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Background and aimsThere is an increased risk of lymphomas in inborn errors of immunity (IEI); however, germline genetic testing is rarely used in oncological patients, even in those with early onset of cancer. Our study focuses on a child with a recombination-activating gene 1 (RAG1) deficiency who was identified through a screening program for Slavic founder genetic variants among patients who died with malignancy at an early age in Belarus.ResultsWe identified one homozygous founder RAG1 variant out of 24 available DNA samples from 71 patients who developed lymphoma aged

Abstract Background The recombination-activating gene 1 (RAG1) protein is essential for the V (variable)-D (diversity)-J (joining) recombination process. Mutations in RAG1 have been reported to be associated with several types of immune disorders. Typical clinical features driven by RAG1 variants include persistent infections, severe lymphopenia, and decreased immunoglobulin levels . Case presentation In this study, a 2-month-24-days-old infant with recurrent fever was admitted to our hospital with multiple infections and absence of T and B lymphocytes. The infant was diagnosed with severe combined immunodeficiency (SCID). A homozygous variation c.2147G>A (NM_000448.2: exonme2: c.2147G>A (p.Arg716Gln)) was identified in the RAG1 gene using whole-exome sequencing and Sanger sequencing. The predicted 3D structure of variant RAG1 indicated altered protein stability. Additionally, decreased expression of variant RAG1 gene was detected at both the mRNA and protein levels. Conclusions Our study identified a novel homozygous variant in RAG1 gene that causes SCID. This finding expands the variant spectrum of RAG1 in SCID and provides further evidence for the clinical diagnosis of SCID.

Mutations in the recombination activating gene 1 (RAG1) and RAG2 in humans are associated with a broad spectrum of clinical phenotypes, from severe combined immunodeficiency to immune dysregulation. Partial (hypomorphic) RAG deficiency (pRD) in particular, frequently leads to hyperinflammation and autoimmunity, with several underlying intrinsic and extrinsic mechanisms causing a break in tolerance centrally and peripherally during T and B cell development. However, the relative contributions of these processes to immune dysregulation remain unclear. In this review, we specifically focus on the recently described tolerance break and B cell abnormalities, as well as consequent molecular and cellular mechanisms of autoantibody production in patients with pRD. [ABSTRACT FROM AUTHOR]

Background: The recombination-activating gene 1 (RAG1) protein is essential for the V (variable)-D (diversity)-J (joining) recombination process. Mutations in RAG1 have been reported to be associated with several types of immune disorders. Typical clinical features driven by RAG1 variants include persistent infections, severe lymphopenia, and decreased immunoglobulin levels. Case presentation: In this study, a 2-month-24-days-old infant with recurrent fever was admitted to our hospital with multiple infections and absence of T and B lymphocytes. The infant was diagnosed with severe combined immunodeficiency (SCID). A homozygous variation c.2147G>A (NM_000448.2: exonme2: c.2147G>A (p.Arg716Gln)) was identified in the RAG1 gene using whole-exome sequencing and Sanger sequencing. The predicted 3D structure of variant RAG1 indicated altered protein stability. Additionally, decreased expression of variant RAG1 gene was detected at both the mRNA and protein levels. Conclusions: Our study identified a novel homozygous variant in RAG1 gene that causes SCID. This finding expands the variant spectrum of RAG1 in SCID and provides further evidence for the clinical diagnosis of SCID. [ABSTRACT FROM AUTHOR]

The emergence, survival, growth and maintenance of autoreactive (AR) B-cell clones, the hallmark of humoral autoimmunity, leave their footprints in B-cell receptor repertoires. Collecting IgH sequences related to polyreactive (PR) ones from adaptive immune receptor repertoire (AIRR) datasets make the reconstruction and analysis of PR/AR B-cell lineages possible. We developed a computational approach, named ImmChainTracer, to extract members and to visualize clonal relationships of such B-cell lineages. Our approach was successfully applied on the IgH repertoires of patients suffering from monogenic hypomorphic RAG1 and 2 deficiency (pRD) or polygenic systemic lupus erythematosus (SLE) autoimmune diseases to identify relatives of AR IgH sequences and to track their fate in AIRRs. Signs of clonal expansion, affinity maturation and class-switching events in PR/AR and non-PR/AR B-cell lineages were revealed. An extension of our method towards B-cell expansion caused by any trigger (e.g., infection, vaccination or antibody development) may provide deeper insight into antigen specific B-lymphogenesis. [ABSTRACT FROM AUTHOR]

Mutations in the recombination activating genes (RAG) cause various forms of immune deficiency. Hematopoietic stem cell transplant (HSCT) is the only cure for patients with severe manifestations of RAG deficiency; however, outcomes are suboptimal with mismatched donors. Gene therapy aims to correct autologous hematopoietic stem and progenitor cells (HSPC) and is emerging as an alternative to allogeneic HSCT. Gene therapy based on viral gene addition exploits viral vectors to add a correct copy of a mutated gene into the genome of HSPCs. Only recently, after a prolonged phase of development, viral gene addition has been approved for clinical testing in RAG1-SCID patients. In the meantime, a new technology, CRISPR/Cas9, has made its debut to compete with viral gene addition. Gene editing based on CRISPR/Cas9 allows to perform targeted genomic integrations of a correct copy of a mutated gene, circumventing the risk of virus-mediated insertional mutagenesis. In this review, we present the biology of the RAG genes, the challenges faced during the development of viral gene addition for RAG1-SCID, and the current status of gene therapy for RAG1 deficiency. In particular, we highlight the latest advances and challenges in CRISPR/Cas9 gene editing and their potential for the future of gene therapy., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: FJT Staal and AC Lankester has patent #RAG1 gene therapy issued to Leiden University Medical Center. none If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

The use of HLA-identical hematopoietic stem cell transplantation (HSCT) demonstrates overall survival rates greater than 75 % for T-B-NK+ severe combined immunodeficiency secondary to pathogenic mutation of recombinase activating genes 1 and 2 (RAG1/2). Limited data exist regarding the use of HSCT in patients with hypomorphic RAG variants marked by greater preservation of RAG activity and associated phenotypes such as granulomatous disease in combination with autoimmunity. We describe a 17-year-old with combined immunodeficiency and immune dysregulation characterized by granulomatous lung disease and autoimmunity secondary to compound heterozygous RAG mutations. A myeloablative reduced toxicity HSCT was completed using an unrelated bone marrow donor. With the increasing cases of immune dysregulation being discovered with hypomorphic RAG variants, the use of HSCT may advance to the forefront of treatment. This case serves to discuss indications of HSCT, approaches to preparative therapy, and the potential complications in this growing cohort of patients with immune dysregulation and RAG deficiency.

Purpose: We described clinical, immunological, and molecular characterization within a cohort of 22 RAG patients focused on the possible correlation between clinical and genetic data. Methods: Immunological and genetic features were investigated by multiparametric flow cytometry and by Sanger or next generation sequencing (NGS) as appropriate. Results: Patients represented a broad spectrum of RAG deficiencies: SCID, OS, LS/AS, and CID. Three novel mutations in RAG1 gene and one in RAG2 were reported. The primary symptom at presentation was infections (81.8%). Infections and autoimmunity occurred together in the majority of cases (63.6%). Fifteen out of 22 (68.2%) patients presented autoimmune or inflammatory manifestations. Five patients experienced severe autoimmune cytopenia refractory to different lines of therapy. Total lymphocytes count was reduced or almost lacking in SCID group and higher in OS patients. B lymphocytes were variably detected in LS/AS and CID groups. Eighteen patients underwent HSCT permitting definitive control of autoimmune/hyperinflammatory manifestations in twelve of them (80%). Conclusion: We reinforce the notion that different clinical phenotype can be found in patients with identical mutations even within the same family. Infections may influence genotype–phenotype correlation and function as trigger for immune dysregulation or autoimmune manifestations. Severe and early autoimmune refractory cytopenia is frequent and could be the first symptom of onset. Prompt recognition of RAG deficiency in patients with early onset of autoimmune/hyperinflammatory manifestations could contribute to the choice of a timely and specific treatment preventing the onset of other complications. [ABSTRACT FROM AUTHOR]

Background and Aims: There is an increased risk of lymphomas in inborn errors of immunity (IEI); however, germline genetic testing is rarely used in oncological patients, even in those with early onset of cancer. Our study focuses on a child with a recombination-activating gene 1 ( RAG1 ) deficiency who was identified through a screening program for Slavic founder genetic variants among patients who died with malignancy at an early age in Belarus., Results: We identified one homozygous founder RAG1 variant out of 24 available DNA samples from 71 patients who developed lymphoma aged <3 years from the Belarusian cancer registry between 1986 and 2023. Our patient had an episode of pneumonia at 3 months of age and was hospitalized for respiratory distress, candida-positive lung disease, and lymphadenopathy at 14 months of age. The diagnosis of Epstein-Barr virus (EBV)-positive diffuse large B-cell lymphoma (DLBCL) was established. The patient had a normal lymphocyte count that decreased over time. One month after chemotherapy initiation, the patient died due to sepsis and multiple organ failure without a genetic diagnosis. In a retrospective analysis, T-cell receptor excision circles (TRECs) and kappa-deleting recombination excision circles (KRECs) were undetectable in peripheral blood., Conclusions: A targeted screening program designed to detect a Slavic founder variant in the RAG1 gene among children revealed a 14-month-old Belarusian male infant with low TREC levels who died of EBV-driven DLBCL and complications of chemotherapy including infections. This case highlights how patients with IEI and recurrent infections may develop serious non-infectious complications, such as fatal malignancy. It also emphasizes the importance of early identification, such as newborn screening for severe combined immune deficiency. Earlier diagnosis of RAG deficiency could have prompted hematopoietic stem cell transplant well before the DLBCL occurrence. This likely would impact the onset and/or management strategies for the cancer., Competing Interests: LN is supported by the Division of Intramural Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA (grant AI001222). JW is supported by National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD, USA (grant # R01AI153830), Jeffrey Modell Foundation, and the Robert A. Good Endowment at the University of South Florida. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (© 2024 Volodashchik, Polyakova, Mikhaleuskaya, Sakovich, Kupchinskaya, Dubrouski, Belevtsev, Dasso, Varabyou, Notarangelo, Walter and Sharapova.)

Vasculitis can be a life-threatening complication associated with high mortality and morbidity among patients with primary immunodeficiencies (PIDs), including variants of severe and combined immunodeficiencies ((S)CID). Our understanding of vasculitis in partial defects in recombination activating gene (RAG) deficiency, a prototype of (S)CIDs, is limited with no published systematic evaluation of diagnostic and therapeutic modalities. In this report, we sought to establish the clinical, laboratory features, and treatment outcome of patients with vasculitis due to partial RAG deficiency. Vasculitis was a major complication in eight (13%) of 62 patients in our cohort with partial RAG deficiency with features of infections and immune dysregulation. Vasculitis occurred early in life, often as first sign of disease (50%) and was complicated by significant end organ damage. Viral infections often preceded the onset of predominately non-granulomatous-small vessel vasculitis. Autoantibodies against cytokines (IFN-α, -ω, and IL-12) were detected in a large fraction of the cases tested (80%), whereas the majority of patients were anti-neutrophil cytoplasmic antibodies (ANCA) negative (>80%). Genetic diagnosis of RAG deficiency was delayed up to 2 years from the onset of vasculitis. Clinical cases with sole skin manifestation responded well to first-line steroid treatment, whereas systemic vasculitis with severe end-organ complications required second-line immunosuppression and/or hematopoietic stem cell transplantation (HSCT) for definitive management. In conclusion, our data suggest that vasculitis in partial RAG deficiency is prevalent among patients with partial RAG deficiency and is associated with high morbidity. Therefore, partial RAG deficiency should be included in the differential diagnosis of patients with early-onset systemic vasculitis. Diagnostic serology may be misleading with ANCA negative findings, and search for conventional autoantibodies should be extended to include those targeting cytokines.

Vasculitis can be a life-threatening complication associated with high mortality and morbidity among patients with primary immunodeficiencies (PIDs), including variants of severe and combined immunodeficiencies ((S)CID). Our understanding of vasculitis in partial defects in recombination activating gene (RAG) deficiency, a prototype of (S)CIDs, is limited with no published systematic evaluation of diagnostic and therapeutic modalities. In this report, we sought to establish the clinical, laboratory features, and treatment outcome of patients with vasculitis due to partial RAG deficiency. Vasculitis was a major complication in eight (13%) of 62 patients in our cohort with partial RAG deficiency with features of infections and immune dysregulation. Vasculitis occurred early in life, often as first sign of disease (50%) and was complicated by significant end organ damage. Viral infections often preceded the onset of predominately non-granulomatous-small vessel vasculitis. Autoantibodies against cytokines (IFN-α, -ω, and IL-12) were detected in a large fraction of the cases tested (80%), whereas the majority of patients were anti-neutrophil cytoplasmic antibodies (ANCA) negative (>80%). Genetic diagnosis of RAG deficiency was delayed up to 2 years from the onset of vasculitis. Clinical cases with sole skin manifestation responded well to first-line steroid treatment, whereas systemic vasculitis with severe end-organ complications required second-line immunosuppression and/or hematopoietic stem cell transplantation (HSCT) for definitive management. In conclusion, our data suggest that vasculitis in partial RAG deficiency is prevalent among patients with partial RAG deficiency and is associated with high morbidity. Therefore, partial RAG deficiency should be included in the differential diagnosis of patients with early-onset systemic vasculitis. Diagnostic serology may be misleading with ANCA negative findings, and search for conventional autoantibodies should be extended to include those targeting cytokines. [ABSTRACT FROM AUTHOR]

The recombination activating genes, including RAG1 and RAG2, are essential for V(D)J somatic recombination in lymphocytes. Leaky severe combined immunodeficiency disorder (SCID) is characterized by normal or intermediate T cells and normal to absent B cells associated with partial T cell and B cell dysfunction. We present a newly found RAG1 deficiency in a 21-year-old boy with leaky SCID. Immunoglobulin levels, flow cytometry, and whole exome sequencing (WES) were evaluated. Flow cytometric analysis revealed a decreased number of CD3+, CD4+, and CD8+ T cells, and B cells whereas NK cell counts were normal. Immunoglobulin levels were also decreased. The WES revealed a newly found homozygous mutation of RAG1 gene (NM_000448: exon 2: c.C2275T). Atypical features, including leukopenia, candidiasis, and low lymphocyte counts in patients with late-onset combined immunodeficiency disorders (CID) such as leaky SCID due to RAG1 deficiency may result in misdiagnosis and inadequate therapy instead of adopting the curative hematopoietic stem cell transplantation in these patients. [ABSTRACT FROM AUTHOR]

Autoimmunity is becoming an increasingly recognized complication in patients with primary immunodeficiencies (PIDs), including a variety of combined immune deficiencies such as Recombination Activating Gene (RAG) defects. The approach to treating autoimmunity in PID patients is complex, requiring a balance between immunosuppression and susceptibility to infection. Inflammatory arthritis is a feature of immune dysregulation in many PIDs, and the optimal treatment may differ from first line therapies that usually consist of disease-modifying anti rheumatic drugs (DMARDs). An example of mechanism-based therapy of arthritis in PID uses blockade of IL-6 signaling with tocilizumab for patients with STAT 3 gain-of-function (GOF) mutation and augmented IL-6 pathway. Herein, we describe two PID cases with arthritis who were found to have defects in RAG. One patient with refractory inflammatory arthritis experienced remarkable improvement in symptoms with tocilizumab therapy. Arthritis can be a clinical feature of immune dysregulation in RAG deficiency, and tocilizumab therapy has been suggested to have utility in treatment of arthritis in RAG deficiency.

Proteins expressed by recombination activating genes 1 and 2 (RAG1/2) are essential in the process of V(D)J recombination that leads to generation of the T and B cell repertoires. Clinical and immunological phenotypes of patients with RAG deficiencies correlate well to the degree of impaired RAG activity and this has been expanding to variants of combined immunodeficiency (CID) or even milder antibody deficiency syndromes. Pathogenic variants that severely impair recombinase activity of RAG1/2 determine a severe combined immunodeficiency (SCID) phenotype, whereas hypomorphic variants result in leaky (partial) SCID and other immunodeficiencies. We report a patient with novel pathogenic compound heterozygous RAG2 variants that result in a CID phenotype with two distinctive characteristics: late-onset progressive hypogammaglobulinemia and highly elevated B cell count. In addition, the patient had early onset of infections, T cell lymphopenia and expansion of lymphocytes after exposure to herpes family viruses. This case highlights the importance of considering pathogenic RAG variants among patients with preserved B cell count and CID phenotype.

The complex interplay between microbiota and immunity is important to human health. To explore how altered adaptive immunity influences the microbiome, we characterize skin, nares, and gut microbiota of patients with recombination-activating gene (RAG) deficiency-a rare genetically defined inborn error of immunity (IEI) that results in a broad spectrum of clinical phenotypes. Integrating de novo assembly of metagenomes from RAG-deficient patients with reference genome catalogs provides an expansive multi-kingdom view of microbial diversity. RAG-deficient patient microbiomes exhibit inter-individual variation, including expansion of opportunistic pathogens (e.g., Corynebacterium bovis, Haemophilus influenzae), and a relative loss of body site specificity. We identify 35 and 27 bacterial species derived from skin/nares and gut microbiomes, respectively, which are distinct to RAG-deficient patients compared to healthy individuals. Underscoring IEI patients as potential reservoirs for viral persistence and evolution, we further characterize the colonization of eukaryotic RNA viruses (e.g., Coronavirus 229E, Norovirus GII) in this patient population., Competing Interests: Declaration of interests The authors declare that they have no competing interests., (Published by Elsevier Inc.)

Mutations in the recombination activating gene 1 ( RAG1 ) and RAG2 in humans are associated with a broad spectrum of clinical phenotypes, from severe combined immunodeficiency to immune dysregulation. Partial (hypomorphic) RAG deficiency (pRD) in particular, frequently leads to hyperinflammation and autoimmunity, with several underlying intrinsic and extrinsic mechanisms causing a break in tolerance centrally and peripherally during T and B cell development. However, the relative contributions of these processes to immune dysregulation remain unclear. In this review, we specifically focus on the recently described tolerance break and B cell abnormalities, as well as consequent molecular and cellular mechanisms of autoantibody production in patients with pRD., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Min, Csomos, Li, Dong, Hu, Meng, Yu, Walter and Wang.)

Thymocytes and thymic epithelial cells (TECs) cross-talk is essential to support T-cell development and preserve thymic architecture and maturation of TECs and Foxp3+ natural regulatory T (nTreg) cells. Accordingly, disruption of thymic lymphostromal cross-talk may have major implications on the thymic mechanisms that govern T cell tolerance. Several genetic defects have been described in humans that affect early stages of T cell development (leading to Severe Combined Immune Deficiency, SCID) or late stages in thymocyte maturation (resulting in combined immunodeficiency). Hypomorphic mutations in SCID-causing genes may allow for generation of a limited pool of T lymphocytes with a restricted repertoire. These conditions are often associated with infiltration of peripheral tissues by activated T cells and immune dysregulation, as best exemplified by Omenn syndrome (OS). In this review, we will discuss our recent findings on abnormalities of thymic microenvironment in OS with a special focus of defective maturation of TECs, altered distribution of thymic dendritic cells (DCs) and impairment of deletional and non-deletional mechanisms of central tolerance. Here, taking advantage of mouse models of OS and atypical SCID, we will discuss how modifications in stromal compartment impact and shape lymphocyte differentiation, and vice versa how inefficient T cell signalling results in defective stromal maturation. These findings are instrumental to understand the extent to which novel therapeutic strategies should act on thymic stroma to achieve full immune reconstitution.

Purpose: Combined immunodeficiency (CID) presents a unique challenge to clinicians. Two patients presented with the prior clinical diagnosis of common variable immunodeficiency (CVID) disorder marked by an early age of presentation, opportunistic infections, and persistent lymphopenia. Due to the presence of atypical clinical features, next generation sequencing was applied documenting RAG deficiency in both patients. Methods: Two different genetic analysis techniques were applied in these patients including whole exome sequencing in one patient and the use of a gene panel designed to target genes known to cause primary immunodeficiency disorders (PIDD) in a second patient. Sanger dideoxy sequencing was used to confirm RAG1 mutations in both patients. Results: Two young adults with a history of recurrent bacterial sinopulmonary infections, viral infections, and autoimmune disease as well as progressive hypogammaglobulinemia, abnormal antibody responses, lymphopenia and a prior diagnosis of CVID disorder were evaluated. Compound heterozygous mutations in RAG1 (1) c256_257delAA, p86VfsX32 and (2) c1835A>G, pH612R were documented in one patient. Compound heterozygous mutations in RAG1 (1) c.1566G>T, p.W522C and (2) c.2689C>T, p. R897X) were documented in a second patient post-mortem following a fatal opportunistic infection. Conclusion: Astute clinical judgment in the evaluation of patients with PIDD is necessary. Atypical clinical findings such as early onset, granulomatous disease, or opportunistic infections should support the consideration of atypical forms of late onset CID secondary to RAG deficiency. Next generation sequencing approaches provide powerful tools in the investigation of these patients and may expedite definitive treatments. [ABSTRACT FROM AUTHOR]

The emergence, survival, growth and maintenance of autoreactive (AR) B-cell clones, the hallmark of humoral autoimmunity, leave their footprints in B-cell receptor repertoires. Collecting IgH sequences related to polyreactive (PR) ones from adaptive immune receptor repertoire (AIRR) datasets make the reconstruction and analysis of PR/AR B-cell lineages possible. We developed a computational approach, named ImmChainTracer, to extract members and to visualize clonal relationships of such B-cell lineages. Our approach was successfully applied on the IgH repertoires of patients suffering from monogenic hypomorphic RAG1 and 2 deficiency (pRD) or polygenic systemic lupus erythematosus (SLE) autoimmune diseases to identify relatives of AR IgH sequences and to track their fate in AIRRs. Signs of clonal expansion, affinity maturation and class-switching events in PR/AR and non-PR/AR B-cell lineages were revealed. An extension of our method towards B-cell expansion caused by any trigger (e.g., infection, vaccination or antibody development) may provide deeper insight into antigen specific B-lymphogenesis.

Thymocytes and thymic epithelial cells (TECs) cross-talk is essential to support T cell development and preserve thymic architecture and maturation of TECs and Foxp3+ natural regulatory T cells. Accordingly, disruption of thymic lymphostromal cross-talk may have major implications on the thymic mechanisms that govern T cell tolerance. Several genetic defects have been described in humans that affect early stages of T cell development [leading to severe combined immune deficiency (SCID)] or late stages in thymocyte maturation (resulting in combined immunodeficiency). Hypomorphic mutations in SCID-causing genes may allow for generation of a limited pool of T lymphocytes with a restricted repertoire. These conditions are often associated with infiltration of peripheral tissues by activated T cells and immune dysregulation, as best exemplified by Omenn syndrome (OS). In this review, we will discuss our recent findings on abnormalities of thymic microenvironment in OS with a special focus of defective maturation of TECs, altered distribution of thymic dendritic cells and impairment of deletional and non-deletional mechanisms of central tolerance. Here, taking advantage of mouse models of OS and atypical SCID, we will discuss how modifications in stromal compartment impact and shape lymphocyte differentiation, and vice versa how inefficient T cell signaling results in defective stromal maturation. These findings are instrumental to understand the extent to which novel therapeutic strategies should act on thymic stroma to achieve full immune reconstitution. [ABSTRACT FROM AUTHOR]

Severe combined immunodeficiency (SCID), including the 'variant' Omenn syndrome (OS), represent a heterogeneous group of monogenic disorders characterized by defect in differentiation of T- and/or B lymphocytes and susceptibility to infections since birth. In the period of 25 years, between January 1986 and December 2010, a total of 21 patients (15 SCID, 6 OS) were diagnosed in Mother & Child Health Institute of Serbia, a tertiary-care teaching University hospital and a national referral center for patients affected with primary immunodeficiency (PID). The diagnoses were based on anamnestic data, clinical findings, and immunological and genetic analysis. The median age at the onset of the first infection was the 2nd month of life. Seven (33 %) patients had positive family history for SCID. Out of five male infants with T-B+NK- SCID phenotype, mutation analysis revealed interleukin-2 (common) gamma-chain receptor (IL2RG) mutations in 3 with positive X-linked family history, and Janus-kinase ( JAK)-3 gene defects in the other two. Six patients had T-B-NK+ SCID phenotype and further 6 features of OS, 11 of which had recombinase-activating gene ( RAG1or RAG2) and 1 Artemis gene mutations. One child with T+B+NK+ SCID phenotype as well had proven RAG mutation. One child each with T-B+NK+ SCID phenotype, CD8 lymphopenia and unknown phenotype remained without known underlying genetic defect. Of the eight patients who underwent hematopoetic stem cell transplant (HSCT) 5 survived, the other 13 died between 2 days and 12 months after diagnosis was made. Early diagnosis of SCID, before onset of severe infections, offers possibility for HSCT and cure. Education of primary-care pediatricians, in particular including awareness of the risk of using live vaccines and non-irradiated blood products, should improve prognosis of SCID in our setting. [ABSTRACT FROM AUTHOR]

Abstract: Mutations in the recombination activating genes (RAG1 or RAG2) can lead to a variety of immunodeficiencies. Herein, we report 5 cases of RAG deficiency from 5 families: 3 of Omenn syndrome, 1 of severe combined immunodeficiency, and 1 of combined immunodeficiency with oligoclonal TCRγδ+ T cells, autoimmunity and cytomegalovirus infection. The genetic defects were heterogeneous and included 6 novel RAG mutations. All missense mutations except for Met443Ile in RAG2 were located in active core regions of RAG1 or RAG2. V(D)J recombination activity of each mutant was variable, ranging from half of the wild type activity to none, however, a significant decrease in average recombination activity was demonstrated in each patient. The reduced recombination activity of Met443Ile in RAG2 may suggest a crucial role of the non-core region of RAG2 in V(D)J recombination. These findings suggest that functional evaluation together with molecular analysis contributes to our broader understanding of RAG deficiency. [Copyright &y& Elsevier]

Mutations in the recombinase-activating genes cause severe immunodeficiency, with a spectrum of phenotypes ranging from severe combined immunodeficiency to immune dysregulation. Hematopoietic stem cell transplantation is the only curative option, but a high risk of graft failure and poor immune reconstitution have been observed in the absence of myeloablation. Our aim was to improve multilineage engraftment; we tested nongenotoxic conditioning with anti-CD45 mAbs conjugated with saporin CD45 (CD45-SAP). Rag1-KO and Rag1-F971L mice, which represent models of severe combined immune deficiency and combined immune deficiency with immune dysregulation, respectively, were conditioned with CD45-SAP, CD45-SAP plus 2 Gy of total body irradiation (TBI), 2 Gy of TBI, 8 Gy of TBI, or no conditioning and treated by using transplantation with lineage-negative bone marrow cells from wild-type mice. Flow cytometry and immunohistochemistry were used to assess engraftment and immune reconstitution. Antibody responses to 2,4,6-trinitrophenyl–conjugated keyhole limpet hemocyanin were measured by ELISA, and presence of autoantibody was detected by microarray. Conditioning with CD45-SAP enabled high levels of multilineage engraftment in both Rag1 mutant models, allowed overcoming of B- and T-cell differentiation blocks and thymic epithelial cell defects, and induced robust cellular and humoral immunity in the periphery. Conditioning with CD45-SAP allows multilineage engraftment and robust immune reconstitution in mice with either null or hypomorphic Rag mutations while preserving thymic epithelial cell homeostasis. [ABSTRACT FROM AUTHOR]

A correction to the article "Rag Defects and Thymic Stroma: Lessons From Animal Models" that was published in a 2014 issue is presented.

Background: V(D)J recombination takes place during lymphocyte development to generate a large repertoire of T- and B-cell receptors. Mutations in recombination-activating gene 1 (RAG1) and RAG2 result in loss or reduction of V(D)J recombination. It is known that different mutations in RAG genes vary in residual recombinase activity and give rise to a broad spectrum of clinical phenotypes. Objective: We sought to study the immunologic mechanisms causing the clinical spectrum of RAG deficiency. Methods: We included 22 patients with similar RAG1 mutations (c.519delT or c.368_369delAA) resulting in N-terminal truncated RAG1 protein with residual recombination activity but presenting with different clinical phenotypes. We studied precursor B-cell development, immunoglobulin and T-cell receptor repertoire formation, receptor editing, and B- and T-cell numbers. Results: Clinically, patients were divided into 3 main categories: T−B− severe combined immunodeficiency, Omenn syndrome, and combined immunodeficiency. All patients showed a block in the precursor B-cell development, low B- and T-cell numbers, normal immunoglobulin gene use, limited B- and T-cell repertoires, and slightly impaired receptor editing. Conclusion: This study demonstrates that similar RAG mutations can result in similar immunobiological effects but different clinical phenotypes, indicating that the level of residual recombinase activity is not the only determinant for clinical outcome. We postulate a model in which the type and moment of antigenic pressure affect the clinical phenotypes of these patients. [Copyright &y& Elsevier]