Rapid Flow Cytometry-Based Assay for the Functional Classification of MEFV Variants

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.

Automated summary

This study established and validated a new functional assay to evaluate the pathogenicity of MEFV variants. Disease-associated MEFV variants induced abnormal pyrin inflammasome activation and pyroptotic cell deaths, leading to pyrin-associated autoinflammatory diseases (PAADs). The pathogenicity analysis of MEFVP257L variants showed consistent results between different assay methods.