Differential methylation of the promoter and first exon of the RASSF1A gene in hepatocarcinogenesis.
OBJECTIVE
Aberrant methylation of the promoter, P2, and the first exon, E1, regions of the tumor suppressor gene RASSF1A, have been associated with hepatocellular carcinoma (HCC), albeit with poor specificity. This study analyzed the methylation profiles of P1, P2 and E1 regions of the gene to identify the region of which methylation most specifically corresponds to HCC and to evaluate the potential of this methylated region as a biomarker in urine for HCC screening.


METHODS
Bisulfite DNA sequencing and quantitative methylation-specific polymerase chain reaction assays were performed to compare methylation of the 56 CpG sites in regions P1, P2 and E1 in DNA isolated from normal, hepatitic, cirrhotic, adjacent non-HCC, and HCC liver tissue and urine samples for the characterization of hypermethylation of the RASSF1A gene as a biomarker for HCC screening.


RESULTS
In tissue, comparing HCC (n = 120) with cirrhosis and hepatitis together (n = 70), methylation of P1 had an area under the receiver operating characteristics curve (AUROC) of 0.90, whereas methylation of E1 and P2 had AUROC of 0.84 and 0.72, respectively. At 90% sensitivity, specificity for P1 methylation was 72.9% versus 38.6% for E1 and 27.1% for P2. Methylated P1 DNA was detected in urine in association with cirrhosis and HCC. It had a sensitivity of 81.8% for α-fetoprotein negative HCC.


CONCLUSIONS
Among the three regions analyzed, methylation of P1 is the most specific for HCC and holds great promise as a DNA marker in urine for screening of cirrhosis and HCC.
JBS Science Inc., Doylestown, Pennsylvania, USA.
Surbhi
S
Jain
JBS Science Inc., Doylestown, Pennsylvania, USA.
Lijia
L
Xie
JBS Science Inc., Doylestown, Pennsylvania, USA.
Batbold
B
Boldbaatar
Department of Microbiology and Immunology, Drexel University College of Medicine, Philadelphia, Pennsylvania, USA.
Selena Y
SY
Lin
Division of Gastroenterology and Hepatology, Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.
James P
JP
Hamilton
Division of Gastroenterology and Hepatology, Department of Medicine, The Johns Hopkins University School of Medicine, Baltimore, Maryland, USA.|Department of Oncology, The Sidney Kimmel Comprehensive Cancer Center, Baltimore, Maryland, USA.
Stephen J
SJ
Meltzer
Department of Microbiology, Medical College, National Cheng Kung University, Tainan, Taiwan, China.
Shun-Hua
SH
Chen
Department of Medicine, Buddhist Tzu Chi General Hospital, Hualien, Taiwan, China.|Tzu Chi University, Hualien, Taiwan, China.
Chi-Tan
CT
Hu
Department of Microbiology and Immunology, Drexel University College of Medicine, Philadelphia, Pennsylvania, USA.
Timothy M
TM
Block
JBS Science Inc., Doylestown, Pennsylvania, USA.
Wei
W
Song
Department of Microbiology and Immunology, Drexel University College of Medicine, Philadelphia, Pennsylvania, USA.
Ying-Hsiu
YH
Su
RASSF1A; biomarkers; cirrhosis; hepatocellular carcinoma; methylation; urine