Rhonda Porche-Sorbet

Laboratory and Clinical Outcomes of Pharmacogenetic vs. Clinical Protocols for Warfarin Initiation in Orthopedic Patients

Summary.  Background: Warfarin is commonly prescribed for prophylaxis and treatment of thromboembolism after orthopedic surgery. During warfarin initiation, out‐of‐range International Normalized Ratio (INR) values and adverse events are common. Methods: In orthopedic patients beginning warfarin therapy, we developed and prospectively validated pharmacogenetic and clinical dose refinement algorithms to revise the estimated therapeutic dose after 4 days of therapy. Results: The pharmacogenetic algorithm used the cytochrome P450 (CYP) 2C9 genotype, smoking status, peri‐operative blood loss, liver disease, INR values and dose history to predict the therapeutic dose. The R2 was 82% in a derivation cohort (n = 86) and 70% when used prospectively (n = 146). The R2 of the clinical algorithm that used INR values and dose history to predict the therapeutic dose was 57% in a derivation cohort (n = 178) and 48% in a prospective validation cohort (n = 146). In 1 month of prospective follow‐up, the percent time spent in the therapeutic range was 7% higher (95% CI: 2.7–11.7) in the pharmacogenetic cohort. The risk of a laboratory or clinical adverse event was also significantly reduced in the pharmacogenetic cohort (Hazard Ratio 0.54; 95% CI: 0.30–0.97). Conclusions: Warfarin dose adjustments that incorporate genotype and clinical variables available after four warfarin doses are accurate. In this non‐randomized, prospective study, pharmacogenetic dose refinements were associated with more time spent in the therapeutic range and fewer laboratory or clinical adverse events. To facilitate gene‐guided warfarin dosing we created a non‐profit website, http://www.WarfarinDosing.org.

Expression of the Regenerating Gene Family in Inflammatory Bowel Disease Mucosa: Reg Iα Upregulation, Processing, and Antiapoptotic Activity

Background The pathophysiology of inflammatory bowel disease (IBD) reflects a balance between mucosal injury related to an ongoing inflammatory process and mucosal reparative mechanisms. Proreparative mucosal factors may offer new therapeutic paradigms. Transcriptional profiling can be applied to identify candidate gene products involved in colonic mucosal regeneration. Methods Resection specimens from patients who underwent colonic resection for IBD or non-IBD indications were analyzed by performing Affymetrix GeneChip hybridization (Affymetrix, Inc., Santa Clara, Calif) and histopathologic scoring. Expression and physiologic processing of Reg Iα, the most highly expressed member of the regenerating (Reg) gene family, was further studied by performing specific immunohistochemistry, protein sequencing, and mass spectroscopy. Results Foregut-derived tissues normally express human Reg proteins with minimal expression in the colon. In the setting of tissue injury associated with IBD, Reg Iα, Reg Iβ, and Reg III mRNA were highly expressed in colonic mucosa. Paired histopathologic scoring demonstrated that Reg expression was not related to the presence or the degree of mucosal inflammation. Studies of the Reg Iα protein revealed evidence of proteolytic cleavage at the N-terminus. In IBD, intact Reg Iα protein was expressed by the metaplastic Paneth granular cell population. Whereas Reg Iα cleaved at the N-terminus, it was also deposited throughout the lamina propria. Reg Iα treatment was shown to reduce epithelial apoptosis that occurred in response to treatment with hydrogen peroxide. Conclusion Ectopic expression, physiologic processing, and directed tissue deposition of Reg Iα are components of the colonic mucosal regenerative response in IBD. Reg Iα may serve to reduce epithelial apoptosis in inflammation.

A Polymorphism in the VKORC1 Regulator Calumenin Predicts Higher Warfarin Dose Requirements in African Americans

Warfarin demonstrates a wide interindividual variability in response that is mediated partly by variants in cytochrome P450 2C9 (CYP2C9) and vitamin K 2,3‐epoxide reductase complex subunit 1 (VKORC1). It is not known whether variants in calumenin (CALU) (vitamin K reductase regulator) have an influence on warfarin dose requirements. We resequenced CALU regions in a discovery cohort of dose outliers: patients with high (>90th percentile, n = 55) or low (<10th percentile, n = 53) warfarin dose requirements (after accounting for known genetic and nongenetic variables). One CALU variant, rs339097, was associated with high doses (P = 0.01). We validated this variant as a predictor of higher warfarin doses in two replication cohorts: (i) 496 patients of mixed ethnicity and (ii) 194 African‐American patients. The G allele of rs339097 (the allele frequency was 0.14 in African Americans and 0.002 in Caucasians) was associated with the requirement for a 14.5% (SD ± 7%) higher therapeutic dose (P = 0.03) in the first replication cohort and a higher‐than‐predicted dose in the second replication cohort (allele frequency 0.14, one‐sided P = 0.03). CALU rs339097 A>G is associated with higher warfarin dose requirements, independent of known genetic and nongenetic predictors of warfarin dose in African Americans.

Performance of Commercial Platforms for Rapid Genotyping of Polymorphisms Affecting Warfarin Dose

Initiation of warfarin therapy is associated with bleeding owing to its narrow therapeutic window and unpredictable therapeutic dose. Pharmacogenetic-based dosing algorithms can improve accuracy of initial warfarin dosing but require rapid genotyping for cytochrome P-450 2C9 (CYP2C9) *2 and *3 single nucleotide polymorphisms (SNPs) and a vitamin K epoxide reductase (VKORC1) SNP. We evaluated 4 commercial systems: INFINITI analyzer (AutoGenomics, Carlsbad, CA), Invader assay (Third Wave Technologies, Madison, WI), Tag-It Mutation Detection assay (Luminex Molecular Diagnostics, formerly Tm Bioscience, Toronto, Canada), and Pyrosequencing (Biotage, Uppsala, Sweden). We genotyped 112 DNA samples and resolved any discrepancies with bidirectional sequencing. The INFINITI analyzer was 100% accurate for all SNPs and required 8 hours. Invader and Tag-It were 100% accurate for CYP2C9 SNPs, 99% accurate for VKORC1 -1639/3673 SNP, and required 3 hours and 8 hours, respectively. Pyrosequencing was 99% accurate for CYP2C9 *2, 100% accurate for CYP2C9 *3, and 100% accurate for VKORC1 and required 4 hours. Current commercial platforms provide accurate and rapid genotypes for pharmacogenetic dosing during initiation of warfarin therapy.

Population variation in VKORC1 haplotype structure.

Recently, improved prediction of maintenance warfarin dose has been linked to single nucleotide polymorphisms (SNPs) in the vitamin K epoxide reductase complex subunit 1 gene (VKORC1) [1–3]. In addition to the individual SNPs, haplotype-dependent predictions for warfarin dosing have been described [1]. The high-dose and low-dose haplotypes can be identified in patients by screening a small number of polymorphisms (haplotype tag SNPs) [1]. Previously, factors such as CYP2C9 genotype have also been described as predictors for warfarin dose [4,5]. VKORC1 haplotype accounts for 21–25% of the variation in warfarin dose, and adding CYP2C9*2 and *3 genotypes improved the prediction model to 31% [4]. As warfarin has a narrow therapeutic index, and overor under-dosing can be lifethreatening, screening of VKORC1 and CYP2C9 prior to initiating therapy are likely to provide essential information for improving dose selection and more cost effective monitoring strategies. Population differences of VKORC1 haplotype and CYP2C9 genotype frequencies have also been suggested from initial studies [1,4,6]. We identified the frequency of genotype combinations across world populations of the four haplotype tag SNPs for VKORC1 described by Reider et al. [1] (861, 5808, 6853, and 9041) and CYP2C9 (*2 and *3) using Pyrosequencing [1,4] in 556 unrelated healthy individuals from the following populations: European–American, African, African–American, Hispanic (Mexican and Peruvian) and Asian (Table 1). Using previously defined criteria to determine high and low dose VKORC1 haplotype groups [1] and incorporating dose-limiting CYP2C9 genetic variation [4], Asian and Caucasian populations had the highest incidence (86% and 55%, respectively) of low dose individuals from either VKORC1 or CYP2C9 variants (from at least one VKORC1 haplotype A or CYP2C9 variant allele). Eighteen percent of the Caucasian population had a combination of at least one VKORC1 haplotype A and at least one CYP2C9 variant, whereas CYP2C9 variant alleles were rare in other populations. In addition, haplotypes of unknown function were prominent in populations of African origin (up to 28%; Table 1). Assessment of the functional consequence of haplotypes not belonging to groups A or B in African populations is warranted. These data are consistent with the observations that Asian patients require a lower average maintenance warfarin dose, and African–Americans a higher average dose to obtain a therapeutic international normalized ratio (INR). However, the low dose requirements for Hispanic patients compared with Caucasian patients are not explained by these variants [7]. With the current focus on utilizing the individual patient’s genetic profile for treatment selection [1–4], care should be taken to identify the predictive SNPs and/or haplotypes in all populations when proposing pharmacogenetic-based dosing regimens for initiation of oral anticoagulation therapy with warfarin.

Gamma-glutamyl carboxylase and its influence on warfarin dose

Via generation of vitamin K-dependent proteins, gamma-glutamyl carboxylase (GGCX) plays a critical role in the vitamin K cycle. Single nucleotide polymorphisms (SNPs) in GGCX, therefore, may affect dosing of the vitamin K antagonist, warfarin. In a multi-centered, cross-sectional study of 985 patients prescribed warfarin therapy, we genotyped for two GGCX SNPs (rs11676382 and rs12714145) and quantified their relationship to therapeutic dose. GGCX rs11676382 was a significant (p=0.03) predictor of residual dosing error and was associated with a 6.1% reduction in warfarin dose (95% CI: 0.6%-11.4%) per G allele. The prevalence was 14.1% in our predominantly (78%) Caucasian cohort, but the overall contribution to dosing accuracy was modest (partial R2 = 0.2%). GGCX rs12714145 was not a significant predictor of therapeutic dose (p = 0.26). GGCX rs11676382 is a statistically significant predictor of warfarin dose, but the clinical relevance is modest. Given the potentially low marginal cost of adding this SNP to existing genotyping platforms, we have modified our non-profit website (www.WarfarinDosing.org) to accommodate knowledge of this variant.

Effect of Genotype-Guided Warfarin Dosing on Clinical Events and Anticoagulation Control Among Patients Undergoing Hip or Knee Arthroplasty: The GIFT Randomized Clinical Trial

Importance Warfarin use accounts for more medication-related emergency department visits among older patients than any other drug. Whether genotype-guided warfarin dosing can prevent these adverse events is unknown. Objective To determine whether genotype-guided dosing improves the safety of warfarin initiation. Design, Setting, and Patients The randomized clinical Genetic Informatics Trial (GIFT) of Warfarin to Prevent Deep Vein Thrombosis included patients aged 65 years or older initiating warfarin for elective hip or knee arthroplasty and was conducted at 6 US medical centers. Enrollment began in April 2011 and follow-up concluded in October 2016. Interventions Patients were genotyped for the following polymorphisms: VKORC1-1639G>A, CYP2C9*2, CYP2C9*3, and CYP4F2 V433M. In a 2 × 2 factorial design, patients were randomized to genotype-guided (n = 831) or clinically guided (n = 819) warfarin dosing on days 1 through 11 of therapy and to a target international normalized ratio (INR) of either 1.8 or 2.5. The recommended doses of warfarin were open label, but the patients and clinicians were blinded to study group assignment. Main Outcomes and Measures The primary end point was the composite of major bleeding, INR of 4 or greater, venous thromboembolism, or death. Patients underwent a screening lower-extremity duplex ultrasound approximately 1 month after arthroplasty. Results Among 1650 randomized patients (mean age, 72.1 years [SD, 5.4 years]; 63.6% women; 91.0% white), 1597 (96.8%) received at least 1 dose of warfarin therapy and completed the trial (n = 808 in genotype-guided group vs n = 789 in clinically guided group). A total of 87 patients (10.8%) in the genotype-guided group vs 116 patients (14.7%) in the clinically guided warfarin dosing group met at least 1 of the end points (absolute difference, 3.9% [95% CI, 0.7%-7.2%], P = .02; relative rate [RR], 0.73 [95% CI, 0.56-0.95]). The numbers of individual events in the genotype-guided group vs the clinically guided group were 2 vs 8 for major bleeding (RR, 0.24; 95% CI, 0.05-1.15), 56 vs 77 for INR of 4 or greater (RR, 0.71; 95% CI, 0.51-0.99), 33 vs 38 for venous thromboembolism (RR, 0.85; 95% CI, 0.54-1.34), and there were no deaths. Conclusions and Relevance Among patients undergoing elective hip or knee arthroplasty and treated with perioperative warfarin, genotype-guided warfarin dosing, compared with clinically guided dosing, reduced the combined risk of major bleeding, INR of 4 or greater, venous thromboembolism, or death. Further research is needed to determine the cost-effectiveness of personalized warfarin dosing. Trial Registration clinicaltrials.gov Identifier: NCT01006733

Definition of a factor Va binding site in factor Xa.

We reported previously that residue 347 in activated fX (fXa) contributes to binding of the cofactor, factor Va (fVa) (Rudolph, A. E., Porche-Sorbet, R. and Miletich, J. P. (2000) Biochemistry 39, 2861–2867). Four additional residues that participate in fVa binding have now been identified by mutagenesis. All five resulting fX species, fXR306A, fXE310N, fXR347N, fXK351A, and fXK414A, are activated and inhibited normally. However, the rate of inhibition by antithrombin III in the presence of submaximal concentrations of heparin is reduced for all the enzymes. In the absence of fVa, all of the enzymes bind and activate prothrombin similarly except fXaE310N, which has a reduced apparent affinity (∼3-fold) for prothrombin compared with wild type fXa (fXaWT). In the absence of phospholipid, fVa enhances the catalytic activity of fXaWT significantly, but the response of the variant enzymes was greatly diminished. On addition of 100 nm PC:PS (3:1) vesicles, fVa enhanced fXaWT, fXaR306A, and fXaE310N similarly, whereas fXaR347N, fXaK351A, and fXaK414Ademonstrated near-normal catalytic activity but reduced apparent affinity for fVa under these conditions. All enzymes function similarly to fXaWT on activated platelets, which provide saturating fVa on an ideal surface. Loss of binding affinity for fVa as a result of the substitutions in residues Arg-347, Lys-351, and Lys-414 was verified by a competition binding assay. Thus, Arg-347, Lys-351, and Lys-414 are likely part of a core fVa binding site, whereas Arg-306 and Glu-310 serve a less critical role.

Factor XSt. Louis II IDENTIFICATION OF A GLYCINE SUBSTITUTION AT RESIDUE 7 AND CHARACTERIZATION OF THE RECOMBINANT PROTEIN

A molecular defect in factor X (fX) results from a point mutation that causes glycine substitution for γ-carboxylated glutamic acid at position 7. The variant (fXSt. Louis II) and wild type (fXWT) proteins were produced in a mammalian expression system and characterized. fXSt. Louis II has <1% and ∼3% of normal clotting activity in modified prothrombin time and partial thromboplastin time assays, respectively. The rate of activation of fXSt. Louis II by factor VIIa and tissue factor is undetectable under conditions that result in complete activation of fXWT; activation by factors VIIIa and IXa is ∼30% of normal activation. The X-activating protein from Russells viper venom activates fXSt. Louis II completely but at a reduced rate. Thrombin generation on phoshopolipid vesicles or activated platelets is ∼30% or ∼5%, respectively. Membrane-dependent autolysis is markedly reduced for fXSt. Louis II. In reactions that are not surface-dependent, fXSt. Louis II is nearly identical to that of fXWT. The rate of inhibition by antithrombin is indistiguishable, as is the rate of thrombin formation in the absence of phospholipid, with or without factor Va.

Heparin and Oversulfated Heparin Byproduct Induce Thrombin Generation Through Contact System Activation in Plasma of Patients With HIT

Heparin-induced thrombocytopenia with thrombosis (HITT) is the most severe side effect of heparin administration. Patients with HITT may die or have permanent sequelae such as stroke or limb amputation. Contaminated heparin is associated with anaphylactic reactions and deaths by activating the contact system. It is also associated with high incidence of HIT via a yet unknown mechanism. This study showed that although oversulfated heparin byproduct induced thrombin activities in both normal and HIT patient plasmas through the contact system activation, authentic heparin induced thrombin activities only in HIT patient plasmas containing autoantibodies against protein/ heparin complex. These data suggest that the negatively charged immunoglobulin G (IgG)/platelet factor 4 (PF4)/heparin complex activate the contact system and produce thrombin in human plasma, and thrombin partially activates the platelets allowing subsequent platelet activation through IgG/Fc receptor II signaling. The newly discovered mechanism of heparin-induced thrombin activity could explain the increased incidence of HIT in patients exposed to contaminated heparin. Furthermore, the assays used in these studies would be valuable for HIT diagnosis, prevention, and treatment.