Microdeletions in the human H19 DMR result in loss of IGF2 imprinting and Beckwith-Wiedemann syndrome

Abstract

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...

(Epi)genotype–phenotype correlations in Beckwith–Wiedemann syndrome

“…The following molecular anomalies were identified: 190 IC2-LoM (184 epigenetic anomalies, 5 already published cases with familial IC2 duplications 21 and 1 IC2 deletion), 87 UPD carriers, 31 IC1-GoM (21 already published cases 22,23 including one IC1 duplication, one translocation, 11 familial microdeletions [24][25][26] ), 10 CDKN1C variants (all unrelated cases, 9 maternally inherited). None of the patients tested was positive for genome-wide UPD.…”

CTCF Regulates Allelic Expression of Igf2 by Orchestrating a Promoter-Polycomb Repressive Complex 2 Intrachromosomal Loop

“…Indeed, maternally transmitted microdeletion of two CTCF sites in the ICR results in biallelic IGF2 expression and H19 silencing in BeckwithWiedemann syndrome (33). Using nuclear transfer, we previously showed that aberrant IGF2 imprinting in human tumor cells was repaired by the imprinting machinery in the normal fibroblast cytoplasm, leading to monoallelic expression of IGF2 in the reconstructed tumor cybrids or hybrids.…”

Epigenetic deregulation of imprinting in congenital diseases of aberrant growth

“…For instance, there are rare micro-deletions at the human H19 ICR that lead to loss of IGF2 imprinting and, hence, BWS. (50,51) One of these small deletions comprised only two of the six CTCF-binding sites at this ICR. Nevertheless, this, somehow, induced hypermethylation along the entire ICR upon maternal transmission, leading to biallelic IGF2 expression and loss of H19 expression.…”

“…Nevertheless, this, somehow, induced hypermethylation along the entire ICR upon maternal transmission, leading to biallelic IGF2 expression and loss of H19 expression. (50) One possible hypothesis to explain this broad effect is that key elements in the ICR, including the CTCF sites, determine the chromatin and nucleosomal organisation of the entire imprinted domain, thus dictating its behaviour in the germline and during development. (52) Future directions As is often the case with exciting novel data, the discovery that the IGF2-H19 locus is involved in the aetiology of SRS raises many questions.…”