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Rare variants in the T-box transcription factor 4 gene (

Respiratory distress syndrome (RDS) may occur in term and near-term infants because of mutations in surfactant-related genes. ATP-binding cassette A3 (ABCA3), a phospholipid carrier specifically expressed in the alveolar epithelium, is the most frequently involved protein. We report the case of a couple of late-preterm fraternal twin infants of opposite sex carrying the same compound heterozygous ABCA3 mutations, one of which has never been previously reported, with different disease severity, suggesting variable penetrance or sex-related differences. ABCA3 deficiency should be considered in term or near-term babies who develop unexplained RDS.

Rare variants in the T-box transcription factor 4 gene (TBX4) have recently been recognised as an emerging cause of paediatric pulmonary hypertension (PH). Their pathophysiology and contribution to persistent pulmonary hypertension in neonates (PPHN) are unknown. We sought to define the spectrum of clinical manifestations and histopathology associated with TBX4 variants in neonates and children with PH.We assessed clinical data and lung tissue in 19 children with PH, including PPHN, carrying TBX4 rare variants identified by next-generation sequencing and copy number variation arrays.Variants included six 17q23 deletions encompassing the entire TBX4 locus and neighbouring genes, and 12 likely damaging mutations. 10 infants presented with neonatal hypoxic respiratory failure and PPHN, and were subsequently discharged home. PH was diagnosed later in infancy or childhood. Three children died and two required lung transplantation. Associated anomalies included patent ductus arteriosus, septal defects, foot anomalies and developmental disability, the latter with a higher prevalence in deletion carriers. Histology in seven infants showed abnormal distal lung development and pulmonary hypertensive remodelling.TBX4 mutations and 17q23 deletions underlie a new form of developmental lung disease manifesting with severe, often biphasic PH at birth and/or later in infancy and childhood, often associated with skeletal anomalies, cardiac defects, neurodevelopmental disability and other anomalies.

Pediatric diffuse lung diseases are rare disorders with an onset in the neonatal period or in infancy, characterized by chronic respiratory symptoms and diffuse interstitial changes on imaging studies. Genetic disorders of surfactant homeostasis represent the main etiology. Surfactant protein B and ABCA3 deficiencies typically cause neonatal respiratory failure, which is often lethal within a few weeks or months. Although heterozygous ABCA3 mutation carriers are mostly asymptomatic, there is growing evidence that monoallelic mutations may affect surfactant homeostasis. Surfactant protein C mutations are dominant or sporadic disorders leading to a broad spectrum of manifestations from neonatal respiratory distress syndrome to adult pulmonary fibrosis. The authors performed pathology and ultrastructural studies in 12 infants who underwent clinical lung biopsy. One carried a heterozygous SP-B mutation, 3 carried SP-C mutations, and 7 carried ABCA3 mutations (5 biallelic and 2 monoallelic). Optical microscopy made it possible to distinguish between surfactant-related disorders and other forms. One of the ABCA3 monoallelic carriers had morphological features of alveolar capillary dysplasia, a genetic disorder of lung alveolar, and vascular development. One patient showed no surfactant-related anomalies but had pulmonary interstitial glycogenosis, a developmental disorder of unknown origin. Electron microscopy revealed specific lamellar bodies anomalies in all SP-B, SP-C, and ABCA3 deficiency cases. In addition, the authors showed that heterozygous ABCA3 mutation carriers have an intermediate ultrastructural phenotype between homozygous carriers and normal subjects. Lung biopsy is an essential diagnostic procedure in unexplained diffuse lung disorders, and electron microscopy should be performed systematically, since it may reveal specific alterations in genetic disorders of surfactant homeostasis.

Pulmonary hypoplasia and hypertension account for significant morbidity and mortality in neonates with congenital diaphragmatic hernia (CDH). Whether CDH is associated with surfactant dysfunction remains controversial. Therefore, we measured disaturated phosphatidylcholine (DSPC) and surfactant protein (SP)-B concentration in tracheal aspirates and their synthesis rate in infants with CDH compared to infants without lung disease. (2)H2O as a precursor of DSPC and 1-(13)C-leucine as a precursor of SP-B were administered to 13 infants with CDH and eight controls matched for gestational age. DSPC and SP-B were isolated from tracheal aspirates, and their fractional synthesis rate was derived from (2)H and (13)C enrichment curves obtained by mass spectrometry. DSPC and SP-B amounts in tracheal aspirates were also measured. In infants with CDH, SP-B fractional synthesis rate and amount were 62±27% and 57±22% lower, respectively, than the value found in infants without lung disease (p

Background Monoallelic mutations of the Surfactant Protein C gene (SFTPC) are associated with Interstitial Lung Disease in children. I73T is the most common mutation, accounting for 30 % of all cases reported. Case presentation We describe three patients carrying the same I73T SPC mutation with very different phenotypes, clinical course (ranging from mild respiratory symptoms to death for respiratory failure) and outcome. Conclusions The disease mechanisms associated with SP-C mutations suggest that the combination of individual genetic background and environmental factors contribute largely to the wide variability of clinical expression. Infants, children and adults with ILD of unknown etiology should be investigated for SP-C genetic abnormalities.

Genetic disorders of the surfactant system are rare diseases with a broad range of clinical manifestations, from fatal respiratory distress syndrome (RDS) in neonates to chronic interstitial lung disease (ILD) in children and adults. ABCA3 [ATP-binding cassette (ABC), subfamily A, member 3] is a lung-specific phospholipid transporter critical for intracellular surfactant synthesis and storage in lamellar bodies (LBs). Its expression is developmentally regulated, peaking prior to birth under the influence of steroids and transcription factors. Bi-allelic mutations of the ABCA3 gene represent the most frequent cause of congenital surfactant deficiency, indicating its critical role in lung function. Mutations affect surfactant lipid and protein processing and LBs’ morphology, leading to partial or total surfactant deficiency. Approximately 200 mutations have been reported, most of which are unique to individuals and families, which makes diagnosis and prognosis challenging. Various types of mutations, affecting different domains of the protein, account in part for phenotype diversity. Disease-causing mutations have been reported in most coding and some non-coding regions of the gene, but tend to cluster in the first extracellular loop and the second nucleotide-binding domain (NBD), leading to defective glycosylation and trafficking defects and interfering with ATP binding and hydrolysis respectively. Mono-allelic damaging and benign variants are often subclinical but may act as disease modifiers in lung diseases such as RDS of prematurity or associate with mutations in other surfactant-related genes. Diagnosis is complex but essential and should combine pathology and ultrastructure studies on lung biopsy with broad-spectrum genetic testing of surfactant-related genes, made possible by recent technology advances in the massive parallel sequencing technology.

SP-C dysfunction is one of the most frequent causes of Interstitial Lung Disease (ILD). We describe three pts with the same SP-C mutation but different phenotypes. Case 1: TD, 3 yrs, persistent cough, dyspnoea, ground glass opacities and subpleural micronodules at CT. SP-C gene analysis positive for the mutation I73T. Hydroxychloroquine was started, 3 months later TD was asymptomatic, CT improved and normalised after 6 months. Case 2: UM, 4 yrs, healthy up to 6 months, when a RSV caused severe respiratory insufficiency. Normal bronchoscopy, CT with ILD. Referred at one yr, O2 and steroid dependent, CT with wide ground glass opacities and “honeycomb”. Despite systemic steroids CT worsened. Lung biopsy showed desquamative interstitial pneumonia (DIP), ultrastructure was consistent with surfactant deficiency. Genetical analysis showed the I73T mutation. After the 6th iv bolus of high dose steroids CT worsened, hydroxychloroquine was started with a reduction of O2 and steroid needs. The last CT is stable, O2 discontinued. Case 3: CR, recurrent pneumonia in the first year, admitted at 15 months (Naples) for acute respiratory distress. Chest Xray with wide opacities and interstitial thickening, CT scan increased parenchymal density and multiple consolidation. Because of prolonged VM tracheostomy was performed. A lung biopsy, examinated in our Hospital, showed ILD with DIP/alveolar proteinosis. Ultrastructurel was consistent with congenital abnormalities of surfactant proteins. Molecular analysis showed the presence of the mutation I73T. Hydroxychloroquine started after the biopsy, the patient died after 2 weeks Conclusion: This study confrims the eterogeneity of SP-C deficiency despite same genetic mutations.

NK2 homeobox-1 (NKX2.1) gene encoding the thyroid transcription factor-1 (TTF-1) plays a critical role in lung, thyroid, and central nervous system morphogenesis and function; mutations cause a rare form of progressive respiratory failure associated with alterations of surfactant synthesis, composition, and homeostasis. Molecular mechanisms are heterogeneous and poorly explored. A 28 days old male, soon after birth, presented respiratory failure requiring oxygen treatment at FiO2 27%, prolonged for 2 weeks. Routine neonatal screenings detected a high thyroid stimulating hormone concentration. On day 27 congenital hypothyroidism was confirmed and substitutive treatment was begun. Since the persistence of respiratory symptoms sweat test, CFTR mutation, lymphocyte subpopulations, and sputum cultures were tested, resulting negative. Brain and cardiac defects were also ruled out. Bronchoscopy and BAL analysis were normal. Computed tomography showed bilateral multiple ground glass attenuation, consolidative areas and diffuse bronchial wall thickening. Based on the severity of symptoms, the exclusion of other causes of respiratory disease and the CT findings of interstitial lung disease, we investigated genes affecting the surfactant homeostasis. Sequencing analysis of the three exons of the TTF1 revealed a heterozygous mutation c.334G > T that results in the replacement of glycine in position 112 with a stop codon, generating a nonsense protein that lacks the correct transactivation domain in the C-terminal region. Genetic analysis of the family showed that the father, who was asymptomatic, carried the mutation. Screening for TTF-1 deletions or mutations should always be considered in children with congenital hypothyroidism and an unexplained neonatal respiratory distress or neurodevelopmental deficits.

© 2015 Macmillan Publishers Limited Genetic defects of surfactant metabolism are associated with a broad range of clinical manifestations, from neonatal respiratory distress syndrome to adult interstitial lung disease. Early therapies may improve symptoms but diagnosis is often delayed owing to phenotype and genotype variability. Our objective was to characterize the cellular/ultrastructural correlates of surfactant protein C (SP-C) mutations in children with idiopathic diffuse lung diseases. We sequenced SFTPC – the gene encoding SP-C – SFTPB and ABCA3, and analyzed morphology, ultrastructure and SP expression in lung tissue when available. We identified eight subjects who were heterozygous for SP-C mutations. Median age at onset and clinical course were variable. None of the mutations were located in the mature peptide-encoding region, but were either in the pro-protein BRICHOS or linker C-terminal domains. Although lung morphology was similar to other genetic surfactant metabolism disorders, electron microscopy studies showed specific anomalies, suggesting surfactant homeostasis disruption, plus trafficking defects in the four subjects with linker domain mutation and protein misfolding in the single BRICHOS mutation carrier in whom material was available. Immunolabeling studies showed increased proSP-C staining in all cases. In two cases, amyloid deposits could be identified. Immunochemistry and ultrastructural studies may be useful for diagnostic purposes and for genotype interpretation.European Journal of Human Genetics advance online publication, 18 March 2015; doi:10.1038/ejhg.2015.45.

To the Editors: The SFTPB gene encodes the hydrophobic pulmonary surfactant protein (SP)-B, which is essential for the build-up of the surfactant layer and lowering of surface tension in the airways. SP-B deficiency was the first reported genetic cause of lethal respiratory distress syndrome (RDS) in infants in 1993 [1]. The phenotype of infants with hereditary SP-B deficiency is of a typically full-term neonate with respiratory failure in the first 24–48 h of life; diagnosis can be delayed, as affected infants may show initially mild symptoms and not require ventilation or further medical support for some time. Chest radiography shows a bilateral, wide ground-glass pattern consistent with a diagnosis of hyaline membrane disease. Typical histological findings are the presence of periodic acid–Schiff-positive eosinophilic material in the alveoli, epithelial cell desquamation, enlarged alveolar macrophages with lamellar inclusions and accumulation of SP-A [2]. Since SP-B was found to be essential for the proteolytic processing of pro-SP-C, newborns with hereditary SP-B deficiency show aberrantly processed SP-C in the intra-alveolar lumen. Lung disease is rapidly progressive and fatal respiratory failure finally sets in within 3–6 months; lung transplantation is suggested as the only effective treatment. To our knowledge, heterozygous mutation has not previously been reported to cause clinical symptoms. A 6-month-old male was admitted to the Bronchopneumology …

Respiratory distress syndrome (RDS) may occur in term and near-term infants because of mutations in surfactant-related genes. ATP-binding cassette A3 (ABCA3), a phospholipid carrier specifically expressed in the alveolar epithelium, is the most frequently involved protein. We report the case of a couple of late-preterm fraternal twin infants of opposite sex carrying the same compound heterozygous ABCA3 mutations, one of which has never been previously reported, with different disease severity, suggesting variable penetrance or sex-related differences. ABCA3 deficiency should be considered in term or near-term babies who develop unexplained RDS.

Background Mutations of genes affecting surfactant homeostasis, such as SFTPB, SFTPC and ABCA3, lead to diffuse lung disease in neonates and children. Haploinsufficiency of NKX2.1, the gene encoding the thyroid transcription factor-1 (TTF-1) - critical for lung, thyroid and central nervous system morphogenesis and function - causes a rare form of progressive respiratory failure designated brain-lung-thyroid syndrome. Molecular mechanisms involved in this syndrome are heterogeneous and poorly explored. We report a novel TTF-1 molecular defect causing recurrent respiratory failure episodes in an infant. Methods The subject was an infant with severe neonatal respiratory distress syndrome followed by recurrent respiratory failure episodes, hypopituitarism and neurological abnormalities. Lung histology and ultrastructure were assessed by surgical biopsy. Surfactant-related genes were studied by direct genomic DNA sequencing and array chromatine genomic hybridization (aCGH). Surfactant protein expression in lung tissue was analyzed by confocal immunofluorescence microscopy. For kinetics studies, surfactant protein B and disaturated phosphatidylcholine (DSPC) were isolated from serial tracheal aspirates after intravenous administration of stable isotope-labeled 2H2O and 13C-leucine; fractional synthetic rate was derived from gas chromatography/mass spectrometry 2H and 13C enrichment curves. Six intubated infants with no primary lung disease were used as controls. Results Lung biopsy showed desquamative interstitial pneumonitis and lamellar body abnormalities suggestive of genetic surfactant deficiency. Genetic studies identified a heterozygous ABCA3 mutation, L941P, previously unreported. No SFTPB, SFTPC or NKX2.1 mutations or deletions were found. However, immunofluorescence studies showed TTF-1 prevalently expressed in type II cell cytoplasm instead of nucleus, indicating defective nuclear targeting. This pattern has not been reported in human and was not found in two healthy controls and in five ABCA3 mutation carriers. Kinetic studies demonstrated a marked reduction of SP-B synthesis (43.2 vs. 76.5 ± 24.8%/day); conversely, DSPC synthesis was higher (12.4 vs. 6.3 ± 0.5%/day) compared to controls, although there was a marked reduction of DSPC content in tracheal aspirates (29.8 vs. 56.1 ± 12.4% of total phospholipid content). Conclusion Defective TTF-1 signaling may result in profound surfactant homeostasis disruption and neonatal/pediatric diffuse lung disease. Heterozygous ABCA3 missense mutations may act as disease modifiers in other genetic surfactant defects.

Background/aims Alveolar capillary dysplasia (ACD) is characterised by pulmonary veins misalignment, capillary paucity and alveolar misdevelopment, and caused by FOXF1 mutations only in 40% of cases. Objectives were 1. to identify known and new gene defects and 2. to correlate them with molecular/cellular mechanisms. Methods We recruited a cohort of 23 pathology-confirmed cases. When DNA was available, genome-wide copy number variation was analysed through Array Comparative Genomic Hybridization (aCGH). Mutations were tested by direct sequencing of FOXF1 and candidate genes identified by aCGH; Molecular pathways were analysed by multi-channel immunofluorescence microscopy of ACD cases compared to human fetal/neonatal lung tissue at various development stages. Results Genomic deletions or mutations were identified in 57% of tested cases. Besides FOXF1, two of the genes involved stand out as potential candidates: MEOX2 and TBX4. ACD cases showed a markedly decreased expression of c-kit, a marker expressed in pulmonary small arteries and capillaries in fetal lung controls. In normal fetal lungs FOXF1 and TBX4 were prevalently expressed at the mesenchymal-epithelial border, and MEOX2 in pulmonary vascular smooth muscle cells (PVSMC). Their expression pattern and intensity were altered in all ACD cases, indicating that decreased FOXF-1 and/or its downstream transcription factor TBX4 disrupt lung micro vessel formation and homing to alveolar epithelium, and that a similar phenotype may derive from dysregulated PVSMC proliferation and angiogenesis related to MEOX2 insufficiency. Conclusion Genetic defects affecting the FOXF1 pathway affect the mesenchymal, endothelial and epithelial cross-talk leading to lung developmental disruption, pulmonary hypertension and hypoxic respiratory failure.

Aims: To determine the role of surfactant-related gene anomalies in neonatal/infantile interstitial lung disease. Methods: 22 infants >32weeks/< 1year with unexplained progressive alveolar/interstitial lung disease. SP-B, SP-C and ABCA3 sequencing. Optical (OM) and electronical (EM) microscopy of biopsy/autopsy lung tissue. Surfactant proteins immunofluorescence. Results: 13/22 infants (59.1%) had lamellar bodies (LB) ultrastructural abnormalities suggesting genetic surfactant deficiency. Eleven infants (50.0%) carried ABCA3 mutations. In the 9 remaining infants (40.9%), one had alveolar proteinosis, one had COX-deficiency, one had Niemann-Pick C disease, two had idiopathic pulmonary fibrosis, four remained cryptogenetic. Conclusions: ABCA3 mutations are the most frequent abnormality. Although EM is indicative in most (92%) cases, molecular mechanisms are elusive in >50% of cases, likely involving complex, multiple surfactant-related gene interactions.

Background: Besides surfactant protein B (SP-B), respiratory distress syndrome in term neonates is associated to mutations of ABCA3, a gene encoding a type II pneumocyte protein essential for intracellular surfactant metabolism. Study goal: To correlate mutations in surfactant-related genes with specific tissue/cell structural anomalies and alterations of surfactant-related protein expression. Methods: 4 neonates with primary, progressive respiratory failure unresponsive to exogenous surfactant. Optical microscopy (OM) on formalin-fixed lung biopsy: HE, PAS, Masson trichrome; immunohistochemestry with CD45, CD68, SP-B and SP-C antibodies; immunofluorescence with ABCA3 antibody. Electron microscopy (EM) on Karnowsky-fixed ultrathin sections. Direct sequencing of SP-B, SP-C and ABCA3 genes on PCR-amplified genomic DNA. Results: Case 1, 2 and 3 were term-born infants dead at 68, 48 and 55d; case 4 was a 32-week preterm alive with severe respiratory failure at 4 months. OM: All cases showed marked interstitial thickening with monocyte infiltrate, type II pneumocyte hyperplasia, intra-alveolar proteinaceous material and macrophage accumulation. SP-B and SP-C protein expression was normal. ABCA3, abundantly expressed with a cytoplasmic granular pattern in normal subjects, was either absent (1), low with a diffuse cytoplasmic pattern (2,4), or apparently normal (3). EM: In all cases, type II pneumocyte lamellar bodies appeared much smaller than normal and filled with homogenous material with an eccentric, denser inclusion. Pseudomyelin structures, corresponding to intracellular surfactant, were virtually absent. Molecular genetics: SP-B and SP-C gene sequences were normal. In the ABCA3 gene, homozygous frameshift (1) or double heterozygote missense mutations (2,3,4) were found. Conclusions: ABCA3 deficiency is a rare but relevant cause of progressive respiratory failure in newborns. Various recessive mutations are associated with this phenotype, leading in most cases to absent or aberrant ABCA3 protein expression and/or function. Whereas OM may be nonspecific, EM shows typical anomalies of the lamellar bodies that may contribute to early diagnosis.