Molecular cloning of immunoglobulin heavy chain (IGH) translocation breakpoints identifies genes of biological importance in the development of normal and malignant B cells. Long-distance inverse PCR (LDI-PCR) was first applied to amplification of IGH gene translocations targeted to the joining (IGHJ) regions. We report here successful amplification of the breakpoint of IGH translocations targeted to switch (IGHS) regions by LDI-PCR. To detect IGHS translocations, Southern blot assays using 5′ and 3′ switch probes were performed. Illegitimate Sμ rearrangements were amplified from the 5′ end (5′Sμ LDI-PCR) from the alternative derivative chromosome, and those of Sγ or Sα were amplified from the 3′ end (3′Sγ or 3′α LDI-PCR) from the derivative chromosome 14. Using a combination of these methods, we have succeeded in amplifying IGHS translocation breakpoints involving FGFR3/MMSET on 4p16, BCL6 on 3q27, MYC on 8q24, IRTA1 on 1q21 and PAX5 on 9p13 as well as BCL11A on 2p13 and CCND3 on 6p21. The combination of LDI-PCR for IGHJ and IGHS allows rapid molecular cloning of almost all IGH gene translocation breakpoints.
Chromosome translocations involving the immunoglobulin heavy chain (IGH) locus on chromosome 14q32.3 are seen in 40–50% of B-lymphoid malignancies, and in particular B-cell non-Hodgkin's lymphoma (B-NHL)1 and myeloma.2 IGH translocations are usually reciprocal and bring genes on other chromosomes into close apposition with the IGH locus, where their expression is deregulated due to the presence of potent B-cell-specific transcriptional enhancers within the IGH locus. Genes involved in IGH translocations play a pivotal role in cell growth, differentiation, apoptosis and signal transduction of both normal and malignant B lymphocytes.3 Many IGH translocation breakpoints have been molecularly cloned; however, several recurrent breakpoints remain to be identified.4 Molecular cloning of IGH translocation breakpoints reveals involvement either of genes of unknown biological functions5, 6 or an unsuspected oncogenic potential of known genes.7, 8, 9
The IGH locus comprises 51 functional variable (V) segments, 27 diverse (D) segments, six joining (J) segments and nine constant (C) segments starting from the telomeric end of 14q32.3 and spans 1.4 megabases (Mb).10 Each C segment, except δ, is preceded by a switch (S) segment that plays a role in isotype class switching. To generate a diversity of the immunoglobulin repertoire, somatic rearrangement of the IGH locus takes place during B-cell differentiation. This process includes VDJ recombination, somatic hypermutation and class switch recombination, all of which involve DNA double-strand cleavage and religation; errors in each process may result in chromosome translocation targeted in IGH locus.11
The IGH translocations commonly take place in either the joining (IGHJ) or the switch (IGHS) segments. We devised a simple and robust method for the rapid molecular cloning of chromosomal translocations targeted to the IGHJ segments: this detects virtually all translocations to the IGHJ segments.12 Since the IGHS regions are another main target of IGH translocation, we have established long-distance inverse PCR (LDI-PCR) for rapid molecular cloning of Sμ, Sγ and Sα translocation breakpoints.
Materials and methods
Cell lines and patient materials
KHM-11 is a multiple myeloma cell line.13 This cell line exhibits a t(4;14)(p16;q32) by FISH (data not shown). CTB-1 is a diffuse large B-cell lymphoma cell (DLBCL) line showing a t(14;22)(q32;q11) cytogenetically, and expresses IgG.14 Karpas 1718 is a cell line derived from the peripheral blood of a patient diagnosed with transformed splenic lymphoma with villous lymphocytes (SLVLs) (Dyer, in preparation). This cell line showed t(8;14)(q24;q32). Patient 1 was diagnosed as a primary gastric diffuse large B-cell lymphoma expressing IgA. This tumor showed t(1;14)(q21;q32), add(4q),−8,+19. Patient 2 was an aggressive B-NHL transformed to leukemic phase with t(1;14)(p22;q32) and a t(9;14)(p13;q32). The JH alleles were cloned previously by LDI-PCR and shown to represent VDJ and DJ recombinations.12
Southern blot assays for IGHS translocation
To detect IGHS translocations, Southern blot assays using series of 5′ and 3′ switch probes were performed.15 Rearranged bands on Southern blot lacking any comigrating band obtained with any other switch probes were designated ‘illegitimate’ rearrangements. The smallest illegitimate bands obtained with various enzyme digestions were amplified using LDI-PCR.
LDI-PCR for switch regions
LDI-PCR and sequence analysis were performed as in a previous report.12 To amplify Sμ, Sγ and Sα rearranged fragments, we designed primer sets at the 5′ flank region of Sμ and the 3′ flank region of Sγ and Sα (Table 1). The Sγ primers anneal to all Sγ regions and the Sα primers to both Sα regions. Based on the position of the primers, we termed these methods as 5′Sμ LDI-PCR, 3′Sγ LDI-PCR and 3′Sα LDI-PCR, respectively. 5′Sμ LDI-PCR amplifies breakpoint sequence on the derivative partner chromosome, whereas the 3′Sγ and 3′Sα LDI-PCRs amplify the breakpoint sequence on the derivative chromosome 14. Figure 1 illustrates the germline configuration of the IGHS segments, a functional class switch recombination and the two types of IGHS translocation. The functional class switch recombination generating isotype switched IG molecules involves two switch regions and deletes the intervening DNA sequence (eg, fusion of Sμ and Sγ with deletion of Cμ for IgG class switching as shown in Figure 1b). In contrast to the functional class switch process, IGHS translocation often involves only one switch region before such S recombination has occurred (Figure 1c).11 However, some IGHS translocations show similarity to functional class switching machinery (Figure 1d). In this case, the 5′Sμ LDI-PCR can amplify breakpoints on the derivative partner chromosome even though the translocation break on the derivative chromosome 14 occurs in Sγ or Sα. In addition, the 5′Sμ LDI-PCR will amplify functional class switch recombination events.
Results and discussion
The KHM-11 cell showed one 5′Sμ illegitimate rearranged band on Southern blot (Figure 2b). A 1.8 kb HindIII rearranged band was amplified as a 1.6 kb band by 5′Sμ LDI-PCR product and sequenced (Figure 2b). Sequence analysis showed Sμ fused to Sα1 region and the Sμ/α hybrid juxtaposed to MMSET (Multiple Myeloma SET domain) gene locus on 4p16.3 (Figure 2d).16 Thus, the t(4;14)(p16;32) arose from two recombination events. Firstly, an Sμ and Sα fusion occurred as in functional class switching for IgA protein synthesis and then the hybrid S region underwent translocation to chromosome 4p16. The 4p16 breakpoint on der(4) of this cell line fell in the first intron of MMSET gene and 1152 bp centromeric to a previous cloned breakpoint from a plasma cell leukemia patient, PCL-1 (Figure 2d).17
A DLBCL cell line, CTB-1, showed three rearranged bands on Southern blot using the 5′Sμ probe (Figure 2c). One of three rearranged bands comigrated with the 3′Sγ probe (data not shown) indicating a functional IgG switch recombination. Other rearranged bands were illegitimate rearrangements. 5′Sμ LDI-PCR succeeded in amplifying a 1.2 kb TaqI illegitimate rearrangement as a 1.0 kb product. The sequence of the PCR product showed fusion of the Sμ and the first intron of the BCL6 gene; the breakpoint sequence has been published elsewhere.18 Subsequent FISH analysis confirmed a complex three-way translocation involving 3q27, 14q32 and 8q24.18
A transformed SLVL cell line, Karpas 1718, showed one 3′Sγ illegitimate rearranged band. A rearranged TaqI fragment detected with the 3′Sγ probe was amplified using LDI-PCR (Figure 3b). Sequence analysis revealed Sγ 4 juxtaposed with the first exon of MYC on 8q24 (Figure 3d).
A gastric DLBCL (patient 1) with t(1;14)(q21;q32) showed a comigration band of Sμ and Sα by the Southern blot assay (data not shown). In addition to the productive switch recombination, one illegitimate 3′Sγ rearranged band was detected. A 2.0 kb SphI rearranged band was amplified by 3′Sγ LDI-PCR and sequenced (Figure 3c). The sequence showed Sγ3 region fused to novel immunoglobulin-like cell surface receptors (IRTAs: immune receptor translocation associated proteins;6, 19 FcRHs: Fc receptor homologs20) cluster region on 1q21, which has been recently cloned from an IGH translocation breakpoint from a myeloma cell line, FR4 (Figure 3e).6 We confirmed that the PCR product was not an artifact by Southern blot (data not shown). This case demonstrates that the IRTAs/FcRHs locus is a recurrent target of IGH translocation. The 1q21 breakpoint of case 1 lies at 96 bp upstream of exon 2 of IRTA1 and 2964 bp telomeric to the breakpoint of the FR4 cell line (Figure 3e). The t(1;14)(q21;q32) in FR4 generated an IRTA1/Cα1 chimeric mRNA and produced a fusion protein comprising the signal peptide and first two amino acids of IRTA1, and transmembrane and intracellular domains of Cα1. Based on this structure, it was suggested that the IRTA1/Cα1 fusion protein inappropriately activated the B-cell receptor (BCR) signaling pathway in a ligand-independent fashion to promote either proliferation or survival.6 The t(1;14)(q21;q32) in case 1 created a fusion of Sγ3 region and the first intron of IRTA1 so that the exon 1 of the IRTA1 gene on der(14) lay in the same transcription orientation of Cγ3. Thus, as suggested in the FR4 cell line, expression of the regulatory elements within IRTA may activate the BCR pathways. Another possible role may be to disrupt the normal function of IRTA1, because both breakpoints separate coding regions of IRTA1. Recent immunohistochemical studies have suggested that IRTA1 is expressed in a subset of marginal zone B cells.20 However, the functions of IRTA1 are unknown.
DNA from a patient with B-NHL in leukemic phase (patient 2) with t(1;14)(p22;q32) and t(9;14)(p21;q32) showed a 3′Sα illegitimate switch rearrangement on the Southern blot assay (Figure 4b). A rearranged 3′Sα HindIII fragment was amplified and sequenced. The non-IGH sequence mapped to a BAC clone (RP11-297B17, accession number AL161781) derived from chromosome 9p13. The 9p13 breakpoint fell only 75 bp telomeric to a previously cloned breakpoint from MZL-1 (Figure 4c).21 Three 9p13 breakpoints (case 2, MZL-1 and #105222) were clustered within the noncoding region of exon 1B of PAX5 gene, indicating that these three cases expressed alternatively spliced PAX5 gene. The t(9;14)(p13;q32) is associated with indolent B-NHLs such as immunoplasmacytoid lymphomas. The two IGH translocations in this case may have contributed to the aggressive clinical course. However, we have failed to clone the other IGH breakpoint, indicating that some breakpoints may not be amenable to the PCR approaches developed here. All the results obtained in this study are summarized in Table 2.
In conclusion, both LDI-PCR protocols for IGHJ and IGHS segments may allow molecular cloning of most IGH translocations from primary clinical material. Cloning of IGHJ translocations is straightforward and can be performed without recourse to Southern blot data; the method can be performed with only very small amounts (20 ng) of high-molecular-weight DNA. In contrast, IGHS translocations are more complex and it is therefore necessary to have large amounts of high-molecular-weight DNA available to allow comprehensive analysis before embarking on LDI-PCR to maximize chances of success. Apart from the translocations described here, we have also used similar methods to clone both BCL11A5 and cyclin D3 (CCND3)7 translocation breakpoints. We have not systematically examined both derivative chromosomes in any of the cases reported here in order to investigate possible mechanisms underlying the genesis of the translocations. However, it should be noted that a combination of 5′Sμ and 3′Sγ or 3′Sα PCR methods may allow both derivative chromosomes to be amplified.
Finally, some IGH breakpoints do not involve either JH or S region. For example, the t(11;14)(q13;q32) in the mantle cell lymphoma cell line NCEB1 falls within an apparently germline IGH locus.12 Breakpoints in cases of BCP-ALL with rare IGH translocations may fall within the Cδ–Cγ3 intron26 (data not shown). Such translocations are not amenable to the LDI-PCR approaches described here, but appear to be uncommon.
Tamura A, Miura I, Iida S, Yokota S, Horiike S, Nishida K et al. Interphase detection of immunoglobulin heavy chain gene translocations with specific oncogene loci in 173 patients with B-cell lymphoma. Cancer Genet Cytogenet 2001; 129: 1–9.
Bergsagel PL, Kuehl WM . Chromosome translocations in multiple myeloma. Oncogene 2001; 20: 5611–5622.
Willis TG, Dyer MJS . The role of immunoglobulin translocations in the pathogenesis of B-cell malignancies. Blood 2000; 96: 808–822.
Cigudosa JC, Parsa NZ, Louie DC, Filippa DA, Jhanwar SC, Johansson B et al. Cytogenetic analysis of 363 consecutively ascertained diffuse large B-cell lymphomas. Genes Chromosomes Cancer 1999; 25: 123–133.
Satterwhite E, Sonoki T, Willis TG, Harder L, Nowak R, Arriola EL et al. The BCL11 gene family: involvement of BCL11A in lymphoid malignancies. Blood 2001; 98: 3413–3420.
Hatzivassiliou G, Miller I, Takizawa J, Palanisamy N, Rao PH, Iida S et al. IRTA1 and IRTA2, novel immunoglobulin superfamily receptors expressed in B cells and involved in chromosome 1q21 abnormalities in B cell malignancy. Immunity 2001; 14: 277–289.
Sonoki T, Harder L, Horsman DE, Karran L, Taniguchi I, Willis TG et al. Cyclin D3 is a target gene of t(6;14)(p21.1;q32.3) of mature B-cell malignancies. Blood 2001; 98: 2837–2844.
Shaughnessy Jr J, Gabrea A, Qi Y, Brents L, Zhan F, Tian E et al. Cyclin D3 at 6p21 is dysregulated by recurrent chromosomal translocations to immunoglobulin loci in multiple myeloma. Blood 2001; 98: 217–223.
Kawamata N, Sakajiri S, Sugimoto KJ, Isobe Y, Kobayashi H, Oshimi K . A novel chromosomal translocation t(1;14)(q25;q32) in pre-B acute lymphoblastic leukemia involves the LIM homeodomain protein gene, Lhx4. Oncogene 2002; 21: 4983–4991.
Honjo T, Matsuta F . Immunoglobulin heavy chain loci of mouse and human. In: Honjo T, Alt FW (eds). Immunoglobulin Genes. London, UK: Academic Press, 1995, pp 145–171.
Kuppers R, Dalla-Favera R . Mechanisms of chromosomal translocations in B cell lymphomas. Oncogene 2001; 20: 5580–5594.
Willis TG, Jadayel DM, Coignet LJ, Abdul-Rauf M, Treleaven JG, Catovsky D et al. Rapid molecular cloning of rearrangements of the IGHJ locus using long-distance inverse polymerase chain reaction. Blood 1997; 90: 2456–2464.
Hata H, Matsuzaki H, Sonoki T, Takemoto S, Kuribayashi N, Nagasaki A et al. Establishment of a CD45-positive immature plasma cell line from an aggressive multiple myeloma with high serum lactate dehydrogenase. Leukemia 1994; 8: 1768–1773.
Uchida Y, Miyazawa K, Yaguchi M, Gotoh A, Iwase O, Ohyashiki K et al. Establishment of novel B-lymphoma cell line, CTB-1, with strong Fas antigen expression having chromosomal translocation t(14;22). Int J Oncol 1997; 10: 1103–1107.
Bergsagel PL, Chesi M, Nardini E, Brents LA, Kirby SL, Kuehl WM . Promiscuous translocations into immunoglobulin heavy chain switch regions in multiple myeloma. Proc Natl Acad Sci USA 1996; 93: 13931–13936.
Chesi M, Nardini E, Lim RS, Smith KD, Kuehl WM, Bergsagel PL . The t(4;14) translocation in myeloma dysregulates both FGFR3 and a novel gene, MMSET, resulting in IgH/MMSET hybrid transcripts. Blood 1998; 92: 3025–3034.
Chesi M, Nardini E, Brents LA, Schrock E, Ried T, Kuehl WM et al. Frequent translocation t(4;14)(p16.3;q32.3) in multiple myeloma is associated with increased expression and activating mutations of fibroblast growth factor receptor 3. Nat Genet 1997; 16: 260–264.
Sanchez-Izquierdo D, Siebert R, Harder L, Marugan I, Gozzetti A, Price HP et al. Detection of translocations affecting the BCL6 locus in B cell non-Hodgkin's lymphoma by interphase fluorescence in situ hybridization. Leukemia 2001; 15: 1475–1484.
Falini B, Tiacci E, Pucciarini A, Bigerna B, Kurth J, Hatzivassiliou G et al. Expression of the IRTA1 receptor identifies intraepithelial and subepithelial marginal zone B cells of the mucosa-associated lymphoid tissue (MALT). Blood 2003; 102: 3684–3692.
Davis RS, Wang YH, Kubagawa H, Cooper MD . Identification of a family of Fc receptor homologs with preferential B cell expression. Proc Natl Acad Sci USA 2001; 98: 9772–9777.
Morrison AM, Jager U, Chott A, Schebesta M, Haas OA, Busslinger M . Deregulated PAX-5 transcription from a translocated IgH promoter in marginal zone lymphoma. Blood 1998; 92: 3865–3878.
Iida S, Rao PH, Nallasivam P, Hibshoosh H, Butler M, Louie DC et al. The t(9;14)(p13;q32) chromosomal translocation associated with lymphoplasmacytoid lymphoma involves the PAX-5 gene. Blood 1996; 88: 4110–4117.
Hamada T, Yonetani N, Ueda C, Maesako Y, Akasaka H, Akasaka T et al. Expression of the PAX5/BSAP transcription factor in haematological tumour cells and further molecular characterization of the t(9;14)(p13;q32) translocation in B-cell non-Hodgkin's lymphoma. Br J Haematol 1998; 102: 691–700.
Busslinger M, Klix N, Pfeffer P, Graninger PG, Kozmik Z . Deregulation of PAX-5 by translocation of the Emu enhancer of the IgH locus adjacent to two alternative PAX-5 promoters in a diffuse large-cell lymphoma. Proc Natl Acad Sci USA 1996; 93: 6129–6134.
Pellet P, Berger R, Bernheim A, Brouet JC, Tsapis A . Molecular analysis of a t(9;14)(p11;q32) translocation occurring in a case of human alpha heavy chain disease. Oncogene 1989; 4: 653–657.
Dyer MJS, Heward JM, Zani VJ, Buccheri V, Catovsky D . Unusual deletions within the immunoglobulin heavy-chain locus in acute leukemias. Blood 1993; 82: 865–871.
This work was supported by Lady Tata Foundation, The Daiwa Anglo-Japanese Foundation, Deutsche Krebshilfe and The Human Resources Support Foundation under Kumamoto City Municipal Centennial Commemorative Project. We thank Dr Abraham Karpas (University of Cambridge, UK) for kindly providing the Karpas 1718 cell line.
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Sonoki, T., Willis, T., Oscier, D. et al. Rapid amplification of immunoglobulin heavy chain switch (IGHS) translocation breakpoints using long-distance inverse PCR. Leukemia 18, 2026–2031 (2004). https://doi.org/10.1038/sj.leu.2403500
- IGH switch region