Diagnostic Biomarkers for Coronary Artery Ectasia and Their Applications

Abstract

The present invention discloses a diagnostic biomarker for coronary artery ectasia and its application. The biomarker for diagnosing coronary artery ectasia is IGSF10. Experiments have proven that by detecting the biomarker provided by the present invention, it is possible to diagnose whether a subject has coronary artery ectasia, with high accuracy, and the present invention has high application value.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to diagnostic biomarkers for coronary artery ectasia and their applications. Background Art

[0002] Coronary artery ectasia (CAE), also known as coronary artery aneurysm-like dilation, refers to a rare abnormal condition in which the coronary artery is diffusely dilated to more than 1.5 times the diameter of the adjacent normal coronary artery. This disease often involves multiple blood vessels, with right coronary artery dilation being more common. CAE can occur singly or multiply and can be cystic or fusiform in shape. 50% of CAE cases are complicated by coronary atherosclerosis. Coronary angiography is the gold standard for the diagnosis of CAE. Simple CAE refers to cases of coronary artery dilation of unknown cause excluding etiologies such as atherosclerosis, vasculitis, Kawasaki disease, infectious diseases, and congenital coronary artery diseases.

[0003] The incidence of CAE in the coronary angiography population is 1.2% - 7.4%. The incidence is slightly higher in men and increases with age. The average age of onset is (55 ± 10) years. The main hazards of CAE are to cause slow coronary blood flow, microcirculation disorders, and promote thrombosis at the dilated site, affecting the normal blood supply of the myocardium, as well as causing a risk of rupture at the dilated site. Its main clinical manifestations include: angina pectoris, myocardial infarction, arrhythmia, sudden death, etc.

[0004] Currently, there are two morphological classification methods for CAE: The first method is to classify according to the diameter of the dilated blood vessel, divided into mild, moderate, and severe degrees of dilation, corresponding to diameters less than 5 mm, diameters of 5 - 8 mm, and diameters greater than 8 mm respectively. This classification method is simple and clear, but it is not easy to accurately classify dilations with multiple lesions. Another classification method is the Markis classification method proposed by Markis et al. in 1976, which is divided into four categories according to the number of blood vessels involved and the degree of diffusion. This classification method can more comprehensively reflect the lesions of CAE. According to the Markis classification method, more than half of CAE cases are single-vessel lesions (i.e., Markis type Ⅳ dilation), with the right coronary artery being the most commonly involved. Summary of the Invention

[0005] In order to make up for the deficiencies of the prior art, one of the purposes of the present invention is to provide biomarkers for the diagnosis of coronary artery ectasia.

[0006] There is an urgent unmet need for diagnostic and progression biomarkers for coronary artery ectasia in order to overcome current obstacles to better clinical trial design by improving the assessment of the progression of coronary artery ectasia across the entire disease spectrum. The inventors of the present invention have identified biomarkers for coronary artery ectasia.

[0007] Accordingly, in the first aspect of the present invention, there is provided an application of a diagnostic reagent in the preparation of a product for diagnosing coronary artery ectasia, and the diagnostic reagent comprises a reagent capable of detecting the expression level of the IGSF10 biomarker in a sample.

[0008] The biomarker IGSF10 (Immunoglobulin Superfamily Member 10, gene ID: 285313) includes the gene, its encoded protein, and its homologs, mutations, and isoforms. This term encompasses full-length, unprocessed biomarkers, as well as any form of biomarker derived from processing in cells. This term encompasses naturally occurring variants of the biomarker (such as splice variants or allelic variants). The Gene ID can be obtained at https: / / www.ncbi.nlm.nih.gov / gene / .

[0009] In the present invention, the sample includes but is not limited to tissue samples, blood such as whole blood or blood components, such as blood cells / cellular components, serum or plasma, urine samples, body fluid samples, or samples from other peripheral sources.

[0010] Furthermore, the sample includes blood, tissue fluid, cerebrospinal fluid, urine, tears, saliva, and sweat.

[0011] Furthermore, the sample is blood.

[0012] Furthermore, the diagnostic reagent is selected from:

[0013] an oligonucleotide probe that specifically recognizes the aforementioned IGSF10 biomarker; or

[0014] a primer that specifically amplifies the aforementioned IGSF10 biomarker.

[0015] In an embodiment of the present invention, the sequence of IGSF10 is as shown in transcript ENST00000282466.4.

[0016] Furthermore, the diagnostic reagent uses high-throughput sequencing methods and / or gene chip methods and / or quantitative PCR methods and / or probe hybridization methods to detect the expression level of the IGSF10 biomarker in a sample.

[0017] In one embodiment, the IGSF10 gene is compared with a corresponding reference level. The above comparison enables determination of whether an individual has coronary artery ectasia.

[0018] Furthermore, the reagent used in the quantitative PCR method includes a primer that specifically amplifies the aforementioned IGSF10 biomarker.

[0019] Furthermore, the primer includes natural oligonucleotides or synthetic oligonucleotides. The primer can be single-stranded or double-stranded and must be long enough to initiate the synthesis of the expected extension product in the presence of an inducer. For example, for diagnostic applications, depending on the complexity of the target sequence, oligonucleotide primers usually contain 15-25 or more nucleotides, although it can contain fewer nucleotides.

[0020] Furthermore, the reagent used in the probe hybridization method includes an oligonucleotide probe that specifically recognizes the IGSF10 marker described above.

[0021] Furthermore, the probe includes a fluorescent probe, an antibody, or an absorbance-based probe. If it is an absorbance-based probe, the chromophore pNA (p-nitroaniline) can be used as a probe for detecting and / or quantifying the target nucleic acid sequence disclosed herein; it can also be a nucleic acid sequence containing a fluorescent molecule or substrate that becomes fluorescent upon exposure to an enzyme, and this nucleic acid sequence is complementary to a fragment of a nucleic acid sequence.

[0022] Furthermore, during the general real-time fluorescence quantitative PCR detection process, the probe is designed to have a melting temperature exceeding that of the forward and reverse primers by 10 °C. The reaction reagents for real-time fluorescence quantitative PCR include, but are not limited to: forward and reverse primers for the target gene target sequence, Taqman fluorescent probe, optimized PCR buffer, deoxynucleotide triphosphates, and a DNA polymerase with 5'-3' exonuclease activity.

[0023] In a preferred embodiment, compared with the normal control, the expression level of IGSF10 is down-regulated in patients with coronary artery ectasia.

[0024] The second aspect of the present invention provides a diagnostic product for coronary artery ectasia, and the diagnostic product includes the diagnostic reagent described above.

[0025] Furthermore, the diagnostic product includes a kit, a chip, and a test strip.

[0026] In some embodiments, the diagnostic reagent includes a reagent for determining 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 the process of confirming the presence and expression degree of the mRNA of the gene for diagnosing coronary artery ectasia in a biological sample isolated from a suspected patient with coronary artery ectasia for the purpose of diagnosing coronary artery ectasia, and is used to measure the expression amount of mRNA.

[0027] Further, the reagent for determining the expression level of the biomarker IGSF10 at the mRNA level includes reagents for determining the mRNA level by means of polymerase chain reaction, real-time fluorescence quantitative reverse transcription polymerase chain reaction, reverse transcription polymerase chain reaction, competitive polymerase chain reaction, nuclease protection assay, in situ hybridization, nucleic acid microarray, RNA blot or DNA chip.

[0028] In some embodiments, the reagent includes a reagent for determining the expression level of the biomarker IGSF10 at the protein level. Determining the expression level of the biomarker IGSF10 at the protein level refers to the process of confirming the presence or absence and the expression level of the protein of the gene for diagnosing coronary artery ectasia in a biological sample isolated from a suspected patient with coronary artery ectasia for the purpose of diagnosing coronary artery ectasia, and is used to measure the expression amount of the protein.

[0029] Further, the reagent for determining the expression level of the biomarker IGSF10 at the protein level includes reagents for detecting the protein level by means of immunoblotting, enzyme-linked immunosorbent assay, radioimmunoassay, immunodiffusion, immunoelectrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, fluorescence-activated cell sorting, mass spectrometry or protein microarray.

[0030] The third aspect of the present invention provides a pharmaceutical composition for preventing or treating coronary artery ectasia, and the pharmaceutical composition includes a reagent for promoting the expression of IGSF10.

[0031] The fourth aspect of the present invention provides a method for screening a candidate drug for preventing or treating coronary artery ectasia for non-therapeutic and non-diagnostic purposes, and the method includes the following steps:

[0032] 1) Treating a system expressing or containing IGSF10 with a test substance;

[0033] 2) Detecting the expression level of IGSF10 in the system described in step 1);

[0034] 3) Selecting a test substance that can reduce the expression level of IGSF10 as a candidate drug.

[0035] The fifth aspect of the present invention provides the application of the IGSF10 biomarker in the preparation of a diagnostic reagent for coronary artery ectasia.

[0036] The sixth aspect of the present invention provides the application of the IGSF10 biomarker in a detection system for diagnosing coronary artery ectasia.

[0037] Further, the detection system includes a relative content detection system and an analysis system for the biomarker.

[0038] Further, the relative content detection system includes the detection of the expression level of a biomarker at the mRNA level and / or the protein level.

[0039] Further, the analysis system includes Linear Regression, Logistic Regression, Polynomial Regression, Stepwise Regression, Ridge Regression, Lasso Regression, ElasticNet Regression.

[0040] The seventh aspect of the present invention provides the use of the IGSF10 biomarker in the preparation of a pharmaceutical composition for preventing or treating coronary artery ectasia.

[0041] The eighth aspect of the present invention provides the use of the IGSF10 biomarker in screening for candidate drugs for preventing or treating coronary artery ectasia.

[0042] 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 absence of a combination when interpreted in the alternative (or).

[0043] The term "diagnosis" as used herein refers to the identification of a disease through the signs and symptoms of the disease or genetic analysis, pathological analysis, histological analysis, etc. Specifically, the term refers to the diagnosis or detection of coronary artery ectasia.

[0044] 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 concentration or relative concentration) of the gene described herein. Also includes scaled amounts or values, normalized amounts or values, or scaled and normalized amounts or values. In some preferred embodiments, the levels determined herein are expression levels.

[0045] The term "expression level" refers to qualitative and / or quantitative differences in transient and / or local nucleic acid molecule expression patterns (e.g., in biological samples, body fluid samples, within and / or between cells, or in the blood). Thus, differentially expressed nucleic acid molecules can qualitatively alter their expression, including, for example, activation or inactivation in samples from diseased subjects relative to samples from healthy subjects. Differences in expression levels in nucleic acid molecule expression can also be quantitative, e.g., because expression is regulated, i.e., upregulated, resulting in an increase in the amount of the nucleic acid molecule; or downregulated, resulting in a decrease in the amount of the nucleic acid molecule. The degree of difference in nucleic acid molecule expression levels only needs to be large enough to be quantified by standard expression profiling techniques, e.g., by quantitative hybridization (e.g., with microarrays, with beads), amplification (PCR, RT-PCR, qRT-PCR, high-throughput RT-PCR), quantitative ELISA, next-generation sequencing (e.g., ABI SOLID, Illumina Genome Analyzer, Roche 454GS FL), flow cytometry (e.g., LUMINEX), etc.

[0046] The terms "biological sample", "biological specimen", "specimen" or "sample" all refer to any sample from an individual or (control) subject that contains a biomarker of the present invention. The biological specimen can be a body fluid sample or a tissue sample. For example, biological samples covered by the present invention are tissue samples, blood (e.g., whole blood or blood components, e.g., blood cells / cellular components, serum or plasma) samples, urine samples, aqueous humor or samples from other peripheral sources. The biological samples can be mixed or combined. The biological samples can be provided by taking a sample from an individual or (control) subject, but can also be provided by using a previously isolated sample. For example, a blood sample can be obtained from an individual or (control) subject by conventional blood collection techniques, or a tissue sample can be obtained from an individual or (control) subject by biopsy. If a biological sample 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, it is designated a "reference biological sample". 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 humans. (Alternatively or additionally) It is also preferred that the measurements 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.

[0047] As used herein, the term "primer" refers to an oligonucleotide, whether naturally occurring in a purified restriction digest or produced synthetically, which can act as a starting point for synthesis when placed under conditions that induce the synthesis of a primer extension product complementary to a nucleic acid strand, i.e., in the presence of nucleotides and an inducing agent such as DNA polymerase and at a suitable temperature and pH. The primer can 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 inducing agent. The exact length of the primer depends on a number of factors, including temperature, primer source, and method of use. For example, for diagnostic applications, depending on the complexity of the target sequence, oligonucleotide primers typically contain 15 - 25 or more nucleotides, although they can contain fewer nucleotides. The factors involved in determining the appropriate length of the primer are readily known to those skilled in the art.

[0048] The term "probe" refers to a substance that can specifically bind to a target substance to be detected in a sample, and is a substance that can confirm the presence of the target substance in the sample through the above-mentioned specific binding. The types of probes are substances commonly used in the art and are not limited. Preferably, they can be peptide nucleic acid (PNA), locked nucleic acid (LNA), peptide, polypeptide, protein, ribonucleic acid or deoxyribonucleic acid, and most preferably peptide nucleic acid. More specifically, the above probes are biological substances, including those derived from biological materials or similar thereto or manufactured in vitro. For example, they can include enzymes, proteins, antibodies, microorganisms, animal and plant cells and organs, nerve cells, deoxyribonucleic acid and ribonucleic acid. Deoxyribonucleic acid includes complementary deoxyribonucleic acid (cDNA), genomic deoxyribonucleic acid, oligonucleotides, and ribonucleic acid includes genomic ribonucleic acid, messenger ribonucleic acid, oligonucleotides. Examples of proteins include antibodies, antigens, enzymes, peptides, etc.

[0049] Advantages of the present invention:

[0050] By detecting the expression level of IGSF10, the present invention can achieve the diagnosis of coronary artery ectasia, increase the sensitivity of detection, improve the detection ability and efficiency, and actively take intervention measures. Brief description of the drawings

[0051] Figure 1 is a protein interaction network diagram;

[0052] Figure 2 is an ROC curve diagram for IGSF10 in diagnosing coronary artery ectasia. Detailed description of the specific embodiments

[0053] The following will further elaborate on the present invention. It should be understood and clearly noted that the foregoing is intended to describe its purpose and not to limit the scope of the present invention.

[0054] As used herein, the term "sensitivity" refers to the number of true positive patients (%) relative to the total number of patients (100%). An individual can be a subject with coronary artery dilation. Sensitivity is calculated by the following formula: Sensitivity = TP / (TP + FN) (TP = true positive; FN = false negative).

[0055] As used herein, the term "specificity" relates to the number of true negative individuals (%) relative to the total number of healthy subjects (100%). Specificity is calculated by the following formula: Specificity = TN / (TN + FP) (TN = true negative; FP = false positive).

[0056] As used herein, the term "AUC" relates to the abbreviation of the area under the curve. In particular, it refers to the area under the receiver operating characteristic (ROC) curve. As used herein, the term "receiver operating characteristic (ROC) curve" refers to a plot of the true positive rate versus the false positive rate for different possible cut-off points of a diagnostic test. It shows the trade-off between sensitivity and specificity, depending on the selected cut-off point (any increase in sensitivity will be accompanied by a decrease in specificity). The area under the ROC curve is a measure of the accuracy of a diagnostic test (the larger the area, the better, with an optimal value of 1, and the ROC curve of a random test lies on the diagonal with an area of 0.5).

[0057] In the present invention, in order to improve the accuracy of diagnosis, it can be analyzed by using statistical methods or algorithms, and analysis methods selected from the group consisting of linear or non-linear regression analysis methods, linear or non-linear classification analysis methods, logistic regression analysis methods, analysis of variance, neural network analysis methods, genetic analysis methods, support vector machine analysis methods, hierarchical analysis or clustering analysis methods, hierarchical algorithms using decision trees or kernel principal component analysis methods, Markov blanket analysis methods, regression feature elimination or entropy-based regression feature elimination analysis methods, forward floating search or backward floating search analysis methods, and combinations thereof can be utilized.

[0058] In a specific embodiment of the present invention, as the above-mentioned statistical method, a logistic regression analysis method is preferably used, but it is not limited thereto.

[0059] Example 1 Screening of Biomarkers Related to Coronary Artery Dilation

[0060] 1. Screening Method

[0061] (1) Data Used for Screening and Pretreatment

[0062] To screen for biomarkers that can be used in the diagnosis of coronary artery ectasia, public gene expression data related to coronary artery ectasia was downloaded from the Gene Expression Omnibus (GEO) database. Dataset GSE87016 was downloaded from the GEO database (http: / / www.ncbi.nlm.nih.gov / geo / ).

[0063] The data downloaded from the Gene Expression Omnibus (GEO) database still needs to be further processed:

[0064] 1) The selected dataset must be genome-wide DNA methylation data;

[0065] 2) These data are from coronary artery ectasia and control blood samples;

[0066] 3) Both standardized or raw datasets are considered in this study.

[0067] (2) High-throughput transcriptome data and preprocessing

[0068] A large amount of paired-end sequencing data of samples was obtained through the Illumina platform. Given the impact of data error rate on the results, Trimmomatic software was used to preprocess the quality of the raw data, and the number of reads during the entire quality control process was statistically summarized.

[0069] The specific steps and order are as follows:

[0070] (1) Remove adapters;

[0071] (2) Remove low-quality reads;

[0072] (3) Remove low-quality bases from the 3' end and 5' end in different ways;

[0073] (4) Statistically analyze the original sequencing volume, effective sequencing volume, Q30, GC content, and conduct a comprehensive evaluation.

[0074] Statistical analysis was performed on the sequences after data quality control.

[0075] (3) Analysis of mRNA gene expression levels

[0076] Using known reference gene sequences and annotation files as databases, the expression abundances of each protein-coding gene in each sample were identified by sequence similarity alignment. The htseq-count software was used to obtain the number of reads mapped to protein-coding genes in each sample. After obtaining the counts by alignment, it is necessary to filter the protein-coding genes and remove genes with zero reads. The number of detected genes in each sample is shown in Table 1. Partial results of the statistical analysis of the number of detected genes in Table 1 are presented

[0077]

[0078]

[0079] The FPKM method can eliminate the influence of protein-coding gene length and sequencing volume differences on the calculation of protein-coding gene expression, and the calculated gene expression level reflects high or low expression.

[0080] (4) Differential analysis of mRNA

[0081] First, filter the genes according to the mean of counts, and only retain the genes with a mean of counts greater than 2 for further analysis. Use DESeq2 to standardize the counts of genes in each sample (estimating the expression level using the BaseMean value), calculate the fold change, and perform a differential significance test using NB (negative binomial distribution test). Finally, screen for differentially expressed protein-coding genes based on the fold change and the results of the differential significance test. The screening conditions for differences are p < 0.05 & |log2foldChange| > 1.

[0082] (5) Differential methylation analysis

[0083] Download the GSE87016 dataset from the GEO database. This dataset contains methylation data of 23 samples (NOR:CAE = 12:11), and use the CHAMP package to perform differential methylation analysis on the methylation data. The set screening criterion is P.Value < 0.05.

[0084] (6) Protein-protein interaction analysis of abnormally methylated modification differentially expressed genes

[0085] To explore the protein-protein interaction relationships between the screened abnormally methylated modification differentially expressed genes, we constructed a PPI network of 20 screened abnormally methylated modification differentially expressed genes using the online database STRING.

[0086] 2. Results

[0087] Use DESeq2 to standardize the counts of genes in each sample (estimating the expression level using the BaseMean value), calculate the fold change, and perform a differential significance test using NB (negative binomial distribution test). Finally, screen for differentially expressed protein-coding genes based on the fold change and the results of the differential significance test. For the mRNA analysis of the high-throughput sequencing transcriptome, 152 differentially expressed genes were obtained, including 93 up-regulated and 59 down-regulated.

[0088] Download the GSE87016 dataset from the GEO database. This dataset contains methylation data of 23 samples (NOR:CAE = 12:11). Use the CHAMP package to perform differential methylation analysis on the methylation data. The set screening criterion is P.Value < 0.05, resulting in 9,377 differentially methylated sites and a total of 4,318 differentially methylated genes, including 2,289 hypermethylated genes and 2,029 hypomethylated genes.

[0089] Take the intersection of the mRNA differentially expressed genes and the differentially methylated genes to obtain the differentially expressed genes regulated by abnormal methylation. Nine genes with down-regulated expression due to hypermethylation modification and 11 genes with up-regulated expression due to hypomethylation modification are obtained.

[0090] To explore the protein-protein interaction relationships among the screened differentially expressed genes with abnormal methylation modifications, we constructed a PPI network of 20 screened differentially expressed genes with abnormal methylation modifications using the online database STRING. Figure 1 The PPI network of 20 differentially expressed genes with abnormal methylation modifications constructed using the STRING database is shown.

[0091] Next, we imported the results obtained from the STRING database into the Cytoscape software (http: / / www.cytoscape.org / ) and used the CytoHubba plugin to screen for core genes. We used a total of 3 algorithms and selected a total of 10 core genes by taking the intersection of the top 10 genes of each algorithm (Table 2).

[0092] Table 2 HUB genes screened for differentially expressed genes with abnormal methylation modifications by three methods

[0093]

[0094] Example 2 Verification and analysis of the diagnostic efficacy of the biomarker IGSF10 for coronary artery ectasia

[0095] Based on the results of the integrated analysis of high-throughput transcriptome data, screen IGSF10 as a candidate gene. Collect blood from patients with coronary artery ectasia and control blood (>15 cases), extract RNA samples, and use fluorescence quantitative PCR (qRT-PCR) to verify the differential expression of the candidate gene in the disease group and the control group.

[0096] Draw the ROC curve of IGSF10 for diagnosing coronary artery ectasia. As Figure 2 shown, IGSF10 shows high diagnostic efficacy in the diagnosis of coronary artery ectasia, with an AUC value of 0.829, a sensitivity of 0.864, and a specificity of 0.812, indicating that IGSF10 can perform a diagnosis with diagnostic efficacy for coronary artery ectasia.

[0097] The description of the above embodiments is only used to understand the method of the present invention and its core idea. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and modifications can be made to the present invention, and these improvements and modifications will also fall within the protection scope of the claims of the present invention.

Claims

1. Use of a diagnostic reagent in the preparation of a product for diagnosing coronary artery dilation, characterized in that, The diagnostic reagent includes a reagent capable of detecting the expression level of the IGSF10 marker in a sample; the sample is blood.

2. The application according to claim 1, characterized in that, The diagnostic reagent is selected from: an oligonucleotide probe that specifically recognizes the IGSF10 marker described in claim 1; or a primer that specifically amplifies the IGSF10 marker described in claim 1.

3. The application according to claim 1, wherein, The diagnostic reagent uses high-throughput sequencing method and / or gene chip method and / or quantitative PCR method and / or probe hybridization method to detect the expression level of the IGSF10 marker in the sample.

4. According to the application described in claim 3, the reagent used in the quantitative PCR method includes a primer that specifically amplifies the IGSF10 marker described in claim 1.

5. According to the application described in claim 3, the reagent used in the probe hybridization method includes an oligonucleotide probe that specifically recognizes the IGSF10 marker described in claim 1.

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