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Abstract Introduction COXPD23 is a rare mitochondrial disease caused by biallelic pathogenic variants in GTPBP3. We report on two siblings with a mild phenotype. Case reports The young boy presented with global developmental delay, ataxic gait and upper limbs tremor, and the older sister with absence seizures and hypertrophic cardiomyopathy. Respiratory chain impairment was confirmed in muscle. Discussion Reviewed cases point toward clustering around two prevalent phenotypes: an early‐onset presentation with severe fatal encephalopathy and a late milder presentation with global developmental delay/ID and cardiopathy, with the latter as, is the main feature. Our patients showed an intermediate phenotype with intrafamilial variability.

BackgroundMitochondria adjust their shape in response to the different energetic and metabolic requirements of the cell, through extremely dynamic fusion and fission events. Several highly conserved dynamin-like GTPases are involved in these processes and, among those, the OPA1 protein is a key player in the fusion of inner mitochondrial membranes. Hundreds of monoallelic or biallelic pathogenic gene variants have been described in OPA1, all associated with a plethora of clinical phenotypes without a straightforward genotype-phenotype correlation.MethodsHere we report two patients harboring novel de novo variants in OPA1. DNA of two patients was analyzed using NGS technology and the pathogenicity has been evaluated through biochemical and morphological studies in patient’s derived fibroblasts and in yeast model.ResultsThe two patients here reported manifest with neurological signs resembling Leigh syndrome, thus further expanding the clinical spectrum associated with variants in OPA1. In cultured skin fibroblasts we observed a reduced amount of mitochondrial DNA (mtDNA) and altered mitochondrial network characterized by more fragmented mitochondria. Modeling in yeast allowed to define the deleterious mechanism and the pathogenicity of the identified gene mutations.ConclusionWe have described two novel-single OPA1 mutations in two patients characterized by early-onset neurological signs, never documented, thus expanding the clinical spectrum of this complex syndrome. Moreover, both yeast model and patients derived fibroblasts showed mitochondrial defects, including decreased mtDNA maintenance, correlating with patients’ clinical phenotypes.

Hypomyelinating leukodystrophies (HLD) are a group of heterogeneous genetic disorders characterized by a deficit in myelin deposition during brain development. Specifically, 4H-Leukodystrophy is a recessive disease due to biallelic mutations in the POLR3A gene, which encodes one of the subunits forming the catalytic core of RNA polymerase III (PolIII). The disease also presents non-neurological signs such as hypodontia and hypogonadotropic hypogonadism. Here, we report the generation of a human induced pluripotent stem cell (hiPSC) line from fibroblasts of the first identified carrier of the biallelic POLR3A variants c.1802 T > A and c.4072G > A.

IntroductionRiboflavin transporter deficiency type 2 (RTD2) is a rare neurodegenerative autosomal recessive disease caused by mutations in the SLC52A2 gene encoding the riboflavin transporters, RFVT2. Riboflavin (Rf) is the precursor of FAD (flavin adenine dinucleotide) and FMN (flavin mononucleotide), which are involved in different redox reactions, including the energetic metabolism processes occurring in mitochondria. To date, human induced pluripotent stem cells (iPSCs) have given the opportunity to characterize RTD2 motoneurons, which reflect the most affected cell type. Previous works have demonstrated mitochondrial and peroxisomal altered energy metabolism as well as cytoskeletal derangement in RTD2 iPSCs and iPSC-derived motoneurons. So far, no attention has been dedicated to astrocytes.Results and discussionHere, we demonstrate that in vitro differentiation of astrocytes, which guarantee trophic and metabolic support to neurons, from RTD2 iPSCs is not compromised. These cells do not exhibit evident morphological differences nor significant changes in the survival rate when compared to astrocytes derived from iPSCs of healthy individuals. These findings indicate that differently from what had previously been documented for neurons, RTD2 does not compromise the morpho-functional features of astrocytes.

Abstract Riboflavin Transporter Deficiency (RTD) is a rare genetic, childhood-onset disease. This pathology has a relevant neurological involvement, being characterized by motor symptoms, ponto-bulbar paralysis and sensorineural deafness. Such clinical presentation is associated with muscle weakness and motor neuron (MN) degeneration, so that RTD is considered part of the MN disease spectrum. Based on previous findings demonstrating energy dysmetabolism and mitochondrial impairment in RTD induced Pluripotent Stem cells (iPSCs) and iPSC-derived MNs, here we address the involvement of intrinsic apoptotic pathways in disease pathogenesis using these patient-specific in vitro models by combined ultrastructural and confocal analyses. We show impaired neuronal survival of RTD iPSCs and MNs. Focused Ion Beam/Scanning Electron Microscopy (FIB/SEM) documents severe alterations in patients’ cells, including deranged mitochondrial ultrastructure, and altered plasma membrane and nuclear organization. Occurrence of aberrantly activated apoptosis is confirmed by immunofluorescence and TUNEL assays. Overall, our work provides evidence of a role played by mitochondrial dysfunction in RTD, and identifies neuronal apoptosis as a contributing event in disease pathogenesis, indicating intrinsic apoptosis pathways as possible relevant targets for more effective therapeutical approaches.

BackgroundCongenital myopathies are a group of heterogeneous inherited disorders, mainly characterized by early-onset hypotonia and muscle weakness. The spectrum of clinical phenotype can be highly variable, going from very mild to severe presentations. The course also varies broadly resulting in a fatal outcome in the most severe cases but can either be benign or lead to an amelioration even in severe presentations. Muscle biopsy analysis is crucial for the identification of pathognomonic morphological features, such as core areas, nemaline bodies or rods, nuclear centralizations and congenital type 1 fibers disproportion. However, multiple abnormalities in the same muscle can be observed, making more complex the myopathological scenario.Case presentationHere, we describe an Italian newborn presenting with severe hypotonia, respiratory insufficiency, inability to suck and swallow, requiring mechanical ventilation and gastrostomy feeding. Muscle biopsy analyzed by light microscopy showed the presence of vacuoles filled with glycogen, suggesting a metabolic myopathy, but also fuchsinophilic inclusions. Ultrastructural studies confirmed the presence of normally structured glycogen, and the presence of minirods, directing the diagnostic hypothesis toward a nemaline myopathy. An expanded Next Generation Sequencing analysis targeting congenital myopathies genes revealed the presence of a novel heterozygous c.965 T > A p. (Leu322Gln) variant in the ACTA1 gene, which encodes the skeletal muscle alpha-actin.ConclusionOur case expands the repertoire of molecular and pathological features observed in actinopathies. We highlight the value of ultrastructural examination to investigate the abnormalities detected at the histological level. We also emphasized the use of expanded gene panels in the molecular analysis of neuromuscular patients, especially for those ones presenting multiple bioptic alterations.

Abstract Beyond motor neuron degeneration, homozygous mutations in the survival motor neuron 1 (SMN1) gene cause multiorgan and metabolic defects in patients with spinal muscular atrophy (SMA). However, the precise biochemical features of these alterations and the age of onset in the brain and peripheral organs remain unclear. Using untargeted NMR-based metabolomics in SMA mice, we identify cerebral and hepatic abnormalities related to energy homeostasis pathways and amino acid metabolism, emerging already at postnatal day 3 (P3) in the liver. Through HPLC, we find that SMN deficiency induces a drop in cerebral norepinephrine levels in overt symptomatic SMA mice at P11, affecting the mRNA and protein expression of key genes regulating monoamine metabolism, including aromatic L-amino acid decarboxylase (AADC), dopamine beta-hydroxylase (DβH) and monoamine oxidase A (MAO-A). In support of the translational value of our preclinical observations, we also discovered that SMN upregulation increases cerebrospinal fluid norepinephrine concentration in Nusinersen-treated SMA1 patients. Our findings highlight a previously unrecognized harmful influence of low SMN levels on the expression of critical enzymes involved in monoamine metabolism, suggesting that SMN-inducing therapies may modulate catecholamine neurotransmission. These results may also be relevant for setting therapeutic approaches to counteract peripheral metabolic defects in SMA.

ABSTRACT Objective We created WiTNNess as a hybrid prospective/cross‐sectional observational study to simulate a clinical trial for infantile‐onset TNNT1 myopathy. Our aims were to identify populations for future trial enrollment, rehearse outcome assessments, specify endpoints, and refine trial logistics. Methods Eligible participants had biallelic pathogenic variants of TNNT1 and infantile‐onset proximal weakness without confounding conditions. The primary endpoint was ventilator‐free survival. “Thriving” was a secondary endpoint defined as the ability to swallow and grow normally without non‐oral feeding support. Endpoints of gross motor function included independent sitting and standing as defined by the Word Health Organization, a novel TNNT1 abbreviated motor score, and video mapping of limb movement. We recorded adverse events, concomitant medications, and indices of organ function to serve as comparators of safety in future trials. Results Sixteen children were enrolled in the aggregate cohort (6 prospective, 10 cross‐sectional; median census age 2.3 years, range 0.5–13.8). Median ventilator‐free survival was 20.2 months and probability of death or permanent mechanical ventilation was 100% by age 60 months. All six children (100%) in the prospective arm failed to thrive by age 12 months. Only 2 of 16 (13%) children in the aggregate cohort sat independently and none stood alone. Novel exploratory motor assessments also proved informative. Laboratory and imaging data suggest that primary manifestations of TNNT1 deficiency are restricted to skeletal muscle. Interpretation WiTNNess allowed us to streamline and economize the collection of historical control data without compromising scientific rigor, and thereby establish a sound operational framework for future clinical trials.

Abstract Background Mitochondrial disease is a degenerative, progressive, heterogeneous group of genetic disorders affecting children and adults. Mitochondrial disease is associated with morbidity and mortality, with predominantly neurological and neuromuscular symptoms including dystonia, weakness, encephalopathy, developmental delay and seizures. Seizures are one of the most common and severe manifestations of mitochondrial disease. These seizures are typically refractory to common anti-seizure therapies. There are no approved disease-modifying treatments for mitochondrial disease. Our objective was to conduct two systematic literature reviews to identify health-related quality of life (HRQoL), utilities, costs and healthcare resource use data in mitochondrial disease with associated seizures. Methods A range of databases and information sources were searched up to July 2022 to identify eligible studies. Search strategies included a range of variant terms for mitochondrial disease and HRQoL, utilities, cost and healthcare resource use outcomes. Two reviewers independently assessed articles against the eligibility criteria; studies were extracted by one reviewer and checked by a second. Risk of bias was assessed for studies reporting HRQoL data. Results were narratively assessed. Results Seven studies were eligible for the HRQoL and utilities review. The studies used different tools to report data, and despite the variability in methods, HRQoL scores across the studies showed moderate/severe disease in patients with mitochondrial disease with associated seizures. Parents of patients with mitochondrial disease with associated seizures were characterised by high total parenting stress. No studies reported utilities data. Two case reports and one retrospective review of medical records of children who died in hospital were eligible for the costs and resource use review. These provided limited information on the duration of hospital stay, in an intensive care unit (ICU), on mechanical ventilation. No studies reported costs data. Conclusion These reviews highlight the limited HRQoL, utilities, costs and resource use data and the variability of instruments used in mitochondrial disease with associated seizures. However, the data available indicate that mitochondrial disease with associated seizures affects patients’ and caregivers’ HRQoL alike. No robust conclusion can be drawn on the impact of mitochondrial disease with associated seizures on hospital or ICU length of stay. Trial registration PROSPERO: CRD42022345005.

Abstract Background The leukodystrophy “Vanishing White Matter” (VWM) is an orphan disease with neurological decline and high mortality. Currently, VWM has no approved treatments, but advances in understanding pathophysiology have led to identification of promising therapies. Several investigational medicinal products are either in or about to enter clinical trial phase. Clinical trials in VWM pose serious challenges, as VWM has an episodic disease course; disease phenotype is highly heterogeneous and predictable only for early onset; and study power is limited by the small patient numbers. To address these challenges and accelerate therapy delivery, the VWM Consortium, a group of academic clinicians with expertise in VWM, decided to develop a core protocol to function as a template for trials, to improve trial design and facilitate sharing of control data, while permitting flexibility regarding other trial details. Overall aims of the core protocol are to collect safety, tolerability, and efficacy data for treatment assessment and marketing authorization. Methods To develop the core protocol, the VWM Consortium designated a committee, including clinician members of the VWM Consortium, family and patient group advocates, and experts in statistics, clinical trial design and alliancing with industries. We drafted three age-specific protocols, to stratify into more homogeneous patient groups, of ages ≥ 18 years, ≥ 6 to

Abstract Background The development of e-health technologies for teleconsultation and exchange of knowledge is one of the core purposes of European Reference Networks (ERNs), including the ERN EURO-NMD for rare neuromuscular diseases. Within ERNs, the Clinical Patient Management System (CPMS) is a web-based platform that seeks to boost active collaboration within and across the network, implementing data sharing. Through CPMS, it is possible to both discuss patient cases and to make patients’ data available for registries and databases in a secure way. In this view, CPMS may be considered a sort of a temporary storage for patients’ data and an effective tool for data sharing; it facilitates specialists’ consultation since rare diseases (RDs) require multidisciplinary skills, specific, and outstanding clinical experience. Following European Union (EU) recommendation, and to promote the use of CPMS platform among EURO-NMD members, a twelve-month pilot project was set up to train the 15 Italian Health Care Providers (HCPs). In this paper, we report the structure, methods, and results of the teaching course, showing that tailored, ERN-oriented, training can significantly enhance the profitable use of the CPMS. Results Throughout the training course, 45 professionals learned how to use the many features of the CPMS, eventually opening 98 panels of discussion—amounting to 82% of the total panels included in the EURO-NMD. Since clinical, genetic, diagnostic, and therapeutic data of patients can be securely stored within the platform, we also highlight the importance of this platform as an effective tool to discuss and share clinical cases, in order to ease both case solving and data storing. Conclusions In this paper, we discuss how similar course could help implementing the use of the platform, highlighting strengths and weaknesses of e-health for ERNs. The expected result is the creation of a “map” of neuromuscular patients across Europe that might be improved by a wider use of CPMS.

Abstract Squamous cell carcinoma antigen recognized by T cells 3 (SART3) is an RNA-binding protein with numerous biological functions including recycling small nuclear RNAs to the spliceosome. Here, we identify recessive variants in SART3 in nine individuals presenting with intellectual disability, global developmental delay and a subset of brain anomalies, together with gonadal dysgenesis in 46,XY individuals. Knockdown of the Drosophila orthologue of SART3 reveals a conserved role in testicular and neuronal development. Human induced pluripotent stem cells carrying patient variants in SART3 show disruption to multiple signalling pathways, upregulation of spliceosome components and demonstrate aberrant gonadal and neuronal differentiation in vitro. Collectively, these findings suggest that bi-allelic SART3 variants underlie a spliceosomopathy which we tentatively propose be termed INDYGON syndrome (Intellectual disability, Neurodevelopmental defects and Developmental delay with 46,XY GONadal dysgenesis). Our findings will enable additional diagnoses and improved outcomes for individuals born with this condition.

Nuzzo, Russo, Errico, D’Amico et al. investigate neuroinflammation in forty-eight pediatric spinal muscular atrophy patients before and after Nusinersen treatment. They find signatures of neuroinflammation that are specifically associated with severe disease and show that Nusinersen therapy has neuro-immunomodulatory effects.

Microtubules are dynamic cytoskeletal structures involved in several cellular functions, such as intracellular trafficking, cell division and motility. More than other cell types, neurons rely on the proper functioning of microtubules to conduct their activities and achieve complex morphologies. Pathogenic variants in genes encoding for α and β-tubulins, the structural subunits of microtubules, give rise to a wide class of neurological disorders collectively known as “tubulinopathies” and mainly involving a wide and overlapping range of brain malformations resulting from defective neuronal proliferation, migration, differentiation and axon guidance. Although tubulin mutations have been classically linked to neurodevelopmental defects, growing evidence demonstrates that perturbations of tubulin functions and activities may also drive neurodegeneration. In this study, we causally link the previously unreported missense mutation p.I384N in TUBA1A, one of the neuron-specific α-tubulin isotype I, to a neurodegenerative disorder characterized by progressive spastic paraplegia and ataxia. We demonstrate that, in contrast to the p.R402H substitution, which is one of the most recurrent TUBA1A pathogenic variants associated to lissencephaly, the present mutation impairs TUBA1A stability, reducing the abundance of TUBA1A available in the cell and preventing its incorporation into microtubules. We also show that the isoleucine at position 384 is an amino acid residue, which is critical for α-tubulin stability, since the introduction of the p.I384N substitution in three different tubulin paralogs reduces their protein level and assembly into microtubules, increasing their propensity to aggregation. Moreover, we demonstrate that the inhibition of the proteasome degradative systems increases the protein levels of TUBA1A mutant, promoting the formation of tubulin aggregates that, as their size increases, coalesce into inclusions that precipitate within the insoluble cellular fraction. Overall, our data describe a novel pathogenic effect of p.I384N mutation that differs from the previously described substitutions in TUBA1A, and expand both phenotypic and mutational spectrum related to this gene.

Summary: Background: Efficacy and safety of onasemnogene abeparvovec (OA) for Spinal Muscular Atrophy infants under 7 months and

Abstract The study reports real world data in type 2 and 3 SMA patients treated for at least 2 years with nusinersen. Increase in motor function was observed after 12 months and during the second year. The magnitude of change was variable across age and functional subgroup, with the largest changes observed in young patients with higher function at baseline. When compared to natural history data, the difference between study cohort and untreated patients swas significant on both Hammersmith Functional Motor Scale and Revised Upper Limb Module both at 12 months and at 24 months.

POLR3B encodes the RPC2 subunit of RNA polymerase III. Pathogenic variants are associated with biallelic hypomyelinating leukodystrophy belonging to the POLR-related disorders. Recently, the association with dominant demyelinating neuropathy, classified as Charcot–Marie–Tooth syndrome type 1I (CMT1I), has been reported as well. Here we report on an additional patient presenting with developmental delay and generalized epilepsy, followed by the onset of mild pyramidal and cerebellar signs, vertical gaze palsy and subclinical demyelinating polyneuropathy. A new heterozygous de novo missense variant, c.1297C > G, p.Arg433Gly, in POLR3B was disclosed via trio-exome sequencing. In silico analysis confirms the hypothesis on the variant pathogenicity. Our research broadens both the genotypic and phenotypic spectrum of the autosomal-dominant POLR3B-related condition.

Abstract People worldwide use SARS-CoV-2 (COVID-19) visualizations to make life and death decisions about pandemic risks. Understanding how these visualizations influence risk perceptions to improve pandemic communication is crucial. To examine how COVID-19 visualizations influence risk perception, we conducted two experiments online in October and December of 2020 (N = 2549) where we presented participants with 34 visualization techniques (available at the time of publication on the CDC’s website) of the same COVID-19 mortality data. We found that visualizing data using a cumulative scale consistently led to participants believing that they and others were at more risk than before viewing the visualizations. In contrast, visualizing the same data with a weekly incident scale led to variable changes in risk perceptions. Further, uncertainty forecast visualizations also affected risk perceptions, with visualizations showing six or more models increasing risk estimates more than the others tested. Differences between COVID-19 visualizations of the same data produce different risk perceptions, fundamentally changing viewers’ interpretation of information.

Since 72% of rare diseases are genetic in origin and mostly paediatrics, genetic newborn screening represents a diagnostic "window of opportunity". Therefore, many gNBS initiatives started in different European countries. Screen4Care is a research project, which resulted of a joint effort between the European Union Commission and the European Federation of Pharmaceutical Industries and Associations. It focuses on genetic newborn screening and artificial intelligence-based tools which will be applied to a large European population of about 25.000 infants. The neonatal screening strategy will be based on targeted sequencing, while whole genome sequencing will be offered to all enrolled infants who may show early symptoms but have resulted negative at the targeted sequencing-based newborn screening. We will leverage artificial intelligence-based algorithms to identify patients using Electronic Health Records (EHR) and to build a repository "symptom checkers" for patients and healthcare providers. S4C will design an equitable, ethical, and sustainable framework for genetic newborn screening and new digital tools, corroborated by a large workout where legal, ethical, and social complexities will be addressed with the intent of making the framework highly and flexibly translatable into the diverse European health systems.

Abstract Background Leigh syndrome (LS) is a progressive neurodegenerative disorder associated with primary or secondary dysfunction of mitochondrial oxidative phosphorylation and is the most common mitochondrial disease in childhood. Numerous reports on the biochemical and molecular profiles of LS have been published, but there are limited studies on genetically confirmed large series. We reviewed the clinical, imaging, biochemical and molecular data of 122 patients with a diagnosis of LS collected in the Italian Collaborative Network of Mitochondrial Diseases database. Results Clinical picture was characterized by early onset of several neurological signs dominated by central nervous system involvement associated with both supra- and sub-tentorial grey matter at MRI in the majority of cases. Extraneurological organ involvement is less frequent in LS than expected for a mitochondrial disorder. Complex I and IV deficiencies were the most common biochemical diagnoses, mostly associated with mutations in SURF1 or mitochondrial-DNA genes encoding complex I subunits. Our data showed SURF1 as the genotype with the most unfavorable prognosis, differently from other cohorts reported to date. Conclusion We report on a large genetically defined LS cohort, adding new data on phenotype-genotype correlation, prognostic factors and possible suggestions to diagnostic workup.

Abstract Background X-linked myotubular myopathy (XLMTM) is a rare congenital myopathy resulting from pathogenic variants in the MTM1 gene. Affected male subjects typically present with severe hypotonia and respiratory distress at birth and they often require intensive supportive care. Long-term survivors are often non-ambulant, ventilator and feeding tube–dependent and they generally show additional organ manifestations, indicating that myotubularin does play a vital role in tissues other than muscle. For XLMTM several therapeutic strategies are under investigation. For XLMTM several therapeutic strategies are under investigation including a study of intravenous MTM1 gene transfer using a recombinant AAV8 vector of which has some concerns arises due to hepatotoxicity. Results We report prospective and retrospective clinical data of 12 XLMTM patients collected over a period of up to 10 years. In particular, we carried out a thorough review of the data about incidence and the course of hepatobiliary disease in our case series. Conclusions We demonstrate that hepatobiliary disease represents a common comorbidity of XLMTM that seems irrespective to age and diseases severity. We recommend to carefully explore and monitor the hepatobiliary function in XLMTM patients. We believe that a better understanding of the pathogenic mechanisms that induce hepatobiliary damage is essential to understand the fatal events that may occur in the gene therapy program.

Abstract Background Spinal muscular atrophy (SMA) is a neuromuscular disorder characterized by muscle atrophy and weakness. SMA type 1 (SMA1) is the most severe form: affected infants are unable to sit unaided; SMA type 2 (SMA2) children can sit, but are not able to walk independently. The Standards of Care has improved quality of life and the increasing availability of disease-modifying treatments is progressively changing the natural history; so, the clinical assessment of nutritional status has become even more crucial. Aims of this multicenter study were to present the growth pattern of treatment-naïve SMA1 and SMA2, and to compare it with the general growth standards. Results Body Weight (BW, kg) and Supine Length (SL, cm) were collected using a published standardized procedure. SMA-specific growth percentiles curves were developed and compared to the WHO reference data. We recruited 133 SMA1 and 82 SMA2 (48.8% females). Mean ages were 0.6 (0.4–1.6) and 4.1 (2.1–6.7) years, respectively. We present here a set of disease-specific percentiles curves of BW, SL, and BMI-for-age for girls and boys with SMA1 and SMA2. These curves show that BW is significantly lower in SMA than healthy peers, while SL is more variable. BMI is also typically lower in both sexes and at all ages. Conclusions These data on treatment-naïve patients point toward a better understanding of growth in SMA and could be useful to improve the clinical management and to assess the efficacy of the available and forthcoming therapies not only on motor function, but also on growth.

Abstract Background Amyotrophic Lateral Sclerosis (ALS) is a rare, progressive, and fatal neurodegenerative disease due to upper and lower motor neuron involvement with symptoms classically occurring in adulthood with an increasing recognition of juvenile presentations and childhood neurodegenerative disorders caused by genetic variants in genes related to Amyotrophic Lateral Sclerosis. The main objective of this study is detail clinical, radiological, neurophysiological, and genetic findings of a Brazilian cohort of patients with a recent described condition known as Spastic Tetraplegia and Axial Hypotonia (STAHP) due to SOD1 deficiency and compare with other cases described in the literature and discuss whether the clinical picture related to SOD1 protein deficiency is a new entity or may be represent a very early-onset form of Amyotrophic Lateral Sclerosis. Methods We conducted a case series report which included retrospective data from five Brazilian patients with SOD1 protein deficiency of a Brazilian reference center for Neuromuscular Disorders. Clinical data were obtained from a review of the medical records and descriptive statistics and variables were summarized using counts and percentages of the total population. Results All 5 patients presented with a childhood-onset neurodegenerative disorders characterized by spastic tetraplegia with axial hypotonia in all cases, with gestational history showing polyhydramnios in 4/5 and intrauterine growth restriction in 3/5 patients, with most patients initially presenting a normal motor development until the six month of life or during the first year followed by a rapidly progressive motor decline with severe dysphagia and respiratory insufficiency in all patients accompanied by cognitive impairment in 3/5 patients. All patients were homozygous for the c.335dupG (p.Cys112Trpfs*11) mutation in the SOD1 gene with completely decreased enzyme activity. Conclusions This case series is the biggest data collection of the new recent clinical entity described as Spastic Tetraplegia and Axial Hypotonia (STAHP) due to SOD1 deficiency.

Abstract Objective We report longitudinal data from 144 type III SMA pediatric and adult patients treated with nusinersen as part of an international effort. Methods Patients were assessed using Hammersmith Functional Motor Scale Expanded (HFMSE), Revised Upper Limb Module (RULM), and 6‐Minute Walk Test (6MWT) with a mean follow‐up of 1.83 years after nusinersen treatment. Results Over 75% of the 144 patients had a 12‐month follow‐up. There was an increase in the mean scores from baseline to 12 months on both HFMSE (1.18 points, p = 0.004) and RULM scores (0.58 points, p = 0.014) but not on the 6MWT (mean difference = 6.65 m, p = 0.33). When the 12‐month HFMSE changes in the treated cohort were compared to an external cohort of untreated patients, in all untreated patients older than 7 years, the mean changes were always negative, while always positive in the treated ones. To reduce a selection bias, we also used a multivariable analysis. On the HFMSE scale, age, gender, baseline value, and functional status contributed significantly to the changes, while the number of SMN2 copies did not contribute. The effect of these variables was less obvious on the RULM and 6MWT. Interpretation Our results expand the available data on the effect of Nusinersen on type III patients, so far mostly limited to data from adult type III patients.

Abstract Background With the increasing number of genomic sequencing studies, hundreds of genes have been implicated in neurodevelopmental disorders (NDDs). The rate of gene discovery far outpaces our understanding of genotype–phenotype correlations, with clinical characterization remaining a bottleneck for understanding NDDs. Most disease-associated Mendelian genes are members of gene families, and we hypothesize that those with related molecular function share clinical presentations. Methods We tested our hypothesis by considering gene families that have multiple members with an enrichment of de novo variants among NDDs, as determined by previous meta-analyses. One of these gene families is the heterogeneous nuclear ribonucleoproteins (hnRNPs), which has 33 members, five of which have been recently identified as NDD genes (HNRNPK, HNRNPU, HNRNPH1, HNRNPH2, and HNRNPR) and two of which have significant enrichment in our previous meta-analysis of probands with NDDs (HNRNPU and SYNCRIP). Utilizing protein homology, mutation analyses, gene expression analyses, and phenotypic characterization, we provide evidence for variation in 12 HNRNP genes as candidates for NDDs. Seven are potentially novel while the remaining genes in the family likely do not significantly contribute to NDD risk. Results We report 119 new NDD cases (64 de novo variants) through sequencing and international collaborations and combined with published clinical case reports. We consider 235 cases with gene-disruptive single-nucleotide variants or indels and 15 cases with small copy number variants. Three hnRNP-encoding genes reach nominal or exome-wide significance for de novo variant enrichment, while nine are candidates for pathogenic mutations. Comparison of HNRNP gene expression shows a pattern consistent with a role in cerebral cortical development with enriched expression among radial glial progenitors. Clinical assessment of probands (n = 188–221) expands the phenotypes associated with HNRNP rare variants, and phenotypes associated with variation in the HNRNP genes distinguishes them as a subgroup of NDDs. Conclusions Overall, our novel approach of exploiting gene families in NDDs identifies new HNRNP-related disorders, expands the phenotypes of known HNRNP-related disorders, strongly implicates disruption of the hnRNPs as a whole in NDDs, and supports that NDD subtypes likely have shared molecular pathogenesis. To date, this is the first study to identify novel genetic disorders based on the presence of disorders in related genes. We also perform the first phenotypic analyses focusing on related genes. Finally, we show that radial glial expression of these genes is likely critical during neurodevelopment. This is important for diagnostics, as well as developing strategies to best study these genes for the development of therapeutics.

Abstract Objective The aim of this paper was to report the 2‐year follow‐up in type I patients treated with Nusinersen and to assess whether possible changes in motor function are related to the subtype, age, or SMN2 copy number. Methods Sixty‐eight patients, with ages ranging from 0.20 to 15.92 years (mean: 3.96; standard deviation: +3.90) were enrolled in the study. All patients were assessed using the Children's Hospital of Philadelphia Infant Test of Neuromuscular Disorders (CHOP INTEND) and the developmental section of the Hammersmith Infant Neurological Examination (HINE‐2) at the time they started treatment and 12 and 24 months after that. Results For both CHOP and HINE‐2 repeated measures analysis of variance showed a significant difference (P

Early onset ataxia represents a group of heterogeneous neurological conditions typically characterized by motor disability. Speech problems are one of the main core features of ataxic syndromes, where automatic and computational characterization of speech impairment might represent a source of biomarkers for early screening and stratification of patients. The main contribution of this paper consists in proposing a novel hierarchical machine learning model (HMLM) to improve detection and assessment of dysarthria from a structured speech disturbance test. Performances are tested on a new audio dataset containing 10 seconds recordings of standardized clinical PATA test for 55 subjects: 18 healthy subjects and 37 with ataxia. Results show that the proposed HMLM achieves performances with an accuracy of about 90% at the first level (healthy vs patients) selecting an optimal subset of conventional features. In cascade, at the second level, speech disturbance severity (Low vs High) is assessed using deep learning feature extraction technique based on a VGG pre-trained network with maximum accuracy of about 80%. Both levels are processed through the majority voting ensemble technique testing Support Vector Machine (SVM), k-Nearest Neighbors (kNN), Decision Tree (DT) and Naïve Bayes (NB). In our results, the use of HMLM considerably outperforms the results achieved with a single machine learning or deep learning modeling. These outcomes demonstrate that the investigation of the PATA speech test through HMLM can be considered very promising. We also observed that the use of conventional feature extraction techniques and machine learning modeling seems to be a good solution for the diagnosis of patients with ataxia, while the deep learning approach is more appropriate for stratification of severity of dysarthria.

Abstract Objective Respiratory insufficiency is a major complication of Duchenne muscular dystrophy (DMD). Its progression shows considerable interindividual variability, which has been less thoroughly characterized and understood than in skeletal muscle. We collected pulmonary function testing (PFT) data from a large retrospective cohort followed at Centers collaborating in the Italian DMD Network. Furthermore, we analyzed PFT associations with different DMD mutation types, and with genetic variants in SPP1, LTBP4, CD40, and ACTN3, known to modify skeletal muscle weakness in DMD. Genetic association findings were independently validated in the Cooperative International Neuromuscular Research Group Duchenne Natural History Study (CINRG‐DNHS). Methods and Results Generalized estimating equation analysis of 1852 PFTs from 327 Italian DMD patients, over an average follow‐up time of 4.5 years, estimated that forced vital capacity (FVC) declined yearly by −4.2%, forced expiratory volume in 1 sec by −5.0%, and peak expiratory flow (PEF) by −2.9%. Glucocorticoid (GC) treatment was associated with higher values of all PFT measures (approximately + 15% across disease stages). Mutations situated 3’ of DMD intron 44, thus predicted to alter the expression of short dystrophin isoforms, were associated with lower (approximately −6%) PFT values, a finding independently validated in the CINRG‐DNHS. Deletions amenable to skipping of exon 51 and 53 were independently associated with worse PFT outcomes. A meta‐analysis of the two cohorts identified detrimental effects of SPP1 rs28357094 and CD40 rs1883832 minor alleles on both FVC and PEF. Interpretation These findings support GC efficacy in delaying respiratory insufficiency, and will be useful for the design and interpretation of clinical trials focused on respiratory endpoints in DMD.

Abstract Background Joubert syndrome is a recessive neurodevelopmental disorder characterized by clinical and genetic heterogeneity. Clinical hallmarks include hypotonia, ataxia, facial dysmorphism, abnormal eye movement, irregular breathing pattern cognitive impairment and, the molar tooth sign is the pathognomonic midbrain-hindbrain malformation on magnetic resonance imaging. The disorder is predominantly caused by biallelic mutations in more than 30 genes encoding proteins with a pivotal role in morphology and function of the primary cilium. Oligogenic inheritance or occurrence of genetic modifiers has been suggested to contribute to the variability of the clinical phenotype. We report on a family with peculiar clinical spectrum Joubert syndrome molecularly and clinically dissecting a complex phenotype, in which hypogonadism, pituitary malformation and growth hormone deficiency occur as major features. Case presentation A 7 year-old male was enrolled in a dedicated “Undiagnosed Patients Program” for a peculiar form of Joubert syndrome complicated by iris and retinochoroidal coloboma, hypogonadism pituitary malformation, and growth hormone deficiency. The molecular basis of the complex phenotype was investigated by whole exome sequencing. The concomitant occurrence of homozygosity for mutations in KIF7 and KIAA0556 was identified, and the assessment of major clinical features associated with mutations in these two genes provided evidence that these two independent events represent the cause underlying the complexity of the present clinical phenotype. Conclusion Beside the clinical variability of Joubert syndrome, co-occurrence of mutations in ciliopathy-associated genes may contribute to increase the clinical complexity of the trait.

Abstract Objective Biallelic variants in RARS1, encoding the cytoplasmic tRNA synthetase for arginine (ArgRS), cause a hypomyelinating leukodystrophy. This study aimed to investigate clinical, neuroradiological and genetic features of patients with RARS1‐related disease, and to identify possible genotype‐phenotype relationships. Methods We performed a multinational cross‐sectional survey among 20 patients with biallelic RARS1 variants identified by next‐generation sequencing techniques. Clinical data, brain MRI findings and genetic results were analyzed. Additionally, ArgRS activity was measured in fibroblasts of four patients, and translation of long and short ArgRS isoforms was quantified by western blot. Results Clinical presentation ranged from severe (onset in the first 3 months, usually with refractory epilepsy and early brain atrophy), to intermediate (onset in the first year with nystagmus and spasticity), and mild (onset around or after 12 months with minimal cognitive impairment and preserved independent walking). The most frequent RARS1 variant, c.5A>G, led to mild or intermediate phenotypes, whereas truncating variants and variants affecting amino acids close to the ArgRS active centre led to severe phenotypes. ArgRS activity was significantly reduced in three patients with intermediate and severe phenotypes; in a fourth patient with intermediate to severe presentation, we measured normal ArgRS activity, but found translation mainly of the short instead of the long ArgRS isoform. Interpretation Variants in RARS1 impair ArgRS activity and do not only lead to a classic hypomyelination presentation with nystagmus and spasticity, but to a wide spectrum, ranging from severe, early‐onset epileptic encephalopathy with brain atrophy to mild disease with relatively preserved myelination.

Abstract Genetic white matter disorders have heterogeneous etiologies and overlapping clinical presentations. We performed a study of the diagnostic efficacy of genome sequencing in 41 unsolved cases with prior exome sequencing, resolving an additional 14 from an historical cohort (n = 191). Reanalysis in the context of novel disease‐associated genes and improved variant curation and annotation resolved 64% of cases. The remaining diagnoses were directly attributable to genome sequencing, including cases with small and large copy number variants (CNVs) and variants in deep intronic and technically difficult regions. Genome sequencing, in combination with other methodologies, achieved a diagnostic yield of 85% in this retrospective cohort.

The aim of this study was to establish the possible effect of age, corticosteroid treatment and brain dystrophin involvement on motor function in young boys affected by Duchenne Muscular Dystrophy who were assessed using the North Star Ambulatory Assessment between the age of 4 and 7 years. The study includes 951 North Star assessments from 226 patients. Patients were subdivided according to age, to the site of mutation and therefore to the involvement of different brain dystrophin isoforms and to corticosteroids duration. There was a difference in the maximum North Star score achieved among patients with different brain dystrophin isoforms (p = 0.007). Patients with the involvement of Dp427, Dp140 and Dp71, had lower maximum NSAA scores when compared to those with involvement of Dp427 and Dp140 or of Dp427 only. The difference in the age when the maximum score was achieved in the different subgroups did not reach statistical significance. Using a linear regression model on all assessments we found that each of the three variables, age, site of mutation and corticosteroid treatment had an influence on the NSAA values and their progression over time. A second analysis, looking at 12-month changes showed that within this time interval the magnitude of changes was related to corticosteroid treatment but not to site of mutation. Our findings suggest that each of the considered variables appear to play a role in the progression of North Star scores in patients between the age of 4 and 7 years and that these should be carefully considered in the trial design of boys in this age range.

Microtubules (MTs) are dynamic components of the cell cytoskeleton involved in several cellular functions, such as structural support, migration and intracellular trafficking. Despite their high similarity, MTs have functional heterogeneity that is generated by the incorporation into the MT lattice of different tubulin gene products and by their post-translational modifications (PTMs). Such regulations, besides modulating the tubulin composition of MTs, create on their surface a “biochemical code” that is translated, through the action of protein effectors, into specific MT-based functions. This code, known as “tubulin code”, plays an important role in neuronal cells, whose highly specialized morphologies and activities depend on the correct functioning of the MT cytoskeleton and on its interplay with a myriad of MT-interacting proteins. In recent years, a growing number of mutations in genes encoding for tubulins, MT-interacting proteins and enzymes that post-translationally modify MTs, which are the main players of the tubulin code, have been linked to neurodegenerative processes or abnormalities in neural migration, differentiation and connectivity. Nevertheless, the exact molecular mechanisms through which the cell writes and, downstream, MT-interacting proteins decipher the tubulin code are still largely uncharted. The purpose of this review is to describe the molecular determinants and the readout mechanisms of the tubulin code, and briefly elucidate how they coordinate MT behavior during critical neuronal events, such as neuron migration, maturation and axonal transport.

Intrathecal delivery of Nusinersen–an antisense oligonucleotide that promotes survival motor neuron (SMN) protein induction–is an approved therapy for spinal muscular atrophy (SMA). Here, we employed nuclear magnetic resonance (NMR) spectroscopy to longitudinally characterize the unknown metabolic effects of Nusinersen in the cerebrospinal fluid (CSF) of SMA patients across disease severity. Modulation of amino acid metabolism is a common denominator of biochemical changes induced by Nusinersen, with distinct downstream metabolic effects according to disease severity. In severe SMA1 patients, Nusinersen stimulates energy-related glucose metabolism. In intermediate SMA2 patients, Nusinersen effects are also related to energy homeostasis but involve ketone body and fatty acid biosynthesis. In milder SMA3 patients, Nusinersen mainly modulates amino acid metabolism. Moreover, Nusinersen modifies the CSF metabolome of a more severe clinical group towards the profile of untreated SMA patients with milder disease. These findings reveal disease severity-specific neurometabolic signatures of Nusinersen treatment, suggesting a selective modulation of peripheral organ metabolism by this CNS-directed therapy in severe SMA patients.

X-linked myotubular myopathy (XLMTM) is a severe form of centronuclear myopathy, characterized by generalized weakness and respiratory insufficiency, associated with pathogenic variants in the MTM1 gene. NGS targeted sequencing on the DNA of a three-month-old child affected by XLMTM identified the novel hemizygous MTM1 c.1261-5T>G intronic variant, which interferes with the normal splicing process, generating two different abnormal transcripts simultaneously expressed in the patient’s muscular cells. The first aberrant transcript, induced by the activation of a cryptic splice site in intron 11, includes four intronic nucleotides upstream of exon 12, resulting in a shift in the transcript reading frame and introducing a new premature stop codon in the catalytic domain of the protein (p.Arg421SerfsTer7). The second aberrant MTM1 transcript, due to the lack of recognition of the 3′ acceptor splice site of intron 11 from the spliceosome complex, leads to the complete skipping of exon 12. We expanded the genotypic spectrum of XLMTM underlying the importance of intron–exons boundaries sequencing in male patients affected by XLMTM.

Duchenne muscular dystrophy (DMD) is a rare genetic disease due to dystrophin gene mutations which cause progressive weakness and muscle wasting. Circadian rhythm coordinates biological processes with the 24-h cycle and it plays a key role in maintaining muscle functions, both in animal models and in humans. We explored expression profiles of circadian circuit master genes both in Duchenne muscular dystrophy skeletal muscle and in its animal model, the mdx mouse. We designed a customized, mouse-specific Fluidic-Card-TaqMan-based assay (Fluid-CIRC) containing thirty-two genes related to circadian rhythm and muscle regeneration and analyzed gastrocnemius and tibialis anterior muscles from both unexercised and exercised mdx mice. Based on this first analysis, we prioritized the 7 most deregulated genes in mdx mice and tested their expression in skeletal muscle biopsies from 10 Duchenne patients. We found that CSNK1E, SIRT1, and MYOG are upregulated in DMD patient biopsies, consistent with the mdx data. We also demonstrated that their proteins are detectable and measurable in the DMD patients’ plasma. We suggest that CSNK1E, SIRT1, and MYOG might represent exploratory circadian biomarkers in DMD.

Autosomal recessive cerebellar ataxias (ARCAs) form an ultrarare yet expanding group of neurodegenerative multisystemic diseases affecting the cerebellum and other neurological or non-neurological systems. With the advent of targeted therapies for ARCAs, disease registries have become a precious source of real-world quantitative and qualitative data complementing knowledge from preclinical studies and clinical trials. Here, we review the ARCA Registry, a global collaborative multicenter platform (>15 countries, >30 sites) with the overarching goal to advance trial readiness in ARCAs. It presents a good clinical practice (GCP)- and general data protection regulation (GDPR)-compliant professional-reported registry for multicenter web-based capture of cross-center standardized longitudinal data. Modular electronic case report forms (eCRFs) with core, extended, and optional datasets allow data capture tailored to the participating site's variable interests and resources. The eCRFs cover all key data elements required by regulatory authorities [European Medicines Agency (EMA)] and the European Rare Disease (ERD) platform. They capture genotype, phenotype, and progression and include demographic data, biomarkers, comorbidity, medication, magnetic resonance imaging (MRI), and longitudinal clinician- or patient-reported ratings of ataxia severity, non-ataxia features, disease stage, activities of daily living, and (mental) health status. Moreover, they are aligned to major autosomal-dominant spinocerebellar ataxia (SCA) and sporadic ataxia (SPORTAX) registries in the field, thus allowing for joint and comparative analyses not only across ARCAs but also with SCAs and sporadic ataxias. The registry is at the core of a systematic multi-component ARCA database cluster with a linked biobank and an evolving study database for digital outcome measures. Currently, the registry contains more than 800 patients with almost 1,500 visits representing all ages and disease stages; 65% of patients with established genetic diagnoses capture all the main ARCA genes, and 35% with unsolved diagnoses are targets for advanced next-generation sequencing. The ARCA Registry serves as the backbone of many major European and transatlantic consortia, such as PREPARE, PROSPAX, and the Ataxia Global Initiative, with additional data input from SPORTAX. It has thus become the largest global trial-readiness registry in the ARCA field.

Abstract Background Hyperornithinemia–hyperammonemia–homocitrullinuria (HHH) syndrome is a rare disorder of urea cycle characterized by progressive pyramidal and cerebellar dysfunction, whose pathophysiology is not yet fully understood. Here we describe the spectrum of the long fibers involvement in HHH syndrome, attempting a correlation between clinical, electrophysiological and neuro-radiological data. Methods Nine HHH patients were longitudinally evaluated by clinical examination, neurophysiological assessment including motor (MEPs), somato-sensory evoked potentials (PESS) and nerve conduction velocity (NCV), brain and spinal cord MRI Results All patients had pyramidal dysfunction and 3/9 an overt spastic paraplegia. Mild to moderate cerebellar signs were found in 7/9, intellectual disability in 8/9. At lower limbs, MEPs resulted abnormal in 7/8 patients and PESS in 2/8; peripheral sensory-motor neuropathy was found in 1/9. MRI documented atrophic changes in supra-tentorial brain regions in 6/9 patients, cerebellum in 6/9, spinal cord in 3/7. Conclusions A predominant corticospinal dysfunction is evident in HHH syndrome, along with milder cerebellar signs, intellectual disability of variable degree and rare peripheral neuropathy. Phenotypical similarities with other disorders affecting the urea cycle (argininemia and pyrroline-5-carboxylate synthetase deficiency) suggest possible common mechanisms contributing in the maintenance of the corticospinal tract integrity. HHH syndrome phenotype largely overlaps with complex Hereditary Spastic Paraplegias (HSPs), in the list of which it should be included, emphasizing the importance to screen all the unsolved cases of HSPs for metabolic biomarkers.

Spinal muscular atrophy (SMA) is a severe neuromuscular disorder caused by biallelic loss or pathogenic variants in the SMN1 gene. Copy number and modifier intragenic variants in SMN2, an almost identical paralog gene of SMN1, are known to influence the amount of complete SMN proteins. Therefore, SMN2 is considered the main phenotypic modifier of SMA, although genotype–phenotype correlation is not absolute. We present eleven unrelated SMA patients with milder phenotypes carrying the c.859G>C-positive modifier variant in SMN2. All were studied by a specific NGS method to allow a deep characterization of the entire SMN region. Analysis of two homozygous cases for the variant allowed us to identify a specific haplotype, Smn2-859C.1, in association with c.859G>C. Two other cases with the c.859G>C variant in their two SMN2 copies showed a second haplotype, Smn2-859C.2, in cis with Smn2-859C.1, assembling a more complex allele. We also identified a previously unreported variant in intron 2a exclusively linked to the Smn2-859C.1 haplotype (c.154-1141G>A), further suggesting that this region has been ancestrally conserved. The deep molecular characterization of SMN2 in our cohort highlights the importance of testing c.859G>C, as well as accurately assessing the SMN2 region in SMA patients to gain insight into the complex genotype–phenotype correlations and improve prognostic outcomes.

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.

Riboflavin transporter deficiency (RTD) is a rare genetic disorder characterized by motor, sensory and cranial neuropathy. This childhood-onset neurodegenerative disease is caused by biallelic pathogenic variants in either SLC52A2 or SLC52A3 genes, resulting in insufficient supply of riboflavin (vitamin B2) and consequent impairment of flavoprotein-dependent metabolic pathways. Current therapy, empirically based high-dose riboflavin supplementation, ameliorates the progression of the disease, even though response to treatment is variable and partial. Recent studies have highlighted concurrent pathogenic contribution of cellular energy dysmetabolism and cytoskeletal derangement. In this context, patient specific RTD models, based on induced pluripotent stem cell (iPSC) technology, have provided evidence of redox imbalance, involving mitochondrial and peroxisomal dysfunction. Such oxidative stress condition likely causes cytoskeletal perturbation, associated with impaired differentiation of RTD motor neurons. In this review, we discuss the most recent findings obtained using different RTD models. Relevantly, the integration of data from innovative iPSC-derived in vitro models and invertebrate in vivo models may provide essential information on RTD pathophysiology. Such novel insights are expected to suggest custom therapeutic strategies, especially for those patients unresponsive to high-dose riboflavin treatments.

Abstract Loss‐of‐function mutations in APOPT1, a gene exclusively found in higher eukaryotes, cause a characteristic type of cavitating leukoencephalopathy associated with mitochondrial cytochrome c oxidase (COX) deficiency. Although the genetic association of APOPT1 pathogenic variants with isolated COX defects is now clear, the biochemical link between APOPT1 function and COX has remained elusive. We investigated the molecular role of APOPT1 using different approaches. First, we generated an Apopt1 knockout mouse model which shows impaired motor skills, e.g., decreased motor coordination and endurance, associated with reduced COX activity and levels in multiple tissues. In addition, by achieving stable expression of wild‐type APOPT1 in control and patient‐derived cultured cells we ruled out a role of this protein in apoptosis and established instead that this protein is necessary for proper COX assembly and function. On the other hand, APOPT1 steady‐state levels were shown to be controlled by the ubiquitination–proteasome system (UPS). Conversely, in conditions of increased oxidative stress, APOPT1 is stabilized, increasing its mature intramitochondrial form and thereby protecting COX from oxidatively induced degradation.

The cytoskeletal network plays a crucial role in the differentiation, morphogenesis, function and homeostasis of the nervous tissue, so that alterations in any of its components may lead to neurodegenerative diseases. Riboflavin transporter deficiency (RTD), a childhood-onset disorder characterized by degeneration of motor neurons (MNs), is caused by biallelic mutations in genes encoding the human riboflavin (RF) transporters. In a patient-specific induced pluripotent stem cells (iPSCs) model of RTD, we recently demonstrated altered cell-cell contacts, energy dysmetabolism and redox imbalance. The present study focuses on cytoskeletal composition and dynamics associated to RTD, utilizing patients' iPSCs and derived MNs. Abnormal expression and distribution of α- and β-tubulin (α- and β-TUB), as well as imbalanced tyrosination of α-TUB, accompanied by an impaired ability to re-polymerize after nocodazole treatment, were found in RTD patient-derived iPSCs. Following differentiation, MNs showed consistent changes in TUB content, which was associated with abnormal morphofunctional features, such as neurite length and Ca2+ homeostasis, suggesting impaired differentiation. Beneficial effects of RF supplementation, alone or in combination with the antioxidant molecule N-acetyl cystine (NAC), were assessed. RF administration resulted in partially improved cytoskeletal features in patients' iPSCs and MNs, suggesting that redundancy of transporters may rescue cell functionality in the presence of adequate concentrations of the vitamin. Moreover, supplementation with NAC was demonstrated to be effective in restoring all the considered parameters, when used in combination with RF, thus supporting the therapeutic use of both compounds.

While rare diseases (RDs) are by definition of low prevalence, the total number of patients suffering from an RD is high, and the majority of them have neurologic manifestations, involving central, peripheral nerve, and muscle. In 2017, 24 European Reference Networks (ERNs), each focusing on a specific group of rare or low-prevalence complex diseases, were formed to improve the care for patients with an RD. One major aim is to have “the knowledge travel instead of the patient,” which has been put into practice by the implementation of the Clinical Patient Management System (CPMS) that enables clinicians to perform pan-European virtual consultations. The European Reference Network for Rare Neurological Diseases (ERN-RND) provides an infrastructure for knowledge sharing and care coordination for patients affected by a rare neurological disease (RND) involving the most common central nervous system pathological conditions. It covers the following disease groups: (i) Cerebellar Ataxias and Hereditary Spastic Paraplegias; (ii) Huntington's disease and Other Choreas; (iii) Frontotemporal dementia; (iv) Dystonia, (non-epileptic) paroxysmal disorders, and Neurodegeneration with Brain Iron Accumulation; (v) Leukoencephalopathies; and (vi) Atypical Parkinsonian Syndromes. At the moment, it unites 32 expert centers and 10 affiliated partners in 21 European countries, as well as patient representatives, but will soon cover nearly all countries of the European Union as a result of the ongoing expansion process. Disease expert groups developed and consented on diagnostic flowcharts and disease scales to assess the different aspects of RNDs. ERN-RND has started to discuss diagnostically unclear patients in the CPMS, is one of four ERNs that serve as foundation of Solve-RD, and has established an RND training and education program. The network will facilitate trial readiness through the establishment of an ERN-RND registry with a minimal data of all patients seen at the ERN-RND centers, thus providing a unique overview of existing genotype-based cohorts. The overall aim of the ERNs is to improve access for patients with RDs to quality diagnosis, care, and treatment. Based on this objective, ERNs are monitored by the European Commission on a regular basis to provide transparency and reassurance to the RD community and the general public.

IntroductionThe aim of this study was to report 36-month longitudinal changes using the North Star Ambulatory Assessment (NSAA) in ambulant patients affected by Duchenne muscular dystrophy amenable to skip exons 44, 45, 51 or 53.Materials and methodsWe included 101 patients, 34 had deletions amenable to skip exon 44, 25 exon 45, 19 exon 51, and 28 exon 53, not recruited in any ongoing clinical trials. Five patients were counted to skip exon 51 and 53 since they had a single deletion of exon 52.ResultsThe difference between subgroups (skip 44, 45, 51 and 53) was significant at 12 (p = 0.043), 24 (p = 0.005) and 36 months (p≤0.001).DiscussionMutations amenable to skip exons 53 and 51 had lower baseline values and more negative changes than the other subgroups while those amenable to skip exon 44 had higher scores both at baseline and at follow up.ConclusionOur results confirm different progression of disease in subgroups of patients with deletions amenable to skip different exons. This information is relevant as current long term clinical trials are using the NSAA in these subgroups of mutations.

PCDH19 clustering epilepsy (PCDH19-CE) is a genetic disease characterized by a heterogeneous phenotypic spectrum ranging from focal epilepsy with rare seizures and normal cognitive development to severe drug-resistant epilepsy associated with intellectual disability and autism. Unfortunately, little is known about the pathogenic mechanism underlying this disease and an effective treatment is lacking. Studies with zebrafish and murine models have provided insights on the function of PCDH19 during brain development and how its altered function causes the disease, but these models fail to reproduce the human phenotype. Induced pluripotent stem cell (iPSC) technology has provided a complementary experimental approach for investigating the pathogenic mechanisms implicated in PCDH19-CE during neurogenesis and studying the pathology in a more physiological three-dimensional (3D) environment through the development of brain organoids. We report on recent progress in the development of human brain organoids with a particular focus on how this 3D model may shed light on the pathomechanisms implicated in PCDH19-CE.

Inositol polyphosphate-5-phosphatase K [INPP5K (MIM: 607875)] acts as a PIP3 5-phosphatase and regulates actin cytoskeleton, insulin, and cell migration. Biallelic pathogenic variants in INPP5K have recently been reported in patients affected by a form of muscular dystrophy with childhood onset. Affected patients have limb girdle muscle weakness, often associated with bilateral cataracts, short stature, and intellectual disability. Here we report four patients affected by INPP5K-related muscle dystrophy, who were apparently unrelated but originated from the same geographical area in South Italy. These patients manifest a recognizable phenotype characterized by early onset muscular dystrophy associated with short stature and intellectual disability. All affected subjects were homozygous or compound heterozygous for the c.67G > A (p.Val23Met) missense change and shared a common haplotype, indicating the occurrence of a founder effect.