POS0883 EVIDENCE FOR SOMATIC MUTATION IN LYMPHOMA DRIVER GENES AT THE ROOT OF CLONAL EVOLUTION OF PATHOGENIC AUTOANTIBODIES IN MAG NEUROPATHY
Authors: S. Kelly, A. Russell, M. Singh, M. Field, K. Jackson, T. Peters, D. Suan, C. Goodnow
Journal: Annals of the Rheumatic Diseases (2024)

<h3>Background:</h3> The precise mechanism by which autoantibody-producing clones develop in patients with autoimmune diseases is incompletely understood. Somatic mutations in lymphoma driver genes that accrue in self-reactive clones have been described by our group in patients with cryoglobulinaemic vasculitis [1], as an explanation for how these clones overcome the tolerance checkpoints that usually prevent autoimmunity. It is uncertain if this applies to autoimmune diseases more broadly. We sought to test this hypothesis in the autoimmune disease myelin-associated glycoprotein (MAG) neuropathy, in which pathogenic autoantibodies targeting MAG leading to a demyelinating polyneuropathy that is often refractory to immune therapies. Almost all patients harbour IgM kappa B-cell clones and somatic mutations in <i>MYD88</i> [2,3] and <i>CXCR4</i> [3] have been identified in these bulk clonal populations. However, it has not been definitively shown that the expanded, somatically mutated clones are the MAG autoantibody-producing clones, and they have not been interrogated at single cell level, nor have their immunoglobulin sequences been published. <h3>Objectives:</h3> To test the hypothesis that somatic mutations in both immunoglobulin and lymphoma driver genes converge on the same B cell to produce expanded, pathogenic clones in MAG neuropathy. <h3>Methods:</h3> Peripheral blood mononuclear cells from the blood of three MAG neuropathy patients were analysed, and single B-lymphocytes were sorted from IgM kappa expansions, along with control B cells. Immunoglobulin sequences, both mutant and germline-reverted, were expressed and tested for MAG-specificity by ELISA. MAG-specific and control B-cells were then interrogated using single-cell multi-omic analysis for cell surface protein expression, gene expression and gDNA sequences [4,5]. <h3>Results:</h3> Multiple, expanded, IgM kappa clones and subclones were identified in all three patients, and at least one MAG-specific clone was confirmed in each patient. All clonal immunoglobulin sequences exhibited high levels of somatic hypermutation with unique V(D)J gene usage specific to each clone. Germline-reverted sequences demonstrated variable MAG binding. All clones harboured a somatic <i>MYD88</i><sup>L265P</sup> gain-of-function mutation, irrespective of MAG-specificity. Strikingly, this mutation was also present in control, non-clonal B cells in all three patients, albeit at a lower frequency. Two different somatic non-sense mutations in <i>CXCR4</i> were also identified in the MAG-specific clones in two patients. Additionally, there were also somatic mutations in IGLL5 and BTG2 present in some of the clones. There were no differences in gene expression or cell surface protein expression between the clones across all three patients, nor between MAG-positive and MAG-negative clones. Our results reveal unexpectedly diverse clonal evolutionary trees and place <i>MYD88</i><sup>L265P</sup> acquisition at the root of these trees, at or before the processes of V(D)J recombination and V-region hypermutation that conferred antibody binding to MAG. <h3>Conclusion:</h3> In this cohort of MAG neuropathy patients, the first published study of interrogation of this disease at a single-cell level, the acquisition of a mutation in a key lymphoma drive gene occurs early in B-cell development, prior to V(D)J rearrangement or acquisition of self-reactivity. This contrasts with our findings in patients with cryoglobulinaemic vasculitis, suggesting that B cells may traverse more than one route of somatic mutagenesis in their evolution towards pathogenic clones. <h3>REFERENCES:</h3> [1] M. Singh, et al. Cell, 180 (5) (2020), pp. 878-894.e19. [2] Vos JM et al. J Neurol Neurosurg Psychiatry. 2018 Sep;89(9):1007-1009. [3]Allain et al. Haematologica. 2018 May;103(5):e207-e210. [4] I.C. Macaulay, et al. Nat. Methods, 12 (6) (2015), pp. 519-522. [5] Ruff, D.W. et al. Methods Mol Biol. 2022:2386:171-188. <h3>Acknowledgements:</h3> National Health and Medical Research Council (NHMRC) grants; APP2010134 &amp; APP1113904, the UNSW Cellular Genomics Futures Institute. <h3>Disclosure of Interests:</h3> <b>None declared.</b>