PDF(Pubmed)
Abstract：
A20 haploinsufficiency (HA20) is an autoinflammatory disease caused by heterozygous loss-of-function variations in TNFAIP3, the gene encoding the A20 protein. Diagnosis of HA20 is challenging due to its heterogeneous clinical presentation and the lack of pathognomonic symptoms. While the pathogenic effect of TNFAIP3 truncating variations is clearly established, that of missense variations is difficult to determine. Herein, we identified a novel TNFAIP3 variation, p.(Leu236Pro), located in the A20 ovarian tumor (OTU) domain and demonstrated its pathogenicity. In the patients\' primary cells, we observed reduced A20 levels. Protein destabilization was predicted in silico for A20_Leu236Pro and enhanced proteasomal degradation was confirmed in vitro through a flow cytometry-based functional assay. By applying this approach to the study of another missense variant, A20_Leu275Pro, for which no functional characterization has been performed to date, we showed that this variant also undergoes enhanced proteasomal degradation. Moreover, we showed a disrupted ability of A20_Leu236Pro to inhibit the NF-κB pathway and to deubiquitinate its substrate TRAF6. Structural modeling revealed that two residues involved in OTU pathogenic missense variations (i.e. Glu192Lys and Cys243Tyr) establish common interactions with Leu236. Interpretation of newly identified missense variations is challenging, requiring, as illustrated here, functional demonstration of their pathogenicity. Together with functional studies, in silico structure analysis is a valuable approach that allowed us (i) to provide a mechanistic explanation for the haploinsufficiency resulting from missense variations and (ii) to unveil a region within the OTU domain critical for A20 function.
摘要：
A20单倍体功能不全（HA20）是由编码A20蛋白的基因TNFAIP3的杂合功能丧失变异引起的自身炎性疾病。HA20的诊断是具有挑战性的，由于其异质性的临床表现和缺乏病理症状。虽然TNFAIP3截断变异的致病作用是明确的，这种错义的变化是很难确定的。在这里，我们发现了一个新的TNFAIP3变异，p.(Leu236Pro)，位于A20卵巢肿瘤（OTU）域并证明其致病性。在患者的原代细胞中，我们观察到A20水平降低。A20_Leu236Pro在计算机上预测蛋白质不稳定，并通过基于流式细胞术的功能测定在体外证实了增强的蛋白酶体降解。通过将这种方法应用于另一种错义变体的研究，A20_Leu275Pro,迄今为止尚未对其进行功能表征，我们表明，该变体也经历了增强的蛋白酶体降解。此外，我们显示了A20_Leu236Pro抑制NF-κB途径和去泛素化其底物TRAF6的能力被破坏。结构模型显示，参与OTU致病错义变异的两个残基（即Glu192Lys和Cys243Tyr）与Leu236建立了共同的相互作用。对新发现的错义变化的解释具有挑战性，要求,如这里所示，其致病性的功能证明。加上功能研究，计算机结构分析是一种有价值的方法，它使我们（i）能够为错义变异导致的单倍体不足提供机械解释，以及（ii）揭示OTU域内对A20功能至关重要的区域。
