VAPB depletion alters neuritogenesis and phosphoinositide balance in motoneuron-like cells: relevance to VAPB-linked amyotrophic lateral sclerosis.

VAPB and VAPA are ubiquitously expressed endoplasmic reticulum membrane proteins that play key roles in lipid exchange at membrane contact sites. A mutant, aggregation-prone, form of VAPB (P56S) is linked to a dominantly inherited form of amyotrophic lateral sclerosis; however, it has been unclear whether its pathogenicity is due to toxic gain of function, to negative dominance, or simply to insufficient levels of the wild-type protein produced from a single allele (haploinsufficiency). To investigate whether reduced levels of functional VAPB, independently from the presence of the mutant form, affect the physiology of mammalian motoneuron-like cells, we generated NSC34 clones, from which VAPB was partially or nearly completely depleted. VAPA levels, determined to be over fourfold higher than those of VAPB in untransfected cells, were unaffected. Nonetheless, cells with even partially depleted VAPB showed an increase in Golgi- and acidic vesicle-localized phosphatidylinositol-4-phosphate (PI4P) and reduced neurite extension when induced to differentiate. Conversely, the PI4 kinase inhibitors PIK93 and IN-10 increased neurite elongation. Thus, for long-term survival, motoneurons might require the full dose of functional VAPB, which may have unique function(s) that VAPA cannot perform.

Buprenorphine may be effective for treatment of paramyotonia congenita.

INTRODUCTION/AIMS: Paramyotonia congenita (PMC) is a skeletal muscle sodium channelopathy characterized by paradoxical myotonia, cold sensitivity, and exercise/cold-induced paralysis. Treatment with sodium-channel-blocking antiarrhythmic agents may expose patients to a risk of arrhythmia or may be poorly tolerated or ineffective. In this study we explored the effectiveness of non-antiarrhythmic sodium-channel blockers in two patients with PMC. METHODS: Earlier treatment with mexiletine was discontinued for gastrointestinal side effects in one of the patients and lack of clinical benefit in the other. One patient received lacosamide, ranolazine, and buprenorphine, and the other was given buprenorphine only. Drug efficacy was assessed by clinical scores, timed tests, and by long and short exercise tests. RESULTS: In both patients, buprenorphine improved pain scores by at least 50%, stiffness and weakness levels, and handgrip/eyelid-opening times. The fall in compound muscle action potential (CMAP) during short exercise normalized in both patients at baseline, and improved after cooling. During long exercise, one patient showed an earlier recovery of CMAP, and the other patient had a less severe decrease (<60%). With buprenorphine, the fall in CMAP induced by cooling normalized in one patient (from -72% to -4%) and improved (from -49% to -37%) in the other patient. DISCUSSION: Buprenorphine showed promising results for the treatment of exercise-induced paralysis and cold intolerance in the two patients assessed. The exercise test may be useful for quantitative assessment of treatment response. Further studies on a larger number of patients, under carefully controlled conditions, should be considered to address the effectiveness and long-term tolerability of this therapeutic option.

Dysregulation of Muscle-Specific MicroRNAs as Common Pathogenic Feature Associated with Muscle Atrophy in ALS, SMA and SBMA: Evidence from Animal Models and Human Patients.

Motor neuron diseases (MNDs) are neurodegenerative disorders characterized by upper and/or lower MN loss. MNDs include amyotrophic lateral sclerosis (ALS), spinal muscular atrophy (SMA), and spinal and bulbar muscular atrophy (SBMA). Despite variability in onset, progression, and genetics, they share a common skeletal muscle involvement, suggesting that it could be a primary site for MND pathogenesis. Due to the key role of muscle-specific microRNAs (myomiRs) in skeletal muscle development, by real-time PCR we investigated the expression of miR-206, miR-133a, miR-133b, and miR-1, and their target genes, in G93A-SOD1 ALS, Δ7SMA, and KI-SBMA mouse muscle during disease progression. Further, we analyzed their expression in serum of SOD1-mutated ALS, SMA, and SBMA patients, to demonstrate myomiR role as noninvasive biomarkers. Our data showed a dysregulation of myomiRs and their targets, in ALS, SMA, and SBMA mice, revealing a common pathogenic feature associated with muscle impairment. A similar myomiR signature was observed in patients' sera. In particular, an up-regulation of miR-206 was identified in both mouse muscle and serum of human patients. Our overall findings highlight the role of myomiRs as promising biomarkers in ALS, SMA, and SBMA. Further investigations are needed to explore the potential of myomiRs as therapeutic targets for MND treatment.

Aging-associated genes and let-7 microRNAs: a contribution to myogenic program dysregulation in oculopharyngeal muscular dystrophy.

Oculopharyngeal muscular dystrophy (OPMD) is a late-onset muscle disease caused by an abnormal (GCN) triplet expansion within the polyadenylate-binding protein nuclear 1 gene and consequent mRNA processing impairment and myogenic defects. Because a reduced cell proliferation potential and the consequent regeneration failure of aging muscle have been shown to be governed by lethal-7 (let-7) microRNA-mediated mechanisms, in the present study, we evaluated the role of let-7 in the pathogenesis of OPMD. By a multidisciplinary approach, including confocal microscopy, Western blot, and quantitative PCR analyses on muscle biopsies from patients and unaffected individuals, we found a significant increase in let-7 expression in OPMD muscles associated with an unusual high percentage of paired box 7-positive satellite cells. Furthermore, IL-6, a cytokine involved in the regulation of satellite cell proliferation and differentiation and a potential target of let-7, was found strongly down-regulated in OPMD compared with control muscles. The decrease in IL-6 transcript levels and protein content was also confirmed in vitro during differentiation of patients' and controls' muscle cells. Overall, our data suggest a key role of let-7 in the regeneration and degeneration process in OPMD muscle and pointed to IL-6 as a potential target molecule for new therapeutic approaches for this disorder.-Cappelletti, C., Galbardi, B., Bruttini, M., Salerno, F., Canioni, E., Pasanisi, M. B., Rodolico, C., Brizzi, T., Mora, M., Renieri, A., Maggi, L., Bernasconi, P., Mantegazza, R. Aging-associated genes and let-7 microRNAs: a contribution to myogenic program dysregulation in oculopharyngeal muscular dystrophy.

MicroRNA signature associated with treatment response in myasthenia gravis: A further step towards precision medicine.

Myasthenia gravis (MG) is an autoimmune disorder affecting neuromuscular transmission currently treated with chronic immunosuppression. Inter-subject variation in treatment response and side effects highlight the need for personalized therapies by identification of biomarkers predictive of drug efficacy in individual patients, still lacking in MG. MicroRNAs (miRNAs) play a key role in immune response and drug metabolism modulation. This study, part of an Italian-Israeli collaborative project, aimed to identify specific miRNAs as biomarkers associated with immunosuppressive treatment response in MG patients. Whole miRNome sequencing, followed by miRNA validation by real-time PCR, was performed in peripheral blood from Italian MG patients (n = 40) classified as responder and non-responder to immunosuppressive therapies. MiRNA sequencing identified 41 miRNAs differentially expressed in non-responder compared to responder Italian MG patients. Validation phase pointed out three miRNAs, miR-323b-3p, -409-3p, and -485-3p, clustered on chromosome 14q32.31, the levels of which were significantly decreased in non-responder versus responder patients, whereas miR-181d-5p and -340-3p showed an opposite trend. ROC curve analysis showed sensitivity and specificity performance results indicative of miR-323b-3p, -409-3p, and -485-3p predictive value for responsiveness to immunosuppressive drugs in MG. Validated miRNAs were further analyzed in blood from responder and non-responder MG patients of the Israeli population (n = 33), confirming a role for miR-323b-3p, -409-3p, -485-3p, -181d-5p and -340-3p as biomarkers of drug efficacy. Gene Ontology enrichment analysis, mRNA target prediction, and in silico modeling for function of the identified miRNAs disclosed functional involvement of the five miRNAs, and their putative target genes, in both immune (i.e. neurotrophin TRK and Fc-epsilon receptor signaling pathways) and drug metabolism processes. Our overall findings thus revealed a blood "miR-323b-3p, -409-3p, -485-3p, -181d-5p, and -340-3p" signature associated with drug responsiveness in MG patients. Its identification sets the basis for precision medicine approaches based on "pharmacomiRs" as biomarkers of drug responsiveness in MG, promising to improve therapeutic success in a cost/effective manner.

Pharmacogenetics of myotonic hNav1.4 sodium channel variants situated near the fast inactivation gate.

Sodium channel myotonia and paramyotonia congenita are caused by gain-of-function mutations in the skeletal muscle voltage-gated sodium channel hNav1.4. The first-line drug is the sodium channel blocker mexiletine; however, some patients show side effects or limited responses. We previously showed that two hNav1.4 mutations, p.G1306E and p.P1158L, reduce mexiletine potency in vitro, whereas another sodium channel blocker, flecainide, is less sensitive to mutation-induced gating defects. This observation was successfully translated to p.G1306E and p.P1158L carriers. Thus, the aim of this study was to perform a pharmacological characterization of myotonic Nav1.4 mutations clustered near the fast inactivation gate of the channel. We chose seven mutations (p.V1293I, p.N1297S, p.N1297K, p.F1298C, p.G1306E, p.I1310N, and p.T1313M) from the database of Italian and French networks for muscle channelopathies. Recombinant hNav1.4 mutants were expressed in HEK293T cells for functional and pharmacological characterization using the patch-clamp technique. All the studied mutations impair the kinetics and/or voltage dependence of fast inactivation, which is likely the main mechanism responsible for myotonia. The severity of myotonia is well-correlated to the enhancement of window currents generated by the intersection of the activation and fast inactivation voltage dependence. Five of the six mutants displaying a significant positive shift of fast inactivation voltage dependence reduced mexiletine inhibition in an experimental condition mimicking myotonia. In contrast, none of the mutations impairs flecainide block nor does p.T1313M impair propafenone block, indicating that class Ic antiarrhythmics may constitute a valuable alternative. Our study suggests that mutation-driven therapy would be beneficial to myotonic patients, greatly improving their quality of life.

Italian recommendations for diagnosis and management of congenital myasthenic syndromes.

Congenital myasthenic syndromes (CMS) are genetic disorders due to mutations in genes encoding proteins involved in the neuromuscular junction structure and function. CMS usually present in young children, but perinatal and adult onset has been reported. Clinical presentation is highly heterogeneous, ranging from mild symptoms to severe manifestations, sometimes with life-threatening respiratory episodes, especially in the first decade of life. Although considered rare, CMS are probably underestimated due to diagnostic difficulties. Because of the several therapeutic opportunities, CMS should be always considered in the differential diagnosis of neuromuscular disorders. The Italian Network on CMS proposes here recommendations for proper CMS diagnosis and management, aiming to guide clinicians in their practical approach to CMS patients.

Myasthenia gravis: from autoantibodies to therapy.

PURPOSE OF REVIEW: The current article reviews the recent advances in the field of myasthenia gravis, which span from autoantibody profiling and pathogenic mechanisms to therapy innovation. The overview is highlighting specifically the data and the needs of targeted treatments in the light of precision medicine in myasthenia gravis. RECENT FINDINGS: Novel data published recently further increased our knowledge on myasthenia gravis. The use of cell-based assays has greatly improved autoantibody detection in myasthenia gravis patients, and the mechanisms of action of these antibodies have been described. The role of Toll-like receptor activation in the generation of thymic alterations and anti-acetylcholine receptor autosensitization has been further investigated implementing our understanding on the relationships between innate immunity and autoimmunity. Additional studies have been focused on the alterations of T-cell/B-cell regulatory mechanisms in thymus and peripheral blood of myasthenia gravis patients. microRNAs and genetic factors are also emerging as key biomarkers in myasthenia gravis pathogenesis and prediction of drug efficacy in individual patients. SUMMARY: The recent immunological and pathological findings in myasthenia gravis promise to improve myasthenia gravis treatment, via the development of more precise and personalized therapies.

Coexistence of CLCN1 and SCN4A mutations in one family suffering from myotonia.

Non-dystrophic myotonias are characterized by clinical overlap making it challenging to establish genotype-phenotype correlations. We report clinical and electrophysiological findings in a girl and her father concomitantly harbouring single heterozygous mutations in SCN4A and CLCN1 genes. Functional characterization of N1297S hNav1.4 mutant was performed by patch clamp. The patients displayed a mild phenotype, mostly resembling a sodium channel myotonia. The CLCN1 c.501C>G (p.F167L) mutation has been already described in recessive pedigrees, whereas the SCN4A c.3890A>G (p.N1297S) variation is novel. Patch clamp experiments showed impairment of fast and slow inactivation of the mutated Nav1.4 sodium channel. The present findings suggest that analysis of both SCN4A and CLCN1 genes should be considered in myotonic patients with atypical clinical and neurophysiological features.

A novel ABCC6 haplotype is associated with azathioprine drug response in myasthenia gravis.

OBJECTIVE: We investigated the association of single nucleotide polymorphisms (SNPs) in drug-metabolizing enzymes and transporters (DMETs) with the response to azathioprine (AZA) in patients affected by myasthenia gravis (MG) to determine possible genotype-phenotype correlations. PATIENTS AND METHODS: Genomic DNA from 180 AZA-treated MG patients was screened through the Affymetrix DMET platform, which characterizes 1931 SNPs in 225 genes. The significant SNPs, identified to be involved in AZA response, were subsequently validated by allelic discrimination and direct sequencing. SNP analysis was carried out using the SNPassoc R package and the haploblocks were determined using haploview software. RESULTS: We studied 127 patients in the discovery phase and 53 patients in the validation phase. We showed that two SNPs (rs8058694 and rs8058696) found in ATP-binding cassette subfamily C member 6, a subfamily member of ATP-binding cassette genes, constituted a new haplotype associated with AZA response in MG patients in the discovery cohort (P=0.011; odds ratio: 0.40; 95% confidence interval: 0.20-0.83) and in the combined cohort (P=0.04; odds ratio: 1.58). CONCLUSION: These findings highlight the role that the ATP-binding cassette subfamily C member 6 haplotype may play in AZA drug response. In view of the significant effects and AZA intolerance, these novel SNPs should be taken into consideration in pharmacogenetic profiling for AZA.

Multidisciplinary study of a new ClC-1 mutation causing myotonia congenita: a paradigm to understand and treat ion channelopathies.

Myotonia congenita is an inherited disease that is characterized by impaired muscle relaxation after contraction caused by loss-of-function mutations in the skeletal muscle ClC-1 channel. We report a novel ClC-1 mutation, T335N, that is associated with a mild phenotype in 1 patient, located in the extracellular I-J loop. The purpose of this study was to provide a solid correlation between T335N dysfunction and clinical symptoms in the affected patient as well as to offer hints for drug development. Our multidisciplinary approach includes patch-clamp electrophysiology on T335N and ClC-1 wild-type channels expressed in tsA201 cells, Western blot and quantitative PCR analyses on muscle biopsies from patient and unaffected individuals, and molecular dynamics simulations using a homology model of the ClC-1 dimer. T335N channels display reduced chloride currents as a result of gating alterations rather than altered surface expression. Molecular dynamics simulations suggest that the I-J loop might be involved in conformational changes that occur at the dimer interface, thus affecting gating. Finally, the gene expression profile of T335N carrier showed a diverse expression of K+ channel genes, compared with control individuals, as potentially contributing to the phenotype. This experimental paradigm satisfactorily explained myotonia in the patient. Furthermore, it could be relevant to the study and therapy of any channelopathy.-Imbrici, P., Altamura, C., Camerino, G. M., Mangiatordi, G. F., Conte, E., Maggi, L., Brugnoni, R., Musaraj, K., Caloiero, R., Alberga, D., Marsano, R. M., Ricci, G., Siciliano, G., Nicolotti, O., Mora, M., Bernasconi, P., Desaphy, J.-F., Mantegazza, R., Camerino, D. C. Multidisciplinary study of a new ClC-1 mutation causing myotonia congenita: a paradigm to understand and treat ion channelopathies.

Identification of a gene expression signature in peripheral blood of multiple sclerosis patients treated with disease-modifying therapies.

Multiple Sclerosis (MS) is an inflammatory disease with neurodegenerative alterations, ultimately progressing to neurological handicap. Therapies are effective in counteracting inflammation but not neurodegeneration. Biomarkers predicting disease course or treatment response are lacking. We investigated whether altered gene and protein expression profiles were detectable in the peripheral blood of 78 relapsing remitting MS (RR-MS) patients treated by disease-modifying therapies. A discovery/validation study on RR-MS responsive to glatiramer acetate identified 8 differentially expressed genes: ITGA2B, ITGB3, CD177, IGJ, IL5RA, MMP8, P2RY12, and S100ß. A longitudinal study on glatiramer acetate, Interferon-ß, or Fingolimod treated RR-MS patients confirmed that 7 out of 8 genes were downregulated with reference to the different therapies, whereas S100ß was always upregulated. Thus, we identified a peripheral gene signature associated with positive response in RR-MS which may also explain drug immunomodulatory effects. The usefulness of this signature as a biomarker needs confirmation on larger series of patients.

Fibrosis and inflammation are greater in muscles of beta-sarcoglycan-null mouse than mdx mouse.

The Sgcb-null mouse, with knocked-down ß-sarcoglycan, develops severe muscular dystrophy as in type 2E human limb girdle muscular dystrophy. The mdx mouse, lacking dystrophin, is the most used model for Duchenne muscular dystrophy (DMD). Unlike DMD, the mdx mouse has mild clinical features and shows little fibrosis in limb muscles. To characterize ECM protein deposition and the progression of muscle fibrosis, we evaluated protein and transcript levels of collagens I, III and VI, decorin, and TGF-ß1, in quadriceps and diaphragm, at 2, 4, 8, 12, 26, and 52 weeks in Sgcb-null mice, and protein levels at 12, 26, and 52 weeks in mdx mice. In Sgcb-null mice, severe morphological disruption was present from 4 weeks in both quadriceps and diaphragm, and included conspicuous deposition of extracellular matrix components. Histopathological features of Sgcb-null mouse muscles were similar to those of age-matched mdx muscles at all ages examined, but, in the Sgcb-null mouse, the extent of connective tissue deposition was generally greater than mdx. Furthermore, in the Sgcb-null mouse, the amount of all three collagen isoforms increased steadily, while, in the mdx, they remained stable. We also found that, at 12 weeks, macrophages were significantly more numerous in mildly inflamed areas of Sgcb-null quadriceps compared to mdx quadriceps (but not in highly inflamed regions), while, in the diaphragm, macrophages did not differ significantly between the two models, in either region. Osteopontin mRNA was also significantly greater at 12 weeks in laser-dissected highly inflamed areas of the Sgcb-null quadriceps compared to the mdx quadriceps. TGF-ß1 was present in areas of degeneration-regeneration, but levels were highly variable and in general did not differ significantly between the two models and controls. The roles of the various subtypes of macrophages in muscle repair and fibrosis in the two models require further study. The Sgcb-null mouse, which develops early fibrosis in limb muscles, appears more promising than the mdx mouse for probing pathogenetic mechanisms of muscle fibrosis and for developing anti-fibrotic treatments. Highlights • The Sgcb-null mouse develops severe muscular dystrophy, the mdx mouse does not. • Fibrosis developed earlier in Sgcb-null quadriceps and diaphragm than mdx. • Macrophages were commoner in mildly inflamed parts of Sgcb-null quadriceps than mdx. • The Sgcb-null model appears more useful than mdx for studying fibrotic mechanisms. • The Sgcb-null model also appears more useful for developing anti-fibrotic treatments.

Innate immunity in myasthenia gravis thymus: pathogenic effects of Toll-like receptor 4 signaling on autoimmunity.

The thymus is the main site of immune sensitization to AChR in myasthenia gravis (MG). In our previous studies we demonstrated that Toll-like receptor (TLR) 4 is over-expressed in MG thymuses, suggesting its involvement in altering the thymic microenvironment and favoring autosensitization and autoimmunity maintenance processes, via an effect on local chemokine/cytokine network. Here, we investigated whether TLR4 signaling may favor abnormal cell recruitment in MG thymus via CCL17 and CCL22, two chemokines known to dictate immune cell trafficking in inflamed organs by binding CCR4. We also investigated whether TLR4 activation may contribute to immunodysregulation, via the production of Th17-related cytokines, known to alter effector T cell (Teff)/regulatory T cell (Treg) balance. We found that CCL17, CCL22 and CCR4 were expressed at higher levels in MG compared to normal thymuses. The two chemokines were mainly detected around medullary Hassall's corpuscles (HCs), co-localizing with TLR4(+) thymic epithelial cells (TECs) and CCR4(+) dendritic cells (DCs), that were present in higher number in MG thymuses compared to controls. TLR4 stimulation in MG TECs increased CCL17 and CCL22 expression and induced the production of Th17-related cytokines. Then, to study the effect of TLR4-stimulated TECs on immune cell interactions and Teff activation, we generated an in-vitro imaging model by co-culturing CD4(+) Th1/Th17 AChR-specific T cells, naïve CD4(+)CD25(+) Tregs, DCs and TECs from Lewis rats. We observed that TLR4 stimulation led to a more pronounced Teff activatory status, suggesting that TLR4 signaling in MG thymic milieu may affect cell-to-cell interactions, favoring autoreactive T-cell activation. Altogether our findings suggest a role for TLR4 signaling in driving DC recruitment in MG thymus via CCL17 and CCL22, and in generating an inflammatory response that might compromise Treg function, favoring autoreactive T-cell pathogenic responses.

A large cohort of myotonia congenita probands: novel mutations and a high-frequency mutation region in exons 4 and 5 of the CLCN1 gene.

Myotonia congenita is a genetic disease characterized by impaired muscle relaxation after forceful contraction (myotonia) and caused by mutations in the chloride channel voltage-sensitive 1 (CLCN1) gene, encoding the voltage-gated chloride channel of skeletal muscle (ClC-1). In a large cohort of clinically diagnosed unrelated probands, we identified 75 different CLCN1 mutations in 106 individuals, among which 29 were novel mutations and 46 had already been reported. Despite the newly described mutations being scattered throughout the gene, in our patients, mutations were mostly found in exons 4 and 5. Most of the novel mutations located in the region comprising the intramembrane helices are involved in the ion-conducting pathway and predicted to affect channel function. We report for the first time that two mutations, inherited on the same allele as a heterozygous trait, abrogate disease expression, although when inherited singularly they were pathogenic. Such a mode of inheritance might explain the incomplete penetrance reported for autosomal dominant mutations in particular families.

Autoimmune mechanisms in myasthenia gravis.

PURPOSE OF REVIEW: This article reviews recent findings on factors and mechanisms implicated in the pathogenesis of myasthenia gravis and briefly summarizes data on therapies acting at various stages of the autoimmune process. RECENT FINDINGS: Data published over the last year promise to improve understanding of pathogenic mechanisms underlying myasthenia gravis. Animal studies have at last shown that antimuscle-specific kinase (MuSK) autoantibodies, like antiacetylcholine receptor (AChR) autoantibodies, are myasthenogenic. A new autoantigen, the low-density lipoprotein receptor-related protein 4 (LRP4), has been identified in variable proportions of otherwise seronegative patients. Anti-LRP4 antibodies may define a new myasthenia gravis subtype, supporting the concept that myasthenia gravis is not a single disease entity, and that different subtypes can differ in aetiology. Genetic and environmental factors are implicated in myasthenia gravis. The finding of persisting viral infection in the thymus of AChR-myasthenia gravis patients, combined with data on chronic inflammation, suggest that pathogens may favour intrathymic AChR-specific autosensitization and maintenance of autoimmunity in genetically susceptible individuals. Defective immunoregulatory mechanisms, involving pathogenic Th17 and regulatory T cells, contribute to tolerance loss and perpetuation of the autoimmune response in myasthenia gravis patients. SUMMARY: The recent identification of mechanisms initiating and perpetuating autoimmunity in myasthenia gravis may stimulate the development of more effective therapies.

Complement Activation Profile in Myasthenia Gravis Patients: Perspectives for Tailoring Anti-Complement Therapy.

The complement system plays a key role in myasthenia gravis (MG). Anti-complement drugs are emerging as effective therapies to treat anti-acetylcholine receptor (AChR) antibody-positive MG patients, though their usage is still limited by the high costs. Here, we searched for plasma complement proteins as indicators of complement activation status in AChR-MG patients, and potential biomarkers for tailoring anti-complement therapy in MG. Plasma was collected from AChR-MG and MuSK-MG patients, and healthy controls. Multiplex immunoassays and ELISA were used to quantify a panel of complement components (C1Q, C2, C3, C4, C5, Factor B, Factor H, MBL, and properdin) and activation products (C4b, C3b, C5a, and C5b-9), of classical, alternative and lectin pathways. C2 and C5 levels were significantly reduced, and C3, C3b, and C5a increased, in plasma of AChR-MG, but not MuSK-MG, patients compared to controls. This protein profile was indicative of complement activation. We obtained sensitivity and specificity performance results suggesting plasma C2, C3, C3b, and C5 as biomarkers for AChR-MG. Our findings reveal a plasma complement "C2, C3, C5, C3b, and C5a" profile associated with AChR-MG to be further investigated as a biomarker of complement activation status in AChR-MG patients, opening new perspectives for tailoring of anti-complement therapies to improve the disease treatment.