SpeckSeq enables high-throughput functional stratification of <i>MEFV</i> variants in autoinflammatory diseases

Variants of uncertain significance (VUS) are a major obstacle in genetic diagnosis, particularly when involving gain-of-function (GoF) mutations that are poorly predicted in silico. MEFV, which encodes the inflammasome sensor pyrin, is mutated in two autoinflammatory diseases, familial Mediterranean fever (FMF) and pyrin-associated autoinflammation with neutrophilic dermatosis (PAAND). Here, we developed SpeckSeq, a method that combines DNA bar-coding, ASC speck-based single-cell sorting and next-generation sequencing to systematically identify hypermorphic MEFV variants in response to different stimuli. SpeckSeq identified 49 GoF mutations separated into two distinct groups containing either PAAND variants or FMF variants. SpeckSeq was validated using patients' cells and supported a reclassification of MEFV variant pathogenicity, leading to novel diagnoses. As a large-scale mutagenesis approach, using human genetics as a guide, SpeckSeq revealed structural and functional pyrin features, including a putative ligand-accommodating cavity in the B30.2 domain. Altogether, SpeckSeq classifies VUS to refine molecular diagnostics and improve our knowledge on the pyrin inflammasome.

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Publication Details

Journal
The Journal of Experimental Medicine
Published
2025-10-29
DOI
https://doi.org/10.1084/jem.20251065
Primary Topic
Inflammasome and immune disorders
Type
article
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article

SpeckSeq enables high-throughput functional stratification of MEFV variants in autoinflammatory diseases

Isabelle Jéru, Sophie Georgin‐Lavialle, Sarah Dalmon, Ignazia Prigione et al.
The Journal of Experimental Medicine
Inflammasome and immune disorders
article

SpeckSeq enables high-throughput functional stratification of MEFV variants in autoinflammatory diseases

Isabelle Jéru, Sophie Georgin‐Lavialle, Sarah Dalmon, Ignazia Prigione, Majdouline El Moussaoui, Martin Broly, Michel Moutschen, Asma Smahi, Guilaine Boursier, Alessandra Carbone, Léa Savey, Alexandre Bélot, Pauline Bronnec, Yvan Jamilloux, Flora Magnotti, Sandrine Hughes, Benjamin Gillet, É. Sbidian, M. Marcotte, Omran Allatif, Florian Milhavet, Christophe Briand, Marco Gattorno, Marine Madrange, Serena Palmeri, Nora Martel, Thomas Henry, H. Bachelez, Gianluca Lombardi, Kevin Barthes, Aysima Atilgan
article en

Abstract

Variants of uncertain significance (VUS) are a major obstacle in genetic diagnosis, particularly when involving gain-of-function (GoF) mutations that are poorly predicted in silico. MEFV, which encodes the inflammasome sensor pyrin, is mutated in two autoinflammatory diseases, familial Mediterranean fever (FMF) and pyrin-associated autoinflammation with neutrophilic dermatosis (PAAND). Here, we developed SpeckSeq, a method that combines DNA bar-coding, ASC speck-based single-cell sorting and next-generation sequencing to systematically identify hypermorphic MEFV variants in response to different stimuli. SpeckSeq identified 49 GoF mutations separated into two distinct groups containing either PAAND variants or FMF variants. SpeckSeq was validated using patients' cells and supported a reclassification of MEFV variant pathogenicity, leading to novel diagnoses. As a large-scale mutagenesis approach, using human genetics as a guide, SpeckSeq revealed structural and functional pyrin features, including a putative ligand-accommodating cavity in the B30.2 domain. Altogether, SpeckSeq classifies VUS to refine molecular diagnostics and improve our knowledge on the pyrin inflammasome.

The Journal of Experimental MedicineVol. 223(2)
Université Claude Bernard Lyon 1 (FR), Délégation Paris 5 (FR), École Normale Supérieure de Lyon (FR), Centre National de la Recherche Scientifique (FR), Central South University (CN), Inserm (FR), Institut Universitaire de France (FR), Université Paris-Est Créteil (FR), Université de Montpellier (FR), Université Paris Cité (FR), Istituto Giannina Gaslini (IT), Centre Hospitalier Universitaire de Nantes (FR), Centre Hospitalier Universitaire Henri-Mondor (FR), Sorbonne Université (FR), Hôpital Femme Mère Enfant (FR), Hôpital de la Croix-Rousse (FR), Biologie Computationnelle, Quantitative et Synthétique (FR), Assistance Publique – Hôpitaux de Paris (FR), Hospices Civils de Lyon (FR), Hôpital Européen (FR), Institut de Génomique Fonctionnelle de Lyon (FR), Hôpitaux Universitaires Henri-Mondor (FR), Centre Hospitalier Universitaire de Liège (BE), Institut du Thorax (FR), Hôpital Saint-Louis (FR), Biostatistical Consulting (United States) (US), Xiangya Hospital Central South University (CN), Centre International de Recherche en Infectiologie (FR), Institut des Maladies Génétiques Imagine (FR), Hôpital Tenon (FR), University of Genoa (IT)
Openalex Percentile: Top 66%
Inflammasome and immune disorders
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