IGLV3-21R110 identifies an aggressive biological subtype of chronic lymphocytic leukemia with intermediate epigenetics.

B-cell receptor (BCR) signaling is crucial for chronic lymphocytic leukemia (CLL) biology. IGLV3-21-expressing B cells may acquire a single point mutation (R110) that triggers autonomous BCR signaling, conferring aggressive behavior. Epigenetic studies have defined 3 CLL subtypes based on methylation signatures reminiscent of naïve-like (n-CLL), intermediate (i-CLL), and memory-like (m-CLL) B cells with different biological features. i-CLL carries a borderline IGHV mutational load and significantly higher use of IGHV3-21/IGLV3-21. To determine the clinical and biological features of IGLV3-21R110 CLL and its relationship to these epigenetic subtypes, we characterized the immunoglobulin gene of 584 CLL cases using whole-genome/exome and RNA sequencing. IGLV3-21R110 was detected in 6.5% of cases: 30 (38%) of 79 i-CLLs, 5 (1.7%) of 291 m-CLLs, and 1 (0.5%) of 189 n-CLLs. All stereotype subset 2 cases carried IGLV3-21R110, whereas 62% of IGLV3-21R110 i-CLL cases had nonstereotyped BCR immunoglobulins. IGLV3-21R110 i-CLL had a significantly higher number of SF3B1 and ATM mutations and total number of driver alterations. However, the R110 mutation was the sole alteration in 1 i-CLL and was accompanied only by del(13q) in 3. Although IGHV mutational status varied, IGLV3-21R110 i-CLL transcriptomically resembled n-CLL/unmutated IGHV CLL with a specific signature including WNT5A/B overexpression. In contrast, i-CLL lacking IGLV3-21R110 mirrored m-CLL/mutated IGHV. Patients with IGLV3-21R110 i-CLL had a short time to first treatment and overall survival similar to those of n-CLL/unmutated IGHV patients, whereas patients with non-IGLV3-21R110 i-CLL had a good prognosis similar to that of patients with m-CLL/mutated IGHV. IGLV3-21R110 defines a CLL subgroup with specific biological features and an unfavorable prognosis independent of IGHV mutational status and epigenetic subtype.

The analysis of organization and diversity mechanism in goat immunoglobulin light chain gene loci.

The genomic organization of goat immunoglobulin light chains (Igλ and Igκ) loci were annotated based on the goat genome database. The goat Igλ chain located on chromosome 17 contains at least 35 Vλ gene fragments (seven potential functional genes, one ORF and 27 pseudogenes), two Jλ-Cλ clusters arranged in a Vλ(35)-Jλ2-Cλ1-Jλ1-Cλ2 pattern, with another Cλ3 on scaffold. The Igκ locus included 11 Vκ (five potential functional genes, two ORFs and four pseudogene fragments), three Jκ genes and a single Cκ gene ordered in Vκ(35)-Jκ(3)-Cκ pattern on chromosome 11. By analyzing the clonies of Igλ and Igκ, we further found Vλ2 (26.23 %) &Vλ3 (73.11 %), Vκ2 (52.07 %) &Vκ4 (46.15 %) were predominately used in the expression of λ and κ chains respectively. λ chain showed more abundance in connective diversity than κ chain. Besides, somatic hypermutation with higher frequency in both immunoglobulin light chains was the major mechanism for the goat repertoire diversity. These results demonstrated goat immunoglobulin light chain variable region genome loci and repertoire diversity.

Contribution of immunoglobulin lambda light chain gene rearrangement analysis in the diagnosis of B-cell neoplasms.

Identification of clonal IGH, IGK and IGL gene rearrangements offers diagnostic adjunct in suspected B-cell neoplasms. However, many centres omit IGL analysis as its value is uncertain. A review of 567 cases with IGH, IGK and IGL rearrangement assessed using BIOMED-2 assays showed clonal immunoglobulin gene rearrangement in 54% of cases, of which 24% had a clonal IGL rearrangement. In two cases, the clonal rearrangement was detected exclusively by IGL analysis. This finding demonstrates the added value of IGL analysis for clonality assessment, especially in suspected B-cell neoplasms in which a clonal IGH and/or IGK rearrangement is not detected or is equivocal.

Immunoglobulin Light Chain Gene Rearrangements, Receptor Editing and the Development of a Self-Tolerant Antibody Repertoire.

Discussion of the antibody repertoire usually emphasizes diversity, but a conspicuous feature of the light chain repertoire is its lack of diversity. The diversity of reported allelic variants of germline light chain genes is also limited, even in well-studied species. In this review, the implications of this lack of diversity are considered. We explore germline and rearranged light chain genes in a variety of species, with a particular focus on human and mouse genes. The importance of the number, organization and orientation of the genes for the control of repertoire development is discussed, and we consider how primary rearrangements and receptor editing together shape the expressed light chain repertoire. The resulting repertoire is dominated by just a handful of IGKV and IGLV genes. It has been hypothesized that an important function of the light chain is to guard against self-reactivity, and the role of secondary rearrangements in this process could explain the genomic organization of the light chain genes. It could also explain why the light chain repertoire is so limited. Heavy and light chain genes may have co-evolved to ensure that suitable light chain partners are usually available for each heavy chain that forms early in B cell development. We suggest that the co-evolved loci of the house mouse often became separated during the inbreeding of laboratory mice, resulting in new pairings of loci that are derived from different sub-species of the house mouse. A resulting vulnerability to self-reactivity could explain at least some mouse models of autoimmune disease.

[Detection and application of bcl-2/IgH gene translocation and immunoglobulin gene rearrangement in follicular lymphoma].

Objective: To evaluate the application of FISH testing of bcl-2/IgH gene translocation and IgH/L gene rearrangement in different stages of follicular lymphoma. Methods: In 32 follicular lymphoma cases, which were collected at Guangdong General Hospital from September 2014 to December 2016, the bcl-2/IgH gene ectopic state was detected by FISH while the IgH/L gene rearrangement was tested using PCR-GeneScan to analyze the relationship between bcl-2/IgH gene translocation, different stages of follicular lymphoma and clonal immunoglobulin (IgH/L) gene rearrangements. Results: From the paraffin sections of all 32 follicular lymphomas, 17 cases showed bcl-2/IgH gene translocation, and the percentages of FL1, FL2 and FL3 translocation were 12/13, 3/5 and 2/14, respectively. Among the 24 cases of IgH/L gene arrangements identified from the total sample, the occurrence rates of FL1, FL2 and FL3 gene arrangement were 7/13, 4/5 and 13/14, respectively. Spearman's rank correlation analysis and χ(2) analysis showed that bcl-2/IgH gene translocation was negatively correlated with follicular lymphoma stage and the association was statistically significant. In more advanced stages of follicular lymphoma, the occurrence of bcl-2/IgH gene translocation tended to decrease with distinct FL1, FL2 and Fl3 gene expression (P<0.05). As IgH/L gene rearrangement in FL3 was higher than that in FL1 and FL2, its detection may be complimentary to FISH test for bcl-2/IgH gene translocation in diagnosing follicular lymphoma. Conclusions: The combined use of FISH and PCR-GeneScan increases the positive rate of follicular lymphoma diagnosis, and this combination is more sensitive than FISH or clonal analysis only to detect the chromosomal abnormality or the gene rearrangement.

Sequence-Based Discovery Demonstrates That Fixed Light Chain Human Transgenic Rats Produce a Diverse Repertoire of Antigen-Specific Antibodies.

We created a novel transgenic rat that expresses human antibodies comprising a diverse repertoire of heavy chains with a single common rearranged kappa light chain (IgKV3-15-JK1). This fixed light chain animal, called OmniFlic, presents a unique system for human therapeutic antibody discovery and a model to study heavy chain repertoire diversity in the context of a constant light chain. The purpose of this study was to analyze heavy chain variable gene usage, clonotype diversity, and to describe the sequence characteristics of antigen-specific monoclonal antibodies (mAbs) isolated from immunized OmniFlic animals. Using next-generation sequencing antibody repertoire analysis, we measured heavy chain variable gene usage and the diversity of clonotypes present in the lymph node germinal centers of 75 OmniFlic rats immunized with 9 different protein antigens. Furthermore, we expressed 2,560 unique heavy chain sequences sampled from a diverse set of clonotypes as fixed light chain antibody proteins and measured their binding to antigen by ELISA. Finally, we measured patterns and overall levels of somatic hypermutation in the full B-cell repertoire and in the 2,560 mAbs tested for binding. The results demonstrate that OmniFlic animals produce an abundance of antigen-specific antibodies with heavy chain clonotype diversity that is similar to what has been described with unrestricted light chain use in mammals. In addition, we show that sequence-based discovery is a highly effective and efficient way to identify a large number of diverse monoclonal antibodies to a protein target of interest.

When monoclonal antibodies are not monospecific: Hybridomas frequently express additional functional variable regions.

Monoclonal antibodies are commonly assumed to be monospecific, but anecdotal studies have reported genetic diversity in antibody heavy chain and light chain genes found within individual hybridomas. As the prevalence of such diversity has never been explored, we analyzed 185 random hybridomas, in a large multicenter dataset. The hybridomas analyzed were not biased towards those with cloning difficulties or known to have additional chains. Of the hybridomas we evaluated, 126 (68.1%) contained no additional productive chains, while the remaining 59 (31.9%) contained one or more additional productive heavy or light chains. The expression of additional chains degraded properties of the antibodies, including specificity, binding signal and/or signal-to-noise ratio, as determined by enzyme-linked immunosorbent assay and immunohistochemistry. The most abundant mRNA transcripts found in a hybridoma cell line did not necessarily encode the antibody chains providing the correct specificity. Consequently, when cloning antibody genes, functional validation of all possible VH and VL combinations is required to identify those with the highest affinity and lowest cross-reactivity. These findings, reflecting the current state of hybridomas used in research, reiterate the importance of using sequence-defined recombinant antibodies for research or diagnostic use.

MDR1A deficiency restrains tumor growth in murine colitis-associated carcinogenesis.

Patients with Ulcerative Colitis (UC) have an increased risk to develop colitis-associated colorectal cancer (CAC). Here, we found that protein expression of ABCB1 (ATP Binding Cassette Subfamily B Member 1) / MDR1 (multidrug resistance 1) was diminished in the intestinal mucosa of patients with active UC with or without CAC, but not in non-UC patients with sporadic colon cancer. We investigated the consequences of ABCB1/MDR1 loss-of-function in a common murine model for CAC (AOM/DSS). Mice deficient in MDR1A (MDR1A KO) showed enhanced intratumoral inflammation and cellular damage, which were associated with reduced colonic tumor size and decreased degree of dysplasia, when compared to wild-type (WT). Increased cell injury correlated with reduced capacity for growth of MDR1A KO tumor spheroids cultured ex-vivo. Gene expression analysis by microarray demonstrated that MDR1A deficiency shaped the inflammatory response towards an anti-tumorigenic microenvironment by downregulating genes known to be important mediators of cancer progression (PTGS2 (COX2), EREG, IL-11). MDR1A KO tumors showed increased gene expression of TNFSF10 (TRAIL), a known inducer of cancer cell death, and CCL12, a strong trigger of B cell chemotaxis. Abundant B220+ B lymphocyte infiltrates with interspersed CD138+ plasma cells were recruited to the MDR1A KO tumor microenvironment, concomitant with high levels of immunoglobulin light chain genes. In contrast, MDR1A deficiency in RAG2 KO mice that lack both B and T cells aggravated colonic tumor progression. MDR1A KO CD19+ B cells, but not WT CD19+ B cells, suppressed growth of colonic tumor-derived spheroids from AOM/DSS-WT mice in an ex-vivo co-culture system, implying that B-cell regulated immune responses contributed to delayed tumor development in MDR1A deficiency. In conclusion, we provide first evidence that loss of ABCB1/MDR1 function may represent an essential tumor-suppressive host defense mechanism in CAC.

Elevated IgA Plasmablast Levels in Subjects at Risk of Developing Rheumatoid Arthritis.

OBJECTIVE: The disease process in rheumatoid arthritis (RA) starts years before the clinical diagnosis is made, and elevated levels of disease-specific autoantibodies can be detected during this period. Early responses to known or novel autoantigens likely drive the eventual production of pathogenic autoimmunity. Importantly, the presence of disease-specific autoantibodies can identify individuals who are at high risk of developing RA but who do not currently have arthritis. The goal of the current study was to characterize plasmablasts from individuals at risk of developing RA. METHODS: We investigated antibody-secreting plasmablasts derived from a well-characterized cohort of individuals who were at risk of developing RA, based on RA-related serum autoantibody positivity, as compared to patients with early (<1 year) seropositive RA as well as healthy control subjects. The plasmablast antibody repertoires of at-risk subjects were analyzed using DNA barcode-based methods with paired heavy- and light-chain gene sequencing. Cells were single-cell sorted, the cell- and plate-specific DNA barcodes were sequentially added, and next-generation sequencing was performed. RESULTS: Total plasmablast levels were similar in the antibody-positive (1%) and control (0.4-1.6%) groups. However, increased frequencies of IgA+ versus IgG+ plasmablasts were observed in the antibody-positive group (39% IgA+ and 37% IgG+) as compared to other groups (1-9% IgA+ and 71-87% IgG+). Paired antibody sequences from antibody-positive subjects revealed cross-isotype clonal families and similar sequence characteristics in the IgA and IgG plasmablast repertoires. Antibody-positive individuals also demonstrated elevated serum levels of IgA isotype anti-cyclic citrullinated peptide 3 antibodies. CONCLUSION: The IgA plasmablast dominance in these antibody-positive individuals suggests that a subset of RA-related autoantibodies may arise from mucosal immune responses and may be involved in early disease pathogenesis in individuals who are at risk of developing RA.

A Comprehensive Analysis of the Phylogeny, Genomic Organization and Expression of Immunoglobulin Light Chain Genes in Alligator sinensis, an Endangered Reptile Species.

Crocodilians are evolutionarily distinct reptiles that are distantly related to lizards and are thought to be the closest relatives of birds. Compared with birds and mammals, few studies have investigated the Ig light chain of crocodilians. Here, employing an Alligator sinensis genomic bacterial artificial chromosome (BAC) library and available genome data, we characterized the genomic organization of the Alligator sinensis IgL gene loci. The Alligator sinensis has two IgL isotypes, λ and κ, the same as Anolis carolinensis. The Igλ locus contains 6 Cλ genes, each preceded by a Jλ gene, and 86 potentially functional Vλ genes upstream of (Jλ-Cλ)n. The Igκ locus contains a single Cκ gene, 6 Jκs and 62 functional Vκs. All VL genes are classified into a total of 31 families: 19 Vλ families and 12 Vκ families. Based on an analysis of the chromosomal location of the light chain genes among mammals, birds, lizards and frogs, the data further confirm that there are two IgL isotypes in the Alligator sinensis: Igλ and Igκ. By analyzing the cloned Igλ/κ cDNA, we identified a biased usage pattern of V families in the expressed Vλ and Vκ. An analysis of the junctions of the recombined VJ revealed the presence of N and P nucleotides in both expressed λ and κ sequences. Phylogenetic analysis of the V genes revealed V families shared by mammals, birds, reptiles and Xenopus, suggesting that these conserved V families are orthologous and have been retained during the evolution of IgL. Our data suggest that the Alligator sinensis IgL gene repertoire is highly diverse and complex and provide insight into immunoglobulin gene evolution in vertebrates.

Organization and genomic complexity of sheep immunoglobulin light chain gene loci.

Sheep is the representative of the artiodactyla and is an agriculturally important animal, but limited knowledge is available with regard to its immunoglobulin genes and their expression mechanisms in the sheep. Based on the recently released sheep genome, we have characterized the genomic organization of the sheep immunoglobulin light gene loci. The sheep Igλ locus, located on chromosome 17, contains 2Cλ segments each preceded by a Jλ, but the Cλ2 appears to be a pseudogene. A total of 42 Vλ segments (14 potentially functional genes, 1 ORF and 27 pseudogenes) were identified. In contrast, the Igκ locus on chromosome 3 contains only a single Cκ gene, 3 Jk segments and 13 Vκ segments (8 potentially functional genes and 5 pseudogenes). Analysis of junctions of the recombined VJ transcripts revealed a restricted Vλ4-Jλ1-Cλ1 recombination and Vk6-Jk3-Cκ recombination, respectively encode most of λ and κ chain antibody repertoire in the sheep despite more potential germline encoded combinatorial diversity. Therefore, the sheep may use gene conversion in combination with somatic hypermutation for antibody repertoire formation.

Proteomic analysis of white and yellow seminal plasma in turkeys (Meleagris gallopavo).

Yellow semen syndrome (YSS) is endemic within domestic turkey populations. Yellow semen is of lower quality and, when used for insemination, results in reduced fertility and hatchability. Little is known about the etiology of YSS. The aim of this study was to compare the proteome of white and yellow seminal plasma of turkeys using 1) 2-dimensional difference gel electrophoresis (2D-DIGE) to quantify seminal plasma proteins and 2) matrix-assisted laser desorption/ionization mass spectrometry to identify the proteins that are differentially abundant in white and yellow seminal plasma. A total of 49 protein spots (30 upregulated and 19 downregulated) were differentially expressed in yellow seminal plasma compared with white seminal plasma. Transthyretin and serum albumin-like showed a 3-fold increase in seminal plasma from males with YSS, and the latter was validated using Western blot analysis. A 3-fold increase was observed for hemopexin-like and immunoglobulin light chain V-J-C region. Pantetheinase-like showed a 1.3-fold increase. Ovotransferrin, hepatocyte growth factor activator, cysteine-rich secretory protein 3-like, and ferritin heavy chain-like showed a significant decrease (at least a 1.3-fold decrease) in yellow semen. Further studies are necessary to evaluate the precise function of the above-mentioned proteins in YSS and to establish quality markers of turkey semen to predict the reproductive potential of individual turkeys.

Human antibody expression in transgenic rats: comparison of chimeric IgH loci with human VH, D and JH but bearing different rat C-gene regions.

Expression of human antibody repertoires in transgenic animals has been accomplished by introducing large human Ig loci into mice and, more recently, a chimeric IgH locus into rats. With human VH, D and JH genes linked to the rat C-region antibody expression was significantly increased, similar to wild-type levels not found with fully human constructs. Here we compare four rat-lines containing the same human VH-region (comprising 22 VHs, all Ds and all JHs in natural configuration) but linked to different rat CH-genes and regulatory sequences. The endogenous IgH locus was silenced by zinc-finger nucleases. After breeding, all lines produced exclusively chimeric human H-chain with near normal IgM levels. However, in two lines poor IgG expression and inefficient immune responses were observed, implying that high expression, class-switching and hypermutation are linked to optimal enhancer function provided by the large regulatory region at the 3' end of the IgH locus. Furthermore, exclusion of Cδ and its downstream interval region may assist recombination. Highly diverse IgG and immune responses similar to normal rats were identified in two strains carrying diverse and differently spaced C-genes.

A critical context-dependent role for E boxes in the targeting of somatic hypermutation.

Secondary B cell repertoire diversification occurs by somatic hypermutation (SHM) in germinal centers following Ag stimulation. In SHM, activation-induced cytidine deaminase mutates the V region of the Ig genes to increase the affinity of Abs. Although SHM acts primarily at Ig loci, low levels of off-target mutation can result in oncogenic DNA damage, illustrating the importance of understanding SHM targeting mechanisms. A candidate targeting motif is the E box, a short DNA sequence (CANNTG) found abundantly in the genome and in many SHM target genes. Using a reporter assay in chicken DT40 B cells, we previously identified a 1928-bp portion of the chicken IgL locus capable of supporting robust SHM. In this article, we demonstrate that mutation of all 20 E boxes in this fragment reduces SHM targeting activity by 90%, and that mutation of subsets of E boxes reveals a functional hierarchy in which E boxes within "core" targeting regions are of greatest importance. Strikingly, when the sequence and spacing of the 20 E boxes are preserved but surrounding sequences are altered, SHM targeting activity is eliminated. Hence, although E boxes are vital SHM targeting elements, their function is completely dependent on their surrounding sequence context. These results suggest an intimate cooperation between E boxes and other sequence motifs in SHM targeting to Ig loci and perhaps also in restricting mistargeting to certain non-Ig loci.

Immunoglobulin light chains in medaka (Oryzias latipes).

The gene segments encoding antibodies have been studied in many capacities and represent some of the best-characterized gene families in traditional animal disease models (mice and humans). To date, multiple immunoglobulin light chain (IgL) isotypes have been found in vertebrates and it is unclear as to which isotypes might be more primordial in nature. Sequence data emerging from an array of fish genome projects is a valuable resource for discerning complex multigene assemblages in this critical branch point of vertebrate phylogeny. Herein, we have analyzed the genomic organization of medaka (Oryzias latipes) IgL gene segments based on recently released genome data. The medaka IgL locus located on chromosome 11 contains at least three clusters of IgL gene segments comprised of multiple gene assemblages of the kappa light chain isotype. These data suggest that medaka IgL gene segments may undergo both intra- and inter-cluster rearrangements as a means to generate additional diversity. Alignments of expressed sequence tags to concordant gene segments which revealed each of the three IgL clusters are expressed. Collectively, these data provide a genomic framework for IgL genes in medaka and indicate that Ig diversity in this species is achieved from at least three distinct chromosomal regions.

Molecular characterization and expression analysis of four isotypes of immunoglobulin light chain genes in orange-spotted grouper, Epinephelus coioides.

To date, many immunoglobulin (Ig) genes have been identified in diverse teleost species, but the contributions of different types of light chain (IgL) to the immune response remain unclear. Screening of a stimulated kidney cDNA library from orange-spotted grouper (Osg, Epinephelus coioides) resulted in the identification of 26 full Ig light chain (OsgIgL) coding sequences. These 26 OsgIgLs encoded peptides from 235 to 248 amino acid residues and could be grouped into five variable (V(L)) and four constant (C(L)) isotypes. The C(L) regions contained three conserved cysteine residues that may participate in intra- or inter-chain disulfide bond formation. The four C(L) isotypes could be sub-grouped into two serological types: κ (C(L)-I, C(L)-II and C(L)-III) and σ (C(L)-IV), by phylogenetic analysis. The OsgIgL genes were found to be expressed in various tissues, with greatest levels of expression observed in the head-kidney and spleen. The major expression type was C(L)-I, which comprised 92% and 91% of total OsgIgL gene expression in the head-kidney and spleen, respectively. Transcription of all four C(L) isotypes was differentially affected in response to various immunostimulators, including lipopolysaccharide (LPS), poly I:C and grouper iridovirus (GIV). Induction of OsgIgL genes in response to immunostimulators was particularly dramatic in the spleen, suggesting this organ holds particular importance for the regulation of OsgIgL expression. Furthermore, vaccination of grouper with formalin-inactivated GIV also induced differential patterns of expression in all four OsgIgL isotypes. In summary, the significant and diverse patterns of transcriptional induction observed for OsgIgL isotypes in the spleen and head-kidney imply that each isotype may have unique roles in the immune response.

Transcriptional analysis of equine λ-light chains in the horse breeds Rhenish-German Coldblood and Hanoverian Warmblood.

The present study analyzed equine λ-light chain genes (IGLV and IGLC) transcribed in the horse breeds Rhenish-German Coldblood (RGC) and Hanoverian Warmblood (HW). Primers were generated for the major expressed IGLV subgroup 8. The significant majority of the sequences represented IGLC6/7. In RGC, IGLC1 and IGLC5 were observed in significant higher frequencies than IGLC4. In HW, significant differences were obtained for the transcription of IGLC1 and IGLC5. IGLC4 was not determined in this breed. Five allotypic IGLC1 variants, four allotypic IGLC5 variants, and three allelic as well as two allotypic IGLC6/7 variants were identified. IGLC1(b, d), IGLC5(c, d), and IGLC6/7(a3, b) were detected in RGC while IGLC1(c) and IGLC5(b) were solely found in HW. Furthermore, 11 out of 144 known IGLV-segments were transcribed of which IGLV15 and IGLV17 were preferred significantly. IGLV25 displayed significant differences in the rearrangement between both breeds. The classified pseudogenes IGLV101ψ and IGLV74ψ were also identified. Rearrangements with IGLC-genes showed significant differences for IGLV15 in both breeds, whereas IGLV25 also revealed significant differences between the breeds. The transcriptional orientation of the functional segments has no influence on the occurrence of the IGLV.

Primary high-grade B-cell lymphoma of the breast with concurrent IGH-BCL2 and MYC-IGL translocations in an adolescent patient.

BCL2 and MYC are oncogenes often deregulated in lymphomas. Concurrent IGH-BCL2 and MYC translocations result in a highly aggressive behavior of these tumors. Both primary breast lymphoma and lymphoma with concurrent BCL2-IGH and MYC translocations are rare and are primarily seen in adult patients. As a result of limited clinician experience and the condition's rarity, it poses a great challenge to pediatric pathologists and oncologists in terms of making an accurate diagnosis and choosing better treatment regimens. In this article, we report a case of an adolescent patient who presented with high-grade breast lymphoma with concurrent BCL2-IGH and MYC-IGL translocations, and we review the clinical, pathological, and genetic features; management strategies; and outcomes associated with this unusual neoplasm.

Targeted annotation of immunoglobulin light chain (IgL) genes in zebrafish from BAC clones reveals kappa-like recombining/deleting elements within IgL constant regions.

Genomic organization, composition, and microsynteny of immunoglobulin light chain (IgL) gene segments in the zebrafish were analyzed through the identification and annotation of overlapping BAC clone insert sequences and an Illumina de novo assembly. The resultant gap-free IgL annotation confirmed a number of previous conclusions about teleost IgL including: suites of (V(L)-J(L)-C(L)) clusters on multiple chromosomes; V(L) in the same or opposite transcriptional orientation as J(L) and C(L); and the apparent absence of lambda IgL in the zebrafish model. In addition, palindromic heptamers (CACAGTG or CACTGTG) within the 3' region of zebrafish C(L) were identified. In mammals, heptamers within J(κ)-C(κ) introns can recombine with downstream kappa deleting elements (Kde) to ablate C(κ) regions prior to rearrangements of V(λ)-J(λ) gene segments. The presence of palindromic heptamers within zebrafish C(L) is intriguing as their recombination with intact RSS might result in the deletion of a large portion of the C(L) thereby permanently silencing C(L) exons within the IgL locus. Given that bony fish have appreciably more C(L) spread over more chromosomes than mice and humans, it is plausible the presence of recombining sequences within C(L) may be tied to a need for heightened mechanisms to facilitate allelic exclusion or receptor editing. Collectively, with this report, gap-free annotations of the heavy and light chain Ig loci have now been completed for Danio rerio, the only teleost for which this has been accomplished, thereby strengthening the overall utility of zebrafish as a model organism for both comparative immunology and biomedical research.

Targets of somatic hypermutation within immunoglobulin light chain genes in zebrafish.

In mammals, somatic hypermutation (SHM) of immunoglobulin (Ig) genes is critical for the generation of high-affinity antibodies and effective immune responses. Knowledge of sequence-specific biases in the targeting of somatic mutations can be useful for studies aimed at understanding antibody repertoires produced in response to infections, B-cell neoplasms, or autoimmune disease. To evaluate potential nucleotide targets of somatic mutation in zebrafish (Danio rerio), an enriched IgL cDNA library was constructed and > 250 randomly selected clones were sequenced and analysed. In total, 55 unique VJ-C sequences were identified encoding a total of 125 mutations. Mutations were most prevalent in V(L) with a bias towards single base transitions and increased mutation in the complementarity-determining regions (CDRs). Overall, mutations were overrepresented at WRCH/DGYW motifs suggestive of activation-induced cytidine deaminase (AID) targeting which is common in mice and humans. In contrast to mammalian models, N and P addition was not observed and mutations at AID hotspots were largely restricted to palindromic WRCH/DGYW motifs. Mutability indexes for di- and trinucleotide combinations confirmed C/G targets within WRCH/DGYW motifs to be statistically significant mutational hotspots and showed trinucleotides ATC and ATG to be mutation coldspots. Additive mutations in VJ-C sequences revealed patterns of clonal expansion consistent with affinity maturation responses seen in higher vertebrates. Taken together, the data reveal specific nucleotide targets of SHM in zebrafish and suggest that AID and affinity maturation contribute to antibody diversification in this emerging immunological model.