Development of a multiplex and cost-effective genotype test toward more personalized medicine for the antiplatelet drug clopidogrel.

There has been a wide range of inter-individual variations in platelet responses to clopidogrel. The variations in response to clopidogrel can be driven by genetic polymorphisms involved in the pathway of absorption, distribution, metabolism, excretion, and the target receptor P2Y12. A set of genetic variants known for causing variations in clopidogrel responses was selected, which included CYP2C19*2, *3, *17, CYP2B6*4, *6, *9, CYP3A4*18, CYP3A5*3, MDR1 2677G>T/A, 3435C>T, and P2Y12 H2 (742T>C). The simultaneous detection of these 10 variants was developed by using a multiplex PCR and single-base extension (MSSE) methodology. The newly developed genotyping test was confirmed by direct DNA sequencing in the representative positive control samples and validated in an extended set of 100 healthy Korean subjects. Genotyping results from the developed MSSE exhibited a perfect concordance with the direct DNA sequencing data and all of variants tested in 100 healthy Korean subjects were in agreement with Hardy-Weinberg equilibrium (p>0.05). The present molecular diagnostic studies provide an accurate, convenient, and fast genotyping method for the detection of multiple variants. This would be helpful for researchers, as well as clinicians, to use genetic information toward more personalized medicine of clopidogrel and other antiplatelet drugs in the future.

Genetic variations in UDP-glucuronosyltransferase 2B15 in a Korean population.

  UDP-glucuronosyltransferase 2B15 (UGT2B15) is involved in the glucoronidation of steroid hormones as well as many drugs. Genetic variations in UGT2B15 have been shown to affect enzyme function and suggested to have a role in human diseases, such as breast and prostate cancers. In the present study, we sequenced genomic DNA from 50 normal Korean subjects to identify single nucleotide polymorphisms (SNPs) in UGT2B15. A total of thirteen genetic variations were found: two in exons, two in introns, seven in the 5'-untranslated region (UTR), and two in the 3'-UTR. The order and frequency distribution of UGT2B15 variations was: -1139T>C (rs9994887), -508G>A (rs1120265), -506T>A (rs1580083), 253T>G (rs1902023) (42%), 23687A>T (rs4148271) (31%), 2635A>T (rs2045100) (28%), -497C>T (14%), -378C>T (14%), 23669C>T (12%), and 23476A>C (rs4148269) (11%), with other minor alleles with a frequency of <10%. Thirteen variations were used to characterize linkage disequilibrium structures at the UGT2B15 locus. Five tag SNPs were identified, and the observed allelic frequencies were compared to those of other ethnic populations. This information describing genetic polymorphisms in UGT2B15 could serve as an important resource for studying individual variations in drug and hormone metabolism in Korean as well as other ethnic populations.

Resolution of a clinical AmpliChip CYP450 Test™ no call: discovery and characterization of novel CYP2D6*1 haplotypes.

A Han Chinese patient failed CYP2D6 genotype analysis with the AmpliChip CYP450 Test™. The CYP2D6 gene locus of the patient and her son were extensively genotyped including copy number variation and gene resequencing. Two SNPs were discovered on the patient's CYP2D6*1 allele, -498C>A and 1661G>C, while the son's CYP2D6*1 allele had -498C>A only. AmpliChip failure was attributed to the presence of a CYP2D6*1 allele carrying the 1661G>C SNP. Functional analyses of -498C>A did not reveal altered activity in vitro or in vivo suggesting that both novel CYP2D6*1 subvariants are functional. The implementation of pharmacogenetics-guided drug therapy relies on accurate clinical-grade genotype analysis. Although the AmpliChip is a reliable platform, numerous allelic (sub)variants and gene arrangements are not detected or may trigger no calls. While such cases may be rare, the clinical/genetic testing community must be aware of the challenges of CYP2D6 testing on the AmpliChip platform and implications regarding accuracy of test results.

Effect of CYP2C19 genetic polymorphism on pharmacokinetics and pharmacodynamics of a new proton pump inhibitor, ilaprazole.

It is well known that the CYP2C19 genetic polymorphism influences the pharmacokinetics and pharmacodynamics of proton pump inhibitors (PPIs), but no report has addressed the effects on ilaprazole, a newly developed PPI. To investigate the effects of the CYP2C19 genetic polymorphism on the disposition and pharmacodynamics of ilaprazole, multiple doses of once-daily 10 mg ilaprazole were repeatedly administered for 7 days to 27 healthy Korean participants, comprising 9 homozygous CYP2C19 extensive metabolizers (homo EMs), 10 heterozygous EMs (hetero EMs), and 8 homozygous poor metabolizers (PMs). The plasma concentration and pharmacodynamic response were measured in the last dose interval. Each genotype group was matched for gender and thus was composed of 4 male and 4 female participants when the analysis was conducted. The pharmacokinetic parameters estimated from the plasma concentrations of ilaprazole and its metabolite ilaprazole sulfone, the serum gastrin level, and the 24-hour intragastric pH were compared among the CYP2C19 genotype groups. No statistically significant differences in the maximum plasma concentration at steady state(C(ss,max)) and the area under the concentration-time curve from zero to 24 hours (AUC(τ)) of ilaprazole and ilaprazole sulfone were observed among the homo EM, hetero EM, and PM CYP2C19 genotypes. In addition, the mean 24-hour intragastric pH, the percentage of time at pH >4, and the AUC(τ) of serum gastrin showed no significant differences among the CYP2C19 genotype groups. The data suggests that the pharmacokinetics and pharmacodynamics of ilaprazole are not significantly influenced by the CYP2C19 genetic polymorphism in healthy participants.

Genetic variations in UDP-glucuronosyltransferase 2B7 gene (UGT2B7) in a Korean population.

Glucuronidation by UDP-glucuronosyltransferase 2B7 (UGT2B7) has been identified as an important pathway for the elimination of its substrate drugs in humans. Alterations in UGT2B7 function or expression may influence individual variations in drug responses. In an effort to screen for UGT2B7 single nucleotide polymorphisms (SNPs) in Koreans, the UGT2B7 gene was directly sequenced in 50 normal subjects. A total of 19 genetic variations were found: seven in exons, eight in introns, and four in the 5'-untranslated region. The order of the frequency distribution of UGT2B7 variations was: -900A>G, -327G>A, -161C>T, 10539A>G, 10711G>C and 10806T>A (40%); 2099T>A, 2100C>T, 2283A>G and 2316A>G (39%); 12029T>A (37%); 10928C>A (33%); 10541G>A (28%); 10897insA (24%); 372A>G (13%) and 211G>T (12%), as well as other minor alleles with less than 10% frequency. Nineteen variations were used to characterize linkage disequilibrium (LD) structures at the UGT2B7 locus. Eight tagging SNPs in UGT2B7 were determined. Identification of UGT2B7 SNPs with LD and the tagging SNPs lays the foundation for investigating UGT2B7-related genotype/phenotype association studies for Koreans as well as other populations.

Auto-interpreter for CYP2D6 SNaPshot genotyping.

CYP2D6 genotyping using SNaPshot method is a very useful tool clinically. However it's hard to interpret the obtained data as a genotype without training. Thus SNaPshot auto-interpreter for the genotype was designed to interpret obtained raw data to a genotype. The auto-interpreter showed good concordance with experts' reading. The validated auto-interpreter of CYP2D6 genotyping using SNaPshot can contribute to accelerating the clinical use.

Identification and functional assessment of BCRP polymorphisms in a Korean population.

The breast cancer resistance protein (BCRP) is a member of the ATP-binding cassette transporters. The aim of the present study was to identify genetic variants of BCRP in Koreans and to assess the functional consequences of BCRP polymorphisms. Twenty single nucleotide polymorphisms (SNP), including four nonsynonymous SNP, were identified by DNA sequencing of the BCRP gene in 92 Korean subjects. BCRP V12M, Q141K, P269S, and Q126Stop were detected at frequencies of 23, 28, 0.2, and 1.9%, respectively. These four coding variants were also screened in Chinese and Vietnamese subjects; the allelic frequencies among the three populations were compared; and predictions were made as to the potential frequency of each variant. In vitro functional analyses of the P269S protein and the promoter SNP -19031C>T (mutated in the hypoxia-inducible factor-1alpha binding site) were performed and compared with those of the wild type. P269S exhibited a 35 to 40% decrease in vesicular uptake of [(3)H]estrone-3-sulfate and [(3)H]methotrexate compared with the wild type. The promoter SNP -19031C>T did not affect BCRP promoter activity in either the presence or absence of chemical-induced hypoxic stress. Our results suggest that the P269S variant could be a functionally altered variant. Genotyping of this variant in clinical studies is needed to address its phenotypic role. Genetic polymorphisms of BCRP were found to be very common in Koreans, as well as in other ethnic groups. Comparative analyses among three Asian populations revealed different frequencies for the four functional BCRP variants.

The CYP3A4*18 allele, the most frequent coding variant in asian populations, does not significantly affect the midazolam disposition in heterozygous individuals.

The objective of this study was to identify CYP3A4 variants in Koreans and to characterize their functional consequences in vitro and in vivo. Four single nucleotide polymorphisms were identified in 50 Koreans by direct DNA sequencing. In an additional genotyping using 248 subjects, CYP3A4(*)18 was confirmed as the most frequent coding variant in Koreans at 1.7%, and its frequency was similar to that of Asians, suggesting that CYP3A4(*)18 would be the highest coding variant in Asians. The recombinant CYP3A4.18 protein prepared in baculovirus expression system showed 67.4% lower Vmax and 1.8-fold higher K(m) for midazolam 1'-hydroxylation compared with the wild type. The mean values of Cmax and area under the concentration curve (AUC) in the CYP3A4(*)1/(*)18 and CYP3A5(*)1/(*)3 subjects (n = 8) were 63% and 32% higher than in CYP3A4(*)1/(*)1 and CYP3A5(*)1/(*)3 carriers (n = 8), respectively. Although the in vitro assay exhibited a significant reduction of the enzyme activity for midazolam, the in vivo differences associated with the CYP3A4(*)1/(*)18 tend to be low (P < 0.07 in Cmax and P < 0.09 in AUC). In summary, the heterozygous CYP3A4(*)1/(*)18 does not appear to cause a significant change of midazolam disposition in vivo; however, the clinical relevance of CYP3A4(*)18/(*)18 remains to be evaluated.

Identification and functional characterization of novel CYP2J2 variants: G312R variant causes loss of enzyme catalytic activity.

CYP2J2 plays important roles in the metabolism of therapeutic drugs, such as astemizole and ebastine, as well as endogenous fatty acids. This study aimed to identify CYP2J2 genetic variants in Koreans and to characterize their functional consequences. From direct sequencing of the CYP2J2 gene, 12 genetic variations, including the two novel nonsynonymous mutations G312R and P351L, were identified from 93 Korean subjects. The two novel CYP2J2 variants were co-expressed with NADPH-cytochrome P450 reductase in Sf9 cells and their catalytic activities were quantified. The recombinant CYP2J2 G312R variant showed almost complete loss of enzymatic activity, as determined by CYP2J2-catalysed astemizole O-demethylation and ebastine hydroxylation. The CYP2J2 P351L variant showed enzymatic activities that were comparable with the wild-type CYP2J2. The reduced CO spectra of the recombinant CYP2J2 proteins suggested no CO binding to the heme in CYP2J2 G312R. In addition, molecular modelling of the three-dimensional structure consistently predicted that there might be spatial hindrance between heme and the bulky side chain of the R312 residue in CYP2J2 G312R variant. The CYP2J2 G312R variant was not found in 192 Chinese, 99 African-Americans, 100 Caucasians and 159 Vietnamese subjects. Two of the 192 Chinese subjects (0.52%) were heterozygous for CYP2J2 P351L. Twelve CYP2J2 variants, including two novel nonsynonymous variants, were identified in a Korean population. The G312R variant is the first nonfunctional CYP2J2 allele to be identified, and is expected to influence the disposition of its substrate therapeutics, as well as endogenous compounds.