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 × 10 −7 , 1.67 × 10 −6 , and 1.87 × 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 × 10 −5 ) and R720W ( p = 9.24 × 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-κB-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.