Hypomorphic Recessive Variants in SUFU Impair the Sonic Hedgehog Pathway and Cause Joubert Syndrome with Cranio-facial and Skeletal Defects.

The Sonic Hedgehog (SHH) pathway is a key signaling pathway orchestrating embryonic development, mainly of the CNS and limbs. In vertebrates, SHH signaling is mediated by the primary cilium, and genetic defects affecting either SHH pathway members or ciliary proteins cause a spectrum of developmental disorders. SUFU is the main negative regulator of the SHH pathway and is essential during development. Indeed, Sufu knock-out is lethal in mice, and recessive pathogenic variants of this gene have never been reported in humans. Through whole-exome sequencing in subjects with Joubert syndrome, we identified four children from two unrelated families carrying homozygous missense variants in SUFU. The children presented congenital ataxia and cerebellar vermis hypoplasia with elongated superior cerebellar peduncles (mild "molar tooth sign"), typical cranio-facial dysmorphisms (hypertelorism, depressed nasal bridge, frontal bossing), and postaxial polydactyly. Two siblings also showed polymicrogyria. Molecular dynamics simulation predicted random movements of the mutated residues, with loss of the native enveloping movement of the binding site around its ligand GLI3. Functional studies on cellular models and fibroblasts showed that both variants significantly reduced SUFU stability and its capacity to bind GLI3 and promote its cleavage into the repressor form GLI3R. In turn, this impaired SUFU-mediated repression of the SHH pathway, as shown by altered expression levels of several target genes. We demonstrate that germline hypomorphic variants of SUFU cause deregulation of SHH signaling, resulting in recessive developmental defects of the CNS and limbs which share features with both SHH-related disorders and ciliopathies.

Negative Regulation of Mitochondrial Antiviral Signaling Protein-Mediated Antiviral Signaling by the Mitochondrial Protein LRPPRC During Hepatitis C Virus Infection.

Hepatitis C virus (HCV) is highly efficient in establishing a chronic infection, having evolved multiple strategies to suppress the host antiviral responses. The HCV nonstructural 5A (NS5A) protein, in addition to its role in viral replication and assembly, has long been known to hamper the interferon (IFN) response. However, the mechanism of this inhibitory activity of NS5A remains partly characterized. In a functional proteomic screening carried out in HCV replicon cells, we identified the mitochondrial protein LRPPRC as an NS5A binding factor. Notably, we found that downregulation of LRPPRC expression results in a significant inhibition of HCV infection, which is associated with an increased activation of the IFN response. Moreover, we showed that LRPPRC acts as a negative regulator of the mitochondrial-mediated antiviral immunity, by interacting with mitochondrial antiviral signaling protein (MAVS) and inhibiting its association with TRAF3 and TRAF6. Finally, we demonstrated that NS5A is able to interfere with MAVS activity in a LRPPRC-dependent manner. Conclusion: Overall, our results indicate that NS5A contributes to the inhibition of innate immune pathways during HCV infection by exploiting the ability of LRPPRC to inhibit MAVS-regulated antiviral signaling.

Impaired urinary concentration ability is a sensitive predictor of renal disease progression in Joubert syndrome.

BACKGROUND: Joubert syndrome (JS) is an inherited ciliopathy characterized by a complex midbrain-hindbrain malformation and multiorgan involvement. Renal disease, mainly juvenile nephronophthisis (NPH), was reported in 25-30% patients although only ∼18% had a confirmed diagnosis of chronic kidney disease (CKD). NPH often remains asymptomatic for many years, resulting in delayed diagnosis. The aim of the study was to identify a biomarker able to quantify the risk of progressive CKD in young children with JS. METHODS: Renal features were investigated in 93 Italian patients, including biochemical tests, ultrasound and 1-deamino-8D-arginine vasopressin test in children with reduced basal urine osmolality. A subset of patients was followed-up over time. RESULTS: At last examination, 27 of 93 subjects (29%) presented with CKD, ranging from isolated urinary concentration defect (UCD) to end-stage renal disease. Both normal and pathological urine osmolality levels remained stable over time, even when obtained at very early ages. Follow-up data showed that the probability of developing CKD can be modelled as a function of the urine osmolality value, exceeding 75% for levels <600 mOsm/kg H2O, and significantly increased in patients with an early diagnosis of isolated UCD. CONCLUSIONS: We conclude that the frequency of CKD in JS increases with age and is higher than previously reported. Urine osmolality represents an early sensitive quantitative biomarker of the risk of CKD progression.

MKS5 and CEP290 Dependent Assembly Pathway of the Ciliary Transition Zone.

Cilia have a unique diffusion barrier ("gate") within their proximal region, termed transition zone (TZ), that compartmentalises signalling proteins within the organelle. The TZ is known to harbour two functional modules/complexes (Meckel syndrome [MKS] and Nephronophthisis [NPHP]) defined by genetic interaction, interdependent protein localisation (hierarchy), and proteomic studies. However, the composition and molecular organisation of these modules and their links to human ciliary disease are not completely understood. Here, we reveal Caenorhabditis elegans CEP-290 (mammalian Cep290/Mks4/Nphp6 orthologue) as a central assembly factor that is specific for established MKS module components and depends on the coiled coil region of MKS-5 (Rpgrip1L/Rpgrip1) for TZ localisation. Consistent with a critical role in ciliary gate function, CEP-290 prevents inappropriate entry of membrane-associated proteins into cilia and keeps ARL-13 (Arl13b) from leaking out of cilia via the TZ. We identify a novel MKS module component, TMEM-218 (Tmem218), that requires CEP-290 and other MKS module components for TZ localisation and functions together with the NPHP module to facilitate ciliogenesis. We show that TZ localisation of TMEM-138 (Tmem138) and CDKL-1 (Cdkl1/Cdkl2/Cdkl3/Cdlk4 related), not previously linked to a specific TZ module, similarly depends on CEP-290; surprisingly, neither TMEM-138 or CDKL-1 exhibit interdependent localisation or genetic interactions with core MKS or NPHP module components, suggesting they are part of a distinct, CEP-290-associated module. Lastly, we show that families presenting with Oral-Facial-Digital syndrome type 6 (OFD6) have likely pathogenic mutations in CEP-290-dependent TZ proteins, namely Tmem17, Tmem138, and Tmem231. Notably, patient fibroblasts harbouring mutated Tmem17, a protein not yet ciliopathy-associated, display ciliogenesis defects. Together, our findings expand the repertoire of MKS module-associated proteins--including the previously uncharacterised mammalian Tmem80--and suggest an MKS-5 and CEP-290-dependent assembly pathway for building a functional TZ.

Loss-of-Function Mutations in APPL1 in Familial Diabetes Mellitus.

Diabetes mellitus is a highly heterogeneous disorder encompassing several distinct forms with different clinical manifestations including a wide spectrum of age at onset. Despite many advances, the causal genetic defect remains unknown for many subtypes of the disease, including some of those forms with an apparent Mendelian mode of inheritance. Here we report two loss-of-function mutations (c.1655T>A [p.Leu552(∗)] and c.280G>A [p.Asp94Asn]) in the gene for the Adaptor Protein, Phosphotyrosine Interaction, PH domain, and leucine zipper containing 1 (APPL1) that were identified by means of whole-exome sequencing in two large families with a high prevalence of diabetes not due to mutations in known genes involved in maturity onset diabetes of the young (MODY). APPL1 binds to AKT2, a key molecule in the insulin signaling pathway, thereby enhancing insulin-induced AKT2 activation and downstream signaling leading to insulin action and secretion. Both mutations cause APPL1 loss of function. The p.Leu552(∗) alteration totally abolishes APPL1 protein expression in HepG2 transfected cells and the p.Asp94Asn alteration causes significant reduction in the enhancement of the insulin-stimulated AKT2 and GSK3ß phosphorylation that is observed after wild-type APPL1 transfection. These findings-linking APPL1 mutations to familial forms of diabetes-reaffirm the critical role of APPL1 in glucose homeostasis.

Mutations in CEP120 cause Joubert syndrome as well as complex ciliopathy phenotypes.

BACKGROUND: Ciliopathies are an extensive group of autosomal recessive or X-linked disorders with considerable genetic and clinical overlap, which collectively share multiple organ involvement and may result in lethal or viable phenotypes. In large numbers of cases the genetic defect remains yet to be determined. The aim of this study is to describe the mutational frequency and phenotypic spectrum of the CEP120 gene. METHODS: Exome sequencing was performed in 145 patients with Joubert syndrome (JS), including 15 children with oral-facial-digital syndrome type VI (OFDVI) and 21 Meckel syndrome (MKS) fetuses. Moreover, exome sequencing was performed in one fetus with tectocerebellar dysraphia with occipital encephalocele (TCDOE), molar tooth sign and additional skeletal abnormalities. As a parallel study, 346 probands with a phenotype consistent with JS or related ciliopathies underwent next-generation sequencing-based targeted sequencing of 120 previously described and candidate ciliopathy genes. RESULTS: We present six probands carrying nine distinct mutations (of which eight are novel) in the CEP120 gene, previously found mutated only in Jeune asphyxiating thoracic dystrophy (JATD). The CEP120-associated phenotype ranges from mild classical JS in four patients to more severe conditions in two fetuses, with overlapping features of distinct ciliopathies that include TCDOE, MKS, JATD and OFD syndromes. No obvious correlation is evident between the type or location of identified mutations and the ciliopathy phenotype. CONCLUSION: Our findings broaden the spectrum of phenotypes caused by CEP120 mutations that account for nearly 1% of patients with JS as well as for more complex ciliopathy phenotypes. The lack of clear genotype-phenotype correlation highlights the relevance of comprehensive genetic analyses in the diagnostics of ciliopathies.

Very mild features of dysequilibrium syndrome associated with a novel VLDLR missense mutation.

Dysequilibrium syndrome (DES) is a non-progressive congenital ataxia characterized by severe intellectual deficit, truncal ataxia and markedly delayed, quadrupedal or absent ambulation. Recessive loss-of-function mutations in the very low density lipoprotein receptor (VLDLR) gene represent the most common cause of DES. Only two families have been reported harbouring homozygous missense mutations, both with a similarly severe phenotype. We report an Italian girl with very mild DES caused by the novel homozygous VLDLR missense mutation p.(C419Y). This unusually benign phenotype possibly relates to a less disruptive effect of the mutation, falling within a domain (EGF-B) not predicted as crucial for the protein function.

Cognitive, adaptive, and behavioral features in Joubert syndrome.

Joubert syndrome (JS) is a recessive neurodevelopmental disorder characterized by a distinctive cerebellar and brainstem malformation recognizable on brain imaging, the so-called molar tooth sign. The full spectrum of cognitive and behavioral phenotypes typical of JS is still far from being elucidated. The aim of this multicentric study was to define the clinical phenotype and neurobehavioral features of a large cohort of subjects with a neuroradiologically confirmed diagnosis of JS. Fifty-four patients aged 10 months to 29 years were enrolled. Each patient underwent a neurological evaluation as well as psychiatric and neuropsychological assessments. Global cognitive functioning was remarkably variable with Full IQ/General Quotient ranging from 32 to 129. Communication skills appeared relatively preserved with respect to both Daily Living and Socialization abilities. The motor domain was the area of greatest vulnerability, with a negative impact on personal care, social, and academic skills. Most children did not show maladaptive behaviors consistent with a psychiatric diagnosis but approximately 40% of them presented emotional and behavioral problems. We conclude that intellectual disability remains a hallmark but cannot be considered a mandatory diagnostic criterion of JS. Despite the high variability in the phenotypic spectrum and the extent of multiorgan involvement, nearly one quarter of JS patients had a favorable long-term outcome with borderline cognitive deficit or even normal cognition. Most of JS population also showed relatively preserved communication skills and overall discrete behavioral functioning in everyday life, independently from the presence and/or level of intellectual disability. © 2016 Wiley Periodicals, Inc.

Oral-facial-digital syndrome type VI: is C5orf42 really the major gene?

Oral-facial-digital type VI syndrome (OFDVI) is a rare phenotype of Joubert syndrome (JS). Recently, C5orf42 was suggested as the major OFDVI gene, being mutated in 9 of 11 families (82 %). We sequenced C5orf42 in 313 JS probands and identified mutations in 28 (8.9 %), most with a phenotype of pure JS. Only 2 out of 17 OFDVI patients (11.7 %) were mutated. A comparison of mutated vs. non-mutated OFDVI patients showed that preaxial and mesoaxial polydactyly, hypothalamic hamartoma and other congenital defects may predict C5orf42 mutations, while tongue hamartomas are more common in negative patients.

A solid quality-control analysis of AB SOLiD short-read sequencing data.

Next generation sequencers have greatly improved our ability to mine polymorphisms and mutations out of entire (or portions of) genomes. The reliability of their outputs, though, showed to be very related to the sequencing chemistry and to deeply affect the quality of the downstream analyses. We focus here on the two-base color code chemistry of AB SOLiD sequencers and propose a comprehensive quality control methodological and software pipeline. We used existing and custom tools to detect and purge short-reads of some common flaws due to sequencing errors and chemical hitches. We apply them to a cohort of SOLiD 4 runs and measure their joint efficacy in terms of the resulting ability to detect the greatest possible number of true variants.

A homozygous PDE6D mutation in Joubert syndrome impairs targeting of farnesylated INPP5E protein to the primary cilium.

Joubert syndrome (JS) is characterized by a distinctive cerebellar structural defect, namely the << molar tooth sign >>. JS is genetically heterogeneous, involving 20 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 mental retardation, obesity, congenital retinal dystrophy, and micropenis 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.

Ex vivo acidic preconditioning enhances bone marrow ckit+ cell therapeutic potential via increased CXCR4 expression.

AIMS: The chemokine receptor CXCR4 modulates endothelial progenitor cell migration, homing, and differentiation, and plays a key role in cardiovascular regeneration. Here we examined the effect of ex vivo acidic preconditioning (AP) on CXCR4 expression and on the regenerative potential of mouse bone marrow (BM) ckit(+) cells. METHODS AND RESULTS: Acidic preconditioning was achieved by exposing BM ckit(+) cells to hypercarbic acidosis (pH 7.0) for 24 h; control cells were kept at pH 7.4. Acidic preconditioning enhanced CXCR4 and stromal cell-derived factor 1 (SDF-1) mRNA levels, as well as CXCR4 phosphorylation. Acidic preconditioning ability to modulate CXCR4 expression depended on cytosolic calcium [Ca(2+)]i mobilization and on nitric oxide (NO), as determined by [Ca(2+)]i buffering with BAPTA, and by treatment with the NO donor (DETA/NO) and the NO synthase inhibitor (L-NAME). Further, AP increased SDF-1-driven chemotaxis, transendothelial migration, and differentiation toward the endothelial lineage in vitro. In a mouse model of hindlimb ischaemia, control and AP ckit(+) cells were transplanted into the ischaemic muscle; AP cells accelerated blood flow recovery, increased capillary, and arteriole number as well as the number of regenerating muscle fibres vs. control. These effects were abolished by treating AP cells with L-NAME. CONCLUSION: Acidic preconditioning represents a novel strategy to enhance BM ckit(+) cell therapeutic potential via NO-dependent increase in CXCR4 expression.

Atypical Childhood-onset Neuroaxonal Dystrophy in an Indian Girl.

A 7-year-old girl presented with progressive walking difficulties, spasticity, and cognitive decline with onset at 3 years of age. No seizures, vision, or hearing impairment were reported. The magnetic resonance imaging of the brain revealed cerebellar atrophy and evidence of iron deposition in the globi pallidi and substantia nigra. The clinico-radiological profile was suggestive of atypical childhood-onset neuroaxonal dystrophy. The patient was found to have compound heterozygous mutations in the PLA2G6 gene confirming the diagnosis.

GNAO1 encephalopathy: Broadening the phenotype and evaluating treatment and outcome.

OBJECTIVE: To describe better the motor phenotype, molecular genetic features, and clinical course of GNAO1-related disease. METHODS: We reviewed clinical information, video recordings, and neuroimaging of a newly identified cohort of 7 patients with de novo missense and splice site GNAO1 mutations, detected by next-generation sequencing techniques. RESULTS: Patients first presented in early childhood (median age of presentation 10 months, range 0-48 months), with a wide range of clinical symptoms ranging from severe motor and cognitive impairment with marked choreoathetosis, self-injurious behavior, and epileptic encephalopathy to a milder phenotype, featuring moderate developmental delay associated with complex stereotypies, mainly facial dyskinesia and mild epilepsy. Hyperkinetic movements were often exacerbated by specific triggers, such as voluntary movement, intercurrent illnesses, emotion, and high ambient temperature, leading to hospital admissions. Most patients were resistant to drug intervention, although tetrabenazine was effective in partially controlling dyskinesia for 2/7 patients. Emergency deep brain stimulation (DBS) was life saving in 1 patient, resulting in immediate clinical benefit with complete cessation of violent hyperkinetic movements. Five patients had well-controlled epilepsy and 1 had drug-resistant seizures. Structural brain abnormalities, including mild cerebral atrophy and corpus callosum dysgenesis, were evident in 5 patients. One patient had a diffuse astrocytoma (WHO grade II), surgically removed at age 16. CONCLUSIONS: Our findings support the causative role of GNAO1 mutations in an expanded spectrum of early-onset epilepsy and movement disorders, frequently exacerbated by specific triggers and at times associated with self-injurious behavior. Tetrabenazine and DBS were the most useful treatments for dyskinesia.

"Fork and bracket" syndrome expands the spectrum of SBF1-related sensory motor polyneuropathies.

Charcot-Marie-Tooth neuropathy type 4 (CMT4) comprises a large group of genetically heterogeneous progressive sensory motor neuropathies characterized by autosomal recessive inheritance. Among these, CMT4B includes 3 forms related to genes of the myotubularin family, namely CMT4B1 (MTMR2), CMT4B2 (MTMR13/SBF2), and CMT4B3 (MTMR5/SBF1).

Clinical, neuroradiological and molecular characterization of cerebellar dysplasia with cysts (Poretti-Boltshauser syndrome).

Cerebellar dysplasia with cysts and abnormal shape of the fourth ventricle, in the absence of significant supratentorial anomalies and of muscular involvement, defines recessively inherited Poretti-Boltshauser syndrome (PBS). Clinical features comprise non-progressive cerebellar ataxia, intellectual disability of variable degree, language impairment, ocular motor apraxia and frequent occurrence of myopia or retinopathy. Recently, loss-of-function variants in the LAMA1 gene were identified in six probands with PBS. Here we report the detailed clinical, neuroimaging and genetic characterization of 18 PBS patients from 15 unrelated families. Biallelic LAMA1 variants were identified in 14 families (93%). The only non-mutated proband presented atypical clinical and neuroimaging features, challenging the diagnosis of PBS. Sixteen distinct variants were identified, which were all novel. In particular, the frameshift variant c.[2935delA] recurred in six unrelated families on a shared haplotype, suggesting a founder effect. No LAMA1 variants could be detected in 27 probands with different cerebellar dysplasias or non-progressive cerebellar ataxia, confirming the strong correlate between LAMA1 variants and PBS.

Cognitive rehabilitation in a child with Joubert Syndrome: Developmental trends and adaptive changes in a single case report.

We report the clinical and rehabilitative follow up of M, a female child carrying a compound heterozygous pathogenic mutations in the TCTN1 gene and affected by Joubert Syndrome (JS). JS is a congenital cerebellar ataxia characterized by "the molar tooth sign" on axial MRI, a pathognomonic neuroradiological malformation involving the cerebellum and brainstem. JS presents with high phenotypic/cognitive variability, and little is known about cognitive rehabilitation programs. We describe the therapeutic settings, intensive rehabilitation targets and outcome indexes in M's cognitive development. Using a single case evidence-based approach, we attempt to distinguish the effectiveness of the intervention from the overall developmental trend. We assume that an adequate amount of focused, goal directed treatment in a relative short period of time can be at least as effective as one provided in longer time, and much less interfering with the child's everyday life. We conclude by discussing specific issues in cognitive development and rehabilitation in JS and, more broadly, in cerebellar malformations.

Functional genome-wide siRNA screen identifies KIAA0586 as mutated in Joubert syndrome.

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.

Mutations in B9D1 and MKS1 cause mild Joubert syndrome: expanding the genetic overlap with the lethal ciliopathy Meckel syndrome.

Joubert syndrome is a clinically and genetically heterogeneous ciliopathy characterized by a typical cerebellar and brainstem malformation (the "molar tooth sign"), and variable multiorgan involvement. To date, 24 genes have been found mutated in Joubert syndrome, of which 13 also cause Meckel syndrome, a lethal ciliopathy with kidney, liver and skeletal involvement. Here we describe four patients with mild Joubert phenotypes who carry pathogenic mutations in either MKS1 or B9D1, two genes previously implicated only in Meckel syndrome.