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Genetic analysis of dystrophin gene for affected male and female carriers with Duchenne/Becker muscular dystrophy in Korea.

ABSTRACT: Duchenne and Becker muscular dystrophy (DMD/BMD) are X-linked recessive disorders caused by mutation in dystrophin gene. We analyzed the results of a genetic test in 29 DMD/BMD patients, their six female relatives, and two myopathic female patients in Korea. As the methods developed, we applied different procedures for dystrophin gene analysis; initially, multiplex polymerase chain reaction was used, followed by multiplex ligation-dependent probe amplification (MLPA). Additionally, we used direct DNA sequencing for some patients who had negative results using the above methods. The overall mutation detection rate was 72.4% (21/29) in DMD/BMD patients, identifying deletions in 58.6% (17/29). Most of the deletions were confined to the central hot spot region between exons 44 and 55 (52.9%, 7/19). The percentage of deletions and duplications revealed by MLPA was 45.5% (5/11) and 27.2% (3/11), respectively. Using the MLPA method, we detected mutations confirming their carrier status in all female relatives and symptomatic female patients. In one patient in whom MLPA revealed a single exon deletion of the dystrophin gene, subsequent DNA sequencing analysis identified a novel nonsense mutation (c.4558G > T; Gln1520X). The MLPA assay is a useful quantitative method for detecting mutation in asymptomatic or symptomatic carriers as well as DMD/BMD patients.

SUBMITTER: Lee BL

PROVIDER: S-EPMC3286774 | biostudies-literature | 2012 Mar

REPOSITORIES: biostudies-literature

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Genetic analysis of dystrophin gene for affected male and female carriers with Duchenne/Becker muscular dystrophy in Korea.

Lee Bo Lyun BL Nam Sook Hyun SH Lee Jun Hwa JH Ki Chang Seok CS Lee Munhyang M Lee Jeehun J

Journal of Korean medical science 20120223 3

Duchenne and Becker muscular dystrophy (DMD/BMD) are X-linked recessive disorders caused by mutation in dystrophin gene. We analyzed the results of a genetic test in 29 DMD/BMD patients, their six female relatives, and two myopathic female patients in Korea. As the methods developed, we applied different procedures for dystrophin gene analysis; initially, multiplex polymerase chain reaction was used, followed by multiplex ligation-dependent probe amplification (MLPA). Additionally, we used direc ...[more]

PMID: 22379338

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Project description:BackgroundDuchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are characterized by muscle wasting leading to loss of ambulation in the first or third decade, respectively. In DMD, the lack of dystrophin hampers connections between intracellular cytoskeleton and cell membrane leading to repeated cycles of necrosis and regeneration associated with inflammation and loss of muscle ordered structure. BMD has a similar muscle phenotype but milder. Here, we address the question whether proteins at variance in BMD compared with DMD contribute to the milder phenotype in BMD, thus identifying a specific signature to be targeted for DMD treatment.MethodsProteins extracted from skeletal muscle from DMD/BMD patients and young healthy subjects were either reduced and solubilized prior two-dimensional difference in gel electrophoresis/mass spectrometry differential analysis or tryptic digested prior label-free liquid chromatography with tandem mass spectrometry. Statistical analyses of proteins and peptides were performed by DeCyder and Perseus software and protein validation and verification by immunoblotting.ResultsProteomic results indicate minor changes in the extracellular matrix (ECM) protein composition in BMD muscles with retention of mechanotransduction signalling, reduced changes in cytoskeletal and contractile proteins. Conversely, in DMD patients, increased levels of several ECM cytoskeletal and contractile proteins were observed whereas some proteins of fast fibres and of Z-disc decreased. Detyrosinated alpha-tubulin was unchanged in BMD and increased in DMD although neuronal nitric oxide synthase was unchanged in BMD and greatly reduced in DMD. Metabolically, the tissue is characterized by a decrement of anaerobic metabolism both in DMD and BMD compared with controls, with increased levels of the glycogen metabolic pathway in BMD. Oxidative metabolism is severely compromised in DMD with impairment of malate shuttle; conversely, it is active in BMD supporting the tricarboxylic acid cycle and respiratory chain. Adipogenesis characterizes DMD, whereas proteins involved in fatty acids beta-oxidation are increased in BMD. Proteins involved in protein/amino acid metabolism, cell development, calcium handling, endoplasmic reticulum/sarcoplasmic reticulum stress response, and inflammation/immune response were increased in DMD. Both disorders are characterized by the impairment of N-linked protein glycosylation in the endoplasmic reticulum. Authophagy was decreased in DMD whereas it was retained in BMD.ConclusionsThe mechanosensing and metabolic disruption are central nodes of DMD/BMD phenotypes. The ECM proteome composition and the metabolic rewiring in BMD lead to preservation of energy levels supporting autophagy and cell renewal, thus promoting the retention of muscle function. Conversely, DMD patients are characterized by extracellular and cytoskeletal protein dysregulation and by metabolic restriction at the level of ?-ketoglutarate leading to shortage of glutamate-derived molecules that over time triggers lipogenesis and lipotoxicity.

Project description:BackgroundBecker muscular dystrophy (BMD) is a genetic neuromuscular disease of growing importance caused by in-frame, partial loss-of-function mutations in the dystrophin (DMD) gene. BMD presents with reduced severity compared with Duchenne muscular dystrophy (DMD), the allelic disorder of complete dystrophin deficiency. Significant therapeutic advancements have been made in DMD, including four FDA-approved drugs. BMD, however, is understudied and underserved-there are no drugs and few clinical trials. Discordance in therapeutic efforts is due in part to lack of a BMD mouse model which would enable greater understanding of disease and de-risk potential therapeutics before first-in-human trials. Importantly, a BMD mouse model is becoming increasingly critical as emerging DMD dystrophin restoration therapies aim to convert a DMD genotype into a BMD phenotype.MethodsWe use CRISPR/Cas9 technology to generate bmx (Becker muscular dystrophy, X-linked) mice, which express an in-frame ~40 000 bp deletion of exons 45-47 in the murine Dmd gene, reproducing the most common BMD patient mutation. Here, we characterize muscle pathogenesis using molecular and histological techniques and then test skeletal muscle and cardiac function using muscle function assays and echocardiography.ResultsOverall, bmx mice present with significant muscle weakness and heart dysfunction versus wild-type (WT) mice, despite a substantial improvement in pathology over dystrophin-null mdx52 mice. bmx mice show impaired motor function in grip strength (-39%, P < 0.0001), wire hang (P = 0.0025), and in vivo as well as ex vivo force assays. In aged bmx, echocardiography reveals decreased heart function through reduced fractional shortening (-25%, P = 0.0036). Additionally, muscle-specific serum CK is increased >60-fold (P < 0.0001), indicating increased muscle damage. Histologically, bmx muscles display increased myofibre size variability (minimal Feret's diameter: P = 0.0017) and centrally located nuclei indicating degeneration/regeneration (P < 0.0001). bmx muscles also display dystrophic pathology; however, levels of the following parameters are moderate in comparison with mdx52: inflammatory/necrotic foci (P < 0.0001), collagen deposition (+1.4-fold, P = 0.0217), and sarcolemmal damage measured by intracellular IgM (P = 0.0878). Like BMD patients, bmx muscles show reduced dystrophin protein levels (~20-50% of WT), whereas Dmd transcript levels are unchanged. At the molecular level, bmx muscles express increased levels of inflammatory genes, inflammatory miRNAs and fibrosis genes.ConclusionsThe bmx mouse recapitulates BMD disease phenotypes with histological, molecular and functional deficits. Importantly, it can inform both BMD pathology and DMD dystrophin restoration therapies. This novel model will enable further characterization of BMD disease progression, identification of biomarkers, identification of therapeutic targets and new preclinical drug studies aimed at developing therapies for BMD patients.

Dataset Information

Genetic analysis of dystrophin gene for affected male and female carriers with Duchenne/Becker muscular dystrophy in Korea.

Publications

Genetic analysis of dystrophin gene for affected male and female carriers with Duchenne/Becker muscular dystrophy in Korea.

Similar Datasets

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