CN114807355A

CN114807355A - DNA methylation marker for evaluating stroke onset risk, primer and application thereof

Info

Publication number: CN114807355A
Application number: CN202210546425.7A
Authority: CN
Inventors: 彭浩; 陈立楠; 刘洋
Original assignee: Suzhou University
Current assignee: Suzhou University
Priority date: 2022-05-19
Filing date: 2022-05-19
Publication date: 2022-07-29
Also published as: WO2023221309A1

Abstract

The invention relates to a DNA methylation marker for evaluating the attack risk of cerebral apoplexy, a primer and application thereof, wherein the DNA methylation marker comprises at least one site of DNA methylation sites Chr4:47840038, Chr4:47839941 and Chr4:47839933 of a CORIN gene promoter region, and a kit for evaluating the attack risk of cerebral apoplexy is provided based on the DNA methylation marker. The CORIN gene DNA methylation marker provided by the invention can be used for predicting the stroke onset risk, can be used as a screening index of stroke high risk groups, and can also be used as an intervention target for preventing and controlling stroke. The invention not only is helpful for explaining the molecular mechanism of Corin participating in the attack of the cerebral apoplexy as a regulator of atrial natriuretic peptide, but also provides important epidemiological evidence for CORIN gene methylation serving as a drug target for preventing and controlling the cerebral apoplexy.

Description

DNA methylation marker for evaluating stroke onset risk, primer and application thereof

Technical Field

The invention relates to the technical field of biological medicines, in particular to a DNA methylation marker for evaluating stroke morbidity risk, a primer and application thereof.

Background

Cerebral apoplexy is a sudden cerebral tissue blood circulation disturbance disease, and is one of the primary diseases affecting human health worldwide at present, the cerebral apoplexy affects 1.01 hundred million people worldwide in 2019, about 655 million people die from the cerebral apoplexy, and the cerebral apoplexy becomes the second leading cause of death worldwide. Therefore, how to effectively prevent and treat cerebral apoplexy is a major challenge in the field of public health, and more potential risk factors and treatment targets are found and clinical transformation of the cerebral apoplexy is promoted.

The major members of the Natriuretic Peptide System (NPs) include Atrial Natriuretic Peptide (ANP) and B-type Natriuretic Peptide (BNP), which play important roles in maintaining blood pressure through Natriuretic, diuretic and vasodilatory effects. Previous studies have shown that increased levels of natriuretic peptides are associated with the risk of stroke. Corin is a type II transmembrane serine protease highly expressed in cardiac myocytes, is the major physiological activator of ANP, and is also capable of activating BNP. Therefore, Corin, an upstream regulator of NPs, may be involved in the pathogenesis of stroke. Basic and epidemiological studies suggest a cardiovascular effect of Corin. For example, overexpression of Corin protein in dilated cardiomyopathy mice can improve cardiac function and delay the progression of heart failure. In contrast, CORIN knockout mice develop hypertension and myocardial hypertrophy. In humans, polymorphisms in the Corin protein-encoding gene Corin are associated with susceptibility to heart failure, myocardial hypertrophy, and hypertension. Some small-scale 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 in this laboratory found that lower serum Corin levels were significantly associated with stroke prevalence and poor stroke prognosis. The research suggests that the Corin protein may be related to the pathogenesis of stroke, and the Corin protein may become a therapeutic target or a candidate drug for preventing and treating stroke. However, no inhibitor of Corin protein is found in human plasma, reducing the safety of clinical transformation. Therefore, there is a need to better understand the molecular mechanisms associated between Corin and stroke. Epigenetic factors serve as interfaces linking genomes and dynamic environments, possibly affecting gene expression and function, and thus have the potential to serve as molecular mechanisms for our needs. DNA methylation, the most common and currently most extensively studied epigenetic modification, has been implicated in the pathological process of disease by affecting the expression of the relevant genes. For example, overall DNA methylation levels associated with stroke, and methylation of the promoters of stroke-associated genes, such as ABCG1, MMP2, era, thrombomodulin and TP53, have been shown to be associated with stroke in several small sample disease control studies. However, no research report about DNA methylation of CORIN genes and stroke exists at present, and a proper methylation marker serving as an intervention target for preventing and controlling stroke is not reported.

Disclosure of Invention

In order to solve the technical problems, the invention provides a DNA methylation marker related to stroke through the research on the correlation between CORIN gene DNA methylation and stroke phenotype, provides a basis for predicting the attack risk of stroke and screening high-risk groups of stroke, and is expected to be used as an intervention target for preventing and controlling stroke.

The first object of the invention is to provide a DNA methylation marker for assessing the risk of stroke onset, wherein the DNA methylation marker comprises at least one of DNA methylation sites Chr4:47840038, Chr4:47839941 and Chr4:47839933 of a CORIN gene promoter region.

Further, the DNA methylation marker comprises a combination of DNA methylation sites of a CORIN gene promoter region, wherein the combination of the DNA methylation sites is any one of the following combinations (1) to (4):

(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: 47839933.

Further, an upstream primer of the amplified DNA methylation marker is shown as SEQ ID NO.1, and a downstream primer is shown as SEQ ID NO. 2.

The second purpose of the invention is to provide the application of the DNA methylation marker in the preparation of a kit for evaluating the attack risk of cerebral apoplexy.

Furthermore, the kit for evaluating the attack risk of the cerebral apoplexy comprises a primer pair for amplifying at least one site of DNA methylation sites Chr4:47840038, Chr4:47839941 and Chr4:47839933 of a CORIN gene promoter region. Preferably, primer pairs are included that simultaneously amplify DNA methylation sites Chr4:47840038, Chr4:47839941 and Chr4: 47839933.

Furthermore, in the primer pair, the upstream primer is shown as SEQ ID NO.1, and the downstream primer is shown as SEQ ID NO. 2. The primer pair can simultaneously amplify Chr4:47840038, Chr4:47839941 and Chr4:47839933 sites, and methylation level of each site is detected when the primer pair is applied, so that the attack risk of stroke is judged.

Further, the application comprises the steps of:

(5) extracting a DNA sample, and carrying out sulfite treatment on the DNA sample;

(6) amplifying the sample treated by the sulfite in the step (1) by using the primer pair to obtain an amplification product;

(7) carrying out transcription and enzyme digestion on the amplification product obtained in the step (2) to obtain a transcription and enzyme digestion product;

(8) and (4) detecting the transcription and enzyme digestion products in the step (3) to obtain the methylation degrees of the Chr4:47840038, Chr4:47839941 and Chr4:47839933 sites in the sample sequence.

Furthermore, the DNA sample is derived from blood, and the sampling is convenient.

The third purpose of the invention is to provide the application of the primer pair for amplifying the DNA methylation marker in the preparation of a kit for evaluating the attack risk of cerebral apoplexy, wherein the upstream primer is shown as SEQ ID NO.1, the downstream primer is shown as SEQ ID NO.2, specifically,

F：GGGTGGGATTTGTAGAGTAGATAA

r: ACTTTAAAAACCCRACTCTACRACAA, wherein R represents a base A/G.

The fourth purpose of the invention is to provide a kit for evaluating the attack risk of cerebral apoplexy, which comprises: primers for amplifying DNA methylation sites Chr4:47840038, Chr4:47839941 and Chr4:47839933 of a CORIN gene promoter region respectively or simultaneously, and a reagent for detecting the methylation level of at least one site of Chr4:47840038, Chr4:47839941 and Chr4: 47839933.

Further, the upstream primer is shown as SEQ ID NO.1, and the downstream primer is shown as SEQ ID NO. 2.

The fifth purpose of the invention is to provide the application of the DNA methylation marker in the preparation of a medicine for treating cerebral apoplexy, the medicine is designed aiming at least one site of DNA methylation sites Chr4:47840038, Chr4:47839941 and Chr4:47839933 of a CORIN gene promoter region, DNA methylation is changeable, and the targeted medicine designed based on the DNA methylation sites can regulate the expression of CORIN gene and the secretion of Corin, thereby achieving the purpose of treating cerebral apoplexy.

By the scheme, the invention at least has the following advantages:

the CORIN gene DNA methylation marker provided by the invention can be used for predicting the attack risk of stroke, provides a basis for screening high-risk groups of stroke, can also be used as an intervention target for preventing and controlling stroke, is not only beneficial to explaining the molecular mechanism of Corin up-regulation acting on stroke, but also provides important epidemiological evidence for the methylation of CORIN gene as a drug target for preventing and controlling stroke.

The foregoing description is only an overview of the technical solutions of the present invention, and in order to make the technical solutions of the present invention more clearly understood and to implement them in accordance with the contents of the description, the following description is made with reference to the preferred embodiments of the present invention and the accompanying detailed drawings.

Drawings

In order that the present disclosure may be more readily and clearly understood, reference will now be made in detail to the present disclosure, examples of which are illustrated in the accompanying drawings.

FIG. 1 shows target sequences and primers for targeted sulfite sequencing, and predicted 9 CpG sites;

FIG. 2 shows the result of detecting the DNA methylation level of 9 CpG sites in stroke patients and healthy controls;

FIG. 3 is a graph of the working characteristics of a subject at CpG 3;

FIG. 4 is a graph of the working characteristics of a subject at CpG 8;

FIG. 5 is a graph of the working characteristics of a subject at CpG 9;

FIG. 6 is a graph of the working characteristics of subjects corresponding to the CpG3+ CpG8 model;

FIG. 7 is a graph of the working characteristics of subjects corresponding to the CpG3+ CpG9 model;

FIG. 8 is a graph of the working characteristics of subjects corresponding to the CpG8+ CpG9 model;

FIG. 9 is a graph of the working characteristics of subjects corresponding to the CpG3+ CpG8+ CpG9 model.

Detailed Description

The present invention is further described below in conjunction with the following figures and specific examples so that those skilled in the art may better understand the present invention and practice it, but the examples are not intended to limit the present invention.

Example 1

A total of 1771 subjects, including 853 subjects with stroke and 918 healthy controls, were included in this experiment. The whole blood DNA sample of each research object is extracted, the methylation level of each CpG site in the CORIN gene promoter region is detected by using a target region sequencing technology, namely an ENSEMBL database is used for inquiring the promoter region of a human CORIN gene (gene number: ENSG00000145244), the region is Chr4: 47840136-. By selection, CORIN methylation obtains methylation levels of 9 CpG sites in total, and 9 CpG sites are shown in fig. 1.

TABLE 1 primer sequences for DNA methylation detection in CORIN Gene promoter region

The specific detection method of DNA methylation comprises the following steps:

first, DNA samples were bisulfite treated with EZ-96DNA methylation kit (Zymo Research, Inc., CA, United States) to convert all cytosine C that was not methylated in the sample DNA to uracil U. Then, multiplex PCR amplification was performed using the primers shown in Table 1 and the bisulfite-treated sample genome as a template. To distinguish between different samples, specific tag sequences compatible with the illumina platform were introduced by PCR amplification to the ends of the library using primers with Index sequences. Finally, all sample Index PCR amplification products are equally mixed, and high-throughput sequencing is carried out on an Illumina Hiseq/Miseq platform in a double-end sequencing mode of 2 × 150bp/2 × 250bp, so as to obtain FastQ data. The methylation level at each CpG site is quantified as the number of reads methylated at that site (i.e., the number of reads in which base C is detected)/total number of reads for that site x 100%.

The results of the study are as follows:

1. clinical characteristics of the subject

853 patients with ischemic stroke (average age 62 years, 53% male) and 918 age-gender-matched healthy controls (average age 61 years, 55% male) were included in this example. Patients with ischemic stroke in the study were found to have more metabolic risk factors than healthy controls such as: hypertension, diabetes, lipids, obesity (all P < 0.05).

TABLE 2 clinical characteristics of the subjects

2. Correlation between CORIN gene promoter region methylation and cerebral arterial thrombosis

The DNA methylation levels of the above 9 CpG sites were examined, and it was found that the DNA methylation levels of the 9 CpG sites examined were lower than those of the healthy controls in all patients with stroke (see FIG. 2, ns: P > 0.05;. P < 0.01;. P < 0.001).

Age, gender, educational level, smoking, drinking, body mass index, ldl cholesterol, hdl cholesterol, diabetes and hypertension were readjusted (see table 3), all 9 sites of methylation were significantly associated with ischemic stroke (P <0.05), and these associations remained after multiple corrections (q < 0.05).

In order to test whether the methylation levels of 9 sites can improve the prediction capability of the traditional risk factors on the ischemic stroke, a working characteristic Curve (Area Under Curve AUC) of a subject is drawn and the areas Under the Curve are compared, and the 9 sites can improve the prediction capability of the traditional risk factors on the ischemic stroke, for example, the methylation levels of 3 sites can improve the prediction capability of the traditional risk factors on the ischemic stroke as shown in FIGS. 3 to 5, namely, Chr4:47840038(0.8578vs 0.8515, P is 0.0076); chr4:47839941(0.8598vs 0.8515, P ═ 0.0019); chr4:47839933 (0.8568vs 0.8515, P ═ 0.0144). The ROC curves of a plurality of methylation site combination models are shown in FIGS. 6 to 9, and the site combination models are found to improve the prediction capability of traditional risk factors on ischemic stroke.

Meanwhile, the calculation results of the net receptance improvement index (NRI) and the integrated discrimination improvement index (IDI) also indicate that the methylation levels of 3 CpG sites can improve the prediction ability of the traditional risk factors on ischemic stroke (table 4).

TABLE 3 correlation between methylation level of CORIN gene promoter region and ischemic stroke

TABLE 4 Net reclassification index and comprehensive discrimination improvement index for individual CpG sites

Example 2

Based on the above research results, we found that methylation of the CORIN gene promoter region may be a predictor of stroke risk, and to verify this conclusion, we performed a prospective cohort study. The inventors included 2498 subjects who did not develop stroke at baseline in 2010, followed stroke events every 2 years and ended the follow-up at 2020. The methylation level of the CORIN gene promoter region was determined as described in example 1.

1. Clinical characteristics of the subject

2498 subjects who did not develop stroke at baseline (mean age 53 years, 39% male) were enrolled, with 88 of them developing stroke during follow-up, which subjects developed stroke with more metabolic risk factors such as hypertension, diabetes, lipids (see Table 5) than subjects who did not develop stroke during follow-up.

TABLE 5 clinical characteristics of the subjects

2. Correlation between CORIN gene promoter region methylation and stroke

In the follow-up course of 10 years, 88 people suffered from stroke, 71 people died from non-stroke reasons and 214 people lost visit (follow-up rate 91.43%). After adjusting the above traditional risk factors, we found that the methylation level of 3 of the 9 CpG sites is associated with low stroke risk, they are 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).

In order to test whether the methylation level of Chr4:47840038, Chr4:47839941 and Chr4:47839933 can improve the prediction capability of the traditional risk factors on the cerebral apoplexy, NRI and IDI are calculated, and the calculation results show that the methylation level of Chr4:47840038 and Chr4:47839933 can improve the prediction capability of the traditional risk factors on the cerebral ischemic stroke (Table 7).

TABLE 6 correlation between methylation level of CORIN gene promoter region and stroke onset

TABLE 7 Absolute reclassification index and comprehensive discrimination improvement index of each CpG site

Example 3 construction of methylation detection kit

Based on the above studies, it can be known that: as shown in figure 1, after methylation occurs at CpG3, CpG8 and CpG9 sites in a promoter region, CORIN gene expression and Corin protein secretion can be inhibited, and then the CORIN protein can participate in the attack of cerebral apoplexy, and can be used as a prediction marker and a potential drug target for the attack risk of cerebral apoplexy. Therefore, the invention constructs a methylation detection kit based on CpG3, CpG8 and CpG9 sites.

The specific detection method comprises the following steps:

firstly, whole blood DNA extraction and quality inspection

a. Agarose gel electrophoresis for genomic DNA integrity: the electrophoresis strip is clearly visible, has no obvious degradation and has no RNA pollution.

Nanodrop 2000 detection of genomic DNA quality: the concentration is more than or equal to 20 ng/mu L, the total amount is more than or equal to 1 mu g, OD260/280 is 1.7-2.0, and OD260/230 is more than or equal to 1.8.

② treatment with sulfite

The qualified DNA samples were sulfite-treated with EZ-96DNA methylation kit (Zymo Research, Orange, Calif.) to convert all unmethylated cytosine C in the sample DNA to uracil U.

③ multiplex PCR amplification

The sulfite-treated sample was then subjected to DNA amplification using the designed primers (F: GGGTGGGATTTGTAGAGTAGATAA; R: ACTTTAAAAACCCRACTCTACRACAA) to obtain an amplification product having a T7 RNA polymerase promoter sequence.

Cutting of CpG fragments

The amplified DNA product is then transcribed into RNA fragments using T7 RNA polymerase, and the resulting RNA fragments are cleaved into CpG-bearing small fragments using RNase A.

Flight mass spectrometry

Finally, within each small RNA fragment, the unmethylated CpG end-product was CpA and the methylated CpG end-product was CpG, and the molecular weight of this end-product was determined using the Agena MassArray flight mass spectrometry system.

Sixthly, calculating methylation level and predicting cerebral apoplexy risk

The methylation level of the CpG sites is quantified as the product mass CpG/(CpG + CpA). times.100%, and the methylation level of each site is brought into a 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 + -0.071986 CpG8 methylation level +0.045531 CpG9 methylation level, the predicted value p is calculated, and when the predicted incidence p is not less than 0.5131, the risk of stroke is high, and the attention is focused and preventive treatment measures are taken.

TABLE 8 comparison of various site methylation combinatorial models for stroke diagnosis

The specific information of each model is as follows:

table 9 details of the model

It should be understood that the above examples are only for clarity of illustration and are not intended to limit the embodiments. Other variations and modifications will be apparent to persons skilled in the art in light of the above description. And are neither required nor exhaustive of all embodiments. And obvious variations or modifications of the invention may be made without departing from the spirit or scope of the invention.

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Claims

1. A DNA methylation marker for assessing the risk of stroke onset, characterized by: the DNA methylation marker comprises at least one of the following DNA methylation sites of the CORIN gene promoter region: chr4:47840038, Chr4:47839941 and Chr4: 47839933.

2. The DNA methylation marker according to claim 1, wherein: the DNA methylation marker comprises a combination of DNA methylation sites of a CORIN gene promoter region; the combination of the DNA methylation sites is any one of the following combinations (1) to (4):

(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: 47839933.

3. The DNA methylation marker according to claim 1, wherein: the upstream primer for amplifying the DNA methylation marker is shown as SEQ ID NO.1, and the downstream primer is shown as SEQ ID NO. 2.

4. Use of the DNA methylation marker of any one of claims 1-3 for the preparation of a kit for assessing the risk of stroke onset.

5. Use according to claim 4, characterized in that: the kit for evaluating the attack risk of the cerebral apoplexy comprises a primer pair for amplifying at least one site of DNA methylation sites Chr4:47840038, Chr4:47839941 and Chr4:47839933 of a CORIN gene promoter region.

6. Use according to claim 5, characterized in that: the upstream primer is shown as SEQ ID NO.1, and the downstream primer is shown as SEQ ID NO. 2.

7. The use according to claim 4, characterized in that it comprises the following steps:

(1) extracting a DNA sample, and carrying out sulfite treatment on the DNA sample;

(2) amplifying the sample treated by the sulfite in the step (1) by using the primer pair to obtain an amplification product;

(3) carrying out transcription and enzyme digestion on the amplification product obtained in the step (2) to obtain a transcription and enzyme digestion product;

(4) and (4) detecting the transcription and enzyme digestion products in the step (3) to obtain the methylation level of the sites Chr4:47840038, Chr4:47839941 or Chr4: 47839933.

8. Use of a primer pair for amplifying the DNA methylation marker of any one of claims 1 to 3 for preparing a kit for assessing the risk of stroke onset, wherein: the upstream primer is shown as SEQ ID NO.1, and the downstream primer is shown as SEQ ID NO. 2.

9. A kit for assessing the risk of stroke onset is characterized in that: the kit for evaluating the attack risk of the cerebral apoplexy comprises: primers for amplifying DNA methylation sites Chr4:47840038, Chr4:47839941 and Chr4:47839933 of a CORIN gene promoter region respectively or simultaneously, and a reagent for detecting the methylation level of at least one site of Chr4:47840038, Chr4:47839941 and Chr4: 47839933.

10. The kit of claim 9, wherein: the upstream primer is shown as SEQ ID NO.1, and the downstream primer is shown as SEQ ID NO. 2.