CN111154864B - Methylation site combination of FOXO3 and TP53 and its application as a diagnostic marker for late-onset asthma - Google Patents
Methylation site combination of FOXO3 and TP53 and its application as a diagnostic marker for late-onset asthma Download PDFInfo
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Abstract
本发明公开了FOXO3和TP53的甲基化位点组合及其作为迟发性哮喘诊断标志物的应用,本发明首次发现了FOXO3和TP53基因的DNA甲基化水平与哮喘严重程度及迟发性哮喘发生发展相关,对于解释迟发性哮喘的发病具有重要意义;本发明提供了一种诊断产品,通过检测外周血中FOXO3和TP53基因的DNA甲基化水平,实现迟发性哮喘的早期无创诊断,以期实现迟发性哮喘的早期治疗。本发明通过检测外周血中FOXO3和TP53基因DNA甲基化水平来用于临床哮喘患者病情的严重程度评估及迟发性哮喘的早期预测。相比现有检测手段而言,具有创伤小,检测便捷,敏感性高,患者接受程度高的优势。
The invention discloses the combination of methylation sites of FOXO3 and TP53 and its application as a diagnostic marker for late-onset asthma. The present invention discovers for the first time that the DNA methylation levels of FOXO3 and TP53 genes are related to asthma severity and late-onset asthma. The occurrence and development of asthma are related, which is of great significance for explaining the onset of delayed asthma; the invention provides a diagnostic product, which can realize the early non-invasive detection of delayed asthma by detecting the DNA methylation levels of FOXO3 and TP53 genes in peripheral blood. Diagnosis with a view to early treatment of late-onset asthma. The invention can be used for the assessment of the severity of clinical asthma patients and the early prediction of late-onset asthma by detecting the DNA methylation levels of FOXO3 and TP53 genes in peripheral blood. Compared with the existing detection methods, it has the advantages of less trauma, convenient detection, high sensitivity and high patient acceptance.
Description
技术领域technical field
本发明涉及一种哮喘诊断标志物,具体涉及一种迟发性哮喘诊断标志物。属于生物诊断技术领域。The invention relates to an asthma diagnostic marker, in particular to a delayed asthma diagnostic marker. It belongs to the technical field of biological diagnosis.
背景技术Background technique
全球哮喘防治指南(Global Initiative for Asthma,GINA)明确指出:迟发性哮喘是一类没有存在持续性呼吸道症状直到成年后才会发病的哮喘亚型。相对于儿童哮喘患者而言,迟发性哮喘患者常伴有肺功能的快速下降,对标准化的哮喘治疗反应差,预后不佳且更易发展为重症哮喘,然而在临床诊断和治疗过程中,迟发性哮喘经常被误诊或被延误治疗。《中国迟发性哮喘流行状况、风险因素与疾病管理现状》研究结果显示中国约有4570万迟发性哮喘患者,其中近70%未确诊,更有95%未规范治疗,给社会与家庭带来了极大的疾病负担,这警示我国亟待加强对迟发性哮喘的规范诊断和治疗。但是,迟发性哮喘的发病机制至今仍不清楚,早期诊断难度大,一旦明确诊断,常规治疗效果通常不明显。因此,通过对迟发性哮喘发病机制及临床特征的深入研究,进而探寻针对迟发性哮喘的早期风险筛查和临床早期诊断的生物学标志物,具有十分重要的意义。The Global Initiative for Asthma (GINA) clearly states that late-onset asthma is a subtype of asthma that does not develop persistent respiratory symptoms until adulthood. Compared with children with asthma, patients with late-onset asthma are often accompanied by rapid decline in lung function, poor response to standardized asthma treatment, poor prognosis and more likely to develop severe asthma. However, in the process of clinical diagnosis and treatment, delayed Paroxysmal asthma is often misdiagnosed or delayed in treatment. The research results of "China's Delayed Asthma Epidemic Situation, Risk Factors and Disease Management Status" show that there are about 45.7 million delayed asthma patients in China, of which nearly 70% are undiagnosed, and 95% have not been treated standardizedly, which has brought great harm to society and families. This has brought a huge burden of disease, which warns that my country urgently needs to strengthen the standardized diagnosis and treatment of delayed asthma. However, the pathogenesis of delayed asthma is still unclear, and early diagnosis is difficult. Once the diagnosis is confirmed, the effect of conventional treatment is usually not obvious. Therefore, it is of great significance to explore biomarkers for early risk screening and early clinical diagnosis of late-onset asthma through in-depth research on the pathogenesis and clinical characteristics of late-onset asthma.
大量的临床流行病学数据和研究结果发现,加速的肺衰老是迟发性哮喘的重要危险因素和关键病理基础。随着年龄的增长,机体肺部结构和功能的减退导致肺部疾病易感性增加,肺部病理表型更加严重,临床预后不佳。在迟发性哮喘患者中,也观察到了更加明显的衰老相关分子信号(例如端粒缩短,表观遗传学改变,ROS积累和免疫衰老等),以上结果强烈提示衰老相关基因的表达和调控异常参与了迟发性哮喘的发生发展。A large number of clinical epidemiological data and research results have found that accelerated lung aging is an important risk factor and key pathological basis for late-onset asthma. With age, the decline of lung structure and function leads to increased susceptibility to lung diseases, more severe lung pathological phenotypes, and poor clinical prognosis. In patients with late-onset asthma, more pronounced senescence-associated molecular signals (such as telomere shortening, epigenetic alterations, ROS accumulation, and immunosenescence, etc.) were also observed, strongly suggesting abnormal expression and regulation of senescence-associated genes Involved in the occurrence and development of delayed asthma.
FOXO3和TP53是两个调节衰老和寿命的经典基因,已被证实与迟发性哮喘的易感性有关,但FOXO3和TP53参与迟发性哮喘发病的机制尚不清楚。最近的一系列证据表明,表观遗传机制可能是衰老相关基因表达调控的主要方式。DNA甲基化是表观遗传调控中研究得最早,也是最重要的一种修饰方式。已有研究证实,DNA甲基化可以作为肿瘤、COPD等疾病发病早期风险预测的生物标志物。另外,DNA甲基化序列是以一种高度稳定的形式存在于人类血清中,能够抵御RNA酶,蛋白酶的消化作用以及细胞的各种应激状态,是一种参与多种疾病高速灵敏的有效生物标志物。FOXO3 and TP53 are two classic genes regulating aging and lifespan, which have been confirmed to be related to the susceptibility to late-onset asthma, but the mechanism by which FOXO3 and TP53 are involved in the pathogenesis of late-onset asthma is still unclear. A series of recent lines of evidence suggests that epigenetic mechanisms may be the primary means by which age-related gene expression is regulated. DNA methylation is the earliest and most important modification method studied in epigenetic regulation. Studies have confirmed that DNA methylation can be used as a biomarker for early risk prediction of tumors, COPD and other diseases. In addition, the DNA methylation sequence exists in human serum in a highly stable form, which can resist the digestion of RNase and protease and various stress states of cells. It is a high-speed, sensitive and effective drug involved in various diseases. Biomarkers.
综上所述,伴随着全球人口老龄化进程,迟发性哮喘的发病率与死亡率逐年增长,但是临床上尚无有效的风险预测标志物。由于血清相对比较容易获取,循环血清中FOXO3和TP53的DNA甲基化生物标志物有望作为非侵入式的风险标志物用于迟发性哮喘发病的早期诊断药物。In summary, with the aging of the global population, the morbidity and mortality of late-onset asthma are increasing year by year, but there is no effective clinical risk prediction marker. Since serum is relatively easy to obtain, DNA methylation biomarkers of FOXO3 and TP53 in circulating serum are expected to be used as non-invasive risk markers for early diagnosis of late-onset asthma.
发明内容Contents of the invention
本发明的目的是为克服上述现有技术的不足,提供一种FOXO3和TP53的甲基化位点组合及其作为迟发性哮喘诊断标志物的应用。The purpose of the present invention is to overcome the shortcomings of the above-mentioned prior art, and provide a combination of methylation sites of FOXO3 and TP53 and its application as a diagnostic marker for delayed asthma.
为实现上述目的,本发明采用下述技术方案:To achieve the above object, the present invention adopts the following technical solutions:
1、FOXO3和TP53的甲基化位点组合,所述位点组合包含:FOXO3基因的位点chr6:108882977和TP53基因的位点chr17:7591672。1. The combination of methylation sites of FOXO3 and TP53, the site combination includes: site chr6: 108882977 of the FOXO3 gene and chr17: 7591672 of the TP53 gene.
2、上述FOXO3和TP53的甲基化位点组合作为迟发性哮喘诊断标志物的应用。2. The application of the combination of methylation sites of FOXO3 and TP53 as a diagnostic marker for delayed asthma.
优选的,迟发性哮喘诊断包括:早期风险筛查和临床早期诊断。Preferably, the diagnosis of delayed asthma includes: early risk screening and clinical early diagnosis.
3、上述FOXO3和TP53的甲基化位点组合在制备迟发性哮喘诊断产品中的应用。3. The application of the combination of methylation sites of FOXO3 and TP53 mentioned above in the preparation of delayed asthma diagnosis products.
优选的,所述产品选自制剂、芯片或试剂盒中的任一种。Preferably, the product is selected from any one of formulations, chips or kits.
优选的,迟发性哮喘诊断包括:早期风险筛查和临床早期诊断。Preferably, the diagnosis of delayed asthma includes: early risk screening and clinical early diagnosis.
4、一种迟发性哮喘诊断标志物,该标志物是上述FOXO3和TP53的甲基化位点组合。4. A diagnostic marker for delayed asthma, which is a combination of the methylation sites of FOXO3 and TP53.
5、一种迟发性哮喘诊断产品,包含:用于扩增上述FOXO3和TP53的甲基化位点组合的引物组。5. A late-onset asthma diagnosis product, comprising: a primer set for amplifying the combination of methylation sites of FOXO3 and TP53.
优选的,所述引物组包含:Preferably, the primer set comprises:
chr6:108882977上游引物:5’-TGTTGTTAGTTCTGGGGTGGGTA-3’,如SEQ ID NO.13所示;chr6: 108882977 upstream primer: 5'-TGTTGTTAGTTCTGGGGTGGGTA-3', as shown in SEQ ID NO.13;
chr6:108882977下游引物:5’-CTACTACAACCTCAACAACCAGCTACCA-3’,如SEQ IDNO.14所示;Chr6: 108882977 downstream primer: 5'-CTACTACAACCTCAACAACCAGCTACCA-3', as shown in SEQ ID NO.14;
chr17:7591672上游引物:5’-AGGTAGAAGATTTTCTGGGAGGAG-3’,如SEQ ID NO.15所示;chr17: 7591672 upstream primer: 5'-AGGTAGAAAGATTTTCTGGGAGGAG-3', as shown in SEQ ID NO.15;
chr17:7591672下游引物:5’-ACAACCACAGAAAAACCCTAAAAC-3’,如SEQ ID NO.16所示。chr17: 7591672 downstream primer: 5'-ACAACCACAGAAAAACCCTAAAAC-3', as shown in SEQ ID NO.16.
6、一种迟发性哮喘诊断试剂盒,包含:用于扩增上述FOXO3和TP53的甲基化位点组合的引物组。6. A diagnostic kit for late-onset asthma, comprising: a primer set for amplifying the above combination of methylation sites of FOXO3 and TP53.
优选的,所述引物组包含:Preferably, the primer set comprises:
chr6:108882977上游引物:5’-TGTTGTTAGTTCTGGGGTGGGTA-3’,如SEQ ID NO.15所示chr6:108882977上游引物:5’-TGTTGTTAGTTCTGGGGTGGGTA-3’,如SEQ ID NO.13所示;chr6: 108882977 upstream primer: 5'-TGTTGTTAGTTCTGGGGTGGGTA-3', as shown in SEQ ID NO.15 chr6: 108882977 upstream primer: 5'-TGTTGTTAGTTCTGGGGTGGGTA-3', as shown in SEQ ID NO.13;
chr6:108882977下游引物:5’-CTACTACAACCTCAACAACCAGCTACCA-3’,如SEQ IDNO.14所示;Chr6: 108882977 downstream primer: 5'-CTACTACAACCTCAACAACCAGCTACCA-3', as shown in SEQ ID NO.14;
chr17:7591672上游引物:5’-AGGTAGAAGATTTTCTGGGAGGAG-3’,如SEQ ID NO.15所示;chr17: 7591672 upstream primer: 5'-AGGTAGAAAGATTTTCTGGGAGGAG-3', as shown in SEQ ID NO.15;
chr17:7591672下游引物:5’-ACAACCACAGAAAAACCCTAAAAC-3’,如SEQ ID NO.16所示。chr17: 7591672 downstream primer: 5'-ACAACCACAGAAAAACCCTAAAAC-3', as shown in SEQ ID NO.16.
优选的,该试剂盒还含有Taq酶聚合酶链式反应体系,具体包含:Taq酶,PCR缓冲液,dNTPs,ddH2O。Preferably, the kit also contains a Taq enzyme polymerase chain reaction system, specifically comprising: Taq enzyme, PCR buffer, dNTPs, ddH 2 O.
7、用于扩增上述FOXO3和TP53的甲基化位点组合的引物组,包含:7. A primer set for amplifying the combination of methylation sites of the above-mentioned FOXO3 and TP53, comprising:
chr6:108882977上游引物:5’-TGTTGTTAGTTCTGGGGTGGGTA-3’,如SEQ ID NO.13所示;chr6: 108882977 upstream primer: 5'-TGTTGTTAGTTCTGGGGTGGGTA-3', as shown in SEQ ID NO.13;
chr6:108882977下游引物:5’-CTACTACAACCTCAACAACCAGCTACCA-3’,如SEQ IDNO.14所示;Chr6: 108882977 downstream primer: 5'-CTACTACAACCTCAACAACCAGCTACCA-3', as shown in SEQ ID NO.14;
chr17:7591672上游引物:5’-AGGTAGAAGATTTTCTGGGAGGAG-3’,如SEQ ID NO.15所示;chr17: 7591672 upstream primer: 5'-AGGTAGAAAGATTTTCTGGGAGGAG-3', as shown in SEQ ID NO.15;
chr17:7591672下游引物:5’-ACAACCACAGAAAAACCCTAAAAC-3’,如SEQ ID NO.16所示。chr17: 7591672 downstream primer: 5'-ACAACCACAGAAAAACCCTAAAAC-3', as shown in SEQ ID NO.16.
本发明的有益效果:Beneficial effects of the present invention:
为了获得一种迟发性哮喘的非侵入式的外周血生物标志物用于迟发性哮喘(Late-onset asthma,LOA)发病风险的早期预测,申请人首先评估了迟发性哮喘病人和健康对照人群外周血中FOXO3和TP53的表达量及其DNA甲基化水平,结果显示迟发性哮喘病人外周血中FOXO3和TP53的表达和甲基化水平变化显著,并进一步筛选出在迟发性哮喘患者中差异表达的8个CpG位点。将8个差异CpG位点的甲基化水平与哮喘病人肺功能指标进行相关性分析,结果显示:FOXO3和TP53各有一个差异CpG位点(FOXO3:chr6:108882977;TP53:chr17:7591672)的甲基化水平与迟发性哮喘的严重程度有关。通过ROC分析和PCA分析证实这两个差异CpG位点的甲基化水平可以有效区分迟发性哮喘患者和健康对照者,为迟发性哮喘的早期诊断提供可能。具体如下:In order to obtain a non-invasive peripheral blood biomarker of late-onset asthma for early prediction of the risk of late-onset asthma (LOA), the applicant first assessed the late-onset asthma patients and healthy The expression levels of FOXO3 and TP53 and their DNA methylation levels in the peripheral blood of the control population, the results showed that the expression and methylation levels of FOXO3 and TP53 in the peripheral blood of patients with delayed asthma changed significantly, and further screened out the patients with delayed asthma Eight CpG sites differentially expressed in asthmatic patients. The correlation analysis of the methylation levels of the 8 differential CpG sites and the pulmonary function indicators of asthmatic patients showed that: FOXO3 and TP53 each have a differential CpG site (FOXO3: chr6: 108882977; TP53: chr17: 7591672) Methylation levels are associated with severity of late-onset asthma. ROC analysis and PCA analysis confirmed that the methylation levels of these two differential CpG sites can effectively distinguish patients with delayed asthma from healthy controls, and provide the possibility for early diagnosis of delayed asthma. details as follows:
1、本发明首次发现了FOXO3和TP53基因的DNA甲基化水平与哮喘严重程度及迟发性哮喘发生发展相关,对于解释迟发性哮喘的发病具有重要意义;1. The present invention discovered for the first time that the DNA methylation levels of FOXO3 and TP53 genes are related to the severity of asthma and the occurrence and development of delayed asthma, which is of great significance for explaining the onset of delayed asthma;
2、本发明提供了一种诊断产品,根据本发明的实验数据,chr6:108882977(FOXO3)和chr17:7591672(TP53)在外周血中的甲基化水平可作为生物标志物用于制备迟发性哮喘的早期诊断试剂盒,通过检测外周血中FOXO3和TP53基因的DNA甲基化水平,实现迟发性哮喘的早期无创诊断,以期实现迟发性哮喘的早期治疗。2. The present invention provides a diagnostic product. According to the experimental data of the present invention, the methylation levels of chr6: 108882977 (FOXO3) and chr17: 7591672 (TP53) in peripheral blood can be used as biomarkers for the preparation of delayed An early diagnosis kit for chronic asthma, by detecting the DNA methylation levels of FOXO3 and TP53 genes in peripheral blood, it can realize the early non-invasive diagnosis of late-onset asthma, so as to realize the early treatment of late-onset asthma.
3、本发明提供了检测FOXO3和TP53基因DNA甲基化水平的产品在制备诊断迟发性哮喘的产品中的应用。所述产品包括制剂、芯片或试剂盒等本领域已知的任何检测基因表达的产品。3. The present invention provides the application of the product for detecting DNA methylation levels of FOXO3 and TP53 genes in the preparation of products for diagnosing delayed asthma. The product includes any gene expression detection product known in the art such as preparation, chip or kit.
4、现有迟发性哮喘的临床诊断依赖于临床表现、肺功能检查或峰流速测量。由于迟发性哮喘的临床表现具有显著的异质性,传统的诊断方法不能早期、明确诊断迟发性哮喘的易感性和严重程度。本发明通过检测外周血中FOXO3和TP53基因DNA甲基化水平来用于临床哮喘患者病情的严重程度评估及迟发性哮喘的早期预测。相比现有检测手段而言,具有创伤小,检测便捷,敏感性高,患者接受程度高的优势。4. The existing clinical diagnosis of delayed asthma relies on clinical manifestations, pulmonary function tests or peak flow measurement. Due to the significant heterogeneity of clinical manifestations of late-onset asthma, traditional diagnostic methods cannot diagnose the susceptibility and severity of late-onset asthma early and clearly. The invention is used for evaluating the severity of clinical asthma patients and early prediction of late-onset asthma by detecting the FOXO3 and TP53 gene DNA methylation levels in peripheral blood. Compared with the existing detection methods, it has the advantages of less trauma, convenient detection, high sensitivity and high patient acceptance.
5、FOXO3和TP53基因DNA甲基化水平的改变不仅体现在哮喘患者外周血中,还体现在哮喘患者诱导痰细胞中。因此,除了外周血外,在诱导痰细胞、支气管肺泡灌洗液、血液、血清、血浆、或者组织活检物(例如肺样本)都能检测到FOXO3和TP53基因DNA甲基化水平变化。5. The changes of DNA methylation levels of FOXO3 and TP53 genes were not only reflected in peripheral blood of asthmatic patients, but also in induced sputum cells of asthmatic patients. Therefore, in addition to peripheral blood, changes in DNA methylation levels of FOXO3 and TP53 genes can be detected in induced sputum cells, bronchoalveolar lavage fluid, blood, serum, plasma, or tissue biopsies (such as lung samples).
6、FOXO3和TP53基因DNA甲基化水平的改变不仅可以通过二代测序检测,还可通过BSP和MSP等本领域内已知的任何方法检测。6. The changes in the DNA methylation levels of FOXO3 and TP53 genes can be detected not only by next-generation sequencing, but also by any method known in the art such as BSP and MSP.
附图说明Description of drawings
图1是FOXO3和TP53基因在健康对照和实验组中的基因表达情况,其中,A为FOXO3在外周血中的表达水平,B为TP53在外周血中的表达水平;Figure 1 is the gene expression of FOXO3 and TP53 genes in the healthy control group and the experimental group, wherein, A is the expression level of FOXO3 in peripheral blood, and B is the expression level of TP53 in peripheral blood;
图2是chr6:108882977和chr17:7591672的甲基化水平与肺功能的相关性分析,其中,A为chr6:108882977(FOXO3)甲基化水平与FEV1%的相关性分析,B为chr6:108882977(FOXO3)甲基化水平与PEF的相关性分析,C为chr17:7591672(TP53)甲基化水平与FEV1的相关性分析,D为chr17:7591672(TP53)甲基化水平与FVC的相关性分析,E为chr17:7591672(TP53)甲基化水平与PEF的相关性分析,F为chr17:7591672(TP53)甲基化水平与FEF25的相关性分析;Figure 2 is the correlation analysis between the methylation level of chr6: 108882977 and chr17: 7591672 and lung function, in which, A is the correlation analysis between the methylation level of chr6: 108882977 (FOXO3) and FEV1%, and B is chr6: 108882977 Correlation analysis between (FOXO3) methylation level and PEF, C is the correlation analysis between chr17:7591672(TP53) methylation level and FEV1, D is the correlation analysis between chr17:7591672(TP53) methylation level and FVC Analysis, E is the correlation analysis between chr17: 7591672 (TP53) methylation level and PEF, F is the correlation analysis between chr17: 7591672 (TP53) methylation level and FEF25;
图3是chr6:108882977和chr17:7591672的甲基化水平区分LOA人群与健康人群的诊断准确性,其中,A为chr6:108882977(FOXO3)甲基化水平联合年龄区分LOA人群与健康人群的ROC曲线,B为chr17:7591672(TP53)联合年龄区分LOA人群与健康人群的ROC曲线,C为chr6:108882977(FOXO3)、chr17:7591672(TP53)联合年龄区分LOA人群与健康人群的ROC曲线,D为chr6:108882977(FOXO3)、chr17:7591672(TP53)联合年龄区分LOA人群与健康人群的PCA聚类分析。Figure 3 shows the diagnostic accuracy of the methylation levels of chr6: 108882977 and chr17: 7591672 to distinguish between LOA population and healthy population, where A is the ROC of chr6: 108882977 (FOXO3) methylation level combined with age to distinguish LOA population from healthy population Curve, B is the ROC curve of chr17: 7591672 (TP53) combined with age to distinguish LOA population and healthy population, C is the ROC curve of chr6: 108882977 (FOXO3), chr17: 7591672 (TP53) combined with age to distinguish LOA population and healthy population, D PCA cluster analysis for chr6: 108882977 (FOXO3), chr17: 7591672 (TP53) combined with age to distinguish the LOA population from the healthy population.
具体实施方式Detailed ways
下面结合附图和实施例对本发明进行进一步的阐述,应该说明的是,下述说明仅是为了解释本发明,并不对其内容进行限定。The present invention will be further described below in conjunction with the accompanying drawings and embodiments. It should be noted that the following description is only for explaining the present invention and not limiting its content.
1.收集临床明确诊断为哮喘病人及健康对照测试者的外周血1. Collect peripheral blood from clinically diagnosed asthma patients and healthy control subjects
迟发性哮喘组选取的对象为中南大学湘雅医院确诊并且符合诊断标准与排除标准的43人(年龄>20岁),对照组选择同一时间段在中南大学湘雅医院体检无明显异常人群60人(年龄>20岁)。所有受试者均告知入组的目的,并且全部都签署了知情同意书,并对两组间的性别、年龄、身高进行比较发现无显著性差异。收集病例的时间为2018年10月到2019年01月。The subjects in the delayed asthma group were 43 people (age > 20 years old) who were diagnosed and met the diagnostic and exclusion criteria of Xiangya Hospital of Central South University. people (age >20 years). All subjects were informed of the purpose of enrollment, and all signed the informed consent, and no significant difference was found in gender, age, and height between the two groups. The cases were collected from October 2018 to January 2019.
支气管哮喘诊断标准:Diagnostic criteria for bronchial asthma:
支气管哮喘防治指南中的支气管哮喘的定义、诊断、治疗和管理方案,具体如下:The definition, diagnosis, treatment and management of bronchial asthma in the guidelines for the prevention and treatment of bronchial asthma are as follows:
(1)反复发作喘息、气急、胸闷或咳嗽,多与接触变应原、冷空气、物理、化学性刺激、病毒性上呼吸道感染、运动等有关。(1) Recurrent episodes of wheezing, shortness of breath, chest tightness, or coughing are mostly related to exposure to allergens, cold air, physical and chemical stimuli, viral upper respiratory tract infections, and exercise.
(2)发作时在双肺可闻及散在或弥漫性,以呼气相为主的哮鸣音,呼气相延长。(2) Scattered or diffuse wheezing, mainly in the expiratory phase, can be heard in both lungs during the attack, and the expiratory phase is prolonged.
(3)上述症状可经治疗缓解或自行缓解。(3) The above symptoms can be relieved by treatment or relieved by themselves.
(4)除外其他疾病所引起的喘息、气急、胸闷或咳嗽。(4) Excluding wheezing, shortness of breath, chest tightness or cough caused by other diseases.
(5)临床症状不典型者(如无明显喘息或体征)应至少具备以下一项试验阳性:(5) Those with atypical clinical symptoms (such as no obvious wheezing or signs) should have at least one of the following positive tests:
a.支气管激发试验或运动试验阳性;a. Positive bronchial provocation test or exercise test;
b.支气管舒张试验阳性(FEV1增加15%以上,且FEV1绝对值增加>200ml);b. Positive bronchodilation test (FEV1 increased by more than 15%, and the absolute value of FEV1 increased by >200ml);
c.PEF日内变异率或昼夜波动率≥20%。c. PEF intraday variation rate or diurnal fluctuation rate ≥ 20%.
符合以上1~4条或4、5条者,可诊断为哮喘。Those who meet the above 1 to 4 or 4 or 5 can be diagnosed as asthma.
根据哮喘的严重程度,哮喘分为轻度哮喘,中度哮喘和重度哮喘。具体的临床标准参考《2019年全球哮喘管理和预防策略》(www.ginasthma.org.)According to the severity of asthma, asthma is divided into mild asthma, moderate asthma and severe asthma. For specific clinical standards, refer to "2019 Global Asthma Management and Prevention Strategy" (www.ginasthma.org.)
2.测定LOA患者及健康对照测试者外周血中FOXO3和TP53的表达水平2. Determination of the expression levels of FOXO3 and TP53 in the peripheral blood of LOA patients and healthy control test subjects
(1)采集LOA患者与健康对照的外周血2ml,1ml外周血用于RNA提取,1ml外周血用于DNA提取;(1) Collect 2ml of peripheral blood from LOA patients and healthy controls, 1ml of peripheral blood is used for RNA extraction, and 1ml of peripheral blood is used for DNA extraction;
(2)提取RNA,具体步骤如下:(2) Extract RNA, the specific steps are as follows:
取1ml外周血通过红细胞裂解液裂解外周血中的红细胞,离心后彻底吸除上层液体,下层即为白细胞沉淀,加入1ml Trizol混匀后转移至1.5ml离心管,室温放置5分钟后,加入0.2ml氯仿,剧烈震荡15s,室温放置3min。4℃,12000g,离心15min。Take 1ml of peripheral blood and use the erythrocyte lysate to lyse the red blood cells in the peripheral blood. After centrifugation, suck out the upper liquid completely, and the lower layer is the white blood cell precipitate. Add 1ml Trizol and mix well, then transfer to a 1.5ml centrifuge tube. After standing at room temperature for 5 minutes, add 0.2 ml chloroform, shake vigorously for 15 seconds, and place at room temperature for 3 minutes. Centrifuge at 12000g for 15min at 4°C.
a.取上层水相,加入0.5ml异丙醇,轻轻震荡5s,室温放置10min。4℃,12000g,离心15min。a. Take the upper aqueous phase, add 0.5ml of isopropanol, shake gently for 5 seconds, and place at room temperature for 10 minutes. Centrifuge at 12000g for 15min at 4°C.
b.体积百分数75%乙醇水溶液洗两次,室温放置至干燥。将RNA溶于50μl无核酶水,用紫外分光光度计测定RNA的浓度。b. Wash twice with 75% ethanol water solution by volume, and place at room temperature until dry. The RNA was dissolved in 50 μl nuclease-free water, and the concentration of RNA was measured with a UV spectrophotometer.
(3)RNA质量检测(3) RNA quality detection
a.RNA的浓度和总量:Qubit精确定量浓度;a. The concentration and total amount of RNA: Qubit accurately quantifies the concentration;
b.RNA的纯度:Nanodrop检测OD260/280和260/230的比值;b. RNA purity: the ratio of OD260/280 and 260/230 detected by Nanodrop;
c.RNA的完整度:琼脂糖凝胶电泳和Agilent 2100Bioanalyzer检测RNA的降解程度;c. RNA integrity: agarose gel electrophoresis and Agilent 2100Bioanalyzer to detect the degree of degradation of RNA;
RNA样品总量在1-4μg之间,260/280比值在1.8-2.0之间。The total amount of RNA sample was between 1-4 μg, and the 260/280 ratio was between 1.8-2.0.
(4)逆转录:使用Sigma公司的反转录试剂盒进行操作,具体步骤如下:取1μgRNA,根据上述已测的RNA浓度,计算出需要加入的RNA体积,应用随机引物1μl Oligo引物和无核酶水配制12μl反应体系,在65℃条件下变性5min。应用4μl 5×Reaction Buffer,2μl dNTP混合液(10mM),1μl AMV逆转录酶和1μl RNase抑制剂在25℃5min,42℃60min,70℃5min条件下进行逆转录反应。(4) Reverse transcription: use the reverse transcription kit of Sigma Company to operate, the specific steps are as follows: take 1 μg RNA, calculate the RNA volume that needs to be added according to the above-mentioned measured RNA concentration, apply
(5)Real-Time qPCR:(5) Real-Time qPCR:
a.引物设计:根据Genebank中FOXO3基因,TP53基因与β-actin编码基因序列设计QPCR扩增引物,由上海生物生工技术服务有限公司合成。具体引物序列如下:a. Primer design: QPCR amplification primers were designed according to the sequences of FOXO3 gene, TP53 gene and β-actin coding gene in Genebank, and were synthesized by Shanghai Biotechnology Service Co., Ltd. The specific primer sequences are as follows:
FOXO3基因:FOXO3 gene:
正向引物为:5’-CGG ACA AAC GGC TCA CTC T-3’,如SEQ ID NO.1所示;The forward primer is: 5'-CGG ACA AAC GGC TCA CTC T-3', as shown in SEQ ID NO.1;
反向引物为:5’-GGA CCC GCA TGA ATC GAC TAT-3’,如SEQ ID NO.2所示;The reverse primer is: 5'-GGA CCC GCA TGA ATC GAC TAT-3', as shown in SEQ ID NO.2;
TP53基因:TP53 gene:
正向引物为:5’-AAG TCT GTG ACT TGC ACG TAC TCC-3’,如SEQ ID NO.3所示;The forward primer is: 5'-AAG TCT GTG ACT TGC ACG TAC TCC-3', as shown in SEQ ID NO.3;
反向引物为:5’-GTC ATG TGC TGT GAC TGC TTG TAG-3’,如SEQ ID NO.4所示;The reverse primer is: 5'-GTC ATG TGC TGT GAC TGC TTG TAG-3', as shown in SEQ ID NO.4;
β-actin基因:β-actin gene:
正向引物为:5’-TTC CAG CCT TCC TTC CTG GG-3’,如SEQ ID NO.5所示;The forward primer is: 5'-TTC CAG CCT TCC TTC CTG GG-3', as shown in SEQ ID NO.5;
反向引物为:5’-TTG CGC TCA GGA GGA GCA AT-3’,如SEQ ID NO.6所示;The reverse primer is: 5'-TTG CGC TCA GGA GGA GCA AT-3', as shown in SEQ ID NO.6;
b.配置25μl反应体系:SYBR green Mix 12.5μl,去离子水8.5μl,正(反)向引物1μl,模板2μl,混合均匀。其中SYBR green Mix购自于TAKARA公司。b. Configure a 25 μl reaction system: SYBR green Mix 12.5 μl, deionized water 8.5 μl, forward (reverse)
c.PCR反应条件:95℃30s,95℃5s,60℃30s,40cycles。以SYBR Green作为荧光标记物,在荧光定量PCR仪上进行PCR反应,通过△△CT法进行相对定量。c. PCR reaction conditions: 95°C for 30s, 95°C for 5s, 60°C for 30s, 40 cycles. Using SYBR Green as a fluorescent marker, the PCR reaction was carried out on a fluorescent quantitative PCR instrument, and the relative quantification was carried out by the ΔΔCT method.
3.结果3. Results
结果如图1所示,相对于健康对照,FOXO3基因在轻度LOA患者外周血中表达上调,在严重LOA患者中表达降低,差异具有统计学差异(P<0.05);相对于健康对照,TP53基因在轻度LOA患者和重度LOA患者中表达均上调,差异具有统计学差异(P<0.05)。The results are shown in Figure 1. Compared with healthy controls, the expression of FOXO3 gene in the peripheral blood of patients with mild LOA was up-regulated, and the expression of FOXO3 gene was decreased in patients with severe LOA. The difference was statistically significant (P<0.05); compared with healthy controls, TP53 The gene expression was up-regulated in mild LOA patients and severe LOA patients, and the difference was statistically significant (P<0.05).
4.测定LOA患者及健康对照测试者外周血中FOXO3和TP53的DNA甲基化水平并分析差异CpG位点4. Determination of DNA methylation levels of FOXO3 and TP53 in peripheral blood of LOA patients and healthy controls and analysis of differential CpG sites
(1)二代甲基化测序(1) Next-generation methylation sequencing
a.取1ml外周血通过外周血基因组DNA提取试剂盒提取DNA。a. Take 1ml of peripheral blood and extract DNA through the peripheral blood genomic DNA extraction kit.
b.样品质控b. Sample quality control
①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。① The integrity of genomic DNA was detected by agarose gel electrophoresis: the electrophoresis bands were clearly visible, no obvious degradation, and no RNA contamination.
②Nanodrop 2000检测基因组DNA质量:浓度≥20ng/μL,总量≥1μg(可供10个多重PCR Panel的检测),OD260/280=1.7~2.0,OD260/230≥1.8;②Nanodrop 2000 to detect the quality of genomic DNA: concentration ≥ 20ng/μL, total amount ≥ 1μg (for detection by 10 multiplex PCR panels), OD260/280=1.7~2.0, OD260/230≥1.8;
c.位于靶基因近端启动子中CpG岛选择c. CpG island selection located in the proximal promoter of the target gene
①最小长度为200bp;①The minimum length is 200bp;
②GC含量≥50%或更高;②GC content ≥ 50% or higher;
③观察到的/预期的二核苷酸CpG的比例大于等于0.60。③ The ratio of observed/expected dinucleotide CpG is greater than or equal to 0.60.
d.引物设计与单位点PCR条件优化d. Primer design and single-site PCR condition optimization
基于软件设计测序引物,挑选能够以经重亚硫酸盐处理的人基因组为模板,扩增获得清晰单一条带的引物用于后续实验。Sequencing primers were designed based on the software, and primers that could amplify a clear single band using the bisulfite-treated human genome as a template were selected for subsequent experiments.
e.多重PCR引物panel优化e. Multiplex PCR primer panel optimization
将经步骤d优化后的引物混合为多重PCR引物panel。并使用多重PCR技术,以标准人基因组为模板进行扩增。基于毛细管电泳的特殊方法,判断多重体系中每对引物是否高效、特异地进行扩增,并以此调整,优化多重PCR panel中的引物组成及浓度。Mix the primers optimized in step d into a multiplex PCR primer panel. And use the multiplex PCR technology to amplify with the standard human genome as the template. Based on the special method of capillary electrophoresis, it is judged whether each pair of primers in the multiplex system can amplify efficiently and specifically, and adjust accordingly to optimize the composition and concentration of primers in the multiplex PCR panel.
f.重亚硫酸盐处理f. Bisulfite treatment
使用EZ DNA Methylation-Gold Kit进行样本处理,将基因组DNA未被甲基化修饰的胞嘧啶C转化为胸腺嘧啶U。Use the EZ DNA Methylation-Gold Kit for sample processing to convert unmethylated cytosine C into thymine U in genomic DNA.
g.样本目标片段多重PCR反应g. Multiplex PCR reaction of sample target fragment
使用优化后的多重PCR引物panel,以转化后的样品基因组为模板,进行多重PCR扩增。经质控后,将以同一个样品基因组DNA为模板的所有多重PCR引物panel的扩增产物混合,并确保每个位点引物扩增产物的量相当。Using the optimized multiplex PCR primer panel, the transformed sample genome is used as a template for multiplex PCR amplification. After quality control, the amplification products of all multiple PCR primer panels using the same sample genomic DNA as a template were mixed, and the amount of primer amplification products at each site was ensured to be equivalent.
h.样本添加特异性标签序列h. Add specific tag sequence to the sample
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。反应采用11个循环数的PCR程序,尽可能降低PCR的倾向性。Using primers with Index sequences, PCR amplification was used to introduce specific tag sequences compatible with the Illumina platform to the end of the library. The reaction uses a PCR program with 11 cycles to reduce the tendency of PCR as much as possible.
i.定量后上机测序i. On-machine sequencing after quantification
将样品Index PCR扩增产物等量混合,并经割胶回收获得最终的MethylTarget测序文库,文库的片段长度分布经Agilent 2100Bioanalyzer验证。文库摩尔浓度精确定量后,最终于Illumina Hiseq/Nextseq平台,以2×150bp的双端测序模式进行高通量测序,获得FastQ数据。The sample Index PCR amplification products were mixed in equal amounts and recovered by tapping to obtain the final MethylTarget sequencing library. The fragment length distribution of the library was verified by Agilent 2100 Bioanalyzer. After the molar concentration of the library was accurately quantified, high-throughput sequencing was finally performed on the Illumina Hiseq/Nextseq platform in a paired-end sequencing mode of 2×150bp to obtain FastQ data.
(2)差异CpG位点分析(2) Differential CpG site analysis
对测序结果进行分析,将质量控制后的测序数据和参考基因组进行比对,对目标片段富集效率,片段有效reads及盐化效率进行统计,分析每个CpG位点在样本中甲基化的比例。对所有甲基化结果,根据样本的分组信息,进行差异显著性分析,分析方法为T检验,单变量逻辑回归与多变量逻辑回归,当p value<0.05时,认为CpG位点甲基化显著差异表达。Analyze the sequencing results, compare the quality-controlled sequencing data with the reference genome, make statistics on the enrichment efficiency of the target fragment, the effective reads of the fragment and the salinization efficiency, and analyze the degree of methylation of each CpG site in the sample Proportion. For all the methylation results, according to the grouping information of the samples, a significant difference analysis is carried out. The analysis method is T test, univariate logistic regression and multivariate logistic regression. When p value<0.05, the methylation of the CpG site is considered to be significant differential expression.
5.结果5. Results
结果如表1所示:相对于健康对照,FOXO3基因有7个差异CpG位点,TP53基因有1个差异CpG位点在LOA患者外周血中甲基化水平发生显著改变,差异具有统计学差异(P<0.05),8个差异CpG位点具体的甲基化引物见表2。The results are shown in Table 1: Compared with healthy controls, there were 7 differential CpG sites in the FOXO3 gene and 1 differential CpG site in the TP53 gene. The methylation levels in the peripheral blood of LOA patients were significantly changed, and the difference was statistically significant. (P<0.05), the specific methylation primers of the 8 differential CpG sites are shown in Table 2.
表1差异CpG位点Table 1 Differential CpG sites
表2差异CpG位点的甲基化引物Table 2 Methylation primers of differential CpG sites
6.通过FOXO3和TO53基因差异CpG位点的甲基化水平与LOA患者临床参数的相关性分析,确定区分LOA人群与健康人群的DNA甲基化生物标志物。6. Through the correlation analysis of the methylation level of the differential CpG sites of FOXO3 and TO53 genes and the clinical parameters of LOA patients, determine the DNA methylation biomarkers that distinguish the LOA population from the healthy population.
根据样品的分组信息,Benjamin-Hochberg方法用于控制错误发现率(FDR),皮尔森相关性用于评估8个差异CpG位点的甲基化水平和哮喘病人的肺功能(FEV1(第1秒用力呼气流量),FEV1%predicted(FEV1预计值),FVC(用力肺活量),FEV1/FVC(FEV1占用力肺活量的百分比),PEF(最大呼气流量),FEF75(用力呼气75%肺活量时的呼气流量),FEF50(用力呼气50%肺活量时的呼气流量)and FEF25(用力呼气25%肺活量时的呼气流量))的关联。统计分析采用R语言和SPSS.19软件完成。p<0.05被认为具有统计学意义。According to the grouping information of the samples, the Benjamin-Hochberg method was used to control the false discovery rate (FDR), and the Pearson correlation was used to evaluate the methylation levels of the 8 differential CpG sites and the lung function (FEV1(1 sec Forced expiratory flow), FEV1% predicted (FEV1 predicted value), FVC (forced vital capacity), FEV1/FVC (percentage of FEV1 occupied force vital capacity), PEF (maximum expiratory flow), FEF75 (forced expiratory 75% of vital capacity Correlation of FEF50 (expiratory flow at 50% of forced expiration) and FEF25 (expiratory flow at 25% of forced expiration)). Statistical analysis was done using R language and SPSS.19 software. p<0.05 was considered statistically significant.
7.结果7. Results
结果如图2所示:chr6:108882977(FOXO3)的甲基化水平与FEV1%和PEF存在负相关关系;chr17:7591672(TP53)的甲基化水平与FEV1,FVC,PEF和FEF25呈正相关关系。提示,chr6:108882977(FOXO3)和chr17:7591672(TP53)可作为生物标记物应用于临床哮喘严重程度及LOA的诊断与疗效评估。The results are shown in Figure 2: the methylation level of chr6: 108882977 (FOXO3) is negatively correlated with FEV1% and PEF; the methylation level of chr17: 7591672 (TP53) is positively correlated with FEV1, FVC, PEF and FEF25 . It is suggested that chr6: 108882977 (FOXO3) and chr17: 7591672 (TP53) can be used as biomarkers in the diagnosis and efficacy evaluation of clinical asthma severity and LOA.
8.大样本量确定2个关键差异CpG位点(chr6:108882977(FOXO3);chr17:7591672(TP53))的甲基化水平作为生物标志物区分LOA人群与健康人群的可行性8. The feasibility of determining the methylation level of two key differential CpG sites (chr6: 108882977 (FOXO3); chr17: 7591672 (TP53)) as a biomarker to distinguish LOA population from healthy population with a large sample size
(1)按照1中的方法收集LOA患者外周血215份,健康对照测试者外周血109份。(1) According to the method in 1, collect 215 samples of peripheral blood from patients with LOA, and 109 samples of peripheral blood from healthy control test subjects.
(2)按照4中的方法检测外周血中2个关键差异CpG位点的甲基化水平。(2) Detect the methylation levels of two key differential CpG sites in peripheral blood according to the method in 4.
(3)ROC曲线与PCA聚类分析评估2个关键差异CpG位点区分LOA人群与健康人群的诊断准确性。(3) ROC curve and PCA cluster analysis evaluated the diagnostic accuracy of two key differential CpG loci in distinguishing LOA population from healthy population.
9.结果9. Results
结果如图3与表1所示:单个关键差异CpG位点的甲基化水平联合年龄区分LOA人群与健康人群的AUC分别为0.878和0.898;2个差异CpG位点的甲基化水平联合年龄区分LOA人群与健康人群的AUC为0.924;PCA分析也显示2个差异CpG位点的甲基化水平联合年龄可以准确区分LOA人群与健康人群,提示chr6:108882977(FOXO3)和chr17:7591672(TP53)可作为生物标记物应用于临床哮喘严重程度及LOA的早期诊断与疗效评估。The results are shown in Figure 3 and Table 1: the AUC of the methylation level of a single key differential CpG site combined with age to distinguish the LOA population from the healthy population was 0.878 and 0.898; the methylation level of 2 differential CpG sites combined with age The AUC for distinguishing the LOA population from the healthy population was 0.924; PCA analysis also showed that the methylation levels of the two differential CpG sites combined with age can accurately distinguish the LOA population from the healthy population, suggesting that chr6: 108882977 (FOXO3) and chr17: 7591672 (TP53 ) can be used as a biomarker in the early diagnosis and curative effect evaluation of clinical asthma severity and LOA.
10.建立与LOA相关的甲基化谱试剂盒10. Establishment of LOA-related methylation profiling kits
根据以上结果,申请人制备了一个可用于LOA早期诊断的试剂盒,试剂盒中含有Taq酶聚合酶链式反应体系和用于扩增所述2个甲基化位点的甲基化PCR引物对,所述Taq酶聚合酶链式反应体系包括:Taq酶0.2μl,10×PCR缓冲液2.5μl,dNTPs 1.5μl,ddH2O 14.8μl。该试剂盒可用于检测亚硫酸氢盐修饰后样本基因组DNA中所述2个甲基化位点的甲基化水平,从而进行LOA的早期诊断。According to the above results, the applicant prepared a kit that can be used for early diagnosis of LOA, which contains Taq enzyme polymerase chain reaction system and methylated PCR primers for amplifying the two methylated sites Yes, the Taq polymerase chain reaction system includes: Taq enzyme 0.2 μl, 10×PCR buffer 2.5 μl, dNTPs 1.5 μl, ddH 2 O 14.8 μl. The kit can be used to detect the methylation levels of the two methylation sites in the genomic DNA of the sample after bisulfite modification, so as to perform early diagnosis of LOA.
上述虽然结合附图对本发明的具体实施方式进行了描述,但并非对本发明保护范围的限制,在本发明的技术方案的基础上,本领域技术人员不需要付出创造性劳动即可做出的各种修改或变形仍在本发明的保护范围以内。Although the specific implementation of the present invention has been described above in conjunction with the accompanying drawings, it does not limit the protection scope of the present invention. On the basis of the technical solution of the present invention, those skilled in the art can make various Modifications or variations are still within the protection scope of the present invention.
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