Functional Genomics of ABCA3 Variants

Abstract

Rare or private, biallelic variants in the ABCA3 (ATP-binding cassette transporter A3) gene are the most common monogenic cause of lethal neonatal respiratory failure and childhood interstitial lung disease. Functional characterization of fewer than 10% of over 200 diseaseassociated ABCA3 variants (majority missense) suggests either disruption of ABCA3 protein trafficking (type I) or of ATPasemediated phospholipid transport (type II). Therapies remain limited and nonspecific. A scalable platform is required for functional characterization of ABCA3 variants and discovery of pharmacologic correctors. To address this need, we first silenced the endogenous ABCA3 locus in A549 cells with CRISPR/Cas9 genome editing. Next, to generate a parent cell line (A549/ABCA3 2/2 ) with a single recombination target site for genomic integration and stable expression of individual ABCA3 missense variant cDNAs, we used lentiviral-mediated integration of a LoxFAS cassette, FACS, and dilutional cloning. To assess the fidelity of this cell-based model, we compared functional characterization (ABCA3 protein processing, ABCA3 immunofluorescence colocalization with intracellular markers, ultrastructural vesicle phenotype) of two individual ABCA3 mutants (type I mutant, p.L101P; type II mutant, p.E292V) in A549/ABCA3 2/2 cells and in both A549 cells and primary, human alveolar type II cells that transiently express each cDNA after adenoviral-mediated transduction. We also confirmed pharmacologic rescue of ABCA3 variant-encoded mistrafficking and vesicle diameter in A549/ABCA3 2/2 cells that express p.G1421R (type I mutant). A549/ABCA3 2/2 cells provide a scalable, genetically versatile, physiologically relevant functional genomics platform for discovery of variant-specific mechanisms that disrupt ABCA3 function and for screening of potential ABCA3 pharmacologic correctors.

Expression and Function of ABC Proteins in Fish Intestine

“…ABCA2 is present in lysosome-related organelles, and its expression correlates with genes involved in cholesterol homeostasis ( Szakacs and Abele, 2020 ). ABCA3 takes part in the regulation of pulmonary surfactants ( Szakacs and Abele, 2020 ; Wambach et al, 2020 ). ABCA4 transports retinoid-lipid complexes ( Cremers et al, 2020 ), and ABCA5 and ABCA7 regulate the amyloid-beta peptide production and transport ( Fu et al, 2015 ; Aikawa et al, 2018 ).…”

Genetic Testing for Neonatal Respiratory Disease

“…Though some infants have severe, early-onset and ultimately fatal disease, others have a milder course, and may not even have lung disease at birth [ 13 , 14 , 15 ]. Presumably, sequence variants associated with milder disease allow for some functional ABCA3 to be produced [ 16 , 17 ]. The exact amount of functional ABCA3 needed to prevent lung disease is not known.…”

First Steps toward Personalized Therapies for ABCA3 Deficiency

“…Among the fewer than 10% of disease-associated variants that have been functionally characterized in vitro, 2 mechanistic classes, disruption of intracellular trafficking or disruption of phospholipid transport into the lamellar bodies, have been described. (7,8).…”

“…The authors then validated the results of the phenotypic cell-based assay using established functional small format assays. (7,8) Importantly, this phenotypic screen was able to distinguish between mutants that disrupted intracellular trafficking and those that impaired phospholipid transport.…”

“…Functional characterization of previously identified pathogenic ABCA3 variants using the 'landing pad' system may reduce the time for developing cell lines that express individual ABCA3 variants. (8). Second, while commonly used to study variants in surfactant genes (e.g., ABCA3, SFTPC), A549 cells are derived from adenorcarcinoma alveolar basal epithelial cells and do not express the full repertoire of AEC2 machinery and cellular characteristics.…”