Impact of the location of CpG methylation within the GSTP1 gene on its specificity as a DNA marker for hepatocellular carcinoma

• Published in: PloS one Vol. 7; no. 4; p. e35789
• Main Authors: Jain, Surbhi, Chen, Sitong, Chang, Kung-Chao, Lin, Yih-Jyh, Hu, Chi-Tan, Boldbaatar, Batbold, Hamilton, James P, Lin, Selena Y, Chang, Ting-Tsung, Chen, Shun-Hua, Song, Wei, Meltzer, Stephen J, Block, Timothy M, Su, Ying-Hsiu
• Format: Journal Article
• Language: English
• Published: United States Public Library of Science 20.04.2012; Public Library of Science (PLoS)
• Subjects: Aged; alpha-Fetoproteins - metabolism; Biology; Biomarkers; Biomarkers, Tumor - genetics; Bisulfite; Cancer genetics; Carcinoma, Hepatocellular - diagnosis; Carcinoma, Hepatocellular - enzymology; Carcinoma, Hepatocellular - genetics; Case-Control Studies; Cell cycle; Cirrhosis; Colorectal cancer; Comparative analysis; CpG Islands; Deoxyribonucleic acid; Diagnosis, Differential; DNA; DNA Methylation; Epigenetic inheritance; Epigenetics; Female; Fetuses; Gastroenterology; Gene expression; Genes; Genetic markers; Genetic research; Glutathione; Glutathione S-Transferase pi - genetics; Glutathione transferase; Hepatitis; Hepatitis - diagnosis; Hepatitis - genetics; Hepatocellular carcinoma; Hepatology; Humans; Immunology; Liver; Liver - enzymology; Liver cancer; Liver cirrhosis; Liver Cirrhosis - diagnosis; Liver Cirrhosis - genetics; Liver diseases; Liver Neoplasms - diagnosis; Liver Neoplasms - enzymology; Liver Neoplasms - genetics; Male; Medicine; Methylation; Middle Aged; Mutation; Organ Specificity; Polymerase chain reaction; Promoter Regions, Genetic; ROC Curve; Sensitivity and Specificity; Sequence Analysis, DNA; Sulfites; Tissues; Transcription; Transcription (Genetics); United States > US; Maryland; Baltimore Maryland; Taiwan; Pennsylvania
• ISSN: 1932-6203; 1932-6203
• DOI: 10.1371/journal.pone.0035789

Hypermethylation of the glutathione S-transferase π 1 (GSTP1) gene promoter region has been reported to be a potential biomarker to distinguish hepatocellular carcinoma (HCC) from other liver diseases. However, reports regarding how specific a marker it is have ranged from 100% to 0%. We hypothesized that, to a large extent, the variation of specificity depends on the location of the CpG sites analyzed. To test this hypothesis, we compared the methylation status of the GSTP1 promoter region of the DNA isolated from HCC, cirrhosis, hepatitis, and normal liver tissues by bisulfite-PCR sequencing. We found that the 5' region of the position -48 nt from the transcription start site of the GSTP1 gene is selectively methylated in HCC, whereas the 3' region is methylated in all liver tissues examined, including normal liver and the HCC tissue. Interestingly, when DNA derived from fetal liver and 11 nonhepatic normal tissue was also examined by bisulfite-PCR sequencing, we found that methylation of the 3' region of the promoter appeared to be liver-specific. A methylation-specific PCR assay targeting the 5' region of the promoter was developed and used to quantify the methylated GSTP1 gene in various diseased liver tissues including HCC. When we used an assay targeting the 3' region, we found that the methylation of the 5'-end of the GSTP1 promoter was significantly more specific than that of the 3'-end (97.1% vs. 60%, p<0.0001 by Fisher's exact test) for distinguishing HCC (n = 120) from hepatitis (n = 35) and cirrhosis (n = 35). Encouragingly, 33.8% of the AFP-negative HCC contained the methylated GSTP1 gene. This study clearly demonstrates the importance of the location of CpG site methylation for HCC specificity and how liver-specific DNA methylation should be considered when an epigenetic DNA marker is studied for detection of HCC.