Identifying heart-specific hypermethylation markers
To screen heart-specific methylation markers, we performed MCTA-seq on genomic DNA samples extracted from normal adult heart tissues (3 pairs of ventricles and atria) and cfDNA samples obtained from the plasma of MI patients after primary PCI (cohort 1, n = 20). The sequencing information are provided in Additional file 3: Table S2. We retrieved our previous MCTA-seq data of WBCs (n = 81), normal plasma (n = 202) and the liver tissue (n = 3) for searching for loci that displayed high methylation values in the heart tissue and the plasma samples after PCI, and low methylation values in normal plasma, WBCs and livers (see “Methods”) [5,6,7]. We also retrieved our previous MCTA-seq data of seven tissues, i.e., the muscle (n = 2), lung (n = 2), stomach (n = 2), colon (n = 2), kidney (n = 2), pancreas (n = 2) and skin (n = 2), for examining the tissue-specificity of the identified loci [7].
We identified six CGCGCGG loci that were located in the CpG islands (CGIs) of CORO6, CACNA1C (two loci), OBSCN, CRIP1 and ZNF503-AS2. Among these markers, CORO6 showed the most specific methylation pattern in the heart. Only CORO6 showed nearly no methylation in the muscle; other loci, including another relatively specific locus, CRIP1, were methylated to various degrees in the muscle. The two CACNA1C loci had the highest methylation values in the heart, but they also showed relatively high methylation levels in other tissues, including the liver and muscle (Fig. 1a and Additional file 4: Table S3).
The methylation values of all six markers were significantly elevated in the plasma from MI patients after PCI compared with the plasma from normal individuals (P < 0.0001, two-tailed Mann–Whitney-Wilcoxon (MWW) test, Fig. 1b–g and Additional file 4: Table S3). CORO6, CACNA1C-1, CACNA1C-2, OBSCN, CRIP1 and ZNF503-AS2 were methylated in 95% (19/20), 100% (20/20), 95% (19/20), 80% (16/20), 65% (13/20), and 55% (11/20) of these MI patients, respectively. The two CACNA1C loci displayed the highest methylation values in the plasma from MI patients; however, these two markers also displayed high methylation frequencies in normal plasma (25.2%, 51 of 202 for CACNA1C-1 and 28.7%, 58 of 202 for CACNA1C-2, Fig. 1c, d). CORO6 ranked second in MI patients, and remarkably, it displayed the lowest methylation frequency in normal plasma (3.0%, 6 of 202) and WBCs (0%, 0 of 81) (Fig. 1b). CRIP1 also displayed a low methylation frequency in normal plasma, similar to CORO6, but it was detected in fewer MI patients than CORO6 (Fig. 1e). The results of the marker analysis in plasma samples were consistent with their methylation patterns in tissues.
Notably, CACNA1C, CORO6 and OBSCN are cardiac myocyte-related genes. CACNA1C is a voltage-dependent calcium channel, and OBSCN is a component of sarcomeres [18,19,20]. CORO6 is an actin-binding protein that has been shown to be highly expressed in both skeletal muscle and the heart and critical for the regulation of acetylcholine receptor clustering in skeletal muscle [21]. We confirmed the heart-enriched gene expression patterns of all three genes using the Human Protein Atlas database (Fig. 1a, right). All CGCGCGG loci were located in the intragenic region of the genes, which was consistent with our previous finding that many tissue-specific hypermethylation markers are located in the intragenic or 3′ CGIs of tissue-specific expressed genes [7].
To further evaluate the specificity of these markers, we examined the MCTA-seq data of the plasma from cancer patients retrieved from our previous studies [6, 7]. CORO6 and CRIP1 were barely detected in the plasma from colorectal cancer (CRC) and hepatocellular carcinoma (HCC) patients (3.9%, 9 of 229 for CRC and 9.5%, 4 of 42 for HCC), suggesting that these two markers were not hypermethylated in cancers (Additional file 1: Fig. S1 and Additional file 4: Table S3). In contrast, other markers were detected at a high frequency in cancer patients.
Together, we used MCTA-seq to identify six hypermethylation markers for detecting heart damage in the blood and CORO6 showed the top performance.
Dynamic changes in heart-derived DNA in MI
We next performed MCTA-seq on a second group of MI patients (cohort 2, n = 20), from whom serial plasma samples were collected at three time points: at hospital admission before PCI (D0), 1 day after PCI (D1), and 2 days after PCI (D2). Sequencing information of theses samples are provided in Additional file 3: Table S2.
The concentration of cfDNA was similar in MI patients at admission and normal individuals (paired two-tailed MWW test, P = 0.21, median 6.5 ng/mL for D0 MI patients and 6.33 ng/mL for the normal individuals, Fig. 2a). Notably, the concentration significantly increased at 1 or 2 days after PCI compared with at admission (median 15.9 ng/mL and 18.8 ng/mL for D1 and D2 cases, respectively, paired two-tailed MWW test, P = 0.02395 for D1 vs. D0 and P = 0.03623 for D2 vs. D0); no significant difference was found between D1 and D2 (paired two-tailed MWW test, P = 0.67) (Fig. 2a and Additional file 5: Table S4). These results were consistent with the previous study showing that the concentration of cfDNA peaks after PCI [16].
We investigated the tissue of origin of the increased cfDNA after PCI. We extended our previously reported deconvolution approach to infer the tissue fractions of the heart and eight other nonhematopoietic tissues (see “Methods”). Notably, the results showed that heart-derived DNA was significantly elevated in the plasma from MI patients at admission compared with the controls (median 1.6% for MI versus 0% for control, P = 1.0168E−11, Fig. 2b). The fraction of heart-derived DNA was clearly elevated on the first day after PCI, while it significantly decreased on the second day after PCI (median 12% and 0.4% for D1 and D2, respectively, Fig. 2b). The level of high-sensitivity troponin (hs-cTn) showed a similar dynamic pattern (median 1.05 for D0 versus 8.46 for D1, P = 0.003652); 3.77 for D2 versus 8.46 for D1, P = 0.3144, Fig. 2c and Additional file 5: Table S4). These dynamic changes were consistent with Zemmour et al.’s study and indicated that MCTA-seq detected true signals of heart injury [14]. Examination of the relationship between the fraction of heart-derived DNA and high-sensitivity troponin showed a correlation coefficient of 0.48 (Additional file 1: Fig. S2).
The data revealed a discordance between the cfDNA concentration and the heart fraction on the second day after PCI: the total cfDNA concentration remained high while the heart fraction decreased (Fig. 2d). Deconvolution analysis showed that the increased cfDNA at D2 was mainly derived from blood cells (Fig. 2d). Also, among the 3130 increased cfDNA counts from D0 to D1 (median values: 2170 and 5300 GE/mL for D0 and D1, respectively), only approximately 20% (median 512 GE/mL) were derived from the heart. The heart-derived DNA amount clearly decreased to a median of 159 GE/mL at D2 (Fig. 2d and Additional file 5: Table S4). The pattern of dynamic changes was confirmed in individual patients (Fig. 3a–i and Additional file 1: Fig. S3). However, there were also exceptions. For example, both the total and heart-derived cfDNA amounts clearly increased in the D2 plasma of patient Pami95, although the hs-cTn level decreased (Fig. 3a); the peak hs-cTn level of that patient was extraordinarily high, suggesting severe heart damage.
Together, these results showed that heart-derived DNA increased in the plasma of MI patients both before and after PCI, while the surge in total cfDNA concentration after PCI was mainly derived from blood cells.
A ddPCR assay for detecting MI
Among the six identified heart methylation markers, the CORO6 locus showed the best heart specificity and lowest frequency in normal plasma. We therefore explored the development of a ddPCR assay for this locus. Two pairs of primers were designed to amplify the methylated and unmethylated states of a 71-bp region (Fig. 4a). Two TaqMan probes were designed to detect three common CpG sites within the amplicon, with a FAM probe for the methylated amplicon and a VIC probe for the unmethylated amplicon (Fig. 4a). A single-tube reaction distinguished the signals of the methylated and unmethylated amplicons.
We first used the assay to examine tissue samples, including the heart, esophagus, kidney, lung, muscle, colon, pancreas, liver, stomach and WBCs. For the heart, methylated molecules accounted for 23% of all amplicons. In contrast, the ratios were 0.79%, 0.39% and 0.015% for the muscle, liver, and WBCs, respectively; slight ratios of 3.61% and 3.14% were detected in kidney and esophagus, respectively (Fig. 4b). To investigate whether the signal of CORO6 was from cardiomyocytes, we enriched cardiomyocytes from a heart tissue sample obtained from human myocardial hypertrophy (HCM) surgery. The CORO6 signal increased to 40% in the cardiomyocyte-enriched portion and remained at 24% in the unenriched portion, suggesting that hypermethylation of CORO6 was cardiomyocyte-specific (Fig. 4b). It was notable that CORO6 gave high heart:WBC and heart:liver ratios, which are two of main sources of cfDNA [7]. The heart:muscle signal ratio was also high, which should be useful for distinguishing between heart and muscle diseases.
Then, we applied the assay to plasma samples from 116 MI patients and 25 control individuals. All plasma samples from MI patients were collected before PCI and within 24 h of the onset of chest pain upon hospital admission. The results showed that the CORO6 methylation signal was significantly higher in MI patients than in controls (median 0.99 [interquartile range (IQR) 0.77–1.98] vs. 0 [IQR: 0–0.91] copies/mL; P = 0.001861) (Fig. 5a and Additional file 6: Table S5). The methylation signal was detected in 54 of 116 MI patients, ranging from 1 to 104 copies/mL, while in contrast, it was detected in 20% (5 of 25) of controls at 1 or 2 copies/mL. The fractional concentration in MI patients was also significantly higher than that in controls (P = 0.005703, Fig. 5b and Additional file 6: Table S5). The area under the curve (AUC) values were 0.6852 (95% confidence interval (CI) 0.59–0.78, P = 0.0037) and 0.6751 (95% CI 0.57–0.78, P = 0.007) for the absolute concentration and for the fractional concentration, respectively (Fig. 5c, d). When one copy of cardiac-specific cfDNA/mL was defined as the cutoff for a positive signal, the diagnostic sensitivity was 46%, and the specificity was 80%. When 0.2% cardiac-specific cfDNA/mL was defined as the cutoff for a positive signal, the diagnostic sensitivity was 47%, and the specificity was 84%.
In summary, we established a methylated CORO6 ddPCR assay for the detection of heart-derived DNA in the blood.