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

Autor: Honda Y; Department of Pediatrics, Faculty of Medicine, Kyoto University Graduate School of Medicine, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, 606-8507, Japan., Maeda Y; Department of Pediatrics, Faculty of Medicine, Kyoto University Graduate School of Medicine, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, 606-8507, Japan., Izawa K; Department of Pediatrics, Faculty of Medicine, Kyoto University Graduate School of Medicine, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, 606-8507, Japan. kizawa@kuhp.kyoto-u.ac.jp., Shiba T; Department of Pediatrics, Faculty of Medicine, Kyoto University Graduate School of Medicine, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, 606-8507, Japan.; Department of Pediatrics, Tenri Hospital, Tenri, Japan., Tanaka T; Department of Pediatrics, Faculty of Medicine, Kyoto University Graduate School of Medicine, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, 606-8507, Japan.; Department of Pediatrics, Otsu Red Cross Hospital, Otsu, Japan., Nakaseko H; Department of Infection and Immunology, Aichi Children's Health and Medical Center, Obu, Japan., Nishimura K; Department of Endocrinology and Rheumatology, Kurashiki Central Hospital, Kurashiki, Japan., Mukoyama H; Department of Rheumatology and Clinical Immunology, Kyoto University Graduate School of Medicine, Kyoto, Japan., Isa-Nishitani M; Department of Pediatrics, Faculty of Medicine, Kyoto University Graduate School of Medicine, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, 606-8507, Japan., Miyamoto T; Department of Pediatrics, Faculty of Medicine, Kyoto University Graduate School of Medicine, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, 606-8507, Japan., Nihira H; Department of Pediatrics, Faculty of Medicine, Kyoto University Graduate School of Medicine, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, 606-8507, Japan., Shibata H; Department of Pediatrics, Faculty of Medicine, Kyoto University Graduate School of Medicine, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, 606-8507, Japan., Hiejima E; Department of Pediatrics, Faculty of Medicine, Kyoto University Graduate School of Medicine, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, 606-8507, Japan., Ohara O; Kazusa DNA Research Institute, Kisarazu, Japan., Takita J; Department of Pediatrics, Faculty of Medicine, Kyoto University Graduate School of Medicine, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, 606-8507, Japan., Yasumi T; Department of Pediatrics, Faculty of Medicine, Kyoto University Graduate School of Medicine, 54 Kawahara-cho, Shogoin, Sakyo-ku, Kyoto, 606-8507, Japan. yasumi@kuhp.kyoto-u.ac.jp., Nishikomori R; Department of Pediatrics and Child Health, Kurume University School of Medicine, Kurume, Japan.
Jazyk: angličtina
Zdroj: Journal of clinical immunology [J Clin Immunol] 2021 Aug; Vol. 41 (6), pp. 1187-1197. Date of Electronic Publication: 2021 Mar 17.
DOI: 10.1007/s10875-021-01021-7
Abstrakt: Purpose: Pathogenic MEFV variants cause pyrin-associated autoinflammatory diseases (PAADs), which include familial Mediterranean fever (FMF), FMF-like disease, and pyrin-associated autoinflammation with neutrophilic dermatosis (PAAND). The diagnosis of PAADs is established by clinical phenotypic and genetic analyses. However, the pathogenicity of most MEFV variants remains controversial, as they have not been functionally evaluated. This study aimed to establish and validate a new functional assay to evaluate the pathogenicity of MEFV variants.
Methods: We transfected THP-1 monocytes with 32 MEFV variants and analyzed their effects on cell death with or without stimulation with Clostridium difficile toxin A (TcdA) or UCN-01. These variants were classified using hierarchical cluster analysis. Macrophages were obtained from three healthy controls and two patients with a novel homozygous MEFV P257L variant, for comparison of IL-1β secretion using a cell-based assay and a novel THP-1-based assay.
Results: Disease-associated MEFV variants induced variable degrees of spontaneous or TcdA/UCN-01-induced cell death in THP-1. Cell death was caspase-1 dependent and was accompanied by ASC speck formation and IL-1β secretion, indicating that pathogenic MEFV variants induced abnormal pyrin inflammasome activation and subsequent pyroptotic cell deaths in this assay. The MEFV variants (n = 32) exhibiting distinct response signatures were classified into 6 clusters, which showed a good correlation with the clinical phenotypes. Regarding the pathogenicity of MEFV P257L variants, the results were consistent between the cell-based assay and the THP-1-based assay.
Conclusion: Our assay facilitates a rapid and comprehensive assessment of the pathogenicity of MEFV variants and contributes to a refined definition of PAAD subtypes.
(© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)
Databáze: MEDLINE
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