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Published on: May 13, 2019
Rapid Flow Cytometry-Based Assay for the Functional Classification of MEFV Variants
Yoshitaka Honda1, Yukako Maeda1, Kazushi Izawa2
1Department of Pediatrics, Faculty of Medicine, Kyoto University Graduate School of Medicine, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, 606-8507, Japan.
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-1β 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-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 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.
Insights
A new functional assay effectively evaluates pathogenic MEFV variants, aiding in diagnosing pyrin-associated autoinflammatory diseases (PAADs) and classifying subtypes. This method clarifies variant pathogenicity for better patient care.
Area of Science:
- Genetics
- Immunology
- Molecular Biology
Background:
- Pathogenic variants in the MEFV gene cause pyrin-associated autoinflammatory diseases (PAADs), including familial Mediterranean fever (FMF).
- Accurate diagnosis relies on clinical and genetic analysis, but the pathogenicity of many MEFV variants remains unconfirmed due to a lack of functional evaluation.
- This uncertainty hinders precise diagnosis and classification of PAAD subtypes.
Purpose of the Study:
- To establish and validate a novel functional assay for assessing the pathogenicity of MEFV variants.
- To correlate functional variant data with clinical phenotypes for improved PAAD classification.
- To provide a reliable tool for evaluating previously uncharacterized MEFV variants.
Main Methods:
- THP-1 monocytes were transfected with 32 MEFV variants and assessed for cell death upon stimulation with TcdA or UCN-01.
- Hierarchical cluster analysis was used to classify variants based on their functional response signatures.
- IL-1β secretion was compared between healthy controls and patients with the MEFV P257L variant using cell-based and THP-1 assays.
Main Results:
- Disease-associated MEFV variants induced varying levels of cell death, dependent on caspase-1 activation, ASC speck formation, and IL-1β secretion.
- The 32 MEFV variants clustered into 6 distinct groups, correlating well with known clinical phenotypes.
- The novel THP-1-based assay demonstrated consistent results with established cell-based methods for evaluating MEFV P257L variant pathogenicity.
Conclusions:
- The developed functional assay provides a rapid and comprehensive method for evaluating MEFV variant pathogenicity.
- This assay aids in refining the classification of pyrin-associated autoinflammatory disease subtypes.
- The findings support improved diagnostic accuracy and personalized treatment strategies for PAAD patients.
