Stefan Winter

Publications of Stefan Winter

• PPARA: A Novel Genetic Determinant of CYP3A4 In Vitro and In Vivo.

  Authors: Kathrin Klein, Maria Thomas, Stefan Winter, Andreas K Nussler, Mikko Niemi, Matthias Schwab, Ulrich M Zanger

  Clinical pharmacology and therapeutics. 04/2012;

  Interindividual variability in cytochrome P450 3A4 (CYP3A4) is believed to be largely heritable; however, predictive genetic factors have remained scarce. Using a candidate-gene approach in a humanInterindividual variability in cytochrome P450 3A4 (CYP3A4) is believed to be largely heritable; however, predictive genetic factors have remained scarce. Using a candidate-gene approach in a human liver bank, we identified single-nucleotide polymorphisms (SNPs) in the Ah-receptor nuclear translocator (ARNT), glucocorticoid receptor (GR), progesterone receptor membrane component 2 (PGRMC2), and peroxisome proliferator-activated receptor-α (PPARA) that are associated with CYP3A4 phenotype. Validation in atorvastatin-treated volunteers confirmed a decrease in atorvastatin-2-hydroxylation in carriers of PPARA SNP rs4253728. Homozygous carriers expressed significantly less PPAR-α protein in the liver. Moreover, shRNA-mediated PPARA gene knockdown in primary human hepatocytes decreased expression levels of the PPAR-α target ACOX1 and of CYP3A4 by more than 50%. In conclusion, this study identified novel genetic determinants of CYP3A4 that, together with nongenetic factors, explained 52, 55, and 33% of hepatic CYP3A4 mRNA, protein, and atorvastatin-2-hydroxylase activity, respectively. These findings have implications for variability in response to drug substrates of CYP3A4.

• 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.

• The postmenopausal hormone replacement therapy-related breast cancer risk is decreased in women carrying the CYP2C19*17 variant.

  Authors: Christina Justenhoven, Ofure Obazee, Stefan Winter, Fergus J Couch, Janet E Olson, Per Hall, Ulf Hannelius, Jingmei Li, Keith Humphreys, Gianluca Severi [......] Arif B Ekici, Ute Hamann, Christian Baisch, Volker Harth, Sylvia Rabstein, Anne Lotz, Beate Pesch, Thomas Brüning, Yon-Dschun Ko, Hiltrud Brauch

  Breast cancer research and treatment. 01/2012; 131(1):347-50.

• 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.

• The earwax-associated SNP c.538G>A (G180R) in ABCC11 is not associated with breast cancer risk in Europeans.

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

  Breast cancer research and treatment. 06/2011; 129(3):993-9.

  Genetic polymorphisms of human ABC-transporter genes have been suggested to modulate breast cancer risk in the general population. In particular ABCC11 (MRP8), which is highly expressed in breastGenetic polymorphisms of human ABC-transporter genes have been suggested to modulate breast cancer risk in the general population. In particular ABCC11 (MRP8), which is highly expressed in breast cancer tissue and involved in the efflux of conjugated estrogen metabolites such as estrone-3-sulfate and estradiol-17beta-glucuronide, has recently been proposed as a potential risk factor for female breast cancer. The wet earwax-associated G-allele of the c.538G>A polymorphism was associated with an increased risk for breast cancer in Japanese women. In contrast, no evidence for such an association could be observed in Caucasian women. We aimed to confirm/refute the association of the c.538G>A variant in ABCC11 with breast cancer risk and/or histo-pathological tumor characteristics in an independent population-based breast cancer case-control study from Germany comprising 1021 cases and 1015 age-matched controls. No association for allele and genotype frequencies of the 538G>A variant in ABCB11 with breast cancer risk was found. Our data suggest that the c.538G>A variation in ABCC11 does not contribute to breast carcinogenesis in women of European descent.

• Paraoxonase (PON1 and PON3) Polymorphisms: Impact on Liver Expression and Atorvastatin-Lactone Hydrolysis.

  Authors: Stephan Riedmaier, Kathrin Klein, Stefan Winter, Ute Hofmann, Matthias Schwab, Ulrich M Zanger

  Frontiers in pharmacology. 01/2011; 2:41.

  Atorvastatin δ-lactone, a major, pharmacologically inactive metabolite, has been associated with toxicity. In a previous study we showed that polymorphisms of UGT1A3 influence atorvastatin δ-lactoneAtorvastatin δ-lactone, a major, pharmacologically inactive metabolite, has been associated with toxicity. In a previous study we showed that polymorphisms of UGT1A3 influence atorvastatin δ-lactone formation. Here we investigated the reverse reaction, atorvastatin δ-lactone hydrolysis, in a human liver bank. Screening of microarray data revealed paraoxonases PON1 and PON3 among 17 candidate esterases. Microsomal δ-lactone hydrolysis was significantly correlated to PON1 and PON3 protein (r(s) = 0.60; r(s) = 0.62, respectively; P < 0.0001). PON1 and PON3 were strongly correlated to each other (r(s) = 0.60) but PON1 was shown to be more extensively glycosylated than PON3. In addition a novel splice-variant of PON3 was identified. Genotyping of 40 polymorphisms within the PON-locus identified PON1 promoter polymorphisms (-108T > C, -832G > A, -1741G > A) and a tightly linked group of PON3 polymorphisms (-4984A > G, -4105G > A, -1091A > G, -746C > T, and F21F) to be associated with changes in atorvastatin δ-lactone hydrolysis and expression of PON1 but not PON3. However, carriers of the common PON1 polymorphisms L55M or Q192R showed no difference in δ-lactone hydrolysis or PON expression. Haplotype analysis revealed decreased δ-lactone hydrolysis in carriers of the most common haplotype *1 compared to carriers of haplotypes *2, *3, *4, and *7. Analysis of non-genetic factors showed association of hepatocellular and cholangiocellular carcinoma with decreased PON1 and PON3 expression, respectively. Increased C-reactive protein and γ-glutamyl transferase levels were associated with decreased protein expression of both enzymes, and increased bilirubin levels, cholestasis, and presurgical exposure to omeprazole or pantoprazole were related to decreased PON3 protein. In conclusion, PON-locus polymorphisms affect PON1 expression whereas non-genetic factors have an effect on PON1 and PON3 expression. This may influence response to therapy or adverse events in statin treatment.

• 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.

• The frameshift polymorphism CYP3A43_74_delA is associated with poor differentiation of breast tumors.

  Authors: Christina Justenhoven, Stefan Winter, Ute Hamann, Susanne Haas, Hans-Peter Fischer, Beate Pesch, Thomas Brüning, Yon-Dschun Ko, Hiltrud Brauch

  Cancer. 12/2010; 116(23):5358-64.

  CYP3A enzymes, due to their role in the metabolism of steroid hormones, are suggested to affect carcinogenesis of hormone-related cancers. The purpose of the present study was to evaluate theCYP3A enzymes, due to their role in the metabolism of steroid hormones, are suggested to affect carcinogenesis of hormone-related cancers. The purpose of the present study was to evaluate the association between polymorphisms located in CYP3A43, breast cancer risk, and tumor characteristics. A 3-plex matrix-assisted laser desorption ionization time of flight mass spectrometry assay has been established for CYP3A43_74_delA (CYP3A43*2A), CYP3A43_1018_C>G (CYP3A43*3), and CYP3A43_1047_C>T (CYP3A43*1B) polymorphisms, and 1021 breast cancer cases and 1015 age-matched, population-based controls from the German GENICA collection have been genotyped. No differences in genotype frequencies between cases and controls were observed, indicating that CYP3A43_74_delA is not associated with breast cancer risk. Subgroup analyses showed an association between the CYP3A43_74_delA allele and high-grade tumors (odds ratio, 1.74; 95% confidence interval, 1.14-2.65 [P=.010 and Ptrend=.012]). The data support the notion that the CYP3A43_74_delA variant may result in decreased protein and/or activity levels, and this may further lead to increased hormone levels to promote tumor cell growth and hinder differentiation.

• Combined UGT1A1 and UGT1A6 genotypes together with a stressful life event increase breast cancer risk.

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

  Breast cancer research and treatment. 11/2010; 124(1):289-92.

• CYP2D6 polymorphisms as predictors of outcome in breast cancer patients treated with tamoxifen: expanded polymorphism coverage improves risk stratification.

  Authors: Werner Schroth, Ute Hamann, Peter A Fasching, Silke Dauser, Stefan Winter, Michel Eichelbaum, Matthias Schwab, Hiltrud Brauch

  Clinical cancer research : an official journal of the American Association for Cancer Research. 09/2010; 16(17):4468-77.

  This study aimed to validate matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS)/Taqman copy number assay (CNA) CYP2D6 genotyping by AmpliChip CYP450 Test forThis study aimed to validate matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS)/Taqman copy number assay (CNA) CYP2D6 genotyping by AmpliChip CYP450 Test for the prediction of tamoxifen metabolizer phenotypes in breast cancer, and to investigate the influence of CYP2D6 variant coverage on genotype-phenotype relationships and tamoxifen outcome. Hormone receptor-positive postmenopausal breast cancer patients (n = 492) treated with adjuvant tamoxifen, previously analyzed by MALDI-TOF MS/CNA, were reanalyzed by AmpliChip CYP450 Test and validated by independent methods. Cox proportional hazard ratios (HR) were calculated for recurrence of poor (PM) relative to extensive metabolizer (EM) phenotypes with increasing numbers of CYP2D6 variants. Kaplan-Meier distributions were calculated for different phenotype classifications. Concordance was 99.2% to 99.5% for CNA and 99.8% to 100% per CYP2D6 allele (*3, *4, *5, *9, *10, and *41). The prevalence of predicted phenotypes was 1.2% for ultrarapid metabolizer (UM), 37.2% for EM without variant, 43.5% for heterozygous EM, 9.7% for intermediate metabolizer (IM), and 8.3% for PM. Approximately, one third of patients were misclassified based on a *4 analysis only, but inclusion of all reduced-function alleles increased the PM-associated HR from 1.33 (P = 0.58) to 2.87 (P = 0.006). Kaplan-Meier analyses showed highest and lowest clinical benefit for UM and PM with respect to both the AmpliChip-based and a redefined phenotype assignment. The latter revealed significant allele-dose-dependent associations (P = 0.011) and largest effect size (HR(PM_EM) = 2.77; 95% confidence interval, 1.31-5.89). MALDI-TOF MS/CNA is suitable for accurate CYP2D6 genotyping. For tamoxifen pharmacogenetics, broad CYP2D6 allele coverage is recommended to reduce phenotype misclassification. Classification based on refined EM and reduced-function metabolizers is advisable. AACR.

• Pathway-Targeted Pharmacogenomics of CYP1A2 in Human Liver.

  Authors: Kathrin Klein, Stefan Winter, Miia Turpeinen, Matthias Schwab, Ulrich M Zanger

  Frontiers in pharmacology. 01/2010; 1:129.

  The human drug metabolizing cytochrome P450 (CYP) 1A2, is one of the major P450 isoforms contributing by about 5-20% to the hepatic P450 pool and catalyzing oxidative biotransformation of up to 10%The human drug metabolizing cytochrome P450 (CYP) 1A2, is one of the major P450 isoforms contributing by about 5-20% to the hepatic P450 pool and catalyzing oxidative biotransformation of up to 10% of clinically relevant drugs including clozapine and caffeine. CYP1A2 activity is interindividually highly variable and although twin studies have suggested a high heritability, underlying genetic factors are still unknown. Here we adopted a pathway-oriented approach using a large human liver bank (n = 150) to elucidate whether variants in candidate genes of constitutive, ligand-inducible, and pathophysiological inhibitory regulatory pathways may explain different hepatic CYP1A2 phenotypes. Samples were phenotyped for phenacetin O-deethylase activity, and the expression of CYP1A2 protein and mRNA was determined. CYP1A2 expression and function was increased in smokers and decreased in patients with inflammation and cholestasis. Of 169 SNPs in 17 candidate genes including the CYP1A locus, 136 non-redundant SNPs with minor allele frequency >5% were analyzed by univariate and multivariate methods. A total of 13 strong significant associations were identified, of which 10 SNPs in the ARNT, AhRR, HNF1α, IL1β, SRC-1, and VDR genes showed consistent changes for at least two phenotypes by univariate analysis. Multivariate linear modeling indicated that the polymorphisms and non-genetic factors together explained 42, 38, and 33% of CYP1A2 variation at activity, protein and mRNA levels, respectively. In conclusion, we identified novel trans-associations between regulatory genes and hepatic CYP1A2 function and expression, but additional genetic factors must be assumed to explain the full extent of CYP1A2 heritability.

• No evidence for glutathione S-transferases GSTA2, GSTM2, GSTO1, GSTO2, and GSTZ1 in breast cancer risk.

  Authors: Irena Andonova, Christina Justenhoven, Stefan Winter, Ute Hamann, Christian Baisch, Sylvia Rabstein, Anne Spickenheuer, Volker Harth, Beate Pesch, Thomas Brüning, Yon-Dschun Ko, Varban Ganev, Hiltrud Brauch

  Breast cancer research and treatment. 10/2009;

  Breast cancer is a complex disease and in recent years a number of breast cancer susceptibility genes have been identified, but the role of low penetrance susceptibility genes has not been completelyBreast cancer is a complex disease and in recent years a number of breast cancer susceptibility genes have been identified, but the role of low penetrance susceptibility genes has not been completely resolved. Glutathione S-transferases (GSTs) are phase II xenobiotic metabolizing enzymes involved in the detoxification of chemical carcinogens and environmental pollutants and play an important role in cell defense mechanisms against oxidative stress. They have been in the spot light for the investigation of a potential association with breast cancer risk but so far, sparse or even no data for a potential contribution of GSTA2, GSTM2, GSTO, and GSTZ to breast cancer risk are available. We genotyped GSTA2_448_C > G (rs2180314), GSTA2_742_A > C (rs6577), GSTM2_-832_T > C (rs638820), GSTO1_-1242_G > A (rs2164624), GSTO1_419_A > C (rs4925), GSTO2_-183_A > G (rs2297235), GSTO2_342_A > G (rs156697), GSTZ1_-4378_A > G (rs1046428), and GSTZ1_94_G > A (rs3177427) by MALDI-TOF MS in the German GENICA breast cancer case-control collection of 1021 cases and 1015 controls and performed breast cancer risk association in general and with respect to the stratifications: menopausal status, family history of breast or ovarian cancer, use of oral contraceptives, use of hormone therapy, body mass index, and smoking as well as histopathological tumor characteristics including hormone receptor status, grade, histology, and node status. We did not observe any breast cancer risk associations and conclude that it is unlikely that glutathione S-transferases GSTA2, GSTM2, GSTO1, GSTO2, and GSTZ1 participate in breast cancer susceptibility.

• Association between CYP2D6 polymorphisms and outcomes among women with early stage breast cancer treated with tamoxifen.

  Authors: Werner Schroth, Matthew P Goetz, Ute Hamann, Peter A Fasching, Marcus Schmidt, Stefan Winter, Peter Fritz, Wolfgang Simon, Vera J Suman, Matthew M Ames [......] Hans Ulrich Ulmer, Julia Boländer, Reiner Strick, Matthias W Beckmann, Heinz Koelbl, Richard M Weinshilboum, James N Ingle, Michel Eichelbaum, Matthias Schwab, Hiltrud Brauch

  JAMA : the journal of the American Medical Association. 10/2009; 302(13):1429-36.

  CONTEXT: The growth inhibitory effect of tamoxifen, which is used for the treatment of hormone receptor-positive breast cancer, is mediated by its metabolites, 4-hydroxytamoxifen and endoxifen. TheCONTEXT: The growth inhibitory effect of tamoxifen, which is used for the treatment of hormone receptor-positive breast cancer, is mediated by its metabolites, 4-hydroxytamoxifen and endoxifen. The formation of active metabolites is catalyzed by the polymorphic cytochrome P450 2D6 (CYP2D6) enzyme. OBJECTIVE: To determine whether CYP2D6 variation is associated with clinical outcomes in women receiving adjuvant tamoxifen. DESIGN, SETTING, AND PATIENTS: Retrospective analysis of German and US cohorts of patients treated with adjuvant tamoxifen for early stage breast cancer. The 1325 patients had diagnoses between 1986 and 2005 of stage I through III breast cancer and were mainly postmenopausal (95.4%). Last follow-up was in December 2008; inclusion criteria were hormone receptor positivity, no metastatic disease at diagnosis, adjuvant tamoxifen therapy, and no chemotherapy. DNA from tumor tissue or blood was genotyped for CYP2D6 variants associated with reduced (*10, *41) or absent (*3, *4, *5) enzyme activity. Women were classified as having an extensive (n=609), heterozygous extensive/intermediate (n=637), or poor (n=79) CYP2D6 metabolism. MAIN OUTCOME MEASURES: Time to recurrence, event-free survival, disease-free survival, and overall survival. RESULTS: Median follow-up was 6.3 years. At 9 years of follow-up, the recurrence rates were 14.9% for extensive metabolizers, 20.9% for heterozygous extensive/intermediate metabolizers, and 29.0% for poor metabolizers, and all-cause mortality rates were 16.7%, 18.0%, and 22.8%, respectively. Compared with extensive metabolizers, there was a significantly increased risk of recurrence for heterozygous extensive/intermediate metabolizers (time to recurrence adjusted hazard ratio [HR], 1.40; 95% confidence interval [CI], 1.04-1.90) and for poor metabolizers (time to recurrence HR, 1.90; 95% CI, 1.10-3.28). Compared with extensive metabolizers, those with decreased CYP2D6 activity (heterozygous extensive/intermediate and poor metabolism) had worse event-free survival (HR, 1.33; 95% CI, 1.06-1.68) and disease-free survival (HR, 1.29; 95% CI, 1.03-1.61), but there was no significant difference in overall survival (HR, 1.15; 95% CI, 0.88-1.51). CONCLUSION: Among women with breast cancer treated with tamoxifen, there was an association between CYP2D6 variation and clinical outcomes, such that the presence of 2 functional CYP2D6 alleles was associated with better clinical outcomes and the presence of nonfunctional or reduced-function alleles with worse outcomes.

• Effect of comedication with proton pump inhibitors (PPIs) on post-interventional residual platelet aggregation in patients undergoing coronary stenting treated by dual antiplatelet therapy.

  Authors: Christine S Zuern, Tobias Geisler, Natalia Lutilsky, Stefan Winter, Matthias Schwab, Meinrad Gawaz

  Thrombosis research. 09/2009;

  INTRODUCTION: Currently, there is an intense debate about whether comedication with proton pump inhibitors (PPIs) weakens the antiplatelet effect of clopidogrel in patients undergoing coronary stentINTRODUCTION: Currently, there is an intense debate about whether comedication with proton pump inhibitors (PPIs) weakens the antiplatelet effect of clopidogrel in patients undergoing coronary stent implantation. Competing mechanisms on the hepatic cytochrome 2C19 level are proposed. The aim of this study was to assess the impact of PPI treatment on clopidogrel response by measuring the ex vivo platelet aggregation in patients with coronary intervention. METHODS: 1425 consecutive patients with symptomatic coronary artery disease undergoing percutaneous coronary intervention were enrolled in this single centre study. PPI comedication was defined as PPI intake >/=1 week prior to a 600 mg clopidogrel loading dose. PPI treatment was based on physician preference. Residual platelet aggregation (RPA) was measured by optical aggregometry. To correct for potential selection bias, propensity score matching was applied. RESULTS: RPA was significantly higher in PPI-treated patients compared with non-PPI-users (final aggregation 34.0% vs. 29.8%, p<0.001). Low responder defined as RPA in the upper tertile were more often found in PPI-users. After adjustment for relevant confounders, PPI treatment was independently associated with higher RPA-levels. DISCUSSION: We demonstrated that peri-procedural co-administration of PPIs significantly decreases the effect of clopidogrel on RPA. To assess if clopidogrel-PPI interaction results in a higher susceptibility for cardiovascular events remains subject to further investigations.

• 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.).

• CYP2C19*17 is associated with decreased breast cancer risk.

  Authors: Christina Justenhoven, Ute Hamann, Christiane B Pierl, Christian Baisch, Volker Harth, Sylvia Rabstein, Anne Spickenheuer, Beate Pesch, Thomas Brüning, Stefan Winter, Yon-Dschun Ko, Hiltrud Brauch

  Breast cancer research and treatment. 05/2009; 115(2):391-6.

  Cytochrome P450 2C19 (CYP2C19) plays an important role in the metabolism of xenobiotics and drugs and contributes to the catabolism of endogenous substrates like estradiol. Genetic variabilityCytochrome P450 2C19 (CYP2C19) plays an important role in the metabolism of xenobiotics and drugs and contributes to the catabolism of endogenous substrates like estradiol. Genetic variability impacts expression and activity of CYP2C19 and therefore can influence catabolism of estrogens. In the present study we analyzed the association of three polymorphisms of CYP2C19 namely CYP2C19*2 (CYP2C19_681_G>A, rs4244285), CYP2C19*3 (CYP2C19_636_G>A, rs57081121) and CYP2C19*17 (CYP2C19_-806_C>T, rs12248560), with breast cancer susceptibility. We genotyped 1,015 breast cancer cases and 1,021 age-matched, population-based controls of the German GENICA study by matrix assisted laser desorption/ionization time-of-flight mass spectrometry. Risk estimates were calculated by logistic regression. All tests were two-sided. We observed a decreased breast cancer risk for carriers of the CYP2C19*17 allele (OR 0.77, 95% CI: 0.65-0.93; P = 0.005). In subgroup analysis we observed a significant decreased breast cancer risk for women using hormone therapy for ten years or longer who were carriers of the CYP2C19*17 allele (OR 0.57, 95% CI: 0.39-0.83; P = 0.003). Since CYP2C19*17 defines an ultra rapid metabolizer phenotype we suggest that an increased catabolism of estrogens by CYP2C19 may lead to decreased estrogen levels and therefore reduces breast cancer risk. This protective effect seems to be stronger in combination with long-term intake of supplemental estrogens during hormone therapy.

• 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.

• 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.

• CYP2C19 and nongenetic factors predict poor responsiveness to clopidogrel loading dose after coronary stent implantation.

  Authors: Tobias Geisler, Elke Schaeffeler, Juergen Dippon, Stefan Winter, Verena Buse, Christian Bischofs, Christine Zuern, Klaus Moerike, Meinrad Gawaz, Matthias Schwab

  Pharmacogenomics. 09/2008; 9(9):1251-1259.

  Aims: To investigate an association of responsiveness to clopidogrel loading dose with genotypes of cytochrome P450 (CYP) 2C19, other CYP isozymes and nongenetic factors in patients with coronaryAims: To investigate an association of responsiveness to clopidogrel loading dose with genotypes of cytochrome P450 (CYP) 2C19, other CYP isozymes and nongenetic factors in patients with coronary artery disease. Materials & methods: Genotyping for CYP2C19 (*2, *3 and *17), CYP3A4*1B and CYP3A5*3 variants was performed in patients (n = 237) who underwent percutaneous coronary intervention. Adenosine diphosphate-induced platelet aggregation was determined after first administration of 600 mg clopidogrel. Results: CYP2C19*2 carriers showed significantly increased residual platelet aggregation (RPA) (OR: 4.6; 95% CI: 2.5-8.7; p < 0.0001) compared with noncarriers. All other polymorphisms had no influence on RPA. For the development of a risk score for better prediction of RPA, CYP2C19*2 genotype and previously identified nongenetic risk factors (age >65 years, Type 2 diabetes mellitus, decreased left ventricular function, renal failure and acute coronary syndrome) were analyzed. Multivariable logistic regression analysis showed a significant correlation of the nongenetic factors (chi (2) = 5.32; p = 0.021) and CYP2C19*2 (chi (2) = 21.31; p < 0.0001) with high RPA, and an even higher association for the combination of both (chi (2) = 25.85; p < 0.0001). Conclusions: Prediction of responsiveness after clopidogrel loading dose may substantially be improved by adding CYP2C19*2 genotype to nongenetic risk factors.