Quantification of DNA methylation using methylation-sensitive restriction enzymes and multiplex digital PCR

Nell, Rogier J.; Steenderen, Debby van; Menger, Nino V.; Weitering, Thomas J.; Versluis, Mieke; Velden, Pieter A. van der

doi:10.1101/816744

Cited by 5 publications

(10 citation statements)

References 25 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The methylation fraction (MF) of the CpG (cg11625005) in position 1,295,737 was determined by an in-house designed ddPCR assay in combination with HgaI methylation-sensitive restriction enzyme (MSRE) that cleaves this CpG when unmethylated, as described by Nell et al [24]. 100ng DNA sample was incubated with HgaI (2U/μl) and appurtenant 10X NEBuffer 1.1 (both from New England Biolabs, Bioké, Leiden, The Netherlands) for 60 minutes at 37°C and 65°C for 20 minutes.…”

Section: Methodsmentioning

confidence: 99%

“…Droplets were analysed through a QX200 droplet reader (Bio-Rad) using QuantaSoft software version 1.7.4 (Bio-Rad). Raw data was uploaded in online digital PCR management and analysis application Roodcom WebAnalysis (version 1.4.2, https://www.roodcom.nl/webanalysis/) [24], in which the MF was calculated by dividing the CNV of the digested sample with that of the paired undigested sample.…”

Section: Methodsmentioning

confidence: 99%

“…In the present study, we aim to elucidate the interaction of genetic and epigenetic mechanisms in regulation of TERT p. We approach this by using novel droplet digital PCR (ddPCR)-based assays [24]. Human-derived benign skin cells (keratinocytes, dermal fibroblasts, melanocytes, skin biopsy samples and naevi) and melanoma cell lines were analyzed.…”

Section: Introductionmentioning

confidence: 99%

See 2 more Smart Citations

Interplay betweenTERTpromoter mutations and methylation culminates in chromatin accessibility andTERTexpression

Salgado

Roelse

Nell

et al. 2019

Preprint

View full text Add to dashboard Cite

AbstractThe telomerase reverse transcriptase (TERT) gene is responsible for telomere maintenance in germline and stem cells, and is re-expressed in 90% of human cancers. Contrary to common concepts, CpG methylation in the TERT promoter (TERTp), was correlated with TERT mRNA expression. Furthermore, two hotspot mutations in TERTp, dubbed C228T and C250T, have been revealed to assist binding of transcription factor ETS/TCF and subsequent TERT expression. This study aimed to elucidate the combined contribution of epigenetic (promoter methylation and higher-order chromatin structure) and genetic (promoter mutations) mechanisms in regulating TERT gene expression in healthy skin and in melanoma cell lines (n=61). We unexpectedly observed that the methylation of TERTp was as high in a subset of healthy skin cells, mainly keratinocytes, as in cutaneous melanoma cell lines. In spite of the high promoter methylation fraction in wild-type (WT) samples, TERT mRNA was only expressed in the melanoma cell lines with high methylation or intermediate methylation in combination with TERT mutations. TERTp methylation was positively correlated with chromatin accessibility and expression in 8 melanoma cell lines. Cooperation between epigenetic and genetic mechanisms were best observed in heterozygous mutant cell lines as chromosome accessibility preferentially concerned the mutant allele. Combined, these results suggest a complex model in which TERT expression requires either a widely open chromatin state throughout the promoter in TERTp-WT samples due to high methylation or a combination of moderate methylation fraction/chromatin accessibility in the presence of the C228T/C250T mutations.Author summaryPvdV and RvD formulated research goals and aims and supervised the overall progress. Wet-lab experiments, preparation of the manuscript and statistical analysis were performed by CS and CR. CS designed the novel assays. RN was involved in the experimental setup. RvD, NG and PvdV were responsible for funding acquisition. CR, RN, NG, RvD and PvdV critically reviewed the manuscript.

show abstract

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Interplay betweenTERTpromoter mutations and methylation culminates in chromatin accessibility andTERTexpression

Salgado

Roelse

Nell

et al. 2019

Preprint

View full text Add to dashboard Cite

show abstract

“…Novel design of a ddPCR assay using methylation-sensitive restriction enzymes (MSREs) to determine TERTp methylation fraction. The methylation fraction (MF) of the CpG (cg11625005) in position 1,295,737 was determined by an in-house designed ddPCR assay in combination with HgaI methylation-sensitive restriction enzyme (MSRE) that cleaves this CpG when unmethylated, as described by Nell et al [24]. 100ng DNA sample was incubated with HgaI (2U/μl) and appurtenant 10X NEBuffer 1.1 (both from New England Biolabs, Bioké, Leiden, The Netherlands) for 60 minutes at 37˚C and 65˚C for 20 minutes.…”

Section: Promoter Methylation Determinationmentioning

confidence: 99%

“…In the present study, we aim to elucidate the interaction of genetic and epigenetic mechanisms in regulation of TERTp. We approach this by using novel droplet digital PCR (ddPCR)based assays [24]. Human-derived benign skin cells (keratinocytes, dermal fibroblasts, melanocytes, skin biopsy samples and naevi) and melanoma cell lines were analysed.…”

Section: Introductionmentioning

confidence: 99%

Interplay between TERT promoter mutations and methylation culminates in chromatin accessibility and TERT expression

et al. 2020

View full text Add to dashboard Cite

The telomerase reverse transcriptase (TERT) gene is responsible for telomere maintenance in germline and stem cells, and is re-expressed in 90% of human cancers. CpG methylation in the TERT promoter (TERTp) was correlated with TERT mRNA expression. Furthermore, two hotspot mutations in TERTp, dubbed C228T and C250T, have been revealed to facilitate binding of transcription factor ETS/TCF and subsequent TERT expression. This study aimed to elucidate the combined contribution of epigenetic (promoter methylation and chromatin accessibility) and genetic (promoter mutations) mechanisms in regulating TERT gene expression in healthy skin samples and in melanoma cell lines (n = 61). We unexpectedly observed that the methylation of TERTp was as high in a subset of healthy skin cells, mainly keratinocytes, as in cutaneous melanoma cell lines. In spite of the high promoter methylation fraction in wild-type (WT) samples, TERT mRNA was only expressed in the melanoma cell lines with either high methylation or intermediate methylation in combination with TERT mutations. TERTp methylation was positively correlated with chromatin accessibility and TERT mRNA expression in 8 melanoma cell lines. Cooperation between epigenetic and genetic mechanisms were best observed in heterozygous mutant cell lines as chromosome accessibility preferentially concerned the mutant allele. Combined, these results suggest a complex model in which TERT expression requires either a widely open chromatin state in TERTp-WT samples due to high methylation throughout the promoter or a combination of moderate methylation fraction/chromatin accessibility in the presence of the C228T or C250T mutations.

show abstract

Selective Enrichment of Hypermethylated DNA by a Multivalent Binding Platform

Kolkman

Segerink

Huskens

2022

Adv Materials Inter

View full text Add to dashboard Cite

One of the key aspects of surviving cancer is to detect the disease as early as possible, thereby enlarging the window of opportunity of curing it. [4,5] A recent trend for early cancer diagnostics is the pre-symptomatic detection of cancer biomarkers in liquid biopsy samples and, therefore, improving the survival rate. [6,7] Hypermethylated DNA (hmDNA) is one of the typical biomarkers found in liquid biopsy samples of cancer patients. [8,9] The presence of hmDNA in blood and urine is correlated to multiple types of cancer, including gastric, lung and ovarian cancer. [10][11][12][13] From a genetic point of view, DNA methylation takes place predominantly at promotor regions with a relative high amount of cytosine bases that are followed directly by a guanine (CpGs) in the 5′-to-3′ direction, the so-called CpG-rich regions. [13] The methylation of a CpG is an epigenetic alternation in which a methyl group is covalently bonded to the cytosine base at the fifth carbon. By CpG methylation, gene expressions are controlled in cells. As a consequence, methylation can play a role in tumor development when tumor suppressor genes are methylated, thereby repressing the transcription and thus silencing these genes. [14] Hypermethylation refers to the situation that methylation of a promotor region occurs, which in a healthy situation does not occur.Early disease detection as well as disease progress and the effectiveness of a therapy can be monitored by measuring the hmDNA concentration over time. [15] Yet, the concentration of hmDNA, especially in early-stage cancers, can be as low as a few DNA copies per liquid biopsy sample. [16][17][18] The current approach to measure hmDNA employs DNA isolation and bisulfite conversion, making it time-consuming and labor intensive. As a result, the method is not widely applicable in the clinic. Typically, hmDNA is detected in bisulfite-treated DNA samples by quantitative polymerase chain reaction (PCR) (qPCR, of the region of interest; can be cancer dependent). Alternative hmDNA detection approaches that may be suitable for use in point-of-care applications, include electrochemistry, [19] CRISPR [20] and isothermal amplification. [21] The detection of specific hmDNA biomarkers by sequencing or biosensing approaches involves the ability to distinguish between hmDNA and non-methylated DNA. [22][23][24] Commonly a preselection step is applied, and three different approaches exist in order to differentiate between hmDNA and non-methylatedThe preselection of hypermethylated DNA (hmDNA) from liquid biopsy samples is key to enable early-stage cancer diagnostics. Due to limited selectivity of the existing preselection approaches, however, wide integration in the clinic is currently prohibited. Here, it is argued that an affinity method on a surface, such as used in affinity chromatography, can be significantly improved by employing the principles of multivalency and superselectivity. In the here proposed method, a methyl binding domain (MBD) protein immobilized at a surface is used as a recepto...

show abstract

Quantification of DNA methylation using methylation-sensitive restriction enzymes and multiplex digital PCR

Cited by 5 publications

References 25 publications

Interplay betweenTERTpromoter mutations and methylation culminates in chromatin accessibility andTERTexpression

Interplay betweenTERTpromoter mutations and methylation culminates in chromatin accessibility andTERTexpression

Interplay between TERT promoter mutations and methylation culminates in chromatin accessibility and TERT expression

Selective Enrichment of Hypermethylated DNA by a Multivalent Binding Platform

Contact Info

Product

Resources

About