Effects of APOC3 Heterozygous Deficiency on Plasma Lipid and Lipoprotein Metabolism.

Objective- Apo (apolipoprotein) CIII inhibits lipoprotein lipase (LpL)-mediated lipolysis of VLDL (very-low-density lipoprotein) triglyceride (TG) and decreases hepatic uptake of VLDL remnants. The discovery that 5% of Lancaster Old Order Amish are heterozygous for the APOC3 R19X null mutation provided the opportunity to determine the effects of a naturally occurring reduction in apo CIII levels on the metabolism of atherogenic containing lipoproteins. Approach and Results- We conducted stable isotope studies of VLDL-TG and apoB100 in 5 individuals heterozygous for the null mutation APOC3 R19X (CT) and their unaffected (CC) siblings.

Genome-wide association analysis of common genetic variants of resistant hypertension.

Resistant hypertension (RHTN), defined as uncontrolled blood pressure (BP) ≥ 140/90 using three or more drugs or controlled BP (<140/90) using four or more drugs, is associated with adverse outcomes, including decline in kidney function. We conducted a genome-wide association analysis in 1194 White and Hispanic participants with hypertension and coronary artery disease from the INternational VErapamil-SR Trandolapril STudy-GENEtic Substudy (INVEST-GENES). Top variants associated with RHTN at p < 10 were tested for replication in 585 White and Hispanic participants with hypertension and subcortical strokes from the Secondary Prevention of Subcortical Strokes GENEtic Substudy (SPS3-GENES).

Multisite Investigation of Outcomes With Implementation of CYP2C19 Genotype-Guided Antiplatelet Therapy After Percutaneous Coronary Intervention.

Objectives: This multicenter pragmatic investigation assessed outcomes following clinical implementation of CYP2C19 genotype-guided antiplatelet therapy after percutaneous coronary intervention (PCI).

Background: CYP2C19 loss-of-function alleles impair clopidogrel effectiveness after PCI.

Methods: After clinical genotyping, each institution recommended alternative antiplatelet therapy (prasugrel, ticagrelor) in PCI patients with a loss-of-function allele.

Pharmacogenetic Associations of β1-Adrenergic Receptor Polymorphisms With Cardiovascular Outcomes in the SPS3 Trial (Secondary Prevention of Small Subcortical Strokes).

Background And Purpose: Functional polymorphisms (Ser49Gly and Arg389Gly) in have been associated with cardiovascular and β-blocker response outcomes. Herein we examined associations of these polymorphisms with major adverse cardiovascular events (MACE), with and without stratification by β-blocker treatment in patients with a history of stroke.

Methods: Nine hundred and twenty-six participants of the SPS3 trial's (Secondary Prevention of Small Subcortical Strokes) genetic substudy with hypertension were included.

Genome-wide analysis of clopidogrel active metabolite levels identifies novel variants that influence antiplatelet response.

Clopidogrel is one of the most commonly used therapeutics for the secondary prevention of cardiovascular events in patients with acute coronary syndromes. However, considerable interindividual variation in clopidogrel response has been documented, resulting in suboptimal therapy and an increased risk of recurrent events for some patients. In this investigation, we carried out the first genome-wide association study of circulating clopidogrel active metabolite levels in 513 healthy participants to directly measure clopidogrel pharmacokinetics.

A systematic analysis and comparison of warfarin initiation strategies.

Objective: Randomized trials have reported inconsistent evidence on the effectiveness of algorithms that use genotypes to initiate warfarin therapy. The Clarification of Optimal Anticoagulation through Genetics (COAG) trial initiated therapy on the basis of predicted maintenance doses, with a pharmacogenetic-guided algorithm in one study group and a clinically guided algorithm in the other. The European Pharmacogenetics of Anticoagulant Therapy (EU-PACT) consortium initiated therapy on the basis of loading doses, with an algorithm-based prediction in one study group and a fixed-dose regimen in the other.

Development of a physiology-directed population pharmacokinetic and pharmacodynamic model for characterizing the impact of genetic and demographic factors on clopidogrel response in healthy adults.

Clopidogrel (Plavix®), is a widely used antiplatelet agent, which shows high inter-individual variability in treatment response in patients following the standard dosing regimen. In this study, a physiology-directed population pharmacokinetic/pharmacodynamic (PK/PD) model was developed based on clopidogrel and clopidogrel active metabolite (clop-AM) data from the PAPI and the PGXB2B studies using a step-wise approach in NONMEM (version 7.2).

Oxylipid Profile of Low-Dose Aspirin Exposure: A Pharmacometabolomics Study.

Background: While aspirin is a well-established and generally effective anti-platelet agent, considerable inter-individual variation in drug response exists, for which mechanisms are not completely understood. Metabolomics allows for extensive measurement of small molecules in biological samples, enabling detailed mapping of pathways involved in drug response.

Methods And Results: We used a mass-spectrometry-based metabolomics platform to investigate the changes in the serum oxylipid metabolome induced by an aspirin intervention (14 days, 81 mg/day) in healthy subjects (n=156).

The Pharmacogenomics of Anti-Platelet Intervention (PAPI) Study: Variation in Platelet Response to Clopidogrel and Aspirin.

Clopidogrel and aspirin are commonly prescribed anti-platelet medications indicated for patients who have experienced, or are at risk for, ischemic cardiovascular events. The Pharmacogenomics of Anti-Platelet Intervention (PAPI) Study was designed to characterize determinants of clopidogrel and dual anti-platelet therapy (DAPT) response in a healthy cohort of Old Order Amish from Lancaster, PA. Following a loading dose, clopidogrel was taken once a day for 7 days.

The Impact of Inpatient Versus Outpatient Initiation on Early Warfarin Dosing.

Background And Objectives: Dosing algorithms for warfarin incorporate clinical and genetic factors but may not account for the numerous comorbidities affecting patients who start warfarin while hospitalized. We aimed to determine whether these algorithms perform differently when warfarin is initiated for inpatients compared with outpatients.

Patients And Methods: We analyzed a prospective cohort of 1015 participants from the Clarification of Optimal Anticoagulation through Genetics (COAG) trial who were randomized to either pharmacogenetically or clinically guided warfarin dosing algorithms.

CYP2C19 metabolizer status and clopidogrel efficacy in the Secondary Prevention of Small Subcortical Strokes (SPS3) study.

Background: The role of the CYP2C19 genotype on clopidogrel efficacy has been studied widely, with data suggesting reduced clopidogrel efficacy in loss-of-function variant carriers taking clopidogrel after percutaneous coronary intervention; however, data are limited regarding the association between CYP2C19 genetic variants and outcomes in stroke patients. We investigated whether CYP2C19 metabolizer status affects the risk of recurrent stroke or major bleeding in subcortical stroke patients taking dual antiplatelet therapy with aspirin and clopidogrel.

Methods And Results: CYP2C19*2 and CYP2C19*17 were genotyped in 522 patients treated with dual antiplatelet therapy from the Secondary Prevention of Small Subcortical Strokes (SPS3) study.

Null mutation in hormone-sensitive lipase gene and risk of type 2 diabetes.

Background: Lipolysis regulates energy homeostasis through the hydrolysis of intracellular triglycerides and the release of fatty acids for use as energy substrates or lipid mediators in cellular processes. Genes encoding proteins that regulate energy homeostasis through lipolysis are thus likely to play an important role in determining susceptibility to metabolic disorders.

Methods: We sequenced 12 lipolytic-pathway genes in Old Order Amish participants whose fasting serum triglyceride levels were at the extremes of the distribution and identified a novel 19-bp frameshift deletion in exon 9 of LIPE, encoding hormone-sensitive lipase (HSL), a key enzyme for lipolysis.

Effectiveness of clopidogrel dose escalation to normalize active metabolite exposure and antiplatelet effects in CYP2C19 poor metabolizers.

Carriers of two copies of the loss-of-function CYP2C19*2 variant convert less clopidogrel into its active metabolite, resulting in diminished antiplatelet responses and higher cardiovascular event rates. To evaluate whether increasing the daily clopidogrel dose in poor metabolizers (PM) overcomes the effect of the CYP2C19 * 2 variant, we enrolled 18 healthy participants in a genotype-stratified, multi-dose, three-period, fixed-sequence crossover study. Six participants with the *1/*1 extensive (EM), *1/*2 intermediate (IM), and *2/*2 poor metabolizer genotypes each received 75 mg, 150 mg, and 300 mg each for 8 days.

Implementation of pharmacogenetics: the University of Maryland Personalized Anti-platelet Pharmacogenetics Program.

Despite a substantial evidence base, implementation of pharmacogenetics into routine patient care has been slow due to a number of non-trivial practical barriers. We implemented a Personalized Anti-platelet Pharmacogenetics Program (PAP3) for cardiac catheterization patients at the University of Maryland Medical Center and the Baltimore Veterans Administration Medical Center Patients' are offered CYP2C19 genetic testing, which is performed in our Clinical Laboratory Improvement Amendment (CLIA)-certified Translational Genomics Laboratory. Results are returned within 5 hr along with clinical decision support that includes interpretation of results and prescribing recommendations for anti-platelet therapy based on the Clinical Pharmacogenetics Implementation Consortium guidelines.

A pharmacogenetic versus a clinical algorithm for warfarin dosing.

Background: The clinical utility of genotype-guided (pharmacogenetically based) dosing of warfarin has been tested only in small clinical trials or observational studies, with equivocal results.

Methods: We randomly assigned 1015 patients to receive doses of warfarin during the first 5 days of therapy that were determined according to a dosing algorithm that included both clinical variables and genotype data or to one that included clinical variables only. All patients and clinicians were unaware of the dose of warfarin during the first 4 weeks of therapy.

Pharmacogenomics of anti-platelet and anti-coagulation therapy.

Arterial thrombosis is a major component of vascular disease, especially myocardial infarction (MI) and stroke. Current anti-thrombotic therapies such as warfarin and clopidogrel are effective in inhibiting cardiovascular events; however, there is great inter-individual variability in response to these medications. In recent years, it has been recognized that genetic factors play a significant role in drug response, and, subsequently, common variants in genes responsible for metabolism and drug action have been identified.

Genetic variation in PEAR1 is associated with platelet aggregation and cardiovascular outcomes.

BACKGROUND- Aspirin or dual antiplatelet therapy with aspirin and clopidogrel is a standard therapy for patients who are at increased risk for cardiovascular events. However, the genetic determinants of variable response to aspirin (alone and in combination with clopidogrel) are not known. METHODS AND RESULTS- We measured ex vivo platelet aggregation before and after dual antiplatelet therapy in individuals (n=565) from the Pharmacogenomics of Anti-Platelet Intervention (PAPI) Study and conducted a genome-wide association study of drug response.

The ABCG8 G574R variant, serum plant sterol levels, and cardiovascular disease risk in the Old Order Amish.

Objective: To determine whether long-term exposure to moderate elevations in plasma plant sterol levels increases risk for atherosclerosis.

Methods And Results: In Old Order Amish participants aged 18 to 85 years, with (n=110) and without (n=181) 1 copy of the ABCG8 G574R variant, we compared mean plasma levels of plant sterols and cholesterol precursors and carotid intima-media wall thickness. Carriers of a single 574R allele had increased plant sterol levels (eg, 35%-37% higher plasma levels of sitosterol, campesterol, and stigmasterol) and increased plant sterol/cholesterol ratios (P<0.

The functional G143E variant of carboxylesterase 1 is associated with increased clopidogrel active metabolite levels and greater clopidogrel response.

Introduction: Carboxylesterase 1 (CES1) is the primary enzyme responsible for converting clopidogrel into biologically inactive carboxylic acid metabolites.

Methods: We genotyped a functional variant in CES1, G143E, in participants of the Pharmacogenomics of Anti-Platelet Intervention (PAPI) study (n=566) and in 350 patients with coronary heart disease treated with clopidogrel, and carried out an association analysis of bioactive metabolite levels, on-clopidogrel ADP-stimulated platelet aggregation, and cardiovascular outcomes.

Results: The levels of clopidogrel active metabolite were significantly greater in CES1 143E-allele carriers (P=0.

Analysis of the gut microbiota in the old order Amish and its relation to the metabolic syndrome.

Obesity has been linked to the human gut microbiota; however, the contribution of gut bacterial species to the obese phenotype remains controversial because of conflicting results from studies in different populations. To explore the possible dysbiosis of gut microbiota in obesity and its metabolic complications, we studied men and women over a range of body mass indices from the Old Order Amish sect, a culturally homogeneous Caucasian population of Central European ancestry. We characterized the gut microbiota in 310 subjects by deep pyrosequencing of bar-coded PCR amplicons from the V1-V3 region of the 16S rRNA gene.

A sensitive and rapid ultra HPLC-MS/MS method for the simultaneous detection of clopidogrel and its derivatized active thiol metabolite in human plasma.

A sensitive, selective, and rapid ultra-high performance liquid chromatography-tandem mass spectrometry (uHPLC-MS/MS) was developed for the simultaneous quantification of clopidogrel (Plavix(®)) and its derivatized active metabolite (CAMD) in human plasma. Derivatization of the active metabolite in blood with 2-bromo-3'-methoxy acetophenone (MPB) immediately after collection ensured metabolite stability during sample handling and storage. Following addition of ticlopidine as an internal standard and simple protein precipitation, the analytes were separated on a Waters Acquity UPLC™ sub-2 μm-C(18) column via gradient elution before detection on a triple-quadrupole MS with multiple-reaction-monitoring via electrospray ionization.

Familial defective apolipoprotein B-100 and increased low-density lipoprotein cholesterol and coronary artery calcification in the old order amish.

Background: Elevated low-density lipoprotein cholesterol (LDL-C) levels are a major cardiovascular disease risk factor. Genetic factors are an important determinant of LDL-C levels.

Methods: To identify single nucleotide polymorphisms associated with LDL-C and subclinical coronary atherosclerosis, we performed a genome-wide association study of LDL-C in 841 asymptomatic Amish individuals aged 20 to 80 years, with replication in a second sample of 663 Amish individuals.

Association of cytochrome P450 2C19 genotype with the antiplatelet effect and clinical efficacy of clopidogrel therapy.

Context: Clopidogrel therapy improves cardiovascular outcomes in patients with acute coronary syndromes and following percutaneous coronary intervention by inhibiting adenosine diphosphate (ADP)-dependent platelet activation. However, nonresponsiveness is widely recognized and is related to recurrent ischemic events.

Objective: To identify gene variants that influence clopidogrel response.

Circulating CD34+ Cell Count is Associated with Extent of Subclinical Atherosclerosis in Asymptomatic Amish Men, Independent of 10-Year Framingham Risk.

Background: Bone-marrow derived progenitor cells (PCs) may play a role in maintaining vascular health by actively repairing damaged endothelium. The purpose of this study in asymptomatic Old Order Amish men (n = 90) without hypertension or diabetes was to determine if PC count, as determined by CD34+ cell count in peripheral blood, was associated with 10-year risk of cardiovascular disease (CVD) and measures of subclinical atherosclerosis.

Methods And Results: CD34+ cell count by fluorescence-activated cell sorting, coronary artery calcification (CAC) by electron beam computed tomography, and CVD risk factors were obtained.