AB0012 Functional Characterization of P.E383k Blau Syndrome-Related Mutation

Background The Blau syndrome (BS) is a rare autosomal dominant autoinflammatory disease clinically characterized by symmetrical arthritis, granulomatous dermatitis and recurrent uveitis. The disease is caused by single mutations in CARD15/NOD2, encoding the NOD2 protein that is known to regulate the defense against pathogens by activating the NF-κB signalling pathway (1). Objectives In vitro and ex vivo functional analysis aimed at characterizing p.E383K mutation found in an Italian family affected by Blau syndrome. Methods For ex vivo studies, peripheral blood mononuclear cells (PBMC) are collected from patients carrying p.E383K mutation, then lysed and analyzed by a protein microarray technique (RPPA) to determine the NF-κB activity. IL1β, IL6, IL8, TNFα, IFNγ, IL12, IL17, IL22, IL23 releases are quantified by ELISA and antibody microarray techniques in PBMC cultured for 7 hours in presence or absence of lipopolysaccharide (LPS) and muramyldipeptide (MDP). In vitro analysis started from the creation and stably transfection of constructs containing human NOD2 cDNA wild-type and mutated p.E383K into HEK293 cells. The cells were cultured for 7 and 24 hours with or without MDP. NF-κB activity was determined by Western blot, evaluating the expression of IKBα and its phosphorylated form in cellular lysates, and also by RPPA. IL8 was assayed in the supernatants of the reported cell cultures through antibody microarray technique. The significance of all the data was tested using non-parametric analysis. Results Confocal microscopy revealed that p.E383K NOD2 resides in cytoplasm of transfected HEK293 cells, as well as wild-type. The expression of p.E383K NOD2, assessed by Western blot, did not present any variations after MDP stimulation. Regarding the NF-κB activity, both in vitro RPPA and Western blot studies showed an inactivation of this pathway, presenting lower expression of phosphorylated IKBα than IKBα in presence or absence of MDP stimulation (p