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Thrombophilia is a condition of enhanced functionality of the haemostatic system with an increased tendency for thrombosis, and it can be a congenital, acquired, or complex defect. Pregnancy can be the cause of acquired transitory thrombophilia, which may lead to complications if inherited thrombophilia is also present.The aim of this study was to determine the genetic structure of the population based on the frequency of the gene variants factor V Leiden G1691A, factor II G20210A, methylenetetrahydrofolate reductase C677T, and plasminogen activator inhibitor-1 4G/5G, as well as to investigate the predictive value of these gene variants in repeated miscarriages.The study included 87 female patients from Central Serbia with an average age of 32.7±4.5 years with inherited thrombophilia and previous miscarriages, with or without intrauterine foetal death. The exclusion criteria included the existence of gynaecological and infectious aetiology and the deficit of factors important for the coagulation process.The resulting genotypes were in Hardy-Weinberg equilibrium. The frequency of genotypes with mutated alleles was significantly higher in this group of patients than in the control group for all variants except factor II G20210A. The most commonly mutated alleles were the plasminogen activator inhibitor-1 4G allele (0.61) and methylenetetrahydrofolate reductase T allele (0.47). Double mutation of plasminogen activator inhibitor-1 4G/5G and methylenetetrahydrofolate reductase C677T was dominant in patients with recurrent pregnancy loss (46.15%).The presence of a combination of genetic variants of the plasminogen activator inhibitor-1 4G/5G and methylenetetrahydrofolate reductase C677T is a significant predictor of spontaneous abortions in women with inherited thrombophilia in Central Serbia.

Ph(-) mijeloproliferativni zloćudni tumori (MPN) dijele se na esencijalnu trombocitozu (ET), policitemiju veru (PV) i primarnu mijelofibrozu (PMF). Tromboza se pojavljuje u 20 – 40 % bolesnika što je čini najčešćom komplikacijom ovih bolesti. Cilj istraživanja bio je utvrditi genetičke i hematološke rizične faktore koji doprinose nastanku tromboze. U tu svrhu, uz detaljnu anamnezu analizirali smo uzorke 258 Ph(-) MPN bolesnika (134 ET, 70 PV i 54 PMF) i 258 zdravih kontrola (bez tromboze) uparenih po dobi i spolu. Odgovarajućom metodom polimerazne lančane reakcije (PCR) ispitane su učestalosti polimorfizama FV Leiden, FII G20210A, ljudskih trombocitnih antigena (HPA-1, -2, -3, -5), P-selektina (P-SEL S290N, N562D, V599L, T715P) i P-selektin-glikoprotein-1-liganda (PSGL-1 M62I). Uz to je bolesnicima pri dijagnozi napravljena krvna slika i PCR metodom s alel specifičnim početnicama utvrđen je status mutacije V617F u genu JAK2 te je u bolesnika pozitivnih na mutaciju PCR metodom u stvarnom vremenu kvantificirano opterećenje mutiranim alelom. Od 79 bolesnika s trombozom, 59 je imalo arterijsku (29 ET, 19 PV i 11 PMF), a 26 bolesnika vensku trombozu (10 ET, 8 PV i 8 PMF). U ET bolesnika leukocitoza, hiperlipidemija i mutacija V617F povećavali su rizik za nastanak tromboze, dok su nositelji alela HPA-5b imali niži rizik. U PV bolesnika rizik za trombozu bio je viši u onih sa šećernom bolesti dok su nositelji alela HPA-2b imali puno niži rizik. U PMF bolesnika rizik za trombozu bio je viši u nositelja alela 290N. Ovo istraživanje doprinijelo je novim saznanjima o etiologiji nastanka tromboze u Ph(-) MPN bolesnika. Ph(-) myeloproliferative neoplasms (MPN) are divided into three different entities including Essential thrombocythemia (ET), Polycythemia vera (PV) and Primary myelofibrosis (PMF). Thrombosis as the most common complication occurs in about 20-40% of patients. Aim of this study was to investigate genetic and hematologic prothrombotic risk factors in Ph(-) MPN patients. For this purpose we analyzed anamnestic status and blood samples of 258 Ph(-) MPN adult patients (134 ET, 70 PV and 54 PMF) and 258 healthy controls (without thrombosis) matched by age and sex. Different types od polymerase chain reactions (PCR) were performed for detection of FV Leiden, FII G20210A, human platelet antigens (HPA-1, -2, -3, -5), P-selectin (P-SEL S290N, N562D, V599L, T715P) and P-selectin glycoprotein 1 ligand (PSGL-1 M62I) polymorphisms. Blood counts were performed at diagnosis as well as detection of V617F mutation in JAK2 gene by allele specific PCR, and for V617F positive patients mutation burden allele was determined by real time quantitative PCR. Seventy-nine patients experienced 109 thrombotic events in total, 59 patients (29 ET, 19 PV and 11 PMF) had arterial thrombosis and 26 patients (10 ET, 8 PV and 8 PMF) venous thrombosis. In the group of ET patients leukocytosis, hyperlipidemia and V617F mutation increased risk for thrombosis, while for carriers of HPA-5b allele this risk was decreased. In PV patients those with diabetes had higher risk for thrombosis while in carriers of HPA-2b risk was much lower. PMF patiens with allele 290N had higher risk for thrombosis. This study brought some new findings of the thrombosis etiology in Ph(-) MPN patients.

Venous thromboembolism is a multifactorial disorder with two manifestations: deep-vein thrombosis and pulmonary embolism. Pulmonary embolism is usually considered as the complication of deep-vein thrombosis, but there are reported cases of isolated pulmonary embolism. FV Leiden and FII G20210A mutations are most common genetic risk factors for the venous thromboembolism. Several studies reported "FV Leiden paradox": lower prevalence of FV Leiden mutation among patients with isolated pulmonary embolism than among those with deep-vein thrombosis. The aim of this study was to determine FV Leiden and FII G20210A mutations frequency in thrombophilic patients in Serbian population. We tested prevalence of these mutations carriers in 1427 individuals divided in three groups of patients (with deep-vein thrombosis, deep-vein thrombosis/ pulmonary embolism and isolated pulmonary embolism) and control group. All subjects were tested for these mutations using PCR-RFLP analysis. Detected frequency of FV Leiden heterozygous carriers in patients with isolated pulmonary embolism was 6.9% (for FII G20210A 11.6%), while in other two groups of patients with deep-vein thrombosis and deep vein thrombosis/pulmonary embolism, frequency was 18.6% (for FII G20210A mutation were 11.6% and 8.3%, respectively). Our results showed that FV Leiden mutation is less frequent in patients with isolated pulmonary embolism than in patients with deep-vein thrombosis or deep-vein thrombosis accompanied with pulmonary embolism, confirming "FV Leiden paradox". On the other hand, detected frequency of FII G20210A mutation carriers was similar in all three groups of patients. [Projekat Ministarstva nauke Republike Srbije, br. 173008]

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.

Cerebrovaskularni poremećaji (CVP) su značajan medicinski problem u djece i odraslih. Cilj istraživanja bio je ispitati razlike u učestalostima polimorfizama gena za HPA (-1, -2, -3, -5), PSEL (S290N, N562D, V599L, T715P), mutacija FV G1691A (FVL) i FII G20210A u bolesnika s CVP-om u odnosu na kontrolnu skupinu, te utvrditi njihovu povezanost s bolešću. Ukupno je ispitano 300 bolesnika s CVP-om (ishemijski moždani udar-iCVI, TIA) (djece-100, odraslih-200) i 200 ispitanika kontrolne skupine (djece-100, odraslih-100), uz podatke za odrasle o standardnim čimbenicima rizika. Polimorfizmi HPA genotipizirani su PCR-SSP-om i PCR-om u stvarnom vremenu, PSEL-a PCR-SSP-om, a FVL i FII G20210A PCR-RFLP-om. U Hrvata alelne učestalosti HPA odgovaraju podacima za Europljane. Multivarijantnom regresijskom analizom u odraslih potvrđena je povezanost anamneze, hipertenzije, šećerne bolesti i alkoholizma s CVP-om, ali ne s alelom HPA-2b. U djece značajno je učestaliji genotip HPA-5a/b i alel HPA-5b u oboljelih od TIA-e u odnosu na iCVI, a u djece s genotipom GA ili s alelom A FVL, 5 puta je veća vjerojatnost oboljenja od CVP-a. Značajno učestaliji genotip GA i alel A FVL dobiven je u djece u odnosu na odrasle s CVP-om, te iCVI-om. Pomoć u predviđanju CVP-a u odraslih imaju anamneza, hipertenzija, šećerna bolest i alkoholizam, a u djece FVL. Cerebrovascular disorders (CVD) are a significant medical problem in children and adults. The aim of this research was to examine differences in the frequency of gene polymorphisms for HPA (-1, -2, -3, -5), PSEL (S290N, N562D, V599L, T715P), FV G1691A (FVL) and FII G20210A mutations in patients with CVD as compared to the control group, and to determine their correlation with the disease. The research included 300 patients with CVD (ischemic stroke-iCVI, TIA) (children-100, adults-200), 200 control subjects (children-100, adult-100), as well as data for standard risk factors for adults. HPA polymorphisms were genotyped using PCR-SSP and real-time PCR, PSEL by PCR-SSP, and the FVL and FII G20210A by PCR-RFLP. The allelic frequencies of HPA in Croats correspond to the data for Europeans. Multivariate regression analysis in adults confirmed the correlation between family history, hypertension, diabetes and alcoholism with CVD, but not with the HPA-2b allele. In children, HPA-5a/b genotype and HPA-5b allele is significantly more frequent in patients with TIA as compared to iCVI, and in children with GA genotype or with A allele FVL, there is a 5 times higher likelihood of CVD disease. Significantly more frequent GA genotype and A allele FVL was obtained in children than in adults with CVD and iCVI. Help in predicting CVD in adults is provided by family history, hypertension, diabetes and alcoholism, and in children by FVL.

Prothrombin G20210A gene variant (FII G20210A) is a risk factor for venous thrombotic disease while conflicting results have been reported for the risk of arterial thrombotic events. However, vascular episodes were absent in up to 40% of the 67 homozygotes for the G20210A described so far, which indicates that the clinical expression depends on additional risk/trigger factors. We describe six homozygotes for the G20210A variant, among which the first pair of siblings (cases n. 3 and 4) reported so far that displayed a strongly heterogeneous clinical outcome. Case 1, a female of 27 years, developed a full thrombosis of common femoral, superficial and popliteal veins. She assumed oral contraceptives in the last two years. Case n. 2, 34 years old, suffered of recurrent pregnancy loss in absence of any causative alteration. Cases n. 3 and n. 5 experienced arterial thrombotic disease, i.e., juvenile myocardial infarction (40 years old) and stroke (48 years old), respectively, in absence of other risk factors. Finally, cases n. 4 and 6 identified as homozygotes for the FII G20210A variant being consanguineous of symptomatic subjects bearing the variant, did not experience any episode of venous nor arterial disease. Both of them have chronic liver disease with an impairement of the prothrombin time INR. Thus, homozygotes for the G20210A are at risk for arterial (in addition to venous) thromobotic events; chronic liver disease might modulate this risk.