Related protein marker for assisting early diagnosis of acute myocardial infarction and application thereof

Abstract

The invention discloses a related protein marker for assisting early diagnosis of acute myocardial infarction and application of the related protein marker, and is characterized in that the protein marker is at least one of GLRX, CA1, EIF4EBP1, MYL4, SOD1, BOLA2BOLA2B, DDT, TNNI3, GH1, ACY1, POMC, AK1, FABP3, YOD1, GLO1, RANBP1, TGM2, PLPBP, OLR1, CEACAM8, CD2AP, AAMDC and QDPR, and the invention further provides application of the related protein marker in preparation of a reagent or a kit for early diagnosis of myocardial infarction. The kit has the advantages that early-stage AMI diagnosis can be conveniently and quickly realized on the molecular level, the detection efficiency is high, the sensitivity is high, the pertinence is strong, and early-stage discovery and timely treatment of AMI patients are facilitated.

Description

Technical Field

[0001] The present invention relates to the technical field of early diagnosis of acute myocardial infarction, and in particular to a related protein marker for assisting early diagnosis of acute myocardial infarction and an application thereof. Background Art

[0002] Acute myocardial infarction (AMI) is a clinical emergency caused by rupture or erosion of coronary atherosclerotic plaques, followed by thrombosis, leading to acute vascular occlusion or severe stenosis, and irreversible myocardial necrosis 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" standard, that is, elevated cardiactroponin (cTn), which is higher than the upper limit of normal (99th percentile of the upper limit of the reference value) at least once, and combined with at least one piece of evidence of acute myocardial ischemia, including: (1) symptoms of acute myocardial ischemia; (2) new ischemic electrocardiogram changes; (3) new pathological Q waves; (4) new ventricular wall motion abnormalities / myocardial activity loss shown by cardiac ultrasound or MRI; (5) confirmed vascular occlusion or thrombosis by coronary angiography or intravascular imaging or autopsy. If the "cTn elevation + one 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 AMI onset. However, this timing is significantly influenced by the analytical sensitivity of the assay (e.g., high-sensitivity troponin assay technology) and individual biological heterogeneity (e.g., renal function and baseline cTn levels). This delayed clinical detection window results in insufficient sensitivity for early diagnosis within 3 hours of AMI onset, a key limiting factor for 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. Restoring coronary blood flow within this period significantly improves the myocardial salvage index and reduces the incidence of major adverse cardiovascular events. Therefore, there is an urgent need for technologies that can enable the early diagnosis and prognosis of AMI.

[0004] Recent studies have demonstrated the multifaceted value of biochemical markers of myocardial injury in the clinical management of acute myocardial infarction (AMI). Core biomarkers commonly used in clinical practice, besides cTn, include creatine kinase isoenzyme (CK-MB) and myoglobin. Myoglobin, an early-release biomarker (detectable within 1-2 hours of onset), has very limited myocardial specificity due to cross-reactivity in pathological conditions such as skeletal muscle injury. While CK-MB is rapid, cost-effective, and effective, it still lacks specificity. Therefore, identifying novel diagnostic markers for the hyperacute phase holds significant clinical value. Summary of the Invention

[0005] The technical problem to be solved by the present invention is to provide a relevant protein marker with high sensitivity and strong 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 technical problems is: a related protein marker for assisting the early diagnosis of acute myocardial infarction, wherein the protein marker 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 marker is 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 marker is a combination of GLRX, CA1, FABP3 and OLR1 proteins.

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

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

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

[0012] Preferably, the protein marker is 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 use of the above-mentioned related protein markers in preparing an early diagnosis reagent or kit for myocardial infarction.

[0017] The present invention also provides use of the above-mentioned reagent for detecting the expression level of the above-mentioned related protein marker in the preparation of an early diagnosis reagent or kit for myocardial infarction.

[0018] Compared with the prior art, the advantages of the present invention are: the present invention provides a protein marker for auxiliary diagnosis of early acute myocardial infarction and its application, and for the first time discloses one or more combination markers 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 for auxiliary diagnosis of early AMI. Therefore, a detection kit based on detecting the protein expression level 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 quickly diagnose early AMI at the molecular level. It has high detection efficiency, high sensitivity, and strong specificity, which is conducive to the early detection and timely treatment of AMI patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] Figure 1 Figure 2 shows the volcano plots of differentially expressed proteins between the early-stage acute myocardial infarction group and the control group. A is the volcano plot of differentially expressed proteins in Olink Explore 384 Cardiometabolic, and B is the volcano plot of differentially expressed proteins in Olink Explore 384 Cardiometabolic II. Note: The plots were segmented based on the significance threshold (p value ≤ 0.05). The y-axis is -log10 (p value). Proteins marked in red are significantly differentially expressed, while proteins marked in blue are not significantly differentially expressed. Figure 2 ROC curve analysis of the early diagnostic value of acute myocardial infarction for the combination of GLRX, CA1, EIF4EBP1, MYL4, SOD1, BOLA2_BOLA2B, DDT, TNNI3, AK1, FABP3, YOD1, GLO1, RANBP1, TGM2, PLPBP, OLR1, CEACAM8, CD2AP, AAMDC and QDPR proteins; Figure 3 This is the ROC curve analysis diagram of the early diagnostic value of acute myocardial infarction by the combination of GLRX, CA1, FABP3 and OLR1 proteins; Figure 4 This is the ROC curve analysis of the early diagnostic value of acute myocardial infarction by the combination of GLRX, CA1, FABP3, OLR1 and TNNI3 proteins; Figure 5 This is the ROC curve analysis diagram of the early diagnostic value of acute myocardial infarction by the combination of SOD1, BOLA2_BOLA2B, DDT, TNNI3 and AK1 proteins; Figure 6 This is the ROC curve analysis diagram of the early diagnostic value of acute myocardial infarction by the combination of YOD1, GLO1, RANBP1, TGM2 and PLPBP proteins; Figure 7 This is the ROC curve analysis of the early diagnostic value of acute myocardial infarction by the combination of OLR1, CEACAM8, CD2AP, AAMDC and QDPR proteins; Figure 8 This is the ROC curve analysis diagram of the early diagnostic value of acute myocardial infarction by the combination of GH1 and ACY1 proteins; Figure 9 This is the ROC curve analysis diagram of the early diagnostic value of acute myocardial infarction by the combination of GH1 and CA1 proteins; Figure 10 This is the ROC curve analysis diagram of the early diagnostic value of the combination of GLRX and POMC proteins for acute myocardial infarction. DETAILED DESCRIPTION

[0020] The present invention will be described in further detail below with reference to the accompanying drawings and embodiments.

[0021] Specific Example 1: Screening of myocardial metabolism-related proteins in the early stage of acute myocardial infarction.

[0022] 1. Clinical Data of Volunteers in the Screening Phase: This study recruited volunteers from the Department of Cardiology at the First Affiliated Hospital of Ningbo University. These included 37 patients with acute myocardial infarction confirmed by coronary angiography and 20 healthy controls. Fasting venous blood samples were collected from all participants for testing of general biochemical parameters such as lipids and blood glucose. Clinical data, including gender, age, smoking and alcohol history, and hypertension, were also recorded. A comparison of the clinical data of the first group of patients with acute myocardial infarction and controls used for differential protein screening is shown in Table 1.

[0023] 2. Plasma Extraction and Biochemical Analysis: During clinical sample collection for this study, approximately 6 mL of fasting blood was drawn from patients upon admission. After centrifugation at 3200 rpm for 15 minutes at 4°C, the blood was separated, and the upper plasma layer and middle buffy coat layer were extracted. Glycated hemoglobin, total cholesterol, triglycerides, high-density lipoprotein (HDL), and low-density lipoprotein (LDL) were quantitatively analyzed using an automated biochemical analyzer (Olympus AU2700, Japan). Apolipoprotein A1 (apoA1), apolipoprotein B (apoB), and serum lipid (LPa) concentrations were measured using turbidimetric immunoassays. The results are shown in Table 1.

[0024] Table 1. Clinical Baseline Characteristics of the Acute Myocardial Infarction Group and the Control Group

[0025] As shown in Table 1, the AMI group had significantly higher male proportions, smoking history, HbAIC, low-density lipoprotein cholesterol, apoA1, and LPa levels compared with the control group, and was younger than the control group. There were no statistically significant differences between the two groups in hypertension, diabetes, alcohol consumption, triglycerides, total cholesterol, high-density lipoprotein cholesterol, and apoB (P>0.05).

[0026] 3. Analysis of myocardial metabolism-related proteins in two blood samples using an ultrasensitive multiplex targeted protein detection assay (Olink): Plasma samples from 37 patients with early acute myocardial infarction and 20 control patients were analyzed using the Olink Explore 384 Cardiometabolic and Olink Explore 384 Cardiometabolic II (Olink Proteomics AB, Sweden) according to the manufacturer's guidelines. This technology involves highly specific binding of target proteins to antibody probes labeled with dual oligonucleotides. These proteins are then detected and quantified using a microfluidic real-time PCR instrument (Biomark HD, USA). The final assay readout is displayed as normalized protein expression values, which are then log2-transformed for use in various biological analyses.

[0027] Based on the Olink Explore 384 Cardiometabolic panel analysis, we found that 125 related proteins were differentially expressed between the two groups of patients. Figure 1As shown in Figure A, based on the -log10 (p value) ranking, we marked GLRX, EIF4EBP1, SOD1, AK1, NPDC1, GLO1, CA1, CD2AP, TGM2, and THOP1 as the 10 most significantly differentially expressed proteins. Based on the Olink Explore 384 Cardiometabolic II panel analysis, we found that 117 related proteins were differentially expressed between the two groups of patients, such as Figure 1 As shown in Figure B, based on the -log10 (p-value) ranking, we marked POMC, MYL4, DDT, AAMDC, BOLA2_BOLA2B, FABP3, TALDO1, NIT1, RANBP1, and INPP5D as the 10 most significantly differentially expressed proteins. Table 2 shows the detailed information of the 20 differentially expressed proteins between the acute myocardial infarction group and the control group.

[0028] Table 2. Detailed information on myocardial metabolism-related proteins that were significantly changed between groups

[0029] Note: The difference in protein expression between the acute myocardial infarction group and the control group was calculated using the t-test with a p-value (P<0.05), indicating that there was a significant difference in protein expression.

[0030] 4. Importance ranking of 22 screened myocardial metabolism-related proteins in the early stage of acute myocardial infarction Univariate ROC analysis was performed on the differentially expressed proteins, and the ranking was based on the area under the univariate curve (AUC). As shown in Table 3, GLRX, CA1, EIF4EBP1, MYL4, SOD1, BOLA2_BOLA2B, DDT, TNNI3, AK1, FABP3, YOD1, GLO1, RANBP1, TGM2, PLPBP, OLR1, CEACAM8, CD2AP, AAMDC, and QDPR all had very high diagnostic value for early acute myocardial infarction.

[0031] Table 3. Specific information of the top 23 proteins in ROC analysis AUC

[0032] In summary, we collected blood from clinical patients with acute myocardial infarction and a control group, and used Olink ultra-sensitive multiplex targeted protein detection and analysis technology to detect 768 myocardial metabolism-related proteins in the patients' plasma. A total of 242 myocardial metabolism-related proteins were found to be differentially expressed between the two groups of patients. 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.

[0033] Specific Example 2: Verify the diagnostic value of the myocardial metabolism-related proteins screened in Specific Example 1 for acute myocardial infarction.

[0034] A second validation set of clinical case and control samples was collected, including 31 patients with acute myocardial infarction confirmed by coronary angiography and 20 healthy controls. Using Olink's ultrasensitive multiplex targeted protein detection and analysis technology combined with 10-fold cross-validation, the expression differences of the 23 most diagnostically effective proteins were verified: 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.

[0035] Table 4. Comparison of clinical data between the acute myocardial infarction case group and the control group in the second validation phase

[0036] As shown in Table 4, the AMI group in the validation cohort had significantly higher male prevalence, smoking history, HbAIC, low-density lipoprotein, apoB, apoA1, and LPa levels compared with 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 high-density lipoprotein (HDL) (P>0.05).

[0037] Table 5. Detailed information of verified proteins between acute myocardial infarction patients and control group

[0038] As shown in Table 5, 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 were significantly different 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) using the ROCR package to generate the ROC curve, combined the 23 indicators using Lightgbm analysis, and through ten-fold cross validation, the results were statistically analyzed. Figure 2 The area under the curve (AUC) values ​​for 23 diagnostic combinations are shown to assess classification performance. AUC confidence intervals were calculated using bootstrap sampling. The AUC value was 0.990, indicating very high diagnostic value, with a 95% confidence interval of 0.962 to 1, 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 multi-marker combinations can improve diagnostic performance. Furthermore, multiple protein combinations demonstrated excellent diagnostic performance for the early diagnosis of acute myocardial infarction.

[0039] like Figure 3 As shown in the figure, the combined AUC value 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.

[0040] like Figure 4 As shown in the results, 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.

[0041] like Figure 5 As shown in the figure, the AUC value of the combination of SOD1, BOLA2_BOLA2B, DDT, TNNI3 and AK1 was 0.990, with a 95% confidence interval of 0.966-1, a sensitivity of 0.968 and a specificity of 1.

[0042] like Figure 6As shown in the results, the AUC value of the combination of YOD1, GLO1, RANBP1, TGM2 and PLPBP was 0.950, with a 95% confidence interval of 0.886-0.995, a sensitivity of 0.935 and a specificity of 0.9.

[0043] like Figure 7 As shown, the AUC value of the combination of OLR1, CEACAM8, CD2AP, AAMDC and QDPR was 0.964, with a 95% confidence interval of 0.910-1, a sensitivity of 1 and a specificity of 0.850.

[0044] like Figure 8 As shown, the AUC value of the combination of GH1 and ACY1 was 0.734, with a 95% confidence interval of 0.572-0.884, a sensitivity of 0.935, and a specificity of 0.55.

[0045] like Figure 9 As shown, the AUC value of the combination of GH1 and CA1 was 0.917, with a 95% confidence interval of 0.831 to 0.984, a sensitivity of 0.871, and a specificity of 0.95.

[0046] like Figure 10 As shown in the figure, the AUC value of the combination of GLRX and POMC was 0.952, with a 95% confidence interval of 0.877 to 1, a sensitivity of 0.903, and a specificity of 1.

[0047] All combinations of the above 23 myocardial metabolism-related proteins belong to the technical solutions of the present invention.

[0048] The above description is not intended to limit the present invention, and the present invention is not limited to the above examples. Any changes, modifications, additions or substitutions made by persons of ordinary skill in the art within the spirit and scope of the present invention shall also fall within the scope of protection of the present invention.

Claims

1. A protein marker for assisting in the early diagnosis of acute myocardial infarction, characterized by: The protein marker 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.

2. A protein marker for assisting early diagnosis of acute myocardial infarction according to claim 1, characterized in that: 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.

3. A protein marker for assisting early diagnosis of acute myocardial infarction according to claim 1, characterized in that: The protein markers are a combination of GLRX, CA1, FABP3 and OLR1 proteins, and / or a combination of GLRX, CA1, FABP3, OLR1 and TNNI3 proteins.

4. A protein marker for assisting early diagnosis of acute myocardial infarction according to claim 1, characterized in that: The protein markers are a combination of SOD1, BOLA2_BOLA2B, DDT, TNNI3 and AK1 proteins.

5. The protein marker for assisting in the early diagnosis of acute myocardial infarction according to claim 1, characterized in that: The protein markers are a combination of YOD1, GLO1, RANBP1, TGM2 and PLPBP proteins.

6. A protein marker for assisting early diagnosis of acute myocardial infarction according to claim 1, characterized in that: The protein markers are a combination of OLR1, CEACAM8, CD2AP, AAMDC and QDPR proteins.

7. A protein marker for assisting early diagnosis of acute myocardial infarction according to claim 1, characterized in that: The protein markers are a combination of GH1 and ACY1 proteins, and / or a combination of GH1 and CA1 proteins.

8. The protein marker for assisting in the early diagnosis of acute myocardial infarction according to claim 1, characterized in that: The protein marker is a combination of GLRX and POMC proteins.

9. Use of the relevant protein marker according to any one of claims 1 to 8 in the preparation of an early diagnostic reagent or kit for myocardial infarction.

10. Use of a reagent for detecting the expression level of a related protein marker according to any one of claims 1 to 8 in the preparation of a reagent or kit for early diagnosis of myocardial infarction.

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