NOD2 Mutations Affect Muramyl Dipeptide Stimulation of Human B Lymphocytes and Interact with Other IBD-Associated Genes

Background Genetic and functional studies have associated variants in the NOD2/CARD15 gene with Crohn's disease. Aims This study aims to replicate the association of three common NOD2 mutations with Crohn's disease, study its effect on NOD2 expression in B cells and its interaction with other IBD-associated genes. Methods A total of 294 IBD patients (179 familial IBD, 115 sporadic IBD) and 298 unrelated healthy controls were from central Pennsylvania. NOD2 mutations were analyzed by primer-specific amplification, PCR based-RFLP, and validated with the ABI SNPlex(M) genotyping system. Gene-gene interaction was studied using a statistical model for epistasis analysis. Results Three common NOD2 mutations are associated with Crohn's disease (p = 5.08 x 10(-7), 1.67 x 10(-6), and 1.87 x 10(-2) for 1007fs, R720W, and G908R, respectively), but not with ulcerative colitis (p = 0.1046, 0.1269, and 0.8929, respectively). For IBD overall, 1007finsC (p = 4.4 x 10(-5)) and R720W (p = 9.24 x 10(-5)) were associated with IBD, but not G908R (p = 0.1198). We revealed significant interactions of NOD2 with other IBD susceptibility genes IL23R, DLG5, and OCTN1. We discovered that NOD2 was expressed in both normal human peripheral blood B cells and in EBV-transformed B cell lines. Moreover, we further demonstrated that muramyl dipeptide (MDP) stimulation of B lymphocytes up-regulated expression of NF-kappa B-p50 mRNA. Conclusion NOD2 is expressed in peripheral B cells, and the up-regulation of NOD2 expression by MDP was significantly impaired by NOD2 mutations. The finding suggests a possible role of NOD2 in the immunological response in IBD pathogenesis.