Autosomal recessive agammaglobulinemia due to defect in µ heavy chain caused by a novel mutation in the IGHM gene.

Agammaglobulinemia is a primary immunodeficiency disorder characterized by profoundly low or absent serum antibodies and low or absent circulating B cells. The most common form is X-linked agammaglobulinemia (XLA) caused by mutations in BTK gene. The remaining cases, clinically similar to XLA, are autosomal recessive agammaglobulinemia (ARA). Nearly 30% of ARA cases present mutations in the µ heavy constant region gene IGHM. Here, we present a 7-month-old patient, born from non-consanguineous parents, who is affected by ARA due to defect in the µ heavy chain. The genetic study showed that the patient is compound heterozygous for an IGHM gene deletion and the novel nonsense mutation X57331.1:g.275C>A (p.Tyr43*) (ClinVar Accession Number: SCV000537868.1). This finding allows for an adequate genetic counseling to the family and also broadens the spectrum of already described point mutations at this locus. The IGHM gene is very complex and it is likely that yet unidentified mutations appear in other patients.

Unravelling the immunopathological mechanisms of heavy chain deposition disease with implications for clinical management.

Randall-type heavy chain deposition disease (HCDD) is a rare disorder characterized by tissue deposition of a truncated monoclonal immunoglobulin heavy chain lacking the first constant domain. Pathophysiological mechanisms are unclear and management remains to be defined. Here we retrospectively studied 15 patients with biopsy-proven HCDD of whom 14 presented with stage 3 or higher chronic kidney disease, with nephrotic syndrome in 9. Renal lesions were characterized by nodular glomerulosclerosis, with linear peritubular and glomerular deposits of γ-heavy chain in 12 patients or α-heavy chain in 3 patients, without concurrent light chain staining. Only 2 patients had symptomatic myeloma. By serum protein electrophoresis/immunofixation, 13 patients had detectable monoclonal gammopathy. However, none of these techniques allowed detection of the nephrotoxic truncated heavy chain, which was achieved by immunoblot and/or bone marrow heavy chain sequencing in 14 of 15 patients. Serum-free kappa to lambda light chain ratio was abnormal in 11 of 11 patients so examined. Immunofluorescence studies of bone marrow plasma cells showed coexpression of the pathogenic heavy chain with light chain matching the abnormal serum-free light chain in all 3 tested patients. Heavy chain sequencing showed first constant domain deletion in 11 of 11 patients, with high isoelectric point values of the variable domain in 10 of 11 patients. All patients received chemotherapy, including bortezomib in 10 cases. Renal parameters improved in 11 patients who achieved a hematological response, as assessed by normalization of the free light chain ratio in 8 cases. Tissue deposition in HCDD relates to physicochemical peculiarities of both variable and constant heavy chain domains. Early diagnosis and treatment with bortezomib-based combinations appear important to preserve renal prognosis. Thus, monitoring of serum-free light chain is an indirect but useful method to evaluate the hematological response.

A mouse model recapitulating human monoclonal heavy chain deposition disease evidences the relevance of proteasome inhibitor therapy.

Randall-type heavy chain deposition disease (HCDD) is a rare disorder characterized by glomerular and peritubular amorphous deposits of a truncated monoclonal immunoglobulin heavy chain (HC) bearing a deletion of the first constant domain (CH1). We created a transgenic mouse model of HCDD using targeted insertion in the immunoglobulin κ locus of a human HC extracted from a HCDD patient. Our strategy allows the efficient expression of the human HC in mouse B and plasma cells, and conditional deletion of the CH1 domain reproduces the major event underlying HCDD. We show that the deletion of the CH1 domain dramatically reduced serum HC levels. Strikingly, even with very low serum level of truncated monoclonal HC, histologic studies revealed typical Randall-type renal lesions that were absent in mice expressing the complete human HC. Bortezomib-based treatment resulted in a strong decrease of renal deposits. We further demonstrated that this efficient response to proteasome inhibitors mostly relies on the presence of the isolated truncated HC that sensitizes plasma cells to bortezomib through an elevated unfolded protein response (UPR). This new transgenic model of HCDD efficiently recapitulates the pathophysiologic features of the disease and demonstrates that the renal damage in HCDD relies on the production of an isolated truncated HC, which, in the absence of a LC partner, displays a high propensity to aggregate even at very low concentration. It also brings new insights into the efficacy of proteasome inhibitor-based therapy in this pathology.

B-cell receptors and heavy chain diseases: guilty by association?

Heavy chain diseases (HCDs) are B-cell proliferative disorders characterized by the production of monoclonal, incomplete, immunoglobulin (Ig) heavy chains (HCs) without associated light chains (LCs). These abnormal HCs are produced as a consequence of HC gene alterations in the neoplastic B cells. HC gene alterations will also impact on surface HC, which is part of the B-cell receptor (BCR), a crucial player in lymphocyte activation by antigen. The selective advantage conferred to mutant cells by abnormal BCR without an antigen-binding domain may be explained by activation of ligand-independent signaling, in analogy to what has been shown for mutated oncogenic growth factor receptors. Here we review data obtained from mouse models showing abnormal, constitutive activity of HCD-BCR, and we discuss the possible mechanism involved, namely, aberrant spontaneous self-aggregation. This self-aggregation might occur as a consequence of escape from the chaperone immunoglobulin binding protein (BiP) and from the anti-aggregation effect of LC association. The concept of misfolding-induced signaling elaborated here may extend to other pathologies termed conformational diseases.

Immunoglobulin heavy chain gene rearrangement in heavy chain deposition disease suggests it is a plasma cell disease: a case report.

Heavy chain deposition disease (HCDD) is characterized by the deposition of truncated monoclonal immunoglobulin heavy chains along glomerular basement membranes. Truncated heavy chains are thought to be associated with plasma cell disease (PCD), but previous bone marrow cytology tests showed that only 30% of HCDD cases are related to PCDs. We report the first known use of immunoglobulin heavy chain (IGH) gene rearrangement to diagnose a patient with γ3-HCDD, although bone marrow morphology test identified no abnormalities. Our findings provide strong evidence for a correlation between PCDs and HCDD, which could help understand the genetic background underlying abnormal heavy chains and assess disease prognosis. Further, concordant with previous findings, bortezomib-based chemotherapy had a good therapeutic effect in our patient. We summarize the experience of diagnosing and treating a case of HCDD, and combine this with a literature review to further explore the correlation between PCDs and HCDD, which has important clinical value.

Screening differentially expressed genes between endometriosis and ovarian cancer to find new biomarkers for endometriosis.

AIM: Endometriosis is one of the most common reproductive system diseases, but the mechanisms of disease progression are still unclear. Due to its high recurrence rate, searching for potential therapeutic biomarkers involved in the pathogenesis of endometriosis is an urgent issue. METHODS: Due to the similarities between endometriosis and ovarian cancer, four endometriosis datasets and one ovarian cancer dataset were downloaded from Gene Expression Omnibus (GEO) database. Differentially expressed genes (DEGs) were identified, followed by gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway and protein-protein interaction (PPI) analyses. Then, we validated gene expression and performed survival analysis with ovarian serous cystadenocarcinoma (OV) datasets in TCGA/GTEx database, and searched for potential drugs in the Drug-Gene Interaction Database. Finally, we explored the miRNAs of key genes to find biomarkers associated with the recurrence of endometriosis. RESULTS: In total, 104 DEGs were identified in the endometriosis datasets, and the main enriched GO functions included cell adhesion, extracellular exosome and actin binding. Fifty DEGs were identified between endometriosis and ovarian cancer datasets including 11 consistently regulated genes, and nine DEGs with significant expression in TCGA/GTEx. Only IGHM had both significant expression and an association with survival, three module DEGs and two significantly expressed DEGs had drug associations, and 10 DEGs had druggability. CONCLUSIONS: ITGA7, ITGBL1 and SORBS1 may help us understand the invasive nature of endometriosis, and IGHM might be related to recurrence; moreover, these genes all may be potential therapeutic targets.KEY MESSAGEThis manuscript used a bioinformatics approach to find target genes for the treatment of endometriosis.This manuscript used a new approach to find target genes by drawing on common characteristics between ovarian cancer and endometriosis.We screened relevant therapeutic agents for target genes in the drug database, and performed histological validation of target genes with both expression and survival analysis difference in cancer databases.

A novel immune prognostic index for stratification of high-risk patients with early breast cancer.

The prognostic value of current multigene assays for breast cancer is limited to hormone receptor-positive, human epidermal growth factor receptor 2-negative early breast cancer. Despite the prognostic significance of immune response-related genes in breast cancer, immune gene signatures have not been incorporated into most multigene assays. Here, using public gene expression microarray datasets, we classified breast cancer patients into three risk groups according to clinical risk and proliferation risk. We then developed the immune prognostic index based on expression of five immune response-related genes (TRAT1, IL2RB, CTLA4, IGHM and IL21R) and lymph node status to predict the risk of recurrence in the clinical and proliferation high-risk (CPH) group. The 10-year probability of disease-free survival (DFS) or distant metastasis-free survival (DMFS) of patients classified as high risk according to the immune prognostic index was significantly lower than those of patients classified as intermediate or low risk. Multivariate analysis revealed that the index is an independent prognostic factor for DFS or DMFS. Moreover, the C-index revealed that it is superior to clinicopathological variables for predicting prognosis. Its prognostic significance was also validated in independent datasets. The immune prognostic index identified low-risk patients among patients classified as CPH, regardless of the molecular subtype of breast cancer, and may overcome the limitations of current multigene assays.

[Genetic diagnosis of patients with primary agammaglobulinemia treated at third level peruvian centers]. / Diagnóstico genético de pacientes con agammaglobulinemia primaria atendidos en centros peruanos de tercer nivel.

Primary agammaglobulinemia result from specific alterations in B cells, which lead to low antibody production. Diagnostic suspicion is established with a history of repeated infections, low immunoglobulins, and absence of CD19+ B lymphocytes. The diagnosis is confirmed by genetic analysis and the detection of a mutation linked to the X or autosomal recessive or dominant chromosome. In Peru, there is no literature on primary agammaglobulinemia and no reports on the genotype of patients with suspected primary agammaglobulinemia. Under this scenario, a study was performed to describe the genotype of patients with suspected primary agammaglobulinemia. Twenty (20) patients were found with mutations in the BTK gene and an autosomal recessive IGHM mutation. Thirteen (13) hereditary mutations and seven de novo mutations were found. It is concluded that the group of primary agammaglobulinemia are mostly mutations in the BTK gene, corresponding to X-linked agammaglobulinemia.

Las agammaglobulinemias primarias (AP) resultan de alteraciones específicas en las células B, lo cual, conduce a baja producción de anticuerpos. La sospecha diagnóstica se establece con el antecedente de infecciones a repetición, inmunoglobulinas bajas y la ausencia linfocitos B CD19+. El diagnóstico se confirma mediante el análisis genético y la detección de una mutación ligada en el cromosoma X o autosómico recesiva o dominante. En Perú, no hay literatura sobre AP ni reportes sobre el genotipo de los pacientes con sospecha de AP. Bajo este escenario, se realizó un estudio que describió el genotipo de pacientes con sospecha de AP. Se encontraron 20 pacientes con mutaciones en el gen BTK y una mutación autosómica recesiva IGHM. Se hallaron 13 mutaciones hereditarias y siete mutaciones de novo. Se concluye que las AP son, en su mayoría, mutaciones en el gen BTK que corresponden con AP ligadas al cromosoma X.

Widespread intronic polyadenylation diversifies immune cell transcriptomes.

Alternative cleavage and polyadenylation (ApA) is known to alter untranslated region (3'UTR) length but can also recognize intronic polyadenylation (IpA) signals to generate transcripts that lose part or all of the coding region. We analyzed 46 3'-seq and RNA-seq profiles from normal human tissues, primary immune cells, and multiple myeloma (MM) samples and created an atlas of 4927 high-confidence IpA events represented in these cell types. IpA isoforms are widely expressed in immune cells, differentially used during B-cell development or in different cellular environments, and can generate truncated proteins lacking C-terminal functional domains. This can mimic ectodomain shedding through loss of transmembrane domains or alter the binding specificity of proteins with DNA-binding or protein-protein interaction domains. MM cells display a striking loss of IpA isoforms expressed in plasma cells, associated with shorter progression-free survival and impacting key genes in MM biology and response to lenalidomide.

Gamma heavy chain disease in man. Genomic sequence reveals two noncontiguous deletions in a single gene.

A genomic clone was isolated from a human lymphoid cell line which synthesized an NH2-terminally deleted gamma 3 heavy chain disease protein. Nucleotide sequence analysis revealed a normal sequence from 310 bp 5' to the initiator ATG through the codon for VH amino acid 14. Amino acid 15 was derived from the codon for the last J4 amino acid. Thus, the clone contained a deletion of the codons for the VH region beyond amino acid 14, as well as those for the entire D region and most of the J coding region. Some sequence abnormalities were observed in the 400 bp after the deletion. Beyond this, there was excellent homology to published J and intervening sequences, including those containing the enhancer elements. The 1,200-bp switch region was abruptly interrupted by a sequence corresponding to the 3' one-third of CH1. Thus, a second deletion eliminated the acceptor splice site at the 5' end of CH1. When splicing of the primary RNA transcript occurred, the truncated VH region was joined via the J4 donor splice site to the next available acceptor site 5' to the first hinge exon. Hence, the aberrant serum protein was the product of two deletions and a splice correction as well as postsynthetic NH2-terminal proteolysis.

Ligand-independent activity of the B cell antigen receptor in physiology and pathology.

The B cell receptor (BCR) is required for stimulation of B cells by antigen, and is also involved in the negative selection of autoreactive B cells. In the past few years, a constitutive ligand-independent signaling activity of the BCR has been demonstrated. In this paper, the various findings are summarized and their interpretation and their significance, both in pathology and in physiology discussed. The constitutive activity of the BCR may be important for tumor formation, at least in the case of heavy-chain diseases, neoplastic proliferations developed from B cells. A large body of evidence suggests that this activity could be required for B cell survival and would play a role in B cell development as a process monitoring BCR functionality. A model explaining signaling in the absence of antigen as a function of dimer formation is proposed. The putative constitutive activity of the pre-BCR is also discussed.

Absence of γ-Chain in Keratinocytes Alters Chemokine Secretion, Resulting in Reduced Immune Cell Recruitment.

Loss-of-function mutations in the common gamma (γc) chain cytokine receptor subunit give rise to severe combined immunodeficiency characterized by lack of T and natural killer cells and infant death from infection. Hematopoietic stem cell transplantation or gene therapy offer a cure, but despite successful replacement of lymphoid immune lineages, a long-term risk of severe cutaneous human papilloma virus infections persists, possibly related to persistent γc-deficiency in other cell types. Here we show that keratinocytes, the only cell type directly infected by human papilloma virus, express functional γc and its co-receptors. After stimulation with the γc-ligand IL-15, γc-deficient keratinocytes show significantly impaired secretion of specific chemokines including CXCL1, CXCL8, and CCL20, resulting in reduced chemotaxis of dendritic cells and CD4+ T cells. Furthermore, γc-deficient keratinocytes also exhibit defective induction of T-cell chemotaxis in a model of stable human papilloma virus-18 infection. These findings suggest that persistent γc-deficiency in keratinocytes alters immune cell recruitment to the skin, which may contribute to the development and persistence of warts in this condition and would require different treatment approaches.

The Expression Pattern of the Pre-B Cell Receptor Components Correlates with Cellular Stage and Clinical Outcome in Acute Lymphoblastic Leukemia.

Precursor-B cell receptor (pre-BCR) signaling represents a crucial checkpoint at the pre-B cell stage. Aberrant pre-BCR signaling is considered as a key factor for B-cell precursor acute lymphoblastic leukemia (BCP-ALL) development. BCP-ALL are believed to be arrested at the pre-BCR checkpoint independent of pre-BCR expression. However, the cellular stage at which BCP-ALL are arrested and whether this relates to expression of the pre-BCR components (IGHM, IGLL1 and VPREB1) is still unclear. Here, we show differential protein expression and copy number variation (CNV) patterns of the pre-BCR components in pediatric BCP-ALL. Moreover, analyzing six BCP-ALL data sets (n = 733), we demonstrate that TCF3-PBX1 ALL express high levels of IGHM, IGLL1 and VPREB1, and are arrested at the pre-B stage. By contrast, ETV6-RUNX1 ALL express low levels of IGHM or VPREB1, and are arrested at the pro-B stage. Irrespective of subtype, ALL with high levels of IGHM, IGLL1 and VPREB1 are arrested at the pre-B stage and correlate with good prognosis in high-risk pediatric BCP-ALL (n = 207). Our findings suggest that BCP-ALL are arrested at different cellular stages, which relates to the expression pattern of the pre-BCR components that could serve as prognostic markers for high-risk pediatric BCP-ALL patients.

Molecular analysis of a t(9;14)(p11;q32) translocation occurring in a case of human alpha heavy chain disease.

We performed the cloning and sequencing of the der(14) breakpoint of a new chromosomal translocation involving the 14q32 immunoglobulin locus. This t(9;14)(p11;q32) translocation was found in a case of malignant lymphoma occurring in human alpha heavy chain disease. A rearranged alpha 1 gene fragment was cloned and shown to contain chromosome 9 information by Southern blotting on sorted chromosomes and by in situ hybridization. Sequence analysis of the junction point region established that the breakage occurred 3' to the heavy chain joining region. In contrast to the data obtained in other translocations affecting 14q32 immunoglobulin locus, the recombination did not involve the immunoglobulin heavy chain locus specific recombination signals on chromosome 14, or homologous sequences on chromosome 9. In the present case, the existence of two almost perfect inverted repeats flanking the junction point suggests that the translocation originated from a local pairing of the two chromosomes 9 and 14. Chromosome 9 fragments sequenced in the vicinity of the breakpoint did not share significant homology with sequences listed in GenBank and EMBL data bases.

Heavy chain diseases.

Heavy chain diseases (HCDs) are rare B-cell lymphoplasma-cell proliferative disorders characterized by production of truncated monoclonal immunoglobulin heavy chains without associated light chains. HCDs involving the three main immunoglobulin classes have been described; alpha-HCD is the most common and has the most uniform presentation, gamma- and mu-HCDs have variable clinical presentations and histopathologic features. HCDs can be thought of as variant types of non-Hodgkin lymphoma: alpha-HCD presents as an extranodal marginal-zone lymphoma of mucosa-associated lymph-node tissue, gamma-HCD as lymphoplasmacytoid non-Hodgkin lymphoma, and mu-HCD as small lymphocytic non-Hodgkin lymphoma or chronic lymphocytic leukemia. Diagnosis of HCD requires documentation of a deleted immunoglobulin heavy chain without a bound light chain in the serum or urine. Prognosis is variable, and no standardized effective treatment programs are available except for alpha-HCD, which in its early stage may respond to antibiotics.

Heterogeneity of the breakpoint localization in malignant cells with a 9p11 chromosomal abnormality.

The p11 band of the short arm of chromosome 9 is involved in various cytogenetic alterations occurring in several malignant diseases. Using probes isolated from the 9p11 band in the study of a case of alpha-heavy-chain disease (MAL) with t(9;14)(p11;q32), we studied the DNA from seven malignant cell samples, including four cases of acute lymphoblastic leukemia with tdic(9;12)(p11;p12). Using pulsed-field electrophoresis analysis we demonstrated that the breakpoints were 3-300 kb distant from the original MAL breakpoint without clustering within the subset of leukemias with the tdic(9;12).

A new extra sequence at the amino terminal of a mu heavy chain disease protein (DAG).

The primary structure of a human mu heavy chain (DAG) protein is described. The native protein is a circular decamer with a molecular weight (Mr) of 500 kDa, each decamer being constituted of the constant domains C mu 2, C mu 3 and C mu4 and interlinked by 15 disulfide bridges. At its NH2-terminal each monomeric chain starts with an "extra sequence". The amino acid sequence of this segment is Arg-Gln-Ser-Asp-Asp-Pro-Val-Leu-Arg-Gly-Thr-Thr-Val-Pro-Val-Thr-Glu and its reinitiation point is located at Val223 (Gal numbering), at the beginning of C mu 2. This sequence has no homology with any other protein included in the present databases.

A pre-translational defect in a case of human mu heavy chain disease.

A patient (BW) was studied with Mu heavy chain disease (mu HCD) in whom a leukemic B-cell clone secreted a shortened monoclonal mu chain without associated light chain. The cells did, however, produce a normal-sized kappa light chain that was detected as urinary Bence-Jones protein. The cytoplasmic and secreted monomeric mu chain had an approximate mol. wt of 58,000. Radiochemical sequence analysis of the biosynthetically labelled mu chain revealed a protein that lacked the entire variable region. The sequence initiated at amino acid position 5 within the first constant region domain (CH1) of C mu. The primary in vitro translation product, the cytoplasmic and secreted proteins were all similarly truncated, thereby excluding extensive postsynthetic degradation. The mu RNA, that directed the synthesis of the truncated mu protein, was about 350 bp smaller than the normal mu RNA. Furthermore, by primer extension analysis it was possible to localize this deletion in the mu RNA to a region 5' of CH1. Thus, a defect at the level of Ig gene structure/assembly that deletes coding information or results in aberrant RNA processing must be responsible for the truncated mu HCD protein BW.