Are Prothrombotic Mutations a Time-to-Event Risk Factor?

BACKGROUND: Deep vein thrombosis (DVT) represents a common disorder involving genetic and acquired risk factors. It has been proposed that acquired risk factors are more important with aging than genetic factors, indicating different prevalence of prothrombotic mutations throughout the lifespan. OBJECTIVE: To determine the role of the most frequent prothrombotic genetic risk factors (Factor V [FV] Leiden and Factor II [FII] G20210A mutations) in first-time DVT etiology in patients of different ages. METHOD: This retrospective study included 701 patients living in Serbia with diagnosed DVT as a first-time thrombotic event. RESULTS: Risk assessment for mutations as age-related markers showed no statistical difference (FV Leiden mutation-OR, 1.027; 95% confidence interval [CI], .87-1.22; P = .76 and FII G20210A mutation-OR, 0.940, 95% CI, .74-1.19; P = .61). Our results show similar mutation prevalence regardless of how old the patients were at the time of the first DVT occurrence. CONCLUSION: Our results indicate that these 2 mutations cannot be used as prognostic marker for time-to-event first DVT in the Serbian population; however, further studies are required.

Economic analysis of pharmacogenomic-guided clopidogrel treatment in Serbian patients with myocardial infarction undergoing primary percutaneous coronary intervention.

INTRODUCTION: Clopidogrel, which is activated by the CYP2C19 enzyme, is among the drugs for which all major regulatory agencies recommend genetic testing to be performed to identify a patient's CYP2C19 genotype in order to determine the optimal antiplatelet therapeutic scheme. The CYP2C19*2 and CYP2C19*3 variants are loss-of-function alleles, leading to abolished CYP2C19 function and thus have the risk of thrombotic events for carriers of these alleles on standard dosages, while the CYP2C19*17 allele results in CYP2C19 hyperactivity. AIMS: Here, we report our findings from a retrospective study to assess whether genotyping for the CYP2C19*2 allele was cost effective for myocardial infarction patients receiving clopidogrel treatment in the Serbian population compared with the nongenotype-guided treatment. RESULTS: We found that 59.3% of the CYP2C19*1/*1 patients had a minor or major bleeding event versus 42.85% of the CYP2C19*1/*2 and *2/*2, while a reinfarction event occurred only in 2.3% of the CYP21C9*1/*1 patients, compared with 11.2% of the CYP2C19*1/*2 and CYP2C19*2/*2 patients. There were subtle differences between the two patient groups, as far as the duration of hospitalization and rehabilitation is concerned, in favor of the CYP2C19*1/*1 group. The mean cost for the CYP2C19*1/*1 patients was estimated at €2547 versus €2799 in the CYP2C19*1/*2 and CYP2C19*2/*2 patients. Furthermore, based on the overall CYP2C19*1/*2 genotype frequencies in the Serbian population, a break-even point analysis indicated that performing the genetic test prior to drug prescription represents a cost-saving option, saving €13 per person on average. CONCLUSION: Overall, our data demonstrate that pharmacogenomics-guided clopidogrel treatment may represent a cost-saving approach for the management of myocardial infarction patients undergoing primary percutaneous coronary intervention in Serbia.

Thrombin generation, D-dimer and protein S in uncomplicated pregnancy.

BACKGROUND: Gestational age-specific reference values are essential for the accurate interpretation of haemostatic tests during pregnancy. METHODS: Our 1-year prospective study included 40 healthy pregnant women with a median age of 30 (range 22-40) years; the subjects were followed in order to establish the gestational age dependent values for endogenous thrombin potential (ETP), D-dimer and protein S (activity and free). RESULTS: During the first trimester 50% of studied women had ETP >100% (reference values out of pregnancy); in the second trimester an ETP over 100% was observed in all women; ETP values remained unchanged during the third trimester. In the first trimester, the median D-dimer concentration of 0.30 mg/L, in the second 0.91 mg/L and in the third of 1.45 mg/L were observed. During the first trimester 14/40 subjects had protein S activity below reference range (<59%, out of pregnancy); the median value of 61.35; interquartile range (IQR) 20.38; in the second 21/37; the median value of 53.1 (IQR 15.65); in the third trimester 28/37 had low level of protein S activity with the median value of 49.0 (IQR 18.8). Free protein S showed a slight decrease from the first trimester; it remained almost stable during the rest of pregnancy, with the equal number of pregnant women with reduced free protein S. CONCLUSIONS: Related to the gestational age, a significant increase of ETP and D-dimer, from the second trimester was observed; the decrease of protein S was observed already from the early pregnancy, with more pronounced variability of protein S activity.

Risk factors for thrombosis in Serbian children.

Venous and arterial thromboses are increasingly encountered in the pediatric population. We present results of a case-control study of inherited and acquired risk factors for thrombosis in 129 pediatric patients from the first day of life to 18 years. The aims of study were to determine the importance of thrombophilic risk factors and comorbidity as a cause of thrombosis in children. Single thrombophilic risk factor was found in 24.4% (n = 21), whereas combined thrombophilic factors were found in 15.1% (n = 13) patients. A total of 87.2% of the children had recognized thrombophilic risk factors for thrombosis and/or additional comorbid risk factors. The single independent risk factors for thrombosis were mutation of factor V Leiden (P = 0.021), lupus anticoagulant antibodies (P = 0.028), and comorbidity (P = 0.000). Mutation of factor V Leiden [odds ratio (OR), 6.2 (95% confidence interval, CI 1.1-38.1, P = 0.048] was found to be a risk factor for venous thrombosis. Lupus anticoagulant antibodies were related to both venous (P = 0.008) and arterial thrombosis (P = 0.016). The frequency of inherited thrombophilic factors were the same in neonates and adolescents (23%). The prothrombotic gene mutations were present in 18.6% (n = 8) of asymptomatic children. Our study confirms that thrombosis in children is a multifactorial disorder, and associated most with the underlying medical disease (comorbidity) for vein thrombosis [OR, 18.6 (95% CI 3.7-93.4), P = 0.000] and for arterial thrombosis [OR, 10.5 (95% CI 2.2-49.9) P = 0.003]. Inherited thrombophilic disorders contributed to the development of thrombosis in children.

Rationalized DNA sequencing-based protocol for genotyping patients receiving coumarin therapy.

During the last decade genetic factors affecting coumarin therapy have been extensively investigated. The most important genes appear to be CYP2C9 and VKORC1, and different studies have shown that DNA testing can dramatically improve the safety and effectiveness of the therapy. However, the implementation of pharmacogenetic testing in everyday practice is still not a reality. Facilities and ability to get results before the start of therapy are very important. The implementation of specific methodology and equipment for particular type of diagnostics can represent a serious, even impossible, financial hurdle to overcome (especially in developing countries). For this reason, the use of every tool that contributes to rationalization of the existing methods can be a considerable asset. Therefore, we set the goal to rationalize our current DNA sequencing based protocol for analysis of the VKORC1 c.-1639G> A, CYP2C9*2 and CYP2C9*3 variant alleles, in order to obtain shorter and easier procedure. Simplification of the protocol was achieved by setting up multiplex PCR and omitting DNA extraction. This rationalization of the existing DNA sequencing based procedure allows getting results in 12 hours. The new protocol was tested on 118 samples. Obtained results have shown full accordance to those obtained with previous, non-modified protocol. Therefore, given the circumstances, we consider that protocol for pharmocogenetic testing should be made more accessible - both to doctors and patients. It is one of the prerequisites in order to make genotyping prior to the therapy common practice.

Pharmacogenetic tests could be helpful in predicting of VKA maintenance dose in elderly patients at treatment initiation.

Several studies have linked mutations in the genes encoding cytochrome P450 2C9 (CYP2C9) and vitamin K epoxide reductase complex 1 (VKORC1) to a reduced VKA dose requirement and an increased risk of bleeding. Nevertheless, implementation of genotyping as a routine practice is still controversial. Our study was conducted in order to investigate the impact of genetic factors, presence of VKORC1-c.1639A, CYP2C*2 and CYP2C*3 among outpatients referred to Anticoagulation Service due to extremely unstable anticoagulant therapy. From 2008 to 2011, 68 patients, mean age 65.9, were included in the study. They were referred from primary care physicians due to inability to sustain the therapeutic range in the period of initiation of anticoagulant therapy. Genotyping results showed that 17 (25%) of them were carriers of both CYP2C9 and VKORC1 variant alleles, 38 (55.9%) were carriers of VKORC1 c.1639AA, 6 (8.8%) were carriers of CYP2C9 variant alleles, while 7 (10.3%) of them were carriers of wild type alleles. INR control upon admission showed that 34 (50%) of them were over-anticoagulated, while 12 (17.6%) of them had subsequent bleeding complications. Among over-anticoagulated patients, 32 were carriers of mutated alleles in both CYP2C9 and VKORC1 gene or VKORC1 alone, while 2 of patients carried wild type alleles. In addition to presence of CYP2C9 or VKORC 1 alleles, older age was an important factor related to a lower dose and risk for over-anticoagulation. Genotype (CYP2C9/VKORC1) and age are the most important factors that could predispose an extreme response and subsequent bleeding complications during the initiation of VKA.

Extreme sensitivity to acenocoumarol therapy in patient with both VKORC.-1639 A/A and CYP2C9*1/*3 genotypes.

The initiation phase of vitamin K antagonist (VKA) is a challenging and demanding process that can result in adverse event such as bleeding. Dosing is influenced by a variety of acquired factors, while another factor that is associated with the optimal dose is the presence of certain genetic variants. We describe a 73 years old male who required extremely low dose of acenocoumarol (0.33 mg/day) to reach the target INR of 2-2.5. During initiation of VKA he started with usually recommended doses of acenocoumarol: 4 mg/day for the first 2 days and 3 mg/day during next 2 days. On fifth day of initiation, massive haematuria occurred and INR level of 10.5 was recorded. Patient was transfused with three doses of fresh frozen plasma in order to stop the bleeding. Acenocoumarol dose was gradually reduced after every INR laboratory monitoring and finally a dose of 0.33 mg/day was required for maintain stable anticoagulation. The genotyping results performed after introducing of VKA showed the patient was homozygous for vitamin K epoxide reductase (VKORC1) c.-1639 G>A, and heterozygous for cytochrome P450 2C9 (CYP2C9)*3 which pointed to extreme sensitivity to acenocoumarol. Our case supports the need for prospective genotyping of CYP2C9 and VKORC1 prior to initiation of VKA where pharmacogenetics, as a good predictor of extreme sensitivity to VKA, could help to tailor optimal VKA dose.

The c.-1639G>A polymorphism of the VKORC1 gene in Serbian population: retrospective study of the variability in response to oral anticoagulant therapy.

A single nucleotide polymorphism c.-1639G>A in the promoter region of vitamin K-epoxide reductase (VKORC1) gene has been found to account for most of the variability in response to oral vitamin K antagonist (VKA). Our aim was to study the effect of c.-1639G>A polymorphism on the acenocoumarol dosage requirements in a group of patients under stable anticoagulation, and to estimate the variability in response to VKA. We conducted a retrospective cohort analysis of 200 stable anticoagulation patients followed from the initiation of VKA. Out of 43 low-dose patients, 40 (93%) carried the A allele. The A allele was less frequent in the group of 30 patients requiring high VKA dose; among these patients 13 (43.3%) carried the A allele in the heterozygous form and none of them carried AA genotype. Patients with GG genotype required 2.6 times higher dose than patients carriers of AA genotype (P < 0.0001). Carriers of AA genotype were more likely to be overanticoagulated during follow-up after initiation of VKA when compared with carriers of the GA and GG genotypes (P < 0.0001). Patients with GG genotype spent more time below therapeutic range compared with patients carriers of AA (P = 0.0328) and GA genotype (P < 0.0001). VKORC1 c.-1639G>A polymorphism significantly influenced VKA dose and represented a good predictor of individuals predisposed to unstable anticoagulation. Pharmacogenetic testing could predict a high risk of overdose among 28.5% of our patients, carriers of AA genotype, before the initiation of anticoagulation.