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Myasthenia gravis (MG) is an autoimmune neuromuscular disease characterized by fatigable skeletal muscle weakness with a fluctuating unpredictable course. One main concern in MG is the lack of objective biomarkers to guide individualized treatment decisions. Specific circulating serum microRNAs (miRNAs) miR-30e-5p, miR-150-5p and miR-21-5p levels have been shown to correlate with clinical course in specific MG patient subgroups. The aim of our study was to better characterize these miRNAs, regardless of the MG subgroup, at an early stage from diagnosis and determine their sensitivity and specificity for MG diagnosis, as well as their predictive power for disease relapse. Serum levels of these miRNAs in 27 newly diagnosed MG patients were compared with 245 healthy individuals and 20 patients with non-MG neuroimmune diseases. Levels of miR-30e-5p and miR-150-5p significantly differed between MG patients and healthy controls; however, no difference was seen compared with patients affected by other neuroimmune diseases. High levels of miR-30e-5p predicted MG relapse (p = 0.049) with a hazard ratio of 2.81. In summary, miR-150-5p is highly sensitive but has low specificity for MG, while miR-30e-5p has the greatest potential as a predictive biomarker for the disease course in MG, regardless of subgroup.

INTRODUCTION/AIMS: The circulating microRNAs (miRNAs) miR-150-5p, miR-30e-5p, and miR-21-5p have been suggested as potential biomarkers for myasthenia gravis (MG); however, the relationships between short-term natural changes of the miRNAs and patient-reported MG outcome scores have not been well-studied. We assessed the short-term fluctuations in miRNA levels and patient-reported outcome measures in MG.

METHODS: This prospective cohort study included 39 MG patients with regular follow-ups and unchanged medications at the Neurology outpatient clinic at Uppsala University Hospital. Patients had weekly follow-up visits for 1 month, at which blood samples were drawn, and scores from MG activities of daily living (MG-ADL), MG quality-of-life-15 (MG-QoL15), and Fatigue Severity Scale (FSS) were assessed. Serum levels of miRNA miR-150-5p, miR-30e-5p, and miR-21-5p were analyzed using quantitative real-time PCR.

RESULTS: Intra-individual levels of miR-30e-5p and miR-150-5p were stable, whereas a significant reduction in miR-21-5p was observed from week 1 to week 2 (p = .0024) and from week 2 to week 3 (p < .0001). There were intra-individual differences over a short time in MG-ADL, with higher scores in female patients (p = .0281) and a significant reduction from the first to the second weeks (p = .0281), whereas MG-QoL15 and FSS scores were stable.

DISCUSSION: The suggested MG biomarkers miR-30e-5p and miR-150-5p were more stable than miR-21-5p over a short time, indicating their short-term stability as biomarkers. Prospective multi-center studies with longer periods of follow-up and matched controls are needed to validate these miRNAs as biomarkers in MG.

There is an unmet need for objective disease-specific biomarkers in the heterogeneous autoimmune neuromuscular disorder myasthenia gravis (MG). This cross-sectional study identified a signature of 23 inflammatory serum proteins with proximity extension assay (PEA) that distinguishes acetylcholine receptor antibody seropositive (AChR+) MG patients from healthy controls (HCs). CCL28, TNFSF14, 4E-BP1, transforming growth factor alpha (TGF-α), and ST1A1 ranked top biomarkers. TGF-β1 and osteoprotegerin (OPG) differed between early- and late-onset MG, whereas CXCL10, TNFSF14, CCL11, interleukin-17C (IL-17C), and TGF-α differed significantly with immunosuppressive treatment. MG patients with moderate to high disease severity had lower uPA. Previously defined MG-associated microRNAs, miR-150-5p, miR-30e-5p, and miR-21-5p, correlated inversely with ST1A1 and TNFSF14. The presented inflammatory proteins that distinguish AChR+ MG are promising serum biomarkers for validation in prospective studies to allow for molecular signatures for patient subgroup stratification and monitoring of treatment response.

Myasthenia Gravis (MG) is an autoimmune neuromuscular disorder where acetylcholine receptor (AChR) antibodies induce membrane attack complex formation at the muscle membrane. The C1-inhibitor (C1-INH) regulates the classical pathway and is a promising marker in other autoimmune disorders. Treatment options for AChR antibody MG include complement inhibitors; nevertheless, the early pathway activation in MG remains unclear. Serum and plasma C1s-C1-INH levels were higher in MG patients than in matched healthy controls, supporting early classical pathway activation in most MG patients. These findings allow prospective validation studies of activated C1s as a putative treatment target and potential accompanying biomarker in MG.

Introduction/Aim

There are no blood biomarkers to monitor treatment effects in myasthenia gravis (MG) or studies visualizing the acetylcholine receptor (AChR) antibody-induced membrane attack complex (MAC) at the human muscle membrane. This study aimed to compare levels of complement activation products and native complement components in MG patients and healthy controls (HCs) and to model the AChR antibody-mediated attacks in human muscle cells.

Methods

We assessed the complement components and activation product levels with enzyme-linked immunosorbent assay and magnetic bead-based sandwich assays in plasma and sera of 23 MG patients and matched HCs. Receiver operator characteristic (ROC) curve analysis evaluated the diagnostic accuracy. Complement levels were correlated with the myasthenia gravis composite (MGC) scores. AChR+ MG modeling in human muscle cells used sera from nine MG patients and three HCs.

Results

MG patients had significantly higher plasma levels of C3a (p < .0001), C5 (p = .0003), and soluble C5b-9 (sC5b-9; p < .0001) than HCs. The ROC curve analysis showed a clear separation between MG patients and HCs for plasma C3a (AUC = 0.9720; p < .0001) and sC5b-9 (AUC = 0.8917, p < .0001). MG patients had higher levels of plasma complement Factor I (FI; p = .0002) and lower properdin levels (p < .0001). The MGC had moderate correlations with plasma Factor B (FB), FI, and Factor H. AChR+ MG patient sera triggered the deposition of MAC and reduced AChRs.

Discussion

We suggest validating plasma C3a and sC5b-9 as blood biomarkers for complement activation in MG. Further, the in vitro study allowed visualization of MAC deposition after applying AChR+ MG sera on human muscle cells.

Background

In the autoimmune disorder myasthenia gravis (MG), the nicotinic acetylcholine receptors (nAChRs) are the primary targets of pathogenic antibodies. While MG mechanisms have been extensively studied in animal models, functional insights into how antibody binding disrupts nAChR-dependent calcium signaling and the effects of complement inhibition in human muscle cells remain limited.

Methods

We used real-time live-cell calcium imaging to assess the effects of cholinergic stimulation or inhibition on human muscle cells with pharmacological agents, sera from patients with acetylcholine receptor antibody seropositive (AChR+) MG, purified recombinant antibodies targeting α- and β-subunits, and a complement C3 inhibitor. Transcriptional changes in nAChR subunits, muscle markers, voltage-gated calcium channels (VGCCs), and complement-related genes were analysed by RT-qPCR. Immunocytochemistry and quantitative image analysis were performed to assess nAChR distribution and membrane attack complex (MAC) deposition.

Findings

Cholinergic stimulation of human muscle cells activated nAChRs, resulting in membrane depolarisation, which in turn led to VGCC opening and calcium transients. MG-associated pathogenic antibodies, particularly those present in sera from patients with AChR + MG and pure recombinant AChR α-subunit-specific monoclonal antibody (mAb), but not β-subunit-specific, abolished choline-induced calcium responses in myotubes. α-subunit-specific mAb also induced transcriptional upregulation of nAChR subunits, muscle structural proteins, and complement components, and were associated with nAChR loss and MAC formation. Importantly, pharmacological inhibition of C3 activation restored calcium signaling, preserved nAChR distribution, and reduced MAC formation induced by α-subunit-specific mAb, implicating complement activation as a key driver of pathogenic effects.

Interpretation

These findings indicate that targeting the α-subunit impairs nAChR-dependent calcium signaling and can induce complement activation. Disrupted signaling and reduced nAChR levels were effectively restored by C3 inhibition, which blocks multiple downstream pathways; thus, terminal complement activation leading to MAC formation is a suggested, but it may not be the only mechanism. Overall, the results highlight C3 as a promising upstream therapeutic target and support combining subunit-specific interventions with proximal complement blockade in MG.