Genetic variants, haplotype determination, and function of novel alleles of CYP2B6 in a Han Chinese population.

Amino acid variants in protein may result in deleterious effects on enzymatic activity. In this study we investigate the DNA variants on activity of CYP2B6 gene in a Chinese Han population for potential use in precision medicine. All exons in CYP2B6 gene from 1483 Chinese Han adults (Zhejiang province) were sequenced using Sanger sequencing. The effects of nonsynonymous variants on recombinant protein catalytic activity were investigated in vitro with Sf12 system. The haplotype of novel nonsynonymous variants with other single nucleotide variants in the same allele was determined using Nanopore sequencing. Of 38 alleles listed on the Pharmacogene Variation Consortium, we detected 7 previously reported alleles and 18 novel variants, of which 11 nonsynonymous variants showed lower catalytic activity (0.00-0.60) on bupropion compared to CYP2B6*1. Further, these 11 novel star-alleles (CYP2B6*39-49) were assigned by the Pharmacogene Variation Consortium, which may be valuable for pharmacogenetic research and personalized medicine.

Characterization of 15 CYP2J2 variants identified in the Chinese Han population on the metabolism of ebastine and terfenadine in vitro.

Genetic polymorphism of the cytochrome P450 (CYP) gene can significantly influence the metabolism of endogenous and xenobiotic compounds. However, few studies have focused on the polymorphism of CYP2J2 and its impact on drug catalytic activity, especially in the Chinese Han population. In this study, we sequenced the promoter and exon regions of CYP2J2 in 1,163 unrelated healthy Chinese Han individuals using the multiplex PCR amplicon sequencing method. Then, the catalytic activities of the detected CYP2J2 variants were evaluated after recombinant expression in S. cerevisiae microsomes. As a result, CYP2J2*7, CYP2J2*8, 13 variations in the promoter region and 15 CYP2J2 nonsynonymous variants were detected, of which V15A, G24R, V68A, L166F and A391T were novel missense variations. Immunoblotting results showed that 11 of 15 CYP2J2 variants exhibited lower protein expression than wild-type CYP2J2.1. In vitro functional analysis results revealed that the amino acid changes of 14 variants could significantly influence the drug metabolic activity of CYP2J2 toward ebastine or terfenadine. Specifically, 4 variants with relatively higher allele frequencies, CYP2J2.8, 173_173del, K267fs and R446W, exhibited extremely low protein expression and defective catalytic activities for both substrates. Our results indicated that a high genetic polymorphism of CYP2J2 could be detected in the Chinese Han population, and most genetic variations in CYP2J2 could influence the expression and catalytic activity of CYP2J2. Our data significantly enrich the knowledge of genetic polymorphisms in CYP2J2 and provide new theoretical information for corresponding individualized medication in Chinese and other Asian populations.

Assessments of CYP­inhibition­based drug-drug interaction between vonoprazan and poziotinib in vitro and in vivo.

CONTEXT: Poziotinib and vonoprazan are two drugs mainly metabolized by CYP3A4. However, the drug-drug interaction between them is unknown. OBJECTIVE: To study the interaction mechanism and pharmacokinetics of poziotinib on vonoprazan. MATERIALS AND METHODS: In vitro experiments were performed with rat liver microsomes (RLMs) and the contents of vonoprazan and its metabolite were then determined with UPLC-MS/MS after incubation of RLMs with vonoprazan and gradient concentrations of poziotinib. For the in vivo experiment, rats in the poziotinib treated group were given 5 mg/kg poziotinib by gavage once daily for 7 days, and the control group was only given 0.5% CMC-Na. On Day 8, tail venous blood was collected at different time points after the gavage administration of 10 mg/kg vonoprazan, and used for the quantification of vonoprazan and its metabolite. DAS and SPSS software were used for the pharmacokinetic and statistical analyses. RESULTS: In vitro experimental data indicated that poziotinib inhibited the metabolism of vonoprazan (IC50 = 10.6 µM) in a mixed model of noncompetitive and uncompetitive inhibition. The inhibitory constant Ki was 0.574 µM and the binding constant αKi was 2.77 µM. In vivo experiments revealed that the AUC(0-T) (15.05 vs. 90.95 µg/mL·h) and AUC(0-∞) (15.05 vs. 91.99 µg/mL·h) of vonoprazan increased significantly with poziotinib pretreatment. The MRT(0-∞) of vonoprazan increased from 2.29 to 5.51 h, while the CLz/F value decreased from 162.67 to 25.84 L/kg·h after pretreatment with poziotinib. CONCLUSIONS: Poziotinib could significantly inhibit the metabolism of vonoprazan and more care may be taken when co-administered in the clinic.

Identification and drug metabolic characterization of four new CYP2C9 variants CYP2C9*72-*75 in the Chinese Han population.

Cytochrome 2C9 (CYP2C9), one of the most important drug metabolic enzymes in the human hepatic P450 superfamily, is required for the metabolism of 15% of clinical drugs. Similar to other CYP2C family members, CYP2C9 gene has a high genetic polymorphism which can cause significant racial and inter-individual differences in drug metabolic activity. To better understand the genetic distribution pattern of CYP2C9 in the Chinese Han population, 931 individuals were recruited and used for the genotyping in this study. As a result, seven synonymous and 14 non-synonymous variations were identified, of which 4 missense variants were designated as new alleles CYP2C9*72, *73, *74 and *75, resulting in the amino acid substitutions of A149V, R150C, Q214H and N418T, respectively. When expressed in insect cell microsomes, all four variants exhibited comparable protein expression levels to that of the wild-type CYP2C9 enzyme. However, drug metabolic activity analysis revealed that these variants exhibited significantly decreased catalytic activities toward three CYP2C9 specific probe drugs, as compared with that of the wild-type enzyme. These data indicate that the amino acid substitution in newly designated variants can cause reduced function of the enzyme and its clinical significance still needs further investigation in the future.

MTH1 suppression enhances the stemness of MCF7 through upregulation of STAT3.

MTH1 protein can sanitize the damaged (d)NTP pool and MTH1 inhibitors have been developed to impede the growth of rapidly proliferating tumor cells; however, the effect of MTH1 inhibition on breast cancer stemness has not been reported yet. Here, we constructed breast cancer cell lines with the stable depletion of MTH1. MTH1 suppression clearly increased the ratio of CD44+CD24-/low subpopulations and promoted the formation of tumorspheres in MCF7 and T47D cells. RNA expression profiling, RT-qPCR and Western blotting showed the upregulation of master stem cell transcription factors Sox2, Oct4 and Nanog in MTH1 knockdown cells. GSEA suggested and Western blotting verified that MTH1 knockdown increased the expression of phosphorylated STAT3 (Tyr705). Furthermore, we indirectly demonstrated that the increased concentration of 8-oxo-dGTP and 8-oxo-GTP in MTH1-knockdown cells and exogenous 8-oxoGTP, rather than 8-oxo-dGTP, could significantly increase the phosphorylation of STAT3. In conclusion, this work indicates that MTH1 inhibition increased the proportion of breast cancer stem cells (BCSCs) and promoted stemness properties in MCF7 cells.

Effects of Simvastatin on the Metabolism of Vonoprazan in Rats Both in vitro and in vivo.

Purpose: To study the potential drug-drug interactions between simvastatin and vonoprazan and to provide the scientific basis for rational use of them in clinical practice. Methods: An incubation system was established with rat liver microsomes, and the main metabolite of vonoprazan M-I was detected by UPLC-MS/MS. The IC50 value of simvastatin was then calculated and its inhibitory mechanism against vonoprazan was also analyzed. Twelve SD rats were randomly divided into 2 groups, then they were given simvastatin or saline for 2 weeks continuously. On the day of the experiment, both groups were intragastrically administered with vonoprazan once, followed by the collection of blood at different time points. Then the plasma concentration of vonoprazan and M-I in rats were detected by UPLC-MS/MS. Results: In vitro experiments revealed that simvastatin could inhibit the metabolism of vonoprazan, and its inhibition type belonged to the mixed non-competitive and competitive inhibition model. In vivo experiments in rats demonstrated that the area under concentration time curve (AUC) of vonoprazan was decreased but the clearance (CLz/F) of it was increased in the simvastatin administrated group, as compared to those of the control group. However, M-I in simvastatin treated group exhibited the higher AUC and lower CLz/F values compared to those in the control group. These data indicated that multiple doses of simvastatin administration could reduce the plasma concentration of vonoprazan and accelerate its metabolic rate in rats. Conclusion: Simvastatin could inhibit the metabolism of vonoprazan in vitro but multiple doses of simvastatin exhibited the opposite effect In vivo. Altogether, our data indicated that an interaction existed between simvastatin and vonoprazan and additional cares might be taken when they were co-administrated in clinic.

Functional characterization of the defective CYP2C9 variant CYP2C9*18.

Cytochrome P450 2C9 (CYP2C9) is one of the most important drugs metabolizing enzymes and accounts for the metabolism of about 13%-17% of clinical drugs. Like other members in CYP2 family, CYP2C9 gene exhibits great genetic polymorphism among different races and individuals. CYP2C9*18 is one CYP2C9 allelic variant identified in a Southeast Asian population and is estimated to cause the amino acid substitutions of I359L and D397A in CYP2C9 enzyme simultaneously. Limited by the low expression level in bacteria and COS-7 cells, no valuable enzyme kinetics have been reported on this CYP2C9 variant. In this study, the baculovirus-based system was used for the high expression of recombinant CYP2C9 s in insect cells. As a result, together with I359L substitution, D397A could significantly decrease the protein expression of CYP2C9.18 in insect cells, although substitution of D397A alone had no effect on the expression of CYP2C9 in vitro. As compared with that of wild-type enzyme, both CYP2C9.18 variant and D397A variant could decrease more than 80% of the catalytic activity of CYP2C9 enzyme toward three probe substrates, suggesting that caution should be exercised when patients carrying CYP2C9*18 taking medicines metabolized by CYP2C9 enzyme with a narrow therapeutic window.

The high expression of MTH1 and NUDT5 promotes tumor metastasis and indicates a poor prognosis in patients with non-small-cell lung cancer.

MutT Homolog 1 (MTH1) is a mammalian 8-oxodGTPase for sanitizing oxidative damage to the nucleotide pool. Nudix type 5 (NUDT5) also sanitizes 8-oxodGDP in the nucleotide pool. The role of MTH1 and NUDT5 in non-small-cell lung cancer (NSCLC) progression and metastasis remains unclear. In the present study, we reported that MTH1 and NUDT5 were upregulated in NSCLC cell lines and tissues, and higher levels of MTH1 or NUDT5 were associated with tumor metastasis and a poor prognosis in patients with NSCLC. Their suppression also restrained tumor growth and lung metastasis in vivo and significantly inhibited NSCLC cell migration, invasion, cell proliferation and cell cycle progression while promoting apoptosis in vitro. The opposite effects were observed in vitro following MTH1 or NUDT5 rescue. In addition, the upregulation of MTH1 or NUDT5 enhanced the MAPK pathway and PI3K/AKT activity. Furthermore, MTH1 and NUDT5 induce epithelial-mesenchymal transition both in vitro and in vivo. These results highlight the essential role of MTH1 and NUDT5 in NSCLC tumor tumorigenesis and metastasis as well as their functions as valuable markers of the NSCLC prognosis and potential therapeutic targets.

Effects of rare CYP2C9 alleles on stable warfarin doses in Chinese Han patients with atrial fibrillation.

Aim: Gene polymorphisms are critical in warfarin dosing variation. Here, the role of rare CYP2C9 alleles on warfarin doses in Chinese Han patients was investigated. Methods: A retrospective study recruited 681 warfarin treated atrial fibrillation patients. The genetic and clinical data were collected. Dose-related variables were selected by univariate analyses and the warfarin-dosing algorithm was derived by multivariate regression analysis. Results: Three rare CYP2C9 alleles (CYP2C9*13, *16 and *60) were associated with lower stable doses. Inclusion of the rare CYP2C9 alleles in the prediction model added an extra 3.7% warfarin dose predictive power. Conclusion:CYP2C9*13, *16 and *60 was associated with lower stable warfarin doses in Chinese patients. The algorithm including rare CYP2C9 alleles tends to more accurately predict stable warfarin doses.

An identification and functional evaluation of a novel CYP2C9 variant CYP2C9*62.

Warfarin is the most commonly used anticoagulant in the clinical treatment of thromboembolic diseases. The dose of warfarin varies significantly within populations, and the dose is closely related to the genetic polymorphisms of the CYP2C9 and VKORC1 genes. In this study, a new CYP2C9 nonsynonymous mutation (8576C > T) was detected after the genetic screening of 162 patients took warfarin. This mutation, named as the new allele CYP2C9*62, can result in an arginine to cysteine amino acid substitution at position 125 of the CYP2C9 protein (R125C). When expressed in insect cells, the protein expression of CYP2C9.62 was significantly lower than that of the wild-type, and its metabolic activity was also significantly decreased after the addition of three typical CYP2C9 probe drugs, suggesting that the new mutant can dramatically affect the metabolism of CYP2C9 drugs in vitro.

The Pol ß variant containing exon α is deficient in DNA polymerase but has full dRP lyase activity.

DNA polymerase (Pol) ß is a key enzyme in base excision repair (BER), an important repair system for maintaining genomic integrity. We previously reported the presence of a Pol ß transcript containing exon α (105-nucleotide) in normal and colon cancer cell lines. The transcript carried an insertion between exons VI and VII and was predicted to encode a ~42 kDa variant of the wild-type 39 kDa enzyme. However, little is known about the biochemical properties of the exon α-containing Pol ß (exon α Pol ß) variant. Here, we first obtained evidence indicating expression of the 42 kDa exon α Pol ß variant in mouse embryonic fibroblasts. The exon α Pol ß variant was then overexpressed in E. coli, purified, and characterized for its biochemical properties. Kinetic studies of exon α Pol ß revealed that it is deficient in DNA binding to gapped DNA, has strongly reduced polymerase activity and higher Km for dNTP during gap-filling. On the other hand, the 5'-dRP lyase activity of the exon α Pol ß variant is similar to that of wild-type Pol ß. These results indicate the exon α Pol ß variant is base excision repair deficient, but does conduct 5'-trimming of a dRP group at the gap margin. Understanding the biological implications of this Pol ß variant warrants further investigation.

Repair pathway for PARP-1 DNA-protein crosslinks.

Poly(ADP-ribose) polymerase-1 (PARP-1) is a regulatory enzyme involved in many different processes of DNA and RNA metabolism, including DNA repair. Previously, PARP-1 was found capable of forming a covalent DNA-protein crosslink (DPC) at the apurinic/apyrimidinic (AP) site in double-stranded DNA. The C1´ atom of the AP site participates in Schiff base formation with a lysine side chain in PARP-1, and a covalent bond is formed upon reduction of the Schiff base. The PARP-1 DPC is formed in vivo where DPC formation correlates with AP site induction by a monofunctional alkylating agent. Here, we examined repair of PARP-1 DPCs in mouse fibroblasts and found that a proteasome inhibitor, MG-132, reduces repair resulting in accumulation of PARP-1 DPCs and increased alkylating agent cytotoxicity. Using a model DNA substrate mimicking the PARP-1 DPC after proteasomal degradation, we found that repair is completed by a sub-pathway of base excision repair (BER). Tyrosyl-DNA phosphodiesterase 1 was proficient in removing the ring-open AP site sugar at the phosphodiester linkage, leaving an intermediate for processing by other BER enzymes. The results reveal proteasomal degradation of the PARP-1 DPC is active in mouse fibroblasts and that a model repair intermediate is processed by the BER machinery.

Transcriptional mutagenesis mediated by 8-oxoG induces translational errors in mammalian cells.

Reactive oxygen species formed within the mammalian cell can produce 8-oxo-7,8-dihydroguanine (8-oxoG) in mRNA, which can cause base mispairing during gene expression. Here we found that administration of 8-oxoGTP in MTH1-knockdown cells results in increased 8-oxoG content in mRNA. Under this condition, an amber mutation of the reporter luciferase is suppressed. Using second-generation sequencing techniques, we found that U-to-G changes at preassigned sites of the luciferase transcript increased when 8-oxoGTP was supplied. In addition, an increased level of 8-oxoG content in RNA induced the accumulation of aggregable amyloid ß peptides in cells expressing amyloid precursor protein. Our findings indicate that 8-oxoG accumulation in mRNA can alter protein synthesis in mammalian cells. Further work is required to assess the significance of these findings under normal physiological conditions.

DNA polymerase ß: A missing link of the base excision repair machinery in mammalian mitochondria.

Mitochondrial genome integrity is fundamental to mammalian cell viability. Since mitochondrial DNA is constantly under attack from oxygen radicals released during ATP production, DNA repair is vital in removing oxidatively generated lesions in mitochondrial DNA, but the presence of a strong base excision repair system has not been demonstrated. Here, we addressed the presence of such a system in mammalian mitochondria involving the primary base lesion repair enzyme DNA polymerase (pol) ß. Pol ß was localized to mammalian mitochondria by electron microscopic-immunogold staining, immunofluorescence co-localization and biochemical experiments. Extracts from purified mitochondria exhibited base excision repair activity that was dependent on pol ß. Mitochondria from pol ß-deficient mouse fibroblasts had compromised DNA repair and showed elevated levels of superoxide radicals after hydrogen peroxide treatment. Mitochondria in pol ß-deficient fibroblasts displayed altered morphology by electron microscopy. These results indicate that mammalian mitochondria contain an efficient base lesion repair system mediated in part by pol ß and thus pol ß plays a role in preserving mitochondrial genome stability.

Effects of CYP2C19 Variants on Fluoxetine Metabolism in vitro.

AIMS: CYP2C19 is an important member of the cytochrome P450 enzyme superfamily. We recently identified 31 CYP2C19 alleles in the Han Chinese population. The aim of this study was to assess the catalytic activities of these allelic isoforms and their effects on the metabolism of fluoxetine in vitro. METHODS: The wild-type and 30 CYP2C19 variants were expressed in insect cells and each variant was characterized using fluoxetine as the substrate. Reactions were performed at 37°C with 20-1,000 µmol/L substrate for 30 min. By using ultra-high performance liquid chromatography-mass spectrometry to detect the products, the kinetic parameters Km, Vmax, and intrinsic clearance (Vmax/Km) of norfluoxetine were determined. RESULTS: Among the CYP2C19 variants tested, T130M showed similar intrinsic clearance (Vmax/Km) values with CYP2C19*1, while the intrinsic clearance values of other variants were significantly decreased (from 9.56 to 77.77%). In addition, CYP2C19*3 and *35FS could not be detected because they have no detectable enzyme activity. CONCLUSION: In China, the assessment of CYP2C19 variants in vitro offers valuable information relevant to the personalized medicine for CYP2C19-metabolized drug.

Systematic screening for CYP3A4 genetic polymorphisms in a Han Chinese population.

AIM: To systematically investigate the genetic polymorphisms of the CYP3A4 gene in a Han Chinese population. MATERIALS & METHODS: The promoter and exons of CYP3A4 gene in 1114 unrelated, healthy Han Chinese subjects were amplified and genotyped by direct sequencing. RESULTS: In total, five previously reported alleles (*1G, *4, *5, *18B and *23) were detected, of which one allele (*23) was reported for the first time in Han Chinese population. Additionally, seven novel exonic variants were also identified and designated as new alleles CYP3A4*28-*34. CONCLUSION: This study provides the most comprehensive data of CYP3A4 polymorphisms in Han Chinese population and detects the largest number of novel CYP3A4 alleles in one ethnic group.

Adipose-specific deletion of Kif5b exacerbates obesity and insulin resistance in a mouse model of diet-induced obesity.

Recent studies have shown that KIF5B (conventional kinesin heavy chain) mediates glucose transporter type 4 translocation and adiponectin secretion in 3T3-L1 adipocytes, suggesting an involvement of KIF5B in the homeostasis of metabolism. However, the in vivo physiologic function of KIF5B in adipose tissue remains to be determined. In this study, adipose-specific Kif5b knockout (F-K5bKO) mice were generated using the Cre-LoxP strategy. F-K5bKO mice had similar body weights to controls fed on a standard chow diet. However, F-K5bKO mice had hyperlipidemia and significant glucose intolerance and insulin resistance. Deletion of Kif5b aggravated the deleterious impact of a high-fat diet (HFD) on body weight gain, hepatosteatosis, glucose tolerance, and systematic insulin sensitivity. These changes were accompanied by impaired insulin signaling, decreased secretion of adiponectin, and increased serum levels of leptin and proinflammatory adipokines. F-K5bKO mice fed on an HFD exhibited lower energy expenditure and thermogenic dysfunction as a result of whitening of brown adipose due to decreased mitochondria biogenesis and down-regulation of key thermogenic gene expression. In conclusion, selective deletion of Kif5b in adipose tissue exacerbates HFD-induced obesity and its associated metabolic disorders, partly through a decrease in energy expenditure, dysregulation of adipokine secretion, and insulin signaling.-Cui, J., Pang, J., Lin, Y.-J., Gong, H., Wang, Z.-H., Li, Y.-X., Li, J., Wang, Z., Jiang, P., Dai, D.-P., Li, J., Cai, J.-P., Huang, J.-D., Zhang, T.-M. Adipose-specific deletion of Kif5b exacerbates obesity and insulin resistance in a mouse model of diet-induced obesity.

Oxidized nucleotide insertion by pol ß confounds ligation during base excision repair.

Oxidative stress in cells can lead to accumulation of reactive oxygen species and oxidation of DNA precursors. Oxidized purine nucleotides can be inserted into DNA during replication and repair. The main pathway for correcting oxidized bases in DNA is base excision repair (BER), and in vertebrates DNA polymerase ß (pol ß) provides gap filling and tailoring functions. Here we report that the DNA ligation step of BER is compromised after pol ß insertion of oxidized purine nucleotides into the BER intermediate in vitro. These results suggest the possibility that BER mediated toxic strand breaks are produced in cells under oxidative stress conditions. We observe enhanced cytotoxicity in oxidizing-agent treated pol ß expressing mouse fibroblasts, suggesting formation of DNA strand breaks under these treatment conditions. Increased cytotoxicity following MTH1 knockout or treatment with MTH1 inhibitor suggests the oxidation of precursor nucleotides.

Effect of 22 CYP2D6 variants found in the Chinese population on tolterodine metabolism in vitro.

Cytochrome P450 2D6 (CYP2D6) is an important member of the cytochrome P450 enzyme superfamily. We recently identified 22 novel variants in the Chinese population using PCR and bidirectional sequencing methods. The aim of this study is to characterize the enzymatic activity of these variants and their effects on the metabolism of the antimuscarinic drug tolterodine in vitro. A baculovirus-mediated expression system was used to express wild-type CYP2D6 and 24 variants (CYP2D6*2, CYP2D6*10, and 22 novel CYP2D6 variants) at high levels. The insect microsomes expressing CYP2D6 proteins were incubated with 0.1-50 µM tolterodine at 37 °C for 30 min and the metabolites were analyzed by high-performance liquid chromatography-tandem mass spectrometry system. Of the 24 CYP2D6 variants tested, 2 variants (CYP2D6*92 and CYP2D6*96) were found to be catalytically inactive, 4 variants (CYP2D6*94, F164L, F219S and D336N) exhibited markedly increased intrinsic clearance values (Vmax/Km) compared with the wild-type (from 66.34 to 99.79%), whereas 4 variants (CYP2D6*10, *93, *95 and E215K) exhibited significantly decreased values (from 49.02 to 98.50%). This is the first report of all these rare alleles for tolterodine metabolism and these findings suggest that more attention should be paid to subjects carrying these infrequent CYP2D6 alleles when administering tolterodine in the clinic.

Effects of CYP2C19 variants on methadone metabolism in vitro.

CYP2C19 is an important member of the cytochrome P450 (CYP450) enzyme super family and is responsible for clearing approximately 10% of commonly used clinical drugs that undergo phase I metabolism. Genetic polymorphisms of CYP2C19 significantly influence the efficacy and safety of some drugs, which might cause undesirable adverse effects or cure failure at standard dosages. The aim of this study was to clarify the catalytic activities of 31 CYP2C19 alleles on the oxidative in vitro metabolism of methadone. Insect microsomes expressing the CYP2C19 alleles were incubated with 50-2000 µM methadone for 30 min at 37 °C and terminated by cooling to -80 °C immediately. Methadone and its metabolite EDDP were analyzed by an ultra performance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) system. Of the 31 tested CYP2C19 allelies variants, CYP2C19*1 is the wild-type. Compared with CYP2C19*1, two CYP2C19 variants (CYP2C19*3 and *35FS) had no detectable enzyme activity, one variant L16F exhibited slightly increased intrinsic clearance values, and one variant N277K showed no significant difference. In addition, 26 variants exhibited significantly decreased values (from 1.48% to 80.40%). These findings suggest that more attention should be paid in clinical administration of methadone to individuals carrying these CYP2C19 alleles. Copyright © 2016 John Wiley & Sons, Ltd.