Residual serum fibrinogen as a universal biomarker for all serotypes of Myasthenia gravis.

Myasthenia Gravis (MG) is an autoimmune disease associated with severe neuromuscular weakness. Diagnostic confirmation of MG is typically delayed and secured in about 85% and 50% of patients with generalized and ocular MG, respectively with serum antibodies. We have identified a sensitive and specific diagnostic biomarker for various MG serotypes with quantitative proteomics. Serum proteomes of 18 individuals (MG patients, healthy controls (HC), Rheumatoid Arthritis (RA) were quantified in a pilot study and occurrence of high residual fibrinogen was validated by immunoblotting and further investigated by targeted mass spectrometry on the sera of 79 individuals (31 MG of various serotypes, 30 HC, 18 RA). Initial proteomic analysis identified high residual fibrinogen in MG patient sera which was then validated by antibody-based testing. Subsequently, a blinded study of independent samples showed 100% differentiation of MG patients from controls. A final serological quantification of 14 surrogate peptides derived from α-, ß-, and γ-subunits of fibrinogen in 79 individuals revealed fibrinogen to be highly specific and 100% sensitive for MG (p < 0.00001), with a remarkable average higher abundance of > 1000-fold over control groups. Our unanticipated discovery of high levels of residual serum fibrinogen in all MG patients can secure rapid bedside diagnosis of MG.

Epidemiological evidence for a hereditary contribution to myasthenia gravis: a retrospective cohort study of patients from North America.

OBJECTIVES: To approximate the rate of familial myasthenia gravis and the coexistence of other autoimmune disorders in the patients and their families. DESIGN: Retrospective cohort study. SETTING: Clinics across North America. PARTICIPANTS: The study included 1032 patients diagnosed with acetylcholine receptor antibody (AChR)-positive myasthenia gravis. METHODS: Phenotype information of 1032 patients diagnosed with AChR-positive myasthenia gravis was obtained from clinics at 14 centres across North America between January 2010 and January 2011. A critical review of the epidemiological literature on the familial rate of myasthenia gravis was also performed. RESULTS: Among 1032 patients, 58 (5.6%) reported a family history of myasthenia gravis. A history of autoimmune diseases was present in 26.6% of patients and in 28.4% of their family members. DISCUSSION: The familial rate of myasthenia gravis was higher than would be expected for a sporadic disease. Furthermore, a high proportion of patients had a personal or family history of autoimmune disease. Taken together, these findings suggest a genetic contribution to the pathogenesis of myasthenia gravis.

Metabolomic profile overlap in prototypical autoimmune humoral disease: a comparison of myasthenia gravis and rheumatoid arthritis.

INTRODUCTION: Myasthenia gravis (MG) and rheumatoid arthritis (RA) are examples of antibody-mediated chronic, progressive autoimmune diseases. Phenotypically dissimilar, MG and RA share common immunological features. However, the immunometabolomic features common to humoral autoimmune diseases remain largely unexplored. OBJECTIVES: The aim of this study was to reveal and illustrate the metabolomic profile overlap found between these two diseases and describe the immunometabolomic significance. METHODS: Metabolic analyses using acid- and dansyl-labelled was performed on serum from adult patients with seropositive MG (n = 46), RA (n = 23) and healthy controls (n = 49) presenting to the University of Alberta Hospital specialty clinics. Chemical isotope labelling liquid chromatography mass spectrometry (CIL LC-MS) methods were utilized to assess the serum metabolome in patients; 12C/13C-dansyl chloride (DnsCl) was used to label amine/phenol metabolites and 12C/13C-p-dimethylaminophenacyl bromide (DmPA) was used for carboxylic acids. Metabolites matching our criteria for significance were selected if they were present in both groups. Multivariate statistical analysis [including principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA)] and biochemical pathway analysis was then conducted to gain understanding of the principal pathways involved in antibody-mediated pathogenesis. RESULTS: We found 20 metabolites dysregulated in both MG and RA when compared to healthy controls. Most prominently, observed changes were related to pathways associated with phenylalanine metabolism, tyrosine metabolism, ubiquinone and other terpenoid-quinone biosynthesis, and pyruvate metabolism. CONCLUSION: From these results it is evident that many metabolites are common to humoral disease and exhibit significant immunometabolomic properties. This observation may lead to an enhanced understanding of the metabolic underpinnings common to antibody-mediated autoimmune disease. Further, contextualizing these findings within a larger clinical and systems biology context could provide new insights into the pathogenesis and management of these diseases.

Beyond the antibodies: serum metabolomic profiling of myasthenia gravis.

INTRODUCTION: Myasthenia gravis (MG) is a chronic, potentially debilitating autoimmune disease characterized by weakness and rapid fatigue of the voluntary muscles that worsens on exertion. Left untreated, MG symptoms may cause significant morbidity or even death. To date, no robust biological marker is available to follow the course of the disease. Therefore, new diagnostic approaches and biological markers are essential not only for improved diagnosis of the disease but for improved outcomes. OBJECTIVES: The present study applied a two-control, multi-label metabolomics profiling approach as a potential strategy for the identification of biomarkers unique to myasthenia gravis (MG). METHODS: Metabolic analyses using acid- and dansyl-labelled serum from seropositive MG (n = 46), rheumatoid arthritis (RA) (n = 23) and healthy controls (HC) (n = 49) were performed on samples from adult patients presenting to the University of Alberta Hospital neuromuscular and rheumatology clinics. Comparisons between patients with MG vs. HC, and RA vs. HC were made using univariate and multivariate statistics. RESULTS: Serum biomarker patterns were statistically significantly different between groups. Principal component analysis (PCA) and partial least squares discriminant analysis (PLS-DA) models exhibited considerable distinction between all groups. Metabolites were then filtered to remove peak pairs common to both disease cohorts. Combined metabolite panels revealed clear separation between MG and HC for both library-matched (AUROC: 0.92 ± 0.03) and highest AUC patients (AUROC: 0.94 ± 0.05). CONCLUSION: In patients presenting to the clinic with seropositive MG, metabolomic profiling is capable of distinguishing patients with disease from those without. These results provide an important first step towards a potential biomarker for improving MG identification.

A genome-wide association study of myasthenia gravis.

IMPORTANCE: Myasthenia gravis is a chronic, autoimmune, neuromuscular disease characterized by fluctuating weakness of voluntary muscle groups. Although genetic factors are known to play a role in this neuroimmunological condition, the genetic etiology underlying myasthenia gravis is not well understood. OBJECTIVE: To identify genetic variants that alter susceptibility to myasthenia gravis, we performed a genome-wide association study. DESIGN, SETTING, AND PARTICIPANTS: DNA was obtained from 1032 white individuals from North America diagnosed as having acetylcholine receptor antibody-positive myasthenia gravis and 1998 race/ethnicity-matched control individuals from January 2010 to January 2011. These samples were genotyped on Illumina OmniExpress single-nucleotide polymorphism arrays. An independent cohort of 423 Italian cases and 467 Italian control individuals were used for replication. MAIN OUTCOMES AND MEASURES: We calculated P values for association between 8,114,394 genotyped and imputed variants across the genome and risk for developing myasthenia gravis using logistic regression modeling. A threshold P value of 5.0×10(-8) was set for genome-wide significance after Bonferroni correction for multiple testing. RESULTS: In the overall case-control cohort, we identified association signals at CTLA4 (rs231770; P=3.98×10(-8); odds ratio, 1.37; 95% CI, 1.25-1.49), HLA-DQA1 (rs9271871; P=1.08×10(-8); odds ratio, 2.31; 95% CI, 2.02-2.60), and TNFRSF11A (rs4263037; P=1.60×10(-9); odds ratio, 1.41; 95% CI, 1.29-1.53). These findings replicated for CTLA4 and HLA-DQA1 in an independent cohort of Italian cases and control individuals. Further analysis revealed distinct, but overlapping, disease-associated loci for early- and late-onset forms of myasthenia gravis. In the late-onset cases, we identified 2 association peaks: one was located in TNFRSF11A (rs4263037; P=1.32×10(-12); odds ratio, 1.56; 95% CI, 1.44-1.68) and the other was detected in the major histocompatibility complex on chromosome 6p21 (HLA-DQA1; rs9271871; P=7.02×10(-18); odds ratio, 4.27; 95% CI, 3.92-4.62). Association within the major histocompatibility complex region was also observed in early-onset cases (HLA-DQA1; rs601006; P=2.52×10(-11); odds ratio, 4.0; 95% CI, 3.57-4.43), although the set of single-nucleotide polymorphisms was different from that implicated among late-onset cases. CONCLUSIONS AND RELEVANCE: Our genetic data provide insights into aberrant cellular mechanisms responsible for this prototypical autoimmune disorder. They also suggest that clinical trials of immunomodulatory drugs related to CTLA4 and that are already Food and Drug Administration approved as therapies for other autoimmune diseases could be considered for patients with refractory disease.