CN116377053A

CN116377053A - Diagnostic biomarker for coronary artery dilatation and application thereof

Info

Publication number: CN116377053A
Application number: CN202310253729.9A
Authority: CN
Inventors: 高学英; 肖冰; 杨秀春
Original assignee: Second Hospital of Hebei Medical University
Current assignee: Second Hospital of Hebei Medical University
Priority date: 2023-03-16
Filing date: 2023-03-16
Publication date: 2023-07-04

Abstract

The invention discloses a diagnosis biomarker for coronary artery expansion disease and application thereof. The biomarker for coronary artery dilation diagnosis is IGSF10. Experiments prove that whether the subject suffers from coronary artery expansion or not can be diagnosed by detecting the biomarker provided by the invention, and the biomarker has higher accuracy and higher application value.

Description

Diagnostic biomarker for coronary artery dilatation and application thereof

Technical Field

The invention belongs to the technical field of biology, and relates to a diagnosis biomarker for coronary artery dilatation and application thereof.

Background

Coronary artery expansion (Coronary Artery Ectasia, CAE), also known as coronary artery tumorous expansion, refers to a rare abnormal condition in which the coronary artery diffusely expands more than 1.5 times the diameter of the adjacent normal coronary artery. This disease often involves multiple blood vessels, often distended by the right coronary artery. CAE can be single or multiple, and has a saccular or shuttle shape. 50% of CAE cases are complicated by coronary atherosclerosis. Coronary angiography is the gold standard for CAE diagnosis. Simple CAE refers to coronary artery dilation cases caused by unknown causes of atherosclerosis, vasculitis, kawasaki disease, infectious diseases, congenital coronary artery disease, etc.

The incidence rate of CAE in coronary angiography crowd is 1.2% -7.4%, the incidence rate of male is slightly high, the incidence rate increases with the increase of age, and the average incidence age is (55+/-10) years. The main hazards of CAE are the slow coronary blood flow, microcirculation disturbance and thrombus formation at the expansion site, the normal blood supply of the cardiac muscle is affected, and the rupture risk at the expansion site is caused, and the main clinical manifestations are: angina pectoris, myocardial infarction, arrhythmia, sudden death, etc.

At present, two morphological classification methods exist for CAE: the first method is to divide the expansion into light expansion, medium expansion and heavy expansion according to the diameter of the expanded blood vessel, wherein the expansion is respectively corresponding to the diameter of less than 5mm, the diameter of 5-8 mm and the diameter of more than 8mm, and the classification method is simple and clear, but is not easy to accurately classify the expansion with a plurality of lesions. Another classification method is Markis et al, and the Markis classification method proposed in 1976 is classified into four types according to the number of affected blood vessels and the degree of diffusion, and can reflect the lesions of CAE more comprehensively. According to the Markis classification, more than half of CAEs are single-branch vascular lesions (i.e., markis type IV expansions), with the right coronary artery most often affected.

Disclosure of Invention

To remedy the deficiencies of the prior art, it is an object of the present invention to provide a biomarker comprising a diagnosis for coronary artery dilation.

In order to overcome the current hurdles to better conduct clinical trial designs by improving the assessment of the progression of coronary artery expansion throughout the disease spectrum, there is an urgent unmet need for diagnostic and progression biomarkers for coronary artery expansion. The inventors identified as biomarkers for coronary artery dilation.

Accordingly, in a first aspect the present invention provides the use of a diagnostic reagent comprising a reagent capable of detecting the level of expression of an IGSF10 marker in a sample in the manufacture of a product for diagnosing coronary artery dilation.

Wherein the biomarker IGSF10 (Immunoglobulin Superfamily Member 10,gene ID:285313) includes genes and encoded proteins and homologs, mutations, and isoforms thereof. The term encompasses full length, unprocessed biomarkers, as well as any form of biomarker derived from processing in a cell. The term encompasses naturally occurring variants (e.g., splice variants or allelic variants) of the biomarker. Gene IDs are available at https:// www.ncbi.nlm.nih.gov/Gene.

In the present invention, the sample includes, but is not limited to, a tissue sample, blood such as whole blood or blood components, e.g., blood cells/cell components, serum or plasma, urine samples, body fluid samples, or other peripherally derived samples.

Further, the sample includes blood, interstitial fluid, cerebrospinal fluid, urine, tears, saliva, sweat.

Further, the sample is blood.

Further, the diagnostic reagent is selected from the group consisting of:

an oligonucleotide probe that specifically recognizes the IGSF10 marker described above; or (b)

Primers specifically amplifying the IGSF10 markers described above.

In an embodiment of the invention, the sequence of IGSF10 is as shown in transcript ENST 00000282466.4.

Further, the diagnostic reagent detects the expression level of the IGSF10 marker in the sample by a high throughput sequencing method and/or a gene chip method and/or a quantitative PCR method and/or a probe hybridization method.

In one embodiment, the IGSF10 gene is compared to a corresponding reference level. The comparison enables a determination to be made as to whether the individual has coronary artery dilation.

Further, the reagents used in the quantitative PCR method include primers that specifically amplify the IGSF10 marker described above.

Further, the primer includes natural oligonucleotides or synthetic oligonucleotides, which may be single-stranded or double-stranded, and must be long enough to prime synthesis of the desired extension product in the presence of an inducer, e.g., for diagnostic applications, an oligonucleotide primer will typically contain 15-25 or more nucleotides, depending on the complexity of the target sequence, although it may contain fewer nucleotides.

Further, the reagents used in the probe hybridization method include oligonucleotide probes that specifically recognize the IGSF10 marker described previously.

Further, the probes include fluorescent probes, antibodies, or absorbance-based probes. In the case of absorbance-based probes, the chromophore pNA (p-nitroaniline) can be used as a probe for detecting and/or quantifying a target nucleic acid sequence disclosed herein; it may also be a nucleic acid sequence comprising a fluorescent molecule or substrate that becomes fluorescent upon exposure to an enzyme and which is complementary to a fragment of a nucleic acid sequence.

Further, the probe is designed to have a melting temperature of 10 ℃ above that of the forward and reverse primers during a typical real-time fluorescent quantitative PCR detection process, and reagents for real-time fluorescent quantitative PCR include, but are not limited to: forward and reverse primers for target sequence of target gene, taqman fluorescent probe, optimized PCR buffer, deoxynucleotide triphosphate, and DNA polymerase with 5'-3' exonuclease activity.

In a preferred embodiment, the expression level of IGSF10 is down-regulated in coronary artery dilated patients compared to normal controls.

In a second aspect the invention provides a diagnostic product for coronary artery dilation, the diagnostic product comprising a diagnostic agent as hereinbefore described.

Further, the diagnostic product comprises a kit, a chip and test paper.

In some embodiments, the diagnostic reagent comprises a reagent that determines the expression level of the biomarker IGSF10 at the mRNA level. Determining the expression level of the biomarker IGSF10 at the mRNA level refers to a process of confirming the presence or absence and the degree of expression of mRNA of a gene for diagnosing coronary artery expansion in a biological sample isolated from a suspected patient of coronary artery expansion in order to diagnose coronary artery expansion, for measuring the expression amount of mRNA.

Further, the reagent for determining the expression level of the biomarker IGSF10 includes a reagent for determining the mRNA level by a polymerase chain reaction, a real-time fluorescent quantitative reverse transcription polymerase chain reaction, a competitive polymerase chain reaction, a nuclease protection assay, an in situ hybridization method, a nucleic acid microarray, a northern blot, or a DNA chip method.

In some embodiments, the agent comprises an agent that determines the level of expression of the biomarker IGSF10 at the protein level. Determining the expression level of the biomarker IGSF10 at the protein level refers to a process of confirming the presence or absence and the degree of expression of a gene for diagnosing coronary artery expansion in a biological sample isolated from a suspected patient of coronary artery expansion in order to diagnose coronary artery expansion, for measuring the expression amount of the protein.

Further, the reagent for determining the expression level of the biomarker IGSF10 at the protein level comprises a reagent for detecting the protein level by immunoblotting, enzyme-linked immunosorbent assay, radioimmunoassay, immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, fluorescence activated cell sorting, mass analysis or protein microarray.

In a third aspect, the invention provides a pharmaceutical composition for preventing or treating coronary artery expansion, the pharmaceutical composition comprising an agent that promotes the expression of IGSF10.

In a fourth aspect, the present invention provides a method for screening candidate drugs for the prevention or treatment of coronary artery expansion for non-therapeutic and non-diagnostic purposes, the method comprising the steps of:

1) Treating a system expressing or containing IGSF10 with a test substance;

2) Detecting the expression level of IGSF10 in the system of step 1);

3) And selecting a substance to be tested capable of reducing the expression level of IGSF10 as a candidate drug.

In a fifth aspect, the invention provides the use of an IGSF10 marker for the preparation of a diagnostic reagent for coronary artery dilation.

In a sixth aspect, the invention provides the use of an IGSF10 marker in a test system for diagnosing coronary artery dilation.

Further, the detection system includes a relative content detection system and an analysis system for the biomarker.

Further, the relative content detection system comprises detection of the expression level of biomarker mRNA levels and/or protein levels.

Further, the analysis system includes Linear Regression linear Regression, logistic Regression logistic Regression, polynomial Regression polynomial Regression, stepwise Regression stepwise Regression, ridge Regression, lasso Regression, elastic net Regression.

In a seventh aspect, the invention provides the use of an IGSF10 marker in the manufacture of a pharmaceutical composition for the prevention or treatment of coronary artery dilation.

In an eighth aspect, the invention provides the use of an IGSF10 marker in the screening of candidate drugs for the prevention or treatment of coronary artery dilation.

The term "and/or" means and includes any and all possible combinations of one or more of the associated listed items, as well as the lack of combinations when interpreted in the alternative.

The term "diagnosis" as used herein refers to the identification of a disease by its signs and symptoms or genetic analysis, pathological analysis, histological analysis, and the like. In particular, the term refers to the diagnosis or detection of coronary artery dilation.

The term "level" refers to the amount (e.g., measured in grams, moles, or counts such as ion or fluorescence counts) or concentration (e.g., absolute or relative concentration) of a gene described herein. Also included are scaled amounts or values, normalized amounts or values, or scaled and normalized amounts or values. In some preferred embodiments, the level determined herein is an expression level.

The term "expression level" refers to qualitative and/or quantitative differences in temporal and/or local expression patterns of nucleic acid molecules (e.g. in biological samples, body fluid samples, intracellular and/or between, or in blood). Thus, a differentially expressed nucleic acid molecule can qualitatively alter its expression, including, for example, activation or inactivation in a sample from a disease subject relative to a sample from a healthy subject. The difference in expression levels in the expression of the nucleic acid molecules may also be quantitative, for example because expression is regulated, i.e. upregulated, resulting in an increased amount of the nucleic acid molecules; or down-regulated, resulting in a decrease in the amount of nucleic acid molecules. The degree of variation in the expression levels of the nucleic acid molecules need only be large enough to be quantified by standard expression characterization techniques, e.g., by quantitative hybridization (e.g., to microarrays, to beads), amplification (PCR, RT-PCR, qRT-PCR, high throughput RT-PCR), quantitative ELISA, next generation sequencing (e.g., ABI sol id, illumina Genome Analyzer, roche 454GS FL), flow cytometry (e.g., lumlnex), and the like.

The terms "biological sample", "sample" or "sample" all refer to any sample from an individual or (control) subject comprising a biomarker of the invention. The biological sample may be a body fluid sample or a tissue sample. For example, biological samples encompassed by the present invention are tissue samples, blood (e.g., whole blood or blood components, such as blood cells/cell components, serum or plasma) samples, urine samples, aqueous humor, or samples from other peripheral sources. The biological samples may be mixed or pooled. The biological sample may be provided by taking a sample from an individual or (control) subject, but may also be provided by using a previously isolated sample. For example, a blood sample may be obtained from an individual or (control) subject by conventional blood collection techniques, or a tissue sample may be obtained from an individual or (control) subject by biopsy (biopsy). A biological sample is designated as a "reference biological sample" if it is obtained from at least one (control) subject, e.g. from at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300, 400, 500 or 1,000 (control) subjects. In some preferred embodiments, the reference biological sample is from the same source as the biological sample of the individual to be tested, e.g., both are blood samples, urine samples, or tissue samples. It is further preferred that both are from the same species, e.g. from a human. It is also preferred (alternatively or additionally) that the measure of the reference biological sample of the (control) subject and the biological sample of the individual to be tested are the same, e.g. both have the same volume. It is particularly preferred that the reference biological sample and the biological sample are from (control) subjects/individuals of the same sex and similar age.

The term "primer" as used herein means an oligonucleotide, whether naturally occurring in a purified restriction digest or synthetically produced, which is capable of acting as a point of origin of synthesis when placed under conditions that induce synthesis of a primer extension product complementary to a nucleic acid strand, i.e., in the presence of a nucleotide and an inducer, such as a DNA polymerase, and at a suitable temperature and pH. The primer may be single-stranded or double-stranded and must be long enough to prime the synthesis of the desired extension product in the presence of the inducer. The exact length of the primer depends on many factors, including temperature, primer source, and method of use. For example, for diagnostic applications, an oligonucleotide primer will typically contain 15-25 or more nucleotides, although it may contain fewer nucleotides, depending on the complexity of the target sequence. Factors involved in determining the appropriate length of the primer will be readily apparent to those skilled in the art.

The term "probe" refers to a substance that can specifically bind to a target substance to be detected in a sample, and refers to a substance that can confirm the presence of the target substance in the sample by the above-mentioned binding specificity. The type of probe is not limited and may be peptide nucleic acid (PNA, peptide nucleic acid), locked nucleic acid (LNA, locked nucleic acid), peptide, polypeptide, protein, ribonucleic acid or deoxyribonucleic acid, and most preferably peptide nucleic acid, which are commonly used in the art. More specifically, the above-mentioned probes are biomass including those derived from or similar to biological materials or produced in vitro, and may include, for example, enzymes, proteins, antibodies, microorganisms, animal and plant cells and organs, nerve cells, deoxyribonucleic acids including complementary deoxyribonucleic acids (cdnas), genomic deoxyribonucleic acids, oligonucleotides, ribonucleic acids including genomic ribonucleic acids, messenger ribonucleic acids, oligonucleotides, and the like, as examples of proteins including antibodies, antigens, enzymes, peptides, and the like.

The invention has the beneficial effects that:

the invention can realize diagnosis of coronary artery expansion by detecting the expression level of IGSF10, increase the sensitivity of detection, improve the detection capability and efficiency and actively take intervention measures.

Drawings

FIG. 1 is a diagram of a protein interaction network;

fig. 2 is a ROC graph of IGSF10 diagnosing coronary artery dilation.

Detailed Description

The invention will be described in further detail below, with the understanding that the description is intended for the purpose of illustration and is not intended to limit the scope of the invention.

As used herein, the term "sensitivity" refers to the number (%) of truly positive patients relative to the total number of patients (100%). The individual may be a subject with coronary artery expansion. Sensitivity was calculated by the following formula: sensitivity = TP/(tp+fn) (TP = true positive; FN = false negative).

As used herein, the term "specificity" relates to the number (%) of true negative individuals relative to the total number of healthy subjects (100%). The specificity was calculated by the following formula: specificity = TN/(tn+fp) (TN = true negative; FP = false positive).

As used herein, the term "AUC" refers to the abbreviation of the area under the curve. In particular, it refers to the area under the subject's operating characteristics (ROC) curve. As used herein, the term "subject operating characteristic (ROC) curve" is a plot of true positive rate versus false positive rate indicating different possible tangent points for a diagnostic test. It shows a trade-off between sensitivity and specificity, depending on the chosen cut-off (any increase in sensitivity is accompanied by a decrease in specificity). The area under the ROC curve is a measure of the accuracy of the diagnostic test (the larger the area, the better, optimally 1, the ROC curve for the random test is located on the diagonal, with an area of 0.5).

In the present invention, in order to improve the accuracy of diagnosis, the analysis may be performed by using a statistical method or algorithm, and an analysis method selected from the group consisting of a linear or nonlinear regression analysis method, a linear or nonlinear classification analysis method, a logistic regression analysis method, a variance analysis, a neural network analysis method, a genetic analysis method, a support vector machine analysis method, a hierarchical analysis or cluster analysis method, a hierarchical algorithm or a kernel principal component analysis method using a decision tree, a markov blanket analysis method, a regression feature elimination or entropy-based regression feature elimination analysis method, a forward floating search or a backward floating search analysis method, and combinations thereof may be used.

In the embodiment of the present invention, as the above statistical method, a logistic regression analysis method is preferably used, but not limited thereto.

Example 1 screening for biomarkers associated with coronary artery dilation

1. Screening method

(1) Data for screening and preprocessing

To screen biomarkers for coronary artery dilation diagnosis, common gene expression data associated with coronary artery dilation is downloaded from a gene expression integrated database (GEO). The dataset GSE87016 is downloaded from the GEO database (http:// www.ncbi.nlm.nih.gov/GEO /).

The data downloaded from the gene expression integrated database (GEO) also requires further processing:

1) The data set selected must be genome-wide DNA methylation data;

2) These data are from coronary artery dilation and control blood samples;

3) The study considered either the normalized or the original dataset.

(2) High throughput transcriptome data and pretreatment

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 carry out quality preprocessing on the original data, and the numbers of reads in the whole quality control process are statistically summarized.

The specific steps and the sequence are as follows:

(1) A dehiscence (adapter);

(2) Removing low quality reads;

(3) Removing low-quality bases from the 3 'and 5' ends in a different manner;

(4) Counting the original sequencing quantity, the effective sequencing quantity, Q30 and GC content, and carrying out comprehensive evaluation.

And carrying out data quantity statistics on the sequence after data quality control.

(3) mRNA Gene expression level analysis

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 genes detected in each sample is shown in Table 1, and the statistical partial results of the number of genes detected in Table 1 are shown

The FPKM method can eliminate the influence of the difference of the length and the sequencing quantity of the protein coding gene on the expression of the calculated protein coding gene, and the calculated gene expression quantity is high or low in reaction expression.

(4) Differential analysis of mRNA

Firstly, filtering genes according to the counts mean value, and only keeping the genes with 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.

(5) 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 is p.value <0.05.

(6) Protein interaction analysis of aberrant methylation modified differentially expressed genes

To investigate the protein interaction relationship between the screened aberrant methylation modified differentially expressed genes we constructed PPI networks of the screened 20 aberrant methylation modified differentially expressed genes using the online database sting.

2. Results

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. Analysis of high throughput sequenced transcriptome mRNA gave 152 differentially expressed genes, including 93 up-regulated and 59 down-regulated.

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.

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.

To investigate the protein interaction relationship between the screened aberrant methylation modified differentially expressed genes we constructed PPI networks of the screened 20 aberrant methylation modified differentially expressed genes using the online database sting. FIG. 1 shows PPI networks of 20 aberrant methylation-modified differentially expressed genes constructed using the STRING database.

Next we import the results obtained in the STRING database into the Cytoscape software (http:// www.cytoscape.org /), and use the CytoHubba plug-in to screen the core genes. We used a total of 3 algorithms, and screened a total of 10 core genes after crossing the first 10 genes of each algorithm (table 2).

TABLE 2 screening of HUB Gene of aberrant methylation modified differentially expressed Gene by three methods

Example 2 coronary artery dilation biomarker IGSF10 diagnostic efficacy validation analysis

Based on the result of high-throughput transcriptome data integration analysis, IGSF10 is screened as a candidate gene, coronary artery expansion patient blood and control blood (15 cases) are collected, RNA samples are extracted, and fluorescent quantitative PCR (qRT-PCR) is utilized to verify the differential expression of the candidate gene in a disease group and a control group.

Drawing an ROC graph of IGSF10 for diagnosing coronary artery expansion, as shown in fig. 2, IGSF10 shows higher diagnostic efficacy in coronary artery expansion diagnosis, AUC value is 0.829, sensitivity is 0.864, specificity is 0.812, suggesting that IGSF10 can diagnose coronary artery expansion with diagnostic efficacy.

The above description of the embodiments is only for the understanding of the method of the present invention and its core ideas. It should be noted that it will be apparent to those skilled in the art that several improvements and modifications can be made to the present invention without departing from the principle of the invention, and these improvements and modifications will fall within the scope of the claims of the invention.

Claims

1. Use of a diagnostic reagent comprising a reagent capable of detecting the expression level of an IGSF10 marker in a sample for the preparation of a product for diagnosing coronary artery dilation;

preferably, the sample comprises blood, interstitial fluid, cerebrospinal fluid, urine, tears, saliva, sweat;

preferably, the sample is blood.

2. The use according to claim 1, wherein the diagnostic agent is selected from the group consisting of:

an oligonucleotide probe that specifically recognizes the IGSF10 marker of claim 1; or (b)

A primer that specifically amplifies the IGSF10 marker of claim 1.

3. The use according to claim 1, wherein the diagnostic reagent detects the expression level of the IGSF10 marker in the sample using a high throughput sequencing method and/or a gene chip method and/or a quantitative PCR method and/or a probe hybridization method;

preferably, the reagents used in the quantitative PCR method comprise primers that specifically amplify the IGSF10 marker of claim 1;

preferably, the reagents used in the probe hybridization method comprise an oligonucleotide probe that specifically recognizes the IGSF10 marker of claim 1.

4. A diagnostic product of coronary artery dilation, characterized in that it comprises the diagnostic agent of any one of claims 1-3;

preferably, the diagnostic product comprises a kit, a chip, and a test paper.

5. A pharmaceutical composition for preventing or treating coronary artery expansion, comprising an agent that promotes IGSF10 expression.

6. A method for screening candidate drugs for the prevention or treatment of coronary artery expansion for non-therapeutic and non-diagnostic purposes, comprising the steps of:

1) Treating a system expressing or containing IGSF10 with a test substance;

2) Detecting the expression level of IGSF10 in the system of step 1);

Use of igsf10 markers for the preparation of a diagnostic reagent for coronary artery expansion.

Use of an igsf10 marker in a detection system for diagnosing coronary artery dilation.

Use of an igsf10 marker for the preparation of a pharmaceutical composition for the prevention or treatment of coronary artery expansion.

Use of an igsf10 marker for screening candidate drugs for the prevention or treatment of coronary artery expansion.