A protein biomarker for assisting in the early diagnosis of acute myocardial infarction and its application

Abstract

This invention discloses a protein biomarker for assisting in the early diagnosis of acute myocardial infarction and its application. The biomarker is characterized by being at least one of GLRX, CA1, EIF4EBP1, MYL4, SOD1, BOLA2_BOLA2B, DDT, TNNI3, GH1, ACY1, POMC, AK1, FABP3, YOD1, GLO1, RANBP1, TGM2, PLPBP, OLR1, CEACAM8, CD2AP, AAMDC, and QDPR. The invention also provides the application of the above-mentioned protein biomarkers in the preparation of early diagnostic reagents or kits for myocardial infarction. The advantages are that it allows for convenient and rapid diagnosis of early AMI at the molecular level, with high detection efficiency, high sensitivity, and strong specificity, which is beneficial for the early detection and timely treatment of AMI patients.

Description

Technical Field

[0001] This invention relates to the field of early diagnosis technology for acute myocardial infarction, and in particular to a relevant protein biomarker for assisting in the early diagnosis of acute myocardial infarction and its application. Background Technology

[0002] Acute myocardial infarction (AMI) is a clinical emergency caused by the rupture or erosion of atherosclerotic plaques in the coronary arteries, followed by thrombosis, leading to acute occlusion or severe stenosis of the blood vessels, and irreversible necrosis of the myocardium due to persistent ischemia and hypoxia. The current diagnosis of AMI is mainly based on the fourth edition of the "Global Definition of Myocardial Infarction" criteria, namely, elevated cardiac troponin (cTn) levels, with at least one reading above the upper limit of normal (99th percentile of the upper limit of the reference value), and at least one piece of evidence of acute myocardial ischemia, including: (1) symptoms of acute myocardial ischemia; (2) new ischemic electrocardiographic changes; (3) new pathological Q waves; (4) new wall motion abnormalities / loss of myocardial activity shown by echocardiography or MRI; (5) vascular occlusion or thrombosis confirmed by coronary angiography or intravascular imaging or autopsy. If "elevated cTn + 1 piece of evidence of ischemia" is met, AMI can be diagnosed.

[0003] As a specific biomarker of myocardial injury, cTn concentrations in peripheral blood typically reach a detectable threshold approximately 3 hours after acute myocardial infarction (AMI). However, the exact timing is significantly influenced by the analytical sensitivity of the detection method (e.g., high-sensitivity troponin assay) and individual biological heterogeneity (e.g., renal function status, baseline cTn levels). This delay in the clinical detection window leads to insufficient sensitivity for early diagnosis within 3 hours of AMI onset, becoming a key limiting factor affecting timely intervention. However, epidemiological studies have confirmed that 1-6 hours after AMI onset is the golden window for intravenous thrombolysis and percutaneous coronary intervention. Achieving coronary blood flow restoration within this period can significantly improve the myocardial salvage index and reduce the incidence of major adverse cardiovascular events. Therefore, there is an urgent need for a technology capable of early diagnosis and prediction of AMI.

[0004] Recent studies have shown that the detection of biochemical biomarkers for myocardial injury has multiple values ​​in the clinical management of acute myocardial infarction (AMI). Currently, commonly used core biomarkers besides cTn include creatine kinase isoenzyme (CK-MB) and myoglobin. Among them, myoglobin, as an early-release biomarker (detectable 1-2 hours after onset), has very limited myocardial specificity due to cross-reactivity in pathological conditions such as skeletal muscle injury. Although CK-MB is rapid, economical, and effective, it still lacks specificity. Therefore, the search for novel diagnostic biomarkers for the hyperacute phase has significant clinical value. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a protein biomarker with high sensitivity and specificity for assisting in the early diagnosis of acute myocardial infarction and its application.

[0006] The technical solution adopted by the present invention to solve the above-mentioned technical problems is as follows: a protein biomarker for assisting in the early diagnosis of acute myocardial infarction, wherein the protein biomarker is at least one of GLRX, CA1, EIF4EBP1, MYL4, SOD1, BOLA2_BOLA2B, DDT, TNNI3, GH1, ACY1, POMC, AK1, FABP3, YOD1, GLO1, RANBP1, TGM2, PLPBP, OLR1, CEACAM8, CD2AP, AAMDC, and QDPR.

[0007] Preferably, the protein markers are a combination of GLRX, CA1, EIF4EBP1, MYL4, SOD1, BOLA2_BOLA2B, DDT, TNNI3, GH1, ACY1, POMC, AK1, FABP3, YOD1, GLO1, RANBP1, TGM2, PLPBP, OLR1, CEACAM8, CD2AP, AAMDC, and QDPR proteins.

[0008] Preferably, the protein markers are a combination of GLRX, CA1, FABP3 and OLR1 proteins.

[0009] Preferably, the protein markers are a combination of GLRX, CA1, FABP3, OLR1, and TNNI3 proteins.

[0010] Preferably, the protein markers are a combination of SOD1, BOLA2_BOLA2B, DDT, TNNI3 and AK1 proteins.

[0011] Preferably, the protein markers are a combination of YOD1, GLO1, RANBP1, TGM2 and PLPBP proteins.

[0012] Preferably, the protein markers are a combination of OLR1, CEACAM8, CD2AP, AAMDC, and QDPR proteins.

[0013] Preferably, the protein marker is a combination of GH1 and ACY1 proteins.

[0014] Preferably, the protein marker is a combination of GH1 and CA1 proteins.

[0015] Preferably, the protein marker is a combination of GLRX and POMC proteins.

[0016] The present invention also provides the application of the above-mentioned related protein biomarkers in the preparation of early diagnostic reagents or kits for myocardial infarction.

[0017] The present invention also provides the application of the above-mentioned reagent for detecting the expression level of the above-mentioned related protein markers in the preparation of early diagnostic reagents or kits for myocardial infarction.

[0018] Compared with the prior art, the advantages of the present invention are as follows: The present invention discloses for the first time a protein biomarker for assisting in the diagnosis of early acute myocardial infarction and its application, which is selected from GLRX, CA1, EIF4EBP1, MYL4, SOD1, BOLA2_BOLA2B, DDT, TNNI3, GH1, ACY1, POMC, AK1, FABP3, YOD1, GLO1, RANBP1, TGM2, PLPBP, OLR1, CEACAM8, CD2AP, AAMDC and QDPR for the auxiliary diagnosis of early AMI, as well as one or more combinations of such biomarkers. Therefore, detection kits based on the detection of protein expression levels of one or more of the following markers—GLRX, CA1, EIF4EBP1, MYL4, SOD1, BOLA2_BOLA2B, DDT, TNNI3, GH1, ACY1, POMC, AK1, FABP3, YOD1, GLO1, RANBP1, TGM2, PLPBP, OLR1, CEACAM8, CD2AP, AAMDC, and QDPR—can conveniently and rapidly diagnose early-stage acute myeloid muscular lesions (AMI) at the molecular level. These kits offer high detection efficiency, high sensitivity, and strong specificity, which is beneficial for the early detection and timely treatment of AMI patients. Attached Figure Description

[0019] Figure 1 This is a volcano plot of differentially expressed proteins between the early stage of acute myocardial infarction and the control group. A is the differentially expressed protein volcano plot in Olink Explore 384 Cardiometabolic, and B is the differentially expressed protein volcano plot in Olink Explore 384 Cardiometabolic II. Note: The division is based on the significance threshold (p value ≤ 0.05), the y-axis is -log10 (p value), red indicates significantly different proteins, and blue indicates proteins with no significant difference.

[0020] Figure 2ROC curve analysis of the early diagnostic value of combinations of proteins GLRX, CA1, EIF4EBP1, MYL4, SOD1, BOLA2_BOLA2B, DDT, TNNI3, AK1, FABP3, YOD1, GLO1, RANBP1, TGM2, PLPBP, OLR1, CEACAM8, CD2AP, AAMDC, and QDPR in acute myocardial infarction;

[0021] Figure 3 ROC curve analysis of the early diagnostic value of the combination of GLRX, CA1, FABP3 and OLR1 proteins in acute myocardial infarction;

[0022] Figure 4 ROC curve analysis of the early diagnostic value of the combination of GLRX, CA1, FABP3, OLR1 and TNNI3 proteins in acute myocardial infarction;

[0023] Figure 5 ROC curve analysis of the early diagnostic value of the combination of SOD1, BOLA2_BOLA2B, DDT, TNNI3 and AK1 proteins in acute myocardial infarction;

[0024] Figure 6 ROC curve analysis of the early diagnostic value of the combination of YOD1, GLO1, RANBP1, TGM2 and PLPBP proteins in acute myocardial infarction.

[0025] Figure 7 ROC curve analysis of the early diagnostic value of OLR1, CEACAM8, CD2AP, AAMDC and QDPR protein combinations for acute myocardial infarction.

[0026] Figure 8 ROC curve analysis of the early diagnostic value of the combination of GH1 and ACY1 proteins in acute myocardial infarction;

[0027] Figure 9 ROC curve analysis of the early diagnostic value of the combination of GH1 and CA1 proteins in acute myocardial infarction;

[0028] Figure 10 ROC curve analysis of the early diagnostic value of the combination of GLRX and POMC proteins in acute myocardial infarction. Detailed Implementation

[0029] The present invention will be further described in detail below with reference to the accompanying drawings and embodiments.

[0030] Specific Implementation Example 1: Screening for myocardial metabolism-related proteins in the early stage of acute myocardial infarction.

[0031] 1. Clinical Data of Volunteers in the Screening Phase: This study collected volunteer data from the Department of Cardiology at the First Affiliated Hospital of Ningbo University. The volunteers included 37 patients diagnosed with acute myocardial infarction via coronary angiography and 20 healthy controls. Fasting venous blood samples were collected from all participants for testing of general biochemical indicators such as blood lipids and blood glucose. Clinical data, including gender, age, smoking and alcohol history, and hypertension, were recorded. The clinical data of the first batch of acute myocardial infarction cases and the control group used for screening for differentially expressed proteins are compared in Table 1 below.

[0032] 2. Plasma Extraction and Biochemical Analysis: During the clinical sample collection process in this study, approximately 6 mL of fasting blood was drawn from each patient upon admission. After centrifugation at 3200 rpm for 15 minutes at 4°C, the blood was separated, and the upper plasma layer and the middle white membrane layer were extracted separately. Quantitative analysis of glycated hemoglobin, total cholesterol, triglycerides, high-density lipoprotein, and low-density lipoprotein was performed using an automated biochemical analyzer (Olympus AU2700, Japan). The concentrations of apolipoprotein A1 (apoA1), apolipoprotein B (apoB), and blood lipids (LPa) were measured using turbidimetric immunoassay. The results are shown in Table 1.

[0033] Table 1. Clinical baseline characteristics of the acute myocardial infarction group and the control group

[0034]

[0035] Table 1 shows that the AMI group had significantly higher rates of male prevalence, smoking history, HbA1c, LDL cholesterol, apoA1, and LPa levels compared to the control group, and a lower age. There were no statistically significant differences between the two groups in hypertension, diabetes, alcohol consumption history, triglycerides, total cholesterol, HDL cholesterol, and apoB (P>0.05).

[0036] 3. Analysis of myocardial metabolism-related proteins in two groups of blood samples using ultrasensitive multiplex targeted protein detection and analysis (Olink): Following the manufacturer's guidelines, plasma samples from 37 patients in the early stages of acute myocardial infarction and 20 control patients were analyzed using Olink Explore 384 Cardiometabolic and Olink Explore 384 Cardiometabolic II (Olink Proteomics AB, Sweden). This technique involves highly specific binding of target proteins to antibody probes labeled with dioliponucleotides, followed by detection and quantification using a microfluidic real-time PCR instrument (Biomark HD, USA). The final detection readings are displayed as normalized protein expression values, which are then log2 transformed for various biological analyses.

[0037] Based on Olink Explore 384 Cardiometabolic panel analysis, we found differential expression of 125 related proteins between the two patient groups. Figure 1 As shown in Figure A, based on -log10 (p-value), we identified GLRX, EIF4EBP1, SOD1, AK1, NPDC1, GLO1, CA1, CD2AP, TGM2, and THOP1 as the 10 most significantly differentially expressed proteins. Using Olink Explore 384 Cardiometabolic II panel analysis, we found 117 related proteins with differential expression between the two patient groups, such as... Figure 1 As shown in Figure B, sorted by -log10 (p-value), we identified POMC, MYL4, DDT, AAMDC, BOLA2_BOLA2B, FABP3, TALDO1, NIT1, RANBP1, and INPP5D as the 10 most significantly different proteins. Table 2 shows detailed information on 20 differentially expressed proteins between the acute myocardial infarction group and the control group.

[0038] Table 2. Detailed information on myocardial metabolism-related proteins with significant changes between groups.

[0039]

[0040] Note: The difference in protein expression between the acute myocardial infarction group and the control group was calculated using a t-test (P<0.05), indicating a significant difference in protein expression.

[0041] 4. Ranking of the importance of 22 myocardial metabolism-related proteins screened in the early stage of acute myocardial infarction.

[0042] Univariate ROC analysis was performed on the differentially expressed proteins. As shown in Table 3, based on the area under the curve (AUC) ranking, GLRX, CA1, EIF4EBP1, MYL4, SOD1, BOLA2_BOLA2B, DDT, TNNI3, AK1, FABP3, YOD1, GLO1, RANBP1, TGM2, PLPBP, OLR1, CEACAM8, CD2AP, AAMDC, and QDPR all have very high diagnostic value for early acute myocardial infarction.

[0043] Table 3. Specific information on the top 23 proteins with AUC from ROC analysis.

[0044]

[0045] In summary, by collecting blood samples from patients with acute myocardial infarction and a control group, we used Olink's ultrasensitive multiplex targeted protein detection and analysis technology to detect 768 myocardial metabolism-related proteins in the patient plasma. We found that 242 myocardial metabolism-related proteins were differentially expressed between the two groups. Among them, the 23 proteins with the highest diagnostic efficacy were GLRX, CA1, EIF4EBP1, MYL4, SOD1, BOLA2_BOLA2B, DDT, TNNI3, GH1, ACY1, POMC, AK1, FABP3, YOD1, GLO1, RANBP1, TGM2, PLPBP, OLR1, CEACAM8, CD2AP, AAMDC, and QDPR.

[0046] Specific Implementation Example 2: Verifying the diagnostic value of myocardial metabolism-related proteins screened in Specific Implementation Example 1 for acute myocardial infarction.

[0047] A second batch of validation set samples was collected from the clinical case group and the control group, including 31 patients with acute myocardial infarction diagnosed by coronary angiography and 20 healthy controls. The expression differentials of the 23 proteins with the highest diagnostic efficacy—GLRX, CA1, EIF4EBP1, MYL4, SOD1, BOLA2_BOLA2B, DDT, TNNI3, GH1, ACY1, POMC, AK1, FABP3, YOD1, GLO1, RANBP1, TGM2, PLPBP, OLR1, CEACAM8, CD2AP, AAMDC, and QDPR—were validated using Olink's ultrasensitive multiplex targeted protein detection and analysis technology combined with ten-fold cross-validation.

[0048] Table 4. Comparison of clinical data between the acute myocardial infarction case group and the control group in the second batch of validation phase.

[0049]

[0050] Table 4 shows that the AMI group in the validation cohort had significantly higher rates of male prevalence, smoking history, HbA1, LDL cholesterol, apoB, apoA1, and LPa levels compared to the control group. There were no statistically significant differences between the two groups in age, hypertension, diabetes, alcohol consumption history, BMI, triglycerides, total cholesterol, and HDL cholesterol (P>0.05).

[0051] Table 5. Detailed information on the validation proteins between acute myocardial infarction and control groups.

[0052]

[0053] Table 5 shows that the myocardial metabolism-related proteins GLRX, CA1, EIF4EBP1, MYL4, SOD1, BOLA2_BOLA2B, DDT, TNNI3, GH1, ACY1, POMC, AK1, FABP3, YOD1, GLO1, RANBP1, TGM2, PLPBP, OLR1, CEACAM8, CD2AP, AAMDC, and QDPR showed significant differences between the acute myocardial infarction group and the control group in the validation cohort, and each protein had good independent diagnostic efficacy. We used R software (Version 4.1.3) to generate ROC curves using the ROCR package, combined the 23 indicators using Lightgbm analysis, and performed ten-fold cross-validation. Figure 2 The study displays the area under the curve (AUC) values ​​of 23 combined diagnostic markers, assessing the classification performance of the data. The AUC confidence interval was calculated using bootstrap sampling, with an AUC value of 0.990, indicating very high diagnostic value. The 95% confidence interval was 0.962–1, with a sensitivity of 0.968 and a specificity of 1. These results highlight the potential of GLRX, CA1, EIF4EBP1, MYL4, SOD1, BOLA2_BOLA2B, DDT, TNNI3, GH1, ACY1, POMC, AK1, FABP3, YOD1, GLO1, RANBP1, TGM2, PLPBP, OLR1, CEACAM8, CD2AP, AAMDC, and QDPR as biomarkers for the early diagnosis of acute myocardial infarction. They also demonstrate that combining multiple markers can improve diagnostic efficacy. Furthermore, several protein combinations exhibit good diagnostic performance for the early diagnosis of acute myocardial infarction.

[0054] like Figure 3 As shown, the combined AUC of GLRX, CA1, FABP3 and OLR1 was 0.998, with a 95% confidence interval of 0.990–1, a sensitivity of 0.968 and a specificity of 1.

[0055] like Figure 4 As shown, the AUC value of the combination of GLRX, CA1, FABP3, OLR1 and TNNI3 was 0.997, with a 95% confidence interval of 0.984 to 1, a sensitivity of 0.968 and a specificity of 1.

[0056] like Figure 5 As shown, the AUC value of the combination of SOD1, BOLA2_BOLA2B, DDT, TNNI3 and AK1 was 0.990, with a 95% confidence interval of 0.966 to 1, a sensitivity of 0.968 and a specificity of 1.

[0057] like Figure 6As shown, the AUC value of the combination of YOD1, GLO1, RANBP1, TGM2 and PLPBP is 0.950, with a 95% confidence interval of 0.886 to 0.995, a sensitivity of 0.935 and a specificity of 0.9.

[0058] like Figure 7 As shown, the AUC value of the combination of OLR1, CEACAM8, CD2AP, AAMDC and QDPR is 0.964, with a 95% confidence interval of 0.910 to 1, a sensitivity of 1, and a specificity of 0.850.

[0059] like Figure 8 As shown, the AUC value of the GH1 and ACY1 combination is 0.734, with a 95% confidence interval of 0.572–0.884, a sensitivity of 0.935, and a specificity of 0.55.

[0060] like Figure 9 As shown, the AUC value of the GH1 and CA1 combination was 0.917, with a 95% confidence interval of 0.831–0.984, a sensitivity of 0.871, and a specificity of 0.95.

[0061] like Figure 10 As shown, the AUC of the GLRX and POMC combination is 0.952, with a 95% confidence interval of 0.877–1, a sensitivity of 0.903, and a specificity of 1.

[0062] All combinations of the above 23 myocardial metabolism-related proteins belong to the technical solutions of this invention.

[0063] The foregoing description is not intended to limit the invention, nor is the invention limited to the examples given. Any changes, modifications, additions, or substitutions made by those skilled in the art within the scope of the invention should also be considered within the protection scope of the invention.

Claims

1. The application of a protein combinatorial biomarker in the preparation of reagents or kits for early diagnosis of myocardial infarction, characterized in that: The protein biomarker consists of GLRX, CA1, FABP3, and OLR1.

2. The application according to claim 1, characterized in that: The protein biomarkers also include TNNI3.

3. A protein biomarker for assisting in the early diagnosis of acute myocardial infarction, characterized in that... The protein biomarker described consists of GLRX, CA1, FABP3, and OLR1.

4. The use of a reagent for detecting the expression level of the protein combination biomarker described in claim 3 in the preparation of a reagent or kit for early diagnosis of myocardial infarction.

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