Gene polymorphic site detection kit for predicting warfarin drug curative effect

Abstract

The invention discloses a gene polymorphic site detection kit for predicting the curative effect of warfarin drugs. The kit comprises a MassARRAY chip and six primers for detecting rs72558192 and rs769942899SNP (Single Nucleotide Polymorphism) sites of a gene CYP2C9, the six primers comprise a forward primer, a reverse primer and an extension primer, the sequences of the forward primer, the reverse primer and the extension primer are respectively shown as SEQ ID No.1-6, and the primers are used for identifying polymorphic sites of warfarin drug effect related genes. The kit disclosed by the invention can be used for carrying out high-throughput detection on the two sites, and has the advantages of high sensitivity, high specificity, stable detection result, high reliability and the like; meanwhile, the method is suitable for gene analysis fields such as clinical disease mutation detection, pharmacogenomics analysis and forensic medicine identification.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of genes, and relates to a gene polymorphism (SNP) site detection kit for predicting the curative effect of warfarin. BACKGROUND

[0002] Warfarin is a chemical structure of 3-(a-phenylpropanone)-4-hydroxycoumarin, and a molecular formula of C 19 H 16 O4. As an oral anticoagulant, it belongs to the category of vitamin K antagonists. Warfarin can interfere with the synthesis of coagulation factors II, VII, IX and X by inhibiting vitamin K reductase and blocking the regeneration of vitamin K. By inhibiting the synthesis of these coagulation factors, warfarin can prolong the clotting time, reduce the formation of thrombus, and reduce the risk of cardiovascular disease.

[0003] Although warfarin is an effective anticoagulant, its therapeutic range is narrow and there is a certain clinical risk. The dosage of warfarin varies greatly among individuals and is affected by many factors, such as food intake, interaction with other drugs, and individual genetic differences, so close monitoring and timely adjustment are required during medication. If the dosage is not appropriate, it may lead to excessive or insufficient coagulation, thereby increasing the risk of bleeding or thrombosis, and also causing liver and kidney damage. Therefore, the study of the pharmacogenomics of warfarin has always been the focus in order to maximize the therapeutic effect and reduce adverse drug reactions.

[0004] Warfarin exists in the form of a racemic mixture, and the efficacy of S-warfarin is 3-5 times that of R-warfarin, and CYP2C9 is the main hydroxyl metabolite. According to statistics, CYP2C9 is involved in the metabolism of 15% of clinically used drugs. In the past few decades, research on individual differences of warfarin has mainly focused on association analysis such as GWAS, and the susceptibility sites discovered in this way are unstable, and most of them are common variations, and most of the genetic difference factors cannot be explained. With the development of sequencing technology, a large number of studies have shown that rare variants have important significance in personalized medicine. Some rare variant sites are located in drug metabolism-related genes, affecting the metabolic pathway of drugs, drug efficacy and the occurrence of adverse reactions, and have a higher risk of causing genetic function defects compared to common variants. Effective detection of individual rare variants can help doctors develop more individualized drug treatment plans, improve drug efficacy and reduce the risk of adverse reactions. For CYP2C9 with high polymorphism, more than 60 alleles have been reported so far, but there are few reports on the occurrence of rare variants of CYP2C9 rs72558192 and rs769942899 and their use as molecular markers for warfarin efficacy.

[0005] The present application researches and finds that CYP2C9 gene rs72558192 and rs769942899 sites are highly related to the efficacy of warfarin drug, and no previous report is found. Therefore, the present application first proposes that CYP2C9 gene rs72558192 and rs769942899 sites have the value of predicting the efficacy of warfarin drug.

[0006] The prior art involves 15% of the metabolism of clinically used drugs by CYP2C9. In the past few decades, the research on individual differences of warfarin mainly focuses on association analysis such as GWAS, and the susceptibility sites found in the research are unstable, and most of them are common variations, and most of the genetic difference factors cannot be explained. With the development of sequencing technology, a large number of studies have shown that rare variations have important significance in individualized medical treatment. Some rare variation sites are located in drug metabolism related genes, affect the metabolism of drugs, drug efficacy and the occurrence of adverse reactions, and have a higher risk of causing gene function defects compared with common variations. SUMMARY

[0007] In view of the defects of the prior art, the purpose of the present application is to provide a genetic polymorphism site detection kit for predicting the efficacy of warfarin drug. The present application lays a foundation for studying the relationship between CYP2C9 gene rs72558192 and rs769942899 genetic polymorphisms and the safety of clinical drug use in Chinese population, provides a theoretical basis for clinical individualized drug use, and also provides a guidance basis for new drug research and development based on the concept of pharmacogenomics.

[0008] The purpose of the present application is achieved by the following technical solutions,

[0009] In a first aspect, the present application provides a primer combination for detecting genetic polymorphism sites related to the efficacy of warfarin drug, and the sequence is shown in Table 1.

[0010] Table 1

[0011]

[0012] In a second aspect, the present application provides a detection kit for predicting genetic polymorphism sites related to the efficacy of warfarin drug, which comprises a primer combination for detecting mutations of CYP2C9 gene rs72558192 and rs769942899 sites in a sample DNA.

[0013] As an embodiment, the primer combination comprises:

[0014] PCR forward primer for rs72558192 site with sequence as shown in SEQ ID NO: 1, PCR reverse primer with sequence as shown in SEQ ID NO: 2, and extension primer with sequence as shown in SEQ ID NO: 3;

[0015] PCR forward primer for rs769942899 site with sequence as shown in SEQ ID NO: 4, PCR reverse primer with sequence as shown in SEQ ID NO: 5, and extension primer with sequence as shown in SEQ ID NO: 6.

[0016] As an embodiment, the kit comprises PCR reaction solution, enzyme cutting reaction solution, and extension reaction solution.

[0017] As an embodiment, the PCR reaction solution comprises deionized water, PCR Buffer, MgCl2, dNTPs, and heat-resistant Taq DNA polymerase. In some embodiments, the ratio of deionized water, PCR Buffer, MgCl2, dNTPs, and heat-resistant Taq DNA polymerase is 18:5:4:1:2.

[0018] As an embodiment, the enzyme cutting reaction solution comprises deionized water, SAP Buffer, and SAP enzyme. In some embodiments, the ratio of deionized water, SAP Buffer, and SAP enzyme is 153:17:30.

[0019] As an embodiment, the extension reaction solution comprises deionized water, Gold Buffer, Termination mix, and high-temperature-resistant extension enzyme. In some embodiments, the ratio of deionized water, Gold Buffer, Termination mix, and high-temperature-resistant UEP extension enzyme is 619:200:200:41.

[0020] As an embodiment, the kit further comprises detection chip, detection carrier, purification required resin, sample spotting, and mass spectrometry required target piece, and human genomic DNA extraction reagent. In some embodiments, the detection chip is MassARRAY chip.

[0021] As an embodiment, the sample comprises peripheral blood.

[0022] In a third aspect, the present application provides a method for non-diagnostic purpose use of a SNP detection kit for genes related to individualized use of warfarin, comprising the following steps:

[0023] S1, using the aforementioned primer composition to perform PCR primer amplification and UEP extension primer dilution;

[0024] S2, PCR reaction;

[0025] S3, SAP digestion reaction;

[0026] S4, UEP extension reaction;

[0027] S5, resin purification.

[0028] As an embodiment, the PCR mixed reaction solution used in step S1 comprises deionized water, PCR Buffer, MgCl2, dNTPs, heat-resistant Taq DNA polymerase, and amplification primer mix.

[0029] As an embodiment, the SAP enzyme mix reaction solution used in step S2 comprises deionized water, SAP Buffer, and SAP enzyme.

[0030] As an embodiment, the UEP extension mix reaction solution used in step S4 comprises deionized water, Gold Buffer, Termination mix, UEP primer mix, and high-temperature-resistant extension enzyme.

[0031] As an embodiment, in step S5, the resin is dropped into the single-base extension product.

[0032] Compared with the prior art, the present application has the following beneficial effects:

[0033] 1) The present application integrates multiple PCR, single-base extension, mass spectrometry detection, etc. technologies, amplifies the detection template while detecting trace samples, and therefore has high detection sensitivity.

[0034] 2) Single-base extension, also known as microsequencing, uses specific probes to recognize DNA, has good specificity, low false positives, etc.

[0035] 3) High-throughput sequencing technology makes the speed fast and the efficiency high, and can complete detection of hundreds of samples in 3-4 hours.

[0036] 4) The operation is relatively simple, and under automatic operation, the occurrence of pollution is reduced.

[0037] 5) The present application can detect multiple patients and can obtain different gene site information at the same time.

[0038] 6) The present application overcomes the high cost defect of the previous technology of detecting a small number of SNP sites at a time.

[0039] 7) The present application provides a theoretical basis for studying the CYP2C9 gene rs72558192 and rs769942899 gene polymorphism of Chinese population and clinical individualized safe medication.

[0040] 8) Compared with the CYP2C9 gene rs1057910 site in the prior art, the Mendelian Clinically Applicable Pathogenicity (M-CAP) score result of the site shows that the variation is possibly benign, so it may have no significant effect on gene function, while the main variations of rs72558192 and rs769942899 in the present application are all shown to be possibly pathogenic, which may cause damage to gene function and affect the efficacy of warfarin drug. BRIEF DESCRIPTION OF DRAWINGS

[0041] Other features, objects, and advantages of the present application will become more apparent from the following detailed description of non-limiting embodiments with reference to the attached drawings:

[0042] Figure 1 Protein structure prediction results of Missense3D for mutations occurring at CYP2C9 rs72558192;

[0043] Figure 2 Protein structure prediction results of Missense3D for mutations occurring at CYP2C9 rs769942899. DETAILED DESCRIPTION

[0044] The present application will be further described in detail with reference to specific examples. It should be understood that these examples are only used to illustrate the present application and not used to limit the scope of the present application. The experimental methods in the following examples, if not specified, are usually carried out according to conventional conditions, for example, the conditions described in Sambrook et al., Molecular Cloning: A Laboratory Manual (New York: Cold Spring Harbor Laboratory Press, 1989), or according to the conditions recommended by the manufacturer.

[0045] Example 1, primer design and synthesis.

[0046] Specific PCR primer sequences (SEQ ID No: 1, SEQ ID No: 2, SEQ ID No: 4, SEQ ID No: 5) and specific extension primer sequences (SEQ ID No: 3, SEQ ID No: 6) were designed for the CYP2C9 gene rs72558192 and rs769942899 sites related to warfarin drug-related genetic polymorphism sites.

[0047] The relevant primers were synthesized by Jierui Biological (Shanghai) Co., Ltd.

[0048] Example 2, sample DNA extraction.

[0049] Collect 5 ml of blood into a vacuum blood collection tube containing 3.2% trisodium citrate and lithium heparin, and let it stand for at least 6 hours. Invert the blood collection tube 3-5 times to ensure the blood is thoroughly mixed with the anticoagulant. DNA extraction was performed using the DNA MiniKit (250) kit; DNA concentration was determined using a Thermo Fisher NanoDrop 2000 ultra-micro UV spectrophotometer; during sample analysis, the concentration, 260 / 280, and 260 / 230 values ​​needed to identify potential contamination causes if the concentration did not meet requirements. This also required aliquoting the DNA samples to minimize freeze-thaw cycles and ensure high-quality DNA. The extracted sample was then diluted with deionized water to 10-20 ng / μL to meet the basic quality control requirements for subsequent genotyping.

[0050] Example 3: Genotyping

[0051] Using an ABI 9700 PCR instrument, the polymorphisms rs72558192 and rs769942899 related to warfarin efficacy were amplified and subsequently detected according to the instruction manual, and the genotyping results of rs72558192 and rs769942899 loci were obtained.

[0052] Table 2

[0053]

[0054] In Table 2, the ratio of deionized water, PCR buffer, MgCl2, dNTPs, and thermostable Taq DNA polymerase in the PCR reaction solution is 18:5:4:1:2; the ratio of deionized water, SAP buffer, and SAP enzyme in the enzyme digestion reaction solution is 153:17:30; and the ratio of deionized water, Gold Buffer, Termination mix, and thermostable UEP extension enzyme in the extension reaction solution is 619:200:200:41.

[0055] 1. PCR reaction conditions: 95℃, 2min; 45 cycles (95℃, 30s; 56℃, 30s; 72℃, 60s); 72℃, 5min.

[0056] 2. SAP digestion reaction conditions: 37℃, 40 min; 85℃, 5 min.

[0057] 3. UEP extended reaction conditions: 94℃, 30s; 40 external cycles (94℃, 5s; 5 internal cycles (52℃, 5s; 80℃, 5s)); 72℃, 3min.

[0058] 4. Purification: Add 16 μΐ of deionized water to each tube of extension product, put into resin until mixed well, centrifuge.

[0059] 5. Spotting: Using micropipette, 1 μΐ of purified product to target spot.

[0060] 6. Instrument detection: Spotting robot 24 needles with NaOH cleaning; spotting; using MassArray time-of-flight mass spectrometry platform for detection.

[0061] Example 4, pathogenicity analysis results

[0062] Accurate pathogenicity assessment of genetic variation data is of great significance for the diagnosis of genetic diseases, and software prediction results are one of the important bases for variation rating. The principle of pathogenicity scoring software is mostly based on the following key steps:

[0063] 1. Data collection and annotation: software collects and integrates a large amount of genomic data, including annotation information of known pathogenic and benign variations. These data from public databases and literature reports provide the basis for evaluating genomic variations; 2. Feature extraction: software uses different algorithms and methods to extract the features of variations. These features may include mutation frequency, sequence conservation, functional annotation, domain information, gene function impact, etc. By analyzing these features, the software can capture different aspects and attributes of the variation; 3. Training and modeling: software uses known pathogenic and benign variation data sets for training and modeling. Machine learning algorithms such as random forest, support vector machine, neural network, etc. are involved. By learning the features of known variations and their corresponding pathogenicity, the software can build a model to predict new unknown variations; 4. Variation evaluation and scoring: according to the model, the software evaluates and scores new unknown variations. According to the extracted features and the weight of the model, the software will give a relative pathogenicity score to predict the possible pathogenicity of the variation.

[0064] Currently, there are a variety of computer software available for evaluating the impact of sequence variations on gene function. These tools use different algorithms, some rely on predicting whether non-synonymous mutations will disrupt protein structure and function, some are based on sequence conservation (because the more important the sequence is to life function, the more genetically stable it is, and mutations at this location are more likely to have serious consequences), and some are based on machine learning of known mutation information using specific algorithms. Common pathogenicity scoring software includes SIFT, PolyPhen-2, CADD, MutationTaster, etc. This invention uses 14 bioinformatics analysis tools to score and predict the rare variations of CYP2C9 rs72558192 and rs769942899. The summary is as follows:

[0065] Table 3, Evaluation of pathogenicity of two sites of CYP2C9 by 14 pathogenicity prediction softwares

[0066]

[0067] Considering the results of various scoring tools, the risk of pathogenic variation at the two sites rs72558192 and rs769942899 of CYP2C9 gene is very high, and therefore has predictive significance for the efficacy of warfarin drug therapy.

[0068] The mutations at the above two sites were predicted for protein structure using Missense3D for characterization and prioritization of missense variants. This software predicts the structural changes introduced by amino acid substitutions based on the three-dimensional structure information of the protein from model or experimental coordinates. Compared with other protein structure prediction tools, the advantages of Missense3D are relatively high accuracy of structure prediction, relatively large database size and support for structure prediction of transmembrane proteins, which can provide more comprehensive information for missense variant analysis. The prediction results are shown in Table 4 and Table 5. Figure 1 、 2

[0069] Further, the (M-CAP) score results of the rs1057910 site of the CYP2C9 gene and the rs72558192 and rs769942899 sites of the present application are compared.

[0070] For the rs1057910 site of the CYP2C9 gene, the Mendelian Clinically Applicable Pathogenicity (M-CAP) score results of the site are as follows:

[0071] rs1057910: GRCh37 / hg19.10: 96,741,053 Reference Allele A

[0072] Alt Allele M-CAP 95% sensitivity TPR G 0.007 Likely Benign T 0.015 Likely Benign

[0073] The results show that the site variation is likely benign, and therefore may have no significant effect on gene function. However, the results for the rs72558192 and rs769942899 sites of the CYP2C9 gene of the present application are as follows:

[0074] rs72558192: GRCh37 / hg19.10: 96,731,936 Reference Allele A

[0075] Alt Allele M-CAP 95% sensitivity TPR C 0.055 Possibly Pathogenic G 0.046 Possibly Pathogenic T 0.024 Likely Benign

[0076] ​rs769942899: GRCh37 / hgl9.10: 96,748,674 Reference Allele G

[0077] Alt Allele M-CAP 95% sensitivity TPR C 0.056 Possibly Pathogenic T 0.056 Possibly Pathogenic

[0078] The results show that the two locus major variations are likely pathogenic, and thus can have a significant impact on gene function.

[0079] In summary, the present application provides a theoretical and prognostic basis for the correlation between CYP2C9 gene rs72558192 and rs769942899 polymorphisms and clinical drug guidance in the cardiovascular population of China, and lays a genetic foundation for individualized pharmacogenomics. The specific embodiments of the present application have been described above. It should be understood that the present application is not limited to the above embodiments, and various modifications or changes can be made by those skilled in the art within the scope of the claims, which does not affect the essential content of the present application.

Claims

1. A detection kit for predicting gene polymorphism sites related to warfarin efficacy, characterized in that, The primer combination comprises primers for detecting mutations at rs72558192 and rs769942899 sites of CYP2C9 gene in sample DNA.

2. The test kit according to claim 1, characterized in that, The primer combination comprises: a PCR forward primer for the rs72558192 site, the sequence of which is shown in SEQ ID NO: 1, a PCR reverse primer, the sequence of which is shown in SEQ ID NO: 2, and an extension primer, the sequence of which is shown in SEQ ID NO: 3; a PCR forward primer for the rs769942899 site, the sequence of which is shown in SEQ ID NO: 4, a PCR reverse primer, the sequence of which is shown in SEQ ID NO: 5, and an extension primer, the sequence of which is shown in SEQ ID NO:

6.

3. The test kit according to claim 1, characterized in that, The kit comprises PCR reaction solution, enzyme digestion reaction solution, and extension reaction solution.

4. The test kit according to claim 3, characterized in that, The PCR reaction solution comprises deionized water, PCR Buffer, MgCl2, dNTPs, and heat-resistant Taq DNA polymerase.

5. The test kit according to claim 3, characterized in that, The enzyme digestion reaction solution comprises deionized water, SAP Buffer, and SAP enzyme.

6. The test kit according to claim 3, characterized in that, The extension reaction solution comprises deionized water, Gold Buffer, Termination mix, and high-temperature-resistant UEP extension enzyme.

7. The test kit according to claim 1 or 3, characterized in that, The kit further comprises a detection chip, a detection carrier, a purification-required resin, a sample spotting, a mass spectrometry-required target piece, and a human genomic DNA extraction reagent.

8. The test kit according to claim 7, characterized in that The detection chip is a MassARRAY chip.

9. The test kit according to claim 1, characterized in that, The sample comprises peripheral blood.

10. The method of non-diagnostic use of the test kit according to claim 1, characterized in that, The method comprises the following steps: S1, PCR primer amplification and UEP extension primer dilution using the primer combination; S2, PCR reaction; S3, SAP digestion reaction; S4, UEP extension reaction; S5, resin purification.

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