Microdeletion of target sites for insulator protein CTCF in a chromosome 11p15 imprinting center in Beckwith-Wiedemann syndrome and Wilms' tumor

Abstract

We have analyzed several cases of Beckwith-Wiedemann syndrome (BWS) with Wilms' tumor in a familial setting, which give insight into the complex controls of imprinting and gene expression in the chromosome 11p15 region. We describe a 2.2-kbp microdeletion in the H19/insulin-like growth factor 2 (IGF2)-imprinting center eliminating three target sites of the chromatin insulator protein CTCF that we believe here is necessary, but not sufficient, to cause BWS and Wilms' tumor. Maternal inheritance of the deletion is associated with IGF2 loss of imprinting and up-regulation of IGF2 mRNA. However, in at least one affected family member a second genetic lesion (a duplication of maternal 11p15) was identified and accompanied by a further increase in IGF2 mRNA levels 35-fold higher than control values. Our results suggest that the combined effects of the H19/IGF2-imprinting center microdeletion and 11p15 chromosome duplication were necessary for manifestation of BWS.

Fig. 1.

IC1 structure in the three-generation BWS family with WT. (A) Pedigree of a large three-generation family with three children affected by BWS and WT. *, Those individuals who inherited the same 11p15 maternal haplotype, along with the IC1 deletion described in this report. The proband (III.1) is indicated by an arrow. (B) Organization of the paternal and maternal IC1 region in members of the BWS/WT pedigree. The integral DMR status on the paternal haplotype of individuals III.1, III.2, III.3, III.4, and III.5 is shown, with B repeats represented by yellow boxes and A repeats denoted by gray boxes. The locations of the CTCF-binding sites are denoted by small orange hatches. The core CTS sequence is shown, with the blackened C or green T, denoting a paternal or maternal single-nucleotide polymorphism (SNP), respectively. Nucleotides specific for the B1 and B5 sequences are marked in red and blue, respectively. The microdeletion generates a chimeric CTS sequence derived from B1 and B5 repeats in the maternal haplotypes of III.1, III.2, III.4, and III.5. The heterozygous nature of the deletion in III.1 was verified by Southern blot analysis (Left).

Fig. 2.

Methylation status of IC1 in children III.1, III.2, III.3, and III.4. Allele-specific methylation was analyzed by bisulfite sequencing of IC1 fragments, including the core CTS in the B1 and B5/B1 repeats (boxed). Paternal and maternal clones, identified by four SNPs (CTAT and TAGG), are grouped. •, methylated CpGs; ○, transformed unmethylated CpGs. Allele-specific paternal methylation was detected for the complete B1, as well as the fused B5/B1 sequences, of all four DNA samples.

Fig. 3.

Chromosome analysis, genomic quantitation of IC1 alleles, and quantitative RT-PCR analysis of allelic IGF2 expression. (A) Metaphase spreads of primary fibroblasts from an affected individual (III.1) and unaffected individual (III.4) were analyzed. High-resolution GTG banding revealed a prolonged 11p15 subtelomeric region (black arrow) on one chromosome 11 of the proband. (B) FISH analysis with a genomic probe for IGF2 shows two signals on one chromosome 11 of the proband (yellow arrow), indicating a duplication of distal 11p. (Inset) An enlarged view of 11p to illustrate the duplication (yellow arrow). (C) Quantitation of IGF2 gene dosage by using real-time PCR quantification. (D) Quantitative RT-PCR analysis, haplotype-specific expression, and promoter usage of the IGF2 gene. The relative IGF2 mRNA amounts are represented by horizontal bars, with colored segments denoting contribution of the parental haplotypes. As well, the relative contribution to IGF2 expression by the P3 or P4 promoters is indicated by darker and lighter shading, respectively. Allelic contribution to expression levels was determined by sequencing cloned transcripts of all individuals heterozygous for a SNP in IGF2 exon 9.

Fig. 4.

Schematic representation of the IC1 microdeletion effects on IGF2 expression. (Upper) The current enhancer insulation model (7). In the analyzed BWS/WT family (Lower), the microdeletion results in LOI of IGF2, although differential methylation is maintained.