Gain-of-function (GOF) variants in human TLR7 have recently been reported in 11 cases, six of which were diagnosed with systemic lupus erythematosus (SLE). We have identified the X-linked L840R TLR7 variant in hemizygosity in a male patient with SLE and in heterozygosity in his clinically asymptomatic mother. The leucine 840 is located at the first amino acid of TLR7 transmembrane domain and is conserved across various species. The L840R substitution is predicted to be deleterious by various scoring algorithms and may therefore affect TLR7 function. Molecular dynamics simulations of TLR7-UNC93B1 interactions revealed that R840 alters nearby amino acids interactions, resulting in increased hydrogen bond between E834 of TLR7 with R157 of UNC93B1. Finally, the L840R TLR7 variant has increased activity compared with WT, as measured with a nuclear factor κB (NF-κB)-specific luciferase reporter upon stimulation with TLR7 agonist R848. Hence, hemizygosity for L840R confers GOF for NF-κB activation and underlies SLE by potentially increasing TLR7 binding to UNC93B1.

Patients with loss-of-function DOCK8 or dominant-negative STAT3 variants have hyper-IgE syndrome, although only DOCK8 deficiency consistently presents with elevated food-specific IgE and symptomatic allergy. We previously found in mice that DOCK8 restricts the differentiation of IL-13+ T follicular helper (Tfh13) cells that drive anaphylactic IgE, although the mechanisms were unclear. Here, we show that DOCK8 promotes STAT3 activity, which inhibits GATA3 in T cells. However, only patients with DOCK8, but not STAT3, deficiency had elevated Tfh13 cells. Cell-specific deletion of either Dock8 (T-Dock8-/-) or Stat3 (T-Stat3-/-) augmented peanut-specific IgE and Tfh13s when oral sensitization was promoted by adjuvants. However, the phenotypes diverged during adjuvant-free oral peanut exposure: only T-Dock8-/- mice developed Tfh13 cells and peanut-specific IgE, accompanied by reduced Foxp3+ Tregs. Treg depletion in T-Stat3-/- mice unmasked Tfh13 induction to oral antigen alone. Thus, DOCK8 and STAT3 cooperate to restrain Tfh13 differentiation to food allergens, and additional Treg impairment in DOCK8 deficiency allows for Tfh13 cell induction and allergy.

Sethumadhavan, Mariasoosai, Yamakawa et al. report a novel TLR7 gain-of-function (GOF) variant associated with autoimmunity in a male patient, which further highlights TLR7's essential role in SLE pathogenesis. The L840R mutation may increase TLR7 binding with UNC93B1, which is a molecular mechanism that was not previously described for other TLR7 GOF or UNC93B1 mutations.

The human integrin lymphocyte function-associated antigen 1 (LFA-1; αLβ2) is broadly expressed on leukocytes and involved in various intercellular adhesions. We report complete LFA-1 deficiency because of inherited αL (CD11a) deficiency in otherwise healthy adults of various ancestries with skin lesions due to commensal papillomaviruses. The patients had no history of invasive infections characteristic of children with inherited deficiency of β2 (CD18), which forms heterodimers with αL, αM (CD11b), αX (CD11c), or αD (CD11d). The development and function of leukocyte subsets are largely preserved in the absence of LFA-1. However, the transendothelial migration of skin-tropic cutaneous lymphocyte antigen (CLA)+ memory T cells is severely impaired, resulting in their selective sequestration in the blood. Conversely, alternative integrins mediate the extravasation of other leukocytes, including other T cell subsets, to other tissues. Human LFA-1 is required for steady-state T cell homing to the skin and control of papillomaviruses but is otherwise largely redundant. Integrin-mediated T cell compartmentalization is thus essential for organ-selective immune surveillance.

Homozygosity for rare loss-of-function IL23R variants abolishes IL-23-dependent IFN-γ production by lymphocytes, including NK and innate-like T cells, thereby underlying clinical disease due to weakly virulent mycobacterial species. We report selective enrichment in homozygosity for four hypomorphic IL23R variants in our cohort of patients with tuberculosis. Three of these IL23R alleles are rare (G300V, G149R, and L372F), with a minor allele frequency (MAF) under 1%, but the fourth (R381Q) is surprisingly common, with an MAF as high as 10.2% in certain populations. The other 15 missense alleles found in the homozygous state in public databases are isomorphic. The four hypomorphic IL-23R variants identified dimerize with IL-12Rβ1 and bind IL-23. However, their function is impaired by low levels of cell surface expression (R381Q, G300V) and/or as a consequence of conformational changes altering agonist efficacy. IFN-γ production in response to IL-23 is impaired in innate-like T cells and NK cells. These data suggest that recessive partial IL-23R deficiency, whether due to rare or common variants, confers a predisposition to tuberculosis while preserving immunity to less virulent mycobacteria.

Homozygosity for rare loss-of-function IL23R variants abolishes IL-23-dependent IFN-γ production by lymphocytes, including NK and innate-like T cells, thereby underlying clinical disease due to weakly virulent mycobacterial species. We report selective enrichment in homozygosity for four hypomorphic IL23R variants in our cohort of patients with tuberculosis. Three of these IL23R alleles are rare (G300V, G149R and L372F), with a minor allele frequency (MAF) under 1%, but the fourth (R381Q) is surprisingly common, with a MAF as high as 10.2% in certain populations. The other 15 missense alleles found in the homozygous state in public databases are isomorphic. The four hypomorphic IL-23R variants identified dimerize with IL-12Rβ1 and bind IL-23. However, their function is impaired by low levels of cell-surface expression (R381Q, G300V) and/or as a consequence of conformational changes altering agonist efficacy. IFN-γ production in response to IL-23 is impaired in innate-like T cells and NK cells. These data suggest that recessive partial IL-23R deficiency, whether due to rare or common variants, confers a predisposition to tuberculosis while preserving immunity to less virulent mycobacteria. One sentence summary:Homozygous hypomorphic IL23R variants impair IL-23-dependent IFN-γ production and underlie tuberculosis.

The pathogenic mechanisms underlying pyoderma gangrenosum (PG) remain unclear. Here we report three patients with PG from two unrelated kindreds with homozygous R57C mutation of the linear deubiquitinase OTULIN. The patients have isolated, pediatric-onset, OTULIN-related PG (ORP). In contrast to OTULIN-related autoinflammatory syndrome (ORAS), caused by mutations affecting the catalytic domain, R57C affects the PUB-interacting motif with distinct biochemical, immunological and clinical consequences. OTULIN-R57C is catalytically active but is unable to bind the linear ubiquitin assembly complex (LUBAC). Patient monocytes show heightened expression of IL1B, and OTULIN-R57C fails to suppress inflammasome activity. Patients have elevated levels of TNF, and their dermal fibroblasts show heightened susceptibility to TNF-dependent cell death. Homozygosity for OTULIN-R57C leads to accumulation of linear ubiquitin and LUBAC autoubiquitination in patients' dermal fibroblasts, consistent with pathogenic LUBAC activity. These findings identify a genetic etiology of isolated PG of childhood. We propose a multifactorial mechanism of ORP, including myeloid IL-1β production and TNF-driven death of skin-resident cells, suggesting that blockade of IL-1β or TNF are therapeutic options in PG.

Langerhans cell histiocytosis (LCH) and Erdheim-Chester disease (ECD) are clonal myeloid disorders associated with mitogen-activated protein (MAP)-kinase-activating mutations and an increased risk of neurodegeneration. We found microglial mutant clones in LCH and ECD patients, whether or not they presented with clinical symptoms of neurodegeneration, associated with microgliosis, astrocytosis, and neuronal loss, predominantly in the rhombencephalon gray nuclei. Neurological symptoms were associated with PU.1+ clone size (p = 0.0003) in patients with the longest evolution of the disease, indicating a phase of subclinical incipient neurodegeneration. Genetic barcoding analysis suggests that clones may originate from definitive or yolk sac hematopoiesis, depending on the patients. In a mouse model, disease topography was attributable to a local clonal proliferative advantage, and microglia depletion by a CSF1R-inhibitor limited neuronal loss and improved survival. These studies characterize a neurodegenerative disease associated with clonal proliferation of inflammatory microglia. The long preclinical stage represents a therapeutic window before irreversible neuronal depletion.

Somatic genetic heterogeneity resulting from post-zygotic DNA mutations is widespread in human tissues and can cause diseases, however, few studies have investigated its role in neurodegenerative processes such as Alzheimer’s disease (AD). Here, we report the selective enrichment of microglia clones carrying pathogenic variants, that are not present in neuronal, glia/stromal cells, or blood, from patients with AD in comparison to age-matched controls. Notably, microglia-specific AD-associated variants preferentially target the MAPK pathway, including recurrent CBL ring-domain mutations. These variants activate ERK and drive a microglia transcriptional program characterized by a strong neuro-inflammatory response, both in vitro and in patients. Although the natural history of AD-associated microglial clones is difficult to establish in humans, microglial expression of a MAPK pathway activating variant was previously shown to cause neurodegeneration in mice, suggesting that AD-associated neuroinflammatory microglial clones may contribute to the neurodegenerative process in patients.

Signal transducer and activator of transcription 3 (STAT3) plays a key role in leukocytic and non-leukocytic cells. Germ line mutations in STAT3, which are mainly found in the SH2, DNA binding and transactivation domains, can be loss- or gain-of-function (LOF and GOF). STAT3 N-terminal domain (NTD) mutations are rare, and their biological effects remain incompletely understood. We explored the significance of STAT3 NTD p.Trp37* variant in a patient with chronic pulmonary aspergillosis and a low Hyper-IgE syndrome (HIES) score. In cell culture models, the expression of full-length p.Trp37* allele showed shorter STAT3 protein expression suggesting a re-initiation (Met99 or Met143). STAT3 activity using luciferase reporter assay showed a twofold-increased activity of the STAT3 p.Trp37* STAT3 protein compared with WT STAT3 at basal level and upon IL-6 stimulation. In contrast, the activity of the short pTrp37* peptide (amino acids 1 to 37) was amorphic but without dominant negative (DN) effect on transcriptional activity or STAT3 Tyr705 phosphorylation. The proteins initiated at Met99 and Met143 were surprisingly hypermorphic. In carriers’ peripheral blood mononuclear cells (PBMCs), both WT and mutated STAT3 mRNA were equally present and the global amount of STAT3 protein was not significantly reduced. In stimulated heterozygous carriers’ PBMCs, however, STAT3 Tyr705 phosphorylation and Th17 were reduced but not completely abolished. This suggests a DN effect of an unknown product of the p.Trp37* allele. Transcriptomics analysis of PBMCs from the index revealed selectively distinct gene expression. We conclude that heterozygosity for the NTD p.Trp37* STAT3 mutation defines a novel allelic form of STAT3 deficiency, associated with a chronic pulmonary aspergillosis and minor signs of HIES.

The last decades have brought a rapid expansion of the number of primary disorders related to the polyubiquitination pathways in humans. Most of these disorders manifest with two seemingly contradictory clinical phenotypes: autoinflammation, immunodeficiency, or both. We provide an overview of the molecular pathogenesis of these disorders, and their role in inflammation and infection. By focusing on data from human genetic diseases, we explore the complexities of the polyubiquitination pathways and the corresponding clinical phenotypes of their deficiencies. We offer a road map for the discovery of new genetic etiologies. By considering the triggers that induce inflammation, we propose autoinflammation and immunodeficiency as continuous clinical phenotypes.

The presence of a second TNFRSF13B mutation, HLA class II markers, or multiple single nucleotide variants in patients helps to explain the incomplete penetrance of immunodeficiency in carriers of TNFRSF13B mutations.

Current methods for variant effect prediction do not differentiate between pathogenic variants resulting in different disease outcomes and are restricted in application due to a focus on variants with a single molecular consequence. We have developed Variant-to-Phenotype (V2P), a multi-task, multi-output machine learning model to predict variant pathogenicity conditioned on top-level Human Phenotype Ontology disease phenotypes (n = 23) for single nucleotide variants and insertions/deletions throughout the human genome. V2P leverages a unique approach for the modeling of variant effect that incorporates resultant disease phenotypes as output and during training to improve the quality of variant disease phenotype and effect predictions, simultaneously. We describe the architecture, training strategy, and biological features contributing to V2P's output, revealing initial characteristics underlying the relationship between disease genotype and phenotype. Moreover, we demonstrate the benefit of incorporating disease phenotypes for variant effect predictions by comparing V2P with several variant effect predictors across various high-quality evaluation datasets from manually curated databases and functional assays. Finally, we examine how V2P's predictions result in the successful identification of pathogenic variants in real and simulated patient sequencing data, outperforming other tested methods in initial comparisons. V2P offers a complete mapping of human genetic variants to disease-phenotypes, offering a uniquely conditioned set of variant effect characterizations.

ABSTRACT:Leukopoiesis is lethally arrested in mice lacking the master transcriptional regulator PU.1. Depending on the animal model, subtotal PU.1 loss either induces acute myeloid leukemia or arrests early B-cell and dendritic-cell development. Although humans with absolute PU.1 deficiency have not been reported, a small cadre of congenital agammaglobulinemia patients with sporadic, inborn PU.1 haploinsufficiency was recently described. To better estimate the penetrance, clinical complications, immunophenotypic features, and malignancy risks of PU.1-mutated agammaglobulinemia (PU.MA), a collection of 134 novel or rare PU.1 variants from publicly available databases, institutional cohorts, previously published reports, and unsolved agammaglobulinemia cases were functionally analyzed. In total, 25 loss-of-function (LOF) variants were identified in 33 heterozygous carriers from 21 kindreds across 13 nations. Of individuals harboring LOF PU.1 variants, 22 were agammaglobulinemic, 5 displayed antibody deficiencies, and 6 were unaffected, indicating an estimated disease penetrance of 81.8% with variable expressivity. In a cluster of patients, disease onset was delayed, sometimes into adulthood. All LOF variants conveyed effects via haploinsufficiency, either by destabilizing PU.1, impeding nuclear localization, or directly interfering with transcription. PU.MA patient immunophenotypes consistently demonstrated B-cell, conventional dendritic-cell, and plasmacytoid dendritic-cell deficiencies. Associated infectious and noninfectious symptoms hewed closely to X-linked agammaglobulinemia and not monogenic dendritic-cell deficiencies. No carriers of LOF PU.1 variants experienced hematologic malignancies. Collectively, in vitro and clinical data indicate heterozygous LOF PU.1 variants undermine humoral immunity but do not convey strong leukemic risks.

Background:CARD11-associated atopy with dominant interference of NF-κB signaling (CADINS) is developed as a result of heterozygous loss-of-function variants in CARD11 that function as strong dominant-negative alleles. In lymphocytes, CARD11 encodes a scaffold protein that links activation of the antigen receptor with downstream signaling. Patients with CADINS generally experience severe atopic dermatitis, asthma, recurrent pneumonia and other upper respiratory tract infections, skin infections, and allergies to a variety of dietary and environmental antigens. Additionally, patients experience elevated levels of serum IgE, but low to normal levels of other immunoglobulin types. Objective:We performed genetic diagnosis of a patient of nonconsanguineous descent presenting at 11 years of age with severe atopic dermatitis, asthma, food allergy, skin and recurrent infections, and an extremely elevated level of serum IgE. Methods:We performed whole genome sequencing of samples obtained from the patient and his entire family. Results:Clinical, laboratory, genetic, and functional findings suggested CADINS. Genetic evaluation revealed a novel heterozygous missense variant (c.2913C>G, p.Cys971Trp) in the CARD11 gene as the potential underlying defect. Expression of CARD11 variant-stimulated constitutive NF-κB activity in T-cell lines demonstrated both loss-of-function and dominant-negative activity. Conclusion:A novel germline heterozygous missense variant (c.2913C>G) in CARD11 potentially leads to CADINS.

Human OTULIN haploinsufficiency predisposes to life-threatening necrosis of the skin and lungs. Disease is triggered by infectious agents, typically Staphylococcus aureus, as well as unknown etiologies. We describe and characterize six unrelated patients who carry rare, predicted deleterious variants of OTULIN in heterozygosity. In addition to staphylococcal infections, the disease in the patients is elicited by previously underappreciated triggers, including mechanical or iatrogenic traumas and pseudomonal or clostridial infections. Severe necrosis of the lungs and/or skin are clinical hallmarks of their disease. By combining in vitro allele characterizations and functional studies in patients’ cells, we demonstrate that the patients suffer from OTULIN haploinsufficiency. We provide guidance for assessing heterozygous OTULIN variants in diagnostic settings by evaluating in silico measures of predicted deleteriousness. The clinical course of the patients expands the genotypic and phenotypic spectrum of OTULIN haploinsufficiency and provides, in the light of a broadening of triggers, leads for therapeutic interventions.

Inborn deficiencies of the alternative pathway (AP) of the complement system have been associated with life-threatening infections, mainly by encapsulated bacteria. Complete factor D (FD) deficiencies have been reported in only seven families in the literature. We report two new cases of biochemically and genetically confirmed complete FD deficiency, including the first in a Down syndrome patient. The index cases respectively suffered from severe H. influenza and N. meningitidis infections. Their FD activity was undetectable but was restored by adding recombinant human FD. FD levels were undetectable in the plasma of both patients using ELISA. Genetic analysis of the CFD gene identified a homozygous missense variant p.M40R in one patient, and compound heterozygous variants-a nonsense mutation p.Cys148* and a splice site variant c.212+2T>G-in the other. Patients with Down syndrome are more susceptible to infections, but this case highlights the importance of investigating the complement system, particularly the AP, even in those with Down syndrome or other secondary immune deficiencies. A familial study should follow if a congenital deficiency is found. The natural history of patients with inherited complete FD deficiency underscores the necessity of preventive measures against encapsulated bacteria for those receiving therapeutic MASP-3 or FD inhibitors.

Human OTULIN haploinsufficiency causes life-threatening staphylococcal disease, but the genotypic and phenotypic spectrum of the disorder remains largely unknown. We describe and characterize six unrelated patients with OTULIN haploinsufficiency, in whom severe necrosis followed infectious and/or traumatic triggers.