Membrane remodeling is required for dynamic cellular processes such as cell division, polarization, and motility. BAR domain proteins and dynamins are key molecules in membrane remodeling that work together for membrane deformation and fission. In striated muscles, sarcolemmal invaginations termed T-tubules are required for excitation-contraction coupling. BIN1 and DNM2, which encode a BAR domain protein BIN1 and dynamin 2, respectively, have been reported to be causative genes of centronuclear myopathy (CNM), a hereditary degenerative disease of skeletal muscle, and deformation of T-tubules is often observed in the CNM patients. However, it remains unclear how BIN1 and dynamin 2 are implicated in T-tubule biogenesis and how mutations in these molecules cause CNM to develop. Here, using an in cellulo reconstitution assay, we demonstrate that dynamin 2 is required for stabilization of membranous structures equivalent to T-tubules. GTPase activity of wild-type dynamin 2 is suppressed through interaction with BIN1, whereas that of the disease-associated mutant dynamin 2 remains active due to lack of the BIN1-mediated regulation, thus causing aberrant membrane remodeling. Finally, we show that in cellulo aberrant membrane remodeling by mutant dynamin 2 variants is correlated with their enhanced membrane fission activities, and the results can explain severity of the symptoms in patients. Thus, this study provides molecular insights into dysregulated membrane remodeling triggering the pathogenesis of DNM2-related CNM. Centronuclear myopathy (CNM) is a congenital myopathy characterized clinically by muscle weakness and pathologically by the presence of centralized nuclei on muscle biopsy (1). Disrupted or disorganized T-tubules or triads in the skeletal muscles are also common pathological observations in CNM tissue (2). BIN1, which encodes an N-terminal amphipathic helix Bin/Amphiphysin/Rvs-homology (N-BAR) domain protein BIN1 (Bridging integrator 1)/Amphiphysin 2, has been identified as one of the causative genes for this disease (3-10). BIN1 generates membrane invagination and recognizes the membrane curvature (11, 12). Among 11 splicing isoforms of BIN1, isoform 8 is specifically expressed in the skeletal muscle (13) and encodes an amphipathic H0 helix, a BAR domain, a phosphoinositide (PI) domain in its N-terminus, and a Src homology 3 (SH3) domain in its C-terminus (14, 15). The C-terminal SH3 domain serves as an interacting site with proline-rich (PR) domain-containing proteins such as dynamin 2 (3, 16). To date, two recessive CNM mutations, p.Q573* and p.K575* (p.Q434* and p.K436* in isoform 8), have been identified in CNM patients, and both mutations cause truncation of the SH3 domain. In fact, the p.K436* mutation was shown to abolish the BIN1-dynamin 2 interaction and recruitment of dynamin 2 to the BIN1-mediated T-tubule-like structures (TLS) (3), while p.Q434* causes defective triad organization ranging from abnormal orientation of the striated structures to membranous aggregation in patient biopsy tissue ...
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.
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. Abbreviations BMD Becker muscular dystrophy DMD Duchenne muscular dystrophy ESE Exonic splicing enhancer ESS Exonic splicing silencer HSF Human Splicing Finder IMD Intermediate muscular dystrophy Remudy Registry of muscular dystrophy
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