A Novel Chromosomal Translocation T(1;14)(Q25;Q32) in Pre-B Acute Lymphoblastic Leukemia Involves the LIM Homeodomain Protein Gene, Lhx4
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Oncogene (2002) 21, 4983 – 4991 ª 2002 Nature Publishing Group All rights reserved 0950 – 9232/02 $25.00 www.nature.com/onc A novel chromosomal translocation t(1;14)(q25;q32) in pre-B acute lymphoblastic leukemia involves the LIM homeodomain protein gene, Lhx4 Norihiko Kawamata*,1, Sakura Sakajiri1, Kei-ji Sugimoto1, Yasushi Isobe1, Hirofumi Kobayashi1,2 and Kazuo Oshimi1 1Division of Hematology, Department of Medicine, Juntendo University School of Medicine, 2-1-1 Hongo, Bunkyo-ku, Tokyo 113-8421, Japan; 2Division of Hematology, Department of Medicine, Saitama Cancer Center, 818 Komuro, Ina, Saitama 362-0806, Japan Chromosome 1q21-25 is one of the hotspots of Introduction chromosomal abnormalities including translocations and duplications in hematological malignancies. This would In acute lymphoblastic leukemia (ALL), several genetic suggest that oncogene(s) reside in this region. We have abnormalities associated with leukemogenesis have cloned the junctional sequence of t(1;14)(q25;q32) in pre- been reported (Look, 1997). In adult ALL of B cell B acute lymphoblastic leukemia cells by an inverse PCR lineage, t(9;22)(q32;q11) resulting in the generation of method. A novel sequence was fused to the joining region an oncogenic fusion gene, BCR – ABL, is most of the immunoglobulin heavy chain gene. We confirmed frequently detected. In addition to BCR – ABL, this rearrangement by Southern blot analysis, genomic ETV6 – AML1, E2A – Pbx, IgH – IL3, E2A – HLF and PCR and fluorescence in situ hybridization. We found a rearrangements involving the MLL gene are detected in coding sequence which is homologous to the mouse Lhx4 ALL of B-cell lineage (Look, 1997; Willis and Dyer, cDNA sequence 17 kb from the breakpoint. The human 2000). However, many oncogenes involved in trans- Lhx4 gene encodes 390 amino-acids, including one locations in ALL remain to be analysed. tandem pair of LIM domains and one homeodomain. In human cancer cells, oncogenes are frequently The human Lhx4 gene consists of six exons. Lhx4 overexpessed by transcriptional deregulation (Lengauer protein is very homologous to human Lhx3 protein et al., 1998; Nowell, 1997; Popescu and Zimonjic, except in the N-terminal region. The transcripts of the 1997). Gene amplification of oncogenes induces Lhx4 gene were not detected in adult multiple tissues overexpression. This amplification is usually detected analysed by Northern blotting, but were detected in the as double minutes or homogeneously stained regions leukemic cells carrying t(1;14)(q25;q32) by reverse- on karyotyping (DeVita et al., 2001). Translocation transcription PCR. The protein expression of Lhx4 in also results in the overexpression of oncogenes, in these leukemic cells was confirmed by Western blot which the regulatory region of the oncogenes is analysis. Lhx4 activated the reporter gene carrying the replaced by the regulatory region, including enhancer mouse a-glycoprotein subunit promoter region, which is elements, of other genes (Willis and Dyer, 2000). In regulated by Lhx3. LIM protein and homeodomain hematological malignancies derived from B lympho- protein genes are frequently involved in translocations of cytes, rearrangements between the immunoglobulin hematological malignancies. The Lhx4 gene is deregu- heavy chain (IgH) gene, which resides on chromo- lated in the leukemic cells and Lhx4 protein may play an some 14q32, and oncogenes are frequently detected important role, possibly as an activator, in leukemogen- (Willis and Dyer, 2000). In these rearrangements, the esis. regulatory region of the oncogenes is replaced by the Oncogene (2002) 21, 4983 – 4991. doi:10.1038/sj.onc. enhancer region of the IgH gene, which induces 1205628 overexpression of the oncogene and is associated with tumorigenesis of the B cell lymphoid malignancies Keywords: inverse PCR; transcriptional factor; immu- (Willis and Dyer, 2000). noglobulin heavy chain gene; RT – PCR; reporter gene Dup(1q21-25) is frequently detected in ALL of B-cell assay lineage (Mitelman et al., 1997). Similarly, lymphomas and myelogenous malignancies often have genetic abnormalities involving this region (Mitelman et al., 1997). 1q21-25 is one of the hotspots, areas of chromosomal alteration including duplications and translocations, frequently detected in hematological malignancies (Mitelman et al., 1997). Although it is not clear whether duplications result in the gene *Correspondence: N Kawamata; E-mail: [email protected] amplification at present, this structural change in Received 6 November 2001; revised 18 April 2002; accepted 26 chromosomes might deregulate the expression pattern April 2002 of the genes. Novel oncogene(s) might reside in this IgH/Lhx4 rearrangement in pre-B ALL N Kawamata et al 4984 region and overexpression of the gene(s) might be using one primer specific to IgH (JH4AS) and one associated with leukemogenesis. novel sequence (WR1). PCR products were obtained T(1;14)(q25,q32) is a novel chromosomal abnorm- from the leukemic cells but not from control DNA ality detected in ALL of B-cell lineage. This (Figure 3a). The sequence of the PCR product was translocation might involve the IgH gene and novel confirmed (data not shown). Further, we performed genes. Overexpression of a novel gene residing on 1q25 Southern blot analysis using one probe specific to IgH and fusion to the IgH gene could be associated with and a novel sequence (w360 probe). Using the IgH leukemogenesis. probe, two rearranged bands were detected on HindIII In this study we cloned a junctional sequence of digestion and one rearranged band on EcoRI digestion, t(1;14)(q25;q32) and found a fusion of the IgH gene and using the w360 probe, a single rearranged band and a LIM homeodomain gene. was detected in the EcoRI and HindIII digest, respectively (Figure 3b). On HindIII digestion, one of the two rearranged bands detected by the IgH probe Results and the rearranged band detected by the W360 probe co-migrated (asterisks in Figure 3b). On EcoRI Clinical sample digestion, the rearranged band detected by the w360 A 53-year-old female was admitted to our hospital probe co-migrated with the germ line band detected by because of fever. She was diagnosed as having acute the IgH probe. This result might suggest that one of lymphoblastic leukemia of pre-B cell type. She was the rearranged bands detected by the IgH probe co- treated with multiple reagent chemotherapy. A migrated with the germ line band on EcoRI digestion, complete remission was achieved after induction and that the rearranged bands detected by the IgH and therapy, and consolidation and maintenance therapy w360 probes also co-migrated on EcoRI digestion. were performed. During the maintenance therapy, the Then we screened the phage genomic library using patient was transferred to another hospital to be the w360 probe and obtained overlapping clones treated by allogeneic bone marrow transplantation. (Figure 4). Using one of the phage clones (lambda Q-banded karyotyping of her leukemic cells were 1212) as a probe, we performed FISH to analyse the performed as previously reported (Sandberg, 1990). chromosomal localization of the novel sequence. The The karyotyping of her leukemic cells at admission signals were detected at 1q23-25 (Figure 5a). These revealed t(9;22)(q23;q11) and t(1;14)(q25;q32) (Figure results confirmed that the junctional sequence we 1a). After complete remission, these chromosomal cloned is derived from t(1;14)(q25;q32). Further, we abnormalities disappeared. Thus, she was diagnosed detected fusion signals between the IgH gene and the with de novo ALL, not blastic crisis from chronic novel sequence in these leukemic cells by the dual color myelogenous leukemia. FISH analysis (Figure 5b) (see below). T(1;14)(q25;q32) was detected in acute lymphoblastic We determined the genomic sequence around the leukemia of pre-B cell type. We employed inverse PCR breakpoint in the novel sequence and compared it with for the DNA from the leukemic cells. We obtained the rearranged sequence. The N-segment of 24 nucleo- several bands (Figure 1b) then we cloned the products tides was inserted between JH4 and the novel sequences into plasmids and sequenced them. Two bands revealed (Figure 2). Further, we sequenced the phage clones and a novel sequence fused to the IgH gene (Figure 2). The searched for a gene around the breakpoint. We found a breakpoint resided in the JH4 region of the IgH gene sequence highly homologous to mouse Lhx4 gene clones (Ravetch et al., 1981). To exclude artifacts during the about 25 kb from the break point (Figure 4). inverse PCR procedure, we performed a genomic PCR To obtain the coding sequence of this putative gene, we performed anchor PCR on HEK293 cells. Then, we assembled the sequences and obtained a 1243-nucleo- tide cDNA sequence which had a single open reading frame of 1170 nucleotides (Figure 6a). The deduced amino-acid sequence was highly homologous to that of mouse Lhx4 (Figure 6b), so we concluded that this gene is a human homolog for mouse Lhx4 and named it the human Lhx4 gene. After our submission of this manuscript, two other groups have reported the cDNA sequence of the human Lhx4 gene (Liu et al., 2002; Machinis et al., 2001). In the sequence reported by Machinis et al. (2001) the nucleotide sequence encoding Figure 1 Chromosomal abnormality and inverse PCR. (a) N-terminal region is missing. The cDNA sequence of T(1;14)(q25;q32) was detected in the leukemic cells. Numbers indi- the human Lhx4 gene reported by Liu et al. (2002) is cate chromosome numbers. Arrows indicate the translocated chro- the same as that reported in this study. mosomes. (b) Inverse PCR was performed on XbaI digested (Xb) The human Lhx4 gene encodes a product of 390 and BglII (Bg) digested DNA (see Materials and methods). Several PCR bands were obtained and all bands were sequenced. Arrow amino-acids, which is homologous to the Lhx family heads indicate the bands having the IgH gene fused to a novel se- proteins (Dawid et al., 1998).