Somatically acquired hypomethylation of IGF2 in breast and colorectal cancer

Yoko Ito, Thibaud Koessler, Ashraf E K Ibrahim, Sushma Rai, Sarah L Vowler, Sayeda Abu-Amero, Ana-Luisa Silva, Ana-Teresa Maia, Joanna E Huddleston, Santiago Uribe-Lewis, Kathryn Woodfine, Maja Jagodic, Raffaella Nativio, Alison Dunning, Gudrun Moore, Elena Klenova, Sheila Bingham, Paul D P Pharoah, James D Brenton, Stephan Beck, Manjinder S Sandhu, Adele Murrell, Yoko Ito, Thibaud Koessler, Ashraf E K Ibrahim, Sushma Rai, Sarah L Vowler, Sayeda Abu-Amero, Ana-Luisa Silva, Ana-Teresa Maia, Joanna E Huddleston, Santiago Uribe-Lewis, Kathryn Woodfine, Maja Jagodic, Raffaella Nativio, Alison Dunning, Gudrun Moore, Elena Klenova, Sheila Bingham, Paul D P Pharoah, James D Brenton, Stephan Beck, Manjinder S Sandhu, Adele Murrell

Abstract

The imprinted insulin-like growth factor 2 (IGF2) gene is expressed predominantly from the paternal allele. Loss of imprinting (LOI) associated with hypomethylation at the promoter proximal sequence (DMR0) of the IGF2 gene was proposed as a predisposing constitutive risk biomarker for colorectal cancer. We used pyrosequencing to assess whether IGF2 DMR0 methylation is either present constitutively prior to cancer or whether it is acquired tissue-specifically after the onset of cancer. DNA samples from tumour tissues and matched non-tumour tissues from 22 breast and 42 colorectal cancer patients as well as peripheral blood samples obtained from colorectal cancer patients [SEARCH (n=case 192, controls 96)], breast cancer patients [ABC (n=case 364, controls 96)] and the European Prospective Investigation of Cancer [EPIC-Norfolk (n=breast 228, colorectal 225, controls 895)] were analysed. The EPIC samples were collected 2-5 years prior to diagnosis of breast or colorectal cancer. IGF2 DMR0 methylation levels in tumours were lower than matched non-tumour tissue. Hypomethylation of DMR0 was detected in breast (33%) and colorectal (80%) tumour tissues with a higher frequency than LOI indicating that methylation levels are a better indicator of cancer than LOI. In the EPIC population, the prevalence of IGF2 DMR0 hypomethylation was 9.5% and this correlated with increased age not cancer risk. Thus, IGF2 DMR0 hypomethylation occurs as an acquired tissue-specific somatic event rather than a constitutive innate epimutation. These results indicate that IGF2 DMR0 hypomethylation has diagnostic potential for colon cancer rather than value as a surrogate biomarker for constitutive LOI.

Figures

Figure 1.
Figure 1.
Pyrosequencing assay to detect methylation levels at the IGF2 DMR0. (A) The human IGF2 gene showing positions of DMRs relative to exons and active promoters on the paternal allele. IGF2 promoters access enhancers downstream of H19 (ovals) when the CTCF binding site within the H19 DMR is methylated. IGF2 and H19 genes are 100 kb apart. The DMR0 region within the IGF2 has been reported to be hypomethylated in colorectal cancer. The expanded region shows the 26 CpGs within this region and the CpGs that we assayed for methylation by pyrosequencing. Our assay encompassed a 255 bp region, which includes six CpGs (CpG12–17 NCBI36 2125904–2126160). CpG15–17 were previously reported to be hypomethylated in colorectal patients with LOI at IGF2 (15). Arrows indicate position and direction of biotinylated, pyrosequencing (PSQ) and sequencing primers. Circles indicate individual CpGs on expanded DMR0. (B) Linearity of pyrosequencing assay: unmethylated template was obtained by PCR amplification of target DMR0 sequence prior to bisulphite conversion. After bisulphite conversion, the unmethylated template was serially diluted with PCR product obtained from bisulphite converted genomic DNA. At 100% genomic DNA, we have close to 50% methylation which is as expected for normal imprinting. (C) Pyrosequencing profiles for allele-specific methylation compared to total methylation. Total methylation levels reliably reflected the average of the two alleles. (D) Methylation diagrams for 12 cloned PCR products from bisulphite-treated DNA showing that alleles separate into methylated and unmethylated alleles and that methylation at CpG13 is not allele-specific. Filled and open circles denote methylated and unmethylated CpGs.
Figure 2.
Figure 2.
Distribution of average CpG methylation across IGF2 DMR0 and population frequency of methylation in a cohort of peripheral blood and placentae. (A) Graph showing average methylation levels of each CpG across the IGF2 DMR0 in peripheral blood, normal colon, normal breast and term placentae samples. (B) Cumulative frequency distribution of average methylation levels for CpG12, 14–17 in the IGF2 DMR0 in placentae and peripheral blood. At the 90% percentile average methylation levels for the DMR0 was 48%; at the 10% percentile, the average methylation level was less than 35%.
Figure 3.
Figure 3.
IGF2 DMR0 methylation levels in colorectal and breast cancers compared to normal tissue from same patients and non-cancer patients: prevalence of hypomethylation in breast and colorectal cancer tumours. (A) DMR0 methylation levels in DNA from colon tissue from patients with no cancer (Colon NC), and colorectal cancer patients' tumour tissue (CRC-T) and matched control biopsies of non-tumour tissues (CRC-NT). Tumours have significantly lower methylation than non-tumour tissue from same patients and this is lower than in colons from patients without cancer. (B) Line plots showing individual data comparisons between colorectal tumour (CRC-T) and matched control biopsies of non-tumour tissues (CRC NT). (C) LOI relative to IGF2 DMR0 methylation in colorectal cancer tissue: colorectal cancer patients with LOI have lower methylation in colon tissue than patients without LOI. Colorectal tumours (CRC-T) have lower methylation than non-tumour tissues (CRC NT) regardless of LOI. (D) DMR0 methylation levels in DNA from breast tissue from individuals with no cancer (Breast NC) and breast cancer patients' tumours (BC-T) and matched control biopsies of non-tumour tissue (BC-NT). Breast cancer tumour tissues have significantly lower methylation levels than non-tumour tissue. Non-tumour tissue from breast cancer patients has a wider variation of methylation levels than tissue from non-cancer patients, but median levels are not significantly different. (E) Line plots showing individual data comparisons between breast tumour (BC-T) and matched control biopsies of non-tumour tissues (BC NT). (Box and whisker plots represent Median and IQR with max and min values. P-values calculated by Wilcoxon Rank sum test.)

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