Functional characterization of four ATP‐binding cassette transporter A3 gene (<i>ABCA3</i>) variants

Hu, June Y.; Yang, Ping; Wegner, Daniel; Heins, Hillary B.; Luke, Cliff J.; Li, Fuhai; White, Frances V.; Silverman, Gary A.; Cole, F. Sessions; Wambach, Jennifer A.

doi:10.1002/humu.24014

Cited by 15 publications

(21 citation statements)

References 40 publications

(122 reference statements)

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“…If the acidic property at this position is well conserved in the ABCA1-A4 subgroup, this is less evident in the second subgroup, to which ABCA3 belongs. Bioinformatics tools all supported a pathogenic effect, consistent with previous in vitro studies which showed partially impaired lipid transport and smaller lamellar bodies [ 23 , 24 ]. This mutation was associated with variable phenotype in homozygous patients, in infants with severe respiratory distress syndrome as well as in an adult with idiopathic pulmonary fibrosis [ 3 , 25 ].…”

Section: Discussionsupporting

confidence: 85%

Structure-Based Understanding of ABCA3 Variants

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Fanen

Callebaut

et al. 2021

IJMS

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ABCA3 is a crucial protein of pulmonary surfactant biosynthesis, associated with recessive pulmonary disorders such as neonatal respiratory distress and interstitial lung disease. Mutations are mostly private, and accurate interpretation of variants is mandatory for genetic counseling and patient care. We used 3D structure information to complete the set of available bioinformatics tools dedicated to medical decision. Using the experimental structure of human ABCA4, we modeled at atomic resolution the human ABCA3 3D structure including transmembrane domains (TMDs), nucleotide-binding domains (NBDs), and regulatory domains (RDs) in an ATP-bound conformation. We focused and mapped known pathogenic missense variants on this model. We pinpointed amino-acids within the NBDs, the RDs and within the interfaces between the NBDs and TMDs intracellular helices (IHs), which are predicted to play key roles in the structure and/or the function of the ABCA3 transporter. This theoretical study also highlighted the possible impact of ABCA3 variants in the cytosolic part of the protein, such as the well-known p.Glu292Val and p.Arg288Lys variants.

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Section: Discussionsupporting

confidence: 85%

Structure-Based Understanding of ABCA3 Variants

Onnée

Fanen

Callebaut

et al. 2021

IJMS

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“…of the different cell lines. We assessed IB, fluorescence-based colocalization, and vesicle ultrastructure of the A549/ABCA3 2/2 cells that stably express individual ABCA3 variants and the adenoviral transduced A549 and ATII cells, as previously described (14,19) (see data supplement). We also assessed vesicle diameter and liposome uptake of the A549/ABCA3 Corrector rescue.…”

Section: Methods Of Functional Characterizationmentioning

confidence: 99%

“…ABCA3 undergoes N-terminal cleavage from a primary translation product to a post-translationally modified product in the multivesicular bodies (34). Mistrafficked ABCA3 proteins that are retained in the endoplasmic reticulum fail to undergo this cleavage, and only the primary translation product is detected (1,(13)(14)(15)(16)(17)(18)(19). IB of mistrafficked ABCA3 mutant proteins demonstrates a single, uncleaved 220-kD band that results from the sum of mobilities of ABCA3 protein (190 kD) and mCherry or GFP (z30 kD).…”

Section: Abca3 Protein Processingmentioning

confidence: 99%

“…Although more than 200 diseaseassociated ABCA3 variants (the majority missense variants) have been identified in symptomatic infants and children, fewer than 10% have been functionally classified as disrupting ABCA3 protein trafficking (type I) or ATPase-mediated phospholipid transport (type II) in cell-based systems (12)(13)(14)(15)(16)(17)(18)(19). The mechanisms of disruption encoded by individual ABCA3 variants are difficult to predict on the basis of location in the gene or the mature protein, and the cell-based models used in these functional studies are not easily scalable for highthroughput screening.…”

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confidence: 99%

“…Although infants with biallelic "null" (nonsense or frameshift) variants present with respiratory failure at birth and die during the first year of life without lung transplantation, the presentation, disease severity, and progression for infants and children with biallelic missense, in-frame insertions or deletions, or splicing variants are variable and difficult to predict (5,21). The basis for this phenotypic variability is not known but may be associated with the specific type of ABCA3 mutant (e.g., disruption of intracellular ABCA3 trafficking or impaired lipid transport by decreased ATP-mediated hydrolysis) (12)(13)(14)(15)(16)(17)(18)(19)22), activation of intracellular stress or degradation pathways (15,23), or other genetic or environmental modifiers.…”

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confidence: 99%

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Functional Genomics of ABCA3 Variants

Wambach

Yang

Wegner

et al. 2020

Am J Respir Cell Mol Biol

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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.

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Biologic characterization of ABCA3 variants in lung tissue from infants and children with ABCA3 deficiency

Wegner

Geurts

et al. 2022

Pediatric Pulmonology

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ABCA3 is a phospholipid transporter protein required for surfactant assembly in lamellar bodies of alveolar type II cells. Biallelic pathogenic ABCA3 variants cause severe neonatal respiratory distress syndrome or childhood interstitial lung disease.However, ABCA3 genotype alone does not explain the diversity in disease presentation, severity, and progression. Additionally, monoallelic ABCA3 variants have been reported in infants and children with ABCA3-deficient phenotypes. The effects of most ABCA3 variants identified in patients have not been characterized at the RNA level. ABCA3 allele-specific expression occurs in some cell types due to epigenetic regulation. We obtained lung tissue at transplant or autopsy from 16 infants and children with ABCA3 deficiency due to compound heterozygous ABCA3 variants for biologic characterization of the predicted effects of ABCA3 variants at the RNA level and determination of ABCA3 allele expression. We extracted DNA and RNA from frozen lung tissue and reverse-transcribed cDNA from mRNA. We performed Sanger sequencing to assess allele-specific expression by comparing the heights of variant nucleotide peaks in amplicons from genomic DNA and cDNA. We found similar genomic and cDNA variant nucleotide peak heights and no evidence of allelespecific expression among explant or autopsy samples with biallelic missense ABCA3 variants (n = 6). We observed allele-specific expression of missense alleles in trans with frameshift (n = 4) or nonsense (n = 1) variants, attributable to nonsensemediated decay. The missense variant c.53 A > G;p.Gln18Arg, located near an exonintron junction, encoded abnormal splicing with skipping of exon 4. Biologic characterization of ABCA3 variants can inform discovery of variant-specific disease mechanisms.

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Functional characterization of four ATP‐binding cassette transporter A3 gene (ABCA3) variants

Cited by 15 publications

References 40 publications

Structure-Based Understanding of ABCA3 Variants

Structure-Based Understanding of ABCA3 Variants

Functional Genomics of ABCA3 Variants

Biologic characterization of ABCA3 variants in lung tissue from infants and children with ABCA3 deficiency

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