Abstract Vestibular migraine (VM) is an underrecognized disorder characterized by episodic vertigo, often accompanied by headache. Despite its substantial prevalence and disease burden, its metabolic features remain poorly characterized, and serum biomarkers for diagnosis or activity assessment are lacking. We performed untargeted metabolomics in discovery (33 VM vs 30 healthy controls [HCs]) and replication (37 VM vs 27 HCs) cohorts, applying machine learning solely in the discovery cohort for marker selection. In the discovery cohort, 510 metabolites exhibited differential abundance (197 upregulated, 313 downregulated). Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis revealed enrichment trends for upregulated metabolites in cysteine/methionine, histidine, and phenylalanine metabolism. Machine learning identified three candidate metabolites─phenylethylamine, N-acetylserotonin, and cysteine-S-sulfate─each achieving an area under the curve > 0.9. In the discovery cohort, cysteine-S-sulfate correlated with vertigo Visual Analog Scale scores, and phenylethylamine correlated with Headache Impact Test-6 and Generalized Anxiety Disorder-7 (GAD-7) scores. In the replication cohort, all three markers maintained differential expression and discriminative performance; additionally, cysteine-S-sulfate correlated with Dizziness Handicap Inventory scores, and phenylethylamine with GAD-7 scores. This study elucidates the metabolic landscape of VM and identifies promising serum biomarkers, though these findings warrant further validation in larger, multicenter cohorts before clinical translation.

Objective:This study aimed to investigate mitochondrial dysfunction and its role in the pathogenesis of Fabry disease (FD) by analyzing circulating cell-free DNA (ccf-DNA) in patients with FD. Methods:Sixty-six FD patients and 21 healthy controls (ctrls) were enrolled. Levels of plasma mitochondrial- (ccf-mtDNA) and nuclear-derived ccf-DNA (ccf-nDNA) were quantified by quantitative reverse-transcription PCR (RT-qPCR), and 14 inflammatory cytokines were measured in treatment-naïve patients. Associations among ccf-DNA levels, cytokine profiles, disease biomarkers, and clinical markers were analyzed, with subgroup analyses stratified by sex, genotype, clinical subtype, and disease severity. Results:Treatment-naïve patients exhibited significantly higher ccf-mtDNA (z=-4.530, P-adj<0.001) and mtDNA/nDNA ratio (z=-2.613, P-adj=0.014) compared with ctrls. In the long-term enzyme replacement therapy (ERT) group (> 12 months), ccf-mtDNA copy number remained elevated (z=-3.141, P-adj=0.006), whereas the mtDNA/nDNA ratio did not differ significantly (z=-1.013, P-adj=0.311). No differences in ccf-nDNA were observed between treatment-naïve patients or the long-term ERT group compared with ctrls. Receiver operating characteristic analysis demonstrated the strong diagnostic performance of ccf-mtDNA (area under the curve=0.860), with 70% sensitivity and 91% specificity at an optimal cut-off value of 1,793,188.04 copies. Both ccf-mtDNA and mtDNA/nDNA ratio correlated positively with inflammatory cytokines including interleukin-17F and tumor necrosis factor-β, with stronger associations observed in male patients with classic FD. No correlations were observed with disease duration, α-galactosidase A activity, plasma globotriaosylsphingosine or clinical markers after adjustment for age and sex. Similarly, ccf-DNA measures did not differ significantly by sex, GLA mutation type (truncated vs. non-truncated), FD subtype (classic vs. non-classic), or across subgroups defined by disease severity or organ involvement (high vs. low MSSI, with or without hypertrophic cardiomyopathy, with or without chronic kidney disease, mild vs. severe white matter lesions, with or without neuralgia, or mild vs. severe pain). Conclusions:Mitochondrial dysfunction, reflected by elevated ccf-mtDNA, is implicated in FD pathogenesis and may be linked to inflammatory activation. ccf-mtDNA represents a promising diagnostic biomarker for FD, potentially offering an additional therapeutic target when combined with ERT.

Neuronal intranuclear inclusion disease (NIID) is a polyglycine disease that primarily affects the neuronal and neuromuscular systems. Here, we developed a novel transgenic mouse model that faithfully recapitulates the multisystemic impairments associated with polyG intranuclear inclusions. Our findings demonstrate that polyG expression induces neurodegeneration, behavioral deficits, and age-dependent accumulation of uN2CpolyG aggregates across multiple tissues. ### Competing Interest Statement The authors have declared no competing interest. Brain Science and Brain-like Intelligence Technology-National Science and Technology Major Project, 2025ZD0217600

Background:Dermatomyositis (DM) is an autoimmune myopathy often linked to aberrant type I interferon (IFN) pathway activation. However, the molecular mediators driving this IFN signature and their utility as biomarkers remain incompletely defined. Methods:We conducted an integrated multi-omics analysis combining plasma proteomics from 14 patients with DM and 5 healthy controls, with transcriptomic profiling of skeletal muscle derived from three publicly available Gene Expression Omnibus datasets (GSE11971, GSE1551, and GSE128470). Selected plasma proteins were further quantified and validated using enzyme-linked immunosorbent assay (ELISA). Results:Proteomic profiling identified 482 differentially expressed proteins (DEPs). Upregulated DEPs were enriched in antiviral responses and IFN-related immune pathways, while downregulated DEPs were associated with extracellular matrix organization. Transcriptomic analysis revealed 156 consistently upregulated differentially expressed genes across datasets, primarily involved in innate immunity, nucleic acid sensing, and antigen presentation. Integrative analysis identified 2'-5'-oligoadenylate synthetase 3 (OAS3) as a central hub within the IFN signaling network. ELISA validation demonstrated significantly elevated plasma OAS3 levels in DM patients (median: 5.073 ng/ml, IQR: 2.93-9.36) compared with controls (median: 2.723 ng/mL, IQR: 1.77-3.34), with a P-value of 0.018. Notably, plasma OAS3 levels showed a positive correlation with serum creatine kinase concentrations (r=0.55, P = 0.044). Conclusions:OAS3 expression was consistently elevated in both plasma and skeletal muscle tissues of individuals with DM. This molecule may act as a key enhancer of type I IFN-mediated pathogenic responses. Our results support the potential of OAS3 as a novel biomarker and therapeutic target in DM.

BackgroundCardiac involvement in dystrophinopathy progresses from diffuse myocardial remodeling to overt systolic failure. Multiparametric cardiac magnetic resonance (CMR) offers simultaneous insights into myocardial mechanics and tissue characterization, yet the optimal diagnostic parameters for tracking disease severity remain to be fully characterized.ObjectiveTo objectively evaluate the descriptive and discriminative performance of integrating strain and quantitative T1 mapping parameters for identifying myocardial fibrosis burden and stratifying systolic dysfunction in patients with dystrophinopathy.Materials and methodsThis retrospective study analyzed 55 patients with genetically confirmed dystrophinopathy and 30 healthy controls who underwent CMR between January 2020 and December 2024. Patients were sequentially stratified by late gadolinium enhancement (LGE) status (Group A: LGE-negative, n = 16; Group B: LGE-positive, n = 39), and further sub-stratified by left ventricular ejection fraction (LVEF) (Group C: LVEF ≥ 50%, n = 23; Group D: LVEF < 50%, n = 16). Least absolute shrinkage and selection operator (LASSO) regression with 10-fold cross-validation was applied for non-zero feature selection, followed by the construction of multivariable Firth penalized logistic regression models. Model comparisons were made using the non-parametric DeLong test.ResultsCompared with Group A, Group B exhibited significant differences in LVEF (p < .001), mid-ventricular circumferential strain (CS_Mid, p < .001), and native T1 in segment 11 (T1map_seg11, mid-inferolateral, p = .004). In single-parameter ROC analysis, CS_Mid, LVEF, and T1map_seg11 exhibited an area under the curve (AUC) of 0.842 [95% confidence interval (CI): 0.735–0.949], 0.828 (95% CI: 0.714–0.942), and 0.715 (95% CI: 0.549–0.880), respectively. The combined model of CS_Mid and T1map_seg11 achieved the AUC of 0.888 (95% CI: 0.793–0.983). Compared Group C with Group D, CS_Global and T1map_seg5 models yielded AUCs of 0.921 (95% CI: 0.822–1.000) and 0.823 (95% CI: 0.694–0.953). The combined model (CS_Global + T1map_seg5) achieved the AUC of 0.951 (95% CI, 0.864–1.000, DeLong p = .567 vs T1map_seg5, DeLong p = .101 vs. T1map_seg5).ConclusionsIn dystrophinopathy, multiparametric CMR enables structural and functional assessment of myocardial remodeling. Segmental native T1, particularly in the basal and mid-inferolateral segments, provides complementary information on regional myocardial tissue abnormalities. In contrast, CS accounts for most of the discrimination of disease severity. These findings suggest that the distinctive contribution of CMR may lie in regional myocardial tissue characterization rather than conventional functional assessment.

OBJECTIVES:Dysferlinopathy is an autosomal recessive muscular dystrophy caused by pathogenic variants of the DYSF gene. Currently, no clinical effective treatment is available. Given the myopathologic and animal model evidence on complement activation in dysferlinopathy, we explored the potential therapeutic effect of complement inhibition. METHODS:We reported a case of teenager-onset dysferlinopathy with progressive proximal weakness, markedly elevated serum creatine kinase levels, and assistance requirement for ambulation when admission. Muscle biopsy showed dysferlin deficiency and marked deposition of complement C5b-9 on nonnecrotic sarcolemma. Based on these findings and previous preclinical studies, the patient received eculizumab (900 mg weekly for 4 weeks) with informed consent. RESULTS:Clinical improvement was observed following complement inhibition therapy. By week 5, all tested muscle groups reached Medical Research Council grade 5/5, and the North Star Assessment for Dysferlinopathy score increased from 28 to 39. The 6-minute walk test (6MWT) improved from 220 m to 363 m. The muscle MRI revealed reduced muscle edema after eculizumab treatment. These benefits were sustained at 13-month follow-up. DISCUSSION:Complement inhibition showed promising clinical improvement in this single case of dysferlinopathy. Further studies with larger sample sizes are needed to investigate the efficacy and safety of complement inhibitors in dysferlinopathy.

ABSTRACT Objective Driven by pathogenic variants in the TTR gene, hereditary transthyretin amyloidosis with polyneuropathy (ATTRv‐PN) presents as a systemic autosomal dominant disorder. This cross‐sectional study aimed to investigate the clinical utility of in vivo corneal confocal microscopy (CCM) for assessing structural changes in corneal nerve fibers among ATTRv‐PN patients and presymptomatic carriers. Methods A total of 41 ATTRv‐PN patients, 15 ATTRv‐PN presymptomatic carriers, and 15 healthy controls underwent CCM assessment. Clinical neurological scores, electrophysiological data, and sural biopsy data from ATTRv‐PN patients and carriers were analyzed. We calculated the corneal nerve fiber length (CNFL), corneal nerve fiber density (CNFD), corneal nerve branch density (CNBD), inferior whorl fiber length (IWFL), inferior whorl fiber density (IWFD), inferior whorl branch density (IWBD), corneal Langerhans cell density (CLCD), and inferior whorl Langerhans cell density (IWLCD). We compared the CCM parameters between patients, carriers, and healthy controls and analyzed their correlations with clinical, electrophysiological, and pathological changes in patients with ATTRv‐PN. Results CCM parameters, including CNFL, CNFD, CNBD, IWFL, CLCD, and IWLCD, were abnormal in ATTRv‐PN patients (p < 0.05). Statistical analysis revealed no significant variation in evaluated CCM parameters between the early‐onset and late‐onset ATTRv‐PN patient subgroups. Similarly, there were not statistically significant differences in these parameters between Stage I patients and Stage II patients. All CCM parameters except CNFD and IWFL were abnormal in presymptomatic carriers (p < 0.05). The CNFL and IWLCD (p < 0.05) were correlated with disease duration. The CNFL, CNFD, and CNBD were positively correlated with sural large myelinated nerve fiber density (p < 0.05), so were CNFL and IWFL with the total myelinated nerve fiber density (p < 0.05) in the sural nerve. Conclusion As an in vivo, noninvasive imaging technique, CCM facilitates the detailed evaluation of corneal nerve fiber morphology in both symptomatic ATTRv‐PN individuals and asymptomatic mutation carriers. We have confirmed the significant abnormalities of CCM parameters of corneal nerve fibers in ATTRv‐PN patients and presymptomatic carriers.

BackgroundThis study aimed to investigate the clinical, serological and myopathological features of Chinese patients with inclusion body myositis (IBM).MethodsThis study retrospectively recruited patients diagnosed with IBM according to the 2024 European Neuromuscular Center (ENMC) criteria at Peking University First Hospital between 2004 and 2024. Clinical features, pathological changes and laboratory data were collected. Subgroups were analyzed by sex, dysphagia, anti-cN1A antibody status, and complement deposition.ResultsForty-three IBM patients (25 males) were included in this cohort. The mean age at onset was 54.9 ± 9.6 years old. All the patients developed weakness in hip flexion, and 81.4% of them with weakness in both knee extension and finger flexion. Dysphagia was reported in 17 patients (39.5%). Endomysial inflammation was observed in all the patients, and 79.1% with rimmed vacuoles, and 76.9% with mitochondrial abnormalities. Female patients more frequently developed dysphagia (P = 0.005) and neck flexion weakness (P < 0.001). Anti-cN1A antibody was positive in 20 patients (66.7%). Seropositive cases were associated with a later age of onset (P = 0.034). Complement deposition was observed in 77.4% of patients and was associated with more severe muscle weakness.ConclusionThis cohort of Chinese IBM patients suggested an earlier age at onset than previously reported. Hip flexors were most commonly affected. Female patients showed a higher frequency of dysphagia and neck flexor weakness. Our study reported a high frequency of complement deposition in muscle tissue. Complement deposition was associated with disease severity, suggesting a potential role of complement in the pathophysiology of IBM.

OBJECTIVES:Anti-signal recognition particle antibody immune-mediated necrotising myopathy (anti-SRP-IMNM) is characterised by prominent muscle weakness and poor neurological outcomes. This study aimed to evaluate the relationship between endomysial fibrosis and the clinical, muscle magnetic resonance imaging (MRI) and myo-pathological features of patients with anti-SRP-IMNM. METHODS:We collected the clinical, imaging, and myo-pathological data of patients diagnosed with anti-SRP-IMNM. Differences between patients with and without increased endomysial fibrosis on muscle biopsy were compared. RESULTS:Ninety-four patients were included in the study, comprising 12 paediatric and 82 adult patients. The mean age at onset was 41.2±17.3 years. The mean serum creatinine kinase concentration was 6885.8±6300.7 IU/L. MRI revealed muscle oedema in 87.3% of patients and fatty infiltration in 77.5%, which was particularly severe in the muscles of the posterior thigh. Endomysial fibrosis was found in 50% of patients and was significantly associated with early onset (p=0.004), paediatric age (p=0.044), muscle fatty infiltration on MRI of the thigh (p=0.045), and inflammatory cell infiltration on pathology (p<0.05). CONCLUSIONS:In anti-SRP-IMNM, endomysial fibrosis may be the pathological basis of fatty infiltration observed on MRI and may be associated with resistance to immunotherapy.

Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disease that primarily affects motor neurons leading to muscle weakness, paralysis, and ultimately, respiratory failure. Extracellular vesicles (EVs) facilitate intercellular communication by mimicking the functions of their parent cells. In this study, we found that repeated administration of neural stem cell-derived extracellular vesicles (NSC-derived EVs) improved motor performance and provided protection to lumbar motor neurons, the neuromuscular junctions, and muscle morphology in the SOD1 G93A transgenic mouse model of ALS. Furthermore, by analyzing the RNA-sequencing of muscle specimens from ALS-SOD1 patients, we demonstrated that the rescue effects of NSC-derived EVs might be linked to the p53 pathway. Compared to the PBS control treatment group, both TP53 and the p53 upregulated modulator of apoptosis (PUMA) were downregulated in the spinal cord of mice treated with NSC-derived EVs. These data provide additional knowledge for the promising use of NSC-derived EVs as a potential therapy for ALS.

BACKGROUND:Anti-complex nucleosomal remodelling histone deacetylase (Mi2)-positive dermatomyositis (DM) is characterized by severe muscle damage. This study investigated muscle magnetic resonance imaging (MRI) features in anti-Mi2-positive DM, compared them with other DM subtypes, and evaluated their associations with clinical features. METHODS:DM patients who underwent thigh MRI were enrolled, including 20 anti-Mi2-positive, 20 anti-TIF1γ-positive, 15 anti-NXP2-positive, and 19 anti-MDA5-positive cases. Clinical and imaging data were collected and compared across subtypes. The relationship between MRI features and clinical characteristics was analysed in the anti-Mi2-positive subgroup. RESULTS:Among the four subtypes, anti-Mi2-positive DM had the highest myofascial oedema frequency in the sartorius and the lowest in the biceps femoris (both p < 0.05). All anti-Mi2-positive patients had muscle oedema - most severe in the quadriceps femoris with relative sparing of the semimembranosus and biceps femoris - and the highest total muscle oedema scores (p < 0.001). Eleven (55%) patients had fatty infiltration, predominantly in the gluteus maximus and hamstrings with sparing of the rectus femoris and adductor longus. In this subgroup, treatment-naïve patients had a higher prevalence of diffuse distribution than treatment-experienced (p = 0.042). Compared with anti-TIF1γ-positive patients, the anti-Mi2-positive subgroup had lower total fatty infiltration scores and milder gluteus maximus, adductor longus, and hamstrings involvement (all p < 0.05). In anti-Mi2-positive DM, total muscle oedema scores correlated negatively with Manual Muscle Testing-8 scores and positively with serum creatine kinase levels (both p < 0.05). Preliminary observations in six anti-Mi2-positive patients followed longitudinally showed that muscle oedema decreased after treatment, resolved during remission, and recurred with relapse. CONCLUSION:This study delineated baseline thigh MRI patterns in anti-Mi2-positive DM and demonstrated that baseline muscle oedema severity correlated with serological and clinical indicators of muscle damage. Additionally, findings from a small longitudinal cohort suggested a potential role for thigh MRI in assessing and monitoring muscle disease activity in this subtype, although these results are preliminary and require validation in larger studies.

BACKGROUND:Iterative decomposition of water and fat with echo asymmetry and least-squares estimation quantitation (IDEAL-IQ), a quantitative 6-point Dixon magnetic resonance imaging (MRI) sequence, has been increasingly used for quantifying muscle fat fraction (FF) in neuromuscular disorders. However, its utility for correlating FF with disease severity and mapping spatiotemporal disease progression in Duchenne muscular dystrophy (DMD) requires further investigation. METHODS:In 10 patients with dystrophinopathies (seven DMD and three BMD) we correlated the muscle FF with the histopathological fatty infiltration. IDEAL-IQ MRI images of 19 individual lower limb muscles were acquired from 133 DMD patients and 41 healthy controls. Ambulatory function tests, including three timed function tests (TFTs) and North Star Ambulatory Assessment (NSAA), were performed. We investigated the spatial distribution of fatty infiltration across five axial MRI slices. Disease progression patterns were analysed using a piecewise linear model and a normal cumulative distribution function model. RESULTS:A significant positive correlation between muscle FF on MRI and histopathologic fatty infiltration was observed (ρ = 0.98, p < 0.001). A mild to moderate correlation was noted for the FF of 19 individual muscles, excluding the sartorius and gracilis muscles, with TFTs (ρ = 0.27-0.60, p < 0.001) and NSAA (ρ = -0.32--0.73, p < 0.001). All the eight individual muscles, except the tibialis posterior muscle, showed an inhomogeneous fatty infiltration pattern with higher FF in muscle ends compared to bellies. Thigh muscle FF aligned well with both piecewise linear and sigmoidal models, showing non-linear increases with disease duration. The piecewise linear model identified an average inflection point for thigh muscle FF at 7.3 (range, 5.2-7.6) years, with average annual FF increase of 1.7% before and 6.7% after the point in DMD. The average μ of DMD patients for thigh muscles was 11.4 years (±2.8), occurring 7.6 years earlier compared to BMD patients. CONCLUSIONS:Our study demonstrates a significant relationship between muscle FF on IDEAL-IQ MRI and histopathologic muscle fatty infiltration, as well as ambulatory clinical function in DMD. DMD patients exhibit a distinct and inhomogeneous fat infiltration pattern of lower limb muscles.

ObjectiveTo analyze the clinical and genetic features of oculopharyngodistal myopathy (OPDM) patients and compare the phenotypic differences among various causative genes.MethodsA total of 65 genetically confirmed OPDM patients from 43 unrelated families, who were admitted to the Department of Neurology, Peking University First Hospital between January 2008 and December 2025, were retrospectively included.The general demographic data, clinical manifestations, laboratory/auxiliary examinations, muscle pathology, and genetic test results were systematically collected and analyzed. The clinical and pathological characteristics among different OPDM subtypes were compared.ResultsAmong the 65 patients(39 male and 26 female), the mean age of onset was (31.20±10.43) years (range: 14 to 63 years). The initial symptom was predominantly distal limb weakness (67.44%), which gradually progressed to involve the extraocular muscles, pharyngeal muscles, facial muscles and proximal limb muscles. Serum creatine kinase levels were mildly to moderately elevated. Muscle pathological examinations revealed rimmed vacuoles and intranuclear inclusions (within muscle fibers). The mean duration from onset to diagnosis was (12.33±7.88) years (range: 1 to 32 years). All probands had negative results on conventional next-generation whole-exome sequencing; pathogenic variants were identified through third-generation long-read sequencing or OPDM-targeted repeat-primed polymerase chain reaction(RP-PCR). Among the 43 families, OPDM2 subtype was the most common genetic subtype (n=25), followed by OPDM4 subtype (n=8), OPDM3 subtype (n=6), OPDM1 subtype (n=3), and OPDM5 subtype (n=1). All OPDM probands subtypes were highly similar in age at onset and muscle pathological changes. However, OPDM3 subtype may be complicated by cerebral white matter lesions and other extra-muscular organ involvement. The proportion of OPDM4 patients who developed distal limb weakness only after 10 years of disease onset(40.0%) was significantly higher than that of other subtypes (all 0.0%, P=0.0122).ConclusionsOPDM2 was the predominant subtype in this study. All subtypes share similar age of onset and muscular pathological changes, yet exhibit distinct disease progression patterns. Future multicenter prospective cohort studies are warranted to further elucidate the clinical characteristics, pathogenetic mechanisms, and prognostic differences among OPDM subtypes.

OBJECTIVES:Mitochondrial encephalomyopathy, lactic acidosis, and stroke-like episodes (MELAS) is a mitochondrial disorder driven by mutations in mitochondrial or nuclear DNA, involving an altered NADH/NAD+-associated redox metabolism as a key pathological mechanism. The traditional metabolomic analyses in MELAS face sensitivity and sample volume limitations, particularly for carboxylic acid metabolites. This study employed a recently established diazo-carboxyl/hydroxylamine-ketone double-click derivatization (DQmB-HA) mass spectrometry method to overcome these barriers, enabling highly sensitive quantification of NADH/NAD+-related serum metabolites in minimal sample volumes. METHODS:Using DQmB-HA mass spectrometry, we analyzed lactate, pyruvate, β-hydroxybutyrate, acetoacetate, α-hydroxybutyrate, and malate in 5-μL serum samples from each of the MELAS patients (n = 70), healthy controls (n = 29), and CPEO patients (n = 17). Individual metabolite levels were quantified, and the lactate/pyruvate ratio and β-hydroxybutyrate/acetoacetate ratio were used as surrogate indicators of cytoplasmic and mitochondrial NADH/NAD+ redox states, respectively. Following this, analyses were performed to assess between-group differences in these indicators and to determine their correlations with disease duration. RESULTS:MELAS patients exhibited significantly elevated lactate, β-hydroxybutyrate, α-hydroxybutyrate, and malate levels, together with increased lactate/pyruvate and β-hydroxybutyrate/acetoacetate ratios compared with healthy controls. Among the evaluated biomarkers, the lactate/pyruvate ratio achieved the highest diagnostic performance (AUC = 0.993, 95% CI = 0.979-1.000), followed by lactate (AUC = 0.976) and β-hydroxybutyrate (AUC = 0.864). Although the β-hydroxybutyrate/acetoacetate ratio showed high sensitivity (95.7%), its overall diagnostic accuracy was limited by lower specificity. However, none of these serum markers show a significant correlation with the disease duration course in MELAS patients. Relative to MELAS, lower concentrations of α-hydroxybutyrate (p < 0.001) and malate (p = 0.026) and elevated lactate/pyruvate ratio (p < 0.001) were observed in CPEO. CONCLUSION:The DQmB-HA method enabled high-sensitivity metabolomic profiling in low-volume clinical samples and revealed broad alterations in metabolites and metabolite ratios associated with NADH/NAD + -related redox metabolism in MELAS, providing a useful framework for metabolomic screening in mitochondrial diseases.

Background:While our previous research showed that a single infusion of recombinant AAV9-mediated GAA gene therapy enabled infants (<1 year) with infantile-onset Pompe disease (IOPD) and minimal prior enzyme replacement therapy (ERT) to remain ventilator- and ERT-free for at least one year, it was unknown whether the same benefits could be sustained in older children with IOPD with prolonged, high-dose ERT exposure after treatment cessation. Methods:In this investigator-initiated, single-arm trial (ChiCTR2200065664), six children with IOPD aged 12.5-50.8 months-all having previously received 10-98 cycles of ERT-were enrolled and administered a single intravenous infusion of AAV9-mediated gene therapy. Over a 12-month period, the following outcomes were evaluated: primary efficacy endpoints (gross motor function, and cardiac structure and function); secondary efficacy endpoints (ventilator-free survival, and the proportion of patients who showed improvement at the end of the trial); exploratory endpoints (IOPD-related skeletal muscle pathological changes); safety and tolerability endpoints; and immunogenicity assessment endpoints. Findings:The intervention was generally well tolerated, with a low incidence of grade ≥3 adverse events (2.31 events per person-time). All six patients remained ventilator-free over the one-year observation period (one patient required transient ventilatory support for pneumonia). However, lung function and cardiac structure showed no further improvement after gene therapy. All children (patient 4 was not documented due to early withdrawal from the study) maintained sustained growth and development throughout follow-up, and overall quality of life showed a trend of improvement (PedsQL 3.0 NMM). During the study period, patient 1 achieved standing with assistance and crawling; patient 2 achieved standing and walking without assistance; and patient 3 achieved walking and sitting without assistance. Muscle biopsies from the three patients revealed relatively high GAA enzyme activity and reduced glycogen accumulation. Interpretation:These preliminary findings suggest that AAV9-mediated GAA gene therapy may reduce reliance on ERT and is associated with improvements in muscle histopathology among biopsied participants. Funding:2023YFC3403300; Z231100004823022.

Fabry disease (FD) exhibits a spectrum of clinical manifestations ranging from mild to severe, posing a diagnostic challenge, particularly in non-classic subtypes. Genetic testing remains a gold standard for a precise diagnosis of FD and is pivotal in genetic counseling. Although conventional approaches such as Sanger sequencing and short-read next-generation sequencing (NGS) have been successfully used to diagnose FD, they often fail to detect deep intronic variants, complex rearrangements, or large deletions or duplications. In contrast, long-read sequencing (LRS) enables comprehensive coverage of intronic and repetitive regions, facilitating precise identification of atypical variants missed by conventional methods. This case series reports two unrelated male patients with clinical, enzymatic, and pathological features consistent with FD, who tested negative for pathogenic variants in the alpha-galactosidase A (GLA) via Sanger sequencing and NGS. LRS identified novel non-coding variants in both patients. Patient 1 carried a ~1.7 kb insertion within intron 4, corresponding to part of a long interspersed nuclear element-1, while RNA sequencing revealed two new GLA transcripts. Patient 2 harbored a ~2.5 kb insertion within a SINE-VNTR-Alu retroposon element located in the 5'-untranslated region, with quantitative real-time PCR showing significantly reduced expression of normal GLA transcripts. These findings reveal non-coding variants that contribute to the missing heritability in FD, highlight this genomic region as a priority for future investigation, and demonstrate the potential utility of LRS in diagnostic workflows for unresolved FD cases.

Aggregation of TDP-43 in neuronal cells is a defining neuropathological hallmark of amyotrophic lateral sclerosis (ALS). Emerging evidence suggests that TDP-43 pathology also occurs in skeletal muscle fibers, but its functional significance in myocytes remains poorly understood. In this study, we utilized the C2C12 myoblast cell to investigate the subcellular localization of TDP-43 during myogenic differentiation. Our findings demonstrate that TDP-43 progressively translocates to mitochondria in parallel with myotube maturation. Notably, increased mitochondrial localization of TDP-43 was also observed in skeletal muscle tissues from patients with ALS, corroborating the clinical relevance of this phenomenon. Functional assays revealed that inhibition of TDP-43 mitochondrial translocation significantly enhances myotube maturation. Collectively, these results support a pathophysiological role for aberrant mitochondrial mislocalization of TDP-43 in regulating myogenic differentiation and contributing to muscle degeneration in TDP-43 proteinopathies.