ARL3 Mutations Cause Joubert Syndrome by Disrupting Ciliary Protein Composition

Abstract

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 JBTS-associated genes. Combined autozygosity mapping of both families highlighted a candidate locus on chromosome 10 (chr10: 101569997-109106128, UCSC Genome Browser 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 localization in cilia. We propose that ARL3 provides a potential hub in the network of proteins implicated in ciliopathies, whereby perturbation of ARL3 leads to the mislocalization of multiple ciliary proteins as a result of abnormal displacement of lipidated protein cargo.

Keywords: ARL13B; ARL3; Joubert syndrome; cilia; guanine nucleotide exchange factor; trafficking.

Figure 1

Clinical and Radiological Images of the Affected Members of the Two Families Included in This Study (A) A pedigree of the two families shows the number of affected siblings in each family and the outcome of segregation analysis (affected, shaded; carriers, half-shaded; and WT, unshaded). The proband in each family is indicated by a black arrow. Genotypes for the proband and their siblings are shown. (B–E) Brain MRI of the four affected individuals (B, II:5 in family 1; C, D, and E, II:1, II:4, and II:5 in family 2) in this study shows evidence of a molar tooth sign, cerebellar vermis hypoplasia, and elongation of the superior cerebellar peduncles (arrowed). (F) Facial photo of the proband (II:5) in family 1 shows dysmorphic features (depressed nasal bridge, upturned nares, ptosis, arched eyebrows, synophrys, telecanthus, and low-set ears). (G and H) Ultrasound scan image of the kidneys of the affected member in family 1 (II:5) shows an echogenic left multicystic dysplastic kidney (G) and an unaffected right kidney (H). (I–R) Retinal imaging, including multicolor scanning laser fundal images of the eyes, of the three affected siblings in family 2 (II:1, II:4, and II:5) shows granular alterations of the retinal pigment epithelium and subtle spicule formation, particularly around the major vascular arcades, and arteriolar attenuation (I, II:1; J, II:4; K, II:5). Autofluorescence images show stippled hypo-autofluorescence areas concentrated around the arcades (L, II:1) and hyper-autofluorescence around fovea (M, II:4; N, II:5). Horizontal optical coherence tomography scans demonstrate thinning of the outer nuclear layer and loss of ellipsoid and external limiting membrane lines with preservation of inner retinal lamination in all three siblings (O, II:1; P, II:4; Q, II:5). A horizontal optical coherence tomography scan of a healthy control individual is shown for comparison (R).

Figure 2

Molecular Genetic Investigations of the Two JBTS-Affected Families (A) Genome-wide homozygosity mapping shows the shared homozygous region between the affected members of the two families on chromosome 10 (blue rectangle). Regions of homozygosity are shown in red, and the position of ARL3 is marked with a black arrow. (B) Schematic representation to ARL3 with the homozygous missense variants located in exon 5. (C) Evolutionary conservation of residue Arg149, which is highly conserved throughout all species shown except D. melanogaster. (D) Sequence chromatograms of the two different ARL3 variants described in this study.

Figure 3

The Human ARL13B-ARL3 Complex Is Predicted to Involve an Interaction between Evolutionarily Conserved Glutamate and Arginine Residues (A) Partial amino acid sequence alignments of the ciliary GEF, ARL13B, and ARL3. Highlighted in red are the evolutionarily conserved glutamate residue located in the switch II domain of ARL13B (E86 [Glu86] in C. reinhardtii ARL13B [CrARL13B]) and the arginine residue in the loop region between the α4 and β6 domains of ARL3 (R148 [Arg148] in CrARL3). (B) Superimposition of the crystal structures of ARL3 (gray) in complex with its known interactors: the effectors UNC119A (salmon; PDB: 4GOJ¹⁵) and BARTL1 (yellow; PDB: 4ZI2²¹), the GAP RP2 (orange; PDB: 3BH6¹⁷), and GEF ARL13B (blue; PDB: 5DI3¹⁹). On the right side is a zoomed-in view of the salt bridge between Glu86 and Arg148 at the CrARL13B-CrARL3 complex interface. (C) Assay of GEF activity for murine WT ARL3 (ARL3^WT) and p.Arg149His ARL3 (ARL3^R149H). Fluorescence polarization was measured for 1 μM mantGDP-loaded ARL3, to which 400 μM GppNHp and 5 μM H. sapiens ARL13B (HsARL13B) were added. Nucleotide exchange was shown by only ARL3^WT. (D) Assay of GEF activity with fluorescence polarization measurements of 0.5 μM mantGDP-loaded CrARL3^WT and CrARL3^R148H, to which 10 μM GppNHp (GTP analog) and 5 μM CrARL13B⋅GppNHp were added at the indicated time points. Only CrARL3^WT showed nucleotide exchange, as indicated by the drop in fluorescence polarization. (E) Assay of GEF activity with fluorescence polarization measurements of 0.5 μM mantGDP-loaded CrARL3^WT and 5 μM CrARL13B⋅GppNHp^WT or CrArl13b^E86R, to which 10 μM GppNHp (GTP analog) was added at the indicated time points. Only CrArl13b^WT showed nucleotide exchange, as indicated by the drop in fluorescence polarization. (F) 30 μg of full-length UNC119A-GST was used to pull down 60 μg of murine ARL3^WT and ARL3^R149H that were loaded with the GTP analog GppNHp. Proteins were detected on immunoblots with anti-GST (red) and anti-His (green) antibodies.

Figure 4

Characterization of Ciliary Phenotype in ARL3-Mutant Fibroblasts from Family 2 (A and C) Affected and control fibroblasts were observed under high-power immunofluorescence for determining ciliary expression of (A) INPP5E and (C) NPHP3. Cilia were localized with anti-ARL13B (red) and anti-PERICENTRIN (magenta) for the identification of the ciliary membrane and the base of cilia, respectively. Scale bars, 10 μm. (B) Quantification of ciliary localization of INPP5E (^∗∗p < 0.0001, unpaired t test, n > 150 cilia for each group). Total cilia INPP5E in control fibroblast (II:3) cilia is higher than in heterozygous fibroblast (I:1 and II:2) cilia. (D) Quantification of ciliary localization of NPHP3 (^∗∗p < 0.0001, unpaired t test, n > 150 cilia for each group).

Figure 5

Model of GSF-Cargo Release in Cilia with WT ARL3 versus p.Arg149 ARL3 Missense Variants ARL13B assists ARL3 in cilia to exchange its bound GDP to GTP. The specific localization of ARL13B in the cilia creates a high concentration of ARL3-GTP. ARL3-GTP in turn can release the cargo bound to its cognate GSF, resulting in ciliary localization. Missense variants of ARL3 (including p.Arg149His and p.Arg149Cys) are not able to interact with ARL13B, and the ARL3GTP concentration is therefore low in the cilia, resulting in inefficient release of GSF cargo in the cilia.