A diagnostic informatics approach for stratifying risk outcome based on combined genotype effects.

BACKGROUND: Diagnostic informatics (DI) in the context of personalized medicine involves the integration of molecular information to provide "actionable" diagnostic and therapeutic strategies. In many cases, retrospective predictions of clinical outcomes affected by multiple genes are complicated by not having the relevant genes measured within the same study. Multiplicative effect modeling is a statistical method for estimating the net effect of ≥ 2 independent variables. The authors demonstrate a DI approach that uses multiplicative-effect modeling to combine genetic information from ≥ 2 independent studies to predict a net clinical outcome. METHODS: As a hypothetical working model, 2 independent studies were selected each reporting on a unique genetic factor proposed to influence the risk of stent thrombosis (ST) among subjects treated with clopidogrel. A multiplicative effect model was used for developing a hypothesis regarding their combined influence on clinical outcome. RESULTS: Application of multiplicative risk modeling yielded a revised estimated risk of outcomes based on combined genotype. In this scenario, combined genotype revised the categorical risk level (high versus low) estimated from single gene effects for 41.5% of the subjects. Further, the maximum relative risk based on single gene effects was increased from 4.54 to 7.84 based on combined genotype. The revised relative risk values in conjunction with combined genotype frequency estimates provides the data necessary to frame a trial hypothesis and conduct appropriate power analysis to estimate the number of subjects needed to test that hypothesis. CONCLUSIONS: This DI approach can be used to generate quantitative hypotheses on multiple gene effects derived from independent genotype studies. This approach is useful for estimating parameters needed in designing future studies to evaluate the net effect of ≥ 2 genetic variants on a common clinical endpoint.

In vivo role of cytochrome P450 2E1 and glutathione-S-transferase activity for acrylamide toxicokinetics in humans.

Acrylamide, a potential food carcinogen in humans, is biotransformed to the epoxide glycidamide in vivo. Both acrylamide and glycidamide are conjugated with glutathione, possibly via glutathione-S-transferases (GST), and bind covalently to proteins and nucleic acids. We investigated acrylamide toxicokinetics in 16 healthy volunteers in a four-period change-over trial and evaluated the respective role of cytochrome P450 2E1 (CYP2E1) and GSTs. Participants ingested self-prepared potato chips containing acrylamide (1 mg) without comedication, after CYP2E1 inhibition (500 mg disulfiram, single dose) or induction (48 g/d ethanol for 1 week), and were phenotyped for CYP2E1 with chlorzoxazone (250 mg, single dose). Unchanged acrylamide and the mercapturic acids N-acetyl-S-(2-carbamoylethyl)-cysteine (AAMA) and N-acetyl-S-(2-hydroxy-2-carbamoylethyl)-cysteine (GAMA) accounted for urinary excretion [geometric mean (percent coefficient of variation)] of 2.9% (42), 65% (23), and 1.7% (65) of the acrylamide dose in the reference period. Hemoglobin adducts increased clearly following the acrylamide test-meal. The cumulative amounts of acrylamide, AAMA, and GAMA excreted and increases in AA adducts changed significantly during CYP2E1 blockade [point estimate (90% confidence interval)] to the 1.34-fold (1.14-1.58), 1.18-fold (1.02-1.36), 0.44-fold (0.31-0.61), and 1.08-fold (1.02-1.15) of the reference period, respectively, but were not changed significantly during moderate CYP2E1 induction. Individual baseline CYP2E1 activity, CYP2E1*6, GSTP1 313A>G and 341T>C single nucleotide polymorphisms, and GSTM1-and GSTT1-null genotypes had no major effect on acrylamide disposition. The changes in acrylamide toxicokinetics upon CYP2E1 blockade provide evidence that CYP2E1 is a major but not the only enzyme mediating acrylamide epoxidation in vivo to glycidamide in humans. No obvious genetic risks or protective factors in xenobiotic-metabolizing enzymes could be determined for exposed subjects.

Relevance of the organic cation transporters 1 and 2 for antiretroviral drug therapy in human immunodeficiency virus infection.

Carrier-mediated transport across cell membranes is an important determinant of activity, resistance, and toxicity of chemotherapeutic agents including antiretroviral (ARV) drugs (ARDs). The organic cation transporters (OCTs) 1 and 2 have been implicated in the translocation of different cationic drugs but so far were insufficiently tested for interactions with ARDs. Here, we assessed among cationic drugs commonly used in human immunodeficiency virus (HIV) therapy inhibitors and substrates of OCTs, and analyzed the tissue distribution of OCTs and their expression in lymph nodes (LNs), the primary intracellular target of HIV and ARDs. Inhibitors were identified by measuring the attenuated uptake of the radiolabeled model substrate 1-methyl-4-phenylpyridinium into OCT-transfected human embryonic kidney-293 cells in the presence of ARDs. Substrates were identified by measuring OCT-specific intracellular accumulation using liquid chromatography/tandem mass spectrometry. Inhibitory drugs were (in order of increasing potency): nelfinavir < ritonavir < saquinavir < indinavir < trimethoprim < pentamidine, with consistently lower IC(50) values determined for OCT1. Substrates with highest transport efficacy (V(max)/K(m)) were lamivudine (OCT1, 8 microl/mg protein/min; OCT2, 4.4 microl/mg protein/min) and zalcitabine (OCT1, 4.1 microl/mg protein/min; OCT2, 2.6 microl/mg protein/min). Using quantitative real-time polymerase chain reaction, a marked expression level of OCT1 was detected in human samples of liver, ovary, prostate, and testis, and of OCT2 in kidney, colon, heart, skeletal muscle, and testis. Expression of OCTs in LNs was low in HIV-negative control individuals but dramatically increased in HIV-infected persons. These data suggest that drug interactions about the OCTs may be relevant for the ARV therapy, in particular by influencing drug accession to infected tissues and hepatic or renal elimination.

Effect of cocoa and tea intake on blood pressure: a meta-analysis.

BACKGROUND: Epidemiological evidence suggests blood pressure-lowering effects of cocoa and tea. We undertook a meta-analysis of randomized controlled trials to determine changes in systolic and diastolic blood pressure due to the intake of cocoa products or black and green tea. METHODS: MEDLINE, EMBASE, SCOPUS, Science Citation Index, and the Cochrane Controlled Trials Register were searched from 1966 until October 2006 for studies in parallel group or crossover design involving 10 or more adults in whom blood pressure was assessed before and after receiving cocoa products or black or green tea for at least 7 days. RESULTS: Five randomized controlled studies of cocoa administration involving a total of 173 subjects with a median duration of 2 weeks were included. After the cocoa diets, the pooled mean systolic and diastolic blood pressure were -4.7 mm Hg (95% confidence interval [CI], -7.6 to -1.8 mm Hg; P = .002) and -2.8 mm Hg (95% CI, -4.8 to -0.8 mm Hg; P = .006) lower, respectively, compared with the cocoa-free controls. Five studies of tea consumption involving a total of 343 subjects with a median duration of 4 weeks were selected. The tea intake had no significant effects on blood pressure. The estimated pooled changes were 0.4 mm Hg (95% CI, -1.3 to 2.2 mm Hg; P = .63) in systolic and -0.6 mm Hg (95% CI, -1.5 to 0.4 mm Hg; P = .38) in diastolic blood pressure compared with controls. CONCLUSION: Current randomized dietary studies indicate that consumption of foods rich in cocoa may reduce blood pressure, while tea intake appears to have no effect.

Dietary sources and antioxidant effects of ergothioneine.

Ergothioneine is a native membrane-impermeable thiol compound that is specifically accumulated in cells via the organic cation transporter OCTN1. In humans, OCTN1 and ergothioneine have been implicated in the etiopathogenesis of autoimmune disorders. However, available evidence about dietary sources and the functional role of ergothioneine in human physiology is scarce. Here, we analyzed the ergothioneine content in common foods using liquid chromatography tandem-mass spectrometry. Additionally, we assessed the protective potency of ergothioneine against various oxidative stressors in OCTN1-expressing cells in comparison with the main intracellular thiol antioxidant glutathione by evaluating cell viability with the MTT reduction assay. Only some food contained ergothioneine with highest concentrations detected in specialty mushrooms, kidney, liver, black and red beans, and oat bran. Ergothioneine exhibited cell protection only against copper(II)-induced toxicity but was far less potent than glutathione, indicting that ergothioneine is not involved in the intracellular antioxidant thiol defense system.

Effects of low habitual cocoa intake on blood pressure and bioactive nitric oxide: a randomized controlled trial.

CONTEXT: Regular intake of cocoa-containing foods is linked to lower cardiovascular mortality in observational studies. Short-term interventions of at most 2 weeks indicate that high doses of cocoa can improve endothelial function and reduce blood pressure (BP) due to the action of the cocoa polyphenols, but the clinical effect of low habitual cocoa intake on BP and the underlying BP-lowering mechanisms are unclear. OBJECTIVE: To determine effects of low doses of polyphenol-rich dark chocolate on BP. DESIGN, SETTING, AND PARTICIPANTS: Randomized, controlled, investigator-blinded, parallel-group trial involving 44 adults aged 56 through 73 years (24 women, 20 men) with untreated upper-range prehypertension or stage 1 hypertension without concomitant risk factors. The trial was conducted at a primary care clinic in Germany between January 2005 and December 2006. INTERVENTION: Participants were randomly assigned to receive for 18 weeks either 6.3 g (30 kcal) per day of dark chocolate containing 30 mg of polyphenols or matching polyphenol-free white chocolate. MAIN OUTCOME MEASURES: Primary outcome measure was the change in BP after 18 weeks. Secondary outcome measures were changes in plasma markers of vasodilative nitric oxide (S-nitrosoglutathione) and oxidative stress (8-isoprostane), and bioavailability of cocoa polyphenols. RESULTS: From baseline to 18 weeks, dark chocolate intake reduced mean (SD) systolic BP by -2.9 (1.6) mm Hg (P < .001) and diastolic BP by -1.9 (1.0) mm Hg (P < .001) without changes in body weight, plasma levels of lipids, glucose, and 8-isoprostane. Hypertension prevalence declined from 86% to 68%. The BP decrease was accompanied by a sustained increase of S-nitrosoglutathione by 0.23 (0.12) nmol/L (P < .001), and a dark chocolate dose resulted in the appearance of cocoa phenols in plasma. White chocolate intake caused no changes in BP or plasma biomarkers. CONCLUSIONS: Data in this relatively small sample of otherwise healthy individuals with above-optimal BP indicate that inclusion of small amounts of polyphenol-rich dark chocolate as part of a usual diet efficiently reduced BP and improved formation of vasodilative nitric oxide. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT00421499.

Absorption, metabolization, and antiplatelet effects of 300-, 600-, and 900-mg loading doses of clopidogrel: results of the ISAR-CHOICE (Intracoronary Stenting and Antithrombotic Regimen: Choose Between 3 High Oral Doses for Immediate Clopidogrel Effect) Trial.

BACKGROUND: For patients undergoing percutaneous coronary intervention, the administration of a clopidogrel loading dose ranging from 300 to 600 mg is currently recommended. It is unknown, though, whether loading doses higher than 600 mg exert additional suppression of platelet function. METHODS AND RESULTS: Sixty patients with suspected or documented coronary artery disease admitted to our hospital for coronary angiography were included in this trial. They were allocated to 1 of 3 clopidogrel loading doses (300, 600, or 900 mg) in a double-blinded, randomized manner. Plasma concentrations of the active thiol metabolite, unchanged clopidogrel, and the inactive carboxyl metabolite of clopidogrel were determined before and serially after drug administration. Optical aggregometry was performed before and 4 hours after administration of clopidogrel. Loading with 600 mg resulted in higher plasma concentrations of the active metabolite, clopidogrel, and the carboxyl metabolite compared with loading with 300 mg (P< or =0.03) and lower values for adenosine diphosphate-induced (5 and 20 micromol/L) platelet aggregation 4 hours after drug administration (P=0.01 and 0.004). With administration of 900 mg, no further increase in plasma concentrations of active metabolite and clopidogrel (P> or =0.38) and no further suppression of adenosine diphosphate-induced (5 and 20 micromol/L) platelet aggregation 4 hours after drug administration was achieved when compared with administration of 600 mg (P=0.59 and 0.39). CONCLUSIONS: Single doses of clopidogrel higher than 600 mg are not associated with an additional significant suppression of platelet function because of limited clopidogrel absorption.

Impact of P-glycoprotein on clopidogrel absorption.

OBJECTIVE: The antiplatelet activity of clopidogrel is characterized by considerable interindividual differences. Variable intestinal absorption is suggested to contribute to the inconsistencies in response. We tested the hypothesis that the intestinal efflux transporter P-glycoprotein (P-gp) limits the oral bioavailability of clopidogrel and that variance in the MDR1 gene encoding P-gp predicts absorption variability. METHODS AND RESULTS: P-gp-mediated transport of clopidogrel was assessed by transflux, influx, and efflux experiments by use of Caco-2 cells. Inhibition of P-gp activity by different modulators increased the absorptive clopidogrel flux across Caco-2 monolayers from 0.51+/-0.19 pmol/cm2 (mean+/-SD) at baseline by a maximum of 5- to 9-fold (P<.001) and the intracellular accumulation from 0.99+/-0.11 pmol/mg protein by a maximum of 2.5-fold (P<.001) in response to 1-micromol/L clopidogrel and decreased clopidogrel efflux to the level of passive diffusion. In 60 patients with coronary artery disease who underwent percutaneous coronary intervention, the peak plasma concentration (Cmax) and the total area under the plasma concentration-time curve (AUC) of clopidogrel and its active metabolite after a single oral loading dose of 300, 600, or 900 mg were tested for correlation with the MDR1 genotype. In the 300-mg and 600-mg groups (but not in the 900-mg group) Cmax and AUC values were lower in subjects homozygous for the MDR1 3435T variant compared with subjects with the 3435C/T and 3435C/C genotypes. After the 600-mg loading dose, Cmax values (mean+/-SD) of clopidogrel and its active metabolite in 3435T/T carriers were 13.3+/-5.2 ng/mL and 2.5+/-1.2 ng/mL, respectively, compared with 49.7+/-41.6 ng/mL (P=.001) and 6.6+/-3.6 ng/mL (P=.011), respectively, in 3435C/T and 3435C/C carriers; AUC values were 1502+/-463 ng/mLxmin for clopidogrel and 209+/-99 ng/mL x min for its active metabolite in 3435T/T carriers compared with 7057+/-5443 ng/mLxmin (P=.0006) and 744+/-541 ng/mLxmin (P=.011), respectively, in 3435C/T and 3435C/C carriers. CONCLUSIONS: Clopidogrel absorption and thereby active metabolite formation are diminished by P-gp-mediated efflux and are influenced by the MDR1 C3435T genotype.

Toxicokinetics of acrylamide in humans after ingestion of a defined dose in a test meal to improve risk assessment for acrylamide carcinogenicity.

High amounts of acrylamide in some foods result in an estimated daily mean intake of 50 microg for a western style diet. Animal studies have shown the carcinogenicity of acrylamide upon oral exposure. However, only sparse human toxicokinetic data is available for acrylamide, which is needed for the extrapolation of human cancer risk from animal data. We evaluated the toxicokinetics of acrylamide in six young healthy volunteers after the consumption of a meal containing 0.94 mg of acrylamide. Urine was collected up to 72 hours thereafter. Unchanged acrylamide, its mercapturic acid metabolite N-acetyl-S-(2-carbamoylethyl)cysteine (AAMA), its epoxy derivative glycidamide, and the respective metabolite of glycidamide, N-acetyl-S-(2-hydroxy-2-carbamoylethyl)cysteine (GAMA), were quantified in the urine by liquid chromatography-mass spectrometry. Toxicokinetic variables were obtained by noncompartmental methods. Overall, 60.3 +/- 11.2% of the dose was recovered in the urine. Although no glycidamide was found, unchanged acrylamide, AAMA, and GAMA accounted for urinary excretion of (mean +/- SD) 4.4 +/- 1.5%, 50.0 +/- 9.4%, and 5.9 +/- 1.2% of the dose, respectively. Apparent terminal elimination half-lives for the substances were 2.4 +/- 0.4, 17.4 +/- 3.9, and 25.1 +/- 6.4 hours. The ratio of GAMA/AAMA amounts excreted was 0.12 +/- 0.02. In conclusion, most of the acrylamide ingested with food is absorbed in humans. Conjugation with glutathione exceeds the formation of the reactive metabolite glycidamide. The data suggests an at least 2-fold and 4-fold lower relative internal exposure for glycidamide from dietary acrylamide in humans compared with rats or mice, respectively. This should be considered for quantitative cancer risk assessment.

The garlic ingredient diallyl sulfide inhibits cytochrome P450 2E1 dependent bioactivation of acrylamide to glycidamide.

Genotoxic effects of acrylamide are supposed to result from oxidative biotransformation to glycidamide. After incubation of rat liver slices with acrylamide we detected free glycidamide using a liquid chromatography tandem mass spectrometric method. Glycidamide formation was diminished in the presence of the cytochrome P450 2E1 inhibitor diallyl sulfide (DAS), which is a specific ingredient of garlic. This may be relevant to human health since the suggested carcinogenic risk of dietary acrylamide may be reduced by concomitant intake of garlic.

Different antioxidative potencies of dihydropyridine calcium channel modulators in various models.

There is evidence that dihydropyridine calcium antagonists (DHP) play a beneficial role during the development of atherosclerosis. Since antioxidative properties of this substance class may be important, we investigated the antioxidative potency of the DHP prototype calcium channel antagonist nifedipine, the long acting calcium channel antagonist lacidipine, the DHP calcium channel agonist Bay K 8644 and the bulky DHP derivate Bay O 5572 (negligible effects on L-type calcium channels) in three different models. Additionally, we examined the potential correlation between lipophilic and antioxidative properties. In an in vitro model, Bay K 8644 was significantly more effective in scavenging superoxide anions (hypoxanthine/xanthine-oxidase-assay) than lacidipine, Bay O 5572 or nifedipine (micro- to millimolar concentration range). Addition of artificial membrane preparations (dimyristoylphosphatidylcholine) to mimic a physiological environment resulted in an enhanced antioxidative effect, with lacidipine being the most effective DHP to quench radicals (low micromolar concentration range). Thirdly, in a more physiological model of hyperglycemia (30 mmol/l) induced release of reactive oxygen species (ROS) from native endothelial cells of porcine coronary arteries, we showed that nifedipine was a significantly more potent antioxidant (therapeutical nanomolar concentration range) than the other DHP. Calculation of the lipophilicity of the four substances (lacidipine>Bay O 5572>Bay K 8644>nifedipine) showed a positive correlation between the antioxidative potency and the lipophilicity in the model with the artificial membranes but not in the other models. We conclude that it seems necessary to access antioxidative properties of substances in physiological models in which we could demonstrate that nifedipine exhibits ROS-quenching properties in a therapeutic concentration range.

Reaction rate constants of superoxide scavenging by plant antioxidants.

Plant phenols may exert protective effects by scavenging superoxide, which is implicated in tissue damage and accelerated inactivation of vasorelaxing nitric oxide. Preventing the interaction of superoxide with tissue biomolecules depends not only on the extent of superoxide scavenging but also on scavenging velocity. However, information on superoxide scavenging kinetics of plant phenols is scarce. We describe an improved lucigenin-based chemiluminescence assay for kinetic analysis. The use of potassium superoxide (KO2) as a nonenzymatic superoxide source allowed simple and reliable determination of the second-order reaction rate constants between superoxide and plant antioxidants at physiologically relevant conditions, avoiding unspecific effects of other reactive oxygen species or superoxide-generating enzymes. We calculated the rate constants for phenols of different structures, ranging from 2.9 x 10(3) mol(-1) l s(-1) for morin to 2.9 x 10(7) mol(-1) l s(-1) for proanthocyanidins. Compounds with pyrogallol or catechol moieties were revealed as the most rapid superoxide scavengers, and the gallate moiety was found to be the minimal essential structure for maximal reaction rate constants with superoxide.

Pharmacokinetics of clopidogrel after administration of a high loading dose.

The adenosine diphosphate (ADP) receptor P2Y12 blocking agent clopidogrel is clinically proven to be efficient in preventing thrombotic events. However, its therapeutic value is limited by an, as yet poorly explained, interindividual heterogeneity in platelet inhibition. To evaluate possible pharmacokinetic determinants of this response variability, we developed a sensitive and specific liquid chromatography tandem mass spectrometry (LC-MS/MS) assay for quantification of unmodified inactive clopidogrel, its inactive carboxyl metabolite, and its active thiol metabolite in plasma. Analyte concentrations and platelet aggregation were assessed in ten healthy volunteers receiving an oral load of 600 mg clopidogrel. Subjects showed marked inter-individual differences in maximal platelet inhibition and in plasma pharmacokinetics. Univariate regression revealed linear correlations between maximal antiplatelet effect and peak plasma concentrations (cmax) of unchanged clopidogrel (r=0.76; p=0.01), of the carboxyl metabolite (r=0.70; p=0.03), and of the thiol metabolite (r=0.73; p=0.02), as well as linear correlations between cmax values of clopidogrel and its metabolites. This indicates that the response variability is predominantly caused by individual differences in clopidogrel absorption and that other factors, such as ADP receptor reactivity or differences in bioactivation of clopidogrel, do not play a major role.

Influence of processing parameters on acrylamide formation during frying of potatoes.

Consistent evidence suggests that the probable human carcinogen acrylamide is formed in starch-rich foodstuffs through heat-induced interaction of asparagine and reducing sugars during Maillard browning. However, information regarding the influence of processing parameters on acrylamide formation is scarce. We investigated the impact of temperature, heating time, browning level, and surface-to-volume ratio (SVR) on acrylamide generation in fried potatoes. Acrylamide content was determined by liquid chromatography (LC) and electrospray ionization tandem mass spectrometry (ESI-MS/MS). In potato shapes with low SVR, acrylamide content consistently increased with increasing temperature and processing times. By contrast, in shapes with intermediate to high SVR, maximal acrylamide formation occurred at 160-180 degrees C, while higher temperatures or prolonged processing times caused a decrease of acrylamide levels. Moreover, browning levels were not a reliable measure of acrylamide content in large-surface products.

Clopidogrel, CYP2C19, and a Black Box.

It has been presumed that CYP2C19 has a major role in the metabolism of clopidogrel. This presumption has been based on in vitro drug metabolism studies using microsomes from baculovirus infected insect cells (BD-Supersomes™). If clopidogrel were primarily a CYP2C19 substrate, a drug/drug interaction with CYP2C19 inhibitors, such as proton pump inhibitors (PPIs), for example, omeprazole and lansoprazole would be anticipated. Several ex vivo studies, using ADP stimulated platelet aggregation, suggested that there was such an interaction. The data from these studies served as a basis for FDA to provide a "Black Box" warning for the clopidogrel label in March of 2010. However, a prospective clinical study, COGENT, and several large meta-analyses have failed to demonstrate a negative effect on major adverse cardiovascular events (MACE), with concomitant administration of clopidogrel and PPIs. The in vitro work using "Supersomes" was revisited. In vitro metabolism using hepatosomes, which resemble the native cytochrome P-450 enzyme expression, has confirmed the earlier work that clopidogrel is primarily a CYP3A substrate. This result correlates better with clinical findings. The absence of an increase in MACE by concomitant administration of PPIs, which are CYP2C19 inhibitors, to a regimen including clopidogrel, is therefore not surprising. A hypothesis is offered to explain why subjects, who carry two reduced function alleles of CYP2C19, may have more reactive platelets.

Impact of CYP2C19 variant genotypes on clinical efficacy of antiplatelet treatment with clopidogrel: systematic review and meta-analysis.

OBJECTIVE: To evaluate the accumulated information from genetic association studies investigating the impact of variants of the cytochrome P450 (CYP) 2C19 genotype on the clinical efficacy of clopidogrel. DESIGN: Systematic review and meta-analysis with a structured search algorithm and prespecified eligibility criteria for retrieval of relevant studies; dominant genetic model assumptions and quantitative methods for calculating summary effect estimates from study level odds ratios; systematic assessment of bias within and between studies; and grading of the cumulative evidence by consensus criteria. DATA SOURCES: Medline, Embase, the Cochrane Library, online databases, contents pages and bibliographies of general medical, cardiovascular, pharmacological, and genetic journals. Eligibility criteria for selecting studies Original full length reports assessing the cumulative incidence of major adverse cardiovascular events or stent thrombosis over a follow-up period of at least a month in association with carrier status for the loss of function or gain of function CYP2C19 allele in adult patients with coronary artery disease and a clinical presentation of acute coronary syndrome or stable angina pectoris who were taking clopidogrel. RESULTS: 15 studies met the inclusion criteria. The random effects summary odds ratio for stent thrombosis in carriers of at least one CYP2C19 loss of function allele versus non-carriers combining nine studies was 1.77 (95% confidence interval 1.31 to 2.40; P < 0.001). This nominally significant odds ratio was subject to considerable bias across the studies (small study effect bias and replication diversity). The adjustment for these quality modifiers tended to abolish the association. The corresponding random effects summary odds ratio of major adverse cardiovascular events for 12 studies combined was 1.11 (0.89 to 1.39; P = 0.36). The random effects summary odds ratio of stent thrombosis in carriers versus non-carriers of at least one CYP2C19*17 gain of function allele for three studies combined was 0.99 (0.60 to 1.62; P = 0.96), and the corresponding odds ratio of major adverse cardiovascular events in five studies was 0.93 (0.75 to 1.14; P = 0.48). The overall quality of epidemiological evidence was graded as low, which excludes reliable clinical assessments. CONCLUSIONS: Accumulated information from genetic association studies does not indicate a substantial or consistent influence of CYP2C19 gene polymorphisms on the clinical efficacy of clopidogrel. The current evidence does not support the use of individualised antiplatelet regimens guided by CYP2C19 genotype.