Comprehensive sequencing method for pharmacokinetic-related genes and primer sets used for the method

Abstract

A method for comprehensively analyzing the sequences of multiple types of pharmacokinetics-related genes derived from a subject, comprising: a first step of carrying out amplification reactions for each of the plurality of pharmacokinetic-related genes separately in a plurality of reaction systems using primer sets designed for each of the plurality of pharmacokinetic-related genes in order to amplify the sequences of the plurality of pharmacokinetic-related genes in a distinguishable manner; a second step of sequencing the gene amplification product obtained in the first step; A method characterized in that each of the primer sets is designed to amplify a region of 260 to 490 bp.

Description

[Technical field]

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

[0002] It is known that individual differences in drug pharmacokinetics, such as absorption from the digestive tract, drug metabolism in the liver, and excretion in bile and urine, cause inter-individual variations in drug blood concentrations, resulting in individual differences in drug efficacy and risk of side effects. Such individual differences in pharmacokinetics are often due to differences in the base sequences of genes related to drug metabolism and biomembrane transport of drugs, and investigating such individual differences in gene sequences will lead to the identification of genomic biomarkers for predicting drug efficacy and side effects.

[0003] However, pharmacokinetic-related genes often exist as families with multiple members that have very similar sequences, so that PCR may amplified each member together. Furthermore, sequence analysis may not be able to distinguish whether a difference in sequence is due to an actual difference in sequence or a misreading (false positive) in the sequence analysis. As described above, many of the pharmacokinetic-related genes are located in complex genomic regions, making it difficult to perform highly accurate and comprehensive genotyping using conventional methods that use PCR or next-generation sequencers. [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Gordon et al., Hum Mol Genet, 2014, Apr. 15, Vol. 23(8):1957-63 [Non-Patent Document 2] Twist et al., NPJ Genom Med, 2016, Jan. 13, 1:15007 Summary of the Invention [Problem to be solved by the invention]

[0005] As described above, methods for comprehensively analyzing the sequences of pharmacokinetic-related genes include whole genome sequencing (WGS) and whole exome sequencing (WES) using a next-generation sequencer. However, WGS and WES are time-consuming and costly, and since pharmacokinetic-related genes are present in complex genomic regions and have high sequence homology, it has been difficult to accurately analyze mutations in pharmacokinetic-related genes. Therefore, an objective of the present invention is to provide a method for efficiently and accurately comprehensively analyzing the sequences of pharmacokinetic-related genes. [Means for solving the problem]

[0006] The present inventors have conducted extensive research to solve the above problems. As a result, they have found that the sequences of multiple pharmacokinetic-related genes derived from a subject can be efficiently and comprehensively analyzed with high accuracy by performing a first step of performing an amplification reaction for each of multiple pharmacokinetic-related genes separately in multiple reaction systems using a primer set designed for each pharmacokinetic-related gene in order to amplify the sequences of the pharmacokinetic-related genes while distinguishing them from one another, the primer set designed to amplify a region of 260 to 490 bp each, and a second step of analyzing the sequences of the gene amplification products obtained in the first step. More specifically, we have compiled a list of 100 pharmacokinetic-related genes, including 60 types of drug-metabolizing enzymes and 37 types of drug transporters that have been reported to be involved in drug metabolism. By targeting the translation regions of these genes and enriching the target regions using a multiplex PCR method that can amplify only specific genomic regions, we have succeeded in developing a targeted resequencing analysis panel that can comprehensively and highly accurately detect genetic polymorphisms and extremely rare mutations (rare variants) that affect the functions of the 100 genes. Based on the above, we have completed the present invention.

[0007] The gist of the present invention is as follows. [1] A method for comprehensively analyzing the sequences of multiple types of pharmacokinetic-related genes derived from a subject, comprising: a first step of performing an amplification reaction for each of the plurality of pharmacokinetic-related genes in a plurality of reaction systems using a primer set designed for each of the plurality of pharmacokinetic-related genes in order to amplify the sequences of the plurality of pharmacokinetic-related genes in a distinguishable manner; and A second step of sequencing the gene amplification product obtained in the first step, The method is characterized in that each of the primer sets is designed to amplify a region of 260 to 490 bp. [2] The method according to [1], wherein an amplification reaction is carried out for multiple regions for each of the multiple types of pharmacokinetic-related genes. [3] The method according to [1] or [2], wherein the multiple pharmacokinetic-related genes to be analyzed are 25 or more genes selected from the 100 genes in Table 1. [4] The method according to [1] or [2], wherein the multiple pharmacokinetic-related genes to be analyzed are 50 or more genes selected from the 100 genes in Table 1. [5] The method according to [1] or [2], wherein the multiple pharmacokinetic-related genes to be analyzed are all 100 genes in Table 1. [6] The method according to any one of [1] to [5], wherein the plurality of reaction systems are 6 to 10 reaction systems. [7] The method according to any one of [1] to [6], using the primer set described in Table 1. [8] A reagent used in a method for comprehensively analyzing the sequences of multiple types of pharmacokinetic-related genes derived from a subject, the reagent comprising multiple primer sets selected from the primer sets listed in Table 2. Effect of the Invention

[0008] According to the present invention, the sequences of pharmacokinetic-related genes can be analyzed efficiently, at low cost, and with high accuracy in a comprehensive manner. For example, by using the analysis method of the present invention to analyze DNA samples from patients who have experienced severe side effects such as liver damage or interstitial pneumonia caused by cancer molecular targeted drugs, functional polymorphisms that can serve as medically useful genomic biomarkers can be identified.

[0009] Furthermore, according to the present invention, it is possible to analyze rare variants that cannot be detected by GWAS. Also, by examining the presence or absence of variants related to severe side effects, it is possible to predict side effects before administering a drug, which is useful for determining a treatment plan. Furthermore, according to the present invention, it is possible to analyze many subject samples with high throughput. [Brief description of the drawings]

[0010] [Figure 1] FIG. 1 is an analysis flow diagram showing one embodiment of the method of the present invention. [Diagram 2] FIG. 1 is a schematic diagram for explaining primer design in the method of the present invention and the concept of dividing reaction vessels in multiple reaction PCR, taking CYP1A1 (A) and CYP1A2 (B) as examples. [Diagram 3] FIG. 1 is a diagram comparing the mutation detection accuracy between the method of the present invention and WES. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] The method of the present invention is a method for comprehensively analyzing sequences of multiple types of pharmacokinetic-related genes derived from a subject, comprising the steps of: a first step of performing an amplification reaction for each of the plurality of pharmacokinetic-related genes in a plurality of reaction systems using a primer set designed for each of the plurality of pharmacokinetic-related genes in order to distinguish and amplify the sequences of the plurality of pharmacokinetic-related genes; The second step involves sequence analysis of the gene amplification product obtained in the first step.

[0012] <Pharmacokinetics-related genes> Pharmacokinetic-related genes include genes encoding drug-metabolizing enzymes or drug transporters, but may also be genes not classified as such, as long as their mutations may be involved in side effects of drugs. For example, NUDT1, NUDT15, VKORC1, etc., listed in Table 1, are also included in the pharmacokinetic-related genes. The pharmacokinetic-related genes are preferably genes expressed in the liver.

[0013] More specifically, pharmacokinetic-related genes include those listed in Table 1. It is preferable to analyze 25 or more types of pharmacokinetic-related genes, more preferably 50 or more types, and even more preferably all 100 types. Among the pharmacokinetic-related genes, there are CYP2D6, CYP2C9, CYP2C19, CYP4F2, CYP2B6, DPYD, TPMT, SLCO1B1, UGT1A1, CYP3A5, etc., whose mutations are known to be involved in side effects of drugs, and it is preferable to analyze at least these.

[0014] Each step will be described below.

[0015] <First step> In the first step, a primer set designed for each pharmacokinetic-related gene is used, and the amplification reaction for each of the multiple types of pharmacokinetic-related genes is carried out in multiple reaction systems.

[0016] <Primer set> A primer set is designed for each of the multiple pharmacokinetic-related genes in order to distinguish and amplify the sequences of the multiple pharmacokinetic-related genes. For example, when analyzing 10 types of genes, A, B, C, D, E, F, G, H, I, and J, 10 primer sets are prepared for specifically amplifying each of the 10 types of genes, A to J. The primer sets for each gene can be set in regions that have low homology with other genes based on known gene sequences.

[0017] In particular, in order to distinguish and amplify genes belonging to the same family, it is preferable to compare the sequences of both genes and design primers at two positions where the sequences differ.

[0018] In addition, many pharmacokinetic-related genes such as cytochrome P450 have pseudogenes or highly similar sequences. Therefore, the read length of about 2x100bp of the commonly used next-generation sequencer (NGS) makes it impossible to distinguish the sequences of each gene, so the primer sets are designed to amplify a region of 260 to 490bp each. By amplifying and analyzing a region of this length, it is possible to amplify the sequence without reducing the amplification efficiency, and furthermore, it is possible to accurately distinguish between highly homologous sequences and perform highly accurate sequence analysis.

[0019] Alternatively, multiple regions of a single pharmacokinetic-related gene may be amplified and sequenced, allowing more mutations to be detected and for more accurate discrimination between genes. The region to be amplified is preferably a coding region (exon), and when multiple regions are amplified, it is preferable to amplify each exon separately. However, multiple regions may be amplified for one exon.

[0020] FIG. 2 shows an example of primer set design using CYP1A1 and CYP1A2 as examples. CYP1A1 and CYP1A2 are genes belonging to the same family (CYP1A family) and have similar sequences. These sequences are compared, and nine regions where the sequences differ between the two are amplified: three in exon 1, one in exon 2, one in exon 3, one in exon 4, one in exon 5, and two in exon 6. Primers F1 (forward primer 1) and R1 (reverse primer 1) for CYP1A1 and primers F1 and R1 for CYP1A2 were designed to amplify similar regions (approximately 300 bp) of CYP1A1 and CYP1A2, respectively, but the former was designed to hybridize only to CYP1A1 and the latter was designed to hybridize only to CYP1A2.

[0021] Examples of primer sets for each gene are shown in Table 2. These primer sets can be appropriately selected and used depending on the type of gene to be analyzed. For example, when analyzing 25 types of genes out of the 100 genes in Table 1, the primer sets shown in Table 2 can be used for the selected 25 types of genes.

[0022] <Amplification reaction> An amplification reaction is carried out for each of the multiple types of pharmacokinetic-related genes. The amplification reaction is carried out in a plurality of reaction systems, the number of which is preferably 4 or more and 15 or less, more preferably 6 or more and 10 or less, and particularly preferably 8.

[0023] For example, when amplifying 8 sites for each of 100 types of genes, a total of 800 reactions will be performed, but these will be divided into multiple reaction systems. For example, 100 reactions using 100 primer sets can be performed per reaction system, making it possible to perform reactions in a total of 8 reaction systems. In one reaction system, for example, 50 to 200 amplification reactions using 50 to 200 primer sets can be performed simultaneously. The number of reactions in each reaction system does not necessarily have to be the same.

[0024] When separating multiple reaction systems in this manner, particularly when genes present in the same family are to be amplified in a differentiated manner, it is preferable to carry out reactions for amplifying corresponding regions of these genes in separate reaction systems. For example, as shown in Figure 2, when CYP1A1 and CYP1A2 are included in the analysis target, in order to ensure that they are amplified separately, the primer sets that amplify the similar regions in CYP1A1 and CYP1A2, in this case F1-R1 of CYP1A1 and F1-R1 of CYP1A2, are added to separate reaction systems and the amplification reactions are carried out separately. This allows similar genes to be amplified separately, ensuring that false positives in sequence analysis are avoided.

[0025] The reaction can be carried out according to normal PCR conditions. 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.

[0026] <Second process> In the second step, the amplification products of each pharmacokinetic-related gene obtained in the first step are subjected to sequence analysis. Prior to sequence analysis, amplification may be performed using primers containing a sequence for sequence analysis in order to add a sequence for sequence analysis. The method of 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 the presence or absence of mutations can be determined by comparing each gene with the wild-type sequence.

[0027] When a mutation is found in a pharmacokinetic-related gene by the sequence analysis method of the present invention, the administration of a drug can be adjusted according to the type of the mutation. For example, when a patient has a high-risk mutation for side effects of a particular drug, the administration of the drug can be stopped or the dosage can be reduced. EXAMPLES

[0028] The present invention will be specifically described below with reference to examples, but the present invention is not limited to the following embodiments.

[0029] The workflow of targeted resequencing using the method of the present invention is shown in Figure 1. It is possible to specifically amplify the coding regions of the 100 pharmacokinetic-related genes listed in Table 1. st PCR primers (1,102 sets) were designed (Table 2). The primer sets were divided into eight groups, and multiplex PCR was performed for each group. The resulting PCR products were then subjected to 2-step PCR to add adapter sequences required for Illumina MiSeq analysis and index sequences for multiple sample identification. nd PCR was performed and the resulting library was sequenced on an Illumina MiSeq (2 × 250 bp). The specific steps are as follows:

[0030] (1) Primer design The total length of the coding regions of the 100 pharmacokinetic-related genes was 162,216 bp. To specifically amplify these coding regions, primers of 15-30 bp were designed to obtain PCR products of 260-490 bp. Finally, 1,102 sets of primers (Table 2) were divided into eight groups, and 1 st PCR was performed and a region of 159,347 bp was amplified.

[0031] (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, 2 μL of 0.1 nM primer solution, and a total reaction solution of 10 μL. Initial heat denaturation was performed at 95°C for 2 minutes, and cycling was performed for 25 cycles of heat denaturation at 95°C for 30 seconds, annealing at 60°C for 90 seconds, and extension reaction at 72°C for 3 minutes, with a final extension at 72°C for 10 minutes.

[0032] 2nd PCR 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), 0.05 μL of primer solution (5 μM equimolar mix of index 1 and index 2), 1 st For 2 μL of PCR product and a total reaction solution of 5 μL, initial thermal denaturation was performed at 98°C for 45 seconds, and cycling consisted of 4 cycles of thermal denaturation at 99°C for 15 seconds, annealing at 65°C for 30 seconds, and extension reaction at 72°C for 30 seconds, with a final extension at 72°C for 1 minute.

[0033] 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 After purification of the PCR product, 2 μL of the product and a total reaction solution of 10 μL were used. 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 reactions at 60°C for 45 seconds, for a total of 40 cycles.

[0034] (3) Library purification and sequencing run 2 nd DNA fragments of the desired size were isolated from the PCR products using AMPure XP (Beckman Coulter), and 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).

[0035] (4) Mutation detection Mutation detection was performed using BWA (Li H et al., Bioinformatics, 2009, 25:1754) to map the sequencing Fastq files to the human reference genome (hg19), and mutation detection and filtering was performed using GATK (McKenna A et al., Genome Res, 2010, 20(9):1297-303). Copy number variation (CNV) detection was performed using BED Tools (Quinlan AR, 2014, 47:11, 12, 1-34).

[0036] As a result, as shown in Figure 3, 163 mutations were detected in one person's genome. When WES was used on the same genome, 162 mutations were detected. When compared with the results of Sanger sequencing, the gold standard for mutation detection, the sensitivity and specificity of the present invention were both 100%. Meanwhile, the sensitivity and specificity of WES were 91.4% and 99.992%, respectively. The breakdown of the discrepancies between the results of WES and Sanger sequencing was 13 false positives and 14 undetected mutations due to insufficient reads.

[0037] [Table 1]

[0038] [Table 2-1] [Table 2-2] [Table 2-3] [Table 2-4] [Table 2-5] [Table 2-6]

Table 2-7

Table 2-8

Table 2-9

Table 2-10

Table 2-11

Table 2-12

Table 2-13

Table 2-14

Table 2-15

Claims

1. A method for comprehensively analyzing sequences of multiple types of pharmacokinetic-related genes derived from a subject, comprising the steps of: a first step of amplifying a plurality of regions of each of the plurality of pharmacokinetic-related genes in eight separate reaction systems using a primer set designed for each of the plurality of pharmacokinetic-related genes in order to distinguish and amplify the sequences of the plurality of pharmacokinetic-related genes from one another; A second step of sequencing the gene amplification product obtained in the first step, The multiple pharmacokinetic-related genes include 100 pharmacokinetic-related genes (ABCB1, ABCB4 , ABCB11, ABCC1, ABCC2, ABCC3, ABCC4, ABCG2, CES1, CES2, CYP1A1, CYP1A2, CYP1B1 , CYP2A6, CYP2A13, CYP2B6, CYP2C8, CYP2C9, CYP2C18, CYP2C19, CYP2D6, CYP2E1, CYP2J2, CYP2S1, CYP2W1, CYP3A4, CYP3A5, CYP3A7, CYP3A43, CYP4A11, CYP4B1, CYP4B2, CYP4F3, CYP4F8, CYP4F12, C YP4Z1, CYP11A1, CYP17A1, CYP19A1, CYP26A1, DPYD, FMO1, FMO2, FMO3, FMO4, FMO5, GSTA1, GSTM1, G STP1, GSTT1, NAT1, NAT2, NUDT1, NUDT15, POR, SLC10A1, SLC10A2, SLC15A1, SLC15A2, SLC16A7, SLC1 9A1, SLC22A1, SLC22A2, SLC22A3, SLC22A4, SLC22A5, SLC22A6, SLC22A8, SLC22A9, SLC22A11, SLC2 2A12, SLC28A1, SLC28A2, SLC28A3, SLC29A1, SLC29A2, SLC29A3, SLC31A1, SLC46A1, SLC47A1, SLC47 A2, SLCO1B1, SLCO1B3, SLCO2B1, SLUT1A1, SLUT1A2, SLUT1E1, SLUT2B1, TPMT, UGT1A1, UGT1A3, UGT1A4, UGT1A5, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B7, VKORC1), Each of the primer sets is designed to amplify a region of 260 to 490 bp, and is selected from a primer set consisting of a forward primer and a reverse primer listed in Table 2; The reaction systems are eight reaction systems described in the Pool column of Table 2, and a plurality of the primer sets described in Table 2 corresponding to each of the 25 or more types of the pharmacokinetic-related genes to be analyzed are used in the reaction systems described in the Pool column of Table 2, respectively, to perform reactions for amplifying corresponding regions between the pharmacokinetic-related genes present in the same family in separate reaction systems. 【Table 1-1】 【Table 1-2】 【Table 1-3】 【Table 1-4】 【Table 1-5】 【Table 1-6】 【Table 1-7】 【Table 1-8】 【Table 1-9】 【Table 1-10】 【Table 1-11】 【Table 1-12】 【Table 1-13】 【Table 1-14】 【Table 1-15】

2. The plurality of pharmacokinetic-related genes to be analyzed are 50 selected from the 100. The method according to claim 1, wherein the gene is one of the above.

3. The plurality of pharmacokinetic-related genes to be analyzed are all of the 100 genes. The method of claim 1.

4. A reagent for use in the method for comprehensively analyzing sequences of multiple types of pharmacokinetic-related genes derived from a subject according to any one of claims 1 to 3, comprising: A reagent comprising a plurality of primer sets described in Table 2 corresponding to each of the pharmacokinetics-related genes to be analyzed, each of the primer sets consisting of a combination of a forward primer and a reverse primer described in Table 2.

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