Immunoglobulin Kappa Locus Research Articles

Vκ polymorphisms in NOD mice are spread throughout the entire immunoglobulin kappa locus and are shared by other autoimmune strains

The diversity of immunoglobulin (Ig) and T cell receptor (TCR) genes available to form the lymphocyte repertoire has the capacity to produce a broad array of both protective and harmful specificities. In type 1 diabetes (T1D), the presence of antibodies to insulin and other islet antigens predicts disease development in both mice and humans, and demonstrate that immune tolerance is lost early in the disease process. Anti-insulin T cells isolated from T1D-prone non-obese diabetic (NOD) mice use polymorphic TCRalpha chains, suggesting that the available T cell repertoire is altered in these autoimmune mice. To probe whether insulin-binding B cells also possess polymorphic V genes, Ig light chains were isolated and sequenced from NOD mice that harbor an Ig heavy chain transgene. Three insulin-binding Vkappa genes were identified, all of which were polymorphic to the closest germline sequence matches present in the GenBank database. Additional analysis of over 300 light chain sequences from multiple sources, including germline DNA, shows that polymorphisms are spread throughout the entire NOD Igkappa locus, as these polymorphic sequences represent 43 distinct Vkappa genes which belong to 14 Vkappa families. Database searches reveal that a majority of polymorphic Vkappa genes identified in NOD are identical to Vkappa genes isolated from SLE-prone NZBxNZW F1 or MRL strains of mice, suggesting that a shared Igkappa haplotype may be present. Predicted amino acid changes preferentially occur in CDR, and thus could alter antigen recognition by the germline B cell repertoire of autoimmune versus non-autoimmune mouse strains.

IGH and IGK gene rearrangements as PCR targets for pediatric Burkitt's lymphoma and mature B-ALL MRD analysis

We recently reported that minimal residual disease (MRD) and minimal disseminated disease (MDD), assessed by long-distance PCR (LD-PCR) for t(8;14), are negative prognostic factors in mature B-cell acute lymphoblastic leukemia (B-ALL) and in Burkitt's lymphoma (BL). However, t(8;14) is detectable in only about 70% of patients, thus preventing MRD studies by this approach in the remaining patients. At present, no molecular assays have been reported for MRD and MDD analysis in t(8;14)-negative patients. The aim of our study was to evaluate the characteristics of patient-specific immunoglobulin (Ig) gene rearrangements as RQ-PCR targets for MRD analysis, in order to extend MRD studies to those patients who are not eligible for the LD-PCR assay. The study was performed according to the guidelines of the European Study Group on MRD detection in ALL (ESG-MRD-ALL). Overall, 36 B-ALL and 19 BL cases were analyzed. Multiple PCR reactions were performed for each sample to identify heavy and kappa light-chain rearrangements. A total of 97 RQ-PCR targets (62 for B-ALL, 35 for BL) were analyzed for sensitivity. The rearrangement pattern identified was similar to that reported for normal peripheral blood lymphocytes. In 88% of the targets, a sensitivity of at least 10−4 was achieved. In 87% of patients (84% of B-ALLs, 95% of BLs) at least one sensitive target was available. All PCR targets identified at diagnosis were preserved at relapse. Our results suggest that MDD and MRD can be successfully studied using a single sensitive Ig target in the great majority of B-ALL and BL cases. The combination of LD-PCR and Ig-based assays will allow MRD analysis in virtually all of the patients.

High Frequency of Clonal Immunoglobulin Receptor Gene Rearrangements in Sporadic Histiocytic/Dendritic Cell Sarcomas

The diagnosis of histiocytic/dendritic cell (H/DC) sarcomas is currently based on morphology and the presence of immunophenotypic features of H/DC differentiation. The issue whether clonal immunoglobulin receptor gene rearrangements are present in H/DC sarcomas has been debated over decades until the recent data by Feldman et al, which provided compelling evidence that patients with follicular lymphoma and concurrent/synchronous H/DC sarcoma share identical genotypic features, suggested the possibility of transdifferentiation or dedifferentiation of 2 otherwise morphologically and immunophenotypically distinctive neoplasms. Here we investigated the molecular characteristics of 23 patients with sporadic H/DC sarcoma. Nine of the 23 cases (39%) showed clonal IGH (+/-IGK) gene rearrangements, whereas 2 (9%) cases showed only clonal IGK gene rearrangements, which were further validated and confirmed by direct DNA sequencing. One histiocytic sarcoma showed t(14;18) by quantitative-polymerase chain reaction, which was confirmed by fluorescence in situ hybridization analysis showing IGH/BCL2 fusions in neoplastic histiocytes. Notably, all IGH/IGK-positive H/DC sarcomas were negative for B-cell-associated transcription factors PAX5 and BOB.1, whereas 4 of 7 IGH/IGK-positive histiocytic sarcoma cases were positive for Oct2. In addition, no evidence of Epstein-Barr virus infection was detected in 8 of 11 IGH/IGK-positive H/DC sarcoma cases by in situ hybridization, suggesting that Epstein-Barr virus infection may not play an important role in the pathogenesis of these tumors. This study provides evidence that clonal immunoglobulin receptor gene rearrangements may be detected at a high frequency in sporadic H/DC sarcomas. The findings suggest that a large subset of H/DC sarcomas have inherited B-cell genotypes, thus providing new insights for the pathogenesis of these rare but aggressive neoplasms.

Violations of the 12/23 rule at the mouse immunoglobulin kappa locus, including Vκ-Vκ rearrangement

Classically, recombination between immunoglobulin gene segments uses a pair of recombination signal sequences (RSSs) with dissimilar spacers (the “12/23 rule”). Using a series of different genotyping assays, four different kinds of atypical rearrangements were identified at the murine kappa locus: (1) Vκ to Vκ, (2) Jκ to Jκ, (3) Vκ to iRS, a heptameric sequence found in the JκCκ intron, and (4) a possible by-product of a rearrangement between a Vκ and the hypothetical 12-RSS side of a pre-existing signal joint. The novel Vκ-Vκ structure prompted further characterization. Sequence analysis of 14 different Vκ-Vκ rearrangements cloned from murine splenocytes and hybridomas revealed a Vκ4 family member as one participant in 13 rearrangements, but no rearrangements contained two Vκ4 genes. The Vκ4 partner in the Vκ-Vκ rearrangement exhibited more trimming of nucleotides at the Vκ-Vκ junction. A signal joint derived from the inversional rearrangement of two neighboring Vκs was also recovered. These data suggest that the Vκ-Vκ structures arise via RAG-mediated, intrachromosomal recombination.

PCR clonality detection in Hodgkin lymphoma

B-cell clonality detection in whole tissue is considered indicative of B-cell non-Hodgkin lymphoma (NHL). We tested frozen tissue of 24 classical Hodgkin lymphomas (cHL) with a varying tumor cell load with the multiplex polymerase chain reaction (PCR) primer sets for IGH and IGK gene rearrangement (BIOMED-2). A clonal population was found in 13 cases with the IGH FR1 and/or FR2/FR3 PCRs. Using the IGK-VJ and IGK-DE PCRs, an additional six cases had a dominant clonal cell population, resulting in a detection rate of 79% in frozen tissue. Of 12 cases, also the formalin-fixed and paraffin-embedded (FFPE) tissue was tested. Surprisingly, in eight of the 12 FFPE cases with acceptable DNA quality (allowing PCR amplification of >200 nt fragments), the IGK multiplex PCRs performed better in detecting clonality (six out of eight clonal IGK rearrangements) than the IGH PCRs (four out of nine clonal rearrangements), despite a rather large amplicon size. There was no evidence of B-cell lymphoma during follow-up of 1 to 6 years and no correlation was found between the presence of a clonal result and Epstein–Barr virus in the tumor cells. Our results indicate that the present routine PCR methods are sensitive enough to detect small numbers of malignant cells in cHL. Therefore, the presence of a clonal B-cell population does not differentiate between cHL and NHL.

OH-2, a Hyperdiploid Myeloma Cell Line without An IGH Translocation, Has a Complex Translocation Juxtaposing MYC near MAFB and the IGK Locus

Despite heterogeneity of phenotypes, multiple myeloma (MM) can be classified into two major groups: hyperdiploid (HRD) tumors with 48–74 chromosomes, which typically have extra copies of at least four of the eight odd chromosomes 3, 5, 7, 9, 11, 15, 19, and 21; and non-hyperdiploid (NHRD) tumors, which usually have immunoglobulin heavy chain (IGH) translocations and <46 or >74 chromosomes. Human myeloma cell lines (HMCL) with a typical HRD genotype are lacking. The OH-2 HMCL was derived from extramedullary myeloma, and retains the same HRD phenotype as the primary tumor, with extra copies of chromosomes 3, 7, 15, 19, and 21, as demonstrated by array comparative genomic hybridization. This provides a unique example of an HMCL and the corresponding primary tumor that share the same HRD phenotype. Also Spectral Karyotyping showed the same HRD phenotype. By fluorescence in situ hybridization it was shown that OH-2 does not have an IGH or Ig lambda light chain translocation. Instead, both the HMCL and the primary tumor have complex translocations involving chromosomes 2, 8, and 20. The translocation juxtaposes MAFB near MYC and the Ig kappa (IGK) enhancer. Both MYC and MAFB are highly expressed, as shown by microarray analysis, and are thought to be dysregulated by the IGK enhancer. Both OH-2 HMCL and the primary tumor cells express high levels of Cyclin D2, an expected consequence of the enhanced MAFB level. OH-2 has a phenotype similar to the 20% of HRD MM tumors that express increased levels of Cyclin D2 but not Cyclin D1 and could be a good in vitro model for studies of this subgroup of MM. OH-2 is dependent on medium containing human serum and growth-promoting cytokines like IL-6 or IL-21, reflecting the critical role of the microenvironment for growth of MM cells.

The Downstream Transcriptional Enhancer, Ed, positively regulates mouse Ig kappa gene expression and somatic hypermutation.

The mouse Igkappa locus has three known transcriptional enhancers: the matrix association region/intronic enhancer, the 3' enhancer (E3'), and the further downstream enhancer (Ed). Previous studies have shown that both matrix association region/intronic and E3' enhancers are required for maximal gene rearrangement of the locus, and that E3' is also required for maximal expression and somatic hypermutation (SHM). To functionally elucidate Ed in vivo, we generated knockout mice with a targeted germline deletion of Ed. Ed deleted homozygous mice (Ed-/-) have moderately reduced numbers of Igkappa expressing B cells and correspondingly increased numbers of Iglambda expressing B cells in spleen. Ed-/- mice also have decreased Igkappa mRNA expression in resting and T cell-dependent activated splenic B cells and reduced Igkappa chains in sera. However, our analysis indicates that Igkappa gene rearrangement is normal in Ed-/- mice. In addition, our results show that Ed-/- mice exhibit reduced SHM in the Igkappa gene J-C intronic region in germinal center B cells from Peyer's patches. We conclude that Ed positively regulates Igkappa gene expression and SHM, but not gene rearrangement.

The downstream enhancer, Ed, positively regulates Ig kappa gene expression and somatic hypermutation

The mouse Ig kappa locus has three known enhancers: the MAR/intronic enhancer (MEi), the 3′enhancer (E3′) and the further downstream enhancer (Ed). Previous studies have shown that both MEi and E3′ enhancers are required for maximal gene rearrangement of the locus, and that E3′ is also required for maximal expression and somatic hypermutation (SHM). To functionally elucidate Ed in vivo, we generated knockout mice with a targeted germ line deletion of Ed. Ed deleted homozygous mice (Ed−/−) have moderately reduced numbers of kappa expressing B cells and correspondingly increased numbers of lambda expressing B cells in spleen. Ed−/− mice also have decreased kappa mRNA expression in resting and T‐cell dependent activated splenic B cells and reduced kappa chains in sera. However, our analysis indicates that kappa gene rearrangement is normal in Ed−/− mice. In addition, our results show that Ed−/− mice exhibit reduced SHM in the kappa gene J‐C intronic region in Peyer's patch germinal center B cells. We conclude that Ed positively regulates Ig kappa gene expression and SHM, but not gene rearrangement.Research supported by grants GM29935 and AI067906 from NIH and I‐0823 from the Robert A. Welch Foundation.

Rearrangement of Mouse Immunoglobulin Kappa Deleting Element Recombining Sequence Promotes Immune Tolerance and Lambda B Cell Production

The recombining sequence (RS) of mouse and its human equivalent, the immunoglobulin (Ig) kappa deleting element (IGKDE), are sequences found at the 3' end of the Ig kappa locus (Igk) that rearrange to inactivate Igk in developing B cells. RS recombination correlates with Ig lambda (Iglambda) light (L) chain expression and likely plays a role in receptor editing by eliminating Igk genes encoding autoantibodies. A mouse strain was generated in which the recombination signal of RS was removed, blocking RS-mediated Igk inactivation. In RS mutant mice, receptor editing and self-tolerance were impaired, in some cases leading to autoantibody formation. Surprisingly, mutant mice also made fewer B cells expressing lambda chain, whereas lambda versus kappa isotype exclusion was only modestly affected. These results provide insight into the mechanism of L chain isotype exclusion and indicate that RS has a physiological role in promoting the formation of lambda L chain-expressing B cells.

Crosslineage T-Cell Receptor Genes Rearrangements and Active Receptor Editing in B-Cell Acute Lymphoblastic Leukemia.

B-cell acute lymphoblastic leukemia (B-ALL) results from clonal expansion of B-lymphocytes derived at different stage of differentiation. Immunoglobulin (Ig) heavy chain genes (IGH), light chain kappa (IGK) and lambda (IGL) genes rearrange during early B-lymphocyte differentiation. T-cell receptor (TCR) genes are considered to rearrange exclusively in normal T lymphocytes, but malignant B lymphoblasts often contain crosslineage rearranged TCR genes. The clonal leukemic cell population, carrying identical copies of rearranged Ig and/or TCR genes, can be identified above 95% of B-ALL patients. In our study Ig/TCR genes rearrangements were detected by multiplex PCR with heteroduplex analysis according to BIOMED-2 protocol. DNA was isolated by column method from mononuclear cells isolated from the peripheral blood/bone marrow samples obtained at initial diagnosis from 36 B-ALL patients. Monoclonal rearrangements of Ig genes were detected in 100% (36/36) of patients. The most frequent rearrangements were observed in IGH genes (94%), including complete IGHV-IGHJ in 83% (30/36) and incomplete IGHD-IGHJ in 22% (8/36) of patients. Among complete IGH rearrangements 2 biallelic rearrangements in IGHV1-7 and IGHJ genes (FR3) were found. Ig light chain genes rearrangements were identified in 26 patients (72%) (including 64% of IGKV-IGKJ, 47% IGKV/intron-Kde, and 22% IGLV-IGLJ) what indicates active receptor editing occurring during B lymphoblasts leukemogenesis. Crosslineage TCR genes rearrangements were found in 97% (35/36) of patients. TCR beta genes rearrangements were detected in 47% (17/36) of patients (complete TRBV-TRBJ in 25% (9/36), TRBD-TRBJ in 6/36 patients - 17%). TRGV-TRGV in 58% (21/36), TRDV-TRDJ in 58% (21/36); 17 monoallelic and 4 biallelic were found. The inactivation of potentially functional IGKV-IGKJ by secondary rearrangements indicates active receptor editing. Our data describe IGK and IGL genes rearrangements incidence, present allelic exclusion and active receptor editing in B-ALL patients. B-ALL lymphoblast undergoes rearrangement on the same IGK allele before IGL genes rearrangement occur. The data may suggest the possible of antigens in B-ALL immunopathogenesis. The results indicate also rearranged IGK, IGL and TCR genes as stable molecular marker for monitoring MRD in B-ALL.

Markers for Minimal Residual Disease (MRD) in B-Lineage Acute Lymphoblastic Leukemia (B-ALL): Analysis of IgH Gene Rearrangement Repertoire (AIRR) vs Conventional Strategy.

Background: Detection of molecular markers for monitoring MRD in B-ALL conventionally involves searching for a variety of immunoglobulin and T-cell receptor rearrangements. However preliminary studies have suggested that AIRR may have advantages. Aim :To directly compare AIRR to the conventional approach for detection of molecular markers.Materials and Methods Marrow samples from a consecutive series of 50 children with B-linage ALL were studied by the conventional approach in Sydney and by AIRR in Adelaide. AIRR involves use of primers specific for individual V and J segments of the IgH gene followed by sequencing of amplified products and calculation of the relative abundance of rearrangements from the quantitative PCR data. IgH rearrangements are regarded as suitable for detecting the major leukemic clone if they comprise > 10% of the rearrangements present. Incomplete D-J rearrangements are detected using primers directed towards genomic regions upstream of the D segments.Results Table 1 shows the number of patients with either 0, 1 or 2 rearrangements suitable for monitoring MRD of the major leukemic clone.The 2 approaches appeared to be equivalent in terms of the number of markers for the major clone, but, of the rearrangements detected conventionally, 49% were IgH rearrangements and 51% involved IgK or various T-cell receptor genes. All of the IgH rearrangements detected in the 50 patients, grouped by abundance, are shown in Table 2.Table 1number of patientsno marker1 marker2 markersconventional approach2939AIRR21236Table 2relative abundance of rearrangement100-10%10-1%<1%detected by both analyses6566conventional only400AIRR only281231High and low abundance rearrangements mark major and minor clones repectively. AIRR detected significantly more rearrangements of both types. Clone sizes and lineage relationships could be inferred from rearrangement abundance and sequence relationships. 68 minor clones were detected at diagnosis in 31 patients. 26 of the 68 showed no sequence relationship to the major clone.Discussion Use of IgH gene rearrangements as markers enables MRD in B-ALL to be sensitively quantified, down to 10−6. The superior ability of AIRR to identify marker IgH rearrangements is therefore likely to enable more sensitive monitoring of the major leukemic clone. Relapse in B-ALL is sometimes due a minor chemoresistant leukemic clone which is already present at diagnosis but is only identified retrospectively. The ability of AIRR to identify IgH markers for minor clones at diagnosis, together with the ability to sensitively monitor clones marked by IgH rearrangements, may enable prospective identification of these minor clones.Conclusions1.AIRR and the conventional strategy are similar in terms of the number of patients with ALL in whom 1 or 2 molecular markers for the major leukemic clone can be detected.2.AIRR detects significantly more IgH markers for the major leukemic clone. This should enable more sensitive quantification of the major clone in many patients.3.AIRR is much superior at identification of markers for minor clones. This may enable chemoresistant minor clones to be detected prospectively.4.The clonal hierarchies of minor clones at diagnosis resemble those observed in patients who relapse.

Immunoglobulin and T-Cell Receptor (Ig/TCR) Gene Rearrangements in Chinese Adult Acute Lymphoblastic Leukemia Patients.

To determine the feasibility of Ig/TCR gene rearrangements as patient-specific targets for the detection of minimal residual disease in Chinese ALL patients, forty adult patients with B-lineage and T cell acute lymphoblastic leukemia(ALL) were enrolled in this study. The frequency and pattern of IGH, IGK, TCRB, TCRG and TCRD gene rearrangement were analyzed by BIOMED-2 multiplex PCR protocol. All DNA samples were obtained from the bone marrow of these patients. IGH, IGK, TCRB, TCRG and TCRD rearrangements were found in 86%,14%,27%,77%,59% of adult patients with B-lineage ALL, respectively. While in T-ALL, clonal TCRB, TCRG and TCRD rearrangements were detected in 83%,78%,39% of patients. There are several features in the pattern of Ig/TCR gene rearrangements in Chinese adult ALL patients. Firstly, IGK rearrangement in adult B-lineage ALL had a lower frequency than in children's pre-B-ALL (14% vs 41%) in reported literature. Secondly, the frequency of TCRG Vrfl-Jr1.3, Vrfl-Jr1.1 and Vr9-Jr1.3 rearrangement was 65%,6%,29% in adult patients with B-lineage ALL, while 60%,27%,13% was the frequency of those in T-ALL, respectively. In conclusion, our data have shown that several special patterns in Ig/TCR gene rearrangements in Chinese adult ALL patients, may provide a guidance for individual treatment protocol designation and MRD-based risk stratification.

Combined BIOMED-2 Clonality Assays for IGH and IGK and Assessment of t(14;18) on Formalin-Fixed, Paraffin Embedded Tissues Effectively Separates Primary Cutaneous B Cell Lymphomas from Reactive Cutaneous Lymphoid Hyperplasia.

Primary cutaneous B-cell lymphomas (PCBCL) are a diverse group of lymphomas that are limited to the skin at the time of diagnosis. Clonally rearranged immunoglobulin (Ig) genes are a key feature of B-cell malignancies. While PCR for Ig rearrangement is well-established and characterized in nodal malignancies, PCR investigations of Ig clonality in PCBCL have been unstandardized and extremely inconsistent. In this study, we analyzed 27 cases of PCBCL (22 cases of PCBCL diagnosed at initial presentation and 5 cases of atypical lymphoid infiltrates (ALI)) for Ig clonality using standardized BIOMED-2 PCR protocols (InVivoScribe Technologies, San Diego, CA). We defined lesions of ALI as those that were clinically suspicious for lymphoma, but could not be confirmed as lymphoma by morphologic and/or immunohistochemical methods. These patients were followed clinically where an eventual diagnosis of lymphoma was made by morphologic and/or immunohistochemical methods. Our primary objective was to evaluate the sensitivity and specificity of the BIOMED-2 method for detecting clonality in PCBCL, including cases initially diagnosed as ALI. Our secondary objective was to assess the role of clonality in the diagnosis of PCBCL. 47 biopsies from 27 cases of PCBCL (7 T1aN0M0, 4 T2aN0M0, 3 T2bN0M0, 3 T3aN0M0, and 10 T3bN0M0) were tested for IGH and IGK gene rearrangements and the (14;18) translocation using BIOMED-2 primers. Select cases from patients with multiple biopsies were further analyzed by sequencing to identify identical clones. We found a clone in 24/27 (89%) cases overall (10/11, 91% marginal zone lymphoma (MZL); 10/11, 91% follicle center lymphoma (FCL); and 4/5 ALI)). Among the 47 biopsies, a clone was detected in 35/47 (75%) cases (13/19, 68% MZL; 15/19, 79% FCL; and 7/9, 78% ALI). We found that clonal detection was improved in cases with multiple biopsies. The t(14;18) translocation was present in 3/11 FCL cases and 1/5 ALI cases, but not detected in any biopsy from MZL cases. Addition of IGK analysis to IGH analysis alone increased sensitivity of clonal detection from 57% to 75%. No clonality was detected in 9 cases of reactive cutaneous lymphoid hyperplasia. While our study is small, the initial results demonstrate that1.the BIOMED-2 protocol shows high sensitivity and specificity in clonality detection in PCBCL2.the (14;18) translocation is rare in PCBCL in general, but can be detected in a minority of cases of FCL3.adding IGK primers to the analysis for clonality may increase its sensitivity over detection of IGH rearrangements alone4.clonality detection may lead to earlier diagnosis of PCBCL in cases otherwise thought to be ALI5.multiple biopsies can lead to a more definitive diagnosis of PCBCL with increased likelihood of clonal detection and6.formalin-fixed paraffin embedded tissues can be used very effectively to establish clonality in PCBCL.To our knowledge, this is the first report of a standardized approach for the detection of clonality in PCBCL. We propose a new algorithm in the diagnosis of PCBCL, where clonality assays are implemented when a definitive diagnosis cannot be made on clinical, morphologic, and/or immunophenotypic grounds. This novel characterization of PCBCL using archival tissue and the BIOMED-2 assay is a powerful strategy for clonality assessment and validation of the diagnosis of PCBCL.

A practical strategy for the routine use of BIOMED‐2 PCR assays for detection of B‐ and T‐cell clonality in diagnostic haematopathology

BIOMED-2 polymerase chain reaction (PCR) assays for clonality analysis of immunoglobulin (IG) and T-cell receptor (TCR) gene rearrangements were evaluated in routine haematopathological practice where paraffin-embedded tissues constitute the majority of specimens. One hundred and twenty-five fresh/frozen and 316 paraffin specimens were analysed for DNA quality and clonality. Seventy-nine per cent of paraffin specimens yielded PCR products of over 300 bp. These specimens and all fresh/frozen specimens were analysed with the complete set of BIOMED-2 reactions for IG (8 reactions) and/or TCR (6 reactions) gene rearrangements. The rate of detection of clonality was 96% in mature B-cell neoplasms and 98% in mature T-cell neoplasms and there were no significant differences in these rates between paraffin and fresh/frozen specimens. As the value of sole use of any individual BIOMED-2 reaction in clonality detection was limited, we assessed combinations of reactions that gave the greatest sensitivity with fewest reactions and were applicable for both fresh/frozen and paraffin specimens. For IG gene rearrangements, three reactions combining one targeting the IG heavy chain framework-2 region and two targeting the IG kappa locus achieved a 91% detection rate. For TCR gene rearrangements, the two TCR gamma reactions gave a 94% detection rate. We therefore recommend this strategy as the first-line assays for routine B- and T-cell clonality analysis in diagnostic haematopathology.

Allelic 'choice' governs somatic hypermutation in vivo at the immunoglobulin κ-chain locus

Monoallelic demethylation and rearrangement control allelic exclusion of the immunoglobulin kappa-chain locus (Igk locus) in B cells. Here, through the introduction of pre-rearranged Igk genes into their physiological position, the critical rearrangement step was bypassed, thereby generating mice producing B cells simultaneously expressing two different immunoglobulin-kappa light chains. Such 'double-expressing' B cells still underwent monoallelic demethylation at the Igk locus, and the demethylated allele was the 'preferred' substrate for somatic hypermutation in each cell. However, methylation itself did not directly inhibit the activation-induced cytidine-deaminase reaction in vitro. Thus, it seems that the epigenetic mechanisms that initially bring about monoallelic variable-(diversity)-joining rearrangement continue to be involved in the control of antibody diversity at later stages of B cell development.

Variant t(2;11)(p11.2;q13) without IGK involvement in a case of mantle cell lymphoma

Mantle cell lymphoma (MCL) is characterized by the t(11;14)(q13;q32) translocation, which leads to overexpression of the cyclin D1 ( CCND1) gene. This translocation is observed in almost all cases of MCL. In this alteration, the involvement of immunoglobulin heavy chain ( IGH) locus plays a key role in the activation of the CCND1 oncogene. Translocations affecting IGH loci are mostly prevalent in B-cell lymphomas, but variant translocations involving immunoglobulin kappa ( IGK) or lambda ( IGL) light chain loci have been observed in a minority of B-lymphoid malignancies. Variant translocations have been reported in only a few cases of MCL, however. This report presents a case of MCL with a variant t(2;11)(p11.2;q13), rearrangement of the CCND1 gene, and overexpression of cyclin D1. To characterize this rearrangement, specific noncommercial probes were used. This set of probes comprises IGK and REL flanking probes and 12 bacterial artificial chromosome (BAC) probes covering the region to be investigated. The results indicated that this alteration has not affected the IGK locus, and the breakpoint was within a 260-kb region located ∼1 Mb telomerically to the IGK gene. It is probable that the KV3J gene localized in this region could deregulate the expression of cyclin D1.

Crosslineage T-Cell Receptor Genes Rearrangements and Active Receptor Editing in B-Cell Acute Lymphoblastic Leukemia.

B-cell acute lymphoblastic leukemia (B-ALL) results from clonal expansion of B-lymphocytes derived at different stage of differentiation. Immunoglobulin (Ig) heavy chain genes (IGH), light chain kappa (IGK) and lambda (IGL) genes rearrange during early B-lymphocyte differentiation. T-cell receptor (TCR) genes are postulated to rearrange exclusively in normal T lymphocytes, but malignant B lymphoblasts often contain crosslineage rearranged TCR genes. The clonal leukemic cell population, carrying identical copies of rearranged Ig and/or TCR genes, can be identified above 95% of B-ALL patients. In our study Ig/TCR genes rearrangements were detected by multiplex PCR with heteroduplex analysis according to BIOMED-2 protocol. DNA was isolated by column method from mononuclear cells isolated from the peripheral blood/bone marrow samples obtained at initial diagnosis from 28 B-ALL patients. Monoclonal rearrangements of Ig genes were detected in 96% (27/28) of patients. The most frequent rearrangements were observed in IGH genes (96%), including complete IGHV-IGHJ in 75% (21/28) and incomplete IGHD-IGHJ in 31% (8/28) of patients. Among complete IGH rearrangements 4 biallelic rearrangements in IGHV1-7 and IGHJ genes (FR3) were found. Ig light chain genes rearrangements were identified in 20 patients (71%) (including 25% of IGKV-IGKJ, 50% of IGKV/intron-Kde, and 25% of IGLV-IGLJ) indicating active receptor editing occurring during B lymphoblasts leukemogenesis. Cross-lineage TCR genes rearrangements were found in 77% (23/28) of patients. TCR beta genes rearrangements were detected in 46% (13/28) of patients (complete TRBV-TRBJ in 32% (9/28), TRBD-TRBJ in 5/28 patients - 18%). TRGV-TRGV were found in 46% (13/28), TRDV-TRDJ in 50% (14/28; 10 monoallelic and 4 biallelic). TCR beta genes rearrangements with presence of TCR gamma genes rearrangements were identified in 25% (7/28) of patients. The identified Ig and TCR rearrangements were stable in patients monitored for minimal residual disease (MRD) and patients with leukemia relapse. The inactivation of potentially functional IGKV-IGKJ by secondary rearrangements indicates active receptor editing. Our data describe IGK and IGL genes rearrangements incidence, present allelic exclusion and active receptor editing in B-ALL patients. B-ALL lymphoblasts undergo many rearrangements on the same IGK allele before they rearrange IGL genes. The data suggest the role of antigen in B-ALL immunopathogenesis. The results indicate also rearranged IGK, IGL and TCR genes as a possible molecular marker for monitoring MRD in B-ALL.

Active Receptor Editing in Chronic Lymphocytic Leukemia B-Lymphocytes - Analysis of Immunoglobulin Light Chain Genes and Kappa Deleting Element Rearrangements.

The immunoglobulin gene rearrangements occur during early lymphoid differentiation in hierarchical order. First the immunoglobulin heavy chain genes (IGH) rearrange, then IGK and in case of IGK deletion rearrange IGL genes. All B-lymphocytes with IGL rearranged genes should have monoallelic or biallelic IGK genes deletions. The human IGK locus contains several variable genes (IGKV), joining genes (IGKJ), genes coding constant region (IGKC), and DNA fragments involved in recombination described as kappa deleting element (Kde). Both IGKV genes and recombination signal sequences (RSS) in the JK-CK region can rearrange to Kde creating respectively IGKV-Kde or intron RSS-Kde rearrangements. Kde rearrangements inactivate IGK allele causing the deletion of either the CK (intron RSS-Kde) or the entire JK-CK region. IGK and IGL rearrangements were analyzed in 48 patients with B-lymphocyte chronic lymphocytic leukemia (B-CLL) from peripheral blood/bone marrow mononuclear cells or enlarged diagnostic lymph nodes. Rearrangements of IGK and IGL were detected in multiplex PCR with heteroduplex analysis according to BIOMED-2 protocol (IGKV-IGKJ, IGKV/intron-Kde, and IGLV-IGLJ). In total, 41 IGK and IGL rearrangements were identified in 46 patients (89.1%). In 5 patients (10.9%) rearrangements were not found - including 4 DNA samples extracted from paraffin embedded tissue and one DNA sample isolated from peripheral blood. IGK rearrangements were detected in 37 of 46 patients (80.4%). In 24 patients (52.2%) IGK rearrangements without presence of IGL rearranged genes were identified, including 18 (39.1%) with only IGKV-IGKJ and 6 (13.1%) with IGKV-IGKJ and also IGKV/intron-Kde. Parallel rearranged IGK and IGL genes were found in 13 (28.3%) patients: 8 (17.4%) patients with IGKV-IGKJ and IGKV/intron - Kde and IGLV-IGLJ, 1 (2.2%) patients with IGKV-IGKJ and IGLV-IGLJ, 4 (8.7%) patients IGKV/intron-Kde and IGLV-IGLJ. In IGKV/intron-Kde rearranged genes group were detected the following rearrangements: IGKV1f/6/IGKV1-Kde (4 of 17); IGKV3f/intron Kde (13 of 17); IGKV2f/IGKV4/IGKV5-Kde (3 of 17; 17 monoallelic and 3 biallelic forms). Rearrangements with Kde always coexisted with IGKV-IGKJ or IGLV-IGLJ. In 4 (8.7%) patients IGL rearrangements were found without any IGK rearranged genes - three (6.5%) DNA samples were obtained from paraffin embedded tissue and the last DNA sample was isolated from peripheral blood after therapy. Lack of IGK monoclonality detection in paraffin embedded tissue samples resulted probably from low DNA quality. The inactivation of potentially functional IGKV-IGKJ by secondary rearrangements indicates active receptor editing. Our data describe IGK and IGL rearrangements incidence and present allelic exclusion and active receptor editing in B-CLL patients. The data provide additional evidence for the role of antigen in B-CLL immunopathogenesis. B-CLL lymphocytes undergo many rearrangements on the same IGK allele before they rearrange IGL genes and produce lambda chains. The results indicate also rearranged IGK/IGL genes as a possible molecular marker for monitoring minimal residual disease in B-CLL.

A Recombination Silencer that Specifies Heterochromatin Positioning and Ikaros Association in the Immunoglobulin κ Locus

Allelic exclusion ensures that individual B lymphocytes produce only one kind of antibody molecule. Previous studies have shown that allelic exclusion of the mouse Igkappa locus occurs by the combination of monoallelic silencing and a low level of monoallelic activation for rearrangement combined with a negative feedback loop blocking additional functional rearrangements. Using yeast artificial chromosome-based single-copy isotransgenic mice, we have identified a cis-acting element that negatively regulates rearrangement in this locus, specifically in B cells. The element, termed Sis, resides in the V-J intervening sequence. Sis specifies the targeting of Igkappa transgenes in pre-B and B cells to centromeric heterochromatin and associates with Ikaros, a repressor protein that also colocalizes with centromeric heterochromatin. Significantly, these are hallmarks of silenced endogenous germline Igkappa genes in B cells. These results lead us to propose that Sis participates in the monoallelic silencing aspect of allelic exclusion regulation.

Mechanism of Activation of the Immunoglobulin Kappa Locus through DNA Looping during B Cell Development

We have investigated changes in the higher-order chromatin structure of the mouse immunoglobulin (Ig) kappa locus upon gene activation during B cell development. Using the technique of chromosome conformation capture (3C), we find that in the fully transcriptionally active state that each of the three enhancers form complexes with themselves and rearranged gene promoters, while the resulting intervening DNA is looped out. These results fit the looping model for the mechanism of enhancer action. The looping interaction between a rearranged gene promoter and one enhancer occurs even before efficient transcription. Using chromatin immunoprecipitation (ChIP) we find that lysine 4 dimethylated histone 3 localizes specifically to two enhancers and a rearranged gene promoter before transcription, while this modification localizes mainly to the promoter and coding region when the gene is fully active. Using ChIP in combination with 3C (ChIP-3C), we demonstrate that lysine 4 dimethylated histone 3 is present in the looped complexes. These results suggest that the function of DNA looping might be to recruit protein factors such as histone methytransferases from the enhancer region to the promoter region to open the chromatin structure to facilitate the association of the transcriptional machine. Research supported by NIH and the Robert A. Welch Foundation.