WO2023206854A1 - Dna hydroxymethylation marker and kit for predicting attack risk of cerebral ischemic stroke - Google Patents

Dna hydroxymethylation marker and kit for predicting attack risk of cerebral ischemic stroke Download PDF

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WO2023206854A1
WO2023206854A1 PCT/CN2022/110851 CN2022110851W WO2023206854A1 WO 2023206854 A1 WO2023206854 A1 WO 2023206854A1 CN 2022110851 W CN2022110851 W CN 2022110851W WO 2023206854 A1 WO2023206854 A1 WO 2023206854A1
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hydroxymethylation
dna
ischemic stroke
marker
kit
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彭浩
姚佳玲
刘洋
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苏州大学
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  • the invention relates to the field of biomedicine technology, and in particular to a DNA hydroxymethylation marker and a kit for predicting the risk of ischemic stroke.
  • ischemic stroke is the main type of stroke, accounting for about 70% to 80% of the total number of strokes.
  • the natriuretic peptide axis is an important cardiac endocrine regulatory system for the body to respond to external environmental stimuli. It plays an important role in maintaining the body's water and sodium balance, blood pressure stability, and energy metabolism balance.
  • the natriuretic peptide axis is mainly composed of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), C-type natriuretic peptide (CNP) and their receptors.
  • the ANP encoding gene NPPA has many genes. The status is significantly related to the susceptibility of hypertension, stroke, myocardial infarction and coronary heart disease; synthetic ANP (recombinant carperitide, valsartan) has been used to treat acute heart failure, but it can cause hypotension and adverse outcomes such as angioedema. It can be seen that synthetic ANP may have major hidden dangers as a drug. A better understanding of the mechanism of ANP involved in ischemic stroke will help develop drugs for ANP and promote the clinical translation of ANP-related research results. However, there have been no reports on the relationship between NPPA gene DNA hydroxymethylation and ischemic stroke, nor has it been reported that a suitable hydroxymethylation marker can be used as an intervention target for ischemic stroke.
  • the present invention provides a DNA hydroxymethylation marker related to ischemic stroke by studying the correlation between NPPA gene DNA hydroxymethylation and ischemic stroke.
  • the degree of methylation indicates the risk of ischemic stroke and helps to explain the molecular mechanism of atrial natriuretic peptide on ischemic stroke. Every 2-fold increase in hydroxymethylation level increases the risk of ischemic stroke. The risk is increased by 39% and the detection sensitivity is high.
  • the first object of the present invention is to provide a DNA hydroxymethylation marker for predicting the risk of ischemic stroke.
  • the DNA hydroxymethylation marker includes a DNA hydroxymethylation site in the promoter region of the NPPA gene. Chr1:11908348, its hydroxymethylation degree indicates the risk of ischemic stroke.
  • hydroxymethylation level of DNA hydroxymethylation site Chr1:11908348 is higher than 2.5%, indicating a higher risk of ischemic stroke.
  • upstream primer for amplifying the above-mentioned DNA hydroxymethylation marker is shown in SEQ ID NO.1
  • the downstream primer is shown in SEQ ID NO.2.
  • detecting the hydroxymethylation level of the above-mentioned DNA hydroxymethylation marker includes the following steps:
  • step (3) Transcribe and digest the amplified product of step (2) to obtain the transcription and enzyme digestion products;
  • step (3) Detect the transcription and enzyme digestion products of step (3) to obtain the degree of hydroxymethylation at Chr1:11908348 in the sequence.
  • the DNA sample is a blood sample.
  • the second object of the present invention is to provide the use of the above-mentioned DNA hydroxymethylation markers in preparing a kit for predicting the risk of ischemic stroke.
  • the kit also includes primers for amplifying the DNA hydroxymethylation site Chr1:11908348 in the promoter region of the NPPA gene.
  • upstream primer is shown in SEQ ID NO.1
  • downstream primer is shown in SEQ ID NO.2.
  • R AAAAATCCTTAATTATCTCACCRCC, where R represents base A/G.
  • the fourth object of the present invention is to provide a kit for predicting the risk of ischemic stroke, which kit includes: a primer for amplifying the DNA hydroxymethylation site Chr1: 11908348 in the promoter region of the NPPA gene.
  • the kit also includes a reagent for detecting the hydroxymethylation level of the DNA hydroxymethylation site Chr1:11908348 in the promoter region of the NPPA gene.
  • the present invention at least has the following advantages:
  • the NPPA gene DNA hydroxymethylation marker provided by the present invention can be used to predict the risk of ischemic stroke, and can also be used as an intervention target to prevent and control ischemic stroke.
  • the detection of the hydroxymethylation marker The sensitivity is significantly better than existing ischemic stroke detection markers, while having good accuracy and specificity.
  • Figure 1 is a schematic diagram of the DNA hydroxymethylation marker of the present invention
  • Figure 3 shows the relationship between NPPA gene DNA hydroxymethylation level and the risk of ischemic stroke
  • Figure 4 is the ROC curve of NPPA gene Chr1:11908348 DNA hydroxymethylation in predicting ischemic stroke
  • Figure 5 is a schematic diagram comparing the prediction of ischemic stroke by two models before and after traditional risk factors plus NPPA gene Chr1:11908348 DNA hydroxymethylation.
  • this region is Chromosome 1:11908117-11908380 (GRCh37.P13, distance from TSS:-540bp to-277bp), 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 Epidesigner
  • the program designs primers for CpG islands and CpG dense region sequences, and selects appropriate primers for DNA hydroxymethylation detection (see Table 1 for primer sequence information). After selection, a total of 9 CpG sites were obtained with hydroxymethylation levels.
  • the 9 CpG sites are shown in Figure 2, namely Chr1:11908353 (CpG1), Chr1:11908348 (CpG2), and Chr1:11908299 (CpG3). , Chr1:11908200(CpG4), Chr1:11908182(CpG5), Chr1:11908178(CpG6), Chr1:11908168(CpG7), Chr1:11908165(CpG8), Chr1:11908142(CpG9).
  • the specific detection methods are:
  • a DNA hydroxymethylation detection kit is used to sequentially treat the DNA sample with T4 ⁇ -glucosyltransferase and APOBEC3A enzyme to label the hydroxymethylated cytosine in the sample DNA with glucose. Pyrimidines are not deaminated by APOBEC3A enzyme, and the remaining cytosine and methylated cytosine are converted into thymine under the action of APOBEC3A enzyme. Then use the primers in Table 1 and use the enzyme-converted 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.
  • 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 hydroxymethylation level of each CpG site was quantified as the number of hydroxymethylated reads at the site (i.e., the number of reads detecting base C)/the total number of reads at the site ⁇ 100%.
  • Table 2 shows the clinical characteristics of the study subjects. It is found that compared with healthy controls, patients with ischemic stroke have more traditional risk factors, such as history of hypertension, diabetes, obesity, hyperglycemia, hyperlipidemia, etc. (all P ⁇ 0.05).
  • Figure 4 shows the ROC curve of the hydroxymethylation level at this site and ischemic stroke.
  • the AUC is 0.53>0.5, indicating that the hydroxymethylation at this site has a certain ability to predict ischemic stroke.
  • NRI net reclassification index
  • IDI comprehensive discriminant index
  • the specific detection methods are:
  • DNA hydroxymethylation detection kit to treat qualified DNA samples with T4 ⁇ -glucosyltransferase and APOBEC3A enzyme to label the hydroxymethylated cytosine in the sample DNA with glucose. Cytosine will not be deaminated by APOBEC3A enzyme, while the remaining cytosine and methylated cytosine will be converted into thymine under the action of APOBEC3A enzyme.
  • the designed primers (F: TTTTGTTTGAGGTTAGAGGTTTGTTTA; R: AAAAATCCTTAATTATCTCACCRCC) were used to amplify the DNA of the enzyme-converted specimen to obtain an amplification product containing the T7 RNA polymerase promoter sequence.
  • 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 hydroxymethylation level of this CpG site is quantified as product quality CpG/(CpG+CpA) ⁇ 100%.
  • the hydroxymethylation level >2.5% indicates a higher risk of ischemic stroke (see Figure 4) and should be closely followed. Pay attention and take preventive treatment measures.
  • the diagnosis of stroke by the CpG2 site methylation model is shown in Table 3.

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Abstract

The present invention relates to a DNA hydroxymethylation marker and a kit for predicting an attack risk of cerebral ischemic stroke. The DNA hydroxymethylation marker comprises a DNA hydroxymethylation site Chr1:11908348 in an NPPA gene promoter region, wherein the degree of hydroxymethylation at the site indicates a degree of the attack risk of cerebral ischemic stroke. The NPPA gene DNA hydroxymethylation marker provided by the present invention can be used for predicting the attack risk of cerebral ischemic stroke, and can also be used as an intervention target for preventing and controlling cerebral ischemic stroke, which not only helps explain the molecular mechanism of atrial natriuretic peptide acting on cerebral ischemic stroke, but also provides important epidemiological evidence for NPPA gene hydroxymethylation as a target for a medicament for preventing and controlling cerebral ischemic stroke.

Description

预测缺血性脑卒中发病风险的DNA羟甲基化标志物及试剂盒DNA hydroxymethylation markers and kits for predicting the risk of ischemic stroke 技术领域Technical field
本发明涉及生物医药技术领域，尤其涉及一种预测缺血性脑卒中发病风险的DNA羟甲基化标志物及试剂盒。The invention relates to the field of biomedicine technology, and in particular to a DNA hydroxymethylation marker and a kit for predicting the risk of ischemic stroke.
背景技术Background technique
近年来虽然我国在心脑血管疾病的预防和治疗方面已取得一定的成绩，但脑卒中发病率、患病率、复发率和死亡率仍然居高不下，是我国居民死亡和残疾的首要原因，且随着社会老龄化和城市化进程加速，居民不健康生活方式流行，心脑血管疾病危险因素普遍暴露，我国脑卒中疾病未来有爆发式增长的态势。脑卒中主要可以分为缺血性脑卒中和出血性卒中两类，我国脑卒中主要以缺血性脑卒中为主，约占脑卒中总数的70％～80％。据全球疾病负担研究(GBD)数据显示，我国缺血性脑卒中的患病率由2010年的1100/10万上升至2019年的1256/10万，出血性卒中的患病率由2010年的232/10万下降至215/10万，脑卒中患病率总体呈上升趋势。另外，《中国卒中报告2019》显示我国每5位死者中至少有1人死于脑卒中，带病生存的脑卒中患者在我国已多达1300万。因此，如何有效地预防和控制脑卒中主要的类型(缺血性脑卒中)，寻找并发现更多缺血性脑卒中的潜在危险因素和干预靶点迫在眉睫。Although my country has made certain achievements in the prevention and treatment of cardiovascular and cerebrovascular diseases in recent years, the incidence, prevalence, recurrence and mortality of stroke remain high and are the primary cause of death and disability among Chinese residents. Moreover, as society ages and urbanization accelerates, residents adopt unhealthy lifestyles, and cardiovascular and cerebrovascular disease risk factors are generally exposed, my country's stroke disease will see explosive growth in the future. Stroke can be mainly divided into two categories: ischemic stroke and hemorrhagic stroke. In my country, ischemic stroke is the main type of stroke, accounting for about 70% to 80% of the total number of strokes. According to data from the Global Burden of Disease Study (GBD), the prevalence of ischemic stroke in my country increased from 1100/100,000 in 2010 to 1256/100,000 in 2019, and the prevalence of hemorrhagic stroke increased from The prevalence rate of stroke dropped from 232/100,000 to 215/100,000, and the prevalence of stroke showed an overall upward trend. In addition, the "China Stroke Report 2019" shows that at least 1 out of every 5 people who die in my country dies from stroke, and there are as many as 13 million stroke patients living with the disease in our country. Therefore, it is urgent to effectively prevent and control the main type of stroke (ischemic stroke) and to find and discover more potential risk factors and intervention targets for ischemic stroke.
利钠肽轴是机体应对外界环境刺激的重要的心脏内分泌调节***，在维持机体水钠平衡、血压稳定和能量代谢平衡方面发挥着重要作用。利钠肽轴主要由心房钠尿肽(ANP)、脑钠尿肽(BNP)、C型钠尿肽(CNP)以及他们的受体构成。当血容量增加时，心肌细胞就会分泌并释放大量的无生物活性的心房钠尿肽前体(pro-ANP)和脑钠尿肽前体(pro-BNP)，pro-ANP、 pro-NNP进一步活化为有活性的ANP和BNP，并与其受体结合激活利钠肽轴，进而促进水钠代谢、降低血容量、扩张血管、促进能量代谢，从而维持心血管稳态。作为利钠肽轴的重要组成之一，ANP可能在缺血性脑卒中的发生发展过程中扮演重要角色，已有大量流行病学证据支持这一假设，例如，ANP的编码基因NPPA的基因多态性与高血压、脑卒中、心肌梗死和冠心病的人群易感性显著相关；人工合成的ANP(重组卡培立肽、缬沙坦)已经被用来治疗急性心衰，但会引起低血压和血管性水肿等不良结局。可见，合成ANP作为药物可能存在较大隐患，更好的理解ANP参与缺血性脑卒中的作用机理将有助于针对ANP的药物研发，促进ANP的相关研究结果向临床转化。但是，尚未见关于NPPA基因DNA羟甲基化与缺血性脑卒中的研究报道，也没有报道一种合适的羟甲基化标志物可作为缺血性脑卒中的干预靶点。The natriuretic peptide axis is an important cardiac endocrine regulatory system for the body to respond to external environmental stimuli. It plays an important role in maintaining the body's water and sodium balance, blood pressure stability, and energy metabolism balance. The natriuretic peptide axis is mainly composed of atrial natriuretic peptide (ANP), brain natriuretic peptide (BNP), C-type natriuretic peptide (CNP) and their receptors. When blood volume increases, myocardial cells secrete and release large amounts of biologically inactive atrial natriuretic peptide precursor (pro-ANP) and brain natriuretic peptide precursor (pro-BNP), pro-ANP, pro-NNP It is further activated into active ANP and BNP, and binds to its receptors to activate the natriuretic peptide axis, thereby promoting water and sodium metabolism, reducing blood volume, dilating blood vessels, and promoting energy metabolism, thereby maintaining cardiovascular homeostasis. As one of the important components of the natriuretic peptide axis, ANP may play an important role in the occurrence and development of ischemic stroke. There is a large amount of epidemiological evidence to support this hypothesis. For example, the ANP encoding gene NPPA has many genes. The status is significantly related to the susceptibility of hypertension, stroke, myocardial infarction and coronary heart disease; synthetic ANP (recombinant carperitide, valsartan) has been used to treat acute heart failure, but it can cause hypotension and adverse outcomes such as angioedema. It can be seen that synthetic ANP may have major hidden dangers as a drug. A better understanding of the mechanism of ANP involved in ischemic stroke will help develop drugs for ANP and promote the clinical translation of ANP-related research results. However, there have been no reports on the relationship between NPPA gene DNA hydroxymethylation and ischemic stroke, nor has it been reported that a suitable hydroxymethylation marker can be used as an intervention target for ischemic stroke.
发明内容Contents of the invention
为解决上述技术问题，本发明通过对NPPA基因DNA羟甲基化与缺血性脑卒中相关性的研究，提供了一种与缺血性脑卒中相关的DNA羟甲基化标志物，其羟甲基化程度指示缺血性脑卒中发病风险，有助于阐释心房利钠肽作用于缺血性脑卒中的分子机制，且羟甲基化水平每增加2倍，缺血性脑卒中的发病风险升高39％，检测灵敏度高。In order to solve the above technical problems, the present invention provides a DNA hydroxymethylation marker related to ischemic stroke by studying the correlation between NPPA gene DNA hydroxymethylation and ischemic stroke. The degree of methylation indicates the risk of ischemic stroke and helps to explain the molecular mechanism of atrial natriuretic peptide on ischemic stroke. Every 2-fold increase in hydroxymethylation level increases the risk of ischemic stroke. The risk is increased by 39% and the detection sensitivity is high.
本发明的第一个目的是提供一种预测缺血性脑卒中发病风险的DNA羟甲基化标志物，该DNA羟甲基化标志物包括NPPA基因启动子区的DNA羟甲基化位点Chr1:11908348，其羟甲基化程度指示缺血性脑卒中发病风险程度。The first object of the present invention is to provide a DNA hydroxymethylation marker for predicting the risk of ischemic stroke. The DNA hydroxymethylation marker includes a DNA hydroxymethylation site in the promoter region of the NPPA gene. Chr1:11908348, its hydroxymethylation degree indicates the risk of ischemic stroke.
进一步地，DNA羟甲基化位点Chr1:11908348的羟甲基化水平高于2.5％指示缺血性脑卒中发病风险较高。Furthermore, the hydroxymethylation level of DNA hydroxymethylation site Chr1:11908348 is higher than 2.5%, indicating a higher risk of ischemic stroke.
进一步地，DNA羟甲基化位点Chr1:11908348的羟甲基化水平每增加2倍，缺血性脑卒中的发病风险升高39％。Furthermore, for every 2-fold increase in the hydroxymethylation level of the DNA hydroxymethylation site Chr1:11908348, the risk of ischemic stroke increased by 39%.
进一步地，扩增上述DNA羟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 the above-mentioned DNA hydroxymethylation marker is shown in SEQ ID NO.1, and the downstream primer is shown in SEQ ID NO.2.
进一步地，检测上述DNA羟甲基化标志物的羟甲基化水平包括以下步骤：Further, detecting the hydroxymethylation level of the above-mentioned DNA hydroxymethylation marker includes the following steps:
(1)提取DNA样本，用T4β-葡糖基转移酶和APOBEC3A酶处理DNA样本；(1) Extract DNA samples and treat the DNA samples with T4β-glucosyltransferase and APOBEC3A enzyme;
(2)用合适的引物对(如上述SEQ ID NO.1和SEQ ID NO.2)扩增步骤(1)处理后的DNA样本，得到扩增产物；(2) Use a suitable primer pair (such as SEQ ID NO.1 and SEQ ID NO.2 above) to amplify the DNA sample processed 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，获取序列中Chr1:11908348位点的羟甲基化程度。(4) Detect the transcription and enzyme digestion products of step (3) to obtain the degree of hydroxymethylation at Chr1:11908348 in the sequence.
进一步地，DNA样本为血液样本。Further, the DNA sample is a blood sample.
本发明的第二个目的是提供上述DNA羟甲基化标志物在制备预测缺血性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 use of the above-mentioned DNA hydroxymethylation markers in preparing a kit for predicting the risk of ischemic stroke.
进一步地，试剂盒中还包括扩增NPPA基因启动子区的DNA羟甲基化位点Chr1:11908348的引物。Furthermore, the kit also includes primers for amplifying the DNA hydroxymethylation site Chr1:11908348 in the promoter region of the NPPA gene.
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.
本发明的第三个目的是提供扩增上述DNA羟甲基化标志物的引物在制备预测缺血性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 primers for amplifying the above-mentioned DNA hydroxymethylation markers in preparing a kit for predicting the risk of ischemic stroke. The upstream primer is as shown in SEQ ID NO.1, and the downstream primer is as shown in SEQ ID NO.1. The primer is shown in SEQ ID NO.2, specifically,
F：TTTTGTTTGAGGTTAGAGGTTTGTTTAF:TTTTGTTTGAGGTTAGAGGTTTGTTTA
R：AAAAATCCTTAATTATCTCACCRCC，其中，R表示碱基A/G。R: AAAAATCCTTAATTATCTCACCRCC, where R represents base A/G.
本发明的第四个目的是提供一种预测缺血性脑卒中发病风险的试剂盒，该试剂盒中包括：扩增NPPA基因启动子区的DNA羟甲基化位点Chr1: 11908348的引物。The fourth object of the present invention is to provide a kit for predicting the risk of ischemic stroke, which kit includes: a primer for amplifying the DNA hydroxymethylation site Chr1: 11908348 in the promoter region of the NPPA gene.
进一步地，试剂盒中还包括检测NPPA基因启动子区的DNA羟甲基化位点Chr1:11908348羟甲基化水平的试剂。Furthermore, the kit also includes a reagent for detecting the hydroxymethylation level of the DNA hydroxymethylation site Chr1:11908348 in the promoter region of the NPPA gene.
本发明的第五个目的是提供上述DNA羟甲基化标志物在制备缺血性脑卒中治疗药物中的应用，以NPPA基因启动子区的DNA羟甲基化位点Chr1:11908348为靶点进行设计，抑制该靶点的羟甲基化水平。The fifth object of the present invention is to provide the application of the above-mentioned DNA hydroxymethylation markers in the preparation of ischemic stroke therapeutic drugs, targeting the DNA hydroxymethylation site Chr1:11908348 in the promoter region of the NPPA gene. Designed to inhibit the hydroxymethylation level of this target.
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NPPA基因DNA羟甲基化标志物可用于预测缺血性脑卒中的发病风险，也可以作为预防和控制缺血性脑卒中的干预靶点，该羟甲基化标志物的检测灵敏度明显优于现有的缺血性脑卒中的检测标志物，同时具有良好的准确度和特异性。The NPPA gene DNA hydroxymethylation marker provided by the present invention can be used to predict the risk of ischemic stroke, and can also be used as an intervention target to prevent and control ischemic stroke. The detection of the hydroxymethylation marker The sensitivity is significantly better than existing ischemic stroke detection markers, while having good accuracy and specificity.
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为本发明的DNA羟甲基化标志物的示意图；Figure 1 is a schematic diagram of the DNA hydroxymethylation marker of the present invention;
图2为检测的NPPA基因启动子区域9个CpG位点的示意图，其中2号位点为本发明的羟甲基化标志物；Figure 2 is a schematic diagram of the nine CpG sites detected in the NPPA gene promoter region, in which site No. 2 is the hydroxymethylation marker of the present invention;
图3为NPPA基因DNA羟甲基化水平与缺血性脑卒中发病风险的关系；Figure 3 shows the relationship between NPPA gene DNA hydroxymethylation level and the risk of ischemic stroke;
图4为NPPA基因Chr1:11908348DNA羟甲基化对缺血性脑卒中预测的ROC曲线；Figure 4 is the ROC curve of NPPA gene Chr1:11908348 DNA hydroxymethylation in predicting ischemic stroke;
图5为传统危险因素加上NPPA基因Chr1:11908348DNA羟甲基化前后两个模型对缺血性脑卒中预测的比较示意图。Figure 5 is a schematic diagram comparing the prediction of ischemic stroke by two models before and after traditional risk factors plus NPPA gene Chr1:11908348 DNA hydroxymethylation.
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
首先从患有缺血性脑卒中且有DNA生物样本的人群中随机选择1000名作为病例，然后从正常且有DNA生物样本的健康社区人群中与病例按照基于年龄和性别的频数匹配选择1000名作为对照，最后符合条件纳入研究的共853名病例和918名对照。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NPPA基因启动子区域各CpG位点羟甲基化水平，即利用ENSEMBL数据库查询人类NPPA基因(基因编号：ENSG00000175206)的启动子区域，该区域为Chromosome 1:11908117-11908380(GRCh37.P13，距TSS:-540bp to-277bp)，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羟甲基化检测(引物序列信息见表1)。经过挑选，共获得9个CpG位点的羟甲基化水平，9个CpG位点如图2所示，分别为Chr1:11908353(CpG1)、Chr1:11908348(CpG2)、Chr1:11908299(CpG3)、Chr1:11908200(CpG4)、Chr1:11908182(CpG5)、Chr1:11908178(CpG6)、Chr1:11908168(CpG7)、Chr1:11908165(CpG8)、Chr1:11908142(CpG9)。First, 1000 cases were randomly selected from the population with ischemic stroke and DNA biological samples were available, and then 1000 cases were frequency matched based on age and gender from the normal healthy community population with DNA biological samples. As controls, a total of 853 cases and 918 controls were finally eligible for inclusion in the study. Whole blood DNA samples of each research subject were extracted, and target region sequencing technology was used to detect the hydroxymethylation level of each CpG site in the NPPA gene promoter region. That is, the ENSEMBL database was used to query the promoter of the human NPPA gene (gene number: ENSG00000175206). Region, this region is Chromosome 1:11908117-11908380 (GRCh37.P13, distance from TSS:-540bp to-277bp), 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 Epidesigner The program designs primers for CpG islands and CpG dense region sequences, and selects appropriate primers for DNA hydroxymethylation detection (see Table 1 for primer sequence information). After selection, a total of 9 CpG sites were obtained with hydroxymethylation levels. The 9 CpG sites are shown in Figure 2, namely Chr1:11908353 (CpG1), Chr1:11908348 (CpG2), and Chr1:11908299 (CpG3). , Chr1:11908200(CpG4), Chr1:11908182(CpG5), Chr1:11908178(CpG6), Chr1:11908168(CpG7), Chr1:11908165(CpG8), Chr1:11908142(CpG9).
表1 NPPA基因启动子区域DNA羟甲基化检测引物序列Table 1 Primer sequences for DNA hydroxymethylation detection in the promoter region of NPPA gene
Figure PCTCN2022110851-appb-000001
Figure PCTCN2022110851-appb-000001
的具体检测方法为：The specific detection methods are:
首先用DNA羟甲基化检测试剂盒对DNA标本依次进行T4β-葡萄糖基转移酶和APOBEC3A酶处理，以葡萄糖基标记样本DNA中的羟甲基化胞嘧啶，被标记了的羟甲基化胞嘧啶不会被APOBEC3A酶脱去氨基，而其余的胞嘧啶和甲基化胞嘧啶则在APOBEC3A酶的作用下转化为胸腺嘧啶。接着用表1中的引物，以酶转化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位点羟甲基化水平量化为该位点羟甲基化的reads数目(即检测到碱基C的reads数目)/该位点总的reads数目×100％。First, a DNA hydroxymethylation detection kit is used to sequentially treat the DNA sample with T4β-glucosyltransferase and APOBEC3A enzyme to label the hydroxymethylated cytosine in the sample DNA with glucose. Pyrimidines are not deaminated by APOBEC3A enzyme, and the remaining cytosine and methylated cytosine are converted into thymine under the action of APOBEC3A enzyme. Then use the primers in Table 1 and use the enzyme-converted 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 hydroxymethylation level of each CpG site was quantified as the number of hydroxymethylated reads at the site (i.e., the number of reads detecting base C)/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
表2表示研究对象的临床特征，发现与健康对照组相比，缺血性脑卒中患者具有更多的传统危险因素，例如高血压史、糖尿病史、肥胖、高血糖、高血脂等(所有P<0.05)。Table 2 shows the clinical characteristics of the study subjects. It is found that compared with healthy controls, patients with ischemic stroke have more traditional risk factors, such as history of hypertension, diabetes, obesity, hyperglycemia, hyperlipidemia, etc. (all P <0.05).
表2研究对象的临床特征Table 2 Clinical characteristics of study subjects
Figure PCTCN2022110851-appb-000002
Figure PCTCN2022110851-appb-000002
Figure PCTCN2022110851-appb-000003
Figure PCTCN2022110851-appb-000003
2、能独立预测缺血性脑卒中发病风险的羟甲基化位点2. Hydroxymethylation sites that can independently predict the risk of ischemic stroke
通过构建Logistic回归模型分析NPPA基因DNA羟甲基化水平与缺血性脑卒中事件的关系，发现能够独立预测缺血性脑卒中发病风险的羟甲基化位点。结果如图3所示，调整传统危险因素及矫正多重检验后，仅有CpG2与缺血性脑卒中发病风险显著相关(P＜0.05且q＜0.05)，该位点羟甲基化水平每增加2倍，缺血性脑卒中发病风险升高39％。By constructing a logistic regression model to analyze the relationship between NPPA gene DNA hydroxymethylation levels and ischemic stroke events, we found hydroxymethylation sites that can independently predict the risk of ischemic stroke. The results are shown in Figure 3. After adjusting for traditional risk factors and correcting for multiple tests, only CpG2 was significantly associated with the risk of ischemic stroke (P<0.05 and q<0.05). Every increase in the hydroxymethylation level of this site 2 times, the risk of ischemic stroke increases by 39%.
NPPA基因Chr1:11908348位点的DNA羟甲基化水平对缺血性脑卒中的预测效能如图4-5所示。图4为该位点羟甲基化水平与缺血性脑卒中的ROC曲线图，AUC为0.53＞0.5，表示该位点羟甲基化具有一定预测缺血性脑卒中的能力。The predictive performance of DNA hydroxymethylation levels at the NPPA gene Chr1:11908348 site for ischemic stroke is shown in Figure 4-5. Figure 4 shows the ROC curve of the hydroxymethylation level at this site and ischemic stroke. The AUC is 0.53>0.5, indicating that the hydroxymethylation at this site has a certain ability to predict ischemic stroke.
进一步计算净重新分类指数NRI和综合判别指数IDI。NRI值为0.2297(0.1226-0.3368)，IDI值为0.0066(0.0018-0.0114)，计算结果及图5均可表明该位点羟甲基化水平能增加传统危险因素对缺血性脑卒中的预测效能且具有统计学意义。同时，采用似然比检验评价构建的两个模型是否有统计学差异，P＝0.0005<0.05，差异具有统计学意义。Further calculate the net reclassification index NRI and the comprehensive discriminant index IDI. The NRI value is 0.2297 (0.1226-0.3368), and the IDI value is 0.0066 (0.0018-0.0114). Both the calculation results and Figure 5 show that the hydroxymethylation level of this site can increase the predictive performance of traditional risk factors for ischemic stroke. and has statistical significance. At the same time, the likelihood ratio test was used to evaluate whether there was a statistical difference between the two constructed models. P=0.0005<0.05, the difference was statistically significant.
实施例2构建羟甲基化检测试剂盒Example 2 Construction of hydroxymethylation detection kit
基于以上研究，可以得知：位于启动子区域的CpG2位点发生羟甲基化之后，可能促进NPPA基因表达和pro-ANP蛋白分泌，进而参与缺血性脑卒中的发病，可以作为缺血性脑卒中发病风险的预测标志物和潜在药物靶点。因此，本发明构建了基于这个CpG2位点的羟甲基化检测试剂盒。Based on the above studies, it can be known that after hydroxymethylation of the CpG2 site located in the promoter region, it may promote NPPA gene expression and pro-ANP protein secretion, thereby participating in the onset of ischemic stroke, and can be used as an ischemic stroke Predictive markers and potential drug targets for stroke risk. Therefore, the present invention constructed a hydroxymethylation detection kit based on this CpG2 site.
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.
②T4β-葡萄糖基转移酶和APOBEC3A酶处理②T4β-glucosyltransferase and APOBEC3A enzyme treatment
用DNA羟甲基化检测试剂盒对质检合格的DNA标本进行T4β-葡萄糖基转移酶和APOBEC3A酶处理，以葡萄糖基标记样本DNA中的羟甲基化胞嘧啶，被标记了的羟甲基化胞嘧啶不会被APOBEC3A酶脱去氨基，而其余的胞嘧啶和甲基化胞嘧啶则在APOBEC3A酶的作用下转化为胸腺嘧啶。Use the DNA hydroxymethylation detection kit to treat qualified DNA samples with T4β-glucosyltransferase and APOBEC3A enzyme to label the hydroxymethylated cytosine in the sample DNA with glucose. Cytosine will not be deaminated by APOBEC3A enzyme, while the remaining cytosine and methylated cytosine will be converted into thymine under the action of APOBEC3A enzyme.
③多重PCR扩增③Multiple PCR amplification
接着用设计好的引物(F：TTTTGTTTGAGGTTAGAGGTTTGTTTA；R：AAAAATCCTTAATTATCTCACCRCC)对酶转化过的标本进行DNA扩增，得到带有T7RNA聚合酶启动子序列的扩增产物。Then, the designed primers (F: TTTTGTTTGAGGTTAGAGGTTTGTTTA; R: AAAAATCCTTAATTATCTCACCRCC) were used to amplify the DNA of the enzyme-converted specimen to obtain an amplification product containing the T7 RNA polymerase promoter sequence.
④CpG片段切割④CpG fragment cleavage
然后运用T7RNA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，胞嘧啶和甲基化的胞嘧啶最终产物为CpA，羟甲基化的胞嘧啶最终产物为CpG，使用Agena MassArray飞行质谱分析***检测这个最终产物的分子量。Finally, within each small RNA fragment, the final product of cytosine and methylated cytosine is CpA, and the final product of hydroxymethylated cytosine is CpG. The Agena MassArray flight mass spectrometry analysis system is used to detect the molecular weight of this final product. .
⑥羟甲基化水平计算和缺血性脑卒中风险预测⑥Hydroxymethylation level calculation and ischemic stroke risk prediction
该CpG位点羟甲基化水平量化为产物质量CpG/(CpG+CpA)×100％，羟甲基化水平>2.5％提示缺血性脑卒中发病风险较高(见图4)，应密切关注 并采取预防性治疗措施。CpG2位点甲基化模型对脑卒中发病诊断情况见表3。The hydroxymethylation level of this CpG site is quantified as product quality CpG/(CpG+CpA) × 100%. The hydroxymethylation level >2.5% indicates a higher risk of ischemic stroke (see Figure 4) and should be closely followed. Pay attention and take preventive treatment measures. The diagnosis of stroke by the CpG2 site methylation model is shown in Table 3.
表3 CpG2位点甲基化模型对脑卒中发病诊断情况Table 3 Diagnosis of stroke by CpG2 site methylation model
Figure PCTCN2022110851-appb-000004
Figure PCTCN2022110851-appb-000004
基于以上研究可以得知，位于启动子区域的Chr1:11908348CpG位点发生羟甲基化之后，可能促进NPPA基因表达和pro-ANP蛋白分泌，进而参与缺血性脑卒中的发病，可以作为缺血性脑卒中发病风险的预测标志物和潜在药物靶点。Based on the above studies, it can be known that after the hydroxymethylation of the Chr1:11908348CpG site located in the promoter region, it may promote NPPA gene expression and pro-ANP protein secretion, thereby participating in the onset of ischemic stroke, and can be used as an ischemic Predictive markers and potential drug targets for stroke risk.
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. 一种预测缺血性脑卒中发病风险的DNA羟甲基化标志物，其特征在于：所述DNA羟甲基化标志物包括NPPA基因启动子区的DNA羟甲基化位点Chr1:11908348，其羟甲基化程度指示缺血性脑卒中发病风险程度。A DNA hydroxymethylation marker for predicting the risk of ischemic stroke, characterized in that: the DNA hydroxymethylation marker includes the DNA hydroxymethylation site Chr1:11908348 in the promoter region of the NPPA gene, The degree of hydroxymethylation indicates the risk of ischemic stroke.
  2. 根据权利要求1所述的DNA羟甲基化标志物，其特征在于：所述DNA羟甲基化位点Chr1:11908348的羟甲基化水平每增加2倍，缺血性脑卒中的发病风险升高39％。The DNA hydroxymethylation marker according to claim 1, characterized in that: for every 2-fold increase in the hydroxymethylation level of the DNA hydroxymethylation site Chr1:11908348, the risk of ischemic stroke increases increased by 39%.
  3. 根据权利要求1所述的DNA羟甲基化标志物，其特征在于：扩增所述DNA羟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 hydroxymethylation marker according to claim 1, characterized in that: the upstream primer for amplifying the DNA hydroxymethylation marker is as shown in SEQ ID NO.1, and the downstream primer is as shown in SEQ ID NO.2 shown.
  4. 根据权利要求1所述的DNA羟甲基化标志物，其特征在于，检测所述DNA羟甲基化标志物的羟甲基化水平包括以下步骤：The DNA hydroxymethylation marker according to claim 1, wherein detecting the hydroxymethylation level of the DNA hydroxymethylation marker includes the following steps:
    (1)提取DNA样本，用T4β-葡糖基转移酶和APOBEC3A酶处理所述DNA样本；(1) Extract a DNA sample and treat the DNA sample with T4β-glucosyltransferase and APOBEC3A enzyme;
    (2)扩增步骤(1)处理后的DNA样本，得到扩增产物；(2) Amplification step (1) After processing the DNA sample, obtain the 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，获取序列中Chr1:11908348位点的羟甲基化程度。(4) Detect the transcription and enzyme digestion products of step (3) to obtain the degree of hydroxymethylation at Chr1:11908348 in the sequence.
  5. 权利要求1-4任一项所述的DNA羟甲基化标志物在制备预测缺血性脑卒中发病风险的试剂盒中的应用。Application of the DNA hydroxymethylation marker according to any one of claims 1 to 4 in the preparation of a kit for predicting the risk of ischemic stroke.
  6. 根据权利要求5所述的应用，其特征在于：所述试剂盒中还包括扩增NPPA基因启动子区的DNA羟甲基化位点Chr1:11908348的引物。The application according to claim 5, characterized in that the kit further includes primers for amplifying the DNA hydroxymethylation site Chr1:11908348 in the promoter region of the NPPA gene.
  7. 扩增权利要求1-4任一项所述的DNA羟甲基化标志物的引物在制备预测缺血性脑卒中发病风险的试剂盒中的应用。Application of a primer for amplifying the DNA hydroxymethylation marker described in any one of claims 1 to 4 in preparing a kit for predicting the risk of ischemic stroke.
  8. 权利要求1-4任一项所述的DNA羟甲基化标志物在制备缺血性脑卒 中治疗药物中的应用，其特征在于：所述缺血性脑卒中治疗药物以NPPA基因启动子区的DNA羟甲基化位点Chr1:11908348为靶点进行设计，抑制其羟甲基化水平。The application of the DNA hydroxymethylation marker according to any one of claims 1 to 4 in the preparation of ischemic stroke therapeutic drugs, characterized in that: the ischemic stroke therapeutic drugs are based on the NPPA gene promoter region The DNA hydroxymethylation site Chr1:11908348 was designed as a target to inhibit its hydroxymethylation level.
  9. 一种预测缺血性脑卒中发病风险的试剂盒，其特征在于：所述试剂盒中包括扩增NPPA基因启动子区的DNA羟甲基化位点Chr1:11908348的引物。A kit for predicting the risk of ischemic stroke, characterized in that the kit includes primers that amplify the DNA hydroxymethylation site Chr1:11908348 in the promoter region of the NPPA gene.
  10. 根据权利要求9所述的试剂盒，其特征在于：所述试剂盒中还包括检测所述NPPA基因启动子区的DNA羟甲基化位点Chr1:11908348羟甲基化水平的试剂。The kit according to claim 9, characterized in that: the kit also includes a reagent for detecting the hydroxymethylation level of the DNA hydroxymethylation site Chr1:11908348 in the promoter region of the NPPA gene.
PCT/CN2022/110851 2022-04-29 2022-08-08 Dna hydroxymethylation marker and kit for predicting attack risk of cerebral ischemic stroke WO2023206854A1 (en)

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