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.

A patient with Owren disease requires pancreatic surgery: A case report.

RATIONALE: Coagulation factor V deficiency is rare, and perioperative management of patients with this condition is particularly important, especially during major abdominal surgery. We present a case of a patient with pancreatic duct stones combined with coagulation factor V deficiency. We share our perioperative management experience. PATIENT CONCERNS: A 31-year-old man presented with recurrent upper abdominal pain for 2 years. DIAGNOSES: The diagnosis of pancreatic duct stones in the patient has been established through abdominal computed tomography and magnetic resonance imaging examinations. The diagnosis of factor V deficiency was initially identified through coagulation function tests, revealing significant prolongation of both aPTT and PT. Subsequent testing of coagulation factors and inhibitors demonstrated that the patient has a deficiency in coagulation factor V. Finally, genetic testing revealed that the factor V deficiency in this case is hereditary. INTERVENTIONS: The patient underwent a partial resection of the pancreatic head, and FFP was infused 1 hour before surgery. 600 mL of FFP was instilled 1 hour before the start of surgery along with 10 U of cryoprecipitate. and 600 ml of FFP were added during surgery. Postoperative treatment included intermittent FFP supplemental infusion in the first 5 days after surgery while monitoring the coagulation function. OUTCOMES: The patient underwent a successful surgery without any abnormal bleeding or oozing during the procedure. The postoperative recovery was smooth, with no abnormal bleeding. LESSONS: Patients with a deficiency of coagulation factor V are not contraindicated for surgery. Appropriate Fresh Frozen Plasma (FFP) replacement therapy can ensure the safe conduct of the surgical procedure. For patients with abnormal blood coagulation function, we recommend testing for coagulation factors and inhibitors, as well as performing genetic testing for abnormal coagulation factors, which can provide guidance on marriage and childbirth.

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.

The Magic of Proteases: From a Procoagulant and Anticoagulant Factor V to an Equitable Treatment of Its Inherited Deficiency.

Proteostasis, i.e., the homeostasis of proteins, responsible for ensuring protein turnover, is regulated by proteases, which also participate in the etiopathogenesis of multiple conditions. The magic of proteases is such that, in blood coagulation, one same molecule, such as coagulation factor V, for example, can perform both a procoagulant and an anticoagulant function as a result of the activity of proteases. However, this magic has an insidious side to it, as it may also prevent the completion of the clinical value chain of factor V deficiency. This value chain encompasses the discovery of knowledge, the transfer of this knowledge, and its translation to clinical practice. In the case of rare and ultra-rare diseases like factor V deficiency, this value chain has not been completed as the knowledge acquisition phase has dragged out over time, holding up the transfer of knowledge to clinical practice. The reason for this is related to the small number of patients afflicted with these conditions. As a result, new indications must be found to make the therapies cost-effective. In the case of factor V, significant research efforts have been directed at developing a recombinant factor V capable of resisting the action of the proteases capable of inactivating this factor. This is where bioethics and health equity considerations come into the equation.

[Autoimmune coagulation factor V/5 deficiency during chronic disseminated intravascular coagulation].

Aortic regurgitation, a thoracoabdominal aortic aneurysm, chronic myeloid leukemia, and chronic kidney disease were all being treated at two hospitals for an 83-year-old man. He was admitted to the Department of Orthopedics at our hospital with a lumbar compression fracture. Later, he experienced melena, for which the Department of Internal Medicine was consulted. Due to the aberrant results of PT-INR (7.1) and a PTT > 200 seconds on a coagulation test, we suspected the presence of an autoimmune coagulation factor deficiency, and prednisolone immunosuppressive therapy medication was started right away. Due to a sharp decline in FV/5 activity, the presence of FV/5 inhibitors, and the presence of anti-FV/5 autoantibodies, a final diagnosis of autoimmune coagulation factor V (FV/5) deficiency was made. After the start of immunosuppressive therapy, the FV/5 inhibitor and anti-FV/5 autoantibodies disappeared, and the FV/5 activity progressively returned to normal. Disseminated intravascular coagulation-which may have been caused by a known aortic aneurysm-worsened while tapering off prednisolone. Due to the patient's advanced age and other problems, the aneurysm was extensive and inappropriate for surgical repair. The coagulation test findings improved gradually upon initiation of warfarin therapy. Herein, the patient had autoimmune FV/5 deficiency, a rare disorder that made diagnosis and therapy difficult because of the patient's several coexisting conditions.

Acquired factor V inhibitor with erythema and eosinophilia in a patient with end-stage renal disease.

Autoimmune factor V deficiency (AiFVD) is a rare bleeding disorder caused by factor V inhibitors. In this report, we present the case of an 89-year-old man who developed bleeding tendency during surgery to create arteriovenous fistula for hemodialysis. The bleeding tendency developed with prolongation of activated partial thromboplastin and prothrombin time, following drug-induced eruption and eosinophilia. Significant reduction in coagulation factor activity and inhibitory pattern in cross-mixing tests suggested the presence of inhibitors to coagulation factors. Subsequently, we detected a factor V inhibitor and anti-factor V autoantibodies was confirmed using enzyme-linked immunosorbent assay with purified human plasma factor V. Thus, the patient was 'definitely diagnosed' with AiFVD in accordance with the diagnostic criteria enacted by the Japanese Ministry of Health, Labor, and Welfare. The bleeding tendency improved after initiating oral prednisolone 50 mg (1 mg/kg) followed by normalization of activated partial thromboplastin time and prothrombin time at the 34th day. After improving the coagulation system prolongation, the inhibitor and autoantibodies has been eradicated. Since it is suggested that drug-induced immune response can cause AiFVD, AiFVD should be considered in patients who undergo hemodialysis and develop failure of hemostasis and drug-induced eruption.

Congenital factor V deficiency in Taiwan: identification of a novel variant p.Tyr1813 ∗ and two variants specific to East Asians.

Congenital coagulation factor V deficiency (FVD) is a rare, autosomal recessive bleeding disorder. We characterized the clinical presentations, laboratory features, and genetic alterations of Taiwanese patients with FVD. From 1983 to 2010, five women, one man, and one boy diagnosed with FVD were enrolled in this study. The factor V coagulant activity was determined using a one-stage prothrombin time-based test. The factor V antigen level was measured in an ELISA. Sanger sequencing was performed for genetic analyses of F5 , the gene responsible for the disease. One novel and de novo F5 genetic variant, p.Tyr1813 ∗ , was identified. Based on the presence of a premature termination codon with a resultant truncated factor V-protein lacking an intact light chain fragment, the variant is pathogenic. In addition, we identified seven variants previously found to cause FVD. Among them, p.Gly420Cys and p.Asp96His were repeatedly detected in five and four patients, respectively. Both variants are found to be specific to the East Asian populations. Various FVD-associated bleeding manifestations were observed, predominantly mucocutaneous bleeding and hypermenorrhea. All patients exhibited very low factor V coagulant activity (<1-2.5 IU/dl, reference range: 60-133 IU/dl). The factor V antigen level was less than 2% in six patients (reference range: 75-157%). The novel F5 genetic variant p.Tyr1813 ∗ and two distinct, East Asians-specific, recurrent variants p.Gly420Cys and p.Asp96His were identified among seven index patients with FVD in Taiwan. Our clinical and laboratory findings support the reported features of FVD.

Recurrent miscarriage in a woman with congenital factor V deficiency: a case report.

BACKGROUND: Factor V deficiency is a rare bleeding disorder that can be either congenital or acquired. Factor V deficiency mostly present with mucosal bleeding. Coagulation factor V does not increase considerably during normal gestation. Since pregnancy can be threatened by blood clotting disorders, abnormal changes in coagulation factors level can pose challenges to pregnant women. CASE PRESENTATION: We report a 40-year-old pregnant woman with prolonged gingival bleeding and epistaxis at 28 weeks of pregnancy. Her past medical history included two unexplained abortions. Physical examination was unremarkable, but the blood test showed elevated PT and PTT with a considerable decrease in factor V activity, while other factors were within normal range. Subsequently, the patient was diagnosed with congenital factor V deficiency. After treatment with fresh frozen plasma, she underwent vaginal delivery and a baby with factor V deficiency was born. CONCLUSIONS: This is the second report of recurrent miscarriage in congenital factor V deficiency patients. Clinicians should consider the possibility of factor V deficiency in women with a history of idiopathic miscarriage even in patients without any symptoms.

Combined Liver-Kidney Transplant for Juvenile Polycystic Kidney Disease and Concomitant Hereditary Factor V Deficiency.

Factor V deficiency is a congenital bleeding diathesis that, in selected cases, may be managed with liver transplant. In this case, we describe the treatment of an adult patient with kidney failure secondary to juvenile onset polycystic kidney disease who received a combined liver-kidney transplant as a method to manage the risks associated with the need for a kidney transplantin the setting of factorV deficiency and high sensitization.

Acute mesenteric thrombosis in a pregnant woman with Factor V Leiden mutation with a history of in vitro fertilization and embryo transfer: case report and literature review / Trombosis mesentérica aguda en gestante con mutación de Factor V Leiden y antecedentes de fertilización in vitro y transferencia de embriones: reporte de caso y revisión de la literature

Objectives: To report the case of a patient diagnosed with acute mesenteric vein thrombosis (AMVT) associated with Factor V Leiden mutation and a history of in vitro fertilization and embryo transfer and review the literature on risk factors and treatments performed for AMVT. Materials and methods: We reported the case of a 37-year-old pregnant woman. A bibliographic search was carried out in Medline/PubMed and LILACS, filtering by type of language (English and Spanish). Primary cohort studies, cases and controls, case reports and case series were included, which addressed the risk factors associated with the development of acute mesenteric thrombosis during pregnancy and treatments performed. Results: The search identified cases and control studies, case reports and case series related to mesenteric ischemia, pregnancy and in vitro fertilization. The literature reported that the main factors associated with mesenteric ischemia are pregnancy itself, genetic factors, drugs, protein C and protein S deficiency and idiopathic causes. Conclusions: SMV thrombosis is a life-threatening and very rarely seen condition that emerges in pregnancies. The literature suggests that, during gestation, the factors associated with the development of acute mesenteric thrombosis are hypercoagulability induced by pregnancy, the administration of oral estrogen during IVF-ET, and other precipitating factors. More studies are required to better understand the possible additional factors and build better optimal treatment algorithms.

Objetivos: presentar el caso de una paciente diagnosticada con trombosis aguda de la vena mesentérica (TAVM) asociada a mutación de Factor V Leiden y antecedente de fertilización in vitro y transferencia de embriones, y hacer una revisión de la literatura sobre los factores de riesgo y los tratamientos realizados en los casos de TAVM. Materiales y métodos: reporte de un caso de mujer gestante de 37 años. Se realizó una búsqueda bibliográfica en las bases de datos Medline/PubMed y LILACS, filtrando por idioma (inglés y español). Se incluyeron estudios de cohortes primarias, casos y controles, reportes de casos y series de casos que examinaran los factores de riesgo asociados con el desarrollo de trombosis mesentérica aguda durante el embarazo y los tratamientos realizados. Resultados: se identificaron estudios de casos y controles, reportes de casos y series relacionados con isquemia mesentérica, embarazo y fertilización in vitro, y se encontró que los principales factores asociados con isquemia mesentérica son el embarazo mismo, factores genéticos, medicamentos, la deficiencia de proteína C y S, y causas idiopáticas. Conclusiones: la trombosis de la vena mesentérica superior es una condición infrecuente que amenaza la vida y ocurre durante el embarazo. La literatura sugiere que, durante la gestación, los factores asociados con la trombosis mesentérica aguda son la hipercoagulabilidad inducida por el embarazo, la administración de estrógeno oral durante el proceso de fertilización in vitro y transferencia de embriones, y otros factores desencadenantes. Es necesario realizar más estudios para comprender mejor los posibles factores adicionales y desarrollar mejores algoritmos para un tratamiento óptimo.

Effectiveness of the scheduled transfusion strategy for factor V deficiency with platelet dysfunction during coronary artery surgery.

BACKGROUND: Factor V deficiency is a rare disease, with an incidence of one in a million. Symptoms are mostly scant, and it is often diagnosed by the presence of an abnormality on PT-INR or APTT. In addition, no established therapy exists and platelet dysfunction is seldom found to be concomitant with this disease CASE PRESENTATION: A 64-year-old man who had both factor V deficiency and platelet dysfunction had angina in the past year. Coronary surgery was required, and we successfully performed coronary artery bypass grafting under strategic planned platelet transfusion with additional adequate cryoprecipitates transfusion. No perioperative problems nor any postoperative major bleeding issues were observed. The postoperative course was also uneventful. CONCLUSION: Strategic planned platelet transfusion with the additional transfusion of an adequate amount of cryoprecipitates is thus considered to be feasible for cases presenting with factor V deficiency and platelet dysfunction.

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.

Combined Factor V and VIII Deficiency with LMAN1 Mutation: A Report of 3 Saudi Siblings.

BACKGROUND Combined factor V and factor VIII deficiency (F5F8D) is a rare bleeding disorder with an incidence of 1: 1 000 000. The identified mutations were observed in LMAN1 and MCFD2 genes. This case report presents the cases of 3 Saudi siblings with the genetic mutation of LMAN1 causing F5F8D, and highlights the challenges in diagnosis and treatment. CASE REPORT Patient X, a 7-year-old boy, was misdiagnosed with hemophilia A after a history of prolonged circumcision bleeding and epistaxis. He was referred to our clinic for pre-operative assessment. Blood workup showed prolonged PT and aPTT, which were normalized by mixing studies. Since his previous diagnosis could not explain a prolonged PT, further investigations were performed, revealing low levels of FVIII and FV. Genetic testing confirmed a c.822G>A homozygous LMAN1 mutation. The other 2 siblings (patient Y and Z), who were 5- and 12-year-old, respectively, girls, were also assessed. They both had a history of epistaxis. The younger sibling also had an episode of bleeding after tooth extraction, and physical examination of this patient revealed a bruise over her left thigh. The older sibling had menorrhagia. Blood workup of both revealed prolonged PT and aPTT, with complete correction by mixing study, and low levels of FV and FVIII. The patients' backgrounds and lab results were highly suggestive of F5F8D. CONCLUSIONS This case report describes an extremely rare bleeding disorder. More attention should be directed toward this disease, and a careful evaluation of suspicious cases should be performed to better diagnose and manage these patients.

[Clinical features of five cases of acquired factor V deficiency].

Acquired factor V deficiency is a rare disease that presents with various bleeding symptoms because of the acquired production of factor V inhibitors and decrease in factor V activity. We have experienced five cases of acquired factor V deficiency diagnosed on the basis of abnormalities in coagulation tests in the last 10 years. All five patients were older men, of whom one had no bleeding symptoms, and three had a history of renal failure and malignant tumors. In the cross-mixing test, two of three cases demonstrated an inhibitor pattern, but one case showed a deficient pattern. In all cases, steroid treatment improved factor V activity as well as prothrombin time and activated partial thromboplastin time. However, patients with intracranial hemorrhage had a poor prognosis. Although this disease is rare, careful management is necessary, especially in the absence of bleeding symptoms and where cross-mixing test does not show an inhibitor pattern.