Early-onset pulmonary and cutaneous vasculitis driven by constitutively active SRC-family kinase HCK.

BACKGROUND: Inborn errors of immunity are genetic disorders characterized by various degrees of immune dysregulation that can manifest as immune deficiency, autoimmunity, or autoinflammation. The routine use of next-generation sequencing in the clinic has facilitated the identification of an ever-increasing number of inborn errors of immunity, revealing the roles of immunologically important genes in human pathologies. However, despite this progress, treatment is still extremely challenging. OBJECTIVE: We sought to report a new monogenic autoinflammatory disorder caused by a de novo activating mutation, p.Tyr515∗, in hematopoietic cell kinase (HCK). The disease is characterized by cutaneous vasculitis and chronic pulmonary inflammation that progresses to fibrosis. METHODS: Whole-exome sequencing, Sanger sequencing, mass spectrometry, and western blotting were performed to identify and characterize the pathogenic HCK mutation. Dysregulation of mutant HCK was confirmed ex vivo in primary cells and in vitro in transduced cell lines. RESULTS: Mutant HCK lacking the C-terminal inhibitory tyrosine Tyr522 exhibited increased kinase activity and enhanced myeloid cell priming, migration and effector functions, such as production of the inflammatory cytokines IL-1ß, IL-6, IL-8, and TNF-α, and production of reactive oxygen species. These aberrant functions were reflected by inflammatory leukocyte infiltration of the lungs and skin. Moreover, an overview of the clinical course of the disease, including therapies, provides evidence for the therapeutic efficacy of the Janus kinase 1/2 inhibitor ruxolitinib in inflammatory lung disease. CONCLUSIONS: We propose HCK-driven pulmonary and cutaneous vasculitis as a novel autoinflammatory disorder of inborn errors of immunity.

Diffuse parenchymal lung disease as first clinical manifestation of GATA-2 deficiency in childhood.

BACKGROUND: GATA-2 transcription factor deficiency has recently been described in patients with a propensity towards myeloid malignancy associated with other highly variable phenotypic features: chronic leukocytopenias (dendritic cell-, monocyto-, granulocyto-, lymphocytopenia), increased susceptibility to infections, lymphatic vasculature abnormalities, and sensorineural deafness. Patients often suffer from opportunistic respiratory infections; chronic pulmonary changes have been found in advanced disease. CASE PRESENTATION: We present a case of a 17-year-old previously healthy Caucasian male who was admitted to the hospital with fever, malaise, headache, cough and dyspnea. A chest X-ray revealed bilateral interstitial infiltrates and pneumonia was diagnosed. Despite prompt clinical improvement under antibiotic therapy, interstitial changes remained stable. A high resolution computer tomography showed severe diffuse parenchymal lung disease, while the patient's pulmonary function tests were normal and he was asymptomatic. Lung tissue biopsy revealed chronic reparative and resorptive reaction with organizing vasculitis. At the time of the initial presentation to the hospital, serological signs of acute infection with Epstein-Barr virus (EBV) were present; EBV viremia with atypical serological response persisted during two-year follow up. No other infectious agents were found. Marked monocytopenia combined with B-cell lymphopenia led to a suspicion of GATA-2 deficiency. Diagnosis was confirmed by detection of the previously published heterozygous mutation in GATA2 (c.1081 C > T, p.R361C). The patient's brother and father were both carriers of the same genetic defect. The brother had no clinically relevant ailments despite leukocyte changes similar to the index patient. The father suffered from spondylarthritis, and apart from B-cell lymphopenia, no other changes within the leukocyte pool were seen. CONCLUSION: We conclude that a diagnosis of GATA-2 deficiency should be considered in all patients with diffuse parenchymal lung disease presenting together with leukocytopenia, namely monocyto-, dendritic cell- and B-lymphopenia, irrespective of severity of the clinical phenotype. Genetic counseling and screening for GATA2 mutations within the patient's family should be provided as the phenotype is highly variable and carriers without apparent immunodeficiency are still in danger of developing myeloid malignancy. A prompt recognition of this rare condition helps to direct clinical treatment strategies and follow-up procedures.

Epithelial basement membrane thickening is related to TGF-Beta 1 expression in children with chronic respiratory diseases.

BACKGROUND: The complex structural changes of bronchial mucosa, known as remodelling, have been considered unique and typical for asthma. However, similar changes were recently found in other chronic respiratory diseases. The aim of this study was to compare basement membrane (BM) thickness and the number of transforming growth factor beta 1 (TGF-ß1) positive epithelial cells in children with asthma, cystic fibrosis (CF), primary ciliary dyskinesia (PCD) and healthy controls. METHODS: A total of 58 children (11.1 ± 3.9 yr, 55% males) were enrolled in this cross-sectional study. Endobronchial biopsy was performed in 27 children with asthma, 12 with CF, 12 with PCD and in 7 control patients. We studied the samples using light microscopy to assess BM width, the number of TGF-ß1 positive epithelial cells and their correlation. RESULTS: We found increased BM thickness (6.65 ± 1.22 µm vs. 2.93 ± 0.75 µm, p < 0.01) and a higher number of TGF-ß1 positive epithelial cells (61.39 ± 19.03 vs. 21.57 ± 12.58, p < 0.01) in children with chronic respiratory diseases compared to controls. There was no difference in these parameters between asthma, CF and PCD. A positive correlation between BM thickness and the number of TGF-ß1 positive cells was observed in all groups including controls (r = 0.84, p < 0.01). CONCLUSIONS: Increased BM thickness and number of TGF-ß1 positive epithelial cells were found in children with asthma, CF and PCD. The number of TGF-ß1 positive cells correlated positively with the BM thickness in all groups. We suggest that this might be a common generic feature of bronchial remodelling in chronic respiratory diseases.

Area under the maximum expiratory flow-volume curve--a sensitive parameter in the evaluation of airway patency.

BACKGROUND: The most frequently used parameters for assessing bronchoconstriction and bronchodilation are forced expiratory volume in 1 s (FEV(1)) and peak expiratory flow (PEF). OBJECTIVES: To assess the sensitivity of other parameters after induced bronchoconstriction and bronchodilation. METHODS: From maximum expiratory flow-volume (MEFV) curves, forced vital capacity, FEV(1), PEF, maximum expiratory flows (MEF) at 25, 50 and 75% of vital capacity and the area under the MEFV curve (A(ex)) were measured in two groups of asthmatic children after induced bronchoconstriction and bronchodilation, and in children with cystic fibrosis (CF) after bronchodilation. RESULTS: In 142 asthmatics without airway obstruction, bronchoconstriction was induced by inhalation of 1% histamine aerosol. The 20% fall in A(ex) compared to baseline was found in all asthmatics, while the 20 and 15% falls in FEV(1) were noted in 36 and 65% of the patients, respectively. Other parameters were less sensitive or interpretation was problematic. Another 110 asthmatics with mild-moderate airway obstruction were treated with various bronchodilators. The 20% increase in A(ex) was observed in all asthmatics, while the 20% increase in FEV(1) was found in only 33% of the patients and the 15% increase in FEV(1) in 51%. In 9 CF children, the pattern of changes in A(ex) and FEV(1) after bronchodilation was similar to that in asthmatics. CONCLUSIONS: A(ex) was a sensitive and less problematic parameter in the evaluation of airway patency in comparison with FEV(1) and other parameters measured from the MEFV curve in our study patients.

An effective combination of sanger and next generation sequencing in diagnostics of primary ciliary dyskinesia.

BACKGROUND: Primary ciliary dyskinesia (PCD) is a multigenic autosomal recessive condition affecting respiratory tract and other organs where ciliary motility is required. The extent of its genetic heterogeneity is remarkable. The aim of the study was to develop a cost-effective pipeline for genetic diagnostics using a combination of Sanger and next generation sequencing (NGS). MATERIALS AND METHODS: Data and samples of 33 families with 38 affected subjects with PCD diagnosed in childhood were collected over the territory of the Czech Republic. A panel of 18 PCD causative or candidate genes was implemented into an Illumina TruSeq Custom Amplicon NGS assay, and three ancestral mutations in SPAG1 were screened by conventional Sanger sequencing, which was also used for the confirmation of the NGS results and for the analysis of familial segregation. RESULTS: The causative gene was DNAH5 in 11/33 (33%) probands, SPAG1 in 8/33 (24%), and DNAI1, CCDC40, LRRC6 in one family each. If the high proportion of subjects with bi-allelic ancestral mutations in SPAG1 is corroborated in other Caucasian populations, a simple Sanger sequencing test for these three mutations may serve as an effective pre-screening step, being followed by an NGS panel for other, much larger, PCD genes. CONCLUSIONS: We present a combination of Sanger sequencing with an NGS panel for known and candidate PCD genes, implemented in a moderate-size national collection of patients. This strategy has proven to be cost-effective, rapid and reliable, and was able to detect the causative gene in two thirds of our PCD patients.

Tracheal diverticulum with decompensation after 8 years of conservative therapy requiring surgical solution.

Tracheal diverticulum is a benign cystic mass in the cervical and mediastinal regions, with an incidence of 1% in post-mortem findings, and 2% in CT findings. The lesion is in most cases completely asymptomatic and is most commonly incidentally detected during a CT examination. We present the case of a young female patient with a tracheal diverticulum who has been followed up for 8 years by pediatric pneumologist. Patient health state deteriorated and she developed stress-induced dyspnea requiring surgical resection. Moreover, we mention differential diagnosis of other mediastinal cystic lesions.

Abnormalities in pulmonary function in infants with high-risk congenital diaphragmatic hernia.

AIMS: The aim of the study was to analyze lung growth and abnormality of infant pulmonary function tests (IPFT) in congenital diaphragmatic hernia (CDH) survivors younger than three years of age with respect to unfavorable prognostic factors. METHODS: Thirty high-risk CDH survivors at the age of 1.32±0.54 years, body weight 9.76±1.25 kg were examined using IPFT: tidal breathing analysis, baby resistance/compliance, whole baby body plethysmography and rapid thoraco-abdominal compression. Gore-Tex patch was used in 13% of patients (GORE group). Pulmonary hypertension was diagnosed and managed in 13% (iNO group). Standard protocols and appropriate reference values were used and obtained data were statistically analysed. RESULTS: High incidence of peripheral airway obstruction (70%), increased value of functional residual capacity (FRCp) 191.3±24.5 mL (126.5±36.9 % predicted; P < 0.0005), increased value of effective airway resistance (Reff) 1.71±0.93 kPa.L(-1).s (144.4±80.1 % predicted; P < 0.01) and decreased specific compliance of the respiratory system (Crs/kg) 14.1±2.3 mL.kPa.kg(-1) (i.e., 76.1±20.1 % predicted, P < 0.0005) was noted in infants with CDH in comparison with reference values. Increased value of FRCp was found in GORE group (165.7±51.9 versus 120.4±31.2, P < 0.02) and in iNO group (183.1±52.6 versus 117.8±25.7 mL; P < 0.0005). CONCLUSION: A high incidence of peripheral airway obstruction, an increased value of FRCp and decreased specific compliance of the respiratory system was noted in infants with CDH. Unfavorable prognostic factors (Gore-Tex patch, pulmonary hypertension) correlate with more severe alteration of pulmonary function in infants.

Impaired Growth during Childhood in Patients with Primary Ciliary Dyskinesia.

Primary ciliary dyskinesia (PCD) leads to recurrent/chronic respiratory infections, resulting in chronic inflammation and potentially in chronic pulmonary disease with bronchiectasis. We analyzed longitudinal data on body length/height and body mass index (BMI) for 29 children and young adults with PCD aging 1.5-24 years (median, 14.5) who had been diagnosed at the age of 0.5-17 years (median, 8). Of these, 10 carried pathogenic mutations in either DNAH5 or DNAI1. In children with PCD, body length/height progressively decreased from +0.40 ± 0.24 SDS (the 1st birthday), +0.16 ± 0.23 SDS (3 years old), and -0.13 ± 0.21 SDS (5 years old) to -0.54 ± 0.19 SDS (7 years old; P = 0.01 versus 0), -0.67 ± 0.21 SDS (9 years old; P = 0.005 versus 0), -0.52 ± 0.24 SDS (11 years old; P = 0.04 versus 0), and -0.53 ± 0.23 SDS (13 years old; P = 0.03 versus 0). These results reflect low growth rates during the childhood growth period. Thereafter, heights stabilized up to the age of 17 years. The growth deterioration was not dependent on sex or disease severity but was more pronounced in DNAH5 or DNAI1 mutation carriers. BMI did not differ from population standards, which suggests that nutritional deficits are not the cause of growth delay. We conclude that PCD leads to chronic deprivation with significant growth deterioration during childhood.

Effectiveness of sequencing selected exons of DNAH5 and DNAI1 in diagnosis of primary ciliary dyskinesia.

INTRODUCTION: Primary ciliary dyskinesia (PCD) is a rare genetically heterogenous condition. Mutations in DNAH5 or DNAI1 genes can be found in about a third of the patients with PCD. Increased occurrence of mutations was described in several exons of these long genes. The objective of the study was to test the sensitivity of sequencing of selected 13 exons (as compared to costly sequencing of all 100 exons of the two genes), and to determine the prevalence of the DNAH5 or DNAI1 mutations in the Czech PCD database. METHODS: The Czech national PCD database has identified 31 pediatric patients, diagnosed based on clinical findings and tests on the ciliated epithelium. Twenty-seven patients from 24 families agreed on genetic testing. In the first step, direct sequencing of selected 13 exons (9 of DNAH5 and 4 of DNAI1) was performed, and then we compared its effectiveness in detecting at least one mutation with results of sequencing all 100 exons of the two genes. RESULTS: The sequencing of all exons identified compound heterozygosity for PCD mutations in nine patients from eight families (DNAH5 in eight and DNAI1 in one patient), and heterozygozity for a DNAH5 mutation of uncertain functional significance in one additional patient. The first step of selected exon sequencing detected a mutation in five out of these eight families, its actual sensitivity being 62.5%, with a high predictive value. The phenotypic and clinical characteristics of all the paediatric patients with PCD are shown. CONCLUSIONS: Selected exon sequencing detects at least one mutated allele in over a half of our patients who have PCD due to DNAH5 or DNAI1 mutations. To lower the costs of the genetic testing, targeted step-wise genetic testing may be a reasonable approach to detect mutations in PCD patients, especially if their phenotype is taken into account.

Dose-related efficacy and safety of formoterol (Oxis) Turbuhaler compared with salmeterol Diskhaler in children with asthma.

To compare the dose-related bronchodilator efficacy and tolerability of formoterol (Oxis) Turbuhaler with salmeterol Diskhaler and placebo in children with asthma. A single-dose, randomized, double-blind, incomplete crossover study of 68 children (7-17 years), with moderate-to-severe asthma, 82% receiving inhaled corticosteroids. Patients received four of six treatments [4.5, 9, 18, or 36 microg formoterol (6, 12, 24 or 48 microg metered doses), 50 microg salmeterol (metered dose) or placebo] at 12-h visits, separated by > or =3 days. Forced expiratory volume in 1 s (FEV1), pulse, blood pressure, electrocardiogram, adverse events and urine formoterol were assessed. The therapeutic ratio of formoterol vs. salmeterol was estimated from the efficacy and systemic effects results. All active treatments significantly improved FEV1 compared with placebo. Formoterol 9-36 microg provided dose-related increases over salmeterol in lung function: average 12-h FEV1 (increases of 4.9-8.7%, p < 0.001) and FEV1 at 12 h post-dose (7.0-12.2%, p < 0.001). The onset of effect of formoterol was also significantly faster than salmeterol for doses > or =9 microg. Salmeterol 50 microg was estimated to be equieffective to 3.3 microg formoterol for 12-h average FEV1 and the estimated equieffective dose for a variety of systemic effects was 7.8-13.5 microg formoterol. All treatments were well tolerated. Formoterol (Oxis) Turbuhaler 4.5-36 microg provided dose-related improvements in bronchodilator efficacy in children with asthma. Formoterol > or =9 microg provided superior bronchodilator efficacy over 12 h compared with salmeterol Diskhaler 50 microg with no increase in systemic effects.