Molecular Mechanisms of Tyrosine Kinase Inhibitor Resistance in Chronic Myeloid Leukemia.

The hematopoietic neoplasm chronic myeloid leukemia (CML) is a rare disease caused by chromosomal reciprocal translocation t(9;22)(q34:q11) with subsequent formation of the BCR-ABL1 fusion gene. This fusion gene encodes a constitutively active tyrosine kinase, which results in malignant transformation of the cells. Since 2001, CML can be effectively treated using tyrosine kinase inhibitors (TKIs) such as imatinib, which prevent phosphorylation of downstream targets by blockade of the BCR-ABL kinase. Due to its tremendous success, this treatment became the role model of targeted therapy in precision oncology. Here, we review the mechanisms of TKI resistance focusing on BCR-ABL1-dependent and -independent mechanisms. These include the genomics of the BCR-ABL1, TKI metabolism and transport and alternative signaling pathways.

CRISPR/Cas9-induced knockout reveals the role of ABCB1 in the response to temozolomide, carmustine and lomustine in glioblastoma multiforme.

Glioblastoma multiforme (GBM) is the most common malignant brain tumor with limited therapeutic options. Besides surgery, chemotherapy using temozolomide, carmustine or lomustine is the main pillar of therapy. However, therapy success is limited and prognosis still is very poor. One restraining factor is drug resistance caused by drug transporters of the ATP-binding cassette family, e.g. ABCB1 and ABCG2, located at the blood-brain barrier and on tumor cells. The active efflux of xenobiotics including drugs, e.g. temozolomide, leads to low intracellular drug concentrations and subsequently insufficient anti-tumor effects. Nevertheless, the role of efflux transporters in GBM is controversially discussed. In the present study, we analyzed the role of ABCB1 and ABCG2 in GBM cells showing that ABCB1, but marginally ABCG2, is relevant. Applying a CRISPR/Cas9-derived ABCB1 knockout, the response to temozolomide was significantly augmented demonstrated by decreased cell number (p < 0.001) and proliferation rate (p = 0.04), while apoptosis was increased (p = 0.04). For carmustine, a decrease of cells in G1-phase was detected pointing to cell cycle arrest in the ABCB1 knockout (p = 0.006). For lomustine, however, loss of ABCB1 did not alter the response to the treatment. Overall, this study shows that ABCB1 is involved in the active transport of temozolomide out of the tumor cells diminishing the response to temozolomide. Interestingly, loss of ABCB1 also affected the response to the lipophilic drug carmustine. These findings show that ABCB1 is not only relevant at the blood-brain barrier, but also in the tumor cells diminishing success of chemotherapy.

Limited Impact of 6-Mercaptopurine on Inflammation-Induced Chemokines Expression Profile in Primary Cultures of Enteric Nervous System.

Increasing evidences indicate that the enteric nervous system (ENS) and enteric glial cells (EGC) play important regulatory roles in intestinal inflammation. Mercaptopurine (6-MP) is a cytostatic compound clinically used for the treatment of inflammatory bowel diseases (IBD), such as ulcerative colitis and Crohn's disease. However, potential impacts of 6-MP on ENS response to inflammation have not been evaluated yet. In this study, we aimed to gain deeper insights into the profile of inflammatory mediators expressed by the ENS and on the potential anti-inflammatory impact of 6-MP in this context. Genome-wide expression analyses were performed on ENS primary cultures exposed to lipopolysaccharide (LPS) and 6-MP alone or in combination. Differential expression of main hits was validated by quantitative real-time PCR (qPCR) using a cell line for EGC. ENS cells expressed a broad spectrum of cytokines and chemokines of the C-X-C motif ligand (CXCL) family under inflammatory stress. Induction of Cxcl5 and Cxcl10 by inflammatory stimuli was confirmed in EGC. Inflammation-induced protein secretion of TNF-α and Cxcl5 was partly inhibited by 6-MP in ENS primary cultures but not in EGC. Further work is required to identify the cellular mechanisms involved in this regulation. These findings extend our knowledge of the anti-inflammatory properties of 6-MP related to the ENS and in particular of the EGC-response to inflammatory stimuli.

Interaction of Phytocompounds of Echinacea purpurea with ABCB1 and ABCG2 Efflux Transporters.

Preparations of Echinacea purpurea (E. purpurea) are widely used for the management of upper respiratory infections, influenza, and common cold, often in combination with other conventional drugs. However, the potential of phytochemical constituents of E. purpurea to cause herb-drug interactions via ABCB1 and ABCG2 efflux transporters remains elusive. The purpose of this study was to investigate the impact of E. purpurea-derived caffeic acid derivatives (cichoric acid and echinacoside) and tetraenes on the mRNA and protein expression levels as well as on transport activity of ABCB1 and ABCG2 in intestinal (Caco-2) and liver (HepG2) cell line models. The safety of these compounds was investigated by estimating EC20 values of cell viability assays in both cell lines. Regulation of ABCB1 and ABCG2 protein in these cell lines were analyzed after 24 h exposure to the compounds at 1, 10, and 50 µg/mL. Bidirectional transport of 0.5 µg/mL Hoechst 33342 and 5 µM rhodamine across Caco-2 monolayer and profiling for intracellular concentrations of the fluorophores in both cell lines were conducted to ascertain inhibition effects of the compounds. Cichoric acid showed no cytotoxic effect, while the EC20 values of tetraenes and echinacoside were 45.0 ± 3.0 and 52.0 ± 4.0 µg/mL in Caco-2 cells and 28.0 ± 4.3 and 62.0 ± 9.9 µg/mL in HepG2 cells, respectively. In general, the compounds showed heterogeneous induction of ABCB1 with the strongest 3.6 ± 1.2-fold increase observed for 10 µg/mL tetraenes in Caco-2 cells (p < 0.001). However, the compounds did not induce ABCG2. None of the phytocompounds inhibited significantly net flux of the fluorophores across Caco-2 monolayers. Overall, tetraenes moderately induced ABCB1 but not ABCG2 in Caco-2 and HepG2 cells while no compound significantly inhibited activity of these transporters at clinically relevant concentration to cause herb-drug interactions.

Involvement of medical students in a surgery congress: impact on learning motivation, decision-making for a career in surgery, and educational curriculum.

During the preclinical period of medical school, the clinical relevance of theoretical knowledge is given little attention. Medical students of the second year were invited to participate in an interdisciplinary congress for robot-assisted and digital surgery. The students had to evaluate the impact of the congress on their learning motivation, decision-making for a career in surgery, and relevance for their educational curriculum. Participation in the congress increased their learning motivation for preclinical subjects, and significantly increased their interest in a surgical career. Most students considered active involvement in medical congresses a valuable supplement to the medical curriculum. Congress participation during the preclinical period was ranked positively by medical students. Greater learning motivation and enthusiasm for the pilot teaching project as well as for surgical disciplines were registered. Thus, early involvement of medical students in scientific congresses should be an integral part of their educational curriculum.

Alternative Polyadenylation of ABC Transporters of the C-Family (ABCC1, ABCC2, ABCC3) and Implications on Posttranscriptional Micro-RNA Regulation.

ATP-binding cassette (ABC) transporters represent a large group of efflux pumps that are strongly involved in the pharmacokinetics of various drugs and nutrient distribution. It was recently shown that micro-RNAs (miRNAs) may significantly alter their expression as proven, e.g., for miR-379 and ABCC2 However, alternative mRNA polyadenylation may result in expression of 3'-untranslated regions (3'-UTRs) with varying lengths. Thus, length variants may result in presence or absence of miRNA binding sites for regulatory miRNAs with consequences on posttranscriptional control. In the present study, we report on 3'-UTR variants of ABCC1, ABCC2, and ABCC3 mRNA. Applying in vitro luciferase reporter gene assays, we show that expression of short ABCC2 3'-UTR variants leads to a significant loss of miR-379/ABCC2 interaction and subsequent upregulation of ABCC2 expression. Furthermore, we show that expression of ABCC2 3'-UTR lengths varies significantly between human healthy tissues but is not directly correlated to the respective protein level in vivo. In conclusion, the presence of altered 3'-UTR lengths in ABC transporters could lead to functional consequences regarding posttranscriptional gene expression, potentially regulated by alternative polyadenylation. Hence, 3'-UTR length variability may be considered as a further mechanism contributing to variability of ABCC transporter expression and subsequent drug variation in drug response. SIGNIFICANCE STATEMENT: micro-RNA (miRNA) binding to 3'-untranslated region (3'-UTR) plays an important role in the control of ATP-binding cassette (ABC)-transporter mRNA degradation and translation into proteins. We disclosed various 3'-UTR length variants of ABCC1, C2, and C3 mRNA, with loss of mRNA seed regions partly leading to varying and tissue-dependent interaction with miRNAs, as proven by reporter gene assays. Alternative 3'-UTR lengths may contribute to variable ABCC transporter expression and potentially explains inconsistent findings in miRNA studies.

Meta-analysis on outcome-worsening comorbidities of COVID-19 and related potential drug-drug interactions.

Drug-drug interactions (DDI) potentially occurring between medications used in the course of COVID-19 infection and medications prescribed for the management of underlying comorbidities may cause adverse drug reactions (ADRs) contributing to worsening of the clinical outcome in affected patients. First, we conducted a meta-analysis to determine comorbidities observed in the course of COVID-19 disease associated with an increased risk of worsened clinical outcome from 24 published studies. In addition, the potential risk of DDI between medications used in the course of COVID-19 treatment in these studies and those for the management of observed comorbidities was evaluated for possible worsening of the clinical outcome. Our meta-analysis revealed an implication cardiometabolic syndrome (e.g. cardiovascular disease, cerebrovascular disease, hypertension, and diabetes), chronic kidney disease and chronic obstructive pulmonary disease as main co-morbidities associated with worsen the clinical outcomes including mortality (risk difference RD 0.12, 95 %-CI 0.05-0.19, p = 0.001), admission to ICU (RD 0.10, 95 %-CI 0.04-0.16, p = 0.001) and severe infection (RD 0.05, 95 %-CI 0.01-0.09, p = 0.01) in COVID-19 patients. Potential DDI on pharmacokinetic level were identified between the antiviral agents atazanavir and lopinavir/ritonavir and some drugs, used in the treatment of cardiovascular diseases such as antiarrhythmics and anti-coagulants possibly affecting the clinical outcome including cardiac injury or arrest because of QTc-time prolongation or bleeding. Concluding, DDI occurring in the course of anti-Covid-19 treatment and co-morbidities could lead to ADRs, increasing the risk of hospitalization, prolonged time to recovery or death on extreme cases. COVID-19 patients with cardiometabolic diseases, chronic kidney disease and chronic obstructive pulmonary disease should be subjected to particular carefully clinical monitoring of adverse events with a possibility of dose adjustment when necessary.

Retinoic acid-induced survival effects in SH-SY5Y neuroblastoma cells.

Neuroblastoma is a malignant childhood cancer arising from the embryonic sympathoadrenal lineage of the neural crest. Retinoic acid (RA) is included in the multimodal therapy of patients with high-risk neuroblastoma to eliminate minimal residual disease. However, the formation of RA-resistant cells substantially lowers 5-year overall survival rates. To examine mechanisms that lead to treatment failure, we chose human SH-SY5Y cells, which are known to tolerate incubation with RA by activating the survival kinases Akt and extracellular signal-regulated kinase 1/2. Characterization of downstream pathways showed that both kinases increased the phosphorylation of the ubiquitin ligase mouse double minute homolog 2 (Mdm2) and thereby enhanced p53 degradation. When p53 signaling was sustained by blocking complex formation with Mdm2 or enhancing c-Jun N-terminal kinase (JNK) activation, cell viability was significantly reduced. In addition, Akt-mediated phosphorylation of the cell-cycle regulator p21 stimulated complex formation with caspase-3, which also contributed to cell protection. Thus, treatment with RA augmented survival signaling and attenuated basal apoptotic pathways in SH-SY5Y cells, which increased cell viability.

Hematopoietic stem cell involvement in BCR-ABL1-positive ALL as a potential mechanism of resistance to blinatumomab therapy.

The bispecific T-cell engager blinatumomab targeting CD19 can induce complete remission in relapsed or refractory B-cell precursor acute lymphoblastic leukemia (BCP-ALL). However, some patients ultimately relapse with loss of CD19 antigen on leukemic cells, which has been established as a novel mechanism to escape CD19-specific immunotherapies. Here, we provide evidence that CD19-negative (CD19-) relapse after CD19-directed therapy in BCP-ALL may be a result of the selection of preexisting CD19- malignant progenitor cells. We present 2 BCR-ABL1 fusion-positive BCP-ALL patients with CD19- myeloid lineage relapse after blinatumomab therapy and show BCR-ABL1 positivity in their hematopoietic stem cell (HSC)/progenitor/myeloid compartments at initial diagnosis by fluorescence in situ hybridization after cell sorting. By using the same approach with 25 additional diagnostic samples from patients with BCR-ABL1-positive BCP-ALL, we identified HSC involvement in 40% of the patients. Patients (6 of 8) with major BCR-ABL1 transcript encoding P210BCR-ABL1 mainly showed HSC involvement, whereas in most of the patients (9 of 12) with minor BCR-ABL1 transcript encoding P190BCR-ABL1, only the CD19+ leukemia compartments were BCR-ABL1 positive (P = .02). Our data are of clinical importance, because they indicate that both CD19+ cells and CD19- precursors should be targeted to avoid CD19- relapses in patients with BCR-ABL1-positive ALL.

Transcriptional and Post-Transcriptional Regulation of Duodenal P-Glycoprotein and MRP2 in Healthy Human Subjects after Chronic Treatment with Rifampin and Carbamazepine.

To predict the outcome of intestinal drug transporter induction on pharmacokinetics, signaling of the DNA message along with messenger RNA (mRNA) transcription and protein translation leading to transporter function must be understood. We quantified the gene expression of PXR and CAR, gene expression and protein abundance of P-glycoprotein (P-gp), multidrug-resistance-associated protein 2 (MRP2) and breast-cancer-resistance protein, the content of 754 microRNAs in human duodenal biopsy specimens, and pharmacokinetics of talinolol and ezetimibe before and after the treatment with rifampin and carbamazepine. Rifampin significantly induced the transcription of ABCB1 and ABCC2 and protein abundance of P-gp but not of MRP2. The abundance of P-gp was significantly correlated to the plasma exposure of ezetimibe and its glucuronide. Carbamazepine induced the mRNA expressions of CAR, ABCB1, and ABCC2 but did not elevate protein abundance. Using in silico prediction tools and luciferase reporter assays, microRNAs were identified that can contribute to ligand-specific regulation of intestinal drug transporters and different changes in drug disposition after induction with rifampin and carbamazepine.

Interaction of herbal products with prescribed medications: A systematic review and meta-analysis.

Although several studies on pharmacokinetic and/or pharmacodynamic herb-drug interactions (HDI) have been conducted in healthy volunteers, there is large uncertainty on the validity of these studies. A qualitative review and a meta-analysis were performed to establish the clinical evidence of these interaction studies. Out of 4026 screened abstracts, 32 studies were included into the qualitative analysis. The meta-analysis was performed on eleven additional studies. St. John's wort (SJW) significantly decreased the AUC (p < 0.0001) and clearance (p = 0.007) of midazolam. Further subgroup analysis identified age to affect Cmax of midazolam (p < 0.01) in the presence of SJW. Echinacea purpurea (EP) significantly increased the clearance of midazolam (p = 0.01). Evidence of publication bias (p > 0.001) was shown on the effect of the herbal products o half-life of midazolam. Green tea (GT) showed significant 85% decrease in plasma concentration of nadolol. The study findings suggest that GT, SJW and EP perpetuate significant interactions with prescribed medications via CYP3A4 or OATP1A2. Our studies show that meta-analyses are important in the area of natural products to provide necessary information on their use in overall medication plans in order to avoid unintended interactions.

Implementation and obstacles of pharmacogenetics in clinical practice: An international survey.

AIMS: Eight years ago, a paper-based survey was administered during the World Pharma 2010 meeting, asking about the challenges of implementing pharmacogenetics (PGx) in clinical practice. The data collected at the time gave an idea about the progress of this implementation and what still needs to be done. Since then, although there have been major initiatives to push PGx forward, PGx clinical implementation may still be facing different challenges in different parts of the world. Our aim was therefore to distribute a follow-up international survey in electronic format to elucidate an overview on the current stage of implementation, acceptance and challenges of PGx in academic institutions around the world. METHODS: This is an online anonymous LimeSurvey-based study launched on 11 November 2018. Survey questions were adapted from the initially published manuscript in 2010. An extensive web search for worldwide scientists potentially involved in PGx research resulted in a total of 1973 names. Countries were grouped based on the Human Development Index. RESULTS: There were 204 respondents from 43 countries. Despite the wide availability of PGx tests, the consistently positive attitude towards their applications and advances in the field, progress of the clinical implementation of PGx still faces many challenges all around the globe. CONCLUSIONS: Clinical implementation of PGx started over a decade ago but there is a gap in progress around the globe and discrepancies between the challenges reported by different countries, despite some challenges being universal. Further studies on ways to overcome these challenges are warranted.

Association of Liver Injury From Specific Drugs, or Groups of Drugs, With Polymorphisms in HLA and Other Genes in a Genome-Wide Association Study.

BACKGROUND & AIMS: We performed a genome-wide association study (GWAS) to identify genetic risk factors for drug-induced liver injury (DILI) from licensed drugs without previously reported genetic risk factors. METHODS: We performed a GWAS of 862 persons with DILI and 10,588 population-matched controls. The first set of cases was recruited before May 2009 in Europe (n = 137) and the United States (n = 274). The second set of cases were identified from May 2009 through May 2013 from international collaborative studies performed in Europe, the United States, and South America. For the GWAS, we included only cases with patients of European ancestry associated with a particular drug (but not flucloxacillin or amoxicillin-clavulanate). We used DNA samples from all subjects to analyze HLA genes and single nucleotide polymorphisms. After the discovery analysis was concluded, we validated our findings using data from 283 European patients with diagnosis of DILI associated with various drugs. RESULTS: We associated DILI with rs114577328 (a proxy for A*33:01 a HLA class I allele; odds ratio [OR], 2.7; 95% confidence interval [CI], 1.9-3.8; P = 2.4 × 10-8) and with rs72631567 on chromosome 2 (OR, 2.0; 95% CI, 1.6-2.5; P = 9.7 × 10-9). The association with A*33:01 was mediated by large effects for terbinafine-, fenofibrate-, and ticlopidine-related DILI. The variant on chromosome 2 was associated with DILI from a variety of drugs. Further phenotypic analysis indicated that the association between DILI and A*33:01 was significant genome wide for cholestatic and mixed DILI, but not for hepatocellular DILI; the polymorphism on chromosome 2 was associated with cholestatic and mixed DILI as well as hepatocellular DILI. We identified an association between rs28521457 (within the lipopolysaccharide-responsive vesicle trafficking, beach and anchor containing gene) and only hepatocellular DILI (OR, 2.1; 95% CI, 1.6-2.7; P = 4.8 × 10-9). We did not associate any specific drug classes with genetic polymorphisms, except for statin-associated DILI, which was associated with rs116561224 on chromosome 18 (OR, 5.4; 95% CI, 3.0-9.5; P = 7.1 × 10-9). We validated the association between A*33:01 terbinafine- and sertraline-induced DILI. We could not validate the association between DILI and rs72631567, rs28521457, or rs116561224. CONCLUSIONS: In a GWAS of persons of European descent with DILI, we associated HLA-A*33:01 with DILI due to terbinafine and possibly fenofibrate and ticlopidine. We identified polymorphisms that appear to be associated with DILI from statins, as well as 2 non-drug-specific risk factors.

MicroRNA-655-3p regulates Echinacea purpurea mediated activation of ABCG2.

1. The aim of this study was to investigate the regulatory effect of Echinacea purpurea (EP) on efflux transporters ABCB1 and ABCG2 and to identify specific microRNAs contributing to their post-transcriptional regulation. 2. ABCB1 and ABCG2 levels were assessed in human hepatoblastoma HepG2 cells treated with 50 µg/mL methanolic extract of commercial EP capsules for different durations. The microRNA expression profile of HepG2 cells after EP treatment was evaluated and in silico target prediction was subsequently conducted to identify specific microRNAs with binding sites in the 3'-UTR of ABCB1 and ABCG2. Luciferase reporter gene assays and site-directed mutagenesis were used to confirm the binding site of identified microRNA within the 3'-UTR of the target gene. 3. EP increased ABCB1 (10-fold ± 3.4, p < 0.001) and ABCG2 (2.7-fold ± 0.5, p < 0.01) mRNA levels after 12 h exposure. Twenty-four microRNAs showed significant expression differences at all durations of exposure to EP. MiR-655-3p showed a 6.79-fold decrease in expression after 12 h exposure compared to 0 h, was predicted in silico to bind ABCG2 3'-UTR and showed a significant negative correlation (p = 0.01) to ABCG2 expression level. The binding of miR-655-3p to ABCG2 3'-UTR was confirmed by reporter gene assays (reduction of reporter gene activity to 60%; p = 0.0001). 4. These results suggest that EP regulates ABCG2 expression via downregulation of miR-655-3p in the liver cells. Thus, miR-655-3p downregulation could be applied to predict EP mediated drug interactions.

Crosstalk control and limits of physiological c-Jun N-terminal kinase activity for cell viability and neurite stability in differentiated PC12 cells.

The c-Jun N-terminal kinases (JNKs) are important mediators of cell viability and structural integrity in postmitotic neurons, which is required for maintaining synaptic connections and neural plasticity. In the present study, we chose differentiated PC12 cells as a well-characterised neuronal model system to selectively examine the regulation of basal JNK activity by extracellular signal-regulated kinase 1/2 (ERK1/2) and Akt. We detected a complex interaction between the kinases to prevent cell death and neurite loss. Especially the appropriate level of JNK activation determined cellular survival. Basal activity of ERK1/2 attenuated the potentiation of JNK phosphorylation and thereby the induction of apoptosis. Importantly, when JNK activity was too low, cell viability and the number of neurite-bearing cells also decreased, even though the activation of ERK1/2 was enhanced. In this case, the JNK-mediated survival signals via activating transcription factor-3 (ATF3) were inhibited. Furthermore, the phosphorylation of ERK1/2 induced by the JNK inhibitor SP600125 inhibited the basal activity of Akt, which normally supported cell viability. Thus, controlling JNK activity is crucial to promote survival and neurite stability of differentiated neuronal cells.

Clinically Relevant Multidrug Transporters Are Regulated by microRNAs along the Human Intestine.

Intestinal drug transporters are crucial determinants for absorption and oral bioavailability of drugs. In healthy tissue donors, a recent study revealed profound discrepancies between mRNA expression and protein abundance as well as differences in the protein content between small and large intestine for clinically relevant multidrug transporters as the ATP binding cassette transporter subfamily B member 1 (ABCB1) and subfamily C member 3 (ABCC3) and the solute carrier family 15 member 1 (SLC15A1, PEPT1). As the mechanisms underlying these observations remained unclear, the aim of the present study was to elucidate the intestinal regiospecific microRNA profile under physiological conditions and identify specific microRNAs contributing to the post-transcriptional regulation of major drug transporters. For this purpose, tissue samples were collected from six intestinal sites obtained from six healthy tissue donors. The expression of 754 microRNAs was determined using qRT-PCR based low density arrays, and microRNA expression levels were correlated with transporter protein abundance quantified by targeted proteomics. A total of 241 microRNA-transporter pairs were identified, showing significant negative correlations to protein abundance (p < 0.05). Out of these, for nine pairs, the binding of the microRNA to the respective transporter 3'-UTR was predicted in silico. Besides the already known interactions of miR-27a-3p-ABCB1 and miR-193a-3p-PEPT1, reporter gene assays confirmed binding of miR-192-5p to the ABCC3 3'-UTR (reduction of reporter gene activity by 31%; p = 0.0012), miR-409-3p to the ABCB1 3'-UTR (reduction by 38%; p = 0.0006), and miR-193b-3p as well as miR-27a-3p to PEPT1 3'-UTR (reduction by 49% (p = 0.0012) and 20% (p = 0.0043), respectively). These results suggest that mucosal microRNA expression contributes to the explanation of discrepancies between mRNA expression and protein abundance as well as site-dependent differences in protein content along the human intestine under physiological conditions, as exemplified for ABCB1, ABCC3, and PEPT1.

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.

SOX11 identified by target gene evaluation of miRNAs differentially expressed in focal and non-focal brain tissue of therapy-resistant epilepsy patients.

MicroRNAs (miRNAs) are small non-coding RNAs that post-transcriptionally control the expression of their target genes via RNA interference. There is increasing evidence that expression of miRNAs is dysregulated in neuronal disorders, including epilepsy, a chronic neurological disorder characterized by spontaneous recurrent seizures. Mesial temporal lobe epilepsy (MTLE) is a common type of focal epilepsy in which disease-induced abnormalities of hippocampal neurogenesis in the subgranular zone as well as gliosis and neuronal cell loss in the cornu ammonis area are reported. We hypothesized that in MTLE altered miRNA-mediated regulation of target genes could be involved in hippocampal cell remodeling. A miRNA screen was performed in hippocampal focal and non-focal brain tissue samples obtained from the temporal neocortex (both n=8) of MTLE patients. Out of 215 detected miRNAs, two were differentially expressed (hsa-miR-34c-5p: mean increase of 5.7 fold (p=0.014), hsa-miR-212-3p: mean decrease of 76.9% (p=0.0014)). After in-silico target gene analysis and filtering, reporter gene assays confirmed RNA interference for hsa-miR-34c-5p with 3'-UTR sequences of GABRA3, GRM7 and GABBR2 and for hsa-miR-212-3p with 3'-UTR sequences of SOX11, MECP2, ADCY1 and ABCG2. Reporter gene assays with mutated 3'-UTR sequences of the transcription factor SOX11 identified two different binding sites for hsa-miR-212-3p and its primary transcript partner hsa-miR-132-3p. Additionally, there was an inverse time-dependent expression of Sox11 and miR-212-3p as well as miR-132-3p in rat neonatal cortical neurons. Transfection of neurons with anti-miRs for miR-212-3p and miR-132-3p suggest that both miRNAs work synergistically to control Sox11 expression. Taken together, these results suggest that differential miRNA expression in neurons could contribute to an altered function of the transcription factor SOX11 and other genes in the setting of epilepsy, resulting not only in impaired neural differentiation, but also in imbalanced neuronal excitability and accelerated drug export.

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.