A novel factor V compound heterozygous mutation associated with thrombosis (Y1961C; FV-Kanazawa, together with 1982_1983del).

BACKGROUND: Factor (F)V is pivotal in both procoagulant and anticoagulant mechanisms. The present report describes a novel F5 mutation in a FV-deficient patient (FV activity, 6 IU/dL; FV antigen, 32 IU/dL) complicated by recurrent deep vein thrombosis. The patient demonstrated activated protein C resistance (APCR) with compound heterozygous mutations consisting of FV-Y1961C (FVKanazawa) and FV-1982_1983del. OBJECTIVES: To clarify thrombotic mechanisms associated with this FV abnormality. METHODS AND RESULTS: Levels of FV-1982_1983del were below the detection sensitivity in our expression experiments using human embryonic kidney 293T cells, and analyses were targeted, therefore, on the FV-Y1961C mutation. Activated partial thromboplastin time-based clotting assays demonstrated that FV-Y1961C exhibited APCR and that the reduced activated protein C (APC) susceptibility in FVa-Y1961C resulted in a marked depression of APC-catalyzed inactivation with delayed cleavage at Arg506 and little cleavage at Arg306 with or without protein S. The APC cofactor activity of FV-Y1961C in APC-catalyzed FVIIIa inactivation promoted by Arg336 cleavage in FVIII was impaired. The binding affinity of FVa-Y1961C to phospholipid membranes was reduced in reactions involving APC/protein S-catalyzed inactivation and in prothrombinase activity. Furthermore, the addition of FVa-Y1961C to plasma failed to inhibit tissue factor-induced procoagulant function. These characteristics were similar to those of FV-W1920R (FVNara) and FV-A2086D (FVBesançon). CONCLUSION: We identified a compound heterozygous FV-Y1961C mutation in the C1 domain representing a novel FV mutation (FVKanazawa) resulting in not only APCR due to impaired FVa susceptibility and FV cofactor activity for APC function but also impaired inhibition of tissue factor-induced procoagulant function. These defects in anticoagulant function associated with FV in FV-Y1961C contributed to a prothrombotic state.

EGFR-TKI-induced Factor V deficiency in a patient with advanced non-small cell lung cancer: The first case report.

Osimertinib, a third-generation epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI), is routinely prescribed as first-line therapy for advanced non-small cell lung cancer, regardless of the presence of the T790M resistance mutation. This study reports a rare case of Factor V inhibitor detection during osimertinib therapy in a patient with lung adenocarcinoma. These findings underscore the importance of vigilant monitoring for coagulation abnormalities during EGFR-TKI therapy.

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.

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.

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.

Hemophilia A and factor V deficiency in a girl with Turner syndrome: a case report.

BACKGROUND: Hemophilia is an X-linked, recessive inherited disease caused by a defect or deficiency of one of the coagulation factors (VIII or IX). It is considered a rare disease in females. One of the reasons that hemophilia affects females is Turner syndrome. Hemophilia with Turner syndrome is a very rare case, but the combination of Turner syndrome, hemophilia, and factor V deficiency is an isolated case that has never been recorded in the medical literature. CASE PRESENTATION: In our case, a 5-year-old Syrian girl presented with hemorrhage of gum, epistaxis, and short stature. The lab tests showed: prolonged activated partial thromboplastin time and prothrombin time with deficiency of factor V (1%) and factor VIII (1%). We diagnosed hemophilia A with factor V deficiency. In addition to short stature, the patient was noted to have spaced nipples and winged neck. We performed karyotyping that showed deletion of one X chromosome (45X0), Turner syndrome. There is no family history of hemophilia or any other genetic disease. CONCLUSIONS: In females affected with hemophilia, karyotyping should be performed. It is very important not to exclude the possibility of a combination of deficiency of more than one clotting factor, and to note that deficiency of more than one factor does not necessarily increase the severity of bleeding compared with deficiency of a single factor.

Clinical Characterization and Molecular Analysis of Fourteen Chinese Patients with Factor V Deficiency.

INTRODUCTION: Coagulation factor V (FV) functions as a vital cofactor that performs procoagulant roles in the coagulation system. We investigated 14 unrelated patients whose plasma FV levels were all below the reference range. METHODS: FV activity (FV:C) and FV antigen were detected by one-stage clotting and ELISA, respectively. All 25 exons of the F5 gene in patients were amplified by the PCR, and they were sequenced directly. Haplotype analysis was performed with different polymorphisms on F5. Protein modeling was applied to analyze the potential molecular mechanisms. RESULTS: Of five patients with higher FV levels (FV:C > 10%), only one had minor bleeding symptoms. In contrast, of the remaining eight patients with lower FV levels (FV:C < 10%), six showed various bleeding manifestations. A total of 10 mutations were detected from 14 patients (6 were novel mutations). Interestingly, the homozygous p.Phe190Ser was found in five pedigrees, and haplotype analysis showed that they shared almost the same haplotype, indicating the common origin rather than a hotspot mutation. In silico analysis preliminarily investigated the potential pathogenic mechanism of the mutation. Modeling analysis showed that all six missense mutations would lead to conformational alterations in the FV protein. Among them, three (p.Gly1715Ser, p.Ser1753Arg, and p.Asp68His) would decrease hydrogen bonds. CONCLUSION: This is the largest genetic analysis of a single cohort of FV deficiency in Chinese. The study demonstrated that FV levels tended to be correlated with the probability of hemorrhage. The identification of a large number of unique FV-deficient pedigrees highlighted the screening for mutations in F5.

[The analysis of a pedigree with hereditary coagulation factor â ¤ deficiency caused by compound heterozygous variation of F5 gene].

Objective: To analyze the gene variation of a genetic coagulation factor Ⅴ (FⅤ) deficiency pedigree and explore the molecular pathogenesis. Methods: The proband was a 32 years old female. The patient was prone to nose bleeding since childhood which was usually self-healed. On March 10, 2021, the proband went to the First Affiliated Hospital of Air Force Medical University for treatment of knee hematoma caused by a fall. None of the family members reported any history of bleeding. The prothrombin time (PT), activated partial thromboplastin time (APTT) and FⅤ activity (FⅤ: C) were detected by clotting method and the FⅤ antigen (FⅤ: Ag) was tested with enzyme-linked immunosorbent assay (ELISA). All exons and flanks of F5 gene were determined by Sanger sequencing. Clustalx-2.1-win, PolyPhen-2 and Swiss-PDBViewer software were used to analyze the conservatism of missense variation sites, whether the variations were harmful and their influences on protein structure and function. MutationTaster and NetGene2 software were used to analyze whether the splice site variation was harmful and its effect on the splice site. Results: The PT and APTT of the proband prolonged to 24.0 s and 69.8 s, respectively. The FⅤ: C and FⅤ: Ag decreased to 6% and 9%, respectively. There were compound heterozygous variations in F5 gene, which included c.911G>A heterozygous missense variation in exon 6 leading to p.Gly276Glu variation and c.5208+1G>A heterozygous missense variation in intron 15. The father and daughter had the p.Gly276Glu heterozygous variation. Her mother and son had the c.5208+1G>A heterozygous variation. Software analysis results of p.Gly276Glu heterozygous variation showed that Gly276 was conserved among homologous species, the variation was harmful, and it could affect the local structure and function of the protein. The c.5208+1G>A heterozygous variation was deleterious and resulted in the disappearance of the splice site, thereby affecting the protein function. Conclusion: The p.Gly276Glu and c.5208+1G>A compound heterozygous variants are deleterious variants associated with the patient's disease and may be the molecular pathogenesis of inherited FⅤ deficiency in this family.

Congenital coagulation factor V deficiency with intracranial hemorrhage.

BACKGROUND: Congenital coagulation factor V (FV) deficiency is a very rare hemorrhagic disease with an incidence of approximately one in a million. The common clinical manifestations of FV deficiency include ecchymosis and mucosal bleeding. Life-threatening intracranial bleeding is rare. It has been reported in several cases. However, the molecular basis has been established in only a few cases. METHODS: We reported a 2-month-old girl with congenital FV deficiency and intracranial hemorrhage. Coagulation screening combined with clinical manifestations was performed to diagnose congenital FV deficiency. Genetic testing was performed to identify the pathogenic genes. A literature review was included to emphasize the clinical manifestation, diagnosis, and treatment for congenital FV deficiency with intracranial bleeding. RESULTS: The coagulation tests revealed a significantly prolonged prothrombin time (PT) of 51 s and an activated partial thromboplastin time (APTT) of 73.7 s. The patient had a plasma FV activity of 0.9%. Genetic testing showed compound heterozygous mutations of the patient's FV gene. A literature review showed that patients with homozygous or compound heterozygous variants of the FV gene were often associated with a severe bleeding phenotype. CONCLUSION: Our study provides a direction for the rapid and accurate diagnosis and treatment for FV deficiency to avoid life-threatening bleeding. Infants with spontaneous cranial hematoma and intracranial hemorrhage should be investigated for underlying hemostatic defects. Congenital coagulation factor deficiency should be considered. Once congenital FV deficiency is diagnosed, fresh frozen plasma (FFP) should be given on a regular basis. Liver transplantation may be performed in severe cases.

Development and Characterization of a Factor V-Deficient CRISPR Cell Model for the Correction of Mutations.

Factor V deficiency, an ultra-rare congenital coagulopathy, is characterized by bleeding episodes that may be more or less intense as a function of the levels of coagulation factor activity present in plasma. Fresh-frozen plasma, often used to treat patients with factor V deficiency, is a scarcely effective palliative therapy with no specificity to the disease. CRISPR/Cas9-mediated gene editing, following precise deletion by non-homologous end-joining, has proven to be highly effective for modeling on a HepG2 cell line a mutation similar to the one detected in the factor V-deficient patient analyzed in this study, thus simulating the pathological phenotype. Additional CRISPR/Cas9-driven non-homologous end-joining precision deletion steps allowed correction of 41% of the factor V gene mutated cells, giving rise to a newly developed functional protein. Taking into account the plasma concentrations corresponding to the different levels of severity of factor V deficiency, it may be argued that the correction achieved in this study could, in ideal conditions, be sufficient to turn a severe phenotype into a mild or asymptomatic one.

Clinical Phenotype and Genetic Analysis of Twins With Congenital Coagulation Factor V Deficiency.

OBJECTIVE: The aim was to investigate the clinical characteristics and molecular pathogenic mechanism of twins with congenital factor V (FV) deficiency. METHODS: We comprehensively analyzed the clinical manifestations and laboratory test results of a set of twins and their parents and performed point mutation analysis with direct high-throughput exon sequencing. RESULTS: The prothrombin time and activated partial thromboplastin time were prolonged for both probands, and the FV activity levels were 13.0% and 9.8%. Next-generation sequencing showed that the affected individuals harbored a paternal c.5113A>C (p.S1705R) and a maternal c.4949C>T (p.A1650V) heterozygous variants in the FV gene, which conformed to an autosomal recessive inheritance pattern. This is the first report of these point mutations. The older boy also had a congenital patent foramen ovale. CONCLUSION: In this set of twins, missense mutations of the FV gene were related to congenital FV deficiency but unrelated to the patent foramen ovale observed in the older boy.

High Mutational Heterogeneity, and New Mutations in the Human Coagulation Factor V Gene. Future Perspectives for Factor V Deficiency Using Recombinant and Advanced Therapies.

Factor V is an essential clotting factor that plays a key role in the blood coagulation cascade on account of its procoagulant and anticoagulant activity. Eighty percent of circulating factor V is produced in the liver and the remaining 20% originates in the α-granules of platelets. In humans, the factor V gene is about 80 kb in size; it is located on chromosome 1q24.2, and its cDNA is 6914 bp in length. Furthermore, nearly 190 mutations have been reported in the gene. Factor V deficiency is an autosomal recessive coagulation disorder associated with mutations in the factor V gene. This hereditary coagulation disorder is clinically characterized by a heterogeneous spectrum of hemorrhagic manifestations ranging from mucosal or soft-tissue bleeds to potentially fatal hemorrhages. Current treatment of this condition consists in the administration of fresh frozen plasma and platelet concentrates. This article describes the cases of two patients with severe factor V deficiency, and of their parents. A high level of mutational heterogeneity of factor V gene was identified, nonsense mutations, frameshift mutations, missense changes, synonymous sequence variants and intronic changes. These findings prompted the identification of a new mutation in the human factor V gene, designated as Jaén-1, which is capable of altering the procoagulant function of factor V. In addition, an update is provided on the prospects for the treatment of factor V deficiency on the basis of yet-to-be-developed recombinant products or advanced gene and cell therapies that could potentially correct this hereditary disorder.

[Combined deficiency of clotting factor V and factor VIII: about three siblings]. / Déficit combiné en facteurs V et VIII de la coagulation: à propos d´une fratrie de trois cas.

Combined deficiency of clotting factor V and factor VIII (DF5F8) is a congenital autosomal recessive disorder. This study involved a family of four children born to consanguineous parents. The eldest daughter was referred for assessment of activated partial thromboplastin time and prothrombin time associated with hemorrhagic manifestations. Coagulation factor dosing showed combined deficiency of factor V and factor VIII as well as normal levels of other coagulation factors. DF5F8 was detected in two girls and a boy. Two protein coding genes LMAN1 (lectin, mannose binding 1) and MCFD2 (multiple coagulation factor deficiency2) were involved in the intracellular passage of Factor V and Factor VIII, including some mutations which caused deficiency of Factor V and VIII. The diagnosis of DF5F8 is routinely possible, especially in patients born to consanguineous parents with a suggestive clinico-biological condition.

Hereditary factor V deficiency from heterozygous mutations with a novel variant p.Pro798Leufs∗13 in the F5 gene.

To explore the causative mutation for autosomal recessive inheritance factor V (FV) deficiency in a Chinese family. Relative coagulation indexes and the FV antigen were tested by the one-stage clotting method and ELISA, respectively. At the same time, the calibrated automated thrombogram (CAT) was used to analyze the mutant protein function. All 25 exons, flanking sequences, 5' and 3' untranslated regions of the F5 were amplified by PCR and sequenced directly, while each suspected variant was verified by reverse sequencing. The possible impact of the mutant was analyzed by the corresponding bioinformatics software. The phenotypic tests showed that the proband's FV activity has decreased to 24%, whereas the FV antigen has also reduced to 28%. The genetic analysis revealed that she was a compound heterozygote for a frameshift variant from small deletion in the exon 13 (c.2390_2390delC, p.Pro798Leufs∗13) and a missense mutation in the exon 25 (c.6665A>G, p.Asp2222Gly). Meanwhile, the online bioinformatics software indicated that the frameshift variant was disease-causing. The pathogenic variant p.Pro798Leufs∗13 and the benign variant p.Asp2222Gly largely account for the decrease of the FV deficiency in this Chinese family, of which the pathogenic variant is firstly reported in the world.

[Clinical and genetic analyses of hereditary factor â ¤ deficiency cases].

Objective: To analyze the clinical phenotype and molecular pathogenesis of nine patients with hereditary factor Ⅴ (FⅤ) deficiency. Methods: Nine patients with hereditary FⅤ deficiency who were admitted to the Institute of Hematology and Blood Diseases Hospital from April 1999 to September 2019 were analyzed. The activated partial thromboplastin time, prothrombin time, and FⅤ procoagulant activity (FⅤ∶C) were measured for phenotypic diagnosis. High-throughput sequencing was employed for the F5 gene mutation screening, Sanger sequencing was adopted to confirm candidate variants and parental carrying status, Swiss-model was used for three-dimensional structure analysis, and ClustalX v.2.1 was used for homologous analysis. Results: The FⅤ∶C of the nine patients ranged from 0.1 to 10.6. Among them, eight had a hemorrhage history, with kin/mucosal bleeding as the most common symptom (three cases, 37.5%) , whereas one case had no bleeding symptom. There were five homozygotes and four compound heterozygotes. A total of 12 pathogenic or likely pathogenic mutations were detected, of which c.6100C>A/p.Pro2034Thr, c.6575T>C/p.Phe2192Ser, c.1600_1601delinsTG/p. Gln534*, c.4713C>A/p.Tyr1571*, and c.952+5G>C were reported for the first time. Conclusion: The newly discovered gene mutations enriched the F5 gene mutation spectrum associated with hereditary FⅤ deficiency. High-throughput sequencing could be an effective method to detect F5 gene mutations.