Heart-specific DNA methylation analysis in plasma for the investigation of myocardial damage

Abstract

Background: Circulating cell-free DNA (cfDNA) can be released when myocardial damage occurs.

Methods: Here, we used the methylated CpG tandem amplification and sequencing (MCTA-seq) method for analyzing dynamic changes in heart-derived DNA in plasma samples from myocardial infarction (MI) patients.

Results: We identified six CGCGCGG loci showing heart-specific hypermethylation patterns. MCTA-seq deconvolution analysis combining these loci detected heart-released cfDNA in MI patients at hospital admission, and showed that the prominently elevated total cfDNA level after percutaneous coronary intervention (PCI) was derived from both the heart and white blood cells. Furthermore, for the top marker CORO6, we developed a digital droplet PCR (ddPCR) assay that clearly detected heart damage signals in cfDNA of MI patients at hospital admission.

Conclusions: Our study provides insights into MI pathologies and developed a new ddPCR assay for detecting myocardial damage in clinical applications.

Keywords: Circulating cell-free DNA; DNA methylation; Myocardial infarction; Sequencing; ddPCR.

Fig. 1

Identification of heart-specific hypermethylation markers with MCTA-seq. a Heatmap of 6 identified heart-specific hypermethylation marker methylation levels and the expression levels in 9 different tissues, including the heart and WBCs. Each column represents one tissue type, and each row represents a marker. The markers (n = 6) are ranked by their methylation levels in the tissue, as calculated by their MePM values of MCTA-seq. In the heatmap, blue indicates low, white and yellow indicate intermediate and red indicates high DNA methylation values, which are shown as log₂(MePM) (left). The expression levels are shown by the log₂(z-score) (right). See the online methods for the identification of heart-specific hypermethylation markers. b–g Comparison of the representative heart-specific marker methylation levels in the plasma of MI patients (n = 20), normal plasma (n = 202), and WBCs (n = 81). MI indicates patients with acute myocardial, PN indicates normal control individuals, and WBC indicates samples of white blood cell. ****P < 0.0001. Two-tailed MWW test

Fig. 2

Comparisons of cfDNA and Tn levels in MI patients and normal individuals. a Comparison of cfDNA concentration (ng/mL plasma) in MI patients and normal individuals. b Ratio of heart-derived DNA in MI patients and normal individuals. c Tn levels in MI patients at different times. d Contribution of different tissues to the plasma of MI patients and normal individuals. D0, D1, D2 indicate MI upon hospital admission before PCI, 1 day after PCI, and 2 days after PCI, respectively. n = 20 for D0/D1/D2 samples and 67 for control individuals. *P < 0.05, **P < 0.01, ****P < 0.0001; ns, no significant difference. Two-tailed MWW test. The statistical values are the median

Fig. 3

Dynamic changes in different tissue-derived DNA and Tn levels during MI and after PCI in representative individual patients. Contribution of different tissues to plasma cfDNA and Tn levels in MI patients based on MCTA-seq deconvolution analysis. D0, D1, D2 indicate MI upon hospital admission before PCI, 1 day after PCI, and 2 days after PCI, respectively. n = 20 for D0/D1/D2 samples. The statistical values are the median. Different colors in the bar graph indicate the contribution of different tissues, and the zig–zag line indicates the change in troponin

Fig. 4

Detection of MI using ddPCR. a Schematic of the approach for the ddPCR-based detection of the methylation status of three CpG sites in the reverse strand of CORO6. b The methylation of the CORO6 locus in different tissues, enriched cardiomyocytes and WBCs. CM indicates cardiomyocytes, WBC indicates white blood cell

Fig. 5

The results of the ddPCR assay for detecting MI. a Measurement of the absolute concentration of methylated molecules of the CORO6 locus in the plasma of MI patients and control individuals. b Comparison of the fractional concentration between MI patients and control individuals. c Receiver operating characteristic (ROC) curve for the diagnosis of MI by the absolute concentration in plasma of MI patients and control individuals. d Receiver operating characteristic (ROC) curve for the diagnosis of MI based on the fractional concentration in the plasma of MI patients (n = 116) and control individuals (n = 25). **P < 0.01. Two-tailed MWW test