LGMD R12 is an autosomal recessive myopathy caused by pathogenic variants in anoctamin-5, a multipass membrane protein that sits in the sarcolemma and sarcoplasmic reticulum of muscle fibers. The protein participates in calcium-activated chloride channel activity, plasma membrane repair, and the maintenance of muscle fiber homeostasis. When both copies of the gene are disabled, patients develop progressive weakness and atrophy of the pelvic and shoulder girdle muscles, though the clinical picture varies widely and serum creatine kinase levels can range from normal to dramatically elevated. No effective therapy currently exists, and the molecular cascade connecting the mutated gene to dying muscle has remained stubbornly obscure. That gap is precisely what the new study set out to close.

The patient at the center of the story arrived at the rheumatology department with nine months of exercise-induced muscle pain and hyperCKemia, creatine kinase levels swinging between 3,079 and 8,386 units per liter against a normal ceiling of 200. He had no rash, normal muscle tone, and full grade V strength, a presentation that easily mimics idiopathic inflammatory myopathy or glycogen storage disease. A right deltoid biopsy showed muscle fiber hypertrophy and necrosis without lymphocytic infiltration, and genetic testing ultimately revealed the culprit: a homozygous duplication in exon 10 of ANO5, designated c.898dupA (p.Ile300fs), a frameshift variant not previously reported in the literature. Both of his parents carried the same mutation in heterozygous form, the classic signature of recessive inheritance. At a two-year follow-up the patient remained able to perform normal daily activities, with creatine kinase hovering near 3,000 to 4,000 units per liter.

The team, working with samples gathered during an earlier study of inflammatory myopathies, sequenced biceps brachii muscle tissue and peripheral blood mononuclear cells from the patient alongside samples from healthy controls. After rigorous quality control, 38,477 muscle cells entered the analysis, 8,994 from the patient and 29,483 from three controls undergoing lipoma surgery, together resolving into fifteen distinct clusters. Cell identity was assigned using canonical marker genes, batch effects were corrected with Harmony, and clusters were visualized through uniform manifold approximation and projection. Consistent with the genetic diagnosis, ANO5 expression was barely detectable at the single-cell RNA level, and the proportion of mature myocytes in the patient was significantly reduced, an early hint that the disease was dismantling the cellular architecture of the muscle from the inside out.

Reclustering the myocytes revealed four subpopulations: muscle progenitor cells, slow-twitch type I fibers, fast-twitch type II fibers, and an intermediate state transitioning from progenitors toward type II fibers. The pattern in the patient was stark. Progenitor cells had multiplied, while type I fibers had all but vanished, and trajectory analysis with Monocle showed the patient’s muscle cells largely frozen in an undifferentiated state. In other words, the regenerative compartment was stocked with raw material that simply could not mature. Immunofluorescence staining of frozen muscle sections confirmed the single-cell story, demonstrating a dramatic loss of both MYH7-positive type I fibers and MYH2-positive type II fibers relative to controls, with laminin staining delineating the shrunken fiber outlines. The contraction-related gene RPL3L was also markedly downregulated, further underscoring the failing contractile machinery.

The metabolic findings may prove just as consequential. Gene ontology and single-cell metabolic pathway analyses, the latter quantifying KEGG-curated pathway activity in each individual cell, showed significant downregulation of glycolytic activity and broader cellular metabolism, spanning nucleotide, energy, carbohydrate, and amino acid pathways. The downregulation was most pronounced in type I fibers, which depend heavily on oxidative phosphorylation, suggesting a maladaptive metabolic shift away from aerobic energy production that could accelerate atrophy. Prior work has shown that ANO5 deficiency impairs myoblast membrane repair through disrupted calcium handling, and the new data connect that biophysical defect to a systemic bioenergetic collapse. Dysregulated energy and biosynthetic processes, the authors argue, likely feed directly into the contractile dysfunction that defines the disease.

Then came the immunological surprise. Although the patient’s biopsy showed no lymphocytic infiltration, the single-cell data told a different story. Within the muscle, B cells had expanded significantly, and gene sets for leukocyte migration and chemotaxis were highly enriched while muscle development pathways withered. Using CellChat, a computational framework that models ligand-receptor signaling between cell types, the researchers mapped heightened immune crosstalk between macrophages and neutrophils, with the LGALS9-CD45/CD44 and ANXA1-FPR1 pathways prominently activated, alongside IL1β-IL1R2 and several chemokine axes. The peripheral blood told a parallel tale: among 8,161 patient PBMCs compared with 25,685 control cells across fourteen cell types, B cells had again expanded, immune regulatory pathways were upregulated, and both the galectin and annexin signaling modules lit up, including NAMPT, IL16, GRN, and CCL5 interactions converging on the same LGALS9 and ANXA1 nodes.

The convergence of those two pathways in both muscle and blood is the study’s most striking molecular claim, positioning LGALS9-CD45/CD44 and ANXA1-FPR1 as candidate pathogenic hubs in LGMD R12. The authors are careful about the apparent contradiction with the clean biopsy, offering two explanations: the sampled region may simply have missed infiltrating immune cells, and mild neurogenic injury detected in the left gastrocnemius by electromyography may have been sufficient to trigger inflammatory signaling. They also acknowledge the study’s central limitation, that the activation of these signaling pathways could not be experimentally verified for want of animal or cell models, and that findings from a single patient, however deeply profiled, cannot establish population-level conclusions.

Still, the work represents a genuine first. By constructing single-cell transcriptomic atlases of both diseased muscle and circulating immune cells from an LGMD R12 patient, the team has generated a mechanistic map that bulk RNA studies could never deliver, one that links a specific frameshift mutation to progenitor cell differentiation failure, fiber type loss, metabolic reprogramming, and systemic immune activation within a single framework. The raw data and analysis scripts have been deposited in public repositories, inviting the community to reanalyze and extend the findings. Whether the expanded muscle progenitor cells can be coaxed to mature, whether the metabolic decline can be pharmacologically slowed, and whether the galectin and annexin signaling hubs can be therapeutically intercepted are now testable questions. For a rare disease with no approved treatment, a single deeply profiled patient may have just supplied the research agenda for the next decade.

Subject of Research: Single-cell transcriptomic profiling of muscle and blood in a patient with ANO5-related limb-girdle muscular dystrophy type R12

Article Title: 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

Article References: Chen, M., Dong, G., Wang, Y., Liu, J., Wang, Z., Bao, D., Huang, Z., Li, X., Li, Y., Cai, Z., & Wei, W. (2026). 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. Frontiers in Genetics, 17, Article 1833196. https://doi.org/10.3389/fgene.2026.1833196

Image Credits: AI Generated

DOI: 10.3389/fgene.2026.1833196

Keywords: LGMD R12, ANO5, single-cell RNA sequencing, muscular dystrophy, muscle progenitor cells, MYH7, metabolism, immunology, LGALS9, ANXA1, genetics, rare disease