Taimour Y. Langaee

Use of Pharmacogenetic and Clinical Factors to Predict the Therapeutic Dose of Warfarin

Initiation of warfarin therapy using trial‐and‐error dosing is problematic. Our goal was to develop and validate a pharmacogenetic algorithm. In the derivation cohort of 1,015 participants, the independent predictors of therapeutic dose were: VKORC1 polymorphism −1639/3673 G>A (−28% per allele), body surface area (BSA) (+11% per 0.25 m2), CYP2C9*3 (−33% per allele), CYP2C9*2 (−19% per allele), age (−7% per decade), target international normalized ratio (INR) (+11% per 0.5 unit increase), amiodarone use (−22%), smoker status (+10%), race (−9%), and current thrombosis (+7%). This pharmacogenetic equation explained 53–54% of the variability in the warfarin dose in the derivation and validation (N= 292) cohorts. For comparison, a clinical equation explained only 17–22% of the dose variability (P < 0.001). In the validation cohort, we prospectively used the pharmacogenetic‐dosing algorithm in patients initiating warfarin therapy, two of whom had a major hemorrhage. To facilitate use of these pharmacogenetic and clinical algorithms, we developed a nonprofit website, http://www.WarfarinDosing.org.

CYP4F2 genetic variant alters required warfarin dose.

Warfarin is an effective, commonly prescribed anticoagulant used to treat and prevent thrombotic events. Because of historically high rates of drug-associated adverse events, warfarin remains underprescribed. Further, interindividual variability in therapeutic dose mandates frequent monitoring until target anticoagulation is achieved. Genetic polymorphisms involved in warfarin metabolism and sensitivity have been implicated in variability of dose. Here, we describe a novel variant that influences warfarin requirements. To identify additional genetic variants that contribute to warfarin requirements, screening of DNA variants in additional genes that code for drug-metabolizing enzymes and drug transport proteins was undertaken using the Affymetrix drug-metabolizing enzymes and transporters panel. A DNA variant (rs2108622; V433M) in cytochrome P450 4F2 (CYP4F2) was associated with warfarin dose in 3 independent white cohorts of patients stabilized on warfarin representing diverse geographic regions in the United States and accounted for a difference in warfarin dose of approximately 1 mg/day between CC and TT subjects. Genetic variation of CYP4F2 was associated with a clinically relevant effect on warfarin requirement.

A genome-wide scan for common genetic variants with a large influence on warfarin maintenance dose

Warfarin dosing is correlated with polymorphisms in vitamin K epoxide reductase complex 1 (VKORC1) and the cytochrome P450 2C9 (CYP2C9) genes. Recently, the FDA revised warfarin labeling to raise physician awareness about these genetic effects. Randomized clinical trials are underway to test genetically based dosing algorithms. It is thus important to determine whether common single nucleotide polymorphisms (SNPs) in other gene(s) have a large effect on warfarin dosing. A retrospective genome-wide association study was designed to identify polymorphisms that could explain a large fraction of the dose variance. White patients from an index warfarin population (n = 181) and 2 independent replication patient populations (n = 374) were studied. From the approximately 550 000 polymorphisms tested, the most significant independent effect was associated with VKORC1 polymorphisms (P = 6.2 x 10(-13)) in the index patients. CYP2C9 (rs1057910 CYP2C9*3) and rs4917639) was associated with dose at moderate significance levels (P approximately 10(-4)). Replication polymorphisms (355 SNPs) from the index study did not show any significant effects in the replication patient sets. We conclude that common SNPs with large effects on warfarin dose are unlikely to be discovered outside of the CYP2C9 and VKORC1 genes. Randomized clinical trials that account for these 2 genes should therefore produce results that are definitive and broadly applicable.

Influence of coagulation factor, vitamin K epoxide reductase complex subunit 1, and cytochrome P450 2C9 gene polymorphisms on warfarin dose requirements

The primary objective of this study was to determine whether variability in warfarin dose requirements is determined by common polymorphisms in genes whose products are involved in the pharmacodynamics and pharmacokinetics of warfarin, namely, the coagulation factors, vitamin K epoxide reductase complex subunit 1 (VKORC1), and cytochrome P450 (CYP) 2C9.

Genetic and Clinical Predictors of Warfarin Dose Requirements in African Americans

The objective of this study was to determine whether, in African‐American patients, additional vitamin K oxidoreductase complex subunit 1 (VKORC1), cytochrome P450 2C9 (CYP2C9), CYP4F2, or apolipoprotein E (APOE) polymorphisms contribute to variability in the warfarin maintenance dose beyond what is attributable to the CYP2C9*2 and *3 alleles and the VKORC1 −1639G>A genotype. In a cohort of 226 African‐American patients, weekly warfarin dose requirements were lower in those with the CYP2C9*8 allele (34 (30–47) mg; P = 0.023) and the CYP2C9 *2, *3, *5, *6, or *11 allele (33(28–40 mg); P < 0.001) as compared with those with the CYP2C9*1/*1 genotype (43 (35–56) mg). The combination of CYP2C9 alleles, VKORC1 −1639G>A genotype, and clinical variables explained 36% of the interpatient variability in warfarin dose requirements. By comparison, a model without the CYP2C9*5, *6, *8, and *11 alleles explained 30% of the variability in dose. No other VKORC1, CYP4F2, or APOE polymorphism contributed to the variance. The inclusion of additional CYP2C9 variants may improve the predictive ability of warfarin dosing algorithms for African Americans.

Two CES1 Gene Mutations Lead to Dysfunctional Carboxylesterase 1 Activity in Man: Clinical Significance and Molecular Basis

The human carboxylesterase 1 (CES1) gene encodes for the enzyme carboxylesterase 1, a serine esterase governing both metabolic deactivation and activation of numerous therapeutic agents. During the course of a study of the pharmacokinetics of the methyl ester racemic psychostimulant methylphenidate, profoundly elevated methylphenidate plasma concentrations, unprecedented distortions in isomer disposition, and increases in hemodynamic measures were observed in a subject of European descent. These observations led to a focused study of the subjects CES1 gene. DNA sequencing detected two coding region single-nucleotide mutations located in exons 4 and 6. The mutation in exon 4 is located in codon 143 and leads to a nonconservative substitution, p.Gly143Glu. A deletion in exon 6 at codon 260 results in a frameshift mutation, p.Asp260fs, altering residues 260-299 before truncating at a premature stop codon. The minor allele frequency of p.Gly143Glu was determined to be 3.7%, 4.3%, 2.0%, and 0% in white, black, Hispanic, and Asian populations, respectively. Of 925 individual DNA samples examined, none carried the p.Asp260fs, indicating it is an extremely rare mutation. In vitro functional studies demonstrated the catalytic functions of both p.Gly143Glu and p.Asp260fs are substantially impaired, resulting in a complete loss of hydrolytic activity toward methylphenidate. When a more sensitive esterase substrate, p-nitrophenyl acetate was utilized, only 21.4% and 0.6% catalytic efficiency (V(max)/K(m)) were determined in p.Gly143Glu and p.Asp260fs, respectively, compared to the wild-type enzyme. These findings indicate that specific CES1 gene variants can lead to clinically significant alterations in pharmacokinetics and drug response of carboxylesterase 1 substrates.

Genetic variants associated with warfarin dose in African-American individuals: a genome-wide association study

Summary Background VKORC1 and CYP2C9 are important contributors to warfarin dose variability, but explain less variability for individuals of African descent than for those of European or Asian descent. We aimed to identify additional variants contributing to warfarin dose requirements in African Americans. Methods We did a genome-wide association study of discovery and replication cohorts. Samples from African-American adults (aged ≥18 years) who were taking a stable maintenance dose of warfarin were obtained at International Warfarin Pharmacogenetics Consortium (IWPC) sites and the University of Alabama at Birmingham (Birmingham, AL, USA). Patients enrolled at IWPC sites but who were not used for discovery made up the independent replication cohort. All participants were genotyped. We did a stepwise conditional analysis, conditioning first for VKORC1 −1639G→A, followed by the composite genotype of CYP2C9*2 and CYP2C9*3. We prespecified a genome-wide significance threshold of p<5×10−8 in the discovery cohort and p<0·0038 in the replication cohort. Findings The discovery cohort contained 533 participants and the replication cohort 432 participants. After the prespecified conditioning in the discovery cohort, we identified an association between a novel single nucleotide polymorphism in the CYP2C cluster on chromosome 10 (rs12777823) and warfarin dose requirement that reached genome-wide significance (p=1·51×10−8). This association was confirmed in the replication cohort (p=5·04×10−5); analysis of the two cohorts together produced a p value of 4·5×10−12. Individuals heterozygous for the rs12777823 A allele need a dose reduction of 6·92 mg/week and those homozygous 9·34 mg/week. Regression analysis showed that the inclusion of rs12777823 significantly improves warfarin dose variability explained by the IWPC dosing algorithm (21% relative improvement). Interpretation A novel CYP2C single nucleotide polymorphism exerts a clinically relevant effect on warfarin dose in African Americans, independent of CYP2C9*2 and CYP2C9*3. Incorporation of this variant into pharmacogenetic dosing algorithms could improve warfarin dose prediction in this population. Funding National Institutes of Health, American Heart Association, Howard Hughes Medical Institute, Wisconsin Network for Health Research, and the Wellcome Trust.

β-Adrenergic Receptor Gene Polymorphisms and β-Blocker Treatment Outcomes in Hypertension

Numerous studies have demonstrated that β1‐ and β2‐adrenergic receptor gene (ADRB1 and ADRB2) variants influence cardiovascular risk and β‐blocker responses in hypertension and heart failure. We evaluated the relationship between ADRB1 and ADRB2 haplotypes, cardiovascular risk (death, nonfatal myocardial infarction (MI), and nonfatal stroke), and atenolol‐based vs. verapamil sustained‐release (SR)‐based antihypertensive therapy in 5,895 coronary artery disease (CAD) patients. After an average of 2.8 years, death rates were higher in patients carrying the ADRB1 Ser49‐Arg389 haplotype (hazard ratio (HR) 3.66, 95% confidence interval (95% CI) 1.68–7.99). This mortality risk was significant in patients randomly assigned to verapamil SR (HR 8.58, 95% CI 2.06–35.8) but not atenolol (HR 2.31, 95% CI 0.82–6.55), suggesting a protective role for the β‐blocker. ADRB2 haplotype associations were divergent within the treatment groups but did not remain significant after adjustment for multiple comparisons. ADRB1 haplotype variation is associated with mortality risk, and β‐blockers may be preferred in subgroups of patients defined by ADRB1 or ADRB2 polymorphisms.

Clinical pharmacogenetics implementation: Approaches, successes, and challenges

Current challenges exist to widespread clinical implementation of genomic medicine and pharmacogenetics. The University of Florida (UF) Health Personalized Medicine Program (PMP) is a pharmacist‐led, multidisciplinary initiative created in 2011 within the UF Clinical Translational Science Institute. Initial efforts focused on pharmacogenetics, with long‐term goals to include expansion to disease‐risk prediction and disease stratification. Herein we describe the processes for development of the program, the challenges that were encountered and the clinical acceptance by clinicians of the genomic medicine implementation. The initial clinical implementation of the UF PMP began in June 2012 and targeted clopidogrel use and the CYP2C19 genotype in patients undergoing left heart catheterization and percutaneous‐coronary intervention (PCI). After 1 year, 1,097 patients undergoing left heart catheterization were genotyped preemptively, and 291 of those underwent subsequent PCI. Genotype results were reported to the medical record for 100% of genotyped patients. Eighty patients who underwent PCI had an actionable genotype, with drug therapy changes implemented in 56 individuals. Average turnaround time from blood draw to genotype result entry in the medical record was 3.5 business days. Seven different third party payors, including Medicare, reimbursed for the test during the first month of billing, with an 85% reimbursement rate for outpatient claims that were submitted in the first month. These data highlight multiple levels of success in clinical implementation of genomic medicine.

Synergistic polymorphisms of β1 and α2C-adrenergic receptors and the influence on left ventricular ejection fraction response to β-blocker therapy in heart failure

Objectives The Arg389Gly polymorphism (Arg389Gly) in the &bgr;1-adrenergic receptor gene (ADRB1) has been associated with improvement in left-ventricular remodeling with &bgr;-blocker treatment. One study of risk for heart failure suggested a synergistic effect of ADRB1 Arg389Gly with the insertion/deletion polymorphism in the &agr;2C-adrenergic receptor gene (ADRA2C). We tested whether the ADRA2C insertion/deletion polymorphism was associated with &bgr;-blocker response in heart failure, either alone or in combination with the ADRB1Arg389Gly polymorphism. Methods Fifty-four &bgr;-blocker naive heart failure patients underwent echocardiography before and after 5–6 months of metoprolol CR/XL therapy. Multivariant linear regression modeling was performed to assess the impact of genotypes and other variables on changes in left-ventricular function in response to metoprolol therapy. Results Deletion carriers had a significantly greater negative chronotropic response. Predictors of the end of study ejection fraction were baseline ejection fraction, deletion carrier status and Arg389Arg genotype. Patients with Arg389Arg/Del-carrier status showed the greatest ejection fraction increase with metoprolol CR/XL. Adjusting for baseline ejection fraction, final S-metoprolol plasma concentration and race, final ejection fraction in patients with this genotype combination was significantly higher than all other genotype combination groups. Conclusion ADRB1 and ADRA2C polymorphisms synergistically influence the ejection fraction response to &bgr;-blocker therapy of heart failure patients.