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. 2009 Jun 12:2:104.
doi: 10.1186/1756-0500-2-104.

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

Supraja Narasimhan  1 , Virginia R Falkenberg, Maung M Khin, Mangalathu S Rajeevan
Affiliations

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

Supraja Narasimhan et al. BMC Res Notes. .

Abstract

Background: Differential 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.

Findings: 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.

Conclusion: 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.

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Figures

Figure 1
Figure 1
The perforin (PRF1) promoter (1411 bp) with locations of 34 CpG and several putative transcription factor binding sites. Ten CpGs are located in the enhancer element region, seven in the methylation-sensitive region (MSR) and 17 in the repetitive element region. "+1" indicates the transcription start site. Lower case letters from 'a -to- l' represent different transcription factor binding sites [14,15]: a, inducer response motif; b, γ-IFN responsive element; c, CRE element; d, AP-2 element; e, TPA-responsive element; f, STAT5-responsive enhancer; g, CCAT box; h, C-fos enhancer; i, 19 homologous repeats; j, three repeats; k, two repeats; l, GC box. Upper case letters from 'A -to- K' represent amplicons (not scaled to size) designed to detect different CpG sites.
Figure 2
Figure 2
Reproducibility of bisulfite treatment determined using amplicon G. Mean methylation level (%) ± SD for four CpG sites 18–21 are shown.
Figure 3
Figure 3
Reproducibility of touchdown PCR determined using amplicon G. Mean methylation level (%) ± SD for four CpG sites 18 to 21 are shown.
Figure 4
Figure 4
The pattern of methylation in 32 CpG sites in 1.4 kb PRF1 promoter using DNA from whole blood and PBMCs from normal subjects. Y-axis, methylation level (%) ± SD. X-axis, CpG sites 1–32. Methylation levels at each CpG site represent mean of 5 normal subjects.
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