Figure 2

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.