Utilising ASC Speck Assay to interrogate the pathogenicity of MEFV variants

Abstract

Objective: To develop a rapid functional assay to validate variants of uncertain significance (VUS) in the MEFV gene. Methods: Overactivity of the pyrin inflammasome pathway and ASC speck oligomerisation in response to stimulation with low concentration of Clostridium difficile toxin A was directly visualised by immunofluorescence microscopy. A semi-automated machine learning algorithm was developed to count cells and ASC specks. Results: The semi-automated ASC speck assay is able to discriminate between healthy controls and patients with Familial Mediterranean Fever (FMF) and pyrin inflammasome overactivity with high sensitivity. It is also able to discriminate pyrin inflammasome overactivity from other autoinflammatory disease controls with high specificity. Conclusion: The semi-automated ASC speck assay may be a useful test to functionally validate VUS in the MEFV gene and screen for pyrin inflammasome overactivity.