CN115386630A

CN115386630A - SNP marker for predicting thrombus treatment effect of antithrombotic drug and application thereof

Info

Publication number: CN115386630A
Application number: CN202211352613.2A
Authority: CN
Inventors: 崔一民; 刘志艳; 向倩; 王哲; 母光妍; 周双; 张涵煦
Original assignee: Peking University First Hospital
Current assignee: Peking University First Hospital
Priority date: 2022-10-31
Filing date: 2022-10-31
Publication date: 2022-11-25
Anticipated expiration: 2042-10-31
Also published as: CN115386630B

Abstract

The invention discloses an SNP marker for predicting the curative effect of antithrombotic drugs on thrombus and application thereof. The invention specifically relates to SNP locus rs10145032 of AHNAK 2. The invention belongs to the field of biological medicine, and provides a method for predicting the curative effect of an antithrombotic drug on thrombus in order to predict the curative effect of the antithrombotic drug on thrombus treatment. The invention has the beneficial effects that: the genotype of the SNP locus rs10145032 is found to be related to the curative effect of the antithrombotic drug on treating thrombus for the first time, and the curative effect of the antithrombotic drug on treating thrombus can be judged by detecting the genotype of the rs10145032.

Description

SNP marker for predicting thrombus treatment effect of antithrombotic drug and application thereof

Technical Field

The invention belongs to the field of biological medicine, and relates to an SNP (single nucleotide polymorphism) site for predicting the curative effect of antithrombotic treatment on thrombus and application thereof, wherein the SNP marker is rs10145032.

Background

The incidence and mortality of thrombotic disease dominate worldwide. The myocardial infarction caused by coronary artery thrombosis and cerebral infarction caused by cerebral embolism are clinically common ischemic stroke, cardiovascular diseases such as deep vein thrombosis, pulmonary embolism and the like are also important pathogenic factors of the thrombosis, the antithrombotic treatment is taken as a core rescue measure of the above-mentioned diseases, and the anticoagulation is an important component of the antithrombotic treatment.

In the therapy and prevention of thromboembolic diseases, various anticoagulant drugs are used, but anticoagulant drugs often present a high risk of bleeding, including cerebral hemorrhage, gastrointestinal bleeding, thrombocytopenia, drug induced alopecia or osteoporosis.

Oral anticoagulant drugs such as rivaroxaban are widely used in the treatment of patients at high risk of thrombosis, but rivaroxaban is still associated with increased risk of bleeding events in clinical applications. In the past, the probability of bleeding events per year of rivaroxaban patients is about 20%, and the patients are at risk of hospitalization and death. Therefore, the research on some pharmacogenomics is beneficial to the clinical individualized medication of the thrombus patients, and the life health of the thrombus patients is effectively guaranteed.

Disclosure of Invention

In view of the above-mentioned shortcomings of the prior art, the present invention aims to provide an SNP marker related to the prediction of thrombus treatment efficacy of antithrombotic drugs, so as to more accurately and comprehensively evaluate the drug efficacy of an individual after using antithrombotic drugs, reduce adverse drug reactions, and realize individuation of clinical medication.

One of the purposes of the invention is to provide a product for predicting the curative effect of antithrombotic drugs on thrombus, wherein the product comprises a reagent for detecting the genotype of the SNP locus rs10145032 in a sample.

Further, the SNP site rs10145032 is the SNP site rs10145032 of the AHNAK2 gene.

The term "SNP" (single nucleotide polymorphism) refers to a DNA sequence polymorphism caused by a variation of a single nucleotide at the genomic level. SNPs are the most common of the human heritable variations, accounting for over 90% of all known polymorphisms. SNPs are widely present in the human genome, with an average of 1 per 300 base pairs. A SNP is a two-state marker, which is caused by a transition or transversion of a single base, and may also be caused by an insertion or deletion of a base. SNPs may be in either the gene sequence or non-coding sequences outside the gene. The naming mode of the SNP locus is named in an 'rs-' mode, and the position, the nucleotide sequence and the like can be accurately found in a database and a related information system through the naming mode.

The term "treatment" refers to the administration of a compound or composition to control the progression of a disease. Control of disease progression is understood to achieve beneficial or desired clinical results, including but not limited to alleviation of symptoms, reduction of disease duration, stabilization of the pathological state (in particular avoidance of additional exacerbations), delay of disease progression, amelioration and remission (both partial and total) of the pathological state. Control of disease progression also involves prolongation of survival compared to expected survival without treatment.

Further, the antithrombotic drug comprises one or more of thrombolytic drugs, anticoagulant drugs and antiplatelet drugs.

Further, the thrombolytic drug comprises urokinase, alteplase, reteplase, or streptokinase.

In the present invention, the term "thrombolytic drug" refers to a thrombolytic drug comprising plasminogen, plasmin, activated proenzyme and inhibitor. The principle of thrombolytic drugs is to dissolve the thrombus by promoting fibrinolysis, where thrombolytic enzymes are direct activators of proteolytic or plasminogen. The fibrinolysis process in vivo is a series of protease catalyzed reactions, firstly, an in vivo activator is activated and converted into an activator of plasminogen; after the treatment of the activator, the plasminogen is converted into plasmin, and finally the plasmin acts on fibrin to dissolve the plasmin.

Further, the anticoagulant comprises heparin, warfarin, fondaparinux sodium, rivaroxaban, apixaban, idoxaban, argatroban, or dabigatran etexilate.

In the present invention, the term "anticoagulant" is also called anticoagulant, and mainly used for preventing and treating an intravascular embolism or thrombosis disease, and preventing stroke or other thrombotic diseases. Anticoagulants are agents that block the clotting process by affecting certain coagulation factors in the clotting process.

Further, the antiplatelet agent comprises one or more of platelet glycoprotein, phosphodiesterase inhibitor, thromboxane A2 inhibitor, adenosine diphosphate P2Y12 receptor antagonist, thrombin receptor antagonist and 5-hydroxytryptamine receptor antagonist.

In the present invention, the term "antiplatelet agent" refers to an agent for inhibiting the growth of cyclooxygenase of platelets. Antiplatelet drugs are mainly classified into two major classes, namely platelet aggregation inhibiting drugs and platelet activation and amplification affecting drugs. Drugs that inhibit platelet aggregation include platelet glycoprotein, phosphodiesterase inhibitors; the medicines influencing the activation and amplification of the blood platelets mainly comprise four types of thromboxane A2 inhibitors, adenosine diphosphate P2Y12 receptor antagonists, thrombin receptor antagonists and 5-hydroxytryptamine receptor antagonists.

Further, the platelet glycoprotein comprises aximab or tirofiban.

Further, the phosphodiesterase inhibitor comprises dipyridamole or cilostazol.

Further, the thromboxane A2 inhibitor comprises aspirin.

Further, the adenosine diphosphate P2Y12 receptor antagonists include thiophenic pyridines (e.g., ticlopidine, clopidogrel, or prasugrel) or non-thiophenic pyridines (e.g., ticagrelor, cangrelor, or ticagrelor).

Further, the thrombin receptor antagonists include Vorapaxar (SCH-530348) or Atopaxar (E5555).

Further, the 5-hydroxytryptamine receptor antagonist comprises sarpogrelate or citalopram.

Further, the thrombus includes one or more of a white thrombus such as a continuation thrombus, a mixed thrombus, a red thrombus, a transparent thrombus.

Further, the mixed thrombus includes a red blood cell-based thrombus, a spherical thrombus or a layered thrombus.

Further, the product comprises one or more of a kit, a chip, a test paper, a high-throughput sequencing and a system.

Further, the agent comprises a nucleic acid ligand having affinity for rs10145032.

Further, the affinity nucleic acid ligand comprises RNA, DNA, PNA, CAN, HNA, LNA or ANA.

Further, the nucleic acid ligand comprises a primer or a probe.

Further, the primer includes 18 or more nucleotides, and 50 or less nucleotides.

Further, the primers or probes may be chemically synthesized using a solid phase support of phosphoryl imine or other well known methods.

Further, the primers or probes modify the nucleic acid sequence using a number of means known in the art.

Further, the plurality of means modifications include methylation, capping, substitution with one or more analogs of a natural nucleotide, or modification between nucleotides.

Further, modifications between the nucleotides include modification of uncharged linkers such as methyl phosphates, phosphotriesters, phosphoimides, carbamates, or modification of charged linkers such as phosphorothioates, phosphorodithioates.

Further, the probes include fragments of any length that can accomplish specific hybridization, specifically binding to the nucleic acid sequence of interest.

Further, the type of the probe includes RNA, DNA, PNA, CAN, HNA, LNA, ANA or other derivatives.

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

In the present invention, the term "probe" refers to a molecule that is capable of binding to a specific sequence or subsequence or other portion of another molecule. Unless otherwise indicated, the term "probe" generally refers to a polynucleotide probe that is capable of binding to another polynucleotide (often referred to as a "target polynucleotide") by complementary base pairing. Depending on the stringency of the hybridization conditions, a probe can bind to a target polynucleotide that lacks complete sequence complementarity to the probe. The probe may be directly or indirectly labeled, and includes within its scope a primer. Hybridization modalities, including but not limited to: solution phase, solid phase, mixed phase or in situ hybridization assays.

The probe is typically labeled directly, e.g., with an isotope, chromophore, luminophore, chromogen, or indirectly, e.g., with biotin to which the streptavidin complex can subsequently bind. Thus, the detectable label used in the assays of the invention may be a primary label (wherein the label comprises a directly detectable element or an element capable of producing a directly detectable element) or a secondary label (wherein a detectable label is associated with a primary label, e.g., as commonly used in immunolabeling). Typically, a labeled signal nucleic acid is used to detect hybridization. The complementary nucleic acid or signal nucleic acid can be labeled by any of several methods commonly used to detect the presence of hybridized polynucleotides. The most common detection method is to use ³ H、 ¹²⁵ I、 ³⁵ S、 ¹⁴ C or ³² Autoradiography of P-labeled probes and the like. Other labels include, for example, ligands that bind to the labeled antibody, fluorophores, chemiluminescent agents, enzymes, and antibodies that are specific binding pair members capable of acting as labeled ligands.

The size of the polynucleotide used as a probe is preferably 18 or more nucleotides, more preferably 20 or more nucleotides, and the entire length of the transcribed region or less. When used as a primer, the polynucleotide is preferably 18 or more nucleotides in size, and 50 or less nucleotides in size.

Further, the product includes a reagent for processing the sample.

In the context of the present invention, the term "sample" as used refers to a composition obtained or derived from a subject comprising cells and/or other molecular entities to be characterized and/or identified, for example, according to 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 the cellular and/or molecular entities to be characterized.

Further, the sample includes, but is not limited to, one of a tissue sample (e.g., a tumor tissue sample), primary or cultured cells or cell lines, cell supernatant, cell lysate, platelets, serum, plasma, vitreous humor, lymph fluid, synovial fluid, follicular fluid, semen, amniotic fluid, milk, whole blood, blood-derived cells, urine, cerebrospinal fluid, saliva, sputum, tears, sweat, mucus, tumor lysate, tissue culture fluid, tissue extract, homogenized tissue, tumor tissue, cell extract, or a combination thereof.

Further, the sample is blood.

Further, the product also comprises a product instruction which records the prediction step of the thrombus curative effect of the antithrombotic drug, and the prediction step comprises the following steps:

1) Contacting nucleic acid from the sample with an agent that detects the rs10145032 genotype;

2) Determining the genotype of rs 10145032;

3) Predicting the efficacy of the subject for treating thrombus with an antithrombotic agent based on the genotype.

Further, when the genotype of rs10145032 is GG, the drug effect of the subject is poor after the subject takes the antithrombotic drug; when the genotype of rs10145032 is GC, the drug effect of the tested subject is better after the tested subject takes the antithrombotic drug; when the genotype of rs10145032 is CC, the test subject has better drug effect and increased bleeding event probability after taking the antithrombotic drug.

The invention also aims to provide application of a reagent for detecting the SNP marker in a sample in preparing a product for predicting the curative effect of the antithrombotic drug on thrombus, wherein the SNP marker comprises the SNP locus rs10145032 of the AHNAK2 gene.

Further, the anticoagulant comprises heparin, warfarin, argatroban, fondaparinux sodium, rivaroxaban, apixaban, idoxaban, or dabigatran etexilate.

Further, the antiplatelet agent comprises one or more of a thromboxane A2 inhibitor, an adenosine diphosphate P2Y12 receptor antagonist, a thrombin receptor antagonist, a 5-hydroxytryptamine receptor antagonist, a platelet glycoprotein and a phosphodiesterase inhibitor.

Further, the platelet glycoprotein comprises axizumab or tirofiban.

Further, the phosphodiesterase inhibitor comprises dipyridamole or cilostazol.

Further, the thromboxane A2 inhibitor comprises aspirin.

Further, when the genotype of rs10145032 is GG, the drug effect of the subject is poor after the subject takes the antithrombotic drug; when the rs10145032 genotype is GC, the drug effect of the subject is better after the subject takes the antithrombotic drug; when the genotype of rs10145032 is CC, the test subject has better drug effect and increased bleeding event probability after taking the antithrombotic drug.

Further, the reagents include reagents for detecting the rs10145032 genotype by direct sequencing, single base extension, allele-specific probe hybridization, allele-specific primer extension, allele-specific amplification, allele-specific nucleotide incorporation, 5' nuclease digestion, molecular beacon assay, oligonucleotide ligation assay, size analysis, and single-stranded conformation polymorphism methods.

Further, the sample is blood.

The invention has the advantages and beneficial effects that:

the invention discovers that the genotype of the SNP locus rs10145032 is related to the curative effect of the antithrombotic drug on thrombus for the first time, and the curative effect of the antithrombotic drug on thrombus can be judged by detecting the genotype of the rs10145032.

Drawings

FIG. 1 is a study design flow diagram;

FIG. 2 is a graph showing the trend of the effect of the AHNAK2 gene polymorphism on the ratio of prothrombin time;

FIG. 3 is a ROC plot of the ratio of AHNAK2 gene polymorphism to prothrombin time prediction;

FIG. 4 is a graph showing the trend of the effect of AHNAK2 gene polymorphism on 1-month bleeding;

FIG. 5 is a ROC plot of AHNAK2 gene polymorphism versus bleeding event prediction.

Detailed Description

The embodiments of the present invention are described below with reference to specific embodiments, and those skilled in the art can easily understand the advantages and functions of the present invention from the disclosure of the present invention. The invention is capable of other and different embodiments and of being practiced or of being carried out in various ways, and its several details are capable of modification in various respects, all without departing from the spirit and scope of the present invention.

The present invention will be described in further detail with reference to the drawings and examples. The following examples are intended to illustrate the invention only and are not intended to limit the scope of the invention. The experimental methods in the examples, in which specific conditions are not specified, are generally carried out under conventional conditions.

Example 1 analysis of pharmacodynamic, pharmacokinetic and biological indicator results after rivaroxaban administration in healthy persons

1. Study subject and sample Collection

Based on the national multicenter clinical bioequivalence test, the research center comprises the first hospital of Beijing university, beijing Huilongguan hospital, liaoning traditional Chinese medicine university subsidiary hospital, qingdao university subsidiary hospital, and the first subsidiary hospital of Nanchang university. The study considers that the health index is 18-26 kg/m and the age is 18-45 years ² Chinese healthy subjects in between. All subjects did not take any medication for at least 4 weeks prior to study initiation. Healthy subjects entered the study the day before dosing, and they were randomly assigned to test or reference groups with no significant difference between groups (p)<0.05 Results are shown in table 1. Apart from the age of the rivaroxaban 15 mg dose group and the 10 mg dose group, there was no difference in the demographic characteristics of the healthy subjects in each dose group.

The subject received a single dose of rivaroxaban, including 10, 15, and 20 mg, under fasting and postprandial conditions. All subjects were discharged from the hospital 48 h or 72 h after rivaroxaban administration. Figure 1 shows the study design of rivaroxaban in healthy volunteers.

The study was conducted according to the clinical practice guidelines and the declaration of Helsinki, with protocols approved by the independent ethics Committee and the first Hospital of Beijing university and all of the institutional review Board of Suntral Hospital participating in the study (lot No.: 2016[1235 ]). The Clinical trial was registered in Clinical Trials under accession number NCT03161496. Before the study began, all subjects were briefly introduced for the purpose, duration and potential risk of the study, and provided written informed consent.

2. Research method

2.1 Blood sample collection

The detection and sampling time of Pharmacodynamic (PD) parameters is 0, 3, 8 and 12 h after administration. Blood samples were collected in 2.7 mL sodium citrate (3.2% v/v) tubes and centrifuged at 2500 g for 15 min at room temperature within 60 min of sampling. Plasma samples were transferred to-70 ℃ for cryopreservation within 6 months after sampling, and were ready for assay.

2.2 PD analysis method

PD parameters (activated partial thromboplastin time (APTT) and Prothrombin Time (PT)) were measured using a Sysmex CS-2100i fully automatic multiparameter hemostasis analyzer (Sysmex, japan). PT and APTT are measured by adopting a verified blood coagulation method detection kit (Thromborel-S and Actin, siemens medical diagnosis products Co., ltd., germany).

2.3 Gene detection analysis method

Extracting genome DNA from peripheral blood by salting-out method, and sequencing with whole exome. Sequencing libraries were constructed by modification of the KAPA library preparation kit (KAPA Biosystems inc., usa). 1. Mu.g of genomic DNA was cut to an average fragment size of 200 bp using Bioruptor (Diagenode, lie' ge, belgium). Fragments were purified using amprexp beads (Beckman Coulter inc., usa). Sequencing libraries were subjected to minimal PCR cycles and quantified using a qubit 2.0 fluorimeter (Thermo Fisher Scientific, USA). The sequencing libraries were pooled into pools for solution phase hybridization using Roche NimbleGen SeqCap EZ exome amplification kit V3 (Roche NimbleGen, inc. The captured sequencing library was analyzed using an agilent 2100 bioanalyzer (agilent technologies, usa) and DNA concentration was measured using a qubit 2.0 fluorometer (Thermo Fisher Scientific, usa) followed by sequencing sent to generate 2 × 150 bp end-to-end reads using the HiSeq 3000 platform (Illumina, inc., usa). In addition to phenotypic differences, the quality control step also found low read depth and coverage, gender mismatches, potential genetic relationships and population stratification.

For the common single nucleotide polymorphism (MAF)>0.01 Using an additive genetic model, performing primary single nucleotide polymorphism association analysis based on chi-square test. Using a genome-wide significance threshold p<1×10 ^-5 The multiplex assay is calibrated. Genome-wide association analysis was performed using PLINK v 1.90. Epacts v 3.2.6 software (https:// genome. Sph. Umich. Edu/wiki/EPACTS) was used for gene detection and screening for rare SNPs (0.001. Sup. Th. SNP)< MAF <0.05). Selecting p in at least 2 Epacts models<0.01 as a candidate gene, was selected according to the following criteria: variant MAF in 1000 genomes<1%, 1000 genome of east Asia ancestry population, exAC and ExAC east Asia ancestry population; the CADD phred score was at least 15 points, which means that there may be destructive variation.

2.4 Statistical analysis of data

All statistical analyses were performed using statistical software package social science 21.0 software (IBM, armonk, usa). Correlation between variables was examined using Pearson correlation analysis. And (3) evaluating the significant difference of PG and PD parameters by adopting a Logistic regression method, and respectively adopting a coarse model and a model adjusted by the baseline characteristic. P <0.05 is statistically significant for the differences. Continuous and categorical variables are expressed as mean ± Standard Deviation (SD), frequency and percentage. And calculating a p value by adopting differential gene hypergeometric distribution test, and calculating the false detection rate by adopting Benjamini-Hochberg multiple test.

3. Results of the study

The study found that the SNP site rs10145032 on the AHNAK2 gene significantly affected the PT ratio of rivaroxaban (p = 0.0004). The different genotype of the SNP locus rs10145032 is GG/GC/CC, the gene distribution of carriers is 40/137/112, and the results are shown in Table 2.

Compared with GC and CC carriers of the AHNAK2 gene rs10145032, the PT ratio of a subject with 40 GG sites carrying the AHNAK2 gene rs10145032 is reduced, and the p value is 0.0004, so that the method has statistical significance. Research results show that PT of a GG gene carrier of the AHNAK2 gene rs10145032 is slightly changed after rivaroxaban is taken, a hypercoagulable state is promoted, the dosage of the medicine needs to be increased according to the medicine taking purpose during clinical medication, and the results are shown in figure 2.

The prediction of SNP rs10145032 on AHNAK2 gene on prothrombin is analyzed by adopting ROC curve, the AUC value is found to be 0.93, the sensitivity is 1.0, the specificity is 0.66, the prediction gene mutation is G gene carrier, the prediction significance on pharmacodynamics can be realized, and the result is shown in figure 3.

Example 2 pharmacodynamic, pharmacokinetic, and biological index results analysis of NVAF patients after taking rivaroxaban

1. Study object selection

The study included non-valvular atrial fibrillation (NVAF) patients who received thrombus prevention over 18 years of age. Exclusion criteria were as follows:

1) Patients with immunodeficiency diseases, viral hepatitis, severe liver dysfunction or renal dysfunction;

2) Patients receiving p-glycoprotein inhibitor combination therapy within 14 days prior to rivaroxaban treatment, including systemic azole antifungal agents ketoconazole, itraconazole, voriconazole, posaconazole, human immunodeficiency virus protease inhibitor ritonavir, or the inducers rifampin, phenytoin, phenobarbital, carbamazepine;

3) Whether rivaroxaban has anaphylaxis and active hemorrhage, whether intracranial or gastrointestinal bleeding history exists in the past 6 months, whether major operation exists in 30 days or not and other contraindications;

4) Do not want to take the medicine on time or donate blood according to regulations.

The study protocol was approved by the independent ethics committee of the first hospital of Beijing university and all of the branch hospitals with clinical registration number NCT03161496. This study followed the moral principles outlined in the declaration of helsinki. All patients participating in the study signed informed consent.

2. Research method

2.1 Gene detection

During the follow-up period, a gene blood sample is collected, and DNA is extracted for genotyping. SNP detection was performed by whole exome sequencing using the SureSelectXT target enrichment system and Illumina NovaSeq 6000 sequencer (Illumina, usa). Strict quality control of the samples and genetic markers is performed after genotyping. Rate of absence<10% minor allele frequency>5%, hardy-Weinberg equilibrium p value>10 ^-6 The Single Nucleotide Polymorphism (SNP) of (1) was included for further analysis.

2.2 Research outcome event

Follow-up was performed by telephone or routine outpatient visit 1 month, 6 months, 1 year and 2 years after the clinical patients were enrolled. The primary safety outcome was bleeding episodes, including major and minor bleedings demarcated by the bleeding academic research consortium. Secondary endpoints are stroke or systemic embolic events. Systemic embolism occurs at sites outside the central nervous system, resulting in acute ischemia of limbs, kidneys and other internal organs, and the diagnosis is based on sudden local pain, accompanied by cold, pulseless limbs or hematuria, and confirmed by angiography, ultrasound examination or computed tomography.

2.3 Statistical analysis of data

Continuous variables are expressed as mean and standard deviation, while categorical variables are expressed as percentages. All statistical analyses were analyzed using R software (http:// www.R-project. Org) and static Package for Social Sciences software, version 21.0 (IBM, armonk, USA). Using the chi-square test, a P value <0.05 indicates a possible deviation from the overall distribution. And calculating a risk ratio by using a Cox proportional risk model, and determining a prognostic factor and a difference between subgroups.

3. Results of the study

257 NVAF patients were enrolled from 6 months to 7 months in 2017 to 2021. At the time of enrollment, 50.97% (131/257) of the patients underwent radiofrequency catheter ablation. Baseline characteristics for inclusion in patients are shown in table 3.

The study explored the effect of genetic variation on clinical outcome (bleeding, embolism and other events) after NVAF patients took rivaroxaban. It was found that SNP rs10145032 on the AHNAK2 gene significantly affected bleeding events at 1 month after drug administration of rivaroxaban (p = 0.03).

The SNP locus rs10145032 has a GG/GC/CC genotype and the carrier gene distribution is 40/117/99, and the results are shown in Table 4.

Compared with GC and GG carriers of the AHNAK2 gene rs10145032, the bleeding event occurrence rate of a CC site NVAF patient carrying the AHNAK2 gene rs10145032 at position 99 is high (10% vs. 30%), the p value is 0.03, and the result is shown in fig. 4.

The prediction of the SNP locus rs10145032 on the AHNAK2 gene on bleeding events is analyzed by adopting an ROC curve, the AUC value is found to be 0.85, the sensitivity is 0.86, the specificity is 0.56, the prediction of gene mutation into a C gene carrier can have prediction significance on pharmacodynamics, and the result is shown in figure 5.

The research proves that the PT change of a person with the AHNAK2 gene rs 10145032C gene mutation after taking rivaroxaban is increased in healthy people, and the bleeding risk is prompted; the bleeding event was also confirmed to be higher in clinical outcome of bleeding events in NVAF patients 1 month after rivaroxaban administration by CC gene carriers of AHNAK2 gene rs10145032.

The above description of the embodiments is only intended to illustrate the method of the invention and its core idea. It should be noted that, for those skilled 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 into the protection scope of the claims of the present invention.

Claims

1. A product for predicting the curative effect of antithrombotic drugs on thrombus, which is characterized by comprising a reagent for detecting the genotype of an SNP site rs10145032 in a sample, wherein the SNP site rs10145032 is the SNP site rs10145032 of an AHNAK2 gene, and the reagent comprises a nucleic acid ligand having affinity with rs10145032, and the nucleic acid ligand comprises a primer or a probe.

2. The product of claim 1, wherein the antithrombotic comprises one or more of a thrombolytic, an anticoagulant, and an antiplatelet agent, wherein the thrombolytic comprises urokinase, alteplase, reteplase, or streptokinase, wherein the anticoagulant comprises heparin, warfarin, argatroban, fondaparinux sodium, rivaroxaban, apixaban, idoxaban, or dabigatran etexilate, wherein the antiplatelet agent comprises one or more of a thromboxane A2 inhibitor, an adenosine P2Y12 receptor antagonist, a thrombin receptor antagonist, a 5-hydroxytryptamine receptor antagonist, platelet glycoprotein, and a phosphodiesterase inhibitor, wherein the thrombus comprises one or more of a white thrombus, a mixed thrombus, a red thrombus, and a clear thrombus, wherein the white thrombus comprises a persistent thrombus, and wherein the mixed thrombus comprises a predominantly red cell thrombus, a spherical thrombus, or a layered thrombus.

3. Use of a reagent for detecting a SNP marker in a sample for the manufacture of a product for predicting the efficacy of an antithrombotic drug in treating thrombosis, wherein the SNP marker comprises the SNP site rs10145032 of the AHNAK2 gene of claim 1.

4. The use of claim 3, wherein the antithrombotic comprises one or more of a thrombolytic, an anticoagulant, and an antiplatelet agent, wherein the thrombolytic comprises urokinase, alteplase, reteplase, or streptokinase, wherein the anticoagulant comprises heparin, warfarin, argatroban, fondaparinux, rivaroxaban, apixaban, idoxaban, or dabigatran etexilate, and wherein the antiplatelet agent comprises one or more of a thromboxane A2 inhibitor, an adenosine diphosphate P2Y12 receptor antagonist, a thrombin receptor antagonist, a 5-hydroxytryptamine receptor antagonist, a platelet glycoprotein, and a phosphodiesterase inhibitor.

5. The use of claim 3, wherein the thrombus comprises one or more of a white thrombus, a mixed thrombus, a red thrombus, a clear thrombus, the white thrombus comprises a continuing thrombus, and the mixed thrombus comprises a predominantly red blood cell thrombus, a spherical thrombus, or a layered thrombus.

6. The product according to any one of claims 1 and 2, wherein the product further comprises product instructions describing a prediction step of the efficacy of the antithrombotic treatment on thrombus, and the prediction step comprises:

2) Determining the genotype of rs 10145032;

3) Predicting the curative effect of the test subject on treating thrombus by using the antithrombotic drug rivaroxaban based on the genotype, wherein when the genotype of rs10145032 is GG, the drug effect of the test subject is poor after the test subject takes the antithrombotic drug; when the rs10145032 genotype is GC, the drug effect of the tested person is better after taking the antithrombotic drug; when the genotype of rs10145032 is CC, the drug effect of the tested person is better after taking the antithrombotic drug, but the bleeding event probability is increased.

7. The use of claim 3, wherein the product comprises one or more of a kit, a chip, a strip, a high throughput sequencing, a system.

8. The use as claimed in claim 3, wherein when the genotype of rs10145032 is GG, the subject has poor drug efficacy after taking the antithrombotic agent; when the rs10145032 genotype is GC, the drug effect of the tested person is better after taking the antithrombotic drug; when the genotype of rs10145032 is CC, the test subject has better drug effect and higher bleeding event probability after taking the antithrombotic drug.

9. The use according to claim 3, wherein the reagents comprise reagents for detection of the rs10145032 genotype by direct sequencing, single base extension, allele-specific probe hybridization, allele-specific primer extension, allele-specific amplification, allele-specific nucleotide incorporation, 5' nuclease digestion, molecular beacon assay, oligonucleotide ligation assay, size analysis and single-stranded conformational polymorphism methods.

10. The use according to any one of claims 3, 4, 5, 7, 8, 9, wherein the sample is blood.