THU0474 Functional Analysis of Peripheral Blood Mononuclear Cells in a Fcas Patient with Novel NLRP3 Mutation

Background Autoinflammatory syndromes manifest with spontaneously recurring attacks of systemic inflammation. Familial cold autoinflammatory syndrome (FCAS) which is characterized by urticarial rash, fever by cold exposure, as well as Muckle-Well syndrome and chronic infantile neurological cutaneous and articular syndrome (CINCA)/ neonatal onset multisystem inflammatory disease ( NOMID ), is caused by mutations in NLRP3 gene which encodes a protein called cryopyrin which release the proinflammatory cytokine IL-1β. Conversion of the proIL-1β precursor to the active protein requires the assembly of intracellular complexes called inflammasomes, which include proteins of the Nod-like receptor family. Mutations of NLRP3 gene lead to excessive production of IL-1β. Objectives Autoinflammatory syndromes manifest with spontaneously recurring attacks of systemic inflammation. Familial cold autoinflammatory syndrome (FCAS) which is characterized by urticarial rash, fever by cold exposure, as well as Muckle-Well syndrome and chronic infantile neurological cutaneous and articular syndrome (CINCA)/ neonatal onset multisystem inflammatory disease ( NOMID ), is caused by mutations in NLRP3 gene which encodes a protein called cryopyrin which release the proinflammatory cytokine IL-1β. Conversion of the proIL-1β precursor to the active protein requires the assembly of intracellular complexes called inflammasomes, which include proteins of the Nod-like receptor family. Mutations of NLRP3 gene lead to excessive production of IL-1β. Methods We report a 32-year-old woman, who has a history of recurrent urticarial rash in response to cold exposure. Genomic DNA from PBMC or whole blood of this FCAS patient and healthy control was obtained. DNA fragment of the NLRP3 gene was amplified by PCR and were sequenced with an ABI 3100 sequencer (Applied Biosystems, Foster City, CA). PBMC were obtained from Heparinized venous blood from this FCAS patient or healthy control using Ficoll-Hypaque, then cultured at 32°C or 37°C with or without LPS stimulation for 3hr. Cytokines including IL-1β were determined in each supernatant by ELISA. In addition, we examined the ASC-dependent NF-κB reporter gene activities of the novel NLRP3 mutant in this patient. Results We report a 32-year-old woman, who has a history of recurrent urticarial rash in response to cold exposure. Genomic DNA from PBMC or whole blood of this FCAS patient and healthy control was obtained. DNA fragment of the NLRP3 gene was a