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Recent studies reported the presence of pre-existing autoantibodies (auto-Abs) neutralizing type I interferons (IFNs) in at least 15% of patients with critical COVID-19 pneumonia. In one study, these auto-Abs were found in almost 20% of deceased patients across all ages. We aimed to assess the prevalence and clinical impact of the auto-Abs to type I IFNs in the Seine-Saint-Denis district, which was one of the most affected areas by COVID-19 in France during the first wave. We tested for the presence of auto-Abs neutralizing type I IFNs in a cohort of patients admitted for critical COVID-19 pneumonia during the first wave in the spring of 2020 in the medicine departments at Robert Ballanger Hospital, Aulnay sous Bois. We found circulating auto-Abs that neutralized 100 pg/mL IFN-α2 and/or IFN-ω in the plasma (diluted 1/10) of 7.9% (11 of 139) of the patients hospitalized for critical COVID-19. The presence of neutralizing auto-Abs was associated with an increased risk of mortality, as these auto-Abs were detected in 21% of patients who died from COVID-19 pneumonia. Deceased patients with and without auto-Abs did not present overt clinical differences. These results confirm both the importance of type I IFN immunity in host defense against SARS-CoV-2 infection and the usefulness of detection of auto-Abs neutralizing type I IFNs in the management of patients.

Genetic variants underlying life-threatening diseases, being unlikely to be transmitted to the next generation, are gradually and selectively eliminated from the population through negative selection. We study the determinants of this evolutionary process in human genes underlying monogenic diseases by comparing various negative selection scores and an integrative approach, CoNeS, at 366 loci underlying inborn errors of immunity (IEI). We find that genes underlying autosomal dominant (AD) or X-linked IEI have stronger negative selection scores than those underlying autosomal recessive (AR) IEI, whose scores are not different from those of genes not known to be disease causing. Nevertheless, genes underlying AR IEI that are lethal before reproductive maturity with complete penetrance have stronger negative selection scores than other genes underlying AR IEI. We also show that genes underlying AD IEI by loss of function have stronger negative selection scores than genes underlying AD IEI by gain of function, while genes underlying AD IEI by haploinsufficiency are under stronger negative selection than other genes underlying AD IEI. These results are replicated in 1,140 genes underlying inborn errors of neurodevelopment. Finally, we propose a supervised classifier, SCoNeS, which predicts better than state-of-the-art approaches whether a gene is more likely to underlie an AD or AR disease. The clinical outcomes of monogenic inborn errors, together with their mode and mechanisms of inheritance, determine the levels of negative selection at their corresponding loci. Integrating scores of negative selection may facilitate the prioritization of candidate genes and variants in patients suspected to carry an inborn error.

Mendelian susceptibility to mycobacterial disease (MSMD) is characterized by a selective predisposition to clinical disease caused by the Bacille Calmette-Guérin (BCG) vaccine and environmental mycobacteria. The known genetic etiologies of MSMD are inborn errors of IFN-γ immunity due to mutations of 15 genes controlling the production of or response to IFN-γ. Since the first MSMD-causing mutations were reported in 1996, biallelic mutations in the genes encoding IFN-γ receptor 1 (IFN-γR1) and IFN-γR2 have been reported in many patients of diverse ancestries. Surprisingly, mutations of the gene encoding the IFN-γ cytokine itself have not been reported, raising the remote possibility that there might be other agonists of the IFN-γ receptor. We describe 2 Lebanese cousins with MSMD, living in Kuwait, who are both homozygous for a small deletion within the IFNG gene (c.354_357del), causing a frameshift that generates a premature stop codon (p.T119Ifs4*). The mutant allele is loss of expression and loss of function. We also show that the patients' herpesvirus Saimiri-immortalized T lymphocytes did not produce IFN-γ, a phenotype that can be rescued by retrotransduction with WT IFNG cDNA. The blood T and NK lymphocytes from these patients also failed to produce and secrete detectable amounts of IFN-γ. Finally, we show that human IFNG has evolved under stronger negative selection than IFNGR1 or IFNGR2, suggesting that it is less tolerant to heterozygous deleterious mutations than IFNGR1 or IFNGR2. This may account for the rarity of patients with autosomal-recessive, complete IFN-γ deficiency relative to patients with complete IFN-γR1 and IFN-γR2 deficiencies.

Inborn errors of TLR3-dependent IFN-α/β- and IFN-λ-mediated immunity in the CNS can underlie herpes simplex virus 1 (HSV-1) encephalitis (HSE). The respective contributions of IFN-α/β and IFN-λ are unknown. We report a child homozygous for a genomic deletion of the entire coding sequence and part of the 3'-UTR of the last exon of IFNAR1, who died of HSE at the age of 2 years. An older cousin died following vaccination against measles, mumps, and rubella at 12 months of age, and another 17-year-old cousin homozygous for the same variant has had other, less severe, viral illnesses. The encoded IFNAR1 protein is expressed on the cell surface but is truncated and cannot interact with the tyrosine kinase TYK2. The patient's fibroblasts and EBV-B cells did not respond to IFN-α2b or IFN-β, in terms of STAT1, STAT2, and STAT3 phosphorylation or the genome-wide induction of IFN-stimulated genes. The patient's fibroblasts were susceptible to viruses, including HSV-1, even in the presence of exogenous IFN-α2b or IFN-β. HSE is therefore a consequence of inherited complete IFNAR1 deficiency. This viral disease occurred in natural conditions, unlike those previously reported in other patients with IFNAR1 or IFNAR2 deficiency. This experiment of nature indicates that IFN-α/β are essential for anti-HSV-1 immunity in the CNS.

The complement system is crucial for defense against pathogens and the removal of dying cells or immune complexes. Thus, clinical indications for possible complete complement deficiencies include, among others, recurrent mild or serious bacterial infections as well as autoimmune diseases (AID). The diagnostic approach includes functional activity measurements of the classical (CH50) and alternative pathway (AP50) and the determination of the C3 and C4 levels, followed by the quantitative analysis of individual components or regulators. When biochemical analysis reveals the causal abnormality of the complement deficiency (CD), molecular mechanisms remains frequently undetermined. Here, using direct sequencing analysis of the coding region we report the pathogenic variants spectrum that underlie the total or subtotal complement deficiency in 212 patients. We identified 107 different hemizygous, homozygous, or compound heterozygous pathogenic variants in 14 complement genes [ β ( = 1), ( = 3), ( = 2), ( = 12), ( = 5), C5 ( = 12), ( = 9), ( = 17), β ( = 7), ( = 3), ( = 7), ( = 18), ( = 10), ( = 2)]. Molecular analysis identified 17 recurrent pathogenic variants in 6 genes ( , and ). More than half of the pathogenic variants identified in unrelated patients were also found in healthy controls from the same geographic area. Our study confirms the strong association of meningococcal infections with terminal pathway deficiency and highlights the risk of pneumococcal and auto-immune diseases in the classical and alternative pathways. Results from this large genetic investigation provide evidence of a restricted number of molecular mechanisms leading to complement deficiency and describe the clinical potential adverse events of anti-complement therapy.

Human inborn errors of IFN-γ underlie mycobacterial disease, due to insufficient IFN-γ production by lymphoid cells, impaired myeloid cell responses to this cytokine, or both. We report four patients from two unrelated kindreds with intermittent monocytosis and mycobacterial disease, including bacillus Calmette–Guérin-osis and disseminated tuberculosis, and without any known inborn error of IFN-γ. The patients are homozygous for ZNFX1 variants (p.S959* and p.E1606Rfs*10) predicted to be loss of function (pLOF). There are no subjects homozygous for pLOF variants in public databases. ZNFX1 is a conserved and broadly expressed helicase, but its biology remains largely unknown. It is thought to act as a viral double-stranded RNA sensor in mice, but these patients do not suffer from severe viral illnesses. We analyze its subcellular localization upon overexpression in A549 and HeLa cell lines and upon stimulation of THP1 and fibroblastic cell lines. We find that this cytoplasmic protein can be recruited to or even induce stress granules. The endogenous ZNFX1 protein in cell lines of the patient homozygous for the p.E1606Rfs*10 variant is truncated, whereas ZNFX1 expression is abolished in cell lines from the patients with the p.S959* variant. Lymphocyte subsets are present at normal frequencies in these patients and produce IFN-γ normally. The hematopoietic and nonhematopoietic cells of the patients tested respond normally to IFN-γ. Our results indicate that human ZNFX1 is associated with stress granules and essential for both monocyte homeostasis and protective immunity to mycobacteria.

Over 500 primary immunodeficiency diseases (PID) have been described, but immunological assessment after a severe infection is not routine. We aimed to evaluate the feasibility of a PID screening protocol and calculate PID prevalence in children admitted for severe infection in a pediatric intensive care unit (PICU). This monocentric retrospective study evaluated the feasibility of a PID monitoring protocol after severe infection in children aged 1 month to 16 years-old hospitalized in the Montpellier University Hospital from January 2018 to December 2020. Follow-up consultations at 3 and 12 months included the three main PID screening scores, comprehensive immunological and genetic screenings. Among 1125 children admitted to the PICU, 46 had severe infections and caused by bacterial (48%), viral (39%) or fungal (2%) pathogens. Before infection, none had completed any screening score recommended by dedicated societies (Jeffrey Modell Foundation, German Patients’ Organization for Primary Immunodeficiencies, French Reference Center for Hereditary Immunodeficiencies). At 3 months, three patients had a PID diagnosis (6.5% prevalence, 95% CI 1.4–17.9). These were associated with a deletion of chromosomal region 22q11.21 (DiGeorge syndrome), ELANE mutation (Elastase deficiency or Severe Congenital Neutropenia 1), and C5 deficiency Forty children (87%) presented immunological anomalies without a formal PID diagnosis. These persisted in only 4/17 children tested at 12 months. The most frequent abnormalities were low NK lymphocytes (41.18%), and abnormal B lymphocyte population distribution (25%). The observed PID prevalence post-severe infection matches previous reports, even with a high rate of viral infections, often overlooked. Systematic PID investigation after severe infection, regardless of the pathogen, should be implemented to improve early detection and treatment.

Haploinsufficiency of cytotoxic T-lymphocyte associated protein 4 (CTLA4), a known cause of inborn errors of immunity, can lead to autoimmunity, inflammation, neoplasia and infections. A previously undescribed CTLA4 variant was identified in a patient who presented with life-threatening cutaneous infection caused by Pseudomonas aeruginosa , severe VZV infection, and Evans syndrome. Our aim was to assess the pathogenicity of the previously undescribed c.379T > G variant in the CTLA4 gene and explore its phenotypic presentation in relatives. We employed genetic and protein-based in silico analyses to evaluate the potential role of the c.379T > G variant in the CTLA4 gene. We subsequently studied CTLA4 expression ex vivo, analyzed the clinical presentation of affected carriers and compared the biological status across affected individuals, healthy carriers and noncarriers. In silico analyses revealed that the c.379T > G mutation, which is located within the ligand binding area of CTLA4, is highly pathogenic. Its clinical manifestations, which are sometimes fatal, include multiple infections, autoimmunity, inflammation of the central nervous system, lymphoproliferation and thymoma with Good’s syndrome. Compared with its expression in healthy donors, the expression of CTLA4 following stimulation in memory regulatory T cells was decreased in affected individuals. Immunophenotyping revealed an increased proportion of immature and double-negative B cells in affected patients compared with their nonmutated relatives. Additionally, a reduction in naive T cells and an increase in CD4 + CD25-Foxp3 + cells were observed in carriers compared with controls. The c.379T > G variant of CTLA4 , resulting in a p.Tyr127Asp substitution, is pathogenic and contributes to the development of a CTLA4 haploinsufficiency phenotype. This finding highlights the heterogeneous presentations of the disease, including oncological and neurological manifestations.

Autoimmune lymphoproliferative syndrome (ALPS) is a primary disorder of lymphocyte homeostasis, leading to chronic lymphoproliferation, autoimmune cytopenia, and increased risk of lymphoma. The genetic landscape of ALPS includes mutations in FAS , FASLG , and FADD , all associated with apoptosis deficiency, while the role of CASP10 defect in the disease remains debated. In this study, we aimed to assess the impact of CASP10 variants on ALPS pathogenesis. We benefit from thousands of genetic analysis datasets performed in our Institute’s genetic platform to identify individuals carrying CASP10 variants previously suspected to be involved in ALPS outcome: p.C401LfsX15, p.V410I and p.Y446C, both at heterozygous and homozygous state. Clinical and laboratory features of the six included subjects were variable but not consistent with ALPS. Two individuals were healthy. Comprehensive analyses of CASP10 protein expression and FAS-mediated apoptosis were conducted and compared to healthy controls and ALPS patients with FAS mutations. Missense CASP10 variants (p.V410I and p.Y446C), which are common in the general population, did not disrupt CASP10 expression, nor FAS-mediated apoptosis. In contrast, homozygous p.C401LfsX15 CASP10 variant lead to a complete abolished CASP10 expression but had no impact on FAS-mediated apoptosis function. At heterozygous state, this p.C401LfsX15 variant lead to a reduced CASP10 protein levels but remained associated with a normal FAS-mediated apoptosis function. These findings demonstrate that CASPASE 10 is dispensable for FAS-mediated apoptosis. In consequences, CASP10 defect unlikely contribute to ALPS pathogenesis, since they did not result in an impairment of FAS-mediated apoptosis nor in clinical features of ALPS in human. Moreover, the absence of FAS expression up-regulation in subjects with CASP10 variants rule out any compensatory mechanisms possibly involved in the normal apoptosis function observed. In conclusion, this study challenges the notion that CASP10 variants contribute to the development of ALPS.

Phospholipase C isozymes (PLCs) hydrolyze phosphatidylinositol 4,5-bisphosphate (PIP 2 ) into inositol 1,4,5-trisphosphate (IP 3 ) and diacylglycerol (DAG), important signaling molecules involved in many cellular processes including Ca 2+ release from the endoplasmic reticulum (ER). PLCG1 encodes the PLCγ1 isozyme that is broadly expressed. Hyperactive somatic mutations of PLCG1 are observed in multiple cancers, but only one germline variant has been reported. Here, we describe seven individuals with heterozygous missense variants in PLCG1 [p.(Asp1019Gly), p.(His380Arg), p.(Asp1165Gly), and p.(Leu597Phe)] who present with hearing impairment (5/7), ocular pathology (4/7), cardiac septal defects (3/6), and various immunological issues (5/7). To model these variants in vivo , we generated the analogous variants in the Drosophila ortholog, small wing ( sl ). We created a null allele sl T2A and assessed its expression pattern. sl is broadly expressed, including wing discs, eye discs, and a subset of neurons and glia. sl T2A mutant flies exhibit wing size reductions, ectopic wing veins, and supernumerary photoreceptors. We document that mutant flies also exhibit a reduced lifespan and age-dependent locomotor defects. Expressing wild-type sl in sl T2A mutant flies rescues the loss-of-function phenotypes, whereas the variants increase lethality. Ectopic expression of an established hyperactive PLCG1 variant, p.(Asp1165His) in the wing pouch causes elevated Ca 2+ activity and severe wing phenotypes. These phenotypes are also observed when the p.(Asp1019Gly) or p.(Asp1165Gly) variants are overexpressed in the wing pouch, arguing that these are gain-of-function variants. However, the wing phenotypes associated with p.(His380Arg) or p.(Leu597Phe) overexpression are either mild or only partially penetrant. Our data suggest that the heterozygous missense variants reported here affect protein function differentially and contribute to the clinical features observed in the affected individuals.