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

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. Here, we describe a novel TNFAIP3 variant in a Dutch family, predominantly presenting with polyautoimmunity rather than autoinflammatory manifestations. We evaluated two patients from a Dutch family with autoimmune symptoms. Whole-exome sequencing (WES) identified a heterozygous c.608T>G (p.Leu203Arg) missense variant in TNFAIP3, located within the OTU domain. Functional analyses included immunoblotting of peripheral blood mononuclear cells (PBMCs) and an overexpression model using transfected HEK293T cells. A20 protein expression was evaluated, while phosphoflow cytometry assessed phosphorylation of key signaling molecules in the NF-κB, STAT and mTOR pathways. Inflammatory cytokine production was measured in cell culture supernatants. Overexpression of this missense A20 variant in HEK293T enhanced NF-κB signaling, reflected by increased TRAF6 expression and IκBα phosphorylation. Patient-derived PBMCs demonstrated reduced A20 expression, increased phosphorylation within the NF-κB, STAT1, and mTOR pathways, and elevated production of pro-inflammatory cytokines. These molecular alterations suggest disrupted immune regulation contributing to the observed autoimmune phenotype. The identification of this 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.