Novel FLCN mutations in Birt-Hogg-Dubé patients and potential intervention of FLCN mRNA

Background: Birt-Hogg-Dubé (BHD) syndrome is an autosomal dominant disorder caused by folliculin ( FLCN ) gene mutations, with ethnically heterogeneous mutational spectra. Current management is primarily supportive and lacks curative therapies. Objectives: This study aimed to characterize the clinical features and genetic variants in two distinct Chinese families with BHD syndrome and to preliminarily explore the feasibility of an mRNA-based protein replacement approach. Design: A family-based prospective cohort study was conducted to identify FLCN mutations in two BHD families carrying rare FLCN variants enrolled in 2023. Methods: Whole-exome sequencing (WES) identified candidate mutations in probands, which were validated by Sanger sequencing (p.W376R and p.Q44*). Structural/functional impacts of p.W376R were analyzed via bioinformatics and qPCR. HEK293T cells were transfected with empty vector, wild-type FLCN , or mutant plasmids (p.W376R and p.Q44*), followed by co-transfection with or without synthetic FLCN mRNA. FLCN expression and mTORC1 signaling were assessed. Results: Affected members in both families predominantly presented with respiratory symptoms, lacking typical skin lesions and kidney tumors. WES identified the FLCN variants p.W376R (previously classified as a Variant of Uncertain Significance, VUS) and a novel nonsense mutation p.Q44*. Genotype–phenotype co-segregation was confirmed for p.W376R. In vitro analyses demonstrated that both mutations reduced FLCN protein expression, leading to mTORC1 hyperactivation. Exogenous FLCN mRNA delivery rescued functional protein expression and reversed mTORC1 dysregulation. Conclusion: We provide co-segregation and functional evidence supporting the reclassification of the FLCN p.W376R variant as pathogenic according to ACMG guidelines and report a novel pathogenic mutation, p.Q44*, expanding the known FLCN mutational spectrum. Importantly, FLCN mRNA supplementation restored FLCN expression in vitro, providing preliminary evidence for mRNA-based therapeutic strategies in BHD syndrome.