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 Table of Contents  
ORIGINAL ARTICLE
Year : 2018  |  Volume : 31  |  Issue : 3  |  Page : 772-779

Circulating cell-free DNA as a sensitive biomarker in patients with acute myocardial infarction


1 Department of Medical Biochemistry, Faculty of Medicine, Menoufia University, Menoufia, Egypt
2 Department of Cardiology, Faculty of Medicine, Menoufia University, Menoufia, Egypt

Date of Submission10-May-2017
Date of Acceptance11-Jun-2017
Date of Web Publication31-Dec-2018

Correspondence Address:
Eman S Arafat
Department of Medical Biochemistry, Menoufia University, Berket El-Sabaa, Menoufia
Egypt
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/mmj.mmj_345_17

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  Abstract 


Objective
The aim of this study was to evaluate the role of circulating cell-free DNA as an early cardiac biomarker in patients with acute myocardial infarction (AMI).
Background
Circulating cell-free DNA mainly originates from programmed cell death or acute cellular injury and reflects the extent of cellular damage.
Patients and methods
This study was carried out on AMI patients (n = 50) and healthy controls (n = 30). All participants were subjected to a full assessment of history, clinical examination, ECG, and echocardiography. Blood samples were taken for cardiac biomarkers, kidney function tests, liver enzymes, lipid profiles, and real-time quantitative PCR for cell-free DNA at the first and the third day of AMI attacks.
Results
AMI patients had significantly higher levels of cardiac markers, total cholesterol, triglyceride, low-density lipoprotein, L1PA2-222, L1PA2-90, and L1PA2-222/L1PA2-90 ratio versus controls. There was a significantly higher peak on the first day of the attack in L1PA2-90 and L1PA2-222 cell-free DNA levels and the L1PA2-222/L1PA2-90 ratio in AMI patients and a gradual decrease on the third day, without a nonsignificant difference between ST-elevation and non-ST-elevation myocardial infarction. There were nonsignificant correlations between the concentration of cell-free DNA parameters and clinical, echocardiography, or laboratory parameters. The diagnostic validity for the L1PA2-222/L1PA2-90 cfDNA ratio on the first day of an AMI attack was significantly higher (P < 0.001) at the cutoff point of 0.61 with a sensitivity of 54% and a specificity of 87%; there was a gradual decrease on the third day of attacks.
Conclusion
Elevated cell-free DNA can be used as an early cardiac biomarker in AMI patients and may complement traditional cardiac troponin-I and creatine kinase-MB biomarkers in the diagnosis of AMI.

Keywords: acute myocardial infarction, cardiac troponin-I, cell-free DNA, creatine kinase-MB, ejection fraction, end diastolic diameter, end systolic diameter, long interspersed element, ST-elevated myocardial infarction


How to cite this article:
Arafat ES, Elmadbouha I, Radwan EEI, Kamal AAM, Badr EA, Ghanayem NM. Circulating cell-free DNA as a sensitive biomarker in patients with acute myocardial infarction. Menoufia Med J 2018;31:772-9

How to cite this URL:
Arafat ES, Elmadbouha I, Radwan EEI, Kamal AAM, Badr EA, Ghanayem NM. Circulating cell-free DNA as a sensitive biomarker in patients with acute myocardial infarction. Menoufia Med J [serial online] 2018 [cited 2019 Jan 22];31:772-9. Available from: http://www.mmj.eg.net/text.asp?2018/31/3/772/248741




  Introduction Top


Acute myocardial infarction (AMI) is a common presentation of coronary artery disease as more than three million individuals have ST-elevated myocardial infarctions (STEMIs) and four million individuals have non-ST-elevated myocardial infarctions (NSTEMIs) per year[1]. The prevalence of coronary heart disease has been increasing in Africa and the Middle East. This increase may reflect increased risk factors for AMI such as smoking, hypertension, dyslipidemia, diabetes, and sedentary lifestyles[2]. AMI is caused by increased myocardial metabolic demand, decreased delivery of oxygen and nutrients to the myocardium, or both[3]. Atherosclerosis is the most common cause of AMI. About 90% of AMI results from an acute thrombus that obstructs an atherosclerotic coronary artery[4]. AMI is diagnosed mainly by elevated blood levels of cardiac markers [creatine kinase-MB (CK-MB) or cardiac troponins (cTn) T and I] and one of the following criteria: the patient has typical chest pain, ECG shows ST-elevation or depression, pathological Q waves, or a coronary intervention had been performed[5],[6].

Most of the nucleic acids are located inside cells, but a small amount of extracellular nucleic acids can be found circulating in the blood stream[7].

Circulating cell-free DNA (cfDNA) mainly originates from programmed cell death or acute cellular injury and detects the extent of cellular damage. cfDNA can be determined in the serum or plasma from healthy individuals at low concentrations ranging from 2.5 to 27.0 ng/ml[8]. High concentrations of cfDNA have been detected widely in a variety of pathological conditions, such as malignancies, trauma, infection, pregnancy-associated disorders, and autoimmune diseases[9].

L1PA2 is a human long interspersed element of the class L1 that is well interspersed throughout the human genome. L1 elements represent about 17% of the human genome[8]. As L1PA2 sequences are distributed over all chromosomes, the amplification of L1PA2 repeats may increase the sensitivity of cfDNA measurements[10]. Oxidative stress and inflammation play an important role in the pathogenesis of coronary artery disease. Reactive oxygen species induce DNA single-strand breakage and activation of the poly-ADP ribose synthetase causes necrotic type cell death because of energy depletion[11].

Therefore, the cfDNA concentration increases in the circulation of patients after AMI and may have prognostic potential[12].

The aim of this study was to evaluate the role of circulating cfDNA (L1PA2-90 and L1PA2-222) as an early biomarker for the diagnosis of patients with AMI.


  Patients and Methods Top


Patients

This study was carried out at Medical Biochemistry and Cardiology Departments, Faculty of Medicine, Menoufia University. A total of 80 individuals were included: 50 AMI patients and 30 age-matched and sex-matched healthy individuals. The patients were recruited from the ICU of the Cardiology Department, Menoufia University Hospital, in the period from May 2015 to May 2016.

AMI patients were diagnosed by clinical examinations, ECG changes, echocardiography, and laboratory cardiac markers according to the European Society of Cardiology/American College of Cardiology Committee Criteria[1],[5]. AMI was defined as detection of initial or 6 h cTn-I level of at least 0.1 ng/ml together with evidence of myocardial ischemia with at least one of the following: (i) symptoms of ischemia; (ii) ECG changes indicative of new ischemia (new ST-T changes or new left bundle branch block); (iii) development of pathological Q waves in the ECG; and (iv) imaging evidence of new loss of viable myocardium or new regional wall motion abnormality. Diagnostic outcomes were first categorized into the following groups: (i) STEMI and (ii) NSTEMI[1].

The participants studied (AMI patients, n = 50) were categorized into the following groups: men (n = 40) and women (n = 10). Their ages ranged from 43 to 87 years, with a mean age of 57.72 ± 9.59 years. This group was subdivided into 34 patients with STEMI (28 men and six women) and 16 patients with NSTEMI (12 men and four women). Controls were selected on the basis of healthy condition without cardiovascular diseases, lung and kidney diseases, with normal blood pressure and ECG, normal serum lipids, and normal blood glucose. Healthy participants served as controls (n = 30), of which 22 were men and eight were women, and their ages ranged from 45 to 81 years, with a mean age of 54.63 ± 10.06 years.

An informed written consent was obtained from every individual who participated in this study and the study was approved by the Ethical Committee of Medical Research, Faculty of Medicine, Menoufia University.

Exclusion criteria for patients were a history of inflammatory diseases, tissue injury, trauma, cancer, and liver and renal diseases.

Transthoracic echocardiography was performed on all participants. Two-dimensional and Doppler echocardiography (Vivid 7; GE Medical Systems, Milwaukee, Wisconsin, USA) was performed in the left lateral decubitus position using a broadband (1.5–4 MHz) phased array transducer at rest. Echo evaluations included left ventricular end diastolic diameter (LV-EDD), left ventricular end systolic diameter (LV-ESD), left ventricular fraction shorting (LV-FS%), left ventricular ejection fraction (LV-EF%), and loss of left ventricular wall motion (akinesia or hypokinesea). Echocardiograms were read centrally in a blinded manner.

Methods

All the participants studied were subjected to the following: full assessment of history, general and cardiological examinations, ECG and echocardiography, and laboratory investigations including cardiac biomarkers such as the cTn-I concentration in serum provided by Ray Biotech Inc. (Norcross, Gerorgia, USA). The CK-MB concentration was determined using an enzyme-linked immunosorbent assay (ELISA) provided by Oxis International Inc. (Beverly Hills, California, USA). Kidney function tests (serum urea and creatinine) were performed, and liver enzymes, and aspartate transaminase (AST) and alanine transaminase (ALT) levels were determined using the Beckman Coulter Synchron CX 9 ALX (Beckman Coulter, Fullerton, California, USA). The lipid profile, such as total cholesterol (TC), triglyceride (TG), and high-density lipoprotein cholesterol (HDL-C) levels, was measured using standard enzymatic colorimetric kits (Spinreact, Girona, Spain) and low-density lipoprotein cholesterol (LDL-C) levels were calculated as TG levels not exceeding 400 mg/dl using the following formula: LDL cholesterol = Total cholesterol − HDL cholesterol−(triglycerides/5).

Specific investigation including quantitative measurement of cfDNA in DNA extracts from plasma using a real-time PCR technique was performed.

Sample collection

Venous blood (5 ml) was withdrawn from every participant after an overnight fast and divided into two parts. The first part was transferred into a plain tube, left at 37°C for 30 min to clot, and then centrifuged for 10 min at 4000 rpm. The serum was obtained for determination of the lipid profile level and cardiac biomarkers (cTn-I and CK-MB) levels. The second part was transferred into an EDTA tube centrifuged for 10 min at 4000 rpm. The plasma obtained for DNA extraction was kept frozen at −20°C till analysis.

Assay methods

Determination of AST, ALT, urea, and creatinine levels was performed using the Beckman Coulter Synchron CX 9 ALX (Beckman Coulter). Lipid profile levels were measured using standard enzymatic colorimetric kits (Spinreact)[13],[14]. Serum LDL-C level was calculated as TG levels not exceeding 400 mg/dl using the following formula: LDL cholesterol = Total cholesterol − HDL cholesterol−(triglycerides/5)[15]. The CK-MB level was determined using an ELISA provided by Oxis International Inc. The cTn-I level was determined using the ELISA kit for the quantitative measurement of human cTn-I concentration in the serum provided from Ray Biotech Inc.

Quantitative measurement of cfDNA was performed using the real-time PCR technique. First, cfDNA was extracted from plasma using the QIA amp Min Elute Virus Spin kit (Qiagen, Hilden, Germany). The real-time PCR step was performed using a Syber green master mix with low Rox; the following primers were used as described previously[16]: for L1PA2 (90): the forward primer (5'-TGCCGCAATAAACATACGTG-3') and the reverse primer (5'-GACCCAG CCATCCCATTAC-3') and for L1PA2 (222): the forward primer (5'-TGCCGCAATAAACAT ACGTG-3') and the reverse primer (5'-AACAAC AGGTGCTG GAGAGG-3'). The reaction mix was prepared by mixing 12.5 μl Syber green master mix, 1 μl of forward primer, 1 μl of reverse primer, and 5.5 μl of nuclease-free water. For each unknown reaction, 5 μl (0.1 μg/μl) of DNA extract was added and for the negative control reaction, 5 μl of nuclease-free water was added. The cycling parameters were set as follows: an initial denaturation step at 98°C for 2 min, a PCR stage involved a melting step at 94°C for 10 s, annealing/collection at 64°C for 40 s, and final extension at 75°C for 10 min. Thermal cycling was run in 96-well plates in a 7500 Real-Time PCR System (Applied Biosystems, Foster City, California, USA). The standard curve analysis method was used and standard curves were created with human genomic DNA, 100 μg (G3041), to determine the concentration of L1PA2 (90) and L1PA2 (222) fragments.

Statistical analysis

Data were analyzed using statistical package for the social science software, version 20 (SPSS Inc., Chicago, Illinois, USA) on an IBM compatible computer. Quantitative data were expressed as mean ± SD and analyzed using a t- test for comparison between two groups of normally distributed variables, whereas for comparison between two groups of non-normally distributed variables, the Mann–Whitney test and the Wilcoxon test were applied. Qualitative data were expressed as number and percentage and analyzed using the χ2-test and Fisher's exact test. An receiver operating characteristic curve was used to determine cutoff points, sensitivity (%), and specificity (%) for quantitative variables of interest. Spearman's correlation was used to assess the correlation between variables parameters. A P value of less than 0.05 was considered to be statistically significant.


  Results Top


There was a nonsignificant statistical difference between the AMI patients and controls in age, sex, and smoking. There was a significant difference between patients and controls in hypertension, previous history of ischemic heart disease, cTn-I level, CK-MB level, TC level, TG level, LDL-C level, and HDL-C level; echocardiography parameters (LV-EF%, LV-EDD, and LV-ESD); and L1PA2-222, L1PA2-90, and L1PA2-222/L1PA2-90 cfDNA ratio (DNA integrity index) [Table 1].
Table 1: Clinical data, echocardiography, biochemical, and PCR analysis in all participants

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There was a significant difference between the first and the third day of AMI attack in L1PA2-90, L1PA2-222, and cfDNA levels and the L1PA2-222/L1PA2-90 cfDNA ratio in AMI patients, whereas there was a nonsignificant difference between STEMI and non-STEMI AMI patients [Table 2].
Table 2: Comparison of L1PA2-222, L1PA2-90 cell-free DNA levels, and L1PA2-222/L1PA2-90 cell-free DNA ratio (integrity index) in acute myocardial infarction patients on the first and the third day of the attack and between ST-elevated myocardial infarction and non-ST-elevated myocardial infarction myocardial infarction patients

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There was a nonsignificant positive correlation between the concentration of cfDNA and LV-EF%, LV-EDD, and LV-ESD parameters. There was a nonsignificant negative correlation between L1PA2-222 and cTn-I levels and the HDL-C level, but a nonsignificant positive correlation with other parameters. There was a nonsignificant negative correlation between L1PA2-90 cfDNA and cTn-I levels, AST level, ALT level, serum creatinine level, and HDL-C level, but a nonsignificant positive correlation existed with other parameters. There was a nonsignificant negative correlation between the L1PA2-222/L1PA2-90 cfDNA ratio and the cTn-I level, CK-MB level, serum urea, and TG levels, but a nonsignificant positive correlation existed with other parameters [Table 3].
Table 3: Correlation coefficient between L1PA2-222, L1PA2-90 levels, and L1PA2-222/L1PA2-90 cell-free DNA ratio (integrity index) with echocardiography and biochemical parameters in the acute myocardial infarction patients

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The receiver operating characteristic curve and diagnostic accuracy of the L1PA2-222/L1PA2-90 cfDNA ratio and its sensitivity and specificity compared with the traditional cardiac parameters cTn-I and CK-MB during the first 3 days of a cardiac attack are shown in detail in [Table 4] and [Figure 1],[Figure 2],[Figure 3]. The L1PA2-222/L1PA2-90 cfDNA ratio peaked on the first day with a cutoff point of 0.61, a sensitivity of 54%, a specificity of 87%, a positive predictive value of 87%, and a negative predictive value of 53% (P < 0.001) [Table 4].
Figure 1: Receiver operating characteristic (ROC) curve and diagnostic accuracy of L1PA2-222/L1PA2-90 cell-free DNA ratio, cardiac troponin-I (cTn-I), and creatine kinase-MB (CK-MB) in acute myocardial infarction patients on the first and third day.

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Figure 2: Standard curve of L1PA2-222.

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Figure 3: Typical amplification plot, showing increases in fluorescence from two samples (sample A and sample B).

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Table 4: Receiver operating characteristic curve and diagnostic validity of LIPA2-222/L1PA2-90 ratio cell-free DNA (integrity index) in parallel to cardiac troponin-I and creatine kinase-MB in the first 3 days for the diagnosis of acute myocardial infarction

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  Discussion Top


AMI is defined as irreversible necrosis of heart muscle because of prolonged ischemia and it is the result of coronary artery disease associated with the apoptosis and death of cardiomyocytes[17]. The membrane of the necrotic cardiomyocytes is disrupted and cellular components leak into the peripheral circulation. This pathway is the mechanism of release of cfDNA in the peripheral circulation in AMI[18].

The present study aims to evaluate the role of circulating cfDNA biomarkers in AMI.

Our results showed that there was a nonsignificant difference between patients and controls regarding age and sex. This was in agreement with previous studies[19]. A strong positive association was observed in many studies between smoking and AMI, indicating that smokers have a higher risk of myocardial infarction than nonsmokers[20].

In contrast, this study and the study by Wang et al.[21] showed a nonsignificant difference between patients and controls in smoking (P > 0.05). This may be explained by the fact that there are certain aspects of other risk factors such as family history, hypertension, obesity, etc., that contribute toward the pathogenesis of AMI and must also be considered.

Our results indicated that patients with AMI showed significantly increased blood pressure and history of ischemic heart disease compared with controls (P < 0.001). This was in consistent with the findings reported by other study[22]. However, there were no significant differences in vascular risk factors, including hypertension and dyslipidemia, between the two groups[21].

In this study, there was a highly significant difference between patients and controls in cardiac markers (cTn-I and CK-MB) (P = 0.001). These results are in agreement with those reported by others[23], who found that AMI patients had a significantly higher level of markers such as CK-MB, cTn-I, and cTn-T.

The present study indicated that AMI patients showed a highly significant increase in TC, TG, and LDL levels (P = 0.001), but a decrease in HDL levels versus controls. These results are in agreement with those reported by another study[24].

In this study, we found that cfDNA levels were significantly higher in patients with AMI than in healthy controls (P < 0.001). These results are in agreement with those reported by another study[25].

The measurement of fragments of cfDNA may be used in routine clinical practice as a useful biomarker of nonspecific tissue damage with a higher predictive value in noncancer individuals with cardiovascular disease[26].

We also found that in serial samples, cfDNA was higher in the first-day specimen than the third-day specimen (P < 0.001). These findings were similar to the results of studies carried out previously[21]. Plasma cfDNA levels were elevated in AMI patients and reverted to normal levels immediately after a percutaneous coronary intervention[21]. The increase in the content of free-circulating DNA in blood is a predictor of lethal outcomes in patients with acute coronary syndrome[21]. These results can be attributed to the fact that a modest amount of cfDNA is constantly present in human blood, originating from programmed cell death, apoptosis, and rupture of blood cells or pathogens. Acute or chronic cell injury contributes toward enhancement of the pool of circulating nucleic acids[27].

In the present study, there was a significant difference between patients and controls in the L1PA2-222/L1PA2-90 ratio. Long DNA fragments, related to the necrosis phenomena, could be distinguished from shorter fragments, related to physiological apoptosis phenomena by PCR. The ratio between L1PA2-222/L1PA2-90 is called the DNA integrity index[28].

In the present study, there was a significant difference between cfDNA on the first and the third day of AMI; these results are in agreement with those reported by a study[21] that found that the concentrations of nuclear and mtDNA were significantly higher in the AMI group on hospital day 1 than on the third day. This result can be explained by the fact that the circulating DNA in plasma is protein-bound (nucleosomal) DNA and circulating DNA has a short half-life (10–15 min) that is removed mainly by the liver. Accumulation of DNA in the circulation can result from an excessive release of DNA caused by massive cell death, inefficient removal of the dead cells, or a combination of both[29].

In this study, the diagnostic accuracy of cfDNA as a diagnostic marker of cell damage and apoptosis in AMI patients on the first day was 66%, with a sensitivity of 54% and a specificity of 87% at a cutoff point of 0.61 ng/ml. These results are in agreement with another study[30] that found that the diagnostic accuracy of cfDNA as a diagnostic marker of cell damage and apoptosis in AMI patients was 89%, with a sensitivity of 94.4% and a specificity of 68.4% at a cutoff point of 0.33 ng/ml.


  Conclusion Top


The cfDNA level is significantly higher in AMI patients than in normal controls. It peaks on the first day of AMI attacks. Therefore, it may play a role in the diagnosis of AMI. Measurement of cfDNA in AMI may be complementary to cTn-I and CK-MB in the diagnosis of AMI in a multiple marker test format. Combining cfDNA and cTn-I and cTn-T and CK-MB provides the best performance for early AMI diagnosis.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
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    Figures

  [Figure 1], [Figure 2], [Figure 3]
 
 
    Tables

  [Table 1], [Table 2], [Table 3], [Table 4]



 

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