Joubert syndrome (JBTS) is a genetically heterogeneous autosomal recessive neurodevelopmental
ciliopathy. We investigated further the underlying genetic etiology of Joubert syndrome by studying
two unrelated families in whom JBTS was not associated with pathogenic variants in known JBTSrelated
genes. Combined autozygosity mapping of both families highlighted a candidate locus on
chromosome 10 (chr10: 101569997-109106128 (hg 19)), and exome sequencing revealed two
missense variants in ARL3 within the candidate locus. The encoded protein, ADP Ribosylation
Factor-Like GTPase 3, ARL3, is a small GTP-binding protein that is involved in directing lipid-modified
proteins into the cilium in a GTP-dependent manner. Both missense variants replace the highly
conserved Arg149 residue, which we show to be necessary for the interaction with its guanine
nucleotide exchange factor ARL13B, such that the mutant protein is associated with reduced INPP5E
and NPHP3 localisation in cilia. We propose that ARL3 provides a potential hub in the network of
encoded ciliopathy genes, whereby perturbation of ARL3 results in the mislocalisation of multiple
ciliary proteins due to abnormal displacement of lipidated protein cargo
