The Role of Dynamic DNA Methylation in Liver Transplant Rejection in Children

Methods: Paired peripheral blood DNA samples were obtained before and after LT from 17 children, including 4 rejectors (Rs) and 13 nonrejectors (NRs), and assayed with MethylC capture sequencing approach covering 5 million CpGs in immune-cell-specific regulatory elements. Differentially methylated CpGs (DMCs) were identified using generalized linear regression models adjusting for sex and age and merged into differentially methylated regions (DMRs) comprising 3 or more DMCs.

Results: Contrasting Rs versus NRs, we identified 2238 DMCs in post-LT and 2620 DMCs in pre-LT samples, which clustered in 216 and 282 DMRs, respectively. DMCs associated with R were enriched in enhancers and depleted in promoters. Among DMRs, the proportion of hypomethylated DMRs increased from 61/282 (22%) in pre-LT to 103/216 (48%, < 0.0001) in post-LT samples. The highest-ranked biological processes enriched in post-LT DMCs were antigen processing and presentation via major histocompatibility complex (MHC) class I, MHC class I complex, and peptide binding ( < 7.92 × 10), respectively. Top-ranked DMRs mapped to genes that mediate B-cell receptor signaling () or regulate several immune cells ) ( < 3.75 × 10). DMRs in MHC class I genes were enriched for single nucleotide polymorphisms (SNPs), which bind transcription factors, affect gene expression and splicing, or alter peptide-binding amino acid sequences.

Conclusions: Dynamic methylation in distal regulatory regions reveals known transplant-relevant MHC-dependent rejection pathways and identifies novel loci for future mechanistic evaluations in pediatric transplant subcohorts.