Application of secreted frizzled-related protein 1 in the preparation and diagnosis of primary dilated cardiomyopathy

Abstract

This invention provides an application of secretory coil-associated protein 1 (SCAP1) in the preparation of diagnostic kits for primary dilated cardiomyopathy (PDM). SCAP1 is not only used in the preparation of diagnostic kits for PDM, but also in the preparation of diagnostic kits for PDM. These kits are used to diagnose, assess, and predict the prognosis of PDM, and the test sample is venous blood. This invention is the first to propose the use of SCAP1 for the diagnosis of human PDM, screening for genes with the most significant differential expression in the cardiac tissue of PDM patients, and verifying the differential expression of this protein in patient serum, as well as its severity assessment and prognosis. This can effectively help with early risk stratification of PDM patients, thereby enabling early specific treatment and improving patient survival rates.

Description

Technical Field

[0001] The invention belongs to the technical field of dilated cardiomyopathy diagnosis, and specifically relates to an application of secretory frizzled-related protein 1 in the preparation and diagnosis of primary dilated cardiomyopathy. Background Art

[0002] Dilated cardiomyopathy is a common cardiomyopathy, which manifests as left ventricular dilatation with systolic dysfunction, and may also be accompanied by right ventricular dilatation and dysfunction. It is the primary cause of heart transplantation and the third cause of heart failure. The prevalence of primary dilated cardiomyopathy is about 40 / 100,000 people. The microscopic molecular mechanism of its onset is unknown, and the abnormal expression of multiple molecules is involved in the pathogenic process of dilated cardiomyopathy. The condition is progressive and the prognosis of the disease is very poor. It is reported that the mortality rate of dilated cardiomyopathy within one year is 20%, and the mortality rate within four years is 56%. Therefore, it is particularly important to diagnose and evaluate dilated cardiomyopathy as soon as possible and accurately predict the outcome, so as to strengthen the disease management of high-risk patients in advance to improve the prognosis. Although imaging examinations, exercise stress tests, gene sequencing, serological examinations and other diagnostic methods are currently used in clinical practice, the combination of these existing methods is not effective as a means of judging the prognosis of dilated cardiomyopathy. Therefore, it is necessary to discover new diagnostic methods to evaluate the condition and predict the prognosis of dilated cardiomyopathy, so as to provide targeted management as soon as possible, which is an effective way to improve the survival rate of the disease.

[0003] Serum marker detection can play an important role in the diagnosis and risk stratification of dilated cardiomyopathy. It can be used alone or in combination with other indicators to form a scoring system to play a role in specific clinical diagnosis and treatment. A clinically valuable biological marker should meet the following criteria: 1) The detection of the indicator can be repeated by a convenient and economical method. 2) Additional information can be obtained based on the results of existing means. 3) It is beneficial to decision-making clinical diagnosis and treatment activities.

[0004] Secreted frizzled-related protein 1 (SFRP1) is a secreted glycoprotein that mainly competitively binds to the frizzled protein receptor with a homologous structure in the Wnt signaling pathway. Through the regulation of the Wnt signaling pathway, it is widely involved in regulating cell phenotypes such as cell proliferation, differentiation, and apoptosis. In the heart, SFRP1 is mainly involved in regulating cardiac hypertrophy, ventricular remodeling, myocardial hypertrophy, and myocardial regeneration. Knockdown of SFRP1 in African clawed frog embryos can inhibit embryonic cardiomyocyte differentiation and cause abnormal reduction in heart development volume; while overexpression of SFRP1 can promote embryonic cardiomyocyte differentiation and cause abnormal increase in heart development volume. In mice with dilated cardiomyopathy caused by lamin a / C gene mutation, the expression level of SFRP1 in the heart is significantly increased, accompanied by a significant downregulation of the WNT / β-catenin pathway. It has been confirmed that intervention with SFRP1 can significantly improve the level of cardiomyocyte hypertrophy caused by phenylproterenol. There is no report on the application of secreted frizzled-related protein 1 in the preparation of dilated cardiomyopathy diagnostic kits. Summary of the invention

[0005] In view of the deficiencies in the prior art, the purpose of the present invention is to provide a secretory frizzled-related protein 1 for use in the preparation and diagnosis of primary dilated cardiomyopathy.

[0006] To achieve the above object, the solution of the present invention is:

[0007] In a first aspect, the present invention provides a use of secreted frizzled-related protein 1 in the preparation and diagnosis of primary dilated cardiomyopathy.

[0008] In a second aspect, the present invention provides a kit for diagnosing primary dilated cardiomyopathy, which includes a reagent for diagnosing primary dilated cardiomyopathy, wherein the reagent is the secretory frizzled-related protein 1 mentioned above.

[0009] Preferably, the test sample of the kit is venous blood, and is used in the preparation of diagnosis, evaluation and prognosis of primary dilated cardiomyopathy.

[0010] In a third aspect, the present invention provides the use of the secretory frizzled-related protein 1 in the preparation of a kit for diagnosing primary dilated cardiomyopathy, wherein the test sample of the kit is venous blood.

[0011] Due to the adoption of the above scheme, the beneficial effects of the present invention are:

[0012] First, the present invention proposes for the first time that secretory frizzled-related protein 1 can be used to diagnose primary human dilated cardiomyopathy, screen out the genes with the most obvious expression differences in the heart tissue of patients with dilated cardiomyopathy, and verify the expression differences and severity of the protein in the patient's serum. The evaluation and prognosis judgment can effectively help the early risk stratification of patients with dilated cardiomyopathy, thereby achieving early specific treatment and improving the survival rate of patients.

[0013] Second, compared with the current complicated and expensive methods for evaluating dilated cardiomyopathy, the detection of secretory frizzled-related protein 1 of the present invention will make the evaluation of the disease simpler, more economical and more efficient, and the value of disease prognosis judgment will be higher. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 A flow chart for preparing SFRP1 of the present invention for diagnosing primary dilated cardiomyopathy.

[0015] Figure 2 This is a Venn diagram of differentially expressed genes in each transcriptome dataset in Example 1 of the present invention.

[0016] Figure 3 This is a diagram of the western blot detection results of human heart tissue in Example 1 of the present invention.

[0017] Figure 4 This is a comparison chart of the serum of dilated cardiomyopathy patients and controls in Example 2 of the present invention.

[0018] Figure 5 This is a correlation diagram between serum SFRP1 levels and clinical indicators in patients with dilated cardiomyopathy in Example 2 of the present invention (Figure A is ejection fraction EF (%), P = 0.047, Figure B is left atrial diameter LAD (mm), P < 0.0001, Figure C is left ventricular end-systolic diameter LVIDs (mm), P = 0.014, and Figure D is left ventricular end-diastolic diameter LVIDd (mm), P = 0.037).

[0019] Figure 6 Graphs showing ROC curves obtained based on multivariate logistics modeling in Example 2 of the present invention (Graph A is the ROC curve for the training set, and Graph B is the ROC curve for the validation set).

[0020] Figure 7 It is a nomogram obtained based on multivariate logistics modeling in Example 2 of the present invention. DETAILED DESCRIPTION

[0021] The present invention provides an application of secretory frizzled-related protein 1 in the preparation and diagnosis of primary dilated cardiomyopathy.

[0022] The technical content of the present invention is further described below in conjunction with the examples. The following examples are illustrative, not restrictive, and the protection scope of the present invention cannot be limited by the following examples. The experimental methods used in the following examples are conventional methods unless otherwise specified. The materials, reagents, etc. used in the following examples, unless otherwise specified, can all be obtained from commercial sources.

[0023] like Figure 1 As shown, the present invention determines SFRP1 as a candidate gene through gene screening, then performs molecular detection, difference comparison and correlation analysis, and then constructs a prediction model, and performs analytical regression through training set modeling and validation set verification.

[0024] Example 1: Screening for the most significant difference molecules

[0025] 1) Search the GEO database for human dilated cardiomyopathy cardiac tissue microarray datasets and download datasets GSE1869, GSE3585, GSE5406, and GSE79962.

[0026] 2) The above four data sets were analyzed for differentially expressed genes using the R package limma, and the screening parameters were set to: |logFC|>1, adjustP<0.05.

[0027] 3) The results of differential gene screening were: GSE1869 had 802 upregulated genes and 234 downregulated genes; GSE3585 had 86 upregulated genes and 57 downregulated genes; GSE5406 had 106 upregulated genes and 131 downregulated genes; GSE79962 had 390 upregulated genes and 255 downregulated genes.

[0028] 4) The most significant genes related to dilated cardiomyopathy screened out from the above data sets were intersected to find the most significant genes based on the current data ( Figure 2 ), it can be seen that there are four common differentially expressed genes in the four data sets, so we will conduct follow-up research on these four genes.

[0029] 5) A total of 4 genes to be studied were found, including SFRP1 (encoding secreted frizzled-related protein 1), ASPN (encoding a secreted matrix protein), AEBP1 (encoding a transcriptional repressor), and FMOD (encoding an interstitial proteoglycan used to aggregate the cell matrix).

[0030] 6) Western blot was used to detect the expression level of human cardiac proteins, and the difference was statistically significant ( Figure 3 ), the results showed that the level of SFRP1 protein in the heart of patients with dilated cardiomyopathy was significantly increased. Finally, SFRP1, a secretory protein, was determined as a serological indicator for subsequent evaluation of the diagnostic value of dilated cardiomyopathy.

[0031] Example 2: Screening for the diagnostic value of dilated cardiomyopathy

[0032] 1) 3 mL of venous blood was drawn from 333 patients with dilated cardiomyopathy and 91 controls, and the supernatant was collected after standing at 4° C. The serum SFRP1 concentration level was detected using the kit for diagnosing dilated cardiomyopathy of the present invention.

[0033] 2) Clinical information of patients with dilated cardiomyopathy was entered, including 51 clinical indicators such as gender, age, hypertension, and diabetes; and the patients' prognosis was followed up by telephone for 2 years.

[0034] 3) SFRP1 expression level ( Figure 4 ) and some clinical indicators such as ejection fraction, left ventricular end-systolic diameter, left ventricular end-diastolic diameter, etc. Figure 5 ), it can be seen that serum SFRP1 levels are correlated with cardiac function indicators such as ejection fraction, left atrial diameter, left ventricular end-systolic diameter, and left ventricular end-diastolic diameter in patients with dilated cardiomyopathy. Figure 4 As shown, the SFRP1 concentration levels in the serum of patients with dilated cardiomyopathy (DCM) and controls (control). It can be seen that the SFRP1 level in the serum of patients with dilated cardiomyopathy was significantly higher than that in the control serum, ****: P<0.0001.

[0035] 4) The patients with dilated cardiomyopathy with prognostic data were randomly divided into a training set and a validation set at a ratio of 2:1. Death within 2 years was used as the outcome. Single-factor logistics modeling was used for all indicators of the training set data, and p < 0.1 was selected for subsequent variable screening.

[0036] 5) Lasso regression analysis was used to screen variables, with λ set to 0.07 and a mean square error of 0.126. Five variables were screened: SFRP1, Coxsackievirus M immunoglobulin (Coxsackievirus IgM), thyroid stimulating hormone (TSH), left atrial diameter (LAD), and plasma brain natriuretic peptide (BNP).

[0037] 6) For the above five variables, multivariate logistic regression analysis was performed with death within 2 years as the outcome. Variables with P < 0.05 were selected, IgM and BNP were excluded, and SFRP1, LAD, and TSH were used to build a multivariate logistic regression model for the training set. The results are shown in Table 1:

[0038] Table 1

[0039] variable p Beta coefficient OR 95％Cl SFRP1 <0.001 0.069 1.063 (1.033,1.101) LAD <0.001 0.094 1.131 (1.062,1.215) TSH 0.011 0.062 1.037 (1.010,1.074)

[0040] As shown in Table 1, elevated levels of SFRP1, LAD and TSH are significantly associated with an increased risk of death within 2 years in patients with dilated cardiomyopathy.

[0041] 7) Use the above multivariate logistic regression model to draw the ROC curve for the training set and validation set data, and calculate the area under the curve ( Figure 6 ).like Figure 6 As shown in Figure A, the area under the ROC curve is 0.898, and the 95% confidence interval is (0.837, 0.959). Figure 6 As shown in Figure B, the area under the ROC curve is 0.813, and the 95% confidence interval is (0.676, 0.951). To evaluate the comprehensive effect of the SFRP1 prediction model on the prognosis of patients with dilated cardiomyopathy, according to the model constructed in our study, the area under the ROC curve of the training set and validation set data is above 0.8, and the area under the ROC curve of the training set data is close to 0.9, so the model has good sensitivity and specificity for the prognosis prediction of patients with dilated cardiomyopathy.

[0042] 8) The concentration of SFRP1, the most significant differential gene in dilated cardiomyopathy, was measured, and the serum SFRP1 concentration level of patients with dilated cardiomyopathy was measured and brought into the nomogram constructed by the constructed logistics regression model ( Figure 7), the nomogram includes three indicators: SFRP1, LAD (left atrial diameter) and TSH (thyroid stimulating hormone), and calculates the score to predict the death of patients with dilated cardiomyopathy within two years. According to the levels of SFRP1, TSH and LAD values, each item and the cumulative score are calculated to predict the possibility of death within two years, and further adopt targeted diagnosis and treatment plans.

[0043] The above description of the embodiments is to facilitate the understanding and use of the present invention by those skilled in the art. It is obvious that those skilled in the art can easily make various modifications to these embodiments and apply the general principles described herein to other embodiments without creative work. Therefore, the present invention is not limited to the above embodiments. Improvements and modifications made by those skilled in the art based on the principles of the present invention without departing from the scope of the present invention should be within the protection scope of the present invention.

Claims

1. Use of a reagent for detecting secreted frizzled-related protein 1 in the preparation of a reagent or a kit for diagnosing primary dilated cardiomyopathy; The reagent for detecting secreted frizzled-related protein 1 detects the concentration level of serum secreted frizzled-related protein 1; The level of SFRP1 protein in cardiac tissue or serum of patients with dilated cardiomyopathy is increased.

Images