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NEOPLASIA
From the Department of Pathology, Leiden University
Medical Center, Leiden, The Netherlands.
Using DNA fiber fluorescence in-situ hybridization
(FISH) and 3-color interphase FISH, 2 cases of follicular lymphoma were identified in which the BCL2 gene was excised from 18q21
and inserted into the immunoglobulin heavy chain (IGH)
locus at 14q32. Both the insertion breakpoint at 14q32 and the deletion
breakpoint at 18q21 were cloned using inverse polymerase chain
reaction. Sequence analysis showed that the JH sequences were
juxtaposed to the 5'-side of BCL2, and the DH sequences
were juxtaposed to the 3'-side of BCL2. There were
breakpoints at both the JH and DH recombination signal sequences, and
N-nucleotides were present at all breakpoint junctions. At the
BCL2 locus, the 3'-breakpoints in both cases were localized
at exactly the same nucleotide position, 6.2 kilobase downstream
of the major breakpoint region, directly adjacent to a complete cryptic
recombination signal sequence (RSS) consisting of a heptamer, a
nonamer, and a 23-base pair (bp) spacer. The BCL2
5'-breakpoints were approximately 600 bp upstream of the gene,
within the CA repeats. Although less evident than for the BCL2
3'-breakpoints, cryptic RSSs were also identified at these
breakpoints, with a 12-bp spacer. On the basis of structural characteristics of these rearrangements, a model is proposed in which
the BCL2 gene is deleted from its locus by recombination activation gene-1/-2 (RAG-1/-2)-mediated excision. The
gene is subsequently inserted into the recombining
IGH locus, a process involving the formation of hybrid
joints between the IGH coding ends and the
BCL2 signal ends.
(Blood. 2000;96:1947-1952) Follicular lymphomas (FLs) are
characterized by a t(14;18)(q32;q21), which juxtaposes the
enhancers of the immunoglobulin heavy chain (IGH) locus at
14q32 to the BCL2 gene at 18q21. Breakpoints at the
IGH locus are located at the J or D segments, and
breakpoints at the BCL2 locus are generally located in the
major breakpoint region (mbr) in the 3'-untranslated region or
downstream of the gene. Using contigs of probes covering the
BCL2 gene and the IG loci, we previously
performed a fiber fluorescence in-situ hybridization (FISH)
study of the BCL2 translocations in FL.1 In 4 of 40 cases, the BCL2 gene was affected by 2 translocation
breakpoints on the same allele: 1 at the 5'-side and 1 at the 3'-side
of the gene. In 2 cases, the IGH locus was juxtaposed to the
3'-side of BCL2 and the immunoglobulin- Tissue samples
Probes for fiber FISH
Probes for interphase FISH BCL2 insertions into the IGH locus were visualized using the following probes: cosmid cosIg6, the 14q-subtelomeric cosmid IgH2 (provided by H. Riethman, The Wistar Institute, Philadelphia, PA), and BCL2 intronic cosmid F6.FISH and fluorescence microscopy Preparations of DNA fibers and interphase nuclei were made as described previously.2,3 The probes were labeled by standard nick-translation with biotin-16-dUTP (uridine 5'-triphosphate), digoxygenin-11-dUTP, or fluorescein-12-dUTP (Roche, Basel, Switzerland). The hybridization solution consisted of 30% formamide; 10% dextransulphate; 50 mmol/L sodium phosphate (pH 7.0); 2 times sodium chloride/sodium citrate (SCC), 3 ng/µL each probe; and a 50-fold excess of human Cot-1 DNA. Hybridization and immunofluorescence detection for dual-color FISH were performed as described previously.3,4 For triple-color FISH, immunofluorescence detection was performed essentially according to Wiegant et al.5 Biotin-labeled probes were first detected with avidin-AMCA (Vector Laboratories, Burlingame, CA); then goat-anti-avidin-biotin (Vector); and finally, avidin-AMCA. Digoxigenin-labeled probes were detected with mouse-antidigoxin (Sigma Chemical, St Louis, MO), and sheep-antimouse-digoxigenin and sheep-antidigoxigenin-TRITC (Roche). Fluorescein-labeled probes were detected with rabbit-anti-FITC (fluorescein isothiocyanate) (Dako, Copenhagen, Denmark) and goat-antirabbit-FITC (Vector).Microscopic images were captured using a COHU 4910 series monochrome CCD camera (COHU, San Diego, CA) attached to a DM fluorescence microscope (Leica, Wetzlar, Germany) equipped with a PL Fluotar 100 times NA 1.30-0.60 objective and I3 and N2.1 filters (Leica) and Leica QFISH software (Leica Imaging Systems, Cambridge, England). The images were processed with Paintshop Pro (JASC, Eden Prairy, CA) and Harvard Graphics (Software Publishing, Santa Clara, CA). Southern blot analysis Southern blotting was performed with IGH probe IGHJ6 (provided by J. J. M. van Dongen, Rotterdam, The Netherlands)6; mbr probe pSp65/18-4RH, a 3-kilobase (kb) EcoRI-HindIII fragment (provided by Y. Tsujimoto, Osaka, Japan); 5'-BCL2 probe pB16, a 1.6-kb EcoRI cDNA fragment (provided by Y. Tsujimoto)7; and probe 5'-5'-BCL2, a 450-base pair (bp) polymerase chain reaction (PCR) product located 1 kb upstream from the BCL2 gene (see Figure 2A). The 5'-5'-BCL2 probe was made with the following primers: forward 5'-CCTATTAAGTAAGCCGCTGTGC-3' (GenBank accession number X51898; 8-29 bp) and reverse 5'-CGTGTCCACCTGAACACCTAG-3' (GenBank X51898; 486-466 bp). Genomic DNA was digested with BamHI, EcoRI, HindIII, and BglII (Roche, Pharmacia), size-fractionated, blotted on nylon filters, and hybridized, as described previously.8Inverse PCR cloning of the 3'-BCL2/5'-BLC2 junction High-molecular-weight genomic DNA was cut with restriction enzymes, and the product was purified by phenylchloroform, chloroform extraction, and ethanol precipitation. Subsequently, 50-100 ng purified digested DNA was self-ligated in a 100-µL volume containing 54 mmol/L Tris HCl (tris[hydroxymethyl] aminomethane hydrochloride) (pH 7.5), 4.5 mmol/L magnesium dichloride (MgCl2), 0.9 mmol/L diothiothreitol (DTT), 0.1 mmol/L adenosine 5'-triphosphate (ATP), and 1 unit T4 DNA ligase (Roche). We used 10 µL of this reaction for the first PCR, with primer 5'-GAAGTCCTCATCGTGTAGCAC-3' (forward; GenBank X51898; 329-349 bp) and primer 5'-GACAAGAGGACAAACAAGTTGC-3' (reverse; GenBank X51898; 119-98 bp). PCR was performed using a touchdown protocol, with the annealing temperature decreasing from 65°C to 60°C. We used 1 µL of this reaction for a second PCR with nested primers 5'-GAGCTGCTGAGTTCTGCATGG-3' (forward; GenBank X51898; 433-453 bp) and 5'-GCACAGCGGCTTACTTAATAGG-3' (reverse; Genbank X51898; 29-8 bp).PCR and sequence analysis The region between the BCL2 mbr and the 3'-breakpoint region of cases FL4104 and FL5117 was amplified using mbr primer 5'-CCTTTAGAGAGTTGCTTTACGT-3' (forward; GenBank M13994; 4412-4433 bp) and 3'-BCL2 primer 5'-CCATGTAGATGGTGTTTGAGTG-3' obtained from inverse PCR and sequencing of the 3'BCL2/5'BCL2 junction. The JH 5'-breakpoints were amplified using a JH6 primer 5'-CTAGAGTGGCCATTCTTACCTG-3' (GenBank X97051; 89841-89820 bp) and a 5'-BCL2 primer 5'-CTGGACCCTTTCTGGCCGTG-3' (GenBank X51898; 856-837 bp). The DH 3'-BCL2 breakpoints were amplified using a DH3 primer 5'-GGTGAGGTCTGTGTCACTGTGG-3' and a 3'-BCL2 primer 5'-TGAATTTGAGGATAGAAAGTGCC-3' (GenBank AF204739; 484-506 bp). All PCRs were performed using standard conditions and protocols, except for the mbr 3'-BCL2 PCR, which was performed according to a long-range PCR protocol using the Expand Long Template PCR System (Roche). The PCR products were directly sequenced using the Big Dye Terminator sequencing kit (Perkin Elmer Biosystems, Foster City, CA) and an ABI PRISM 377 automated sequencer (Perkin Elmer Biosystems). The sequences were compared with the GenBank database using the Basic Logical Alignment Search Tool (BLAST) program with CENSOR, a database of repetitive sequences.9
BCL2 insertion into the IGH locus in 2 cases of FL In 2 cases of FL (from a series of 401), fiber FISH with BCL2 and IGH bar codes revealed copies of 18q21 with deletion of the entire BCL2 gene (Figure 1). In the same preparations, we found copies of the BCL2 gene that had the immunoglobulin constant region juxtaposed to their 5'-side, while part of cosmid U2-2, representing the DH region, was present at the 3'-side. These results suggested that the BCL2 gene had been excised from its original location on chromosome 18 and inserted into the IGH locus on chromosome 14. Using 3-color interphase FISH with a probe for the BCL2 gene, a probe for the IGH constant region, and a subtelomeric chromosome 14 probe, we were able to colocalize these 3 probes and confirm the presence of a BCL2 insertion into the IGH locus (not shown).
Localization of the BCL2 breakpoints Southern blots of BamHI, EcoRI, and HindIII digests of tumor DNA were hybridized with the 5'-BCL2 probe (pB16) and the 5'-5'-BCL2 probe (Figure 2A). Rearranged bands were observed in several digests: FL4104 with the 5'-BCL2 and 5'-5'-BCL2 probes: BamHI, EcoRI, and HindIII; FL5117 with a 5'-BCL2 probe: HindIII; and FL5117 with a 5'-5'-BCL2 probe: BamHI. The bands obtained with the 5'-5'-BCL2 probe were different from those obtained with the 5'-BCL2 probe. Because the enzymes used did not cut in the region between the 2 probes, in both tumors, the breakpoint should have been located between these 2 probes. Hybridization with the mbr probe (Figures 2B,C) showed rearranged bands in tumor DNA digested with SacI (both FL 4104 and FL 5117) and XbaI (FL 5117), but not with EcoRI and HindIII, enzymes which normally detect mbr breakpoints. This suggested that the 3'-BCL2 breakpoints were not located within the mbr, but were several kb 3' of the gene.
Cloning of the 5'-BCL2/3'-BCL2 junctions To clone the 5'-BCL2/3'-BCL2 junction at derivative chromosome 18 in both tumors, an inverse PCR was performed with primers 5'-side of the supposed location of the 5'-BCL2 breakpoints (Figure 2A) on self-ligation products of tumor DNA digested with several restriction enzymes. In FL4104, inverse PCR on a HindIII digest yielded a product of approximately 2 kb. In FL5117, the PCR products were obtained with Sau3AI (2 kb) and TaqI (1.5 kb). Sequencing of the products in FL4104 and FL5117 showed that the breakpoints were 639 bp and 596 bp upstream of the BCL2 gene. Both breakpoints were present in 2 different CA repeats (Figure 3; Genbank X51898). The sequence that was juxtaposed to the 5'-BCL2 region was for a large part identical in FL4104 and FL5117, indicating that the 3'-BCL2 breakpoints of FL4104 and FL5117 were located close to each other. The sequence did not show homology to known BCL2 breakpoint regions.
To determine whether the sequence cloned by inverse PCR was indeed from the 3'-side of the BCL2 gene, fiber FISH was performed on normal cells with a 3'-BCL2 cosmid probe, the mbr probe labeled in green, and the 1.5-kb FL5117-TaqI inverse PCR product labeled in red. The inverse PCR product gave a hybridization signal a few kb 3'-side of the mbr (not shown). Based on this observation, PCR was performed according to a long-range PCR protocol, with a forward primer just 5'-side of the mbr and a reverse primer 456 bp 3'-side of the 3'-BCL2 breakpoints of both FL4104 and FL5117. The PCR was performed on normal placenta DNA and on cosmid F11 containing the 3'-end of the BCL2 gene. A product of 7 kb was obtained with both templates, indicating that the breakpoints were located approximately 6.5 kb 3'-side of the mbr, as estimated upon gel analysis. Using this PCR product as a template, 1022 bp of sequence surrounding the breakpoints was determined (GenBank AF204739). Sequence analysis showed that the breakpoints were located 2 bp from each other within a MER1-like repeat unit named Charlie2 (according to the CENSOR database). The sequence showed no similarity with a previously identified breakpoint region far 3'-side of the mbr.10 The presence of a HindIII site 193 bp 5'-side of the breakpoints and the presence of a 66-bp overlap with a recently published sequence of the 3'-MBR region of BCL2 (GenBank AF217803; 2852-2918 bp) enabled us to map both breakpoints 6.2 kb 3'-side of the MBR of BCL2 (Figure 2B). At the 5'-BCL2/3'-BCL2 junction, N-nucleotides were present in both cases (Figure 3). Cloning of the 3'-BCL2/DH and 5'-BCL2/JH junctions On the fiber FISH level, the 3'-side of BCL2 colocalized with cosmid U2-2, which covered most of the DH region. To clone the breakpoints, PCRs were performed with a primer 5'-side of the 3'-BCL2 breakpoints and 1 of 3 different primers specific for the 5'-recombination signal sequences of the D2, D3, and D7-27 (DHQ52) families, respectively.11 Products of 1600 bp (FL4104) and 500 bp (FL5117) were obtained with the D3-specific primer. Sequence analysis with a primer in the BCL2 breakpoint region revealed a juxtaposition of the BCL2 gene to D4-23 in FL4104 and a juxtaposition to D3-9 in FL5117. N-nucleotides were present at the breakpoint junctions (Figure 3). In addition, comparison with the 5'-BCL2/3'-BCL2 junctions and the germline sequence showed that 6 and 4 nucleotides of the sequence had been deleted in FL4104 and FL5117, respectively. Taking these deletions into account, the BCL2 breakpoints at the 3'-BCL2/DH junction were in both cases at the same nucleotide position. On the 3'-side, the breakpoints were flanked by a cryptic IG recombination signal sequence (RSS) consisting of a heptamer and a nonamer separated by a 23-bp spacer. The RSS shows the identity of 11 of 16 nucleotides to the consensus IG RSS12 (Figure 3C). The heptamer showed only 1 mismatch, and the pentamer contained 5 matches including adenosines at positions 5, 6, and 7 of the nonamer, in which most of the nonamer's RSS function resides.12,13To amplify the junction between the 5'-BCL2 and JH locus, a PCR was performed on FL4104 and FL5117 with a primer 3'-side of the JH genes and another immediately 3'-side of the 5'-BCL2 breakpoints of both tumors. Products of 1100 bp (FL4104) and 1000 bp (FL5117) were obtained. Sequence analysis revealed that in both cases, the BCL2 gene was juxtaposed to the 5'-side of the JH4 gene, and N-nucleotides were present at the breakpoint junctions (Figure 3). In both cases a cryptic RSS with a 12-bp spacer was identified flanking the 5'-BCL2 breakpoint (Figure 3). Although less similar to the consensus RSS, both shared the first 4 nucleotides of the heptamer (CACA) with the consensus. In FL4104, adenosines were present at positions 5, 6, and 7 of the nonamer. In FL5117, the nonamer was less well defined and contained part of the CA repeat; still 6 of 9 bp were identical to the consensus sequence. The presence of these cryptic RSS elements strongly suggested that the BCL2 deletion had been mediated by the RAG-1 and RAG-2 proteins.
We identified 2 cases of FL with excision of the BCL2 gene from 18q21 and insertion of the gene into the IGH locus. Sequencing of the BCL2/IGH junctions revealed that the IGH breakpoints were at the JH and DH RSS elements, and N-nucleotides were present at all breakpoints. This demonstrates that the insertion of BCL2 into IGH took place during V(D)J recombination in precursor B cells, just like t(14;18) found in most other FLs. In the case of the usual t(14;18), the mechanism of chromosome breakage at 14q32 is clearly a RAG-1/-2-mediated recombination, because IGH breakpoints are consistently located at RSSs. A normal RSS consists of conserved heptamer and nonamer sequences separated by a spacer of either 12 or 23 bp in length. The consensus sequence is CACAGTG-12/23-ACAAAAACC,12 with the nonamer being less conserved than the heptamer. Apart from these natural RSSs, RAG-1/-2-mediated cleavage occasionally occurs at sites other than the IG or T-cell receptor (TCR) VDJ genes.14 A comparison of these so-called cryptic RSS elements shows a great variety of sequences. The only feature shared by all these sequences is a CAC trinucleotide at the first 3 positions of the heptamer, with frequently an A at the fourth position. Experiments with plasmid substrates have also suggested that the CAC trinucleotide is the only indispensable part of the RSS, although nucleotides at other positions can influence recombination efficiency by several orders of magnitude. An example is the presence of one or more adenosines at positions 6, 7, and 8 of the nonamer, on which most of the nonamer's activity depends.12,13 Rearrangements involving cryptic RSSs at non-IG, non-TCR loci have been described in T-cell leukemia and in normal T cells, include deletions of HPRT,15,16 TAL1,17,18 and MTS1 genes19 and t(7;9),20 t(1;7),21 and t(10;14).22 In the deletion cases, cryptic RSSs, at least consisting of the sequence CACA, were present at the deleted side of both deletion breakpoints in the following configuration: 5'-break-CACA...TGTG-break-3'. Two breakpoint regions, at the MTS1 and HPRT loci, were located in or at the border of a CA repeat. Other translocations with disrupted CA repeats have been described in a variety of leukemias and lymphomas.23-25 Strikingly, the 5'- and 3'-breakpoint configuration of the BCL2 gene excision in the present FL cases was identical to the above described RAG-1/-2-mediated deletions of TAL1, HPRT, and MTS1. This strongly suggests that the BCL2 gene has also been deleted from its locus by the RAG-1/-2-mediated recombination. Noteworthy, the BCL2 excision breakpoints in the 2 cases described here were not located at the common t(14;18) breakpoint clusters. Although the latter has been extensively searched for possible cryptic RSS sequences, even the CAC trinucleotide has not been found at the breakpoint sites. It is therefore unlikely that normal t(14;18) breakage of chromosome 18q21 is induced by RAG-1/-2-mediated cleavage. Recently, a model for RAG-1/-2-mediated induction of the BCL1 and BCL2 breakpoints, which does not require RSS elements to be present at the oncogene locus, was proposed. In cell-free systems, it was demonstrated that blunt-ended DNA fragments in complex with truncated RAG-1 and RAG-2 proteins can act as transposons by invading intact plasmid DNA, thereby resulting in either complete insertion of the blunt-ended fragment or in a single-sided strand exchange.26,27 As a mechanism for insertion of the excised BCL2 gene into
chromosome 14q32 in the present FL cases, one might initially think of
this strand invasion model. The breakpoints at the IGH locus were, however, located at DH and JH RSS heptamer borders, with deletion
of intervening sequences. It is therefore much more likely that the
synaptic complex formation and excision of the DJ sequences occurred
prior to the insertion event. This would imply that the RSS signal ends
of the excised BCL2 gene were ligated to the DH and JH
coding ends of the IGH locus, thereby forming a hybrid joint. A model for the transposition of BCL2 to the
IGH locus shows excision of the BCL2 gene
followed by diffusion to its target localization and insertion in the
IgH locus (Figure 4).
In a variant of this model, both BCL2 ends are not
excised synchronously but metachronously, and both BCL2
signal ends are subsequently excised and fused to the IgH coding ends.
According to this variant model, the BCL2 gene is not freely
diffusing from one to the other chromosome; however, this model
needs a complex sequence of events at both sides of the
BCL2 and IGH loci.
In normal B and T cells, hybrid joints can be found in the form of inversions in the V(D)J region.28,29 In cell-free systems, hybrid joints can be produced by a mixture of truncated RAG proteins and HMG1.30 Furthermore, their formation in vivo was shown to be independent of Ku86,31 in contrast to both signal joint and coding joint formation. On the basis of these results it was proposed that the mechanism of hybrid joint formation is RAG-1/-2-mediated strand invasion of the coding end hairpin by the blunt signal end in the synaptic complex, essentially identical to the RAG-1/-2-mediated insertion reactions observed in cell-free systems. However, a mechanism in which an intact coding end hairpin is directly attacked by the signal end would predict hybrid joints without N-nucleotides and without deletions because both are the result of coding end processing after opening of the hairpin. In the case of our BCL2 insertions, as well as in previously studied hybrid joints,32 extensive N-regions and deletions were present at the hybrid joints. This implies that opening of the coding end hairpin and subsequent processing by terminal deoxynucleotidyl transferase (TdT) and exonucleases preceded the joining reaction. Therefore, the actual joining must have involved 2 blunt ends, which is not compatible with the strand invasion hypothesis. This suggests that the joining reaction was mediated by double-stranded DNA (dsDNA) repair proteins, just like normal coding joint and signal joint formation. The presently described cases of FL have a distinct genomic configuration compared with FL with the usual t(14;18). Whereas concurrent 5'- and 3'-BCL2 breakpoints have been described previously,33,34 our cases contained a unique 3'-breakpoint 6.2 kb from the mbr in a region not previously sequenced. This implies that this breakpoint may have been missed in previous studies, especially because the configuration described will not lead to a cytogenetically detectable t(14;18). Furthermore, in contrast to the present cases, earlier studies did not indicate any resemblance to the RSS elements in both the mbr and minor breakpoint cluster (mcr) region of BCL2, and even the CAC trinucleotide was not found, indicating that these t(14;18) breakpoints are not directly mediated by RAG-1/-2. For these breakpoints, the RAG-1/-2-mediated DNA strand-attack mechanism described above may be an attractive model.
Submitted November 19, 1999; accepted May 5, 2000.
Supported by the Dutch Cancer Society, grant 95-1047.
The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734.
Reprints: Philip M. Kluin, Department of Pathology, Leiden University Medical Center, PO Box 9600, Gebouw 1, L1-Q 2300RC Leiden, The Netherlands; e-mail: pkluin{at}pat.azl.nl.
1. Vaandrager JW, Schuuring E, Raap AK, Philippo K, Kleiverda JK, Kluin P. Interphase FISH detection of BCL2 rearrangement in follicular lymphoma using breakpoint-flanking probes. Genes Chromosom Cancer. 2000;27:85-94[Medline] [Order article via Infotrieve].
2.
Vaandrager JW, Schuuring E, Kluin Nelemans JC, Dyer MJS, Raap AK, Kluin PM.
DNA fiber FISH analysis of immunoglobulin class switching in B cell neoplasia: abberrant CH gene rearrangements in follicle center cell lymphoma.
Blood.
1998;92:2871-2878
3.
Vaandrager JW, Schuuring E, Zwikstra E, et al.
Direct visualization of dispersed 11q13 chromosomal translocations in mantle cell lymphoma by multi-color DNA fiber FISH.
Blood.
1996;88:1177-1182
4.
Florijn RJ, Blonden LAJ, Vrolijk H, et al.
High-resolution DNA Fiber-FISH for genomic DNA mapping and colour bar-coding of large genes.
Hum Mol Genet.
1995;4:831-836
5.
Wiegant J, Ried T, Nederlof PM, Van der Ploeg M, Tanke HJ, Raap AK.
In situ hybridization with fluoresceinated DNA.
Nucleic Acids Res.
1991;19:3237-3241 6. Beishuizen A, Verhoeven MA, Mol EJ, Breit TM, Wolvers Tettero IL, van Dongen JJ. Detection of immunoglobulin heavy-chain gene rearrangements by Southern blot analysis: recommendations for optimal results. Leukemia. 1993;7:2045-2053[Medline] [Order article via Infotrieve].
7.
Tsujimoto Y, Croce CM.
Analysis of the structure, transcripts, and protein products of BCL-2, the gene involved in human follicular lymphoma.
Proc Natl Acad Sci U S A.
1986;83:5214-5218
8.
De Boer CJ, Loyson S, Kluin PM, et al.
Multiple breakpoints within the BCL-1 locus in B-cell lymphoma: rearrangements of the cyclin D1 gene.
Cancer Research.
1993;53:4148-4152
9.
Jurka J, Klonowski P, Dagman V, Pelton P.
CENSOR 10. Akasaka T, Akasaka H, Yonetani N, et al. Refinement of the BCL2/immunoglobulin heavy chain fusion gene in t(14;18)(q32;q21) by polymerase chain reaction amplification for long targets. Genes Chromosomes Cancer. 1998;21:17-29[Medline] [Order article via Infotrieve]. 11. Corbett SJ, Tomlinson IM, Sonnhammer ELL, Buck D, Winter G. Sequence of the human immunoglobulin diversity (D) segment locus: a systematic analysis provides no evidence for the use of DIR segments, inverted D segments, "minor" D segments or D-D recombination. J Mol Biol. 1997;270:587-597[Medline] [Order article via Infotrieve].
12.
Hesse JE, Lieber MR, Mizuuchi K, Gellert M.
V(D)J recombination: a functional definition of the joining signals.
Genes Dev.
1989;3:1053-1061 13. Akamatsu Y, Tsurushita N, Nagawa F, et al. Essential residues in V(D)J recombination signals. J.Immunol. 1994;153:4520-4529[Abstract]. 14. Lewis SM, Agard E, Suh S, Czyzyk L. Cryptic signals and the fidelity of V(D)J joining. Mol Cell Biol. 1997;17:3125-3136[Abstract].
15.
Fuscoe JC, Zimmerman LJ, Lippert MJ, Nicklas JA, O'Neill JP, Albertini RJ.
V(D)J recombinase-like activity mediates hprt gene deletion in human fetal T-lymphocytes.
Cancer Research.
1991;51:6001-6005 16. Fuscoe JC, Zimmerman LJ, Harrington BK, et al. V(D)J recombinase-mediated deletion of the hprt gene in T-lymphocytes from adult humans. Mutat Res. 1992;283:13-20[Medline] [Order article via Infotrieve].
17.
Breit TM, Mol EJ, Wolvers TI, Ludwig WD, Van Wering ER, van Dongen JJ.
Site-specific deletions involving the tal-1 and sil genes are restricted to cells of the T cell receptor alpha/beta lineage: T cell receptor delta gene deletion mechanism affects multiple genes.
J Exp Med.
1993;177:965-977
18.
Aplan PD, Lombardi DP, Ginsberg AM, Cossman J, Bertness VL, Kirsch IR.
Disruption of the human SCL locus by "Illegitimate" V-(D)- J recombinase activity.
Science.
1990;250:1426-1429
19.
Cayuela JM, Gardie B, Sigaux F.
Disruption of the multiple tumor suppressor gene MTS1/p16(INK4a)/CDKN2 by illegitimate V(D)J recombinase activity in T-cell acute lymphoblastic leukemias.
Blood.
1997;90:3720-3726
20.
Tycko B, Reynolds TC, Smith SD, Sklar J.
Consistent breakage between consensus recombinase heptamers of chromosome 9 DNA in a recurrent chromosomal translocation of human T cell leukemia.
J Exp Med.
1989;169:369-377
21.
Burnett RC, Thirman MJ, Rowley JD, Diaz MO.
Molecular analysis of the T-cell acute lymphoblastic leukemia-associated t(1;7)(p34;q34) that fuses LCK and TCRB.
Blood.
1994;84:1232-1236
22.
Kagan J, Joe YS, Freireich EJ.
Joining of recombination signals on the der 14q- chromosome in T-cell acute leukemia with t(10;14) chromosome translocation.
Cancer Research.
1994;54:226-230 23. Boehm T, Mengle-Gaw L, Kees UR, et al. Alternating purine-pyrimidine tracts may promote chromosomal translocations seen in a variety of human lymphoid tumours. EMBO J. 1989;8:2621-2631[Medline] [Order article via Infotrieve].
24.
Thandla SP, Ploski JE, Raza-Egilmez SZ, et al.
ETV6-AML1 translocation breakpoints cluster near a purine/pyrimidine repeat region in the ETV6 gene.
Blood.
1999;93:293-299
25.
Wiemels JL, Greaves M.
Structure and possible mechanisms of TEL-AML1 gene fusions in childhood acute lymphoblastic leukemia.
Cancer Research.
1999;59:4075-4082 26. Hiom K, Melek M, Gellert M. DNA transposition by the RAG1 and RAG2 proteins: a possible source of oncogenic translocations [see comments]. Cell. 1998;94:463-470[Medline] [Order article via Infotrieve]. 27. Agrawal A, Eastman QM, Schatz DG. Transposition mediated by RAG1 and RAG2 and its implications for the evolution of the immune system [see comments]. Nature. 1998;394:744-751[Medline] [Order article via Infotrieve]. 28. Sollbach AE, Wu GE. Inversions produced during V(D)J rearrangement at IgH, the immunoglobulin heavy-chain locus. Mol Cell Biol. 1995;15:671-681[Abstract].
29.
Alexandre D, Chuchana P, Roncarolo MG, et al.
Reciprocal hybrid joints demonstrate successive V-J rearrangements on the same chromosome in the human TCR gamma locus.
Int Immunol.
1991;3:973-982
30.
Melek M, Gellert M, van Gent DC.
Rejoining of DNA by the RAG1 and RAG2 proteins.
Science.
1998;280:301-303 31. Han JO, Steen SB, Roth DB. Ku86 is not required for protection of signal ends or for formation of nonstandard V(D)J recombination products. Mol Cell Biol. 1997;17:2226-2234[Abstract]. 32. Lewis SM, Hesse JE, Mizuuchi K, Gellert M. Novel strand exchanges in V(D)J recombination. Cell. 1988;55:1099-1107[Medline] [Order article via Infotrieve]. 33. Nomdedeu JF, Baiget M, Gaidano G, et al. p53 mutation in a case of blastic transformation of follicular lymphoma with double bcl-2 rearrangement (MBR and VCR). Leuk Lymphoma. 1998;29:595-605[Medline] [Order article via Infotrieve]. 34. Mikraki V, Ladanyi M, Chaganti RS. Structural alterations in the 5' region of the BCL2 gene in follicular lymphomas with BCL2-MBR or BCL2-MCR rearrangements. Genes Chromosomes Cancer. 1991;3:117-121[Medline] [Order article via Infotrieve]. 35. Yabumoto K, Akasaka T, Muramatsu M, et al. Rearrangement of the 5' cluster region of the BCL2 gene in lymphoid neoplasm: a summary of nine cases. Leukemia. 1996;10:970-977[Medline] [Order article via Infotrieve]. 36. V BASE. Available at http://www.mrc-cpe.cam.ac.uk/imt-doc/restricted/ok.html. Tomlinson I, MRC, Center for Protein Engineering, Cambridge, UK. Accessed October 6, 1998.
© 2000 by The American Society of Hematology.
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  T. Katzenberger, G. Ott, T. Klein, J. Kalla, H. K. Muller-Hermelink, and M. M. Ott Cytogenetic Alterations Affecting BCL6 Are Predominantly Found in Follicular Lymphomas Grade 3B with a Diffuse Large B-Cell Component Am. J. Pathol., August 1, 2004; 165(2): 481 - 490. [Abstract] [Full Text] [PDF]
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 S. L. Barrans, P. A.S. Evans, S. J.M. O'Connor, R. G. Owen, G. J. Morgan, and A. S. Jack The Detection of t(14;18) in Archival Lymph Nodes: Development of a Fluorescence in Situ Hybridization (FISH)-Based Method and Evaluation by Comparison with Polymerase Chain Reaction J. Mol. Diagn., August 1, 2003; 5(3): 168 - 175. [Abstract] [Full Text] [PDF]
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 P L Roddam, S Rollinson, M O'Driscoll, P A Jeggo, A Jack, and G J Morgan Genetic variants of NHEJ DNA ligase IV can affect the risk of developing multiple myeloma, a tumour characterised by aberrant class switch recombination J. Med. Genet., December 1, 2002; 39(12): 900 - 905. [Abstract] [Full Text] [PDF]
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F. Jiang, F. Lin, R. Price, J. Gu, L. J. Medeiros, H. Z. Zhang, S.-S. Xie, N. P. Caraway, and R. L. Katz Rapid Detection of IgH/BCL2 Rearrangement in Follicular Lymphoma by Interphase Fluorescence in Situ Hybridization with Bacterial Artificial Chromosome Probes J. Mol. Diagn., August 1, 2002; 4(3): 144 - 149. [Abstract] [Full Text] [PDF]
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 G. Ott, T. Katzenberger, A. Lohr, S. Kindelberger, T. Rudiger, M. Wilhelm, J. Kalla, A. Rosenwald, J. G. Muller, M. M. Ott, et al. Cytomorphologic, immunohistochemical, and cytogenetic profiles of follicular lymphoma: 2 types of follicular lymphoma grade 3 Blood, May 15, 2002; 99(10): 3806 - 3812. [Abstract] [Full Text] [PDF]
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A. Albinger-Hegyi, B. Hochreutener, M.-T. Abdou, I. Hegyi, M. T. Dours-Zimmermann, M. O. Kurrer, P. U. Heitz, and D. R. Zimmermann High Frequency of t(14;18)-Translocation Breakpoints Outside of Major Breakpoint and Minor Cluster Regions in Fol |
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Abstract
Introduction
(IGL)
locus to the 5'-side, indicating 2 separate translocation events. In
the other 2 cases, the BCL2 3'- and 5'-flanking regions were
juxtaposed to each other (3'-BCL2/5'-BCL2). Juxtaposition of the IGH constant genes to the 5'-side and
the DH gene region to the 3'-side of BCL2
suggested that the BCL2 gene had been excised from its locus
at chromosome 18q21 and inserted into the IGH locus at
chromosome 14q32, an orientation that is the opposite of the
orientation in normal t(14;18). In this report, these variant
BCL2 rearrangements are analyzed in more detail and give
evidence of a mechanism that involves the recombination activation
gene-1/-2 (RAG-1/-2)-mediated excision of BCL2
and the formation of hybrid joints between BCL2 and
IGH.
, and Bcl2+).
) genes (gift of P. Leder, Boston, MA).
a program for identification and elimination of repetitive elements from DNA sequences.
Comput Chem.
1996;20:119-122