Discovery of NUP98/NSD1 fusion in 2 CN-AML cases. (A) Array-CGH profiles of chromosome 11p15 showing the ratio of tumor DNA/control DNA (blue tracing) versus the inverted experiment (red tracing). The profile of patient 3495 (top panel) shows a part of the 0.4-Mb duplication involving the 5′ part of NUP98 (indicated by the blue vertical arrow), and the profile of patient 4716 shows the 0.1-Mb deletion involving the 3′ part of NUP98 (indicated by the red vertical bar). The horizontal arrows indicate the bar representing the NUP98 gene. (B) Split-signal FISH analysis of NUP98 for patients 3495 and 4716 using a partly overlapping green and red probe located in NUP98 (cen indicates probe situated more centromeric; tel indicates probe situated more telomeric. (C) RT-PCR analysis using NUP98- and NSD1-specific primers, and GAPDH primers as internal control, reveals a specific NUP98/NSD1 fusion transcript in patients 3495 and 4716. The reciprocal NSD1-NUP98 transcript was not detected in both patients (pos indicates positive control, [patient 5007]; neg indicates negative control [normal bone marrow]; ntc indicates non-template control). (D) Sequence analysis confirmed an identical in-frame fusion between NUP98 exon 12 and NSD1 exon 6 in both patients. (E) The NUP98/NSD1 fusion protein will harbor the GLFG-repeats of NUP98, and among others the PHD fingers and SET domain of NSD1. (F) Dual-color FISH analysis using a green probe for NUP98 and a red probe for NSD1 confirmed the fusion of NUP98 and NSD1 at the chromosomal level.