Epigenetic biomarkers of T-cells in human glioma

Wiencke, John K.; Accomando, William P.; Zheng, Shichun; Patoka, Joe; Dou, Xiaoqin; Hsuang, George; Christensen, Brock C.; Houseman, E. Andrés; Koestler, Devin C.; Bracci, Paige M.; Wrensch, Margaret; Nelson, Heather H.; Kelsey, Karl T.

doi:10.4161/epi.22675

Cited by 31 publications

(33 citation statements)

References 54 publications

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“…37,38 Purified CD3 T cells were obtained from healthy blood donors using antibody based negative selection and magnetic bead technology (Miltenyi, San Diego, CA). 5 Purity of the T cells was confirmed by FACS analysis. T cell DNA was bisulfite converted, quantified with C-less assay, and diluted to create a standard curve.…”

Section: Cd3z Demethylation Real Time Qpcrmentioning

confidence: 99%

“…5 Copy number of the target locus in each sample was determined by reference to a 4-point standard curve, which was based on known copies of bisulfite converted template. Quantification of total bisulfite DNA copies for the standard and in each sample was determined by reference to the C-Less assay as described previously.…”

Section: Cd3z Demethylation Real Time Qpcrmentioning

confidence: 99%

“…2,3 Our earlier experiments showed that cell lineage-specific differentially methylated regions (DMRs) distinguish normal human leukocyte subsets and can be used to detect and quantify these subsets in peripheral blood. [4][5][6] Methylation based detection of immune cells can be applied to many cohorts of archival blood samples that would otherwise be useless for immune cell enumeration by conventional FACS analyses. 2 types of assays characterize immune subsets using DNA methylation: one is based on single-gene DNA methylation events quantified by methylation specific (MS) real time PCR, 5,7 hereafter referred to as qPCR.…”

Section: Introductionmentioning

confidence: 99%

“…[4][5][6] Methylation based detection of immune cells can be applied to many cohorts of archival blood samples that would otherwise be useless for immune cell enumeration by conventional FACS analyses. 2 types of assays characterize immune subsets using DNA methylation: one is based on single-gene DNA methylation events quantified by methylation specific (MS) real time PCR, 5,7 hereafter referred to as qPCR. A second assay uses off-the-shelf or custom DNA methylation microarrays and captures many cell type DMRs simultaneously.…”

Section: Introductionmentioning

confidence: 99%

“…DMR targets in CpG sites within the promoter of the CD3Z gene estimate total T cells. 5 The CD3Z protein is a specific T cell lineage marker and associates with the T cell receptor and functions to generate activation signals in T lymphocytes. In T cells, the CD3Z gene promoter is demethylated, but this promoter is densely methylated in other immune cell subsets.…”

Section: Introductionmentioning

confidence: 99%

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A comparison of DNA methylation specific droplet digital PCR (ddPCR) and real time qPCR with flow cytometry in characterizing human T cells in peripheral blood

et al. 2014

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Quantitating the copy number of demethylated CpG promoter sites of the CD3Z gene can be used to estimate the numbers and proportions of T cells in human blood and tissue. Quantitative methylation specific PCR (qPCR) is useful for studying T cells but requires extensive calibration and is imprecise at low copy numbers. Here we compared the performance of a new digital PCR platform (droplet digital PCR or ddPCR) to qPCR using bisulfite converted DNA from 157 blood specimens obtained from ambulatory care controls and patients with primary glioma. We compared both ddPCR and qPCR with conventional flow cytometry (FACS) evaluation of CD3 positive T cells. Repeated measures on the same blood sample revealed ddPCR to be less variable than qPCR. Both qPCR and ddPCR correlated significantly with FACS evaluation of peripheral blood CD3 counts and CD3/total leukocyte values. However, statistical measures of agreement showed that linear concordance was stronger for ddPCR than for qPCR and the absolute values were closer to FACS for ddPCR. Both qPCR and ddPCR could distinguish clinically significant differences in T cell proportions and performed similarly to FACS. Given the higher precision, greater accuracy, and technical simplicity of ddPCR, this approach appears to be a superior DNA methylation based method than conventional qPCR for the assessment of T cells.

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Section: Cd3z Demethylation Real Time Qpcrmentioning

confidence: 99%

Section: Cd3z Demethylation Real Time Qpcrmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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A comparison of DNA methylation specific droplet digital PCR (ddPCR) and real time qPCR with flow cytometry in characterizing human T cells in peripheral blood

et al. 2014

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The fate is not always written in the genes: Epigenomics in epidemiologic studies

Langevin

Kelsey

2013

Environ and Mol Mutagen

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Cost-effective, high-throughput epigenomic technologies have begun to emerge, rapidly replacing the candidate gene approach to molecular epidemiology and offering a comprehensive strategy for the study of epigenetics in human subjects. Epigenome-wide association studies (EWAS) provide new opportunities for advancing our understanding of epigenetic changes associated with complex disease states. However, such analyses are complicated by the dynamic nature of DNA methylation. In contrast to genomic studies, where genotype is essentially constant across somatic cells, EWAS present a new set of challenges, largely due to differential DNA methylation across distinct cell types, particularly for studies involving heterogeneous tissue sources, and changes in the epigenetic profile that occur over time. This review describes potential applications of EWAS from the viewpoint of the molecular epidemiologist, along with special considerations and pitfalls involved in the design of such studies.

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DNA Methylation and Cell-Type Distribution

Houseman

2015

Translational Bioinformatics

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Epigenetic processes form the principal mechanisms by which cell differentiation occurs. Consequently, DNA methylation measurements are strongly influenced by the DNA methylation profiles of constituent cell types as well as by their mixing proportions, raising the potential for confounding of direct molecular associations at single CpG dinucleotides by associations between overall cell-type distribution with phenotype or exposure. In this chapter we review the literature on epigenetics and cell mixture; we then present techniques for deconvolution of DNA methylation measurements, either in the presence or in the absence of reference data. Finally, we present several data analysis examples.

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Epigenetic biomarkers of T-cells in human glioma

Cited by 31 publications

References 54 publications

A comparison of DNA methylation specific droplet digital PCR (ddPCR) and real time qPCR with flow cytometry in characterizing human T cells in peripheral blood

A comparison of DNA methylation specific droplet digital PCR (ddPCR) and real time qPCR with flow cytometry in characterizing human T cells in peripheral blood

The fate is not always written in the genes: Epigenomics in epidemiologic studies

DNA Methylation and Cell-Type Distribution

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