Gastric Cancer
Copyright ©The Author(s) 2003. Published by Baishideng Publishing Group Inc. All rights reserved.
World J Gastroenterol. Jan 15, 2003; 9(1): 26-29
Published online Jan 15, 2003. doi: 10.3748/wjg.v9.i1.26
Silencing-specific methylation and single nucleotide polymorphism of hMLH1 promoter in gastric carcinomas
Da-Jun Deng, Jin Zhou, Bu-Dong Zhu, Jia-Fu Ji, Jeffrey C Harper, Steven M Powell
Da-Jun Deng, Jin Zhou, Bu-Dong Zhu, Jia-Fu Ji, Peking University Health Science Center and Beijing Institute for Cancer Research, Beijing 100034, China
Jeffrey C Harper, Steven M Powell, University of Virginia Health Science Center, Charlottesville, VA 22908-0708, United States
Author contributions: All authors contributed equally to the work.
Supported by grant (2000-A-29) from Peking University Center for Human Disease Genomics, grant (0106) from Peking University Cancer Research Center, grant (3171045) from National Natural Science Foundation of China, and by NIH Grant CA67900
Correspondence to: Professor Da-Jun Deng, Department of Cancer Etiology, Peking University School of Oncology and BICR, Da-Hong-Luo-Chang Street, Western District, Beijing 100034, China. dengdajun@sina.com
Telephone: +86-10-66162978 Fax: +86-10-66175832
Received: September 13, 2002
Revised: September 25, 2002
Accepted: October 18, 2002
Published online: January 15, 2003

Abstract

AIM: To investigate CpG methylation and single nucleotide polymorphism (SNP) of a specific promoter region of hMLH1 in primary gastric carcinoma.

METHODS: Primary gastric carcinomas (n = 80), their corresponding normal mucosal samples, and gastric mucosal biopsies from normal/gastritis control patients (n = 54) were used. Hypermethylation at -253 nt and -251 nt in relation with the translational start site and SNP of a silencing specific region (-339 nt-46 nt) in the hMLH1 promoter were analyzed by Bst UI-combined bisulfite assay (COBRA), denaturing high performance liquid chromatogram (DHPLC), and sequencing.

RESULTS: (A) The specific methylation at -253 nt and -251 nt was observed in 2 of 60 primary gastric carcinomas, but neither in all of the corresponding mucosa nor in normal/gastritis samples, by Bst UI-COBRA and DHPLC. (B) The hMLH1 promoter was methylated homogeneously in the xenograft of the primary gastric carcinoma with the methylated and unmethylated hMLH1. (C) The pattern of SNP at -93 nt of the hMLH1 promoter in 54 Chinese patients with gastric carcinoma was the same as that in the control patients: 51% was A/G heteroalleles, 34% and 15% were A/A and G/G homoalleles, respectively.

CONCLUSION: Biallelic inactivation of hMLH1 by epigenetic silencing existed in human primary gastric carcinoma homogeneously. Hypermethylation of hMLH1 may play a role in the early stage of development of a few gastric carcinomas. The SNP at -93 nt is not related to the susceptibility of gastric carcinomas.




INTRODUCTION

Hpermethylation of CpG islands in upstream is an epigenetic mechanism of lost of functions of tumor suppressor genes, DNA repair genes, etc[1-3]. The methylated hMLH1 was observed in most of primary gastric carcinomas with the microsatellite instability-H phenotype (MSI-H)[4-12]. Silencing of hMLH1 by CpG methylation may play an important role in the development of MSI-H tumors. However, the hMLH1-methylated proportions in MSI-L and MSI-stable sporadic gastric carcinoma varied greatly (0%-75%) in previous reports. Nakajma et al[13] reported that hMLH1 methylation was detected in 8 of 100 primary gastric carcinoma cases, but not detected in their corresponding normal mucosa or in intestine metaplastic mucosa. Kang et al[14] reported a much higher rate of hMLH1 methylation in gastric carcinoma (20.3%), adenoma (9.8%), and intestine metaplastic mucosa (6.3%). Different results might result from applications of both different markers used to classify MSI tumors, and different approaches or primers used to detect methylation of CpG island of hMLH1. It was reported recently that the methylation of CpGs in a small C-region (-270 nt ~ -199 nt) of the hMLH1 promoter was invariably correlated with the absence of gene expression[15-17]. Thus, it is of interest to further explore the silencing specific methylation of the hMLH1 promoter in primary gastric carcinomas. In the present study, we analyzed the silencing specific hMLH1 methylation at -253 nt and -251 nt in the C-region in primary gastric carcinoma and normal/gastritis control.

Deng et al[18,19] established a novel approach to detect CpG methylation by denaturing high performance liquid chromatography (DHPLC), which was further developed to quantify CpG methylation and SNP of CpG islands simultaneously[20,21]. It was reported that there is a SNP at -93 nt of the hMLH1 promoter[22]. In order to evaluate the possible role of the SNP in the gastric carcinogenesis, the pattern of the SNP in patients with gastric carcinoma was also compared with that in control patients without malignant diseases using DHPLC.

MATERIALS AND METHODS
Gastric samples

60 primary gastric carcinomas and their corresponding normal gastric mucosa were collected: 34 from the Beijing Institute for Cancer Research (BICR) surgically (28 males and 6 females, 29-59 years old, average age: 47.7 years old), and 26 from the University of Virginia Health System (Uva) (18 males and 8 females, 41-84 years old, the average age: 63.8 years old). Three Uva xenografted human GC were obtained from nude mice as described[23]. In addition, 56 biopsy samples of gastric epithelial tissues were collected from normal/gastritis patients at BICR (50 males and 4 female, 18-47 years old, the average age: 29.7 years old). All samples were used in the analysis of both methylation and SNP. Additional 18 corresponding normal gastric mucosal samples from BICR (Chinese patients, 13 males and 5 females, 26-78 years old, average age: 48.7 years old) with gastric carcinoma were used in SNP analysis in order to have the same number of cases as control. All clinical samples and histopathological information for each case were obtained according to approved institutional guidelines.

DNA extraction and bisulfite modification

Genomic DNA of tissue samples was isolated with QIAGen DNA Purification Kits. Two mg genomic DNA was treated with sodium bisulfite in order to convert the unmethylated C to U (T in PCR products) as described[24].

Design of Primers and PCR Conditions

Primers were designed according to the specific region (-339 nt ~ -46 nt in relation to the translational start site) of CpG islands of the sense strand of hMLH1 (GenBank accession number U83845, gi: 2511457) as described[18,20]. The strandspecific primers for the modified CpG islands included hMLH1-mF (5’-gtatttttgtttttattggttggata-3’) and hMLH1-mR (5’-aataccttcaaccaatcacctcaata-3’). Primers for the templates without bisulfite-treatment included hMLH1-wF (5’-gcatctctgctcctattggctggata-3’) and hMLH1-wR (5’-agtgccttcagccaatcacctcagtg-3’). Hot-started touchdown PCR (-1.0 °C per cycle, total 35 cycles) was used to amplify hMLH1 without bisulfite-treatment (72 °C → 58 °C), and the sense strand templates with bisulfite-treatment (ssPCR, 65 °C → 50 °C for hMLH1)[18,20].

Detection of CpG methylation by combined bisulfite restriction analysis (COBRA) and DHPLC

The specific region of the methylated CpG island contain a BstUI restriction site (CGCG) that is converted to UGUG in the unmethylated CpG island after bisulfite modification. Hence the methylation of the bisulfite-modified hMLH1 could be analyzed directly by BstUI-COBRA. In the confirmation study, methylation status was detected further by DHPLC. Basic mechanism to detect methylation by DHPLC is that the retention time of the methylated PCR products is longer than that of the unmethylated ones, because of higher denaturing temperature of the methylated sequence resulted from higher G+C content after bisulfite modification[20].

Analysis for SNP by DHPLC and sequencing

The SNP at -93 nt in the corresponding normal mucosal samples and the gastric biopsies from the control patients was detected by DHPLC and confirmed by sequencing as described[20]. Because all control normal/gastritis samples were collected from Chinese patients, therefore only corresponding normal samples from Chinese cases with gastric carcinomas hospitalized in BICR were used in the SNP analysis.

RESULTS AND DISCUSSION
Silencing-specific methylation of hMLH1 promoter

One BstUI restriction site (CGCG) exists in the silencing-specific C-region of the CpG island that was invariably correlated with the absence of gene expression[15]. This site remains only in the methylated templates but not in the unmethylated ones after bisulfite modification. Therefore, BstUI-COBRA was used to detect the methylation of the specific region[18]. If the template is methylated, its ssPCR product (294 bp) is digested into a 206 bp and an 88 bp fragments. If not methylated, not digested. The specific CpG methylation of the hMLH1 promoter was observed in only 3.3% (2 of 60) of primary gastric carcinoma cases by this assay (Figure 1-T1, T2). T1 sample was distal adenocarcinoma (mod-poor differentiation) from BICR, and T2 was gastro-esoph junction adenocarcinoma (poor differentiation) from UVa. Such methylation was neither observed in all 60 corresponding normal gastric mucosa (Figure 1-N1, N2) nor in 54 normal/gastritis samples from the BICR control patients. The unmethylated hMLH1 was detectable in all tested human samples (Figure 1-T1, T2, N1, N2). The same result was observed by DHPLC analysis (Figure 2-A, B).

Figure 1
Figure 1 Detection of hMLH1 methylation by BstUI-COBRA assay. PCR products and methylated ssPCR products (294 bp) were digested into two small fragments (206 bp and 88 bp). Unmethylated ssPCR products were not digested by BstUI. T1, T2: primary gastric carcinomas; N1, N2: corresponding normal gastric mucosal samples; X2: xenograft of primary gastric carcinomas (T2) with hMLH1 methylation; NC1, NC2: negative control xenografts of primary gastric carcinomas without hMLH1 methylation; PC: PCR products of the hMLH1 templates not treated by bisulfite
Figure 2
Figure 2 DHPLC Chromatograms of the specific methylation of the hMLH1 promoter ssPCR products were analyzed at partial denaturing temperature 54 °C, point mutation mode. T1 and T2, primary gastric carcinomas; N1 and N2, the corresponding normal gastric mucosal samples; X2, the xenograft of T2 in nude mouse

The positive rate (3.3%) of the specific CpG hypermethylation of hMLH1 in the present study was lower than those reported by others. Nakajima et al[13] reported that hMLH1 methylation was observed in 8 of 100 primary gastric carcinomas (8%) by RsaI-COBRA (restriction site GTAC). Although the specific C-region was also included in the ssPCR products in Nakajima’s work, the RsaI restriction site GTAC is at -76 nt in the D-region where the correlation between CpG methylation and gene expression is not as well as the C-region[15]. Kang et al[14] reported that hMLH1 methylation was detected in 20.3% of gastric carcinoma cases. The possible reason for the higher positive rate might result from detection of CpG methylation in A-region of the hMLH1 promoter that are methylated partially even in cells expressing hMLH1[15]. Another reason was that they used a very sensitive assay, methylation-specific PCR (MSP)[24], which would result in a positive result even if 0.1% of testing cells were methylated. Detection of methylation by BstUI-COBRA used in the present study reflects the exact status of the hMLH1 methylation in the testing samples. Therefore, the low methylation rate in the present study most likely represents the true state of these gastric cancers.

Only the methylated hMLH1 was detectable in the xenograft of primary gastric carcinoma

It was reported that bialleles of hMLH1 were inactivated by CpG methylation in cell lines[25]. Unlike in cell lines, both the methylated and unmethylated hMLH1 were observed in primary gastric carcinomas (Figure 1-T1, T2). In order to confirm that hMLH1 is methylated homogeneously in malignant cells, we detected the status of hMLH1 methylation in UVa xenografts originated from hMLH1-methylated and -unmethylated primary gastric carcinomas by BstUI-COBRA. No hMLH1 methylation was detected in two xenografts from two hMLH1-unmethylated primary gastric carcinoma (Figure 1-NC1, NC2). Only methylated hMLH1 was observed in the X2 xenograft from primary gastric carcinoma T2, in which both the methylated and unmethylated hMLH1 were observed (Figure 1-X2, T2). This result was also confirmed by results of DHPLC (Figure 2-B). These results suggested that bialleles of hMLH1 was methylated homogeneously in all of the malignant cells. The unmethylated hMLH1 in T2 should come from normal cells such as fibrocytes, fibroblasts, and lymphocytes comprising the primary carcinoma. To best of our knowledge, this is the first report to describe the distribution of methylation status of CpG islands in primary carcinoma.

Thus, all of the malignant cells in the primary gastric cancer T2 appear originate from a single initiated cell with biallelic aberrant methylation of the hMLH1 promoter. It is useful to study whether certain extent of methylation in the specific C-region of hMLH1 CpG island is detectable in precancerous gastric lesions by sensitive assays such as MSP[24] and Methylight[26]. Taken together, the hMLH1 methyaltion may play an important role in the initiation stage of a few gastric carcinomas. In addition to inactivation of hMLH1 by germ-line defects[27-32], silencing of hMLH1 by CpG methylation is an alternative way to inactivate hMLH1.

SNP at -93 of the hMLH1 promoter is not correlated with gastric carcinoma

There is a SNP at -93 of the hMLH1 promoter[22], which is located within the D-region tested in the present study. In order to evaluate the correlation of the SNP with risk of gastric cancer, we compared the pattern of the SNP in 54 Chinese patients with gastric carcinoma with that in 56 Chinese control patients through DHPLC and sequencing. Similar percentages of homoalleles (G/G and A/A) and heteroalleles (A/G) were observed in samples from both groups (Table 1). The result suggested that this SNP was not correlated with the risk of gastric carcinoma. Similar result was observed in hereditary nonpolyposis colorectal cancer (HNPCC) and non-HNPCC populations[22].

Table 1 Pattern of the SNP at -93 nt of the hMLH1 promoter in patients with and without gastric carcinoma.
PatientsnG/GA/GA/A
Without gastric carcinoma569 (16.1%)29 (51.8%)18 (32.1%)
With gastric carcinoma548 (14.9%)27 (50.0%)19 (35.2%)
Total11017 (15.5%)56 (50.9%)37 (33.6%)
Footnotes

Edited by Xu XQ

References
1.  Bird A. DNA methylation de novo. Science. 1999;286:2287-2288.  [PubMed]  [DOI]
2.  Jones PA, Laird PW. Cancer epigenetics comes of age. Nat Genet. 1999;21:163-167.  [PubMed]  [DOI]
3.  Yakoob J, Fan XG, Hu GL, Zhang Z. DNA methylation and carcinogenesis in digestive neoplasms. World J Gastroenterol. 1998;4:174-177.  [PubMed]  [DOI]
4.  Leung SY, Yuen ST, Chung LP, Chu KM, Chan AS, Ho JC. hMLH1 promoter methylation and lack of hMLH1 expression in sporadic gastric carcinomas with high-frequency microsatellite instability. Cancer Res. 1999;59:159-164.  [PubMed]  [DOI]
5.  Fleisher AS, Esteller M, Wang S, Tamura G, Suzuki H, Yin J, Zou TT, Abraham JM, Kong D, Smolinski KN. Hypermethylation of the hMLH1 gene promoter in human gastric cancers with microsatellite instability. Cancer Res. 1999;59:1090-1095.  [PubMed]  [DOI]
6.  Kang GH, Shim YH, Ro JY. Correlation of methylation of the hMLH1 promoter with lack of expression of hMLH1 in sporadic gastric carcinomas with replication error. Lab Invest. 1999;79:903-909.  [PubMed]  [DOI]
7.  Bevilacqua RA, Simpson AJ. Methylation of the hMLH1 promoter but no hMLH1 mutations in sporadic gastric carcinomas with high-level microsatellite instability. Int J Cancer. 2000;87:200-203.  [PubMed]  [DOI]
8.  Pinto M, Oliveira C, Machado JC, Cirnes L, Tavares J, Carneiro F, Hamelin R, Hofstra R, Seruca R, Sobrinho-Simões M. MSI-L gastric carcinomas share the hMLH1 methylation status of MSI-H carcinomas but not their clinicopathological profile. Lab Invest. 2000;80:1915-1923.  [PubMed]  [DOI]
9.  Jung HY, Jung KC, Shim YH, Ro JY, Kang GH. Methylation of the hMLH1 promoter in multiple gastric carcinomas with microsatellite instability. Pathol Int. 2001;51:445-451.  [PubMed]  [DOI]
10.  Sakata K, Tamura G, Endoh Y, Ohmura K, Ogata S, Motoyama T. Hypermethylation of the hMLH1 gene promoter in solitary and multiple gastric cancers with microsatellite instability. Br J Cancer. 2002;86:564-567.  [PubMed]  [DOI]
11.  Oue N, Sentani K, Yokozaki H, Kitadai Y, Ito R, Yasui W. Promoter methylation status of the DNA repair genes hMLH1 and MGMT in gastric carcinoma and metaplastic mucosa. Pathobiology. 2001;69:143-149.  [PubMed]  [DOI]
12.  Baek MJ, Kang H, Kim SE, Park JH, Lee JS, Paik YK, Kim H. Expression of hMLH1 is inactivated in the gastric adenomas with enhanced microsatellite instability. Br J Cancer. 2001;85:1147-1152.  [PubMed]  [DOI]
13.  Nakajima T, Akiyama Y, Shiraishi J, Arai T, Yanagisawa Y, Ara M, Fukuda Y, Sawabe M, Saitoh K, Kamiyama R. Age-related hypermethylation of the hMLH1 promoter in gastric cancers. Int J Cancer. 2001;94:208-211.  [PubMed]  [DOI]
14.  Kang GH, Shim YH, Jung HY, Kim WH, Ro JY, Rhyu MG. CpG island methylation in premalignant stages of gastric carcinoma. Cancer Res. 2001;61:2847-2851.  [PubMed]  [DOI]
15.  Deng G, Chen A, Hong J, Chae HS, Kim YS. Methylation of CpG in a small region of the hMLH1 promoter invariably correlates with the absence of gene expression. Cancer Res. 1999;59:2029-2033.  [PubMed]  [DOI]
16.  Deng G, Peng E, Gum J, Terdiman J, Sleisenger M, Kim YS. Methylation of hMLH1 promoter correlates with the gene silencing with a region-specific manner in colorectal cancer. Br J Cancer. 2002;86:574-579.  [PubMed]  [DOI]
17.  Kang YH, Bae SI, Kim WH. Comprehensive analysis of promoter methylation and altered expression of hMLH1 in gastric cancer cell lines with microsatellite instability. J Cancer Res Clin Oncol. 2002;128:119-124.  [PubMed]  [DOI]
18.  Deng DJ, Deng GR, Zhou J, Xin HJ. Detection of CpG methylations in human mismatch repair gene hMLH1 promoter by dena-turing high-performance liquid chromatography (DHPLC). Chin J Cancer Res. 2000;12:171-191.  [PubMed]  [DOI]
19.  Deng D, Deng G, Lü Y. [Analysis of the methylation in CpG island by denaturing high-performance liquid chromatography]. Zhonghua Yixue Zazhi. 2001;81:158-161.  [PubMed]  [DOI]
20.  Deng D, Deng G, Smith MF, Zhou J, Xin H, Powell SM, Lu Y. Simultaneous detection of CpG methylation and single nucleotide polymorphism by denaturing high performance liquid chromatography. Nucleic Acids Res. 2002;30:E13.  [PubMed]  [DOI]
21.  Deng D, El-Rifai W, Ji J, Zhu B, Trampont P, Li J, Smith MF, Powel SM. Hypermethylation of metallothionein-3 CpG island in gastric carcinoma. Carcinogenesis. 2003;24:25-29.  [PubMed]  [DOI]
22.  Ito E, Yanagisawa Y, Iwahashi Y, Suzuki Y, Nagasaki H, Akiyama Y, Sugano S, Yuasa Y, Maruyama K. A core promoter and a frequent single-nucleotide polymorphism of the mismatch repair gene hMLH1. Biochem Biophys Res Commun. 1999;256:488-494.  [PubMed]  [DOI]
23.  El-Rifai W, Frierson HF, Harper JC, Powell SM, Knuutila S. Expression profiling of gastric adenocarcinoma using cDNA array. Int J Cancer. 2001;92:832-838.  [PubMed]  [DOI]
24.  Herman JG, Graff JR, Myöhänen S, Nelkin BD, Baylin SB. Methylation-specific PCR: a novel PCR assay for methylation status of CpG islands. Proc Natl Acad Sci USA. 1996;93:9821-9826.  [PubMed]  [DOI]
25.  Veigl ML, Kasturi L, Olechnowicz J, Ma AH, Lutterbaugh JD, Periyasamy S, Li GM, Drummond J, Modrich PL, Sedwick WD. Biallelic inactivation of hMLH1 by epigenetic gene silencing, a novel mechanism causing human MSI cancers. Proc Natl Acad Sci U S A. 1998;95:8698-8702.  [PubMed]  [DOI]
26.  Eads CA, Danenberg KD, Kawakami K, Saltz LB, Blake C, Shibata D, Danenberg PV, Laird PW. MethyLight: a high-throughput assay to measure DNA methylation. Nucleic Acids Res. 2000;28:E32.  [PubMed]  [DOI]
27.  Leach FS, Nicolaides NC, Papadopoulos N, Liu B, Jen J, Parsons R, Peltomäki P, Sistonen P, Aaltonen LA, Nyström-Lahti M. Mutations of a mutS homolog in hereditary nonpolyposis colorectal cancer. Cell. 1993;75:1215-1225.  [PubMed]  [DOI]
28.  Kinzler KW, Vogelstein B. Lessons from hereditary colorectal cancer. Cell. 1996;87:159-170.  [PubMed]  [DOI]
29.  Eshleman JR, Markowitz SD. Microsatellite instability in inherited and sporadic neoplasms. Curr Opin Oncol. 1995;7:83-89.  [PubMed]  [DOI]
30.  Marra G, Boland CR. Hereditary nonpolyposis colorectal cancer: the syndrome, the genes, and historical perspectives. J Natl Cancer Inst. 1995;87:1114-1125.  [PubMed]  [DOI]
31.  Zhao B, Wang ZJ, Xu YF, Wan YL, Li P, Huang YT. Report of 16 kindreds and one kindred with hMLH1 germline mutation. World J Gastroenterol. 2002;8:263-266.  [PubMed]  [DOI]
32.  Cai Q, Sun MH, Lu HF, Zhang TM, Mo SJ, Xu Y, Cai SJ, Zhu XZ, Shi DR. Clinicopathological and molecular genetic analysis of 4 typical Chinese HNPCC families. World J Gastroenterol. 2001;7:805-810.  [PubMed]  [DOI]