Dataset Information

Methylation status of individual CpG sites within Alu elements in the human genome and Alu hypomethylation in gastric carcinomas.

ABSTRACT: BACKGROUND: Alu methylation is correlated with the overall level of DNA methylation and recombination activity of the genome. However, the maintenance and methylation status of each CpG site within Alu elements (Alu) and its methylation status have not well characterized. This information is useful for understanding natural status of Alu in the genome and helpful for developing an optimal assay to quantify Alu hypomethylation. METHODS: Bisulfite clone sequencing was carried out in 14 human gastric samples initially. A Cac8I COBRA-DHPLC assay was developed to detect methylated-Alu proportion in cell lines and 48 paired gastric carcinomas and 55 gastritis samples. DHPLC data were statistically interpreted using SPSS version 16.0. RESULTS: From the results of 427 Alu bisulfite clone sequences, we found that only 27.2% of CpG sites within Alu elements were preserved (4.6 of 17 analyzed CpGs, A approximately Q) and that 86.6% of remaining-CpGs were methylated. Deamination was the main reason for low preservation of methylation targets. A high correlation coefficient of methylation was observed between Alu clones and CpG site J (0.963), A (0.950), H (0.946), D (0.945). Comethylation of the sites H and J were used as an indicator of the proportion of methylated-Alu in a Cac8I COBRA-DHPLC assay. Validation studies showed that hypermethylation or hypomethylation of Alu elements in human cell lines could be detected sensitively by the assay after treatment with 5-aza-dC and M.SssI, respectively. The proportion of methylated-Alu copies in gastric carcinomas (3.01%) was significantly lower than that in the corresponding normal samples (3.19%) and gastritis biopsies (3.23%). CONCLUSIONS: Most Alu CpG sites are deaminated in the genome. 27% of Alu CpG sites represented in our amplification products. 87% of the remaining CpG sites are methylated. Alu hypomethylation in primary gastric carcinomas could be detected with the Cac8I COBRA-DHPLC assay quantitatively.

SUBMITTER: Xiang S

PROVIDER: S-EPMC2834620 | biostudies-literature | 2010

REPOSITORIES: biostudies-literature

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Methylation status of individual CpG sites within Alu elements in the human genome and Alu hypomethylation in gastric carcinomas.

Xiang Shengyan S Liu Zhaojun Z Zhang Baozhen B Zhou Jing J Zhu Bu-Dong BD Ji Jiafu J Deng Dajun D

BMC cancer 20100217

<h4>Background</h4>Alu methylation is correlated with the overall level of DNA methylation and recombination activity of the genome. However, the maintenance and methylation status of each CpG site within Alu elements (Alu) and its methylation status have not well characterized. This information is useful for understanding natural status of Alu in the genome and helpful for developing an optimal assay to quantify Alu hypomethylation.<h4>Methods</h4>Bisulfite clone sequencing was carried out in 1 ...[more]

PMID: 20163738

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Project description:BackgroundThe complex composition of different cell types within a tissue can be estimated by deconvolution of bulk gene expression profiles or with various single-cell sequencing approaches. Alternatively, DNA methylation (DNAm) profiles have been used to establish an atlas for multiple human tissues and cell types. DNAm is particularly suitable for deconvolution of cell types because each CG dinucleotide (CpG site) has only two states per DNA strand-methylated or non-methylated-and these epigenetic modifications are very consistent during cellular differentiation. So far, deconvolution of DNAm profiles implies complex signatures of many CpGs that are often measured by genome-wide analysis with Illumina BeadChip microarrays. In this study, we investigated if the characterization of cell types in tissue is also feasible with individual cell type-specific CpG sites, which can be addressed by targeted analysis, such as pyrosequencing.ResultsWe compiled and curated 579 Illumina 450k BeadChip DNAm profiles of 14 different non-malignant human cell types. A training and validation strategy was applied to identify and test for cell type-specific CpGs. We initially focused on estimating the relative amount of fibroblasts using two CpGs that were either hypermethylated or hypomethylated in fibroblasts. The combination of these two DNAm levels into a "FibroScore" correlated with the state of fibrosis and was associated with overall survival in various types of cancer. Furthermore, we identified hypomethylated CpGs for leukocytes, endothelial cells, epithelial cells, hepatocytes, glia, neurons, fibroblasts, and induced pluripotent stem cells. The accuracy of this eight CpG signature was tested in additional BeadChip datasets of defined cell mixtures and the results were comparable to previously published signatures based on several thousand CpGs. Finally, we established and validated pyrosequencing assays for the relevant CpGs that can be utilized for classification and deconvolution of cell types.ConclusionThis proof of concept study demonstrates that DNAm analysis at individual CpGs reflects the cellular composition of cellular mixtures and different tissues. Targeted analysis of these genomic regions facilitates robust methods for application in basic research and clinical settings.

Dataset Information

Methylation status of individual CpG sites within Alu elements in the human genome and Alu hypomethylation in gastric carcinomas.

Publications

Methylation status of individual CpG sites within Alu elements in the human genome and Alu hypomethylation in gastric carcinomas.

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