Mutations in the cilia gene ARL13B lead to the classical form of Joubert syndrome

Abstract

Joubert syndrome (JS) and related disorders are a group of autosomal-recessive conditions sharing the "molar tooth sign" on axial brain MRI, together with cerebellar vermis hypoplasia, ataxia, and psychomotor delay. JS is suggested to be a disorder of cilia function and is part of a spectrum of disorders involving retinal, renal, digital, oral, hepatic, and cerebral organs. We identified mutations in ARL13B in two families with the classical form of JS. ARL13B belongs to the Ras GTPase family, and in other species is required for ciliogenesis, body axis formation, and renal function. The encoded Arl13b protein was expressed in developing murine cerebellum and localized to the cilia in primary neurons. Overexpression of human wild-type but not patient mutant ARL13B rescued the Arl13b scorpion zebrafish mutant. Thus, ARL13B has an evolutionarily conserved role mediating cilia function in multiple organs.

Figure 1

Mapping JSRD Family MTI-001 Identified the JBTS8 Locus Containing the ARL13B Gene, Mutation of which Produces Altered GTP Binding (A) MTI-001 consists of a double first cousin marriage where each of two brothers were married twice (generation II, double hash refers to divorce); one had two half-brothers and one had two half-sisters, and each of those subsequently married his/her respective cousin (generation III). These two couples each produced an affected offspring (filled symbols in generation IV) as well as healthy siblings. One affected girl from branch 1 died from related medical complications (single hash line). (B) Whole-genome analysis of linkage results with chromosomal position (x axis) and multipoint LOD scores (y axis) showing a significant LOD score (>3) on chr. 3. No other peaks reached this threshold for significance. (C) Expanded view of the candidate interval showing that the linkage signal is composed of one major peak defined by recombinations at rs1005343 and rs938988 SNP markers (red), thus defining a minimal candidate interval from 107 to 112 cM. (D) Expanded view of the candidate interval on chr. 3p12.3-q12.3, encompassing the centromere, containing 41 candidate genes, including ARL6 and ARL13B (red). Transcriptional direction indicated by arrowheads. Genomic structure of ARL13B consists of 10 coding exons extending over about 70 KB of genomic sequence. (E) Homology modeling of wild-type ARL13B showing R (green) residue and predicted hydrogen bond (red dashed line) with D (blue) residue that engages in GTP binding. (F) The p.R79Q mutation is a basic polar to neutral polar substitution (red) and is predicted to disrupt this hydrogen bond (absent dashes indicated by red arrow). (G) GTP binding assay via recombinant wild-type ARL13B (green) or p.R79Q mutant (red) showing altered kinetics and binding capacity. Experiment performed in triplicate. Error bars represent SEM. Negative control was denatured wild-type ARL13B (black). Statistical differences observed between wild-type and mutant at 500, 1000, and 2000 nM GTP concentration (^∗p < 0.05, one-way ANOVA).

Figure 2

Human Mutations in ARL13B Occur in Conserved Residues and Interfere with Evolutionarily Conserved Functions (A) Domain structure of ARL13B protein, with GTP binding, coiled-coil, and proline-rich domains. Red indicates conserved regions involved in GTP binding. Mutations are indicated along the top. (B) Evolutionary conservation of ARL13B, showing that each mutated residues is conserved at least to Danio rerio (zebrafish). (C–F) Wild-type zebrafish have straight tails and absence of cystic kidney. Noninjected arl13b^sco/sco embryos have curved tails (red arrow) and cystic kidneys (double red arrow). The arl13b^sco/sco embryos injected with human wild-type ARL13B RNA show rescue of the curved tail and absence of cystic kidney. The human p.R79Q and p.R200C mutant ARL13B RNA failed to rescue either phenotype in the majority of embryos, although about 15%–24% showed evidence of rescued phenotype, suggesting a hypomorphic allele. A control amino acid substitution (p.S371I) in ARL13B showed a rescue effect similar to wild-type (not shown).

Figure 3

Arl13b Localization in Cilia within Kidney, Retina, and Developing Cerebellar Granule Neurons Boxed region indicates location of high-power field shown in the third panel in (A) and (B). (A) Kidney tubule shows Arl13b-positive cilia protruding into the lumen (arrows) in close approximation to the epithelial nuclei (blue, DAPI). (B) Retina shows intense Arl13b staining in photoreceptor connecting cilia layer (CC, arrow). (C) External granule layer (EGL) of P0 cerebellum from EGFP-Centrin 2 transgenic mouse stained with Arl13b. EGFP-labeled centrioles (green dots, arrow, left) are evident in many cells, defining the position of the basal body. An Arl13b-positive cilium projects adjacent to the basal body in many of these cells (arrows, right). (D) Acutely dissociated CGN showing cilia (arrow) stained with acetylated tubulin (green) costains with Arl13b (red). Acetylated tubulin also stains the microtubule cytoskeleton (arrowhead), which is negative for Arl13b. Abbreviations: Co, cortex; os, outer stripe of medulla; is, inner stripe of medulla; OM, outer medulla; IM, inner medulla; GCL, ganglion cell layer; IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer; CC, connecting cilia; OS, outer segment; P, postnatal day. Scale bar represents 20 μm in (A)–(C) and 5 μm in (D).