Impact of the location of CpG methylation within the GSTP1 gene on its specificity as a DNA marker for hepatocellular carcinoma - PubMed (original) (raw)

doi: 10.1371/journal.pone.0035789. Epub 2012 Apr 20.

Sitong Chen, Kung-Chao Chang, Yih-Jyh Lin, Chi-Tan Hu, Batbold Boldbaatar, James P Hamilton, Selena Y Lin, Ting-Tsung Chang, Shun-Hua Chen, Wei Song, Stephen J Meltzer, Timothy M Block, Ying-Hsiu Su

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

Surbhi Jain et al. PLoS One. 2012.

Abstract

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.

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Conflict of interest statement

Competing Interests: The authors have the following competing interest: Wei Song is employed by a commercial company, JBS Science Inc.,Doylestown, PA. This does not alter the authors' adherence to all the PLoS ONE policies on sharing data and materials.

Figures

Figure 1

Figure 1. Methylation profiles of the sense and antisense strands of the GSTP1 gene by BSP sequencing of DNA isolated from normal liver and diseased liver tissues.

(A) Diagram of the locations of bisulfite sequencing primers and the CpG sites, indicated by vertical bars, in the promoter and the first exon regions of the GSTP1 gene (Genbank accession #M24485, nt. 999–1387). The transcription start site (TSS) is also indicated. The CpG sites are bracketed by the bisulfite sequencing primers for the forward (F) and reverse (R) sense strands (GSTP1_S_F and GSTP1_S_R) and the antisense strands (GSTP1_AS_F and GSTP1_AS_R). (B) Methylation status of each CpG site in both sense (S) and antisense (AS) strands of the promoter and the first exon regions of the GSTP1 gene from −28 to +4 on the basis of the sense strand 5′ to 3′ direction relative to TSS in hepatocellular carcinoma (HCC, n = 20) tissue, matched adjacent non-HCC liver tissue (Adj Non-HCC, n = 20), and normal (n = 6), hepatitis (n = 5), and cirrhosis (n = 5) tissues. The filled boxes indicate methylation detected and open boxes indicate no methylation detected. (C) Analysis of the extent of methylation at each CpG site of the sense strand GSTP1 gene by BS-PCR sequencing of DNA isolated from normal liver and diseased liver tissues. CpG site locations, BS-PCR sequencing assay, and DNA samples are the same as in panel B. The filled boxes indicate a high level of methylation detected (more than 50%); hatched boxes indicate a low level of methylation detected (50% or less); and open boxes indicate no methylation detected.

Figure 2

Figure 2. Methylation status of the sense (S) and antisense (AS) strands of the promoter and the first exon region of the GSTP1 gene (Genebank accession #M24485, nt. 999–1387) of the DNA isolated from normal adult livers, fetal liver, and normal nonliver tissues.

The open boxes indicate unmethylated CpG sites; the filled boxes indicate methylation detected.

Figure 3

Figure 3. Comparison of the specificity of the 5′-end and the 3-end of the mGSTP1 as a biomarker to distinguish HCC samples from tissue samples of other liver diseases, as determined by MSP assays.

(A) Locations of forward (F) and reverse (R) primers and TaqMan probe (P) of the 5′-end MSP (5′-MSP) (including the TaqMan probe) and 3′-end MSP (3′-MSP) SybrGreen assays. The CpG sites (vertical bars) and the transcription start site (TSS) are indicated. Receiver operating characteristic (ROC) curves of the methylated GSTP1 gene as a marker to discriminate HCC (n = 120) from non-HCC liver tissues including hepatitis (n = 35) and cirrhosis (n = 35) (B), or hepatitis, cirrhosis, and adjacent non-HCC (C), generated by 5′-end MSP and 3′-end MSP assays, respectively, as indicated. The amount of methylated DNA was the average of two duplicate MSP assays as detailed in Materials and Methods. The area under the curve of each ROC (AUROC) curve and the specificity and sensitivity determined by the cutoff of 10 copies per input of 300 copies of DNA are shown in the inserted table. Note that the CpG sites included in each primer and probe are as follows; 5′-end MSP (F: −27 to −24; P: −23 to −19; R: −11 to −10) and 3′-end MSP (F: −4 to −2; R: +4 to +7).

Figure 4

Figure 4. Scatter plot distribution of serum AFP levels (x-axis) and the amount of methylated 5′-end of the GSTP1 DNA (mGTSP1) (y-axis) for 115 HCC samples.

Each circle represents the value for an individual HCC case. A vertical reference line intersects at an AFP value of 20 ng/ml. A horizontal reference line intersects right above the MSP value of 0 as the reference for undetectable (ND), which is less than 10 copies per assay. The number of HCC cases and the percent of the total HCC in each of four areas are indicated.

References

    1. Cancer Facts and Figures 2010. American Cancer Society: Atlanta: American Cancer Society; 2010.
    1. Jemal A, Siegel R, Ward E, Hao Y, Xu J, et al. Cancer Statistics, 2008. CA: A Cancer Journal for Clinicians. 2008;58:71–96. - PubMed
    1. Bruix J, Sherman M. Management of hepatocellular carcinoma. Hepatology. 2005;42:1208–1236. - PubMed
    1. Nomoto S, Kinoshita T, Kato K. Hypermethylation of multiple genes as clonal markers in multicentric hepatocellular carcinoma. Br J Cancer. 2007;97(9):1260–1265. - PMC - PubMed
    1. Hanahan D, Weinberg RA. The Hallmarks of Cancer. Cell. 2000;100:57–70. - PubMed

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