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.

A longitudinal prospective study of active tuberculosis in a Western Europe setting: insights and findings.

PURPOSE: This study investigates the potential of inflammatory parameters (IP), symptoms, and patient-related outcome measurements as biomarkers of severity and their ability to predict tuberculosis (TB) evolution. METHODS: People with TB were included prospectively in the Stage-TB study conducted at five clinical sites in Barcelona (Spain) between April 2018 and December 2021. Data on demographics, epidemiology, clinical features, microbiology, and Sanit George Respiratory Questionnaire (SGRQ) and Kessler-10 as Health-Related Quality of Life (HRQoL) were collected at three time points during treatment. C-reactive protein (CRP), erythrocyte sedimentation rate (ESR), neutrophil/lymphocyte, and monocyte/lymphocyte ratios (NLR and MLR), complement factors C3, C4, and cH50, clinical and microbiological data, and HRQoL questionnaires were assessed at baseline, 2 months, and 6 months. Their ability to predict sputum culture conversion (SCC) and symptom presence after 2 months of treatment was also analysed. RESULTS: The study included 81 adults and 13 children with TB. The CRP, ESR, NLR, and MLR values, as well as the presence of symptoms, decreased significantly over time in both groups. Higher IP levels at baseline were associated with greater bacillary load and persistent symptoms. Clinical severity at baseline predicted a delayed SCC. Kessler-10 improved during follow-up, but self-reported lung impairment (SGRQ) persisted in all individuals after 6 months. CONCLUSIONS: IP levels may indicate disease severity, and sustained high levels are linked to lower treatment efficacy. Baseline clinical severity is the best predictor of SCC. Implementing health strategies to evaluate lung function and mental health throughout the disease process may be crucial for individuals with TB.

Molecular Challenges in the Diagnosis of X-Linked Chronic Granulomatous Disease: CNVs, Intronic Variants, Skewed X-Chromosome Inactivation, and Gonosomal Mosaicism.

Chronic granulomatous disease (CGD) is a prototypical inborn error of immunity affecting phagocytes, in which these cells are unable to produce reactive oxygen species. CGD is caused by defects in genes encoding subunits of the NADPH oxidase enzyme complex (CYBA, CYBB, CYBC1, NCF1, NCF2, NCF4); inflammatory responses are dysregulated, and patients are highly susceptible to recurrent severe bacterial and fungal infections. X-linked CGD (XL-CGD), caused by mutations in the CYBB gene, is the most common and severe form of CGD. In this study, we describe the analytical processes undertaken in 3 families affected with XL-CGD to illustrate several molecular challenges in the genetic diagnosis of this condition: in family 1, a girl with a heterozygous deletion of CYBB exon 13 and skewed X-chromosome inactivation (XCI); in family 2, a boy with a hemizygous deletion of CYBB exon 7, defining its consequences at the mRNA level; and in family 3, 2 boys with the same novel intronic variant in CYBB (c.1151 + 6 T > A). The variant affected the splicing process, although a small fraction of wild-type mRNA was produced. Their mother was a heterozygous carrier, while their maternal grandmother was a carrier in form of gonosomal mosaicism. In summary, using a variety of techniques, including an NGS-based targeted gene panel and deep amplicon sequencing, copy number variation calling strategies, microarray-based comparative genomic hybridization, and cDNA analysis to define splicing defects and skewed XCI, we show how to face and solve some uncommon genetic mechanisms in the diagnosis of XL-CGD.

Performance of QuantiFERON-TB Gold Plus assays in paediatric tuberculosis: a multicentre PTBNET study.

RATIONALE: In 2016, a new interferon-gamma release assay (IGRA) was introduced, QuantiFERON-TB Gold Plus (QFT-Plus), claimed to have improved sensitivity in active tuberculosis (TB). OBJECTIVES: This study aimed to determine the performance of QFT-Plus, compared with previous generation IGRAs and the tuberculin skin test (TST), in children with TB in Europe. METHODS: Multicentre, ambispective cohort study within the Paediatric Tuberculosis Network European Trials Group (ptbnet), a dedicated paediatric TB research network comprising >300 members, capturing TB cases <18 years-of-age diagnosed between January 2009 and December 2019. MEASUREMENTS AND MAIN RESULTS: 1001 TB cases from 16 countries were included (mean age (IQR) 5.6 (2.4-12.1) years). QFT-Plus was performed in 358, QFT Gold in-Tube (QFT-GIT) in 600, T-SPOT.TB in 58 and TST in 636 cases. The overall test sensitivities were: QFT-Plus 83.8% (95% CI 80.2% to 87.8%), QFT-GIT 85.5% (95% CI 82.7% to 88.3%), T-SPOT.TB 77.6% (95% CI 66.9% to 88.3%) and TST (cut-off ≥10 mm) 83.3% (95% CI 83.3% to 86.2%). There was a trend for tests to have lower sensitivity in patients with miliary and/or central nervous system (CNS) TB (73.1%, 70.9%, 63.6% and 43.5%, respectively), and in immunocompromised patients (75.0%, 59.6%, 45.5% and 59.1%, respectively). CONCLUSIONS: The results indicate that the latest generation IGRA assay, QFT-Plus, does not perform better than previous generation IGRAs or the TST in children with TB disease. Overall, tests performed worse in CNS and miliary TB, and in immunocompromised children. None of the tests evaluated had sufficiently high sensitivity to be used as a rule-out test in children with suspected TB.

Activation-induced deaminase is critical for the establishment of DNA methylation patterns prior to the germinal center reaction.

Activation-induced deaminase (AID) initiates antibody diversification in germinal center B cells by deaminating cytosines, leading to somatic hypermutation and class-switch recombination. Loss-of-function mutations in AID lead to hyper-IgM syndrome type 2 (HIGM2), a rare human primary antibody deficiency. AID-mediated deamination has been proposed as leading to active demethylation of 5-methycytosines in the DNA, although evidence both supports and casts doubt on such a role. In this study, using whole-genome bisulfite sequencing of HIGM2 B cells, we investigated direct AID involvement in active DNA demethylation. HIGM2 naïve and memory B cells both display widespread DNA methylation alterations, of which ∼25% are attributable to active DNA demethylation. For genes that undergo active demethylation that is impaired in HIGM2 individuals, our analysis indicates that AID is not directly involved. We demonstrate that the widespread alterations in the DNA methylation and expression profiles of HIGM2 naïve B cells result from premature overstimulation of the B-cell receptor prior to the germinal center reaction. Our data support a role for AID in B cell central tolerance in preventing the expansion of autoreactive cell clones, affecting the correct establishment of DNA methylation patterns.

Impact of JAK Inhibitors in Pediatric Patients with STAT1 Gain of Function (GOF) Mutations-10 Children and Review of the Literature.

INTRODUCTION: Since the first description of gain of function (GOF) mutations in signal transducer and activator of transcription (STAT) 1, more than 300 patients have been described with a broad clinical phenotype including infections and severe immune dysregulation. Whilst Jak inhibitors (JAKinibs) have demonstrated benefits in several reported cases, their indications, dosing, and monitoring remain to be established. METHODS: A retrospective, multicenter study recruiting pediatric patients with STAT1 GOF under JAKinib treatment was performed and, when applicable, compared with the available reports from the literature. RESULTS: Ten children (median age 8.5 years (3-18), receiving JAKinibs (ruxolitinib (n = 9) and baricitinib (n = 1)) with a median follow-up of 18 months (2-42) from 6 inborn errors of immunity (IEI) reference centers were included. Clinical profile and JAKinib indications in our series were similar to the previously published 14 pediatric patients. 9/10 (our cohort) and 14/14 patients (previous reports) showed partial or complete responses. The median immune deficiency and dysregulation activity scores were 15.99 (5.2-40) pre and 7.55 (3-14.1) under therapy (p = 0.0078). Infection, considered a likely adverse event of JAKinib therapy, was observed in 1/10 patients; JAKinibs were stopped in 3/10 children, due to hepatotoxicity, pre-HSCT, and absence of response. CONCLUSIONS: Our study supports the potentially beneficial use of JAKinibs in patients with STAT1 GOF, in line with previously published data. However, consensus regarding their indications and timing, dosing, treatment duration, and monitoring, as well as defining biomarkers to monitor clinical and immunological responses, remains to be determined, in form of international prospective multicenter studies using established IEI registries.

Accuracy of Xpert Ultra for the diagnosis of paediatric tuberculosis in a low TB burden country: a prospective multicentre study.

INTRODUCTION: Childhood pulmonary tuberculosis (TB) remains a diagnostic challenge. This study aimed to evaluate the performance of Xpert Ultra for the diagnosis of pulmonary TB in children in a low TB prevalence setting. METHODS: Prospective, multicentre, diagnostic accuracy study. Children with clinical or radiological suspicion of pulmonary TB were recruited at 11 paediatric units in Spain. Up to three gastric or sputum specimens were taken on 3 consecutive days, and analysed by Xpert MTB/RIF, Xpert Ultra and culture in parallel. RESULTS: 86 children were included (median age 4.9 years, IQR 2.0-10.0; 51.2% male). The final diagnosis was pulmonary TB in 75 patients (87.2%); 33 (44.0%) were microbiologically confirmed. A total of 219 specimens, comprising gastric aspirates (n=194; 88.6%) and sputum specimens (n=25; 11.4%), were analysed. Using culture as reference standard and comparing individual specimens, the sensitivity was 37.8% (14/37) for Xpert MTB/RIF and 81.1% (30/37) for Xpert Ultra (p<0.001); specificity was 98.4% (179/182) and 93.4% (170/182), respectively (p=0.02). In the per-patient analysis, considering positive results on any specimen, the sensitivity was 42.9% (9/21) for Xpert MTB/RIF and 81.0% for Xpert Ultra (17/21, p=0.01); specificity was 96.9% (63/65) and 87.7% (57/65, p=0.07), respectively. CONCLUSIONS: In children with pulmonary TB in a low burden setting, Xpert Ultra has significantly higher sensitivity than the previous generation of Xpert assay and only marginally lower specificity. Therefore, in children undergoing evaluation for suspected pulmonary TB, Xpert Ultra should be used in preference to Xpert MTB/RIF whenever possible.

Seletalisib for Activated PI3Kδ Syndromes: Open-Label Phase 1b and Extension Studies.

Mutations in two genes can result in activated PI3Kδ syndrome (APDS), a rare immunodeficiency disease with limited therapeutic options. Seletalisib, a potent, selective PI3Kδ inhibitor, was evaluated in patients with APDS1 and APDS2. In the phase 1b study (European Clinical Trials Database 2015-002900-10) patients with genetic and clinical confirmation of APDS1 or APDS2 received 15-25 mg/d seletalisib for 12 wk. Patients could enter an extension study (European Clinical Trials Database 2015-005541). Primary endpoints were safety and tolerability, with exploratory efficacy and immunology endpoints. Seven patients (median age 15 years; APDS1 n = 3; APDS2 n = 4) received seletalisib; five completed the phase 1b study. For the extension study, four patients entered, one withdrew consent (week 24), three completed ≥84 wk of treatment. In the phase 1b study, patients had improved peripheral lymphadenopathy (n = 2), lung function (n = 1), thrombocyte counts (n = 1), and chronic enteropathy (n = 1). Overall, effects were maintained in the extension. In the phase 1b study, percentages of transitional B cells decreased, naive B cells increased, and senescent CD8 T cells decreased (human cells); effects were generally maintained in the extension. Seletalisib-related adverse events occurred in four of seven patients (phase 1b study: hepatic enzyme increased, dizziness, aphthous ulcer, arthralgia, arthritis, increased appetite, increased weight, restlessness, tendon disorder, and potential drug-induced liver injury) and one of four patients had adverse events in the extension (aphthous ulcer). Serious adverse events occurred in three of seven patients (phase 1b study: hospitalization, colitis, and potential drug-induced liver injury) and one of four patients had adverse events in the extension (stomatitis). Patients with APDS receiving seletalisib had improvements in variable clinical and immunological features, and a favorable risk-benefit profile was maintained for ≤96 wk.

Impact of cytomegalovirus infection prior to hematopoietic stem cell transplantation in children with inborn errors of immunity.

The presence of active viral infections has an impact on the prognosis of patients undergoing hematopoietic stem cell transplantation (HSCT). Nevertheless, the number of reports of cytomegalovirus infection in patients with inborn errors of immunity (IEI) who undergo HSCT is relatively low. To analyze the effect of cytomegalovirus infection acquired prior to curative treatment on patient survival in 123 children with IEI. An observational and retrospective study was performed with patients younger than 18 years diagnosed with IEI who were candidates for HSCT, gene therapy, or thymus transplantation at five hospitals in Spain between 2008 and 2019. We included 123 children, 25 infected by cytomegalovirus prior to undergoing curative treatment (20.3%). At IEI diagnosis, 24 of the patients were already infected, 21 of whom had symptomatic cytomegalovirus disease (87%), while the other three patients developed disease before undergoing curative treatment. The patients with cytomegalovirus infection had higher mortality than those without (p = 0.006). Fourteen patients developed refractory cytomegalovirus infection (56%), all of whom died, while no patients with non-refractory infection died (p = 0.001) All deaths that occurred before curative treatment and three of the five after the treatment were attributed to cytomegalovirus. Patients with refractory cytomegalovirus disease had the highest pre-HSCT mortality rate (64.3%), compared with the non-infected children and those with non-refractory cytomegalovirus disease (10.1%) (p < 0.0001). CONCLUSION: Prevention and prompt control of cytomegalovirus infection, together with early HSCT/gene therapy, are crucial for improving the prognosis in children with IEI. WHAT IS KNOWN: • Cytomegalovirus is the most frequent viral infection in children with inborn errors of immunity who are candidates to hematopoietic stem cell transplantation (HSCT). • Active viral infections at the time of HSCT lead to worse prognosis. WHAT IS NEW: • In children with inborn errors of immunity and indication of HSCT, refractory cytomegalovirus disease is associated with a very high mortality rate, compared with non-infected children and those with non-refractory cytomegalovirus disease. • In patients with novel transplantation indications, the presence and treatment response of CMV infection should be considered to decide the best possible moment for HSCT.

Atypical Inflammatory Syndrome Triggered by SARS-CoV-2 in Infants with Down Syndrome.

While adults with Down syndrome (DS) are at increased risk of severe COVID-19 pneumonia, little is known about COVID-19 in children with DS. In children without DS, SARS-CoV-2 can rarely cause severe COVID-19 pneumonia, or an even rarer and more typically pediatric condition, multisystem inflammatory syndrome in children (MIS-C). Although the underlying mechanisms are still unknown, MIS-C is thought to be primarily immune-mediated. Here, we describe an atypical, severe form of MIS-C in two infant girls with DS who were hospitalized for over 4 months. Immunological evaluation revealed pronounced neutrophilia, B cell depletion, increased circulating IL-6 and IL-8, and elevated markers of immune activation ICAM1 and FcÉ£RI. Importantly, uninfected children with DS presented with similar but less stark immune features at steady state, possibly explaining risk of further uncontrolled inflammation following SARS-CoV-2 infection. Overall, a severe, atypical form of MIS-C may occur in children with DS.

Health-Related Quality of Life and Multidimensional Fatigue Scale in Children with Primary Immunodeficiencies.

PURPOSE: Patients with primary immunodeficiency disease (PID) have an increased risk of experiencing physical activity limitations, social difficulties, and psychological problems due to their chronic condition. Evaluation of their health-related quality of life (HRQOL) and fatigue is crucial in these patients to help understand their complex disease and provide adequate medical care. METHODS: In this study, we evaluated HRQOL and fatigue in pediatric and young adult patients with PID attending our center. Participants completed the Pediatric Quality of Life Inventory (PedsQL), version 4.0, and the PedsQL multidimensional fatigue module, standard version. RESULTS: Fifty-three PID patients were recruited (age range: 2-23 years). The mean HRQOL score obtained was 66.61 (SD: 18.73) out of 100, and the emotional and work/school dimensions were the ones most highly affected. There were no significant differences in reported quality of life between patients and their caregivers. The mean patient-reported fatigue value was 68.81 (SD: 17.80) out of 100, and the rest-related dimension was the one most highly affected. In the caregivers' assessment, general fatigue was the most highly affected dimension. CONCLUSIONS: The results of this study show that quality of life is poor and fatigue measures are considerably increased in our young adult and pediatric patients with PIDs. These findings can indicate areas requiring more intensive interventions, and they will serve as a basis for comparison of future results.

Severe Autoinflammatory Manifestations and Antibody Deficiency Due to Novel Hypermorphic PLCG2 Mutations.

Autoinflammatory diseases (AIDs) were first described as clinical disorders characterized by recurrent episodes of seemingly unprovoked sterile inflammation. In the past few years, the identification of novel AIDs expanded their phenotypes toward more complex clinical pictures associating vasculopathy, autoimmunity, or immunodeficiency. Herein, we describe two unrelated patients suffering since the neonatal period from a complex disease mainly characterized by severe sterile inflammation, recurrent bacterial infections, and marked humoral immunodeficiency. Whole-exome sequencing detected a novel, de novo heterozygous PLCG2 variant in each patient (p.Ala708Pro and p.Leu845_Leu848del). A clear enhanced PLCγ2 activity for both variants was demonstrated by both ex vivo calcium responses of the patient's B cells to IgM stimulation and in vitro assessment of PLC activity. These data supported the autoinflammation and PLCγ2-associated antibody deficiency and immune dysregulation (APLAID) diagnosis in both patients. Immunological evaluation revealed a severe decrease of immunoglobulins and B cells, especially class-switched memory B cells, with normal T and NK cell counts. Analysis of bone marrow of one patient revealed a reduced immature B cell fraction compared with controls. Additional investigations showed that both PLCG2 variants activate the NLRP3-inflammasome through the alternative pathway instead of the canonical pathway. Collectively, the evidences here shown expand APLAID diversity toward more severe phenotypes than previously reported including dominantly inherited agammaglobulinemia, add novel data about its genetic basis, and implicate the alternative NLRP3-inflammasome activation pathway in the basis of sterile inflammation.

Diagnostic Accuracy of QuantiFERON-TB Gold Plus Assays in Children and Adolescents with Tuberculosis Disease.

In 2016, a new interferon-gamma release assay, QuantiFERON-TB Gold Plus, was introduced. We conducted a cross-sectional multicenter study, involving 158 children and adolescents with tuberculosis disease. The overall sensitivity of the assay was 82.9% (IQR 77.0%-88.8%), indicating that in children this test does not have higher sensitivity than previous generation interferon-gamma release assays.

Epidemiology of congenital Chagas disease 6 years after implementation of a public health surveillance system, Catalonia, 2010 to 2015.

BackgroundChagas disease is endemic in Latin America and affects 8 million people worldwide. In 2010, Catalonia introduced systematic public health surveillance to detect and treat congenital Chagas disease.AimThe objective was to evaluate the health outcomes of the congenital Chagas disease screening programme during the first 6 years (2010-2015) after its introduction in Catalonia.MethodsIn a surveillance system, we screened pregnant women and newborns and other children of positive mothers, and treated Chagas-positive newborns and children. Diagnosis was confirmed for pregnant women and children with two positive serological tests and for newborns with microhaematocrit and/or PCR at birth or serology at age 9 months.ResultsFrom 2010 to 2015, the estimated screening coverage rate increased from 68.4% to 88.6%. In this period, 33,469 pregnant women were tested for Trypanosoma cruzi and 937 positive cases were diagnosed. The overall prevalence was 2.8 cases per 100 pregnancies per year (15.8 in Bolivian women). We followed 82.8% of newborns until serological testing at age 9-12 months and 28 were diagnosed with Chagas disease (congenital transmission rate: 4.17%). Of 518 siblings, 178 (34.3%) were tested and 14 (7.8%) were positive for T. cruzi. Having other children with Chagas disease and the heart clinical form of Chagas disease were maternal risk factors associated with congenital T. cruzi infection (p < 0.05).ConclusionThe increased screening coverage rate indicates consolidation of the programme in Catalonia. The rate of Chagas disease congenital transmission in Catalonia is in accordance with the range in non-endemic countries.

Laboratory evaluation of the IFN-γ circuit for the molecular diagnosis of Mendelian susceptibility to mycobacterial disease.

The integrity of the interferon (IFN)-γ circuit is necessary to mount an effective immune response to intra-macrophagic pathogens, especially Mycobacteria. Inherited monogenic defects in this circuit that disrupt the production of, or response to, IFN-γ underlie a primary immunodeficiency known as Mendelian susceptibility to mycobacterial disease (MSMD). Otherwise healthy patients display a selective susceptibility to clinical disease caused by poorly virulent mycobacteria such as BCG (bacille Calmette-Guérin) vaccines and environmental mycobacteria, and more rarely by other intra-macrophagic pathogens, particularly Salmonella and M. tuberculosis. There is high genetic and allelic heterogeneity, with 19 genetic etiologies due to mutations in 10 genes that account for only about half of the patients reported. An efficient laboratory diagnostic approach to suspected MSMD patients is important, because it enables the establishment of specific therapeutic measures that will improve the patient's prognosis and quality of life. Moreover, it is essential to offer genetic counseling to affected families. Herein, we review the various genetic and immunological diagnostic approaches that can be used in concert to reach a molecular and cellular diagnosis in patients with MSMD.

TNFAIP3 haploinsufficiency is the cause of autoinflammatory manifestations in a patient with a deletion of 13Mb on chromosome 6.

There is scarce literature about autoinflammation in syndromic patients. We describe a patient who, in addition to psychomotor and growth delay, presented with fevers, neutrophilic dermatosis, and recurrent orogenital ulcers. Comparative Genomic Hybridization (CGH) array permitted to identify a 13.13Mb deletion on chromosome 6, encompassing 53 genes, and including TNFAIP3 gene (A20). A20 is a potent inhibitor of the NF-kB signalling pathway and restricts inflammation via its deubiquitinase activity. Western blotting and immunoprecipitation assays showed decreased A20 expression and increased phosphorylation of p65 and IkBa. Patient's cells displayed increased levels of total K63-linked ubiquitin and increased levels of ubiquitinated RIP and NEMO after stimulation with TNF. We describe the molecular characterization of an autoinflammatory disease due to a large chromosomal deletion and review the phenotypes of patients with A20 haploinsufficiency. CGH arrays should be the first diagnostic method for comprehensive analysis of patients with syndromic features and immune dysregulation.

Th1-skewed profile and excessive production of proinflammatory cytokines in a NFKB1-deficient patient with CVID and severe gastrointestinal manifestations.

Monoallelic loss-of-function mutations in NFKB1 were recently recognized as the most common monogenic cause of common variable immunodeficiency (CVID). The prototypic clinical phenotype of NFKB1-deficient patients includes common CVID features, such as hypogammaglobulinaemia and sinopulmonary infections, plus other highly variable individual manifestations. Here, we describe a patient with a profound CVID phenotype and severe gastrointestinal manifestations, including chronic and recurrent diarrhoea. Using an NGS customized panel of 323 genes related to primary immunodeficiencies, we identified a novel monoallelic loss-of-function mutation in NFKB1 leading to a truncated protein (c.1149delT/p.Gly384Glu ∗ 48). Interestingly, we also found a rare variant in NOD2 previously associated with Crohn's disease (p.His352Arg). Our patient had hypogammaglobulinaemia with a small number of B cells, most of which were naïve. The most noteworthy findings included marked skewing towards a Th1 phenotype in peripheral blood T cells and excessive production of proinflammatory cytokines (IL-1ß, TNFα). The patient's 6-year-old daughter, a carrier of the NFKB1 mutation, is clinically asymptomatic, but has started to show cellular and molecular changes. This case of NFKB1 deficiency appears to be a combination of immunodeficiency and a hyperinflammatory state. The current situation of the patient's daughter provides a glimpse of the preclinical phase of the condition.

Age-specific pediatric reference ranges for immunoglobulins and complement proteins on the Optilite™ automated turbidimetric analyzer.

BACKGROUND: Measurement of immunoglobulins and complement proteins are frontline tests used in the assessment of immune system integrity, and reference values can vary with age. Their measurement provides an insight into the function of the innate and adaptive immune systems. METHODS: We generated pediatric reference ranges for IgG, IgA, IgM, IgD, the IgG and IgA subclasses, and C3 and C4 using the Optilite™ turbidimetric analyzer. RESULTS: The concentrations of IgG, IgA, and IgD showed an increase with age, as expected, while IgM remained stable between the age groups. For the IgG subclasses, no significant differences were observed in IgG1 or IgG3, while IgG2 and IgG4 concentrations increased steadily with age. The concentration of IgG2 plateaued at 15-18 years, while IgG4 plateaued at 10-14 years. The trend of concentrations across all groups was IgG1 > IgG2 > IgG3 > IgG4. For both IgA1 and IgA2, concentrations increased significantly with age, plateauing at 15-18 years. The median IgA1 concentration was greater than IgA2 across all groups. There was a good correlation between the total IgG or IgA concentration and summation of their subclasses (R2  = 0.89, P < .0001, slope y = 0.98x + 14.51 mg/dL and R2  = 0.91, P < .0001, slope y = 1.35x-3.28 mg/dL, respectively). The concentration of C3 and C4 remained stable across the groups, with no significant differences observed. CONCLUSION: We have generated age-specific reference ranges in healthy children for C3, C4, IgG, IgA, IgM, IgD and the IgG and IgA subclasses using the Optilite™ turbidimetric analyzer. These ranges will help identify individuals with abnormal concentrations, thus will aid in the diagnosis of both primary and secondary immunological disorders.

Early Versus Late Diagnosis of Complement Factor I Deficiency: Clinical Consequences Illustrated in Two Families with Novel Homozygous CFI Mutations.

The complement system is an important effector arm of innate immunity and plays a crucial role in the defense against common pathogens. But effective defense and maintenance of homeostasis requires a careful balance between complement activation and regulation. Factor I (FI) is one of the most important regulators of the complement system. Complete FI deficiency is a rare autosomal recessive disorder typically resulting in severe, recurrent infection by encapsulated bacteria. In the present study, we describe two patients from unrelated families with complete FI deficiency diagnosed at very different ages: Patient 1 is a 60-year-old man who had experienced several severe infections (pneumonia, meningitis, sepsis) since childhood, one of which caused significant and permanent neurologic sequelae. In contrast, patient 2 was diagnosed at the age of 4 years after a single infectious episode (otitis media) and through detection of a flat beta2 peak on serum protein electrophoresis. This early diagnosis of FI deficiency enabled prompt implementation of a therapeutic intervention consisting of vaccination with encapsulated bacteria and prophylactic antibiotics. The two patients had novel homozygous mutations in the CFI gene (p.Gly162Asp and p.His380Arg) that disrupted protein function. Interestingly, p.His380Arg is the first mutation described affecting a residue of the highly conserved FI catalytic triad (His380, Asp429, and Ser525). This study illustrates the importance of early versus late diagnosis of FI deficiency and, in general, highlights the clinical relevance of prompt detection of complement system deficiencies.

Clinical and structural impact of mutations affecting the residue Phe367 of FOXP3 in patients with IPEX syndrome.

Immune dysregulation, polyendocrinopathy, enteropathy, X-linked (IPEX) syndrome is a monogenic autoimmune disease characterized by early-onset life-threatening multisystemic autoimmunity. This rare hereditary disorder is caused by loss-of-function mutations in the gene encoding the forkhead box P3 (FOXP3) transcription factor, which plays a key role in the differentiation and function of CD4(+)CD25(+) natural regulatory T cells (Tregs), essential for the establishment and maintenance of natural tolerance. We identified a novel mutation in the FOXP3 gene affecting the Phe367 residue of the protein (F367V) in a family with three male siblings affected by IPEX. Two other mutations affecting the FOXP3 Phe367 residue (F367L and F367C) have been described previously. This unique situation of three mutations affecting the same residue in FOXP3 led us to study the molecular impact of these mutations on the structure of FOXP3 protein. Structure analysis showed that Phe367 is involved in a rich interaction network related to both monomer and dimer structure stabilization, and is crucial for FOXP3 regulatory activity. The relevance of this location is confirmed by the results of SIFT and PolyPhen-2 pathogenicity predictions for F367V mutation. In summary, as assessment of the pathogenicity of a novel mutation is crucial to achieve a proper molecular diagnosis, we analysed the impact of mutations affecting the Phe367 residue using a combined approach that provides a mechanistic view of their pathogenic effect.