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The Wiskott-Aldrich syndrome related protein WAVE2 is implicated in the regulation of actin-cytoskeletal reorganization downstream of the small Rho GTPase, Rac. We inactivated the WAVE2 gene by gene-targeted mutation to examine its role in murine development and in actin assembly. WAVE2-deficient embryos survived until approximately embryonic day 12.5 and displayed growth retardation and certain morphological defects, including malformations of the ventricles in the developing brain. WAVE2-deficient embryonic stem cells displayed normal proliferation, whereas WAVE2-deficient embryonic fibroblasts exhibited severe growth defects, as well as defective cell motility in response to PDGF, lamellipodium formation and Rac-mediated actin polymerization. These results imply a non-redundant role for WAVE2 in murine embryogenesis and a critical role for WAVE2 in actin-based processes downstream of Rac that are essential for cell movement. [ABSTRACT FROM AUTHOR]

Importance: Case reports that externalize expert diagnostic reasoning are utilized for clinical reasoning instruction but are difficult to search based on symptoms, final diagnosis, or differential diagnosis construction. Computational approaches that uncover how experienced diagnosticians analyze the medical information in a case as they formulate a differential diagnosis can guide educational uses of case reports., Objective: To develop a "reasoning-encoded" case database for advanced clinical reasoning instruction by applying natural language processing (NLP), a sub-field of artificial intelligence, to a large case report library., Design: We collected 2525 cases from the New England Journal of Medicine (NEJM) Clinical Pathological Conference (CPC) from 1965 to 2020 and used NLP to analyze the medical terminology in each case to derive unbiased (not prespecified) categories of analysis used by the clinical discussant. We then analyzed and mapped the degree of category overlap between cases., Results: Our NLP algorithms identified clinically relevant categories that reflected the relationships between medical terms (which included symptoms, signs, test results, pathophysiology, and diagnoses). NLP extracted 43,291 symptoms across 2525 cases and physician-annotated 6532 diagnoses (both primary and related diagnoses). Our unsupervised learning computational approach identified 12 categories of medical terms that characterized the differential diagnosis discussions within individual cases. We used these categories to derive a measure of differential diagnosis similarity between cases and developed a website ( universeofcpc.com ) to allow visualization and exploration of 55 years of NEJM CPC case series., Conclusions: Applying NLP to curated instances of diagnostic reasoning can provide insight into how expert clinicians correlate and coordinate disease categories and processes when creating a differential diagnosis. Our reasoning-encoded CPC case database can be used by clinician-educators to design a case-based curriculum and by physicians to direct their lifelong learning efforts., (© 2022. The Author(s), under exclusive licence to Society of General Internal Medicine.)

Background: Recognition of viral nucleic acids is one of the primary triggers for a type I interferon-mediated antiviral immune response. Inborn errors of type I interferon immunity can be associated with increased inflammation and/or increased susceptibility to viral infections as a result of dysbalanced interferon production. NFX1-type zinc finger-containing 1 (ZNFX1) is an interferon-stimulated double-stranded RNA sensor that restricts the replication of RNA viruses in mice. The role of ZNFX1 in the human immune response is not known., Objective: We studied 15 patients from 8 families with an autosomal recessive immunodeficiency characterized by severe infections by both RNA and DNA viruses and virally triggered inflammatory episodes with hemophagocytic lymphohistiocytosis-like disease, early-onset seizures, and renal and lung disease., Methods: Whole exome sequencing was performed on 13 patients from 8 families. We investigated the transcriptome, posttranscriptional regulation of interferon-stimulated genes (ISGs) and predisposition to viral infections in primary cells from patients and controls stimulated with synthetic double-stranded nucleic acids., Results: Deleterious homozygous and compound heterozygous ZNFX1 variants were identified in all 13 patients. Stimulation of patient-derived primary cells with synthetic double-stranded nucleic acids was associated with a deregulated pattern of expression of ISGs and alterations in the half-life of the mRNA of ISGs and also associated with poorer clearance of viral infections by monocytes., Conclusion: ZNFX1 is an important regulator of the response to double-stranded nucleic acids stimuli following viral infections. ZNFX1 deficiency predisposes to severe viral infections and a multisystem inflammatory disease., (Copyright © 2021. Published by Elsevier Inc.)

TNFRSF13B encodes the transmembrane activator and CAML interactor (TACI) receptor, which drives plasma cell differentiation. Although TNFRSF13B supports host defense, dominant-negative TNFRSF13B alleles are common in humans and other species and only rarely associate with disease. We reasoned that the high frequency of disruptive TNFRSF13B alleles reflects balancing selection, the loss of function conferring advantage in some settings. Testing that concept, we investigated how a common human dominant-negative variant, TNFRSF13B A181E, imparts resistance to enteric pathogens. Mice engineered to express mono- or biallelic A144E variants of tnrsf13B, corresponding to A181E, exhibited a striking resistance to pathogenicity and transmission of Citrobacter rodentium, a murine pathogen that models enterohemorrhagic Escherichia coli, and resistance was principally owed to natural IgA deficiency in the intestine. In WT mice with gut IgA and in mutant mice reconstituted with enteric IgA obtained from WT mice, IgA induces LEE expression of encoded virulence genes, which confer pathogenicity and transmission. Taken together, our results show that C. rodentium and most likely other enteric organisms appropriated binding of otherwise protective antibodies to signal induction of the virulence program. Additionally, the high prevalence of TNFRSF13B dominant-negative variants reflects balancing selection.

Human nude SCID is a rare autosomal recessive inborn error of immunity (IEI) characterized by congenital athymia, alopecia, and nail dystrophy. Few cases have been reported to date. However, the recent introduction of newborn screening for IEIs and high-throughput sequencing has led to the identification of novel and atypical cases. Moreover, immunological alterations have been recently described in patients carrying heterozygous mutations. The aim of this paper is to describe the extended phenotype associated with FOXN1 homozygous, compound heterozygous, or heterozygous mutations. We collected clinical and laboratory information of a cohort of 11 homozygous, 2 compound heterozygous, and 5 heterozygous patients with recurrent severe infections. All, except one heterozygous patient, had signs of CID or SCID. Nail dystrophy and alopecia, that represent the hallmarks of the syndrome, were not always present, while almost 50% of the patients developed Omenn syndrome. One patient with hypomorphic compound heterozygous mutations had a late-onset atypical phenotype. A SCID-like phenotype was observed in 4 heterozygous patients coming from the same family. A spectrum of clinical manifestations may be associated with different mutations. The severity of the clinical phenotype likely depends on the amount of residual activity of the gene product, as previously observed for other SCID-related genes. The severity of the manifestations in this heterozygous family may suggest a mechanism of negative dominance of the specific mutation or the presence of additional mutations in noncoding regions.

Background: Myxoma virus (MYXV) is an oncolytic poxvirus that lacks the gene for 1 of the subunits of ribonucleotide reductase (RR), a crucial DNA synthesis and repair enzyme. The overexpression of RR has been implicated in the invasiveness of several cancers, including soft tissue sarcomas (STS). The purpose of the study was to investigate the oncolytic efficacy of MYXV in STS with different levels of RR expression., Methods: The oncolytic effect of recombinant MYXV was evaluated in 4 human STS cell lines, LS141 (a dedifferentiated liposarcoma), DDLS8817 (a dedifferentiated liposarcoma), RDD2213 (recurrent dedifferentiated liposarcoma), and HSSYII (a synovial sarcoma) using infectivity and cytotoxicity assays. Following the overexpression of RRM2 by cDNA transfection and silencing of RRM2 by siRRM2 in these STS cell lines, the RRM2 expression levels were analyzed by Western blot., Results: We observed a direct correlation between viral oncolysis and RRM2 mRNA levels ( R  = 0.96) in STS. Higher RRM2 expression was associated with a more robust cell kill. Silencing the RRM2 gene led to significantly greater cell survival (80%) compared with the control group ( P  = .003), whereas overexpression of the RRM2 increased viral oncolysis by 33% ( P  < .001)., Conclusions: Our results show that the oncolytic effects of MYXV correlate directly with RR expression levels and are enhanced in STS cell lines with naturally occurring or artificially induced high expression levels of RR. Myxoma virus holds promise in the treatment of advanced soft tissue cancer, especially in tumors overexpressing RR., Competing Interests: Declaration of conflicting interests:The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (© The Author(s) 2021.)

LRRC8 family proteins on the plasma membrane play a critical role in cellular osmoregulation by forming volume-regulated anion channels (VRACs) necessary to prevent necrotic cell death. We demonstrate that intracellular LRRC8 proteins acting within lysosomes also play an essential role in cellular osmoregulation. LRRC8 proteins on lysosome membranes generate large lysosomal volume-regulated anion channel (Lyso-VRAC) currents in response to low cytoplasmic ionic strength conditions. When a double-leucine L 706 L 707 motif at the C terminus of LRRC8A was mutated to alanines, normal plasma membrane VRAC currents were still observed, but Lyso-VRAC currents were absent. We used this targeting mutant, as well as pharmacological tools, to demonstrate that Lyso-VRAC currents are necessary for the formation of large lysosome-derived vacuoles, which store and then expel excess water to maintain cytosolic water homeostasis. Thus, Lyso-VRACs allow lysosomes of mammalian cells to act as the cell`s "bladder." When Lyso-VRAC current was selectively eliminated, the extent of necrotic cell death to sustained stress was greatly increased, not only in response to hypoosmotic stress, but also to hypoxic and hypothermic stresses. Thus Lyso-VRACs play an essential role in enabling cells to mount successful homeostatic responses to multiple stressors., Competing Interests: The authors declare no competing interest.

Background: An increasing number of NFKB1 variants are being identified in patients with heterogeneous immunologic phenotypes., Objective: To characterize the clinical and cellular phenotype as well as the management of patients with heterozygous NFKB1 mutations., Methods: In a worldwide collaborative effort, we evaluated 231 individuals harboring 105 distinct heterozygous NFKB1 variants. To provide evidence for pathogenicity, each variant was assessed in silico; in addition, 32 variants were assessed by functional in vitro testing of nuclear factor of kappa light polypeptide gene enhancer in B cells (NF-κB) signaling., Results: We classified 56 of the 105 distinct NFKB1 variants in 157 individuals from 68 unrelated families as pathogenic. Incomplete clinical penetrance (70%) and age-dependent severity of NFKB1-related phenotypes were observed. The phenotype included hypogammaglobulinemia (88.9%), reduced switched memory B cells (60.3%), and respiratory (83%) and gastrointestinal (28.6%) infections, thus characterizing the disorder as primary immunodeficiency. However, the high frequency of autoimmunity (57.4%), lymphoproliferation (52.4%), noninfectious enteropathy (23.1%), opportunistic infections (15.7%), autoinflammation (29.6%), and malignancy (16.8%) identified NF-κB1-related disease as an inborn error of immunity with immune dysregulation, rather than a mere primary immunodeficiency. Current treatment includes immunoglobulin replacement and immunosuppressive agents., Conclusions: We present a comprehensive clinical overview of the NF-κB1-related phenotype, which includes immunodeficiency, autoimmunity, autoinflammation, and cancer. Because of its multisystem involvement, clinicians from each and every medical discipline need to be made aware of this autosomal-dominant disease. Hematopoietic stem cell transplantation and NF-κB1 pathway-targeted therapeutic strategies should be considered in the future., (Copyright © 2020. Published by Elsevier Inc.)

When migratory T cells encounter antigen-presenting cells (APCs), they arrest and form radially symmetric, stable intercellular junctions termed immunological synapses which facilitate exchange of crucial biochemical information and are critical for T-cell immunity. While the cellular processes underlying synapse formation have been well characterized, those that maintain the symmetry, and thereby the stability of the synapse, remain unknown. Here we identify an antigen-triggered mechanism that actively promotes T-cell synapse symmetry by generating cytoskeletal tension in the plane of the synapse through focal nucleation of actin via Wiskott-Aldrich syndrome protein (WASP), and contraction of the resultant actin filaments by myosin II. Following T-cell activation, WASP is degraded, leading to cytoskeletal unraveling and tension decay, which result in synapse breaking. Thus, our study identifies and characterizes a mechanical program within otherwise highly motile T cells that sustains the symmetry and stability of the T cell-APC synaptic contact., (© 2020 The Authors. Published under the terms of the CC BY 4.0 license.)

Mutations affecting recombination activation genes RAG1 and RAG2 are associated with variable phenotypes, depending on the residual recombinase activity. The aim of this study is to describe a variety of clinical phenotypes in RAG-deficient patients from the highly consanguineous Egyptian population. Thirty-one patients with RAG mutations (from 28 families) were included from 2013 to 2017. On the basis of clinical, immunological and genetic data, patients were subdivided into three groups; classical T - B - severe combined immunodeficiency (SCID), Omenn syndrome (OS) and atypical SCID. Nineteen patients presented with typical T - B - SCID; among these, five patients carried a homozygous RAG2 mutation G35V and five others carried two homozygous RAG2 mutations (T215I and R229Q) that were detected together. Four novel mutations were reported in the T - B - SCID group; three in RAG1 (A565P, N591Pfs*14 and K621E) and one in RAG2 (F29S). Seven patients presented with OS and a novel RAG2 mutation (C419W) was documented in one patient. The atypical SCID group comprised five patients. Two had normal B cell counts; one had a previously undescribed RAG2 mutation (V327D). The other three patients presented with autoimmune cytopaenias and features of combined immunodeficiency and were diagnosed at a relatively late age and with a substantial diagnostic delay; one patient had a novel RAG1 mutation (C335R). PID disorders are frequent among Egyptian children because of the high consanguinity. RAG mutations stand behind several variable phenotypes, including classical SCID, OS, atypical SCID with autoimmunity and T - B + CID., (© 2018 British Society for Immunology.)

Background: Cytotoxic T-lymphocyte antigen 4 (CTLA-4) is a negative immune regulator. Heterozygous CTLA4 germline mutations can cause a complex immune dysregulation syndrome in human subjects., Objective: We sought to characterize the penetrance, clinical features, and best treatment options in 133 CTLA4 mutation carriers., Methods: Genetics, clinical features, laboratory values, and outcomes of treatment options were assessed in a worldwide cohort of CTLA4 mutation carriers., Results: We identified 133 subjects from 54 unrelated families carrying 45 different heterozygous CTLA4 mutations, including 28 previously undescribed mutations. Ninety mutation carriers were considered affected, suggesting a clinical penetrance of at least 67%; median age of onset was 11 years, and the mortality rate within affected mutation carriers was 16% (n = 15). Main clinical manifestations included hypogammaglobulinemia (84%), lymphoproliferation (73%), autoimmune cytopenia (62%), and respiratory (68%), gastrointestinal (59%), or neurological features (29%). Eight affected mutation carriers had lymphoma, and 3 had gastric cancer. An EBV association was found in 6 patients with malignancies. CTLA4 mutations were associated with lymphopenia and decreased T-, B-, and natural killer (NK) cell counts. Successful targeted therapies included application of CTLA-4 fusion proteins, mechanistic target of rapamycin inhibitors, and hematopoietic stem cell transplantation. EBV reactivation occurred in 2 affected mutation carriers after immunosuppression., Conclusions: Affected mutation carriers with CTLA-4 insufficiency can present in any medical specialty. Family members should be counseled because disease manifestation can occur as late as 50 years of age. EBV- and cytomegalovirus-associated complications must be closely monitored. Treatment interventions should be coordinated in clinical trials., (Copyright © 2018 American Academy of Allergy, Asthma & Immunology. All rights reserved.)

Background: Diagnostic error is a major problem in health care, yet there are few medical school curricula focused on improving the diagnostic process and decreasing diagnostic errors. Effective strategies to teach medical students about diagnostic error and diagnostic safety have not been established., Methods: We designed, implemented and evaluated a virtual patient module featuring two linked cases involving diagnostic errors. Learning objectives developed by a consensus process among medical educators in the Society to Improve Diagnosis in Medicine (SIDM) were utilized. The module was piloted with internal medicine clerkship students at three institutions and with clerkship faculty members recruited from listservs. Participants completed surveys on their experience using the case and a qualitative analysis was performed., Results: Thirty-five medical students and 25 faculty members completed the survey. Most students found the module to be relevant and instructive. Faculty also found the module valuable for students but identified insufficient curricular time as a barrier to implementation., Conclusions: Medical students and faculty found a prototype virtual patient module about the diagnostic process and diagnostic error to be educational.

[This corrects the article on p. 798 in vol. 8, PMID: 28769923.].

Mutations in Sp110 are the underlying cause of veno-occlusive disease with immunodeficiency (VODI), a combined immunodeficiency that is difficult to treat and often fatal. Because early treatment is critically important for patients with VODI, broadly usable diagnostic tools are needed to detect Sp110 protein deficiency. Several factors make establishing the diagnosis of VODI challenging: (1) Current screening strategies to identify severe combined immunodeficiency are based on measuring T cell receptor excision circles (TREC). This approach will fail to identify VODI patients because the disease is not associated with severe T cell lymphopenia at birth; (2) the SP110 gene contains 17 exons, making it a challenge for Sanger sequencing. The recently developed next-generation sequencing (NGS) platforms that can rapidly determine the sequence of all 17 exons are available in only a few laboratories; (3) there is no standard functional assay to test for the effects of novel mutations in Sp110; and (4) it has been difficult to use flow cytometry to identify patients who lack Sp110 because of the low level of Sp110 protein in peripheral blood lymphocytes. We report here a novel flow cytometric assay that is easily performed in diagnostic laboratories and might thus become a standard assay for the evaluation of patients who may have VODI. In addition, the assay will facilitate investigations directed at understanding the function of Sp110.

Background: Inducible co-stimulator (ICOS) deficiency was the first monogenic defect reported to cause common variable immunodeficiency (CVID)-like disease in 2003. Since then, 16 patients have been reported worldwide with an increasing range of clinical phenotypes., Objective: We sought to compare the clinical and immunological phenotype and provide clinical follow-up and therapeutic approaches for treating ICOS-deficient patients., Methods: We describe the clinical and laboratory data of 15 patients with available clinical data. Previous publications and clinical assessment were used as data sources., Results: The observed ICOS gene mutations were all deletions leading to undetectable protein expression. The clinical phenotype of ICOS deficiency is much broader than initially anticipated and includes not only CVID-like disease but an increased susceptibility to viral and opportunistic infections, as well as cancer. Impaired B-cell development led to decreased memory B-cells in all patients, and hypogammaglobulinemia in all but one patient. Circulating CXCR5 + CD4 + follicular T-helper-cell numbers were also reduced in all patients. Treatment included immunoglobulin replacement, regular antibiotic prophylaxis, corticosteroids, and steroid-sparing agents. Three patients underwent hematopoietic stem cell transplantation; one of them died due to capillary leak syndrome on day 5 posttransplantation., Conclusion: The disease spectrum of ICOS deficiency is expanding from solely B-cell to combined B- and T-cell immunodeficiency, suggesting genetic and environmental modifiers. Genetic diagnosis is the only tool to distinguish ICOS deficiency from other immunological defects. Patients with antibody deficiency, autoimmunity, and combined immunodeficiency should be screened for ICOS mutations.

Mutations of the recombinase-activating genes 1 and 2 ( RAG1 and RAG2 ) in humans are associated with a broad range of phenotypes. For patients with severe clinical presentation, hematopoietic stem cell transplantation (HSCT) represents the only curative treatment; however, high rates of graft failure and incomplete immune reconstitution have been observed, especially after unconditioned haploidentical transplantation. Studies in mice have shown that Rag -/- natural killer (NK) cells have a mature phenotype, reduced fitness, and increased cytotoxicity. We aimed to analyze NK cell phenotype and function in patients with mutations in RAG and in non-homologous end joining (NHEJ) genes. Here, we provide evidence that NK cells from these patients have an immature phenotype, with significant expansion of CD56 bright CD16 -/int CD57 - cells, yet increased degranulation and high perforin content. Correlation was observed between in vitro recombinase activity of the mutant proteins, NK cell abnormalities, and in vivo clinical phenotype. Addition of serotherapy in the conditioning regimen, with the aim of depleting the autologous NK cell compartment, may be important to facilitate engraftment and immune reconstitution in patients with RAG and NHEJ defects treated by HSCT.