Potjewijd Judith, Koenen Hans J P M, van der Made Caspar I, van Rijssen Esther, He Xuehui, Ysermans Renee, Ungethum Liset, Theunissen Ruud, Schurgers Leon J, Damoiseaux Jan, van Paassen Pieter, Smeets Ruben L
Department of Clinical and Experimental Immunology, Maastricht University Medical Center, Maastricht, The Netherlands.
Department of Laboratory Medicine, Laboratory of Medical Immunology, Radboud University Medical Center, Nijmegen, the Netherlands.
Clin Exp Immunol. 2025 Jul 25. doi: 10.1093/cei/uxaf048.
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.
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.
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.
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.
TNFAIP3基因杂合功能缺失突变导致A20单倍体不足（HA20）。A20蛋白是NF-κB信号通路的负反馈调节因子。传统上，HA20与白塞病样症状相关，然而，最近的研究结果表明，它也可能表现为更广泛的自身免疫性疾病。本研究在一个荷兰家族中引入了一种新的TNFAIP3变体，主要表现为多自身免疫性而非自身炎症特征。
我们评估了一个患有自身免疫症状的荷兰家族中的两名患者。全外显子组测序（WES）确定了基因变体。对患者外周血单个核细胞（PBMC）进行免疫印迹分析，并使用转染的HEK293T细胞进行过表达模型评估A20表达。磷酸化流式细胞术分析了NF-κB、STAT和mTOR通路中关键信号分子的磷酸化情况。细胞培养上清液中的细胞因子水平评估了炎症反应。
在TNFAIP3中鉴定出一种新的杂合c.608T>G（p.Leu203Arg）变体，影响OTU结构域。这种错义A20变体在HEK293T中的过表达增强了NF-κB信号通路，表现为TRAF6表达增加和IκBα磷酸化。功能分析显示患者PBMC中A20表达降低，NF-κB、STAT1和mTOR通路磷酸化增加，促炎细胞因子产生升高。这些分子改变表明免疫调节紊乱导致了自身免疫表型。
导致HA20的新型TNFAIP3变体的发现扩大了临床谱，包括主要的自身免疫表现。除了NF-κB和STAT1激活外，我们还发现了mTOR通路激活，为A20的功能和自身免疫进展提供了新的线索。此外，mTOR通路的参与也提供了新的治疗可能性。
