The retina contains several ciliated cell types, including the retinal pigment epithelium (RPE) and photoreceptor cells. The photoreceptor cilium is one of the most highly modified sensory cilia in the human body. The outer segment of the photoreceptor is a highly elaborate primary cilium, containing stacks or folds of membrane where the photopigment molecules are located. Perhaps unsurprisingly, defects in cilia often lead to retinal phenotypes, either as part of syndromic conditions involving other organs, or in isolation in the so-called retinal ciliopathies. The study of retinal ciliopathies has been limited by a lack of retinal cell lines. RPE1 retinal pigment epithelial cell line is commonly used in such studies, but the existence of a photoreceptor cell line has largely been neglected in the retinal ciliopathy field. 661W cone photoreceptor cells, derived from mouse, have been widely used as a model for studying macular degeneration, but not described as a model for studying retinal ciliopathies such as retinitis pigmentosa. Here, we characterize the 661W cell line as a model for studying retinal ciliopathies. We fully characterize the expression profile of these cells, using whole transcriptome RNA sequencing, and provide this data on Gene Expression Omnibus for the advantage of the scientific community. We show that these cells express the majority of markers of cone cell origin. Using immunostaining and confocal microscopy, alongside scanning electron microscopy, we show that these cells grow long primary cilia, reminiscent of photoreceptor outer segments, and localize many cilium proteins to the axoneme, membrane and transition zone. We show that siRNA knockdown of cilia genes Ift88 results in loss of cilia, and that this can be assayed by high-throughput screening. We present evidence that the 661W cell line is a useful cell model for studying retinal ciliopathies.

Cilia are highly specialized cellular organelles that serve multiple functions in human development and health. Their central importance in the body is demonstrated by the occurrence of a diverse range of developmental disorders that arise from defects of cilia structure and function, caused by a range of different inherited mutations found in more than 150 different genes. Genetic analysis has rapidly advanced our understanding of the cell biological basis of ciliopathies over the past two decades, with more recent technological advances in genomics rapidly accelerating this progress. The 100,000 Genomes Project was launched in 2012 in the UK to improve diagnosis and future care for individuals affected by rare diseases like ciliopathies, through whole genome sequencing (WGS). In this review we discuss the potential promise and medical impact of WGS for ciliopathies and report on current progress of the 100,000 Genomes Project, reviewing the medical, technical and ethical challenges and opportunities that new, large scale initiatives such as this can offer.

Primary ciliopathies represent a group of inherited disorders due to defects in the primary cilium, the 'cell's antenna'. The 100,000 Genomes Project was launched in 2012 by Genomics England (GEL), recruiting National Health Service (NHS) patients with eligible rare diseases and cancer. Sequence data were linked to Human Phenotype Ontology (HPO) terms entered by recruiting clinicians.

Nephronophthisis-related ciliopathies (NPHP-RC) are degenerative recessive diseases that affect kidney, retina, and brain. Genetic defects in NPHP gene products that localize to cilia and centrosomes defined them as "ciliopathies." However, disease mechanisms remain poorly understood. Here, we identify by whole-exome resequencing, mutations of MRE11, ZNF423, and CEP164 as causing NPHP-RC. All three genes function within the DNA damage response (DDR) pathway. We demonstrate that, upon induced DNA damage, the NPHP-RC proteins ZNF423, CEP164, and NPHP10 colocalize to nuclear foci positive for TIP60, known to activate ATM at sites of DNA damage. We show that knockdown of CEP164 or ZNF423 causes sensitivity to DNA damaging agents and that cep164 knockdown in zebrafish results in dysregulated DDR and an NPHP-RC phenotype. Our findings link degenerative diseases of the kidney and retina, disorders of increasing prevalence, to mechanisms of DDR.

The 100 000 Genomes Project (100K) recruited National Health Service patients with eligible rare diseases and cancer between 2016 and 2018. PanelApp virtual gene panels were applied to whole genome sequencing data according to Human Phenotyping Ontology (HPO) terms entered by recruiting clinicians to guide focused analysis.

Ciliopathies are a broad range of inherited developmental and degenerative diseases associated with structural or functional defects in motile or primary non-motile cilia. There are around 200 known ciliopathy disease genes and whilst genetic testing can provide an accurate diagnosis, 24-60% of ciliopathy patients who undergo genetic testing do not receive a genetic diagnosis. This is partly because following current guidelines from the American College of Medical Genetics and the Association for Molecular Pathology, it is difficult to provide a confident clinical diagnosis of disease caused by missense or non-coding variants, which account for more than one-third of cases of disease. Mutations in PRPF31 are the second most common cause of the degenerative retinal ciliopathy autosomal dominant retinitis pigmentosa. Here, we present a high-throughput high-content imaging assay providing quantitative measure of effect of missense variants in PRPF31 which meets the recently published criteria for a baseline standard in vitro test for clinical variant interpretation. This assay utilizes a new PRPF31+/- human retinal cell line generated using CRISPR gene editing to provide a stable cell line with significantly fewer cilia in which novel missense variants are expressed and characterised. We show that high-content imaging of cells expressing missense variants in a ciliopathy gene on a null background can allow characterisation of variants according to the cilia phenotype. We hope that this will be a useful tool for clinical characterisation of PRPF31 variants of uncertain significance, and can be extended to variant classification in other ciliopathies.

Defects in primary cilium biogenesis underlie the ciliopathies, a growing group of genetic disorders. We describe a whole-genome siRNA-based reverse genetics screen for defects in biogenesis and/or maintenance of the primary cilium, obtaining a global resource. We identify 112 candidate ciliogenesis and ciliopathy genes, including 44 components of the ubiquitin-proteasome system, 12 G-protein-coupled receptors, and 3 pre-mRNA processing factors (PRPF6, PRPF8 and PRPF31) mutated in autosomal dominant retinitis pigmentosa. The PRPFs localize to the connecting cilium, and PRPF8- and PRPF31-mutated cells have ciliary defects. Combining the screen with exome sequencing data identified recessive mutations in PIBF1, also known as CEP90, and C21orf2, also known as LRRC76, as causes of the ciliopathies Joubert and Jeune syndromes. Biochemical approaches place C21orf2 within key ciliopathy-associated protein modules, offering an explanation for the skeletal and retinal involvement observed in individuals with C21orf2 variants. Our global, unbiased approaches provide insights into ciliogenesis complexity and identify roles for unanticipated pathways in human genetic disease.

The ciliopathies are a group of heterogeneous diseases with considerable variations in phenotype for allelic conditions such as Meckel-Gruber syndrome (MKS) and Joubert syndrome (JBTS) even at the inter-individual level within families. In humans, mutations in TMEM67 (also known as MKS3) cause both MKS and JBTS, with TMEM67 encoding the orphan receptor meckelin (TMEM67) that localizes to the ciliary transition zone. We now describe the Tmem67(tm1(Dgen/H)) knockout mouse model that recapitulates the brain phenotypic variability of these human ciliopathies, with categorization of Tmem67 mutant animals into two phenotypic groups. An MKS-like incipient congenic group (F6 to F10) manifested very variable neurological features (including exencephaly, and frontal/occipital encephalocele) that were associated with the loss of primary cilia, diminished Shh signalling and dorsalization of the caudal neural tube. The 'MKS-like' group also had high de-regulated canonical Wnt/β-catenin signalling associated with hyper-activated Dishevelled-1 (Dvl-1) localized to the basal body. Conversely, a second fully congenic group (F > 10) had less variable features pathognomonic for JBTS (including cerebellar hypoplasia), and retention of abnormal bulbous cilia associated with mild neural tube ventralization. The 'JBTS-like' group had de-regulated low levels of canonical Wnt signalling associated with the loss of Dvl-1 localization to the basal body. Our results suggest that modifier alleles partially determine the variation between MKS and JBTS, implicating the interaction between Dvl-1 and meckelin, or other components of the ciliary transition zone. The Tmem67(tm1(Dgen/H)) line is unique in modelling the variable expressivity of phenotypes in these two ciliopathies.

Ciliopathies are a group of developmental disorders that manifest with multi-organ anomalies. Mutations in TMEM67 (MKS3) cause a range of human ciliopathies, including Meckel-Gruber and Joubert syndromes. In this study we describe multi-organ developmental abnormalities in the Tmem67(tm1Dgen/H1) knockout mouse that closely resemble those seen in Wnt5a and Ror2 knockout mice. These include pulmonary hypoplasia, ventricular septal defects, shortening of the body longitudinal axis, limb abnormalities, and cochlear hair cell stereociliary bundle orientation and basal body/kinocilium positioning defects. The basal body/kinocilium complex was often uncoupled from the hair bundle, suggesting aberrant basal body migration, although planar cell polarity and apical planar asymmetry in the organ of Corti were normal. TMEM67 (meckelin) is essential for phosphorylation of the non-canonical Wnt receptor ROR2 (receptor-tyrosine-kinase-like orphan receptor 2) upon stimulation with Wnt5a-conditioned medium. ROR2 also colocalises and interacts with TMEM67 at the ciliary transition zone. Additionally, the extracellular N-terminal domain of TMEM67 preferentially binds to Wnt5a in an in vitro binding assay. Cultured lungs of Tmem67 mutant mice failed to respond to stimulation of epithelial branching morphogenesis by Wnt5a. Wnt5a also inhibited both the Shh and canonical Wnt/β-catenin signalling pathways in wild-type embryonic lung. Pulmonary hypoplasia phenotypes, including loss of correct epithelial branching morphogenesis and cell polarity, were rescued by stimulating the non-canonical Wnt pathway downstream of the Wnt5a-TMEM67-ROR2 axis by activating RhoA. We propose that TMEM67 is a receptor that has a main role in non-canonical Wnt signalling, mediated by Wnt5a and ROR2, and normally represses Shh signalling. Downstream therapeutic targeting of the Wnt5a-TMEM67-ROR2 axis might, therefore, reduce or prevent pulmonary hypoplasia in ciliopathies and other congenital conditions.

Dyneins are highly complex, multicomponent, microtubule-based molecular motors. These enzymes are responsible for numerous motile behaviors in cytoplasm, mediate retrograde intraflagellar transport (IFT), and power ciliary and flagellar motility. Variants in multiple genes encoding dyneins, outer dynein arm (ODA) docking complex subunits, and cytoplasmic factors involved in axonemal dynein preassembly (DNAAFs) are associated with human ciliopathies and are of clinical interest. Therefore, clear communication within this field is particularly important. Standardizing gene nomenclature, and basing it on orthology where possible, facilitates discussion and genetic comparison across species. Here, we discuss how the human gene nomenclature for dyneins, ODA docking complex subunits, and DNAAFs has been updated to be more functionally informative and consistent with that of the unicellular green alga Chlamydomonas reinhardtii, a key model organism for studying dyneins and ciliary function. We also detail additional nomenclature updates for vertebrate-specific genes that encode dynein chains and other proteins involved in dynein complex assembly.

Primary ciliary defects cause a group of developmental conditions known as ciliopathies. Here, we provide mechanistic insight into ciliary ubiquitin processing in cells and for mouse model lacking the ciliary protein Mks1. In vivo loss of Mks1 sensitises cells to proteasomal disruption, leading to abnormal accumulation of ubiquitinated proteins. We identified UBE2E1, an E2 ubiquitin-conjugating enzyme that polyubiquitinates β-catenin, and RNF34, an E3 ligase, as novel interactants of MKS1. UBE2E1 and MKS1 colocalised, and loss of UBE2E1 recapitulates the ciliary and Wnt signalling phenotypes observed during loss of MKS1. Levels of UBE2E1 and MKS1 are co-dependent and UBE2E1 mediates both regulatory and degradative ubiquitination of MKS1. We demonstrate that processing of phosphorylated β-catenin occurs at the ciliary base through the functional interaction between UBE2E1 and MKS1. These observations suggest that correct β-catenin levels are tightly regulated at the primary cilium by a ciliary-specific E2 (UBE2E1) and a regulatory substrate-adaptor (MKS1).

The cilium is an essential organelle at the surface of mammalian cells whose dysfunction causes a wide range of genetic diseases collectively called ciliopathies. The current rate at which new ciliopathy genes are identified suggests that many ciliary components remain undiscovered. We generated and rigorously analyzed genomic, proteomic, transcriptomic and evolutionary data and systematically integrated these using Bayesian statistics into a predictive score for ciliary function. This resulted in 285 candidate ciliary genes. We generated independent experimental evidence of ciliary associations for 24 out of 36 analyzed candidate proteins using multiple cell and animal model systems (mouse, zebrafish and nematode) and techniques. For example, we show that OSCP1, which has previously been implicated in two distinct non-ciliary processes, causes ciliogenic and ciliopathy-associated tissue phenotypes when depleted in zebrafish. The candidate list forms the basis of CiliaCarta, a comprehensive ciliary compendium covering 956 genes. The resource can be used to objectively prioritize candidate genes in whole exome or genome sequencing of ciliopathy patients and can be accessed at http://bioinformatics.bio.uu.nl/john/syscilia/ciliacarta/.

Cilia are microtubule-based organelles with important functions in motility and sensation. They contribute to a broad spectrum of developmental disorders called ciliopathies and have recently been linked to common conditions such as cancers and congenital heart disease. There has been increasing interest in the biology of cilia and their contribution to disease over the past two decades. In 2013 we published a "Gold Standard" list of genes confirmed to be associated with cilia. This was published as part of the SYSCILIA consortium for systems biology study dissecting the contribution of cilia to human health and disease, and was named the Syscilia Gold Standard (SCGS). Since this publication, interest in cilia and understanding of their functions have continued to grow, and we now present an updated SCGS version 2. This includes an additional 383 genes, more than doubling the size of SCGSv1. We use this dataset to conduct a review of advances in understanding of cilia biology 2013- 2021 and offer perspectives on the future of cilia research. We hope that this continues to be a useful resource for the cilia community.

Ciliopathies are clinically and genetically heterogeneous diseases. We studied three patients from two independent families presenting with features of Joubert syndrome: abnormal breathing pattern during infancy, developmental delay/intellectual disability, cerebellar ataxia, molar tooth sign on magnetic resonance imaging scans, and polydactyly. We identified biallelic loss-of-function (LOF) variants in CBY1, segregating with the clinical features of Joubert syndrome in the families. CBY1 localizes to the distal end of the mother centriole, contributing to the formation and function of cilia. In accordance with the clinical and mutational findings in the affected individuals, we demonstrated that depletion of Cby1 in zebrafish causes ciliopathy-related phenotypes. Levels of CBY1 transcript were found reduced in the patients compared with controls, suggesting degradation of the mutated transcript through nonsense-mediated messenger RNA decay. Accordingly, we could detect CBY1 protein in fibroblasts from controls, but not from patients by immunofluorescence. Furthermore, we observed reduced ability to ciliate, increased ciliary length, and reduced levels of the ciliary proteins AHI1 and ARL13B in patient fibroblasts. Our data show that CBY1 LOF-variants cause a ciliopathy with features of Joubert syndrome.

Primary cilia defects result in a group of related pleiotropic malformation syndromes known as ciliopathies, often characterised by cerebellar developmental and foliation defects. Here, we describe the cerebellar anatomical and signalling defects in the Tmem67tm1(Dgen)/H knockout mouse. At mid-gestation, Tmem67 mutant cerebella were hypoplastic and had aberrantly high canonical Wnt/β-catenin signalling, proliferation and apoptosis. Later in development, mutant cerebellar hemispheres had severe foliation defects and inferior lobe malformation, characterized by immature Purkinje cells (PCs). Early postnatal Tmem67 mutant cerebellum had disrupted ciliogenesis and reduced responsiveness to Shh signalling. Transcriptome profiling of Tmem67 mutant cerebella identified ectopic increased expression of homeobox-type transcription factors (Hoxa5, Hoxa4, Hoxb5 and Hoxd3), normally required for early rostral hindbrain patterning. HOXB5 protein levels were increased in the inferior lobe, and increased canonical Wnt signalling, following loss of TMEM67, was dependent on HOXB5. HOXB5 occupancy at the β-catenin promoter was significantly increased by activation of canonical Wnt signalling in Tmem67-/- mutant cerebellar neurones, suggesting that increased canonical Wnt signalling following mutation or loss of TMEM67 was directly dependent on HOXB5. Our results link dysregulated expression of Hox group genes with ciliary Wnt signalling defects in the developing cerebellum, providing new mechanistic insights into ciliopathy cerebellar hypoplasia phenotypes.

The clinical spectrum of ciliopathies affecting motile cilia spans impaired mucociliary clearance in the respiratory system, laterality defects including heart malformations, infertility and hydrocephalus. Using linkage analysis and whole exome sequencing, we identified two recessive loss-of-function MNS1 mutations in five individuals from four consanguineous families: 1) a homozygous nonsense mutation p.Arg242* in four males with laterality defects and infertility and 2) a homozygous nonsense mutation p.Gln203* in one female with laterality defects and recurrent respiratory infections additionally carrying homozygous mutations in DNAH5. Consistent with the laterality defects observed in these individuals, we found Mns1 to be expressed in mouse embryonic ventral node. Immunofluorescence analysis further revealed that MNS1 localizes to the axonemes of respiratory cilia as well as sperm flagella in human. In-depth ultrastructural analyses confirmed a subtle outer dynein arm (ODA) defect in the axonemes of respiratory epithelial cells resembling findings reported in Mns1-deficient mice. Ultrastructural analyses in the female carrying combined mutations in MNS1 and DNAH5 indicated a role for MNS1 in the process of ODA docking (ODA-DC) in the distal respiratory axonemes. Furthermore, co-immunoprecipitation and yeast two hybrid analyses demonstrated that MNS1 dimerizes and interacts with the ODA docking complex component CCDC114. Overall, we demonstrate that MNS1 deficiency in humans causes laterality defects (situs inversus) and likely male infertility and that MNS1 plays a role in the ODA-DC assembly.

Initially identified in DNA damage repair, ATM-interactor (ATMIN) further functions as a transcriptional regulator of lung morphogenesis. Here we analyse three mouse mutants, Atmin(gpg6/gpg6), Atmin(H210Q/H210Q) and Dynll1(GT/GT), revealing how ATMIN and its transcriptional target dynein light chain LC8-type 1 (DYNLL1) are required for normal lung morphogenesis and ciliogenesis. Expression screening of ciliogenic genes confirmed Dynll1 to be controlled by ATMIN and further revealed moderately altered expression of known intraflagellar transport (IFT) protein-encoding loci in Atmin mutant embryos. Significantly, Dynll1(GT/GT) embryonic cilia exhibited shortening and bulging, highly similar to the characterised retrograde IFT phenotype of Dync2h1. Depletion of ATMIN or DYNLL1 in cultured cells recapitulated the in vivo ciliogenesis phenotypes and expression of DYNLL1 or the related DYNLL2 rescued the effects of loss of ATMIN, demonstrating that ATMIN primarily promotes ciliogenesis by regulating Dynll1 expression. Furthermore, DYNLL1 as well as DYNLL2 localised to cilia in puncta, consistent with IFT particles, and physically interacted with WDR34, a mammalian homologue of the Chlamydomonas cytoplasmic dynein 2 intermediate chain that also localised to the cilium. This study extends the established Atmin-Dynll1 relationship into a developmental and a ciliary context, uncovering a novel series of interactions between DYNLL1, WDR34 and ATMIN. This identifies potential novel components of cytoplasmic dynein 2 and furthermore provides fresh insights into the molecular pathogenesis of human skeletal ciliopathies.

Many genetic diseases have been linked to the dysfunction of primary cilia, which occur nearly ubiquitously in the body and act as solitary cellular mechanosensory organelles. The list of clinical manifestations and affected tissues in cilia-related disorders (ciliopathies) such as nephronophthisis is broad and has been attributed to the wide expression pattern of ciliary proteins. However, little is known about the molecular mechanisms leading to this dramatic diversity of phenotypes. We recently reported hypomorphic NPHP3 mutations in children and young adults with isolated nephronophthisis and associated hepatic fibrosis or tapetoretinal degeneration. Here, we chose a combinatorial approach in mice and humans to define the phenotypic spectrum of NPHP3/Nphp3 mutations and the role of the nephrocystin-3 protein. We demonstrate that the pcy mutation generates a hypomorphic Nphp3 allele that is responsible for the cystic kidney disease phenotype, whereas complete loss of Nphp3 function results in situs inversus, congenital heart defects, and embryonic lethality in mice. In humans, we show that NPHP3 mutations can cause a broad clinical spectrum of early embryonic patterning defects comprising situs inversus, polydactyly, central nervous system malformations, structural heart defects, preauricular fistulas, and a wide range of congenital anomalies of the kidney and urinary tract (CAKUT). On the functional level, we show that nephrocystin-3 directly interacts with inversin and can inhibit like inversin canonical Wnt signaling, whereas nephrocystin-3 deficiency leads in Xenopus laevis to typical planar cell polarity defects, suggesting a role in the control of canonical and noncanonical (planar cell polarity) Wnt signaling.