Application of ITGA2 mutation site rs35235 in diagnosis of coronary artery CTO

Abstract

The invention relates to the technical field of medical biological detection, provides novel application of ITGA2 gene mutation site rs35235, and particularly relates to application of ITGA2 gene mutation site rs35235 in preparation of a diagnostic reagent or a diagnostic kit for detecting coronary artery CTO (chronic complete occlusion) susceptible conditions. The invention further provides a biological preparation and a detection kit which are used for diagnosis of coronary artery CTO susceptible conditions by means of quantitative PCR (polymerase chain reaction) detection aiming at the ITGA2 gene mutation site rs35235. The kit and the detection method disclosed by the invention are simple, convenient, reliable, short in period, high in specificity and easy to clinically popularize.

Description

Technical Field

[0001] The present invention relates to the field of medical biological detection technology, and particularly to the use of the ITGA2 gene mutation site rs35235 as a molecular marker in the diagnosis of coronary CTO susceptibility, and more particularly to the use of the ITGA2 gene mutation site rs35235 in the preparation of a reagent or kit for diagnosing coronary CTO susceptibility. Background Art

[0002] Coronary CTO (Chronic Total Occlusion) is a serious cardiovascular disease, accounting for 15% to 25% of patients diagnosed with coronary heart disease by coronary angiography. CTO lesions usually occur in important branches of the coronary arteries, resulting in complete blockage of blood flow (100% occlusion) and the occlusion time is at least 3 months. [1-2] This type of lesion is the most complex and difficult to treat type of coronary artery disease. Successful recanalization of CTO vessels can significantly improve myocardial blood supply and prognosis, but recanalization is challenging and requires extremely high technical and equipment expertise. In advanced stages, CTO vessels experience prolonged occlusion, altered vessel wall structure, and reduced elasticity, leading to low success rates and poor treatment outcomes for interventional therapies (such as PCI).

[0003] China is a country with a high incidence of cardiovascular accidents. The incidence and mortality of cardiovascular diseases continue to rise, bringing a huge burden to the health of the people. According to relevant data, cardiovascular disease has become the leading cause of death among Chinese residents, accounting for more than 40% of the total number of deaths. The "China Cardiovascular Health and Disease Report 2023" shows that the number of people with cardiovascular disease in my country has reached 330 million, and 2 out of every 5 deaths are due to cardiovascular disease. [3] Therefore, predicting the risk of disease and intervening in advance will not only help patients recover, but also improve the overall health of the population, which is of great significance for promoting the individualized treatment and prevention of cardiovascular diseases in my country.

[0004] SNP (single nucleotide polymorphism) refers to DNA sequence polymorphism caused by the variation of a single nucleotide in a gene, including single base conversion, insertion and deletion. Human phenotypic differences, drug reactions and the development of diseases may be related to SNP polymorphism. [4] Different SNPs cause individuals to respond differently to medications, environmental exposures, and other factors. SNPs may be the genetic basis for varying degrees of response to drugs. Therefore, SNPs have unique value in drug screening and efficacy evaluation, and can promote the development of personalized medicine in my country.

[0005] The ITGA2 gene encodes the integrin α2 subunit, which is an important molecule for cell adhesion and signal transduction and participates in the interaction between cells and extracellular matrix. [5-6]Studies have shown that certain SNPs in the ITGA2 gene may be associated with the risk of cardiovascular disease. [7] By detecting SNPs genotypes to predict a patient's risk of developing CTO lesions, clinicians can more effectively intervene in patients and potential patients, thereby contributing to the patient's recovery and improving the overall health of the population.

[0006] Currently, there are multiple methods for detecting genetic polymorphisms. Sanger sequencing is a traditional method for detecting SNPs. During the Sanger sequencing method, the detection starts from a fixed point of the nucleotide, randomly terminates at a specific base, and performs fluorescent labeling after each base, generating a series of nucleotides of four different lengths ending with A, T, C, and G. The sequence is then detected by electrophoresis on a urea-denatured PAGE gel to obtain a visible DNA base sequence. This method has high accuracy, but it requires strong equipment support, which is difficult for general laboratories to have. Correspondingly, the principle of the fluorescent quantitative PCR method is that DNA fragments with different GC contents have different Tm values, so the base composition type of the DNA fragment containing the SNP site can be determined based on the melting curve. Compared with the Sanger method, fluorescent quantitative PCR only requires PCR for detection, and most laboratories have such conditions.

[0007] Currently, there is still a lack of a simple and accurate detection method for predicting coronary CTO. Certain SNPs in the ITGA2 gene may be associated with the risk of cardiovascular disease. By detecting SNP genotypes to predict a patient's risk of CTO lesions, clinicians can more effectively intervene in patients and potential patients, thereby contributing to patient recovery and improving the overall health of the population. If SNPs associated with coronary CTO can be identified as biomarkers and corresponding diagnostic kits developed, it will surely strongly promote the development of personalized antiplatelet therapy in my country, provide data support for clinicians' medication decisions, and improve patient compliance and long-term prognosis. Summary of the Invention

[0008] The present invention is directed to the above-mentioned problems. The first purpose is to provide a gene mutation site that causes coronary CTO, the SNP rs35235 site of the ITGA2 gene; the second purpose is to provide a use of the gene mutation site, specifically as a diagnostic marker for coronary CTO susceptibility; the third purpose is to provide a biological preparation for detecting the genotype of the SNP site rs35235 that causes coronary CTO; and the fourth purpose is to provide a detection kit for coronary CTO.

[0009] The research idea of ​​this invention is as follows: by isolating and studying single nucleotide polymorphisms in the peripheral blood DNA of patients with coronary CTO, a highly specific and sensitive SNP site that is highly correlated with coronary CTO is found - the SNP rs35235 site on the ITGA2 gene, and a coronary CTO detection kit that is easy to use in clinical practice is developed to provide data support for clinicians to judge coronary CTO, thereby accurately optimizing the patient's treatment plan.

[0010] Experiments have confirmed that the SNP rs35235 site on the ITGA2 gene is a susceptibility gene for the CC mutation type of coronary artery CTO, further confirming that the rs35235 can be used to detect coronary artery CTO, providing data support for clinicians to use drugs rationally.

[0011] In a first aspect, the present invention provides the use of a reagent for detecting the ITGA2 gene mutation site rs35235 in the preparation of a diagnostic kit for coronary CTO susceptibility. The kit is also a kit for detecting coronary CTO genotypes.

[0012] Preferably, the diagnostic kit comprises a reagent for detecting the genotype of the ITGA2 gene mutation site rs35235 in a biological sample.

[0013] The reagent for detecting the content of the ITGA2 gene mutation site rs35235 in a biological sample is selected from PCR primers that are specific for the ITGA2 gene, and detects the polymorphism of the ITGA2 gene rs35235 site. The primer sequences are shown in Table 1 below:

[0014] Table 1 Primers specific for detecting the ITGA2 gene mutation site rs35235

[0015]

[0016] Preferably, the biological sample is obtained from the peripheral blood of the subject.

[0017] The second aspect of the present invention provides a biological preparation for detecting the coronary CTO genotype, including a primer pair for detecting the ITGA2 gene, a premixed quantitative PCR reaction solution, a positive quality control product, and a negative quality control product.

[0018] The third aspect of the present invention provides a kit for detecting the coronary CTO genotype, which contains reagents for detecting the genotype of the ITGA2 gene mutation site rs35235 in a biological sample, and specifically consists of a primer system, an amplification system (premixed quantitative PCR reaction solution) and positive and negative quality control products.

[0019] In the above-mentioned biological preparations or detection kits:

[0020] The primer sequences are shown in Table 1 above, and the purification method is HPLC purification.

[0021] The premixed quantitative PCR reaction solution (2×SYBR Green) contains the buffer, magnesium ions, dNTPs, SYBR Green and other substances required for the quantitative PCR reaction and can be purchased commercially.

[0022] The method for obtaining positive quality control products is to construct and synthesize plasmids based on the sequence information of the rs776746 site published in the NCBI database to obtain homozygous DNA sequences with genotyping of CC or TT, namely CC-type positive quality control products and TT-type positive quality control products.

[0023] The negative control was DEPC-treated deionized water.

[0024] A fourth aspect of the present invention provides a method for diagnosing coronary CTO susceptibility using the above-mentioned biological detection preparation or detection kit, as follows:

[0025] A. Whole Blood DNA Extraction: Use a standardized whole blood DNA extraction kit to extract genomic DNA.

[0026] B. Real-time fluorescence quantitative PCR was used to quantitatively detect the copy number of the ITGA2 gene mutation site rs35235. The primers used in the detection process are shown in SEQ ID NOs. 1-2.

[0027] C. Interpretation of results

[0028] After the reaction is completed, the melting curves of the positive control and the sample to be tested are retrieved to interpret the results. Figure 1 ) and positive control product TT ( Figure 2 ) and other melting curve characteristics are single peak curves. The heterozygous genotype is CT, and its melting curve characteristics are double peaks ( Figure 3 ), using this as the key point of interpretation, the CT type can be interpreted by comparing with the positive quality control product ( Figure 4 、 Figure 5 ), the key points for judging CC and TT types are T m The value is greater than T of TT type m When the genotype is determined to be CC genotype, the patient is considered to have a risk of coronary CTO.

[0029] The beneficial protection and effects of the present invention are as follows:

[0030] The inventor obtained a large number of blood samples from patients who underwent percutaneous coronary intervention for chronic coronary artery occlusion from the hospital where he worked, which provided strong support for the research of the present invention.

[0031] Currently, the causes of coronary CTOs remain unclear, hindering their prediction and prevention. This study, based on extensive data, has found that the SNP rs35235 detected by this kit is highly correlated with the occurrence of coronary CTOs. Preemptive screening using the ITGA2 gene SNP rs35235 helps predict a patient's risk of CTO, providing an important reference for clinicians and enabling more effective intervention for patients and potential patients. This study provides a basis for diagnosing coronary CTOs, helping clinicians make personalized decisions, optimize treatment plans, and improve patient compliance.

[0032] Technically, testing for the rs35235 mutation in the ITGA2 gene is essentially a quantitative PCR test of the blood genome. It boasts ease of use, high sensitivity, specificity, and reproducibility, and is increasingly being used in clinical testing. Laboratories only need a common quantitative PCR instrument to perform the test, making it more convenient and efficient than Sanger sequencing, providing faster results. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is the melting curve of CC type positive quality control product;

[0034] Figure 2 is the melting curve of TT type positive quality control product;

[0035] Figure 3 is the melting curve of CT-type positive quality control product;

[0036] Figure 4 This is a comparison of the melting curves of CC and CT types, and the two have obvious differences in morphology;

[0037] Figure 5 Comparison of CT and TT melting curves. The two melting curves have different shapes and T m (TT)< T m (CT);

[0038] Figure 6 Comparison of CC and TT melting curves. m Different values, T m (TT)< T m (CC). DETAILED DESCRIPTION

[0039] The present invention will now be described in detail with reference to the embodiments and accompanying drawings, but the implementation of the present invention is not limited thereto.

[0040] The reagents and raw materials used in the present invention are all commercially available or can be prepared according to literature methods. Experimental methods in the following examples where specific conditions are not specified are generally carried out under conventional conditions or conditions recommended by the manufacturers.

[0041] Example 1 Determination of the ITGA2 gene mutation site rs35235

[0042] 1. Sample collection and sample data collation

[0043] The inventors collected clinical data from patients undergoing PCI at the First Affiliated Hospital of the Naval Medical University of the Chinese People's Liberation Army from August to December 2024 for a statistical analysis. Enrollment was closed on December 1, 2024. A total of 500 patients were initially enrolled in this study, including 264 in the experimental group and 236 in the control group. The study was approved by the Ethics Committee of Changhai Hospital (CHEC2024-320).

[0044] The study subjects met the following inclusion criteria.

[0045] Inclusion criteria for the experimental group: ① Patients who underwent PCI interventional treatment at the First Affiliated Hospital of the Naval Medical University of the Chinese People's Liberation Army from August 2024 to December 2024; ② Patients with complete occlusive lesions in at least two vessels, TIMI grade 0, confirmed by selective percutaneous coronary angiography (CAG); or occlusion of more than two vessels ≥80% and ≤99%; ③ Occlusion duration ≥3 months based on patient medical history and relevant imaging data; ④ Previous history of stent implantation and in-stent restenosis.

[0046] Inclusion criteria for the control group: ① Patients who underwent PCI interventional treatment at the First Affiliated Hospital of the Naval Medical University of the Chinese People's Liberation Army from August 2024 to December 2024; ② Negative results confirmed by selective percutaneous coronary angiography (CAG), or the degree of occlusion of one vessel was less than 20%.

[0047] Exclusion criteria: ① acute or chronic infection; ② malignant tumor; ③ autoimmune disease; ④ receiving immunosuppressive therapy; ⑤ severe liver and kidney disease; ⑥ occlusion time ≤ 3 months based on the patient's medical history and relevant imaging data.

[0048] 2. Extraction and purification of peripheral blood genomic DNA:

[0049] 1. Blood sample processing

[0050] (1) Use a blood collection tube containing EDTA anticoagulant to collect 1-2 ml of peripheral venous blood. The blood sample should be stored at 4°C for no more than one week and below -20°C for no more than three months;

[0051] (2) Add 15 μL of Proteinase K (20 mg / ml) solution to a 15 mL centrifuge tube, add 1 mL of blood sample, and mix thoroughly.

[0052] (3) Add 2.4 ml of buffer GE to the centrifuge tube containing the blood sample and shake for 30 seconds to mix;

[0053] (4) Place at 65°C for 10 minutes, shake once every 3 minutes to facilitate lysis, and briefly centrifuge to collect water droplets on the inner wall of the tube cap;

[0054] (5) Add 2 ml of anhydrous ethanol to the sample and mix well;

[0055] (6) Transfer the solution obtained in the previous step to the adsorption column CB5, centrifuge at 3000 rpm for 3 min, and discard the waste liquid;

[0056] (7) Add 2 ml of buffer GD to the adsorption column CB5, centrifuge at 5000 rpm for 1 min, and discard the waste liquid;

[0057] (8) Add 2 ml of buffer PW to the adsorption column CB5, centrifuge at 5000 rpm for 1 min, and discard the waste liquid;

[0058] (9) Add 2 ml of buffer PW to the adsorption column CB5, centrifuge at 5000 rpm for 15 min, discard the collection tube, and place the adsorption column CB5 in a new 15 ml centrifuge tube;

[0059] (10) Add 300 μL of elution buffer TB to the middle part of the adsorption membrane, let it stand at room temperature for 5 minutes, centrifuge at 5000 rpm for 2 minutes, and collect the solution in a centrifuge tube.

[0060] 2. Whole Blood DNA Extraction

[0061] Genomic DNA was extracted using the TIANamp Blood DNA Kit (TIANGEN), a standardized whole blood DNA extraction kit. It should be noted that to ensure the kit's detection rate and accuracy, the resulting genomic DNA concentration should be no less than 1 ng / μL, and the OD260 / 280 should be between 1.7 and 1.9. The recommended DNA concentration is 100-500 ng / μL.

[0062] 3. Genotyping of rs35235

[0063] (1) Obtain DNA samples from subjects;

[0064] (2) Design specific amplification primers for a single SNP;

[0065] (3) Perform PCR reaction and recover the product for Sanger sequencing;

[0066] (4) Compare the clinical data of patients diagnosed with CTO and non-CTO after coronary angiography, and compare the distribution differences of rs35235 genotypes between the two groups.

[0067] Table 2 shows the rs35235 genotype and the incidence of coronary artery occlusion in 500 patients:

[0068] Table 2 Summary of rs35235 genotypes and the incidence of coronary artery occlusion

[0069]

[0070] Statistical analysis showed that patients with CC genotype of rs35235 were more likely to suffer from coronary CTO than those with CT and TT genotypes. =17.39, =2, <0.001).

[0071] Patients with the CC genotype have a significantly higher incidence of coronary CTO than those with the TT and CT genotypes, providing an important reference for clinicians when assessing their patients' coronary CTO risk. By identifying patients with the CC genotype, physicians can better predict their susceptibility to coronary CTO, thereby optimizing treatment plans and improving patient compliance and treatment outcomes.

[0072] Example 2 CTO genotype detection

[0073] 1. Extraction of DNA from whole blood samples

[0074] The extraction steps are the same as in Example 1.

[0075] 2. Quantitative PCR reaction

[0076] 1. PCR reaction system configuration

[0077] Configure the real-time fluorescence quantitative PCR reaction system according to the standards shown in Table 3 below:

[0078] Table 3 Real-time fluorescence quantitative PCR reaction system

[0079]

[0080] 2. Real-time fluorescence quantitative PCR reaction

[0081] Real-time fluorescence quantitative PCR reaction was performed according to the following procedure

[0082] (1) Pre-denaturation conditions: 95°C, 5 minutes;

[0083] (2) The amplification stage consisted of 45 cycles with the following conditions: denaturation: 95°C, 10 seconds; annealing: 60°C, 10 seconds; extension and fluorescence signal acquisition: 72°C, 10 seconds;

[0084] (3) The final melting curve is obtained according to the program set by each instrument.

[0085] 3. Interpretation of experimental results

[0086] After the reaction is completed, the melting curves of the positive control and the sample to be tested are retrieved to interpret the results. Figure 1 ) and positive control product TT ( Figure 2 ) and other melting curve characteristics are single peak curves. The heterozygous genotype is CT, and its melting curve characteristics are double peaks ( Figure 3 ), using this as the key point of interpretation, the CT type can be interpreted by comparing with the positive quality control product ( Figure 4 、 Figure 5 ), the key points for judging CC and TT types are T m The value is greater than T of TT type m value( Figure 6 ).

[0087] This invention provides the first SNP-based kit for screening coronary CTOs. Based on the aforementioned research, the inventors have developed a novel approach to predicting coronary CTOs, using genetic sequence changes to predict a patient's risk of developing a coronary CTO. This approach provides data support for clinicians to proactively intervene and treat the condition, optimize treatment plans for coronary CTOs, and improve patient compliance and long-term prognosis.

[0088] The references cited in the background technology of the present invention are as follows:

[0089] [1]LIBBY P, THEROUX P. Pathophysiology of coronary artery disease[J]. Circulation, 2005, 111(25): 3481-8.

[0090] [2]YBARRA LF, RINFRET S, BRILAKIS ES, et al. Definitions andClinical Trial Design Principles for Coronary Artery Chronic Total OcclusionTherapies: CTO-ARC Consensus Recommendations [J]. Circulation, 2021, 143(5):479-500.

[0091] [3] China Cardiovascular Health and Disease Report Editorial Group. China Cardiovascular Health and Disease Report 2023[R]. Beijing: Science Press, 2023.

[0092] [4]SHASTRY B S. SNPs: impact on gene function and phenotype [J]. Methods Mol Biol, 2009, 578(3-22.

[0093] [5]WANG Y, FU W, XIE F, et al. Common polymorphisms in ITGA2, PON1and THBS2 are associated with coronary atherosclerosis in a candidate geneassociation study of the Chinese Han population [J]. J Hum Genet, 2010, 55(8): 490-4.

[0094] [6]CAMPBELL ID, HUMPHRIES M J. Integrin structure, activation, and interactions [J]. Cold Spring Harb Perspect Biol, 2011, 3(3):

[0095] [7]UTHAMALINGAM S, PATVARDHAN EA, SUBRAMANIAN S, et al. Utility of the neutrophil to lymphocyte ratio in predicting long-term outcomes in acutedecompensated heart failure [J]. Am J Cardiol, 2011, 107(3): 433-8.

[0096] The basic principles, main features, and advantages of the present invention are shown and described above. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The above embodiments and descriptions are merely illustrative of the principles of the present invention. Various changes and modifications may be made to the present invention without departing from the spirit and scope of the present invention. Such changes and modifications are intended to fall within the scope of the present invention. The scope of protection claimed in the present invention is defined by the appended claims and their equivalents.

Claims

1. Application of reagents for detecting the ITGA2 gene mutation site rs35235 in the preparation of diagnostic kits for detecting coronary CTO susceptibility.

2. The use according to claim 1, characterized in that The reagent for detecting the ITGA2 gene mutation site rs35235 is a reagent for detecting the genotype of the ITGA2 gene mutation site rs35235 in a biological sample.

3. The use according to claim 2, characterized in that The diagnostic kit comprises a reagent for detecting the genotype of the ITGA2 gene mutation site rs35235 in a biological sample.

4. The use according to claim 2 or 3, characterized in that The reagent for detecting the genotype of the ITGA2 gene mutation site rs35235 in a biological sample is selected from PCR primers with detection specificity for the ITGA2 gene, and the sequences of the primers are shown in SEQ ID NOs. 1-2.

5. The use according to claim 1, characterized in that The biological sample is obtained from the peripheral blood of the subject.

6. A biological agent for detecting coronary CTO susceptibility, characterized in that: The method comprises a primer pair for detecting the ITGA2 gene, a premixed quantitative PCR reaction solution, a positive quality control product, and a negative quality control product. The sequence of the primer pair is shown in SEQ ID NO. 1-2.

7. A kit for detecting a susceptibility condition of coronary artery CTO, the kit comprising a reagent for detecting the genotype of the ITGA2 gene mutation site rs35235 in a biological sample.

8. The kit according to claim 7, characterized in that The diagnostic kit is composed of a primer system, an amplification system, and positive and negative quality control products. The primer system includes PCR primers as shown in SEQ ID NO.1~2.

9. The kit according to claim 7, characterized in that The method for obtaining the positive quality control product is to construct and synthesize the plasmid according to the sequence information of the rs35235 site published in the NCBI database to obtain a DNA sequence with a genotype of TT or CC, namely a TT-type positive quality control product and a CC-type positive quality control product; the negative quality control product is DEPC-treated deionized water.

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