A family with autism and rare copy number variants disrupting the Duchenne/Becker muscular dystrophy gene DMD and TRPM3.
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SUBMITTER: Pagnamenta AT
PROVIDER: S-EPMC3105230 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description: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.
| S-EPMC4931045 | biostudies-literature
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.
| S-EPMC7113522 | biostudies-literature
Project description:Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) are caused by mutations in the DMD gene. The aim of this study is to identify pathogenic DMD variants in probands and reduce the risk of recurrence of the disease in affected families. Variations in 100 unrelated DMD/BMD patients were detected by multiplex ligation-dependent probe amplification (MLPA) and next-generation sequencing (NGS). Pathogenic variants in DMD were successfully identified in all cases, and 11 of them were novel. The most common mutations were intragenic deletions (69%), with two hotspots located in the 5' end (exons 2-19) and the central of the DMD gene (exons 45-55), while point mutations were observed in 22% patients. Further, c.1149+1G>A and c.1150-2A>G were confirmed by hybrid minigene splicing assay (HMSA). This two splice site mutations would lead to two aberrant DMD isoforms which give rise to severely truncated protein. Therefore, the clinical use of MLPA, NGS, and HMSA is an effective strategy to identify variants. Importantly, eight embryos were terminated pregnancies according to prenatal diagnosis and a healthy boy was successfully delivered by preimplantation genetic diagnosis (PGD). Early and accurate genetic diagnosis is essential for prenatal diagnosis/PGD to reduce the risk of recurrence of DMD in affected families.
| S-EPMC6700351 | biostudies-literature
Project description:To improve assessment of dystrophinopathy, the aim of this study was to identify whether serum creatinine (Crn) level reflects disease severity.Biochemical, Vignos score, and genetic data were collected on 212 boys with dystrophinopathy.Serum Crn level had a strong inverse correlation with Vignos score by simple correlation (r = -0.793) and partial correlation analysis after adjustment for age, height, and weight (r = -0.791; both P < 0.01). Serum Crn level was significantly higher in patients with in-frame than out-of-frame mutations (Z = -4.716, ?P < 0.01) and in Becker muscular dystrophy (BMD) patients than Duchenne muscular dystrophy (DMD) patients at ages 4, 5, 7, and 9?yr (all P < 0.0125). After adjusting for age, height, and weight, BMD patients still had a significantly higher serum Crn level than DMD patients (? = 7.140, ?t = 6.277, ?P < 0.01).Serum Crn level reflected disease severity and may serve as a supplemental index to distinguish DMD from BMD in clinical practice.
| S-EPMC4380093 | biostudies-literature
Project description:Duchenne and Becker muscular dystrophies (DBMD) are allelic disorders caused by mutations in dystrophin. Adults with DBMD develop life-threatening cardiomyopathy. Inhibition of phosphodiesterase 5 (PDE5) improves cardiac function in mouse models of DBMD. To determine whether the PDE5-inhibitor sildenafil benefits human dystrophinopathy, we conducted a randomized, double-blind, placebo-controlled trial (ClinicalTrials.gov, number NCT01168908).Adults with DBMD and cardiomyopathy (ejection fraction???50%) were randomized to receive sildenafil (20mg 3× daily) or placebo for 6 months. All subjects received an additional 6 months of open-label sildenafil. The primary endpoint was change in left ventricular end-systolic volume (LVESV) on cardiac magnetic resonance imaging. Secondary cardiac endpoints, skeletal muscle function, and quality of life were also assessed.An interim analysis (performed after 15 subjects completed the blinded phase) revealed that 29% (4 of 14) of subjects had a ?10% increase in LVESV after 6 months of sildenafil compared to 13% (1 of 8) of subjects receiving placebo. Subjects with LVESV?>?120ml at baseline were more likely to worsen at 12 months regardless of treatment assignment (p?=?0.035). Due to the higher number of subjects worsening on sildenafil, the data and safety monitoring board recommended early termination of the study. There were no statistically significant differences in outcome measures between treatment arms.Due to the small sample size, comparisons between groups must be interpreted with caution. However, this trial suggests that sildenafil is unlikely to improve cardiac function in adults with DBMD.
| S-EPMC4191982 | biostudies-literature
Project description:The prevalence of urinary incontinence in Duchenne and Becker muscular dystrophy (D/BMD) is reported to be between 15-29%, this however includes ages across the lifespan, and with no description of impact on daily life. The present study, aimed to determine the prevalence of urinary incontinence in men with D/BMD, and to identify which aspects of daily life were impacted by urinary incontinence. Twenty-seven adult males, 11 with BMD and 16 with DMD, aged 20-57 years, volunteered to participate in this study. Six questionnaires were completed to provide an overview of participant mobility, urinary incontinence and distress caused by urinary incontinence. These included: The Barthel index of disability, International Consultation on Incontinence Questionnaire-Urinary Incontinence Form, Incontinence Impact Questionnaire Short Form, The Urogenital Distress Inventory, and the Brooke and Vignos scale. The prevalence of urinary incontinence within the present men with D/BMD was 37%, assessed as urine leakage of once a week or more. Those with urinary incontinence all reported only a "small amount" of urine leakage, with urinary incontinence frequency of once a week in 5/10 participants, two or three times a week in 2/10 participants, and once a day in 3/10 participants. Of those with urinary incontinence 8/10 experienced some impact on their daily life from urine leakage including travel (4/10), social aspects (5/10), and emotional aspects (8/10). All participants with urinary incontinence were bothered by some aspect, including urine leakage (9/10), and frequent urination (4/10). In conclusion, 37% of the present men with D/BMD experience urinary incontinence on a weekly or daily basis and negatively impacted aspects of life related to travel, social and emotional wellbeing. Urine leakage, and frequent urination should be considered a meaningful problem by care providers, and discussed openly with those with D/BMD.
| S-EPMC7259643 | biostudies-literature
Project description:Purpose: DMD pathogenic variants for Duchenne and Becker muscular dystrophy are detectable with high sensitivity by standard clinical exome analyses of genomic DNA. However, up to 7% of DMD mutations are deep intronic and analysis of muscle-derived RNA is an important diagnostic step for patients who have negative genomic testing but abnormal dystrophin expression in muscle. In this study, muscle biopsies were evaluated in 19 patients with clinical features of a dystrophinopathy, but negative clinical DMD mutation analysis. Methods: Reverse transcription PCR (RT-PCR) or high-throughput RNA sequencing (RNA-Seq) methods identified 19 mutations with one of three pathogenic pseudoexon types: deep intronic point mutations, deletions or insertions, and translocations. Results: In association with point mutations creating intronic splice acceptor sites, we observed the first examples of DMD pseudo 3’-terminal exon mutations causing high efficiency transcription termination within introns. This connection between splicing and premature transcription termination is reminiscent of U1 snRNP-mediating telescripting in sustaining RNA polymerase II elongation across large genes, such as DMD. Conclusions: We propose a novel classification of three distinct types of mutations identifiable by muscle RNA analysis, each of which differ in potential treatment approaches. Recognition and appropriate characterization may lead to therapies directed toward full-length dystrophin expression for some patients.
2022-05-06 | GSE175861 | GEO
Project description:BACKGROUND:Duchenne Muscular Dystrophy (DMD) is the most common muscle disease in children, and there are no effective therapies for DMD or Becker Muscular Dystrophy (BMD). Currently, targeted gene therapy treatments have emerged. As a result, genetic diagnosis is the basis of treatment. In addition, genetic and prenatal diagnosis significantly reduces their incidence rates. This study combines the application of multiplex ligation-dependent probe amplification technology (MLPA) and "next-generation" sequencing technology (NGS) as the most economical and efficient method of diagnosis. Therefore, in the diagnosis of DMD/BMD, patients' MLPA data are first used to detect DMD gene deletions or duplications, and NGS and Sanger sequencing are then applied to exclude MLPA-negative samples. Meanwhile, polymerase chain reaction (PCR) is used to detect single exon deletions to exclude false-positives in MLPA caused by point mutations. METHODS:In this study, we recruited 1051 proband families of DMD from 2016 to 2018 and had access to information that could identify individual participants during or after data collection. Patients who were diagnosed with DMD were first tested by MLPA. MLPA results with single exon deletions were validated with PCR amplification and Sanger sequencing. The negative results of MLPA were further analysed with NGS and validated by Sanger sequencing. For novel missense mutations, phenotype-genotype correlations were analysed using PolyPhen2 and mutation taster. All methods were performed in accordance with the relevant guidelines and regulations. RESULTS:DMD mutations were identified in 1029 families (97.91%, 1029/1051). Large deletions, duplications, and small mutations accounted for 70.41% (740/1051), 8.28% (87/1051), and 19.12% (201/1051) of all cases, respectively. There were 205 small mutation types, 53 of which were novel. The rate of de novo mutations was 39.45% (187/474) and was higher in large duplications (49.53%, 157/317). Among 68 asymptomatic patients (<?3?years old) with unexplained persistent hyperCKaemia upon conventional physical examination, 63 were diagnosed as DMD/BMD according to genetic diagnosis. CONCLUSION:Our results expand the spectrum of DMD mutations, which could contribute to the treatment of DMD/BMD and provide an effective diagnosis method. Thus, the combination of MLPA, NGS and Sanger sequencing is of great significance for family analysis, gene diagnosis and gene therapy.
| S-EPMC6694523 | biostudies-literature
Project description:Cardiomyopathy is a leading cause of early mortality in Duchenne muscular dystrophy (DMD). There is a need to gain a better understanding of the molecular pathogenesis for the development effective therapies. Exosomes (exo) are secreted vesicles and exert effects via their RNA, lipid and protein cargo. The role of exosomes in disease pathology is unknown. Exosomes derived from stem cells have demonstrated cardioprotection in the murine DMD heart. However, it is unknown how the disease status of the donor cell type influences exosome function. Here, we sought to determine the phenotypic responses of DMD cardiomyocytes (DMD-iCMs) after long-term exposure to DMD cardiac exosomes (DMD-exo). DMD-iCMs were vulnerable to stress, evidenced by production of reactive oxygen species, the mitochondrial membrane potential and cell death levels. Long-term exposure to non-affected exosomes (N-exo) was protective. By contrast, long-term exposure to DMD-exo was not protective, and the response to stress improved with inhibition of DMD-exo secretion in vitro and in vivo The microRNA (miR) cargo, but not exosome surface peptides, was implicated in the pathological effects of DMD-exo. Exosomal surface profiling revealed N-exo peptides associated with PI3K-Akt signaling. Transcriptomic profiling identified unique changes with exposure to either N- or DMD-exo. Furthermore, DMD-exo miR cargo regulated injurious pathways, including p53 and TGF-beta. The findings reveal changes in exosomal cargo between healthy and diseased states, resulting in adverse outcomes. Here, DMD-exo contained miR changes, which promoted the vulnerability of DMD-iCMs to stress. Identification of these molecular changes in exosome cargo and effectual phenotypes might shed new light on processes underlying DMD cardiomyopathy.This article has an associated First Person interview with the first author of the paper.
| S-EPMC7673361 | biostudies-literature
Project description:BackgroundThe goal of this study is to identify the hub genes for Duchenne muscular dystrophy (DMD) and Becker muscular dystrophy (BMD) via weighted correlation network analysis (WGCNA).MethodsThe gene expression profile of vastus lateralis biopsy samples obtained in 17 patients with DMD, 11 patients with BMD and 6 healthy individuals was downloaded from the Gene Expression Omnibus (GEO) database (GSE109178). After obtaining different expressed genes (DEGs) via GEO2R, WGCNA was conducted using R package, modules and genes that highly associated with DMD, BMD, and their age or pathology were screened. Gene Ontology (GO) and Kyoto Encyclopaedia of Genes and Genomes (KEGG) enrichment analysis and protein-protein interaction (PPI) network analysis were also conducted. Hub genes and highly correlated clustered genes were identified using Search Tool for the Retrieval of Interacting Genes (STRING) and Cystoscape software.ResultsOne thousand four hundred seventy DEGs were identified between DMD and control, with 1281 upregulated and 189 downregulated DEGs. Four hundred and twenty DEGs were found between BMD and control, with 157 upregulated and 263 upregulated DEGs. Fourteen modules with different colors were identified for DMD vs control, and 7 modules with different colors were identified for BMD vs control. Ten hub genes were summarized for DMD and BMD respectively, 5 hub genes were summarized for BMD age, 5 and 3 highly correlated clustered genes were summarized for DMD age and BMD pathology, respectively. In addition, 20 GO enrichments were found to be involved in DMD, 3 GO enrichments were found to be involved in BMD, 3 GO enrichments were found to be involved in BMD age.ConclusionIn DMD, several hub genes were identified: C3AR1, TLR7, IRF8, FYB and CD33(immune and inflammation associated genes), TYROBP, PLEK, AIF1(actin reorganization associated genes), LAPTM5 and NT5E(cell death and arterial calcification associated genes, respectively). In BMD, a number of hub genes were identified: LOX, ELN, PLEK, IKZF1, CTSK, THBS2, ADAMTS2, COL5A1(extracellular matrix associated genes), BCL2L1 and CDK2(cell cycle associated genes).
| S-EPMC8684282 | biostudies-literature