Biomarkers for dilated cardiomyopathy and their applications

Abstract

This invention discloses the application of Foxc2 as a biomarker for dilated cardiomyopathy and related diseases. Foxc2 as a biomarker has high sensitivity and specificity in the diagnosis and prediction of dilated cardiomyopathy and related diseases, providing a new detection method and scheme for the diagnosis and prediction of dilated cardiomyopathy and related diseases.

Description

Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to a biomarker for dilated cardiomyopathy and its application. Background Technology

[0002] The "Summary of the 2022 Report on Cardiovascular Health and Disease in China" published in the Chinese Journal of Circulation in 2023 indicates that the number of people with cardiovascular diseases in my country has reached 330 million, with cardiovascular disease being the leading cause of death, and its prevalence is increasing year by year, with the age of onset becoming younger. Dilated cardiomyopathy (DCM) is a type of cardiomyopathy characterized by left ventricular or biventricular enlargement accompanied by systolic dysfunction. When the volume of the left ventricle increases and its sphericity worsens, the movement of the mitral valve leaflets is restricted, the valve annulus dilates, and ultimately leads to leaflet junction defects and "functional" regurgitation (FMR). Despite advances in medical and surgical treatments, functional FMR remains associated with a high incidence and mortality rate of DCM and DCM-related heart failure. Therefore, we urgently need to identify subjects with DCM and FMR early, intervene in a timely manner, and reduce the incidence of in-hospital and long-term adverse events.

[0003] In recent years, there has been an increasing number of studies on biomarkers for the diagnosis of DCM, mainly including genetic biomarkers and immune biomarkers, such as anticardiomyocyte antibody (ANT), myocardial infarction gene (TTN), Bcl2-associated immortalized gene 3 (BAG3), brain natriuretic peptide (BNP), and troponin I / T (cTnI, cTnT). These genes have a certain predictive ability with cardiomyopathy phenotypes, but lack long-term prognostic monitoring, sensitivity and specificity. Summary of the Invention

[0004] In order to solve the technical problems existing in the prior art, the present invention provides the following technical solution:

[0005] The first aspect of this invention provides the use of a substance for detecting biomarkers in a test sample in the preparation of a product, wherein the biomarker includes Foxc2; and the product includes any one of the following:

[0006] Products for diagnosing or assisting in the diagnosis of dilated cardiomyopathy or related diseases;

[0007] Products that predict or assist in the prediction of dilated cardiomyopathy or related diseases.

[0008] Forkhead box C2 (Foxc2), also known as mesenchyme forkhead1 (MHF1), is a transcription factor belonging to the "C" subfamily of the forkhead / winged helix family.

[0009] In some implementations, the sample includes at least one of blood, plasma, and serum.

[0010] In some implementations, the associated diseases include dilated cardiomyopathy with mitral regurgitation.

[0011] In some implementations, the substance used to detect biomarkers in the sample includes a substance that detects the expression level of a biomarker gene or a substance that detects the level of a biomarker protein.

[0012] In some implementations, the substance used to detect biomarkers in the sample comprises substances selected from one or more detection techniques or methods chosen from the group consisting of: PCR reaction, RT-PCR derivatization reaction, 3SR amplification, LCR, SDA, NASBA, TMA, SYBR Green, TaqMan probe, molecular beacon, two-hybrid probe, composite probe, ISH, microarray, Southern blotting, Northern blotting, multiple analyte assay, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, immunofluorescence assay, enzyme immunoassay, immunoprecipitation assay, chemiluminescence assay, immunohistochemistry assay, dot blot assay, or narrow-line blot assay.

[0013] In some implementations, the substance used to detect biomarkers in the sample is selected from one or more of the following: substances specific to biomarkers, probes specific to biomarkers, gene chips, and PCR primers.

[0014] In some embodiments, the substance specific to the biomarker includes any one of antibodies that specifically bind to the biomarker, ligand proteins or peptides that specifically bind to the biomarker, and non-protein compounds that specifically recognize the biomarker.

[0015] In some embodiments, the antibody comprises at least one of polyclonal antibody, monoclonal antibody, single-chain antibody, functional antibody fragment, antibody Fab region, nanobody, chimeric antibody, and multispecific antibody.

[0016] In some implementations, the product comprises at least one of reagents, kits, test strips, chips, and systems.

[0017] A second aspect of the present invention provides a product comprising a substance for detecting Foxc2;

[0018] The products include those for diagnosing or assisting in diagnosing whether a sample to be tested is a sample of dilated cardiomyopathy or a related disease, those for diagnosing or assisting in diagnosing whether a person to be tested is a patient with dilated cardiomyopathy or a related disease, and those for predicting or assisting in predicting whether a sample to be tested is a sample of dilated cardiomyopathy or a related disease, or those for predicting or assisting in predicting whether a person to be tested is a patient with dilated cardiomyopathy or a related disease.

[0019] In some implementations, the associated diseases include dilated cardiomyopathy with mitral regurgitation.

[0020] In some embodiments, the substance for detecting Foxc2 includes a substance for detecting the expression level of the Foxc2 gene or a substance for detecting the level of the Foxc2 protein.

[0021] In some embodiments, the substance used to detect Foxc2 comprises substances selected from one or more detection techniques or methods chosen from the group consisting of: PCR reaction, RT-PCR derivatization reaction, 3SR amplification, LCR, SDA, NASBA, TMA, SYBR Green, TaqMan probe, molecular beacon, two-hybrid probe, composite probe, ISH, microarray, Southern blotting, Northern blotting, multiple analyte assay, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, immunofluorescence assay, enzyme immunoassay, immunoprecipitation assay, chemiluminescence assay, immunohistochemistry assay, dot blot assay, or narrow-line blot assay.

[0022] In some embodiments, the substance used to detect Foxc2 is selected from one or more of the following: substances specific to Foxc2, Foxc2-specific probes, gene chips, and PCR primers.

[0023] In some embodiments, the substance specific to Foxc2 comprises any one of an antibody that specifically binds to Foxc2, a ligand protein or peptide that specifically binds to Foxc2, and a non-protein compound that specifically recognizes Foxc2.

[0024] In some embodiments, the antibody comprises at least one of polyclonal antibody, monoclonal antibody, single-chain antibody, functional antibody fragment, antibody Fab region, nanobody, chimeric antibody, and multispecific antibody.

[0025] In some implementations, the subject of the test includes humans or non-human mammals.

[0026] In some implementations, the product comprises at least one of reagents, kits, test strips, chips, and systems.

[0027] In some implementations, the kit includes a gene detection kit and a protein detection kit.

[0028] In some implementations, the gene detection kit includes primers, reagents, or chips for detecting the transcriptional level of the Foxc2 gene.

[0029] In some implementations, the protein detection kit includes reagents, a chip, antibodies, and ligands for detecting the protein level of Foxc2 expression.

[0030] A third aspect of the present invention provides the use of biomarkers in constructing computer models for the diagnosis or assistance in the diagnosis, prediction or assistance in the prediction of dilated cardiomyopathy or related diseases, said biomarkers including Foxc2.

[0031] In some implementations, the associated diseases include dilated cardiomyopathy with mitral regurgitation.

[0032] A fourth aspect of the present invention provides the use of biomarkers in screening candidate drugs for the treatment of dilated cardiomyopathy or related diseases, said biomarkers including Foxc2.

[0033] In some implementations, the associated diseases include dilated cardiomyopathy with mitral regurgitation.

[0034] In some implementations, screening candidate drugs for the treatment of dilated cardiomyopathy or related diseases includes administering the candidate drug to animal models of dilated cardiomyopathy or related diseases and confirming whether the candidate drug can be used to treat dilated cardiomyopathy or related diseases by detecting the expression level of Foxc2 in the plasma of the animal models.

[0035] The fifth aspect of the present invention provides an early medical auxiliary diagnostic system for dilated cardiomyopathy or related diseases, the diagnostic system including a result determination module for comparing the processed value of a biomarker obtained by detecting the gene or protein expression level of Foxc2 with a set value.

[0036] In some implementations, the diagnostic system includes a processing module for detecting and processing the expression levels of biomarker-related genes or proteins in a sample to obtain processed values.

[0037] In some implementations, the diagnostic system includes an output module for outputting the obtained diagnostic results.

[0038] In some implementations, the associated diseases include dilated cardiomyopathy with mitral regurgitation.

[0039] A sixth aspect of the present invention provides a scoring device for assessing the risk of a subject having dilated cardiomyopathy or a related disease, the scoring device comprising the following units:

[0040] Detection unit: Detects the expression level of genes or proteins such as Foxc2 in a sample;

[0041] Analysis Unit: The gene or protein expression level of the detected biomarkers is used as an input variable and input into the computer model of the third aspect for analysis;

[0042] Scoring unit: Outputs the risk value of the subject for dilated cardiomyopathy or related diseases.

[0043] In some implementations, the associated diseases include dilated cardiomyopathy with mitral regurgitation.

[0044] The seventh aspect of this invention provides a method for diagnosing and predicting dilated cardiomyopathy or related diseases, comprising the following steps:

[0045] 1) Collect samples from the object to be tested;

[0046] 2) Detect the expression level of Foxc2 in the sample;

[0047] 3) Based on the comparison of expression level results with the expression level of healthy controls, it is determined whether the subject has dilated cardiomyopathy or related diseases or the risk of having dilated cardiomyopathy or related diseases.

[0048] In some implementations, the associated diseases include dilated cardiomyopathy with mitral regurgitation.

[0049] In some implementations, step 2) of detecting the expression level of Foxc2 in the sample includes detecting the expression level of the Foxc2 gene in the sample or detecting the level of the Foxc2 protein in the sample.

[0050] In some implementations, the detection of Foxc2 expression level in the sample in step 2) includes detection by PCR reaction, RT-PCR derivatization, 3SR amplification, LCR, SDA, NASBA, TMA, SYBR Green, TaqMan probe, molecular beacon, two-hybrid probe, composite probe, ISH, microarray, Southern blotting, Northern blotting, multiple analyte assay, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, immunofluorescence assay, enzyme immunoassay, immunoprecipitation assay, chemiluminescence assay, immunohistochemistry assay, dot blot assay, or narrow-line blot assay.

[0051] In some implementations, detecting the Foxc2 gene expression level in a sample also includes normalizing the data using an internal reference gene.

[0052] In some implementations, the internal reference gene includes U6.

[0053] The beneficial effects of this invention are: This invention provides the application of Foxc2 as a biomarker for dilated cardiomyopathy and dilated cardiomyopathy complicated with mitral regurgitation. Foxc2 as a biomarker has high sensitivity and specificity in the diagnosis and prediction of dilated cardiomyopathy and dilated cardiomyopathy complicated with mitral regurgitation, providing a new detection method and scheme for the diagnosis and prediction of dilated cardiomyopathy and dilated cardiomyopathy complicated with mitral regurgitation. Attached Figure Description

[0054] Figure 1 This study investigated the expression of Foxc2 in the plasma of control group and DCM subjects. (A) qRT-PCR was used to detect the expression of Foxc2 mRNA in plasma. (B) Western blot was used to detect the protein expression levels of Foxc2 and transferrin in plasma. (C) Gray-scale quantitative statistical analysis was used to determine the protein expression level of Foxc2. ***P<0.01, ****P<0.001.

[0055] Figure 2 ROC curve plot showing the predictive value of Foxc2 for the occurrence of DCM.

[0056] Figure 3 Foxc2 was used as a risk factor for predicting DCM and DCM with FMR. (A) qRT-PCR was used to detect the expression of Foxc2 mRNA in the plasma of subjects with DCM and DCM with FMR. (B) The predictive value of Foxc2 for DCM with FMR - ROC curve. ****P<0.001.

[0057] Figure 4 The predictive value of Foxc2 for DCM combined with FMR disease - ROC curve. Detailed Implementation

[0058] The DCM biomarkers described in this invention are discovered using molecular biology techniques (such as Western blot and RT-qPCR) to analyze plasma samples. Generally, plasma samples are derived from biological samples (plasma) from one or more human subjects clinically diagnosed with DCM, as well as from other human subjects without DCM (control groups with DCM). Biological samples from human subjects with DCM are simultaneously analyzed using relevant molecular biology assays along with those from other human subjects without DCM. Molecules differentially expressed in biological samples from subjects with DCM compared to non-DCM subjects (control subjects with dilated disease), including those differing at statistically significant levels, are identified as biomarkers to distinguish the control group.

[0059] Identifying (or assisting in) the diagnosis of DCM using biomarkers involves identifying a subject exhibiting one or more symptoms consistent with the presence of DCM, including making a preliminary diagnosis of subjects not previously identified as having DCM and diagnosing recurrence of the disease in subjects previously diagnosed with DCM. Methods for diagnosing (or assisting in) whether a subject has DCM include: (1) comparing the level of the biomarker in the sample to a DCM-positive and / or DCM-negative reference level for the biomarker to diagnose (or assist in) whether the subject has DCM; and (2) diagnosing (or assisting in) whether the subject has DCM based on relevant imaging data and biological indicators. Biomarkers used include B-type natriuretic peptide (BNP) and cardiac troponin I (cTnI). Imaging-related biological indicators include cardiac enlargement, left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), and left ventricular end-diastolic diameter (LVEDd). When such a method is used to aid in the diagnosis of DCM, the results of the method can be used in conjunction with other methods (or their results) that can be used clinically to determine whether a subject has DCM.

[0060] The present invention will be further described in detail below through specific embodiments. Unless otherwise specified, the raw materials, reagents, or apparatus used in the embodiments can be obtained from conventional commercial sources or by existing technical methods. Unless otherwise specified, the experimental or testing methods are conventional methods in the art.

[0061] Example 1

[0062] I. Subject Group

[0063] This study collected clinical cases from 92 participants, who were grouped by gender (male / female) and disease type (non-dilated cardiomyopathy (control) / dilated cardiomyopathy (DCM) / dilated cardiomyopathy with mitral regurgitation (DCM with FMR)). Participants were hospital-acquired patients. Inclusion criteria for DCM participants: meeting the diagnostic criteria for DCM as outlined in the "Chinese Guidelines for the Diagnosis and Treatment of Dilated Cardiomyopathy"; age over 18 years. Participants with incomplete three-dimensional echocardiographic findings were excluded. A total of 53 participants were selected for the DCM group and 39 for the non-DCM group. All participants signed informed consent forms.

[0064] Table 1 Basic Information of Cases

[0065]

[0066] II. Detection and Statistical Analysis of Physiological and Biochemical Indicators

[0067] Physiological, biochemical, and echocardiographic parameters of the subjects were collected through subject case studies: B-type natriuretic peptide (BNP), cardiac troponin I (cTnI), left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), and left ventricular end-diastolic diameter (LVEDd).

[0068] Statistical analysis was performed between the non-valvular heart disease group and the control group using physiological and biochemical indicators, including B-type natriuretic peptide (BNP), cardiac troponin I (cTnI), left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), and left ventricular end-diastolic diameter (LVEDd). All data are expressed as mean ± standard deviation (x±s), and independent samples t-tests were used to compare the means of two samples.

[0069] The results showed that, compared with the control group, the EF and FS indices in the DCM group were significantly downregulated, while the BNP, troponin I, and LVDd indices were significantly upregulated, which all indicate that the clinical diagnostic criteria for DCM are met (refer to the "Guidelines for the Diagnosis and Treatment of Dilated Cardiomyopathy in China").

[0070] Table 2 Comparative analysis of physiological and biochemical indicators between DCM and the control group

[0071]

[0072] Statistical analysis was performed between the DCM group and the DCM combined with FMR group using physiological and biochemical indicators, including B-type natriuretic peptide (BNP), cardiac troponin I (cTnI), left ventricular ejection fraction (LVEF), left ventricular fractional shortening (LVFS), and left ventricular end-diastolic diameter (LVEDd). All data are expressed as (x±s), and independent samples t-tests were used to compare the means of two samples.

[0073] The results showed that, compared with the DCM group, there were no significant differences in EF, FS, BNP, troponin I, and LVDd in the DCM combined with FMR group.

[0074] Table 3 Comparative analysis of physiological and biochemical indicators between DCM and DCM combined FMR

[0075]

[0076] III. Extraction of total RNA from plasma

[0077] (1) Collect 1-2 mL of fresh blood from the subject and place it in an anticoagulant tube (containing EDTA or heparin sodium), then invert and mix well;

[0078] (2) Centrifuge at 3000 rpm for 10 minutes at low temperature (4℃) and collect the upper light yellow plasma layer;

[0079] (3) Preparation for plasma mRNA extraction: Whole blood / plasma / serum total RNA extraction kit (Xinhai Gene), catalog number: B0901; Self-prepared reagents: isopropanol, chloroform;

[0080] (4) Take 250 μL of the above plasma and add it to 750 μL of TRIzol LS reagent. Then immediately mix by vigorously inverting the wrist and let it stand for 5 minutes.

[0081] (5) Add 0.2 mL of chloroform to the above solution, shake vigorously with your wrist for 15 seconds, and let stand at room temperature for 2 minutes;

[0082] (6) Centrifuge at 13,000 rpm for 5 minutes at room temperature, and transfer 500 μL of the supernatant to the adsorption column core;

[0083] (7) Add 300 μL of isopropanol to the solution in the adsorption column core, cover the adsorption column tube, and mix up and down 2-3 times.

[0084] (8) Centrifuge at 13,000 rpm for 15 seconds at room temperature and discard the waste liquid;

[0085] (9) Add 500 μL Washing Buffer to the adsorption column and centrifuge at 13,000 rpm for 15 seconds at room temperature;

[0086] (10) Repeat step (9) once;

[0087] (11) Place the adsorption column back into the centrifuge and centrifuge at 13,000 rpm for 2 minutes;

[0088] (12) Place the adsorption column into a 1.5 mL collection tube and add 30 μL of Nuclease Free H2O to the core of the adsorption column;

[0089] (13) Let stand at room temperature for 2 minutes, centrifuge at 13000 rpm for 1 minute, and the elution buffer is the RNA product;

[0090] (14) The concentration and purity of total RNA in plasma samples were determined by an ultra-micro spectrophotometer.

[0091] IV. Detection of Foxc2 mRNA expression level in the plasma of subjects

[0092] 1. Reverse transcription synthesizes the first strand of cDNA into mRNA.

[0093] (1) Prepare the reagent kit: PrimeScript TM RT Master Mix (Perfect Real Time), Product No.: RR036A;

[0094] (2) Prepare the RT reaction solution according to the following composition (the reaction solution is prepared on ice):

[0095] Table 4 Reverse transcription reaction system

[0096]

[0097] (3) Mix gently and proceed with the reverse transcription reaction as follows:

[0098] Table 5 Reverse Transcription Procedure

[0099]

[0100] 2. Real-time quantitative PCR

[0101] (1) Foxc2 and U6 primers were designed using online primer design software. The primers were then synthesized by Sangon Biotech Co., Ltd. The specific primer sequences are as follows:

[0102] Foxc2 forward primer: 5'-CCTCCTGGTATCTCAACCACA-3' (SEQ ID No. 1);

[0103] Foxc2 reverse primer: 5'-GAGGGTCGAGTTCTCAATCCC-3' (SEQ ID No. 2);

[0104] U6 forward primer: 5'-GGAACGATACAGAGAAGATTAGC-3' (SEQ ID No. 3);

[0105] U6 reverse primer: 5'-TGGAACGCTTCACGAATTTGCG-3' (SEQ ID No. 4).

[0106] (2) Prepare the PCR reaction solution according to the following composition (the reaction solution should be prepared on ice):

[0107] Table 6 PCR Reaction System

[0108]

[0109] (3) Perform amplification and analysis according to the following two-step PCR reaction procedure:

[0110] Table 7 PCR reaction procedure

[0111]

[0112] The results showed that, compared with the control group, the mRNA expression level of Foxc2 in the plasma of subjects in the DCM group was significantly decreased. Figure 1 (A)

[0113] V. Detection of Foxc2 protein expression level in the plasma of subjects

[0114] (1) Prepare the BCA protein quantification kit: Pierce TM BCA Protein Assay Kits (ThermoFisher), catalog number: 23227;

[0115] (2) Prepare BCA working solution, Reagent A:Reagent B = 50:1 (volume ratio);

[0116] (3) Plotting the standard curve: Add 200 μL of BCA working solution and standard (the concentration gradient of the standard is: 0, 0.125, 0.25, 0.5, 1, 2 mg / ml) to a 96-well plate, mix well and incubate in a 37℃ incubator for 30 minutes. Detect the absorbance at a wavelength of 562 nm using an ELISA reader and plot the standard curve of absorbance versus protein concentration.

[0117] (4) Determine the concentration of the test sample: Add 200 μL of BCA working solution and diluted test sample (the dilution factor is generally 10) to a 96-well plate, mix well and incubate in a 37℃ incubator for 30 minutes. Detect the absorbance at 562 nm wavelength using an ELISA reader and calculate the concentration of the test sample based on the above standard curve.

[0118] (5) Prepare protein samples: Adjust the loading volume according to the protein concentration determined by the BCA method, unify the loading volume with 6x Loading Buffer and ddH2O, mix well, bathe in a 100℃ metal bath for 5 minutes, centrifuge briefly at low speed, dispense and store at -20℃ for later use.

[0119] (6) Prepare SDS-PAGE gel: Prepare according to the instructions of the PAGE gel rapid preparation kit (Yamei Biotechnology, catalog number: PG112);

[0120] (7) Electrophoresis: Slowly add the prepared protein sample to the gel loading well, add Running Buffer (1×) to the electrophoresis tank, and run at a constant voltage of 120V for 1 hour;

[0121] (8) Transfer: Soak filter paper in pre-cooled semi-dry transfer buffer (Guangzhou Daoyi Science and Technology Co., Ltd., item number: 20019522, anhydrous ethanol: deionized water: 5× semi-dry transfer buffer = 1:3:1), and soak the PVDF membrane in methanol beforehand to make a "sandwich" structure (from top to bottom: filter paper, gel, PVDF membrane, filter paper), constant current 1.3A / piece, 9 minutes (Guangzhou Daoyi Science and Technology Co., Ltd., item number: 20019501);

[0122] (9) Sealing: Incubate the PVDF membrane with 5% skim milk powder for 1 hour;

[0123] (10) Incubate with primary antibody overnight at 4℃ (Foxc2: CST, catalog number: 12974; Transferrin: proteintech, catalog number: 17435-1-AP);

[0124] (11) Wash 3 times with 0.1% TBST at room temperature, 5 minutes each time;

[0125] (12) Incubate with secondary antibody at room temperature for 1 hour;

[0126] (13) Wash 3 times with 0.1% TBST at room temperature, 5 minutes each time;

[0127] (14) ECL chromogenic solution is used to display protein bands.

[0128] Statistical analysis using ImageJ software showed that, compared with the control group, the expression level of Foxc2 protein in plasma was significantly reduced in the DCM group. Figure 1 (B, C)

[0129] VI. Correlation Analysis of Foxc2 and DCM Merged FMR

[0130] Based on the mRNA expression levels of Foxc2 in the plasma of subjects in the control group and DCM group obtained in step four, independent risk factor analysis was performed, and ROC curves were plotted (e.g., ...). Figure 2 As shown in the figure, the results show that AUC ROC =0.933 (>0.9), 95% CI: 0.886-0.980, indicating that Foxc2 has practical significance for the prediction and diagnosis of DCM subjects.

[0131] To further verify whether Foxc2 can serve as a risk factor for DCM and FMR, this embodiment used SPSS 24.0 to perform independent risk factor analysis on the mRNA in the plasma of subjects with DCM and DCM combined with FMR, and compared and evaluated them using ROC curves (e.g., Figure 3 As shown in the figure, the results show that AUC ROC =0.928 (>0.9), 95% CI: 0.859-0.996, which indicates that Foxc2 has the potential value in predicting, identifying and diagnosing DCM combined with FMR.

[0132] Table 8 Analysis of Foxc2 as an independent risk factor for predicting DCM

[0133]

[0134]

[0135] Table 9. Analysis of Foxc2 as an independent risk factor for predicting DCM combined with FMR

[0136]

[0137] Example 2

[0138] Verify the accuracy of Foxc2 in diagnosing DCM combined with FMR.

[0139] Peripheral blood was collected upon outpatient admission to separate plasma. Foxc2 expression was detected by qRT-PCR to diagnose and screen the number of patients with febrile malignant muscular dystrophy (FMR) among diabetic cerebral muscular dystrophy (DCM) patients. The results were then compared with the subsequent echocardiographic examination results of DCM patients to evaluate the accuracy of Foxc2 in diagnosing DCM complicated with FMR.

[0140] I. Study subjects: Peripheral venous blood was collected from 40 outpatients with DCM. Baseline data are shown in Table 10.

[0141] Table 10 Baseline data of outpatients admitted to DCM

[0142]

[0143] II. Experimental Procedure

[0144] 1. Blood collection method

[0145] All patients had 3 mL of peripheral venous blood collected upon admission.

[0146] 2. Peripheral blood plasma separation and total RNA extraction

[0147] Plasma was separated from peripheral blood by centrifugation, using the same method as in Example 1; total RNA was extracted using the Trizol method, also using the same method as in Example 1.

[0148] 3. The accuracy of reverse transcription and qRT-PCR detection of Foxc2 in diagnosing DCM combined with FMR

[0149] Reverse transcription and quantitative real-time PCR were performed using the same methods and systems as in Example 1. -△△ The Ct method was used with U6 as an internal reference for relative quantitative analysis of Foxc2. A Foxc2 > 0.494 was used as the diagnostic cutoff value, and the results were compared with echocardiographic findings in patients with DCM to assess the accuracy of Foxc2 in diagnosing DCM complicated with FMR.

[0150] III. Test Results

[0151] The results of the experiment are shown in Table 11. Using Foxc2>0.494 as the diagnostic cutoff value, 18 out of 30 DCM patients were found to have DCM combined with FMR, and 2 were found to have DCM alone. Echocardiography, which is the "gold standard" for diagnosis, showed that among the 30 patients with chest pain, 20 had DCM combined with FMR and 10 had DCM alone.

[0152] Using Foxc2 > 0.494 as the diagnostic cutoff value and comparing it with echocardiographic results, it was found that using Foxc2 testing as a diagnostic test, 18 patients were "true positives" and 9 patients were "true negatives," with an accuracy (Acc) of 90% (the proportion of true positives and true negatives in the total number of cases). This was evaluated using ROC curves (e.g., Figure 4 As shown in the figure, the results show that AUC ROC =0.963 (>0.9), indicating that Foxc2 has good accuracy in diagnosing DCM combined with FMR.

[0153] Table 11. Consistency between diagnostic test and echocardiography results

[0154]

[0155] In summary, this embodiment provides the plasma content of Foxc2 as a diagnostic efficacy for detecting DCM combined with FMR. Foxc2 can serve as a novel biomarker for diagnosing (or assisting in the diagnosis) of DCM combined with FMR, which is helpful for predicting, diagnosing and monitoring the occurrence and development of DCM.

[0156] Example 3

[0157] A real-time PCR kit for diagnosing DCM combined with FMR contains: Foxc2 and U6 primers (10 μM, sequences shown in the table below), TB Green fluorescent dye (5×) and ddH2O.

[0158] Table 12 Foxc2 and U6 primers

[0159]

[0160] The preparation method for the PCR kit reaction system is shown in the table below:

[0161] Table 13 Reaction System

[0162]

[0163] The PCR kit reaction conditions are as follows: pre-denaturation: 95℃ for 30 seconds (1 cycle); PCR reaction: 95℃ for 5 seconds, 60℃ for 34 seconds (40 cycles).

[0164] PCR kit result analysis used 2 -△△ The Ct method uses U6 as an internal reference for relative quantitative analysis of Foxc2.

[0165] The above embodiments are preferred embodiments of the present invention, but the embodiments of the present invention are not limited to the above embodiments. Any changes, modifications, substitutions, combinations, or simplifications made without departing from the spirit and principle of the present invention shall be considered equivalent substitutions and shall be included within the protection scope of the present invention.

Claims

1. Use of a substance for detecting the expression level of Foxc2 in a sample to be measured in the manufacture of a product, characterized in that, The product is any one of the following: A product for diagnosing or aiding in the diagnosis of dilated cardiomyopathy or dilated cardiomyopathy with mitral regurgitation; A product for predicting or aiding in the prediction of dilated cardiomyopathy or dilated cardiomyopathy with mitral regurgitation.

2. Use according to claim 1, characterized in that, The substance for detecting the Foxc2 expression level in the sample to be tested comprises a substance for detecting the Foxc2 gene expression level or a substance for detecting the Foxc2 protein level.

3. Use according to claim 2, characterized in that, The substance for detecting the Foxc2 expression level in the sample to be tested comprises a substance for one or more detection techniques or methods selected from the group consisting of RT-PCR reaction, ISH, microarray, Northern blot, enzyme-linked immunosorbent assay, radioimmunoassay, immunofluorescence assay, and slot blot assay.

4. Use according to claim 3, characterized in that, The substance for detecting the Foxc2 expression level in the sample to be tested comprises a Foxc2-specific probe or PCR primer.

5. Use of the substance for detecting the Foxc2 expression level in constructing a computer model for diagnosing or aiding in the diagnosis, predicting or aiding in the prediction of dilated cardiomyopathy or dilated cardiomyopathy with mitral regurgitation.

6. An early medical aid diagnosis system for dilated cardiomyopathy or dilated cardiomyopathy with mitral regurgitation, characterized by, The early medical auxiliary diagnosis system comprises a result determination module for comparing the processed value of Foxc2 obtained by detecting the gene or protein expression level of Foxc2 with a set value, wherein when the processed value is lower than the set value, it is determined that the subject has dilated cardiomyopathy or dilated cardiomyopathy with mitral regurgitation; The diagnosis system comprises a processing module for processing the detection result of the gene or protein expression level of Foxc2 in the sample to obtain a processed value; The diagnosis system comprises an output module for outputting the obtained diagnosis result.

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