Predicting HLA alleles from high-resolution SNP data in three Southeast Asian populations.

The major histocompatibility complex (MHC) containing the classical human leukocyte antigen (HLA) Class I and Class II genes is among the most polymorphic and diverse regions in the human genome. Despite the clinical importance of identifying the HLA types, very few databases jointly characterize densely genotyped single nucleotide polymorphisms (SNPs) and HLA alleles in the same samples. To date, the HapMap presents the only public resource that provides a SNP reference panel for predicting HLA alleles, constructed with four collections of individuals of north-western European, northern Han Chinese, cosmopolitan Japanese and Yoruba Nigerian ancestry. Owing to complex patterns of linkage disequilibrium in this region, it is unclear whether the HapMap reference panels can be appropriately utilized for other populations. Here, we describe a public resource for the Singapore Genome Variation Project with: (i) dense genotyping across ∼ 9000 SNPs in the MHC; (ii) four-digit HLA typing for eight Class I and Class II loci, in 96 southern Han Chinese, 89 Southeast Asian Malays and 83 Tamil Indians. This resource provides population estimates of the frequencies of HLA alleles at these eight loci in the three population groups, particularly for HLA-DPA1 and HLA-DPB1 that were not assayed in HapMap. Comparing between population-specific reference panels and a cosmopolitan panel created from all four HapMap populations, we demonstrate that more accurate imputation is obtained with population-specific panels than with the cosmopolitan panel, especially for the Malays and Indians but even when imputing between northern and southern Han Chinese. As with SNP imputation, common HLA alleles were imputed with greater accuracy than low-frequency variants.

Bernard Lerer: recipient of the 2014 inaugural Werner Kalow Responsible Innovation Prize in Global Omics and Personalized Medicine (Pacific Rim Association for Clinical Pharmacogenetics).

This article announces the recipient of the 2014 inaugural Werner Kalow Responsible Innovation Prize in Global Omics and Personalized Medicine by the Pacific Rim Association for Clinical Pharmacogenetics (PRACP): Bernard Lerer, professor of psychiatry and director of the Biological Psychiatry Laboratory, Hadassah-Hebrew University Medical Center, Jerusalem, Israel. The Werner Kalow Responsible Innovation Prize is given to an exceptional interdisciplinary scholar who has made highly innovative and enduring contributions to global omics science and personalized medicine, with both vertical and horizontal (transdisciplinary) impacts. The prize is established in memory of a beloved colleague, mentor, and friend, the late Professor Werner Kalow, who cultivated the idea and practice of pharmacogenetics in modern therapeutics commencing in the 1950s. PRACP, the prize's sponsor, is one of the longest standing learned societies in the Asia-Pacific region, and was founded by Kalow and colleagues more than two decades ago in the then-emerging field of pharmacogenetics. In announcing this inaugural prize and its winner, we seek to highlight the works of prize winner, Professor Lerer. Additionally, we contextualize the significance of the prize by recalling the life and works of Professor Kalow and providing a brief socio-technical history of the rise of pharmacogenetics and personalized medicine as a veritable form of 21(st) century scientific practice. The article also fills a void in previous social science analyses of pharmacogenetics, by bringing to the fore the works of Kalow from 1995 to 2008, when he presciently noted the rise of yet another field of postgenomics inquiry--pharmacoepigenetics--that railed against genetic determinism and underscored the temporal and spatial plasticity of genetic components of drug response, with invention of the repeated drug administration (RDA) method that estimates the dynamic heritabilities of drug response. The prize goes a long way to cultivate transgenerational capacity and broader cognizance of the concept and practice of responsible innovation as an important criterion of 21(st) century omics science and personalized medicine. A new call is presently in place for the 2016 PRACP Werner Kalow prize. Nominations can be made in support of an exceptional individual interdisciplinary scholar, or alternatively, an entire research team, from any region in the world with a record of highly innovative contributions to global omics science and/or personalized medicine, in the spirit of responsible innovation. The application process is straightforward, requiring a signed, 1500-word nomination letter (by the applicant or sponsor) submitted not later than May 31, 2015.

Ensuring good quality RNA for quantitative real-time PCR isolated from renal proximal tubular cells using laser capture microdissection.

BACKGROUND: In order to provide gene expression profiles of different cell types, the primary step is to isolate the specific cells of interest via laser capture microdissection (LCM), followed by extraction of good quality total RNA sufficient for quantitative real-time polymerase chain reaction (qPCR) analysis. This LCM-qPCR strategy has allowed numerous gene expression studies on specific cell populations, providing valuable insights into specific cellular changes in diseases. However, such strategy imposed challenges as cells of interests are often available in limited quantities and quality of RNA may be compromised during long periods of time spent on collection of cells and extraction of total RNA; therefore, it is crucial that protocols for sample preparation should be optimised according to different cell populations. FINDINGS: We made several modifications to existing protocols to improve the total RNA yield and integrity for downstream qPCR analyses. A modified condensed hematoxylin and eosin (H&E) staining protocol was developed for the identification of rat renal proximal tubular cells (PTCs). It was then determined that a minimal of eight thousands renal PTCs were required to meet the minimal total RNA yield required for downstream qPCR. RNA integrity was assessed using at every progressive step of sample preparation. Therefore, we decided that the shortened H&E staining, together with microdissection should be performed consecutively within twenty minutes for good quality for gene expression analysis. These modified protocols were later applied on six individual rat samples. A panel of twenty rat renal drug transporters and five housekeeping genes showed Ct values below thirty-five, confirming the expression levels of these drug transporters can be detected. CONCLUSIONS: We had successfully optimized the protocols to achieve sufficient good quality total RNA from microdissected rat renal PTCs for gene expression profiling via qPCR. This protocol may be suitable for researchers who are interested in employing similar applications for gene expression studies.

Pharmacogenetic effects of regulatory nuclear receptors (PXR, CAR, RXRα and HNF4α) on docetaxel disposition in Chinese nasopharyngeal cancer patients.

PURPOSE: This exploratory study was aimed at elucidating the pharmacogenetics of regulatory nuclear receptors (PXR, CAR, RXRα and HNF4α) and their implications on docetaxel pharmacokinetics and pharmacodynamics in local Chinese nasopharyngeal cancer patients. METHODS: A total of 59 single nucleotide polymorphisms (SNPs), including tag-SNPs and functionally relevant SNPs of the genes encoding these regulatory nuclear receptors (PXR/NR1I2, CAR/NR1I3, RXRα/NR2B1 and HNF4α/NR2A1), were profiled in the patients enrolled in our study by direct sequencing (N = 50). The generalized linear model was employed to estimate the haplotypic effects on the pharmacokinetics and pharmacodynamics of the patients. RESULTS: The pharmacokinetic profiles of docetaxel in these patients were characterized by marked interindividual variability, with approximately four- to sixfold variations observed in Cmax, AUC0-∞ and CL. Individual SNP association tests revealed that polymorphisms in NR2B1 and NR2A1 were significantly correlated with altered docetaxel pharmacokinetics. Subsequent haplotype association analysis identified the NR2B1 LD block 2 AG haplotype [*+4458G>A(rs3132291) and *+4988A>G(rs4842198)] to be significantly associated with altered pharmacokinetics, in which patients carrying two copies of the AG haplotype had approximately a 20 % decreased Cmax and AUC0-∞ and a 21 % increased CL compared to those who carried only one copy or no copies of the haplotype. A number of SNPs in NR1I2, NR1I3, NR2B1 and NR2A1 were also associated with a significant decrease in blood counts from baseline. No haplotype was found to exert any effects on the pharmacodynamics parameters. CONCLUSIONS: The present exploratory study identified several SNPs in the genes encoding regulatory nuclear receptors which may account for the interpatient variability in docetaxel pharmacokinetics and pharmacodynamics. These findings highlight the important role of regulatory nuclear receptors on the disposition of docetaxel.

P-glycoprotein and vacuolar ATPase synergistically confer anthracycline resistance to fission yeast and human cells.

Drug resistance is a major hurdle to the success of chemotherapy. The permeability glycoprotein (P-gp) is an important factor dictating drug access to the cells, as it controls the efflux of chemotherapeutic agents against the concentration gradient. Pmd1, a P-gp-like protein, was recently isolated as a doxorubicin resistance gene in fission yeast. Although the null mutant of pmd1 (Δpmd1) exhibited sensitivity to doxorubicin, it showed an unexpectedly high resistance to the drug at relatively high concentrations. The data presented here suggest that this is due to the presence of cooperative processes that can complement and counteract drug cytotoxicity in the absence of Pmd1. One such factor, Rav1, is an essential factor in controlling the assembly of the pH-regulating transporter vacuolar-ATPase (V-ATPase) in fission yeast. The simultaneous disruption of Pmd1 and Rav1 resulted in a prominent accumulation of doxorubicin in the cytoplasm of cells, accompanied by a decline in cell viability. With concurrent treatment of pharmacological inhibitors in human cervical cancer cells, P-gp and V-ATPase were further shown to act synergistically to sensitize cells to doxorubicin also in the human cells. Furthermore, a novel Cornichon-like protein SPAC2C4.05 (herein named as Cor1) was demonstrated for the first time to be involved in the interaction with P-gp and V-ATPase to counteract doxorubicin-dependent cytotoxicity. Therefore this study identified a molecular cooperation between multiple membrane transporter proteins that confers chemoresistance to cells against the chemical insult of doxorubicin. Interestingly, this network exhibited differential effects to doxorubicin as compared with its close epimeric analog epirubicin, suggestive of the intricacy of the drug response regulated by this synergistic interaction. A model is discussed on how the versatility of this network can differentiate closely related chemical drug structures yet allow for the robustness to counteract a vast range of drugs.

Genetic variations of NR2A1 in Asian populations: implications in pharmacogenetics studies.

HNF4α (encoded by gene NR2A1) is a dominant transcriptional regulator of various drug disposition genes. It forms a circuitry of molecular cross-talk with other nuclear receptors such as PXR and CAR to synergistically initiate transcription. This study reports on the frequency, linkage disequilibrium pattern and tag-SNP selection of NR2A1 polymorphisms in three local Asian populations, namely Chinese, Malays and Indians (n = 56 subjects each). A total of 69 polymorphisms were identified in the genomic region of NR2A1, of which thirty-three were novel polymorphisms with low allelic frequencies (<0.02). The exonic region of NR2A1 was highly conserved with only 4 novel and 1 reported SNPs identified at low allelic frequencies of less than 0.02. Based on the criteria of MAF ≥ 0.05 and R(2) ≥ 0.80, there were 19, 20 and 22 tag-SNPs selected to represent the genetic polymorphisms of NR2A1 in Chinese, Malays and Indians, respectively. In-silico predictions suggested that some of these polymorphic variants may exert functional effects through affecting the binding sites of transcription and splicing factors. Our study provides valuable information on the genetic variability of NR2A1 which would be useful for pharmacogenetics studies in the local Asian populations.

Genetic variations of NR1I3 and NR2B1 in Asian populations.

Several nuclear receptors are being increasingly recognized for their role as master xenosensors. Among them, CAR-RXRα heterodimer, as encoded by NR1I3 and NR2B1, responds to the presence of drug compounds and regulates the transcription of a wide array of genes involved in their disposition. To investigate the frequency distribution and linkage disequilibrium patterns of NR1I3 and NR2B1 genetic variations, these genes were screened in 168 healthy local Asian subjects, namely Chinese, Malays, and Indians (n=56 subjects each). A total of 38 and 88 SNPs were identified in NR1I3 and NR2B1, respectively. Among them, there were 13 and 43 novel SNPs present at low allelic frequencies (<10%) in NR1I3 and NR2B1, respectively. Notably, the genetic variations in the NR1I3 and NR2B1 genes were mainly confined to the introns whilst the exons were highly conserved across the ethnic populations. Indians harboured distinct frequency distributions from Chinese and Malays in both genes. Based on the linkage disequilibrium patterns of both genes, a number of tag-SNPs were selected for each population (n=8-13 for NR1I3; n=12-18 for NR2B1). In-silico prediction analyses revealed a number of possible functional SNPs. Our data would be valuable for future pharmacogenetic studies on the drug substrates of CAR-RXRα target genes.

Influence of SLCO1B3 haplotype-tag SNPs on docetaxel disposition in Chinese nasopharyngeal cancer patients.

WHAT IS ALREADY KNOWN ABOUT THIS SUBJECT: SLCO1B3 is an influx transporter located at the hepatocyte basolateral membrane and it is involved in the uptake of a broad range of drug substrates including docetaxel. The pharmacogenetics of SLCO1B3 is not well characterized and previous in vivo and in vitro studies reported conflicting results with regards to the functional effects of the limited number of SLCO1B3 polymorphisms that were studied. Docetaxel displays a wide interindividual variability in its pharmacokinetics and pharmacodynamics and an understanding of SLCO1B3 pharmacogenetics might provide clinical benefits in guiding docetaxel dosing. WHAT THIS STUDY ADDS: The SLCO1B3 gene was comprehensively screened in the local healthy Asian populations (n= 168). A strong linkage disequilibrium pattern was detected across a total of 88 polymorphisms and 15 haplotype-tag SNPs (htSNPs) were identified. These htSNPs were profiled in a cohort of Chinese nasopharyngeal cancer (NPC) patients (n= 50). Genotypic-phenotypic analysis showed that a haplotypic construct comprising of four variants [IVS4+76G>A, 699G>A(Met233Ile), IVS12-5676A>G, and *347_*348insA] was the critical determinant of docetaxel disposition. This study suggests that the comprehensive screening and haplotypic linkage analysis of SLCO1B3 can better elucidate its pharmacogenetic effects on interpatient variability of docetaxel and other putative drug substrates. Further studies are warranted in cancer patients belonging to other ethnic groups. AIMS To completely screen the SLCO1B3 gene in three distinct healthy Asian populations (Chinese, Malay and Indian, n= 168) and investigate the influence of haplotype-tag SNPs (htSNPs) on docetaxel disposition in 50 nasopharyngeal carcinoma patients. METHODS: Genomic DNA of individuals was screened for SLCO1B3 polymorphisms by direct sequencing. htSNPs were derived based on the sequence clustering algorithm and profiled in the patients. Population based genetic association analysis was performed using Haplostats package implemented in R and PLINK. RESULTS: A strong linkage disequilibrium pattern was detected across a total of 88 polymorphisms and 15-htSNPs were identified. The SLCO1B3 haplotypic region comprising seven htSNPs was found to be significantly associated with docetaxel clearance (P= 0.003). Conditional haplotype analyses revealed that the haplotypic constructs comprising the IVS4+76G>A, 699G>A(Met233Ile), IVS12-5676A>G, and *347_*348insA polymorphisms were critical determinants of variability in docetaxel disposition [clearance and area under the plasma concentration-time curve (AUC(0,∞)): r(2) = 29% and 22%, respectively]. Patients harbouring the GAG*347insA haplotype were significantly associated with a 30% decrease in clearance and a 40% increase in AUC(0,∞) of docetaxel compared with patients harbouring the reference haplotype, GGA*347wt (P= 0.025 and 0.018, respectively). In contrast, a 50% higher clearance was observed in patients carrying the GAG*347wt haplotype compared with those with the reference haplotype (P= 0.002). The functional SLCO1B3 haplotypic constructs included the widely studied Met233Ile variant and *347_*348insA located in the putative miR-890 binding site in the 3'-untranslated region which may influence the transport characteristics of SLCO1B3. CONCLUSIONS: This study highlights the importance of SLCO1B3 polymorphic variations in influencing docetaxel disposition in nasopharyngeal carcinoma patients.

Impact of CYP2D6, CYP3A5, CYP2C9 and CYP2C19 polymorphisms on tamoxifen pharmacokinetics in Asian breast cancer patients.

AIM: To investigate the impact of genetic polymorphisms in CYP2D6, CYP3A5, CYP2C9 and CYP2C19 on the pharmacokinetics of tamoxifen and its metabolites in Asian breast cancer patients. METHODS: A total of 165 Asian breast cancer patients receiving 20 mg tamoxifen daily and 228 healthy Asian subjects (Chinese, Malay and Indian; n= 76 each) were recruited. The steady-state plasma concentrations of tamoxifen and its metabolites were quantified using high-performance liquid chromatography. The CYP2D6 polymorphisms were genotyped using the INFINITI™ CYP450 2D6I assay, while the polymorphisms in CYP3A5, CYP2C9 and CYP2C19 were determined via direct sequencing. RESULTS: The polymorphisms, CYP2D6*5 and *10, were significantly associated with lower endoxifen and higher N-desmethyltamoxifen (NDM) concentrations. Patients who were *1/*1 carriers exhibited 2.4- to 2.6-fold higher endoxifen concentrations and 1.9- to 2.1-fold lower NDM concentrations than either *10/*10 or *5/*10 carriers (P < 0.001). Similarly, the endoxifen concentrations were found to be 1.8- to 2.6-times higher in *1/*5 or *1/*10 carriers compared with *10/*10 and *5/*10 carriers (P≤ 0.001). Similar relationships were observed between the CYP2D6 polymorphisms and metabolic ratios of tamoxifen and its metabolites. No significant associations were observed with regards to the polymorphisms in CYP3A5, CYP2C9 and CYP2C19. CONCLUSIONS: The present study in Asian breast cancer patients showed that CYP2D6*5/*10 and *10/*10 genotypes are associated with significantly lower concentrations of the active metabolite of tamoxifen, endoxifen. Identifying such patients before the start of treatment may be useful in optimizing therapy with tamoxifen. The role of CYP3A5, CYP2C9 and CYP2C19 seem to be minor.

The effects of CYP3A4, CYP3A5, ABCB1, ABCC2, ABCG2 and SLCO1B3 single nucleotide polymorphisms on the pharmacokinetics and pharmacodynamics of docetaxel in nasopharyngeal carcinoma patients.

PURPOSE: This exploratory study aimed to explain the interindividual variabilities of docetaxel pharmacokinetics and pharmacodynamics in Asian nasopharyngeal carcinoma patients (n = 54) through the genotyping of CYP3A4, CYP3A5, ABCB1, ABCC2, ABCG2 and SLCO1B3 genes. METHODS: Docetaxel was administered over 1 h on days 1, 8, and 15 every 28 days at 30 mg/m(2)/dose. Genomic DNA was isolated from peripheral blood and genotyped for the selected polymorphisms in the candidate genes. Docetaxel pharmacokinetic parameters were estimated by non-compartmental modelling. RESULTS: Patients homozygous for the variant allele (GG) of SLCO1B3 rs11045585 (IVS12-5676A > G) had significantly higher area under the plasma concentration-time curve of docetaxel (P = 0.026) and lower clearance (P = 0.036) compared to patients with AA/AG genotypes. Patients harbouring the heterozygous genotype (GA + GT + TA) for ABCB1 rs2032582 (2677G > T/A) had the highest percentage decrease in nadir haemoglobin from cycle 1 baseline compared to those with GG/TT genotypes (P = 0.006). Similar trend was observed for ABCB1 rs1045642 (3435C > T) with heterozygotes (CT) having the highest percentage decrease in nadir haemoglobin from cycle 1 baseline compared to those with CC/TT genotypes (P = 0.066). CONCLUSIONS: This study suggests that the cooperative influence of functional polymorphisms in SLCO1B3 and ABCB1 genes may be responsible for the interindividual variability in docetaxel disposition in Asian nasopharyngeal cancer patients.

Pharmacogenetics of CYP1A2, novel polymorphisms and haplotypes in three distinct Asian populations.

CYP1A2 play an important role in the metabolism of many carcinogens and clinically important drugs. CYP1A2 activity has been found to be influenced by the presence of polymorphic variants which were reported to display wide interethnic variation. This study investigates the frequency distribution and linkage disequilibrium patterns of CYP1A2 genetic polymorphisms, and characterize their haplotype structures in three healthy Asian populations in Singapore (Chinese, Malay, and Indian). The entire CYP1A2 gene was screened in 126 healthy subjects from all three ethnic groups (N=42 each). A total of 25 polymorphisms was identified, of which nine were novel. The polymorphisms, -2467delT and -163C>A were detected at high frequencies in all Asian ethnic groups. Significant interethnic differences were observed in the genotypic frequency distribution of IVS2-99G>A (P<0.01) and 1548C>T (P=0.05) across the three ethnic groups while -163C>A (P=0.02) was found to differ between Chinese and Malays. Haplotype analyses revealed four to six major haplotypes in each ethnic population which accounted for more than 60% of the cumulative haplotype frequencies. Future studies should be done to investigate the functional roles of these haplotypes.

Genomic copy number variations in three Southeast Asian populations.

Research on the role of copy number variations (CNVs) in the genetic risk of diseases in Asian populations has been hampered by a relative lack of reference CNV maps for Asian populations outside the East Asians. In this article, we report the population characteristics of CNVs in Chinese, Malay, and Asian Indian populations in Singapore. Using the Illumina Human 1M Beadchip array, we identify 1,174 CNV loci in these populations that corroborated with findings when the same samples were typed on the Affymetrix 6.0 platform. We identify 441 novel loci not previously reported in the Database of Genomic Variations (DGV). We observe a considerable number of loci that span all three populations and were previously unreported, as well as population-specific loci that are quite common in the respective populations. From this we observe the distribution of CNVs in the Asian Indian population to be considerably different from the Chinese and Malay populations. About half of the deletion loci and three-quarters of duplication loci overlap UCSC genes. Tens of loci show population differentiation and overlap with genes previously known to be associated with genetic risk of diseases. One of these loci is the CYP2A6 deletion, previously linked to reduced susceptibility to lung cancer.

Oral sulfasalazine as a clinical BCRP probe substrate: pharmacokinetic effects of genetic variation (C421A) and pantoprazole coadministration.

This study evaluated the utility of oral sulfasalazine as a probe substrate for Breast Cancer Resistance Protein (BCRP; ABCG2) activity by assessing the impact of genetic variation or coadministration of an inhibitor (pantoprazole) on plasma and urine pharmacokinetics of sulfasalazine and metabolites. Thirty-six healthy male subjects prescreened for ABCG2 421CC (reference activity), CA, and AA (lower activity) genotypes (N = 12 each) received a single 500 mg oral dose of enteric coated sulfasalazine alone, with 40 mg pantoprazole, or with 40 mg famotidine (gastrointestinal pH control) in a 3-period, single fixed sequence, crossover design. No significant difference in sulfasalazine or metabolite pharmacokinetics in 421AA or CA compared to 421CC subjects was found; however, high inter-subject variability was observed. Geometric mean (95% CI) sulfasalazine plasma AUC((0-infinity)) values were 32.1 (13.2, 78.1), 16.8 (7.15, 39.6) and 62.7 (33.4, 118) microg h/mL, and C(max) were 4.01 (1.62, 9.92), 1.70 (0.66, 4.40), and 6.86 (3.61, 13.0) microg/mL for CC, CA, and AA subjects, respectively. Pantoprazole and famotidine did not affect sulfasalazine pharmacokinetics in any genotypic cohort. These results suggest that the pharmacokinetics of oral, enteric-coated 500 mg sulfasalazine are not sufficiently sensitive to ABCG2 genetic variation or inhibitors to be useful as a clinical probe substrate of BCRP activity.

Singapore Genome Variation Project: a haplotype map of three Southeast Asian populations.

The Singapore Genome Variation Project (SGVP) provides a publicly available resource of 1.6 million single nucleotide polymorphisms (SNPs) genotyped in 268 individuals from the Chinese, Malay, and Indian population groups in Southeast Asia. This online database catalogs information and summaries on genotype and phased haplotype data, including allele frequencies, assessment of linkage disequilibrium (LD), and recombination rates in a format similar to the International HapMap Project. Here, we introduce this resource and describe the analysis of human genomic variation upon agglomerating data from the HapMap and the Human Genome Diversity Project, providing useful insights into the population structure of the three major population groups in Asia. In addition, this resource also surveyed across the genome for variation in regional patterns of LD between the HapMap and SGVP populations, and for signatures of positive natural selection using two well-established metrics: iHS and XP-EHH. The raw and processed genetic data, together with all population genetic summaries, are publicly available for download and browsing through a web browser modeled with the Generic Genome Browser.

CBR1 and CBR3 pharmacogenetics and their influence on doxorubicin disposition in Asian breast cancer patients.

The present study aimed to identify polymorphic genes encoding carbonyl reductases (CBR1, CBR3) and investigate their influence on doxorubicin disposition in Asian breast cancer patients (n = 62). Doxorubicin (60 mg/m(2)) was administered every 3 weeks for four to six cycles and the pharmacokinetic parameters were estimated using non-compartmental analysis (WinNonlin). The Mann-Whitney U-test was used to assess genotypic-phenotypic correlations. Five CBR1 (-48G>A, c.219G>C, c.627C>T, c.693G>A, +967G>A) and CBR3 (c.11G>A, c.255C>T, c.279C>T, c.606G>A, c.730G>A) polymorphisms were identified. The CBR1 D2 diplotypes were characterized by the presence of at least one variant allele at the c.627C>T and +967G>A loci. Patients in the CBR1 D1 diplotype group had significantly higher clearance (CL) normalized to body surface area (BSA) (CL/BSA[L/h/m(2)]: median 25.09; range 16.44-55.66) and significantly lower exposure levels; area under curve (AUC(0-infinity)/dose/BSA [h/m(5)]; median 15.08; range 6.18-38.03) of doxorubicin compared with patients belonging to the CBR1 D2 diplotype group (CL/BSA[L/h/m(2)]; median 20.88; range 8.68-31.79, P = 0.014; and AUC(0-infinity)/dose/BSA[h/m(5)]; median 21.35; range 9.82-67.17, P = 0.007 respectively). No significant influence of CBR3 polymorphisms on the pharmacokinetics of doxorubicin were observed in Asian cancer patients. The present exploratory study shows that CBR1 D2 diplotypes correlate with significantly higher exposure levels of doxorubicin, suggesting the possibility of lowered intracellular conversion to doxorubicinol in these patients. Further evaluation of carbonyl reductase polymorphisms in influencing the treatment efficacy of doxorubicin-based chemotherapy in Asian cancer patients are warranted.

PXR pharmacogenetics: association of haplotypes with hepatic CYP3A4 and ABCB1 messenger RNA expression and doxorubicin clearance in Asian breast cancer patients.

PURPOSE: To characterize pregnane X receptor (PXR) polymorphic variants in healthy Asian populations [Chinese, Malay and Indian (n=100 each)], and to investigate the association between PXR haplotypes and hepatic mRNA expression of PXR and its downstream target genes, CYP3A4 and ABCB1, as well as their influence on the clearance of doxorubicin in Asian breast cancer patients. EXPERIMENTAL DESIGN: PXR genotyping was done by direct DNA sequencing, and PXR haplotypes and haplotype clusters were derived by expectation-maximization algorithm. Genotype-phenotype correlations were done using Mann-Whitney U test and Kruskal-Wallis test. RESULTS: Significant interethnic variations were observed in PXR pharmacogenetics among the three Asian ethnic groups. The expression of PXR mRNA in liver tissues harboring the PXR*1B haplotype clusters was 4-fold lower compared with the non-PXR*1B (*1A + *1C) haplotype clusters [PXR*1B versus PXR*1A; P=0.015; PXR*1B versus PXR*1C; P=0.023]. PXR*1B-bearing liver tissues were associated with significantly lower expression of CYP3A4 (PXR*1B versus non-PXR*1B, P=0.030) and ABCB1 (PXR*1B versus non-PXR*1B, P=0.060) compared with non-PXR*1B-bearing liver tissues. Doxorubicin clearance in breast cancer patients harboring the PXR*1B haplotypes was significantly lower compared with patients carrying the non-PXR*1B haplotypes [PXR*1B versus non-PXR*1B, CL/BSA (L h(-1) m(-2)): 20.84 (range, 8.68-29.24) versus 24.85 (range, 13.80-55.66), P=0.022]. CONCLUSIONS: This study showed that PXR*1B was associated with reduced hepatic mRNA expression of PXR and its downstream targets, CYP3A4 and ABCB1. Genotype-phenotype correlates in breast cancer patients showed PXR*1B to be significantly associated with lower doxorubicin clearance, suggesting that PXR haplotype constitution could be important in influencing interindividual and interethnic variations in disposition of its putative drug substrates.

Influence of ABCB1 and ABCG2 polymorphisms on doxorubicin disposition in Asian breast cancer patients.

The influence of three high frequency ABCB1 polymorphisms (c.1236C>T, c.2677G>A/T, and c.3435C>T) and the ABCG2 c.421C>A polymorphism on the disposition of doxorubicin in Asian breast cancer patients receiving adjuvant chemotherapy was investigated in the present study. The allelic frequency of the ABCB1 c.1236T, c.2677T, c.2677A, and c.3435T variants were 60%, 38%, 7%, and 22%, respectively, and the frequency of the ABCG2 c.421A allele was 23%. Pairwise analysis showed increased exposure levels to doxorubicin in patients harboring at least one ABCB1 c.1236T allele (P = 0.03). Patients homozygous for the CC-GG-CC genotype had significantly lower doxorubicin exposure levels compared to the patients who had CT-GT-CT (P = 0.02) and TT-TT-TT genotypes (P = 0.03). Significantly increased clearance of doxorubicin was also observed in patients harboring CC-GG-CC genotypes when compared to patients harboring the CT-GT-CT genotype (P = 0.01). Patients harboring the CC-GG-CC genotypes had significantly lower peak plasma concentrations of doxorubicinol compared to patients who had TT-TT-TT genotypes (P = 0.03). No significant influences on doxorubicin pharmacokinetic parameters were observed in relation to the ABCG2 c.421C>A polymorphism. In conclusion, the present exploratory study suggests that the three high frequency linked polymorphisms in the ABCB1 gene might be functionally important with regards to the altered pharmacokinetics of doxorubicin in Asian breast cancer patients, resulting in significantly increased exposure levels and reduced clearance.

The ABCG2 C421A polymorphism does not affect oral nitrofurantoin pharmacokinetics in healthy Chinese male subjects.

AIMS: A number of drugs are substrates or inhibitors of the efflux transporter breast cancer resistance protein (BCRP; ABCG2), which can limit systemic exposure by reducing absorption and/or increasing biliary elimination. The identification of a BCRP-selective clinical probe drug would provide a useful tool to understand the effect of genetic polymorphisms and transporter-based drug interactions on drug pharmacokinetics. The aim of this study was to assess the utility of nitrofurantoin as a clinical probe substrate for BCRP activity by evaluating the impact of genetic variation on nitrofurantoin pharmacokinetics. METHODS: Nitrofurantoin pharmacokinetics were studied in an open-label, single-oral dose (100 mg) study in 36 male Chinese subjects who were pre-screened for ABCG2 421 CC, CA and AA genotypes (n = 12 each). Plasma and urine concentrations of nitrofurantoin were determined by LC/MS/MS and LC/UV respectively. anova was used to compare pharmacokinetic parameters among genotypes. RESULTS: There were no significant differences in nitrofurantoin pharmacokinetics among the genotypic cohorts. The geometric mean nitrofurantoin plasma AUC((0-infinity)) (95% confidence interval) values were 2.21 (2.00, 2.45), 2.42 (2.11, 2.78) and 2.32 (1.99, 2.70) microg h ml(-1) and half-life values were 0.79 (0.59, 1.0), 0.76 (0.64, 0.89) and 0.72 (0.62, 0.84) h for ABCG2 421 genotypes CC, CA and AA, respectively. The percentage of dose excreted unchanged in the urine was 43, 44 and 39%, respectively. CONCLUSIONS: The ABCG2 C421A polymorphism had no effect on nitrofurantoin plasma and urine pharmacokinetic parameters in healthy Chinese subjects. These results indicate that nitrofurantoin is not a suitable clinical probe substrate for assessing BCRP activity.

Mutation screening in KCNQ1, HERG, KCNE1, KCNE2 and SCN5A genes in a long QT syndrome family.

INTRODUCTION: Long QT syndrome (LQTS), an inherited cardiac arrhythmia, is a disorder of ventricular repolarisation characterised by electrocardiographic abnormalities and the onset of torsades de pointes leading to syncope and sudden death. Genetic polymorphisms in 5 well-characterised cardiac ion channel genes have been identified to be responsible for the disorder. The aim of this study is to identify disease-causing mutations in these candidate genes in a LQTS family. MATERIALS AND METHODS: The present study systematically screens the coding region of the LQTS-associated genes (KCNQ1, HERG, KCNE1, KCNE2 and SCN5A) for mutations using DNA sequencing analysis. RESULTS: The mutational analysis revealed 7 synonymous and 2 non-synonymous polymorphisms in the 5 ion channel genes screened. CONCLUSION: We did not identify any clear identifiable genetic marker causative of LQTS, suggesting the existence of LQTS-associated genes awaiting discovery.