Precision and Performance Characteristics of Bisulfite Conversion and Real-Time PCR (MethyLight) for Quantitative DNA Methylation Analysis

Ogino, Shuji; Kawasaki, Takako; Brahmandam, Mohan; Cantor, Mami; Kirkner, Gregory J.; Spiegelman, Donna; Makrigiorgos, G. Mike; Weisenberger, Daniel J.; Laird, Peter W.; Loda, Massimo; Fuchs, Charles S.

doi:10.2353/jmoldx.2006.050135

Cited by 374 publications

(436 citation statements)

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“…28 Cancers were considered methylated when PMR values were 44.0, as this threshold has previously been shown to correlate with loss of gene expression in a study that correlated the distribution of PMR to protein expression by immunohistochemistry. 29 For normal colorectal mucosa samples, PMR values were analysed as continuous variables given that DNA methylation is likely to occur at lower levels within an apparently normal field of colorectal crypts. An analysis of CIMP status in colorectal cancer samples was performed using MethyLight on a set of five CIMP markers as previously described.…”

Section: Methylation Analysesmentioning

confidence: 99%

Methylation of the 3p22 region encompassing MLH1 is representative of the CpG island methylator phenotype in colorectal cancer

et al. 2011

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Epigenetic silencing of cancer-related genes by promoter methylation is a frequent event in sporadic colorectal cancer. The CpG island methylator phenotype (CIMP þ ), in which discrete genes throughout the genome are simultaneously methylated, and long-range epigenetic silencing, whereby multiple genes within contiguous chromosomal regions are methylated, have been described in subsets of colorectal cancer. We previously reported the concurrent methylation of the mismatch repair gene MLH1 with a cluster of flanking genes in chromosome region 3p22 in sporadic colorectal carcinoma exhibiting microsatellite instability and the BRAF V600E mutation. Herein, we aimed to determine whether methylation of MLH1 and neighbouring 3p22 genes, singly or concomitantly, correlate with the germline c.-93G4A SNP within the MLH1 promoter, CIMP þ and other clinicopathological and molecular features of the tumours. By studying a cohort of 946 sporadic colorectal cancer cases, we show a strong association between concordant methylation of Z3 of five 3p22 genes with CIMP þ and the BRAF V600E mutation (Po0.001). These associations were independent of microsatellite instability, as concomitant methylation of 3p22 genes other than MLH1 was found in microsatellite stable cancers. These findings show that long-range epigenetic silencing across 3p22 occurs in the context of CIMP þ and the BRAF V600E mutation, and only gives rise to microsatellite instability when this process encompasses MLH1. Furthermore, the strong relationship between long-range epigenetic silencing of 3p22 and CIMP þ provides further evidence that these two purportedly distinct epigenetic phenotypes represent a single entity with a common aetiology. Low-level methylation of MLH1 and flanking 3p22 genes, as well as the BRAF V600E mutation, were detected in the apparently normal colonic mucosa of a small number of cases whose tumours showed a similar molecular profile, suggesting that these concurring genetic and epigenetic events can occur as a field defect in neoplastic development.

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Section: Methylation Analysesmentioning

confidence: 99%

Methylation of the 3p22 region encompassing MLH1 is representative of the CpG island methylator phenotype in colorectal cancer

et al. 2011

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“…17,31 Bisulfite Conversion and Real-Time PCR (MethyLight) for Quantitative DNA Methylation Analysis Sodium bisulfite treatment on tumor tissue samples and genomic DNA was performed as described previously. 24 We did not use laser capture microdissection because ample amounts of DNA were critical for reliable quantification of DNA methylation. 24 Utilizing MethyLight technology (real-time PCR) 20 and ABI 7300 (Applied Biosystems, Foster City, CA, USA), we quantified methylation in 8 promoters (CACNA1G (calcium channel, voltagedependent, T-type a-1G subunit), CDKN2A (cyclindependent kinase inhibitor 2A (p16/INK4A)), CRABP1 (cellular retinoic acid binding protein 1), IGF2 (insulin-like growth factor 2), MLH1, NEU-ROG1 (neurogenin 1), RUNX3 (runt-related transcription factor 3) and SOCS1 (suppressor of cytokine signaling 1)), all of which have been selected based on screening of 195 CpG islands.…”

Section: Sequencing Of Kras and Brafmentioning

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“…24 We did not use laser capture microdissection because ample amounts of DNA were critical for reliable quantification of DNA methylation. 24 Utilizing MethyLight technology (real-time PCR) 20 and ABI 7300 (Applied Biosystems, Foster …”

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confidence: 99%

“…MethyLight assays (quantitative methylation-specific PCR) are robust and reproducible in quantifying methylation in DNA from paraffinembedded tumor tissue. [21][22][23][24][25][26][27] Low index of methylation in MethyLight analysis is not associated with gene silencing, 24 indicating that low methylation index in MethyLight indeed reflects low-level DNA methylation.…”

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CpG island methylator phenotype-low (CIMP-low) colorectal cancer shows not only few methylated CIMP-high-specific CpG islands, but also low-level methylation at individual loci

et al. 2008

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The CpG island methylator phenotype (CIMP or CIMP-high) with widespread promoter methylation is a distinct phenotype in colorectal cancer. However, the concept of CIMP-low with less extensive CpG island methylation is still evolving. Our aim is to examine whether density of methylation in individual CpG islands was different between CIMP-low and CIMP-high tumors. Utilizing MethyLight technology and 889 population-based colorectal cancers, we quantified DNA methylation (methylation index, percentage of methylated reference) at 14 CpG islands, including 8 CIMP-high-specific loci (CACNA1G, CDKN2A (p16), CRABP1, IGF2, MLH1, NEUROG1, RUNX3 and SOCS1). Methylation positivity in each locus was defined as methylation index44. Low-level methylation (methylation index40, o20) in each CIMP-high-specific locus was significantly more common in 340 CIMP-low tumors (1/8-5/8 methylation-positive loci) than 133 CIMP-high tumors (Z6/8 methylation-positive loci) and 416 CIMP-0 tumors (0/8 methylation-positive loci) (Pr0.002). In the other six loci (CHFR, HIC1, IGFBP3, MGMT, MINT31 and WRN), which were not highly specific for CIMP-high, low-level methylation, was not persistently more prevalent in CIMP-low tumors. In conclusion, compared to CIMP-high and CIMP-0 tumors, CIMP-low colorectal cancers show not only few methylated CIMP-high-specific CpG islands, but also more frequent low-level methylation at individual loci. Our data may provide supporting evidence for a difference in pathogenesis of DNA methylation between CIMP-low and CIMP-high tumors. Modern Pathology (2008) 21, 245-255; doi:10.1038/modpathol.3800982; published online 18 January 2008 Keywords: colon cancer; CpG island methylator phenotype; DNA methylation; promoter; MethyLight; CIMP-low Epigenetic aberrations are important mechanisms in human carcinogenesis. 1 A number of tumor suppressor genes are silenced by promoter methylation in colorectal cancers. 1,2 A subset of colorectal cancers have been shown to exhibit widespread promoter methylation, which is referred to as the CpG island methylator phenotype (CIMP). 1,3 CIMPhigh colorectal tumors have a distinct clinical, pathologic and molecular profile, such as associations with proximal tumor location, female sex, poor tumor differentiation, inactive WNT/b-catenin (CTNNB1) and high BRAF and low TP53 mutation rates, 4-6 independent of microsatellite instability status. 7-10 Although controversial, CIMP may have prognostic implications in colorectal cancer. [11][12][13][14] In contrast to CIMP-high in colorectal cancer, the concept of CIMP-low (with less widespread CIMPspecific promoter methylation) is still emerging. 15,16 While CIMP-high colorectal cancer is associated with female sex and BRAF mutation, CIMP-low is associated with KRAS mutation. 17 Thus, CIMP-low cannot be explained by a simple mixture of CIMPhigh and CIMP-0 (CIMP-negative). 17 We have also demonstrated a possible link between CIMP-low, microsatellite instability-low, MGMT methylation and KRAS mutation in colorectal cancer. 18 18q loss of heterozy...

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Predictors of Lymph Node Count in Colorectal Cancer Resections

Morikawa

Tanaka²,

Kuchiba³

et al. 2012

Arch Surg

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Precision and Performance Characteristics of Bisulfite Conversion and Real-Time PCR (MethyLight) for Quantitative DNA Methylation Analysis

Cited by 374 publications

References 37 publications

Methylation of the 3p22 region encompassing MLH1 is representative of the CpG island methylator phenotype in colorectal cancer

Methylation of the 3p22 region encompassing MLH1 is representative of the CpG island methylator phenotype in colorectal cancer

CpG island methylator phenotype-low (CIMP-low) colorectal cancer shows not only few methylated CIMP-high-specific CpG islands, but also low-level methylation at individual loci

Predictors of Lymph Node Count in Colorectal Cancer Resections

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