PID Comes Full Circle: Applications of V(D)J Recombination Excision Circles in Research, Diagnostics and Newborn Screening of Primary Immunodeficiency Disorders

The vast majority of patients suffering from a primary immunodeficiency (PID) have defects in their T- and/or B-cell compartments. Despite advances in molecular diagnostics, in many patients no underlying genetic defect has been identified. B- and T-lymphocytes are unique in their ability to create a receptor by genomic rearrangement of their antigen receptor genes via V(D)J recombination. During this process, stable circular excision products are formed that do not replicate when the cell proliferates. Excision circles can be reliably quantified using real-time quantitative (RQ-)PCR techniques. Frequently occurring δREC–ψJα T-cell receptor excision circles (TRECs) have been used to assess thymic output and intronRSS–Kde recombination excision circles (KREC) to quantify B-cell replication history. In this perspective, we describe how TRECs and KRECs are formed during precursor – T- and B-cell differentiation, respectively. Furthermore, we discuss new insights obtained with TRECs and KRECs and specifically how these excision circles can be applied to support therapy monitoring, patient classification and newborn screening of PID

Clinical management of ovarian small-cell carcinoma of the hypercalcemic type: A proposal for conservative surge

Ovarian small-cell carcinoma of the hypercalcemic type is a rare and highly malignant tumor. In two thirds of the patients, the tumor is associated with asymptomatic paraneoplastic hypercalcemia. The diagnosis may be impeded; the tumor must be distinguished

The 11q Terminal Deletion Disorder Jacobsen Syndrome is a Syndromic Primary Immunodeficiency

Background: Jacobsen syndrome (JS) is a rare contiguous gene syndrome caused by partial deletion of the long arm of chromosome 11. Clinical features include physical and mental growth retardation, facial dysmorphism, thrombocytopenia, impaired platelet function and pancytopenia. In case reports, recurrent infections and impaired immune cell function compatible with immunodeficiency were described. However, Jacobsen syndrome has not been recognized as an established syndromic primary immunodeficiency. Goal: To evaluate the presence of immunodeficiency in a series of 6 patients with JS. Methods: Medical history of 6 patients with JS was evaluated for recurrent infections. IgG, IgA, IgM and specific antibodies against S. pneumoniae were measured. Response to immunization with a polysaccharide vaccine (Pneumovax) was measured and B and T lymphocyte subset analyses were performed using flowcytometry. Results: Five out of 6 patients suffered from recurrent infections. These patients had low IgG levels and impaired response to S. pneumoniae polysaccharide vaccination. Moreover, we also found a significant decrease in the absolute number of memory B cells, suggesting a defective germinal center function. In a number of patients, low numbers of T lymphocytes and NK cells were found. Conclusions: Most patients with JS suffer from combined immunodeficiency in the presence of recurrent infections. Therefore, we consider JS a syndromic primary immunodeficiency. Early detection of immunodeficiency may reduce the frequency and severity of infections. All JS patients should therefore undergo immunological evaluation. Future studies in a larger cohort of patients will more precisely define the pathophysiology of the immunodeficiency in JS

Comparative analysis of Ig and TCR gene rearrangements at diagnosis and at elapse of childhood precursor-B–ALL provides improved strategies for selection of stable PCR targets for monitoring of minimal residual disease

Immunoglobulin (Ig) and T-cell receptor (TCR) gene rearrangements are excellent patient-specific polymerase chain reaction (PCR) targets for detection of minimal residual disease (MRD) in acute lymphoblastic leukemia (ALL), but they might be unstable during the disease course. Therefore, we performed detailed molecula

IgM Augments Complement Bactericidal Activity with Serum from a Patient with a Novel CD79a Mutation

Antibody replacement therapy for patients with antibody deficiencies contains only IgG. As a result, concurrent IgM and IgA deficiency present in a large proportion of antibody deficient patients persists. Especially patients with IgM deficiency remain at risk for recurrent infections of the gastrointestinal and respiratory tract. The lack of IgM in the current IgG replacement therapy is likely to contribute to the persistence of these mucosal infections because this antibody class is especially important for complement activation on the mucosal surface. We evaluated whether supplementation with IgM increased serum bactericidal capacity in vitro. Serum was collected from a patient with agammaglobulinemia and supplemented with purified serum IgM to normal levels. Antibody and complement deposition on the bacterial surface was determined by multi-color flow cytometry. Bacterial survival in serum was determined by colony-forming unit counts. We present a patient previously diagnosed with agammaglobulinemia due to CD79A (Igα) deficiency revealing a novel pathogenic insertion variant in the CD79a gene (NM_001783.3:c.353_354insT). Despite IgG replacement therapy and antibiotic prophylaxis, this patient developed a Campylobacter jejuni spondylodiscitis of lumbar vertebrae L4–L5. We found that serum IgM significantly contributes to complement activation on the bacterial surface of C. jejuni. Furthermore, supplementation of serum IgM augmented serum bactericidal activity significantly. In conclusion, supplementation of intravenous IgG replacement therapy with IgM may potentially offer greater protection against bacterial infections, also in the context of increasing antibiotic resistance

ImmunoGlobulin galaxy (IGGalaxy) for simple determination and quantitation of immunoglobulin heavy chain rearrangements from NGS

Background: Sequence analysis of immunoglobulin heavy chain (IGH) gene rearrangements and frequency analysis is a powerful tool for studying the immune repertoire, immune responses and immune dysregulation in health and disease. The challenge is to provide user friendly, secure and reproducible analytical services that are available for both small and large laboratories which are determining VDJ repertoire using NGS technology. Results: In this study we describe ImmunoGlobulin Galaxy (IGGalaxy)- a convenient web based application for analyzing next-generation sequencing results and reporting IGH gene rearrangements for both repertoire and clonality studies. IGGalaxy has two analysis options one using the built in igBLAST algorithm and the second using output from IMGT; in either case repertoire summaries for the B-cell populations tested are available. IGGalaxy supports multi-sample and multi-replicate input analysis for both igBLAST and IMGT/HIGHV-QUEST. We demonstrate the technical validity of this platform using a standard dataset, S22, used for benchmarking the performance of antibody alignment utilities with a 99.9 % concordance with previous results. Re-analysis of NGS data from our samples of RAG-deficient patients demonstrated the validity and user friendliness of this tool. Conclusions: IGGalaxy provides clinical researchers with detailed insight into the repertoire of the B-cell population per individual sequenced and between control and pathogenic genomes. IGGalaxy was developed for 454 NGS results but is capable of analyzing alternative NGS data (e.g. Illumina, Ion Torrent). We demonstrate the use of a Galaxy virtual machine to determine the VDJ repertoire for reference data and from B-cells taken from immune deficient patients. IGGalaxy is available as a VM for download and use on a desktop PC or on a server

Exhaustion of the CD8+ T cell compartment in patients with mutations in phosphoinositide 3-kinase delta

Pathogenic gain-of-function mutations in the gene encoding phosphoinositide 3-kinase delta (PI3Kδ) cause activated PI3Kδ syndrome (APDS), a disease characterized by humoral immunodeficiency, lymphadenopathy, and an inability to control persistent viral infections including Epstein-Barr virus (EBV) and cytomegalovirus (CMV) infections. Understanding the mechanisms leading to impaired immune response is important to optimally treat APDS patients. Immunosenescence of CD8+ T cells was suggested to contribute to APDS pathogenesis. However, the constitutive activation of T cells in APDS may also result in T cell exhaustion. Therefore, we studied exhaustion of the CD8+ T cell compartment in APDS patients and compared them with healthy controls and HIV patients, as a control for exhaustion. The subset distribution of the T cell compartment of APDS patients was comparable with HIV patien

Overview of 15-year severe combined immunodeficiency in the Netherlands: towards newborn blood spot screening

Severe combined immune deficiency (SCID) is a fatal primary immunodeficiency usually presenting in the first months of life with (opportunistic) infections, diarrhea, and failure to thrive. Hematopoietic stem cell transplantation (HSCT) and gene therapy (GT) are curative treatment options. The objective of the study was to assess the morbidity, mortality, and diagnostic and therapeutic delay in children with SCID in the Netherlands in the last 15 years. These data may help to judge whether SCID should be considered to be included in our national neonatal screening program. In the period 1998–2013, 43 SCID patients were diagnosed in the Netherlands, 11 of whom were atypical SCID (presentation beyond the first year). The median interval between the first symptom and diagnosis was 2 months (range 0–1173 months). The total mortality was 42 %. In total, 32 patients were treated with HSCT of whom 8 were deceased. Nine patients died due to severe infectious complications before curative treatment could be initiated. Conclusion: Because of a high mortality of patients with SCID before HSCT could be initiated, only a national newborn screening program and pre-emptive HSCT or GT will be able to improve survival of these patients

Decreased IL7Rα and TdT expression underlie the skewed immunoglobulin repertoire of human B-cell precursors from fetal origin

Newborns are unable to mount antibody responses towards certain antigens. This has been related to the restricted repertoire of immunoglobulin (Ig) genes of their B cells. The mechanisms underlying the restricted fetal Ig gene repertoire are currently unresolved. We here addressed this with detailed molecular and cellular analysis of human precursor-B cells from fetal liver, fetal bone marrow (BM), and pediatric BM. In the absence of selection processes, fetal B-cell progenitors more frequently used proximal V, D and J genes in complete IGH gene rearrangements, despite normal Ig locus contraction. Fewer N-nucleotides were added in IGH gene rearrangements in the context of low TdT and XRCC4 expression. Moreover, fetal progenitor-B cells expressed lower levels of IL7Rα than their pediatric counterparts. Analysis of progenitor-B cells from IL7Rα-deficient patients revealed that TdT expression and N-nucleotides additions in Dh-Jh junctions were dependent on functional IL7Rα. Thus, IL7Rα affects TdT expression, and decreased expression of this receptor underlies at least in part the skewed Ig repertoire formation in fetal B-cell precursors. These new insights provide a better understanding of the formation of adaptive immunity in the developing fetus

Autoimmune lymphoproliferative syndrome (ALPS) in a child from consanguineous parents: a dominant or recessive disease?

Autoimmune lymphoproliferative syndrome (ALPS) is characterized by autoimmune features and lymphoproliferations and is generally caused by defective Fas-mediated apoptosis. This report describes a child with clinical features of ALPS without detectable Fas expression on freshly isolated blood leukocytes. Detection of FAS transcripts via real-time quantitative PCR made a severe transcriptional defect unlikely. Sequencing of the FAS gene revealed a 20-nucleotide duplication in the last exon affecting the cytoplasmic signaling domain. The patient was homozygous for this mutation, whereas the consanguineous parents and the siblings were heterozygous. The patient reported here is a human homologue of the Fas-null mouse, inasmuch as she carries an autosomal homozygous mutation in the FAS gene and she shows the severe and accelerated ALPS phenotype. The heterozygous family members did not have the ALPS phenotype, indicating that the disease-causing FAS mutation in this family is autosomal recessive