Abstract
Abstract
Rationale
Most patients with nonheritable forms of pulmonary arterial hypertension (PAH) lack identifiable pathogenic variants. Increasing evidence suggests that, in addition to high-impact predicted loss-of-function variants, rare missense coding variants may also contribute to disease pathogenesis.
Objectives
We identified ultra-rare missense variants in FOXF1 in two index cases with PAH. FOXF1 encodes a highly conserved transcription factor essential for lung vascular development. We sought to define the frequency of FOXF1 variants, characterize FOXF1 expression, and assess the functional relevance of FOXF1 alterations in PAH.
Methods
Three independent international PAH cohorts were analyzed for predicted deleterious FOXF1 variants. FOXF1 expression was evaluated using bulk and single-cell RNA sequencing and immunofluorescence. Bioinformatic and chromatin-profiling approaches were used to identify potential downstream transcriptional targets, followed by promoter-reporter assays in vitro.
Measurements and Main Results
Nine novel and one rare missense variants, one start-site variant, and one in-frame deletion in FOXF1 were identified across the three cohorts. FOXF1 expression was reduced in PAH lungs and localized predominantly to the endothelial cell compartment. Histopathological analysis revealed heterogeneous FOXF1 expression, with the lowest levels in phenotypically abnormal endothelial cells within complex vascular lesions. Single-cell analyses confirmed reduced endothelial FOXF1 expression in PAH, whereas broad FOXF1 downregulation across endothelial populations was not observed in other chronic lung diseases. Integration of CUT&Tag and ATAC-seq identified a canonical FOXF1-binding motif within the BMPR2 promoter. Promoter-reporter assays further supported FOXF1-dependent transcriptional regulation of BMPR2 and demonstrated reduced transcriptional activity of PAH-associated FOXF1 variants.
Conclusions
Large-scale genomic and transcriptomic analyses support an association between FOXF1 dysregulation and PAH and identify BMPR2 as a potential downstream transcriptional target.