DNA hypermethylation/boundary control loss identified in retinoblastomas associated with genetic and epigenetic inactivation of the RB1 gene promoter.

DNA hypermethylation events occur frequently in human cancers, but less is known of the mechanisms leading to their initiation. Retinoblastoma, an intraocular cancer affecting young children, involves bi-allelic inactivation of the RB1 gene (RB-/- ). RB1 encodes a tumour suppressing, cell cycle regulating transcription factor (pRB) that binds and regulates the RB1 core and other E2F responsive promoters with epigenetic functions that include recruitment of histone deacetylases (HDACs). Evidence suggests that bi-allelic epigenetic inactivation/hypermethylation of the RB1 core promoter (PrE-/E- ), is specific to sporadic retinoblastomas (frequency~10%), whereas heritable RB1 promoter variants (Pr-/+ , frequency~1-2%) are not associated with known epigenetic phenomena. We report heritable Pr-/- retinoblastomas with the expected loss of pRB expression, in which hypermethylation consistent with distal boundary displacement (BD) relative to normal peripheral blood DNAs was detected in 4/4 cases. In contrast, proximal BD was identified in 16/16 RB-/- retinoblastomas while multiple boundaries distal of the core promoter was further identified in PrE-/E- and PrE-/E+ retinoblastomas. However, weak or no DNA hypermethylation/BD in peripheral blood DNA was detected in 8/9 Pr-/+ patients, with the exception, a carrier of a microdeletion encompassing several RB1 promoter elements. These findings suggest that loss of boundary control may be a critical step leading to epigenetic inactivation of the RB1 gene and that novel DNA methylation boundaries/profiles identified in the RB1 promoter of Pr-/- retinoblastomas, may be the result of epigenetic phenomena associated with epimutation in conjunction with loss of pRB expression/binding and/or RB1 promoter interactions with boundary control elements.