CN111321219A - Use of ACTA2 methylation as a diagnostic marker for asthma - Google Patents
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Abstract
The invention discloses application of ACTA2 methylation as a diagnostic marker of asthma. The methylation site of ACTA2 is selected from one or more of cg19791409, cg17967429, cg09822170 and cg 17133119. Compared with healthy control, the methylation level of the methylation site of the asthma patient is obviously increased, and the methylation site can be used as a molecular marker for diagnosing asthma according to the correlation between the change of the methylation site and the asthma. The invention also discloses a kit for diagnosing asthma, and the kit has good clinical application prospect.
Description
Technical Field
The invention relates to the field of biotechnology, in particular to application of ACTA2 methylation as a diagnostic marker of asthma.
Background
Asthma is a common chronic respiratory disease characterized by more complex respiratory symptoms and reversible airflow limitation. Asthma has high incidence worldwide, but has great difference in phenotype and pathogenesis [ Mcracken J L, Veeranki S P, Ameredes B T, et al. diagnosis and Management of asthma in additives: A Review [ J ] JAMA,2017,318(3):279-290], brings certain difficulties to diagnosis and treatment, and causes great economic loss due to the consequences of error work and study, and the like. Therefore, finding more reliable and intuitive diagnostic indicators becomes a hot spot in asthma research. It is widely believed that asthma is a disease simultaneously affected by both environmental and genetic effects [ Yang I V, Lozupone C A, Schwartz D A. the environmental, epigenome, and andasthma [ J ]. Journal of Allergy and Clinical Immunology,2017,140(1):14-23], and recent epigenetic development brings new ideas for asthma diagnosis.
DNA methylation is a common epigenetic (epigenetic) modification, under the catalysis of DNA methyltransferase, the 5 th carbon atom of cytosine is methylated by using the methyl provided by S-adenosylmethionine, so that cytosine is converted into 5-methylcytosine, which plays a crucial role in the regulation of gene expression. DNA methylation as an important component of epigenetics has been well documented as being closely related to The development of asthma [ Yang IV. DNA methylation of asthma and asthma [ J ]. Lancet. respiratory medicine,2019,7(4): 289-290; tanday s. epoxy student identities linked to inactive and equivalent [ J ]. The lancet. respiratory medicine 2015,3(4): 274; morales E1, Bustaminate M, Vilahur N, et al DNA methylation at ALOX12 is associated with genetic knowledge in children [ J ]. American journel of respiratory and cognitive card media, 2012,185(9):937-43 ]. The method provides a new idea for diagnosing asthma by detecting the DNA methylation level of nasal mucosa epithelial cells of asthma patients, searching genes related to asthma attack and verifying the genes in another attack queue.
Disclosure of Invention
The invention aims to provide a methylation site as an early asthma diagnosis marker, so as to solve the technical problem that the marker in the prior art cannot conveniently and accurately diagnose early asthma.
According to one aspect of the present invention, the present invention provides use of a reagent for detecting the methylation level of the methylation site of the ACTA2 gene in the preparation of a kit for diagnosing asthma.
Further, the methylation sites are selected from one or more of cg19791409, cg17967429, cg09822170, cg 17133119.
In a particular embodiment of the invention, the methylation site is cg 19791409.
Further, the reagents include those used in the methods employed to detect methylation levels; the methods include pyrosequencing, bisulfite sequencing, quantitative and/or qualitative methylation specific polymerase chain reaction, quantitative and/or qualitative bisulfite specific polymerase chain reaction, digital polymerase chain reaction, targeted sequencing in combination with bisulfite, southern blotting, restriction landmark genomic scanning, single nucleotide primer extension, CpG island microarray, single nucleotide primer extension SNUPE, combined sodium bisulfite restriction endonuclease analysis, mass spectrometry.
According to another aspect of the present invention, there is provided a kit for diagnosing asthma, which comprises a reagent for detecting the methylation level of the methylation site of the ACTA2 gene in a sample to be tested.
Further, the methylation sites are selected from one or more of cg19791409, cg17967429, cg09822170, cg 17133119.
Further, the reagents include those used in the methods employed to detect methylation levels; the methods include pyrosequencing, bisulfite sequencing, quantitative and/or qualitative methylation specific polymerase chain reaction, quantitative and/or qualitative bisulfite specific polymerase chain reaction, digital polymerase chain reaction, targeted sequencing in combination with bisulfite, southern blotting, restriction landmark genomic scanning, single nucleotide primer extension, CpG island microarray, single nucleotide primer extension SNUPE, combined sodium bisulfite restriction endonuclease analysis, mass spectrometry.
Further, the sample to be tested is from nasal mucosal epithelial tissue.
According to yet another aspect of the present invention, there is provided a device for asthma diagnosis, risk assessment, the device comprising a module for detecting methylation level of methylation site of ACTA2 gene of a subject; the methylation sites are selected from one or more of cg19791409, cg17967429, cg09822170, cg 17133119.
Further, the apparatus comprises an assessment module for determining that the subject is an asthmatic patient or that the subject is at high risk of suffering from asthma when the methylation level of one or more of the methylation sites selected from cg19791409, cg17967429, cg09822170, cg17133119 is increased in the subject's test value compared to a normal sample test value or a normal reference value.
According to yet another aspect of the present invention, there is provided a method for drug screening for the treatment of asthma, the method comprising the steps of detecting the methylation level of the methylation site of the ACTA2 gene in a subject; a reduced level of methylation at the methylation site when compared to prior to use of the screening drug indicates that the screening drug is effective in the subject; the methylation sites are selected from one or more of cg19791409, cg17967429, cg09822170, cg 17133119.
According to yet another aspect of the present invention, there is provided a biomarker for early diagnosis of asthma, risk assessment of disease and drug screening, the biomarker being methylation site of ACTA2 gene selected from one or more of cg19791409, cg17967429, cg09822170 and cg 17133119.
According to yet another aspect of the present invention, there is provided a method for early diagnosis of asthma, risk assessment, the method comprising the steps of detecting the methylation level of the methylation site of the ACTA2 gene in a subject; the methylation site is selected from one or more of cg19791409, cg17967429, cg09822170, cg 17133119; comparing the subject test value to a normal sample test value or a normal reference value, when the methylation level of one or more methylation sites selected from cg19791409, cg17967429, cg09822170, cg17133119 is increased, the subject is judged as an asthmatic patient or is judged as at high risk of suffering from asthma.
In the present invention, "diagnosis" and "disease risk assessment" have meanings known in the art, and for example, "diagnosis" is a judgment as to whether or not the disease is affected, and "disease risk assessment" is an assessment as to the magnitude of the disease risk and the magnitude of the risk of recurrence after treatment.
In the present invention, "methylation" refers to methylation of CpG sites.
In the present invention, methylation levels can be expressed using methods known in the art, for example, by the ratio of methylated cytosine C detected during sequencing to total C detected, specifically β (beta value) values ranging from 0 to 1 or 0 to 100%.
According to a typical embodiment of the invention, the reagents comprise oligonucleotide primers for amplifying nucleotide sequences containing specific methylation sites.
In the present invention, the kit may further comprise one or more reagents selected from the group consisting of: dntps, buffers, DNA polymerases, restriction endonucleases (including methyl specific endonucleases) and labels (including fluorescent labels, chemiluminescent labels, radioactive labels, etc.).
The invention has the advantages and beneficial effects that:
the invention establishes a method and a standard for screening specific methylation sites related to diseases, and screens the methylation sites related to early diagnosis of asthma by taking asthma as an example. The screened methylation sites as early diagnosis markers of asthma can be used for rapid diagnosis of diseases.
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FIG. 1 shows a significant differential methylation site chromosome profile;
FIG. 2 shows a differential methylation site volcano plot;
FIG. 3 shows a statistical plot of the distribution of significantly different methylation sites over each functional region;
figure 4 shows a statistical plot of gene methylation levels for each functional partition of the ACTA2 gene, where a: cg 19791409; b: a 5' -UTR region;
FIG. 5 shows a statistical plot of the methylation levels of the cg19791409 gene ACTA 2;
FIG. 6 shows a ROC plot.
Detailed Description
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 study of asthma-related methylation
1. Study object
The study cohort (cohort a) selected 2017-10 to 2018-10 asthmatic adult patients who were seen at outpatient clinic of department of respiration in affiliated hospital of Jiangsu university, 6 patients, and 5 healthy physical examiners who were in the same period, in the same residence, in the same age, in the same sex ratio, and have no direct relationship with asthmatic patients, and the general data of the patients are shown in table 1. The validation cohort (cohort B) selected 2018-10 to 2019-10 of 12 asthmatic adult patients who were treated at the department of respiration in the department of hospital affiliated to Jiangsu university, and 12 healthy physical examiners who were in the same period, in the same residence, in the same age, in the same sex ratio, and had no direct relationship with asthmatic patients, and the general data of the patients are shown in table 2. The study was approved by the ethical committee of the affiliated hospital of the university of Jiangsu, and all subjects had signed informed consent for clinical studies.
Grouping standard: (1) age 18-65 years; (2) the patient had a clear history of asthma diagnosis, and conformed to the guidelines for bronchial asthma prevention and treatment [ the respiratory disease division asthma group of the chinese medical society, the guidelines for bronchial asthma prevention and treatment (the definition, diagnosis, treatment, and management protocols of bronchial asthma) [ J ]. the journal of tuberculosis and respiration of china, 2008, 31 (3): 177-185], and has obvious respiratory system symptoms such as chest distress or wheezing when the medicine is taken; (3) the patients have no history of allergic rhinitis, no obvious nasal symptoms such as running nose, nasal obstruction and the like when the patients are taken into the nasal cavity, and no history of nasal cavity administration is available in recent time.
Exclusion criteria: (1) the history of using tumor and chemotherapy drugs is clear; (2) the history of upper respiratory tract infection in the last month; (3) denying cardiovascular and cerebrovascular diseases; (4) a pregnancy status.
2. Materials and methods
2.1 materials
Curette (American ASI corporation); collagenase type I (Sig in USA)ma corporation); animal tissue/cell genome DNA extraction kit (Solambio, China company)
2.2 specimen sampling
Cleaning nasal cavity, moistening nasal cavity with small amount of normal saline, repeatedly scraping with nasal curette at inferior turbinate position for 5-10 times, placing the lower end of the nasal curette into 0.1% collagenase, shaking for several minutes, and digesting in water bath at 37 deg.C for 1 hr.
2.3DNA extraction and treatment
Centrifuging the cells obtained after digestion, extracting DNA according to the kit use instruction, detecting the qualified DNA, and treating the DNA by using a bisulfate method. Primer design was performed on the post-treatment sequences using primer 3:
forward primer sequence: 5'-AGTGATAGATGTAAAATATAGGGTGATGG-3' (SEQ ID NO.1), reverse primer sequence: 5'-ACTAAACACACTAAAAATTTCAATATTCCTTT-3' (SEQ ID NO. 2). Then the sample is processed by steps of amplification, incubation, precipitation, hybridization, elution and the like, and then is analyzed by Illumina Infinium methylation EPIC BeadChip. The chip experiment and sequencing were completed by Shanghai sky Biotechnology Ltd.
2.4 statistical analysis
The statistical software SPSS 23.0 software is used for analyzing the experimental data and quantifying the dataAnd (6) carrying out statistics. The average number of the two samples is compared by adopting a t test, and the difference is statistically significant when p is less than 0.05.
3. Results
3.1 two queue general data comparison
The difference in sex, age, smoking history, BMI, peripheral blood leukocyte count ratio among patients in the group was not statistically significant (p all > 0.05). The differences of FEV1/FVC (%), FEV 1% pred in the asthma group are lower than those in the healthy control group, and the total IgE is higher than that in the healthy control group, and the differences are all statistically significant (p is less than 0.05). See table 1 and table 2.
TABLE 1 general data comparison between asthmatic and healthy control groups in cohort A
Table 2 general data comparison between asthmatic and healthy control in cohort B
3.2 study cohort (cohort A) Whole genome methylation assay results
3.2.1 Overall distribution of differential methylation
This co-detection consisted of 22 autosomes except X, Y chromosomes, and the number of differentially methylated sites (DMP) and Differentially Methylated Regions (DMR) are shown in Table 3. The distribution of DMP in each chromosome is shown in FIG. 1, and the trend pairs are shown in FIG. 2.
TABLE 3 methylation significant Change site number statistics
3.2.2 distribution of methylated regions in functional regions
The differentially methylated regions detected in this study were mostly concentrated in the genomic and intergenic regions, and were expressed in the promoter region, as shown in FIG. 3 (note: 1. stExon "means in the first exon region of the gene," 3'UTR "means in the 3' UTR sequence," 5'UTR "means in the 5' UTR sequence," Body "means in the gene region," ExonBnd "means in the exon region," IGR "means in the intergenic region," TSS1500 "means in the 200bp-1500bp region upstream of the transcription start site, and TSS 200" means in the 200bp region upstream of the transcription start site).
The ACTA2 gene promoter region cg19791409 and 5' -UTR region (cg17967429, cg09822170 and cg17133119) in the asthma group were significantly hypermethylated compared with the healthy control group, and the differences were statistically significant (p < 0.05). (FIG. 4).
3.3 validation cohort (cohort B) clinical validation analysis
Results as shown in fig. 5, quantitative determination of methylation level of the enlarged sample for the cg19791409 gene of ACTA2 found that: the methylation level in the asthma group (A) was generally higher than that in the healthy control group (NA), and the difference was statistically significant (p < 0.05).
The diagnostic efficacy of cg19791409 on asthma was analyzed using a subject working curve (ROC curve) with an AUC value of 0.889 and a sensitivity of 75% and specificity of 100% when the cutoff value was 0.1354, the ROC curve being shown in fig. 6.
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.
Sequence listing
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Claims (10)
1. Application of a reagent for detecting methylation level of methylation sites of ACTA2 gene in preparation of a kit for diagnosing asthma.
2. The use according to claim 1, wherein the methylation sites are selected from one or more of cg19791409, cg17967429, cg09822170, cg 17133119.
3. Use according to claim 1 or 2, wherein the reagents comprise reagents used in the methods employed for detecting methylation levels; the methods include pyrosequencing, bisulfite sequencing, quantitative and/or qualitative methylation specific polymerase chain reaction, quantitative and/or qualitative bisulfite specific polymerase chain reaction, digital polymerase chain reaction, targeted sequencing in combination with bisulfite, southern blotting, restriction landmark genomic scanning, single nucleotide primer extension, CpG island microarray, single nucleotide primer extension SNUPE, combined sodium bisulfite restriction endonuclease analysis, mass spectrometry.
4. A kit for diagnosing asthma is characterized by comprising a reagent for detecting the methylation level of the methylation sites of the ACTA2 gene in a sample to be detected.
5. The kit of claim 4, wherein the methylation site is selected from one or more of cg19791409, cg17967429, cg09822170, cg 17133119.
6. The kit of claim 4 or 5, wherein the reagents comprise reagents used in the method for detecting methylation levels; the methods include pyrosequencing, bisulfite sequencing, quantitative and/or qualitative methylation specific polymerase chain reaction, quantitative and/or qualitative bisulfite specific polymerase chain reaction, digital polymerase chain reaction, targeted sequencing in combination with bisulfite, southern blotting, restriction landmark genomic scanning, single nucleotide primer extension, CpG island microarray, single nucleotide primer extension SNUPE, combined sodium bisulfite restriction endonuclease analysis, mass spectrometry.
7. The kit according to any one of claims 4 to 6, wherein the sample to be tested is derived from nasal mucosal epithelial tissue.
8. An apparatus for asthma diagnosis and risk assessment, which comprises a module for detecting methylation level of methylation site of ACTA2 gene of a subject; the methylation sites are selected from one or more of cg19791409, cg17967429, cg09822170, cg 17133119.
9. The apparatus of claim 8, further comprising an assessment module for determining that the subject is an asthmatic patient or that the subject is at high risk of developing asthma when the methylation level of one or more methylation sites selected from cg19791409, cg17967429, cg09822170, cg17133119 is increased in the subject's test value compared to the normal sample test value or normal reference value.
10. A method for drug screening for the treatment of asthma, comprising the steps of detecting the methylation level of the methylation site of the ACTA2 gene in a subject; a reduced level of methylation at the methylation site when compared to prior to use of the screening drug indicates that the screening drug is effective in the subject; the methylation sites are selected from one or more of cg19791409, cg17967429, cg09822170, cg 17133119.
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