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In Vitro Analysis of the Functional Effects of an NLRP3 G809S Variant with the co-Existence of MEFV Haplotype Variants in Atypical Autoinflammatory Syndrome

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Hereditary periodic fever syndromes have been considered monogenic diseases. However, some recent reports have described patients with co-existence of recurrent fever responsible genes. This study assessed whether a rare variant, found in Japanese children showing atypical autoinflammatory syndrome, located in the leucine-rich repeat domain of Nod-like receptor family, pyrin domain containing 3 (NLRP3) with co-existence of Mediterranean fever (MEFV) haplotype variants may contribute to a proinflammatory phenotype using a systematic approach.


Cytokine production in serum or from peripheral blood monocytes was measured by ELISA. DNA sequence analysis of genes including NLRP3, MEFV, mevalonate kinase (MVK), and tumor necrosis factor receptor superfamily, member 1A (TNFRSF1A) were performed on patient samples. In vitro functional assays determined the effects of the NLRP3 variants and pyrin using NF-κB activation and speck formation assays.


A heterozygous genetic variant of NLRP3, G809S, was found in samples from both patients. Additionally the previously reported heterozygous MEFV variants (P369S-R408Q or E148Q-P369S-R408Q) were also detected in both patients. Serum IL-1ra and sTNFR1 levels increased in the attack phase of the disease in both patients. The production levels of IL-1β from monocytes isolated from both cases were elevated following LPS and IFN-γ stimulation. The NLRP3 G809S variant demonstrated no increase of NF-κB activity following monosodium urate stimulation, whereas it significantly increased speck formation by interacting with apoptosis-associated speck-like protein with caspase recruitment domain.


The phenotype of atypical autoinflammatory disease in patients could be modified by a synergistic effect with two other variants of autoinflammatory-associated genes.

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Apoptosis-associated speck-like protein containing a CARD


Cryopyrin-associated periodic syndrome


Chronic infantile neurologic cutaneous, articular


Familial cold-induced autoinflammatory syndrome


Familial Mediterranean fever


Human embryonic kidney




Mediterranean fever


Mevalonate kinase


Muckle–Wells syndrome


Monosodium urate


Nucleotide-binding site


Nod-like receptor family pyrin domain containing 3


Neonatal-onset multisystem inflammatory disease


Pathogen-associated molecular patterns


Peripheral blood mononuclear cells


Tumor necrosis factor receptor superfamily member 1A


Tumor necrosis factor receptor-associated periodic syndrome


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We thank the members of the families who agreed to participate in the study. We thank Dr. Ozaki T for the initial treatment of case 2. We thank K. Kasahara and M. Yamamoto for their technical help. This work was supported by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan, and by Health and Labour Science Research Grants for Research on Intractable Diseases from the Ministry of Health, Labour and Welfare.

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The authors have declared no conflicts of interest.

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Correspondence to Hidenori Ohnishi.

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Kubota, K., Ohnishi, H., Teramoto, T. et al. In Vitro Analysis of the Functional Effects of an NLRP3 G809S Variant with the co-Existence of MEFV Haplotype Variants in Atypical Autoinflammatory Syndrome. J Clin Immunol 33, 325–334 (2013).

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