Loss of DNA methylation at imprinted loci is a frequent event in hepatocellular carcinoma and identifies patients with shortened survival

Abstract

Background: Aberrant DNA methylation at imprinted loci is an important molecular mechanism contributing to several developmental and pathological disorders including cancer. However, knowledge about imprinting defects due to DNA methylation changes is relatively limited in hepatocellular carcinoma (HCC), the third leading cause of cancer death worldwide. Therefore, comprehensive quantitative DNA methylation analysis at imprinted loci showing ~50 % methylation in healthy liver tissues was performed in primary HCC specimens and the peritumoural liver tissues.

Results: We found frequent and extensive DNA methylation aberrations at many imprinted loci in HCC. Unsupervised cluster analysis of DNA methylation patterns at imprinted loci revealed subgroups of HCCs with moderate and severe loss of methylation. Hypomethylation at imprinted loci correlated significantly with poor overall survival (log-rank test, p = 0.02). Demethylation at imprinted loci was accompanied by loss of methylation at LINE-1, a commonly used marker for global DNA methylation levels (p < 0.001). In addition, we found that loss of methylation at imprinted loci correlated with the presence of a CTNNB1 mutation (Fisher's exact test p = 0.03). Re-analysis of publically available genome-wide methylation data sets confirmed our findings. The analysis of benign liver tumours (hepatocellular adenoma (HCA) and focal nodular hyperplasia (FNH)), the corresponding adjacent liver tissues, and healthy liver tissues showed that aberrant DNA methylation at imprinted loci is specific for HCC.

Conclusions: Our analyses demonstrate frequent and widespread DNA methylation aberrations at imprinted loci in human HCC and identified a hypomethylated subgroup of patients with shorter overall survival.

Keywords: DNA methylation; Hepatocellular carcinoma; Imprinting; Pyrosequencing.

Fig. 1

Aberrant DNA methylation at multiple imprinted loci in primary HCC. All individual methylation values quantified with high-resolution pyrosequencing from 40 primary HCC specimens, 34 adjacent liver specimens, and 5 healthy liver tissues are displayed with scatter plots. Black triangles represent HCCs and grey triangles the adjacent liver tissues. The results of the Mann Whitney U test are indicated: **p < 0.01; ***p < 0.001. Additional file 1: Table S1 contains a complete compilation of the results of all statistical calculations

Fig. 2

Cluster analysis of methylation levels at imprinted loci. DNA methylation patterns at imprinted loci separate HCC into three subgroups. Unsupervised clustering of methylation levels at imprinted loci was performed. Upper panel: red = gain of methylation, blue = moderate loss of methylation, and green = severe loss of methylation. Lower panel: distribution of CTNNB1 (β-catenin) mutations

Fig. 3

Aberrations of DNA methylation at imprinted loci and global DNA methylation levels. Loss of methylation at imprinted loci is accompanied by reduced DNA methylation at LINE-1 sequences which serves as a surrogate marker for global DNA methylation level [21]. DNA methylation at LINE-1 sequences is significantly lower in the subgroup with subtle loss of methylation. The difference is even more pronounced in the subgroup displaying severe loss of methylation at imprinted loci. ***p < 0.001

Fig. 4

Survival analysis. The overall survival of HCC patients displaying hypomethylation at imprinted loci is significantly lower than in patients with retention of imprint methylation. The Kaplan-Meier plot shows survival of two groups (loss and gain of methylation at imprinted loci), log-rank test p = 0.02, median survival 41 and 156 weeks, respectively