Development of Novel Mass Spectrum-Based Assay for Simultaneous Detection of 36 Variants in the 14 Pharmacogenetic Genes for the Japanese Population

Abstract

Pharmacogenetics (PGx) enhances personalized care, often reducing medical costs, and improving patients’ QOL. Unlike single variant analysis, multiplex PGx panel tests can result in applying comprehensive PGx-guided medication to maximize drug efficacy and minimize adverse reactions. Among PGx genes, drug-metabolizing enzymes and drug transporters have significant roles in the efficacy and safety of various pharmacotherapies. In this study, a genotyping panel has been developed for the Japanese population called PGx_JPN panel comprising 36 variants in 14 genes for drug-metabolizing enzymes and drug transporters using a mass spectrometry-based genotyping method, in which all the variants could be analyzed in two wells for multiplex analysis. The verification test exhibited good concordance with the results analyzed using the other standard genotyping methods (microarray, TaqMan assay, or another mass spectrometry-based commercial kit). However, copy number variations such as CYP2D6*5 could not apply to this system. In this study, we demonstrated that the mass spectrometry-based multiplex method could be useful for in the simultaneous genotyping of more than 30 variants, which are essential among the Japanese population in two wells, except for copy number variations. Further study is needed to assess our panel to demonstrate the clinical use of pharmacogenomics for precision medicine in the Japanese population.

INTRODUCTION

Pharmacogenetics (PGx) is the study field of the interactions between genetic variability and the responses to drugs. Until now, several variations have been reported in the genes associated with drug metabolism (e.g., CYP), disposition/excretion (e.g., drug transporters), and responses (human leukocyte antigens (HLAs)).¹⁾ PGx-related genes (pharmacogenes) have many features: A) contribution of associated genes depends on the metabolizing and transporting pathway of target drugs. B) A gene contains numerous variations associated with pharmacokinetics and responses. C) the frequency of each variation differs considerably among different populations. Some researchers stated that PGx is necessary to enhance personalized care, reduce medical costs, improve patients’ QOL, and develop “omics” for precision medicine.²⁾ ICH E18 guideline strongly encourages genomic sample acquisition in all phases and studies of clinical development.³⁾ European Medical Agency also issued a “Guideline on the use of pharmacogenetic methodologies in the pharmacokinetic assessment of medicinal products” for variant assessment of drug-metabolizing enzymes in non-clinical and clinical phases.⁴⁾ In Japan, the Japanese Society of Clinical Pharmacology and Therapeutics has made a proposal that defines pharmacogenetic testing and lists the genes involved in such testing.⁵⁾

There are several published reports on allele frequencies of the functional variations in the Japanese general population on PGx-related genes such as CYP2D6, UGT1A1, and so on.^6,7) The c.415C > T (p.R139C) in the NUDT15 gene is also associated with thiopurine tolerance, especially in Asian populations.⁸⁾ Currently, there are three genes (UGT1A1 for irinotecan safety, NUDT15 for thiopurines safety, and CYP2C9 for siponimod administration) that their variant analyses are covered by national medical insurance in Japan.

To apply PGx information, several projects, including the Dutch Pharmacogenetics Working Group (DPWG)⁹⁾ and Clinical Pharmacogenetics Implementation Consortium (CPIC),¹⁰⁾ or Ubiquitous Pharmacogenomics (U-PGx)¹¹⁾ have already been developed. They have been collecting multiple PGx data with clinical evidence and establishing the guidelines to use PGx information for therapeutic recommendations, so-called “PGx-Guided Pharmacotherapy.” In Japan, the “Japan PGx Data Science Consortium (JPDSC)” collected 2994 clinical samples and constructed a database to help find the actionable PGx variations.¹²⁾

For single variant genotyping in PGx genes, TaqMan assay, PCR-restriction fragment length polymorphism, and PCR-single strand conformation polymorphism are useful because they are easy to develop and cost effective. However, they are unsuitable for detecting multiple variations at a time to realize pharmacotherapy because several PGx molecules are often involved in a drug’s pharmacokinetics and responses. Several studies have already revealed that multiplex analysis improves patients’ medication management.¹³⁾ There will also be increased demands for precise actions of several drugs in elderly patients (polypharmacy) in Japan. Next Generation Sequencing (NGS) or microarray-based assay may be used for a multiplex assay, but these are time-, labor-, and cost-consuming as clinical tests.¹⁴⁾

In this study, the specific panel has been designed for the Japanese population, and the results were verified using clinical samples with a mass spectrometry-based assay system (MassARRAY system).¹⁵⁾ It is based on a single extension for the polymorphic site and discriminates the genotypes by the molecular weights of the extended products (extended A, T, G, or C). Thus, this method would be suitable for multiplex assay if the primer sequence lengths (molecular weights) were different among the target variation sites. This study is the first challenge to apply multiplex genotyping of PGx gene variant genes using a mass spectrometry-based assay system, which is relatively frequent in Japanese.

MATERIALS AND METHODS

Panel Design

For creating a Japanese-specific panel, named “PGx_JPN” panel, pharmacogenes and their variations were selected with the following criteria; 1) Genes with genetic variations were listed in drug package inserts in Japan, and 2) The allele frequency in the Japanese general population was reported to be over 0.3%, 3) Guidelines were publicized from CPIC or DPWG, or 4) Important variations with low frequency but have frequently been assessed by clinical studies. Tandem variations like TA in UGT1A1 (UGT1A1*28), and gene deletions such as CYP2D6*5 were finally excluded from the PGx_JPN panel because these were difficult to design the primer sequences in the panel development stage. Finally, 36 variations in 14 pharmacogenes were selected^16,17) (Table 1). PCR and extension primers were designed to detect variations with Assay Design Suite version 2.2 software provided by Agena Bioscience (Part of Mesa Laboratories, Inc., San Diego, CA, U.S.A.). The panel comprises 23-plex and 13-plex wells for detecting all the variations. Many PCR products contained proximal 2 or 3 variations (Supplementary Table 1). The designed oligonucleotides were purchased from Eurofins Genomics K.K. (Tokyo, Japan), and mixed to prepare a PCR primer mix and extension (EXT) primer mix for each well (Supplementary Table 1).

Table 1. Targeted Mutations in This Study

Gene name	Allele name	Rsid	Variation site	Allele frequency in Japanese population16)	Expected enzyme activity16)	Related drugs in DPWG11,17)	Related drugs in CPIC17)	Related drugs listed by JSCPT proposal5)
ABCG2/BCRP		rs2231142	421C > A	31.3	Decreased			Erlotinib, gefitinib, imatinib, sunitinib
ALDH2	*2	rs671	1510G > A (also known as 1459G > A)	26.7	None
CYP2B6	*4	rs2279343	785A > G	9.3	Increased	Efavirenz	Efavirenz	Efavirenz
	*5	rs3211371	1459C > T	1.1	Normal
	*9	rs3745274	516G > T	N.D.	Decreased
	(*6)		*4 + *9	16.4	Decreased
	(*7)		*4 + *5 + *9	N.D.	Decreased
CYP2C9	*2	rs1799853	430C > T	N.D.	Decreased	Phenytoin, siponimod, warfarin	Celecoxib, flurbiprofen, ibuprofen, lornoxicam, meloxicam, phenytoin, piroxicam, tenoxicam, warfarin	Siponimod, warfarin
	*3	rs1057910	1075A > C	2.9	Decreased
CYP2C19	*2	rs4244285	681G > A	26.7–29.0	None	Amitriptyline, citalopram, clomipramine, clopidogrel, doxepine, escitalopram, imipramine, lansoprazole, omeprazole, pantoprazole, sertraline, voriconazole	Amitriptyline, citalopram, clomipramine, clopidogrel, doxepine, escitalopram, imipramine, sertraline, trimipramine, voriconazole	Clopidogrel, ecitalopram, proton pump inhibitor
	*3	rs4986893	636G > A	10.8–12.8	None
	*17	rs12248560	807C > T	1.0	Increased
CYP3A4	*16	rs12721627	554C > G	1.4	Decreased
CYP3A5	*3	rs776746	6986A > G	71–85	Decreased	Tacrolimus	Tacrolimus	Tacrolimus
CYP2D6	*2	rs1135840, rs16947	4180G > C, 2850C > T	N.D.	Normal	Amitriptyline, aripiprazole, atomoxetine, brexpiprazole, clomipramine, codeine, doxepine, eliglustat, flecainide, haloperidol, imipramine, metoprolol, nortriptyline, paroxetine, pimozide, propafenone, risperidone, tamoxifen, tramadol, venlafaxine, zuclopenthixol	Amitriptyline, atomoxetine, codeine, clomipramine, fluvoxamine, ondansetron, paroxetine,tamoxifen, tricyclic antidepressants, tropisetron	Atomoxetine, brexpiprazole
	*3	rs35742686	775delA	N.D.	None
	*4	rs3892097	1846G > A	0.3	None
	*5	ND	Whole gene deletion	4.1–7.2	None
	*9	rs5030656	2616delAAG	N.D.	Decreased
	*10	rs1065852	100C > T	33.3–40.8	Decreased
	*14	rs5030865	1758G > A	0.3	Decreased
	*18	rs765776661	4125_4133dupGTGCCCACT	0.5	None
	*21	rs72549352	2579_2580insC	0.6	None
	*36(*10C)	rs28371735	4156C > T	Most *10 allele holders	None
	*41	rs28371725	2988G > A	0–1.6	Decreased
	*49	rs1135822	1612T > A	N.D.	Decreased
NAT2	*5	rs1801280	341T > C	1.4	Decreased			Isoniazid
	*6	rs1799930	590G > A	20.5	Decreased
	*7	rs1799931	857G > A	8.8	Decreased
NUDT15	*2/*3	rs116855232	415C > T	N.D.	Decreased	Azathioprine, mercaptopurine, thioguanine	Azathioprine, mercaptopurine, thioguanine	Azathioprine, mercaptopurine
	*4	rs147390019	416G > A	N.D.
SLCO1B1	*5	rs4149056	521T > C	13.9	Decreased	Atorvastatin, simvastatin	Simvastatin	Rosuvastatin, simvastatin
	*15/*17	rs2306283	388A > G;521T > C		Decreased
SLCO1B3		rs4149117	334T > G	N.D.	N.D.
UGT1A1	*6	rs4148323	211G > A	13–19	Decreased	Irinotecan	Atazanivir	Irinotecan
	*60	rs4124874	3279T > G	14–26	Decreased
	*28/*93a)	rs10929302	3156G > A	9–13/1.2	Decreased
VKORC1	*2	rs9923231	1639G > A	N.D.	Decreased	Acenocoumarol, phenprocoumon, warfarin	Warfarin	Warfarin

a) 3156G > A is linked with *28 allele in Japanese population.

Clinical Samples for Verification Tests

Fifty-two DNA samples were used for the assay. Forty samples were from healthy individuals by JPDSC with written informed consent from each participant,¹²⁾ which were now transferred and stored at the National Institutes of Health Sciences. The ethics committee approved this usage of the National Institutes of Health Sciences (No. 290-2). The samples contain genotype data, including about 1.4-M single-nucleotide polymorphisms (SNPs) analyzed using microarray (HumanOmni2.5-8 BeadChip kits; Illumina). Additionally, 12 DNA samples in 1000 Genome Project and Human Variation Panel,¹⁸⁾ were purchased from the Coriell Institute for Medical Research under the approval of the ethics committee (No. 290-3). The purchased DNA samples confirmed that the appropriate informed consent was obtained at the collection sites. All DNA samples were determined for DNA concentrations with NanoDrop to adjust their concentration to 5 ng/µL with sterilized water.

Data Collection to Compare with the PGx_JPN Panel

All DNA samples were applied for MassARRAY (Agena Bioscience) assay with the PGx_JPN Panel. PGx_JPN panel was processed according to the manufacturer’s instruction for iPLEX assay. The MassARRAY assay uses “iPLEX Pro chemistry” comprises 4 steps, 1) multiplex PCR, 2) 1 base extension and MS, 3) MS measurememt, 4) data Analysis.¹⁹⁾ The instrument can assess known variations as the difference in MS peak shift, and the raw data is processed immediately into variation data within nine hours.

TaqMan SNP Genotyping Assays were conducted to detect rs116855232 in the NUDT15, rs2279343 and rs3211371 in the CYP2B6, and rs12721627 in the CYP3A4 genes, according to the instructions from the manufacturer (Thermo Fisher Scientific; Waltham, MA, U.S.A.). We did not any comparison tests for several SNPs that mutant allele was not found in the samples used in this study or there was not TaqMan probe. Commercial panels, VeriDose Core Panel, VeriDose CYP2D6 CNV panel,²⁰⁾ and required reagents were purchased from Agena Bioscience. The established MassARRAY assay for VeriDose Panels was also performed following the manufacturer’s instructions (Agena Bioscience). The target variants for the three reference methods are shown in Table 2. Coriell’s samples were used as deduced allele data comparison (not for each SNP data comparison) for each gene, using published genotyping data for ten genes (CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP3A4, CYP3A5, NAT2, SLCO1B1, UGT1A1, and VKORC1), which had been analyzed in a previous research¹⁸⁾ (Supplementary Table 2).

Table 2. The Result of Verification Test for PGx_JPN Panel

Gene name	Rsid	Mutation site	Well number	The number of samples analyzed with PGx_JPN panel			The number of samples (concordance rate in each sample, %)
				Wild	Wild/ variant	Variant	VeriDose Core Panel (Total N = 52)	Microarray (Total N = 40)	TaqMan (Total N = 52)
ABCG2/BCRP	rs2231142	421C > A	1	22	20	10	—	40 (100%)	—
ALDH2	rs671	1510G > A (also known as 1459G > A)	1	19	28	5	—	40 (100%)	—
CYP2B6	rs2279343	785A > G	1	16	28	8	—	—	52 (100%)
	rs3211371	1459C > T	1	49	3	0	—	—	52 (100%)
	rs3745274	516G > T	2	25	24	3	52 (100%)	—	—
CYP2C9	rs1799853	430C > T	2	50	2	0	52 (100%)	—	—
	rs1057910	1075A > C	1	45	7	0	52 (100%)	37 (100%)b)	—
CYP2C19	rs4244285	681G > A	1	26	19	7	52 (100%)	40 (100%)	—
	rs4986893	636G > A	1	36	13	3	52 (100%)	40 (100%)	—
	rs12248560	807C > T	1	15	1	1	17 (100%)a)	—	—
CYP3A4	rs12721627	554C > G	1	49	3	0	—	—	52 (100%)
CYP3A5	rs776746	6986A > G	1	8	26	18	52 (100%)	40 (100%)	—
CYP2D6	rs16947	2850C > T	1	37	13	2	52 (100%)	—	—
	rs1135840	4180G > C	1	3	7	7	17 (100%)a)	—	—
	rs35742686	775delA	2	51	1	0	52 (100%)	—	—
	rs3892097	1846G > A	1	50	1	1	52 (100%)	—	—
	rs5030656	2616delAAG	2	51	1	0	52 (100%)	—	—
	rs1065852	100C > T	1	6	7	4	17 (100%)a)	—	—
	rs5030865	1758G > A	1	51	1	0	52 (100%)	—	—
	rs765776661c)	4125_4133dupGTGCCCACT	1	17	0	0	17 (100%)a)	—	—
	rs72549352	2579_2580insC	2	51	1	0	—	—	—
	rs28371735	4156C > T	1	5	12	0	6 (35.3%)a)	—	—
	rs28371725	2988G > A	2	49	3	0	52 (100%)	—	—
	rs1135822d)	1612T > A	2	52	0	0	—	—	—
NAT2	rs1801280	341T > C	1	44	6	2	—	40 (100%)	—
	rs1799930	590G > A	2	23	24	5	—	40 (100%)	—
	rs1799931	857G > A	1	38	12	2	—	40 (100%)	—
NUDT15	rs116855232	415C > T	2	44	4	4	—	—	52 (100%)
	rs147390019c)	416G > A	2	52	0	0	—	—	—
SLCO1B1	rs4149056	521T > C	2	25	24	3	52 (100%)	40 (100%)	—
	rs2306283	388A > G	1	8	24	20	—	40 (100%)	—
SLCO1B3	rs4149117	334T > G	2	10	32	10	—	40 (100%)	—
UGT1A1	rs4148323	211G > A	1	30	17	5	—	40 (100%)	—
	rs4124874	3279T > G	1	18	23	11	—	40 (100%)	—
	rs10929302	3156G > A	1	33	13	6	—	40 (100%)	—
VKORC1	rs9923231	1639G > A	2	6	16	30	52 (100%)	40 (100%)	—

a) Data from the second test with 17 samples. b) Three of 40 samples have been excluded due to no information. c) Data comparison was not performed since the variants were not found in the current samples.

Data Comparison

The data obtained from the PGx_JPN panel were processed using Typer software v4.1 with MassARRAY (Agena Bioscience). Each data was grouped into “homozygous wild type,” “heterozygous variation,” and “homozygous variation” and the degree to which the data from the PGx-JPN panel matched against the ones from any other assays (Illumina BeadChip and TaqMan assay) were calculated. Since 2 SNPs, rs147390019 for NUDT15*3 and rs1135822 for CYP2D6*49, were absent in our samples, the data comparison step was excluded.

RESULTS

The detected SNPs with the PGx_JPN panel are shown in Table 2. All results obtained from the PGx_JPN panel were finally consistent with any other panels and assays (Illumina BeadChip, VeriDose Core Panel, and TaqMan assays), except for CYP2D6*36 (rs28371735). During this evaluation, first, three variations (rs12248560 for CYP2C19, rs1065852 and rs28371735 for CYP2D6) failed to analyze their correct genotypes in the first test using 52 samples. Therefore, the panel was slightly modified with EXT primer for rs12248560 and PCR primers for rs1065852 and rs28371735. With this modification, the panel design for the following variations was changed: rs12248560, rs1065852, rs1135840, rs28371735, and rs765776661 in well 1. The second test was conducted with 17 samples for well 1 only to check the accuracy of the modified PCR/EXT primers. Only one variation site, which recognizes CYP2D6*36 (rs28371735) failed to genotype the variation even in the second test and was not concordant with the result from the VeriDose CYP2D6 CNV panel (data not shown). Three of the 35 variation sites (rs147390019 in NUDT15, rs765776661 and rs1135822 in CYP2D6) exhibited the homozygous wild type in all samples used in this study, so it could not be confirmed whether these variations could be found correctly using the clinical samples.

DISCUSSION

Several hurdles for implementing the precision medicine are currently known using pharmacogenetic information: low predictability, the cost-effectiveness of the genotyping, and method validation for reliable data. The most important issue is the low predictability of a single genetic variation to pharmacokinetics, efficacy, and safety of the drugs, and polygenic score analysis using several involved genetic variations has been increasingly attempted and reported.^21–23) For this purpose, a streamlined genotyping system, which can analyze several related variants simultaneously, is strongly desired.

In this study, a newly developed Japanese-specific PGx panel (a multiplex assay) was evaluated using a mass spectrometry-based genotyping system and compared with other techniques. The single-base extension variation in the mass spectrometry can qualitatively recognize variant sites clearly. Also, it takes a short time to transform raw data into visible results and easily estimate each allele. Our test reported that the new PGx panel could detect almost all 36 designed variations with relatively high frequency in the Japanese population correctly in 48 samples at once. This shows that it will be useful for clinical doctors to receive overall PGx information for a patient when they consider PGx-based drug selection and treatment.

Regarding Coriell’s samples for allele comparison, the result from our PGx_JPN panel coincided well with those published data for the target ten genes.¹⁸⁾ But one allele was disconcordant in the samples NA18540 and NA18617. NA18540, a sample from Chinese, failed to detect UGT1A1*28 with rs10929302. This result might be obtained from population differences in linkage disequilibrium between Chinese and Japanese, in which the linkage between 3156G > A and *28 was reported only in Japanese.²⁴⁾ NA18617 has a heterozygous GA allele (G allele: CYP2B6*4) in this mass spectrometry analysis, while data from Coriell (Hap Map project) was reported to be homozygous wild type (AA) in this rs2279343. Pratt and colleagues assigned the allele by determining consensus genotype from the data with many assay platforms, but there was only one platform to detect *4.¹⁸⁾ It is possible that our designed panel found this variant incorrect; otherwise, their assignment criteria may have poorly judged this allele to *1 instead of *4. Further verification study with any other methods will be required to determine which result is correct for this sample. CYP2D6 allele inconsistency was obtained from incorrect typing of CYP2D6*36.

There were three variants in CYP2D6 (rs765776661, *18; rs1135822, *49) and NUDT15 (rs147390019, *4) detected only wild type in the samples used in our tests. The possible reason is that these variations are relatively low frequency in the Japanese population.^25–27) To resolve this case, more samples need to be obtained and confirmed to establish meaningful data so that the panel can show a clear benefit of implementing PGx information to clinical treatment.

There are two limitations to using a MS-based system. Firstly, this mass spectrometry-based assay has a restriction in that it cannot apply for the copy number and tandem-repeat variations based on its genotyping mechanisms. Thus, combining other methods is necessary to complete the genotyping of several pharmacogenes, including CYP2D6, CYP2A6, and UGT1A1. Additionally, our PGx_JPN panel found that it was difficult to genotype CYP2D6*36. This allele is often found as a CYP2D6*36-CYP2D6*10 tandem-type arrangement and accounts for a high rate in the Japanese population.²⁷⁾ It is supposed that the EXT primer might bind to a similar sequence found in CYP2D7P, which is a pseudo gene but has a homologous sequence to CYP2D6. Secondly, the system cannot be used for unknown/undefined variations other than variants in the panel, unlike the NGS assay.

In conclusion, we succeeded in developing a mass spectrometry-based multiplex method of the PGx_JPN panel for the first time. For applying clinical application, high-resolution results, simplified workflow and easy data management are critical. Our panel can identify more than 30 variants in 2 wells by using simple workflow and short hands-on time. In addition, it takes short time to transform raw data to SNP information with the software in order to send back SNP results quickly. Therefore, our multiplex genotyping system will contribute to the implementation of precision medicine in clinical testing

Acknowledgments

This study was supported in part by AMED under Grant No. JP21ak0101073j0005. The authors are grateful to Drs. Kenichi Ishikawa, Masatomo Mirura, Ryosuke Nakamura, Taisei Mushiroda, and Daiki Tsuji for the advice about developing our PGx panel. The authors also thank the Field Application Team, Agena Bioscience for supporting the panel development and modification.

Conflict of Interest

Nozomi Yamamoto, Yuji Tanno, and Yuya Yokozawa are employees of Veritas Corporation Corporation.

Supplementary Materials

This article contains supplementary materials.

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