A primary human muscle cell-based assay for detecting myasthenia gravis autoantibody binding and assessing AChR cluster impairment

Abstract

Myasthenia gravis (MG) is an autoimmune disease caused by pathogenic autoantibodies against proteins at the neuromuscular junction (NMJ). The diagnosis and clinical management of MG patients largely relies on the detection of antigen-specific autoantibodies targeting acetylcholine receptor (AChR) or muscle-specific kinase (MuSK). Yet a subset of patients remains seronegative for known MG autoantibodies, highlighting a critical need for alternative approaches to identify pathogenic NMJ antibodies. We established a new human in vitro model of the NMJ based on primary human muscle cells that recapitulates key features of the NMJ: differentiation to myotubes, expression of key NMJ proteins and formation of postsynaptic AChR clusters in response to agrin stimulation. The model allows new insights into myogenesis and genetic muscle diseases, and the new muscle cell-based assay (CBA) detected autoantibodies in sera from patients with AChR- and MuSK-positive MG with 96.43% sensitivity and 100% specificity, while healthy control sera showed no reactivity. Incubation with patient sera significantly reduced AChR clustering compared to controls, demonstrating functional pathogenic effects. Thus, we established a physiologically relevant human NMJ model that enables detection and functional characterization of neuromuscular autoantibodies. This novel approach addresses a key limitation of current antigen-specific diagnostics and provides a method for improved detection and characterization of MG antibodies, independent of antigen specificity. One Sentence SummaryWe established a postsynaptic human in vitro neuromuscular junction model to assess binding and pathogenicity of MG autoantibodies. Key messagesO_ST_ABSWhat is already known on this topic?C_ST_ABSCurrent diagnosis of myasthenia gravis (MG) relies largely on the detection of antigen-specific autoantibodies against AChR and MuSK, leaving a clinically relevant subset of patients seronegative. What are the new findings?We established a physiologically relevant human in vitro neuromuscular junction model based on primary human muscle cells and developed a novel muscle cell-based assay (CBA) for the detection of neuromuscular autoantibodies. How might this impact on clinical practice or future developments?The CBA detected autoantibodies in patients with AChR- or MuSK-positive MG with high sensitivity and specificity and demonstrated their functional pathogenic effects on AChR clustering. This antigen-independent approach may improve the detection and functional characterization of MG autoantibodies, particularly in patients who are seronegative in current diagnostic assays. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=130 SRC="FIGDIR/small/743478v1_ufig1.gif" ALT="Figure 1000"> View larger version (38K): org.highwire.dtl.DTLVardef@c2e1fforg.highwire.dtl.DTLVardef@8336fforg.highwire.dtl.DTLVardef@8fa2b0org.highwire.dtl.DTLVardef@1d94e_HPS_FORMAT_FIGEXP M_FIG C_FIG