5‐Hydroxymethylcytosine as a clinical biomarker: Fluorescence‐based assay for high‐throughput epigenetic quantification in human tissues

Margalit, Sapir; Avraham, Sigal; Shahal, Tamar; Michaeli, Yael; Gilat, Noa; Magod, Prerna; Caspi, Michal; Loewenstein, Shelly; Lahat, Guy; Friedmann‐Morvinski, Dinorah; Kariv, Revital; Rosin‐Arbesfeld, Rina; Zirkin, Shahar; Ebenstein, Yuval

doi:10.1002/ijc.32519

Cited by 26 publications

(38 citation statements)

References 44 publications

(135 reference statements)

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“…Studies on the functional role of 5hmC have been heavily focused on change in chromosome-wide global 5hmC density or concentration, or regulation of transcription in the promoter region, or loss of 5hmC across many types of cancer [ 12 , 13 ]. Unlike the uniform distribution of 5mC outside of the promoter regions, satellites, and repeat DNA sequences [ 14 ], 5hmC has distinct distributions across different functional regions, and its abundance varies across different tissues and cell types [ 15 – 17 ].…”

Section: Discussionmentioning

confidence: 99%

5-Hydroxymethylcytosine (5hmC) at or near cancer mutation hot spots as potential targets for early cancer detection

Lu¹,

Lu²

2022

BMC Res Notes

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Objective Universal noninvasive genomic screening to detect cancer and/or fetal DNA in plasma at all stages of development is highly warranted. Since 5-hydroxymethylcytosine (5hmC) emerged as an intermediate metabolite in active DNA demethylation, there have been increasing efforts to elucidate its function as a stable modification of the genome. In the current study, we demonstrate that discrete 5hmC sites within 80 bp hotspot regions exist in a greater proportion of cancer versus normal cells. Result 5hmC was detected in 16 of 17 known hotspots having C to T or G to A mutations. The results show the presence of two characteristically distinct 5hmC groups: Tier 1 Group with 3 to eightfold more 5hmCs detected in tumor-cells than in normal-cell derived DNA (as observed in 6 of 11 CpG sites). Tier 2 group with equal allele frequency of 5hmC among normal and tumor-cell derived DNA at 5 CpG hotspot sites as well as 5 non-CpG hotspots. Thus, detection and quantification of the Tier 1 group of 5hmC sites or its prevalence at or near cancer mutation hot spots in cells may enable early detection, screening and potentially prediction of the likelihood of cancer occurrence or the severity of the cancer.

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Section: Discussionmentioning

confidence: 99%

5-Hydroxymethylcytosine (5hmC) at or near cancer mutation hot spots as potential targets for early cancer detection

Lu¹,

Lu²

2022

BMC Res Notes

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“…First, the enzyme β-glucosyltransferase (β-GT) is used to attach an azide modified glucose moiety from a synthetic cofactor (UDP-6-N3-Glu), to the hydroxyl group of 5-hmC. In the second step, a copper-free click reaction is used to connect a fluorophore-alkyne to the azide-labeled 5-hmC ( Figure 4B,ii) [101][102][103][104][105][106]. Gabrieli et al presented a whole-genome optical 5-hmC map of human peripheral blood cells, revealing variable regions and long-range information that were not accessible by sequencing [106].…”

Section: Optical Mapping Of Epigeneticsmentioning

confidence: 99%

Single-molecule optical genome mapping in nanochannels: multidisciplinarity at the nanoscale

Jeffet

Margalit

Michaeli

et al. 2021

Essays in Biochemistry

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The human genome contains multiple layers of information that extend beyond the genetic sequence. In fact, identical genetics do not necessarily yield identical phenotypes as evident for the case of two different cell types in the human body. The great variation in structure and function displayed by cells with identical genetic background is attributed to additional genomic information content. This includes large-scale genetic aberrations, as well as diverse epigenetic patterns that are crucial for regulating specific cell functions. These genetic and epigenetic patterns operate in concert in order to maintain specific cellular functions in health and disease. Single-molecule optical genome mapping is a high-throughput genome analysis method that is based on imaging long chromosomal fragments stretched in nanochannel arrays. The access to long DNA molecules coupled with fluorescent tagging of various genomic information presents a unique opportunity to study genetic and epigenetic patterns in the genome at a single-molecule level over large genomic distances. Optical mapping entwines synergistically chemical, physical, and computational advancements, to uncover invaluable biological insights, inaccessible by sequencing technologies. Here we describe the method’s basic principles of operation, and review the various available mechanisms to fluorescently tag genomic information. We present some of the recent biological and clinical impact enabled by optical mapping and present recent approaches for increasing the method’s resolution and accuracy. Finally, we discuss how multiple layers of genomic information may be mapped simultaneously on the same DNA molecule, thus paving the way for characterizing multiple genomic observables on individual DNA molecules.

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“…For To test non-methylation labelling efficiency, DNA was labelled with eM.SssI as described above, with or without MTAN. All DNA samples were purified from excess fluorophores, then applied on a custom epoxy-covered multi-well slide and imaged using commercial slide scanner, as described in the work of Margalit et al 53 .…”

Section: Genetic Barcoding and Non-methylated Cpg Labelingmentioning

confidence: 99%

Chemoenzymatic labeling of DNA methylation patterns for single-molecule epigenetic mapping

Gabrieli

Michaeli

Avraham

et al. 2021

Preprint

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DNA methylation, specifically, methylation of cytosine (C) nucleotides at the 5-carbon position (5-mC), is the most studied and among the most significant epigenetic modifications. Here we developed a chemoenzymatic procedure to fluorescently label non-methylated cytosines in the CpG context allowing epigenetic profiling of single DNA molecules spanning hundreds of thousands of base pairs. For this method, a CpG methyltransferase was used to transfer an azide to cytosines from a synthetic S-adenosyl-l-methionine cofactor analog. A fluorophore was then clicked onto the DNA, reporting on the amount and position of non-methylated CpGs. We found that labeling efficiency was increased two-fold by the addition of a nucleosidase that degrades the inactive by-product of the azide-cofactor after labeling, and prevents its inhibitory effect. We first used the method to determine the decline in global DNA methylation in chronic lymphocytic leukemia patients and then performed whole genome methylation mapping of the model plant Arabidopsis thaliana. Our genome maps show high concordance with published methylation maps produced by bisulfite sequencing. Although mapping resolution is limited by optical detection to 500-1000 base pairs, the labeled DNA molecules produced by this approach are hundreds of thousands of base pairs long, allowing access to long repetitive and structurally variable genomic regions.

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5‐Hydroxymethylcytosine as a clinical biomarker: Fluorescence‐based assay for high‐throughput epigenetic quantification in human tissues

Cited by 26 publications

References 44 publications

5-Hydroxymethylcytosine (5hmC) at or near cancer mutation hot spots as potential targets for early cancer detection

5-Hydroxymethylcytosine (5hmC) at or near cancer mutation hot spots as potential targets for early cancer detection

Single-molecule optical genome mapping in nanochannels: multidisciplinarity at the nanoscale

Chemoenzymatic labeling of DNA methylation patterns for single-molecule epigenetic mapping

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