P268 Decoding the transcriptome of Duchenne muscular dystrophy to the single nuclei level reveals clinical-genetic correlations

Duchenne muscular dystrophy (DMD) is characterized by early onset muscle weakness leading to irreversible severe disability. The process of muscle degeneration in DMD involves a complex interplay between muscle fibers, muscle resident cells and, circulating cells invading the muscle. Despite considerable progress in the understanding of this process, there is still a lack of knowledge of what are the cellular and molecular consequences of the absence of dystrophin in humans. We aimed to study changes in the gene expression profile to the single nuclei level of muscle samples from patients with DMD and age/gender matched controls. We have performed single nuclei RNA sequencing analysis of 7 samples of the quadriceps muscle of DMD patients aged 2 to 4 years before steroids were started and 5 samples from age and gender matched healthy controls. Bioinformatic analysis was performed using packages developed for the analysis of single cell/nuclei RNA sequencing in R and python. A total of 30857 nuclei from controls and 25817 nuclei from DMD were analyzed. Using Seurat we identified 19 different nuclei clusters that were reduced to 11 putative identities after differentially expressed gene signatures were investigated including slow and fast myofibers, regenerative fibers, satellite cells, endothelial cells, smooth muscle cells, fibroadipogenic progenitor cells (FAPs), adipocytes, macrophages and, lymphocytes. We observed significant differences in the proportion of cell populations in the DMD samples versus controls, characterized by a reduction in the number of nuclei from muscle fibers and smooth muscle cells and an increase in the nuclei from regenerative fibers, satellite cells and FAPs. Patients with a better muscle function at the time of the biopsy had a significant higher number of regenerative fibers, while those with a worse muscle function had a significant increase in FAPs. Analysis of gene expression profile revealed significant differences in genes expressed in several clusters. In the case of myonuclei we observe a increase in gene involved in myogenesis, muscle growth, axon guidance and linking of muscle fibers to cytoskeletal with a reduced expression of genes involved in metabolic pathways such as glycolysis and oxidative phosphorylation. FAPs from DMD patients were characterized by an increase expression of genes coding for components of the extracellular matrix and genes involved in cell dividion. A deep analysis of the FAP cluster allowed us to identify seven different population, which proportion varied from controls to DMD. Two of these clusters were exclusively present in DMD samples, and were characterized by the expression of genes involved in cell division and proliferation. The population of these later FAP subtypes was increased in patients with worse muscle function at baseline. We have observed substantial differences in the population of cells present in skeletal muscle samples of DMD patients compared with controls even at earlier stages of disease progression. Moreover, we have identified a large number of genes which expression is dysregulated in DMD cell population pointing towards an enhanced regenerative activity in DMD patients associated with an increase proliferative activity of FAPS, which produce high levels of extracellular matrix components. Duchenne muscular dystrophy (DMD) is characterized by early onset muscle weakness leading to irreversible severe disability. The process of muscle degeneration in DMD involves a complex interplay between muscle fibers, muscle resident cells and, circulating cells invading the muscle. Despite considerable progress in the understanding of this process, there is still a lack of knowledge of what are the cellular and molecular consequences of the absence of dystrophin in humans. We aimed to study changes in the gene expression profile to the single nuclei level of muscle samples from patients with DMD and age/gender matched controls. We have performed single nuclei RNA sequencing analysis of 7 samples of the quadriceps muscle of DMD patients aged 2 to 4 years before steroids were started and 5 samples from age and gender matched healthy controls. Bioinformatic analysis was performed using packages developed for the analysis of single cell/nuclei RNA sequencing in R and python. A total of 30857 nuclei from controls and 25817 nuclei from DMD were analyzed. Using Seurat we identified 19 different nuclei clusters that were reduced to 11 putative identities after differentially expressed gene signatures were investigated including slow and fast myofibers, regenerative fibers, satellite cells, endothelial cells, smooth muscle cells, fibroadipogenic progenitor cells (FAPs), adipocytes, macrophages and, lymphocytes. We observed significant differences in the proportion of cell populations in the DMD samples versus controls, characterized by a reduction in the number of nuclei from muscle fibers and smooth muscle cells and an increase in the nuclei from regenerative fibers, satellite cells and FAPs. Patients with a better muscle function at the time of the biopsy had a significant higher number of regenerative fibers, while those with a worse muscle function had a significant increase in FAPs. Analysis of gene expression profile revealed significant differences in genes expressed in several clusters. In the case of myonuclei we observe a increase in gene involved in myogenesis, muscle growth, axon guidance and linking of muscle fibers to cytoskeletal with a reduced expression of genes involved in metabolic pathways such as glycolysis and oxidative phosphorylation. FAPs from DMD patients were characterized by an increase expression of genes coding for components of the extracellular matrix and genes involved in cell dividion. A deep analysis of the FAP cluster allowed us to identify seven different population, which proportion varied from controls to DMD. Two of these clusters were exclusively present in DMD samples, and were characterized by the expression of genes involved in cell division and proliferation. The population of these later FAP subtypes was increased in patients with worse muscle function at baseline. We have observed substantial differences in the population of cells present in skeletal muscle samples of DMD patients compared with controls even at earlier stages of disease progression. Moreover, we have identified a large number of genes which expression is dysregulated in DMD cell population pointing towards an enhanced regenerative activity in DMD patients associated with an increase proliferative activity of FAPS, which produce high levels of extracellular matrix components.