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.

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.

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.

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.