Abstracts

Day 1: Monday, May 21, 2012

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Session I: Structure and Function of the Neuromuscular Junction: Recent Advances

Structure of the Neuromuscular Junction: Function and Cooperative Mechanisms in Synapse
Masaharu Takamori, MD, Kanazawa-Nishi Hospital and Kanazawa University

Acetylcholine (ACh)-mediated signal transmission in the neuromuscular junction depends on coordinated interaction between the presynaptic active zone where synaptic vesicles fuse and the postsynaptic differentiation where ACh receptors (AChRs) are clustered. As cooperative mechanisms in the presynapse, the activation of G protein-coupled receptor (M1 muscarinic AChR) and tyrosine kinase B receptor helps on the Ca²⁺ influx mediated by voltage-gated Ca²⁺ channel (P/Q-type) and non-voltage-gated Ca²⁺ channel (Transient receptor potential canonical, TRPCs 3, 6 and 7). These signals are mediated through phospholipase C activation and lead to the Ca²⁺-triggered exocytosis and also the fast-mode of endocytosis which requires larger influx of external Ca²⁺. The synaptic vesicle recycling is thus promoted and potentially compensates synaptic fatigue. In the postsynapse, the Dok7-stimulated MuSK contributes to AChR clustering via its interacting sites for the signals including agrin/Lrp4 (via MuSK Ig1/2 domains), Wnts/Dishevelled (via MuSK Frizzled-like cysteine-rich domain), Tid1s (helps rapsyn stabilization via Hsps 70 and 90β), neuregulin/ErbB, and collagen Q (C-terminus). In the muscle contraction, the TRPC3 collaborates with ryanodine receptor-1 for sarcoplasmic Ca²⁺ release, refilling Ca²⁺ after the store depletion in cooperation with Orai1 (Ca²⁺ influx channel) and STIM1 (Ca²⁺ sensor). The IP3-dependent dissociation of the TRPC-Homer1-IP3R complex allows STIM1 access to TRPC. The various sites in these structures are immunologically targeted in Lambert-Eaton myasthenic syndrome and myasthenia gravis.
 

Presynaptic Organization of Neuromuscular Junction
Hiroshi Nishimune, PhD, University of Kansas Medical School

Presynaptic active zones play essential roles for the function and pathology of neuromuscular junctions (NMJs) because they are synaptic vesicle release sites. We recently reported that the number of active zones increases as the size of mouse NMJs increases 3-fold during postnatal development, however, the density of active zones remains constant from postnatal day 0 to 54. Interestingly, the active zone density decreases at the NMJs of aged mice. These results suggest that NMJs maintain the density of synaptic vesicle release sites while they mature, but this density becomes impaired during aging. What is the molecular mechanism for organizing active zones? Recent findings in the central and peripheral nervous systems demonstrate that synaptic channel proteins are involved in the formation and maintenance of synapses by interacting directly with synapse organizers. We identified recently that presynaptic voltage-dependent calcium channels (VDCC) of NMJs interact extracellularly with the muscle-derived synapse organizer laminin β2 and intracellularly with active zone proteins, Bassoon, CAST/Erc2, and ELKS. These results suggest that VDCCs function as a scaffolding protein to organize the active zones. Consistent with this model, double knock out mice for P/Q- and N-type VDCCs has a significantly reduced number of active zones at NMJs and features an attenuation of the active-zone proteins. These molecular mechanisms of active zone organization and the loss of active zones in VDCC knock out mice are consistent with the pathological conditions observed in Lambert-Eaton myasthenic syndrome cause by autoantibodies against VDCCs and Pierson syndrome caused by laminin β2 mutation.
 

Dok7, MuSK and the Development of Neuromuscular Junction
Yuji Yamanashi, PhD, The University of Tokyo

Skeletal muscle contraction is controlled by motor neurons, which contact the muscle at the neuromuscular junction (NMJ). Defective neuromuscular transmission at NMJs gives rise to the fatigable muscle weakness known as myasthenia. The formation of the NMJ is orchestrated by the muscle-specific receptor tyrosine kinase MuSK and by neural agrin, which can activate MuSK via the coreceptor Lrp4. Previously, we demonstrated that the muscle protein Dok-7 is essential for neuromuscular synaptogenesis, and also demonstrated in collaboration with David Beeson and his colleagues that mutations in DOK7 underlie a limb-girdle type of congenital myasthenic syndrome, DOK7 myasthenia, which is associated with abnormally small and simplified NMJs. In addition, we showed that Dok-7 directly interacts with the cytoplasmic portion of MuSK and activates the receptor tyrosine kinase, and that neural agrin requires Dok-7 in order to activate MuSK in myotubes. In vivo overexpression of exogenous Dok-7 indeed increased MuSK activation and promoted NMJ formation in mice, which did not exhibit defects in motor function. Interestingly, the overexpression of Dok-7 promoted NMJ formation in the appropriate location, namely, the central region of the skeletal muscle. These observations indicate that Dok-7 positively regulates the development of NMJs by controlling MuSK activity and its responsiveness to neural agrin, identifying the Dok-7-MuSK signaling pathway as a new therapeutic target not only for DOK7 myasthenia but also other NMJ synaptopathies.
 

Myasthenogenicity of the Main Immunogenic Region
Jon Martin Lindstrom, PhD, Medical School of the University of Pennsylvania

In myasthenia gravis (MG) and experimental autoimmune MG (EAMG), many pathologically significant autoantibodies are directed at the main immunogenic region (MIR), a conformation-dependent region at the extracellular tip of α1 subunits of muscle nicotinic acetylcholine receptors (AChRs). Human muscle AChR α1 MIR sequences were integrated into Aplesia ACh-binding protein (AChBP). The chimera was potent at inducing both acute and chronic EAMG, though less potent than Torpedo electric organ AChR. Wild-type AChBP also induced EAMG but was even less potent, and weakness developed slowly without an acute phase. AChBP is more closely related in sequence to neuronal α7 AChR, however, autoimmune responses were induced to muscle AChR, but not to neuronal AChR subtypes. The greater accessibility of muscle AChRs to antibodies, compared to neuronal AChRs, may allow muscle AChRs to induce self-sustaining autoimmune responses. The human α1 subunit MIR is a potent immunogen for producing pathologically significant autoantibodies. Additional epitopes in this region or other parts of the AChR extracellular domain contribute significantly to myasthenogenicity. We show that an AChR-related protein can induce EAMG. Thus, in principle, an AChR-related protein could induce MG. AChBP is a water-soluble protein resembling the extracellular domain of AChRs, yet rats immunized with chimeras developed autoantibodies to both extracellular and cytoplasmic domains of muscle AChRs. We propose that an initial autoimmune response directed at the MIR on the extracellular surface leads to an autoimmune response sustained by muscle AChRs. Autoimmune stimulation sustained by endogenous muscle AChR may be a target for specific immunosuppression.
 

Session II: Advances in Immunology and their Relationship to Myasthenia Gravis

Etiology of Autoimmune Diseases
Jean-François Bach, MD, DSc, Necker Hospital

There is not a single cause for autoimmune diseases but a combination of genetic and acquired predisposing factors. Genetic determinism is complex involving many genes. A growing number of genes or more often genetic regions have been identified but their pertinence to the disease etiology is made uncertain by the low or very low relative risk associated with a given gene polymorphisms. The only exception is that of HLA genes whose involvement in disease pathogenesis is, however, not as clearly understood as generally assumed. Environmental factors are very diverse. Some are clearly identified: specific virus or bacteria in a small number of autoimmune diseases. Several candidate infectious agents, notably viruses, have been incriminated as in the case of multiple sclerosis (EBV) or of Type 1 diabetes (T1D) (Coxsackie virus) without any definite evidence perhaps due to the long time elapsed between viral infection and disease onset (hit and run hypothesis). Other factors have been considered such as defective vitamin D exposure, chemical modification of auto-antigenes or polyclonal B or T cell stimulation as in the case of some drug induce auto-immunity. The problem is complicated by the fact that the environment often has a protective rather than a triggering role. A correlation has been observed, over the last decades, between the decline of certain infectious diseases and the increase in autoimmune diseases. The causal relationship between these two observations has been directly demonstrated in animal models and indirectly in man. The mechanisms underlying the protective effect of infections are becoming better known with, in particular, the involvement of homeostatic competition regulatory T cells and Toll-like receptors with a possible influence of microbiota.
 

Defects of Immunoregulatory Mechanisms in MG
Sonia Berrih-Aknin, PhD, University Pierre et Marie Curie & Institut National de la Santé et de la Recherche Médicale

Regulatory CD4⁺CD25⁺ T cells prevent the activation of auto-reactive T cells and play a key role in the induction of peripheral tolerance. Using purified CD4⁺CD25⁺ (Treg) and CD4⁺CD25^- (Tconv) cells from Myasthenia Gravis (MG) thymus, we previously showed a severe defect in the autologous suppression assay compared to control cells. Cross-experiments (using MG Treg cells and healthy T conv, and conversely) showed that not only MG Treg cells were defective in suppressing healthy Tconv cell proliferation, but also that MG Tconv cells were resistant to suppression by healthy Treg cells. The resistance of MG Tconv cells to suppression was not due to their over-proliferation. However, after activation, MG Tconv cells expressed a lower expression of FoxP3 compared to CTRL cells, and secreted higher levels of IL-6 and IL-17, two cytokines implicated in Treg regulation and auto-immunity. These results were corroborated by the transcriptomic analysis of Treg and Tconv subsets, which pointed out a dysregulation of TH17 related genes (IL17A, IL17F, IL17RA, IL21, IL22, IL23R) in MG cells compared to controls.
 
Altogether, our results demonstrate that the defects of immunoregulation observed in MG patients are due to both Treg and Tconv cells, and that IL-17 and related cytokines could play an important role in this defect. This work was supported by FP7 FIGHT-MG project.
 

A Journey from the Thymus to the Endplate
Marc De Baets, MD, PhD, Maastricht University
Abstract pending

Functional Defect in Regulatory T Cells in Autoimmune Myasthenia Gravis
Matthew N.Meriggioli, MD, FAAN, University of Illinois College of Medicine

Published studies examining the role of regulatory T cells (Tregs) in the pathogenesis of myasthenia gravis (MG) have reported conflicting results. In these investigations, isolation of Tregs was achieved based on expression of CD25 alone, likely resulting in the isolation of impure and/or heterogenous human "Treg" populations. In this study, we used surface CD4, CD25^high, and CD127^low expression to isolate a relatively pure population of Tregs, and then investigated whether there was a deficiency and/or a functional defect in these cells in the peripheral blood of MG patients.
 
We collected blood samples from 24 MG patients and 22 age-matched healthy control subjects. Peripheral blood mononuclear cells (PBMCs) were isolated, and the expression of surface CD4, CD25, and CD127, and intracellular FOXP3 was analyzed by flow cytometry, and the function of Tregs (CD4+CD25^highCD127^low/- cells) was assessed by co-culture experiments with CD25-negative autologous responder T cells (Tresp). Expression of intracellular FOXP3 was assessed by flow cytometry and RT-PCR, and cytokine profiles from T cell co-culture supernatants were analyzed.
 
No alteration in the relative numbers of Tregs within the peripheral CD4+ T cell pool was seen in MG patients. In vitro proliferation assays demonstrated that Treg-mediated suppression of responder T cells (Tresp) was impaired, both for polyclonal activation (anti-CD3 Abs) and activation with specific antigen (acetylcholine receptor peptides). This defect was associated with a reduced cellular expression of FOXP3 at both the protein and mRNA level. Both polyclonal and AChR-activated Tresp cells from MG patients could be effectively suppressed using Tregs isolated from healthy controls, while polyclonal-activated Tresp cells from controls were not suppressed using Tregs isolated from MG patients. Cytokine profiles revealed altered levels of IL-6, IL-17, IFN-?, and IL-10 in MG patients.
 
Our findings indicate a clinically relevant Treg-intrinsic defect in immune regulation in MG that may reveal a novel therapeutic target. Study supported by: This work was supported by the NIH (National Institute of Neurologic Disorders and Stroke, K08NS058800, and the Muscular Dystrophy Association (MDA); and National Institute of Allergy and Infectious Diseases, RO1 AI 058190, BSP.
 

Neuronal Acetylcholine Receptor Autoimmunity
Steven Vernino, MD, PhD, University of Texas Southwestern Medical Center

Autoimmunity against acetylcholine receptors (AChR) at the neuromuscular junction is the usual cause of myasthenia gravis. Neuronal nicotinic AChRs that are highly homologous to the neuromuscular AChR are found throughout the nervous system. The ganglionic neuronal AChR mediates fast synaptic transmission in sympathetic, parasympathetic and enteric autonomic ganglia. Impaired ganglionic synaptic transmission is one important cause of autonomic failure.
 
Ganglionic AChR antibodies are found in many patients with autoimmune autonomic ganglionopathy (AAG). Patients with high levels of ganglionic AChR antibodies typically present with rapid onset of severe autonomic failure, with orthostatic hypotension, gastrointestinal dysmotility, anhidrosis, bladder dysfunction and sicca symptoms. A characteristic impairment of the pupillary light reflex is often seen. Lower levels of ganglionic AChR antibodies may be found in patients with other disorders including those with malignancy. However, AAG is only rarely associated with malignancy such as thymoma, lung cancer or lymphoma.
 
Like myasthenia gravis, AAG is an antibody-mediated neurological disorder. Antibodies from patients with AAG inhibit synaptic transmission in autonomic ganglia. Animal models of AAG provide additional evidence that AAG is an antibody-mediated disorder caused by impairment of synaptic transmission in autonomic ganglia. AAG patients improve clinically when treated with therapies to reduce antibody levels (such as plasma exchange, IVIG, corticosteroids, or immunsuppression). Although symptoms improve, most patients continue to have objective evidence of severe autonomic failure.
 
Autoimmunity against other neuronal nicotinic or muscarinic AChR subtypes may also be important, and studies are ongoing in this area.
 

The Role of B Cell Activating Factor in Autoimmune Myasthenia Gravis
Robert P. Lisak, MD, Wayne State University

Autoimmune myasthenia gravis (MG) is a disease mediated by antibodies directed against molecules of the post-synaptic portion of the neuromuscular junction (NMJ), most often skeletal muscle nicotinic acetylcholine receptor (AChR). In addition, many patients with MG also have increased incidence of other organ specific and non-organ specific autoantibodies as well as other autoimmune diseases. This supports the hypothesis that MG patients have increased activity of B cells and their progeny, plasmablasts and plasma cells. B cell activating factor (BAFF) is a member of the tumor necrosis factor superfamily and is an important factor in B cell development, activation, and survival. Increased serum levels of BAFF have been reported by several groups in MG as well as in some other autoimmune diseases. BAFF is found in germinal follicles at sites of inflammation along with the chemokine CXCL13, a major chemotactic factor for B cells. Thus it is not surprising that BAFF has been found in germinal follicles within the hyperplastic thymus of patients with MG along with increased expression of CXCL13. Production of anti-AChR as well as antibodies to other antigens by cells from the thymus of patients with MG has also been reported. Several BAFF antagonists exist and treatment of patients with these might offer an additional approach to treatment of patients with MG.
 

Session III: Myasthenia Gravis: Clinical and Laboratory Developments

Population Differences in the Clinical Presentation and Serology of MG
Janice M. Massey, MD, Duke University Medical Center

Clinical classification of MG, epidemiologic data and serologic status define various subsets of MG. Previous studies showed 16% of patients persist with restricted ocular disease but 25% of untreated patients with generalized weakness died. Thymoma is found in 15% of patients. MG has an incidence of 14-20/100,000 and appears to be increasing in part due to better diagnosis in an aging population. While women predominate in younger and men in older populations, each increase with age, as does the incidence of MG. AChR antibodies are present in 50-80% of patients with a higher sensitivity in more severe patients. of seronegative patients, 20-40% have antibodies to MuSK. Patients with MuSK MG (MMG) are often female and may have distinct clinical and electrophysiologic patterns. While some MMG patients are indistinguishable from AChR+ MG, others show facial and bulbar weakness with marked muscle atrophy while another group has prominent neck extensor, shoulder or trunk weakness and frequent crisis. Furthermore, there are differences in response to certain treatments. Cholinesterase inhibitors may produce worsening, while a sustained improvement may occur with rituximab. The recognition of these clinical patterns among patients with MG is evolving. Further delineation by serotyping or genotyping may allow for targeting phenotypes for specific or novel therapies and provide further insights in questions of basic pathophysiology of MG.
 

Cell-Based Assays in Myasthenia Gravis
Angela Vincent, FRS, University of Oxford

The increasing number of antibody-mediated diseases, and the diversity of target antigens now known, make it imperative to establish diagnostic antibody assays that are sensitive, informative and distinguish between different forms of a disease or syndrome. In myasthenia gravis, antibodies to the AChR are still measured mainly by radioimmunoprecipitation as first reported by Lindstrom et al in 1976. This assay, although sensitive and specific, does not usually distinguish between antibodies to the fetal and adult forms of the AChR which can be relevant to fetal or neonatal disorders. Moreover, a proportion of those that are negative for AChR antibodies have antibodies to MuSK and a few to LRP4, so these also need to be measured separately in many cases. And there are other potential antigens at the neuromuscular junction that need to be explored.
 
We have performed cell-based assays for each of these antibodies. These involve expressing the antigen on the surface of live human embryonic kidney (HEK) cells by DNA transfection, and measurement of antibody binding by indirect immunofluorescence. By clustering the AChRs using co-transfection with rapsyn, we have improved the sensitivity for AChR antibodies in both generalized (Leite et al 2008) and ocular MG and shown that these antibodies are pathogenic (Jacob, et al 2012). We have also used the same approach to look for antibodies to other neuromuscular junction proteins in cohorts of patients previously negative for AChR or MuSK antibodies.
 
The cell-based approach offers not only increased sensitivity but also the knowledge that the antibodies are binding to the extracellular domain of the antigen and are therefore potentially pathogenic; antibodies that bind to intracellular epitopes will not be detected on the unpermeabilised live cells. In addition, they have the potential to be used for multiple antigen (multiplex) testing; mosaics of fixed HEK cells expressing different antigens can be placed together for testing each serum (as already being done for some of the CNS antigens by Euroimmun AG, Luebeck, Germany). In this manner it should be possible in the future to provide simultaneous sensitive assays for antibodies to fetal and adult AChRs, MuSK, LRP4 and other antigens in a single test.
 

Concentric Needle Jitter Studies
Erik Stålberg, MD, PhD, Pro.em, University Hospital Uppsala

The presence of abnormal neuromuscular jitter is the most sensitive electrophysiologic evidence of disturbed neuromuscular transmission. Jitter has usually been measured using SFEMG needle electrodes (SFE). Recently, many countries do not allow reusable electrodes, thus an alternative to the SFE must be found. The best surrogate at the moment is the smallest available concentric electrode (CNE), a so-called facial electrode. It has a recording surface area three times that of the SFE, and thus usually records from more than one muscle fiber. There is a significant risk of obtaining a summation signal from more than one muscle fiber in a motor unit. It is however possible to obtain reasonable signals mainly representing single fiber action potentials, called "apparent single muscle fiber action potentials "(ASFAP), which can be used for jitter analysis.
 
This method is more difficult than SFEMG in muscles with large or dense motor units, e.g. large limb muscles, and in reinnervation. In facial muscles it is easier to obtain acceptable signals.
 
Reference CNE jitter values have been obtained in extensor digitorumn, orbicularis and frontalis muscles. The CNE reference limits are somewhat lower than with the SFEMG electrode. The diagnostic sensitivity of CNE in MG is similar to that of SFEMG, thus very high.
 
The CNE can be used for jitter studies. Until more reference values have been obtained using standardized equipment, signal and measurement criteria, borderline findings should be interpreted with caution.
 

Management Challenges in Muscle-Specific Tyrosyne Kinase MG
Amelia Evoli, MD, Catholic University, Roma

Myasthenia gravis with antibodies to muscle-specific tyrosine kinase (MuSK-MG) is generally considered a severe disease on account of weakness distribution with prevalent involvement of bulbar muscles and a rapidly progressive course with early respiratory crises. Its treatment can be unrewarding owing to poor response to acetyl-cholinesterase inhibitors in most patients, disease relapses in spite of high-dose immunosuppression and development of permanent bulbar weakness. In a population of 75 MuSK-MG patients treated in our Institution for at least 2 years (follow-up ranging 2-35 years), the maximum MGFA clinical class was IIIb or greater in 89%, with 32% rate of respiratory crises. High-dose prednisone plus plasma-exchange, as in other forms of MG, is the recommended approach in treating rapidly progressive bulbar weakness. Maximum disease severity did not predict the disease course. Twenty patients (26.6%) suffered from two or more disease relapses while under combined immunosuppressive therapy, and 15 (20%) developed fixed facial and oro-pharyngeal weakness (most patients who developed permanent weakness also had relapsing disease). In the management of these patients, oral steroids proved effective through the whole disease course, and repeated plasma-exchange produced clear albeit short-term improvement, while conventional immunosuppressants resulted comparatively less effective. Rituximab is a very promising treatment for refractory MuSK-MG, as all treated patients, reported so far, achieved significant improvement with substantial decrease of medication. It is yet to be clarified whether the early use of rituximab could prevent the permanent oro-pharyngeal weakness which constitutes a relevant disability in these patients.
 

Daniel B. Drachman, MD, Johns Hopkins School of Medicine
Abstract pending

Session IV: Myasthenia Gravis: Outcome Measurements and Clinical Trial Development

MG Activities of Daily Living (ADL) Profile
Srikanth Muppidi, MD, University of Texas Southwestern Medical Center

The MG-specific Activities of Daily Living scale (MG-ADL) was developed to assess the status of symptoms and activities in MG. MG-ADL is an eight-item patient-reported questionnaire that can be completed in a few minutes without need for equipment or training. Previously in 254 MG patients, MG-ADL score was compared to quantitative MG score (QMG). MG-ADL correlates well with Quantitative MG score (r= 0.583, P < 0.001). Recently, we analyzed the performance of MG-ADL during a multicenter, prospective scale validation study. Eighty-seven patients completed the MG-ADL, MG Composite and MG-QOL15 on the first visit and 76 returned for the second visit. The mean initial MG-ADL score was 4.89 (±3.54) and improved to 3.59 (±3.3) at the second visit. At the first visit, there was a strong positive correlation between the MG-ADL and MGC (r=0.85, P <0.0001) and the MG-QOL15 (r=0.76, P<0.0001). Correlation of the change in MG-ADL score and physician impression of change between the two visits was also strong (r= 0.70, P <0.0001). Test-retest analysis demonstrated a high reliability coefficient. Sensitivity/specificity analysis revealed that a 2-point improvement in the MG-ADL best predicted clinical improvement. The MG-ADL correlates strongly with QMG, other newer validated MG outcome measures and a 2-point improvement in MG-ADL indicates clinical improvement. The MG-ADL is useful as a secondary outcome measure in MG treatment trials and was responsive to change MG status in recent MG treatment trials. Because of the simplicity and ease of use, MG-ADL can also be used in routine clinical management.
 

Michael Rose, MD, FRCP, King's College Hospital
Abstract pending

The MG Composite
Ted M. Burns, MD, University of Virginia

The "MG Composite" (MGC) scale consists of test items that measure symptoms and signs of MG, with weighted response options. The individual test items of the MGC were selected from existing MG-specific scales based on their performance during two randomized, controlled clinical trials of mycophenolate. Test items were selected so as to be meaningful to both the physician and the patient, frequently abnormal in patients with active disease, responsive to clinical change and appropriately weighted. The MGC was validated in a 175-subject study (11 sites). Total MGC scores showed excellent concurrent validity with other scales. Analyses of sensitivities and specificities of the MGC for identifying clinical improvement revealed that a 3-point improvement in MGC score was optimal for signifying clinical improvement. A 3-point improvement in the MGC appeared to be meaningful to the patient, as indicated by improved MG-QOL15 scores. The test-retest reliability coefficient was 98%, indicating excellent test-retest reliability. Rasch analysis of the MGC was also performed to investigate additional properties, including its unidimensionality and the appropriateness of the weights assigned to the response categories for the MGC items. The fit statistics indicated that the items belong together and can be summated for a total score. There was an overall absence of item order distortion between response categories. The Rasch model expected category response values were compatible with item weights previously assigned. Our Rasch analysis suggested that: 1) the score can be summated to estimate an overall disease severity score; 2) the response options of the ten items are not significantly distorted; and, 3) the assigned weights of the response options are appropriate.
 

Patient Registries: Useful Tools for Clinical Research in Myasthenia Gravis
Fulvio Baggi, PhD, Neurological Institute "Carlo Besta"

Clinical trials in MG are challenging, not least because of the numerous disease subcategories that must be considered: AChR-MG, MuSK-MG, Double Negative-MG, Ocular-MG, Generalized-MG, Childhood-MG, Early-and Late-Onset MG, and Thymoma-MG. MG Registries can facilitate clinical trials in several ways, including as a source of clinical, biological and immunological data on large numbers of patients, and as a source of referrals.
 
A European-MG database (European MG Network, Grant EU-2005105, DG SANCO) has been developed, and the Myasthenia Gravis Foundation of America is developing a patient-driven registry, to be used in support of research, advocacy and public awareness.
 
Physician-derived registries have the advantage of incorporating diagnostic and treatment data that may allow comparison of outcomes from different therapeutic approaches, which can be supplemented with patient self-reported data. By analyzing data on disease progression and responses to different disease management strategies, registries may help to improve disease outcomes.
 
We present an ongoing collaborative project involving two large physician-derived registries, the Duke MG Patient Registry (US) and the INNCB MG Registry (Italy), and our efforts to develop and implement a common platform with a core of Common Data Elements (CDE) that are concordant with an ongoing NINDS project to establish MG-specific CDEs. Registries have inherent ethical issues about privacy and data use that must be clearly discussed and presented to patients via informed consent. MG Patient Associations should play a pivotal role in disseminating information about registries and encouraging patient participation.
 

Abstracts

Day 3: Wednesday, May 23, 2012

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Session VII: Congenital Myasthenic Syndromes

Synaptic Basal-Lamina Associated Congenital Myasthenic Syndromes
Ricardo A. Maselli, MD, University of California, Davis

Proteins associated with the basal lamina (BL) participate in complex signal transduction processes that are essential for the development, maintenance and regeneration of the neuromuscular junction (NMJ). Major components of the junctional BL are: 1. β2 laminins; 2. Collagens, like collagen IV chains (α3-6), collagen XIII and ColQ; 3. Heparan sulfate proteoglycans, such as perlecan and agrin; and 4. Nidogen-2. Important interactions include β2 laminins and agrin with α-dystroglycan; agrin with Lrp4, which activates the agrin-MuSK signaling complex; and ColQ with perlecan and MuSK, which anchors ColQ-AChE at the endplate. Mice lacking ColQ (ColQ^-/-), laminin β2 (Lamb2^-/-) and collagen XIII (Col13a1^-/-) show immature nerve terminals enwrapped by Schwann cell projections invaginated into the synaptic cleft, which decrease contact surface for neurotransmission. So far in humans there are identified mutations in COLQ resulting in deficiency of endplate AChE, which is a frequent congenital myasthenic syndrome (CMS) (~12% of CMSs), and mutations in AGRN and LAMB2 resulting in deficiency of agrin and laminin-β2 respectively, which are infrequent CMS variants. The ultrastructure of the NMJ in these syndromes shows striking resemblance to that of mice lacking the protein encoded by the mutant gene. In addition, in deficiency of endplate AChE there is subsynaptic degeneration, which was also unexpectedly found in a severe case of agrin deficiency. The response of these syndromes to pharmacologic interventions is often disappointing, but the extracellular localization of the mutant proteins may provide favorable conditions for replacement strategies, including gene therapy and stem cells.
 

DOK7 Mutations Underlie Neuromuscular Junction Synaptopathy
Jacqueline Palace, MD, Oxford University Hospital

Congenital myasthenic syndromes (CMS) are a heterogeneous group of genetically determined disorders caused by neuromuscular transmission dysfunction. Post-synaptic mutations constitute over 80% of the UKs' genetically identified congenital myasthenic syndromes and DOK-7 mutations account for around ¼ of these.
 
DOK-7 CMS, like other CMS, presents early in life with fatigable muscle weakness and has an autosomal recessive pattern of inheritance.
 
However in contrast to the classical CMS picture it typically presents after infancy and most patients have normal early motor milestones with onset around 2-3 years. The pattern of weakness is of a limb girdle phenotype and the extra-ocular muscles are spared. of note it does not respond or gets worse with regular pyridostigmine and only sometimes improves with 3,4-diaminopyridine. Progressive weakness over decades may be associated with an end plate myopathy and respiratory involvement may require ventilatory support. Treatment with ephedrine or salbutamol improves weakness and function in the majority of patients over weeks to months, and is presumed to be related to signalling that stabilises the muscle end-plate structures . Other useful distinguishing clinical features include tongue wasting and stridor.
 

Synaptic Dysfunction in Congenital Myasthenic Syndromes
David Beeson, PhD, University of Oxford

Congenital myasthenic syndromes (CMS) are hereditary disorders of neuromuscular transmission characterised by fatigable muscle weakness. The number of cases recognised is increasing with improved diagnosis. To date we have identified over 300 different mutations present in over 350 unrelated kinships. The underlying genetic defects are diverse involving a series of different genes with a variety of different phenotypes. Typically patients present soon after birth with feeding difficulties, a weak cry, and more seriously, difficulties in breathing requiring resuscitation, although some syndromes may not manifest until childhood or even adulthood. The type of treatment and their effectiveness will depend on the underlying pathogenic mechanism. We aim to define the molecular mechanism for each mutation identified and feed this information back to the clinic as a basis for a rational strategy for treatment. Here, we highlight a new mechanism for disruption of AChR function where a mutation in the AChR ?-subunit gene causes reduced ion channel conductance. We analyse a series of variants in DOK7 in order to define mutations that impair AChR cluster formation, and we investigate mutations in the newly identified CMS-associated gene GFPT1. The study of these disorders is proving highly informative for understanding the diverse molecular mechanisms that can underlie synaptic dysfunction.
 

Congenital Myasthenic Syndromes (CMS) in 2012
Andrew G. Engel, MD, Mayo Clinic

An increasing number CMS patients, disease genes, and clinical entities are being identified worldwide. Among 342 patients investigated by us, pathogenic mutations in AChR, rapsyn, ColQ, Dok7, ChAT and GFPT1 were identified in 48, 14, 14, 10, 5 and 3%, respectively. This presentation will focus on kinetic mutations that impair closed-to-open isomerization of AChR after agonist occupancy, functional consequences and structural interpretation of mutations in ChAT, the clinical and structural heterogeneity of GFPT1 deficiency, and the pathophysiologic basis of the neuromuscular transmission defect in plectin deficiency and centronuclear myopathy.
 

Clinical and Electrodiagnostic Obsverations in Congenital Myasthenic Syndromes (CMS)
Charles (Mike) Harper, MD, Mayo Clinic College of Medicine

Clinical and electrodiagnostic observations have been correlated with findings on morphological, microelectrode, and molecular genetic studies in a wide spectrum of CMS subtypes. A number of unique pattern have been observed that permit accurate diagnosis and treatment of CMS in many cases based on clinical and electrodiagnostic data alone. 1) Slow Channel CMS: AD pattern of inheritance, severe weakness of neck and distal upper limb muscles, exacerbation of symptoms by cholinesterase inhibitors (CEI), repetitive CMAPs (R-CMAP), which increase in number and amplitude after CEI, and a rate-dependent CMAP decrement. 2) Endplate Acetylcholine Esterase deficiency: AR inheritance, generalized muscle hypotrophy, impaired pupillary reflexes, skeletal deformities, lack of clinical benefit from CIE, R-CMAPs that do not change after administration of CIE, and a rate dependent CMAP decrement. 3) Fast Channel Syndrome: AR inheritance, generalized weakness, mild decrement on repetitive stimulation studies, no R-CMAP and excellent response to a combination of CEI and 3.4-DAP. 4) DOK7-CMS: AR inheritance, limb-girdle predominant weakness, decrement on spinal accessory-trapezius repetitive stimulation. 5) Choline acetyl transferase (ChAT) deficiency: AR inheritance, bimodal age of presentation with bulbar-respiratory crises early, may required prolonged repetitive stimulation or exercise to elicit decrement with delayed recovery in between exacerbations. Additional examples will be discussed.
 

Session VIII: Lambert-Eaton Myasthenic Syndrome

Treatment in Lambert-Eaton Myasthenic Syndrome
Paul Maddison, MD, FRCP, Nottingham University Hospitals, Queen's Medical Centre

Lambert-Eaton myasthenic syndrome (LEMS) is a presynaptic autoimmune disorder of neuromuscular transmission characterised by impaired quantal release of acetylcholine that principally causes proximal limb weakness and autonomic dysfunction. Over 50% of LEMS patients have an underlying tumour, most often small-cell lung cancer. Tumour surveillance and cancer treatment remain the principal aim following initial LEMS diagnosis.
 
Almost all patients will benefit initially from symptomatic treatment with 3,4-diaminopyridine (3,4-DAP), a potassium channel blocking agent, also capable of interacting with calcium channel subunits, which is well tolerated with few side effects. In a recent meta-analysis of four randomised placebo-controlled trials of 3,4-DAP in a total of 53 LEMS patients, treatment with 3,4-DAP significantly improved quantitative myasthenia gravis scores, and resting compound muscle action potential amplitudes. All four trials demonstrated significant increases in muscle strength scores following 3,4-DAP treatment compared with placebo.
 
Often, additional treatment is required in the form of immunosuppression. One randomised cross-over trial demonstrated a significant improvement in myometric limb strength following short course intravenous immunoglobulin (IVIg) infusions, but there are no data to suggest that IVIg is an effective long-term treatment for LEMS. Oral steroids (prednisolone) has been shown to be effective from observational studies, but patients often require significant maintenance doses.
 
Plasma exchange has been used effectively as acute treatment for severe LEMS symptoms. In patients with symptoms refractory to oral immunosuppression, where multiple courses of plasma exchange or IVIg are required, novel treatment approaches may be beneficial, such as the use of the anti-CD20 monoclonal antibody therapy Rituximab.
 

SOX1 in Lambert-Eaton Myasthenic Syndrome and Screening for Small Cell Lung Cancer
Maarten J. Titulaer, MD, PhD, University of Pennsylvania

SOX1 belongs to the Sry-like high mobility group superfamily of developmental transcription factors, an intranuclear protein probably involved in prevention of differentiation of neural progenitor cells. It was shown to be the antigen recognized by anti-glial nuclear antibodies. SOX1 antibodies are specific serological markers for small cell lung cancer (SCLC) with or without the Lambert-Eaton myasthenic syndrome (LEMS). SOX antibodies have diagnostic value in discriminating SCLC-LEMS from non-tumor LEMS, but no clinical or pathogenic effect has been shown in LEMS or SCLC.
 
As half of the LEMS patients have a SCLC, detection is an important issue for patient and physician. SOX1 is a specific marker (95%), however sensitivity is unfortunately only moderate (65%). To improve prediction of the risk for SCLC, we recently described the DELTA-P score, which uses 6 simple clinical features within 3 months form onset. These features are Dysartria/bulbar signs, Erectile dysfunction, Loss of weight (>5%), Tobacco (smoking) at onset, Age (>50 years) and Karnofsky Performance Status (<70, dependent in activities in daily living), each scoring 1 point if present. A score of 0-1 relates to a very low risk of SCLC (0-2.5%), while a score of 3-6 is linked to very high suspicion of SCLC (84-100%). The DELTA-P score is easy and reliable in predicting the presence of SCLC in LEMS. Addition of SOX1 antibodies only adds 2% of accuracy to this clinical model, without direct impact on screening strategy. As SOX antibody assays are not generally available and time-consuming, the purely clinical score is preferable.
 

Session IX: Thymus, Thymectomy and the MGTX Trial

MGTX: Update on the Thymectomy Trial in Non-Thymomatous Myasthenia Gravis
Gil I. Wolfe, MD, University at Buffalo School of Medicine and Biomedical Sciences
Gary R. Cutter, PhD, University of Alabama School of Public Health

MGTX is a multi-center, single-blind, randomized trial supported by the NIH/NINDS,which aims to answer 3 questions: Does extended transsternal thymectomy (ETTX) combined with a strictly defined prednisone protocol, when compared with the prednisone protocol alone (1) result in a greater improvement in myasthenic weakness, (2) result in a lower total dose of prednisone, and (3) enhance the quality of life by reduction of adverse events and manifestations associated with medical therapies? Inclusion criteria are MG Foundation of America Class 2 to 4, positive acetylcholine receptor antibody, age >18.0 and <65.0 years, and MG history <5 years. Patients are followed for a minimum of 3 years. As of February 2012, 119 of the target 150 patients were enrolled. A total of 50 centers in 17 countries are currently enrolling patients. Recruitment has been slower than desired due to the rarity of MG, institutional obstacles, and pre-existing patient perceptions about surgery and/or medication. The drop-out rate has been lower than predicted. Despite challenges posed by a multinational study that randomizes patients to surgical or non-surgical arms, MGTX investigators remain enthusiastic about completing the trial. Enrollment will conclude in November 2012 with the sample size accrued at that time. A result favoring thymectomy would establish its benefits unequivocally; failure to show a difference would suggest that thymectomy is an unnecessary procedure in this non-thymomatous population and should lead to cost savings. Thus the results will impact on clinical practice.
 

Biomarker Development for Myasthenia Gravis
Henry J. Kaminski, MD, George Washington University

Biomarkers are best defined as characteristics (proteins, RNA, SNP, imaging) that are objectively measured and evaluated as an indicator of pathogenic processes or pharmacologic responses to a therapeutic intervention. A predictive biomarker may identify a patient subgroup that has a greater potential for benefit or be at risk for an adverse effect for a specific intervention. Biomarkers are important in clinical trials, particularly Phase II trials in rare disorders, where the robust biomarker reflects the underlying disease process in a sensitive and reliable manner. Acetylcholine receptor and muscle specific kinase antibodies, as well as single fiber electromyography, serve as excellent biomarkers for diagnosis but do not adequately substitute for clinical evaluations to predict treatment response. Biomarker discovery has not been explored in MG and the lack of biomarkers is a significant deficiency for assessment of novel therapeutics. New technologies are emerging that enable broad biomarker discovery in biological fluids, including circulating microRNA, mRNA profiling of blood and component cells, nanoparticle proteomics, and highly parallel FACS bead assays of cytokines. Biomarker evaluation is ideally done in the context of longitudinal clinical trials. The MGTX trial has collected over 5000 specimens, including serum for RNA and protein analysis and thymus, which will allow robust biomarker discovery. The ultimate goal for biomarker discovery for MG will be to identify a sensitive and reliable substitute for a clinically meaningful endpoint that is a direct measure of the effectiveness of a therapy in the context of a continuum of natural history of MG and a patient's overall well-being. (Supported by NINDS U01 NS042685)
 

Thymus Pathology Observed in the MGTX Trial
Alexander Marx, MD, University of Heidelberg

Evaluation of the histomorphology of prospectively collected thymectomy specimens comple-ments the analysis of biomarkers obtained during the course of the Thymectomy in Nonthymo-matous Myasthenia Gravis (MG) Trial (MGTX Trial). Thymectomy specimens were obtained following a standardized (extended) surgical procedure, and size and weight were recorded. Standardized work-up and processing comprised sampling at several defined levels of the resection specimens after 24h of fixation in 4% buffered formalin, and semi-automated morpho-metric measurement of i) thymic versus extrathymic (mostly adipose) tissue; ii) cortical versus medullary compartments; and iii) immunohistochemically defined expression of CD20 (B-cells), CD10/Bcl6 (germinal center cells), CD23/CD35+ networks (follicular dendritic cells) and desmin (myoid cells). While still blinded with respect to clinical data, we received and analyzed the first 30% of the expected resection specimens, allowing to conclude the following: a) the material yields excellent morphology in ~90% of cases; b) grading of thymic lymphofollicular hyperplasia (TFH) with CD10+ germinal centers shows a spectrum from complete lack (11%), mild (41%), moderate (24%) to severe (24%) TFH; c) no or slight cortical involution was observed in 35% of cases and there was a strong correlation with thymic tissue content (i.e. the degree of atrophy); d) Heterogeneity of morphological findings (e.g. focal atrophy; cyst formation) was encountered in 85% of cases, showing that a minimum number of histological sections will be necessary to achieve representative results. The study shows that the provided thymectomy material will be adequate to correlate quantita-tative morphological data with clinical parameters of MGTX, and with genomic and proteomic profiles of thymus samples and blood obtained by the "Biomarkers in MG Study."
 

Minimally Invasive Thymectomy: An Update
Dan M. Meyer, MD, University of Texas Southwestern Medical Center

Surgical management of nonthymomatous myasthenia gravis has evolved from an open transsternal technique to markedly less invasive options. The results of the studies using progressively less invasive techniques, from full maximal transsternal thymectomy to robotic techniques will be compared. Approaches include full and partial sternotomy, extended transcervical thymectomy, combined transcervcal/transsternal, video-thoracoscopic (unilateral or extended, VATETl), and more recently robotically-assisted procedures will be discussed. Moreover, in concert with the evolution of these techniques, controversy exists over the importance of complete resection of associated mediastinal tissue. Series in the English literature with keywords related to thymectomy for myasthenia gravis will be assessed, comparing studies that demonstrate intermediate- and long-term complete stable remission rates. Moreover, quantitative analysis using the Myasthenia Gravis Foundation of America severity score is an important aspect to compare the impact of the various techniques. Technical concerns related to the specific operative procedure will also be reviewed. Until the results of the MGTX international randomized trial become available, surgery will continue to be an important management option in the treatment of myasthenia gravis. Decisions regarding the approach of the surgical procedure remain controversial but will continue to be defined based upon improved follow-up from the less invasive surgical options.
 

Thymectomy for Myasthenia Gravis Patients—the Robotic Approach
Pier Cristoforo Giulianotti, MD, University of Illinois at Chicago

Thymectomy for Myasthenia Gravis Patients has traditionally been performed through a trans-sternal open approach because of the excellent exposure that the median sternotomy provides, and the need of recognizing all thymic tissue that can be present in all anterior mediastinum and neck. The open sternotomy has negatives consequences and morbidity that are the main reason for the resistance of patients and neurologist to accept the procedure as a mainstay of myasthenia treatment. The development of minimally invasive surgery has led to a greater acceptance of thymectomy. This is due to sufficient radicality achieved in the resection of thymic tissue and the decreased morbidity and mortality, even though the standard thoracoscopic approach is technically challenging and sometimes needs a sterna lifter and bilateral approaches. The introduction of the da Vinci® surgical system (Intuitive Surgical, Inc, Sunnyvale, California) was the next step in the development of minimally invasive radical thymectomy. The robotic thoracoscopic thymectomy allows for a radical resection with the advantages of preserving the benefits of the minimally invasive surgery in terms of less trauma, less pain, less wound complications and faster recovery.
 

The MGTX International Trial: Methods For Management And Lessons Learned
Inmaculada B. Aban, PhD, and Greg Minisman, MA, University of Alabama School of Public Health

The importance of conducting medical research on a global or international platform cannot be overemphasized in current times. Sponsors are encouraging international clinical trials for a number of reasons. Globally, clinical trials continue to be under increasing pressure to meet patient recruitment goals quickly and efficiently, at times with very limited resources. Conducting clinical trials in multiple countries increases access to potentially eligible study patients, especially for rare diseases such as MG, but also involves many unique complexities. From a Data Coordinating Center (DCC) perspective, we present lessons learned from planning, launching and conducting the ongoing multicenter, multinational, NIH/NINDS-sponsored study: "Thymectomy in Non-Thymomatous MG Patients Receiving Prednisone (MGTX)." The responsibilities of the MGTX DCC are not limited to study design, data management, and statistical analysis but also include, among other things, maintenance of complex and variable domestic and international regulatory approvals, contractual agreements, site invoicing and remuneration, study supply distribution, and addressing questions regarding protocol issues. We highlight the data management tools and systems that have been employed to accommodate the many challenges brought about by the inclusion of sites from multiple countries that operate under various healthcare delivery systems. In addition, we present the methods used to effectively communicate with centers and address various problems when they arise. Successful implementation of extended-duration international trials requires a dynamic organizational structure that has the ability to adapt to inevitable changes.