Bi-allelic variants in SNF8 cause a disease spectrum ranging from severe developmental and epileptic encephalopathy to syndromic optic atrophy.

The endosomal sorting complex required for transport (ESCRT) machinery is essential for membrane remodeling and autophagy and it comprises three multi-subunit complexes (ESCRT I-III). We report nine individuals from six families presenting with a spectrum of neurodevelopmental/neurodegenerative features caused by bi-allelic variants in SNF8 (GenBank: NM_007241.4), encoding the ESCRT-II subunit SNF8. The phenotypic spectrum included four individuals with severe developmental and epileptic encephalopathy, massive reduction of white matter, hypo-/aplasia of the corpus callosum, neurodevelopmental arrest, and early death. A second cohort shows a milder phenotype with intellectual disability, childhood-onset optic atrophy, or ataxia. All mildly affected individuals shared the same hypomorphic variant, c.304G>A (p.Val102Ile). In patient-derived fibroblasts, bi-allelic SNF8 variants cause loss of ESCRT-II subunits. Snf8 loss of function in zebrafish results in global developmental delay and altered embryo morphology, impaired optic nerve development, and reduced forebrain size. In vivo experiments corroborated the pathogenicity of the tested SNF8 variants and their variable impact on embryo development, validating the observed clinical heterogeneity. Taken together, we conclude that loss of ESCRT-II due to bi-allelic SNF8 variants is associated with a spectrum of neurodevelopmental/neurodegenerative phenotypes mediated likely via impairment of the autophagic flux.

Responsiveness of the Scale for the Assessment and Rating of Ataxia and Natural History in 884 Recessive and Early Onset Ataxia Patients.

OBJECTIVE: The Scale for the Assessment and Rating of Ataxia (SARA) is the most widely applied clinical outcome assessment (COA) for genetic ataxias, but presents metrological and regulatory challenges. To facilitate trial planning, we characterize its responsiveness (including subitem-level relations to ataxia severity and patient-focused outcomes) across a large number of ataxias, and provide first natural history data for several of them. METHODS: Subitem-level correlation and distribution-based analysis of 1,637 SARA assessments in 884 patients with autosomal recessive/early onset ataxia (370 with 2-8 longitudinal assessments) were complemented by linear mixed effects modeling to estimate progression and sample sizes. RESULTS: Although SARA subitem responsiveness varied between ataxia severities, gait/stance showed a robust granular linear scaling across the broadest range (SARA < 25). Responsiveness was diminished by incomplete subscale use at intermediate or upper levels, nontransitions ("static periods"), and fluctuating decreases/increases. All subitems except nose-finger showed moderate-to-strong correlations to activities of daily living, indicating that metric properties-not content validity-limit SARA responsiveness. SARA captured mild-to-moderate progression in many genotypes (eg, SYNE1-ataxia: 0.55 points/yr, ataxia with oculomotor apraxia type 2: 1.14 points/yr, POLG-ataxia: 1.56 points/yr), but no change in others (autosomal recessive spastic ataxia of Charlevoix-Saguenay, COQ8A-ataxia). Whereas sensitivity to change was optimal in mild ataxia (SARA < 10), it substantially deteriorated in advanced ataxia (SARA > 25; 2.7-fold sample size). Use of a novel rank-optimized SARA without subitems finger-chase and nose-finger reduces sample sizes by 20 to 25%. INTERPRETATION: This study comprehensively characterizes COA properties and annualized changes of the SARA across and within a large number of ataxias. It suggests specific approaches for optimizing its responsiveness that might facilitate regulatory qualification and trial design. ANN NEUROL 2023;94:470-485.

Child-to-adult transition: a survey of current practices within the European Reference Network for Rare Neurological Diseases (ERN-RND).

BACKGROUND: Transition from child-centered to adult-centered healthcare is a gradual process that addresses the medical, psychological, and educational needs of young people in the management of their autonomy in making decisions about their health and their future clinical assistance. This transfer is challenging across all chronic diseases but can be particularly arduous in rare neurological conditions. AIM: To describe the current practice on the transition process for young patients in centers participating in the European Reference Network for Rare Neurological Diseases (ERN-RND). METHODS: Members of the ERN-RND working group developed a questionnaire considering child-to-adult transition issues and procedures in current clinical practice. The questionnaire included 20 questions and was sent to members of the health care providers (HCPs) participating in the network. RESULTS: Twenty ERN-RND members (75% adult neurologists; 25% pediatricians; 5% nurses or study coordinators) responded to the survey, representing 10 European countries. Transition usually occurs between 16 and 18 years of age, but 55% of pediatric HCPs continue to care for their patients until they reach 40 years of age or older. In 5/20 ERN-RND centers, a standardized procedure managing transition is currently adopted, whereas in the remaining centers, the transition from youth to adult service is usually assisted by pediatricians as part of their clinical practice. CONCLUSIONS: This survey demonstrated significant variations in clinical practice between different centers within the ERN-RND network. It provided valuable data on existing transition programs and highlighted key challenges in managing transitions for patients with rare neurological disorders.

An Integrated Phenotypic and Genotypic Approach Reveals a High-Risk Subtype Association for EBF3 Missense Variants Affecting the Zinc Finger Domain.

OBJECTIVE: Collier/Olf/EBF (COE) transcription factors have distinct expression patterns in the developing and mature nervous system. To date, a neurological disease association has been conclusively established for only the Early B-cell Factor-3 (EBF3) COE family member through the identification of heterozygous loss-of-function variants in individuals with autism spectrum/neurodevelopmental disorders (NDD). Here, we identify a symptom severity risk association with missense variants primarily disrupting the zinc finger domain (ZNF) in EBF3-related NDD. METHODS: A phenotypic assessment of 41 individuals was combined with a literature meta-analysis for a total of 83 individuals diagnosed with EBF3-related NDD. Quantitative diagnostic phenotypic and symptom severity scales were developed to compare EBF3 variant type and location to identify genotype-phenotype correlations. To stratify the effects of EBF3 variants disrupting either the DNA-binding domain (DBD) or the ZNF, we used in vivo fruit fly UAS-GAL4 expression and in vitro luciferase assays. RESULTS: We show that patient symptom severity correlates with EBF3 missense variants perturbing the ZNF, which is a key protein domain required for stabilizing the interaction between EBF3 and the target DNA sequence. We found that ZNF-associated variants failed to restore viability in the fruit fly and impaired transcriptional activation. However, the recurrent variant EBF3 p.Arg209Trp in the DBD is capable of partially rescuing viability in the fly and preserved transcriptional activation. INTERPRETATION: We describe a symptom severity risk association with ZNF perturbations and EBF3 loss-of-function in the largest reported cohort to date of EBF3-related NDD patients. This analysis should have potential predictive clinical value for newly identified patients with EBF3 gene variants. ANN NEUROL 2022;92:138-153.

Expanding the natural history of CASK-related disorders to the prenatal period.

AIM: To assess whether microcephaly with pontine and cerebellar hypoplasia (MICPCH) could manifest in the prenatal period in patients with calcium/calmodulin-dependent serine protein kinase (CASK) gene disorders. METHOD: In this international multicentre retrospective study, we contacted a CASK parents' social media group and colleagues with expertise in cerebellar malformations and asked them to supply clinical and imaging information. Centiles and standard deviations (SD) were calculated according to age by nomograms. RESULTS: The study consisted of 49 patients (44 females and 5 males). Information regarding prenatal head circumference was available in 19 patients; 11 out of 19 had a fetal head circumference below -2SD (range -4.1SD to -2.02SD, mean gestational age at diagnosis 20 weeks). Progressive prenatal deceleration of head circumference growth rate was observed in 15 out of 19. At birth, 20 out of 42 had a head circumference below -2SD. A total of 6 out of 15 fetuses had a TCD z-score below -2 (range -5.88 to -2.02). INTERPRETATION: This study expands the natural history of CASK-related disorders to the prenatal period, showing evidence of progressive deceleration of head circumference growth rate, head circumference below -2SD, or small TCD. Most cases will not be diagnosed according to current recommendations for fetal central nervous system routine assessment. Consecutive measurements and genetic studies are advised in the presence of progressive deceleration of head circumference growth rates or small TCD. WHAT THIS PAPER ADDS: Progressive deceleration of fetal head circumference growth rate can be observed. A small transcerebellar diameter is an additional important manifestation. Most cases will not be diagnosed according to current recommendations for fetal central nervous system routine assessment. Consecutive measurements are advised when measurements are within the low range of norm.

CAPRIN1P512L causes aberrant protein aggregation and associates with early-onset ataxia.

CAPRIN1 is a ubiquitously expressed protein, abundant in the brain, where it regulates the transport and translation of mRNAs of genes involved in synaptic plasticity. Here we describe two unrelated children, who developed early-onset ataxia, dysarthria, cognitive decline and muscle weakness. Trio exome sequencing unraveled the identical de novo c.1535C > T (p.Pro512Leu) missense variant in CAPRIN1, affecting a highly conserved residue. In silico analyses predict an increased aggregation propensity of the mutated protein. Indeed, overexpressed CAPRIN1P512L forms insoluble ubiquitinated aggregates, sequestrating proteins associated with neurodegenerative disorders (ATXN2, GEMIN5, SNRNP200 and SNCA). Moreover, the CAPRIN1P512L mutation in isogenic iPSC-derived cortical neurons causes reduced neuronal activity and altered stress granule dynamics. Furthermore, nano-differential scanning fluorimetry reveals that CAPRIN1P512L aggregation is strongly enhanced by RNA in vitro. These findings associate the gain-of-function Pro512Leu mutation to early-onset ataxia and neurodegeneration, unveiling a critical residue of CAPRIN1 and a key role of RNA-protein interactions.

SUFU haploinsufficiency causes a recognisable neurodevelopmental phenotype at the mild end of the Joubert syndrome spectrum.

BACKGROUND: Joubert syndrome (JS) is a recessively inherited ciliopathy characterised by congenital ocular motor apraxia (COMA), developmental delay (DD), intellectual disability, ataxia, multiorgan involvement, and a unique cerebellar and brainstem malformation. Over 40 JS-associated genes are known with a diagnostic yield of 60%-75%.In 2018, we reported homozygous hypomorphic missense variants of the SUFU gene in two families with mild JS. Recently, heterozygous truncating SUFU variants were identified in families with dominantly inherited COMA, occasionally associated with mild DD and subtle cerebellar anomalies. METHODS: We reanalysed next generation sequencing (NGS) data in two cohorts comprising 1097 probands referred for genetic testing of JS genes. RESULTS: Heterozygous truncating and splice-site SUFU variants were detected in 22 patients from 17 families (1.5%) with strong male prevalence (86%), and in 8 asymptomatic parents. Patients presented with COMA, hypotonia, ataxia and mild DD, and only a third manifested intellectual disability of variable severity. Brain MRI showed consistent findings characterised by vermis hypoplasia, superior cerebellar dysplasia and subtle-to-mild abnormalities of the superior cerebellar peduncles. The same pattern was observed in two out of three tested asymptomatic parents. CONCLUSION: Heterozygous truncating or splice-site SUFU variants cause a novel neurodevelopmental syndrome encompassing COMA and mild JS, which likely represent overlapping entities. Variants can arise de novo or be inherited from a healthy parent, representing the first cause of JS with dominant inheritance and reduced penetrance. Awareness of this condition will increase the diagnostic yield of JS genetic testing, and allow appropriate counselling about prognosis, medical monitoring and recurrence risk.

Refining the mutational spectrum and gene-phenotype correlates in pontocerebellar hypoplasia: results of a multicentric study.

BACKGROUND: Pontocerebellar hypoplasias (PCH) comprise a group of genetically heterogeneous disorders characterised by concurrent hypoplasia of the pons and the cerebellum and variable clinical and imaging features. The current classification includes 13 subtypes, with ~20 known causative genes. Attempts have been made to delineate the phenotypic spectrum associated to specific PCH genes, yet clinical and neuroradiological features are not consistent across studies, making it difficult to define gene-specific outcomes. METHODS: We performed deep clinical and imaging phenotyping in 56 probands with a neuroradiological diagnosis of PCH, who underwent NGS-based panel sequencing of PCH genes and MLPA for CASK rearrangements. Next, we conducted a phenotype-based unsupervised hierarchical cluster analysis to investigate associations between genes and specific phenotypic clusters. RESULTS: A genetic diagnosis was obtained in 43 probands (77%). The most common causative gene was CASK, which accounted for nearly half cases (45%) and was mutated in females and occasionally in males. The European founder mutation p.Ala307Ser in TSEN54 and pathogenic variants in EXOSC3 accounted for 18% and 9% of cases, respectively. VLDLR, TOE1 and RARS2 were mutated in single patients. We were able to confirm only few previously reported associations, including jitteriness and clonus with TSEN54 and lower motor neuron signs with EXOSC3. When considering multiple features simultaneously, a clear association with a phenotypic cluster only emerged for EXOSC3. CONCLUSION: CASK represents the major PCH causative gene in Italy. Phenotypic variability associated with the most common genetic causes of PCH is wider than previously thought, with marked overlap between CASK and TSEN54-associated disorders.

Clinical variability at the mild end of BRAT1-related spectrum: Evidence from two families with genotype-phenotype discordance.

Biallelic mutations in the BRAT1 gene, encoding BRCA1-associated ATM activator 1, result in variable phenotypes, from rigidity and multifocal seizure syndrome, lethal neonatal to neurodevelopmental disorder, and cerebellar atrophy with or without seizures, without obvious genotype-phenotype associations. We describe two families at the mildest end of the spectrum, differing in clinical presentation despite a common genotype at the BRAT1 locus. Two siblings displayed nonprogressive congenital ataxia and shrunken cerebellum on magnetic resonance imaging. A third unrelated patient showed normal neurodevelopment, adolescence-onset seizures, and ataxia, shrunken cerebellum, and ultrastructural abnormalities on skin biopsy, representing the mildest form of NEDCAS hitherto described. Exome sequencing identified the c.638dup and the novel c.1395G>A BRAT1 variants, the latter causing exon 10 skippings. The p53-MCL test revealed normal ATM kinase activity. Our findings broaden the allelic and clinical spectrum of BRAT1-related disease, which should be suspected in presence of nonprogressive cerebellar signs, even without a neurodevelopmental disorder.

De Novo and Dominantly Inherited SPTAN1 Mutations Cause Spastic Paraplegia and Cerebellar Ataxia.

BACKGROUND: Pathogenic variants in SPTAN1 have been linked to a remarkably broad phenotypical spectrum. Clinical presentations include epileptic syndromes, intellectual disability, and hereditary motor neuropathy. OBJECTIVES: We investigated the role of SPTAN1 variants in rare neurological disorders such as ataxia and spastic paraplegia. METHODS: We screened 10,000 NGS datasets across two international consortia and one local database, indicative of the level of international collaboration currently required to identify genes causative for rare disease. We performed in silico modeling of the identified SPTAN1 variants. RESULTS: We describe 22 patients from 14 families with five novel SPTAN1 variants. Of six patients with cerebellar ataxia, four carry a de novo SPTAN1 variant and two show a sporadic inheritance. In this group, one variant (p.Lys2083del) is recurrent in four patients. Two patients have novel de novo missense mutations (p.Arg1098Cys, p.Arg1624Cys) associated with cerebellar ataxia, in one patient accompanied by intellectual disability and epilepsy. We furthermore report a recurrent missense mutation (p.Arg19Trp) in 15 patients with spastic paraplegia from seven families with a dominant inheritance pattern in four and a de novo origin in one case. One further patient carrying a de novo missense mutation (p.Gln2205Pro) has a complex spastic ataxic phenotype. Through protein modeling we show that mutated amino acids are located at crucial interlinking positions, interconnecting the three-helix bundle of a spectrin repeat. CONCLUSIONS: We show that SPTAN1 is a relevant candidate gene for ataxia and spastic paraplegia. We suggest that for the mutations identified in this study, disruption of the interlinking of spectrin helices could be a key feature of the pathomechanism. © 2022 International Parkinson and Movement Disorder Society.

Postnatal microcephaly and retinal involvement expand the phenotype of RPL10-related disorder.

Hemizygous missense variants in the RPL10 gene encoding a ribosomal unit are responsible for an X-linked syndrome presenting with intellectual disability (ID), autism spectrum disorder, epilepsy, dysmorphic features, and multiple congenital anomalies. Among 15 individuals with RPL10-related disorder reported so far, only one patient had retinitis pigmentosa and microcephaly was observed in approximately half of the cases. By exome sequencing, three Italian and one Spanish male children, from three independent families, were found to carry the same hemizygous novel missense variant p.(Arg32Leu) in RPL10, inherited by their unaffected mother in all cases. The variant, not reported in gnomAD, is located in the 28S rRNA binding region, affecting an evolutionary conserved residue and predicted to disrupt the salt-bridge between Arg32 and Asp28. In addition to features consistent with RPL10-related disorder, all four boys had retinal degeneration and postnatal microcephaly. Pathogenic variants in genes responsible for inherited retinal degenerations were ruled out in all the probands. A novel missense RPL10 variant was detected in four probands with a recurrent phenotype including ID, dysmorphic features, progressive postnatal microcephaly, and retinal anomalies. The presented individuals suggest that retinopathy and postnatal microcephaly are clinical clues of RPL10-related disorder, and at least the retinal defect might be more specific for the p.(Arg32Leu) RPL10 variant, suggesting a specific genotype/phenotype correlation.

Heterozygous KIF1A variants underlie a wide spectrum of neurodevelopmental and neurodegenerative disorders.

BACKGROUND: Dominant and recessive variants in the KIF1A gene on chromosome 2q37.3 are associated with several phenotypes, although only three syndromes are currently listed in the OMIM classification: hereditary sensory and autonomic neuropathy type 2 and spastic paraplegia type 30, both recessively inherited, and mental retardation type 9 with dominant inheritance. METHODS: In this retrospective multicentre study, we describe the clinical, neuroradiological and genetic features of 19 Caucasian patients (aged 3-65 years) harbouring heterozygous KIF1A variants, and extensively review the available literature to improve current classification of KIF1A-related disorders. RESULTS: Patients were divided into two groups. Group 1 comprised patients with a complex phenotype with prominent pyramidal signs, variably associated in all but one case with additional features (ie, epilepsy, ataxia, peripheral neuropathy, optic nerve atrophy); conversely, patients in group 2 presented an early onset or congenital ataxic phenotype. Fourteen different heterozygous missense variants were detected by next-generation sequencing screening, including three novel variants, most falling within the kinesin motor domain. CONCLUSION: The present study further enlarges the clinical and mutational spectrum of KIF1A-related disorders by describing a large series of patients with dominantly inherited KIF1A pathogenic variants ranging from pure to complex forms of hereditary spastic paraparesis/paraplegias (HSP) and ataxic phenotypes in a lower proportion of cases. A comprehensive review of the literature indicates that KIF1A screening should be implemented in HSP regardless of its mode of inheritance or presentations as well as in other complex neurodegenerative or neurodevelopmental disorders showing congenital or early onset ataxia.

Superior Cerebellar Atrophy: An Imaging Clue to Diagnose ITPR1-Related Disorders.

The inositol 1,4,5-triphosphate receptor type 1 (ITPR1) gene encodes an InsP3-gated calcium channel that modulates intracellular Ca2+ release and is particularly expressed in cerebellar Purkinje cells. Pathogenic variants in the ITPR1 gene are associated with different types of autosomal dominant spinocerebellar ataxia: SCA15 (adult onset), SCA29 (early-onset), and Gillespie syndrome. Cerebellar atrophy/hypoplasia is invariably detected, but a recognizable neuroradiological pattern has not been identified yet. With the aim of describing ITPR1-related neuroimaging findings, the brain MRI of 14 patients with ITPR1 variants (11 SCA29, 1 SCA15, and 2 Gillespie) were reviewed by expert neuroradiologists. To further evaluate the role of superior vermian and hemispheric cerebellar atrophy as a clue for the diagnosis of ITPR1-related conditions, the ITPR1 gene was sequenced in 5 patients with similar MRI pattern, detecting pathogenic variants in 4 of them. Considering the whole cohort, a distinctive neuroradiological pattern consisting in superior vermian and hemispheric cerebellar atrophy was identified in 83% patients with causative ITPR1 variants, suggesting this MRI finding could represent a hallmark for ITPR1-related disorders.

Clinico-Genetic, Imaging and Molecular Delineation of COQ8A-Ataxia: A Multicenter Study of 59 Patients.

OBJECTIVE: To foster trial-readiness of coenzyme Q8A (COQ8A)-ataxia, we map the clinicogenetic, molecular, and neuroimaging spectrum of COQ8A-ataxia in a large worldwide cohort, and provide first progression data, including treatment response to coenzyme Q10 (CoQ10). METHODS: Cross-modal analysis of a multicenter cohort of 59 COQ8A patients, including genotype-phenotype correlations, 3D-protein modeling, in vitro mutation analyses, magnetic resonance imaging (MRI) markers, disease progression, and CoQ10 response data. RESULTS: Fifty-nine patients (39 novel) with 44 pathogenic COQ8A variants (18 novel) were identified. Missense variants demonstrated a pleiotropic range of detrimental effects upon protein modeling and in vitro analysis of purified variants. COQ8A-ataxia presented as variable multisystemic, early-onset cerebellar ataxia, with complicating features ranging from epilepsy (32%) and cognitive impairment (49%) to exercise intolerance (25%) and hyperkinetic movement disorders (41%), including dystonia and myoclonus as presenting symptoms. Multisystemic involvement was more prevalent in missense than biallelic loss-of-function variants (82-93% vs 53%; p = 0.029). Cerebellar atrophy was universal on MRI (100%), with cerebral atrophy or dentate and pontine T2 hyperintensities observed in 28%. Cross-sectional (n = 34) and longitudinal (n = 7) assessments consistently indicated mild-to-moderate progression of ataxia (SARA: 0.45/year). CoQ10 treatment led to improvement by clinical report in 14 of 30 patients, and by quantitative longitudinal assessments in 8 of 11 patients (SARA: -0.81/year). Explorative sample size calculations indicate that ≥48 patients per arm may suffice to demonstrate efficacy for interventions that reduce progression by 50%. INTERPRETATION: This study provides a deeper understanding of the disease, and paves the way toward large-scale natural history studies and treatment trials in COQ8A-ataxia. ANN NEUROL 2020;88:251-263.

Novel KCND3 Variant Underlying Nonprogressive Congenital Ataxia or SCA19/22 Disrupt KV4.3 Protein Expression and K+ Currents with Variable Effects on Channel Properties.

KCND3 encodes the voltage-gated potassium channel KV4.3 that is highly expressed in the cerebellum, where it regulates dendritic excitability and calcium influx. Loss-of-function KV4.3 mutations have been associated with dominant spinocerebellar ataxia (SCA19/22). By targeted NGS sequencing, we identified two novel KCND3 missense variants of the KV4.3 channel: p.S347W identified in a patient with adult-onset pure cerebellar syndrome and p.W359G detected in a child with congenital nonprogressive ataxia. Neuroimaging showed mild cerebellar atrophy in both patients. We performed a two-electrode voltage-clamp recording of KV4.3 currents in Xenopus oocytes: both the p.G345V (previously reported in a SCA19/22 family) and p.S347W mutants exhibited reduced peak currents by 50%, while no K+ current was detectable for the p.W359G mutant. We assessed the effect of the mutations on channel gating by measuring steady-state voltage-dependent activation and inactivation properties: no significant alterations were detected in p.G345V and p.S347W disease-associated variants, compared to controls. KV4.3 expression studies in HEK293T cells showed 53% (p.G345V), 45% (p.S347W) and 75% (p.W359G) reductions in mutant protein levels compared with the wildtype. The present study broadens the spectrum of the known phenotypes and identifies additional variants for KCND3-related disorders, outlining the importance of SCA gene screening in early-onset and congenital ataxia.

A Novel KCNA2 Variant in a Patient with Non-Progressive Congenital Ataxia and Epilepsy: Functional Characterization and Sensitivity to 4-Aminopyridine.

Kv1.2 channels, encoded by the KCNA2 gene, are localized in the central and peripheral nervous system, where they regulate neuronal excitability. Recently, heterozygous mutations in KCNA2 have been associated with a spectrum of symptoms extending from epileptic encephalopathy, intellectual disability, and cerebellar ataxia. Patients are treated with a combination of antiepileptic drugs and 4-aminopyridine (4-AP) has been recently trialed in specific cases. We identified a novel variant in KCNA2, E236K, in a Serbian proband with non-progressive congenital ataxia and early onset epilepsy, treated with sodium valproate. To ascertain the pathogenicity of E236K mutation and to verify its sensitivity to 4-AP, we transfected HEK 293 cells with Kv1.2 WT or E236K cDNAs and recorded potassium currents through the whole-cell patch-clamp. In silico analysis supported the electrophysiological data. E236K channels showed voltage-dependent activation shifted towards negative potentials and slower kinetics of deactivation and activation compared with Kv1.2 WT. Heteromeric Kv1.2 WT+E236K channels, resembling the condition of the heterozygous patient, confirmed a mixed gain- and loss-of-function (GoF/LoF) biophysical phenotype. 4-AP inhibited both Kv1.2 and E236K channels with similar potency. Homology modeling studies of mutant channels suggested a reduced interaction between the residue K236 in the S2 segment and the gating charges at S4. Overall, the biophysical phenotype of E236K channels correlates with the mild end of the clinical spectrum reported in patients with GoF/LoF defects. The response to 4-AP corroborates existing evidence that KCNA2-disorders could benefit from variant-tailored therapeutic approaches, based on functional studies.

Defective kinesin binding of TUBB2A causes progressive spastic ataxia syndrome resembling sacsinopathy.

Microtubules participate in fundamental cellular processes, including chromosomal segregation and cell division, migration and intracellular trafficking. Their proper function is required for correct central nervous system development and operative preservation, and mutations in genes coding tubulins, the constituting units of microtubules, underlie a family of neurodevelopmental and neurodegenerative diseases, collectively known as 'tubulinopathies', characterized by a wide range of neuronal defects resulting from defective proliferation, migration and function. Here, we causally link a previously unreported missense mutation in TUBB2A (c.1249G>A, p.D417N), encoding one of the neuron-specific ß-tubulin isotype II, to a disorder characterized by progressive spastic paraplegia, peripheral sensory-motor polyneuropathy and ataxia. Asp417 is a highly conserved solvent-exposed residue at the site mediating binding of kinesin superfamily motors. Impaired binding to KIF1A, a neuron-specific kinesin required for transport of synaptic vesicle precursors of the disease-associated TUBB2A mutant, was predicted by structural analyses and confirmed experimentally in vitro. We show that overexpression of TUBB2AD417N disrupts the mitotic spindle bipolarity and morphology and affects the M phase entry and length. Differently from the TUBB2AN247K and TUBB2AA248V, two mutants previously identified to affect neurodevelopment, TUBB2AD417N retains the ability to assemble into microtubules. Consistent with the differential clinical and structural impact, TUBB2AA248V does not drastically affect TUBB2A binding to KIF1A, nor mitotic spindle bipolarity. Overall, our data demonstrate a pathogenic role of the p.D417N substitution that is different from previously reported TUBB2A mutations and expand the phenotypic spectrum associated with mutations in this gene.

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.

Clinical and Genetic Overview of Paroxysmal Movement Disorders and Episodic Ataxias.

Paroxysmal movement disorders (PMDs) are rare neurological diseases typically manifesting with intermittent attacks of abnormal involuntary movements. Two main categories of PMDs are recognized based on the phenomenology: Paroxysmal dyskinesias (PxDs) are characterized by transient episodes hyperkinetic movement disorders, while attacks of cerebellar dysfunction are the hallmark of episodic ataxias (EAs). From an etiological point of view, both primary (genetic) and secondary (acquired) causes of PMDs are known. Recognition and diagnosis of PMDs is based on personal and familial medical history, physical examination, detailed reconstruction of ictal phenomenology, neuroimaging, and genetic analysis. Neurophysiological or laboratory tests are reserved for selected cases. Genetic knowledge of PMDs has been largely incremented by the advent of next generation sequencing (NGS) methodologies. The wide number of genes involved in the pathogenesis of PMDs reflects a high complexity of molecular bases of neurotransmission in cerebellar and basal ganglia circuits. In consideration of the broad genetic and phenotypic heterogeneity, a NGS approach by targeted panel for movement disorders, clinical or whole exome sequencing should be preferred, whenever possible, to a single gene approach, in order to increase diagnostic rate. This review is focused on clinical and genetic features of PMDs with the aim to (1) help clinicians to recognize, diagnose and treat patients with PMDs as well as to (2) provide an overview of genes and molecular mechanisms underlying these intriguing neurogenetic disorders.

TUBB Variants Underlying Different Phenotypes Result in Altered Vesicle Trafficking and Microtubule Dynamics.

Tubulinopathies are rare neurological disorders caused by alterations in tubulin structure and function, giving rise to a wide range of brain abnormalities involving neuronal proliferation, migration, differentiation and axon guidance. TUBB is one of the ten ß-tubulin encoding genes present in the human genome and is broadly expressed in the developing central nervous system and the skin. Mutations in TUBB are responsible for two distinct pathological conditions: the first is characterized by microcephaly and complex structural brain malformations and the second, also known as "circumferential skin creases Kunze type" (CSC-KT), is associated to neurological features, excess skin folding and growth retardation. We used a combination of immunocytochemical and cellular approaches to explore, on patients' derived fibroblasts, the functional consequences of two TUBB variants: the novel mutation (p.N52S), associated with basal ganglia and cerebellar dysgenesis, and the previously reported variant (p.M73T), linked to microcephaly, corpus callosum agenesis and CSC-KT skin phenotype. Our results demonstrate that these variants impair microtubule (MT) function and dynamics. Most importantly, our studies show an altered epidermal growth factor (EGF) and transferrin (Tf) intracellular vesicle trafficking in both patients' fibroblasts, suggesting a specific role of TUBB in MT-dependent vesicular transport.