Mutations in CSPP1 lead to classical Joubert syndrome

Abstract

Joubert syndrome and related disorders (JSRDs) are genetically heterogeneous and characterized by a distinctive mid-hindbrain malformation. Causative mutations lead to primary cilia dysfunction, which often results in variable involvement of other organs such as the liver, retina, and kidney. We identified predicted null mutations in CSPP1 in six individuals affected by classical JSRDs. CSPP1 encodes a protein localized to centrosomes and spindle poles, as well as to the primary cilium. Despite the known interaction between CSPP1 and nephronophthisis-associated proteins, none of the affected individuals in our cohort presented with kidney disease, and further, screening of a large cohort of individuals with nephronophthisis demonstrated no mutations. CSPP1 is broadly expressed in neural tissue, and its encoded protein localizes to the primary cilium in an in vitro model of human neurogenesis. Here, we show abrogated protein levels and ciliogenesis in affected fibroblasts. Our data thus suggest that CSPP1 is involved in neural-specific functions of primary cilia.

Figure 1

CSPP1 Is Recurrently Mutated in JBTS (A) Whole-exome sequencing results summarized for 287 unique probands indicate genes demonstrating evidence of mutations. The most commonly mutated genes were CEP290, C5orf42, and AHI1. Six unique probands displayed mutations in CSPP1 (light green). In more than half of affected individuals, a causative mutation could not be identified. (B) CSPP1 mutation locations relative to the genomic exon locations in hg19. Compound-heterozygous mutations are shown on the top, and homozygous mutations are shown on the bottom. (C) CSPP1 alteration locations relative to the predicted protein. Blocks represent coiled-coil domains, green indicates the nuclear localization signal, and asterisks indicate predicted phosphorylation sites.

Figure 2

Brain Imaging Demonstrates Molar Tooth Sign in Individuals with CSPP1 Muations Axial MRI (upper panels) and parasagittal MRI (lower panels, where available). Images show molar tooth sign (red circle) with elongated superior cerebellar peduncles, deepened interhemispheric fossa, and cerebellar vermis hypoplasia. Lower panels show thick and horizontally oriented superior cerebellar peduncles in all affected children (red arrows) and thin corpus callosum in MTI-136, MTI-1561, and MTI-2109.

Figure 3

CSPP1 Is Expressed Predominantly in Neural Tissues Localized to Cilia, and the Truncating Mutation Abrogates Ciliogenesis (A) RNA from human tissue RNA collection (Clontech) was retrotranscribed to cDNA with the SuperScript III First-Strand Synthesis System for RT-PCR (Invitrogen) with oligodT. PCR with primers specific to CSPP1 demonstrated that human neural tissues, and especially the cerebellum, have the highest expression. (B) Immunoblot with CSPP1 antibody (Proteintech) (arrow) in MTI-2109 control and affected fibroblasts showed severely reduced protein levels in the presence of the mutation. GAPDH (Millipore) was used as a loading control. (C) CSPP1 immunostaining (red) in human-embryonic-stem-cell-derived neural rosettes showed localization in the apical region (close to the lumen), suggestive of colocalization with primary cilium. (D) CSPP1 (red) localized to the base of the primary cilium (green) in control fibroblasts and was absent in MTI-2109 affected fibroblasts. The scale bar represents 5 μm. (E) Acetylated tubulin (AcTUB) (Sigma) immunostaining (upper panel, green) and ARL13B immunostaining (middle panel, red) in MTI-2109 control and affected fibroblasts indicated a ciliary defect in mutated cells. γ-tubulin (γ-TUB) immunostaining (lower panel, red) indicated that the centrosome was equally present in affected and control cells. Graphs show the percentage of fibroblasts (n = 105–256) with AcTUB-positive (upper panel) and ARL13B-positive (middle panel) cilia and a γ-TUB-positive centrosome (lower panel). Scale bars represent 25 μm. DAPI (blue) was used for labeling DNA.