A genome-wide association scan identifies the hepatic cholesterol transporter ABCG8 as a susceptibility factor for human gallstone disease.

With an overall prevalence of 10-20%, gallstone disease (cholelithiasis) represents one of the most frequent and economically relevant health problems of industrialized countries. We performed an association scan of >500,000 SNPs in 280 individuals with gallstones and 360 controls. A follow-up study of the 235 most significant SNPs in 1,105 affected individuals and 873 controls replicated the disease association of SNP A-1791411 in ABCG8 (allelic P value P(CCA) = 4.1 x 10(-9)), which was subsequently attributed to coding variant rs11887534 (D19H). Additional replication was achieved in 728 German (P = 2.8 x 10(-7)) and 167 Chilean subjects (P = 0.02). The overall odds ratio for D19H carriership was 2.2 (95% confidence interval: 1.8-2.6, P = 1.4 x 10(-14)) in the full German sample. Association was stronger in subjects with cholesterol gallstones (odds ratio = 3.3), suggesting that His19 might be associated with a more efficient transport of cholesterol into the bile.

Metabolic activation and analgesic effect of flupirtine in healthy subjects, influence of the polymorphic NAT2, UGT1A1 and GSTP1.

AIMS: The rare association of flupirtine with liver injury is most likely caused by reactive quinone diimines and their oxidative formation may be influenced by the activities of N-acetyltransferases (NAT) that conjugate the less toxic metabolite D13223, and by glucuronosyltransferases (UGT) and glutathione S-transferases (GST) that generate stable terminal glucuronides and mercapturic acid derivatives, respectively. The influence of genetic polymorphisms of NAT2, UGT1A1 and GSTP1 on generation of the terminal mercapturic acid derivatives and analgesic effects was evaluated to identify potential genetic risk factors for hepatotoxicity of flupirtine. METHODS: Metabolic disposition of flupirtine was measured after intravenous administration (100 mg), after swallowing an immediate-release (IR) tablet (100 mg) and after repeated administration of modified release (MR) tablets (400 mg once daily 8 days) in 36 selected healthy subjects. Analgesic effects were measured using pain models (delayed onset of muscle soreness, electric pain). RESULTS: Flupirtine IR was rapidly but incompletely absorbed (∼ 72%). Repeated administration of flupirtine MR showed lower bioavailability (∼ 60%). Approximately 12% of bioavailable flupirtine IR and 8% of bioavailable flupiritine MR was eliminated as mercapturic acid derivatives into the urine independent of the UGT1A1, NAT2 and GSTP1 genotype. Carriers of variant GSTP1 alleles showed lower bioavailability but increased intestinal secretion of flupirtine and increased efficiency in experimental pain. Flupirtine was not a substrate for ABCB1 and ABCC2. CONCLUSIONS: Formation of mercapturic acid derivatives is a major elimination route for flupirtine in man. However, the theoretically toxic pathway is not influenced by the frequent polymorphisms of UGT1A1, NAT2 and GSTP1.

Bi-allelic and tri-allelic 5-HTTLPR polymorphisms and triptan non-response in cluster headache.

BACKGROUND: Triptans are only effective in terminating cluster headache (CH) attacks in 70-80% of patients. Pharmacogenetic aspects of the serotonin metabolism, specifically variation in the 5-HTTLPR may be involved. METHODS: Genetic association study in a well-defined cohort of 148 CH patients with information on drug response to triptans. CH was diagnosed according to the criteria of the International Headache Society. Genotypes of the 43-bp insdel (rs4795541) and A > G (rs25531) polymorphisms in the 5-HTTLPR promoter region were detected by restriction fragment length polymorphism analysis. We used logistic regression analysis to investigate the association between bi-allelic and tri-allelic genotypes and triptan non-response with genotype models. RESULTS: Mean age at study entry among patients was 44.6 ± 10.5 years, 77.7% were men. The genotype distribution both for the bi-allelic and the tri-allelic polymorphism was in Hardy-Weinberg equilibrium. We did not find an association of the bi-allelic polymorphism with triptan non-response. While the effect estimates for the S variant of the tri-allelic polymorphisms suggested increased odds of triptan non-response in CH patients (multivariable-adjusted odds ratio [95% confidence interval]: L*L* genotype-reference; L*S* genotype-1.33 [0.53-3.32]; S*S* genotype-1.46 [0.54-3.98]), the results were not statistically significant. CONCLUSIONS: Data from our study do not indicate a role of bi-allelic and tri-allelic genotypes of the 5-HTTLPR polymorphism in triptan non-response in CH.

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

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

Two new Loci for body-weight regulation identified in a joint analysis of genome-wide association studies for early-onset extreme obesity in French and german study groups.

Meta-analyses of population-based genome-wide association studies (GWAS) in adults have recently led to the detection of new genetic loci for obesity. Here we aimed to discover additional obesity loci in extremely obese children and adolescents. We also investigated if these results generalize by estimating the effects of these obesity loci in adults and in population-based samples including both children and adults. We jointly analysed two GWAS of 2,258 individuals and followed-up the best, according to lowest p-values, 44 single nucleotide polymorphisms (SNP) from 21 genomic regions in 3,141 individuals. After this DISCOVERY step, we explored if the findings derived from the extremely obese children and adolescents (10 SNPs from 5 genomic regions) generalized to (i) the population level and (ii) to adults by genotyping another 31,182 individuals (GENERALIZATION step). Apart from previously identified FTO, MC4R, and TMEM18, we detected two new loci for obesity: one in SDCCAG8 (serologically defined colon cancer antigen 8 gene; p = 1.85x10(-8) in the DISCOVERY step) and one between TNKS (tankyrase, TRF1-interacting ankyrin-related ADP-ribose polymerase gene) and MSRA (methionine sulfoxide reductase A gene; p = 4.84x10(-7)), the latter finding being limited to children and adolescents as demonstrated in the GENERALIZATION step. The odds ratios for early-onset obesity were estimated at approximately 1.10 per risk allele for both loci. Interestingly, the TNKS/MSRA locus has recently been found to be associated with adult waist circumference. In summary, we have completed a meta-analysis of two GWAS which both focus on extremely obese children and adolescents and replicated our findings in a large followed-up data set. We observed that genetic variants in or near FTO, MC4R, TMEM18, SDCCAG8, and TNKS/MSRA were robustly associated with early-onset obesity. We conclude that the currently known major common variants related to obesity overlap to a substantial degree between children and adults.

Associations between the oxytocin receptor gene (OXTR) and "mind-reading" in humans--an exploratory study.

BACKGROUND/AIMS: The application of intranasal oxytocin enhances facial emotion recognition in normal subjects and in subjects with autism spectrum disorders (ASD). In addition, various features of social cognition have been associated with variants of the oxytocin receptor gene (OXTR). Therefore, we tested for associations between mind-reading, a measure for social recognition and OXTR polymorphisms. METHODS: 76 healthy adolescents and young adults were tested for associations between OXTR rs53576, rs2254298, rs2228485 and mind-reading using the "Reading the Mind in the Eyes Test" (RMET). RESULTS: After Bonferroni correction for multiple comparisons, rs2228485 was associated with the number of incorrect answers when subjects evaluated male faces (P =0.000639). There were also associations between OXTR rs53576, rs2254298 and rs2228485 and other RMET dimensions according to P <0.05 (uncorrected). CONCLUSION: This study adds further evidence to the hypothesis that genetic variations in the OXTR modulate mind-reading and social behaviour.

A genome-wide association study identifies three loci associated with mean platelet volume.

Mean platelet volume (MPV) is increased in myocardial and cerebral infarction and is an independent and strong predictor for postevent morbidity and mortality. We conducted a genome-wide association study (GWAS), the KORA (Kooperative Gesundheitsforschung in der Region Augsburg) F3 500K study, and found MPV to be strongly associated with three common single-nucleotide polymorphisms (SNPs): rs7961894 located within intron 3 of WDR66 on chromosome 12q24.31, rs12485738 upstream of the ARHGEF3 on chromosome 3p13-p21, and rs2138852 located upstream of TAOK1 on chromosome 17q11.2. We replicated all three SNPs in another GWAS from the UK and in two population-based samples from Germany. In a combined analysis including 10,048 subjects, the SNPs had p values of 7.24 x 10(-48) for rs7961894, 3.81 x 10(-27) for rs12485738, and 7.19 x 10(-28) for rs2138852. These three quantitative trait loci together accounted for 4%-5% of the variance in MPV. In-depth sequence analysis of WDR66 in 382 samples from the extremes revealed 20 new variants and a haplotype with three coding SNPs and one SNP at the transcription start site associated with MPV (p = 6.8 x 10(-5)). In addition, expression analysis indicated a direct correlation of WDR66 transcripts and MPV. These findings may not only enhance our understanding of platelet activation and function, but may also provide a focus for several novel research avenues.

Meta-analysis of the INSIG2 association with obesity including 74,345 individuals: does heterogeneity of estimates relate to study design?

The INSIG2 rs7566605 polymorphism was identified for obesity (BMI> or =30 kg/m(2)) in one of the first genome-wide association studies, but replications were inconsistent. We collected statistics from 34 studies (n = 74,345), including general population (GP) studies, population-based studies with subjects selected for conditions related to a better health status ('healthy population', HP), and obesity studies (OB). We tested five hypotheses to explore potential sources of heterogeneity. The meta-analysis of 27 studies on Caucasian adults (n = 66,213) combining the different study designs did not support overall association of the CC-genotype with obesity, yielding an odds ratio (OR) of 1.05 (p-value = 0.27). The I(2) measure of 41% (p-value = 0.015) indicated between-study heterogeneity. Restricting to GP studies resulted in a declined I(2) measure of 11% (p-value = 0.33) and an OR of 1.10 (p-value = 0.015). Regarding the five hypotheses, our data showed (a) some difference between GP and HP studies (p-value = 0.012) and (b) an association in extreme comparisons (BMI> or =32.5, 35.0, 37.5, 40.0 kg/m(2) versus BMI<25 kg/m(2)) yielding ORs of 1.16, 1.18, 1.22, or 1.27 (p-values 0.001 to 0.003), which was also underscored by significantly increased CC-genotype frequencies across BMI categories (10.4% to 12.5%, p-value for trend = 0.0002). We did not find evidence for differential ORs (c) among studies with higher than average obesity prevalence compared to lower, (d) among studies with BMI assessment after the year 2000 compared to those before, or (e) among studies from older populations compared to younger. Analysis of non-Caucasian adults (n = 4889) or children (n = 3243) yielded ORs of 1.01 (p-value = 0.94) or 1.15 (p-value = 0.22), respectively. There was no evidence for overall association of the rs7566605 polymorphism with obesity. Our data suggested an association with extreme degrees of obesity, and consequently heterogeneous effects from different study designs may mask an underlying association when unaccounted for. The importance of study design might be under-recognized in gene discovery and association replication so far.

Moderation of adult depression by the serotonin transporter promoter variant (5-HTTLPR), childhood abuse and adult traumatic events in a general population sample.

The impact of the promoter polymorphisms of the serotonin transporter (5-HTTLPR) on mood has been studied by two-way interaction models comprising one environmental factor and genotype variants. However, childhood abuse is assumed to be associated with different psychobiological long-term effects than adult traumatic events. Both types of trauma may interact on an individual basis throughout the lifespan moderating the impact of the 5-HTTLPR s allele on depressive disorders. Therefore, the hypothesis of a three-way interaction among the 5-HTTLPR, childhood abuse and adult traumatic experience was tested. Caucasian subjects (1,974) from the general population in Germany (Study of Health in Pomerania (SHIP)) were analyzed. Depressive symptoms were measured with the Beck Depression Inventory (BDI-II). Childhood abuse was assessed with the Childhood Trauma Questionnaire. Adult traumatic events were derived from the SCID interview (DSM-IV) on posttraumatic stress disorder (PTSD). Global three-way interactions among the 5-HTTLPR, adult traumatic experiences and childhood abuse (P = 0.0007) were found. Carriers of the ss or sl genotypes who had been exposed to childhood abuse and to more than two adult traumatic events had higher mean BDI-II scores (16.0 [95% CI 8.4-23.6]) compared to those carrying the ll genotype (7.6 [4.5-10.7]). These results were supported using a second, more severe definition of childhood abuse (P = 0.02). No two-way interactions were observed (P > 0.05). Childhood abuse and adult traumatic events may act synergistically in interaction with the s allele of the 5-HTTLPR to increase the risk for depressive symptoms independently from the lifetime diagnosis of PTSD.

Aminoglycoside-induced suppression of CYP2C19*3 premature stop codon.

BACKGROUND: CYP2C19 is a polymorphic enzyme that plays a pivotal role in the metabolism of 10% of clinically used drugs worldwide. The CYP2C19*3 allele is characterized by a premature stop codon that leads to a truncated nonfunctional protein and consequently a poor metabolizer phenotype. Aminoglycoside antibiotics have been shown to induce readthrough of premature stop codons and partial restoration of protein function. We investigated the ability of the aminoglycosides gentamicin and G418 to induce readthrough of CYP2C19*3 premature stop codon in human cells. METHODS: A CYP2C19*3 expression model in HeLa cells was used in all experiments. CYP2C19-EGFP expression was assessed by flow cytometry, immunoblotting, and fluorescence microscopy, whereas CYP2C19 enzymatic activity was quantified by hydroxylation of 3-cyano-7-ethoxycoumarin. RESULTS: G418 and gentamicin promoted readthrough of the CYP2C19*3 premature stop codon in a concentration-dependent manner. Flow cytometry revealed a maximum 23% protein restoration with the highest aminoglycoside concentrations tested, namely 300 mg/ml G418 and 1000 mg/ml gentamicin. At these concentrations, G418 was more effective than gentamicin in restoring CYP2C19 expression in immunoblotting and fluorescence microscopy assays, as well as in restoring CYP2C19 enzymatic activity. CONCLUSION: This is the first demonstration of readthrough of a stop codon in a pharmacogenetic target of clinical relevance, namely CYP2C19*3. The experimental models may be adapted to explore readthrough of stop codons in other genes of pharmacogenetic interest.

Role of MRP4 (ABCC4) in platelet adenine nucleotide-storage: evidence from patients with delta-storage pool deficiencies.

We previously showed that the MRP4 (ABCC4) transporter is expressed in human platelet delta-granules and may be involved in ADP transport. We now demonstrate by immunoblotting and immunofluorescence microscopy that platelet MRP4 is absent in two patients with a platelet delta-storage pool deficiency (delta-SPD)-like phenotype with reduced platelet adenine nucleotide (AN) but normal serotonin levels, whereas their other membrane marker proteins of platelet granules were normally expressed and localized. In these patients, MRP4 was present in lymphocytes, and the coding region of their MRP4/ABCC4 gene did not show any mutation that explained the lack of expression. In platelets with "classic" delta-SPD (low AN and serotonin levels), MRP4 was quantitatively (immunoblot) normal, but, like other delta-granules membrane marker proteins (eg, LAMP2), was mostly displaced from delta-granules to patches at the plasma membrane, suggesting that platelets with classic delta-SPD have an abnormality that impairs the assembly of normal delta-granules. Thus, defective expression of platelet MRP4 is associated with selective defect in AN storage. The genetic basis of the new delta-SPD phenotype remains to be elucidated.

Drug efflux transporter multidrug resistance-associated protein 5 affects sensitivity of pancreatic cancer cell lines to the nucleoside anticancer drug 5-fluorouracil.

Pancreatic adenocarcinoma is one of the malignancies that is highly resistant to therapy and among the leading causes of cancer-related death. Several factors may influence pancreatic cancer resistance, and expression of ATP-binding cassette transport proteins is one of the major mechanisms of drug resistance. Members of this family's C-branch, also referred to as multidrug resistance-associated proteins (MRPs), might be of particular interest because they are able to efflux nucleoside analogs used in the treatment of pancreatic cancer. Expression of MRP1, MRP3, MRP4, and MRP5 in human pancreas and pancreatic carcinoma has been reported. However, contributions of MRPs to chemoresistance of pancreatic cancer are not fully understood. MRP5 mRNA expression in pancreatic adenocarcinoma cell lines correlated significantly with cellular sensitivity to 5-fluorouracil (5-FU) (r = 0.738, p < 0.05). Long-term treatment with 5-FU increased expression of MRP5 by 2.4-fold and was associated with significant drug resistance [IC(50) values for control and 5-fluorouracil (5-FU)-resistant Patu-T cell lines were 11.3 ± 5.3 and 33.2 ± 6.9 µM, respectively (p < 0.05)]. Consequently, overexpression of MRP5 in Colo-357 cells resulted in significantly reduced accumulation of 5-FU related radioactivity and 5-FU cytotoxicity. Knockdown of MRP5 significantly increased cellular cytotoxicity of 5-FU to Patu-02 cells and enhanced accumulation of radioactivity related to 5-FU and its metabolites. Our results suggest that MRP5 is expressed and functionally active and contributes to variable sensitivities of pancreatic adenocarcinoma cell lines to 5-FU. Further investigations using models that resemble human pancreas tumors are necessary to prove a causative relation between expression and activity of MRP5 and tumor resistance to 5-FU.

MTHFR 677C>T polymorphism and cluster headache.

BACKGROUND: An association between the 677C>T polymorphism (rs1801133) in the methylenetetrahydrofolate reductase gene (MTHFR) and cluster headache is plausible, but has not been investigated. OBJECTIVE: To investigate this association among Caucasians. METHODS: Case-control study among 147 cluster headache patients and 599 population-based age- and gender-matched controls. Cluster headache was diagnosed according to the criteria of the International Headache Society. Genotypes of the MTHFR 677C>T polymorphism were detected by restriction fragment length polymorphism analysis. We used logistic regression analysis to investigate the association between cluster headache and genotypes with additive, dominant, and recessive models. We considered a Bonferroni-corrected P value <.004 as significant. RESULTS: Mean age at study entry among patients was 44.9 years (SD 11.4), of whom 76.2% were men. The genotype distribution among controls and patients was in Hardy-Weinberg equilibrium. The genotype and allele distribution did not differ between patients with any cluster headache and controls. We also did not find an association when assuming additive, dominant or recessive genetic models. When we looked at subgroups, the effect estimates suggested an increased risk for chronic cluster headache (dominant model: odds ratio = 2.82; 99.6% confidence interval = 0.72-11.07; P = .03). CONCLUSIONS: Data from our case-control study do not indicate an association between genotypes of the MTHFR 677C>T polymorphism and cluster headache overall. Subgroup analyses suggested that carriers of the MTHFR 677T allele may have an increased risk for chronic cluster headache. This may be regarded as hypothesis-generating and should be further investigated in independent studies.

Pharmacogenomics of G protein-coupled receptor ligands in cardiovascular medicine.

Agonists and antagonists of G protein-coupled receptors are important drugs for the treatment of cardiovascular disease, but the therapeutic response of any given patient remains difficult to predict because of large interindividual variability. Among the factors potentially contributing to such variability, we have reviewed the evidence for a role of pharmacodynamic pharmacogenetics (i.e., polymorphisms in the cognate receptors for such drugs as well as other proteins potentially modifying their action). Based upon the availability of data and the prevalence of use, we have focused on ligands at adrenergic and angiotensin II receptors (including the indirectly acting angiotensin-converting enzyme inhibitors). The vast majority of gene polymorphisms reviewed here have shown only inconsistent effects on drug action, which does not make these polymorphisms useful genetic markers to predict treatment responses. We conclude that considerable additional research, partly involving other types of study than those available now, will be necessary to allow a definitive judgment whether pharmacodynamic pharmacogenetic markers are useful in an individualized approach to cardiovascular therapy. Moreover, we predict that even such additional research will result in only few cases where the promise of tailored treatment can be fulfilled; however, some of these few cases may be of major clinical relevance.

Hepatic steatosis is associated with low serum testosterone and high serum DHEAS levels in men.

Obesity and metabolic syndrome are associated with low serum testosterone levels. Hepatic steatosis contributes to the metabolic syndrome and might be regarded as its hepatic manifestation. In this study, we sought to investigate the relationship between hepatic steatosis, serum testosterone and dehydroepiandrosterone sulphate (DHEAS) levels in men. This is a cross-sectional population-based study. We used data of 1912 men recruited for the population-based Study of Health in Pomerania, which was conducted in a region with high prevalence of metabolic syndrome and related diseases. Hepatic steatosis was defined according to sonographic criteria. The relationship of hepatic steatosis with serum testosterone and DHEAS levels was analysed by multivariable logistic regression. Men with low serum testosterone levels had a higher risk of hepatic steatosis than men with high serum testosterone levels. Adjustment for age and further confounders attenuated this association, but did not affect statistical significance (odds ratio 2.36; 95% confidence interval 1.66-3.37; p < 0.05). In the full model, the highest risk of hepatic steatosis was found in subjects with the highest serum DHEAS levels (odds ratio 1.59; 95% confidence interval 1.04-2.43; p < 0.05). Exclusion of men with high alcohol consumption did not affect these results substantially. Hepatic steatosis is associated with low serum testosterone and high serum DHEAS levels. These associations are independent of alcohol consumption.

Survey of Neonates in Pomerania (SNiP): a population-based birth study--objectives, design and population coverage.

Neonatal health is of major concern to parents, midwives, physicians and society as a whole, yet a prospective population-based birth cohort to collect comprehensive data on multiple issues including medical, social, environmental and genetic aspects remains to be established in Germany. The survey of newborns in Pomerania (SNiP) described in this paper attempts to take up this goal. The objectives of SNiP are to establish (a) a population-based birth cohort providing detailed information about neonatal health, morbidity and mortality, (b) a biobank with newborn DNA and serum from cord blood, placenta tissue samples and DNA obtained from oral mucosal swabs of the mothers, (c) a prospective study design by re-examination of the SNiP population prior to attendance at primary school. From March 2003 until November 2008 all childbearing mothers in a well-defined region in North-Eastern Germany were asked to participate with their newborns. Detailed data on health status of the newborn, pregnancy, medical and family history, socio-economic status and maternal life style were obtained via face-to-face interview, standardised questionnaires and medical records. Placental tissue samples, cord blood plasma and DNA were continuously collected; sampling of maternal DNA from mouth swabs started in 2007. As a result, during the study period n = 6747 births and n = 6828 babies were enrolled. A population coverage of 95% was achieved. The active participation rate was 75%. A non-responder analysis revealed no meaningful selection bias. Thus, SNiP is a population-based, representative study in Germany that is able to describe the health and living conditions of newborns and their families comprehensively. It can contribute to existing knowledge and to similar cohort studies since data are accessible by researchers.

Inhibition of glycogen synthase kinase 3beta prevents peroxide-induced collapse of mitochondrial membrane potential in rat ventricular myocytes.

1. Preconditioning has been proposed to protect the myocardium by inhibiting glycogen-synthase kinase (GSK) 3beta. The aim of the present study was to test whether transfection of ventricular myocytes with inactive GSK3 beta would mimic preconditioning and whether a constitutively active form of GSK3 beta would prevent protection by an opioid receptor agonist. 2. Isolated ventricular myocytes from adult rats were infected with live adenovirus containing either a wild-type (wtGSK), constitutively active (caGSK) or dominant-negative (dnGSK) GSK3 beta plasmid. Cells were loaded with tetramethylrhodamine ethyl ester (TMRE) and exposed to H(2)O(2) (100 micromol/L) for 40 min before mitochondrial membrane potential (Delta Psi(m)) was assessed using flow cytometric analysis. 3. Fluorescence intensity was reduced in H(2)O(2)-treated cells compared with untreated cells, presumably because oxidant injury opened mitochondrial permeability transition pores, causing mitochondria to lose TMRE. The selective GSK3 beta inhibitor SB216763, as well as the delta-opioid receptor agonist [d-Ala(2)-D-Leu(5)]-enkephalin (DADLE) (1 micromol/L), protected cells against peroxide-induced loss of Delta Psi(m). 4. Cells transfected with dnGSK (1 micromol/L) were equally protected against peroxide stress, when given throughout the TMRE and H(2)O(2) treatment, confirming a protective effect of GSK3 beta with a highly selective inhibition. Cells transfected with wtGSK did not show any difference in responses to H(2)O(2), SB216763 or DADLE compared with untransfected cells, suggesting that adenovirus infection itself had no effect. In contrast, caGSK-transfected myocytes could no longer be protected with DADLE, suggesting a role for GSK3 beta between the surface receptor and the mitochondria. 5. These experiments confirm that inhibition of GSK3 beta protects the myocytes, but also that the preconditioning mimetic DADLE loses its protective effect when a constitutively active GSK3 beta is present.

Gastric inhibitory polypeptide receptor: association analyses for obesity of several polymorphisms in large study groups.

BACKGROUND: Gastric inhibitory polypeptide (GIP) is postulated to be involved in type 2 diabetes mellitus and obesity. It exerts its function through its receptor, GIPR. We genotyped three GIPR SNPs (rs8111428, rs2302382 and rs1800437) in German families with at least one obese index patient, two case-control studies and two cross-sectional population-based studies. METHODS: Genotyping was performed by MALDI-TOF, ARMS-PCR and RFLP. The family-study: 761 German families with at least one extremely obese child or adolescent (n = 1,041) and both parents (n = 1,522). Case-control study: (a) German obese children (n = 333) and (b) obese adults (n = 987) in comparison to 588 adult lean controls. The two cross-sectional population-based studies: KORA (n = 8,269) and SHIP (n = 4,310). RESULTS: We detected over-transmission of the A-allele of rs2302382 in the German families (pTDT-Test = 0.0089). In the combined case-control sample, we estimated an odd ratio of 1.54 (95%CI 1.09;2.19, pCA-Test = 0.014) for homozygotes of the rs2302382 A-allele compared to individuals with no A-allele. A similar trend was found in KORA where the rs2302382 A-allele led to an increase of 0.12 BMI units (p = 0.136). In SHIP, however, the A-allele of rs2302382 was estimated to contribute an average decrease of 0.27 BMI units (p-value = 0.031). CONCLUSION: Our data suggest a potential relevance of GIPR variants for obesity. However, additional studies are warranted in light of the conflicting results obtained in one of the two population-based studies.

Human platelets express organic anion-transporting peptide 2B1, an uptake transporter for atorvastatin.

Statins are widely used to treat dyslipidemia. Effects of statins in addition to low-density lipoprotein lowering include altered platelet aggregation, requiring drug uptake into platelets. Possible candidates for mediating intraplatelet accumulation of statins include members of the organic anion-transporting polypeptide family such as OATP2B1 (SLCO2B1), a high-affinity uptake transporter for atorvastatin. Therefore, we analyzed OATP expression, localization, and function in human platelets. OATP2B1, but not OATP1B1, was detected in platelets and megakaryocytes on transcript and protein levels. Protein localization was almost exclusively confined to the plasma membrane. Moreover, we could demonstrate significant inhibition of estrone sulfate uptake into platelets by atorvastatin as well as direct transport of atorvastatin into platelets using a liquid chromatography-tandem mass spectrometry method. As a consequence of OATP2B1-mediated uptake of atorvastatin, we observed significant atorvastatin-mediated reduction of thrombin-induced Ca(2+) mobilization in platelets (37.3 +/- 6.7% of control at 15 microM atorvastatin), mechanistically explainable by reduced lipid modification of signal proteins. This effect was reversed by addition of mevalonate. Finally, we demonstrated expression of HMG-CoA reductase, the primary target of atorvastatin, in platelet cytosol. In conclusion, OATP2B1 is an uptake transporter expressed in platelets and is involved in statin-mediated alteration of platelet aggregation.

Exploring warfarin pharmacogenomics with the extreme-discordant-phenotype methodology: impact of FVII polymorphisms on stable anticoagulation with warfarin.

PURPOSE: To explore the pharmacogenomics of warfarin using the extreme-discordant-phenotype (EDP) methodology. METHODS: The target phenotype was the stable warfarin dose prescribed to 353 patients. Pharmacogenetic polymorphisms assessed were coagulation factor VII (FVII) -401G>T and FVII -402G>A, VKORC1 3673G>A, and CYP2C9*2, *3, *5, and *11 alleles. The EDP analyses contrasted the frequencies of these polymorphisms at different cutoff points (5th through 30th percentiles of the warfarin dose distribution) at opposite ends of the warfarin dose distribution. RESULTS: Significant differences existed in FVII -402G>A genotype frequency at the 5th percentile with an over-representation of the wildtype GG genotype at low warfarin doses and in VKORC1 3673G>A and CYP2C9 polymorphisms at all cutoff points where the variant alleles were overrepresented at low warfarin doses. CONCLUSION: The EDP methodology provides increased statistical power for detection of small contributions of genetic polymorphisms to multiple drug-response phenotypes, such as warfarin dose requirement for adequate anticoagulation.