Colorectal Cancer
Copyright ©2008 The WJG Press and Baishideng. All rights reserved.
World J Gastroenterol. Dec 28, 2008; 14(48): 7329-7334
Published online Dec 28, 2008. doi: 10.3748/wjg.14.7329
MLH1 promoter germline-methylation in selected probands of Chinese hereditary non-polyposis colorectal cancer families
Heng-Hua Zhou, Shi-Yan Yan, Xiao-Yan Zhou, Xiang Du, Tai-Ming Zhang, Xu Cai, Yong-Ming Lu, San-Jun Cai, Da-Ren Shi
Heng-Hua Zhou, Xiao-Yan Zhou, Xiang Du, Tai-Ming Zhang, Xu Cai, Yong-Ming Lu, Da-Ren Shi, Department of Pathology, Cancer Hospital/Institute, Fudan University; Department of Oncology, Shanghai Medical College, Fudan University, Shanghai 200032, China
Shi-Yan Yan, Department of Gastroenterology, Xinhua Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200092, China
San-Jun Cai, Department of Abdominal Surgery, Cancer Hospital/Institute, Fudan University, Shanghai 200032, China
Author contributions: Zhou HH and Yan SY participated in the treatment of patients, analyzed the data, performed the research and wrote the manuscript; Zhou XY, Du X and Shi DR participated in the treatment of patients, collected the data and designed the research; Cai SJ participated in the treatment of patients, collected the data; Other authors contributed to the new reagents/analytic tools.
Supported by Shanghai Medical Development Fund for Major Projects, No. 05III004 and Shanghai Pujiang Projects for Talents, No. 06PJ14019
Correspondence to: Da-Ren Shi, Department of Pathology, Cancer Hospital, Fudan University, 270 Dong An Road, Shanghai 200032, China. shidaren2000@yahoo.com
Telephone: +86-21-64046008 Fax: +86-21-64046008
Received: October 8, 2008
Revised: November 2, 2008
Accepted: November 9, 2008
Published online: December 28, 2008
Abstract

AIM: To detect the MLH1 gene promoter germline-methylation in probands of Chinese hereditary nonpolyposis colorectal cancer (HNPCC), and to evaluate the role of methylation in MLH1 gene promoter and molecular genetics in screening for HNPCC.

METHODS: The promoter germline methylation of MLH1 gene was detected by methylation-specific PCR (MSP) in 18 probands from unrelated HNPCC families with high microsatellite-instability (MSI-H) phenotype but without germline mutations in MSH2, MLH1 and MSH6 genes. At the same time, 6 kindreds were collected with microsatellite-stability (MSS) phenotype but without germline mutations in MSH2, MLH1 and MSH6 genes as controls. The results of MSP were confirmed by clone sequencing. To ensure the reliability of the results, family H65 with nonsense germline mutation at c.2228C > A in MSH2 gene was used as the negative control and the cell line sw48 was used as the known positive control along with water as the blank control. Immunochemical staining of MLH1 protein was performed with Envision two-step method in those patients with aberrant methylation to judge whether the status of MLH1 gene methylation affects the expression of MLH1 protein.

RESULTS: Five probands with MLH1 gene promoter methylation were detected in 18 Chinese HNPCC families with MSI-H phenotype but without germline mutations in MSH2, MLH1 and MSH6 genes. Two of the five probands from families H10 and H29 displayed exhaustive-methylation, fulfilling the Japanese criteria (JC) and the Amsterdam criteria (AC), respectively. The other 3 probands presented part-methylation fulfilling the AC. Of the 13 probands with unmethylation phenotype, 8 fulfilled the JC and the Bethesda guidelines (BG), 5 fulfilled the AC. The rate of aberrant methylation in MLH1 gene in the AC group (22.2%, 4/18) was higher than that in the JC/BG groups (5.6%, 1/18) in all HNPCC families with MSI-H phenotype but without germline mutations in MSH2, MLH1 and MSH6 genes. However, no proband with methylation in MLH1 gene was found in the families with MSS phenotype and without germline mutations in MSH2, MLH1 and MSH6 genes. No expression of MLH1 protein was found in tumor tissues from two patients with exhaustive-methylation phenotype, whereas positive expression of MLH1 protein was observed in tumor tissues from patients with partial methylation phenotype (excluding family H42 without tumor tissue), indicating that exhaustive-methylation of MLH1 gene can cause defective expression of MLH1 protein.

CONCLUSION: Methylation phenotype of MLH1 gene is correlated with microsatellite phenotype of MMR genes, especially with MSI-H. Exhaustive-methylation of MLH1 gene can silence the expression of MLH1 protein. MLH1 promoter methylation analysis is a promising tool for molecular genetics screening for HNPCC.

Keywords: Hereditary non-polyposis colorectal cancer; MLH1; Methylation; Germline; Methylation-specific PCR; Microsatellite phenotype