Michael J. Higgins
Roswell Park Cancer Institute
Changes in the expression of 11p15.5 genes can result in Beckwith-Wiedemann syndrome (BWS) and tumors. We have identified a CpG-island exhibiting maternal specific methylation in an intron of the KvLQT1 gene. Sequence identity with ESTs and RT-PCR indicated the presence of a transcript including the CpG-island and oriented in the opposite direction (i.e. antisense) with respect to KvLQT1. This transcript is at least 3.2 kb long and shows no evidence of introns or a significant ORF. SSCP and REF failed to detect polymorphisms over the transcribed region suggesting a high degree of conservation. The likely murine homologue was sequenced and exhibits 74-98% identity over 400 bp with the human transcript. SSCP analysis using F1 fetal liver cDNA from interspecific crosses suggests that the mouse transcript is expressed exclusively from the paternal allele. This transcript is analogous to the antisense RNA initiating from an intronic CpG-island in the Igf2 receptor (Igf2r) gene, the expression of which was found to be necessary for the imprinting of lgf2r itself. Although differential methylation at the human CpG-island was observed in DNA samples from 18 normal individuals, absence or reduction in the methylated allele was observed in DNA from cytogenetically normal BWS patients that show normal methylation and expression at H19. Furthermore the methylated allele was absent in fibroblasts derived from a fetus with suspected BWS, an inv(11) (p13p15.5), and loss of imprinting (LOI) at IGF2. However, normal methylation was preserved in BWS patients with H19 hypermethylation and IGF2 LOI. These findings are consistent with there being two distinct imprinting control centers in 1lp15.5. These results will be presented in the context of the expression competition model of genomic imprinting and BWS as a model disorder for elucidating the mechanisms of IGF2 imprinting. (Funded by NIH CA63333)