Brain Advance Access originally published online on June 16, 2008
Brain 2008 131(7):1684-1685; doi:10.1093/brain/awn131
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Scientific Commentary |
‘Seronegative’ myasthenia gravis is no longer seronegative
High-affinity IgG autoantibodies to muscle nicotinic acetylcholine receptors (AChRs) were discovered to cause myasthenia gravis (MG) and its animal model more than 30 years ago (Patrick and Lindstrom, 1973
; Lindstrom et al., 1976a, b; Vincent et al., 2006), and the antigenic structure of muscle AChRs is still being actively investigated (Kalamida et al., 2007; Lindstrom et al., 2008). Immune precipitation of AChRs tagged in their acetylcholine-binding sites with 125I-labelled 
–bungarotoxin provided a sensitive immunodiagnostic assay for MG (Lindstrom et al., 1976b). However, up to 20% of those who appeared to have autoimmune MG, because they benefited from plasmapheresis or exhibited antibodies bound to their neuromuscular junctions, did not have autoantibodies detectable by the conventional assay (Vincent et al., 2006).
Angela Vincent and her co-workers discovered that about half of the putative seronegative MG patients actually had autoantibodies to muscle-specific kinase (MuSK) (Hoch et al., 2001; Vincent and Leite, 2005). These MuSK-MG patients exhibited distinct clinical features and their neuromuscular transmission was impaired indirectly through disrupted signalling by MuSK released by the nerve ending that trophically mediates localization of AChRs and other post-synaptic components (Vincent and Leite, 2005).
In classical seropositive MG, autoantibodies bound to the AChRs impair neuromuscular transmission by three mechanisms (Lindstrom, 2000; Engel and Hohlfeld, 2004; Vincent et al., 2006):
- fixation of complement that causes focal lysis of the post-synaptic membrane that reduces the number of AChRs and disrupts their localization next to sites of ACh release;
- cross-linking of AChRs by antibodies increases their rate of endocytosis, which causes loss of AChRs by the process of antigenic modulation; and
- rarely, autoantibodies directly impair AChR function either competitively or non-competitively.
The discovery of autoantibodies to MuSK still left 
12% of MG patients as seronegative, yet clinically and by thymic and endplate pathology appearing much like seropositive MG patients (Vincent and Leite, 2005). Occasional hints suggested that IgM or low-affinity antagonist IgG antibodies to AChRs might explain why the remaining patients were seronegative using the conventional assay (Yamamoto et al., 1991; Barrett-Jolley et al., 1994; Bufler et al., 1998).
In this issue of Brain, Angela Vincent and coworkers report that they have, at last, devised an assay which detects low-affinity IgG autoantibodies to AChRs in 66% of the remaining seronegative patients. The trick was to aggregate the AChRs on the surface of the transfected cells so that high concentrations of AChRs could compensate for the low affinity of the autoantibodies. Bound autoantibodies were detected by microscopy using fluorescently labelled antibodies to IgG. Cotransfection of human embryonic kidney cells with human muscle AChR and rapsyn, the protein which anchors AChRs to the cytoskeleton in muscle, provided the aggregate that permitted detection of the low-affinity autoantibodies.
Problems with the assay are that it provides subjective qualitative results evaluated by microscopy rather than objective quantitative assay values in moles of toxin-labelled AChRs bound per litre of serum obtained by 
counting in the conventional assay for high-affinity autoantibodies; and the transient transfection and evaluation of binding are very laborious compared to an immunoprecipitation assay. One possible solution to these problems would be to grow transfected cell lines in microwell cultures and measure the amount of bound autoantibody using 125I-labelled anti-IgG. If successful, this would eliminate microscopy, speed throughput and permit objective quantification of the amount of autoantibodies in moles of autoantibody per litre of serum. Further upregulation of the amount of AChR in the cell lines by growth in nicotine (Kuryatov et al., 2005) might enhance the sensitivity of the assay. Another approach to detection of these antibodies could involve the use of heavily loaded blots of bacterially expressed AChR subunit constructs, since it was observed that the autoantibodies could be adsorbed using bacterially expressed subunits.
These experiments have illuminated the status of ‘seronegative’ MG patients:
- autoantibodies to AChRs are present;
- these are directed at the extracellular surface, predominantly at subunits, usually away from the ACh-binding site, and are detectable despite apparently low-affinity binding because they can cross-link tightly packed AChRs; thus they might be able to cause AChR loss through antigenic modulation; however, their low affinity and dependence on aggregation by rapsyn may prevent this;
- these autoantibodies are complement-fixing IgG1 antibodies, thus they should be able to impair transmission by disrupting synaptic architecture and causing AChR loss through focal lysis;
- thymic pathology in these patients resembles that in ‘seropositive’ MG patients with lymphocytic infiltrates, germinal centres and myoid cells showing deposits of complement.
Department of Neuroscience, Medical School of the University of Pennsylvania, Pennsylvania, PA, USA
E-mail: jslkk{at}mail.med.upenn.edu
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References |
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 Top References |
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Barrett-Jolley R, Byrne N, Vincent A, Newsom-Davies J. Plasma from patients with seronegative myasthenia gravis inhibit nAChR responses in the TE671/RD cell line. Pflugers Arch (1994) 428:492–8.[CrossRef][Web of Science][Medline]
Bufler J, Pitz R, Czep M, Wick M, Franke C. Purified IgG from seropositive and seronegative patients with mysasthenia gravis reversibly blocks currents through nicotinic acetylcholine receptor channels. Ann Neurol (1998) 43:458–64.[CrossRef][Web of Science][Medline]
Engel A, Hohlfeld R. Acquired autoimmune myasthenia gravis. In: Myology—Engel A, Franzini-Armstrong C, eds. (2004) 3rd. New York: McGraw-Hill. 1755–90.
Hoch W, McConville J, Helms S, Newsom-Davis J, Melms A, Vincent A. Auto-antibodies to the receptor tyrosine kinase MuSK in patients with myasthenia gravis without acetylcholine receptor antibodies. Nat Med (2001) 7:365–8.[CrossRef][Web of Science][Medline]
Kalamida D, Poulas K, Avramopoulou V, Fostieri E, Lagoumintzis G, Lazaridis K, et al. Muscle and neuronal nicotinic acetylcholine receptors. Structure, function and pathogenicity. FEBS J. (2007) 274:3799–845.[CrossRef][Medline]
Kuryatov A, Luo J, Cooper J, Lindstrom J. Nicotine acts as a pharmacological chaperone to up-regulate human 4β2 acetylcholine receptors. Mol Pharmacol (2005) 68:1839–51.
Lindstrom J. Acetylcholine receptors and myasthenia. Muscle Nerve (2000) 23:453–77.[CrossRef][Web of Science][Medline]
Lindstrom J, Einarson B, Lennon V, Seybold M. Pathological mechanisms in experimental autoimmune myasthenia gravis. I. Immunogenicity of syngeneic muscle acetylcholine receptor and quantitative extraction of receptor and antibody-receptor complexes from muscles of rats with experimental automimmune myasthenia gravis. J Exp Med (1976a) 144:726–38.
Lindstrom J, Luo J, Kuryatov A. MG and the tops and bottoms of AChRs. Ann NY Acad Sci. (2008) in press.
Lindstrom J, Seybold M, Lennon V, Whittingham S, Duane D. Antibody to acetylcholine receptor in myasthenia gravis. Prevalence, clinical correlates, and diagnostic value. Neurology (1976b) 26:1054–9.
Patrick J, Lindstrom J. Autoimmune response to acetylcholine receptor. Science (1973) 180:871–2.
Vincent A, Lang B, Kleopa K. Autoimmune channelopathies and related neurological disorders. Neuron (2006) 52:123–38.[CrossRef][Web of Science][Medline]
Vincent A, Leite M. Neuromuscular junction autoimmune disease: muscle specific kinase antibodies and treatments for myasthenia gravis. Curr Opin Neurol (2005) 18:519–25.[Web of Science][Medline]
Yamamoto T, Vincent A, Ciulla TA, Lang B, Johnston I, Newsom-Davis J. Seronegative myasthenia gravis: a plasma factor inhibiting agonist-induced acetylcholine receptor function copurifies with IgM. Ann Neurol (1991) 30:550–7.[CrossRef][Web of Science][Medline]
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