Annals of Clinical and Translational Neurology

Background and Objectives: Deep sequencing of brain tissue in the research setting has established that mosaic variants are a major cause of malformations of cortical development (MCDs) and epilepsy. However, genetic testing in the clinical setting primarily detects germline variants using clinically accessible samples. We aimed to determine the diagnostic yield and clinical utility of deep sequencing in the clinical setting to identify pathogenic mosaic variants for this population. Methods: We performed a retrospective cohort analysis of individuals at Boston Children's Hospital with MCDs with or without epilepsy who received clinical deep sequencing between September 2017 and February 2026. Demographic, clinical, and genetic testing data were abstracted from the medical record. For individuals without systemic features, we classified brain tissue as an affected tissue sample. For individuals with systemic features, we classified brain or relevant non-brain tissue as affected. The primary outcome was the diagnostic yield of clinical deep sequencing performed using affected vs unaffected tissue samples. The secondary outcome was the clinical utility of genetic diagnoses. Results: Our cohort included 37 individuals (19/37 (51%) female, 18/37 (49%) male) with MCDs, of whom 35/37 (95%) had epilepsy (25 with brain tissue samples available from epilepsy surgery) and 8/37 (22%) had systemic features. Most (35/37 (95%)) had dysplasia phenotypes on MRI and 12/27 (44%) with pathology available had Focal Cortical Dysplasia Type I or II. The diagnostic yield was 53% (17/32; 16 mosaic and 1 germline variant) when clinical deep sequencing was performed using an affected tissue sample vs 0% (0/6) using an unaffected tissue sample (p=0.016). Of the diagnosed cases, 13/17 (76%) had testing performed on brain tissue (1 with systemic features) and 4/17 (24%) on non-brain tissue (3 buccal and 1 duodenal tissue, all with systemic features). All but one diagnosis involved the mTOR pathway. All diagnoses had clinical utility. Discussion: Clinical deep sequencing, when performed using an affected tissue sample, has high diagnostic yield and clinical utility for individuals with MCDs, especially dysplasia phenotypes, and epilepsy. Our findings support implementation of clinical deep sequencing for this population, especially as the genetic diagnoses have implications for emerging precision therapies.

Objective: In KCNQ2-related disorders (KCNQ2-RD), neurodevelopmental outcome remains variable despite established genotype-phenotype correlations. Our aim is to improve counselling, by developing and internally validating models predicting neurodevelopmental outcomes based on early clinical and genetic features, universally available to clinicians. Methods: We conducted a multicentric retrospective cohort study including 277 individuals carrying a (likely) pathogenic variant in the KCNQ2 gene, with a minimum follow-up age of three years. Mosaic variants were excluded. The cohort was randomly split into training (70%) and validation (30%) sets. Ten expert selected parameters with minimal missing data were used to train random forest models to predict (i) dichotomous outcomes and (ii) three-category outcomes for cognition, language, and gross motor milestones. Results: Models incorporated seven clinical (neonatal hypotonia, EEG characteristics, age at seizure onset, seizure type, and seizure frequency at onset, prematurity, and sex) and three genetic variables (de novo status, exon localisation, and position within known KCNQ2-developmental and epileptic encephalopathy (DEE) hotspot regions). Dichotomous models showed the highest predictive performance, with accuracies of 0.83 for normal vs. mild-profound intellectual disability (ID), 0.83 for achievement of first words, and 0.86 for achievement of independent walking. Three category models remained clinically informative: accuracies were 0.79 for normal vs. mild vs. moderate-profound ID, 0.70 for first words [≤]16 months vs. >16 months vs. never, and 0.71 for independent walking [≤]18 months vs. >18 months vs. never. The strongest predictors for adverse neurodevelopmental outcomes were presence of hypotonia at birth, seizure onset within the first day of life, multiple seizures per day at onset, tonic seizures at onset, a burst-suppression pattern on EEG at onset, the presence of a de novo variant, and variant location within exons 6-7. Significance: These prediction models demonstrate the feasibility of early prognostication in KCNQ2-RD and support future prospective external validation. They enable more accurate individualised counselling by integrating clinical and genetic information readily available at time of genetic diagnosis and provide an objective foundation for early intervention planning and future precision medicine trial stratification.

Background Heterozygous GAA-TTC repeat expansions in the FGF14 gene cause spinocerebellar ataxia 27B (SCA27B), a late-onset cerebellar ataxia (LOCA) increasingly recognized in populations of European ancestry. Objective To characterize the clinical and genetic features of SCA27B and compare its phenotype and progression with those of unexplained idiopathic LOCA (ILOCA) in a large multicenter cohort. Methods We conducted a nationwide multicenter study of patients with unexplained LOCA recruited from 21 Spanish hospitals. FGF14 GAA-TTC repeat expansions were analyzed using a standardized molecular protocol. Clinical, neuroimaging, and longitudinal data were compared between patients with genetically confirmed SCA27B and those with ILOCA. Longitudinal changes in the Scale for the Assessment and Rating of Ataxia (SARA) were analyzed using linear mixed-effects models. Results Among 430 patients with unexplained LOCA, 111 (25.8%) carried FGF14 expansions >250 repeats, and 16 additional patients (3.7%) had intermediate expansions of 200-249 repeats. Including 90 externally recruited genetically confirmed cases, the SCA27B cohort comprised 217 patients. Compared with 283 patients with ILOCA, SCA27B patients had a later age at onset (median 63 vs 55 years, p<0.001), more frequent episodic symptoms (45.2% vs 12.7%), and more frequent oculomotor abnormalities (82.5% vs 71.6%). Downbeat nystagmus was particularly frequent in SCA27B (67.4% vs 32.9%). Conversely, extracerebellar features, including pyramidal and sensory signs, were more common in ILOCA. Among patients receiving 4-aminopyridine (4-AP), subjective improvement was reported more frequently in SCA27B than ILOCA (72.5% vs 36.0%). Longitudinal SARA analysis showed similar rates of progression between groups (SCA27B slope 0.37 vs ILOCA 0.35 points/year; interaction p=0.56). Conclusions SCA27B was a frequent genetic cause of unexplained LOCA in this nationwide Spanish cohort and showed a distinct clinical phenotype characterized by later onset, episodic symptoms, oculomotor abnormalities, and frequent subjective response to 4-AP. Disease progression was similar to that observed in ILOCA. These findings support systematic FGF14 testing in patients with late-onset cerebellar ataxia, particularly those with characteristic clinical features.

Background and Objectives: Facioscapulohumeral muscular dystrophy (FSHD) is characterised by asynchronous muscle degeneration and marked pathological heterogeneity. While conventional MRI can detect fatty replacement and oedema-like abnormalities, it provides limited insight into other relevant biological processes such as extracellular matrix remodelling and tissue perfusion. This study investigated whether multispectral optoacoustic tomography (MSOT), an emerging imaging technique that non-invasively interrogates tissue composition through endogenous optical absorbers, can provide complementary molecular information on muscle involvement in FSHD. Methods: MSOT was performed on 568 muscles from 15 genetically confirmed FSHD patients and 9 healthy volunteers using a standardized protocol. The analysis included single-wavelength signal intensity and spectrally unmixed signals related to lipid, collagen, deoxygenated haemoglobin, oxygenated haemoglobin, total haemoglobin, and tissue oxygen saturation. Patients also underwent conventional muscle MRI together with clinical strength and disease severity assessments. Feasibility, reproducibility, discrimination between patients and controls, and associations with MRI and clinical measures were evaluated. Results: MSOT acquisition was well tolerated and highly reproducible (intra- and inter-rater ICC = 0.88). FSHD muscles displayed a distinct optoacoustic profile characterised by reduced signal intensity at wavelengths below 900 nm, increased lipid signal and reduced oxygenated and total haemoglobin signals. Lipid signal discriminated FSHD from control muscles (AUC 0.830, p = 0.008) and increased with MRI-defined fatty replacement (p < 0.0001). Short tau inversion recovery (STIR)-positive muscles on MRI exhibited a distinct molecular phenotype, with higher collagen-associated and lipid signals together with increased deoxygenated and total haemoglobin signals, consistent with active tissue remodelling. Although FSHD muscles classified as normal by conventional imaging showed group-level optoacoustic profiles comparable to controls, 13 of 141 (9%) demonstrated at least one abnormal MSOT-derived parameter. Reduced haemoglobin-related signals correlated with greater global disease severity, whereas increased lipid signal was associated with lower muscle strength. Discussion: MSOT is a feasible and reproducible imaging technique in FSHD that provides complementary molecular information beyond conventional structural imaging. Its ability to detect signatures related to fatty replacement, STIR positivity, functional impairment, and isolated abnormalities in radiologically normal muscles supports further longitudinal studies of MSOT-derived readouts as potential biomarkers of disease activity, progression, and therapeutic response.

INTRODUCTION Myasthenia gravis (MG) and Lambert-Eaton myasthenic syndrome (LEMS) are autoimmune diseases of the neuromuscular junction resulting in fatigable muscle weakness. Rituximab (RTX) is used to treat patients refractory to standard immunosuppression, but evidence for its efficacy remains inconsistent. Here, we analyzed real-world data on the clinical course and side effects of RTX in MG and LEMS patients. METHODS This was a single-center study of all patients diagnosed with MG (n=64) or LEMS (n=5) treated with RTX from 2011 until 2021. Outcomes of RTX treatment were recorded retrospectively with Myasthenia Gravis Foundation of America Post-Intervention Status (MGFA-PIS), number of rescue therapies, myasthenic crises, and steroid dose at 1-year and 2-year follow-ups. RESULTS MGFA-PIS improved at both 1-year (y) and 2-y follow-up compared with baseline. Incidence rates of rescue therapies per 100 person-months (95% CI) decreased from 15.0 (11.8-18.8) at baseline to 7.5 (4.7-12.3) at 1-y and 4.3 (2.5-7.8) at 2y-follow-up. The number of patients without myasthenic crises within one year increased from baseline (49, 86.0%) to 1y-follow-up (55, 96.5%). Median (IQR) daily steroid dose decreased from 10 (5-22.5) mg/d at baseline to 4 (0-10) mg/d at 1y-follow-up, and to 2.5 (0-10) mg/d at 2y-follow-up. CONCLUSION This study indicates that RTX was associated with a stabilized clinical course and decreased steroid use in patients with autoimmune myasthenic syndromes, including those with thymoma-associated MG. Our data suggest that therapeutic benefit is apparent within the first year of treatment and is maintained through two years.

Background: Hereditary spastic paraplegia (HSP) comprises a heterogeneous group of neurodegenerative disorders characterized by progressive lower-extremity weakness and spasticity, with or without additional neurological symptoms. Objectives: To characterize the molecular genetic spectrum, inheritance patterns, molecular finding rate, and clinical characteristics of HSP within a multicenter Canadian cohort. Methods: We investigated 671 patients from 423 families who met clinical criteria for HSP identified through the Canadian HSP (CanHSP) registry from 7 medical centers across Canada. Patients were evaluated using known HSP and HSP mimic gene panels, whereas unsolved and more recent patients underwent whole-exome sequencing (WES). 435 individuals underwent gene-panel testing and 485 underwent WES. Copy-number variants (CNVs) were assessed using ExomeDepth, and candidate CNVs using multiplex ligation-dependent probe amplification (MLPA). Results: Molecular findings were identified in 426 of 671 individuals (63.5%): 351 (52.3%) had HSP-associated findings, 70 (10.4%) HSP-mimicking disorders, and five (0.7%) other molecular findings; 245 (36.5%) remained unresolved. Candidate variants were found in 80 relevant genes, 36 of which were HSP-associated genes. The most frequently implicated genes were SPAST (22.5%), SPG7 (5.7%), ATL1 (5.2%), SPG11 (3.6%), and SPG8 (3.3%). Phenotypically, SPAST, ATL1, and SPG8 w[zg1.1]ere predominantly associated with pure HSP, whereas SPG7 and SPG11 more often showed complicated phenotypes, with prominent cerebellar involvement in SPG7 and cognitive and developmental features in SPG11. Conclusion: Patients in CanHSP present with genetic and phenotypic heterogeneity. Reanalysis and complementary approaches, including whole-genome, RNA, and long-read sequencing, may increase diagnostic yield.

Background: Serial glial fibrillary acidic protein (GFAP) trajectories have become an important framework for contextualizing evolving secondary-injury pathophysiology after moderate-to-severe traumatic brain injury (msTBI). However, total GFAP pools release and clearance signals that may be less useful for longitudinal bedside decisions than a proteoform-resolved assay. We compared total GFAP with neoGFAP, defined here as calpain-generated GFAP proteoforms intended to index active astroglial proteolysis during the subacute phase. Methods: We analyzed 651 serial serum samples from 95 msTBI patients from a previously described single-site cohort. Total GFAP and neoGFAP were measured on the same MSD platform from 6 to 240 hours after injury. Early (6 to 72 h) and late (96 to 240 h) windows, data-derived tertiles, and serial trajectory summaries were calculated directly from serial samples. Models were benchmarked against age plus admission post-resuscitation Glasgow Coma Scale (GCS) and the admission IMPACT extended risk score using five-fold stratified cross-validation. Outcomes were unfavorable outcome (GOSE 1 to 4), less-than-good recovery (GOSE 1 to 6), Disability Rating Scale (DRS) [&ge;]15, mortality, and neuroimaging worsening at 6 months. Results: The cohort contributed 95 serial biomarker profiles, with 90 participants evaluable for 6-month GOSE and 89 for DRS. Unfavorable outcome occurred in 57/90 (63.3%), and less-than-good recovery in 79/90 (87.8%). For unfavorable outcome, IMPACT plus early neoGFAP reached AUROC 0.85 versus 0.84 for IMPACT plus early total GFAP and 0.81 for IMPACT alone. For less-than-good recovery, IMPACT plus late neoGFAP achieved AUROC 0.90 versus 0.84 for late total GFAP and 0.82 for IMPACT alone. Secondary analyses for DRS, mortality, and neuroimaging worsening showed smaller differences. Conclusions: In this retrospective analysis, neoGFAP provided clearer incremental value than total GFAP for recovery-oriented monitoring, especially when late-window reassessment of patients who remained at risk for less-than-good recovery was required. Results support prospective testing of neoGFAP as a pathophysiology-informed adjunct to serial bedside decision making, repeat-assessment thresholds, and recovery stratification.

Spinocerebellar ataxia type 7 (SCA7) is a rare neurodegenerative disorder characterized by progressive cerebellar ataxia and visual impairment. We investigated longitudinal changes in resting-state functional connectivity and their clinical associations. Resting-state functional MRI was acquired from 16 individuals with SCA7 and 16 age- and sex-matched healthy controls across three visits over 24 months. Network-to-network functional connectivity was quantified, and machine-learning models were trained using functional connectivity features. SCA7 showed lower MoCA (p = 0.045) and MMSE (p = 0.025) scores and progressive worsening of ataxia (SARA, p < 0.001). Significant Group x Visit interactions were observed for Visual-Default Mode (p = 0.012) and Somatomotor-Cerebellar Dorsal Attention connectivity (p = 0.038). Functional connectivity abnormalities involved cortical, cortico-cerebellar, and cerebellar networks that became more widespread at the final follow-up assessment, with the visual network emerging as the most consistently affected system across analyses. Functional connectivity abnormalities were associated with cognitive performance (MMSE: r = -0.62, p = 0.01) and disease severity (SARA: r = 0.589, p = 0.016). Functional connectivity features accurately classified SCA7 and healthy controls (accuracy = 96.4%, F1 = 0.969). These findings support resting-state functional connectivity as a candidate biomarker warranting further validation in larger, independent cohorts.

ABSTRACT Introduction: Incomplete penetrance and variable expressivity characterize autosomal dominant neurological disorders, but their genetic determinants remain largely unknown. DYT-TOR1A dystonia provides a model for investigating incomplete penetrance. We aimed to identify modifiers of disease penetrance and severity beyond the known TOR1A c.646G>C modifier. Methods: In a prospective genotype-phenotype association study, we phenotyped Ashkenazi Jewish carriers of TOR1A c.907_909del using a prespecified five-level severity score. The primary analysis compared participants with no or minimal dystonia (scores 0-1) and severe dystonia (scores 3-4), after excluding c.646G>C carriers. Findings were assessed in an independent retrospective cohort. Sanger, exome, and short-read and long-read whole-genome sequencing were used for genotyping. Results: The prospective cohort included 61 carriers: 21 had severe dystonia and 28 had no or minimal dystonia, six of whom carried c.646G>C. Among the 43 participants in the primary analysis, haplotype A was identified in 33 (77%) and haplotype B in eight (19%). All haplotype B carriers had no or minimal dystonia (p=0.003). Haplotype B remained associated with a milder phenotype after including 12 participants with intermediate severity (p=0.006). The retrospective cohort included 147 carriers, 16 of whom carried c.646G>C. After their exclusion, dystonia manifested in 12 (38%) of 32 participants with haplotype B and 63 (64%) of 99 without it (p=0.009). Haplotype B was also associated with lower severity scores (p=0.011). Together, c.646G>C and local haplotype status predicted the phenotype of more than two-thirds of carriers. In silico analyses linked haplotype B to lower TOR1A and higher TOR1B expression in dystonia-relevant brain regions. Conclusion: A common local haplotype in trans to the disease-causing TOR1A variant was strongly associated with reduced penetrance and severity. These findings establish DYT-TOR1A dystonia as a model for investigating how local genetic variation contributes to incomplete penetrance in autosomal dominant neurological disorders.

Progressive multiple sclerosis (PMS) lacks blood-based biomarkers of the neurodegenerative processes driving disability. We measured synaptopodin, a postsynaptic protein, in neuronal-enriched extracellular vesicles (NEVs) across two independent cohorts: the SPRINT-MS trial (n=184) and a real-world validation cohort (n=268). A one SD increase in baseline synaptopodin was associated with ~32% lower risk of confirmed disability progression in PMS (p=0.011), with the strongest effect in secondary PMS (SPMS) (~57% lower odds, p=0.019). This SPMS association was replicated in the validation cohort (~60% lower odds, p=0.009). Higher baseline synaptopodin was associated with future slower whole-brain atrophy and preserved microstructural integrity across MRI metrics (p=0.007-0.013). Longitudinal changes in NEV synaptopodin were associated with disability worsening (p=0.007) and paralleled MRI microstructural changes. NEV synaptopodin, but not NfL, was associated with confirmed disability progression in PMS participants with both biomarkers measured (HR 0.71 vs 1.12, p=0.007 vs 0.22 for NEV synaptopodin and NfL, respectively) and SPMS (HR 0.58 vs 1.10, p=0.003 vs 0.50). Adding synaptopodin, but not NfL, to a base model improved discrimination for disability progression in SPMS in both cohorts (e.g. validation AUC 0.755 to 0.838, p=0.005). These findings identify NEV synaptopodin as a prognostic and longitudinal, blood biomarker of synaptic integrity in PMS, particularly SPMS, complementing NfL.

Background: Multiple sclerosis (MS) is a chronic neurodegenerative disorder in which inflammatory demyelinating lesions affect the brain, optic nerve and spinal cord. MS lesion formation is accompanied by profound changes to blood vessels, including the density of PDGFR{beta}+ mural cells, historically identified as pericytes. Intriguingly, in recent years, single-cell and lineage tracing studies have shown that the PDGFR{beta}+ cell population is heterogeneous, comprising both pericytes and perivascular fibroblasts. Yet, due to their overlapping expression profiles, the spatial distribution of these cell populations in MS lesions remains poorly understood. Methods: We employed multiplex immunohistochemistry for endothelial cells (CD31), basement membrane (laminin), fibroblasts (PDGFR{beta}, COL1A1, SMA), pericytes (PDGFR{beta}, SLC6A12) and immune cells (CD45, CD68) to characterize the spatial localization of fibroblasts and pericytes in MS lesions, and how this relates to perivascular space enlargement and immune cell presence. Results: We analysed 17633 individual vessels across 5 control white matter, 5 normal-appearing white matter, 4 active and 4 chronic active MS lesions. By carefully delineating endothelium and perivascular compartments, we find that perivascular space area but not number of vessels is increased in MS lesions. Through mining of publicly available sequencing datasets, we confirm COL1A1 and SLC6A12 as fibroblast and pericyte markers, respectively, in the human brain. COL1A1+ and SLCA12+ vessels were largely distinct of one another. Unsupervised clustering of the expression profile of PDGFR{beta}, COL1A1 and SLC6A12 in individual vessels distinguished three partially overlapping vessel clusters. Of these, the fibroblast-associated vessel type (COL1A1 high, SLC6A12 low) was increased in chronic active lesion rim and center. Importantly, fibroblast-associated vessels were related to increased perivascular space enlargement and more accumulation of immune cells. Conclusion: We identify distinct fibroblast- and pericyte-associated vascular phenotypes in human white matter. Notably, fibroblast-associated vessels are increased in chronic active lesions, where they are related to immune cell cuffs. These findings provide a spatial link between perivascular fibroblasts and chronic inflammation in MS.

Myasthenia gravis (MG) is an autoimmune disease caused by pathogenic autoantibodies against proteins at the neuromuscular junction (NMJ). The diagnosis and clinical management of MG patients largely relies on the detection of antigen-specific autoantibodies targeting acetylcholine receptor (AChR) or muscle-specific kinase (MuSK). Yet a subset of patients remains seronegative for known MG autoantibodies, highlighting a critical need for alternative approaches to identify pathogenic NMJ antibodies. We established a new human in vitro model of the NMJ based on primary human muscle cells that recapitulates key features of the NMJ: differentiation to myotubes, expression of key NMJ proteins and formation of postsynaptic AChR clusters in response to agrin stimulation. The model allows new insights into myogenesis and genetic muscle diseases, and the new muscle cell-based assay (CBA) detected autoantibodies in sera from patients with AChR- and MuSK-positive MG with 96.43% sensitivity and 100% specificity, while healthy control sera showed no reactivity. Incubation with patient sera significantly reduced AChR clustering compared to controls, demonstrating functional pathogenic effects. Thus, we established a physiologically relevant human NMJ model that enables detection and functional characterization of neuromuscular autoantibodies. This novel approach addresses a key limitation of current antigen-specific diagnostics and provides a method for improved detection and characterization of MG antibodies, independent of antigen specificity. One Sentence SummaryWe established a postsynaptic human in vitro neuromuscular junction model to assess binding and pathogenicity of MG autoantibodies. Key messagesO_ST_ABSWhat is already known on this topic?C_ST_ABSCurrent diagnosis of myasthenia gravis (MG) relies largely on the detection of antigen-specific autoantibodies against AChR and MuSK, leaving a clinically relevant subset of patients seronegative. What are the new findings?We established a physiologically relevant human in vitro neuromuscular junction model based on primary human muscle cells and developed a novel muscle cell-based assay (CBA) for the detection of neuromuscular autoantibodies. How might this impact on clinical practice or future developments?The CBA detected autoantibodies in patients with AChR- or MuSK-positive MG with high sensitivity and specificity and demonstrated their functional pathogenic effects on AChR clustering. This antigen-independent approach may improve the detection and functional characterization of MG autoantibodies, particularly in patients who are seronegative in current diagnostic assays. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=130 SRC="FIGDIR/small/743478v1_ufig1.gif" ALT="Figure 1000"> View larger version (38K): org.highwire.dtl.DTLVardef@18ed154org.highwire.dtl.DTLVardef@151036corg.highwire.dtl.DTLVardef@1b7ab34org.highwire.dtl.DTLVardef@1490fe9_HPS_FORMAT_FIGEXP M_FIG C_FIG

Background: Idiopathic normal pressure hydrocephalus (iNPH) is a potentially treatable neurological disorder whose molecular basis remains incompletely understood. Metabolomic studies in iNPH are scarce and have focused largely on comparisons with other neurological disorders or on cerebrospinal fluid (CSF) alone. Consequently, the broader metabolic phenotype of iNPH and the extent to which CSF alterations are reflected in peripheral blood remain unclear. We therefore compared paired CSF and plasma metabolomic profiles from patients with iNPH and controls. Methods: Paired CSF and plasma samples from 10 patients with iNPH and 10 controls were analyzed via hydrophilic interaction liquid chromatography-mass spectrometry. After quality control and deduplication, 134 CSF and 149 plasma metabolites were quantified. Protein-normalized abundances were log2-transformed and analyzed via age-adjusted ordinary least-squares regression as the primary model, with group-only regression and Mann-Whitney U tests used for sensitivity analyses. Significance was defined as Benjamini-Hochberg q < 0.05 and |log2-fold change| larger or equal to log2(1.5). Results: Age-adjusted analysis revealed 56 altered CSF metabolites in iNPH patients, 34 of which were increased and 22 of which were decreased; 47 were also significantly altered according to the Mann-Whitney U test. The CSF phenotype encompassed energy and carnitine/acylcarnitine metabolism, redox and one-carbon/transsulfuration-associated metabolism, tryptophan-kynurenine metabolism, amino acid and nucleotide turnover, and neuronal- or membrane-associated metabolites. Prominent changes included increased succinate, 3-hydroxybutyrate, oxidized glutathione, cystathionine, kynurenine and kynurenic acid, together with decreased carnitine, short-chain acylcarnitines, methionine and ergothioneine. In plasma, 27 metabolites met the age-adjusted significance criteria, but only allantoin, N-formylmethionine, 3-hydroxybutyrate, N-acetylmethionine and kynurenic acid were also significant according to the Mann-Whitney U test. Global CSF-plasma effect-size concordance was moderate. The most consistent cross-compartment features were 3-hydroxybutyrate, N-formylmethionine and N-acetylmethionine. Conclusions: iNPH was associated with a broad and comparatively consistent CSF-centered metabolic phenotype, whereas plasma showed a smaller, partial and model-sensitive reflection of these alterations. These findings support altered energy substrate handling, redox and sulfur-associated metabolism, tryptophan-kynurenine metabolism and neuronal or membrane-associated metabolite handling as components of iNPH biology. These exploratory findings require validation in larger, longitudinal cohorts.

ObjectiveThe current International League Against Epilepsy (ILAE) guidelines for Lennox-Gastaut syndrome (LGS) mandate the presence of generalized paroxysmal fast activity (GPFA) on EEG. However, GPFA is not necessarily restricted to LGS and has been described in, among others, idiopathic generalized epilepsies. A clear definition for GPFA is currently lacking, hampering research into its utility as a diagnostic or treatment biomarker. Here, we assessed expert agreement in the interpretation of GPFA to explore feasibility of a consensus-based definition. MethodsWe internationally distributed a two-part survey on GPFA to EEG experts. First, participants completed thirteen questions gauging their preferred clinical and EEG criteria for GPFA, including duration, frequency, amplitude and topography. Second, participants classified the presence versus absence of GPFA in eleven EEG clips, while blinded to clinical context, from patients with LGS, other epilepsies, and physiological variants. Results162 respondents, predominantly from North America and Europe, completed the survey. 87% considered GPFA to also occur in epilepsies other than LGS. Considerable heterogeneity was present on defining duration, spectral frequency, amplitude and topographic distribution of GPFA. Particularly poor agreement was seen for lower limits of duration and frequency, with no single response option receiving a majority consensus (36% and 30% agreement, respectively). Similarly, the degree of agreement on interpretations was highly variable across real-world EEG clips. SignificanceMarked discrepancies exist in the EEG identification and clinical interpretation of GPFA, undermining its utility as a potential biomarker in research and clinical trials of LGS and other epilepsies. The findings also highlight the need to establish a consensus-based and eventually evidence-informed definition to formally operationalize the clinical and EEG criteria for GPFA. Key pointsO_LIGeneralized paroxysmal fast activity (GPFA) is variably defined in the literature, limiting its utility as a diagnostic or treatment biomarker C_LIO_LIIn this international survey (n = 162 respondents, 25 countries) we found limited agreement on GPFAs defining features and on interpretation of fast activity on EEG clips C_LIO_LI87% of respondents considered GPFA to occur in epilepsies other than Lennox-Gastaut syndrome C_LIO_LIPending further elucidation of the pathophysiology underlying GPFA or its computational characterization (i.e., to inform a biologically-or evidence-based definition), we underscore the need for an interim consensus-based definition C_LI

ObjectivePlasma metabolomics offers insight into multiple sclerosis (MS) pathophysiology, but existing studies are limited by small sample sizes and incomplete clinical data. MethodsWe conducted plasma metabolomic profiling of 411 deeply phenotyped patients with MS and 46,443 controls, analyzing 162 metabolites in 30 biologically related metabolite groups. We characterized associations with MS diagnosis, disability, disease subtype, and inflammatory disease activity using regression and differential network enrichment analysis. We additionally examined 25 pre-diagnosis individuals whose samples were collected before their first demyelinating event. ResultsFourteen of 30 metabolite groups were associated with MS after false discovery rate correction, with the strongest positive associations observed for atherogenic lipoproteins, glycine, cholines, and saturated fatty acids, and the strongest negative associations for aromatic amino acids, branched-chain amino acids, alanine, and citrate. Differential network enrichment analysis identified two dysregulated subnetworks encompassing amino acid and energy metabolism and lipid and lipoprotein metabolism. Five metabolite groups were negatively associated with disability: small high-density lipoprotein particles, histidine, branched-chain amino acids, albumin, and aromatic amino acids. The omega-6/omega-3 fatty acid ratio was significantly associated with recent relapse (OR = 1.92, FDR-p = 0.030) and nominally associated with future MRI activity, especially in patients on moderate or high-efficacy disease-modifying therapy. The MS metabolic signature was not detectable in pre-diagnosis samples. InterpretationThese findings highlight coordinated dysregulation of amino acid and lipoprotein metabolism as hallmarks of established MS and identify a novel association of the omega-6/omega-3 ratio with inflammatory disease activity.

Parkinsons disease (PD) arises through disruption of multiple interconnected cellular processes, but the genetic contributions to these processes may differ across ancestries. We investigated functional convergence among genes harboring pathogenic or likely pathogenic (P/LP) variants and variants of uncertain significance (VUS) in a multicenter Indian cohort recruited through the Genetics of Parkinsons Disease in India Young Onset Parkinsons Disease project (GOPI YOPD). The cohort included 668 participants (463 males 69.3%) with a mean age at motor onset of 39.4+/-8.8 years. P/LP variants and VUS identified through previously reported whole-exome or whole genome sequencing were retained as separate evidential categories. The P/LP-associated gene set comprised 11 unique genes and the VUS associated set comprised 40 unique genes. Separate STRING functional-enrichment analyses evaluated Gene Ontology Biological Process, Molecular Function and Cellular Component terms, KEGG pathways, WikiPathways and STRING local network clusters. Terms meeting a Benjamini Hochberg false discovery rate threshold of <0.05 were organized into eight non-mutually-exclusive ontology/pathway categories. Gene to pathway mappings were subsequently projected to individual participants to estimate pathway representation and examine clinical associations. At least one reportable P/LP variant or VUS was identified in 336/668 participants (50.3%): 35 had a P/LP variant alone, 282 had VUS alone and 19 had a P/LP variant together with VUS in one or more additional genes. The most frequently represented categories were mitochondrial organization (247/336, 73.5%), autophagy related processes (228/336, 67.9%) and regulation of synaptic vesicle transport (201/336, 59.8%). PRKN was the most frequent P/LP-associated gene, occurring in 29/54 P/LP carriers, followed by PLA2G6 and PINK1. Lysosomal transport was represented exclusively by VUS-associated genes, particularly GBA1, VPS13C and LRRK2. Among P/LP carriers, additional VUS in distinct genes were not associated with age at onset (P = 0.81) or family history (52.6% versus 31.4%; P = 0.15). No pathway phenotype association remained significant after correction for multiple testing. Genetic findings in this Indian cohort converged across an interconnected mitochondrial autophagic lysosomal vesicular network, with different contributions from P/LP-associated and VUS associated gene sets. This study provides the first pathway resolved South Asian genetic profile and a framework for comparative studies across populations.

Background: Amyotrophic lateral sclerosis (ALS) is a rare neurodegenerative disease with no known cure. Disease progression in people living with ALS is heterogeneous, hindering personalized treatment development. The current gold standard for measuring disease progression in ALS, the ALS Functional Rating Scale - Revised (ALSFRS-R), is widely used but based on subjective measurements. Blood-based neurofilament light (NfL) has been studied as a diagnostic and prognostic biomarker but less information exists on its utility as a disease progression biomarker. Methods: We present results from blood draws of 300 participants in the FDA-funded Clinic-Based Multi-Site ALS Natural History and Biofluid study of the ALS Natural History Consortium (NHC). Plasma NfL levels were measured and analyzed against different disease progression metrics based on the ALSFRS-R. Results: NfL levels were found to be correlated with the ALSFRS-R average rate of change (r=-0.53, 95% CI -0.62 to -0.42). This association differed at a cutoff value of 61 pg/mL, with stronger correlations below this cutoff (r=-0.51 vs r=-0.18). Survival differed stratifying by this cutoff value, with participants under the cutoff having higher survival probabilities. The predictive value of NfL when predicting time to death was higher compared with the first ALSFRS-R across different event horizons. A model including both was better when predicting events up to 2 years after diagnosis. Conclusions: These results highlight the utility of NfL as a disease progression biomarker in ALS alongside ALSFRS-R based disease progression metrics. The cutoff value can aid in clinical trial stratification, pragmatic trial planning, and clinical care.

Background and objectives: Cerebral Autosomal Dominant Arteriopathy with Subcortical Infarcts and Leukoencephalopathy (CADASIL) is the most common monogenic cerebral small vessel disease. Though neuropsychiatric symptoms (NPS) of CADASIL profoundly impact function and quality of life, these symptoms remain understudied. This analysis characterizes the prevalence, severity, and profile of NPS among individuals with CADASIL-causing NOTCH3 variants and non-carrier biological family controls in the United States CADASIL Consortium (USCC). Methods: This cross-sectional analysis used multimodal baseline data from 12 enrollment sites across the country. Statistical analyses used nonparametric tests and regression models to compare clinical and neuropsychiatric outcomes across CADASIL risk tiers and controls, adjusting for age, sex, race, and education where appropriate. The primary outcome measure was the Neuropsychiatric Inventory Questionnaire (NPI-Q), an informant-reported measure which assesses 12 categories of NPS. NPI-Q item severity and distress were evaluated for individuals with confirmed cysteine-altering NOTCH3 variants and compared with controls. Results: Four hundred fifty-nine USCC participants were included in the analytic cohort: 343 cases [256 high-risk (HR); 63 medium-risk (MR); 24 low-risk (LR)]; and 116 controls. Among participants with available NPI-Q data, at least one neuropsychiatric symptom was reported by 72.6% (n = 228/314) of cases compared with 53.2% (n = 58/109) of controls. Reported symptoms were more common among CADASIL cases than controls, occurring in 75.0%, 71.2%, and 72.7% of participants in the LR, MR, and HR groups, respectively, compared with 53.2% of controls. Compared with controls, participants in the MR group and HR group had higher NPI-Q severity scores. HR participants also had higher NPI-Q distress scores than controls. Discussion: Our findings support the notion that NPS are an important clinical manifestation of CADASIL. Beyond previously reported symptoms, additional neuropsychiatric manifestations were identified. Given the higher burden and variability in the neuropsychiatric presentation of CADASIL, the relationships among neuropsychiatric, neurological, and patient-reported outcomes merit further investigation. The NPS profile may enhance the evaluation of the safety and efficacy of different treatments to target these symptoms for CADASIL patients. Trial Registration Information: The study is registered at ClinicalTrials.gov (NCT05677880, 12/12/2022).

Introduction/Aims To evaluate the usefulness of the Goal Attainment Scale (GAS) light for assessing response to risdiplam in patients with SMA aged [&ge;]15 years. Methods In this population-based, longitudinal, ambispective study, patients were evaluated before and at 12 and 24 months after risdiplam initiation using motor scales (SMA Functional Composite Score Revised [SMA-FCR]), pinch strength (MyoPinch), functional scales (EK2, ALSFRS-R), patient and clinician global impression of change (PGIC and CGIC), and GAS light. Longitudinal changes were assessed using linear mixed-effects models. The minimal detectable change (MDC) and minimal clinically important change (MCIC) of GAS light were calculated. Results Forty-four patients (median age 32 years; 56.8% female) were included: 31.8% non-sitters, 56.8% sitters, and 11.4% walkers. GAS light priorities differed across functional subgroups, with patients prioritising moderately affected domains. After 24 months of risdiplam treatment, motor outcomes showed non-significant improvements in walkers, whereas functional scales improved significantly only in non-sitters. In contrast, GAS light detected significant, increasing improvements across all functional subgroups. The MCIC and MDC for GAS light were 6.5 and 10.65 points, respectively. According to the CGIC, 58% of patients improved slightly, 29% remained stable, and 13% worsened slightly at 24 months. Using the MCIC threshold, 64.5% achieved clinically meaningful goal improvement. Discussion GAS light is a feasible, sensitive, patient-centred tool that may complement standardised outcome measures when evaluating treatment response in adults with SMA. These findings further support risdiplam as a valuable therapeutic option in this population.

The glymphatic system is the waste clearance pathway in the central nervous system. Although its dysfunction has been associated with several neurological disorders, its role in multiple sclerosis (MS) remains poorly understood. This system is thought to be particularly active during sleep, yet the relationship between sleep metrics and MRI-derived markers of glymphatic activity has been scarcely explored. In this study, we aimed to perform a multimodal magnetic resonance imaging (MRI) assessment of glymphatic function in MS and investigate its association with sleep parameters, clinical disability, disease duration and MRI-derived structural markers. The study included 22 patients (34.3{+/-}10.1 years; 16 female) with relapsing-remitting MS and 26 healthy controls (32.2{+/-}5.7 years; 18 female), all of whom underwent 3.0 Tesla MRI. Glymphatic function was assessed using diffusion tensor image analysis along the perivascular space (DTI-ALPS) index and global blood-oxygen-level-dependent (BOLD) signal-cerebrospinal fluid (CSF) coupling. In addition, sleep micro- and macrostructural features, clinical disability, disease duration, along with mean diffusivity (MD), lateral ventricular, choroid plexus volumes and lesion load were evaluated. Patients showed reduced DTI-ALPS index, though this difference became only a trend after controlling for MD. No significant group differences were observed in global BOLD signal-CSF coupling. However, in patients, the weaker coupling was associated with longer total sleep time, lower arousal index, greater disability and lateral ventricle enlargement, whereas the DTI-ALPS index showed no significant correlations with any of the measures. Together, these findings challenge the assumption that DTI-ALPS can serve as a reliable marker of glymphatic function in demyelinating diseases such as MS, where disease-related alterations in white matter microstructure can confound diffusion-based measures. In contrast, gBOLD-CSF coupling showed associations with sleep, clinical disability and structural changes in patients, despite the absence of a significant group-level difference. This finding suggests that gBOLD-CSF coupling may capture individual variability in glymphatic-related dynamics relevant to sleep and the clinical expression of MS.