Pyrin variant E148Q potentiates inflammasome activation and the effect of pathogenic mutations in cis.

The p.E148Q variant in pyrin is present in different populations at a frequency of up to 29%, and has been associated with diseases, including vasculitis and FMF. The pathogenicity of p.E148Q in FMF is unclear, even when observed in cis or in trans to a single, typically recessive, pathogenic mutation. We performed functional validation to determine whether p.E148Q increases the ability of pyrin to form an active inflammasome complex in cell lines. We interrogated the Australian Autoinflammatory Disease RegistrY (AADRY) to find candidate inheritance patterns for the p.E148Q variant in pyrin. Different pyrin variant combinations were tested in HEK293T cells stably expressing the adaptor protein apoptosis-associated speck-like (ASC), which were analysed by flow cytometry to visualize inflammasome formation, with and without stimulation by Clostridioides difficile toxin B (TcdB). Inflammasome-dependent cytokine secretion was also quantified by ELISA of supernatants from THP-1 cells transduced with lentiviral expression vectors. In AADRY, we observed the p.E148Q allele in individuals with autoinflammatory diseases alone or in conjunction with other pyrin variants. Two FMF families harboured the allele p.E148Q-M694I in cis with dominant heritability. In vitro, p.E148Q pyrin could spontaneously potentiate inflammasome formation, with increased IL-1β and IL-18 secretion. p.E148Q in cis to classical FMF mutations provided significant potentiation of inflammasome formation. The p.E148Q variant in pyrin potentiates inflammasome activation in vitro. In cis, this effect is additive to known pathogenic FMF mutations. In some families, this increased effect could explain why FMF segregates as an apparently dominant disease.

Reygaerts T ，Laohamonthonkul P ，Hrovat-Schaale K ，Moghaddas F ，Baker PJ ，Gray PE ，Masters SL ... -  《-》

Defect of suppression of inflammasome-independent interleukin-8 secretion from SW982 synovial sarcoma cells by familial Mediterranean fever-derived pyrin mutations.

Sugiyama R ，Agematsu K ，Migita K ，Nakayama J ，Mokuda S ，Ogura F ，Haraikawa K ，Okumura C ，Suehiro S ，Morikawa S ，Ito Y ，Masumoto J ... -  《-》

Familial Mediterranean fever mutations are hypermorphic mutations that specifically decrease the activation threshold of the Pyrin inflammasome.

FMF is the most frequent autoinflammatory disease and is associated in most patients with bi-allelic MEFV mutations. MEFV encodes Pyrin, an inflammasome sensor activated following RhoGTPase inhibition. The functional consequences of MEFV mutations on the ability of Pyrin variants to act as inflammasome sensors are largely unknown. The aim of this study was to assess whether MEFV mutations affect the ability of Pyrin to detect RhoGTPase inhibition and other inflammasome stimuli. IL-1β and IL-18 released by monocytes from healthy donors (HDs) and FMF patients were measured upon specific engagement of the Pyrin, NLRP3 and NLRC4 inflammasomes. Cell death kinetics following Pyrin activation was monitored in real time. Monocytes from FMF patients secreted significantly more IL-1β and IL-18 and died significantly faster than HD monocytes in response to low concentrations of Clostridium difficile toxin B (TcdB), a Pyrin-activating stimulus. Monocytes from patients bearing two MEFV exon 10 pathogenic variants displayed an increased Pyrin inflammasome response compared with monocytes from patients with a single exon 10 pathogenic variant indicating a gene-dosage effect. Using a short priming step, the response of monocytes from FMF patients to NLRP3- and NLRC4-activating stimuli was normal indicating that MEFV mutations trigger a specific hypersensitivity of monocytes to low doses of a Pyrin-engaging stimulus. Contrary to the NLRP3 mutations described in cryopyrin-associated periodic syndrome, FMF-associated MEFV mutations do not lead to a constitutive activation of Pyrin. Rather, FMF-associated mutations are hypermorphic mutations that specifically decrease the activation threshold of the Pyrin inflammasome without affecting other canonical inflammasomes.

Jamilloux Y ，Lefeuvre L ，Magnotti F ，Martin A ，Benezech S ，Allatif O ，Penel-Page M ，Hentgen V ，Sève P ，Gerfaud-Valentin M ，Duquesne A ，Desjonquères M ，Laurent A ，Rémy-Piccolo V ，Cimaz R ，Cantarini L ，Bourdonnay E ，Walzer T ，Py BF ，Belot A ，Henry T ... -  《-》

Familial Mediterranean fever mutations lift the obligatory requirement for microtubules in Pyrin inflammasome activation.

Van Gorp H ，Saavedra PH ，de Vasconcelos NM ，Van Opdenbosch N ，Vande Walle L ，Matusiak M ，Prencipe G ，Insalaco A ，Van Hauwermeiren F ，Demon D ，Bogaert DJ ，Dullaers M ，De Baere E ，Hochepied T ，Dehoorne J ，Vermaelen KY ，Haerynck F ，De Benedetti F ，Lamkanfi M ... -  《-》

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

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. 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. 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. Our assay facilitates a rapid and comprehensive assessment of the pathogenicity of MEFV variants and contributes to a refined definition of PAAD subtypes.

Honda Y ，Maeda Y ，Izawa K ，Shiba T ，Tanaka T ，Nakaseko H ，Nishimura K ，Mukoyama H ，Isa-Nishitani M ，Miyamoto T ，Nihira H ，Shibata H ，Hiejima E ，Ohara O ，Takita J ，Yasumi T ，Nishikomori R ... -  《-》