VARS2 deficiency drives seronegative myasthenia gravis pathology via mitochondrial, immune, and neuromuscular dysregulation

Seronegative myasthenia gravis (SNMG) is a clinically heterogeneous subtype of myasthenia gravis (MG) that lacks the classical pathogenic autoantibodies used in routine diagnosis, leaving the underlying mechanisms poorly defined. Integrative genetic and single-cell transcriptomic analyses identified VARS2 as an MG-associated candidate gene with particular relevance to SNMG. VARS2 dysregulation was predominantly observed in CD4+ T cells, especially in the SNMG subgroup. To evaluate its in vivo relevance to SNMG, we established a zebrafish vars2 knockdown model. vars2 deficiency induced a myasthenia-like phenotype characterized by impaired locomotor activity, abnormal skeletal muscle development, defective motor neuron formation, and reduced expression of neuromuscular junction-related genes. Furthermore, the loss of vars2 disrupted mitochondrial homeostasis, evidenced by mitochondrial structural damage, membrane potential loss, increased reactive oxygen species accumulation, and suppression of metabolic pathways. These changes were accompanied by immune dysregulation, marked by CD4+ T cell/Th17-associated inflammatory activation, reduced expression of Treg-related genes, and increased neutrophil and macrophage recruitment. Crucially, pharmacological interventions partially reversed these defects through mitochondrial support (CoQ10), acetylcholinesterase inhibition (Neostigmine), and targeted immune suppression (ML604440 for Th17 differentiation, GSK805 for Th17 function, and UNBS5162 for CXCL-related signaling). Together, these findings suggest that VARS2 links mitochondrial dysfunction to immune imbalance and neuromuscular injury in SNMG. Our study highlights mitochondrial immunometabolism as a candidate mechanistic axis and identifies VARS2 as a potential biomarker for SNMG-related immune remodeling.