CN116287208A

CN116287208A - Method for noninvasively diagnosing coronary artery expansion disease

Info

Publication number: CN116287208A
Application number: CN202310253726.5A
Authority: CN
Inventors: 宗义君; 杨秀春; 肖冰
Original assignee: Hebei College Of Traditional Chinese Medicine
Current assignee: Hebei College Of Traditional Chinese Medicine
Priority date: 2023-03-16
Filing date: 2023-03-16
Publication date: 2023-06-23

Abstract

The invention belongs to the field of biological medicine, and particularly relates to a method for noninvasively diagnosing coronary artery expansion. Specifically, the invention provides the use of an agent for detecting the expression level and/or methylation degree of USP18 in the preparation of a product for diagnosing coronary artery expansion.

Description

Method for noninvasively diagnosing coronary artery expansion disease

Technical Field

The invention belongs to the field of biological medicine, and particularly relates to a method for noninvasively diagnosing coronary artery expansion.

Background

Coronary artery dilation (coronary artery ectasia, CAE) is a rare but easily identifiable anatomical morphological abnormality. It is generally referred to as a diffuse expansion of the epicardial coronary artery, more than 1.5 times that of the adjacent normal segment, and more than 2 times more than that of the limiting expansion, and is generally referred to as a coronary aneurysm. CAE can be single-shot or multiple-shot, and can be in a saccular shape (the transverse diameter of a tumor body is larger than the long diameter) or a fusiform shape (the transverse diameter of the tumor body is smaller than the long diameter). 50% of CAE patients have combined coronary atherosclerosis. Simple CAE refers to a disease that excludes the unknown cause of atherosclerosis, vasculitis, kawasaki disease, infectious disease, congenital coronary artery disease, etc.

The etiology mechanism of CAE is not completely defined. The pathological manifestations are mainly the destruction of the middle layer structure of the coronary artery vessel wall, and the degradation of elastic fiber is mainly caused by the following possible causes: 1. atherosclerosis, a variant of the obstructive coronary disease, is considered by the learner as CAE. 2. The CAE of children and adolescents is often an advanced complication of kawasaki disease, either autoimmune or inflammatory response. Both connective tissue diseases systemic arteritis (such as nodular arteritis, large arteritis) and equine syndrome can lead to CAE. 3. Vascular infectious diseases such as fungal or septic emboli, syphilis, borreliosis, etc. 4. The simple CAE causes are unknown, and may be related to gene susceptibility (such as special HLAT genotypes, matrix metalloproteinase gene variation), over-expression of angiotensin converting enzyme and the like.

The gold standard for CAE diagnosis is coronary angiography. Coronary angiography suggests that the degree of coronary lumen expansion meets the CAE standard with or without incorporation of coronary atherosclerotic plaque, stenosis, and thrombosis. Meanwhile, the medicine does not accompany the cases of Kawasaki disease, systemic vasculitis (lupus erythematosus, polyarteritis nodosa, behcet disease and the like), syphilis, intervention treatment complications such as coronary rotational abrasion, stent implantation and the like. The coronary angiography greatly improves the detection rate, and the incidence rate in the crowd receiving the coronary angiography is 1.2-9.9%, wherein the detection rate of patients with coronary heart disease and abdominal aortic aneurysm is higher. The detection rate of the simple CAE is 0.1-0.32%.

The rapid development and application of the high-throughput sequencing technology provide a more comprehensive and rapid analysis means for researching pathogenesis of coronary artery expansion, and also provide a new idea for further treatment schemes of coronary artery expansion.

Disclosure of Invention

According to the invention, the gene information of a coronary artery expansion disease patient is obtained through sequencing, and a methylation analysis result is combined, so that a diagnosis target USP18 capable of diagnosing coronary artery expansion disease is provided, the expression quantity change of the diagnosis target USP18 in a patient sample is consistent with the data analysis result through fluorescent quantitative PCR verification, and the application value of the USP18 in diagnosis is proved through the verification of a subject working characteristic curve.

In a first aspect, the present invention provides the use of an agent for detecting the expression level and/or degree of methylation of USP18 in the manufacture of a product for diagnosing coronary artery dilation.

Preferably, the USP18 has a low degree of methylation and high expression in patients with coronary artery expansion.

Preferably, the "high" means that the expression level of USP18 in a patient is at least 1.1-fold, specifically at least 1.1-fold, 1.2-fold, 1.3-fold, 1.4-fold, 1.5-fold, 1.6-fold, 1.7-fold, 1.8-fold, 1.9-fold, 2.0-fold, 2.1-fold, 2.2-fold, 2.3-fold, 2.4-fold, 2.5-fold, 2.6-fold, 2.7-fold, 2.8-fold, 2.9-fold, 3.0-fold, 3.1-fold, 3.2-fold, 3.3-fold, 3.4-fold or 3.5-fold or more relative to the control expression level in a healthy control population.

Preferably, the "low" means that the USP18 has a degree of methylation in the patient that is less than the expression level in a healthy control population, e.g., about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% of the control expression level.

Preferably, the detection is performed on a subject sample. The sample comprises: tissue, blood urine, saliva, semen, milk, cerebrospinal fluid, tears, sputum, mucus, lymph, cytosol, ascites, pleural effusions, amniotic fluid, bladder irrigation fluid and bronchoalveolar lavage fluid.

In particular, the healthy control is a population identified as not suffering from coronary artery dilation.

Most preferably, the sample is blood.

Preferably, the expression level includes an mRNA expression level and/or a protein expression level.

Preferably, the reagent for detecting the expression amount of mRNA includes a reagent used in the following method: quantitative detection methods based on PCR, southern hybridization, northern hybridization, dot hybridization, fluorescence In Situ Hybridization (FISH), DNA microarrays, ASO methods, high throughput sequencing platforms.

Specifically, the reagent for detecting mRNA expression level, such as specific primer and/or probe, uses fluorescent quantitative PCR to quantitatively detect USP18 in the specific embodiment of the present invention.

Preferably, the probe may be a DNA, RNA, DNA-RNA chimera, PNA or other derivative. The length of the probe is not limited, and any length may be used as long as it specifically hybridizes to the target nucleotide sequence and binds thereto. As used herein, the term "hybridization" shall include "the process of binding a nucleic acid strand to a complementary strand via base pairing" as performed in polymerase chain reaction techniques.

When detecting mRNA, reverse transcription-polymerase chain reaction (RT-PCR) can also be used for detection; specifically, RNA in a sample to be tested is extracted using an RNA extraction technique (for example, using acidic phenol/guanidine isothiocyanate) or a finished kit, reverse transcribed into DNA, and then the amount of expression of the DNA is detected.

Preferably, the aforementioned PCR-based detection method may also be replaced by other amplification methods, such as: strand displacement amplification (Strand displacement amplification, SDA), nucleic acid sequence dependent amplification (Nucleic acid sequence-based amplification, NASBA), transcription dependent amplification system (Transcript-based amplification system, TAS), Q replicase (Q-beta replicase) catalyzes RNA amplification, rolling circle amplification (Rolling circle amplification, RCA), loop-mediated isothermal amplification (Loop mediated isothermal amplification, LAMP).

Preferably, the reagent for detecting the protein expression level includes a reagent used in the following method: western Blot (Western Blot), enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), sandwich assay, immunohistochemical staining, mass spectrometry, immunoprecipitation assay, complement fixation assay, flow cytometry fluorescence assay, and protein chip.

Specifically, common reagents for detecting the expression level of a protein include specific antibodies. Preferably, the antibody comprises an intact antibody molecule, any fragment of an antibody or an antibody with modifications, in particular, the antibody comprises a chimeric antibody, scFv, fab, F (ab') 2, fv, etc. Provided that the fragment is capable of retaining the ability to bind to the protein. The preparation of antibodies for detecting protein levels is well known to those skilled in the art, and any method may be used in the present invention to prepare the antibodies.

The detection result in any of the foregoing detection methods may be embodied by a label, specifically, the label includes: radiolabels, enzymatic labels, chemiluminescent labels, fluorescent labels, and other suitable labels.

Preferably, the product comprises a kit, a diagnostic device.

In another aspect, the invention also provides a kit for diagnosing coronary artery expansion, comprising reagents for detecting the expression level of USP 18.

Preferably, the kit includes the aforementioned reagent for detecting the mRNA expression level.

Preferably, the kit includes the reagent for detecting the protein expression level.

Preferably, the kit can also comprise an mRNA expression quantity auxiliary detection reagent, a protein expression quantity auxiliary detection reagent and a detection instrument.

Preferably, the mRNA expression level-aiding detection reagent includes, but is not limited to: reagents for visualizing the amplicon corresponding to the primer, for example, reagents for visualizing the amplicon by agarose gel electrophoresis, enzyme-linked gel method, chemiluminescence method, in situ hybridization method, fluorescence detection method, or the like; an RNA extraction reagent; a reverse transcription reagent; cDNA amplification reagents; preparing a standard substance used for a standard curve; a positive control; negative control.

More specifically, the positive control includes a sample taken from a patient diagnosed with coronary artery expansion, and the negative control includes a sample taken from a healthy control to confirm that the patient is not coronary artery expansion. The sample includes blood taken directly from a subject, and also includes a treated sample such as a DNA sample obtained by transcription of RNA for easy preservation, and the like.

Preferably, the protein expression level-assisted detection reagent includes, but is not limited to: blocking solution, antibody diluent, washing buffer, chromogenic stop solution and standard substance for preparing standard curve.

Preferably, the detection device comprises a real-time quantitative PCR instrument, a high-throughput sequencing platform, a detection chip and a chip signal reader.

The appropriate reagents, controls, instructions, etc. of the invention are packaged together in a suitable container into a kit.

On the other hand, the invention also provides application of the kit in preparing a product for diagnosing coronary artery expansion.

In another aspect, the present invention provides a method for diagnosing coronary artery expansion, comprising comparing the result of detection of the expression level and/or the result of detection of the methylation degree of USP18 with a threshold value, and determining whether the subject suffers from coronary artery expansion according to the comparison result.

More specifically, the USP18 expression level detection result and methylation degree detection result are obtained by detecting a sample from a subject.

Preferably, the sample is blood.

More specifically, the judgment method is to compare the detection result of the expression amount of USP18 with the threshold value, and if the input expression amount of USP18 is higher than the threshold value, the disease is represented; otherwise, the patient is not ill; if the methylation level of the input USP18 is below a threshold, it is indicative of a disease; otherwise, the patient is not ill;

the term "threshold" according to the present invention may also be referred to as a critical value, a cut-off value, a cutoff value, and a threshold value obtained under different detection methods and detection reagents, and the method for determining the threshold value is well known to those skilled in the art. The threshold value comprises an expression quantity threshold value and a methylation degree threshold value.

The method of determining the "threshold" is common in the art. The threshold values obtained by different detection programs are different, and sometimes the threshold values measured by reagents of different lot numbers of the same manufacturer are different, so that the threshold values need to be adjusted at any time according to the variation of experimental reagents and detection methods in clinical application.

In a specific embodiment, the detection method to obtain the threshold value needs to be the same as the detection method for the subject sample.

The term "coronary artery expansion syndrome" as used herein, coronary Artery Ectasia (CAE), means that the coronary artery is restricted or diffusely expanded for various reasons by more than 1.5 times or more than the diameter of the adjacent normal coronary artery.

The gold standard for CAE diagnosis, as described herein, is coronary angiography. Coronary angiography suggests that the degree of coronary lumen expansion meets the CAE standard with or without incorporation of coronary atherosclerotic plaque, stenosis, and thrombosis. Meanwhile, the medicine does not accompany the cases of Kawasaki disease, systemic vasculitis (lupus erythematosus, polyarteritis nodosa, behcet disease and the like), syphilis, coronary rotational abrasion, stent implantation and the like, and the like.

Drawings

FIG. 1 is a volcanic chart obtained by differential analysis.

Fig. 2 is a heat map obtained by the difference analysis.

FIG. 3 is a volcanic plot of differential methylation sites.

FIG. 4 is a Manhattan diagram of differential methylation sites.

FIG. 5 is a graph of the working characteristics of USP18 in diagnosing coronary artery dilation.

Detailed Description

The present invention is further described in terms of the following examples, which are given by way of illustration only, and not by way of limitation, of the present invention, and any person skilled in the art may make any modifications to the equivalent examples using the teachings disclosed above. Any simple modification or equivalent variation of the following embodiments according to the technical substance of the present invention falls within the scope of the present invention.

Example 1: data analysis and verification

Step 1, sample collection and sequencing data processing

The collected blood samples were sequenced, and the sample information according to the present invention is shown in table 1. Sequencing a cDNA library using an Illumina Hiseq high throughput sequencing platform based on sequencing-by-synthesis (Sequencing By Synthesis, SBS) technology can yield a large number of high quality Reads, and these Reads or bases produced by the sequencing platform are referred to as Raw Data (Raw Data). A large amount of sample double-ended sequencing data was obtained by the Illumina platform. In view of the influence of the data error rate on the result, the trimmonic software is adopted to perform quality preprocessing on the original data, and the numbers of reads in the whole quality control process are counted and summarized, and the summarized result is shown in table 2.

TABLE 1 sample information for normal control and CAE patients

TABLE 2 sequencing data statistics Table

Note that: (1) Sample: sample name; (2) Rawreads: the number of original reads; (3) Rawbases: original sequencing quantity, i.e., number of bases; (4) clearreads: the number of clearready obtained after filtration; (5) clearbases: sequencing amount and base number obtained after filtering; (6) ValidBases: effective base percentage (7) Q30: calculating the percentage of bases with Phred values greater than 30 to total bases; (8) GC: calculating the percentage of the sum of the numbers of bases G and C to the total number of bases

Of the transcriptome sequencing data, only data aligned to the reference genome can be used for subsequent analysis. Thus, reads aligned to a designated reference genome are referred to as Mapped Reads.

TABLE 3 statistics of mapping alignment efficiency for each sample

Note that: sample: sample name; total_reads: the number of clearready after quality control of the sequencing data; total_map: number of reads aligned to genome and percentage thereof; read1_map: comparing the number of reads 1 to a reference genome and a percentage thereof; read2_map: the number of reads 2 aligned to the reference genome and the percentage thereof; the slice_map: the split versus number of reads onto the genome and percent thereof; unsplice_map: undivided ratio versus number of reads onto genome and percent property_map: paired read1 and read2 simultaneously aligned reads to the genome and percentages thereof

And (3) using known reference gene sequences and annotation files as databases, and adopting a sequence similarity comparison method to identify the expression abundance of each protein coding gene in each sample. The number of reads aligned to the protein-encoding gene in each sample was obtained using the htseq-count software. After the counts are obtained by comparison, the protein coding genes need to be filtered to remove the genes with the reads of zero number. The number of detected genes in each sample is shown in Table 4.

TABLE 4 statistical partial results display of the number of detected genes

And carrying out differential analysis on the sequencing results of the two groups of mRNA, filtering genes according to the counts mean value, and only keeping the genes with the counts mean value larger than 2 for further analysis. And (3) carrying out standardization treatment on the count number of each sample gene by using DESeq2 (using BaseMean value to estimate the expression quantity), calculating a difference multiple, carrying out difference significance test by using NB (negative binomial distribution test), and finally screening the difference protein coding genes according to the difference multiple and the difference significance test result. The condition for screening the difference is p <0.05 +|log2foldchange| >1. 152 differentially expressed genes, including 93 up-regulated and 59 down-regulated, were analyzed. The volcanic and thermal charts obtained by the difference analysis are shown in fig. 1 and 2.

Step 2, differential methylation analysis

GSE87016 dataset containing 23 samples of methylation data (NOR: cae=12:11) was downloaded from GEO database and differential methylation analysis was performed on methylation data using the CHAMP package. The set screening criteria were p.value <0.05, yielding 9377 differential methylation sites, 4318 total differential methylation genes, including 2289 hypermethylation genes, 2029 hypomethylation genes. Volcanic and manhattan diagrams of the differential methylation sites are shown in figures 3 and 4.

Step 3, screening differential expression genes modified by abnormal methylation

Intersection of the mRNA differential expression gene and the differential methylation gene to obtain differential expression genes with abnormal methylation regulation, and 9 genes with downregulated expression with hypermethylation modification and 11 genes with upregulated expression with hypomethylation modification are obtained.

Step 4, fluorescent quantitative PCR verification

Based on the results of the foregoing assays, USP18 was hypomethylated and highly expressed in CAE patients.

Blood from patients with coronary artery expansion and control blood (> 15 cases) were collected using USP18 as candidate gene (case group vs control group), RNA samples were extracted, and differential expression of candidate gene in disease group and control group was verified by fluorescent quantitative PCR (qRT-PCR).

Step 5, ROC curve verification

The working characteristic curve (receiver operating characteristic curve) of the subject is called as ROC curve for short, and is called as susceptibility curve (sensitivity curve); the points on the ROC curve reflect the same sensitivity, the upper limit, the lower limit, the group distance and the cutoff point of the measured value are determined by analyzing the measured results of the disease group and the reference group, an accumulated frequency distribution table is listed according to the selected group distance interval, the true positive rate (sensitivity), the specificity and the false positive rate (1-specificity) of all the cutoff points are calculated respectively, and the ROC curve is plotted. Sensitivity (sensitivity), which is the proportion of patients that can be correctly determined by the screening method. Specificity (specificity) refers to the proportion of patients that can be correctly determined as non-patients by the screening method.

The AUC value was 0.76, the sensitivity was 0.769,1-specific was 0.25 in the ROC curve obtained according to USP18, as shown in fig. 5.

The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.

Claims

1. Use of an agent for detecting the expression level and/or the methylation level of USP18 for the preparation of a product for diagnosing coronary artery dilation.

2. The use of claim 1, wherein said USP18 has a low degree of methylation and high expression in patients suffering from coronary artery expansion.

3. The use of claim 1, wherein the test is performed on a sample from a subject, the sample being blood.

4. The use according to claim 1, wherein the expression level comprises mRNA expression level and/or protein expression level.

5. The use according to claim 4, wherein the reagent for detecting mRNA expression level comprises a reagent used in the following method: quantitative detection methods based on PCR, southern hybridization, northern hybridization, dot hybridization, fluorescence in situ hybridization, DNA microarrays, ASO methods, high throughput sequencing platforms.

6. The method according to claim 5, wherein the reagent for detecting mRNA expression level comprises a specific primer and/or probe.

7. The use according to claim 4, wherein the reagent for detecting the protein expression level comprises a reagent used in the following method: western blot, enzyme-linked immunosorbent assay, radioimmunoassay, sandwich assay, immunohistochemical staining, mass spectrometry, immunoprecipitation assay, complement fixation assay, flow cytometry and protein chip.

8. The use according to claim 7, wherein the reagent for detecting the expression level of a protein comprises a specific antibody.

9. A kit for diagnosing coronary artery expansion, comprising a reagent for detecting the expression level of USP 18;

preferably, the kit comprises the reagent for detecting mRNA expression level according to claim 5;

preferably, the kit comprises the reagent for detecting protein expression level according to claim 7;

preferably, the kit can also comprise an mRNA expression quantity auxiliary detection reagent, a protein expression quantity auxiliary detection reagent and a detection instrument;

preferably, the mRNA expression level-assisting detection reagent includes: a reaction reagent, an RNA extraction reagent, a reverse transcription reagent, a cDNA amplification reagent, a standard substance used for preparing a standard curve, a positive control substance and a negative control substance for visualizing the amplicon corresponding to the primer;

preferably, the protein expression level auxiliary detection reagent comprises: sealing liquid, antibody diluent, washing buffer solution, chromogenic stop solution and standard substance for preparing standard curve;

10. Use of the kit of claim 9 for the preparation of a product for diagnosing coronary artery dilation.