Pathogenic RAB34 variants impair primary cilium assembly and cause a novel oral-facial-digital syndrome

Abstract

Oral-facial-digital syndromes (OFDS) are a group of clinically and genetically heterogeneous disorders characterized by defects in the development of the face and oral cavity along with digit anomalies. Pathogenic variants in over twenty genes encoding ciliary proteins have been found to cause OFDS through deleterious structural or functional impacts on primary cilia. We identified by exome sequencing bi-allelic missense variants in a novel disease-causing ciliary gene RAB34 in four individuals from three unrelated families. Affected individuals presented a novel form of OFDS (OFDS-RAB34) accompanied by cardiac, cerebral, skeletal, and anorectal defects. RAB34 encodes a member of the Rab GTPase superfamily and was recently identified as a key mediator of ciliary membrane formation. Unlike many genes required for cilium assembly, RAB34 acts selectively in cell types that use the intracellular ciliogenesis pathway, in which nascent cilia begin to form in the cytoplasm. We find that the protein products of these pathogenic variants, which are clustered near the RAB34 C-terminus, exhibit a strong loss of function. Although some variants retain the ability to be recruited to the mother centriole, cells expressing mutant RAB34 exhibit a significant defect in cilium assembly. While many Rab proteins have been previously linked to ciliogenesis, our studies establish RAB34 as the first small GTPase involved in OFDS and reveal the distinct clinical manifestations caused by impairment of intracellular ciliogenesis.

Synchronized Temporal-spatial Analysis via Microscopy and Phoshoproteomics (STAMP) of Quiescence

“…The copyright holder for this preprint this version posted June 26, 2024. ; https://doi.org/10.1101/2024.06. 24.600542 doi: bioRxiv preprint 3 human, non-cancerous, adherent, mononucleated cells, 2) a commonly used model system for studies of ciliogenesis, and 3) readily tractable for several different methodologies and perturbations.…”

“…Note that some of these key signals, including RAB34 and TULP3, have transient localizations inside the primary cilia and/or basal body. 24,25 These findings highlight the value of using synchronized model systems to track temporal-spatial signaling, as such transient signaling events may be missed in other contexts. For example, this is the first evidence, to our knowledge, that IFT-B recruits to basal bodies before IFT-A and that cilia appear to need to be fully formed before GPCR entry.…”

A disease-associated PPP2R3C-MAP3K1 phospho-regulatory module controls centrosome function

“…Silent mutations for sgRNA resistance and/or to encode the L193S mutation were introduced by gene synthesis and Gibson assembly. Stable expression of PPP2R3C-GFP was achieved by replacement of Rab34 with PPP2R3C-GFP in pCW-Pgk-Rab34_hPgk-miRFP-670-Centrin2 70 (used in HeLa cells) and by replacement of Rab8 with PPP2R3C-GFP in pCW-Pgk-mScarlet-Rab8_hPgk-NeoR 71 (used in RPE1 cells). For tetracycline-inducible expression of Cas9-BFP, pCW-Tet-Cas9-BFP was prepared by Gibson assembly from pCW-Cas9 (Addgene #50661).…”