Exome capture reveals ZNF423 and CEP164 mutations, linking renal ciliopathies to DNA damage response signaling.

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.

PCARE and WASF3 regulate ciliary F-actin assembly that is required for the initiation of photoreceptor outer segment disk formation.

The outer segments (OS) of rod and cone photoreceptor cells are specialized sensory cilia that contain hundreds of opsin-loaded stacked membrane disks that enable phototransduction. The biogenesis of these disks is initiated at the OS base, but the driving force has been debated. Here, we studied the function of the protein encoded by the photoreceptor-specific gene C2orf71, which is mutated in inherited retinal dystrophy (RP54). We demonstrate that C2orf71/PCARE (photoreceptor cilium actin regulator) can interact with the Arp2/3 complex activator WASF3, and efficiently recruits it to the primary cilium. Ectopic coexpression of PCARE and WASF3 in ciliated cells results in the remarkable expansion of the ciliary tip. This process was disrupted by small interfering RNA (siRNA)-based down-regulation of an actin regulator, by pharmacological inhibition of actin polymerization, and by the expression of PCARE harboring a retinal dystrophy-associated missense mutation. Using human retinal organoids and mouse retina, we observed that a similar actin dynamics-driven process is operational at the base of the photoreceptor OS where the PCARE module and actin colocalize, but which is abrogated in Pcare-/- mice. The observation that several proteins involved in retinal ciliopathies are translocated to these expansions renders it a potential common denominator in the pathomechanisms of these hereditary disorders. Together, our work suggests that PCARE is an actin-associated protein that interacts with WASF3 to regulate the actin-driven expansion of the ciliary membrane at the initiation of new outer segment disk formation.

Homozygous loss-of-function mutations in MNS1 cause laterality defects and likely male infertility.

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.

Recurrent De Novo Mutations Disturbing the GTP/GDP Binding Pocket of RAB11B Cause Intellectual Disability and a Distinctive Brain Phenotype.

The Rab GTPase family comprises ∼70 GTP-binding proteins, functioning in vesicle formation, transport and fusion. They are activated by a conformational change induced by GTP-binding, allowing interactions with downstream effectors. Here, we report five individuals with two recurrent de novo missense mutations in RAB11B; c.64G>A; p.Val22Met in three individuals and c.202G>A; p.Ala68Thr in two individuals. An overlapping neurodevelopmental phenotype, including severe intellectual disability with absent speech, epilepsy, and hypotonia was observed in all affected individuals. Additionally, visual problems, musculoskeletal abnormalities, and microcephaly were present in the majority of cases. Re-evaluation of brain MRI images of four individuals showed a shared distinct brain phenotype, consisting of abnormal white matter (severely decreased volume and abnormal signal), thin corpus callosum, cerebellar vermis hypoplasia, optic nerve hypoplasia and mild ventriculomegaly. To compare the effects of both variants with known inactive GDP- and active GTP-bound RAB11B mutants, we modeled the variants on the three-dimensional protein structure and performed subcellular localization studies. We predicted that both variants alter the GTP/GDP binding pocket and show that they both have localization patterns similar to inactive RAB11B. Evaluation of their influence on the affinity of RAB11B to a series of binary interactors, both effectors and guanine nucleotide exchange factors (GEFs), showed induction of RAB11B binding to the GEF SH3BP5, again similar to inactive RAB11B. In conclusion, we report two recurrent dominant mutations in RAB11B leading to a neurodevelopmental syndrome, likely caused by altered GDP/GTP binding that inactivate the protein and induce GEF binding and protein mislocalization.

Missense mutations in the WD40 domain of AHI1 cause non-syndromic retinitis pigmentosa.

BACKGROUND: Recent findings suggesting that Abelson helper integration site 1 (AHI1) is involved in non-syndromic retinal disease have been debated, as the functional significance of identified missense variants was uncertain. We assessed whether AHI1 variants cause non-syndromic retinitis pigmentosa (RP). METHODS: Exome sequencing was performed in three probands with RP. The effects of the identified missense variants in AHI1 were predicted by three-dimensional structure homology modelling. Ciliary parameters were evaluated in patient's fibroblasts, and recombinant mutant proteins were expressed in ciliated retinal pigmented epithelium cells. RESULTS: In the three patients with RP, three sets of compound heterozygous variants were detected in AHI1 (c.2174G>A; p.Trp725* and c.2258A>T; p.Asp753Val, c.660delC; p.Ser221Glnfs*10 and c.2090C>T; p.Pro697Leu, c.2087A>G; p.His696Arg and c.2429C>T; p.Pro810Leu). All four missense variants were present in the conserved WD40 domain of Jouberin, the ciliary protein encoded by AHI1, with variable predicted implications for the domain structure. No significant changes in the percentage of ciliated cells, nor in cilium length or intraflagellar transport were detected. However, expression of mutant recombinant Jouberin in ciliated cells showed a significantly decreased enrichment at the ciliary base. CONCLUSIONS: This report confirms that mutations in AHI1 can underlie autosomal recessive RP. Moreover, it structurally and functionally validates the effect of the RP-associated AHI1 variants on protein function, thus proposing a new genotype-phenotype correlation for AHI1 mutation associated retinal ciliopathies.

Interactome analysis reveals that FAM161A, deficient in recessive retinitis pigmentosa, is a component of the Golgi-centrosomal network.

Defects in FAM161A, a protein of unknown function localized at the cilium of retinal photoreceptor cells, cause retinitis pigmentosa, a form of hereditary blindness. By using different fragments of this protein as baits to screen cDNA libraries of human and bovine retinas, we defined a yeast two-hybrid-based FAM161A interactome, identifying 53 bona fide partners. In addition to statistically significant enrichment in ciliary proteins, as expected, this interactome revealed a substantial bias towards proteins from the Golgi apparatus, the centrosome and the microtubule network. Validation of interaction with key partners by co-immunoprecipitation and proximity ligation assay confirmed that FAM161A is a member of the recently recognized Golgi-centrosomal interactome, a network of proteins interconnecting Golgi maintenance, intracellular transport and centrosome organization. Notable FAM161A interactors included AKAP9, FIP3, GOLGA3, KIFC3, KLC2, PDE4DIP, NIN and TRIP11. Furthermore, analysis of FAM161A localization during the cell cycle revealed that this protein followed the centrosome during all stages of mitosis, likely reflecting a specific compartmentalization related to its role at the ciliary basal body during the G0 phase. Altogether, these findings suggest that FAM161A's activities are probably not limited to ciliary tasks but also extend to more general cellular functions, highlighting possible novel mechanisms for the molecular pathology of retinal disease.

Disruption of the basal body protein POC1B results in autosomal-recessive cone-rod dystrophy.

Exome sequencing revealed a homozygous missense mutation (c.317C>G [p.Arg106Pro]) in POC1B, encoding POC1 centriolar protein B, in three siblings with autosomal-recessive cone dystrophy or cone-rod dystrophy and compound-heterozygous POC1B mutations (c.199_201del [p.Gln67del] and c.810+1G>T) in an unrelated person with cone-rod dystrophy. Upon overexpression of POC1B in human TERT-immortalized retinal pigment epithelium 1 cells, the encoded wild-type protein localized to the basal body of the primary cilium, whereas this localization was lost for p.Arg106Pro and p.Gln67del variant forms of POC1B. Morpholino-oligonucleotide-induced knockdown of poc1b translation in zebrafish resulted in a dose-dependent small-eye phenotype, impaired optokinetic responses, and decreased length of photoreceptor outer segments. These ocular phenotypes could partially be rescued by wild-type human POC1B mRNA, but not by c.199_201del and c.317C>G mutant human POC1B mRNAs. Yeast two-hybrid screening of a human retinal cDNA library revealed FAM161A as a binary interaction partner of POC1B. This was confirmed in coimmunoprecipitation and colocalization assays, which both showed loss of FAM161A interaction with p.Arg106Pro and p.Gln67del variant forms of POC1B. FAM161A was previously implicated in autosomal-recessive retinitis pigmentosa and shown to be located at the base of the photoreceptor connecting cilium, where it interacts with several other ciliopathy-associated proteins. Altogether, this study demonstrates that POC1B mutations result in a defect of the photoreceptor sensory cilium and thus affect cone and rod photoreceptors.

CCDC151 mutations cause primary ciliary dyskinesia by disruption of the outer dynein arm docking complex formation.

A diverse family of cytoskeletal dynein motors powers various cellular transport systems, including axonemal dyneins generating the force for ciliary and flagellar beating essential to movement of extracellular fluids and of cells through fluid. Multisubunit outer dynein arm (ODA) motor complexes, produced and preassembled in the cytosol, are transported to the ciliary or flagellar compartment and anchored into the axonemal microtubular scaffold via the ODA docking complex (ODA-DC) system. In humans, defects in ODA assembly are the major cause of primary ciliary dyskinesia (PCD), an inherited disorder of ciliary and flagellar dysmotility characterized by chronic upper and lower respiratory infections and defects in laterality. Here, by combined high-throughput mapping and sequencing, we identified CCDC151 loss-of-function mutations in five affected individuals from three independent families whose cilia showed a complete loss of ODAs and severely impaired ciliary beating. Consistent with the laterality defects observed in these individuals, we found Ccdc151 expressed in vertebrate left-right organizers. Homozygous zebrafish ccdc151(ts272a) and mouse Ccdc151(Snbl) mutants display a spectrum of situs defects associated with complex heart defects. We demonstrate that CCDC151 encodes an axonemal coiled coil protein, mutations in which abolish assembly of CCDC151 into respiratory cilia and cause a failure in axonemal assembly of the ODA component DNAH5 and the ODA-DC-associated components CCDC114 and ARMC4. CCDC151-deficient zebrafish, planaria, and mice also display ciliary dysmotility accompanied by ODA loss. Furthermore, CCDC151 coimmunoprecipitates CCDC114 and thus appears to be a highly evolutionarily conserved ODA-DC-related protein involved in mediating assembly of both ODAs and their axonemal docking machinery onto ciliary microtubules.

Active transport and diffusion barriers restrict Joubert Syndrome-associated ARL13B/ARL-13 to an Inv-like ciliary membrane subdomain.

Cilia are microtubule-based cell appendages, serving motility, chemo-/mechano-/photo- sensation, and developmental signaling functions. Cilia are comprised of distinct structural and functional subregions including the basal body, transition zone (TZ) and inversin (Inv) compartments, and defects in this organelle are associated with an expanding spectrum of inherited disorders including Bardet-Biedl syndrome (BBS), Meckel-Gruber Syndrome (MKS), Joubert Syndrome (JS) and Nephronophthisis (NPHP). Despite major advances in understanding ciliary trafficking pathways such as intraflagellar transport (IFT), how proteins are transported to subciliary membranes remains poorly understood. Using Caenorhabditis elegans and mammalian cells, we investigated the transport mechanisms underlying compartmentalization of JS-associated ARL13B/ARL-13, which we previously found is restricted at proximal ciliary membranes. We now show evolutionary conservation of ARL13B/ARL-13 localisation to an Inv-like subciliary membrane compartment, excluding the TZ, in many C. elegans ciliated neurons and in a subset of mammalian ciliary subtypes. Compartmentalisation of C. elegans ARL-13 requires a C-terminal RVVP motif and membrane anchoring to prevent distal cilium and nuclear targeting, respectively. Quantitative imaging in more than 20 mutants revealed differential contributions for IFT and ciliopathy modules in defining the ARL-13 compartment; IFT-A/B, IFT-dynein and BBS genes prevent ARL-13 accumulation at periciliary membranes, whereas MKS/NPHP modules additionally inhibit ARL-13 association with TZ membranes. Furthermore, in vivo FRAP analyses revealed distinct roles for IFT and MKS/NPHP genes in regulating a TZ barrier to ARL-13 diffusion, and intraciliary ARL-13 diffusion. Finally, C. elegans ARL-13 undergoes IFT-like motility and quantitative protein complex analysis of human ARL13B identified functional associations with IFT-B complexes, mapped to IFT46 and IFT74 interactions. Together, these findings reveal distinct requirements for sequence motifs, IFT and ciliopathy modules in defining an ARL-13 subciliary membrane compartment. We conclude that MKS/NPHP modules comprise a TZ barrier to ARL-13 diffusion, whereas IFT genes predominantly facilitate ARL-13 ciliary entry and/or retention via active transport mechanisms.

Mutations in the gene encoding the basal body protein RPGRIP1L, a nephrocystin-4 interactor, cause Joubert syndrome.

Protein-protein interaction analyses have uncovered a ciliary and basal body protein network that, when disrupted, can result in nephronophthisis (NPHP), Leber congenital amaurosis, Senior-Løken syndrome (SLSN) or Joubert syndrome (JBTS). However, details of the molecular mechanisms underlying these disorders remain poorly understood. RPGRIP1-like protein (RPGRIP1L) is a homolog of RPGRIP1 (RPGR-interacting protein 1), a ciliary protein defective in Leber congenital amaurosis. We show that RPGRIP1L interacts with nephrocystin-4 and that mutations in the gene encoding nephrocystin-4 (NPHP4) that are known to cause SLSN disrupt this interaction. RPGRIP1L is ubiquitously expressed, and its protein product localizes to basal bodies. Therefore, we analyzed RPGRIP1L as a candidate gene for JBTS and identified loss-of-function mutations in three families with typical JBTS, including the characteristic mid-hindbrain malformation. This work identifies RPGRIP1L as a gene responsible for JBTS and establishes a central role for cilia and basal bodies in the pathophysiology of this disorder.

Mutations in C8orf37, encoding a ciliary protein, are associated with autosomal-recessive retinal dystrophies with early macular involvement.

Cone-rod dystrophy (CRD) and retinitis pigmentosa (RP) are clinically and genetically overlapping heterogeneous retinal dystrophies. By using homozygosity mapping in an individual with autosomal-recessive (ar) RP from a consanguineous family, we identified three sizeable homozygous regions, together encompassing 46 Mb. Next-generation sequencing of all exons, flanking intron sequences, microRNAs, and other highly conserved genomic elements in these three regions revealed a homozygous nonsense mutation (c.497T>A [p.Leu166(∗)]) in C8orf37, located on chromosome 8q22.1. This mutation was not present in 150 ethnically matched control individuals, single-nucleotide polymorphism databases, or the 1000 Genomes database. Immunohistochemical studies revealed C8orf37 localization at the base of the primary cilium of human retinal pigment epithelium cells and at the base of connecting cilia of mouse photoreceptors. C8orf37 sequence analysis of individuals who had retinal dystrophy and carried conspicuously large homozygous regions encompassing C8orf37 revealed a homozygous splice-site mutation (c.156-2A>G) in two siblings of a consanguineous family and homozygous missense mutations (c.529C>T [p.Arg177Trp]; c.545A>G [p.Gln182Arg]) in siblings of two other consanguineous families. The missense mutations affect highly conserved amino acids, and in silico analyses predicted that both variants are probably pathogenic. Clinical assessment revealed CRD in four individuals and RP with early macular involvement in two individuals. The two CRD siblings with the c.156-2A>G mutation also showed unilateral postaxial polydactyly. These results underline the importance of disrupted ciliary processes in the pathogenesis of retinal dystrophies.

FAM161A, associated with retinitis pigmentosa, is a component of the cilia-basal body complex and interacts with proteins involved in ciliopathies.

Retinitis pigmentosa (RP) is a retinal degenerative disease characterized by the progressive loss of photoreceptors. We have previously demonstrated that RP can be caused by recessive mutations in the human FAM161A gene, encoding a protein with unknown function that contains a conserved region shared only with a distant paralog, FAM161B. In this study, we show that FAM161A localizes at the base of the photoreceptor connecting cilium in human, mouse and rat. Furthermore, it is also present at the ciliary basal body in ciliated mammalian cells, both in native conditions and upon the expression of recombinant tagged proteins. Yeast two-hybrid analysis of binary interactions between FAM161A and an array of ciliary and ciliopathy-associated proteins reveals direct interaction with lebercilin, CEP290, OFD1 and SDCCAG8, all involved in hereditary retinal degeneration. These interactions are mediated by the C-terminal moiety of FAM161A, as demonstrated by pull-down experiments in cultured cell lines and in bovine retinal extracts. As other ciliary proteins, FAM161A can also interact with the microtubules and organize itself into microtubule-dependent intracellular networks. Moreover, small interfering RNA-mediated depletion of FAM161A transcripts in cultured cells causes the reduction in assembled primary cilia. Taken together, these data indicate that FAM161A-associated RP can be considered as a novel retinal ciliopathy and that its molecular pathogenesis may be related to other ciliopathies.

The ciliopathy-associated protein homologs RPGRIP1 and RPGRIP1L are linked to cilium integrity through interaction with Nek4 serine/threonine kinase.

Recent studies have established ciliary dysfunction as the underlying cause of a broad range of multi-organ phenotypes, known as 'ciliopathies'. Ciliopathy-associated proteins have a common site of action in the cilium, however, their overall importance for ciliary function differs, as implied by the extreme variability in ciliopathy phenotypes. The aim of this study was to gain more insight in the function of two ciliopathy-associated protein homologs, RPGR interacting protein 1 (RPGRIP1) and RPGRIP1-like protein (RPGRIP1L). Mutations in RPGRIP1 lead to the eye-restricted disease Leber congenital amaurosis, while mutations in RPGRIP1L are causative for Joubert and Meckel syndrome, which affect multiple organs and are at the severe end of the ciliopathy spectrum. Using tandem affinity purification in combination with mass spectrometry, we identified Nek4 serine/threonine kinase as a prominent component of both the RPGRIP1- as well as the RPGRIP1L-associated protein complex. In ciliated cells, this kinase localized to basal bodies, while in ciliated organs, the kinase was predominantly detected at the ciliary rootlet. Down-regulation of NEK4 in ciliated cells led to a significant decrease in cilium assembly, pointing to a role for Nek4 in cilium dynamics. We now hypothesize that RPGRIP1 and RPGRIP1L function as cilium-specific scaffolds that recruit a Nek4 signaling network which regulates cilium stability. Our data are in line with previously established roles in the cilium of other members of the Nek protein family and define NEK4 as a ciliopathy candidate gene.

PDE6D Mediates Trafficking of Prenylated Proteins NIM1K and UBL3 to Primary Cilia.

Mutations in PDE6D impair the function of its cognate protein, phosphodiesterase 6D (PDE6D), in prenylated protein trafficking towards the ciliary membrane, causing the human ciliopathy Joubert Syndrome (JBTS22) and retinal degeneration in mice. In this study, we purified the prenylated cargo of PDE6D by affinity proteomics to gain insight into PDE6D-associated disease mechanisms. By this approach, we have identified a specific set of PDE6D-interacting proteins that are involved in photoreceptor integrity, GTPase activity, nuclear import, or ubiquitination. Among these interacting proteins, we identified novel ciliary cargo proteins of PDE6D, including FAM219A, serine/threonine-protein kinase NIM1 (NIM1K), and ubiquitin-like protein 3 (UBL3). We show that NIM1K and UBL3 localize inside the cilium in a prenylation-dependent manner. Furthermore, UBL3 also localizes in vesicle-like structures around the base of the cilium. Through affinity proteomics of UBL3, we confirmed its strong interaction with PDE6D and its association with proteins that regulate small extracellular vesicles (sEVs) and ciliogenesis. Moreover, we show that UBL3 localizes in specific photoreceptor cilium compartments in a prenylation-dependent manner. Therefore, we propose that UBL3 may play a role in the sorting of proteins towards the photoreceptor outer segment, further explaining the development of PDE6D-associated retinal degeneration.

OFD1 is mutated in X-linked Joubert syndrome and interacts with LCA5-encoded lebercilin.

We ascertained a multi-generation Malaysian family with Joubert syndrome (JS). The presence of asymptomatic obligate carrier females suggested an X-linked recessive inheritance pattern. Affected males presented with mental retardation accompanied by postaxial polydactyly and retinitis pigmentosa. Brain MRIs showed the presence of a "molar tooth sign," which classifies this syndrome as classic JS with retinal involvement. Linkage analysis showed linkage to Xpter-Xp22.2 and a maximum LOD score of 2.06 for marker DXS8022. Mutation analysis revealed a frameshift mutation, p.K948NfsX8, in exon 21 of OFD1. In an isolated male with JS, a second frameshift mutation, p.E923KfsX3, in the same exon was identified. OFD1 has previously been associated with oral-facial-digital type 1 (OFD1) syndrome, a male-lethal X-linked dominant condition, and with X-linked recessive Simpson-Golabi-Behmel syndrome type 2 (SGBS2). In a yeast two-hybrid screen of a retinal cDNA library, we identified OFD1 as an interacting partner of the LCA5-encoded ciliary protein lebercilin. We show that X-linked recessive mutations in OFD1 reduce, but do not eliminate, the interaction with lebercilin, whereas X-linked dominant OFD1 mutations completely abolish binding to lebercilin. In addition, recessive mutations in OFD1 did not affect the pericentriolar localization of the recombinant protein in hTERT-RPE1 cells, whereas this localization was lost for dominant mutations. These findings offer a molecular explanation for the phenotypic spectrum observed for OFD1 mutations; this spectrum now includes OFD1 syndrome, SGBS2, and JS.

Usher syndrome and Leber congenital amaurosis are molecularly linked via a novel isoform of the centrosomal ninein-like protein.

Usher syndrome (USH) and Leber congenital amaurosis (LCA) are autosomal recessive disorders resulting in syndromic and non-syndromic forms of blindness. In order to gain insight into the pathogenic mechanisms underlying retinal degeneration, we searched for interacting proteins of USH2A isoform B (USH2A(isoB)) and the LCA5-encoded protein lebercilin. We identified a novel isoform of the centrosomal ninein-like protein, hereby named Nlp isoform B (Nlp(isoB)), as a common interactor. Although we identified the capacity of this protein to bind calcium with one of its three EF-hand domains, the interacton with USH2A(isoB) did not depend on this. Upon expression in ARPE-19 cells, recombinant Nlp(isoB), lebercilin and USH2A(isoB) were all found to co-localize at the centrosomes. Staining of retinal sections with specific antibodies against all three proteins revealed their co-localization at the basal bodies of the photoreceptor-connecting cilia. Based on this subcellular localization and the nature of their previously identified binding partners, we hypothesize that the pathogenic mechanisms for LCA and USH show significant overlap and involve defects in ciliogenesis, cilia maintenance and intraflagellar and/or microtubule-based transport. The direct association of Nlp(isoB) with USH2A(isoB) and lebercilin indicates that Nlp can be considered as a novel candidate gene for USH, LCA and allied retinal ciliopathies.

CC2D2A is mutated in Joubert syndrome and interacts with the ciliopathy-associated basal body protein CEP290.

Joubert syndrome and related disorders (JSRD) are primarily autosomal-recessive conditions characterized by hypotonia, ataxia, abnormal eye movements, and intellectual disability with a distinctive mid-hindbrain malformation. Variable features include retinal dystrophy, cystic kidney disease, and liver fibrosis. JSRD are included in the rapidly expanding group of disorders called ciliopathies, because all six gene products implicated in JSRD (NPHP1, AHI1, CEP290, RPGRIP1L, TMEM67, and ARL13B) function in the primary cilium/basal body organelle. By using homozygosity mapping in consanguineous families, we identify loss-of-function mutations in CC2D2A in JSRD patients with and without retinal, kidney, and liver disease. CC2D2A is expressed in all fetal and adult tissues tested. In ciliated cells, we observe localization of recombinant CC2D2A at the basal body and colocalization with CEP290, whose cognate gene is mutated in multiple hereditary ciliopathies. In addition, the proteins can physically interact in vitro, as shown by yeast two-hybrid and GST pull-down experiments. A nonsense mutation in the zebrafish CC2D2A ortholog (sentinel) results in pronephric cysts, a hallmark of ciliary dysfunction analogous to human cystic kidney disease. Knockdown of cep290 function in sentinel fish results in a synergistic pronephric cyst phenotype, revealing a genetic interaction between CC2D2A and CEP290 and implicating CC2D2A in cilium/basal body function. These observations extend the genetic spectrum of JSRD and provide a model system for studying extragenic modifiers in JSRD and other ciliopathies.

A CEP104-CSPP1 Complex Is Required for Formation of Primary Cilia Competent in Hedgehog Signaling.

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.

CiliaCarta: An integrated and validated compendium of ciliary genes.

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/.

Versatile screening for binary protein-protein interactions by yeast two-hybrid mating.

Identification of binary protein-protein interactions is a crucial step in determining the molecular context and functional pathways of proteins. State-of-the-art proteomics techniques provide high-throughput information on the content of proteomes and protein complexes, but give little information about transient interactions, about the binary protein pairs, or about the interacting epitopes. A powerful method to reveal this information is the yeast two-hybrid system. We have employed an optimized GAL4-based yeast two-hybrid system to dissect the photoreceptor cilium-associated protein complex around the retinitis pigmentosa GTPase regulator (RPGR) in mammalian photoreceptors. This enabled us to identify associating protein partners that, similar to RPGR, were also associated with a heterogeneous group of inherited retinal degenerations arising from ciliary defects. We describe how to generate high content pretransformed cDNA libraries, and perform an efficient yeast mating screen for protein-protein interactions with any bait protein of interest.