Genomic imprinting is an epigenetic phenomenon restricting gene expression in a manner dependent on parent of origin. Imprinted gene products are critical regulators of growth and development, and imprinting disorders are associated with both genetic and epigenetic mutations, including disruption of DNA methylation within the imprinting control regions (ICRs) of these genes. It was recently reported that some patients with imprinting disorders have a more generalised imprinting defect, with hypomethylation at a range of maternally methylated ICRs. We report a cohort of 149 patients with a clinical diagnosis of Beckwith -Wiedemann syndrome (BWS), including 81 with maternal hypomethylation of the KCNQ1OT1 ICR. Methylation analysis of 11 ICRs in these patients showed that hypomethylation affecting multiple imprinted loci was restricted to 17 patients with hypomethylation of the KCNQ1OT1 ICR, and involved only maternally methylated loci. Both partial and complete hypomethylation was demonstrated in these cases, suggesting a possible postzygotic origin of a mosaic imprinting error. Some ICRs, including the PLAGL1 and GNAS/NESPAS ICRs implicated in the aetiology of transient neonatal diabetes and pseudohypoparathyroidism type 1b, respectively, were more frequently affected than others. Although we did not find any evidence for mutation of the candidate gene DNMT3L, these results support the hypotheses that trans-acting factors affect the somatic maintenance of imprinting at multiple maternally

The overgrowth-and tumor-associated Beckwith-Wiedemann syndrome results from dysregulation of imprinted genes on chromosome 11p15.5. Here we show that inherited microdeletions in the H19 differentially methylated region (DMR) that abolish two CTCF target sites cause this disease. Maternal transmission of the deletions results in hypermethylation of the H19 DMR, biallelic IGF2 expression, H19 silencing and Beckwith-Wiedemann syndrome, indicative of loss of function of the IGF2-H19 imprinting control element.A subclass of individuals with Beckwith-Wiedemann syndrome (BWS; OMIM 130650) show hypermethylation and silencing of the maternal H19 allele and activation of both parental IGF2 alleles 1,2 . Upregulation of IGF2 is important for the somatic overgrowth and tumor predisposition of BWS; the role of the putative tumor-suppressor gene H19 is uncertain 3,4 . No mutation has yet been associated with these epigenetic alterations. In mice, a targeted deletion showed that imprinting of the paternally expressed Igf2 and maternally expressed H19 genes is controlled by a 2-kb DMR located 5¢ of H19 (ref. 5). The DMR contains a chromatin boundary that is methylated on the paternal chromosome and interacts with the zinc finger protein CTCF on the maternal chromosome 6-9 . This binding is required to maintain the unmethylated status and the insulator function of the maternal DMR 10-12 .We analyzed seven individuals with BWS with H19 hypermethylation for the presence of deletions in the H19 DMR. The H19 DMR sequence differs from the homologous mouse region in that it is organized into two repeat units, each consisting of two types of direct repeats, and contains seven potential binding sites for CTCF (Fig. 1a) 13 . We analyzed by Southern blotting a region spanning all the repeats and found an allele with a 1.8-kb deletion in two individuals (Fig. 1b). We then amplified DNA fragments encompassing the deletion's breakpoints by PCR and sequenced them. The two mutations were very similar, each deleting 2.5 copies of the B repeat and one copy of A repeat and abolishing two CTCF sites (Fig. 1a). Both deletions were generated by recombination of the B6 repeat with the B3 repeat and differed only with respect to the cross-over breakpoints, which were 20 bp apart. The sister of proband 1 died prenatally with signs of BWS. By PCR amplification of an 88-bp fragment encompassing the mutation from an autopsy specimen, we found that she also inherited the 1.8-kb deletion from her mother (data not shown). We then looked for the presence of the deletion in the relatives of the probands who had no features of BWS. In family 1, we found the mutation in the mother, maternal grandfather, an uncle and his daughters (Fig. 1b). In family 2, we found the mutation in the mother, maternal aunt and maternal grandfather, indicating that the 1.8-kb deletion is associated with the BWS phenotype only when maternally transmitted (Fig. 1b). We did not detect the 1.8-kb deletion in any of 14 individuals with BWS with defects other than H19 hypermethylation or...

BackgroundDifferentially methylated regions (DMRs) are associated with many imprinted genes. In mice methylation at a DMR upstream of the H19 gene known as the Imprint Control region (IC1) is acquired in the male germline and influences the methylation status of DMRs 100 kb away in the adjacent Insulin-like growth factor 2 (Igf2) gene through long-range interactions. In humans, germline-derived or post-zygotically acquired imprinting defects at IC1 are associated with aberrant activation or repression of IGF2, resulting in the congenital growth disorders Beckwith-Wiedemann (BWS) and Silver-Russell (SRS) syndromes, respectively. In Wilms tumour and colorectal cancer, biallelic expression of IGF2 has been observed in association with loss of methylation at a DMR in IGF2. This DMR, known as DMR0, has been shown to be methylated on the silent maternal IGF2 allele presumably with a role in repression. The effect of IGF2 DMR0 methylation changes in the aetiology of BWS or SRS is unknown.Methodology/Principal FindingsWe analysed the methylation status of the DMR0 in BWS, SRS and Wilms tumour patients by conventional bisulphite sequencing and pyrosequencing. We show here that, contrary to previous reports, the IGF2 DMR0 is actually methylated on the active paternal allele in peripheral blood and kidney. This is similar to the IC1 methylation status and is inconsistent with the proposed silencing function of the maternal IGF2 allele. Beckwith-Wiedemann and Silver-Russell patients with IC1 methylation defects have similar methylation defects at the IGF2 DMR0, consistent with IC1 regulating methylation at IGF2 in cis. In Wilms tumour, however, methylation profiles of IC1 and IGF2 DMR0 are indicative of methylation changes occurring on both parental alleles rather than in cis.Conclusions/SignificanceThese results support a model in which DMR0 and IC1 have opposite susceptibilities to global hyper and hypomethylation during tumorigenesis independent of the parent of origin imprint. In contrast, during embryogenesis DMR0 is methylated or demethylated according to the germline methylation imprint at the IC1, indicating different mechanisms of imprinting loss in neoplastic and non-neoplastic cells.