Iva Pruner

A novel prothrombin mutation in two families with prominent thrombophilia--the first cases of antithrombin resistance in a Caucasian population.

V. DJORDJEV IC ,* M. KOVAC,†‡ P . MIL J IC ,†§ M. MURATA, ¶ A. TAKAGI , ¶ I . PRUNER ,* D . FRANCUSK I , * T . KOJ IMA ¶ and D . RADOJKOVIC* *Institute of Molecular Genetics and Genetic Engineering, University of Belgrade; †Faculty of Medicine, University of Belgrade; ‡Hemostasis Department, Blood Transfusion Institute of Serbia; §Clinic of Hematology, University Clinical Center, Belgrade, Serbia; and ¶Department of Pathophysiological Laboratory Sciences, Nagoya University Graduate School of Medicine, Higashi-ku, Nagoya, Japan

Factor V Leiden mutation and high FVIII are associated with an increased risk of VTE in women with breast cancer during adjuvant tamoxifen — Results from a prospective, single center, case control study

BACKGROUND Estimates of the risk ratio of tamoxifen-associated venous thromboembolism (VTE) in breast cancer patients range from 2.4 to 7.1. The occurrence of thrombosis in patients with breast cancer complicates the clinical condition and causes a change of treatment. Our study was conducted in order to investigate the influence of patient-related risk factors for thrombosis development in breast cancer patients whose treatment included adjuvant tamoxifen. METHODS The prospective, single center, case control study included 150 breast cancer women, 50 whom developed venous thrombosis during adjuvant tamoxifen and 100 whom did not have thrombosis, as a control group. Patient-related risk factors such as: age, body mass index, previous VTE, varicose veins, concomitant diseases, the presence of prothrombotic mutations (FV Leiden, FII G20210A) and FVIII activity were evaluated in both groups. RESULTS In respect of prothrombotic mutations, the FV Leiden mutation was present in a higher number of women from the VTE group (10/50 vs 7/100; P=0.020). Additionally, FVIII activity was significantly higher in the VTE group; median (IQR), of 1.79 (0.69) vs 1.45 (0.55); P<0.001 and more women in this group (24/50 vs 34/100) had increased FVIII activity; P=0.020. In those women with FVIII>1.5IU/ml, who were carriers of prothrombotic mutations, an OR of 3.76 (CI 95% 1.276-11.096; P=0.016) was obtained for VTE. CONCLUSION The results of our study showed that the factor V Leiden mutation and high FVIII are associated with an increased risk of VTE in women with breast cancer during adjuvant tamoxifen.

+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.

Clinical and biochemical characterization of the prothrombin Belgrade mutation in a large Serbian pedigree: new insights into the antithrombin resistance mechanism

Essentials Prothrombin Belgrade mutation leads to antithrombin resistance. Clinical and biochemical phenotypes in a large family with this mutation were investigated. In carriers, we detected decreased factor II activity and increased endogenous thrombin potential. Prothrombin Belgrade mutation represents a strong prothrombotic risk factor.

The 3′End Prothrombin Gene Variants in Patients With Different Thrombotic Events

BACKGROUND Prothrombin (FII) A19911G and C20221T gene variants are associated with increased prothrombin levels and potentially represent thrombotic risk factors. OBJECTIVE To determine the frequency of A19911G and C20221T FII gene variants in patients with thrombotic disorders and in women who have experienced pregnancy loss (PL). METHODS We determined the frequency of these variants in 133 patients with deep venous thrombosis (DVT), 80 patients with isolated pulmonary embolism (PE), 101 patients with idiopathic PL, and 180 control individuals. RESULTS The FII A19911G variant was more prevalent in patients with DVT and with PL compared with controls; however, these differences were not statistically significant. The 19911GG genotype was associated with increased risk of PE (odds ratio, 1.91; 95% confidence interval, 1.04-3.51). We did not detect carriers of the FII C20221T gene variant in this study. CONCLUSIONS This is the first study, to our knowledge, that demonstrates the FII 19911GG genotype may represent a risk factor for isolated PE. Also, our results show that the FII C20221T is a rare variant in this population and therefore, routine thrombophilia screening should not include screening for this genotype.

Determination of transgene copy number in stably transfected mammalian cells by PCR-capillary electrophoresis assay.

The quantitative determination of transgene copy number in stably transfected mammalian cells has been traditionally estimated by Southern blot analysis. Recently, other methods have become available for appraisal of gene copy number, such as real-time PCR. Herein we describe a new method based on a fluorescently labeled PCR, followed by capillary electrophoresis. We amplified our target gene (prothrombin) and the internal control originating from genomic DNA (18S rRNA) in the same PCR tube and calculated the mean peak height ratio of the target:control gene for every cell clone sample. With this approach we identified stably transfected cell clones bearing the same transgene copy number. The results of our assay were confirmed by real-time PCR. Our method proves to be fast, low-cost, and reproducible compared with traditionally used methods. This assay can be used as a rapid screening tool for the determination of gene copy number in gene expression experiments.

THE PREVALENCE OF PAI-1 4G/5G POLYMORPHISM IN WOMEN WITH FETAL LOSS – FIRST DATA FOR A SERBIAN POPULATION / UČESTALOST POLIMORFIZMA PAI-1 4G/5G KOD ŽENA SA SPONTANIM POBAČAJEM - PRVI PODACI ZA SRPSKU POPULACIJU

Summary Background: Plasminogen activator inhibitor 1 (PAI-1) is an inhibitor of fibrinolysis. The PAI-1 4G/5G polymorphism is associated with elevated plasma levels of PAI-1. Over- expression of PAI-1 and impaired fibrinolysis in homozygous carriers of the 4G/4G PAI polymorphism may lead to abnor- mal placental formation and increased risk of fetal loss (FL). The aim of our study was to determine the frequency of this polymorphism in patients with FL in a Serbian population. Methods: The study was carried out in a group of 203 women (91 controls and 112 women with FL). The presence of PAI-1 4G/5G polymorphism was detected by PCR-RFLP analysis. Results: Slightly increased frequency of the PAI-1 4G/4G genotype was observed in the study group compared to the controls (32.1% vs. 30.8%). The frequency of PAI-1 was highest in women experiencing FL in the second trimester of pregnancy (50%), but this difference was not statistically sig- nificant. Conclusions: Our findings suggest that PAI-1 4G/4G might be a risk factor for FL occurring in the second trimester of pregnancy. Further studies are required in order to determine the role of PAI-1 4G/5G polymorphism in the etiology of FL. Kratak sadržaj Uvod: Inhibitor aktivatora plazminogena 1 (PAI-1) igra zna- čajnu ulogu u procesu inhibicije fibrinolize. Pokazano je da je PAI-1 4G/5G polimorfizam povezan sa povišenim nivoom PAI-1 proteina u plazmi. Povećana ekspresija PAI-1 i sma- njena fibrinoliza kod homozigotnih nosilaca PAI-1 4G/5G polimorfizma može dovesti do poremećaja tokom formiranja placente i povećanja rizika za spontane pobačaje (SP). U okviru ove studije analizirali smo učestalost PAI-1 4G/5G polimorfizma kod pacijentkinja sa spontanim pobačajima. Metode: Studija je obuhvatila grupu od 203 žene (91 u kon- trolnoj grupi i 112 žena sa SP). Prisustvo PAI-1 4G/5G poli- morfizama je detektovano PCR-RFLP analizom. Rezultati: Detektovana učestalost homozigotnih nosilaca PAI-1 4G/5G polimorfizma je bila nešto visa u grupi paci- jentkinja u odnosu na kontrolnu grupu (32,1% vs. 30,8%). Najviša učestalost je detektovana kod žena koje su imale SP u drugom trimestru trudnoće (50%), ali ova razlika nije bila statistički značajna. Zaključak: Rezultati naše studije ukazuju da PAI-1 4G/4G može biti faktor rizika za pojavu SP u drugom trimestru trud- noće. Potrebna su dalja ispitivanja u cilju određivanja uloge PAI-1 4G/4G polimorfizma u etiologiji spontanih pobačaja.

The effect of FII c.1787G>A (prothrombin Belgrade) mutation on prothrombin gene expression in vitro

The FII c.1787G>A (prothrombin Belgrade) is a novel prothrombotic mutation which leads to impaired inhibition of thrombin by antithrombin (antithrombin resistance). So far, the mechanism of this variant has not been fully elucidated. To investigate the effect of FII c.1787G>A mutation on the prothrombin gene expression, its functional analysis was performed in vitro. By Real-Time PCR, expression levels of FII gene variants were evaluated in Cos-7 cells transiently transfected with c.1787G (wild-type) and c.1787A prothrombin expression vectors, with no differences observed. The relative quantification of prothrombin protein amounts was accomplished by Western blot analysis, also with no differences observed. Therefore, the mechanism of FII c.1787G>A mutation does not alter prothrombin expression profile.

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

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

The 3’ End Prothrombin Gene Variants in Serbian Patients with Idiopathic Thrombophilia

Abstract Thrombophilia is a multifactorial disorder that arises from the interaction of acquired and genetic risk factors. Despite the significant efforts made to understand the etiology of this disease, there are still a certain number of patients suffering from idiopathic thrombophilia. The aim of this study was to screen the 3’ end of the prothrombin (FII) gene, which is susceptible to gain-of-function mutations due to its non canonical architecture, in patients with idiopathic thrombophilia and to determine its eventual role in the pathogenesis of thrombophilia. This study was carried out in 100 patients with idiopathic thrombophilia and 100 healthy controls. DNA variants in the 715 bp long region of the 3’ end of the prothrombin gene were identified by sequencing. In our study, we detected two variants: A19911G and C20068T. The frequency of the A19911G gene variant was slightly increased in the group of patients compared to controls, however with no statistically significant difference compared to controls [odds ratio (OR) = 1.06; 95% confidence interval (95% CI) 0.53-2.13]. Heterozygous carriers of the FII C20068T gene variant were four times more frequent in patients (4.0%) than in controls (1.0%), but this difference did not reach statistical significance (OR = 4.12; 95% CI 0.45-37.57). Our findings suggest that variant A19911G is not a significant risk factor, while C20068T may represent a potential risk factor for idiopathic thrombophilia. To confirm our results, further studies should be conducted in a larger cohort of patients.