Novel TMEM67 mutations and genotype‐phenotype correlates in meckelin‐related ciliopathies

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 TMEM67mutated 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. © 2010 Wiley‐Liss, Inc.     

Genomic analysis of Meckel–Gruber syndrome in Arabs reveals marked genetic heterogeneity and novel candidate genes

Meckel–Gruber syndrome (MKS, OMIM #249000) is a multiple congenital malformation syndrome that represents the severe end of the ciliopathy phenotypic spectrum. Despite the relatively common occurrence of this syndrome among Arabs, little is known about its genetic architecture in this population. This is a series of 18 Arab families with MKS, who were evaluated clinically and studied using autozygome-guided mutation analysis and exome sequencing. We show that autozygome-guided candidate gene analysis identified the underlying mutation in the majority (n=12, 71%). Exome sequencing revealed a likely pathogenic mutation in three novel candidate MKS disease genes. These include C5orf42, Ellis–van-Creveld disease gene EVC2 and SEC8 (also known as EXOC4), which encodes an exocyst protein with an established role in ciliogenesis. This is the largest and most comprehensive genomic study on MKS in Arabs and the results, in addition to revealing genetic and allelic heterogeneity, suggest that previously reported disease genes and the novel candidates uncovered by this study account for the overwhelming majority of MKS patients in our population.     

A missense mutation in TMEM67 causes Meckel-Gruber syndrome type 3 (MKS3): a family from China

Meckel-Gruber syndrome (MKS) is a lethal autosomal recessive condition characterized by renal cysts and variably associated features, including developmental anomalies of the central nervous system (typically encephalocele), hepatic ductal dysplasia and cysts, and polydactyly. Genetic heterogeneity has been demonstrated at eleven loci, MKS1-11. Here, we present the clinical and molecular characteristics of a Chinese MKS3 family with occipital encephalocele and kidney enlargement. DNA sequencing of affected fetuses revealed a homozygous c.1645C>T substitution in exon 16 of TMEM67, leading to a p.R549C substitution in meckelin. The R549 residue is highly conserved across human, rat, mouse, zebrafish, chicken, wolf and platypus genomes. Hha I restriction analysis demonstrated that the c.1645C>T mutation was absent in 200 unrelated control chromosomes of Chinese background, supporting the hypothesis that it represents causative mutation, not rare polymorphism. Our data provide additional molecular and clinical information for establishing a better genotype-phenotype understanding of MKS.     

A novel murine allele of Intraflagellar Transport Protein 172 causes a syndrome including VACTERL-like features with hydrocephalus

The primary cilium is emerging as a crucial regulator of signaling pathways central to vertebrate development and human disease. We identified atrioventricular canal 1 (avc1), a mouse mutation that caused VACTERL association with hydrocephalus, or VACTERL-H. We showed that avc1 is a hypomorphic mutation of intraflagellar transport protein 172 (Ift172), required for ciliogenesis and Hedgehog (Hh) signaling. Phenotypically, avc1 caused VACTERL-H but not abnormalities in left-right (L-R) axis formation. Avc1 resulted in structural cilia defects, including truncated cilia in vivo and in vitro. We observed a dose-dependent requirement for Ift172 in ciliogenesis using an allelic series generated with Ift172(avc1) and Ift172(wim), an Ift172 null allele: cilia were present on 42% of avc1 mouse embryonic fibroblast (MEF) and 28% of avc1/wim MEFs, in contrast to >90% of wild-type MEFs. Furthermore, quantitative cilium length analysis identified two specific cilium populations in mutant MEFS: a normal population with normal IFT and a truncated population, 50% of normal length, with disrupted IFT. Cells from wild-type embryos had predominantly full-length cilia, avc1 embryos, with Hh signaling abnormalities but not L-R abnormalities, had cilia equally divided between full-length and truncated, and avc1/wim embryos, with both Hh signaling and L-R abnormalities, were primarily truncated. Truncated Ift172 mutant cilia showed defects of the distal ciliary axoneme, including disrupted IFT88 localization and Hh-dependent Gli2 localization. We propose a model in which mutation of Ift172 results in a specific class of abnormal cilia, causing disrupted Hh signaling while maintaining L-R axis determination, and resulting in the VACTERL-H phenotype.     

A homozygous deleterious CDK10 mutation in a patient with agenesis of corpus callosum,retinopathy, and deafness

The primary cilium is a key organelle in numerous physiological and developmental processes. Genetic defects in the formation of this non‐motile structure, in its maintenance and function, underlie a wide array of ciliopathies in human, including craniofacial, brain and heart malformations, and retinal and hearing defects. We used exome sequencing to study the molecular basis of disease in an 11‐year‐old female patient who suffered from growth retardation, global developmental delay with absent speech acquisition, agenesis of corpus callosum and paucity of white matter, sensorineural deafness, retinitis pigmentosa, vertebral anomalies, patent ductus arteriosus, and facial dysmorphism reminiscent of STAR syndrome, a suspected ciliopathy. A homozygous variant, c.870_871del, was identified in the CDK10 gene, predicted to cause a frameshift, p.Trp291Alafs*18, in the cyclin‐dependent kinase 10 protein. CDK10 mRNAs were detected in patient cells and do not seem to undergo non‐sense mediated decay. CDK10 is the binding partner of Cyclin M (CycM) and CDK10/CycM protein kinase regulates ciliogenesis and primary cilium elongation. Notably, CycM gene is mutated in patients with STAR syndrome. Following incubation, the patient cells appeared less elongated and more densely populated than the control cells suggesting that the CDK10 mutation affects the cytoskeleton. Upon starvation and staining with acetylated‐tubulin, γ‐tubulin, and Arl13b, the patient cells exhibited fewer and shorter cilia than control cells. These findings underscore the importance of CDK10 for the regulation of ciliogenesis. CDK10 defect is likely associated with a new form of ciliopathy phenotype; additional patients may further validate this association.     

A TCTN2 mutation defines a novel Meckel Gruber syndrome locus

Meckel Gruber syndrome (MKS) is an autosomal recessive multisystem disorder that represents a severe form of ciliopathy in humans and is characterized by significant genetic heterogeneity. In this article, we describe the identification of a novel MKS locus MKS8 that we map to TCTN2, in a multiplex consanguineous family. TCTN2 is a paralog of the recently identified Tectonic 1, which has been shown to modulate sonic hedgehog signaling. Expression analysis at different developmental stages of the murine ortholog revealed a spatial and temporal pattern consistent with the MKS phenotype observed in our patient. The exclusion of this and the other seven MKS genes in our collection of consanguineous Arab MKS families confirms the existence of two additional MKS loci.     

oleed,a medaka Polycomb group gene,regulates ciliogenesis and left–right patterning

Left‐right (LR) patterning is an essential part of the animal body plan. Primary cilia are known to play a pivotal role in this process. In humans, genetic disorders of ciliogenesis cause serious congenital diseases. A comprehensive mechanism that regulates ciliogenesis has not been proposed so far. Here, we show that EED, a core member of the Polycomb group (PcG) genes and a presumed player in many epigenetic processes, is required for ciliogenesis and subsequent LR patterning in the medaka fish, Oryzias latipes. Moderate knockdown of oleed, a medaka homolog of EED, preferentially caused situs inversus. In the affected embryo, the cilia in Kupffer’s vesicle showed various defects in their structure, position and motility. Furthermore, we demonstrated that oleed maintains the expression of Noto, which, in mice, regulates ciliogenesis and LR patterning. This study provides the first evidence for the involvement of epigenetic plasticity in LR patterning through ciliogenesis.     

Two novel B9D1 variants causing Joubert syndrome: Utility of mRNA and splicing studies

The primary cilium is an organelle which plays an important role in the transduction of signals in the Wnt and Sonic hedgehog pathways. Abnormal or absent primary cilia result in various neurodevelopmental, retinal, renal, hepatic and musculoskeletal abnormalities. Joubert syndrome (JS) is a ciliopathy with a prevalence estimated to be between 1:80 000 and 1:100 000. JS occurs due to bi-allelic mutations in one of the 34 identified genes, all of which encode for protein components of the primary cilia. The presentation of JS is highly variable, however a clinical diagnosis can be established by the presence of the molar tooth sign on axial brain MRI, hypotonia in infancy, and developmental delay. JS is less severe than Meckel syndrome (MKS), which is another recessive, and often lethal, ciliopathy. This report outlines an interesting case of JS, in which two novel mutations in B9D1 were identified. This gene is not commonly associated with JS, and is often implicated in MKS. Functional mRNA study was helpful in delineating the pathogenic role of novel variants in this case.     

Convergent extension movements and ciliary function are mediated by ofd1, a zebrafish orthologue of the human oral-facial-digital type 1 syndrome gene

In humans, OFD1 is mutated in oral-facial-digital type I syndrome leading to prenatal death in hemizygous males and dysmorphic faces and brain malformations, with polycystic kidneys presenting later in life in heterozygous females. To elucidate the function of Ofd1, we have studied its function during zebrafish embryonic development. In wild-type embryos, ofd1 mRNA is widely expressed and Ofd1-green fluorescent protein (GFP) fusion localizes to the centrosome/basal body. Disrupting Ofd1 using antisense morpholinos (MOs) led to bent body axes, hydrocephalus and oedema. Laterality was randomized in the brain, heart and viscera, likely a consequence of shorter cilia with disrupted axonemes and perturbed intravesicular fluid flow in Kupffer's vesicle. Embryos injected with ofd1 MOs also displayed convergent extension (CE) defects, which were enhanced by loss of Slb/Wnt11 or Tri/Vangl2, two proteins functioning in a non-canonical Wnt/Planar Cell Polarity (PCP) pathway. Pronephric glomerular midline fusion was compromised in vangl2 and ofd1 loss of function embryos and we suggest this anomaly may be a novel CE defect. Thus, Ofd1 is required for ciliary motility and function in zebrafish, supporting data showing that Ofd1 is essential for primary cilia function in mice. In addition, our data show that Ofd1 is important for CE during gastrulation, consistent with data linking primary cilia and non-canonical Wnt/PCP signalling.     

Ciliary Genes TBC1D32/C6orf170 and SCLT1 are Mutated in Patients with OFD Type IX

Clinical syndromes caused by defects in the primary cilium are heterogeneous but there are recurrent phenotypic manifestations that define them as a collective group known as ciliopathies. Dozens of genes have been linked to various ciliopathies but large patient cohorts have clearly revealed the existence of additional genetic heterogeneity, which is yet to be fully appreciated. In our search for novel ciliopathy‐linked genes through the study of unmapped ciliopathy phenotypes, we have identified two simplex cases with a severe ciliopathy phenotype consistent with oro‐facio‐digital syndrome type IX featuring midline cleft, microcephaly, and colobomatous microphathalmia/anophthalmia. In addition, there was variable presence of polydactyly, absent pituitary, and congenital heart disease. The autozygome of each index harbored a single novel truncating variant as revealed by exome sequencing, and the affected genes (SCLT1 and TBC1D32/C6orf170) have established roles in centrosomal biology and ciliogenesis. Our findings suggest a previously unrecognized role of SCLT1 and TBC1D32 in the pathogenesis of ciliopathy in humans.     

Insufficiency of BUBR1, a mitotic spindle checkpoint regulator, causes impaired ciliogenesis in vertebrates

Budding uninhibited by benzimidazole-related 1 (BUBR1) is a central molecule of the spindle assembly checkpoint. Germline mutations in the budding uninhibited by benzimidazoles 1 homolog beta gene encoding BUBR1 cause premature chromatid separation (mosaic variegated aneuploidy) [PCS (MVA)] syndrome, which is characterized by constitutional aneuploidy and a high risk of childhood cancer. Patients with the syndrome often develop Dandy-Walker complex and polycystic kidneys; implying a critical role of BUBR1 in morphogenesis. However, little is known about the function of BUBR1 other than mitotic control. Here, we report that BUBR1 is essential for the primary cilium formation, and that the PCS (MVA) syndrome is thus a novel ciliopathy. Morpholino knockdown of bubr1 in medaka fish also caused ciliary dysfunction characterized by defects in cerebellar development and perturbed left-right asymmetry of the embryo. Biochemical analyses demonstrated that BUBR1 is required for ubiquitin-mediated proteasomal degradation of cell division cycle protein 20 in the G0 phase and maintains anaphase-promoting complex/cyclosome-CDC20 homolog 1 activity that regulates the optimal level of dishevelled for ciliogenesis.     

A disease causing deletion of 29 base pairs in intron 15 in the MKS1 gene is highly associated with the campomelic variant of the Meckel-Gruber syndrome

Meckel-Gruber syndrome (MKS) is an autosomal recessive disorder causing severe defects in the developing central nervous system and other organs. Recently, mutations in the MKS1 gene have been identified as disease causing in individuals of Finnish MKS families. The primary aim of the present study was to assess the frequency of the 'Finnish founder mutation' (29 bp IVS15-7_35) in the MKS1 gene in 20 aborted fetuses with a diagnosis of MKS. The secondary aim was to screen for novel mutations in the coding sequence of the MKS1 gene of MKS fetuses and to obtain genotype-phenotype correlations where possible. Furthermore, we evaluated the carrier rate of a deletion of 29 bp in intron 15 of the MKS1 gene in a German population. To identify and characterize mutations in the MKS1 gene, sequence analyses and quantitative real time polymerase chain reaction studies were performed. We could identify the same type of mutation, a deletion of 29 bp in intron 15 of the MKS1 gene, in 8 out of the 20 cases studied. Six out of the eight cases with such a mutation displayed the campomelic variant of MKS. The carrier frequency among 519 healthy German individuals was 1:260. This deletion in the MKS1 gene is highly associated with a distinct subtype of the MKS, namely the campomelic variant. In individuals of European origin suffering from the campomelic MKS variant, the described deletion is highly likely to be causative. Regarding the results of our study, the incidence of MKS in Germany can be estimated as 1:135,000. In families with a known mutation in the MKS1 gene, it is now possible to offer an early prenatal testing, for example with chorionic villus sampling and mutation analysis.     

Assessing the pathogenic potential of human Nephronophthisis disease-associated NPHP-4 missense mutations in C. elegans

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.     

Alström Syndrome protein ALMS1 localizes to basal bodies of cochlear hair cells and regulates cilium-dependent planar cell polarity

Alstr?m Syndrome is a life-threatening disease characterized primarily by numerous metabolic abnormalities, retinal degeneration, cardiomyopathy, kidney and liver disease, and sensorineural hearing loss. The cellular localization of the affected protein, ALMS1, has suggested roles in ciliary function and/or ciliogenesis. We have investigated the role of ALMS1 in the cochlea and the pathogenesis of hearing loss in Alstr?m Syndrome. In neonatal rat organ of Corti, ALMS1 was localized to the basal bodies of hair cells and supporting cells. ALMS1 was also evident at the basal bodies of differentiating fibrocytes and marginal cells in the lateral wall. Centriolar ALMS1 expression was retained into maturity. In Alms1-disrupted mice, which recapitulate the neurosensory deficits of human Alstr?m Syndrome, cochleae displayed several cyto-architectural defects including abnormalities in the shape and orientation of hair cell stereociliary bundles. Developing hair cells were ciliated, suggesting that ciliogenesis was largely normal. In adult mice, in addition to bundle abnormalities, there was an accelerated loss of outer hair cells and the progressive appearance of large lesions in stria vascularis. Although the mice progressively lost distortion product otoacoustic emissions, suggesting defects in outer hair cell amplification, their endocochlear potentials were normal, indicating the strial atrophy did not affect its function. These results identify previously unrecognized cochlear histopathologies associated with this ciliopathy that (i) implicate ALMS1 in planar cell polarity signaling and (ii) suggest that the loss of outer hair cells causes the majority of the hearing loss in Alstr?m Syndrome.     

The links between axin and carcinogenesis

The products of the two mammalian Axin genes (Axin1 and its homologue Axin2) are essential for the degradation of beta catenin, a component of Wnt signalling that is frequently dysregulated in cancer cells. Axin is a multidomain scaffold protein that has many functions in biological signalling pathways. Overexpression of mutant [corrected] axin results in axis duplication in mouse embryos. Wnt signalling activity determines dorsal-ventral axis formation in vertebrates, implicating axin as a negative regulator of this signalling pathway. In addition, Wnts modulate pattern formation and the morphogenesis of most organs by influencing and controlling cell proliferation, motility, and fate. Defects in different components of the Wnt signalling pathway promote tumorigenesis and tumour progression. Recent biochemical studies of axins indicate that these molecules are the primary limiting components of this pathway. This review explores the intriguing connections between defects in axin function and human diseases.     

Control of the Wnt pathways by nephrocystin-4 is required for morphogenesis of the zebrafish pronephros

Nephronophthisis is a hereditary nephropathy characterized by interstitial fibrosis and cyst formation. It is caused by mutations in NPHP genes encoding the ciliary proteins, nephrocystins. In this paper, we investigate the function of nephrocystin-4, the product of the nphp4 gene, in vivo by morpholino-mediated knockdown in zebrafish and in vitro in mammalian kidney cells. Depletion of nephrocystin-4 results in convergence and extension defects, impaired laterality, retinal anomalies and pronephric cysts associated with alterations in early cloacal morphogenesis. These defects are accompanied by abnormal ciliogenesis in the cloaca and in the laterality organ. We show that nephrocystin-4 is required for the elongation of the caudal pronephric primordium and for the regulation of cell rearrangements during cloaca morphogenesis. Moreover, depletion of either inversin, the product of the nphp2 gene, or of the Wnt-planar cell polarity (PCP) pathway component prickle2 increases the proportion of cyst formation in nphp4-depleted embryos. Nephrocystin-4 represses the Wnt-β-catenin pathway in the zebrafish cloaca and in mammalian kidney cells in culture. In these cells, nephrocystin-4 interacts with inversin and dishevelled, and regulates dishevelled stability and subcellular localization. Our data point to a function of nephrocystin-4 in a tight regulation of the Wnt-β-catenin and Wnt-PCP pathways, in particular during morphogenesis of the zebrafish pronephros. Moreover, they highlight common signalling functions for inversin and nephrocystin-4, suggesting that these two nephrocystins are involved in common physiopathological mechanisms.     

Cardiac malformations and midline skeletal defects in mice lacking filamin A

The X-linked gene filamin A (Flna) encodes a widely expressed actin-binding protein that crosslinks actin into orthogonal networks and interacts with a variety of other proteins including membrane proteins, integrins, transmembrane receptor complexes and second messengers, thus forming an important intracellular signalling scaffold. Heterozygous loss of function of human FLNA causes periventricular nodular heterotopia in females and is generally lethal (cause unknown) in hemizygous males. Missense FLNA mutations underlie a spectrum of disorders affecting both sexes that feature skeletal dysplasia accompanied by a variety of other abnormalities. Dilp2 is an X-linked male-lethal mouse mutation that was induced by N-ethyl-N-nitrosourea. We report here that Dilp2 is caused by a T-to-A transversion that converts a tyrosine codon to a stop codon in the Flna gene (Y2388X), leading to absence of the Flna protein and male lethality because of incomplete septation of the outflow tract of the heart, which produces common arterial trunk. A proportion of both male and female mutant mice have other cardiac defects including ventricular septal defect. In addition, mutant males have midline fusion defects manifesting as sternum and palate abnormalities. Carrier females exhibit milder sternum and palate defects and misshapen pupils. These results define crucial roles for Flna in development, demonstrate that X-linked male lethal mutations can be recovered from ENU mutagenesis screens and suggest possible explanations for lethality of human males hemizygous for null alleles of FLNA.