TCTN3 mutations cause Mohr-Majewski syndrome

Abstract

Orofaciodigital syndromes (OFDSs) consist of a group of heterogeneous disorders characterized by abnormalities in the oral cavity, face, and digits and associated phenotypic abnormalities that lead to the delineation of 13 OFDS subtypes. Here, by a combined approach of homozygozity mapping and exome ciliary sequencing, we identified truncating TCTN3 mutations as the cause of an extreme form of OFD associated with bone dysplasia, tibial defect, cystic kidneys, and brain anomalies (OFD IV, Mohr-Majewski syndrome). Analysis of 184 individuals with various ciliopathies (OFD, Meckel, Joubert, and short rib polydactyly syndromes) led us to identify four additional truncating TCTN3 mutations in unrelated fetal cases with overlapping Meckel and OFD IV syndromes and one homozygous missense mutation in a family with Joubert syndrome. By exploring roles of TCTN3 in human ciliary related functions, we found that TCTN3 is necessary for transduction of the sonic hedgehog (SHH) signaling pathway, as revealed by abnormal processing of GLI3 in patient cells. These results are consistent with the suggested role of its murine ortholog, which forms a complex at the ciliary transition zone with TCTN1 and TCTN2, both of which are also implicated in the transduction of SHH signaling. Overall, our data show the involvement of the transition zone protein TCTN3 in the regulation of the key SHH signaling pathway and that its disruption causes a severe form of ciliopathy, combining features of Meckel and OFD IV syndromes.

Figure 1

Phenotype of Fetuses and TCTN3 Mutation Identification (A–D) Pictures of fetus 10047, showing lobulated tongue (A), postaxial polydactyly of the hand (B), bowing of long bones, a trident appearance of acetabular bones but no short ribs (C), and severe tibia hypoplasia (D). (E–H) Pictures of fetus 11385, showing lingual hamartoma (E), postaxial polydactyly of the hand (F), moderate bowing of long bones (G), and slightly thickened tibia bones (H). (I) After homozygosity mapping with Affymetrix 250 SNP microarrays, a ciliary gene exome sequencing and filtering of the data identified a unique nonsense mutation in one of the targeted homozygous regions.

Figure 2

Analysis of Ciliogenesis and SHH Pathway in TCTN3 Mutants (A) Analysis of the primary cilia in kidneys of two affected fetuses aged 14.5 and 19 gestation weeks (gw), respectively (cases 860 and 10047), compared to a matched control, costained with acetylated α tubulin (red; Sigma) and pericentrin (green; AbCam). Confocal images were taken with a Leica SP5 confocal microscope. (B) TCTN3-mutated patient fibroblasts failed to upregulate GLI1 and PTCH1 after stimulation with Smoothened Agonist (SAG) for 18 hr, as compared to controls. Primers used for quantitative RT-PCR are listed in Table S5. Values were normalized to GAPDH and presented as relative expression levels ± SEM. ^∗∗p < 0.05, ^∗∗∗p < 0.001 (Student’s two-tailed t test). (C) Immunoblot analysis with the use of a GLI3 antibody (Santa Cruz Biotechnology) showed that TCTN3 processing of GLI3 into the repressor form, GLI3R, is increased in TCTN3-mutated fibroblasts as compared to controls. A graphical evaluation of the GLI3-FL:GLI3-R ratio using actin as a loading control and ImageJ software for densitometry is presented.