A novel missense variant in TNFAIP3 associated with autoimmunity reveals the contribution of STAT1/ mTOR pathways.

Introduction: Heterozygous loss-of-function mutations in the TNFAIP3 gene lead to A20 haploinsufficiency (HA20). A20 protein is a negative feedback regulator of NF-κB signaling. Traditionally, HA20 is associated with Behçet's disease-like symptoms, however, recent findings suggest it may also manifest with a broader array of autoimmune diseases. This study introduces a novel TNFAIP3 variant within a Dutch family, predominantly exhibiting polyautoimmunity rather than autoinflammatory characteristics.

Methods: We evaluated two patients from a Dutch family with autoimmune symptoms. Whole-exome sequencing (WES) identified genetic variants. Immunoblotting on peripheral blood mononuclear cells (PBMCs) from the patients and an overexpression model using transfected HEK293T cells assessed A20 expression. Phosphoflow cytometry analyzed phosphorylation of key signaling molecules in the NF-κB, STAT and mTOR pathways. Cytokine levels in cell culture supernatants evaluated inflammatory responses.

Results: A novel heterozygous c.608T>G (p.Leu203Arg) variant in TNFAIP3 was identified, affecting the OTU domain. Overexpression of this missense A20 variant in HEK293T enhanced NF-κB signaling, reflected by increased TRAF6 expression and IκBα phosphorylation. Functional assays revealed reduced A20 expression in patient PBMCs, increased NF-κB, STAT1 and mTOR pathway phosphorylation, and elevated pro-inflammatory cytokine production. These molecular alterations suggest disrupted immune regulation contributing to the autoimmune phenotype.

Conclusion: The discovery of a novel TNFAIP3 variant contributing to HA20 expands the clinical spectrum to include predominant autoimmune manifestations. In addition to NF-κB and STAT1 activation, we discovered mTOR pathway activation, shedding new light on A20's function and progression toward autoimmunity. Furthermore, the involvement of mTOR pathway also provides new therapeutic possibilities.