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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.

Vascular complications are the leading cause of increased morbidity and mortality of diabetic patients. It has been postulated that matrix metalloproteinases MMP-2 and MMP-9, zinc-dependent endopeptidases through remodeling of the extracellular matrix, can contribute to the onset and progression of diabetic vascular complications. The aim of our study was to assess whether there is a major difference in single nucleotide polymorphisms in the MMP-2 (at position -1306C˃T) and MMP-9 (at position -1562C˃T) gene in type 2 diabetic patients and healthy controls and to determine whether there is an association of these gene variants with the presence of microvascular complications in diabetic patients. Our study included 102 type 2 diabetes patients and a control group which was comprised of 56 healthy controls. All diabetic patients were screened for microvascular diabetes complications. Genotypes were detected by polymerase chain reactions followed by restriction analyses with specific endonucleases and their frequencies were determined. The MMP-2 variant -1306C>T showed a negative correlation with type 2 diabetes (p=0.028). It was also shown that the presence of the -1306C allele increases the probability of developing type 2 diabetes. This was a 2.2 fold increase and that the -1306 T allele has a protective role in regards to

Uvod/Cilj Terapija kumarinima predstavlja jedan od najboljih modela za primenu farmakogenetike. Doprinos faktora koji utiču na terapiju kumarinima može značajno da varira između etničkih grupa, što opravdava sprovođenje studija specifičnih za populaciju. Cilj ove studije je bio da se analizira uticaj najvažnijih genetičkih faktora (geni VKORC1 i CYP2C9) koji utiču na terapiju kumarinima kod bolesnika iz Srbije. Metode Sprovedena je retrospektivna studija koja je obuhvatila 207 bolesnika na terapiji acenokumarolom. Genetičke analize su vršene direktnim sekvenciranjem. Analiziran je uticaj na dozu acenokumarola varijanti (VKORC1*2, CYP2C9*2, CYP2C9*3) koje izazivaju preosetljivost i varijanti gena VKORC1 koje izazivaju rezistenciju na kumarine. Višestruka regresiona analiza je korišćena u cilju dizajniranja matematičkog modela za predviđanje individualne doze leka na osnovu kliničko-demografskih i genetičkih podataka. Rezultati Studija je potvrdila značajan uticaj analiziranih genetičkih faktora na održavanje doze acenokumarola. Dizajniran je matematički model za predviđanje individualne doze acenokumarola i njegov nekorigovani R2 je bio 61,8. Prilikom testiranja, naš model je dao R2 vrednost od 42,6 i pokazao bolje predviđanje u poređenju sa modelom koji su dali drugi autori. Kod analiziranih bolesnika pronađeno je devet različitih varijanti u kodirajućem regionu gena VKORC1. Među nosiocima ovih varijanti 78% je bilo potpuno rezistentno, te nije bilo moguće postići terapeutski efekat čak ni sa visokim dozama acenokumarola. Zaključci Populacioni model za predviđanje individualne doze acenokumarola može pokazati prednosti u odnosu na modele koji se koriste na generalizovan način. Takođe, VKORC1 varijante koje izazivaju rezistenciju na kumarin treba uzeti u obzir prilikom planiranja terapije., Introduction/Objective Coumarin therapy represents one of the best models for applying pharmacogenetics. The contribution of factors influencing coumarin therapy can vary significantly between ethnic groups, which justifies conducting population-specific studies. The aim of this study was to analyze the influence of the most important genetic factors (VKORC1 and CYP2C9 genes) that affect coumarin therapy in patients from Serbia. Methods A retrospective study involving 207 patients on acenocoumarol therapy was conducted. Genetic analyses were performed by direct sequencing. Influence on acenocoumarol dose of variants (VKORC1, CYP2C9*2, CYP2C9*3) causing hypersensitivity and VKORC1 variants causing resistance to acenocoumarol were analyzed. Multiple regression analysis was used to design a mathematical model for predicting individual drug dosage based on clinical-demographic and genetic data. Results The study confirmed significant influence of the analyzed genetic factors on acenocoumarol maintenance dose. We designed mathematical model for predicting individual acenocoumarol dose and its unadjusted R2 was 61.8. In the testing cohort, our model gave R2 value of 42.6 and showed better prediction in comparison with model given by other authors. In the analyzed patients, nine different variants in the VKORC1 coding region were found. Among carriers of these variants 78% were completely resistant, and it was not possible to achieve therapeutic effect even with high doses of acenocoumarol. Conclusions Population-specific model for prediction individual dose of acenocoumarol, may show advantages over protocols that are used in a generalized manner. Also, VKORC1 variants which cause coumarin resistance should be considered when planning therapy.

Kardiovaskularne bolesti (KVB) su klasa oboljenja koja obuhvataju srce i/ili krvne sudove i nastaju složenim sadejstvom genetičkih i stečenih faktora. Razvoj molekularne biologije omogućio je nove uvide u fundamentalne mehanizme koji dovode do KVB, kao i napredak u dijagnostici, prognostici i lečenju. Novi pristupi u dijagnostici i lečenju KVB podstaknuti su istraživanjima koja se odnose na nekodirajuće RNK - duge nekodirajuće RNK i kratke nekodirajuće RNK. Ova klasa molekula, ne samo da se povezuje sa različitim mehanizmima koji dovode do razvoja KVB, nego se prepoznaje njihov potencijal kao biomarkera, farmakogenetičkih faktora, novih meta lekova i novih alatki u lečenju bolesti. Upotreba nekih od njih u lečenju ljudi je i odobrena od strane relevantnih agencija. Posebnu pažnju privlače studije koje se odnose na nekodirajuće RNK poreklom iz ekstracelularnih vezikula, dodatno potvrđujući potencijal nekodirajuće RNK kao leka budućnosti, ne samo u tretmanu KVB., Cardiovascular diseases (CVDs) are group of diseases which encompass heart and/or blood vessels and they originate from complex coaction of genetic and acquired factors. Development of molecular biology has enabled new insights into fundamental mechanisms which lead to CVDs, as well as progress in diagnostics, prognosis and treatment. New approaches to diagnostics and treatment of CVDs have been encouraged by researches which are related to non-coding RNA, long non-coding and short non-coding RNA. Not only is this group of molecules being associated with different mechanisms which lead to CVDs, but their potential to be biomarkers, pharmacogenetic factors, new drug targets and new treatment tools is being recognised. Relevant agencies have approved some of them to be used for human treatments. Studies related to non-coding RNAs deriving from extracellular vesicles are getting special attention, additionally confirming non-coding RNAs potential as drug of the future, not limited just to CVDs.

Introduction. Despite proven clinical effect of clopidogrel, a considerable number of patients do not have an adequate response to this type of medication. Problems during therapy occur in the form of resistance, which is present in 11% of patients or bleeding that occurs in about 9% of patients. Pharmacogenomics studies demonstrated that variants of the CYP2C19 gene significantly influence the interindividual heterogenity of the clopidogrel response. The American Heart Association, US Food and Drug Administration and the European Medicines Agency, cite the CYP2C19 gene as a significant factor which influences patients response to clopidogrel. Further, it has been shown that the contribution of genetic and non-genetic factors affecting clopidogrel therapy may vary between patients from different populations, which justifies conducting population-specific studies. The aim. The aim of our study was to examine the significance of the CYP2C19*2 and the CYP2C19*17 variants in the individual response to clopidogrel, in Serbian patients. Methods. The study involved 108 patients with carotid artery stenosis who underwent endarterectomy and received clopidogrel for at least 30 days after the intervention. Also, 120 patients with myocardial infarction receiving clopidogrel after PCI (percutaneous coronary intervention) were included. Commercial tests were used for standard laboratory testing. Allelic discrimination was performed after Sanger sequencing. Results were analysed using statistical tests. Results. In patients undergoing endarterectomy CYP2C19*2 carriers had a higher risk for being clopidogrel low-responder in comparison with non-carriers (1.250, 95% CI 1.695–1.658, P<0.01). In the group of patients undergoing PCI, risk for reinfarction in patients who were carriers of CYP2C19*2 was higher compared to patients with wild type genotype (OR 5.355, 95% CI 0.955-31.08; P=0.038). Variant CYP2C19*17 showed no association with variations in response to clopidogrel therapy.

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.

Aterosklerotske promene karotidnih arterija su najčešći uzrok moždane ishemije. Karotidna endarterektomija (KE) predstavlja efektivan tretman stenoze karotidnih arterija i danas je najčešći operativni zahvat za prevenciju moždanog udara. Još uvek se kod 2%-5% bolesnika razvija tromboza karotidnih arterija u perioperativnom periodu i dvojna antitrombocitna terapija (aspirin i klopidogrel) značajno smanjuje rizik od razvoja moždanog udara posle KE. Međutim, i pored dokazane kliničke efikasnosti ovih lekova, značajan broj bolesnika nema dobar terapijski odgovor na aspirin i/ili klopidogrel. U literaturi, prevalenca rezistencije na klopidogrel varira od 5% do 44% i najveća je kod bolesnika sa moždanim udarom. CYP2C19 enzim je uključen u oba oksidaciona procesa metabolizma klopidogrela. Nekoliko farmakogenetičkih studija je pokazalo da osobe koje su nosioci CYP2C19*2 alela imaju smanjeno stvaranje aktivnog metabolita klopidogrela i visoku reaktivnost trombocita, što dovodi do povećanja rizika od razvoja neželjenih kardiovaskularnih događaja. Međutim, postoje studije koje nisu ukazale na uticaj CYP2C19 genotipa na klinički efekat klopidogrela niti na prediktivnu vrednost CYP2C19*2 varijantne gena. Glavni cilj ove studije je bio da se ispita povezanost terapijskog odgovora na antitrombocitni lek klopidogrel, koji je praćen laboratorijskim ispitivanjem agregacije trombocita, i prisustva CYP2C19*2 varijante gena i da se utvrdi uticaj genetskih i negenetskih faktora na reaktivnost trombocita kod bolesnika sa stenozom karotidnih arterija kod kojih je izvršena karotidna endarterektomija. U istraživanju efekat antiagregacijskog leka klopidogrela praćen je ADP-indukovanom agregacijom trombocita. Vrednosti testa očekivano su opadale tokom prvih mesec dana tretmana i pokazana je statistički značajna razlika između vrednosti ADP-indukovane agregacije trombocita dobijene pre hirurške intervencije, dvadeset četiri časa nakon uzimanja prve doze klopidogrela (75 mg), sedmog i tridesetog, Carotid artery atherosclerosis is one of the many causes of acute ischemic stroke. Carotid endarterectomy (KE) is the standard treatment for carotid stenosis and is the most frequently performed operation to prevent stroke. Perioperative carotid thrombosis remains a significant problem in 2%-5% of patients and dual antiplatelet therapy, including aspirin and clopidogrel, has a potential role in reducing the risk of stroke after KE. Despite their proven clinical effect, a considerable number of patients don’t have an adequate response to aspirin, clopidogrel or both. According to published data, the prevalence of clopidogrel resistance varies from 5% and may be as high as 44% in patients with acute ischemic stroke. The CYP2C19 enzyme is involved in both oxidative steps of clopidogrel metabolism. Several pharmacogenomic studies have demonstrated that individuals who are carriers of CYP2C19*2 allele have a reduced conversion of clopidogrel into its active metabolite and higher platelet reactivity, which increases the risk of adverse cardiovascular events. However, there are contrasting conclusions in the literature regarding the predictive value of the CYP2C19*2 variant allele The aim of this study was to determine the prevalence of low responsiveness to clopidogrel and to identify risk factors, both genetic (CYP2C19*2) and non-genetic, for low responsiveness based on platelet function testing in patients with carotid artery stenosis undergoing KE. ADP-induced platelet aggregation was used to analyze the effect of clopidogrel. ADP-induced platelet aggregation showed a tendency of decline during the first month of treatment and the values of platelet aggregation obtained before the KE. Twenty four hours after the first dose of the clopidogrel, 7 and 30 days of taking the drug were statistical significantly different (P<0,001). In the study group, the number of patients with high platelet reactivity declined from 79.5% after 24 h to about a quarter of the patients after

Aterosklerotske promene karotidnih arterija su najčešći uzrok moždane ishemije. Karotidna endarterektomija (KE) predstavlja efektivan tretman stenoze karotidnih arterija i danas je najčešći operativni zahvat za prevenciju moždanog udara. Još uvek se kod 2%-5% bolesnika razvija tromboza karotidnih arterija u perioperativnom periodu i dvojna antitrombocitna terapija (aspirin i klopidogrel) značajno smanjuje rizik od razvoja moždanog udara posle KE. Međutim, i pored dokazane kliničke efikasnosti ovih lekova, značajan broj bolesnika nema dobar terapijski odgovor na aspirin i/ili klopidogrel. U literaturi, prevalenca rezistencije na klopidogrel varira od 5% do 44% i najveća je kod bolesnika sa moždanim udarom. CYP2C19 enzim je uključen u oba oksidaciona procesa metabolizma klopidogrela. Nekoliko farmakogenetičkih studija je pokazalo da osobe koje su nosioci CYP2C19*2 alela imaju smanjeno stvaranje aktivnog metabolita klopidogrela i visoku reaktivnost trombocita, što dovodi do povećanja rizika od razvoja neželjenih kardiovaskularnih događaja. Međutim, postoje studije koje nisu ukazale na uticaj CYP2C19 genotipa na klinički efekat klopidogrela niti na prediktivnu vrednost CYP2C19*2 varijantne gena.Glavni cilj ove studije je bio da se ispita povezanost terapijskog odgovora na antitrombocitni lek klopidogrel, koji je praćen laboratorijskim ispitivanjem agregacije trombocita, i prisustva CYP2C19*2 varijante gena i da se utvrdi uticaj genetskih i negenetskih faktora na reaktivnost trombocita kod bolesnika sa stenozom karotidnih arterija kod kojih je izvršena karotidna endarterektomija.U istraživanju efekat antiagregacijskog leka klopidogrela praćen je ADP-indukovanom agregacijom trombocita. Vrednosti testa očekivano su opadale tokom prvih mesec dana tretmana i pokazana je statistički značajna razlika između vrednosti ADP-indukovane agregacije trombocita dobijene pre hirurške intervencije, dvadeset četiri časanakon uzimanja prve doze klopidogrela (75 mg), sedmog i tridesetog da, Carotid artery atherosclerosis is one of the many causes of acute ischemic stroke. Carotid endarterectomy (KE) is the standard treatment for carotid stenosis and is the most frequently performed operation to prevent stroke. Perioperative carotid thrombosis remains a significant problem in 2%-5% of patients and dual antiplatelet therapy, including aspirin and clopidogrel, has a potential role in reducing the risk of stroke after KE. Despite their proven clinical effect, a considerable number of patients don’t have an adequate response to aspirin, clopidogrel or both. According to published data, the prevalence of clopidogrel resistance varies from 5% and may be as high as 44% in patients with acute ischemic stroke. The CYP2C19 enzyme is involved in both oxidative steps of clopidogrel metabolism. Several pharmacogenomic studies have demonstrated that individuals who are carriers of CYP2C19*2 allele have a reduced conversion of clopidogrel into its active metabolite and higher platelet reactivity, which increases the risk of adverse cardiovascular events. However, there are contrasting conclusions in the literature regarding the predictive value of the CYP2C19*2 variant alleleThe aim of this study was to determine the prevalence of low responsiveness to clopidogrel and to identify risk factors, both genetic (CYP2C19*2) and non-genetic, for low responsiveness based on platelet function testing in patients with carotid artery stenosis undergoing KE.ADP-induced platelet aggregation was used to analyze the effect of clopidogrel. ADP-induced platelet aggregation showed a tendency of decline during the first month of treatment and the values of platelet aggregation obtained before the KE. Twenty four hours after the first dose of the clopidogrel, 7 and 30 days of taking the drug were statistical significantly different (P<0,001). In the study group, the number of patients with highplatelet reactivity declined from 79.5% after 24 h to about a quarter of the patients after 30

Klopidogrel je antiagregacioni lek koji je sa aspirinom, u sklopu dvojne antiagregacione terapije, indikovan u stanjima koja nose rizik od ishemijskih događaja i tromboembolijskih komplikacija. Postoji velika interindividualna varijabilnost u odgovoru na klopidogrel. Značajan broj pacijenata kao posledicu neadekvatnog odgovora na terapiju može iskusiti različita neželjena dejstva. Kod oko 9% pacijenata koji su na terapiji klopidogrelom javlja se krvarenje kao neželjeni efekat. Ono samo po sebi može biti životno ugrožavajuće, a sekundarno može biti povezano sa pojavom ishemijskih događaja. Uzroci varijabilnosti u odgovoru na terapiju klopidogrelom, mogu biti kako stečeni, tako i posledica genetičkih faktora. Među genetičkim faktorima najznačajniji doprinos ima gen za enzim CYP2C19 koji je neophodan u procesu konverzije klopidogrela u aktivnu formu leka. Zbog toga su varijante CYP2C19 gena najčešće razmatrane u okviru farmakogenetike klopidogrela. Od posebnog kliničkog značaja su varijante rs4244285 (c.681G>A; CYP2C19*2) i rs4986893 (c.636G>A; CYP2C19*3), koje su povezane sa oslabljenim odgovorom na klopidogrel. Promotorska varijanta rs12248560 (c.-806C>T, CYP2C19*17) se dovodi u vezu sa pojačanim stepenom inhibicije trombocita i povišenim rizikom od krvarenja kod pacijenata na terapiji klopidogrelom. U fokusu ove studije bilo je ispitivanje uticaja CYP2C19 gena na pojavu pojačanog odgovora na klopidogrel koje vodi povišenom riziku od krvarenja. Istraživanje je sprovedeno u grupi pacijenata koji su pretrpeli infarkt miokarda i bili podvrgnuti perkutanoj koronarnoj intervenciji (PCI), kao i grupi pacijenata sa stenozom karotida koji su podvrgnuti karotidnoj endarterektomiji (CEA). U studiji su analizirane promotorske varijante rs12248560 (c.-806C>T, CYP2C19*17) i rs11568732 (c.-889T>G, CYP2C19*20) za koje su literaturni podaci i naši preliminarni rezultati pokazali da mogu biti povezane sa pojačanim odgovorom na klopidogrel. Dodatno su analizirane egzonske varijante rs, Wide inter-individual variability in clopidogrel response has been reported. Significant number of patients with inadequate response to clopidogrel experience different adverse effects. Bleeding as an adverse event occurs in about 9% of patients on clopidogrel therapy. It can be life threatening and secondarily, related to higher risk of ischemic events. Variability in clopidogrel response may be explained by different non-genetic and genetic factors. Among genetic factors, the most important role has CYP2C19 gene encoding enzyme responsible for conversion of clopidogrel to its active form. Hence, CYP2C19 gene variants are the most commonly investigated genetic variants related to clopidogrel pharmacogenetics. Clinically, the most relevant variants are rs4244285 (c.681G>A; CYP2C19*2) and rs4986893 (c.636G>A; CYP2C19*3) which lead to clopidogrel poor response. Additionally, promoter variant rs12248560 (c.-806C>T, CYP2C19*17) is associated with elevated platelet inhibition and increased risk of bleeding in patients on clopidogrel. This study was focused on investigating role of CYP2C19 gene in clopidogrel hyper-responsiveness which increases the risk of bleeding. This study was conducted in a group of patients with acute myocardial infarction who underwent percutaneous coronary intervention (PCI) as well as in patients with carotid stenosis who underwent carotid endarterectomy (CEA). In this study we examined two promoter rs12248560 (CYP2C19*17) and rs11568732 (CYP2C19*20) variants, for which literature and our preliminary results showed to be associated with elevated clopidogrel response. Additionally, we analyzed exonic variants rs4244285 (c.681G>A; CYP2C19*2) and rs4986893 (c.636G>A, CYP2C19*3) considering their potential influence on the promoter variants’ effects. Since literature data on rs11568732 (c.-889T>G, CYP2C19*20) variant is scarce, we aimed to investigate its functional significance. Further, one of our aims was the anlysis of the relevant CYP2C19 varia

The correct Author names are shown in this paper.

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). 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 gt T, rs12248560), rs11568732 (c.-889T gt G, CYP2C19*20), CYP2C19*2 (c.681G gt A; rs4244285) and CYP2C19*3 (c.636G gt 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. Association between bleeding (BARC type ae lt yen gt 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. 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.

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 (sic)2547 versus (sic)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 (sic)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.

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.

Uvod: I pored dokazanog kliničkog efekta oralne antiagregacijske terapije, značajan broj pacijenata nema adekvatan odgovor na primenjeni klopidogrel. Cilj naše studije je bio da se utvrdi uticaj prisutne CYP2C19*2 varijante gena na terapijski odgovor u toku primene klopidogrela kod pacijenata sa stenozom karotidne arterije. Metode: U jednogodišnju prospektivnu studiju uključeno je 112 pacijenata sa stenozom karotidne arterije kod kojih je izvršena endarterektomija. Posle operativnog zahvata, pacijenti su primali 75 mg dnevno klopidogrela u trajanju od najmanje mesec dana. Svi ispitanici su praćeni od momenta prijema. Za CYP2C19 genotipizaciju korišćen je TaqMan test. Uticaj CYP2C19*2 alela na trombocitnu reaktivnost ispitivan je primenom multiple-electrode aggregometry (MEA). Rezultati: Rezultati genotipizacije su pokazali da su 82 (73,2%) ispitanika homozigoti za wild-type, 29 (25,9%) heterozigoti za CYP2C19*2 alel, dok je 1 (0,9%) bio homozigot za CYP2C19*2. Nakon 24 sata, u grupi sa wild-type genotipom 29,3% ispitanika dali su odgovor na klopidogrel, a u grupi sa CYP2C19*2 varijantom gena 10% ispitanika. U grupi sa wild-type genotipom, 74,4% ispitanika su imali terapijski odgovor nakon 7 dana, odnosno 82,9% nakon 30 dana od primene klopidogrela. U grupi sa CYP2C19*2 alelom broj ispitanika sa terapijskim odgovorom raste do 46,7% nakon 7 dana, odnosno do 53,3% nakon 30 dana od primene klopidogrela. Rizik za slab odgovor je veći kod nosilaca CYP2C19*2 alela u odnosu na nenosioce (wild-type) (OR 4,250, 95% CI 1 .695 -10.658 , P lt 0,01). Zaključak: CYP2C19*2 varijanta gena značajno utiče na terapijski odgovor u toku primene klopidogrela kod bolesnika sa stenozom karotidne arterije kod kojih je izvršena endarterektomija., 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 c Yp 2C 19*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 c Yp 2C 19*2 allele vs. wildtype (OR 4.250, 95% CI 1.695 -10.658 , P lt 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.

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 lt 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.

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.

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.

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.

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 gt 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.

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 lt .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.

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-beta 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.

Tromboze dubokih vena (TDV) su multifaktorijalno oboljenje koje se javlja sa učestalošću 1/1000 stanovnika godišnje. Mutacije FV Leiden, FII G20210A i MTHFR C677T predstavljaju genetske faktore za nastanak venskih tromboza. Cilj ove studije je da se utvrdi učestalost FV Leiden, FII G20210A i MTHFR C677T mutacija kod bolesnika sa TDV gornjih ili donjih ekstremiteta. Studija je obuhvatila 119 bolesnika podeljenih u dve grupe. Grupa bolesnika sa TDV donjih ekstremiteta brojila je 77, a grupa sa TDV gornjih ekstremiteta 42 bolesnika. Prisustvo FV Leiden, FII G20210A i MTHFR C677T mutacija dokazivano je umnožavanjem odgovarajućeg fragmenta gena pomoću lančane reakcije umnožavanja polimerazom i digestijom dobijenih fragmenata restrikcionim enzimima (PCR-RFLP).Učestalost FV Leiden mutacije, u grupi bolesnika sa TDV donjih ekstremiteta, iznosila je 26,0% u heterozigotnom obliku i 1,3% u homozigotnom obliku. U grupi bolesnika sa TDV gornjih ekstremiteta, učestalost heterozigotnih nosilaca iznosila je 7,2%. Mutacija FII G20210A bila je prisutna kod 15,6% ispitanika u grupi bolesnika sa TDV donjih ekstremiteta u heterozigotnom obliku, dok je u grupi sa TDV gornjih ekstremiteta ova mutacija bila zastupljena sa 7,2% u heterozigotnom i 2,3% u homozigotnom obliku. Učestalost MTHFR C677T mutacije kod bolesnika sa TDV donjih ekstremiteta iznosila je 42,8% u heterozigotnom obliku i 13% u homozigotnom obliku, a kod TDV gornjih ekstremiteta 52,4% u heterozigotnom obliku i 9,5% u homozigotnom obliku.Mutacije FV Leiden i FII G20210A predstavljaju značajne faktore rizika za nastanak TDV donjih ekstremiteta. Kod TDV gornjih ekstremiteta ove mutacije su manje zastupljene i potrebno je sprovesti dalja istraživanja koja obuhvataju veći broj bolesnika. MTHFR C677T mutacija je manje značajan faktor rizika i treba ga razmatrati samo u slučajevima kada se pojavljuje u kombinaciji sa drugim faktorima rizika., Deep vein thrombosis (DVT) is a multifactorial disease that occurs with frequency of 1/1000 per year. The FV Leiden, FII G20210A and MTHFR C677T mutations represent genetic factors for the occurrence of vein thrombosis. The goal of this study was to determine the frequency of these mutations in patients with DVT of upper and lower limbs. The study encompassed 119 patients divided in two groups. The group of patients with the lower limbs thrombosis included 77 patients, while the upper limbs thrombosis group included 42 patients. The presence of FV Leiden, FII G20210A and MTHFR C677T mutations was detected by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis. In patients with DVT of lower limbs, the frequency of FV Leiden mutation was 26,0% in heterozygous form and 1,3% in homozygous form. In the group of patients with DVT of upper limbs, the frequency of heterozygous carriers was 7.2%. In patients with DVT of lower limbs, FII G20210A mutation occurred in heterozygous form in 15.6% subjects, and in the group with DVT of upper limbs the frequency was 7.2% in heterozygous and 2.3% in homozygous form. The frequency of MTHFR C677T mutation in patients with lower limbs DVT was 42.8% in heterozygous form and 13% in homozygous form, while in the group of patients with upper limbs DVT, the frequency was 52.4% in heterozygous form and 9.5% in homozygous form. The FV Leiden and FII G20210A mutations represent significant risk factors for the occurrence of DVT of lower limbs. These mutations are less frequent in DVT of upper limbs and more extensive further studies are needed to determine their potential role. The MTHFR C677T mutation represents less significant risk factor for lower limb DVT and should be taken into account only in cases when it occurs in combination with other risk factors.

Uvod. Prisustvo urođene trombofilije predstavlja dodatni faktor rizika od nastanka venskog tromboembolizma u trudnoći, ali i komplikacija same trudnoće kao što su ponavljani gubitak trudnoće, abrupcija placente, intrauterino zaostajanje u rastu i razvoju, te rana preeklampsija. Trudnice sa trombofilijom, a posebno one sa nedostatkom antitrombina, u riziku su od nastanka obe vrste komplikacija u trudnoći. Prikaz bolesnice. U radu je prikazana trudnica sa urođenim nedostatkom antitrombina u toku njene prve trudnoće, čija majka je četiri puta imala duboke venske tromboze, tokom trudnoće i puerperijuma. Uz redovno laboratorijsko praćenje hemostaznih parametara i redovne akušerske kontrole sa praćenjem važnog parametra placentne vaskularizacije, čitava trudnoća protekla je bez komplikacija. Profilaktička terapija niskomolekularnim heparinom uvedena je od 20. nedelje trudnoće, a supstituciona terapija primenom koncentrata antitrombina, neposredno pre porođaja. Porođaj je protekao bez komplikacija i u 37. nedelji rođena je zdrava muška beba telesne težine 3,6 kg i dužine 52 cm, sa Abgar skorom 9/10. Zaključak. Pravovremeno postavljena dijagnoza trombofilije, redovne akušerske kontrole i praćenje hemostaznih parametara u toku trudnoće, kao i primena adekvatne profilaktičke i supstitucione terapije, doprinele su prevenciji mogućih maternalnih ili komplikacija same trudnoće kod bolesnice sa urođenim nedostatkom antitrombina., Background. Presence of inherited thrombophilia is an additional risk factor for maternal thromboembolism and certain adverse pregnancy outcomes, including recurrent fetal loss, placental abruption, intrauterine growth restriction and earlyonset severe preeclampsia. Pregnant women with thrombophilia, especially those with antithrombin (AT) deficiency, are at high risk of both kinds of complications. Case report. We presented a pregnant women with congenital antithrombin deficiency in the first pregnancy, whose mother had had four times pregnancy-related deep vein thrombosis, and antithrombin deficiency. With the regular laboratory monitoring of hemostatic parameters and gynaecology surveillance including the follow-up of placental vascular flow, the whole pregnancy proceeded without complications. The prophylactic therapy with low molecular weight heparin was introduced from the 20th week of gestation and one dose of substitution therapy with antithrombin concentrate was administrated before delivery. Pregnancy and labour were terminated without complications at the 37th week of gestation, resulting in the delivery of a healthy male newborn of 3.6 kg body weight, 52 cm long, and with the Apgar scores of 9/10. Conclusion. A timely made diagnosis of thrombophilia, accompanied with regular obstetrics check-ups and follow-ups of hemostatic parameters during pregnancy, as well as the use of adequate prophylactic and substitution therapy, are the successful tools for the prevention of possible maternal complications and pregnancy itself in our patient with congenital AT deficiency.

MARP family members CARP, Ankrd2 and DARP are expressed in the striated muscle, while DARP protein is also detected in brown adipose tissue (BAT). Taking into account recent findings concerning the common origin of muscle and brown fat, expression of CARP and Ankrd2 in mouse BAT was investigated. We demonstrated Ankrd2 expression in both inactive and thermogenically active BAT, while CARP expression was not detected. Our findings suggest that the expression of Ankrd2 in BAT could be a part of the 'myogenic transcriptional signature', further supporting the evidence that muscle and brown adipose cells arise from the same myoblastic precursor.

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 gt 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.

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 R1040C/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. Blood Coagul Fibrinolysis 21: 679-682

Backgound/Aims: Existing data regarding the prevalence of thrombophilia in women with pregnancy complications are conflicting. Methods: To investigate the relationship between pregnancy-associated complications and the presence of thrombophilia, we studied the records of 453 women with pregnancy-associated complications. In 55 women, intrauterine fetal death ( fetus mortus in utero, FMU) after 20 weeks of gestation was recorded, in 231 two or more consecutive recurrent fetal losses (RFL) were recorded, while 167 had a venous thromboembolism (VTE) during one of their pregnancies. The control group consisted of 128 healthy women, with no previous history of thrombotic events or miscarriages. Results: In the FMU group we found 54.5% of women had thrombophilia, in the RFL group 38%, and in the VTE group 52.7%. The most frequent thrombophilia in the VTE group was the FV Leiden ( OR 17.9, 95% CI 4.2-75.9). The most frequent thrombophilia in the FMU group was the FII G20210A ( OR 7.09, 95% CI 1.8-27.9). Statistical difference between RFL and the control group was observed only for FV Leiden ( OR 6.8, 95% CI 1.6-29.7). Conclusion: Thrombophilia was found to be considerably more common in women with pregnancy-associated complications in comparison with the women with normal pregnancies, most frequently in patients with VTE or FMU.

Factor V Leiden (FVLeiden) and prothrombin G20210A are the most common genetic causes of thrombophilia and established risk factors for different clinical manifestations of venous thromboembolism (VTE). This study investigated whether the clinical manifestation of VTE, the extension of deep vein thrombosis (DVT) and the presence of transient risk factors at the time of the first VTE, differed among patients with mutations (97 with FVLeiden; 33 with prothrombin G20210A) and in 109 patients without thrombophilia. Isolated pulmonary embolism (PE) was less prevalent in patients with FVLeiden (6%) and no thrombophilia (6%) than in those with prothrombin G20210A (15%). No difference was found in the incidence of distal DVT. Regarding the extension of proximal DVT, the lowest incidence for isolated popliteal vein and the highest for iliofemoral vein were observed in patients with prothrombin G20210A. No difference was observed between groups of patients with or without thrombophilia by unprovoked VTE. The pregnancy/puerperium was the most prevalent risk factor in carriers of prothrombin G20210A. Among FVLeiden carriers, the most prevalent risk factor was surgery, and in patients without thrombophilia, it was trauma (P lt .05). Thrombosis of the upper limb was more frequent in a group without thrombophilia than in patients with mutations (P lt .01). Transverse sinus venous thrombosis was present only in patients with prothrombin G20210A. Carriers of prothrombin G20210A have an increased risk of developing isolated PE and more severe clinical manifestations than those with FVLeiden or without thrombophilia.

A single nucleotide polymorphism c.-1639G gt 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 gt 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 lt 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 lt 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 lt 0.0001). VKORC1 c.-1639G gt 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. Blood Coagul Fibrinolysis 21:558-563

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.

Background: The excess of matrix metalloproteinases (MMPs) might be associated with the airways destruction or dilatation in bronchiectasis. The functional promoter polymorphisms of MMPI and MMP9 genes, involved in the extracellular matrix remodeling, might increase the expression of MMPs leading to the development of bronchiectasis. Methods: Detection of MMP1 G-1607GG and MMP9 C-1562T gene variants was performed on 37 patients with idiopathic disseminated bronchiectasis and 102 control subjects. We also described a novel method for simple and rapid detection of MMP1 G-1607GG polymorphism. Results: The frequency of -1607GG allele was significantly higher in the group of patients than in control subjects (P = 0.014). The heterozygote genotype showed association with bronchiectasis (odds ratio, 5.3; 95% confidence intervals, 1.4-20.0). The association was even stronger in homozygotes for -1607GG allele (odds ration, 8.7; 95% confidence intervals, 1.9-41.0). The allelic and genotype frequencies of MMP9 C-1562T variant did not show significant differences between the groups. Conclusions: This is the first report concerning a role of MMP1 G-1607GG and MMP9 C-1562T variants in pathogenesis of idiopathic disseminated bronchiectasis. The results of our study revealed the association of -1607GG allele and the lack of association of MMP9 C-1562T variant with the disease.

In order to investigate the influence of genetic factors in childhood stroke, we compared the distributions of mutations/polymorphisms affecting hemostasis and/or endothelial function (factor V [FV] Leiden, factor II [FII] G20210A, methylenetetrahydrofolate reductase [MTHFR] C677T, angiotensin-converting enzyme [ACE] insertion/deletion [1D], and endothelial nitric oxide synthase [eNOS] G894T) among children with stroke and controls. A total number of 26 children with arterial ischemic stroke and a control group of 50 healthy children were included in the study. No statistically significant differences in allelic and genotypic distribution were detected in comparisons between groups. However, when combined genotypes were analyzed, statistical significance was observed for the association of MTHFR CT and eNOS IT gene variants. The results Of our study Suggest that this genotype combination represents a risk factor of 7.2 (P =. 017) for arterial ischemic stroke in children.

Uvod/Cilj. Pojedinačni nukleotidni polimorfizam c.- 1639G gt A u promotorskom regionu gena za vitamin K epoksid- reduktazu (VKORC1), odgovoran je za varijabilnost odgovora u toku primene oralnih antikoagulanasa (OA). Cilj našeg istraživanja bio je da utvrdimo učestalost polimorfizma c.- 1639G gt A i njegov uticaj na dozu antikoagulansa acenokumarola, te da procenimo povezanost varijabilnosti u odgovoru na terapiju sa prisutnim polimorfizmom. Metode. U ispitivanje je bilo uključeno 200 bolesnika koji su primali OA (43 malu dozu, 127 srednju i 30 veliku dozu). Rezultati. Kod 40 (93%) bolesnika lečenih malom dozom OA, dokazano je prisustvo A-alela. U grupi koja je primala veliku dozu OA, 13 (43,3%) bolesnika bili su nosioci A-alela, heterozigoti (GA genotip), i nijedan od njih nije bio nosilac homozigotne varijante AA genotipa. Posmatrano u celini, u grupi sa AA genotipom doza održavanja OA bila je 10 mg nedeljno, sa GA 19 mg i sa GG genotipom 26 mg. Kod nosilaca GG genotipa bile su potrebne 2,6 puta veće doze antikoagulansa za postizanje terapijskog raspona INR u odnosu na nosioce AA genotipa (p lt 0,0001). Retrospektivnom analizom utvrđeno je da je 33 (16,5%) bolesnika u toku uvođenja terapije bilo predozirano, a kod 11 (5,5%) bolesnika predoziranost je bila udružena sa pojavom krvarenja. Od 33 predozirana bolesnika, 27 su bili nosioci AA genotipa, a šest nosioci GA genotipa (p lt 0,000001). Zaključak. Na individualnu osetljivost na antikoagulanse VKORC1 ima značajan uticaj. Nosiocima AA genotipa potrebne su 2,6 puta manje doze antikoagulansa za održavanje terapijskog raspona INR u odnosu na nenosioce. Farmakogenetski testovi mogli su da ukažu na visok rizik od predoziranja kod 28,5% naših bolesnika, nosioca AA genotipa, pre uvođenja terapije OA., Background/Aim. A single nucleotide polymorphism c.- 1639G gt 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 anticoagulants (OA). The aim of the study was to determine the incidence and the effect of c.-1639G gt A polymorphism on the acenocoumarol dosage requirements in the group of patients under stable anticoagulation, and to estimate the variability in response to OA. Methods. Our study included 200 consecutive patients requiring low (n = 43), medium (n = 127) and high (n = 30) acenocoumarol dose. Results. 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 dose: among these patients 13 (43.3%) carried the A allele in the heterozygous form and none of them carried AA genotype. The patients with GG genotype required 2.6 times higher dose than the patients carriers of AA genotype (p lt 0.0001). In 33 patients (16.5%) the overdose occurred during the initiation of anticoagulant therapy and in 11 patients (5.5%) it was associated with bleeding. Out of the group of 33 overdosed patients, 27 and 6 patients carried AA and GA genotype, respectively (p lt 0.000001). Conclusion. VKORC1 significantly influenced OA dose and predicted individuals predisposed to unstable anticoagulation. The carriers of AA genotype required 2.6 time lower doses of OA than the carriares of GG genotype. Pharmacogenetic testing could predict a high risk of overdose among 28.5 % of our patients - carriers of AA genotype, before anticoagulation therapy initiation.

The absolute rate of recurrence of venous thromboembolism (VTE) is approximately 5% per year. There is a lower rate of recurrence in provoked VTE, and higher in idiopathic one. So far, there is no consensus whether hereditary thrombophilia should be considered as a persistent risk factor, and whether it requires long-term anticoagulant therapy. The aim of our study was to estimate the risk of recurrent VTE in patients carrying FV Leiden mutation in Serbian population. In retrospective study (1994-2006), we have evaluated the risk of recurrent VTE in 56 patients who are carriers of FV Leiden mutation, in comparison to group consisting of 56 patients non-carriers of FV Leiden mutation. Patients with FII G20210A and MTHFR C677T mutations, antiphospholipid antibodies, antithrombin III, protein C or protein S deficiency, malignancies and diabetes were excluded from the study. Recurrent VTE occurred in 44.6% of the patients, carriers of the FV Leiden mutations, vs. 26.7% in non-carriers group (P lt 0.05). The incidence rate was 3.7 and 2.2% per year, respectively. The estimated relative risk of recurrence for FV Leiden carriers was 1.67 (95% CI 0.99-2.81, P = 0.049). The 60% of patients with mutation and only 13% without mutation develop rethrombosis during first year after discontinuance of therapy (P lt 0.01). In our study patients with symptomatic VTE who are carriers of the FV Leiden gene mutations have a higher risk of recurrent VTE than non-carriers. Our data suggest the importance of the FV Leiden mutation detection and the estimation of the clinical condition for successful secondary prophylaxis of VTE.