Application of ADAMTS-5 as biomarker of calcified aortic valve disease

Abstract

The invention relates to the technical field of molecular markers, in particular to application of ADAMTS-5 as a biomarker of calcified aortic valve diseases. Through comparison of ADAMTS-5 levels in serum of a control group (a patient without CAVD) and serum of a patient in a CAVD group, the ADAMTS-5 level in serum of a CAVD patient is found to be remarkably reduced, and the ADAMTS-5 level in serum of a severe CAVD group is particularly remarkably reduced, so that a biomarker for CAVD diagnosis is developed on the basis, and then ADAMTS-5 is confirmed to be an independent predictive factor of CAVD through verification and is not influenced by levels of blood fat, renal function and the like. In addition, a working characteristic curve of a subject confirms that the under-curve area (AUC) of the ADAMTS-5 in CAVD diagnosis is remarkably improved to 0.799 (95% confidence interval: 0.750-0.848, Plt, 0.001), and the ADAMTS-5 has good sensitivity and specificity.

Description

Technical Field

[0001] This invention relates to the field of molecular biomarker technology, specifically to the application of ADAMTS-5 as a biomarker for calcific aortic valve disease. Background Technology

[0002] Calcific aortic valve disease (CAVD) is a progressive age-related disease with high morbidity and mortality. The main pathophysiological change is the fibrosis and calcification of the aortic valve leaflets, leading to valve hardening and hemodynamic changes that affect cardiac function. Previous views considered CAVD an age-related degenerative disease, a process of valvular tissue degeneration and hardening with age. However, recent basic research indicates that CAVD is an active progressive process involving complex pathological changes such as endothelial damage, inflammatory cell infiltration, extracellular matrix remodeling, and osteoblastic differentiation of valvular interstitial cells. The cells in valvular tissue mainly include valvular endothelial cells, valvular interstitial cells, and valvular progenitor cells. Studies have confirmed that increased calcium salts due to osteoblastic differentiation of valvular interstitial cells may be an important initiating factor in valvular calcification. The clinical symptoms of CAVD vary depending on the severity of the disease. Patients may not experience any obvious symptoms in the early stages, but as the disease progresses, symptoms such as shortness of breath, chest pain, palpitations, syncope, dizziness, fatigue, and edema may appear. These symptoms can gradually worsen as CAVD becomes more severe and affect quality of life. Therefore, early diagnosis and treatment are crucial for CAVD. Summary of the Invention

[0003] To overcome the above problems, this invention provides the application of ADAMTS-5 as a biomarker for calcific aortic valve disease.

[0004] To achieve the above technical objectives, the present invention adopts the following technical solution: A first aspect of the invention provides the use of a biomarker or a substance for detecting said biomarker in any of the following: (a1) To prepare products for the diagnosis or auxiliary diagnosis of calcific aortic valve disease; (a2) Prepare products for screening calcific aortic valve disease; (a3) To prepare products for predicting or assessing the risk of calcific aortic valve disease; (a4) Prepare products for prognostic assessment of calcific aortic valve disease; The biomarker is depolymerized protein-like metalloproteinase containing platelet-reactive protein 5 (ADAMTS-5).

[0005] The amino acids of ADAMTS-5 described in this invention are shown in SEQ ID NO: 1.

[0006] In one or more embodiments, the substance used to detect the biomarker is a reagent for detecting ADAMTS-5 content.

[0007] Preferably, the method for detecting the biomarker includes: enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay, radioimmunoassay, immunoprecipitation assay, Western blotting, high performance liquid chromatography (HPLC), capillary gel electrophoresis, near-infrared spectroscopy, mass spectrometry, immunochemiluminescence assay, colloidal gold immunochromatography, fluorescence immunochromatography, surface plasmon resonance (SPR), immuno-PCR, or biotin-avidin assay.

[0008] In one or more embodiments, the product includes a reagent kit, a protein chip, an immunochromatographic diagnostic strip, a high-throughput sequencing platform, or a biosensor.

[0009] A second aspect of the present invention provides a composition comprising a reagent for detecting ADAMTS-5 content.

[0010] A third aspect of the invention provides the use of the composition described in the second aspect in any of the following: (b1) To prepare products for the diagnosis or auxiliary diagnosis of calcific aortic valve disease; (b2) To prepare products for screening calcific aortic valve disease; (b3) To prepare products for predicting or assessing the risk of calcific aortic valve disease; (b4) Prepare products for prognostic assessment of calcific aortic valve disease.

[0011] A fourth aspect of the present invention provides a kit comprising the composition described in the second aspect.

[0012] A fifth aspect of the invention provides the use of the kit described in the fourth aspect in any of the following: (c1) Prepare products for the diagnosis or auxiliary diagnosis of calcific aortic valve disease; (c2) Prepare products for screening calcific aortic valve disease; (c3) Prepare products for predicting or assessing the risk of calcific aortic valve disease; (c4) Prepare products for prognostic assessment of calcific aortic valve disease.

[0013] In one or more embodiments, the test sample of the kit is plasma.

[0014] A sixth aspect of the invention provides an apparatus for the diagnosis, auxiliary diagnosis, screening, risk prediction, or prognostic assessment of calcific aortic valve disease, comprising a reagent for detecting ADAMTS-5 levels and a computer-readable storage medium storing a computer program that causes a computer to perform the following steps: ADAMTS-5 levels can be used for the diagnosis, auxiliary diagnosis, screening, risk prediction, or prognostic assessment of calcific aortic valve disease.

[0015] The beneficial effects of this invention are as follows: (1) By comparing the serum ADAMTS-5 levels of the control group (patients without CAVD) and the CAVD group, this invention found that the serum ADAMTS-5 level of CAVD patients was significantly reduced, and the reduction was particularly significant in the severe CAVD group. Based on this, a biomarker for the diagnosis of CAVD was developed. Subsequently, it was verified that ADAMTS-5 is an independent predictor of CAVD and is not affected by blood lipids, renal function, etc. Furthermore, receiver operating characteristic (ROC) curves confirmed that the area under the curve (AUC) of ADAMTS-5 in the diagnosis of CAVD significantly increased to 0.799 (95% confidence interval: 0.750 to 0.848, P<0.001); the AUC of ADAMTS-5 in the diagnostic ability of CAVD severity significantly increased to 0.792 (95% confidence interval: 0.736 to 0.848, P<0.001); and the AUC of ADAMTS-5 in the diagnostic ability of severe CAVD significantly increased to 0.823 (95% confidence interval: 0.780 to 0.865, P<0.001), demonstrating that ADAMTS-5 has good sensitivity and specificity as a biomarker for calcific aortic valve disease.

[0016] (2) This invention provides novel biomarkers for the auxiliary diagnosis, screening, risk prediction or prognosis assessment of CAVD, and provides theoretical basis and technical support for the precision diagnosis and targeted intervention of CAVD in the future. It has broad prospects for clinical translation and industrial application potential. Attached Figure Description

[0017] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0018] Figure 1 The values ​​represent the serum ADAMTS-5 levels in the control group, the non-severe CAVD group, and the severe CAVD group; where * indicates P≤0.05, ** indicates P≤0.01, and *** indicates P≤0.001. Figure 2 For smooth curve fitting; where the red solid line represents the probability of CAVD occurrence, and the orange line represents the 95% confidence interval curve; Figure 3 Correlation plot of serum ADAMTS-5 and CAVD among different subgroups; Figure 4 The graph shows the receiver operating characteristic (ROC) analysis results for diagnosing the incidence and severity of CAVD; where A is the ROC curve for diagnosing the incidence of CAVD; B is the ROC curve for diagnosing the severity of CAVD; and C is the ROC curve for diagnosing the incidence of severe CAVD. Model 1: serum ADAMTS-5 level; Model 2: CRP, CR, UA, eGFR, TG, FFA, PLA2, NTproBNP; Model 3: ADAMTS-5 added to Model 2. Detailed Implementation

[0019] It should be noted that the following detailed descriptions are exemplary and intended to provide further illustration of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0020] It should be noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of exemplary embodiments according to the invention. As used herein, the singular form is intended to include the plural form as well, unless the context clearly indicates otherwise. Furthermore, it should be understood that when the terms "comprising" and / or "including" are used in this specification, they indicate the presence of features, steps, operations, devices, components, and / or combinations thereof.

[0021] Clinically, the diagnosis of CAVD includes physical examination, echocardiography, and cardiac CT scans. However, physical examination findings are not unique to CAVD; many heart diseases present with similar signs, making the results highly sensitive, with mild to moderate lesions showing almost no positive signs. It is also highly subjective and relies heavily on the physician's experience. Echocardiography image quality is affected by the patient's body size; measurements may vary between different operators. During cardiac CT scans, both patients and medical staff cannot avoid radiation exposure; contrast agents are contraindicated in some patients; and the lesion may be underestimated in valves that are only thickened and fibrotic but not yet calcified.

[0022] This invention compares the serum ADAMTS-5 levels of patients in the control group (non-CAVD patients) with those in the CAVD group and finds that the serum ADAMTS-5 level of CAVD patients is significantly reduced, and the reduction is particularly significant in the severe CAVD group. Based on this, a biomarker for the diagnosis of CAVD was developed. Subsequently, it was verified that ADAMTS-5 is an independent predictor of CAVD and is not affected by blood lipids, renal function, or other levels. Furthermore, the area under the curve (AUC) of ADAMTS-5 in the diagnosis of CAVD was significantly increased to 0.799 (95% confidence interval: 0.750 to 0.848, P<0.001) by the receiver operating characteristic (ROC) curve; the AUC of ADAMTS-5 in the diagnostic ability of CAVD severity was significantly increased to 0.792 (95% confidence interval: 0.736 to 0.848, P<0.001) by the ROC curve; and the AUC of ADAMTS-5 in the diagnostic ability of severe CAVD was significantly increased to 0.823 (95% confidence interval: 0.780 to 0.865, P<0.001) by the ROC curve. These results indicate that serum ADAMTS-5 has significant independent and additive diagnostic value for both the incidence and severity of CAVD and the incidence of severe CAVD. Therefore, the level of ADAMTS-5 in serum can be used for the diagnosis, auxiliary diagnosis, screening, risk prediction, or prognostic assessment of CAVD.

[0023] Terminology Explanation: AR - Aortic regurgitation; BAV - Bicuspid aortic valve; SBP - Systolic blood pressure; DBP - Diastolic blood pressure; BMI - Body mass index; CAD - Coronary atherosclerotic heart disease; ACEI - Angiotensin-converting enzyme inhibitor; ARB - Angiotensin receptor blocker; ARNI - Angiotensin receptor neprilysin inhibitor; CRP - Cranial reactive protein; ALT - Alanine aminotransferase; AST - Aspartate aminotransferase; UA - Uric acid; eGFR - Estimated glomerular filtration rate; FBG - Fasting blood glucose; HCY - Homocysteine; TG - Triglycerides; TC - Total cholesterol; HDL-C - High-density lipoprotein cholesterol; LDL-C - Low-density lipoprotein cholesterol; FFA - Free fatty acids; PLA2 - Phospholipase a2; LP(a) - Lipoprotein(a); NTproBNP - Pro-brain natriuretic peptide; Creatinine - Creatinine; r s - Spearman's rho coefficient; CI - confidence interval; AUC - area under the curve; NRI - net weight classification improvement index; IDI - comprehensive discrimination improvement index; ESR - erythrocyte sedimentation rate; LVEF - left ventricular ejection fraction.

[0024] To enable those skilled in the art to better understand the technical solution of the present invention, the technical solution of the present invention will be described in detail below with reference to specific embodiments.

[0025] Example 1 A total of 361 participants were included (mean age 69.96 ± 7.61 years, 213 males, 59.0%), of whom 125 (34.6%) were in the control group, 108 (29.9%) in the non-severe CAVD group, and 128 (35.5%) in the severe CAVD group. There were no significant differences among the control, non-severe CAVD, and severe CAVD groups in terms of age, sex, mean blood pressure, smoking history, hypertension, diabetes, and coronary heart disease.

[0026] The clinical characteristics of the three groups of subjects are shown in Table 1.

[0027] Table 1 Clinical characteristics of the subjects

[0028] As shown in Table 1, the detection rate of BAV increased significantly with the increase of CAVD severity (0.8% in the control group vs. 8.3% in the non-severe CAVD group vs. 19.5% in the severe CAVD group, P<0.001), and the detection rate of AR was significantly increased in the case group (P<0.001). The SBP level in the non-severe CAVD group was higher than that in the other two groups.

[0029] Table 1 also shows that the levels of CRP, ESR, HCY, FFA, and LP(a) in patients in the non-severe CAVD group and the severe CAVD group were significantly higher than those in the control group, while the red blood cell count was lower in the latter group. Patients in the severe CAVD group exhibited more severe myocardial damage, cardiac workload, and renal insufficiency, with a stepwise significant increase in NT-proBNP levels (P<0.001), significantly elevated UA levels, and significantly decreased eGFR (P<0.001). Furthermore, there were significant differences in TG, creatinine, and PLA2 levels among the three groups. No significant differences were observed in other laboratory indicators.

[0030] Furthermore, regarding echocardiographic parameters, with increasing severity of CAVD, the left atrial and left ventricular diameters significantly increased (P<0.001), while LVEF significantly decreased in the severe group (P = 0.002). As a grouping criterion, the aortic valve orifice area significantly decreased with increasing disease severity, while the maximum flow velocity, maximum transvalvular pressure gradient, and mean transvalvular pressure gradient significantly increased (all P<0.001) (Table 1).

[0031] Figure 1The levels of ADAMTS-5 in the serum of patients in the control group, non-severe CAVD group, and severe CAVD group were measured from... Figure 1 As can be seen, compared with the control group, the serum ADAMTS-5 level of CAVD patients was significantly reduced, and the reduction was particularly significant in the severe CAVD group (control group: 248.63 ± 56.45 μg / mL; non-severe CAVD group: 232.68 ± 44.50 μg / mL; severe CAVD group: 196.23 ± 40.04 μg / mL; P<0.001).

[0032] Example 2 Correlation between serum ADAMTS-5 levels and CAVD: To further explore the potential factors influencing CAVD, univariate and multivariate ordered logistic regression analyses were performed. The results are shown in Table 2.

[0033] Table 2. Results of univariate and multivariate logistic regression analyses of the independent factors of CAVD.

[0034] The univariate analysis results in Table 2 showed that, regarding clinical characteristics and medication history, BAV (OR=6.067, P<0.001), diuretics (OR=5.028, P<0.001), hypoglycemic agents (OR=1.517, P=0.034), and AR (OR=0.072, P<0.001) were significantly associated with disease severity. Regarding laboratory indicators, elevated levels of CRP (P=0.008), neutrophil percentage (P=0.040), Creatinine (P=0.008), UA (P<0.001), FFA (P=0.003), NTproBNP (P<0.001), eGFR (P<0.001), TG (P=0.003), and ADAMTS-5 (P<0.001) were significantly associated with disease severity.

[0035] To control for confounding factors, a multivariate analysis model ( Figure 2 The study included age, male sex, SBP, BMI, CRP, eGFR, LDL-C, TG, FFA, PLA2, NT-proBNP, BAV, and ADAMTS-5. Given the high collinearity between TC and LDL-C (variance inflation factor (VIF): 8.26), LDL-C was included in the model based on clinical significance.

[0036] Multivariate analysis showed that BAV (OR=6.780, 95% CI: 2.779-16.540, P<0.001), SBP (OR=1.012, 95% CI: 1.000-1.024, P=0.043), CRP (OR=1.010, 95% CI: 1.002-1.018, P=0.021), and NT-proBNP (P=0.024) were independent risk factors for increased severity of CAVD. ADAMTS-5 level (OR=0.983, 95% CI: 0.977-0.989, P<0.001) was an independent protective predictor of CAVD severity. Furthermore, regarding renal function, eGFR showed a marginally significant negative correlation with CAVD severity (OR=0.986, 95% CI: 0.973-1.000, P=0.054), suggesting that impaired renal function (decreased eGFR) may be a potential factor contributing to disease progression. TG showed a marginally significant protective trend in the multivariate model (OR=0.715, P=0.055), but did not reach statistical significance; however, this does not imply a protective effect from high triglycerides, but is more likely attributable to the effects of lipid-lowering therapy.

[0037] Furthermore, the relationship between serum ADAMTS-5 and CAVD was assessed using smooth curve fitting, and the results showed a non-linear correlation ( Figure 2 The results of the threshold effect analysis are detailed in Table 3. When the serum ADAMTS-5 level was greater than 179.81 μg / mL, the risk of CAVD decreased by 1.6% for every 1 unit increase (P<0.05).

[0038] Table 3 Results of the Logistic Regression Model

[0039] To further validate the robustness of the association between ADAMTS-5 and CAVD risk, subgroup analyses were performed based on TG, LDL-C, LP(a), PLA2, eGFR, and CRP levels. This analysis compared normally distributed variables grouped by their means and skewed distributed variables grouped by their medians. Results are as follows: Figure 3As shown, ADAMTS-5 was significantly positively correlated with CAVD risk in all pre-specified subgroups (P < 0.05 for all subgroups), indicating that the association between elevated ADAMTS-5 levels and reduced CAVD risk remained largely consistent across different subgroups. No significant interactions were observed between serum ADAMTS-5 and TG, LDL-C, LP(a), PLA2, eGFR, or CRP (P > 0.05). This suggests that serum ADAMTS-5 remains an independent predictor of CAVD in different subgroups, unaffected by levels of blood lipids, renal function, etc.

[0040] Example 3 Independent and additional diagnostic properties of serum ADAMTS-5: Three diagnostic models were constructed: Model 1 included serum ADAMTS-5 levels; Model 2 selected variables with P-values ​​<0.05 in univariate ordered analysis (including CRP, CR, UA, eGFR, TG, FFA, PLA2, and NTproBNP, excluding ADAMTS-5); Model 3 integrated Model 1 and Model 2, i.e., adding ADAMTS-5 to Model 2. To evaluate the diagnostic efficacy of serum ADAMTS-5 levels for CAVD, enrolled patients were divided into a control group and a CAVD group (including non-severe and severe CAVD patients); the AUC of Model 1 (serum ADAMTS-5) was 0.697 (95% confidence interval: 0.639 - 0.756, P <0.001), and the optimal cutoff value was determined to be 230.25 μg / mL, with a sensitivity of 69.07% and a specificity of 64.80%. Compared to Model 2 (AUC = 0.773, 95% confidence interval: 0.723 to 0.824, P < 0.001), when serum ADAMTS-5 was included in Model 3 ( Figure 4 When A), the AUC significantly increased to 0.799 (95% confidence interval: 0.750 to 0.848, P<0.001). Subsequently, patients with non-severe and severe CAVD were included to assess the diagnostic ability of serum ADAMTS-5 levels for CAVD severity; the AUC of Model 1 was 0.733 (95% confidence interval: 0.668 to 0.798, P<0.001); the optimal cutoff value for serum ADAMTS-5 was determined to be 224.54 μg / mL, with a sensitivity of 82.03% and a specificity of 60.19% (95% confidence interval: 56.41%-79.06%); compared with Model 2 (AUC = 0.704, 95% confidence interval: 0.639 to 0.770, P<0.001), the AUC of Model 3 significantly increased to 0.792 (95% confidence interval: 0.736 to 0.848, P<0.001) after the addition of serum ADAMTS-5. Figure 4(B) Finally, controls, patients with mild to moderate and severe CAVD were included to assess the diagnostic ability of serum ADAMTS-5 levels for severe CAVD; the AUC of Model 1 (serum ADAMTS-5) was 0.761 (95% confidence interval: 0.711 to 0.811, P<0.001), and the optimal cutoff value was determined to be 230.25 μg / mL, with a sensitivity of 82.03% and a specificity of 64.81%; compared with Model 2 (AUC = 0.755, 95% confidence interval: 0.704 to 0.807, P<0.001), when serum ADAMTS-5 was included in Model 3 ( Figure 4 When the AUC was in the middle (C), it significantly increased to 0.823 (95% confidence interval: 0.780 to 0.865, P<0.001).

[0041] Furthermore, the discrimination and reclassification effects of the serum-included / serum-without ADAMTS-5 model in diagnosing the incidence and severity of CAVD were evaluated. Results showed that the introduction of serum ADAMTS-5 significantly improved AUC, NRI, and IDI (Table 4). These findings indicate that serum ADAMTS-5 has significant independent and additive diagnostic value for both the incidence and severity of CAVD and the incidence of severe CAVD.

[0042] Table 4. Prognostic potential of serum ADAMTS-5 increments

[0043] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. The use of a biomarker or a substance for detecting said biomarker in any of the following: (a1) To prepare products for the diagnosis or auxiliary diagnosis of calcific aortic valve disease; (a2) Prepare products for screening calcific aortic valve disease; (a3) To prepare products for predicting or assessing the risk of calcific aortic valve disease; (a4) Prepare products for prognostic assessment of calcific aortic valve disease; in, The biomarker is ADAMTS-5.

2. The application as described in claim 1, characterized in that, The substance used to detect the biomarker is a reagent for detecting ADAMTS-5 content.

3. The application as described in claim 2, characterized in that, Methods for detecting the biomarkers include: enzyme-linked immunosorbent assay (ELISA), immunofluorescence assay, radioimmunoassay, immunoprecipitation assay, Western blotting, high performance liquid chromatography (HPLC), capillary gel electrophoresis, near-infrared spectroscopy, mass spectrometry, immunochemiluminescence assay, colloidal gold immunochromatography, fluorescence immunochromatography, surface plasmon resonance (SPR), immuno-PCR, or biotin-avidin assay.

4. The application as described in claim 1, characterized in that, The products include reagent kits, protein chips, immunochromatographic diagnostic strips, high-throughput sequencing platforms, or biosensors.

5. A composition, characterized in that, This includes reagents for detecting ADAMTS-5 levels.

6. The use of the composition of claim 5 in any of the following: (b1) To prepare products for the diagnosis or auxiliary diagnosis of calcific aortic valve disease; (b2) To prepare products for screening calcific aortic valve disease; (b3) To prepare products for predicting or assessing the risk of calcific aortic valve disease; (b4) Prepare products for prognostic assessment of calcific aortic valve disease.

7. A reagent kit, characterized in that, Includes the composition of claim 5.

8. The use of the kit according to claim 7 in any of the following: (c1) Prepare products for the diagnosis or auxiliary diagnosis of calcific aortic valve disease; (c2) Prepare products for screening calcific aortic valve disease; (c3) Prepare products for predicting or assessing the risk of calcific aortic valve disease; (c4) Prepare products for prognostic assessment of calcific aortic valve disease.

9. The application as described in claim 8, characterized in that, The test sample for this kit is plasma.

10. A device for the diagnosis, auxiliary diagnosis, screening, risk prediction, or prognostic assessment of calcific aortic valve disease, characterized in that, Includes reagents for detecting ADAMTS-5 levels and a computer-readable storage medium storing a computer program that causes a computer to perform the following steps: ADAMTS-5 levels can be used for the diagnosis, auxiliary diagnosis, screening, risk prediction, or prognostic assessment of calcific aortic valve disease.

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