The ciliopathies are an expanding group of disorders caused by mutations in genes implicated in the biogenesis and function of primary cilia. Bardet-Biedl syndrome (BBS) is a model ciliopathy characterized by progressive retinal degeneration, obesity, polydactyly, cognitive impairment, kidney anomalies and hypogonadism. Mutations in SDCCAG8(NPHP10) were described recently in patients with nephronophthisis and retinal degeneration (Senior-Loken syndrome; SLS). Given the phenotypic and genetic overlap between known ciliopathy genes, we hypothesized that mutations in SDCCAG8 might also contribute alleles to more severe, multisystemic ciliopathies. We performed genetic and phenotypic analyses of 2 independent BBS cohorts. Subsequent to mutation screening, we made a detailed phenotypic analysis of 5 families mutated for SDCCAG8 (3 homozygous and 2 compound heterozygous mutations) and conducted statistical analyses across both cohorts to examine possible phenotype-genotype correlations with mutations at this locus. All patients with mutations in SDCCAG8 fulfilled the diagnostic criteria for BBS (retinal degeneration, obesity, cognitive defects, renal failure, hypogonadism). Interestingly, none of the patients with primary SDCCAG8 mutations had polydactyly, a frequent but not obligatory BBS feature. In contrast, the same patients displayed early-onset renal failure, obesity, as well as recurrent pulmonary and ENT infections. Comparison of the phenotypes of these families with our entire BBS cohort indicated that renal impairment and absent polydactyly correlated significantly with causal SDCCAG8 mutations. Thus, SDCCAG8 mutations are sufficient to cause BBS in 1–2% of our combined cohorts, and define this gene as the sixteenth BBS locus (BBS16). The absence of polydactyly and the concomitant, apparently fully penetrant association with early kidney failure represents the first significant genotype-phenotype correlation in BBS that potentially represents an indicator for phenotype-driven priority screening and informs specific patient management.
Bardet-Biedl; Ciliopathy; Nephronophthisis; Polydactyly; SDCCAG8
Jeune's Asphyxiating Thoracic Dystrophy (JATD) is an autosomal recessive disorder with symptoms of retinal degeneration, kidney cysts, and chondrodysplasia and results from mutations in the ift80 gene. This study was conducted to characterize zebrafish lacking ift80 function for photoreceptor degeneration and defects in ciliogenesis in order to establish zebrafish as a vertebrate model for visual dysfunction in JATD and to determine if ift80 interacts genetically with Bardet-Biedl Syndrome (BBS) genes.
Zebrafish were injected with morpholinos (MOs) targeted to the ift80 gene. Retinas were analyzed by histology, transmission electron microscopy, and immunohistochemistry. Ear and kidney cilia were analyzed by whole-mount immunostaining. Intraflagellar Transport (IFT) particle composition was analyzed by Western blotting. Genetic interactions were tested by co-injection of MOs against ift80 and bbs4 or bbs8 followed by in situ hybridization.
Zebrafish lacking ift80 function exhibited defects in photoreceptor outer segment formation, and photoreceptor death. Staining with opsin antibodies revealed opsin mislocalization in both rods and cones. Ultrastructural analysis showed abnormal disk stacking and shortened photoreceptor outer segments. The kinocilia of the ear and motile cilia in the kidney were shorter and reduced in number. Western blotting revealed a slight increase in the stability of other IFT proteins. Co-injection of MOs against ift80 and BBS genes led to convergent-extension defects.
Zebrafish lacking ift80 exhibited defects characteristic of Jeune's Asphyxiating Thoracic Dystrophy. Because the developing outer segments degenerated, Ift80 could possibly act as a maintenance factor for the IFT particle.
The causes and mechanisms leading to loss of visual function in Jeune syndrome have not been extensively explored. The authors show that zebrafish lacking ift80, a gene responsible for a subset of Jeune syndrome, undergo photoreceptor degeneration in a mechanism consistent with defects in cilia maintenance.
Jeune's asphyxiating thoracic dystrophy (JATD) is an autosomal recessive disorder with symptoms of retinal degeneration, kidney cysts, and chondrodysplasia and results from mutations in the ift80 gene. This study was conducted to characterize zebrafish lacking ift80 function for photoreceptor degeneration and defects in ciliogenesis to establish zebrafish as a vertebrate model for visual dysfunction in JATD and to determine whether ift80 interacts genetically with Bardet-Biedl syndrome (BBS) genes.
Zebrafish were injected with morpholinos (MOs) targeted to the ift80 gene. Retinas were analyzed by histology, transmission electron microscopy, and immunohistochemistry. Ear and kidney cilia were analyzed by whole-mount immunostaining. Intraflagellar transport (IFT) particle composition was subjected to Western blot analysis. Genetic interactions were tested by coinjection of MOs against ift80 and bbs4 or bbs8 followed by in situ hybridization.
Zebrafish lacking ift80 function exhibited defects in photoreceptor outer segment formation and photoreceptor death. Staining with opsin antibodies revealed opsin mislocalization in both rods and cones. Ultrastructural analysis showed abnormal disc stacking and shortened photoreceptor outer segments. The kinocilia of the ear and motile cilia in the kidney were shorter and reduced in number. Western blot analysis revealed a slight increase in the stability of other IFT proteins. Coinjection of MOs against ift80 and BBS genes led to convergent-extension defects.
Zebrafish lacking ift80 exhibited defects characteristic of JATD. Because the developing outer segments degenerated, Ift80 could possibly act as a maintenance factor for the IFT particle.
Photoreceptor degeneration is a common feature of ciliopathies, owing to the importance of the highly specialized ciliary structure of these cells. Absence of AHI1, which encodes a cilium-localized protein, has been shown to cause a form of Joubert syndrome highly penetrant for retinal degeneration1,2. We show that Ahi1 knockout mice fail to form outer segments (OS), and show abnormal distribution of opsin throughout photoreceptors. Apoptotic cell death occurs rapidly between 2-4 weeks of age and is significantly delayed by reduced dosage of opsin. This phenotype also displays dosage-sensitive genetic interactions with Nphp1, another ciliopathy gene. Although not a primary cause of retinal blindness in humans, an allele of AHI1 modifies the relative risk of retinal degeneration greater than 7 fold within a nephronophthisis cohort. Our data support context-specific roles for AHI1 as a contributor to retinopathy and may explain a proportion of the variability of retinal phenotypes observed in nephronophthisis.
Eight proteins, defects in which are associated with Meckel-Gruber syndrome and nephronophthisis ciliopathies, work together as two functional modules at the transition zone to establish basal body/transition zone connections with the membrane and barricade entry of non-ciliary components into this organelle.
Meckel-Gruber syndrome (MKS), nephronophthisis (NPHP), and related ciliopathies present with overlapping phenotypes and display considerable allelism between at least twelve different genes of largely unexplained function. We demonstrate that the conserved C. elegans B9 domain (MKS-1, MKSR-1, and MKSR-2), MKS-3/TMEM67, MKS-5/RPGRIP1L, MKS-6/CC2D2A, NPHP-1, and NPHP-4 proteins exhibit essential, collective functions at the transition zone (TZ), an underappreciated region at the base of all cilia characterized by Y-shaped assemblages that link axoneme microtubules to surrounding membrane. These TZ proteins functionally interact as members of two distinct modules, which together contribute to an early ciliogenic event. Specifically, MKS/MKSR/NPHP proteins establish basal body/TZ membrane attachments before or coinciding with intraflagellar transport–dependent axoneme extension and subsequently restrict accumulation of nonciliary components within the ciliary compartment. Together, our findings uncover a unified role for eight TZ-localized proteins in basal body anchoring and establishing a ciliary gate during ciliogenesis, and suggest that disrupting ciliary gate function contributes to phenotypic features of the MKS/NPHP disease spectrum.
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, hitherto not implicated in ciliopathies. 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. We identify TTBK2, CCDC92, NPHP3 and DVL3 as novel CEP164 interaction partners. Our findings link degenerative diseases of kidney and retina, disorders of increasing prevalence, to mechanisms of DDR.
In addition to their role in motility, eukaryotic cilia serve as a distinct compartment for signal transduction and regulatory sequestration of biomolecules. Recent genetic and biochemical studies have revealed an extraordinary diversity of protein complexes involved in the biogenesis of cilia during each cell cycle. Mutations in components of these complexes are at the heart of human ciliopathies such as Nephronophthisis (NPHP), Meckel-Gruber syndrome (MKS), Bardet-Biedl syndrome (BBS) and Joubert syndrome (JBTS). Despite intense studies, proteins in some of these complexes, such as the NPHP1-4-8 and the MKS, remain poorly understood. Using a combination of computational analyses we studied these complexes to identify novel domains in them which might throw new light on their functions and evolutionary origins. First, we identified both catalytically active and inactive versions of transglutaminase-like (TGL) peptidase domains in key ciliary/centrosomal proteins CC2D2A/MKS6, CC2D2B, CEP76 and CCDC135. These ciliary TGL domains appear to have originated from prokaryotic TGL domains that act as peptidases, either in a prokaryotic protein degradation system with the MoxR AAA+ ATPase, the precursor of eukaryotic dyneins and midasins, or in a peptide-ligase system with an ATP-grasp enzyme comparable to tubulin-modifying TTL proteins. We suggest that active ciliary TGL proteins are part of a cilia-specific peptidase system that might remove tubulin modifications or cleave cilia- localized proteins, while the inactive versions are likely to bind peptides and mediate key interactions during ciliogenesis. Second, we observe a vast radiation of C2 domains, which are key membrane-localization modules, in multiple ciliary proteins, including those from the NPHP1-4-8 and the MKS complexes, such as CC2D2A/MKS6, RPGRIP1, RPGRIP1L, NPHP1, NPHP4, C2CD3, AHI1/Jouberin and CEP76, most of which can be traced back to the last eukaryotic ancestor. Identification of these TGL and C2 domains aid in the proper reconstruction of the Y-shaped linkers, which are key structures in the transitional zone of cilia, by allowing precise prediction of the multiple membrane-contacting and protein-protein interaction sites in these structures. These findings help decipher key events in the evolutionary separation of the ciliary and nuclear compartments in course of the emergence of the eukaryotic cell.
ciliogenesis; transglutaminase-like; membrane; tubulin-tyrosine ligase; C2; transition zone; Y-shaped linkers; evolution; origin of eukaryotes; ciliopathy
IFT80, a protein component of intraflagellar transport (IFT) complex B, is required for the formation, maintenance and functionality of cilia. Mutations in IFT80 cause Jeune asphyxiating thoracic dystrophy (JATD) and short rib polydactyly (SRP) type III. Both diseases are autosomal recessive chondrodysplasias and share clinical and radiological similarities, including shortening of the long bones and constriction of the thoracic cage. A murine Ift80 gene-trap line was used to investigate the role of Ift80 during development. The homozygote appears hypomorphic rather than a true null due to low level wild-type transcript production by alternative splicing around the gene-trap cassette. Hypomorphic levels of Ift80 result in embryonic lethality highlighting a key role for Ift80 in development. In rare cases, gene-trap homozygotes survive to postnatal stages and phenocopy both JATD and SRP type III by exhibiting growth retardation, shortening of the long bones, constriction of the ribcage and polydactyly. Mouse embryonic fibroblasts made from this line showed a significant reduction in hedgehog pathway activation in response to Hedgehog analog treatment. This defective signalling was not accompanied by the loss or malformation of cilia as seen in some knockout models of other IFT component genes. Phenotypes indicative of defects in cilia structure or function such as situs inversus, cystic renal disease and retinal degeneration were not observed in this line. These data suggest that there is an absolute requirement for Ift80 in hedgehog signalling, but low level expression permits ciliogenesis indicating separate but linked roles for this protein in formation and function.
Nephronophthisis (NPHP) is the most common genetic cause of end-stage renal disease in children and young adults. In Chlamydomonas reinhardtii, Caenorhabditis elegans, and mammals, the NPHP1 and NPHP4 gene products nephrocystin-1 and nephrocystin-4 localize to basal bodies or ciliary transition zones (TZs), but their function in this location remains unknown. We show here that loss of C. elegans NPHP-1 and NPHP-4 from TZs is tolerated in developing cilia but causes changes in localization of specific ciliary components and a broad range of subtle axonemal ultrastructural defects. In amphid channel cilia, nphp-4 mutations cause B tubule defects that further disrupt intraflagellar transport (IFT). We propose that NPHP-1 and NPHP-4 act globally at the TZ to regulate ciliary access of the IFT machinery, axonemal structural components, and signaling molecules, and that perturbing this balance results in cell type–specific phenotypes.
Despite rapid advances in disease gene identification, the predictive power of the genotype remains limited, in part due to poorly understood effects of second-site modifiers. Here we demonstrate that a polymorphic coding variant of RPGRIP1L (retinitis pigmentosa GTPase regulator-interacting protein-1 like), a ciliary gene mutated in Meckel-Gruber (MKS) and Joubert (JBTS) syndromes, is associated with the development of retinal degeneration in patients with ciliopathies caused by mutations in other genes. As part of our resequencing efforts of the ciliary proteome, we identified several putative loss of function RPGRIP1L mutations, including one common variant, A229T. Multiple genetic lines of evidence showed this allele to be associated with photoreceptor loss in ciliopathies. Moreover, we show that RPGRIP1L interacts biochemically with RPGR, loss of which causes retinal degeneration, and that the 229T-encoded protein significantly compromises this interaction. Our data represent an example of modification of a discrete phenotype of syndromic disease and highlight the importance of a multifaceted approach for the discovery of modifier alleles of intermediate frequency and effect.
Nephronophthisis (NPHP), a recessive cystic kidney disease, is the most frequent genetic cause of end-stage kidney disease in children and young adults. Positional cloning of nine genes (NPHP1-9) and functional characterization of their encoded proteins (nephrocystins) has contributed to a unifying theory that defines cystic kidney diseases as “ciliopathies”. The theory is based on the finding that all proteins mutated in cystic kidney diseases of humans or animal models are expressed in primary cilia or centrosomes of renal epithelial cells. Primary cilia are sensory organelles that connect mechanosensory, visual, and other stimuli to mechanisms of epithelial cell polarity and cell cycle control. Mutations in NPHP genes cause defects in signaling mechanisms that involve the non-canonical Wnt signaling pathway and the sonic hedgehog signaling pathway, resulting in defects of planar cell polarity and tissue maintenance. The ciliary theory explains the multiple organ involvement in NPHP, which includes retinal degeneration, cerebellar hypoplasia, liver fibrosis, situs inversus, and mental retardation. Positional cloning of dozens of unknown genes that cause NPHP will elucidate further signaling mechanisms involved. Nephrocystins are highly conserved in evolution, thus allowing the use of animal models to develop future therapeutic approaches.
nephronophthisis; cystic kidney disease; planar cell polarity; wnt signaling; hedgehog signaling; ciliopathies
Bardet–Biedl syndrome (BBS; OMIM no. 209 900) and Alström syndrome (ALMS; OMIM no. 203 800) are rare, multisystem genetic disorders showing both a highly variable phenotype and considerable phenotypic overlap; they are included in the emerging group of diseases called ciliopathies. The genetic heterogeneity of BBS with 14 causal genes described to date, serves to further complicate mutational analysis. The development of the BBS–ALMS array which detects known mutations in these genes has allowed us to detect at least one mutation in 40.5% of BBS families and in 26.7% of ALMS families validating this as an efficient and cost-effective first pass screening modality. Furthermore, using this method, we found two BBS families segregating three BBS alleles further supporting oligogenicity or modifier roles for additional mutations. We did not observe more than two mutations in any ALMS family.
Bardet–Biedl syndrome; BBS; Alström syndrome; ALMS1; arrayed primer extension; mutation analysis
Nephronophthisis-related ciliopathies (NPHP-RC) are recessive disorders featuring dysplasia or degeneration preferentially in kidney, retina, and cerebellum. Here we combine homozygosity mapping with candidate gene analysis by performing “ciliopathy candidate exome capture” followed by massively-parallel sequencing. We detect 12 different truncating mutations of SDCCAG8 in 10 NPHP-RC families. We demonstrate that SDCCAG8 is localized at both centrioles and directly interacts with NPHP-RC-associated OFD1. Depletion of sdccag8 causes kidney cysts and a body axis defect in zebrafish and induces cell polarity defects in 3D renal cell cultures. This work identifies SDCCAG8 loss of function as a novel cause of a retinal-renal ciliopathy and validates exome capture analysis for broadly heterogeneous single-gene disorders.
Human ciliopathies are hereditary conditions caused by defects of proteins expressed at the primary cilium. Among ciliopathies, Joubert syndrome and related disorders (JSRD), Meckel syndrome (MKS) and nephronophthisis (NPH) present clinical and genetic overlap, being allelic at several loci. One of the most interesting gene is TMEM67, encoding the transmembrane protein meckelin. We performed mutation analysis of TMEM67 in 341 probands, including 265 JSRD representative of all clinical subgroups and 76 MKS fetuses. We identified 33 distinct mutations, of which 20 were novel, in 8/10 (80%) JS with liver involvement (COACH phenotype) and 12/76 (16%) MKS fetuses. No mutations were found in other JSRD subtypes, confirming the strong association between TMEM67 mutations and liver involvement. Literature review of all published TMEM67 mutated cases was performed to delineate genotype-phenotype correlates. In particular, comparison of the types of mutations and their distribution along the gene in lethal versus non lethal phenotypes showed in MKS patients a significant enrichment of missense mutations falling in TMEM67 exons 8 to 15, especially when in combination with a truncating mutation. These exons encode for a region of unknown function in the extracellular domain of meckelin.
TMEM67; MKS3; Joubert syndrome; Meckel syndrome; congenital hepatic fibrosis; COACH syndrome
Disorders within the “ciliopathy” spectrum include Joubert (JS), Bardet-Biedl syndromes (BBS) and nephronophthisis (NPHP). Although mutations in single ciliopathy genes can lead to these different syndromes between families, there have been no reports of phenotypic discordance within a single family. We report on two consanguineous families with discordant ciliopathies in sibling. In Ciliopathy-672, the older child displayed dialysis-dependent NPHP whereas the younger displayed the pathognomonic molar tooth MRI sign (MTS) of JS. A second branch displayed two additional children with NPHP. In Ciliopathy-1491, the oldest child displayed classical features of BBS whereas the two younger children displayed the MTS. Importantly, the children with BBS and NPHP lacked MTS, whereas children with JS lacked obesity or NPHP, and the child with BBS lacked MTS and NPHP. Features common to all three disorders included intellectual disability, postaxial polydactyly, and visual reduction. The variable phenotypic expressivity in this family suggests that genetic modifiers may determine specific clinical features within the ciliopathy spectrum.
Molar-tooth; polydactyly; intellectual disability; retinal blindness; obesity; nephronophthisis
An emerging group of human genetic diseases termed ‘ciliopathies’ are caused by dysfunction of two functionally and physically associated organelles, the centrosome and cilium. These organelles are central to perception of the physical environment through detection of a diverse variety of extracellular signals such as growth factors, chemicals, light and fluid flow. Many of the described ciliopathies display multi-organ involvement, with renal and retina being the most commonly affected. Nephronophthisis is a recessive disorder of the kidney that is the leading cause of end-stage renal failure in children. Through positional cloning, many of the causative mutations have been mapped to genes involved in centrosome and cilia function. In this review, we discuss the identified causative mutations that give rise to nephronophthisis and how these are related to the disease etiology in both the kidney and other organs.
Nephronophthisis; NPHP genes; Joubert syndrome; Meckel-Gruber syndrome; Senior-Løken syndrome
Ciliopathies encompass a broad array of clinical findings associated with genetic defects in biogenesis and/or function of the primary cilium, a ubiquitous organelle involved in the transduction of diverse biological signals. Degeneration or dysfunction of retinal photoreceptors is frequently observed in diverse ciliopathies. The sensory cilium in a photoreceptor elaborates into unique outer segment discs that provide extensive surface area for maximal photon capture and efficient visual transduction. The daily renewal of approximately 10% of outer segments requires a precise control of ciliary transport. Here, we review the ciliopathies with associated retinal degeneration, describe the distinctive structure of the photoreceptor cilium, and discuss mouse models that allow investigations into molecular mechanisms of cilia biogenesis and defects. We have specifically focused on two ciliary proteins – CEP290 and RPGR – that underlie photoreceptor degeneration and syndromic ciliopathies. Mouse models of CEP290 and RPGR disease, and of their multiple interacting partners, have helped unravel new functional insights into cell type-specific phenotypic defects in distinct ciliary proteins. Elucidation of multifaceted ciliary functions and associated protein complexes will require concerted efforts to assimilate diverse datasets from in vivo and in vitro studies. We therefore discuss a possible framework for investigating genetic networks associated with photoreceptor cilia biogenesis and pathology.
Ciliopathy; Retinal degeneration; Primary cilium; Sensory cilia; CEP290; RPGR; Bardet–Biedl syndrome; Leber congenital amaurosis; Joubert syndrome; Nephronophthisis
'Ciliopathies' are an emerging class of genetic multisystemic human disorders that are caused by a multitude of largely unrelated genes that affect ciliary structure/function. They are unified by shared clinical features, such as mental retardation, cystic kidney, retinal defects and polydactyly, and by the common localization of the protein products of these genes at or near the primary cilium of cells. With the realization that many previously disparate conditions are a part of this spectrum of disorders, there has been tremendous interest in the function of cilia in developmental signaling and homeostasis. Ciliopathies are mostly inherited as simple recessive traits, but phenotypic expressivity is under the control of numerous genetic modifiers, putting these conditions at the interface of simple and complex genetics. In this review, we discuss the ever-expanding ciliopathy field, which has three interrelated goals: developing a comprehensive understanding of genes mutated in the ciliopathies and required for ciliogenesis; understanding how the encoded proteins work together in complexes and networks to modulate activity and structure-function relationships; and uncovering signaling pathways and modifier relationships.
Ciliopathies are pleiotropic and genetically heterogeneous disorders caused by defective development and function of the primary cilium. Bardet-Biedl syndrome (BBS) proteins localize to the base of cilia and undergo intraflagellar transport, and the loss of their functions leads to a multisystemic ciliopathy. Here we report the identification of mutations in guanylate cyclases (GCYs) as modifiers of Caenorhabditis elegans bbs endophenotypes. The loss of GCY-35 or GCY-36 results in suppression of the small body size, developmental delay, and exploration defects exhibited by multiple bbs mutants. Moreover, an effector of cGMP signalling, a cGMP-dependent protein kinase, EGL-4, also modifies bbs mutant defects. We propose that a misregulation of cGMP signalling, which underlies developmental and some behavioural defects of C. elegans bbs mutants, may also contribute to some BBS features in other organisms.
Bardet-Biedl syndrome (BBS) is a genetically heterogeneous, multisystemic disorder. Defects to the cilium, an evolutionarily conserved organelle, cause ciliopathies, a growing class of diseases that includes BBS. BBS proteins are involved in the vesicular transport of proteins to the cilium and in the process of intraflagellar transport. Here we show that, in addition to sensory defects, Caenorhabditis elegans bbs mutants exhibit reduced body size and delayed developmental timing. The reduced body size phenotype is not fully recapitulated by IFT mutants, suggesting that BBS proteins may have additional functions beyond bridging IFT motors. We further identified that the loss of function mutations in the soluble guanylate cyclase complex, GCY-35/GCY-36, results in a suppression of these defects. Interestingly, GCY-35/GCY-36 influences the body size through a cGMP-dependent protein kinase EGL-4 in a group of body cavity neurons. BBS proteins, on the other hand, function through a non-overlapping set of ciliated sensory neurons to influence cGMP signalling in the body cavity neurons. In conclusion, this study reveals a non-cell autonomous role for sensory cilia in regulating cGMP signalling during development. We propose that aberrant cGMP signalling, essential for a number of cellular processes, may also contribute to some ciliopathy features in other systems.
Nephronophthisis is the most common genetic cause of end-stage renal failure during childhood and adolescence. Genetic studies have identified disease-causing mutations in at least 11 different genes (NPHP1–11), but the function of the corresponding nephrocystin proteins remains poorly understood. The two evolutionarily conserved proteins nephrocystin-1 (NPHP1) and nephrocystin-4 (NPHP4) interact and localize to cilia in kidney, retina, and brain characterizing nephronophthisis and associated pathologies as result of a ciliopathy. Here we show that NPHP4, but not truncating patient mutations, negatively regulates tyrosine phosphorylation of NPHP1. NPHP4 counteracts Pyk2-mediated phosphorylation of three defined tyrosine residues of NPHP1 thereby controlling binding of NPHP1 to the trans-Golgi sorting protein PACS-1. Knockdown of NPHP4 resulted in an accumulation of NPHP1 in trans-Golgi vesicles of ciliated retinal epithelial cells. These data strongly suggest that NPHP4 acts upstream of NPHP1 in a common pathway and support the concept of a role for nephrocystin proteins in intracellular vesicular transport.
Centrosome; Epithelium; Golgi; Protein-tyrosine Kinase (Tyrosine Kinase); Vesicles; Pyk2; Cilium; Cystic Kidney Disease; Nephrocystin
Intraflagellar transport (IFT), the motor-dependent movement of IFT particles along the axoneme, is critical for the assembly, maintenance, and function of motile and sensory cilia, and, consequently, this process underlies ciliary motility, cilium-based signaling, and ciliopathies. Here, I present my perspective on IFT as a model system for studying motor-driven cargo transport. I review evidence that kinesin-2 motors physically transport IFT particles as cargo and hypothesize that several accessory kinesins confer cilia-specific functions by augmenting the action of the two core IFT motors, kinesin-2 and dynein 1b, which assemble the cilium foundation.
Conserved intraflagellar transport (IFT) particle proteins and IFT-associated motors are needed to assemble most eukaryotic cilia and flagella. Proteins in an IFT-A subcomplex are generally required for dynein-driven retrograde IFT, from the ciliary tip to the base. We describe novel structural and functional roles for IFT-A proteins in chordotonal organs, insect mechanosensory organs with cilia that are both sensory and motile.
The reduced mechanoreceptor potential A (rempA) locus of Drosophila encodes the IFT-A component IFT140. Chordotonal cilia are shortened in rempA mutants and an IFT-B protein accumulates in the mutant cilia, consistent with a defect in retrograde IFT. A functional REMPA-YFP fusion protein concentrates at the site of the ciliary dilation (CD), a highly structured axonemal inclusion of hitherto unknown composition and function. The CD is absent in rempA mutants, and REMPA-YFP is undetectable in the absence of another IFT-A protein, IFT122. In a mutant lacking the IFT dynein motor, the CD is disorganized and REMPA-YFP is mislocalized. A TRPV ion channel, required to generate sensory potentials and regulate ciliary motility, is normally localized in the cilia, proximal to the CD. This channel spreads into the distal part of the cilia in dynein mutants, and is undetectable in rempA mutants.
IFT-A proteins are located at and required by the ciliary dilation, which separates chordotonal cilia into functionally distinct zones. A requirement for IFT140 in stable TRPV channel expression also suggests that IFT-A proteins may mediate preciliary transport of some membrane proteins.
A spectrum of complex oligogenic disorders called the ciliopathies have been connected to dysfunction of cilia. Among the ciliopathies are Nephronophthisis (NPHP), characterized by cystic kidney disease and retinal degeneration, and Meckel–Gruber syndrome (MKS), a gestational lethal condition with skeletal abnormalities, cystic kidneys and CNS malformation. Mutations in multiple genes have been identified in NPHP and MKS patients, and an unexpected finding has been that mutations within the same gene can cause either disorder. Further, there is minimal genotype–phenotype correlation and despite recessive inheritance, numerous patients were identified as having a single heterozygous mutation. This has made it difficult to determine the significance of these mutations on disease pathogenesis and led to the hypothesis that clinical presentation in an individual will be determined by genetic interactions between mutations in multiple cilia-related genes. Here we utilize Caenorhabditis elegans and cilia-associated behavioral and morphologic assays to evaluate the pathogenic potential of eight previously reported human NPHP4 missense mutations. We assess the impact of these mutations on C. elegans NPHP-4 function, localization and evaluate potential interactions with mutations in MKS complex genes, mksr-2 and mksr-1. Six out of eight nphp-4 mutations analyzed alter ciliary function, and three of these modify the severity of the phenotypes caused by disruption of mksr-2 and mksr-1. Collectively, our studies demonstrate the utility of C. elegans as a tool to assess the pathogenicity of mutations in ciliopathy genes and provide insights into the complex genetic interactions contributing to the diversity of phenotypes associated with cilia disorders.
The autosomal recessive kidney disease nephronophthisis (NPHP) constitutes the most frequent genetic cause of terminal renal failure in the first 3 decades of life. Ten causative genes (NPHP1–NPHP9 and NPHP11), whose products localize to the primary cilia-centrosome complex, support the unifying concept that cystic kidney diseases are “ciliopathies”. Using genome-wide homozygosity mapping, we report here what we believe to be a new locus (NPHP-like 1 [NPHPL1]) for an NPHP-like nephropathy. In 2 families with an NPHP-like phenotype, we detected homozygous frameshift and splice-site mutations, respectively, in the X-prolyl aminopeptidase 3 (XPNPEP3) gene. In contrast to all known NPHP proteins, XPNPEP3 localizes to mitochondria of renal cells. However, in vivo analyses also revealed a likely cilia-related function; suppression of zebrafish xpnpep3 phenocopied the developmental phenotypes of ciliopathy morphants, and this effect was rescued by human XPNPEP3 that was devoid of a mitochondrial localization signal. Consistent with a role for XPNPEP3 in ciliary function, several ciliary cystogenic proteins were found to be XPNPEP3 substrates, for which resistance to N-terminal proline cleavage resulted in attenuated protein function in vivo in zebrafish. Our data highlight an emerging link between mitochondria and ciliary dysfunction, and suggest that further understanding the enzymatic activity and substrates of XPNPEP3 will illuminate novel cystogenic pathways.
Bardet-Biedl syndrome (BBS) is a pleiotropic recessive disorder that belongs to the rapidly growing family of ciliopathies. It shares phenotypic traits with other ciliopathies, such as Alström syndrome (ALMS), nephronophthisis (NPHP) or Joubert syndrome. BBS mutations have been detected in 16 different genes (BBS1-BBS16) without clear genotype-to-phenotype correlation. This extensive genetic heterogeneity is a major concern for molecular diagnosis and genetic counselling. While various strategies have been recently proposed to optimise mutation detection, they either fail to detect mutations in a majority of patients or are time consuming and costly.
We tested a targeted exon-capture strategy coupled with multiplexing and high-throughput sequencing on 52 patients: 14 with known mutations as proof-of-principle and 38 with no previously detected mutation. Thirty genes were targeted in total including the 16 BBS genes, the 12 known NPHP genes, the single ALMS gene ALMS1 and the proposed modifier CCDC28B.
This strategy allowed the reliable detection of causative mutations (including homozygous/heterozygous exon deletions) in 68% of BBS patients without previous molecular diagnosis and in all proof-of-principle samples. Three probands carried homozygous truncating mutations in ALMS1 confirming the major phenotypic overlap between both disorders. The efficiency of detecting mutations in patients was positively correlated with their compliance with the classical BBS phenotype (mutations were identified in 81% of ‘classical’ BBS patients) suggesting that only a few true BBS genes remain to be identified. We illustrate some interpretation problems encountered due to the multiplicity of identified variants.
This strategy is highly efficient and cost effective for diseases with high genetic heterogeneity, and guarantees a quality of coverage in coding sequences of target genes suited for diagnosis purposes.
Targeted sequencing; ciliopathies; Bardet-Biedl syndrome; multiplexing; diagnosis