Background/Purpose: Using whole exome sequencing, we discovered a de novo heterozygous germline mutation in MYD88 (myeloid differentiation primary response 88) (c.666T>G, p.S222R) in a child with destructive small-to-medium joint polyarticular juvenile arthritis (JA) exhibiting persistent neutrophil-predominant synovial infiltrates and rash. MyD88 is a critical adaptor protein that connects Toll-like and IL-1 receptor signaling to activation of NF-κB. Germline loss-of-function mutations in MyD88 cause immunodeficiency, while somatic gain-of-function mutations have been linked to lymphoma. We asked whether the MyD88 S222R mutation causes gain-of-function effects that could contribute to the development of destructive arthritis.

Methods: The patient was evaluated for peripheral blood immunophenotype, cytokine and chemokine production, and monocyte osteoclastogenesis, compared to family and other healthy controls. Functional studies in monocytes and dermal fibroblasts included gene/protein expression, quantitation of neutrophil chemotaxis, and siRNA-mediated knockdown of MyD88 and p65. Wild type (WT) or S222R MyD88-AU1 fusion proteins were re-expressed in MyD88-deficient THP-1 cells. NF-κB activity was measured via p65 subunit phosphorylation and using a reporter system. Effects on MyD88 structure were predicted via molecular dynamics modeling, and mechanistic studies were performed to assess the capacity of S222R MyD88 to oligomerize, which is necessary for signaling, using IF microscopy and proximity ligation assay (PLA).

Results: MyD88 S222R results in increased NF-κB p65 phosphorylation and NF-κB reporter activity in THP-1 cells compared to WT MyD88. IF microscopy and PLA demonstrated increased MyD88-S222R oligomerization compared to WT MyD88, indicating a behavior similar to MyD88-L265P, which is the most common lymphoma-associated MyD88 gain-of-function mutation. Immunophenotyping showed a persistent absence of CD16 on monocytes, an expansion of CD4+ Th17 T cells, and the presence of a previously uncharacterized CD123+CD11c+ dendritic cell population, as well as markedly increased basal and stimulated p-STAT3 in monocytes and T and B lymphocytes in the patient. Peripheral monocytes exhibited baseline interferon and chemokine gene expression signatures, while monocyte-derived osteoclasts exhibited enhanced survival and were morphologically larger than those cultured from a control. Fibroblasts exhibited significantly greater baseline expression of CXCL chemokines compared to controls, which abated upon MyD88 or p65 knockdown.

Conclusion: This is the first description of a de novo germline MyD88 mutation (S222R) associated with severe polyarticular JA. We demonstrate clear gain-of-function effects of the S222R mutation using THP-1 cells which are consistent with biologic effects in hematopoietic and non-hematopoietic cells derived from the patient, and likely to be a consequence of increased oligomerization. These effects offer plausible mechanisms for neutrophil-predominant, destructive arthritis, and support a role for single gene defects contributing to extreme JA phenotypes.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/severe-juvenile-arthritis-associated-with-a-de-novo-gain-of-function-germline-mutation-in-myd88-3/