Prediction of phenprocoumon maintenance dose and phenprocoumon plasma concentration by genetic and non-genetic parameters.

PURPOSE: The anticoagulation response to vitamin K antagonists is characterised by high inter-individual variability. The impact of single nucleotide polymorphisms (SNPs) in several genes of enzymes involved in the vitamin K cycle on phenprocoumon dose variability and phenprocoumon plasma concentrations is still under investigation. METHODS: We assessed the influence of VKORC1 c.-1639G>A, CYP2C9*2, CYP2C9*3, CYP4F2 c.1297G>A, CALU c.*4A>G, EPHX1 c.337T>C, GGCX c.214+597G>A, F7 c.-402G>A, F7 c.-401G>T, PROC c.-228C>T and PROC c.-215G>A along with clinical and demographic parameters on steady-state phenprocoumon therapy in 75 patients. A prediction model was developed for total phenprocoumon plasma concentrations and daily phenprocoumon doses required for therapeutic anticoagulation. RESULTS: The VKORC1 c.-1639 genotype was the main predictor of the phenprocoumon daily dose (adjusted R(2) = 37.6%) and the total phenprocoumon concentration (adjusted R(2) = 38.3%). CYP2C9 affected the phenprocoumon concentration, but not the dose requirements. SNPs in the other genes of the vitamin K cycle, concomitant medication, nicotine use and alcohol consumption did not predict phenprocoumon concentrations and phenprocoumon dose requirements in a multiple linear regression model. Phenprocoumon concentrations were predicted by VKORC1 c.-1639, CYP2C9 genotype, age and BMI. The final prediction model for the daily phenprocoumon dose requirements comprised VKORC1 c.-1639 genotype, age and height accounting for 48.6% of the inter-individual variability. CONCLUSIONS: A rough prediction of phenprocoumon maintenance doses can be achieved by a limited set of parameters (VKORC1, age, height). The investigated SNPs in CYP4F2, CALU, EPHX1, GGCX, F7, and PROC did not improve the predictive value of a pharmacogenetic-based dosing equation for phenprocoumon.

VKORC1 haplotypes and their impact on the inter-individual and inter-ethnical variability of oral anticoagulation.

In order to elucidate the role of VCORC1 sequence variants in warfarin sensitivity, we established a complete SNP map of the VKORC1 gene locus in 200 blood donors from Western Germany. Nearly all of the genetic variability of the VKORC1 gene in Europeans is reflected by three main haplotypes. Recently described polymorphisms associated with low warfarin dose requirement (dbSNP:rs9934438; dbSNP:rs17878363) were found in complete linkage disequilibrium with the VKORC1*2 haplotype. In two patient cohorts of European origin with either increased coumarin sensitivity (n= 14) or partial coumarin resistance (n=36) the VKORC1*2 frequency varied highly significant between the two groups and also when compared to 200 blood donor controls (coumarin sensitive 96%, coumarin resistant 7%, controls 42%) thus demonstrating a strong association between these two phenotypes and the VKORC1 haplotype (p = 1.6 x 10(-8) for coumarin sensitive and p = 1.9 x 10(-8) for coumarin resistant). Analysis of database derived VKORC1 genotypes of African Americans and Chinese revealed that haplotype frequencies in these populations differ significantly from the European sample (for VKORC1*2: Europeans 42%, Chinese 95%, African Americans 14%). These observations suggest VKORC1 as principal genetic modulator of the ethnic differences in warfarin response. Since hereditary pharmacodynamic (VKORC1) and pharmacokinetic (CYP2C9) factors account for up to 50% of the inter-individual variability of the warfarin response, these genetic markers may serve as clinically relevant predictors of warfarin dosing in future studies.

Validation of a rapid and inexpensive allele-specific amplification (ASA)-PCR genotyping assay for vitamin K antagonist pharmacogenomics.

BACKGROUND: Variant alleles of vitamin K epoxide reductase complex subunit 1 gene (VKORC1), the target molecule of vitamin K antagonists, and of cytochrome P450 (CYP) 2C9, an enzyme involved in coumarin metabolism, affect the anticoagulant response of coumarins, which have a narrow therapeutic window. Genotyping for these variants allows for prediction of therapeutic drug doses. The discussion of the clinical role of genotype-guided coumarin dosing is ongoing. For pharmacogenetic information to be useful, results must be available quickly. METHODS: Here we report on the establishment of an allele-specific amplification (ASA)-PCR assay for the three most relevant polymorphisms for coumarin pharmacogenetics. The assay was validated against sequencing data on 100 random samples from Caucasian blood donors, incorporating all genotypes. Divergent results were confirmed by repeating the analysis with both methods. One hundred percent congruence with DNA sequencing was determined as the 'pass' criterion for the assay. RESULTS: The ASA-PCR assay reproducibly identified the three informative single nucleotide polymorphisms. Discrepancies between ASA-PCR and sequencing were clarified by retrospective analysis as being due to erroneous analysis or documentation. In summary, the congruence of sequencing and duplex ASA-PCR was 100%. CONCLUSION: ASA-PCR is significantly faster and less expensive than sequencing. We expect that pharmacogenetics-based dosing decisions may reduce the frequency of over- and undertreatment with vitamin K antagonists, especially during drug initiation, and thus improve patient safety.

"Disfibrinogenemia Jujuy", alteración asociada a trastornos hemorrágicos / "Dysfibrinogenemia Jujuy", associated to bleeding disorders / "Disfibrinogenemia Jujuy", alteração associada a distúrbios hemorrágicos

El objetivo del presente trabajo fue estudiar a una joven paciente con manifestaciones hemorrágicas, caracterizar su fibrina plasmática e identificar la posible alteración molecular del fibrinógeno de la paciente y sus familiares directos. Se diagnosticó una disfibrinogenemia en la paciente, su madre y el medio-hermano, ambos asintomáticos. En estos individuos, la formación y lisis de la fibrina plasmática difirieron de los controles. La fase lag prolongada indicó la liberación lenta y defectuosa de los fibrinopéptidos; la pendiente menor que la del control sugirió una polimerización dificultada. La DOMax inferior mostró una fibrina compuesta por fibras delgadas. La fibrinolisis inducida por estreptoquinasa resultó más rápida que la correspondiente control. La secuenciación del ADN reveló una deleción homocigota que condujo a la ausencia de la glicina 14 de la cadena Aa del fibrinógeno (AaGly14del según nomenclatura de http://www.geht.org/databaseang/fibrinogen). La madre y el medio hermano resultaron heterocigotas para la misma mutación. Esta alteración no descripta previamente, que se ha denominado fibrinógeno Jujuy, podría no interaccionar correctamente con el sitio activo de la trombina, provocando que los fibrinopéptidos se hidrolicen y liberen lentamente, originando fibras de fibrina más delgadas y degradables que las del control. Este mecanismo explicaría el sangrado moderado de la paciente.

The aim of this work was to study a young female with moderate bleeding symptoms, to characterize the plasma fibrin and to identify the possible molecular alteration in the fibrinogen of the patient and her family. A dysfibrinogenemia was diagnosed in the patient, the mother and the half-brother, both the latter asymptomatic. Kinetic parameters obtained from fibrin formation and lysis assays of the patient’s plasma samples were significantly different compared to the ones obtained with control plasma. A prolonged lag phase indicated slow and defective fibrinopeptide releases, whereas a minor slope suggested an impaired fibrin assembly. A lower ODMax revealed a fibrin network composed of thinner fibers. Fibrinolysis induced by streptokinase resulted faster than control. DNA sequencing showed a homozygous deletion leading to AaGly14del (according to http://www.geht.org/databaseang/fibrinogen). The mother and the half-brother resulted heterozygous for the same mutation. This previously undescribed alteration was named fibrinogen Jujuy. The mutate fibrinogen might not be correctly fixed to the active site of thrombin resulting in slow cleavage and release of fibrinopeptides, rendering thinner fibers, more susceptible to lysis than control. This mechanism may explain the moderate bleeding symptoms of the patient.

O objetivo do presente trabalho foi estudar uma jovem paciente com manifestações hemorrágicas, caracterizar sua fibrina plasmática e identificar a possível alteração molecular do fibrinogênio da paciente e seus familiares diretos. Foi diagnosticada uma disfibrinogenemia na paciente, sua mãe e o meio-irmão, ambos assintomáticos. Nestes indivíduos, a formação e lise da fibrina plasmática diferiram dos controles. A fase lag prolongada indicou a liberação lenta e defeituosa dos fibrinopeptídeos; a pendente menor que a do controle sugeriu uma polimerização dificultada. A DOMax inferior mostrou uma fibrina composta por fibras delgadas. A fibrinólise induzida por estreptoquinase resultou mais rápida que a correspondente controle. A sequenciação do DNA revelou uma deleção homozigótica que conduziu à ausência da glicina 14 da cadeia Aa do fibrinogênio (AaGly14del conforme nomenclatura de http://www.geht.org/databaseang/fibrinogen). A mãe e o meio irmão resultaram heterozigotos para a mesma mutação. Esta alteração não descrita previamente, que denominamos fibrinogênio Jujuy, poderia não interagir corretamente com o sítio ativo da trombina, provocando que os fibrinopeptídeos se hidrolisem e liberem lentamente, originando fibras de fibrina mais finas e degradáveis que as do controle. Este mecanismo explicaria o sangramento moderado da paciente.

Impact of pharmacokinetic (CYP2C9) and pharmacodynamic (VKORC1, F7, GGCX, CALU, EPHX1) gene variants on the initiation and maintenance phases of phenprocoumon therapy.

Compared to warfarin, little is known about the effect of pharmacogenomics on the inter-individual variability of phenprocoumon therapy. In a retrospective cohort study, we investigated the impact of VKORC1 c.-1639G>A; CYP2C9*2 , CYP2C9*3 ; GGCX c.214+597G>A; CALU c.*4A>G; EPHX1 c.337T>C; F7 c.-402G>A, and F7 c.-401G>T on the initiation (n=54) and maintenance phases (n=91) of phenprocoumon therapy. We assessed the following outcome parameters: time to stable international normalised ratio (INR), time to first supra-therapeutic INR, time out of INR range, probability of over-anticoagulation, number of anticoagulation clinic visits. During the initiation phase, homozygotes for the VKORC1 c.-1639 A and G alleles achieved stable INRs later (p<0.001), spent more time at supra-therapeutic INRs (p<0.001), had increased risks of over-anticoagulation (odds ratio 19.83, p=0.003 and 4.45, p=0.045, respectively), and had higher frequencies of anticoagulation clinic visits (p<0.001) compared to GA carriers. CYP2C9*2, *3 carriers reached stable INRs faster (p=0.024) with fewer anticoagulation clinic visits (p=0.001) than wild-type carriers. EPHX1 c.337 C carrier spent significantly more time above range in the initiation phase (p=0.023). GGCX , CALU , and F7 gene variants did not affect outcome parameters of the initiation phase and none of the genotypes had an impact on maintenance phase parameters. Compared to the VKORC1 genotype, early INR values were less informative in the prediction of outcome parameters such as time to stable INR and time above the INR range. Our study is limited by the retrospective study design with no standardised protocol in a usual care setting. Therefore, our findings should be validated in a larger, controlled prospective study.