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

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.