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.

"Complex" Vasovagal Syncope: A Zebra Among Horses.

Background: Vasovagal syncope (VVS) occurs due to cerebral hypoperfusion from a fall in blood pressure, with accompanying bradycardia in most cases. Seizure and/or asystole may accompany VVS, though their prediction within the VVS cohort remains elusive. Objective: To further characterize VVS and to find predictive features of "complex" VVS (defined as VVS associated with seizures and/or asystole). Methods: We reviewed medical records of all patients who were referred for orthostatic intolerance and had a definite VVS during the head-up tilt table testing (HUTT). The following variables were recorded: cardiovascular indices during HUTT, autonomic testing results, and semiology of asystole and/or seizure when present. Simple frequency and correlation analysis were performed using the ANOVA. Results: A total of 78 independent VVS were recorded in 60 patients of which 24% were not preceded by presyncope. Vasodepressor (45%) and mixed (38%) VVS were the most prevalent types. Eighteen (23%) were complex VVS; five had an associated seizure (SySz), nine were accompanied by asystole (SyAs), and four had both (SySzAs). Males were significantly more likely to have complex VVS. Mean asystole duration was somewhat longer in the SyAsSz group. The severity of bradycardia significantly correlated with complex VVS and was a predictor of SySz. Autonomic abnormalities were frequent but did not distinguish the two VVS subgroups. Seizures had multiple distinguishing features from those typically associated with epileptic seizures. Conclusions: The underlying pathophysiologic mechanisms of complex VVS remain unclear, but the severity of cerebral hypoperfusion due to bradycardia likely plays a key role in seizure generation.

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.

Diagnostic Criteria for Small Fiber Neuropathy.

OBJECTIVES: Despite its relative common occurrence, definitive diagnosis of small fiber neuropathy (SFN) remains problematic. In practice, patients with pain, numbness, and/or paresthesias in their lower limbs are diagnosed with SFN if found to have dissociated sensory loss in their feet, that is, impaired pinprick perception (PP) but relatively preserved vibration. We sought to assess the sensitivity and specificity of clinical examination and various diagnostic tools available for screening SFN. METHODS: Medical records of 56 patients diagnosed with SFN were reviewed. Diagnosis was based on symptoms, detailed neurological examination that included PP, and abnormal results on at least one testing modality-quantitative sudomotor axon reflex (sweat) test (QSART), quantitative sensory testing (QST), and heart rate variability (HRV) testing. RESULTS: Sensitivity of PP was relatively consistent between modalities of about 63% in presence of appropriate sensory symptoms. Laboratory testing diagnosed 88% of patients when both QSART and QST are employed. QST was most sensitive for detection of SFN with the heat-pain testing having higher sensitivity than cooling. Heart rate variability testing revealed low correlation across all groups. CONCLUSIONS: The diagnostic yield for SFN increases by combining clinical features with various testing modalities. In symptomatic patients, we propose the following diagnostic criteria for diagnosis of SFN: Definite SFN-abnormal neurological examination and both QSART and QST; Probable SFN-abnormal neurological examination, and either QSART or QST; Possible SFN-abnormal neurological exam, QSART, or QST.

Pinprick Testing in Small Fiber Neuropathy: Accuracy and Pitfalls.

OBJECTIVES: To assess the sensitivity, specificity, and predictive value of pinprick perception and its concordance with established laboratory measures in small fiber neuropathy. METHODS: Fifty-six patients suspected of SFN underwent detailed evaluation and standardized quantitative sensory and autonomic testing. Sensitivity, specificity, positive predictive value (PPV), and negative predictive value of pinprick sensation were determined. RESULTS: Pinprick perception had relatively consistent results between modalities with moderate sensitivity (70%); combining the testing modalities increased the diagnostic yield. PPV of pinprick approached 89%; however, negative predictive value was 19%. CONCLUSIONS: As a screening tool, pinprick has high PPV for SFN, but many patients are likely to be missed if they report unimpaired subjective perception of pinprick sensation. Multimodal testing is recommended in clinically suspected cases to positively rule out SFN.

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.

Blood volume expansion and cardiorespiratory function: effects of training modality.

PURPOSE: To evaluate the effects of different modalities of aerobic (i.e., interval (INT) and continuous (CONT)) training on cardiorespiratory function and the importance of training-induced blood volume (BV) expansion on aerobic power and LV function. We hypothesized that if modality-mediated differences in cardiorespiratory function exist after INT and CONT, they would be related directly to differences in training-induced hypervolemia. METHODS: We examined the effects of 12 wk of CONT and INT on BV, volume-regulatory hormones (angiotensin II, aldosterone, atrial natriuretic peptide), and cardiorespiratory function in 20 untrained males (mean age 30 +/- 4 (SD)). Participants were stratified (mass and VO2max) and randomly assigned to control, CONT, or INT. RESULTS: There were no significant changes in cardiorespiratory function or BV in the control group. Twelve weeks of continuous and interval training, respectively, resulted in significant changes in VO2max (23 +/- 18 vs 21 +/- 10%), peak stroke volume (20 +/- 18 vs 11 +/- 18%), and BV (12 +/- 9 vs 10 +/- 6%). Changes in VO2max were directly related to changes in BV (r = 0.47). Angiotensin II significantly increased after 1 wk of CONT and INT and thereafter returned to baseline values. There was no significant difference between the CONT and INT groups with regard to changes in vascular volumes, volume-regulatory hormones, and/or cardiorespiratory function. CONCLUSIONS: These data indicate that: 1) 12 wk of CONT and INT result in similar improvements in VO2max, and LV function and 2) training-induced hypervolemia accounts for approximately 47% of the changes in VO2max after CONT and INT.

Myocardial response to incremental exercise in endurance-trained athletes: influence of heart rate, contractility and the Frank-Starling effect.

Recent evidence indicates that endurance-trained athletes are able to increase their stroke volume (SV) throughout incremental upright exercise, probably due to a progressively greater effect of the Frank-Starling mechanism. This is contrary to the widely held belief that SV reaches a plateau at a submaximal heart rate (irrespective of fitness level), owing to a limitation in the time for diastolic filling. The purpose of this investigation was to evaluate whether endurance-trained athletes rely on a progressively greater effect of the Frank-Starling mechanism throughout incremental exercise. A secondary purpose was to evaluate the effects of postural position on the cardiovascular responses to incremental exercise. Ten male cyclists participated in this investigation. Left ventricular function was assessed throughout incremental exercise in the supine and upright positions (counterbalanced) using radionuclide ventriculography. Stroke volume increased in a linear fashion during incremental exercise in both the upright and supine positions. The increases in cardiac output (Q) throughout incremental to maximal exercise (in both the supine and upright positions) were significantly related to changes in heart rate, myocardial contractility and the Frank-Starling mechanism. Percentage changes in end-diastolic volume and SV were significantly greater in the upright position versus the supine position, reflecting an increased reliance on the Frank-Starling effect to increase Q. We conclude from this investigation that highly trained endurance athletes are able to make progressively increasing usage of the Frank-Starling effect throughout incremental exercise. Postural position has a significant effect on the relative contribution of heart rate, myocardial contractility and the Frank-Starling mechanism to the increase in Q during exercise conditions.