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DMD gene pathogenic variations cause a spectrum of phenotypes, ranging from severe Duchenne muscular dystrophy, the Becker milder cases, the intermediate or very mild muscle phenotypes invariably characterized by high CK, and the ultrarare fully-asymptomatic cases. Besides these phenotypes, X-linked dilated cardiomyopathy is also caused by DMD mutations. Males carrying DMD deletions with absent or very mild phenotypes have been sparsely described. We performed a horizon scan on public datasets to enroll males with the above phenotypes and carrying DMD deletions to delineatemyopathic genotype-phenotype relationships. We inventoried 81 males, who were divided into the following clinical categorization: fully-asymptomatic males aged >43 years (A, N = 22); isolated hyperCKemia (CK, N = 35); and mild weakness (any age) with or without high CK (WCK, N = 24). In all cases, deleted intervals were exons 2 to 55, and no downstream exons were ever involved, apart from an exon 78 deletion in a WCK patient. All deletions were in-frame apart from the known exception to the rule of exon 2 and exon 78. We correlated the mild phenotypes (A and CK) to deleted exons, intronic breakpoints, exon-exon junctions, 3' isoforms rule, and protein epitopes, and we found that some genetic profiles are exclusively/mainly occurring in A/CK phenotypes, suggesting they are compatible with a quasi-normal muscular performance. We discussed diverse pathogenic mechanisms thatmay contribute tomild dystrophinopathic phenotypes, and we tried to address some "critical" genetic configurations or exon content needed to preserve a semi-functional DMD gene. [ABSTRACT FROM AUTHOR]

Duchenne muscular dystrophy (DMD) is a rare neuromuscular disease caused by pathogenic variations in the DMD gene. There is a need for robust DMD biomarkers for diagnostic screening and to aid therapy monitoring. Creatine kinase, to date, is the only routinely used blood biomarker for DMD, although it lacks specificity and does not correlate with disease severity. To fill this critical gap, we present here novel data about dystrophin protein fragments detected in human plasma by a suspension bead immunoassay using two validated anti-dystrophin-specific antibodies. Using both antibodies, a reduction of the dystrophin signal is detected in a small cohort of plasma samples from DMD patients when compared to healthy controls, female carriers, and other neuromuscular diseases. We also demonstrate the detection of dystrophin protein by an antibody-independent method using targeted liquid chromatography mass spectrometry. This last assay detects three different dystrophin peptides in all healthy individuals analysed and supports our finding that dystrophin protein is detectable in plasma. The results of our proof-of-concept study encourage further studies in larger sample cohorts to investigate the value of dystrophin protein as a low invasive blood biomarker for diagnostic screening and clinical monitoring of DMD. [ABSTRACT FROM AUTHOR]

Molecular diagnosis for Duchenne and Becker muscular dystrophies (DMD/BMD) involves a two‐tiered approach for detection of deletions/duplications using MLPA or array CGH, followed by sequencing of coding and flanking intronic regions to detect sequence variants, which is time‐consuming and expensive. We have developed a comprehensive next‐generation sequencing (NGS)‐based single‐step assay to sequence the entire 2.2 Mb of the DMD gene to detect all copy number and sequence variants in both index males and carrier females. Assay validation was 100% concordant with other methodologies. A total of 772 samples have been tested, of which 62% (N = 480) were index cases with a clinical suspicion of DMD. Carrier testing females account for 38% (N = 292). Molecular diagnosis was confirmed in 86% (N = 413) of the index cases. Intragenic deletions and duplications (single‐exon or multi‐exon) were detected in 60% (N = 247) and 14% (N = 58) of the index cases, respectively. Full‐sequence analysis of the entire gene allows for detection of deep intronic pathogenic variants and accurate breakpoint detection of CNVs involving similar exons, which could have an impact on the outcome of clinical trials. This comprehensive assay is highly sensitive for diagnostic testing for DMD and is also suitable for confirmatory testing for newborn screening for DMD. [ABSTRACT FROM AUTHOR]

Background: Duchenne 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. Methods: Proteins 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. Results: Proteomic 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. Conclusions: The 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 α‐ketoglutarate leading to shortage of glutamate‐derived molecules that over time triggers lipogenesis and lipotoxicity. [ABSTRACT FROM AUTHOR]

Duchenne muscular dystrophy is the most common form of childhood muscular dystrophy. A mutation in the DMD gene disrupts dystrophin (protein) production, causing damage to muscle integrity, weakness, loss of ambulation, and cardiopulmonary compromise by the second decade of life. Life expectancy has improved from mid-teenage years to mid-20s with the use of glucocorticoids and beyond the third decade with ventilator support and multidisciplinary care. However, Duchenne muscular dystrophy is associated with comorbidities and is a fatal disease. Glucocorticoids prolong ambulation, but their side effects are significant. Emerging investigational therapies have surfaced over the past decade and have rapidly been tested in clinical trials. Gene-specific strategies include nonsense readthrough, exon skipping, gene editing, utrophin modulation, and gene replacement. Other mechanisms include muscle regeneration, antioxidants, and antifibrosis and anti-inflammatory pathways. With potential therapies emerging, early diagnosis is needed to initiate treatment early enough to minimize morbidity and mortality. Newborn screening can be used to significantly improve early diagnosis, especially for gene-specific therapeutics. [ABSTRACT FROM AUTHOR]

Background: Duchenne muscular dystrophy is a lethal disease caused by lack of dystrophin. Skipping of exons adjacent to out-of-frame deletions has proven to restore dystrophin expression in Duchenne patients. Exon 51 has been the most studied target in both preclinical and clinical settings and the availability of standardized procedures to quantify exon skipping would be advantageous for the evaluation of preclinical and clinical data. Objective: To compare methods currently used to quantify antisense oligonucleotide–induced exon 51 skipping in the DMD transcript and to provide guidance about the method to use. Methods: Six laboratories shared blinded RNA samples from Duchenne patient-derived muscle cells treated with different amounts of exon 51 targeting antisense oligonucleotide. Exon 51 skipping levels were quantified using five different techniques: digital droplet PCR, single PCR assessed with Agilent bioanalyzer, nested PCR with agarose gel image analysis by either ImageJ or GeneTools software and quantitative real-time PCR. Results: Differences in mean exon skipping levels and dispersion around the mean were observed across the different techniques. Results obtained by digital droplet PCR were reproducible and showed the smallest dispersion. Exon skipping quantification with the other methods showed overestimation of exon skipping or high data variation. Conclusions: Our results suggest that digital droplet PCR was the most precise and quantitative method. The quantification of exon 51 skipping by Agilent bioanalyzer after a single round of PCR was the second-best choice with a 2.3-fold overestimation of exon 51 skipping levels compared to digital droplet PCR. [ABSTRACT FROM AUTHOR]

Duchenne muscular dystrophy is an X-linked neuromuscular disease, affecting mainly males, with an incidence of about 1: 3500-5000. The first symptoms usually appear at the age of 2-3 years, when boys do not run as quickly as their peers, have problems with climbing stairs, and fall more often. The typical biochemical abnormalities include increased activity of CK and AST/ALT. Subsequently, with the progressive weakening of muscles, boys lose the ability to walk around the age of 10-13 years. Later on, during adolescence, the respiratory function deteriorates and cardiomyopathy may appear. Steroid therapy, gold standard therapy, diminishes muscle tissue inflammation, prolongs the period of independent walking, as well as delays the deterioration of pulmonary and cardiac function. Mutations of the dystrophin gene lead to the impaired production of dystrophin protein - an important component of dystrophin-associated complex (DAPC), that connects the cytoskeleton of a muscle fiber to the surrounding extracellular matrix. Different types of mutations generally result in severe DMD (out-of-frame) and benign BMD (in-frame) phenotypes, although there have been exceptions described. Research regarding the role of different dystrophin isoforms, produced due to alternative splicing and found in other than muscular, tissues are conducted in relation to autism or other developmental disorders. Also, the regulatory role of non-coding RNA molecules in the course of DMD is currently being analyzed. [ABSTRACT FROM AUTHOR]

Objective: Duchenne muscular dystrophy (DMD) is characterised by progressive muscle weakness. It has recently been reported that single nucleotide polymorphisms (SNPs) located in the SPP1 and LTBP4 loci can account for some of the inter-individual variability observed in the clinical disease course. The validation of genetic association in large independent cohorts is a key process for rare diseases in order to qualify prognostic biomarkers and stratify patients in clinical trials.Methods: Duchenne patients from five European neuromuscular centres were included. Information about age at wheelchair dependence and steroid use was gathered. Melting curve analysis of PCR fragments or Sanger sequencing were used to genotype SNP rs28357094 in the SPP1 gene in 336 patients. The genotype of SNPs rs2303729, rs1131620, rs1051303 and rs10880 in the LTBP4 locus was determined in 265 patients by mass spectrometry. For both loci, a multivariate analysis was performed, using genotype/haplotype, steroid use and cohort as covariates.Results: We show that corticosteroid treatment and the IAAM haplotype of the LTBP4 gene are significantly associated with prolonged ambulation in patients with DMD. There was no significant association between the SNP rs28357094 in the SPP1 gene and the age of ambulation loss.Conclusions: This study underlines the importance of replicating genetic association studies for rare diseases in large independent cohorts to identify the most robust associations. We anticipate that genotyping of validated genetic associations will become important for the design and interpretation of clinical trials. [ABSTRACT FROM AUTHOR]

Dystrophin is a subsarcolemmal protein that, by linking the actin cytoskeleton to the extracellular matrix via dystroglycans, is critical for the integrity of muscle fibers. Here, we report that epidermal melanocytes, obtained from conventional skin biopsy, express dystrophin with a restricted localization to the plasma membrane facing the dermal-epidermal junction. In addition the full-length muscle isoform mDp427 was clearly detectable in melanocyte cultures as assessed by immunohistochemistry, RNA, and Western blot analysis. Melanocytes of Duchenne muscular dystrophy (DMD) patients did not express dystrophin, and the ultrastructural analysis revealed typical mitochondrial alterations similar to those occurring in myoblasts from the same patients. Mitochondria of melanocytes from DMD patients readily accumulated tetramethylrhodamine methyl ester, indicating that they are energized irrespective of the presence of dystrophin but, at variance from mitochondria of control donors, depolarized upon the addition of oligomycin, suggesting that they are affected by a latent dysfunction unmasked by inhibition of the ATP synthase. Pure melanocyte cultures can be readily obtained by conventional skin biopsies and may be a feasible and reliable tool alternative to muscle biopsy for functional studies in dystrophinopathies. The mitochondrial dysfunction occurring in DMD melanocytes could represent a promising cellular biomarker for monitoring dystrophinopathies also in response to pharmacological treatments. J. Cell. Physiol. 228: 1323-1331, 2013. © 2012 Wiley Periodicals, Inc. [ABSTRACT FROM AUTHOR]

The 2.2 Mb long dystrophin (DMD) gene, the largest gene in the human genome, corresponds to roughly 0.1% of the entire human DNA sequence. Mutations in this gene cause Duchenne muscular dystrophy and other milder X-linked, recessive dystrophinopathies. Using a custom-made tiling array, specifically designed for the DMD locus, we identified a variety of novel long non-coding RNAs (lncRNAs), both sense and antisense oriented, whose expression profiles mirror that of DMD gene. Importantly, these transcripts are intronic in origin and specifically localized to the nucleus and are transcribed contextually with dystrophin isoforms or primed by MyoD-induced myogenic differentiation. Furthermore, their forced ectopic expression in both human muscle and neuronal cells causes a specific and negative regulation of endogenous dystrophin full length isoforms and significantly down-regulate the activity of a luciferase reporter construct carrying the minimal promoter regions of the muscle dystrophin isoform. Consistent with this apparently repressive role, we found that, in muscle samples of dystrophinopathic female carriers, lncRNAs expression levels inversely correlate with those of muscle full length DMD isoforms. Overall these findings unveil an unprecedented complexity of the transcriptional pattern of the DMD locus and reveal that DMD lncRNAs may contribute to the orchestration and homeostasis of the muscle dystrophin expression pattern by either selective targeting and down-modulating the dystrophin promoter transcriptional activity. [ABSTRACT FROM AUTHOR]

Elucidation of new biomarkers and potential drug targets from high-throughput profiling data is a challenging task due to a limited number of available biological samples and questionable reproducibility of differential changes in cross-dataset comparisons. In this paper we propose a novel computational approach for drug and biomarkers discovery using comprehensive analysis of multiple expression profiling datasets. The new method relies on aggregation of individual profiling experiments combined with leave-one-dataset-out validation approach. Aggregated datasets were studied using Sub-Network Enrichment Analysis algorithm (SNEA) to find consistent statistically significant key regulators within the global literature-extracted expression regulation network. These regulators were linked to the consistent differentially expressed genes. We have applied our approach to several publicly available human muscle gene expression profiling datasets related to Duchenne muscular dystrophy (DMD). In order to detect both enhanced and repressed processes we considered up- and down-regulated genes separately. Applying the proposed approach to the regulators search we discovered the disturbance in the activity of several muscle-related transcription factors (e.g. MYOG and MYOD1), regulators of inflammation, regeneration, and fibrosis. Almost all SNEA-derived regulators of down-regulated genes (e.g. AMPK, TORC2, PPARGC1A) correspond to a single common pathway important for fast-to-slow twitch fiber type transition. We hypothesize that this process can affect the severity of DMD symptoms, making corresponding regulators and downstream genes valuable candidates for being potential drug targets and exploratory biomarkers. INSET: Author Summary. [ABSTRACT FROM AUTHOR]

In Duchenne muscular dystrophy, the exon-skipping approach has obtained proof of concept in animal models, myogenic cell cultures, and following local and systemic administration in Duchenne patients. Indeed, we have previously demonstrated that low doses (7.5 mg/Kg/week) of 2′-O-methyl-phosphorothioate antisense oligoribonucleotides (AONs) adsorbed onto ZM2 nanoparticles provoke widespread dystrophin restoration 7 days after intraperitoneal treatment in mdx mice. In this study, we went on to test whether this dystrophin restoration was still measurable 90 days from the end of the same treatment. Interestingly, we found that both western blot and immunohistochemical analysis (up to 7% positive fibres) were still able to detect dystrophin protein in the skeletal muscles of ZM2-AON-treated mice at this time, and the level of exon-23 skipping could still be assessed by RT real-time PCR (up to 10% of skipping percentage). In contrast, the protein was undetectable by western blot analysis in the skeletal muscles of mdx mice treated with an identical dose of naked AON, and the percentage of dystrophin-positive fibres and exon-23 skipping were reminiscent of those of untreated mdxmice. Our data therefore demonstrate the long-termresidual efficacy of this systemic low-dose treatment and confirm the protective effect nanoparticles exert on AON molecules. [ABSTRACT FROM AUTHOR]

Duchenne muscular dystrophy is caused by mutations in the DMD gene that disrupt the open reading frame and prevent the full translation of its protein product, dystrophin. Restoration of the open reading frame and dystrophin production can be achieved by exon skipping using antisense oligonucleotides targeted to splicing elements. This approach aims to transform the Duchenne muscular dystrophy phenotype to that of the milder disorder, Becker muscular dystrophy, typically caused by in-frame dystrophin deletions that allow the production of an internally deleted but partially functional dystrophin. There is ongoing debate regarding the functional properties of the different internally deleted dystrophins produced by exon skipping for different mutations; more insight would be valuable to improve and better predict the outcome of exon skipping clinical trials. To this end, we have characterized the clinical phenotype of 17 patients with Becker muscular dystrophy harbouring in-frame deletions relevant to on-going or planned exon skipping clinical trials for Duchenne muscular dystrophy and correlated it to the levels of dystrophin, and dystrophin-associated protein expression. The cohort of 17 patients, selected exclusively on the basis of their genotype, included 4 asymptomatic, 12 mild and 1 severe patient. All patients had dystrophin levels of >40% of control and significantly higher dystrophin (P = 0.013), β-dystroglycan (P = 0.025) and neuronal nitric oxide synthase (P = 0.034) expression was observed in asymptomatic individuals versus symptomatic patients with Becker muscular dystrophy. Furthermore, grouping the patients by deletion, patients with Becker muscular dystrophy with deletions with an end-point of exon 51 (the skipping of which could rescue the largest group of Duchenne muscular dystrophy deletions) showed significantly higher dystrophin levels (P = 0.034) than those with deletions ending with exon 53. This is the first quantitative study on both dystrophin and dystrophin-associated protein expression in patients with Becker muscular dystrophy with deletions relevant for on-going exon skipping trials in Duchenne muscular dystrophy. Taken together, our results indicate that all varieties of internally deleted dystrophin assessed in this study have the functional capability to provide a substantial clinical benefit to patients with Duchenne muscular dystrophy. [ABSTRACT FROM PUBLISHER]

Duchenne muscular dystrophy (DMD) is the most common childhood muscular dystrophy affecting ~1:5000 live male births. Following the identification of pathogenic variations in the dystrophin gene in 1986, the underlining genotype/phenotype correlations emerged and the role of the dystrophin protein was elucidated in skeletal, smooth, and cardiac muscles, as well as in the brain. When the dystrophin protein is absent or quantitatively or qualitatively modified, the muscle cannot sustain the stress of repeated contractions. Dystrophin acts as a bridging and anchoring protein between the sarcomere and the sarcolemma, and its absence or reduction leads to severe muscle damage that eventually cannot be repaired, with its ultimate substitution by connective tissue and fat. The advances of an understanding of the molecular pathways affected in DMD have led to the development of many therapeutic strategies that tackle different aspects of disease etiopathogenesis, which have recently led to the first successful approved orphan drugs for this condition. The therapeutic advances in this field have progressed exponentially, with second-generation drugs now entering in clinical trials as gene therapy, potentially providing a further effective approach to the condition. [ABSTRACT FROM AUTHOR]

• We reviewed Duchenne muscular dystrophy (DMD) genetic bases, and novel personalized therapeutical options. • We underlined the high allelic heterogeneity of the DMD mutations. • We summarized the different clinical characteristics which can be associated with the predominant skeletal muscle phenotype. • We described the implications of these features for the clinical definition and the design of novel therapies. • We also anticipated some novelties about new treatments still in the pre-clinical phase, and we reasoned about genotype-phenotype relationship. Duchenne muscular dystrophy (DMD) is a severe X-linked disease characterized by progressive muscle weakness. It is caused by a variety of DMD gene pathogenic variations (large deletions or duplications, and small mutations) which leads to the absence or to a decreased amount of dystrophin protein. The allelic Becker muscular dystrophy is characterized by later onset and milder muscle involvement, and other rarer phenotypes might also be associated, such as dilated cardiomyopathy, cognitive impairment, and other neurological signs. Following the identification of the genetic cause and the disease pathophysiology, innovative personalized therapies emerged. These can be categorized into two main groups: (1) therapies aiming at the restoration of dystrophin at the sarcolemma; (2) therapeutics dealing with secondary consequences of dystrophin deficiency. In this review we provide an overview about DMD genotype-phenotype correlation, and on main approaches to restore dystrophin as stop codon read-through, exon skipping, vector-mediated gene therapy, and genome-editing strategies, some of these are based on approved orphan drugs. Finally, we present the clinical potential of novel strategies combining therapies to correct the genetic defect and other approaches, targeting secondary downstream pathological cascade due to dystrophin deficiency. [ABSTRACT FROM AUTHOR]

Antisense therapy is a powerful tool for inducing post-transcriptional modifications and thereby regulating target genes associated with disease. There are several classes of antisense oligonucleotides (AONs) with therapeutic use, such as double-stranded RNAs (interfering RNAs, utilized for gene silencing, and single-stranded AONs with various chemistries, which are useful for antisense targeting of micro-RNAs and mRNAs. In particular, the use of AONs for exon skipping, by targeting pre-mRNA, is proving to be a highly promising therapy for some genetic disorders like Duchenne muscular dystrophy and spinal muscular atrophy. However, AONs are unable to cross the plasma membrane unaided, and several other obstacles still remain to be overcome, in particular their instability due to their nuclease sensitivity and their lack of tissue specificity. Various drug delivery systems have been explored to improve the bioavailability of nucleic acids, and nanoparticles (NPs) have been suggested as potential vectors for DNA/RNA. This review describes the recent progress in AON conjugation with natural and synthetic delivery systems, and provides an overview of the efficacy of NP-AON complexes as an exon-skipping treatment for Duchenne muscular dystrophy. [ABSTRACT FROM AUTHOR]

Duchenne muscular dystrophy (DMD) is a severe hereditary neuromuscular disorder caused by mutations in the dystrophin gene. Antisense-mediated targeted exon skipping has been shown to restore dystrophin expression both in DMD patients and in the mdx mouse, the murine model of DMD, but the ineffective delivery of these molecules limits their therapeutic use. We demonstrated that PMMA/N-isopropil-acrylamide (ZM2) nanoparticles (NPs), administered both intraperitoneally and orally, were able to deliver 2′OMePS antisense inducing various extents of dystrophin restoration in the mdx mice. Defining NP biodistribution is crucial to improve effects on target and dose regimens; thus, we performed in vivo studies of novel ZM4 NPs. ZM4 are conjugated with NIR fluorophores as optical probes suitable for studies on the Odyssey Imaging System. Our results indicate that NPs are widely distributed in all body muscles, including skeletal muscles and heart, suggesting that these vehicles are appropriate to deliver antisense oligonucleotides for targeting striated muscles in the DMD animal model, thus opening new horizons for Duchenne therapy. [ABSTRACT FROM AUTHOR]

We describe a 29-year-old patient who complained of left thigh muscle weakness since he was 23 and of moderate proximal weakness of both lower limbs with difficulty in climbing stairs and running since he was 27. Mild weakness of iliopsoas and quadriceps muscles and muscle atrophy of both the distal forearm and thigh were observed upon clinical examination. He harboured a novel c.1150-3C>G substitution in the DMD gene, affecting the intron 10 acceptor splice site and causing exon 11 skipping and an out-of-frame transcript. However, protein of normal molecular weight but in reduced amounts was observed on Western Blot analysis. Reverse transcription analysis on muscle RNA showed production, via alternative splicing, of a transcript missing exon 11 as well as a low abundant full-length transcript which is enough to avoid the severe Duchenne phenotype. Our study showed that a reduced amount of full length dystrophin leads to a mild form of Becker muscular dystrophy. These results confirm earlier findings that low amounts of dystrophin can be associated with a milder phenotype, which is promising for therapies aiming at dystrophin restoration. [ABSTRACT FROM AUTHOR]

A ready source of autologous myogenic cells is of vital importance for drug screening and functional genetic studies in Duchenne muscular dystrophy (DMD), a rare disease caused by a variety of dystrophin gene mutations. As stem cells (SCs) can be easily and noninvasively obtained from urine specimens, we set out to determine whether they could be myogenically induced and useful in DMD research. To this end, we isolated stem cells from the urine of two healthy donors and from one patient with DMD, and performed surface marker characterization, myogenic differentiation (MyoD), and then transfection with antisense oligoribonucleotides to test for exon skipping and protein restoration. We demonstrated that native urine-derived stem cells express the full-length dystrophin transcript, and that the dystrophin mutation was retained in the cells of the patient with DMD, although the dystrophin protein was detected solely in control cells after myogenic transformation according to the phenotype. Notably, we also showed that treatment with antisense oligoribonucleotide against dystrophin exon 44 induced skipping in both native and MyoD-transformed urine-derived stem cells in DMD, with a therapeutic transcript-reframing effect, as well as visible protein restoration in the latter. Hence MyoD-transformed cells may be a good myogenic model for studying dystrophin gene expression, and native urine stem cells could be used to study the dystrophin transcript, and both diagnostic procedures and splicing modulation therapies in both patients and control subjects, without invasive and costly collection methods. New, bankable bioproducts from urine stem cells, useful for prescreening studies and therapeutic applications alike, are also foreseeable after further, more in-depth characterization. [ABSTRACT FROM AUTHOR]

Abstract: Collagen VI is a major extracellular matrix (ECM) protein with a critical role in maintaining skeletal muscle functional integrity. Mutations in COL6A1, COL6A2 and COL6A3 genes cause Ullrich Congenital Muscular Dystrophy (UCMD), Bethlem Myopathy, and Myosclerosis. Moreover, Col6a1 −/− mice and collagen VI deficient zebrafish display a myopathic phenotype. Recently, two additional collagen VI chains were identified in humans, the α5 and α6 chains, however their distribution patterns and functions in human skeletal muscle have not been thoroughly investigated yet. By means of immunofluorescence analysis, the α6 chain was detected in the endomysium and perimysium, while the α5 chain labeling was restricted to the myotendinous junctions. In normal muscle cultures, the α6 chain was present in traces in the ECM, while the α5 chain was not detected. In the absence of ascorbic acid, the α6 chain was mainly accumulated into the cytoplasm of a sub-set of desmin negative cells, likely of interstitial origin, which can be considered myofibroblasts as they expressed α-smooth muscle actin. TGF-β1 treatment, a pro-fibrotic factor which induces trans-differentiation of fibroblasts into myofibroblasts, increased the α6 chain deposition in the extracellular matrix after addition of ascorbic acid. In order to define the involvement of the α6 chain in muscle fibrosis we studied biopsies of patients affected by Duchenne Muscular Dystrophy (DMD). We found that the α6 chain was dramatically up-regulated in fibrotic areas where, in contrast, the α5 chain was undetectable. Our results show a restricted and differential distribution of the novel α6 and α5 chains in skeletal muscle when compared to the widely distributed, homologous α3 chain, suggesting that these new chains may play specific roles in specialized ECM structures. While the α5 chain may have a specialized function in tissue areas subjected to tensile stress, the α6 chain appears implicated in ECM remodeling during muscle fibrosis. [Copyright &y& Elsevier]