The Silence Speaks, but We Do Not Listen: Synonymous c.1824C>T Gene Variant in the Last Exon of the Prothrombin Gene as a New Prothrombotic Risk Factor.

BACKGROUND: Thrombosis is a major global disease burden with almost 60% of cases related to underlying heredity and most cases still idiopathic. Synonymous single nucleotide polymorphisms (sSNPs) are considered silent and phenotypically neutral. Our previous study revealed a novel synonymous FII c.1824C>T variant as a potential risk factor for pregnancy loss, but it has not yet been associated with thrombotic diseases. METHODS: To determine the frequency of the FII c.1824C>T variant we have sequenced patients' DNA. Prothrombin RNA expression was measured by quantitative PCR. Functional analyses included routine hemostasis tests, western blotting and ELISA to determine prothrombin levels in plasma, and global hemostasis assays for thrombin and fibrin generation in carriers of the FII c.1824C>T variant. Scanning electron microscopy was used to examine the structure of fibrin clots. RESULTS: Frequency of the FII c.1824C>T variant was significantly increased in patients with venous thromboembolism and cerebrovascular insult. Examination in vitro demonstrated increased expression of prothrombin mRNA in FII c.1824T transfected cells. Our ex vivo study of FII c.1824C>T carriers showed that the presence of this variant was associated with hyperprothrombinemia, hypofibrinolysis, and formation of densely packed fibrin clots resistant to fibrinolysis. CONCLUSION: Our data indicate that FII c.1824C>T, although a synonymous variant, leads to the development of a prothrombotic phenotype and could represent a new prothrombotic risk factor. As a silent variant, FII c.1824C>T would probably be overlooked during genetic screening, and our results show that it could not be detected in routine laboratory tests.

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

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

Predictive genetic markers of coagulation, inflammation and apoptosis in Perthes disease­Serbian experience.

UNLABELLED: Perthes disease is one of the most common forms of pediatric femoral head osteonecrosis with an unknown etiology. Coagulation factors were the first genetic factors suspected to have a role in the pathogenesis of this disease, but studies showed inconsistent results. It is described that inflammation is present during early stages of Perthes disease, but its genetic aspect has not been studied extensively. Little is known regarding the status of apoptotic factors during the repair process that leads to the occurrence of hip deformity in patients. Therefore, the aim of this study was to analyze major mediators involved in coagulation, inflammation, and apoptotic processes as possible causative factors of Perthes disease. The study cohort consisted of 37 patients. Gene variants of TNF-α, FV, FII, and MTHFR genes were determined by PCR-RFLP, while IL-3 and PAI-1 were genotyped by direct sequencing. The expression level of Bax, Bcl-2, Bcl2L12, Fas and FasL was analyzed by quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) technique. Our results showed a significantly increased level of expression of pro-apoptotic factor Bax along with significantly higher Bax/Bcl-2 ratio in the patient group. CONCLUSION: The results presented indicate that apoptosis could be one of the factors contributing to the lack of balanced bone remodeling process in Perthes patients.

Prothrombin 3'end gene variants in isolated pulmonary embolism--the first report of FIIc.*64_*66del and FIIc.*303T>C variants.

OBJECTIVE: Pulmonary embolism is usually considered as a complication of deep vein thrombosis, but there are still a number of cases of isolated pulmonary embolism. We aimed to investigate whether prothrombin 3'end gene variants might play a significant role in the pathogenesis of isolated pulmonary embolism. METHODS AND RESULTS: In this study 100 patients with isolated pulmonary embolism and 100 controls were screened by DNA sequencing. Screening included last intron, last exon, 3'UTR and part of the 3'FR region of the prothrombin gene. Our results have shown that heterozygous carriers of the FIi G2021 OA variant have a significantly higher risk of isolated pulmonary embolism (OR 4.83; 95% CI 1.33-17.52; P=0.02). Carriers of the Ili 19911GG genotype (OR 1.41; 95% CI 0.72-2.73; P=0.31) and FII 20068CT genotype (OR 3.06; 95% CI 0.31-29.95; P=0.34) were more frequent in patients with isolated pulmonary embolism compared to controls. We also detected the novel gene variants, FIIc.*64_*66del and FII c.*303T>C, in two patients. CONCLUSIONS: Our results suggest that FII G20210A represents a significant risk factor for isolated pulmonary embolism. The FII G19911A and FII C20068T are potentially associated with an increased risk for the occurrence of isolated pulmonary embolism, but the results did not reach statistical significance. This is the first study in which the two novel 3'end prothrombin gene variants, FIIc.*64_*66del and FlI c.*303T>C, were reported.

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.

Effect of prothrombin Belgrade mutation, causing antithrombin resistance, on fibrin clot properties.

INTRODUCTION: Prothrombin Belgrade mutation is the result of the c.1787G>A substitution in the prothrombin gene. It is located in the antithrombin and sodium binding site and leads to impaired inactivation of thrombin by antithrombin, resulting in antithrombin resistance and thrombotic disorders. However, it negatively affects sodium binding and may have hypocoagulant effects. Considering that prothrombin Belgrade mutation mechanism is still not fully elucidated and that sodium binding is important for thrombin affinity towards fibrinogen, our aim was to determine whether this mutation affects fibrin clot formation and lysis. METHODS: Using HEK293T cell line, recombinant wild type and mutated prothrombin were generated by transient transfection. Samples that correspond to plasma of a non-carrier, heterozygous and homozygous carriers were reconstituted using prothrombin deficient plasma and recombinant proteins. Reconstituted samples were used in OHP assay (Overall Hemostasis Potential) to determine kinetic profiles of coagulation and fibrinolysis. Clot turbidity assay was performed to observe kinetics of clot formation and lysis more closely. Fibrin clots formed in reconstituted plasma samples were analyzed by confocal microscopy to determine density of fibrin network. Fibrin clots were additionally observed using electron microscopy to determine thickness of individual fibrin fibers. RESULTS: No significant difference found in OHP, OCP, OFP, and fibrin network density between wild type, heterozygous, and homozygous carrier reconstituted plasma samples. There were significant differences between samples for slope and slope time parameters in kinetic profiles and fibrin fiber thickness. CONCLUSIONS: Results indicate that prothrombin Belgrade mutation has no significant impact on fibrinolysis, however it may affect kinetics of clot formation and its architecture.

Genome sequence diversity of SARS-CoV-2 in Serbia: insights gained from a 3-year pandemic study.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), responsible for the COVID-19 pandemic, has been evolving rapidly causing emergence of new variants and health uncertainties. Monitoring the evolution of the virus was of the utmost importance for public health interventions and the development of national and global mitigation strategies. Here, we report national data on the emergence of new variants, their distribution, and dynamics in a 3-year study conducted from March 2020 to the end of January 2023 in the Republic of Serbia. Nasopharyngeal and oropharyngeal swabs from 2,398 COVID-19-positive patients were collected and sequenced using three different next generation technologies: Oxford Nanopore, Ion Torrent, and DNBSeq. In the subset of 2,107 SARS-CoV-2 sequences which met the quality requirements, detection of mutations, assignment to SARS-CoV-2 lineages, and phylogenetic analysis were performed. During the 3-year period, we detected three variants of concern, namely, Alpha (5.6%), Delta (7.4%), and Omicron (70.3%) and one variant of interest-Omicron recombinant "Kraken" (XBB1.5) (<1%), whereas 16.8% of the samples belonged to other SARS-CoV-2 (sub)lineages. The detected SARS-CoV-2 (sub)lineages resulted in eight COVID-19 pandemic waves in Serbia, which correspond to the pandemic waves reported in Europe and the United States. Wave dynamics in Serbia showed the most resemblance with the profile of pandemic waves in southern Europe, consistent with the southeastern European location of Serbia. The samples were assigned to sixteen SARS-CoV-2 Nextstrain clades: 20A, 20B, 20C, 20D, 20E, 20G, 20I, 21J, 21K, 21L, 22A, 22B, 22C, 22D, 22E, and 22F and six different Omicron recombinants (XZ, XAZ, XAS, XBB, XBF, and XBK). The 10 most common mutations detected in the coding and untranslated regions of the SARS-CoV-2 genomes included four mutations affecting the spike protein (S:D614G, S:T478K, S:P681H, and S:S477N) and one mutation at each of the following positions: 5'-untranslated region (5'UTR:241); N protein (N:RG203KR); NSP3 protein (NSP3:F106F); NSP4 protein (NSP4:T492I); NSP6 protein (NSP6: S106/G107/F108 - triple deletion), and NSP12b protein (NSP12b:P314L). This national-level study is the most comprehensive in terms of sequencing and genomic surveillance of SARS-CoV-2 during the pandemic in Serbia, highlighting the importance of establishing and maintaining good national practice for monitoring SARS-CoV-2 and other viruses circulating worldwide.

A phenotype driven integrative framework uncovers molecular mechanisms of a rare hereditary thrombophilia.

Antithrombin resistance is a rare subtype of hereditary thrombophilia caused by prothrombin gene variants, leading to thrombotic disorders. Recently, the Prothrombin Belgrade variant has been reported as a specific variant that leads to antithrombin resistance in two Serbian families with thrombosis. However, due to clinical data scarcity and the inapplicability of traditional genome-wide association studies (GWAS), a broader perspective on molecular and phenotypic mechanisms associated with the Prothrombin Belgrade variant is yet to be uncovered. Here, we propose an integrative framework to address the lack of genomic samples and support the genomic signal from the full genome sequences of five heterozygous subjects by integrating it with subjects' phenotypes and the genes' molecular interactions. Our goal is to identify candidate thrombophilia-related genes for which our subjects possess germline variants by focusing on the resulting gene clusters of our integrative framework. We applied a Non-negative Matrix Tri-Factorization-based method to simultaneously integrate different data sources, taking into account the observed phenotypes. In other words, our data-integration framework reveals gene clusters involved with this rare disease by fusing different datasets. Our results are in concordance with the current literature about antithrombin resistance. We also found candidate disease-related genes that need to be further investigated. CD320, RTEL1, UCP2, APOA5 and PROZ participate in healthy-specific or disease-specific subnetworks involving thrombophilia-annotated genes and are related to general thrombophilia mechanisms according to the literature. Moreover, the ADRA2A and TBXA2R subnetworks analysis suggested that their variants may have a protective effect due to their connection with decreased platelet activation. The results show that our method can give insights into antithrombin resistance even if a small amount of genetic data is available. Our framework is also customizable, meaning that it applies to any other rare disease.

Clinical characteristics and type of thrombophilia in women with pregnancy-related venous thromboembolic disease.

BACKGROUND: Normal pregnancy is characterized by numerous changes in the hemostatic system, creating the hypercoagulable state which increases the risk of venous thromboembolic event (VTE) occurrence. The risk is further increased by the presence of inherited or acquired thrombophilia. OBJECTIVE: In this study, we aimed to determine the prevalence of different types of thrombophilia in women with pregnancy-related VTE, and to investigate the possible connection between the type of thrombophilia and localization of VTE as well as the gestational age of VTE occurrence. PARTICIPANTS AND METHODS: Two hundred and two women with the first episode of pregnancy-related VTE and 130 controls were investigated. The antithrombin, protein C and protein S activity, APC resistance, FVG1691A, and FIIG20210A were determined. None of the investigated women was pregnant at the time of thrombophilia testing, and none was using oral contraceptives. RESULTS: Thrombophilia was diagnosed in 95 patients (47%) and 7 controls (5.4%). The prevalence of FV Leiden, FIIG20210A mutations, antithrombin, PC and PS deficiencies taken together and combined thrombophilia was 22.3, 10.4, 6.9 and 6.9%, respectively. Significantly more frequent antepartum occurrence of VTE (11 vs. 3, p < 0.05) was found in women with natural coagulation inhibitor deficiency. Pulmonary embolism occurred more frequently in nonthrombophilic women (25 vs. 3, p < 0.001). CONCLUSION: Inherited thrombophilia was found to be considerably more frequently present in women with pregnancy- and puerperium-related VTE compared to healthy controls. Women with thrombophilia are at higher risk of developing thromboses localized in the iliacofemoral region, and women without thrombophilia are at higher risk of developing pulmonary embolism. Deficiency in natural coagulation inhibitors is associated with antepartum VTE occurrence.

Prothrombin 3'end Gene Variants in Patients With Sporadic Colon Adenocarcinoma.

BACKGROUND/AIM: Thrombin plays significant roles in various types of cancer. However, the expression levels of prothrombin, the thrombin precursor, in cancer remain unclear. Variants of the 3'end of the prothrombin gene lead to increased prothrombin expression. This study aimed to analyze prothrombin 3'end gene variants in colon tumor and adjacent normal tissue samples. MATERIALS AND METHODS: The study group consisted of 93 patients suffering from colon adenocarcinoma. The 3'end of the prothrombin gene was analyzed by DNA sequencing. RESULTS: Three variants, all previously associated with increased prothrombin expression were detected. Frequency of the FII 19911G allele was 46.77% and 47.85% in tumor and normal tissue, respectively. For the FII 20210A allele, the detected frequencies were 2.15% and 1.61%, respectively. The frequency of the FII c.1824T allele was 0.54% in both tissues. Four patients showed different genotypes in tumor and normal tissue. CONCLUSION: Prothrombin 3' end gene variants may play a role in colorectal cancer.

MTHFR C677T polymorphism in chronic pancreatitis and pancreatic adenocarcinoma.

Chronic pancreatitis and pancreatic adenocarcinoma are extensively studied as common and potentially lethal disorders. However, their causes and genetic background in most cases remain unclear. The C677T polymorphism in 5',10'-methylenetetrahydrofolate reductase (MTHFR) gene may modulate the risk of pancreatic disorders. In this study, we tested whether MTHFR C677T polymorphism is associated with chronic pancreatitis and pancreatic adenocarcinoma in the Serbian population. DNA was extracted from blood samples of 51 chronic pancreatitis patients, 21 pancreatic adenocarcinoma patients, and a control group consisting of 50 healthy smokers. The MTHFR C677T polymorphism was analyzed by polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) technique. Although, no statistically significant differences were observed in the distribution of MTHFR genotype or allele frequencies between patients and control groups, the results showed an increased frequency of homozygotes for MTHFR C677T polymorphism in chronic pancreatitis patients (14%) and a decreased frequency in pancreatic adenocarcinoma patients (5%) in comparison to the control group (8%). We speculate that the MTHFR C677T polymorphism could act as a possible risk factor for chronic pancreatitis and a possible protective factor in pancreatic adenocarcinoma. This observation needs further investigation in prospective studies on a larger number of patients, in which the effect of other genetic and environmental factors should also be taken into consideration.

Influence of decreased fibrinolytic activity and plasminogen activator inhibitor-1 4G/5G polymorphism on the risk of venous thrombosis.

: Objective of our study is to determine whether decreased fibrinolytic activity or plasminogen activator inhibitor (PAI)-1 4G/5G polymorphism influence the risk of venous thrombosis.Our case-control study included 100 patients with venous thrombosis, and 100 random controls. When patients were compared with random controls, unconditional logistic regression was used to calculate odds ratios (ORs) with 95% confidence intervals (CIs).Decreased fibrinolytic activity yielded a 2.7-fold increase in risk for venous thrombosis than physiological fibrinolytic activity (OR 2.70; 95% CI 1.22-5.98), when comparing patients with random controls. Adjustment for several putative confounders did not change the estimate (OR 3.02; 95% CI 1.26-7.22). Analysis of venous thrombotic risk influenced by PAI-1 genotype, showed no influence of PAI-1 4G/5G gene variant in comparison with 5G/5G genotype (OR 0.57 95% CI; 0.27-1.20).Decreased fibrinolytic activity increased, whereas PAI-1 4G/5G polymorphism did not influence venous thrombosis risk in this study.

Arterial ischemic stroke in a child with beta-thalassemia trait and methylentetrahydrofolate reductase mutation.

Genetic and acquired disorders that foster a procoagulable state represent risk factors for stroke in childhood. Although an increased incidence of thromboembolic complications has been reported in patients with thalassemia, severe cerebral thromboembolism has rarely been observed in patients with beta-thalassemia minor. This article describes a case study of a 1-year-old boy who presented with left-sided hemiparesis, seizures, microcytic anemia, and recent infection with reactive thrombocytosis. Ischemic infarction in the territory of the right middle cerebral artery was confirmed by magnetic resonance imaging and magnetic resonance angiography. Genetic tests showed that the patient was heterozygous for the beta(degrees) -thalassemia IVS-I-1 mutation and homozygous for the methylentetrahydrofolate reductase C677T mutation. Based on these findings, it was concluded that the synergistic effects of multiple, genetic, and acquired prothrombotic risk factors brought about the hypercoagulable state that resulted in overt stroke in a thalassemic patient in early childhood.

Early onset of abdominal venous thrombosis in a newborn with homozygous type II heparin-binding site antithrombin deficiency.

: The overall incidence of thromboembolic events in the neonatal period is 5 per 100 000 births, wherein more than 40% of all such manifestations are symptomatic renal vein thromboses. We describe the case of a newborn female who developed extensive thrombosis, which filled the inferior vena cava and renal vein and was diagnosed in the first weeks of life. A homozygous type II heparin-binding site antithrombin deficiency (c. 391C>T, p. Leu131Phe) was detected in the background. Despite the timely diagnosis and appropriate treatment, clinical signs of renal insufficiency, because of left kidney atrophy and arterial hypertension, were observed. Our case demonstrates the seriousness of the consequences arising after early onset of venous thrombosis caused by homozygous type II heparin-binding site antithrombin deficiency. In addition to prompt diagnosis, of huge importance is the determination of inherited thrombophilia, as it significantly affects therapeutic treatment and indicates that long-term follow-up is mandatory.

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.

Screening of alpha-1-antitrypsin gene by denaturing gradient gel electrophoresis (DGGE).

Alpha-1-antitrypsin (AAT) is a serine protease inhibitor whose deficiency could cause emphysema and liver disease and, as recently described, could be a risk factor for lung cancer development. Alpha-1-antitrypsin inhibits a variety of proteases but its primary target is neutrophil elastase, an extracellular endopeptidase capable of degrading most protein components of the extracellular matrix. Inhibition of neutrophil elastase by AAT has an important role in maintaining the integrity of connective tissue. The gene encoding for AAT spans over 12.2 kb, consists of seven exons and is highly polymorphic. Therefore several methods for mutation screening of alpha-1-antitrypsin gene have been developed. Method described here is based on denaturing gradient gel electrophoresis (DGGE). This method is highly efficient and reliable and allows rapid analysis of entire coding region of alpha-1-antitrypsin gene, including splice junction sites. Previously described DGGE based analysis of AAT gene included overnight electrophoresis of individually amplified fragments. The optimization of the method described in this paper is directed towards the shortening of the duration of electrophoresis and amplification of fragments in multiplex reaction in order to make the analysis less time-consuming and therefore more efficient.

Gene-environment interaction between the MMP9 C-1562T promoter variant and cigarette smoke in the pathogenesis of chronic obstructive pulmonary disease.

The aetiology of chronic obstructive pulmonary disease (COPD) is complex. While cigarette smoking is a well-established cause of COPD, a myriad of assessed genetic factors has given conflicting data. Since gene-environment interactions are thought to be implicated in aetiopathogenesis of COPD, we aimed to examine the matrix metalloproteinase (MMP) 9 C-1562T (rs3918242) functional variant and cigarette smoke in the pathogenesis of this disease. The distribution of the MMP9 C-1562T variant was analyzed in COPD patients and controls with normal pulmonary function from Serbia. Interaction between the C-1562T genetic variant and cigarette smoking was assessed using a case-control model. The response of the C-1562T promoter variant to cigarette smoke condensate (CSC) exposure was examined using a dual luciferase reporter assay. The frequency of T allele carriers was higher in the COPD group than in smoker controls (38.4% vs. 20%; OR = 2.7, P = 0.027). Interaction between the T allele and cigarette smoking was identified in COPD occurrence (OR = 4.38, P = 0.005) and severity (P = 0.001). A functional analysis of the C-1562T variant demonstrated a dose-dependent and allele-specific response (P < 0.01) to CSC. Significantly higher MMP9 promoter activity following CSC exposure was found for the promoter harboring the T allele compared to the promoter harboring the C allele (P < 0.05). Our study is the first to reveal an interaction between the MMP9-1562T allele and cigarette smoke in COPD, emphasising gene-environment interactions as a possible cause of lung damage in the pathogenesis of COPD. Environ. Mol. Mutagen. 57:447-454, 2016. © 2016 Wiley Periodicals, Inc.

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.

Thrombotic events in acute promyelocytic leukemia.

INTRODUCTION: Thrombotic events (TE) appear to be more common in acute promyelocytic leukemia (APL) than in other acute leukemias, with reported prevalence ranging from 2 to 10-15%. MATERIALS AND METHODS: We retrospectively analyzed the data on TE appearance in 63 APL patients. RESULTS: TE occured in 13 (20.6%) cases, four arterial (6.3%) and nine venous (14.3%). TE were more frequently diagnosed after initiation of weekly D-dimer monitoring (7 TE during 20 months vs 6 during 76 months, P=0.032). Patients with and without venous thrombosis were significantly different regarding female/male ratio (P=0.046), PT (P=0.022), aPTT (P=0.044), ISTH DIC score (P=0.001), bcr3 (P=0.02) and FLT3-ITD (P=0.028) mutation. The most significant risk factor for venous TE occurrence in multivariate analysis was FLT3-ITD mutation (P=0.034). PAI-1 4G/4G polymorphism was five times more frequent in patients with venous TE than without it (P=0.05). Regarding risk factors for arterial TE we failed to identify any. CONCLUSIONS: We have demonstrated that APL-related TE rate is higher than previously reported and that weekly D-dimer monitoring might help to identify patients with silent thrombosis. Moreover, our study suggests a possible relationship between venous TE occurrence and several laboratory findings (PT, aPTT, ISTH DIC score, bcr3 isoform, FLT3-ITD mutation and PAI 4G/4G). Prophylactic use of heparin might be considered in patients with ISTH DIC score<5, bcr3 isoform, FLT3-ITD mutation and PAI 4G/4G.

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.