ABCA3 Mutations Research Articles

Fatal Familial Lung Disease Caused by ABCA3 Deficiency without Identified ABCA3 Mutations

To test the hypothesis that some functionally significant variants in the gene encoding member A3 of the ATP Binding Cassette family (ABCA3) are not detected using exon-based sequencing approaches. The first of 2 female siblings who died from neonatal respiratory failure was examined for mutations with sequence analysis of all ABCA3 exons and known regulatory elements within the 5' untranslated region. Lung tissue from both siblings was immunostained for ABCA3 and examined with electron microscopy. Segregation of ABCA3 alleles was determined with analysis of polymorphisms in the parents and all children. No mutations were identified with ABCA3 sequence analysis in the first affected infant. Affected siblings were concordant for their ABCA3 alleles, but discordant from those of their unaffected siblings. ABCA3 protein was not detectable with immunostaining in lung tissue samples from both affected infants. Electron microscopy demonstrated small, dense lamellar bodies, characteristically seen with ABCA3 mutations. The segregation of ABCA3 alleles, absence of ABCA3 immunostaining, lung pathology, and ultrastructural findings support genetic ABCA3 deficiency as the cause of lung disease in these 2 infants, despite the lack of an identified genetic variant.

ABCA3 mutations influence the stress level and immunological function of lung epithelial A549 cells

Einleitung: ABCA3 is an ABC-transporter in the outer membrane of lamellar bodies (LBs) in alveolar epithelial type II cells (ATII) involved in the surfactant lipid transport. Mutations in the ABCA3 gene have been associated with genetic interstitial lung disease (ILD). Next to being essential for surfactant secretion, ATII are an active part of the lung immunological system and involved in lung repair. We investigate how ABCA3 mutations influence ATII cell homeostasis, primary the stress level and immunological function, and potential involvement of ATII cells in epithelial-mesenchymal transition (EMT). Methoden: A549 cells, a widely accepted ATII model, were stably transfected with vectors expressing HA-tagged WT ABCA3 protein and Q215K and E292V mutations. Initially, protein localization and processing features were analysed by immunofluorescence, immunoblotting and deglycosylation assay. Cellular stress was assayed by BiP immunoblotting and RT-PCR of XBP-1 mRNA splicing. To mimic viral and bacterial respiratory infections, cells were infected with respiratory syncitial virus (RSV) or treated with P. aeruginosa LPS. The immunological answer was measured by quantitative PCR of TLR3 and TLR4 receptors and associated cytokines (IL-6, IL-8, RANTES and GM-CSF). Ergebnisse: While WT and E292V proteins localized to LBs, Q215K protein was retained inside of the ER, indicating a misfolding defect. Also, ABCA3 mutations increased intracellular stress level. Compared to WT, mutants displayed a changed immunological status already in the absence of infectious agents with significantly lower gene expression of TLR3 and TLR4. After exposure to RSV and LPS, mutant lines were unable to upregulate TLR receptors, failing to become sensitized to further infections. Moreover, cytokine mRNA expression prior to infection and cytokine answer after exposure to infections were changed in mutants. Interestingly, RSV infection triggered a morphological transformation of Q215K and E292V cells from epithelial cells to fibroblasts. Diskussion: Besides elevating ER stress, we propose that non-functional ABCA3 protein can affect several important functions of ATII. Our initial data suggest that ABCA3 mutations can alter the immunological answer of ATII and make them more prone to EMT, leading to the speculation that stressed and non-functional ATII cells are a possible origin of fibrotic foci in genetic ILD. If all described is a secondary phenomenon to ABCA3, and if and to which extent raised ER stress level might be a connection between mutated ABCA3 and our immunology/EMT-related observations remains to be investigated. In addition, it would be necessary to confirm these data in patient samples and prove the significance on disease onset and progression.

Inherited Surfactant Deficiency Caused by Uniparental Disomy of Rare Mutations in the Surfactant Protein-B and ATP Binding Cassette, Subfamily A, Member 3 Genes

To characterize inheritance of homozygous, rare, recessive loss-of-function mutations in surfactant protein-B (SFTPB) or ATP binding cassette, subfamily A, member 3 (ABCA3) genes in newborns with lethal respiratory failure. We resequenced genes from parents whose infants were homozygous for mutations in SFTPB or ABCA3. For infants with only 1 heterozygous parent, we performed microsatellite analysis for chromosomes 2 (SFTPB) and 16 (ABCA3). We identified 1 infant homozygous for the g.1549C > GAA mutation (121ins2) in SFTPB for whom only the mother was heterozygous and 3 infants homozygous for mutations in ABCA3 (p.K914R, p.P147L, and c.806_7insGCT) for whom only the fathers were heterozygous. For the SP-B-deficient infant, microsatellite markers confirmed maternal heterodisomy with segmental isodisomy. Microsatellite analysis confirmed paternal isodisomy for the 3 ABCA3-deficient infants. Two ABCA3-deficient infants underwent lung transplantation at 3 and 5 months of age, respectively, and 2 infants died. None exhibited any nonpulmonary phenotype. Uniparental disomy should be suspected in infants with rare homozygous mutations in SFTPB or ABCA3. Confirmation of parental carrier status is important to provide recurrence risk and to monitor expression of other phenotypes that may emerge through reduction to homozygosity of recessive alleles.

Serum KL-6 differentiates neuroendocrine cell hyperplasia of infancy from the inborn errors of surfactant metabolism

Background:The study was conducted in order to determine if the glycoprotein KL-6 is a useful biomarker in differentiating neuroendocrine cell hyperplasia of infancy (NEHI), a benign form of children’s interstitial...

Clinical, radiological and pathological features of ABCA3 mutations in children

Clinical, radiological and pathological features of ABCA3 mutations in children

Clinical, radiological and pathological features of ABCA3 mutations in children

Clinical, radiological and pathological features of ABCA3 mutations in children

Aberrant catalytic cycle and impaired lipid transport into intracellular vesicles in ABCA3 mutants associated with nonfatal pediatric interstitial lung disease

The ATP-binding cassette transporter ABCA3 mediates uptake of choline-phospholipids into intracellular vesicles and is essential for surfactant metabolism in lung alveolar type II cells. We have shown previously that ABCA3 mutations in fatal surfactant deficiency impair intracellular localization or ATP hydrolysis of ABCA3 protein. However, the mechanisms underlying the less severe phenotype of patients with ABCA3 mutation are unclear. In this study, we characterized ABCA3 mutant proteins identified in pediatric interstitial lung disease (pILD). E292V (intracellular loop 1), E690K (adjacent to Walker B motif in nucleotide binding domain 1), and T1114M (8th putative transmembrane segment) mutant proteins are localized mainly in intracellular vesicle membranes as wild-type protein. Lipid analysis and sucrose gradient fractionation revealed that the transport function of E292V mutant protein is moderately preserved, whereas those of E690K and T1114M mutant proteins are severely impaired. Vanadate-induced nucleotide trapping and photoaffinity labeling of wild-type and mutant proteins using 8-azido-[(32)P]ATP revealed an aberrant catalytic cycle in these mutant proteins. These results demonstrate the importance of a functional catalytic cycle in lipid transport of ABCA3 and suggest a pathophysiological mechanism of pILD due to ABCA3 mutation.

Population and Disease-Based Prevalence of the Common Mutations Associated With Surfactant Deficiency

The prevalence of the common mutations in the surfactant protein-B (121ins2), surfactant protein-C (I73T), and ATP-binding cassette member A3 (E292V) genes in population-based or case-control cohorts of newborn respiratory distress syndrome (RDS) is unknown. We determined the frequencies of these mutations in ethnically diverse population and disease-based cohorts using restriction enzyme analysis (121ins2 and E292V) and a 5' nuclease assay (I73T) in DNA samples from population-based cohorts in Missouri, Norway, South Korea, and South Africa, and from a case-control cohort of newborns with and without RDS (n = 420). We resequenced the ATP-binding cassette member A3 gene (ABCA3) in E292V carriers and computationally inferred ABCA3 haplotypes. The population-based frequencies of 121ins2, E292V, and I73T were rare (<0.4%). E292V was present in 3.8% of newborns with RDS, a 10-fold greater prevalence than in the Missouri cohort (p < 0.001). We did not identify other loss of function mutations in ABCA3 among patients with E292V that would account for their RDS. E292V occurred on a unique haplotype that was derived from a recombination of two common ABCA3 haplotypes. E292V was over-represented in newborns with RDS suggesting that E292V or its unique haplotype impart increased genetic risk for RDS.

Cerebropulmonary dysgenetic syndrome

Ventilatory treatment of neonatal respiratory distress often results in bronchopulmonary dysplasia from congenital surfactant deficiency due to mutants of transporter protein ABCA3. Association of this condition with other severe disorders in premature newborns has not heretofore been reported. A neonatal autopsy included an in vivo whole blood sample for genetic testing. Autopsy revealed severe interstitial pulmonary fibrosis at age 8 days with heterozygotic mutation p.E292V of ABCA3 and severe dystrophic retardation of cerebral cortex and cerebellum. Subsequently, 1300 archival neonatal autopsies, 1983–2006, were reviewed for comparable concurrent findings and bronchopulmonary dysplasia or retarded cerebral dystrophy lacking the other principal feature of this syndrome. Archival review revealed four similar cases and eight less so, without gene analysis. Further review for bronchopulmonary dysplasia revealed 59 cases, 1983–2006. Several other examples of similar retarded migration of germinal matrix and underdevelopment of cortical mantle, without pulmonary lesions of this type, were identified. The determination of an ABCA3 mutation in one case of severe pulmonary fibrosis with significant dystrophy of the brain and the identification of four highly similar archival cases and eight others with partial pathological findings supports the designation of an independent disorder, here referred to as the cerebropulmonary dysgenetic syndrome.

Hereditary disorders of surfactant homeostasis cause acute and chronic lung disease in infancy

In this issue of Thorax , Doan et al 1 report the clinical and pathological findings from nine paediatric patients with severe acute and chronic lung disease caused by mutations...

Clinical, radiological and pathological features of ABCA3 mutations in children

Background:Mutations in the ABCA3 gene can result in fatal surfactant deficiency in term newborn infants and chronic interstitial lung disease in older children. Previous studies on ABCA3 mutations have focused...

Ultrastructural and molecular analysis in fatal neonatal interstitial pneumonia caused by a novel ABCA3 mutation

Pulmonary surfactant is essential to maintain alveolar patency, and invariably fatal neonatal lung disease has been recognized to involve mutations in the genes encoding surfactant protein-B or ATP-binding cassette transporter family member ABCA3. The lipid transporter ABCA3 targets surfactant phospholipids to lamellar bodies that are lysosomal-derived organelles of alveolar type II cells. ABCA3−/− mice have grossly reduced surfactant phosphatidyl glycerol levels and die of respiratory failure soon after birth. We studied lung biopsy samples of two siblings with a novel homozygous ABCA3 mutation at nucleotide position 578 (c.578C>G), leading to a Pro193Arg amino-acid exchange, who died at 55 and 105 days of age. Light microscopy revealed thickened alveolar septa with abundant myxoid interstitial matrix, marked hyperplasia of type II pneumocytes, desquamation of alveolar macrophages and focal alveolar proteinosis. Surfactant protein-B was detected by immunohistochemistry after antigen retrieval. Transmission electron microscopy showed rare cytoplasmic inclusions with concentric membranes and eccentrically placed electron-dense aggregates. These ‘fried-egg'-appearing lamellar bodies differed both from normal lamellar bodies and the larger, poorly formed composite bodies with multiple vesicular inclusions observed in surfactant protein-B deficiency. In conclusion, our findings underscore that the implications of interstitial lung disease in infant lungs differ from those in adults. In infants with a desquamative interstitial pneumonitis pattern, surfactant or ABCA3 mutations should be evaluated. Importantly, these findings support the notion that electron microscopy is useful in distinguishing between surfactant protein-B and ABCA3 deficiency, and has an important role in evaluating biopsies or autopsies of term infants with unexplained severe respiratory failure and interstitial lung disease.

Diffuse lung disease in young children: application of a novel classification scheme.

Considerable confusion exists regarding nomenclature, classification, and management of pediatric diffuse lung diseases due to the relative rarity and differences in the spectrum of disease between adults and young children. A multidisciplinary working group was formed to: (1) apply consensus terminology and diagnostic criteria for disorders presenting with diffuse lung disease in infancy; and (2) describe the distribution of disease entities, clinical features, and outcome in young children who currently undergo lung biopsy in North America. Eleven centers provided pathologic material, clinical data, and imaging from all children less than 2 years of age who underwent lung biopsy for diffuse lung disease from 1999 to 2004. Multidisciplinary review categorized 88% of 187 cases. Disorders more prevalent in infancy, including primary developmental and lung growth abnormalities, neuroendocrine cell hyperplasia of infancy, and surfactant-dysfunction disorders, constituted the majority of cases (60%). Lung growth disorders were often unsuspected clinically and under-recognized histologically. Cases with known surfactant mutations had characteristic pathologic features. Age at biopsy and clinical presentation varied among categories. Pulmonary hypertension, presence of a primary developmental abnormality, or ABCA3 mutation was associated with high mortality, while no deaths occurred in cases of pulmonary interstitial glycogenosis, or neuroendocrine cell hyperplasia of infancy. This retrospective cohort study identifies a diverse spectrum of lung disorders, largely unique to young children. Application of a classification scheme grouped clinically distinct patients with variable age of biopsy and mortality. Standardized terminology and classification will enhance accurate description and diagnosis of these disorders.

Heterozygosity for ABCA3 Mutations Modifies the Severity of Lung Disease Associated with a Surfactant Protein C Gene (SFTPC) Mutation

Heterozygous SFTPC mutations have been associated with adult and pediatric interstitial lung disease (pILD). Inheritance is autosomal dominant, but de novo mutations may cause sporadic disease. SFTPC mutations have been associated with variable onset of symptoms, ranging from early infancy to late adulthood. The underlying mechanisms for this variability are unknown. Recently, mutations in ABCA3 (encoding member A3 of the adenosine triphosphate-binding cassette family of transporters) were identified as a cause of pILD. To test the hypothesis that ABCA3 mutations modify the severity of lung disease in individuals with SFTPC mutations, we sequenced ABCA3 from four symptomatic infants with the same SFTPC mutation, a substitution of isoleucine by threonine in codon 73 (I73T). Each infant developed respiratory symptoms by 2 mo of age and inherited the mutation from an asymptomatic parent. Three of the four infants were also heterozygous for an ABCA3 mutation, which was inherited from the parent without SFTPC I73T. The finding of heterozygosity for ABCA3 mutations in severely affected infants with SFTPC I73T, and independent inheritance from disease-free parents supports that ABCA3 acts as a modifier gene for the phenotype associated with an SFTPC mutation.

Heterozygous ABCA3 mutation associated with non-fatal evolution of respiratory distress

A boy without symptoms up to 12 months of age started with persisting cough followed by respiratory failure at 18 months of age, resulting in mechanical ventilation because of alveolar proteinosis. Lung biopsy showed PAS-positive material. PCR was negative for CMV, Pneumocystis jiroveci and adenovirus. BALF showed mature SP-B. Analysis of the ATP-binding cassette transporter A3 (ABCA3; OMIM 601615) gene showed a compound heterozygous mutation from paternal W1148X and maternal T1114A. Alveolar lavage with 720 mg of bovine surfactant allowed weaning from ventilator support. Heterozygous mutation in the ABCA3 gene could be associated with a milder evolution as compared to the homozygous frequently lethal evolution.

Targeted inactivation of the murine Abca3 gene leads to respiratory failure in newborns with defective lamellar bodies

Mutations in the human ABCA3 gene, encoding an ABC-transporter, are associated with respiratory failure in newborns and pediatric interstitial lung disease. In order to study disease mechanisms, a transgenic mouse model with a disrupted Abca3 gene was generated by targeting embryonic stem cells. While heterozygous animals developed normally and were fertile, individuals homozygous for the altered allele ( Abca3−/−) died within one hour after birth from respiratory failure, ABCA3 protein being undetectable. Abca3−/− newborns showed atelectasis of the lung in comparison to a normal gas content in unaffected or heterozygous littermates. Electron microscopy demonstrated the absence of normal lamellar bodies in type II pneumocytes. Instead, condensed structures with apparent absence of lipid content were found. We conclude that ABCA3 is required for the formation of lamellar bodies and lung surfactant function. The phenotype of respiratory failure immediately after birth corresponds to the clinical course of severe ABCA3 mutations in human newborns.

Alteration of the Pulmonary Surfactant System in Full-Term Infants with Hereditary ABCA3 Deficiency

ABCA3 mutations are known to cause fatal surfactant deficiency. We studied ABCA3 protein expression in full-term newborns with unexplained respiratory distress syndrome (URDS) as well as the relevance of ABCA3 mutations for surfactant homeostasis. Lung tissue of infants with URDS was analyzed for the expression of ABCA3 in type II pneumocytes. Coding exons of the ABCA3 gene were sequenced. Surfactant protein expression was studied by immunohistochemistry, immunoelectron microscopy, and Western blotting. ABCA3 protein expression was found to be greatly reduced or absent in 10 of 14 infants with URDS. Direct sequencing revealed distinct ABCA3 mutations clustering within vulnerable domains of the ABCA3 protein. A strong expression of precursors of surfactant protein B (pro-SP-B) but only low levels and aggregates of mature surfactant protein B (SP-B) within electron-dense bodies in type II pneumocytes were found. Within the matrix of electron-dense bodies, we detected precursors of SP-C (pro-SP-C) and cathepsin D. SP-A was localized in small intracellular vesicles, but not in electron-dense bodies. SP-A and pro-SP-B were shown to accumulate in the intraalveolar space, whereas mature SP-B and SP-C were reduced or absent, respectively. Our data provide evidence that ABCA3 mutations are associated not only with a deficiency of ABCA3 but also with an abnormal processing and routing of SP-B and SP-C, leading to severe alterations of surfactant homeostasis and respiratory distress syndrome. To identify infants with hereditary ABCA3 deficiency, we suggest a combined diagnostic approach including immunohistochemical, ultrastructural, and mutation analysis.

Functional and Trafficking Defects in ATP Binding Cassette A3 Mutants Associated with Respiratory Distress Syndrome

Members of the ATP binding cassette (ABC) protein superfamily actively transport a wide range of substrates across cell and intracellular membranes. Mutations in ABCA3, a member of the ABCA subfamily with unknown function, lead to fatal respiratory distress syndrome (RDS) in the newborn. Using cultured human lung cells, we found that recombinant wild-type hABCA3 localized to membranes of both lysosomes and lamellar bodies, which are the intracellular storage organelles for surfactant. In contrast, hABCA3 with mutations linked to RDS failed to target to lysosomes and remained in the endoplasmic reticulum as unprocessed forms. Treatment of those cells with the chemical chaperone sodium 4-phenylbutyrate could partially restore trafficking of mutant ABCA3 to lamellar body-like structures. Expression of recombinant ABCA3 in non-lung human embryonic kidney 293 cells induced formation of lamellar body-like vesicles that contained lipids. Small interfering RNA knockdown of endogenous hABCA3 in differentiating human fetal lung alveolar type II cells resulted in abnormal, lamellar bodies comparable with those observed in vivo with mutant ABCA3. Silencing of ABCA3 expression also reduced vesicular uptake of surfactant lipids phosphatidylcholine, sphingomyelin, and cholesterol but not phosphatidylethanolamine. We conclude that ABCA3 is required for lysosomal loading of phosphatidylcholine and conversion of lysosomes to lamellar body-like structures.

166 The E292V Variant in ABCA3 is Over-Represented in Newborns with Respiratory Distress Syndrome (RDS)

Background: ABCA3 is an ATP Binding Cassette protein localized to the limiting membrane of lamellar bodies in alveolar Type II cells. Recessive mutations in ABCA3 disrupt surfactant composition and function and cause lethal respiratory distress in newborns. A valine for glutamic acid substitution at codon 292 (E292V) has been found in unrelated individuals with chronic respiratory insufficiency.Objective: To determine the prevalence of the E292V variant in infants with and without RDS. Design/Methods: We obtained genomic DNA and clinical data from an unselected cohort of newborns with RDS (RDS, n=125, gestational age [GA] 31±5 wks), newborns with RDS referred for evaluation of familial or atypical RDS (REF, n=56, GA 34±5 wks), and normal newborns (noRDS, n=167, GA 39±2 wks). We amplified a 637 base pair product that included codon 292 and subjected amplicons to BsrG1 restriction analysis and agarose gel electrophoresis.Results: Excluding 1 each of concordant twins in the REF and noRDS groups, 3 RDS (2.4%), 5 REF (9.1%), and 2 noRDS (1.2%) infants were heterozygous for E292V (P=0.009). Males and females were equally represented. One infant with E292V in the noRDS group was Black, all others with E292V were White. Symptomatic infants (REF+RDS) with E292V were more mature than those without E292V (35±3 vs 32±5 wks, P=0.007). Duration of mechanical ventilation, and need for supplemental oxygen were not significantly different. Infants with RDS and E292V developed pneumothoraces more frequently (78%) than infants with RDS but without E292V (9%) (P<0.001). Six infants had resolution of symptoms within 1 month of birth; the noRDS individuals with E292V remained asymptomatic.Conclusions: The E292V variant in ABCA3 is significantly more prevalent in infants with RDS than in asymptomatic controls. Patients heterozygous for E292V come to clinical attention by presenting with RDS that is more severe than anticipated for gestational age. E292V may be a genetic influence for the risk and severity of RDS.

ABCA3 mutations associated with pediatric interstitial lung disease.

ABCA3 is a member of the ATP-binding cassette family of proteins that mediate the translocation of a wide variety of substrates, including lipids, across cellular membranes. Mutations in the gene encoding ABCA3 were recently identified in full-term neonates with fatal surfactant deficiency. To test the hypothesis that ABCA3 mutations are not always associated with fatal neonatal lung disease but are a cause of pediatric interstitial lung disease. DNA samples were obtained from 195 children with chronic lung disease of unknown etiology. The 30 coding exons of the ABCA3 gene were sequenced in four unrelated children with a referring diagnosis of desquamative interstitial pneumonitis and who were older than 10 years at the time of enrollment. Three of four patients (ages 16, 23, and 11 years) with desquamative interstitial pneumonitis had ABCA3 mutations identified on both alleles. All three had the same missense mutation (E292V) and a second unique mutation. The E292V mutation was not found on 200 control alleles from adults without lung disease, but seven additional patients of the remaining study patients had the E292V mutation on one allele. Immunohistochemical analysis of surfactant protein expression in three patients revealed a specific staining pattern for surfactant protein-B, which was the same pattern observed in several infants with fatal lung disease due to ABCA3 mutations. ABCA3 mutations cause some types of interstitial lung disease in pediatric patients.