Effect of Nod2/Card15 Mutations On Ileal Gene Expression Profiles in Crohn’S Disease
Casey K. Mccullough2, Christina M. Hamm2, Elizabeth Gorbe2, Jonathan T. Unkart2, Ellen Li2, Qing Qing Gong2, Candace R. Miller1, Thaddeus S. Stappenbeck3, Christian Stone2, David W. Dietz4, Steven R. Hunt*1
1Department of Surgery, Washington University, Saint Louis, MO; 2Division of Gastroenterology, Washington University, Saint Louis, MO; 3Department of Pathology and Immunology, Washington University, Saint Louis, MO; 4Department of Colon and Rectal Surgery, Cleveland Clinic Foundation, Cleveland, OH
Background: Three mutations (Leu1007InsC, R702W, G908R) in the nucleotide-binding oligomerization domain (NOD)2/caspase recruitment domain (CARD)15 have been associated with an increased risk of Crohn’s disease (CD) and ileal disease location. Analysis of the effect of NOD2/CARD15 mutations on ileal gene expression could provide further insight on the pathogenesis of ileal CD.
Methods: We performed microarray analysis on normal ileal mucosa from three NOD2- (none of the three mutations) normal control subjects, and on inflamed and uninflamed ileal mucosa from three NOD2- CD patients, and three NOD2+ (at least one of three mutations) CD patients. All patients were nonsmokers. In the six CD patients, both inflamed and macroscopically uninflamed tissue biopsies were obtained from fresh pathologic specimens at the time of their initial ileocolic resection. The probes prepared from the ileal RNA and the common reference ileal RNA (from a fourth NOD2- control subject) were hybridized with the Agilent Whole Human Genome array using the two-color protocol. The data was analyzed using Statistical Analysis of Microarrays (SAM) software.
Results: SAM analysis detected no significant difference between NOD2+ and NOD2- uninflamed mucosa. We identified a number of genes that were upregulated in the NOD2+ inflamed ileal mucosa relative to NOD2- inflamed ileal mucosa. One of the identified target genes, CYBB, encodes cytochrome b-245, beta polypeptide, which is a component of the microbicidal oxidase system in phagocytes (q=0.04). In addition, we observed altered expression of genes (q<0.05) in the uninflamed mucosa in CD patients compared to control subjects that was independent of NOD2 status.
Conclusions: We have identified target genes that are upregulated in inflamed ileal mucosal biopsies from NOD2+ CD patients relative to inflamed mucosa from NOD2- CD patients. One of these genes, CYBB, plays an integral role in mucosal defense. These experiments have also detected altered expression of genes in macroscopically normal ileal mucosa from CD patients compared to control subjects, prior to upregulation of pro-inflammatory genes associated with CD activity.
