Coronary heart disease methylation markers and uses thereof

Abstract

The application belongs to the technical field of biomedicine, and particularly relates to a coronary heart disease methylation marker and application thereof. The coronary heart disease methylation marker is disclosed for the first time, and can be used for diagnosis, screening or risk stratification of coronary heart disease.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to methylation markers for coronary heart disease and their applications. Background Technology

[0002] Coronary artery disease (CAD) is an ischemic heart disease characterized by a group of clinical syndromes caused by narrowing of the coronary arteries due to atherosclerosis. These syndromes can lead to myocardial ischemia, dysfunction, and even irreversible myocardial necrosis. CAD and its associated acute clinical events, such as acute coronary syndrome and acute myocardial infarction, are among the leading causes of death worldwide. Although interventions targeting traditional risk factors such as hypertension and hyperlipidemia have significantly improved the prevention and control of CAD over the past few decades, early detection of CAD remains a serious challenge.

[0003] Currently, clinical assessment of coronary artery disease (CAD) primarily relies on imaging examinations (such as coronary CT angiography) and invasive functional examinations (such as coronary angiography). While these methods have high diagnostic value in mid-to-late-stage lesions with significant structural stenosis or occlusion, their application still has the following significant limitations: ① Invasive examinations carry certain risks: Invasive procedures such as angiography may cause complications such as bleeding, embolism, or vascular damage, and are not suitable for routine screening of asymptomatic individuals.

[0004] ② High cost and inconvenient operation: The purchase and maintenance costs of high-end imaging equipment are high, and the cost of a single examination is expensive, making it difficult to promote and apply in large-scale population screening or dynamic follow-up.

[0005] ③ Insufficient early warning capability: Existing imaging technologies often only show positive results when organic changes have already occurred in blood vessels (such as plaque formation or significant luminal stenosis), making it difficult to issue early warnings in the early stages of molecular and cellular dysfunction, thus limiting their role in disease prevention and early intervention.

[0006] ④ Difficulty in dynamic monitoring: Due to factors such as operational complexity and radiation exposure, existing methods are difficult to achieve low-cost, non-invasive, and continuous tracking of disease progression, treatment response, and prognosis.

[0007] In terms of biomarkers, although clinical practice has developed markers such as high-sensitivity troponin for the acute diagnosis of myocardial injury, and B-type natriuretic peptide (BNP) and N-terminal pro-B-type natriuretic peptide (NT-proBNP) as predictive indicators of acute heart failure, these markers have limited efficacy in screening for chronic diseases and cannot yet meet the clinical needs for early warning.

[0008] In recent years, epigenetic markers, especially DNA methylation, have shown great potential in the field of early disease diagnosis. DNA methylation is a stable and heritable epigenetic modification that participates in gene transcription regulation, cell differentiation, and embryonic development, playing a crucial role in mammalian gene regulation, genome stability, and development. In eukaryotes, methylation occurs only in cytosine, primarily at the cytosine-guanine (CpG) dinucleotide site. Each cell type in the human body has a unique DNA methylation profile, which is highly consistent across individuals and remains stably inherited, thus serving as an organ / cell-specific biomarker. Liquid biopsy technology has gained increasing attention in recent years due to its low invasiveness and high accuracy. Cell-free DNA (cfDNA) is a highly fragmented DNA molecule released into the circulatory system during cell apoptosis and necrosis. When the body is in a disease state, damaged organs / cells release more cfDNA into the bloodstream. Furthermore, cfDNA has a short half-life (approximately 5-150 minutes), allowing it to reflect the body's state in real time. This "global snapshot" characteristic makes it an increasingly desirable candidate biomarker for various diseases. Therefore, it is necessary to develop methylation markers for coronary artery disease. Summary of the Invention

[0009] The first aspect of the present invention is to provide methylation markers for coronary heart disease.

[0010] A second aspect of the present invention aims to provide the use of the markers of the first aspect of the present invention and / or substances for detecting the markers of the first aspect of the present invention in the preparation of products.

[0011] The third aspect of this invention is to provide a product.

[0012] A fourth aspect of the present invention is to provide an apparatus or system.

[0013] The fifth aspect of this invention is to provide a computer device.

[0014] The sixth aspect of this invention aims to provide a computer-readable storage medium.

[0015] The seventh aspect of this invention aims to provide a method.

[0016] To achieve the above objectives, the technical solution adopted by the present invention is as follows: A first aspect of the invention provides coronary artery disease methylation markers, which are combinations of coronary artery endothelial cell-specific methylation markers as shown in Table 1, including chr1:3045481-3045688; chr1:3196023-3196052; chr1:34019230-34019443; chr1:40298127-40298264; chr1:100965219-100965287; chr1:213901453-213901543; chr1:231999517-231999679; chr2:64316559-64316838; chr2:22490 0663-224900746;chr2:235951775-235951814;chr3:10967863-10968066 ;chr3:52477516-52477789;chr3:56043538-56043590;chr3:128823882- 128824117;chr3:180123736-180123801;chr4:164662770-164662968;ch r5:17067087-17067182;chr5:84922317-84922406;chr5:149925793-149 926083;chr5:149981480-149982147;chr5:160886646-160886716;chr6: 105555868-105555947;chr7:52686677-52686832;chr7:157665939-1576 66012;chr8:3435911-3435978;chr8:62210048-62210149;chr8:7038497 9-70385008;chr8:70385369-70385679;chr8:132104431-132104575;chr 9:116078328-116078449;chr9:124937302-124937468;chr9:134431394- 134431498;chr10:125586229-125586257;chr11:31401880-31402155;ch r11:109523177-109523222;chr11:128271128-128271168;chr12:317692 31-31769353;chr12:71057085-71057151;chr12:130137908-130138144;chr13:32030728-32030917;chr13:47619482-47619579;chr14:78404624-78404661;chr15: 88448898-88449006;chr16:51058219-51058394;chr16:87647423-87647532;chr16:889263 07-88926360;chr17:45087517-45087598;chr18:66167758-66167811;chr19:54222400-54222520;chr20:42777237-42777248;chr20:46277625-46277765;andchr21:32849714-32849811.

[0017] In some embodiments, the coronary methylation markers are chr1:3045481-3045688;chr1:3196023-3196052;chr1:34019230-34019443;chr1:40298127-40298264;chr1:100965219-100965287;chr1:213901453-213901543;chr1:231999517-231999679;chr2:64316559-64316838;chr2:224900663-224900746;chr2:235951775 -235951814;chr3:10967863-10968066;chr3:52477516-52477789;chr3 :56043538-56043590;chr3:128823882-128824117;chr3:180123736-180 123801;chr4:164662770-164662968;chr5:17067087-17067182;chr5:8 4922317-84922406;chr5:149925793-149926083;chr5:149981480-14998 2147;chr5:160886646-160886716;chr6:105555868-105555947;chr7:5 2686677-52686832;chr7:157665939-157666012;chr8:3435911-3435978 ;chr8:62210048-62210149;chr8:70384979-70385008;chr8:70385369- 70385679;chr8:132104431-132104575;chr9:116078328-116078449;chr 9:124937302-124937468;chr9:134431394-134431498;chr10:12558622 9-125586257;chr11:31401880-31402155;chr11:109523177-109523222; chr11:128271128-128271168;chr12:31769231-31769353;chr12:710570 85-71057151;chr12:130137908-130138144;chr13:32030728-32030917;chr13:47619482-47619579;chr14:78404624-78404661;chr15:88448898-88449006; chr16:51058219-51058394;chr16:87647423-87647532;chr16:88926307-88926360; chr17:45087517-45087598;chr18:66167758-66167811;chr19:54222400-54222520;chr20:42777237-42777248;chr20:46277625-46277765; andchr21:32849714-32849811.

[0018] In some embodiments, the reference genome for the methylation marker is GRCh37.

[0019] In some embodiments, the methylation marker is used for the diagnosis, screening, or risk stratification of coronary artery disease; preferably for the diagnosis or screening of coronary artery disease.

[0020] In this invention, the diagnosis, screening or risk stratification of coronary heart disease is the diagnosis or screening of coronary heart disease; preferably, it includes distinguishing between test subjects who do not have coronary heart disease and test subjects who have coronary heart disease.

[0021] In this invention, the non-coronary heart disease test subject refers to the test subject whose coronary artery stenosis is less than 50%.

[0022] In this invention, the subject of the test suffering from coronary heart disease refers to the subject whose coronary artery stenosis is greater than or equal to 50%.

[0023] A second aspect of the invention provides the use of the methylation markers of the first aspect of the invention and / or substances that detect the methylation markers of the first aspect of the invention in the preparation of products for the diagnosis, screening or risk stratification of coronary heart disease.

[0024] In some embodiments, the substance comprises a substance for use in one or more detection techniques or methods selected from the group consisting of: methylation-specific PCR, bisulfite sequencing, methylation-specific microarray, whole-genome methylation sequencing (e.g., WGBS, EM-seq, GM-seq), pyrosequencing, methylation-specific high-performance liquid chromatography, digital PCR, methylation-specific high-resolution melting curve analysis, methylation-sensitive restriction endonuclease assay, and quantitative real-time PCR.

[0025] In some embodiments, the substance is a reagent.

[0026] In some embodiments, the product is a reagent, kit, test plate, test strip, device, system, or chip.

[0027] In some embodiments, the test sample of the product includes at least one of body fluids and excrement; more specifically, body fluids.

[0028] In some embodiments, the body fluid includes at least one of blood, lymph, pleural fluid, cerebrospinal fluid, synovial fluid, ascites, saliva, and internal fluid accumulation; more specifically, blood.

[0029] In some embodiments, the blood includes at least one of serum, plasma, dried blood spots, and whole blood; more specifically, plasma.

[0030] In some embodiments, the excrement includes at least one of urine, feces, tears, and sweat.

[0031] In some implementations, the test sample is derived from the object to be tested.

[0032] In this invention, the test object includes animals.

[0033] In this invention, the animals include mammals such as humans, non-human primates (e.g., orangutans, apes), rodents (e.g., rats, mice, guinea pigs), pets (e.g., cats, dogs), and livestock (e.g., horses, cattle, sheep, pigs, rabbits); and further, animals derived from humans.

[0034] In some embodiments, the product also includes an instruction manual that describes methods for diagnosing, screening, or risk stratifying the coronary artery disease.

[0035] In some embodiments, the method for diagnosing, screening, or risk stratifying coronary artery disease includes the following steps: Detect the methylation status of cfDNA molecules in the test sample; Obtain the relative content of cfDNA in the test sample, and then use it for the diagnosis, screening, or risk stratification of coronary artery disease (and then perform the diagnosis, screening, or risk stratification of coronary artery disease): The relative content of cfDNA is the proportion of the number of cfDNA fragments with positive signals to the total number of cfDNA fragments. The number of cfDNA fragments with positive signals is the number of cfDNA fragments that meet the following conditions: 1) part or all of the cfDNA is within the region of the methylation marker of the first aspect of the present invention, and the part or all of the cfDNA is referred to as fragment A; and 2) all sites of fragment A are unmethylated and contain at least 3 CpG sites.

[0036] In some implementations, the method further includes the step of outputting information based on the obtained analysis results.

[0037] In some implementations, when the relative content of cfDNA in the test sample is significantly or extremely significantly different from that in a sample from an animal that does not have coronary heart disease (e.g., a healthy animal) (preferably when the relative content of cfDNA in the test sample is higher than that in a sample from an animal that does not have coronary heart disease (e.g., a healthy animal), the result is determined to be that the test sample came from an animal with coronary heart disease.

[0038] A third aspect of the invention provides a product comprising the methylation marker of the first aspect of the invention and / or a substance for detecting the methylation marker of the first aspect of the invention.

[0039] In some embodiments, the substance comprises a substance for use in one or more detection techniques or methods selected from the group consisting of: methylation-specific PCR, bisulfite sequencing, methylation-specific microarray, whole-genome methylation sequencing (e.g., WGBS, EM-seq, GM-seq), pyrosequencing, methylation-specific high-performance liquid chromatography, digital PCR, methylation-specific high-resolution melting curve analysis, methylation-sensitive restriction endonuclease assay, and quantitative real-time PCR.

[0040] In some embodiments, the substance is a reagent.

[0041] In some implementations, the product is used for the diagnosis, screening, or risk stratification of coronary artery disease.

[0042] In some embodiments, the product is a reagent, kit, test plate, test strip, device, system, or chip.

[0043] In some embodiments, the test sample of the product includes at least one of body fluids and excrement; more specifically, body fluids.

[0044] In some embodiments, the body fluid includes at least one of blood, lymph, pleural fluid, cerebrospinal fluid, synovial fluid, ascites, saliva, and internal fluid accumulation; more specifically, blood.

[0045] In some embodiments, the blood includes at least one of serum, plasma, dried blood spots, and whole blood; more specifically, plasma.

[0046] In some embodiments, the excrement includes at least one of urine, feces, tears, and sweat.

[0047] In some implementations, the test sample is derived from the object to be tested.

[0048] In some implementations, the test subject includes animals.

[0049] In some embodiments, the product also includes an instruction manual that describes methods for diagnosing, screening, or risk stratifying the coronary artery disease.

[0050] In some embodiments, the method for diagnosing, screening, or risk stratifying coronary artery disease includes the following steps: Detect the methylation status of cfDNA molecules in the test sample; Obtain the relative content of cfDNA in the test sample, and then use it for the diagnosis, screening, or risk stratification of coronary artery disease (and then perform the diagnosis, screening, or risk stratification of coronary artery disease): The relative content of cfDNA is the proportion of the number of cfDNA fragments with positive signals to the total number of cfDNA fragments. The number of cfDNA fragments with positive signals is the number of cfDNA fragments that meet the following conditions: 1) part or all of the cfDNA is within the region of the methylation marker of the first aspect of the present invention, and the part or all of the cfDNA is referred to as fragment A; and 2) all sites of fragment A are unmethylated and contain at least 3 CpG sites.

[0051] In some implementations, the method further includes the step of outputting information based on the obtained analysis results.

[0052] In some implementations, when the relative content of cfDNA in the test sample is significantly or extremely significantly different from that in a sample from an animal without coronary heart disease (e.g., a healthy animal) (preferably when the relative content of cfDNA in the test sample is higher than that in a sample from an animal without coronary heart disease (e.g., a healthy animal), the result is determined to be that the test sample came from an animal with coronary heart disease.

[0053] A fourth aspect of the present invention provides an apparatus or system comprising: Detection module: used to detect the methylation status of cfDNA molecules in the test sample; and Analysis module: Used to obtain the relative content of cfDNA in the test sample, and then to diagnose, screen or risk stratify coronary heart disease (and then diagnose, screen or risk stratify coronary heart disease): The relative content of cfDNA is the proportion of the number of cfDNA fragments with positive signals to the total number of cfDNA fragments. The number of cfDNA fragments with positive signals is the number of cfDNA fragments that meet the following conditions: 1) part or all of the cfDNA is within the region of the methylation marker of the first aspect of the present invention, and the part or all of the cfDNA is referred to as fragment A; and 2) all sites of fragment A are unmethylated and contain at least 3 CpG sites.

[0054] In some implementations, the device or system is used for the diagnosis, screening, or risk stratification of coronary artery disease.

[0055] In this invention, the methylation status of cfDNA molecules in a test sample is detected by one or more detection techniques or methods selected from the group consisting of: methylation-specific PCR, bisulfite sequencing, methylation-specific microarray, whole-genome methylation sequencing (e.g., WGBS, EM-seq, GM-seq), pyrosequencing, methylation-specific high-performance liquid chromatography, digital PCR, methylation-specific high-resolution melting curve method, methylation-sensitive restriction endonuclease method, and quantitative real-time PCR.

[0056] In some embodiments, the test sample is derived from at least one of the body fluids, tissues, cells, and excretions of the subject; more specifically, it is a body fluid.

[0057] In some embodiments, the bodily fluids and excrement are the bodily fluids and excrement described in the second aspect of the present invention.

[0058] In some embodiments, the apparatus or system further includes an output module that outputs information based on the analysis results obtained by the analysis module.

[0059] In some implementations, when the relative content of cfDNA in the test sample is significantly or extremely significantly different from that in a sample from an animal without coronary heart disease (e.g., a healthy animal) (preferably when the relative content of cfDNA in the test sample is higher than that in a sample from an animal without coronary heart disease (e.g., a healthy animal), the result is determined to be that the test sample came from an animal with coronary heart disease.

[0060] A fifth aspect of the present invention provides a computer device, the computer device including a memory and a processor, the memory storing a program, the processor executing the program to implement the following method: S1: Obtain the methylation status of cfDNA molecules in the test sample; and S2: Obtain the relative content of cfDNA in the test sample, and then perform diagnosis, screening or risk stratification for coronary heart disease (and then perform diagnosis, screening or risk stratification for coronary heart disease): The relative content of cfDNA is the proportion of the number of cfDNA fragments with positive signals to the total number of cfDNA fragments. The number of cfDNA fragments with positive signals is the number of cfDNA fragments that meet the following conditions: 1) part or all of the cfDNA is within the region of the methylation marker of the first aspect of the present invention, and the part or all of the cfDNA is referred to as fragment A; and 2) all sites of fragment A are unmethylated and contain at least 3 CpG sites.

[0061] In some embodiments, the test sample is the test sample of the fourth aspect of the present invention.

[0062] In some implementations, the method further includes the step of outputting information based on the obtained analysis results.

[0063] In some implementations, when the relative content of cfDNA in the test sample is significantly or extremely significantly different from that in a sample from an animal without coronary heart disease (e.g., a healthy animal) (preferably when the relative content of cfDNA in the test sample is higher than that in a sample from an animal without coronary heart disease (e.g., a healthy animal), the result is determined to be that the test sample came from an animal with coronary heart disease.

[0064] A sixth aspect of the present invention provides a computer-readable storage medium comprising a stored computer program; wherein, when the computer program is executed, the computer-readable storage medium controls the implementation of the method of the sixth aspect of the present invention.

[0065] A seventh aspect of the present invention provides a method for diagnosing, screening, or risk stratifying coronary heart disease, comprising the following steps: Detect the methylation status of cfDNA molecules in the test sample; Obtain the relative content of cfDNA in the test sample, and then use it for the diagnosis, screening, or risk stratification of coronary artery disease (and then perform the diagnosis, screening, or risk stratification of coronary artery disease): The relative content of cfDNA is the proportion of the number of cfDNA fragments with positive signals to the total number of cfDNA fragments. The number of cfDNA fragments with positive signals is the number of cfDNA fragments that meet the following conditions: 1) part or all of the cfDNA is within the region of the methylation marker of the first aspect of the present invention, and the part or all of the cfDNA is referred to as fragment A; and 2) all sites of fragment A are unmethylated and contain at least 3 CpG sites.

[0066] In some implementations, the method further includes the step of outputting information based on the obtained analysis results.

[0067] In some implementations, when the relative content of cfDNA in the test sample is significantly or extremely significantly different from that in a sample from an animal without coronary heart disease (e.g., a healthy animal) (preferably when the relative content of cfDNA in the test sample is higher than that in a sample from an animal without coronary heart disease (e.g., a healthy animal), the result is determined to be that the test sample came from an animal with coronary heart disease.

[0068] In some embodiments, the test sample is the test sample of the fourth aspect of the present invention.

[0069] The beneficial effects of this invention are: This invention discloses for the first time a methylation marker for coronary heart disease, which can be used for the diagnosis, screening or risk stratification of coronary heart disease, and is particularly effective in distinguishing between non-coronary heart disease subjects and coronary heart disease patients. Attached Figure Description

[0070] Figure 1 The amount of cfDNA in coronary artery endothelial cells of non-coronary artery disease subjects and coronary artery disease patients is shown (cohort 1).

[0071] Figure 2 The ROC curves (cohort 1) showing the amount of cfDNA in coronary endothelial cells distinguishing between non-coronary artery disease subjects and coronary artery disease patients are presented.

[0072] Figure 3 The amount of cfDNA in coronary artery endothelial cells of non-coronary artery disease subjects and coronary artery disease patients is shown (cohort 2).

[0073] Figure 4The ROC curves (cohort 2) showing the amount of cfDNA in coronary endothelial cells distinguishing between non-coronary artery disease subjects and coronary artery disease patients are presented. Detailed Implementation

[0074] The present invention will be further described in detail below through specific embodiments.

[0075] It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention.

[0076] Unless otherwise specified, experimental methods in the following examples are generally performed under standard conditions or as recommended by the manufacturer. Unless otherwise specified, the materials and reagents used in these examples are commercially available. For reagents whose manufacturers are listed, similar products from other manufacturers are substituted.

[0077] Example 1. Methylation Detection and Methylation Signal Processing System for Samples In this embodiment, ~60x GMseq whole-genome methylation sequencing was performed on 8 coronary artery endothelial cell samples and 30 volunteer plasma samples; ~50x WGBS whole-genome methylation sequencing was performed on 77 volunteer plasma samples. Cell samples were used to identify methylation marker regions specific to coronary artery endothelial cells.

[0078] The entire methylation detection process includes the following steps: 1. Plasma / cell sample extraction For whole blood, plasma separation should be performed promptly (within 4 hours for EDTA anticoagulant tubes; within 72 hours for Streck tubes). The separation steps are as follows: ① Centrifuge at 1600g for 10 minutes at 4℃. After centrifugation, aliquot the upper plasma into multiple 1.5mL or 2.0mL centrifuge tubes. When aspirating the plasma, be careful not to aspirate the white blood cells in the middle layer.

[0079] ② Centrifuge at 16000g for 10 minutes at 4℃ to remove residual cells, and transfer the supernatant into a new 1.5mL or 2.0mL centrifuge tube (be careful not to aspirate the white blood cells at the bottom of the tube) to obtain the required plasma.

[0080] Plasma cfDNA was extracted according to the instructions of the MagMAX™ Cell-Free DNA Isolation Kit. Cell gDNA was extracted according to the instructions of the BUDM-KF96 extraction reagent.

[0081] 2. Construction of methylated libraries ①GM sequencing: gDNA / cfDNA was constructed using the Hieff NGS® Ultima Pro DNA Library Prep Kit for Illumina, followed by TET enzyme oxidation and pyridine borane reduction, and then PCR amplification to construct the pretext library.

[0082] ②WGBS sequencing: The extracted cfDNA was first converted to bisulfite using the EpiArt Magnetic DNA Methylation Bisulfite Kit-EM103, and then the converted cfDNA was used to construct a library using the Hieff NGS® Methyl-seq ssDNALibrary Prep Kit for Illumina V2, followed by PCR amplification to construct the pretext library.

[0083] 3. Hybridization and Sequencing Whole-genome methylation sequencing does not require hybridization; however, targeted methylation sequencing requires prior hybridization capture of the library before sequencing using the Gene+seq sequencer or other sequencers based on the same principle. Sequencing procedures should be performed according to the manufacturer's instructions.

[0084] 4. Methylation signal processing system (this step is only included in whole blood / plasma samples) In this embodiment, a positive signal pattern is defined as containing at least 3 CpG sites, all of which are unmethylated. Based on the methylation status and positive signal pattern in the cfDNA molecule, the amount of cfDNA derived from target cells in the biological sample (plasma) is determined (relative cfDNA content). The amount of cfDNA derived from target cells is the proportion of the number of positive signal fragments (the number of positive signal fragments, i.e., the number of cfDNA fragments that meet the following conditions: 1) part or all of the cfDNA is within the region of the methylation markers derived from target cells in Table 1, and the part or all of the cfDNA within the region of the methylation markers derived from target cells in Table 1 is referred to as fragment A; 2) all sites of fragment A are unmethylated and contain at least 3 CpG sites) to the total number of cfDNA fragments.

[0085] Example 2. Combination of methylation markers specific to coronary artery endothelial cells The target cells in this embodiment are coronary artery endothelial cells. First, data acquisition: In this embodiment, 8 coronary artery endothelial cells were selected, and their methylation data were obtained using the method described in Example 1. Additionally, whole-genome methylation data (GSE186458) of 205 cell samples from various organs throughout the body, including coronary smooth muscle cells, aortic endothelial cells, and great saphenous vein endothelial cells, were downloaded from the GEO database as background cells.

[0086] Secondly, methylation regions (blocks) were segmented. The samples were divided into two groups: coronary artery endothelial cells were classified as target cells, and other cells were merged into a non-target cell (background cell) group. Block segmentation was performed using wgbstools segment, with the specific rules being: the Pearson correlation coefficient between any two adjacent CpG sites in the same block region was greater than 0.9, and the number of CpG sites in the same methylation region was greater than 3.

[0087] Next, the methylation level is calculated based on the pre-defined block regions, and the average methylation difference between the two groups of samples is calculated. In this embodiment, we mainly focus on markers of specific hypomethylation. The average methylation difference between the two groups of samples is the difference between the 2.5 quartile of the methylation level of all samples in the background cell group and the upper quartile (75th quartile) of the methylation level of all samples in the coronary endothelial cell group. The threshold for the average methylation difference is set to 0.3, the hypermethylation threshold is set to 0.66, and the hypomethylation threshold is set to 0.33. This yields potential methylation markers for coronary endothelial cells (i.e., when the average methylation level of all cell samples in the coronary endothelial cell group is less than the hypomethylation threshold of 0.33, and the average methylation level of all samples in the background cell group is greater than the hypermethylation threshold of 0.66, and the difference between the 2.5 quartile of the methylation level of all samples in the background cell group and the upper quartile of the methylation level of all samples in the coronary endothelial cell group is >0.3, then the methylated region is determined to be a specific hypomethylation marker for coronary endothelial cells).

[0088] Finally, based on the methylation characteristics at the cell sequencing fragment level, the coronary endothelial cell-specific methylation markers retained from the initial screening were further screened. First, the distance between every two adjacent CpG sites in each block region was calculated, and boundary optimization was performed for sites with a distance greater than 150 bp. Second, the methylation characteristics at the sequencing fragment level were compared between the coronary endothelial cell group and the non-coronary endothelial cell (background cell) group. The specific method was as follows: For the coronary endothelial cell-specific methylation markers retained from the initial screening, the proportion of fragments with all CpG sites in an unmethylated state in all sequencing fragments under each marker (the total U fragment ratio) was compared between the coronary endothelial cell group and the non-coronary endothelial cell (background cell) group, such that the difference in the total U fragment ratio between the coronary endothelial cell group and the non-coronary endothelial cell (background cell) group was greater than 0.2; at the same time, the total U fragment ratio of non-coronary endothelial cells (background cells) under each marker was <0.1. In addition, considering factors such as target marker depth, CpG density, and coefficient of variation (CV) of the total U fragment ratio, the minimum depth of coronary endothelial cell markers was set to be greater than 5, the CpG density of the marker was less than 0.1, and the coefficient of variation (CV) of the total U fragment ratio of the coronary endothelial cell-specific marker was less than 0.5, resulting in a final combination of coronary endothelial cell-specific methylation markers, totaling 52 methylation markers (Table 1).

[0089] Table 1. Combination of methylation markers specific to coronary artery endothelial cells

[0090] Note: The reference genome for the above methylation markers is Genome Reference Consortium HumanBuild 37 (GRCh37).

[0091] Example 3. The markers can be used to distinguish between subjects without coronary artery disease and patients with coronary artery disease. This embodiment is based on plasma samples from clinical volunteers. It includes subjects aged 18 years and older who underwent coronary angiography at Fuwai Hospital, Chinese Academy of Medical Sciences, due to suspected coronary artery disease caused by chest pain. The samples were divided into a control group and a disease group according to the degree of vascular stenosis assessed by coronary angiography. The control group consisted of non-coronary artery disease subjects with coronary artery stenosis <50%; the disease group consisted of coronary artery disease patients with coronary artery stenosis ≥50%. Two retrospective cohort studies were constructed. Cohort ① underwent WGBS sequencing, and Cohort ② underwent GMseq sequencing. Cohort ①: Plasma samples were collected from 77 volunteers. The samples were divided into a control group and a disease group according to the degree of vascular stenosis assessed by coronary angiography, with 18 and 59 samples in the two groups, respectively. Cohort ②: Plasma samples were collected from 30 volunteers. The samples were divided into a control group and a disease group according to the degree of vascular stenosis assessed by coronary angiography, with 10 and 20 samples in the two groups, respectively. Following the methylation detection procedure described in Example 1 and based on the coronary endothelial cell-specific methylation marker combination in Example 2, the methylation signal extraction and quantification of coronary endothelial cell cfDNA (i.e., quantification of cfDNA derived from coronary endothelial cells) were completed using the methylation signal processing system described in Example 1. Based on the quantification results, the Youden index was calculated, and the threshold for the amount of coronary endothelial cell cfDNA was determined when the Youden index reached its maximum.

[0092] Queue ① ( Figures 1-2 The amount of coronary endothelial cell cfDNA in the disease group was significantly higher than that in the control group; the threshold for coronary endothelial cell cfDNA was 0.011, the specificity of the control group and the disease group samples was 77.8%, the sensitivity was 78%, and the AUC value was 0.774. Cohort ② ( Figures 3-4 The amount of coronary endothelial cell cfDNA in the disease group was significantly higher than that in the control group; the threshold for coronary endothelial cell cfDNA was 0.029, the specificity of the control group and the disease group samples was 70%, the sensitivity was 100%, and the AUC value was 0.853. These results indicate that the coronary endothelial cell-specific methylation markers provided by this invention can identify patients with coronary heart disease and vascular stenosis.

[0093] The technical solutions of the present invention are not limited to the specific embodiments described above. Any technical modifications made in accordance with the technical solutions of the present invention fall within the protection scope of the present invention.

Claims

1. Methylation markers for coronary artery disease, namely chr1:3045481-3045688; chr1:3196023-3196052; chr1:34019230-34019443; chr1:40298127-40298264; chr1:100965219-100965287; chr1:213901453-213901543; chr1:231999517-231999679; chr2:64316559-64316838; chr2:224900663-224900746; chr2:235951775-2359 51814;chr3:10967863-10968066;chr3:52477516-52477789;chr3:5604 3538-56043590;chr3:128823882-128824117;chr3:180123736-1801238 01;chr4:164662770-164662968;chr5:17067087-17067182;chr5:84922 317-84922406;chr5:149925793-149926083;chr5:149981480-149982147 ;chr5:160886646-160886716;chr6:105555868-105555947;chr7:52686677-52686832;chr7:157665939-157666012;chr8:3435911-3435978;ch r8:62210048-62210149;chr8:70384979-70385008;chr8:70385369-703 85679;chr8:132104431-132104575;chr9:116078328-116078449;chr9: 124937302-124937468;chr9:134431394-134431498;chr10:125586229- 125586257;chr11:31401880-31402155;chr11:109523177-109523222;c hr11:128271128-128271168;chr12:31769231-31769353;chr12:710570 85-71057151;chr12:130137908-130138144;chr13:32030728-32030917;chr13:47619482-47619579;chr14:78404624-78404661;chr15:88448898-88449006; chr16:51058219-51058394;chr16:87647423-87647532;chr16:88926307-88926360;c hr17:45087517-45087598;chr18:66167758-66167811;chr19:54222400-54222520;chr20:42777237-42777248;chr20:46277625-46277765;andchr21:32849714-32849811;; The reference genome for the methylation marker is GRCh37.

2. The use of the methylation marker of claim 1 and / or the substance that detects the methylation marker of claim 1 in the preparation of products for the diagnosis or screening of coronary heart disease.

3. The application according to claim 2, characterized in that, The substance comprises substances selected from one or more detection techniques or methods chosen from the group consisting of: methylation-specific PCR, bisulfite sequencing, methylation-specific microarray, whole-genome methylation sequencing, pyrosequencing, methylation-specific high-performance liquid chromatography, digital PCR, methylation-specific high-resolution melting curve method, methylation-sensitive restriction endonuclease method, and quantitative real-time PCR. and / or The product is a reagent, kit, test plate, test strip, device, system, or chip; and / or The test samples for the product include at least one of body fluids and excrement.

4. The application according to claim 3, characterized in that, The body fluids include at least one of the following: blood, lymph, pleural effusion, cerebrospinal fluid, synovial fluid, ascites, saliva, and effusions in the body; and / or The test sample is from the object to be tested.

5. The application according to claim 4, characterized in that, The bodily fluid is blood; and / or The test subjects include animals; and / or The product also includes an instruction manual that describes the methods for diagnosing or screening for coronary heart disease; The method for diagnosing or screening for coronary heart disease includes the following steps: Detect the methylation status of cfDNA molecules in the test sample; Obtain the relative content of cfDNA in the test sample, and then use it for diagnosis or screening of coronary heart disease: Wherein, the relative content of cfDNA is the proportion of the number of cfDNA fragments with positive signals to the total number of cfDNA fragments, and the number of cfDNA fragments with positive signals is the number of cfDNA fragments that meet the following conditions: 1) part or all of the cfDNA is within the region of the methylation marker described in claim 1, and the part or all of the cfDNA is referred to as fragment A; 2) all sites of fragment A are unmethylated and contain at least 3 CpG sites.

6. A product for the diagnosis or screening of coronary heart disease, comprising the methylation marker of claim 1 and / or a substance for detecting the methylation marker of claim 1.

7. The product according to claim 6, characterized in that, The substance comprises substances selected from one or more detection techniques or methods chosen from the group consisting of: methylation-specific PCR, bisulfite sequencing, methylation-specific microarray, whole-genome methylation sequencing, pyrosequencing, methylation-specific high-performance liquid chromatography, digital PCR, methylation-specific high-resolution melting curve method, methylation-sensitive restriction endonuclease method, and quantitative real-time PCR. and / or The product is a reagent, kit, test plate, test strip, device, system, or chip; and / or The test samples for the product include at least one of body fluids and excrement.

8. The product according to claim 7, characterized in that, The body fluids include at least one of the following: blood, lymph, pleural effusion, cerebrospinal fluid, synovial fluid, ascites, saliva, and effusions in the body; and / or The test sample is from the object to be tested.

9. The product according to claim 8, characterized in that, The bodily fluid is blood; and / or The test subjects include animals; and / or The product also includes an instruction manual that describes the methods for diagnosing or screening for coronary heart disease; The method for diagnosing or screening for coronary heart disease includes the following steps: Detect the methylation status of cfDNA molecules in the test sample; Obtain the relative content of cfDNA in the test sample, and then use it for diagnosis or screening of coronary heart disease: Wherein, the relative content of cfDNA is the proportion of the number of cfDNA fragments with positive signals to the total number of cfDNA fragments, and the number of cfDNA fragments with positive signals is the number of cfDNA fragments that meet the following conditions: 1) part or all of the cfDNA is within the region of the methylation marker described in claim 1, and the part or all of the cfDNA is referred to as fragment A; 2) all sites of fragment A are unmethylated and contain at least 3 CpG sites.

10. An apparatus or system comprising: Detection module: Used to detect the methylation status of cfDNA molecules in the test sample; and Analysis module: Used to obtain the relative content of cfDNA in the test sample, and then to diagnose or screen for coronary heart disease. Wherein, the relative content of cfDNA is the proportion of the number of cfDNA fragments with positive signals to the total number of cfDNA fragments, and the number of cfDNA fragments with positive signals is the number of cfDNA fragments that meet the following conditions: 1) part or all of the cfDNA is within the region of the methylation marker described in claim 1, and the part or all of the cfDNA is referred to as fragment A; 2) all sites of fragment A are unmethylated and contain at least 3 CpG sites.

11. The apparatus or system according to claim 10, characterized in that, The device or system further includes: an output module, which outputs information based on the analysis results obtained by the analysis module.

12. A computer device, the computer device comprising a memory and a processor, the memory storing a program, the processor executing the program to implement the following method: S1: Obtain the methylation status of cfDNA molecules in the test sample; and S2: Obtain the relative content of cfDNA in the test sample, and then use it for diagnosis or screening of coronary heart disease. in, The relative content of cfDNA is the proportion of the number of cfDNA fragments with positive signals to the total number of cfDNA fragments. The number of cfDNA fragments with positive signals is the number of cfDNA fragments that meet the following conditions: 1) part or all of the cfDNA is within the region of the methylation marker described in claim 1, and the part or all of the cfDNA is referred to as fragment A; 2) all sites of fragment A are unmethylated and contain at least 3 CpG sites.

13. The computer device according to claim 12, characterized in that: The method further includes the following step: outputting information based on the obtained analysis results.

14. A computer-readable storage medium comprising a stored computer program; wherein, When the computer program is executed, it controls the computer-readable storage medium to implement the method of any one of claims 12-13.

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