Genome-wide DNA methylation analysis on testicular embryonal carcinoma

Genomic DNA from 6 pure EC and 2 normal testis was fragmented and immunoprecipitated with anti-5mC monoclonal antibodies by MeDIP. After IP, DNA was purified and amplified using Whole Genome Amplification. Subsequently, DNA was biotin-labeled and hybridized to Human Tiling Array 2.0R Chips (Affymetrix) according to the manufacturer’s instruction. Raw data (CEL files) were normalized and analyzed by TAS (Affymetrix). Technical replicate of MeDIP-chip procedure was performed. Testicular embryonic carcinoma (EC) is a major subtype of non-seminomatous germ cell tumors (NSGCTs) in males. To identify epigenetic changes during testicular tumorigenesis, we profiled the DNA methylation of 6 ECs. These samples represent different stages (stage I and stage III) and different extent of invasiveness. Non-cancerous testicular tissues were included. A total of 1167 tumor-hypermethylated differential methylated regions (DMRs) were identified across the genome. Among them, 40 genes/ncRNA were found to have hypermethylated promoters. Interestingly, we found several hypermethylated sex-linked genes, including X-linked genes STAG2, SPANXD/E, MIR1184, Y-linked genes RBMY1A1/1B/1D and FAM197Y2P. Expression of RBMY1A was confirmed by immunohistochemistry in normal germ cells, but lost in EC and seminoma. Our genome-wide analysis identified methylation changes in several previously unknown genes for testicular ECs, which might provide insight into the crosstalk between normal germ cell development and carcinogenesis. A total of 8 DNA samples, including 6 EC and 2 normal testicular tissues