The MOG antibody non-P42 epitope is predictive of a relapsing course in MOG antibody-associated disease

Background Myelin oligodendrocyte glycoprotein (MOG) IgG seropositivity is a prerequisite for MOG antibody-associated disease (MOGAD) diagnosis. While a significant proportion of patients experience a relapsing disease, there is currently no biomarker predictive of disease course. We aim to determine whether MOG-IgG epitopes can predict a relapsing course in MOGAD patients. Methods MOG-IgG-seropositive confirmed adult MOGAD patients were included (n=202). Serum MOG-IgG and epitope binding were determined by validated flow cytometry live cell-based assays. Associations between epitopes, disease course, clinical phenotype, Expanded Disability Status Scale and Visual Functional System Score at onset and last review were evaluated. Results Of 202 MOGAD patients, 150 (74%) patients had MOG-IgG that recognised the immunodominant proline42 (P42) epitope and 115 (57%) recognised histidine103/serine104 (H103/S104). Fifty-two (26%) patients had non-P42 MOG-IgG and showed an increased risk of a relapsing course (HR 1.7; 95% CI 1.15 to 2.60, p=0.009). Relapse-freedom was shorter in patients with non-P42 MOG-IgG (p=0.0079). Non-P42 MOG-IgG epitope status remained unchanged from onset throughout the disease course and was a strong predictor of a relapsing course in patients with unilateral optic neuritis (HR 2.7, 95% CI 1.06 to 6.98, p=0.038), with high specificity (95%, 95% CI 77% to 100%) and positive predictive value (85%, 95% CI 45% to 98%). Conclusions Non-P42 MOG-IgG predicts a relapsing course in a significant subgroup of MOGAD patients. Patients with unilateral optic neuritis, the most frequent MOGAD phenotype, can reliably be tested at onset, regardless of age and sex. Early detection and specialised management in these patients could minimise disability and improve long-term outcomes.

][3] The MOG P42S mutant contained a mutation at position 42, where the Proline substituted for Serine. 3,4 he MOG H103A/S104E mutant consisted of full-length human MOG with the histidine and serine at positions 103 and 104 substituted with alanine (103A) and glutamic acid (104E). 46][7] MOG WT, MOG P42S and MOG H103A/S104E cell surface expression was controlled in each experiment, were similarly high as described. 3The P42 and H103/S104 MOG-IgG epitope statuses were calculated originally using a control cohort as previously described. 3Briefly, MOG-IgG binding to MOG WT, MOG P42S and MOG H103A/S104E was determined by the delta median fluorescence intensity (ΔMFI): MOG WT, or MOG P42S, or MOG H103A/S104E ΔMFI = MOG MFI -CTL MFI.Epitope status of each patient serum was first calculated using control sera to establish a control reference range by calculating the 3SD above and below the control P42 MOG-IgG or H103/S104 MOG-IgG mean.MOG-IgG were assigned as P42 or H103/S104 MOG-IgG (above mean + 3SD) and non-P42 or non-H103/S104 MOG-IgG (below mean + 3SD).Three independent experiments were repeated, and reported samples remained in the same category in at least two of three independent experiments with low inter-assay variability.Then, to remove the need for a control cohort and facilitate global adoption of the method by diagnostic providers, a receiver-operating-characteristic (ROC) analysis was used to determine optimal thresholds for P42 and MOG H103/S104 MOG-IgG binding (online supplemental figure 1A-D).These thresholds were utilised for calculation of epitopes in this study.Flow cytometry data was acquired on the LSRII flow cytometer (BD Biosciences) and analysed using FlowJo v10 (TreeStar) software and Microsoft Excel.

Supplementary Tables
Supplemental Table 1.Severity characteristics of MOGAD patients stratified by P42 epitope serostatus.BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) to the reference epitope status for each patient as determined using a control cohort, and results were highly concordant between analyses methods.(E) The vast majority of patient sera positive for MOG-IgG using a secondary anti-human IgG (H+L) antibody were also positive for MOG-IgG1 using a secondary anti-human IgG1 antibody in a flow cytometry live cell-based assay.(F) Epitope status was determined using a secondary anti-human IgG1 antibody in 45 patient sera that displayed a non-P42 MOG-IgG when using secondary antihuman IgG (H+L) antibody.Most patients (41/45) displayed the same non-P42 epitope when tested with IgG1.Of these 41 patients, 34 (83%) exhibited a relapsing course.Abbreviations: AUC, area under the curve.

Supplemental figure 2. Patient sex and age distribution across MOG-IgG epitopes.
Bar plots showing the number of male and female patients within the MOGAD cohort (n=202) stratified by MOG-IgG P42 (left) and H103/S104 (right) epitope status (A).Boxand-whisker plots showing the distribution of patient ages at the date of sera collection stratified by MOG-IgG P42 (left) and H103/S104 (right) epitope status (B).The time from onset to first sample collection was not different between P42 (median 2.2 months, IQR 0.17 -40.0) and non-P42 MOG-IgG epitope groups (median 5.7 months, IQR 0.16 -59.9), p = 0.278.Abbreviations: F, female; M, male.
BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s)

Supplemental figure 4 .
Supplemental figure3.MOG-IgG epitopes were not associated with severity in MOGAD patients.EDSS (A) and VFSS (B) scores at disease onset (top graphs), last followup date (middle graphs), and difference between onset and last follow up date (delta, bottom graphs) were compared between patients who presented with the four combinations of the MOG-IgG epitopes.There was no significant association between severity scores (delta) and epitope groups (Kruskal-Wallis test).Abbreviations: EDSS, Expanded Disability Status Scale; VFSS, Visual Functional Systems Score.BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) J Neurol Neurosurg Psychiatry doi: 10.1136/jnnp-2023Treatment during the first clinical episode in patients with P42 and non-P42 MOG-IgG.Administration of acute corticosteroid immunotherapy or lack thereof were compared between patients with P42 MOG-IgG (n=39 total; Treated: n=15 BON, n=8 UON, n=4 ON/TM, n=4 TM, n=2 ON NOS, and n=1 Mixed; Untreated: n=4 UON, and n=1 ON NOS) and non-P42 MOG-IgG (n=9 total; Treated: n=3 BON, n=2 UON, n=1 TM; Untreated: n=1 BON, n=1 UON, and n=1 TM).Acute corticosteroid immunotherapy included intravenous pulsed methylprednisolone and oral corticosteroid taper, or intravenous pulsed methylprednisolone only.There was no significant association between acute corticosteroid immunotherapy during the first clinical episode and the two epitope groups (Chi-square and Fisher's exact Test).Abbreviations: T, treated; U, untreated.Supplemental figure 5. Non-P42 MOG-IgG was the strongest predictor of a relapsing course in patients with UON at onset.Univariate Cox Proportional Hazard Model showing the risk of a relapsing course in patients with non-42 MOG-IgG and the same clinical phenotype throughout the course of their MOGAD disease.Reference groups were patients with P42 MOG-IgG with the same clinical phenotype throughout MOGAD course.Abbreviations: BON, bilateral optic neuritis; ON, optic neuritis; UON, unilateral optic neuritis; TM, transverse myelitis.BMJ Publishing Group Limited (BMJ) disclaims all liability and responsibility arising from any reliance Supplemental material placed on this supplemental material which has been supplied by the author(s) J Neurol Neurosurg Psychiatry doi: 10.1136/jnnp-2023both sides of the survival curves.Abbreviations: UON, unilateral optic neuritis.
J Neurol Neurosurg Psychiatry doi: 10.1136/jnnp-2023-332851 -553.a number of patients for whom paired data was available.Supplemental b number of patients with ON, TM, Brain, and Mixed phenotypes for whom the data was available.c MOG-IgG titer was calculated for patient sera collected at onset.