Elucidating the Landscape of Aberrant DNA Methylation in Hepatocellular Carcinoma

<div><p>Background</p><p>Hepatocellular carcinoma (HCC) is one of the most common cancers and frequently presents with an advanced disease at diagnosis. There is only limited knowledge of genome-scale methylation changes in HCC.</p> <p>Methods and Findings</p><p>We performed genome-wide methylation profiling in a total of 47 samples including 27 HCC and 20 adjacent normal liver tissues using the Illumina HumanMethylation450 BeadChip. We focused on differential methylation patterns in the promoter CpG islands as well as in various less studied genomic regions such as those surrounding the CpG islands, i.e. shores and shelves. Of the 485,577 loci studied, significant differential methylation (DM) was observed between HCC and adjacent normal tissues at 62,692 loci or 13% (p<1.03e-07). Of them, 61,058 loci (97%) were hypomethylated and most of these loci were located in the intergenic regions (43%) or gene bodies (33%). Our analysis also identified 10,775 differentially methylated (DM) loci (17% out of 62,692 loci) located in or surrounding the gene promoters, 4% of which reside in known Differentially Methylated Regions (DMRs) including reprogramming specific DMRs and cancer specific DMRs, while the rest (10,315) involving 4,106 genes could be potential new HCC DMR loci. Interestingly, the promoter-related DM loci occurred twice as frequently in the shores than in the actual CpG islands. We further characterized 982 DM loci in the promoter CpG islands to evaluate their potential biological function and found that the methylation changes could have effect on the signaling networks of Cellular development, Gene expression and Cell death (p = 1.0e-38), with <i>BMP4</i>, <i>CDKN2A</i>, <i>GSTP1</i>, and <i>NFATC1</i> on the top of the gene list.</p> <p>Conclusion</p><p>Substantial changes of DNA methylation at a genome-wide level were observed in HCC. Understanding epigenetic changes in HCC will help to elucidate the pathogenesis and may eventually lead to identification of molecular markers for liver cancer diagnosis, treatment and prognosis.</p> </div

Top 20 hypomethylated and hypermethylated loci in HCC based on Delta-beta values.

a<p>Bonferroni Corrected p-value ^b DMR ^c Enhancer.</p

Dot plots of Beta values for 5 hypo- and hypermethylated loci among the 20 top based on the Delta-beta values from the tumor (n = 27, red) versus adjacent normal comparison (n = 20, blue).

<p>Each point represents the Beta value for an individual. The median Beta value for each locus and tissue type is indicated by a line inside each box-and-whisker within the graph. Paired samples are connected by a line.</p

Biological functions of 10 IPA networks of the genes harboring 982 differentially methylated (DM) loci in the promoter CpG islands.

<p>Italic and bold formatted genes represent hypo- and hypermethylated genes, respectively, with the bold italic formatted gene (<i>GNAS</i>) in network 7 having both hypo- and hypermethylated loci. The other genes are typically found in these networks in other studies, but not in our data set.</p

Distribution of promoter methylation levels.

<p>Genomic surroundings of the 10,775 promoter DM loci excluding the Open seas are shown <b>A.</b> in normals and <b>B.</b> in HCC tumors. The illustrative box plots present the median by a line in the box with the 25th percentile, 75th percentile and the range of the Beta values. Outlier values are shown with yellow color dots extending above or below the range markers. Density of functional loci on each genomic region is indicated on the top part of the figure. Averages of the Beta values are shown on each box plot. *previously known DMRs, cDMRs, rDMRs, Enhancers.</p

Methylation profiles of 10,775 promoter region DM loci by DMRs and in relation to CpG islands.

<p><b>A.</b> Distribution of DM loci in CpG islands and the surrounding shore (0–2 kb from promoter CpG islands), shelf (2–4 kb from promoter CpG islands) and Open sea (other regions in promoter) DM areas. <b>B.</b> DM locus distribution by known differentially methylated regions (DMRs), reprogramming specific DMRs (rDMRs), cancer specific DMRs (cDMRs) and potential novel DMRs in HCC. <b>C.</b> Unsupervised hierarchical clustering of beta values for 702 Shelves, 1,952 Shores, 982 CpG island, and 7,139 Open seas loci (rows) in 47 samples (columns). Blue and red blocks on top of the maps represent 20 adjacent normal and 27 HCC tissues, respectively, while red for the loci represents hypermethylation and blue hypomethylation.</p

Validation results of 8 DM loci by Pyrosequencing.

<p>Gene name, Target ID (locus) by Illumina, and the correlation coefficients (r) are presented. Beta values for each individual HCC (red) and normal (blue) samples are presented by dots. The x-axis and y-axis indicate the Beta value from the Methylation450 BeadChip analysis and the methylation level by Pyrosequencing, respectively.</p

Clinical and Pathological Characteristics of 27 HCC cases.

*<p>A person had more than 2 drinks a day for 10 years or more.</p

Anaphalis sinica Hance var. viscosissima Kitam.

原著和名: クリヤマハハコ科名: キク科 = Compositae採集地: 栃木県 塩谷郡 栗山村 (下野 栗山村)採集日: 1969/8/31採集者: 萩庭丈壽整理番号: JH027401国立科学博物館整理番号: TNS-VS-97740

Distinct Plasma Bile Acid Profiles of Biliary Atresia and Neonatal Hepatitis Syndrome

Biliary atresia (BA) is a severe chronic cholestasis disorder of infants that leads to death if not treated on time. Neonatal hepatitis syndrome (NHS) is another leading cause of neonatal cholestasis confounding the diagnosis of BA. Recent studies indicate that altered bile acid metabolism is closely associated with liver injury and cholestasis. In this study, we systematically measured the bile acid metabolome in plasma of BA, NHS, and healthy controls. Liver bile acids were also measured using biopsy samples from 48 BA and 16 NHS infants undergoing operative cholangiography as well as 5 normal adjacent nontumor liver tissues taken from hepatoblastoma patients as controls. Both BA and NHS samples had significantly elevated bile acid levels in plasma compared to normal controls. BA patients showed a distinct bile acid profile characterized by the higher taurochenodeoxycholic acid (TCDCA) level and lower chenodeoxycholic acid (CDCA) level than those in NHS patients. The ratio of TCDCA to CDCA in plasma was significantly higher in BA compared to healthy infants (<i>p</i> < 0.001) or NHS (<i>p</i> < 0.001). The area under receiver operating characteristic curve for TCDCA/CDCA to differentiate BA from NHS was 0.923 (95% CI: 0.862–0.984). These findings were supported by significantly altered expression levels of bile acid transporters and nuclear receptors in liver including farnesoid X receptor (FXR), small heterodimer partner (SHP), bile salt export pump (BSEP), and multidrug resistant protein 3 (MDR3) in BA compared to NHS. Taken together, the plasma bile acid profiles are distinct in BA, NHS, and normal infants, as characterized by the ratio of TCDCA/CDCA differentially distributed among the three groups of infants