CN116622834A - DNA methylation site related to new acute coronary syndrome and application thereof - Google Patents
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Abstract
The invention relates to a DNA methylation site related to new acute coronary syndrome and application thereof, belonging to the field of medical molecular biology. The DNA methylation sites in the invention are 26 novel ACS related DNA methylation sites such as cg00660626, cg24395386, cg01057742, cg18157738 and the like, and the application prospect of the DNA methylation sites in ACS etiology mechanism, risk prediction and early warning is researched, and the novel ACS related DNA methylation sites and corresponding target genes provided by the invention are as follows: providing a new etiology mechanism and a potential prevention and treatment target point of the acute coronary syndrome; the disease risk prediction efficiency of the acute coronary syndrome is remarkably improved, and high-risk individuals can be conveniently identified; as an early screening marker of acute coronary syndrome.
Description
Technical Field
The invention belongs to the field of medical molecular biology, and particularly relates to a DNA methylation site related to new-onset coronary syndrome and application thereof, in particular to a DNA methylation molecular marker related to the onset of new-onset coronary syndrome and application thereof.
Background
According to statistics of Chinese cardiovascular health and disease report in 2020, the number of patients suffering from coronary heart disease in China is about 1139 ten thousand, and the death cause rank is high and is the second most important, thus bringing heavy burden to national health and social economy. Acute coronary syndromes (acute coronary disease, ACS), including unstable angina (unstable angina pectoris, UAP), non-ST elevation myocardial infarction (non-ST-elevation myocardial infarction, NSTEMI) and ST elevation myocardial infarction (ST-elevation myocardial infarction, STEMI), are the most common and severe coronary heart disease subtypes, frequently occurring emergently and with a poor prognosis, which severely jeopardizes national health. Therefore, making countermeasures for preventing and controlling ACS is a great need for advancing healthy chinese construction. ACS is a complex disease caused by long-term co-action of environment and organism (genes and epigenetic), and although the existing research searches for the etiology mechanism of the complex disease in different aspects, the etiology mechanism of the complex disease is still not completely elucidated, is not easy to be found in early stage, is difficult to be used for early risk early warning, and needs to find new molecular markers.
DNA methylation is an epigenetic modification regulated by genes and by the environment that can serve important biological functions by regulating gene transcription. Previous studies have shown that DNA methylation may play an important role in the development of coronary heart disease. However, previous studies have mostly employed cross-sectional case-control study designs to discover methylation changes associated with disease states by detecting methylation levels of corresponding target organs in individuals who have developed the disease and control individuals. Coronary heart disease is a chronic progressive disease caused by multiple factors, and in exploring the association of DNA methylation and coronary heart disease, case-control study design cannot determine whether methylation change of positive sites occurs before or after onset, so that the etiology mechanism is difficult to infer, and the method cannot be used for predicting the risk of the disease. Therefore, to find methylation changes associated with risk of future disease occurrence, the best approach is to employ prospective research designs. However, there has not been a prospective study to discover and verify DNA methylation changes associated with risk of ACS in chinese population. In view of the above, the invention discovers and verifies the differential methylation sites related to the new ACS in two prospective queue groups by a high-throughput DNA methylation chip technology, and further functional discussion and prediction efficacy evaluation of the differential methylation sites show that the differential methylation sites can be used as a prevention and treatment target and a risk prediction marker of the new ACS.
Disclosure of Invention
In order to meet the above defects or improvement demands of the prior art, the invention provides a method for screening and verifying DNA methylation sites related to the risk of new acute coronary syndrome based on a whole genome DNA methylation chip technology, and aims to search etiology mechanisms related to ACS occurrence and potential predictive early warning markers from an epigenetic layer by means of prospective queue research, thereby solving the problems of complex ACS etiology and lack of primary preventive measures.
According to a first aspect of the present invention there is provided a DNA methylation site associated with new acute coronary syndrome, the DNA methylation site is 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, TTC33 gene methylation site cg12455300, TRIM27 gene methylation site cg27100266, HDDC2 gene methylation site cg12853539, MYO1G gene methylation site cg22111043, TSTYL 5 gene methylation site cg13249519, EMC2 gene methylation site cg22293416, HMCN2 gene methylation site cg14341771, KLF6 gene methylation site cg19347588 at least one of 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, FZD2 gene methylation site cg23053625, PLCD3 gene methylation site cg14633020, DYNLL2 gene methylation site cg20953894, CSNK1D gene methylation site cg20953894, MLLT1 gene methylation site cg20953894, RELB gene methylation site cg20953894 and PSMF1 gene methylation site cg 20953894.
According to another aspect of the invention there is provided the use of said DNA methylation site as a biomarker for new acute coronary syndrome.
According to another aspect of the invention, the application of the DNA methylation site in screening of drug targets for preventing or treating acute coronary syndrome is provided.
According to another aspect of the invention, the application of the DNA methylation site as a target for researching the etiology mechanism of the acute coronary syndrome is provided.
According to another aspect of the invention, there is provided the use of a reagent for detecting the methylation level of the DNA methylation site in the preparation of a reagent for detecting the risk of developing acute coronary syndrome, or in the preparation of a kit for detecting the risk of developing acute coronary syndrome.
According to another aspect of the invention, there is provided the use of a reagent for detecting the methylation level of a DNA methylation site in the preparation of a reagent for screening for acute coronary syndrome, or in the preparation of a kit for screening for acute coronary syndrome.
Preferably, the detection method adopted by the kit is a methylation chip, methylation specific PCR, bisulfite sequencing, restriction enzyme analysis combined with sodium bisulfite, fluorescence quantification or high throughput sequencing.
According to a further aspect of the present invention there is provided a kit for detecting the risk of developing acute coronary syndrome, the kit comprising reagents for detecting the methylation level of the DNA methylation site and specific primers for amplifying the cg00660626, cg24395386, cg01057742, cg18157738, cg03609847, cg12455300, cg27100266, cg12853539, cg22111043, cg13249519, cg22293416, cg14341771, cg19347588, cg16749093, cg04869583, cg14317273, cg27392564, cg04517903, cg01550915, cg23053625, cg14633020, cg20953894, cg07733728, cg11702503, cg20089365 or cg01680988 sites.
According to another aspect of the invention there is provided a kit for screening for early stage acute coronary syndrome, the kit comprising reagents for detecting the methylation level of the DNA methylation site and specific primers for amplifying the cg00660626, cg24395386, cg01057742, cg18157738, cg03609847, cg12455300, cg27100266, cg12853539, cg22111043, cg13249519, cg22293416, cg14341771, cg19347588, cg16749093, cg04869583, cg14317273, cg27392564, cg04517903, cg01550915, cg23053625, cg14633020, cg20953894, cg07733728, cg11702503, cg20089365 or cg01680988 sites.
In general, the above technical solutions conceived by the present invention have the following beneficial effects compared with the prior art:
due to the combination of two-stage prospective cohort design and high throughput DNA methylation chip technology, DNA methylation sites associated with newly developed ACS can be discovered and validated, and potential biological mechanisms discussed. The novel ACS-related DNA methylation site and the corresponding target gene provided by the invention are as follows: (1) Providing a new etiology mechanism and a potential prevention and treatment target point of the acute coronary syndrome; (2) The disease risk prediction efficiency of the acute coronary syndrome is remarkably improved, and high-risk individuals can be conveniently identified; (3) As an early screening marker of acute coronary syndrome, more effective early screening and early warning are realized through noninvasive blood detection.
Drawings
FIG. 1 is a technical roadmap of the invention.
FIG. 2 is a Manhattan, QQ and volcanic plot of the results of a full-epigenetic genomic association study of newly developed ACS in a discovery stage population.
FIG. 3 is a forest graph of the correlation between the methylation levels of 26 different methylation sites and their annotated gene mRNA expression levels.
Detailed Description
The present invention will be described in further detail with reference to the drawings and examples, in order to make the objects, technical solutions and advantages of the present invention more apparent. It should be understood that the specific embodiments described herein are for purposes of illustration only and are not intended to limit the scope of the invention. In addition, the technical features of the embodiments of the present invention described below may be combined with each other as long as they do not collide with each other.
To achieve the above object, the present invention provides a method for finding a DNA methylation site associated with verifying a risk of newly developing ACS (technical scheme see FIG. 1), comprising the steps of:
(1) Whole blood leukocyte DNA from subjects in the discovery phase and validation phase was extracted.
(2) And (3) performing quality control on the extracted DNA sample, and re-extracting the unqualified sample.
(3) And performing bisulfite conversion on the DNA sample with qualified quality control.
(4) Detection of DNA methylation was performed according to the instructions of Infinium Human Methylation EPIC chip (Illumina, usa); the file scanned by the iScan scanner is imported into Genome Studio software, and the scanned data is converted into data in an IDAT format.
(5) The original methylation chip data is subjected to pretreatment, quality control and standardization, and unqualified DNA methylation probes and samples are removed.
(6) The whole genome DNA methylation correlation analysis of the new ACS was performed in the discovery phase population, resulting in 72 DNA methylation sites associated with the new ACS (FDR < 0.05), and the correlation of 26 DNA methylation sites with the new ACS (consistent orientation, FDR < 0.05) was verified in the verification phase population.
(7) The potential biological functions of the verification site are discussed through annotation analysis of gene functions and related pathways and document retrieval to explore and explain the results.
(8) In combination with the gene expression data, the correlation between the verification sites and the annotated gene expression levels thereof was explored, and three of the 26 verification sites were found to be significantly inversely correlated with the annotated gene expression levels thereof, namely, cg03609847 and PIGG gene expression, cg12853539 and HDDC2 gene expression, cg16749093 and EHBP1L1 gene expression levels were negatively correlated, respectively.
(9) Exploring the predictive role of methylation risk scores constructed from DNA methylation sites on risk of ACS occurrence in both the discovery phase and the validation phase populations.
According to one aspect of the present invention there is provided a DNA methylation site associated with a newly developed ACS, the DNA methylation sites include at least one of 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, HMCN2 gene cg14341771, KLF6 gene cg 673, EHBP1L1 gene cg16749093, DNM1L gene cg04869583, NRXN3 gene cg14317273, KIF7 gene cg27392564, TELO2 gene cg04517903, ABCA3 gene cg01550915, FZD2 gene cg23053625, PLCD3 gene cg14633020, dyl 2 gene cg20953894, CSNK1D gene cg20953894, MLLT1 gene cg20953894, ref 37, and PSMF1 gene cg 20953894.
Furthermore, the invention provides the application of the reagent for detecting the methylation level of the DNA methylation site in preparing a reagent for treating the risk of acute coronary syndrome, or the application of the reagent for preparing a reagent for treating the risk of acute coronary syndrome.
The invention also provides application of the reagent for detecting the methylation level of the DNA methylation site in preparation of a reagent for screening the acute coronary syndrome or in preparation of a kit for screening the acute coronary syndrome.
Preferably, the kit may be a reagent for detecting the site-specific DNA methylation level using any technique known in the art, as long as it is capable of detecting the leukocyte DNA methylation level at the DNA methylation sites cg00660626, cg24395386, cg01057742, cg18157738, cg03609847, cg12455300, cg27100266, cg12853539, cg22111043, cg13249519, cg22293416, cg14341771, cg19347588, cg16749093, cg04869583, cg14317273, cg27392564, cg04517903, cg01550915, cg23053625, cg14633020, cg20953894, cg07733728, cg11702503, cg20089365 and cg01680988 sites in the sample. Including but not limited to the embodiments listed below. Also included in the kit are, but not limited to, specific primers for amplifying the cg00660626, cg24395386, cg01057742, cg18157738, cg03609847, cg12455300, cg27100266, cg12853539, cg22111043, cg13249519, cg22293416, cg14341771, cg19347588, cg16749093, cg04869583, cg14317273, cg27392564, cg04517903, cg01550915, cg23053625, cg14633020, cg20953894, cg07733728, cg11702503, cg20089365 or cg01680988 sites. The primer can be designed by adopting MethPrimer software; such reagents include PCR kits and the usual reagents required for the corresponding PCR technique, such as: dNTPs, mgCl2, double distilled water, taq enzyme and the like.
In a first embodiment, the kit comprises reagents for detecting the level of leukocyte DNA methylation at the cg00660626, cg24395386, cg01057742, cg18157738, cg03609847, cg12455300, cg27100266, cg12853539, cg22111043, cg13249519, cg22293416, cg14341771, cg19347588, cg16749093, cg04869583, cg14317273, cg27392564, cg04517903, cg01550915, cg23053625, cg14633020, cg20953894, cg07733728, cg11702503, cg20089365 and cg01680988 sites in a sample using target region DNA methylation sequencing (Targeted Bisulfite Sequencing, TBS). TBS satisfies detection of several to hundreds of genes/DNA methylation sites, has the advantages of high accuracy, high throughput, low cost and fast cycle, and is widely used for screening, verifying and clinically transforming methylation markers of multiple sites of clinical samples. Firstly, the construction of a library is required to be completed, and the design and synthesis of the BS-PCR primer are completed aiming at a target region or site. And (3) simultaneously extracting sample DNA, carrying out bisulphite conversion (EZ DNA Methylation GoldKit, zymo Research) on the sample DNA after the sample DNA is detected to be qualified, carrying out BSP amplification on a template subjected to bisulphite conversion treatment by using high-fidelity U-base-resistant DNA polymerase, mixing BSP amplification products from the same sample, carrying out label primer amplification, carrying out Illumina sequencing joint on the amplified products, finally obtaining sequencing libraries of each sample with different labels, and carrying out machine sequencing on each sample library after purification, quantification and multi-text library mixing and quality inspection. And after the library is qualified, carrying out the sequence of an Illumina platform according to the effective concentration and the requirement of the target off-machine data volume on different libraries, and predicting the occurrence risk of ACS according to the DNA methylation level of the sites.
In a second embodiment, the kit includes reagents for detecting the level of leukocyte DNA methylation at the cg00660626, cg24395386, cg01057742, cg18157738, cg03609847, cg12455300, cg27100266, cg12853539, cg22111043, cg13249519, cg22293416, cg14341771, cg19347588, cg16749093, cg04869583, cg14317273, cg27392564, cg04517903, cg01550915, cg23053625, cg14633020, cg20953894, cg07733728, cg11702503, cg20089365 and cg01680988 sites in a sample using Pyrosequencing (Pyrosequencing). Pyrosequencing is a technique well known in the art and is a "gold standard" method for detecting the methylation status of a particular gene, followed by transformation with bisulfite and pyrosequencing. Those skilled in the art can select the selection according to the needs, and the details are not repeated here. By using the kit, the methylation level of the DNA methylation sites in the sample can be directly measured by a pyrosequencing method, and the occurrence risk of ACS can be predicted according to the DNA methylation levels of the sites.
In a third embodiment, the kit comprises a kit for detecting a nucleic acid sequence using a DNA microarray assay (e.g., infinium Human Methylation EPIC methylation chip, for specific procedures, see the instruction manual of the company Illumina, inc. official working procedure) to detect cg00660626, cg24395386, cg01057742, cg18157738, cg03609847, cg12455300, cg27100266, cg12853539, cg22111043, cg13249519, cg22293416, cg14341771, a reagent for the methylation level of leukocyte DNA at the cg19347588, cg16749093, cg04869583, cg14317273, cg27392564, cg04517903 and cg04517903 sites, the risk of ACS occurrence is predicted based on the DNA methylation levels at these sites.
Furthermore, the invention also provides application of the DNA methylation site or the susceptibility gene in screening drug targets for preventing or treating acute coronary syndrome.
The invention also provides application of the DNA methylation site or the susceptibility gene as a target for researching etiology and mechanism of the acute coronary syndrome.
Preferably, DNA methylation sites and susceptibility gene sites related to novel ACS are provided, wherein the DNA methylation sites participate in the occurrence and development processes of diseases perhaps by changing gene expression, DNA methylation is a reversible epigenetic modification, and the purpose of preventing or treating diseases can be achieved by changing the methylation level of specific sites influenced by life style, administration and the like. Therefore, the invention provides a valuable target for preventing and treating ACS, and can be used for drug development.
The following are specific examples
The invention is further illustrated below in conjunction with specific examples, which are intended to illustrate the invention and are not to be construed as limiting the invention. One of ordinary skill in the art can appreciate that: many variations, modifications, substitutions, and alterations are possible in these embodiments without departing from the principles and spirit of the invention.
Example 1 discovery and validation of differential methylation sites associated with Cryptographic New syndromes
1 subject of study
The discovery stage: subjects were based on new ACS nest type cases in Dongfeng-Tongji (DFTJ) cohort-control study population. The DFTJ cohort baseline was established from 9 in 2008 to 6 in 2010, incorporating 27,009 retirement workers from eastern wind motor company. The staff of the unit can seek medical attention in the affiliated staff hospitals of the unit, and the electronic medical records, medical records and death records of the study objects can be tracked in detail through the electronic system. After baseline investigation, follow-up monitoring, including mortality, morbidity and postnatal, etc. of various common chronic diseases will be performed once every 5 years. The cohort was first followed in 2013, 4-12 months, with 14,120 subjects newly enrolled, except for 2008 baseline (a follow-up rate of 96.5%), and a total of 38,295 subjects were enrolled. Two fasting blood samples were collected from each subject in situ (1 tube is EDTA anticoagulant tube and 1 tube is procoagulant tube). After blood collection, the blood was centrifuged at 3000rpm for 15 minutes in a cryocentrifuge and 4 tubes of plasma, serum, anticoagulated whole blood, procoagulant whole blood were separated, each tube of approximately 450 μl, and stored in a-80 ℃ freezer for subsequent testing. On the first follow-up group, the participants with coronary heart disease (n= 6457), stroke (n=2406), cancer (n=2686), severe electrocardiographic abnormalities (n=838), and 2013 blood deficiency (n=3626) were excluded, the remaining 24,415 participants. New ACS cases are defined as AMI encoded as ICD-10I21 and UAP encoded as ICD-10I 20. Diagnosis was performed by an expert panel according to the ACCF/AHA guidelines, based on symptoms and clinical examinations. 785 new ACS cases were selected from baseline 2013 to 31 months 2018. Controls were matched according to baseline age (+ -1 year), gender, time of blood collection (+ -6 months) 1:1. 34 ACS cases and controls were excluded from the quality control process of methylation data. Finally, we included a total of 751 pairs of new ACS case-control samples as the discovery stage population.
Verification: the study subjects were based on a coronary heart disease nest type case control study population established in a chinese chronic disease prospective cohort (China Kadoorie Biobank, CKB) cohort. CKB is a prospective cohort comprising adults between 30 and 79 years of age 512,724 who were recruited from 10 different regions of china (5 cities and 5 rural areas) in 2004-2008. DNA methylation was measured from whole blood samples at baseline in 494 new coronary heart disease cases and 494 matched controls diagnosed during the follow-up period of 12 months 31 years 2015. New cases of coronary heart disease include fatal ischemic heart disease encoded as ICD-10I20-I25 and non-fatal AMI encoded as I21. Controls were matched 1:1 based on year of birth (+ -3 years), baseline age (+ -3 years), gender, study area, and fasting time (0-6, 6-8, 8-10, and ≡10 hours) prior to blood withdrawal. All participants did not suffer from coronary heart disease, stroke, or cancer at baseline. To keep pace with the discovery stage population, we excluded 20 non-ACS cases and their controls. In addition, 8 pairs of ACS cases and controls were excluded from the quality control process of methylation data. Thus, we retained a total of 476 pairs of new ACS case-control samples as the verification stage population.
2 main reagents and instruments
The main instrument is as follows: PCR Gene amplification apparatus (2720 Thermal Cycler, U.S. Applied Biosystem), high-speed microplate shaker (U.S. Illumina), micro-UV spectrometer (NANODROP 1000, U.S. Thermo Fisher Scientific), high precision tube sheet heating system (Hybex, U.S. SciGene), heat sealing film machine (ALPS 25, U.S. Thermo Fisher Scientific), hybridization oven (U.S. Illumina), high throughput genotyping system (iScan, U.S. Illumina), 96 deep well plate (0.8 mL, U.S. Thermo Fisher Scientific), chip pad (U.S. Illumina) and PCR octant (0.2 mL, hung Siderurgh Biotechnology Co., ltd.). The primer is designed and synthesized by Shenzhen Aisi Gene technology Co.
The main reagent comprises: whole blood genomic DNA extraction kit (DP 1002, beijing Baitaike Biotechnology Co., ltd.), erythrocyte lysate (R1010, beijing Soilebao technology Co., ltd.), isopropyl alcohol (analytical grade, china medicine group chemical reagent Co., ltd.), EZ DNA Methylation-Gold conversion kit (D5006, U.S. Zymo Co., ltd.), infinium HumanMethylationEPIC methylation chip kit (U.S. Illumina Co., ltd.), sodium hydroxide (NaOH; analytical grade, china medicine group chemical reagent Co., ltd.), and absolute ethyl alcohol (analytical grade, china medicine group chemical reagent Co., ltd.).
3 Experimental method
3.1 whole blood leukocyte DNA extraction: taking out blood sample from-80deg.C refrigerator, thawing in 37 deg.C water bath for 30min, and standing at room temperature. Sequentially adding erythrocyte lysate to lyse erythrocyte by adopting an artificial method, lysing leucocyte nuclei by using the cell nucleus lysate to release DNA, removing protein by using protein precipitation liquid, precipitating DNA by isopropanol, rinsing by using ethanol, adding DNA dissolution liquid, placing into a 55 ℃ incubator overnight, and storing in a refrigerator at-20 ℃ after the DNA is completely dissolved.
3.2DNA sample quality control: the DNA after extraction needs to be measured on a NANODROP quantitative instrument for DNA concentration and purity, the DNA concentration is more than 50 ng/. Mu.L, the purity A260/280 is between 1.8 and 2.0, and the quality of the DNA sample is qualified, otherwise, concentration or purification treatment is needed until the DNA sample meets the requirements. The concentration of the DNA sample to be detected required by the chip experiment is 50 ng/. Mu.L, so that the sample with the concentration higher than the concentration range needs to be added with DNA dissolving liquid for dilution, the target volume of the DNA sample to be detected is 15 mu.L, and the target concentration is 50 ng/. Mu.L. In addition, DNA agarose gel electrophoresis is needed, whether the DNA to be detected is degraded or not is checked through the condition of the DNA agarose gel electrophoresis, and if the DNA to be detected is degraded, the DNA is extracted again.
3.3DNA bisulfite conversion: DNA samples were subjected to bisulfite conversion and sample purification according to the instructions of the EZ DNA bisulfite conversion kit (Zymo company, usa).
3.4DNA methylation detection: experiments were performed according to the instructions of Infinium HumanMethylationEPIC chip (Illumina, usa). The files scanned by the iScan scanner are imported into the genome studio software, and the scanned data are converted into data in an IDAT format.
4 statistical analysis
4.1 methylation chip data pretreatment, quality control, and normalization (for example, discovery stage population)
Whole genome methylation data of 1502 subjects were read using "bigmelon" and then quality control was performed according to the following criteria:
quality control for CpG probes:
a. probes excluding non-CpG sites (n=59);
b. probes with detection P values >0.01 or bead count <3 in >5% of samples were excluded (n=3960);
c. excluding probes that cross-hybridized to other genomic sites (n= 43254);
SNP related probes: single base extension site of type I probe (n=399); (n=80156); within the probe target region overlapping SNPs with asian populations MAF >0.01 (n=26826);
e. probes on sex chromosomes (n= 17616).
Quality control for samples:
a. outlier samples (n=0) displayed by multi-dimensional scale analysis (multi-dimensional scaling, MDS) plots were excluded;
b. samples with a probe loss rate >1% were excluded (n=1);
c. samples excluding gender mismatch (n=10);
d. exclusion is a mixed sample deduced from quality control SNPs in the individual genotype data and DNA methylation data (n=28).
After the samples failed in quality control were paired out, a total of 1502 samples and 777513 CPGs probes were quality controlled. The quality controlled samples and CpGs probes were normalized using the "dasen" method in the "bigmelon" package and the experimental batch was corrected using the "Combat" method for further analysis.
4.2 Whole-Gene DNA methylation correlation analysis of novel ACS
The correlation between the methylation level of CpG sites and the risk of ACS in the discovery stage is calculated by adopting a conditional logistic regression model, and the M value is taken as an independent variable to correct the proportions of white blood cells predicted by gender, smoking state, drinking state, BMI, hypertension, dyslipidemia, diabetes and six whole blood, wherein the significance level is defined as FDR <0.05 (whole genome significance level). The correlation between CpG site methylation level and ACS occurrence risk in the verification stage is calculated by adopting a conditional logistic regression model, and the M value is taken as an independent variable to correct the leukocyte ratio of age, sex, smoking state, drinking state, BMI, region, hypertension, dyslipidemia, diabetes and six whole blood predictions, wherein the significance level in the verification stage is defined as FDR <0.05.
Annotating the DNA methylation sites to genes according to the manifest annotation file provided by Illumina officials network, querying the found sites and genes for relation to cardiovascular disease or cardiovascular related phenotypes by means of the public databases MRC-IEU EWAS catalyst, metabank database, GWAS catalyst and PhenoScanner database. And carrying out KEGG and GO gene set enrichment analysis on the gene where the ACS related DNA methylation site discovered in the discovery stage is located.
4.3 analysis of correlation of DNA methylation with Gene expression
For the 26 DNA methylation sites verified, we extracted 156 healthy physical subjects with white blood cell mRNA expression levels. We then examined the correlation between methylation levels and gene expression levels using Pearson correlation.
Results 4 results
4.1 differential methylation sites associated with novel ACS
After correction of major risk factors and the proportion of 6 leukocyte subtypes in ACS during the discovery phase, 72 DNA methylation sites (FDR) associated with new ACS were discovered<0.05, fig. 2). For significant sites of the discovery phase, further validation was performed in a validation phase population, in which 26 DNA methylation sites were correlated with newly ACS in a consistent relationship with the direction of the discovery phase (FDR<0.05). Meta analysis of the discovery dataset and replication dataset further confirmed the robustness of these 26 DNA methylation sites to association with ACS (P meta Are all<2.1×10 -7 See table 1 below.
TABLE 1
Enrichment analysis is carried out on the Top genes of the whole genome methylation correlation analysis results in the discovery stage. The significantly enriched KEGG pathway includes hedgehog signaling pathway, phosphoinositide metabolism, circadian rhythm, phosphatidylinositol signaling system, endoplasmic reticulum protein processing, type II diabetes, kinesin, and Glycosyl Phosphatidylinositol (GPI) anchored biosynthesis. In addition, many significantly enriched GO pathways are closely related to phosphoinositides, including phosphatidylinositol-3, 4-diphosphate binding, inositol tetraphosphate kinase activity, inositol 1,3, 4-triphosphate 6-kinase activity, inositol 1,3, 4-triphosphate 5-kinase activity, inositol tetraphosphate 6-kinase activity, and the like.
4.2 correlation of DNA methylation sites with Gene expression
To further understand the potential gene expression regulatory function of the validation site, we assessed the relationship between the expression levels of 26 validated DNA methylation sites and their annotated genes. The studies found that cg03609847 and PIGG gene expression, cg12853539 and HDDC2 gene expression, cg16749093 and EHBP1L1 gene expression levels were negatively correlated (P <0.05, fig. 3). Indicating that the differential methylation site can participate in the occurrence and development processes of the acute coronary syndrome by changing the expression level of the target gene.
EXAMPLE 2 predictive efficacy assessment of differential methylation sites against risk of ACS occurrence
1. Experimental method
1.1 predictive efficacy of single differential methylation sites on risk of ACS occurrence
The performance of individual differential methylation sites in predicting risk of ACS occurrence was assessed based on logistic regression models in the DFTJ cohort and CKB cohort, respectively. The reference model is a traditional cardiovascular risk factor including age, sex, BMI, smoking status, drinking status, hypertension, dyslipidemia, and diabetes. Then, the improvement in performance when single differential methylation sites were added to the reference model was assessed. We performed a subject work feature (receiver operating characteristic, ROC) curve analysis and calculated the area under the ROC curve (the area under the ROC curves, AUC) and corresponding 95% CI using the "pROC" package.
1.2 predictive efficacy of multiple differential methylation sites in combination against risk of ACS occurrence
And evaluating the performance of the combination of a plurality of different methylation sites on the aspect of predicting ACS occurrence risk based on a logistic regression model in the DFTJ queue and the CKB queue group respectively. The reference model is a traditional cardiovascular risk factor including age, sex, BMI, smoking status, drinking status, hypertension, dyslipidemia, and diabetes. Then, the improvement in performance when 26 differential methylation sites were added simultaneously to the reference model was evaluated.
2 experimental results
2.1 predictive efficacy of single differential methylation sites on risk of ACS occurrence
After adding single differential methylation sites into the traditional risk factor reference model, the risk prediction AUC value of ACS of the DFTJ queue group is obviously improved from 0.639 to 0.650-0.704 except cg07733728 sites (P values are all <0.05, delong test); in addition to cg07733728 and cg01680988 sites, the risk prediction AUC values for ACS in the CKB cohort group increased significantly from 0.660 to 0.672-0.700 (P values <0.05, deltang test). It is demonstrated that most of the differential methylation sites provided by the present invention have some efficacy in improving ACS risk prediction (see table 2 below).
2.2 predictive efficacy of multiple differential methylation sites in combination against risk of ACS occurrence
As shown in table 2, after 26 differential methylation sites are added to the conventional risk factor reference model, the risk prediction AUC value of ACS in the DFTJ cohort is significantly increased from 0.639 to 0.752 (P < 0.0001), and the risk prediction AUC value of ACS in the CKB cohort is significantly increased from 0.660 to 0.720 (P < 0.0001), which indicates that the model constructed by combining the 26 differential methylation sites provided by the present invention with the conventional risk factors has the best effect of improving ACS risk prediction.
TABLE 2
In summary, the invention discloses 26 specific methylation markers related to the occurrence of acute coronary syndrome, which can be used for preparing an early screening and predictive warning kit for acute coronary syndrome. The kit has the value that only peripheral blood is needed without other tissue samples, DNA methylation is detected through the most simplified and specific primer pair, and then ACS occurrence risk is predicted in an auxiliary way through the DNA methylation level, so that the kit is convenient to detect, accurate and capable of greatly improving the sensitivity and the specificity of early disease prediction, and therefore, the kit can help to guide diagnosis and more effective individuation treatment.
It will be readily appreciated by those skilled in the art that the foregoing description is merely a preferred embodiment of the invention and is not intended to limit the invention, but any modifications, equivalents, improvements or alternatives falling within the spirit and principles of the invention are intended to be included within the scope of the invention.
Claims (9)
1. DNA methylation sites associated with new acute coronary syndrome characterized in that, the DNA methylation site is 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, TTC33 gene methylation site cg12455300, TRIM27 gene methylation site cg27100266, HDDC2 gene methylation site cg12853539, MYO1G gene methylation site cg22111043, TSTYL 5 gene methylation site cg13249519, EMC2 gene methylation site cg22293416, HMCN2 gene methylation site cg14341771, KLF6 gene methylation site cg19347588 at least one of 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, FZD2 gene methylation site cg23053625, PLCD3 gene methylation site cg14633020, DYNLL2 gene methylation site cg20953894, CSNK1D gene methylation site cg20953894, MLLT1 gene methylation site cg20953894, RELB gene methylation site cg20953894 and PSMF1 gene methylation site cg 20953894.
2. Use of the DNA methylation site of claim 1 as a biomarker for new acute coronary syndrome.
3. Use of the DNA methylation site according to claim 1 for screening for drug targets for preventing or treating acute coronary syndrome.
4. Use of the DNA methylation site according to claim 1 as a target for studying the causative mechanism of acute coronary syndrome.
5. The use of a reagent for detecting the methylation level of a DNA methylation site according to claim 1 in the preparation of a reagent for detecting the risk of developing acute coronary syndrome, or in the preparation of a kit for detecting the risk of developing acute coronary syndrome.
6. Use of a reagent for detecting the methylation level of a DNA methylation site according to claim 1 in the preparation of a reagent for screening for acute coronary syndrome, or in a kit for screening for acute coronary syndrome.
7. The use according to claim 5 or 6, wherein the detection method used by the kit is methylation chip, methylation specific PCR, bisulfite sequencing, restriction enzyme assay combined with sodium bisulfite, fluorescent quantitation or high throughput sequencing.
8. A kit for detecting the risk of developing acute coronary syndrome, comprising a detection reagent for the methylation level of the DNA methylation site of claim 1, and further comprising a specific primer for amplifying the cg00660626, cg24395386, cg01057742, cg18157738, cg03609847, cg12455300, cg27100266, cg12853539, cg22111043, cg13249519, cg22293416, cg14341771, cg19347588, cg16749093, cg04869583, cg14317273, cg27392564, cg04517903, cg01550915, cg23053625, cg14633020, cg20953894, cg07733728, cg11702503, cg20089365 or cg01680988 sites.
9. A kit for screening for early acute coronary syndrome, comprising a detection reagent for the methylation level of the DNA methylation site of claim 1, and further comprising a specific primer for amplifying the cg00660626, cg24395386, cg01057742, cg18157738, cg03609847, cg12455300, cg27100266, cg12853539, cg22111043, cg13249519, cg22293416, cg14341771, cg19347588, cg16749093, cg04869583, cg14317273, cg27392564, cg04517903, cg01550915, cg23053625, cg14633020, cg20953894, cg07733728, cg11702503, cg20089365 or cg01680988 sites.
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