A semi-automated ASC speck assay to evaluate pyrin inflammasome activation

Pei Dai; Oliver Skinner; Xufeng Lin; Aiden Telfser; Stephanie Ruiz-Diaz; Rohit G. Saldanha; Katie Frith; Ming Wei Lin; Kahn Preece; Paul E. Gray; Alberto Pinzon-Charry; Anna Sullivan; Stephen Adelstein; Winnie W.Y. Tong; Matthew J.S. Parker; Laila Girgis; Brynn Wainstein; Samar Ojami; Elissa K. Deenick; Leonard D. Goldstein; Michael J. Rogers; Tri Giang Phan

doi:10.1002/cti2.70054

A semi-automated ASC speck assay to evaluate pyrin inflammasome activation

Pei Dai
, Oliver Skinner
, Xufeng Lin
, Aiden Telfser
, Stephanie Ruiz-Diaz
, Rohit G. Saldanha
, Katie Frith
, Ming Wei Lin
, Kahn Preece
, Paul E. Gray
, Alberto Pinzon-Charry
, Anna Sullivan
, Stephen Adelstein
, Winnie W.Y. Tong
, Matthew J.S. Parker
, Laila Girgis
, Brynn Wainstein
, Samar Ojami
, Elissa K. Deenick
, Leonard D. Goldstein

Michael J. Rogers, Tri Giang Phan

School of Medicine

Garvan Institute of Medical Research
University of New South Wales
Nepean Hospital
University of Melbourne
Sydney Children's Hospital
Bankstown-Lidcombe Hospital
Westmead Hospital
Westmead Millennium Institute for Medical Research
The University of Sydney
Hunter New England Health
Children’s Health Queensland
Royal Prince Alfred Hospital
St. Vincent's Hospital Sydney
Monash Health
Monash University

Research output: Contribution to journal › Article › peer-review

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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 concentrations of Clostridium difficile toxin A was directly visualised by immunofluorescence microscopy. A semi-automated 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.

Original language	English
Article number	e70054
Number of pages	9
Journal	Clinical and Translational Immunology
Volume	14
Issue number	10
DOIs	https://doi.org/10.1002/cti2.70054
Publication status	Published - 2025

Keywords

ASC speck
functional testing
inflammasome
MEFV
pyrin
VUS

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Dai, P., Skinner, O., Lin, X., Telfser, A., Ruiz-Diaz, S., Saldanha, R. G., Frith, K., Lin, M. W., Preece, K., Gray, P. E., Pinzon-Charry, A., Sullivan, A., Adelstein, S., Tong, W. W. Y., Parker, M. J. S., Girgis, L., Wainstein, B., Ojami, S., Deenick, E. K., ... Phan, T. G. (2025). A semi-automated ASC speck assay to evaluate pyrin inflammasome activation. Clinical and Translational Immunology, 14(10), Article e70054. https://doi.org/10.1002/cti2.70054

@article{670540f7e11246888aee29cbecd985c3,

title = "A semi-automated ASC speck assay to evaluate pyrin inflammasome activation",

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 concentrations of Clostridium difficile toxin A was directly visualised by immunofluorescence microscopy. A semi-automated 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.",

keywords = "ASC speck, functional testing, inflammasome, MEFV, pyrin, VUS",

author = "Pei Dai and Oliver Skinner and Xufeng Lin and Aiden Telfser and Stephanie Ruiz-Diaz and Saldanha, \{Rohit G.\} and Katie Frith and Lin, \{Ming Wei\} and Kahn Preece and Gray, \{Paul E.\} and Alberto Pinzon-Charry and Anna Sullivan and Stephen Adelstein and Tong, \{Winnie W.Y.\} and Parker, \{Matthew J.S.\} and Laila Girgis and Brynn Wainstein and Samar Ojami and Deenick, \{Elissa K.\} and Goldstein, \{Leonard D.\} and Rogers, \{Michael J.\} and Phan, \{Tri Giang\}",

year = "2025",

doi = "10.1002/cti2.70054",

language = "English",

volume = "14",

journal = "Clinical and Translational Immunology",

issn = "2050-0068",

publisher = "John Wiley \& Sons",

number = "10",

}

Dai, P, Skinner, O, Lin, X, Telfser, A, Ruiz-Diaz, S, Saldanha, RG, Frith, K, Lin, MW, Preece, K, Gray, PE, Pinzon-Charry, A, Sullivan, A, Adelstein, S, Tong, WWY, Parker, MJS, Girgis, L, Wainstein, B, Ojami, S, Deenick, EK, Goldstein, LD, Rogers, MJ & Phan, TG 2025, 'A semi-automated ASC speck assay to evaluate pyrin inflammasome activation', Clinical and Translational Immunology, vol. 14, no. 10, e70054. https://doi.org/10.1002/cti2.70054

TY - JOUR

T1 - A semi-automated ASC speck assay to evaluate pyrin inflammasome activation

AU - Dai, Pei

AU - Skinner, Oliver

AU - Lin, Xufeng

AU - Telfser, Aiden

AU - Ruiz-Diaz, Stephanie

AU - Saldanha, Rohit G.

AU - Frith, Katie

AU - Lin, Ming Wei

AU - Preece, Kahn

AU - Gray, Paul E.

AU - Pinzon-Charry, Alberto

AU - Sullivan, Anna

AU - Adelstein, Stephen

AU - Tong, Winnie W.Y.

AU - Parker, Matthew J.S.

AU - Girgis, Laila

AU - Wainstein, Brynn

AU - Ojami, Samar

AU - Deenick, Elissa K.

AU - Goldstein, Leonard D.

AU - Rogers, Michael J.

AU - Phan, Tri Giang

PY - 2025

Y1 - 2025

N2 - 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 concentrations of Clostridium difficile toxin A was directly visualised by immunofluorescence microscopy. A semi-automated 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.

AB - 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 concentrations of Clostridium difficile toxin A was directly visualised by immunofluorescence microscopy. A semi-automated 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.

KW - ASC speck

KW - functional testing

KW - inflammasome

KW - MEFV

KW - pyrin

KW - VUS

UR - https://www.scopus.com/pages/publications/105019703246

U2 - 10.1002/cti2.70054

DO - 10.1002/cti2.70054

M3 - Article

AN - SCOPUS:105019703246

SN - 2050-0068

VL - 14

JO - Clinical and Translational Immunology

JF - Clinical and Translational Immunology

IS - 10

M1 - e70054

ER -

A semi-automated ASC speck assay to evaluate pyrin inflammasome activation

Abstract

Keywords

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