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November 2002, Volume 16, Number 11, Pages 2205-2212
Table of contents    Previous  Article  Next   [PDF]
Original Manuscript
Disruption of the RanBP17/Hox11L2 region by recombination with the TCRdelta locus in acute lymphoblastic leukemias with t(5;14)(q34;q11)
T E Hansen-Hagge1,5,a, M Schäfer2,a, H Kiyoi1,6,a, S W Morris3, J A Whitlock4, P Koch2, I Bohlmann2, C Mahotka1,7, C R Bartram1,2 and J W G Janssen1,2

1University of Ulm, Section of Molecular Biology, Department of Pediatrics II, Ulm, Germany

2University of Heidelberg, Institute of Human Genetics, Heidelberg, Germany

3St Jude Children's Research Hospital, Department of Pathology, Memphis, TN, USA

4Vanderbilt University Medical Center, Division of Pediatric Hematology-Oncology, Nashville, TN, USA

5Humboldt University, Medical Faculty-Charité, Department of Dermatology, Schumannstrasse 20/21, D-10117 Berlin, Germany

6Nagoya University School of Medicine, Department of Infectious Diseases, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8560, Japan

7Institut für Pathologie, Medizinische Einrichtungen der Heinrich Heine Universität, Moorenstr 5, Geb 14.79, Raum 316, D-40225 Düsseldorf, Germany

Correspondence to: J W G Janssen, University of Heidelberg, Institute of Human Genetics, Im Neuenheimer Feld 328, D-69120 Heidelberg, Germany; Fax: 49-6221-565155

aThe first three authors contributed equally to this work

Abstract

The t(5;14)(q33-34;q11) translocation constitutes a recurrent rearrangement in acute lymphoblastic leukemia involving the T cell receptor (TCR) delta locus on chromosome 14. Breakpoint sequences of the derivative chromosome 5 were isolated by application of a ligation-mediated PCR technique using TCR delta-specific primers to amplify genomic DNA from the leukemic cells of a patient with t(5;14). Through exon trap analysis, we identified various putative exons of the chromosome 5 target gene of the translocation; compilation of sequence information of trapped exons and available expressed sequence tags (ESTs) from the GenBank database allowed us to assemble 1.2 kb of the cDNA. Full-length cDNAs were isolated from a human testis cDNA library and sequence analysis predicted a putative Ran binding protein, a novel member of the importin-beta superfamily of nuclear transport receptors, called RanBP17. The t(5;14) breakpoint maps to the 3' coding region of the gene. The breakpoint of a second t(5;14) positive patient was mapped about 8 kb downstream of the most 3' RanBP17 exon and 2 kb upstream of the first exon of the orphan homeobox gene, Hox11L2. In both cases TCR delta enhancer sequences are juxtaposed downstream of the truncated or intact RanBP17 gene, respectively on the derivative chromosome.

Leukemia (2002) 16, 2205-2212. doi:10.1038/sj.leu.2402671

Keywords

chromosomal translocation; acute lymphoblastic leukemia; RanBP17; Hox11L2; T cell receptor delta

Introduction

Recurrent chromosomal aberrations, such as translocations and inversions involving the immunoglobulin (Ig) and T cell receptor (TCR) loci, are nonrandomly associated with lymphoid malignancies. It is now generally accepted that 'illegitimate' V(D)J recombinase activity during early B or T cell development or 'class switching' in the late B cell life cycle constitutes the underlying mechanism for these aberrations.1 Molecular studies of a number of B and T cell translocations has allowed the identification of genes involved in lymphoid leukemogenesis. Frequently, juxtaposition of these genes with regulatory elements of the Ig and TCR genes leads to the inappropriate expression of the translocation partners (Refs 2, 3, 4 and references therein). Many genes located at the chromosomal breakpoints of Ig- and TCR loci encode for transcription factors, but other classes of signal transducers are also involved, including apoptosis control factors BCL2 and BCL10, the cell cycle protein cyclinD1, and tyrosine kinase receptors such as FGFR3.2,3,5,6,7,8 Chromosome translocations in T-lymphoid malignancies frequently involve TCR genes, in particular the TCR alpha/delta locus on chromosome 14q11.2

We previously described a new recurring chromosomal abnormality, t(5;14)(q33-q34;q11), in the leukemic cells of four children with acute lymphoblastic leukemias (ALL) of B- or T-lineage.9 In the current study, we have identified the aberrant TCR delta rearrangements in two ALL patients with t(5;14) and localized their breakpoints within and immediately 3' of the RanBP17 gene, respectively upstream of the Hox11L2 gene.

Materials and methods

DNA and RNA purification and analysis

High molecular weight DNA was prepared and analyzed by Southern blotting, as described previously.10 The TCRdelta-specific JdeltaS16 probe was kindly provided by Drs T Boehm and TH Rabbitts (MRC Laboratory of Molecular Biology, Cambridge, UK).11 Prior to blot hybridization, a pre-reassociation of the probe with an excess of competing, unlabeled human DNA was performed.12 Total RNA was isolated and purified by acid guanidinium isothiocyanate/phenol-chloroform extraction, as described.13 Northern blot analyses were performed as described by Shackleford and Varmus.14 Briefly, various amounts of poly A+ RNA were resolved on 1.0% agarose formaldehyde gels, blotted on to Hybond-N membranes (Amersham Pharmacia Biotech, Freiburg, Germany), then hybridized as described for Southern blotting.10

Isolation of the t(5;14) breakpoint region

The breakpoint sequences were isolated by using a ligation-mediated PCR technique.15 Briefly, peripheral blood leukemic cell DNA from a patient with early B-ALL carrying the t(5;14) (case 2 in Whitlock et al9) was digested with BglII and EcoRI and ligated to plasmid pTZ19R DNA (Amersham Pharmacia Biotech) that had been digested with BamHI and SalI, then purified by repeated precipitation of the plasmid DNA with 0.5 M NaClO4 and 50% isopropanol. As BamHI and BglII restricted DNA have compatible cohesive ends and EcoRI and SalI DNA ends are not compatible, this results in a linear ligation product of plasmid DNA with genomic DNA fragments from the t(5;14) patient. The ligation products were amplified with a set of primers consisting of vector and TCR delta-specific pimer, respectively: M13-O (5'-GGT TTT CCC AGT CAC GAC-3') and Tdelta-O(5'-AAT GCT AGC TAT TTC ACC CA-3'). Two microliters of the first PCR reaction mixture (see below for detailed PCR conditions) were reamplified by a semi-nested PCR approach with primers M13-O and Tdelta-M (5'-CAT TTC AGG ATT ATT TCT GCC-3'). Two mul of the resulting PCR products were then used for a third round of PCR with primers M13-I (5'-GTT GTA AAA CGA CGG CCA GT-3') and Tdelta-I (5'-GGA GGA TCC GAG TTA CTT ACT TGG TTC CAC-3', the BamHI site is underlined). The PCR products were digested with EcoRI and BamHI and cloned into pTZ19R. The breakpoint sequences of the second patient with T-ALL carrying the t(5;14) (case 4 in Whitlock et al9) was cloned using a similar approach. Peripheral blood leukemic cell DNA from the patient was digested with SacI and EcoRI and ligated to plasmid pTZ19R DNA that had been digested with SacI and SalI. Ligation products were amplified with the following set of primers: T7-3L (5'-CAC ACA GGA AAC AGC TAT GAC C-3') and TD6L (5'-TCT TCC CAG GAG TCC TCC TAA ATG-3') for the first round of PCR; T7L (5'-CGA TTT AAT ACG ACT CAC TAT AGG G-3') and TD5L (5'-CCC ATT TCA GGA TTA TTT CTG CCT C-3') for the second round of PCR; T7L and TD4L (5'-GAG TTA CTT ACT TGG TTC CAC AGT C-3') for the third round of PCR. PCR products were ligated into a T-vector and sequenced.

Genomic library construction and P1 clone isolation

A genomic DNA library was prepared from DNA of mononuclear cells from peripheral blood of a healthy donor. The library preparation was performed essentially as described.16 Bacteriophage EMBL-3 DNA was digested with BamHI and EcoRI, and the small BamHI/EcoRI linker fragments were specifically removed by repeated precipitation of phage DNA with 0.5 M NaClO4 and 50% isopropanol (v/v). Genomic DNA was partially digested with Sau3AI, size selected by sucrose gradient fractionation (7-20 kb), ligated into EMBL 3 phage arms, and packaged with Gigapack III gold packaging extract (Stratagene, La Jolla, CA, USA).

Previously, we published the cloning and characterization of pseudogenic sequences of a gene encoding a deubiquitinating enzyme UBH1 near the t(5;14) breakpoint.17 Using two pseudo-UBH1-specific PCR primers PUBH-5 (5'-CAA GAA AAA CAA AAT GGC CGC-3') and PUBH-3 (5'-CAT CAC GGT TCC CCC TCA A-3') a human genomic P1 library was screened (Genome Systems, St Louis, MO, USA) and two overlapping P1 clones spanning the chromosome 5 breakpoint region were isolated.

Exon trapping and isolation of cDNA clones

Exon trapping was performed as described by Auch and Reth.18 DNA of the two P1 clones containing chromosome 5 breakpoint sequences was partially digested with Sau3AI, cloned into the BamHI site of the lambda phage vector for genomic exon trapping (lambdaGET) and converted into plasmid clones using the cre recombinase-positive bacteria, BNN132.19

DNA from pools of 10 plasmid clones was transiently transfected into COS-7 cells using DOTAP (Roche Molecular Biochemicals, Mannheim, Germany). Subsequently, RNA was isolated and possible exons were identified by RT-PCR using the following primers: Ex5 (5'-CCA GGC TTT TGT CAA ACA G-3') and Ex3 (5'-GGT GCA GCA CTG ATC CAC-3'). The resulting PCR products, representing trapped exons, were used to screen a human testis 5'-stretch plus cDNA library HL3024b and a human testis large insert cDNA library HL55034 (Clontech Laboratories, Palo Alto, CA, USA), as recommended by the manufacturer.

RT-PCR and PCR assays

Total RNA (5 mug) was transcribed into cDNA using random hexamers and Superscript RT, as recommended by the manufacturer (Life Technologies, Eggenstein, Germany). PCR was performed essentially as described by Saiki et al.20 Each 100 mul reaction mixture contained the following: 1 mug of genomic DNA, 100 ng of plasmid DNA, or 1/10 of the cDNA synthesis reaction mixture; 100 pmoles of each 5' and 3' oligodeoxynucleotide; 200 muM deoxynucleotide triphosphates (dNTPs); 10 mM Tris-HCl, pH 8.3; 50 mM KCl; 3 mM MgCl2; 0.001% gelatin (w/v); and 1 U Taq polymerase (Perkin Elmer; Langen, Germany). Each PCR consisted of 35 cycles in an automatic PCR processor (BioMed, Theres, Germany) with 45 s at 92°C, 1.5 min at 56°C, and 2 min at 72°C. Before the first cycle double-stranded DNA or DNA/RNA hybrids were denatured for 10 min at 92°C, and after the last cycle the reaction mixture was incubated for 15 min at 72°C. For nested PCR, 2 mul of the preceding PCR reaction was used for the next amplification.

DNA sequencing

For sequence analysis of PCR products or cDNA clones, DNA molecules were purified and cloned as described.21 DNA fragments were separated by agarose gel electrophoresis, purified using diethyl aminoethyl paper (Schleicher and Schuell, Dassel, Germany), and subcloned into pTZ19R or pT7T3 (Amersham Pharmacia Biotech). Plasmids were sequenced either with a Thermosequenase fluorescent-labelled primer cycle sequencing kit with 7-deaza-dGTP (Amersham Pharmacia Biotech) on a LI-COR long Readir 4200 DNA sequencer (MWG-Biotech, Ebersberg, Germany) or with a T7 DNA polymerase sequencing kit with 7-deaza-dGTP or a dye terminator cycle sequencing kit on an ALF Express II (Amersham Pharmacia Biotech).

Computer search and programs

Sequences were evaluated with the University of Wisconsin Genetics Computer Group (GCG) sequence analysis package (version 10.0 - Unix) and with server facilities of the National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD, USA (www.ncbi.nlm.nih.gov), as well as with the psort program of Kenta Nakai, Institute for Molecular and Cellular Biology, Osaka University, Japan (http://psort.nibb.ac.jp/).

GenBank accession numbers

Human RanBP17 cDNA sequence encoded the largest 1088 amino acid translation product, AJ271459, and alternatively spliced cDNA sequences, AJ288952, AJ288953, AJ288954, AJ288955; ~1.1-kb genomic breakpoint sequence of case 2, AF022791, ~1150-bp genomic breakpoint sequence of case 4, AJ296280.

Results

Isolation of breakpoint sequences

In the course of a Southern blot analysis of T cell receptor (TCR) delta rearrangements in ALL patients, we identified one case showing a recombination which could not be explained by any known assembly of the limited number of TCR delta elements.17 A cytogenetic analysis revealed that this patient's leukemic cells harbored a t(5;14) (q34;q11) translocation. The results of both cytogenetic and Southern blot analyses implied a Ddelta2Ddelta3 rearrangement at the TCR delta locus on 14q11, which was joined to unknown sequences from chromosome 5. We assumed that this chromosomal aberration was most likely caused by illegitimate activity of the recombination machinery, as has been observed in various other translocations associated with B and T cell leukemias.2

Taking advantage of our detailed Southern blot analysis of the t(5;14)-positive patient, we were able to clone the breakpoint sequences by a ligated mediated PCR technique. BglII restriction sites were localized to positions ~0.8 kb upstream of a regular Vdelta1Ddelta3Jdelta1 recombination event on one allele and ~1.0 kb upstream of a 'X'-Ddelta2Ddelta3Jdelta1 rearrangement on the second allele. To isolate the chromosome 5 sequences flanking Ddelta2, genomic DNA of leukemic cells from the patient was digested with the restriction enzymes BglII and EcoRI. Then, the BglII-cleaved DNA ends were ligated to complementary BamHI overhangs generated in BamHI-, SalI-digested pTZ19R plasmid DNA. After three rounds of nested PCR with this ligation reaction using three sets of TCR delta-and pTZ19R-specific primers, we obtained two PCR products of approximately 1.0 and 1.2 kb. (Figure 1a). Sequence analysis of the 1.0-kb fragment confirmed the Vdelta1Ddelta3Jdelta1 recombination on one allele (data not shown). As expected, the second fragment consisted of a rearranged Ddelta2Ddelta3Jdelta1 segment that was joined to unknown sequences derived from chromosome 5 (Figure 1a and b).

A computer search of the putative 5q breakpoint sequences revealed a stretch of ~610 bp with sequence similarity to a long interspersed element (LINE)22 directly adjacent to the Ddelta2 followed by 430 bp of unknown sequence from chromosome 5. Despite the repetitive character of this breakpoint probe, we were still able to demonstrate by Southern blot analysis that this fragment hybridized to a genomic band of the same size as the illegitimately rearranged chromosome 5-Ddelta2Ddelta3Jdelta1 fragment detected with a TCR delta-derived probe (Figure 2, panels a and b). Subsequently, a human genomic EMBL-3 phage library was screened using the 1.2-kb PCR fragment as a probe. Two overlapping clones covering approximately 16 kb of the breakpoint region were obtained. Chromosomal in situ hybridization using a 7.5-kb PvuII genomic breakpoint probe confirmed that the clones were derived from 5q34 (data not shown).17

Isolation and characterization of the RanBP17 gene

The human genomic DNA phage clones encompassing the breakpoint region were subcloned and screened for expressed sequences by exon trapping.18,19 Using this approach, we identified a 79-bp candidate exon from chromosome 5. PCR amplification of a human fetal liver cDNA library using primers derived from this putative exon resulted in a DNA fragment of the predicted size. Therefore, this cDNA library was screened with the 79-bp fragment, leading to the isolation of a 657-bp cDNA clone that was used again to screen the library and eventually lead to a complete cDNA coding for a novel deubiquitinating enzyme, UBH1.17 Further characterization of cDNA and genomic DNA around the chromosomal breakpoint on chromosome 5 revealed the presence of a pseudogenic variant of this novel deubiquitinating enzyme on the translocation chromosome. This pseudogenic variant is highly homologous, but not identical to the transcribed UBH1 cDNA, contains several premature termination codons and an insertion of an additional 63 bases at position 932 and therefore was omitted as a candidate gene for the t(5;14) chromosomal rearrangement. Hybridization of DNA from the t(5;14) patient with an UBH1 cDNA probe showed hybridization with the functional UBH1 gene and cross hybridization with the pseudo-UBH1 gene on the 'X'-Ddelta2Ddelta3Jdelta1 fragment (Figure 2, panel c). Since various initial attempts to isolate additional gene-specific exons from the 16 kb long breakpoint region failed, we decided to screen a human genomic P1 library (Genomic Systems) for clones spanning this chromosome 5 region. Two overlapping P1 clones with a length of approximately 90 kb containing breakpoint sequences were isolated by amplification with a pair of UBH-1 pseudogene-specific primers and used for exon trapping. Five different exons were isolated, sequenced, and compared to the GenBank sequence database. Various ESTs with homology to the trapped exons were detected, thus enabling us to assemble a partial 1.2-kb cDNA sequence of a novel gene. In light of its high expression in testis, we screened two different testis-specific cDNA libraries.23 Eventually, we succeeded in the cloning and sequencing of a 4450-bp cDNA with a 3264 bp open reading frame encoding for a predicted translation product of 1088 amino acids that possesses a calculated molecular weight of approximately 120 kDa. Finally, by using a human testis large insert cDNA library, we were able to isolate various full-length cDNAs. Sequence analyses of a large number of independent cDNA clones revealed the presence of three different polyadenylation sites (Figure 3). One of the polyadenylation sites is generated because the splice donor site of exon 24 is occasionally not recognized and the mRNA then proceeds into 3' intron sequences. In addition, we observed a low number of alternatively spliced mRNAs with exon 9 missing which had no effect on the open reading frame, and various alternatively spliced RNAs that resulted in premature stops of the large open reading frame (Figure 3; exons 14b-e).

Sequence and predicted function of the RanBP17 gene

The 5' end of the analyzed sequence is embedded in a 'favorable' (AAGatgG) Kozak consensus motif for translation initiation.24 A GenBank computer search showed a 66.8% identity in a 1088 amino acid overlap of our protein sequence with a RAN binding protein 16 form 1 protein (Acc. No. AF064729) suggesting that the protein belongs to a novel family of RAN binding proteins and was therefore called, RanBP17.23 Common to other Ran-binding proteins, RanBP17 contains an importin-beta N-terminal domain, has an acidic isoelectric point of 6.4 (range 4.6-5.9) and a size of 124 kDa (range 90-130 kDa). Prior to the cloning of human RanBP16 and RanBP17 the importin-beta family comprised at least 21 family members in humans.25

Blast searches solely revealed significant similarity (39.1% identity) over almost the entire protein sequence to the hypothetical protein C35A5.8 of C. elegans (Acc. No. T19745).26 As expected from the homology of RanBP17 with the hypothetical C. elegans gene C35A5.8 by database searches, zoo-blot analysis using a RanBP17 cDNA probe from the 3' region (exons 23-25) revealed hybridization to single bands in DNA of several species (rodents, canine, bovine, rabbit and chicken DNA) (data not shown). These data confirm that the RanBP17 gene is very well conserved among different species and lacks homology to other Ran-binding proteins.

Computer analysis also revealed homology of the RanBP17 cDNA sequence with various genomic contigs from this region of chromosome 5 that are in the process of being sequenced and assembled in the framework of the human genome sequencing project (Sequencing of human chromosome 5, DOE Joint genomic Institute). This genomic information confirmed and complemented our efforts to determine the exon/intron boundaries of the complete cDNA sequence (Figure 3 and Table 1) and enabled us to draw a map of the region spanning the breakpoint sequences, the UBH1 pseudogene and the RanBP17 exons located in the vicinity of the breakpoint (Figure 4). The t(5;14) breakpoint is located in the intron separating exons 24a and 25 of the RanBP17 gene. An analysis of cDNA, breakpoint and genomic sequence data unveiled that exon 24 of RanBP17 is joined to the Ddelta2Ddelta3Jdelta1 breakpoint sequence containing the delta enhancer sequence located between the Jdelta1 and Cdelta elements.

Determination of the breakpoint in a second t(5;14)-positive patient

Southern blot analysis of a second t(5;14)-positive patient revealed the presence of a SacI restriction enzyme site ~1 kb upstream of Jdelta1. To isolate the chromosome 5 sequences flanking Jdelta1, genomic DNA of leukemic cells from the patient was digested with the restriction enzymes SacI and EcoRI and ligated to SacI, SalI-digested pTZ19R plasmid DNA. After three rounds of nested PCR using TCRdelta and plasmid-specific primers, we obtained two PCR products of approximately 1280 and 1150 bp (data not shown). Southern blot analysis of genomic DNA of leukemic cells of the patient analysis had revealed that the 1280 bp fragment represents a Vdelta2DdeltaJdelta1 rearrangement. A comparison of the sequence of the second rearranged allele with the Genbank database showed a rather complex pattern. Bases 4 to 849 showed homology to two clones (AC021307 and CNS01R17) that are derived from chromosome 14, but do not belong to the TCR alpha/delta region. Bases 850 to 870 showed no homology and bases 871 to 989 showed homology to a clone (AC10454) that is derived from chromosome 5 and locates ~8 kb downstream of the most 3' mapped RanBP17 exon (exon 28). These data suggest an illegitimate recombination of TCR delta with sequences from chromosome 14 and 5 (Figure 4).

Computer analyses of the genomic sequences surrounding the second breakpoint revealed the presence of some ESTs upstream and a HOX11-related homeobox gene, HOX11L2 downstream of the breakpoint, respectively (Figure 4).

Discussion

We previously described a recurrent, albeit rare, translocation t(5;14)(q33-34;q11) in patients with B- or T-lineage ALL involving the T cell receptor delta locus.9,17 We now describe the mapping of the breakpoints of two t(5;14) cases in the 3' end and downstream of a gene encoding a putative Ran binding protein and upstream of an orphan homeobox gene, Hox11L2.

Sequence comparisons revealed the localization of a member of the human LINE-1 repeat subfamily L1PA4 at the translocation boundary on chromosome 5q33-q34. Involvement of LINE-1 sequences has been reported for rearrangements mediated by the IG/TCR recombinase at the DEK/CAN translocation breakpoints in the t(6;9) in acute myeloid leukemia (AML) and at the sites of molecular rearrangements in various human genetic diseases such as familial adenomatous polyposis, Ehlers-Danlos syndrome, familial aniridia, hemophilia A, X-linked chronic granulomatosis, and Alport syndrome-diffuse leiomyomatosis.27,28,29,30,31,32,33,34,35,36 However, the role of LINE sequences in these recombination processes, if any, remains to be elucidated.

A GenBank sequence database search revealed homology of the gene located at the chromosome 5 breakpoint to a gene encoding for the human Ran binding protein 16 form 1 (Acc. No. AF064729) and was therefore called RanBP17 (see also Ref. 23). RAN proteins comprise a distinct subset of small GTPases that belong to the RAS superfamily. Various observations have corroborated a role for RAN in nuclear import, including the discovery of the RAN binding proteins, several of which are known to be components of the nuclear transport machinery.37,38 The significant homology (~67% identity) of RanBP17 to a RAN binding protein strongly supports a role of RanBP17 in nuclear protein transport. There are two other examples of nuclear pore complex protein that are involved in leukemogenesis, namely the CAN and NUP98 proteins. The CAN protein, a putative oncogene product asssociated with the t(6;9) DEK-CAN fusion in myeloid leukemogenesis, has been reported to function as a nuclear pore complex protein and alternatively termed NUP214 (nucleoporin of 214 kDa).39 The N-terminal domain of the nucleoporin protein NUP98 has been shown to be fused to the proteins HOXA9, HOXD13, DDX10, or PMX1 in AML containing chromosome translocations t(7;11)(p15;p15), t(2;11) (q35;p15), inv(11)(p15;q22), or t(1;11)(q23;p15), respectively, and to RAP1GDS1 in T cell ALL with a recurrent translocation t(4;11)(q21;p15).40,41,42,43,44,45

The sequences of the breakpoints and the 5' centromere-3' telomere orientation of the RanBP17 and TCR delta genes on 5q35 and 14q11, respectively, insinuates a head-to-tail juxtaposition of RanBP17 with the TCR delta gene. This translocation event brings the delta enhancer sequences in the immediate vicinity of the RanBP17 gene and should result in an inappropriate RanBP17 activation (Figure 5). As a result this would give rise to an increased amount of C-terminal truncated or complete RanBP17 protein in the first and second patient, respectively. Unfortunately we do not have any cell material or RNA available for the t(5;14)-positive patients reported here and are therefore not able to determine the exact mechanism(s) of gene activation in these patients. A comparison of testis RNA, RNA of the pre-B ALL cell line Nalm-6, and RNA from a third t(5;14)-positive patient revealed increased RanBP17 expression in the leukemic cells of the patient, while the RanBP17 transcript pattern closely resembled RNAs from various t(5;14)-negative ALL patients analyzed by us (data not shown). However, the poor quality of this Northern blot and the limited amount of patient material prevented us from making final judgments about a possible qualitative or quantitative difference compared to the RanBP17 RNAs from normal tissues.

All nuclear transport receptors have an N-terminal RanGTP-binding domain, a C-terminal cargo-binding domain, and the capacity to bind components of the nuclear pore complex (NPC). It appears noteworthy that we observed a low number of spliced transcripts in testis RNA with a premature termination or a different polyadenylation site that results in a C-terminally truncated RanBP17 protein (Figure 3, exons 14b-e and 24 3' intron). However, the mechanisms by which these translocations contribute to leukemogenesis remain enigmatic. Aberrant expression of RanBP17 due to juxtaposition to the TCR delta enhancer may presumably activate cell growth. Whether loss of the C-terminal domain in the first patient may prohibit the binding of RanBP17 substrates or interfere with physiological RanBP17 activity in a dominant-negative mode to contribute to the development of ALL remains to be explored.

It is also noteworthy that a set of ESTs and a HOX11L2 gene are located 3' of the RanBP17 gene (Figure 4). Whether these ESTs also belong to the RanBP17 gene and represent part of the two larger RanBP17 transcripts observed by Northern blot analysis is as yet not solved.23 Homeobox (HOX) genes, an evolutionarily highly conserved family of transcription factors, are involved in murine and human leukemogenesis (reviewed in Ref. 46). We have already mentioned the role of HoxA9 and HoxD13 in human leukemogenesis. HOX11 (Tlx-1; tcl-3), together with HOX11L1 (Enx, Tlx-2) and HOX11L2 (Rnx, Tlx-3) members of the HOX11-family, is involved in the t(10;14)(q24;q11) translocation in T cell acute leukemia (reviewed in Ref. 2). Therefore a role for Hox11L2 in leukemogenesis remains an alternative possibility. In fact (Bernard et al47) recently showed activation of the Hox11L2 gene in T-ALL patients harboring a t(5;14)(q35;q32). In several patients the translocation breakpoints were mapped between introns 21 and 24 of the RanBP17 gene on 5q35 and several hundred kb from the CTIP2/hRIT/BCL11B gene on chromosome 14 (Figure 5). They postulated transcriptional activation of the Hox11L2 gene by the influence of the CTIP2 gene. In the t(5;14)(q35;q11) described herein, breakpoints in 5q35 are linked to TCR delta sequences on chromosome 14q11 (Figure 5). Similar to the chromosomal translocation involving LMO2 (Rhombotin2/TTG-2) t(11;14(p13;q11)) the loss of putative 5' regulatory sequences of the Hox11L2 gene may lead to its deregulated transcription.48,49 As indicated above we have no material of the t(5;14)-positive patients reported here to analyze Hox11L2 expression in these patients or check the reciprocal chromosome for presence of the Hox11L2 gene by Southern blot analyses. Further fluorescence in situ hybridization (FISH) studies and molecular biological studies of additional leukemic patients should elucidate the frequency and mode of RanBP17 and/or Hox11L2 alteration in human leukemias. During revision of this manuscript Ferrando et al50 reported on the importance of Hox11L2 in leukemogenesis based on gene expression profiling analysis.

Acknowledgements

This study was supported by grants from the European Community (EH 5th Framework Program-Quality of Life and Management of Living Resources; The Consortium Haematopoiesis & Cancer), from the Dr Mildred Scheel Stiftung für Krebsforschung (10-1253) to JWGJ and from the Deutsche Forschungsgemeinschaft to JWGJ and CRB, respectively. Supported also by National Cancer Institute grants CA76301 and CA69129 (to SWM) and Cancer Center Support (core) grant CA27165, and by the American Lebanese Syrian Associated Charities, St Jude Children's Research Hospital. We are grateful to Christel Tell, Ulrike Spadinger, Daniela Becke, Magda Dietl, Dorothé Erz, Xiaoli Cui, and Yvonne Stark for their excellent technical assistance.

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Figures

Figure 1 (a) Analysis of PCR products obtained by ligation-mediated PCR using DNA from a t(5;14)-positive ALL patient. Amplification products acquired after three rounds of nested PCR were electrophorized on a 1.5% agarose gel and stained with ethidium bromide. The two bands representing the two allelic T cell receptor delta rearrangements are depicted and specified on the right. (b) Partial sequence analysis of the PCR product representing the illegitimately recombined chromosome 5-Ddelta2Ddelta3Jdelta1 segment. The Ddelta2, Ddelta3 and Jdelta1 elements are presented in italics. Insertion of additional bases (N) are indicated by bold letters.

Figure 2 Southern blot analysis to confirm the cloning of a t(5;14)-specific DNA fragment that encompasses the illegitimate recombination. Patient's DNA (lanes P) and peripheral blood cell DNA of a healthy donor (lanes C) were doubly digested with HindIII and EcoRI, blotted, and hybridized with the TCR delta-derived probe JdeltaS16 (panel a). The same blot was rehybridized with the 1.2 kb-PCR breakpoint probe (panel b) and, subsequently, with a 803-bp 3' PvuII-SalI fragment of the UBH1 cDNA (panel c). Fragments of either known size (Jdelta1 and Vdelta1Ddelta3Jdelta1) or deduced from the comparison of the blots (pseudo-UBH1, UBH1 and t(5;14)) are indicated by arrows.

Figure 3 Structure of the RanBP17 cDNA and its alternatively spliced RNA products. The upper bar depicts the structure of the largest cDNA isolated. The relative size and position of the various exons are indicated by vertical lines. Exons discussed in the text are marked by numbers. The three polyadenylation sites, as well as the breakpoint in the t(5;14)-positive patient, are indicated. White regions mark 5' and 3' untranslated sequences, while grey boxes represent the open reading frame. Alternatively spliced RNAs with their respective ORFs are indicated below.

Figure 4 Genomic map illustrating the location of the t(5;14) breakpoints in relation to the exons of RanBP17, the UBH1 pseudogene and Hox11L2. The amino acid numbers of the RanBP17 predicted protein sequence are indicated above respective exons. The map has been drawn to scale and a 1-kb size marker is indicated.

Figure 5 Schematic representation of the translocation events of both t(5;14)-positive patients. The map of the region containing the RanBP17 and Hox11L2 genes on chromosome 5q34-35, the breakpoints of the two t(5;14)(q34;q11)-positive patients described herein and the breakpoints observed in t(5;14)(q35;q32) patients47 are shown at the top. The next line represents the TCR delta locus with its various V, D and J elements, constant region (Cdelta) and transcription enhancer element (deltaenh) and the location of the breakpoints of the two t(5;14)(q34;q11)-positive patients are depicted. The bottom of the figure shows the situation of the derivative chromosomes in the cases with t(5;14)(q34;q11). Exon and intron sizes are not drawn to scale.

Tables

Table 1 Exon-intron junctions of the human RanBP17 gene

Received 5 November 2001; accepted 29 May 2002
November 2002, Volume 16, Number 11, Pages 2205-2212
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