DNA hypermethylation/boundary control loss identified in retinoblastomas associated with genetic and epigenetic inactivation of the <i>RB1</i> 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 <i>RB1</i> gene (<i>RB^−/-</i>). <i>RB1</i> encodes a tumour suppressing, cell cycle regulating transcription factor (<i>pRB)</i> that binds and regulates the <i>RB1</i> core and other <i>E2F</i> responsive promoters with epigenetic functions that include recruitment of histone deacetylases (<i>HDACs</i>). Evidence suggests that bi-allelic epigenetic inactivation/hypermethylation of the <i>RB1</i> core promoter (<i>Pr^E-/E-</i>), is specific to sporadic retinoblastomas (frequency~10%), whereas heritable <i>RB1</i> promoter variants (<i>Pr^−/+</i>, frequency~1-2%) are not associated with known epigenetic phenomena. We report heritable <i>Pr^−/-</i> retinoblastomas with the expected loss of <i>pRB</i> 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 <i>BD</i> was identified in <i>16/16 RB^−/-</i> retinoblastomas while multiple boundaries distal of the core promoter was further identified in <i>Pr^E-/E-</i>and <i>Pr^E-/E+</i> retinoblastomas. However, weak or no DNA hypermethylation/<i>BD</i> in peripheral blood DNA was detected in 8/9 <i>Pr^−/+</i> patients, with the exception, a carrier of a microdeletion encompassing several <i>RB1</i> promoter elements. These findings suggest that loss of boundary control may be a critical step leading to epigenetic inactivation of the <i>RB1</i> gene and that novel DNA methylation boundaries/profiles identified in the <i>RB1</i> promoter of <i>Pr^−/-</i> retinoblastomas, may be the result of epigenetic phenomena associated with epimutation in conjunction with loss of <i>pRB</i> expression/binding and/or <i>RB1</i> promoter interactions with boundary control elements.