Dilated cardiomyopathy biomarker and application thereof

Abstract

The invention discloses application of Foxc2 as a biomarker of dilated cardiomyopathy and related diseases thereof, the Foxc2 as the biomarker has high sensitivity and specificity in diagnosis and prediction of dilated cardiomyopathy and related diseases thereof, and a new detection means and scheme are provided for diagnosis and prediction of dilated cardiomyopathy and related diseases thereof.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and particularly relates to a biomarker for dilated cardiomyopathy and its application. Background Art

[0002] "Summary of Cardiovascular Health and Disease in China 2022" released by Chinese Circulation Journal in 2023 pointed out that the number of people with cardiovascular diseases in China has reached 330 million. Its fatality rate ranks first, and the prevalence rate shows an increasing trend year by year, and the onset age is also getting younger. Dilated cardiomyopathy (DCM) is a type of cardiomyopathy characterized by the enlargement of the left ventricle or both ventricles and accompanied by systolic dysfunction. When the volume of the left ventricle expands and its sphericity intensifies, the movement of the mitral valve leaf is restricted, the mitral annulus dilates, and ultimately leads to leaflet engagement defects and "functional" regurgitation (FMR). Despite the progress of medical and surgical treatments, functional FMR is still associated with a high incidence and mortality of DCM and DCM-related heart failure. Therefore, it is urgent for us to identify DCM subjects with FMR at an early stage, intervene in a timely manner, and reduce the incidence of in-hospital and long-term adverse events.

[0003] In recent years, there have been more and more studies on biomarkers for the etiological diagnosis of DCM, mainly including genetic biomarkers and immune biomarkers, such as anti-myocardial antibody (ANT), titin (TTN), Bcl2-associated athanogene 3 (BAG3), brain natriuretic peptide (BNP), cardiac troponin I / T (cTnI, cTnT), etc. There is a certain predictability between these genes and the cardiomyopathy phenotype, but they 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 solutions:

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

[0006] A product for diagnosing or assisting in diagnosing dilated cardiomyopathy or its related diseases;

[0007] A product for predicting or assisting in predicting dilated cardiomyopathy or its related diseases.

[0008] Forkhead box-C2 (Foxc2), also known as mesenchyme forkhead 1 (MHF1), is a transcription factor belonging to the "C" subfamily of the forkhead / winged helix family.

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

[0010] In some embodiments, the related disease thereof includes dilated cardiomyopathy with mitral regurgitation.

[0011] In some embodiments, the substance for detecting a biomarker in a sample to be tested includes a substance for detecting the gene expression level of the biomarker or a substance for detecting the protein level of the biomarker.

[0012] In some embodiments, the substance for detecting a biomarker in a sample to be tested comprises a substance for one or more detection techniques or methods selected from the group consisting of: PCR reaction, RT-PCR-derived reaction, 3SR amplification, LCR, SDA, NASBA, TMA, SYBR Green, TaqMan probe, molecular beacon, dual hybridization probe, composite probe, ISH, microarray, Southern blot, Northern blot, multi-analyte profiling test, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, immunofluorescence assay, enzyme immunoassay, immunoprecipitation assay, chemiluminescence assay, immunohistochemical assay, dot blot assay, or slot blot assay.

[0013] In some embodiments, the substance for detecting a biomarker in a sample to be tested is selected from: a substance specific for the biomarker, a biomarker-specific probe, a gene chip, one or more of PCR primers.

[0014] In some embodiments, the substance specific for the biomarker comprises any one of an antibody that specifically binds to the biomarker, a ligand protein or polypeptide that specifically binds to the biomarker, and a non-protein compound that specifically recognizes the biomarker.

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

[0016] In some embodiments, the product includes at least one of a reagent, a kit, a test strip, a chip, and a system.

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

[0018] The products include products for diagnosing or assisting in diagnosing whether a test sample is a sample of dilated cardiomyopathy or a related disease thereof, products for diagnosing or assisting in diagnosing whether a test subject is a patient with dilated cardiomyopathy or a related disease thereof, products for predicting or assisting in predicting whether a test sample is a sample of dilated cardiomyopathy or a related disease thereof, and products for predicting or assisting in predicting whether a test subject is a patient with dilated cardiomyopathy or a related disease thereof.

[0019] In some embodiments, the related disease includes dilated cardiomyopathy complicated with mitral regurgitation.

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

[0021] In some embodiments, the substance for detecting Foxc2 includes substances for one or more detection techniques or methods selected from the group consisting of: PCR reaction, RT-PCR-derived reaction, 3SR amplification, LCR, SDA, NASBA, TMA, SYBRGreen, TaqMan probe, molecular beacon, dual hybridization probe, composite probe, ISH, microarray, Southern blot, Northern blot, multi-analyte profiling test, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, immunofluorescence assay, enzyme immunoassay, immunoprecipitation assay, chemiluminescence assay, immunohistochemical assay, dot blot assay or slot blot assay.

[0022] In some embodiments, the substance for detecting Foxc2 is selected from: a substance specific for Foxc2, a Foxc2-specific probe, a gene chip, one or more PCR primers.

[0023] In some embodiments, the substance specific for Foxc2 includes any one of an antibody specifically binding to Foxc2, a ligand protein or polypeptide specifically binding to Foxc2, and a non-protein compound specifically recognizing Foxc2.

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

[0025] In some embodiments, the test subject includes a human or a non-human mammal.

[0026] In some embodiments, the product includes at least one of a reagent, a kit, a test strip, a chip, and a system.

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

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

[0029] In some embodiments, the protein detection kit contains reagents, chips, antibodies, or ligands for detecting the protein level of Foxc2 expression.

[0030] The third aspect of the present invention provides the use of a biomarker in constructing a computer model for diagnosing or assisting in diagnosing, predicting or assisting in predicting dilated cardiomyopathy or its related diseases, and the biomarker includes Foxc2.

[0031] In some embodiments, the related diseases include dilated cardiomyopathy complicated with mitral regurgitation.

[0032] The fourth aspect of the present invention provides the use of a biomarker in screening candidate drugs for treating dilated cardiomyopathy or its related diseases, and the biomarker includes Foxc2.

[0033] In some embodiments, the related diseases include dilated cardiomyopathy complicated with mitral regurgitation.

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

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

[0036] In some embodiments, the diagnosis system includes a processing module for detecting and processing the expression level of the gene or protein related to the biomarker in the sample to obtain a processed value.

[0037] In some embodiments, the diagnosis system includes an output module for outputting the obtained diagnosis result.

[0038] In some embodiments, the related diseases include dilated cardiomyopathy complicated with mitral regurgitation.

[0039] The sixth aspect of the present invention provides a scoring device for evaluating the risk of a subject having dilated cardiomyopathy or a related disease thereof, and the scoring device includes the following units:

[0040] A detection unit: detecting the gene or protein expression level of, such as Foxc2, in a sample;

[0041] An analysis unit: using the gene or protein expression level of the detected biomarker as an input variable and inputting it into the computer model of the third aspect for analysis;

[0042] A scoring unit: outputting the risk value of the subject corresponding to the sample having dilated cardiomyopathy or a related disease thereof.

[0043] In some embodiments, the related disease thereof includes dilated cardiomyopathy complicated with mitral regurgitation.

[0044] The seventh aspect of the present invention provides a method for diagnosing and predicting dilated cardiomyopathy or a related disease thereof, which includes the following steps:

[0045] 1) Collecting a sample of an object to be detected;

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

[0047] 3) Comparing the expression level result with the expression level of a healthy control to determine whether the object has dilated cardiomyopathy or a related disease thereof or the risk magnitude of having dilated cardiomyopathy or a related disease thereof.

[0048] In some embodiments, the related disease thereof includes dilated cardiomyopathy complicated with mitral regurgitation.

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

[0050] In some embodiments, detecting the expression level of Foxc2 in the sample in step 2) includes detection by PCR reaction, RT-PCR-derived reaction, 3SR amplification, LCR, SDA, NASBA, TMA, SYBR Green, TaqMan probe, molecular beacon, dual hybridization probe, composite probe, ISH, microarray, Southern blot, Northern blot, multi-analyte profiling test, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, immunofluorescence assay, enzyme immunoassay, immunoprecipitation assay, chemiluminescence assay, immunohistochemical assay, dot blot assay or slot blot assay.

[0051] In some embodiments, detecting the expression level of the Foxc2 gene in a sample further includes performing data normalization using a reference gene.

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

[0053] The beneficial effects of the present invention are as follows: The present invention provides the application of Foxc2 as a biomarker for dilated cardiomyopathy and dilated cardiomyopathy complicated with mitral regurgitation. Foxc2 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. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 Expression of Foxc2 in plasma of control group and DCM subjects. (A) Detection of Foxc2 mRNA expression in plasma by qRT-PCR. (B) Detection of protein expression levels of Foxc2 and Transferrin in plasma by Western blot. (C) Quantitative statistical analysis of the protein expression level of Foxc2 by gray value. ***P<0.01, ****P<0.001.

[0055] Figure 2 ROC curve graph for the predictive value of Foxc2 in the occurrence of DCM disease.

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

[0057] Figure 4 ROC curve graph for the predictive value of Foxc2 in DCM subjects complicated with FMR disease. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0058] The DCM biomarkers described in the present invention were discovered by analyzing plasma samples using molecular biology techniques (such as Western blot, RT-qPCR). Generally, the plasma samples were biological samples (plasma) from one or more human subjects clinically diagnosed with DCM and other non-DCM human subjects (control subjects with DCM). The biological samples from human subjects with DCM were analyzed synchronously with those from other non-DCM human subjects in relevant molecular biology tests. Molecules that were differentially expressed in the biological samples from subjects with DCM compared to non-DCM subjects (control subjects with dilated diseases), including those that differed at a statistically significant level, were identified as biomarkers to distinguish the control group.

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

[0060] The content of the present invention will be further described in detail below through specific examples. The raw materials, reagents or devices used in the examples can be obtained from conventional commercial channels or can be obtained by methods of existing technologies without special instructions. Unless otherwise specified, the test or measurement methods are conventional methods in the art.

[0061] Example 1

[0062] I. Test Population

[0063] A total of 92 clinical cases of subjects were collected in this embodiment, and the subjects were grouped according to gender (male / female) and disease type (non-dilated cardiomyopathy (control) / dilated cardiomyopathy (DCM) / dilated cardiomyopathy with mitral regurgitation (DCM with FMR)). The subjects were from those diagnosed and treated in the hospital. Inclusion criteria for DCM subjects: meeting the DCM diagnostic criteria in the "Chinese Guidelines for the Diagnosis and Treatment of Dilated Cardiomyopathy"; being over 18 years old. Subjects with incomplete cardiac three-dimensional echocardiogram indexes were excluded. A total of 53 cases were selected in the DCM group and 39 cases in the non-DCM group. All subjects signed the informed consent form.

[0064] Table 1 Basic information table of cases

[0065]

[0066] II. Detection and statistical analysis of physiological and biochemical indexes

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

[0068] Statistical analysis was performed on the physiological and biochemical indexes 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) between the DCM non-valvular heart disease group and the control group. All data were expressed as (x±s), and an independent samples t-test was used between the means of two samples.

[0069] The results showed that compared with the control group, the EF and FS indexes in the DCM group were significantly down-regulated, while the BNP, troponin I, and LVDd indexes were significantly up-regulated, all of which indicated that they met the clinical diagnostic requirements of DCM (refer to the "Chinese Guidelines for the Diagnosis and Treatment of Dilated Cardiomyopathy").

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

[0071]

[0072] Statistical analysis was performed on the physiological and biochemical indexes 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) between the DCM group and the DCM with FMR group. All data were expressed as (x±s), and an independent samples t-test was used between the means of two samples.

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

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

[0075]

[0076] III. Extraction of total plasma RNA

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

[0078] (2) Centrifuge at 3000 rpm for 10 minutes at low temperature (4°C), and collect the upper pale yellow plasma.

[0079] (3) Prepare for plasma mRNA extraction: Total RNA Extraction Kit for Whole Blood / Plasma / Serum (Xinhai Gene), product number: B0901; Reagents to be prepared by yourself: isopropanol, chloroform.

[0080] (4) Add 250 μL of the above plasma to 750 μL of TRIzol LS reagent, then immediately invert and mix well by wrist shaking, and let it stand for 5 minutes.

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

[0082] (6) Centrifuge at 13000 rpm for 5 minutes at room temperature, and pipette 500 μL of the upper supernatant into the adsorption column core.

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

[0084] (8) Centrifuge at 13000 rpm for 15 seconds at room temperature, and discard the waste liquid.

[0085] (9) Add 500 μL of Washing Buffer to the adsorption column, centrifuge at 13000 rpm for 15 seconds at room temperature.

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

[0087] (11) Put the adsorption column back into the centrifuge and centrifuge at 13000 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 adsorption column core.

[0089] (13) Let it stand at room temperature for 2 minutes, centrifuge at 13,000 rpm for 1 minute, and the eluate is the RNA product;

[0090] (14) Use a ultra-micro spectrophotometer to detect the concentration and purity of total RNA in plasma samples.

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

[0092] 1. Reverse transcription to synthesize the first strand of cDNA of mRNA

[0093] (1) Prepare the kit: PrimeScript TM RT Master Mix (Perfect Real Time), product number: RR036A;

[0094] (2) Prepare the RT reaction solution according to the following components (the reaction solution preparation is carried out on ice):

[0095] Table 4 Reverse transcription reaction system

[0096]

[0097] (3) Gently mix well and carry out the reverse transcription reaction according to the following program:

[0098] Table 5 Reverse transcription reaction program

[0099]

[0100] 2. Real-time fluorescence quantitative PCR

[0101] (1) Use an online primer design software to design Foxc2 and U6 primers. After primer design, they are 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 components (the reaction solution should be prepared on ice):

[0107] Table 6 PCR reaction system

[0108]

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

[0110] Table 7 PCR reaction program

[0111]

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

[0113] 5. Detection of Foxc2 protein expression level in subjects’ plasma

[0114] (1) Prepare BCA protein quantification kit: Pierce TM BCA Protein Assay Kits (ThermoFisher), Cat. No. 23227;

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

[0116] (3) Drawing a 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°C incubator for 30 min. Detect the absorbance at a wavelength of 562 nm using an ELISA reader and draw a standard curve between absorbance and protein concentration.

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

[0118] (5) Preparation of protein samples: Adjust the loading volume according to the protein concentration determined by the BCA method, unify the loading volume through 6x Loading Buffer and ddH2O, mix well, incubate in a metal bath at 100 °C for 5 minutes, centrifuge briefly at low speed, aliquot and store at -20 °C for later use;

[0119] (6) Preparation of SDS-PAGE gel: Prepare according to the operation instructions of the PAGE gel rapid preparation kit (Yaenzyme Biotech, product number: PG112);

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

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

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

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

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

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

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

[0127] (14) ECL developing solution was used to visualize protein bands.

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

[0129] VI. Analysis of the association between Foxc2 and DCM complicated with FMR

[0130] Independent risk factor analysis was performed on the mRNA expression levels of Foxc2 in the plasma of subjects in the control group and the DCM group obtained in step four, and an ROC curve was drawn (asFigure 2 As shown in the figure, the result shows that the 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] In order to further verify whether Foxc2 can be used as a risk factor for DCM and FMR, in this embodiment, SPSS 24.0 is used to perform independent risk factor analysis on the mRNA in the plasma of the DCM group and the subjects with DCM complicated with FMR, and a ROC curve is used to compare and evaluate it (as Figure 3 shown in the figure), the result shows that the AUC ROC = 0.928 (> 0.9), 95% CI: 0.859 - 0.996, indicating that Foxc2 has the potential value for predicting, identifying and diagnosing the disease of DCM complicated 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 complicated with FMR

[0136]

[0137] Example 2

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

[0139] Collect peripheral blood from outpatients admitted to the hospital and isolate plasma. Detect the expression of Foxc2 by qRT-PCR, diagnose and screen the number of patients with FMR among DCM patients, and then compare with the subsequent echocardiogram examination results of DCM patients to evaluate the accuracy of Foxc2 in diagnosing DCM complicated with FMR.

[0140] I. Research objects: Collect peripheral venous blood from 40 DCM patients admitted to the hospital as outpatients. The baseline data are shown in Table 10.

[0141] Table 10 Baseline data of DCM patients admitted to the hospital as outpatients

[0142]

[0143] II. Experimental steps

[0144] 1. Blood collection method

[0145] Peripheral venous blood (3 mL) was collected from all patients at the time of admission.

[0146] 2. Isolation of peripheral blood plasma and extraction of total RNA

[0147] The same method as in Example 1 was used, i.e., plasma was isolated from peripheral blood by centrifugation; the same method as in Example 1 was used, i.e., total RNA was extracted by the Trizol method.

[0148] 3. Accuracy of diagnosing DCM complicated with FMR by reverse transcription and qRT-PCR detection of Foxc2

[0149] The reverse transcription reaction was carried out using the same method and system as in Example 1, and the fluorescence quantitative PCR reaction was carried out using the same method and system as in Example 1. The 2 -△△ Ct method was used with U6 as the internal reference for relative quantitative analysis of Foxc2. Taking Foxc2 > 0.494 as the diagnostic cut-off value, it was compared with the cardiac ultrasound results of DCM patients to evaluate the accuracy of Foxc2 in diagnosing DCM complicated with FMR.

[0150] III. Test results

[0151] The test results are shown in Table 11. Taking Foxc2 > 0.494 as the diagnostic cut-off value, among 30 DCM patients, 18 were DCM patients complicated with FMR and 2 were simple DCM patients; cardiac ultrasound, regarded as the "gold standard" for diagnosis, showed that among 30 chest pain patients, 20 were DCM patients complicated with FMR and 10 were simple DCM patients.

[0152] Taking Foxc2 > 0.494 as the diagnostic cut-off value and comparing it with the cardiac ultrasound results, it was found that using Foxc2 detection as a diagnostic test, 18 "true positive" patients and 9 "true negative" patients were detected. Its accuracy (Acc) was 90% (the proportion of true positive and true negative in the total number of cases examined). It was evaluated by the ROC curve (as Figure 4 shown), and the results showed that AUC ROC = 0.963 (> 0.9), indicating that Foxc2 has good accuracy in diagnosing DCM complicated with FMR.

[0153] Table 11 Concordance between diagnostic test and cardiac ultrasound examination results

[0154]

[0155] In summary, this example provides the diagnostic efficacy of the content of Foxc2 in plasma for detecting DCM complicated with FMR. Foxc2 can be used as a new biomarker for diagnosing (or assisting in diagnosing) DCM complicated with FMR, which helps to predict, diagnose, and monitor the occurrence and development of DCM.

[0156] Example 3

[0157] A fluorescence quantitative PCR kit for diagnosing DCM complicated with FMR, which contains: Foxc2 and U6 primers (10 μM, the sequences are shown in the following table), TB Green fluorescent dye (5×) and ddH2O.

[0158] Table 12 Foxc2 and U6 primers

[0159]

[0160] The preparation method of the reaction system of the PCR kit is as shown in the following table:

[0161] Table 13 Reaction system

[0162]

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

[0164] The result analysis of the PCR kit adopts 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 other changes, modifications, substitutions, combinations, and simplifications made without departing from the spirit and principle of the present invention shall be equivalent replacement methods and are all included in the protection scope of the present invention.

Claims

1. Use of a substance for detecting biomarkers in a sample to be tested in preparing a product, characterized in that: The biomarker includes Foxc2; the product includes any one of the following: Products for diagnosing or assisting in the diagnosis of dilated cardiomyopathy or its related diseases; Products that predict or assist in predicting dilated cardiomyopathy or its related diseases.

2. The use according to claim 1, characterized in that: The related diseases include dilated cardiomyopathy combined with mitral regurgitation.

3. The use according to claim 1, characterized in that: The substance for detecting the biomarker in the sample to be tested includes a substance for detecting the expression level of the biomarker gene or a substance for detecting the level of the biomarker protein.

4. The use according to claim 3, characterized in that: The material for detecting the biomarker in the sample to be tested comprises a material used in one or more detection techniques or methods selected from the following group: PCR reaction, RT-PCR derivatization reaction, 3SR amplification, LCR, SDA, NASBA, TMA, SYBR Green, TaqMan probe, molecular beacon, double hybridization probe, composite probe, ISH, microarray, Southern blot, Northern blot, multi-analyte spectrum test, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay, immunofluorescence assay, enzyme immunoassay, immunoprecipitation assay, chemiluminescence assay, immunohistochemistry assay, dot blot assay or slot blot assay; Preferably, the substance for detecting the biomarker in the sample to be tested is selected from: one or more of a substance specific to the biomarker, a probe specific to the biomarker, a gene chip, and a PCR primer.

5. A product, characterized in that: The product includes a substance for detecting Foxc2; The products include products for diagnosing or assisting in diagnosing whether a sample to be tested is a sample of dilated cardiomyopathy or a disease related thereto, products for diagnosing or assisting in diagnosing whether a subject to be tested is a patient of dilated cardiomyopathy or a disease related thereto, products for predicting or assisting in predicting whether a sample to be tested is a sample of dilated cardiomyopathy or a disease related thereto, and products for predicting or assisting in predicting whether a subject to be tested is a patient of dilated cardiomyopathy or a disease related thereto; Preferably, the related diseases include dilated cardiomyopathy combined with mitral regurgitation.

6. The product according to claim 5, characterized in that The substance for detecting Foxc2 includes a substance for detecting the expression level of Foxc2 gene or a substance for detecting the level of Foxc2 protein; Preferably, the subject includes a human or a non-human mammal; Preferably, the product comprises at least one of a reagent, a kit, a test paper, a chip, and a system; Preferably, the kit includes a gene detection kit and a protein detection kit.

7. Use of biomarkers in constructing a computer model for diagnosis or auxiliary diagnosis, prediction or auxiliary prediction of dilated cardiomyopathy or its related diseases, characterized in that: The biomarkers include Foxc2; Preferably, the related diseases include dilated cardiomyopathy combined with mitral regurgitation.

8. Use of a biomarker in screening candidate drugs for treating dilated cardiomyopathy or diseases related thereto, characterized in that: The biomarkers include Foxc2; Preferably, the related diseases include dilated cardiomyopathy combined with mitral regurgitation.

9. An early medical auxiliary diagnosis system for dilated cardiomyopathy or related diseases, characterized in that: The diagnostic system includes a result determination module for comparing the processed value of the biomarker obtained by detecting the gene or protein expression level of Foxc2 with a set value; Preferably, the diagnostic system includes a processing module for detecting and processing the expression level of a gene or protein associated with a biomarker in a sample to obtain a processed value; Preferably, the diagnostic system comprises an output module for outputting the obtained diagnostic results; Preferably, the related diseases include dilated cardiomyopathy combined with mitral regurgitation.

10. A scoring device for assessing a subject's risk of suffering from dilated cardiomyopathy or a disease related thereto, characterized in that: The scoring device comprises the following units: Detection unit: detect the gene or protein expression level of Foxc2 in the sample; Analysis unit: using the detected gene or protein expression level of the biomarker as an input variable and inputting it into the risk prediction model described in claim 7 for analysis; Scoring unit: outputs the risk value of the subject suffering from dilated cardiomyopathy or its related diseases corresponding to the sample; Preferably, the related diseases include dilated cardiomyopathy combined with mitral regurgitation.

Images