ATP7A is a P-type ATPase copper transporter and a central component of the intracellular network to maintain copper (Cu) homeostasis, trafficking between the trans-Golgi network (TGN) and the cell periphery in response to intracellular Cu levels. Pathogenic ATP7A variants cause Menkes disease (MNK), occipital horn syndrome (OHS) and X-linked distal hereditary motor neuropathy (HMNX), with the clinical severity inversely related to residual ATP7A function. In MNK and OHS, variants result in loss of ATP7A function due to absent (MNK) or markedly reduced (OHS) protein levels, leading to abolished ATP7A trafficking and/or impaired Cu transport capacity. HMNX-associated variants are thought to retain partial Cu transport activity, although functional data remain limited. To date, three missense variants (p.T994I, p.P1386S, p.A991D) are established causes of HMNX, but the number of ATP7A variants reported in patients with a distal motor neuropathy phenotype is increasing, creating a need for functional assessment of variants of uncertain significance (VUS). We have developed a functional framework for evaluating HMNX-associated ATP7A variants. Using three patient fibroblast lines carrying the confirmed variants, we demonstrate reduced ATP7A TGN localization under low-Cu conditions and impaired capacity of these cell lines to maintain intracellular Cu levels. By applying these assays to five VUS (p.R703H, p.Y760C, p.A768G, p.Q990P, p.M1311V) identified in individuals from unsolved peripheral neuropathy families, we identify p.Q990P as a novel ATP7A variant in a patient with progressive peripheral motor neuropathy. This approach enables comparison of Cu trafficking and handling across ATP7A alleles, providing a functional framework to support diagnostic variant classification.

Background The classification of idiopathic inflammatory myopathies (IIMs) has evolved substantially with the identification of myositis-specific autoantibodies (MSAs). However, how contemporary classification frameworks are applied in routine clinical practice remains variable. This study provides a clinicopathological and serological characterization of IIMs from a national neuromuscular referral center in Cyprus. Methods We conducted a retrospective, single-center cohort study of adult patients diagnosed with IIM between 2007 and 2025. Patients were classified using a clinicopathological and serological approach into dermatomyositis (DM), immune-mediated necrotizing myopathy (IMNM), antisynthetase syndrome (ASyS), overlap myositis (OM), inclusion body myositis (IBM), and polymyositis (PM), with the 2017 EULAR/ACR criteria used as a reference framework and historical diagnoses re-evaluated. Results 36 patients were included. Final subtype distribution: IMNM in 14 patients (39%), DM in 7 (19%), IBM in 4 (11%), OM in 5 (14%), ASyS in 3 (8%), PM in 2 (6%). Only one patient remained non-classifiable. Autoantibody testing was performed in 26/36 patients; MSAs were detected in 19/26 tested (73%; 53% of the total cohort), most commonly anti-HMGCR, while anti-NT5C1A antibodies were detected in three additional patients. Muscle biopsy was performed in the majority of patients, reflecting historical diagnostic practices. Conclusion This study demonstrates real-world application of contemporary IIM classification, enabling accurate disease subtyping with direct implications for malignancy surveillance and interstitial lung disease monitoring, as well as reclassification of cases, most notably those previously labelled as PM. The findings further underscore the role of muscle biopsy, particularly when serology is negative, discordant, or unavailable.

OBJECTIVES:Newly diagnosed acetylcholine receptor (AChR)-positive generalized myasthenia gravis (gMG) is typically managed with corticosteroids, but long-term exposure carries substantial adverse effects. We evaluated the safety and effectiveness of rituximab (RTX) as the first and the only add-on to steroid therapy in AChR-positive gMG within two years from diagnosis. MATERIALS AND METHODS:We studied 24 adults who received RTX within 24 months from gMG diagnosis (mean 9.3 ± 7.1 months). This was a retrospective, single-center observational cohort study including consecutive eligible patients. Clinical outcomes (MG-ADL, MGFA-PIS), corticosteroid doses, and AChR antibody titers were assessed at baseline and 3, 6, and 12 months. RESULTS:Mean age at RTX initiation was 63 ± 15 years; 10 patients (41.7%) were ≥ 65 and 4 (17%) had thymoma. Follow-up averaged 15.5 ± 6.9 months. Patients were stratified as steroid responders (n = 16), partial responders (n = 5), and refractory (n = 3). MG-ADL scores improved from 1.9 ± 2.4-0.32 ± 0.78 at 6-months and 0.0 at 12 months. Corticosteroid dose decreased from 22.4 ± 9.0 mg/day at initiation to 11.4 ± 4.7 mg/day at 6 months and 7.7 ± 2.2 mg/day at 12 months. At 6-months, 61% achieved or maintained pharmacological remission and 30% minimal manifestations; at 12-months, 64% were in remission and 36% had minimal manifestations. AChR antibody titers declined by ~70% at both time points. No serious RTX-related adverse events requiring treatment discontinuation were observed. CONCLUSIONS:Our study highlights RTX's dual role both as a steroid-sparing maintenance therapy and as an early immunosuppressive option for steroid partial responders in addition to refractory AChR-positive gMG patients.

Introduction Efgartigimod, an FcRn antagonist that reduces circulating immunoglobulin G (IgG), is approved for the treatment of acetylcholine receptor (AChR) antibody-positive generalized myasthenia gravis (gMG). While its efficacy has been demonstrated in randomized clinical trials, real-world data describing longitudinal use and management of efgartigimod in routine clinical practice are still emerging.Methods We conducted a retrospective observational study across two neuromuscular referral centers, including adults with AChR antibody-positive gMG treated with intravenous efgartigimod between April 2023 and August 2025. Treatment was initiated across a range of real-world clinical scenarios, including refractory disease, non-crisis symptom relapse, corticosteroid tapering, maintenance of minimal-symptom expression, and bridging before thymectomy or initiation of conventional immunosuppression. Retreatment intervals were individualized based on clinical course. Clinical outcomes, corticosteroid use, serological markers, adjunctive therapies, and safety events were analyzed descriptively.Results Thirteen patients received a total of 52 efgartigimod treatment cycles. Most patients experienced repeated improvement in MG-Activities of Daily Living (MG-ADL) during treatment cycles, although clinical status varied over time. Corticosteroid doses were reduced in the majority of patients during follow-up. Episodes of clinically significant worsening occurred outside the setting of myasthenic crisis and were managed with established rescue therapies, after which efgartigimod could often be resumed. Treatment was generally well tolerated.Conclusion This study describes how efgartigimod is integrated into routine clinical practice as part of longitudinal disease management for AChR antibody-positive gMG. Real-world use encompassed diverse clinical scenarios and required individualized retreatment decisions and ongoing reassessment. These findings highlight the role of FcRn blockade as a flexible therapeutic option that complements established therapy in everyday care.

Serum antibodies to muscle specific kinase (MuSK) are present in a proportion of patients with acetylcholine receptor antibody seronegative myasthenia gravis (SNMG), but their reported frequencies in different populations vary. From 2002, serum samples were sent to Oxford for MuSK antibody testing from 35 centres in 6 continents. MuSK antibodies were identified in 143/465 (30.8%) previously untested SNMG sera. They were not identified in 147 adult acetylcholine receptor antibody positive patients or in 63 adult ocular MG patients. As expected from subsequent reports, the MuSK antibody positive patients differed in gender, clinical severity, bulbar predominance and treatment requirements from the 322/465 (69.2%) MuSK antibody negative SNMG patients, but they were improved following the more aggressive treatments received, although still showing bulbar predominance. Similar features were seen in the 42 childhood cases tested (17/42 (40%) MuSK-Ab positive from 18 centres); although numbers were small there was bulbar predominance that persisted after the treatments that were more extensive than those given to the juvenile SNMG patients. Although found in each continent, MuSK-MG showed an unexpected and surprising distribution. The frequency of MuSK-Abs within individual centres varied in the Northern Hemisphere with a clear north-south gradient from 0% to 47% in Europe and North America. There were fewer cohorts from Asia but data from 11 publications, including five from East and South East Asia, confirmed a latitudinal distribution of MuSK-Ab frequency consistent with a Gaussian curve peaking at 40 degrees North, and trending towards very low frequencies above this latitude in Europe and below this latitude in South East Asia. Thus, MuSK-Abs, that are predominantly IgG4 rather than IgG1 subclass, show a striking and unexpected relationship with latitude in the Northern Hemisphere. The results raise interesting questions regarding the environmental and genetic factors involved, with possible relevance also for the increasing number of IgG4 antibody-mediated neurological disorders, including forms of neuropathy and autoimmune encephalitis.

Charcot-Marie-Tooth disease type 4C is a demyelinating neuropathy caused by loss of function mutations in the SH3TC2 gene, that is highly expressed in myelinating Schwann cells. We generated and tested a clinical stage vector with a minimal human MPZ promoter driving expression of SH3TC2. Groups of 1-month old Sh3tc2-/- mice were treated with 3 different doses of AAV9-hMPZmini.SH3TC2.SV40pA or the formulation buffer by lumbar intrathecal injection. Outcomes were compared 8 weeks post injection by behavioral, electrophysiological, proteomics, morphological analysis and evaluation of tissue integrity and inflammatory responses. Vector biodistribution to the peripheral nerves and high rates of cell-specific therapeutic gene expression in Schwann cells resulted in significant therapeutic benefits in the CMT4C model. Treated mice showed improved motor performance in grip strength and motor nerve conduction velocities. Morphological analysis revealed significant improvement in g-ratios, myelin thickness and ratios of demyelinated fibers in lumbar roots and femoral nerves of treated mice. Proteomic profiles showed correction of muscle denervation associated pathobiochemical processes in treated mice. Not observed tissue toxicity or immune reactions in neural tissues or peripheral organs. This study provides proof of principle for dose-dependent effectiveness and safety of intrathecal AAV9-mediated gene replacement paving the way for clinical translation.

Gene therapy for neurological disorders is a rapidly evolving field with many preclinical studies and some successful clinical trials. Selectivity of cell specific targeting, efficiency and safety of administration route, and vector dosing are some of the most challenging aspects that need to be addressed for a successful treatment approach. In clinical practice, intravenous delivery is the most commonly used route of administration of viral vectors but adverse events led to the development of intrathecal delivery as a safer delivery method. Preclinical studies confirm the efficacy of intrathecally injected viral vectors for targeting both the central and peripheral nervous system at lower doses than those systemically-administered, limiting toxicity. Clinical applications of intrathecal gene therapy confirm efficacy and safety in patients. Further preclinical studies are needed to improve current vector capsid and payload technologies, while insights from ongoing clinical trials help to optimise patient selection and immunosuppressive protocols.

Charcot-Marie-Tooth (CMT) Type 1A, the most common inherited demyelinating peripheral neuropathy, is caused by PMP22 gene duplication, leading to over-production of PMP22 protein in Schwann cells. To treat CMT1A we developed a PMP22 gene silencing therapy using adeno-associated viral vectors (AAV9) to deliver a therapeutic miRNA expression cassette (U6.miR871) via lumbar intrathecal administration. A single injection produced long-term miR871 expression, triggered selective RNA interference against the PMP22 mRNA and subsequently lowered protein levels and improved disease manifestations in a humanized CMT1A model. To support clinical translation, we confirmed on-target specificity of miR871 for PMP22 in vitro, identified a safe and effective dosing range in mice, demonstrated absence of significant toxicity in rodents and non-human primates (NHPs), and performed a detailed AAV biodistribution study in a large animal model. We found vector biodistribution and miR871 expression in distal peripheral nerves, PMP22 target engagement in mice and NHPs, and silencing to levels expected to support normal myelination in humans. We identified the minimally efficacious to maximum tolerated dose range of AAV9.U6.miR871 in mice, and confirmed safety range in NHPs for extrapolation to anticipated clinical trials. Our study supports the scale-up potential of gene therapy to treat CMT1A and other demyelinating peripheral neuropathies.

Dominantly inherited intronic GAA repeat expansions in the fibroblast growth factor 14 gene have recently been shown to cause spinocerebellar ataxia 27B. Currently, the pathogenic threshold of (GAA)>= 300 repeat units is considered highly penetrant, while (GAA)250-299 is likely pathogenic with reduced penetrance. This study investigated the frequency of the GAA repeat expansion and the phenotypic profile in a Cypriot cohort with unresolved late-onset cerebellar ataxia. We analysed this trinucleotide repeat in 155 patients with late-onset cerebellar ataxia and 227 non-neurological disease controls. The repeat locus was examined by long-range PCR followed by fragment analysis using capillary electrophoresis, agarose gel electrophoresis and automated electrophoresis. A comprehensive comparison of all three electrophoresis techniques was conducted. Additionally, bidirectional repeat-primed PCRs and Sanger sequencing were carried out to confirm the absence of any interruptions or non-GAA motifs in the expanded alleles. The (GAA)>= 250 repeat expansion was present in 12 (7.7%) patients. The average age at disease onset was 60 +/- 13.5 years. The earliest age of onset was observed in a patient with a (GAA)287 repeat expansion, with ataxia symptoms appearing at 25 years of age. All patients with spinocerebellar ataxia 27B displayed symptoms of gait and appendicular ataxia. Nystagmus was observed in 41.7% of the patients, while 58.3% exhibited dysarthria. Our findings indicate that spinocerebellar ataxia 27B represents the predominant aetiology of autosomal dominant cerebellar ataxia in the Cypriot population, as this is the first dominant repeat expansion ataxia type detected in this population. Given our results and existing research, we propose including fibroblast growth factor 14 GAA repeat expansion testing as a first-tier genetic diagnostic approach for patients with late-onset cerebellar ataxia. The GAA repeat expansions in the FGF14 gene cause SCA27B. Livanos et al. found that 7.7% of unresolved ataxia Cypriot patients had the GAA repeat expansion. The primary clinical symptoms included gait, appendicular ataxia, nystagmus, and dysarthria. SCA27B is now considered one of the predominant causes of ataxia in Cyprus.

X-linked Charcot-Marie-Tooth disease (CMT1X) is an inherited demyelinating neuropathy caused by loss-of-function mutations in the GJB1 gene, encoding the gap junction protein connexin32 (Cx32). Cx32 plays a critical role in Schwann cell function and myelin formation in the peripheral nervous system. We have developed a gene replacement therapeutic approach using a humanized AAVrh10 vector construct expressing GJB1 under the control of the Schwann cell-specific human myelin protein zero (MPZ) promoter. Lumbar intrathecal injection of increasing AAVrh10-hMPZ.GJB1 doses (low: 1 ​× ​1011 vg, standard: 2 ​× ​1011 vg and high: 1 ​× ​1012 vg) into Gjb1-null mice resulted in adequate, dose-dependent biodistribution of the vector in anterior lumbar roots and peripheral nerves, as well as high rates of Schwann cell-specific Cx32 expression in the standard- and high-dose groups. Both standard and high vector doses provided significant therapeutic benefit evaluated by behavioural, electrophysiological and morphological outcomes. Intrathecal delivery of AAVrh10-hMPZ.GJB1 induced the production of anti-AAVrh10 antibodies at 6 weeks post-injection. However, no histopathological or inflammatory changes were observed in neural or peripheral tissues, besides a mild increase in inflammatory cell numbers in sciatic nerves of mice treated with the high dose only. This study provides proof of concept for a clinically translatable AAVrh10-mediated gene therapy approach for CMT1X.

BACKGROUND:Neuromuscular diseases (NMDs) comprise a range of genetic and acquired rare disorders that affect motor neurons, peripheral nerves, neuromuscular junctions and skeletal muscles, leading to significant impairments such as muscle weakness and fatigue resulting in functional limitations. This study aims to investigate the prevalence and severity of disease-related symptoms in adult patients with NMDs registered in the European Reference Network (ERN) EURO-NMD. A cross-sectional electronic survey was conducted with 1,253 participants who reported the severity of 28 symptoms, which were scored using multi-criteria decision analysis (MCDA). RESULTS:The results identified muscle fatigue, weakness and impaired physical function/activity as the most severe and prevalent symptoms in all NMD groups, followed by coordination and/or balance problems, muscle stiffness, mental fatigue, and pain. Notably, the analysis highlighted differences in symptom severity between disease subtypes and underlined the need for standardised patient-reported outcome measures (PROMs) to address the broad heterogeneity of NMDs. CONCLUSIONS:The findings stress the critical importance of capturing patient perspectives to guide clinical care, research priorities and therapeutic development. This work argues for the development of uniform PROMs to better assess disease impact, natural history and treatment efficacy, contributing to improved patient-centred care across diverse NMD populations.

Background and objectives Myasthenia gravis (MG) is a chronic antibody-mediated autoimmune neuromuscular disorder causing fluctuating muscle weakness due to autoantibodies that target the acetylcholine receptor (AChR) in the neuromuscular junction (NMJ). Despite extensive research, MG remains unpredictable and heterogeneous, underscoring the need for better insight into disease pathogenesis and clinical course. The objective of this study was to identify clinical and immunologic disease-related parameters that correlate with disease severity. Methods This large-scale, multicenter, cross-sectional study included prospectively recruited patients with MG and MG serum samples procured from reputable biobanks. Inclusion criteria required an MG clinical presentation and a positive anti-AChR immunoglobulin G (IgG) titer (>= 0.5 nM), confirmed by 2 independent tests. Enrolled patients underwent profiling of subunit-specific anti-AChR IgG titers. Disease severity patterns and their relation to age, sex, disease onset, thymic involvement, anti-AChR titer, and anti-AChR subunit immunodominance were investigated. Results The study included 513 patients with MG (50.5% female) with a median age of 64 years (range 14-98.5). Extended clinical data were available for 232 patients. Anti-AChR titers increased monotonically with disease severity (rho = 0.31, 95% CI 0.17-0.44, p < 0.0001) and were higher in female patients (median 14.6 nM vs 7.7 nM, 95% CI for Delta 2.1-7.3 nM, p < 0.0001) in whom higher antibody titers decreased monotonically with age. Male patients' titers were predominantly alpha-immunodominant (male 23.2% vs female 16.6%, 95% CI for Delta 0%-7.6%, p = 0.031), while female patients' titers were gamma-immunodominant (female 30% vs male 24.5%, 95% CI for Delta 2%-11%, p = 0.0027). Gamma immunodominance correlated with higher anti-AChR titer (rho = 0.25, 95% CI 0.16-0.33, p < 0.0001) and severe (Myasthenia Gravis Foundation of America IV-V) disease (44% vs 27%, 95% CI for Delta 4%-34%, p = 0.0141). Unsupervised principal component analysis disclosed 2 distinct disease endotypes: female patients with moderate to severe early-onset MG, gamma subunit immunodominance, high anti-AChR titers, and frequent thymic hyperplasia (endotype A) and the remaining patients, mostly patients with late-onset MG, alpha subunit immunodominance, and low anti-AChR titers (endotype B). Discussion Two distinct MG endotypes emerge based on sex, age, thymic involvement, autoantibody titer, and immunoglobulin AChR subunit immunodominance-suggesting different underlying etiologies with potential implications for sex-dependent precision medicine, women's health, and the development of new therapeutic modalities for MG.

Charcot-Marie-Tooth (CMT) disease refers to a diverse group of inherited and progressive neuropathies for which no approved treatments currently exist and management strategies remain limited to symptomatic interventions. Recent advances in gene therapy offer promising strategies to address CMT neuropathies. This review highlights key progress in developing gene silencing, replacement, or editing therapies for representative CMT types, and summarizes preclinical successes and translational challenges. Delivery technologies such as AAV vectors and nanoparticle systems have shown promise, but delivery limitations across the blood-nerve and -brain barriers, immune reactions and other potential toxicities, and scalability remain challenging. Advancing into the era of CMT treatments requires clinical readiness, which depends on optimizing therapeutic delivery, enhancing safety, and developing biomarkers for treatment monitoring.

CMT1A is the most common inherited demyelinating peripheral neuropathy, affecting ∼1.5 million people worldwide. CMT1A is caused by PMP22 gene duplication, leading to PMP22 protein over-expression, myelin sheath destabilization in Schwann cells, secondary axonal loss, and disability. Thus, Schwann cells are the primary target for CMT1A therapies, and we propose that the most direct route to CMT1A treatment will involve normalizing PMP22 levels in Schwann cells of peripheral nerves. To do this, we developed an RNAi gene therapy that reduces PMP22 expression and improves neuropathy in a CMT1A mouse model. Our therapeutic approach involves lumbar intrathecal delivery of an AAV9 vector carrying an engineered, PMP22-targeted miRNA (scAAV9-U6-miR871). This approach requires that AAV9 travel to Schwann cells at the distal peripheral nerves. While mouse nerves are only a few millimeters long, AAV9 would have to travel greater distances from the site of injection in a human (perhaps 1 m). Prior to this study, the ability of AAV to transduce Schwann cells of longer distal nerves following lumbar intrathecal delivery was unknown. To address this question, we performed a biodistribution and target engagement study in 20 non-human primates. We found the first evidence of an IT-delivered AAV9 construct effectively transducing the Schwann cells of 4 different distal nerves in a large animal model, and importantly, we measured PMP22 knockdown at both the transcript and protein levels at two timepoints (6 and 12 wks), demonstrating target engagement of our therapeutic, PMP22-targeted miR871 miRNA. We also found no significant adverse events in treated NHPs or off-targets in human cells. Our data support biodistribution to peripheral nerves, target engagement, safety, tolerability, and the therapeutic potential of scAAV9-U6-miR871. This study supports translation of CMT1A gene therapy and establishes a strategy to treat other demyelinating neuropathies affecting Schwann cells.