Peripheral Blood Gene Expression Analysis in Small Bowel Transplantation: a Pilot Study to Detect Novel Biomarkers of Graft Rejection

Abstract

Introduction: Long term survival of intestinal transplant patients is hampered by rejection episodes. Currently protocol endoscopy of the graft and biopsies are the only way to predict a rejection episode. Symptoms and clinical findings frequently appear when injury to the graft is irreversible. In this report we present our initial data of putative candidate biomarkers of graft rejection in peripheral blood of intestinal transplant patients. Materials and methods: Gene expression analysis was performed in peripheral blood of intestinal transplant patients. The results were matched with concurrent graft biopsies using bioinformatics. Peripheral blood samples of intestinal transplant patients were collected in Tempus Blood RNA tubes. Concurrent graft endoscopies were performed and graft biopsies were obtained at the time of the blood samples, to match the results of the gene expression analysis with the state of the graft. Whole genome microarray analysis using the Illumina-HT12 Expression beadchip microarray was performed. The gene expression levels in patient samples were compared to those in a pool of healthy volunteers. Bioinformatics analysis was performed using the MetaCore 6.3 software from GeneGo Inc. and the Pathway Studio 7.1 software from Ariadne Genomics. Results: Peripheral blood samples (n=11), of 3 adult patients [transplant day (n=1), no rejection (n=1), minimal rejection (n=2), mild rejection (n=5) and severe rejection (n=2)] were collected. Bioinformatics: Enrichment Analysis: The three most affected pathways differentially expressed in rejection versus a pool of healthy volunteers were related to protein translation: translation initiation, translation elongation termination, and translation in mitochondria, with p-values for all rejection stages in all patients in the 10-4 to 10-18 range. No significant enrichment was observed for these categories in the day of transplant sample. In addition to translation, significant enrichment of several immune response categories was observed in rejection samples. Subsequent gene set enrichment analysis verified these results. The level of enrichment was very high (p-values of 10-5 - 10-60) and increased with the level of rejection in all patients. Genes significantly down-regulated in translation related gene sets included ribosomal proteins RPL13A, RP L22, RPS23, RPL13 and RPL10A, that could be used as potential biomarkers for future experiments. These results were further verified with the use of additional samples of 2 more small bowel transplant patients. Conclusion: In this pilot study we found a list of genes (involved in translation) significantly down-regulated in the peripheral blood of intestinal transplant patients during rejection. Our hypothesis, derived from this project, is that down-regulation of translation in peripheral blood mononuclear cells occurs early during the course of rejection and persists and progresses with the severity of the rejection episode as determined by concurrent graft biopsies.