Molecular genetics and cell biology of ciliopathies

Defects in cilia structure and/or function are now known to be the cause of an important group of Mendelian developmental conditions called ciliopathies. Meckel-Gruber syndrome (MKS) and Joubert syndrome-related disorders (JBTS) are the focus of this work. The research comprised genetic screening of an established MKS/JSRD patient cohort for mutations in seven known genes, and different approaches to identify new causes for these disorders. The latter included whole exome sequencing (WES) of mutation-negative patients, and a high-throughput whole genome siRNA-based reverse genetics screen to identify novel ciliopathy genes and genes implicated in the process of ciliogenesis.
Mutation screening in the University of Leeds MKS/JSRD patient cohort showed that about 50% patients (n=29/65) were mutation-negative for known genes and confirmed mutations in TMEM67 as a major cause of MKS/JSRD. WES gave a conclusive molecular diagnosis for n=4/7 families. WES allowed the identification of mutations in TMEM237 as a new cause of JSRD. In vitro assays showed that the TMEM237 protein is required for correct cilia formation and function. Loss of the protein in patient fibroblasts and after transcript knockdown caused defects in ciliogenesis and the Wnt signaling pathway.
The whole genome reverse genetics screen identified new functional modules that were not previously linked to cilia (components of the spliceosome and proteasome) or had a poorly characterized ciliary function (several neuroactive GPCRs). Cross-comparison of screen hits with available WES data allowed the prioritisation and confirmation of mutations in PIBF1 and C21orf2 as new causes of JBTS and the skeletal ciliopathy Jeune syndrome, respectively.
In summary, the multiple approaches presented in this work have allowed further insights into the structure and function of the primary cilia, as well as the disease mechanisms of human ciliopathies.