A new pharmacogenetic algorithm to predict the most appropriate dosage of acenocoumarol for stable anticoagulation in a mixed Spanish population

This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.There is a strong association between genetic polymorphisms and the acenocoumarol dosage requirements. Genotyping the polymorphisms involved in the pharmacokinetics and pharmacodynamics of acenocoumarol before starting anticoagulant therapy would result in a better quality of life and a more efficient use of healthcare resources. The objective of this study is to develop a new algorithm that includes clinical and genetic variables to predict the most appropriate acenocoumarol dosage for stable anticoagulation in a wide range of patients. We recruited 685 patients from 2 Spanish hospitals and 1 primary healthcare center. We randomly chose 80% of the patients (n = 556), considering an equitable distribution of genotypes to form the generation cohort. The remaining 20% (n = 129) formed the validation cohort. Multiple linear regression was used to generate the algorithm using the acenocoumarol stable dosage as the dependent variable and the clinical and genotypic variables as the independent variables. The variables included in the algorithm were age, weight, amiodarone use, enzyme inducer status, international normalized ratio target range and the presence of CYP2C9∗2 (rs1799853), CYP2C9∗3 (rs1057910), VKORC1 (rs9923231) and CYP4F2 (rs2108622). The coefficient of determination (R2) explained by the algorithm was 52.8% in the generation cohort and 64% in the validation cohort. The following R2 values were evaluated by pathology: atrial fibrillation, 57.4%; valve replacement, 56.3%; and venous thromboembolic disease, 51.5%. When the patients were classified into 3 dosage groups according to the stable dosage (<11 mg/week, 11-21 mg/week, >21 mg/week), the percentage of correctly classified patients was higher in the intermediate group, whereas differences between pharmacogenetic and clinical algorithms increased in the extreme dosage groups. Our algorithm could improve acenocoumarol dosage selection for patients who will begin treatment with this drug, especially in extreme-dosage patients. The predictability of the pharmacogenetic algorithm did not vary significantly between diseases.This study was funded by a grant from the Spanish Ministry of Health and Social Policy (Instituto de Salud Carlos III, PI07/0710) and the Andalusian Regional Ministry of Health (Progress and Health Foundation, PI-0717-2013

Translational pharmacogenomics: a study of warfarin dosing

2014 - 2015Warfarin is one of the most commonly used oral anticoagulants worldwide and is highly efficacious for the treatment and prevention of thromboembolic disorders. However, due to its narrow therapeutic index, large inter-individual variability in dose requirements, and extensive drug and food interactions, warfarin remains a challenging drug to prescribe. Genetic factors (CYP2C9 and VKORC1), together with clinical factors (age and body weight), account for up to 60% of warfarin dose variance, whereas ~40% variability remains poorly understood. Several warfarin dosing algorithms, comprising genetic and non-genetic covariates have been published over the years. However, none of the published algorithms included patients from Southern Italy. We therefore conducted a candidate-gene study to develop an algorithm for predicting warfarin maintenance dose in patients from the Campania Region (n=266) in Southern Italy. Our pharmacogenetic dosing algorithm consisted of six variables (age, body surface area, amiodarone intake, CYP2C9*2, CYP2C9*3, and VKORC1 -1639G>A). It led to the accurate prediction of warfarin maintenance dose in 44% of patients (mean absolute error 7.41 mg/week). The prediction accuracy of the pharmacogenetic algorithm was superior to three previously published pharmacogenetic algorithms derived from patients in Northern and Central Italy. Given that previous studies suggested a role for miR-133a in warfarin response, we conducted a pilot study comparing baseline serum levels of miRNA in patients who achieved warfarin stable dose (n=10) to those who did not achieve warfarin stability (n=10), using the Affymetrix miRNA array. No association was found between miRNA-133a and warfarin response. Interestingly, circulating levels of miR-548a-3p were observed to be higher in patients who did not achieve warfarin stability (P=0.0053, fold change =1.66) compared to patients who achieved stable dose. In silico analyses showed that several target genes of miR-548a-3p are involved in the coagulation pathway. Work is currently underway to validate and replicate these findings in a larger cohort of prospectively recruited patients initiated onto warfarin therapy (n=980) using TaqMan miRNA real-time quantitative PCR. Pharmacogenetic algorithms have shown that common variants in CYP2C9 and VKORC1 genes cannot fully explain the extreme dose requirements in individuals sensitive and resistant to warfarin. To investigate the role of other genetic variants in these patients with extreme phenotypes, we performed a genome-wide association study (GWAS) comprising of warfarin sensitive patients (≤1.5 mg/day, n=55), warfarin resistant patients (≥10 mg/day, n=51), and healthy controls from the National Blood Service (NBS, n=2,501). Our results suggested that an intergenic variant on chromosome 10, rs4918797, could be involved in warfarin sensitivity. Intronic SNPs in MIR6873 on chromosome 6 (rs114213056) and PIGN on chromosome 18 (rs10163900, rs76455916, rs77118150, and rs79434376) showed suggestive association with warfarin resistance. The findings of this thesis showed that a multitude of factors affect warfarin dosing, some of which still need further investigations. Insights of the roles of other factors such as non-coding RNA and rare genetic variants will hopefully improve dose prediction and drug efficacy and ultimately patient outcomes. The work being undertaken with warfarin acts as a pathfinder project, the concepts from which could be applied to other drugs with variable dose requirement. [edited by author]XIV n.s

Profiling of warfarin pharmacokinetics‐associated genetic variants: Black Africans portray unique genetic markers important for an African specific warfarin pharmacogenetics‐dosing algorithm

Background: Warfarin dose variability observed in patients is attributed to variation in genes involved in the warfarin metabolic pathway. Genetic variation in CYP2C9 and VKORC1 has been the traditional focus in evaluating warfarin dose variability, with little focus on other genes. Objective: We set out to evaluate 27 single nucleotide polymorphisms (SNPs) in the CYP2C cluster loci and 8 genes (VKORC1, ABCB1, CYP2C9, CYP2C19, CYP2C8, CYP1A2, CYP3A4, and CYP3A5) involved in pharmacokinetics of warfarin. Patients/methods: 503 participants were recruited among black Africans and Mixed Ancestry population groups, from South Africa and Zimbabwe, and a blood sample taken for DNA. Clinical parameters were obtained from patient medical records, and these were correlated with genetic variation. Results: Among black Africans, the SNPs CYP2C rs12777823G>A, CYP2C9 c.449G>A (*8), CYP2C9 c.1003C>T (*11) and CYP2C8 c.805A>T (*2) were significantly associated with warfarin maintenance dose. Conversely, CYP2C9 c.430C>T (*2), CYP2C8 c.792C>G (*4) and VKORC1 g.-1639G>A were significantly associated with maintenance dose among the Mixed Ancestry. The presence of CYP2C8*2 and CYP3A5*6 alleles was associated with increased mean warfarin maintenance dose, whereas CYP2C9*8 allele was associated with reduced warfarin maintenance dose. Conclusion: African populations present with a diversity of variants that are important in predicting pharmacogenetics-based warfarin dosing in addition to those reported in CYP2C9 and VKORC1. It is therefore important, to include African populations in pharmacogenomics studies to be able to identify all possible biomarkers that are potential predictors for drug response

Individualización del tratamiento con acenocumarol: desarrollo de nuevos algoritmos farmacogenéticos y su validación en enfermedad tromboembólica venosa

Tesis doctoral inédita leída en la Universidad Autónoma de Madrid, Facultad de Medicina, Departamento de Cirugía. Fecha de lectura: 24-01-202

Acenocoumarol Pharmacogenetic Dosing Algorithm versus Usual Care in Patients with Venous Thromboembolism: A Randomised Clinical Trial

Patients with venous thromboembolism (VTE) require immediate treatment with anticoagulants such as acenocoumarol. This multicentre randomised clinical trial evaluated the effectiveness of a dosing pharmacogenetic algorithm versus a standard-of-care dose adjustment at the beginning of acenocoumarol treatment. We included 144 patients with VTE. On the day of recruitment, a blood sample was obtained for genotyping (CYP2C9*2, CYP2C9*3, VKORC1, CYP4F2, APOE). Dose adjustment was performed on day 3 or 4 after the start of treatment according to the assigned group and the follow-up was at 12 weeks. The principal variable was the percentage of patients with an international normalised ratio (INR) within the therapeutic range on day 7. Thirty-four (47.2%) patients had an INR within the therapeutic range at day 7 after the start of treatment in the genotype-guided group compared with 14 (21.9%) in the control group (p = 0.0023). There were no significant differences in the time to achieve a stable INR, the number of INRs within the range in the first 6 weeks and at the end of study. Our results suggest the use of a pharmacogenetic algorithm for patients with VTE could be useful in achieving target INR control in the first days of treatment

Learning Optimal Prescriptive Trees from Observational Data

We consider the problem of learning an optimal prescriptive tree (i.e., an interpretable treatment assignment policy in the form of a binary tree) of moderate depth, from observational data. This problem arises in numerous socially important domains such as public health and personalized medicine, where interpretable and data-driven interventions are sought based on data gathered in deployment -- through passive collection of data -- rather than from randomized trials. We propose a method for learning optimal prescriptive trees using mixed-integer optimization (MIO) technology. We show that under mild conditions our method is asymptotically exact in the sense that it converges to an optimal out-of-sample treatment assignment policy as the number of historical data samples tends to infinity. Contrary to existing literature, our approach: 1) does not require data to be randomized, 2) does not impose stringent assumptions on the learned trees, and 3) has the ability to model domain specific constraints. Through extensive computational experiments, we demonstrate that our asymptotic guarantees translate to significant performance improvements in finite samples, as well as showcase our uniquely flexible modeling power by incorporating budget and fairness constraints

Genetic determinants of response to aspirin and warfarin and development of silicon nanowire based genotyping

PhD ThesisChronic diseases such as cardiovascular diseases and colorectal cancer are the leading cause of mortality worldwide. Commonly used drugs such as aspirin and warfarin are shown to effective at reducing the risk of chronic diseases but have a narrow therapeutic window and are associated with adverse drug reactions, particularly, hemorrhage. Identification of pharmacogenetic markers such as single nucleotide polymorphisms (SNPs) that could help deliver personalized dose could help improve the risk-benefit ratio. Furthermore, development of a rapid point of care genotyping device consisting of a pharmacogenetic SNP panel for aspirin and warfarin could help implement personalized medicine in the clinical setting. Analysis of candidate SNPs in aspirin’s pharmacokinetic and pharmacodynamic pathways was carried out to explain variation in aspirin’s colorectal chemopreventive efficacy using two large population based case-control datasets. Associations and interactions were tested using logistic regression models and meta-analysis of the 2 datasets. A novel sitespecific association for rs1799853 (OR=0.73, 95% CI=0.60-0.90, P=0.003) and rs1105879 (OR=1.16, 95% CI=1.02-1.32, P=0.03) with colon cancer risk was observed. Furthermore, stratification by aspirin use showed increased risk of colorectal cancer in aspirin users but not in non-users carrying variant allele of the SNPs rs4936367 and rs7112513 in PAFAH1B2 gene and rs2070959 and rs1105879 in UGT1A6 gene (Pinteraction<0.05 for all). These results provide insight into aspirin’s differential chemopreventive efficacy and the neoplastic transformation of cells in colon and rectum. Utility of clinically validated pharmacogenetic dosing algorithms consisting of three warfarin dose associated SNPs from the European population needs to tested in the Gujarati Indians, an Indian sub-population. Dose prediction accuracy of the algorithms was compared between Gujarati Indian and European population. Mean squared difference of both pharmacogenetic algorithms was higher in Gujarati Indian compared to European population (Klein et al 2009, 216.3 v/s 160.7, P=0.05; Gage et al 2008, 170.6 v/s 143.2, P=0.07). Poor prediction accuracy could be explained by the presence of study subjects requiring dose for target INR range 2.5-3.5 and low frequency of the VKORC1 rs9923231 variant, which is the most important genetic determinant of warfarin dosing in Europeans. Therefore, the SNP panel and dosing algorithms developed from European populations cannot be assumed to have utility in the Gujarati Indian population. Finally, to help develop a rapid, point-of care, silicon nanowire (SiNW) based SNP genotyping device, a panel of isothermal melting probes were designed to genotype three warfarin dose associated SNPs. Testing of hybridization and washing conditions to have optimal hybridization kinetics between the probe and target DNA and high target sequence specificity was carried out using custom designed microarray platform. Accurate genotype calls for all 3 SNPs in 2 anonymised samples using empirically optimized hybridization and washing conditions was carried out successfully. Current work highlighted associations between probe characteristics and hybridization parameters, which would be useful in designing and testing probes on the SiNW platform. Identification, validation and testing of clinical utility of population specific pharmacogenetic markers along with development and deployment of ultra-rapid point of care genotyping technologies could help deliver personalized risk-benefit ratio for aspirin and warfarin

Warfarin e PEG-IFN/RBV: sviluppo di test farmacogenetici per la personalizzazione della terapia farmacologica

The aim of this study was the development and the validation of two pharmacogenetic tests for determination of the personalized dosage of drugs used in clinical settings (Warfarin and PEG-Interferone/Ribavirin) in which genetic testing is necessary and recommended. Warfarin (Coumadin®) is a widely prescribed anticoagulant used for treatment and prevention of thrombotic disorders. Although highly efficacious, Warfarin’s narrow therapeutic index and wide inter-individual variability in dose response make its dosing difficult. The standard procedure to define the personalized dose is an iterative process that can take weeks or months and exposes the patients to an increased risk of over- or underanticoagulation and thus to serious side effects like thromboembolism or bleeding. The anticoagulation effect of the Coumadin® drug therapy is influenced by combination of genetic and non-genetic factors , which account for more than half of the inter-individual variation in Warfarin doses required to achieve a therapeutic level of anticoagulation or therapeutic INR (Prothrombin International Normalized Ratio). Since 2011, the FDA requires the product label to include a recommended daily Warfarin dose (mg/day) for therapeutic INR, which is to be based on the genotypes of CYP2C9 and VKORC1. In addition, Genome-Wide Association Studies (GWAS) have confirmed that CYP2C9, VKORC1 and CYP4F2 are the main genetic factors determining the effective Warfarin dose in Caucasians. Therefore, knowledge of the genetic profile provides a means to assess the differential risk of adverse reactions and allows the personalization of the therapy, e.g. lowering the dosage for patients with the alleles CYP2C9 *2 and *3 or that are homozygous for the A-allele (A/A) of the rs9923231 polymorphism in the VKORC1 gene. The goal of this study was to develop an innovative genotyping system assessing the gene polymorphisms that mainly determine the clinical response to Warfarin. Four polymorphisms were investigated: rs9923231 in the VKORC1 gene, rs1799853 and rs1057910 in the CYP2C9 gene and rs2108622 in the CYP4F2 gene. To evaluate already available systems and decide on the method to be used for the test we performed a benchmarking of the tests present in the market. The genotyping is performed using a multiplex PCR in combination with a Reverse Line Blot hybridization assay (mPCR/RLB). To ensure specific amplification the test uses the SSP-PCR (Sequence Specific Primer PCR) approach. The high specificity of this PCR method was confirmed by direct sequencing. The test identifies and differentiates SNPs using ASO probes (Allele Specific Oligonucleotide) that recognize the different alleles. The hybrid formed between the amplified target DNA sequence and the complementary probes is detected by a colorimetric reaction using biotin-streptavidin and alkaline phosphatase. In the first part of the study several parameters like assay temperature, typology of hybridization and washing solutions, incubation time and probe design were analyzed in order to assess their influence on the assay’s performance. Next, the amplification protocol was defined and the conditions of the RLB assay were set up. The kit prototype was then validated on 125 samples, which had been previously genotyped with a standard method (TaqMan® Drug Metabolism Genotyping Assay, Life Technologies), and the analytical sensitivity of the assay was determined. All tested samples were accurately genotyped (100% diagnostic specificity and sensitivity) and the method efficiently worked in a broad range of DNA concentrations. Sensitivity tests demonstrated that the method developed in this study, provided reliable results within a DNA concentration range of 1 ng/reaction to 500 ng/reaction. The shelf life of the reagents was determined to be at least twelve months. In addition, the assay was shown to work on/with the following combinations of probe support materials (a nylon membrane and a nitrocellulose blotting membrane) and PCR master mixes (three commercial available master mixes). A software was developed that automates and facilitates interpretation of the RLB (strip) patterns. After completion, the system, GENEQUALITY AB WARFARIN TYPE cod. 04-74A-20 (AB ANALITICA), was notified to the Ministero della Salute and was CE IVD marked for commercialization in the European Community. As this system is the only assay on the market that analyzes the CYP4F2 gene in combination with the CYP2C9 and the VKORC1 gene, it represents the most complete approach for determining the genetic factors influencing Warfarin dosage in Caucasians. The Hepatitis C virus (HCV) infection is a major health problem with more than 170 million infected individuals worldwide (2-2.5% of the world’s population). Approximately 30% of the infections are cleared spontaneously, whereas 40% of the patients develop a chronic infection that can lead to cirrhosis, hepatocellular carcinoma and end-stage liver disease. The current standard of care is a combination treatment with pegylated interferon (PEG-INF) and Ribavirin (RBV), but its efficacy and tolerability are limited. Many efforts have been made to identify the factors that influence host resistance to HCV infection and treatment outcome. Several Genome-Wide Association Studies (GWAS) have demonstrated independently that variations in the IL28B (Interleukin 28B) gene are strongly associated with spontaneous clearance of the virus, early viral kinetics and with the varying PEG-INF/RBV therapy response in different populations. Particularly, polymorphism IL28B rs12979860 influences the prospect of recovery from an HCV infection. Patients homozygous for allele rs12979860-C show a two- to threefold higher SVR (Sustained Virological Response) rate compared to persons with a non-CC genotype when undergoing Peg-INF/RBV treatment, have a higher probability of spontaneous clearance of the virus and, during therapy, display a stronger reduction of virus RNA levels, which is reflected in higher RVR (Rapid Virological Response) and EVR (Early Virological Response) rates. Other studies reported an association of certain functional variants of the ITPA (Inosine Triphosphatase) protein with the reduction of hemoglobin during Peg-INF/RBV therapy. These allelic variants of the ITPA gene decrease the enzymatic activity of ITPA, thus protecting from RBV-induced anemia. The aim was to develop an innovative system to genotype the most significant genetic polymorphisms important for the prediction of treatment response and risk of adverse reactions in patients infected with HCV. Four polymorphisms were investigated in this study: rs12979860 and rs8099917 in the IL28B gene and rs7270101 and rs1127354 in the ITPA gene. Human genomic DNA was extracted from peripheral blood using an automated system (EZ1, Qiagen). Genotyping of the genetic variants was performed combining multiplex PCR with a Reverse Line Blot hybridization assay (mPCR/RLB). For amplification (PCR) of the target sequences, biotinylated primers were used. The primers were designed and analyzed in silico using bioinformatics alignment tools like “blat” (UCSC) and ClustalW, excluding genomic sequences that contain SNPs, SINEs/LINEs or other repetitive elements and all primer binding sites that show a sequence similarity of more than 95% with other genomic sequences. Due to the fact that a sequence that is highly homologous to the target region in IL28B gene is located on the same chromosome 15.000 bp upstream, the SSP-PCR approach was adopted in order to ensure a high specificity of the amplification. To evaluate the efficacy of the SPP-PCR, the amplification products were analyzed by direct sequencing. Subsequently, the protocol for the multiplex amplification was set up. Probes recognizing the different alleles of each investigated SNP (two ASO, Allele Specific Oligonucleotide, per SNP) were designed and their specificity analyzed under varying experimental conditions. This allowed estimation of the effect of parameters like temperature and time of incubation on the assay performance. The analytical sensitivity of the assay was determined using a range of DNA concentrations between 0.5 ng/µl and 200 ng/µl. The diagnostic specificity of the test was assessed analyzing 160 samples of a known genotype. The test provided a result within a DNA concentration of 1 ng/reaction to 400 ng/reaction and all tested samples were accurately genotyped. This underlines the reliability and specificity of the assay. The shelf life of the reagents was determined to be at least eight months and the compatibility of the assay with other amplification master mixes or probe supports (membranes) was tested. The system, GENEQUALITY IL28B-ITPA TYPE cod. 04-47A-20 (AB ANALITICA), was notified to the Ministero della Salute and CE IVD marked for commercialization in the European Community. RLB is a convenient way to identify up to 8 targets in 20 individual specimens simultaneously. It is more flexible and less costly than DNA microarrays while providing the same specificity and sensitivity. The system developed in this study, GENEQUALITY IL28B-ITPA TYPE, code 04-47A-20 (AB ANALITICA), allows analysis of the most important host genetic factors influencing the efficacy of the Peg-INF/RBV therapy and determining the risk of RBV-induced anemia. It is the only assay on the market that analyzes ITPA and IL28B polymorphisms together and therefore represents the most complete approach for determining the genetic constitution in regard to the IL28B and the ITPA gene for personalized management of patients with Hepatitis C. A genotype-phenotype correlation analysis was performed to evaluate the association between IL28B and ITPA polymorphisms and the therapy outcome in a sub-group of patients (N=52). Genetic analysis of the IL28B polymorphism rs12979860 revealed that 27% of the patients carried the C/C genotype, whereas 56% and 17% carried the C/T and the T/T genotype, respectively. The T/T genotype of the IL28B polymorphism rs8099917 was in 40% of patients, whereas 48% carried T/G and 12% the G/G. No association was found between variants of the IL28B polymorphisms and SVR rates: 57% of patients with IL28B rs12979860-CC and 52% with IL28B rs8099917-TT achieved SVR and 43% and 48%, respectively, failed to clear the HCV virus. For analysis of the influence of ITPA, the patients were subdivided into the following three groups: normal ITPase activity (100% ITPA), mild ITPase deficiency (60% ITPA- 60% ITPase activity) and moderate to severe ITPase deficiency (≤30% ITPA, ≤30% ITPase activity). These groups reflected the individual Composite ITPase Deficiency Variable based on the genotypes of the ITPA polymorphisms rs7270101 and rs1127354. Patients with normal ITPase activity were compared to the groups of patients with mild ITPase deficiency and moderate to severe ITPase deficiency by statistical analysis. The results showed that the predicted ITPase deficiency correlated to a reduction of median Hb levels at 4, 12 and 24 weeks of treatment. A statistically significant difference was observed between the groups with normal ITPase activity and moderate to severe ITPase deficiency (≤30% ITPA) at 4 and 24 weeks (Dunnett’s Multiple Comparison Test: T4, p < 0.0001; T24, p < 0.0451). This clearly shows that patients with an at least by 70% decreased ITPase activity are protected from anemia throughout the treatment