Factor V Protein Research Articles

Identification of a Novel Factor V A2 Domain Tyrosine Deletion Mutation in a Patient with Factor V Deficiency and Bleeding.

The gene for coagulation Factor V (FV) is located on chromosome 1q23. FV deficiency shows an autosomal recessive mode of inheritance; heterozygotes are generally not clinically affected. The homozygous clinical phenotype occurs in approximately 1 per million individuals with variable severity of bleeding. Thus, genotype-phenotype correlations are likely to shed light on functionally important residues of FV. Here we describe a case of FV deficiency with a severe bleeding phenotype. The proband is a male infant from Argentina. His parents are unrelated. He was born healthy with no bleeding from the umbilical stump or other symptoms. He presented at eight months with a CNS hemorrhage, then suffered a second massive subdural bleed at nine months of age. Both episodes required surgical drainage and treatment with fresh frozen plasma He continues to receive prophylactic FFP infusions and has some residual neurologic impairment. The proband's FV activity ranges from 2–14%. Two siblings are unaffected. His father's FV activity is 50% and his mother's is 70%. We performed DNA sequencing spanning the entire coding region of the proband's FV gene and found two heterozygous mutations: a heterozygous single base pair deletion, del 2952T in exon 13, located in the B-domain of the FV protein, causing a frameshift mutation followed by a premature termination codon 3 amino acids downstream; and a novel 3-bp deletion in exon 10. This deletion is in-frame and results in the deletion of Y478. The del 2952T frameshift mutation was present in the father, while the del Y478 mutation was present in the mother. Y478 is in the A2 domain of FV and adjacent to another tyrosine, Y477. Evidence suggests that these tyrosine residues are important for co-factor function. Tyrosine residue sulfation has been shown to be required for full activity of the homologous co-factor, FVIII, as well as for hirudin. These sulfated tyrosines and surrounding acidic amino acids have been proposed to be important in interactions with the thrombin anion binding exosite; in the case of hirudin, sulfation of a carboxy-terminal tyrosine increases the affinity for thrombin 10-fold. The homologous tyrosines, Y718 and Y719 appear to be sulfated in FVIII. FV has been shown to be sulfated, but the precise location of the FV sulfation sites has not yet been determined. One of this patient's FV alleles is nonfunctional due to a frameshift and a premature trancation of translation. With respect to the other allele, we hypothesize that, like FVIII, one or both of FV tyrosines 477 and 478 is sulfated, and that deletion of Y478 may result in disruption of FV co-factor function. In vitro mutagenesis and expression studies to characterize the functional consequences of the del Y478 and/or del Y477 are in progress.

Identification of three F5 gene mutations associated with inherited coagulation factor V deficiency in two Chinese pedigrees.

To investigate the molecular defects in two Chinese pedigrees with inherited factor V (FV) deficiency. A 37-year-old male (proband 1) and an 18-month-old boy (proband 2) were diagnosed as inherited coagulation FV deficiency by severely reduced plasma levels of FV activity and antigen. All 25 exons and their flanking sequence of F5 gene were amplified by polymerase chain reaction (PCR) for both probands and the PCR products were directly sequenced. Total RNA was extracted from the peripheral lymphocytes of proband 1 for detecting the changes at mRNA level. The homozygous deletion IVS8 -2A>G was identified in the F5 gene of proband 1 and complementary DNA (cDNA) analysis revealed the abolishment of the canonical splicing site by the mutation and the activation of the cryptic acceptor site 24 bp upstream instead. The insertion introduced eight additional amino acids (AA) into the FV protein. Two heterozygous mutations of F5 gene were discovered in proband 2. The 2238-9del AG in exon 13 introduced a premature termination code at 689 AA and the substitution of G6410 by T in exon 23 lead to the missense mutation Gly2079Val. Three F5 gene mutations, IVS8 -2A>G, 2238-9del AG and G6410T, have been identified in two Chinese pedigree with congenital FV deficiency, respectively.

Severe factor V deficiency: exon skipping in the factor V gene causing a partial deletion of the C1 domain

Severe factor V (FV) deficiency is a rare coagulation disorder, characterized by very low or unmeasurable plasma levels of functional and immunoreactive FV. Among rare inherited coagulopathies, FV deficiency is the least characterized from a molecular point of view (only 12 mutations have been reported). The aim of this work was to investigate, at the molecular level, the pathogenetic mechanisms responsible for a case of severe FV deficiency. A 19-year-old Iranian man showing unmeasurable FV activity and severely reduced FV antigen level in plasma was studied. Mutation screening was performed by sequencing. The effect of the identified mutation was investigated both at the mRNA and at the protein level. Molecular analysis of the factor V (FV) gene identified a novel homozygous A-->T transversion at position + 3 of the donor splice site of intron 19 (IVS19 + 3A-->T). Production of mutant mRNA in HeLa cells demonstrated that this mutation causes the entire exon 19 to be skipped from the FV mRNA. The mutant processed transcript codes for a deleted FV, lacking the first 24 amino acids of the C1 domain. Expression of the mutant FV protein in COS-1 cells showed that the deleted protein was synthesized but not secreted; moreover, the intracellular amount of deleted FV was reduced compared to wild type, suggesting intracellular degradation of mutant FV. This work reports the molecular characterization of the first mutation causing a partial deletion in the FV molecule, resulting in a severe impairment of protein secretion.

Identification of a large deletion and three novel mutations in exon 13 of the factor V gene in a Spanish family with normal factor V coagulant and anticoagulant properties.

As part of the GAIT (genetic analysis of idiopathic thrombophilia) project, we analyzed polymorphisms in the factor V (FV) gene to assess their role as genetic determinants of normal phenotypic variation of hemostasis-related traits in a Spanish population. During the analysis of exon 13 polymorphisms, we detected an abnormal PCR-amplified fragment in some members of the GAIT19 family. Direct sequence analysis revealed a deletion of 108 bp in eight out of 20 individuals in this family. This deletion removes exactly 36 amino acids from the B domain of FV; thus it does not alter the reading frame of the sequence. Among the deleted amino acids there is the 4070A>G polymorphism (H1299R), which could affect the level or function of FV. In addition, in the same family we identified three novel DNA variants (L1257I, Q1317Q and T1327T) in exon 13 of the F5 gene. Despite these variants, we did not detect any differences either in the coagulant or anticoagulant traits, or in the plasma protein levels involved in the blood coagulation cascade, between the carriers compared with their non-carrier relatives. From these results, we can conclude that the mutant allele is expressed and the resultant protein is functional. Moreover, it is unlikely that the 4070A>G polymorphism, within the deletion, and the novel DNA variants alter the functional properties of the mature FV protein. Further analyses of this naturally occurring mutation and the novel DNA variants should yield useful information for the understanding of the function of the B domain of FV.

Prothrombin G20210A gene mutation and further prothrombotic risk factors in childhood thrombophilia.

Risk factors for venous thrombosis in adults are the prothrombin G20210A and the factor V (FV) G1691A mutations and hereditary deficiencies of protein C, protein S and antithrombin. However, data are limited on the relevance of these risk factors for thrombosis in children and adolescents. We therefore investigated 261 patients aged 0 to 18 (median 5.7 years, 48% male) with venous thrombosis and controls (n=370) for the presence of prothrombotic risk factors including the prothrombin G20210A mutation. The following frequencies of hereditary risk factors (patients versus controls), odds ratios (OR) and 95% confidence intervals (CI), or results of Fisher's exact test, respectively, were found: prothrombin G20210A, 4.2% versus 1.1%, OR/CI 4.1/1.3 to 12.8; FV G1691A, 31.8% versus 4. 1%, OR/CI 11.0/6.2 to 19.7; protein C deficiency, 9.2% versus 0.8%, OR/CI 12.4/3.7 to 41.6, protein S deficiency, 5.7% versus 0.8%, OR/CI 7.5/2.1 to 26.0; antithrombin deficiency in 3.4% in the patients, but not in the controls, P=0.0003. The prothrombin mutation was combined with the heterozygous FV G1691A mutation (2. 3%) or protein C deficiency (0.3%) in the patients, but not in the controls (prothrombin and FV mutation, P=0.0048; prothrombin and protein C deficiency, not significant). The carrier frequencies and ORs of all hereditary risk factors showed a non-significant trend toward higher prevalences in patients suffering spontaneous thrombosis, compared with those with an additional underlying disease. In conclusion, the prothrombin G20210A and the FV G1691A mutation, deficiencies of protein C, protein S, and antithrombin are important risk factors for venous thrombosis during childhood and adolescence.