Background: Epigenetics modifications in the graft may influence injury severity and function post liver transplantation (LT). This study aimed to interrogate the effect of different graft DNA methylation patterns on gene expression profiles that associate with graft injury after LT.

Methods: The study included 22 deceased donor LT patients with severe (SI, n=11; AST>500 IU/L) and mild (MI, n=11; AST<500 IU/L) early graft injury at 1-day post-LT. Tissue biopsies were collected at pre-implantation (L1) and at post-reperfusion (L2). Genomic DNA was extracted from L1 biopsies; bisulfite converted and used in Infinium 450K methylation arrays. Raw data was normalized by SWAN method and analyzed with R bioconductor. Beta scores were converted to M-values. F-test was fit for significant demethylated CpG sites (q<0.05). RNA was isolated from all biopsies, labeled and used in gene expression microarrays (L1 and L2). Probeset summaries were obtained using RMA algorithm. Unpaired ANOVA was fit for deregulated probesets (p<0.001, FDR <5%). Molecular pathways were evaluated by IPA tool. CpGs (Methylight) and genes (RT-PCR) were validated.

Results: There was not difference between groups in demographics, graft preservation type, and cold- and warm-ischemia times. In total, 3663 CpG sites (2574 hypomethylated; 1089 hypermethylated) were significantly demethylated and mapping within genic regions. Interestingly, 2251 CpGs (92% hypomethylated, p<0.0001) mapped within GC-islands located at promoter regions (1971 genes) in SI grafts. Molecular pathway analysis based on CpGs methylation identified apoptosis activation signaling (TP53, BIM, BAD, BAX, DIABLO, APAF1, CAD, FADD, CASP2, CASP3), ubiquitin protein degradation, and cell cycle regulation (Rb, p27KIP, p18INK4C, CDK, CHK1) (p<0.0001). Genes with multiple hypomethylated CpGs anticipated increases in liver cell death (BCL2L11, HSPD1, APP, INHA, TRIM27, BAD) and G1/S cell cycle check-point (SKP2, CDC25A, SIN3A, PA2G4). No genes were differentially expressed at pre-implantation. A unique significantly deregulated molecular signature (94 genes) correlating with CpG demethylation coincided with liver damage (KRT18), apoptosis and cell cycle regulation (PAWR, KRAS, DUSP4, PTPR3) at post-reperfusion in SI grafts. Genes were validated by RT-qPCR.

Conclusion: Hypomethylation of genes involved in the regulation of cell death pathways may induce increases in graft injury post-LT.

Preemptive intervention by pathway-specific blockage may reduce injury and related complications.

CITATION INFORMATION: Gehrau R, Bontha S, Mas V, Maluf D. DNA Methylation Patterns and Association with Graft Injury Severity Post Liver Transplantation. Am J Transplant. 2016;16 (suppl 3).

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