SNP locus related to risk of liver injury caused by methotrexate and application of SNP locus

Abstract

The invention discloses an SNP (Single Nucleotide Polymorphism) site related to the risk of liver injury caused by methotrexate and application of the SNP site. The SNP is located at 31, 356 and 966bp positions of a No.6 chromosome of a human GRCh38 genome, and the basic group of the SNP is C or G. According to the scheme, the SNP site related to the methotrexate liver injury is provided, the risk of the liver injury caused by methotrexate can be effectively predicted, and the SNP site serves as a marker for predicting the liver injury caused by methotrexate.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of biotechnology, and particularly relates to a SNP site related to the risk of liver injury caused by methotrexate and application thereof. BACKGROUND

[0002] Methotrexate (MTX) is a cell cycle-specific drug widely used in the treatment of hematological tumors, rheumatoid arthritis and other autoimmune diseases. Although methotrexate is used in anti-tumor and immunoregulatory therapy, it can also cause a series of side effects, such as: ① liver damage, ② gastrointestinal reactions, ③ bone marrow suppression, ④ skin adverse reactions, etc.

[0003] The HLA (human leukocyte antigen) gene complex is located on the short arm of human chromosome 6 (6p21.31), and is divided into three types according to structure and function: class I, class II, and class III. With the rapid development of genetic pharmacology, more and more studies have confirmed that HLA genotypes are strongly associated with adverse drug reactions, and different types of HLA genotypes can be used as genetic markers to predict immune-specific liver injury caused by different drugs. By detecting HLA genotypes associated with adverse drug reactions, doctors can select the most suitable drugs and doses for patients, improve treatment effectiveness, and reduce the occurrence of adverse drug reactions.

[0004] At present, HLA genotype detection mainly uses first-generation sequencing and second-generation sequencing methods, which have long detection cycles, high detection costs, and complex operation and analysis methods, which are not conducive to the wide promotion of this detection. SUMMARY

[0005] The present application aims to at least solve one of the technical problems existing in the prior art. To this end, the present application proposes a SNP site related to the risk of liver injury caused by methotrexate.

[0006] The present application also proposes a primer set and / or a probe for detecting the above-mentioned SNP site.

[0007] The present application also proposes a kit.

[0008] The present application also proposes a gene chip.

[0009] The present application also proposes the application of the above-mentioned SNP site, primer set and / or probe, kit and / or gene chip.

[0010] The present application also proposes a system for predicting the risk of adverse drug reactions in response to methotrexate or the risk of liver injury caused by methotrexate.

[0011] The present invention also provides a method for developing a drug for treating adverse drug reactions in response to methotrexate or liver damage caused by methotrexate.

[0012] According to a first aspect of the present invention, a SNP site associated with the risk of liver damage caused by methotrexate is proposed. The SNP site is located in the human GRCh38 genome, at 31,356,966 bp on chromosome 6, and its base is C or G.

[0013] According to a second aspect of the present invention, a primer set and / or probe for amplifying the above-mentioned SNP site is proposed.

[0014] In some embodiments of the present invention, the primer set includes: the primer set includes a forward primer sequence such as SEQ ID NO: 1 and a reverse primer sequence such as SEQ ID NO: 2;

[0015] In some embodiments of the present invention, the sequence of the probe is shown in SEQ ID NO: 3.

[0016] In some embodiments of the present invention, the 5' end of the probe is labeled with a fluorescent reporter group, and the 3' end is labeled with a fluorescent quencher group;

[0017] In some embodiments of the present invention, the fluorescent reporter group includes but is not limited to any one of FAM, ROX, CY5, HEX, JOE, CY3, NED, TAMRA, TAXAS RED, VIC and TET;

[0018] In some embodiments of the present invention, the fluorescence quenching group includes but is not limited to any one of super Quenther1, BHQ, TAMRA, MGB and DABCYL.

[0019] According to a third aspect of the present invention, a kit is provided, comprising the above-mentioned primer set and / or probe.

[0020] In some embodiments of the present invention, the kit further comprises a primer probe set for detecting an internal reference gene, wherein the primer sequences of the primer probe set are shown in SEQ ID NO: 4 and SEQ ID NO: 5, and the probe sequence is shown in SEQ ID NO: 6.

[0021] In some embodiments of the present invention, the kit further comprises a PCR reaction solution; the PCR reaction solution comprises a stabilizer, DNA polymerase, Mg 2+ and at least one of dNTPs.

[0022] In some embodiments of the present application, the PCR reaction solution comprises 2x SuperFast Star Universal Probe Mixture (UNG).

[0023] According to a fourth aspect of the present application, a gene chip is provided, which comprises the primer set and / or probe described above.

[0024] According to a fifth aspect of the present application, the SNP site, primer set and / or probe, kit or gene chip described above is used in any of the following:

[0025] 1) to prepare a product for detecting or evaluating the risk of a human subject developing an adverse drug reaction in response to methotrexate;

[0026] 2) to prepare a product for detecting or evaluating the risk of a human subject developing liver injury caused by methotrexate;

[0027] 3) to prepare a product for screening a high-risk subject of liver injury caused by methotrexate;

[0028] 4) to develop and / or identify and / or screen a drug for treating an adverse drug reaction in response to methotrexate;

[0029] 5) to develop and / or identify and / or screen a drug for treating liver injury caused by methotrexate.

[0030] In some embodiments of the present application, the adverse drug reaction in response to methotrexate is liver injury caused by methotrexate.

[0031] According to a sixth aspect of the present application, a system for predicting the risk of developing an adverse drug reaction in response to methotrexate or the risk of developing liver injury caused by methotrexate is provided, which comprises:

[0032] a data storage device for storing the typing detection result of the SNP site in the sample;

[0033] a data analysis device for calculating the risk of developing an adverse drug reaction in response to methotrexate or the risk of developing liver injury caused by methotrexate in the sample based on the typing detection result of the SNP site;

[0034] a result output device for outputting the result of the data analysis device.

[0035] In some embodiments of the present application, if the genotype detected by the SNP site is GG, it is determined that the risk of developing an adverse drug reaction in response to methotrexate or the risk of developing liver injury caused by methotrexate is low; if the genotype detected by the SNP site is GC or CC, it is determined that the risk of developing an adverse drug reaction in response to methotrexate or the risk of developing liver injury caused by methotrexate is high.

[0036] According to a seventh aspect of the present application, the present application further provides a method for developing a drug for treating adverse reactions to methotrexate or methotrexate-induced liver damage, comprising the following steps: using the above-mentioned SNP site as a marker or target to develop and / or identify and / or screen a drug for treating adverse reactions to methotrexate or methotrexate-induced liver damage.

[0037] According to some embodiments of the present application, at least the following advantages are achieved: the present application provides a SNP site related to methotrexate-induced liver damage, which can effectively predict the risk of methotrexate-induced liver damage as a marker for predicting methotrexate-induced liver damage. The present application uses SNP site detection to replace the existing technology of predicting methotrexate-induced liver damage by using complex HLA whole gene typing, which has the advantages of time advantage, cost economy, simple operation and easy data analysis, and is expected to become an important member of the routine detection project in various scale medical institutions across the country. DETAILED DESCRIPTION

[0038] The concept and technical effects of the present application will be described below in conjunction with the embodiments to fully understand the purpose, features and effects of the present application. Obviously, the described embodiments are only a part of the embodiments of the present application, but not all embodiments. Based on the embodiments of the present application, other embodiments obtained by those skilled in the art without creative labor are within the scope of protection of the present application.

[0039] The experimental methods in the following examples not specified in the specific conditions are usually carried out according to the conventional conditions or according to the conditions recommended by the manufacturer. The materials, reagents and the like used in the present embodiment are commercially available reagents and materials unless otherwise specified.

[0040] Example 1 A SNP site for detecting methotrexate-induced liver damage

[0041] The present embodiment provides a SNP site (rs41563412) for detecting methotrexate-induced liver damage, which is located at the 214th position from the 5' end of the HLA-B gene, i.e. at the position of 31,356,966 bp on chromosome 6 in the GRCh38 genome (GRCh38 / hg38 coordinates: chr6: 31,356,966), and the mutation type is c.214G>C. In the population, the proportion of patients with liver damage who take methotrexate will be significantly higher than that of people who do not carry this mutation if the base C mutation of rs41563412 occurs in the above-mentioned mutation. The screening and verification of the SNP site for detecting methotrexate-induced liver damage are as follows:

[0042] 1. SNP site screening

[0043] The SNP site screening method is as follows:

[0044] For 20 patients taking methotrexate (10 patients with methotrexate-induced liver damage, 10 patients without liver toxicity (liver damage)), the NimbleGen SeqCap target region capture kit is used to hybridize and capture the collected patient sample DNA, the captured region is the 6p21.3 region (chr6: 28477797-33448354), which contains the traditional HLA region (about 3.37 Mb) and its flanking region (about 1.6 Mb), and the sequencing depth is 100x. After GATK recognizes the SNP, the sites with MAF>0.05 and in accordance with the Hardy-Weinberg equilibrium are screened, and the significance threshold is set to 5x10 -6 Under this significance threshold, only the distribution frequency of SNP site rs41563412 on the HLA-B gene is significantly different between the methotrexate-induced liver damage group and the non-liver damage group. Rs41563412 is located at the 214th position from the 5' end of the HLA-B gene, and the mutation is mainly from base G to base C.

[0045] 2, verification phase

[0046] In order to further verify the correlation between rs41563412 and methotrexate-induced liver damage, QPCR method is used to genotype rs41563412 site for another 94 patients taking methotrexate samples (50 patients with methotrexate-induced liver damage, 44 patients without liver damage) completely different from the above 20 patients. The genotyping results are consistent with the sequencing results. The genotyping results are analyzed for correlation using Fisher's exact test method.

[0047] The steps of QPCR method are as follows:

[0048] 1, screening of primer probe composition for detecting SNP site rs41563412

[0049] The present application screens a plurality of primer probes by using biological software Oligo 7.60 to design primer probes for SNP site rs41563412, and then comprehensively analyzes the plurality of primer probes screened to finally screen a primer probe group. The specific sequences are shown in Table 1.

[0050] Table 1

[0051]

[0052]

[0053] The blood sample of the sample to be detected is extracted in the anticoagulant blood to obtain the template DNA as the template, and the following reaction system and reaction procedure are used for detection.

[0054] The reaction system is shown in Table 2.

[0055] Table 2

[0056]

[0057] The fluorescence PCR reaction cycle parameters are: 95℃ 2min; then enter the cycle stage: 95℃ 10sec, 62℃ 35s, 72℃ 35s, 40 cycles.

[0058] The result judgment is shown in Table 3:

[0059] Table 3

[0060]

[0061]

[0062] In the embodiment of the present application, the research data of 94 patients taking methotrexate are shown in Table 4.

[0063] Table 4

[0064]

[0065] Further, the genotype of rs41563412 is statistically analyzed by Fisher's exact test to verify the difference in distribution of the site genotype (GC+CC vs GG), 38.0% (19 / 50) of the methotrexate liver damage group carries rs41563412 base C (genotype is GC+CC), which is much higher than 4.55% (2 / 44) of the non-liver damage group, OR=12.87, p=2.4x10 -6 .

[0066] The patient carrying rs41563412 base C taking methotrexate to cause liver damage is about 13 times that of the non-carrier, which is an independent risk factor for methotrexate-induced liver damage, so rs41563412 can be used as a genetic marker for evaluating the risk of methotrexate-induced liver damage.

[0067] The above has made a detailed description of the embodiments of the present application, but the present application is not limited to the above embodiments, and various changes can be made within the knowledge range possessed by those skilled in the art without departing from the purpose of the present application. In addition, the embodiments of the present application and the features in the embodiments can be combined with each other without conflict.

Claims

1. A SNP site associated with the risk of liver injury caused by methotrexate, characterized in that, The SNP site is located at 31,356,966 bp on chromosome 6 of the human GRCh38 genome, and the base is C or G.

2. A primer set and / or a probe for amplifying the SNP site according to claim 1.

3. The primer set and / or probe according to claim 2, characterized in that, The primer set comprises a forward primer sequence as shown in SEQ ID NO: 1 and a reverse primer sequence as shown in SEQ ID NO: 2; and / or, the probe has a sequence as shown in SEQ ID NO:

3.

4. A kit characterized in that, The kit comprises the primer set and / or the probe according to any one of claims 2-3.

5. A gene chip, characterized by The gene chip comprises the primer set and / or the probe according to any one of claims 2-3.

6. Use of the SNP site according to claim 1, the primer set and / or the probe according to any one of claims 2-3, the kit according to claim 4, or the gene chip according to claim 5 in any one of the following: 1) preparing a product for detecting or evaluating the risk of a human subject developing an adverse drug reaction in response to methotrexate; 2) preparing a product for detecting or evaluating the risk of a human subject developing liver injury caused by methotrexate; 3) preparing a product for screening a high-risk subject of liver injury caused by methotrexate; 4) developing and / or identifying and / or screening a drug for treating an adverse drug reaction in response to methotrexate; 5) developing and / or identifying and / or screening a drug for treating liver injury caused by methotrexate.

7. Use according to claim 6, characterized in that, The adverse drug reaction in response to methotrexate is liver injury caused by methotrexate.

8. A system for predicting the risk of a subject developing an adverse drug reaction in response to methotrexate or the risk of a subject developing liver injury caused by methotrexate, comprising: a data storage device for storing the typing detection result of the SNP site according to claim 1 in a sample; a data analysis device for calculating the risk of a sample developing an adverse drug reaction in response to methotrexate or the risk of a sample developing liver injury caused by methotrexate based on the typing detection result; a result output device for outputting the result of the data analysis device.

9. The system of claim 8, wherein, If the genotype obtained by typing detection of the SNP site is GG, it is determined that the risk of a subject developing an adverse drug reaction in response to methotrexate or the risk of a subject developing liver injury caused by methotrexate is low; if the genotype obtained by typing detection of the SNP site is GC or CC, it is determined that the risk of a subject developing an adverse drug reaction in response to methotrexate or the risk of a subject developing liver injury caused by methotrexate is high.

10. A method for developing a drug for treating adverse reactions to a drug responsive to methotrexate or developing a drug for methotrexate-induced liver damage, characterized by, comprising the step of using the SNP site according to claim 1 as a marker or a target to develop and / or identify and / or screen a drug for treating an adverse drug reaction in response to methotrexate or liver injury caused by methotrexate.

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