ArticleBMC research notes2009
Determination of quantitative and site-specific DNA methylation of perforin by pyrosequencing.
Article in BMC research notes, 2009. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 8 papers.
What it found
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The abstract states no effect estimate the extractor could read, or names no intervention and outcome on the map, so this paper lights no cell and moves no belief. It is still indexed, cited and linked below.
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Trials whose registry record cites this paper, or whose number appears in the abstract. A trial that started after this paper was published is citing it as background, not reporting it.
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Who cites it
8 citing papers in PubMed, 10 citations in OpenAlex.
- Epigenetic mechanisms and steroid-resistant nephrotic syndrome: The future potential for SRNS diagnosis.iScience · 2026Review
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- Cord blood PRF1 methylation patterns and risk of lower respiratory tract infections in infants: findings from the Ulm Birth Cohort.Medicine · 2015Article
- DNA methylation within the I.4 promoter region correlates with CYPl19A1 gene expression in human ex vivo mature omental and subcutaneous adipocytes.BMC medical genetics · 2013Observational
- Acute psychosocial stress-mediated changes in the expression and methylation of perforin in chronic fatigue syndrome.Genetics & epigenetics · 2013Article
- Article
- IL-2R common gamma-chain is epigenetically silenced by nucleophosphin-anaplastic lymphoma kinase (NPM-ALK) and acts as a tumor suppressor by targeting NPM-ALK.Proceedings of the National Academy of Sciences of the United States of America · 2011Article
Corrections and comments
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Authors and funding
4 authors at 2 institutions in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
backgroundDifferential expression of perforin (PRF1), a gene with a pivotal role in immune surveillance, can be attributed to differential methylation of CpG sites in its promoter region. A reproducible method for quantitative and CpG site-specific determination of perforin methylation is required for molecular epidemiologic studies of chronic diseases with immune dysfunction.
findingsWe developed a pyrosequencing based method to quantify site-specific methylation levels in 32 out of 34 CpG sites in the PRF1 promoter, and also compared methylation pattern in DNAs extracted from whole blood drawn into PAXgene blood DNA tubes (whole blood DNA) or DNA extracted from peripheral blood mononuclear cells (PBMC DNA) from the same normal subjects. Sodium bisulfite treatment of DNA and touchdown PCR were highly reproducible (coefficient of variation 1.63 to 2.18%) to preserve methylation information. Application of optimized pyrosequencing protocol to whole blood DNA revealed that methylation level varied along the promoter in normal subjects with extremely high methylation (mean 86%; range 82-92%) in the distal enhancer region (CpG sites 1-10), a variable methylation (range 49%-83%) in the methylation sensitive region (CpG sites 11-17), and a progressively declining methylation level (range 12%-80%) in the proximal promoter region (CpG sites 18-32) of PRF1. This pattern of methylation remained the same between whole blood and PBMC DNAs, but the absolute values of methylation in 30 out of 32 CpG sites differed significantly, with higher values for all CpG sites in the whole blood DNA.
conclusionThis reproducible, site-specific and quantitative method for methylation determination of PRF1 based on pyrosequencing without cloning is well suited for large-scale molecular epidemiologic studies of diseases with immune dysfunction. PBMC DNA may be better suited than whole blood DNA for examining methylation levels in genes associated with immune function.
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Registered trials
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