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

Abstract

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.

Keywords: A20 haploinsufficiency; NF-κB; STAT1 and mTOR pathway; TNFAIP3; polyautoimmunity.

Graphical Abstract

Figure 1:

PEDIGREE of the family (A), P1 mother and P2 daughter. Brother of P1 passed away at the age of 40, had been residing abroad, and had a known medical history of rheumatism and thyroid disease. (B) The locations of the missense variants on the domain structure of the A20 protein. The panel includes all previously reported missense mutations and the variant evaluated in this study [8, 12].

Figure 2:

reduced A20 expression in patient-derived PBMCs and in a mutant overexpression model. (A) Immunoblot analyses of A20 and β-actin in PBMCs before and after TNFα stimulation. The expression levels of A20 in D001 (patient 1) and D002 (patient 2) were reduced compared to HCs and L001 (CVID patient control with TNFAIP3 LoF mutation c.1309del (p.Ala437Profs)). A20 positive control: PBMC lysate with high A20 expression. A20 negative control: granulocyte lysate. Exposure times: 20 minutes for A20, 10 seconds for β-actin. (B) Immunoblot of HEK293T cells transfected with wild-type (WT) or mutant (Mut) A20 constructs, or with empty vector (EV), with and without TNFα stimulation. Cells expressing the mutant construct show reduced A20 levels and increased expression of TRAF6 and phosphorylated IκBα (phospho-IκBα), indicating enhanced NF-κB pathway activation. β-actin was used as a loading control. Exposure times: 30 seconds for A20, 3 seconds for β-actin, 120 seconds for TRAF6 and 900 seconds for phosphorylated IκBα.

Figure 3:

increased phosphorylation of NF-κB and IκBα in patient T cells. Flow cytometry analysis showed depletion of CD19 + B cells in P1 (A). Phosphoflow cytometry of p65 NF-κB (B) and IκBα (C) in CD4+, CD8 + and CD14 + gated cells (indicated above histograms) from HA20 patients 1 (P1, mother) and 2 (P2, daughter) and healthy control (HC). The mean fluorescence intensity (MFI) is depicted in each histogram. SI, stimulation index.

Figure 4:

increased phosphorylation of IκBα in stimulated patient B cells. Flow cytometry analysis (A) showed the constitution of CD19 + B cells in P1. Phosphoflow cytometry of CD19 + B cells (indicated above histograms) of HA20 patient 1 (P1, mother) and 2 (P2, daughter) and age-matched healthy controls (H1, H2). The mean fluorescence intensity (MFI) is depicted in each histogram. SI: stimulation index.

Figure 5:

concentration of innate cytokines in culture supernatants of in vitro stimulated peripheral blood mononuclear cells. Isolated PBMCs were stimulated for 24 hours with poly I:C (10 μg/mL) and LPS (10 ng/mL) or left unstimulated in normal RPMI medium. In addition, cells were treated with the JAK inhibitor tofacitinib, p38 inhibitor or vehicle (0.1% DMSO). Concentrations of TNFα (A) and IL-1β (B) were measured in the culture supernatants using ELISA.

Figure 6:

increased pro-inflammatory cytokines in patient serum. Serum profiling using multiplex Luminex technology of HA20 patients 1 (P1, mother) and 2 (P2, daughter) versus healthy controls. Log10-transformed data are shown.

Figure 7:

increased baseline phosphorylation of STAT1, mTOR, and p70S6K in patients. Phosphoflow cytometry of (A) STAT1, (B) mTOR and (C) p70S6K in CD4+ and CD8+ T cells, CD19+ B cells, CD14+ monocytes and CD56+ NK cells (indicated above histograms) of HA20 patients 1 (P1, mother) and 2 (P2, daughter) and age-matched healthy controls (H1, H2). The mean fluorescence intensity (MFI) is depicted in each histogram. P, patient; H, healthy control.