alexa Loss of Imprinting of the Insulin-Like Growth Factor II Gene Occurs by Biallelic Methylation in a Core Region of H19-Associated CTCF-binding Sites in Colorectal Cancer


Journal of Gastrointestinal & Digestive System

Author(s): Nakagawa H, Chadwick RB

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We hypothesize that loss of imprinting (LOI) of the insulin-like growth factor II (IGF2) gene is associated with a predisposition to sporadic colorectal cancer. We confirmed a previously known strong correlation between LOI and microsatellite instability and showed that LOI was not a consequence of microsatellite instability or mismatch repair deficiency. LOI of IGF2 correlated strongly with biallelic hypermethylation of a core of five CpG sites in the insulator region of IGF2/H19, which is a known CTCF-binding element. As this methylation-dependent LOI was present in both tumors and normal colonic mucosa, it is possible that hypermethylation creates a field defect predisposing to cancer. A methylator phenotype has been postulated to account for a subset of colorectal cancers (CRCs). In these sporadic, mostly right-sided tumors, hypermethylation of the CpG islands in the MLH1 promoter is associated with the absence of MLH1 protein, defective mismatch repair, and widespread microsatellite instability (MSI) (1–5). Among all CRCs the MSI-positive tumors account for some 10–15% (6, 7). Hereditary nonpolyposis colorectal cancer (HNPCC), caused by inherited germline mutations in MLH1 and MSH2, accounts for some 3% (6, 7). Among the manifestations of the methylator phenotype, promoter methylation frequently down-regulates other genes, notably p16 (8), and many other CpG islands methylated in colon cancer are also methylated in normal colonic mucosa (8). This generalized methylation increases with age (8) and may or may not be related to cancer. Globally, CpG island methylation shows a high degree of nonrandomness in distribution and tumor specificity (9). Recently the growth-regulating insulin-like growth factor II (IGF2) gene was implicated in CRCs belonging to the MSI/methylator phenotype. Loss of imprinting (LOI) of IGF2 was found in 10 of 11 cancers with MSI but only 2 of 16 cancers without MSI (10). That LOI might characterize a cancer-prone state could be suggested based on the fact that it occurred in the unaffected colonic mucosa of most of the patients whose tumors showed LOI and in a small number of individuals without cancer (10). These findings raise the important question of whether LOI is also a manifestation of the methylator phenotype or an independent event. Genomic imprinting is defined as an epigenetic change leading to differential expression of the two parental alleles in somatic cells and usually involves allele-specific methylation of certain regions. IGF2 is one of many imprinted genes where only its paternal allele is expressed and the maternal allele is silent (11, 12). LOI is said to occur when the normally silenced allele is activated. LOI of IGF2 is observed frequently in a variety of childhood and adult tumors (13–16) and has been implicated in Beckwith–Wiedemann syndrome, a congenital overgrowth disorder that predisposes to embryonal tumors (17). The occurrence of LOI theoretically doubles the active gene dosage (18) and might contribute to tumor growth through its autocrine or endocrine effects. Recent studies in knock-out mice demonstrated that the supply of IGF2 could modify the growth of intestinal adenomas (19), thus implicating it in colorectal tumor progression and evolution. In this study we addressed the question of whether LOI of IGF2 is a consequence of deficient mismatch repair and MSI, and whether it is associated with allele-specific methylation in the IGF2 region. To provide an easy and reproducible quantitative allele-specific assay of imprinting, we developed and validated a simple primer extension assay. Our results support the concept that sporadic CRC with MSI is associated with hypermethylation that causes loss of MLH1 function, loss of function of other genes such as p16, and altered expression of IGF2. Importantly, we demonstrate that the normal colonic mucosa shows aberrant hypermethylation and LOI of IGF2 as a sign of a field defect.

This article was published in National Academy of Sciences and referenced in Journal of Gastrointestinal & Digestive System

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