DNA methylation sites associated with new-onset acute coronary syndrome and uses thereof

Abstract

The present application relates to DNA methylation sites related to new acute coronary syndrome and application thereof, and belongs to the field of medical molecular biology. The DNA methylation sites in the present application are 26 new ACS-related DNA methylation sites such as cg00660626, cg24395386, cg01057742 and cg18157738, and the application prospect thereof in ACS pathogenesis, risk prediction and early warning is studied. The new ACS-related DNA methylation sites and corresponding target genes provided by the present application will: provide new pathogenesis and potential prevention and treatment targets of acute coronary syndrome; significantly improve the risk prediction efficiency of acute coronary syndrome, facilitate the identification of high-risk individuals; and serve as an early screening marker for acute coronary syndrome.

Description

Technical Field

[0001] This invention belongs to the field of medical molecular biology, and more specifically, relates to DNA methylation sites associated with new-onset acute coronary syndrome and their applications, particularly to a DNA methylation molecular marker associated with the pathogenesis of new-onset acute coronary syndrome and its applications. Background Technology

[0002] According to the 2020 "Report on Cardiovascular Health and Disease in China," approximately 11.39 million people in my country currently suffer from coronary heart disease, which ranks second among causes of death, placing a heavy burden on national health and the socio-economic situation. Acute coronary syndrome (ACS), including unstable angina pectoris (UAP), non-ST-segment elevation myocardial infarction (NSTEMI), and ST-segment elevation myocardial infarction (STEMI), is the most common and serious subtype of coronary heart disease. It often has a rapid onset and poor prognosis, seriously endangering national health. Therefore, developing strategies for the prevention and control of ACS is a major requirement for promoting the construction of a healthy China. ACS is a complex disease caused by the long-term interaction between the environment and the organism (genes and epigenetics). Although existing research has sought its pathogenesis from different aspects, the pathogenesis of this complex disease is still not fully understood, and it is not easy to detect early and make early risk warnings. There is an urgent need to discover new molecular markers.

[0003] DNA methylation is an epigenetic modification regulated by genes and the environment, playing an important biological function by regulating gene transcription. Previous studies have shown that DNA methylation may play an important role in the development and progression of coronary artery disease (CAD). However, previous studies have mostly used cross-sectional case-control designs, detecting methylation levels in corresponding target organs in individuals with disease and controls to identify methylation changes associated with disease status. CAD is a multifactorial, chronic, progressive disease. When exploring the association between DNA methylation and CAD, case-control designs cannot determine whether methylation changes at positive sites occur before or after the onset of disease, making it difficult to infer the pathogenesis and often unsuitable for predicting disease risk. Therefore, the best approach to identifying methylation changes associated with future disease risk is to use a prospective study design. However, there are currently no prospective studies in the Chinese population that have identified and validated DNA methylation changes associated with the risk of ACS (acute coronary syndrome). In view of this, the present invention uses high-throughput DNA methylation microarray technology to discover and validate differentially methylated sites associated with new ACS in two prospective cohort populations. Further functional exploration and predictive efficacy evaluation of the differentially methylated sites show that these differentially methylated sites can serve as new targets for the prevention and treatment of ACS, as well as risk predictive biomarkers. Summary of the Invention

[0004] To address the above-mentioned deficiencies or improvement needs of existing technologies, this invention provides a method for screening and validating DNA methylation sites associated with the risk of new-onset acute coronary syndrome (ACS) based on whole-genome DNA methylation microarray technology. The aim is to use prospective cohort studies to identify the etiological mechanisms and potential predictive and early warning biomarkers related to the occurrence of ACS at the epigenetic level, thereby solving the problem of the complex etiology of ACS and the lack of primary prevention measures.

[0005] According to a first aspect of the present invention, DNA methylation sites associated with new-onset acute coronary syndrome are provided, wherein the DNA methylation sites are PRKCZ gene methylation site cg00660626, PRDM16 gene methylation site cg24395386, LCE5A gene methylation site cg01057742, IGFN1 gene methylation site cg18157738, PIGG gene methylation site cg03609847, and T The methylation sites for the following genes are listed: TC33 (cg12455300), TRIM27 (cg27100266), HDDC2 (cg12853539), MYO1G (cg22111043), TSPYL5 (cg13249519), EMC2 (cg22293416), and HMCN2 (cg14341771). KL... The following gene methylation sites are listed: F6 gene methylation site cg19347588, EHBP1L1 gene methylation site cg16749093, DNM1L gene methylation site cg04869583, NRXN3 gene methylation site cg14317273, KIF7 gene methylation site cg27392564, TELO2 gene methylation site cg04517903, ABCA3 gene methylation site cg01550915, and FZD2. At least one of the following gene methylation sites: cg23053625, cg14633020, cg20953894, cg07733728, cg11702503, cg20089365, and cg01680988.

[0006] According to another aspect of the present invention, the application of the aforementioned DNA methylation sites as biomarkers for newly diagnosed acute coronary syndrome is provided.

[0007] According to another aspect of the present invention, the application of the aforementioned DNA methylation sites for screening drug targets for the prevention or treatment of acute coronary syndrome is provided.

[0008] According to another aspect of the present invention, the DNA methylation site is provided as a target for studying the etiology and mechanism of acute coronary syndrome.

[0009] According to another aspect of the present invention, the application of the reagent for detecting the methylation level of the DNA methylation site is provided in the preparation of a reagent for detecting the risk of acute coronary syndrome, or in the preparation of a kit for detecting the risk of acute coronary syndrome.

[0010] According to another aspect of the present invention, the application of the reagent for detecting the methylation level of the DNA methylation site is provided in the preparation of reagents for screening acute coronary syndrome, or in the preparation of kits for screening acute coronary syndrome.

[0011] Preferably, the detection method used in the kit is methylation chip, methylation-specific PCR, bisulfite sequencing, restriction endonuclease analysis combined with sodium bisulfite, quantitative fluorescence method, or high-throughput sequencing.

[0012] According to another aspect of the present invention, a kit for detecting the risk of acute coronary syndrome is provided, the kit comprising a detection reagent for the methylation level of the DNA methylation sites, and further comprising amplification of cg00660626, cg24395386, cg01057742, cg18157738, cg03609847, cg12455300, cg27100266, cg12853539, cg22111043, cg13249519, c Specific primers for the following loci: g22293416, cg14341771, cg19347588, cg16749093, cg04869583, cg14317273, cg27392564, cg04517903, cg01550915, cg23053625, cg14633020, cg20953894, cg07733728, cg11702503, cg20089365, or cg01680988.

[0013] According to another aspect of the present invention, a kit for early screening of acute coronary syndrome is provided, the kit comprising a detection reagent for the methylation level of the DNA methylation sites, and further comprising amplification of cg00660626, cg24395386, cg01057742, cg18157738, cg03609847, cg12455300, cg27100266, cg12853539, cg22111043, cg13249519, cg Specific primers for the following loci: 22293416, cg14341771, cg19347588, cg16749093, cg04869583, cg14317273, cg27392564, cg04517903, cg01550915, cg23053625, cg14633020, cg20953894, cg07733728, cg11702503, cg20089365, or cg01680988.

[0014] In summary, the technical solutions conceived by this invention have the following beneficial effects compared with the prior art:

[0015] By combining a two-stage prospective cohort study design with high-throughput DNA methylation microarray technology, DNA methylation sites associated with new-onset ACS can be discovered and validated, and potential biological mechanisms can be explored. The DNA methylation sites and corresponding target genes associated with new-onset ACS provided by this invention will: (1) provide new etiological mechanisms and potential prevention and treatment targets for acute coronary syndrome; (2) significantly improve the predictive efficacy of acute coronary syndrome risk, making it easier to identify high-risk individuals; and (3) serve as early screening biomarkers for acute coronary syndrome, enabling more effective early screening and warning through non-invasive blood testing. Attached Figure Description

[0016] Figure 1 This is a technical roadmap for the present invention.

[0017] Figure 2 Manhattan plot, QQ plot, and volcano plot showing the results of a full epigenome association study of newly diagnosed ACS in the population during the discovery phase.

[0018] Figure 3 Forest plot showing the correlation between the methylation levels of 26 differentially methylated sites and the mRNA expression levels of their annotated genes. Detailed Implementation

[0019] To make the objectives, technical solutions, and advantages of this invention clearer, the invention will be further described in detail below with reference to the accompanying drawings and embodiments. It should be understood that the specific embodiments described herein are merely illustrative and not intended to limit the invention. Furthermore, the technical features involved in the various embodiments of this invention described below can be combined with each other as long as they do not conflict with each other.

[0020] To achieve the above objectives, this invention provides a method for discovering and verifying DNA methylation sites associated with the risk of new-onset ACS (see technical roadmap). Figure 1 The method includes the following steps:

[0021] (1) Extract DNA from whole blood leukocytes of research subjects in the discovery and validation phases.

[0022] (2) Perform quality control on the extracted DNA samples, and re-extract samples that do not meet the requirements.

[0023] (3) Perform bisulfite conversion on DNA samples that have passed quality control.

[0024] (4) DNA methylation was detected according to the operating instructions of the Infinium Human Methylation EPIC chip (Illumina, USA); the scanned files were imported into Genome Studio software and the scanned data was converted into IDAT format.

[0025] (5) Perform preprocessing, quality control and standardization on the raw methylation chip data to exclude DNA methylation probes and samples that do not meet quality standards.

[0026] (6) In the discovery phase population, whole-genome DNA methylation association analysis of newly diagnosed ACS was performed, and 72 DNA methylation sites associated with newly diagnosed ACS were obtained (FDR<0.05). In the validation phase population, the association between 26 DNA methylation sites and newly diagnosed ACS was validated (in the same direction, FDR<0.05).

[0027] (7) The results were explored and interpreted through gene function and related pathway annotation analysis and literature search, and the potential biological functions of the verification sites were investigated.

[0028] (8) Combining gene expression data, we explored the correlation between validation sites and the expression levels of their annotated genes. We found that three of the 26 validation sites were significantly negatively correlated with the expression levels of their annotated genes: cg03609847 and PIGG gene expression, cg12853539 and HDDC2 gene expression, and cg16749093 and EHBP1L1 gene expression levels were negatively correlated.

[0029] (9) Explore the predictive role of a methylation risk score constructed from DNA methylation sites in the risk of ACS in the discovery and validation phases.

[0030] According to one aspect of the invention, DNA methylation sites associated with newly diagnosed ACS are provided, said DNA methylation sites including PRKCZ gene cg00660626, PRDM16 gene cg24395386, LCE5A gene cg01057742, IGFN1 gene cg18157738, PIGG gene cg03609847, TTC33 gene cg12455300, TRIM27 gene cg27100266, HDDC2 gene cg12853539, MYO1G gene cg22111043, TSPYL5 gene cg13249519, EMC2 gene cg22293416, and HMCN2 gene cg14341771. At least one of the following gene loci: KLF6 gene cg19347588, EHBP1L1 gene cg16749093, DNM1L gene cg04869583, NRXN3 gene cg14317273, KIF7 gene cg27392564, TELO2 gene cg04517903, ABCA3 gene cg01550915, FZD2 gene cg23053625, PLCD3 gene cg14633020, DYNLL2 gene cg20953894, CSNK1D gene cg07733728, MLLT1 gene cg11702503, RELB gene cg20089365, and PSMF1 gene cg01680988.

[0031] Furthermore, the present invention provides the application of the reagent for detecting the methylation level of the DNA methylation site in the preparation of a reagent for assessing the risk of acute coronary syndrome, or in the preparation of a kit for assessing the risk of acute coronary syndrome.

[0032] The present invention also provides the application of the reagent for detecting the methylation level of the DNA methylation site in the preparation of reagents for screening acute coronary syndrome, or in the preparation of kits for screening acute coronary syndrome.

[0033] Preferably, the kit can be any reagent known in the art for detecting site-specific DNA methylation levels, as long as it can detect the following DNA methylation sites in the sample: cg00660626, cg24395386, cg01057742, cg18157738, cg03609847, cg12455300, cg27100266, cg12853539, cg22111043, cg13249519, and cg2229341. 6. Leukocyte DNA methylation levels at the following loci: cg14341771, cg19347588, cg16749093, cg04869583, cg14317273, cg27392564, cg04517903, cg01550915, cg23053625, cg14633020, cg20953894, cg07733728, cg11702503, cg20089365, and cg01680988. This includes, but is not limited to, the embodiments listed below. The kit also includes, but is not limited to, reagents for amplifying cg00660626, cg24395386, cg01057742, cg18157738, cg03609847, cg12455300, cg27100266, cg12853539, cg22111043, cg13249519, cg22293416, cg14341771, and cg19347. Specific primers for the loci 588, cg16749093, cg04869583, cg14317273, cg27392564, cg04517903, cg01550915, cg23053625, cg14633020, cg20953894, cg07733728, cg11702503, cg20089365, or cg01680988. These primers can be designed using MethPrimer software. The reagents include PCR kits and commonly used reagents required for the corresponding PCR techniques, such as dNTPs, MgCl2, double-distilled water, and Taq polymerase.

[0034] In a first embodiment, the kit includes the detection of cg00660626, cg24395386, cg01057742, cg18157738, cg03609847, cg12455300, cg27100266, cg12853539, cg22111043, cg13249519, cg22293416, cg14341771, and cg1934758 in a sample using Targeted Bisulfite Sequencing (TBS). 8. A reagent for detecting leukocyte DNA methylation levels at the following loci: cg16749093, cg04869583, cg14317273, cg27392564, cg04517903, cg01550915, cg23053625, cg14633020, cg20953894, cg07733728, cg11702503, cg20089365, and cg01680988. TBS can detect several to hundreds of gene / DNA methylation sites, offering advantages such as high accuracy, high throughput, low cost, and fast turnaround time. It is widely used for screening, validating, and translating methylation markers at multiple sites in clinical samples. The first step is to construct the library and design and synthesize BS-PCR primers for the target region or site. Simultaneously, sample DNA was extracted and, after passing testing, the sample DNA underwent bisulfite conversion (EZ DNA Methylation Gold Kit, Zymo Research). BSP amplification was performed on the bisulfite-converted template using a high-fidelity U-base-resistant DNA polymerase. BSP amplification products from the same sample were mixed and amplified with tagged primers, attaching Illumina sequencing adapters to obtain sequencing libraries for each sample with different tags. Each sample library underwent purification, quantification, and multi-library mixing, quality control, and sequencing. After library approval, different libraries were pooled according to effective concentration and target data volume requirements and then sequenced on the Illumina platform. The risk of ACS was predicted based on the DNA methylation levels at these sites.

[0035] In a second embodiment, the kit includes the use of pyrosequencing to detect cg00660626, cg24395386, cg01057742, cg18157738, cg03609847, cg12455300, cg27100266, cg12853539, cg22111043, cg13249519, cg22293416, and cg143417 in the sample. A reagent for determining the methylation levels of leukocyte DNA at loci cg19347588, cg16749093, cg04869583, cg14317273, cg27392564, cg04517903, cg01550915, cg23053625, cg14633020, cg20953894, cg07733728, cg11702503, cg20089365, and cg01680988. Pyrosequencing is a well-known technique in the art and is the "gold standard" method for detecting the methylation status of specific genes. It involves first converting the DNA with bisulfite, followed by pyrosequencing. Those skilled in the art can choose this method according to their needs, and will not be elaborated further here. Using this kit, the methylation level of the DNA methylation sites in the sample can be directly measured by pyrosequencing, and the risk of ACS can be predicted based on the DNA methylation level of these sites.

[0036] In the third embodiment, the kit includes the use of DNA microarray detection methods (such as the Infinium HumanMethylation EPIC methylation chip, for details please refer to the Illumina website's operation manual) to detect cg00660626, cg24395386, cg01057742, cg18157738, cg03609847, cg12455300, cg27100266, cg12853539, cg22111043, cg13249519, cg22293416, cg14341771, cg19347588, cg A reagent for determining the DNA methylation levels of leukocytes at sites 16749093, cg04869583, cg14317273, cg27392564, cg04517903, cg01550915, cg23053625, cg14633020, cg20953894, cg07733728, cg11702503, cg20089365, and cg01680988, and using the DNA methylation levels at these sites to predict the risk of ACS.

[0037] Furthermore, the present invention also provides the application of the aforementioned DNA methylation sites or susceptibility genes in screening drug targets for the prevention or treatment of acute coronary syndrome.

[0038] This invention also provides the application of the aforementioned DNA methylation sites or susceptibility genes as targets for studying the etiology and mechanism of acute coronary syndrome.

[0039] Preferably, the invention provides DNA methylation sites and susceptibility gene sites associated with newly diagnosed ACS. These DNA methylation sites may participate in the disease's development by altering gene expression. DNA methylation is a reversible epigenetic modification; the methylation level at specific sites can be influenced by lifestyle changes and medication use, thereby achieving the goal of disease prevention or treatment. Therefore, this invention provides highly valuable targets for the prevention and treatment of ACS, which can be used in drug development.

[0040] The following are specific embodiments.

[0041] The present invention will be further illustrated below with reference to specific embodiments. These embodiments are for illustrative purposes only and should not be construed as limiting the invention. Those skilled in the art will understand that various changes, modifications, substitutions, and variations can be made to these embodiments without departing from the principles and spirit of the invention.

[0042] Example 1: Discovery and validation of differentially methylated sites associated with new-onset acute coronary syndrome

[0043] 1. Research Subjects

[0044] Discovery Phase: The study participants were based on a newly diagnosed ACS nested case-control population from the Dongfeng-Tongji (DFTJ) cohort. The DFTJ cohort baseline was established from September 2008 to June 2010, enrolling 27,009 retired employees of Dongfeng Motor Corporation. All employees of this unit received medical treatment at the affiliated employee hospital, allowing for detailed tracking of electronic medical records, medical records, and death records through an electronic system. Following the baseline survey, follow-up monitoring was conducted every 5 years, including mortality, incidence and outcomes of various common chronic diseases. The cohort underwent its first follow-up from April to December 2013, including follow-up of those at the 2008 baseline (follow-up rate 96.5%), and the addition of 14,120 new participants, ultimately resulting in a total of 38,295 participants. Two fasting blood samples were collected from each participant on-site (one tube for EDTA anticoagulation and one tube for procoagulation). After blood collection, the blood was centrifuged at 3000 rpm for 15 minutes in a low-temperature centrifuge, and then aliquoted into four tubes each of plasma, serum, anticoagulated whole blood, and procoagulated whole blood, approximately 450 μL per tube, and stored at -80°C for subsequent testing. Based on the initial follow-up population, participants with coronary artery disease (n=6457), stroke (n=2406), cancer (n=2686), severe electrocardiographic abnormalities (n=838), and those with insufficient blood samples in 2013 (n=3626) were excluded, leaving 24,415 participants. Newly diagnosed ACS cases were defined as AMI (ICD-10I21) and UAP (ICD-10I20). Diagnosis was made by an expert panel based on symptoms and clinical examination, according to ACCF / AHA guidelines. From baseline in 2013 to December 31, 2018, 785 newly diagnosed ACS cases were selected. Controls were matched 1:1 based on baseline age (±1 year), sex, and blood collection time (±6 months). Thirty-four ACS case-control pairs were excluded during quality control of methylation data. Finally, a total of 751 newly diagnosed ACS case-control samples were included as the discovery phase population.

[0045] Validation Phase: The study participants were based on a nested case-control study population for coronary artery disease (CAD) established within the China Kadoorie Biobank (CKB) cohort. CKB is a prospective cohort comprising 512,724 adults aged 30 to 79 years, recruited from 10 different regions in China (5 urban and 5 rural) between 2004 and 2008. DNA methylation was measured from baseline whole blood samples in 494 newly diagnosed CAD cases and 494 matched controls during the follow-up period ending December 31, 2015. Newly diagnosed CAD cases included fatal ischemic heart disease coded as ICD-10I20-I25 and non-fatal acute myocardial infarction (AMI) coded as I21. Controls were matched 1:1 based on year of birth (±3 years), baseline age (±3 years), sex, study region, and pre-blood draw fasting time (0–6, 6–8, 8–10, and ≥10 hours). All participants were free of coronary artery disease, stroke, or cancer at baseline. To maintain consistency with the discovery phase population, we excluded 20 non-ACS cases and their controls. Additionally, 8 ACS case-control pairs were excluded during quality control of the methylation data. Therefore, we retained a total of 476 newly diagnosed ACS case-control pairs as the validation phase population.

[0046] 2. Main Reagents and Instruments

[0047] Major instruments included: a PCR gene amplification instrument (2720 Thermal Cycler, Applied Biosystems, USA), a high-speed microplate shaker (Illumina, USA), a micro-volume UV spectrophotometer (NANODROP 1000, Thermo Fisher Scientific, USA), a high-precision tube-plate heating system (Hybex, SciGene, USA), a heat-sealing machine (ALPS 25, Thermo Fisher Scientific, USA), a hybridization oven (Illumina, USA), a high-throughput genotyping system (iScan, Illumina, USA), 96-well deep-well plates (0.8 mL, Thermo Fisher Scientific, USA), chip pads (Illumina, USA), and PCR eight-tube strips (0.2 mL, Hangzhou Aisijin Biotechnology Co., Ltd., China). Primers were designed and synthesized by Shenzhen Aisigen Technology Co., Ltd.

[0048] Main reagents: Whole blood genomic DNA extraction kit (DP1002, Beijing Biotech Biotechnology Co., Ltd., China), red blood cell lysis buffer (R1010, Beijing Solarbio Science & Technology Co., Ltd., China), isopropanol (analytical grade, China National Pharmaceutical Group Chemical Reagent Co., Ltd.), EZ DNA Methylation–Gold conversion kit (D5006, Zymo Biotech, USA), Infinium Human Methylation EPIC methylation chip matching reagent (Illumina, USA), sodium hydroxide (NaOH; analytical grade, China National Pharmaceutical Group Chemical Reagent Co., Ltd.), and anhydrous ethanol (analytical grade, China National Pharmaceutical Group Chemical Reagent Co., Ltd.).

[0049] 3 Experimental Methods

[0050] 3.1 Whole blood leukocyte DNA extraction: Blood samples were removed from a -80℃ freezer and thawed in a 37℃ water bath for 30 minutes. They were then removed and allowed to stand at room temperature. Red blood cells were lysed manually by adding red blood cell lysis buffer, followed by leukocyte nucleus lysis buffer to release DNA. Protein precipitation buffer was used to remove proteins, and isopropanol was used to precipitate DNA. After rinsing with ethanol, DNA dissolving solution was added, and the mixture was incubated overnight at 55℃. Once the DNA was completely dissolved, it was stored at -20℃.

[0051] 3.2 DNA Sample Quality Control: Extracted DNA must be analyzed for concentration and purity using a Nanodrop quantifier. A DNA concentration >50 ng / μL and an A260 / 280 purity between 1.8 and 2.0 are considered acceptable. Otherwise, concentration or purification is required until the requirements are met. The required DNA concentration for the microarray experiment is 50 ng / μL. Therefore, samples with concentrations higher than this range need to be diluted with DNA dissolving buffer. The target volume for the DNA sample is 15 μL, and the target concentration is 50 ng / μL. Furthermore, DNA agarose gel electrophoresis is required. The results of DNA agarose gel electrophoresis indicate whether the DNA has degraded. If degradation has occurred, the DNA must be extracted again.

[0052] 3.3 DNA bisulfite conversion: The DNA sample was converted to bisulfite and purified according to the instructions of the EZ DNA bisulfite conversion kit (Zymo, USA).

[0053] 3.4 DNA Methylation Detection: The experiment was conducted according to the operating instructions of the Infinium HumanMethylation EPIC chip (Illumina, USA). The scanned files from the iScan scanner were imported into GenomeStudio software, and the scanned data was converted to IDAT format.

[0054] 4. Statistical Analysis

[0055] 4.1 Methylation chip data preprocessing, quality control and standardization (taking the discovery phase population as an example)

[0056] Whole-genome methylation data from 1502 study subjects were read using BigMelon, and then quality control was performed according to the following criteria:

[0057] Quality control of CpG probes:

[0058] a. Exclude probes that are not CpG sites (n=59);

[0059] b. Exclude probes with a detection P-value > 0.01 or a bead count < 3 in > 5% of the samples (n = 3960);

[0060] c. Exclude probes that cross-hybrid with other genomic sites (n = 43254);

[0061] d. SNP-related probes: Single base extension sites of type I probes (n=399); (n=80156); Probe target regions overlapping with SNPs in Asian populations with MAF≥0.01 (n=26826);

[0062] e. Probes on sex chromosomes (n = 17616).

[0063] Regarding sample quality control:

[0064] a. Exclude outlier samples (n=0) shown in the multi-dimensional scaling (MDS) plot;

[0065] b. Exclude samples with a probe missing rate >1% (n=1);

[0066] c. Exclude samples with gender mismatch (n=10);

[0067] d. Exclude mixed samples (n=28) inferred from quality control SNPs in individual genotype data and DNA methylation data.

[0068] After excluding samples that failed quality control in pairs, a total of 1502 samples and 777513 CpG probes passed the quality control. The quality-controlled samples and CpG probes were standardized using the "dasen" method in the "bigmelon" package, and the experimental batches were calibrated using the "Combat" method for further analysis.

[0069] 4.2 Genome-wide DNA methylation association analysis of newly diagnosed ACS

[0070] In the discovery phase, the association between CpG site methylation levels and the risk of ACS was calculated using a conditional logistic regression model, with M as the independent variable. The model was adjusted for factors such as sex, smoking status, alcohol consumption, BMI, hypertension, dyslipidemia, diabetes, and the predicted white blood cell percentages from six whole blood tests. A significance level was defined as FDR < 0.05 (genome-wide significance). In the validation phase, the association between CpG site methylation levels and the risk of ACS was calculated using a conditional logistic regression model, with M as the independent variable. The model was adjusted for factors such as age, sex, smoking status, alcohol consumption, BMI, region, hypertension, dyslipidemia, diabetes, and the predicted white blood cell percentages from six whole blood tests. A significance level in the validation phase was defined as FDR < 0.05.

[0071] DNA methylation sites were annotated to genes using the manifest annotation file provided on the Illumina website. Using public databases such as the MRC-IEU EWAS Catalog, MethBank, GWAS Catalog, and PhenoScanner, the identified sites and genes were queried to determine if they were associated with cardiovascular disease or cardiovascular-related phenotypes. KEGG and GO gene set enrichment analyses were performed on genes containing ACS-related DNA methylation sites identified during the discovery phase.

[0072] 4.3 Association analysis between DNA methylation and gene expression

[0073] For the 26 validated DNA methylation sites, we extracted the mRNA expression levels from leukocytes of 156 healthy individuals. We then used a Pearson correlation test to examine the correlation between methylation levels and gene expression levels.

[0074] 4 Results

[0075] 4.1 Differentially methylated sites associated with newly diagnosed ACS

[0076] During the discovery phase, after adjusting for major risk factors for ACS and the proportions of six leukocyte subtypes, 72 DNA methylation sites associated with new-onset ACS were identified (FDR < 0.05, with additional methylation sites below 0.05). Figure 2 For the significant sites identified in the discovery phase, further validation was conducted in the validation population. The association between 26 DNA methylation sites and newly diagnosed ACS was consistent with the association observed in the discovery phase (FDR < 0.05). Meta-analysis of the discovery and replication datasets further confirmed the robustness of the association between these 26 DNA methylation sites and ACS (P < 0.05). meta All <2.1×10 -7 See Table 1 below.

[0077] Table 1

[0078]

[0079]

[0080] Enrichment analysis was performed on the top genes identified in the genome-wide methylation association analysis (GWAS) during the discovery phase. Significantly enriched KEGG pathways included the hedgehog signaling pathway, phosphatidylinositol metabolism, circadian rhythms, the phosphatidylinositol signaling system, endoplasmic reticulum protein processing, type II diabetes, motor proteins, and glycosylphosphatidylinositol (GPI)-anchored biosynthesis. Furthermore, many significantly enriched GO pathways were closely related to phosphatidylinositol, including phosphatidylinositol-3,4-bisphosphate binding, inositol tetraphosphate kinase activity, inositol 1,3,4-triphosphate 6-kinase activity, inositol 1,3,4-triphosphate 5-kinase activity, and inositol tetraphosphate 6-kinase activity.

[0081] 4.2 Association between DNA methylation sites and gene expression

[0082] To further understand the potential gene expression regulatory functions of validation sites, we assessed the relationship between 26 validated DNA methylation sites and the expression levels of their annotated genes. The study found a negative association between the expression levels of cg03609847 and PIGG genes, cg12853539 and HDDC2 genes, and cg16749093 and EHBP1L1 genes (P < 0.05). Figure 3 This suggests that differentially methylated sites may participate in the development and progression of acute coronary syndrome by altering the expression levels of target genes.

[0083] Example 2: Evaluation of the predictive power of differentially methylated sites on the risk of ACS

[0084] 1. Experimental Methods

[0085] 1.1 Predictive power of single differentially methylated sites for the risk of ACS

[0086] We evaluated the performance of individual differentially methylated sites in predicting the risk of acute coronary syndrome (ACS) using logistic regression models in the DFTJ and CKB cohorts, respectively. The reference model included conventional cardiovascular risk factors such as age, sex, BMI, smoking status, alcohol consumption, hypertension, dyslipidemia, and diabetes. We then assessed the performance improvement when individual differentially methylated sites were added to the reference model. We performed receiver operating characteristic (ROC) curve analysis and calculated the area under the ROC curves (AUC) and corresponding 95% CI using the "pROC" package.

[0087] 1.2 Predictive efficacy of multiple differentially methylated sites combined for ACS risk

[0088] The combined effect of multiple differentially methylated sites in predicting the risk of acute coronary syndrome (ACS) was evaluated using a logistic regression model in both the DFTJ and CKB cohorts. The reference model included traditional cardiovascular risk factors such as age, sex, BMI, smoking status, alcohol consumption, hypertension, dyslipidemia, and diabetes. The performance improvement when 26 differentially methylated sites were simultaneously added to the reference model was then assessed.

[0089] 2 Experimental Results

[0090] 2.1 Predictive power of single differentially methylated sites for the risk of ACS

[0091] After adding a single differential methylation site to the traditional risk factor reference model, except for the cg07733728 site, the predictive AUC value of ACS risk in the DFTJ cohort significantly improved from 0.639 to 0.650–0.704 (all P values ​​< 0.05, Delong test); except for the cg07733728 and cg01680988 sites, the predictive AUC value of ACS risk in the CKB cohort significantly improved from 0.660 to 0.672–0.700 (all P values ​​< 0.05, Delong test). This indicates that most of the differential methylation sites provided by this invention have a certain efficacy in improving ACS risk prediction (see Table 2 below).

[0092] 2.2 Predictive efficacy of multiple differentially methylated sites combined for ACS risk

[0093] As shown in Table 2, after adding 26 differential methylation sites to the traditional risk factor reference model, the AUC value for predicting ACS risk in the DFTJ cohort population significantly increased from 0.639 to 0.752 (P<0.0001), and the AUC value for predicting ACS risk in the CKB cohort population significantly increased from 0.660 to 0.720 (P<0.0001). This indicates that the model constructed by combining the 26 differential methylation sites provided in this invention with traditional risk factors has the best efficacy in improving ACS risk prediction.

[0094] Table 2

[0095]

[0096]

[0097] In summary, this invention discloses 26 specific methylation markers associated with the occurrence of acute coronary syndrome (ACS), which can be used to prepare an early screening and predictive kit for ACS. The value of this kit lies in its requirement only of peripheral blood samples, eliminating the need for other tissue samples. It detects DNA methylation using the most concise and specific primer pairs, and then uses DNA methylation levels to help predict the risk of ACS. The detection is convenient and accurate, significantly improving the sensitivity and specificity of early disease prediction. Therefore, putting this kit into practice can help guide diagnosis and more effective personalized treatment.

[0098] Those skilled in the art will readily understand that the above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. DNM1L The application of a reagent for detecting the methylation level of gene methylation site cg04869583 in the preparation of reagents for detecting the risk of acute coronary syndrome, or in the preparation of a kit for detecting the risk of acute coronary syndrome, is characterized by, The risk assessment for acute coronary syndrome is based on the methylation level of cg04869583 and traditional cardiovascular risk factors, which include age, sex, BMI, smoking status, alcohol consumption, hypertension, dyslipidemia, and diabetes.

2. DNM1L The application of a reagent for detecting the methylation level of gene methylation site cg04869583 in the preparation of reagents for screening acute coronary syndrome, or in the preparation of a kit for screening acute coronary syndrome, is characterized by, The acute coronary syndrome screening assesses the methylation level of cg04869583 and traditional cardiovascular risk factors, which include age, sex, BMI, smoking status, alcohol consumption, hypertension, dyslipidemia, and diabetes.

3. The application as described in claim 1 or 2, characterized in that, The detection methods used in the kit are methylation chip, methylation-specific PCR, bisulfite sequencing, or restriction endonuclease analysis combined with sodium bisulfite.

4. The application as described in claim 3, characterized in that, The methylation-specific PCR was performed using a real-time fluorescence method.

5. The application as described in claim 3, characterized in that, The bisulfite sequencing method is a high-throughput sequencing method.

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