Characterization of genes encoding for proteins in cystic kidney disease and ciliopathies

Polycystic kidney disease (PKD) is the most common heritable kidney disease affecting between 1:400 to 1:1000 people1. It poses a major challenge for healthcare professionals and the healthcare system since therapeutic strategies are often limited, and renal replacement therapy remains the only option for many patients. The disease is a common clinical feature of syndromic ciliopathies and causative of most of their morbidity and mortality. Ciliopathies are caused by defects in cilia formation and/or function. Previous research studies from our lab have shown that the three human genes TMEM218, CBY1 and PATJ encoding proteins localized to the evolutionary conserved ciliary transition zone (TZ) subdomain and are mutated in patients presenting with a wide range of ciliopathyassociated features such as Bardet-Biedl, Joubert and Meckel-Gruber syndrome. Functional analysis of the three proteins showed that they interact with various big players in PKD, such as Polycystin-2 and Nephrocystin-12,3. To further understand the role and function of the ciliary TZ and the different modules it consists of, as well as driving factors leading to clinical features, we investigated these three well described genes and the proteins they encode. By creating fusion constructs of those proteins with a biotin ligase (miniTurboID), we used a proximity biotinylation based mass spectrometry approach to map their respective interactome. Using a split-nanoluciferase based protein-protein-interaction assay, we were able to confirm many of these newly discovered interactions. In this work, we selected and analysed the three interesting candidate proteins Cingulin (CGN), leucine zipper putative tumour suppressor 2 (LZTS2) and valosin-containing protein (VCP)-interacting protein 1 (VCPIP1). Immunofluorescence (IF) microscopy studies confirmed co-localization of some novel interaction partners. In addition, respective functional knock-down analyses revealed ciliopathy-associated phenotypes in the in vivo model organism zebrafish (Danio rerio). By creating IMCD3-knock out cells for Tmem218, we investigated its function in ciliogenesis and analysed differentially expressed genes and assessed their impact on a cellular level via RNA-sequencing. These novel insights might provide a base for the identification of novel therapeutic targets and strategies for the management of PKD and related ciliopathies.