Department of Pediatrics, Faculty of Medicine, Kyoto University Graduate School of Medicine, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, 606-8507, Japan.
Department of Pediatrics, Tenri Hospital, Tenri, Japan.
J Clin Immunol. 2021 Aug;41(6):1187-1197. doi: 10.1007/s10875-021-01021-7. Epub 2021 Mar 17.
Pathogenic MEFV variants cause pyrin-associated autoinflammatory diseases (PAADs), which include familial Mediterranean fever (FMF), FMF-like disease, and pyrin-associated autoinflammation with neutrophilic dermatosis (PAAND). The diagnosis of PAADs is established by clinical phenotypic and genetic analyses. However, the pathogenicity of most MEFV variants remains controversial, as they have not been functionally evaluated. This study aimed to establish and validate a new functional assay to evaluate the pathogenicity of MEFV variants.
We transfected THP-1 monocytes with 32 MEFV variants and analyzed their effects on cell death with or without stimulation with Clostridium difficile toxin A (TcdA) or UCN-01. These variants were classified using hierarchical cluster analysis. Macrophages were obtained from three healthy controls and two patients with a novel homozygous MEFV variant, for comparison of IL-1β secretion using a cell-based assay and a novel THP-1-based assay.
Disease-associated MEFV variants induced variable degrees of spontaneous or TcdA/UCN-01-induced cell death in THP-1. Cell death was caspase-1 dependent and was accompanied by ASC speck formation and IL-1β secretion, indicating that pathogenic MEFV variants induced abnormal pyrin inflammasome activation and subsequent pyroptotic cell deaths in this assay. The MEFV variants (n = 32) exhibiting distinct response signatures were classified into 6 clusters, which showed a good correlation with the clinical phenotypes. Regarding the pathogenicity of MEFV variants, the results were consistent between the cell-based assay and the THP-1-based assay.
Our assay facilitates a rapid and comprehensive assessment of the pathogenicity of MEFV variants and contributes to a refined definition of PAAD subtypes.
致病性 MEFV 变异导致 pyrin 相关自身炎症性疾病（PAAD），包括家族性地中海热（FMF）、FMF 样疾病和 pyrin 相关自身炎症伴中性粒细胞皮肤病（PAAND）。PAAD 的诊断通过临床表型和遗传分析确立。然而，由于大多数 MEFV 变异尚未经过功能评估，其致病性仍存在争议。本研究旨在建立和验证一种新的功能测定法，以评估 MEFV 变异的致病性。
我们将 32 种 MEFV 变异体转染至 THP-1 单核细胞中，并在有无艰难梭菌毒素 A（TcdA）或 UCN-01 刺激的情况下分析其对细胞死亡的影响。采用层次聚类分析对这些变体进行分类。从 3 名健康对照者和 2 名携带新型纯合 MEFV 变异体的患者中获取巨噬细胞，比较细胞基测定法和新型 THP-1 基测定法中 IL-1β 的分泌。
与疾病相关的 MEFV 变异体在 THP-1 中诱导自发或 TcdA/UCN-01 诱导的细胞死亡，其程度不同。细胞死亡依赖于半胱天冬酶-1，伴随着 ASC 斑点形成和 IL-1β 的分泌，表明致病性 MEFV 变异体在此测定中诱导异常的 pyrin 炎性小体激活和随后的细胞焦亡。表现出不同反应特征的 MEFV 变异体（n=32）被分为 6 个簇，与临床表型有很好的相关性。关于 MEFV 变异体的致病性，细胞基测定法和 THP-1 基测定法的结果一致。
我们的测定法促进了对 MEFV 变异体致病性的快速和全面评估，并有助于对 PAAD 亚型进行更精细的定义。
