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Abstract Background Butane is an aliphatic hydrocarbon used in various commercial products. While numerous reports of sudden cardiac-related deaths from butane inhalation have been described, butane-associated acute encephalopathy has rarely been reported. Case presentation A 38-year-old man presented with cognitive dysfunction after butane gas inhalation. Neuropsychological test results showed impairments in verbal and visual memory, and frontal executive function. Diffusion weighted MRI revealed symmetric high-signal changes in the bilateral hippocampus and globus pallidus. FDG-PET demonstrated decreased glucose metabolism in the bilateral precuneus and occipital areas and the left temporal region. At the 8-month follow-up, he showed still significant deficits in memory and frontal functions. Diffuse cortical atrophy with white matter hyperintensities and extensive glucose hypometabolism were detected on follow-up MRI and FDG-PET, respectively. Brain autopsy demonstrated necrosis and cavitary lesions in the globus pallidus. Conclusions Only a few cases of butane encephalopathy have been reported to date. Brain lesions associated with butane encephalopathy include lesions in the bilateral thalamus, insula, putamen, and cerebellum. To the best of our knowledge, this is the first report on bilateral hippocampal and globus pallidal involvement in acute butane encephalopathy. The pathophysiology of central nervous system complications induced by butane intoxication is not yet fully understood. However, the direct toxic effects of butane or anoxic injury secondary to cardiac arrest or respiratory depression have been suggested as possible mechanisms of edematous changes in the brain after butane intoxication.

GNE myopathy, caused by biallelic mutations in the GNE gene, is characterized by initial ankle dorsiflexor weakness and rimmed vacuoles in the muscle histopathology, resulting in reduced sialic acid production. Sialyllactose is a source of sialic acid. We performed a pilot clinical trial to analyze the pharmacokinetic properties of 6′-sialyllactose (6SL) and evaluated the safety, and efficacy of oral 6SL in patients with GNE myopathy. Ten participants were in the pharmacokinetic study, and 20 in the subsequent clinical trial. For the pharmacokinetic study, participants were administered either 3 g (low-dose) or 6 g (high-dose) of 6SL in a single dose. Plasma concentrations of 6SL, sialic acid, and sialic acid levels on the surface of red blood cells were periodically assessed in blood samples. Patients were randomly allocated to test (low- and high-dose groups) or placebo groups for the trial. Motor function, ambulation, plasma 6SL and sialic acid concentrations, GNE myopathy-functional activity scale scores, and MRI findings were assessed. 6SL was well tolerated, except for self-limited gastrointestinal discomfort. Free sialic acid in both low- and high-dose groups significantly increased at 6 and 12 weeks, but not in the placebo group. In the high-dose group, proximal limb powers improved with daily 6SL. Considering the fat fraction on muscle MRI, results in the high-dose group were superior to those in the low-dose group. 6SL may be a good candidate for GNE myopathy therapeutics as it induces an increase or reduces the decrease in limb muscle power, attenuates muscle degeneration, and improves the biochemical properties of sialic acid.

We propose a digital biomarker related to muscle strength and muscle endurance (DB/MS and DB/ME) for the diagnosis of muscle disorders based on a multi-layer perceptron (MLP) using stimulated muscle contraction. When muscle mass is reduced in patients with muscle-related diseases or disorders, measurement of DBs that are related to muscle strength and endurance is needed to suitably recover damaged muscles through rehabilitation training. Furthermore, it is difficult to measure DBs using traditional methods at home without an expert; moreover, the measuring equipment is expensive. Additionally, because traditional measurements depend on the subjectb’s volition, we propose a DB measurement technique that is unaffected by the subjectb’s volition. To achieve this, we employed an impact response signal (IRS) based on multi-frequency electrical stimulation (MFES) using an electromyography sensor. The feature vector was then extracted using the signal. Because the IRS is obtained from stimulated muscle contraction, which is caused by electrical stimulation, it provides biomedical information about the muscle. Finally, to estimate the strength and endurance of the muscle, the feature vector was passed through the DB estimation model learned through the MLP. To evaluate the performance of the DB measurement algorithm, we collected the MFES-based IRS database for 50 subjects and tested the model with quantitative evaluation methods using the reference for the DB. The reference was measured using torque equipment. The results were compared with the reference, indicating that it is possible to check for muscle disorders which cause decreased physical performance using the proposed algorithm.

Mutations in the manganese transporter gene SLC39A14 lead to inherited disorders of manganese metabolism. Chelation therapy with edetate calcium disodium (SLC39A14 deficiencyTA) is known to effectively reduce manganese deposition. We describe the first identified Korean case of SLC39A14-associated manganism and the treatment response to a 5-year chelation therapy. An 18-year-old female presented with childhood-onset dystonia. Magnetic resonance imaging showed T1 hyperintensity throughout the basal ganglia, brainstem, cerebellum, cerebral and cerebellar white matter, and pituitary gland. Blood manganese levels were elevated, and whole-exome sequencing revealed compound heterozygous mutations in SLC39A14. Treatment with intravenous CaNa2EDTA led to a significant reduction in serum manganese levels and T1 hyperintensities. However, her dystonia improved insignificantly. Hence, early diagnosis of this genetic disorder is essential because it is potentially treatable. Even though our treatment did not significantly reverse the establish deficits, chelation therapy could have been more effective if it was started at an earlier stage of the disease.

Abstract Background A single cycle (two repeated treatments) with intrathecal autologous bone marrow-derived mesenchymal stem cells (BM-MSCs, 26-day interval) showed safety and provided therapeutic benefit lasting 6 months in patients with ALS but did not demonstrate long-term efficacy. This phase III clinical trial (ALSUMMIT) protocol was developed to evaluate the long-term efficacy and safety of the combined protocol of single-cycle intrathecal therapy and three additional booster injections of BM-MSC (Lenzumestrocel) treatment in patients with ALS. Methods ALSUMMIT is a multicentre, randomized, double-blind, parallel-group, sham procedure-controlled, phase III trial for ALS. The 115 subjects will be randomized (1:2:2) into three groups: (1) study Group 1 (single-cycle, two repeated injections with 26-day interval), (2) study Group 2 (single-cycle + three additional booster injections at 4, 7, and 10 months), and (3) the control group. Participants who have an intermediate rate of disease progression will be included in this trial to reduce clinical heterogeneity. The primary endpoint will be evaluated by combined assessment of function and survival (CAFS), also known as joint rank scores (JRS), at 6 months (study Group 1 vs. control) and 12 months (study Group 2 vs. control) after the first Lenzumestrocel or placebo administration. Safety assessment will be performed throughout the study period. Additionally, after the 56-week main study, a long-term follow-up observational study will be conducted to evaluate the long-term efficacy and safety up to 36 months. Discussion Lenzumestrocel is the orphan cell therapy product for ALS conditionally approved by the South Korea Ministry of Food and Drug Safety (MFDS). This ALSUMMIT protocol was developed for the adoption of enrichment enrolment, add-on design, and consideration of ethical issues for the placebo group. Trial registration ClinicalTrials.gov NCT04745299 . Registered on Feb 9, 2021. Clinical Research Information Service (CRIS) KCT0005954 . Registered on Mar 4, 2021.

A 49-year-old man developed recurrent myalgia and hyperCKemia during acute attacks of neuromyelitis optica. Muscle biopsy was performed, and the pathological findings were analyzed. Predominant myofibrillar pathology was observed, which constitutes a unique finding that has not been reported before. This case result shows that neuromyelitis optica-associated hyperCKemia can produce variable pathologic phenotypes. Further studies are needed to elucidate the relationship between myofibril destruction and aquaporin 4 autoimmunity.

Biallelic mutations in the dysferlin gene cause limb-girdle muscular dystrophy 2B or Miyoshi distal myopathy. We found that nonsense mutations are the most common mutation type among Korean patients with dysferlinopathy; more than half of the patients have at least one nonsense allele, which may be amenable to readthrough therapy. We generated a knockin mouse, dqx, harboring DYSF p.Q832∗ mutation. Homozygous dqx mice lacked dysferlin in skeletal muscle, while 2 weeks of oral ataluren restored dysferlin expression and ameliorated skeletal muscle pathology. Their physical performance improved, and protection against eccentric contractions was noted. The improvement was most evident in mice treated with oral ataluren of 0.9 mg/mL. These improvements were sustained for 8 weeks in ataluren-treated dqx mice, while the parameters of A/J mice treated with ataluren over the same period did not improve. These results support that readthrough therapy by oral ataluren may also be applicable to dysferlinopathy patients with nonsense mutation.

Background Brainstem encephalitis is a rare, severe, and potentially life-threatening inflammation of the central nervous system, exhibiting various treatment responses and outcomes owing to multiple etiologies. Case Report We describe the favorable outcome of salvage immunotherapy using a combination of infliximab and methotrexate in a 62-year-old woman with refractory brainstem encephalitis. The patient was initially presumed to be at a subacute stage of medullary infarction but showed progressively worsening conditions involving cervical myelopathy, despite having completed the schedule of subsequent immunotherapy with intravenous methylprednisolone, immunoglobulin, and rituximab. After completion of four sessions of weekly rituximab injection, she was treated with 5 mg/kg of infliximab, scheduled at 0, 2, and 6 weeks, along with methotrexate (weekly 12.5 mg). After completion of infliximab injection and maintenance with methotrexate treatment, she showed an improving course of quadriplegia. Conclusion This case report provides evidence for the potential efficacy of infliximab with methotrexate in cases of refractory brainstem encephalitis.

Abstract Among the three isoforms encoded by Rtn4, Nogo-A has been intensely investigated as a central nervous system inhibitor. Although Nogo-A expression is increased in muscles of patients with amyotrophic lateral sclerosis, its role in muscle homeostasis and regeneration is not well elucidated. In this study, we discovered a significant increase in Nogo-A expression in various muscle-related pathological conditions. Nogo−/− mice displayed dystrophic muscle structure, dysregulated muscle regeneration following injury, and altered gene expression involving lipid storage and muscle cell differentiation. We hypothesized that increased Nogo-A levels might regulate muscle regeneration. Differentiating myoblasts exhibited Nogo-A upregulation and silencing Nogo-A abrogated myoblast differentiation. Nogo-A interacted with filamin-C, suggesting a role for Nogo-A in cytoskeletal arrangement during myogenesis. In conclusion, Nogo-A maintains muscle homeostasis and integrity, and pathologically altered Nogo-A expression mediates muscle regeneration, suggesting Nogo-A as a novel target for the treatment of myopathies in clinical settings.

To establish an efficient, safe immunosuppressive regimen of adeno-associated vector (AAV)-mediated gene therapy for Duchenne muscular dystrophy (DMD), we evaluated the effect of tacrolimus (FK506) on skeletal muscle transduction with AAV8 and AAV9 vectors expressing the LacZ and microdystrophin (M3) genes labeled by FLAG. We utilized 3- to 4-year-old Macaca fascicularis, screened for neutralizing antibodies against AAV. 3 days before AAV injection and throughout the experiment, 0.06 mg/kg tacrolimus was intravenously administered. A viral suspension of 1 × 1013 viral genomes/muscle was intramuscularly injected bilaterally at the tibialis anterior and biceps brachii muscles, which were biopsied at 8, 16, 24, and 42 weeks after injection. Without tacrolimus, AAV8- and AAV9-mediated LacZ expression disappeared 8 and 16 weeks after transduction, respectively. With tacrolimus, AAV8/9-mediated LacZ expression persisted for at least 42 weeks after injection. At 42 weeks after AAV8CMVLacZ and AAV9CMVLacZ injection, nearly 50% and 17% of muscle fibers were positive for β-galactosidase, respectively. AAV8/9-mediated M3-FLAG expression lasted for up to 42 weeks using tacrolimus. No significant generalized toxicity was observed in any monkey. These results indicate that tacrolimus administration regulated the immune response to transgenes and truncated microdystrophin in normal primates and may enhance the benefits of AAV-mediated gene therapy for DMD.

Low serum progranulin (PGRN) is known to be associated with granulin (GRN) gene mutation and T alleles of GRN rs5848 polymorphism. However, there have been only a few Asian studies exploring these. We investigated the serum PGRN levels, rs5848 genotypes, and their relations with cerebrospinal fluid (CSF) Alzheimer's disease (AD) biomarkers in the Korean population. Serum PGRN levels, GRN rs5848 polymorphism, and GRN mutations were evaluated in 239 participants (22 cognitively unimpaired participants and 217 patients with neurodegenerative diseases). CSF AD biomarkers were also evaluated in 214 participants. There was no significant difference in the serum PGRN levels among the diagnostic groups. We could not find any GRN mutation carrier in our sample. The differences in the frequencies of the rs5848 genotypes among the clinical groups or the effects of the rs5848 genotypes on serum PGRN were not observed. There was no correlation between the serum PGRN level or rs5848 genotype and CSF AD biomarkers. Neither the T allele nor the TT genotype had an effect on the development of AD. Our results showed that serum PGRN levels were not associated with rs5848 genotypes, indicating that multiple single nucleotide polymorphisms might affect PGRN concentrations in an ethnicity-specific manner.

Objectives: Vestibular migraine (VM) is a common vestibular disorder, and familial aggregation of VM with autosomal-dominant inheritance has been described, which supports a genetic background. This study aimed to describe the clinical phenotype of a family with VM, and identify a candidate gene for VM.Methods: We recruited six individuals (four affected and two unaffected) from three consecutive generations of a Korean family with VM, and performed whole-exome sequencing to search for candidate genes.Results: All affected individuals presented with recurrent vertigo, headache, and nausea/vomiting that fulfilled the diagnostic criteria of VM. Two individuals also experienced transient hemiparesis or dysarthria during the episodes. The symptoms were triggered by physical or emotional stress. Interictal examinations showed uni- or bi-directional horizontal gaze-evoked nystagmus in three of the individuals. They had no causative mutations in genes causing familial hemiplegic migraine or episodic ataxia. Through whole-exome sequencing from three affected individuals, we identified a nonsense mutation c.3526C>T in TRPM7 that encodes a cation channel selective to Ca2+ and Mg2+.Conclusions: Alterations in intracellular Ca2+ and Mg2+ homeostasis by TRPM7 mutation may contribute to the development of the VM phenotype. Our result suggest that TRPM7 is a novel candidate gene for VM.

Objectives: The cause of Meniere's disease (MD) is unclear but likely involves genetic and environmental factors. The aim of this study was to investigate the genetic basis underlying MD by screening putative candidate genes for MD.Methods: Sixty-eight patients who met the diagnostic criteria for MD of the Barany Society were included. We performed targeted gene sequencing using next generation sequencing (NGS) panel composed of 45 MD-associated genes. We identified the rare variants causing non-synonymous amino acid changes, stop codons, and insertions/deletions in the coding regions, and excluded the common variants with minor allele frequency >0.01 in public databases. The pathogenicity of the identified variants was analyzed by various predictive tools and protein structural modeling.Results: The average read depth for the targeted regions was 1446.3-fold, and 99.4% of the targeted regions were covered by 20 or more reads, achieving the high quality of the sequencing. After variant filtering, annotation, and interpretation, we identified a total of 15 rare heterozygous variants in 12 (17.6%) sporadic patients. Among them, four variants were detected in familial MD genes (DTNA, FAM136A, DPT), and the remaining 11 in MD-associated genes (PTPN22, NFKB1, CXCL10, TLR2, MTHFR, SLC44A2, NOS3, NOTCH2). Three patients had the variants in two or more genes. All variants were not detected in our healthy controls (n = 100). No significant differences were observed between patients with and without a genetic variant in terms of sex, mean age of onset, bilaterality, the type of MD, and hearing threshold at diagnosis.Conclusions: Our study identified rare variants of putative candidate genes in some of MD patients. The genes were related to the formation of inner ear structures, the immune-associated process, or systemic hemostasis derangement, suggesting the multiple genetic predispositions in the development of MD.

Duchenne muscular dystrophy is a progressive and lethal X-linked recessive neuromuscular disease caused by mutations in the dystrophin gene. It has a high rate of diagnostic delay; early diagnosis and treatment are often not possible due to delayed recognition of muscle weakness and lack of effective treatments. Current treatments based on genetic therapy can improve clinical results, but treatment must begin as early as possible before significant muscle damage. Therefore, early diagnosis and rehabilitation of Duchenne muscular dystrophy are needed before symptom aggravation. Creatine kinase is a diagnostic marker of neuromuscular disorders. Herein, the authors report a case of an infant patient with Duchenne muscular dystrophy with a highly elevated creatine kinase level but no obvious symptoms of muscle weakness. The patient was diagnosed with Duchenne muscular dystrophy via next-generation sequencing and chromosomal microarray analysis to identify possible inherited metabolic and neuromuscular diseases related to profound hyperCKemia. The patient is enrolled in a rehabilitation program and awaits the approval of the genetic treatment in Korea. This is the first report of an infantile presymptomatic Duchenne muscular dystrophy diagnosis using next-generation sequencing and chromosomal microarray analysis.

Nogo-A (Rtn 4A), a member of the reticulon 4 (Rtn4) protein family, is a neurite outgrowth inhibitor protein that is primarily expressed in the central nervous system (CNS). However, previous studies revealed that Nogo-A was upregulated in skeletal muscles of Amyotrophic lateral sclerosis (ALS) patients. Additionally, experiments showed that endoplasmic reticulum (ER) stress marker, C/EBP homologous protein (CHOP), was upregulated in gastrocnemius muscle of a murine model of ALS. We therefore hypothesized that Nogo-A might relate to skeletal muscle diseases. According to our knocking down and overexpression results in muscle cell line (C2C12), we have found that upregulation of Nogo-A resulted in upregulation of CHOP, pro-inflammatory cytokines such as interleukin (IL)-6 and tumor necrosis factor (TNF)-α, while downregulation of Nogo-A led to downregulation of CHOP, IL-6 and TNF-α. Immunofluorescence results showed that Nogo-A and CHOP were expressed by myofibers as well as tissue macrophages. Since resident macrophages share similar functions as bone marrow-derived macrophages (BMDM), we therefore, isolated macrophages from bone marrow to study the role of Nogo-A in activation of these cells. Lipopolysaccharide (LPS)-stimulated BMDM in Nogo-KO mice showed low mRNA expression of CHOP, IL-6 and TNF-α compared to BMDM in wild type (WT) mice. Interestingly, Nogo knockout (KO) BMDM exhibited lower migratory activity and phagocytic ability compared with WT BMDM after LPS treatment. In addition, mice experiments data revealed that upregulation of Nogo-A in notexin- and tunicamycin-treated muscles was associated with upregulation of CHOP, IL-6 and TNF-α in WT group, while in Nogo-KO group resulted in low expression level of CHOP, IL-6 and TNF-α. Furthermore, upregulation of Nogo-A in dystrophin-deficient (mdx) murine model, myopathy and Duchenne muscle dystrophy (DMD) clinical biopsies was associated with upregulation of CHOP, IL-6 and TNF-α. To the best of our knowledge, this is the first study to demonstrate Nogo-A as a regulator of inflammation in diseased muscle and bone marrow macrophages and that deletion of Nogo-A alleviates muscle inflammation and it can be utilized as a therapeutic target for improving muscle diseases.

The muscular dystrophies are a group of devastating genetic disorders that affect both skeletal and cardiac muscle. An effective gene therapy for these diseases requires bodywide muscle delivery. Tyrosine mutant adeno-associated virus (AAV) has been considered as a class of highly potent gene transfer vectors. Here, we tested the hypothesis that systemic delivery of tyrosine mutant AAV can result in bodywide muscle transduction in newborn dogs. Three tyrosine mutant AAV vectors (Y445F/Y731F AAV-1, Y445F AAV-6, and Y731F AAV-9) were evaluated. These vectors expressed the alkaline phosphatase reporter gene under transcriptional regulation of either the muscle-specific Spc5-12 promoter or the ubiquitous Rous sarcoma virus promoter. Robust skeletal and cardiac muscle transduction was achieved with Y445F/Y731F AAV-1. However, Y731F AAV-9 only transduced skeletal muscle. Surprisingly, Y445F AAV-6 resulted in minimal muscle transduction. Serological study suggests that the preexisting neutralization antibody may underlie the limited transduction of Y445F AAV-6. In summary, we have identified Y445F/Y731F AAV-1 as a potentially excellent systemic gene transfer vehicle to target both skeletal muscle and the heart in neonatal puppies. Our findings have important implications in exploring systemic neonatal gene therapy in canine models of muscular dystrophy.

Epitope-specific monoclonal antibodies can provide unique insights for studying cellular proteins. Dystrophin is one of the largest cytoskeleton proteins encoded by 79 exons. The absence of dystrophin results in Duchenne muscular dystrophy (DMD). Over the last two decades, dozens of exon-specific human dystrophin monoclonal antibodies have been developed and successfully used for DMD diagnosis. Unfortunately, the majority of these antibodies have not been thoroughly characterized in dystrophin-deficient dogs, an outstanding large animal model for translational research. To fill the gap, we performed a comprehensive study on 65 dystrophin monoclonal antibodies in normal and dystrophic dogs (heart and skeletal muscle) by immunofluorescence staining and western blot. For comparison, we also included striated muscles from normal BL10 and dystrophin-null mdx mice. Our analysis revealed distinctive species, tissue and assay-dependent recognition patterns of different antibodies. Importantly, we identified 15 antibodies that can consistently detect full-length canine dystrophin in both immunostaining and western blot. Our results will serve as an important reference for studying DMD in the canine model.

The dystrophin-deficient dog is excellent large animal model for testing novel therapeutic modalities for Duchenne muscular dystrophy (DMD). Despite well-documented descriptions of dystrophic symptoms in these dogs, very few quantitative studies have been performed. Here, we developed a comprehensive set of non-invasive assays to quantify dog gait (stride length and speed), joint angle and limb mobility (for both forelimb and hind limb), and spontaneous activity at night. To validate these assays, we examined three 8-m-old mix-breed dystrophic dogs. We also included three age-matched siblings as the normal control. High-resolution video recorders were used to digitize dog walking and spontaneous movement at night. Stride speed and length were significantly decreased in affected dogs. The mobility of the limb segments (forearm, front foot, lower thigh, rear foot) and the carpus and hock joints was significantly reduced in dystrophic dogs. There was also a significant reduction of the movement in affected dogs during overnight monitoring. In summary, we have established a comprehensive set of outcome measures for clinical phenotyping of DMD dogs. These non-invasive end points would be valuable in monitoring disease progression and therapeutic efficacy in translational studies in the DMD dog model.

Loss of muscle force is a salient feature of Duchenne muscular dystrophy (DMD), a fatal disease caused by dystrophin deficiency. Assessment of force production from a single intact muscle has been considered as the gold standard for studying physiological consequences in murine models of DMD. Unfortunately, equivalent assays have not been established in dystrophic dogs. To fill the gap, we developed a novel in situ protocol to measure force generated by the extensor carpi ulnaris (ECU) muscle of a dog. We also determined the muscle length to fiber length ratio and the pennation angle of the ECU muscle. Muscle pathology and contractility were compared between normal and affected dogs. Absence of dystrophin resulted in marked histological damage in the ECU muscle of affected dogs. Central nucleation was significantly increased and myofiber size distribution was altered in the dystrophic ECU muscle. Muscle weight and physiological cross sectional area (PCSA) showed a trend of reduction in affected dogs although the difference did not reach statistical significance. Force measurement revealed a significant decrease of absolute force, and the PCSA or muscle weight normalized specific forces. To further characterize the physiological defect in affected dog muscle, we conducted eccentric contraction. Dystrophin-null dogs showed a significantly greater force loss following eccentric contraction damage. To our knowledge, this is the first convincing demonstration of force deficit in a single intact muscle in the canine DMD model. The method described here will be of great value to study physiological outcomes following innovative gene and/or cell therapies.

Nitrosative stress compromises force generation in Duchenne muscular dystrophy (DMD). Both inducible nitric oxide synthase (iNOS) and delocalized neuronal NOS (nNOS) have been implicated. We recently demonstrated that genetic elimination of nNOS significantly enhanced specific muscle forces of the extensor digitorum longus (EDL) muscle of dystrophin-null mdx4cv mice (Li D et al J. Path. 223:88-98, 2011). To determine the contribution of iNOS, we generated iNOS deficient mdx4cv mice. Genetic elimination of iNOS did not alter muscle histopathology. Further, the EDL muscle of iNOS/dystrophin DKO mice yielded specific twitch and tetanic forces similar to those of mdx4cv mice. Additional studies suggest iNOS ablation did not augment nNOS expression neither did it result in appreciable change of nitrosative stress markers in muscle. Our results suggest that iNOS may play a minor role in mediating nitrosative stress-associated force reduction in DMD.