CpG Methylation Analysis—Current Status of Clinical Assays and Potential Applications in Molecular Diagnostics

Sepulveda, Antonia R.; Jones, Dan; Ogino, Shuji; Samowitz, Wade S.; Gulley, Margaret L.; Edwards, Robin; Levenson, Victor; Pratt, Victoria M.; Yang, Bin; Nafa, Khédoudja; Yan, Liying; Vitazka, Patrick

doi:10.2353/jmoldx.2009.080125

Cited by 47 publications

(21 citation statements)

References 139 publications

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“…17,32,[37][38][39][40][41][42][43][44][45] Genome-wide DNA hypomethylation plays an important role in genomic instability by reactivating transposable DNA sequences and, therefore, contributes to colorectal carcinogenesis. 1,3-6,35,39,46 -48 Previous studies have reported that most carcinomas of colon, stomach, breast, lung, prostate, liver, and esophagus show hypomethylation of LINE-1 compared with matched normal tissues.…”

Section: Discussionmentioning

confidence: 99%

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Precision of Pyrosequencing Assay to Measure LINE-1 Methylation in Colon Cancer, Normal Colonic Mucosa, and Peripheral Blood Cells

Irahara

Nosho

Baba

et al. 2010

The Journal of Molecular Diagnostics

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134

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Genome-wide DNA hypomethylation plays an important role in epigenomic and genomic instability and colorectal carcinogenesis. DNA methylation in the long interspersed nucleotide element-1 , L1 (LINE-1) repetitive element is a good indicator of global DNA methylation level. In addition , LINE-1 hypomethylation in blood cells has been associated with colorectal adenoma risk , and LINE-1 hypomethylation in colorectal cancer is related with prognosis and linearly predicts shorter patient survival. However , no study has comprehensively evaluated the precision of sodium bisulfite conversion and PCR-pyrosequencing to measure LINE-1 methylation. Using 10 paraffin-embedded colon cancers, 5 matched normal colon mucosa, and 5 unrelated peripheral blood buffy coat leukocyte specimens, we enriched tumor DNA by macrodissection and laser capture microdissection. LINE-1 methylation was calculated as an average of 100 * C/(C ؉ T) at 4 CpG sites after bisulfite-PCR-pyrosequencing. The LINE-1 methylation value in colon cancers varied, ranging approximately from 30 to 80. To measure assay precision, we performed bisulfite conversion on seven different DNA specimen aliquots and repeated PCRpyrosequencing seven times. Run-to-run (between-run) SD ranged from 1.3 to 4.4 (median, 3.0) in macrodissected colon cancers; 1.1 to 10.5 (median, 3.8) in laser capture microdissection specimens; 1.3 to 2.5 (median, 1.9) in normal colon; and 1.5 to 3.4 (median, 1.9) in leukocyte DNA. In conclusion, bisulfite conversion and PCR-pyrosequencing assay can measure LINE-1 methylation in macrodissected colon cancer, normal colon, and blood DNA , and may be useful in clinical and research settings.

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Section: Discussionmentioning

confidence: 99%

“…Assays to measure DNA methylation, which are important in epigenetic research and clinical diagnostics, 17 typically rely on conversion of unmethylated cytosine to uracil by sodium bisulfite. However, no previous study has evaluated the precision of bisulfite conversion to measure LINE-1 methylation.…”

mentioning

confidence: 99%

Precision of Pyrosequencing Assay to Measure LINE-1 Methylation in Colon Cancer, Normal Colonic Mucosa, and Peripheral Blood Cells

Irahara

Nosho

Baba

et al. 2010

The Journal of Molecular Diagnostics

Self Cite

134

132

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“…6,9,10 CpG methylation is a central mechanism of epigenetic gene regulation, and occurs early in gastric cancer development, affecting genes known to have roles in gastric carcinogenesis such as MLH1, CDKN2A (p16), CDH1 (E-cadherin), RUNX3, COX-2, MGMT, and others. [11][12][13][14][15][16][17][18] Altered CpG methylation is already detected in mucosa with gastritis, is associated with chronic inflammation in response to H. pylori infection, and has been shown to be partially reversible after eradication of H. pylori infection. [19][20][21] In our study we used next-generation sequencing of bisulfite-treated DNA, which allows for sensitive quantification of the level of methylation at each individual CpG site present on the sequenced region.…”

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confidence: 99%

High-definition CpG methylation of novel genes in gastric carcinogenesis identified by next-generation sequencing

et al. 2016

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Gastric cancers are the most frequent gastric malignancy and usually arise in the sequence of Helicobacter pylori-associated chronic gastritis. CpG methylation is a central mechanism of epigenetic gene regulation affecting cancer-related genes, and occurs early in gastric carcinogenesis. DNA samples from non-metaplastic gastric mucosa with variable levels of gastritis (non-metaplastic mucosa), intestinal metaplasia, or gastric cancer were screened with methylation arrays for CpG methylation of cancer-related genes and 30 gene targets were further characterized by high-definition bisulfite next-generation sequencing. In addition, data from The Cancer Genome Atlas were analyzed for correlation of methylation with gene expression. Overall, 13 genes had significantly increased CpG methylation in gastric cancer vs non-metaplastic mucosa (BRINP1, CDH11, CHFR, EPHA5, EPHA7, FGF2, FLI1, GALR1, HS3ST2, PDGFRA, SEZ6L, SGCE, and SNRPN). Further, most of these genes had corresponding reduced expression levels in gastric cancer compared with intestinal metaplasia, including novel hypermethylated genes in gastric cancer (FLI1, GALR1, SGCE, and SNRPN), suggesting that they may regulate neoplastic transformation from non-malignant intestinal metaplasia to cancer. Our data suggest a tumor-suppressor role for FLI1 in gastric cancer, consistent with recently reported data in breast cancer. For the genes with strongest methylation/expression correlation, namely FLI1, the expression was lowest in microsatellite-unstable tumors compared with other gastric cancer molecular subtypes. Importantly, reduced expression of hypermethylated BRINP1 and SGCE was significantly associated with favorable survival in gastric cancer. In summary, we report novel methylation gene targets that may have functional roles in discrete stages of gastric carcinogenesis and may serve as biomarkers for diagnosis and prognosis of gastric cancer.

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“…The first generation sequencing used to characterize the glioblastoma landscape captures the most prevalent mutations. They did not analyze the deeper heterogeneity of low prevalence mutations that have been found in several tumor types, including colon cancer [129]. Efforts to examine whether such sub-clonal diversity exist in glioblastoms using highly sensitive techniques [130] have not identified the presence of low-prevalence mutations.…”

Section: Mutationsmentioning

confidence: 99%

Genetic Profiling: Searching for Novel Genetic Aberrations in Glioblastoma

Jamshidi¹,

Chen²

2013

Oncogenomics and Cancer Proteomics - Novel Approaches in Biomarkers Discovery and Therapeutic Targets in Cancer

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CpG Methylation Analysis—Current Status of Clinical Assays and Potential Applications in Molecular Diagnostics

Abstract: Methylation of CpG islands in gene promoter

Cited by 47 publications

References 139 publications

Precision of Pyrosequencing Assay to Measure LINE-1 Methylation in Colon Cancer, Normal Colonic Mucosa, and Peripheral Blood Cells

Precision of Pyrosequencing Assay to Measure LINE-1 Methylation in Colon Cancer, Normal Colonic Mucosa, and Peripheral Blood Cells

High-definition CpG methylation of novel genes in gastric carcinogenesis identified by next-generation sequencing

Genetic Profiling: Searching for Novel Genetic Aberrations in Glioblastoma

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