Quantitation of DNA methylation in Epstein-Barr virus-associated nasopharyngeal carcinoma by bisulfite amplicon sequencing

BMC Cancer. 2017 Jul 17;17(1):489. doi: 10.1186/s12885-017-3482-3.

Abstract

Background: Epigenetic changes, including DNA methylation, disrupt normal cell function, thus contributing to multiple steps of carcinogenesis. Nasopharyngeal carcinoma (NPC) is endemic in southern China and is highly associated with Epstein-Barr virus (EBV) infection. Significant changes of the host cell methylome are observed in EBV-associated NPC with cancer development. Epigenetic marks for NPC diagnosis are urgently needed. In order to explore DNA methylation marks, we investigated DNA methylation of candidate genes in EBV-associated nasopharyngeal carcinoma.

Methods: We first employed methyl-capture sequencing and cDNA microarrays to compare the genome-wide methylation profiles of seven NPC tissues and five non-cancer nasopharyngeal epithelium (NNE) tissues. We found 150 hypermethylated CpG islands spanning promoter regions and down-regulated genes. Furthermore, we quantified the methylation rates of seven candidate genes using bisulfite amplicon sequencing for nine NPC and nine NNE tissues.

Results: All seven candidate genes showed significantly higher methylation rates in NPC than in NNE tissues, and the ratios (NPC/NNE) were in descending order as follows: ITGA4 > RERG > ZNF671 > SHISA3 > ZNF549 > CR2 > RRAD. In particular, methylation levels of ITGA4, RERG, and ZNF671 could distinguish NPC patients from NNE subjects.

Conclusions: We identified the DNA methylation rates of previously unidentified NPC candidate genes. The combination of genome-wide and targeted methylation profiling by next-generation sequencers should provide useful information regarding cancer-specific aberrant methylation.

Keywords: Bisulfite amplicon sequencing; DNA methylation; Epigenetic mark; Methyl-capture sequencing; Nasopharyngeal carcinoma.

MeSH terms

  • Adult
  • Aged
  • Carcinoma / diagnosis
  • Carcinoma / genetics*
  • Carcinoma / pathology
  • Carcinoma / virology
  • Cell Line, Tumor
  • CpG Islands / genetics
  • DNA Methylation / genetics*
  • Diagnosis, Differential
  • Epigenesis, Genetic / genetics
  • Epithelium / metabolism
  • Epstein-Barr Virus Infections / complications
  • Epstein-Barr Virus Infections / genetics*
  • Epstein-Barr Virus Infections / virology
  • Female
  • GTP Phosphohydrolases / genetics*
  • Herpesvirus 4, Human / genetics
  • Herpesvirus 4, Human / pathogenicity
  • High-Throughput Nucleotide Sequencing
  • Humans
  • Integrin alpha6 / genetics*
  • Male
  • Middle Aged
  • Nasopharyngeal Carcinoma
  • Nasopharyngeal Neoplasms / diagnosis
  • Nasopharyngeal Neoplasms / genetics*
  • Nasopharyngeal Neoplasms / pathology
  • Nasopharyngeal Neoplasms / virology
  • Nasopharynx / metabolism
  • Tumor Suppressor Proteins / genetics*

Substances

  • ITGA6 protein, human
  • Integrin alpha6
  • Tumor Suppressor Proteins
  • ZNF671 protein, human
  • GTP Phosphohydrolases
  • RERG protein, human