Pei Dai^1,2,3, Oliver Skinner^4,5, Xufeng Lin⁶, Aiden Telfser¹, Stephanie Ruiz-Diaz¹, Rohit G. Saldanha^7,8,9, Katie Frith^9,10, Ming-Wei Lin^11,12,13,14, Kahn Preece¹⁵, Paul E. Gray^10,16, Alberto Pinzo¹⁷, Anna Sullivan¹⁷, Stephen Adelstein^18,19, Winnie W. Y. Tong^2,20, Matthew J.S. Parker^19,21, Laila Girgis^2,22, Brynn Wainstein^8,9, Samar Ojami^23,24,25, Elissa K. Deenick^1,26, Leonard D. Goldstein^2,6, Michael J. Rogers⁴, Tri Giang Phan<sup>1,2

1</sup>Precision Immunology Program, Garvan Institute of Medical Research, Sydney, NSW, Australia
²St Vincent’s Healthcare Clinical Campus, School of Clinical Medicine, Faculty of Medicine and Health, UNSW Sydney, Sydney, NSW, Australia
³Department of Immunology, Nepean Hospital, Sydney, NSW, Australia
⁴Immune Biotherapies Program, Garvan Institute of Medical Research, Sydney, NSW, Australia
⁵Department of Microbiology and Immunology, The University of Melbourne at The Peter Doherty Institute for Infection and Immunity, Melbourne, VIC, Australia
⁶Computational Biology Group, Data Science Platform, Garvan Institute of Medical Research, Sydney, NSW, Australia.
⁷Department of General Paediatrics, Sydney Children’s Hospital, Sydney, NSW, Australia
⁸Department of Paediatrics, Bankstown-Lidcombe Hospital, Sydney, NSW, Australia
⁹Randwick Clinical Campus, School of Clinical Medicine, Faculty of Medicine and Health, UNSW Sydney, Sydney, NSW, Australia
¹⁰Department of Immunology, Sydney Children’s Hospital, Sydney, NSW, Australia
¹¹Department of Clinical Immunology, Westmead Hospital, Sydney, NSW, Australia
¹²Department of Immunopathology, ICPMR, Westmead Hospital, Sydney, NSW, Australia
¹³Centre of Immunology and Allergy Research, Westmead Institute of Medical Research, Sydney, NSW, Australia
¹⁴Western Clinical School, Faculty of Medicine and health, University of Sydney, Sydney, NSW, Australia
¹⁵Paediatric Immunology Department, John Hunter Children’s Hospital, Newcastle, NSW, Australia
¹⁶Western Sydney University School of Medicine, Sydney, NSW, Australia
¹⁷Queensland Children’s Hospital, Brisbane, QLD, Australia
¹⁸Department of Clinical Immunology and Allergy and Institute of Academic Medicine, Royal Prince Alfred Hospital, Sydney, NSW, Australia
¹⁹Central Clinical School, Faculty of Medicine and Health, University of Sydney, Sydney, NSW, Australia
²⁰HIV and Immunology Department, St Vincent’s Hospital, Sydney, NSW Australia
²¹Department of Rheumatology, Royal Prince Alfred Hospital, Sydney, NSW, Australia
²²Department of Rheumatology, St Vincent’s Hospital, Sydney, NSW Australia
²³Monash Pathology, Monash Health, Melbourne, VIC, Australia
²⁴Monash Infectious Diseases, Monash Health, Melbourne, VIC, Australia
²⁵Department of Medicine, Monash University, Melbourne, VIC, Australia
²⁶Kirby Institute, Faculty of Medicine and Health, UNSW Sydney, Sydney, NSW Australia

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.