DNA and RNA Molecules as a Foundation of Therapy Strategies for Treatment of Cardiovascular Diseases.

There has always been a tendency of medicine to take an individualised approach to treating patients, but the most significant advances were achieved through the methods of molecular biology, where the nucleic acids are in the limelight. Decades of research of molecular biology resulted in setting medicine on a completely new platform. The most significant current research is related to the possibilities that DNA and RNA analyses can offer in terms of more precise diagnostics and more subtle stratification of patients in order to identify patients for specific therapy treatments. Additionally, principles of structure and functioning of nucleic acids have become a motive for creating entirely new therapy strategies and an innovative generation of drugs. All this also applies to cardiovascular diseases (CVDs) which are the leading cause of mortality in developed countries. This review considers the most up-to-date achievements related to the use of translatory potential of DNA and RNA in treatment of cardiovascular diseases, and considers the challenges and prospects in this field. The foundations which allow the use of translatory potential are also presented. The first part of this review focuses on the potential of the DNA variants which impact conventional therapies and on the DNA variants which are starting points for designing new pharmacotherapeutics. The second part of this review considers the translatory potential of non-coding RNA molecules which can be used to formulate new generations of therapeutics for CVDs.

Pharmacogenetics of Clopidogrel Therapy and Neurointerventional Procedures: We Need Precision Data for Precision Medicine.

Genetics has given a real boost to personalized and precision medicine, providing data used either for precise diagnostics, prediction of the course of illness, or for selecting therapy and tailoring it. Pharmacogenetics, as a discipline researching connection between genetic background of an individual and the effect of a certain drug, has created new possibilities in medicine. One of the most researched drugs in pharmacogenetics is certainly clopidogrel.

Correction to: Analysis of the CYP2C19 genotype associated with bleeding in Serbian STEMI patients who have undergone primary PCI and treatment with clopidogrel.

The correct Author names are shown in this paper.

Analysis of the CYP2C19 genotype associated with bleeding in Serbian STEMI patients who have undergone primary PCI and treatment with clopidogrel.

PURPOSE: Bleeding is one of the possible adverse events during clopidogrel therapy. The CYP2C19 gene is the most significant genetic factor which influences response to clopidogrel treatment. We aimed to examine the contribution of the CYP2C19 gene to bleeding occurrence during clopidogrel therapy in Serbian patients with ST segment elevation myocardial infarction (STEMI) undergoing primary percutaneous coronary intervention (PCI). METHODS: This case-control study included 53 patients who experienced bleeding and 55 patients without bleeding. Bleeding events were defined and classified using the Bleeding Academic Research Consortium (BARC) criteria. All patients were prescribed daily doses of clopidogrel during the 1-year follow-up after PCI. The CYP2C19*17 (c.-806C>T, rs12248560), rs11568732 (c.-889T>G, CYP2C19*20), CYP2C19*2 (c.681G>A; rs4244285) and CYP2C19*3 (c.636G>A; rs4986893) variants were analysed in all 108 patients. Additionally, sequencing of all nine exons, 5'UTR and 3'UTR in the rs11568732 carriers was performed. RESULTS: Association between bleeding (BARC type ≥ 2) and the CYP2C19*17 variant was not observed [odds ratio (OR), 0.53; 95% confidence interval (CI), 0.2-1.1; p = 0.107). The rs11568732 variant showed significant association with bleeding (OR, 3.7; 95% CI, 1.12-12.44; p = 0.025). Also, we found that the rs11568732 variant appears independently of haplotype CYP2C19*3B, which is contrary to the previous findings. CONCLUSIONS: Our results indicate the absence of CYP2C19*17 influence and turn the attention to the potential significance of the rs11568732 variant in terms of adverse effects of clopidogrel. However, it is necessary to conduct an independent conformation study in order to verify this finding. Also, an analysis of the functional implication of the rs11568732 variant is necessary in order to confirm the significance of this variant, both in relation to its influence on gene expression and in relation to its medical significance.

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.

Clopidogrel High On-Treatment Platelet Reactivity in Patients with Carotid Artery Stenosis Undergoing Endarterectomy. A Pilot Study.

OBJECTIVES: A considerable number of patients do not achieve an adequate response to clopidogrel. Our study aimed to evaluate genetic and non-genetic factors as possible risks for clopidogrel high on-treatment platelet reactivity (HTPR) in patients (n=112) with carotid artery stenosis undergoing endarterectomy (CEA). METHODS: Using multiple-electrode impedance aggregometry (MEA) the antiplatelet effectiveness of clopidogrel was measured after 24 h, 7 and 30 days of clopidogrel treatment, which was introduced after elective CEA at a dose of 75 mg daily, for at least 30 days. RESULTS: HTPR was observed among 25% patients after clopidogrel therapy for 30 days. Further analysis showed that 53.3% of patients carrying the CYP2C19*2 gene variant had clopidogrel-HTPR, while in the wild type group there were 14.6% (p<0.001). Multivariate logistic regression analysis identified the CYP2C19*2 variant allele (OR 4.384; 95% CI 1.296-14.833, p=0.017) and high total cholesterol level (OR 2.090; 95% CI 1.263-3.459, p=0.004) as the only independent risk factors for clopidogrel-HTPR. CONCLUSION: The CYP2C19*2 gene variant and high total cholesterol level were major factors for clopidogrel- HTPR in patients with carotid artery stenosis undergoing CEA.

Differential expression and localization of Ankrd2 isoforms in human skeletal and cardiac muscles.

Four human Ankrd2 transcripts, reported in the Ensembl database, code for distinct protein isoforms (360, 333, 327 and 300 aa), and so far, their existence, specific expression and localization patterns have not been studied in detail. Ankrd2 is preferentially expressed in the slow fibers of skeletal muscle. It is found in both the nuclei and the cytoplasm of skeletal muscle cells, and its localization is prone to change during differentiation and upon stress. Ankrd2 has also been detected in the heart, in ventricular cardiomyocytes and in the intercalated disks (ICDs). The main objective of this study was to distinguish between the Ankrd2 isoforms and to determine the contribution of each one to the general profile of Ankrd2 expression in striated muscles. We demonstrated that the known expression and localization pattern of Ankrd2 in striated muscle can be attributed to the isoform of 333 aa which is dominant in both tissues, while the designated cardiac and canonical isoform of 360 aa was less expressed in both tissues. The 360 aa isoform has a distinct nuclear localization in human skeletal muscle, as well as in primary myoblasts and myotubes. In contrast to the isoform of 333 aa, it was not preferentially expressed in slow fibers and not localized to the ICDs of human cardiomyocytes. Regulation of the expression of both isoforms is achieved at the transcriptional level. Our results set the stage for investigation of the specific functions and interactions of the Ankrd2 isoforms in healthy and diseased human striated muscles.

Influence of Cyp2c19*2 Gene Variant on Therapeutic Response During Clopidogrel Treatment in Patients with Carotid Artery Stenosis.

BACKGROUND: Despite the proven clinical effect of oral antiplatelet drugs, a considerable number of patients do not have an adequate response to clopidogrel. The aim of our study was to determine the influence of CYP2C19*2 loss-of-function variant allele on clopidogrel responsiveness in patients with carotid artery stenosis. METHODS: One hundred and twelve patients with carotid artery stenosis undergoing endarterectomy were included in this one-year prospective study. All of them received clopidogrel (75 mg daily) for at least 30 days after the intervention. They were followed from the moment of hospital admission. CYP2C19*2 genotyping was performed by TaqMan Assay. The influence of CYP2C19*2 variant allele on clopidogrel platelet reactivity was determined using multiple-electrode aggregometry (MEA). RESULTS: Genotyping results showed that 82 (73.2%) patients were homozygous for wild type, 29 (25.9%) were heterozygous for the CYP2C19*2 allele and 1 (0.9%) was CYP2C19*2 homozygous. After 24 hours, among those with the wild type 29.3% were clopidogrel responders, and in those with the CYP2C19*2 alleles 10%. In the wild type group, 74.4% were clopidogrel responders after 7 days of taking the drug; 82.9% after 30 days of clopidogrel introduction, respectively. In patients with the CYP2C19*2 alleles the number of responders increased up to 46.7% after 7 days; 53.3% after 30 days of taking the drug, respectively. The risk for being a low-responder is higher for the patients heterozygous for the CYP2C19*2 allele vs. wild-type (OR 4.250, 95% CI 1.695-10.658, P<0.01). CONCLUSIONS: The CYP2C19*2 loss-of-function variant allele has significant influence on clopidogrel response in patients with carotid artery stenosis undergoing endarterectomy.

Cardiac transcription factor Nkx2.5 interacts with p53 and modulates its activity.

Transcription factor Nkx2.5, essential for heart development, regulates cardiomyocyte-specific gene expression through combinatorial interactions with other cardiac-restricted (GATA4 and dHAND) or ubiquitous (p300) transcription regulators. Here we demonstrate that Nkx2.5 and p53 synergistically activate the promoter of the striated muscle stress responsive transcriptional cofactor Ankrd2, involved in coordination of proliferation and apoptosis during myogenic differentiation. Moreover, the p53 protein is able to interact with both wild type Nkx2.5 and its mutant ΔNkx2.5 (aa 1-198) found in patients with diverse cardiac malformations. Nkx2.5 interaction site of p53 maps to the C terminal region, while p53 binding site on Nkx2.5 lies outside its C terminus. In addition, overexpression of Nkx2.5 has a modulatory, promoter dependent effect on p53 transactivation, while the mutant significantly abolished p53 activity on the Mdm2, p21(WAF1/CIP1) and Bax promoters. Their physical interaction contributes to the observed behavior in the case of the Mdm2 promoter. Our data provide a new evidence for the role of p53 in cardiac function through interaction with Nkx2.5.

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.

Does anticoagulant therapy improve pregnancy outcome equally, regardless of specific thrombophilia type?

The study was conducted to evaluate the effect of anticoagulant therapy in women with thrombophilia and to detect the possible differences among carriers of mutations (factor V [FV] Leiden and FIIG20210) and those with natural anticoagulant deficiency. The 4-year prospective investigation included 85 pregnant women, with a history of recurrent fetal loss (RFL). They were treated with prophylactic doses of low-molecular-weight heparin (nadroparin) starting from 6 to 8 weeks of gestation. Pregnancy outcomes were evaluated based on the thrombophilia type. Carriers of thrombophilic mutations had a live birth rate of 93%, compared to 41.6% for women with natural anticoagulant deficiencies. Significant differences between the groups were also observed for intrauterine fetal death, intrauterine growth restriction, and postpartum thrombosis. The optimal therapy for women with natural anticoagulant deficiency and RFL remains unclear and future prospective study with a large number of patients is required to determine the best treatment for these severe thrombophilic conditions.

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.

Prothrombotic genetic risk factors in stroke: a possible different role in pediatric and adult patients.

The role of thrombophilia in the pathogenesis of stroke is still controversial, especially in the pediatric stroke. In order to examine the role of common thrombophilic mutations in children and adults with stroke, a case-control study was carried out in a group of 80 children and 73 younger adult patients. The control groups encompassed 100 healthy children and 120 healthy blood donors. Our results showed no significant differences in the frequency of factor V (FV) Leiden, FII G20210A, and methylenetetrahydrofolate reductase (MTHFR) C677T variants between patient groups and corresponding controls. According to our results, carriers of 677CT genotype have 3.62 higher risks to develop stroke in children than in adults (P < .001). The obtained data indicate that heterozygosity for MTHFR C677T variant represents a possible important risk factor for pediatric stroke and suggest a different role of this gene variant in etiology of stroke in pediatric and adult patients.

Multi-tasking role of the mechanosensing protein Ankrd2 in the signaling network of striated muscle.

BACKGROUND: Ankrd2 (also known as Arpp) together with Ankrd1/CARP and DARP are members of the MARP mechanosensing proteins that form a complex with titin (N2A)/calpain 3 protease/myopalladin. In muscle, Ankrd2 is located in the I-band of the sarcomere and moves to the nucleus of adjacent myofibers on muscle injury. In myoblasts it is predominantly in the nucleus and on differentiation shifts from the nucleus to the cytoplasm. In agreement with its role as a sensor it interacts both with sarcomeric proteins and transcription factors. METHODOLOGY/PRINCIPAL FINDINGS: Expression profiling of endogenous Ankrd2 silenced in human myotubes was undertaken to elucidate its role as an intermediary in cell signaling pathways. Silencing Ankrd2 expression altered the expression of genes involved in both intercellular communication (cytokine-cytokine receptor interaction, endocytosis, focal adhesion, tight junction, gap junction and regulation of the actin cytoskeleton) and intracellular communication (calcium, insulin, MAPK, p53, TGF-ß and Wnt signaling). The significance of Ankrd2 in cell signaling was strengthened by the fact that we were able to show for the first time that Nkx2.5 and p53 are upstream effectors of the Ankrd2 gene and that Ankrd1/CARP, another MARP member, can modulate the transcriptional ability of MyoD on the Ankrd2 promoter. Another novel finding was the interaction between Ankrd2 and proteins with PDZ and SH3 domains, further supporting its role in signaling. It is noteworthy that we demonstrated that transcription factors PAX6, LHX2, NFIL3 and MECP2, were able to bind both the Ankrd2 protein and its promoter indicating the presence of a regulatory feedback loop mechanism. CONCLUSIONS/SIGNIFICANCE: In conclusion we demonstrate that Ankrd2 is a potent regulator in muscle cells affecting a multitude of pathways and processes.

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.

+1040 C/T polymorphism in coding region of thrombin-activatable fibrinolysis inhibitor gene and the risk of idiopathic recurrent fetal loss.

Recurrent fetal loss (RFL) is common health problem affecting up to 5% of women of reproductive age. It has been shown that plasma thrombin-activatable fibrinolysis inhibitor (TAFI) concentrations increase during pregnancy and return to baseline levels soon after delivery. The +1040C/T single nucleotide polymorphism in coding region of TAFI gene is associated with TAFI blood levels. The aim of our study was to investigate the relationship between +1040C/T polymorphism in TAFI gene and idiopathic RFL. Study was carried out in a group of 120 women (61 controls and 59 women with idiopathic RFL). The +1040C/T polymorphism was detected by restriction fragment length polymorphism PCR. Increased frequency of +1040T/T genotype was observed in a study group, but without statistically significant difference. Carriers of T/T genotype have increased risk of fetal loss by 1.23-fold, compared with carriers of C/C (95% CI 0.462-3.277; P = 0.7) and 1.34-fold compared with carriers of C/T genotype (95% CI 0.501-3.601; P = 0.6). C allele is associated with reduced risk of recurrent fetal loss compared with T allele (OR 0.91; 95% CI 0.545-1.533; P = 0.7). In conclusion, we observed increased frequency of +1040T/T genotype in a patient group, suggesting that this genotype could be potential risk factor for idiopathic RFL. Further investigation should be carried out in order to establish the role of this polymorphism in the etiology of idiopathic recurrent miscarriages.

A novel role for cardiac ankyrin repeat protein Ankrd1/CARP as a co-activator of the p53 tumor suppressor protein.

The muscle ankyrin repeat protein (MARP) family member Ankrd1/CARP is a part of the titin-mechanosensory signaling complex in the sarcomere and in response to stretch it translocates to the nucleus where it participates in the regulation of cardiac genes as a transcriptional co-repressor. Several studies have focused on its structural role in muscle, but its regulatory role is still poorly understood. To gain more insight into the regulatory function of Ankrd1/CARP we searched for transcription factors that could interact and modulate its activity. Using protein array methodology we identified the tumor suppressor protein p53 as an Ankrd1/CARP interacting partner and confirmed their interaction both in vivo and in vitro. We demonstrate a novel role for Ankrd1/CARP as a transcriptional co-activator, moderately up regulating p53 activity. Furthermore, we show that p53 operates as an upstream effector of Ankrd1/CARP, by up regulating the proximal ANKRD1 promoter. Our findings suggest that, besides acting as a transcriptional co-repressor, Ankrd1/CARP could have a stimulatory effect on gene expression in cultured skeletal muscle cells. It is probable that Ankrd1/CARP has a role in the propagation of signals initiated by myogenic regulatory factors (MRFs) during myogenesis.

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.

An overview of genetic risk factors in thrombophilia.

Thrombophilia is a multifactorial disorder, involving both genetic and acquired risk factors that affect the balance between procoagulant and anticoagulant factors and lead to increased tendency to thrombosis. The concept that thrombophilia could be associated with genetic defects was first proposed in 1965 after the discovery of familiar antihrombin III deficiency. Further family studies showed that deficiency of protein C or protein S also increased thrombotic risk. In the coming years the advent in DNA technology, especially the invention of PCR reaction, played an important role in the identification of the exact nature of these deficiencies and opened new possibilities in the genetic research of thrombophilia. The breakthrough came with the discovery of activated protein C resistance and Factor V Leiden mutation. Shortly afterwards a mutation in the 3' untranslated region of Factor II gene (FII G20210A) associated with increased concentration of factor II in plasma, was described. Large epidemiologic studies have conformed that these two common mutations represent significant risk factors for thrombophilia. In the last decade several prothrombotic genetic risk factors have been described, including genes variants associated with increased levels of coagulation factors, defects of natural coagulation inhibitors, defects of the fibrinolytic system and hyperhomocysteinemia. These genetic defects or their combination have been extensively studied in an attempt to elucidate the possible association with increased thrombotic tendency. The large-scale DNA analysis systems are now becoming available, opening a new era in the genetic studies of thrombophilia. New technology will enable many genes to be studied in a single patient bringing us closer to the "personalized" medicine.