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Degenerative and regenerative pathways underlying Duchenne muscular dystrophy revealed by single-nucleus RNA sequencing.

ABSTRACT: Duchenne muscular dystrophy (DMD) is a fatal muscle disorder characterized by cycles of degeneration and regeneration of multinucleated myofibers and pathological activation of a variety of other muscle-associated cell types. The extent to which different nuclei within the shared cytoplasm of a myofiber may display transcriptional diversity and whether individual nuclei within a multinucleated myofiber might respond differentially to DMD pathogenesis is unknown. Similarly, the potential transcriptional diversity among nonmuscle cell types within dystrophic muscle has not been explored. Here, we describe the creation of a mouse model of DMD caused by deletion of exon 51 of the dystrophin gene, which represents a prevalent disease-causing mutation in humans. To understand the transcriptional abnormalities and heterogeneity associated with myofiber nuclei, as well as other mononucleated cell types that contribute to the muscle pathology associated with DMD, we performed single-nucleus transcriptomics of skeletal muscle of mice with dystrophin exon 51 deletion. Our results reveal distinctive and previously unrecognized myonuclear subtypes within dystrophic myofibers and uncover degenerative and regenerative transcriptional pathways underlying DMD pathogenesis. Our findings provide insights into the molecular underpinnings of DMD, controlled by the transcriptional activity of different types of muscle and nonmuscle nuclei.

SUBMITTER: Chemello F

PROVIDER: S-EPMC7703557 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundDuchenne Muscular Dystrophy (DMD) is an X-linked recessive disorder with its primary insult on the skeletal muscle. Severe muscle wasting, chronic inflammation and fibrosis characterize dystrophic muscle. Here we identify dysregulated pathways in DMD utilizing a co-expression network approach as described in Weighted Gene Co-expression Network Analysis (WGCNA). Specifically, we utilize WGCNA's "preservation" statistics to identify gene modules that exhibit a weak conservation of network topology within healthy and dystrophic networks. Preservation statistics rank modules based on their topological metrics such as node density, connectivity and separability between networks.MethodsRaw data for DMD was downloaded from Gene Expression Omnibus (GSE6011) and suitably preprocessed. Co-expression networks for each condition (healthy and dystrophic) were generated using the WGCNA library in R. Preservation of healthy network edges was evaluated with respect to dystrophic muscle and vice versa using WGCNA. Highly exclusive gene pairs for each of the low preserved modules within both networks were also determined using a specificity measure.ResultsA total of 11 and 10 co-expressed modules were identified in the networks generated from 13 healthy and 23 dystrophic samples respectively. 5 out of the 11, and 4 out of the 10 modules were identified as exhibiting none-to-weak preservation. Functional enrichment analysis identified that these weakly preserved modules were highly relevant to the condition under study. For instance, weakly preserved dystrophic module D2 exhibited the highest fraction of genes exclusive to DMD. The highly specific gene pairs identified within these modules were enriched for genes activated in response to wounding and affect the extracellular matrix including several markers such as SPP1, MMP9 and ITGB2.ConclusionThe proposed approach allowed us to identify clusters of genes that are non-randomly associated with the disease. Furthermore, highly specific gene pairs pointed to interactions between known markers of disease and identification of putative markers likely associated with disease. The analysis also helped identify putative novel interactions associated with the progression of DMD.

Project description:Dystrophinopathies cover a spectrum of rare progressive X-linked muscle diseases, arising from DMD mutations. They are among the most common pediatric muscular dystrophies, being Duchenne muscular dystrophy (DMD) the most severe form. Despite the fact that there is still no cure for these serious diseases, unprecedented advances are being made for the development of therapies for DMD. Some of which are already conditionally approved: exon skipping and premature stop codon read-through. The present work aimed to characterize the mutational spectrum of DMD in an Argentinian cohort, to identify candidates for available pharmacogenetic treatments and finally, to conduct a comparative analysis of the Latin American (LA) frequencies of mutations amenable for available DMD therapies. We studied 400 patients with clinical diagnosis of dystrophinopathy, implementing a diagnostic molecular algorithm including: MLPA/PCR/Sanger/Exome and bioinformatics. We also performed a meta-analysis of LA's metrics for DMD available therapies. The employed algorithm resulted effective for the achievement of differential diagnosis, reaching a detection rate of 97%. Because of this, corticosteroid treatment was correctly indicated and validated in 371 patients with genetic confirmation of dystrophinopathy. Also, 20 were eligible for exon skipping of exon 51, 21 for exon 53, 12 for exon 45 and another 70 for premature stop codon read-through therapy. We determined that 87.5% of DMD patients will restore the reading frame with the skipping of only one exon. Regarding nonsense variants, UGA turned out to be the most frequent premature stop codon observed (47%). According to the meta-analysis, only four LA countries (Argentina, Brazil, Colombia and Mexico) provide the complete molecular algorithm for dystrophinopathies. We observed different relations among the available targets for exon skipping in the analyzed populations, but a more even proportion of nonsense variants (∼40%). In conclusion, this manuscript describes the theragnosis carried out in Argentinian dystrophinopathy patients. The implemented molecular algorithm proved to be efficient for the achievement of differential diagnosis, which plays a crucial role in patient management, determination of the standard of care and genetic counseling. Finally, this work contributes with the international efforts to characterize the frequencies and variants in LA, pillars of drug development and theragnosis.