Yoshihiko Saito

Myotonic dystrophy type 1 is caused by the expression of expanded CTG repeats in the DMPK gene and the resulting loss of function of MBNL protein. Affected skeletal muscle displays abundant centrally located nuclei despite limited immune-cell–associated fibre necrosis, complicating interpretation of muscle damage and remodelling mechanisms. Here we show that muscle stem cells are activated and fuse with existing muscle fibres in myotonic dystrophy type 1. Single-nucleus transcriptomics in patient’s muscle identifies increased activated muscle stem cells and distinct myonuclear populations exhibiting transitional transcriptional states, including muscle stem cell associated markers and elevated DMPK expression. Myofibre-specific MBNL knockdown mouse models demonstrate that muscle stem cell fusion contributes to central nucleation, whereas their deletion does not improve myotonia or muscle strength. Together, these findings indicate that loss of MBNL function in muscle drives myonuclear accretion through stem cell-mediated fusion, giving rise to myonuclei with immature states in myotonic dystrophy type 1.

Tubulinopathies encompass a spectrum of disorders resulting from variants in genes encoding α- and β-tubulins, the key components of microtubules. While previous studies have linked de novo or dominantly inherited TUBA4A missense variants to neurodegenerative phenotypes, including amyotrophic lateral sclerosis, frontotemporal dementia, spastic ataxia, and recently, an isolated congenital myopathy, the full phenotypic and genotypic spectrum of TUBA4A-related disorders remains incompletely characterised. In this multi-centre study, we identified one previously reported and 12 novel TUBA4A missense variants in 31 individuals from 19 unrelated families. Remarkably, individuals in 17 families presented with a myopathy without any CNS involvement or history of such disease. In the remaining two families, we observed probands with cerebellar ataxia and epilepsy accompanying proximal and axial muscle weakness along with protein aggregation. The coexistence of neuromuscular and neurodegenerative features with protein aggregation defines a multisystem proteinopathy. These two families thus establish the first association between TUBA4A and multisystem proteinopathy. Our cohort exhibited diverse genotypes and inheritance patterns: four families demonstrated autosomal dominant transmission through heterozygous variants in TUBA4A, three probands had recessive inheritance due to homozygous variants, while the respective heterozygous carriers were asymptomatic; five probands carried de novo variants, and nine probands with heterozygous variants were classified as sporadic cases. Clinical phenotypes ranged from mild to severe myopathy, predominantly affecting the axial and paraspinal muscles. We observed a range of disease onset, from congenital to late adulthood. Creatine kinase levels were variable, ranging from normal to highly elevated. Cardiac function remained preserved across the cohort. Muscle biopsies showed heterogenous myopathic changes, including myofibre size variation, nemaline bodies, core-like regions, and internal nuclei. Immunohistochemical analysis revealed protein accumulations positive for TDP-43 (n=2), p62 (n=5), and TUBA4A (n=6). Complementary in silico and in vitro investigations suggested that the identified TUBA4A variants cause significant protein abnormalities and may differentially impact microtubule dynamics. Correlation analyses integrating clinical severity, variant location, and mechanistic readouts further demonstrated that domain specificity within TUBA4A influences both the pattern of muscle involvement and the extent of microtubule disruption. Our findings establish myo-tubulinopathies as distinct clinical entities, encompassing both primary myopathies and multisystem proteinopathies with muscle involvement. This study broadens the phenotypic and genotypic spectrum of TUBA4A-related disorders beyond autosomal dominant or de novo mechanisms and neurodegenerative presentations. These results underscore the importance of considering TUBA4A variants in the differential diagnosis of axial myopathies and multisystem proteinopathies, regardless of central nervous system (CNS) involvement.

X-linked myotubular myopathy (XLMTM) due to MTM1 mutations is a rare and often lethal congenital myopathy. Its downstream molecular and cellular mechanisms are currently incompletely understood. The most abundant protein in muscle, myosin, has been implicated in the pathophysiology of other congenital myopathies. Hence, in the present study, we aimed to define whether myosin is also dysfunctional in XLMTM and whether it, thus, may constitute a potential drug target. To this end, we used skeletal muscle tissue from patients and canine/mouse models; we performed Mant-ATP chase experiments coupled with x-ray diffraction analyses and LC/MS-based proteomics studies. In patients with XLMTM, we found that myosin molecules are structurally disordered and preferably adopt their ATP-consuming biochemical state. This phosphorylation-related (mal)adaptation was mirrored by a striking remodeling of the myofiber energetic proteome in XLMTM dogs. In line with these, we confirmed an accrued myosin ATP consumption in mice lacking MTM1. Hence, we treated these with a myosin ATPase inhibitor, mavacamten. After a 4-week treatment period, we observed a partial restoration of the myofiber proteome, especially proteins involved in cytoskeletal, sarcomeric, and energetic pathways. Altogether, our study highlights myosin inhibition as a potentially new drug mechanism for the complex XLMTM muscle phenotype.

In recent years, whole-exome and whole-genome sequencing have been increasingly applied for the genetic diagnosis of muscle diseases. However, standard short-read sequencing often fails to detect pathogenic variants in some inherited muscle diseases, such as Duchenne/Becker muscular dystrophy (DMD/BMD), facioscapulohumeral muscular dystrophy (FSHD), oculopharyngeal muscular dystrophy (OPMD), and oculopharyngodistal myopathy (OPDM). This review outlines the genetic diagnostic approaches for these conditions, with a particular focus on novel analytical approaches for genetic diagnosis.

Whether skeletal muscle in myotonic dystrophy type 1 (DM1) maintains its regenerative potential or shows inherent muscle stem cell (MuSC) defects remains unresolved. Here, we examine MuSC function in DM1 by combining an adult-onset Mbnl1/2 knockout mouse model with single-nucleus RNA sequencing (snRNA-seq) of human DM1 muscle biopsies. Loss of Mbnl in mouse myofibres triggers MuSC activation and fusion, resulting in central nuclear accumulation independent of degeneration. Lineage tracing confirms direct MuSC contribution to these nuclei, a process abolished upon MuSC ablation. In human DM1 specimens, we identify activated MuSCs and a myonuclear population with an immature transcriptional signature including expression of MuSC markers, and elevated DMPK levels. These findings imply that MuSCs in DM1 contribute to muscle repair by fusing into myofibres but give rise to immature myonuclei, likely due to toxic RNA effects on transcriptional reprogramming. Our study emphasises a previously overlooked role of MuSCs in DM1 pathology and identifies potential targets to support muscle regeneration.

Valosin Containing Protein-associated multisystem proteinopathy (VCP-MSP) is a progressive, autosomal dominant disorder caused by pathogenic variants in the VCP gene, resulting in a heterogeneous clinical presentation. Muscle biopsy findings are characteristic but not pathognomonic. This study aimed to comprehensively analyse VCP-related myopathology and explore correlations with clinical phenotypes, genetic variants, and disease progression. Muscle biopsy images and data were collected retrospectively from adults (≥18 years) with pathogenic or likely pathogenic VCP variants enrolled in the VCP Multicentre International Study. Biopsy data were standardized using the “Common Data Elements for Muscle Biopsy Reporting.” Variations in biopsy findings were analysed by biopsy site, time from disease onset, the four most common VCP variants, and clinical phenotypes. A total of 112 muscle biopsies were included. Most individuals were male (66.0%). The mean age at biopsy was 53.3 years (SD 10.0), with a mean disease duration of 6.5 years (SD 4.5). The most frequent VCP variant was c.464G>A (p.Arg155His) (18.8%). The top clinical phenotypes were isolated myopathy (37.5%), myopathy with Paget disease of bone (17.9%), and myopathy with motor neuron involvement (13.4%). The vastus lateralis was the most common biopsy site (34.8%), and 91% were open biopsies. Histopathologic findings included atrophic fibres (87.5%), rimmed vacuoles (72.3%), endomysial fibrosis (58.0%), and protein aggregates (51.8%), primarily p62 (60.3%) and VCP (36.2%). Degeneration niches with fibrofatty replacement and atrophic fibres were seen in 33.3% of biopsies without frequency differences by clinical phenotypes. There were no differences in biopsy findings among the 4 most common VCP gene variants, except for the absence of degeneration niches in muscle biopsies of 12 patients with c.277C>T (p.Arg93Cys). MRI data from 30 patients showed fat pockets corresponding to these niches and STIR hyperintensity correlated with inflammatory infiltrates in 42.9%. Concordance between clinical phenotype, biopsy, and neurophysiology was observed in only 49.4% of cases, indicating significant heterogeneity. VCP-MSP muscle biopsies consistently show myopathic or mixed patterns with rimmed vacuoles and p62/VCP-positive inclusions, regardless of clinical phenotype, age, or progression. Some lack vacuoles, challenging diagnosis. Discrepancies between clinical, neurophysiology, and biopsy findings should prompt consideration of VCP-MSP to improve detection and management.

Facioscapulohumeral muscular dystrophy (FSHD) is a genetic muscle disease caused by ectopic expression of the toxic protein DUX4, resulting in muscle weakness. However, the mechanism by which DUX4 exerts its toxicity remains unclear. In this study, we observed abnormal iron accumulation in muscles of patients with FSHD and in mice with muscle-specific DUX4 expression (DUX4-Tg mice). Treatment with iron chelators, an iron-deficient diet, and genetic modifications inhibiting intracellular uptake of iron did not improve but rather exacerbated FSHD pathology in DUX4-Tg mice. Unexpectedly, however, iron supplementation, from either a high-iron diet or intravenous iron administration, resulted in remarkable improvement in grip strength and running performance in DUX4-Tg mice. Iron supplementation suppressed abnormal iron accumulation and the ferroptosis-related pathway involving increased lipid peroxidation in DUX4-Tg muscle. Muscle-specific DUX4 expression led to retinal vasculopathy, a part of FSHD pathology, which was prevented by iron administration. Furthermore, high-throughput compound screening of the ferroptosis pathway identified drug candidates including ferrostatin-1 (Fer-1), a potent inhibitor of lipid peroxidation. Treatment with Fer-1 dramatically improved physical function in DUX4-Tg mice. Our findings demonstrate that DUX4-provoked toxicity is involved in the activation of the ferroptosis-related pathway and that supplementary iron could be a promising and readily available therapeutic option for FSHD.

Tubulinopathies encompass a wide spectrum of disorders resulting from variants in genes encoding α- and β-tubulins, the key components of microtubules. While previous studies have linked de novo or dominantly inherited TUBA4A missense variants to neurodegenerative phenotypes, including amyotrophic lateral sclerosis, frontotemporal dementia, hereditary spastic ataxia, and more recently, an isolated report of congenital myopathy, the full phenotypic and genotypic spectrum of TUBA4A -related disorders remains incompletely characterised. In this multi-centre study, we identified 13 novel TUBA4A missense variants in 31 individuals from 19 unrelated families. Remarkably, affected individuals in 17 families presented with a primary axial myopathy without any identified CNS involvement or history of such disease. In the remaining two families, we observed probands with cerebellar ataxia and epilepsy accompanying proximal and axial muscle weakness, establishing the first documented association between TUBA4A variants and multisystem proteinopathy. Our cohort exhibited diverse genotypes and associated inheritance patterns: four families demonstrated autosomal dominant transmission through heterozygous variants in TUBA4A , three probands had homozygous TUBA4A variants, where the biallelic genotype was found to be associated with the disease, and the heterozygous carriers were asymptomatic; five probands carried de novo variants, and nine probands with heterozygous TUBA4A variants were classified as "isolated-sporadic cases" where parental samples were unavailable. Clinical phenotypes ranged from mild to severe myopathy, predominantly affecting the axial and paraspinal muscles. We observed a range of disease onset, from congenital to late adulthood. Creatine kinase levels were also variable, ranging from normal to highly elevated. Cardiac function remained preserved across the cohort. Muscle biopsies revealed a range of pathologies, including myofibre size variation, myofibre atrophy, nemaline bodies, core-like regions, internal nuclei, and endomysial fibrosis. Immunohistochemical staining showed evidence of proteinopathy, with autophagic features and TUBA4A accumulation in patient myofibres. Complementary in silico and in vitro investigations suggested that the identified TUBA4A substitutions cause significant protein abnormalities and may differentially impact microtubule dynamics. Our findings establish myo-tubulinopathies as distinct clinical entities, encompassing both primary myopathies and multisystem proteinopathies with muscle involvement. This study broadens the phenotypic and genotypic spectrum of TUBA4A -related disorders beyond autosomal dominant or de novo mechanisms and neurodegenerative presentations. These results underscore the importance of considering TUBA4A variants in the differential diagnosis of axial myopathies and multisystem proteinopathies, regardless of central nervous system (CNS) involvement.

Transposon-like human element 1B (THE1B) originates from ancient retroviral sequences integrated into the primate genome approximately 50 million years ago, now accounting for at least 27,233 copies in the human genome, suggesting their extensive influence on human genomic architecture. Here we report identification of 19 THE1B fusion transcripts through short- and long-read RNA-seq analysis, 15 of which are previously unmapped, showing elevated expression in 16 individuals with sarcoid myopathy (SM), as compared to 400 controls with various other muscle diseases. Analysis of publicly available RNA-seq data indicated a correlation between the reduced expression of eight THE1B fusion transcripts and clinical improvement in individuals with cutaneous sarcoidosis receiving tofacitinib treatment. Single-cell or single-nucleus RNA-seq analyses of sarcoidosis not only confirmed these transcripts but also revealed a novel read-through transcript, SIRPB1-SIRPD, and TREM2.1, predominantly in granuloma-associated macrophages. The expression profiles of THE1B fusion transcripts in tuberculosis (TB) significantly differed from SM in single-cell RNA-seq data, suggesting that the differences between TB’s caseous granulomas and sarcoidosis’s non-caseous granulomas might be linked to disparate expression patterns of THE1B fusion transcripts. Our retrotrans-genomics approach has not only identified the genomic landscape of sarcoidosis but also provided new insights into its etiology.

Rigid spine syndrome is a rare childhood-onset myopathy characterised by slowly progressive or non-progressive scoliosis, neck and spine contractures, hypotonia, and respiratory insufficiency. Biallelic variants in SELENON account for most cases of rigid spine syndrome, however, the underlying genetic cause in some patients remains unexplained. We used exome and genome sequencing to investigate the genetic basis of rigid spine syndrome in patients without a genetic diagnosis. In five patients from four unrelated families, we identified biallelic variants in HMGCS1 (3-hydroxy-3-methylglutaryl-coenzyme A synthase). These included six missense variants and one frameshift variant distributed throughout HMGCS1. All patients presented with spinal rigidity primarily affecting the cervical and dorsolumbar regions, scoliosis, and respiratory insufficiency. Creatine kinase levels were variably elevated. The clinical course worsened with intercurrent disease or certain drugs in some patients; one patient died from respiratory failure following infection. Muscle biopsies revealed irregularities in oxidative enzyme staining with occasional internal nuclei and rimmed vacuoles. HMGCS1 encodes a critical enzyme of the mevalonate pathway and has not yet been associated with disease. Notably, biallelic hypomorphic variants in downstream enzymes including HMGCR and GGPS1 are associated with muscular dystrophy resembling our cohort’s presentation. Analyses of recombinant human HMGCS1 protein and four variants (p.S447P, p.Q29L, p.M70T, p.C268S) showed that all mutants maintained their dimerization state. Three of the four mutants exhibited reduced thermal stability, and two mutants showed subtle changes in enzymatic activity compared to the wildtype. Hmgcs1 mutant zebrafish displayed severe early defects, including immobility at 2 days and death by day 3 post-fertilisation and were rescued by HMGCS1 mRNA. We demonstrate that the four variants tested (S447P, Q29L M70T, and C268S) have reduced function compared to wildtype HMGCS1 in zebrafish rescue assays. Additionally, we demonstrate the potential for mevalonic acid supplementation to reduce phenotypic severity in mutant zebrafish. Overall, our analyses suggest that these missense variants in HMGCS1 act through a hypomorphic mechanism. Here, we report an additional component of the mevalonate pathway associated with disease and suggest biallelic variants in HMGCS1 should be considered in patients presenting with an unresolved rigid spine myopathy phenotype. Additionally, we highlight mevalonoic acid supplementation as a potential treatment for patients with HMGCS1-related disease.