Method for sequencing genes related to drug side effects and primer sets used therefor

Abstract

To provide a method for analyzing sequences of side effect-related HLA genes and pharmacokinetic-related 14 genes efficiently, accurately and comprehensively.SOLUTION: The present invention provides a data acquisition (gene sequence analysis) method for determining a side effect expression risk of a drug. The method includes the steps of: preparing a plurality of primer sets for amplifying 261-483bp regions including a pharmacokinetic mutation for each of human leukocyte antigen (HLA) genes, including HLA-A gene, HLA-B gene, HLA-DRB1 gene and HLA-DQA1 gene and specific 14 genes; using the plurality of primer sets to perform an amplification reaction in a plurality of reaction systems for each of the four types of HLA genes derived from a subject and pharmacokinetic-related 14 genes; and analyzing the sequences of resultant gene amplification products.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to genetic analysis, and more particularly to research, diagnosis, medical treatment, etc., that utilize genetic analysis. [Background technology]

[0002] There are many types of human leukocyte antigen (HLA) genes that determine human leukocyte antigens, and each HLA gene has several dozen different types (alternatives). HLA is closely related to immunity and plays a role in the occurrence of side effects such as drug rash and agranulocytosis. It has been reported that certain alleles are risk factors. Individual differences in drug pharmacokinetics, such as absorption from the gastrointestinal tract, drug metabolism in the liver, and excretion in bile and urine, cause interindividual variations in drug blood concentrations, resulting in individual differences in drug efficacy and the risk of side effects. These individual differences in pharmacokinetics are often due to differences in the base sequences of genes related to drug metabolism and biomembrane transport, and investigating these individual differences in gene sequences can help predict the risk of drug side effects.

[0003] However, because HLA genes have very similar sequences, PCR can sometimes amplify each component together, and sequence analysis can sometimes make it impossible to distinguish whether a difference in sequence is due to an actual sequence difference or a misreading (false positive) in the sequence analysis. As HLA genes are present in complex genomic regions, it has been difficult to perform highly accurate and comprehensive genotyping using conventional methods such as PCR or next-generation sequencers. Non-patent document 1 describes a method for typing HLA genes using sequence-specific oligonucleotide probes. A comparison between a method using a DNA probe and a method using a next-generation sequencer is disclosed.

[0004] Patent Document 1 discloses a comprehensive analysis method for pharmacokinetic-related genes. However, the analysis does not include HLA genes. Furthermore, Patent Document 1 analyzes as many as 100 types of genes, and there has been a demand for a method for efficiently analyzing drug side effects using a smaller number of genes. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2020 / 145351 [Non-patent literature]

[0006] [Non-Patent Document 1] Smith AG et al., HLA.2019;1-11. Summary of the Invention [Problem to be solved by the invention]

[0007] The present invention efficiently identifies the sequences of side effect-related genes, particularly HLA genes and pharmacokinetic-related genes. Another object of the present invention is to provide a method for efficiently obtaining information for predicting side effects of drugs through accurate and comprehensive analysis. [Means for solving the problem]

[0008] The present inventors have conducted extensive research to solve the above problems. As a result, in order to amplify the sequences of multiple types of side effect-related HLA genes in a distinguishable manner, a primer set designed to amplify a region of 261 to 483 bp for each gene was used, and a primer set for each HLA gene was used. It has been found that the sequences of multiple types of side effect-related HLA genes derived from subjects can be analyzed efficiently and comprehensively with high accuracy by performing a first step in which the amplification reaction for each gene is carried out in multiple reaction systems, and a second step in which the gene amplification products obtained in the first step are sequenced. I put it out.

[0009] More specifically, we listed four HLA genes (HLA-A, HLA-B, HLA-DRB1, and HLA-DQA1) that have been reported to be involved in side effects. We targeted the coding regions of these genes and used multiplex PCR, which can amplify only specific genomic regions. Targeted resequencing enables comprehensive and highly accurate detection of alleles and rare variants associated with specific side effects through enrichment. We succeeded in developing an analytical panel. In addition, we used similarly designed primer sets for 14 pharmacokinetic-related genes and performed amplification reactions in the same multiple reaction systems as for HLA genes. We found that by performing this and analyzing the sequences, we could efficiently obtain more information about the risk of drug side effects. Based on the above, the present invention has been completed.

[0010] The gist of the present invention is as follows. [1] A method for obtaining data (gene sequence analysis) to determine the risk of adverse drug reactions; Human leukocyte antigen (HLA) genes, including HLA-A gene, HLA-B gene, HLA-DRB1 gene, and preparing a plurality of primer sets for amplifying a 261 to 483 bp region containing a mutation associated with a side effect for each of the HLA-DQA1 and HLA-DQA2 genes; performing an amplification reaction for each of the four types of HLA genes derived from a subject in a plurality of reaction systems using the plurality of primer sets; and a step of sequencing the obtained gene amplification product; A method comprising: [2] The method according to [1], wherein the plurality of reaction systems are the reaction systems of 4. [3] The method according to [1] or [2], using the primer set listed in Table 1. [Table 1] (SEQ ID NOs: 1 to 74) [4] Furthermore, CYP (Cytochrome P450) 2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP3A5, DPYD (dihydropyrimidine dehydrogenase), NAT2 (N-acetyltransferases 2), NUDT15 (Nudix hydrolase 15), TPMT (Thiopurine S-methyltransferase), UGT1A1 (Uridine diphosphate glucuronosyltransferase 1A1), SLCO1B1 (solute carrier organic anion transporter family member 1B1), ABCG2 (ATP binding cassette subfamily G member 2), and VKORC1 (vitamin K epoxide reductase complex subunit 1). Multiple primer sets were prepared to amplify the 261-483 bp region containing the mutation associated with side effects. The method according to any one of [1] to [3], wherein in the step of amplifying the HLA genes, the amplification reaction of 14 genes is carried out in the same reaction system as the amplification reaction of the HLA genes. [5] The method according to [4], wherein the amplification reaction of the 14 genes is carried out using the primer sets listed in Table 2-1 and Table 2-2. [6] A reagent used in a method for comprehensively analyzing the sequences of HLA-A genes, HLA-B genes, HLA-DRB1 genes, and HLA-DQA1 genes derived from a subject, the reagent comprising multiple primer sets listed in Table 1. [7] Furthermore, the subjects' CYP (Cytochrome P450) 2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP3A5, DPYD (dihydropyrimidine dehydrogenase), NAT2 (N-acetyltransferases 2), NUDT15 (Nudix hydrolase 15), TPMT (Thiopurine S-methyltransferase), UGT1A1 (Uridine diphosphate glucuronosyltransferase 1A1), SLCO1B1 (solute carrier organic anion transporter family member 1B1), ABCG2 (ATP binding cassette subunit), and ATP-binding cassette subunits were analyzed. The reagent described in [6] is used in a method for comprehensively analyzing 14 genes, namely, mitochondrial G member 2, and VKORC1 (vitamin K epoxide reductase complex subunit 1), and includes multiple primer sets described in Table 2-1 and Table 2-2. [Table 2-1] (SEQ ID NOs: 75 to 218) [Table 2-2] (SEQ ID NOs: 219 to 358) [Effects of the Invention]

[0011] According to the present invention, the sequences of side effect-related HLA genes can be efficiently, inexpensively, and accurately comprehensively identified. This allows for the acquisition of data (information) for determining the risk of adverse drug reactions. For example, data obtained by the analytical method of the present invention can be used to predict the risk of serious adverse drug reactions, such as drug rash or agranulocytosis, caused by drugs for treating psychiatric and neurological disorders, before starting drug treatment, and can be used to determine treatment strategies. In addition, the analysis of the CYP (Cytochrome P450) 2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP3A5, DPYD (dihydropyrimidine dehydrogenase), NAT2 (N-acetyltransferase 2), and NUDT15 (Nudix hydrolase 15) can be used to predict the risk of serious adverse drug reactions, such as drug rash or agranulocytosis, caused by drugs for treating psychiatric and neurological disorders, before starting drug treatment, and can be used to determine treatment strategies. ), TPMT (Thiopurine S-methyltransferase), UGT1A1 (Uridine diphosphate glucuronosyltransferase 1A1), SLCO1B1 (solute carrier organic anion transporter family member 1B1), ABCG2 (ATP binding cassette subfamily G member 2), and VKORC1 (vitamin K epoxide reductase complex subunit 1) were analyzed in combination. This allows for more efficient prediction of a wider range of side effect risks. Furthermore, the present invention enables analysis of unknown variants that are not registered in PharmVar (https: / / www.pharmvar.org / ), a public database of drug-metabolizing enzyme gene mutations, and enables high-throughput analysis of many subject samples. [Brief explanation of the drawings]

[0012] [Figure 1] 1 is an analytical flow diagram showing one embodiment of the method of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0013] The method of the present invention is a gene sequence analysis method for determining the risk of adverse drug reactions; Human leukocyte antigen (HLA) genes, including HLA-A gene, HLA-B gene, HLA-DRB1 gene, and preparing a plurality of primer sets for amplifying a 261 to 483 bp region containing a mutation associated with a side effect for each of the HLA-DQA1 and HLA-DQA2 genes; performing an amplification reaction for each of the four types of HLA genes derived from a subject in a plurality of reaction systems using the plurality of primer sets; and a step of sequencing the obtained gene amplification product; Includes.

[0014] The HLA genes HLA-A, HLA-B, HLA-DRB1, and HLA-DQA1 are associated with various drugs as shown in Table 3 below. It is known that there are alleles associated with side effects to drugs. Therefore, by amplifying and sequencing the region containing the mutation (allele) of each HLA gene using the method of the present invention, it is possible to detect side effects of drugs. Data can be obtained to determine the risk of adverse reactions.

[0015] [Table 3] 8) HLA Research Institute (http: / / hla.or.jp / med / frequency_search / ja / allele / )

[0016] Each step will be described below.

[0017] <First step> In the first step, a primer set (primer pair) designed to amplify a 261-483 bp region containing a mutation associated with drug side effects was used for each of the four side effect-related HLA genes. The amplification reaction for each type of gene is carried out in multiple reaction systems.

[0018] <Primer set> Primer sets are designed for each of the four side effect-related HLA gene sequences to differentiate and amplify their sequences. The primer set for each gene can be set based on known gene sequence information, and can be located on the 5' and 3' sides of the region containing the mutation, in regions with little homology to other genes. The length of the primers can be, for example, 15 to 30 bp.

[0019] In addition, there are many pseudogenes and highly similar sequences in the HLA gene region. Because the read length of approximately 2 x 100 bp of next-generation sequencers makes it impossible to distinguish the sequences of each gene, the primer sets are designed to amplify a region of 261 to 483 bp each. By amplifying and analyzing regions of this length range, sequences can be amplified without reducing amplification efficiency, and highly homologous sequences can be accurately distinguished, enabling highly accurate sequence analysis.

[0020] For each HLA gene associated with side effects, the region containing the mutation associated with drug side effects was amplified and sequenced. This allows for more mutations to be detected and for genes to be distinguished more accurately. The region to be amplified is preferably a coding region (exon), and when multiple regions are to be amplified, it is preferable to amplify each exon separately. Multiple regions may be amplified by using the same method.

[0021] Examples of primer sets for each gene are shown in Table 1. Primer sets can be selected as appropriate from these primer sets. For each of the HLA-A, HLA-B, HLA-DRB1, and HLA-DQA1 genes, it is preferable to use two or more, four or more, or five or more primer sets from the primer sets in Table 1, and it is particularly preferable to use all of the primer sets in Table 1.

[0022] <Amplification reaction> For each of the four types of side effect-related HLA genes, an amplification reaction is carried out using the primer set selected above. For example, a sample containing genomic DNA from a subject (saliva, blood, hair, etc.) and a reaction reagent containing DNA polymerase can be placed in a reaction vessel together with the above primer set to carry out the reaction. The amplification reaction is carried out in multiple reaction systems, preferably 2 or more, 3 or more, or 4 or more, and preferably 10 or less, 8 or less, or 6 or less, and particularly preferably 4.

[0023] For example, if six locations (six regions) of each of four types of genes are to be amplified, a total of 24 reactions will be performed, but these can be divided into multiple reaction systems. For example, six reactions using six primer sets can be performed per reaction system, resulting in a total of four reaction systems. The number of reactions in each reaction system does not necessarily have to be the same. For example, in Table 1, the reaction is divided into Pool 1 to Pool 4, and each primer set is placed in a reaction tube in each Pool to carry out the reaction.

[0024] The reaction can be carried out according to the conditions of ordinary PCR. For example, 20 to 30 cycles of thermal denaturation, annealing, and amplification are performed. The temperature of each step can be appropriately set depending on the Tm value of the primers, the enzyme used, and the like.

[0025] <Second process> In the second step, the amplification products of each side effect-related HLA gene obtained in the first step are sequenced. Do the following. Before sequence analysis, the DNA may be amplified using a primer containing a sequence for sequence analysis in order to add the sequence for sequence analysis. The method for sequence analysis is not particularly limited, but it is preferable to use a next-generation sequencer or the like. Comprehensive analysis can be performed using next-generation sequencers and comparing each gene with the wild-type sequence to determine whether or not there are any mutations.

[0026] As a result, information can be obtained regarding which HLA gene a subject has which mutation, and as a result, which drug was administered when the risk of side effects is high.

[0027] Based on the information obtained by the sequence analysis method of the present invention, the type (allele) of the HLA gene related to side effects allows for the change of the type of drug and the adjustment of the dosage. For example, when having a high-risk type allele for side effects against a specific drug, the administration of that drug can be discontinued or the dosage can be reduced.

[0028] <Drug metabolism-related genes other than HLA> In the method of the present invention, 14 drug metabolism-related genes of CYP (Cytochrome P450) 2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP3A5, DPYD (dihydropyrimidine dehydrogenase), NAT2 (N-acetyltransferases 2), NUDT15 (Nudix hydrolase 15), TPMT (Thiopurine S-methyltransferase), UGT1A1 (Uridine diphosphate glucuronosyltransferase 1A1), SLCO1B1 (solute carrier organic anion transporter family member 1B1), ABCG2 (ATP binding cassette subfamily G member 2), and VKORC1 (vitamin K epoxide reductase complex subunit 1) may be analyzed simultaneously.

[0029] These genes are known to have mutations associated with various pharmacokinetics, as shown in Table 4. These genes are also disclosed in Patent Document 1, but this is a group of genes selected to efficiently predict the pharmacokinetics (blood drug concentrations) of a wide range of drugs with a smaller number of genes.

[0030] [Table 4]

[0031] 1)Kumondai et al. Biol Pharm Bull. 2016;39(1):84-9. 2) Nakajima et al. Pharmacogenet Genomics. 2007 Jun;17(6):431-45. 3) Mushiroda at al. J Hum Genet. 2006;51(3):249-53. 4) Tamura et al. J Clin Oncol. 2020 Feb 20;38(6):558-566. 5) NBDC Human Database (https: / / humandbs.biosciencedbc.jp / hum0163-v2#hum0163.v1.freq.v1) 6)Kakuta et al. J Gastorenterol. 2018 Sep;53(9):1065-1078. 7)Hikino et al. J Hum Genet. 2019 Dec;64(12):1195-1202.

[0032] For each of these 14 genes, multiple primer sets are prepared to amplify the 261-483 bp region containing the mutation. The amplification reaction using these primer sets was also the same as the amplification reaction of the HLA gene described above. Conducted in the same multiple reaction systems. Examples of primer sets for each gene are shown in Tables 2-1 and 2-2. Primer sets can be selected appropriately from these sets depending on the type of gene to be analyzed. For each of the above 14 types of genes, it is preferable to use two or more, four or more, or five or more primer sets from the primer sets in Tables 2-1 and 2-2, and it is particularly preferable to use all of the primer sets in Tables 2-1 and 2-2.

[0033] For example, if six regions of each of the four HLA genes and the 14 genes listed above are to be amplified, a total of 18 genes, a total of 108 reactions will be performed, but these will be divided into multiple reaction systems. For example, 27 reactions using 27 primer sets can be performed per reaction system, resulting in a total of four reaction systems. Note that the number of reactions in each reaction system does not necessarily have to be the same. For example, in Tables 2-1 and 2-2, the reaction is carried out by dividing the reaction mixture into Pool 1 to Pool 4 and placing each primer set in a reaction tube in each Pool. [Example]

[0034] The present invention will be specifically described below with reference to examples, but the present invention is not limited to the following embodiments.

[0035] The workflow for targeted resequencing using the method of the present invention is shown in Figure 1. It can specifically amplify the coding regions of four HLA genes related to side effects (HLA-A, HLA-B, HLA-DRB1, and HLA-DQA1) and 14 pharmacokinetic-related genes. st PCR primer Primers (182 sets) are designed (Table 1, Table 2-1 and Table 2-2). The subjects were divided into four groups, and multiplex PCR was performed in each group. The PCR products were then subjected to 2 PCR reactions to add the adapter sequences required for Illumina MiSeq analysis and index sequences for multiple sample identification.nd PCR is performed and the resulting library is sequenced on an Illumina MiSeq (2 × 250 bp). The specific steps are as follows:

[0036] (1) Primer design The total length of the translated regions of the 18 genes was 67,505 bp. To specifically amplify these translated regions, primers of 16-29 bp were designed to obtain PCR products of 261-483 bp. In summary, 182 sets of primers (Table 1, Table 2-1 and Table 2-2) were divided into four groups (pools listed in each table), and then st PCR was performed to amplify a region of 67,505 bp.

[0037] (2) PCR 1 st PCR was performed using a thermal cycler (GeneAmp PCR System 9700, Thermo Fisher Scientific) with 2 μL of 5 ng / μL template DNA, 5 μL of 2× Platinum Multiplex PCR Master Mix (Thermo Fisher Scientific), 1 μL of GC enhancer, and 2 μL of 0.1 nM primer solution. The total volume of the reaction solution was 10 μL. The initial heat denaturation was performed at 95°C for 2 minutes. The cycling was performed at 95°C for 2 minutes. ℃, 30 seconds, annealing at 60℃, 90 seconds, extension reaction at 72℃, 3 minutes, 25 cycles A final extension was carried out at 72°C for 10 min.

[0038] 2 ndPCR was performed using a thermal cycler (GeneAmp PCR System 9700, Thermo Fisher Scientific) with 2 μL of 5× KAPA HiFi Fidelity Buffer (Kapa Biosystems), 0.3 μL of 10 mM dNTPs, 0.2 U KAPA HiFi HotStart DNA Polymerase (Kapa Biosystems), and primer solution (5 μM equimolar mix of index 1 and index 2) 0.05 μL, 1 st For 2 μL of PCR product and a total reaction volume of 5 μL, initial heat denaturation was performed at 98°C for 45 seconds, and cycling was performed at 99°C for 15 seconds. The annealing was performed at 65°C for 30 seconds, followed by four cycles of extension at 72°C for 30 seconds, with a final extension at 72°C for 1 minute.

[0039] qPCR was performed using a real-time PCR system (Applied Biosystems 7900HT Fast Real-Time PCR System, Thermo Fisher Scientific) with 5 μL of 2× KAPA SYBR FAST qPCR Master Mix (Kapa Biosystems), 2 nd For 2 μL of purified PCR product and a total reaction volume of 10 μL, initial heat denaturation was performed at 95°C for 5 minutes, and cycling was performed at 95°C for 30 seconds, followed by annealing and extension. The reaction was carried out under the conditions of 60°C, 45 seconds, and 40 cycles.

[0040] (3) Library purification and sequencing run 2 nd DNA fragments of the desired size were collected from the PCR products using AMPure XP (Beckman Coulter). The libraries were purified. The libraries from each group were diluted to 4 nM, mixed in equal amounts, and sequenced using the Illumina MiSeq system and MiSeq Reagent Kit v2. (2 × 250 bp, 500 cycles).

[0041] (4) Mutation detection The detection of HLA alleles was performed by using the Fastq files obtained by sequencing as HLA typing sequences. HLA-HD is a genetic algorithm (Kawaguchi S et al, Hum Mutat, 2017, 38(7): This was done by analyzing using the NIRS 20 ...

[0042] As a result, the regions amplified by the above 182 primer sets were sequenced separately. This enabled us to efficiently detect mutations related to pharmacokinetics or side effects in 18 genes.

Claims

1. A gene sequence analysis method for determining a risk of adverse drug reactions, comprising: Human leukocyte antigen (HLA) genes: HLA-A gene, HLA-B gene, and HLA-DRB1 gene and HLA-DQA1 gene, respectively, providing a plurality of primer sets for amplifying a region of 261 to 483 bp in length containing a mutation associated with a side effect; performing an amplification reaction using the plurality of primer sets for each of the four types of HLA genes derived from a subject in four separate reaction systems; and a step of sequencing the obtained gene amplification product; Including, The primer set is selected from the primer sets consisting of a forward primer and a reverse primer listed in Table 1, The reaction systems are four reaction systems described in the Pool column of Table 1, and the multiple primer sets described in Table 1 corresponding to each of the four types of HLA genes to be analyzed are used in the reaction systems described in the Pool column of Table 1, respectively, to perform reactions for amplifying corresponding regions of the four types of HLA genes in separate reaction systems. A method characterized by: Table 1 (SEQ ID NOs: 1 to 74) (Table 1)

2. In addition, CYP (Cytochrome P450) 2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP3A5, DPYD (dihydropyrimidine dehydrogenase), NAT2 (N-acetyltransferases 2), NUDT15 (Nudix hydrolase 15), TPMT (Thiopurine S-methyltransferase), UGT1A1 (Uridine diphosphate glucuronosyltransferase 1A1), SLCO1B1 (solute carrier organic anion transporter family member 1B1), ABCG2 (ATP binding cassette subfamily G member 2 ), and VKORC1 (vitamin K epoxide reductase complex subunit 1) Regarding that, Multiple primer sets were prepared to amplify regions of 261 to 483 bp in length that contained pharmacokinetic-related mutations. In the HLA gene amplification step, the amplification reaction of 14 genes is carried out in multiple reaction systems that are the same as the HLA gene amplification reaction. The method of claim 1.

3. The method described in claim 2, wherein the amplification reaction of the 14 genes is carried out using the primer sets listed in Tables 2-1 and 2-2. Table 2-1 (SEQ ID NOs: 75 to 218) (Table 2-1) Table 2-2 (SEQ ID NOs: 219 to 358) (Table 2-2)

4. A reagent for use in a method for comprehensively analyzing the sequences of HLA-A genes, HLA-B genes, HLA-DRB1 genes, and HLA-DQA1 genes derived from a subject, comprising: A plurality of primer sequences listed in Table 1 corresponding to each of the HLA genes to be analyzed A reagent comprising a plurality of primer sets, each consisting of a combination of a forward primer and a reverse primer as set forth in Table 1. Table 3 (SEQ ID NOs: 1 to 74) (Table 1)

5. Furthermore, subject-derived CYP (Cytochrome P450) 2A6, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP3A5, DPYD (dihydropyrimidine dehydrogenase), NAT2 (N-acetyltransferase 2 ), NUDT15 (Nudix hydrolase 15), TPMT (Thiopurine S-methyltransferase), UGT1A1 (Uridine diphosphate glucuronosyltransferase 1A1), SLCO1B1 (solute carrier organic anion transporter family member 1B1), ABCG2 (ATP binding cassette subfamily G member 2), and VKORC1 (vitamin K epoxide reductase complex subunit 1) The reagent according to claim 4, which is used in a method for comprehensively analyzing 14 genes, and comprises a plurality of primer sets listed in Tables 2-1 and 2-2. Table 4-1 (SEQ ID NOs: 75 to 218) (Table 2-1) Table 4-2 (SEQ ID NOs: 219 to 358) (Table 2-2)

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