Background Free radicals generated during ischemia reperfusion injury (IRI) might result in epigenetic modifications and gene expression (GE) changes affecting graft outcomes. Integrative analyses of DNA methylation (DNAm), miRNA and GE were done to identify epigenetic modifications and its influence on graft injury post-liver transplantation (LT).

Methods Graft biopsies were collected from LT patients at pre (L1) and post-LT (L2). Donor and recipient had similar clinical and demographic characteristics. Paired liver samples from grafts with mild injury (MI) and severe injury (SI) (AST / ALT > or <500 IU/L) post-LT were tested for DNAm (Illumina Infinium 450K methylation arrays), mRNA (GeneChip® HG-U133A v2.0) and microRNA(GeneChip® miRNA v4.0 array). Data and enrichment analyses were performed using Bioconductor packages minfi (scanned DNAm arrays);oligo (scanned GE arrays) and GenomeRunner, respectively.

Results 3323 CpG sites were differentially methylated (1820 hypomethylated, 1503 hypermethylated) between L1 and L2 (FDR<0.05), of which 2869 CpG sites were associated to 2480 genes. Genes encoding for epigenetic regulators such as DNA-m enzymes (DNMT1,DNMT3A), histone modifying enzymes (EP300, KDM1A,HDAC5) and miRNAs (MIR1204, MIR1258, MIR320A, MIR663) were differentially methylated at the promoter region. Comparative analysis of MI vs. SI (L1/L2) subgroups revealed 327 unique SI genes involved in cellular functions such as apoptosis activation (BNIP3, CRADD, SH2GLB1), oxidative stress (GSTZ1), immune response (CXCL16, CD3EAP, CISH, LAT, TLR9), hypoxia (ARNT), angiogenesis stimulator (TYMP), oxidative phosphorylation (COXBA, NUBPL), ubiquitination (UBEE3C, UBE53) and proteasomal degradation (PSMA7, PSMD8). Transcriptome analysis of the L1 vs. L2 showed 94% down and 6% upregulated genes. Evaluation of the MI and SI subgroups revealed 35 genes and several miRNAs unique to SI. Molecular characterization of the GE and DNAm data represented several overlapping pathways such as apoptosis,cellular stress, immune regulation, ECM formation, ubiquitin proteasome system, hepatotoxicity and the methylome genes were further involved in hypoxia,angiogenesis,oxidative phosphorylation, P53 and ERBB2-ERBB3 signaling.

Conclusions Epigenetic modifications of donor DNA have not been previously investigated in IRI pos-LT, but represents a great potential to modify gene expression and pathways related to graft injury.

CITATION INFORMATION: Bagchi D, Bontha S, Dozmorov M, Fernandez A, Mas V, Maluf D. Effect of Epigenetic Modifications in Ischemia-Reperfusion-Injury Post Liver Transplantation: An Integrative Approach. Am J Transplant. 2017;17 (suppl 3).

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