Mutations in CSPP1, encoding a core centrosomal protein, cause a range of ciliopathy phenotypes in humans

Abstract

Ciliopathies are characterized by a pattern of multisystem involvement that is consistent with the developmental role of the primary cilium. Within this biological module, mutations in genes that encode components of the cilium and its anchoring structure, the basal body, are the major contributors to both disease causality and modification. However, despite rapid advances in this field, the majority of the genes that drive ciliopathies and the mechanisms that govern the pronounced phenotypic variability of this group of disorders remain poorly understood. Here, we show that mutations in CSPP1, which encodes a core centrosomal protein, are disease causing on the basis of the independent identification of two homozygous truncating mutations in three consanguineous families (one Arab and two Hutterite) affected by variable ciliopathy phenotypes ranging from Joubert syndrome to the more severe Meckel-Gruber syndrome with perinatal lethality and occipital encephalocele. Consistent with the recently described role of CSPP1 in ciliogenesis, we show that mutant fibroblasts from one affected individual have severely impaired ciliogenesis with concomitant defects in sonic hedgehog (SHH) signaling. Our results expand the list of centrosomal proteins implicated in human ciliopathies.

Figure 1

Identification of Ciliopathy Phenotypes in Three Consanguineous Families (A) Pedigrees of the three study families are shown. The proband is indicated in each pedigree by an arrow, and asterisks denote individuals whose DNA was available for analysis. Please note that the degree of consanguinity between I:1 and I:2 in families 2 and 3 is uncertain but that the average relationship is first cousins once removed. (B and C) Prenatal sonographic images of V:1 in family 1 show echogenic kidneys (B) and occipital encephalocele (C). (D and E) Prenatal sonographic images of V:3 in family 1 show echogenic kidneys (D) and occipital encephalocele (E). (F) A skeletal survey of V:3 in family 1 shows a severe skull defect. (G and H) A side view (G) and top view (H) of MRI of the family 2 proband show typical cerebellar involvement of JBTS.

Figure 2

Identification of Two Homozygous Truncating Mutations in CSPP1 Schematic of the CSPP and CSPP-L isoforms. The locations of the two truncating CSPP1 alterations identified in the three families are indicated along with the CSPP1 sequence chromatograms. Red boxes indicate potential coiled-coil regions.

Figure 3

CSPP1-Related Ciliopathy Is Associated with Impaired SHH Signaling and Ciliogenesis Defects (A) mRNA expression of GLI1 was markedly lower in fibroblasts from V:3 in family 1 than in control fibroblasts in response to SAG stimulation of 100 nM for 18 hr (p < 0.0084 on the basis of three independent experiments each performed in triplicate). Error bars represent the SEM. (B and C) Immunofluorescence images of serum-starved fibroblasts from the affected individual in family 3 (B) and control fibroblasts (C) stained for the ciliary marker acetylated α-tubulin (Sigma-Aldrich) (green) and DNA (blue). Compared to controls, fibroblasts from V:3 in family 1 showed a marked ciliogenesis defect at 40× magnification. Scale bars represent 50 μm.

Figure 4

Abnormal Localization of RPGRIP1L in Cells with a CSPP1 Mutation Immunofluorescence images of serum-starved fibroblasts from the affected individual in family 3 (A) and control fibroblasts (B) stained for the ciliary marker acetylated α-tubulin (green), RPGRIP1L (Proteintech, catalog no. 55160-1-AP) (red), and DNA (blue). (A) The localization of RPGRIP1L at the basal body was lost in mutant fibroblasts. (B) RPGRIP1L localized normally at the basal body of the cilia in control fibroblasts.