Single-cell transcriptome analysis of muscle and blood tissues from a patient with a rare homozygous ANO5 variant causing limb-girdle muscular dystrophy type R12

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Background Limb-girdle muscular dystrophy type R12 (LGMD R12) is a recessive genetic myopathy, typically caused by pathogenic variants in anoctamin-5 (ANO5) , which encodes a multipass membrane protein localized to the sarcolemma and sarcoplasmic reticulum of muscle fibers. Effective therapeutic strategies for LGMD R12 remain elusive. A comprehensive analytical framework is required to dissect the cellular and molecular changes underlying this disease. Methods The ANO5 variant in the patient and his family members was validated by Sanger sequencing. We performed single-cell RNA sequencing analysis on biceps brachii muscle tissue (MUS) and peripheral blood mononuclear cells (PBMCs) from healthy controls (HCs) and a patient who was homozygous for the pathogenic variant in exon 10 of ANO5 (c.898dupA, p.Ile300fs). Immunofluorescence staining was conducted to validate key findings from the single-cell transcriptome analysis. Results In the MUS cell atlas , we observed a significant loss of type I muscle fibers in the patient with LGMD R12. Accordingly, immunofluorescence staining revealed a dramatic loss of MYH7 + cells (MYH7 + , a marker of type I muscle fibers) compared with those in controls. Glycolytic pathway activity and cellular metabolic activity were significantly downregulated, suggesting that they are two transcriptomic signatures in the muscle tissue of this patient. In addition, muscle progenitor cells from the patient exhibited impaired differentiation capacity, failing to generate mature myocytes. In both MUS and PBMCs, we also observed heightened immune crosstalk accompanied by significant activation of pathways such as LGALS9–CD45/CD44 and ANXA1–FPR1, suggesting immunological activation in this patient. Conclusion LGMD R12 with pathogenic ANO5 variants is a rare genetic disease. Our transcriptome and experimental analyses revealed dramatic cellular and molecular changes in affected tissue at the single-cell resolution in a patient with LGMD R12. This study provides valuable insights into the underlying mechanisms and potential development of interventions for this recessive genetic myopathy.