WO2023221309A1 - Dna methylation marker for evaluating onset risk of cerebral stroke, primer, and use thereof - Google Patents

Dna methylation marker for evaluating onset risk of cerebral stroke, primer, and use thereof Download PDF

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WO2023221309A1
WO2023221309A1 PCT/CN2022/111781 CN2022111781W WO2023221309A1 WO 2023221309 A1 WO2023221309 A1 WO 2023221309A1 CN 2022111781 W CN2022111781 W CN 2022111781W WO 2023221309 A1 WO2023221309 A1 WO 2023221309A1
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chr4
dna methylation
stroke
primer
risk
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彭浩
陈立楠
刘洋
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苏州大学
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  • the present invention relates to the field of biomedicine technology, and in particular to a DNA methylation marker, primer and application for assessing the risk of stroke.
  • Stroke is a sudden disorder of blood circulation in the brain tissue. It has become one of the leading diseases affecting human health worldwide. In 2019, the number of stroke patients worldwide reached 101 million, and about 6.55 million people died from it. Stroke has become the second leading cause of death in the world. There are great differences in the incidence and prevalence of stroke around the world. my country is an area with a high incidence of stroke. In 2019, the total number of people suffering from stroke in my country reached 28.76 million, and 2.19 million people died of stroke. This has brought great changes to our country’s medical system. Therefore, how to effectively prevent and treat stroke is a major challenge facing my country's public health field. It is urgent to discover more potential risk factors and treatment targets and promote their clinical transformation.
  • NPs Natriuretic Peptide system
  • ANP atrial natriuretic peptide
  • BNP B-type natriuretic peptide
  • Corin is a type II transmembrane serine protease highly expressed in cardiomyocytes. It is the main physiological activator of ANP and can also activate BNP. Therefore, Corin, as an upstream regulator of NPs, may be involved in the pathogenesis of stroke. Basic research and epidemiological studies suggest the cardiovascular effects of Corin.
  • CORIN knockout mice developed hypertension and cardiac hypertrophy.
  • polymorphisms in the gene encoding Corin protein, CORIN are associated with susceptibility to heart failure, cardiac hypertrophy, and hypertension.
  • Corin protein may be related to the pathogenesis of stroke, and Corin protein may become a therapeutic target or drug candidate for the prevention and treatment of stroke.
  • no inhibitors of Corin protein have been found in human plasma, which reduces the safety of its clinical translation. Therefore, there is a need to better understand the molecular mechanisms underlying the association between Corin and stroke.
  • Epigenetic factors serve as interfaces between the genome and the dynamic environment and may influence gene expression and function, thus having the potential to serve as the molecular mechanisms we need.
  • DNA methylation has been considered to be involved in the pathological process of disease by affecting the expression of related genes.
  • the present invention provides a DNA methylation marker related to stroke through research on the correlation between CORIN gene DNA methylation and stroke phenotype, which can be used to predict the risk of stroke and brain disease. It provides a basis for screening high-risk groups for stroke and is also expected to serve as an intervention target for the prevention and control of stroke.
  • the first object of the present invention is to provide a DNA methylation marker for assessing the risk of stroke.
  • the DNA methylation marker includes the DNA methylation site Chr4 in the promoter region of the CORIN gene: At least one site among 47840038, Chr4:47839941 and Chr4:47839933.
  • DNA methylation markers include a combination of DNA methylation sites in the promoter region of the CORIN gene, and the combination of DNA methylation sites is any one of the following combinations (1)-(4):
  • upstream primer for amplifying DNA methylation markers is shown in SEQ ID NO.1
  • downstream primer is shown in SEQ ID NO.2.
  • the second object of the present invention is to provide the application of the above-mentioned DNA methylation markers in preparing a kit for assessing the risk of stroke.
  • the kit for assessing the risk of stroke includes a primer pair that amplifies at least one of the DNA methylation sites Chr4:47840038, Chr4:47839941 and Chr4:47839933 in the CORIN gene promoter region.
  • a primer pair that simultaneously amplifies the DNA methylation sites Chr4:47840038, Chr4:47839941 and Chr4:47839933 is included.
  • the upstream primer is as shown in SEQ ID NO.1
  • the downstream primer is as shown in SEQ ID NO.2.
  • This primer pair can simultaneously amplify the Chr4:47840038, Chr4:47839941 and Chr4:47839933 sites. When used, the methylation level of each site is separately detected to determine the risk of stroke.
  • DNA samples are derived from blood, making sampling easy.
  • the third object of the present invention is to provide the application of a primer pair for amplifying the above-mentioned DNA methylation marker in preparing a kit for assessing the risk of stroke.
  • the upstream primer is as shown in SEQ ID NO.1
  • the downstream primer is as shown in SEQ As shown in ID NO.2, specifically,
  • R ACTTAAAAACCCRACTCTACRACAA, where R represents base A/G.
  • the fourth object of the present invention is to provide a kit for assessing the risk of stroke, which kit includes: amplifying the DNA methylation sites Chr4:47840038 and Chr4:47839941 in the promoter region of the CORIN gene, respectively or simultaneously. and Chr4:47839933, and reagents for detecting the methylation level of at least one site in Chr4:47840038, Chr4:47839941 and Chr4:47839933.
  • upstream primer is shown in SEQ ID NO.1
  • downstream primer is shown in SEQ ID NO.2.
  • the fifth object of the present invention is to provide the application of the above-mentioned DNA methylation markers in the preparation of stroke therapeutic drugs.
  • the therapeutic drugs target the DNA methylation sites Chr4:47840038, Chr4:47839941 and By designing at least one site in Chr4:47839933, DNA methylation is changeable.
  • Targeted drugs designed based on the above DNA methylation sites can regulate the expression of the CORIN gene and the secretion of Corin, thereby achieving the goal of treating brain disease. purpose of stroke.
  • the present invention at least has the following advantages:
  • the CORIN gene DNA methylation markers provided by the present invention can be used to predict the risk of stroke, provide a basis for the screening of high-risk groups for stroke, and can also be used as intervention targets for the prevention and control of stroke, which not only helps to explain The molecular mechanism by which Corin upregulates stroke will also provide important epidemiological evidence for CORIN gene methylation as a drug target for the prevention and control of stroke.
  • Figure 1 shows the target sequence and primers for targeted sulfite sequencing, as well as the predicted 9 CpG sites;
  • Figure 2 shows the detection results of DNA methylation levels of 9 CpG sites in stroke patients and healthy controls
  • Figure 3 shows the receiver operating characteristic curve corresponding to the CpG3 site
  • Figure 4 shows the receiver operating characteristic curve corresponding to the CpG8 site
  • Figure 5 shows the receiver operating characteristic curve corresponding to the CpG9 site
  • Figure 6 shows the receiver operating characteristic curve corresponding to the CpG3+CpG8 model
  • Figure 7 shows the receiver operating characteristic curve corresponding to the CpG3+CpG9 model
  • Figure 8 shows the receiver operating characteristic curve corresponding to the CpG8+CpG9 model
  • Figure 9 shows the receiver operating characteristic curve corresponding to the CpG3+CpG8+CpG9 model.
  • the region is Chr4:47840136-47839906 (GRCh37.p13, distance from TSS: -13bp to 217bp), intercept the nucleic acid sequence of this region on NCBI, import the nucleic acid sequence into EMBOSS Cpgplot software to predict CpG islands, and then use the Epidesigner program to map CpG Design primers based on the sequences of islands and CpG dense regions, and select appropriate primers for DNA methylation detection (see Table 1 for primer sequence information). After selection, CORIN methylation obtained a total of methylation levels of 9 CpG sites. The 9 CpG sites are shown in Figure 1.
  • the DNA sample was treated with bisulfite using the EZ-96 DNA Methylation Kit (Zymo Research, Inc., CA, United States) to convert all the unmethylated cytosine C in the sample DNA into uracil. U.
  • the primers in Table 1 use the primers in Table 1 and use the bisulfite-treated sample genome as a template to perform multiplex PCR amplification.
  • primers with Index sequences are used to introduce specific tag sequences compatible with the Illumina platform to the end of the library through PCR amplification.
  • the Index PCR amplification products of all samples were mixed in equal amounts, and high-throughput sequencing was performed on the Illumina Hiseq/Miseq platform in the paired-end sequencing mode of 2 ⁇ 150bp/2 ⁇ 250bp to obtain FastQ data.
  • the methylation level of each CpG site was quantified as the number of methylated reads at the site (i.e., the number of reads with base C detected)/the total number of reads at the site ⁇ 100%.
  • This example included 853 ischemic stroke patients (average age 62 years old, 53% males) and 918 age and gender-matched healthy controls (average age 61 years, 55% males). It was found that ischemic stroke patients in the study had more metabolic risk factors such as hypertension, diabetes, lipids, and obesity than healthy controls (all P ⁇ 0.05).
  • the receiver operating characteristic curve (receiver operating characteristic curve ROC) was drawn and the area under the curve (Area Under Curve AUC), it was found that all 9 sites can improve the prediction ability of traditional risk factors for ischemic stroke.
  • Figures 6 to 9 show the ROC curves of the combined model of multiple methylation sites. It is found that the combined model can also improve the prediction ability of traditional risk factors for ischemic stroke.
  • methylation in the CORIN gene promoter region may be a predictor of stroke risk.
  • we conducted a prospective cohort study The inventors enrolled 2,498 research subjects who did not have stroke at baseline in 2010, followed up on stroke events every 2 years, and ended the follow-up in 2020.
  • the method for detecting the methylation level of the CORIN gene promoter region is as described in Example 1.
  • the present invention constructs a methylation detection kit based on CpG3, CpG8 and CpG9 sites.
  • the specific detection methods are:
  • the EZ-96 DNA methylation kit (Zymo Research, Orange, CA) was used to perform sulfite treatment on the DNA samples that passed the quality inspection, and all the unmethylated cytosine C in the sample DNA was converted into uracil U.
  • the designed primers F: GGGTGGGATTTGTAGAGTAGATAA; R: ACTTAAAAACCCRACTCTACRACAA
  • F GGGTGGGATTTGTAGAGTAGATAA
  • R ACTTAAAAACCCRACTCTACRACAA
  • the amplified DNA product is then transcribed into an RNA fragment using T7 RNA polymerase, and the resulting RNA fragment is cut into small fragments containing CpG using RNase A.
  • the final product of unmethylated CpG is CpA
  • the final product of methylated CpG is CpG.
  • the molecular weight of this final product is detected using the Agena MassArray flight mass spectrometry analysis system.

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Abstract

Provided is a DNA methylation marker for evaluating the onset risk of cerebral stroke, a primer, and use thereof. The DNA methylation marker comprises at least one of DNA methylation sites Chr4:47840038, Chr4:47839941, and Chr4:47839933 of the CORIN gene promoter region. Also provided is a kit for evaluating the onset risk of cerebral stroke based on the DNA methylation marker.

Description

一种评估脑卒中发病风险的DNA甲基化标志物、引物及其应用A DNA methylation marker, primer and application for assessing the risk of stroke 技术领域Technical field
本发明涉及生物医药技术领域,尤其涉及一种评估脑卒中发病风险的DNA甲基化标志物、引物及其应用。The present invention relates to the field of biomedicine technology, and in particular to a DNA methylation marker, primer and application for assessing the risk of stroke.
背景技术Background technique
脑卒中是一类突发性的脑组织血液循环障碍疾病,目前已经成为全球范围内影响人类健康的首要疾病之一,2019年全球脑卒中患病达到1.01亿人,约有655万人死于脑卒中,脑卒中已经成为全球第二大死因。全球脑卒中发病率、患病率出现较大差异,我国属于脑卒中高发地区,2019年我国脑卒中患病总人数达到2876万人,219万人死于脑卒中,这为我国的医疗系统带来了巨大的负担,因此如何有效地防治脑卒中是我国公共卫生领域面临的重大挑战,发现更多的潜在危险因素与治疗靶点并促进其临床转化刻不容缓。Stroke is a sudden disorder of blood circulation in the brain tissue. It has become one of the leading diseases affecting human health worldwide. In 2019, the number of stroke patients worldwide reached 101 million, and about 6.55 million people died from it. Stroke has become the second leading cause of death in the world. There are great differences in the incidence and prevalence of stroke around the world. my country is an area with a high incidence of stroke. In 2019, the total number of people suffering from stroke in my country reached 28.76 million, and 2.19 million people died of stroke. This has brought great changes to our country’s medical system. Therefore, how to effectively prevent and treat stroke is a major challenge facing my country's public health field. It is urgent to discover more potential risk factors and treatment targets and promote their clinical transformation.
利钠肽系统(Natriuretic Peptide system,NPs)的主要成员包括心房利钠肽(ANP)和B型利钠肽(BNP),通过利钠、利尿和血管舒张在维持血压方面发挥重要作用。已有的研究表明,利钠肽水平的增加与脑卒中的发病风险有关。Corin是一种在心肌细胞中高表达的II型跨膜丝氨酸蛋白酶,是ANP的主要生理激活因子,也能够激活BNP。因此,Corin作为NPs的上游调节因子可能参与脑卒中的发病机制。基础研究和流行病学研究提示Corin对心血管的影响。例如,在扩张型心肌病小鼠中使Corin蛋白过表达可改善心功能并延缓心力衰竭的进展。相反,CORIN基因敲除小鼠则发生高血压和心肌肥厚。在人类中,Corin蛋白的编码基因CORIN的多态性与心衰、心肌肥厚和高血压的易感性相关。一些小型横断面临床研究发现,可溶性Corin与心血管疾病相关,如房颤,心力衰竭,心肌梗死。本实验室先前的研究发现,较低的血清Corin水平与脑卒中的患病率以及不良脑卒中预后显著相关。 上述研究提示了Corin蛋白可能与脑卒中的发病机制有关,Corin蛋白可能成为脑卒中防治的治疗靶点或候选药物。然而,在人血浆中未发现Corin蛋白的抑制剂,降低了其临床转化的安全性。因此,有必要更好地了解Corin与脑卒中之间关联的分子机制。表观遗传因子作为联系基因组和动态环境的界面,可能影响基因的表达和功能,从而具有作为我们需要的分子机制的潜力。DNA甲基化作为最常见和目前研究最为深入的表观遗传修饰,已被认为通过影响相关基因的表达参与了疾病的病理过程。例如,总体DNA甲基化水平与脑卒中有关,以及脑卒中相关基因启动子的甲基化,如ABCG1,MMP2,ERα,血栓调节蛋白和TP53在一些小样本病例对照研究中被证明与脑卒中有关。但目前尚未见关于CORIN基因DNA甲基化与脑卒中的研究报道,也没有报道一种合适的甲基化标志物可作为预防和控制脑卒中的干预靶点。The main members of the Natriuretic Peptide system (NPs) include atrial natriuretic peptide (ANP) and B-type natriuretic peptide (BNP), which play an important role in maintaining blood pressure through natriuresis, diuresis and vasodilation. Existing studies have shown that increased natriuretic peptide levels are related to the risk of stroke. Corin is a type II transmembrane serine protease highly expressed in cardiomyocytes. It is the main physiological activator of ANP and can also activate BNP. Therefore, Corin, as an upstream regulator of NPs, may be involved in the pathogenesis of stroke. Basic research and epidemiological studies suggest the cardiovascular effects of Corin. For example, overexpressing the protein Corin in mice with dilated cardiomyopathy improves cardiac function and delays the progression of heart failure. In contrast, CORIN knockout mice developed hypertension and cardiac hypertrophy. In humans, polymorphisms in the gene encoding Corin protein, CORIN, are associated with susceptibility to heart failure, cardiac hypertrophy, and hypertension. Some small cross-sectional clinical studies have found that soluble Corin is associated with cardiovascular diseases, such as atrial fibrillation, heart failure, and myocardial infarction. Previous studies from our laboratory found that lower serum Corin levels were significantly associated with the prevalence of stroke and poor stroke prognosis. The above studies suggest that Corin protein may be related to the pathogenesis of stroke, and Corin protein may become a therapeutic target or drug candidate for the prevention and treatment of stroke. However, no inhibitors of Corin protein have been found in human plasma, which reduces the safety of its clinical translation. Therefore, there is a need to better understand the molecular mechanisms underlying the association between Corin and stroke. Epigenetic factors serve as interfaces between the genome and the dynamic environment and may influence gene expression and function, thus having the potential to serve as the molecular mechanisms we need. As the most common and currently most intensively studied epigenetic modification, DNA methylation has been considered to be involved in the pathological process of disease by affecting the expression of related genes. For example, overall DNA methylation levels are associated with stroke, and methylation of the promoters of stroke-related genes, such as ABCG1, MMP2, ERα, thrombomodulin, and TP53, have been shown to be associated with stroke in some small case-control studies. related. However, there has been no research report on CORIN gene DNA methylation and stroke, nor has it been reported that a suitable methylation marker can be used as an intervention target to prevent and control stroke.
发明内容Contents of the invention
为解决上述技术问题,本发明通过对CORIN基因DNA甲基化与脑卒中表型相关性的研究,提供一种与脑卒中相关的DNA甲基化标志物,为预测脑卒中的发病风险、脑卒中高危人群的筛查提供依据,也有望作为预防和控制脑卒中的干预靶点。In order to solve the above technical problems, the present invention provides a DNA methylation marker related to stroke through research on the correlation between CORIN gene DNA methylation and stroke phenotype, which can be used to predict the risk of stroke and brain disease. It provides a basis for screening high-risk groups for stroke and is also expected to serve as an intervention target for the prevention and control of stroke.
本发明的第一个目的是提供一种用于评估脑卒中发病风险的DNA甲基化标志物,所述的DNA甲基化标志物包括CORIN基因启动子区的DNA甲基化位点Chr4:47840038、Chr4:47839941和Chr4:47839933中的至少一个位点。The first object of the present invention is to provide a DNA methylation marker for assessing the risk of stroke. The DNA methylation marker includes the DNA methylation site Chr4 in the promoter region of the CORIN gene: At least one site among 47840038, Chr4:47839941 and Chr4:47839933.
进一步地,DNA甲基化标志物包括CORIN基因启动子区的DNA甲基化位点的组合,DNA甲基化位点的组合为以下组合(1)-(4)中的任意一种:Further, the DNA methylation markers include a combination of DNA methylation sites in the promoter region of the CORIN gene, and the combination of DNA methylation sites is any one of the following combinations (1)-(4):
(1)Chr4:47840038和Chr4:47839941,(2)Chr4:47840038和Chr4:47839933,(3)Chr4:47839941和Chr4:47839933,(4)Chr4:47840038、Chr4:47839941和Chr4:47839933。(1) Chr4:47840038 and Chr4:47839941, (2) Chr4:47840038 and Chr4:47839933, (3) Chr4:47839941 and Chr4:47839933, (4) Chr4:47840038, Chr4:47839941 and Chr4:47 839933.
进一步地,扩增DNA甲基化标志物的上游引物如SEQ ID NO.1所示,下游引物如SEQ ID NO.2所示。Further, the upstream primer for amplifying DNA methylation markers is shown in SEQ ID NO.1, and the downstream primer is shown in SEQ ID NO.2.
本发明的第二个目的是提供上述DNA甲基化标志物在制备评估脑卒中发病风险的试剂盒中的应用。The second object of the present invention is to provide the application of the above-mentioned DNA methylation markers in preparing a kit for assessing the risk of stroke.
进一步地,评估脑卒中发病风险的试剂盒中包括扩增CORIN基因启动子区的DNA甲基化位点Chr4:47840038、Chr4:47839941和Chr4:47839933中至少一个位点的引物对。优选地,包括同时扩增DNA甲基化位点Chr4:47840038、Chr4:47839941和Chr4:47839933的引物对。Furthermore, the kit for assessing the risk of stroke includes a primer pair that amplifies at least one of the DNA methylation sites Chr4:47840038, Chr4:47839941 and Chr4:47839933 in the CORIN gene promoter region. Preferably, a primer pair that simultaneously amplifies the DNA methylation sites Chr4:47840038, Chr4:47839941 and Chr4:47839933 is included.
进一步地,所述引物对中,上游引物如SEQ ID NO.1所示,下游引物如SEQ ID NO.2所示。该引物对可同时扩增出Chr4:47840038、Chr4:47839941和Chr4:47839933位点,应用时再分别检测每个位点的甲基化水平,进而判断脑卒中发病风险。Further, in the primer pair, the upstream primer is as shown in SEQ ID NO.1, and the downstream primer is as shown in SEQ ID NO.2. This primer pair can simultaneously amplify the Chr4:47840038, Chr4:47839941 and Chr4:47839933 sites. When used, the methylation level of each site is separately detected to determine the risk of stroke.
进一步地,所述应用包括以下步骤:Further, the application includes the following steps:
(5)提取DNA样本,并对DNA样本进行亚硫酸盐处理;(5) Extract DNA samples and perform sulfite treatment on the DNA samples;
(6)用上述引物对扩增步骤(1)经亚硫酸盐处理后的样品,得到扩增产物;(6) Use the above primer pair to amplify the sample treated with sulfite in step (1) to obtain an amplification product;
(7)对步骤(2)的扩增产物进行转录和酶切,得到转录和酶切产物;(7) Carry out transcription and enzyme digestion on the amplification product of step (2) to obtain the transcription and enzyme digestion products;
(8)对步骤(3)的转录和酶切产物进行检测,获取样品序列中Chr4:47840038、Chr4:47839941和Chr4:47839933位点的甲基化程度。(8) Detect the transcription and enzyme digestion products of step (3) to obtain the methylation degree of Chr4:47840038, Chr4:47839941 and Chr4:47839933 sites in the sample sequence.
进一步地,DNA样本来源于血液,取样方便。Furthermore, DNA samples are derived from blood, making sampling easy.
本发明的第三个目的是提供扩增上述DNA甲基化标志物的引物对在制备评估脑卒中发病风险的试剂盒中的应用,上游引物如SEQ ID NO.1所示,下游引物如SEQ ID NO.2所示,具体地,The third object of the present invention is to provide the application of a primer pair for amplifying the above-mentioned DNA methylation marker in preparing a kit for assessing the risk of stroke. The upstream primer is as shown in SEQ ID NO.1, and the downstream primer is as shown in SEQ As shown in ID NO.2, specifically,
F:GGGTGGGATTTGTAGAGTAGATAAF: GGGTGGGATTTGTAGAGTAGATAA
R:ACTTTAAAAACCCRACTCTACRACAA,其中,R表示碱基A/G。R: ACTTAAAAACCCRACTCTACRACAA, where R represents base A/G.
本发明的第四个目的是提供一种评估脑卒中发病风险的试剂盒,该试剂盒中包括:分别或同时扩增CORIN基因启动子区的DNA甲基化位点Chr4:47840038、Chr4:47839941和Chr4:47839933的引物,以及检测Chr4:47840038、Chr4:47839941和Chr4:47839933中至少一个位点甲基化水平的试剂。The fourth object of the present invention is to provide a kit for assessing the risk of stroke, which kit includes: amplifying the DNA methylation sites Chr4:47840038 and Chr4:47839941 in the promoter region of the CORIN gene, respectively or simultaneously. and Chr4:47839933, and reagents for detecting the methylation level of at least one site in Chr4:47840038, Chr4:47839941 and Chr4:47839933.
进一步地,上游引物如SEQ ID NO.1所示,下游引物如SEQ ID NO.2所示。Further, the upstream primer is shown in SEQ ID NO.1, and the downstream primer is shown in SEQ ID NO.2.
本发明的第五个目的是提供上述DNA甲基化标志物在制备脑卒中治疗药物中的应用,该治疗药物针对CORIN基因启动子区的DNA甲基化位点Chr4:47840038、Chr4:47839941和Chr4:47839933中的至少一个位点进行设计,DNA甲基化是可改变的,基于上述DNA甲基化位点设计的靶向药物,可以调控CORIN基因的表达和Corin的分泌,进而达到治疗脑卒中的目的。The fifth object of the present invention is to provide the application of the above-mentioned DNA methylation markers in the preparation of stroke therapeutic drugs. The therapeutic drugs target the DNA methylation sites Chr4:47840038, Chr4:47839941 and By designing at least one site in Chr4:47839933, DNA methylation is changeable. Targeted drugs designed based on the above DNA methylation sites can regulate the expression of the CORIN gene and the secretion of Corin, thereby achieving the goal of treating brain disease. purpose of stroke.
借由上述方案,本发明至少具有以下优点:Through the above solutions, the present invention at least has the following advantages:
本发明提供的CORIN基因DNA甲基化标志物可用于预测脑卒中的发病风险,为脑卒中高危人群的筛查提供依据,也可以作为预防和控制脑卒中的干预靶点,不仅有助于阐释Corin上调作用于脑卒中的分子机制,也将为CORIN基因甲基化作为预防和控制脑卒中的药物靶点提供重要的流行病学证据。The CORIN gene DNA methylation markers provided by the present invention can be used to predict the risk of stroke, provide a basis for the screening of high-risk groups for stroke, and can also be used as intervention targets for the prevention and control of stroke, which not only helps to explain The molecular mechanism by which Corin upregulates stroke will also provide important epidemiological evidence for CORIN gene methylation as a drug target for the prevention and control of stroke.
上述说明仅是本发明技术方案的概述,为了能够更清楚了解本发明的技术手段,并可依照说明书的内容予以实施,以下以本发明的较佳实施例并配合详细附图说明如后。The above description is only an overview of the technical solutions of the present invention. In order to have a clearer understanding of the technical means of the present invention and implement them according to the content of the description, the preferred embodiments of the present invention are described below with detailed drawings.
附图说明Description of the drawings
为了使本发明的内容更容易被清楚的理解,下面根据本发明的具体实施例并结合附图,对本发明作进一步详细的说明。In order to make the content of the present invention easier to understand clearly, the present invention will be described in further detail below based on specific embodiments of the present invention and in conjunction with the accompanying drawings.
图1为靶向亚硫酸盐测序的目标序列和引物,以及预测的9个CpG位点;Figure 1 shows the target sequence and primers for targeted sulfite sequencing, as well as the predicted 9 CpG sites;
图2为脑卒中患者和健康对照中9个CpG位点的DNA甲基化水平检测结果;Figure 2 shows the detection results of DNA methylation levels of 9 CpG sites in stroke patients and healthy controls;
图3为CpG3位点对应的受试者工作特征曲线;Figure 3 shows the receiver operating characteristic curve corresponding to the CpG3 site;
图4为CpG8位点对应的受试者工作特征曲线;Figure 4 shows the receiver operating characteristic curve corresponding to the CpG8 site;
图5为CpG9位点对应的受试者工作特征曲线;Figure 5 shows the receiver operating characteristic curve corresponding to the CpG9 site;
图6为CpG3+CpG8模型对应的受试者工作特征曲线;Figure 6 shows the receiver operating characteristic curve corresponding to the CpG3+CpG8 model;
图7为CpG3+CpG9模型对应的受试者工作特征曲线;Figure 7 shows the receiver operating characteristic curve corresponding to the CpG3+CpG9 model;
图8为CpG8+CpG9模型对应的受试者工作特征曲线;Figure 8 shows the receiver operating characteristic curve corresponding to the CpG8+CpG9 model;
图9为CpG3+CpG8+CpG9模型对应的受试者工作特征曲线。Figure 9 shows the receiver operating characteristic curve corresponding to the CpG3+CpG8+CpG9 model.
具体实施方式Detailed ways
下面结合附图和具体实施例对本发明作进一步说明,以使本领域的技术人员可以更好地理解本发明并能予以实施,但所举实施例不作为对本发明的限定。The present invention will be further described below in conjunction with the accompanying drawings and specific examples, so that those skilled in the art can better understand and implement the present invention, but the examples are not intended to limit the present invention.
实施例1Example 1
本实验中共纳入了1771名研究对象,包括脑卒中患者853人和健康对照918人。提取每一位研究对象的全血DNA标本,运用目标区域测序技术检测CORIN基因启动子区域各CpG位点甲基化水平,即利用ENSEMBL数据库查询人类CORIN基因(基因编号:ENSG00000145244)的启动子区域,该区域为Chr4:47840136-47839906(GRCh37.p13,距TSS:-13bp to 217bp),在NCBI上截取该区域的核酸序列,将核酸序列导入EMBOSS Cpgplot软件预测CpG岛,然后运用Epidesigner程序对CpG岛及CpG密集区域序列进行引物设计,挑选合适的引物进行DNA甲基化检测(引物序列信息见表1)。经过挑选,CORIN甲基化共获得9个CpG位点的甲基化水平,9个CpG位点如图1所示。A total of 1,771 research subjects were included in this experiment, including 853 stroke patients and 918 healthy controls. Whole blood DNA samples of each research subject were extracted, and target region sequencing technology was used to detect the methylation level of each CpG site in the promoter region of the CORIN gene. That is, the ENSEMBL database was used to query the promoter region of the human CORIN gene (gene number: ENSG00000145244). , the region is Chr4:47840136-47839906 (GRCh37.p13, distance from TSS: -13bp to 217bp), intercept the nucleic acid sequence of this region on NCBI, import the nucleic acid sequence into EMBOSS Cpgplot software to predict CpG islands, and then use the Epidesigner program to map CpG Design primers based on the sequences of islands and CpG dense regions, and select appropriate primers for DNA methylation detection (see Table 1 for primer sequence information). After selection, CORIN methylation obtained a total of methylation levels of 9 CpG sites. The 9 CpG sites are shown in Figure 1.
表1 CORIN基因启动子区域DNA甲基化检测引物序列Table 1 Primer sequences for DNA methylation detection in CORIN gene promoter region
Figure PCTCN2022111781-appb-000001
Figure PCTCN2022111781-appb-000001
DNA甲基化的具体检测方法为:The specific detection methods for DNA methylation are:
首先用EZ-96DNA甲基化试剂盒(Zymo Research,Inc.,CA,United States)对DNA标本进行重亚硫酸盐处理,将样本DNA中没有甲基化修饰的胞嘧啶C全部转化为尿嘧啶U。接着用表1中的引物,以重亚硫酸盐处理后的样品基因组为模板,进行多重PCR扩增。为区分不同样品,利用带有Index序列的引物,通过PCR扩增向文库末端引入和illumina平台兼容的特异性标签序列。最终,将所有样品Index PCR扩增产物等量混合,在Illumina Hiseq/Miseq平台,以2×150bp/2×250bp的双端测序模式进行高通量测序,获得FastQ数据。各CpG位点甲基化水平量化为该位点甲基化的reads数目(即检测到碱基C的reads数目)/该位点总的reads数目×100%。First, the DNA sample was treated with bisulfite using the EZ-96 DNA Methylation Kit (Zymo Research, Inc., CA, United States) to convert all the unmethylated cytosine C in the sample DNA into uracil. U. Then use the primers in Table 1 and use the bisulfite-treated sample genome as a template to perform multiplex PCR amplification. In order to distinguish different samples, primers with Index sequences are used to introduce specific tag sequences compatible with the Illumina platform to the end of the library through PCR amplification. Finally, the Index PCR amplification products of all samples were mixed in equal amounts, and high-throughput sequencing was performed on the Illumina Hiseq/Miseq platform in the paired-end sequencing mode of 2×150bp/2×250bp to obtain FastQ data. The methylation level of each CpG site was quantified as the number of methylated reads at the site (i.e., the number of reads with base C detected)/the total number of reads at the site × 100%.
研究结果如下:The research results are as follows:
1、研究对象的临床特征1. Clinical characteristics of the research subjects
本实施例中包含了853名缺血性脑卒中患者(平均年龄62岁,男性占53%)和918名年龄性别匹配的健康对照(平均年龄61岁,男性占55%)。发现研究中缺血性脑卒中患者相比于健康对照有更多的代谢风险因素如:高血压、糖尿病、脂质、肥胖(all P<0.05)。This example included 853 ischemic stroke patients (average age 62 years old, 53% males) and 918 age and gender-matched healthy controls (average age 61 years, 55% males). It was found that ischemic stroke patients in the study had more metabolic risk factors such as hypertension, diabetes, lipids, and obesity than healthy controls (all P<0.05).
表2 研究对象的临床特征Table 2 Clinical characteristics of study subjects
Figure PCTCN2022111781-appb-000002
Figure PCTCN2022111781-appb-000002
Figure PCTCN2022111781-appb-000003
Figure PCTCN2022111781-appb-000003
2、CORIN基因启动子区域甲基化与缺血性脑卒中的关系2. The relationship between methylation of CORIN gene promoter region and ischemic stroke
对以上9个CpG位点的DNA甲基化水平进行检测,发现检测的9个CpG位点的DNA甲基化水平在脑卒中患者中均低于健康对照(见图2,ns:P>0.05;*:P<0.05;**:P<0.01;***:P<0.001)。The DNA methylation levels of the above 9 CpG sites were detected, and it was found that the DNA methylation levels of the 9 CpG sites tested were lower in stroke patients than in healthy controls (see Figure 2, ns: P>0.05 ;*:P<0.05; **:P<0.01; ***:P<0.001).
再调整了年龄、性别、教育水平、吸烟、饮酒、体质指数、低密度脂蛋白胆固醇、高密度脂蛋白胆固醇、糖尿病和高血压(见表3),所有9个甲基化位点均与缺血性脑卒中显著相关(P<0.05),并且在进行多重校正后这些关联依旧存在(q<0.05)。After adjusting for age, gender, education level, smoking, alcohol consumption, body mass index, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, diabetes and hypertension (see Table 3), all 9 methylation sites were associated with deficiencies. Hemorrhagic stroke was significantly associated (P<0.05), and these associations remained after multiple correction (q<0.05).
为了检验9个位点的甲基化水平能否提升传统危险因素对缺血性脑卒中的预测能力,绘制了受试者工作特征曲线(receiver operating characteristic curve ROC)并比较了曲线下面积(Area Under Curve AUC),发现9个位点均可以提升传统危险因素对缺血性脑卒中的预测能力,如图3至图5展示了3个位点的甲基化水平提升传统危险因素对缺血性脑卒中的预测能力:Chr4:47840038(0.8578vs 0.8515,P=0.0076);Chr4:47839941(0.8598vs 0.8515,P=0.0019);Chr4:47839933(0.8568vs 0.8515,P=0.0144)。图6至图9展示了多个甲基化位点组合模型的ROC曲线,发现位点组合后的模型也能提升传统危险因素对缺血性脑卒中的预测能力。In order to test whether the methylation levels of 9 sites can improve the predictive ability of traditional risk factors for ischemic stroke, the receiver operating characteristic curve (receiver operating characteristic curve ROC) was drawn and the area under the curve (Area Under Curve AUC), it was found that all 9 sites can improve the prediction ability of traditional risk factors for ischemic stroke. Figures 3 to 5 show that the methylation levels of 3 sites improve the prediction ability of traditional risk factors for ischemia. Predictive ability of stroke: Chr4:47840038 (0.8578vs 0.8515, P=0.0076); Chr4:47839941 (0.8598vs 0.8515, P=0.0019); Chr4:47839933 (0.8568vs 0.8515, P=0.01 44). Figures 6 to 9 show the ROC curves of the combined model of multiple methylation sites. It is found that the combined model can also improve the prediction ability of traditional risk factors for ischemic stroke.
同时,净重新分类指数(net reclassification improvement,NRI)和综合判别改善指数(integrated discrimination index,IDI)的计算结果也表明,3个CpG位点的甲基化水平可以提升传统危险因素对缺血性脑卒中的预测能力(表4)。At the same time, the calculation results of the net reclassification improvement (NRI) and the integrated discrimination index (IDI) also show that the methylation levels of the three CpG sites can improve the impact of traditional risk factors on ischemic disease. Predictive ability of stroke (Table 4).
表3 CORIN基因启动子区域甲基化水平与缺血性脑卒中之间的关系Table 3 Relationship between methylation level of CORIN gene promoter region and ischemic stroke
Figure PCTCN2022111781-appb-000004
Figure PCTCN2022111781-appb-000004
表4 各CpG位点净重新分类指数和综合判别改善指数Table 4 Net reclassification index and comprehensive discrimination improvement index of each CpG site
Figure PCTCN2022111781-appb-000005
Figure PCTCN2022111781-appb-000005
实施例2Example 2
根据以上的研究结果,我们发现CORIN基因启动子区域甲基化可能是脑卒中发病风险的预测因子,为了验证这一结论,我们进行了一项前瞻性的 队列研究。发明人于2010年纳入了2498名基线时未发生脑卒中的研究对象,每2年进行一次脑卒中事件的随访,并在2020结束随访。CORIN基因启动子区域甲基化水平检测方法如实施例1所述。Based on the above research results, we found that methylation in the CORIN gene promoter region may be a predictor of stroke risk. In order to verify this conclusion, we conducted a prospective cohort study. The inventors enrolled 2,498 research subjects who did not have stroke at baseline in 2010, followed up on stroke events every 2 years, and ended the follow-up in 2020. The method for detecting the methylation level of the CORIN gene promoter region is as described in Example 1.
1、研究对象的临床特征1. Clinical characteristics of the research subjects
纳入了基线时未发生脑卒中的研究对象2498名(平均年龄53岁,男性占39%),其中88人在随访期间发生了脑卒中,相比于随访期间未发生脑卒中的研究对象,发生脑卒中的研究对象有更多的代谢危险因素,如高血压、糖尿病、脂质(见表5)。A total of 2,498 research subjects who did not suffer from stroke at baseline (average age 53 years, 39% male) were included in the study, and 88 of them developed stroke during the follow-up period. Compared with the research subjects who did not suffer from stroke during the follow-up period, Stroke research subjects have more metabolic risk factors, such as hypertension, diabetes, and lipids (see Table 5).
表5 研究对象的临床特征Table 5 Clinical characteristics of study subjects
Figure PCTCN2022111781-appb-000006
Figure PCTCN2022111781-appb-000006
2、CORIN基因启动子区域甲基化与脑卒中的关系2. The relationship between methylation of CORIN gene promoter region and stroke
姑苏队列10年的随访过程中88人发生了脑卒中,71人死于非脑卒中原因,214人失访(随访率91.43%)。在调整了上述传统危险因素后,我们发现9个CpG位点中的3个位点甲基化水平与低脑卒中发病风险有关,他们是CpG3(位于Chr4:47840038,HR=0.74,P=0.015)、CpG8(位于Chr4:47839941,HR=0.80,P=0.047)、CpG9(Chr4:47839933,HR=0.82,P=0.050))(见表6)。During the 10-year follow-up of the Gusu cohort, 88 people suffered from stroke, 71 died of non-stroke causes, and 214 were lost to follow-up (follow-up rate 91.43%). After adjusting for the above traditional risk factors, we found that the methylation level of three of the nine CpG sites was associated with a lower risk of stroke. They were CpG3 (located at Chr4:47840038, HR=0.74, P=0.015 ), CpG8 (located at Chr4:47839941, HR=0.80, P=0.047), CpG9 (Chr4:47839933, HR=0.82, P=0.050)) (see Table 6).
为了检验Chr4:47840038、Chr4:47839941、Chr4:47839933甲基化水平能否提升传统危险因素对脑卒中的预测能力,计算了NRI和IDI,计算结果表明,Chr4:47840038和Chr4:47839933的甲基化水平可以提升传统危险因素对缺血性脑卒中的预测能力(表7)。In order to test whether the methylation levels of Chr4:47840038, Chr4:47839941, and Chr4:47839933 can improve the prediction ability of traditional risk factors for stroke, the NRI and IDI were calculated. The calculation results showed that the methylation levels of Chr4:47840038 and Chr4:47839933 The level of risk factors can improve the prediction ability of traditional risk factors for ischemic stroke (Table 7).
表6 CORIN基因启动子区域甲基化水平与脑卒中发病的关系Table 6 The relationship between the methylation level of CORIN gene promoter region and the incidence of stroke
Figure PCTCN2022111781-appb-000007
Figure PCTCN2022111781-appb-000007
表7 各CpG位点净重新分类指数和综合判别改善指数Table 7 Net reclassification index and comprehensive discrimination improvement index of each CpG site
Figure PCTCN2022111781-appb-000008
Figure PCTCN2022111781-appb-000008
实施例3构建甲基化检测试剂盒Example 3 Construction of methylation detection kit
基于以上研究,可以得知:如图1所示,位于启动子区域的CpG3、CpG8、CpG9位点发生甲基化之后,可能抑制CORIN基因表达和Corin蛋白分泌,进而参与脑卒中的发病,可以作为脑卒中发病风险的预测标志物和潜在药物靶点。因此,本发明构建了基于CpG3、CpG8和CpG9位点的甲基化检测试剂盒。Based on the above studies, it can be known that: as shown in Figure 1, after the CpG3, CpG8, and CpG9 sites located in the promoter region are methylated, it may inhibit CORIN gene expression and Corin protein secretion, and then participate in the pathogenesis of stroke. As a predictive marker and potential drug target for stroke risk. Therefore, the present invention constructs a methylation detection kit based on CpG3, CpG8 and CpG9 sites.
具体检测方法为:The specific detection methods are:
①全血DNA提取并质检① Whole blood DNA extraction and quality inspection
a.琼脂糖凝胶电泳检测基因组DNA完整性:电泳条带清晰可见,无明显降解,且无RNA污染。a. Agarose gel electrophoresis detects the integrity of genomic DNA: the electrophoresis band is clearly visible, with no obvious degradation and no RNA contamination.
b.Nanodrop 2000检测基因组DNA质量:浓度≥20ng/μL,总量≥1μg,OD260/280=1.7~2.0,OD260/230≥1.8。b. Nanodrop 2000 detects the quality of genomic DNA: concentration ≥20ng/μL, total amount ≥1μg, OD260/280=1.7~2.0, OD260/230≥1.8.
②亚硫酸盐处理②Sulfite treatment
用EZ-96DNA甲基化试剂盒(Zymo Research,Orange,CA)对质检合格的DNA标本进行亚硫酸盐处理,将样本DNA中没有甲基化的胞嘧啶C全部转化为尿嘧啶U。The EZ-96 DNA methylation kit (Zymo Research, Orange, CA) was used to perform sulfite treatment on the DNA samples that passed the quality inspection, and all the unmethylated cytosine C in the sample DNA was converted into uracil U.
③多重PCR扩增③Multiple PCR amplification
接着用设计好的引物(F:GGGTGGGATTTGTAGAGTAGATAA;R:ACTTTAAAAACCCRACTCTACRACAA)对亚硫酸盐处理过的标本进行DNA扩增,得到带有T7 RNA聚合酶启动子序列的扩增产物。Then, the designed primers (F: GGGTGGGATTTGTAGAGTAGATAA; R: ACTTAAAAACCCRACTCTACRACAA) were used to amplify the DNA of the sulfite-treated specimen, and an amplification product containing the T7 RNA polymerase promoter sequence was obtained.
④CpG片段切割④CpG fragment cleavage
然后运用T7 RNA聚合酶将扩增的DNA产物转录为RNA片段,用RNase A将所得的RNA片段切割成带有CpG的小片段。The amplified DNA product is then transcribed into an RNA fragment using T7 RNA polymerase, and the resulting RNA fragment is cut into small fragments containing CpG using RNase A.
⑤飞行质谱分析⑤Flight mass spectrometry analysis
最终,在每一个小的RNA片段内,未甲基化的CpG最终产物为CpA, 甲基化的CpG最终产物为CpG,使用Agena MassArray飞行质谱分析系统检测这个最终产物的分子量。Finally, within each small RNA fragment, the final product of unmethylated CpG is CpA, and the final product of methylated CpG is CpG. The molecular weight of this final product is detected using the Agena MassArray flight mass spectrometry analysis system.
⑥甲基化水平计算和脑卒中风险预测⑥Calculation of methylation levels and prediction of stroke risk
该CpG位点甲基化水平量化为产物质量CpG/(CpG+CpA)×100%,将各位点甲基化水平带入到预测模型:Logit(p)=-9.421495+0.041777*年龄+-0.507642*性别+2.063699*教育水平+0.063120*吸烟+0.036780*饮酒+0.238716*体质指数+-0.327117*LDL+0.327616*HDL+1.935708*糖尿病+1.594185*高血压+-0.028011*CpG3甲基化水平+-0.071986*CpG8甲基化水平+0.045531*CpG9甲基化水平中计算预测值p,当预测发病率p≥0.5131则提示脑卒中发病风险较高,应密切关注并采取预防性治疗措施。The methylation level of the CpG site is quantified as product quality CpG/(CpG+CpA)×100%, and the methylation level of each site is brought into the prediction model: Logit(p)=-9.421495+0.041777*Age+-0.507642 *Gender+2.063699*Education level+0.063120*Smoking+0.036780*Drinking+0.238716*Body mass index+-0.327117*LDL+0.327616*HDL+1.935708*Diabetes+1.594185*Hypertension+-0.028011*CpG3 methylation level+-0.0719 86 Calculate the predicted value p from *CpG8 methylation level + 0.045531 * CpG9 methylation level. When the predicted incidence rate p ≥ 0.5131, it indicates a higher risk of stroke, and you should pay close attention and take preventive treatment measures.
表8 各种位点甲基化组合模型对脑卒中发病诊断的比较Table 8 Comparison of various site methylation combination models for the diagnosis of stroke
Figure PCTCN2022111781-appb-000009
Figure PCTCN2022111781-appb-000009
各模型具体信息如下:The specific information of each model is as follows:
表9 模型的具体信息Table 9 Specific information of the model
Figure PCTCN2022111781-appb-000010
Figure PCTCN2022111781-appb-000010
Figure PCTCN2022111781-appb-000011
Figure PCTCN2022111781-appb-000011
Figure PCTCN2022111781-appb-000012
Figure PCTCN2022111781-appb-000012
Figure PCTCN2022111781-appb-000013
Figure PCTCN2022111781-appb-000013
显然,上述实施例仅仅是为清楚地说明所作的举例,并非对实施方式的限定。对于所属领域的普通技术人员来说,在上述说明的基础上还可以做出其它不同形式变化或变动。这里无需也无法对所有的实施方式予以穷举。而由此所引申出的显而易见的变化或变动仍处于本发明创造的保护范围之中。Obviously, the above-mentioned embodiments are only examples for clear explanation and are not intended to limit the implementation. For those of ordinary skill in the art, other changes or modifications may be made based on the above description. An exhaustive list of all implementations is neither necessary nor possible. The obvious changes or modifications derived therefrom are still within the protection scope of the present invention.

Claims (10)

  1. 一种用于评估脑卒中发病风险的DNA甲基化标志物,其特征在于:所述DNA甲基化标志物包括CORIN基因启动子区的以下DNA甲基化位点中的至少一个:Chr4:47840038、Chr4:47839941、Chr4:47839933。A DNA methylation marker for assessing the risk of stroke, characterized in that: the DNA methylation marker includes at least one of the following DNA methylation sites in the CORIN gene promoter region: Chr4: 47840038, Chr4:47839941, Chr4:47839933.
  2. 根据权利要求1所述的DNA甲基化标志物,其特征在于:所述DNA甲基化标志物包括CORIN基因启动子区的DNA甲基化位点的组合;所述DNA甲基化位点的组合为以下组合(1)-(4)中的任意一种:The DNA methylation marker according to claim 1, characterized in that: the DNA methylation marker includes a combination of DNA methylation sites in the CORIN gene promoter region; the DNA methylation site The combination is any one of the following combinations (1)-(4):
    (1)Chr4:47840038和Chr4:47839941,(1)Chr4:47840038 and Chr4:47839941,
    (2)Chr4:47840038和Chr4:47839933,(2)Chr4:47840038 and Chr4:47839933,
    (3)Chr4:47839941和Chr4:47839933,(3)Chr4:47839941 and Chr4:47839933,
    (4)Chr4:47840038、Chr4:47839941和Chr4:47839933。(4) Chr4:47840038, Chr4:47839941 and Chr4:47839933.
  3. 根据权利要求1所述的DNA甲基化标志物,其特征在于:扩增所述DNA甲基化标志物的上游引物如SEQ ID NO.1所示,下游引物如SEQ ID NO.2所示。The DNA methylation marker according to claim 1, characterized in that: the upstream primer for amplifying the DNA methylation marker is as shown in SEQ ID NO.1, and the downstream primer is as shown in SEQ ID NO.2 .
  4. 权利要求1-3任一项所述的DNA甲基化标志物在制备评估脑卒中发病风险的试剂盒中的应用。Application of the DNA methylation marker according to any one of claims 1 to 3 in the preparation of a kit for assessing the risk of stroke.
  5. 根据权利要求4所述的应用,其特征在于:所述评估脑卒中发病风险的试剂盒中包括扩增CORIN基因启动子区的DNA甲基化位点Chr4:47840038、Chr4:47839941和Chr4:47839933中至少一个位点的引物对。The application according to claim 4, characterized in that: the kit for assessing stroke risk includes DNA methylation sites Chr4:47840038, Chr4:47839941 and Chr4:47839933 that amplify the promoter region of the CORIN gene. Primer pairs for at least one site in .
  6. 根据权利要求5所述的应用,其特征在于:上游引物如SEQ ID NO.1所示,下游引物如SEQ ID NO.2所示。The application according to claim 5, characterized in that: the upstream primer is as shown in SEQ ID NO.1, and the downstream primer is as shown in SEQ ID NO.2.
  7. 根据权利要求4所述的应用,其特征在于,所述的应用包括以下步骤:The application according to claim 4, characterized in that the application includes the following steps:
    (1)提取DNA样本,对DNA样本进行亚硫酸盐处理;(1) Extract DNA samples and perform sulfite treatment on the DNA samples;
    (2)用所述引物对扩增步骤(1)经亚硫酸盐处理后的样品,得到扩增产物;(2) Use the primer pair to amplify the sample treated with sulfite in step (1) to obtain an amplification product;
    (3)对步骤(2)的扩增产物进行转录和酶切,得到转录和酶切产物;(3) Transcribe and digest the amplified product of step (2) to obtain the transcription and enzyme digestion products;
    (4)对步骤(3)的转录和酶切产物进行检测,获取Chr4:47840038、Chr4:47839941或Chr4:47839933位点的甲基化水平。(4) Detect the transcription and enzyme digestion products of step (3) to obtain the methylation level of Chr4:47840038, Chr4:47839941 or Chr4:47839933.
  8. 扩增权利要求1-3任一项所述的DNA甲基化标志物的引物对在制备评估脑卒中发病风险的试剂盒中的应用,其特征在于:上游引物如SEQ ID NO.1所示,下游引物如SEQ ID NO.2所示。The application of a primer pair for amplifying the DNA methylation marker described in any one of claims 1-3 in the preparation of a kit for assessing the risk of stroke, characterized in that: the upstream primer is as shown in SEQ ID NO.1 , the downstream primer is shown in SEQ ID NO.2.
  9. 一种评估脑卒中发病风险的试剂盒,其特征在于:所述评估脑卒中发病风险的试剂盒中包含:分别或同时扩增CORIN基因启动子区的DNA甲基化位点Chr4:47840038、Chr4:47839941和Chr4:47839933的引物,以及检测Chr4:47840038、Chr4:47839941和Chr4:47839933中至少一个位点甲基化水平的试剂。A kit for assessing the risk of stroke, characterized in that: the kit for assessing the risk of stroke includes: amplifying the DNA methylation sites Chr4:47840038 and Chr4 in the promoter region of the CORIN gene, respectively or simultaneously :47839941 and Chr4:47839933 primers, and reagents for detecting the methylation level of at least one site in Chr4:47840038, Chr4:47839941 and Chr4:47839933.
  10. 根据权利要求9所述的试剂盒,其特征在于:上游引物如SEQ ID NO.1所示,下游引物如SEQ ID NO.2所示。The kit according to claim 9, characterized in that: the upstream primer is as shown in SEQ ID NO.1, and the downstream primer is as shown in SEQ ID NO.2.
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