Search Results (11)

Factor V (FV) Leiden and F2 G20210A are inherited genetic risk factors that are in the first line of laboratory tests for thromboembolic diseases. Their detection relies on PCR assays, which are subject to contamination, as well as pipetting error, when manually performed and require individual assays for each gene. In this article, we report an improved PCR and restriction endonuclease assay for the simultaneous detection of the FV Leiden and F2 G20210A variants, based on multiplex amplification with fluorescent primers, digestion control, identity monitoring, and contamination tracking. Full article

Background and Objectives: Thrombophilia is a prothrombotic disorder that increases the risk of blood clotting and can pose serious health problems. It is considered a condition of gene–gene or gene–environment interactions. Variation in the prevalence of thrombophilia mutations and their interaction among populations necessitates localized genetic assessments. However, population-based genetic data remains limited for developing effective preventive strategies. Materials and Methods: This cross-sectional observational study was conducted over five years (2020–2024) at a tertiary university hospital in Northern Greece. A total of 2961 individuals aged 18–85 years (mean: 50.5) were registered based on family or medical history of venous thromboembolism (VTE) or clinical symptoms of VTE. The final analysis included 2078 participants comprising 1143 males (55%) and 935 females (45%), who met all the inclusion criteria. Inclusion criteria were absence of acute illness or malignancy, informed consent, and an adequate DNA quantity for genotyping, whereas excluded criteria included incomplete laboratory data, active inflammatory or malignant disease, and cognitive or psychiatric conditions. Peripheral blood samples were collected in 2 mL K₃-EDTA tubes, and genomic DNA was analyzed using real-time polymerase chain reaction (PCR) with melting curve analysis and hybridization probes (LightMix^® in vitro diagnostics, TIB MolBiol, Berlin, Germany). Five thrombophilia-related polymorphisms, Factor V Leiden (F5 G1691A), prothrombin (F2 G20210A), methylenetetrahydrofolate reductase (MTHFR C677T and MTHFR A1298C), and Plasminogen Activator Inhibitor-1 (PAI-1) 4G/5G, were examined for allele and genotype frequencies, Hardy–Weinberg equilibrium testing, pairwise linkage disequilibrium (D′ and r²), and power analysis. For subjects tested for Factor V Leiden (n = 1476), the activated protein C resistance (APC) ratio was additionally evaluated using the ACL TOP 750 analyzer. Results: Allele frequencies were 7.3% for FV Leiden and 3.7% for FII. The PAI-1 allele was distributed at 44%, while the MTHFR (C677T and A1298C) alleles were each present at 33%. Significant linkage disequilibrium was identified between MTHFR (C677T and A1298C) and between MTHFR A1298C and PAI-1. No evolutionary pressure or demographic bias was found in the Hardy–Weinberg equilibrium. The APC ratio demonstrated a high sensitivity (99.2%) and specificity (96.6%), indicating that it may serve as a reliable screening method. Conclusions: Our findings highlight informative patterns in the genetic predisposition to thrombophilia, which may help develop rule-based strategies for implementing thromboprophylaxis guidelines and personalized medical interventions. Full article

Background/Objectives: Cancer progression and the hemostatic system are closely linked. Coagulation factor V (FV) has a key function in coagulation, with both pro- and anticoagulant functions. FV gene (F5) expression and F5 variants have been linked to breast cancer progression. The direct impact of F5 variants on FV expression and functional effects in breast cancer are unknown. We aimed to investigate whether the F5 variants FV Leiden (F5 rs6025) and F5 rs6028 influenced FV expression, coagulant activity, and apoptosis in breast cancer cells. Methods: MDA-MB-231 cells were transfected with overexpression plasmids containing F5 wild type, F5 rs6025 or F5 rs6028. We investigated the functional impact of the F5 variants on F5 mRNA, FV protein, FV coagulant activity, and apoptosis in vitro, and examined the potential of the variants as transcriptional regulators of F5 expression in silico. Results: Increased F5 mRNA, FV protein, and apoptosis were observed in cells transfected with F5 wild-type overexpression plasmid compared to empty vector. F5 mRNA, protein, coagulant activity, and apoptosis were further increased with the F5 rs6025 and F5 rs6028 variants compared to F5 wild type. Cis-expression quantitative trait loci analyses indicated a regulatory effect of F5 rs6028, and putative transcription factor binding sites for several transcription factors overlapped with the position of F5 rs6025. Conclusions: Our study demonstrated that F5 rs6025 and F5 rs6028 have a regulatory effect on FV synthesis that might affect apoptosis in breast cancer. The F5 variants might therefore enhance the tumor suppressor function of FV in breast cancer. Full article

Inherited defects in the genes of blood coagulation essentially express the severity of the clinical phenotype that is directly correlated to the number of mutated alleles of the candidate leader gene (e.g., heterozygote vs. homozygote) and of possible additional coinherited traits. The F5 gene, which codes for coagulation factor V (FV), plays a two-faced role in the coagulation cascade, exhibiting both procoagulant and anticoagulant functions. Thus, defects in this gene can be predisposed to either bleeding or thrombosis. A Sanger sequence analysis detected a premature stop-codon in exon 13 of the F5 gene (c.3481C>T; p.R1161Ter) in several members of a family characterised by low circulating FV levels and contrasting clinical phenotypes. The propositus, a 29 y.o. male affected by recurrent haemorrhages, was homozygous for the F5 stop-codon and for the F5 c.1691G>A (p.R506Q; FV-Leiden) inherited from the heterozygous parents, which is suggestive of combined cis-segregation. The homozygous condition of the stop-codon completely abolished the F5 gene expression in the propositus (FV:Ag < 1%; FV:C < 1%; assessed by ELISA and PT-based one-stage clotting assay respectively), removing, in turn, any chance for FV-Leiden to act as a prothrombotic molecule. His father (57 y.o.), characterised by severe recurrent venous thromboses, underwent a complete molecular thrombophilic screening, revealing a heterozygous F2 G20210A defect, while his mother (56 y.o.), who was negative for further common coagulation defects, reported fully asymptomatic anamnesis. To dissect these conflicting phenotypes, we performed the ProC^®Global (Siemens Helthineers) coagulation test aimed at assessing the global pro- and anticoagulant balance of each family member, investigating the responses to the activated protein C (APC) by means of an APC-sensitivity ratio (APC-sr). The propositus had an unexpectedly poor response to APC (APC-sr: 1.09; n.v. > 2.25), and his father and mother had an APC-sr of 1.5 and 2.0, respectively. Although ProC^®Global prevalently detects the anticoagulant side of FV, the exceptionally low APC-sr of the propositus and his discordant severe–moderate haemorrhagic phenotype could suggest a residual expression of mutated FV p.506QQ through a natural readthrough or possible alternative splicing mechanisms. The coagulation pathway may be physiologically rebalanced through natural and induced strategies, and the described insights might be able to track the design of novel treatment approaches and rebalancing molecules. Full article

Background: FV Leiden is an autosomal dominant disease, representing one of the most prevalent genetic causes for hereditary thrombophilia manifested by venous thromboembolism. Methods: We report a case of a 30-year-old patient who was admitted for enrollment in phase II cardiac rehabilitation. The cardiovascular disease onset was five years ago when the patient was diagnosed with superficial vein thrombosis, for which anticoagulant treatment was recommended. However, he discontinued the prescribed treatment independently, which resulted in the development of deep vein thrombosis. A screening for risk factors associated with venous thromboembolism was conducted, leading to the identification of a heterozygous mutation of factor V Leiden. Later, the patient was hospitalized for acute coronary syndrome necessitating stent implantation. Following this procedure, the patient started a cardiac rehabilitation program, where the patient received multidisciplinary counseling. Conclusions: At the end of the cardiac rehab, significant improvements were observed in clinical and hemodynamic parameters. Consequently, the patient was advised to continue rehabilitation treatment in the outpatient setting. Also, for patients with suboptimal maintenance of the therapeutic range of INR, the use of apixaban might be considered. Furthermore, the utilization of a reduced dosage of apixaban has demonstrated its effectiveness in preventing further venous thromboembolism. Full article

Factor V (FV) Leiden and prothrombin G20210A are the most common hereditary thrombophilias. While their role in venous thromboembolism is well known, there are still uncertainties regarding their relationship with arterial thrombotic events, especially coronary ones. Our research, based on an in-depth analysis of the available literature, provides up-to-date information on the relationship between FV Leiden and prothrombin G20210A and acute myocardial infarction. FV Leiden and prothrombin G20210A screening should be implemented only in select cases, such as acute coronary syndrome in young individuals and/or in the absence of traditional cardiovascular risk factors and/or in the absence of significant coronary artery stenosis at angiography. Their identification should be followed by the implementation of optimal control of modifiable traditional cardiovascular risk factors to reduce the risk of recurrent events and genotyping and genetic counseling of all family members of affected cases for proper prophylaxis. An extended dual antiplatelet therapy (DAPT) may be considered, given the lower risk of bleeding under DAPT conferred by FV Leiden. Full article

Antiphospholipid syndrome (APS) is a hypercoagulable state accompanied by the presence of heterogeneous antiphospholipid antibodies (aPL), which nonspecifically affect hemostasis by the presence of lupus anticoagulans (LA), anticardiolipin antibodies (aCL), antibodies against β2-glycoprotein-I (anti-β2GPI), but also non-criteria antibodies such as antibodies against β2-glycoprotein-I domain I (anti-DI), anti-phosphatidylserine/prothrombin (anti-PS/PT), anti-annexin V, and many others. The main target of the antibodies is the activated protein C (APC) system, the elimination of which can manifest itself as a thrombotic complication. The aim of this study was to determine the thrombogenicity of antibodies using a modified protein C-activated thrombin generation assay (TGA) on a group of 175 samples suspected of APS. TGA was measured with/without APC and the ratio of both measurements was evaluated (as for APC resistance), where a cut-off was calculated ≤4.5 (90th percentile) using 21 patients with heterozygous factor V Leiden mutation (FV Leiden heterozygous). Our study demonstrates the well-known fact that multiple positivity of different aPLs is a more severe risk for thrombosis than single positivity. Of the single antibody positivity, LA antibodies are the most serious (p value < 0.01), followed by aCL and their subgroup anti-DI (p value < 0.05). Non-criteria antibodies anti-annexin V and anti-PT/PS has a similar frequency occurrence of thrombogenicity as LA antibodies but without statistical significance or anti-β2GPI1 positivity. The modified TGA test can help us identify patients in all groups who are also at risk for recurrent thrombotic and pregnancy complications; thus, long-term prophylactic treatment is appropriate. For this reason, it is proving increasingly beneficial to include the determination antibodies in combination with modified TGA test. Full article

Venous thromboembolism (VTE) constitutes a serious and potentially fatal disease, often complicated by pulmonary embolism and is associated with inherited or acquired factors risk. A series of risk factors are known to predispose to venous thrombosis, and these include mutations in the genes that encode anticoagulant proteins as antithrombin, protein C and protein S, and variants in genes that encode instead pro-coagulant factors as factor V (FV Leiden) and factor II (FII G20210A). However, the molecular causes responsible for thrombotic events in some individuals with evident inherited thrombosis remain unknown. An improved knowledge of risk factors, as well as a clear understanding of their role in the pathophysiology of VTE, are crucial to achieve a better identification of patients at higher risk. Moreover, the identification of genes with rare variants but a large effect size may pave the way for studies addressing new antithrombotic agents in order to improve the management of VTE patients. Over the past 20 years, qualitative or quantitative genetic risk factors such as inhibitor proteins of the hemostasis and of the fibrinolytic system, including fibrinogen, thrombomodulin, plasminogen activator inhibitor-1, and elevated concentrations of factors II, FV, VIII, IX, XI, have been associated with thrombotic events, often with conflicting results. The aim of this review is to evaluate available data in literature on these genetic variations to give a contribution to our understanding of the complex molecular mechanisms involved in physiologic and pathophysiologic clot formation and their role in clinical practice. Full article

Inherited thrombophilia (e.g., venous thromboembolism, VTE) is due to rare loss-of-function mutations in anticoagulant factors genes (i.e., SERPINC1, PROC, PROS1), common gain-of-function mutations in procoagulant factors genes (i.e., F5, F2), and acquired risk conditions. Genome Wide Association Studies (GWAS) recently recognized several genes associated with VTE though gene defects may unpredictably remain asymptomatic, so calculating the individual genetic predisposition is a challenging task. We investigated a large family with severe, recurrent, early-onset VTE in which two sisters experienced VTE during pregnancies characterized by a perinatal in-utero thrombosis in the newborn and a life-saving pregnancy-interruption because of massive VTE, respectively. A nonsense mutation (CGA > TGA) generating a premature stop-codon (c.1171C>T; p.R391*) in the exon 6 of SERPINC1 gene (1q25.1) causing Antithrombin (AT) deficiency and the common missense mutation (c.1691G>A; p.R506Q) in the exon 10 of F5 gene (1q24.2) (i.e., FV Leiden; rs6025) were coinherited in all the symptomatic members investigated suspecting a cis-segregation further confirmed by STR-linkage-analyses [i.e., SERPINC1 IVS5 (ATT)_5–18, F5 IVS2 (AT)_6–33 and F5 IVS11 (GT)_12–16] and SERPINC1 intragenic variants (i.e., rs5878 and rs677). A multilocus investigation of blood-coagulation balance genes detected the coexistence of FV Leiden (rs6025) in trans with FV HR2-haplotype (p.H1299R; rs1800595) in the aborted fetus, and F11 rs2289252, F12 rs1801020, F13A1 rs5985, and KNG1 rs710446 in the newborn and other members. Common selected gene variants may strongly synergize with less common mutations tuning potential life-threatening conditions when combined with rare severest mutations. Merging classic and newly GWAS-identified gene markers in at risk families is mandatory for VTE risk estimation in the clinical practice, avoiding partial risk score evaluation in unrecognized at risk patients. Full article

A carbon nanofiber enriched 8-channel screen-printed electrochemical array was used for the impedimetric detection of SNP related to Factor V Leiden (FV Leiden) mutation, which is the most common inherited form of thrombophilia. FV Leiden mutation sensing was carried out in three steps: solution-phase nucleic acid hybridization between zip nucleic acid probe (Z-probe) and mutant type DNA target, followed by the immobilization of the hybrid on the working electrode area of array, and measurement by electrochemical impedance spectroscopy (EIS). The selectivity of the assay was tested against mutation-free DNA sequences and synthetic polymerase chain reaction (PCR) samples. The developed biosensor was a trustful assay for FV Leiden mutation diagnosis, which can effectively discriminate wild type and mutant type even in PCR samples. Full article

In the present study, a sensitive and selective impedimetric detection of solution-phase nucleic acid hybridization related to Factor V Leiden (FV Leiden) mutation was performed by carbon nanofibers (CNF) modified screen printed electrodes (SPE). The microscopic and electrochemical characterization of CNF-SPEs was explored in comparison to the unmodified electrodes. Since the FV Leiden mutation is a widespread inherited risk factor predisposing to venous thromboembolism, this study herein aimed to perform the impedimetric detection of FV Leiden mutation by a zip nucleic acid (ZNA) probe-based assay in combination with CNF-SPEs. The selectivity of the assay was then examined against the mutation-free DNA sequences as well as the synthetic PCR samples. Full article