*Purpose: Transcriptional regulation of LT rejection may reveal novel predictive and therapeutic targets. To identify signaling pathways which are differentially regulated by DNA methylation in children with biopsy-proven acute cellular rejection (rejectors, R) after LT.

*Methods: Paired peripheral blood DNA samples were obtained before and after LT within the first 60 days from 21 children, 8R and 13 NR. Samples from 8 R were obtained before the diagnosis of rejection. Genome-wide methylation was assayed with a customized methylation capture sequencing (MCC-Seq) technology covering about 5 million CpGs located in regulatory or hypomethylated regions of DNA from whole blood. Differentially methylated CpGs (DMCs) upon different treatment responses were identified using generalized linear regression models (GLM) by adjusting sex and age.

*Results: Mean+/-SD age was 6.4+/-7.8 years, male: female gender was 9: 12. Rejectors demonstrated 3375 DMCs in pre-LT and 9302 DMCs in post-LT samples. The distribution of hypomethylated: hypermethylated DMCs increased significantly in post-LT samples compared with pre-LT samples, from 1693: 1682 to 6821: 2481 (p<0.0001). Pathways enriched among hypomethylated DMCs in post-LT samples from rejectors (p<0.005, FDR<0.05, Webgestalt, Hallmark H50) included Reactive oxygen species, TNF signaling via NFKB, IL2-STAT5 signaling and inflammatory response. Differentially methylated DNA loci within 1500 base pair upstream and 200 bp downstream of the transcription start sites (TSS) revealed a similar enrichment. Among rejectors, hypomethylated: hypermethylated DMCs increased significantly from 128: 148 pre-LT to 400: 213 post-LT, p=0.0001. The enriched top-ranked pathways in and near the TSS were complement, allograft rejection and peroxisome signaling (p<0.05, FDR>0.5, Webgestalt, Hallmark H50). Hypomethylated DMCs in complement pathways were CD59, FCN1, GCA, ITIH1, and SRC, and allograft rejection signaling were ELANE, FGR, FYB1 which regulates IL2 expression, SRGN, TLR6, which regulates cellular immune responses, and ZAP70, the zeta chain of the T-cell receptor associated protein kinase 70. Interestingly, some pathways enriched in post-LT samples were also enriched in pre-LT samples, albeit at p-values not adjusted for false discovery, likely because of the modest sample size. These pathways included IL2-STA5 signaling among genome-wide DMGs, and allograft rejection and peroxisome signaling in and near TSS.

*Conclusions: Hypomethylated DNA loci in immune signaling genes in peripheral blood leukocytes identify children at risk for early liver allograft rejection.

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