Biomarker for cardiomyopathy prognosis and application thereof

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to a biomarker and application thereof. The invention finds that when the CECR1 in a biological sample of a hypertrophic cardiomyopathy HCM patient is obviously reduced, the prognosis is poor. The biomarker disclosed by the invention is simple and convenient to sample and can be detected in tissues and blood, and compared with prediction through a model in the prior art, the biomarker disclosed by the invention has the advantages of few detection indexes and quick report result. The biomarker disclosed by the invention is high in accuracy, wherein the blood plasma CECR1 level in a screening queue predicts that AUCs of cardiovascular death of HCM patients and AUCs of SCD are 0.720 (95% CI 0.657-0.783) and 0.798 (95% CI 0.725-0.872) respectively; the blood plasma CECR1 level in the verification queue predicts that the AUCs of the cardiovascular death of the HCM patient and the AUCs of the SCD are 0.757 (95% CI 0.654-0.860) and 0.738 (95% CI 0.587-0.889) respectively; the method can predict cardiovascular death of HCM patients and poor prognosis of SCD, has universality and generalizability, is high in clinical acceptance, improves patient compliance, improves diagnosis efficiency, and provides technical support for precision medical treatment.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of biological medicine, and particularly relates to a biomarker and application thereof. BACKGROUND

[0002] Hypertrophic cardiomyopathy (HCM) is the most common genetic cardiovascular disease, with a prevalence of 1:500-1:200. The annual cardiovascular mortality rate of HCM is 1-2%, and the main feature is myocardial hypertrophy that cannot be explained by other cardiac load abnormalities such as hypertension, valvular disease, etc. alone or in combination. HCM has a wide clinical manifestation and a hidden course, and a considerable proportion of patients are ignored due to mild symptoms, while some patients have severe symptoms such as heart failure (HF, heart failure) and sudden cardiac death (SCD, sudden cardiac death).

[0003] In order to better predict the prognosis of HCM patients, the HCM Risk-SCD model was proposed by the European Society of Cardiology in 2014 to assess the risk of SCD in patients. For the high-risk population of the HCM Risk-SCD model, the guidelines recommend implanting an implantable cardioverter defibrillator (ICD, implantable cardioverter defibrillator) to reduce the incidence of SCD. The HCM Risk-SCD model is based on a series of patient characteristics, myocardial structure and electrocardiogram indicators, including maximum left ventricular wall thickness, non-sustained ventricular tachycardia, age, etc. However, there is still a lot of controversy about the effectiveness of the HCM Risk-SCD model. In 2020, the American Heart Association and the American College of Cardiology jointly issued the "2020 AHA / ACC Hypertrophic Cardiomyopathy Diagnosis and Treatment Guidelines", which added three risk factors of left ventricular ejection fraction <50%, late gadolinium enhancement and left ventricular apical aneurysm to the risk factors of the HCM Risk-SCD model. However, its verification effect is still not accurate, and the verification of the HCM patient population is not ideal. More importantly, the current prognosis management of HCM only considers the risk of SCD, but does not consider the risk of non-sudden death and other cardiovascular adverse events.

[0004] Therefore, there is an urgent need for a marker with high specificity and sensitivity to accurately predict the adverse prognosis of HCM and guide clinical diagnosis and treatment. SUMMARY

[0005] The present application finds that the expression level of CECR1 is related to the prognosis of hypertrophic cardiomyopathy (HCM), and when the expression level of CECR1 in the biological sample of a patient with hypertrophic cardiomyopathy is significantly reduced, the prognosis of the subject is poor. Based on this, the present application is completed.

[0006] In a first aspect, the present application provides a marker for predicting prognosis of hypertrophic cardiomyopathy (HCM), wherein the marker is CECR1.

[0007] Further, when the expression level of CECR1 in the biological sample of the subject is significantly reduced, the prognosis of the subject is poor.

[0008] Further, when the expression level of CECR1 in the biological sample of the subject is less than 1738 ng / mL, the prognosis of the subject is poor.

[0009] Further, the poor prognosis includes cardiovascular death and sudden cardiac death.

[0010] Further, the subject is a patient with hypertrophic cardiomyopathy.

[0011] Further, the biological sample of the subject is blood and / or tissue.

[0012] Further, the blood sample of the subject is at least one of peripheral blood, plasma and / or serum.

[0013] In a second aspect, the present application provides use of a reagent for detecting the expression level of the marker of the first aspect of the present application in the preparation of a reagent for predicting the prognosis of hypertrophic cardiomyopathy, wherein the marker is CECR1.

[0014] Further, when the expression level of CECR1 in the biological sample of the subject is significantly reduced, the prognosis of the subject is poor.

[0015] Further, when the expression level of CECR1 in the biological sample of the subject is less than 1738 ng / mL, the prognosis of the subject is poor.

[0016] Further, the poor prognosis includes cardiovascular death and sudden cardiac death.

[0017] Further, the subject is a patient with hypertrophic cardiomyopathy.

[0018] Further, the biological sample of the subject is blood and / or tissue.

[0019] Further, the blood sample of the subject is at least one of peripheral blood, plasma and / or serum.

[0020] In a third aspect, the present application provides a kit for predicting the prognosis of hypertrophic cardiomyopathy, wherein the kit contains a reagent for detecting the expression level of the marker of the first aspect of the present application, wherein the marker is CECR1.

[0021] Further, when the expression level of CECR1 in the biological sample of the subject is significantly reduced, the prognosis of the subject is poor.

[0022] Further, when the expression level of CECR1 in the biological sample of the subject is less than 1738 ng / mL, the subject has poor prognosis.

[0023] Further, the poor prognosis comprises cardiovascular death and sudden cardiac death.

[0024] Further, the subject is a patient with hypertrophic cardiomyopathy.

[0025] Further, the biological sample of the subject is blood and / or tissue.

[0026] Further, the blood sample of the subject is at least one of peripheral blood, plasma and / or serum.

[0027] Beneficial effects The biomarker of the present application is simple to sample, can be detected in tissue, and can be detected in peripheral blood. Compared with the prior art, the detection index is less, and the report result is fast. The biomarker of the present application has high accuracy: the AUCs of plasma CECR1 level in the screening cohort to predict cardiovascular death and SCD of HCM patients are 0.720 (95% CI 0.657-0.783) and 0.798 (95% CI 0.725-0.872), respectively; the AUCs of plasma CECR1 level in the verification cohort to predict cardiovascular death and SCD of HCM patients are 0.757 (95% CI 0.654-0.860) and 0.738 (95% CI 0.587-0.889), respectively; the biomarker can predict the poor prognosis of cardiovascular death and SCD of HCM patients, has universality and generalizability, has high acceptance in clinic, improves patient compliance, improves diagnosis efficiency, and provides technical support for precision medicine. BRIEF DESCRIPTION OF DRAWINGS

[0028] Figure 1 Figure 246 is a result of multiple survival analysis of 246 HCM patients.

[0029] Note: 1A is a Manhattan plot of the correlation between HCM gene expression level and cardiovascular death, and it is found that CECR1 is the most significantly related gene to HCM cardiovascular death; 1B is a ROC curve of CECR1 level about cardiovascular death.

[0030] Figure 2 Figure 20 is a result of Western Blot and qPCR test of 20 tissue samples.

[0031] Note: 2A is the statistical chart of the difference between groups of the relative expression level of CECR1 mRNA (n=10 in each group); 2B is the representative chart of the difference between groups of the relative expression level of CECR1 protein (n=10 in each group); 2C is the statistical chart of the difference between groups of the relative expression level of CECR1 protein (n=10 in each group).

[0032] Figure 3 Figure 2D is the correlation scatter plot of the expression level of CECR1 in myocardial tissue and plasma of 43 HCM patients.

[0033] Figure 4 Figure 3D is the correlation scatter plot of the expression level of CECR1 in myocardial tissue and plasma of 43 HCM patients.

[0034] Note: 4A is the distribution result of the expression level of plasma CECR1; 4B is the ROC curve of plasma CECR1 predicting HCM cardiovascular death; 4C is the KM curve of plasma CECR1 level about HCM cardiovascular death; 4D is the ROC curve of plasma CECR1 predicting cardiac sudden death; 4E is the KM curve of plasma CECR1 level about cardiac sudden death.

[0035] Figure 5 Figure 5D is the ROC curve of plasma CECR1 predicting cardiac sudden death; 5E is the KM curve of plasma CECR1 level about cardiac sudden death.

[0036] Note: 5A is the distribution result of the expression level of plasma CECR1; 5B is the ROC curve of plasma CECR1 predicting HCM cardiovascular death; 5C is the KM curve of plasma CECR1 level about HCM cardiovascular death; 5D is the ROC curve of plasma CECR1 predicting cardiac sudden death; 5E is the KM curve of plasma CECR1 level about cardiac sudden death. DETAILED DESCRIPTION

[0037] The specific embodiments of the present application are described below. It should be noted that the description of the embodiments is for the purpose of helping understand the present application, but does not constitute a limitation on the present application. In addition, the technical features involved in the following described embodiments can be combined with each other as long as they do not conflict with each other.

[0038] The experimental methods in the following examples are all conventional methods unless otherwise specified. The experimental materials used in the following examples are all commercially available unless otherwise specified.

[0039] Explanation of terms KM (Kaplan-Meier) Survival Curve: A powerful statistical tool used to analyze survival time data. It reveals the probability of a specific event (such as death, recurrence, or treatment failure) occurring within a certain time frame.

[0040] Log-Rank Test: A non-parametric statistical method used to compare differences between survival curves of two or more groups. It is one of the core tools in survival analysis, often used in conjunction with the Kaplan-Meier curve. The main purpose of the Log-Rank test is to determine whether there are significant differences in survival curves between different groups through hypothesis testing. The null hypothesis (H0) is that the survival functions of all groups are the same, and the alternative hypothesis (H1) is that at least one group is different.

[0041] Log-Rank One-Sided p-Value: A one-sided p-value is a result of hypothesis testing for a specific direction, indicating the probability of observing the current data under the assumption that one group has a higher or lower survival rate.

[0042] Example 1 - Screening criteria Inclusion criteria: 1) Clinical diagnosis of HCM; diagnostic criteria: echocardiography or magnetic resonance imaging left ventricular end-diastolic thickness ≥ 15 mm at any site can be diagnosed, and positive pathogenic gene detection or genetic involvement family members can also be diagnosed.

[0043] 2) Accepting interventricular septum resection and retaining frozen tissue.

[0044] Exclusion criteria: 1) Known or suspected to have a phenotype disease that can also cause myocardial hypertrophy, such as amyloid myocardial disease.

[0045] 2) Poor sequencing quality.

[0046] 3) Loss of follow-up.

[0047] Example 2 - Screening trial (tissue) A. Trial method The total RNA of myocardial tissue was extracted using Qiagen's RNeasy Mini kit. The concentration and A260 / A280 ratio of the extracted total RNA were measured using a NanoDrop spectrophotometer. The quality and RNA integrity (RNA integrity number, RIN) of the RNA were detected using the Qubit RNA IQ assay kit Qubit 4 fluorometer. The samples with A260 / A280 ratio of 1.8-2.0 and RIN value >7.5 were subjected to cDNA library construction and subsequent mRNA sequencing.

[0048] The library construction was performed according to the standard protocol using Roche's KAPA mRNA Hyper Prep kit. Sequencing was performed on Illumina's PE150 model Hiseq X-ten platform. Nine libraries were loaded on randomly selected lanes, each with more than 30M paired-end reads. The basic principle of sequencing is sequencing by synthesis. Using Trimmonmatic (v0.36), adapter sequence data (reads), low-quality reads, and N-containing (N represents undetermined base information) reads were removed to obtain clean reads. Using TopHat (v2.1.1) and Bowtie2 (v2.3.4), clean reads were aligned to the reference genome (GRCH37 / hg19). Using Cufflinks (v2.2.1), transcript assembly and FPKM calculation were performed. The log-rank value of the relationship between the FPKM value of each gene in the cohort and cardiovascular death was calculated.

[0049] B. Trial results As shown in Figure 1 , mRNA transcriptome sequencing was performed on myocardial tissue after interventricular septal resection in 246 HCM patients, and multiple survival analysis showed that CECR1 the expression level of the gene was most closely related to the prognosis of HCM patients (log-rank P = 9.6 x 10 -5 ( Figure 1 A). At the tissue level, the ROC curve of CECR1 level with respect to cardiovascular death is shown in Figure 1 B, and the AUC of CECR1 for predicting cardiovascular death in HCM patients was 0.801 (95% CI 0.723-0.880).

[0050] Example 3 - Wet test to validate the relationship between CECR1 and HCM cardiovascular death (tissue) A. Trial method Tissue from 10 HCM patients who had cardiovascular death were selected, which were different from the samples selected in Example 2. Tissue from 10 HCM patients who had no cardiovascular death (i.e. survived) were matched according to age and gender. The CECR1 protein and mRNA expression levels of the 20 patients were determined by Western Blot and qPCR. The specific experimental methods are as follows: Western Blot: The myocardial tissue was immediately frozen in liquid nitrogen after excision. The frozen myocardial tissue was ground and pulverized in liquid nitrogen, and resuspended in 500 μl of RIPA lysis buffer (Bi Yun Tian) containing protease and phosphatase inhibitors (Roche), and incubated on ice for 60 minutes to ensure complete extraction. The tissue homogenate was centrifuged at 15,000g for 30 minutes at 4°C. The supernatant was transferred to a new tube, and the total protein concentration of each sample was determined using the BCA protein detection kit (Thermo Fisher Scientific). 5x SDS-PAGE loading buffer was added to the total protein, mixed and incubated for 5 minutes to denature the protein. 10 μg of protein was loaded into a 4%-12% polyacrylamide gel (Applygen) for electrophoresis, and then transferred to a methanol-activated polyvinylidene fluoride membrane (Bio-Rad) at a current of 300 mA. The membrane was blocked with 5% skim milk at room temperature for 3 hours, and then incubated with different primary antibodies (CECR1, GAPDH) at 4°C overnight. After incubation with the secondary antibody, the immunoreactive bands were developed using the ultra-sensitive chemiluminescent substrate (Thermo Fisher Scientific). The band intensity was analyzed by ImageJ software and calibrated with the intensity of GAPDH in each membrane.

[0051] qPCR analysis: 50 mg of frozen myocardial tissue was added with 1 mL of Trizol (Ambion) for homogenization. Then 200 μl of chloroform was added to the homogenate, mixed thoroughly and incubated on ice for 30 minutes. After centrifugation at 15,000g for 30 minutes at 4°C, the supernatant was taken and 500 μl of isopropanol was added, and the sample was incubated at -20°C overnight. The RNA was obtained by centrifugation at 15,000g for 30 minutes at 4°C, and washed twice with 75% ethanol. The RNA concentration, purity and integrity were evaluated by the foregoing method. The total RNA was reverse transcribed using the RT kit (Takara). Real-time quantitative PCR was performed using the TB Green PCR reagent kit (Takara). The relative expression of the target gene in each group was calculated by the 2 −ΔΔCt method, and the expression of GAPDH was standardized.

[0052] B. Trial results like Figure 2 As shown, Western blot and qPCR experiments confirmed that the relative mRNA expression level of CECR1 in the myocardial tissue of HCM patients who experienced cardiovascular death ( Figure 2 A) and protein ( Figure 2 The relative expression levels of B and C were significantly lower than those in HCM patients who did not experience cardiovascular death (relative mRNA expression level: p =0.0024; Relative protein expression level: p =0.0056). The above results demonstrate a clear relationship between the relative expression level of CECR1 and HCM patients who died from cardiovascular disease, namely, the relative expression level of CECR1 was significantly reduced in HCM patients who died from cardiovascular disease.

[0053] Example 4 - Screening trial (plasma and tissue correlation of expression) A. Trial method Forty-three HCM patients with both tissue mRNA transcriptome sequencing results and plasma samples were selected, representing cases entirely different from those selected in Examples 2 and 3. Plasma CECR1 concentrations in these 43 patients were measured using ELISA.

[0054] ELISA Method: Plasma CECR1 levels were detected using an enzyme-linked immunosorbent assay (ELISA) kit (NOVUS), strictly following the manufacturer's instructions. Human plasma samples were collected upon enrollment with the patient's written consent and stored at -80°C until use. Before testing, plasma samples were thawed and thoroughly mixed. For CECR1 detection, plasma samples were diluted 200-fold with sample diluent and incubated at 37°C for 90 minutes; subsequently, they were incubated sequentially with the biotinylated detection antibody for 60 minutes and with the HRP conjugate for 30 minutes. Substrate reagents were added sequentially at a constant rate, and stop solution was added in the same manner after complete reaction. Colorimetric intensity was measured at 340 nm using a spectrophotometer (TECAN). CECR1 concentration was calculated based on the standard curve and dilution factor.

[0055] B. Trial results like Figure 3 As shown, the tissue expression level and plasma expression level of CECR1 in 43 patients were significantly correlated (r=0.317; p=0.038).

[0056] Example 5 - Screening trial (plasma) A. Trial method Plasma samples were collected from 1238 HCM patients in Fu Wai Hospital, Chinese Academy of Medical Sciences, which were completely different from the selected samples in Examples 2-4. After a median follow-up of 2.5 years, 53 patients had cardiovascular death, of which 28 were sudden cardiac death (SCD). The plasma of these patients was detected for CECR1 concentration.

[0057] ELISA method: The plasma CECR1 level was detected by enzyme-linked immunosorbent assay (ELISA) kit (NOVUS), and the operation strictly followed the manufacturer's instructions. Human plasma samples were collected after obtaining the written consent of the patients at the time of enrollment, and were stored at -80°C until use. Before detection, the plasma samples were thawed and thoroughly mixed. When used for CECR1 detection, the plasma samples were diluted 200 times with sample diluent and incubated at 37°C for 90 minutes; then sequentially incubated with biotinylated detection antibody for 60 minutes, and with HRP conjugate for 30 minutes. The substrate reagent was added at a constant rate, and after sufficient reaction, the stop solution was added in the same way. The color development intensity was detected by spectrophotometer (TECAN) at 340 nm wavelength. The CECR1 concentration was calculated according to the standard curve and dilution factor.

[0058] B. Trial results As shown in Figure 4 , the expression level of CECR1 in patient plasma was detected by ELISA. The expression level of plasma CECR1 was 2073.1 (1581.1-2848.6) ng / mL, and the expression distribution was as shown in Figure 4 A. The decrease of plasma CECR1 expression level was associated with increased risk of cardiovascular death (per 1000 ng / mL decrease: adjusted HR 3.310, 95% CI 2.052-5.339, P < 0.001) and SCD (per 1000 ng / mL decrease: adjusted HR 6.394, 95% CI 2.891-14.145, P < 0.001).

[0059] As shown in Figure 4 B, the AUC of plasma CECR1 expression level for predicting cardiovascular death in HCM patients was 0.720 (95% CI 0.657-0.783), and the optimal threshold was defined as 1738 ng / mL by the Youden index. As shown in Figure 4 C, the KM curve showed that the risk of cardiovascular death in HCM patients with plasma CECR1 concentration below 1738 ng / mL was significantly increased (log-rank P<0.001), proving that the threshold is reliable.

[0060] For SCD outcome, the AUC of plasma CECR1 level to predict SCD in HCM patients was 0.798 (95% CI 0.725-0.872) (Fig. 1A). Figure 4 D). KM curve showed that the risk of SCD was also significantly increased when plasma CECR1 concentration was lower than 1738 ng / mL in HCM patients (log-rank P < 0.001) (Fig. 1D). Figure 4 E).

[0061] Example 6 - Validation trial (plasma) A. Trial method A total of 402 HCM patients from the First Affiliated Hospital of the Fourth Military Medical University (Xijing Hospital) were enrolled, which were completely different from the samples selected in Examples 2-5, with a median follow-up of 4.8 years, of which 16 cases of cardiovascular death and 10 cases of SCD. The plasma of these patients was detected for CECR1 concentration.

[0062] ELISA method: The plasma CECR1 level was detected by enzyme-linked immunosorbent assay (ELISA) kit (NOVUS), and the operation strictly followed the manufacturer's instructions. Human plasma samples were collected after obtaining the written consent of the patients at the time of enrollment, and were stored at -80°C until use. Before detection, the plasma samples were thawed and thoroughly mixed. When used for CECR1 detection, the plasma samples were diluted 200 times with sample diluent, incubated at 37°C for 90 minutes; then sequentially incubated with biotinylated detection antibody for 60 minutes, and with HRP conjugate for 30 minutes. The substrate reagent was added at a constant rate, and after sufficient reaction, the termination liquid was added in the same way. The color development intensity was measured by spectrophotometer (TECAN) at 340 nm wavelength. The CECR1 concentration was calculated according to the standard curve and dilution factor.

[0063] B. Trial results As shown in Figure 5 , in this validation cohort, the plasma expression level of CECR1 was 1965.2 (1549.6-2531.3) ng / mL, and the expression distribution was as shown in Figure 5 A. The decrease of plasma CECR1 level was associated with increased risk of cardiovascular death (per 1000 ng / mL decrease: adjusted HR 6.551, 95% CI 2.247-19.097, P < 0.001) and SCD (per 1000 ng / mL decrease: adjusted HR 4.822, 95% CI 1.471-15.801, P = 0.009).

[0064] The results showed that in this validation cohort, with a threshold of 1738 ng / mL, the AUCs of plasma CECR1 levels for predicting cardiovascular death and SCD in HCM patients were 0.757 (95% CI 0.654-0.860) and 0.738 (95% CI 0.587-0.889), respectively Figure 5 B-5C). KM curves showed that the risk of cardiovascular death (log-rank P < 0.001) and SCD (log-rank P = 0.003) was significantly increased when the plasma CECR1 concentration in HCM patients was lower than 1738 ng / mL Figure 5 D-5E). The above results proved the universality and generalizability of the index.

Claims

1. A marker for the prognosis of hypertrophic cardiomyopathy (HCM), the marker being CECR1; when CECR1 is significantly decreased in a biological sample of a subject, the prognosis of the subject is poor.

2. The marker of claim 1, wherein the subject is a patient with hypertrophic cardiomyopathy; the biological sample of the subject is blood and / or tissue; the poor prognosis includes cardiovascular death and sudden cardiac death.

3. Use of a reagent for detecting the expression level of the marker of claim 1 in the preparation of a reagent for predicting the prognosis of hypertrophic cardiomyopathy; the marker is CECR1.

4. The use of claim 3, wherein when CECR1 is significantly decreased in a biological sample of a subject, the prognosis of the subject is poor.

5. The use of claim 4, wherein the subject is a patient with hypertrophic cardiomyopathy; the biological sample of the subject is blood and / or tissue; the poor prognosis includes cardiovascular death and sudden cardiac death.

6. A kit for predicting the prognosis of hypertrophic cardiomyopathy, the kit containing a reagent for detecting the expression level of the marker of claim 1; the marker is CECR1.

7. The kit of claim 6, wherein when CECR1 is significantly decreased in a biological sample of a subject, the prognosis of the subject is poor.

8. The kit of claim 7, wherein the subject is a patient with hypertrophic cardiomyopathy; the biological sample of the subject is blood and / or tissue; the poor prognosis includes cardiovascular death and sudden cardiac death.

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