HLA-DRB1*16: 01-DQB1*05: 02 is a novel genetic risk factor for flupirtine-induced liver injury.

OBJECTIVE: Flupirtine is a nonopioid analgesic with regulatory approval in a number of European countries. Because of the risk of serious liver injury, its use is now limited to short-term pain management. We aimed to identify genetic risk factors for flupirtine-related drug-induced liver injury (DILI) as these are unknown. MATERIALS AND METHODS: Six flupirtine-related DILI patients from Germany were included in a genome-wide association study (GWAS) involving a further 614 European cases of DILI because of other drugs and 10,588 population controls. DILI was diagnosed by causality assessment and expert review. Human leucocyte antigen (HLA) and single nucleotide polymorphism genotypes were imputed from the GWAS data, with direct HLA typing performed on selected cases to validate HLA predictions. Four replication cases that were unavailable for the GWAS were genotyped by direct HLA typing, yielding an overall total of 10 flupirtine DILI cases. RESULTS: In the six flupirtine DILI cases included in the GWAS, we found a significant enrichment of the DRB1*16:01-DQB1*05:02 haplotype compared with the controls (minor allele frequency cases 0.25 and minor allele frequency controls 0.013; P=1.4 × 10(-5)). We estimated an odds ratio for haplotype carriers of 18.7 (95% confidence interval 2.5-140.5, P=0.002) using population-specific HLA control data. The result was replicated in four additional cases, also with a haplotype frequency of 0.25. In the combined cohort (six GWAS plus four replication cases), the haplotype was also significant (odds ratio 18.7, 95% confidence interval 4.31-81.42, P=6.7 × 10(-5)). CONCLUSION: We identified a novel HLA class II association for DILI, confirming the important contribution of HLA genotype towards the risk of DILI generally.

Which Genetic Determinants Should be Considered for Tacrolimus Dose Optimization in Kidney Transplantation? A Combined Analysis of Genes Affecting the CYP3A Locus.

BACKGROUND: Tacrolimus is established as immunosuppressant after kidney transplantation. Polymorphism of the cytochrome P450 3A5 (CYP3A5) gene contributes significantly to tacrolimus dose requirements. Recently, CYP3A4*22 was reported to additionally affect tacrolimus pharmacokinetics (PK). In addition, there are further polymorphic genes, possibly influencing CYP3A activity [pregnane x receptor NR1I2, P450 oxidoreductase (POR), and peroxisome proliferator-activator receptor alpha (PPARA)]. We aimed to investigate combined effects of these gene variants on tacrolimus maintenance dose and PK in patients with stable kidney transplantation of 2 study centers. METHODS: A total of 223 white patients (German cohort, 136; Danish cohort, 87) was included and genotyped for CYP3A5 (rs776746), CYP3A4 (rs35599367), NR1I2 (rs2276707), POR (rs1057868), and PPARA (rs4253728). Dosage and trough concentration/dose ratios were considered separately. A subset was investigated for comprehensive PK parameters. RESULTS: Tacrolimus dose, trough concentration, and trough concentration/dose ratio did not differ between the German and Danish cohort. CYP3A5*3 and CYP3A4*22 contributed to dose requirements only in the German and in the total cohort. Homozygous carriers of both variants required 4.8 ± 3.1 mg, whereas carriers of the wild types required 165% higher mean tacrolimus doses (12.5 ± 7.7 mg, P = 1.4 × 10). The PK investigation revealed only nonsignificant impact of CYP3A4 genotypes on AUC12h in CYP3A5 nonexpressers (P = 0.079, power = 57%). For the entire sample, the final multiple linear regression model for trough concentration/dose ratio included CYP3A5, CYP3A4, and age. It explained 18.3% of the interindividual variability of tacrolimus trough concentration/dose ratios (P = 8.8 × 10). CONCLUSIONS: Therapeutic drug monitoring remains essential in clinical care of patients with kidney transplantation. Genotyping of CYP3A5 and CYP3A4, however, could facilitate rapid dose finding to adapt the appropriate immunosuppressant dose, whereas other genetic factors had only little or no effect.

Genetic variants may play an important role in mRNA-miRNA interaction: evidence for haplotype-dependent downregulation of ABCC2 (MRP2) by miRNA-379.

BACKGROUND: The functional influence of single-nucleotide polymorphisms (SNPs) of the ATP-binding cassette (ABC) transporter ABCC2 (MRP2) has been characterized in numerous studies. The aim of this study was to address the question of whether distinct ABCC2 haplotypes, which differ in their mRNA secondary structures, show an influence on the degree of mRNA and protein downregulation through miRNA interaction. METHODS: A model using human peripheral blood monocytic cells (PBMCs) isolated from healthy Caucasian volunteers, with three defined ABCC2 haplotypes comprising the 5'-UTR SNP -24C>T, the 1249G>A SNP (V417I), and the silent 3972C>T SNP, was outlined. Cells were transiently transfected with miRNA-379, already known to target ABCC2 in HepG2 cells. RESULTS: ABCC2 was downregulated through miR-379 in a haplotype-dependent manner: the wild-type CGC/CGC was modestly affected (mRNA: -12.7±4.2%, protein: -9.9±0.1%), whereas variant haplotypes were more strongly suppressed: CGT/CGT (mRNA: -36.7±2.4%, protein: -21.6±0.4%) and TGT/TGT (mRNA: -55.7±1.2%, protein: -46.3±4.0%). In addition, glutathione-methylfluorescein efflux was significantly reduced in miR-379-transfected peripheral blood monocytic cells corresponding to ABCC2 protein expression. CONCLUSION: This observation may suggest a differential suppression of ABCC2 by miR-379 caused by haplotype-dependent differences in mRNA secondary structures, resulting in changes in mRNA target accessibility or mRNA stability.

MicroRNAs and their relevance to ABC transporters.

MicroRNAs (miRNAs) are small noncoding RNAs, which regulate the expression of their target genes post-transcriptionally by RNA interference. They are involved in almost all cellular processes, including proliferation, differentiation, apoptosis, cell survival and the maintenance of tissue specificity. Recent findings also suggest that efflux pumps of the ABC (ATP-binding cassette) transporter family are subject to miRNA-mediated gene regulation. Moreover, it seems that ABC transporters are embedded in a concerted and miRNA-guided network of concurrently regulated proteins that mediate altered drug transport and cell survival in changing environmental conditions. In this review, we summarize recent findings of miRNAs interacting with ABC transporters, which have been connected with drug distribution as well as with drug resistance. Additionally, we specify findings of complex miRNA-protein pathways conferring increased drug export and cell survival.

Epigenetic modulation of the drug resistance genes MGMT, ABCB1 and ABCG2 in glioblastoma multiforme.

BACKGROUND: Resistance of the highly aggressive glioblastoma multiforme (GBM) to drug therapy is a major clinical problem resulting in a poor patient's prognosis. Beside promoter methylation of the O6-methylguanine-DNA-methyltransferase (MGMT) gene the efflux transporters ABCB1 and ABCG2 have been suggested as pivotal factors contributing to drug resistance, but the methylation of ABCB1 and ABCG2 has not been assessed before in GBM. METHODS: Therefore, we evaluated the proportion and prognostic significance of promoter methylation of MGMT, ABCB1 and ABCG2 in 64 GBM patient samples using pyrosequencing technology. Further, the single nucleotide polymorphisms MGMT C-56 T (rs16906252), ABCB1 C3435T (rs1045642) and ABCG2 C421A (rs2231142) were determined using the restriction fragment length polymorphism method (RFLP). To study a correlation between promoter methylation and gene expression, we analyzed MGMT, ABCB1 and ABCG2 expression in 20 glioblastoma and 7 non-neoplastic brain samples. RESULTS: Despite a significantly increased MGMT and ABCB1 promoter methylation in GBM tissue, multivariate regression analysis revealed no significant association between overall survival of glioblastoma patients and MGMT or ABCB1 promoter methylation. However, a significant negative correlation between promoter methylation and expression could be identified for MGMT but not for ABCB1 and ABCG2. Furthermore, MGMT promoter methylation was significantly associated with the genotypes of the MGMT C-56 T polymorphism showing a higher methylation level in the T allele bearing GBM. CONCLUSIONS: In summary, the data of this study confirm the previous published relation of MGMT promoter methylation and gene expression, but argue for no pivotal role of MGMT, ABCB1 and ABCG2 promoter methylation in GBM patients' survival.

Challenges in pharmacogenetics.

The attempt to optimize drug treatment of patients by using evidenced-based medicine considering individual physiological and disease-related conditions is standard of modern medicine. Pharmacogenetics (PGx) has contributed to individualization considering hereditary genetic information; however, increasingly, pharmacogenomics is becoming essential, particularly in relation to modern oncology. New technologies such as next-generation sequencing and rapid development of computational and information sciences will help to better elucidate the consequences of genetic variation, considering also epigenetics and gene-environmental interactions and their translation into clinically relevant individual phenotypes. This review highlights the current challenging and most promising examples of PGx.

Down-regulation of ATP-binding cassette C2 protein expression in HepG2 cells after rifampicin treatment is mediated by microRNA-379.

microRNAs (miRNAs), which contribute to the post-transcriptional processing through 3'-untranslated region-interference, have been shown to be involved in the regulation of ATP-binding cassette (ABC) membrane transporters. The aim of this study was to investigate whether ABCC2, an important efflux transporter for various endogenous and exogenous compounds at several compartment barriers, is subject to miRNA-mediated post-transcriptional gene regulation. We screened the expression of 377 human miRNAs in HepG2 cells after 48 h of treatment with 5 µM rifampicin [a pregnane X receptor (PXR) ligand] or vehicle using reverse transcription-polymerase chain reaction-based low-density arrays. Specific miRNA, ABCC2 mRNA, and protein expression were monitored in HepG2 cells undergoing rifampicin treatment for 72 h. Loss- and gain-of-function experiments and reporter gene assays were performed for further confirmation. Highly deregulated miRNAs compared with in silico data revealed miRNA (miR) 379 as candidate miRNA targeting ABCC2 mRNA. Under rifampicin treatment, ABCC2 mRNA increased significantly, with a maximal fold change of 1.56 ± 0.43 after 24 h. In addition, miR-379 increased (maximally 4.10 ± 1.33-fold after 48 h), whereas ABCC2 protein decreased with a maximal fold change of 0.47 ± 0.08 after 72 h. In contrast, transfection of miR-379 inhibitor led to an elevation of ABCC2 protein expression after rifampicin incubation for 48 h. We identify a miRNA negatively regulating ABCC2 on the post-transcriptional level and provide evidence that this miRNA impedes overexpression of ABCC2 protein after a PXR-mediated external transcriptional stimulus in HepG2 cells.

Implications of genetic variation of common Drug Metabolizing Enzymes and ABC Transporters among the Pakistani Population.

Genetic polymorphism of drug metabolizing enzymes and transporters may influence drug response. The frequency varies substantially between ethnicities thus having implications on appropriate selection and dosage of various drugs in different populations. The distribution of genetic polymorphisms in healthy Pakistanis has so far not been described. In this study, 155 healthy adults (98 females) were included from all districts of Karachi. DNA was extracted from saliva and genotyped for relevant SNVs in CYP1A1, CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP3A4 and CYP3A5 as well as ALDH3A1, GSTA1, ABCB1 and ABCC2. About 64% of the participants were born to parents who were unrelated to each other. There was generally a higher prevalence (p < 0.05) of variant alleles of CYP450 1A2, 2B6, 2C19, 3A5, ALDH3A1, GSTM1 as well as ABCB1 and ABCC2 in this study cohort than in other ethnicities reported in the HapMap database. In contrast, the prevalence of variant alleles was lower in GSTA1. Therefore, in the Pakistani population sample from Karachi a significantly different prevalence of variant drug metabolizing enzymes and ABC transporters was observed as compared to other ethnicities, which could have putative clinical consequences on drug efficacy and safety.

Advances and challenges in hereditary cancer pharmacogenetics.

INTRODUCTION: Cancer pharmacogenetics usually considers tumor-specific targets. However, hereditary genetic variants may interfere with the pharmacokinetics of antimetabolites and other anti-cancer drugs, which may lead to severe adverse events. Areas covered: Here, the impact of hereditary genes considered in drug labels such as thiopurine S-methyltransferase (TPMT), UDP-glucuronosyltransferase 1A1 (UTG1A1) and dihydropyrimidine dehydrogenase (DPYD) are discussed with respect to guidelines of the Clinical Pharmacogenetics Implementation Consortium (CPIC). Moreover, the association between genetic variants of drug transporters with the clinical outcome is comprehensively discussed. Expert opinion: Precision therapy in the field of oncology is developing tremendously. There are a number of somatic tumor genetic markers that are indicative for treatment with anti-cancer drugs. By contrast, for some hereditary variants, recommendations have been developed. Although we have vast knowledge on the association between drug transporter variants and clinical outcome, the overall data is inconsistent and the predictability of the related phenotype is low. Further developments in research may lead to the discovery of rare, but functionally relevant single nucleotide polymorphisms and a better understanding of multiple genomic, epigenomic as well as phenotypic factors, contributing to drug response in malignancies.

Correction: A European Spectrum of Pharmacogenomic Biomarkers: Implications for Clinical Pharmacogenomics.

[This corrects the article DOI: 10.1371/journal.pone.0162866.].

A European Spectrum of Pharmacogenomic Biomarkers: Implications for Clinical Pharmacogenomics.

Pharmacogenomics aims to correlate inter-individual differences of drug efficacy and/or toxicity with the underlying genetic composition, particularly in genes encoding for protein factors and enzymes involved in drug metabolism and transport. In several European populations, particularly in countries with lower income, information related to the prevalence of pharmacogenomic biomarkers is incomplete or lacking. Here, we have implemented the microattribution approach to assess the pharmacogenomic biomarkers allelic spectrum in 18 European populations, mostly from developing European countries, by analyzing 1,931 pharmacogenomics biomarkers in 231 genes. Our data show significant inter-population pharmacogenomic biomarker allele frequency differences, particularly in 7 clinically actionable pharmacogenomic biomarkers in 7 European populations, affecting drug efficacy and/or toxicity of 51 medication treatment modalities. These data also reflect on the differences observed in the prevalence of high-risk genotypes in these populations, as far as common markers in the CYP2C9, CYP2C19, CYP3A5, VKORC1, SLCO1B1 and TPMT pharmacogenes are concerned. Also, our data demonstrate notable differences in predicted genotype-based warfarin dosing among these populations. Our findings can be exploited not only to develop guidelines for medical prioritization, but most importantly to facilitate integration of pharmacogenomics and to support pre-emptive pharmacogenomic testing. This may subsequently contribute towards significant cost-savings in the overall healthcare expenditure in the participating countries, where pharmacogenomics implementation proves to be cost-effective.

Targeting gene expression during the early bone healing period in the mandible: A base for bone tissue engineering.

PURPOSE: Although bone tissue engineering techniques have become more and more sophisticated than in the past, natural bone healing mechanisms have not been sufficiently considered for further improvement of these techniques so far. We used an established animal model with transcriptome analysis to generate an unbiased picture of early bone healing to support tissue engineering concepts. MATERIAL AND METHODS: In 30 Wistar rats, a 3-mm bone defect was created in the mandibular angle. Tissue was sampled at 5, 10, and 15 days, and the former defect area was excised to undergo transcriptome analysis after RNA extraction. Five differentially expressed genes were further evaluated with reverse transcription-polymerase chain reaction (rt-PCR). RESULTS: Transcriptome analysis revealed 2467 significantly over- and under-expressed transcripts after 5 days and 2265 after 15 days of bone healing, respectively. Validation via rt-PCR confirmed overexpression of osteoactivin, angiopoietin-like factor-4, and metallomatrix proteinase-9 and underexpression of mastcellprotease-10 and proteoglycane-2 in comparison to values in the control group. CONCLUSION: This systematic genome-wide transcriptome analysis helps to decipher the physiological mechanisms behind physiological bone healing. The exemplary depiction of 5 genes demonstrates the great complexity of metabolic processes during early bone healing. Here, BMP-2 signaling pathways and local hypoxia play decisive roles in bone formation.