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Background: In 2014, germline signal transducer and activator of transcription (STAT) 3 gain-of-function (GOF) mutations were first described to cause a novel multisystem disease of early-onset lymphoproliferation and autoimmunity. Objective: This pivotal cohort study defines the scope, natural history, treatment, and overall survival of a large global cohort of patients with pathogenic STAT3 GOF variants. Methods: We identified 191 patients from 33 countries with 72 unique mutations. Inclusion criteria included symptoms of immune dysregulation and a biochemically confirmed germline heterozygous GOF variant in STAT3. Results: Overall survival was 88%, median age at onset of symptoms was 2.3 years, and median age at diagnosis was 12 years. Immune dysregulatory features were present in all patients: lymphoproliferation was the most common manifestation (73%); increased frequencies of double-negative (CD4−CD8−) T cells were found in 83% of patients tested. Autoimmune cytopenias were the second most common clinical manifestation (67%), followed by growth delay, enteropathy, skin disease, pulmonary disease, endocrinopathy, arthritis, autoimmune hepatitis, neurologic disease, vasculopathy, renal disease, and malignancy. Infections were reported in 72% of the cohort. A cellular and humoral immunodeficiency was observed in 37% and 51% of patients, respectively. Clinical symptoms dramatically improved in patients treated with JAK inhibitors, while a variety of other immunomodulatory treatment modalities were less efficacious. Thus far, 23 patients have undergone bone marrow transplantation, with a 62% survival rate. Conclusion:: STAT3 GOF patients present with a wide array of immune-mediated disease including lymphoproliferation, autoimmune cytopenias, and multisystem autoimmunity. Patient care tends to be siloed, without a clear treatment strategy. Thus, early identification and prompt treatment implementation are lifesaving for STAT3 GOF syndrome.

International audience; PurposeX-linked inhibitor of apoptosis protein (XIAP) deficiency, also known as the X-linked lymphoproliferative syndrome of type 2 (XLP-2), is a rare immunodeficiency characterized by recurrent hemophagocytic lymphohistiocytosis, splenomegaly, and inflammatory bowel disease. Variants in XIAP including missense, non-sense, frameshift, and deletions of coding exons have been reported to cause XIAP deficiency. We studied three young boys with immunodeficiency displaying XLP-2-like clinical features. No genetic variation in the coding exons of XIAP was identified by whole-exome sequencing (WES), although the patients exhibited a complete loss of XIAP expression.MethodsTargeted next-generation sequencing (NGS) of the entire locus of XIAP was performed on DNA samples from the three patients. Molecular investigations were assessed by gene reporter expression assays in HEK cells and CRISPR-Cas9 genome editing in primary T cells.ResultsNGS of XIAP identified three distinct non-coding deletions in the patients that were predicted to be driven by repetitive DNA sequences. These deletions share a common region of 839 bp that encompassed the first non-coding exon of XIAP and contained regulatory elements and marks specific of an active promoter. Moreover, we showed that among the 839 bp, the exon was transcriptionally active. Finally, deletion of the exon by CRISPR-Cas9 in primary cells reduced XIAP protein expression.ConclusionsThese results identify a key promoter sequence contained in the first non-coding exon of XIAP. Importantly, this study highlights that sequencing of the non-coding exons that are not currently captured by WES should be considered in the genetic diagnosis when no variation is found in coding exons.

Background Autosomal recessive (AR) complete IRF8 deficiency is a rare severe inborn error of immunity underlying an absence of blood myeloid mononuclear cells, intracerebral calcifications, and multiple infections. Only three unrelated patients have been reported. Materials and Methods We studied an Argentinian child with multiple infectious diseases and severe pulmonary alveolar proteinosis (PAP). We performed whole-exome sequencing (WES) and characterized his condition by genetic, immunological, and clinical means. Results The patient was born and lived in Argentina. He had a history of viral pulmonary diseases, disseminated disease due to bacillus Calmette-Guérin (BCG), PAP, and cerebral calcifications. He died at the age of 10 months from refractory PAP. WES identified two compound heterozygous variants in IRF8: c.55del and p.R111*. In an overexpression system, the p.R111* cDNA was loss-of-expression, whereas the c.55del cDNA yielded a protein with a slightly lower molecular weight than the wild-type protein. The mutagenesis of methionine residues downstream from c.55del revealed a re-initiation of translation. However, both variants were loss-of-function in a luciferase assay, suggesting that the patient had AR complete IRF8 deficiency. The patient had no blood monocytes or dendritic cells, associated with neutrophilia, and normal counts of NK and other lymphoid cell subsets. Conclusion We describe the fourth patient with AR complete IRF8 deficiency. This diagnosis should be considered in children with PAP, which is probably due to the defective development or function of alveolar macrophages.

Inherited bone marrow failure syndromes (IBMFSs) are a group of disorders typified by impaired production of 1 or several blood cell types. The telomere biology disorders dyskeratosis congenita (DC) and its severe variant, Høyeraal-Hreidarsson (HH) syndrome, are rare IBMFSs characterized by bone marrow failure, developmental defects, and various premature aging complications associated with critically short telomeres. We identified biallelic variants in the gene encoding the 5′-to-3′ DNA exonuclease Apollo/SNM1B in 3 unrelated patients presenting with a DC/HH phenotype consisting of early-onset hypocellular bone marrow failure, B and NK lymphopenia, developmental anomalies, microcephaly, and/or intrauterine growth retardation. All 3 patients carry a homozygous or compound heterozygous (in combination with a null allele) missense variant affecting the same residue L142 (L142F or L142S) located in the catalytic domain of Apollo. Apollo-deficient cells from patients exhibited spontaneous chromosome instability and impaired DNA repair that was complemented by CRISPR/Cas9-mediated gene correction. Furthermore, patients’ cells showed signs of telomere fragility that were not associated with global reduction of telomere length. Unlike patients’ cells, human Apollo KO HT1080 cell lines showed strong telomere dysfunction accompanied by excessive telomere shortening, suggesting that the L142S and L142F Apollo variants are hypomorphic. Collectively, these findings define human Apollo as a genome caretaker and identify biallelic Apollo variants as a genetic cause of a hitherto unrecognized severe IBMFS that combines clinical hallmarks of DC/HH with normal telomere length.

Isolated congenital asplenia (ICA) is the only known human developmental defect exclusively affecting a lymphoid organ. In 2013, we showed that private deleterious mutations in the protein-coding region of RPSA, encoding ribosomal protein SA, caused ICA by haploinsufficiency with complete penetrance. We reported seven heterozygous protein-coding mutations in 8 of the 23 kindreds studied, including 6 of the 8 multiplex kindreds. We have since enrolled 33 new kindreds, 5 of which are multiplex. We describe here eleven new heterozygous ICA-causing RPSA protein-coding mutations, and the first two mutations in the 5’-UTR of this gene, which disrupt mRNA splicing. Overall, 40 of the 73 ICA patients (55%) and 23 of the 56 kindreds (41%) carry mutations located in translated or untranslated exons of RPSA. Eleven of the 43 kindreds affected by sporadic disease (26%) carry RPSA mutations, whereas 12 of the 13 multiplex kindreds (92%) carry RPSA mutations. We also report that six of eighteen (33%) protein-coding mutations and the two (100%) 5’-UTR mutations display incomplete penetrance. Three mutations were identified in 2 independent kindreds, due to a hotspot or a founder effect. Lastly, RPSA ICA-causing mutations were demonstrated to be de novo in 7 of the 23 probands. Mutations in RPSA exons can affect the translated or untranslated regions and can underlie ICA with complete or incomplete penetrance.

Monogenic causes for serious manifestations of common allergic disease inform our mechanistic understanding of the pathogenesis of atopy. However, infections and other syndromic phenotypes often accompany such disorders. We performed next-generation sequencing on a cohort of patients with severe atopic dermatitis with evidence of familial inheritance, regardless of comorbidities. We discovered 9 individuals from 5 families harboring distinct, novel heterozygous mutations in CARD11, a lymphocyte scaffolding protein that facilitates NF-κB and mTOR signaling following antigen receptor (AgR) engagement. Significant infections beyond the skin, and non-immunologic comorbidities, were documented in some but not all patients. Improvement of skin and infectious history were noted in the majority of the patients. Each CARD11 mutant exhibited attenuated AgR-driven signaling to NF-κB and mTORC1, and dominantly interfered with the ability of WT CARD11 to activate these pathways in transfected T cell lines. Primary patient T cells also showed defects in AgR-induced activation of NF-κB and mTORC1, which is critical for promoting Th1 and preventing Th2 responses. Impaired proliferation, mTORC1 signaling and IFN-γ production were partially rescued by supplementing with excess glutamine, which requires CARD11 for import into T cells. In contrast to B cell lymphoproliferative disease associated with gain-of-function CARD11 mutations, and severe combined immunodeficiency associated with null CARD11 mutations, our new findings indicate single hypomorphic mutations in CARD11 can cause potentially correctable cellular defects that lead to severe atopy.

BACKGROUND In previous clinical trials involving children with X-linked severe combined immunodeficiency (SCID-X1), a Moloney murine leukemia virus–based γ-retrovirus vector expressing interleukin-2 receptor γ-chain (γc) complementary DNA successfully restored immunity in most patients but resulted in vector-induced leukemia through enhancermediated mutagenesis in 25% of patients. We assessed the efficacy and safety of a self-inactivating retrovirus for the treatment of SCID-X1. METHODS We enrolled nine boys with SCID-X1 in parallel trials in Europe and the United States to evaluate treatment with a self-inactivating (SIN) γ-retrovirus vector containing deletions in viral enhancer sequences expressing γc (SIN-γc). RESULTS All patients received bone marrow–derived CD34+ cells transduced with the SIN-γc vector, without preparative conditioning. After 12.1 to 38.7 months of follow-up, eight of the nine children were still alive. One patient died from an overwhelming adenoviral infection before reconstitution with genetically modified T cells. Of the remaining eight patients, seven had recovery of peripheral-blood T cells that were functional and led to resolution of infections. The patients remained healthy thereafter. The kinetics of CD3+ T-cell recovery was not significantly different from that observed in previous trials. Assessment of insertion sites in peripheral blood from patients in the current trial as compared with those in previous trials revealed significantly less clustering of insertion sites within LMO2, MECOM, and other lymphoid proto-oncogenes in our patients. CONCLUSIONS This modified γ-retrovirus vector was found to retain efficacy in the treatment of SCID-X1. The long-term effect of this therapy on leukemogenesis remains unknown. (Funded by the National Institutes of Health and others; ClinicalTrials.gov numbers, NCT01410019, NCT01175239, and NCT01129544.)

We investigated a T-cell activation deficiency in a 3-month-old boy with protracted diarrhea, serious cytomegalovirus pneumonia, and a family history (in a brother) of cytomegalovirus infection and toxoplasmosis. In spite of detection of normal number of peripheral lymphocytes, T cells did not proliferate after activation by anti-CD3 and anti-CD2 antibodies, although proliferation induced by antigens was detectable. We sought to determine the origin of this defect as it potentially represented a valuable tool to analyze T-cell physiology. T- cell activation by anti-CD3 antibody or phytohemagglutinin (PHA) led to reduced interleukin-2 (IL-2) production and abnormal nuclear factor- activated T cell (NF-AT; a complex regulating the IL-2 gene transcription) binding activity to a specific oligonucleotide. T-cell proliferation was restored by IL-2. Early events of T-cell activation, such as anti-CD3 antibody-induced cellular protein tyrosine phosphorylation, p59fyn and p56lck kinase activities, and phosphoinositide turnover, were found to be normal. In contrast, anti- CD3 antibody-induced Ca2+ flux was grossly abnormal. Release from endoplasmic reticulum stores was detectable as tested in the presence of anti-CD3 antibody or thapsigargin after cell membrane depolarization in a K+ rich medium, whereas extracellular entry of Ca2+ was defective. The latter abnormality was not secondary to defective K+ channel function, which was found to be normal. A similar defect was found in other hematopoietic cell lineages and in fibroblasts as evaluated by both cytometry and digital video imaging experiments at a single-cell level. This primary T-cell immunodeficiency appears, thus, to be due to defective Ca2+ entry through the plasma membrane. The same abnormality did not alter B-cell proliferation, platelet function, and polymorphonuclear neutrophil (PMN) function. Elucidation of the mechanism underlying this defect would help to understand the physiology of Ca2+ mobilization in T cells.

Mutations in the transmembrane activator and calcium-modulating cyclophilin ligand interactor (TACI) were previously found to be associated with hypogammaglobulinemia in humans. It has been shown that proliferation inducing ligand (APRIL) elicits class switch recombination (CSR) by inducing recruitment of MyD88 to a TACI highly conserved cytoplasmic domain (THC). We have identified a patient with hypogammaglobulinemia carrying a missense mutation (S231R) predicted to affect the THC. Aiming to evaluate the relevance of this novel mutation of TACI in CSR induction, we tested the ability of TACI, TLR9, or/and CD40 ligands to trigger CSR in naive B cells and B-cell lines carrying S231R. IgG secretion was impaired when triggered by TACI or/and TLR9 ligands on S231R-naive B cells. Likewise, these stimuli induced less expression of activation-induced cytidine deaminase, I(γ)1-C(μ), and I(γ)1-C(μ), while induction by optimal CD40 stimulation was indistinguishable from controls. These cells also showed an impaired cooperation between TACI and TLR9 pathways, as well as a lack of APRIL-mediated enhancement of CD40 activation in suboptimal conditions. Finally, after APRIL ligation, S231R-mutated TACI failed to colocalize with MyD88. Collectively, these results highlight the requirement of an intact MyD88-binding site in TACI to trigger CSR.

Orthotopic liver transplantation is the only definitive mode of therapy for children with end-stage liver disease. However, it remains challenging because of the necessity to prevent long-term complications. The aim of this study was to analyze the evolution of transplanted patients with more than one year of follow up. Between November 1992 and November 2001, 238 patients underwent 264 liver transplantations. A total of 143 patients with more than one year of follow up were included. The median age of patients +/- SD was 5.41 years +/- 5.26 (r: 0.58-21.7 years). All children received primary immunosuppression with cyclosporine. The indications for liver replacement were: fulminant hepatic failure (n: 50), biliary atresia (n: 38), cirrhosis (n: 37), chronic cholestasis (n: 13) and miscellaneous (n: 5). The indications for liver re-transplantation were: biliary cirrhosis (n: 7), hepatic artery thrombosis (n: 4) and chronic rejection (n: 3). Reduced-size liver allografts were used in 73/157 liver transplants, 14 of them were from living-related donors and 11 were split-livers. Patient and graft survival rates were 93% and 86% respectively. Death risk was statistically higher in retransplanted and reduced-size grafted patients. Growth retardation and low bone density were recovered before the first 3 years post-transplant. The incidence of lymphoproliferative disease was 7.69%. De novo hepatitis B was diagnosed in 7 patients (4.8%). Social risk did not affect the outcome of our population. The prevention, detection and early treatment of complications in the long-term follow up contributed to improve the outcome.