Duchenne Muscular Dystrophy Transcript Research Articles

Differential expression of DMD transcripts as a novel prognostic biomarker in histologically diverse mesotheliomas.

The identification of prognostic biomarkers is crucial for guiding treatment strategies in mesothelioma patients. The Duchenne muscular dystrophy (DMD) gene and its specific transcripts have been associated with patient survival in various tumours. In this study, we aimed to investigate the prognostic potential of DMD gene expression and its transcripts in mesothelioma patients. We analysed The Cancer Genome Atlas (TCGA) mesothelioma RNAseq, mutation, and clinical data to assess the association between DMD gene expression and its transcripts (Dp427, Dp71 splice variants) and mesothelioma survival. We also evaluated the specific Dp71 transcript as a unique prognostic biomarker across mesothelioma subtypes. Additionally, we performed differential gene expression analysis between high and low DMD gene/transcript expression groups. The analysis included 57 epithelioid, 23 biphasic, two sarcomatoid, and five not otherwise specified (NOS) histological subtypes of mesothelioma samples. Univariate analysis revealed that high expression of the DMD gene and its Dp71 transcript was significantly associated with shorter survival in mesothelioma patients (P=0.003 and P<0.001, respectively). In a multivariate analysis, the association between Dp71 expression and survival remained significant [hazard ratio (HR) 2.29, 95% confidence interval (CI): 1.24-4.23, P=0.008] across all mesothelioma patients, and also among patients with mesotheliomas without deep CDKN2A deletions (HR 3.58, 95% CI: 1.31-9.80, P=0.01). Pathway analysis revealed enrichment of cell cycle (P=3.01×10-4) and homologous recombination (P=0.01) pathways in differentially expressed genes (DEGs) between high and low Dp71 groups. Furthermore, there were correlations between Dp71 transcript expression and tumour microenvironment (TME) cells, including a weak positive correlation with macrophages (R=0.32, P=0.002) specifically M2 macrophages (R=0.34, P=0.001). Our findings indicate that the differential expression of specific DMD transcripts is associated with poor survival in mesothelioma patients. The specific Dp71 transcript can serve as a potential biomarker for predicting patient survival in diverse histological subtypes of mesothelioma. Further studies are needed to understand the role of specific dystrophin transcripts in cancer and TME cells, and their implications in the pathogenesis and progression of mesothelioma. Identifying patients at risk of poor survival based on DMD transcript expression can guide treatment strategies in mesothelioma, informing decisions regarding treatment intensity, follow-up schedules, eligibility for clinical trials, and ultimately, end-of-life care planning.

Challenges of Assessing Exon 53 Skipping of the Human DMD Transcript with Locked Nucleic Acid-Modified Antisense Oligonucleotides in a Mouse Model for Duchenne Muscular Dystrophy.

Antisense oligonucleotide (AON)-mediated exon skipping is a promising therapeutic approach for Duchenne muscular dystrophy (DMD) patients to restore dystrophin expression by reframing the disrupted open reading frame of the DMD transcript. However, the treatment efficacy of the already conditionally approved AONs remains low. Aiming to optimize AON efficiency, we assessed exon 53 skipping of the DMD transcript with different chemically modified AONs, all with a phosphorothioate backbone: 2'-O-methyl (2'OMe), locked nucleic acid (LNA)-2'OMe, 2'-fluoro (FRNA), LNA-FRNA, αLNA-FRNA, and FANA-LNA-FRNA. Efficient exon 53 skipping was observed with the FRNA, LNA-FRNA, and LNA-2'OMe AONs in human control myoblast cultures. Weekly subcutaneous injections (50 mg/kg AON) for a duration of 6 weeks were well tolerated by hDMDdel52/mdx males. Treatment with the LNA-FRNA and LNA-2'OMe AONs resulted in pronounced exon 53 skip levels in skeletal muscles and heart up to 90%, but no dystrophin restoration was observed. This discrepancy was mainly ascribed to the strong binding nature of LNA modifications to RNA, thereby interfering with the amplification of the unskipped product resulting in artificial overamplification of the exon 53 skip product. Our study highlights that treatment effect on RNA and protein level should both be considered when assessing AON efficiency.

MRNA in situ hybridization exhibits unbalanced nuclear/cytoplasmic dystrophin transcript repartition in Duchenne myogenic cells and skeletal muscle biopsies

To gain insight on dystrophin (DMD) gene transcription dynamics and spatial localization, we assayed the DMD mRNA amount and defined its compartmentalization in myoblasts, myotubes, and skeletal muscle biopsies of Duchenne muscular dystrophy (DMD) patients. Using droplet digital PCR, Real-time PCR, and RNAscope in situ hybridization, we showed that the DMD transcript amount is extremely reduced in both DMD patients’ cells and muscle biopsies and that mutation-related differences occur. We also found that, compared to controls, DMD transcript is dramatically reduced in the cytoplasm, as up to 90% of it is localized in nuclei, preferentially at the perinuclear region. Using RNA/protein colocalization experiments, we showed that about 40% of nuclear DMD mRNA is localized in the nucleoli in both control and DMD myogenic cells. Our results clearly show that mutant DMD mRNA quantity is strongly reduced in the patients’ myogenic cells and muscle biopsies. Furthermore, mutant DMD mRNA compartmentalization is spatially unbalanced due to a shift in its localization towards the nuclei. This abnormal transcript repartition contributes to the poor abundance and availability of the dystrophin messenger in cytoplasm. This novel finding also has important repercussions for RNA-targeted therapies.

DMD antisense oligonucleotide mediated exon skipping efficiency correlates with flanking intron retention time and target position within the exon

ABSTRACT Mutations in the DMD gene are causative for Duchenne muscular dystrophy (DMD). Antisense oligonucleotide (AON) mediated exon skipping to restore disrupted dystrophin reading frame is a therapeutic approach that allows production of a shorter but functional protein. As DMD causing mutations can affect most of the 79 exons encoding dystrophin, a wide variety of AONs are needed to treat the patient population. Design of AONs is largely guided by trial-and-error, and it is yet unclear what defines the skippability of an exon. Here, we use a library of phosphorodiamidate morpholino oligomer (PMOs) AONs of similar physical properties to test the skippability of a large number of DMD exons. The DMD transcript is non-sequentially spliced, meaning that certain introns are retained longer in the transcript than downstream introns. We tested whether the relative intron retention time has a significant effect on AON efficiency, and found that targeting an out-of-frame exon flanked at its 5’-end by an intron that is retained in the transcript longer (‘slow’ intron) leads to overall higher exon skipping efficiency than when the 5’-end flanking intron is ‘fast’. Regardless of splicing speed of flanking introns, we find that positioning an AON closer to the 5’-end of the target exon leads to higher exon skipping efficiency opposed to targeting an exons 3’-end. The data enclosed herein can be of use to guide future target selection and preferential AON binding sites for both DMD and other disease amenable by exon skipping therapies.

Downregulation of Dystrophin Expression Occurs across Diverse Tumors, Correlates with the Age of Onset, Staging and Reduced Survival of Patients.

Altered dystrophin expression was found in some tumors and recent studies identified a developmental onset of Duchenne muscular dystrophy (DMD). Given that embryogenesis and carcinogenesis share many mechanisms, we analyzed a broad spectrum of tumors to establish whether dystrophin alteration evokes related outcomes. Transcriptomic, proteomic, and mutation datasets from fifty tumor tissues and matching controls (10,894 samples) and 140 corresponding tumor cell lines were analyzed. Interestingly, dystrophin transcripts and protein expression were found widespread across healthy tissues and at housekeeping gene levels. In 80% of tumors, DMD expression was reduced due to transcriptional downregulation and not somatic mutations. The full-length transcript encoding Dp427 was decreased in 68% of tumors, while Dp71 variants showed variability of expression. Notably, low expression of dystrophins was associated with a more advanced stage, older age of onset, and reduced survival across different tumors. Hierarchical clustering analysis of DMD transcripts distinguished malignant from control tissues. Transcriptomes of primary tumors and tumor cell lines with low DMD expression showed enrichment of specific pathways in the differentially expressed genes. Pathways consistently identified: ECM-receptor interaction, calcium signaling, and PI3K-Akt are also altered in DMD muscle. Therefore, the importance of this largest known gene extends beyond its roles identified in DMD, and certainly into oncology.

Clinical, muscle imaging, and genetic characteristics of dystrophinopathies with deep-intronic DMD variants.

Phenotypic heterogeneity within or between families with a same deep-intronic splice-altering variant in the DMD gene has never been systematically analyzed. This study aimed to determine the phenotypic and genetic characteristics of patients with deep-intronic DMD variants. Of 1338 male patients with a suspected dystrophinopathy, 38 were confirmed to have atypical pathogenic DMD variants via our comprehensive genetic testing approach. Of the 38 patients, 30 patients from 22 unrelated families with deep-intronic DMD variants underwent a detailed clinical and imaging assessment. Nineteen different deep-intronic DMD variants were identified in the 30 patients, including 15 with Duchenne muscular dystrophy (DMD), 14 with Becker muscular dystrophy (BMD), and one with X-linked dilated cardiomyopathy. Of the 19 variants, 15 were single-nucleotide variants, 2 were structural variants (SVs), and 2 were pure-intronic large-scale SVs causing aberrant inclusion of other protein-coding genes sequences into the mature DMD transcripts. The trefoil with single fruit sign was observed in 18 patients and the concentric fatty infiltration pattern was observed in 2 patients. Remarkable phenotypic heterogeneity was observed not only in skeletal but also cardiac muscle involvement in 2 families harboring a same deep-intronic variant. Different skeletal muscle involvement between families with a same variant was observed in 4 families. High inter-individual phenotypic heterogeneity was observed within two BMD families and one DMD family. Our study first highlights the variable phenotypic expressivity of deep-intronic DMD variants and demonstrates a new class of deep-intronic DMD variants, i.e., pure-intronic SVs involving other protein-coding genes.

VP.58 Golodirsen induced DMD transcripts localization and dystrophin production in MyoD-converted fibroblasts from 4053-101 clinical trial patients

Antisense oligonucleotides (AONs) are short, synthetic nucleic acid sequences that work by modulating exon incorporation at the level of pre-mRNA. In Duchenne muscular dystrophy (DMD), a fatal muscle degenerative disorder caused by mutations in the DMD gene, AONs skip specific exons to correct the reading frame, producing an internally shortened but partly functional dystrophin protein. Golodirsen is an approved AON phosphorodiamidate morpholino oligomer (PMO) that specifically targets DMD exon 53. In the clinical study 4053-101, we demonstrated that intravenous golodirsen administration induces an unequivocal exon skipping and protein restoration in all the treated patients, but with inter-patient variability. We used fibroblasts isolated from the patients in this clinical trial, that were induced to undergo myogenic differentiation in vitro by expression of MyoD, to better understand the reasons behind the observed variability. We evaluated the amount and the molecular weight of dystrophin protein in treated and non-treated patient cells, by an automated capillary-based immunoassay (WES) system. In these in-vitro studies we demonstrated that the amount of protein was comparable to the previous in-vivo study and that the size of the restored protein was compatible with the different genomic deletions carried by patients. Next, we used an in-situ RNA hybridization technique, BaseScope, to investigate the sub-cellular localization of the DMD transcript in treated and non-treated differentiated patient-derived myogenic cells in vitro, which allowed us to assess the ratio of skipped and unskipped products. Our study provides additional information on the dynamics of DMD mRNA in patients and may help to better understand the biological reasons underpinning variability in dystrophin restoration that can be seen in AON clinical trials.

RNA-seq in DMD urinary stem cells recognized muscle-related transcription signatures and addressed the identification of atypical mutations by whole-genome sequencing.

SummaryUrinary stem cells (USCs) are a non-invasive, simple, and affordable cell source to study human diseases. Here we show that USCs are a versatile tool for studying Duchenne muscular dystrophy (DMD), since they are able to address RNA signatures and atypical mutation identification. Gene expression profiling of DMD individuals’ USCs revealed a profound deregulation of inflammation, muscle development, and metabolic pathways that mirrors the known transcriptional landscape of DMD muscle and worsens following USCs’ myogenic transformation. This pathogenic transcription signature was reverted by an exon-skipping corrective approach, suggesting the utility of USCs in monitoring DMD antisense therapy. The full DMD transcript profile performed in USCs from three undiagnosed DMD individuals addressed three splicing abnormalities, which were decrypted and confirmed as pathogenic variations by whole-genome sequencing (WGS). This combined genomic approach allowed the identification of three atypical and complex DMD mutations due to a deep intronic variation and two large inversions, respectively. All three mutations affect DMD gene splicing and cause a lack of dystrophin protein production, and one of these also generates unique fusion genes and transcripts. Further characterization of USCs using a novel cell-sorting technology (Celector) highlighted cell-type variability and the representation of cell-specific DMD isoforms. Our comprehensive approach to USCs unraveled RNA, DNA, and cell-specific features and demonstrated that USCs are a robust tool for studying and diagnosing DMD.

DEVELOPING AN EFFECTIVE EXONS 45-55 SKIPPING THERAPY FOR DUCHENNE MUSCULAR DYSTROPHY

Duchenne muscular dystrophy (DMD) is an X-linked recessive disorder characterized by progressive body-wide muscle weakness and deterioration, ultimately resulting in death by the 20’s due to cardiac/respiratory malfunction. Since advancements in ventilation assistance, cardiac failure is increasingly becoming the leading cause of death in patients. However, there is no cure for DMD. DMD is caused mostly by out-of-frame deletions in the dystrophin gene (DMD). Exon skipping is a promising therapy using antisense oligonucleotides (AOs) to remove incompatible exons from the DMD transcript, restoring the reading frame and production of truncated, partially functional dystrophin in muscle.

DMD – ANIMAL MODELS & PRECLINICAL TREATMENT: P.212 Systemic delivery of PPMO restores the full-length dystrophin protein in the Dup2 Mouse

The gene corrective therapy resulting in a selective exclusion of target exon(s) is an approach that is already in clinical use to treat patients with Duchenne muscular dystrophy (DMD). Two currently approved therapies alter splicing to exclude exon 51 or exon 53 from the mature mRNA, restoring translation of a truncated, yet functionally active protein. However, the antisense PMO therapies allow to restore the wild-type (WT) DMD transcript and full-length dystrophin in patients with a single exon duplication.

Premature termination codons in the DMD gene cause reduced local mRNA synthesis

Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene leading to the presence of premature termination codons (PTC). Previous transcriptional studies have shown reduced DMD transcript levels in DMD patient and animal model muscles when PTC are present. Nonsense-mediated decay (NMD) has been suggested to be responsible for the observed reduction, but there is no experimental evidence supporting this claim. In this study, we aimed to investigate the mechanism responsible for the drop in DMD expression levels in the presence of PTC. We observed that the inhibition of NMD does not normalize DMD gene expression in DMD. Additionally, in situ hybridization showed that DMD messenger RNA primarily localizes in the nuclear compartment, confirming that a cytoplasmic mechanism like NMD indeed cannot be responsible for the observed reduction. Sequencing of nascent RNA to explore DMD transcription dynamics revealed a lower rate of DMD transcription in patient-derived myotubes compared to healthy controls, suggesting a transcriptional mechanism involved in reduced DMD transcript levels. Chromatin immunoprecipitation in muscle showed increased levels of the repressive histone mark H3K9me3 in mdx mice compared to wild-type mice, indicating a chromatin conformation less prone to transcription in mdx mice. In line with this finding, treatment with the histone deacetylase inhibitor givinostat caused a significant increase in DMD transcript expression in mdx mice. Overall, our findings show that transcription dynamics across the DMD locus are affected by the presence of PTC, hinting at a possible epigenetic mechanism responsible for this process.

Single Exon Skipping Can Address a Multi-Exon Duplication in the Dystrophin Gene.

Duchenne muscular dystrophy (DMD) is a severe muscle wasting disease typically caused by protein-truncating mutations that preclude synthesis of a functional dystrophin. Exonic deletions are the most common type of DMD lesion, however, whole exon duplications account for between 10–15% of all reported mutations. Here, we describe in vitro evaluation of antisense oligonucleotide-induced splice switching strategies to re-frame the transcript disrupted by a multi-exon duplication within the DMD gene. Phosphorodiamidate morpholino oligomers and phosphorodiamidate morpholino oligomers coupled to a cell penetrating peptide were evaluated in a Duchenne muscular dystrophy patient cell strain carrying an exon 14–17 duplication. Two strategies were employed; the conventional approach was to remove both copies of exon 17 in addition to exon 18, and the second strategy was to remove only the first copy of exon 17. Both approaches result in a larger than normal but in-frame DMD transcript, but surprisingly, the removal of only the first exon 17 appeared to be more efficient in restoring dystrophin, as determined using western blotting. The emergence of a normal sized DMD mRNA transcript that was not apparent in untreated samples may have arisen from back splicing and could also account for some of the dystrophin protein being produced.

Challenges associated with homologous directed repair using CRISPR-Cas9 and TALEN to edit the DMDgenetic mutation in canine Duchenne muscular dystrophy.

Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene that abolish the expression of dystrophin protein. Dogs with the genetic homologue, golden retriever muscular dystrophy dog (GRMD), have a splice site mutation that leads to skipping of exon 7 and a stop codon in the DMD transcript. Gene editing via homology-directed repair (HDR) has been used in the mdx mouse model of DMD but not in GRMD. In this study, we used clustered regularly interspaced short palindromic repeats (CRISPR) and transcription activator-like effector nucleases (TALEN) to restore dystrophin expression via HDR in myoblasts/myotubes and later via intramuscular injection of GRMD dogs. In vitro, DNA and RNA were successfully corrected but dystrophin protein was not translated. With intramuscular injection of two different guide arms, sgRNA A and B, there was mRNA expression and Sanger sequencing confirmed inclusion of exon 7 for all treatments. On Western blot analysis, protein expression of up to 6% of normal levels was seen in two dogs injected with sgRNA B and up to 16% of normal in one dog treated with sgRNA A. TALEN did not restore any dystrophin expression. While there were no adverse effects, clear benefits were not seen on histopathologic analysis, immunofluorescence microscopy, and force measurements. Based on these results, methods must be modified to increase the efficiency of HDR-mediated gene repair and protein expression.

P.141PPMO-mediated skipping therapy of duplicated exon 2 in the DMD gene

Duchenne muscular dystrophy (DMD) is caused by fatal mutations that disrupt the reading frame of the DMD gene, leading to lack of dystrophin protein. The only approved gene corrective therapy is an antisense phosphorodiamidate morpholino oligomer (PMO) that alters splicing to exclude exon 51 from the mRNA, restoring translation of a shortened but functionally active protein. Use of antisense PMO skipping in the presence of single exon duplications, in contrast, may restore a wild-type DMD transcript and dystrophin protein. Here we report the application of cell-penetrating peptide-conjugated PMO, or PPMO, to target the DMD exon 2 splice acceptor site in Dup2 mice, which carry a duplication of exon 2 (the most common single duplication mutation in DMD patients, accounting for around 10% of all duplication mutations). In the presence of an exon 2 duplication, skipping can result in two therapeutic transcripts. Skipping of a single copy of exon 2 results in a wild-type transcript, whereas skipping both copies results in a transcript lacking exon 2 entirely (Del2), but from which functionally active dystrophin protein can be expressed via utilization of an exon 5 internal ribosome entry site (IRES). A single intravenous injection of PPMO was delivered to 12 week old mice at three dose levels; saline injection and scrambled PPMO were used as controls. All mice (n=5-6 each) were sacrificed one week post-injection. Five muscles–tibialis anterior (TA), gastrocnemius (Gas), quadriceps (Qua), diaphragm (Dia) and heart were harvested for analysis of DMD mRNA transcripts by RT-PCR, and for dystrophin protein expression by western blot and immunofluorescence staining. The single injection resulted in a clear dose response of exon skipping, maximal at the highest dose tested; robust skipping of the duplicated exon 2 (both WT and Del2 transcripts) was seen in skeletal muscles, including TA (40.8%), Gas (39.7%), Qua (53.5%), and Dia (53.1%), with minimal skipping observed in heart muscle (8%). Dystrophin protein restoration was seen at levels of up to 13% in Dia and up to 15% in Gas, and at lower levels in other tissues, likely reflecting the single injection and short time period allowed for expression. We are evaluating longer term expression, with a goal of developing a meaningful therapy for patients with exon 2 duplications and potentially other 5′ DMD mutations.

A fusion peptide directs enhanced systemic dystrophin exon skipping and functional restoration in dystrophin-deficient mdx mice.

Duchenne muscular dystrophy (DMD) is caused by mutations in the DMD gene that abolish the synthesis of dystrophin protein. Antisense oligonucleotides (AOs) targeted to trigger excision of an exon bearing a mutant premature stop codon in the DMD transcript have been shown to skip the mutated exon and partially restore functional dystrophin protein in dystrophin-deficient mdx mice. To fully exploit the therapeutic potential of this method requires highly efficient systemic AO delivery to multiple muscle groups, to modify the disease process and restore muscle function. While systemic delivery of naked AOs in DMD animal models requires high doses and is of relatively poor efficiency, we and others have recently shown that short arginine-rich peptide-AO conjugates can dramatically improve in vivo DMD splice correction. Here we report for the first time that a chimeric fusion peptide (B-MSP-PMO) consisting of a muscle-targeting heptapeptide (MSP) fused to an arginine-rich cell-penetrating peptide (B-peptide) and conjugated to a morpholino oligomer (PMO) AO directs highly efficient systemic dystrophin splice correction in mdx mice. With very low systemic doses, we demonstrate that B-MSP-PMO restores high-level, uniform dystrophin protein expression in multiple peripheral muscle groups, yielding functional correction and improvement of the mdx dystrophic phenotype. Our data demonstrate proof-of-concept for this chimeric peptide approach in DMD splice correction therapy and is likely to have broad application.

RNA-Seq Analysis of an Antisense Sequence Optimized for Exon Skipping in Duchenne Patients Reveals No Off-Target Effect.

Non-coding uridine-rich small nuclear RNAs (UsnRNAs) have emerged in recent years as effective tools for exon skipping for the treatment of Duchenne muscular dystrophy (DMD), a degenerative muscular genetic disorder. We recently showed the high capacity of a recombinant adeno-associated virus (rAAV)-U7snRNA vector to restore the reading frame of the DMD mRNA in the muscles of DMD dogs. We are now moving toward a phase I/II clinical trial with an rAAV-U7snRNA-E53, carrying an antisense sequence designed to hybridize exon 53 of the human DMD messenger. As observed for genome-editing tools, antisense sequences present a risk of off-target effects, reflecting partial hybridization onto unintended transcripts. To characterize the clinical antisense sequence, we studied its expression and explored the occurrence of its off-target effects in human in vitro models of skeletal muscle and liver. We presented a comprehensive methodology combining RNA sequencing and in silico filtering to analyze off-targets. We showed that U7snRNA-E53 induced the effective exon skipping of the DMD transcript without inducing the notable deregulation of transcripts in human cells, neither at gene expression nor at the mRNA splicing level. Altogether, these results suggest that the use of the rAAV-U7snRNA-E53 vector for exon skipping could be safe in eligible DMD patients.

Myoblasts and macrophages are required for therapeutic morpholino antisense oligonucleotide delivery to dystrophic muscle

Exon skipping is a promising therapeutic strategy for Duchenne muscular dystrophy (DMD), employing morpholino antisense oligonucleotides (PMO-AO) to exclude disruptive exons from the mutant DMD transcript and elicit production of truncated dystrophin protein. Clinical trials for PMO show variable and sporadic dystrophin rescue. Here, we show that robust PMO uptake and efficient production of dystrophin following PMO administration coincide with areas of myofiber regeneration and inflammation. PMO localization is sustained in inflammatory foci where it enters macrophages, actively differentiating myoblasts and newly forming myotubes. We conclude that efficient PMO delivery into muscle requires two concomitant events: first, accumulation and retention of PMO within inflammatory foci associated with dystrophic lesions, and second, fusion of PMO-loaded myoblasts into repairing myofibers. Identification of these factors accounts for the variability in clinical trials and suggests strategies to improve this therapeutic approach to DMD.

P.498 - Dystrophin Dp427 is lost due to multiple DMD intron retentions in rhabdomyosarcoma CRL-2061 cells

Dystrophin inactivation, responsible for Duchenne muscular dystrophy (DMD), is recently implicated as an anti-tumor suppressor factor in cancers with myogenic characteristics. Rhabdomyosarcoma (RMS), a mesodermal cancer, shows myogenic features and occurs at a higher frequency in mdx mouse, a DMD model mouse. In humans, as the clinical management of DMD advances to increase the life expectancy of patients, a subsequent issue is anticipated to be that of addressing other complications such as RMS. RMS, although displays myogenic properties, has not yet been fully evaluated for dystrophin expression. Here, we used reverse transcription PCR and western blotting to analyze dystrophin in RMS CRL-2061 cells. The 14 kb DMD transcript was fully amplified as 20 fragments as in skeletal muscle. Alternatively, spliced transcripts including 3 intron retentions and 2 exons skipping events were disclosed. In one fragment extending from exon 70 to 79, no normally spliced product was obtained. Instead, 6 alternatively spliced products including a new product deleting exon 73 were identified, and exon 78 deleted products was most abundant. However, dystrophin Dp427 was not detected by western blotting. Remarkably, analysis of 9 short DMD introns indicated retention of introns 40, 58 and 70. These intron retentions occupied 75, 15 and 20 % of the total PCR amplified products, respectively, indicating the possibility of every DMD transcript to carry an intron retention event. Multiple intron retentions in the DMD transcript are suggested to hamper translation of the Dp427 isoform and promote tumorigenesis in RMS. This study exemplifies the important significance of DMD intron retention in tumorigenesis in RMS.

Targeted RNA-Seq profiling of splicing pattern in the DMD gene: exons are mostly constitutively spliced in human skeletal muscle

We have analysed the splicing pattern of the human Duchenne Muscular Dystrophy (DMD) transcript in normal skeletal muscle. To achieve depth of coverage required for the analysis of this lowly expressed gene in muscle, we designed a targeted RNA-Seq procedure that combines amplification of the full-length 11.3 kb DMD cDNA sequence and 454 sequencing technology. A high and uniform coverage of the cDNA sequence was obtained that allowed to draw up a reliable inventory of the physiological alternative splicing events in the muscular DMD transcript. In contrast to previous assumptions, we evidenced that most of the 79 DMD exons are constitutively spliced in skeletal muscle. Only a limited number of 12 alternative splicing events were identified, all present at a very low level. These include previously known exon skipping events but also newly described pseudoexon inclusions and alternative 3′ splice sites, of which one is the first functional NAGNAG splice site reported in the DMD gene. This study provides the first RNA-Seq-based reference of DMD splicing pattern in skeletal muscle and reports on an experimental procedure well suited to detect condition-specific differences in this low abundance transcript that may prove useful for diagnostic, research or RNA-based therapeutic applications.

Dp412e: a novel human embryonic dystrophin isoform induced by BMP4 in early differentiated cells.

BackgroundDuchenne muscular dystrophy (DMD) is a devastating X-linked recessive genetic myopathy. DMD physiopathology is still not fully understood and a prenatal onset is suspected but difficult to address.MethodsThe bone morphogenetic protein 4 (BMP4) is a critical signaling molecule involved in mesoderm commitment. Human induced pluripotent stem cells (hiPSCs) from DMD and healthy individuals and human embryonic stem cells (hESCs) treated with BMP4 allowed us to model the early steps of myogenesis in normal and DMD contexts.ResultsUnexpectedly, 72h following BMP4 treatment, a new long DMD transcript was detected in all tested hiPSCs and hESCs, at levels similar to that found in adult skeletal muscle. This novel transcript named “Dp412e” has a specific untranslated first exon which is conserved only in a sub-group of anthropoids including human. The corresponding novel dystrophin protein of 412-kiloDalton (kDa), characterized by an N-terminal-truncated actin-binding domain, was detected in normal BMP4-treated hiPSCs/hESCs and in embryoid bodies. Finally, using a phosphorodiamidate morpholino oligomer (PMO) targeting the DMD exon 53, we demonstrated the feasibility of exon skipping validation with this BMP4-inducible hiPSCs model.ConclusionsIn this study, the use of hiPSCs to analyze early phases of human development in normal and DMD contexts has led to the discovery of an embryonic 412 kDa dystrophin isoform. Deciphering the regulation process(es) and the function(s) associated to this new isoform can contribute to a better understanding of the DMD physiopathology and potential developmental defects. Moreover, the simple and robust BMP4-inducible model highlighted here, providing large amount of a long DMD transcript and the corresponding protein in only 3 days, is already well-adapted to high-throughput and high-content screening approaches. Therefore, availability of this powerful cell platform can accelerate the development, validation and improvement of DMD genetic therapies.Electronic supplementary materialThe online version of this article (doi:10.1186/s13395-015-0062-6) contains supplementary material, which is available to authorized users.