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Increased susceptibility to develop severe forms of Epstein-Barr virus (EBV) infection in early age is a significant hallmark of an underlying primary immunodeficiency (PID). Here, we present immunologic and genetic evaluations of a 3-year-old child who was born to first-cousins parents and presented with recurrent infections, failure to thrive, and severe EBV-related infection and proliferation. A diagnosis of diffuse large B cell lymphoma was made and the immunological workup was suggestive of T cell immunodeficiency. Unfortunately, the patient succumbed to EBV-related lymphoma. Whole-exome sequencing revealed a novel homozygous mutation, c.991del.C; p. Q331Sfs*6 in the SLP76 gene. The SLP76 protein, a TCR signaling molecule, was recently linked to a human disease of the immune system. In order to examine the effect of this new SLP76 mutation on T cell signaling, a SLP76-deficient Jurkat-derived T cell line was transduced either with wild-type (WT), or with the specific SLP76 mutant, or with a mock vector. Downstream TCR signaling events, including ERK1/2 phosphorylation, CD69 expression, and Ca2 + mobilization, were reduced in cells harboring the reported mutation, linking this novel mutation to the expected immunological outcome. SLP76 deficiency should be added to the growing list of monogenetic diseases that predispose affected individuals to acquire severe and uncontrolled EBV infections and to develop substantial complications. This case further links mutations in the SLP76 gene to a significant human immunodeficiency and extends its clinical phenotype.

BackgroundPatients with primary immunodeficiency disorders (PIDs) often suffer from recurrent infections because of their inappropriate immune response to both common and less common pathogens. These patients may present with unique and severe cutaneous infectious manifestations that are not common in healthy individuals and may be more challenging to diagnose and treat.ObjectiveTo describe a cohort of patients with PIDs with atypical presentations of skin infections, who posed a diagnostic and/or therapeutic challenge.MethodsThis is a retrospective study of pediatric patients with PID with atypical presentations of infections, who were treated at the immunodeficiency specialty clinic and the pediatric dermatology clinic at the Sheba Medical Center between September 2012 and August 2022. Epidemiologic data, PID diagnosis, infectious etiology, presentation, course, and treatment were recorded.ResultsEight children with a diagnosis of PID were included, five of whom were boys. The average age at PID diagnosis was 1.7 (±SD 3.2) years. The average age of cutaneous infection was 6.9 (±SD 5.9) years. Three patients were born to consanguineous parents. The PIDs included the following: common variable immunodeficiency, severe combined immunodeficiency, DOCK8 deficiency, ataxia telangiectasia, CARD11 deficiency, MALT1 deficiency, chronic granulomatous disease, and a combined cellular and humoral immunodeficiency syndrome of unknown etiology. The infections included the following: ulcerative-hemorrhagic varicella-zoster virus (two cases) atypical fungal and bacterial infections, resistant Norwegian scabies, giant perianal verrucae (two cases), and diffuse molluscum contagiosum.ConclusionsIn this case series, we present unusual manifestations of infectious skin diseases in pediatric patients with PID. In some of the cases, recognition of the infectious process prompted life-saving treatment. Increasing familiarity with these dermatological manifestations, as well as keeping a high index of suspicion, is important to enabling early diagnosis of cutaneous infections in PIDs and initiation of prompt suitable treatment.

X-linked agammaglobulinemia (XLA) is caused by mutations in the Bruton tyrosine kinase) BTK) gene. Affected patients have severely reduced amounts of circulating B cells. Patients with atypical XLA may have residual circulating B cells, and there are few studies exploring these cells' repertoire. We aimed to study the B cell repertoire of a novel hypomorphic mutation in the BTK gene, using the next generation sequencing (NGS) technology. Clinical data was collected from our clinical records. Real-time PCR was used to determine KREC copies, and NGS was used to determine the immunoglobulin (Ig) heavy chain (IgH) repertoire diversity. Both patients had a relatively mild clinical and laboratory phenotype, residual BTK protein expression, and the same novel mutation in the BTK gene, c.1841 T C, p. L614P. Signal-joint kappa-deleting recombination excision circles (sj-KREC) for both patients were completely absent reflecting lack of naïve B cells. The intron RSS-Kde coding joints (cj) were significantly reduced, reflecting residual replicating B cells. NGS displayed restricted IgH repertoire with highly uneven distribution of clones, especially for Pt2. We report a novel BTK mutation, c.1841 T C (p. L614P) that is associated with a relatively mild phenotype. We conclude that the IgH repertoire in atypical XLA is restricted with highly uneven distribution of clones. This phenomenon may be explained by extremely reduced to non-existent levels of BTK in B cells. This report sheds further light on atypical cases of XLA.

Background Cutaneous manifestations of dedicator of cytokinesis 8 gene (DOCK8) deficiency, a combined type of T and B cell immunodeficiency, previously designated as autosomal recessive hyper IgE syndrome, includes dermatitis and skin infections. There are limited treatment options for dermatitis related to the syndrome. Objective To describe a cohort of patients with DOCK8 deficiency with a focus on the treatment of their cutaneous manifestations. Methods A retrospective study on all children with the genetic diagnosis of DOCK8 deficiency treated at the Sheba Medical Center between 1/1/2003 and 1/1/2021 was preformed. Collected data included: demographic features, family history, laboratory, genetic testing, skin manifestations, treatment, and disease course. Description of two cases of severe recalcitrant dermatitis treated with dupilumab is detailed. Results Nine children with a genetic diagnosis of DOCK8 deficiency were included, of whom six were girls (66%) with a median age of 8.5 (±2.2 SD) years. The median age at diagnosis was 2.8 (±2.6 SD) years. Six patients were born to consanguineous parents. Five out of six patients who received hematopoietic stem cell transplantation (HSCT) had a complete response, and one was recently transplanted. Of note, two patients, while awaiting HSCT, were treated with dupilumab for their severe dermatitis resulting in a marked improvement of the cutaneous manifestations and pruritus. Conclusions Hematopoietic stem cell transplantation is the gold standard and most effective therapy for patients with DOCK8 deficiency. Dupilumab, a biological therapy indicated for atopic dermatitis and other Th2 derived dermatoses, is an excellent option for dermatitis in patients with DOCK8 deficiency and can be used as a bridge before HSCT. Larger studies are needed to confirm this observation.

Genetic diagnostic tools including whole-exome sequencing (WES) have advanced our understanding in human diseases and become common practice in diagnosing patients with suspected primary immune deficiencies. Establishing a genetic diagnosis is of paramount importance for tailoring adequate therapeutic regimens, including identifying the need for hematopoietic stem cell transplantation (HSCT) and genetic-based therapies. Here, we genetically studied two adult patients who were clinically diagnosed during infancy with severe combined immune deficiency (SCID). Two unrelated patients, both of consanguineous kindred, underwent WES in adulthood, 2 decades after their initial clinical manifestations. Upon clinical presentation, immunological workup was performed, which led to a diagnosis of SCID. The patients presented during infancy with failure to thrive, generalized erythematous rash, and recurrent gastrointestinal and respiratory tract infections, including episodes of Pneumocystis pneumonia infection and Candida albicans fungemia. Hypogammaglobulinemia and T-cell lymphopenia were detected. Both patients were treated with a 10/10 HLA matched sibling donor unconditioned HSCT. Retrospective genetic workup revealed homozygous bi-allelic mutations in IL7RA in one patient and in RAG2 in the other. Our study exemplifies the impact of retrospectively establishing a genetic diagnosis. Pinpointing the genetic cause raises several issues including optimized surveillance and treatment, understanding disease mechanisms and outcomes, future family planning, and social and psychological considerations.

Biallelic mutations in the zeta-associated protein 70 (ZAP70) gene cause combined immunodeficiency (CID). Neonatal screening for severe CID in Israel is implemented since 2015. We report on clinical, flow cytometry, and genetic data of an unusual ZAP70 deficiency patient. A 10-week-old Bedouin female presented with severe autoimmune hemolytic anemia. Cytomegalovirus (CMV) negative packed cell therapy was given without improvement; indexes of hemolysis worsened. At this time, thrombocytopenia was noted. The patient was treated with single dose of 1 g/kg intravenous immunoglobulin with rapid resolution of hemolysis. Serum immunoglobulin concentrations were normal; flow cytometry revealed severe CD8 lymphocytopenia. Lymphocyte proliferation test demonstrated reduced response to concanavalin A and phytohemagglutinin. Gated T cells were negative for intracellular ZAP70. A genetic analysis revealed a missense homozygous c.1388C > T (p.A463V) mutation, confirming the diagnosis of ZAP70 deficiency. She later on developed urinary tract infection due to ESBL producing E. coli treated with amikacin and severe CMV infection that partially responded to ganciclovir therapy and at 7 months of age, she successfully underwent allogeneic hematopoietic stem cell transplantation. Neonatal screening by T cell receptor excision circles (TRECs) for SCID was normal, yet very low TRECs were recorded at the time of CID diagnosis. Normal neonatal screening for SCID does not rule out the diagnosis of CID due to ZAP70 deficiency. This type of CID can present with autoimmunity as the sole initial manifestation of the disease.

Summary Capping protein regulator and myosin 1 linker 2 (CARMIL2) deficiency is characterized by impaired T cell activation, which is attributed to defective CD28-mediated co-signaling. Herein, we aimed to analyze the effect of exogenous interleukin (IL)-2 on in-vitro T cell activation and proliferation in a family with CARMIL2 deficiency. This study included four children (one male and three females; aged 2·5–10 years at presentation). The patients presented with inflammatory bowel disease and recurrent viral infections. Genetic analysis revealed a novel homozygous 25-base pairs deletion in CARMIL2. Immunoblotting demonstrated the absence of CARMIL2 protein in all four patients and confirmed the diagnosis of CARMIL2 deficiency. T cells were activated in-vitro with the addition of IL-2 in different concentrations. CD25 and interferon (IFN)-γ levels were measured after 48 h and 5 days of activation. CD25 surface expression on activated CD8+ and CD4+ T cells was significantly diminished in all patients compared to healthy controls. Additionally, CD8+ T cells from all patients demonstrated significantly reduced IFN-γ production. When cells derived from CARMIL2-deficient patients were treated with IL-2, CD25 and IFN-γ production increased in a dose-dependent manner. T cell proliferation, as measured by Cell Trace Violet, was impaired in one patient and it was also rescued with IL-2. In conclusion, we found that IL-2 rescued T cell activation and proliferation in CARMIL2-deficient patients. Thus, IL-2 should be further studied as a potential therapeutic modality for these patients.

Clathrin-mediated endocytosis (CME) is critical for internalisation of molecules across cell membranes. The FCH domain only 1 (FCHO1) protein is key molecule involved in the early stages of CME formation. The consequences of mutations in FCHO1 in humans were unknown. We identify ten unrelated patients with variable T and B cell lymphopenia, who are homozygous for six distinct mutations in FCHO1. We demonstrate that these mutations either lead to mislocalisation of the protein or prevent its interaction with binding partners. Live-cell imaging of cells expressing mutant variants of FCHO1 provide evidence of impaired formation of clathrin coated pits (CCP). Patient T cells are unresponsive to T cell receptor (TCR) triggering. Internalisation of the TCR receptor is severely perturbed in FCHO1-deficient Jurkat T cells but can be rescued by expression of wild-type FCHO1. Thus, we discovered a previously unrecognised critical role of FCHO1 and CME during T-cell development and function in humans. FCH domain only 1 (FCHO1) is a key molecule involved in clathrin-mediated endocytosis (CME). Here, the authors report homozygous FCHO1 mutations in individuals with variable T and B cell lymphopenia, which are associated with loss-of-function of FCHO1 and impaired formation of clathrin-coated pits in T cells.

During the process of generating diverse T and B cell receptor (TCR and BCR, respectively) repertoires, double-strand DNA breaks are produced. Subsequently, these breaks are corrected by a complex system led by the non-homologous end-joining (NHEJ). Pathogenic variants in genes involved in this process, such as theTo provide new clinical and immunological insights on NHEJ1 deficiency arising from a newly diagnosed patient with severe immunodeficiency.A male infant, born to consanguineous parents, suspected of having primary immunodeficiency underwent immunological and genetic workup. This included a thorough assessment of T cell phenotyping and lymphocyte activation by mitogen stimulation tests, whole-exome sequencing (WES), TCR repertoire Vβ repertoireClinical findings included microcephaly, recurrent pneumonia, and failure to thrive. An immune workup revealed lymphopenia, reduced T cell function, and hypogammaglobulinemia. Skewed TCR Vβ repertoire, TCR gamma (TRG) repertoire, and BCR repertoire were determined in the patient. Genetic analysis identified a novel homozygous missense pathogenic variant inA novel

Dysregulated immune responses are essential underlying causes of a plethora of pathologies including cancer, autoimmunity, and immunodeficiency. We here investigated 4 patients from unrelated families presenting with immunodeficiency, autoimmunity, and malignancy. We identified 4 distinct homozygous mutations in TNFRSF9 encoding the tumor necrosis factor receptor superfamily member CD137/4-1BB, leading to reduced, or loss of, protein expression. Lymphocytic responses crucial for immune surveillance, including activation, proliferation, and differentiation, were impaired. Genetic reconstitution of CD137 reversed these defects. CD137 deficiency is a novel inborn error of human immunity characterized by lymphocytic defects with early-onset Epstein-Barr virus (EBV)-associated lymphoma. Our findings elucidate a functional role and relevance of CD137 in human immune homeostasis and antitumor responses.

Background Infantile cortical hyperostosis (ICH)/Caffey disease is an inflammatory collagenopathy of infancy, manifested by subperiosteal bone hyperplasia. Genetically, ICH was linked with heterozygosity for an R836C mutation in the COL1A1 gene. Although an autosomal-recessive trait is also suspected, it has not been proven thus far. Methods A case of an infant male born to consanguineous parents is reported, presenting with classical findings, course, and clinical outcome of ICH. Whole-exome sequencing (WES) was performed in order to identify a possible underlying genetic defect. Results WES analysis revealed a novel homozygous nonsense mutation in lysine 2 of fetuin-A, encoded by the ALPHA-2-HS-GLYCOPROTEIN (AHSG) gene (c.A4T; p.K2X). Fetuin-A is an important regulator of bone remodeling and an inhibitor of ectopic mineralization. By enzyme-linked immunosorbent assay (ELISA), we show a complete deficiency of this protein in the patient’s serum, compared to controls. Conclusion A novel homozygous nonsense mutation in AHSG gene has been found in ICH patient with a typical phenotype, resulting in fetuin-A deficiency. This finding postulates an autosomal-recessive mode of inheritance in ICH, which, unlike the autosomal-dominant inheritance associated with COL1A1, is associated with AHSG and fetuin-A deficiency.

Caspase-8 (CASP8) is a protease that initiates apoptosis and regulates inflammation and immune responses. We identified germline mutations in CASP8 in 3 unrelated patients with infant-onset inflammatory bowel disease: 2 patients were homozygous for the mutation 710A>G, p.Q237R, which resulted in reduced protein expression, and 1 patient carried the mutation 793C>T, p.R265W. We isolated peripheral blood mononuclear cells from our index patient and observed defects in T- and B-cell maturation, proliferation, and/or activation. Macrophages from 1 patient with CASP8 deficiency and monocytic BLaER1 cells with knockout of CASP8 or overexpression of CASP8 with the 710A>G mutation had altered inflammasome activity on stimulation with lipopolysaccharide. Patient-derived intestinal organoids and colon carcinoma cells with knockout of CASP8 had defects in cell death processes that involved loss of TRAIL signaling and increased necroptosis. These findings indicate that CASP8 controls inflammation, innate and adaptive immunity, and intestinal barrier integrity in humans.

Benign neutropenia, observed in different ethnic groups, is the most common form of neutropenia worldwide. A specific single nucleotide polymorphism, rs2814778, located at the promoter of the ACKR1 (previously termed DARC) gene, which disrupts a binding site for the GATA1 erythroid transcription factor, resulting in a ACKR1-null phenotype, was found to serve as a predictor of low white blood cell and neutrophil counts in African-Americans and Yemenite Jews. Individuals with benign neutropenia due to the ACKR1-null allele have been found to have an increased susceptibility to human immunodeficiency virus infection and, on the other hand, a protective effect against malaria. The associated protective effect may explain the spread of the ACKR1-null allele by natural selection. The reviewed relationships between ACKR1 polymorphism and various pathological states may have important clinical implications to individuals with and without benign neutropenia. Potential mechanisms for ACKR1 (previously termed DARC) modulation during neutrophil recruitment to inflammation, and chemokine bioavailability in the circulation and in local tissue are reviewed and discussed.

Signal transducer and activator of transcription (STAT)1 heterozygous gain-of-function (GOF) mutations are known to induce immune dysregulation and chronic mucocutaneous candidiasis (CMCC). Previous reports suggest an association between demodicosis and STAT1 GOF. However, immune characterization of these patients is lacking. Here, we present a retrospective analysis of patients with immune dysregulation and STAT1 GOF who presented with facial and ocular demodicosis. In-depth immune phenotyping and functional studies were used to characterize the patients. We identified five patients (three males) from two non-consanguineous Jewish families. The mean age at presentation was 11.11 (range = 0.58–24) years. Clinical presentation included CMCC, chronic demodicosis and immune dysregulation in all patients. Whole-exome and Sanger sequencing revealed a novel heterozygous c.1386C>A; p.S462R STAT1 GOF mutation in four of the five patients. Immunophenotyping demonstrated increased phosphorylated signal transducer and activator of transcription in response to interferon-α stimuli in all patients. The patients also exhibited decreased T cell proliferation capacity and low counts of interleukin-17-producing T cells, as well as low forkhead box protein 3+ regulatory T cells. Specific antibody deficiency was noted in one patient. Treatment for demodicosis included topical ivermectin and metronidazole. Demodicosis may indicate an underlying primary immune deficiency and can be found in patients with STAT1 GOF. Thus, the management of patients with chronic demodicosis should include an immunogenetic evaluation.

Diagnosis of primary complement deficiencies requires a high index of suspicion. Thus, susceptible patients are often underdiagnosed and untreated. Here, we present a multi-center experience with three novel inborn errors of the classical complement system. This is a retrospective multicenter analysis of computerized medical records of children (> 18 years) admitted in the period between 2003 and 2018 at Shaare Zedek Medical Center in Jerusalem and Edmond and Lily Safra Children's Hospital, Tel-Hashomer Medical Center, in Ramat Gan, Israel. Patients were genetically diagnosed by a complimentary immune work-up. We identified 5 patients (3 males) from four different consanguineous families harboring three novel mutations in the complement components C6-C8. Genetic mutations were identified by whole exome sequencing. Clinical manifestations consisted of meningitis with or without meningococcemia. The immune work-up demonstrated nearly absent levels of CH50, compatible with a complement pathway defect. The mean diagnosis delay was 10.56 (0–30) years. Conclusion: Invasive meningococcal infections may be life-threatening and cause severe neurological sequela. Awareness of risk factors for primary complement deficiencies, even at the firs infectious episode, should facilitate prompt immune and genetic investigations, commencing diagnosis and proper treatment.

SLP76 is a key molecule involved in the T cell signaling pathway; therefore, SLP76 deficiency can lead to a severe type of immunodeficiency. By reporting on a patient with germline mutation in SLP76, deficiency in this gene is linked, for the first time, to a human disease., The T cell receptor (TCR) signaling pathway is an ensemble of numerous proteins that are crucial for an adequate immune response. Disruption of any protein involved in this pathway leads to severe immunodeficiency and unfavorable clinical outcomes. Here, we describe an infant with severe immunodeficiency who was found to have novel biallelic mutations in SLP76. SLP76 is a key protein involved in TCR signaling and in other hematopoietic pathways. Previous studies of this protein were performed using Jurkat-derived human leukemic T cell lines and SLP76-deficient mice. Our current study links this gene, for the first time, to a human immunodeficiency characterized by early-onset life-threatening infections, combined T and B cell immunodeficiency, severe neutrophil defects, and impaired platelet aggregation. Hereby, we characterized aspects of the patient's immune phenotype, modeled them with an SLP76-deficient Jurkat-derived T cell line, and rescued some consequences using ectopic expression of wild-type SLP76. Understanding human diseases due to SLP76 deficiency is helpful in explaining the mixed T cell and neutrophil defects, providing a guide for exploring human SLP76 biology.

Biallelic mutations in the zeta-associated protein 70 (ZAP70) gene cause combined immunodeficiency (CID). Neonatal screening for severe CID in Israel is implemented since 2015. We report on clinical, flow cytometry, and genetic data of an unusual ZAP70 deficiency patient. A 10-week-old Bedouin female presented with severe autoimmune hemolytic anemia. Cytomegalovirus (CMV) negative packed cell therapy was given without improvement; indexes of hemolysis worsened. At this time, thrombocytopenia was noted. The patient was treated with single dose of 1 g/kg intravenous immunoglobulin with rapid resolution of hemolysis. Serum immunoglobulin concentrations were normal; flow cytometry revealed severe CD8 lymphocytopenia. Lymphocyte proliferation test demonstrated reduced response to concanavalin A and phytohemagglutinin. Gated T cells were negative for intracellular ZAP70. A genetic analysis revealed a missense homozygous c.1388C T (p.A463V) mutation, confirming the diagnosis of ZAP70 deficiency. She later on developed urinary tract infection due to ESBL producing E. coli treated with amikacin and severe CMV infection that partially responded to ganciclovir therapy and at 7 months of age, she successfully underwent allogeneic hematopoietic stem cell transplantation. Neonatal screening by T cell receptor excision circles (TRECs) for SCID was normal, yet very low TRECs were recorded at the time of CID diagnosis. Normal neonatal screening for SCID does not rule out the diagnosis of CID due to ZAP70 deficiency. This type of CID can present with autoimmunity as the sole initial manifestation of the disease.

Mutations in the common gamma chain of the interleukin 2 receptor (IL2RG) or the associated downstream signaling enzyme Janus kinase 3 (JAK3) genes are typically characterized by a T cell-negative, B cell-positive, natural killer (NK) cell-negative (T-B+NK-) severe combined immunodeficiency (SCID) immune phenotype. We report clinical course, immunological, genetic and proteomic work-up of two patients with different novel mutations in the IL-2-JAK3 pathway with a rare atypical presentation of T-B+NK- SCID. Lymphocyte subpopulation revealed significant T cells lymphopenia, normal B cells, and NK cells counts (T-B+NK+SCID). Despite the presence of B cells, IgG levels were low and IgA and IgM levels were undetectable. T-cell proliferation in response to mitogens in patient 1 was very low and T-cell receptor V-beta chain repertoire in patient 2 was polyclonal. Whole-exome sequencing revealed novel mutations in both patients (patient 1-c.923delC frame-shift mutation in the IL2RG gene, patient 2-c.G172A a homozygous missense mutation in the JAK3 gene). Bioinformatic analysis of the JAK3 mutation indicated deleterious effect and 3D protein modeling located the mutation to a surface exposed alpha-helix structure. Our findings help to link between genotype and phenotype, which is a key factor for the diagnosis and treatment of SCID patients.

Severe combined immunodeficiency (SCID) is a fatal disorder resulting from various genetic defects. In the Middle East, where consanguineous marriage is prevalent, autosomal recessive mutations in recombination-activating genes (RAG) are a leading cause of SCID. We present a large cohort of SCID patients due to RAG1 or RAG2 mutations. Twenty-six patients with RAG1 or RAG2 deficiency, diagnosed at Sheba Medical Center, were retrospectively investigated. Clinical presentation, immunologic phenotype, genetic analysis, treatment, and outcome were analyzed. Majority of patients were referred from the Palestinian Authority. Most patients were males of Muslim Arab descent, 77% were born to consanguineous parents, and 65% had family history of immunodeficiency. Nearly all patients suffered from various infections before turning 2 months old, eight patients (31%) presented with Omenn and Omenn-like syndrome, and three patients (11%) had maternal engraftment. Notably, seven patients (27%) suffered from vaccine-derived infections, including a rare case of measles encephalitis. Nineteen patients underwent hematopoietic stem cell transplantation (HSCT) at a median age of 6 months, with a successful outcome for 72% of them. Genetic analysis revealed 11 different mutations (7 RAG2, 4 RAG1), two of them novel. Consanguineous marriages account for a genetic “founder effect.” SCID is a pediatric emergency that dictates immediate precautions and curative treatment with HSCT. Due to lack of newborn screening for SCID within the Palestinian population, most patients in this cohort were diagnosed upon clinical symptoms, which led to a delayed diagnosis, harmful administration of contra-indicated live vaccines, delay in HSCT, and poor outcome.

Newborn screening (NBS) programs for severe combined immunodeficiency (SCID), using the TREC-based assay, have enabled early diagnosis, prompt treatment, and eventually changed the natural history of affected infants. Nevertheless, it was believed that some affected infants with residual T cell, such as patients with MHC II deficiency, will be misdiagnosed by this assay. A full immune workup and genetic analysis using direct Sanger sequencing and whole exome sequencing have been performed to a patient that was identified by the Israeli NBS program for SCID. The patient was found to have severe CD4 lymphopenia with an inverted CD4/CD8 ratio, low TREC levels in peripheral blood, abnormal response to mitogen stimulation, and a skewed T cell receptor repertoire. HLA-DR expression on peripheral blood lymphocytes was undetectable suggesting a diagnosis of MHC II deficiency. Direct sequencing of the RFX5 gene revealed a stop codon change (p. R239X, c. C715T), which could cause the patient's immune phenotype. His parents were found to be heterozygote carriers for the mutation. Whole exome sequencing could not identify other potential mutations to explain his immunodeficiency. The patient underwent successful conditioned hematopoietic stem cell transplantation from healthy matched unrelated donor and is currently well and alive with full chimerism. Infants with MHC class II deficiency can potentially be identified by the TREC-based assay NBS for SCID. Therefore, MHC II molecules (e.g., HLA-DR) measurement should be part of the confirmatory immune-phenotyping for patients with positive screening results. This will make the diagnosis of such patients straightforward.

Neural progenitor cells undergo somatic retrotransposition events, mainly involving L1 elements, which can be potentially deleterious. Here, we analyze the whole genomes of 20 brain samples and 80 non-brain samples, and characterized the retrotransposition landscape of patients affected by a variety of neurodevelopmental disorders including Rett syndrome, tuberous sclerosis, ataxia-telangiectasia and autism. We report that the number of retrotranspositions in brain tissues is higher than that observed in non-brain samples and even higher in pathologic vs normal brains. The majority of somatic brain retrotransposons integrate into pre-existing repetitive elements, preferentially A/T rich L1 sequences, resulting in nested insertions. Our findings document the fingerprints of encoded endonuclease independent mechanisms in the majority of L1 brain insertion events. The insertions are "non-classical" in that they are truncated at both ends, integrate in the same orientation as the host element, and their target sequences are enriched with a CCATT motif in contrast to the classical endonuclease motif of most other retrotranspositions. We show that L1Hs elements integrate preferentially into genes associated with neural functions and diseases. We propose that pre-existing retrotransposons act as "lightning rods" for novel insertions, which may give fine modulation of gene expression while safeguarding from deleterious events. Overwhelmingly uncontrolled retrotransposition may breach this safeguard mechanism and increase the risk of harmful mutagenesis in neurodevelopmental disorders.

Background The SRP54 (signal recognition protein 54) is a conserved component of the ribonucleoprotein complex that mediates cotranslational targeting and translocation of proteins to the endoplasmic reticulum. In 2017, mutations in the gene have been described as a cause of congenital neutropenia with or without pancreatic insufficiency, and since then, only limited cases were added to the literature. Methods Two patients with neutropenia underwent hematological, immunological, and genetic work-up, including lymphocyte phenotyping, immunoglobulins, and complement levels, antineutrophil and antinuclear antibodies, bone marrow FISH panel for myelodysplastic syndrome, whole-exome sequencing, and in silico proteomic analysis. Results Clinical findings in the two families revealed a wide spectrum of immunological and clinical manifestations, ranging from mild asymptomatic neutropenia during febrile illnesses to severe neutropenia and life-threatening infection requiring leg amputation. Immunological and hematological work-up showed isolated neutropenia with normal lymphocyte subpopulations, immunoglobulin and complement levels, and negative autoimmune tests. Bone marrow aspirations showed variability ranging from normal myelopoiesis to myeloid maturation arrest at the promyelocytic stage, with normal FISH panel for myelodysplastic syndrome. Genetic analysis identified a novel, de novo, in-frame deletion in the SRP54 gene, c.342-344delAAC, p.T115del. In silico proteomic analysis suggested impaired SRP54 protein function due to reduced GTP activity and stability. Conclusions We describe congenital neutropenia with variable clinical presentation in novel mutation of the SRP54 gene.

Allogeneic hematopoietic stem cell transplantation (HSCT) is the effective mean of immune restoration in severe combined immunodefiency (SCID). Usually, HSCT without cytoreductive conditioning is attempted. Nevertheless, conditioning procedures are still preferred in a subset of patients. Herein, we describe the immunological outcome in a cohort of conditioned and unconditioned patients, from diagnosis, through transplantation, to follow-up. This retrospective study was conducted on 17 patients with SCID (10 conditioned, 7 unconditioned) who later underwent HSCT. Immune reconstitution was assessed in the post-transplant year by quantification of T cell receptor excision circles (TRECs) and kappa-deleting recombination excision circles (KRECs), among additional laboratory and clinical evaluations. Unconditioned patients were diagnosed and transplanted earlier. TREC and KREC quantification showed a gradual increase in both groups, with higher levels in the conditioned group. Engraftment percentages differed drastically between groups, favoring the conditioned group. Unconditioned patients were significantly more dependent on intravenous immunoglobulins (IVIGs). One patient from each group succumbed to disease complications. Conditioning demonstrated superior laboratorial outcomes. Patients with unique characteristics (i.e., consanguinity, Bacillus Calmette-Guerin vaccination, impaired access to IVIG) may require personalized considerations. The effort to implement secondary prevention of SCID with newborn screening should continue.

Severe combined immunodeficiency (SCID) is a fatal disorder resulting from various genetic defects. In the Middle East, where consanguineous marriage is prevalent, autosomal recessive mutations in recombination-activating genes (RAG) are a leading cause of SCID. We present a large cohort of SCID patients due to RAG1 or RAG2 mutations.Twenty-six patients with RAG1 or RAG2 deficiency, diagnosed at Sheba Medical Center, were retrospectively investigated. Clinical presentation, immunologic phenotype, genetic analysis, treatment, and outcome were analyzed.Majority of patients were referred from the Palestinian Authority. Most patients were males of Muslim Arab descent, 77% were born to consanguineous parents, and 65% had family history of immunodeficiency. Nearly all patients suffered from various infections before turning 2 months old, eight patients (31%) presented with Omenn and Omenn-like syndrome, and three patients (11%) had maternal engraftment. Notably, seven patients (27%) suffered from vaccine-derived infections, including a rare case of measles encephalitis. Nineteen patients underwent hematopoietic stem cell transplantation (HSCT) at a median age of 6 months, with a successful outcome for 72% of them. Genetic analysis revealed 11 different mutations (7 RAG2, 4 RAG1), two of them novel.Consanguineous marriages account for a genetic "founder effect." SCID is a pediatric emergency that dictates immediate precautions and curative treatment with HSCT. Due to lack of newborn screening for SCID within the Palestinian population, most patients in this cohort were diagnosed upon clinical symptoms, which led to a delayed diagnosis, harmful administration of contra-indicated live vaccines, delay in HSCT, and poor outcome.

Immunodeficiency, centromeric instability, and facial anomalies (ICF) syndrome is an extremely rare autosomal recessive disease. The immune phenotype is characterized by hypogammaglobulinemia in the presence of B cells. T cell lymphopenia also develops in some patients. We sought to further investigate the immune defect in an ICF patient with a novel missense mutation in DNMT3B and a severe phenotype. Patient lymphocytes were examined for subset counts, immunoglobulin levels, T and B cell de novo production (via excision circles) and receptor repertoire diversity. Mutated DNMT3B protein structure was modeled to assess the effect of a mutation located outside of the catalytic region on protein function. A novel homozygous missense mutation, Ala585Thr, was found in DNMT3B. The patient had decreased B cell counts with hypogammaglobulinemia, and normal T cell counts. CD4+ T cells decreased over time, leading to an inversion of the CD4+ to CD8+ ratio. Excision circle copy numbers were normal, signifying normal de novo lymphocyte production, but the ratio between naive and total B cells was low, indicating decreased in vivo B cell replication. T and B cell receptor repertoires displayed normal diversity. Computerized modeling of the mutated Ala585 residue suggested reduced thermostability, possibly affecting the enzyme kinetics. Our results highlight the existence of a T cell defect that develops over time in ICF patient, in addition to the known B cell dysfunction. With intravenous immunoglobulin (IVIG) treatment ameliorating the B cell defect, the extent of CD4+ lymphopenia may determine the severity of ICF immunodeficiency.

// Amos Toren 1 , Tatyana Pismenyuk 1 , Michal Yalon 1 , Shani Freedman 1 , Amos J. Simon 1 , Tamar Fisher 1 , Itai Moshe 1 , Juergen K.V. Reichardt 2 , Shlomi Constantini 3 , Yael Mardor 4 , David Last 4 , David Guez 4 , Dianne Daniels 4 , Moria Assoulin 4 , Ruty Mehrian-Shai 1 1 Pediatric Hemato-Oncology, Edmond and Lilly Safra Children's Hospital and Cancer Research Center, Sheba Medical Center, Tel Hashomer Affiliated to The Sackler School of Medicine, Tel-Aviv University, Tel Aviv, Israel 2 YachayTech University, Urcuqui, Ecuador 3 Department of Pediatric Neurosurgery, Dana Children’s Hospital, Tel-Aviv-Sourasky Medical Center, Israel 4 The Advanced Technology Center, Sheba Medical Center, Tel Hashomer Affiliated to The Sackler School of Medicine, Tel-Aviv University, Tel Aviv, Israel Correspondence to: Ruty Mehrian-Shai, email: Ruty.Shai@sheba.health.gov.il Keywords: glioblastoma multiforme, temozolomide, chemosensitivity, proliferation, zinc Abbreviations: GBM: Glioblastoma multiforme; TMZ: Temozolomide; ZnCl 2 : Zinc chloride Received: May 30, 2016 Accepted: July 19, 2016 Published: August 19, 2016 ABSTRACT Temozolomide (TMZ) is an alkylating agent that has become the mainstay treatment of the most malignant brain cancer, glioblastoma multiforme (GBM). Unfortunately only a limited number of patients positively respond to it. It has been shown that zinc metal reestablishes chemosensitivity but this effect has not been tested with TMZ. Using both in vitro and in vivo experimental approaches, we investigated whether addition of zinc to TMZ enhances its cytotoxicity against GBM. In vitro cell viability analysis showed that the cytotoxic activity of TMZ was substantially increased with addition of zinc and this response was accompanied by an elevation of p21, PUMA, BAX and Caspase-3 expression and a decrease in growth fraction as manifested by low ki67 and lower colony formation. Analysis of GBM as intracranial xenografts in athymic mice and administration of concurrent TMZ and zinc yielded results consistent with those of the in vitro analyses. The co-treatment resulted in significant reduction in tumor volume in TMZ/zinc treated mice relative to treatment with TMZ alone. Our results suggest that zinc may serve as a potentiator of TMZ therapy in GBM patients.

Monosomy 21 is an extremely rare genetic disorder presenting with a wide array of symptoms. Recurrent infections, some life threatening, have been reported in several monosomy 21 patients and attributed to an, as of yet, undefined immunodeficiency. Here we report on a 3-year-old boy with mosaic monosomy 21 who presented with clinical and laboratory evidence of immunodeficiency. Despite suffering from infections highly suggestive of a cell-mediated immune defect, the patient's T cells displayed normal counts, subsets and proliferation capability. T cell receptor repertoire was diverse, and de novo T cell production was intact. Consistent with earlier case reports, our patient displayed mildly low B cell counts with hypogammaglobulinemia. B cell subsets demonstrated mainly naïve and marginal zone B cells that have not undergone class switch. Subsequently, IgG, IgA and IgE levels were near absent, whereas IgM level was normal. De novo B cell production and B cell receptor diversity were normal. Together, these results are indicative of a defect in immunoglobulin class switching as the principal cause of immunodeficiency in monosomy 21. A better understanding of the immunodeficiency in this syndrome will enable targeted treatment and prevention of infections in order to prevent morbidity and mortality in these patients.

Primary immunodeficiencies (PIDs) are a heterogeneous group of monogenic inborn errors of immunity. The genetic causes of these diseases can be identified using whole exome sequencing (WES). Here, DNA samples from 106 patients with a clinical suspicion of PID were subjected to WES in order to test the diagnostic yield of this test in a highly consanguineous community. A likely genetic diagnosis was achieved in 70% of patients. Several factors were considered to possibly influence the diagnostic rate of WES among our cohort including early age, presence of consanguinity, family history suggestive of PID, the number of family members who underwent WES and the clinical phenotype of the patient. The highest diagnostic rate was in patients with combined immunodeficiency or with a syndrome. Notably, WES findings altered the clinical management in 39% (41/106) of patients in our cohort. Our findings support the use of WES as an important diagnostic tool in patients with suspected PID, especially in highly consanguineous communities.

Clathrin-mediated endocytosis (CME) is critical for internalisation of molecules across cell membranes. The FCH domain only 1 (FCHO1) protein is key molecule involved in the early stages of CME formation. The consequences of mutations in FCHO1 in humans were unknown. We identify ten unrelated patients with variable T and B cell lymphopenia, who are homozygous for six distinct mutations in FCHO1. We demonstrate that these mutations either lead to mislocalisation of the protein or prevent its interaction with binding partners. Live-cell imaging of cells expressing mutant variants of FCHO1 provide evidence of impaired formation of clathrin coated pits (CCP). Patient T cells are unresponsive to T cell receptor (TCR) triggering. Internalisation of the TCR receptor is severely perturbed in FCHO1-deficient Jurkat T cells but can be rescued by expression of wild-type FCHO1. Thus, we discovered a previously unrecognised critical role of FCHO1 and CME during T-cell development and function in humans., FCH domain only 1 (FCHO1) is a key molecule involved in clathrin-mediated endocytosis (CME). Here, the authors report homozygous FCHO1 mutations in individuals with variable T and B cell lymphopenia, which are associated with loss-of-function of FCHO1 and impaired formation of clathrin-coated pits in T cells.

MALT1 (mucosa-associated lymphoid tissue lymphoma-translocation gene 1) is an intracellular signaling protein that activates NFκB and is crucial for both the adaptive and innate immune responses. Only 6 patients with immune deficiencies secondary to inherited mutations in the MALT1 gene have been described. To provide clinical and immunological insights from 2 patients diagnosed with MALT1 immunodeficiency syndrome due to a novel MALT1 mutation. Two cousins with suspected combined immunodeficiency underwent immunological and genetic work-up, including lymphocyte phenotyping, lymphocyte activation by mitogen stimulation, and next-generation sequencing (NGS) of T cell receptor gamma chain (TRG) repertoire. Whole exome sequencing was performed to identify the underlying genetic defect. Clinical findings included recurrent infections, failure to thrive, lymphadenopathy, dermatitis, and autoimmunity. Immune work-up revealed lymphocytosis, low to normal levels of immunoglobulins, absence of regulatory T cells, and low Th17 cells. A normal proliferative response was induced by phytohemagglutinin and IL-2 but was diminished with anti-CD3. TRG repertoire was diverse with a clonal expansion pattern. Genetic analysis identified a novel autosomal recessive homozygous c.1799T>A; p. I600N missense mutation in MALT1. MALT1 protein expression was markedly reduced, and in vitro IL-2 production and NFκB signaling pathway were significantly impaired. Two patients harboring a novel MALT1 mutation presented with signs of immune deficiency and dysregulation and were found to have an abnormal T cell receptor repertoire. These findings reinforce the link between MALT1 deficiency and combined immunodeficiency. Early diagnosis is crucial, and curative treatment by hematopoietic stem cell transplantation may be warranted.

Background Acute MI induces leptin expression in the heart, however the role of myocardial leptin in cardiac ischemia and reperfusion (IR) remains unknown. To shed light on the effects of elevated levels of leptin in the myocardium, we overexpressed cardiac leptin and assessed local remodeling and myocardial function in this context. Methods and results Cardiac leptin overexpression was stimulated in mice undergoing IR by a single intraperitoneal injection of leptin antagonist (LepA). All mice exhibited a normal pattern of body weight gain. A rapid, long-term upregulation of leptin mRNA was demonstrated in the heart, adipose, and liver tissues in IR/LepA-treated mice. Overexpressed cardiac leptin mRNA extended beyond postoperative day (POD) 30. Plasma leptin peaked 7.5 hours postoperatively, especially in IR/LepA-treated mice, subsiding to normal levels by 24 hours. On POD-30 IR/LepA-treated mice demonstrated cardiomyocyte hypertrophy and perivascular fibrosis compared to IR/saline controls. Echocardiography on POD-30 demonstrated eccentric hypertrophy and systolic dysfunction in IR/LepA. We recorded reductions in Ejection Fraction (p

Infants with severe combined immunodeficiency (SCID) are at risk of developing severe life-threatening infections if they are inadvertently given attenuated live vaccines. Concomitant appearance of two live vaccine-associated complications in one person is rarely reported. In this study, we present two SCID infants, who received BCG and oral polio vaccines according to their local immunization schedule early in life, before the diagnosis of immunodeficiency was made. Their clinical presentation, extensive immunological workup, genetic tests, and clinical disease course are presented. Both patients developed localized and disseminated infections originating from the BCG vaccine (BCGitis and BCGiosis, respectively) and in addition suffered from diarrhea and chronic fecal secretion of vaccine-derived poliovirus. Alarmingly, in case 2, the poliovirus was a type 2 vaccine-derived poliovirus in which both neurovirulence attenuation sites reverted to the neurovirulent genotype. These cases highlight the importance of early recognition of SCID by neonatal screening or thorough family anamnesis, and the need to further defer the timing of administration of attenuated live vaccines.

RAS guanyl-releasing protein 1 (RASGRP1) deficiency has recently been shown to cause a primary immunodeficiency (PID) characterized by CD4+ T cell lymphopenia and Epstein-Barr virus (EBV)-associated B cell lymphoma. Our report of three novel patients widens the scope of RASGRP1 deficiency by providing new clinical and immunological insights on autoimmunity, immune cell development, and predisposition to lymphoproliferative disease. One patient of Turkish origin (P1) and two Palestinian patients (P2, P3) were evaluated for immunodeficiency. To decipher the molecular cause of disease, whole exome sequencing was conducted. Identified mutations were validated by immunological and biochemical assays. We report three patients presenting with similar clinical characteristics of immunodeficiency and EBV-associated lymphoproliferative disease. In addition, P2 and P3 exhibited overt autoimmune manifestations. Genetic screening identified two novel loss-of-function mutations in RASGRP1. Immunoblotting and active Ras pull-down assays confirmed perturbed ERK1/2 signaling and reduced Ras-GTPase activity in heterologous Jurkat cells with ectopic expression of RASGRP1 mutants. All three patients had CD4+ T cell lymphopenia. P2 and P3 showed decreased mitogen-induced lymphocyte proliferation, reduced T cell receptor excision circles, abnormal T cell receptor (TCR) Vβ repertoires, and increased frequencies of TCRγδ cells. TCR gamma repertoire diversity was significantly reduced with a remarkable clonal expansion. RASGRP1 deficiency is associated with life-threatening immune dysregulation, severe autoimmune manifestations, and susceptibility to EBV-induced B cell malignancies. Early diagnosis is critical and hematopoietic stem cell transplantation might be considered as curative treatment.

Leukocyte adhesion deficiency type 1 (LAD-1) is an autosomal recessive primary immunodeficiency, hallmarked by defective polymorphonuclear transmigration. It is caused by mutations in the gene encoding CD18, which interfere with the CD18/CD11 heterodimerization and expression on leukocyte cell surface. LAD-1 diagnosis rests primarily on the measurement of CD18 expression. However, CD18 measurement entails its pitfalls. Here we present a cohort of ten LAD patients and a review of the relevant literature illustrating the difficulties in sole reliance on CD18 measurement for initial diagnosis. These include normal range expression in some mutations, great variability between patients with the same mutation and subjective interpretation of results. We think there is a need for additional markers as part of the initial LAD diagnostic algorithm. We suggest CD11a expression, which was near absent in all patients in our cohort. The dual use of CD18 and CD11a can increase testing sensitivity and prevent delayed diagnosis of LAD-1.

VPS45 mutations cause severe congenital neutropenia (SCN). We report on a girl with SCN and neurological impairment harboring a homozygous p. E238K mutation in VPS45 (vacuolar sorting protein 45). She successfully underwent hematopoietic stem cell transplantation. Our findings delineate the phenotype and indicate a possible genotype-phenotype correlation for neurological involvement. (C) 2015 Wiley Periodicals, Inc.

Regulation of the actin cytoskeleton is crucial for normal development and function of the immune system, as evidenced by the severe immune abnormalities exhibited by patients bearing inactivating mutations in the Wiskott–Aldrich syndrome protein (WASP), a key regulator of actin dynamics. WASP exerts its effects on actin dynamics through a multisubunit complex termed Arp2/3. Despite the critical role played by Arp2/3 as an effector of WASP-mediated control over actin polymerization, mutations in protein components of the Arp2/3 complex had not previously been identified as a cause of immunodeficiency. Here, we describe two brothers with hematopoietic and immunologic symptoms reminiscent of Wiskott–Aldrich syndrome (WAS). However, these patients lacked mutations in any of the genes previously associated with WAS. Whole-exome sequencing revealed a homozygous 2 bp deletion, n.c.G623DEL-TC (p.V208VfsX20), in Arp2/3 complex component ARPC1B that causes a frame shift resulting in premature termination. Modeling of the disease in zebrafish revealed that ARPC1B plays a critical role in supporting T cell and thrombocyte development. Moreover, the defects in development caused by ARPC1B loss could be rescued by the intact human ARPC1B ortholog, but not by the p.V208VfsX20 variant identified in the patients. Moreover, we found that the expression of ARPC1B is restricted to hematopoietic cells, potentially explaining why a mutation in ARPC1B has now been observed as a cause of WAS, whereas mutations in other, more widely expressed, components of the Arp2/3 complex have not been observed.

Summary Ras-associated lymphoproliferative disease (RALD) is an autoimmune lymphoproliferative syndrome (ALPS)-like disease caused by mutations in Kirsten rat sarcoma viral oncogene homologue (KRAS) or neuroblastoma RAS viral (V-Ras) oncogene homologue (NRAS). The immunological phenotype and pathogenesis of RALD have yet to be studied extensively. Here we report a thorough immunological investigation of a RALD patient with a somatic KRAS mutation. Patient lymphocytes were analysed for phenotype, immunoglobulin levels and T cell proliferation capacity. T and B cell receptor excision circles (TREC and KREC, respectively), markers of naive T and B cell production, were measured serially for 3 years. T and B cell receptor repertoires were studied using both traditional assays as well as next-generation sequencing (NGS). TREC and KREC declined dramatically with time, as did T cell receptor diversity. NGS analysis demonstrated T and B clonal expansions and marked restriction of T and B cell receptor repertoires compared to healthy controls. Our results demonstrate, at least for our reported RALD patient, how peripheral T and B clonal expansions reciprocally limit lymphocyte production and restrict the lymphocyte receptor repertoire in this disease. Decreased naive lymphocyte production correlated with a clinical deterioration in our patient's immune status, suggesting that TREC and KREC may be used as an aid in monitoring disease progression. Both the methodologies used here and the conclusions regarding immune homeostasis may be applicable to the research of ALPS and other immune dysregulation syndromes.

Mutations in the dedicator of cytokinesis 8 (DOCK8) gene cause a combined immunodeficiency usually diagnosed as autosomal recessive hyper IgE syndrome. We sought to reveal the varying manifestations in patients with a unique mutation in DOCK8 gene by a retrospective medical record review. Ten patients from five consanguineous families and three tribes were included. Seven patients were homozygous for the c.C5134A, p.S1711X mutation, and the remaining three patients were their siblings manifesting hyper IgE syndrome features without a genetic diagnosis. Prior to the genetic diagnosis, the clinical diagnosis was “hyper IgE syndrome” in six patients and “anti-pneumococcal antibody deficiency,” “recurrent pneumonia with bronchiectasis,” and “asthma with hypereosinophilic syndrome” each diagnosed once. One additional patient was diagnosed due to family history. The age of presentation varied from 1 to 16 months. Eczema was diagnosed in all patients, food allergies in three, and severe herpes keratitis or malignancy or autoimmunity in two patients. Elevated IgE was recorded in nine patients; however, in six patients, the initial serum IgE concentration was equal to or less than three times the normal concentration for age, and in these patients, the median age at IgE evaluation was 7.5 months compared with 21.5 months in patients with an initial IgE concentration above three times the normal concentration for age (P = 0.067). The spectrum of disease manifestations in patients with a unique mutation in DOCK8 is variable. The genotype-phenotype correlations may be modified by genetic and/or epigenetic modifiers beyond the monogenic effect. Younger patients tend to have lower IgE concentrations at the initial measurement of IgE.

The thymus is a highly specialized organ for T cell receptor (TCR) rearrangement and selection mechanisms that ensure the formation of functional and self-tolerant cells. Little is known about how peripheral blood assessment of thymic function reflects thymus activity during infancy. We compared thymic function-related markers in the thymus with those in peripheral blood in order to check their correlations. We concomitantly blood samples from immunocompetent infants who underwent cardiac surgery that involved thymectomy. The studied thymic markers included TCR excision circles (TRECs), four different TCRD (TCR delta chain) gene rearrangements, the TCR repertoire, regulatory T cells (Tregs, defined as the CD4+CD25+FOXP3+ cell population) and real-time quantitative polymerase chain reaction (RQ-PCR) mRNA expression of forkhead box P3 (FOXP3). Twenty patients were enrolled in this study. Their mean age at the time of the surgery was 3 months/5 days ± 3 months/18 days. There was a significant correlation between thymic and peripheral blood levels of TREC, all four TCRD gene rearrangements and the amount of Tregs. The levels of these parameters were significantly higher in the thymus than those detected in the peripheral blood. The TCR repertoire distribution in both samples was similar. FOXP3 mRNA levels in the thymus and peripheral blood correlated well. Our findings demonstrated a strong and significant correlation between peripheral blood and intra-thymic activity parameters during infancy. Assessment of these parameters in peripheral blood can be used to accurately estimate different intra-thymic capacities for assessing T cell function in health and disease.

Background: Understanding genetic predisposition to cancer is of paramount importance to tailor therapeutic strategies. Several defects in immune checkpoint regulators have been discovered in patients with EBV-induced lymphoma (e.g. CD27, PRKCD, RASGRP1, MAGT1, SH2D1A, ITK). Here, we describe the clinical and immune phenotype of 2 unrelated patients from consanguineous families presenting with a primary immune deficiency (PID) and EBV-associated lymphoproliferation due to biallelic mutations in CD137 (TNFRSF9/4-1BB). Methods: One patient of Turkish origin (P1) and Palestinian origin (P2), respectively were evaluated. Genetic analysis using whole exome sequencing was conducted. Immunological and biochemical assays were performed on primary patient material. Results: Clinical findings included recurrent sinopulmonary and herpes virus infections from childhood on. One patient suffered from auto-immunity (AIHA and auto-immune thrombocytopenia). Abnormal immunoglobulin levels were documented (IgG 413-1670, IgM 105-714, IgA 49-68 mg/dL). Both patients developed EBV-associated lymphoproliferative disorders: P1 developed Burkitt lymphoma and was treated according to NHL-BFM2000 regimen in combination with rituximab. He is currently in remission. P2 had monoclonal EBV-positive lymphoproliferation and was successfully treated with immunosuppressive therapy (e.g. cellcept, glucocorticoids). To shed light on the underlying genetic etiology, we performed whole exome sequencing. A large homozygous deletion in CD137 (c.1_545+1716del) was identified for P1, while P2 harbored a homozygous missense mutation (c.C452T, p.Thr151Met). Impaired anti-CD3 T cell lymphocyte activation and proliferation were observed, amenable to correction upon addition of anti-CD28 monoclonal antibodies. In an attempt to provide definitive proof that the CD137 gene variant causes the activation defect of T-cells, we designed a genetic rescue experiment in patient T cells. Upon retrovirus-mediated recombinant expression of WT CD137, proliferation and activation defects in T cells were restored. Conclusions: In sum, we here show that a genetic defect in the immune checkpoint molecule CD137 causes a new primary immunodeficiency disorder with susceptibility to EBV-induced lymphomagenesis. Disclosures No relevant conflicts of interest to declare.

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 child's 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 ?1 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.).

Background MHC class II (MHC-II) molecules play a pivotal role in the development, activation, and homeostasis of CD4 + T H cells in the thymus. The absence of MHC-II molecules causes severe T-cell immunodeficiency. Objective We sought to study thymic function, including T-cell receptor excision circle (TREC) quantification, in patients with MHC-II deficiency. Methods Eight MHC-II–deficient patients underwent a thorough T-cell immunologic work-up, including thymic activity, which was estimated based on TREC levels and T-cell receptor (TCR) genes, as well as analysis of several sequential human TCR gene rearrangements. Results In vitro responses to mitogens were normal or only slightly reduced, and flow cytometric evaluations of the TCR-Vβ repertoires of total CD3 + lymphocytes were normal in all patients. However, both the flow cytometric evaluation of the TCR-Vβ repertoire on CD4 + cells and spectratyping evaluation of the TCR-Vγ repertoire on total CD3 + lymphocytes showed clonal abnormalities. TRECs were present in all patients in both total lymphocytes and sorted CD4 + cells. Additionally, TRECs were detected in genomic DNA obtained from Guthrie cards with dried blood spots. Quantitative RT-PCR assessment of different TCR gene rearrangement events revealed lower levels in MHC-II–deficient patients compared with levels seen in healthy control subjects. This was irrespective of the total lymphocyte numbers, suggesting a reduced global thymic activity. Conclusions Our report highlights potential pitfalls in diagnosing MHC-II deficiency and emphasizes the probable importance of MHC-II molecules in the normal thymic maturation process of T cells. Patients with MHC-II deficiency have detectable TRECs and might therefore be missed by a TREC-based newborn screening program.