Characterization of zebrafish coagulation cofactors Fviii and Fv mutants and modeling hemophilia A and factor V deficiency.

The aim of this study is to characterize zebrafish coagulation cofactors fviii and fv mutant fish and assess if they phenocopy classical hemophilia A and factor V deficiency in humans. The embryos from fviii and fv zebrafish heterozygote mutants generated by ENU mutagenesis were purchased from the ZIRC repository. They were reared to adulthood and genotyped. The heterozygote male and female were crossed to get homozygote, heterozygote, and wild-type fish. Functional kinetic coagulation assays and bleeding assays were performed on normal and mutant adult fish, and venous laser injury assays were performed on the larvae. The DNA from fviii and fv mutants were sequenced to confirm if they have a premature stop codon in exon 19, and in exon 2, respectively, and in both mutants, the amino acid glutamine is replaced with a stop codon. Homozygous and heterozygous 5 days post fertilization (dpf) larvae for fviii and fv deficient mutants exhibited prolonged time to occlusion after venous laser injury compared to wild-type controls. The homozygous and heterozygous fviii adult mutants showed modest bleeding and delayed fibrin formation in the kinetic partial thromboplastin time (kPTT) assay with their plasma. fv homozygous larvae had poor survival beyond 12 dpf. However, heterozygous fv mutants exhibited heavy bleeding and prolonged fibrin formation in the kPTT and kPT assay compared with wild-type siblings. Our characterization showed fviii and fv mutants from ZIRC phenocopied to a considerable extent classical hemophilia A and factor V deficiency in humans, respectively. These models should be useful in studying and developing novel drugs that reverse the phenotype and in generating suppressor mutations to identify novel factors that compensate for these deficiencies.

Comparison of an Unlabeled Probe High-Resolution Melting Analysis Assay (HRMA) for Factor V Leiden 1691 G/A Mutation to a Fluorogenic 5' Nuclease PCR Hydrolysis Assay.

BACKGROUND: The clinically significant Factor V Leiden (FVL) point mutation (1691 G/A) causes replacement of Arg with Gln (glutamine), preventing activated protein C from inactivating Factor V leading to a lengthened clotting process. Individuals with the Factor V Leiden mutations have an increased risk for venous thrombosis. The aim of this study is to compare an unlabeled probe high-resolution melting analysis (HRMA) assay for Factor V Leiden mutation to a TaqMan hydrolysis assay (fluorogenic 5' nuclease PCR hydrolysis assay). HRMA is a post-PCR, homogenous, closed-tube system for the detection of sequence variants. Post-PCR, the amplicons are heated gradually until the melting temperature is reached and the fluorescent dye unbinds from the amplicon and exhibits low fluorescence. A melt-curve analysis is generated that is characteristic of a particular sequence variant. Therefore, HRMA allows for comparison of one base changes in genetic sequences based on their differences in melting rate. METHODS: Blood samples were collected in EDTA tubes and DNA extracted using the Roche MagNaPure. Reactions of both HRMA and TaqMan were carried out on 3 controls (1691 G/G, 1691 G/A, and 1691 G/G and G/A) and 20 samples. RESULTS: The genotypes for 3 reference controls purchased from Coriell (F5 1691 G/G, FVL 1691 G/A, and Heterozygote 1691 G/G and G/A) were confirmed by both the HRMA and TaqMan FVL assays. All 20 samples were confirmed to be F5 1691 G/G by both HRMA and TaqMan assays. CONCLUSIONS: Comparing the results of the unlabeled probe HRMA FVL assay with a real-time TaqMan probe end point genotyping assay resulted in 100% sensitivity and 100% specificity for both assays.

The Etiology of the Thrombotic Phenomena Involved in the Process of Coronary Artery Disease-What Is the Role of Thrombophilic Genes in the Development of This Pathology?

Cardiovascular diseases, among which includes coronary artery disease, represent one of the most important causes of mortality and morbidity worldwide. Research aimed at determining the risk factors involved recognizes a group of "traditional" risk factors, but also more recent studies identified over 100 "novel" ones which may have a role in the disease. Among the latter is the thrombophilia profile of a patient, a pathology well-established for its involvement in venous thromboembolism, but with less studied implications in arterial thrombosis. This paper reviews the literature, explaining the pathophysiology of the thrombophilia causes associated most with coronary thrombosis events. Results of several studies on the subject, including a meta-analysis with over 60,000 subjects, determined the significant involvement of factor V Leiden, prothrombin G20210A mutation, plasminogen activator inhibitor-1 and antiphospholipid syndrome in the development of coronary artery disease. The mechanisms involved are currently at different stages of research, with some already established and used as therapeutic targets.

Gene Dosage of F5 c.3481C>T Stop-Codon (p.R1161Ter) Switches the Clinical Phenotype from Severe Thrombosis to Recurrent Haemorrhage: Novel Hypotheses for Readthrough Strategy.

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.

Risk Factors of Thrombophilia-Related Mutations for Early and Late Pregnancy Loss.

Background and Objectives: This retrospective cohort study investigates the role of genetic thrombophilia in pregnant women experiencing early pregnancy loss compared to those with late pregnancy loss. Materials and Methods: Participants were categorized into early and late pregnancy loss groups based on gestational age. A total of 156 patients were included, out of which 103 had early-trimester pregnancy losses and 96 had multiple miscarriages. Results: The study revealed a synergistic effect of Factor V Leiden (FVL G1691A) and Methylenetetrahydrofolate Reductase (MTHFR C677T) mutations (coefficient 3.42). Prothrombin (PT) G20210A and ß-Fibrinogen 455 G>A mutations exhibited a significant interaction (coefficient 1.98). Additionally, MTHFR A1298C and Plasminogen Activator Inhibitor-1 (PAI-1 4G/5G) mutations showed a significant interaction (coefficient 1.65). FVL G1691A and Endothelial Protein C Receptor (EPCR) allele A1/A2 mutations also demonstrated a significant association (coefficient 2.10). Lastly, MTHFR C677T and Glycoprotein IIb/IIIa T1565C mutations interacted significantly (coefficient 1.77). Risk factor analysis identified several mutations associated with early pregnancy loss, including PAI-1 4G/5G homozygous (OR 3.01), FVL G1691A heterozygous (OR 1.85), and MTHFR A1298C heterozygous (OR 1.55). Both homozygous and heterozygous MTHFR C677T mutations were significant risk factors (OR 2.38; OR 2.06), as was PT G20210A homozygous mutation (OR 1.92). The PAI-1 4G/4G homozygous variant posed a risk (OR 1.36). Late pregnancy loss was associated with MTHFR A1298C homozygous mutation (OR 3.79), ß-Fibrinogen 455 G>A heterozygous mutation (OR 2.20), and MTHFR A1298C heterozygous mutation (OR 2.65). Factor XIII G1002T heterozygous mutation (OR 1.18) and PAI-1 4G/5G homozygous mutation (OR 2.85) were also significant risk factors. EPCR allele A1/A2 (OR 1.60) and A2/A3 (OR 1.73) mutations were identified as significant risk factors for late pregnancy loss. Furthermore, FVL G1691A homozygous mutation, PT G20210A homozygous mutation, MTHFR C677T heterozygous mutation, MTHFR A1298C heterozygous mutation, and EPCR allele A1/A2 were identified as significant risk factors for multiple miscarriage. Conclusions: This study highlights significant interactions and risk factors related to genetic thrombophilia mutations in different types of pregnancy loss, contributing valuable insights for miscarriage management guidelines.

Thrombosis risk in single- and double-heterozygous carriers of factor V Leiden and prothrombin G20210A in FinnGen and the UK Biobank.

ABSTRACT: The factor V Leiden (FVL; rs6025) and prothrombin G20210A (PTGM; rs1799963) polymorphisms are 2 of the most well-studied genetic risk factors for venous thromboembolism (VTE). However, double heterozygosity (DH) for FVL and PTGM remains poorly understood, with previous studies showing marked disagreement regarding thrombosis risk conferred by the DH genotype. Using multidimensional data from the UK Biobank (UKB) and FinnGen biorepositories, we evaluated the clinical impact of DH carrier status across 937 939 individuals. We found that 662 participants (0.07%) were DH carriers. After adjustment for age, sex, and ancestry, DH individuals experienced a markedly elevated risk of VTE compared with wild-type individuals (odds ratio [OR] = 5.24; 95% confidence interval [CI], 4.01-6.84; P = 4.8 × 10-34), which approximated the risk conferred by FVL homozygosity. A secondary analysis restricted to UKB participants (N = 445 144) found that effect size estimates for the DH genotype remained largely unchanged (OR = 4.53; 95% CI, 3.42-5.90; P < 1 × 10-16) after adjustment for commonly cited VTE risk factors, such as body mass index, blood type, and markers of inflammation. In contrast, the DH genotype was not associated with a significantly higher risk of any arterial thrombosis phenotype, including stroke, myocardial infarction, and peripheral artery disease. In summary, we leveraged population-scale genomic data sets to conduct, to our knowledge, the largest study to date on the DH genotype and were able to establish far more precise effect size estimates than previously possible. Our findings indicate that the DH genotype may occur as frequently as FVL homozygosity and may confer a similarly increased risk of VTE.

Thrombophilia genetic mutations and their relation to disease severity among patients with COVID-19.

OBJECTIVES: Patients with COVID-19 infection appear to develop virus-induced hypercoagulability resulting in numerous thrombotic events. The aim of the present study was to determine the relationship between the thrombophilia genes mutations (prothrombin G20210A, factor V Leiden, and methyltetrahydrofolate reductase (MTHFR)) and the severity of COVID-19 patients. DESIGN: Prospective cross-sectional study. METHOD: One hundred and forty patients (80 adults and 60 children) were included in the current study. They were divided into the severe COVID-19 group and the mild COVID-19 group, with each group comprising 40 adults and 30 children. The patients were assessed for FV R506Q, FV R2H1299R, MTHFR A1298C, MTHFR C677T, and prothrombin gene G20210A polymorphisms. CBC, D-dimer, renal and liver function tests, hs-CRP, ferritin, and LDH were also assessed. Thrombotic events were clinically and radiologically documented. RESULTS: Severe COVID-19 cases were significantly more frequent to have a heterozygous mutation for all the studied genes compared to mild COVID-19 cases (p<0.05 for all). Being mutant to gene FV R506Q carried the highest risk of developing a severe disease course (p<0.0001). Patients with abnormally high D-dimer levels were significantly more frequent to be heterozygous for FV R506Q, FV R2H1299R, and prothrombin gene G20210A (p = 0.006, 0.007, and 0.02, respectively). CONCLUSION: We concluded that there is an evident relationship between severe COVID-19 and inherited thrombophilia. In the current study, FV R506Q gene mutation carried the highest risk of developing a severe COVID-19 disease course.

Coagulation factor V in breast cancer: a p53-regulated tumor suppressor and predictive marker for treatment response to chemotherapy.

BACKGROUND: Patients with cancer are at an increased risk of developing coagulation complications, and chemotherapy treatment increases the risk. Tumor progression is closely linked to the hemostatic system. Breast cancer tumors express coagulation factor V (FV), an essential factor in blood coagulation. The functional role of FV during treatment with chemotherapy is poorly understood and was explored in this study. OBJECTIVES: We aimed to investigate the role of FV in breast cancer progression by exploring associations with treatment response, gene regulation, and the functional effects of FV. METHODS: The receiver operating characteristic plotter was used to explore the predictive value of FV mRNA (F5) expression for treatment with FEC (5-fluorouracil, anthracycline, and cyclophosphamide). Breast cancer cohorts were analyzed to study treatment response to FEC. The effect of chemotherapy on F5 expression, the regulation of F5, and the functional effects of FV dependent and independent of chemotherapy were studied in breast cancer cell lines. RESULTS: F5 tumor expression was significantly higher in responders to FEC than in nonresponders. In vitro experiments revealed that anthracycline treatment increased the expression of F5. Inhibition and knockdown of p53 reduced the anthracycline-induced F5 expression. Mutation of a p53 half-site (c.158+1541/158+1564) in a luciferase plasmid reduced luciferase activity, suggesting that p53 plays a role in regulating F5. FV overexpression increased apoptosis and reduced proliferation slightly during anthracycline treatment. CONCLUSION: Our study identified F5 as a p53-regulated tumor suppressor candidate and a promising marker for response to chemotherapy. FV may have functional effects that are therapeutically relevant in breast cancer.

KLF5 inhibits the migration and invasion in cervical cancer cell lines by regulating SNAI1.

BACKGROUND: Epithelial-mesenchymal transition (EMT) is an important biological process by which malignant tumor cells to acquire migration and invasion abilities. This study explored the role of KLF5 in the EMT process of in cervical cancer cell lines. OBJECTIVE: Krüpple-like factor 5 (KLF5) is a basic transcriptional factor that plays a key role in cell-cycle arrest and inhibition of apoptosis. However, the molecular mechanism by which KLF5 mediates the biological functions of cervical cancer cell lines has not been elucidated. Here, we focus on the potential function of ELF5 in regulating the EMT process in in vitro model of cervical cancer cell lines. METHOD: Western-blot and real-time quantitative PCR were used to detect the expression of EMT-related genes in HeLa cells. MTT assays, cell scratch and Transwell assays were used to assess HeLa cells proliferation and invasion capability. Using the bioinformatics tool JASPAR, we identified a high-scoring KLF5-like binding sequence in the SNAI1 gene promoter. Luciferase reporter assays was used to detect transcriptional activity for different SNAI1 promoter truncates. RESULT: After overexpressing the KLF5 gene in HeLa cells, KLF5 not only significantly inhibited the invasion and migration of HeLa cells, but also increased the expression of E-cadherin and decreased the expression of N-cadherin and MMP9. In addition, the mRNA expression of upstream regulators of E-cadherin, such as SNAI1, SLUG, ZEB1/2 and TWIST1 was also decreased. Furthermore, KLF5 inhibiting the expression of the SNAI1 gene via binding its promoter region, and the EMT of Hela cells was promoted after overexpression of the SNAI1 gene. CONCLUSION: These results indicate that KLF5 can downregulate the EMT process of HeLa cells by decreasing the expression of the SNAI1 gene, thereby inhibiting the migration and invasion of HeLa cervical cancer cells.

Inherited thrombophilia gene mutations and risk of venous thromboembolism in patients with cancer: A systematic review and meta-analysis.

In the general population, individuals with an inherited thrombophilia have a higher risk of thrombosis, but the effect of inherited thrombophilia on the risk of cancer-associated venous thromboembolism (VTE) remains controversial. Our objective was to determine the risk of VTE in cancer patients with inherited thrombophilia. We conducted a systematic review and meta-analysis of studies reporting on VTE after a cancer diagnosis in adult patients who were tested for inherited thrombophilia. In September 2022, we searched Medline, EMBASE, and Cochrane Central. Two reviewers screened the abstracts/full texts and assessed study quality using the Quality in Prognostic Studies tool. We used Mantel-Haenszel random-effects models to estimate pooled odds ratios (OR) of VTE and 95% confidence intervals (95%CI). We included 37 and 28 studies in the systematic review and meta-analysis, respectively. Most studies focused on specific cancer types and hematologic malignancies were rare. The risk of VTE was significantly higher in cancer patients with non-O (compared with O) blood types (OR: 1.56 [95% CI: 1.28-1.90]), Factor V Leiden, and Prothrombin Factor II G20210A mutations compared with wild types (OR: 2.28 [95% CI: 1.51-3.48] and 2.14 [95% CI: 1.14-4.03], respectively). Additionally, heterozygous and homozygous methylenetetrahydrofolate reductase C677T had ORs of 1.50 (95% CI: 1.00-2.24) and 1.38 (95% CI: 0.87-2.22), respectively. Among those with Plasminogen-Activator Inhibitor-1 4G/5G, Vascular Endothelial Growth Factor (VEGF) A C634G, and VEGF C2578A mutations, there was no significant association with VTE. In conclusion, this meta-analysis provided evidence that non-O blood types, Factor V Leiden, and Prothrombin Factor II G20210A mutations are important genetic risk factors for VTE in cancer patients.

Determination of vWF, ADAMTS-13 and Thrombospondin-1 in Venous Thromboembolism and Relating Them to the Presence of Factor V Leiden Mutation.

Thrombophilia in venous thromboembolism (VTE) is multifactorial. Von Willebrand factor (vWF) plays a major role in primary hemostasis. While elevated vWF levels are well documented in VTE, findings related to its cleaving protease (ADAMTS-13) are contradicting. The aim of this study was to determine vWF, ADAMTS-13, and the multifactorial Thrombospondin-1 (TSP-1) protein levels in patients after 3-6 months following an unprovoked VTE episode. We also explored a possible association with factor V Leiden (FVL) mutation. vWF, ADAMTS-13 and TSP-1 were analyzed using ELISA kits in 60 VTE patients and 60 controls. Patients had higher levels of vWF antigen (P = .021), vWF collagen-binding activity (P = .008), and TSP-1 protein (P < .001) compared to controls. ADAMTS-13 antigen was lower in patients (P = .046) compared to controls but ADAMTS-13 activity was comparable between the two groups (P = .172). TSP-1 showed positive correlation with vWF antigen (rho = 0.303, P = .021) and negative correlation with ADAMTS-13 activity (rho = -0.244, P = .033) and ADAMTS-13 activity/vWF antigen ratio (rho = -0.348, P = .007). A significant association was found between the presence of FVL mutation and VTE (odds ratio (OR): 9.672 (95% confidence interval (CI) 2.074-45.091- P = .004), but no association was found between the mutation and the studied proteins (P > .05). There appears to be an imbalance between vWF and ADAMTS-13 in VTE patients even after 3-6 months following the onset of VTE. We report that the odds of developing VTE in carriers of FVL mutation are 9.672 times those without the mutation, but the presence of this mutation is not associated with the studied proteins.

In vitro and ex vivo rescue of a nonsense mutation responsible for severe coagulation factor V deficiency.

BACKGROUND: Coagulation factor V (FV) deficiency is a rare bleeding disorder that is usually managed with fresh-frozen plasma. Patients with nonsense mutations may respond to treatment with readthrough agents. OBJECTIVES: To investigate whether the F5 p.Arg1161Ter mutation, causing severe FV deficiency in several patients, would be amenable to readthrough therapy. METHODS: F5 mRNA and protein expression were evaluated in a F5 p.Arg1161Ter-homozygous patient. Five readthrough agents with different mechanisms of action, i.e. G418, ELX-02, PTC-124, 2,6-diaminopurine (2,6-DAP), and Amlexanox, were tested in in vitro and ex vivo models of the mutation. RESULTS: The F5 p.Arg1161Ter-homozygous patient showed residual F5 mRNA and functional platelet FV, indicating detectable levels of natural readthrough. COS-1 cells transfected with the FV-Arg1161Ter cDNA expressed 0.7% FV activity compared to wild-type. Treatment with 0-500 µM G418, ELX-02, and 2,6-DAP dose-dependently increased FV activity up to 7.0-fold, 3.1-fold, and 10.8-fold, respectively, whereas PTC-124 and Amlexanox (alone or in combination) were ineffective. These findings were confirmed by thrombin generation assays in FV-depleted plasma reconstituted with conditioned media of treated cells. All compounds except ELX-02 showed some degree of cytotoxicity. Ex vivo differentiated megakaryocytes of the F5 p.Arg1161Ter-homozygous patient, which were negative at FV immunostaining, turned positive after treatment with all 5 readthrough agents. Notably, they were also able to internalize mutant FV rescued with G418 or 2,6-DAP, which would be required to maintain the crucial platelet FV pool in vivo. CONCLUSION: These findings provide in vitro and ex vivo proof-of-principle for readthrough-mediated rescue of the F5 p.Arg1161Ter mutation.

The risk of venous thromboembolism in oral contraceptive users: the role of genetic factors-a prospective cohort study of 240,000 women in the UK Biobank.

BACKGROUND: More than 150 million women worldwide use oral contraceptives. Women with inherited thrombophilia and carriers of certain thrombophilia gene variants, such as factor V Leiden and the prothrombin, are at an increased risk for venous thromboembolism, especially when combined with oral contraceptive use. Venous thromboembolism is a complex disorder involving many genetic risk factors, and recently, polygenic risk scores have been proposed to capture a significant proportion of the genetic risk of venous thromboembolism. OBJECTIVE: The aim of this study was to estimate the risk for developing venous thromboembolism when initiating oral contraceptive use (first 2 years) and during continued use among women with a high genetic liability. STUDY DESIGN: We used a prospective study design in which 244,420 participants from the UK Biobank were followed from birth. The effect of oral contraceptive use during the first 2 years and in the remaining years of oral contraceptive use on the risk of developing venous thromboembolism was estimated using a Cox regression with a time-dependent exposure variable. Women were stratified according to their polygenic risk scores and whether they were carriers of factor V Leiden and/or prothrombin variants. RESULTS: When genetic risk was not considered, an increased risk for venous thromboembolism was observed during the first 2 years of oral contraceptive use (hazard ratio, 3.09; 95% confidence interval, 3.00-3.20) but not during continued use (hazard ratio, 0.92; 95% confidence interval, 0.80-1.05). However, when genetic risk was considered, women in the highest polygenic risk score category had a more pronounced risk of developing a venous thromboembolism during the first 2 years of oral contraceptive use (hazard ratio, 6.35; 95% confidence interval, 4.98-8.09), and a high risk was also observed among factor V Leiden (hazard ratio, 5.73; 95% confidence interval, 5.31-6.17) and prothrombin variant carriers (hazard ratio, 5.23; 95% confidence interval, 4.67 - 5.87). A high polygenic risk score in combination with being a factor V Leiden and prothrombin variant carrier conferred the highest risk for developing a venous thromboembolism during the first 2 years of oral contraceptive use (hazard ratio, 14.8; 95% confidence interval, 9.28-23.6). Women with a high genetic liability also had an increased risk during continued use but it was less pronounced, and the highest risk was conferred to carriers of both factor V Leiden and the prothrombin variant (hazard ratio, 4.93; 95% confidence interval, 3.16-7.7). CONCLUSION: Evaluating polygenic risk can identify additional venous thromboembolism risk that is not captured in the commonly investigated genes for inherited thrombophilia. Our results indicate that oral contraceptive use is associated with an increased risk for developing a venous thromboembolism, particularly among women with a high genetic predisposition, and that oral contraceptive use dramatically increases the risk thereof short after initiation of use, which decreases with continued use. This suggests that the polygenic risk score could be used to identify women who are at high risk for developing a venous thromboembolism and advise them on alternative methods of contraception.

Molecular and clinical characterization of two unrelated families with factor V deficiency, including a novel nonsense variant (p.Gln1532*).

BACKGROUND: Factor V (FV) is an essential cofactor in the coagulation cascade. The characterization of novel mutations is advantageous for the clinical management of FV-deficient patients. METHODS: Coagulation screening and thrombin generation assay were performed with the plate-poor plasma. All 25 exons of the F5 gene were amplified and sequenced. The ClustalX-2.1 software was applied to the multiple sequence alignment. The possible adverse effects of mutations were investigated with online bioinformatics software and protein modeling. RESULTS: Two unrelated families with FV deficiency were under investigation. Proband A was an 18-year-old youth with recurrent epistaxis. Proband B was a 29-year-old woman who did not present with any bleeding symptoms. Three heterozygous mutations (p.Gln1532*, p.Phe218Ser, and p.Asp2222Gly) were detected. Interestingly, they were compound heterozygotes and both contained the p.Asp2222Gly, a polymorphism. The thrombin generation assay showed that both patients had impaired ability of thrombin generation, and in particular, proband A was more severe. Conservation, pathogenicity and protein modeling studies all indicated that these three mutations could cause deleterious effects on the function and structure of FV. CONCLUSION: These three mutations are responsible for the FV-deficient in two pedigrees. Moreover, the nonsense variant p.Gln1532* is first reported in the world.

The relevance of prothrombotic genetic variants in women who experienced pregnancy loss or embryo implantation failure: A retrospective analysis of 1922 cases.

OBJECTIVE: The aim of our study was that to assess the allelic and genotype frequencies of nine prothrombotic gene variants in patients with a history of pregnancy loss and recurrent pregnancy loss (RPL). Women who underwent assisted reproductive technology (ART) with ongoing pregnancy and those with recurrent implantation failure (RIF) were also included. METHODS: Nine prothrombotic gene variants were evaluated: factor V Leiden (FVL), factor V, H1299R variant (FVR2), factor II (FII) G20210A, methylene-tetrahydrofolate reductase (MTHFR) C677T and A1298C, beta-fibrinogen -455G>A, factor XIII (FXIII) V34L, human platelet antigen-1 (HPA-1) L33P variants, and plasminogen activator inhibitor-1 (PAI-1) 4G/5G. The following study groups were assessed: (1) women who experienced one (n = 334) or two (n = 264) episodes of pregnancy loss; (2) 468 women who experienced RPL; (3) 214 women who underwent ART followed by ongoing pregnancies; and (4) 282 women who experienced RIF after ART, that is, three or more consecutive implantation failures following high-quality embryo transfers to the uterus with an appropriate endometrium. As control group, 430 subjects from the general population were enrolled. RESULTS: FVL, the -455G>A variant of beta-fibrinogen, and PAI-1 4G were associated with a higher risk of developing RPL compared with the general population. Furthermore, FVL, FVR2, FII G20210A and MTHFR C677T conferred a significantly higher risk of RIF in women who performed ART compared with the general population. No statistical differences between the general population and other study groups were observed. CONCLUSIONS: Specific prothrombotic genetic variants are more frequently expressed in women with RPL and RIF, supporting their role in the development of polimicrothrombosis and impairing the invasion during embryo implantation.

Spontaneous miscarriage driven by maternal genetic mutation at position of PAI-1-844G/A: shed light on a race-specific genetic polymorphism.

OBJECTIVE: Association between a genetic polymorphism and disease, either positively or negatively, within a population may not necessarily predict association in other race-ethnic populations. The aim of this study was to genotype well recognized thrombophilia associated polymorphisms as common risk factors for miscarriage and investigate their benefit to use as risk factors in southwest region of Iran females (Khuzestan) in the Arabs ethnic minority group with spontaneous miscarriage. We developed a Reverse Dot Blot Assay for the genotyping of four polymorphisms. RESULTS: There were significant differences in the genotype distribution and allelic frequencies of the MTHFR 1298 A > C, MTHFR 677 C > T, Factor V Leiden 1691 G > A, PAI-1-844G > A polymorphisms between the case and control groups. The MTHFR 1298 A > C, MTHFR 677 C > T and Factor V Leiden 1691 G > A polymorphisms were significantly associated with spontaneous miscarriage risk. Unlike some other race-ethnic populations, PAI-1-844G > A polymorphism was associated with risk of developing unplanned miscarriage in Iranian Arabs ethnic minority group females.

E74-like Factor 5 Promoter Methylation in Circulating Tumor DNA as a Potential Prognostic Marker in Breast Cancer Patients.

BACKGROUND: Epigenetic alternations, such as DNA methylation, play a crucial role in breast tumor initiation and progression. The identification of noninvasive prognostic biomarkers has great importance in cancer management. Methylated cell-free DNA (cfDNA), circulating in the blood as a convenient tumor-associated DNA marker, can be used as a minimally invasive cancer biomarker. This study aimed to evaluate the promoter methylation status of E74-like factor 5 (ELF5) tumor suppressor gene in both tumors and plasma cell-free DNA of 80 breast cancer patients, compared with normal controls. METHODS: Plasma cfDNA concentrations were measured using quantitative real-time PCR, and methylation pattern in the ELF5 gene promoter region was performed using methylation-specific polymerase chain reaction (MS-PCR) technique. RESULTS: The data revealed a statistically significant increase in cfDNA concentrations in breast cancer patients, particularly in those with higher stages of the disease, triple-negative status, and metastasis (p<0.001). ELF5 promoter region hypermethylation was observed in 70% of breast cancer patients in both plasma cfDNA and tumor tissues. Notably, all patients with lymph node involvement and distant metastatic exhibited promoter hypermethylation in the ELF5 gene. CONCLUSION: Our findings suggest that ELF5 promoter methylation in circulating DNA could serve as a potential non-invasive prognostic molecular marker in breast cancer patients. However, further studies are warranted to evaluate its diagnostic value.

[Elucidation of an altered anticoagulant function due to Factor V abnormality and development of a simple screening assay for thrombophilia].

Coagulation factor V (FV) is both procoagulant and anticoagulant functions. Congenital FV abnormality, which are caused by mutations in the FV gene, are characterized by a tendency to bleed. However, FV-R506Q (FVLeiden) is the most common FV abnormality that eliminates an activated protein C (APC) cleavage site, resulting in the occurrence of deep venous thrombosis (DVT). In Japan, the thrombotic predisposition caused by FVLeiden and FV molecular abnormalities was believed to be nonexistent. We did, however, report the first case in Japan of a young patient with FV abnormality-related thrombosis. The recurrent DVT in this case was caused by a novel mutation of FV-W1920R (FVNara), located in the C1 domain and far from the APC cleavage sites. We considered the possibility that there were cases of FV-related thrombotic predisposition that had gone undetected in Japan. We thoroughly examined FV-related anticoagulant function to understand the pathogenesis of thrombosis caused by FV abnormality. Furthermore, using recombinant thrombomodulin, we successfully developed a novel assay with clot waveform analysis for the rapid detection of FV deficiency with APC resistance. Other FV abnormality-related thrombosis has been reported in Japan in recent years, and we hope to further clarify the FV-related thrombotic predisposition in the future.