Joubert syndrome: congenital cerebellar ataxia with the molar tooth

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1 Joubert syndrome: congenital cerebellar ataxia with the molar tooth Marta Romani, Alessia Micalizzi, Enza Maria Valente Lancet Neurol 2013; 12: Published Online July 17, S (13) Neurogenetics Unit, Mendel Laboratory, IRCCS Casa Sollievo della Sofferenza, San Giovanni Rotondo, Italy (M Romani PhD, A Micalizzi BSc, Prof E M Valente MD); and Department of Medicine and Surgery, University of Salerno, Salerno, Italy (E M Valente) Correspondence to: Prof Enza Maria Valente, Neurogenetics Unit, CSS-Mendel Institute, Viale Regina Margherita 261, Rome, Italy e.valente@css-mendel.it Joubert syndrome is a congenital cerebellar ataxia with autosomal recessive or X-linked inheritance, the diagnostic hallmark of which is a unique cerebellar and brainstem malformation recognisable on brain imaging the so-called molar tooth sign. Neurological signs are present from the neonatal period and include hypotonia progressing to ataxia, global developmental delay, ocular motor apraxia, and breathing dysregulation. These signs are variably associated with multiorgan involvement, mainly of the retina, kidneys, skeleton, and liver. 21 causative genes have been identified so far, all of which encode for proteins of the primary cilium or its apparatus. The primary cilium is a subcellular organelle that has key roles in development and in many cellular functions, making Joubert syndrome part of the expanding family of ciliopathies. Notable clinical and genetic overlap exists between distinct ciliopathies, which can co-occur even within families. Such variability is probably explained by an oligogenic model of inheritance, in which the interplay of mutations, rare variants, and polymorphisms at distinct loci modulate the expressivity of the ciliary phenotype. Introduction Joubert syndrome is an inherited congenital cerebellar ataxia that is characterised by an unusual midbrain hindbrain malformation, the molar tooth sign (figure 1), and variable organ involvement. The pathogenic basis of this clinically and genetically heterogeneous disorder relates to the dysfunction of a subcellular organelle, the primary cilium, which makes Joubert syndrome part of an expanding group of disorders collectively called ciliopathies. The recent advent of next-generation sequencing strategies has enabled impressive progress in our knowledge of Joubert syndrome, with several genes being identified and characterised at the pathophysiological level. In turn, this progress has led to a dissection of the complexity of the primary cilium, to explain how the malfunction of this ubiquitous organelle could result in such a variety of phenotypes, ranging from developmental malformations to progressive defects. In this Review, we discuss the clinical range and genetic basis of Joubert syndrome, the overlap with other ciliopathies, and the evidence supporting oligogenic inheritance. Finally, we review the multifaceted roles of primary cilia in CNS development and in the function of neuronal cells. Primary cilium The primary cilium is an immotile organelle that protrudes from the surface of nearly all cell types. Although long thought to be a vestigial remnant without a relevant function, this organelle has recently become the focus of intensive research that has drawn attention to its many key roles in embryonic development, inherited human diseases, and even tumorigenesis. Primary cilia emerge from the basal body, a modified centriolar structure anchored to the plasma membrane. Their structural core is the axoneme, composed of nine doublets of microtubules, which is surrounded by a membrane contiguous with the cell plasma membrane but expresses specific signalling molecules. The basal body is joined to the axoneme by the y-shaped fibres of the transition zone, a recently described region that works as a functional ciliary gate, which regulates and restricts the flux of specific proteins to the cilium, to maintain it as a compartmentalised organelle (figure 2). 1 3 In many adult tissues, primary cilia work as sensors for extracellular signals, and transduce them within cells to regulate tissue maintenance, polarity, or proliferation. The disruption of these sensory functions in specialised cells such as the kidney and bile duct epithelium or the retinal photoreceptors explains many of the organ defects seen in ciliopathies. Moreover, studies have shown that most neuronal cell types possess a primary cilium, which draws attention to its many roles in brain development and function. 4 6 Clinical range of Joubert syndrome Epidemiology Reliable epidemiological data for Joubert syndrome are scarce. A prevalence of between 1 per and 1 per livebirths is reported by many investigators, but this is probably an underestimate that is indicative of the low awareness of the molar tooth sign in historical texts. 7,8 In the Askhenazi Jewish population, the predicted prevalence of Joubert syndrome is as high as one per people because of the presence of the founder mutation Arg73Lys in TMEM216, which has a carrier frequency of 1 per 90 to 1 per 100 of the population. 9,10 Similarly, in the Hutterite population, about one in 17 healthy people carry the founder mutation Arg18X in TMEM237, leading to a predicted disease prevalence of about one per 150 of the population. 11 Neurological features and multiorgan involvement Joubert syndrome can be clinically suspected from as early as the first few months of life, upon the observance of hypotonia, abnormal ocular movements (mainly ocular motor apraxia, nystagmus, and strabismus), and occasionally changes in respiratory pattern, characterised Vol 12 September 2013

2 A B C D * Figure 1: Neuroimaging findings in a 2-year-old child with pure Joubert syndrome (upper panels) compared with a healthy control child (lower panels) (A) Midsagittal T1-weighted image shows a moderate hypoplasia and dysplasia of the cerebellar vermis (arrows) with secondary distortion and enlargement of the fourth ventricle with rostral shifting of the fastigium (arrowhead). A deepened interpeduncular fossa is also noted (asterisk). (B) Parasagittal T1-weighted image shows the thickened, elongated, and horizontally orientated superior cerebellar peduncles (arrow). (C) Axial T1-weighted image at the pontomesencephalic junction shows the molar tooth sign with a deepened interpeduncular fossa (arrowhead) and elongated, thickened, and horizontally orientated superior cerebellar peduncles (arrows). Additionally, the cerebellar vermis seems to be hypoplastic and its remnants dysplastic. (D) Coronal T1-weighted image reveals the thickened superior cerebellar peduncles (arrows). by short alternate episodes of apnoea and tachypnoea, or episodic tachypnoea alone. About 10 15% of affected neonates also have polydactyly, which can be pre-axial, meso-axial, or post-axial, and involve the hands and feet variably. 12 Later, delay in the acquisition of developmental steps and intellectual disability of variable severity are seen in nearly all children, with expressive speech usually affected more than comprehension because of concurrent oromotor apraxia; 13 nevertheless, rarely, patients with normal cognitive function have also been reported. 14 Facial dysmorphisms, including prominent forehead, ptosis, prognathia, arched eyebrows, lower lip eversion with trapezoid-shaped mouth, and tongue protrusion, are often present. However, facial features do not aid the diagnosis, since in many patients they can be entirely normal. 15 About 50% of children learn to walk independently and develop ataxia with a broad-based, unsteady gait, and have difficulties in running or climbing stairs. 12 The clinical picture of Joubert syndrome can be complicated by a large range of associated organ defects, which can manifest at different ages. Among these defects, the most common are retinal defects (that range in severity from Leber congenital amaurosis to slowly progressive retinopathies with partially preserved vision), renal defects (nephronophthisis or cystic dysplastic kidneys), and congenital liver fibrosis. Rarer features include chorioretinal or optic nerve colobomas, congenital heart malformations, situs inversus, severe scoliosis, skeletal dysplasia, Hirschsprung disease, and midline oral and facial defects, such as cleft lip, cleft palate, or both, notched upper lip, lobulated tongue with multiple frenula, and lingual or oral soft tumours. In particular, the association of Joubert syndrome with polydactyly and midline orofacial defects defines the so-called orofaciodigital type VI syndrome, which is also part of the phenotype of Joubert syndrome. 16 This extreme phenotypic variability has caused much confusion in the classification of Joubert syndrome, with several acronyms used to describe overlapping disorders sharing the molar tooth sign, which were collectively termed Joubert syndrome and related disorders. 17 However, increasing evidence suggests that the variable clinical manifestations associated with the molar tooth sign do not comprise distinct clinical syndromes, but are rather part of the wide phenotypic range that is characteristic of Joubert syndrome. In view of the emerging genetic complexity of this disorder, the distinction between Joubert syndrome and Joubert syndrome and related disorders now seems to be blurred and does not contribute to the diagnostic definition of patients (which is simply based on the presence of the molar tooth sign), and simultaneously creates confusion, especially for families. For these reasons, we propose to abandon the term Joubert syndrome and related disorders in favour of the classic term Joubert syndrome to encompass all molar tooth sign-related disorders, and to adopt a descriptive classification that defines the disease s clinical subgroups on the basis of the extent of organ involvement (figure 3). Vol 12 September

3 AHI1 Wnt pathway OFD1 Anterograde transport Kinesin-2 plus IFT complex B plus cargo Shh pathway KIF7 RPGRIP1L Transition zone BBSome TTC21B Basal body Retrograde transport Dynein plus IFT complex A plus cargo ARL13B, INPP5E NPHP1, RPGRIP1L, other NPHP proteins CEP290, TMEM67, CC2D2A, AHI1, TCTN1, TCTN2, TCTN3, TMEM216, TMEM237, TMEM231, TMEM138, TMEM17, MKS1 Figure 2: Schematic representation of the structure of the primary cilium and its protein complexes Most proteins that are mutated in Joubert syndrome and Meckel syndrome cluster in large complexes at the basal body or the transition zone of the cilium. These complexes participate in the regulation of ciliogenesis, control the trafficking of specific pools of molecules targeted to the cilium, and are implicated in signalling pathways mediated by the cilium. Other Joubert syndrome proteins are also found along the ciliary axoneme or interact with Shh or Wnt pathways, which have crucial roles in embryonic development. IFT=intraflagellary transport. Neuroimaging and pathology After the first description of four siblings with Joubert syndrome by Marie Joubert in 1969, 18 several other patients were reported with the core features of the syndrome, but only in 1997 did Maria and colleagues delineate the characteristic cerebellar and brainstem malformation that is the hallmark of this syndrome, which they called the molar tooth sign. 19 The molar tooth sign arises from the combination of cerebellar vermis hypodysplasia; horizontalised, thickened, and elongated superior cerebellar peduncles; and an abnormally deep interpeduncular fossa at the level of the isthmus and upper pons, which creates the appearance of a molar tooth on axial brain images (figure 1). At the neuropathological level, this malformation results from the hypoplasia and dysplasia of the cerebellar vermis and of pontine and medullary structures, and the absence of decussation of the superior cerebellar peduncles and the pyramidal tracts. 7,20,21 This failure of selected tracts to cross the brainstem midline has been further confirmed by diffusion tensor imaging studies, 22,23 and implies an underlying defect of axon guidance that is unique to Joubert syndrome among all ciliopathies, and still remains unexplained. 24 Diagnosis In the presence of neurological features suggestive of Joubert syndrome, the diagnosis is easily confirmed upon demonstration of the molar tooth sign on brain imaging. Brain imaging will also reveal possibly associated CNS defects, which can include ventriculomegaly, occipital meningo-encephalocele, polymicrogyria, periventricular nodular heterotopia, hypothalamic hamartoma, absence of the pituitary gland, corpus callosum defects, hippocampal malformations, and morphological brainstem abnormalities, which affect mostly the midbrain and tectum. An enlarged posterior fossa, as seen in the Dandy- Walker malformation, has been described in nearly half of patients with Joubert syndrome, but none of them matched the diagnostic criteria for Dandy-Walker malformation (cystic dilatation of the fourth ventricle associated with upwards rotation of the hypoplastic cerebellar vermis). 25 In rare cases where a clear diagnosis cannot be made, diffusion tensor imaging can help the diagnosis by showing the absence of crossing fibres in the midbrain. 26 Patients with Joubert syndrome need to enter a diagnostic workflow and undergo regular follow-up examinations to ensure proper assessment and management of multiorgan complications. 12 In neonates and infants, particular care should be taken to manage episodes of abnormal breathing since prolonged episodes of apnoea can be life threatening and need assisted ventilation. Respiratory defects tend to improve spontaneously and sometimes disappear with age, although sleep-related breathing disorders can persist beyond childhood in some patients. 27 Appropriate rehabilitation protocols can help young patients to overcome delays in the acquisition of developmental milestones and cognition. Ophthalmological investigations should include assessment of ocular movements, visual acuity, and fundus oculi; in selected patients, electroretinogram and slit lamp examination are needed to diagnose particular retinopathies with normal fundus appearance and abnormalities of the anterior segment of the eye, respectively. Neuro-ophthalmological rehabilitation is useful to improve fixation and pursuit in children with ocular motor apraxia and to optimise residual visual function in those with notable visual impairment Vol 12 September 2013

4 Nephrological assessment is particularly important, especially in young children. About 20 30% of patients with Joubert syndrome might develop juvenile nephronophthisis, a slowly progressive disease characterised by the formation of small cysts at the cortico-medullary junction and interstitial fibrosis. 8 Nephronophthisis can remain asymptomatic for several years, until acute or chronic renal insufficiency manifests in the late first or early second decade, eventually necessitating dialysis or kidney transplant. Infantile nephronophthisis is rarer and more severe than the juvenile form, and manifests in the first few years of life. Renal ultrasound might reveal small cysts and loss of cortico-medullary differentiation, or can remain negative. Thus, monitoring of defects in urinary concentration ability in young children (first morning void urine analysis, polydipsia, polyuria, or a combination of these), which generally precede impairment of renal function, is important. Additionally, children with nephro nophthisis often have early onset of anaemia. Timely diagnosis of nephronophthisis is essential to initiate supportive treatment of chronic renal failure and to ensure appropriate fluid intake. Such measures can prevent the development of complications, such as renal osteodystrophy and poor growth, and are likely to delay progression to end-stage renal disease. Congenital liver fibrosis might be suspected in the presence of hepatomegaly, raised concentrations of liver serum enzymes, or abnormal liver echogenicity, and can be confirmed by liver MRI and eventually by liver biopsy. Patients might remain asymptomatic or can develop severe complications such as portal hypertension and oesophageal varices, which need to be addressed appropriately. Finally, a thorough examination should disclose rare associated disorders that might need treatment, such as congenital heart defects, scoliosis, or Hirschsprung disease. On the basis of the recognition of the molar tooth sign, no real differential diagnoses exist for Joubert syndrome. However, clinical differential diagnosis should include other syndromes with malformation of the cerebellum and brainstem that can also present with hypotonia, abnormal eye movements, and developmental delay, and other ciliopathies with similar organ involvement to Joubert syndrome, such as Senior-Løken and Bardet- Biedl syndromes, and cerebro-oculo-renal disorders. 5,28,29 In fetuses with Joubert syndrome, ultrasound examination from the 20th or 21st week of gestation can detect hypoplasia of the cerebellar vermis, which can be associated, in a subset of foetuses, with polydactyly, occipital encephalocele, or both. If a cerebellar malformation is suspected, foetal MRI will confirm the diagnosis, often allowing recognition of the molar tooth sign In families with a genetic diagnosis of Joubert syndrome in a previously affected child, prenatal diagnosis becomes possible in the first trimester of pregnancy based on genetic testing of a chorionic villous Liver Colobomas Scoliosis, skeletal dysplasia Congenital heart defects Other CNS malformations Kidney Congenital liver fibrosis Nephronophthisis Joubert syndrome+liver Joubert syndrome +liver +kidney Joubert syndrome +kidney Cystic dysplastic kidneys Brain (molar tooth sign) Pure Joubert syndrome Hypotonia/ataxia, developmental delay, oculomotor apraxia, breathing abnormalities Joubert syndrome+ retina+ kidney (COR) OFDVI Joubert syndrome+retina sample. Because of the high carrier frequency in the Ashkenazi Jews and Hutterites, prenatal screening is now common in these populations. Genetic bases of Joubert syndrome The genetic bases of Joubert syndrome are extremely complex and only partly understood, despite the tremendous acceleration in gene discovery enabled by next-generation sequencing techniques. So far, 21 causative genes have been identified, with autosomal or X-linked recessive inheritance (table 1). Whole exome sequencing studies in Joubert syndrome families led to the estimation that mutations in known genes account for only about half of patients, which suggests further genetic heterogeneity (Gleeson JG, University of California, CA, USA, personal communication). 61,72 Several research groups have undertaken next-generation sequencing-based targeted resequencing in various ciliopathies, 33,76,77 but comprehensive mutation screenings for all known Joubert syndrome genes in large cohorts of patients are still scarce. However, significant correlations have emerged between mutations in particular genes and specific Joubert syndrome subgroups, whereas other genes seem to be associated with a much wider phenotypic range. Genotype phenotype correlations The most relevant genotype phenotype correlation has been established between TMEM67 and the subgroup of people with Joubert syndrome with liver involvement. Midline defects (tongue tumours, multiple frenula, cleft lip/palate, etc) Leber congenital amaurosis Other retinopathy Orofacial Polydactyly Dysmorphic features Situs inversus Hirschsprung disease Retina Figure 3: Range of organ involvement in Joubert syndrome and classification in clinical subgroups (in bold) Chorioretinal colobomas are found more frequently in the subgroup of patients with Joubert syndrome with liver involvement, but can also be present in other subgroups. Similarly, polydactyly (especially pre-axial or meso-axial) is invariably present in the orofaciodigital type VI subgroup, but post-axial polydactyly is also frequently reported in association with other Joubert syndrome phenotypes. Other clinical features outside the circles occur rarely, without a specific association with a clinical subgroup. COR=cerebello-ocolo-renal. OFDVI=orofaciodigital type VI syndrome. Vol 12 September

5 Mutations in TMEM67 account for about 80% of patients with this rare phenotype, whereas only a few have mutations in other genes such as CC2D2A or RPGRIP1L This subgroup includes the disorder known by the acronym COACH (cerebellar vermis hypoplasia, oligophrenia, ataxia, coloboma, and hepatic fibrosis), 78 and the Gentile syndrome, in which patients present with Joubert syndrome and liver fibrosis in the absence of other clinical features. 79 Another relevant correlation involves CEP290, which is mutated in about 50% of patients with Joubert syndrome, with the cerebello-oculo-renal phenotype, characterised by the co-occurrence of retinopathy (usually Leber congenital amaurosis) and juvenile nephronophthisis. 45 Several genes (AHI1, INPP5E, ARL13B, and CC2D2A) are nearly exclusively mutated in patients presenting with either pure Joubert syndrome or Joubert syndrome with retinal involvement, 34 37,60 and in particular those patients with CC2D2A mutations show a significantly higher frequency of ventriculomegaly and seizures than patients Locus Gene Protein Prevalent Joubert syndrome phenotypes Mutation frequency in Joubert syndrome* (%) Genetic overlap with other ciliopathies References JBTS1 9q34 INPP5E Inositol polyphosphate-5- phosphatase JBTS2 11q12 TMEM216 Transmembrane protein 216 Joubert syndrome with or without retinal involvement; (Joubert syndrome with liver involvement ) Joubert syndrome with or without kidney involvement; (Joubert syndrome with liver involvement, OFDVI) ~3% MORM ~3% MKS2 9,10,33 JBTS3 6q23 AHI1 Jouberin Joubert syndrome with or without retinal ~7 11% 33,36 41 involvement JBTS4 2q13 NPHP1 Nephrocystin 1 Joubert syndrome with kidney involvement ~4 7% NPHP1, SLSN1 33,38,42 44 JBTS5 12q21 CEP290 Centrosomal protein 290kDa Joubert syndrome with retinal and kidney involvement ~7 16% MKS4, NPHP6, SLSN6, BBS14, LCA10 33,41,45 51 JBTS6 8q22 TMEM67 Meckelin Joubert syndrome with liver involvement ~6 10% MKS3, NPHP11, BBS 33,52 55 JBTS7 16q12 FTM (RPGRIP1L) RPGRIP1L Joubert syndrome with kidney involvement; ~1 2% MKS5, NPHP8 33,41,54,56 59 (Joubert syndrome with liver involvement ) JBTS8 3q11 ARL13B ADP-ribosylation factorlike Joubert syndrome ~1 2% 33,60 13B JBTS9 4p15 CC2D2A Coiled-coil and C2 Joubert syndrome with or without retinal ~6 10% MKS6 33,54,61 63 domain-containing protein 2A involvement; (Joubert syndrome with liver involvement ) JBTS10 Xp22 CXOrf5 (OFD1) Oralfaciodigital Variable phenotypes <1% OFD1, SGB2 64,65 syndrome 1 JBTS11 2q24 TTC21B Tetratricopeptide repeat NA MKS, NPHP12, ATD4 33,66 protein 21B JBTS12 15q26 KIF7 Kinesin-like protein 7 Joubert syndrome (OFDVI) NA ACS, HLS2, MEDF 67,68 JBTS13 12q24 TCTN1 Tectonic-1 Joubert syndrome with or without retinal 1 mutated family only 41,69 involvement JBTS14 2q33 TMEM237 Transmembrane protein Joubert syndrome with kidney involvement ~1% MKS 11, JBTS15 7q32 CEP41 Centrosomal protein Joubert syndrome <1% MKS 70 41kDa JBTS16 11q12 TMEM138 Transmembrane protein Variable phenotypes ~1 2% MKS JBTS17 5p13 C5Orf42 C5Orf42 Joubert syndrome with or without retinal NA MKS 41,61,72 involvement JBTS18 10q24 TCTN3 Tectonic-3 Joubert syndrome with or without OFD NA OFD4 and MKS features 73 features JBTS19 16q12 ZNF423 Zinc finger protein 423 Joubert syndrome with kidney involvement 1 mutated family only NPHP14 74 JBTS20 16q23 TMEM231 Transmembrane protein 231 Joubert syndrome with retinal and kidney involvement NA MKS 61 JBTS 12q24 TCTN2 Tectonic-2 Joubert syndrome NA MKS8 75 The terms in the first column are the standard nomenclature for Joubert syndrome genetic loci. Cited references refer to papers in which mutations in patients with Joubert syndrome are reported. MORM=mental retardation, truncal obesity, retinal dystrophy, and micropenis. MKS=Meckel syndrome. OFD=orofaciodigital syndrome. NPHP=nephronophthisis. SLSN=Senior-Løken syndrome. BBS=Bardet-Biedl syndrome. LCA=Leber congenital amaurosis. SGB=Simpson-Golabi-Behmel syndrome. NA=not assessable. ATD=asphyxiating thoracic dystrophy (Jeune syndrome). ACS=acrocallosal syndrome. HLS=hydrolethalus syndrome. MEDF=macrocephaly, multiple epiphyseal dysplasia, and distinctive facial appearance. *Only screenings of at least 100 Joubert syndrome probands unselected for specific phenotypes have been included in the calculation of frequencies. Kidney involvement is present in a subset of patients. Only heterozygous mutations reported. Locus number unassigned. Table 1: Genes and loci causative of Joubert syndrome Vol 12 September 2013

6 without CC2D2A mutations. 80 Finally, mutations in NPHP1, RPGRIP1L, and TMEM237 are almost invariably associated with Joubert syndrome with renal involvement. 11,42,56 Despite this evidence, genotype phenotype correlations remain problematic. For example, the same founder mutation Arg73Cys in TMEM216 causes a Joubert syndrome phenotype that can be pure or variably associated with polydactyly, renal disease, orofaciodigital features, and retinopathy. 10 Although essential for diagnosis, the molar tooth sign is not helpful for the classification of patients with Joubert syndrome into subtypes, since there are almost no neuroimaging genotype correlations, with the possible exception of thinner superior cerebellar peduncles seen in patients with the NPHP1 homozygous deletion. 42 Additionally, high intrafamilial variability exists in neuroimaging findings. 25 Shared features among ciliopathies The variable expressivity of Joubert syndrome goes beyond what is expected in classic autosomal recessive inheritance, and gains a further degree of complexity when we consider the striking degree of clinical and genetic overlap with other ciliopathies, such as isolated nephronophthisis, Senior-Løken syndrome, Meckel syndrome, Bardet-Biedl syndrome, some orofaciodigital syndromes, and skeletal ciliopathies such as short rib polydactylies. Shared clinical features variably include retinal dystrophy, nephronophthisis or cystic dysplastic kidneys, congenital liver fibrosis, polydactyly, situs inversus, occipital encephalocele, and midline oral and facial defects (table 2). This clinical overlap is mirrored by notable genetic overlap, with allelism at many gene loci (table 1). The most striking example is Meckel syndrome, a severe malformative condition that is often lethal in utero, which presents with encephalocele and other posterior fossa anomalies, ductal plate malformation of the liver, polycystic kidneys, and polydactyly. Meckel syndrome and Joubert syndrome are indeed allelic disorders; so far, 11 genes are known to cause both syndromes, and it is not uncommon to find affected siblings carrying the same homozygous mutation who present with either Meckel syndrome or Joubert syndrome. 10,53,71 Oligogenic inheritance and mutational load How mutations in one gene can cause such wide phenotypic variability, often encompassing different syndromes of varying severity, remains an unanswered question. Correlations between the mutation type and the ciliary phenotype have been recorded for some genes such as TMEM67, RPGRIP1L, CC2D2A, and TMEM216, with null mutations and hypomorphic missense mutations being significantly enriched in patients with lethal (Meckel syndrome) and non-lethal (Joubert syndrome, nephronophthisis, and Bardet-Biedl syndrome) disorders, respectively. 10,33,53,57,62,80,81 However, the real situation is far more complex. For example, pathogenic mutations in CEP290 have been associated with the whole range of ciliopathies, without any obvious genotype phenotype correlation. 46 Even more strikingly, the same homozygous deletion of a 290 kb genomic region on chromosome 2 encompassing the entire NPHP1 gene causes various phenotypes, ranging from isolated nephronophthisis to Senior-Løken syndrome (nephronophthisis plus retinal dystrophy) to Joubert syndrome. 42,43 In 2001, Katsanis and colleagues 82 described for the first time a triallelic digenic mode of inheritance in Bardet-Biedl syndrome, a ciliopathy characterised by retinal dystrophy, obesity, polydactyly, intellectual impairment, renal dysfunction, and hypogonadism. Like other ciliopathies, Bardet-Biedl syndrome has wide genetic heterogeneity, with 17 gene loci identified so far. Autosomal recessive mutations in one Bardet-Biedl syndrome gene were not fully penetrant in some families, and only the co-occurrence of a third heterozygous mutation in another Bardet-Biedl syndrome gene resulted in disease. 82,83 This oligogenic inheritance, characterised by the concurrent effect of two or more distinct genes on the resulting phenotype, was subsequently confirmed in other ciliopathies such as nephronophthisis, 84 and gained further complexity with the finding that not only pathogenic mutations but also rare variants or even common polymorphisms in other ciliary genes could interact genetically with the main recessive mutations to modulate the expressivity of the ciliary phenotype. For example, the Arg229Thr polymorphism in RPGRIP1L was associated with photoreceptor loss in various ciliopathies, 58 whereas the Arg830Trp polymorphism in AHI1 correlated, in two distinct studies, with an increased risk of patients with NPHP1 whole-gene deletion developing neurological symptoms and retinopathy, respectively. 38,39 Moreover, rare variants in TTC21B, a ciliary gene mutated in nephronophthisis, were significantly enriched in cohorts of patients with ciliopathies compared with healthy patients, which suggests that they could contribute to increased penetrance of mutations in other genes. 66 In line with these observations, mutation screenings of ciliary genes in large cohorts of patients with Joubert syndrome have often identified single heterozygous variants, the pathogenic significance of which remain unclear. 33,46,62,67,70,74,80 These variants could be hypothesised to represent genetic modifiers of the phenotype, and mutations in more than one gene have indeed been reported in some patients with Joubert syndrome who underwent several screenings or whole-exome sequencing. 38,46,68,70 Intriguingly, investigators have reported the characterisation of Tmem67-knockout mice and showed that differences in the genetic background of the transgenic animals could substantially affect the severity of the phenotype, which resembled either Meckel Vol 12 September

7 Joubert syndrome Meckel syndrome Orofaciodigital syndromes Skeletal ciliopathies* Nephro nophthisis or Senior-Løken syndrome CNS Developmental delays, intellectual disability ++ NA Hypotonia, ataxia ++ NA CVH Molar tooth sign ++ OFDVI Occipital encephalocele Other posterior fossa abnormalities Other CNS defects Eye Retinal dystrophy (SLS) ++ Colobomas + + Kidney Nephronophthisis Cystic kidneys Liver Congenital fibrosis Skeletal Polydactyly Scoliosis + + Bones shortening/bowing Other skeletal defects + ++ Facial Oral defects Facial defects Other defects Laterality defects Congenital heart defects Hirschsprung disease + NA + Obesity NA ++ Genital abnormalities Diabetes mellitus NA + Anosmia NA + Bardet-Biedl syndrome +Means that the feature might be present. ++Indicates obligate or highly frequent features. Blank cells indicate features not yet reported in association with that syndrome. NA=not applicable. CVH=cerebellar vermis hypoplasia. OFDVI=orofaciodigital type VI syndrome. SLS=Senior-Løken syndrome. *Including Jeune syndrome (or asphyxiating thoracic dystrophy), other short rib polydactylies, and Sensenbrenner syndrome (cranioectodermal dysplasia). Table 2: Clinical overlap between Joubert syndrome and other ciliopathies For the Cildb database see syndrome or Joubert syndrome. 85 All these findings support the concept of mutational load, which refers to the sum of all mutations and genomic variations contributing to define the penetrance and expressivity of ciliopathies. 86 Novel pathogenetic mechanisms related to the effect of variations residing within non-coding regulatory sequences of gene expression have also been identified. For example, we have recently shown that two causative genes of Joubert syndrome, TMEM216 and TMEM138, are aligned head-to-tail on chromosome 11, and form a cluster in which the expression of both genes is coregulated by a conserved regulatory element lying in the intergenic region. This coregulation is crucial to mediate the coordinated role of both proteins in transporting tethered pools of vesicles to the cilium, and genetic variations affecting this conserved element could have detrimental effects on the expression of both genes. 71 Pathogenetic mechanisms The finding that all causative genes for Joubert syndrome encode proteins of the primary cilium or its apparatus, and the striking clinical and genetic overlap with other ciliopathies, has put this subcellular organelle at the centre of intense research over the past decade. To date, the so-called ciliome is predicted to comprise at least 3000 proteins (eg, see the Cildb database), 87 suggesting a complex organisation that well represents the multifaceted roles of primary cilia in different cells and tissues, and the pleiotropic effect resulting from ciliary mutations Vol 12 September 2013

8 Functional networks of ciliary proteins Recent data have shown that ciliary proteins interact within functional networks that largely relate to specific ciliopathy phenotypes. For example, two motor complexes of proteins (intraflagellary transport complex B and A) mediate the anterograde and retrograde intraflagellary transport systems that bidirectionally move ciliary proteins along the axoneme with use of the microtubule motor proteins kinesins and dyneins, respectively. 88 Mutations in the intraflagellary transport proteins are a major cause of skeletal ciliopathies, including Sensenbrenner syndrome, Jeune syndrome, and other short rib polydactyly syndromes. 5 The correct assembly of intraflagellary transport complexes at the ciliary base, and the turnaround from anterograde to retrograde transport at the ciliary tip, are mediated by another protein complex, the BBSome, that consists of several proteins that are mutated in Bardet-Biedl syndrome (figure 2). 89 Protein complexes at the ciliary transition zone The characterisation of a growing number of proteins implicated in Joubert syndrome, Meckel syndrome, and nephronophthisis using state-of-the-art proteomic approaches has shown that most of these proteins localise at the basal body or at the ciliary transition zone, where they form a large network that is broken down into complexes with shared functions. 75 The most relevant complex for Joubert syndrome pathogenesis is the B9 or tectonic complex, which includes many proteins (B9D1, B9D2, TCTN1, TCTN2, TCTN3, CC2D2A, CEP290, TMEM216, TMEM138, TMEM67, TMEM237, TMEM231, TMEM17, MKS1, and AHI1), mutations in any of which, with the exception of TMEM17, can cause Joubert syndrome, Meckel syndrome, or both. Two other complexes are formed that include mostly nephronophthisis proteins, but also Joubert syndrome proteins such as RPGRIP1L (figure 2). Several functions have been ascribed to proteins of the tectonic complex: they limit the diffusion rate of plasma membrane proteins into the cilium, maintain the connection between the transition zone and the ciliary membrane, and control specific pools of vesicles targeted to the cilium. Consequently, mutations in or knockdown of these proteins result in malfunctioning of the whole complex and disruption of the integrity of the ciliary barrier, which causes various anomalies such as reduced cilia formation, abnormal ciliary morphology and trafficking, and changed localisation of signalling receptors. 11,71,75,90 A redundancy in the function of proteins within each complex, and functional interaction between complexes, has been shown by analysis of Caenorhabditis elegans double mutants, which showed pronounced phenotypes only when mutations affected two proteins belonging to distinct networks. 2,91 Primary cilia in development: implications for the neurological defects of Joubert syndrome In embryonic development, increasing evidence suggests a key role for primary cilia in regulating main pathways, such as Shh, Wnt, and planar cell polarity, which are implicated in left right axis formation, limb development, and neurogenesis. Shh pathway By contrast with Drosophila, in vertebrates many core components of the Shh pathway, such as Ptch1, Smo, Sufu, and Gli transcription factors, localise to the cilia, and ciliary malfunction leads to severe disruption of the Shh pathway. 92,93 Indeed, mutations in several Joubert syndrome-related genes have been linked to altered Shh signalling, and knockout mice for these genes show a range of Shh-related phenotypes that include polydactyly, microphthalmia, cleft palate, laterality defects, and various brain developmental abnormalities, such as neural tube dorsalisation and closure defects, holoprosencephaly, malformed hindbrain, and exencephaly. 69,70,73,75,90,94 96 In the CNS, the Shh pathway also has a major role in cerebellar development, which suggests an intriguing link between mutations in ciliary genes, abnormal Shh signalling, and cerebellar defects. Shh is expressed by Purkinje cells starting from embryonic day 17 5, and strongly drives the proliferation of granule cell precursors from progenitors in the rhombic lip. In the mouse cerebellum, targeted ablation of ciliary genes induced a failure of expansion of cerebellar progenitors in response to Shh, resulting in cerebellar hypoplasia and foliation abnormalities. 97,98 In line with these findings, reduced proliferation of granule cell progenitors associated with cerebellar hypoplasia has been shown in some mouse models of Joubert syndrome (eg, Rpgrip1l conditional mutants and ZNF423/Zpf423-knockout mice). 57,99 A study of human foetal samples showed that cerebellar granule cell progenitors have a primary cilium and proliferate in response to Shh signalling; this proliferation was found to be dramatically reduced after 16 gestational weeks in the cerebellum of patients with Joubert syndrome, Meckel syndrome, or Jeune syndrome, caused by distinct genetic mutations. 100 Nevertheless, these findings are difficult to reconcile with the normal size of cerebellar hemispheres seen in most patients with these ciliopathies, which suggests that other pathological events occurring earlier during development might be responsible for the selective vermal hypoplasia that is typical of Joubert syndrome. A direct link has also been shown between primary cilia, Shh signalling, and human stem cells. Undifferentiated embryonic stem cells contained primary cilia that expressed key components of the Shh signalling pathway essential for early development. 101 Moreover, primary cilia were implicated in the Shh-mediated control of adult neural stem cell formation within the Vol 12 September

9 hippocampal dentate gyrus. In mouse models, the conditional removal of ciliary genes (such as Rpgrip1l, which can cause Joubert syndrome) or of genes essential for the Shh pathway (such as Smo) led to faulty development of these neuronal precursors and notable hypotrophy of the dentate gyrus. 102 Hippocampal malformations have been described in a subset of patients with Joubert syndrome, 25,103 leading to speculation of a potential link with the learning and memory deficits in these patients. However, this idea is just one of many possible mechanisms that could explain the occurrence of intellectual disability in Joubert syndrome. For example, in a recent study involving Arl13b conditional knockout mice, the investigators showed that primary cilia also have a guiding role in the migration of postmitotic interneurons in the developing brain. These findings suggest that the abnormal cortical neuronal development associated with ciliary dysfunction could partly underlie the cognitive defects in Joubert syndrome. 104 Canonical Wnt pathway Recent studies of Ahi1-knockout and Cep290-knockout mice have shown a cerebellar phenotype characterised by vermis hypoplasia and a midline fusion defect with expansion of the roof plate, which was correlated with the disruption of another key developmental pathway, the canonical Wnt signalling cascade. 105 The activation of this complex pathway eventually leads to the stabilisation and nuclear localisation of β-catenin, with subsequent activation of target genes. 106 The role of primary cilia in canonical Wnt signalling has long been debated, with conflicting evidence. Lancaster and colleagues 107 showed that jouberin, encoded by AHI1, is a positive regulator of canonical Wnt signalling by facilitating β-catenin nuclear translocation, and that the primary cilium constrains this pathway by sequestering jouberin and β-catenin along the cilium and limiting their nuclear entry. In line with these findings, Ahi1-mutant mice displayed a specific decrease in Wnt activity and reduced cell proliferation in the developing cerebellum at the midline anlage, which could be partially rescued by lithium, a Wnt pathway agonist. 105 Similarly, Tmem67-knockout mouse embryos that presented with cerebellar vermis hypoplasia and deep interpeduncular fossa that highly resembled the molar tooth sign showed abnormal cilia and no nuclear β-catenin accumulation after Wnt stimulation and a decrease of Wnt downstream targets. 85 These findings provide a direct link between dysregulated canonical Wnt signalling and vermian hypoplasia seen in Joubert syndrome. Non-canonical Wnt (planar cell polarity) pathway The planar cell polarity pathway (or non-canonical Wnt signalling pathway) relates to the ability of cells to orient along an axis along the plane of the tissue, and is implicated in several developmental processes, including neural tube closure and left right patterning. 108 A close correlation exists between the planar cell polarity pathway and primary cilia, in that mutations in key planar cell polarity pathway effectors result in abnormal ciliogenesis, and vice versa. Mutations in several Joubert syndromerelated genes, such as TMEM237, TMEM216, and TMEM67, were found to substantially disrupt this pathway, leading to hyperactivation of downstream effectors such as dishevelled-1 and RhoA signalling, and defective ciliary formation and function. 10,11,109 Conclusions and future directions The area of ciliopathies is expanding at an impressively fast pace as the complexity of the human ciliome is being unravelled and an increasing number of heterogeneous human disorders are being causally related to mutations in ciliary genes. The concept of mutational load invariably needs a shift in the focus of genetic testing from single gene mutation analysis to more extensive and systematic screenings that would capture the genetic variability of every patient, to offer personalised counselling and prognostic indications. The advent of next-generation sequencing technologies is now making this shift possible, since high-throughput simultaneous resequencing of several target genes or even the whole exome are being offered as clinical diagnostic tests by many genetic laboratories, some of which provide their services directly to consumers through the online market. The use of such techniques on very large cohorts of well phenotyped patients will probably help to delineate the genetic modifiers contributing to specific clinical manifestations of Joubert syndrome. However, such a vast amount of generated data inevitably raises major interpretation challenges, especially regarding the functional effect of common and rare variants on disease penetrance and expressivity. This challenge is particularly relevant in the clinical setting, since we are expected to translate the bulk of genetic data into meaningful information that would improve the diagnosis, prognosis, and counselling for patients and their families. Standards for appropriate design, quality control, analysis, and interpretation of sequencing data, and ethical regulations, are clearly needed soon, before next-generation sequencing techniques can be applied safely to routine diagnostic protocols. Most of the existing knowledge about Joubert syndrome pathogenesis is derived from the study of zebrafish or mouse models. However, although extremely useful, these models often do not recapitulate their human counterpart in terms of organ specificity and severity. In particular, morpholino-based silencing of selected ciliary genes in zebrafish, which is a widely adopted strategy to model human ciliopathies, could produce different results in comparison to germline mutants, and might have toxic side-effects that could be difficult to distinguish from gene-specific effects. 110 A promising research method is now offered by induced pluripotent stem cell Vol 12 September 2013

10 Search strategy and selection criteria References for this Review were identified through searches of PubMed for articles published between 1969 and June, 2013 with the search terms Joubert syndrome, ciliopathies, primary cilium, and molar tooth sign. Articles were also identified by searches of the authors own files. Only papers published in English were reviewed. The final reference list was generated on the basis of originality and relevance to the broad scope of this Review. technology. Induced pluripotent stem cells can be generated from somatic cell types through ectopic expression of a set of transcription factors, to acquire the features of embryonic stem cells and thus have the potential to give rise to virtually any cell type, including inaccessible tissues such as neurons. Patient-derived induced pluripotent stem cells are proving an ideal source to study diseases in vitro, and have laid the basis for many applications, such as patient-specific correction of gene mutations and drug tailoring. 111 Moreover, since human induced pluripotent stem cells maintain the genetic background of the patient, they have the unique advantage to enable exploitation of the complex relation between pathogenic mutations, genetic modifiers, and phenotype that exists in oligogenic disorders such as ciliopathies. No therapies are yet available to cure Joubert syndrome, or to improve prognosis by mitigating the disease expressivity. However, the rapid rate of genetic discoveries and the use of innovative technologies are expected to greatly improve our understanding of the pathobiology of primary cilia, and will bridge the gap towards the identification of potential targets for treatment. Contributors MR, AM, and EMV all contributed to the literature search, figures, and writing of the Review. Conflicts of interest We declare that we have no conflicts of interest. Acknowledgments Research in the Valente laboratory is supported by grants from the European Research Council (ERC Starting Grant no ), the Italian Ministry of Health (Ricerca Corrente 2013, Ricerca Finalizzata Malattie Rare 2008), and the US National Institute of Health (R01NS048453). We thank Eugen Boltshauser, Joseph Gleeson, Andrea Poretti, and Francesco Emma for critical reading of the manuscript and valuable suggestions, and Daniel Doherty for his helpful comments about Joubert syndrome nomenclature. Andrea Poretti also provided the brain imaging in figure 1. References 1 Pazour GJ, Bloodgood RA. Targeting proteins to the ciliary membrane. Curr Top Dev Biol 2008; 85: Williams CL, Li C, Kida K, et al. 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