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.

[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.

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.

[Analysis of a pedigree with inherited factor V deficiency caused by compound heterozygous mutation].

Objective: To explore the molecular pathogenesis of a family with hereditary factor Ⅴ (FⅤ) deficiency. Methods: All the exons, flanking sequences, 5' and 3' untranslated regions of the F5 of the proband, and the corresponding mutation sites of the family members were analyzed via direct DNA sequencing. The CAT measurement was used to detect the amount of thrombin produced. The ClustalX software was used to analyze the conservation of mutation sites. The online bioinformatics software, Mutation Taster, PolyPhen-2, PROVEAN, LRT, and SIFT were applied to predict the effects of mutation sites on protein function. The Swiss-PdbViewer software was used to analyze the changes in the protein model and intermolecular force before and after amino acid variation. Results: The proband had a heterozygous missense mutation c.1258G>T (p.Gly392Cys) in exon 8 of the F5, and a heterozygous deletion mutation c.4797delG (p.Glu1572Lys fsX19) in exon 14, which results in a frameshift and produces a truncated protein. Her grandfather and father had p.Gly392Cys heterozygous variation, whereas her maternal grandmother, mother, little aunt, and cousin all had p.Glu1572LysfsX19 heterozygous variation. The ratio of proband's thrombin generation delay to peak time was significantly increased. Conservation analysis results showed that p.Gly392 was located in a conserved region among the 10 homologous species. Five online bioinformatics software predicted that p.Gly392Cys was pathogenic, and Mutation Taster also predicted p.Glu1572Lys fsX19 as a pathogenic variant. Protein model analysis showed that the replacement of Gly392 by Cys392 can lead to the extension of the original hydrogen bond and the formation of a new steric hindrance, which affected the stability of the protein structure. Conclusion: The c.1258G>T heterozygous missense mutation in exon 8 and the c.4797delG heterozygous deletion mutation in exon 14 of the F5 may be responsible for the decrease of FⅤ levels in this family.

Congenital factor V deficiency from compound heterozygous mutations with a novel variant c.2426del (p.Pro809Hisfs*2) in the F5 gene: A case report.

INTRODUCTION: Congenital factor V deficiency (FVD) is a rare bleeding disorder characterized by low or undetectable plasma factor V (FV) levels leading to mild to severe bleeding symptoms. Currently, more than 100 mutations have been reported in F5. We herein report a patient with FVD from mutations in the F5 gene. PATIENT CONCERNS: A 52-year-old man with prolonged prothrombin time and activated partial thromboplastin time corrected by mixing test on preoperative screening. His past medical or family history was not remarkable. DIAGNOSIS: Factor assays revealed a markedly reduced FV activity at 7%. Other factors were not decreased. DNA sequencing analysis to detect F5 gene mutations showed the patient was compound heterozygous for c.286G>C (p.Asp96His) and c.2426del (p.Pro809Hisfs*2). Asp96His was previously described missense mutation and Pro809Hisfs*2 was a novel deleterious mutation. INTERVENTIONS: Fresh-frozen plasma was administered to supplement FV before surgery. OUTCOMES: Subsequent factor assays revealed temporarily increased FV activity at 33%. CONCLUSION: As was the case in our patient, genotype-phenotype correlations are poor in FVD, and molecular genetic test is necessary to confirm the diagnosis.

The relationship between the levels and function of endothelial progenitor cells and factor V Leiden and protein C deficiency in patients with primary Budd-Chiari syndrome.

OBJECTIVE: Budd-Chiari syndrome (BCS) is a life-threatening hepatic disease characterized by hepatic venous obstruction at the level of hepatic vein, hepatic venules, or inferior vena cava. No evidence reported the relationship between the endothelial progenitor cells and the deficiency of factor V Leiden and protein C in patients with primary Budd-Chiari syndrome. PATIENTS AND METHODS: We recruited participants between June 2014 and July 2015. For primary BCS group, 28 patients were collected. 20 patients were included in the NAFLD group. Another 73 healthy participants were recruited into the control group. None of the patients and participants had received interventional therapy or had undergone surgery prior to being recruited. Levels and functions of endothelial progenitor cells (EPCs) were examined. The factor V Leiden mutation, protein C deficiency and protein S deficiency were evaluated. Finally, the relationship between the levels and function of endothelial progenitor cells and factor V Leiden and protein C deficiency in patients with primary Budd-Chiari syndrome was analyzed. RESULTS: The results showed that no significant differences were found between the BCS (and NAFLD) and control group considering age, sex, BMI, smoking (p>0.05 for variables). However, significant differences were observed in TG, TC, HDL-C, white blood cells, hemoglobin, ALT, AST, ALP, γ-GT, total bilirubin, and albumin (p<0.05 for variables). Compared with the healthy participants, significant downregulation was found in BCS and NAFLD patients regarding CD34+/CD45-, late outgrowth endothelial cells (OECs) colonies, OECs proliferation, and OECs tubulogenesis (p<0.001 for variables). Among the 28 BCS patients, factor V Leiden mutation (n=10, 35.71%, OR 12.67, 95% CI 5.24-27.93) and hereditary protein C deficiency (n=4, 14.29%, OR 7.48, 95% CI 2.02-21.43) were more prevalent than those in the control group. These results suggested that factor V Leiden mutation and protein C deficiency were major risk factors for BCS. Finally, we demonstrated that factor V Leiden and protein C deficiency may negatively regulate the OECs levels and functions in BCS patients. CONCLUSIONS: It's important to improve the OECs levels and functions, and to prevent the deficiency of factor V Leiden and protein C in the treatment of BCS.

Antisense-based RNA therapy of factor V deficiency: in vitro and ex vivo rescue of a F5 deep-intronic splicing mutation.

Antisense molecules are emerging as a powerful tool to correct splicing defects. Recently, we identified a homozygous deep-intronic mutation (F5 c.1296+268A>G) activating a cryptic donor splice site in a patient with severe coagulation factor V (FV) deficiency and life-threatening bleeding episodes. Here, we assessed the ability of 2 mutation-specific antisense molecules (a morpholino oligonucleotide [MO] and an engineered U7 small nuclear RNA [snRNA]) to correct this splicing defect. COS-1 and HepG2 cells transfected with a F5 minigene construct containing the patient's mutation expressed aberrant messenger RNA (mRNA) in excess of normal mRNA. Treatment with mutation-specific antisense MO (1-5 µM) or a construct expressing antisense U7snRNA (0.25-2 µg) dose-dependently increased the relative amount of correctly spliced mRNA by 1 to 2 orders of magnitude, whereas control MO and U7snRNA were ineffective. Patient-derived megakaryocytes obtained by differentiation of circulating progenitor cells did not express FV, but became positive for FV at immunofluorescence staining after administration of antisense MO or U7snRNA. However, treatment adversely affected cell viability, mainly because of the transfection reagents used to deliver the antisense molecules. Our data provide in vitro and ex vivo proof of principle for the efficacy of RNA therapy in severe FV deficiency, but additional cytotoxicity studies are warranted.

Inherited hematological disorders due to defects in coat protein (COP)II complex.

Many diseases attributed to trafficking defects are primary disorders of protein folding and assembly. However, an increasing number of disease states are directly attributable to defects in trafficking machinery. In this context, the cytoplasmic coat protein (COP)II complex plays a pivotal role: it mediates the anterograde transport of correctly folded secretory cargo from the endoplasmic reticulum towards the Golgi apparatus. This review attempts to describe the involvement of COPII complex alteration in the pathogenesis of human genetic disorders; particularly, we will focus on two disorders, the Congenital Dyserythropoietic Anemia type II and the Combined Deficiency of Factor V and VIII.

Quebec platelet disorder: update on pathogenesis, diagnosis, and treatment.

Quebec platelet disorder (QPD) is an autosomal dominant bleeding disorder associated with reduced platelet counts and a unique gain-of-function defect in fibrinolysis due to increased expression and storage of urokinase plasminogen activator (uPA) by megakaryocytes. QPD increases risks for bleeding and its key clinical feature is delayed-onset bleeding, following surgery, dental procedures or trauma, which responds only to treatment with fibrinolytic inhibitors. The genetic cause of the disorder is a tandem duplication mutation of the uPA gene, PLAU, which upregulates uPA expression in megakaryocytes by an unknown mechanism. The increased platelet stores of uPA trigger plasmin-mediated degradation of QPD α-granule proteins. The gain-of-function defect in fibrinolysis is thought to be central to the pathogenesis of QPD bleeding as the activation of QPD platelets leads to release of uPA from α-granules and accelerated clot lysis. The purpose of this review is to summarize current knowledge on QPD pathogenesis and the recommended approaches to QPD diagnosis and treatment.

Inherited and acquired factor V deficiency.

The clotting factor V, also known as proaccelerin or labile factor, is synthesized by the liver and possibly by the megakaryocytes. Factor V exerts a pivotal role in hemostasis, as it participates in both procoagulant and anticoagulant pathways, being an essential cofactor of the prothrombinase complex in the former case and participating in the inactivation of factor VIII (FVIII) in the latter. Isolated factor V deficiency due to mutations in the F5 gene is a rare inherited coagulopathy typically associated with a broad spectrum of bleeding symptoms, ranging from easy bruising, delayed bleeding after haemostatic challenges such as trauma or surgery to more severe joint bleeds. The combined deficiency of factor V and FVIII, commonly known as F5F8D, is a recessive disorder not attributable to the association of isolated factor V and FVIII deficiencies, but rather to defective intracellular processing of both proteins due to mutations involving the LMAN1 and MCFD2 genes, which encode two proteins forming an essential cargo receptor complex. Overall, patients affected by F5F8D do not bleed more in terms of both frequency and severity than those carrying specific deficiencies of both factors and the bleeding phenotype is generally mild. Although now increasingly rare, inhibitors directed against factor V may also develop in individuals of any age and are characterized by a very heterogeneous clinical phenotype. The aim of the current review is to provide an overview on the physiopathology, diagnostics, and clinical management of both inherited and acquired factor V deficiency.

A premature infant with fetal myocardial and abdominal calcifications and factor V Leiden homozygosity.

We present a premature male neonate with confirmed factor V Leiden deficiency diagnosed prenatally with cardiac and abdominal calcifications. Our patient's findings suggest that clinicians consider thromboembolic conditions when multiple fetal calcifications are visualized.

The risk of symptomatic pulmonary embolism due to proximal deep venous thrombosis differs in patients with different types of inherited thrombophilia.

It is uncertain whether the presence of inherited thrombophilia influences the risk of developing symptomatic pulmonary embolism (PE) and whether different thrombophilic alterations are associated with different risks of symptomatic PE. To investigate such issue, we retrospectively studied 920 patients with proximal deep vein thrombosis (DVT) of the legs with or without symptomatic PE referred for thrombophilia screening; patients with overt cancer or antiphospholipid antibodies had been excluded. Three hundred fifty-four patients (38.5%) had deficiency of antithrombin (AT, n = 16), protein C (PC, n = 26), protein S (PS, n = 22), factor V Leiden (FVL, n = 168), prothrombin G20210A (PT-GA, n = 87), or multiple abnormalities (n = 35), and 566 had none of the studied thrombophilic abnormalities. Symptomatic PE complicated the first DVT in 242 patients (26%); the risk of PE was increased in patients with AT deficiency (relative risk [RR] 2.4, 95% confidence interval [CI] 1.6-3.6) or with PT-GA (RR 1.5, 95%CI 1.1-2.0) and decreased in those with FVL (RR 0.7, 95%CI 0.5-1.0) in comparison with those with unknown inherited defect. These data suggest that patients with proximal DVT have different risks of symptomatic PE according to the type of inherited thrombophilia.

[Therapeutic consequences of thrombophilic testing]. / Conséquences thérapeutiques de la mise en évidence d'une thrombophilie.

OBJECTIVE: The objectives of this article are to review the data about the consequences of thrombophilia testing and to think about its indications. CURRENT KNOWLEDGE AND KEY POINTS: The indications of congenital thrombophilic testing have extended since the discovery of prevalent abnormalities, such as mutations of factor V or II genes. However, thrombophilia does not result in a significant increase in the risk of recurrence unlike the spontaneous occurrence of thrombotic events. The factor V Leiden mutation is associated with a moderate increase in recurrence rate, while the G20210A mutation of factor II is not associated with a significant increase in recurrence. Regarding the decrease in natural anticoagulants is concerned, there is no definite conclusion, although the decrease in antithrombin is suspected of being associated with an increase in recurrence. FUTURE PROSPECTS AND PROJECTS: Finally, identification of a constitutional thrombophilia most often do not influence the therapeutic decisions unless some rare abnormalities are found, such as a decrease in antithrombin, homozygous mutations in factors V or II genes or associations of thrombophilia. One must remember that antiphospholipid antibodies must be searched because their impact on recurrences is well-known. Diagnostic work-up for thrombophilia is not useful after a distal or a superficial venous thrombosis (except for antiphospholipid antibodies in case of distal venous thrombosis).

Factor V deficiency: a concise review.

Factor V (FV; proaccelerin or labile factor) is the plasma cofactor for the prothrombinase complex that activates prothrombin to thrombin. FV deficiency can be caused by mutations in the FV gene or in genes encoding components of a putative cargo receptor that transports FV (and factor VIII) from the endoplasmic reticulum to the Golgi. Because FV is present in platelet alpha-granules as well as in plasma, low FV levels are also seen in disorders of platelet granules. Additionally, acquired FV deficiencies can occur in the setting of rheumatologic disorders, malignancies, and antibiotic use and, most frequently, with the use of topical bovine thrombin. FV levels have limited correlation with the risk of bleeding, but overall, FV-deficient patients appear to have a less severe phenotype than patients with haemophilia A or B. The most commonly reported symptoms are bleeding from mucosal surfaces and postoperative haemorrhage. However, haemarthroses and intramuscular and intracranial haemorrhages can also occur. Because no FV-specific concentrate is available, fresh frozen plasma remains the mainstay of treatment. Antifibrinolytics can also provide benefit, especially for mucosal bleeding. In refractory cases, or for patients with inhibitors, prothrombin complex concentrates, recombinant activated FVIIa, and platelet transfusions have been successfully used. Some patients with inhibitors may also require immunosuppression.

Arterial thrombosis associated with heterozygous factor V Leiden disorder, hyperhomocysteinemia, and peripheral arterial disease: importance of synergistic factors.

A 47-year-old man with heterozygous factor V Leiden disorder and intermittent hyperhomocysteinemia developed spontaneous acute popliteal artery thrombosis. Homocysteine levels were above normal limits at presentation. Intra-arterial thrombolysis was used successfully to treat the acute thrombosis; long-term treatment included anticoagulation, folic acid, and risk factor modification. Although factor V Leiden is strongly associated with deep venous thrombosis, additional cofactors such as hyperhomocysteinemia may predispose to an increased risk of acute arterial thrombosis in areas of pre-existing peripheral arterial disease.

Modulation of factor V levels in plasma by polymorphisms in the C2 domain.

OBJECTIVE: Functional polymorphisms contributing to coagulation factor levels are preferential markers for association studies aimed at identifying prothrombic genetic components. METHODS AND RESULTS: Factor V (FV) microsatellite genotypes were found to be associated with FV levels (P=0.003). Single nucleotide polymorphisms analysis and sequencing of the promoter and of coding regions identified two polymorphisms (Met2120Thr, Asp2194Gly) present in 20% of the population (n=1013) that are responsible for genotype-phenotype associations. The effect of the Met2120Thr polymorphism, both in plasma (mean reduction of FV level in the heterozygous condition: 25%) and in recombinant FV studies (34% reduction), was comparable to that of the Asp2194Gly change (20% and 34%, respectively). The study of 10 subjects with a rare genotype indicated that the Asp2194Gly substitution is the functional determinant of the reduced FV levels associated with the FVHR2 haplotype. Among Leiden carriers, the doubly heterozygous condition for FV2120Thr was found to be associated with a significantly increased activated protein-C resistance (APCR) (P<0.05), and the doubly heterozygous condition for FV2194Gly was found to be more frequent (P=0.009) in symptomatic than in asymptomatic subjects. CONCLUSIONS: Extensive analysis of FV polymorphisms indicated that changes in the C2 domain modulate FV levels and might increase APCR and thrombotic risk in FV Leiden carriers through a pseudohomozygous mechanism.

Type I coagulation factor V deficiency caused by compound heterozygous mutation of F5 gene.

A 16-year-old Chinese female with prolonged bleeding after surgery has been studied. Routine clotting tests revealed a prolonged activated partial thromboplastin time (APTT; 126.6 s) and prothrombin time (PT; 42.8 s). The coagulation factors activities were normal except for factor V, which was only 0.3% of normal. DNA analysis of the FV gene revealed five nucleotide substitutions in exons, including two silent mutations (G327A and A5112G), one polymorphism (G1628A), a G1348T missense mutation and 4887 approximately 8delG. These abnormalities were associated with her FV deficiency, perhaps by causing a Gly392Cys substitution in FV amino acid sequence or by introducing a premature stop codon at amino acid position 1390. This is the third case in which FV deficiency is caused by compound heterozygous mutation of F5 gene, and is the first report from a Chinese family.

Arg2074Cys missense mutation in the C2 domain of factor V causing moderately severe factor V deficiency: molecular characterization by expression of the recombinant protein.

Factor V (FV) deficiency is a rare bleeding disorder whose genetic basis has been described in a relatively small number of cases. Among a total of 12 genetic defects reported in severely or moderately severe deficient patients, 3 were missense mutations and in no case was the mechanism underlying the deficiency explored at the molecular level. In this study, a homozygous missense mutation at cDNA position 6394 in exon 23 of the FV gene was identified in a 22-year-old Italian patient. This mutation causes the replacement of arginine 2074 with a cysteine residue (Arg2074Cys) in the C2 domain of the protein. The effect of the Arg2074Cys mutation on FV secretion, stability, and activity was investigated. Site-directed mutagenesis of FV cDNA was used to introduce the identified mutation, and wild-type as well as mutant FV proteins were expressed by transient transfection in COS-1 cells. An enzyme immunoassay detected low FV antigen levels both in the conditioned media of cells expressing the mutant protein and in cell lysates. Metabolic labeling and pulse-chase experiments confirmed that the mutation caused an impaired secretion of FV associated with rapid intracellular degradation. In addition, evaluation of wild-type and mutant coagulant activity demonstrated that the FV molecules carrying the Arg2074Cys mutation have reduced activity. These findings, beside confirming the structural and functional importance of the arginine 2074 residue, demonstrate that its substitution with a cysteine impairs both FV secretion and activity.