Abstract Rationale Platelet-derived growth factor alpha (PDGFRα) is a receptor tyrosine kinase expressed by fibroblasts and smooth muscle cells, including a short-lived progenitor cell, secondary crest myofibroblasts, which may help explain its crucial role in alveolar septation and elastogenesis during lung development. We identified a parent (affected 1)-child (affected 2) pair with a shared monoallelic variant of unknown significance (VUS) in PDGFRA, c.2084GT; p.Ser695Ile and these phenotypic features: neonatal respiratory insufficiency, early-onset pulmonary emphysema and hemorrhage, pulmonary failure requiring a lung transplant (parent, fourth decade). Lung tissue pathology showed poor alveolarization, dilated distal airways, and elastic fragmentation. We hypothesized that the VUS in PDGFRA mechanistically contributes to early-onset, severe, progressive lung disease via a dominant negative (DN) or atypical gain-of-function (GOF) effect in pulmonary myofibroblasts. Methods Fixed lung tissue was obtained for both cases. Dermal fibroblasts were harvested from cases, grown in culture (standard methods) to subconfluency and serum-starved. Age-matched commercial control dermal fibroblasts (4 per case) were obtained (Corriell). Protein quantification, sample preparation, and gel electrophoresis were performed using conventional methods (Neptune Lab). Immunoblotting was performed with phospho-AKT, phospho-ERK, and α-actinin/GAPDH. Protein expression was quantified using ImageJ. Cell viability was assessed via Alamar Blue assay via microplate reader. Cell migration was assessed using a scratch assay after treatment with PDGF-A, with light microscopy photos taken at various time points, with and without Dasatanib (tyrosine kinase inhibitor) treatment. Immunocytochemistry was done to assess PDGFRα abundance, subcellular localization and cell proportions employing confocal microscopy on lung samples and dermal fibroblasts. Results Dermal fibroblasts from cases and controls all showed robust AKT and ERK activation with PDGFA treatment. Cases showed moderate basal AKT and ERK activation (not evident in 4/4pediatric controls and 3/4adult controls. Dermal fibroblasts from both cases show abnormal basal localization of PDGFRα. Ligand-induced survival of PDGFRα-expressing fibroblasts was reduced for both cases compared with controls. Wounding assays revealed reduced ligand-induced cell migration in both cases compared with controls. Dastanib inhibition reduced cell migration in a dose-dependent manner for both cases and reduced ligand-induced survival of PDGFRα-expressing fibroblasts. Conclusion We have identified abnormalities in receptor localization, cell signaling, cell survival, and wound repair in dermal fibroblasts, providing evidence that the genetic variant PDGFRA, c.2084GT; p.Ser695Ile is contributing to the pulmonary phenotype, possibly through a dominant negative mechanism. This provides a mechanistic basis for abnormalities that are initiated during alveolar formation and enable later progression to severe emphysema. Figure: Proposed consequences of PDGFRA VUS This abstract is funded by: Doris Duke Foundation
Sadreameli et al. (Fri,) studied this question.