CN107058527B

CN107058527B - KCNE3 and NAT2 gene-based SNP marker related to hepatotoxicity reaction of antituberculosis drugs, kit and application

Info

Publication number: CN107058527B
Application number: CN201710174453.XA
Authority: CN
Inventors: 贺兆辉; 申阿东; 曹诗琴; 晋家骧; 王纯刚
Original assignee: Beijing Guangda Longtai Technology Co Ltd
Current assignee: Beijing Guangda Longtai Technology Co Ltd
Priority date: 2017-03-22
Filing date: 2017-03-22
Publication date: 2020-03-31
Anticipated expiration: 2037-03-22
Also published as: CN107058527A

Abstract

The invention relates to an SNP marker related to hepatotoxicity reaction of antituberculosis drugs, the SNP marker comprises a combination of KCNE3 gene SNP locus and NAT2 gene SNP locus, the KCNE3 gene SNP locus is rs12272502, and the NAT2 gene SNP locus is one or more of rs4921914, rs10103029 and rs 7816847. The invention also provides a kit for evaluating the risk of hepatotoxicity reaction of the antituberculous drug, which is used for detecting the SNP marker related to the hepatotoxicity reaction of the antituberculous drug. The SNP marker related to the hepatotoxicity reaction of the antituberculosis drug and the application thereof can accurately and genetically mark the hepatotoxicity reaction of the drug caused by the antituberculosis drug, thereby carrying out molecular marker-assisted selection and drug prediction, realizing individual drug administration and reducing adverse drug reactions. In the research process, a large sample is researched, the sample size has enough representativeness, and the accuracy of the research result is ensured.

Description

KCNE3 and NAT2 gene-based SNP marker related to hepatotoxicity reaction of antituberculosis drugs, kit and application

Technical Field

The invention relates to a Single Nucleotide Polymorphism (SNP) marker, a kit and application, in particular to an undisclosed SNP marker related to drug hepatotoxicity reaction caused by antituberculosis drugs, a combination thereof, a kit and application thereof, belonging to the field of biological genetic engineering.

Background

Tuberculosis can be the biggest disaster in infectious diseases, which is fatal to millions of people every year worldwide and is one of the major diseases currently causing death of residents in developing countries, especially children. Pharmacotherapy is currently the main approach to the treatment of tuberculosis, and the commonly used drugs are Isoniazid (INH), Rifampin (RFP), Pyrazinamide (PZA) and Ethambutol (EMB). Although chemotherapy has a good clinical treatment effect on tuberculosis, Adverse Drug Reactions (ADRs) occurring during the treatment process affect the anti-tuberculosis treatment effect and the health of patients. Among them, the anti-tubercular drug hepatotoxic reaction (ATDH) is the most serious common anti-tubercular ADR. The previous researches suggest that factors such as age, weight, sex, drug combination, nutritional status and the like influence the onset of ATDH. With the development of pharmacogenomics in the genetic field, Single Nucleotide Polymorphisms (SNPs) are receiving attention in studies of revealing different phenotypic traits of individuals and different responses to factors such as environmental exposure and drug therapy, and studies on the relationship between SNPs and ATDH individual differences are getting deeper and deeper, and focus on N-acetyltransferase 2 gene (N-acetyltransferase 2, NAT 2).

NAT2 is the main metabolic enzyme in the metabolic process of Isoniazid (INH), a commonly used drug for the treatment of tuberculosis. Genetic studies have shown that the acetylated metabolic patterns (fast, medium and slow acetylated metabolic patterns) of NAT2 are mainly determined by the functional polymorphic sites of the coding regions and the haplotypes thereof (fast, medium and slow acetylated metabolic patterns). Functional experiments prove that the amino acid change of the SNP mutant allele of a partial coding region relative to the wild type allele reduces the expression quantity of NAT2 and/or the activity of the acetylase of the SNP mutant allele.

Relevant studies that have been published so far are:

"research on relationship between polymorphism of N-acetyltransferase 2 and glutathione S-transferase M1 genes and hepatic injury caused by antitubercular drugs", anhui ru et al, "journal of china tabe-nordic, 2014 1 month, volume 36, stage 1, page 14-20) disclose that polymorphic sites of rs1805158, rs1801280, rs72554617, rs1799930, and rs1799931 of NAT2 are related to hepatic injury caused by antitubercular drugs, the counted patients are patients who have hepatic injury after receiving isoniazid + rifampin + ethambutol + pyrazinamide combination treatment, the used method is to analyze the relevance of each SNP site and hepatic injury individually, and there is no disclosure on whether the relevance of hepatic injury is higher when some SNPs are combined into a group;

WO 2015109608a1 discloses that rs1041983, rs1495741 SNPs of NAT2 and combinations thereof are associated with liver damage caused by any one or a combination of anti-tubercular drugs isoniazid, rifampicin, pyrazinamide, and discloses the association of some combinations comprising NAT2rs1041983 or rs1495741 and XO gene rs2295475 or rs1884752 SNPs with drug liver damage;

CN 106119363A discloses that the combination of 7 SNP loci (rs1801279, rs1041983, rs1801280, rs1799929, rs1799930, rs1208 and rs1799931) of N-acetyltransferase 2 gene (N-acetyltransferase 2, NAT2) can be used for detecting the susceptibility genotype of anti-tubercular drugs for liver injury.

Disclosure of Invention

The invention aims to provide an undisclosed SNP marker related to hepatotoxicity reaction of an antituberculosis drug, so that the hepatotoxicity reaction of the drug caused by the antituberculosis drug is more comprehensively and accurately molecularly and genetically marked, molecular marker-assisted selection and drug prediction are carried out, individual drug administration is realized, and adverse drug reactions are reduced.

The invention also aims to provide the non-diagnostic application of the SNP marker in the hepatotoxicity reaction of the antituberculosis drugs.

The invention also provides a kit for evaluating the risk of the hepatotoxicity reaction of the antituberculous drug, which is used for more comprehensively and accurately evaluating the risk of the hepatotoxicity reaction of the antituberculous drug by accurately detecting the SNP marker related to the hepatotoxicity reaction of the antituberculous drug.

In order to achieve the purpose, the technical scheme of the invention is as follows:

the invention provides an SNP marker related to hepatotoxicity reaction of antituberculosis drugs, the SNP marker comprises a combination of KCNE3 gene SNP locus and NAT2 gene SNP locus, the KCNE3 gene SNP locus is rs12272502, and the NAT2 gene SNP locus is one or more of rs4921914, rs10103029 and rs 7816847.

Furthermore, the SNP marker combination is combined with one or more of BLNK gene SNP locus rs2290714, PPP2R2B gene SNP locus rs1031915, FLT3 gene SNP locus rs9319408, VAV2 gene SNP locus rs679106, XO gene SNP locus rs1429372 and IL6 gene SNP locus rs2069852, so that the aim of more accurately marking drug hepatotoxicity caused by antituberculosis drugs can be fulfilled.

Furthermore, all the above cases can be combined with other SNP sites of NAT2 gene related to hepatotoxicity reaction of anti-tuberculosis drugs and their combination (such as: SNP site rs1041983 or rs1495741 of NAT2 gene, or their combination), which can more accurately mark anti-tuberculosis drugs to cause hepatotoxicity reaction of drugs.

The primer sequences of the specific amplification primers of the SNP markers can be respectively as follows:

the primer sequence of rs12272502 is SEQ ID No.1 and SEQ ID No. 2;

the primer sequences of rs2290714 are SEQ ID No.4 and SEQ ID No. 5;

the primer sequences of rs1031915 are SEQ ID No.7 and SEQ ID No. 8;

the primer sequence of rs9319408 is SEQ ID No.10 and SEQ ID No. 11;

the primer sequence of rs679106 is SEQ ID No.13 and SEQ ID No. 14;

the primer sequence of rs1429372 is SEQ ID No.16 and SEQ ID No. 17;

the primer sequence of rs2069852 is SEQ ID No.19 and SEQ ID No. 20;

the primer sequences of rs4921914 are SEQ ID No.22 and SEQ ID No. 23;

the primer sequence of rs10103029 is SEQ ID No.25 and SEQ ID No. 26;

the primer sequence of rs7816847 is SEQ ID No.28 and SEQ ID No. 29;

the primer sequence of rs1041983 is SEQ ID No.31 and SEQ ID No. 32;

the primer sequence of rs1495741 is SEQ ID No.34 and SEQ ID No. 35.

Further, the primer sequences of the specific extension primers corresponding to the specific amplification primers for the SNP markers may be:

the primer sequence of rs12272502 is SEQ ID No. 3; the primer sequence of rs2290714 is SEQ ID No. 6; the primer sequence of rs1031915 is SEQ ID No. 9; the primer sequence of rs9319408 is SEQ ID No. 12; the primer sequence of rs679106 is SEQ ID No. 15; the primer sequence of rs1429372 is SEQ ID No. 18; the primer sequence of rs2069852 is SEQ ID No. 21; the primer sequence of rs4921914 is SEQ ID No. 24; the primer sequence of rs10103029 is SEQ ID No. 27; the primer sequence of rs7816847 is SEQ ID No. 30; the primer sequence of rs1041983 is SEQ ID No. 33; the primer sequence of rs1495741 is SEQ ID No. 36.

The invention also provides non-diagnostic application of the SNP marker in the hepatotoxicity reaction of the anti-tuberculosis drugs, such as application in preparing a kit for evaluating the risk of the hepatotoxicity reaction of the anti-tuberculosis drugs.

The invention also provides a kit for evaluating the risk of hepatotoxicity reaction of the antituberculous drug, which is used for detecting at least the following SNP markers related to the hepatotoxicity reaction of the antituberculous drug: the SNP marker comprises a combination of KCNE3 gene SNP locus and NAT2 gene SNP locus, the KCNE3 gene SNP locus is rs12272502, and the NAT2 gene SNP locus is one or more of rs4921914, rs10103029 and rs 7816847.

Optionally, the kit contains the following specific amplification primer sequences:

the primer sequences of the SNP locus rs12272502 are SEQ ID No.1 and SEQ ID No. 2;

the primer sequences of the SNP locus s4921914 are SEQ ID No.22 and SEQ ID No. 23;

the primer sequences of the SNP locus rs10103029 are SEQ ID No.25 and SEQ ID No. 26;

the primer sequences of the SNP locus rs7816847 are SEQ ID No.28 and SEQ ID No. 29.

Furthermore, the specific extension primer sequence corresponding to the specific amplification primer sequence contained in the kit is as follows:

the primer sequence of the SNP locus rs12272502 is SEQ ID No. 3;

the primer sequence of the SNP locus rs4921914 is SEQ ID No. 24;

the primer sequence of the SNP locus rs10103029 is SEQ ID No. 27;

the primer sequence of the SNP locus rs7816847 is SEQ ID No. 30.

Furthermore, the kit for evaluating the risk of hepatotoxicity reaction of the antituberculosis drug is also used for detecting one or more of BLNK gene SNP locus rs2290714, PPP2R2B gene SNP locus rs1031915, FLT3 gene SNP locus rs9319408, VAV2 gene SNP locus rs679106, XO gene SNP locus rs1429372 and IL6 gene SNP locus rs2069852 related to hepatotoxicity reaction of the antituberculosis drug.

In response to this detection, the kit optionally contains specific amplification primer sequences as follows:

the primer sequences of the SNP locus rs2290714 are SEQ ID No.4 and SEQ ID No. 5;

the primer sequences of the SNP locus rs1031915 are SEQ ID No.7 and SEQ ID No. 8;

the primer sequences of the SNP locus rs9319408 are SEQ ID No.10 and SEQ ID No. 11;

the primer sequences of the SNP locus rs679106 are SEQ ID No.13 and SEQ ID No. 14;

the primer sequences of the SNP locus rs1429372 are SEQ ID No.16 and SEQ ID No. 17;

the primer sequences of the SNP locus rs2069852 are SEQ ID No.19 and SEQ ID No. 20.

In response to the detection, the specific extension primer sequence corresponding to the specific amplification primer sequence contained in the kit may be:

the primer sequence of the SNP locus rs2290714 is SEQ ID No. 6;

the primer sequence of the SNP locus rs1031915 is SEQ ID No. 9;

the primer sequence of the SNP locus rs9319408 is SEQ ID No. 12;

the primer sequence of the SNP locus rs679106 is SEQ ID No. 15;

the primer sequence of the SNP locus rs1429372 is SEQ ID No. 18;

the primer sequence of the SNP locus rs2069852 is SEQ ID No. 21.

Furthermore, the kit for evaluating the risk of the anti-tubercular drug hepatotoxicity reaction is also used for detecting the locus rs1495741 and/or rs1041983 of the NAT2 gene related to the anti-tubercular drug hepatotoxicity reaction.

the primer sequences of the SNP locus rs1041983 are SEQ ID No.31 and SEQ ID No. 32;

the primer sequences of the SNP locus rs1495741 are SEQ ID No.34 and SEQ ID No. 35.

the primer sequence of the SNP locus rs1041983 is SEQ ID No. 33;

the primer sequence of the SNP locus rs1495741 is SEQ ID No. 36.

Optionally, the kit may further comprise common reagents required by the corresponding PCR technology, such as dNTPs, MgCl₂Double distilled water, Taq enzyme, etc., which are well known to those skilled in the art, and in addition, there may be a standard and a control (e.g., a genotype-determining standard and a blank, etc.).

In conclusion, the technical scheme of the invention provides the undisclosed SNP marker combination based on KCNE3 and NAT2 genes and related to the hepatotoxicity reaction of the antituberculosis drug, so that the drug hepatotoxicity reaction caused by the antituberculosis drug is more comprehensively and accurately marked by molecular genetic markers;

in addition, the undisclosed SNP marker combination is applied to a kit for evaluating the hepatotoxicity reaction risk of the antituberculosis drug, so that the detection is carried out more comprehensively and accurately, the molecular marker assisted selection and the medication prediction are carried out, the individual medication is realized, and the adverse drug reactions are reduced.

Detailed Description

The present invention is further illustrated by the following specific examples.

Example 1

Description of the study subjects:

the study object of the invention is that China Han nationality tuberculosis children with the ages of 0-16 years are treated in hospital in Beijing children hospital affiliated to the university of capital medicine in 2005-2014 (under the premise that the children and parents agree with the information), and the Chinese Han nationality tuberculosis children completely meet the following 3 standards of ① clinical diagnosis of tuberculosis, ② receives antitubercular chemotherapy for more than 2 weeks according to standard chemotherapeutic dose and scheme, and ③ has normal basic liver and kidney functions.

The tuberculosis diagnosis standard is used for diagnosing tuberculosis according to clinical diagnosis standards and treatment schemes (trial) of child pulmonary tuberculosis, which are set by adult and child tuberculosis diagnosis and grading standards set by the American thoracic society and the respiratory group of the Chinese medical society of pediatrics, wherein the clinical diagnosis standards of tuberculosis are ① active tuberculosis contact history, ② PPD test positive, ③ anti-tuberculosis treatment effective, ④ excludes other diseases, meets any 2 items from ① to ④, has typical clinical symptoms and imaging evidence, and is clinically diagnosed as tuberculosis.

The ATDH determination standard is that the ATDH can be diagnosed by meeting any 1 of the following 4 items, namely ① serum ALT content > (2 × ULN), ALT represents alanine aminotransferase, ULN corresponding to serum ALT is 40IU/L, ② serum DBil content > (2 × ULN), DBil represents direct bilirubin, ULN corresponding to serum DBil is 6.8 μmol/L, ③ serum AST content, serum ALP content and serum Tbil content are all > (1 × ULN), wherein n1 item (2 × ULN), n1 is more than or equal to 1, AST represents aspartate aminotransferase, ALP represents alkaline phosphatase, Tbil represents total bilirubin, ULN corresponding to serum AST is 40IU/L, ULN corresponding to serum ALP is 220IU/L, ULN corresponding to serum Tbil is 19.0 μmol/L, ④ meets the standard a and the standard b:

standard a: serum ALT content, serum DBil content, serum AST content, serum ALP content and serum Tbil content, wherein n2 (1 × ULN) in the above 5 items, n2 is not less than 1;

standard b: has one of the following suspected symptoms of hepatotoxicity: yellow stain of skin and sclera, discomfort of liver region, nausea, emesis, anorexia, fever, rash, pruritus and other liver toxicity suspected symptoms.

The grouping standard of ATDH is ① ATDH after anti-tuberculosis chemotherapy is started, ② ATDH caused by virus infection, neonatal jaundice, liver and kidney basic diseases and other diseases is excluded, ③ ATDH symptom is relieved after tuberculosis medicine is reduced or stopped, ④ possibility of ATDH caused by other medicines is excluded, and the ATDH can be diagnosed when ① to ④ are met.

Control group entry criteria: after antituberculous chemotherapy, liver function is still normal.

In this example 1, large sample data was screened using the whole-genome association assay (GWAS). Based on the standard of the study object, 385 tuberculosis children are divided into ATDH group and non-ATDH control group, 77 ATDH group and 308 control group.

1. Regarding GWAS whole genome chip:

GWAS whole genome chip data come from previous tuberculosis susceptibility research of Chinese Han nationality population, and finally a typing result of 691388 SNP sites is obtained through quality control analysis. The chip implementation is as follows:

1) GWAS chip type: HumanOmni ZhongHua-8Bead Chip, Illumina;

2) the data analysis method of the GWAS chip comprises the following steps: plink software.

The chip obtains the parting result of 900, 015 SNP loci altogether, and 691388 SNP loci are finally obtained for subsequent analysis after the quality control such as the detection rate, Hardy-Weinberger balance (HWE), sample genetic relationship, main component analysis and the like.

(1) Detection rate criteria set and HWE analysis: SNP typing data can be included in the subsequent analysis only if they meet the following criteria: the detection rate of the sample is more than 99 percent, the detection rate of the SNP locus is more than 95 percent, the P value of HWE of a control group is more than 0.0001, and the minimum allele frequency is more than 0.01. In addition, the genome studio v3.0(Illumina) software is adopted to screen the SNP locus scatter diagram, and the SNP loci with poor clustering are discarded.

(2) Analyzing sample genetic relationship: to determine whether there is a consanguineous relationship between individuals, a probability distribution of consanguineous homology (IBD) alleles is used for analysis. In the analysis, the case group and the control group respectively have a PI-HAT > 0.05 of IBD of a pair of samples, therefore, deletion is given to the cases and controls with blood relation and the corresponding control and case samples. In addition, 15 samples were randomly selected for reproducibility test, and the genotyping coincidence rate was 99.99%, demonstrating that: the chip has stable detection result and reliable SNP locus genotyping data.

(3) And (3) main component analysis: integrating data of African people (YRI), European people (CEU), Japanese People (JPT), northern China people (CHB) and southern China people (CHS) in the thousand-person genome plan and data of the chip, and performing principal component analysis on the integrated data to ensure that case and control group samples detected by the chip have the genetic characteristics of Chinese people and have consistent genetic background.

(4) Performing crowd layering analysis on data of the chip: to study population stratification of the samples, the expansion coefficient (λ) was determined_GC) Analysis of (2). Lambda [ alpha ]_GCAt 1.017, it shows that we have well reduced the impact of crowd stratification on the results.

2. Data analysis was performed on large samples using the GWAS whole genome chip described above:

and (3) carrying out correlation analysis on the data of the chip: the data were retrieved for the SNP site typing results of the 385 ATDH cases and the control samples. And (3) carrying out genotype comparison analysis on 691388 SNP sites of 385 cases of ATDH and non-ATDH control children by adopting Plunk software, correcting data by adopting age and gender, and verifying the data by combining a discrete association decision algorithm to obtain a result of susceptibility correlation between the SNP sites and the ATDH.

The OR value and the P value of the selected ATDH related SNP sites are shown in the table 1:

table 1: OR value and P value of ATDH related SNP site

SNP site	Gene	OR value (95% CI)	P value
				rs6696544	CAMTA1	2.412(1.612-3.611)	1.87E-05
rs12272502	KCNE3	2.228(1.494-3.324)	8.63E-05
				rs2290714	BLNK	2.281(1.535-3.391)	4.54E-05
rs1031915	PPP2R2B	2.197(1.479-3.263)	9.7E-05
				rs9319408	FLT3	2.832(1.455-5.51)	2.18E-03
rs679106	VAV2	2.938(1.779-4.852)	2.56E-05
				rs1429372	XO	1.821(1.231-2.693)	2.68E-03
rs2069852	IL6	1.540(1.053-2.254)	2.62E-02
				rs4921914	NAT2	3.544(2.289-5.489)	1.42E-08
rs10103029	NAT2	3.589(2.296-5.611)	2.09E-08
				rs7816847	NAT2	3.408(2.188-5.307)	5.82E-08
rs1041983	NAT2	3.434(2.236-5.273)	1.73E-08
				rs1495741	NAT2	3.522(2.277-5.446)	1.52E-08

As can be seen from Table 1, the results of the detection involved 13 SNP sites of 9 genes in total. The SNP locus rs6696544 of the CAMTA1 gene, the SNP locus rs12272502 of the KCNE3 gene, the SNP locus rs2290714 of the BLNK gene, the SNP locus rs1031915 of the PPP2R2B gene, the SNP locus rs9319408 of the FLT3 gene, the SNP locus rs679106 of the VAV2 gene, the SNP locus rs1429372 of the XO gene, the SNP locus rs2069852 of the IL6 gene, the SNP locus rs4921914, rs10103029 and rs7816847 of the NAT2 gene are SNP loci related to the hepatotoxicity reaction of the antitubercular drugs discovered for the first time.

Example 2:

the study subject is described in more detail in example 1.

In this example 2, 582 samples (not overlapping with 385 samples in the above example 1) were additionally selected for 13 SNP sites obtained in example 1 (122 samples in the ATDH group and 460 samples in the non-ATDH control group), and mass spectrometry was performed.

1. The experimental steps are as follows:

1) designing and synthesizing a primer:

according to the SNP locus sequence information, PCR reaction and single base extension primer are designed by using primer design software Assay design3.1 and are handed to biological companies for synthesis. The primers corresponding to each SNP site are shown in Table 2. Wherein, Forward Primer is an upstream Primer, Reverse Primer is a downstream Primer, and Extended Primer is an extension Primer.

Table 2: primers corresponding to the 13 SNP sites obtained in example 1:

2) DNA extraction:

and extracting DNA in blood samples, tissues, cells and saliva by using a finished product kit. Detecting OD value by using a NanoDrop2000 instrument, detecting by using 1.25% agarose gel electrophoresis, transferring the qualified DNA to a 96-well plate, and storing at-20 ℃ for later use.

3) A systematic genotyping step:

① PCR amplification reaction:

polymerase Chain Reaction (PCR) is used to amplify a region of DNA located between known sequences at both ends. The reaction mix was incubated at three different temperatures for template denaturation, primer annealing and primer extension sequentially for each PCR cycle. The process can be automated using a programmable temperature thermal cycler.

② alkaline phosphatase treatment of product:

after the PCR reaction was completed, the PCR product was treated with SAP (shrimp alkaline phosphatase) to remove free dNTPs from the system.

③ Single base extension reaction:

after the alkaline phosphatase treatment was completed, the single-base extension reaction was carried out in a total volume of 9. mu.l.

④ resin purification:

⑤ chip spotting:

the resin purified extension product was transferred to a SpectroCHIP bioarray using a MassARRAY Nanodispenser RS1000 spotter.

⑥ mass spectrometric detection and data output:

analyzing the spotted SpectroCHIP chip by using a MALDI-TOF mass spectrometer, acquiring original data and a genotyping chart by using TYPER4.0 software according to a detection result, checking the integrity and correctness of a data file, storing the result and analyzing.

① - ⑥ are conventional methods for mass spectrometric detection in the art and are not described in detail herein.

2. And (3) multi-site joint risk assessment:

the above 582 samples were subjected to multi-site joint risk assessment calculation using the odds ratio weighted genetic riskscore (OR-GRS) method with OR values as weights.

The method can be roughly divided into two steps:

a) OR values for all SNP sites the number of mutant genes, then summed, i.e.:

in the formula, G represents a set vector of risk allele factors for a set of genetic susceptibility loci (G)_iNumber of at-risk alleles representing the ith genetic susceptibility site); b) sensitivity and specificity were calculated for the summed values of all samples, ROC curves were plotted, and the area under the curve was calculated as: AUC values.

For the principles and scoring methods related to OR-GRS, reference is made in particular to the "genetic risk scoring principles and methods" of the literature (Wang Cheng, Dajuncheng, Sun Yi, Shelan, Panhan Liang, Lexus and Han, Shenhong Bing, 2015, [ J ]. J. Chinese journal of epidemiology, 36 (10): 1062-1064).

The results of AUC values for the relevant SNP site combinations are as follows:

1) the AUC values of the combination of KCNE3 gene SNP site rs12272502 and any at least one of NAT2 gene SNP sites rs4921914, rs10103029 and rs7816847 calculated according to the above method, and the AUC values of the combination and at least one of the other 6 gene SNP sites, as shown in table 3. Wherein, the SNP loci of other 6 genes are BLNK gene locus (rs2290714), PPP2R2B gene locus (rs1031915), FLT3 gene locus (rs9319408), VAV2 gene locus (rs679106), XO gene locus (rs1429372) and IL6 gene locus (rs 2069852).

The schemes in table 3 are:

scheme 1: KCNE3 gene SNP locus rs12272502 is combined with any at least one of NAT2 gene SNP loci rs4921914, rs10103029 and rs 7816847;

scheme 2: the combination of the KCNE3 gene SNP locus and the NAT2 gene SNP locus in the scheme 1 and the combination of any one of other 6 SNP loci;

scheme 3: the combination of the KCNE3 gene SNP locus and the NAT2 gene SNP locus in the scheme 1 is combined with any two loci in other 6 SNP loci;

scheme 4: the combination of the KCNE3 gene SNP locus and the NAT2 gene SNP locus in the scheme 1 is combined with any three loci in other 6 SNP loci;

scheme 5: the combination of the KCNE3 gene SNP locus and the NAT2 gene SNP locus in the scheme 1 is combined with any four loci in other 6 SNP loci;

scheme 6: the combination of the KCNE3 gene SNP locus and the NAT2 gene SNP locus in the scheme 1 and the combination of any five loci in other 6 SNP loci;

scheme 7: the combination of the SNP site of the KCNE3 gene and the SNP site of the NAT2 gene in the scheme 1 is combined with all other six SNP sites.

Shown in table 3 are the maximum AUC values (the combination with max subscript on the AUC values in table 3), the minimum AUC values (the combination with min subscript on the AUC values in table 3), and the AUC values for several randomly drawn combinations between the maximum and minimum values, measured for each combination regimen.

Table 3: the AUC values based on the combination of SNP sites of KCNE3 and NAT2 genes, and the combination with SNP sites of the other 6 genes:

2) results of AUC values of the above-described combinations of related SNP sites 1) AUC values of all combinations with NAT2 gene site rs1495741 and/or rs1041983 (indicated as NAT2 in table 4), as shown in table 4.

The schemes in table 4 are:

scheme 1: all combinations covered by scheme 1 in the above result 1) in combination with NAT2 gene site rs1495741 and/or rs 1041983;

scheme 2: all combinations covered by schemes 2-7 in the above result 1) in combination with NAT2 gene site rs1495741 and/or rs 1041983;

shown in table 4 are the maximum AUC values (the combination with the "max" subscript on the AUC values in table 4), the minimum AUC values (the combination with the "min" subscript on the AUC values in table 4), and the AUC values for several randomly drawn combinations between the maximum and minimum values, measured for each combination regimen. Table 4: results of AUC values of the above related SNP site combinations 1) AUC values of all cases combined with NAT2 gene site rs1495741 and/or rs 1041983:

3) comparative example:

the comparative examples are shown in Table 5. Shown in table 5 are AUC values of each of 5 SNP sites related to anti-tubercular drug hepatotoxicity reaction of NAT2 gene and combinations thereof detected in the present invention, and AUC values of combinations of SNP sites of NAT2 gene and SNP sites of XO gene and/or IL6 gene.

Table 5: AUC values as comparative examples:

based on the KCNE3 gene SNP site rs12272502 as the SNP site related to the anti-tubercular drug hepatotoxicity reaction found for the first time, and in the comparison of the AUC values of scheme 1 and Table 5 in tables 3 and 4, the AUC value of the combination of the SNP sites of KCNE3 and NAT2 genes is obviously higher than the AUC values of each of the 5 SNP sites related to the anti-tubercular drug hepatotoxicity reaction of NAT2 gene and the combination thereof, it can be seen that the combination of the SNP sites of KCNE3 and NAT2 genes provides possibility for more comprehensive and accurate assessment of the risk of the anti-tubercular drug hepatotoxicity reaction.

As can be seen from the comparison of the AUC values of the schemes 2 to 7 in Table 3 with those in Table 5, further, the AUC value of the combination of the SNP sites of the KCNE3 and NAT2 genes with at least one of the SNP sites of the other 6 genes in the invention is significantly higher than that of the comparative example (Table 5), and particularly, when the combination scheme is the combination of the SNP site of the KCNE3 gene with the SNP site of the NAT2 gene with at least any two sites of the other 6 SNP sites (schemes 3 to 7 in Table 3), the AUC value is significantly higher than that of the comparative example (Table 5), thereby showing that these schemes can achieve the purpose of more comprehensively and accurately evaluating the risk of hepatotoxicity reaction of anti-tuberculosis drugs.

Further, as can be seen from the comparison of the AUC values of scheme 2 in table 4 and table 5, in the combinations formed by combining with the NAT2 gene locus rs1495741 and/or rs1041983 (i.e., scheme 2 in table 4) based on all combinations covered by schemes 2-7 in table 3, some combinations can achieve higher AUC values to more accurately assess the risk of anti-tubercular drugs causing drug hepatotoxicity reactions.

Example 3:

in this example 3, a kit for evaluating the risk of hepatotoxicity reaction of an antituberculous drug is prepared and used to detect SNP locus rs12272502 of KCNE3 gene and SNP locus rs4921914 of NAT2 gene.

The kit contains a specific amplification primer and a reagent of a specific extension primer for detecting the SNP locus, wherein the specific amplification primer sequence of the SNP locus rs12272502 is SEQ ID No.1 and SEQ ID No. 2; the sequence of the specific extension primer is SEQ ID No. 3; the specific amplification primer sequences of the SNP locus rs4921914 are SEQ ID No.22 and SEQ ID No. 23; the sequence of the specific extension primer is SEQ ID No. 24; the kit also comprises common reagents required by corresponding PCR technology: (e.g., dNTPs, MgCl₂Double distilled water, Taq enzyme, and the like, and in addition, a standard substance and a control (such as a standard substance for determining a genotype, a blank control, and the like) can be provided. For example:

this implementationThe kit reagents in example 3 include: dNTPs (25mM), MgCl₂(25mM), Primer Mix (500nM), HotStar Taq (5U/. mu.l), double distilled water.

The amplification reaction system comprises: 1.85. mu.L of double distilled water, 0.1. mu.L of dNTP (25mM), 0.325. mu.L of MgCl₂(25mM), 1. mu.L of Primer Mix (500nM), 0.625. mu.L of PCR Buffer (1.25X), 0.1. mu.L of HotStar Taq (5U/. mu.L), and 1. mu.L of template DNA was added to carry out PCR amplification reaction. Reaction conditions for PCR amplification: denaturation at 94 deg.C for 5 min; at 94 ℃ for 20s, at 57 ℃ for 30s, at 72 ℃ for 1min, for 45 cycles; extension at 72 ℃ for 3 min. After the PCR reaction was completed, the PCR product was treated with SAP (Shamp alkalinephsphatase, shrimp alkaline phosphatase) to remove free dNTPs from the system. Extension reaction system: 0.041. mu.L LipLEX enzyme (1X), 0.94. mu.L Primer Mix, 0.619. mu.L double distilled water, 0.2. mu.L iPLEX Termination Mix (1X), 0.2. mu.L iPLEX Buffer Plus (0.222X), 7. mu.L SAP treated PCR product. And (3) extension reaction conditions: denaturation at 94 ℃ for 30 s; 5s at 94 ℃; 5s at 52 ℃ and 5s at 80 ℃ for 40 cycles; extension at 72 ℃ for 3 min. The purified product was genotyped using the SequenomMassArray system.

Alternatively, the reaction system for PCR amplification consists of (50. mu.L) 0.50ng of genomic DNA, 30pmol of the forward primer, 30pmol of the reverse primer, 25. mu.L of 2 XTAQ plantatum PCR MasterMix and deionized water. 2 × Taq plant primer PCR MasterMix was purchased from Tiangen Biochemical technology (Beijing) Ltd, cat # KT 204. Reaction conditions for PCR amplification: denaturation at 94 deg.C for 5 min; at 94 ℃ for 20s, at 57 ℃ for 30s, at 72 ℃ for 1min, for 45 cycles; extension at 72 ℃ for 3 min. Sequencing the PCR amplification product.

The PCR reaction system, the reaction conditions, and the subsequent genotyping and evaluating methods may be varied or substituted according to the common knowledge or the conventional techniques in the art. The value of the kit lies in that: and evaluating the hepatotoxicity reaction risk of the antituberculous drugs, thereby performing molecular marker-assisted selection and medication prediction, realizing individualized medication and reducing adverse drug reactions.

Example 4:

in this example 4, a kit for evaluating the risk of hepatotoxicity reaction of an antituberculous drug is prepared and used to detect SNP site rs12272502 of KCNE3 gene, SNP site rs4921914 of NAT2 gene and SNP site rs1031915 of PPP2R2B gene.

The reagent of the kit is basically the same as that in the embodiment 3, and the difference is that the kit also contains a reagent of a specific amplification primer and a specific extension primer for detecting the SNP site rs1031915 of the PPP2R2B gene, wherein the specific amplification primer sequence of rs1031915 is SEQ ID No.7 and SEQ ID No. 8; the sequence of the specific extension primer is SEQ ID No. 9.

The corresponding PCR reaction system and reaction conditions were substantially the same as in example 3.

Example 5:

in this example 5, a kit for evaluating the risk of hepatotoxicity reaction of an antituberculous drug is prepared and used to detect KCNE3 gene SNP site rs12272502, NAT2 gene SNP site rs4921914, BLNK gene SNP site rs2290714, PPP2R2B gene SNP site rs1031915, VAV2 gene SNP site rs679106, FLT3 gene SNP site rs9319408, XO gene SNP site rs1429372, IL6 gene SNP site rs2069852, and NAT2 gene SNP site rs 1981043.

Correspondingly, the reagent of the kit contains the above reagent of the specific amplification primer and the specific extension primer of the SNP site of the gene to be detected, wherein the specific amplification primer and the specific extension primer of the SNP site of the gene to be detected are respectively as follows:

the specific amplification primer sequences of rs12272502 are SEQ ID No.1 and SEQ ID No. 2; the sequence of the specific extension primer is SEQ ID No. 3;

the specific amplification primer sequences of rs4921914 are SEQ ID No.22 and SEQ ID No. 23; the sequence of the specific extension primer is SEQ ID No. 24;

the sequences of the specific amplification primers of rs2290714 are SEQ ID No.4 and SEQ ID No. 5; the sequence of the specific extension primer is SEQ ID No. 6;

the sequences of the specific amplification primers of rs1031915 are SEQ ID No.7 and SEQ ID No. 8; the sequence of the specific extension primer is SEQ ID No. 9;

the specific amplification primer sequences of rs679106 are SEQ ID No.13 and SEQ ID No. 14; the sequence of the specific extension primer is SEQ ID No. 15;

the sequences of the specific amplification primers of rs9319408 are SEQ ID No.10 and SEQ ID No. 11; the sequence of the specific extension primer is SEQ ID No. 12;

the specific amplification primer sequence of rs1429372 is SEQ ID No.16 and SEQ ID No. 17; the sequence of the specific extension primer is SEQ ID No. 18;

rs2069852 has the specific amplification primer sequence of SEQ ID No.19 and SEQ ID No. 20; the sequence of the specific extension primer is SEQ ID No. 21;

rs1041983 specific amplification primer sequence of SEQ ID No.31 and SEQ ID No. 32; the sequence of the specific extension primer is SEQ ID No. 33.

Example 6:

in this example 6, a kit for evaluating the risk of hepatotoxicity reaction of an antituberculous drug is prepared and used to detect KCNE3 gene SNP site rs12272502, NAT2 gene SNP site rs4921914, BLNK gene SNP site rs2290714, PPP2R2B gene SNP site rs1031915, VAV2 gene SNP site rs679106, FLT3 gene SNP site rs9319408, XO gene SNP site rs1429372, IL6 gene SNP site rs2069852, and NAT2 gene SNP site rs 1495741.

Compared with example 5, the SNP marker to be detected based on the kit of this example 6 is different from it only in that the SNP locus of the NAT2 gene detected in this example 6 is rs1495741, not rs 1041983.

Accordingly, the reagents of the kit are essentially the same as in example 5, except that: the kit contains a specific amplification primer and a specific extension primer of the SNP locus rs1495741, and replaces a related reagent of rs 1041983. Wherein, the specific amplification primer sequence of rs1495741 is SEQ ID No.34 and SEQ ID No. 35; the sequence of the specific extension primer is SEQ ID No. 36.

Example 7:

in this example 7, a kit for evaluating the risk of hepatotoxicity reaction of an antituberculous drug is prepared and used to detect SNP site rs12272502 of KCNE3 gene, SNP site rs7816847 and rs4921914 of NAT2 gene, SNP site rs2290714 of BLNK gene, SNP site rs1031915 of PPP2R2B gene, SNP site rs2069852 of IL6 gene, and SNP site rs1495741 of NAT2 gene.

the specific amplification primer sequences of rs7816847 are SEQ ID No.28 and SEQ ID No. 29; the sequence of the specific extension primer is SEQ ID No. 30;

the specific amplification primer sequence of rs1495741 is SEQ ID No.34 and SEQ ID No. 35; the sequence of the specific extension primer is SEQ ID No. 36.

The above-described embodiments are only arbitrary combinations of the technical features of the present invention, and for the sake of brevity, all possible combinations of the technical features in the above-described embodiments are not described, however, as long as there is no contradiction between the combinations of the technical features, the embodiments should be considered as the scope of the present description.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

SEQUENCE LISTING

<110> Beijing Guangdong Tai science and technology Limited liability company

<120> KCNE3 and NAT2 gene-based SNP marker related to hepatotoxicity reaction of antituberculosis drug, kit and application

<160>36

<170>PatentIn version 3.5

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Claims

1. An SNP marker related to hepatotoxicity of antituberculous drugs based on KCNE3 and NAT2 genes, which is characterized by comprising the KCNE3 gene SNP site rs12272502 and one or more of NAT2 gene SNP sites rs4921914, rs10103029 and rs 7816847.

2. The SNP marker related to hepatotoxicity of antituberculous drugs according to claim 1, further comprising one or more of SNP site rs2290714 of BLNK gene, SNP site rs1031915 of PPP2R2B gene, SNP site rs9319408 of FLT3 gene, SNP site rs679106 of VAV2 gene, SNP site rs1429372 of XO gene and SNP site 206rs 9852 of IL6 gene.

3. The SNP marker associated with a hepatotoxicity response to an anti-tubercular drug according to claim 1 or 2, further comprising SNP site rs1041983 and/or rs1495741 of the NAT2 gene.

4. Use of a SNP marker associated with anti-tubercular drug hepatotoxicity according to claims 1 or 2, in non-diagnostic applications of anti-tubercular drug hepatotoxicity reactions.

5. The non-diagnostic use of a SNP marker associated with hepatotoxic response to anti-tubercular drugs according to claim 3.

6. Use of a SNP marker associated with a hepatotoxicity response of an anti-tubercular drug for the manufacture of a kit for assessing the risk of a hepatotoxicity response of an anti-tubercular drug, wherein the SNP marker associated with a hepatotoxicity response of an anti-tubercular drug comprises SNP site rs12272502 of KCNE3 gene and comprises one or more of SNP sites rs4921914, rs10103029 and rs7816847 of NAT2 gene.

7. A kit for assessing the risk of hepatotoxicity of an anti-tubercular drug, comprising specific amplification primers specific for the SNP marker set forth in claim 1, or comprising said specific amplification primers and specific extension primers specific for the SNP marker set forth in claim 1.

8. The kit for assessing the risk of hepatotoxicity of antituberculous drugs according to claim 7, wherein said specific amplification primers contained therein comprise:

the specific amplification primer sequence of the SNP locus rs12272502 is SEQ ID No.1 and SEQ ID No. 2.

9. The kit for assessing the risk of hepatotoxicity of antituberculous drugs according to claim 8, wherein said specific extension primers contained therein comprise:

the sequence of the specific extension primer of the SNP locus rs12272502 is SEQ ID No. 3.

10. The kit for assessing the risk of hepatotoxicity of antituberculous drugs according to claim 8 or 9, wherein in said specific amplification primers contained:

the specific amplification primer sequences of the SNP locus s4921914 are SEQ ID No.22 and SEQ ID No. 23;

the specific amplification primer sequences of the SNP locus rs10103029 are SEQ ID No.25 and SEQ ID No. 26;

the specific amplification primer sequences of the SNP locus rs7816847 are SEQ ID No.28 and SEQ ID No. 29.

11. The kit for assessing the risk of hepatotoxicity of antituberculous drugs according to claim 10, wherein in said specific extension primers contained:

the sequence of the specific extension primer of the SNP locus rs4921914 is SEQ ID No. 24;

the sequence of the specific extension primer of the SNP locus rs10103029 is SEQ ID No. 27;

the sequence of the specific extension primer of the SNP locus rs7816847 is SEQ ID No. 30.

12. Use of the SNP markers associated with anti-tubercular drug hepatotoxicity responses for the manufacture of a kit for assessing the risk of anti-tubercular drug hepatotoxicity responses according to claim 6, characterized in that said SNP markers associated with anti-tubercular drug hepatotoxicity responses further comprise one or more of BLNK gene SNP site rs2290714, PPP2R2B gene SNP site rs1031915, FLT3 gene SNP site rs9319408, VAV2 gene SNP site rs679106, XO gene SNP site rs 9372 and IL6 gene SNP site rs 2069852.

13. The kit for assessing the risk of hepatotoxicity of antituberculous drugs according to claim 11, further comprising specific amplification primers specific for the SNP marker according to claim 2, or comprising said specific amplification primers and specific extension primers specific for the SNP marker according to claim 2.

14. The kit for assessing the risk of a hepatotoxic response to an anti-tubercular drug according to claim 13, wherein said specific amplification primers contained therein are selected from the group consisting of:

the specific amplification primer sequences of the SNP locus rs2290714 are SEQ ID No.4 and SEQ ID No. 5;

the specific amplification primer sequence of the SNP locus rs1031915 is SEQ ID No.7 and SEQ ID No. 8;

the specific amplification primer sequence of the SNP locus rs9319408 is SEQ ID No.10 and SEQ ID No. 11;

the specific amplification primer sequence of the SNP locus rs679106 is SEQ ID No.13 and SEQ ID No. 14;

the specific amplification primer sequence of the SNP locus rs1429372 is SEQ ID No.16 and SEQ ID No. 17;

the specific amplification primer sequences of the SNP locus rs2069852 are SEQ ID No.19 and SEQ ID No. 20.

15. The kit for assessing the risk of hepatotoxicity of an anti-tubercular drug according to claim 14, wherein said specific extension primers contained therein:

the sequence of the specific extension primer of the SNP locus rs2290714 is SEQ ID No. 6;

the sequence of the specific extension primer of the SNP locus rs1031915 is SEQ ID No. 9;

the sequence of the specific extension primer of the SNP locus rs9319408 is SEQ ID No. 12;

the sequence of the specific extension primer of the SNP locus rs679106 is SEQ ID No. 15;

the sequence of the specific extension primer of the SNP locus rs1429372 is SEQ ID No. 18;

the sequence of the specific extension primer of the SNP locus rs2069852 is SEQ ID No. 21.

16. Use of a SNP marker associated with anti-tubercular drug hepatotoxicity responses for the manufacture of a kit for assessing the risk of anti-tubercular drug hepatotoxicity responses according to claim 12, characterized in that it also comprises NAT2 gene locus rs1495741 and/or rs 1041983.

17. The kit for assessing the risk of hepatotoxicity of antituberculous drugs according to claim 15, further comprising specific amplification primers specific for the SNP marker according to claim 3, or the specific amplification primers and specific extension primers specific for the SNP marker according to claim 3.

18. The kit for assessing the risk of a hepatotoxic response to an anti-tubercular drug according to claim 17, wherein said specific amplification primers contained therein are selected from the group consisting of:

the specific amplification primer sequence of the SNP locus rs1041983 is SEQ ID No.31 and SEQ ID No. 32;

the specific amplification primer sequences of the SNP locus rs1495741 are SEQ ID No.34 and SEQ ID No. 35.

19. The kit for assessing the risk of a hepatotoxic response to an anti-tubercular drug according to claim 18, wherein said specific extension primers contained therein are selected from the group consisting of:

the sequence of the specific extension primer of the SNP locus rs1041983 is SEQ ID No. 33;

the sequence of the specific extension primer of the SNP locus rs1495741 is SEQ ID No. 36.

20. The kit for assessing the risk of hepatotoxic response to antitubercular drug according to claim 19, wherein the kit is used for detecting SNP site rs12272502 of KCNE3 gene, SNP site rs4921914 of NAT2 gene, SNP site rs2290714 of BLNK gene, SNP site rs1031915 of PPP2R2B gene, SNP site rs679106 of VAV2 gene, SNP site rs9319408 of FLT3 gene, SNP site rs1429372 of XO gene, SNP site rs2069852 of IL6 gene and SNP site rs1041983 of NAT2 gene;

the kit comprises the specific amplification primer and the specific extension primer of the SNP site of the gene to be detected, and the specific amplification primer and the specific extension primer of the SNP site of the gene to be detected are respectively as follows:

21. The kit for assessing the risk of hepatotoxic response to antitubercular drug according to claim 19, wherein the kit is used for detecting SNP site rs12272502 of KCNE3 gene, SNP site rs4921914 of NAT2 gene, SNP site rs2290714 of BLNK gene, SNP site rs1031915 of PPP2R2B gene, SNP site rs679106 of VAV2 gene, SNP site rs9319408 of FLT3 gene, SNP site rs1429372 of XO gene, SNP site rs2069852 of IL6 gene and SNP site rs1495741 of NAT2 gene;

22. The kit for assessing the risk of hepatotoxic response to antituberculous drugs according to claim 19, wherein the kit is used for detecting SNP site rs12272502 of KCNE3 gene, SNP sites rs7816847 and rs4921914 of NAT2 gene, SNP site rs2290714 of BLNK gene, SNP site rs1031915 of PPP2R2B gene, SNP site rs2069852 of IL6 gene and SNP site rs1495741 of NAT2 gene;

23. Use of a SNP marker associated with anti-tubercular drug hepatotoxicity responses for the manufacture of a kit for assessing the risk of anti-tubercular drug hepatotoxicity responses according to claim 6, characterized in that it also comprises NAT2 gene locus rs1495741 and/or rs 1041983.