RAB23 loss-of-function mutation causes context-dependent ciliopathy in Carpenter syndrome

The primary cilium is a signal transduction organelle whose dysfunction clinically causes ciliopathies in humans. RAB23 is a small GTPase known to regulate the Hedgehog signalling pathway and ciliary trafficking. Mutations of RAB23 in humans lead to Carpenter syndrome (CS), an autosomal recessive disorder clinically characterized by craniosynostosis, polysyndactyly, skeletal defects, obesity, and intellectual disability. Although the clinical features of CS bear some resemblance to those of ciliopathies, the exact relationship between the pathological manifestations of CS and the ciliary function of RAB23 remains ambiguous. Besides, the in vivo ciliary functions of RAB23 remain poorly characterised. Here, we demonstrate in vivo and in vitro Rab23 loss-of-function mutants modelling CS, including Rab23 conditional knockout (CKO) mouse mutants, CS patient-derived induced pluripotent stem cells (iPSCs), and zebrafish morphants. The Rab23-CKO mutants exhibit multiple developmental and phenotypical traits recapitulating the clinical features of human ciliopathies and CS, indicating a causal link between the loss of Rab23 and ciliopathy. In line with the ciliopathy-like phenotypes, all three different vertebrate mutant models consistently show a perturbation of primary cilia formation, intriguingly, in a context-dependent manner. In vivo examination of primary cilia in Rab23-CKO mutants reveals profound cell-type specific ciliary abnormalities in chondrocytes and neocortical neurons, but not in epithelial cells, cerebellar granule cells and hippocampus neurons. A profound reduction in ciliation frequency and/or shortening of primary cilia was observed in the neurons and neural progenitor cells derived from CS patient iPSCs. Furthermore, Rab23-KO neural progenitor cells were desensitized to primary cilium-dependent activation of the Hedgehog signaling pathway. Collectively, these findings indicate that the absence of RAB23 causes dysfunctional primary cilia in a cell-type distinctive manner, which underlies the pathological manifestations of CS. Our findings present the first in vivo evidence validating the unique context-specific function of RAB23 in the primary cilium. Through the use of patient-derived iPSCs differentiated cells, we present direct evidence of primary cilia anomalies in CS, thereby confirming CS as a ciliopathy disorder.