NOD2, a Gene Responsible for Inherited Uveitis, Alters Gene Expression as Analyzed by Microarray

Abstract

Abstract: : Purpose: Mutations in the nucleotide-binding region of the NOD2 gene have been found in patients with the autosomal dominantly inherited uveitis, Blau/Jabs syndrome. NOD2’s role in the immune system is not well defined yet, but it is thought to be involved in the innate immune response to intracellular pathogens. In order to evaluate the function and interactions of NOD2 further, we performed an Affymetrix array on mRNA collected from U937 cells overexpressing wild-type NOD2 (wtNOD2) and NOD2 with the R334Q mutation (Blau NOD2). Methods: RT-PCR was used to generate cDNA for the NOD2 gene from human peripheral blood. Using a commercial kit (Stratagene), site-directed mutagenesis was performed to create Blau NOD2 (a G1001A nucleotide transition to change an arginine to a glutamine codon - R334Q). Wild type NOD2 and BlauNOD2 were subcloned into pGC, a retroviral expression vector containing a yellow fluorescent protein (YFP) reporter. U937 cells, a human monocyte line, were stably transduced with pGC packaged in retrovirus containing wtNOD2, BlauNOD2 and an empty vector control (VC). Using the YFP reporter, cells were cloned by cell sorting and high expressing clones were established for use in all studies. mRNA was extracted from the U937 cells using the RNeasy (Qiagen) kit. Affymetrix (HG-U133) arrays with approximately 33,000 genes were then run on the three samples in duplicate. In order for a transcript to be considered upregulated or downregulated, concordance had to be present between the duplicate arrays and between the multiple probes used for each gene, and the expression must have been changed at least two fold. Results: The overexpression of NOD2 resulted in increased expression of multiple transcripts including IL-18 and MCP-1 in both wtNOD2 and BlauNOD2 as compared with the vector control. Genes regulated by either the Blau or wt NOD2 are critical to understanding the biology of NOD2 but presumably not responsible for the pathogenesis of Blau syndrome. At least 16 genes, most coding for histones, were differentially affected by BlauNOD2 vs. wtNOD2. Conclusion: Microarray is a powerful technique to analyze alterations in gene expression. The NOD2 gene markedly affects expression of many genes within a monocyte-like cell line.

Keywords: gene microarray • inflammation • immunomodulation/immunoregulation