Generation of a P4hbY393C mouse model of cole-carpenter syndrome and therapeutic proof-of-concept.

Abstract

Cole-Carpenter syndrome (CCS) is a rare autosomal dominant skeletal disorder characterized by severe bone fragility, recurrent fractures, craniosynostosis, and distinctive craniofacial abnormalities. Pathogenic variants in P4HB, encoding protein disulfide isomerase A1 (PDIA1), represent the most frequent cause of CCS. The recurrent missense mutation p.Tyr393Cys (Y393C) has been identified in unrelated patients, yet the in vivo consequences of this mutation on skeletal biology and its therapeutic implications remain poorly understood. Here, we generated a mouse model carrying the mouse homolog P4hbY393C mutation. Comprehensive skeletal phenotyping revealed pronounced osteopenia and reduced local bone quality in mutant mice across multiple ages. Consistently, CCS mice exhibited reduced circulating levels of procollagen type I N-terminal propeptide (PINP I), indicating decreased type I collagen biosynthesis. At the cellular level, primary osteoblasts isolated from CCS mice showed lower expression and secretion of type I collagen, along with decreased expression of osteocalcin and higher mineralization. On the other hand, osteoclast differentiation was increased. Given the lack of disease-modifying therapies for CCS, we explored different therapeutic strategies. We performed a functional screen of an FDA-approved drug library identifying four compounds that significantly enhanced type I collagen secretion. Finally, we developed an allele-specific RNA interference (siRNAs) approach and identified siRNA sequences capable of selectively silencing the mutant P4hb allele in vitro and ex vivo without cytotoxic effects. Collectively, our study establishes a robust preclinical model for P4hb-related CCS, defines defective type I collagen biosynthesis as a central pathogenic mechanism, and identifies promising therapeutic strategies with translational potential for this currently untreatable skeletal disorder.