Putative Imprinting Control Regions with Aberrant Blood-Based DNA Methylation are Associated with Hepatocellular...

Site-specific 5-methylcytosine levels measured in blood can be suitable surrogate markers for epigenetic alterations in inaccessible tissues and are associated with hepatocellular carcinoma (HCC) risk. However, replication has been limited due to prior low genomic coverage and the tissue-specific nature of methylation at many CpG sites. One exception is imprinting control regions (ICRs), which use stable, early-onset methylation to regulate monoallelic expression across somatic tissues, implying aberrant methylation at ICRs detected in blood may be relevant to liver disease.

Leveraging primary HCC samples, we aimed to identify putative ICRs with aberrant DNA methylation associated with HCC to inform development of a blood-based risk stratification panel for earlier detection and clinical triage.

Whole genome bisulfite sequencing (WGBS) was performed on leukocyte-derived DNA of 10 primary HCC cases and 51 controls to identify differential methylation. An independent comparison of 29 primary HCC cases and 36 controls was used to validate differential methylation measured in leukocytes using the novel Human Imprintome Methylation Array.

We identified 1,519 differentially methylated regions associated with HCC by WGBS (FDR p < 0.05), which mapped to 81 putative ICRs, the majority of which had not previously been linked to HCC. Comparison with published studies further established 16 putative ICRs that have previously been associated with HCC or precancerous chronic liver disease states for a total of 97 loci. Validation using the Human Imprintome Methylation Array replicated 46 (57%) of these regions (FDR p < 0.05). Nearest genes were enriched for pathways related to liver disease including liver hyperplasia, hepatitis, steatosis, and HCC.

Our comprehensive genome profiling identified aberrant methylation at putative ICRs in blood that were associated with HCC. These findings support the hypothesis that ICRs, which exhibit relatively stable methylation across tissues, warrant further investigation as early blood-based HCC biomarkers.