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Hypomorphic IL2RG mutations may lead to milder phenotypes than X-SCID, named variably as atypical X-SCID or X-CID. We report an 11-year-old boy with a novel c. 172C>T;p.(Pro58Ser) mutation in IL2RG, presenting with atypical X-SCID phenotype. We also review the growing number of hypomorphic IL2RG mutations causing atypical X-SCID. We studied the patient’s clinical phenotype, B, T, NK, and dendritic cell phenotypes, IL2RG and CD25 cell surface expression, and IL-2 target gene expression, STAT tyrosine phosphorylation, PBMC proliferation, and blast formation in response to IL-2 stimulation, as well as protein-protein interactions of the mutated IL2RG by BioID proximity labeling. The patient suffered from recurrent upper and lower respiratory tract infections, bronchiectasis, and reactive arthritis. His total lymphocyte counts have remained normal despite skewed T and B cells subpopulations, with very low numbers of plasmacytoid dendritic cells. Surface expression of IL2RG was reduced on his lymphocytes. This led to impaired STAT tyrosine phosphorylation in response to IL-2 and IL-21, reduced expression of IL-2 target genes in patient CD4+ T cells, and reduced cell proliferation in response to IL-2 stimulation. BioID proximity labeling showed aberrant interactions between mutated IL2RG and ER/Golgi proteins causing mislocalization of the mutated IL2RG to the ER/Golgi interface. In conclusion, IL2RG p.(Pro58Ser) causes X-CID. Failure of IL2RG plasma membrane targeting may lead to atypical X-SCID. We further identified another carrier of this mutation from newborn SCID screening, lost to closer scrutiny. Electronic supplementary material The online version of this article (10.1007/s10875-020-00745-2) contains supplementary material, which is available to authorized users.

Background The telomere biology disorders (TBDs) include a range of multisystem diseases characterized by mucocutaneous symptoms and bone marrow failure. In dyskeratosis congenita (DKC), the clinical features of TBDs stem from the depletion of crucial stem cell populations in highly proliferative tissues, resulting from abnormal telomerase function. Due to the wide spectrum of clinical presentations and lack of a conclusive laboratory test it may be challenging to reach a clinical diagnosis, especially if patients lack the pathognomonic clinical features of TBDs. Methods Clinical sequencing was performed on a cohort of patients presenting with variable immune phenotypes lacking molecular diagnoses. Hypothesis-free whole-exome sequencing (WES) was selected in the absence of compelling diagnostic hints in patients with variable immunological and haematological conditions. Results In four patients belonging to three families, we have detected five novel variants in known TBD-causing genes (DKC1, TERT and RTEL1). In addition to the molecular findings, they all presented shortened blood cell telomeres. These findings are consistent with the displayed TBD phenotypes, addressing towards the molecular diagnosis and subsequent clinical follow-up of the patients. Conclusions Our results strongly support the utility of WES-based approaches for routine genetic diagnostics of TBD patients with heterogeneous or atypical clinical presentation who otherwise might remain undiagnosed. Electronic supplementary material The online version of this article (10.1186/s13023-018-0864-9) contains supplementary material, which is available to authorized users.

With Mars colonisation becoming a reality for the near future, it is of importance to analyse how crew and cargo can be transported between Earth and a colony on Mars. This article is a feasibility and design study of a launch vehicle whose mission is to shuttle crew and cargo from Low Mars Orbit to a colony on the Martian surface. A single-stage reusable rocket has been selected to fulfil this mission, code-named Charon. The mission profile of such a vehicle was created, leading to a Maximum Growth Allowance (MGA) Delta-V budget of 6.2 km/s. With the mission profile in mind, each subsystem underwent a preliminary design. With reliability and maintainability in mind, subsystems were designed for redundancy and modularity, and an abort system was included for an added level of safety. The iterative design process resulted in a vehicle with a MGA mass of 198.7 tons, capable of transporting 1200 kg of cargo and a crew of 6 people to a 500 km orbit and back. The preliminary design of the vehicle is deemed safe. Following a fault tree analysis, the Single Launch Loss of Mission, Vehicle and Crew (SL-LOM, SL-LOV, SL-LOC) probabilities are computed to be of 0.975%, 0.12%, and 0.079%. Finally, from the vehicle’s constraints on the base, the feasibility of the project has been reflected upon. It is deemed that such a concept is of high interest only when the base is already operational, due to the launch and maintenance infrastructure that it requires, as well as the power it requires from the Martian base.

Antibody class-switch recombination and somatic hypermutation critically depend on the function of activation-induced cytidine deaminase (AID). Rare variants in its gene AICDA have been reported to cause autosomal recessive AID deficiency (autosomal recessive hyper-IgM syndrome type 2 (HIGM2)). Exome sequencing of a multicase Finnish family with an HIGM2 phenotype identified a rare, homozygous, variant (c.416T > C, p.(Met139Thr)) in the AICDA gene, found to be significantly enriched in the Finnish population compared with other populations of European origin (38.56-fold, P

ObjectivesTNFAIP3encodes A20 that negatively regulates nuclear factor kappa light chain enhancer of activated B cells (NF-κB), the major transcription factor coordinating inflammatory gene expression.TNFAIP3polymorphisms have been linked with a spectrum of inflammatory and autoimmune diseases and recently, loss-of-function mutations in A20 were found to cause a novel inflammatory disease ‘haploinsufficiency of A20’ (HA20). Here we describe a family with HA20 caused by a novelTNFAIP3loss-of-function mutation and elucidate the upstream molecular mechanisms linking HA20 to dysregulation of NF-κB and the related inflammasome pathway.MethodsNF-κB activation was studied in a mutation-expressing cell line using luciferase reporter assay. Physical and close-proximity protein-protein interactions of wild-type andTNFAIP3p.(Lys91*) mutant A20 were analyzed using mass spectrometry. NF-κB –dependent transcription, cytokine secretion, and inflammasome activation were compared in immune cells of the HA20 patients and control subjects.ResultsThe protein-protein interactome of p.(Lys91*) mutant A20 was severely impaired, including inter-actions with proteins regulating NF-κB activation, DNA repair responses, and the NLR family pyrin domain containing 3 (NLRP3) inflammasome. The p.(Lys91*) mutant A20 failed to suppress NF-κB signaling, which led to increased NF-κB –dependent proinflammatory cytokine transcription. Functional experiments in the HA20 patients’ immune cells uncovered a novel caspase-8-dependent mechanism of NLRP3 inflammasome hyperresponsiveness that mediated the excessive secretion of interleukin-1β and -18.ConclusionsThe current findings significantly deepen our understanding of the molecular mechanisms underlying HA20 and other diseases associated with reduced A20 expression or function, paving the way for future therapeutic targeting of the pathway.

Background: The nuclear factor kappa light-chain enhancer of activated B cells (NF-kappa B) signaling pathway is a key regulator of immune responses. Accordingly, mutations in several NF-kappa B pathway genes cause immunodeficiency. Objective: We sought to identify the cause of disease in 3 unrelated Finnish kindreds with variable symptoms of immunodeficiency and autoinflammation. Methods: We applied genetic linkage analysis and next-generation sequencing and functional analyses of NFKB1 and its mutated alleles. Results: In all affected subjects we detected novel heterozygous variants in NFKB1, encoding for p50/p105. Symptoms in variant carriers differed depending on the mutation. Patients harboring a p.I553M variant presented with antibody deficiency, infection susceptibility, and multiorgan autoimmunity. Patients with a p.H67R substitution had antibody deficiency and experienced autoinflammatory episodes, including aphthae, gastrointestinal disease, febrile attacks, and small-vessel vasculitis characteristic of Behc, et disease. Patients with a p.R157X stop-gain experienced hyperinflammatory responses to surgery and showed enhanced inflammasome activation. In functional analyses the p.R157X variant caused proteasome-dependent degradation of both the truncated and wild-type proteins, leading to a dramatic loss of p50/p105. The p.H67R variant reduced nuclear entry of p50 and showed decreased transcriptional activity in luciferase reporter assays. The p.I553M mutation in turn showed no change in p50 function but exhibited reduced p105 phosphorylation and stability. Affinity purification mass spectrometry also demonstrated that both missense variants led to altered protein-protein interactions. Conclusion: Our findings broaden the scope of phenotypes caused by mutations in NFKB1 and suggest that a subset of autoinflammatory diseases, such as Behcet disease, can be caused by rare monogenic variants in genes of the NF-kappa B pathway.

Objective: the aim of this work was to evaluate the possible different impacts of genetic and environmental factors in childhood deafness in northern Cameroon. GJB2 mutations are responsible for more than half of all cases of prelingual nonsyndromic recessive deafness in Caucasians, representing the most important deafness-causing factor in the industrialized world. Other genes such as MTRNR1 are also involved. In sub-Saharan Africa, environmental factors seem to dominate genetic contributions, but few studies on the etiology of deafness in Africa are available for comparison. Design: prospective cross sectional study. Study Sample: we performed a molecular screen of the GJB2 and MTRNR1 genes in 70 deaf children and 67 unaffected controls in Maroua (Cameroon) and a literature analysis focused on deafness epidemiology in developing countries. Results: no GJB2 mutations emerged, and only a single MTRNR1 variant that may be pathogenic was found. Conclusion: environmental factors turn out to be more r...

The alpha-synuclein gene (SNCA) multiplication causes autosomal dominant Parkinson Disease (PD): triplication is associated with early-onset rapidly progressing parkinsonism with a strong likelihood of developing dementia, while duplication is associated with a less severe phenotype similar to idiopathic PD. We tested for SNCA multiplication 144 unrelated PD patients with a dominant family history. We identified one patient with SNCA duplication (0.7%). The SNCA-duplicated patient was a woman of 45 years of age with PD onset at 41 years of age. She experienced a rapidly progressive disease with early motor complications (on/off fluctuations and dyskinesias). Medical records confirmed that the proband's mother developed PD at 47 years of age and died at 63 with dementia. She experienced rapid progression in both motor and cognitive symptoms: development of dementia at 54 years of age, 7 years after onset. Although SNCA duplication is an unusual cause of familial PD testing for it is worthwhile. The clinical presentation of duplicated cases may be more aggressive than usual.

The signal transducer and activator of transcription (STAT) family of transcription factors orchestrate hematopoietic cell differentiation. Recently, mutations in STAT1, STAT5B, and STAT3 have been linked to development of immunodysregulation polyendocrinopathy enteropathy X-linked-like syndrome. Here, we immunologically characterized 3 patients with de novo activating mutations in the DNA binding or dimerization domains of STAT3 (p.K392R, p.M394T, and p.K658N, respectively). The patients displayed multiorgan autoimmunity, lymphoproliferation, and delayed-onset mycobacterial disease. Immunologically, we noted hypogammaglobulinemia with terminal B-cell maturation arrest, dendritic cell deficiency, peripheral eosinopenia, increased double-negative (CD4(-)CD8(-)) T cells, and decreased natural killer, T helper 17, and regulatory T-cell numbers. Notably, the patient harboring the K392R mutation developed T-cell large granular lymphocytic leukemia at age 14 years. Our results broaden the spectrum of phenotypes caused by activating STAT3 mutations, highlight the role of STAT3 in the development and differentiation of multiple immune cell lineages, and strengthen the link between the STAT family of transcription factors and autoimmunity.

The Grb10-Interacting GYF Protein-2 (GIGYF2) gene has been proposed as the Parkinson-disease (PD) gene underlying the PARK11 locus. However, association of GIGYF2 with PD has been challenged and a functional validation of GIGYF2 mutations is lacking. In this frame, we performed a mutational screening of GIGYF2 in an Italian PD cohort. Exons containing known mutations were analyzed in 552 cases and 552 controls. Thereafter, a subset of 184 familial PD cases and controls were subjected to a full coding-exon screening. These analyses identified 8 missense variations in 9 individuals (4 cases, 5 controls). Furthermore, we developed a zebrafish model of gigyf2 deficiency. Abrogation of gigyf2 function in zebrafish embryos did not lead to a drastic cell loss in diencephalic dopaminergic (DA) neuron clusters, suggesting that gigyf2 is not required for DA neuron differentiation. Notably, gigyf2 functional abrogation did not increase diencephalic DA neurons susceptibility to the PD-inducing drug MPP+. These data, together with those recently reported by other groups, suggest that GIGYF2 is unlikely to be the PARK11 gene.

Mutations in the GJB2 gene, which encodes the gap-junction protein connexin 26, are the most common cause of nonsyndromic hearing loss (NSHL) and account for about 32% of cases. We analyzed 734 patients and identified mutations in 474/1468 chromosomes. Thirty-six different mutations and five polymorphisms were found in 269 NSHL subjects. Our data confirm 35delG as the most frequent GJB2 mutation in the Italian population, accounting for about 68% of all the mutated GJB2 alleles analyzed. We also identified two novel variants: the V156I mutation and the C>A change at nucleotide 684 in the 3'UTR of the gene. The GJB6 gene deletion, del(GJB6-D13S1830), which can cause HL in combination with GJB2 mutations in trans, was identified in three patients, while the del(GJB6-D13S1854) was not observed in our cohort of patients. We collected audiometric data from 200 patients with biallelic DFNB1 mutations or with dominant mutation in GJB2 to determine the degree of HL to correlate the genotypes with the audiological phenotypes.

Objectives: Mutations in the GJB2 gene, encoding Connexin 26, can cause nonsyndromic recessive deafness or dominant hearing loss (HL) with or without keratoderma. The objective was to perform a molecular evaluation to establish the inherited pattern of deafness in the sporadic cases afferent to our center. Methods: The subject was a 2-year-old Italian girl with nonsyndromic early onset HL. We performed DNA sequencing of the GJB2 gene and deletion analysis of the GJB6 gene in all family members. Results: Direct sequencing of the gene showed a heterozygous C3G transition at nucleotide 172 resulting in a proline to alanine amino acid substitution at codon 58 (P58A). The analyses indicate that the P58A mutation appeared de novo in the proband with a possible dominant effect. Conclusions: This mutation occurs in the first extracellular domain (EC1), which seems to be very important for connexonconnexon interaction and for the control of voltage gating of the channel. The de novo occurrence of an EC1 mutation in a sporadic case of deafness is consistent with the assumption that P58A can cause dominant HL. Key Words: Connexin 26, dominant deafness, de novo mutation, P58A. Laryngoscope, 117:821–824, 2007

The NFkB signaling pathway is a master regulator of the immune response. Recently, dominant mutations in NFKB2 have shown to cause antibody deficiency with adrenal insufficiency. We investigated two families with antibody deficiency and autoinflammatory features, with onset in teenage years and early adulthood (Fig. 1 and Table 1). In family 1, all patients available to study presented with respiratory tract infections and B cell dysfunction marked by hypogammaglobulinemia, poor antibody response to vaccines, or low switched memory B cell counts. Several experienced recurrent episodes of aphthous mucositis in upper gastrointestinal and genital area, sometimes accompanied by abdominal pain, monoarthritis, or fever with elevated inflammatory markers (peripheral blood leukocytes >10x106 cells/ml, C reactive protein >100 mg/L). A lesional biopsy revealed lymphocytic small cell vasculitis reminiscent of Behcet's disease. In family 2, all patients presented with respiratory tract infections, hypogammaglobulinemia, and poor vaccine responses. Based on the pedigrees, we assumed autosomal dominant inheritance. For family 1, we performed genotyping and linkage analysis coupled with whole genome sequencing to pinpoint the causative variant. Linkage analysis showed a 15.4 Mb haplotype on chr4 segregating with the phenotype in all affected individuals. When focusing our search on novel, heterozygous variants negatively affecting conserved residues, we identified only one variant in this genomic location, localizing to NFKB1 gene (p.H67R). In family 2, we exome sequenced one affected individual and filtered the data for novel heterozygous variants negatively affecting conserved residues. This resulted in 17 variants, out of which the I553M NFKB1 variant was considered the best candidate. Several functional analyses showed that the mutations had a negative impact on NFKB1 function. The H67R variant increased NFKB1 affinity to NEMO and delayed protein entry to nucleus, when assessed by affinity purification mass spectrometry and immunofluorescence microscopy. The mutation also decreased NFKB1 transcriptional activity, when expressed together with an NFkB-responsive luciferase reporter, and showed a neomorphic DNA binding pattern on chromatin immunoprecipitation-sequencing. Furthermore, the cell growth was hampered when the mutant NFKB1 was overexpressed in cultured HEK293 cells. The I553M showed a similar negative effect on cultured cell proliferation. Furthermore, the I553M variant negatively affected serine S907 and S893 phosphorylation, leading to defective posttranslational processing of the full-length NFKB1 upon TNF stimulation. To conclude, we show that hypogammaglobulinemia and Behcet's disease-like autoinflammatory syndrome can be caused by mutations in NFKB1. This suggests that a subset of Behcet's disease cases may be of monogenic origin, and highlights the role of NFkB complex in B cell maturation and innate immune regulation. Table 1. Patient characteristics Patient 1 2 3 4 5 6 7 8 9 10 11 12 Age 55 43# 25 29 40 30 37 7 10 60# 61 32 Sex F M F F F F M F M M F M Mutation status H67R ND H67R H67R H67R H67R H67R H67R H67R ND I553M I553M Immunodeficiency Infections URTI RTI URTI - URTI URTI URTI URTI - RTI RTI RTI Antibody deficiency Hypogammaglobulinemia, SAD ND Hypogammaglobulinemia, SAD - IgG subclass deficiency, SAD Hypogammaglobulinemia, SAD Hypogammaglobulinemia, SAD Hypogammaglobulinemia Hypogammaglobulinemia Hypogammaglobulinemia Hypogammaglobulinemia, SAD SAD Immunoglobulin replacement + - + - - + + - - - + - Immune dysregulation Febrile attacks - + + + - - + + - - - - Complex aphtae Mouth, genitalia ND Mouth, genitalia Mouth, genitalia - Mouth Esophagus Mouth, esophagus - - - - Arthritis Monoarthritis - Monoarthritis Monoarthritis - - - - - - Oligoarthritis - Gut disease Periodic abdominal pain, chronic idiopathic diarrhea ND Periodic abdominal pain, microscopic colitis Periodic abdominal pain - - - Periodic abdominal pain - ND Chronic idiopathic diarrhea Coeliac disease Other - Hyperinflammatory response and death after gallbladder surgery - Iritis, Hyperinflammatory response to tooth excision Kidney tumor - Rudimentary left kidney - - - Astma, hypothyreosis - URTI, upper respiratory tract infections; RTI, respiratory tract infections including recurrent pneumonia; SAD, Specific antibody deficiency; ND, No data; #Death age Figure 1. Family pedigrees. Figure 1. Family pedigrees. Disclosures Mustjoki: Sigrid Juselius Foundation: Research Funding; Signe and Ane Gyllenberg Foundation: Research Funding; the Finnish Cancer Societies: Research Funding; Novartis: Honoraria, Research Funding; Academy of Finland: Research Funding; Finnish Cancer Institute: Research Funding; Pfizer: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding. Saarela:Roche: Honoraria.

BackgroundSignificant efforts have been focused on investigating the contribution of common variants to Parkinson disease (PD) risk. Several independent GWAS and metanalysis studies have shown a genome-wide significant association of single nucleotide polymorphisms (SNPs) in the α-synuclein (SNCA) and microtubule-associated protein tau (MAPT) regions. Here we investigated the role of SNCA and MAPT as PD susceptibility genes in a large Italian population of 904 patients and 891 controls. An evaluation of gene–gene and gene-environment interactions in association with PD was also attempted.MethodsThe SNCA Rep1 microsatellite was genotyped by a fluorescent PCR assay, whereas the SNPlex genotyping system was used to genotype 12 additional markers across the SNCA gene, and 2 SNPs tagging the risk MAPT H1 haplotype.ResultsSingle-marker analysis demonstrated nominal evidence of association for: i) the 261-bp-long allele of Rep1; ii) 7 SNPs in the SNCA region (top SNP: rs356186, P = 3.08 × 10−04, intron 4); iii) both SNPs identifying the MAPT H1 haplotype (P = 4.63 × 10−04 and P = 4.23 × 10−04 for rs1800547 and rs9468, respectively). Moreover, we found a highly significant protective haplotype spanning ∼83 kb from intron 4 to the 3′ end of SNCA (P = 1.29 × 10−05).ConclusionsOur findings strongly confirm SNCA and MAPT as major PD susceptibility genes for idiopathic PD in the Italian population. Interaction analyses did not evidence either epistatic effects between the two loci or gene-environment interactions.

Large granular lymphocyte (LGL) leukemia is a group of rare lymphoproliferative disorders which involve inappropriate clonal expansion of either cytotoxic T-lymphocytes (CTLs) or natural killer cells (NK). The usual age at onset is between 40 to 60 years. We here describe four very young patients showing LGL infiltration in the bone marrow (BM) and all diagnosed with primary immune deficiency. Patient 1 is a 13-year old male who presented with gingivostomatitis, but no severe infections, autoimmune disease or allergies. He had persistent leukopenia (1.9x109/L), neutropenia ( Patient 2 is a 15-year old female who has neonatal diabetes, autoimmune desquamative interstitial pulmonary disease, autoimmune enteropathy, exocrine pancreatic insufficiency and delayed growth. She developed transfusion-dependent, Coombs-negative anemia at age of 15. In the PB, 50% of the lymphocytes were LGLs. BM showed pure red cell aplasia plus LGL infiltration of TCRgd positive T cells (CD3+, CD2+, CD5+, CD8-/+, CD4-/+, CD57+) at 43% of BM cellularity. Clonal TCRg rearrangement was detected. Hypogammaglobulinemia presented at age of 7. T, B and NK cell counts were low normal, but DCs were non-existent. WES and functional studies revealed a germline gain-of-function mutation of STAT3 gene. Patient 3 is 42-year old female who has had severe infections (bacterial, viral and protozoan) starting at the age of 4 months. From age of 9, autoimmune manifestations occurred with immune thrombocytopenia, neutropenia and anemia, and intensively positive rheumatoid factor. Since age of 17, CD8+ LGLs were detected in PB reaching level of 50% of lymphocytes. In the BM, CD3+CD5-CD56- T -lymphocyte infiltration with clonal TCRd rearrangement was detected. In her 30ies she developed pulmonal arterial hypertension and hypogammaglobulinemia (0.8 g/L). She has low counts of B cells (0.02x109/L), and monocytoid (1.9x106/L) and plasmacytoid (0) DCs. WES identified a homozygous germline missense mutation in CECR1 gene, leading to total loss-of-function of the encoded adenosine deaminase 2 (ADA2) protein. Patient 4 presented with waxing and waning skin nodules from age of 10 years, but no severe infections or allergies. At age of 26, she was diagnosed with hemolytic anemia with spherocytosis, splenomegaly and cholelithiasis. Five years later remarkable hepatomegaly, and clinical autoimmune manifestations with SLE-type skin lesions, autoimmune hemolytic anemia and iritis were observed. Complement C3 (0.17 g/l) and C4 ( We conclude that LGL lymphoproliferation in young patients is often a sign of underlying primary immunodeficiency, highlighting the need for detailed genetic studies in these cases. Table 1. Patient Age at LGL LGL in PB(109/L) LGL immune-phenotype Clonality BM LGL infiltration Clinical PID 1 13 0.38 NK NA 40% N,THepatomegaly GATA2 deficiency 2 15 1.79 CTL TCRg 43% AutoimmunityAHepatosplenomegaly STAT3 hyperactivation 3 17 1,49 CTL TCRd NA N,T, AAutoimmunityPulmonary arterial hypertension ADA2 deficiency 4 31 NA CTL TCRg weak 40% AutoimmunityHepatosplenomegalyHepatopulmonal syndrome ADA2 deficiency A, anemia; N, neutropenia, CTL, cytotoxic T cell; LGL, large granular lymphocyte; NK, natural killer cell; PID, primary immune deficiency; TCR, T cell receptor; T, thrombocytopenia Disclosures Mustjoki: Signe and Ane Gyllenberg Foundation: Research Funding; Finnish Cancer Institute: Research Funding; Sigrid Juselius Foundation: Research Funding; Academy of Finland: Research Funding; the Finnish Cancer Societies: Research Funding; Pfizer: Honoraria, Research Funding; Bristol-Myers Squibb: Honoraria, Research Funding; Novartis: Honoraria, Research Funding. Siitonen:Pzizer: Other: charges of EAHAD congress 2015; Novartis: Other: charges of EHA congress 2015. Saarela:Roche: Honoraria.