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 Hidewaki Nakagawa*, Robert B. Chadwick*, Pa¨ ivi Peltoma¨ ki*, Christoph Plass*, Yusuke Nakamura†, and Albert de la Chapelle*‡ *Division of Human Cancer Genetics, Comprehensive Cancer Center, Ohio State University, 420 West 12th Avenue, Columbus, OH 43210; and †Laboratory of Molecular Medicine, Human Genome Center, Institute of Medical Science, University of Tokyo, Minato-ku, Tokyo 108-8639, Japan Contributed by Albert de la Chapelle, November 6, 2000 We hypothesize that loss of imprinting (LOI) of the insulin-like dosage (18) and might contribute to tumor growth through its growth factor II (IGF2) gene is associated with a predisposition to autocrine or endocrine effects. Recent studies in knock-out mice sporadic colorectal cancer. We confirmed a previously known demonstrated that the supply of IGF2 could modify the growth strong correlation between LOI and microsatellite instability and of intestinal adenomas (19), thus implicating it in colorectal showed that LOI was not a consequence of microsatellite instabil- tumor progression and evolution. ity or mismatch repair deficiency. LOI of IGF2 correlated strongly In this study we addressed the question of whether LOI of with biallelic hypermethylation of a core of five CpG sites in the IGF2 is a consequence of deficient mismatch repair and MSI, insulator region of IGF2͞H19, which is a known CTCF-binding and whether it is associated with allele-specific methylation in element. As this methylation-dependent LOI was present in both the IGF2 region. To provide an easy and reproducible quanti- tative allele-specific assay of imprinting, we developed and tumors and normal colonic mucosa, it is possible that hypermeth- validated a simple primer extension assay. Our results support ylation creates a field defect predisposing to cancer. the concept that sporadic CRC with MSI is associated with hypermethylation that causes loss of MLH1 function, loss of methylator phenotype has been postulated to account for function of other genes such as p16, and altered expression of Aa subset of colorectal cancers (CRCs). In these sporadic, IGF2. Importantly, we demonstrate that the normal colonic mostly right-sided tumors, hypermethylation of the CpG islands mucosa shows aberrant hypermethylation and LOI of IGF2 as a in the MLH1 promoter is associated with the absence of MLH1 sign of a field defect. protein, defective mismatch repair, and widespread microsatel- Materials and Methods lite instability (MSI) (1–5). Among all CRCs the MSI-positive Tissue Samples and DNA͞RNA Extraction. tumors account for some 10–15% (6, 7). Hereditary nonpol- We randomly selected sporadic CRC patients from whom samples of freshly frozen yposis colorectal cancer (HNPCC), caused by inherited germline tumor and matched normal colonic mucosa were available. The mutations in MLH1 and MSH2, accounts for some 3% (6, 7). site from which the normal colonic mucosa was obtained was not Among the manifestations of the methylator phenotype, pro- immediately adjacent to the tumor. Furthermore, we selected moter methylation frequently down-regulates other genes, no- tumor and normal mucosa from HNPCC patients with previ- tably p16 (8), and many other CpG islands methylated in colon ously identified germline mutations of MLH1. The MSI status of cancer are also methylated in normal colonic mucosa (8). This all tumors had been determined previously (6, 7, 20). Among the generalized methylation increases with age (8) and may or may sporadic tumors we chose, 51 were MSI(ϩ) and 89 were MSI(Ϫ). not be related to cancer. Globally, CpG island methylation shows All 20 HNPCC tumors were MSI(ϩ), and the patients had a high degree of nonrandomness in distribution and tumor germline mutation of MLH1 or MSH2 as described (6, 7). All specificity (9). patients gave informed consent before sample collection, ac- Recently the growth-regulating insulin-like growth factor II cording to institutional guidelines. DNA was extracted as de- (IGF2) gene was implicated in CRCs belonging to the MSI͞ scribed (6). We selected informative samples in which heterozy- methylator phenotype. Loss of imprinting (LOI) of IGF2 was gosity for the IGF2 ApaI polymorphism allowed imprinting found in 10 of 11 cancers with MSI but only 2 of 16 cancers analysis by the previously described method (13, 14). RNA was GENETICS extracted by using TRIzol according to its manufacturer’s in- without MSI (10). That LOI might characterize a cancer-prone structions (GIBCO͞BRL). state could be suggested based on the fact that it occurred in the unaffected colonic mucosa of most of the patients whose tumors Preparation of RNA-Specific PCR Product Containing IGF2 Polymor- showed LOI and in a small number of individuals without cancer phism. It is critical to avoid genomic DNA contamination of RNA (10). These findings raise the important question of whether LOI when examining the imprinting pattern. To eliminate genomic is also a manifestation of the methylator phenotype or an independent event. Genomic imprinting is defined as an epige- netic change leading to differential expression of the two pa- Abbreviations: LOI, loss of imprinting; ROI, retention of imprinting; IGF2, insulin-like rental alleles in somatic cells and usually involves allele-specific growth factor II; MSI, microsatellite instability; CRC, colorectal cancer; HNPCC, hereditary nonpolyposis colorectal cancer; SNuPE, single nucleotide primer extension; MSP, methyl- methylation of certain regions. IGF2 is one of many imprinted ation-specific PCR; DMR, differential methylated region. genes where only its paternal allele is expressed and the maternal See commentary on page 392. allele is silent (11, 12). LOI is said to occur when the normally ‡To whom reprint requests should be addressed. E-mail: [email protected]. silenced allele is activated. LOI of IGF2 is observed frequently The publication costs of this article were defrayed in part by page charge payment. This in a variety of childhood and adult tumors (13–16) and has been article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. implicated in Beckwith–Wiedemann syndrome, a congenital §1734 solely to indicate this fact. overgrowth disorder that predisposes to embryonal tumors (17). Article published online before print: Proc. Natl. Acad. Sci. USA, 10.1073͞pnas.011528698. The occurrence of LOI theoretically doubles the active gene Article and publication date are at www.pnas.org͞cgi͞doi͞10.1073͞pnas.011528698 PNAS ͉ January 16, 2001 ͉ vol. 98 ͉ no. 2 ͉ 591–596 Downloaded by guest on September 30, 2021 DNA contamination, total RNA was treated with RNase-free DNase I (Ambion, Austin, TX) according to its manufacturer’s instructions. After inactivation of DNase, total RNA was treated with Superscript II (GIBCO͞BRL) to produce cDNA, by using random hexamers according to its manufacturer’s instructions. We amplified a RNA-specific PCR product (1.3 kb) containing the IGF2 exon 9 ApaI polymorphism from cDNA by using an exon-connecting primer pair P1 5Ј-GACACCCTCCAGT- TCGTCTGT-3Ј (exon 7) and P2 5Ј-CGGGGATGCATAAAG- TATGAG-3Ј (exon 9). This RNA-specific product could be clearly distinguished from the genomic DNA-specific product (3.3 kb) on 1% agarose gels. To further eliminate genomic DNA contamination, the RNA-specific product was excised from the agarose gel after electrophoresis and purified with a QIAEXII gel extraction kit (Qiagen, Chatsworth, CA). Then this RNA- specific product was subjected to a second round of PCR with P3 5Ј-CTTGGACTTTGAGTCAAATTGG-3Ј and P4 5Ј- GGTCGTGCCAATTACATTTCA-3Ј (20–25 cycles). The sec- ond PCR product was treated with exonuclease I (usb, Cleve- land) and shrimp alkaline phosphatase (usb) before the primer extension reaction. Fig. 1. Evaluation of fluorescent SNuPE assays with insert sequences display- ing A and G at the IGF polymorphism in serial mixtures of two plasmids. The Quantitative Analysis of IGF2 Imprinting by Single Nucleotide Primer A plasmid containing the A allele of the IGF2 A͞G polymorphism and the G Extension (SNuPE) Assays. Fluorescent SNuPE assays were carried plasmid containing the G allele were mixed in known proportions as indi- out in 20 ␮l containing 50 ng PCR product, 50 ␮M ddATP, 50 cated. The mixtures were subjected to nested PCR and SNuPE assay. The ␮M dCTP, 50 ␮M dGTP, 0.2 pmol 6-carboxyfluorescein-labeled chromatograms showed three fluorescent peaks. The peak at the left at 23 bp primer, and 0.64 units Themo Sequenase (Amersham Pharma- is 6-carboxyfluorescein-primer, the peak at 26 bp is the primer extension cia) in the buffer provided by the manufacturer. The extension product from the A allele, and the peak at 31 bp is the primer extension primer 5Ј-6-carboxyfluorescein-CTGAACCAGCAAA- product from the G allele. The expected values of A͞(A ϩ G), expressed as a GAGAAAAGAA-3Ј was purchased from Perkin–Elmer. The percentage, are compared with those observed by SNuPE assay. reaction was carried out in a thermal cycler (Perkin–Elmer) with an initial denaturation step of 2 min at 95°C followed by 25 cycles forward primer 5Ј-GGACGGAATTGGTTGTAGTT-3Ј and re- of 95°C for 30 s, 55°C for 30 s, and 72°C for 30 s. The products verse primer 5Ј-AGGCAATTGTCAGTTCAGTAA-3Ј.This of the primer extension reaction were analyzed on an ABI 377 PCR product was directly sequenced by the reverse primer to sequencer with a 9% denaturing polyacrylamide