A non-invasive method for diagnosing coronary artery ectasia

Abstract

The present invention belongs to the field of biomedicine, and particularly relates to a method for non-invasive diagnosis of coronary artery ectasia. Specifically, the present invention provides the use of a reagent for detecting the expression level and / or methylation degree of USP18 in the preparation of a product for diagnosing coronary artery ectasia.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine, and particularly relates to a method for non-invasive diagnosis of coronary artery ectasia. Background Art

[0002] Coronary artery ectasia (CAE) is a rare but easily recognizable anatomical and morphological abnormality. Generally, it refers to the diffuse dilation of the epicardial coronary artery, exceeding 1.5 times that of the adjacent normal segment. Localized dilation exceeding 2 times is generally called coronary artery aneurysm. CAE can occur singly or multiply, and can be cystic (the transverse diameter of the aneurysm body is greater than the longitudinal diameter) or fusiform (the transverse diameter of the aneurysm body is less than the longitudinal diameter). 50% of CAE patients are complicated with coronary atherosclerosis. Simple CAE refers to those caused by unknown reasons excluding etiologies such as atherosclerosis, vasculitis, Kawasaki disease, infectious diseases, and congenital coronary artery diseases.

[0003] The etiological mechanism of CAE has not been fully clarified. The pathological manifestations are mainly the destruction of the middle layer structure of the coronary artery wall and the degradation of elastic fibers. The possible etiologies are mainly as follows: 1. Atherosclerosis. Some scholars believe that CAE is a variant of obstructive coronary artery disease. 2. Autoimmune or inflammatory reactions. CAE in children and adolescents is usually a late complication of Kawasaki disease. Connective tissue diseases such as systemic arteritis (such as polyarteritis nodosa, giant cell arteritis) and Marfan syndrome can both cause CAE. 3. Vascular infectious diseases such as fungal or septic emboli, syphilis, borreliosis, etc. 4. The etiology of simple CAE is unknown and may be related to gene susceptibility (such as special HLA class I genotypes, matrix metalloproteinase gene mutations), overexpression of angiotensin-converting enzyme, etc.

[0004] The gold standard for CAE diagnosis is coronary angiography. Coronary angiography indicates that the degree of dilation of the coronary artery lumen reaches the CAE standard, with or without coronary atherosclerotic plaques, stenosis, and thrombosis. At the same time, it is not accompanied by Kawasaki disease, systemic vasculitis (such as lupus erythematosus, Takayasu arteritis, polyarteritis nodosa, and Behcet's disease), syphilis, and complications of interventional treatments such as coronary rotational atherectomy and stent implantation. Coronary angiography has greatly improved the detection rate, and the incidence rate in the population undergoing coronary angiography is 1.2% - 9.9%. Among them, the detection rates in patients with coronary heart disease and abdominal aortic aneurysm are relatively high. The detection rate of simple CAE is 0.1% - 0.32%.

[0005] The rapid development and application of high-throughput sequencing technology have provided a more comprehensive and rapid analysis method for the study of the pathogenesis of coronary artery ectasia, and also provided new ideas for further treatment plans for coronary artery ectasia. Summary of the Invention

[0006] The present invention obtains the gene information of patients with coronary artery ectasia through sequencing, and in combination with the results of methylation analysis, provides a diagnostic target USP18 for diagnosing coronary artery ectasia. The change in the expression level of the diagnostic target USP18 in patient samples is verified by fluorescence quantitative PCR to be consistent with the data analysis results, and is further verified by the receiver operating characteristic curve, demonstrating the application value of USP18 of the present invention in diagnosis.

[0007] In a first aspect, the present invention provides the use of a reagent for detecting the expression level and / or methylation degree of USP18 in the preparation of a product for diagnosing coronary artery ectasia.

[0008] Preferably, the USP18 has a low methylation degree and a high expression level in patients with coronary artery ectasia.

[0009] Preferably, the "high" means that the expression level of the USP18 in the patient body is greater than the expression level in the healthy control group, and is at least 1.1 times higher than the control expression level. Specifically, at least 1.1 times, 1.2 times, 1.3 times, 1.4 times, 1.5 times, 1.6 times, 1.7 times, 1.8 times, 1.9 times, 2.0 times, 2.1 times, 2.2 times, 2.3 times, 2.4 times, 2.5 times, 2.6 times, 2.7 times, 2.8 times, 2.9 times, 3.0 times, 3.1 times, 3.2 times, 3.3 times, 3.4 times or 3.5 times or more.

[0010] Preferably, the "low" means that the methylation degree of the USP18 in the patient body is less than the expression level in the healthy control group, for example, about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90% of the control expression level.

[0011] Preferably, the detection is performed on a sample of the subject. The sample includes: tissue, blood, urine, saliva, semen, milk, cerebrospinal fluid, tears, sputum, mucus, lymph, cytosol, ascites, pleural effusion, amniotic fluid, bladder irrigation fluid, and bronchoalveolar lavage fluid.

[0012] Specifically, the healthy control is a group of people confirmed not to have coronary artery ectasia.

[0013] Most preferably, the sample is blood.

[0014] Preferably, the expression level includes mRNA expression level and / or protein expression level.

[0015] Preferably, the reagents for detecting the mRNA expression level include the reagents used in the following methods: PCR-based quantitative detection method, Southern hybridization, Northern hybridization, dot hybridization, fluorescence in situ hybridization (FISH), DNA microarray, ASO method, high-throughput sequencing platform.

[0016] Specifically, the reagents for detecting the mRNA expression level are, for example, specific primers and / or probes. As in the specific embodiments of the present invention, fluorescence quantitative PCR was used to quantitatively detect USP18.

[0017] Preferably, the probe can be DNA, RNA, DNA-RNA chimera, PNA or other derivatives. There is no limitation on the length of the probe, and any length is acceptable as long as it can complete specific hybridization and specifically bind to the target nucleotide sequence. As used herein, the term "hybridization" should include "the process in which a nucleic acid strand binds to a complementary strand via base pairing" and the amplification process as implemented in polymerase chain reaction technology.

[0018] When detecting mRNA, reverse transcription-polymerase chain reaction (RT-PCR) can also be used for detection; specifically, RNA extraction technology (such as using acidic phenol / isothiocyanate guanidine) or a commercial kit is used to extract RNA from the sample to be tested, reverse transcribed into DNA, and then the expression level of DNA is detected.

[0019] Preferably, the aforementioned PCR-based detection method can also be replaced by other amplification methods, such as: Strand displacement amplification (SDA), Nucleic acid sequence-based amplification (NASBA), Transcript-based amplification system (TAS), Q-beta replicase-catalyzed RNA amplification, Rolling circle amplification (RCA), Loop mediated isothermal amplification (LAMP).

[0020] Preferably, the reagents for detecting the protein expression level include the reagents used in the following methods: Western Blot, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), sandwich assay, immunohistochemical staining, mass spectrometry, immunoprecipitation analysis, complement fixation analysis, flow cytometry fluorescence side scatter technology, and protein chip.

[0021] Specifically, common reagents for detecting protein expression levels include specific antibodies. Preferably, the antibodies include intact antibody molecules, any fragment of an antibody, or an antibody with modifications. Specifically, the antibodies include chimeric antibodies, scFv, Fab, F(ab’)2, Fv, etc. As long as the fragment can retain the binding ability to the protein. The preparation of antibodies for detecting protein levels is well-known to those skilled in the art, and the present invention can use any method to prepare the antibodies.

[0022] The detection results in any of the aforementioned detection methods can be reflected by markers. Specifically, the markers include: radioactive markers, enzyme markers, chemiluminescent markers, fluorescent markers, and other suitable markers.

[0023] Preferably, the product includes a kit and a diagnostic device.

[0024] On the other hand, the present invention also provides a kit for diagnosing coronary artery ectasia, and the kit includes a reagent for detecting the expression level of USP18.

[0025] Preferably, the kit includes the aforementioned reagent for detecting mRNA expression level.

[0026] Preferably, the kit includes the aforementioned reagent for detecting protein expression level.

[0027] Preferably, the kit may further include an auxiliary detection reagent for mRNA expression level, an auxiliary detection reagent for protein expression level, and a detection instrument.

[0028] Preferably, the auxiliary detection reagent for mRNA expression level includes, but is not limited to: reaction reagents for visualizing the amplicon corresponding to the primer, such as reagents for visualizing the amplicon by agarose gel electrophoresis, enzyme-linked gel method, chemiluminescence method, in situ hybridization method, fluorescence detection method, etc.; RNA extraction reagent; reverse transcription reagent; cDNA amplification reagent; standard product for preparing a standard curve; positive control product; negative control product.

[0029] More specifically, the positive control product includes a sample taken from a patient diagnosed with coronary artery ectasia, and the negative control product includes a sample taken from a healthy control, and the healthy control is confirmed not to be a patient with coronary artery ectasia. The sample includes blood directly taken from a subject, and also includes a processed sample, such as a DNA sample obtained by RNA transcription for easy storage.

[0030] Preferably, the auxiliary detection reagent for protein expression level includes, but is not limited to: blocking solution, antibody dilution solution, washing buffer, color development termination solution, and standard product for preparing a standard curve.

[0031] Preferably, the detection device includes a real-time quantitative PCR instrument, a high-throughput sequencing platform, a detection chip, and a chip signal reader.

[0032] The suitable reagents, controls, instructions, etc. of the present invention are packaged together in a suitable container into a kit.

[0033] On the other hand, the present invention also provides the use of the aforementioned kit in the preparation of a product for diagnosing coronary artery ectasia.

[0034] On the other hand, the present invention provides a method for diagnosing coronary artery ectasia, the method comprising comparing the detection result of the expression level of USP18 and / or the detection result of the methylation degree with a threshold value, and judging whether the subject has coronary artery ectasia according to the comparison result.

[0035] More specifically, the detection result of the expression level of USP18 and the detection result of the methylation degree are obtained by detecting a sample from the subject.

[0036] Preferably, the sample is blood.

[0037] More specifically, the method of judgment is to compare the detection result of the expression level of USP18 with the threshold value. If the input expression level of USP18 is higher than the threshold value, it represents the subject is diseased; otherwise, the subject is not diseased. If the input methylation degree of USP18 is lower than the threshold value, it represents the subject is diseased; otherwise, the subject is not diseased.

[0038] The term "threshold value" in the present invention can also be referred to as a critical value, a cut-off value, a cutoff value. The threshold values obtained under different detection methods and detection reagents are different. The method for determining the threshold value is well known to those skilled in the art. For example, the method used in the present invention is to collect a large number of healthy control and patient samples, obtain the detection data, and then draw an ROC curve to obtain the threshold value under this detection method. The threshold values in the present invention include an expression level threshold value and a methylation degree threshold value.

[0039] The method for determining the "threshold value" is common in the art. The threshold values obtained by different detection procedures are different. Sometimes, the threshold values measured by reagents of different batches from the same manufacturer are also different. Therefore, in clinical applications, the threshold value needs to be adjusted at any time according to the changes in experimental reagents and detection methods.

[0040] In a specific embodiment, the detection method for obtaining the threshold value needs to be the same as the detection method for the sample of the subject.

[0041] The "coronary artery ectasia" in the present invention is coronary artery ectasia (CAE), which refers to local or diffuse dilation of the coronary artery caused by various reasons, exceeding 1.5 times or more of the diameter of the adjacent normal coronary artery.

[0042] As described in the present invention, the gold standard for CAE diagnosis is coronary angiography. Coronary angiography indicates that the degree of dilation of the coronary artery lumen reaches the CAE standard with or without coronary atherosclerotic plaques, stenosis, or thrombosis. At the same time, it is not accompanied by Kawasaki disease, systemic vasculitis (such as lupus erythematosus, Takayasu arteritis, polyarteritis nodosa, and Behçet's disease), syphilis, or individual treatment complications such as coronary rotational atherectomy and stent implantation. Brief Description of the Drawings

[0043] Figure 1 is a volcano plot obtained by differential analysis.

[0044] Figure 2 is a heat map obtained by differential analysis.

[0045] Figure 3 is a volcano plot of differentially methylated sites.

[0046] Figure 4 is a Manhattan plot of differentially methylated sites.

[0047] Figure 5 is the receiver operating characteristic curve of USP18 in the diagnosis of coronary artery dilation. Detailed Embodiments

[0048] The present invention will be further described below in conjunction with embodiments. The following description is only for the preferred embodiments of the present invention and does not limit the present invention in other forms. Any person skilled in the relevant art may use the disclosed technical content to make equivalent changes to equivalent embodiments. Any simple modification or equivalent change made to the following embodiments based on the technical essence of the present invention without departing from the content of the present invention's solution falls within the protection scope of the present invention.

[0049] Example 1: Data Analysis and Verification

[0050] Step 1: Sample collection and sequencing data processing

[0051] Sequencing was performed on the collected blood samples. The sample information involved in the present invention is shown in Table 1. Based on the Sequencing By Synthesis (SBS) technology, the cDNA library was sequenced using the Illumina HiSeq high-throughput sequencing platform, which can generate a large number of high-quality Reads. These Reads or bases produced by the sequencing platform are called raw data (Raw Data). Through the Illumina platform, a large amount of paired-end sequencing data of samples was obtained. Considering the influence of the 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. The summary results are shown in Table 2.

[0052] Table 1. Sample Information of Normal Controls and CAE Patients

[0053]

[0054]

[0055] Table 2. Statistical Table of Sequencing Data

[0056]

[0057] Note: (1) Sample: Sample name; (2) Rawreads: Number of raw reads; (3) Rawbases: Raw sequencing volume, i.e., number of bases; (4) CleanReads: Number of clean reads obtained after filtering; (5) CleanBases: Sequencing volume obtained after filtering, number of bases; (6) ValidBases: Percentage of valid bases; (7) Q30: Percentage of bases with Phred value greater than 30 in the total bases; (8) GC: Percentage of the total number of bases G and C in the total number of bases

[0058] In the transcriptome sequencing data, only the data mapped to the reference genome can be used for subsequent analysis. Therefore, the Reads mapped to the specified reference genome are called Mapped Reads.

[0059] Table 3. Statistical Results of Mapping Alignment Efficiency of Each Sample

[0060]

[0061]

[0062] Note: sample: sample name; total_reads: the number of clean reads after quality control of the sequencing data; total_map: the number of reads mapped to the genome and its percentage; read1_map: the number of read1 mapped to the reference genome and its percentage; read2_map: the number of read2 mapped to the reference genome and its percentage; splice_map: the number of reads mapped to the genome after split alignment and its percentage; unsplice_map: the number of reads mapped to the genome without split alignment and its percentage; proper_map: the number of paired read1 and read2 that are simultaneously mapped to the genome and its percentage

[0063] Using the 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 the protein-coding genes in each sample. After obtaining the counts by alignment, it is necessary to filter the protein-coding genes to remove genes with zero reads. The number of detected genes in each sample is shown in Table 4

[0064] Table 4. Partial results of the statistical display of the number of detected genes

[0065]

[0066] Differential analysis was performed on the sequencing results of the two groups of mRNAs. First, the genes were filtered according to the mean value of counts, and only genes with a mean value of counts greater than 2 were retained for the next step of analysis. DESeq2 was used to normalize the counts of each sample gene (using the BaseMean value to estimate the expression level), calculate the fold change, and perform a differential significance test using NB (negative binomial distribution test). Finally, differential protein-coding genes were screened according to the fold change and the results of the differential significance test. The screening condition for differences was p < 0.05 & |log2foldChange| > 1. 152 differentially expressed genes were obtained through analysis, including 93 up-regulated and 59 down-regulated. The volcano plot and heat map obtained from the differential analysis are as shown in Figure 1 、 2 shown

[0067] Step 2. Differential methylation analysis

[0068] Download the GSE87016 dataset from the GEO database. This dataset contains methylation data of 23 samples (NOR:CAE = 12:11). The CHAMP package was used to perform differential methylation analysis on the methylation data. The screening criteria set was P.Value < 0.05, resulting in 9377 differentially methylated sites and a total of 4318 differentially methylated genes, including 2289 hypermethylated genes and 2029 hypomethylated genes. The volcano plot and Manhattan plot of the differentially methylated sites are shown in Figure 3 , 4 as shown.

[0069] Step 3: Screen differentially expressed genes with abnormal methylation modifications

[0070] Take the intersection of the mRNA differentially expressed genes and the differentially methylated genes to obtain the differentially expressed genes regulated by abnormal methylation, resulting in 9 genes with downregulated expression due to hypermethylation modification and 11 genes with upregulated expression due to hypomethylation modification.

[0071] Step 4: Verification by fluorescence quantitative PCR

[0072] Based on the above analysis results, USP18 is hypomethylated and highly expressed in CAE patients.

[0073] Using USP18 as a candidate gene (case group vs control group), 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.

[0074] Step 5: Verification by ROC curve

[0075] The receiver operating characteristic curve is abbreviated as the ROC curve, also known as the sensitivity curve; each point on the ROC curve reflects the same sensitivity. By analyzing the measurement results of the disease group and the reference group, determine the upper and lower limits, group interval, and cut-off point of the measured values, list the cumulative frequency distribution table at the selected group interval, and calculate the true positive rate (sensitivity), specificity, and false positive rate (1 - specificity) for all cut-off points, and plot the ROC curve. Sensitivity, that is, the sensitivity, refers to the proportion of the screening method that can correctly determine the actual diseased people as patients. Specificity refers to the proportion of the screening method that can correctly determine the actual disease-free people as non-patients.

[0076] In the ROC curve obtained according to USP18, the AUC value is 0.76, the sensitivity is 0.769, and 1 - specificity is 0.25, as specifically shown in Figure 5 the following.

[0077] The technical features of the above - described embodiments can be combined arbitrarily. For the sake of brevity of description, not all possible combinations of the technical features in the above - described embodiments are described. However, as long as there is no contradiction in the combination of these technical features, it should be considered to be within the scope described in this specification.

Claims

1. Use of a reagent for detecting the expression level of USP18 in the preparation of a product for diagnosing coronary artery ectasia.

2. The use according to claim 1, wherein the detection is performed on a sample of a subject, and the sample is blood.

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

4. The use according to claim 3, wherein the reagent for detecting the mRNA expression level is a specific primer and / or probe.

5. The use according to claim 3, wherein the reagent for detecting the protein expression level includes a specific antibody.

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