The Human Retinoblastoma Gene Is Imprinted

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Abstract
Genomic imprinting is an epigenetic process leading to parent-of-origin–specific DNA methylation and gene expression. To date, ∼60 imprinted human genes are known. Based on genome-wide methylation analysis of a patient with multiple imprinting defects, we have identified a differentially methylated CpG island in intron 2 of the retinoblastoma (RB1) gene on chromosome 13. The CpG island is part of a 5′-truncated, processed pseudogene derived from the KIAA0649 gene on chromosome 9 and corresponds to two small CpG islands in the open reading frame of the ancestral gene. It is methylated on the maternal chromosome 13 and acts as a weak promoter for an alternative RB1 transcript on the paternal chromosome 13. In four other KIAA0649 pseudogene copies, which are located on chromosome 22, the two CpG islands have deteriorated and the CpG dinucleotides are fully methylated. By analysing allelic RB1 transcript levels in blood cells, as well as in hypermethylated and 5-aza-2′-deoxycytidine–treated lymphoblastoid cells, we have found that differential methylation of the CpG island skews RB1 gene expression in favor of the maternal allele. Thus, RB1 is imprinted in the same direction as CDKN1C, which operates upstream of RB1. The imprinting of two components of the same pathway indicates that there has been strong evolutionary selection for maternal inhibition of cell proliferation. Genomic imprinting is an epigenetic process leading to parent-of-origin–specific DNA methylation and gene expression. Defects in this process lead to abnormal development, growth, or behavior. It is still unclear why and how imprinting evolved and how many human genes are imprinted. Based on genome-wide DNA methylation analysis in a patient with a generalized imprinting defect, we have found that the paradigmatic retinoblastoma 1 (RB1) gene on chromosome 13 is imprinted. Imprinting of RB1 is linked to the insertion of a DNA sequence derived by retrotransposition from a gene on chromosome 9. Part of the inserted DNA sequence has evolved into a differentially methylated alternative RB1 promoter. Differential methylation of this sequence skews expression of the RB1 gene in favour of the maternal allele. The direction of the imprint imposed on the RB1 gene is the same as of the maternally expressed CDKN1C gene, which operates upstream of RB1. The imprinting of two components of the same pathway indicates that there has been strong evolutionary selection for maternal inhibition of cell proliferation.