CN114959006A - Application of plasma cfDNA in diagnosis of coronary artery stenosis of suspected coronary artery disease patient - Google Patents

Abstract

The invention relates to the technical field of free DNA application, in particular to application of plasma cfDNA in diagnosis of coronary artery stenosis of a suspected coronary artery disease patient. The invention provides a method for indirectly judging the coronary stenosis condition according to the blood plasma cfDNA level, provides more coronary stenosis evaluation basis for suspected coronary artery disease patients who are proposed to carry out coronary angiography, and reduces unnecessary invasive coronary angiography; the method can also be used for screening patients with high risk coronary heart disease, plays a certain early warning role in coronary heart disease, improves the diagnosis accuracy and reduces unnecessary medical expenses.

Description

Application of plasma cfDNA in the diagnosis of coronary stenosis in patients with suspected coronary artery disease

technical field

The invention relates to the technical field of cell-free DNA application, in particular to the application of plasma cfDNA in diagnosing coronary artery stenosis in patients with suspected coronary artery disease.

Background technique

Coronary artery disease (CAD) is a disease that seriously threatens health, and its pathological characteristics are the accumulation of atherosclerotic plaques in the coronary walls, leading to narrowing or even blockage of the coronary arteries and reduced blood supply to the heart. As the most common type of heart disease, CAD is the leading cause of death in the world. CAD may cause acute cardiovascular events, such as myocardial infarction, and may also progress to heart failure over time. Early diagnosis and accurate assessment of coronary artery stenosis in patients with suspected CAD are important for implementing appropriate medical care to reduce cardiac ischemic injury and improve patient outcomes.

At present, imaging methods such as non-invasive coronary computed tomography (CCTA) and invasive coronary angiography (CAG) can be used to directly assess the presence and degree of coronary stenosis in clinical practice. However, CCTA and CAG have certain risks and limitations. For example, iodinated contrast media used in CCTA procedures may cause severe allergic reactions in some patients. In addition, extremely obese patients and patients with abnormal heart rhythms such as atrial fibrillation are not suitable for evaluation of their coronary arteries with CCTA due to disturbed image quality. In addition, vascular calcification is common in elderly patients, and it is difficult to identify the actual degree of stenosis under CCTA. In the case of CAG, it directly determines the location and severity of coronary stenosis and is currently considered the gold standard technique for diagnosing CAD. In addition to the use of contrast agents, CAG procedures involve catheterization and are therefore invasive. Complications and adverse reactions may occur in CAG surgery, including but not limited to infection, catheter arterial injury, excessive bleeding, arrhythmia, myocardial infarction, stroke, etc. Given the inherent limitations and potential risks of current forms of CCTA and CAG imaging modalities, there is a need to research and develop new non-invasive diagnostic methods to complement and improve the clinical assessment of coronary artery stenosis in patients with suspected CAD.

Circulating cell-free DNA (cfDNA) describes the extracellular DNA fragments present in blood and other types of bodily fluids. Since its discovery in the plasma of healthy subjects in 1948, cfDNA has gained increasing attention as a non-invasive biomarker for disease diagnosis and prognosis. Elevated levels of circulating cfDNA are seen in various diseases such as cancer, autoimmune rheumatism, aging, and stroke. The relationship between changes in circulating cfDNA levels and cardiac diseases such as myocardial infarction and heart failure has also been confirmed. However, the use of circulating cfDNA as a non-invasive biomarker in the diagnostic assessment of coronary stenosis has not been reported.

SUMMARY OF THE INVENTION

In order to solve the above problems, the purpose of the present invention is to provide an application of plasma cfDNA in diagnosing coronary artery stenosis in patients with suspected coronary artery disease. The present invention proposes that coronary artery stenosis can be indirectly judged according to the level of plasma cfDNA, provides more coronary artery stenosis assessment basis for patients with suspected coronary artery disease who are going to undergo coronary angiography, and reduces unnecessary invasive coronary angiography; Screening high-risk patients with coronary heart disease can play a certain early warning role in coronary heart disease, improve the accuracy of diagnosis, and reduce unnecessary medical expenses.

The object of the present invention can be realized through the following technical solutions:

The first object of the present invention is to provide the application of plasma cfDNA in the preparation of reagents for early diagnosis of coronary stenosis, and the level of plasma cfDNA is 1258-1259 ng/ml as the cut-off value for diagnosing coronary stenosis.

In one embodiment of the present invention, when the level of plasma cfDNA is lower than 1258-1259ng/ml, the coronary artery is normal; when the level of plasma cfDNA is higher than 1258-1259ng/ml, it is coronary artery stenosis; when the plasma cfDNA level is higher than 1258-1259ng/ml When the level is 1258-1259ng/ml, it is suspected coronary stenosis.

The second object of the present invention is to provide the application of plasma cfDNA in the preparation of a kit for early diagnosis of coronary stenosis, and the level of plasma cfDNA is 1258-1259 ng/ml as a cutoff value for diagnosing coronary stenosis.

In one embodiment of the present invention, when the level of plasma cfDNA is lower than 1258-1259ng/ml, the coronary artery is normal; when the level of plasma cfDNA is higher than 1258-1259ng/ml, it is coronary artery stenosis; when the plasma cfDNA level is higher than 1258-1259ng/ml When the level is 1258-1259ng/ml, it is suspected coronary stenosis.

The third object of the present invention is to provide the application of a substance for detecting the level of plasma cfDNA in the preparation of a reagent for early diagnosis of coronary stenosis.

In one embodiment of the present invention, when the level of plasma cfDNA is lower than 1258-1259ng/ml, the coronary artery is normal; when the level of plasma cfDNA is higher than 1258-1259ng/ml, it is coronary artery stenosis; when the plasma cfDNA level is higher than 1258-1259ng/ml When the level is 1258-1259ng/ml, it is suspected coronary stenosis.

The fourth object of the present invention is to provide the application of a substance for detecting plasma cfDNA level in the preparation of a kit for early diagnosis of coronary stenosis.

In one embodiment of the present invention, when the level of plasma cfDNA is lower than 1258-1259ng/ml, the coronary artery is normal; when the level of plasma cfDNA is higher than 1258-1259ng/ml, it is coronary artery stenosis; when the plasma cfDNA level is higher than 1258-1259ng/ml When the level is 1258-1259ng/ml, it is suspected coronary stenosis.

The fifth object of the present invention is to provide the application of plasma cfDNA as a therapeutic target in the preparation of anti-coronary stenosis drugs.

The sixth object of the present invention is to provide the application of plasma cfDNA inhibitors in the preparation of anti-coronary stenosis drugs.

Compared with the prior art, the present invention has the following beneficial effects:

(1) The present invention collects patients with suspected coronary artery disease who are admitted for coronary angiography, and analyzes the differences in plasma cfDNA levels when the baseline data of patients in different groups are consistent by grouping the angiography results;

(2) The present invention utilizes a statistical method to evaluate the specificity and sensitivity of the plasma cfDNA level for diagnosing the presence or absence of coronary stenosis.

Description of drawings

Figure 1 shows the plasma cfDNA levels of suspected CAD patients in Example 2 of the present invention. The Quant-iT PicoGreendsDNA detection kit was used to analyze the circulating cfDNA levels of plasma samples from 183 patients. Figure 1A shows the plasma cfDNA of Group A (non-CAD, n=51, including 19 male and 32 female patients) and Group B (mild and severe CAD, n=132, including 75 male and 57 female patients) Levels were compared in male patients, female patients, and total patients; Plasma cfDNA levels (including 18 male and 19 female patients) were compared in male patients, female patients, and all patients, respectively. Data are presented as box plots (median in interquartile range). P values were determined by the Mann-Whitney U test.

FIG. 2 is the ROC curve of the plasma cfDNA of the suspected CAD patient in Example 2 of the present invention. After the plasma cfDNA level of the patient is detected, the method of Hanley and McNeil is used for ROC analysis. Figure 2A shows the ROC curves of circulating cfDNA drawn from the analysis of Category A including patients in Group A (non-CAD) and Group B (mild and severe CAD); Drawn by Category B analysis of Group (non-CAD) and Group C (mild CAD) patients. Labels in the figure: ROC: receiver operating characteristic analysis; AUC: area under the ROC curve.

Detailed ways

The invention provides the application of plasma cfDNA in preparing reagent for early diagnosis of coronary stenosis, and the level of plasma cfDNA is 1258-1259 ng/ml as the cut-off value for diagnosing coronary stenosis.

In one embodiment of the present invention, when the level of plasma cfDNA is lower than 1258-1259ng/ml, the coronary artery is normal; when the level of plasma cfDNA is higher than 1258-1259ng/ml, it is coronary artery stenosis; when the plasma cfDNA level is higher than 1258-1259ng/ml When the level is 1258-1259ng/ml, it is suspected coronary stenosis.

The invention provides the application of plasma cfDNA in the preparation of an early diagnosis kit for coronary stenosis, and the level of plasma cfDNA is 1258-1259 ng/ml as a cutoff value for diagnosing coronary stenosis.

In one embodiment of the present invention, when the level of plasma cfDNA is lower than 1258-1259ng/ml, the coronary artery is normal; when the level of plasma cfDNA is higher than 1258-1259ng/ml, it is coronary artery stenosis; when the plasma cfDNA level is higher than 1258-1259ng/ml When the level is 1258-1259ng/ml, it is suspected coronary stenosis.

The invention provides the application of a substance for detecting the level of plasma cfDNA in the preparation of a reagent for early diagnosis of coronary stenosis.

In one embodiment of the present invention, when the level of plasma cfDNA is lower than 1258-1259ng/ml, the coronary artery is normal; when the level of plasma cfDNA is higher than 1258-1259ng/ml, it is coronary artery stenosis; when the plasma cfDNA level is higher than 1258-1259ng/ml When the level is 1258-1259ng/ml, it is suspected coronary stenosis.

The invention provides the application of a substance for detecting plasma cfDNA level in preparing a kit for early diagnosis of coronary artery stenosis.

In one embodiment of the present invention, when the level of plasma cfDNA is lower than 1258-1259ng/ml, the coronary artery is normal; when the level of plasma cfDNA is higher than 1258-1259ng/ml, it is coronary artery stenosis; when the plasma cfDNA level is higher than 1258-1259ng/ml When the level is 1258-1259ng/ml, it is suspected coronary stenosis.

The invention provides the application of plasma cfDNA as a therapeutic target in the preparation of anti-coronary stenosis drugs.

The invention provides the application of plasma cfDNA inhibitor in the preparation of anti-coronary artery stenosis medicine.

The present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

This study analyzed 183 adult patients admitted to the Department of Cardiology, Yueyang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine. All patients had symptoms of chest discomfort and underwent CAG to assess the degree of coronary stenosis. The patient had no diagnosis of acute coronary syndrome and no history of systemic inflammatory disease, myocarditis, pericarditis, active infection, or cancer. This study complied with the ethical standards of the Declaration of Helsinki and was approved by the Ethics Review Committee of Yueyang Hospital Affiliated to Shanghai University of Traditional Chinese Medicine (approval number: 2021-025).

Example 1

1. Clinical and Laboratory Measurements

All participants underwent a complete medical history and thorough physical examination. Age, smoking and drinking habits, and comorbidities were recorded by structured questionnaires. Body temperature and cardiovascular risk factors were collected before CAG. In this study, smokers were defined as those who smoked ≥1 cigarette per day for at least 6 months. After an overnight fast, liver, kidney function, total cholesterol, triglycerides, and low-density lipoprotein cholesterol (LDL-C) were determined using an automated chemistry analyzer (AU5800 series clinical chemistry analyzer, Beckman Coulter). and high-density lipoprotein cholesterol (HDL-C, high-density lipoprotein cholesterol). High performance liquid chromatography (HCL-723, Tosoh Bioscience) was used to determine HbA1c. High-sensitivity CRP (hsCRP) was determined by particle-enhanced immunoturbidimetry (CRP-M100, Shenzhen Mingde Biomedical Electronics Co., Ltd.). D-dimer (DDi) was determined using a Sysmex CS-5100 (Siemens Healthineers). White blood cell (WBC) counts were determined by Mindray 6800plus (Shenzhen Mingde Biomedical Electronics Co., Ltd.).

2. Circulating cfDNA Measurements

Arterial blood was collected into 3.2% citric acid tubes by K2 EDTA vacutainers (Jiangsu Kangjian Medical Equipment Co.) before CAG surgery and processed within 1 hour after collection. Plasma was separated from EDTA-treated blood by centrifugation at 2000 xg for 10 min at room temperature. Plasma samples were stored at -80°C prior to analysis. Due to the potential impact of hemolysis on cfDNA levels, prior to cfDNA analysis, plasma samples were visually inspected for hemolysis and those with detectable hemolysis were excluded. Plasma cfDNA was quantified using the Quant-iT ^™ PicoGreen ^™ dsDNA Detection Kit (Cat#P11496, Lot#1911829, Molecular Probes, Invitrogen) as described. Samples were measured in triplicate using a fluorescence microplate reader (BioTek Epoch) at excitation and emission wavelengths of 480 nm and 520 nm, respectively.

3. Coronary angiography

CAG is routinely performed through the radial artery. In the event of unsuccessful puncture or radial vasospasm, the femoral artery is chosen as an alternative route. According to the American Heart Association, basic angiographic views include left anterior oblique, spider, anteroposterior plus foot, and right anterior oblique views of the left coronary artery; left anterior oblique, left anterior oblique plus head, and right anterior oblique views of the right coronary artery Oblique position, if necessary, adopt other positions. Lobital (Xenetix, Guerbet) was used as a contrast medium in patients with normal renal function, and lodisanol (Vispaque, GE Healthcare Ireland Limited Company) in patients with impaired renal function. The equipment for digital subtraction angiography was IGS 320 from General Electric Company. Interpretation of coronary angiography was performed independently by two attending cardiologists. After CAG, patients were assigned to different groups for designated comparisons. Group A included patients with no significant stenosis or luminal stenosis <30%, i.e. non-CAD. Group B included patients with luminal stenosis ≥30%, including mild (luminal stenosis ≥30% but <50%) and severe (luminal stenosis ≥50%) CAD. Group C included only patients with mild CAD. Category A refers to the comparison of groups A and B, and category B refers to the comparison of groups A and C.

4. Data Analysis

SAS software (version 9.4) was used for data analysis. Qualitative variables use frequency counts and proportions (percentages), and quantitative variables use mean and standard deviation or median and interquartile range (IQR) to describe data. Differences between quantitative variables were compared using Student's t-test, Mann-Whitney U test, or one-way ANOVA test. Differences between categorical data (binary or ordinal) were analyzed using Pearson's chi-square test (χ ² ). The linear correlation between different quantitative variables was evaluated according to the Pearson or Spearman correlation coefficient. Robust logistic regression analysis was performed to explore the relationship between plasma cfDNA levels and degree of luminal stenosis, and relative risk (OR) and 95% confidence interval (CI) were calculated accordingly. Receiver operating characteristic (ROC) analysis was used to determine cut-off points and their incremental values for circulating cfDNA predicting coronary stenosis. The area under the curve (AUC) of the ROC was compared using the method of Hanley and McNeil. P values less than 0.05 were considered statistically significant.

Example 2

1. Patient demographics and clinical characteristics

The demographics, clinical characteristics, and blood chemistry parameters of the 183 patients with symptoms of chest discomfort are summarized in Table 1. According to the degree of coronary stenosis defined by CAG examination, 183 patients were assigned to Group A (non-CAD, patients with no luminal stenosis or luminal stenosis <30%), Group B (mild and severe CAD, luminal stenosis ≥ 30%) 30% of patients) or Group C (mild CAD, luminal stenosis ≥30% but <50% of patients). The mean age of the patients was 65.4±10.4 years. Compared with group A, the age of patients in group B increased. Among all patients, 94 males (51.4%) and 89 females (48.6%). Among the 183 patients, the incidences of smoking and drinking were 24.0% and 25.7%, respectively. There were no significant differences in smoking and drinking between groups. Compared with group A, group B included more hypertensive patients. Except for hypertension, no differences in the distribution of diabetes, dyslipidemia, or cerebral infarction were found when comparing groups A and B or A and C. In addition, group B had higher levels of creatinine and DDi compared to group A. No differences were found between groups A and B or between groups A and C in HbA1c, HDL, LDL, cholesterol, triglycerides, leukocytes, hsCRP, BNP, albumin, or EF levels.

Table 1. Demographic and clinical characteristics of patients with suspected CAD

A组和B组之间的差异有统计学意义(P<0.05)。

The difference between group A and group B was statistically significant (P<0.05).

Abbreviations: BNP, brain natriuretic peptide; CAG, coronary angiography; hsCRP, high-sensitivity C-reactive protein; DDi, D-dimer; EF, ejection fraction; HbA1c, glycosylated hemoglobin; HDL, high-density lipoprotein; IQR, four Quantile range; LDL, low-density lipoprotein; PCI, percutaneous coronary intervention; SD, standard deviation; WBC, white blood cells.

2. Elevated plasma cfDNA levels in patients with coronary artery stenosis

To explore the potential of plasma cfDNA as a biomarker for assessing coronary stenosis, we further analyzed patients' plasma cfDNA levels. As shown in Table 2, compared with group A, plasma cfDNA levels in group B were significantly increased. In addition, given that Group B consisted of patients with mild and severe luminal stenosis, to assess whether plasma cfDNA levels differed in patients with only mild CAD from those with a CAD diagnosis excluded, patients with mild coronary stenosis ( Group C) was compared with the patients of Group A. Results showed that compared with patients in group A, patients in group C had significantly higher plasma cfDNA levels, even after adjusting for confounders. Meanwhile, higher plasma cfDNA levels were observed in male patients, hypertensive patients, and patients without a history of PCI. In addition, although no statistically significant difference was found, compared with group A, there was a trend for increased plasma cfDNA levels in male patients in groups B and C, but not in females (Figure 1). These results not only demonstrate elevated plasma cfDNA levels in patients with coronary artery stenosis defined by CAG, but also suggest that circulating cfDNA has the potential to be useful in distinguishing mild CAD from non-CAD in patients with chest discomfort.

Table 2. Plasma cfDNA levels in patients with suspected CAD

组间差异有统计学意义(P<0.05)。Robust regression analysis of differences in plasma cfDNA levels between groups A and B and between groups A and C, adjusted for sex (mutually adjusted covariates in the regression were selected by the directed acyclic graph method) .

The difference between groups was statistically significant (P<0.05).

3. Diagnostic performance of circulating cfDNA in patients with coronary artery stenosis

Next, patients in groups A and B were classified into category A, and patients in groups A and C into category B, followed by ROC analysis to further evaluate the diagnostic performance of plasma cfDNA in assessing coronary stenosis in patients with suspected CAD. As shown in Table 3 and Figure 2, ROC analysis showed that the optimal cutoff value (ie, Optimal cutoff value, the same as below) of plasma cfDNA in group B patients was 1258.39 ng/ml (AUC: 0.604; 95% CI: [0.508] -0.701]; p=0.029) with a sensitivity of 72% and a specificity of 51%. Meanwhile, in patients with category B, the optimal cutoff value of plasma cfDNA in group C was 1258.84 ng/mL (AUC: 0.627; 95% CI: [0.510-0.745]; P=0.042), and its sensitivity was 76%, specificity Sex is 51%. These results suggest that, in this study system, plasma cfDNA levels of 1258-1259 ng/ml can be used as a cut-off value for identifying coronary stenosis, supporting the diagnostic accuracy of plasma cfDNA in predicting coronary stenosis in patients with suspected CAD.

Table 3. ROC analysis of plasma cfDNA from patients with suspected CAD

Category A includes patients in Groups A and B; Category B includes patients in Groups A and C. AUC: area under the ROC curve; CI: confidence interval; ROC: receiver operating characteristic.

4. Validation of the diagnostic accuracy of plasma cfDNA

To verify the diagnostic accuracy of plasma cfDNA in assessing coronary stenosis, the percentile method was further applied. Plasma cfDNA levels were divided into 19 intervals (from P5 to P95), and then ROC analysis was performed to evaluate the detection effect of the 19 cfDNA values. As shown in Table 4, the level of 1258.85 ng/mL corresponding to P35 proved to be the cutoff value for plasma cfDNA in patients with Category A with a sensitivity of 78.99% and a specificity of 40.63%. Likewise, the cut-off value for Category B patients was 1259.80 ng/mL, corresponding to P40, with a sensitivity of 75.68% and a specificity of 50.98%. These results confirm that plasma cfDNA levels of 1258-1259 ng/mL can predict the presence of CAG-defined coronary stenosis in the patients included in this study.

The present study demonstrates that plasma cfDNA levels are elevated in patients with coronary artery stenosis, laying a preliminary basis for the development of plasma cfDNA-based assays to facilitate non-invasive or minimally invasive evaluation of coronary artery stenosis in patients with suspected CAD.

Table 4. Validation of the diagnostic performance of plasma cfDNA for assessing coronary stenosis in patients with suspected CAD

Category A includes patients in groups A and B, and category B includes patients in groups A and C.

The foregoing description of the embodiments is provided to facilitate understanding and use of the invention by those of ordinary skill in the art. It will be apparent to those skilled in the art that various modifications to these embodiments can be readily made, and the generic principles described herein can be applied to other embodiments without inventive step. Therefore, the present invention is not limited to the above-mentioned embodiments, and improvements and modifications made by those skilled in the art according to the disclosure of the present invention without departing from the scope of the present invention should all fall within the protection scope of the present invention.

Claims (10)

1. The application of the plasma cfDNA in the preparation of the reagent for early diagnosis of coronary artery stenosis is characterized in that the level of the plasma cfDNA is used as a boundary value for diagnosing the coronary artery stenosis when 1258-1259 ng/ml.

2. The use of the plasma cfDNA in the preparation of an agent for the early diagnosis of coronary stenosis according to claim 1, wherein when the level of the plasma cfDNA is lower than 1258-1259ng/ml, the coronary artery is normal; coronary artery stenosis when plasma cfDNA levels were above 1258-1259 ng/ml; when the plasma cfDNA level was 1258-1259ng/ml, it was suspected coronary stenosis.

3. The application of the plasma cfDNA in the preparation of the kit for early diagnosis of coronary artery stenosis is characterized in that the level of the plasma cfDNA is used as a boundary value for diagnosing the coronary artery stenosis when 1258-1259 ng/ml.

4. The use of the plasma cfDNA in the preparation of a kit for the early diagnosis of coronary stenosis as claimed in claim 3, wherein when the plasma cfDNA level is lower than 1258-1259ng/ml, the coronary artery is normal; coronary artery stenosis when plasma cfDNA levels were above 1258-1259 ng/ml; when the plasma cfDNA level was 1258-1259ng/ml, it was suspected coronary stenosis.

5. The application of the substance for detecting the plasma cfDNA level in the preparation of the reagent for early diagnosis of coronary artery stenosis is characterized in that the plasma cfDNA level is used as a boundary value for diagnosing the coronary artery stenosis when 1258-1259 ng/ml.

6. The use of the substance for detecting the plasma cfDNA level in the preparation of the reagent for the early diagnosis of coronary artery stenosis as claimed in claim 5, wherein when the plasma cfDNA level is lower than 1258-1259ng/ml, the coronary artery is normal; coronary artery stenosis when plasma cfDNA levels were above 1258-1259 ng/ml; when the plasma cfDNA level was 1258-1259ng/ml, it was suspected coronary stenosis.

7. The application of the substance for detecting the plasma cfDNA level in the preparation of the kit for early diagnosis of coronary artery stenosis is characterized in that the plasma cfDNA level is used as a cut-off value for diagnosing the coronary artery stenosis when 1258-1259 ng/ml.

8. The use of the substance for detecting the plasma cfDNA level in the preparation of the kit for the early diagnosis of coronary artery stenosis as claimed in claim 7, wherein when the plasma cfDNA level is lower than 1258-1259ng/ml, the coronary artery is normal; coronary artery stenosis when plasma cfDNA levels were above 1258-1259 ng/ml; when the plasma cfDNA level was 1258-1259ng/ml, it was suspected coronary stenosis.

9. Application of blood plasma cfDNA as a therapeutic target in preparation of anti-coronary artery stenosis drugs.

10. Application of a plasma cfDNA inhibitor in preparation of a coronary artery stenosis resisting drug.

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