Anti-ABCA3 polyclonal antibody

Mutations in genes encoding surfactant protein B (SP-B), ATP-binding cassette transporter A3 (ABCA3) and surfactant protein C (SP-C) can result in neonatal and pediatric lung disease. We retrospectively reviewed 391 molecular analyses of genes encoding SP-B (SFTPB), SP-C (SFTPC) and ABCA3 (ABCA3) performed in our laboratory from 2000 to 2015 in term and preterm newborn infants with severe respiratory distress syndrome (RDS), infants and children with interstitial lung disease (ILD), chorionic villi for prenatal diagnosis, parents and siblings of affected infants. Direct sequencing of SFTPB, SFTPC and ABCA3 was performed on genomic DNA extracted from peripheral blood. Histopathologic, immunohistochemical and ultrastructural analyses were performed when lung tissue was available. Genetic variants in SFTPB, SFTPC, ABCA3 were identified in 71 of 181 (39%) term and preterm newborn infants tested for severe and unexplained RDS and in 38 of 74 (51%) infants and children with ILD. A higher mortality rate was recorded among term newborn infants with homozygous or compound heterozygous mutations in SFTPB and ABCA3. Light microscopy and immunohistochemical analysis of the lung tissue were performed in 11 infants and electron microscopy in 8. Prenatal diagnosis was performed in 8 women with a previous child who died because of ABCA3 deficiency; 2 fetuses affected, 5 carriers and 1 normal were identified. Surfactant dysfunction was identified in a significant number of newborn infants with severe unexplained respiratory failure and children with ILD, indicating the importance of genetic studies in infants and children with this phenotype. While actual treatment is primarily supportive, early identification is important to establish appropriate management and evaluation of treatment options and to offer genetic counselling and prenatal diagnosis.

In the metabolism of pulmonary surfactant, the ATP-binding cassette sub-family A member 3 (ABCA3) is a crucial protein in the formation of the storage compartment for surfactant, the lamellar body (LB), and the transport of phospholipids in it. Mutations in ABCA3 not only disturb surfactant metabolism but also cause chronic interstitial lung diseases. Assays for ABCA3 transport function are needed to investigate pathophysiology of the mutations and treatment options for the patients. We metabolically labeled choline (Cho) head phospholipids with the Cho analogue, propargyl-Cho. The universal incorporation of propargyl-Cho was confirmed by mass spectrometry and labeled lipids were visualized in confocal microscopy by click reaction with an azide fluorophore. After pulse-labeling propargyl-Cho labeled lipids accumulated in ABCA3 + vesicles in a time and concentration dependent manner. When treated with the choline kinase inhibitor MN58b during the first 12 h, the lipids intensity inside ABCA3 + vesicles decreased, whereas intensity was unchanged when treated after 12 h. Miltefosine, a substrate of ABCA3, decreased the incorporation of labeled lipids in ABCA3 + vesicles at all time points. The lipids intensity inside the mutated (p.N568D or p.L1580P) ABCA3 + vesicles was decreased compared to wild type, while the intensity outside of vesicles showed no difference. Propargyl-Cho can metabolically pulse-label Cho phospholipids. Visualization and quantification of fluorescence intensity of the labeled lipids inside ABCA3 + vesicles at equilibrium can specifically assess the transport function of ABCA3.