematic representation of the NUP98, POU1F1, and the NUP98-POU1F1 fusion proteins showing the relevant domains of the partner and chimeric proteins. D) Genomic breakpoint analysis with the NUP98_Fint11H sense primer in combination with the POU1F1_Rint4B and POU1F1_Rint4A antisense primers gave origin to amplification products of 603 and 412 bp, respectively (lanes 1 and 2). Lane 3: 100 bp molecular marker. E) Sequencing of the amplification products showed that the breakpoint was located 7490 bp downstream of NUP98 exon 11 and 129 bp downstream of the start of POU1F1 exon 4 (arrow). F) Schematic representation of the genomic DNA breakpoint (arrow) and nucleotide sequence of the genomic breakpoint of the translocation t(3;11) and corresponding normal chromosomes 3 and 11.
1476-4598-12-5-2.jpg
PMC3567982_01_1476-4598-12-5-2.jpg
F2
RT-PCR with an antisense primer located on POU1F1 exon 5 and three NUP98 sense primers located on exons 9, 10, and 11 (Additional file 1) showed the presence of PCR fragments suggestive of an NUP98-POU1F1 rearrangement resulting from fusion of NUP98 exon 11 with POU1F1 exon 5 (Figure 2A). Additional RT-PCR analysis with sense primers located on NUP98 exons 9, 10, 11 and an antisense primer located on POU1F1 exon 4 and with a sense primer located on NUP98 exon 12 and antisense primers located on POU1F1 exons 4 and 5, gave additional support to this hypothesis, since no amplification was observed (Figure 2A). NUP98 exon 11 was fused in-frame with nucleotide 730 of the POU1F1 transcript (GenBank accession no. NM_000306) (Figure 2B). This fusion is expected to give rise to a chimeric fusion protein where the N terminus of NUP98 is fused to the C terminus of POU1F1 (Figure 2C). POU1F1_Rint4A and POU1F1_Rint4B antisense primers, amplification products of 412 bp and 603 bp were observed, respectively (Figure 2D). Partial sequencing of the amplification products showed that the breakpoint was located 7490 bp downstream of NUP98 exon 11 and, interestingly, within POU1F1 exon 4, 129 bp downstream of the start of POU1F1 exon 4, and no evidence of mutation or deletion was detected in the breakpoint region (Figure 2A and F).