CN111500702B

CN111500702B - Application of cg00843506 site methylation of RPN1 gene in diagnosing asthma

Info

Publication number: CN111500702B
Application number: CN202010340821.5A
Authority: CN
Inventors: 钱粉红; 张黎莎
Original assignee: Affiliated Hospital of Jiangsu University
Current assignee: Affiliated Hospital of Jiangsu University
Priority date: 2020-04-26
Filing date: 2020-04-26
Publication date: 2021-04-20
Anticipated expiration: 2040-04-26
Also published as: CN111500702A

Abstract

The invention discloses an application of cg008843506 locus methylation of an RPN1 gene in diagnosing asthma. According to the invention, clinical patients are collected to analyze the methylation state of the cg008843506 gene of the RPN1, and the methylation level of the cg008843506 gene of an asthma patient is remarkably reduced compared with that of a healthy control, and ROC curve analysis proves that the methylation diagnosis of the cg008843506 gene is high in asthma efficacy, and can be used as a molecular marker for clinical diagnosis of asthma.

Description

Application of cg00843506 site methylation of RPN1 gene in diagnosing asthma

Technical Field

The invention relates to the technical field of biology, in particular to application of cg00843506 locus methylation of RPN1 gene in diagnosing asthma.

Background

Bronchial asthma (hereinafter, asthma) is a chronic inflammation of respiratory tract, and symptoms such as wheezing and cough can appear during attack, and the condition of the disease is aggravated at night or in the early morning. With the popularization of asthma knowledge and the improvement of asthma understanding, the work of treating and preventing asthma is advanced to a certain extent. But asthma can not be cured radically so far, and the treatment effect is not ideal, thus causing great economic burden and psychological burden to patients. Adult asthma and childhood asthma have been investigated to show an increasing trend in their mortality and morbidity over 30 years, accounting for 1% of the global disease burden. The pathogenesis of the asthma is not clear, so that the research on the asthma is still significant and necessary.

Traditional Chinese medicine has long been documented in the area of "asthma". Although the name of asthma is not explicitly mentioned in Huangdi's classic, there are similar descriptions of asthma symptoms. Zhang Zhongjing of Han Dynasty (golden lack essence, consumptive lung disease, lung abscess, cough and qi-ascending syndrome and treatment) says that the main causes of cough and qi-ascending, water chicken in throat and belamcanda rhizome and ephedra decoction. It is indicated that the throat emits a frog-like cry sound during asthma attack, similar to wheezing during asthma attack in western medicine, and a prescription for treatment is proposed, in which the indicated symptoms are similar to those of the acute attack of bronchial asthma in modern medicine. The first time of the primordial generation of Zhudanxi suggested the name of asthma, with the research, later doctors gradually differentiated the difference between asthma and asthma based on the deep understanding of the etiology and pathogenesis. Asthma in traditional Chinese medicine belongs to the category of 'asthma', wheezing sounds can be produced in throats during a grand attack, shortness of breath is felt, dyspnea is accompanied, and even asthma can not lie flat. The pathogenesis of the disease is invasion of exogenous pathogenic factors, improper diet, emotional stimulation and body weakness. According to traditional Chinese medicine, the pathogenesis of the disease is that phlegm is formed due to the accumulation of body fluids caused by dysfunction of viscera, and the phlegm is stored in the lung, which becomes the root cause of the pathogenesis. The lung is the main disease location of asthma and is closely related to spleen and kidney. In remission, it is also felt short breath, fatigue, with symptoms of asthma, but mildly, making it difficult to get all of these symptoms to disappear. The asthma is acute, complicated and complicated, and may cause "dyspnea" with the syndrome of pathogenic excess complicated with deficiency of vital qi. Asthma is a complex disease, which can occur repeatedly, and has a long and difficult course and complex symptoms. The first part of asthma is lung, which affects spleen and kidney, and its lung function is affected by repeated attack, and finally lung distension may be severe, and the disease may be complicated disease of multiple viscera. The disease condition is complicated, so that physicians have been studying the disease from ancient times to now.

DNA methylation refers to covalent binding of a methyl group to the cytosine carbon atom number 5 of a genomic CpG dinucleotide, and the methyl group is mainly involved in the expression regulation of the gene. Methylation of many genes has been shown to be closely related to various clinical diseases, and some may even be identified as an independent factor in the pathogenesis of the disease. DNA methylation abnormality is closely related to diseases such as tumor. Therefore, methylated genes can be used as not only markers for molecular diagnosis of diseases, but also targets for molecular therapy (reference: value of DNA methylation markers in molecular diagnosis and therapy, Vol.1, No. 3 of 2009, 9).

The present application is intended to study DNA methylation associated with asthma in order to discover biomarkers that can be used in clinical diagnosis.

Disclosure of Invention

The research idea of the invention is as follows: according to the early stage methylation chip result of the subject group, screening a cg00843506 locus located in the RPN1 gene promoter region as a Target gene locus to carry out quantitative detection of Methyl Target with enlarged sample size, and carrying out correlation analysis on clinical data of a patient. The results show that the methylation level of cg00843506 locus in the promoter region of the RPN1 gene is significantly different between healthy control and asthmatic patients, so that whether the subjects suffer from asthma can be judged according to the methylation state of cg00843506 locus.

Specifically, the invention provides the following technical scheme:

in a first aspect, the present invention provides a kit for detecting asthma, the kit comprising: and (3) detecting the methylation level of cg00843506 locus of promoter region of RPN1 gene.

The reagents include reagents used in methods for detecting DNA methylation.

As a specific example of the reagent, the reagent includes a primer and/or a probe against RPN1 gene.

Furthermore, the kit also comprises a reagent for detecting the internal reference gene.

The reagent for detecting the internal reference gene comprises a primer and/or a probe aiming at the internal reference gene.

Internal controls were used in the kit to indicate the quality of DNA extraction and modification.

Still further, the kit further comprises: DNA polymerase, dNTPs, Mg²⁺Ions.

Still further, the kit further comprises: bisulfite, bisulfite or hydrazine salts.

Still further, the kit further comprises (but is not limited to): quality control, negative control and/or instructions.

In a second aspect, the invention provides application of the cg00843506 locus of the promoter region of the RPN1 gene in preparing a reagent for diagnosing asthma.

Further, the reagents include primers and/or probes.

In a third aspect, the invention provides application of a reagent for detecting the methylation level of the cg00843506 locus of the promoter region of the RPN1 gene in preparing a kit for diagnosing asthma.

The kit is as described above.

In a fourth aspect, the present invention provides a method for non-diagnostically detecting DNA methylation of the RPN1 gene in vitro, the method comprising:

(1) processing a sample to be detected by bisulfite or hydrazine to obtain a modified sample to be detected;

(2) and (3) detecting the modified sample to be detected in the step (1) by using the kit, and obtaining the DNA methylation condition of the RPN1 gene.

As used herein, a "test sample" refers to a nucleic acid sample to be tested, which contains a nucleic acid or nucleic acids, in which it is desired to know whether or not a target nucleic acid is present. In the present invention, the sample to be tested may be a tissue or a body fluid.

As used herein, "target nucleic acid" refers to a nucleic acid fragment of interest that is a DNA-specific fragment of the RPN1 gene methylation.

As used herein, a "probe" refers to a single-stranded nucleic acid having a known nucleotide sequence, which has a nucleotide sequence structure substantially complementary to a target nucleic acid and can form a double strand with the "target nucleic acid". The "probe" may carry a label. For example, the label can be attached to the 5 'end or the 3' end of the probe.

The fluorescent group labeled by the detection probe can be VIC, ROX, FAM, Cy5, HEX, TET, JOE, NED and the like; the quencher group can be TAMRA, BHQ, MGB, Dabcyl. The multi-channel PCR detection system is suitable for a common multi-channel PCR detection system for clinical detection at present, and realizes multi-color fluorescence detection in one reaction tube.

Methods for assessing DNA methylation are well known. For example, Laird (2010) Nature Reviews Genetics11:191-203 provides a review of DNA methylation analysis. In some embodiments, methods of assessing methylation include random shearing or random fragmentation of genomic DNA, cleavage of DNA with a methylation dependent or methylation sensitive restriction enzyme, followed by selective identification and/or analysis of cleaved or uncleaved DNA. Selective identification can include, for example, separating cleaved and uncleaved DNA (e.g., by size) and quantifying the cleaved or uncleaved sequence of interest. See, for example, U.S. patent No.7,186,512. In some embodiments, the methods may encompass amplifying intact DNA after restriction enzyme digestion, thereby amplifying only DNA that is not cleaved by the restriction enzyme in the amplified region. See, for example, U.S. patent application nos. 10/971,986; 11/071,013, respectively; and 10/971,339. In some embodiments, amplification may be performed using gene-specific primers. Alternatively, adaptors can be added to the ends of randomly fragmented DNA, which can be digested with methylation dependent or methylation sensitive restriction enzymes, and the entire DNA can be amplified using primers that hybridize to the adaptor sequences. In some embodiments, a second step may be performed to determine the presence, absence, or amount of a particular gene in the amplification pool of DNA. In some embodiments, real-time quantitative PCR is used to amplify DNA.

Other methods for detecting DNA methylation may involve genome sequencing before and after bisulfite treatment of DNA. See, for example, Frommer, Proc. Natl. Acad. Sci. USA89:1827-1831 (1992). When sodium bisulfite is contacted with DNA, unmethylated cytosines are converted to uracil, while methylated cytosines are not modified.

In some embodiments, restriction enzyme digestion of PCR products amplified from bisulfite converted DNA is used to detect DNA methylation. See, e.g., Sadri & Hornsby, Nucl. acids Res.24:5058-5059 (1996); xiong & Laird, Nucleic Acids Res.25:2532-2534 (1997).

In some embodiments, the MethyLight assay is used to detect DNA methylation, alone or in combination with other methods (see EAds et al, Cancer Res.59:2302-2306 (1999)).

In some embodiments, Ms-SNuPE (Methylation-sensitive Single Nucleotide Primer Extension) is used alone or in combination with other methods to detect DNA Methylation (see Gonzalgo & Jones Nucleic Acids Res.25:2529-2531 (1997)).

In some embodiments, methylation-specific PCR ("MSP") reactions are used, alone or in combination with other methods, to detect DNA methylation. The MSP assay must first modify the DNA by sodium bisulfite, convert all unmethylated but not methylated cytosines to uracil, and then amplify using primers specific for methylated versus unmethylated DNA. See, Herman, Proc.Natl.Acad.Sci.USA93: 9821-; U.S. Pat. No.5,786,146.

In some embodiments, high throughput DNA methylation analysis is used to determine the methylation status of a gene. Briefly, genomic DNA is isolated from a cell or tissue sample (e.g., a tissue sample or blood sample) and converted in a sodium bisulfite reaction (the bisulfite method converts unmethylated cytosine residues to uracil), using standard assays in the art. Bisulfite converted DNA products are amplified, fragmented and hybridized to an array containing CpG sites from the entire genome using standard assays in the art. After hybridization, the array is imaged and processed using standard assays in the art for analysis of DNA methylation status.

In some embodiments, bisulfite sequencing pcr (bsp) is used to identify the methylation state of a gene. Briefly, genomic DNA is isolated from a cell or tissue sample (e.g., a tissue sample or a blood sample) and converted in a sodium bisulfite reaction (the bisulfite method converts unmethylated cytosine residues to uracil), using standard assays in the art. The bisulfite-converted DNA products are amplified with primers designed to be specific for bisulfite-converted DNA (e.g., bisulfite-specific primers) and ligated into vectors for transformation into host cells using standard assays in the art. After selecting host cells containing PCR amplified, bisulfite converted, DNA products of interest, the DNA products are isolated and sequenced to determine the site of methylation, using standard assays in the art.

In some embodiments, quantitative methylation specific pcr (qmsp) is used to identify the methylation state of a gene. Briefly, genomic DNA is isolated from a cell or tissue sample and converted in a sodium bisulfite reaction (the bisulfite method converts unmethylated cytosine residues to uracil), using standard assays in the art.

Drawings

FIG. 1 shows a statistical plot of the methylation level of cg00843506 of RPN1 gene;

FIG. 2 shows a ROC plot;

FIG. 3 shows a graph of correlation analysis of cg00843506 methylation of RPN1 gene with clinical symptoms;

FIG. 4 shows a graph of correlation analysis of cg00843506 methylation of RPN1 gene with lung function;

FIG. 5 shows a graph of correlation analysis of cg00843506 methylation of RPN1 gene with total serum IgE.

Detailed description of the preferred embodiments

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. The experimental procedures, for which specific conditions are not noted in the following examples, are generally performed according to conventional conditions such as those described in J. SammBruk et al, molecular cloning protocols, third edition, scientific Press, 2002, or according to the manufacturer's recommendations.

Example 1 asthma-associated methylation site study

1. Research population

The study population of the present application consisted of asthmatic patients (n-13) and healthy controls (n-13) collected during 2017 to 2019, month 9.

Patient inclusion criteria included: (1) current (last 12 months) respiratory symptoms; (2) previous doctor diagnosis of asthma; (3) spirometry during an asthma attack indicates a decrease in lung function.

Exclusion criteria: respiratory infections have occurred recently (over the past month); complications, such as cancer or other respiratory diseases, including but not limited to COPD; a history of autoimmune disease; diabetes and other underlying diseases.

Blood was collected. The following information is collected at the same time: clinical features, complete blood cell count results, serum total IgE and asthma control. All participants provided written informed consent and the study was approved by the ethical committee of human research at the subsidiary hospital of Jiangsu university.

2. DNA extraction and DNA methylation assay

Using eBioscience^TM1 XRBC lysis buffer (Thermo Fisher Scientific, Shanghai, China) leukocytes were isolated from peripheral blood and centrifuged.

Genomic DNA was extracted from leukocytes using a general genomic DNA extraction kit (Solarbio, Beijing, China), and then EZ DNA Methylation was used according to the manufacturer's protocol^TMGOLD kit (Zymo Research, CA, USA) for bisulfite conversion. After PCR amplification (hotstarttaq polymerase kit, TAKARA, tokyo, japan) and library construction, samples were sequenced using paired-end sequencing protocol according to the manufacturer's guidelines (Illumina MiSeq benchmark sequence, CA, usa). The DNA methylation levels of specific CpG sites were determined by methyl target sequencing (Genesky biotechnology inc., Shanghai, China) using a next generation sequencing based multi-objective CpG methylation analysis method.

3. Statistical analysis

Prior to statistical analysis, a normal distribution survey was performed on all variables. The continuous variables of the normal distribution are expressed as mean ± standard deviation and analyzed by independent sample t-test. The continuous variable of the anomaly distribution is represented as a median value. Univariate analysis of continuous variables was performed using Kruskal-Wallis test and Mann-Whitney U test to calculate differences in methylation between groups. Spearman correlation analysis was used to analyze the relationship between RPN1 promoter methylation and changes in lung function and IgE levels. Statistical analysis was performed using IBM SPSS Statistics Version 23(IBM Corp, Armonk, NY), all of which were bi-directional. For the statistical methods described above, a p-value <0.05 is considered statistically significant.

4. Results

(1) Feature(s)

Table 1 lists the characteristics of asthma patients and healthy controls. There were no differences in age, Body Mass Index (BMI) and white blood cell count between asthmatic and healthy controls. However, other indices, including serum total IgE, FEV 1% pred and FEV1/FVC, were significantly different between the two groups (p < 0.001).

TABLE 1 basic data of the study population

(2) Difference in methylation

The present study detected hypomethylation of the RPN1 gene cg00843506 in asthmatic patients compared to healthy controls (p <0.05, figure 1). The diagnosis efficacy of cg00843506 on asthma was analyzed by using a receiver operating curve (ROC curve), the AUC value was 0.817, the cutoff value was 0.3084, the sensitivity was 76.9%, the specificity was 76.9%, and the detailed results are shown in table 2 and fig. 2.

TABLE 2 ROC Curve statistics

(3) Symptom correlation analysis

As a total of 7 patients in 13 patients in the group complain of obvious respiratory symptoms such as chest distress, wheezing and the like, 6 patients have no obvious respiratory symptoms recently. Therefore, the present study was divided into symptomatic group (S) and asymptomatic group (NS) according to the presence or absence of distinct respiratory symptoms in patients, and the methylation level differences were compared. FIG. 3 shows that methylation changes in cg00843506 were not associated with the presence of clinical symptoms (p > 0.05).

(4) Lung function correlation analysis

The correlation between the methylation level of cg00843506 site in asthmatic patients and the percentage of forced expiratory volume in the expected value at 1 second (FEV 1% prep) and the ratio of forced expiratory volume in the first second (FEV1/FVC) was compared by Spearman correlation analysis. Specific correlation values are as follows: FEV 1% pred correlation coefficient rs is 0.731, and p is 0.005 in cg00843506 locus in RPN1 gene; the correlation coefficient rs of FEV1/FVC is 0.599, p is 0.031; the above results indicate that cg00843506 methylation level is significantly positively correlated with lung function (fig. 4).

(5) Correlation analysis of serum Total IgE levels

The correlation between cg00843506 site methylation levels and serum total IgE levels in asthma group patients was compared by Spearman correlation analysis, respectively. The specific correlations are as follows: the correlation coefficient rs is-0.599 and p is 0.031 at the cg00843506 locus in the RPN1 gene; the above results indicate that cg00843506 methylation levels were significantly negatively correlated with serum total IgE levels (fig. 5).

The invention verifies that the RPN1 gene-related locus has differential methylation in asthma patients by sequencing the methylation level of a target region, and suggests that the gene can be used as a DNA methylation molecular marker for assisting asthma diagnosis by performing correlation analysis on clinical data of the patients.

Claims

1. A kit for detecting asthma, comprising: and (3) detecting the methylation level of cg00843506 locus of promoter region of RPN1 gene.

2. The kit of claim 1, wherein the reagents comprise primers and/or probes for the RPN1 gene.

3. The kit of claim 2, wherein the kit further comprises a reagent for detecting an internal reference gene.

4. The kit according to claim 3, wherein the reagent for detecting the internal reference gene comprises a primer and/or a probe for the internal reference gene.

5. The kit of any one of claims 1-4, wherein the kit further comprises: DNA polymerase, dNTPs, Mg²⁺Ions.

6. The kit of claim 5, further comprising: bisulfite, bisulfite or hydrazine salts.

7. The application of the reagent for detecting the methylation level of the cg00843506 locus of the promoter region of the RPN1 gene is characterized in that the reagent is used for preparing a kit for diagnosing asthma.

8. Use according to claim 7, wherein the kit is a kit according to any one of claims 1 to 6.