title: Functional Characterization of ATP-Binding Cassette Transporter A3 Mutations from Infants with Respiratory Distress Syndrome.
abstract: Mutations in the ATP-binding cassette transporter A3 gene (ABCA3) result in severe neonatal respiratory distress syndrome and childhood interstitial lung disease. As most ABCA3 mutations are rare or private, determination of mutation pathogenicity is often based on results from in silico prediction tools, identification in unrelated diseased individuals, statistical association studies, or expert opinion. Functional biologic studies of ABCA3 mutations are needed to confirm mutation pathogenicity and inform clinical decision making. Our objective was to functionally characterize two ABCA3 mutations (p.R288K and p.R1474W) identified among term and late-preterm infants with respiratory distress syndrome with unclear pathogenicity in a genetically versatile model system. We performed transient transfection of HEK293T cells with wild-type or mutant ABCA3 alleles to assess protein processing with immunoblotting. We used transduction of A549 cells with adenoviral vectors, which concurrently silenced endogenous ABCA3 and expressed either wild-type or mutant ABCA3 alleles (p.R288K and p.R1474W) to assess immunofluorescent localization, ATPase activity, and organelle ultrastructure. Both ABCA3 mutations (p.R288K and p.R1474W) encoded proteins with reduced ATPase activity but with normal intracellular localization and protein processing. Ultrastructural phenotypes of lamellar body-like vesicles in A549 cells transduced with mutant alleles were similar to wild type. Mutant proteins encoded by ABCA3 mutations p.R288K and p.R1474W had reduced ATPase activity, a biologically plausible explanation for disruption of surfactant metabolism by impaired phospholipid transport into the lamellar body. These results also demonstrate the usefulness of a genetically versatile, human model system for functional characterization of ABCA3 mutations with unclear pathogenicity.
authors.affiliation: 1 Edward Mallinckrodt Department of Pediatrics, and.
authors.forename: Jennifer A
authors.initials: JA
authors.lastname: Wambach
authors.affiliation: 1 Edward Mallinckrodt Department of Pediatrics, and.
authors.forename: Ping
authors.initials: P
authors.lastname: Yang
authors.affiliation: 1 Edward Mallinckrodt Department of Pediatrics, and.
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Wegner
authors.affiliation: 1 Edward Mallinckrodt Department of Pediatrics, and.
authors.forename: Hillary B
authors.initials: HB
authors.lastname: Heins
authors.affiliation: Departments of 2 Radiation Oncology and.
authors.forename: Lyudmila N
authors.initials: LN
authors.lastname: Kaliberova
authors.affiliation: Departments of 2 Radiation Oncology and.
authors.forename: Sergey A
authors.initials: SA
authors.lastname: Kaliberov
authors.affiliation: Departments of 2 Radiation Oncology and.
authors.forename: David T
authors.initials: DT
authors.lastname: Curiel
authors.affiliation: 3 Pathology, Washington University School of Medicine, St. Louis, Missouri; and.
authors.forename: Frances V
authors.initials: FV
authors.lastname: White
authors.affiliation: 4 Department of Pediatrics, Northwestern University Feinberg School of Medicine, Chicago, Illinois.
authors.forename: Aaron
authors.initials: A
authors.lastname: Hamvas
authors.affiliation: 1 Edward Mallinckrodt Department of Pediatrics, and.
authors.forename: Brian P
authors.initials: BP
authors.lastname: Hackett
authors.affiliation: 1 Edward Mallinckrodt Department of Pediatrics, and.
authors.forename: F Sessions
authors.initials: FS
authors.lastname: Cole
mesh_terms: D000072283:A549 Cells; D018528:ATP-Binding Cassette Transporters / Q000235:genetics*; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000256:Adenoviridae / Q000378:metabolism; D005455:Fluorescent Antibody Technique; D057809:HEK293 Cells; D006801:Humans; D015151:Immunoblotting; D007223:Infant; D050505:Mutant Proteins / Q000378:metabolism; D009154:Mutation / Q000235:genetics*; D015388:Organelles / Q000378:metabolism / Q000648:ultrastructure; D012127:Respiratory Distress Syndrome, Newborn / Q000235:genetics*; D013347:Subcellular Fractions / Q000378:metabolism
chemical_list: C441524:ABCA3 protein, human; D018528:ATP-Binding Cassette Transporters; D050505:Mutant Proteins; D000251:Adenosine Triphosphatases
keywords: childhood interstitial lung disease; neonatal respiratory distress; respiratory distress syndrome; surfactant