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Journal of Clinical Immunology
Published in: Journal of Clinical Immunology 6/2021

01-08-2021 | Familial Mediterranean Fever | Original Article

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

Authors: Yoshitaka Honda, Yukako Maeda, Kazushi Izawa, Takeshi Shiba, Takayuki Tanaka, Haruna Nakaseko, Keisuke Nishimura, Hiroki Mukoyama, Masahiko Isa-Nishitani, Takayuki Miyamoto, Hiroshi Nihira, Hirofumi Shibata, Eitaro Hiejima, Osamu Ohara, Junko Takita, Takahiro Yasumi, Ryuta Nishikomori

Published in: Journal of Clinical Immunology | Issue 6/2021

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Abstract

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 MEFVP257L 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 MEFVP257L 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.
Appendix
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Metadata
Title
Rapid Flow Cytometry-Based Assay for the Functional Classification of MEFV Variants
Authors
Yoshitaka Honda
Yukako Maeda
Kazushi Izawa
Takeshi Shiba
Takayuki Tanaka
Haruna Nakaseko
Keisuke Nishimura
Hiroki Mukoyama
Masahiko Isa-Nishitani
Takayuki Miyamoto
Hiroshi Nihira
Hirofumi Shibata
Eitaro Hiejima
Osamu Ohara
Junko Takita
Takahiro Yasumi
Ryuta Nishikomori
Publication date
01-08-2021
Publisher
Springer US
Published in
Journal of Clinical Immunology / Issue 6/2021
Print ISSN: 0271-9142
Electronic ISSN: 1573-2592
DOI
https://doi.org/10.1007/s10875-021-01021-7

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