Functional Studies of Cryopyrin V200M Mutation Identified in Behçet’s Disease Patients

1. PP-078-10 Elif Eren, Yetiş Gültekin, Şahru Yüksel and Nesrin Özören Boğaziçi University, Department of Molecular Biology and Genetics, Kuzey Park Binası, 34342 Bebek Istanbul-TURKEY Phone: +  90 212 359 76 29  Fax: +  90 212 287 24 68, e-mails: eliferen2@gmail.com, yetisgultekin@yahoo.com Functional Studies of Cryopyrin V200M Mutation Identified in Behçet’s Disease Patients FIGURE 2: Genetic Studies FIGURE 1: Inflammasome and Behçet’s Disease • Inflammasome: cryopyrin + ASC + pro-caspase-1 • Pathogens infection: pro-caspase-1  caspase-1  IL1β secretion  inflammation • Screening of Behçet’s patients cryopyrin and ASC genes for mutations. • 1344 GA G/A heterozygote • 200 GTG  ATG • Valine Methionine • Identification of cryopyrin V200M mutation in 3 patients among 103. • Not found in 50 controls. • Nucleotide conserved among species •  important role for protein function • Pedigree analysis: From Mariathasan S., ASC, Ipaf and Cryopyrin/Nalp3: bona fide intracellular adapters of caspase-1 inflammasome, Microbes and Infection 9 (2007) 664-671. No mutation found in healthy family members. • Behçet’s disease: auto-inflammatory disease • Activation of inflammasome even in absence of pathogens. • Hypothesis: • Mutation in inflammasome component •  over-activation of inflammasome ABSTRACT The inflammasome is a cytoplasmic complex composed of ASC, NALP3 (cryopryrin) and caspase-1, which are assembled in response to pathogens. This assembly leads to caspase-1 cleavage. Active caspase-1 then cleaves pro-IL1β into mature IL-1β that can be secreted and can initiate innate responses against the pathogen. Active inflammasomes can also activate the NF-κB pathway. It has been previously shown that mutations in components of this complex cause auto-inflammatory diseases such as familial cold autoinflammatory syndrome (FACS), Muckle-Wells syndrome (MWS), and neonatal-onset multiple-system inflammatory disease (NOMID). Behçet’s disease is also an auto-inflammatory disease of unknown etiology characterized by recurrent inflammatory episodes. Aberrant secretion of cytokines is observed in Behçet’s disease. Thus, we hypothesized that mutations in inflammasome components may result in a constitutively active complex. We have identified cryopyrin V200M mutation in a higher frequency (3/103 patients, not found in 50 controls) than previously reported. To test if cryopyrin V200M mutation results in a constitutively active inflammasome, we have cloned the mutation into vector and we are investigating whether this mutation over-activates NF-κB pathway with luciferase reporter gene assays. • Polymorphism analysis of cryopyrin exon 3 FIGURE 3: Transfection of HEK293FT cells with different plasmids Highly polymorphic exon HEK293FT cells were transfected via Ca3(PO4)2 with 70% efficiency FIGURE 4: The effect of V200M mutation on NF-kB activity- Preliminary Results FIGURE 5: Effect of V200M Mutation on Inflammasome Assembly and Function Interaction with ASC Caspase-1 Activation In order to determine if V200M mutation affects interaction between Cryopyrin and ASC, HEK293T cells are transfected with different plasmids expressing these proteins and co-immunoprecipitation is performed. Caspase-1 activation is compared by Western blot between HEK293T cells transfected with Caspase-1, ASC and WT or mutant Cryopyrin. • CONCLUSIONS • Cryopyrin V200M mutation was found in 3/103 Behcet’s disease patients. • Many polymorphisms were identified in Cryopyrin exon 3 in the general Turkish population. • Preliminary results show that NF-kB activity increases 2 fold with V200M Cryopyrin compared to WT. • Experiments are ongoing to test if mutant protein’s interaction with its partners ASC and caspase-1 increases by co-ip. ACKNOWLEDGMENTS This project was supported by TÜBA-GEDIP award, EMBO-YIP-SDIG 1468 to N.Ö and BAP-06HB103 to Ş.Y. Y.G is supportedbyTurkishEducationFoundationScholarship (T.E.V) Congress attendance was partly funded by Boğaziçi University Arts and Science Faculty and The Institute of Science.