Atar Lev

A congenital neutrophil defect syndrome associated with mutations in VPS45

BACKGROUND Neutrophils are the predominant phagocytes that provide protection against bacterial and fungal infections. Genetically determined neutrophil disorders confer a predisposition to severe infections and reveal novel mechanisms that control vesicular trafficking, hematopoiesis, and innate immunity. METHODS We clinically evaluated seven children from five families who had neutropenia, neutrophil dysfunction, bone marrow fibrosis, and nephromegaly. To identify the causative gene, we performed homozygosity mapping using single-nucleotide polymorphism arrays, whole-exome sequencing, immunoblotting, immunofluorescence, electron microscopy, a real-time quantitative polymerase-chain-reaction assay, immunohistochemistry, flow cytometry, fibroblast motility assays, measurements of apoptosis, and zebrafish models. Correction experiments were performed by transfecting mutant fibroblasts with the nonmutated gene. RESULTS All seven affected children had homozygous mutations (Thr224Asn or Glu238Lys, depending on the childs ethnic origin) in VPS45, which encodes a protein that regulates membrane trafficking through the endosomal system. The level of VPS45 protein was reduced, as were the VPS45 binding partners rabenosyn-5 and syntaxin-16. The level of β1 integrin was reduced on the surface of VPS45-deficient neutrophils and fibroblasts. VPS45-deficient fibroblasts were characterized by impaired motility and increased apoptosis. A zebrafish model of vps45 deficiency showed a marked paucity of myeloperoxidase-positive cells (i.e., neutrophils). Transfection of patient cells with nonmutated VPS45 corrected the migration defect and decreased apoptosis. CONCLUSIONS Defective endosomal intracellular protein trafficking due to biallelic mutations in VPS45 underlies a new immunodeficiency syndrome involving impaired neutrophil function. (Funded by the National Human Genome Research Institute and others.).

Timely and spatially regulated maturation of B and T cell repertoire during human fetal development

Immunocompetence in the developing fetus is temporally and spatially regulated. Developing Immunity The adaptive immune response plays a critical role in protecting the body from both foreign pathogens and internal dangers such as cancer. However, little is known about how the immune system develops during human gestation. Rechavi et al. analyzed differences in B and T lymphocyte ontogeny from 12 to 26 weeks of gestational age. They found that B cell development precedes T cell development and that repertoire maturation is both temporally and spatially regulated. These data can be used as a baseline to improve immune function in developing fetuses and to assess the effects of therapeutic interventions. Insights into the ontogeny of the human fetal adaptive immune system are of great value for understanding immunocompetence of the developing fetus. However, to date, this has remained largely uncharted territory, in large part because blood samples from healthy, early gestation fetuses have been hard to come by. In a comprehensive study, we analyzed levels of T cell receptor excision circles (TRECs), signal-joint κ receptor excision circles (sjKRECs), and intron recombination signal sequence–K-deleting element (iRSS-Kde) rearrangement, and T and B lymphocyte repertoire clonality in human fetuses from 12 to 26 weeks of gestational age. Using next-generation sequencing, we analyzed the diversity and complexity of T cell receptor β (TRB) and immunoglobulin heavy chain (IGH) repertoires in four fetuses at 12, 13, 22, and 26 weeks of gestation and in healthy full-term infants. We report the progressive increase of TREC, sjKREC, and iRSS-Kde levels over time and confirm that B cell development precedes T cell development in the human fetus. Temporally and spatially regulated maturation of B and T cell repertoire diversity and complexity during human fetal development was observed, including evidence that immunoglobulin somatic hypermutation and class switch recombination occur already during intrauterine life. Our results help define physiological levels of immunodeficiency in premature infants and may serve as a reference for future studies aimed at investigating the impact of intrauterine pathologies on fetal immune development and function.

Reduced central tolerance in Omenn syndrome leads to immature self-reactive oligoclonal T cells.

BACKGROUND Omenn syndrome (OS) is characterized by a peculiar severe T-cell immune deficiency associated with autoimmunelike manifestations. Dysregulations of the central and peripheral immune tolerance, mediated by the protein autoimmune regulator (AIRE) and regulatory T cells, respectively, were proposed as possible mechanisms of this aberrant inflammatory process. OBJECTIVE We studied mechanisms of central and peripheral tolerance in patients with OS and also examined the gene expression profile associated with OS features. METHODS T-cell receptor diversity, DNA rearrangement, and the expression of AIRE and forkhead box P3 mRNA as well as the expression of regulatory T cells in cells obtained from patients with OS were studied. Characterization of gene expression in these cells was carried out by using the TaqMan Low-Density Array. RESULTS Transcript expression of peripheral blood AIRE but not forkhead box P3 was reduced in patients with OS. The expression of natural killer T and regulatory T cells was normal, although the latter showed an abnormal CD4-negative population. Patients with OS have oligoclonal T cells with limited DNA recombination activity, including the presence of early but not late T-cell maturation events, regardless of the genetic defect underlying the syndrome. The transcriptional profile associated with OS features reveals significant changes in 25.5% of the tested genes compared with normal control. CONCLUSION Our findings suggest that T-cell oligoclonal expansion in OS emanates from an incomplete block before the maturation stage of negative selection, which may explain escape of autoreactive T cells from the thymus. Dysregulated genes in patients with OS are closely involved with self-tolerance and autoimmunity.

Mutations in STN1 cause Coats plus syndrome and are associated with genomic and telomere defects

Somech and colleagues identify two new mutations in STN1 that causes Coats plus syndrome and telomere abnormalities in human, recapitulated in a zebra fish model.

Molecular assessment of thymus capabilities in the evaluation of T-cell immunodeficiency.

T-cell immunodeficiency may pose a diagnostic challenge to clinicians, especially when the basic T-cell immune workup is not sufficiently informative. An intensive assessment of thymus capabilities that involves either measuring the recent thymic emigrant cells or analyzing the T-cell receptor (TCR) repertoire is often required to estimate the severity and nature of the immune disorder. A comprehensive T-cell immune workup, including TCR excision circles (TRECs) and TCR repertoire analyses, was performed in three patients with various degrees of severity of T-cell immunodeficiency. All three patients had normal peripheral CD3+ T lymphocytes. TCR repertoire analysis revealed oligoclonal (patient 1), restricted (patient 2), and near-normal (patient 3) patterns. TREC quantification was significantly reduced in patients 1 and 2 but normal in patient 3. Based on clinical features at presentation and at follow-up, and supported by the results of immunologic studies, patients 1 and 2 were diagnosed as having significant T-cell immunodeficiency and patient 3 as having T-cell immunocompetence. Assessment of thymus capabilities by TRECs and TCR repertoire analyses is helpful in diagnosing patients with T-cell immunodeficiency and should be part of the evaluation of every patient suspected of having that condition.

Purine nucleoside phosphorylase deficiency presenting as severe combined immune deficiency

Purine nucleoside phosphorylase (PNP) deficiency is an autosomal recessive genetic disorder of the purine salvage pathway, associated with a variable extent of immunodeficiency. Here, we report a PNP-deficient patient who presented early in life with clinical and laboratory characteristics of severe combined immunodeficiency, including severe infections, marked T-and B-cell deficiency, lack of lymphocyte response to mitogenic stimulation, monoclonal T-cell receptors representation and the absence of T-cell receptor excision circles and Kappa-receptor excision circles. The patient carried homozygote mutation at the PNP gene that putatively led to aberrant splicing, allowing normal and abnormally spliced products from the mutant alleles. We suggest that the aberrant slice site was used preferentially over the normal slice site in some cells correlating with the severity of disease.

Characterizing T Cells in SCID Patients Presenting with Reactive or Residual T Lymphocytes

Introduction. Patients with severe combined immunodeficiency (SCID) may present with residual circulating T cells. While all cells are functionally deficient, resulting in high susceptibility to infections, only some of these cells are causing autoimmune symptoms. Methods. Here we compared T-cell functions including the number of circulating CD3+ T cells, in vitro responses to mitogens, T-cell receptor (TCR) repertoire, TCR excision circles (TREC) levels, and regulatory T cells (Tregs) enumeration in several immunodeficinecy subtypes, clinically presenting with nonreactive residual cells (MHC-II deficiency) or reactive cells. The latter includes patients with autoreactive clonal expanded T cell and patients with alloreactive transplacentally maternal T cells. Results. MHC-II deficient patients had slightly reduced T-cell function, normal TRECs, TCR repertoires, and normal Tregs enumeration. In contrast, patients with reactive T cells exhibited poor T-cell differentiation and activity. While the autoreactive cells displayed significantly reduced Tregs numbers, the alloreactive transplacentally acquired maternal lymphocytes had high functional Tregs. Conclusion. SCID patients presenting with circulating T cells show different patterns of T-cell activity and regulatory T cells enumeration that dictates the immunodeficient and autoimmune manifestations. We suggest that a high-tolerance capacity of the alloreactive transplacentally acquired maternal lymphocytes represents a toleration advantage, yet still associated with severe immunodeficiency.

T-cell compartment in synovial fluid of pediatric patients with JIA correlates with disease phenotype

IntroductionJuvenile idiopathic arthritis (JIA) is an autoimmune disease where T cells are key players. It can be classified into two main clinical diseases: polyarticular and pauciarticular, based on the number of joints involved. Oligoarthritis, which is considered a pauciarticular subtype since it involves up to four joints upon presentation, is further divided into persistent or extended forms based on disease progression.Methodology/Principal FindingsHere we assessed the T-cell compartment in synovial fluid obtained from 33 JIA patients with active disease and correlated the analyzed parameters with the patients’ clinical characteristics. The T-cell compartment was determined by the representation of T-cell receptor (TCR) repertoires and the amount of TCR excision circles (TRECs).ResultsPatients with polyarticular disease have more a clonal pattern of their TCR repertoire. These findings were consistent in all tested TCR-Vγ consensus primers. Similarly, patients with polyarticular disease had lower TREC levels than patients with pauciarticular disease. A predictive value of TRECs may be suggested, as lower TREC levels were observed in patients in whom disease modifying anti rheumatic drugs were initiated subsequently during the follow-up.ConclusionIn pediatric JIA patients, we showed an alteration in the T cells from synovial fluid, which correlated with disease phenotype, assumedly secondary to enhanced proliferation, clonal TCR restriction, and reduced T-cell production, possibly reflecting a different disease or a different course of disease progression.

G23D: Online tool for mapping and visualization of genomic variants on 3D protein structures

BackgroundEvaluation of the possible implications of genomic variants is an increasingly important task in the current high throughput sequencing era. Structural information however is still not routinely exploited during this evaluation process. The main reasons can be attributed to the partial structural coverage of the human proteome and the lack of tools which conveniently convert genomic positions, which are the frequent output of genomic pipelines, to proteins and structure coordinates.ResultsWe present G23D, a tool for conversion of human genomic coordinates to protein coordinates and protein structures. G23D allows mapping of genomic positions/variants on evolutionary related (and not only identical) protein three dimensional (3D) structures as well as on theoretical models. By doing so it significantly extends the space of variants for which structural insight is feasible. To facilitate interpretation of the variant consequence, pathogenic variants, functional sites and polymorphism sites are displayed on protein sequence and structure diagrams alongside the input variants. G23D also provides modeling of the mutant structure, analysis of intra-protein contacts and instant access to functional predictions and predictions of thermo-stability changes. G23D is available at http://www.sheba-cancer.org.il/G23D.ConclusionsG23D extends the fraction of variants for which structural analysis is applicable and provides better and faster accessibility for structural data to biologists and geneticists who routinely work with genomic information.

Molecular assessment of thymic capacities in patients with Schimke immuno-osseous dysplasia

Schimke immuno-osseous dysplasia (SIOD) is caused by SMARCAL1 deficiency and characterized by defective T-cell immunity. The immunodeficiency and the role of thymic function in SIOD patients are not clearly understood. We performed thymic evaluations by assessing T-cell receptor (TCR) diversity, rearrangement, and excision circles in family members with different disease severity carrying the same bi-allelic mutation and in a heterozygous carrier. The expression of SMARCAL1 mRNA in a normal thymic sample was measured using real-time quantitative polymerase chain reaction. Thymus functions were significantly reduced in SIOD patients, and these findings were highly correlated with the clinical phenotype. Quantification of SMARCAL1 mRNA transcript was 3.86-fold higher than normal values for adult kidneys. Genotype alone apparently does not define phenotype, and analysis of TCR diversity, rearrangement, and thymus output can quantify the extent of T-cell immunodeficiency. High thymic expression of SMARCAL1 mRNA raises the possibility of its importance in thymus maintenance and function.