Platelet mitochondrial DNA methylation: a potential new marker of cardiovascular disease

Abstract

Background: Platelets are critical in the etiology of cardiovascular disease (CVD), and the mitochondria in these cells serve as an energy source for platelet function. Epigenetic factors, especially DNA methylation, have been employed as markers of CVD. Unlike nuclear DNA methylation, mitochondrial DNA (mtDNA) methylation has not been widely studied, in part, due to debate about its existence and role. In this study, we examined platelet mtDNA methylation in relation to CVD.

Results: We measured mtDNA methylation in platelets by bisulfite-PCR pyrosequencing and examined associations of CVD with methylation in mitochondrial genes; cytochrome c oxidase (MT-CO1, MT-CO2, and MT-CO3); tRNA leucine 1 (MT-TL1); ATP synthase (MT-ATP6 and MT-ATP8); and NADH dehydrogenase (MT-MD5). We report that CVD patients have significantly higher mtDNA methylation than healthy controls in MT-CO1 (18.53%, P < 0.0001), MT-CO2 (3.33%, P = 0.0001), MT-CO3 (0.92%, P < 0.0001), and MT-TL1 (1.67%, P = 0.0001), which are involved in ATP synthesis. Platelet mtDNA methylation was not related with age, BMI, and race in this study.

Conclusions: Our results suggest that platelet mtDNA methylation, which could serve as non-invasive and easy-to-obtain markers, may be implicated in the etiology of CVD.

Keywords: Cardiovascular disease; DNA methylation; Mitochondrial epigenetics; Platelet.

Figure 1

Distribution of percentage mtDNA methylation in healthy individuals and CVD patients. Distribution of percentage mtDNA methylation in healthy individuals and CVD patients for (a) MT-CO1, (b) MT-CO2, (c) MT-CO3, (d) MT-TL1, (e) MT-ATP6, (f) MT-ATP8, and (g) MT-ND5 are shown. The values from all individuals are displayed, with lines corresponding to the mean and 95% confidence interval values. ****P < 0.0001;***P = 0.0001.

Figure 2

The predictive probability of platelet mtDNA methylation as a CVD biomarker. Nonparametric receiver operating characteristic (ROC) curves are shown for (a) MT-CO1, (b) MT-CO2, (c) MT-CO3, and (d) MT-TL1. Areas under ROC (AUROC) curves with sensitivity, specificity, and positive (PPV) and negative (NPV) predictive values are below the graph.

Figure 3

A representative gel following PCR amplification of mitochondrial and nuclear DNA sequences. Amplification of MT-CO1, CDH1, CDKN2A, and LINE-1 in extracted mtDNA (lanes 1 to 4) and control DNA from human adult normal peripheral blood leukocyte (Biochain, Newark, CA, USA) (lanes 6 to 9) were visualized on a 2% agarose gel. The fifth lane contains a DNA size marker.

Figure 4

Technical replicates and quantitative validation of mtDNA pyrosequencing assays. Spearman correlation coefficients (r) of the technical replicates (replicates 1 and 2) for the designed pyrosequencing assays were (a) 0.97 for MT-CO1, (b) 0.93 for MT-CO2, and (c) 0.76 for MT-CO3. The plot curve is displayed with a 95% confidence band. The quantitative validation of 0% and 100% methylated DNA for the (d) MT-CO1, (e) MT-CO2, and (f) MT-CO3 assays (red circle) were compared with 0% and 100% control oligonucleotides (black rectangle).