Hypermethylation of CpG island loci of multiple tumor suppressor genes in retinoblastoma.

Epigenetic silencing of tumor suppressor genes by methylation of discrete regions of the CpG islands is a major mechanism underlying tumorigenesis. Methylation of at least three of five specific genes may represent a distinct trait, termed the CpG island methylator phenotype (CIMP). Positive CIMP is associated with BRAF mutations. The present study sought to investigate the presence of BRAF mutations in human retinoblastoma and the role of epigenetic silencing of multiple tumor suppression genes in a search for methylation phenotype. Twenty-five archival retinoblastoma samples were analyzed for BRAF mutations with polymerase chain reaction, Mutector assay, and direct sequencing. Nineteen samples were also analyzed for the promoter methylation status of eight candidate cancer-related genes using real-time quantitative methylation-specific polymerase chain reaction after sodium bisulfite modification. The CIMP status was determined. No BRAF mutations were found. The frequencies of cancer-related gene methylation were as follows: 89% for RASSF1A, 52% for NEUROG1, 5% for DAP-kinase, RUNX3 and CACNA1G, and 0 for RAR-beta2, SOCS-1 and IGF-2. The lack of BRAF mutations in retinoblastoma is in accord with the negative CIMP status and the high hypermethylation rate for RASSF1A. The high methylation status for NEUROG1 may point to an alternative pathway in the development and progression of retinoblastoma, but further studies are needed.