Multipoint analysis of human chromosome 11p15/ mouse distal chromosome 7: inclusion of H19/IGF2 in the minimal WT2 region, gene specificity of H19 silencing in Wilms' tumorigenesis and methylation hyper-dependence of H19 imprinting

WT2 is defined by maternal-specific loss of heterozygosity (LOH) on chromos ome 11p15.5 in Wilms' tumors (WTs). The imprinted H19 gene, in this region, is silenced and hypermethylated in most WTs, and this is linked to patholo gical biallelic expression of IGF2. However, H19 and IGF2 lie within a larg er imprinted domain, and the gene specificity of H19 epimutation has been a persistent question. To address this, we assessed LOH, gene expression and DNA methylation at multiple sites in and around the imprinted domain. LOH mapping showed that the entire domain, including IGF2/H19, is within the mi nimal WT2 region. Genes within the domain, including IPL/TSSC3/BWR1C, IMPT1 /ORCTL2/BWR1A/TSSC5, KvLQT1/KCNA9 and TAPA1/CD81, as well as the zinc finge r gene ZNF195/ZNFP104 near the centromeric border, were expressed persisten tly in many WTs. DNA hypermethylation was not detected with 5' upstream pro bes for IPL, IMPT1, KvLQT1 and ZNF195 in WTs or WT-associated kidneys. Full y developed WTs showed variable hypomethylation at an imprinted CpG island in a KvLQT1 intron, but this was only complete in the cases with LOH and wa s not observed in pre-neoplastic WT-associated kidneys with H19 epimutation . Analysis of the corresponding region of mouse chromosome 7 using methyltr ansferase-hypomorphic mice showed that the H19 imprint was fully erased, bu t that the allelic bias at Ipl, Impt1, p57(Kip2) and, to a lesser extent, K v1qt1, persisted. Pre-existing massive allelic asymmetry for DNA methylatio n and hyper-dependence of transcription on methylation status may underlie the mechanism of gene-specific silencing of H19 in Wilms' tumorigenesis.