New bioinformatics approach to analyze gene expressions and signaling pathways reveals unique purine gene dysregulation profiles that distinguish between CD and UC

Abstract

Background: Expression of purine genes is modulated by inflammation or experimental colitis and altered expression leads to disrupted gut function. We studied purine gene dysregulation profiles in inflammatory bowel disease (IBD) and determined whether they can distinguish between Crohn's disease (CD) and ulcerative colitis (UC) using Pathway Analysis and a new Comparative Analysis of Gene Expression and Selection (CAGES) method.

Methods: Raw datasets for 22 purine genes and 36 probe-sets from National Center for Biotechnology Information (NCBI) GEO (Gene Expression Omnibus) (http://www.ncbi.nlm.nih.gov/projects/geo/) were analyzed by National Cancer Institute (NCI) Biological Resources Branch (BRB) array tools for random-variance of multiple/36 t-tests in colonic mucosal biopsies or peripheral blood mononuclear cells (PBMCs) of CD, UC or control subjects. Dysregulation occurs in 59% of purine genes in IBD including ADORA3, CD73, ADORA2A, ADORA2B, ADAR, AMPD2, AMPD3, DPP4, P2RY5, P2RY6, P2RY13, P2RY14, and P2RX5.

Results: In CD biopsies, expression of ADORA3, AMPD3, P2RY13, and P2RY5 were negatively correlated with acute inflammatory score, Crohn's Disease Activity Index (CDAI) or disease chronicity; P2RY14 was positively correlated in UC. In mucosal biopsies or PBMCs, CD and UC were distinguished by unique patterns of dysregulation (up- or downregulation) in purine genes. Purine gene dysregulation differs between PBMCs and biopsies and possibly between sexes for each disease. Ingenuity Pathway Analysis (IPA) revealed significant associations between alterations in the expression of CD73 (upregulation) or ADORA3 (downregulation) and inflammatory or purine genes (<or=10% of 57 genes) as well as G-protein coupled receptors, cAMP-dependent, and inflammatory pathways; IPA distinguishes CD from UC.

Conclusion: CAGES and Pathway Analysis provided novel evidence that UC and CD have distinct purine gene dysregulation signatures in association with inflammation, cAMP, or other signaling pathways. Disease-specific purine gene signature profiles and pathway associations may be of therapeutic, diagnostic, and functional relevance.

Figure 1

A negative correlation exists between duration of CD and mRNA expression of (A) ADORA3 and (B) P2RY14 in mucosal biopsy. Error bars = S.D.

Figure 2

Purine Gene dysregulation signature profiles clearly distinguish between UC and CD. (A) Purine gene dysregulation profiles in mucosal biopsies. (B) Purine gene dysregulation profiles in PBMCs. CAGES analysis was done as described in methods, and all alterations in gene expressions depicted in the histograms are statistically significant at p<0.001 – p<0.00001. Data is derived from Tables 2, Supplement Tables 1-3.

Figure 3

Canonical pathway analysis reveals novel signaling pathways associated with CD73 / NT5E or ADORA3 mRNA gene dysregulation that distinguish between CD and UC. (A, B) NT5E pathway analysis reveals that NT5E gene dysregulation is associated with acute phase response signaling, cAMP-mediated signaling, NRF2-mediated oxidative stress response and G-protein coupled receptor signaling (i.e. bars above threshold line for significant association). A distinct profile is observed in UC (B). ADORA3 pathway analysis reveals that ADORA3 gene dysregulation is associated with acute phase response signaling, cAMP-mediated signaling, G-protein coupled receptor signaling, IGF-1 signaling and IL-10 signaling. A distinct profile is observed in UC (D). Bars – The significance calculated for each function/Canonical Pathway is a measurement of the likelihood that the function is associated with the dataset by random chance. On the y-axis of the diagram, the significance is expressed as the minus log (10-based) of the p-values calculated from Ingenuity pathway analysis and Fischer's exact test. Threshold set at 0.05 for p-value, in the chart it corresponds to −log (0.05)). Note: the higher the bar, the lower the p-value and hence the more significant for the enrichment of the group/pathway/network. Ratio - The ratio between the number of genes in our list and the total number of genes in the defined functional group or pathway or network; curve just links all the data points for the ratio.