Elke Schaeffeler

Publications of Elke Schaeffeler

• Organic Anion Transporters and Their Implications in Pharmacotherapy.

  Authors: Arian Emami Riedmaier, Anne T Nies, Elke Schaeffeler, Matthias Schwab

  Pharmacological reviews. 03/2012;

  Organic anion transporters play an essential role in the distribution and excretion of numerous endogenous metabolic products and exogenous organic anions, including a host of widely prescribedOrganic anion transporters play an essential role in the distribution and excretion of numerous endogenous metabolic products and exogenous organic anions, including a host of widely prescribed drugs. The expression and activity of these transporters is influenced by several conditions, including transcriptional regulation, gender-dependent regulation, and genetic variation. In addition, the interaction of these transporters with several drugs and endogenous substrates has been well documented and may play a significant role in drug disposition and development of various disease states, such as nephrotoxicity and familial idiopathic hypouricemia. Members of this family of transporters have been localized mainly to the renal epithelia of various species. Much of the early research in this field has focused on their role in renal drug transport, yet increasing research on this family of transporters has localized them to various other epithelial tissues, including liver, brain, and placenta. Thus, an understanding of the role of these transporters in drug interaction and disposition in the kidney and other tissues may help in the determination of individual drug response, susceptibility to drug toxicity, and chemical carcinogenesis. This review seeks to summarize current knowledge of the molecular function and substrate profile of cloned organic anion transporters and to discuss recent progress in the understanding of the impact of interindividual variability, transcriptional regulation, and tissue distribution on individual drug response.

• Simultaneous quantification of eleven thiopurine nucleotides by liquid chromatography-tandem mass spectrometry.

  Authors: Ute Hofmann, Georg Heinkele, Sieglinde Angelberger, Elke Schaeffeler, Cornelia Lichtenberger, Simon Jaeger, Walter Reinisch, Matthias Schwab

  Analytical chemistry. 02/2012; 84(3):1294-301.

  The prodrugs azathioprine and 6-mercaptopurine, which are well-established anticancer and immunosuppressive agents, are extensively metabolized by activating and inactivating enzymes. Whereas theThe prodrugs azathioprine and 6-mercaptopurine, which are well-established anticancer and immunosuppressive agents, are extensively metabolized by activating and inactivating enzymes. Whereas the 6-thioguanine nucleotides (TGN) are currently being considered as major active metabolites, methylthioinosine nucleotides seem to contribute to the cytotoxic effect as well. Thiopurine-related adverse drug reactions and thiopurine failure are frequent. Thus, therapeutic monitoring of TGN and methylthioinosine derivatives has been suggested to improve thiopurine therapy, however with limited success. To elucidate systematically underlying molecular mechanisms as potential explanation for interindividual variability of thiopurine response, we developed a novel highly specific and sensitive liquid chromatography-tandem mass spectrometry (LC-MS/MS) method for simultaneous quantitation of eleven mono-, di-, and triphosphates of thioguanosine, methylthioinosine, methylthioguanosine, and thioinosine. Using stable isotope-labeled analogues as internal standards obtained by chemical synthesis, an intra- and interassay variability below 8% and an accuracy of 92% to 107% were achieved in spiked quality control samples with known standards. All eleven metabolites could be determined in red blood cells from patients with inflammatory bowel diseases and long-term azathioprine therapy. Thus, our novel method opens a new avenue for the understanding of the thiopurine metabolism by quantitation of all important thiopurine nucleotide metabolites in one run.

• Association of a Functional Variant in the Wnt Co-Receptor LRP6 with Early Onset Ileal Crohn's Disease.

  Authors: Maureen J Koslowski, Zora Teltschik, Julia Beisner, Elke Schaeffeler, Guoxing Wang, Irmgard Kübler, Michael Gersemann, Rachel Cooney, Derek Jewell, Walter Reinisch, Séverine Vermeire, Paul Rutgeerts, Matthias Schwab, Eduard F Stange, Jan Wehkamp

  PLoS genetics. 02/2012; 8(2):e1002523.

  Ileal Crohn's Disease (CD), a chronic small intestinal inflammatory disorder, is characterized by reduced levels of the antimicrobial peptides DEFA5 (HD-5) and DEFA6 (HD-6). Both of these α-defensinsIleal Crohn's Disease (CD), a chronic small intestinal inflammatory disorder, is characterized by reduced levels of the antimicrobial peptides DEFA5 (HD-5) and DEFA6 (HD-6). Both of these α-defensins are exclusively produced in Paneth cells (PCs) at small intestinal crypt bases. Different ileal CD-associated genes including NOD2, ATG16L1, and recently the β-catenin-dependant Wnt transcription factor TCF7L2 have been linked to impaired PC antimicrobial function. The Wnt pathway influences gut mucosal homeostasis and PC maturation, besides directly controlling HD-5/6 gene expression. The herein reported candidate gene study focuses on another crucial Wnt factor, the co-receptor low density lipoprotein receptor-related protein 6 (LRP6). We analysed exonic single nucleotide polymorphisms (SNPs) in a large cohort (Oxford: n = 1,893) and prospectively tested 2 additional European sample sets (Leuven: n = 688, Vienna: n = 1,628). We revealed an association of a non-synonymous SNP (rs2302685; Ile1062Val) with early onset ileal CD (OR 1.8; p = 0.00034; for homozygous carriers: OR 4.1; p = 0.00004) and additionally with penetrating ileal CD behaviour (OR 1.3; p = 0.00917). In contrast, it was not linked to adult onset ileal CD, colonic CD, or ulcerative colitis. Since the rare variant is known to impair LRP6 activity, we investigated its role in patient mucosa. Overall, LRP6 mRNA was diminished in patients independently from the genotype. Analysing the mRNA levels of PC product in biopsies from genotyped individuals (15 controls, 32 ileal, and 12 exclusively colonic CD), we found particularly low defensin levels in ileal CD patients who were carrying the variant. In addition, we confirmed a direct relationship between LRP6 activity and the transcriptional expression of HD-5 using transient transfection. Taken together, we identified LRP6 as a new candidate gene in ileal CD. Impairments in Wnt signalling and Paneth cell biology seem to represent pathophysiological hallmarks in small intestinal inflammation and should therefore be considered as interesting targets for new therapeutic approaches.

• PXR variants and artemisinin use in Vietnamese subjects: Frequency distribution and impact on the inter-individual variability of CYP3A induction by artemisinin.

  Authors: Rita Piedade, Elke Schaeffeler, Stefan Winter, Sara Asimus, Matthias Schwab, Michael Ashton, Oliver Burk, José P Gil

  Antimicrobial agents and chemotherapy. 01/2012;

  Artemisinins induce drug metabolism through the activation of pregnane x receptor (PXR) in vitro. Herein, we report the re-sequencing and genotyping of PXR variants in 75 Vietnamese individualsArtemisinins induce drug metabolism through the activation of pregnane x receptor (PXR) in vitro. Herein, we report the re-sequencing and genotyping of PXR variants in 75 Vietnamese individuals previously characterized for CYP3A enzyme activity after artemisinin exposure. A total of 31 PXR variants were identified, including five novel SNPs, and we identified significantly different allele frequencies relative to other ethnic groups. A trend of significance was observed between the level of CYP3A4 induction by artemisinin and two PXR variants, 8118 C>T (Y328Y) and 10719 A>G.

• DNA methylation is associated with downregulation of the organic cation transporter OCT1 (SLC22A1) in human hepatocellular carcinoma.

  Authors: Elke Schaeffeler, Claus Hellerbrand, Anne T Nies, Stefan Winter, Stephan Kruck, Ute Hofmann, Heiko van der Kuip, Ulrich M Zanger, Hermann Koepsell, Matthias Schwab

  Genome medicine. 12/2011; 3(12):82.

  ABSTRACT: BACKGROUND: Organic cation transporters (OCTs) determine not only physiological processes but are also involved in the cellular uptake of anticancer agents. Based on microarray analyses inABSTRACT: BACKGROUND: Organic cation transporters (OCTs) determine not only physiological processes but are also involved in the cellular uptake of anticancer agents. Based on microarray analyses in hepatocellular carcinoma (HCC), SLC22A1/OCT1 mRNA seems to be downregulated, but systematic protein expression data are currently missing. Moreover, the underlying molecular mechanisms responsible for altered SLC22A1 expression in HCC are not fully understood. Therefore, we investigated the role of DNA methylation in the transcriptional regulation of the family members SLC22A1/OCT1, SLC22A2/OCT2 and SLC22A3/OCT3 in HCC. METHODS: Semiquantitative immunohistochemistry of SLC22A1 protein expression was performed in paired HCC and histological normal adjacent liver tissues (n = 71) using tissue microarray analyses, and the results were correlated with clinicopathological features. DNA methylation, quantified by MALDI-TOF mass spectrometry and gene expression of SLC22A1, SLC22A2 and SLC22A3 were investigated using fresh-frozen HCC (n = 22) and non-tumor adjacent liver tissues as well as histologically normal liver samples (n = 120) from a large-scale liverbank. RESULTS: Based on tissue microarray analyses, we observed a significant downregulation of SLC22A1 protein expression in HCC compared to normal adjacent tissue (P < 0.0001). SLC22A1 expression was significantly inverse correlated with expression of the proliferation marker MIB1/Ki-67 (rs = -0.464, P < 0.0001). DNA methylation of SLC22A1 was significantly higher in HCC compared with non-tumor adjacent liver tissue and was lowest in histologically normal liver tissue. Methylation levels for SLC22A1 in combination with RASSF1A resulted in a specificity of > 90% and a sensitivity of 82% for discriminating HCC and tumor-free liver tissue. CONCLUSIONS: DNA methylation of SLC22A1 is associated with downregulation of SLC22A1 in HCC and might be a new biomarker for HCC diagnosis and prognosis. Moreover, targeting SLC22A1 methylation by demethylating agents may offer a novel strategy for anticancer therapy of HCC.

• Genetic polymorphism of cytochrome P450 2D6 determines oestrogen receptor activity of the major infertility drug clomiphene via its active metabolites.

  Authors: Thomas E Mürdter, Reinhold Kerb, Miia Turpeinen, Werner Schroth, Boian Ganchev, Gabriele M Böhmer, Svitlana Igel, Elke Schaeffeler, Ulrich Zanger, Hiltrud Brauch, Matthias Schwab

  Human molecular genetics. 11/2011; 21(5):1145-54.

  Clomiphene citrate is the most used drug for the treatment of female infertility, a common condition in western societies and developing countries. Despite dose escalation, up to 30% of women do notClomiphene citrate is the most used drug for the treatment of female infertility, a common condition in western societies and developing countries. Despite dose escalation, up to 30% of women do not respond. Since clomiphene shares structural similarities with tamoxifen, which is predominantly bioactivated by the polymorphic cytochrome P450 (CYP) 2D6, we systematically explored clomiphene metabolism and action in vitro and in vivo by pharmacogenetic, -kinetic and -dynamic investigations. Human liver microsomes were incubated with clomiphene citrate and nine metabolites were identified by mass spectrometry and tested at the oestrogen receptor for their antagonistic capacity. (E)-4-hydroxyclomiphene and (E)-4-hydroxy-N-desethylclomiphene showed strongest inhibition of the oestrogen receptor activity with 50% inhibitory concentrations of 2.5 and 1.4 nm, respectively. CYP2D6 has been identified as the major enzyme involved in their formation using recombinant CYP450 isozymes as confirmed by inhibition experiments with CYP monoclonal antibodies. We correlated the CYP2D6 genotype of 30 human liver donors with the microsomal formation rate of active metabolites and observed a strong gene-dose effect. A healthy female volunteer study confirmed our in vitro data that the CYP2D6 polymorphism substantially determines the formation of the active clomiphene metabolites. Comparison of the C(max) of (E)-4-hydroxyclomiphene and (E)-4-hydroxy-N-desethylclomiphene showed 8 and 12 times lower concentrations in subjects with non-functional CYP2D6 alleles. Our results highlight (E)-4-hydroxyclomiphene and (E)-4-hydroxy-N-desethylclomiphene as the active clomiphene metabolites, the formation of which strongly depends on the polymorphic CYP2D6 enzyme. Our data provide first evidence of a biological rationale for the variability in the response to clomiphene treatment.

• Mammalian MATE (SLC47A) transport proteins: impact on efflux of endogenous substrates and xenobiotics.

  Authors: Katja Damme, Anne T Nies, Elke Schaeffeler, Matthias Schwab

  Drug metabolism reviews. 09/2011; 43(4):499-523.

  Multidrug and toxin extrusion proteins (MATEs; SLC47A) are mammalian transporters being predominately expressed in the brush-border membrane of proximal tubule epithelial cells in the kidney and theMultidrug and toxin extrusion proteins (MATEs; SLC47A) are mammalian transporters being predominately expressed in the brush-border membrane of proximal tubule epithelial cells in the kidney and the canalicular membrane of hepatocytes. Functionally, MATEs act as efflux transporters for organic compounds, thereby mediating the elimination process. Two isoforms, MATE1 and 2, have been identified, and, so far, only a limited number of substrates, including clinically used drugs such as metformin and cimetidine, are known. A knockout mouse model has been established, as well, and is a valuable tool for further systematic pharmacokinetic analyses. In this review, we summarize the progress in MATE research on structural, molecular, functional, and pathophysiological aspects. Consequences of genetic variants for pharmacokinetic alterations and drug therapy are discussed.

• Polymorphisms of the nuclear receptor pregnane X receptor and organic anion transporter polypeptides 1A2, 1B1, 1B3, and 2B1 are not associated with breast cancer risk.

  Authors: Christina Justenhoven, Elke Schaeffeler, Stefan Winter, Christian Baisch, Ute Hamann, Volker Harth, Sylvia Rabstein, Anne Spickenheuer, Beate Pesch, Thomas Brüning, Yon-Dschun Ko, Matthias Schwab, Hiltrud Brauch

  Breast cancer research and treatment. 01/2011; 125(2):563-9.

  Organic anion transporter polypeptides (OATPs, SLCOs) are involved in the uptake of conjugates steroid hormones such as estrone-3-sulfate. It has been suggested that the expression of OATPs in breastOrganic anion transporter polypeptides (OATPs, SLCOs) are involved in the uptake of conjugates steroid hormones such as estrone-3-sulfate. It has been suggested that the expression of OATPs in breast tissues could impact breast carcinogenesis and tumor pathology. The nuclear receptor pregnane X receptor (PXR) is involved in the regulation of SLCO1A2 expression. We investigated 31 variants located in PXR, SLCO1A2, SLCO1B1, SLCO1B3, and SLCO2B1 for an association with breast cancer risk and/or histo-pathological tumor characteristics. Polymorphisms were selected on the basis of a known or potential functional consequence and an allele frequency >2%. Genotyping was performed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry using the GENICA population-based breast cancer case-control collection comprising 1,021 cases and 1,015 age-matched controls. Statistical analysis was performed by SAS, and all tests were two-sided. None of the 31 analyzed transporter and PXR polymorphisms showed an association with breast cancer risk or tumor characteristics. Our data suggest that among the many known transporters common variations of PXR, SLCO1A2, SLCO1B1, SLCO1B3, and SLCO2B1 do not contribute to breast carcinogenesis.

• Proton pump inhibitors inhibit metformin uptake by organic cation transporters (OCTs).

  Authors: Anne T Nies, Ute Hofmann, Claudia Resch, Elke Schaeffeler, Maria Rius, Matthias Schwab

  PloS one. 01/2011; 6(7):e22163.

  Metformin, an oral insulin-sensitizing drug, is actively transported into cells by organic cation transporters (OCT) 1, 2, and 3 (encoded by SLC22A1, SLC22A2, or SLC22A3), which are tissueMetformin, an oral insulin-sensitizing drug, is actively transported into cells by organic cation transporters (OCT) 1, 2, and 3 (encoded by SLC22A1, SLC22A2, or SLC22A3), which are tissue specifically expressed at significant levels in various organs such as liver, muscle, and kidney. Because metformin does not undergo hepatic metabolism, drug-drug interaction by inhibition of OCT transporters may be important. So far, comprehensive data on the interaction of proton pump inhibitors (PPIs) with OCTs are missing although PPIs are frequently used in metformin-treated patients. Using in silico modeling and computational analyses, we derived pharmacophore models indicating that PPIs (i.e. omeprazole, pantoprazole, lansoprazole, rabeprazole, and tenatoprazole) are potent OCT inhibitors. We then established stably transfected cell lines expressing the human uptake transporters OCT1, OCT2, or OCT3 and tested whether these PPIs inhibit OCT-mediated metformin uptake in vitro. All tested PPIs significantly inhibited metformin uptake by OCT1, OCT2, and OCT3 in a concentration-dependent manner. Half-maximal inhibitory concentration values (IC(50)) were in the low micromolar range (3-36 µM) and thereby in the range of IC(50) values of other potent OCT drug inhibitors. Finally, we tested whether the PPIs are also transported by OCTs, but did not identify PPIs as OCT substrates. In conclusion, PPIs are potent inhibitors of the OCT-mediated metformin transport in vitro. Further studies are needed to elucidate the clinical relevance of this drug-drug interaction with potential consequences on metformin disposition and/or efficacy.

• Effect of CYP2B6, ABCB1, and CYP3A5 polymorphisms on efavirenz pharmacokinetics and treatment response: an AIDS Clinical Trials Group study.

  Authors: Heather J Ribaudo, Huan Liu, Matthias Schwab, Elke Schaeffeler, Michel Eichelbaum, Alison A Motsinger-Reif, Marylyn D Ritchie, Ulrich M Zanger, Edward P Acosta, Gene D Morse, Roy M Gulick, Gregory K Robbins, David Clifford, David W Haas

  The Journal of infectious diseases. 09/2010; 202(5):717-22.

  In AIDS Clinical Trials Group protocols 384, A5095, and A5097s, we characterized relationships between 22 polymorphisms in CYP2B6, ABCB1, and CYP3A5; plasma efavirenz exposure; and/or treatmentIn AIDS Clinical Trials Group protocols 384, A5095, and A5097s, we characterized relationships between 22 polymorphisms in CYP2B6, ABCB1, and CYP3A5; plasma efavirenz exposure; and/or treatment responses. A stepwise logistic regression procedure selected polymorphisms associated with reduced drug clearance adjusted for body mass index and the composite CYP2B6 516/983 genotype. Relationships between selected polymorphisms and treatment responses were characterized by competing risk methodology. Association analyses involved 821 individuals (317 for pharmacokinetics and 643 for treatment response). Models that included CYP2B6 516/983 genotype best predicted pharmacokinetics. Slow-metabolizer genotypes were associated with increased central nervous system events among white participants and decreased virologic failure among black participants.

• Azathioprine versus mesalazine for prevention of postoperative clinical recurrence in patients with Crohn's disease with endoscopic recurrence: efficacy and safety results of a randomised, double-blind, double-dummy, multicentre trial.

  Authors: Walter Reinisch, Sieglinde Angelberger, Wolfgang Petritsch, Olga Shonova, Milan Lukas, Simon Bar-Meir, Alexander Teml, Elke Schaeffeler, Matthias Schwab, Karin Dilger, Roland Greinwald, Ralph Mueller, Eduard F Stange, Klaus R Herrlinger

  Gut. 06/2010; 59(6):752-9.

  The aim of the study was to compare azathioprine versus mesalazine tablets for the prevention of clinical recurrence in patients with postoperative Crohn's disease (CD) with moderate or severeThe aim of the study was to compare azathioprine versus mesalazine tablets for the prevention of clinical recurrence in patients with postoperative Crohn's disease (CD) with moderate or severe endoscopic recurrence. This was a 1 year, double-blind, double-dummy, randomised study which took place in 21 gastroenterology centres in Austria, the Czech Republic, Germany and Israel. The study participants were 78 adults with CD who had undergone resection with ileocolonic anastomosis in the preceding 6-24 months without subsequent clinical recurrence and with a Crohn's disease activity index (CDAI) score <200, but with moderate or severe endoscopic recurrence. The study drugs were azathioprine 2.0-2.5 mg/kg/day or mesalazine 4 g/day over 1 year. The primary end point was therapeutic failure during 1 year, defined as a CDAI score > or = 200 and an increase of > or = 60 points from baseline, or study drug discontinuation due to lack of efficacy or intolerable adverse drug reaction. Treatment failure occurred in 22.0% (9/41) of azathioprine-treated patients and 10.8% (4/37) of mesalazine-treated patients, a difference of 11.1% (95% CI -5.0% to 27.3%, p=0.19). Clinical recurrence was significantly less frequent with azathioprine versus mesalazine (0/41 (0%) vs 4/37 (10.8%), p=0.031), whereas study drug discontinuation due to adverse drug reactions only occurred in azathioprine-treated patients (9/41 (22.0%) vs 0%, p=0.002). The proportion of patients showing > or = 1 point reduction in Rutgeerts score between baseline and month 12 was 63.3% (19/30) and 34.4% (11/32) in the azathioprine and mesalazine groups, respectively (p=0.023). In this population of patients with postoperative CD at high risk of clinical recurrence, superiority for azathioprine versus mesalazine could not be demonstrated for therapeutic failure.

• Determination of 6-thioguanosine diphosphate and triphosphate and nucleoside diphosphate kinase activity in erythrocytes: novel targets for thiopurine therapy?

  Authors: Susanne Karner, Shaojun Shi, Christine Fischer, Elke Schaeffeler, Markus F Neurath, Klaus R Herrlinger, Ute Hofmann, Matthias Schwab

  Therapeutic drug monitoring. 03/2010; 32(2):119-28.

  6-Thioguanine nucleotides are the sum of 6-thioguanosine 5'-monophosphate (TGMP), -diphosphate (TGDP), and -triphosphate (TGTP) representing essential metabolites involved in drug action of6-Thioguanine nucleotides are the sum of 6-thioguanosine 5'-monophosphate (TGMP), -diphosphate (TGDP), and -triphosphate (TGTP) representing essential metabolites involved in drug action of thiopurines. Elevated levels of TGDP have been associated with poor response to azathioprine therapy in patients with inflammatory bowel disease. The conversion of TGDP to TGTP is supposed to be catalyzed by nucleoside diphosphate kinase (NDPK). The aim of this work was to investigate simultaneously individual 6-thioguanosine phosphate levels and NDPK activity in red blood cells (RBCs) of patients on azathioprine therapy. Ion-pair high-performance liquid chromatography methods with fluorescence and ultraviolet detection were applied to quantify individual levels of 6-thioguanosine 5'-phosphates and NDPK activity, respectively, in RBCs. Recombinantly expressed NDPK isoforms A and B were unequivocally identified to catalyze the formation of TGTP (30.6 +/- 3.88 nmol x min x mg for NDPK A versus 41.2 +/- 1.05 nmol x min x mg for NDPK B). Comprehensive analyses on the stability of TGMP, TGDP, and TGTP and the reproducibility of NDPK activity in RBCs were performed to provide a reliable sampling protocol for clinical practice. Of note, isolation of RBCs within 6 hours followed by immediate storage at -80 degrees C is crucial for prevention of degradation of 5'-phosphates. In a clinical study of 37 patients on azathioprine, TGTP was the predominant 6-thioguanosine phosphate in RBCs. In contrast, three patients showed TGTP/(TGDP + TGTP) ratios of 57.2%, 64.3%, and 66% corresponding to elevated TGDP levels. NDPK activity ranged from 4.1 to 11.3 nmol x min x mg hemoglobin. No correlation between NDPK activity and the 6-thioguanosine phosphate levels was found. The question whether interindividual variability of NDPK activity may explain differences in 6-thioguanosine 5'-phosphates levels has to be investigated in a prospective large-scale study.

• Thiopurine methyltransferase genetics is not a major risk factor for secondary malignant neoplasms after treatment of childhood acute lymphoblastic leukemia on Berlin-Frankfurt-Munster protocols.

  Authors: Martin Stanulla, Elke Schaeffeler, Anja Moricke, Sally A Coulthard, Gunnar Cario, Andre Schrauder, Peter Kaatsch, Michael Dordelmann, Karl Welte, Martin Zimmermann, Alfred Reiter, Michel Eichelbaum, Hansjorg Riehm, Martin Schrappe, Matthias Schwab

  Blood. 07/2009;

  Thiopurine methyltransferase (TPMT) is involved in the metabolism of thiopurines such as 6-mercaptopurine and 6-thioguanine. TPMT activity is significantly altered by genetics, and heterozygous andThiopurine methyltransferase (TPMT) is involved in the metabolism of thiopurines such as 6-mercaptopurine and 6-thioguanine. TPMT activity is significantly altered by genetics, and heterozygous and even more homozygous variant individuals reveal substiantially decreased TPMT activity. Treatment for childhood acute lymphoblastic leukemia (ALL) regularly includes the use of thiopurine drugs. Importantly, childhood ALL patients with low TPMT activity have been considered to be at increased risk of developing therapy-associated acute myeloid leukemia and brain tumors. In the present study, we genotyped 105 out of 129 patients who developed a secondary malignant neoplasm (SMN) after ALL treatment on seven consecutive German Berlin-Frankfurt-Munster (BFM) trials for all functionally relevant TPMT variants. Frequencies of TPMT variants were similarly distributed in SMN patients and the overall ALL patient population of 814 patients. Thus, TPMT does not play a major role in the etiology of SMN after treatment for childhood ALL according to BFM strategies.

• Expression of organic cation transporters OCT1 (SLC22A1) and OCT3 (SLC22A3) is affected by genetic factors and cholestasis in human liver.

  Authors: Anne T Nies, Hermann Koepsell, Stefan Winter, Oliver Burk, Kathrin Klein, Reinhold Kerb, Ulrich M Zanger, Dietrich Keppler, Matthias Schwab, Elke Schaeffeler

  Hepatology (Baltimore, Md.). 05/2009;

  An important function of hepatocytes is the biotransformation and elimination of various drugs, many of which are organic cations and are taken up by organic cation transporters (OCTs) of the soluteAn important function of hepatocytes is the biotransformation and elimination of various drugs, many of which are organic cations and are taken up by organic cation transporters (OCTs) of the solute carrier family 22 (SLC22). Because interindividual variability of OCT expression may affect response to cationic drugs such as metformin, we systematically investigated genetic and nongenetic factors of OCT1/SLC22A1 and OCT3/SLC22A3 expression in human liver. OCT1 and OCT3 expression (messenger RNA [mRNA], protein) was analyzed in liver tissue samples from 150 Caucasian subjects. Hepatic OCTs were localized by way of immunofluorescence microscopy. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and genome-wide single-nucleotide polymorphism microarray technology served to genotype 92 variants in the SLC22A1-A3/OCT1-3 gene cluster. Transport of metformin by recombinant human OCT1 and OCT3 was compared using transfected cells. OCT1 mRNA and protein expression varied 113- and 83-fold, respectively; OCT3 mRNA expression varied 27-fold. OCT1 transcript levels were on average 15-fold higher compared with OCT3. We localized the OCT3 protein to the basolateral hepatocyte membrane and identified metformin as an OCT3 substrate. OCT1 and OCT3 expression are independent of age and sex but were significantly reduced in liver donors diagnosed as cholestatic (P </= 0.01). Several haplotypes for OCT1 and OCT3 were identified. Multivariate analysis adjusted for multiple testing showed that only the OCT1-Arg61Cys variant (rs12208357) strongly correlated with decreased OCT1 protein expression (P < 0.0001), and four variants in OCT3 (rs2292334, rs2048327, rs1810126, rs3088442) were associated with reduced OCT3 mRNA levels (P = 0.03). Conclusion: We identified cholestasis and genetic variants as critical determinants for considerable interindividual variability of hepatic OCT1 and OCT3 expression. This indicates consequences for hepatic elimination of and response to OCT substrates such as metformin. (HEPATOLOGY 2009.).

• Pharmacogenomics of human liver cytochrome P450 oxidoreductase: multifactorial analysis and impact on microsomal drug oxidation.

  Authors: Ana M Gomes, Stefan Winter, Kathrin Klein, Miia Turpeinen, Elke Schaeffeler, Matthias Schwab, Ulrich M Zanger

  Pharmacogenomics. 04/2009; 10(4):579-99.

  AIMS: NADPH:CYP oxidoreductase (POR) is an essential component of several enzyme systems, including the microsomal CYP monooxygenases. We investigated genetic and nongenetic POR variability and itsAIMS: NADPH:CYP oxidoreductase (POR) is an essential component of several enzyme systems, including the microsomal CYP monooxygenases. We investigated genetic and nongenetic POR variability and its impact on drug-oxidation activities in human liver microsomes. MATERIAL AND METHODS: POR mRNA, protein and activity, as well as ten major drug-oxidation activities, were measured in the microsomes of 150 Caucasian surgical liver samples. Matrix-assisted laser desorption/ionisation-time of flight mass spectrometric assays were established to determine the frequency of 46 selected POR SNPs. Multivariate log-linear regression models, including main effects and two-way interaction terms, and analyses of variance were used to identify statistically significant relationships. RESULTS: POR phenotypes were less variable within the study population as compared with CYP phenotypes. Intronic SNPs g.18557G>A (intron 2), g.25676C>T (intron 3) and g.30986 G>A (intron 10) were associated with various drug-oxidation activities. The common allele POR*28 (A503V) was not associated with any activity or expression changes. Haplotype analysis identified two novel composite alleles POR*36 (P228L plus A503V) and POR*37 (A503V plus V631I). CONCLUSION: Models that integrate POR and microsomal CYP function are complex and depend on the CYP isozyme, the substrate and numerous genetic and nongenetic factors. Intronic POR variants may influence microsomal CYP activities. These data provide a basis for further studies towards inclusion of POR polymorphisms in pharmacogenomic strategies.

• Multidrug resistance-related protein 2 genotype of the donor affects kidney graft function.

  Authors: Olaf Grisk, Antje Steinbach, Sabine Ciecholewski, Torsten Schlüter, Ingrid Klöting, Helmut Schmidt, Eike Dazert, Elke Schaeffeler, Leif Steil, Stefan Gauer, Gabriele Jedlitschky, Matthias Schwab, Gerd Geisslinger, Ingeborg Hauser, Uwe Völker, Heyo Kroemer, Rainer Rettig

  Pharmacogenetics and genomics. 03/2009;

  OBJECTIVES: We tested the effect of kidney-specific multidrug resistance-related protein (MRP2, ABCC2) deficiency on renal organic solute disposition as well as on renal protein and gene expression.OBJECTIVES: We tested the effect of kidney-specific multidrug resistance-related protein (MRP2, ABCC2) deficiency on renal organic solute disposition as well as on renal protein and gene expression. Furthermore, we investigated whether a particular kidney donor ABCC2 genotype is associated with delayed graft function in patients. METHODS: A new MRP2-deficient rat strain was established. Renal cross-transplantations were performed between congenic MRP2-deficient and wild-type rats. Renal disposition of MRP2 substrates was investigated in native and transplanted rats. Proteomic analyses and transcriptional profiling were performed in rat kidney graft cortices. Ninety-eight human kidney donor-recipient pairs were genotyped for five ABCC2 polymorphisms. The relationship between delayed graft function and ABCC2 genetic variants in donors and recipients was analyzed by backward stepwise logistic regression. RESULTS: In rats, the absence of renal MRP2 reduced renal bilirubin glucuronide excretion at pathologic plasma concentrations, modified renal p-aminohippurate excretion and did not affect renal morphine-6-glucuronide excretion. Renal MRP2 deficiency led to renal cortical protein or mRNA upregulation of glutathione transferase isoenzymes, glutaredoxin 2, and heme oxygenase-1. In patients, a particular donor ABCC2 genotype was associated with an increased incidence of delayed graft function. CONCLUSION: Kidney graft-specific MRP2 deficiency has mild effects on the renal excretion of some organic solutes under experimental conditions and induces a protein and gene expression pattern indicative of activated antioxidant defense mechanisms. This suggests that MRP2 is a determinant of the redox status in tubular epithelial cells and thus of the susceptibility to renal damage under conditions of treatment with multiple drugs and increased oxygen radical formation.

• Mutation screening of apical sodium-dependent bile acid transporter (SLC10A2): novel haplotype block including six newly identified variants linked to reduced expression.

  Authors: Olga Renner, Simone Harsch, Elke Schaeffeler, Matthias Schwab, Dietmar Klass, Wolfgang Kratzer, Eduard Stange

  Human genetics. 02/2009;

  The apical sodium-dependent bile acid transporter (SLC10A2) plays a key role in the reabsorption of luminal bile acids into the enterohepatic circulation. Rare variations in SLC10A2 have beenThe apical sodium-dependent bile acid transporter (SLC10A2) plays a key role in the reabsorption of luminal bile acids into the enterohepatic circulation. Rare variations in SLC10A2 have been reported to be associated with Crohn's disease, primary bile acid malabsorption and familial hypertriglyceridemia; however, variants associated with reduced SLC10A2 expression have not been reported to date. In this study, we have performed a sequence analysis of SLC10A2 using genomic DNA of 93 individuals. A new haplotype structure was identified including ten variants with complete linkage disequilibrium (LD' = 1.0, r (2) = 1.0) of which six polymorphisms were novel. The sequence variants were confirmed in three independent cohorts (n = 1,290) by a recently established MALDI-TOF MS iPLEXtrade mark assay. Remarkably, haplotype carriers with the minor allele exhibited significant reduced ileal SLC10A2 expression on mRNA levels (2.6-fold, P = 0.0009) and protein levels (2.4-fold, P = 0.0157). In future studies a single tag SNP selected of this haplotype block will provide reliable genetic testing to investigate systemically the influence of the SLC10A2 haplotype for disease susceptibility and/or drug response.

• Genetic variants of Wnt transcription factor TCF-4 (TCF7L2) putative promoter region are associated with small intestinal Crohn's disease.

  Authors: Maureen J Koslowski, Irmgard Kübler, Mathias Chamaillard, Elke Schaeffeler, Walter Reinisch, Guoxing Wang, Julia Beisner, Alexander Teml, Laurent Peyrin-Biroulet, Stefan Winter, Klaus R Herrlinger, Paul Rutgeerts, Séverine Vermeire, Rachel Cooney, Klaus Fellermann, Derek Jewell, Charles L Bevins, Matthias Schwab, Eduard F Stange, Jan Wehkamp

  PLoS ONE. 02/2009; 4(2):e4496.

  Reduced expression of Paneth cell antimicrobial alpha-defensins, human defensin (HD)-5 and -6, characterizes Crohn's disease (CD) of the ileum. TCF-4 (also named TCF7L2), a Wnt signalling pathwayReduced expression of Paneth cell antimicrobial alpha-defensins, human defensin (HD)-5 and -6, characterizes Crohn's disease (CD) of the ileum. TCF-4 (also named TCF7L2), a Wnt signalling pathway transcription factor, orchestrates Paneth cell differentiation, directly regulates the expression of HD-5 and -6, and was previously associated with the decrease of these antimicrobial peptides in a subset of ileal CD. To investigate a potential genetic association of TCF-4 with ileal CD, we sequenced 2.1 kb of the 5' flanking region of TCF-4 in a small group of ileal CD patients and controls (n = 10 each). We identified eight single nucleotide polymorphisms (SNPs), of which three (rs3814570, rs10885394, rs10885395) were in linkage disequilibrium and found more frequently in patients; one (rs3814570) was thereby located in a predicted regulatory region. We carried out high-throughput analysis of this SNP in three cohorts of inflammatory bowel disease (IBD) patients and controls. Overall 1399 healthy individuals, 785 ulcerative colitis (UC) patients, 225 CD patients with colonic disease only and 784 CD patients with ileal involvement were used to determine frequency distributions. We found an association of rs3814570 with ileal CD but neither with colonic CD or UC, in a combined analysis (allele positivity: OR 1.27, 95% CI 1.07 to 1.52, p = 0.00737), which was the strongest in ileal CD patients with stricturing behaviour (allele frequency: OR 1.32, 95% CI 1.08 to1.62, p = 0.00686) or an additional involvement of the upper GIT (allele frequency: OR 1.38, 95% CI 1.03 to1.84, p = 0.02882). The newly identified genetic association of TCF-4 with ileal CD provides evidence that the decrease in Paneth cell alpha-defensins is a primary factor in disease pathogenesis.

• A Variant of the SLC10A2 Gene Encoding the Apical Sodium-Dependent Bile Acid Transporter Is a Risk Factor for Gallstone Disease.

  Authors: Olga Renner, Simone Harsch, Elke Schaeffeler, Stefan Winter, Matthias Schwab, Marcin Krawczyk, Jonas Rosendahl, Henning Wittenburg, Frank Lammert, Eduard F Stange

  PloS one. 01/2009; 4(10):e7321.

  BACKGROUND: Cholelithiasis is a multifactorial process and several mechanisms of gallstone formation have been postulated. As one of these mechanisms, a decreased expression of the ileal apicalBACKGROUND: Cholelithiasis is a multifactorial process and several mechanisms of gallstone formation have been postulated. As one of these mechanisms, a decreased expression of the ileal apical sodium-dependent bile acid transporter gene SLC10A2 in gallstone carriers was described previously. In this study the SLC10A2 gene was investigated to identify novel genetic variants and their association with gallstone formation. METHODOLOGY/PRINCIPAL FINDINGS: Study subjects were selected with the presence or absence of gallstones confirmed by ultrasound and medical history. Genomic DNA was obtained from blood leukocytes. Sequence analysis was performed of all six exonic and flanking regions as well as of 2,400 base pairs of the SLC10A2 promoter in a cohort of gallstone carriers and control subjects from Stuttgart, Germany. Genotype frequencies of newly identified genetic variants (n = 6) and known single nucleotide polymorphisms (n = 24) were established using MALDI-TOF mass spectrometry. Six new genetic variants were found within the SLC10A2 gene. Although none of the variants was linked to gallstone disease in the Stuttgart cohort overall, the minor allele of SNP rs9514089 was more prevalent in male non-obese gallstone carriers (p = 0.06680, OR = 11.00). In a separate population from Aachen, Germany, the occurrence of rs9514089 was two-fold higher in gallstone patients (22%) than in corresponding controls (11%) (p = 0.00995, OR = 2.19). In the pooled Aachen/Stuttgart cohort rs9514089 was highly significantly linked to cholelithiasis (p = 0.00767, OR = 2.04). A more frequent occurrence was observed for male gallstone carriers (22%) compared to controls (9%) (p = 0.01017, OR = 2.99), for the total normal weight group (p = 0.00754, OR = 2.90), and for male non-obese gallstone patients (p = 0.01410, OR = 6.85). Moreover, for the minor allele of rs9514089 an association with low plasma cholesterol levels was found especially in gallstone carriers (p = 0.05). CONCLUSIONS/SIGNIFICANCE: We have identified SLC10A2 as a novel susceptibility gene for cholelithiasis in humans. Comprehensive statistical analysis provides strong evidence that rs9514089 is a genetic determinant especially in male non-obese gallstone carriers. The minor allele of rs9514089 is related to differences in plasma cholesterol levels among the subjects.

• Highly Multiplexed Genotyping of Thiopurine S-Methyltransferase Variants Using MALDI-TOF Mass Spectrometry: Reliable Genotyping in Different Ethnic Groups.

  Authors: Elke Schaeffeler, Ulrich M Zanger, Michel Eichelbaum, Steven Asante-Poku, Jae-Gook Shin, Matthias Schwab

  Clinical chemistry. 09/2008;

  BACKGROUND: To avoid severe hematotoxicity in patients, determination of the TPMT (thiopurine S-methyltransferase) genotype before commencing thiopurine therapy has become accepted. METHODS: We usedBACKGROUND: To avoid severe hematotoxicity in patients, determination of the TPMT (thiopurine S-methyltransferase) genotype before commencing thiopurine therapy has become accepted. METHODS: We used MALDI-TOF mass spectrometry to develop novel multiplexing assays for comprehensive diagnostic testing of TPMT. Two iPLEX Gold assays, a 16-plex and a 7-plex assay, consisting of multiplex PCR, shrimp alkaline phosphatase digestion, primer extension, and MALDI-TOF MS analysis, allow detection of all currently known functionally relevant 24 TPMT alleles (TPMT*2 to *18, *20 to *23). Previously identified variant DNA samples and newly constructed synthetic templates were used as quality controls. RESULTS: Assay evaluation performed on a panel of 586 genomic DNA samples previously genotyped by other methods (denaturing HPLC, sequencing) resulted in 100% agreement. Analyses of the distribution of TPMT alleles in 116 samples from a Ghanaian population revealed a TPMT*8 allele frequency of 3.4%. In a Korean population of 118 unrelated individuals, we found a TPMT*6 allele frequency of 1.3%. CONCLUSIONS: The newly developed multiplex MALDI-TOF MS assay allows efficient genotyping for all currently known functional TPMT variants. To achieve the most accurate prediction of TPMT phenotype, molecular diagnosis of TPMT should include all these variants.