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The mode of inheritance of X-linked myotubular myopathy (XL-MTM) is still considered recessive, although female carriers are frequently symptomatic. We aimed to characterize the phenotypic spectrum and burden of disease in a carrier cohort. We performed a cross-sectional study among XL-MTM carriers in Germany, recruiting predominantly through a patient association. We used a custom-made questionnaire on medical and family history, the Checklist Individual Strength (CIS20-R), Short Form 12 (SF-12) health survey, Pain Detect, Short-Form McGill Pain Questionnaire (SF-MPQ-D), Graded Chronic Pain Scale (GCPS), and Depression Anxiety Stress Scale (DASS21). All participants received a neurologic examination including manual muscle testing (MMT), a 6-minute walking test (6MWT), and timed up-and-go (TUG) test. Where possible, we performed ultrasound of the abdomen and vastus lateralis muscles, echocardiography, pulmonary function testing, blood count, and further laboratory testing. Carriers were classified as manifesting based on the degree of muscular weakness and ambulatory status. In our study population (n=14), most carriers were classified as manifesting with a mild (n=8; independent ambulation, with limb or axial weakness) or minimal phenotype (n=3; only facial muscle weakness). We consider only the minority non-manifesting (n=3). Frequent features include a high-arched palate, fatigue, muscle pain and stiffness, and pulmonary problems. Many carriers are caregivers of boys with XL-MTM (n=11). Families are burdened with stillbirth and death in early childhood (n=8). The majority of female carriers of XL-MTM show neuromuscular and additional symptoms. This should be considered in diagnostics, clinical care, and future clinical trials. Supporting grant DFG FU 356/12-2 [ABSTRACT FROM AUTHOR]

Pathogenic variants in CHRNE encoding the epsilon subunit of acetyl choline receptor (AChR) result in impaired neuromuscular transmission and congenital myasthenic syndromes (CMS). Clinical manifestations include facial, ocular and limb fatigability and weakness, whereby severity of symptoms may vary in patients harboring the same pathogenic variant. Although the underlying pathophysiology is well-known, blood biomarkers enabling a patient-stratification are lacking. Previous studies revealed CHRNE protein in white blood cells (WBC) rendering these cells suitable for the study of cellular and minimal-invasive marker proteins. This retrospective two center-study includes 19 patients carrying biallelic pathogenic variants in CHRNE identified by molecular genetic testing. The patients were clinically characterized according to clinical severity (grade 1-4). Biomarker discovery was carried out on blood samples: proteomic profiling was performed on WBC (n =12) and on extracellular vesicles (EVs) purified from serum samples (n =7) in addition to amino acid profiling (n = 9) and miRNA screening (n = 18) on sera. In 19 patients (mean age 14.8 years) from 13 families seven known pathogenic variants in CHRNE were identified in addition to one variant previously not reported (c.1032+2_1032+3delTAinsGT). Whereas 15 patients were classified as mildly affected (group A: grade 1 and 2), 4 patients were classified as moderate or severely affected (group B: grade 3 and 4). Proteomics unveiled a significant increase of 7 and a decrease of 36 proteins in WBC in comparison to normal, age matched controls. GO-term based in silico studies of these dysregulated proteins indicated affection of secretory granules and the extracellular space. When comparing proteomic signatures of CHRNE-patients with a mild (A) versus moderate to severe phenotypes (B), 2 proteins (SCAMP2 and SNX2) were significantly increased in second group (B). These two proteins are involved in vesical transport. Prompted by this finding, proteomic profiling of EVs was initiated revealing a significant dysregulation of 20 proteins (7 are increased and 13 decreased). Three of these proteins (TARSH, ATRN & PLEC) are known to be important for synaptogenesis and synaptic function. Metabolic profiling of sera showed a decrease of 7 amino acids/ amino acid metabolites (aspartic and glutamic acids, phosphoserine, amino adipate, citrulline, ornithine & 1-methyhistidine) in terms of a metabolic fingerprint in CHRNE-related CMS patients. Moreover, miRNA screening unveiled increase of miR−365b−3p and miR−483−3p and decrease of miR-4433b-3p when comparing sera of CHRNE patients versus healthy controls. Moreover, a comparison of CHRNE patients versus other CMS-subtypes showed increase of miR−205−5p, miR−10b−5p, and miR−483−5 and decrease of miR−1290. Our combined data introduce a panel of minimal-invasive biomarkers on protein, metabolic and miRNA level with some of those play different roles along the neuromuscular axis. In the latter regard, impaired EV homeostasis impacting on synaptic function seems to be a molecular disease driver and two proteins in WBC correlate with disease severity. Our miRNA profiles introduce four miRNAs which may open new avenues in CMS patient stratification. [ABSTRACT FROM AUTHOR]

Congenital myasthenic syndromes (CMS) are inherited disorders of defective neuromuscular transmission. The clinical phenotype and treatment response vary between the over 30 genetic subtypes. Currently, there is limited published large cohort data in this population. A multicentre international study was undertaken to identify the genetic spectrum, clinical data, treatments, and standardised outcome measures in genetically confirmed CMS patients. Cross sectional data was collected at the time of routine clinic appointment within the last 12-months. Clinical, genetic information and three outcome measures (Myasthenia Gravis Quantitative Score - QMG, Myasthenia Gravis Activities of Daily Living - MG-ADL and Sit to stand in 1 minute - STS1M) were collected. Cohort data analysis was performed on complete data sets, with sub analysis of common subtypes. 187 patients were included, with AChR deficiency 29%, DOK7 20%, RAPSN 17%, COLQ 7%, and Slow Channel 6%. Median age at clinic was 25 years (range 1-83 years). Mean age at symptom onset (n=172) was 2.8 years. Median age at genetic diagnosis (n=174) was 8.4 years. Median age at treatment initiation (n=169) was 5 years. Total QMG score (n=132) was median 12/39 (range 0-34), in AChR deficiency-13 (range 3-34), DOK7-10 (range 0-29), RAPSN-8 (range 2-17). MG-ADL score (n=174), median 5/24 (range 0-20), in AChR deficiency-5 (range 0-18), DOK7-7 (range 0-14), RAPSN-3.5 (range 0-10) and STS1M (n=146), median-22 (range 0-50), AChR deficiency-21.5(range 0-38), DOK7-17.5 (range 4-45) and RAPSN-25 (range 9-50). This is the largest reported CMS cohort including genetics, clinical data, and standardised outcome measures. Significant variability in the outcome measures was noted within individual CMS subtypes. This highlights the wide spectrum but also provides pivotal information for clinical trial designs. STS1M was identified as a robust easy deliverable outcome measure of fatigable weakness across all ages. [ABSTRACT FROM AUTHOR]

Spinal muscular atrophy (SMA) ranks as the second most prevalent neurodegenerative condition in childhood, characterized by a deficiency in the survival of motor neuron (SMN) protein. This deficiency primarily results in the degeneration of alpha motor neurons, leading to progressive muscle weakness and atrophy. However, motor neuron loss in SMA may not solely result from SMN deficiency. Retrograde signals from skeletal muscles and neuromuscular junctions likely play a significant role in motor neuron vulnerability, influencing the overall clinical presentation. Despite some biomarkers, such as SMN2 copy number, being associated with disease severity, there is a notable absence of therapeutic biomarkers directly linked to neuromuscular transmission, which could indicate the impact of appropriate functioning neuromuscular junctions in SMA. In light of this, we explored the potential of Thrombospondin-4 (TSP4) as a cerebrospinal fluid (CSF) biomarker, with potential implications for indicating treatment response. We used untargeted proteomic analyses to determine clinical-relevant biomarkers in CSF samples derived from symptomatic (n=4) versus non-symptomatic (n=6) (identified via newborn screening) pediatric SMA patients. The identified biomarker candidate, TSP4, was next quantified in CSF samples derived from further pediatric and adult SMA patients (n=32), as well as non-disease (n=18) and disease controls (n=20) by enzyme-linked immunosorbent assay (ELISA). Our proteomic findings unveiled dysregulation of TSP4 in CSF derived from pediatric SMA patients, indicating a distinction between pre-symptomatic and symptomatic patients. Further analysis by ELISA demonstrated a TSP4-decrease in pediatric SMA patients, but no alteration in adult SMA patients or pediatric patients with other neurological disorders (serving as disease controls). Of note, TSP4 levels in CSF increased following the administration of Nusinersen in pediatric SMA patients. TSP4, as a neuromuscular junction resident protein, holds the potential to serve as a CSF biomarker in pediatric (but not adult) SMA patients and hereby not only indicates clinical manifestation but may even serve as a therapy marker under administration of Nusinersen. Further studies are needed to investigate the exact role of the protein in the etiology of SMA. [ABSTRACT FROM AUTHOR]

Spinal muscular atrophy (SMA) is a progressive, recessive neuromuscular disease characterized by significant reduction of SMN protein. This causes degeneration of lower motor neurons in the spinal cord and brainstem leading to weakness and muscle atrophy. Clinical severity is categorized in different subtypes (type 0-3), which is in turn influenced by residual SMN level. While the genetic basis of SMA is well described, the tissue-specific molecular pathways underlying SMA are still not fully understood and identification of marker proteins in human vulnerable tissue such as skeletal muscle is still lacking. To elucidate molecular markers in SMA-diseased muscle, we performed unbiased proteomics and transcriptomics on muscle biopsies derived from 3 SMA type 3 (walker) patients in addition to immunofluorescence including 5 additional cases and SMA myoblasts. Combined proteomic and transcriptomic studies unraveled FUS (a DNA/RNA-binding protein and aggregation marker) as a potential molecular marker increased in SMA muscle and SMA myoblasts. Results of our immunofluorescence studies showed increased FUS protein abundance in both accompanied by perimyonuclear disposition of the protein in a proportion of myofibres compared to the immunoreactivity obtained in control samples. To identify proteins that were associated with FUS we performed an interactome analysis, the results are pending. Our data classified FUS as a protein involved in SMA-based myopathology and support the concept of skeletal muscle as a primary tissue target of SMA. Further studies are crucial to define the role of mis-localized FUS in SMA muscle. [ABSTRACT FROM AUTHOR]

Spinal muscular atrophy (SMA) is a neuromuscular disorder caused by recessive pathogenic variants affecting the survival of motor neuron (SMN1) gene (localized on 5q). In consequence, cells lack expression of the corresponding protein. This pathophysiological condition is clinically associated with motor neuron (MN) degeneration leading to severe muscular atrophy. Additionally, vulnerability of other cellular populations and tissues including skeletal muscle has been demonstrated. Although the therapeutic options for SMA have considerably changed, treatment responses may differ thus underlining the persistent need for validated biomarkers. To address this need and to identify novel marker proteins for SMA, we performed unbiased proteomic profiling on cerebrospinal fluid derived (CSF) from genetically proven SMA type 1-3 cases and afterwards performed ELISA studies on CSF and serum samples to validate the potential of a novel biomarker candidates in both body fluids. To further decipher the pathophysiological impact of this biomarker, immunofluorescence studies were carried out on spinal cord and skeletal muscle derived from a 5q-SMA mouse model. Proteomics revealed an increase of LARGE1 in CSF derived from adult patients showing a clinical response upon treatment with nusinersen. Moreover, LARGE1 levels were validated in CSF samples of further SMA patients (type 1-3) by ELISA. These studies also unveiled a distinguishment between groups in improvement of motor skills: adult patients do present with lowered level per se at baseline visit while no elevation upon treatment in the pediatric cohort can be observed. ELISA-based studies of serum samples showed no changes in the pediatric cohort but unraveled elevated level in adult patients responding to future intervention with nusinersen, while non-responders did not show a significant increase. Additional immunofluorescence studies of LARGE1 in MN and skeletal muscle of a SMA type 3 mouse model revealed an increase of LARGE1 during disease progression. Our combined data unraveled LARGE1 as a protein dysregulated in serum and CSF of SMA-patients (and in MN and skeletal muscle of SMA mice) holding the potential to serve as a disease marker for SMA and enabling to differentiate between patients responding and non-responding to therapy with nusinersen. [ABSTRACT FROM AUTHOR]

GNE myopathy (GNEM) is a rare autosomal recessive distal myopathy characterized by early adult-onset, slow to moderately progressive distal muscle weakness that preferentially affects the tibialis anterior muscle and that usually spares the quadriceps femoris. Muscle biopsy reveals presence of rimmed vacuoles (inclusion body myopathy 2) and fibrosis seen in scattered areas. The disease is caused by bi-allelic variants in the GNE gene encoding the bifunctional enzyme UDP-N-acetylglucosamine 2-epimerase/N-acetylmannosamine kinase. The pathophysiology of GNE myopathy presumably involves aberrant protein sialylation. Thus far, the proteomic signature of GNE-mutant muscle has not been studied to determine a biochemical fingerprint serving as pathogenicity markers. Moreover, except myostatin, easy-accessible minimal-invasive biomarkers are still lacking for GNE myopathy. Exome sequencing was applied to identify the genetic cause of a myopathy present in three adolescents (all developed symptoms at approximately 15 years of age) of a consanguineous family from Afghanistan. In silico tools were applied to evaluate the pathogenicity of a candidate variant. Moreover, untargeted proteomics was performed to determine the proteinogenic signature of muscle biopsies derived from three additional unrelated GNEM patients with different grades of myopathology and immunostaining was completed to validate the findings and to define marker proteins of pathophysiological relevance. Additionally, proteomic profiling and ELISA were performed on serum derived from 24 GNE patients to identify novel minimal-invasive biomarkers. Genetic testing unveiled the novel homozygous p.(Val569Leu) missense variant in the 3 siblings. In silico studies confirmed a pathogenic effect of this amino acid substitution. Histological studies showed pathology including presence of vacuoles in quadriceps muscle derived from one of the patients carrying this variant. Muscle proteomics on three biopsies with other different GNEM genotypes unraveled 176 proteins (161 increased and 15 decreased) commonly affected in GNE patient-derived muscle. This defined catalogue of dysregulated proteins served to confirm the pathogenicity of the novel missense variant – in comparison to further missense variants with well-known pathogenicity – by immunostaining of paradigmatic proteins serving as tissue disease markers. Serum profiling enabled the definition of two proteins (serglycin and periostin) significantly decreased in patients. Pathophysiological relevance of altered periostin was confirmed by immunostaining on muscle biopsies showing increase in fibrotic scars. Our combined clinical, genetic and histology data extend the current genetic landscape of GNE myopathy by introducing a variant of early-onset and proximal muscle vulnerability. Muscle proteomics enabled a defined catalogue of tissue markers holding the potential to evaluate the pathogenicity of novel variants as exemplified on p.(Val569Leu) mutant muscle. Studies on serum derived from GNEM patients led to the identification of two novel blood biomarkers, serglycin and periostin, whereby an impact of periostin in fibrotic remodeling of muscle was demonstrated thus underlying the pathophysiological relevance. [ABSTRACT FROM AUTHOR]

Delandistrogene moxeparvovec, an rAAVrh74-based gene transfer therapy designed to address absent functional dystrophin by delivering a transgene encoding engineered micro-dystrophin, is approved (US, UAE, Qatar, Kuwait, Bahrain, Oman; Mar 2024) for ambulatory pediatric patients (pts) aged 4 through 5 years with Duchenne muscular dystrophy (DMD) with a confirmed DMD mutation. We report Part 1 (Week [Wk] 52) results of the two-part EMBARK trial (NCT05096221). Key inclusion criteria: Ambulatory males aged ≥4 <8 years with a confirmed DMD mutation within exons 18–79 (inclusive); screening North Star Ambulatory Assessment (NSAA) score >16 <29; rAAVrh74 antibody titers <1:400; stable daily oral corticosteroid dose (≥12 wks pre-screening). Pts received single-dose delandistrogene moxeparvovec (1.33×10¹⁴ vg/kg, intravenous) or placebo. Primary endpoint: change from baseline to Wk 52 in NSAA total score. At Wk 52 (N=125), the primary endpoint was not statistically significant—least-squares mean between-group difference for delandistrogene moxeparvovec (n=63) vs placebo (n=61) was 0.65 points (P=0.2441). Time to rise, 10-meter walk/run, stride velocity 95th centile and time to ascend 4 steps favored the treatment group (P<0.05, nominal). A prespecified global statistical test on a composite of functional endpoints supported treatment benefit (P=0.0044). Micro-dystrophin was expressed in the treatment group at Wk 12 (western blot). Mean creatine kinase levels decreased from baseline with delandistrogene moxeparvovec vs placebo (P=0.0002, nominal). Treatment-related treatment-emergent adverse events (AEs) included vomiting (54.0%), nausea (31.7%) and decreased appetite (27.0%), with no study discontinuations, deaths or complement-mediated AEs. Based on the totality of functional assessments including timed function tests, treatment with delandistrogene moxeparvovec indicates beneficial modification of disease trajectory. Safety was manageable and consistent with prior experience. [ABSTRACT FROM AUTHOR]

X-linked myotubular myopathy (XLMTM) is a rare neuromuscular disorder caused by mutations in the myotubularin (MTM1) gene, affecting 1:50000 male births. The clinical severity varies, however the need for ventilatory support is often inevitable. At present, no approved therapies are available for XLMTM. Patient management is mainly supportive. A first MTM1 gene replacement therapy tested in a clinical trial showed notable improvements on ventilator dependence and motor function but raised serious safety concerns. Forty patients have been enrolled in our European prospective natural history study of patients with XL-MTM designed to characterize the disease course by using standardized evaluations adjusted for age, ambulatory and respiratory status. At 3-year follow-up, data were available for 26 patients (65%) with a mean age of 14.3 (min 3.4, max 53.8) years. Drop-outs were due to loss to follow-up (n=1), death (n=1) and early withdrawal from the study (n=3). Nineteen percent (5/26) of patients were under the age of 6 years old. Among non-ambulant patients at baseline (n=19), only 1 acquired the ability to walk during the study. Respiratory function, strength and motor function did not statistically change. Muscle strength measured by MyoPinch and MyoGrip showed a mean decline of -1,04 kg (n=20) and -0.33kg (n=20) respectively. MFM D1 showed the most important decline of -2.98% at 36 months. Nine patients presented with a mean increase of grip strength of 1.5kg (SD 4.18) (n=13). Ten patients showed a mean increase of the pinch strength of 0.16kg (SD 0.71) (n=14). Correlation data will be presented. This first multicentric longitudinal natural history in all types of patients with XL-MTM demonstrates clearly that patients experience a decline over years that can be reliably measured. We have identified several strength-, functional and respiratory outcomes that can be used to build robust clinical trials in this population. [ABSTRACT FROM AUTHOR]

Pathogenic variants in the DNM2 gene can cause an autosomal dominant form of centronuclear myopathy (AD-CNM). The phenotypical spectrum is highly variable in severity and time of onset ranging from childhood to adulthood. Currently there are no approved treatments and patients mainly rely on supportive therapies. We set up an international, prospective natural history study in order to characterize the disease course in this patient cohort and to identify prognostic variables of the disease. At predefined intervals over 36 months, assessments of respiratory and motor function, as well as activity and muscle strength, were carried out using standardized measures. Medical files were assessed for cardiac function. Hepatic function was evaluated using ultrasound and blood analysis. We administered measures for quality of life and patient reported outcomes. 15 patients with AD-CNM caused by variants in the DNM2 gene were included for participation (female n=9 vs. male n=6; ambulant n=10 vs. non-ambulant n=5; mean age 41,5 years ±23,3 SD). 9 patients were available for assessment after 36 months (dropout n=6). Grip and pinch strength showed an age-related trajectory with peaks at 50 years of age, while the total strength assessed by the MFM32 reached its maximum just below 40 years, although with higher variability. Overall, patients remained largely stable for muscle strength and function, as well as pulmonary function over 36 months. No patient's ambulatory status changed over the 36 months period. Conclusive longitudinal data will be presented. This multicentric natural history study describes the disease trajectory of a cohort of patients with AD-CNM over 36 months. We identified outcomes and measures that can be used to assess several bodily functions relevant to both patients and investigators, as needed for the solid design of clinical trials [ABSTRACT FROM AUTHOR]

Stride velocity 95th centile (SV95C) is a wearable-derived endpoint representing the 5% fastest strides taken during everyday living. In July 2023, SV95C received European Medicines Agency (EMA) qualification for use as a primary endpoint in trials of patients with Duchenne muscular dystrophy (DMD) aged ≥ 4 years-becoming the first digital endpoint to receive such qualification. We present the data supporting this qualification, providing insights into the evidentiary basis of qualification as a digital clinical outcome assessment. Clinical trials, natural history studies, and patient surveys (ages 5 - 14 years) showed that SV95C is accurate, valid, reliable, sensitive, and clinically meaningful. SV95C significantly correlated with traditional DMD assessments, increased rapidly after steroid initiation (0.090 m/s 3 months post-treatment), and declined steadily in patients on stable steroid regimens. Compared with traditional assessments, SV95C demonstrated earlier sensitivity to disease progression (3 vs 9 months) and greater sensitivity at 12 months. Distribution- and anchor-based approaches revealed a change of - 0.10 to - 0.20 m/s as clinically meaningful. The EMA qualification of SV95C illustrates the willingness of regulators to accept novel digital endpoints for drug approval, setting an important precedent for the evidentiary basis of regulatory digital endpoint qualification that could transform clinical development in disorders affecting movement., Competing Interests: Declarations. Competing interests: L.S. is a member of scientific advisory boards for Novartis Gene Therapies (formerly AveXis), Biogen, Biophytis, Cytokinetics, Dynacure, F. Hoffmann-La Roche Ltd, GeneTx Biotherapeutics, REGENXBIO, Santhera Pharmaceuticals, and Sarepta Therapeutics, Inc., has consulted for Pfizer and Affinia, conducts research funded by Novartis Gene Therapies (formerly AveXis), Biogen, and F. Hoffmann-La Roche Ltd, holds part of the patent WO2017129890A1 with no financial interest, and has provided consultancy services to SYSNAV. P.S. is an employee of and hold stocks in F. Hoffmann-La Roche Ltd. M.P. reports no disclosures relevant to the manuscript. A.M. reports no disclosures relevant to the manuscript. N.B. reports no disclosures relevant to the manuscript. V.A.S. provides intellectual consultancies and teaching activities for Biogen, F. Hoffmann-La Roche Ltd, Novartis, Lupin, Dyne Therapeutics, and PTC Therapeutics. C.V. reports participation in scientific advisory boards for Novartis Gene Therapies (formerly AveXis), Biogen, PTC Therapeutics, F. Hoffmann-La Roche Ltd, Italfarmaco, and Sarepta Therapeutics, Inc., and is involved in research funded by Novartis Gene Therapies (formerly AveXis), Biogen, Sarepta Therapeutics, Inc., and F. Hoffmann-La Roche Ltd. U.S.S. is a member of scientific advisory boards for Novartis Gene Therapies (formerly AveXis), Biogen, F. Hoffmann-La Roche Ltd, Pfizer, Santhera Pharmaceuticals, Sarepta Therapeutics, Inc., Italfarmaco, and PTC Therapeutics, and has received honoraria for invited talks or chair positions in scientific symposia from Novartis Gene Therapies (formerly AveXis), Biogen, F. Hoffmann-La Roche Ltd, Pfizer, Santhera Pharmaceuticals, Sarepta Therapeutics, Inc., Italfarmaco, and PTC Therapeutics. M.S. has provided consultancy services for and received honoraria (as a member of scientific advisory boards) from Biogen, F. Hoffmann-La Roche Ltd, and Novartis Gene Therapies (formerly AveXis). A.M.S. reports no disclosures relevant to the manuscript. S.C.P. reports participation in scientific advisory boards for EspeRare Foundation, Wave Life Sciences Ltd, Argenx, and Sarepta Therapeutics, Inc., and has provided consultancy service for Alia Therapeutics and LSC Lifesciences. M.T. has participated in scientific advisory boards for Biogen, PTC Therapeutics, F. Hoffmann-La Roche Ltd, and Sarepta Therapeutics, Inc., and has received honoraria for invited lectures from Biogen, Sarepta Therapeutics, Inc., and PTC Therapeutics. A.N. reports participation in scientific advisory boards for Novartis Gene Therapies (formerly AveXis), Biogen, PTC Therapeutics, F. Hoffmann-La Roche Ltd, Italfarmaco, Pfizer, Dyne Therapeutics, and Sarepta Therapeutics, Inc., and is involved in research funded by Novartis Gene Therapies (formerly AveXis) and Biogen. P.F. reports no disclosures relevant to the manuscript. T. S. is an employee of Sarepta Therapeutics, Inc. and has stock and stock options. R.D.D. is Head of Clinical Development at Solid Biosciences, was previously employed at F. Hoffmann-La Roche Ltd, Santhera Pharmaceuticals, and Novartis, and has stock in Solid Biosciences and F. Hoffmann-La Roche Ltd. N.G. reports activities as a Data and Safety Monitoring Board member for Pfizer, Antisense Therapeutics, Wave Life Sciences Ltd, and Genethon. E.M. has served on clinical steering committees and/or as a consultant and received compensation from Italfarmaco, PTC Therapeutics, Sarepta Therapeutics, Inc., Santhera Pharmaceuticals, Pfizer Inc., F. Hoffmann-La Roche Ltd, Wave Life Sciences, NS Pharma, and Dyne Therapeutics, and is involved in research funded by Novartis Gene Therapies (formerly AveXis), Biogen, Sarepta Therapeutics, Inc., and F. Hoffmann-La Roche Ltd. V.S. has served on scientific advisory boards for Astellas Gene Therapies, Biogen, Edgewise Therapeutics, Ipsen, Kate Therapeutics, ML Bio Solutions, Novartis Gene Therapies, PepGen, F. Hoffmann-La Roche Ltd, Sanofi, Sarepta Therapeutics, Inc., Vertex Pharmaceuticals, and Wave Therapeutics, has received speaker honoraria from Pfizer, F. Hoffmann-La Roche Ltd, Sanofi, and Sarepta Therapeutics, Inc., has received grants for clinical research from Sarepta Therapeutics, Inc. and Sanofi, and has received support from the NIHR Newcastle Biomedical Research Centre. M.G.O. is an employee of and hold stocks in F. Hoffmann-La Roche Ltd. J.B. is an employee of and hold stocks in F. Hoffmann-La Roche Ltd. F.M. reports participation in scientific advisory boards for Novartis Gene Therapies (formerly AveXis), Biogen, F. Hoffmann-La Roche Ltd, Italfarmaco, Pfizer, Dyne Therapeutics, and Sarepta Therapeutics, Inc., and is involved in research funded by Novartis Gene Therapies (formerly AveXis), Biogen, Sarepta Therapeutics, Inc., and F. Hoffmann-La Roche Ltd. A.T. has nothing to disclose other than his employment at SYSNAV, a company that collaborated with the Institute of Myology to create ActiMyo®. M.A. was an employee of SYSNAV at the time that this manuscript was developed. D.E. has nothing to disclose other than his employment at SYSNAV, a company that collaborated with the Institute of Myology to create ActiMyo®., (© 2024. The Author(s).)

This study explores the potential of 1 H-NMR spectroscopy-based metabolic profiling in various biofluids as a diagnostic and predictive modality to assess disease severity in individuals with 5q spinal muscular atrophy. A total of 213 biosamples (urine, plasma, and CSF) from 153 treatment-naïve patients with SMA across five German centers were analyzed using 1 H-NMR spectroscopy. Prediction models were developed using machine learning algorithms which enabled the patients with SMA to be grouped according to disease severity. A quantitative enrichment analysis was employed to identify metabolic pathways associated with disease progression. The results demonstrate high sensitivity (84-91%) and specificity (91-94%) in distinguishing treatment-naïve patients with SMA from controls across all biofluids. The urinary and plasma profiles differentiated between early-onset (type I) and later-onset (type II/III) SMA with over 80% accuracy. Key metabolic differences involved alterations in energy and amino acid metabolism. This study suggests that 1 H-NMR spectroscopy based metabolic profiling may be a promising, non-invasive tool to identify patients with SMA and for severity stratification, potentially complementing current diagnostic and prognostic strategies in SMA management.

Duchenne muscular dystrophy (DMD) is a rare, X-linked neuromuscular disease caused by pathogenic variants in the DMD gene that result in the absence of functional dystrophin, beginning at birth and leading to progressive impaired motor function, loss of ambulation and life-threatening cardiorespiratory complications. Delandistrogene moxeparvovec, an adeno-associated rh74-viral vector-based gene therapy, addresses absent functional dystrophin in DMD. Here the phase 3 EMBARK study aimed to assess the efficacy and safety of delandistrogene moxeparvovec in patients with DMD. Ambulatory males with DMD, ≥4 years to <8 years of age, were randomized and stratified by age group and North Star Ambulatory Assessment (NSAA) score to single-administration intravenous delandistrogene moxeparvovec (1.33 × 10 14 vector genomes per kilogram; n = 63) or placebo (n = 62). At week 52, the primary endpoint, change from baseline in NSAA score, was not met (least squares mean 2.57 (delandistrogene moxeparvovec) versus 1.92 (placebo) points; between-group difference, 0.65; 95% confidence interval (CI), -0.45, 1.74; P = 0.2441). Secondary efficacy endpoints included mean micro-dystrophin expression at week 12: 34.29% (treated) versus 0.00% (placebo). Other secondary efficacy endpoints at week 52 (between-group differences (95% CI)) included: Time to Rise (-0.64 (-1.06, -0.23)), 10-meter Walk/Run (-0.42 (-0.71, -0.13)), stride velocity 95th centile (0.10 (0.00, 0.19)), 100-meter Walk/Run (-3.29 (-8.28, 1.70)), time to ascend 4 steps (-0.36 (-0.71, -0.01)), PROMIS Mobility and Upper Extremity (0.05 (-0.08, 0.19); -0.04 (-0.24, 0.17)) and number of NSAA skills gained/improved (0.19 (-0.67, 1.06)). In total, 674 adverse events were recorded with delandistrogene moxeparvovec and 514 with placebo. There were no deaths, discontinuations or clinically significant complement-mediated adverse events; 7 patients (11.1%) experienced 10 treatment-related serious adverse events. Delandistrogene moxeparvovec did not lead to a significant improvement in NSAA score at week 52. Some of the secondary endpoints numerically favored treatment, although no statistical significance can be claimed. Safety was manageable and consistent with previous delandistrogene moxeparvovec trials. ClinicalTrials.gov: NCT05096221., (© 2024. The Author(s).)

Background and Purpose: Spinal muscular atrophy (SMA) as the second most common neurodegenerative disorder in childhood is characterized by the deficiency of survival of motor neuron (SMN) protein leading predominantly to degeneration of alpha motor neurons and consequently to progressive muscle weakness and atrophy. Besides some biomarkers like SMN2 copy number therapeutic biomarkers for SMA with known relevance for neuromuscular transmission are lacking. Here, we examined the potential of Thrombospondin-4 (TSP4) to serve as a cerebrospinal fluid (CSF) biomarker, which may also indicate treatment response., Methods: We used untargeted proteomic analyses to determine biomarkers in CSF samples derived from pediatric pre-symptomatic (n = 6) and symptomatic (n = 4) SMA patients. The identified biomarker TSP4 was then validated in additional 68 CSF samples (9 adult and 24 pediatric SMA patients, 5 adult and 13 pediatric non-disease controls in addition to 17 pediatric disease controls) by enzyme-linked immunosorbent assay (ELISA) as an additional analytical approach., Results: Untargeted proteomic analyses of CSF identified a dysregulation of TSP4 and revealed a difference between pre-symptomatic SMA patients and patients identified after the onset of first symptoms. Subsequent ELISA-analyses showed that TSP4 is decreased in pediatric but not adult SMA patients. CSF of pediatric patients with other neurological disorders demonstrated no alteration of TSP4 levels. Furthermore, CSF TSP4 levels of pediatric SMA patients increased after first dose of Nusinersen., Conclusions: We found that TSP4 levels are exclusively reduced in CSF of pediatric SMA patients and increase after treatment, leading us to the hypothesis that TSP4 could serve as a CSF biomarker with the potential to monitor treatment response in pediatric SMA patients. Moreover, TSP4 enable to distinguish pre-symptomatic and symptomatic patients suggesting a potential to serve as a stratification marker., (© 2024. The Author(s).)

Background: TCF4 acts as a transcription factor that binds to the immunoglobulin enhancer Mu-E5/KE5 motif. Dominant variants in TCF4 are associated with the manifestation of Pitt-Hopkins syndrome, a rare disease characterized by severe mental retardation, certain features of facial dysmorphism and, in many cases, with abnormalities in respiratory rhythm (episodes of paroxysmal tachypnea and hyperventilation, followed by apnea and cyanosis). Frequently, patients also develop epilepsy, microcephaly, and postnatal short stature. Although TCF4 is expressed in skeletal muscle and TCF4 seems to play a role in myogenesis as demonstrated in mice, potential myopathological findings taking place upon the presence of dominant TCF4 variants are thus far not described in human skeletal muscle., Method: To address the pathological effect of a novel deletion affecting exons 15 and 16 of TCF4 on skeletal muscle, histological and immunofluorescence studies were carried out on a quadriceps biopsy in addition to targeted transcript studies and global proteomic profiling., Results: We report on muscle biopsy findings from a Pitt-Hopkins patient with a novel heterozygous deletion spanning exon 15 and 16 presenting with neuromuscular symptoms. Microscopic characterization of the muscle biopsy revealed moderate fiber type I predominance, imbalance in the proportion of fibroblasts co-expressing Vimentin and CD90, and indicate activation of the complement cascade in TCF4-mutant muscle. Protein dysregulations were unraveled by proteomic profiling. Transcript studies confirmed a mitochondrial vulnerability in muscle and confirmed reduced TCF4 expression., Conclusion: Our combined findings, for the first time, unveil myopathological changes as phenotypical association of Pitt-Hopkins syndrome and thus expand the current clinical knowledge of the disease as well as support data obtained on skeletal muscle of a mouse model., (© 2024. The Author(s).)

Importance: There is increasing evidence that early diagnosis and treatment are key for outcomes in infants with spinal muscular atrophy (SMA), and newborn screening programs have been implemented to detect the disease before onset of symptoms. However, data from controlled studies that reliably confirm the benefits of newborn screening are lacking., Objective: To compare data obtained on patients with SMA diagnosed through newborn screening and those diagnosed after clinical symptom onset., Design, Setting, and Participants: This nonrandomized controlled trial used data from the SMARTCARE registry to evaluate all children born between January 2018 and September 2021 with genetically confirmed SMA and up to 3 SMN2 copies. The registry includes data from 70 participating centers in Germany, Austria, and Switzerland. Data analysis was performed in February 2023 so that all patients had a minimal follow-up of 18 months., Exposure: Patients born in 2 federal states in Germany underwent screening in a newborn screening pilot project. All other patients were diagnosed after clinical symptom onset. All patients received standard care within the same health care system., Main Outcomes: The primary end point was the achievement of motor milestones., Results: A total of 234 children (123 [52.6%] female) were identified who met inclusion criteria and were included in the analysis: 44 (18.8%) in the newborn screening cohort and 190 children (81.2%) in the clinical symptom onset cohort. The mean (SD) age at start of treatment with 1 of the approved disease-modifying drugs was 1.3 (2.2) months in the newborn screening cohort and 10.7 (9.1) months in the clinical symptom onset cohort. In the newborn screening cohort, 40 of 44 children (90.9%) gained the ability to sit independently vs 141 of 190 (74.2%) in the clinical symptom onset cohort. For independent ambulation, the ratio was 28 of 40 (63.6%) vs 28 of 190 (14.7%)., Conclusions and Relevance: This nonrandomized controlled trial demonstrated effectiveness of newborn screening for infants with SMA in the real-world setting. Functional outcomes and thus the response to treatment were significantly better in the newborn screening cohort compared to the unscreened clinical symptom onset group., Trial Registration: German Clinical Trials Register: DRKS00012699.

Newborn screening for 5qSMA offers the potential for early, ideally pre-symptomatic, therapeutic intervention. However, limited data exist on the outcomes of individuals with 4 copies of SMN2, and there is no consensus within the SMA treatment community regarding early treatment initiation in this subgroup. To provide evidence-based insights into disease progression, we performed a retrospective analysis of 268 patients with 4 copies of SMN2 from the SMArtCARE registry in Germany, Austria and Switzerland. Inclusion criteria required comprehensive baseline data and diagnosis outside of newborn screening. Only data prior to initiation of disease-modifying treatment were included. The median age at disease onset was 3.0 years, with a mean of 6.4 years. Significantly, 55% of patients experienced symptoms before the age of 36 months. 3% never learned to sit unaided, a further 13% never gained the ability to walk independently and 33% of ambulatory patients lost this ability during the course of the disease. 43% developed scoliosis, 6.3% required non-invasive ventilation and 1.1% required tube feeding. In conclusion, our study, in line with previous observations, highlights the substantial phenotypic heterogeneity in SMA. Importantly, this study provides novel insights: the median age of disease onset in patients with 4 SMN2 copies typically occurs before school age, and in half of the patients even before the age of three years. These findings support a proactive approach, particularly early treatment initiation, in this subset of SMA patients diagnosed pre-symptomatically. However, it is important to recognize that the register will not include asymptomatic individuals., (© 2024. The Author(s).)

Bi-allelic variants in VWA1, encoding Von Willebrand Factor A domain containing 1 protein localized to the extracellular matrix (ECM), were linked to a neuromuscular disorder with manifestation in child- or adulthood. Clinical findings indicate a neuromyopathy presenting with muscle weakness. Given that pathophysiological processes are still incompletely understood, and biomarkers are still missing, we aimed to identify blood biomarkers of pathophysiological relevance: white blood cells (WBC) and plasma derived from six VWA1-patients were investigated by proteomics. Four proteins, BET1, HNRNPDL, NEFM and PHGDH, known to be involved in neurological diseases and dysregulated in WBC were further validated by muscle-immunostainings unravelling HNRNPDL as a protein showing differences between VWA1-patients, healthy controls and patients suffering from neurogenic muscular atrophy and BICD2-related neuromyopathy. Immunostaining studies of PHGDH indicate its involvement in apoptotic processes via co-localisation with caspase-3. NEFM showed an increase in cells within the ECM in biopsies of all patients studied. Plasma proteomics unravelled dysregulation of 15 proteins serving as biomarker candidates among which a profound proportion of increased ones (6/11) are mostly related to antioxidative processes and have even partially been described as blood biomarkers for other entities of neuromuscular disorders before. CRP elevated in plasma also showed an increase in the extracellular space of VWA1-mutant muscle. Results of our combined studies for the first time describe pathophysiologically relevant biomarkers for VWA1-related neuromyopathy and suggest that VWA1-patient derived blood might hold the potential to study disease processes of clinical relevance, an important aspect for further preclinical studies., (© 2024 The Authors. Journal of Cellular and Molecular Medicine published by Foundation for Cellular and Molecular Medicine and John Wiley & Sons Ltd.)

Background: Duchenne muscular dystrophy, the most common childhood muscular dystrophy, is caused by dystrophin deficiency. Preclinical and phase 2 study data have suggested that givinostat, a histone deacetylase inhibitor, might help to counteract the effects of this deficiency. We aimed to evaluate the safety and efficacy of givinostat in the treatment of Duchenne muscular dystrophy., Methods: This multicentre, randomised, double-blind, placebo-controlled, phase 3 trial was done at 41 tertiary care sites in 11 countries. Eligible participants were ambulant, male, and aged at least 6 years, had a genetically confirmed diagnosis of Duchenne muscular dystrophy, completed two four-stair climb assessments with a mean of 8 s or less (≤1 s variance), had a time-to-rise of at least 3 s but less than 10 s, and had received systemic corticosteroids for at least 6 months. Participating boys were randomly assigned (2:1, allocated according to a list generated by the interactive response technology provider) to receive either oral givinostat or matching placebo twice a day for 72 weeks, stratified by concomitant steroid use. Boys, investigators, and site and sponsor staff were masked to treatment assignment. The dose was flexible, based on weight, and was reduced if not tolerated. Boys were divided into two groups on the basis of their baseline vastus lateralis fat fraction (VLFF; measured by magnetic resonance spectroscopy): group A comprised boys with a VLFF of more than 5% but no more than 30%, whereas group B comprised boys with a VLFF of 5% or less, or more than 30%. The primary endpoint compared the effects of givinostat and placebo on the change in results of the four-stair climb assessment between baseline and 72 weeks, in the intention-to-treat, group A population. Safety was assessed in all randomly assigned boys who received at least one dose of study drug. When the first 50 boys in group A completed 12 months of treatment, an interim futility assessment was conducted, after which the sample size was adapted using masked data from the four-stair climb assessments. Furthermore, the starting dose of givinostat was reduced following a protocol amendment. This trial is registered with ClinicalTrials.gov, NCT02851797, and is complete., Findings: Between June 6, 2017, and Feb 22, 2022, 359 boys were assessed for eligibility. Of these, 179 were enrolled into the study (median age 9·8 years [IQR 8·1-11·0]), all of whom were randomly assigned (118 to receive givinostat and 61 to receive placebo); 170 (95%) boys completed the study. Of the 179 boys enrolled, 120 (67%) were in group A (81 givinostat and 39 placebo); of these, 114 (95%) completed the study. For participants in group A, comparing the results of the four-stair climb assessment at 72 weeks and baseline, the geometric least squares mean ratio was 1·27 (95% CI 1·17-1·37) for boys receiving givinostat and 1·48 (1·32-1·66) for those receiving placebo (ratio 0·86, 95% CI 0·745-0·989; p=0·035). The most common adverse events in the givinostat group were diarrhoea (43 [36%] of 118 boys vs 11 [18%] of 61 receiving placebo) and vomiting (34 [29%] vs 8 [13%]); no treatment-related deaths occurred., Interpretation: Among ambulant boys with Duchenne muscular dystrophy, results of the four-stair climb assessment worsened in both groups over the study period; however, the decline was significantly smaller with givinostat than with placebo. The dose of givinostat was reduced after an interim safety analysis, but no new safety signals were reported. An ongoing extension study is evaluating the long-term safety and efficacy of givinostat in patients with Duchenne muscular dystrophy., Funding: Italfarmaco., Competing Interests: Declaration of interests EM declares payment or honoraria for lectures and symposia from Sarepta Therapeutics, PTC Therapeutics, and Roche; and participation on advisory boards for Sarepta Therapeutics, NS Pharma, Santhera, PTC Therapeutics, Roche, Pfizer, WAVE Life Sciences, Italfarmaco, and Dyne Therapeutics, all outside the scope of this manuscript. JJV declares grants from PTC Therapeutics; consulting fees from Santhera, Sarepta Therapeutics, and PTC Therapeutics; and payment for lectures, presentations, speakers bureaus, manuscript writing or educational events from PTC Therapeutics, all outside the scope of this manuscript. OB-T declares grants to her institution from Roche, Novartis, Biogen, Genethon, and Metafora Biosystems; payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing or educational events from Novartis; support for attending meetings and/or travel from Novartis; participation on a data safety monitoring board or advisory board for Minoryx Therapeutics; and unpaid leadership roles with AFM-Téléthon (scientific board president) and Société Francophone de Neurogenetique, all outside the scope of this manuscript. CMZ declares grants or contracts from Biogen and Novartis; consulting fees from Sarepta Therapeutics; payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing or educational events from Sarepta Therapeutics and Optum; support for attending meetings and/or travel from Sarepta Therapeutics and Optum; and participation on a data safety monitoring board or advisory board for Sarepta Therapeutics, all outside the scope of this manuscript. JKM declares research grants to her institution from Italfarmaco, Biogen, Novartis, NS Pharma, Pfizer, PTC Therapeutics, ReveraGen Biopharma, Roche, Sarepta Therapeutics, and Alberta Children's Hospital Foundation, all outside the scope of this manuscript. NG declares payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing or educational events from Biogen; and participation on a data safety monitoring board or advisory board for Pfizer, Biogen, and WAVE Life Sciences, all outside the scope of this manuscript. WM-F declares payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing or educational events from Biogen, Roche, Sarepta Therapeutics, Sanofi-Aventis, and Novartis; payment to his institution for expert testimony from Astellas Pharma; participation on a data safety monitoring board or advisory board with payment to himself from PTC Therapeutics, Roche, and Novartis, and to his institution from Astellas Pharma; and unpaid leadership roles for the Deutsche Gesellschaft für Muskelkranke and Glykogenose Deutschland, all outside the scope of this manuscript. EHN declares grants to his institution from Pfizer and PTC Therapeutics; and consulting fees to his institution from Pfizer, Edgewise Therapeutics, Sarepta Therapeutics, Epirium Bio, Regenxbio, Janssen Pharmaceuticals, all outside the scope of this manuscript. US-S declares consulting fees from Santhera, PTC Therapeutics, Sarepta Therapeutics, and Pfizer, all outside the scope of this manuscript. EB declares participation on advisory boards for Pfizer, Roche, Biogen, and Novartis, all outside the scope of this manuscript. GPC declares payment for a lecture from Biogen; participation on advisory boards for PTC Therapeutics, Roche, and Sanofi Genzyme; and an unpaid leadership role (president) of the Italian Association of Myology, all outside the scope of this manuscript. KDM declares grants to her institution from Italfarmaco, Sarepta Therapeutics, PTC Therapeutics, FibroGen, Capricor Therapeutics, Pfizer, Edgewise Therapeutics, Biomarin Pharmaceutical, Centers for Disease Control and Prevention, and the National Institutes of Health; an honorarium to cover travel costs from the Muscular Dystrophy Association; reimbursement to her institution of support to attend meetings and/or travel from the Parent Project Muscular Dystrophy; participation on a data safety monitoring board or advisory board for Dyne Therapeutics, NS Pharma, TRiNDS, and Sarepta Therapeutics; and payment to her institution for participation in a grant review board for the Muscular Dystrophy Association, all outside the scope of this manuscript. LS declares grants to his institution from Roche, Novartis, Biogen, Sysnav Healthcare, Zentech, and Perkin Elmer; consulting fees from Pfizer, Santhera, Dynacure, Dyne Therapeutics, Audentes Therapeutics, Zentech, Sarepta Therapeutics, Roche, Novartis, Biohaven, Biogen Digital Health, Scholar Rock, Regenexbio, and Evox Therapeutics; payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing or educational events from Roche, Biogen, and Novartis; payment for expert testimony from Audentes Therapeutics; participation on a data safety monitoring board or advisory board for FibroGen, Lupin Therapeutics, and Illumina; and unpaid leadership roles for the World Muscle Society, SMA Europe, and the Association Belge contre les Maladies neuro-Musculaires , all outside the scope of this manuscript. KV declares a research service agreement for the current clinical trial. Outside of this manuscript, she declares grants from the National Institutes of Health; and research service support from Sarepta Therapeutics, Catabasis Pharmaceuticals, PTC Therapeutics, Summit Therapeutics, Astellas Pharma, ML Bio/VCU, and Edgewise Therapeutics, all directed to the University of Florida, Gainesville, FL, USA. JJ declares consulting fees from Biogen, AveXis, Sarepta Therapeutics, and Roche; and receipt of travel and speaker fees from PTC Therapeutics, all outside the scope of this manuscript. SM declares payment or honoraria for lectures, presentations, speakers bureaus, manuscript writing or educational events from Roche, Biogen, and Novartis, all outside the scope of this manuscript. SS declares payments for participation in ad-hoc advisory boards for PTC Medical, Sarepta Therapeutics, Pfizer, and Roche; and payment for chairing a symposium sponsored by Sarepta Therapeutics at the British Neuropsychiatry Association 2019 annual meeting, all outside the scope of this manuscript. LM declares study funding and payments to her institution for provision of study materials in the current clinical trial. Outside the current manuscript she declares grants to her institution from Anavex Life Sciences. KS declares grants or contracts from Biogen (principal investigator and clinical trial site for a nusinersen study), ReveraGen-Santhera (principal investigator for a vamorolone study), and PTC Therapeutics (principal investigator for an ataluren study); payment to her institution from Roche for a teaching and educational seminar on spinal muscular atrophy; an honorarium from Novartis for a TREAT NMD talk on spinal muscular atrophy; and attendance at advisory boards for Biogen and Roche (including travel), all outside the scope of this manuscript. CGL declares contracts (as principal investigator) from Sarepta Therapeutics, Dyne Therapeutics, Avidity Biosciences, FibroGen, Scholar Rock, and Biohaven; consulting fees from Sarepta Therapeutics (payments to her institution), NS Pharma (payments to herself), and Avidity (payments to her institution and to herself); payments for participation in a speakers bureau from Biogen; and support for attending meetings and/or travel from the Muscular Dystrophy Association, CureCMD, and CureSMA, all outside the scope of this manuscript. KC declares payments directly to KJC Statistics for statistical support of the current trial. SC, NC, and PB are employees of Italfarmaco, the sponsor of the current trial. CMM reports receiving grants or research support from Astellas Pharma, BioMarin Pharmaceutical, Capricor Therapeutics, Catabasis Pharmaceuticals, Edgewise Therapeutics, Italfarmaco, Pfizer, PTC Therapeutics, and Santhera Pharmaceuticals; and consulting fees from Sarepta Therapeutics, Astellas Pharma, Avidity Biosciences, BioMarin Pharmaceutical, Bristol Myers Squib, Capricor Therapeutics, Catabasis Pharmaceuticals, Edgewise Therapeutics, Eli Lilly, Epirium Bio, Entrada Therapeutics, Gilead Sciences, Halo Therapeutics, Italfarmaco, Novartis, PepGen, Pfizer, PTC Therapeutics, Prosensa, and Santhera Pharmaceuticals, all outside the scope of this manuscript. BB and GZ declare no competing interests., (Copyright © 2024 Elsevier Ltd. All rights reserved.)

Spinal muscular atrophy (SMA) is a neuromuscular disorder caused by recessive pathogenic variants affecting the survival of motor neuron (SMN1) gene (localized on 5q). In consequence, cells lack expression of the corresponding protein. This pathophysiological condition is clinically associated with motor neuron (MN) degeneration leading to severe muscular atrophy. Additionally, vulnerability of other cellular populations and tissues including skeletal muscle has been demonstrated. Although the therapeutic options for SMA have considerably changed, treatment responses may differ thus underlining the persistent need for validated biomarkers. To address this need and to identify novel marker proteins for SMA, we performed unbiased proteomic profiling on cerebrospinal fluid derived (CSF) from genetically proven SMA type 1-3 cases and afterwards performed ELISA studies on CSF and serum samples to validate the potential of a novel biomarker candidates in both body fluids. To further decipher the pathophysiological impact of this biomarker, immunofluorescence studies were carried out on spinal cord and skeletal muscle derived from a 5q-SMA mouse model. Proteomics revealed increase of LARGE1 in CSF derived from adult patients showing a clinical response upon treatment with nusinersen. Moreover, LARGE1 levels were validated in CSF samples of further SMA patients (type 1-3) by ELISA. These studies also unveiled a distinguishment between groups in improvement of motor skills: adult patients do present with lowered level per se at baseline visit while no elevation upon treatment in the pediatric cohort can be observed. ELISA-based studies of serum samples showed no changes in the pediatric cohort but unraveled elevated level in adult patients responding to future intervention with nusinersen, while non-responders did not show a significant increase. Additional immunofluorescence studies of LARGE1 in MN and skeletal muscle of a SMA type 3 mouse model revealed an increase of LARGE1 during disease progression. Our combined data unraveled LARGE1 as a protein dysregulated in serum and CSF of SMA-patients (and in MN and skeletal muscle of SMA mice) holding the potential to serve as a disease marker for SMA and enabling to differentiate between patients responding and non-responding to therapy with nusinersen., (© 2024. The Author(s).)