gene is known to be involved in multiple rearrangements in haematological malignancy [3
]. The 11p15 breakpoint in our patient suggested possible disruption of the NUP98
gene. We therefore attempted a 3'RACE approach to test for the presence of a fusion mRNA using a method similar to that used to identify RAP1GDS1 as a fusion partner of NUP98 [9
]. As the most 5' break in NUP98
known at the time occurred in the intron after exon 10 [9
], we used a NUP98
forward primer from exon 9 for 3'RACE. This approach resulted in a number of RT-PCR products from the t(9;11) patient that were different in size from the NUP98
3'RACE products amplified from normal individuals. However sequence analysis of these products showed that they resulted from partially spliced NUP98
mRNAs rather than novel fusion mRNAs (results not shown).
Subsequently, an AML patient was reported in which the t(9;11)(p22;p15) resulted in an in-frame fusion of the NUP98
]. This involved fusion of NUP98
exon 9 to LEDGF
exon 6. As RACE of our patient had failed to detect any fusions including exon 9 of NUP98,
we used a NUP98
primer from exon 8 with a LEDGF
reverse primer from exon 6 for RT-PCR (Figure ). An RT-PCR product was obtained in which exon 8 of NUP98
was fused in-frame to exon 2 of LEDGF.
breakpoint thus maps to the 5.5 kb intron between exons 8 and 9 and is the most 5' NUP98
breakpoint reported to date. The LEDGF
breakpoint in our patient is more 5' than that found in the patient reported by Ahuja et al [2
] and occurs within the 3.5 kb intron between exons 1 and 2.
Figure 1 RT-PCR analysis of NUP98-LEDGF expression. RT-PCR for the NUP98-LEDGF fusion was performed as outlined in the materials and methods. RNA samples are from peripheral blood mononuclear cells taken from a normal donor (lane 1), bone marrow mononuclear cells (more ...)
We also used RT-PCR to assess expression of the NUP98-LEDGF fusion mRNA in remission peripheral blood taken twenty months after presentation. Using standard PCR conditions with 35 cycles of PCR (Figure ), there was no fusion transcript visible. Even after first round PCR with 45 cycles followed by fully nested PCR with 45 cycles we did not detect NUP98-LEDGF fusion mRNA in the remission sample (data not shown). However, RT-PCR of the relapse specimen showed that the NUP98-LEDGF transcript was re-expressed consistent with the association of the translocation with the disease.
We were able to amplify the reciprocal LEDGF-NUP98
fusion transcript at presentation and relapse. Sequencing of this product showed an in-frame fusion of exon 2 of LEDGF
to exon 9 of NUP98
as expected. The reciprocal transcript is unlikely to be important in the pathogenesis of the disease as the previous report did not observe a reciprocal fusion transcript [2
]. However, it is possible that the reciprocal fusion transcript may modulate the course of the disease particularly in the light of the less aggressive form of the disease seen in our patient. Similar patient to patient differences in whether or not the reciprocal transcript is expressed have also been noted for other fusions including BCR-ABL and NUP98-RAP1GDS1
We were also able to obtain RNA using fixed leukemic cells from the first reported t(9;11) patient [1
]. This RNA was highly degraded and had an average length of 200 bases. Despite being able to amplify a 400 bp NUP98
RT-PCR product, we were unable to amplify a a smaller product corresponding to either of the 2 known NUP98-LEDGF
fusions nor any of a number of other potential NUP98-LEDGF
fusions that we tried.
The previously reported NUP98-LEDGF
] encodes a protein fusing the amino terminus of the NUP98
gene containing 28 of the 38 FG repeats with exon 6 onwards of p52/75. FG repeats in NUP98 fusion proteins have been shown to act as transactivation domains which recruit CBP/p300 [8
]. The minimum number of FG repeats which are important for the transforming properties of NUP98 fusion proteins has not yet been defined. The fusion in our patient contains 23 FG repeats from NUP98.
Interestingly, just after the breakpoint LEDGF
exon 2 codes for an additional FG residue. It is unknown whether this residue is important for the function of the NUP98-LEDGF fusion protein.
gene codes for 2 transcriptional co-activators, p75 (LEDGF:
Lens Epithelium Derived Growth Factor) and p52 which have different 3' ends generated by alternative splicing [15
]. Both NUP98
-p52 and NUP98
-p75 fusion mRNAs were seen in our patient at presentation and relapse (data not shown) as well as in the previously characterised t(9;11) patient [2
]. It remains to be determined whether one transcript is more important in the leukemogenic process.
p52 and p75 both contain a PWWP domain at their amino terminus [14
]. PWWP is the core motif of a 70 amino acid domain found in a variety of nuclear proteins [14
]. The PWWP domain was lost in the NUP98-LEDGF
fusion described by Ahuja et al [2
] and disrupted in the fusion described here. Interestingly, the PWWP domain is also found in NSD1, the most recently identified NUP98
partner gene and is absent in the NUP98-NSD1
fusion transcript [10
The three AML patients with the t(9;11)(p22;p15) vary in their clinical picture (Table ). At this stage, it is too early to determine which clinicopathological features are a hallmark of this translocation, especially as it is not clear as to whether the first patient's translocation has a similar molecular basis to the other two. Patients 2 and 3 have a more mature myeloid phenotype than that seen in patient 1. All three presented with the t(9;11)(p22;p15) as their sole cytogenetic abnormality. At relapse, the leukemic cells of both patients 2 and 3 had the same karyotype as at presentation whereas the relapse karyotype of patient 1 no longer showed the translocation.
Clinical features, immunophenotype of patients with the t(9;11)(p22p15).
translocations are associated with secondary leukemias that occur after treatment with topoisomerase II inhibitors [3
]. The patient in this report had not received chemotherapy prior to her disease and prior chemotherapy was not mentioned in either of the other two case reports [1
]. It seems that t(9;11)(p22;p15) is preferentially associated with de-novo AML rather than therapy related AML.