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

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

doi:10.1007/s10875-021-01021-7

Cited by 15 publications

(14 citation statements)

References 41 publications

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“…4D). Thus, in agreement with Honda and colleagues 23 , we observed that different mutations within MEFV, including p.M694V, lead to different level of enhancement of TcdA-triggered responses. This suggests that different CHS-targeting mutations may cause different level of deregulation of the pyrin in ammasome.…”

Section: B302 Domain Is Dispensable For Pyrin Activation In Response ...supporting

confidence: 93%

“…While most of the research on pyrin has focused on the B30.2 domain, our results indicate that the CHS domain is a central regulator of pyrin in ammasome. Furthermore, this study and others 21,23 demonstrate that various mutations in the CHS are pathogenic and validate that MEFV mutations outside exon 10 (i.e. affecting other domains than the B30.2 domain) can cause FMF.…”

Section: Discussionsupporting

confidence: 79%

“…In contrast, all the other domain deletions strongly decreased (coiled-coil domain deletion) or abolished IL-18 secretion in response to TcdA. Cells expressing the p.M694V pyrin variant secreted higher level of IL-18 than cells expressing WT pyrin in agreement with the hyper-reactivity of primary monocytes from FMF patients to clostridial toxins 19,23 . Undifferentiated U937 cells are de cient for IL-1β production.…”

Section: B302 Domain Is Dispensable For Pyrin Activation In Response ...supporting

confidence: 52%

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Mutations in the B30.2 and the Central Helical scaffold domains of pyrin differentially affect inflammasome activation

Chirita

Magnotti

Bronnec

et al. 2022

Preprint

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Familial Mediterranean Fever (FMF) is the most common monogenic autoinflammatory disorder. FMF is caused by mutations in the MEFV gene, encoding pyrin, an inflammasome sensor. The best characterized pathogenic mutations associated with FMF cluster in exon 10. Yet, mutations have been described along the whole MEFV coding sequence. Exon 10 encodes the B30.2 domain of the pyrin protein, but the function of this human-specific domain remains unclear. Pyrin is an inflammasome sensor detecting RhoA GTPase inhibition following exposure to bacterial toxins such as TcdA. Here, we demonstrate that the B30.2 domain is dispensable for pyrin inflammasome activation in response to this toxin. Deletion of the B30.2 domain mimics the most typical FMF-associated mutation and confers spontaneous inflammasome activation in response to pyrin dephosphorylation. Our results indicate that the B30.2 domain is a negative regulator of the pyrin inflammasome that acts independently from and downstream of pyrin dephosphorylation. Additionally, we identify the central helical scaffold (CHS) domain of pyrin, which lies immediately upstream of the B30.2 domain as a second regulatory domain. Mutations affecting the CHS domain mimic pathogenic mutations in the B30.2 domain and render the pyrin inflammasome activation under the sole control of the dephosphorylation. In addition, specific mutations in the CHS domain strongly increase the cell susceptibility to steroid catabolites, recently described to activate pyrin, in both a cell line model and in monocytes from genotype-selected FMF patients. Taken together, our work reveals the existence of two distinct regulatory regions at the C-terminus of the pyrin protein, that act in a distinct manner to regulate positively or negatively inflammasome activation. Furthermore, our results indicate that different mutations in pyrin regulatory domains have different functional impacts on the pyrin inflammasome which could contribute to the diversity of pyrin-associated autoinflammatory diseases.

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Section: B302 Domain Is Dispensable For Pyrin Activation In Response ...supporting

confidence: 93%

Section: Discussionsupporting

confidence: 79%

Section: B302 Domain Is Dispensable For Pyrin Activation In Response ...supporting

confidence: 52%

See 1 more Smart Citation

Mutations in the B30.2 and the Central Helical scaffold domains of pyrin differentially affect inflammasome activation

Chirita

Magnotti

Bronnec

et al. 2022

Preprint

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“…THP-1 nucleofection and cell death assays were performed as described previously ( Fujisawa et al, 2007 ; Honda et al, 2021 ) with some modifications. THP-1 cells (1 × 10 6 ) were transfected with 500 ng plasmid encoding pAcGFP-fused CDC42 variants or hMEFV -pAcGFP1-t2a- CDC42 using 4D-Nucleofector and the SG Cell Line 4D-Nucleofector X Kit (Lonza).…”

Section: Methodsmentioning

confidence: 99%

Trapping of CDC42 C-terminal variants in the Golgi drives pyrin inflammasome hyperactivation

Nishitani‐Isa

Mukai

Honda

et al. 2022

Journal of Experimental Medicine

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Mutations in the C-terminal region of the CDC42 gene cause severe neonatal-onset autoinflammation. Effectiveness of IL-1β–blocking therapy indicates that the pathology involves abnormal inflammasome activation; however, the mechanism underlying autoinflammation remains to be elucidated. Using induced-pluripotent stem cells established from patients carrying CDC42R186C, we found that patient-derived cells secreted larger amounts of IL-1β in response to pyrin-activating stimuli. Aberrant palmitoylation and localization of CDC42R186C protein to the Golgi apparatus promoted pyrin inflammasome assembly downstream of pyrin dephosphorylation. Aberrant subcellular localization was the common pathological feature shared by CDC42 C-terminal variants with inflammatory phenotypes, including CDC42*192C*24 that also localizes to the Golgi apparatus. Furthermore, the level of pyrin inflammasome overactivation paralleled that of mutant protein accumulation in the Golgi apparatus, but not that of the mutant GTPase activity. These results reveal an unexpected association between CDC42 subcellular localization and pyrin inflammasome activation that could pave the way for elucidating the mechanism of pyrin inflammasome formation.

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“…Mutant pyrin causes hyperactivation of inflammasomes and interleukin-1b (IL-1b) hypersecretion (1). Incidentally, FMF diagnosis might be difficult since it exhibits diverse clinical manifestations, and the majority of MEFV variants are classified as variants of unknown significance (2,3).…”

Section: Introductionmentioning

confidence: 99%

Case Report: A Pediatric Case of Familial Mediterranean Fever Concurrent With Autoimmune Hepatitis

Aoki

Izawa²,

Tanaka³

et al. 2022

Front. Immunol.

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Familial Mediterranean fever (FMF) is a hereditary, autoinflammatory disease that causes recurrent fever, arthritis, and serositis. The diagnosis of FMF is based on the presentation of typical clinical symptoms and the Mediterranean fever gene (MEFV) test. However, the challenge lies in diagnosing atypical cases. In this report, we have described a pediatric patient with complex FMF whose diagnosis required trio-whole exome sequencing (WES) and functional validation of a rare MEFV variant. A 3-year-old boy presented with recurrent episodes of elevated liver enzymes and arthralgia. He was diagnosed with autoimmune hepatitis (AIH), and his liver enzymes improved rapidly with steroid treatment. However, he exhibited recurrent arthralgia and severe abdominal attacks. Trio-WES identified compound heterozygous mutations in MEFV (V726A and I692del). Ex vivo functional assays of the patient’s monocytes and macrophages, which had been pre-treated with Clostridium difficile toxin A (TcdA) and colchicine, were comparable to those of typical FMF patients, thereby confirming the diagnosis of FMF. Although he was intolerant to colchicine because of liver toxicity, subsequent administration of canakinumab successfully ameliorated his abdominal attacks. However, it was ineffective against liver injury, which recurred after steroid tapering. Therefore, in this case, the pathogenesis of AIH was probably interleukin-1β (IL-1β)-independent. In fact, AIH might have been a concurrent disease with FMF, rather than being one of its complications. Nevertheless, further studies are necessary to determine whether FMF-induced inflammasome activation contributes to AIH development. Moreover, we must consider the possibility of mixed phenotypes in such atypical patients who present distinct pathologies simultaneously.

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

Cited by 15 publications

References 41 publications

Mutations in the B30.2 and the Central Helical scaffold domains of pyrin differentially affect inflammasome activation

Mutations in the B30.2 and the Central Helical scaffold domains of pyrin differentially affect inflammasome activation

Trapping of CDC42 C-terminal variants in the Golgi drives pyrin inflammasome hyperactivation

Case Report: A Pediatric Case of Familial Mediterranean Fever Concurrent With Autoimmune Hepatitis

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