Published online 4 July 2008
Haematologica, Vol 93, Issue 9, 1398-1401 doi:10.3324/haematol.12945
Copyright © 2008 by Ferrata Storti Foundation
Paolo Gorello1 ,
Lucia Brandimarte1 ,
Roberta La Starza1 ,
Valentina Pierini1 ,
Loredana Bury1 ,
Roberto Rosati1 ,
Massimo F. Martelli1 ,
Peter Vandenberghe2 ,
Iwona Wlodarska2 ,
Cristina Mecucci1
In a case of acute myeloid leukemia we report molecular cytogenetic findings of a t(3;11)(q12;p15), characterized as a new NUP98 translocation rearranging with LOC348801 at chromosome 3. NUP98 involvement was detected by fluorescence in situ hybridization. 3’-RACE-PCR showed nucleotide 1718 (exon 13) of NUP98 was fused in-frame with nucleotide 1248 (exon 2) of LOC348801. RT-PCR and cloning experiments detected two in-frame spliced NUP98-LOC348801 transcripts and the reciprocal LOC348801-NUP98. A highly specific double-color double-fusion FISH assay reliably detects NUP98-LOC348801.
Chromosome translocations are recurrent features in hematologic malignancies. In primary and therapy-related acute myeloid leukemia (AML) and in T-cell acute lymphoblastic leukemia (T-ALL), the NUP98 gene, a member of the nucleoporin gene family which maps to chromosome 11p15.5, is a frequent target in chromosomal translocations. To date, 22 diverse NUP98 partners with different characteristics have been described and grouped into homeobox and non-homeobox genes.1–5 Here we characterize for the first time fusion of NUP98 and LOC348801 in a case of primary acute myeloid leukemia (AML) with t(3;11)(q12;p15).
Case report
A 28 year-old man was referred because of fatigue, fever and bleeding. Clinical examination revealed petechia, enlarged liver, spleen and lymph nodes. The peripheral blood count was: Hb 9.1 gr/dL, PLT 30x109/L, WBC 86.4x109/L with 41% blasts. Acute myeloid leukemia M2, according to the FAB classification, was diagnosed on bone marrow. The karyotype was: 46,XY,t(3;11)(q12;p15). The patient achieved hematologic remission after chemotherapy with the HOVON protocol.6 He relapsed seven months later. Second-line therapy with cytosine arabinoside and mitoxantrone failed. He died 23 months after diagnosis.
In this first case of primary AML with t(3;11)(q12;p15) characterized by NUP98-LOC348801 fusion, the 5’-region of NUP98 gene encoding GLFG repeats motifs and the GLEBS-like motif was fused in-frame with the 3’-region of LOC348801 gene (Figure 1). The reciprocal LOC348801-NUP98 fusion transcript was also present. LOC348801 is the 23rd gene to be described as a NUP98 fusion partner. It maps to chromosome 3q12.2 and contains four exons encoding for a protein with 178 aminoacids still lacking functional characterization.
The interphase break-apart FISH assay indicated NUP98 was involved in t(3;11)(q12;p15) (data not shown). Our 3’-RACE-PCR experiments showed that nucleotide 1718 (exon 13) of NUP98 was fused in-frame with nucleotide 1248 (exon 2) of LOC348801 (Figure 1). The genomic breakpoints appeared to fall within intron 13 of NUP98 and intron 1 of LOC348801. Double-color double-fusion FISH which gave one green signal, one red signal, and two fusion signals (Figure 2), further confirmed the reciprocal translocation t(3;11)(q12;p15) produced the NUP98-LOC348801 fusion gene.
Interestingly, in LOC348801 nucleotide 1248 is located 33 nucleotides upstream to the ATG start codon (1281–1283) (Figure 1). Thus, the predicted protein fuses the NUP98 FG repeat motifs and GLEBS-like motif to the entire LOC348801 through an 11 bridging peptide translated from non-coding sequence at the start of LOC348801 exon 2. RT-PCR and cloning experiments detected two in-frame alternatively spliced transcripts. Isoform 1 had NUP98 exon 12, upstream to the breakpoint region while in isoform 2 it was eliminated by alternative splicing (Figure 1). Alternative splicing mechanisms were reported in other NUP98 fusions.8–12 In mammalians splicing physiologically produces NUP98 or the NUP98-NUP96 mRNA which encodes a precursor protein of 186 kDa. The precursor is then proteolytically cleaved to produce NUP98 and NUP96 proteins.8,13 Interestingly, in several oncogenes aberrant and alternative splicing defects may underlie susceptibility to tumor development and progression.14,15 Our case emphasizes that alternative splicing is a frequent event in NUP98 leukemic recombinations.
the authors wish to thank Dr. Geraldine Boyd for assistance in the preparation of the manuscript. Funding: AIRC (Associazione Italiana Ricerca sul Cancro), MIUR (Ministero per l'Istruzione, l'Università e la Ricerca Scientifica); Fondazione Cassa di Risparmio, Perugia, Italy, FIRB, Italy and Associazione "Sergio Luciani", Fabriano, Italy. IAP (Interuniversity Attraction Poles, University of Leuven, Belgium) B.C. is supported by a grant from FIRC (Fondazione Italiana Ricerca sul Cancro). BAC clones were kindly provided by Dr Mariano Rocchi (DAPEG Sez. di Genetica, University of Bari, Italy).
PG and LB shared authorship.
Haematologica, Vol 93, Issue 9, 1398-1401 doi:10.3324/haematol.12945
Copyright © 2008 by Ferrata Storti Foundation
Acute Myeloid Leukemia |
t(3;11)(q12;p15)/NUP98-LOC348801 fusion transcript in acute myeloid leukemia
1 IbiT Foundation, Fondazione IRCCS Biotecnologie nel Trapianto, Hematology, University of Perugia, Perugia, Italy
2 Center for Human Genetics, University of Leuven, Leuven, Belgium
Correspondence: Cristina Mecucci, University of Perugia, IBiT Foundation, Fondazione IRCCS Biotecnologie nel Trapianto, Policlinico Monteluce, via Brunamonti 51, 06122 Perugia, Italy. E-mail:crimecux{at}unipg.it
ABSTRACT
 TOP ABSTRACT IntroductionDesign and MethodsResults and DiscussionReferences |
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In a case of acute myeloid leukemia we report molecular cytogenetic findings of a t(3;11)(q12;p15), characterized as a new NUP98 translocation rearranging with LOC348801 at chromosome 3. NUP98 involvement was detected by fluorescence in situ hybridization. 3’-RACE-PCR showed nucleotide 1718 (exon 13) of NUP98 was fused in-frame with nucleotide 1248 (exon 2) of LOC348801. RT-PCR and cloning experiments detected two in-frame spliced NUP98-LOC348801 transcripts and the reciprocal LOC348801-NUP98. A highly specific double-color double-fusion FISH assay reliably detects NUP98-LOC348801.
Key words: acute myeloid leukemia, NUP98, translocation partners.
Introduction
|
TOPABSTRACTIntroductionDesign and MethodsResults and DiscussionReferences |
|---|
Chromosome translocations are recurrent features in hematologic malignancies. In primary and therapy-related acute myeloid leukemia (AML) and in T-cell acute lymphoblastic leukemia (T-ALL), the NUP98 gene, a member of the nucleoporin gene family which maps to chromosome 11p15.5, is a frequent target in chromosomal translocations. To date, 22 diverse NUP98 partners with different characteristics have been described and grouped into homeobox and non-homeobox genes.1–5 Here we characterize for the first time fusion of NUP98 and LOC348801 in a case of primary acute myeloid leukemia (AML) with t(3;11)(q12;p15).
Design and Methods
|
TOPABSTRACTIntroductionDesign and MethodsResults and DiscussionReferences |
|---|
Case report
A 28 year-old man was referred because of fatigue, fever and bleeding. Clinical examination revealed petechia, enlarged liver, spleen and lymph nodes. The peripheral blood count was: Hb 9.1 gr/dL, PLT 30x109/L, WBC 86.4x109/L with 41% blasts. Acute myeloid leukemia M2, according to the FAB classification, was diagnosed on bone marrow. The karyotype was: 46,XY,t(3;11)(q12;p15). The patient achieved hematologic remission after chemotherapy with the HOVON protocol.6 He relapsed seven months later. Second-line therapy with cytosine arabinoside and mitoxantrone failed. He died 23 months after diagnosis.
Fluorescence in situ hybridization (FISH)
Interphase break-apart FISH assay was performed with clone RP11-348A20 spanning the 5’ region and exons 1–27 of NUP98/11p15, and clone CTD-3234F16 spanning the rest of NUP98 and flanking its 3’ region.7 Once the new NUP98 partner was identified at 3q12, we designed a specific double-color, double-fusion FISH assay by combining RP11-348A20/CTD-3234F16 for NUP98 (in green) with RP11-683B14, encompassing LOC348801 (in red).
3’-RACE- and RT-PCR
Total RNA was extracted by Trizol (Invitrogen) from the patient’s cryopreserved bone marrow cells and 1µg was reverse transcribed using 3’-RACE kit (Invitrogen). cDNA was amplified in semi-nested PCR (Expand extra long PCR system; Roche Applied Science, Penzberg, Germany) using NUP_1083_1106F (exon 8) as the first gene specific primer, NUP_1400_1419F (exon 11) as the second and AUAP (Abridged Universal Amplification Primer, Invitrogen) as reverse primer in both steps. The PCR product was sub-cloned into the pGEM-T easy vector (Promega), sequenced and analyzed using the BLAST program (NCBI, http://www.ncbi.nim.nih.gov/) and BLAT Genome Search (http://genome.ucsc.edu/cgi-bin/hgBlat) programs.
To confirm the NUP98-LOC348801 fusion transcript we performed RT-PCR experiments using primers NUP_1284_1303F (exon 10) and LOC_1843_1824R (exon 4 ) for the first amplification round and primers NUP_1400_1419F (exon 11) and LOC_1787_1768_R (exon 4) for the second.
Primers LOC_1171_1190F (exon 1) and NUP1861_1843R (exon 14) for the first round and primers LOC_1219_1238F (exon 1) and NUP1861_1843R (exon 14) for the second were used to search for the reciprocal fusion transcript.
Results and Discussion
|
TOPABSTRACTIntroductionDesign and MethodsResults and DiscussionReferences |
|---|
In this first case of primary AML with t(3;11)(q12;p15) characterized by NUP98-LOC348801 fusion, the 5’-region of NUP98 gene encoding GLFG repeats motifs and the GLEBS-like motif was fused in-frame with the 3’-region of LOC348801 gene (Figure 1). The reciprocal LOC348801-NUP98 fusion transcript was also present. LOC348801 is the 23rd gene to be described as a NUP98 fusion partner. It maps to chromosome 3q12.2 and contains four exons encoding for a protein with 178 aminoacids still lacking functional characterization.
 View larger version (29K): [in a new window] [Download PPT slide] |
Figure 1. NUP98/LOC348801 fusion transcript and sequencing. In-frame fusion of NUP98 and LOC348801, joining nucleotide 1718 (exon 13) of NUP98 to nucleotide 1248 (exon 2) of LOC348801. The in-frame fusion inserts 11 aminoacids (lower panel) derived from the LOC348801 exon 2 non-coding sequence (nt.1248-1280). Two in-frame splicing NUP98 isoforms (upper left) maintained the same fusion sequence. The NUP98 exon 12 was present in Isoform 1 and absent in isoform 2. (Sequence numbers refer to GenBank accessions NM_139131.1 for NUP98 and NM_001085451.1 for LOC348801) CDS: coding sequence.
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 View larger version (21K): [in a new window] [Download PPT slide] |
Figure 2. Double-color double-fusion FISH assay. Schematic representation of parts of chromosome 3 long arm and chromosome 11 short arm with localization/orientation of LOC348801 and NUP98 genes. DNA clones used for FISH studies are shown with their relative position and size (upper panel). In interphase nuclei, RP11-348A20/CTD-3234F16 for NUP98 (green) and RP11-683B14 for LOC348801 (red) gave one green signal, one red signal, and two fusion signals (lower panel).
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In this patient, a reciprocal LOC348801-NUP98 fusion transcript was also found as nucleotide 1247 (exon 1) of LOC348801 was fused with nucleotide 1719 (exon 14) of NUP98 (data not shown). Reciprocal fusion transcripts were reported in NUP98 translocations with different partners. However, since fusion with a partner may or may not produce the reciprocal transcripts generation does not seem to depend upon the characteristics of the partner gene.16–18 Whatever the mechanism, any possible biological and clinical significance of different NUP98 fusion isoforms and/or reciprocal transcripts is still not understood.
To date, t(3;11)(q12;p15) has been reported in two females, one with myelodysplastic syndrome and the other with T-ALL. In both cases NUP98 involvement was proven but partner(s) were not characterized.1 In another case of primary T-ALL/AML a t(3;11) recombination, which cytogenetically resembled translocation of our patient, was really a 3q translocation/inversion in which NUP98 recombined with the IQCG gene at 3q29.4
In conclusion NUP98, like MLL, is another promiscuous genes, that rearranges with many partners. In MLL-leukemia the translocation partner may influence clinical and phenotype features.19 Here we describe a new recombination between NUP98 and LOC348801 at 3q12 resulting in two in-frame spliced products and a reciprocal transcript. In our view, molecular characterization of new partners is a step towards a better understanding of the pathogenesis and mechanism of NUP98-positive leukemias. The highly specific double-color double-fusion FISH we set up for the NUP98-LOC348801 fusion provides the differential diagnosis between NUP98-IQCG and NUP98-LOC348801 and rapidly confirms genomic rearrangements, particularly in the event of ambiguous results with conventional cytogenetics.
Acknowledgments
the authors wish to thank Dr. Geraldine Boyd for assistance in the preparation of the manuscript. Funding: AIRC (Associazione Italiana Ricerca sul Cancro), MIUR (Ministero per l'Istruzione, l'Università e la Ricerca Scientifica); Fondazione Cassa di Risparmio, Perugia, Italy, FIRB, Italy and Associazione "Sergio Luciani", Fabriano, Italy. IAP (Interuniversity Attraction Poles, University of Leuven, Belgium) B.C. is supported by a grant from FIRC (Fondazione Italiana Ricerca sul Cancro). BAC clones were kindly provided by Dr Mariano Rocchi (DAPEG Sez. di Genetica, University of Bari, Italy).
Footnotes
PG and LB shared authorship.
PG: designed molecular studies and wrote the paper; LB: performed molecular studies and sequencing analyses; RLS: designed FISH studies and wrote the paper; VP: selected DNA clones and performed FISH experiments; LB: performed molecular studies and sequencing analyses; RR: supervised molecular studies; MFM: supervised clinical and experimental findings; PV: performed cytogenetic analysis and was involved in the management of the patient; IW: performed FISH experiments and provided clinical data; CM: was responsible for the conception and supervision of the study.
The authors reported no potential conflicts of interest.
Received for publication February 20, 2008. Revision received March 19, 2008. Accepted for publication April 10, 2008.
References
|
TOPABSTRACTIntroductionDesign and MethodsResults and DiscussionReferences |
|---|
- Romana SP, Radford-Weiss I, Ben Abdelali R, Schluth C, Petit A, et al. NUP98 rearrangements in hematopoietic malignancies: a study of the Groupe Francophone de Cytogénetique Hématologique. Leukemia 2006;20:679-706.
- Reader JC, Meekins JS, Gojo I, Ning Y. A novel NUP98-PHF23 fusion resulting from a cryptic translocation t(11;17)(p15;p13) in acute myeloid leukemia. Leukemia 2007;21:842-4.[Web of Science][Medline]
- Panagopoulos I, Kerndrup G, Carlsen N, Strombeck B, Isaksson M, Johansson B. Fusion of NUP98 and the SET binding protein 1 (SETBP1) gene in a pediatric acute T cell lymphoblastic leukaemia with t(11;18)(p15;q12). Br J Haematol 2007;136:294-6.[CrossRef][Web of Science][Medline]
- Pan Q, Zhu Y-J, Gu B-W, Cai X, Bai X-T, Yun H-Y, et al. A new fusion gene NUP98-IQCG identified in an acute T-lymphoid/myeloid leukemia with a t(3;11) (q29q13;p15) del(3)(q29) translocation. Oncogene 2008;27:3414-23.[CrossRef][Web of Science][Medline]
- Moore MAS, Chung KY, Plasilova M, Schuringa JJ, Shieh J-H, Zhou P, et al. NUP98 dysregulation in myeloid leukemogenesis. Ann N Y Acad Sci 2007;1106:114-42.[CrossRef][Web of Science][Medline]
- Daenen S, Löwenberg B, Sonneveld P, van Putten WL, Verhoef G, Verdonck LF, et al. Efficacy of etoposide and mitoxantrone in patients with acute myelogenous leukemia refractory to standard induction therapy and intermediate-dose cytarabine with amsidine. Dutch Hematology-Oncology Working Group for Adults (HOVON). Leukemia 1994;8:6-10.[Web of Science][Medline]
- Nebral K, Konig M, Schmidt H, Lutz D, Sperr WR, Kalwak K, et al. Screening for NUP98 rearrangements in hematopoietic malignancies by fluorescence in situ hybridisation. Haematologica 2005;90:746-52.
[Abstract/Free Full Text] - Lam DH, Aplan PD. NUP98 gene fusions in hematologic malignancies. Leukemia 2001;15:1689-95.[Web of Science][Medline]
- La Starza R, Gorello P, Rosati R, Riezzo A, Veronese A, Terrazzi E, et al. Cryptic insertion producing two NUP98/NSD1 chimeric transcript in adult refractory anemia with excess of blasts. Genes Chromosom Cancer 2004;41:395-9.[CrossRef][Web of Science][Medline]
- Kasper LH, Brindle PL, Schnabel CA, Pritchard CE, Cleary ML, Deursen JM. CREB binding protein interacts with nucleoporin – specific FG repeats that activate transcription and mediate NUP98-HOXA9 oncogenicity. Mol Cell Biol 1999;19:764-76.
[Abstract/Free Full Text] - Arai Y, Kyo T, Miwa H, Arai K, Kamada N, Kita K, et al. Heterogeneous fusion transcript involving the NUP98 gene and HOXD13 gene activation in a case of acute myeloid leukaemia with the t(2;11)(q31;p15) translocation. Leukemia 2000;14:1621-9.[CrossRef][Web of Science][Medline]
- Hatano Y, Miura I, Nakamura T, Yamazaki Y, Takahashi N, Miura AB. Molecular heterogenity of the NUP98/HOXA fusion transcript in myelodysplastic syndromes associated with t(7;11)(p15;p15). Br J Haematol 1999;107:600-4.[CrossRef][Web of Science][Medline]
- Fontoura MA, Blodel G, Matunis MJ. A conserved biogenesis pathway for nucleoporins: proteolytic processing of a 186-kilodalton precursor generates Nup98 and a novel nucleoporin, Nup96. J Cell Biol 1999;144:1097-112.
[Abstract/Free Full Text] - Kalnina Z, Zayakin P, Silina K, Line A. Alteration of Pre-mRNA Splicing in Cancer. Genes, Chromosome and Cancer 2005;42:342-57.[CrossRef]
- Venables JP. Aberrant and alternative splicing in cancer. Cancer Res 2004;64:7647-54.
[Abstract/Free Full Text] - Hussey DJ, Nicola M, Moore S, Peters GB, Dobrovic A. The (4;11) (q21;p15) translocation fuses the NUP98 and RAP1GDS1 genes and is recurrent in T-cell acute lymphocytic leukemia. Blood 1999;94:2072-9.
[Abstract/Free Full Text] - Iwase S, Akiyama N, Sekikawa T, Saito S, Arakawa Y, Horiguchi-Yamada J, et al. Both NUP98/TOP1 and TOP1/NUP98 transcript are detected in a de novo AML with t(11;20)(p15;q11). Genes Chromosomes Cancer 2003;38:102-5.[CrossRef][Web of Science][Medline]
- Ahuja HG, Felix AC, Aplan PD. The t(11;20)(p15;q11) chromosomal translocation associated with therapy-related myelodysplastic syndrome results in a NUP98-TOP1 fusion. Blood 1999;94:3258-61.
[Abstract/Free Full Text] - Krivtsov AV, Armstrong SA. MLL translocation, histone modifications and leukaemia stem-cell development. Nat Rev Cancer 2007;7:823-33.[CrossRef][Web of Science][Medline]
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