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Age-related Hypermethylation of the 5' Region of MLH1 in Normal Colonic Mucosa Is Associated with Microsatellite-unstable Colorectal Cancer Development¹

Division of Human Cancer Genetics, Comprehensive Cancer Center [H. N., A. d. l. C.], Department of Pathology [G. J. N.], and Division of Surgical Oncology [E. E. Z., E. W. M.], The Ohio State University, Columbus, Ohio 43210, and Department of Medical Genetics, Haartman Institute, University of Helsinki, FIN-00014 Helsinki, Finland [R. S., L. A. A.]

	ABSTRACT

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Hypermethylation of the MLH1 promoter underlies most sporadic colorectal cancers with microsatellite instability (MSI). To investigate the role of hypermethylation in the normal colonic mucosa as a possible precursor lesion, we studied 700 bp upstream of MLH1 covering 51 CpG sites. We found partially methylated alleles in 15 of 34 (44%) patients <60 years of age and 20 of 24 (83%) patients 80 years of age (P = 0.0026). Fully methylated alleles were present in 18 of 33 (55%) patients with MSI+ tumors but in only 18 of 90 (20%) patients with MSI- tumors (P = 0.00019). By in situ analysis, methylation was patchy and located mainly in the cryptal regions close to the lumen. We conclude that the spread of methylation in the MLH1 promoter in the normal colonic mucosa is closely associated with age and the development of sporadic MSI+ colorectal cancers.

	Introduction

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Colorectal tumors that show MSI³ share several distinct features that differ from those of tumors belonging to the chromosome instability pathway (1) . In this study, we address questions pertaining to MSI+ CRCs, which account for some 15% of all CRCs. A minority of these MSI+ cases, perhaps up to 5%, occur in individuals with the HNPCC syndrome, in which affected individuals are germ-line heterozygous for heritable mutation in one of the mismatch repair genes (2, 3, 4) . Most other MSI+ tumors, which account for some 10% of all CRCs, are sporadic and caused by hypermethylation of the promoter region of the MLH1 gene, leading to loss of the MLH1 protein (5, 6, 7, 8) . Both types of MSI+ tumors share distinct phenotypic features such as preferential location in the right-sided colon, histological features of poorly differentiated adenocarcinomas or mucinous carcinomas, a Crohn-like lymphocytic infiltration, and relatively favorable prognosis (2 , 3 , 9 , 10) . The molecular basis of these features is unknown. Moreover, the mechanisms involved in methylation and associated loss of MLH1 function leading to cancer are poorly understood. Here we address the role of methylation in normal colonic mucosa. Based on previous evidence of hypermethylation of the MLH1 promoter region in normal colonic mucosa from some CRC patients (11) , we postulated that these changes represent precursor lesions preceding the development of cancer. Previous authors have studied different regions of the MLH1 promoter, such as its 5'-most part (6) , its 3'-most part (12) , a small number of CpG sites (11) , or combinations of the above (13) . To glean a more global understanding of the role of promoter methylation, we analyzed a large series of normal colonic mucosa samples from CRC patients for most of the 700-bp promoter region, verified the results by studying all 51 CpG sites in selected cases, verified the in-solution results by in situ methylation analysis, and correlated these findings with clinical parameters. Our results support the concept that partial methylation of the MLH1 promoter is a common age-related event in the normal mucosa of CRC patients largely unrelated to the MSI status of the tumor, whereas fully methylated alleles are observed much more frequently in the normal mucosa of MSI+ patients than in that of MSI- patients and increase with age. Moreover, methylation appears to be patchy and to preferentially affect colonic crypt cells close to the lumen. This supports a model in which the molecular events leading to MSI+ CRC comprise a gradual, age-related, and perhaps environment-related spreading of methylation over most of the MLH1 promoter region.

	Materials and Methods

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Tissue Samples.
We selected 111 MSI- CRCs, 43 sporadic MSI+ CRCs, and 10 HNPCC cancers with identified germ-line mutations and studied their normal colonic mucosa for methylation of MLH1. The MSI status of the tumors and clinical parameters of the patients had been determined previously (4 , 14) . All normal mucosa samples were derived from sites apparently separate from the tumors, not from the tumor margin, and were evaluated histopathologically before DNA extraction. In addition, we obtained multiple normal-appearing mucosa samples from fresh surgical specimens of 17 patients with sporadic CRC who underwent surgery at the Ohio State University James Cancer Hospital. Multiple normal-appearing mucosa samples were harvested at sites 10 cm proximal, 2 cm proximal, 2 cm distal, and 10 cm distal from the tumor. To avoid contamination of normal tissue with tumor cells, the normal specimens were taken first, and different instruments were used for normal mucosa and tumors. Institutional Review Board rules including informed consent were strictly followed. Genomic DNA was extracted by standard methods.

COBRA.
Bisulfite treatment of genomic DNA was carried out as described previously (15 , 16) . Briefly, 1 µg of genomic DNA was denatured in 0.3 N NaOH at 80°C for 15 min. The denatured DNA solution was incubated in 3 M sodium bisulfite and 500 µM hydroquinone at 50°C for 16 h. The bisulfite-treated DNA was purified using a Qiagen gel purification kit, treated with 0.3 N NaOH for 10 min at room temperature, precipitated with ethanol, and resuspended in 20 µl of TE buffer. Bisulfite PCR was performed to amplify methylated alleles and unmethylated alleles of the 5' region 700 bp upstream of MLH1. With primer sequences P1 (5'-TTTTTAGGAGTGAAGGAGGAGGTT-3') and P2 (5'-ACCAATCAAATTTCTCAACTCTATAAA-3'), PCR was carried out in a 50 µl volume containing 50 ng of bisulfite-treated DNA, 10 mM Tris-HCl (pH 8.3), 50 mM KCl, 0.2 mM nucleotide triphosphate, 500 µM primers, and 2 units of AmpliTaq Gold DNA polymerase (Perkin-Elmer) for 40 cycles (30 s at 95°C, 30 s at 54°C, and 60 s at 72°C, followed by a 5-min extension at 72°C). The PCR products were purified using the Qiagen purification kit and digested by BstUI (BioLab) for 5 h at 60°C, run on 2% agarose gels, and transferred to membranes. A 310-bp probe for hybridization was generated by bisulfite-PCR with primers 5'-TTTCGTAGTGGCGTTTGACGTC-3' and 5'-AACTTATATACCTCTACTAAAATCTAA-3'. The purified PCR product was labeled by using the AlkPhos Direct Labeling Kit (Amersham) according to the manufacturer’s instructions. Hybridization was performed at 50°C overnight, and the membranes were washed twice at 50°C for 10 min. The signals were detected by the CDP-star detection kit (Amersham).

Bisulfite Sequencing.
Methylated alleles spanning the 700-bp upstream 5' region of MLH1 were preferentially amplified by methylation-specific primers. First, the unmethylated and methylated alleles were amplified from bisulfite-treated DNA by sense primer P1 and antisense primer P3 (5'-ACCTTCAACCAATCACCTCAATA-3') that did not contain any CpG sites. Nested PCR was then carried out to preferentially amplify the methylated allele by the methylation-specific primer Pm (5'-ACGTAGACGTTTTATTAGGGTCGC-3') and P3. Potential methylation sites are underlined. The purified PCR products were cloned into TA vector pCR2.1 (Invitrogen), and at least 10 insert-positive plasmid clones were extracted by the QIAprep Spin Miniprep kit (Qiagen) and sequenced using the ABI sequencing system (Perkin-Elmer Applied Biosystems).

MSP-ISH.
MSP-ISH was carried out as described previously (17) . Serial 4–6-µm sections on silane-coated slides were prepared from paraffin-embedded tissues that showed methylation of MLH1 by COBRA in both normal and tumor tissues. After deparaffinization, digestion by pepsin, and treatment with sodium bisulfite, PCR in situ was performed on these sections. The primer sequences for PCR in situ were 5'-ACGTAGACGTTTTATTAGGGTCGC-3' and 5'-CTCATGGTAACTACCCGCG-3', and after denaturing at 94°C for 3 min, 35 cycles were conducted at 55°C for 1.5 min and 94°C for 1 min. After amplification, in situ hybridization was performed according to the previously published protocol (17) , using as a probe a biotin-labeled plasmid containing the 110-bp methylation-specific PCR product generated in solution phase by the same primers. After washing, the slides were incubated with streptavidin-alkaline phosphatase conjugate (1:200; Enzo Diagnostics) and then exposed to the chromogen (Enzo Diagnostics), nitroblue tetrazolium and 5-bromo-4-chloro-3-indolyl phosphate (Enzo Diagnostics). The counterstain was nuclear fast red, which colors the negative nuclei a light pink in comparison with the blue signal of the chromogen.

Statistical Analysis.
Statistical comparisons were performed by Fisher’s exact test or ² test.

	Results

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Detection of Methylation of the 5' MLH1 Region in Normal Colonic Mucosa by COBRA.
COBRA is a highly sensitive method that allows infrequent cells bearing methylated alleles to be detected among a majority of cells with unmethylated alleles. For an extensive allele-specific study of the methylation, we amplified a region covering 700 bp upstream of MLH1. As shown in Fig. 1a , the 5'-most part of this sequence has a high density of CpG sites that have been extensively analyzed before (6) . Other investigators (12) demonstrated that methylation of CpG sites in a more proximal small region invariably correlated with the absence of MLH1 expression. Our analysis covered these two regions, and by combining COBRA and bisulfite sequencing analysis, we determined whether there were partially or fully methylated alleles. Our region contains five BstUI recognition sites (CGCG), and COBRA shows a 370-bp band when all these sites are methylated in the same alleles. Partially methylated alleles show up as bands between 600 and 370 bp (Fig. 1b) .

View larger version (36K): [in this window] [in a new window] [Download PPT slide]   Fig. 1. Methylation analysis of MLH1 in normal colonic mucosa. a, map of the 5' promoter region and exon 1 of MLH1. Individual CpG sites are represented by vertical lines; BstUI sites (CGCG) for COBRA are shown as B. The location of primers designed for COBRA and bisufite sequencing, the expected PCR products, and the probes for COBRA and MSP-ISH are shown. b, COBRA by BstUI digestion demonstrating partially (P) and fully (F) methylated alleles in some normal colonic mucosa samples. pc is a positive control in which the bisulfite-PCR product from a tumor with fully methylated alleles was studied. c, bisulfite sequencing of the 5' MLH1 region in six normal mucosa samples; the presence of fully methylated alleles had been shown by COBRA in cases 1 and 2, whereas cases 3–6 had shown partial methylation. This region is defined by primers Pm and P3 (a) and covers 51 CpG sites, including five BstUI sites shown as B. Each horizontal compartment represents one cloned allele. Unmethylated CpG sites are represented as open boxes, and methylated sites are represented as filled boxes.

MSP-ISH.
COBRA is highly sensitive and therefore subject to potential contamination by DNA with methylated alleles from tumors or other specimens. To eliminate this possibility and to determine the cellular location of MLH1 methylation, we performed MSP-ISH (17) on two normal colonic tissue samples showing the presence of methylated alleles by COBRA and bisulfite sequencing. We detected methylation of MLH1 in areas of normal glandular epithelium in both specimens (Fig. 2) . Staining positive for methylation was observed in the nuclei in focal populations of colonic crypts. A patchy distribution of hypermethylation was noted involving from 1–10% of the normal colonic epithelium. The phenomenon was clearly more evident near the surface of the crypts than at the bottom of the crypts.

View larger version (73K): [in this window] [in a new window] [Download PPT slide]   Fig. 2. In situ detection of methylated alleles of MLH1 in normal colonic epithelium by MSP-ISH. a shows the lumenal aspect of the colonic epithelium, and b shows the basal aspect of the crypt from the same glands (a and b, x400). Note that the signal (blue) is evident in many more normal-appearing colonic epithelial cells toward the lumenal aspect than in those toward the base of the gland. c (x200) shows the results of PCR in situ hybridization for hypermethylation from a serial section of the same area where the PCR amplification step was omitted; an equivalent lack of signal was noted when the sodium bisulfite treatment step was omitted.

View larger version (65K): [in this window] [in a new window] [Download PPT slide]   Fig. 3. Incidence of MLH1 methylation in normal colon mucosa in relation to patient age and the MSI status of the tumors. The incidence of methylated alleles from patients >80 years old is significantly higher than that in patients <60 years old, both overall (P = 0.0026) and in patients with MSI- tumors (P = 0.016). However, in each age group, there are no significant differences between samples from patients with MSI+ tumors and patients with MSI- tumors.

View larger version (39K): [in this window] [in a new window] [Download PPT slide]   Fig. 4. Methylation status of different samples of normal colonic mucosa from the same patient. a, four different normal colon tissues were studied per patient. Lane 1, 10 cm proximal of the tumor; Lane 2, 2 cm proximal of the tumor; Lane 3, 2 cm distal of the tumor; Lane 4, 10 cm distal of the tumor; Lane T, tumor; Lane pc, positive control. Case 1 is a 41-year-old patient with left-sided rectal cancer. Case 2 is a 45-year-old patient with left-sided sigmoid cancer. Case 3 is a 77-year-old patient with right-sided colon cancer. Case 4 is an 85-year-old patient with right-sided colon cancer. b, summary of methylation at four different sites of colon tissue from 17 patients. Each vertical group of four symbols represents the four sites from a single patient, which were placed on the X-axis according to the age of the patient. •, full methylation; , partial methylation; , no methylation. These data illustrate the increase of methylation with age, the preferential methylation in the right-sided colon, and the patchy distribution of methylation.

	Discussion

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After it became known that methylation of the promoter region of MLH1 underlies most sporadic MSI+ CRCs (5) , the phenomenon was actively studied. Most researchers have studied cancer rather than normal tissue, and the preferred method has been the highly sensitive methylation-specific PCR (6) . By this method, it was shown that methylation of CpG sites in the 5'-most part of the region correlates with loss of expression at the RNA and protein levels. Importantly, demethylation with 5-aza-2'-deoxycytidine in vitro was shown to bring about reexpression of MLH1 (6) . Thus a direct link between methylation, loss of expression, and loss of protein was established. Interestingly, in another study, Deng et al. (12) used cell lines to show that methylation of a small region closer to the transcription start site (more 3') invariably correlated with the absence of MLH1 expression. In a study of numerous colorectal tumors, we confirmed this finding (13) .

Here we addressed the role of MLH1 methylation in the normal colonic mucosa of patients with CRC. Theoretically and conceptually, such methylation might well represent a precursor phenomenon to cancer. Previous studies had provided somewhat conflicting results. In a study of the 5' MLH1 region, Toyota et al. (27) found a correlation between "CpG island methylator phenotype" and the occurrence of MSI+ tumors, but the methylation of MLH1 was cancer specific and was not observed in normal colon. By contrast, Kuismanen et al. (11) , studying just four CpG sites, concluded that there existed a reverse pattern in that many MSI- cases showed hypomethylation of the MLH1 promoter in the tumor but hypermethylation in normal mucosa. Hypothesizing that these apparently contradictory results were due to the fact that different CpG sites were studied, we here analyzed the entire 700-bp region upstream of MLH1 in a large series of normal mucosa samples from cancer patients. Our results show that methylation of MLH1 increases with age, in many cases spreading over almost all 51 CpG sites, and correlates with the occurrence of MSI+ tumors. By in situ methylation analysis, we show that in at least two cases, the methylation observed in solution by the highly sensitive COBRA method is not an artifact. Moreover, methylation occurs in some but not all samples, at multiple sites of the colon, is distributed in patches, and preferentially affects cells closer to the colonic lumen than those at the bottom of the crypts. Most cases of methylation of the normal mucosa and MSI+ tumors are right-sided and affect old (>60 years) or very old (80 years) people. Despite this, our data show that the age-dependent increase in methylation of the normal mucosa is relatively independent of the MSI status of the tumor. Taken together, we interpret these findings to support a model in which methylation increases with age in most patients, can spread over the entire promoter region, and is most commonly associated with right-sided MSI+ tumors in very old patients. Given these facts, we speculate that, perhaps as a result of environmental influences from the chemical composition of the colonic content (28) , MSI+ tumors arise when the spread of methylation (29) reaches a threshold where a decrease or abrogation of MLH1 expression occurs. This is in keeping with data on immunohistochemical staining for MLH1 in our samples. In data not shown, we have observed a common pattern in which the tumor stains negative for MLH1, whereas the normal tissue is invariably generally positive, but with numerous patches or single crypts in which the staining is weak or absent.

An important caveat to these conclusions is that our study (as well as others) has been done on cancer patients rather than normal healthy individuals. If an acceptable protocol could be established, it would be of great interest to study the methylation of MLH1, its distribution in the promoter region, its distribution in the colon, and its age dependence in normal individuals, especially those who go on to develop cancer. Such a study would have the power to determine more convincingly whether the methylation described here is a precursor of cancer.

	ACKNOWLEDGMENTS

 
We thank Denise Soble for recruiting patients to the study and Drs. P. Peltomäki and C. Plass for fruitful discussions.

	FOOTNOTES

 
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ Supported by NIH Grants CA67941 and CA16058. H. N. is a postdoctoral fellow from Japan Society for Promoting of Science and is supported by Sumitomo Life Social Welfare Services Foundation, The Osaka Medical Research Foundation for Incurable Disease, and Yamanouchi Foundation for Research on Metabolic Disorders.

² To whom requests for reprints should addressed, at Human Cancer Genetics Program, Comprehensive Cancer Center, The Ohio State University, Medical Research Facility, Room 646, 420 West 12th Avenue, Columbus, OH 43210. Phone: (614) 688-4781; Fax: (614) 688-4772; E-mail: delachapelle-1{at}medctr.osu.edu

³ The abbreviations used are: MSI, microsatellite instability; CRC, colorectal cancer; HNPCC, hereditary nonpolyposis colorectal cancer; COBRA, combined bisulfite restriction analysis; MSP-ISH, methylation-specific PCR in situ hybridization.

Received 6/15/01. Accepted 8/15/01.

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