Centronuclear myopathy (CNM) is a congenital myopathy characterised by centralised nuclei in skeletal myofibers. T-tubules, sarcolemmal invaginations required for excitation-contraction coupling, are ...disorganised in the skeletal muscles of CNM patients. Previous studies showed that various endocytic proteins are involved in T-tubule biogenesis and their dysfunction is tightly associated with CNM pathogenesis.
and
are two causative genes for CNM that encode essential membrane remodelling proteins in endocytosis, dynamin 2 and BIN1, respectively. In this review, we overview the functions of dynamin 2 and BIN1 in T-tubule biogenesis and discuss how their dysfunction in membrane remodelling leads to CNM pathogenesis.
Duchenne muscular dystrophy (DMD) is caused by a nonsense or frameshift mutation in the
DMD
gene, while its milder form, Becker muscular dystrophy (BMD) is caused by an in-frame deletion/duplication ...or a missense mutation. Interestingly, however, some patients with a nonsense mutation exhibit BMD phenotype, which is mostly attributed to the skipping of the exon containing the nonsense mutation, resulting in in-frame deletion. This study aims to find BMD cases with nonsense/frameshift mutations in
DMD
and to investigate the exon skipping rate of those nonsense/frameshift mutations. We searched for BMD cases with nonsense/frameshift mutations in
DMD
in the Japanese Registry of Muscular Dystrophy. For each
DMD
mutation identified, we constructed minigene plasmids containing one exon with/without a mutation and its flanking intronic sequence. We then introduced them into HeLa cells and measured the skipping rate of transcripts of the minigene by RT-qPCR. We found 363 cases with a nonsense/frameshift mutation in
DMD
gene from a total of 1497 dystrophinopathy cases in the registry. Among them, 14 had BMD phenotype. Exon skipping rates were well correlated with presence or absence of dystrophin, suggesting that 5% exon skipping rate is critical for the presence of dystrophin in the sarcolemma, leading to milder phenotypes. Accurate quantification of the skipping rate is important in understanding the exact functions of the nonsense/frameshift mutations in
DMD
and for interpreting the phenotypes of the BMD patients.
Duchenne and Becker muscular dystrophies (DMD/BMD) are the most common inherited neuromuscular disease. The genetic diagnosis is not easily made because of the large size of the dystrophin gene, ...complex mutational spectrum and high number of tests patients undergo for diagnosis. Multiplex ligation-dependent probe amplification (MLPA) has been used as the initial diagnostic test of choice. Although MLPA can diagnose 70% of DMD/BMD patients having deletions/duplications, the remaining 30% of patients with small mutations require further analysis, such as Sanger sequencing. We applied a high-throughput method using Ion Torrent next-generation sequencing technology and diagnosed 92% of patients with DMD/BMD in a single analysis. We designed a multiplex primer pool for DMD and sequenced 67 cases having different mutations: 37 with deletions/duplications and 30 with small mutations or short insertions/deletions in DMD, using an Ion PGM sequencer. The results were compared with those from MLPA or Sanger sequencing. All deletions were detected. In contrast, 50% of duplications were correctly identified compared with the MLPA method. Small insertions in consecutive bases could not be detected. We estimated that Ion Torrent sequencing could diagnose ~92% of DMD/BMD patients according to the mutational spectrum of our cohort. Our results clearly indicate that this method is suitable for routine clinical practice providing novel insights into comprehensive genetic information for future molecular therapy.
Abstract
Mutations in the type 1 ryanodine receptor (RyR1), a Ca
2+
release channel in skeletal muscle, hyperactivate the channel to cause malignant hyperthermia (MH) and are implicated in severe ...heat stroke. Dantrolene, the only approved drug for MH, has the disadvantages of having very poor water solubility and long plasma half-life. We show here that an oxolinic acid-derivative RyR1-selective inhibitor, 6,7-(methylenedioxy)-1-octyl-4-quinolone-3-carboxylic acid (Compound 1, Cpd1), effectively prevents and treats MH and heat stroke in several mouse models relevant to MH. Cpd1 reduces resting intracellular Ca
2+
, inhibits halothane- and isoflurane-induced Ca
2+
release, suppresses caffeine-induced contracture in skeletal muscle, reduces sarcolemmal cation influx, and prevents or reverses the fulminant MH crisis induced by isoflurane anesthesia and rescues animals from heat stroke caused by environmental heat stress. Notably, Cpd1 has great advantages of better water solubility and rapid clearance in vivo over dantrolene. Cpd1 has the potential to be a promising candidate for effective treatment of patients carrying RyR1 mutations.
To identify the characteristic pathologic features of dermatomyositis (DM) associated with anti-Mi-2 autoantibodies (anti-Mi-2 DM).
We reviewed 188 muscle biopsies from patients (1) pathologically ...diagnosed with DM through the sarcoplasmic expression for the myxovirus-resistant protein A and (2) serologically positive for 1 of 5 DM-specific autoantibodies (DMSAs) (anti-Mi-2, n = 30; other DMSAs, n = 152) or negative for all 5 DMSAs (n = 6). We then compared the histopathologic and immunohistochemical features of patients with anti-Mi-2 DM to those with non-Mi-2 DM and patients with anti-synthetase syndrome (ASS) (n = 212) using the
test, Fisher exact test, and a logistic regression model.
Patients with anti-Mi-2 DM showed significantly higher severity scores in muscle fiber and inflammatory domains than non-Mi-2 DM patients. The presence of perifascicular necrosis, increased perimysial alkaline phosphatase activity, and sarcolemmal membrane attack complex deposition was more frequent in patients with anti-Mi-2 DM (
< 0.01). After Bonferroni correction, there were no significant differences in the percentages of the features mentioned above between the patients with anti-Mi-2 DM and those with ASS (
> 0.01).
Perifascicular necrosis and perimysial pathology, features previously reported in ASS, are common in patients with anti-Mi-2 DM. Our findings not only assist in differentiating anti-Mi-2 DM from other DM subtypes but also suggest the possibility of an overlapping mechanism between anti-Mi-2 DM and ASS.
This study provides Class II evidence that the muscle biopsies of DM patients with anti-Mi-2 autoantibodies are more likely to demonstrate higher severity scores in muscle fiber and inflammatory domains.
Abstract
Duchenne muscular dystrophy (DMD) is an X-linked genetic disorder characterized by progressive muscular weakness because of the loss of dystrophin. Extracellular Ca2+ flows into the ...cytoplasm through membrane tears in dystrophin-deficient myofibers, which leads to muscle contracture and necrosis. Sarco/endoplasmic reticulum Ca2+-ATPase (SERCA) takes up cytosolic Ca2+ into the sarcoplasmic reticulum, but its activity is decreased in dystrophic muscle. Here, we show that an allosteric SERCA activator, CDN1163, ameliorates dystrophic phenotypes in dystrophin-deficient mdx mice. The administration of CDN1163 prevented exercise-induced muscular damage and restored mitochondrial function. In addition, treatment with CDN1163 for 7 weeks enhanced muscular strength and reduced muscular degeneration and fibrosis in mdx mice. Our findings provide preclinical proof-of-concept evidence that pharmacological activation of SERCA could be a promising therapeutic strategy for DMD. Moreover, CDN1163 improved muscular strength surprisingly in wild-type mice, which may pave the new way for the treatment of muscular dysfunction.
Dystrophinopathy is caused by alterations in
DMD
. Approximately 1% of patients remain genetically undiagnosed, because intronic variations are not detected by standard methods. Here, we combined ...laboratory and in silico analyses to identify disease-causing genomic variants in genetically undiagnosed patients and determine the regulatory mechanisms underlying abnormal
DMD
transcript generation.
DMD
transcripts from 20 genetically undiagnosed dystrophinopathy patients in whom no exon variants were identified, despite dystrophin deficiency on muscle biopsy, were analyzed by transcriptome sequencing. Genome sequencing captured intronic variants and their effects were interpreted using in silico tools. Targeted long-read sequencing was applied in cases with suspected structural genomic abnormalities. Abnormal
DMD
transcripts were detected in 19 of 20 cases; Exonization of intronic sequences in 15 cases, exon skipping in one case, aberrantly spliced and polyadenylated transcripts in two cases and transcription termination in one case. Intronic single nucleotide variants, chromosomal rearrangements and nucleotide repeat expansion were identified in
DMD
gene as pathogenic causes of transcript alteration. Our combined analysis approach successfully identified pathogenic events. Detection of diseasing-causing mechanisms in
DMD
transcripts could inform the therapeutic options for patients with dystrophinopathy.
Collagen VI-related dystrophy spans a clinical continuum from severe Ullrich congenital muscular dystrophy to milder Bethlem myopathy. This disease is caused by causative variants in COL6A1, COL6A2, ...or COL6A3. Most reported causative variants are de novo; therefore, to identify possible associated causative variants, comprehensive large cohort studies are required for different ethnicities. We retrospectively reviewed clinical information, muscle histology, and genetic analyses from 147 Japanese patients representing 130 families, whose samples were sent for diagnosis to the National Center of Neurology and Psychiatry between July 1979 and January 2020. Genetic analyses were conducted by gene-based resequencing, targeted panel resequencing, and whole exome sequencing, in combination with cDNA analysis. Of a total of 130 families with 1-5 members with collagen VI-related dystrophy, 120 had mono-allelic and 10 had bi-allelic variants in COL6A1, COL6A2, or COL6A3. Among them, 60 variants were in COL6A1, 57 in COL6A2, and 23 in COL6A3, including 37 novel variants. Mono-allelic variants were classified into four groups: missense (69, 58%), splicing (40, 33%), small in-frame deletion (7, 6%), and large genomic deletion (4, 3%). Variants in the triple helical domains accounted for 88% (105/120) of all mono-allelic variants. We report the causative variant profile of a large set of Japanese cases of collagen VI-related dystrophy. This dataset can be used as a reference to support genetic diagnosis and variant-specific treatment.
Thermoregulation is an important aspect of human homeostasis, and high temperatures pose serious stresses for the body. Malignant hyperthermia (MH) is a life-threatening disorder in which body ...temperature can rise to a lethal level. Here we employ an optically controlled local heat-pulse method to manipulate the temperature in cells with a precision of less than 1 °C and find that the mutants of ryanodine receptor type 1 (RyR1), a key Ca
2+
release channel underlying MH, are heat hypersensitive compared with the wild type (WT). We show that the local heat pulses induce an intracellular Ca
2+
burst in human embryonic kidney 293 cells overexpressing WT RyR1 and some RyR1 mutants related to MH. Fluorescence Ca
2+
imaging using the endoplasmic reticulum–targeted fluorescent probes demonstrates that the Ca
2+
burst originates from heat-induced Ca
2+
release (HICR) through RyR1-mutant channels because of the channels’ heat hypersensitivity. Furthermore, the variation in the heat hypersensitivity of four RyR1 mutants highlights the complexity of MH. HICR likewise occurs in skeletal muscles of MH model mice. We propose that HICR contributes an additional positive feedback to accelerate thermogenesis in patients with MH.
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