Autoantibodies in Japanese patients with ocular myasthenia gravis

Abstract Introduction The majority of patients with myasthenia gravis (MG) initially present with ocular symptoms, but it is difficult to predict which cases will remain as ocular MG (OMG) or will progress to generalized MG. Herein we evaluated the serologic profile of Japanese OMG and its relationship with clinical features. Methods Seventy‐three patients with OMG from five Japanese myasthenia gravis (MG) centers were enrolled. Live cell‐based assays (CBAs) were used to determine the presence of autoantibodies (Abs) to clustered adult (2α, β, δ, ε) and fetal (2α, β, δ, γ) acetylcholine receptor (AChR) isoforms, muscle‐specific receptor tyrosine kinase (MuSK), and lipoprotein receptor–related protein‐4 (LRP4). Results Thirty‐four of 73 (46.5%) serum samples were positive for Abs against both the adult‐type and fetal‐type AChR, as expected, but 7 (9.6%) and 2 (2.7%) were positive only for fetal or adult AChR‐Abs, respectively. Four (5.4%) samples were positive for MuSK‐Abs, but two of these also contained antibodies to fetal AChR or LRP4. Twenty‐six (35.6%) samples were seronegative. Discussion Abs against fetal‐specific AChR, MuSK, and LRP4 are found in some patients with OMG. Future studies attempting to predict conversion from ocular symptoms to generalized MG may benefit from measurement of these antibodies.


Funding information
Ministry of Health, Labor and Welfare of Japan (Health and Labor Sciences research grants on Rare and Intractable Diseases); Wellcome Trust, Grant Number: 104079/Z/14/Z (to S.R. I.) Results: Thirty-four of 73 (46.5%) serum samples were positive for Abs against both the adult-type and fetal-type AChR, as expected, but 7 (9.6%) and 2 (2.7%) were positive only for fetal or adult AChR-Abs, respectively. Four (5.4%) samples were positive for MuSK-Abs, but two of these also contained antibodies to fetal AChR or LRP4.
Discussion: Abs against fetal-specific AChR, MuSK, and LRP4 are found in some patients with OMG. Future studies attempting to predict conversion from ocular symptoms to generalized MG may benefit from measurement of these antibodies. the majority initially present with ocular symptoms, and it is difficult to predict which of these cases will remain ocular MG (OMG) or progress to generalized MG (GMG). Acetylcholine receptor antibodies (AChR-Abs) are found in 80% to 90% of GMG and usually around 50% of OMG cases 1,2 ; however, in one Japanese study (that included 22% OMG cases), 79.8% of MG patients had AChR-Abs, whereas 12.9% of GMG and no OMG patients had muscle-specific receptor tyrosine kinase (MuSK)-Abs. 3 Other reports have described MuSK-Abs in individual OMG patients. [4][5][6][7] In human muscle, the AChR subunits largely switch from the fetal type (2α, β, δ, γ) to the adult type (2α, β, δ, ε) by birth. However, γ-subunit expression persists into adulthood in the extraocular muscles 8 and it is possible that OMG patients have antibodies to fetal as well as adult AChRs.
Recently, live cell-based assays (CBAs) have been established to detect autoantibody binding to native clustered AChR, MuSK, and lipoprotein receptor-related protein-4 (LRP4). These live CBAs offer better sensitivity than fixed CBAs or radioimmunoassays (RIAs), [9][10][11] because they detect binding of divalent immunoglobulin G (IgG) antibodies to adjacent native antigens anchored on the cell surface and do not detect binding to irrelevant intracellular epitopes. [12][13][14][15] In this study, we used live CBAs to test MG-associated antibodies, including adult and fetal AChR subtypes, in 73 Japanese OMG patients.

| METHODS
We obtained approval from the ethics committee of Nagasaki Kawatana Medical Center (Nagasaki, Japan) and permission from the ethics committee of each institution that participated in this study. All subjects provided informed consent.

| Statistical analysis
Statistical analysis was performed using GraphPad Prism 8 version 8.2.1 (GraphPad, La Jolla, California). The Mann-Whitney U test was used for analyses according to the age of onset. The significance of statistical differences between subgroups was evaluated using Fisher's exact test. Differences were considered significant at P < .05.

| DISCUSSION
We used CBAs to evaluate the serologic profiles of Japanese OMG patients, 61.6% of whom had undergone a variety of immunotherapies. As expected, 46.6% of the patients were positive for both clustered adult and fetal AChR-Abs, but another 17.8% had antibodies only binding to fetal AChR, adult AChR, or MuSK (2 of the latter also with fetal AChR or LRP4 antibodies).
Most patients with MG have antibodies that bind effectively to solubilized mixtures of adult and fetal AChRs by RIA, but, increasingly, cellbased assays are used to measure antibodies for increased sensitivity and specificity. 13 These CBAs have the advantages that only extracellular epitopes are exposed to the antibodies, the antigen is expressed on the membrane in native confirmation, and high expression of the antigen allows antibodies to bind divalently to adjacent targets, as is highly relevant at synapses such as the neuromuscular junctions. 13 It was surprising to find seven patients with only fetal AChR antibodies, although, in one previous study, 17  antibodies were seen in 12 of 200 (6%) OMG patients. The role of fetal AChR antibodies is poorly understood; unusually, the extraocular muscle fibers have both adult (epsilon subunit containing) and fetal (gamma subunit containing) AChRs, suggesting that the fetal AChR antibodies could be pathogenic, but expression of the epsilon subunit is much higher than the gamma subunit. 18 Moreover, mothers with predominant fetal AChR antibodies do not have severe ocular symptoms and some are asymptomatic. 19 Therefore, an alternative explanation is that relatively low levels of adult AChR-Abs are present initially, but are adsorbed by the adult AChRs in the extraocular and levator muscles, leaving the fetal AChR antibodies in the circulation. In patients with higher levels of antibodies, typical of generalized MG, adsorption would be less likely to deplete adult AChR-Abs.
MuSK-Abs were first described in patients with severe generalized symptoms who often presented with bulbar and respiratory failure, 20 but some present first with ocular symptoms only. In a recent study, 15