Amount and distribution of 5-methylcytosine in human DNA from different types of tissues or cells

Ehrlich, Melanie; Gama-Sosa, Miguel A.; Huang, Lan; Midgett, Rose Marie; Kuo, Kenneth C.; McCune, Roy A.; Gehrke, Charles W.

doi:10.1093/nar/10.8.2709

Cited by 918 publications

(605 citation statements)

References 41 publications

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“…Because approximately 70% of cytosine residues at CpG dinucleotides are methylated, these findings are consistent with deamination of 5-methylcytosine and subsequent conversion to a thymine after enzymatic replication. 27 These findings support previous work demonstrating that formalin fixation increases the rate of deamination 28 and suggests that methylated cytosines are especially susceptible to this effect. Despite these differences, examination of the total base changes revealed that all types of high-quality discrepancies were rare and, as described above, did not affect the ability to make high-quality consensus variant calls.…”

Section: Evaluation Of Formalin Fixationeinduced Sequencing Artifactssupporting

confidence: 88%

Comparison of Clinical Targeted Next-Generation Sequence Data from Formalin-Fixed and Fresh-Frozen Tissue Specimens

Spencer

Sehn

Abel

et al. 2013

The Journal of Molecular Diagnostics

179

156

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Next-generation sequencing (NGS) has emerged as a powerful technique for the detection of genetic variants in the clinical laboratory. NGS can be performed using DNA from FFPE tissue, but it is unknown whether such specimens are truly equivalent to unfixed tissue for NGS applications. To address this question, we performed hybridization-capture enrichment and multiplexed Illumina NGS for 27 cancerrelated genes using DNA from 16 paired fresh-frozen and routine FFPE lung adenocarcinoma specimens and conducted extensive comparisons between the sequence data from each sample type. This analysis revealed small but detectable differences between FFPE and frozen samples. Compared with frozen samples, NGS data from FFPE samples had smaller library insert sizes, greater coverage variability, and an increase in C to T transitions that was most pronounced at CpG dinucleotides, suggesting interplay between DNA methylation and formalin-induced changes; however, the error rate, library complexity, enrichment performance, and coverage statistics were not significantly different. Comparison of base calls between paired samples demonstrated concordances of >99.99%, with 96.8% agreement in the single-nucleotide variants detected and >98% accuracy of NGS data when compared with genotypes from an orthogonal single-nucleotide polymorphism array platform. This study demonstrates that routine processing of FFPE samples has a detectable but negligible effect on NGS data and that these samples can be a reliable substrate for clinical NGS testing. Next-generation sequencing (NGS) has recently emerged as a cost-effective method for identifying clinically actionable genetic variants across many genes in a single test. This approach has now been successfully applied to detect somatic mutations in hematopoietic malignant tumors, solid tumors, and constitutional mutations in genes associated with inherited cancer predisposition syndromes, among other clinical testing applications. 1e5 Further, numerous studies have demonstrated that NGS techniques are able to detect the full range of DNA variation, including single-nucleotide variants (SNVs), insertions/deletions, translocations, and copy number changes, 4,6e11 in the research setting. Thus, clinical NGS-based diagnostics offer an improvement over current molecular methods, such as PCR and Sanger sequencing, by which only a limited spectrum of mutations can be identified at a single genomic locus.Implementing NGS methods for routine clinical testing requires validation across the range of variables encountered in a clinical diagnostics laboratory, including the various specimen types that may be submitted for testing. The preferred specimen for most molecular tests is fresh tissue (eg, blood anticoagulated with EDTA or fresh tissue from a surgical biopsy or excision in saline) because this minimizes processing and Supported by the

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Section: Evaluation Of Formalin Fixationeinduced Sequencing Artifactssupporting

confidence: 88%

Comparison of Clinical Targeted Next-Generation Sequence Data from Formalin-Fixed and Fresh-Frozen Tissue Specimens

Spencer

Sehn

Abel

et al. 2013

The Journal of Molecular Diagnostics

179

156

View full text Add to dashboard Cite

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“…Human DNA was purified and M. luteus DNA (Sigma) was repurified as described previously (18). They were enriched in m5C in a mostly strandasymmetrical fashion by nick translation after pre-nicking as above except that m5dCTP (P-L Biochemicals), dGTP, dTTP, and dATP were the triphosphates used and the DNase digestion was for 10 min to give "'4-20 kb length products.…”

Section: Methodsmentioning

confidence: 99%

“…The normally programmed occurrence of de novo methylation is also evidenced by the considerably lower methylation level of the highly repeated sequence fraction of human sperm DNA than that of somatic tissue DNAs (18).…”

Section: Introductionmentioning

confidence: 99%

Human placental DNA methyltransferase: DNA substrate and DNA binding specificity

1984

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We have partially purified a DNA methyltransferase from human placenta using a novel substrate for a highly sensitive assay of methylation of hemimethylated DNA. This substrate was prepared by extensive nick translation of bacteriophage XP12 DNA, which normally5 has virtually all of its cytosine residues replaced by 5-methylcytosine (m C). Micrococcus luteus DNA wgs just as good a substrate if it was first similarly nick translated with m dCTP instead of dCTP in the polymerization mixture. At different stages in purification and under various conditions (including in the _presence or absence of high mobility group proteins), the methylation of m C-deficient DVA and that of hemimethylated DNA were compared. Although hemimethylated, m C-rich DNAs were much better substrates than were m C-deficient DNAs and normal XP12 DNA could not be methylated, all of these DNAs were bound equally well by the enzyme. In contrast, from the same placental extraSt, a DNAbinding protein of unknown functon was isolated which binds to m C-rich DNA in preference to the analogous m C-poor DNA.

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“…Between 60% and 90% of all CpGs are methylated in mammals [Ehrlich et al, 1982]. Methylation near gene promoters (with or without a CpG island) varies considerably depending on cell type, with higher promoter methylation correlating with low or no transcription [Suzuki and Bird, 2008].…”

Section: Dna Methylation and Demethylationmentioning

confidence: 99%

Epigenetics, fragile X syndrome and transcriptional therapy

Tabolacci

Chiurazzi

2013

American J of Med Genetics Pt A

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Epigenetics refers to the study of heritable changes in gene expression that occur without a change in DNA sequence. Epigenetic mechanisms therefore include all transcriptional controls that determine how genes are expressed during development and differentiation, but also in individual cells responding to environmental stimuli. The purpose of this review is to examine the basic principles of epigenetic mechanisms and their contribution to human disorders with a particular focus on fragile X syndrome (FXS), the most common monogenic form of developmental cognitive impairment. FXS represents a prototype of the so-called repeat expansion disorders due to "dynamic" mutations, namely the expansion (known as "full mutation") of a CGG repeat in the 5 0 UTR of the FMR1 gene. This genetic anomaly is accompanied by epigenetic modifications (mainly DNA methylation and histone deacetylation), resulting in the inactivation of the FMR1 gene. The presence of an intact FMR1 coding sequence allowed pharmacological reactivation of gene transcription, particularly through the use of the DNA demethylating agent 5 0 -aza-2 0 -deoxycytydine and/or inhibitors of histone deacetylases. These treatments suggested that DNA methylation is dominant over histone acetylation in silencing the FMR1 gene. The importance of DNA methylation in repressing FMR1 transcription is confirmed by the existence of rare unaffected males carrying unmethylated full mutations. Finally, we address the potential use of epigenetic approaches to targeted treatment of other genetic conditions. Ó 2013 Wiley Periodicals, Inc.Key words: fragile X syndrome; epigenetics; transcriptional therapy INTRODUCTION When in 1942 Conrad H. Waddington coined the term "epigenetics," the exact nature of genes and their role in heredity and in transcriptional regulation was not known; he used it as a conceptual model of how genes might interact with their surroundings to produce a phenotype [Waddington, 1942]. The term is a portmanteau of the words epigenesis (differentiation of cells from their initial totipotent state in embryonic development) and genetics. Only in 2008 at a Cold Spring harbor meeting, consensus was reached on the definition of epigenetic trait, as "stably heritable phenotype resulting from changes in a chromosome without alterations in the DNA sequence" [Berger et al., 2009]. The greek prefix epi-refers to features that are "on top of" genetics, that is, all those stable but reversible changes to DNA, RNA and proteins that regulate gene expression and allow cells with the same DNA to do different things at different times.Broadly speaking, epigenetic mechanisms include all transcriptional controls that regulate gene expression, whether during development and differentiation or in mature cells responding to the environment. Epigenetic changes can be inherited (e.g., imprinting) and be relatively stable (e.g., chromosome X inactivation), but are often rapidly imposed or removed on a given locus according to cell needs. Four major layers of epigenetic controls have ...

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Amount and distribution of 5-methylcytosine in human DNA from different types of tissues or cells

Cited by 918 publications

References 41 publications

Comparison of Clinical Targeted Next-Generation Sequence Data from Formalin-Fixed and Fresh-Frozen Tissue Specimens

Comparison of Clinical Targeted Next-Generation Sequence Data from Formalin-Fixed and Fresh-Frozen Tissue Specimens

Human placental DNA methyltransferase: DNA substrate and DNA binding specificity

Epigenetics, fragile X syndrome and transcriptional therapy

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