Autosomal CpG Sites Research Articles

Abstract 4996: Genome-wide DNA methylation profiles in hepatocellular carcinoma

Abstract Alterations in DNA methylation frequently occur in hepatocellular cancer (HCC). We have previously demonstrated that hypermethylation in candidate genes can be detected in plasma DNA prior to HCC diagnosis. To identify with a genome-wide approach additional genes hypermethylated in HCC that could be used for more accurate analysis of plasma DNA for early diagnosis, we analyzed tumor and adjacent non-tumor tissues from 62 Taiwanese HCC cases using Illumina methylation arrays that screen 26,486 autosomal CpG sites. After Bonferroni adjustment, a total of 2,324 CpG sites significantly differed in methylation level, with 684 CpG sites significantly hypermethylated and 1,640 hypomethylated in tumor compared to non-tumor tissues. Array data were validated with pyrosequencing in a subset of 5 of these genes; correlation coefficients ranged from 0.92 to 0.97. Analysis of plasma DNA from 38 cases demonstrated that 37% to 63% of cases had detectable hypermethylated DNA (≥5% methylation) for these 5 genes individually. At least one of these genes was hypermethylated in 87% of cases, suggesting that measurement of DNA methylation in plasma samples is feasible. The panel of methylated genes indentified in the current study will be further tested in large cohort of prospectively collected samples to determine their utility as early biomarkers of hepatocellular carcinoma. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4996. doi:1538-7445.AM2012-4996

DNA methylation patterns in alcoholics and family controls

To assess whether DNA methylation patterns in chronic alcoholics are different from non-alcoholic sibling controls. We examined the methylation patterns in DNA samples from 25 chronic alcoholics and 22 matched siblings as controls (one per family). DNA was extracted from peripheral blood and analyzed for differences in the methylation patterns after bisulfite-conversion. We used the Illumina GoldenGate Methylation Cancer Panel I (Illumina, San Diego, CA), which probes the methylation profile at 1505 CpG sites from 807 cancer related genes. We excluded the 84 X-chromosome CpG sites and 134 autosomal CpG sites that failed to show a within sample reliability score of at least 95% for all samples, leaving 1287 autosomal CpG sites (associated with 743 autosomal genes) with reliable signals for all samples. A methylation score was calculated as the average methylation for the 1287 CpG sites examined. Differences were assessed by a two-sample t-test. We also examined the average sib pair differences in methylation scores at each of the 1287 sites. All analyses were performed using SPSS, version 9.0, P < 0.05 was considered significant. Methylation levels at the 1287 CpG sites averaged 28.2% for both alcoholics and controls. The mean difference in methylation scores between alcoholic and non-alcoholic sibs by CpG site was < 1% with small inter-individual variances; and only 5 CpG sites had an average sib difference > 5%. Subgroup analysis showed that methylation scores were significantly lower for the alcoholic-dependent subjects who smoked compared to their non-smoking unaffected siblings. Specifically, among smokers who are alcoholic, global methylation indices were significantly lower than in non-alcoholic sib controls, whereas among non-smoking alcoholics, the global indices were significantly higher (P = 0.008). Although we observed no effect of alcoholism alone on DNA methylation, there is a decrease in alcoholics who smoke, suggesting a mechanism for alcohol-tobacco synergy for carcinogenesis.

Abstract 3004: Low levels of methylation in normal DNA mark a large subset of genes primed for hypermethylation in cancer

Abstract Cytosine methylation of DNA is a vital component of epigenetic memory. Complex changes of DNA methylation in cancer permanently disturb epigenetic regulation and participate in neoplastic development. To characterize changes in methylome in myeloid neoplasms, we analyzed DNA methylation in 8 patients with myelodysplastic syndrome, 2 patients with acute myeloid leukemia (MDS/AML) and 3 healthy controls. Using Digital Restriction Enzyme Analysis of Methylation (DREAM), we performed quantitative mapping of DNA methylation status at 40,000 autosomal CpG sites. To measure methylation, we first create distinct DNA signatures at unmethylated or methylated CCCGGG sites by sequential restriction digests of gDNA with the SmaI and XmaI endonucleases and then resolve these signatures by massively parallel sequencing. The sequences are mapped back to the genome and methylation levels for individual CpG sites are calculated based on the numbers of sequencing reads with unmethylated or methylated signatures. We found that DNA methylation status had a binomial distribution with the majority of CpG sites showing either very low or very high methylation. CpG islands (CGI) were generally unmethylated, while non-CGI were predominantly methylated with the exception of gene transcription start sites (TSS). When we compared average methylation in MDS/AML with average methylation in controls, the picture was different in CGI and in non-CGI. DNA methylation was increased in MDS/AML in CGI at all distances from gene transcription start sites (TSS) by 2-3%. In contrast, methylation in non-CGI was decreased in MDS/AML by approximately 3% with the exception of regions within 1 kb from TSS. Interestingly, low levels of CGI methylation detectable in blood DNA from healthy controls were associated with a propensity for hypermethylation in leukemia. Methylation levels 0-1% in controls were observed in 5,680 genes. Of those, 51 genes (1%) showed average methylation &amp;gt;20% in MDS/AML. Methylation 1-5% in controls was seen in 1,912 genes. Of those, 192 genes (10%) showed average methylation &amp;gt;20% in MDS/AML. Methylation 5-10% affected 364 genes in controls, while 131 (36%) of these genes had &amp;gt;20% methylation in MDS/AML (P=8.3E-240). The same pattern was observed when we restricted our analysis to methylation in CGI close to TSS (−1 kb to +1 kb). Methylation 0-1% in controls was found in 4,743 genes. Of those, only 24 genes (0.5%) showed average methylation &amp;gt;20% in MDS/AML. Methylation 1-5% in controls was detected in 1,204 genes. Of those, 94 genes (8%) showed average methylation &amp;gt;20% in MDS/AML. Methylation 5-10% was observed in 158 genes in controls, while 55 (35%) of these had &amp;gt;20% methylation in MDS/AML (P=7.7E-172). We conclude that low levels of methylation in CGI present in DNA of healthy individuals mark a large subset of genes destined for hypermethylation in cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3004. doi:10.1158/1538-7445.AM2011-3004

Cell specific patterns of methylation in the human placenta

Epigenetic processes, such as DNA methylation, are known to regulate tissue specific gene expression. We explored this concept in the placenta to define whether DNA methylation is cell-type specific. Cytotrophoblasts and fibroblasts were isolated from normal midtrimester placentas. Using immunocytochemistry, we demonstrated 95% purity for cytotrophoblasts and 60-70% for fibroblasts. We compared DNA methylation profiles from cytotrophoblasts, fibroblasts and whole placental villi using bisulfite modified genomic DNA hybridized to the Illumina Methylation27 array. Euclidean cluster analysis of the DNA methylation profiles showed 2 main clusters, one containing cytotrophoblasts and placenta, the other fibroblasts. Differential methylation analysis identified 442 autosomal CpG sites that differed between cytotrophoblasts and fibroblasts, 315 between placenta and fibroblasts and 61 between placenta and cytotrophoblasts. Three candidate methylation differences were validated by targeted pyrosequencing assays. Pyrosequencing assays were developed for CpG sites less methylated in cytotrophoblasts than fibroblasts mapping to the promoter region of the beta subunit of human chorionic gonadotropin 5 (CGB5), as well as 2 CpG sites mapping to each of 2 tumor suppressor genes. Our data suggest that epigenetic regulation of gene expression is likely to be a key factor in the functional specificity of cytotrophoblasts. These data are proof of principle for cell-type specific epigenetic regulation in placenta and demonstrate that the methylation profile of placenta is mainly driven by cytotrophoblasts.

DNA Methylation Profiles of Ovarian Epithelial Carcinoma Tumors and Cell Lines

BackgroundEpithelial ovarian carcinoma is a significant cause of cancer mortality in women worldwide and in the United States. Epithelial ovarian cancer comprises several histological subtypes, each with distinct clinical and molecular characteristics. The natural history of this heterogeneous disease, including the cell types of origin, is poorly understood. This study applied recently developed methods for high-throughput DNA methylation profiling to characterize ovarian cancer cell lines and tumors, including representatives of three major histologies.Methodology/Principal FindingsWe obtained DNA methylation profiles of 1,505 CpG sites (808 genes) in 27 primary epithelial ovarian tumors and 15 ovarian cancer cell lines. We found that the DNA methylation profiles of ovarian cancer cell lines were markedly different from those of primary ovarian tumors. Aggregate DNA methylation levels of the assayed CpG sites tended to be higher in ovarian cancer cell lines relative to ovarian tumors. Within the primary tumors, those of the same histological type were more alike in their methylation profiles than those of different subtypes. Supervised analyses identified 90 CpG sites (68 genes) that exhibited ‘subtype-specific’ DNA methylation patterns (FDR<1%) among the tumors. In ovarian cancer cell lines, we estimated that for at least 27% of analyzed autosomal CpG sites, increases in methylation were accompanied by decreases in transcription of the associated gene.SignificanceThe significant difference in DNA methylation profiles between ovarian cancer cell lines and tumors underscores the need to be cautious in using cell lines as tumor models for molecular studies of ovarian cancer and other cancers. Similarly, the distinct methylation profiles of the different histological types of ovarian tumors reinforces the need to treat the different histologies of ovarian cancer as different diseases, both clinically and in biomarker studies. These data provide a useful resource for future studies, including those of potential tumor progenitor cells, which may help illuminate the etiology and natural history of these cancers.