Biallelic human SHARPIN loss of function induces autoinflammation and immunodeficiency.

The linear ubiquitin assembly complex (LUBAC) consists of HOIP, HOIL-1 and SHARPIN and is essential for proper immune responses. Individuals with HOIP and HOIL-1 deficiencies present with severe immunodeficiency, autoinflammation and glycogen storage disease. In mice, the loss of Sharpin leads to severe dermatitis due to excessive keratinocyte cell death. Here, we report two individuals with SHARPIN deficiency who manifest autoinflammatory symptoms but unexpectedly no dermatological problems. Fibroblasts and B cells from these individuals showed attenuated canonical NF-κB responses and a propensity for cell death mediated by TNF superfamily members. Both SHARPIN-deficient and HOIP-deficient individuals showed a substantial reduction of secondary lymphoid germinal center B cell development. Treatment of one SHARPIN-deficient individual with anti-TNF therapies led to complete clinical and transcriptomic resolution of autoinflammation. These findings underscore the critical function of the LUBAC as a gatekeeper for cell death-mediated immune dysregulation in humans.

A variant in human AIOLOS impairs adaptive immunity by interfering with IKAROS.

In the present study, we report a human-inherited, impaired, adaptive immunity disorder, which predominantly manifested as a B cell differentiation defect, caused by a heterozygous IKZF3 missense variant, resulting in a glycine-to-arginine replacement within the DNA-binding domain of the encoded AIOLOS protein. Using mice that bear the corresponding variant and recapitulate the B and T cell phenotypes, we show that the mutant AIOLOS homodimers and AIOLOS-IKAROS heterodimers did not bind the canonical AIOLOS-IKAROS DNA sequence. In addition, homodimers and heterodimers containing one mutant AIOLOS bound to genomic regions lacking both canonical motifs. However, the removal of the dimerization capacity from mutant AIOLOS restored B cell development. Hence, the adaptive immunity defect is caused by the AIOLOS variant hijacking IKAROS function. Heterodimeric interference is a new mechanism of autosomal dominance that causes inborn errors of immunity by impairing protein function via the mutation of its heterodimeric partner.

The Role of Interferon-γ in Autoimmune Polyendocrine Syndrome Type 1.

BACKGROUND: Autoimmune polyendocrine syndrome type 1 (APS-1) is a life-threatening, autosomal recessive syndrome caused by autoimmune regulator (AIRE) deficiency. In APS-1, self-reactive T cells escape thymic negative selection, infiltrate organs, and drive autoimmune injury. The effector mechanisms governing T-cell-mediated damage in APS-1 remain poorly understood. METHODS: We examined whether APS-1 could be classified as a disease mediated by interferon-γ. We first assessed patients with APS-1 who were participating in a prospective natural history study and evaluated mRNA and protein expression in blood and tissues. We then examined the pathogenic role of interferon-γ using Aire-/-Ifng-/- mice and Aire-/- mice treated with the Janus kinase (JAK) inhibitor ruxolitinib. On the basis of our findings, we used ruxolitinib to treat five patients with APS-1 and assessed clinical, immunologic, histologic, transcriptional, and autoantibody responses. RESULTS: Patients with APS-1 had enhanced interferon-γ responses in blood and in all examined autoimmunity-affected tissues. Aire-/- mice had selectively increased interferon-γ production by T cells and enhanced interferon-γ, phosphorylated signal transducer and activator of transcription 1 (pSTAT1), and CXCL9 signals in multiple organs. Ifng ablation or ruxolitinib-induced JAK-STAT blockade in Aire-/- mice normalized interferon-γ responses and averted T-cell infiltration and damage in organs. Ruxolitinib treatment of five patients with APS-1 led to decreased levels of T-cell-derived interferon-γ, normalized interferon-γ and CXCL9 levels, and remission of alopecia, oral candidiasis, nail dystrophy, gastritis, enteritis, arthritis, Sjögren's-like syndrome, urticaria, and thyroiditis. No serious adverse effects from ruxolitinib were identified in these patients. CONCLUSIONS: Our findings indicate that APS-1, which is caused by AIRE deficiency, is characterized by excessive, multiorgan interferon-γ-mediated responses. JAK inhibition with ruxolitinib in five patients showed promising results. (Funded by the National Institute of Allergy and Infectious Diseases and others.).

Dominant-activating germline mutations in the gene encoding the PI(3)K catalytic subunit p110δ result in T cell senescence and human immunodeficiency.

The p110δ subunit of phosphatidylinositol-3-OH kinase (PI(3)K) is selectively expressed in leukocytes and is critical for lymphocyte biology. Here we report fourteen patients from seven families who were heterozygous for three different germline, gain-of-function mutations in PIK3CD (which encodes p110δ). These patients presented with sinopulmonary infections, lymphadenopathy, nodular lymphoid hyperplasia and viremia due to cytomegalovirus (CMV) and/or Epstein-Barr virus (EBV). Strikingly, they had a substantial deficiency in naive T cells but an over-representation of senescent effector T cells. In vitro, T cells from patients exhibited increased phosphorylation of the kinase Akt and hyperactivation of the metabolic checkpoint kinase mTOR, enhanced glucose uptake and terminal effector differentiation. Notably, treatment with rapamycin to inhibit mTOR activity in vivo partially restored the abundance of naive T cells, largely 'rescued' the in vitro T cell defects and improved the clinical course.

Mutations that prevent caspase cleavage of RIPK1 cause autoinflammatory disease.

RIPK1 is a key regulator of innate immune signalling pathways. To ensure an optimal inflammatory response, RIPK1 is regulated post-translationally by well-characterized ubiquitylation and phosphorylation events, as well as by caspase-8-mediated cleavage1-7. The physiological relevance of this cleavage event remains unclear, although it is thought to inhibit activation of RIPK3 and necroptosis8. Here we show that the heterozygous missense mutations D324N, D324H and D324Y prevent caspase cleavage of RIPK1 in humans and result in an early-onset periodic fever syndrome and severe intermittent lymphadenopathy-a condition we term 'cleavage-resistant RIPK1-induced autoinflammatory syndrome'. To define the mechanism for this disease, we generated a cleavage-resistant Ripk1D325A mutant mouse strain. Whereas Ripk1-/- mice died postnatally from systemic inflammation, Ripk1D325A/D325A mice died during embryogenesis. Embryonic lethality was completely prevented by the combined loss of Casp8 and Ripk3, but not by loss of Ripk3 or Mlkl alone. Loss of RIPK1 kinase activity also prevented Ripk1D325A/D325A embryonic lethality, although the mice died before weaning from multi-organ inflammation in a RIPK3-dependent manner. Consistently, Ripk1D325A/D325A and Ripk1D325A/+ cells were hypersensitive to RIPK3-dependent TNF-induced apoptosis and necroptosis. Heterozygous Ripk1D325A/+ mice were viable and grossly normal, but were hyper-responsive to inflammatory stimuli in vivo. Our results demonstrate the importance of caspase-mediated RIPK1 cleavage during embryonic development and show that caspase cleavage of RIPK1 not only inhibits necroptosis but also maintains inflammatory homeostasis throughout life.

IKAROS gain of function disease: Allogeneic hematopoietic cell transplantation experience and expanded clinical phenotypes.

IKAROS, encoded by IKZF1, is a tumor suppressor and a key hematopoietic transcription factor responsible for lymphoid and myeloid differentiation. IKZF1 mutations result in inborn errors of immunity presenting with increased susceptibility to infections, immune dysregulation, and malignancies. In particular, patients carrying IKZF1 gain-of-function (GOF) mutations mostly exhibit symptoms of immune dysregulation and polyclonal plasma cell proliferation. Herein, we describe seven new IKAROS GOF cases from two unrelated families, presenting with novel infectious, immune dysregulation and hematologic diseases. Two of the patients underwent allogeneic hematopoietic cell transplantation (HCT) due to poorly responsive complications. HCT was well-tolerated achieving full engraftment in both patients receiving reduced intensity, matched unrelated donor grafts, with no severe acute or chronic graft-vs-host-disease, and in remission from their diseases 2.5 and 4 years post-HCT, respectively. These results suggest that HCT is a valid and curative option in patients with IKAROS GOF disease and severe clinical manifestations.

A novel IKZF1 variant in a family with autosomal dominant CVID: A case for expanding exon coverage in inborn errors of immunity.

Common variable immune deficiency (CVID) is a heterogenous group of disorders characterized by varying degrees of hypogammaglobulinemia, recurrent infections, and autoimmunity. Currently, pathogenic variants are identified in approximately 20-30% of CVID cases. Here we report a 3-generation family with autosomal dominant Common Variable Immunodeficiency (CVID) diagnosed in 9 affected individuals. Although primary immune deficiency panels and exome sequencing were non-diagnostic, whole genome sequencing revealed a novel, pathogenic c.499C > T: p.His167Tyr variant in IKZF1, a critical regulator of B cell development. Functional testing done through pericentromeric heterochromatin localization and light shift chemiluminescent electrophoretic mobility shift assay confirmed the variant's deleterious effect via a haploinsufficiency mechanism. Our findings expand the spectrum of known IKZF1 mutations and contribute to a more comprehensive understanding of CVID's genetic heterogeneity. Furthermore, this case underscores the importance of considering whole genome sequencing for comprehensive genetic diagnosis when concern for a monogenic inborn errors of immunity is high.

Biallelic PI4KA Mutations Disrupt B-Cell Metabolism and Cause B-Cell Lymphopenia and Hypogammaglobulinemia.

PURPOSE: PI4KA-related disorder is a highly clinically variable condition characterized by neurological (limb spasticity, developmental delay, intellectual disability, seizures, ataxia, nystagmus) and gastrointestinal (inflammatory bowel disease and multiple intestinal atresia) manifestations. Although features consistent with immunodeficiency (autoimmunity/autoinflammation and recurrent infections) have been reported in a subset of patients, the burden of B-cell deficiency and hypogammaglobulinemia has not been extensively investigated. We sought to describe the clinical presentation and manifestations of patients with PI4KA-related disorder and to investigate the metabolic consequences of biallelic PI4KA variants in B cells. METHODS: Clinical data from patients with PI4KA variants were obtained. Multi-omics analyses combining transcriptome, proteome, lipidome and metabolome analyses in conjunction with functional assays were performed in EBV-transformed B cells. RESULTS: Clinical and laboratory data of 13 patients were collected. Recurrent infections (7/13), autoimmune/autoinflammatory manifestations (5/13), B-cell deficiency (8/13) and hypogammaglobulinemia (8/13) were frequently observed. Patients' B cells frequently showed increased transitional and decreased switched memory B-cell subsets. Pathway analyses based on differentially expressed transcripts and proteins confirmed the central role of PI4KA in B cell differentiation with altered B-cell receptor (BCR) complex and signalling. By altering lipids production and tricarboxylic acid cycle regulation, and causing increased endoplasmic reticulum stress, biallelic PI4KA mutations disrupt B cell metabolism inducing mitochondrial dysfunction. As a result, B cells show hyperactive PI3K/mTOR pathway, increased autophagy and deranged cytoskeleton organization. CONCLUSION: By altering lipid metabolism and TCA cycle, impairing mitochondrial activity, hyperactivating mTOR pathway and increasing autophagy, PI4KA-related disorder causes a syndromic inborn error of immunity presenting with B-cell deficiency and hypogammaglobulinemia.

TCF3 haploinsufficiency defined by immune, clinical, gene-dosage, and murine studies.

BACKGROUND: TCF3 is a transcription factor contributing to early lymphocyte differentiation. Germline monoallelic dominant negative and biallelic loss-of-function (LOF) null TCF3 mutations cause a fully penetrant severe immunodeficiency. We identified 8 individuals from 7 unrelated families with monoallelic LOF TCF3 variants presenting with immunodeficiency with incomplete clinical penetrance. OBJECTIVE: We sought to define TCF3 haploinsufficiency (HI) biology and its association with immunodeficiency. METHODS: Patient clinical data and blood samples were analyzed. Flow cytometry, Western blot analysis, plasmablast differentiation, immunoglobulin secretion, and transcriptional activity studies were conducted on individuals carrying TCF3 variants. Mice with a heterozygous Tcf3 deletion were analyzed for lymphocyte development and phenotyping. RESULTS: Individuals carrying monoallelic LOF TCF3 variants showed B-cell defects (eg, reduced total, class-switched memory, and/or plasmablasts) and reduced serum immunoglobulin levels; most but not all presented with recurrent but nonsevere infections. These TCF3 LOF variants were either not transcribed or translated, resulting in reduced wild-type TCF3 protein expression, strongly suggesting HI pathophysiology for the disease. Targeted RNA sequencing analysis of T-cell blasts from TCF3-null, dominant negative, or HI individuals clustered away from healthy donors, implying that 2 WT copies of TCF3 are needed to sustain a tightly regulated TCF3 gene-dosage effect. Murine TCF3 HI resulted in a reduction of circulating B cells but overall normal humoral immune responses. CONCLUSION: Monoallelic LOF TCF3 mutations cause a gene-dosage-dependent reduction in wild-type protein expression, B-cell defects, and a dysregulated transcriptome, resulting in immunodeficiency. Tcf3+/- mice partially recapitulate the human phenotype, underscoring the differences between TCF3 in humans and mice.

A Heterozygous Gain-of-Function Variant in IKBKB Associated with Autoimmunity and Autoinflammation.

PURPOSE: Biallelic loss-of-function variants in IKBKB cause severe combined immunodeficiency. We describe a case of autoimmunity and autoinflammation in a male infant with a heterozygous gain-of-function (GOF) IKBKB variant. METHODS: Case report and review of the literature. We performed in silico variant analysis, measurement of plasma soluble biomarkers associated with immune activation, functional stimulation of patient peripheral blood mononuclear cells, and functional validation of variants transduced in Jurkat cells. RESULTS: A patient with two heterozygous IKBKB variants (E518K and T559M) presents with previously undescribed autoimmune cytopenias and autoinflammation. He had decreased TNF-α-induced IkBα degradation in vitro, and had increased serum biomarkers associated with macrophage recruitment and activation. Jurkat cells transduced with the IKKb T559M variant showed increased basal levels of phosphorylation of IKKα/b and p65, and higher degradation of IkBα suggesting a GOF mechanism. No significant changes were observed in Jurkat cells transduced with the E518K variant. CONCLUSIONS: A GOF variant in IKBKB may associate with autoinflammation and autoimmunity highlighting a novel clinical phenotype.

Inborn errors of human IKAROS: LOF and GOF variants associated with primary immunodeficiency.

IKAROS/IKZF1 plays a pivotal role in lymphocyte differentiation and development. Germline mutations in IKZF1, which have been shown to be associated with primary immunodeficiency, can be classified through four different mechanisms of action depending on the protein expression and its functional defects: haploinsufficiency, dimerization defective, dominant negative, and gain of function. These different mechanisms are associated with variable degrees of susceptibility to infectious diseases, autoimmune disorders, allergic diseases, and malignancies. To date, more than 30 heterozygous IKZF1 germline variants have been reported in patients with primary immunodeficiency. Here we review recent discoveries and clinical/immunological characterization of IKAROS-associated diseases that are linked to different mechanisms of action in IKAROS function.

Germline IKAROS dimerization haploinsufficiency causes hematologic cytopenias and malignancies.

IKAROS is a transcription factor forming homo- and heterodimers and regulating lymphocyte development and function. Germline mutations affecting the IKAROS N-terminal DNA binding domain, acting in a haploinsufficient or dominant-negative manner, cause immunodeficiency. Herein, we describe 4 germline heterozygous IKAROS variants affecting its C-terminal dimerization domain, via haploinsufficiency, in 4 unrelated families. Index patients presented with hematologic disease consisting of cytopenias (thrombocytopenia, anemia, neutropenia)/Evans syndrome and malignancies (T-cell acute lymphoblastic leukemia, Burkitt lymphoma). These dimerization defective mutants disrupt homo- and heterodimerization in a complete or partial manner, but they do not affect the wild-type allele function. Moreover, they alter key mechanisms of IKAROS gene regulation, including sumoylation, protein stability, and the recruitment of the nucleosome remodeling and deacetylase complex; none affected in N-terminal DNA binding defects. These C-terminal dimerization mutations are largely associated with hematologic disorders, display dimerization haploinsufficiency and incomplete clinical penetrance, and differ from previously reported allelic variants in their mechanism of action. Dimerization mutants contribute to the growing spectrum of IKAROS-associated diseases displaying a genotype-phenotype correlation.

SASH3 variants cause a novel form of X-linked combined immunodeficiency with immune dysregulation.

Sterile alpha motif (SAM) and Src homology-3 (SH3) domain-containing 3 (SASH3), also called SH3-containing lymphocyte protein (SLY1), is a putative adaptor protein that is postulated to play an important role in the organization of signaling complexes and propagation of signal transduction cascades in lymphocytes. The SASH3 gene is located on the X-chromosome. Here, we identified 3 novel SASH3 deleterious variants in 4 unrelated male patients with a history of combined immunodeficiency and immune dysregulation that manifested as recurrent sinopulmonary, cutaneous, and mucosal infections and refractory autoimmune cytopenias. Patients exhibited CD4+ T-cell lymphopenia, decreased T-cell proliferation, cell cycle progression, and increased T-cell apoptosis in response to mitogens. In vitro T-cell differentiation of CD34+ cells and molecular signatures of rearrangements at the T-cell receptor α (TRA) locus were indicative of impaired thymocyte survival. These patients also manifested neutropenia and B-cell and natural killer (NK)-cell lymphopenia. Lentivirus-mediated transfer of the SASH3 complementary DNA-corrected protein expression, in vitro proliferation, and signaling in SASH3-deficient Jurkat and patient-derived T cells. These findings define a new type of X-linked combined immunodeficiency in humans that recapitulates many of the abnormalities reported in mice with Sly1-/- and Sly1Δ/Δ mutations, highlighting an important role of SASH3 in human lymphocyte function and survival.

A Point Mutation in IKAROS ZF1 Causes a B Cell Deficiency in Mice.

IKZF1 (IKAROS) is essential for normal lymphopoiesis in both humans and mice. Previous Ikzf1 mouse models have demonstrated the dual role for IKZF1 in both B and T cell development and have indicated differential requirements of each zinc finger. Furthermore, mutations in IKZF1 are known to cause common variable immunodeficiency in patients characterized by a loss of B cells and reduced Ab production. Through N-ethyl-N-nitrosourea mutagenesis, we have discovered a novel Ikzf1 mutant mouse with a missense mutation (L132P) in zinc finger 1 (ZF1) located in the DNA binding domain. Unlike other previously reported murine Ikzf1 mutations, this L132P point mutation (Ikzf1L132P ) conserves overall protein expression and has a B cell-specific phenotype with no effect on T cell development, indicating that ZF1 is not required for T cells. Mice have reduced Ab responses to immunization and show a progressive loss of serum Igs compared with wild-type littermates. IKZF1L132P overexpressed in NIH3T3 or HEK293T cells failed to localize to pericentromeric heterochromatin and bind target DNA sequences. Coexpression of wild-type and mutant IKZF1, however, allows for localization to pericentromeric heterochromatin and binding to DNA indicating a haploinsufficient mechanism of action for IKZF1L132P Furthermore, Ikzf1+/L132P mice have late onset defective Ig production, similar to what is observed in common variable immunodeficiency patients. RNA sequencing revealed a total loss of Hsf1 expression in follicular B cells, suggesting a possible functional link for the humoral immune response defects observed in Ikzf1L132P/L132P mice.

Cushing syndrome and glucocorticoids: T-cell lymphopenia, apoptosis, and rescue by IL-21.

BACKGROUND: Pediatric endogenous Cushing syndrome (eCs) is mainly caused by pituitary corticotropin-producing adenomas, and most glucocorticoid-dependent effects progressively regress upon tumor removal. eCs reproduces long-term, high-dose glucocorticoid therapy, representing a clean, natural, and unbiased model in which to study glucocorticoid bona fide effects on immunity. OBJECTIVE: We performed extensive immunologic studies in otherwise healthy pediatric patients with eCs before and 6 to 13 months after tumor resection, as well as in in vitro glucocorticoid-treated control cells. METHODS: Flow cytometry, immunoblotting, enzyme-linked immunosorbent assay, real-time quantitative PCR, and RNA-Seq techniques were used to characterize patients' and in vitro glucocorticoid treated cells. RESULTS: Reduced thymic output, decreased naive T cells, diminished proliferation, and increased T-cell apoptosis were detected before surgery; all these defects eventually normalized after tumor removal in patients. In vitro studies also showed increased T-cell apoptosis, with correspondingly diminished NF-κB signaling and IL-21 levels. In this setting, IL-21 addition upregulated antiapoptotic BCL2 expression and rescued T-cell apoptosis in a PI3K pathway-dependent manner. Similar and reproducible findings were confirmed in eCs patient cells as well. CONCLUSIONS: We identified decreased thymic output and lymphocyte proliferation, together with increased apoptosis, as the underlying causes to T-cell lymphopenia in eCs patients. IL-21 was decreased in both natural and in vitro long-term, high-dose glucocorticoid environments, and in vitro addition of IL-21 counteracted the proapoptotic effects of glucocorticoid therapy. Thus, our results suggest that administration of IL-21 in patients receiving long-term, high-dose glucocorticoid therapy may contribute to ameliorate lymphopenia and the complications associated to it.

TNF inhibition in vasculitis management in adenosine deaminase 2 deficiency (DADA2).

BACKGROUND: Deficiency of adenosine deaminase 2 (DADA2) is a recessively inherited autoinflammatory disorder caused by a loss of functional ADA2 protein. TNF inhibition (TNFi) has proven to be highly effective in treating inflammatory manifestations. OBJECTIVE: We sought to explore the pathophysiology and the underlying mechanisms of TNF-inhibitor response in these patients. METHODS: We performed Sanger sequencing of the ADA2 gene. We used flow cytometry, intracellular cytokine staining, transcriptome analysis, immunohistochemistry, and cell differentiation experiments to define an inflammatory signature in patients with DADA2 and studied their response to TNF-inhibitor treatment. RESULTS: We demonstrated increased inflammatory signals and overproduction of cytokines mediated by IFN and nuclear factor kappa B pathways in patients' primary cells. Treatment with TNFi led to reduction in inflammation, rescued the skewed differentiation toward the proinflammatory M1 macrophage subset, and restored integrity of endothelial cells in blood vessels. We also report 8 novel disease-associated variants in 7 patients with DADA2. CONCLUSIONS: Our data explore the cellular mechanism underlying effective treatment with TNFi therapies in DADA2. DADA2 vasculitis is strongly related to the presence of activated myeloid cells, and the endothelial cell damage is rescued with anti-TNF treatment.

CARD9 Expression Pattern, Gene Dosage, and Immunodeficiency Phenotype Revisited.

BACKGROUND: CARD9 deficiency is an autosomal recessive primary immunodeficiency underlying increased susceptibility to fungal infection primarily presenting as invasive CNS Candida and/or cutaneous/invasive dermatophyte infections. More recently, a rare heterozygous dominant negative CARD9 variant c.1434 + 1G > C was reported to be protective from inflammatory bowel disease. OBJECTIVE: We studied two siblings carrying homozygous CARD9 variants (c.1434 + 1G > C) and born to heterozygous asymptomatic parents. One sibling was asymptomatic and the other presented with candida esophagitis, upper respiratory infections, hypogammaglobulinemia, and low class-switched memory B cells. METHODS AND RESULTS: The CARD9 c.1434 + 1G > C variant generated two mutant transcripts confirmed by mRNA and protein expression: an out-of-frame c.1358-1434 deletion/ ~ 55 kDa protein (CARD9Δex.11) and an in-frame c.1417-1434 deletion/ ~ 61 kDa protein (CARD9Δ18 nt.). Neither transcript was able to form a complete/functional CBM complex, which includes TRIM62. Based on the index patient's CVID-like phenotype, CARD9 expression was tested and detected in lymphocytes and monocytes from humans and mice. The functional impact of different CARD9 mutations and gene dosage conditions was evaluated in heterozygous and homozygous c.1434 + 1 G > C members of the index family, and in WT (two WT alleles), haploinsufficiency (one WT, one null allele), and null (two null alleles) individuals. CARD9 gene dosage impacted lymphocyte and monocyte functions including cytokine generation, MAPK activation, T-helper commitment, transcription, plasmablast differentiation, and immunoglobulin production in a differential manner. CONCLUSIONS: CARD9 exon 11 integrity is critical to CBM complex function. CARD9 is expressed and affects particular T and B cell functions in a gene dosage-dependent manner, which in turn may contribute to the phenotype of CARD9 deficiency.

Case report and review of the literature: immune dysregulation in a large familial cohort due to a novel pathogenic RELA variant.

OBJECTIVE: To explore and define the molecular cause(s) of a multi-generational kindred affected by Bechet's-like mucocutaneous ulcerations and immune dysregulation. METHODS: Whole genome sequencing and confirmatory Sanger sequencing were performed. Components of the NFκB pathway were quantified by immunoblotting, and function was assessed by cytokine production (IL-6, TNF-α, IL-1ß) after lipopolysaccharide (LPS) stimulation. Detailed immunophenotyping of T-cell and B-cell subsets was performed in four patients from this cohort. RESULTS: A novel variant in the RELA gene, p. Tyr349LeufsTer13, was identified. This variant results in premature truncation of the protein before the serine (S) 536 residue, a key phosphorylation site, resulting in enhanced degradation of the p65 protein. Immunoblotting revealed significantly decreased phosphorylated [p]p65 and pIκBα. The decrease in [p]p65 may suggest reduced heterodimer formation between p50/p65 (NFκB1/RelA). Immunophenotyping revealed decreased naïve T cells, increased memory T cells, and expanded senescent T-cell populations in one patient (P1). P1 also had substantially higher IL-6 and TNF-α levels post-stimulation compared with the other three patients. CONCLUSION: Family members with this novel RELA variant have a clinical phenotype similar to other reported RELA cases with predominant chronic mucocutaneous ulceration; however, the clinical phenotype broadens to include Behçet's syndrome and IBD. Here we describe the clinical, immunological and genetic evaluation of a large kindred to further expand identification of patients with autosomal dominant RELA deficiency, facilitating earlier diagnosis and intervention. The functional impairment of the canonical NFκB pathway suggests that this variant is causal for the clinical phenotype in these patients.

Severe SARS-CoV-2 disease in the context of a NF-κB2 loss-of-function pathogenic variant.

BACKGROUND: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is a novel coronavirus that emerged recently and has created a global pandemic. Symptomatic SARS-CoV-2 infection, termed coronavirus disease 2019 (COVID-19), has been associated with a host of symptoms affecting numerous organ systems, including the lungs, cardiovascular system, kidney, central nervous system, gastrointestinal tract, and skin, among others. OBJECTIVE: Although several risk factors have been identified as related to complications from and severity of COVID-19, much about the virus remains unknown. The host immune response appears to affect the outcome of disease. It is not surprising that patients with intrinsic or secondary immune compromise might be particularly susceptible to complications from SARS-CoV-2 infection. Pathogenic loss-of-function or gain-of-function heterozygous variants in nuclear factor-κB2 have been reported to be associated with either a combined immunodeficiency or common variable immunodeficiency phenotype. METHODS: We evaluated the functional consequence and immunologic phenotype of a novel NFKB2 loss of function variant in a 17-year-old male patient and describe the clinical management of SARS-CoV-2 infection in this context. RESULTS: This patient required a 2-week hospitalization for SARS-CoV-2 infection, including 7 days of mechanical ventilation. We used biologic therapies to avert potentially fatal acute respiratory distress syndrome and treat hyperinflammatory responses. The patient had an immunologic phenotype of B-cell dysregulation with decreased switched memory B cells. Despite the underlying immune dysfunction, he recovered from the infection with intense management. CONCLUSIONS: This clinical case exemplifies some of the practical challenges in management of patients with SARS-CoV-2 infection, especially in the context of underlying immune dysregulation.

IKAROS-Associated Diseases in 2020: Genotypes, Phenotypes, and Outcomes in Primary Immune Deficiency/Inborn Errors of Immunity.

IKAROS, encoded by IKZF1, is a zinc finger transcription factor and a critical regulator of hematopoiesis. Mutations in IKZF1 have been implicated in immune deficiency, autoimmunity, and malignancy in humans. Somatic IKZF1 loss-of-function mutations and deletions have been shown to increase predisposition to the development of B cell acute lymphoblastic leukemia (B-ALL) and associated with poor prognosis. In the last 4 years, germline heterozygous IKZF1 mutations have been reported in primary immune deficiency/inborn errors of immunity. These allelic variants, acting by either haploinsufficiency or dominant negative mechanisms affecting particular functions of IKAROS, are associated with common variable immunodeficiency, combined immunodeficiency, or primarily hematologic phenotypes in affected patients. In this review, we provide an overview of genetic, clinical, and immunological manifestations in patients with IKZF1 mutations, and the molecular and cellular mechanisms that contribute to their disease as a consequence of IKAROS dysfunction.