Background Mutations in ciliary genes cause a spectrum of both overlapping and distinct clinical syndromes (ciliopathies). CEP120 and CC2D2A are paradigmatic examples for this genetic heterogeneity and pleiotropy as mutations in both cause Joubert syndrome but are also associated with skeletal ciliopathies and Meckel syndrome, respectively. The molecular basis for this phenotypical variability is not understood but basal exon skipping likely contributes to tolerance for deleterious mutations via tissue-specific preservation of the amount of expressed functional protein. Methods We systematically reviewed and annotated genetic variants and clinical presentations reported in CEP120- and CC2D2A-associated disease and we combined in silico and ex vivo approaches to study tissue-specific transcripts and identify molecular targets for exon skipping. Results We confirmed more severe clinical presentations associated with truncating CC2D2A mutations. We identified and confirmed basal exon skipping in the kidney, with possible relevance for organ-specific disease manifestations. Finally, we proposed a multimodal approach to classify exons amenable to exon skipping. By mapping reported variants, 14 truncating mutations in 7 CC2D2A exons were identified as potentially rescuable by targeted exon skipping, an approach that is already in clinical use for other inherited human diseases. Conclusion Genotype-phenotype correlations for CC2D2A support the deleteriousness of null alleles and CC2D2A, but not CEP120, offers potential for therapeutic exon skipping approaches.

Background: Joubert syndrome and related disorders (JSRD) and Jeune syndrome are multisystem ciliopathy disorders with overlapping phenotypes. There are a growing number of genetic causes for these rare syndromes, including the recently described genes ARL3 and CEP120.Methods: We sought to explore the developmental expression patterns of ARL3 and CEP120 in humans to gain additional understanding of these genetic conditions. We used an RNA in situ detection technique called RNAscope to characterise ARL3 and CEP120 expression patterns in human embryos and foetuses in collaboration with the MRC-Wellcome Trust Human Developmental Biology Resource.Results: Both ARL3 and CEP120 are expressed in early human brain development, including the cerebellum and in the developing retina and kidney, consistent with the clinical phenotypes seen with pathogenic variants in these genes.Conclusions: This study provides insights into the potential pathogenesis of JSRD by uncovering the spatial expression of two JSRD-causative genes during normal human development.

Nephronophthisis-related ciliopathies (NPHP-RC) are a group of inherited genetic disorders that share a defect in the formation, maintenance or functioning of the primary cilium complex, causing progressive cystic kidney disease and other clinical manifestations. Mutations in centrosomal protein 164 kDa (CEP164), also known as NPHP15, have been identified as a cause of NPHP-RC. Here we have utilised the MRC-Wellcome Trust Human Developmental Biology Resource (HDBR) to perform immunohistochemistry studies on human embryonic and foetal tissues to determine the expression patterns of CEP164 during development. Notably expression is widespread, yet defined, in multiple organs including the kidney, retina and cerebellum. Murine studies demonstrated an almost identical Cep164 expression pattern. Taken together, these data support a conserved role for CEP164 throughout the development of numerous organs, which, we suggest, accounts for the multi-system disease phenotype of CEP164-mediated NPHP-RC.

Joubert syndrome (JBTS) is an incurable multisystem ciliopathy syndrome. The most commonly mutated gene in JBTS patients with a cerebello-retinal-renal phenotype is CEP290 (alias JBTS5). The encoded CEP290 protein localises to the proximal end of the primary cilium, in the transition zone, where it controls ciliary protein composition and signalling. We examined primary cilium structure and composition in fibroblast cells derived from homozygous and compound heterozygous JBTS5 patients with nonsense mutations in CEP290 and show that elongation of cilia, impaired ciliogenesis and ciliary composition defects are typical features in JBTS5 cells. Targeted skipping of the mutated exon c.5668 G > T using antisense oligonucleotide (ASO) therapy leads to restoration of CEP290 protein expression and functions at the transition zone in homozygous and compound heterozygous JBTS5 cells, allowing a rescue of both cilia morphology and ciliary composition. This study, by demonstrating that targeted exon skipping is able to rescue ciliary protein composition defects, provides functional evidence for the efficacy of this approach in the treatment of JBTS.

CEP104 is an evolutionarily conserved centrosomal and ciliary tip protein. CEP104 loss-of-function mutations are reported in patients with Joubert syndrome, but their function in the etiology of ciliopathies is poorly understood. Here, we show that cep104 silencing in zebrafish causes cilia-related manifestations: shortened cilia in Kupffer's vesicle, heart laterality, and cranial nerve development defects. We show that another Joubert syndrome-associated cilia tip protein, CSPP1, interacts with CEP104 at microtubules for the regulation of axoneme length. We demonstrate in human telomerase reverse transcriptase-immortalized retinal pigmented epithelium (hTERT-RPE1) cells that ciliary translocation of Smoothened in response to Hedgehog pathway stimulation is both CEP104 and CSPP1 dependent. However, CEP104 is not required for the ciliary recruitment of CSPP1, indicating that an intra-ciliary CEP104-CSPP1 complex controls axoneme length and Hedgehog signaling competence. Our in vivo and in vitro analyses of CEP104 define its interaction with CSPP1 as a requirement for the formation of Hedgehog signaling-competent cilia, defects that underlie Joubert syndrome.

Significance Our current understanding of genetic disease is often inadequate, largely due to genetic background effects that modify disease presentation. This is particularly challenging for rare diseases that lack sufficient numbers of patients for genome-wide association studies. We show in a series of experiments using a murine model of Joubert syndrome, a multisystem ciliopathy, that a single locus is a modifier of cystic kidney disease. We go on to show that the human homolog plays a similar role in disease using a cohort of patients. These findings make a significant contribution to the underplayed (and often ignored) role of genetic background in murine models and how this can be exploited to understand further rare inherited disease.

Joubert syndrome (JBTS) is a genetically heterogeneous autosomal-recessive neurodevelopmental ciliopathy. We investigated further the underlying genetic etiology of Joubert syndrome by studying two unrelated families in whom JBTS was not associated with pathogenic variants in known JBTS-associated genes. Combined autozygosity mapping of both families highlighted a candidate locus on chromosome 10 (chr10: 101569997-109106128, UCSC Genome Browser hg 19), and exome sequencing revealed two missense variants in ARL3 within the candidate locus. The encoded protein, ADP ribosylation factor-like GTPase 3 (ARL3), is a small GTP-binding protein that is involved in directing lipid-modified proteins into the cilium in a GTP-dependent manner. Both missense variants replace the highly conserved Arg149 residue, which we show to be necessary for the interaction with its guanine nucleotide exchange factor ARL13B, such that the mutant protein is associated with reduced INPP5E and NPHP3 localization in cilia. We propose that ARL3 provides a potential hub in the network of proteins implicated in ciliopathies, whereby perturbation of ARL3 leads to the mislocalization of multiple ciliary proteins as a result of abnormal displacement of lipidated protein cargo.

Genetic treatments of renal ciliopathies leading to cystic kidney disease would provide a real advance in current therapies. Mutations in CEP290 underlie a ciliopathy called Joubert syndrome (JBTS). Human disease phenotypes include cerebral, retinal, and renal disease, which typically progresses to end stage renal failure (ESRF) within the first two decades of life. While currently incurable, there is often a period of years between diagnosis and ESRF that provides a potential window for therapeutic intervention. By studying patient biopsies, patient-derived kidney cells, and a mouse model, we identify abnormal elongation of primary cilia as a key pathophysiological feature of CEP290-associated JBTS and show that antisense oligonucleotide (ASO)-induced splicing of the mutated exon (41, G1890*) restores protein expression in patient cells. We demonstrate that ASO-induced splicing leading to exon skipping is tolerated, resulting in correct localization of CEP290 protein to the ciliary transition zone, and restoration of normal cilia length in patient kidney cells. Using a gene trap Cep290 mouse model of JBTS, we show that systemic ASO treatment can reduce the cystic burden of diseased kidneys in vivo. These findings indicate that ASO treatment may represent a promising therapeutic approach for kidney disease in CEP290-associated ciliopathy syndromes.

The majority of multi-exon genes undergo alternative splicing to produce different mRNA transcripts and this may occur in a tissue-specific manner. Assessment of mRNA transcripts isolated from blood samples may sometimes be unhelpful in determining the affect on function of putative splice-site variants affecting kidney-specific mRNA transcripts. Here we present data demonstrating the power of using human urine-derived renal epithelial cells (hUREC) as a source of kidney RNA. We report clinical and molecular genetic data from three affected cases from two families all with end-stage renal disease by 15 years of age. In both families, heterozygous variants which are predicted to effect function in NPHP3 were found on one allele, in combination with a synonymous SNV (c.2154C>T; p.Phe718=), 18 base pairs from the exon–intron boundary within exon 15 of NPHP3. The only mRNA transcript amplified from wild-type whole blood showed complete splicing out of exon 15. Urine samples obtained from control subjects and the father of family 2, who carried the synonymous SNV variant, were therefore used to culture hUREC and allowed us to obtain kidney-specific mRNA. Control kidney mRNA showed retention of exon 15, while the mRNA from the patient's father confirmed evidence of a heterozygous alternate splicing of exon 15 of NPHP3. Analysis of RNA derived from hUREC allows for a comparison of kidney-specific and whole-blood RNA transcripts and for assessment of the effect on function of putative splice variants leading to end-stage kidney disease.

Zebrafish are a valuable vertebrate model in which to study development and characterize genes involved in cystic kidney disease. Zebrafish embryos and larvae are transparent, allowing non-invasive imaging during their rapid development, which takes place over the first 72 hours post fertilisation. Gene-specific knockdown of nephronophthisis-associated genes leads to ciliary phenotypes which can be assessed in various developmental structures. Here we describe in detail the methods used for imaging cilia within Kupffer’s vesicle to assess nephronophthisis and related ciliopathy phenotypes.

Zebrafish are a valuable vertebrate model in which to study development and characterize genes involved in cystic kidney disease. Zebrafish embryos and larvae are transparent, allowing non-invasive imaging during their rapid development, which takes place over the first 72 hours post fertilisation. Gene-specific knockdown of nephronophthisis-associated genes leads to ciliary phenotypes which can be assessed in various developmental structures. Here we describe in detail the methods used for imaging cilia within Kupffer’s vesicle to assess nephronophthisis and related ciliopathy phenotypes.

Joubert syndrome (JBTS) is the archetypal ciliopathy caused by mutation of genes encoding ciliary proteins leading to multisystem phenotypes, including a cerebello-retinal-renal syndrome. JBTS is genetically heterogeneous, however mutations in CEP290 are a common underlying cause. The renal manifestation of JBTS is a juvenile-onset cystic kidney disease, known as nephronophthisis, typically progressing to end-stage renal failure within the first two decades of life, thus providing a potential window for therapeutic intervention. In order to increase understanding of JBTS and its associated kidney disease and to explore potential treatments, we conducted a comprehensive analysis of primary renal epithelial cells directly isolated from patient urine (human urine-derived renal epithelial cells, hURECs). We demonstrate that hURECs from a JBTS patient with renal disease have elongated and disorganized primary cilia and that this ciliary phenotype is specifically associated with an absence of CEP290 protein. Treatment with the Sonic hedgehog (Shh) pathway agonist purmorphamine or cyclin-dependent kinase inhibition (using roscovitine and siRNA directed towards cyclin-dependent kinase 5) ameliorated the cilia phenotype. In addition, purmorphamine treatment was shown to reduce cyclin-dependent kinase 5 in patient cells, suggesting a convergence of these signalling pathways. To our knowledge, this is the most extensive analysis of primary renal epithelial cells from JBTS patients to date. It demonstrates the feasibility and power of this approach to directly assess the consequences of patient-specific mutations in a physiologically relevant context and a previously unrecognized convergence of Shh agonism and cyclin-dependent kinase inhibition as potential therapeutic targets.

Heterozygous mutations in UMOD encoding the urinary protein uromodulin are the most common genetic cause of autosomal dominant tubulointerstitial kidney disease (ADTKD). We describe the exceptional case of a patient from a consanguineous family carrying a novel homozygous UMOD mutation (p.C120Y) affecting a conserved cysteine residue within the EGF-like domain III of uromodulin. Comparison of heterozygote and homozygote mutation carriers revealed a gene dosage effect with unprecedented low levels of uromodulin and aberrant uromodulin fragments in the urine of the homozygote proband. Despite an amplified biological effect of the homozygote mutation, the proband did not show a strikingly more severe clinical evolution nor was the near absence of urinary uromodulin associated with urinary tract infections or kidney stones.

Heterozygous mutations in UMOD encoding the urinary protein uromodulin are the most common genetic cause of autosomal dominant tubulointerstitial kidney disease (ADTKD). We describe the exceptional case of a patient from a consanguineous family carrying a novel homozygous UMOD mutation (p.C120Y) affecting a conserved cysteine residue within the EGF-like domain III of uromodulin. Comparison of heterozygote and homozygote mutation carriers revealed a gene dosage effect with unprecedented low levels of uromodulin and aberrant uromodulin fragments in the urine of the homozygote proband. Despite an amplified biological effect of the homozygote mutation, the proband did not show a strikingly more severe clinical evolution nor was the near absence of urinary uromodulin associated with urinary tract infections or kidney stones.

Mutations that give rise to premature termination codons are a common cause of inherited genetic diseases. When transcripts containing these changes are generated, they are usually rapidly removed by the cell through the process of nonsense-mediated decay. Here we discuss observed changes in transcripts of the centrosomal protein CEP290 resulting not from degradation, but from changes in exon usage. We also comment on a landmark paper (Drivas . Sci Transl Med. 2015) where modelling this process of exon usage may be used to predict disease severity in ciliopathies, and how understanding this process may potentially be used for therapeutic benefit in the future.

Mutations that give rise to premature termination codons are a common cause of inherited genetic diseases. When transcripts containing these changes are generated, they are usually rapidly removed by the cell through the process of nonsense-mediated decay. Here we discuss observed changes in transcripts of the centrosomal protein CEP290 resulting not from degradation, but from changes in exon usage. We also comment on a landmark paper (Drivas et al. Sci Transl Med. 2015) where modelling this process of exon usage may be used to predict disease severity in CEP290 ciliopathies, and how understanding this process may potentially be used for therapeutic benefit in the future.

The hedgehog (Hh) signalling pathway is conserved throughout metazoans and plays an important regulatory role in both embryonic development and adult homeostasis. Many levels of regulation exist that control the release, reception, and interpretation of the hedgehog signal. The fatty nature of the Shh ligand means that it tends to associate tightly with the cell membrane, and yet it is known to act as a morphogen that diffuses to elicit pattern formation. Heparan sulfate proteoglycans (HSPGs) play a major role in the regulation of Hh distribution outside the cell. Inside the cell, the primary cilium provides an important hub for processing the Hh signal in vertebrates. This review will summarise the current understanding of how the Hh pathway is regulated from ligand production, release, and diffusion, through to signal reception and intracellular transduction.

This protocol describes a method to visualise ligands distributed across a field of cells. The ease of expressing exogenous proteins, together with the large size of their cells in early embryos, make Xenopus laevis a useful model for visualising GFP-tagged ligands. Synthetic mRNAs are efficiently translated after injection into early stage Xenopus embryos, and injections can be targeted to a single cell. When combined with a lineage tracer such as membrane tethered RFP, the injected cell (and its descendants) that are producing the overexpressed protein can easily be followed. This protocol describes a method for the production of fluorescently tagged Wnt and Shh ligands from injected mRNA. The methods involve the micro dissection of ectodermal explants (animal caps) and the analysis of ligand diffusion in multiple samples. By using confocal imaging, information about ligand secretion and diffusion over a field of cells can be obtained. Statistical analyses of confocal images provide quantitative data on the shape of ligand gradients. These methods may be useful to researchers who want to test the effects of factors that may regulate the shape of morphogen gradients.

The study of primary cilia is of broad interest both in terms of disease pathogenesis and the fundamental biological role of these structures. Murine models of ciliopathies provide valuable tools for the study of these diseases. However, it is important to consider the precise phenotype of murine models and how dependant it is upon genetic background. Here we compare and contrast murine models of Cep290, a frequent genetic cause of Joubert syndrome in order to refine our concept of genotype-phenotype correlations.