[Clinical phenotype and immunological features of a patient with A20 haploinsufficiency].

Objective: To explore the clinical phenotype, immunological features, pathogenesis and gene variation of a case with A20 haploinsufficiency (HA20). Methods: A patient diagnosed with tumor necrosis factor α-induced protein 3 (TNFAIP3) mutated HA20 was admitted into Shenzhen Children's Hospital in May,2019.The clinical data was analyzed. Flow cytometry was used to detect the patient's peripheral blood lymphocyte subsets, and also, the percentage of follicular helper T cell (TFH) cells in the patient and thirteen healthy controls. After the construction of empty vector, wild-type and mutant plasmid vectors, a wild-type or mutant overexpression system of the TNFAIP3 gene was established in 293T cells and Hela cells. Then, the expression level of A20 in 293T cells and the expression of inhibitor K binding α (IKBα) in green fluorescent protein (GFP)+Hela cells before and after tumor necrosis factor α (TNF-α) stimulation were measured, to verify the pathogenicity of this variation. Results: A 5 years and 11 months old boy, presented with recurrent oral ulcer, abdominal pain, joint swelling and arthralgia. Oral ulcer, chronic skin rashes, knee joint swelling were observed. The levels of inflammatory markers were increased. Colonoscopy showed congestion of mucosa and multiple ulcers in terminal ileum and ileocecus. The absolute number of naive B cells was 124×10(6) cells/L (reference range 147×10(6)-431×10(6) cells/L), accounting for 0.430 of the total B cells (reference range 0.484-0.758). Compared to healthy controls (0.016-0.071), the percentage of TFH cells in CD4(+)T cells was much lower (0.008).A heterozygous mutation of TNFAIP3 gene (c.909_913 del, p.L303fs) was identified by genetic analysis. In vitro study showed that truncated A20 protein was expressed in TNFAIP3 mutant overexpressed 293T cells, which verified the pathogenicity of this variation. Besides, after TNF-α stimulation, the degradation rate of IkBα protein in mutant overexpressed Hela cells (35%) was between the other two groups (15% in the wild-type group and 57% in the non-loaded group). Conclusions: This case with HA20 due to a de novo TNFAIP3 gene mutation presents with early onset Behcet-like autoinflammatory syndrome. This variation leads to expression of truncated A20 protein, enhanced degradation of IkBα, and further activation of nuclear factor κB signaling pathway.目的： 探讨A20单倍剂量不足（HA20）患儿的临床特征、肿瘤坏死因子α诱导蛋白3（TNFAIP3）基因突变特点、免疫表型及免疫学致病机制。 方法： 回顾性分析2019年5月深圳市儿童医院收治的1例TNFAIP3基因突变所致HA20患儿的临床特征；运用流式细胞术进行患儿外周血精细淋巴细胞分型检测以及患儿和13名健康对照滤泡辅助性T细胞（TFH）比例检测；构建空载体、野生型及突变型质粒载体；在293T和Hela细胞系中建立TNFAIP3基因野生型或突变体过表达体系，分别检测A20蛋白和肿瘤坏死因子α（TNF-α）刺激前后绿色荧光蛋白（GFP）阳性Hela细胞中抑制性卡巴蛋白α（IKBα）表达水平，以验证该变异位点的致病性。 结果： 患儿男，5岁11月龄，以反复口腔溃疡、腹痛、关节肿痛为主要表现，阳性体征包括口腔溃疡、双下肢暗红色陈旧性皮疹、双膝关节肿胀伴压痛，疾病活动期时炎症指标升高，肠镜显示回肠末及回盲部黏膜充血肿胀，见多发溃疡；基因测序分析显示TNFAIP3基因杂合突变[c.909_913 del(p.L303fs）]；外周血淋巴细胞精细分型显示初始B细胞124×10(6)个/L(参考范围147×10(6)~431×10(6)个/L)，占总B细胞的0.430（参考范围0.484~0.758）;TFH占CD4(+)T细胞0.008(健康对照组范围为0.016~0.071)。体外实验显示突变体过表达293T和Hela细胞中均存在A20截短型蛋白表达，TNF-α刺激后TNFAIP3基因野生型过表达组IkBα蛋白降解率为15%，空载体过表达组Hela细胞中IkBα蛋白降解率较高，为57%，而突变体过表达组IkBα蛋白降解率介于两组之间，为35%。 结论： 该HA20患儿以早发性白塞病样自身炎症反应综合征为主要临床特征，存在TNFAIP3基因新发移码突变。该突变引起A20截短型蛋白表达和剂量相对不足，导致IkBα蛋白降解增加，核因子κB信号通路活化增强。.