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1.  Functional genome-wide siRNA screen identifies KIAA0586 as mutated in Joubert syndrome 
eLife  null;4:e06602.
Defective primary ciliogenesis or cilium stability forms the basis of human ciliopathies, including Joubert syndrome (JS), with defective cerebellar vermis development. We performed a high-content genome-wide small interfering RNA (siRNA) screen to identify genes regulating ciliogenesis as candidates for JS. We analyzed results with a supervised-learning approach, using SYSCILIA gold standard, Cildb3.0, a centriole siRNA screen and the GTex project, identifying 591 likely candidates. Intersection of this data with whole exome results from 145 individuals with unexplained JS identified six families with predominantly compound heterozygous mutations in KIAA0586. A c.428del base deletion in 0.1% of the general population was found in trans with a second mutation in an additional set of 9 of 163 unexplained JS patients. KIAA0586 is an orthologue of chick Talpid3, required for ciliogenesis and Sonic hedgehog signaling. Our results uncover a relatively high frequency cause for JS and contribute a list of candidates for future gene discoveries in ciliopathies.
DOI: http://dx.doi.org/10.7554/eLife.06602.001
eLife digest
Joubert syndrome is a rare disorder that affects the brain and causes physical, mental, and sometimes visual impairments. In individuals with this condition, two parts of the brain called the cerebellar vermis and the brainstem do not develop properly. This is thought to be due to defects in the development and maintenance of tiny hair-like structures called cilia, which are found on the surface of cells.
Currently, mutations in 25 different genes are known to be able to cause Joubert syndrome. However, these mutations only account for around 50% of the cases that have been studied, and the ‘unexplained’ cases suggest that mutations in other genes may also cause the disease.
Here, Roosing et al. used a technique called a ‘genome-wide siRNA screen’ to identify other genes regulating the formation of cilia that might also be connected with Joubert syndrome. This approach identified almost 600 candidate genes. The data from the screen were combined with gene sequence data from 145 individuals with unexplained Joubert syndrome. Roosing et al. found that individuals with Joubert syndrome from 15 different families had mutations in a gene called KIAA0586. In chickens and mice, this gene—known as Talpid3—is required for the formation of cilia.
Roosing et al.'s findings reveal a new gene that is involved in Joubert syndrome and also provides a list of candidate genes for future studies of other conditions caused by defects in the formation of cilia. The next challenges are to find out what causes the remaining unexplained cases of the disease and to understand what roles the genes identified in this study play in cilia.
DOI: http://dx.doi.org/10.7554/eLife.06602.002
doi:10.7554/eLife.06602
PMCID: PMC4477441  PMID: 26026149
Joubert syndrome; ciliopathy; siRNA; high-content screen; KIAA0586; Talpid3; human
2.  Whole Exome Sequencing identifies a splicing mutation in NSUN2 as a cause of a Dubowitz-like syndrome 
Journal of medical genetics  2012;49(6):380-385.
Dubowitz Syndrome is an autosomal recessive disorder characterized by the constellation of mild microcephaly, growth and mental retardation, eczema and peculiar facies, but causes are still unknown. We studied a multiplex consanguineous family with many features of Dubowitz syndrome using whole exome sequencing and identified a splice mutation in NSUN2, encoding a conserved RNA methyltransferase. NSUN2 has been implicated in Myc-induced cell proliferation and mitotic spindle stability, which might help explain the varied clinical presentations that can include chromosomal instability and immunological defects. Patient cells displayed loss of NSUN2-specific methylation at two residues of the aspartate tRNA. Our findings establish NSUN2 as the first causal gene with relationship to the Dubowitz syndrome spectrum phenotype.
doi:10.1136/jmedgenet-2011-100686
PMCID: PMC4771841  PMID: 22577224
Dubowitz; NSUN2; MISU; RNA methylation; microcephaly
3.  PATHOGENETIC MECHANISMS OF FOCAL CORTICAL DYSPLASIA 
Epilepsia  2014;55(7):970-978.
SUMMARY
Objective
Focal cortical dysplasias (FCDs) constitute a prevalent cause of intractable epilepsy in children, and one of the leading conditions requiring epilepsy surgery. Despite the recent advances on the cellular and molecular biology of these conditions, the pathogenetic mechanisms of FCDs remain largely unknown. The purpose if this work is to review the molecular underpinnings of FCDs and to highlight potential therapeutic targets.
Methods
A systematic review of the literature regarding the histological, molecular, and electrophysiological aspects of FCDs was conducted.
Results
Disruption of the mTOR signaling comprises a common pathway underlying the structural and electrical disturbances of some FCDs. Other mechanisms such as viral infections, prematurity, head trauma, and brain tumors are also posited. mTOR inhibitors (i.e., rapamycin) have shown positive results on seizure management in animal models and in a small cohort of patients with FCD.
Significance
Encouraging progresses have been achieved on the molecular and electrophysiological basis of constitutive cells in the dysplastic tissue. Despite the promising results of mTOR inhibitors, large-scale randomized trials are in need to evaluate their efficacy and side effects, along with additional mechanistic studies for the development of novel, molecular-based diagnostic and therapeutic approaches.
doi:10.1111/epi.12650
PMCID: PMC4107035  PMID: 24861491
4.  An siRNA-based functional genomics screen for the identification of regulators of ciliogenesis and ciliopathy genes 
Wheway, Gabrielle | Schmidts, Miriam | Mans, Dorus A. | Szymanska, Katarzyna | Nguyen, Thanh-Minh T. | Racher, Hilary | Phelps, Ian G. | Toedt, Grischa | Kennedy, Julie | Wunderlich, Kirsten A. | Sorusch, Nasrin | Abdelhamed, Zakia A. | Natarajan, Subaashini | Herridge, Warren | van Reeuwijk, Jeroen | Horn, Nicola | Boldt, Karsten | Parry, David A. | Letteboer, Stef J.F. | Roosing, Susanne | Adams, Matthew | Bell, Sandra M. | Bond, Jacquelyn | Higgins, Julie | Morrison, Ewan E. | Tomlinson, Darren C. | Slaats, Gisela G. | van Dam, Teunis J. P. | Huang, Lijia | Kessler, Kristin | Giessl, Andreas | Logan, Clare V. | Boyle, Evan A. | Shendure, Jay | Anazi, Shamsa | Aldahmesh, Mohammed | Al Hazzaa, Selwa | Hegele, Robert A. | Ober, Carole | Frosk, Patrick | Mhanni, Aizeddin A. | Chodirker, Bernard N. | Chudley, Albert E. | Lamont, Ryan | Bernier, Francois P. | Beaulieu, Chandree L. | Gordon, Paul | Pon, Richard T. | Donahue, Clem | Barkovich, A. James | Wolf, Louis | Toomes, Carmel | Thiel, Christian T. | Boycott, Kym M. | McKibbin, Martin | Inglehearn, Chris F. | Stewart, Fiona | Omran, Heymut | Huynen, Martijn A. | Sergouniotis, Panagiotis I. | Alkuraya, Fowzan S. | Parboosingh, Jillian S. | Innes, A Micheil | Willoughby, Colin E. | Giles, Rachel H. | Webster, Andrew R. | Ueffing, Marius | Blacque, Oliver | Gleeson, Joseph G. | Wolfrum, Uwe | Beales, Philip L. | Gibson, Toby | Doherty, Dan | Mitchison, Hannah M. | Roepman, Ronald | Johnson, Colin A.
Nature cell biology  2015;17(8):1074-1087.
Defects in primary cilium biogenesis underlie the ciliopathies, a growing group of genetic disorders. We describe a whole genome siRNA-based reverse genetics screen for defects in biogenesis and/or maintenance of the primary cilium, obtaining a global resource. We identify 112 candidate ciliogenesis and ciliopathy genes, including 44 components of the ubiquitin-proteasome system, 12 G-protein-coupled receptors, and three pre-mRNA processing factors (PRPF6, PRPF8 and PRPF31) mutated in autosomal dominant retinitis pigmentosa. The PRPFs localise to the connecting cilium, and PRPF8- and PRPF31-mutated cells have ciliary defects. Combining the screen with exome sequencing data identified recessive mutations in PIBF1/CEP90 and C21orf2/LRRC76 as causes of the ciliopathies Joubert and Jeune syndromes. Biochemical approaches place C21orf2 within key ciliopathy-associated protein modules, offering an explanation for the skeletal and retinal involvement observed in individuals with C21orf2-variants. Our global, unbiased approaches provide insights into ciliogenesis complexity and identify roles for unanticipated pathways in human genetic disease.
doi:10.1038/ncb3201
PMCID: PMC4536769  PMID: 26167768
cilia; ciliopathies; reverse genetics; whole-genome siRNA screen; Jeune syndrome; Joubert syndrome
5.  Inactivating Mutations in MFSD2A, Required for Omega-3 Fatty Acid Transport in Brain, Cause a Lethal Microcephaly Syndrome 
Nature genetics  2015;47(7):809-813.
Docosahexanoic acid (DHA) is the most abundant omega-3 fatty acid in brain, and although considered essential, deficiency has not been linked to disease1,2. Despite the large mass of DHA in phospholipids, the brain does not synthesize it. DHA is imported across the blood-brain barrier (BBB) through the Major Facilitator Superfamily Domain 2a (Mfsd2a)3. Mfsd2a transports DHA as well as other fatty acids in the form of lysophosphatidylcholine (LPC). We identify two families displaying MFSD2A mutations in conserved residues. Patients exhibited a lethal microcephaly syndrome linked to inadequate uptake of LPC lipids. The MFSD2A mutations impaired transport activity in a cell-based assay. Moreover, when expressed in mfsd2aa zebrafish morphants, mutants failed to rescue microcephaly, BBB breakdown and lethality. Our results establish a link between transport of DHA and LPCs by MFSD2A and human brain growth and function, presenting the first evidence of monogenic disease related to transport of DHA in humans.
doi:10.1038/ng.3311
PMCID: PMC4547531  PMID: 26005868
6.  Biallelic mutations in SNX14 cause a syndromic form of cerebellar atrophy and lysosome-autophagosome dysfunction 
Nature genetics  2015;47(5):528-534.
Pediatric-onset ataxias often present clinically with developmental delay and intellectual disability, with prominent cerebellar atrophy as a key neuroradiographic finding. Here we describe a novel clinically distinguishable recessive syndrome in 12 families with cerebellar atrophy together with ataxia, coarsened facial features and intellectual disability, due to truncating mutations in sorting nexin 14 (SNX14), encoding a ubiquitously expressed modular PX-domain-containing sorting factor. We found SNX14 localized to lysosomes, and associated with phosphatidyl-inositol (3,5)P2, a key component of late endosomes/lysosomes. Patient cells showed engorged lysosomes and slower autophagosome clearance rate upon starvation induction. Zebrafish morphants showed dramatic loss of cerebellar parenchyma, accumulated autophagosomes, and activation of apoptosis. Our results suggest a unique ataxia syndrome due to biallelic SNX14 mutations, leading to lysosome-autophagosome dysfunction.
doi:10.1038/ng.3256
PMCID: PMC4414867  PMID: 25848753
7.  Aberrant methylation of tRNAs links cellular stress to neuro-developmental disorders 
The EMBO Journal  2014;33(18):2020-2039.
Mutations in the cytosine-5 RNA methyltransferase NSun2 cause microcephaly and other neurological abnormalities in mice and human. How post-transcriptional methylation contributes to the human disease is currently unknown. By comparing gene expression data with global cytosine-5 RNA methylomes in patient fibroblasts and NSun2-deficient mice, we find that loss of cytosine-5 RNA methylation increases the angiogenin-mediated endonucleolytic cleavage of transfer RNAs (tRNA) leading to an accumulation of 5′ tRNA-derived small RNA fragments. Accumulation of 5′ tRNA fragments in the absence of NSun2 reduces protein translation rates and activates stress pathways leading to reduced cell size and increased apoptosis of cortical, hippocampal and striatal neurons. Mechanistically, we demonstrate that angiogenin binds with higher affinity to tRNAs lacking site-specific NSun2-mediated methylation and that the presence of 5′ tRNA fragments is sufficient and required to trigger cellular stress responses. Furthermore, the enhanced sensitivity of NSun2-deficient brains to oxidative stress can be rescued through inhibition of angiogenin during embryogenesis. In conclusion, failure in NSun2-mediated tRNA methylation contributes to human diseases via stress-induced RNA cleavage.
doi:10.15252/embj.201489282
PMCID: PMC4195770  PMID: 25063673
5-methylcytidine; Misu; NSun2; RNA modification
8.  Cilia in the nervous system: linking cilia function and neurodevelopmental disorders 
Current opinion in neurology  2011;24(2):98-105.
Purpose of review
Ciliopathies are genetic disorders caused by defects of primary ciliary structure and/or function and are characterized by pleiotropic clinical features. The ciliopathies include several partially overlapping syndromes such as Joubert syndrome, Bardet–Biedl syndrome and Meckel–Gruber syndrome, all of which have pronounced neurodevelopmental features. Here we focus on potential roles of cilia in central nervous system function, to explore how impairments may cause brain malformation and neurodevelopmental disease.
Recent findings
Cilia have long been considered as ‘sensory cellular antennae’, responding as chemo-sensors, mechano-sensors and thermo-sensors, although their roles in development were not well understood until recently. The surprising finding that disparate syndromes are all due to defects of the primary cilia, along with the recent advances in genetics, has helped elucidate further roles of primary cilia beyond sensory functions. Several molecules that are associated with key signaling pathways have been discovered in primary cilia. These include sonic hedgehog, wingless, planar cell polarity and fibroblast growth factor, which are essential for many cellular processes. Additionally, mutations in ‘ciliome’ genes have largely shown developmental defects such as abnormal body axis and brain malformation, implying disrupted cilia-related signaling pathways. Accordingly, the emerging theme is that primary cilia may play roles as modulators of signal transduction to help shape cellular responses within the environmental context during both development and homeostasis.
Summary
The link between cilia and signal pathways has become a framework for understanding the pathogenesis of ciliopathies. Despite recent progress in ciliary biology, fundamental questions remain about how cilia regulate neuronal function in the central nervous system. Therefore, investigation of ciliary function in the nervous system may reveal cilia-modulating mechanisms in neurodevelopmental processes, as well as suggest new treatments for disease.
doi:10.1097/WCO.0b013e3283444d05
PMCID: PMC3984876  PMID: 21386674
brain; central nervous system; cilia; ciliopathy; Joubert syndrome; neuron; signaling pathways
9.  The Sacred Disease: The Puzzling Genetics of Epileptic Disorders 
Neuron  2013;80(1):9-11.
In the September 12, 2013 issue of Nature, the Epi4K consortium and the Epilepsy Phenome/Genome Project (EPGP) reported sequencing 264 patient trios with epileptic encephalopathies. The consortia focused on genes exceptionally intolerant to sequence variations and found substantial interconnections with autism and intellectual disability gene networks.
doi:10.1016/j.neuron.2013.09.019
PMCID: PMC3984878  PMID: 24094099
10.  The ciliopathies in neuronal development: a clinical approach to investigation of Joubert syndrome and Joubert syndrome-related disorders 
A group of disorders with disparate symptomatology, including congenital cerebellar ataxia, retinal blindness, liver fibrosis, polycystic kidney disease, and polydactyly, have recently been united under a single disease mechanism called ‘ciliopathies’. The ciliopathies are due to defects of the cellular antenna known as the primary cilium, a microtubule-based extension of cellular membranes found in nearly all cell types. Key among these ciliopathies is Joubert syndrome, displaying ataxia, oculomotor apraxia, and mental retardation* with a pathognomonic ‘molar tooth sign’ on brain magnetic resonance imaging. The importance of ciliary function in neuronal development has been appreciated only in the last decade with the classification of Joubert syndrome as a ciliopathy. This, together with the identification of many of the clinical features of ciliopathies in individuals with Joubert syndrome and the localization of Joubert syndrome’s causative gene products at or near the primary cilium, have defined a new class of neurological disease. Cilia are involved in diverse cellular processes including protein trafficking, photoreception, embryonic axis patterning, and cell cycle regulation. Ciliary dysfunction can affect a single tissue or manifest as multi-organ involvement. Ciliary defects have been described in retinopathies such as retinitis pigmentosa and Leber congenital amaurosis (defects in photoreceptor ciliary protein complexes), renal syndromes with nephronophthisis and cystic dysplastic kidneys, and liver conditions such as fibrosis and biliary cirrhosis. Recognizing the diverse presentations of the ciliopathies and screening strategies following diagnosis is an important part of the treatment plan of children with cilia-related disorders.
doi:10.1111/j.1469-8749.2011.04021.x
PMCID: PMC3984879  PMID: 21679365
11.  Stem Cells and Regeneration: Special Review Issue 
Human molecular genetics  2008;17(0):R1-R2.
doi:10.1093/hmg/ddn186
PMCID: PMC4489689  PMID: 18632689
12.  Off-target effect of doublecortin-family shRNA on neuronal migration associated with endogenous microRNA dysregulation 
Neuron  2014;82(6):1255-1262.
SUMMARY
Acute gene inactivation using short hairpin RNA (shRNA, knockdown) in developing brain is a powerful technique to study genetic function, however, discrepancies between knockdown and knockout murine phenotypes have left unanswered questions. For example, doublecortin (Dcx) knockdown but not knockout shows a neocortical neuronal migration phenotype. Here we report that in utero electroporation of shRNA, but not siRNA or miRNA to Dcx demonstrates a migration phenotype in Dcx knockouts akin to the effect in wildtype mice, suggesting shRNA-mediated off-target toxicity. This effect was not limited to Dcx, as it was observed in Dclk1 knockouts, as well as with a fraction of scrambled shRNAs, suggesting a sequence-dependent but not sequence-specific effect. Profiling RNAs from electroporated cells showed a defect in endogenous let7 miRNA levels, and disruption of let7 or Dicer recapitulated the migration defect. The results suggest that shRNA-mediated knockdown can produce untoward migration effects by altering endogenous miRNA pathways.
doi:10.1016/j.neuron.2014.04.036
PMCID: PMC4086250  PMID: 24945770
doublecortin; dcx; shRNA; miRNA; off-target; migration
13.  Joubert syndrome: report of 11 cases 
The Turkish journal of pediatrics  2012;54(6):605-611.
SUMMARY
Joubert syndrome (JS) is an autosomal recessive condition characterized by hypotonia, ataxia, psychomotor delay, and variable occurrence of oculomotor apraxia and neonatal breathing abnormalities. The 11 cases were searched according to their clinic, radiologic, and mutation analysis findings, according to which they were diagnosed as JS. Physical, neurological and fundus examinations were performed in all patients. Cerebral magnetic resonance imaging scan, abdominal ultrasonography, and if necessary, echocardiography were performed. CC2D2A and ARL13B mutations were analyzed in our 11 JS patients. The mean age was 31.09±37.49 months (range: 1 month – 10 years). Two of the cases were siblings. Nine of the cases had a history of episodic hyperpnea. The other findings were hypotonia, ataxia, psychomotor retardation, and nystagmus. In all patients, the “molar tooth sign” was observed with scanning methods. In addition, cerebellar cortical dysplasia was established in one of the cases. Macrocephaly (1 patient), multiple renal cysts (1 patient), ocular coloboma (2 patients), ptosis (1 patient), congenital heart disease (1 patient), polydactyly (2 patients), and congenital hip dislocation (2 patients) were also determined. We identified mutation (c.C4452T → p.R1518W) in CC2D2A in two patients. JS can show heterogeneity clinically, neuroradiologically and genetically. Determination of the symptoms, early diagnosis and genetic consultation are the goals for decision-making to begin treatment and rehabilitation programs.
PMCID: PMC4442635  PMID: 23692786
Joubert syndrome; clinical and radiological findings; mutation analysis
14.  The Very Low Density Lipoprotein Receptor–Associated Pontocerebellar Hypoplasia and Dysmorphic Features in Three Turkish Patients 
Journal of child neurology  2012;28(3):379-383.
Pontocerebellar hypoplasia consists of a rare heterogeneous group of congenital neurodevelopmental disorders characterized by hypoplasia and atrophy of the cerebellar cortex, dentate and pontine nuclei, and inferior olives. The very low density lipoprotein receptor protein is an integral part of the reelin signaling pathway, which guides neuroblast migration in the cerebral cortex and cerebellum. Mutations in this receptor cause nonprogressive cerebellar ataxia, mental retardation, and cerebellar hypoplasia. In this report, we present 3 patients from 2 different families displaying very low density lipoprotein receptor–associated pontocerebellar hypoplasia, cortical dysplasia, mental retardation, and bipedal gait. One of the siblings has also displayed dysmorphic features, as we previously reported before the identification of the genetic defect in this family.
doi:10.1177/0883073812441065
PMCID: PMC4442636  PMID: 22532556
pontocerebellar hypoplasia; VLDLR mutation; dysmorphic features; reelin
15.  Exome Sequencing Can Improve Diagnosis and Alter Patient Management 
Science translational medicine  2012;4(138):138ra78.
The translation of “next-generation” sequencing directly to the clinic is still being assessed but has the potential for genetic diseases to reduce costs, advance accuracy, and point to unsuspected yet treatable conditions. To study its capability in the clinic, we performed whole-exome sequencing in 118 probands with a diagnosis of a pediatric-onset neurodevelopmental disease in which most known causes had been excluded. Twenty-two genes not previously identified as disease-causing were identified in this study (19% of cohort), further establishing exome sequencing as a useful tool for gene discovery. New genes identified included EXOC8 in Joubert syndrome and GFM2 in a patient with microcephaly, simplified gyral pattern, and insulin-dependent diabetes. Exome sequencing uncovered 10 probands (8% of cohort) with mutations in genes known to cause a disease different from the initial diagnosis. Upon further medical evaluation, these mutations were found to account for each pro-band's disease, leading to a change in diagnosis, some of which led to changes in patient management. Our data provide proof of principle that genomic strategies are useful in clarifying diagnosis in a proportion of patients with neurodevelopmental disorders.
doi:10.1126/scitranslmed.3003544
PMCID: PMC4442637  PMID: 22700954
16.  Primary cilia in the developing and mature brain 
Neuron  2014;82(3):511-521.
Primary cilia were the largely neglected non-motile counterparts of their better-known cousin, the motile cilia. For years these non-motile cilia were considered evolutionary remnants of little consequence to cellular function. Fast-forward 10 years and we now recognize primary cilia as key integrators of extracellular ligand-based signaling and cellular polarity, which regulate neuronal cell fate, migration differentiation, as well as a host of adult behaviors. Important future questions will focus on structure-function relationships, their roles in signaling and disease, and as areas of target for treatments.
doi:10.1016/j.neuron.2014.04.024
PMCID: PMC4104280  PMID: 24811376
primary cilium; ciliopathies; nervous system; development; treatment
17.  De novo somatic mutations in components of the PI3K-AKT3-mTOR pathway cause hemimegalencephaly 
Nature genetics  2012;44(8):941-945.
De novo somatic mutations in focal areas are well documented in diseases such as neoplasia but are rarely reported in malformation of the developing brain. Hemimegalencephaly (HME) is characterized by overgrowth of either one of the two cerebral hemispheres. The molecular etiology of HME remains a mystery. The intractable epilepsy that is associated with HME can be relieved by the surgical treatment hemispherectomy, allowing sampling of diseased tissue. Exome sequencing and mass spectrometry analysis in paired brain-blood samples from individuals with HME (n = 20 cases) identified de novo somatic mutations in 30% of affected individuals in the PIK3CA, AKT3 and MTOR genes. A recurrent PIK3CA c.1633G>A mutation was found in four separate cases. Identified mutations were present in 8–40% of sequenced alleles in various brain regions and were associated with increased neuronal S6 protein phosphorylation in the brains of affected individuals, indicating aberrant activation of mammalian target of rapamycin (mTOR) signaling. Thus HME is probably a genetically mosaic disease caused by gain of function in phosphatidylinositol 3-kinase (PI3K)-AKT3-mTOR signaling.
doi:10.1038/ng.2329
PMCID: PMC4417942  PMID: 22729223
18.  Mutations in CENPE define a novel kinetochore-centromeric mechanism for Microcephalic Primordial Dwarfism 
Human genetics  2014;133(8):1023-1039.
Defects in centrosome, centrosomal-associated and spindle-associated proteins are the most frequent cause of Primary Microcephaly (PM) and Microcephalic Primordial Dwarfism (MPD) syndromes in humans. Mitotic progression and segregation defects, microtubule spindle abnormalities and impaired DNA damage-induced G2-M cell cycle checkpoint proficiency have been documented in cell lines from these patients. This suggests that impaired mitotic entry, progression and exit strongly contribute to PM and MPD. Considering the vast protein networks involved in coordinating this cell cycle stage, the list of potential target genes that could underlie novel developmental disorders is large. One such complex network, with a direct microtubule-mediated physical connection to the centrosome, is the kinetochore. This centromeric-associated structure nucleates microtubule attachments onto mitotic chromosomes. Here, we described novel compound heterozygous variants in CENPE in two siblings who exhibit a profound MPD associated with developmental delay, simplified gyri and other isolated abnormalities. CENPE encodes centromere-associated protein E (CENP-E), a core kinetochore component functioning to mediate chromosome congression initially of misaligned chromosomes and in subsequent spindle microtubule capture during mitosis. Firstly, we present a comprehensive clinical description of these patients. Then, using patient cells we document abnormalities in spindle microtubule organisation, mitotic progression and segregation, before modeling the cellular pathogenicity of these variants in an independent cell system. Our cellular analysis shows that a pathogenic defect in CENP-E, a kinetochore-core protein, largely phenocopies PCNT-mutated Microcephalic Osteodysplastic Primordial Dwarfism type II patient cells. PCNT encodes a centrosome-associated protein. These results highlight a common underlying pathomechanism. Our findings provide the first evidence for a kinetochore-based route to MPD in humans.
doi:10.1007/s00439-014-1443-3
PMCID: PMC4415612  PMID: 24748105
Microcephaly; growth restriction; centromere; kinetochore; CENP-E; spindle microtubules
19.  CLP1 Founder Mutation Links tRNA Splicing and Maturation to Cerebellar Development and Neurodegeneration 
Cell  2014;157(3):651-663.
SUMMARY
Neurodegenerative diseases can occur so early as to affect neurodevelopment. From a cohort of over 2000 consanguineous families with childhood neurological disease, we identified a founder mutation in four independent pedigrees in cleavage and polyadenylation factor I subunit (CLP1). CLP1 is a multifunctional kinase implicated in tRNA, mRNA and siRNA maturation. Kinase activity of the CLP1 mutant protein was defective, and the tRNA endonuclease complex (TSEN) was destabilized, resulting in impaired pre-tRNA cleavage. Germline clp1 null zebrafish showed cerebellar neurodegeneration that was rescued by wild type but not mutant human CLP1 expression. Patient-derived induced neurons displayed both depletion of mature tRNAs and accumulation of unspliced pre-tRNAs. Transfection of partially processed tRNA fragments into patient cells exacerbated an oxidative stress-induced reduction in cell survival. Our data links tRNA maturation to neuronal development and neurodegeneration through defective CLP1 function in humans.
doi:10.1016/j.cell.2014.03.049
PMCID: PMC4128918  PMID: 24766810
CLP1; tRNA splicing endonuclease complex; TSEN; ataxia; neurodegeneration
21.  The ciliary proteins Meckelin and Jouberin are required for retinoic acid-dependent neural differentiation of mouse embryonic stem cells 
The dysfunction of the primary cilium, a complex, evolutionarily conserved, organelle playing an important role in sensing and transducing cell signals, is the unifying pathogenetic mechanism of a growing number of diseases collectively termed “ciliopathies”, typically characterized by multiorgan involvement. Developmental defects of the central nervous system (CNS) characterize a subset of ciliopathies showing clinical and genetic overlap, such as Joubert syndrome (JS) and Meckel syndrome (MS). Although several knock-out mice lacking a variety of ciliary proteins have shown the importance of primary cilia in the development of the brain and CNS-derived structures, developmental in vitro studies, extremely useful to unravel the role of primary cilia along the course of neural differentiation, are still missing.
Mouse embryonic stem cells (mESCs) have been recently proven to mimic brain development, giving the unique opportunity to dissect the CNS differentiation process along its sequential steps. In the present study we show that mESCs express the ciliary proteins Meckelin and Jouberin in a developmentally-regulated manner, and that these proteins co-localize with acetylated tubulin labeled cilia located at the outer embryonic layer. Further, mESCs differentiating along the neuronal lineage activate the cilia-dependent sonic hedgehog signaling machinery, which is impaired in Meckelin knockout cells but results unaffected in Jouberin-deficient mESCs. However, both lose the ability to acquire a neuronal phenotype. Altogether, these results demonstrate a pivotal role of Meckelin and Jouberin during embryonic neural specification and indicate mESCs as a suitable tool to investigate the developmental impact of ciliary proteins dysfunction.
doi:10.1016/j.diff.2014.02.005
PMCID: PMC4355919  PMID: 24613594
Embryonic stem cells; Neural differentiation; Primary cilium
22.  Expanding CEP290 mutational spectrum in ciliopathies 
Ciliopathies are an expanding group of rare conditions characterised by multiorgan involvement, that are caused by mutations in genes encoding for proteins of the primary cilium or its apparatus. Among these genes, CEP290 bears an intriguing allelic spectrum, being commonly mutated in Joubert syndrome and related disorders (JSRD), Meckel syndrome (MKS), Senior-Loken syndrome and isolated Leber congenital amaurosis (LCA). Although these conditions are recessively inherited, in a subset of patients only one CEP290 mutation could be detected.
To assess whether genomic rearrangements involving the CEP290 gene could represent a possible mutational mechanism in these cases, exon dosage analysis on genomic DNA was performed in two groups of CEP290 heterozygous patients, including five JSRD/MKS cases and four LCA, respectively. In one JSRD patient, we identified a large heterozygous deletion encompassing CEP290 C-terminus, that resulted in marked reduction of mRNA expression. No copy number alterations were identified in the remaining probands.
The present work expands the CEP290 genotypic spectrum to include multiexon deletions. Although this mechanism does not appear to be frequent, screening for genomic rearrangements should be considered in patients in whom a single CEP290 mutated allele was identified.
doi:10.1002/ajmg.a.33025
PMCID: PMC4340070  PMID: 19764032
Joubert syndrome and related disorders; Meckel syndrome; CEP290; genomic rearrangement
23.  Sun Proteins Enlighten Nuclear Movement in Development 
Neuron  2009;64(2):147-149.
Regulation of nuclear movement is a critical event in neurogenesis and neuronal migration during brain development. In this issue of Neuron, Zhang et al. identify a role for SUN and the KASH-domain-containing nuclear membrane proteins as the long-sought linker between microtubules and the nucleus during brain development.
doi:10.1016/j.neuron.2009.10.010
PMCID: PMC4340071  PMID: 19874779
24.  A homozygous PDE6D mutation in Joubert syndrome impairs targeting of farnesylated INPP5E protein to the primary cilium 
Human mutation  2014;35(1):137-146.
Joubert syndrome (JS) is characterized by a distinctive cerebellar structural defect, namely the « molar tooth sign ». JS is genetically heterogeneous, involving 18 genes identified to date, which are all required for cilia biogenesis and/or function. In a consanguineous family with JS associated with optic nerve coloboma, kidney hypoplasia and polydactyly, combined exome sequencing and mapping identified a homozygous splice site mutation in PDE6D, encoding a prenyl-binding protein. We found that pde6d depletion in zebrafish leads to renal and retinal developmental anomalies and wild-type but not mutant PDE6D is able to rescue this phenotype. Proteomic analysis identified INPP5E, whose mutations also lead to JS or MORM syndromes, as novel prenyl-dependent cargo of PDE6D. Mutant PDE6D shows reduced binding to INPP5E, which fails to localize to primary cilia in patient fibroblasts and tissues. Furthermore, mutant PDE6D is unable to bind to GTP-bound ARL3, which acts as a cargo-release factor for PDE6D-bound INPP5E. Altogether, these results indicate that PDE6D is required for INPP5E ciliary targeting and suggest a broader role for PDE6D in targeting other prenylated proteins to the cilia. This study identifies PDE6D as a novel JS disease gene and provides the first evidence of prenyl-binding dependent trafficking in ciliopathies.
doi:10.1002/humu.22470
PMCID: PMC3946372  PMID: 24166846
Joubert syndrome; primary cilia; PDE6D; INPP5E; prenylation
25.  Mutation spectrum of Joubert syndrome and related disorders among Arabs 
Human Genome Variation  2014;1:14020-.
Joubert syndrome (JS) is a rare autosomal recessive (AR), neurological condition characterized by dysgenesis of the cerebellar vermis with the radiological hallmark of molar tooth sign, oculomotor apraxia, recurrent hyperventilation and intellectual disability. Most cases display a broad spectrum of additional features, including polydactyly, retinal dystrophy and renal abnormalities, which define different subtypes of JS-related disorders (JSRDs). To date, 23 genes have been shown to cause JSRDs, and although most of the identified genes encode proteins involved in cilia function or assembly, the molecular mechanisms associated with ciliary signaling remain enigmatic. Arab populations are ethnically diverse with high levels of consanguinity (20–60%) and a high prevalence of AR disorders. In addition, isolated communities with very-high levels of inbreeding and founder mutations are common. In this article, we review the 70 families reported thus far with JS and JSRDs that have been studied at the molecular level from all the Arabic countries and compile the mutations found. We show that JS and the related JSRDs are genetically heterogeneous in Arabs, with 53 mutations in 15 genes. Thirteen of these mutations are potentially founder mutations for the region.
doi:10.1038/hgv.2014.20
PMCID: PMC4785524  PMID: 27081510

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