Investigating Embryonic Expression Patterns and Evolution of AHI1 and CEP290 Genes,Implicated in Joubert Syndrome

Joubert syndrome and related diseases (JSRD) are developmental cerebello-oculo-renal syndromes with phenotypes including cerebellar hypoplasia, retinal dystrophy and nephronophthisis (a cystic kidney disease). We have utilised the MRC-Wellcome Trust Human Developmental Biology Resource (HDBR), to perform in-situ hybridisation studies on embryonic tissues, revealing an early onset neuronal, retinal and renal expression pattern for AHI1. An almost identical pattern of expression is seen with CEP290 in human embryonic and fetal tissue. A novel finding is that both AHI1 and CEP290 demonstrate strong expression within the developing choroid plexus, a ciliated structure important for central nervous system development. To test if AHI1 and CEP290 may have co-evolved, we carried out a genomic survey of a large group of organisms across eukaryotic evolution. We found that, in animals, ahi1 and cep290 are almost always found together; however in other organisms either one may be found independent of the other. Finally, we tested in murine epithelial cells if Ahi1 was required for recruitment of Cep290 to the centrosome. We found no obvious differences in Cep290 localisation in the presence or absence of Ahi1, suggesting that, while Ahi1 and Cep290 may function together in the whole organism, they are not interdependent for localisation within a single cell. Taken together these data support a role for AHI1 and CEP290 in multiple organs throughout development and we suggest that this accounts for the wide phenotypic spectrum of AHI1 and CEP290 mutations in man.     

CEP290 mutations are frequently identified in the oculo-renal form of Joubert syndrome-related disorders

Joubert syndrome–related disorders (JSRDs) are a group of clinically and genetically heterogeneous conditions that share a midbrain-hindbrain malformation, the molar tooth sign (MTS) visible on brain imaging, with variable neurological, ocular, and renal manifestations. Mutations in the CEP290 gene were recently identified in families with the MTS-related neurological features, many of which showed oculo-renal involvement typical of Senior-Löken syndrome (JSRD-SLS phenotype). Here, we performed comprehensive CEP290-mutation analysis on two nonoverlapping cohorts of JSRD-affected patients with a proven MTS. We identified mutations in 19 of 44 patients with JSRD-SLS. The second cohort consisted of 84 patients representing the spectrum of other JSRD subtypes, with mutations identified in only two patients. The data suggest that CEP290 mutations are frequently encountered and are largely specific to the JSRD-SLS subtype. One patient with mutation displayed complete situs inversus, confirming the clinical and genetic overlap between JSRDs and other ciliopathies.     

The Ciliopathy Protein CC2D2A Associates with NINL and Functions in RAB8-MICAL3-Regulated Vesicle Trafficking

Ciliopathies are a group of human disorders caused by dysfunction of primary cilia, ubiquitous microtubule-based organelles involved in transduction of extra-cellular signals to the cell. This function requires the concentration of receptors and channels in the ciliary membrane, which is achieved by complex trafficking mechanisms, in part controlled by the small GTPase RAB8, and by sorting at the transition zone located at the entrance of the ciliary compartment. Mutations in the transition zone gene CC2D2A cause the related Joubert and Meckel syndromes, two typical ciliopathies characterized by central nervous system malformations, and result in loss of ciliary localization of multiple proteins in various models. The precise mechanisms by which CC2D2A and other transition zone proteins control protein entrance into the cilium and how they are linked to vesicular trafficking of incoming cargo remain largely unknown. In this work, we identify the centrosomal protein NINL as a physical interaction partner of CC2D2A. NINL partially co-localizes with CC2D2A at the base of cilia and ninl knockdown in zebrafish leads to photoreceptor outer segment loss, mislocalization of opsins and vesicle accumulation, similar to cc2d2a-/- phenotypes. Moreover, partial ninl knockdown in cc2d2a-/- embryos enhances the retinal phenotype of the mutants, indicating a genetic interaction in vivo, for which an illustration is found in patients from a Joubert Syndrome cohort. Similar to zebrafish cc2d2a mutants, ninl morphants display altered Rab8a localization. Further exploration of the NINL-associated interactome identifies MICAL3, a protein known to interact with Rab8 and to play an important role in vesicle docking and fusion. Together, these data support a model where CC2D2A associates with NINL to provide a docking point for cilia-directed cargo vesicles, suggesting a mechanism by which transition zone proteins can control the protein content of the ciliary compartment.     

Molecular Genetics and Pathogenic Mechanisms for the Severe Ciliopathies: Insights into Neurodevelopment and Pathogenesis of Neural Tube Defects

Meckel–Gruber syndrome (MKS) is a severe autosomal recessively inherited disorder characterized by developmental defects of the central nervous system that comprise neural tube defects that most commonly present as occipital encephalocele. MKS is considered to be the most common syndromic form of neural tube defect. MKS is genetically heterogeneous with six known disease genes: MKS1, MKS2/TMEM216, MKS3/TMEM67, RPGRIP1L, CEP290, and CC2D2A with the encoded proteins all implicated in the correct function of primary cilia. Primary cilia are microtubule-based organelles that project from the apical surface of most epithelial cell types. Recent progress has implicated the involvement of cilia in the Wnt and Shh signaling pathways and has led to an understanding of their role in normal mammalian neurodevelopment. The aim of this review is to provide an overview of the molecular genetics of the human disorder, and to assess recent insights into the etiology and molecular cell biology of severe ciliopathies from mammalian animal models of MKS.     

Whole exome sequencing identified a homozygous novel variant in CEP290 gene causes Meckel syndrome

Meckel syndrome (MKS) is a pre‐ or perinatal multisystemic ciliopathic lethal disorder with an autosomal recessive mode of inheritance. Meckel syndrome is usually manifested with meningo‐occipital encephalocele, polycystic kidney dysplasia, postaxial polydactyly and hepatobiliary ductal plate malformation. Germline variants in CEP290 cause MKS4. In this study, we investigated a 35‐years‐old Chinese female who was 17+1 weeks pregnant. She had a history of adverse pregnancy of having foetus with multiple malformations. We performed ultrasonography and identified the foetus with occipital meningoencephalocele and enlarged cystic dysplastic kidneys. So, she decided to terminate her pregnancy and further genetic molecular analysis was performed. We identified the aborted foetus without postaxial polydactyly. Histological examination of foetal kidney showed cysts in kidney and thinning of the renal cortex with glomerular atrophy. Whole exome sequencing identified a novel homozygous variant (c.2144T>G; p.L715^*) in exon 21 of the CEP290 in the foetus. Sanger sequencing confirmed that both the parents of the foetus were carrying this variant in a heterozygous state. This variant was not identified in two elder sisters of the foetus as well as in the 100 healthy individuals. Western blot analysis showed that this variant leads to the formation of truncated CEP290 protein with the molecular weight of 84 KD compared with the wild‐type CEP290 protein of 290 KD. Hence, it is a loss‐of‐function variant. We also found that the mutant cilium appears longer in length than the wild‐type cilium. Our present study reported the first variant of CEP290 associated with MKS4 in Chinese population.     

CEP290 tethers flagellar transition zone microtubules to the membrane and regulates flagellar protein content

Mutations in human CEP290 cause cilia-related disorders that range in severity from isolated blindness to perinatal lethality. Here, we describe a Chlamydomonas reinhardtii mutant in which most of the CEP290 gene is deleted. Immunoelectron microscopy indicated that CEP290 is located in the flagellar transition zone in close association with the prominent microtubule–membrane links there. Ultrastructural analysis revealed defects in these microtubule–membrane connectors, resulting in loss of attachment of the flagellar membrane to the transition zone microtubules. Biochemical analysis of isolated flagella revealed that the mutant flagella have abnormal protein content, including abnormal levels of intraflagellar transport proteins and proteins associated with ciliopathies. Experiments with dikaryons showed that CEP290 at the transition zone is dynamic and undergoes rapid turnover. The results indicate that CEP290 is required to form microtubule–membrane linkers that tether the flagellar membrane to the transition zone microtubules, and is essential for controlling flagellar protein composition.     

Selective loss of RPGRIP1-dependent ciliary targeting of NPHP4, RPGR and SDCCAG8 underlies the degeneration of photoreceptor neurons

The retinitis pigmentosa GTPase regulator (RPGR) and nephrocystin-4 (NPHP4) comprise two key partners of the assembly complex of the RPGR-interacting protein 1 (RPGRIP1). Mutations in RPGR and NPHP4 are linked to severe multisystemic diseases with strong retinal involvement of photoreceptor neurons, whereas those in RPGRIP1 cause the fulminant photoreceptor dystrophy, Leber congenital amaurosis (LCA). Further, mutations in Rpgrip1 and Nphp4 suppress the elaboration of the outer segment compartment of photoreceptor neurons by elusive mechanisms, the understanding of which has critical implications in uncovering the pathogenesis of syndromic retinal dystrophies. Here we show RPGRIP1 localizes to the photoreceptor connecting cilium (CC) distally to the centriole/basal body marker, centrin-2 and the ciliary marker, acetylated-α-tubulin. NPHP4 abuts proximally RPGRIP1, RPGR and the serologically defined colon cancer antigen-8 (SDCCAG8), a protein thought to partake in the RPGRIP1 interactome and implicated also in retinal–renal ciliopathies. Ultrastructurally, RPGRIP1 localizes exclusively throughout the photoreceptor CC and Rpgrip1^nmf247 photoreceptors present shorter cilia with a ruffled membrane. Strikingly, Rpgrip1^nmf247 mice without RPGRIP1 expression lack NPHP4 and RPGR in photoreceptor cilia, whereas the SDCCAG8 and acetylated-α-tubulin ciliary localizations are strongly decreased, even though the NPHP4 and SDCCAG8 expression levels are unaffected and those of acetylated-α-tubulin and γ-tubulin are upregulated. Further, RPGRIP1 loss in photoreceptors shifts the subcellular partitioning of SDCCAG8 and NPHP4 to the membrane fraction associated to the endoplasmic reticulum. Conversely, the ciliary localization of these proteins is unaffected in glomeruli or tubular kidney cells of Rpgrip1^nmf247, but NPHP4 is downregulated developmentally and selectively in kidney cortex. Hence, RPGRIP1 presents cell type-dependent pathological effects crucial to the ciliary targeting and subcellular partitioning of NPHP4, RPGR and SDCCAG8, and acetylation of ciliary α-tubulin or its ciliary targeting, selectively in photoreceptors, but not kidney cells, and these pathological effects underlie photoreceptor degeneration and LCA.     

The connecting cilium inner scaffold provides a structural foundation that protects against retinal degeneration

Inherited retinal degeneration due to loss of photoreceptor cells is a leading cause of human blindness. These cells possess a photosensitive outer segment linked to the cell body through the connecting cilium (CC). While structural defects of the CC have been associated with retinal degeneration, its nanoscale molecular composition, assembly, and function are barely known. Here, using expansion microscopy and electron microscopy, we reveal the molecular architecture of the CC and demonstrate that microtubules are linked together by a CC inner scaffold containing POC5, CENTRIN, and FAM161A. Dissecting CC inner scaffold assembly during photoreceptor development in mouse revealed that it acts as a structural zipper, progressively bridging microtubule doublets and straightening the CC. Furthermore, we show that Fam161a disruption in mouse leads to specific CC inner scaffold loss and triggers microtubule doublet spreading, prior to outer segment collapse and photoreceptor degeneration, suggesting a molecular mechanism for a subtype of retinitis pigmentosa.

Inherited retinal degeneration due to loss of photoreceptor cells is a leading cause of human blindness. Ultrastructure expansion microscopy on mouse retina reveals the presence of a novel structure inside the photoreceptor connecting cilium, the inner scaffold, that protects the outer segment against degeneration.     

Joubert Syndrome in French Canadians and Identification of Mutations in CEP104

Joubert syndrome (JBTS) is a primarily autosomal-recessive disorder characterized by a distinctive mid-hindbrain and cerebellar malformation, oculomotor apraxia, irregular breathing, developmental delay, and ataxia. JBTS is a genetically heterogeneous ciliopathy. We sought to characterize the genetic landscape associated with JBTS in the French Canadian (FC) population. We studied 43 FC JBTS subjects from 35 families by combining targeted and exome sequencing. We identified pathogenic (n = 32 families) or possibly pathogenic (n = 2 families) variants in genes previously associated with JBTS in all of these subjects, except for one. In the latter case, we found a homozygous splice-site mutation (c.735+2T>C) in CEP104. Interestingly, we identified two additional non-FC JBTS subjects with mutations in CEP104; one of these subjects harbors a maternally inherited nonsense mutation (c.496C>T [p.Arg166^∗]) and a de novo splice-site mutation (c.2572−2A>G), whereas the other bears a homozygous frameshift mutation (c.1328_1329insT [p.Tyr444fs^∗3]) in CEP104. Previous studies have shown that CEP104 moves from the mother centriole to the tip of the primary cilium during ciliogenesis. Knockdown of CEP104 in retinal pigment epithelial (RPE1) cells resulted in severe defects in ciliogenesis. These observations suggest that CEP104 acts early during cilia formation by regulating the conversion of the mother centriole into the cilia basal body. We conclude that disruption of CEP104 causes JBTS. Our study also reveals that the cause of JBTS has been elucidated in the great majority of our FC subjects (33/35 [94%] families), even though JBTS shows substantial locus and allelic heterogeneity in this population.     

Genetic Defects in TAPT1 Disrupt Ciliogenesis and Cause a Complex Lethal Osteochondrodysplasia

The evolutionarily conserved transmembrane anterior posterior transformation 1 protein, encoded by TAPT1, is involved in murine axial skeletal patterning, but its cellular function remains unknown. Our study demonstrates that TAPT1 mutations underlie a complex congenital syndrome, showing clinical overlap between lethal skeletal dysplasias and ciliopathies. This syndrome is characterized by fetal lethality, severe hypomineralization of the entire skeleton and intra-uterine fractures, and multiple congenital developmental anomalies affecting the brain, lungs, and kidneys. We establish that wild-type TAPT1 localizes to the centrosome and/or ciliary basal body, whereas defective TAPT1 mislocalizes to the cytoplasm and disrupts Golgi morphology and trafficking and normal primary cilium formation. Knockdown of tapt1b in zebrafish induces severe craniofacial cartilage malformations and delayed ossification, which is shown to be associated with aberrant differentiation of cranial neural crest cells.     

Zebrafish mutations affecting cilia motility share similar cystic phenotypes and suggest a mechanism of cyst formation that differs from pkd2 morphants

Zebrafish are an attractive model for studying the earliest cellular defects occurring during renal cyst formation because its kidney (the pronephros) is simple and genes that cause cystic kidney diseases (CKD) in humans, cause pronephric dilations in zebrafish. By comparing phenotypes in three different mutants, locke, swt and kurly, we find that dilations occur prior to 48 hpf in the medial tubules, a location similar to where cysts form in some mammalian diseases. We demonstrate that the first observable phenotypes associated with dilation include cilia motility and luminal remodeling defects. Importantly, we show that some phenotypes common to human CKD, such as an increased number of cells, are secondary consequences of dilation. Despite having differences in cilia motility, locke, swt and kurly share similar cystic phenotypes, suggesting that they function in a common pathway. To begin to understand the molecular mechanisms involved in cyst formation, we have cloned the swt mutation and find that it encodes a novel leucine rich repeat containing protein (LRRC50), which is thought to function in correct dynein assembly in cilia. Finally, we show that knock-down of polycystic kidney disease 2 (pkd2) specifically causes glomerular cysts and does not affect cilia motility, suggesting multiple mechanisms exist for cyst formation.     

Jagged2a-notch signaling mediates cell fate choice in the zebrafish pronephric duct

Pronephros, a developmental model for adult mammalian kidneys (metanephros) and a functional kidney in early teleosts, consists of glomerulus, tubule, and duct. These structural and functional elements are responsible for different kidney functions, e.g., blood filtration, waste extraction, salt recovery, and water balance. During pronephros organogenesis, cell differentiation is a key step in generating different cell types in specific locations to accomplish designated functions. However, it is poorly understood what molecules regulate the differentiation of different cell types in different parts of the kidney. Two types of epithelial cells, multi-cilia cells and principal cells, are found in the epithelia of the zebrafish distal pronephric duct. While the former is characterized by at least 15 apically localized cilia and expresses centrin2 and rfx2, the latter is characterized by a single primary cilium and sodium pumps. Multi-cilia cells and principal cells differentiate from 17.5 hours post-fertilization onwards in a mosaic pattern. Jagged2a-Notch1a/Notch3-Her9 is responsible for specification and patterning of these two cell types through a lateral inhibition mechanism. Furthermore, multi-cilia cell hyperplasia was observed in mind bomb mutants and Mind bomb was shown to interact with Jagged2a and facilitate its internalization. Taken together, our findings add a new paradigm of Notch signaling in kidney development, namely, that Jagged2a-Notch signaling modulates cell fate choice in a nephric segment, the distal pronephric duct.     

Mutations in KIAA0586 Cause Lethal Ciliopathies Ranging from a Hydrolethalus Phenotype to Short-Rib Polydactyly Syndrome

KIAA0586, the human ortholog of chicken TALPID3, is a centrosomal protein that is essential for primary ciliogenesis. Its disruption in animal models causes defects attributed to abnormal hedgehog signaling; these defects include polydactyly and abnormal dorsoventral patterning of the neural tube. Here, we report homozygous mutations of KIAA0586 in four families affected by lethal ciliopathies ranging from a hydrolethalus phenotype to short-rib polydactyly. We show defective ciliogenesis, as well as abnormal response to SHH-signaling activation in cells derived from affected individuals, consistent with a role of KIAA0586 in primary cilia biogenesis. Whereas centriolar maturation seemed unaffected in mutant cells, we observed an abnormal extended pattern of CEP290, a centriolar satellite protein previously associated with ciliopathies. Our data show the crucial role of KIAA0586 in human primary ciliogenesis and subsequent abnormal hedgehog signaling through abnormal GLI3 processing. Our results thus establish that KIAA0586 mutations cause lethal ciliopathies.     

Mutations in ARL2BP,Encoding ADP-Ribosylation-Factor-Like 2 Binding Protein,Cause Autosomal-Recessive Retinitis Pigmentosa

Retinitis pigmentosa (RP) is a genetically heterogeneous retinal degeneration characterized by photoreceptor death, which results in visual failure. Here, we used a combination of homozygosity mapping and exome sequencing to identify mutations in ARL2BP, which encodes an effector protein of the small GTPases ARL2 and ARL3, as causative for autosomal-recessive RP (RP66). In a family affected by RP and situs inversus, a homozygous, splice-acceptor mutation, c.101−1G>C, which alters pre-mRNA splicing of ARLBP2 in blood RNA, was identified. In another family, a homozygous c.134T>G (p.Met45Arg) mutation was identified. In the mouse retina, ARL2BP localized to the basal body and cilium-associated centriole of photoreceptors and the periciliary extension of the inner segment. Depletion of ARL2BP caused cilia shortening. Moreover, depletion of ARL2, but not ARL3, caused displacement of ARL2BP from the basal body, suggesting that ARL2 is vital for recruiting or anchoring ARL2BP at the base of the cilium. This hypothesis is supported by the finding that the p.Met45Arg amino acid substitution reduced binding to ARL2 and caused the loss of ARL2BP localization at the basal body in ciliated nasal epithelial cells. These data demonstrate a role for ARL2BP and ARL2 in primary cilia function and that this role is essential for normal photoreceptor maintenance and function.     

The carboxyl-terminal region of SDCCAG8 comprises a functional module essential for cilia formation as well as organ development and homeostasis

In humans, ciliary dysfunction causes ciliopathies, which present as multiple organ defects, including developmental and sensory abnormalities. Sdccag8 is a centrosomal/basal body protein essential for proper cilia formation. Gene mutations in SDCCAG8 have been found in patients with ciliopathies manifesting a broad spectrum of symptoms, including hypogonadism. Among these mutations, several that are predicted to truncate the SDCCAG8 carboxyl (C) terminus are also associated with such symptoms; however, the underlying mechanisms are poorly understood. In the present study, we identified the Sdccag8 C-terminal region (Sdccag8-C) as a module that interacts with the ciliopathy proteins, Ick/Cilk1 and Mak, which were shown to be essential for the regulation of ciliary protein trafficking and cilia length in mammals in our previous studies. We found that Sdccag8-C is essential for Sdccag8 localization to centrosomes and cilia formation in cultured cells. We then generated a mouse mutant in which Sdccag8-C was truncated (Sdccag8^ΔC/ΔC mice) using a CRISPR-mediated stop codon knock-in strategy. In Sdccag8^ΔC/ΔC mice, we observed abnormalities in cilia formation and ciliopathy-like organ phenotypes, including cleft palate, polydactyly, retinal degeneration, and cystic kidney, which partially overlapped with those previously observed in Ick- and Mak-deficient mice. Furthermore, Sdccag8^ΔC/ΔC mice exhibited a defect in spermatogenesis, which was a previously uncharacterized phenotype of Sdccag8 dysfunction. Together, these results shed light on the molecular and pathological mechanisms underlying ciliopathies observed in patients with SDCCAG8 mutations and may advance our understanding of protein–protein interaction networks involved in cilia development.     

Mutations in CSPP1, Encoding a Core Centrosomal Protein,Cause a Range of Ciliopathy Phenotypes in Humans

Ciliopathies are characterized by a pattern of multisystem involvement that is consistent with the developmental role of the primary cilium. Within this biological module, mutations in genes that encode components of the cilium and its anchoring structure, the basal body, are the major contributors to both disease causality and modification. However, despite rapid advances in this field, the majority of the genes that drive ciliopathies and the mechanisms that govern the pronounced phenotypic variability of this group of disorders remain poorly understood. Here, we show that mutations in CSPP1, which encodes a core centrosomal protein, are disease causing on the basis of the independent identification of two homozygous truncating mutations in three consanguineous families (one Arab and two Hutterite) affected by variable ciliopathy phenotypes ranging from Joubert syndrome to the more severe Meckel-Gruber syndrome with perinatal lethality and occipital encephalocele. Consistent with the recently described role of CSPP1 in ciliogenesis, we show that mutant fibroblasts from one affected individual have severely impaired ciliogenesis with concomitant defects in sonic hedgehog (SHH) signaling. Our results expand the list of centrosomal proteins implicated in human ciliopathies.     

Cby1 promotes Ahi1 recruitment to a ring-shaped domain at the centriole–cilium interface and facilitates proper cilium formation and function

Defects in centrosome and cilium function are associated with phenotypically related syndromes called ciliopathies. Cby1, the mammalian orthologue of the Drosophila Chibby protein, localizes to mature centrioles, is important for ciliogenesis in multiciliated airway epithelia in mice, and antagonizes canonical Wnt signaling via direct regulation of β-catenin. We report that deletion of the mouse Cby1 gene results in cystic kidneys, a phenotype common to ciliopathies, and that Cby1 facilitates the formation of primary cilia and ciliary recruitment of the Joubert syndrome protein Arl13b. Localization of Cby1 to the distal end of mature centrioles depends on the centriole protein Ofd1. Superresolution microscopy using both three-dimensional SIM and STED reveals that Cby1 localizes to an ∼250-nm ring at the distal end of the mature centriole, in close proximity to Ofd1 and Ahi1, a component of the transition zone between centriole and cilium. The amount of centriole-localized Ahi1, but not Ofd1, is reduced in Cby1^−/− cells. This suggests that Cby1 is required for efficient recruitment of Ahi1, providing a possible molecular mechanism for the ciliogenesis defect in Cby1^−/− cells.     

Mutations of CEP83 Cause Infantile Nephronophthisis and Intellectual Disability

Ciliopathies are a group of hereditary disorders associated with defects in cilia structure and function. The distal appendages (DAPs) of centrioles are involved in the docking and anchoring of the mother centriole to the cellular membrane during ciliogenesis. The molecular composition of DAPs was recently elucidated and mutations in two genes encoding DAPs components (CEP164/NPHP15, SCLT1) have been associated with human ciliopathies, namely nephronophthisis and orofaciodigital syndrome. To identify additional DAP components defective in ciliopathies, we independently performed targeted exon sequencing of 1,221 genes associated with cilia and 5 known DAP protein-encoding genes in 1,255 individuals with a nephronophthisis-related ciliopathy. We thereby detected biallelic mutations in a key component of DAP-encoding gene, CEP83, in seven families. All affected individuals had early-onset nephronophthisis and four out of eight displayed learning disability and/or hydrocephalus. Fibroblasts and tubular renal cells from affected individuals showed an altered DAP composition and ciliary defects. In summary, we have identified mutations in CEP83, another DAP-component-encoding gene, as a cause of infantile nephronophthisis associated with central nervous system abnormalities in half of the individuals.