CN111334572A

CN111334572A - New application of CTSZ gene methylation

Info

Publication number: CN111334572A
Application number: CN202010340801.8A
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: 2020-06-26
Anticipated expiration: 2040-04-26
Also published as: CN111334572B

Abstract

The invention discloses a new application of CTSZ gene methylation, and particularly discloses an application of cg01623438 and cg02744249 methylation in diagnosing asthma. The present study demonstrated that the combined diagnostic effect of cg01623438 and cg02744249 was higher than that of cg01623438 or cg02744249 alone, with AUC values of 0.781, sensitivity of 71.4%, and specificity of 73.3%. The results of the present study indicate that cg01623438 and cg02744249 methylation can be used as biomarkers for asthma diagnosis.

Description

New application of CTSZ gene methylation

Technical Field

The invention relates to the technical field of biology, in particular to a new application of CTSZ gene methylation.

Background

Asthma is the most prevalent chronic disease worldwide, affecting more than 3 hundred million people. Many cells and cellular components, such as mast cells, eosinophils, T lymphocytes, macrophages, neutrophils and epithelial cells, play an important role in asthma. Asthma is a genetic disease that is well accepted, but genetic factors are not the only cause of increased morbidity. Previous studies have shown that environmental exposure to allergens, air pollution or second hand smoke can also lead to asthma attacks. Epigenetic processes translate environmental exposure associated with risk of disease into chromatin regulation and ultimately lead to disease. Theory on epigenetics has recently developed rapidly. DNA methylation is an important component of epigenetic regulation involving transcription factor-mediated regulation and upstream signaling pathways that ultimately affect immune cell function. DNA methylation also plays an important role in cell differentiation and function by regulating the activity state of genes at an early stage of cell differentiation. In most cases, methylation and demethylation lead to gene silencing and reactivation, respectively. The consequences of DNA methylation changes have been demonstrated in different neoplastic and autoimmune diseases.

Alterations in promoter methylation levels are associated with a variety of diseases. For example, the methylation status of the MGMT promoter is very important in glioma patients. The level of methylation of the IFI44L promoter is a sensitive and specific diagnostic marker for SLE. Previous studies have also shown that changes in promoter methylation in SMAD3, TET1, and Vanin-1(VNN1) are associated with the pathogenesis and treatment of asthma. All of the above findings suggest that promoter methylation may play an important role in asthma.

Cathepsin z (ctsz) is a cathepsin family member, and the gene is located in a frequently amplified region of 20q13.2 (15). CTSZ is involved in the development of many diseases such as breast cancer, acute cutaneous delayed-type hypersensitivity and primary cholangitis, and tuberculosis. CTSZ plays an important role in host immune defense through the following mechanisms: migration of immune cells (e.g., monocytes, macrophages and dendritic cells); phagocytosis; signal transduction; communicating with a cell; and (5) proliferation. CTSZ regulates protein expression, e.g., can regulate MMP2 and MMP 9. MMP9 is produced primarily by epithelial cells, smooth muscle cells, macrophages and neutrophils, all of which are critical in airway wall remodeling.

The present application compares the differences in CTSZ promoter methylation in leukocytes of adult asthmatic patients and healthy controls and analyzes the correlation between changes in methylation and various current diagnostic criteria (e.g., symptom onset, lung function and total serum IgE) to assess the importance of CTSZ promoter methylation in asthma diagnosis.

Disclosure of Invention

The invention provides a methylation site for asthma diagnosis, wherein the methylation site is the methylation site of a CTSZ gene.

Further, the methylation sites are selected from one or two of cg01623438 and cg 02744249.

The present invention provides reagents for detecting the aforementioned methylation sites.

The "reagent" used in the present invention may be any form of preparation that can be used for detecting the methylation level, including but not limited to organic reagents, inorganic reagents, probes, primers, chips, and the like.

Knowing the methylation sites for asthma diagnosis as disclosed herein, one skilled in the art can design, synthesize corresponding probes or primers to detect the methylation levels of these methylation sites using routine techniques. If necessary, the chip can be designed.

In the present invention, the method for measuring methylation of a gene derived from a clinical sample may be selected from the group consisting of PCR, methylation specific PCR, real-time methylation specific PCR, PCR using a methylated DNA-specific binding protein, quantitative PCR, Pyrosequencing (Pyrosequencing), and disulfide bond sequencing.

As used herein, the term "sample" is meant in its broadest sense and includes any biological sample obtained from an individual, a body fluid, a cell line, a tissue culture, or other source, depending on the type of analysis to be performed. Methods for obtaining body fluids and tissue biopsies from mammals are generally widely known.

The invention also provides a kit for detecting the methylation sites.

Further, the kit comprises the reagents described above.

Furthermore, the kit also comprises a transformation solution, a binding solution, a washing solution, a purification solution, an eluent, a DNA extraction reagent and a PCR amplification reagent.

The conversion solution comprises an ammonium bisulfite solution.

The binding solution comprises guanidine hydrochloride.

The washing solution comprises purified water.

The purified liquid comprises isopropanol, ethanol or sodium hydroxide.

The eluent was a TE solution.

The PCR amplification reagent is a ddPCR amplification reagent.

The invention also provides the application of the methylation site in preparing an asthma diagnostic reagent.

The invention also provides the application of the methylation sites in the preparation of asthma diagnostic kits.

The invention also provides the application of the reagent in preparing an asthma diagnosis kit.

The invention also provides a method of diagnosing asthma, comprising contacting a sample containing nucleic acids from a subject with an agent that determines the methylation status of the nucleic acids in the sample, and identifying the methylation status of at least one region of at least one nucleic acid, wherein a methylation status of at least one region of at least one nucleic acid that is different from the methylation status of the same region of the same nucleic acid in a subject without asthma is indicative of the subject having asthma.

The methods of the invention comprise determining the methylation state of one or more regions of one or more nucleic acids isolated from a subject. The phrases "nucleic acid" or "nucleic acid sequence" as used herein refer to an oligonucleotide, nucleotide, polynucleotide or a fragment of either of these, or to genomic or synthetically derived DNA or RNA that may be single-or double-stranded, or to genomic or synthetically derived DNA or RNA that may represent a sense or antisense strand, Peptide Nucleic Acid (PNA), or DNA-like or RNA-like material of natural or synthetic origin. As will be appreciated by those skilled in the art, when the nucleic acid is RNA, deoxyribonucleoside A, G, C and T are replaced by ribonucleoside A, G, C and U, respectively.

Any nucleic acid sample, in purified or unpurified form, can be used according to the invention, provided it contains or is suspected of containing nucleic acid sequences that include the target site (e.g., CpG-containing nucleic acids). One nucleic acid region capable of being differentially methylated is a CpG island, a nucleic acid sequence having an increased density compared to other nucleic acid regions of the dinucleotide CpG. CpG dinucleotides (doubls) occur only about 20% frequently in vertebrate DNA, which can be expected from a fraction of G C base pairs. In some regions, the density of CpG dinucleotides reaches predictive values; it was increased 10-fold relative to the remaining genome. CpG islands have an average content of G C of about 60% and typically DNA has an average content of G C of about 40%. The islands take the form of extensions typically about 1 to 2 kilobases long. There are approximately 45,000 such islands in the human gene.

In many genes, CpG islands start immediately upstream of the promoter and extend downstream into the transcribed region. Methylation of CpG islands in the promoter often prevents gene expression. The island may also surround the 5 'region of the coding region of the gene and the 3' region of the coding region. Thus, a CpG island may be found in various regions of a nucleic acid sequence upstream of a coding sequence including a regulatory region, including a promoter region, a coding region (e.g., an exon), downstream of a coding region, such as an enhancer region and an intron.

Generally, CpG-containing nucleic acids are DNA. However, the methods of the invention may employ, for example, samples containing DNA or containing both DNA and RNA, including messenger RNA, where the DNA or RNA may be single-stranded or double-stranded, or DNA-RNA hybrids may be included in the sample.

Mixtures of nucleic acids may also be employed. The specific nucleic acid sequence to be detected may be a fragment of a macromolecular sequence or may exist as discrete molecules, such that the specific sequence constitutes the entire nucleic acid. The nucleic acid sequence need not be present initially in pure form, and the nucleic acid sequence may be present in very small fragments of a complex mixture, such as included in the entire human DNA. Nucleic acid-containing samples for determining the methylation status of nucleic acids contained in the sample or for detecting methylated CpG islands can be extracted by various techniques, such as those described by Sambrook et al (Molecular Cloning: A laboratory Manual, Cold spring harbor, N.Y., 1989; the entire contents of which are incorporated herein by reference).

Nucleic acids may contain regulatory regions that are regions of DNA that encode information or control transcription of the nucleic acid. The regulatory region includes at least one promoter. A "promoter" is a minimal sequence sufficient for direct transcription such that promoter-dependent gene expression can control cell-type specificity, tissue specificity, or can be induced by an external signal or agent. Promoters may be located in the 5 'or 3' regions of a gene. For CpG island methylation sites, all or part of the promoter region of some nucleic acids can be examined. Furthermore, it is generally considered that methylation of a promoter of a target gene proceeds naturally from the outer boundary inward. Thus, the early stages of cell transformation can be detected by methylation assays in the outer regions of the promoter region.

Detailed explanation of methylation determination method

Methylation specific PCR

When genomic DNA is treated with bisulfite, methylated cytosines in the 5 '-CpG-3' region remain unchanged and unmethylated cytosines become uracil. Thus, PCR primers corresponding to the region containing the 5 '-CpG-3' base sequence were constructed for the base sequence modified with bisulfite. Here, two types of primers corresponding to the methylated case and the unmethylated case were designed. When genomic DNA is bisulfite modified and then PCR is performed using two types of primers, in the case where DNA is methylated, the PCR product is derived from DNA in which a primer corresponding to the sequence using methylated bases is used. In contrast, in the case where the gene is unmethylated, the PCR product is obtained from DNA in which a primer corresponding to the unmethylated base sequence is used. DNA methylation can be quantified using agarose gel electrophoresis.

Real-time methylation specific PCR

Real-time methylation specific PCR is a real-time assay modified from methylation specific PCR and involves treating genomic DNA with bisulfite, designing PCR primers corresponding to the methylation status, and performing real-time PCR using the primers. Here, the method for measuring methylation includes two methods: a method of performing an assay using a TanMan probe complementary to the amplified base sequence, and a method of performing an assay using Sybergreen. Thus, real-time methylation-specific PCR selectively quantitatively analyzes only DNA. Here, a standard curve is prepared using an in vitro methylated DNA sample, and as a standard, a gene having no 5 '-CpG-3' sequence in the base sequence can also be amplified as a negative control group, and the degree of methylation is quantitatively analyzed.

Pyrophosphoric acid sequencing

Pyrosequencing is a real-time sequencing method modified from the bisulfite sequencing method. In the same manner as bisulfite sequencing, genomic DNA was modified by bisulfite treatment, and primers corresponding to regions without 5 '-CpG-3' sequences were constructed. After treatment of genomic DNA with bisulfite, it was amplified using PCR primers, followed by real-time sequence analysis using sequencing primers. The amount of cytosine and thymine in the 5 '-CpG-3' region was quantitatively analyzed, and the degree of methylation was expressed as the methylation index.

PCR or quantitative PCR using methylated DNA-specific binding protein and DNA chip

In the method of PCR or DNA chip using a methylated DNA specific binding protein, when a protein that specifically binds only to methylated DNA is mixed with DNA, the protein specifically binds only to methylated DNA, and thus only methylated DNA can be isolated. In the present invention, genomic DNA is mixed with methylated DNA-specific binding protein, and then only methylated DNA is selectively isolated. The isolated DNA was amplified using PCR primers corresponding to its promoter region, and then methylation of the DNA was determined by agarose gel electrophoresis.

In addition, DNA methylation can also be measured by quantitative PCR methods. In particular, methylated DNA isolated using a methylated DNA specific binding protein can be labeled with a fluorescent dye and hybridized to a DNA chip into which complementary probes are integrated, thereby determining methylation of the DNA. Here, the methylated DNA-specific binding protein is not limited to McrBt.

Drawings

Fig. 1 shows a statistical map of methylation levels of methylation sites of CTSZ gene promoter, wherein a: cg01623438, B: cg 02744249;

FIG. 2 shows a ROC plot of the CTSZ gene promoter cg01623438 methylation diagnosis of asthma;

FIG. 3 shows ROC plots for methylation of CTSZ gene cg02744249 in diagnosis of asthma;

FIG. 4 shows ROC plots for the combined diagnosis of asthma of cg01623438 methylation and cg02744249 in the CTSZ gene;

fig. 5 shows a map of the correlation analysis of CTSZ gene promoter methylation with clinical symptoms, where a: cg01623438, B: cg 02744249;

FIG. 6 shows a graph of correlation analysis of CTSZ gene promoter methylation with FEV 1% pred, where A: cg01623438, B: cg 02744249;

FIG. 7 shows a graph of correlation analysis between CTSZ gene promoter methylation and FEV1/FVC, wherein A: cg01623438, B: cg 02744249;

fig. 8 shows a graph of CTSZ gene promoter methylation correlated with serum total IgE analysis, wherein a: cg01623438, B: cg 02744249.

Detailed description of the preferred embodiments

Embodiments of the present invention will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

Example 1

1. Research population

The study population of the present application consisted of asthma patients between 18-65 years of age (n-14) and healthy controls (n-15) 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, total serum 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 miseqbenchtopsequence, 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 CTSZ 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, sex, 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 study characteristics

(2) Difference in methylation

The present study detected two methylation sites in the CTSZ promoter (cg01623438 and cg02744249), both hypomethylated in asthmatic patients (p <0.05, figure 1).

The diagnostic efficacy of cg01623438 on asthma was analyzed using a receiver operating curve (ROC curve) with an AUC value of 0.757, detailed results are shown in table 2 and figure 2.

TABLE 2 cg01623438ROC curve statistics

The diagnostic efficacy of cg02744249 on asthma was analyzed using the receiver operating curve (ROC curve) with an AUC value of 0.724, and the detailed results are shown in table 3 and figure 3.

TABLE 3 cg02744249ROC curve statistics

The diagnostic efficacy of the combination of cg01623438 and cg02744249 on asthma was analyzed using the receiver operating curve (ROC curve) with an AUC value of 0.781, a sensitivity of 71.4% and a specificity of 73.3%, detailed results are shown in table 4 and figure 4.

TABLE 4 Combined ROC Curve statistics

(3) Clinical symptoms

When blood samples were taken from 14 patients, 9 patients experienced symptoms including chest pain, cough and wheezing. No significant discomfort was experienced in 5 patients. These patients were divided into two groups according to the presence or absence of the above symptoms. Figure 5 shows that methylation changes in cg01623438 and cg02744249 are independent of the presence of clinical symptoms (p > 0.05).

(4) Level of lung function and serum total IgE

In addition to clinical symptoms, pulmonary function tests and total serum IgE are also widely used for clinical diagnosis. The results show that the methylation level of cg01623438 is negatively correlated with IgE levels (rs ═ 0.579, P ═ 0.001), and positively correlated with changes in FEV 1% pred (rs ═ 0.575, P ═ 0.001) and FEV1/FVC (rs ═ 0.440) (P ═ 0.017). cg02744249 has a similar trend (IgE: rs ═ 0.419, p ═ 0.024; FEV 1% pred: rs ═ 0.487, p ═ 0.007; FEV 1/FVC: rs ═ 0.420, p ═ 0.023), detailed results are shown in fig. 6-8.

Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims

1. Methylation sites for asthma diagnosis, which are methylation sites of the CTSZ gene.

2. The methylation site of claim 1, wherein the methylation site is selected from one or both of cg01623438 and cg 02744249.

3. A reagent for detecting the methylation site according to claim 1 or 2.

4. The reagent of claim 3 comprising specific primers and/or probes that amplify the methylation sites.

5. A kit for detecting the methylation site of claim 1 or 2.

6. A kit according to claim 5 comprising the reagent of claim 3 or 4.

7. The kit according to claim 5, further comprising a transformation solution, a binding solution, a washing solution, a purification solution, an elution solution, a DNA extraction reagent, and a PCR amplification reagent.

8. Use of the methylation sites of claim 1 or 2 for the preparation of a diagnostic agent for asthma.

9. Use of the methylation sites of claim 1 or 2 for the preparation of a kit for the diagnosis of asthma.

10. Use of the reagent of claim 3 or 4 for the preparation of a kit for asthma diagnosis.