CN114113602A - Immunoglobulin-associated coagulation factor X as biomarker for acute myocardial infarction - Google Patents
Immunoglobulin-associated coagulation factor X as biomarker for acute myocardial infarction Download PDFInfo
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Abstract
The invention provides an immunoglobulin-associated coagulation factor X as a biomarker of acute myocardial infarction. In particular, the invention provides the use of a reagent for detecting coagulation factor X (F10) specifically binding to immunoglobulins in a sample from a test subject for the preparation of a test system for assessing the risk of an individual for suffering from acute myocardial infarction and/or coronary artery disease. The present invention also provides a method for identifying biomarkers from blood samples using immunoglobulin-bound proteomes (IgAP), which can reliably detect differential levels of immunoglobulin-associated F10 in AMI and CAD patients.
Description
Technical Field
The invention relates to a diagnostic marker of acute myocardial infarction and related application, in particular to application of a reagent for detecting immunoglobulin-related coagulation factor X (F10) in a sample from an individual to be detected in preparing a composition and/or a kit and/or a diagnostic system for evaluating the risk of the individual suffering from acute myocardial infarction and/or coronary artery disease.
Background
Atherosclerosis is the basis for the pathological development of Coronary Artery Disease (CAD), the most common and life-threatening cardiovascular disease. Immune activity and chronic inflammation play a key role in the development of atherosclerosis, CAD, and susceptibility to undesirable tachycardias such as acute myocardial infarction. At the cellular level, atherosclerotic lesions are characterized by the deposition of Low Density Lipoproteins (LDL), leading to a series of inflammatory responses and a continuum of innate immune cells (e.g., monocytes/macrophages) and adaptive immune cells (e.g., T and B cells). Activated T cells are known to mediate adaptive immunity and release cytokines that regulate atherosclerotic plaque growth, instability and rupture. In addition to the widely studied T lymphocytes, there is increasing evidence that B cells are another important branch of adaptive immunity and also play an important role in the pathophysiology of atherosclerosis [ Tsiantoursas, D.D., Diehl, C.J., Witztum, J.L., and Binder, C.J. (2014.) B cells and 365 human immunity in atherocrosclerosis. Circuit Res 114,1743-1756 ]. The main function of B cells is to secrete immunoglobulins that bind to the immunogen and elicit various inflammatory responses. Human immunoglobulins are distinguished by their fragment crystallizable (Fc) into five major isotypes, a, D, E, G and M. Ig isoforms differ in atherosclerosis and CAD. For example, studies have shown that IgM has anti-atherosclerotic effects and clears apoptotic cells by neutralizing pro-inflammatory factors [ Kyaw, t., Tay, c., krishnamthi, s., kanella kis, p., Agrotis, a., taping, p., Bobik, a., and Toh, B.H, (2011). B1a B lymphocyte, aryl ether protective by nuclear protective IgM that is involved in apoptosis IgM detection and recovery of nuclear stress in atherogenic delivery. circ Res 109, 830. 840 ]. In contrast, elevated serum IgA, IgE and IgG levels in dyslipidemic male patients are associated with a high risk of myocardial infarction. However, the function and mechanism of different Ig classes is still elusive in the development of atherosclerosis and CAD. The core function of immunoglobulins is to bind to specific antigens that trigger a downstream immune cascade. More and more immune responses have been proposed to modulate atherosclerosis by antigen stimulation. For example, low density lipoproteins in atherosclerotic/CAD patients often exhibit a large number of oxidation-specific epitopes (OSE) that result from oxidative modification of the lipid moiety, which are known to be immunogenic. It has been reported that IgM and IgG re-recognize this oxidized LDL (oxLDL) molecule has a protective or promoting effect on the progression of atherosclerosis [ Tsiantoulas, D., Diehl, C.J., Witztum, J.L., and Binder, C.J. (2014). B cells and humoral immunity in atherocross. Circ Res 114,1743-1756], respectively ]. In addition, tyrosine nitration is a modification of oxidative proteins, which is frequently observed in atherosclerotic lesions in animals and patients. Thomson et al reported that CAD patients had significantly increased plasma levels of reactive Immunoglobulins against proteins with nitrotyrosine modifications [ Thomson, l., Tenopoulou, m., Lightfoot, r., Tsika, e., parasitodis, i., Martinez, m., groco, t.m., Doulias, p.t., Wu, y., Tang, w.h., et al (2012), Immunoglobulins against systemic diseases-mediated epitopes in coronar area disease. circulation 126, 2392. zones 2401 ]. These studies point to a key role for immunoglobulins in the recognition of atherosclerosis-associated antigenic determinants and in the modulation of immune responses associated with CAD progression. The identification of CAD-specific immunoglobulins and related antigens would aid in the development of novel disease monitoring biomarkers. However, little is known about the identity of antigens other than the atherosclerotic epitopes recognized by immunoglobulins. Furthermore, the question as to whether and how the homologous features of immunoglobulins evolve during the occurrence of adverse cardiovascular events remains elusive.
Acute Myocardial Infarction (AMI) is the most common cause of death worldwide. Reliable assessment of the risk of developing AMI in patients with Coronary Artery Disease (CAD) is critical to reducing mortality and improving quality of life.
Disclosure of Invention
It is an object of the present invention to find new and reliable markers for assessing the risk of developing AMI in patients with coronary artery disease.
The present inventors have found that AMI patients have significantly elevated levels of coagulation factor X (F10) bound to immunoglobulins, a major component of B cell-mediated adaptive immunity. On this basis, the present invention has developed an assay that is capable of measuring F10 specifically binding to immunoglobulins using a small volume (20 μ L) of serum sample. The assay of the invention can reliably detect differential levels of immunoglobulin-associated F10 in AMI and CAD patients, a method that has potential use in AMI risk assessment.
According to a particular embodiment of the invention, the invention integrates the purification of immunoglobulins from a small serum sample with an immunoassay targeting F10. Briefly, 20 μ L of serum was first used and the G-protein cross-linked agarose bead technique was relied upon to separate immunoglobulins and binding proteins. The separated protein was eluted from the beads with 0.1M glycine at pH 3. The protein concentration of the eluate is measured to normalize the protein content for downstream determination. The eluted protein was then subjected to a standard immunoassay to determine the level of F10. Preliminary results of the present invention show that AMI patients have higher levels of immunoglobulin bound F10 (n 25, mean 29.05pg/μ g, 95% CI 20.08-38.02 pg/μ g of input immunoglobulin binding protein) than CAD (n 38, mean 18.08pg/μ g, 95% CI 15.02-21.15 pg/μ g of input immunoglobulin binding protein) and healthy individuals (n 30, mean 7.86/μ g, 95% CI 6.9-8.83 pg/μ g of input immunoglobulin binding protein).
Thus, in one aspect, the present invention provides the use of an agent for detecting coagulation factor X (F10) that specifically binds to an immunoglobulin in a sample from a test subject in the preparation of a test system for assessing the risk of an individual for suffering from an acute myocardial infarction and/or coronary artery disease.
According to a specific embodiment of the present invention, the reagent for detecting coagulation factor X specifically binding to immunoglobulin in the present invention includes a reagent material and/or an apparatus for use in an immunoassay method.
According to a specific embodiment of the present invention, the reagent for detecting coagulation factor X specifically bound to immunoglobulin in the present invention includes a reagent material and/or equipment used in a label-free quantitative LC-MS/MS analysis method.
According to a particular embodiment of the invention, the detection of coagulation factor X specifically binding to immunoglobulins in a sample from a test individual may be carried out by a method comprising:
separating immunoglobulin and binding protein in a sample by adopting a G protein cross-linked agarose bead technology;
eluting the separated protein from the microbeads by using glycine solution with pH of 3 to obtain eluent;
the protein in the eluate was subjected to standard immunoassay or label-free quantitative LC-MS/MS analysis to determine the level of F10 therein.
According to a particular embodiment of the invention, the sample is a serum sample.
According to a particular embodiment of the invention, the individual is a patient with coronary artery disease. The methods of the invention may be used for risk assessment of AMI in CAD patients.
According to a particular embodiment of the invention, the level of coagulation factor X specifically binding to immunoglobulins in the sample is increased and the individual is at increased risk of suffering from an acute myocardial infarction and/or coronary artery disease. AMI risk was positively correlated with F10 expression levels.
In another aspect, the present invention provides a method for detecting coagulation factor X (F10) specifically binding to immunoglobulin in a sample from a test subject, the method comprising:
separating immunoglobulin and binding protein in a sample by adopting a G protein cross-linked agarose bead technology;
eluting the separated protein from the microbeads in glycine solution with pH of 3 to obtain an eluate;
standard immunoassay or label-free quantitative LC-MS/MS analysis was performed on the protein of the eluate to determine the level of F10 in the sample.
According to a particular embodiment of the invention, the method of the invention further comprises, before the standard immunoassay is performed on the eluate, a process of measuring the protein concentration in the eluate in order to normalize the protein content.
In another aspect, the present invention also provides a system for risk assessment of acute myocardial infarction and/or coronary artery disease, comprising a detection unit comprising a reagent material and/or a device for detecting coagulation factor X (F10) specifically binding to immunoglobulin in a sample from an individual to be tested.
According to a specific embodiment of the present invention, the system for assessing acute myocardial infarction risk and/or coronary artery disease further comprises an analysis unit, wherein the analysis unit is used for analyzing the detection result of the detection unit to assess the risk of acute myocardial infarction and/or coronary artery disease of the individual.
The system for assessing the risk of acute myocardial infarction and/or coronary artery disease can be a virtual device as long as the functions of the detection unit and the data analysis unit can be realized. The detection unit may include various detection reagent materials and/or detection instrument devices, etc. The data analysis unit may be any computing device, module or virtual device that can analyze and process the detection result of the detection unit to obtain a pre-determination result of the risk of acute myocardial infarction and/or coronary artery disease of the subject to be detected, for example, the data analysis unit may be a computing device that stores computing program instructions based on an algorithm model in advance, and the analysis pre-determination result or classification result of the risk of acute myocardial infarction and/or coronary artery disease of the subject to be detected can be obtained by inputting the detection result of the detection unit into the computing device.
In summary, the present invention provides immunoglobulin-associated coagulation factor X (F10) as a biomarker for acute myocardial infarction for risk assessment of AMI in CAD patients. Also provided are methods for identifying biomarkers from blood samples using immunoglobulin-bound proteomes (IgAP), which can reliably detect differential levels of immunoglobulin-associated F10 in AMI and CAD patients.
Drawings
FIG. 1 is a flow chart of the IgAP assay.
FIG. 2 shows the respondent IgAP protein levels, graphically, as the content of all respondent-isolated IgAP proteins, in log2 transformed peak intensities. Error bars represent Standard Deviation (SD).
Fig. 3A and 3B are graphs of levels of F10 in igap (a) and total serum (B) of different patients.
Figure 4 shows a receiver operating characteristic curve (ROC) depicting the ability to predict AMI from CAD using the IgAP protein F10 (n 58, 38 AMI and 20 CAD), with the shaded area indicating the 95% confidence interval.
Fig. 5 is a diagram of violins expressed by AMI and CAD patient F10. The x-axis represents the actual patient cohort and the y-axis corresponds to the log2 intensity value of F10.
Fig. 6 shows a confusion matrix for predicting AMI from CAD using the IgAP protein F10. F10 has a prediction accuracy of 0.86 (95% confidence interval 0.75% -0.94), a specificity of 0.75 (95% confidence interval: 0.84-1), and a sensitivity of 0.92 (95% confidence interval: 0.55-0.95).
Detailed Description
The technical solutions of the present invention will be described in detail below in order to clearly understand the technical features, objects, and advantages of the present invention, but the present invention is not limited to the practical scope of the present invention.
The following tests and procedures not specified in the examples were carried out according to the usual operating conditions in the field or as suggested by the instructions of the equipment manufacturers.
The main test materials and methods used in each example were as follows:
1.1 reagents and materials
Protein G agarose, N-hydroxysuccinimide (NHS) -activated agarose, immobilized pepsin resin and a Zeba spin column were purchased from Pierce (Thermo Fisher Scientific, Rockford, IL.). Incomplete Freund's Adjuvant (IFA), TRIS hydrochloride (Tris-HCl), ammonium bicarbonate (NH) were obtained from4HCO3) 2,2, 2-Trifluoroethanol (TFE), Dithiotrimethylbenzene (DTT), Iodoacetamide (IAM) and Iodoethanol (IE) Sigma-Aldrich (St. Louis, Mo.). Urea and AG-5O1-X8 resin were purchased from Bio-Rad (Hellci, Calif.). For the LC-MS/MS sample preparation, Millipore (Bedford, MA) Microcon 10kDa MWCO (Microcon-10) centrifugal filter columns and Thermo Scientific (IL. Rockford) Hypersep SpinTip C18 column (C18-SpinTips) -MS grade water, Acetonitrile (ACN) and EMD (Millerica, Mass.) formic acid were used。PierceTMC18 pipette, 10 μ L bed from Thermo Scientific (Rockford, IL).
1.2 clinical samples
The serum samples were provided by the cardiovascular department of general hospital of people's liberation force (301) in China, approved by ethics. The IgAP survey included a panel of CAD (n-21), AMI (n-37) and non-CAD/AMI control individuals (n-37). The ELISA assay of F10 included a separate CAD cohort (n-38), AMI (n-25) and healthy control group (n-30). Prior to the experiment, the serum was stored in aliquots of polyethylene tubes at-80 ℃ until use.
1.3IgAP isolation
To isolate IgAP, 20. mu.L of serum was diluted with 500. mu.L of Phosphate Buffered Saline (PBS) and incubated with 10. mu.L of Protein G agarose beads (Sigma-Aldrich, St. Louis, Mo.) for 3 hours at 4 ℃. After washing, the beads were resuspended in 20. mu.L of 50mM Tris-HCl (pH 8.0) and stored at 4 ℃ prior to treatment.
1.4 bead digestion and desalination
The beads were resuspended in 50. mu.L of buffer I (50mM Tris-HCl pH 8.0; 2M urea; 10. mu.g/ml sequencing grade trypsin; 1mM DTT) and incubated for 1 hour at 400rpm in a 30 ℃ hot mixer. After preserving the supernatant, the beads were washed twice with 25 μ L of buffer II (50mM Tris-HCl pH 8.0, 2M urea, 5mM iodoacetamide) and the supernatants were pooled together and protected from light. After an additional 250ng of trypsin was added, the reaction was continued overnight at 400rpm in a hot mixer at 30 ℃. The following day, the ratio of v: v ratio 10% formic acid was added to the reaction to stop the digestion. The samples were then desalted using a C18 pipette according to the manufacturer's protocol. The peptide obtained was dried in vacuo, redissolved in 12. mu.L of 0.1% formic acid and stored at-20 ℃.
1.5LC-MS/MS and proteome analysis
All LC-MS/MS analyses were performed by Easy-nLC 1200 system and Q active HF (Thermo Scientific). For each sample, 6. mu.L of the peptide mixture was injected and separated on a reversed-phase C18 column (75. mu. m.times.15 cm) at a flow rate of 250 nL/min. Mobile phase a (0.1% formic acid in ultrapure water) and mobile phase B (0.1% formic acid/80% acetonitrile in ultrapure water) were used) A linear gradient of 7-25% mobile phase B was established over 50 minutes. The ionization voltage of the electrospray was set to 2.3 kV. The mass spectrometer was operated in positive ion mode at 120000 resolution with MS spectrum m/z 350-6An Automatic Gain Control (AGC) target. High energy collision dissociation (HCD) fragments were mass analysed with the first 12 strongest ions of normalised collision energy 27. AGC target of MS/MS spectrogram is 1 x 105The resolution was 30000. The dynamic exclusion time was set to 30 seconds. Raw data were acquired using XCalibur 4.0.27(Thermo Scientific) software and processed against the UniProt human refseq database using sequenst HT with the Proteome Discover (PD) software suite 2.2(Thermo Scientific). The mass tolerances for the precursor and the fragments were set to 10ppm and 0.02Da, respectively. Up to two missing trypsin cleavage sites are allowed. Modification options were set to include carbonamido methylation (C) as the static group, and oxidation (M) and acetyl (N-terminal) as the dynamic template. The Percolator algorithm was used to determine the False Discovery Rate (FDR) for peptide profile matching and peptide identification, and the threshold was set to 1% based on the q-value. Label-free quantification of proteins was performed using the protome discover 2.2.21, where Minora feature alignment and feature mapping were used to calculate the abundance of peptides in MS1 scans.
1.6 ELISA analysis of F10 in IgAP
After isolation of IgAP, the G protein beads were resuspended in 50 μ L of elution buffer (0.1M glycine-HCl, pH 3.0) and incubated for 5 min at 4 ℃. The reaction was then titrated to pH 7 with 1M Tris-HCl (pH 11) and the supernatant collected by centrifugation, the concentration of which was determined by Bradford assay. The concentration of F10 in the eluted IgAP was determined using an ELISA kit and normalized to the amount of input protein according to the manufacturer's protocol.
Example 1 quantitative analysis of immunoglobulin-related proteomes (IgAP)
Serum samples from 38 AMI patients, 20 stable CAD patients and 37 individuals diagnosed with non-CAD/AMI (nca) disease were combined into a discovery cohort. Table 1 summarizes clinical features of CAD and AMI patients.
TABLE 1 demographic characteristics of MS data sets
± denotes Standard Error (SE). And/represents a data vacancy.
To study immunoglobulin associated proteomes (IgAP), this example used an IgAP assay that integrated purification of immunoglobulin complexes with label-free quantitative LC-MS/MS, as shown in figure 1, comprising:
the immunoglobulin-associated Protein group with G Protein agarose beads was isolated and purified from 20. mu.L of serum of each individual using Protein G magnetic beads according to the procedure described in "1.3 IgAP isolation";
the separated proteins were eluted from the magnetic beads and then trypsinized and desalted by the procedure described in "1.4 bead digestion and desalting" to obtain desalted peptide mixture samples;
the desalted peptide mixture sample was analyzed by label-free quantitative LC-MS/MS analysis according to the procedure described in "1.5 LC-MS/MS and proteome analysis" above.
In this example, the average abundance of IgAP protein was examined and no significant fluctuations among individuals were observed, indicating good technical reproducibility (fig. 2).
Example 2 validation of F10 as an IgAP differential protein using ELISA
Autoantibodies to coagulation factors have been implicated in a range of clinical manifestations from minimal signs/symptoms to life-threatening diseases. The studies of the present invention indicate that enhanced IgAP proteins in AMI patients (as compared to CAD and NCA patients) include coagulation factor X (F10), which is a key component of the intrinsic and extrinsic coagulation pathways. Next, the present invention chooses to use ELISA-based assays to verify this enrichment. A set of validated patients was collected, including 25 AMI and 38 CAD patients. To obtain a more homogeneous control group, 30 healthy individuals (HEA group) were included for comparison. Protein G mediated immunoglobulin purification was performed prior to acidic elution. The eluate was then subjected to ELISA analysis of F10 absolute concentration using a commercially available kit. The results show that the AMI patients had significantly higher levels of immunoglobulin binding F10 (mean 29.05pg/μ g and 95% CI 20.08-38.02 pg/μ g of input IgAP protein) than CAD (mean 18.08pg/μ g and 95% CI 15.02-21.15 pg/μ g of input IgAP protein) and healthy individuals (mean 7.86pg/μ g and 95% CI 6.9-8.83 pg/μ g of input IgAP protein) (fig. 3A). This is consistent with the findings in LC-MS/MS based analysis. Moreover, the level of F10 bound to Ig was significantly lower in healthy populations than in CAD and AMI patients (fig. 3A). At the same time, there was no difference in the total serum F10 levels of the CAD, AMI and healthy groups (fig. 3B). The levels of immunoglobulin-associated F10 are increasing in healthy, CAD and AMI patients, suggesting that B cell immunity recognizes the epitope of F10 during the progression of atherosclerotic disease.
Example 3 prediction of AMI from CAD Using IgAP protein F10
The predicted performance of the IgAP protein F10 was examined using 58 samples (of which 38 AMIs and 20 CAD).
As shown in fig. 4, a receiver operating characteristic curve (ROC) was applied to evaluate the ability of the IgAP protein F10 to predict AMI from CAD (n-58, 38 AMI and 20 CAD).
[ instruction: when the threshold value of the model (positive and negative demarcation points are predicted) is changed, different specificities and sensitivities can be obtained, the specificity and the sensitivities are drawn in a coordinate system with the abscissa as 1-specificity and the ordinate as sensitivity, the obtained curve formed by connecting a series of points is an application subject operating characteristic curve (ROC), and the area under the curve (AUC) is used for measuring the accuracy of the model classification. The area under the curve (AUC) is 0.88, reflecting the higher accuracy of the detection method. The shaded area represents the 95% confidence interval for AUC, from 0.78 to 0.99.
Fig. 5 shows a violin diagram expressed by AMI and CAD patient F10. The x-axis represents the actual patient cohort and the y-axis corresponds to the log2 intensity value of F10. Horizontal line 21.74 represents the optimal threshold for classifying AMI and CAD, where AMI is predicted to be at high risk when the F10 expression value is greater than 21.74, as determined by the jordan index (calculate sensitivity + difference-1 for each point on the AUC curve, and the threshold when this value is the maximum is the optimal threshold). The violin diagram shows the distribution state and density of the F10 expression values.
As shown in fig. 6, a confusion matrix for predicting AMI from CAD using the IgAP protein F10 is shown. In the test results, the number of True Positives (TP) was 35, the number of False Positives (FP) was 5, the number of False Negatives (FN) was 3, and the number of True Negatives (TN) was 15. According to the calculation formula: the accuracy (TP + TN)/(TP + TN + FP + FN), specificity (TN/(TN + FP), and sensitivity (TP/(TP + FN) allowed the prediction of F10 to be 0.86 (95% confidence interval 0.75% -0.94), specificity to be 0.75 (95% confidence interval: 0.84-1), and sensitivity to be 0.92 (95% confidence interval: 0.55-0.95).
Furthermore, the specific relationship between the values of the expression level of F10 and the AMI risk profile may be reflected by single-factor analysis and multi-factor analysis, in addition to those described in fig. 4 and 5. According to the one-way logistic regression analysis, the OR value of F10 is 4.25, namely the expression value of F10 is increased by one unit, and the probability of suffering from AMI is increased by 4.25 times. The combined effect of factors F10, age, sex, BMI, etc. was analyzed by multifactor logistic regression analysis to give a corrected F10 OR value of 4.15.
Claims (10)
1. Use of a reagent for the detection of coagulation factor X (F10) that specifically binds to immunoglobulins in a sample from a test subject for the preparation of a test system for assessing the risk of an individual for suffering from an acute myocardial infarction and/or a coronary artery disease.
2. The use according to claim 1, wherein the reagent for detecting coagulation factor X specifically binding to immunoglobulin comprises a reagent material and/or a device for use in an immunoassay.
3. The use according to claim 1, wherein the reagent for detecting coagulation factor X specifically bound to immunoglobulins comprises the reagent materials and/or equipment used in label-free quantitative LC-MS/MS analysis methods.
4. Use according to claim 2 or 3, wherein the detection of coagulation factor X specifically bound to immunoglobulins in a sample from an individual to be tested is carried out by a method comprising:
separating immunoglobulin and binding protein in a sample by adopting a G protein cross-linked agarose bead technology;
eluting the separated protein from the microbeads by using glycine solution with pH of 3 to obtain eluent;
the protein in the eluate was subjected to standard immunoassay or label-free quantitative LC-MS/MS analysis to determine the level of F10 therein.
5. The use of claim 1, wherein the sample is a serum sample.
6. The use of claim 1, wherein the subject is a coronary artery disease patient.
7. Use according to claim 1, wherein the level of coagulation factor X specifically binding to immunoglobulins in the sample is increased and the individual is at increased risk of suffering from an acute myocardial infarction and/or coronary artery disease.
8. A method of detecting coagulation factor X (F10) specifically binding to an immunoglobulin in a sample from a test individual, the method comprising:
separating immunoglobulin and binding protein in a sample by adopting a G protein cross-linked agarose bead technology;
eluting the separated protein from the microbeads by using glycine solution with pH of 3 to obtain eluent;
performing a standard immunoassay or a label-free quantitative LC-MS/MS analysis on the protein of the eluate to determine the level of F10 in the sample;
preferably, the eluate further comprises a process of measuring the protein concentration in the eluate to normalise the protein content before performing the standard immunoassay.
9. A system for risk assessment of acute myocardial infarction and/or coronary artery disease, the system comprising a detection unit comprising reagent material and/or means for detecting coagulation factor X (F10) specifically binding to immunoglobulins in a sample from an individual to be tested.
10. The acute myocardial infarction and/or coronary artery disease risk assessment system according to claim 9, further comprising an analysis unit for analyzing the detection result of the detection unit to assess the risk of acute myocardial infarction and/or coronary artery disease of the individual.
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