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In 1969, Marie Joubert could hardly have predicted the importance of the obscure autosomal recessive disorder now named Joubert syndrome (JS).1,2 Almost 30 years after the first description of hypotonia, ataxia, abnormal eye movements and alternating hyperpnea/apnea in four French Canadian siblings, the “molar tooth sign,” was identified as the key feature on magnetic resonance imaging (MRI).3 Seven years later, the first causal genes were identified (NPHP1 and AHI1),4–6 followed quickly by CEP290, RPGRIP1L, TMEM67/MKS3, ARL13B and CC2D2A.7–11 All of these genes and their gene products have been associated with the primary cilium and basal body (PC/BB), subcellular organelles whose central roles in diverse cellular processes have only recently been appreciated (for reviews see Yoder, 2008),12 making JS one of the rapidly expanding group of disorders termed ciliopathies.13 In addition to its importance for understanding the genetic networks underlying normal and abnormal brain development, JS represents a relatively simple model for more common, genetically complex disorders, in which multiple genetic and environmental factors contribute to the phenotype seen in a given patient.
Many features of JS (mid-hindbrain malformation, retinal dystrophy, cystic renal disease, congenital hepatic fibrosis and polydactyly) overlap with ciliopathies in general and Meckel syndrome (MKS) in particular (Table 1). Recent evidence indicates that these overlapping phenotypic features reflect a shared molecular pathophysiology involving the PC/BB organelle (see “Clinical and genetic overlap with other disorders” below).
The term molar tooth sign was coined by Maria et al. (1997) to refer to the appearance of long, thick superior cerebellar peduncles, a deep interpeduncular fossa and vermis hypoplasia/aplasia in the same axial slice on brain MRI (Fig 1A, D).3 When these features are not captured in the same slice, the following imaging can be features useful in confirming a JS diagnosis. Vermis hypoplasia is best assessed on sagittal views (Fig 1B, E, H); however, this can be difficult when the hemispheres impinge on the midline. The acquisition of thinner slices has made this less of a problem for more recent clinical imaging studies. Vermis size must also be assessed on axial and coronal views, paying particular attention to whether cerebrospinal fluid is present in the midline between the hemispheres (Fig 1A, C, D, F, G). The vermis hypoplasia in JS is unlikely to be due to atrophy, since the vermis has been noted to be small on the earliest possible prenatal imaging at 14–16 weeks gestation (D. Doherty, personal observations). Evaluation of the 4th ventricle on sagittal views is also informative. In patients with JS, the roof of the 4th ventricle is oriented more horizontally than usual, even when the vermis as a whole is not rotated superiorly (Fig 1B, H). Finally, although not entirely specific for JS, the 4th ventricle opens widely immediately inferior to the tectum, rather than remaining quite narrow for several millimeters (Fig 1B). When the vermis is completely absent and a large posterior fossa cyst is present, the radiologic diagnosis of JS can be difficult. A variety of additional imaging features have been observed, including agenesis of the corpus callosum (Fig 1K),14,15 encepahlocele (Fig 1I),1,16,17 hydrocephalus,14,18 posterior fossa cysts (often labeled Dandy-Walker malformation or Dandy-Walker variant),19 cerebellar17 and cortical heterotopias (Fig 1J), and polymicrogyria.5,16
Neuropathology findings have been reported in at least 15 individuals with JS, ranging from 18 weeks gestation to 31 years of age (Table 2).1,3,8,20–30 Lack of standardization limits the conclusions that can be drawn from these reports; nonetheless, a number of the findings have been reported in more than one case. In addition to vermis hypoplasia, the most frequent structures noted to be abnormal are: the deep cerebellar nuclei, the inferior olivary nuclei, multiple cranial nerve nuclei and the decussations of the superior cerebellar peduncles and pyramidal tracts. It is not clear from the pathology which abnormalities are primary and which are secondary. Diffusion tensor imaging and tractography are consistent with defects in the decussation of the superior cerebellar peduncles.31–33
Abnormal eye movements are uniformly present in JS though variable in severity.1–3,34–38 The presence of nystagmus, saccades instead of smooth pursuit and tracking/acquiring targets with head movements rather than eye movements are the predominant clinical signs. Quantitative eye movement recordings demonstrate that the gains (target velocity/eye velocity) for smooth pursuit, saccades, optokinetic nystagmus and vestibuloocular reflex are variably reduced.35,39,40 Each of these oculomotor deficits reflect abnormalities of specific neural ensembles in the cerebellar vermis (oculomotor, nodulus, uvula), flocculus/paraflocculus, deep cerebellar nuclei, vestibular nuclei, pontine nuclei and/or inferior olives. Retinal dystrophy (Fig 2A) with visual loss occurs in a subset of patients with JS. Early onset of the retinal dystrophy shares overlapping clinical features with Leber congenital amaurosis, and preservation of vision despite markedly abnormal electroretinogram (ERG) findings supports more severe involvement of rod versus cone photoreceptors in some cases.34,41 Chorioretinal coloboma (Fig 2B), identified in 19% of our cohort, is associated with significant visual impairment if the macula or optic nerve are involved. Strabismus and ptosis are additional ocular findings that may be corrected with surgical intervention.
While most reports indicate that patients with JS have substantial cognitive impairment, the range of ability is quite broad.25,42,43 A convenience sample of 32 patients aged 1–17 years (mean 6 years) evaluated with the Child Development Inventory displayed a mean developmental age equivalent of 19 months.44 Developmental and intelligence quotients between 30 and 80 have been reported in patients with JS;38,42 however, the substantial motor and speech impairments make it difficult to accurately measure underlying cognitive ability. Similarly, autism has been reported in children with JS,45–47 but the severe oral-motor dyspraxia and oculomotor issues in JS cause speech delay and abnormal eye contact, complicating the assessment of two core features of the diagnosis (communication and social interaction). Takahashi et al. (2006) reported that none of 31 patients with JS exceeded the Autism Behavior Checklist cutoff for autism, and they did not find increased load of neuropsychiatric issues seen in families of individuals with autism.48 In our experience, many patients with JS are very interested in social interaction, engage in pretend play and exhibit theory of mind, features inconsistent with autism.
Similar to the retinal disease seen in JS, the renal disease ranges in severity, from cystic renal dysplasia overlapping with MKS (Fig 2D) to classic nephronophthisis (Fig 2E) with onset in late childhood or later.49,50 The first sign of later onset renal disease is often failure to concentrate urine (salt-losing renal insufficiency) followed by echogenic kidneys on ultrasound and eventual renal failure. In patients with AHI1 mutations, onset of renal disease has been reported in young adults.51,52 Saraiva and Baraitser (1992) reported renal disease in 30% of cases in the literature,17 similar to the 23% in our cohort.
COACH syndrome (Cerebellar vermis hypoplasia, Oligophrenia - developmental delay/mental retardation, Ataxia, Colobomas, and Hepatic fibrosis) is considered a sub-type of JS.16,53,54 Clinically, the liver disease can be asymptomatic or present with mildly elevated serum transaminases, but more often, it is identified by liver imaging or signs of portal hypertension (varices, hepatosplenomegaly, ascites and rarely, upper GI bleeding). Biopsy findings are in the spectrum of the ductal plate malformation (Fig 2H) and congenital liver fibrosis (Fig 2I), and the fibrosis is progressive at least in some cases.55 Severe disease requires porto-systemic shunting or liver transplantation and can result in death. Liver disease occurs in 18% of our cohort; however, this may be an over-estimate of the true prevalence since we have focused on recruiting patients with liver disease.
Polydactyly is seen in 19% of our cohort and is a feature of many ciliopathies. Most frequently, it is post-axial (Fig 2F), although it can be pre-axial or very rarely, mesaxial. In general, polydactyly is not functionally significant, and surgical correction is at the discretion of the patient/family. As in many children with hypotonia, scoliosis is a common complication and requires close monitoring, especially during puberty.
While many patients with JS display dysmorphic facial features, no easily recognizable facial appearance has been described,56,57 in contrast to well-known disorders like Down and Williams syndromes. Oral frenulae, tongue hamartomas, micropenis and pituitary dysfunction have also been reported in small numbers of patients.16,17 Despite the presence of obvious breathing abnormalities in most infants with JS, the prevalence of central and obstructive sleep apnea in older children is likely under appreciated.
Guidelines for the evaluation and management of patients with JS were developed by a consensus panel convened by the Joubert syndrome Foundation and Related Cerebellar Disorders, and have been published elsewhere.58 These recommendations include yearly ophthalmologic evaluation, urinalysis, renal and liver ultrasounds, as well as serum transaminases, BUN, and creatinine to monitor for and allow early treatment of the medical complications described above. Lifelong monitoring for obstructive and central sleep apnea is also warranted. Specific developmental and behavioral supports for JS do not exist, and interventions are tailored to the needs of each individual.
Although the prevalence of JS has been estimated by Flannery and Hudson (1994)59 and Parisi and Glass (2007)58 to be 1/100,000–1/250,000, no population-based prevalence data exist. Based on the 30 patients we follow in our center that draws patients from a region of ~10 million people, a very conservative estimate of the minimum prevalence is ~1/300,000.
Mutations in seven genes (NPHP1, AHI1, CEP290, RPGRIP1L, MKS3/TMEM67, CC2D2A and ARL13B) and 2 additional loci (9q34, 11p12-11q13.3) have been associated with JS.4–11,60–65 In our cohort, NPHP1, AHI1, RPGRIP1L, ARL13B, CC2D2A and MKS3/TMEM67 account for <50% of subjects. Due to its large size (54 exons), we have not sequenced CEP290 in most of our cohort, but it is reported to be responsible for JS in at least 7% of cases.11,66
With the advent of improved sequencing technologies, it will likely become possible to sequence all of the JS-related genes simultaneously; however, clinical testing currently involves sequencing each gene individually, so strategies to prioritize testing are important to save time and money. The strongest genotype-phenotype correlation to date is between MKS3 mutations and clinically apparent liver disease (elevated transaminases, portal hypertension and/or liver fibrosis on biopsy). Thus, all patients with JS and liver disease should be tested first for MKS3 mutations. Genotype-phenotype correlations involving the other genes are not as strong (Table 3). For instance, patients with NPHP1 deletions invariably have renal disease and less severe brain malformation. Most subjects with AHI1 mutations have retinal dystrophy, but very few have renal disease. In contrast, CEP290 mutations cause a spectrum of phenotypes, from isolated JS, to JS with retinal and renal disease, to severe MKS. RPGRIP1lL mutations also cause a broad spectrum of disease with renal and liver involvement, but only rarely retinal dystrophy. Based on these observations, gene sequencing can be prioritized based on the clinical scenario (Fig 3).
Although similarities between MKS and JS had been noted as early as 1987,67 it was not until CEP290 mutations were found in subjects with JS11 and MKS68 that the allelic nature of these syndromes was revealed. Since that time, mutations in RPGRIP1L, CC2D2A and MKS3/TMEM67 have been shown to cause both MKS and JS.7,8,10,14,69,70 Similarly, NPHP1 mutations can cause isolated nephronophthisis71 or mild forms of JS.4,72 The most striking example is CEP290, where mutations can cause JS,11 MKS68,73 and BBS74, as well as isolated LCA75,76 and nephronophthisis.77 This is in keeping with work in humans and model systems showing that loss of function in genes with related functions cause similar phenotypes (reviewed in Brunner et al. 2004).78
As with many disorders, the spectrum of phenotypes associated with JS has expanded due to increased diagnosis in patients with related phenotypes, as well as advances in molecular testing. With the expanded phenotypes has come increasing overlap with other ciliopathies, particularly MKS and BBS, making the clinical classification of individual patients challenging. Not surprisingly in retrospect, these disorders now share molecular causes at least at the level of the genes (if not the specific mutations) involved.
Further complicating matters is emerging evidence for oligogenic inheritance, whereby full penetrance of a phenotype requires mutations in more than one gene. Thus, genetic modifiers may account for the often remarkable variability that is observed in ciliopathies.74,79–82 This has been most clearly demonstrated in BBS;81 however, a higher than expected prevalence of heterozygous CEP290 and AHI1 sequence variants was detected in patients with JS caused by mutations in both copies of NPHP1.83 In summary, clinical categorizations and causal mutations provide complementary information. Clinical categories remain important for directing diagnostic testing and providing prognostic information, while identifying the mutations involved allows for diagnostic, carrier and prenatal testing, as well as precise recurrence risk counseling.
Cilia are specialized, membrane-bound, hair-like structures that project from the cell surface (Fig 4). Each cilium is composed of a microtubule cytoskeleton (the axoneme) surrounded by a specialized membrane and anchored in the cell by the basal body. Motile cilia have doublet microtubules arranged in a 9+2 concentric pattern and are present on a variety of cell types including respiratory epithelia, brain ependymal lining cells and sperm. Primary cilia lack the central two microtubules (9+0 axoneme) and are usually non-motile, with the important exception of the cilia of the primitive node that rotate clockwise to generate the leftward nodal fluid flow required for establishing left-right asymmetry in vertebrates (reviewed in Shiratori and Hamada 2006 as well as Hirokawa et al., 2006).84–90
Primary cilia or modifications thereof are present in most cell types, including renal tubule epithelial cells, retinal photoreceptors, chondrocytes, fibroblasts and neurons. In general, the microtubules that form the axoneme are nucleated by the basal body that forms from the mother centriole when it docks at the plasma membrane after cell division (reviewed in Marshall, 2008).91 Intraflagellar transport (IFT) proteins including dynein and kinesin motors are required for the assembly and function of cilia (reviewed in Pedersen and Rosenbaum).92
Ciliary membranes contain receptors and ion channel proteins mediating mechano/chemo-sensation and other types of cell signaling. Mounting evidence supports a role for the PC/BB in the sonic hedgehog (SHH), WNT and PDGFα signaling pathways that control diverse processes such as cell division, differentiation, migration and planar cell polarity. SHH binding to its transmembrane receptor, patched (PTCH), results in increased localization of smoothened (SMO) to the primary cilium. This binding abolishes the inhibitory effect of PTCH on SMO and transduces signals to the nucleus via the GLI transcription factors, resulting in de-repression and activation of SHH target genes (reviewed in Satir and Christensen 2007).93 SHH signaling is required for at least two major processes in hindbrain development: early for dorsal-ventral patterning of the neural tube, and later for proliferation of the cerebellar granule cells.94,95
In light of the phenotypic overlap between JS and other ciliopathies, it is not surprising that the proteins encoded by all of the JS genes have been shown to localize to the PC/BB or directly interact with components of the PC/BB (Table 4).7,10,11,14,61,65,69,96–98 In addition, mutations in the mouse orthologs of two JS genes (rpgrip1l and arl13b) disrupt cilium number/morphology and cause aberrant dorsal-ventral patterning of the early neural tube, 96,99 but later defects in brain development have not been explored. It is unclear whether the human RPGRIP1L and ARL13B phenotypes are milder because the mutations identified in humans have less severe effects on overall PC/BB function, effects on a subset of PC/BB functions or effects outside the PC/BB. Disruption of cilium function by loss of ift88 function after E13 in the mouse cerebellum results in markedly decreased granule cell proliferation without disruption in overall patterning of the cerebellum or brainstem.100,101 Purkinje cells are also abnormal, potentially secondary to the granule cell defect. Conditional loss of kif3a function generates a similar phenotype, supporting a specific role for the cilium. Given the requirement for IFT proteins in granule cell proliferation, it would not be surprising if RPGRIP1L and ARL13B were also required for granule cell proliferation, likely via effects on SHH signaling.
The JS genes are highly conserved across all vertebrates, with orthologs in additional species as distantly related as sea urchin, nematodes and insects. NPHP1 encodes nephrocystin-1 which interacts with AHI1, other NPHP proteins and components of cell-cell and cell-matrix signaling pathways. Nephrocystin-1 is localized to the transition zone of the PC/BB in renal epithelia and also to the adherens junctions and focal adhesions in a cell cycle dependent manner (reviewed in Hildebrandt and Zhou, 2007).102
RPGRIP1L encodes the RPGRIPlL protein which, like its retinally-expressed homolog RPGRIP, interacts with the NPHP4 gene product, nephrocystin-4, a ciliary protein defective in some cases of isolated nephronophthisis and Senior-Løken syndrome (nephronophthisis plus retinal dystrophy/LCA).7 Mutations in both RPGRIPlL and NPHP4 disrupt this interaction. RPGRIP1L localizes to the basal body and also colocalizes with CEP290 in brain and kidney. Knockout of the murine ortholog of RPGRIP1L, results in ciliary assembly/functional defects, abnormal left-right asymmetry and preaxial polydactyly, all likely resulting from disturbed Shh signaling.99
The protein encoded by CEP290 localizes to centrosomes and the mitotic spindle in a cell cycle dependent manner and has been shown to directly activate the ATF4 transcription factor in cultured cells.11,103 A homozygous in-frame deletion found in a murine retinal dysplasia model (rd16) abolished interaction with RPGR, a microtubule transport protein, suggesting a specific role for CEP290 in microtubule-based ciliary transport.104 More recently, CEP290 has been implicated in vesicle transport and cilium formation/maintenance through its interactions with PCM1105 and CP110106 as well as its role in transport of G-proteins into olfactory cilia.107
TMEM67/MKS3 encodes the meckelin protein which localizes to the primary cilium and to the plasma membrane. MKS3 interacts with MKS1 and knockdown of either MKS1 or MKS3 by siRNA in ciliated renal epithelia blocked migration of the basal body to the plasma membrane and primary cilium formation.61 In contrast to these results, cilia are present, albeit elongated and dysmorphic, in a recently published mks3 knockout mouse.108
AHI1 encodes a protein with WD40 and SH3 domains and is highly expressed in human fetal brain and kidney. In postnatal mouse brain, expression is detected at higher levels in the cerebellar dentate nuclei and the deep cortical neurons that form the corticospinal tract, potentially correlating with the failure of decussation observed in functional and neuropathological studies of JS.5,6 AHI1 localizes to the cell junctions and centrosomes in cultured cells and interacts with NPHP1 and HAP1.65,109
ARL13B is an ADP ribosylation factor (Arf) related gene in the Ras superfamily of small GTPases, some of which have been implicated in cytoskeletal dynamics, lipid metabolism and vesicle trafficking. ARL13B localizes to cilia in the mouse node, neural tube and fibroblasts, and an arl13b null mutation in mice causes neural tube defects, polydactyly and aberrant Shh signaling in the neural tube.96,110 Loss of function in the zebrafish scorpion mutant results in abnormal body shape and pronephric (kidney) cysts.111
CC2D2A encodes a protein with similar overall structure to RPGRIP1L including coiled-coil domains, a C2 domain and an overlapping centrosomal protein related domain. CC2D2A physically interacts with CEP290 and loss of Cc2d2a function in the zebrafish sentinel mutant results in abnormal body shape and pronephric (kidney) cysts that is strongly exacerbated by knockdown of Cep290 function.14
Recently, the BBS proteins have been shown to form a complex (the BBSome) that associates with PCM1 and rab8, and is required for sorting proteins to the cilium.112 CEP290 has been shown to be part of the complex and is required for transporting olfactory receptors to the olfactory cilium.105,107 Other BBS proteins share homology with chaperonins and are likely required to help build the BBSome complex (reviewed in Tobin and Beales, 2007).113 The JS gene products share protein domains with BBS gene products including coiled coils (RPGRIP1L, CC2D2A, CEP290, ARL13B, and BBS2, 4, 7, 9), WD40 repeats (AHI1 and BBS1, 2, 7) and Rab GTPase motifs (ARL13B and ARL6), so it is tempting to hypothesize that the JS proteins also function as a multi-protein complex with members that play similar roles to the BBS proteins.
Despite remarkable advances in the genetics of JS, little is known about how specific gene defects result in abnormal brain development. A few hypotheses can be generated based on the limited data available. As described above, the brain malformation in JS comprises at least several components: 1) decreased vermis size (likely due to decreased cell numbers); 2) aberrant axonal pathfinding (disrupted decussation of the superior cerebellar peduncles and pyramids); and 3) possible abnormal neuronal migration (fragmented dentate nuclei, cerebellar and cortical heterotopias, “pachygyric:” inferior olives).
One theory proposes that the vermis hypoplasia in JS is due to decreased granule cell proliferation caused by aberrant SHH signaling via defective cilia; however, conditional knockout of kif3a and ift88 in the developing mouse cerebellum results in markedly decreased size of the cerebellar hemispheres and vermis,100,101 so somehow, mutations that cause JS would have to affect the vermis preferentially. Alternatively, defective dorsal-ventral patterning could result in alterations to the cells fated to become the cerebellar vermis and deep cerebellar nuclei. To date, defective SHH signaling in the developing cerebellum has not been reported in the existing mouse models for JS. An alternative theory is that subtle alterations in specification of the mid-hindbrain boundary and resulting rhombomere identities could underlie JS. SHH signaling115,116 and cilium function117 are required for normal specification of the mid-hindbrain boundary and altered segment identity could affect the birth, cell fate, persistence or migration of cerebellar cell types. Consistent with this theory is the observation that the vermis develops from the most rostral portion of rhombomere 1 after morphogenetic movements transform the rostral-caudal axis into a medial-lateral orientation.118 Tissue-specific elimination of JS gene function at various stages of mid-hindbrain development may clarify the mechanisms, but given the complexity of PC/BB function, it is likely that none of the theories above will fully explain the mid-hindbrain phenotype in JS.
Despite great progress in the understanding of JS, many questions remain unanswered. Clinically, prenatal diagnosis remains an issue for >50% of families in which the genetic cause has not been identified. Prognostic information in the literature is limited by small numbers of patients, diverse ascertainment strategies, short duration of follow-up and lack of standardized assessments. We do not yet have accurate information on the easiest outcomes to measure: lifespan and cause of death. The neuropathological findings in humans and model organisms are incompletely characterized, and while the known genes are implicated in the PC/BB, the precise molecular function of these genes remains elusive and many of the players have yet to be identified. Furthermore, the details of how PC/BB dysfunction results in brain malformation and organ dysfunction in JS remain to be explored.
Technological breakthroughs may help with some of these issues (e.g. re-sequencing many genes to identify genetic modifiers that more fully explain JS phenotypes), while other issues will require time-consuming, detail-oriented patient phenotyping. Autopsies (pre- and post-natal) have the potential to reveal the most information about the human brain malformation in JS and should be performed by experienced neuropathologists whenever possible. Finally, high-throughput technologies to evaluate protein-protein interaction networks combined with genetic approaches in model systems to validate the protein-protein interactions will yield a more accurate view of the genetic/protein networks underlying JS as well as normal brain development.
The participation of the Joubert Syndrome Foundation and Related Cerebellar Disorders and families with JS was invaluable for this work. The following colleagues provided images and/or important comments on the manuscript: Laura Finn, Ian Glass, Meral Gunay-Aygun, Robert Hevner, Dana Knutzen, Dennis Shaw, Ekaterini Tsilou, and Avery Weiss. D.D. was supported by the National Institutes of Health (NCRR 5KL2RR025015), The Arc of Washington State Trust Fund and the March of Dimes Endowment for Healthier Babies at Seattle Children’s Hospital.
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