Inactivating GNAS variants impair GPCR signaling and cause multiple suture craniosynostosis in humans and zebrafish

Abstract G protein α-subunit (Gαs), encoded by GNAS, mediates GPCR signaling through cAMP second messenger pathways, and plays a pivotal role in craniofacial morphogenesis and osteoblast differentiation. Craniosynostosis, one of the most prevalent craniofacial developmental anomalies, is characterized by the premature fusion of cranial sutures. Here, we identify germline heterozygous variants in GNAS as a novel genetic cause of craniosynostosis. Affected individuals presented with multiple-suture synostosis, recognizable dysmorphic features, brachydactyly, short stature, with or without hormone resistance. We identified three de novo missense variants (c.286A>G;p.K96E, c.758A>G;p.Y253C, and c.691C>T;p.R231C) and one maternally inherited splicing variant (c.1039-2A>G). Functional analyses using bioluminescence resonance energy transfer (BRET) assays compared these variants to well-characterized activating variants p.R201H and p.Q227L. All tested variants impaired trimeric G protein assembly to varying degrees and exhibited reduced coupling with PTHR1. While the p.R201H and p.Q227L variants induced excessive cAMP production, the craniosynostosis-associated variants either displayed decreased basal cAMP levels or reduced agonist-induced cAMP production compared to wild-type, suggesting an inactivating nature. In zebrafish models, heterozygous gnas inactivation recapitulated human phenotypes, including multiple-suture synostosis, craniofacial abnormalities, and short stature. Mechanistically, GNAS haploinsufficiency in human mesenchymal stem cells promoted osteogenic differentiation through disrupted cAMP-CREB signaling, which relieved SMAD6-mediated repression of RUNX2 transcription. This study establishes inactivating GNAS variants as a genetic cause of craniosynostosis, uncovers a disease mechanism linking G protein inactivation to craniosynostosis through defective GPCR signal transduction.