Application of ADAM12 biomarker in the diagnosis of coronary artery ectasia

Abstract

The present invention discloses the application of ADAM12 biomarker in the diagnosis of coronary artery ectasia, and the present invention can more accurately judge coronary artery ectasia. Experiments have shown that by detecting the biomarker ADAM12 of the present invention, it can be judged whether the subject suffers from coronary artery ectasia, which has high practical application value.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology and relates to the application of ADAM12 biomarker in the diagnosis of coronary artery ectasia. Background Art

[0002] Coronary artery ectasia (CAE) refers to a disease in which the coronary artery is locally or diffusely dilated by more than 1.5 times the diameter of the adjacent normal coronary artery due to various reasons. It often involves multiple blood vessels, with right coronary artery dilation being more common. It is a rare coronary artery abnormality, more common in men than in women. In 2018, it was included in the "First Batch of Rare Disease Catalogs" jointly formulated by 5 departments including the National Health Commission.

[0003] The pathogenesis of coronary artery ectasia is still unclear. The degradation and loss of elastic fibers in the coronary artery media are considered to be the central link of the disease. Multiple factors such as hyperhomocysteinemia, hyperinsulinemia, Mycoplasma pneumoniae infection, and excessive exposure to nitric oxide can lead to enhanced activities of extracellular matrix metalloproteinases, intracellular cysteine proteases, and serine proteases, causing degradation of the coronary artery media and internal and external elastic membranes, resulting in the diffusion of inflammatory cells into the vascular media, promoting the proliferation and migration of local vascular smooth muscle cells and the generation of extracellular matrix, and ultimately leading to the dilated remodeling of the coronary artery.

[0004] The clinical manifestations of coronary artery ectasia are usually asymptomatic and are only accidentally discovered during a heart examination. Stable angina pectoris is the most common symptom, and malignant arrhythmias or even sudden death can also occur when spontaneous dissection forms. Due to its difficult-to-detect nature, the early diagnosis and prediction of coronary artery ectasia become extremely important. Summary of the Invention

[0005] The purpose of the present invention is to provide the application of ADAM12 biomarker in the diagnosis of coronary artery ectasia, which can more accurately judge coronary artery ectasia in view of the deficiencies of the prior art.

[0006] To achieve the above purpose, the present invention adopts the following technical solutions:

[0007] The present invention provides the application of a reagent for detecting ADAM12 biomarker in a sample in the preparation of a product for diagnosing coronary artery ectasia.

[0008] Further, the reagent is selected from: a probe that specifically recognizes the ADAM12 gene, or a primer that specifically amplifies the ADAM12 gene, or a binder that specifically binds to the protein encoded by the ADAM12 gene.

[0009] In the present invention, the binder that specifically binds to the protein encoded by the ADAM12 gene is in the form of, for example, a receptor for protein ADAM12, a lectin that binds to protein ADAM12, an antibody against protein ADAM12, a peptide antibody against protein ADAM12, a bispecific binder or a bispecific antibody. Specific examples of the specific binder are peptides, peptidomimetics, aptamers, spiegelmers, darpins, ankyrin repeat proteins, Kunitz-type domains, antibodies, single-domain antibodies, and monovalent antibody fragments.

[0010] Furthermore, the test sample includes, but is not limited to, body fluids (such as blood, plasma, serum, peripheral blood, cerebrospinal fluid, synovial fluid, urine, sweat, semen, feces, sputum, tears, mucus, amniotic fluid, etc.), exudates, bone marrow, ascites, pelvic lavage fluid, pleural fluid, spinal fluid, lymph fluid, eye fluid, extracts from nasal, laryngeal or genital swabs, cell suspensions of digestive tissues, or extracts of fecal matter, as well as tissue and organ samples from humans, animals (such as non-human mammals), and processed samples derived therefrom.

[0011] Furthermore, the test sample is peripheral blood.

[0012] Furthermore, the reagent can be formulated in a single dosage form, or alternatively, the individual reagents can be formulated separately and provided together in the form of an optional kit with its instructions for use.

[0013] Furthermore, the product includes a chip, a kit, a test strip, or a nucleic acid membrane strip.

[0014] Furthermore, the chip includes a gene chip and a protein chip. The gene chip includes oligonucleotide probes for ADAM12 gene for detecting the transcriptional level of the ADAM12 gene, and the protein chip includes specific antibodies or ligands for ADAM12 protein; the kit includes a gene detection kit and a protein detection kit. The gene detection kit includes reagents or chips for detecting the transcriptional level of the ADAM12 gene, and the protein detection kit includes reagents or chips for detecting the expression level of the ADAM12 protein; the test strip includes a gene detection test strip and a protein detection test strip.

[0015] Furthermore, the membrane strip includes a substrate and a probe that specifically recognizes ADAM12 fixed on the substrate; the substrate can be any substrate suitable for fixing oligonucleotide probes, such as nylon membrane, nitrocellulose membrane, polypropylene membrane, glass slide, silica wafer, microscale magnetic beads, etc.

[0016] Furthermore, the probe can be DNA, RNA, DNA-RNA chimera, PNA, or other derivatives.

[0017] Further, the length of the probe can be as short as 25, 20, 15, 13 or 10 base lengths. Similarly, the length of the probe can be as long as 60, 80, 100, 150, 300 base pairs or longer, even the entire gene.

[0018] Further, the length of the probe is generally at least 14 base pairs and usually does not exceed 30 base pairs, and the length complementary to the target nucleotide sequence is optimally 15 - 25 base pairs.

[0019] Further, the self - complementary sequence of the probe is preferably less than 4 base pairs to avoid affecting the hybridization efficiency.

[0020] Further, the kit includes reagents for detecting the expression level of ADAM12 gene or protein by Western blotting, ELISA, radioimmunoassay, immunodiffusion, Ouchterlony immunodiffusion, rocket electrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS, protein chip detection method.

[0021] Further, the kit includes reagents for detecting ADAM12 gene or protein, and one or more substances selected from the following group: containers, instruction manuals, positive controls, negative controls, buffers, adjuvants or solvents.

[0022] The kit of the present invention may also be accompanied by an instruction manual for using the kit, which describes how to perform the detection using the kit and how to use the detection results to judge the tumor development and select the treatment plan.

[0023] In certain embodiments, provided herein is a kit for detecting the mRNA level of a biomarker. In certain embodiments, the kit contains one or more probes that specifically bind to the mRNA of a biomarker. In certain embodiments, the kit further contains a washing solution. In certain embodiments, the kit further contains reagents for performing hybridization assays, mRNA isolation or purification tools, detection tools, and positive and negative controls. In certain embodiments, the kit further contains an instruction manual for using the kit. The kit can be customized for home use, clinical use, or research use.

[0024] The present invention also provides a product for in vitro detecting the expression level of ADAM12 in a sample, and the product includes reagents capable of analyzing and detecting the expression level of the biomarker described above.

[0025] Further, the sample includes but is not limited to body fluids (such as blood, plasma, serum, peripheral blood, cerebrospinal fluid, synovial fluid, urine, sweat, semen, feces, sputum, tears, mucus, amniotic fluid, etc.), exudates, bone marrow, ascites, pelvic lavage fluid, pleural fluid, spinal fluid, lymph fluid, eye fluid, extracts from nasal, throat or genital swabs, cell suspensions of digestive tissues, or extracts of fecal matter, as well as tissue and organ samples from humans, animals (such as non-human mammals), and processed samples derived therefrom.

[0026] In the context of the present invention, the term "sample" refers to a composition obtained from or derived from a subject, which contains cells and / or other molecular entities to be characterized and / or identified according to, for example, physical, biochemical, chemical, and / or physiological characteristics. For example, a sample refers to any sample derived from a subject that is expected or known to contain cells and / or molecular entities to be characterized.

[0027] Further, the product includes a chip, a kit, a test strip, or a nucleic acid membrane strip.

[0028] Further, the chip includes a gene chip and a protein chip.

[0029] Further, the gene chip includes oligonucleotide probes specific for the ADAM12 gene for detecting the transcription level of the ADAM12 gene.

[0030] Further, the probe can be DNA, RNA, a DNA-RNA chimera, PNA, or other derivatives.

[0031] Further, the length of the probe can be as short as 25, 20, 15, 13, or 10 base lengths. Similarly, the length of the probe can be as long as 60, 80, 100, 150, 300 base pairs or longer, even the entire gene.

[0032] Further, the length of the probe is usually at least 14 base pairs, and generally does not exceed 30 base pairs at most. The length complementary to the target nucleotide sequence is optimally 15 - 25 base pairs.

[0033] Further, the self-complementary sequence of the probe is preferably less than 4 base pairs to avoid affecting the hybridization efficiency.

[0034] Further, the protein chip includes specific antibodies or ligands of the ADAM12 protein.

[0035] Further, the kit includes a gene detection kit and a protein detection kit.

[0036] Further, the gene detection kit includes reagents or chips for detecting the transcription level of the ADAM12 gene.

[0037] Furthermore, the protein detection kit includes reagents or chips for detecting the expression level of ADAM12 protein.

[0038] Furthermore, the test strip includes a gene detection test strip and a protein detection test strip.

[0039] Furthermore, the kit includes reagents for detecting the expression level of ADAM12 gene or protein by Western blotting, ELISA, radioimmunoassay, immunodiffusion, Ouchterlony immunodiffusion, rocket electrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS, protein chip detection method.

[0040] In the present invention, those skilled in the art determine the expression level of biomarkers by known assays, and the specific assays include multi - analyte profiling test, enzyme - linked immunosorbent assay (ELISA), radioimmunoassay, Western blotting assay, immunofluorescence assay, enzyme immunoassay, immunoprecipitation assay, chemiluminescence assay, immunohistochemical assay, dot blot assay or slot blot assay.

[0041] The present invention also provides the use of the aforementioned product in the preparation of a tool for diagnosing coronary artery ectasia.

[0042] The present invention also provides the use of ADAM12 in the construction of a computational model for predicting coronary artery ectasia.

[0043] In certain embodiments, as used herein, "subject" refers to a mammal, such as a rodent, feline, canine, and primate. Specifically, the subject according to the present invention is a human.

[0044] The gene ID of ADAM12 is 8038. In the present invention, ADAM12 includes wild - type, mutant or fragments thereof. In general sequencing analysis in the art, the original sequencing results are aligned to the human reference genome. Therefore, the ADAM12 in the screening results may contain different transcripts, as long as it can be aligned to ADAM12 (gene ID: 8038) on the reference genome. There are multiple subtypes of ADAM12 that have been publicly reported.

[0045] In the context of the present invention, the term "biomarker" refers to a gene that is differentially present (i.e., increased or decreased) in a biological sample from a subject or group of subjects having a first phenotype (e.g., having a disease) compared to a biological sample from a subject or group of subjects having a second phenotype (e.g., not having the disease). The term generally refers to the concentration or amount of a gene or the concentration or amount of two or more genes.

[0046] The term "probe" refers to a nucleic acid oligomer or aptamer that specifically hybridizes to a target sequence in a nucleic acid or its complement under conditions that promote hybridization, thereby allowing detection of the target sequence or its amplified nucleic acid. Detection can be direct (i.e., produced by a probe that hybridizes directly to the target or amplified sequence) or indirect (i.e., produced by a probe that hybridizes to an intermediate molecular structure that links the probe and the target or amplified sequence).

[0047] Advantages and beneficial effects of the present invention:

[0048] The present invention has for the first time discovered a new molecular marker for coronary artery ectasia - the ADAM12 gene. By detecting the expression level of ADAM12 in the peripheral blood of a subject, it is possible to determine whether the subject has coronary artery ectasia, thereby guiding clinicians to optimize the prevention and treatment plans for the subject; at the same time, using molecular markers to achieve the diagnosis and treatment of diseases has higher specificity and sensitivity compared to traditional means. BRIEF DESCRIPTION OF THE DRAWINGS

[0049] Figure 1 is a protein - protein interaction network diagram;

[0050] Figure 2 is a ROC curve graph for ADAM12 in diagnosing coronary artery ectasia. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0051] The present invention will be further described in detail below with reference to the drawings and embodiments. The following embodiments are only used to illustrate the present invention and are not used to limit the scope of the present invention. For experimental methods without specific conditions noted in the embodiments, they are generally carried out under conventional conditions or according to the conditions recommended by the manufacturer.

[0052] In practice, the receiver operating characteristic curve (ROC curve) is typically calculated by plotting the variable values against their relative frequencies in "normal" (e.g., apparently healthy individuals without prenatal disorders or conditions) and "disease" populations (similarly, e.g., two different states such as viral and bacterial RTIs or typical and atypical pneumonias). For any particular marker, the distributions of marker levels in subjects with and without the disease / condition may overlap. Under such conditions, the test cannot absolutely distinguish normal from disease with 100% accuracy, and the overlapping region may indicate where the test fails to distinguish normal from disease. A threshold is selected such that tests below the threshold are considered "abnormal", tests above the threshold are considered "normal", or tests below or above the threshold indicate a particular condition. The area under the ROC curve (AUC) is a measure of the probability that a perceptual measurement will allow correct identification of the condition. Even if the test results do not necessarily provide an accurate number, the ROC curve can be used. As long as the results can be ranked, an ROC curve can be created.

[0053] The horizontal axis of the ROC curve represents (1 - specificity), which increases as the false positive rate increases. The vertical axis of the curve represents sensitivity, which increases as the true positive rate increases. Thus, for a selected specific cut-off value, the value of (1 - specificity) can be determined, and the corresponding sensitivity can be obtained. The area under the ROC curve is a measure of the probability that the measured biomarker level allows for the correct identification of a disease or condition. Thus, the area under the ROC curve (AUC) can be used to determine the effectiveness of a test.

[0054] As outlined above, in addition to the specific biomarker or panel of biomarkers to be detected in aspects of the present invention, other parameters can be considered for a specific diagnosis or differential diagnosis. As used herein, a parameter is a characteristic, feature, or measurable factor that can help define a specific system. Parameters are important elements in health and physiological related assessments, such as disease / disorder / clinical condition risk. In addition, a parameter is defined as a characteristic of an objective measurement and evaluated as an indicator of a normal biological process, a pathogenic process, or a pharmacological response to a therapeutic intervention.

[0055] Example 1 Screening of Biomarkers Related to Coronary Artery Ectasia

[0056] 1. Screening Method

[0057] (1) Data Used for Screening and Preprocessing

[0058] To screen for biomarkers that can be used for the diagnosis of coronary artery ectasia, public gene expression data related to coronary artery ectasia were downloaded from the Gene Expression Omnibus (GEO) database. Dataset GSE87016 was downloaded from the GEO database (http: / / www.ncbi.nlm.nih.gov / geo / ).

[0059] The data downloaded from the Gene Expression Omnibus (GEO) database also needed to be further processed:

[0060] 1) The selected dataset must be genome-wide DNA methylation data;

[0061] 2) These data are from coronary artery ectasia and control blood samples;

[0062] 3) Both standardized or raw datasets were considered in this study.

[0063] (2) High-Throughput Transcriptome Data and Preprocessing

[0064] Through the Illumina platform, a large amount of paired-end sequencing data of samples was obtained. Given the impact of data error rate on the results, the 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.

[0065] The specific steps and sequence are as follows:

[0066] (1) Remove the adaptor;

[0067] (2) Remove low-quality reads;

[0068] (3) Remove low-quality bases from the 3' end and 5' end in different ways;

[0069] (4) Count the original sequencing volume, effective sequencing volume, Q30, GC content, and conduct a comprehensive evaluation.

[0070] Count the data volume of the sequences after data quality control.

[0071] (3) Analysis of mRNA gene expression levels

[0072] Use the known reference gene sequences and annotation files as databases, and adopt the method of sequence similarity alignment to identify the expression abundances of each protein-coding gene in each sample. Use the htseq-count software to obtain the number of reads mapped to the protein-coding genes in each sample. After obtaining the counts by alignment, it is necessary to filter the protein-coding genes and remove the genes with zero reads. The number of detected genes in each sample is shown in Table 1. Part of the results of the statistical number of detected genes in Table 1 are presented

[0073]

[0074] The FPKM method can eliminate the influence of protein-coding gene length and sequencing volume differences on calculating the expression of protein-coding genes. The calculated gene expression levels reflect high or low expression.

[0075] (4) Differential analysis of mRNA

[0076] First, filter the genes according to the mean value of counts, and only retain the genes with a mean value of counts greater than 2 for the next step of analysis. Use DESeq2 to standardize the counts of genes in each sample (using the BaseMean value to estimate the expression level), calculate the fold change, and conduct a differential significance test using NB (negative binomial distribution test). Finally, screen the differentially expressed protein-coding genes according to the fold change and the results of the differential significance test. The screening condition is p < 0.05 & |log2foldChange| > 1.

[0077] (5) Differential methylation analysis

[0078] 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 screening criterion set is P.Value < 0.05.

[0079] (6) Protein - protein interaction analysis of differentially expressed genes with abnormal methylation modifications

[0080] To explore the protein - protein interaction relationships among the screened differentially expressed genes with abnormal methylation modifications, we constructed a PPI network of the 20 screened differentially expressed genes with abnormal methylation modifications using the online database STRING.

[0081] 2. Results

[0082] Use DESeq2 to standardize the counts of genes in each sample (estimate the expression level using the BaseMean value), calculate the fold change, and perform differential significance testing using NB (negative binomial distribution test). Finally, screen for differentially expressed protein - coding genes based on the fold change and differential significance test results. Through the mRNA analysis of the high - throughput sequencing transcriptome, 152 differentially expressed genes were obtained, including 93 up - regulated and 59 down - regulated genes.

[0083] 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 screening criterion set is P.Value < 0.05, obtaining 9377 differentially methylated sites and a total of 4318 differentially methylated genes, including 2289 hypermethylated genes and 2029 hypomethylated genes.

[0084] Take the intersection of the mRNA differentially expressed genes and the differentially methylated genes to obtain the differentially expressed genes regulated by abnormal methylation, obtaining 9 genes with down - regulated expression due to hypermethylation modification and 11 genes with up - regulated expression due to hypomethylation modification.

[0085] To explore the protein - protein interaction relationships among the screened differentially expressed genes with abnormal methylation modifications, we constructed a PPI network of the 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.

[0086] 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 adopted a total of 3 algorithms, and after taking the intersection of the top 10 genes of each algorithm, a total of 10 core genes were screened out (Table 2).

[0087] Table 2 HUB genes for screening abnormally methylated differentially expressed genes by 3 methods

[0088]

[0089] Example 2 Verification and analysis of the diagnostic efficacy of the biomarker ADAM12 for coronary artery ectasia

[0090] Based on the results of the integrated analysis of high-throughput transcriptome data, ADAM12 was screened as a candidate gene. Blood samples from patients with coronary artery ectasia and control blood samples (>15 cases) were collected, RNA samples were extracted, and the differential expression of the candidate gene in the disease group and the control group was verified using fluorescence quantitative PCR (qRT-PCR).

[0091] The ROC curve for ADAM12 in diagnosing coronary artery ectasia was plotted. As Figure 2 shown, ADAM12 showed relatively high diagnostic efficacy in the diagnosis of coronary artery ectasia, with an AUC value of 0.752, a sensitivity of 0.692, and a specificity of 0.812, indicating that ADAM12 can perform a diagnostic test with diagnostic efficacy for coronary artery ectasia.

[0092] The description of the above embodiments is only for understanding the method and its core idea of the present invention. 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 reagent for detecting the ADAM12 biomarker in a sample in the preparation of a product for diagnosing coronary artery ectasia; the test sample is blood, plasma, serum, or peripheral blood.

2. The use according to claim 1, wherein the test sample is peripheral blood.

3. The use according to claim 1, characterized in that: The reagent is selected from: a probe that specifically recognizes the ADAM12 gene, or a primer that specifically amplifies the ADAM12 gene, or a binding agent that specifically binds to the protein encoded by the ADAM12 gene.

4. The use according to claim 1, characterized in that: The products include chips, test kits, test strips or nucleic acid membrane strips.

5. The use according to claim 4, characterized in that: The chip includes a gene chip and a protein chip. The gene chip includes an oligonucleotide probe targeting the ADAM12 gene for detecting the transcription level of the ADAM12 gene, and the protein chip includes a specific antibody or ligand for the ADAM12 protein; the kit includes a gene detection kit and a protein detection kit. The gene detection kit includes a reagent or chip for detecting the transcription level of the ADAM12 gene, and the protein detection kit includes a reagent or chip for detecting the expression level of the ADAM12 protein; the test paper includes a gene detection test paper and a protein detection test paper.

6. The use according to claim 4, characterized in that: The kit includes reagents for detecting the expression level of ADAM12 gene or protein by western blotting, ELISA, radioimmunoassay, radioimmunodiffusion, octotron immunodiffusion, rocket electrophoresis, tissue immunostaining, immunoprecipitation assay, complement fixation assay, FACS, and protein chip detection.

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