Wiskott-Aldrich syndrome: a study of 577 patients defines the genotype as a biomarker for disease severity and survival.

ABSTRACT: Wiskott-Aldrich syndrome (WAS) is a multifaceted monogenic disorder with a broad disease spectrum and variable disease severity and a variety of treatment options including allogeneic hematopoietic stem cell transplantation (HSCT) and gene therapy (GT). No reliable biomarker exists to predict disease course and outcome for individual patients. A total of 577 patients with a WAS variant from 26 countries and a median follow-up of 8.9 years (range, 0.3-71.1), totaling 6118 patient-years, were included in this international retrospective study. Overall survival (OS) of the cohort (censored at HSCT or GT) was 82% (95% confidence interval, 78-87) at age 15 years and 70% (61-80) at 30 years. The type of variant was predictive of outcome: patients with a missense variant in exons 1 or 2 or with the intronic hot spot variant c.559+5G>A (class I variants) had a 15-year OS of 93% (89-98) and a 30-year OS of 91% (86-97), compared with 71% (62-81) and 48% (34-68) in patients with any other variant (class II; P < .0001). The cumulative incidence rates of disease-related complications such as severe bleeding (P = .007), life-threatening infection (P < .0001), and autoimmunity (P = .004) occurred significantly later in patients with a class I variant. The cumulative incidence of malignancy (P = .6) was not different between classes I and II. It confirms the spectrum of disease severity and quantifies the risk for specific disease-related complications. The class of the variant is a biomarker to predict the outcome for patients with WAS.

Sensitization to Aeroallergens in Patients with Primary Immunodeficiencies.

BACKGROUND: Primary immunodeficiencies (PID) are a heterogeneous group of diseases that cause alterations in the immune system, leading to the increased susceptibility to infections, autoimmune diseases, and malignancies. The aim was to evaluate the presence of clinical manifestations and diagnoses of asthma, rhinitis, and atopic dermatitis in patients diagnosed with PID, and correlate these with allergen sensitization verified by skin prick testing (SPT). MATERIAL AND METHODS: This cross-sectional, descriptive, and observational study was conducted from February 2015 to February 2016. We performed a medical report analysis and SPT for aeroallergens in patients with a PID diagnosis. RESULTS: Thirty-one patients with a PID diagnosis were included. The mean age of the participants was 15.41 years. From the 31 patients, 28 had symptoms suggestive of allergic disease (asthma, rhinitis, and atopic dermatitis) and only 7 presented positive SPT for at least 1 aeroallergen. CONCLUSION: The frequency of allergenic sensitization in the group of patients with PID and symptoms suggestive of asthma, rhinitis, or atopic dermatitis is lower than that found in the general population, probably due the impairment of IgE formation secondary to their immunologic alterations.

Adult Food Allergy Prevalence: Reducing Questionnaire Bias.

BACKGROUND: Food allergy (FA) prevalence has increased in the last decades, but epidemiologic studies could show overestimated results. The objective of this study is to estimate the prevalence of immediate FA in adults in a region of Central Brazil, using a questionnaire to try to reduce misperceptions about FA reaction. METHODS: A cross-sectional study was conducted, enrolling an adult population aged 18-65 years comprised of families in a Central Brazilian city. In the first phase, participants answered a self-administered questionnaire for FA screening. In the second phase, the participants who reported an FA in the first questionnaire were visited to complete the second questionnaire applied by trained researchers. RESULTS: Of the 4,916 adults visited, 1,583 returned a completed questionnaire. Reported FA occurred in 171 (10.8%) subjects, and the more frequent citations were cow's milk, pork, fruits, shrimp, and vegetables. One hundred and four of these individuals completed the second questionnaire, and 15 (1.0%) were considered to have an FA diagnosis. The main foods were fruits, followed by cow's milk, shrimp, pork, and vegetables. CONCLUSION: After use of a specific questionnaire to recognize possible IgE-mediated FAs, a low frequency of FA was considered in this population. Use of a directed questionnaire administered by trained researchers could be an alternative for epidemiological IgE-mediated FA studies to achieve more accurate results.

Serum and salivary IgE, IgA, and IgG4 antibodies to Dermatophagoides pteronyssinus and its major allergens, Der p1 and Der p2, in allergic and nonallergic children.

Allergic rhinitis (AR) is a public health problem with high prevalence worldwide. We evaluated levels of specific IgE, IgA, and IgG4 antibodies to the Dermatophagoides pteronyssinus (Dpt) house dust mite and to its major allergens (Der p1 and Der p2) in serum and saliva samples from allergic and nonallergic children. A total of 86 children were analyzed, from which 72 had AR and 14 were nonallergic healthy children. Serum IgE and serum/salivary IgG4 levels to Dpt, Der p1, and Der p2 were higher in allergic children whereas serum/salivary IgA levels to all allergens were higher in nonallergic children. IgE levels positively correlated with IgG4 and IgA to all allergens in allergic children, while IgA levels negatively correlated with IgG4 to Dpt and Der p1 in nonallergic children. In conclusion, mite-specific IgA antibodies predominate in the serum and saliva of nonallergic children whereas mite-specific IgE and IgG4 are prevalent in allergic children. The presence of specific IgA appears to have a key role for the healthy immune response to mucosal allergens. Also, specific IgA measurements in serum and/or saliva may be useful for monitoring activation of tolerance-inducing mechanisms during allergen specific immunotherapeutic procedures, especially sublingual immunotherapy.

Multiplexed Proteomic Analysis for Diagnosis and Screening of Five Primary Immunodeficiency Disorders From Dried Blood Spots.

Early detection of Primary Immunodeficiencies Disorders (PIDDs) is of paramount importance for effective treatment and disease management. Many PIDDs would be strong candidates for newborn screening (NBS) if robust screening methods could identify patients from dried blood spots (DBS) during the neonatal period. As majority of congenital PIDDs result in the reduction or absence of specific proteins, direct quantification of these target proteins represents an attractive potential screening tool. Unfortunately, detection is often limited by the extremely low protein concentrations in blood cells and limited blood volume present in DBS. We have recently developed a robust novel method for quantification of low abundance proteins in DBS for PIDDs using peptide immunoaffinity enrichment coupled to selected reaction monitoring (immuno-SRM). Here, we further generated a multiplexed Immuno-SRM panel for simultaneous screening of eight signature peptides representing five PIDD-specific and two cell-type specific proteins from DBS. In samples from 28 PIDD patients including two carriers, representing X-Linked Agammaglobulinemia (XLA), Wiskott-Aldrich Syndrome (WAS), X-Linked Chronic Granulomatous Disease (XL-CGD), DOCK8 Deficiency and ADA deficiency, peptides representing each disease are significantly reduced relative to normal controls and patient identification had excellent agreement with clinical and molecular diagnosis. Also included in the multiplex panel are cell specific markers for platelets (CD42) and Natural Killer Cells (CD56). In patients with WAS, CD42 levels were found to be significantly reduced consistent with characteristic thrombocytopenia. A patient with WAS analyzed before and after bone marrow transplant showed normalized WAS protein and platelet CD42 after treatment highlighting the ability of immuno-SRM to monitor the effects of PIDD treatment. The assay was readily reproduced in two separate laboratories with similar analytical performance and complete agreement in patient diagnosis demonstrating the effective standardized methods. A high-throughput Immuno-SRM method screens PIDD-specific peptides in a 2.5-min runtime meeting high volume NBS workflow requirements was also demonstrated in this report. This high-throughput method returned identical results to the standard Immuno-SRM PIDD panel. Immuno-SRM peptide analysis represents a robust potential clinical diagnostic for identifying and studying PIDD patients from easily collected and shipped DBS and supports a significant potential for early PIDD diagnosis through newborn screening.

Abnormal brown adipose tissue mitochondrial structure and function in IL10 deficiency.

BACKGROUND: Inflammation is the most relevant mechanism linking obesity with insulin-resistance and metabolic disease. It impacts the structure and function of tissues and organs involved in metabolism, such as the liver, pancreatic islets and the hypothalamus. Brown adipose tissue has emerged as an important component of whole body energy homeostasis, controlling caloric expenditure through the regulation of non-shivering thermogenesis. However, little is known about the impact of systemic inflammation on the structure and function of brown adipose tissue. METHODS: The relations between IL10 and mitochondria structure/function and also with thermogenesis were evaluated by bioinformatics using human and rodent data. Real-time PCR, immunoblot, fluorescence and transmission electron microscopy were employed to determine the effect of IL10 in the brown adipose tissue of wild type and IL10 knockout mice. FINDINGS: IL10 knockout mice, a model of systemic inflammation, present severe structural abnormalities of brown adipose tissue mitochondria, which are round-shaped with loss of cristae structure and increased fragmentation. IL10 deficiency leads to newborn cold intolerance and impaired UCP1-dependent brown adipose tissue mitochondrial respiration. The reduction of systemic inflammation with an anti-TNFα monoclonal antibody partially rescued the structural but not the functional abnormalities of brown adipose tissue mitochondria. Using bioinformatics analyses we show that in both humans and mice, IL10 transcripts correlate with mitochondrial lipid metabolism and caspase gene expression. INTERPRETATION: IL10 and systemic inflammation play a central role in the regulation of brown adipose tissue by controlling mitochondrial structure and function. FUND: Sao Paulo Research Foundation grant 2013/07607-8.

Rapid Multiplexed Proteomic Screening for Primary Immunodeficiency Disorders From Dried Blood Spots.

Background: Primary immunodeficiency disorders (PIDD) comprise a group of life-threatening congenital diseases characterized by absent or impaired immune responses. Despite the fact that effective, curative treatments are available with optimal clinical outcomes when diagnosed early, newborn screening does not exist for the majority of these diseases due to the lack of detectable, specific biomarkers or validated methods for population-based screening. Peptide immunoaffinity enrichment coupled with selected reaction monitoring mass spectrometry (immuno-SRM) is a sensitive proteomic assay, involving antibody-mediated peptide capture, that allows for concurrent quantification of multiple analytes. This assay has promise for use in potential newborn screening of PIDDs that lead to diminished or absent target proteins in the majority of cases. Objective: To determine and evaluate if a multiplex assay based on immuno-SRM is able to reliably and precisely distinguish affected patients with X-linked agammaglobulinemia (XLA), Wiskott-Aldrich Syndrome (WAS), and CD3ϵ-associated severe combined immunodeficiency (SCID) from one another and from unaffected normal control dried blood spot (DBS) samples. Methods: We performed a blinded, multiplexed analysis of proteolytically-generated peptides from WASp, BTK, and CD3ϵ (for WAS, XLA, and SCID, respectively) in DBS samples from 42 PIDD patients, 40 normal adult controls, and 62 normal newborns. The peptide ATPase copper transporting protein (ATP7B) 1056 was simultaneously monitored for quality assurance purposes. Results: The immuno-SRM assays reliably quantified the target peptides in DBS and accurately distinguished affected patients from normal controls. Analysis of signature peptides found statistically significant reduction or absence of peptide levels in affected patients compared to control groups in each case (WASp and BTK: p = 0.0001, SCID: p = 0.05). Intra and inter-assay precision ranged from 11 to 22% and 11 to 43% respectively; linearity (1.39-2000 fmol peptide), and stability (≤ 0.09% difference in 72 h) showed high precision for the multiplexed assay. Inter-laboratory assay comparison showed high concordance for measured peptide concentrations, with R2 linearity ≥ 0.97 for the WASp 274, CD3ϵ 197, BTK 407, and ATP7B 1056 peptides. Conclusion: Immuno-SRM-based quantification of proteotypic peptides from WASp, BTK, and CD3ϵ in DBS distinguishes relevant PIDD cases from one another and from controls, raising the possibility of employing this approach for large-scale multiplexed newborn screening of selective PIDDs.

Clinical, Immunological, and Molecular Heterogeneity of 173 Patients With the Phenotype of Immune Dysregulation, Polyendocrinopathy, Enteropathy, X-Linked (IPEX) Syndrome.

Background: Immune Dysregulation, Polyendocrinopathy, Enteropathy, X-linked (IPEX) Syndrome is a rare recessive disorder caused by mutations in the FOXP3 gene. In addition, there has been an increasing number of patients with wild-type FOXP3 gene and, in some cases, mutations in other immune regulatory genes. Objective: To molecularly asses a cohort of 173 patients with the IPEX phenotype and to delineate the relationship between the clinical/immunologic phenotypes and the genotypes. Methods: We reviewed the clinical presentation and laboratory characteristics of each patient and compared clinical and laboratory data of FOXP3 mutation-positive (IPEX patients) with those from FOXP3 mutation-negative patients (IPEX-like). A total of 173 affected patients underwent direct sequence analysis of the FOXP3 gene while 85 IPEX-like patients with normal FOXP3 were investigated by a multiplex panel of "Primary Immune Deficiency (PID-related) genes." Results: Forty-four distinct FOXP3 variants were identified in 88 IPEX patients, 9 of which were not previously reported. Among the 85 IPEX-like patients, 19 different disease-associated variants affecting 9 distinct genes were identified. Conclusions: We provide a comprehensive analysis of the clinical features and molecular bases of IPEX and IPEX-like patients. Although we were not able to identify major distinctive clinical features to differentiate IPEX from IPEX-like syndromes, we propose a simple flow-chart to effectively evaluate such patients and to focus on the most likely molecular diagnosis. Given the large number of potential candidate genes and overlapping phenotypes, selecting a panel of PID-related genes will facilitate a molecular diagnosis.

Corrigendum: A Novel Homozygous JAK3 Mutation Leading to T-B+NK- SCID in Two Brazilian Patients.

[This corrects the article DOI: 10.3389/fped.2018.00230.].

A Novel Homozygous JAK3 Mutation Leading to T-B+NK- SCID in Two Brazilian Patients.

We report a novel homozygous JAK3 mutation in two female Brazilian SCID infants from two unrelated kindreds. Patient 1 was referred at 2 months of age due to a family history of immunodeficiency and the appearance of a facial rash. The infant was screened for TRECs (T-cell receptor excision circles) and KRECs (kappa-deleting recombination excision circles) for the assessment of newly formed naïve T and B cells respectively, which showed undetectable TRECs and normal numbers of KRECs. Lymphocyte immunophenotyping by flow cytometry confirmed the screening results, revealing a T-B+NK- SCID. The patient underwent successful HSCT. Patient 2 was admitted to an intensive care unit at 8 months of age with severe pneumonia, BCGosis, and oral moniliasis; she also had a positive family history for SCID but newborn screening was not performed at birth. At 10 months of age she was diagnosed as a T-B+NK- SCID and underwent successful HSCT. JAK3 sequencing revealed the same homozygous missense mutation (c.2350G>A) in both patients. This mutation affects the last nucleotide of exon 17 and it is predicted to disrupt the donor splice site. cDNA sequencing revealed skipping of exon 17 missing in both patients, confirming the predicted effect on mRNA splicing. Skipping of exon 17 leads to an out of frame deletion of 151 nucleotides, frameshift and creation of a new stop codon 60 amino acids downstream of the mutation resulting in a truncated protein which is likely nonfunctional.

Dried Blood Spots, an Affordable Tool to Collect, Ship, and Sequence gDNA from Patients with an X-Linked Agammaglobulinemia Phenotype Residing in a Developing Country.

Background: New sequencing techniques have revolutionized the identification of the molecular basis of primary immunodeficiency disorders (PID) not only by establishing a gene-based diagnosis but also by facilitating defect-specific treatment strategies, improving quality of life and survival, and allowing factual genetic counseling. Because these techniques are generally not available for physicians and their patients residing in developing countries, collaboration with overseas laboratories has been explored as a possible, albeit cumbersome, strategy. To reduce the cost of time and temperature-sensitive shipping, we selected Guthrie cards, developed for newborn screening, to collect dried blood spots (DBS), as a source of DNA that can be shipped by regular mail at minimal cost. Method: Blood was collected and blotted onto the filter paper of Guthrie cards by completely filling three circles. We enrolled 20 male patients with presumptive X-linked agammaglobulinemia (XLA) cared for at the Vietnam National Children's Hospital, their mothers, and several sisters for carrier analysis. DBS were stored at room temperature until ready to be shipped together, using an appropriately sized envelope, to a CLIA-certified laboratory in the US for sequencing. The protocol for Sanger sequencing was modified to account for the reduced quantity of gDNA extracted from DBS. Result: High-quality gDNA could be extracted from every specimen. Bruton tyrosine kinase (BTK) mutations were identified in 17 of 20 patients studied, confirming the diagnosis of XLA in 85% of the study cohort. Type and location of the mutations were similar to those reported in previous reviews. The mean age when XLA was suspected clinically was 4.6 years, similar to that reported by Western countries. Two of 15 mothers, each with an affected boy, had a normal BTK sequence, suggesting gonadal mosaicism. Conclusion: DBS collected on Guthrie cards can be shipped inexpensively by airmail across continents, providing sufficient high-quality gDNA for Sanger sequencing overseas. By using this method of collecting gDNA, we were able to confirm the diagnosis of XLA in 17 of 20 Vietnamese patients with the clinical diagnosis of agammaglobulinemia.

Guia prático de atualização: medicamentos biológicos no tratamento da asma, doenças alérgicas e imunodeficiências / Practical update guide: biological drugs in the treatment of asthma, allergic diseases and immunodeficiencies

O presente guia apresenta revisão extensa sobre imunobiológicos utilizados, liberados e ainda sob estudo, para o tratamento da asma, doenças alérgicas e imunodeficiências. Além das características físico-químicas de alguns desses fármacos, são revisadas as indicações e os resultados de estudos clínicos realizados para avaliar eficácia e segurança. Separados por doença específica, são apresentados os principais agentes disponíveis e aprovados para utilização segundo as normas regulatórias nacionais.

This guide presents an extensive review of immunobiological drugs used, approved and/or under investigation for the treatment of asthma, allergic diseases and immunodeficiencies. In addition to the physicochemical characteristics of some of these drugs, their indications and results of clinical studies evaluating efficacy and safety are reviewed. The main agents available and approved for use in each specific disease according to national regulatory standards are presented.

Spirometric parameters and levels of interferon gamma and IL-5 in induced sputum from patients with allergic rhinitis or asthma.

BACKGROUND: This study was designed to analyze spirometric parameters and cytokine (interferon [IFN] gamma and IL-5) levels in induced sputum from patients with asthma or allergic rhinitis (AR) and nonatopic subjects. METHODS: Thirty-three subjects aged 18-60 years were enrolled in the present study. Eight patients had asthma without AR symptoms, 16 had AR without asthma symptoms or history, and both groups had positive skin-prick test (SPT) to aeroallergens. The nine remaining subjects were healthy nonatopic subjects with negative SPT to aeroallergens. Spirometry was performed by evaluating the forced vital capacity (FVC), forced expiratory volume in the 1st second (FEV1), and forced expiratory flow between 25 and 75% (FEF25-75) of FVC before and after bronchodilator use. Induced sputum samples were also collected for measuring cytokine (IFN-gamma and IL-5) levels by enzyme-linked immunosorbent assay. RESULTS: Significant pre- and postbronchodilator change was observed only for FEV1 with higher variation values in asthmatic subjects compared with patients with AR (p < 0.05) and nonatopic subjects (p < 0.01). There was no significant difference in pre- and postbronchodilator spirometric parameters among the three groups, although asthmatic patients showed lower prebronchodilator FEF25-75 values (p = 0.065). IL-5 levels were higher in induced sputum from patients with asthma and AR compared with nonatopic subjects (p = 0.020 and p = 0.032, respectively), but IFN-gamma levels showed no significant difference between the groups. CONCLUSION: Spirometric measurements were not able to show any occurrence of bronchial constriction in patients with AR, but predominant IL-5 levels in induced sputum from these patients reinforce the role of Th2-type immune response in lower respiratory airways that could contribute to the concept of "one airway, one disease."

Spirometric parameters and levels of interferon gamma and IL-5 in induced sputum from patients with allergic rhinitis or asthma.

BACKGROUND: This study was designed to analyze spirometric parameters and cytokine (interferon [IFN] gamma and IL-5) levels in induced sputum from patients with asthma or allergic rhinitis (AR) and nonatopic subjects. METHODS: Thirty-three subjects aged 18-60 years were enrolled in the present study. Eight patients had asthma without AR symptoms, 16 had AR without asthma symptoms or history, and both groups had positive skin-prick test (SPT) to aeroallergens. The nine remaining subjects were healthy nonatopic subjects with negative SPT to aeroallergens. Spirometry was performed by evaluating the forced vital capacity (FVC), forced expiratory volume in the 1st second (FEV(1)), and forced expiratory flow between 25 and 75% (FEF(25-75)) of FVC before and after bronchodilator use. Induced sputum samples were also collected for measuring cytokine (IFN-gamma and IL-5) levels by enzyme-linked immunosorbent assay. RESULTS: Significant pre- and postbronchodilator change was observed only for FEV(1) with higher variation values in asthmatic subjects compared with patients with AR (p < 0.05) and nonatopic subjects (p < 0.01). There was no significant difference in pre- and postbronchodilator spirometric parameters among the three groups, although asthmatic patients showed lower prebronchodilator FEF(25-75) values (p = 0.065). IL-5 levels were higher in induced sputum from patients with asthma and AR compared with nonatopic subjects (p = 0.020 and p = 0.032, respectively), but IFN-gamma levels showed no significant difference between the groups. CONCLUSION: Spirometric measurements were not able to show any occurrence of bronchial constriction in patients with AR, but predominant IL-5 levels in induced sputum from these patients reinforce the role of Th2-type immune response in lower respiratory airways that could contribute to the concept of "one airway, one disease."

Phenotype-genotype analysis of cryopyrin-associated periodic syndromes (CAPS): description of a rare non-exon 3 and a novel CIAS1 missense mutation.

We describe in this paper the phenotype-genotype analysis of a Brazilian cohort of patients with cryopyrin-associated periodic syndromes (CAPS). Patient 1 presented with an urticarial rash and recurrent fever exacerbated by cold weather, arthritis, and anterior uveitis, thus, receiving a clinical diagnosis of familial cold autoinflammatory syndrome. CIAS1 sequencing identified the T436I mutation, previously associated to a clinical phenotype of chronic infantile neurological cutaneous and articular/neonatal onset multisystem inflammatory disease. Patient 2 developed a papular exanthema with daily fever shortly after birth, frontal bossing, patellae enlargement, and cognitive and motor impairments. Sequencing identified the exceedingly rare G755R CIAS1 mutation in exon 4. Patient 3 developed skin rash and articular symptoms 6 h after birth, followed by aseptic meningitis. He was found to have the novel C148Y missense mutation in CIAS1. This report expands the spectrum of CIAS1 mutations associated to clinical disease, suggests that the same mutation can be associated with different clinical syndromes, and supports the evidence that CAPS patients should always be screened for mutations outside exon 3.

Detection of immunoglobulin G antibodies to Neospora caninum in humans: high seropositivity rates in patients who are infected by human immunodeficiency virus or have neurological disorders.

Considering that little is known about the epidemiology of Neospora caninum infection in humans, particularly in populations with high Toxoplasma gondii infection rates, the present study aimed to investigate the presence of antibodies to N. caninum in T. gondii-seropositive and -seronegative individuals. A total of 256 serum samples divided into four groups (61 samples from human immunodeficiency virus [HIV]-positive patients, 50 samples from patients with neurological disorders, 91 samples from newborns, and 54 samples from healthy subjects) were assessed for N. caninum and T. gondii serologies by indirect fluorescent-antibody test, enzyme-linked immunosorbent assay, and immunoblotting (IB). Immunoglobulin G antibodies to N. caninum were predominantly detected in HIV-infected patients (38%) and patients with neurological disorders (18%), while newborns and healthy subjects showed lower seropositivity rates (5% and 6%, respectively). Seropositivity to N. caninum was significantly associated with seropositivity to T. gondii in both HIV-infected patients and patients with neurological disorders. Seroreactivity to N. caninum was confirmed by IB, with positive sera predominantly recognizing the 29-kDa antigen of N. caninum. The results of this study indicate the presence of N. caninum infection or exposure in humans, particularly in HIV-infected patients or patients with neurological disorders, who could have opportunistic and concurrent infections with T. gondii. These findings may bring a new concern for the unstable clinical health of HIV-infected patients and the actual role of N. caninum infection in immunocompromised patients.