Temporal analyses reveal a pivotal role for sense and antisense enhancer RNAs in coordinate immunoglobulin lambda locus activation.

Transcription enhancers are essential activators of V(D)J recombination that orchestrate non-coding transcription through complementary, unrearranged gene segments. How transcription is coordinately increased at spatially distinct promoters, however, remains poorly understood. Using the murine immunoglobulin lambda (Igλ) locus as model, we find that three enhancer-like elements in the 3' Igλ domain, Eλ3-1, HSCλ1 and HSE-1, show strikingly similar transcription factor binding dynamics and close spatial proximity, suggesting that they form an active enhancer hub. Temporal analyses show coordinate recruitment of complementary V and J gene segments to this hub, with comparable transcription factor binding dynamics to that at enhancers. We find further that E2A, p300, Mediator and Integrator bind to enhancers as early events, whereas YY1 recruitment and eRNA synthesis occur later, corresponding to transcription activation. Remarkably, the interplay between sense and antisense enhancer RNA is central to both active enhancer hub formation and coordinate Igλ transcription: Antisense Eλ3-1 eRNA represses Igλ activation whereas temporal analyses demonstrate that accumulating levels of sense eRNA boost YY1 recruitment to stabilise enhancer hub/promoter interactions and lead to coordinate transcription activation. These studies therefore demonstrate for the first time a critical role for threshold levels of sense versus antisense eRNA in locus activation.

A rare case of IgA lambda multiple myeloma in a 32-year-old woman with t(14;16) translocation associated with kidney injury and non-albumin proteinuria.

BACKGROUND: Multiple myeloma (MM) is a malignant disorder characterized by monoclonal differentiated plasma cells. While it is more commonly diagnosed in elderly individuals, it can also affect younger populations, though with a lower incidence. CASE PRESENTATION: Here, we present the case of a 32-year-old woman diagnosed with IgA lambda MM. She presented with fatigue, nausea, acute kidney injury (AKI) with a rapid increase in creatinine, and anemia. A kidney biopsy was done to rule out a rapidly progressive glomerular disease and a diagnosis was thus reached. A genetic workup revealed t(14;16) translocation and an extra copy of TP53. The patient received aggressive intravenous steroids and intravenous fluid resuscitation, resulting in an improvement in renal function. Treatment with daratumumab in combination with bortezomib, thalidomide, and dexamethasone was initiated and well tolerated. Despite the generally poor prognosis of IgA MM, our case emphasizes the importance of considering MM in young patients with unexplained kidney injury. CONCLUSION: Early recognition and prompt intervention are essential in managing MM patients, especially in those with high-risk cytogenetic abnormalities. This case serves as a reminder for clinicians to maintain a high index of suspicion for MM, even in younger populations, when presented with unexplained kidney injury.

Characterization of the immunoglobulin lambda chain locus from diverse populations reveals extensive genetic variation.

Immunoglobulins (IGs), crucial components of the adaptive immune system, are encoded by three genomic loci. However, the complexity of the IG loci severely limits the effective use of short read sequencing, limiting our knowledge of population diversity in these loci. We leveraged existing long read whole-genome sequencing (WGS) data, fosmid technology, and IG targeted single-molecule, real-time (SMRT) long-read sequencing (IG-Cap) to create haplotype-resolved assemblies of the IG Lambda (IGL) locus from 6 ethnically diverse individuals. In addition, we generated 10 diploid assemblies of IGL from a diverse cohort of individuals utilizing IG-Cap. From these 16 individuals, we identified significant allelic diversity, including 36 novel IGLV alleles. In addition, we observed highly elevated single nucleotide variation (SNV) in IGLV genes relative to IGL intergenic and genomic background SNV density. By comparing SNV calls between our high quality assemblies and existing short read datasets from the same individuals, we show a high propensity for false-positives in the short read datasets. Finally, for the first time, we nucleotide-resolved common 5-10 Kb duplications in the IGLC region that contain functional IGLJ and IGLC genes. Together these data represent a significant advancement in our understanding of genetic variation and population diversity in the IGL locus.

IGLV3-21*01 is an inherited risk factor for CLL through the acquisition of a single-point mutation enabling autonomous BCR signaling.

The prognosis of chronic lymphocytic leukemia (CLL) depends on different markers, including cytogenetic aberrations, oncogenic mutations, and mutational status of the immunoglobulin (Ig) heavy-chain variable (IGHV) gene. The number of IGHV mutations distinguishes mutated (M) CLL with a markedly superior prognosis from unmutated (UM) CLL cases. In addition, B cell antigen receptor (BCR) stereotypes as defined by IGHV usage and complementarity-determining regions (CDRs) classify ∼30% of CLL cases into prognostically important subsets. Subset 2 expresses a BCR with the combination of IGHV3-21-derived heavy chains (HCs) with IGLV3-21-derived light chains (LCs), and is associated with an unfavorable prognosis. Importantly, the subset 2 LC carries a single-point mutation, termed R110, at the junction between the variable and constant LC regions. By analyzing 4 independent clinical cohorts through BCR sequencing and by immunophenotyping with antibodies specifically recognizing wild-type IGLV3-21 and R110-mutated IGLV3-21 (IGLV3-21R110), we show that IGLV3-21R110-expressing CLL represents a distinct subset with poor prognosis independent of IGHV mutations. Compared with other alleles, only IGLV3-21*01 facilitates effective homotypic BCR-BCR interaction that results in autonomous, oncogenic BCR signaling after acquiring R110 as a single-point mutation. Presumably, this mutation acts as a standalone driver that transforms IGLV3-21*01-expressing B cells to develop CLL. Thus, we propose to expand the conventional definition of CLL subset 2 to subset 2L by including all IGLV3-21R110-expressing CLL cases regardless of IGHV mutational status. Moreover, the generation of monoclonal antibodies recognizing IGLV3-21 or mutated IGLV3-21R110 facilitates the recognition of B cells carrying this mutation in CLL patients or healthy donors.

Compound screening identified gossypetin and isoquercitrin as novel inhibitors for amyloid fibril formations of Vλ6 proteins associated with AL amyloidosis.

AL amyloidosis is a life-threatening disease characterized by the deposition of amyloidogenic immunoglobulin light chain secreted from clonal plasma cells. Here we established an in-vitro screening system of amyloid inhibition of a variable domain in λ6 light chain mutant (Vλ6), Wil, and screened a food-additive compound library to identify compounds inhibiting the fibril formation. We found gossypetin and isoquercitrin as novel inhibitors. NMR analysis showed that both compounds directly interacted with natively-folded Wil, and proteolysis experiments demonstrated that these compounds conferred proteolytic resistance, suggesting that the compounds enhance the kinetic stability of Wil. Since gossypetin and isoquercitrin specifically interacted with the protein at micromolar concentrations, these compounds could be used as lead to further develop inhibitors against AL amyloidosis.

Murine gammaherpesvirus infection is skewed toward Igλ+ B cells expressing a specific heavy chain V-segment.

One of the defining characteristics of the B cell receptor (BCR) is the extensive diversity in the repertoire of immunoglobulin genes that make up the BCR, resulting in broad range of specificity. Gammaherpesviruses are B lymphotropic viruses that establish life-long infection in B cells, and although the B cell receptor plays a central role in B cell biology, very little is known about the immunoglobulin repertoire of gammaherpesvirus infected cells. To begin to characterize the Ig genes expressed by murine gammaherpesvirus 68 (MHV68) infected cells, we utilized single cell sorting to sequence and clone the Ig variable regions of infected germinal center (GC) B cells and plasma cells. We show that MHV68 infection is biased towards cells that express the Igλ light chain along with a single heavy chain variable gene, IGHV10-1*01. This population arises through clonal expansion but is not viral antigen specific. Furthermore, we show that class-switching in MHV68 infected cells differs from that of uninfected cells. Fewer infected GC B cells are class-switched compared to uninfected GC B cells, while more infected plasma cells are class-switched compared to uninfected plasma cells. Additionally, although they are germinal center derived, the majority of class switched plasma cells display no somatic hypermutation regardless of infection status. Taken together, these data indicate that selection of infected B cells with a specific BCR, as well as virus mediated manipulation of class switching and somatic hypermutation, are critical aspects in establishing life-long gammaherpesvirus infection.

Immunoglobulin variable domain high-throughput sequencing reveals specific novel mutational patterns in POEMS syndrome.

Polyneuropathy, organomegaly, endocrinopathy, monoclonal gammopathy, and skin changes (POEMS) syndrome is a rare multisystem disease resulting from an underlying plasma cell (PC) dyscrasia. The pathophysiology of the disease remains unclear, but the role of the monoclonal immunoglobulin (Ig) light chain (LC) is strongly suspected because of the highly restrictive usage of 2 λ variable (V) domains (IGLV1-40 and IGLV1-44) and the general improvement of clinical manifestations after PC clone-targeted treatment. However, the diagnostic value of Ig LC sequencing, especially in the case of incomplete forms of the disease, remains to be determined. Using a sensitive high-throughput Ig repertoire sequencing on RNA (rapid amplification of cDNA ends-based repertoire sequencing [RACE-RepSeq]), we detected a λ LC monoclonal expansion in the bone marrow (BM) of 83% of patients with POEMS syndrome, including some in whom BM tests routinely performed to diagnose plasma cell dyscrasia failed to detect λ+ monoclonal PCs. Twenty-four (83%) of the 29 LC clonal sequences found were derived from the IGLV1-40 and IGLV1-44 germline genes, as well as 2 from the closely related IGLV1-36 gene, and all were associated with an IGLJ3*02 junction (J) gene, confirming the high restriction of VJ region usage in POEMS syndrome. RACE-RepSeq VJ full-length sequencing additionally revealed original mutational patterns, the strong specificity of which might crucially help establish or eliminate the diagnosis of POEMS syndrome in uncertain cases. Thus, RACE-RepSeq appears as a sensitive, rapid, and specific tool to detect low-abundance PC clones in BM and assign them to POEMS syndrome, with all the consequences for therapeutic options.

An expanded genetic code facilitates antibody chemical conjugation involving the lambda light chain.

Most of the currently approved therapeutic antibodies are of the immunoglobulin gamma (IgG) κ isotype, leaving a vast opportunity for the use of IgGλ in medical treatments. The incorporation of designer amino acids into antibodies enables efficient and precise manufacturing of antibody chemical conjugates. Useful conjugation sites have been explored in the constant domain of the human κ-light chain (LCκ), which is no more than 38% identical to its LCλ counterpart in amino acid sequence. In the present study, we used an expanded genetic code for site-specifically incorporating Nε-(o-azidobenzyloxycarbonyl)-l-lysine (o-Az-Z-Lys) into the antigen-binding fragment (Fab) of an IgGλ, cixutumumab. Ten sites in the LCλ constant domain were found to support efficient chemical conjugation exploiting the bio-orthogonal azido chemistry. Most of the identified positions are located in regions that differ between the two light chain isotypes, thus being specific to the λ isotype. Finally, o-Az-Z-Lys was incorporated into the Fab fragments of cixutumumab and trastuzumab to chemically combine them; the resulting bispecific Fab-dimers showed a strong antagonistic activity against a cancer cell line. The present results expand the utility of the chemical conjugation method to the whole spectrum of humanized antibodies, including the λ isotype.

Development of immunoglobulin lambda-chain-positive B cells, but not editing of immunoglobulin kappa-chain, depends on NF-kappaB signals.

By genetically ablating IkappaB kinase (IKK)-mediated activation of the transcription factor NF-kappaB in the B cell lineage and by analyzing a mouse mutant in which immunoglobulin lambda-chain-positive B cells are generated in the absence of rearrangements in the locus encoding immunoglobulin kappa-chain, we define here two distinct, consecutive phases of early B cell development that differ in their dependence on IKK-mediated NF-kappaB signaling. During the first phase, in which NF-kappaB signaling is dispensable, predominantly kappa-chain-positive B cells are generated, which undergo efficient receptor editing. In the second phase, predominantly lambda-chain-positive B cells are generated whose development is ontogenetically timed to occur after rearrangements of the locus encoding kappa-chain. This second phase of development is dependent on NF-kappaB signals, which can be substituted by transgenic expression of the prosurvival factor Bcl-2.

KSHV induces immunoglobulin rearrangements in mature B lymphocytes.

Kaposi sarcoma herpesvirus (KSHV/HHV-8) is a B cell tropic human pathogen, which is present in vivo in monotypic immunoglobulin λ (Igλ) light chain but polyclonal B cells. In the current study, we use cell sorting to infect specific B cell lineages from human tonsil specimens in order to examine the immunophenotypic alterations associated with KSHV infection. We describe IL-6 dependent maturation of naïve B lymphocytes in response to KSHV infection and determine that the Igλ monotypic bias of KSHV infection in vivo is due to viral induction of BCR revision. Infection of immunoglobulin κ (Igκ) naïve B cells induces expression of Igλ and isotypic inclusion, with eventual loss of Igκ. We show that this phenotypic shift occurs via re-induction of Rag-mediated V(D)J recombination. These data explain the selective presence of KSHV in Igλ B cells in vivo and provide the first evidence that a human pathogen can manipulate the molecular mechanisms responsible for immunoglobulin diversity.

Analytical evaluation of the clonoSEQ Assay for establishing measurable (minimal) residual disease in acute lymphoblastic leukemia, chronic lymphocytic leukemia, and multiple myeloma.

BACKGROUND: The clonoSEQ® Assay (Adaptive Biotechnologies Corporation, Seattle, USA) identifies and tracks unique disease-associated immunoglobulin (Ig) sequences by next-generation sequencing of IgH, IgK, and IgL rearrangements and IgH-BCL1/2 translocations in malignant B cells. Here, we describe studies to validate the analytical performance of the assay using patient samples and cell lines. METHODS: Sensitivity and specificity were established by defining the limit of detection (LoD), limit of quantitation (LoQ) and limit of blank (LoB) in genomic DNA (gDNA) from 66 patients with multiple myeloma (MM), acute lymphoblastic leukemia (ALL), or chronic lymphocytic leukemia (CLL), and three cell lines. Healthy donor gDNA was used as a diluent to contrive samples with specific DNA masses and malignant-cell frequencies. Precision was validated using a range of samples contrived from patient gDNA, healthy donor gDNA, and 9 cell lines to generate measurable residual disease (MRD) frequencies spanning clinically relevant thresholds. Linearity was determined using samples contrived from cell line gDNA spiked into healthy gDNA to generate 11 MRD frequencies for each DNA input, then confirmed using clinical samples. Quantitation accuracy was assessed by (1) comparing clonoSEQ and multiparametric flow cytometry (mpFC) measurements of ALL and MM cell lines diluted in healthy mononuclear cells, and (2) analyzing precision study data for bias between clonoSEQ MRD results in diluted gDNA and those expected from mpFC based on original, undiluted samples. Repeatability of nucleotide base calls was assessed via the assay's ability to recover malignant clonotype sequences across several replicates, process features, and MRD levels. RESULTS: LoD and LoQ were estimated at 1.903 cells and 2.390 malignant cells, respectively. LoB was zero in healthy donor gDNA. Precision ranged from 18% CV (coefficient of variation) at higher DNA inputs to 68% CV near the LoD. Variance component analysis showed MRD results were robust, with expected laboratory process variations contributing ≤3% CV. Linearity and accuracy were demonstrated for each disease across orders of magnitude of clonal frequencies. Nucleotide sequence error rates were extremely low. CONCLUSIONS: These studies validate the analytical performance of the clonoSEQ Assay and demonstrate its potential as a highly sensitive diagnostic tool for selected lymphoid malignancies.

Concurrent immunoglobulin G-lambda type multiple myeloma and mixed cellularity classical Hodgkin lymphoma: A case report.

A 66-year-old man with a swollen right inguinal lymph node (LN) had pain on the lower side of the back. Computed tomography revealed bone disease in the back and swollen right inguinal LNs. Laboratory studies showed anemia and serum immunoglobulin G-lambda (IgG-λ) type monoclonal protein. The bone marrow contained 39.6% plasma cells. He was diagnosed with IgG-λ type multiple myeloma (MM). However, the pathological findings of the right inguinal LN were mixed cellular classical Hodgkin lymphoma (HL). The administration of melphalan, prednisone, and bortezomib (MPB) was started for MM; however, swelling in the right inguinal LN increased. After three cycles of MPB, the administration of doxorubicin, bleomycin, vinblastine, and dacarbazine (ABVD) was started for HL. However, HL was refractory to ABVD. Pancytopenia subsequently progressed and rapid swelling occurred in his LNs. He died 7 months after diagnosis. Multiple myeloma was diagnosed, based on the typical symptoms, although the pathological findings of the LN indicated a diagnosis of HL. We analyzed the molecular relationship between MM and HL cells using a direct sequencing method. The sequencing results demonstrated that the variable-diversity-joining (VDJ) region of the IgH gene was identified with 94.4% of IGLV3-32*01 in the bone marrow sample at diagnosis. Furthermore, clonotypic IgH sequence was identified in CD30-positive cells from the LN. These results suggested that the clonal HL cells were derived from the same source as the clonal MM cells and demonstrated that MM and HL in this patient may have originated from the same B cell progenitor.

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.

Zebrafish model of amyloid light chain cardiotoxicity: regeneration versus degeneration.

Cardiac dysfunction is the most frequent cause of morbidity and mortality in amyloid light chain (AL) amyloidosis caused by a clonal immunoglobulin light chain (LC). Previously published transgenic animal models of AL amyloidosis have not recapitulated the key phenotype of cardiac dysfunction seen in AL amyloidosis, which has limited our understanding of the disease mechanisms in vivo, as well as the development of targeted AL therapeutics. We have developed a transgenic zebrafish model in which a λ LC derived from a patient with AL amyloidosis is conditionally expressed in the liver under the control of the Gal4 upstream activation sequence enhancer system. Circulating LC levels of 125 µg/ml in these transgenic zebrafish are comparable to median pathological serum LC levels. Functional analysis links abnormal contractile function with evidence of cellular and molecular proteotoxicity in the heart, including increased cell death and autophagy. However, despite pathological and functional phenotypes analogous to human AL, the lifespan of the transgenic fish is comparable to control fish without the expressed AL-LC transgene. Nuclear labeling experiments suggest increased cardiac proliferation in the transgenic fish, which can be counteracted by treatment with a small molecule proliferation inhibitor leading to increased zebrafish mortality because of cardiac apoptosis and functional deterioration. This transgenic zebrafish model provides a platform to study underlying AL disease mechanisms in vivo further. NEW & NOTEWORTHY Heart failure is a major cause of mortality in amyloid light (AL) amyloidosis, yet it has been difficult to model in animals. We report the generation of a transgenic zebrafish model for AL amyloidosis with pathological concentration of circulating human light chain protein that results in cardiac dysfunction. The light chain toxicity triggers regeneration in the zebrafish heart resulting in functional compensation early in life, but with age develops into cardiac dysfunction.

Localized Lichen Myxedematosus With Plasma Cell Light Chain Restriction: Is It the Exception or the Rule?

Lichen myxedematosus is a chronic cutaneous mucinosis that can present on a spectrum from localized cutaneous lesions to systemic disease of scleromyxedema. The clinical presentation of localized cutaneous lichen myxedematosus is waxy lichenoid papules, nodules, and/or plaques that have histopathologic findings of mucin deposition and a variable degree of fibroblast proliferation. There is an absence of serum paraproteins, and there are no other systemic causes of cutaneous mucinosis such as thyroid disease. The pathogenesis of lichen myxedematosus is unknown. We report 3 cases of localized cutaneous lichen myxedematosus with a light chain-restricted plasmacytic component by in situ hybridization. Our findings deliver an insight for disease pathogenesis and highlight for the first time, the significance of plasma cells in lesions of localized cutaneous lichen myxedematosus. We suggest that plasma cell light chain restriction could represent a clue to distinguish localized cutaneous disease from systemic disease.

Ultrasensitive automated RNA in situ hybridization for kappa and lambda light chain mRNA detects B-cell clonality in tissue biopsies with performance comparable or superior to flow cytometry.

The assessment of B-cell clonality is a critical component of the evaluation of suspected lymphoproliferative disorders, but analysis from formalin-fixed, paraffin-embedded tissues can be challenging if fresh tissue is not available for flow cytometry. Immunohistochemical and conventional bright field in situ hybridization stains for kappa and lambda are effective for evaluation of plasma cells but are often insufficiently sensitive to detect the much lower abundance of light chains present in B-cells. We describe an ultrasensitive RNA in situ hybridization assay that has been adapted for use on an automated immunohistochemistry platform and compare results with flow cytometry in 203 consecutive tissues and 104 consecutive bone marrows. Overall, in 203 tissue biopsies, RNA in situ hybridization identified light chain-restricted B-cells in 85 (42%) vs 58 (29%) by flow cytometry. Within 83 B-cell non-Hodgkin lymphomas, RNA in situ hybridization identified restricted B-cells in 74 (89%) vs 56 (67%) by flow cytometry. B-cell clonality could be evaluated in only 23/104 (22%) bone marrow cases owing to poor RNA preservation, but evaluable cases showed 91% concordance with flow cytometry. RNA in situ hybridization allowed for recognition of biclonal/composite lymphomas not identified by flow cytometry and highlighted unexpected findings, such as coexpression of kappa and lambda RNA in 2 cases and the presence of lambda light chain RNA in a T lymphoblastic lymphoma. Automated RNA in situ hybridization showed excellent interobserver reproducibility for manual evaluation (average K=0.92), and an automated image analysis system showed high concordance (97%) with manual evaluation. Automated RNA in situ hybridization staining, which can be adopted on commonly utilized immunohistochemistry instruments, allows for the interpretation of clonality in the context of the morphological features in formalin-fixed, paraffin-embedded tissues with a clinical sensitivity similar or superior to flow cytometry.

Localized conformational changes trigger the pH-induced fibrillogenesis of an amyloidogenic λ light chain protein.

Solvent conditions modulate the expression of the amyloidogenic potential of proteins. In this work the effect of pH on the fibrillogenic behavior and the conformational properties of 6aJL2, a model protein of the highly amyloidogenic variable light chain λ6a gene segment, was examined. Ordered aggregates showing the ultrastructural and spectroscopic properties observed in amyloid fibrils were formed in the 2.0-8.0 pH range. At pH <3.0 a drastic decrease in lag time and an increase in fibril formation rate were found. In the 4.0-8.0 pH range there was no spectroscopic evidence for significant conformational changes in the native state. Likewise, heat capacity measurements showed no evidence for residual structure in the unfolded state. However, at pH <3.0 stability is severely decreased and the protein suffers conformational changes as detected by circular dichroism, tryptophan and ANS fluorescence, as well as by NMR spectroscopy. Molecular dynamics simulations indicate that acid-induced conformational changes involve the exposure of the loop connecting strands E and F. These results are compatible with pH-induced changes in the NMR spectra. Overall, the results indicate that the mechanism involved in the acid-induced increase in the fibrillogenic potential of 6aJL2 is profoundly different to that observed in κ light chains, and is promoted by localized conformational changes in a region of the protein that was previously not known to be involved in acid-induced light chain fibril formation. The identification of this region opens the potential for the design of specific inhibitors.

Clonal immunoglobulin λ light-chain gene rearrangements detected by next generation sequencing in POEMS syndrome.

Polyneuropathy, organomegaly, endocrinopathy, M-protein, and skin changes (POEMS) syndrome is a rare plasma cell dyscrasia characterized by polyneuropathy, organomegaly, endocrinopathy, extravascular fluid overload, M protein, and a myriad of skin changes. The pathogenesis is poorly understood, but monoclonal plasma cells are λ-restricted and these immunoglobulin λ light chain variable (IGLV) region genes are derived from only two germlines, either IGLV1-44 or 1-40. Here we analyzed the clonal IGLV gene rearrangements of genomic DNA samples of bone marrow mononuclear cells using next-generation sequencing (NGS) to understand the clonal composition of IGLV genes in patients with POEMS syndrome (n = 30). The dominant IGLV gene rearrangement of POEMS syndrome-specific germline sequences were significantly increased in 11 POEMS patients (36.7%; IGLV1-44: n = 9, IGLV1-40: n = 2). In some cases, IGLV gene rearrangement clone was not detected as significant increase but was detected using cDNA samples by heteroduplex (HD) analysis and Sanger sequencing, suggesting that the quite small number of monoclonal plasma cells may produce large quantity of mRNA of monoclonal proteins. However, significant increase of dominant clone sizes was not directly linked to the initial disease status. On the other hand, in cases with significantly increased dominant clones, they decreased and increased accompanying with disease remission and relapse. These data demonstrate that monoclonal plasma cells are related to the pathogenesis of POEMS syndrome.

Convergent mechanisms favor fast amyloid formation in two lambda 6a Ig light chain mutants.

Extracellular deposition as amyloids of immunoglobulin light chains causes light chain amyloidosis. Among the light chain families, lambda 6a is one of the most frequent in light chain amyloidosis patients. Its germline protein, 6aJL2, and point mutants, R24G and P7S, are good models to study fibrillogenesis, because their stability and fibril formation characteristics have been described. Both mutations make the germline protein unstable and speed up its ability to aggregate. To date, there is no molecular mechanism that explains how these differences in amyloidogenesis can arise from a single mutation. To look into the structural and dynamical differences in the native state of these proteins, we carried out molecular dynamics simulations at room temperature. Despite the structural similarity of the germline protein and the mutants, we found differences in their dynamical signatures that explain the mutants' increased tendency to form amyloids. The contact network alterations caused by the mutations, though different, converge in affecting two anti-aggregation motifs present in light chain variable domains, suggesting a different starting point for aggregation in lambda chains compared to kappa chains.

Targeting of somatic hypermutation by immunoglobulin enhancer and enhancer-like sequences.

Somatic hypermutation (SH) generates point mutations within rearranged immunoglobulin (Ig) genes of activated B cells, providing genetic diversity for the affinity maturation of antibodies. SH requires the activation-induced cytidine deaminase (AID) protein and transcription of the mutation target sequence, but how the Ig gene specificity of mutations is achieved has remained elusive. We show here using a sensitive and carefully controlled assay that the Ig enhancers strongly activate SH in neighboring genes even though their stimulation of transcription is negligible. Mutations in certain E-box, NFκB, MEF2, or Ets family binding sites--known to be important for the transcriptional role of Ig enhancers--impair or abolish the activity. Full activation of SH typically requires a combination of multiple Ig enhancer and enhancer-like elements. The mechanism is evolutionarily conserved, as mammalian Ig lambda and Ig heavy chain intron enhancers efficiently stimulate hypermutation in chicken cells. Our results demonstrate a novel regulatory function for Ig enhancers, indicating that they either recruit AID or alter the accessibility of the nearby transcription units.