Detection and measurement of PCR bias in quantitative methylation analysis of bisulphite-treated DNA

Warnecke, P.; Stirzaker, Clare; Melki, John; Millar, Douglas; Paul, Cheryl L.; Clark, Susan J.

doi:10.1093/nar/25.21.4422

Cited by 351 publications

(260 citation statements)

References 9 publications

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“…The sequence divergence between methylated and unmethylated alleles after bisulfite conversion may lead to an amplification bias towards one product, i.e. the unmethylated T‐rich allele 36. In addition, alleles with incomplete bisulfite conversion may mimick hypermethylated alleles.…”

Section: Discussionmentioning

confidence: 99%

Single CpG hypermethylation, allele methylation errors, and decreased expression of multiple tumor suppressor genes in normal body cells of mutation‐negative early‐onset and high‐risk breast cancer patients

Böck

Appenzeller

Haertle

et al. 2018

Intl Journal of Cancer

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To evaluate the role of constitutive epigenetic changes in normal body cells of BRCA1/BRCA2‐mutation negative patients, we have developed a deep bisulfite sequencing assay targeting the promoter regions of 8 tumor suppressor (TS) genes (BRCA1, BRCA2, RAD51C, ATM, PTEN, TP53, MLH1, RB1) and the estrogene receptor gene (ESR1), which plays a role in tumor progression. We analyzed blood samples of two breast cancer (BC) cohorts with early onset (EO) and high risk (HR) for a heterozygous mutation, respectively, along with age‐matched controls. Methylation analysis of up to 50,000 individual DNA molecules per gene and sample allowed quantification of epimutations (alleles with >50% methylated CpGs), which are associated with epigenetic silencing. Compared to ESR1, which is representative for an average promoter, TS genes were characterized by a very low (< 1%) average methylation level and a very low mean epimutation rate (EMR; < 0.0001% to 0.1%). With exception of BRCA1, which showed an increased EMR in BC (0.31% vs. 0.06%), there was no significant difference between patients and controls. One of 36 HR BC patients exhibited a dramatically increased EMR (14.7%) in BRCA1, consistent with a disease‐causing epimutation. Approximately one third (15 of 44) EO BC patients exhibited increased rates of single CpG methylation errors in multiple TS genes. Both EO and HR BC patients exhibited global underexpression of blood TS genes. We propose that epigenetic abnormalities in normal body cells are indicative of disturbed mechanisms for maintaining low methylation and appropriate expression levels and may be associated with an increased BC risk.

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Section: Discussionmentioning

confidence: 99%

Single CpG hypermethylation, allele methylation errors, and decreased expression of multiple tumor suppressor genes in normal body cells of mutation‐negative early‐onset and high‐risk breast cancer patients

Böck

Appenzeller

Haertle

et al. 2018

Intl Journal of Cancer

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“…26 Biased amplification of remaining DNA (sequence-, strand-, and level of methylation-dependent bias) has been reported. 27 Although these problems may not be significant for homogeneous or ample specimens, they can be critical for heterogeneous clinical specimens and may produce inaccurate results, especially if DNA degradation is specific to certain sequences. In addition, degradation of the major part of a limited clinical sample may prevent its comprehensive analysis, which will be also reflected in reduced analytical sensitivity.…”

Section: Technical Approachmentioning

confidence: 99%

Array-Based Multiplex Analysis of DNA Methylation in Breast Cancer Tissues

Melnikov

Scholtens

Wiley

et al. 2008

The Journal of Molecular Diagnostics

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Abnormal DNA methylation is well established for cancer cells , but a methylation-based diagnostic test is yet to be developed. One of the problems is insufficient accuracy of cancer detection in heterogeneous clinical specimens when only a single gene is analyzed. A new technique was developed to produce a multigene methylation signature in each sample, and its potential for selection of informative genes was tested using DNA from formalin-fixed , paraffin-embedded breast cancer tissues. Fifty-six promoters were analyzed in each of 138 clinical specimens by a microarray-based modification of the previously developed technique. Specific methylation signatures were identified for atypical ductal hyperplasia , ductal carcinoma in situ, and invasive ductal carcinoma. Informative promoters selected by Fisher's exact test were used for composite biomarker design using naïve Bayes algorithm. All informative promoters were unmethylated in disease compared with normal tissue. Cross-validation showed 72.4% sensitivity and 74.7% specificity for detection of ductal carcinoma in situ and invasive ductal carcinoma, and 87.5% sensitivity and 95% specificity for detection of atypical ductal hyperplasia. These results indicate that informative cancer-specific methylation signatures can be detected in heterogeneous tissue specimens , suggesting that a diagnostic assay can then be developed. (J Mol

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“…The strand-speci®c nested primers used for ampli®cation of bisulphite-treated DNA are indicated in Table 1. The primer sets ampli®ed methylated and unmethylated sequences without signi®cant PCR bias when tested on control DNA, methylated to de®ned levels, as described in Warnecke et al (1997). The location of the primers is indicated according to the GSTP1 sequence (GenBank Accession number M24485).…”

Section: Methylation Analysismentioning

confidence: 99%

Detailed methylation analysis of the glutathione S-transferase π (GSTP1) gene in prostate cancer

et al. 1999

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Glutathione-S-Transferases (GSTs) comprise a family of isoenzymes that provide protection to mammalian cells against electrophilic metabolites of carcinogens and reactive oxygen species. Previous studies have shown that the CpG-rich promoter region of the p-class gene GSTP1 is methylated at single restriction sites in the majority of prostate cancers. In order to understand the nature of abnormal methylation of the GSTP1 gene in prostate cancer we undertook a detailed analysis of methylation at 131 CpG sites spanning the promoter and body of the gene. Our results show that DNA methylation is not con®ned to speci®c CpG sites in the promoter region of the GSTP1 gene but is extensive throughout the CpG island in prostate cancer cells. Furthermore we found that both alleles are abnormally methylated in this region. In normal prostate tissue, the entire CpG island was unmethylated, but extensive methylation was found outside the island in the body of the gene. Loss of GSTP1 expression correlated with DNA methylation of the CpG island in both prostate cancer cell lines and cancer tissues whereas methylation outside the CpG island in normal prostate tissue appeared to have no e ect on gene expression.

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Detection and measurement of PCR bias in quantitative methylation analysis of bisulphite-treated DNA

Cited by 351 publications

References 9 publications

Single CpG hypermethylation, allele methylation errors, and decreased expression of multiple tumor suppressor genes in normal body cells of mutation‐negative early‐onset and high‐risk breast cancer patients

Single CpG hypermethylation, allele methylation errors, and decreased expression of multiple tumor suppressor genes in normal body cells of mutation‐negative early‐onset and high‐risk breast cancer patients

Array-Based Multiplex Analysis of DNA Methylation in Breast Cancer Tissues

Detailed methylation analysis of the glutathione S-transferase π (GSTP1) gene in prostate cancer

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