Unraveling the spatial landscape of Dystrophinopathies: a transcriptomic approach to Becker and Duchenne muscular dystrophies

Abstract Dystrophinopathies are caused by pathogenic variants in the DMD gene resulting in partial (Becker) or complete loss (Duchenne) of dystrophin. Becker (BMD) and Duchenne muscular dystrophy (DMD), are characterized by progressive muscle wasting, fatty replacement, fibrosis, and loss of function. To study histopathological changes, we used spatial transcriptomics to profile skeletal muscle biopsies of BMD, DMD patients and healthy controls (N = 4 per group). We estimated the proportion of cell types and their spatial localization across samples applying a deconvolution strategy using single-nuclei RNA-sequencing data. We identified genes enriched in fat patches and cell types such as fibroadipogenic progenitor cells (FAPs) in areas of active pathology. Using expression data of ligand receptor pairs, we highlight cell-cell communications leading to fibrotic and adipogenic lesions. Finally, analysis of gene expression gradients in areas of adjacent muscle and fat, allowed the identification of genes associated with muscle areas committed to become fat. Significance statement This study investigates the cellular and molecular changes that occur in muscles affected by Becker and Duchenne muscular dystrophy (BMD and DMD). These diseases are caused by mutations in the DMD gene, leading to muscle degeneration and the replacement of muscle tissue with fibrotic and fatty tissue causative for an early death. By using spatial transcriptomics, the researchers analyzed muscle biopsies from BMD, DMD patients, and healthy controls. They identified specific genes and cell types, such as fibroadipogenic progenitor cells, that are involved in disease progression. The study also revealed how different cells communicate with each other to drive muscle degeneration and fat accumulation. These findings provide new insights into the mechanisms of disease and potential targets for future therapies. Competing Interest Statement The authors have declared no competing interest. Funding Statement We would like to thank the AFM-Telethon support with grant n. 23629 to P. Spitali and L.G.M. Heezen. Healthy control biopsies from the Netherlands were supported the Netherlands Organization for Scientific Research (NWO, under research program VIDI, Grant # 917.164.90). Author Declarations I confirm all relevant ethical guidelines have been followed, and any necessary IRB and/or ethics committee approvals have been obtained. Yes The details of the IRB/oversight body that provided approval or exemption for the research described are given below: The LUMC Toetsing Commissie for Neurologische ziekten evaluated the research protocol (B20.001) and approved with number 3.4258/010/FB/jr. Informed consent forms were obtained from one DMD patient and his legal representatives. The healthy controls included in this study from the Netherlands were obtained in The Hague, obtained from individuals undergoing anterior cruciate ligament (ACL) surgery. For these subjects (N = 3), the study was approved by the local Medical Ethical Review Board of The Hague Zuid-West and the Erasmus Medical Centre and conducted in accordance with the ethical standards stated in the 1964 Declaration of Helsinki and its later amendments (ABR number: NL54081.098.16). All other subjects (N = 1 healthy control, N = 3 DMD and N = 4 BMD) were included in the United States of America and this study was approved by the Nationwide Childrens Hospital Institutional Review Board (0502HSE046 and IRB14-00719). All subjects or their guardians provided written informed consent prior to participation. Patients were included in their respective dystrophinopathy group based on clinical diagnosis. I confirm that all necessary patient/participant consent has been obtained and the appropriate institutional forms have been archived, and that any patient/participant/sample identifiers included were not known to anyone (e.g., hospital staff, patients or participants themselves) outside the research group so cannot be used to identify individuals. Yes I understand that all clinical trials and any other prospective interventional studies must be registered with an ICMJE-approved registry, such as ClinicalTrials.gov. I confirm that any such study reported in the manuscript has been registered and the trial registration ID is provided (note: if posting a prospective study registered retrospectively, please provide a statement in the trial ID field explaining why the study was not registered in advance). Yes I have followed all appropriate research reporting guidelines, such as any relevant EQUATOR Network research reporting checklist(s) and other pertinent material, if applicable. Yes Data and code availability Spatial transcriptomics count data will be available on a public server upon publication. All original code has been deposited on GitHub https://github.com/Qirongmao97/NMDhuman_spatial) and is publicly available as of date of publication. The used snRNAseq data is available upon reasonable request through the corresponding author of that dataset (Suárez-Calvet et al., 2023).