Impact of the Location of CpG Methylation within the GSTP1 Gene on Its Specificity as a DNA Marker for Hepatocellular Carcinoma

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

Biochemical identification of nonmethylated DNA by BioCAP-Seq

CpG islands are regions of vertebrate genomes that often function as gene regulatory elements and are associated with most gene promoters. CpG island elements usually contain nonmethylated CpG dinucleotides, while the remainder of the genome is pervasively methylated. We developed a biochemical approach called biotinylated CxxC affinity purification (BioCAP) to unbiasedly isolate regions of the genome that contain nonmethylated CpG dinucleotides. The resulting highly pure nonmethylated DNA is easily analyzed by quantitative PCR to interrogate specific loci or via massively parallel sequencing to yield genome-wide profiles

Epigenetics in renal cell cancer: mechanisms and clinical applications

Renal cell carcinoma (RCC) is characterized by an infrequent number of somatic mutations. By contrast, epigenetic aberrations are commonly found in RCC, indicating that epigenetic reprogramming is an important event in RCC development. Epigenetic alterations comprise several different aberrations, such as changes in histone modifications, DNA methylation, and microRNA levels, and occur in the most important signalling pathways in RCC, such as the von Hippel-Lindau disease tumour suppressor (VHL)-hypoxia-inducible factor (HIF) pathway, the WNT-beta-catenin pathway, and pathways involved in epithelial-mesenchymal transition. Owing to their involvement in these pathways and frequent occurrence in RCC, epigenetic alterations are regarded as potential biomarkers for the early detection of disease and for prediction of prognosis and treatment response. In addition, most of these alterations are potentially reversible, so they also provide new targets for therapy. At the moment, epigenetic biomarkers for RCC are not being used in clinical practice, but targeted epigenetic therapies are under investigation. Understanding the extent of epigenetic changes occurring in RCC and the mechanisms by which they influence disease progression and treatment response, as well as knowledge of current research on biomarkers and treatments, is crucial to successful clinical translation of epigenetics in RCC