*Purpose: We investigated global DNA methylation (DNAm) patterns during ischemia reperfusion (I/R) injury in liver transplant (LT) patients, and their role on regulating gene expression and pathways associated with graft injury and graft outcomes.

*Methods: Ninety two paired biopsies (46 LT recipients, 38 deceased donors (DD) and 8 (living donors (LD)) LT patients were collected at pre-implantation (Pre-Imp) and 90 minutes post-reperfusion (Post-Rep). DD LT patients were sub-classified based on I/R injury severity post-LT as: high (HI, n=21) or low (LI, n=17) I/R injury based on AST/ALT >2,000 IU/L or <2,000 IU/L, respectively. LDLT recipients were used as controls. Methylome and transcriptome were interrogated and interactions were analyzed.

*Results: From the analyses of differentially methylated (Dme) CpGs at Pre-Imp from LD and DD LT, 18 Dme CpGs were identified (FDR<0.05), 6 CpGs (FDR<0.05 & Δb>0.10), respectively. For Post-Rep biopsies, the analyses of Dme CpGs at Post-Rep from LD and DD liver transplants, showed 1 Dme CpG (FDR<0.05), and 1 Dme CpG (FDR<0.05 & Δb>0.10), respectively. Among HI DDLT recipients there were 6334 Dme CpG sites Pre-Imp and Post-Rep using an FDR<0.01. Among LI DDLT recipients, there were 336 Dme CpG sites between Pre-Imp and Post-Rep (FDR<0.05). From gene expression (GE) using same samples, 2730 DE probe sets between Pre-Imp and Post-Rep samples were observed (FDR<0.01). When restricting attention to the LI group, there were 1338 DE probe sets Pre-Imp and Post-Rep samples (FDR<0.01). CGI regions were categorized into CGIs, CGI shelf, shores and non-CGI regions. Distribution of the Dme CpGs was analyzed showing 43% CGI with equal distribution (32%) of Dme CpGs on the promoter and gene body regions. Genes mapped to the Dme CpGs were related to the global GE microarray data. A total of 208 genes in the LI group and 45 genes in the HI group were common between the DNAm and GE datasets. The analysis of these genes showed activation of fibroblast cell proliferation, cell survival, cell cycle progression and inhibition of cell death in LI while cell movement of neutrophils, phagocytosis of blood cells, and cell viability were affected in the HI group. From the integrative analysis, ALOX5AP and S100P genes associated to the Highinjury group were identified as hypo-methylated at TSS site in the DNAm analyses and concurrently up-regulated (logFC >1; FDR<0.1) in the corresponding GE data set. Expression of ALOX5AP and S100P genes were also validated by RT-PCR. ALOX5AP showed significant up-regulation (HI Pre-Imp vs. Post-Rep: Adjusted P-value 0.003, LI Pre-Imp vs. Post-Rep: Adjusted P-value 0.0003). S100P followed the trend of up-regulation with less statistical power of significance P = 0.058 . MMP9 gene expression validated by RT-PCR showed significant up-regulation during reperfusion within high injury group (adjusted P-value =0.0001) and also between high and low injury group (adjusted P-value =0.002).

*Conclusions: Hereby, a key effect of ischemia on graft DNAm patterns was identified. For Pre-Imp biopsies, it was observed that DNAm modifications occurred mainly in DD samples. From integrative analyses it was observed that DNAm patterns play a key role in I/R injury by inducing differential GE changes affecting severity of graft injury and outcome.

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