Determination of quantitative and site-specific DNA methylation of perforin by pyrosequencing

We 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.This 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.Perforin (PRF1), a major immune surveillance gene, is a candidate gene for the identification of methylation markers, and for investigating the pathophysiology of a number of chronic diseases with suspected immune dysfunction [1-9]. Earlier studies to assess PRF1 promoter methylation used bisulfite sequencing of cloned PCR products [5]. Cloning PCR products for quantitative site-specific determination of CpG methylation is