Application of biomarker cyclic dinucleotide cGAMP in diagnosis and risk prediction of myocardial infarction

Abstract

The application provides an application of a biomarker cyclic dinucleotide cGAMP in diagnosis and risk prediction of myocardial infarction. The application specifically relates to an application of the biomarker cyclic dinucleotide cGAMP in preparation of a myocardial infarction diagnosis tool, an application in preparation of a myocardial infarction risk prediction product and an application in construction of a myocardial infarction risk prediction model. Experimental results show that a cGAMP concentration in plasma of a coronary heart disease patient has a significant U-shaped relationship with a risk of suffering from myocardial infarction, and the cGAMP concentration higher or lower than a critical concentration will increase the risk of myocardial infarction, and the critical concentration is 1352 ng / L. No matter whether the plasma concentration is greater than or less than the critical concentration, the cGAMP has a medium diagnosis capacity for myocardial infarction.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to the application of a biomarker cyclic di-nucleotide cGAMP in the diagnosis and risk prediction of myocardial infarction. BACKGROUND

[0002] Myocardial infarction (MI) is a type of coronary artery disease that occurs when blood flow to a part of the heart is blocked for a long enough time that part of the heart muscle is damaged or dies. According to the pathological mechanism and electrocardiogram, myocardial infarction can be divided into ST-segment elevation myocardial infarction (STEMI) and non-ST-segment elevation myocardial infarction (NSTEMI). Typical symptoms include severe chest pain, chest tightness, radiating pain (such as left shoulder, jaw or back), and accompanying shortness of breath, nausea, cold sweat, etc. However, its clinical manifestations have significant heterogeneity. Some patients (especially the elderly, diabetic patients or women) may only show atypical symptoms such as fatigue, syncope or upper abdominal discomfort, or even no symptoms at all (i.e. "silent myocardial infarction"), making early identification difficult. In addition, chest pain symptoms are easily confused with gastroesophageal reflux, pulmonary embolism or musculoskeletal diseases, further increasing the risk of misdiagnosis.

[0003] Currently, the diagnosis of myocardial infarction mainly relies on the joint analysis of clinical manifestations, dynamic evolution of electrocardiogram and myocardial injury markers (such as troponin, creatine kinase isoenzyme). Although electrocardiogram is the preferred tool for rapid screening in emergency, it may lack specific changes in the hyperacute phase, and individual anatomical variations (such as left bundle branch block) interfere with interpretation. Although myocardial injury markers (such as high-sensitivity troponin) significantly improve the diagnostic sensitivity, their levels usually do not significantly increase until several hours after the onset of symptoms, which may lead to early diagnosis delay. Imaging techniques (such as echocardiography, coronary CTA or angiography) can assist in assessing the extent of myocardial ischemia and vascular lesions, but have limitations such as radiation exposure, invasive procedures (such as angiography) or device dependence, making it difficult to be widely used for rapid screening in pre-hospital or resource-poor areas.

[0004] Traditional risk assessment models (such as GRACE score, TIMI score) are mainly based on age, medical history, electrocardiogram and laboratory indicators, but their prediction performance is limited by the lack of coverage of new biomarkers (such as inflammatory factors, genetic markers) and individual pathological mechanisms. In addition, high-risk groups of myocardial infarction (such as patients with chronic kidney disease, diabetes) often have multiple system diseases, which further interfere with the accuracy of risk assessment. Since myocardial infarction is closely related to fatal complications such as malignant arrhythmia and cardiogenic shock, and the reperfusion treatment time window directly affects the prognosis of patients, early and accurate identification and risk stratification are of great significance to improve clinical outcomes and reduce medical costs. Therefore, exploring new non-invasive and efficient biomarkers or artificial intelligence assisted diagnosis models has become a research hotspot in the current cardiovascular field. SUMMARY

[0005] The purpose of the present application is to provide a new biomarker cyclic dinucleotide cGAMP for risk prediction and disease diagnosis of myocardial infarction in patients with coronary heart disease, and to provide a new way for diagnosis and risk prediction of myocardial infarction.

[0006] In order to achieve the above-mentioned purpose, the present application provides the use of a reagent for detecting the biomarker cyclic dinucleotide cGAMP in the preparation of a myocardial infarction diagnosis tool.

[0007] Preferably, the reagent is used to detect the concentration of cyclic dinucleotide cGAMP in the plasma of patients with coronary heart disease.

[0008] Preferably, the reagent is an ELISA kit.

[0009] More preferably, the reagent detects the concentration of cGAMP in the plasma of patients with coronary heart disease by using a double antibody sandwich method.

[0010] The double antibody sandwich method specifically includes: using purified cGAMP antibody to coat a microplate to prepare a solid phase antibody, sequentially adding cGAMP to the microhole coated with the monoclonal antibody, and combining the cGAMP with a HRP-labeled cGAMP antibody to form an antibody-antigen-enzyme-labeled antibody complex, after thorough washing, adding a substrate TMB for color development. TMB is converted into blue under the catalysis of HRP enzyme, and is converted into the final yellow under the action of acid. The color depth is positively correlated with the cGAMP in the sample. The absorbance is measured at 450 nm wavelength by using an enzyme-labeled instrument, and the concentration of cGAMP in the sample is calculated through a standard curve.

[0011] The present application also provides the use of the biomarker cyclic dinucleotide cGAMP in the preparation of a myocardial infarction risk prediction product.

[0012] Preferably, the risk of suffering from myocardial infarction is predicted by detecting the concentration of cyclic dinucleotide cGAMP in the plasma of patients with coronary heart disease.

[0013] Preferably, the product for detecting the concentration of cyclic dinucleotide cGAMP in the plasma of a patient with coronary heart disease is an ELISA kit.

[0014] Preferably, the myocardial infarction includes ST-segment elevation myocardial infarction and non-ST-segment elevation myocardial infarction.

[0015] Preferably, the concentration of cyclic dinucleotide cGAMP in the plasma of a patient with coronary heart disease is significantly related to the risk of myocardial infarction in a U-shaped curve, and both higher and lower concentrations of cGAMP relative to the critical concentration of 1352 ng / L increase the risk of myocardial infarction.

[0016] Preferably, when the concentration of cyclic dinucleotide cGAMP is 1352 ng / L, the risk of myocardial infarction in a patient with coronary heart disease is low; when the concentration of cyclic dinucleotide cGAMP is < 1352 ng / L, the concentration of cyclic dinucleotide cGAMP is inversely related to the risk of myocardial infarction; when the concentration of cyclic dinucleotide cGAMP is > 1352 ng / L, the concentration of cyclic dinucleotide cGAMP is positively related to the risk of myocardial infarction.

[0017] The application also provides the use of the biomarker cyclic dinucleotide cGAMP in constructing a risk prediction model for myocardial infarction.

[0018] Preferably, the input variables of the risk prediction model include the plasma concentration of cyclic dinucleotide cGAMP.

[0019] Preferably, the input variables of the risk prediction model also include age, gender, hypertension, diabetes, smoking, alcohol consumption, body mass index, systolic blood pressure, left ventricular ejection fraction, serum creatinine, cardiac troponin T, N-terminal pro-brain natriuretic peptide, high-density lipoprotein, apolipoprotein A1, and free fatty acids.

[0020] In the present application, the diagnosis of myocardial infarction and the prediction of the risk of myocardial infarction by the biomarker cyclic dinucleotide cGAMP comprises the following steps: step 1), collecting the venous blood of the patient after fasting using an anticoagulant tube; step 2), separating the plasma from the venous blood and performing cGAMP quantitative detection; step 3), determining the risk of myocardial infarction in a patient with coronary heart disease based on the results of cGAMP quantitative detection combined with a U-shaped curve, which is a curve describing the relationship between the concentration of cyclic dinucleotide cGAMP and the risk of myocardial infarction.

[0021] The beneficial effects of the present application at least include:

[0022] One, the present application first discovers that the concentration of cGAMP in the plasma of patients with coronary heart disease has a significant U-shaped relationship with the risk of suffering from myocardial infarction, so that cGAMP can be used as a biomarker for myocardial infarction to assist in the diagnosis or risk prediction of myocardial infarction, providing a new way for the diagnosis and risk prediction of myocardial infarction, and also providing a convenient and low-cost diagnostic tool for clinicians.

[0023] Two, cGAMP as a single biomarker for diagnosing myocardial infarction has high diagnostic value. Specifically, when the level of cGAMP is <1352 ng / L, the AUC is 0.66, the sensitivity is 56%, and the specificity is 68%; when the level of cGAMP is ≥1352 ng / L, the AUC is 0.62, the sensitivity is 47%, and the specificity is 71%.

[0024] Three, the detection of the biomarker cGAMP concentration of the present application can be completed by blood detection, and can be implemented in routine clinical examination, which is simple to operate, has high repeatability and reliability, and is suitable for large-scale popularization and application. Compared with traditional imaging examination, it has the advantages of low cost and non-invasiveness, improves the acceptance and comfort of patients. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 As a U-shaped curve of the risk of myocardial infarction in patients with coronary heart disease, the cyclic dinucleotide cGAMP as a biomarker;

[0026] Figure 2 As a ROC curve constructed by cGAMP as a single biomarker for diagnosing myocardial infarction when the cGAMP concentration of the coronary heart disease patients in the examples is ≥1352 ng / L;

[0027] Figure 3 As a ROC curve constructed by cGAMP as a single biomarker for diagnosing myocardial infarction when the cGAMP concentration of the coronary heart disease patients in the examples is <1352 ng / L. DETAILED DESCRIPTION

[0028] The present application will be described in detail below in combination with the drawings and examples, but the present application can be implemented in various different ways limited and covered by the claims.

[0029] The experimental methods not specified in the examples are usually carried out according to the conventional conditions or the conditions recommended by the manufacturers. The materials, reagents, etc. used in the following examples can be obtained from commercial channels unless otherwise specified.

[0030] Examples

[0031] Verification of the risk prediction and diagnostic performance of the single biomarker cGAMP on myocardial infarction

[0032] 1.1 Case selection

[0033] Coronary heart disease patients who visited the Second Xiangya Hospital of Central South University due to chest pain were selected. All patients were adults aged 35-75 years old, and had undergone coronary angiography to confirm the presence or absence of myocardial infarction. In this embodiment, a total of 270 coronary heart disease patients were selected.

[0034] 1.2 Case data

[0035] The relevant clinical background information of the patients was collected, including but not limited to the general condition of the patients (such as age, gender, weight, height, etc.), medical history (smoking history, drinking history, hypertension, diabetes, atrial fibrillation, etc.), laboratory tests (including blood routine, liver function, kidney function, electrolytes, blood lipids, etc.), echocardiogram, and drug use, etc. The specific statistical information of the case data is shown in Table 1.

[0036] 1.3 Detection of cyclic di-nucleotide cGAMP value

[0037] Sample collection: venous blood samples were taken from the patients for subsequent laboratory tests. The blood samples were centrifuged at room temperature to separate the plasma for detection of cGAMP concentration.

[0038] In this application, the plasma was separated by centrifugation at 2500 rpm for 5 minutes.

[0039] cGAMP detection: The human cyclic guanosine monophosphate (cGAMP) enzyme-linked immunoassay kit (provided by Fankewei Company, item number: F11293-A) was used to quantitatively analyze the cGAMP concentration in the plasma. The experimental operation was carried out according to the kit instructions, and the standard curve was established using known concentration of cGAMP standard solution. The operation process ensured strict quality control, and the experimental repeatability was good.

[0040] 1.4 Data statistics

[0041] According to the cGAMP concentration in the plasma of the coronary heart disease patients, the patients were divided into three groups, namely group 1, group 2 and group 3, and the baseline clinical data were compared, which are shown in detail in Table 1.

[0042] Table 1 Case clinical data

[0043]

[0044]

[0045]

[0046] 1.5 Data analysis

[0047] In this embodiment, among the 270 patients, 212 (78.52%) were male, with a median age of (61.83±10.78) years and a mean plasma cGAMP concentration of (1352.58±106.02 ng / L).

[0048] As shown in Table 1, compared with patients in group 2, more patients in groups 1 and 3 were diagnosed with myocardial infarction. Other variables showed no significant differences. Specifically, 19 patients in group 2 were diagnosed with myocardial infarction, 36 patients in group 1, and 34 patients in group 3. The myocardial infarction mentioned includes ST-segment elevation myocardial infarction (STEMI, corresponding to STEMI in Table 1) and non-ST-segment elevation myocardial infarction (NSTEMI, corresponding to NIST in Table 1).

[0049] Of the 270 patients, 89 (32.96%) were diagnosed with myocardial infarction. The results of the univariate logistic regression model are shown in Table 2. Elevated levels of HDL, apoA1, and LVEF were negatively correlated with the occurrence of MI, suggesting a protective effect. On the other hand, higher levels of free fatty acids, cTNT, NT-proBNP, creatinine, and a history of hypertension were significantly positively correlated with an increased risk of myocardial infarction in patients with coronary heart disease.

[0050] Table 2. Variables selected for the univariate logistic regression model.

[0051]

[0052] Please refer to Table 3. With group 2 as the control group, the risk of myocardial infarction in patients in group 1 was 167% higher than that in group 2 (adjusted OR: 2.67, 95% CI: 1.23-5.78, P = 0.013); the risk of myocardial infarction in patients in group 3 was 155% higher than that in group 2 (adjusted OR: 2.55, 95% CI: 1.17-5.55, P = 0.018).

[0053] Table 3. Relationship between cGAMP and myocardial infarction

[0054]

[0055] This trend can be visually illustrated using RCS curves, such as... Figure 1 As shown, from Figure 1It can be seen that the concentration of the cyclic dinucleotide cGAMP in the plasma of the coronary heart disease patients has a significant U-shaped relationship with the risk of suffering from myocardial infarction, and when the concentration of the cyclic dinucleotide cGAMP is around 1352 ng / L, the risk of the coronary heart disease patients suffering from myocardial infarction is low; and the risk of myocardial infarction is increased when the concentration of cGAMP is higher or lower than the critical concentration (1352 ng / L), and the risk of the coronary heart disease patients suffering from myocardial infarction is high when the concentration of the cyclic dinucleotide cGAMP is extremely high (greater than 1450 ng / L) or extremely low (less than 1250 ng / L).

[0056] That is, when the concentration of the cyclic dinucleotide cGAMP is 1352 ng / L, the risk of the coronary heart disease patients suffering from myocardial infarction is low; when the concentration of cGAMP is less than 1352 ng / L, the risk of the coronary heart disease patients suffering from myocardial infarction is reduced (reverse correlation) as the concentration of the cyclic dinucleotide cGAMP in the plasma of the coronary heart disease patients increases; and when the concentration of cGAMP is greater than or equal to 1352 ng / L, the risk of the coronary heart disease patients suffering from myocardial infarction is increased (positive correlation) as the concentration of the cyclic dinucleotide cGAMP in the plasma of the coronary heart disease patients increases.

[0057] The cyclic dinucleotide cGAMP is used as a single biomarker for diagnosing whether the coronary heart disease patients have myocardial infarction, and the results of the ROC curve analysis are shown in Figure 2 and Figure 3 From Figure 2 and Figure 3 It can be seen that no matter whether the concentration of cGAMP in the plasma of the coronary heart disease patients is greater than or less than the critical concentration 1352 ng / L, cGAMP has a moderate diagnostic ability for myocardial infarction. Specifically, when the level of cGAMP is less than 1352 ng / L, the AUC is 0.66, the sensitivity is 56%, and the specificity is 68%; and when the level of cGAMP is greater than or equal to 1352 ng / L, the AUC is 0.62, the sensitivity is 47%, and the specificity is 71%.

[0058] The above is a further detailed description of the present application in combination with specific preferred embodiments, and the specific implementation of the present application should not be limited to these descriptions. For ordinary skilled persons in the technical field to which the present application belongs, a number of simple deductions and substitutions can be made without departing from the concept of the present application, and all of them should be considered as falling within the protection scope of the present application.

Claims

1. Application of reagents for detecting the biomarker cyclic dinucleotide cGAMP in the preparation of diagnostic kits for myocardial infarction.

2. Application of the biomarker cyclic dinucleotide cGAMP in the preparation of a myocardial infarction risk prediction kit.

3. The application according to claim 2, characterized in that, The risk of myocardial infarction can be predicted by detecting the concentration of cyclic dinucleotide cGAMP in the plasma of patients with coronary artery disease.

4. The application according to claim 3, characterized in that, The kit used to detect the concentration of cyclic dinucleotide cGAMP in the plasma of patients with coronary heart disease is an ELISA kit.

5. The application according to claim 2, characterized in that, The myocardial infarction includes ST-segment elevation myocardial infarction and non-ST-segment elevation myocardial infarction.

6. The application according to any one of claims 2 to 5, characterized in that, The concentration of the cyclic dinucleotide cGAMP in the plasma of patients with coronary heart disease has a U-shaped relationship with the risk of myocardial infarction. Both excessively high and excessively low cGAMP concentrations relative to a critical concentration of 1352 ng / L will increase the risk of myocardial infarction.

7. The application according to claim 6, characterized in that, When the concentration of cyclic dinucleotide cGAMP is 1352 ng / L, it predicts a low risk of myocardial infarction in patients with coronary artery disease; when the concentration of cyclic dinucleotide cGAMP is <1352 ng / L, the concentration of cyclic dinucleotide cGAMP is inversely correlated with the risk of myocardial infarction; when the concentration of cyclic dinucleotide cGAMP is >1352 ng / L, the concentration of cyclic dinucleotide cGAMP is positively correlated with the risk of myocardial infarction.

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