A NPPB gene DNA hydroxymethylation marker, primer and application thereof

Abstract

The present invention relates to a NPPB gene DNA hydroxymethylation marker, a primer and an application thereof. The NPPB gene DNA hydroxymethylation marker comprises at least one of the DNA hydroxymethylation sites Chr1:11919160 (CpG1), Chr1:11919144 (CpG2), Chr1:11919133 (CpG5) and Chr1:11919011 (CpG10) in the promoter region of the NPPB gene, and the degree of hydroxymethylation thereof indicates the risk of stroke. The NPPB gene DNA hydroxymethylation marker provided by the present invention can be used to predict the risk of stroke, and provides a new direction for the prevention and treatment of stroke. At the same time, a stroke-specific DNA hydroxymethylation marker combination is provided based on the above DNA hydroxymethylation sites, and detection based on multiple DNA hydroxymethylation sites overcomes the problem of low single DNA hydroxymethylation signal, and improves the detection sensitivity, specificity and accuracy.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine technology, and in particular to a NPPB gene DNA hydroxymethylation marker, a primer and an application thereof. Background Art

[0002] B-type natriuretic peptide (BNP) is a member of the natriuretic peptide family and is a polypeptide composed of 32 amino acid residues. When the ventricular load increases, ventricular myocytes synthesize and secrete BNP to participate in the regulation of cardiovascular homeostasis. Many basic studies have found that BNP can participate in the regulation of cardiovascular homeostasis and the occurrence and development of cardiovascular diseases and their risk factors by relaxing vascular smooth muscle—dilation of blood vessels, blocking the sympathetic nervous system—inhibition of adrenocortical hormone release, and inhibiting the renin angiotensin aldosterone system—diuresis and sodium excretion. In clinical practice, BNP has become a biomarker for heart failure, used for screening, diagnosis and differential diagnosis of heart failure, and is also a useful predictor of the future outcome of patients with heart failure. Nesiritide, a recombinant human B-type natriuretic peptide synthesized according to the physiological characteristics of BNP, has been used clinically for patients with heart failure. However, it is worth noting that many clinical studies have found that nesiritide can cause dose-dependent hypotension and increase the mortality rate of patients with chronic heart failure and acute renal failure. Therefore, further exploration of the molecular mechanism of BNP's involvement in regulating cardiovascular diseases will help develop new drugs and thus prevent and control cardiovascular diseases. Diseases such as left ventricular dysfunction, atrial fibrillation, heart failure, and atherosclerosis are known causes of elevated plasma BNP levels, and the high incidence of these cardiovascular diseases ultimately leads to stroke. A large number of epidemiological studies have shown that plasma BNP levels are associated with stroke, and plasma BNP levels at admission can predict in-hospital mortality in stroke patients. Stroke is an acute cerebrovascular disease, a group of diseases that cause brain tissue damage due to sudden rupture of cerebral blood vessels or blockage of blood vessels that prevent blood from flowing into the brain, including ischemic and hemorrhagic strokes. The morbidity and mortality of stroke remain high. How to effectively prevent and treat stroke is a major challenge facing my country's public health field.

[0003] At present, the main diagnostic methods for stroke include cranial CT, cranial MRI, carotid color Doppler ultrasound CTA and angiography. These methods are complicated and cumbersome, and imaging examinations have limited diagnostic ability for small lesions, low detection sensitivity and specificity, and the diagnostic results are somewhat subjective. Therefore, new diagnostic and treatment strategies are urgently needed in the clinic, and exploring more pathogenesis and therapeutic targets will make a significant contribution to human health. So far, there have been no reports on the relationship between NPPB gene DNA hydroxymethylation and stroke, nor have there been reports on a suitable hydroxymethylation marker that can be used as an intervention target for the prevention and control of stroke. Summary of the invention

[0004] To solve the above technical problems, the present invention provides a DNA hydroxymethylation marker associated with stroke by studying the correlation between NPPB gene DNA hydroxymethylation and stroke phenotype, and further combines them to provide a stroke-specific DNA hydroxymethylation marker combination. The detection based on multiple DNA hydroxymethylation sites overcomes the problem of low single DNA hydroxymethylation signal, improves detection sensitivity, specificity and accuracy, and is also safer.

[0005] The first object of the present invention is to provide a DNA hydroxymethylation marker for predicting the risk of stroke, wherein the DNA hydroxymethylation marker includes the DNA hydroxymethylation sites Chr1:11919160 (CpG1) (CpG1), Chr1:11919144 (CpG2) (CpG2), Chr1:11919133 (CpG5) (CpG5) or Chr1:11919011 (CpG10) in the promoter region of the NPPB gene.

[0006] Further, the DNA hydroxymethylation marker is selected from a combination of at least two sites among Chr1:11919160 (CpG1), Chr1:11919144 (CpG2), Chr1:11919133 (CpG5) and Chr1:11919011 (CpG10).

[0007] Preferably, the DNA hydroxymethylation marker is any one of the following combinations (1)-(10):

[0008] (1) Chr1:11919160 and Chr1:11919144,

[0009] (2) Chr1:11919160 and Chr1:11919133,

[0010] (3) Chr1:11919160 and Chr1:11919011,

[0011] (4) Chr1:11919144 and Chr1:11919133,

[0012] (5) Chr1:11919144 and Chr1:11919011,

[0013] (6) Chr1:11919133 and Chr1:11919011,

[0014] (7) Chr1:11919160, Chr1:11919144 and Chr1:11919133,

[0015] (8) Chr1:11919160, Chr1:11919144 and Chr1:11919011,

[0016] (9) Chr1:11919160, Chr1:11919133 and Chr1:11919011,

[0017] (10) Chr1:11919144, Chr1:11919133 and Chr1:11919011,

[0018] (11) Chr1:11919160, Chr1:11919144, Chr1:11919133 and Chr1:11919011.

[0019] Furthermore, detecting the hydroxymethylation level of the above DNA hydroxymethylation marker comprises the following steps:

[0020] (1) extracting DNA samples and treating the DNA samples with T4 β-glucosyltransferase and APOBEC3A enzyme;

[0021] (2) amplifying the sample treated in step (1) to obtain an amplified product;

[0022] (3) transcribing and digesting the amplified product of step (2) to obtain transcription and digestion products;

[0023] (4) Detecting the transcription and enzyme digestion products of step (3) to obtain the hydroxymethylation level of Chr1:11919160 (CpG1), Chr1:11919144 (CpG2), Chr1:11919133 (CpG5) or Chr1:11919011 (CpG10) in the sequence.

[0024] Furthermore, the DNA sample is a blood sample.

[0025] Furthermore, the upstream primer for amplifying the DNA hydroxymethylation marker is shown as SEQ ID NO.1, and the downstream primer is shown as SEQ ID NO.2.

[0026] The second object of the present invention is to provide the use of the above-mentioned DNA hydroxymethylation marker in the preparation of a kit for predicting the risk of stroke.

[0027] Furthermore, the kit also includes primers for amplifying at least one of the DNA hydroxymethylation sites Chr1:11919160 (CpG1), Chr1:11919144 (CpG2), Chr1:11919133 (CpG5) and Chr1:11919011 (CpG10) in the promoter region of the NPPB gene.

[0028] Furthermore, the upstream primer is shown in SEQ ID NO. 1, and the downstream primer is shown in SEQ ID NO. 2. The primer pair can simultaneously amplify the four sites, and then detect the hydroxymethylation level of each site respectively, thereby determining the risk of stroke.

[0029] The third object of the present invention is to provide a primer for amplifying the above DNA hydroxymethylation marker in the preparation of a kit for predicting the risk of stroke, wherein the upstream primer is shown in SEQ ID NO.1, and the downstream primer is shown in SEQ ID NO.2. Specifically,

[0030] F: GGTTTATTTTTATATAAGGTYGGTTTTGTT

[0031] R:ACRTCCRAATTTACTTCCCACCTAC, where R represents the base A / G.

[0032] The fourth object of the present invention is to provide a kit for predicting the risk of stroke, which includes: primers for amplifying at least one of the DNA hydroxymethylation sites Chr1:11919160 (CpG1), Chr1:11919144 (CpG2), Chr1:11919133 (CpG5) and Chr1:11919011 (CpG10) in the promoter region of the NPPB gene.

[0033] Furthermore, the kit also contains a reagent for detecting the hydroxymethylation level of at least one site among Chr1:11919160 (CpG1), Chr1:11919144 (CpG2), Chr1:11919133 (CpG5) and Chr1:11919011 (CpG10), the reagent corresponds to the primer, and the site detected by the reagent at least covers the site amplified by the primer.

[0034] Furthermore, the upstream primer is shown as SEQ ID NO.1, and the downstream primer is shown as SEQ ID NO.2.

[0035] By means of the above scheme, the present invention has at least the following advantages:

[0036] The NPPB gene DNA hydroxymethylation marker provided by the present invention can be used to predict the risk of stroke, provide a basis for screening of high-risk populations for stroke, and can also be used as an intervention target for preventing and controlling stroke. At the same time, the detection based on multiple DNA hydroxymethylation sites overcomes the problem of low single DNA hydroxymethylation signal and improves the detection sensitivity, specificity and accuracy.

[0037] The above description is only an overview of the technical solution of the present invention. In order to more clearly understand the technical means of the present invention and implement it according to the contents of the specification, the following is a description of the preferred embodiments of the present invention in conjunction with detailed drawings. BRIEF DESCRIPTION OF THE DRAWINGS

[0038] In order to make the contents of the present invention more clearly understood, the present invention is further described in detail below based on specific embodiments of the present invention in conjunction with the accompanying drawings.

[0039] Figure 1 Target sequences and primers for NGS-based multi-target CpG hydroxymethylation sequencing, as well as 11 predicted CpG sites;

[0040] Figure 2 The correlation between DNA hydroxymethylation levels of 11 CpG sites and stroke in stroke patients and healthy controls;

[0041] Figure 3-13 It is a comparison diagram of the ROC curves of the traditional model only and each CpG site combined with the traditional model;

[0042] Figure 14-24 This is a comparison chart of the ROC curves of the traditional model combined with various combination models of CpG sites. DETAILED DESCRIPTION

[0043] The present invention is further described below in conjunction with the accompanying drawings and specific embodiments so that those skilled in the art can better understand the present invention and implement it, but the embodiments are not intended to limit the present invention.

[0044] Example 1

[0045] In the CATIS (China Antihypertensive Trial for Acute Ischemic Stroke) population, 1771 people were included, including 853 stroke patients and 918 healthy controls. Whole blood DNA samples were extracted from each research subject, and the hydroxymethylation level of each CpG site in the promoter region of the NPPB gene was detected using the NGS-based multi-target CpG hydroxymethylation sequencing technology, that is, the promoter region of the human NPPB gene was queried using the ENSEMBL database, which is Chr1:11919190-11918953 (from TSS: -168bp to 3bp), and the nucleic acid sequence of this region was intercepted on NCBI, and the nucleic acid sequence was imported into the EMBOSS Cpgplot software to predict the CpG island, and then the Epidesigner program was used to design primers for the CpG island and CpG dense region sequences, and appropriate primers were selected for DNA hydroxymethylation detection (primer sequence information is shown in Table 1). After selection, the hydroxymethylation levels of 11 CpG sites in NPPB hydroxymethylation were obtained. The 11 CpG sites were as follows: Figure 1 shown.

[0046] Table 1 Primer sequences for detecting DNA hydroxymethylation in the promoter region of NPPB gene

[0047]

[0048] The specific detection method of DNA hydroxymethylation is:

[0049] First, the DNA sample was treated with T4β-glucosyltransferase and APOBEC3A enzyme in sequence using a DNA hydroxymethylation kit to label the hydroxymethylated cytosine in the sample DNA with glucose. The labeled hydroxymethylated cytosine will not be deaminated by the APOBEC3A enzyme, while the remaining cytosine and methylated cytosine are converted into thymine under the action of the APOBEC3A enzyme. Then, multiple PCR amplification was performed using the primers in Table 1 and the sample genome after enzyme conversion as a template. In order to distinguish different samples, primers with Index sequences were used to introduce specific label sequences compatible with the Illumina platform to the end of the library through PCR amplification. Finally, all sample Index PCR amplification products were mixed in equal amounts and high-throughput sequenced on the Illumina HiSeq / Miseq platform in a 2x150bp / 2x250bp double-end sequencing mode to obtain FastQ data. The hydroxymethylation level of each CpG site was quantified as the number of reads with hydroxymethylation at the site (ie, the number of reads in which base C was detected) / the total number of reads at the site × 100%.

[0050] The results of the study are as follows:

[0051] 1. Clinical characteristics of the subjects

[0052] Table 2 shows the clinical characteristics of the study subjects, including 853 ischemic stroke patients (mean age 62 years, 53% male) and 918 age- and sex-matched healthy controls (mean age 61 years, 55% male). Ischemic stroke patients had more metabolic risk factors such as hypertension, diabetes, lipids, and obesity than healthy controls (P<0.05).

[0053] Table 2 Clinical characteristics of the subjects

[0054]

[0055]

[0056] 2. The relationship between DNA hydroxymethylation in the promoter region of NPPB gene and ischemic stroke As shown in Table 3, the DNA hydroxymethylation levels of the 11 CpG sites we detected were higher in stroke patients than in healthy controls (q<0.05). Figure 2 As shown, after adjusting for age, sex, education level, smoking, drinking, body mass index, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, hypertension and diabetes, CpG1 (Chr1:11919160), CpG2 (Chr1:11919144), CpG5 (Chr1:11919133), CpG9 (Chr1:11919019), CpG10 (Chr1:11 919011), CpG11 (Chr1:11918989) were significantly correlated with ischemic stroke (P<0.05). After multiple corrections, CpG1 (Chr1:11919160), CpG2 (Chr1:11919144), CpG5 (Chr1:11919133), and CpG10 (Chr1:11919011) were still associated with stroke (q<0.05).

[0057] Table 3 Median levels of NPPB promoter hydroxymethylation in ischemic stroke patients and healthy controls

[0058]

[0059] In order to test whether the methylation level of CpG sites can improve the predictive ability of traditional risk factors for stroke, the receiver operating characteristic curve (ROC) was drawn and the area under the curve (AUC) was compared. The results are as follows: Figure 3-13 As shown in Table 4 and Figure 3-13As shown, the area under the ROC curve: AUC (traditional risk factors + CpG1 hydroxymethylation level) > AUC (traditional risk factors + CpG2 hydroxymethylation level) = AUC (traditional risk factors + CpG5 hydroxymethylation level) > AUC (traditional risk factors + CpG10 hydroxymethylation level) > AUC (traditional risk factors + CpG9 hydroxymethylation level) > AUC (traditional risk factors + CpG 4 traditional risk factors + CpG 4 hydroxymethylation level) = AUC (traditional risk factors + CpG7 hydroxymethylation level) = AUC (traditional risk factors + CpG11 hydroxymethylation level) > AUC (traditional risk factors + CpG3 hydroxymethylation level) = AUC (traditional risk factors + CpG6 hydroxymethylation level) = AUC (traditional risk factors + CpG8 hydroxymethylation level) = AUC (traditional risk factors only); combined Figure 2 Based on the results (CpG1, CpG2, CpG5, and CpG10 were still associated with stroke after correction for multiple tests (q<0.05)), four loci were screened out: CpG1, CpG2, CpG5, and CpG10.

[0060] Table 4 The diagnosis of ischemic stroke using the traditional model combined with each CpG site

[0061]

[0062] The traditional model included age, sex, education level, smoking, alcohol consumption, body mass index, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, history of hypertension, and history of diabetes.

[0063] As shown in Table 5 and Figure 14-24 As shown, the model with the largest area under the ROC curve (AUC) is: traditional model + CpG1 + CpG2 + CpG5 + CpG10, with an AUC of 0.866 and a P value less than 0.05.

[0064] Table 5 The diagnosis of ischemic stroke using the traditional model combined with the CpG site combination model

[0065]

[0066] The traditional model included age, sex, education level, smoking, alcohol consumption, body mass index, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, history of hypertension, and history of diabetes.

[0067] Table 6 shows the predictive value of CpG1 combined with CpG2, CpG5, and CpG10 hydroxymethylation levels for the risk of ischemic stroke. On the basis of the traditional model, after incorporating the hydroxymethylation levels of CpG1, CpG2, CpG5, and CpG10, the predictive level of the traditional model + CpG1 + CpG2 + CpG5 + CpG10 model for the risk of ischemic stroke was improved (NRI 0.0475%, P < 0.05, IDI 0.0326%).

[0068] Table 6 Predictive value of CpG1 combined with CpG2, CpG5, and CpG10 hydroxymethylation levels for the risk of ischemic stroke

[0069]

[0070]

[0071] NRI: Net Reclassification Improvement Index; IDI: Overall Identification Index

[0072] The traditional model includes age, sex, education level, smoking, drinking, body mass index, low-density lipoprotein cholesterol, high

[0073] low-density lipoprotein cholesterol, history of hypertension, and history of diabetes mellitus.

[0074] As shown in Table 7, the calculation formula of the optimal stroke prediction model was: Logit(P)=-12.509147+0.031318*age+0.558676*sex+2.022532*education level+0.399809*smoking+0.082706*drinking+0.260029*body mass index-0.356943*low-density lipoprotein cholesterol+0.353775*high-density lipoprotein cholesterol+1.600649*history of hypertension+1.767850*history of diabetes+0.542035*CpG1 hydroxymethylation level+0.425184*CpG2 hydroxymethylation level+0.847899*CpG5 hydroxymethylation level+0.679995*CpG10 hydroxymethylation level. The cutoff value of this model is: P = 0.4935596.

[0075] Table 7 Logistic regression analysis of influencing factors of ischemic stroke

[0076]

[0077]

[0078] Logit(P)=-12.509147+0.031318*age+0.558676*sex+2.022532*education level+0.399809*smoking+0.082706*drinking+0.260029*body mass index-0.356943*low-density lipoprotein cholesterol+0.353775*high-density lipoprotein cholesterol+1.600649*history of hypertension+1.767850*history of diabetes+0.542035*CpG1 hydroxymethylation level+0.425184*CpG2 hydroxymethylation level+0.847899*CpG5 hydroxymethylation level+0.679995*CpG10 hydroxymethylation level

[0079] Example 2 Construction of a Hydroxymethylation Detection Kit

[0080] Based on the above research, it can be learned that after hydroxymethylation occurs at the CpG1 (Chr1: 11919160), CpG2 (Chr1: 11919144), CpG5 (Chr1: 11919133) and CpG10 (Chr1: 11919011) sites located in the promoter region, NPPB gene expression may be promoted and thus participate in the onset of stroke, and can be used as a predictive marker and potential drug target for the risk of stroke. Therefore, the present invention constructs a hydroxymethylation detection kit based on CpG1 (Chr1: 11919160), CpG2 (Chr1: 11919144), CpG5 (Chr1: 11919133) and CpG10 (Chr1: 11919011) sites.

[0081] The specific detection method is:

[0082] ① Whole blood DNA extraction and quality inspection

[0083] a. Detection of genomic DNA integrity by agarose gel electrophoresis: The electrophoresis bands were clearly visible, with no obvious degradation and no RNA contamination.

[0084] b.Nanodrop 2000 detection of genomic DNA quality: concentration ≥ 20 ng / μL, total amount ≥ 1 μg, OD260 / 280 = 1.7-2.0, OD260 / 230 ≥ 1.8.

[0085] ②T4β-glucosyltransferase and APOBEC3A enzyme treatment

[0086] The DNA samples that passed the quality inspection were treated with T4β-glucosyltransferase and APOBEC3A enzyme using a DNA hydroxymethylation kit to convert all the remaining cytosine and methylated cytosine in the sample DNA into thymine.

[0087] ③Multiplex PCR amplification

[0088] Then, the designed primers (F: GGTTTATTTTTATATAAGGTYGGTTTTGTT; R: ACRTCCRAATTTACTTCCCACCTAC) were used to amplify the DNA of the enzyme-treated specimen to obtain an amplification product with a T7 RNA polymerase promoter sequence.

[0089] ④CpG fragment cutting

[0090] The amplified DNA product is then transcribed into RNA fragments using T7 RNA polymerase, and the resulting RNA fragments are cut into small fragments containing CpG using RNase A.

[0091] ⑤ Flight mass spectrometry

[0092] Finally, in each small RNA fragment, the final product of unhydroxymethylated CpG is CpA, and the final product of hydroxymethylated CpG is CpG. The molecular weight of this final product is detected using the Agena MassArray flight mass spectrometry system.

[0093] ⑥ Calculation of hydroxymethylation level and prediction of stroke risk

[0094] The hydroxymethylation level of the CpG site is quantified as product mass CpG / (CpG+CpA)×100%. When the data is brought into the stroke prediction model: Logit(P)=-12.509147+0.031318*age+0.558676*sex+2.022532*education level+0.399809*smoking+0.082706*drinking+0.260029*body mass index-0.356943*low-density lipoprotein cholesterol+ 0.353775*high-density lipoprotein cholesterol+1.600649*history of hypertension+1.767850*history of diabetes+0.542035*CpG1 hydroxymethylation level+0.425184*CpG2 hydroxymethylation level+0.847899*CpG5 hydroxymethylation level+0.679995*CpG10 hydroxymethylation level. When the predicted model P value is calculated to be >0.4935596, it indicates a high risk of stroke and should be closely monitored and preventive treatment measures should be taken.

[0095] Obviously, the above embodiments are merely examples for clear explanation and are not intended to limit the implementation methods. For those skilled in the art, other different forms of changes or modifications can be made based on the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from these are still within the protection scope of the invention. Sequence Listing <110> Soochow University <120> A NPPB gene DNA hydroxymethylation marker, primer and application thereof <160> 2 <170> SIPOSequenceListing 1.0 <210> 1 <211> 30 <212> DNA <213> (Artificial sequence) <400> 1 ggtttatttt tatataaggt yggttttgtt 30 <210> 2 <211> 25 <212> DNA <213> (Artificial sequence) <400> 2 acrtccraatttacttcccacctac 25

Claims

1. Use of primers for amplifying DNA hydroxymethylation markers in the preparation of a kit for predicting the risk of stroke, characterized in that: The DNA hydroxymethylation marker is NPPB The combination of DNA hydroxymethylation sites Chr1:11919160, Chr1:11919144, Chr1:11919133 and Chr1:11919011 in the gene promoter region, the kit is used to detect the methylation levels of the four DNA hydroxymethylation sites.

2. The use according to claim 1, characterized in that: The method for detecting the hydroxymethylation level of a site comprises the following steps: (1) extracting a DNA sample, and treating the DNA sample with T4 β-glucosyltransferase and APOBEC3A enzyme; (2) amplifying the sample treated in step (1) to obtain an amplification product; (3) performing transcription and enzyme digestion on the amplified product of step (2) to obtain transcription and enzyme digestion products; (4) Detecting the transcription and enzyme digestion products of step (3) to obtain the hydroxymethylation levels of sites Chr1:11919160, Chr1:11919144, Chr1:11919133 and Chr1:11919011 in the sample sequence.

3. The use according to claim 1, characterized in that: The upstream primer for amplifying the DNA hydroxymethylation marker is shown in SEQ ID NO.1, and the downstream primer is shown in SEQ ID NO.2.

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