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

Abstract

BackgroundEpigenetic 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.MethodsWe 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.ResultsAll 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.ConclusionsWe 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.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-017-3482-3) contains supplementary material, which is available to authorized users.

Inflammation and cancer

“…However, it remains expensive to achieve sufficient sequencing depth for high accuracy, and it is difficult to apply this technique to small samples. We recently explored methylated tumor suppressor genes using affinity-based enrichment with a methyl-CpG-binding domain protein, followed by a next-generation sequencer (methyl-capture sequencing) [ 46 ]. This method can be applied to relatively small samples at less cost.…”

“…The BLUEPRINT consortium evaluated BAS as one of the best all-round methods for use in DNA methylation assays in large-scale validation studies, biomarker development, and clinical diagnostics [ 48 ]. We found several differentially methylated candidate genes in nasopharyngeal carcinoma tissues compared to normal nasopharynx tissues that may be useful biomarkers for cancer screening [ 46 ]. Among these candidates, we measured the methylation rates of RERG in nasopharynx biopsy specimens by using restriction enzyme-based real-time PCR, which is more convenient than BAS.…”

DNA methylation biomarkers for nasopharyngeal carcinoma

“…DNA methylation is closely related to gene expression regulation [10], embryonic development [11], X chromosome inactivation [12], genomic stability [13] and genomic imprinting [14]. DNA methylation is more common in patients with EB virus-associated NPC [15]. Abnormal DNA methylation affects the function of key genes, especially tumour suppressor genes, and thus participates in various processes of nasopharyngeal carcinoma development.…”

Epstein-Barr Virus-Induced Epigenetic Pathogenesis of Viral-Associated Lymphoepithelioma-Like Carcinomas and Natural Killer/T-Cell Lymphomas

“…Using genome-wide techniques, epigenomes (CpG methylomes) of EBV-associated tumors have been established, with the discovery of novel and known methylated genes involved in EBV-associated tumorigenesis. NPC methylomes have been established using methylated DNA immunoprecipitation coupled with microarrays (MeDIP-chip) [ 27 ], HumanMethylation450 (analyzing 485,000 CpG sites per genome) BeadChip [ 28 , 29 ], and MethylCap-sequencing [ 30 ]. EBVaGC methylomes have been profiled using Infinium HumanMethylation27 (analyzing 27,000 CpG sites per genome) and HumanMethylation450 BeadChips [ 31 , 32 , 33 , 34 ].…”