Distinct methylation changes at the IGF2-H19 locus in congenital growth disorders and cancer

Abstract

Background: Differentially 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 findings: We 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/significance: These 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.

Figure 1. IGF2 DMR0 Methylation levels in Beckwith-Wiedemann Syndrome (BWS) and Silver-Russell Syndrome (SRS) patients.

A. Location of DMR0 within the IGF2 gene relative to the paternally methylated imprinting control region IC1 which is upstream of H19. Pyrosequencing was used to analyse methylation at six CpGs within the DMR0 region (NCBI 36: 11, 2125904–2126160, containing SNP rs3741210). Bisulphite (BSQ) and pyrosequencing (PSQ) primer positions are as indicated. This region contains the CpGs (numbered 15,16,17) marked in grey which have previously been published to be hypomethylated in Colorectal cancer . Vertical lines indicate MspI/HpaII sites in the DMR0. B. Box plot showing medians, IQR, max. and min. values for IGF2 DMR0 methylation levels as measured by pyrosequencing of bisulphite converted DNA obtained from peripheral blood samples from BWS and SRS patients. Methylation percentages reflect the proportion of bisulphite converted cytosines at the CpGs measured in the assay. Normal methylation levels for imprinted DMRs are 45–55% (see C). Categories of BWS patients included those with: no imprinting defect at the IC1 region and the IC2 region (NM); low methylation (LM) at the IC2 (IC2-LM); increased paternal 11p15 through paternal duplication or uniparental disomy (pUPD); and hypermethylation at IC1 (IC1-HM). SRS patients included two each of maternal duplication of 11p15 (Mat Dup) and hypomethylation at IC1 (IC1-LM). BWS with hypermethylation at the IC1, also have hypermethylation at DMR0, while SRS with hypomethylation IC1 have hypomethylation at DMR0. C. Parent of origin specific methylation in normal, BWS and SRS patients. The BWS and SRS patients have imprinting defects at IC1. Bisulphite converted DNA was amplified with allele-specific primers for the rs3741210 polymorphism in IGF2 DMR0 prior to pyrosequencing for methylation. The T allele is shown by squares and C alleles shown by triangles in the plots. Circles show the percentage of total methylation of the CpGs assayed. The paternal allele is more methylated at DMR0 than the maternal allele. Both alleles are hypermethylated in BWS and both alleles are hypomethylated in SRS.

Figure 2. Methylation levels at the IGF2 DMR0 region in Wilms tumour patients. A.

Box plot showing medians, IQR, max. and min. values for IGF2 DMR0 methylation levels as measured by pyrosequencing of peripheral blood (open box), normal kidney (hatched box) and tumour biopsies (grey box) from Wilms Tumour patients that do not have BWS. Methylation percentages reflect the proportion of bisulphite converted cytosines at the CpGs measured in the assay. Normal methylation levels for imprinted DMRs are 45–55% (see D). Wilms Tumour patient samples include peripheral blood and tumour DNA from patients with normal methylation at IC1 in their tumours (NM); peripheral blood, normal kidney and tumour DNA samples from patients with hypomethylation of IC1 in their tumours (IC1-HM); and tumour DNA samples from patients with loss of heterozygosity (LOH) in their tumours. Strikingly, Wilms Tumour patients with hypermethylation at IC1 have hypomethylation at DMR0 in their tumours. B Methylation levels in peripheral blood, unaffected kidney tissue and Wilms tumour kidney tissue from two BWS patients (one with IC1 hypermethylation and one with pUPD). Both patients have hypomethylation of IGF2 DMR0 in their tumours. C Linear regression curve showing inverse relationship between methylation levels at the IC1 and DMR0 in Wilms Tumour. Tumours with hypermethylation at the IC1 have loss of methylation at DMR0. D. Peripheral blood and kidney tissue from a patient with Wilms Tumour that does not have hypermethylation at IC1. The C-allele is hypermethylated, while the T-allele is hypomethylated in both the blood and kidney–indicating similar parent of origin methylation in these tissues.

Figure 3. Summary of IGF2 DMR0 Methylation relative to IC1 in normal, congenital growth disorders and Wilms tumour.

In normal individuals, IGF2 expression is from the paternal allele and H19 is from the maternal allele. IC1 and DMR0 are methylated on the paternal chromosome. In BWS, the epigenotype and expression pattern of the maternal chromosome are converted to paternal and in SRS, the epigenotype and expression pattern of the paternal chromosome are converted to maternal (imprint switching). In the Wilms tumour, IGF2 activation and H19 silencing are associated with disruption of both maternal and paternal imprints (somatic loss of imprinting).