HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Langerak, A. W. Articles by van Dongen, J. J. M. Search for Related Content PubMed PubMed Citation Articles by Langerak, A. W. Articles by van Dongen, J. J. M.

Unraveling the Consecutive Recombination Events in the Human IGK Locus¹

Anton W. Langerak^2,*, Bertrand Nadel, Anneke de Torbal^*, Ingrid L. M. Wolvers-Tettero^*, Ellen J. van Gastel-Mol^*, Brenda Verhaaf^*, Ulrich Jäger and Jacques J. M. van Dongen^*

^* Department of Immunology, Erasmus MC, University Medical Center Rotterdam, Rotterdam, The Netherlands; and Department of Internal Medicine I, Division of Hematology, University of Vienna, Vienna, Austria

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
In addition to the classical V-J, V- deleting element (Kde), and intron-Kde gene rearrangements, atypical recombinations involving J recombination signal sequence (RSS) or intronRSS elements can occur in the Ig (IGK) locus, as observed in human B cell malignancies. In-depth analysis revealed that atypical JRSS-intronRSS, V-intronRSS, and JRSS-Kde recombinations not only occur in B cell malignancies, but rather reflect physiological gene rearrangements present in normal human B cells as well. Excision circle analysis and recombination substrate assays can discriminate between single-step vs multistep rearrangements. Using this combined approach, we unraveled that the atypical V-intronRSS and JRSS-Kde pseudohybrid joints most probably result from ongoing recombination following an initial aberrant JRSS-intronRSS signal joint formation. Based on our observations in normal and malignant human B cells, a model is presented to describe the sequential (classical and atypical) recombination events in the human IGK locus and their estimated relative frequencies (0.2–1.0 vs <0.03). The initial JRSS-intronRSS signal joint formation (except for J1RSS-intronRSS) might be a side event of an active V(D)J recombination mechanism, but the subsequent formation of V-intronRSS and JRSS-Kde pseudohybrid joints can represent an alternative pathway for IGK allele inactivation and allelic exclusion, in addition to classical C deletions. Although usage of this alternative pathway is limited, it seems essential for inactivation of those IGK alleles that have undergone initial aberrant recombinations, which might otherwise hamper selection of functional Ig L chain proteins.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
To form a large variety of unique Ag-recognizing Ig molecules, human B cells undergo recombination between variable (V), diversity (D), and joining (J) gene segments of the Ig genes. The resulting V(D)J exons encode the variable domains of Ig chains. In the bone marrow, VDJ recombination of Ig H chain (IGH)³ genes starts at the progenitor B cell stage, followed by VJ recombination in Ig (IGK) or Ig (IGL) L chain loci during the small precursor B cell or precursor B-II cell stage (1, 2). Pairing of Ig H and Ig or Ig -chains allows further differentiation into immature and subsequently mature surface membrane Ig- or Ig-positive B cells.

Previous studies have shown that most Ig-positive B cells retain their IGL genes in germline configuration (3, 4). In contrast, the vast majority of Ig-positive B cells have one or two rearranged IGK alleles in addition to their rearranged IGL allele(s), in line with hierarchical Ig L chain recombination (4, 5). In Ig-positive B cells, the IGK rearrangements mainly concern deletional rearrangements involving the -deleting element (Kde), which is positioned downstream of the constant (C) gene segment (6) (Fig. 1). Kde can either recombine to a V gene segment upstream of a previously formed V-J rearrangement or to an isolated heptamer recombination signal sequence (RSS) in the intron between the J gene segments and the C exon (intronRSS heptamer) (6) (Fig. 1). Recombination to the intronRSS heptamer results in the deletion of C and the IGK intronic enhancer (Ei), whereas rearrangement to one of the V gene segments deletes the entire J-C area; both types of recombinations prevent expression of the IGK allele, and it is believed that such events participate in the regulation of allelic and isotypic exclusion.

View larger version (19K): [in this window] [in a new window]   FIGURE 1. Schematic overview of classical rearrangements in the human IGK locus. IGK recombination mostly starts with a V-J rearrangement. The functionality of this rearrangement can be disrupted by rearrangement of the Kde. Recombination of Kde to an isolated heptamer in the intron between the J and C segments (intron-Kde rearrangement) results in deletion of the C region, whereas recombination between Kde and a V gene segment deletes the entire (V)J-C region. Both types of Kde rearrangements also result in deletion of the IGK enhancers (iE and 3'E), probably precluding further rearrangements in the human IGK locus.

Based on extensive Southern blot and PCR screening, we identified a series of human B cell malignancies with uncommon IGK locus hybridization patterns and/or extended PCR product sizes, suspicious of the presence of the before mentioned atypical IGK gene rearrangements. These malignancies constitute single-cell model systems that enable the study of atypical IGK recombinations in full detail, i.e., within the context of the entire IGK/IGL L chain gene configuration. More importantly, we also analyzed and quantified the occurrence of these IGK recombinations in normal human peripheral blood and tonsillar B lymphocytes. The analysis of intermediate circular excision products, as well as functional data obtained in recombination substrate assays, allowed us to investigate whether specific rearrangements are formed via single-step or multistep rearrangements. Based on these data, we now propose a comprehensive and integrated model of the step-wise consecutive recombinations in the human IGK locus, including the here-described atypical IGK recombinations. The implications of these recombination pathways on Ig protein expression and allelic exclusion of human IGK genes are discussed.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Cell samples

Several leukemia/lymphoma cell samples were selected on the basis of an initial suspicion of atypically rearranged IGK genes, as deduced from initial Southern blot hybridization patterns and/or extended PCR product sizes (10). These included the precursor B cell lines 380 and RCH-ACV, bone marrow (BM), or lymph node samples from precursor B cell acute lymphoblastic leukemia (precursor B-ALL; n = 5), B cell non-Hodgkin’s lymphoma (n = 4), B cell chronic lymphocytic leukemia (B-CLL; n = 2), one unclassified B cell proliferation, and an acute myeloid leukemia (AML-M5) showing illegitimate atypical IGK recombination. BM mononuclear cells (MNC) were isolated by Ficoll-Paque (density, 1.077 g/ml; Amersham Biosciences, Buckinghamshire, U.K.) density centrifugation. BM-MNC fractions and lymph node suspensions were subsequently used for DNA and RNA isolation. Tonsils, peripheral blood (PB)-MNC, and (regenerating) BM-MNC from healthy controls were included as a source of normal B lymphocytes.

DNA isolation and Southern blot analysis

DNA isolation and Southern blot analysis was performed as previously described (11). In short, 15–20 µg genomic DNA was digested with restriction enzymes, separated in 0.7% agarose gels, and vacuum blotted. The configuration of the IGK locus was determined using BglII and BamHI/HindIII digests and ³²P-labeled probes specific for the areas upstream (IGKJU) or downstream (IGKJ5) of the J segments, or specific for the C (IGKC) and the Kde (IGKDE) regions (10). IGL rearrangements were studied in EcoRI/HindIII digests using a general IGL probe (IGLC3) or probes specific for the J1-C1 (IGLC1D) or J2-C2 and J3-C3 (IGLJ2) areas in combination with BglII digests (12, 13).

Primer design

To detect recombinations between JRSS elements and the intronRSS heptamer, new primer sets were designed (Fig. 2, and Table I). Using the germline IGK sequence (accession no. X67858) and OLIGO 6.2 software, a J consensus primer (J2-5-F1-EMC) was designed to detect rearrangements between J2RSS, J3RSS, J4RSS, or J5RSS and the intronRSS. The J2-5-F1-EMC primer recognizes a homologous sequence in each J gene segment upstream of the JRSS that rearranges to intronRSS. To detect J1RSS rearrangements a separate primer was designed that recognizes a sequence upstream of J1 (J1-F1-EMC). A primer downstream of intronRSS (intron-R1-EMC) was chosen as reverse primer. JRSS-Kde recombinations were analyzed using the J1-F1-EMC and J2-5-F1-EMC primers together with a Kde primer. For detection of V-intronRSS rearrangements, V family primers as well as a V consensus primer were used in combination with the earlier mentioned intron-R1-EMC primer (Fig. 2, and Table I). Inversional V-intronRSS rearrangements were analyzed using the same V family primers, in combination with a primer recognizing a sequence upstream of intronRSS (intronRSS primer) (Fig. 2, and Table I).

View larger version (15K): [in this window] [in a new window]   FIGURE 2. Schematic diagram of primer sets for detection of atypical IGK gene rearrangements such as JRSS-intronRSS, V-intronRSS and JRSS-Kde rearrangements. Approximate position of the specially designed primers is indicated; the sequences of the primers are given in Table I. Classical IGK rearrangements as well as atypical JRSS-intronRSS, V-intronRSS, and JRSS-Kde rearrangements are shown schematically.

PCR analysis and sequencing

PCR amplification of JRSS-intronRSS, V-intronRSS, and JRSS-Kde recombinations, as well as of inversional V-intronRSS rearrangements and J-intronRSS circular excision products was performed using the relevant primer sets (see Primer design and Table I). A 50-µl reaction volume contained 50 ng genomic DNA, 1.5 mM MgCl₂ (Applied Biosystems, Foster City, CA), 0.2 mM dNTP (Amersham Biosciences), 6.25 pmol of each primer, and 1 U of AmpliTaq Gold DNA polymerase in buffer II (Applied Biosystems). The PCR consisted of 10 min preactivation at 94°C, followed by 40 cycles of 45 s denaturation at 94°C, 90 s annealing at 60°C, and 2 min extension at 72°C, followed by 10 min final extension at 72°C. PCR products were electrophoresed on a 1% agarose gel and visualized with ethidium bromide.

After amplification, the PCR products were further analyzed by heteroduplex analysis to determine the monoclonal or polyclonal character (15). In short, following denaturation at 94°C for 5 min and reannealing at 4°C for 1 h, the PCR products were separated in 6% polyacrylamide gels in 0.5x Tris-boric acid-EDTA buffer.

PCR products derived from monoclonal rearrangements were directly sequenced. To sequence the polyclonal recombinations from tonsil and MNC samples from healthy controls, the PCR products were first cloned, using the pGEM-T Easy vector kit according to the manufacturer’s instructions (Promega, Madison, WI). After transformation to competent cells, positive colonies were grown and plasmid DNA was isolated for further sequencing.

Sequencing was performed on the ABI 377 fluorescent sequencer (Applied Biosystems), using the dye terminator cycle sequencing kit and AmpliTaqFS DNA polymerase (Applied Biosystems). Sequencing primers were identical with those used for PCR amplification. Sequencing was performed using either 60 ng of monoclonal PCR product and 3.2 pmol primer, or 500 ng of plasmid DNA with cloned PCR products from healthy controls and 6 pmol primer, in combination with 5 µl of dye terminator mix. The cycling protocol consisted of 25 cycles of 30 s, 96°C, followed by 4 min, 60°C.

Analysis of the obtained sequences was performed using the germline IGK genomic sequence (accession no. X67858). V and J gene segments were identified using DNAPLOT software (W. Müller and H.-H. Althaus, University of Cologne, Cologne, Germany) by searching for homology with all known human germline IGK sequences obtained from the VBASE directory of human Ig genes (http://www.mrc-cpe.cam.ac.uk/imt-doc/) and/or ImmunoGenetics (IMGT) (http://imgt.cines.fr:8104).

Real-time quantitative PCR (RQ-PCR) analysis of different types of IGK gene recombinations

RQ-PCR of different IGK recombinations was essentially performed as described previously using ABI Prism 7700 equipment (Applied Biosystems) (16). As clonal control DNA for the analyzed IGK recombinations, the following sources with high percentages (90–100%) of clonal cells were selected: cell line U698 (V1-J1), a B-CLL sample (V1-J4), cell line ROS15 (V1-Kde), cell line Nalm1 (intron-Kde), B-CLL sample 91-062 (J1RSS-intronRSS), ALL sample 5381 (J4RSS-intronRSS), cell line RCH-ACV (V1-intronRSS), and AML sample 95-058 (J4RSS-Kde); no clonal control DNA was available for J1RSS-Kde. The applied forward/reverse primers and TaqMan probes are listed in Table I.

All control cell lines and leukemic DNA samples were serially diluted in HeLa DNA as nontemplate control. A standard albumin RQ-PCR was performed to normalize the amount of input DNA of the nondiluted control DNA and DNA from tonsil, PB-MNC, and (regenerating) BM-MNC using serially diluted reference buffy DNA. In a typical 25-µl RQ-PCR, 5 µl of genomic DNA (20 ng/µl), 12.5 µl of 2x master mix, 22.5 pmol specific forward primer, 22.5 pmol specific reverse primer, and 2.5 pmol specific TaqMan probe were used. In two independent experiments, reactions were always performed in duplo. The protocol consisted of 2 min at 50°C and 10 min at 95°C, followed by 50 cycles of 15 s at 95°C and 1 min at 60°C. Ten-fold serial dilutions of each clonal control DNA (500–0.5 ng of template DNA) were used to make a standard curve for that particular type of IGK recombination RQ-PCR. C_T values of tonsil, PB-MNC, and BM-MNC DNA for a particular IGK RQ-PCR were subsequently plotted on the respective standard curve, and expressed as percentage recombination relative to the respective clonal control DNA.

Recombination substrate assay

The recombination substrate assay was essentially performed as described previously (17). In short, recombination plasmids with the chloramphenicol acetyltransferase (CAT) gene under control of the Ptac promoter were used; in these constructs, CAT expression is prevented by a termination signal (OOP) upstream of the CAT coding sequence (see also Fig. 5 for further explanation). In eukaryotic cells, recombination between sequences in the upstream and downstream cassettes, which flank the OOP termination signal, results in removal of the latter, thereby giving rise to CAT expression when transformed in Escherichia coli. Cassettes for analysis of human IGK RSS-like elements (see also Fig. 5) were obtained by PCR amplification from genomic DNA, using specific primers with specific restriction sites (Mlul and SalI or NotI for the upstream cassette; Spel and BglII for the downstream cassette) for easy cloning into the recombination backbone vector: (Mlul)-VA2/2D-29-(Notl)-VA27/3-20-(SalI)//(Spel)-J1-(BglII) (17). Primers used for constructing the various cassettes are listed in Table I. Cloning of the intron RSS motif cassette was performed as follows: PCR amplification on DNA from cells with germline IGK genes using Ki-1A (SpeI) and Ki-2B (BgIII) primers, subcloning in pPCR-Script AmpSK(+) (Stratagene, La Jolla, CA) in the correct orientation, and finally digestion with Notl/SalI and further cloning using Notl and SalI restriction sites into the appropriate recombination substrates. The Kde cassette was produced by PCR amplification of germline IGK DNA using CKde (SpeI) and Kde-A (BglII) primers, and subsequently cloned via Spel and BgIll restriction sites into the appropriate recombination substrates. Finally, to produce the signal joint cassettes, the J1RSS-intronRSS and J3RSS-intronRSS signal joints were PCR amplified from leukemic cells harboring these elements using primers J1-F1-EMC or J2-5-F1-EMC together with intron-R1-EMC. PCR products were subcloned in pPCR-Script AmpSK(+), and taken out BssHI/NotI. Because BssHI has a compatible overhang with Mlul, the fragment could directly be cloned as an Mlul/NotI cassette into the appropriate recombination substrates.

View larger version (42K): [in this window] [in a new window]   FIGURE 5. Recombination substrate assay. Sequences of clones obtained in recombination substrate assay, using different constructs (for a description see also Materials and Methods). A, Configurations of signal joints and coding joints formed upon recombination between the J1 gene segment and the intronRSS heptamer. Perfect JRSS-intronRSS signal joints are formed, whereas the coding joints show a variable level of deletion and insertion of nucleotides. B, Configurations of pseudohybrid joints formed upon recombination between VA2/2D-29 or VA27/3–20 and the J1RSS-intronRSS signal joint, showing both deletion and insertion of nucleotides. C, Configurations of pseudohybrid joints formed upon recombination between the J3RSS-intronRSS signal joint and Kde, with evidence for deletion and insertion of nucleotides.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Atypical IGK recombinations detected in human B-lineage lymphoproliferations

In the process of screening human B cell lymphoproliferations by IGK Southern blot and PCR analysis, we have come across cases with IGK recombinations other than the classical V-J, V-Kde, and/or intron-Kde joinings (Fig. 1). These atypical IGK recombinations were further studied by Southern blot analysis using four different probes to determine the exact configuration of both IGK alleles (Fig. 3, and Table II).

View larger version (14K): [in this window] [in a new window]   FIGURE 3. Southern blot analysis of human IGK gene rearrangements. A, Restriction map of part of the human IGK locus. The location of relevant BglII (Bg), BamHI (B), EcoRI (E), and HindIII (H) restriction sites as well as the IGKJU, IGKJ5, IGKC, and IGKDE Southern blot probes are indicated. B, Example of interpretation of hybridization patterns of the complete set of IGK Southern blot probes in combination with BglII restriction digests.

Because deletional rearrangements in the IGK locus mostly coincide with IGL gene rearrangements, we also checked this coincidental occurrence for atypical IGK recombinations. In 8 of the 13 cases, both IGL alleles were in germline configuration. Of the five cases showing IGL rearrangements (Table II), three contained at least one classical Kde recombination; in the other two (4511 and 5301), V-intronRSS rearrangements were observed. However, because other cases (among others, case 95-074 and cell line RCH-ACV) displayed isolated V-intronRSS recombinations without V-J rearrangements, a complete association between atypical IGK recombinations and the start of IGL gene recombination seems unlikely.

Detailed characterization of the detected atypical IGK recombinations

The vast majority of Southern blot deduced atypical IGK rearrangements could be confirmed via PCR heteroduplex analysis and sequencing (Fig. 2, and Table II). The two JRSS-intronRSS recombinations (91-062 and F-7) both involved J1RSS. Of the many samples with presumed V-intronRSS recombinations, only two indeed contained direct V-intronRSS couplings (95-074 and cell line RCH-ACV). In six others (4882, 94-101, 95-060, G-10, 4511, and 5301), the unusually long V-intronRSS PCR products cases revealed a V-J and JRSS-intronRSS configuration. PCR and sequencing in sample 5381 further showed an inversional V2D-28-J3 rearrangement upstream of a J4RSS-intronRSS coupling. Finally, in four other samples (92-051, 5712, 95-058, and cell line 380) the unusually long V-Kde PCR products appeared to represent V-J and JRSS-Kde rearrangements rather than direct V-Kde couplings.

Remarkably, in most cases with V-J and JRSS-intronRSS or V-J and JRSS-Kde configurations neighboring J segments were involved in the two couplings. Exceptions concerned 4511 and 5301 with V-J1 and V-J2, respectively, upstream of J4RSS-intronRSS. All J (except J5) gene segments were found to be involved in the atypical IGK recombinations. V segments in the upstream V-J couplings were derived from the three large V families (V1, V2, V3) and even concerned the most J proximal V4-1 segment. The few cases with direct V-intronRSS couplings all concerned V1 segments.

Detailed junctional region analysis revealed a signal joint configuration with two perfectly joined RSS elements in only four of the nine JRSS-intronRSS atypical recombinations. The other five showed processed signal joints, i.e., nucleotide deletion from one or both RSS elements and even nucleotide insertion. Such a configuration is remarkable in view of the orientation of the JRSS and intronRSS elements in the germline configuration, which in principle should lead to perfect signal joints. Coding jointlike configurations with deleted and inserted nucleotides were observed in all V-intronRSS and JRSS-Kde recombinations. Given their hybrid jointlike configuration, these were termed pseudohybrid joints. Analysis of V-J junctions upstream of JRSS-intronRSS rearrangements revealed out-of-frame V-J fusions in five samples (94-101, G-10, 4511, 5301, and 5381), but in-frame alleles in two others (4882 and 95-060). Although no membrane Ig expression was observed (precursor B cell leukemia), in-frame V2-30-J1-C transcripts were found to be present in case 4882 (data not shown). In 95-060, membrane Ig protein expression was observed that could only result from the V1-39-J2 and J3RSS-intronRSS allele, because the other allele contained a deletional rearrangement. Hence, the presence of a JRSS-intronRSS recombination (except for J1RSS-intronRSS) does not seem to block expression of an in-frame upstream V-J rearrangement.

Occurrence of atypical IGK recombinations in normal human B cells

To exclude that the atypical IGK rearrangements are only formed aberrantly in leukemic cells or (virally) transformed cells, tonsillar DNA from a healthy individual was analyzed. Using the same IGK primers as for the clonal cell samples, the tonsillar DNA was amplified and per type of rearrangement 5–15 cloned PCR products were sequenced (Table III). V-intronRSS and JRSS-intronRSS sequences both could readily be amplified. Many of the V-intronRSS recombinations concerned couplings with heterogeneous V segment usage and showed pseudohybrid joints with a comparable extent of nucleotide deletion and insertion as seen in the clonal cell samples. Part of the seemingly V-intronRSS recombinations in tonsillar DNA actually concerned V-J and JRSS-intronRSS configurations with heterogeneous V family and J gene segment usage. Interestingly, in contrast to the clonal cell samples, the vast majority (11/13 sequences) of tonsillar JRSS-intronRSS rearrangements displayed perfect signal joints. Finally, heterogeneous (i.e., diverse with deletion and insertion of nucleotides) JRSS-Kde recombinations could also be identified in tonsillar B cells, though the J usage seemed to be less diverse in the small number of sequences analyzed in detail. Taken together, these data suggest that the atypical IGK recombinations that we initially identified in transformed cell samples do represent physiological events given their occurrence in healthy control tonsillar B cells.

RQ-PCR was applied to quantify the different types of IGK recombinations in (regenerating) BM-MNC, PB-MNC, and tonsil. For this purpose V1, J1(RSS), J4(RSS), Kde, and the intronRSS heptamer were chosen as representative elements involved in the various rearrangements. Following normalization of input DNA relative to an albumin standard curve, individual RQ-PCR were performed for these selected IGK recombination types. Using standard curves based on 10-fold serial dilutions of clonal control DNA samples, levels in tonsil, PB-MNC, and (regenerating) BM-MNC DNA were expressed as percentage recombination relative to the respective clonal control DNA. It is important to note that the various IGK recombination levels as measured in the normal tissues reflect relative and no absolute values, which formally cannot be used for direct comparisons between different types of recombinations in the human IGK locus. With this in mind, we nevertheless tried to roughly compare the frequencies of these different IGK recombinations (Table IV). In tonsil, intron-Kde rearrangements were most abundant and were arbitrarily set to 1.0; intron-Kde levels in PB and BM, being roughly 5- and 30-fold lower, respectively, than those observed in tonsil, were also set to 1.0. V1-J1, V1-J4, and V1-Kde rearrangements appeared to be relatively frequent, with levels roughly 0.20–0.45 relative tothose of intron-Kde. Atypical J1RSS-intronRSS and J4RSS-intronRSS rearrangements could also be found in tonsil, PB, and BM, although the levels appeared to be much lower than those of the classical IGK rearrangements, ranging from (almost) undetectable to roughly 0.06 (as compared with intron-Kde levels) to maximally 0.1 (as compared with V-J and V-Kde levels). Finally, recombination levels of the V1-intronRSS and J4RSS-Kde rearrangements were of the same order of magnitude as JRSS-intronRSS. Due to the lack of a clonal control DNA sample, J1RSS-Kde rearrangement levels could not be quantified; however, the C_T values were not that different from those obtained for J4RSS-Kde rearrangements, suggesting a similar frequency for both rearrangements. Taken together, these data further confirm that atypical IGK rearrangements do indeed occur in BM, PB, and tonsillar B cells, albeit at frequencies of 0.06 to 0.1 relative to the most abundant IGK gene rearrangements in these cells.

Next, we addressed the exact mechanism by which these atypical IGK recombinations are formed via detection of excision circles, which are formed during the rearrangement process. Because excision circles are not replicated upon further cell division, it is impossible to study these in clonally transformed cell samples. For that reason, we restricted our excision circle studies to tonsil DNA. Because virtually all JRSS-intronRSS recombinations in tonsil DNA concerned perfect signal joints (Table III), it seemed logical to assume that excision circles would contain coding joints involving the J gene segment and the region upstream of the intronRSS heptamer. Using a J consensus primer as reverse primer in combination with a primer upstream of intronRSS as forward primer, PCR products can only be formed through amplification of such excision circles (Fig. 4A). Indeed, in tonsillar DNA, such J-intron upstream PCR products could be identified (Fig. 4B, and Table III), strongly suggesting that JRSS-intronRSS coupling occurs via direct recombination, resulting in a signal joint on the chromosome and a coding joint on the circular excision product.

View larger version (63K): [in this window] [in a new window]   FIGURE 4. Atypical JRSS-intronRSS recombination. A, Schematic drawing of a JRSS-intronRSS signal joint as well as the parallel excision circle with the coding joint. Indicated are the primers that can be used for amplification of these signal and coding joints: 1, J1-F1-EMC or J2-5-F1-EMC; 2, intron-R1-EMC; 3, J1-4; and 4, intronRSS (see also Fig. 2 and Table I). B, Agarose gel electrophoresis of PCR-amplified JRSS-intronRSS signal joints and parallel coding joints with the primers as described in A.

Unraveling the mechanism of atypical IGK recombination via recombination substrate assay

To unravel the exact mechanism of V-intronRSS and JRSS-Kde recombinations, an in vitro recombination substrate assay was used. IGK RSS elements of interest together with their flanking sequences were cloned as upstream and downstream cassettes in a vector that contains an intermediate stop element for blocking CAT gene expression. Upon transfection in a precursor B cell line, proper recombination between RSS elements in the upstream and downstream cassettes results in removal of the stop element, thereby enabling CAT expression. Sequencing of colonies, selectively grown on chloramphenicol, allows determining the exact recombination breakpoints.

We first analyzed a construct in which the J1 gene segment with its RSS was cloned in one cassette and the intronRSS heptamer with flanking DNA in the other (Fig. 5A). Sequence analysis revealed perfect signal joints between the J1RSS and intronRSS elements, whereas analysis of the excision products showed fusion of the J1 coding sequence and the upstream intron area with deletion as well as insertion of P and N nucleotides (Fig. 5A). This result nicely confirmed the observations from our excision circle analyses. We subsequently cloned the J1RSS-intronRSS signal joint and flanking DNA from one of the leukemic cell samples, and tested it for its ability to recombine with the RSS of the frequently used VA2/2D-29 and VA27/3-20 gene segments (Fig. 5B), which was assumed to be the ongoing recombination step leading to V-intronRSS pseudohybrid joint formation. Of the many colonies obtained in the assay, all 10 sequenced showed that V(D)J recombination between V and the J1RSS-intronRSS signal joint indeed occurs. Moreover, the sequences resembled the V-intronRSS pseudohybrid joints as obtained from tonsil DNA, being diverse with deletion of nucleotides at both the V and the signal joint sides as well as insertion of occasional N nucleotides and formation of P nucleotides (Fig. 5B). Finally, to test the functional properties of JRSS-intronRSS signal joints in the other direction, we made a construct in which a J3RSS-intronRSS signal joint was tested against the KdeRSS (Fig. 5C). Similarly, all sequenced constructs showed pseudohybrid joints. In all cases, J3RSS was coupled to the Kde sequence, with diversity of deletion and insertion of both N and P nucleotides (Fig. 5C). Collectively, these results show that recombination between JRSS and intronRSS leads to signal joint formation and that the composition and position of this signal joint is such that it can be involved in secondary recombination to either one of the V gene segments or Kde, finally giving rise to V-intronRSS and JRSS-Kde pseudohybrid joints.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
The human IGK locus contains several RSS elements in addition to the RSS flanking V and J gene segments, allowing other rearrangements than the common V-J rearrangements to occur. Firstly, the presence of a 23-bp RSS flanking the Kde element at the very 3' end of the locus provides an alternative partner for the 12-bp VRSS elements, resulting in V-Kde rearrangements. Secondly, the Kde element can also rearrange to an isolated heptamer in the J-C intron (intronRSS), resulting in intron-Kde rearrangements (Fig. 1). Thirdly, additional nonclassical recombinations involving VRSS, JRSS, intronRSS, and KdeRSS elements have been described by Feddersen et al. and Seriu et al. (7, 8, 9). Several of these rearrangements cannot be formed directly, either because of the inverted positions of their respective RSS (JRSS and KdeRSS, and V and intronRSS) or because their RSS spacer lengths do not obey the 12/23 rule (J and Kde both contain RSS with 23-bp spacers). In this study, we describe the detailed characterization of three atypical JRSS-intronRSS, V-intronRSS, and JRSS-Kde recombinations in human B cells and show how they fit in a scheme of sequential IGK recombinations.

Our data show that all three types of atypical rearrangements occur in B cell malignancies as well as in normal human tonsillar B cells. Semiquantitative analysis revealed that the classical V-J, V-Kde, and intronRSS-Kde rearrangements are relatively frequent events, and that JRSS-intronRSS and V-intronRSS and JRSS-Kde rearrangements are less predominant (<5% of intron-Kde rearrangement levels, and 10% of the V-J and V-Kde rearrangement levels) (Table IV). Based on this quantitation as well as the data from excision circle analysis and recombination substrate assays, a comprehensive and integrated model is proposed for the consecutive recombination events as they can occur in the IGK locus (Fig. 6). In this model, initial V-J recombination occurs once or multiple times until an in-frame combination is formed. If no in-frame rearrangement is obtained, two major inactivation pathways are available: 1) intron-Kde (C deletion); or 2) V-Kde (J-C deletion) recombination. Alternatively, in a minor pathway, JRSS-intronRSS signal joints might be formed. Perfect JRSS-intronRSS signal joints (i.e., without deleted nucleotides) can undergo subsequent rearrangement, either to an upstream RSS element of a V segment (V-intronRSS pseudohybrid joints) or to the downstream RSS of the Kde (JRSS-Kde pseudohybrid joints). Finally, by analogy to intronRSS-Kde formation, ongoing recombination from V-intronRSS rearrangements can theoretically result in V-Kde recombinations, unless the intronRSS became damaged during the V to JRSS-intronRSS rearrangement. As this often proved to be the case (see V-intronRSS pseudohybrid joint sequences in Table III), this recombination step is depicted with dotted lines in Fig. 6. We consider the possibility of ongoing recombination of JRSS-Kde pseudohybrid joints into V-Kde rearrangements not very likely either (dotted lines), because JRSS-Kde couplings delete both enhancers (iE and 3'E) that are known to be important for IGK recombination (21, 22, 23). Although V-Kde rearrangements are very prominent, they thus probably are not end-stage rearrangements in each IGK inactivation pathway (see Fig. 6).

View larger version (20K): [in this window] [in a new window]   FIGURE 6. Tentative model describing the consecutive recombination events as they can occur in the human IGK locus. Starting from a germline allele, most recombination events concern V-J rearrangements, which in case no functional IGK allele can be expressed might be replaced by secondary V-J rearrangements, as long as upstream V and downstream J gene segments are available for recombination. Major consecutive rearrangements (thick lines) in the IGK locus are the classical Kde rearrangements: intron-Kde rearrangements downstream of a preserved V-J recombination, or V-Kde rearrangements that loop out a pre-existing V-J coupling. Apart from these major classical rearrangements, also less frequent atypical rearrangements can occur. Recombination between any of the J segments and the intronRSS results in JRSS-intronRSS signal joints, which, except for the case that J1RSS is involved, will be preceded by a V-J coupling. This signal joint, as shown in this study, can further recombine in two different ways: 1) recombination between a V segment and a perfect JRSS-intronRSS signal joint results in a V-intronRSS coupling; and 2) recombination between Kde and a perfect JRSS-intronRSS signal joint results in a JRSS-Kde coupling. Finally, as long as the intronRSS of the V-intronRSS pseudohybrid joint is still intact, further recombination to the RSS of Kde, leading to a V-Kde coupling, might occur; however, because in many cases the intronRSS is actually damaged (see also sequences in Table III), this will often prevent further recombination, leaving the V-intronRSS pseudohybrid joint as end-stage configuration. One might argue that in a similar way the undamaged JRSS of a JRSS-Kde coupling might still rearrange to the RSS of an upstream V segment (as long as present); however, loss of both IGK enhancers in the (V-J) + JRSS-Kde configuration implies that such a V-Kde coupling most probably cannot be formed anymore, leaving the JRSS-Kde pseudohybrid joints as end-stage configuration. For this reason, these latter recombination steps are presented as dotted lines.

An important issue concerns the functional implications of the atypical IGK recombinations. When J1RSS, being the most upstream JRSS, is recombined to the intronRSS heptamer, V-J joints can no longer occur and hence no functional Ig expression is possible from that allele. In that sense, J1RSS-intronRSS recombinations function as deletional rearrangements. The same holds for V-intronRSS and JRSS-Kde rearrangements, which both most probably occur as secondary events following initial JRSS-intronRSS signal joint formation. The lack of intact J gene segments prohibits production of potentially functional V-J recombinations in cis in both cases. However, the functional implications are much less clear for V-J and J2-5RSS-intronRSS recombinations. In theory, splicing of an upstream in-frame V-J exon to the C exon could result in Ig expression in such a configuration, unless the loss of important regulatory elements in the region between the J segments and the intronRSS heptamer would prevent this. So far, human and mouse IGK enhancer sequences (iE) have only been identified in the 3' part of the intron downstream of the intronRSS heptamer and not in the 5' intron part (21, 22, 23). This is supported by our observation that JRSS-intronRSS atypical joints did not block V-J-C transcription and/or translation. Apparently, the initial occurrence of JRSS-intronRSS signal joints (J1RSS-intronRSS excluded) might be a simple side event of an active V(D)J recombinase system, but ongoing recombination to V-intronRSS and JRSS-Kde pseudohybrid joints leads to IGK allele inactivation.

In summary, in this study, we show that in addition to the dominant classical rearrangements in the human IGK locus (V-J, intron-Kde and V-Kde), atypical IGK recombinations (JRSS-intronRSS, V-intronRSS, and JRSS-Kde) also occur, albeit at frequencies of <5% of intron-Kde and 10% of V-J and V-Kde (Table IV). As illustrated in the model (Fig. 6), initial V-J couplings can be followed by downstream JRSS-intronRSS recombinations on the same allele. Because we could not identify functional implications of JRSS-intronRSS couplings, we believe that such recombinations should be considered as occasional intermediates of an active V(D)J recombinase; J1RSS-intronRSS recombinations are exceptional in that they do not allow functional V-J recombination and expression. Remarkably, in tonsils, the JRSS-intronRSS couplings mostly, though not always, concerned perfect signal joints, whereas B cell malignancies (in particular precursor B-ALL) often showed damaged signal joints (Tables II and III). This can probably be explained by counterselection on perfect JRSS-intronRSS signal joints for ongoing recombination in precursor B-ALL cells that are renowned for having a highly active V(D)J recombinase. Undamaged JRSS-intronRSS signal joints are likely to undergo further recombination to V-intronRSS or JRSS-Kde pseudohybrid joints, as shown in recombination substrate assays (Fig. 5). The formation of both types of pseudohybrid joints might represent an alternative pathway of allele inactivation and allelic exclusion for those IGK alleles that have undergone initial aberrant recombinations: in case of V-intronRSS recombination, no J segments are left for functional IGK expression, while JRSS-Kde recombination deletes the C region and enhancers, thereby preventing expression of an Ig -chain. Theoretically, the V-intronRSS pseudohybrid joint could be involved in further recombination to Kde (V-Kde coupling). However, damaged intronRSS (see also Table III) frequently preclude this and consequently the V-intronRSS pseudohybrid joint will often be the end-stage rearrangement (Fig. 6). Analogously, the undamaged JRSS of a JRSS-Kde coupling might theoretically rearrange to the RSS of an upstream V segment, but the loss of both IGK enhancers in the (V-J) + JRSS-Kde configuration probably blocks this, leaving the (V-J) + JRSS-Kde rearrangement as end-stage configuration (Fig. 6). Although the usage of the here-described alternative IGK recombination pathway is limited, this pathway is essential for inactivation of IGK alleles with aberrant recombinations, which otherwise might hamper selection of functional Ig L chain proteins.

	Acknowledgments

 
We thank members of the Molecular Immunology unit (Department of Immunology, Erasmus Medical Center) for stimulating discussions and valuable comments; V. H. J. van der Velden and M. van der Burg for critical reading of the manuscript; and the BIOMED-2 Concerted Action BMH4-CT98-3936 (coordinator J. J. M. van Dongen) for kindly providing two samples with atypical IGK recombinations for further analysis.

	Footnotes

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ A.W.L. was supported by the Haak-Bastiaanse Kuneman Foundation; and U.J. and B.N. were supported by the Center of Molecular Medicine of the Austrian Academy of Sciences Grant 20010.

² Address correspondence and reprint requests to Dr. Anton W. Langerak, Molecular Immunology Unit, Department of Immunology, Erasmus MC, University Medical Center Rotterdam, Dr. Molewaterplein 50, 3015 GE, Rotterdam, The Netherlands. E-mail address: a.langerak{at}erasmusmc.nl

³ Abbreviations used in this paper: IGH, Ig H chain locus; IGK, Ig locus; IGL, Ig locus; Kde, -deleting element; ALL, acute lymphoblastic leukemia; AML, acute myeloid leukemia; BM, bone marrow; CAT, chloramphenicol acetyltransferase; CLL, chronic lymphocytic leukemia; MNC, mononuclear cells; PB, peripheral blood; RQ-PCR, real-time quantitative PCR; RSS, recombination signal sequence.

Received for publication March 3, 2004. Accepted for publication July 13, 2004.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Ghia, P., E. ten Boekel, A. G. Rolink, F. Melchers. 1998. B-cell development: a comparison between mouse and man. Immunol. Today 19:480.[Medline]
2. Ghia, P., E. ten Boekel, E. Sanz, A. de la Hera, A. Rolink, F. Melchers. 1996. Ordering of human bone marrow B lymphocyte precursors by single-cell polymerase chain reaction analyses of the rearrangement status of the immunoglobulin H and L chain gene loci. J. Exp. Med. 184:2217.[Abstract/Free Full Text]
3. Gorman, J. R., F. W. Alt. 1998. Regulation of immunoglobulin light chain isotype expression. Adv. Immunol. 69:113.[Medline]
4. Van der Burg, M., T. Tumkaya, M. Boerma, S. de Bruin-Versteeg, A. W. Langerak, J. J. van Dongen. 2001. Ordered recombination of immunoglobulin light chain genes occurs at the IGK locus but seems less strict at the IGL locus. Blood 97:1001.[Abstract/Free Full Text]
5. Korsmeyer, S. J., P. A. Hieter, J. V. Ravetch, D. G. Poplack, T. A. Waldmann, P. Leder. 1981. Developmental hierarchy of immunoglobulin gene rearrangements in human leukemic pre-B-cells. Proc. Natl. Acad. Sci. USA 78:7096.[Abstract/Free Full Text]
6. Siminovitch, K. A., A. Bakhshi, P. Goldman, S. J. Korsmeyer. 1985. A uniform deleting element mediates the loss of genes in human B cells. Nature 316:260.[Medline]
7. Feddersen, R. M., D. J. Martin, B. G. Van Ness. 1990. Novel recombinations of the IG-locus that result in allelic exclusion. J. Immunol. 145:745.[Abstract]
8. Feddersen, R. M., D. J. Martin, B. G. Van Ness. 1990. The frequency of multiple recombination events occurring at the human Ig L chain locus. J. Immunol. 144:1088.[Abstract]
9. Seriu, T., T. E. Hansen-Hagge, Y. Stark, C. R. Bartram. 2000. Immunoglobulin gene rearrangements between the deleting element and J recombination signal sequences in acute lymphoblastic leukemia and normal hematopoiesis. Leukemia 14:671.[Medline]
10. Beishuizen, A., M.-A. J. Verhoeven, E. J. Mol, J. J. M. Van Dongen. 1994. Detection of immunoglobulin light chain gene rearrangement patterns by Southern blot analysis. Leukemia 8:2228.[Medline]
11. Van Dongen, J. J. M., I. L. M. Wolvers-Tettero. 1991. Analysis of immunoglobulin and T cell receptor genes. I. Basic and technical aspects. Clin. Chim. Acta 198:1.[Medline]
12. Tumkaya, T., W. M. Comans-Bitter, M. A. Verhoeven, J. J. van Dongen. 1995. Southern blot detection of immunoglobulin light chain gene rearrangements for clonality studies. Leukemia 9:2127.[Medline]
13. Tümkaya, T., A. Beishuizen, I. L. M. Wolvers-Tettero, J. J. M. Van Dongen. 1996. Identification of immunoglobulin isotype gene rearrangements by Southern blot analysis. Leukemia 10:1834.[Medline]
14. Van Dongen, J. J. M., A. W. Langerak, M. Bruggemann, P. A. S. Evans, M. Hummel, F. L. Lavender, E. Delabesse, F. Davi, E. Schuuring, R. Garcia-Sanz, et al 2003. Design and standardization of PCR primers and protocols for detection of clonal immunoglobulin and T-cell receptor gene recombinations in suspect lymphoproliferations: report of the BIOMED-2 Concerted Action BMH4-CT98-3936. Leukemia 17:2257.[Medline]
15. Langerak, A. W., T. Szczepanski, M. Van der Burg, I. L. M. Wolvers-Tettero, J. J. M. Van Dongen. 1997. Heteroduplex PCR analysis of rearranged T cell receptor genes for clonality assessment in suspect T cell proliferations. Leukemia 11:2192.[Medline]
16. Van der Velden, V. H., M. J. Willemse, C. E. van der Schoot, K. Hahlen, E. R. van Wering, J. J. van Dongen. 2002. Immunoglobulin deleting element rearrangements in precursor-B acute lymphoblastic leukemia are stable targets for detection of minimal residual disease by real-time quantitative PCR. Leukemia 16:928.[Medline]
17. Marculescu, R., T. Le, P. Simon, U. Jaeger, B. Nadel. 2002. V(D)J-mediated translocations in lymphoid neoplasms: a functional assessment of genomic instability by cryptic sites. J. Exp. Med. 195:85.[Abstract/Free Full Text]
18. Hesse, J. E., M. R. Lieber, M. Gellert, K. Mizuuchi. 1987. Extrachromosomal DNA substrates in pre-B cells undergo inversion or deletion at immunoglobulin V-(D)-J joining signals. Cell 49:775.[Medline]
19. Lewis, S., A. Gifford, D. Baltimore. 1984. Joining of V to J gene segments in a retroviral vector introduced into lymphoid cells. Nature 308:425.[Medline]
20. Lewis, S., A. Gifford, D. Baltimore. 1985. DNA elements are asymmetrically joined during the site-specific recombination of immunoglobulin genes. Science 228:677.[Abstract/Free Full Text]
21. Xu, Y., L. Davidson, F. W. Alt, D. Baltimore. 1996. Deletion of the Ig light chain intronic enhancer/matrix attachment region impairs but does not abolish V J rearrangement. Immunity 4:377.[Medline]
22. Mostoslavsky, R., N. Singh, A. Kirillov, R. Pelanda, H. Cedar, A. Chess, Y. Bergman. 1998. -chain monoallelic demethylation and the establishment of allelic exclusion. Genes Dev. 12:1801.[Abstract/Free Full Text]
23. Inlay, M., F. W. Alt, D. Baltimore, Y. Xu. 2002. Essential roles of the light chain intronic enhancer and 3' enhancer in rearrangement and demethylation. Nat. Immunol. 3:463.[Medline]
24. Langerak, A. W., I. L. Wolvers-Tettero, E. J. van Gastel-Mol, M. E. Oud, J. J. van Dongen. 2001. Basic helix-loop-helix proteins E2A and HEB induce immature T-cell receptor rearrangements in nonlymphoid cells. Blood 98:2456.[Abstract/Free Full Text]
25. Szczepanski, T., M. J. Pongers-Willemse, A. W. Langerak, J. J. van Dongen. 1999. Unusual immunoglobulin and T-cell receptor gene rearrangement patterns in acute lymphoblastic leukemias. Curr. Top. Microbiol. Immunol. 246:205.[Medline]
26. Marculescu, R., K. Vanura, T. Le, P. Simon, U. Jager, B. Nadel. 2003. Distinct t(7;9)(q34;q32) breakpoints in healthy individuals and individuals with T-ALL. Nat. Genet. 33:342.[Medline]
27. Pongers-Willemse, M. J., T. Seriu, F. Stolz, E. d’Aniello, P. Gameiro, P. Pisa, M. Gonzalez, C. R. Bartram, E. R. Panzer-Gruemayer, A. Biondi, et al 1999. Primers and protocols for standardized detection of minimal residual disease in acute lymphoblastic leukemia using immunoglobulin and T cell receptor gene rearrangements and TAL1 deletions as PCR targets: report of the BIOMED-1 Concerted Action: investigation of minimal residual disease in acute leukemia. Leukemia 13:110.[Medline]
28. Feeney, A. J., G. Lugo, G. Escuro. 1997. Human cord blood repertoire. J. Immunol. 158:3761.[Abstract]

This article has been cited by other articles:

				M. C. van Zelm, T. Szczepanski, M. van der Burg, and J. J.M. van Dongen Replication history of B lymphocytes reveals homeostatic proliferation and extensive antigen-induced B cell expansion J. Exp. Med., March 19, 2007; 204(3): 645 - 655. [Abstract] [Full Text] [PDF]

				W. A. Dik, K. Pike-Overzet, F. Weerkamp, D. de Ridder, E. F.E. de Haas, M. R.M. Baert, P. van der Spek, E. E.L. Koster, M. J.T. Reinders, J. J.M. van Dongen, et al. New insights on human T cell development by quantitative T cell receptor gene rearrangement studies and gene expression profiling J. Exp. Med., June 6, 2005; 201(11): 1715 - 1723. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Request Permissions Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Langerak, A. W. Articles by van Dongen, J. J. M. Search for Related Content PubMed PubMed Citation Articles by Langerak, A. W. Articles by van Dongen, J. J. M.