Rapid Flow Cytometry-Based Assay for the Functional Classification of MEFV Variants
Rapid Flow Cytometry-Based Assay for the Functional Classification of MEFV Variants
复制标题
DOI：
10.1007/s10875-021-01021-7
复制
发表时间：
2021-03-17
影响因子：
9.1
通讯作者：
Nishikomori, Ryuta
中科院分区：
文献类型：
--
作者：
Honda, Yoshitaka;Maeda, Yukako;Nishikomori, Ryuta
目的致病性MEFV变异体可引起芘相关性自身炎症性疾病（PAAD），包括家族性地中海热（FMF）、FMF样疾病和芘相关性自身炎症伴嗜中性粒细胞性皮肤病（PAAND）。PAAD的诊断是通过临床表型和遗传分析建立的。然而，大多数MEFV变体的致病性仍然存在争议，因为它们尚未进行功能评估。本研究旨在建立和验证一种新的功能检测方法来评估MEFV变异株的致病性。方法我们用32种MEFV变体转染THP-1单核细胞，并分析它们在有或没有艰难梭菌毒素A（TcdA）或UCN-01刺激的情况下对细胞死亡的影响。这些变体使用分层聚类分析进行分类。从三名健康对照和两名具有新型纯合MEFVP 257 L变体的患者获得巨噬细胞，用于使用基于细胞的测定和基于新型THP-1的测定比较IL-1 β分泌。结果疾病相关的MEFV变异体诱导THP-1中不同程度的自发或TcdA/UCN-01诱导的细胞死亡。细胞死亡是半胱天冬酶-1依赖性的，并伴随着ASC斑点形成和IL-1 β分泌，表明致病性MEFV变体在该测定中诱导异常的pyrin炎性小体活化和随后的pyroptotic细胞死亡。表现出不同应答特征的MEFV变体（n = 32）被分为6个簇，其显示出与临床表型的良好相关性。关于MEFVP 257 L变体的致病性，基于细胞的测定和基于THP-1的测定之间的结果是一致的。结论该方法可快速、全面地评价MEFV变异株的致病性，并有助于PAAD亚型的精确定义。
Purpose 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. Methods 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 MEFVP257L variant, for comparison of IL-1 beta secretion using a cell-based assay and a novel THP-1-based assay. Results 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-1 beta 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 MEFVP257L variants, the results were consistent between the cell-based assay and the THP-1-based assay. Conclusion Our assay facilitates a rapid and comprehensive assessment of the pathogenicity of MEFV variants and contributes to a refined definition of PAAD subtypes.