Bardet-Biedl syndrome (BBS) is a pleiotropic ciliopathy caused by dysfunction of the BBSome, a cargo adaptor essential for export of transmembrane receptors from cilia. Although actin-dependent ectocytosis has been proposed to compensate defective cargo retrieval, its molecular basis remains unclear, especially in relation to BBS pathology. In this study, we investigated how actin polymerization and ectocytosis are regulated within the cilium. Our findings reveal that ciliary CDC42, a RHO-family GTPase triggers in situ actin polymerization, ciliary ectocytosis, and cilia shortening in BBSome-deficient cells. Activation of the Sonic Hedgehog pathway further enhances CDC42 activity specifically in BBSome-deficient cilia. Inhibition of CDC42 in BBSome-deficient cells decreases the frequency and duration of ciliary actin polymerization events, causing buildup of G protein coupled receptor 161 (GPR161) in bulges along the axoneme during Sonic Hedgehog signaling. Overall, our study identifies CDC42 as a key trigger of ciliary ectocytosis. Hyperactive ciliary CDC42 and ectocytosis and the resulting loss of ciliary material might contribute to BBS disease severity.

• Keywords
• Actin, Bardet-Biedl Syndrome, CDC42, Cilium, Ectocytosis,
• MeSH
• Actins * metabolism   MeSH
• Bardet-Biedl Syndrome * metabolism   genetics   pathology   MeSH
• cdc42 GTP-Binding Protein * metabolism   genetics   MeSH
• Cilia * metabolism   MeSH
• Humans MeSH
• Mice MeSH
• Hedgehog Proteins metabolism   MeSH
• Receptors, G-Protein-Coupled metabolism   genetics   MeSH
• Signal Transduction MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Names of Substances
• Actins * MeSH
• cdc42 GTP-Binding Protein * MeSH
• Hedgehog Proteins MeSH
• Receptors, G-Protein-Coupled MeSH

Bardet-Biedl syndrome (BBS) is an archetypal ciliopathy caused by dysfunction of primary cilia. BBS affects multiple tissues, including the kidney, eye and hypothalamic satiety response. Understanding pan-tissue mechanisms of pathogenesis versus those which are tissue-specific, as well as gauging their associated inter-individual variation owing to genetic background and stochastic processes, is of paramount importance in syndromology. The BBSome is a membrane-trafficking and intraflagellar transport (IFT) adaptor protein complex formed by eight BBS proteins, including BBS1, which is the most commonly mutated gene in BBS. To investigate disease pathogenesis, we generated a series of clonal renal collecting duct IMCD3 cell lines carrying defined biallelic nonsense or frameshift mutations in Bbs1, as well as a panel of matching wild-type CRISPR control clones. Using a phenotypic screen and an unbiased multi-omics approach, we note significant clonal variability for all assays, emphasising the importance of analysing panels of genetically defined clones. Our results suggest that BBS1 is required for the suppression of mesenchymal cell identities as the IMCD3 cell passage number increases. This was associated with a failure to express epithelial cell markers and tight junction formation, which was variable amongst clones. Transcriptomic analysis of hypothalamic preparations from BBS mutant mice, as well as BBS patient fibroblasts, suggested that dysregulation of epithelial-to-mesenchymal transition (EMT) genes is a general predisposing feature of BBS across tissues. Collectively, this work suggests that the dynamic stability of the BBSome is essential for the suppression of mesenchymal cell identities as epithelial cells differentiate.

• Keywords
• Bardet–Biedl syndrome, Wnt signalling, collecting duct cells, epithelial-to-mesenchymal transition, fibrosis, kidney, primary cilia,
• MeSH
• Bardet-Biedl Syndrome * genetics   metabolism   pathology   MeSH
• Cilia metabolism   MeSH
• Humans MeSH
• Mice, Knockout MeSH
• Mice MeSH
• Microtubule-Associated Proteins metabolism   MeSH
• Proteins metabolism   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Bbs1 protein, human MeSH Browser
• Bbs1 protein, mouse MeSH Browser
• Microtubule-Associated Proteins MeSH
• Proteins MeSH

Bardet-Biedl Syndrome (BBS) is a pleiotropic genetic disease caused by the dysfunction of primary cilia. The immune system of patients with ciliopathies has not been investigated. However, there are multiple indications that the impairment of the processes typically associated with cilia may have influence on the hematopoietic compartment and immunity. In this study, we analyze clinical data of BBS patients and corresponding mouse models carrying mutations in Bbs4 or Bbs18. We find that BBS patients have a higher prevalence of certain autoimmune diseases. Both BBS patients and animal models have altered red blood cell and platelet compartments, as well as elevated white blood cell levels. Some of the hematopoietic system alterations are associated with BBS-induced obesity. Moreover, we observe that the development and homeostasis of B cells in mice is regulated by the transport complex BBSome, whose dysfunction is a common cause of BBS. The BBSome limits canonical WNT signaling and increases CXCL12 levels in bone marrow stromal cells. Taken together, our study reveals a connection between a ciliopathy and dysregulated immune and hematopoietic systems.

• Keywords
• Bardet-Biedl Syndrome, CXCL12, ciliopathy, immunity, obesity,
• MeSH
• Autoimmune Diseases * MeSH
• Bardet-Biedl Syndrome * complications   genetics   MeSH
• Cilia MeSH
• Hematopoiesis * genetics   MeSH
• Humans MeSH
• Disease Models, Animal MeSH
• Mutation MeSH
• Mice MeSH
• Microtubule-Associated Proteins genetics   MeSH
• Animals MeSH
• Check Tag
• Humans MeSH
• Mice MeSH
• Animals MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• BBS4 protein, mouse MeSH Browser
• Microtubule-Associated Proteins MeSH

Bardet-Biedl syndrome (BBS) is a pleiotropic ciliopathy caused by dysfunction of primary cilia. More than half of BBS patients carry mutations in one of eight genes encoding for subunits of a protein complex, the BBSome, which mediates trafficking of ciliary cargoes. In this study, we elucidated the mechanisms of the BBSome assembly in living cells and how this process is spatially regulated. We generated a large library of human cell lines deficient in a particular BBSome subunit and expressing another subunit tagged with a fluorescent protein. We analyzed these cell lines utilizing biochemical assays, conventional and expansion microscopy, and quantitative fluorescence microscopy techniques: fluorescence recovery after photobleaching and fluorescence correlation spectroscopy. Our data revealed that the BBSome formation is a sequential process. We show that the pre-BBSome is nucleated by BBS4 and assembled at pericentriolar satellites, followed by the translocation of the BBSome into the ciliary base mediated by BBS1. Our results provide a framework for elucidating how BBS-causative mutations interfere with the biogenesis of the BBSome.

• Keywords
• BBSome, Bardet-Biedl Syndrome, Bardet-Biedl syndrome, assembly, ciliopathy, cilium, genetic disease, microscopic imaging, primary cilium, protein assembly, protein sorting,
• MeSH
• Bardet-Biedl Syndrome genetics   metabolism   pathology   MeSH
• Cell Line MeSH
• Cilia metabolism   MeSH
• CRISPR-Cas Systems genetics   MeSH
• Cytoplasm metabolism   MeSH
• Gene Editing MeSH
• Microscopy, Fluorescence MeSH
• Fluorescence Recovery After Photobleaching MeSH
• Humans MeSH
• Mutation MeSH
• Protein Subunits genetics   metabolism   MeSH
• Microtubule-Associated Proteins deficiency   genetics   metabolism   MeSH
• Check Tag
• Humans MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Names of Substances
• Bbs1 protein, human MeSH Browser
• BBS4 protein, human MeSH Browser
• Protein Subunits MeSH
• Microtubule-Associated Proteins MeSH

Cystic kidney diseases are a very heterogeneous group of chronic kidney diseases. The diagnosis is usually based on clinical and ultrasound characteristics and the final diagnosis is often difficult to be made. Next-generation sequencing (NGS) may help the clinicians to find the correct final diagnosis. The aim of our study was to test the diagnostic yield of NGS and its ability to improve the diagnosis precision in a heterogeneous group of children with cystic kidney diseases. Next-generation sequencing of genes responsible for the formation of cystic kidneys was performed in 31 unrelated patients with various clinically diagnosed cystic kidney diseases gathered at the Department of Pediatrics of Motol University Hospital in Prague between 2013 and 2018. The underlying pathogenic variants were detected in 71% of patients (n = 22), no or only one (in case of autosomal recessive inheritance) pathogenic variant was found in 29% of patients (n = 9). The result of NGS correlated with the clinical diagnosis made before the NGS in 55% of patients (n = 17), in the remaining 14 children (45%) the result of NGS revealed another type of cystic kidney disease that was suspected clinically before or did not find causal mutation in suspected genes. The most common unexpected findings were variants in nephronophthisis (NPHP) genes in children with clinically suspected autosomal recessive polycystic kidney disease (ARPKD, n = 4). Overall, 24 pathogenic or probably pathogenic variants were detected in the PKHD1 gene, 8 variants in the TMEM67 gene, 4 variants in the PKD1 gene, 2 variants in the HNF1B gene and 2 variants in BBS1 and NPHP1 genes, respectively. NGS is a valuable tool in the diagnostics of various forms of cystic kidney diseases. Its results changed the clinically based diagnoses in 16% (n = 5) of the children.

• MeSH
• Adaptor Proteins, Signal Transducing genetics   MeSH
• Kidney Diseases, Cystic diagnosis   genetics   MeSH
• Cytoskeletal Proteins genetics   MeSH
• Child MeSH
• Genetic Predisposition to Disease genetics   MeSH
• Hepatocyte Nuclear Factor 1-beta genetics   MeSH
• TRPP Cation Channels genetics   MeSH
• Infant MeSH
• Humans MeSH
• Mutation * MeSH
• Infant, Newborn MeSH
• Polycystic Kidney, Autosomal Recessive diagnosis   genetics   MeSH
• Child, Preschool MeSH
• Microtubule-Associated Proteins genetics   MeSH
• Receptors, Cell Surface genetics   MeSH
• High-Throughput Nucleotide Sequencing methods   MeSH
• Check Tag
• Child MeSH
• Infant MeSH
• Humans MeSH
• Male MeSH
• Infant, Newborn MeSH
• Child, Preschool MeSH
• Female MeSH
• Publication type
• Journal Article MeSH
• Research Support, Non-U.S. Gov't MeSH
• Geographicals
• Czech Republic MeSH
• Names of Substances
• Adaptor Proteins, Signal Transducing MeSH
• Bbs1 protein, human MeSH Browser
• Cytoskeletal Proteins MeSH
• Hepatocyte Nuclear Factor 1-beta MeSH
• HNF1B protein, human MeSH Browser
• TRPP Cation Channels MeSH
• NPHP1 protein, human MeSH Browser
• PKHD1 protein, human MeSH Browser
• polycystic kidney disease 1 protein MeSH Browser
• Microtubule-Associated Proteins MeSH
• Receptors, Cell Surface MeSH