Biotechnical Methods Section (BTS)

Detection of illegitimate rearrangements within the immunoglobulin light chain loci in B cell malignancies using end sequenced probes

Abstract

Translocations involving the immunoglobulin loci are recurring events of B cell oncogenesis. The majority of translocations involve the immunoglobulin heavy chain (IGH) locus, while a minor part involves the immunoglobulin light chain loci consisting of the kappa light chain (IGK) located at 2p11.2 and the lambda light chain (IGL) located at 22q11.2. We characterised BAC clones, spanning the IGK and IGL loci, for detection of illegitimate rearrangements by fluorescence in situ hybridisation (FISH). Within the IGL region we have identified six end sequenced probes (22M5, 1152K19, 2036J16, 3188M21, 3115E23 and 274M7) covering the variable (IGLV) cluster and two probes (165G5 and 31L9) covering the constant (IGLC) cluster. Within the IGK region four probes (969D7, 316G9, 122B6 and 2575M21) have been identified covering the variable (IGKV) cluster, and one probe (1021F11) covering the IGK constant (IGKC) cluster. A series of 24 cell lines of different origin have been analysed for the presence of translocations involving the immunoglobulin light chain loci by dual-colour FISH where the split of the variable cluster and the constant cluster indicated a translocation. Probes established in this study can be used for universal screening of illegitimate rearrangements within the immunoglobulin light chain loci in B cell malignancies.

Introduction

Immunoglobulin molecules are composed of a heavy chain and a light chain, each consisting of a variable and a constant part.1 The heavy chain is encoded by the gene segments within the immunoglobulin heavy chain (IGH) locus located within a large region of 1.2 Mb in chromosome band 14q32.3.2 The light chain is encoded by one of the two types of immunoglobulin light chains: lambda (IGL) or kappa (IGK). IGL is located within a 1 Mb locus at the chromosome band 22q11.2 and it consists of 38 potentially active variable (IGLV) gene segments, 35 pseudogenes and seven IGL constant gene segments, each with a joining (J)-segment IGL (J–C) gene segments together with a VPREB gene, seven non-immunoglobulin-related genes (TGFβ1, 5′OY11.1, 3′OY11.1, BCRL4, POM121, GGT and GGT-rel), three Alu-rich clusters, minisatellite clusters and α-satellite clusters.3 IGK is located within a 1.8 Mb locus at the chromosome band 2p11.2 and it has a largely duplicated structure with the duplicate gene regions being 96–100% identical.4,5 The IGK proximal copy is present in a 542 kb contig with 22 potentially functional variable (IGKV) gene segments and 18 pseudogenes plus five joining (J)-segments and one IGK constant (IGKC) gene segment. The IGK distal copy is a 433 kb contig with 21 potentially functional IGKV gene segments and 15 pseudogenes.6 The two contigs that contain 33 pairs of homologous IGKV gene segments and 10 solitary genes are separated by a DNA sequence of 800 kb without any IGKV gene segments.5

During B cell development the IGH (V-D-J) (a hyphen indicates the genomic DNA in germline configuration between genes and segments; otherwise the genomic DNA is in rearranged configuration) cluster is rearranged when the B cell selects an IGHV gene segment, an IGH diversity (D)-segment, and an IGH joining (J)-segment, all of which are located within a 957 kb large region.2 After generation of the functional IGH gene, an immunoglobulin light chain variable gene segment is juxtaposed to an immunoglobulin light chain joining (J)-segment to form a mature immunoglobulin light chain gene. The gene product of the heavy chain and the light chain is merged to form a functional immunoglobulin molecule.1 Each time the B cell rearranges the immunoglobulin loci by DNA double strand breaks, an error can occur and a proto-oncogene can be translocated into the immunoglobulin loci. These DNA double strand breaks occur when the B cell chooses a diversity (D)-segment and a joining (J)-segment of the IGH locus to form an IGH (V-DJ-C) cluster. The joining is mediated by recombination signal sequences (RSS), which reside adjacent to both variable gene segments and joining (J)-segments of the IGH, the IGK and the IGL loci. When the B cell has selected an IGHV gene segment, which is then juxtaposed to the IGH (DJ-C), the IGH (VDJ) cluster is again subject to double strand breaks during somatic hypermutation before the double strand breaks during the isotypic switch. The immunoglobulin light chain loci are also subject to DNA double strand breaks, when selecting a variable gene segment that is juxtaposed to an immunoglobulin light chain joining (J)-segment before DNA double strand breaks during somatic hypermutation. Even though most of the major translocations involve the heavy chain of the immunoglobulin locus, the two light chains, IGL and IGK loci, are just as important since their involvement in the translocations with the oncogene c-MYC can lead to the variant form of Burkitt's lymphoma.7,8 It is know that all Burkitt's lymphoma contain either a t(8;14)(q24;q32.3) or the variant t(2;8)(p11.2;q24) and t(8;22)(q24;q11.2) forms. In follicular lymphomas, 80% of the cases contain a t(14;18)(q32.3;q21) involving the BCL2 gene but the variant translocations t(2;18)(p11.2;q21) and t(18;22)(q21;q11.2) have also been identified.9,10 The large majority (95%) of mantle cell lymphomas carries a t(11;14)(q13;q32.3) involving the CCND1 gene and the variant translocation t(11;22)(q13;q11.2) has likewise been identified.9,11

It is well known that translocations within the IGH locus are a recurring event in most B cell malignancies. We have previously published a set of IGHV and IGHC probes, which can be used for universal screening of illegitimate rearrangement within the IGH locus by reliable interphase FISH analysis.12 In this study, we have extended this to the immunoglobulin light chain loci by identifying BAC probes covering both the variable and the constant clusters of the IGK and IGL loci.

Materials and methods

Cell line preparations

The cell lines LP-1, NCI-H929, RPMI-8226, and KG-1 were purchased from DSMZ (Braunschweig, Germany); OPM-2, U266B1, SKO-007 J3, HS Sultan, RS-4;11, IM-9, CCRF-CEM, Raji, MOLT-3, CA46, RPMI-7666, SUP-B15, DOHH-2, SD-1, KASUMI-1, ST486, and SKO-007 were purchased from ATCC (Rockville, MD, USA); BL2, BL60, and LY91 were kindly obtained from Prof Gilber Lenoir, IARC (Lyon, France). Cell lines were grown (37°C, 5% CO2) in RPMI 1640, 25 mM Hepes, 1 mM glutamax (Life Technologies, Gaithersburg, MD, USA) supplemented with 20% foetal calf serum (Life Technologies), 100 μg/ml streptomycin, and 100 U/ml penicillin (Life Technologies). For analysis, cell lines were incubated in hypotonic 0.075 M KCl, fixed in Carnoy's solution (3:1 (vol/vol) methanol: glacial acetic acid), dropped on to HCl-ethanol cleaned slides, and air dried at room temperature (RT).

Immunoglobulin light chain-specific probes

A 1500 kb part of the contig NT_011520, including the sequences for the known IGL gene segments (from 1 500 000 bp to 3 000 000 bp) was chosen for analysis. End-sequenced BAC probes for the IGL locus were identified by analysis of the 1500 kb DNA sequence using the STC-BAC search computer at TIGR.13 Clones with only one sequenced end were end-sequenced to obtain the other end. BLAST analysis with both BAC end sequences was used to choose 24 BAC probes with 5′ end and 3′ end sequences consisting of more than 200 bp and with over 94% identity for further analysis by fiber-, interphase- and metaphase FISH. Two PAC clones for the IGLC cluster that were found at http://www.biologia.uniba.it/rmc/ were sequenced at both ends and included in the analysis. Two contigs containing 541 592 bp and 433 305 bp of the IGK locus were assembled from the published sequence.4 Analysis of the two DNA contigs separately using the STC-BAC search program at TIGR identified four end-sequenced BAC probes for the IGK locus.13 Fifteen BAC probes with 5′ end and 3′ end sequences consisting of more than 94% identity over 200 bp were chosen by BLAST analysis with both BAC end sequences, for further analysis by fiber-, interphase- and metaphase FISH. One fully sequenced BAC clone (316G9) was also included. Four end-sequenced BAC clones with only one end located inside the 542 kb DNA sequence were also included. One PAC clone for the IGKC cluster was found at http://www.biologia.uniba.it/rmc/ and end-sequenced before it was included in the analysis.

Growth of clones, DNA purification, and DNA labelling

Growth of clones and DNA purification were performed as described using QIAGEN 500 tips (Qiagen, Hilden, Germany).12 The probes were labelled by nick translation with biotin-16-dUTP (Roche Diagnostics, Mannheim, Germany) or digoxigenin-11-dUTP (Roche Diagnostics) for indirect labelling, and fluorescein-12-dUTP (Roche Diagnostics) or tetramethylrhodamine-5-dUTP (Roche Diagnostics) for direct labelling, as described by the manufacturer (Roche Diagnostics). A 10-fold excess of each of Cot-1-DNA (Roche Diagnostics), E. coli-tRNA (Roche Diagnostics), and Salmon sperm DNA (Roche Diagnostics) were added to the labelled probe. The probe was ethanol precipitated and dissolved in hybridisation mixture (50% formamide, 300 mM NaCl, 30 mM sodium citrate, 10% dextran sulphate, 50 mM sodium phosphate, pH 7).

FISH

FISH was carried out as described earlier.12 The variable cluster probes were labelled with either fluorescein-12-dUTP (Roche Diagnostics) or biotin-16-dUTP (Roche Diagnostics) detected with fluorescein isothiocyanate (FITC)-conjugated antibodies (Vector Laboratories, Burlingame, CA, USA). The constant cluster probes were labelled with either tetramethylrhodamine-5-dUTP (Roche Diagnostics) or digoxigenin-11-dUTP (Roche Diagnostics) detected with tetramethylrhodamine isothiocyanate (TRITC) conjugated antibodies (Vector Laboratories). Slides were mounted in Vectashield (Vector Laboratories) containing 4′-6′-diamidino-2-phenylindole (DAPI). The signals were scored using an Axiovert 135M microscope (Carl Zeiss, Oberkochen, Germany) equipped with a SenSys charge-coupled device camera (Photometrics, Tucson, AZ, USA), and IPLAB Spectrum Quips FISH software (Applied Imaging International, Newcastle, UK). Five metaphases from each cell line were analysed with each probe set. Also, fifty interphase nuclei on the same slide were examined for co-localisation of the two probe sets located on the variable and the constant cluster.

Fiber-FISH

DNA fiber preparations were made as described by Erdel et al14 2 × 106 cells of the progenitor cell line KG-1, were used and probes were labelled with either biotin-16-dUTP or digoxigenin-11-dUTP, as described above. Hybridisation was performed as described before.12

DNA sequencing

Fluorescent DNA sequencing using primers SP6 and T7 was performed with a Dye Terminator Cycle Sequencing Kit (Perkin Elmer, Warrington, UK) and an ABI 310 Prism Genetic Analyser (Perkin Elmer) according to the manufacturer's instructions except for the use of 2 μg DNA per sample and 99 cycles.

Computer search and programs

Clone identification was performed by using the STC-BAC end-sequencing program at http://www.tigr.org/tdb/humgen/bac_end_search/bac_end_search.html, Resources for Molecular Cytogenetics (RMC) at http://www.biologia.uniba.it/rmc/ and BLAST at http://www.ncbi.nlm.nih.gov/BLAST/. The nomenclature was used according to the international ImMunoGeneTics database (IMGT) at http://imgt.cines.fr:8104.15

GenBank accession numbers

The end sequences of 1117G4_SP6, 1117G4_T7, 1019H10_SP6, 1019H10_T7, 869I1_SP6, 869I1_T7, 165G5_SP6, and 969D7_SP6 have been deposited at the GenBank under the accession numbers AZ757842, AZ757843, AZ757844, AZ757845, AZ757846, AZ757847, AZ757848 and BH817039, respectively.

Results

Identification of IGK probes

The human immunoglobulin IGK locus has a duplicated structure, consisting of a proximal copy, and a distal copy, separated by 800 kb without any IGKV gene segments. The DNA sequences of the IGK proximal copy and the IGK distal copy were assembled from the published sequences (AP001234, AP001215, AP001233, AP001248, AP001211, AP001240, AP001244, AP001241, AP001230, AP001209, AP001231, AP001249, AP001228, AP001237, AP001243, AP001222, AP001238, AP001246, AP001226, AP001219, AP001217, AP001239, AP001225, AC009958, AP001243 and AF017732) and (AP001247, AP001221, AP001218, AP001220, AP001235, AP001224, AP001216, AP001232, AP001212, AP001236, AP001242, AP001227, AP001213, AP001210, AP001223, AP001245, AP001229 and AP001214), respectively. We identified 19 end-sequenced BAC clones with the STC-BAC search computer at TIGR, one fully sequenced BAC probe (316G9) at NCBI, and one PAC clone at http://www.biologia.uniba.it/rmc/. In silico analysis of the 21 clones showed that one of the BAC clones (479P12) was located just outside the IGKV proximal cluster. Fifteen BAC probes (969D7, 1023H23, 75N1, 1097F16, 62I20, 2063D22, 316G9, 99G6, 97F19, 3159C20, 122B6, 1061B13, 943H14, 2575M21, 2341E11) cover the IGKV proximal cluster. The end-sequenced PAC clone (1117G4) and two of the BAC clones (15J7 and 2366J12) were shown to cover the most proximal IGKV gene segments, the entire J segment cluster and the IGKC cluster. Two BAC clones (1021F11 and 157D12) were located within the IGKC cluster and extended telomeric to the IGK locus (see Figure 1). The 21 in silico identified BAC and PAC clones were used to build two contigs, which spanned the IGK proximal locus. Seven BAC clones (479P12, 969D7, 316G9, 122B6, 15J7, 2575M7 and 1021F11) covering the IGKV proximal cluster, IGKC cluster, and extend outside the IGK gene region were chosen for analysis using fiber-, interphase- and metaphase-FISH. No attempt was made to identify BAC clones for the IGK distal cluster due to the fact that the IGK distal cluster is largely duplicated, with the duplicated gene regions being 96–100% identical.4,5 The BAC end-sequences of the 2575M21 probe displayed no identity with the IGK distal contig. Analysis of the DNA sequence of 2575M21 showed that one BAC end was located outside the IGK distal contig, while the other BAC end was located within an area which had presumably been deleted during the insertion of the extra IGK distal cluster segment during evolution. This segment contained the IGKV (1D–43) and IGKV (1D-42) gene segments. The overlap between the selected BAC clones ranged from 6454 bp (between 122B6 and 15J7 at the distal copy) to 62164 bp (between 969D7 and 316G9 at the proximal copy) (see Figure 1). A gap of 9034 bp between the IGKC cluster and IGKV cluster was identified when replacing the clone 15J7 with 2575M21. It is within this gap between 2575M21 and 1021F11 that the expected translocations occur.16,17 The BAC and PAC clone names, supplier and position on the two IGK contigs are listed in Table 1.

Figure 1
figure1

Fiber-FISH analysis of the IGK locus. DNA fiber of KG-1 hybridised with probe sets (a) 479P12, 969D7, 316G9 and 122B6; (b) 316G9, 122B6, 15J7 and 1021F11. Overlaps between the green and red labelled BAC probes are seen as yellow regions ranging between 10 825 bp and 62 184 bp. All sizes are calculated from data in Table 1. A black line indicates the extent of the probes. The locations of IGKV gene segments, joining (J)-segments, and IGKC gene segments are illustrated on the outermost bar. A green box indicates functional IGKV gene segments. A yellow box indicates open reading frames. A yellow line indicates joining (J)-segments. The blue box indicates IGKC gene segment. A red box indicates pseudogene segments. The blue line indicates the extent of probe 2575M21, which is used instead of 15J7 in the interphase- and metaphase-FISH assay. The outermost bar at the top represents the distal copy of the IGK and the proximal copy is represented at the bottom. The dashed line indicates the 800 kb spacing between the proximal and distal copy of IGK. The outermost bar is derived (with permission) from the IMGT database.6

Table 1 BAC and PAC clones

Identification of IGL probes

A 1.5 Mb part of the contig NT_011520 was used to identify BAC and PAC probes within the human IGL locus. They cover the 5′IGLV and 3′IGLC cluster sequences sufficiently for reliable interphase FISH. The contig included the sequences of the IGL locus gene segments (GenBank accession number D86989-D87024 and D88268-D88271) and sequences outside the IGL locus (GenBank accession number AP000555, AP000360, AP000361, AP000362, AC000029 and U07000). We identified 24 end-sequenced BAC clones by using the STC-BAC search computer at TIGR and found two PAC clones at http://www.biologia.uniba.it/rmc/. In silico analysis of the 26 clones showed that one BAC clone (99N17) was located just outside the IGLV cluster. Sixteen BAC probes (179H3, 22M5, 1040J16, 1152K19, 646D5, 2540L3, 2036J16, 2523F21, 3188M21, 114D2, 757F24, 3115E23, 2650N21, 2650N19, 274M7 and 126O14) covered the IGLV cluster and extend outside the IGL gene region with 146 kb. Two of the end-sequenced PAC clones (1019H10 and 869I1) and the two BAC clones (2305N6 and 2507C12) were shown to cover the three most proximal IGLV gene segments and the entire IGLC cluster. One BAC clone (165G5) was located within the IGLC cluster and extended telomeric to the IGL locus (see Figure 2). Four BAC clones (3062A1, 2191F9, 2521E17 and 31L9) were shown to be located just outside the IGLC cluster. The 26 in silico identified BAC and PAC clones were used to build two BAC contigs, which covered the entire IGL locus. Ten of the BAC clones (22M5, 1040J16, 646D5, 2036J16, 3118M21, 3115E23, 274M7, 2507C12, 165G5 and 31L9) covering the IGLV and IGLC clusters were chosen for FISH analysis on DNA fibers, interphase nuclei, and metaphase chromosomes. Later the BAC clone 1040J16 was exchanged with the BAC clone 1152K19 due to hybridisation to chromosome 3 instead of the IGL locus on chromosome 22. The overlap between the selected BAC clones ranged from 3375 bp (between 646D5 and 2036J16) to 36 689 bp (between 1152K19 and 646D5) (Figure 2).

Figure 2
figure2

Fiber-FISH analysis of the IGL locus. DNA fiber of KG-1 hybridised with probe sets (a) 22M5, 1152K19, 646D5 and 2036J16: (b) 646D5, 2036J16, 3188M21 and 3115E23; (c) 3188M21, 3115E23, 274M7 and 2507C12; and (d) 274M7, 2507C12, 165G5 and 31L9. Overlaps between the BAC probes are seen as yellow regions of different sizes ranging from 6454 bp to 62 164 bp. All sizes are calculated from data in Table 1. A black line indicates the extent of the probes which were used. The locations of IGLV gene segments, joining (J)-segments, and IGLC gene segments are illustrated on the top bar. A green box indicates functional IGLV gene segments. A yellow box indicates open reading frames. A yellow line indicates joining (J)-segments. A blue box indicates IGLC gene segments. A red box indicates pseudogene segments. The top bar is derived (with permission) from the IMGT database.6

A gap of 35 194 bp between the IGLC cluster and IGLV cluster was identified when omitting the clone 2507C12. It is within this gap that known translocations occur.16,17 The BAC and PAC clone names, supplier, and position on NT_0011520 are listed in Table 1.

BAC probes which cross-hybridise to other loci than the IGL and IGK

Dual colour metaphase- and interphase-FISH analyses using BAC probes containing the IGLV cluster were shown to co-localise with the probes containing the IGLC cluster while BAC probes containing the IGKV cluster were shown to co-localise with the probes containing the IGKC cluster. The co-localised signal for IGL probes was located at 22q11.2, while the signals for the IGK probes were located at 2p11.2, as expected. However, two BAC probes, 479P12 and 646D5, were shown to cross-hybridise to chromosomal loci other than 22q11.2 or 2p11.2. The IGLV probe 646D5, containing IGLV (9–49, 1–50, 1–51, 5–52, VPREB, (IV)–53, 10–54, 11–55, (I)–56, 6–57, (V)–58, (IV)–59, and 4–60) gene segments, TGFβ1, repetitive elements, minisatellite and α-satellite clusters showed multiple cross-hybridisation patterns to chromosome bands 1p11, 2p12, 8q11, 15q11, and 16p11 (see Figure 3b). The IGKV probe 479P12 partly overlaps clone 969D7 that does not show any cross-hybridisation. The non-overlapped DNA sequence of 479P12 does not include any IGKV gene segments and showed cross-hybridisation to chromosome bands on 1p11, 9p11, 15q11, 16p11, and 22q11 (see Figure 4b). The exclusion of the 479P12 clone leaves >10.4 kb and >10.8 kb of DNA located outside the last IGKV (2–40 and 2D–40) gene segments on the proximal and distal IGK copy, respectively. We excluded these two probes (646D5 and 479P12) to avoid extra signals in interphase FISH analysis caused by the cross-hybridisation.

Figure 3
figure3

Interphase and metaphase FISH analysis of the IGL locus. (a) Cell line KG-1: Hybridisation of the IGLV probes 22M5, 1152K19, 2036J16, 3188M21, 3115E23 and 274M7 labelled with green (Fluorescein) together with the IGLC probes 165G5 and 31L9 labelled with red (Rhodamine). These show normal IGL configurations on metaphases with a yellow signal located on both chromosome 22, band q11.2. The two yellow signals on interphase cells indicate two normal IGL loci. (b) Cell line KG-1: Hybridisation of the IGLV probe 646D5 labelled with red (Rhodamine) together with the IGLV probe 2036J16 labelled with green (Fluorescein). Cross-hybridisation to chromosomes other than 22q11.2 is observed at 1p11, 2p12, 8q11, 15q11, and 16p11 with the probe 646D5, while the probe 2036J16 is specifically hybridising to 22q11.2. This is shown by normal IGL configurations on metaphases with a yellow signal located on both chromosomes 22, band q11.2 together with red signals located on both chromosomes 1, 2, 8, 15, and 16. (c) Cell line BL60: Hybridisation of the IGLV probes 22M5, 1152K19, 2036J16, 3188M21, 3115E23 and 274M7 labelled with green (Fluorescein) together with the IGLC probes 165G5 and 31L9 labelled with red (Rhodamine). Hybridisation on the metaphase chromosomes shows one yellow signal located on 22q11.2 indicating a normal IGL configuration on one allele and a split of the IGLC and IGLV probes on the other allele. The derivative chromosome 22 contains a red signal while the derivative partner chromosome 8 contains a green signal. Hybridisation on the interphase nucleus shows a yellow signal indicating a normal IGL allele and a split red and green signal indicating the translocation.

Figure 4
figure4

Interphase and metaphase FISH analysis of the IGK locus. (a) Cell line KG-1: Hybridisation of the IGKV probes 969D7, 316G9, 122B6 and 257M21 labelled with green (Fluorescein) together with the IGKC probe 1021F11 labelled with red (Rhodamine). These show normal IGK configurations on metaphases with a yellow signal located on both chromosome 2, band p11.2. The two yellow signals, seen on interphase cells, indicate two normal IGK loci. (b) Cell line KG-1: Hybridisation of the IGKV probe 479P12 labelled with green (Fluorescein) together with the IGKV probe 22M5 labelled with red (Rhodamine). Cross-hybridisation to chromosomes other than 2p11.2 is observed at 1p11, 9p11, 15q11, 16p11 and 22q11.2 with the probe 479P12, while the probe 22M5 is specifically hybridising to 2p11.2. This is shown by normal IGK configurations on metaphases with a yellow signal located on both chromosomes 2, band p11.2 together with green signals located on both homologues of chromosomes 1, 9, 15, 16, and 22. (c) Cell line LY91: Hybridisation of the IGKV probes 969D7, 316G9, 122B6 and 257M21 labelled with green (Fluorescein) together with the IGKC probe 1021F11 labelled with red (Rhodamine). Hybridisation on the metaphase chromosomes shows one yellow signal located on 2p11.2 indicating a normal IGL configuration on one allele and a split of the IGKC and IGKV probes on the other allele. A derivative chromosome 2 contains a red signal, while the derivative partner chromosome contains a green signal. Hybridisation on the interphase nucleus shows a yellow signal indicating a normal IGK allele, and a split red and green signal indicates the translocation.

Fiber-FISH

Four probes were labelled in a biotin(green)–digoxigenin(red)–biotin(green)–digoxigenin(red) pattern, in each experiment. Two experiments for the IGK locus and four experiments for the IGL locus were performed. This strategy allowed identification of the orientation and overlap of the four probes in each experiment. Fiber-FISH mapping with four large BAC clones sometimes resulted in DNA fibers, which were too stretched to capture in one picture. We therefore looked for low-resolution DNA fibers that could be captured in a single frame. We have shown that the probes in the order 479P12-969D7-316G9-122B6-15J7-1021F11 would cover the IGK proximal locus and presumably also the IGK distal locus (Figure 1) and the probes in the order 22M5-1152K19-646D5-2036J16-3188M21-3115E23-274M7-2507C12-31L9 would cover the IGL locus (Figure 2). Yellow overlapping regions caused by the colocalized green and red fluorescence were of different sizes, ranging from 3 kb between 646D5 and 2036J16 to 105 kb between 2507C12 and 165G9. The high resolution of the stretched DNA fibers did not permit us to see both the distal and proximal copies of the IGK simultaneously on a single fiber.

Detection of translocations within the IGL and the IGK loci in different B cell lines

In order to clarify the translocation pattern in 24 cell lines of different origin, we assayed for illegitimate rearrangement within the IGL and IGK loci. In the IGL translocation assay, probes 31L9 and 165G9, covering the IGLC cluster were labelled directly with a red fluorochrome (Rhodamine), and probes 22M5, 1152K19, 2036J16, 3188M21, 3115E23, 274M7 containing the IGLV cluster sequence were labelled with a green fluorochrome (Fluorescein). In the IGK translocation assay, probe 1021F11 containing the IGKC cluster was labelled red (Rhodamine), while probes 969D7, 316G9, 122B6, and 2575M21 were labelled green (Fluorescein). Localisation of a green and a red signal together, forming a yellow signal, indicated a normal IGL and IGK locus. Split signals from the probes covering the variable cluster (Green) and the probes covering the constant cluster (Red) were evaluated as an illegitimate rearrangement within the IGL and IGK loci. Data from all cell lines are shown in Table 2.

Table 2 Karyotype data and illegitimate rearrangements involving the immunoglobulin light chain loci detected in haematopoetic cell lines by dual colour FISH

Illegitimate rearrangement within the IGL or IGK loci was detected in one out of eight myeloma cell lines, and in three out of seven lymphoma cell lines. The myeloma cell line, RPMI-8226, had lost two green signals when using the IGL translocation assay, indicating either deletion of the IGLV gene segments or loss of the partner derivative chromosomes. Two clearly derivative chromosomes 22 were shown in the metaphase FISH analysis. Two lymphoma cell lines (BL2 and BL60) showed translocations within the IGL locus, while one lymphoma cell line (LY91) showed translocations within the IGK locus. Five cell lines, OPM-2, SKO-007 J3, LP-1, MOLT-3 and SD-1 had signal patterns that demonstrated chromosome duplications of chromosome 22, whereas seven cell lines OPM-2, SKO-007, SKO-007 J3, RPMI-8226, RAJI, MOLT-3 and SD-1, contained duplicated chromosome 2. Representative illustrations are shown in Figures 3 and 4.

Discussion

This study was initiated to detect the involvement of the immunoglobulin light chain loci at 2p11.2 and 22q11.2 in translocations in B cell malignancies by reliable interphase FISH, using BAC probes covering the variable and constant clusters of the IGK and IGL loci. The IGK locus contains 76 IGKV gene segments, but only 43 of the IGKV gene segments contain an open reading frame (Figure 1). A large part of the IGK locus is duplicated, giving rise to an IGK-proximal copy and a similar, distal copy of 542 kb and 433 kb, respectively. The duplication of the IGK locus is believed to be a relatively late evolutionary event since the duplicated genes are 96–100% identical. Furthermore, most restriction sites occur in both copies at the same positions and the DNA sequences are almost identical.4 The two duplicated copies are arranged in opposite 5′,3′-polarity and are separated by an 800-kb apparently IGKV gene segment-free stretch of DNA. During the development of B-cells, the proximal copy of the IGKV gene segments is rearranged by deletion of the DNA, sometimes involving several hundred kilobases, in between the IGKV gene segments and the IGK (J-C) gene segments whereas the distal copy is rearranged by inversion.18 The high level of identity of the distal and proximal parts of IGK makes it highly likely that the same probes can be used for both parts of the IGKV clusters. The cross-hybridisations caused by the IGKV probe 479P12 to 1p11, 9p11, 15q11, and 16p11 are generated from the sequences located between the distal copy and proximal copy of the IGK gene region. Excluding this probe for interphase FISH still leaves >10 kb of FITC-labelled probe generated from the 969D7 probe which generates enough signal to be evaluated. The IGLV probe 646D5, which cross-hybridises to 1p11, 2p12, 8q11, 15q11, and 16p11 is generated from the sequences of 646D5 between the flanking probes 2036J36 and 1152K19. These sequences of 146 383 bp includes multiple IGLV gene segments, a large number of repetitive elements, minisatellite clusters, and α-satellite clusters, all of which could be responsible for the cross-hybridisation. IGLV orphons have been described at 8q11 at IMGT, however, the cross-hybridisation to 2p12, 15q11 and 16p11 has not been reported before. These cross-hybridisations could be caused by IGLV orphons, as is the case for the IGHV and IGKV which have orphons on 1p11, 2p12, 15q11 and 16p11. However, further investigation is required before any definite conclusions can be made.

In conclusion, we have identified a set of IGKV and IGKC probes and a set of IGLV and IGLC probes, which can be used for universal screening of illegitimate rearrangement within the immunoglobulin light chain loci. The two assays can detect: (1) translocations involving the IGK (V–J) recombination cluster: (2) translocations involving the IGL (V–J) recombination cluster: and (3) deletions and duplications.

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Acknowledgements

We thank Dr Mariano Rocchi (RMC) for providing the IGKC and IGLC probes, Prof Gilber Lenoir (IARC) for providing the cell lines; Erik Kjærsgaard, Ulla Høy Davidsen and Dr Charles Medom Hansen for reading the manuscript and Marianne Lodahl for help with sequencing. This work was supported by the Danish Cancer Society and the Danish National Research Foundation.

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Correspondence to AN Silahtaroglu.

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Poulsen, T., Silahtaroglu, A., Gisselø, C. et al. Detection of illegitimate rearrangements within the immunoglobulin light chain loci in B cell malignancies using end sequenced probes. Leukemia 16, 2148–2155 (2002). https://doi.org/10.1038/sj.leu.2402648

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Keywords

  • immunoglobulin light chain
  • IGL
  • IGK
  • B cell malignancies
  • translocation
  • FISH

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