The frequency of factor V Leiden and concomitance of factor V Leiden with prothrombin G20210A mutation and methylene tetrahydrofolate reductase C677T gene mutation in healthy population of Denizli, Aegean region of Turkey.

Factor V Leiden causing activated protein C resistance is the most common inherited form of thrombophilia leading to thrombosis. Its frequency shows great ethnic and geographic variations. The aim of this study was to determine the frequency of FV Leiden and coinheritance of FV Leiden with two other frequent hereditary thrombophilia causes, namely, prothrombin G20210A and methylene-tetrahydrofolate reductase (MTHFR) C677T mutation in the Aegean region of Turkey. The study population consisted of 1030 (500 men and 530 women) apparently healthy subjects. Functional resistance to activated protein C (APC) was measured by using the test kit STA staclot APC-R ((Diagnostica Stago, Asnieres, France, Cat. No. 00721). In subjects with APC resistance, molecular analyses of FV Leiden and of prothrombin G20210A and MTHFR C677T mutation were performed by using FV-PTH-MTHFR StripA (Vienna Lab, Labordiagnostika GmbH, Austria) kit, which was based on hybridization of polymerase chain reaction (PCR) amplified DNA products with mutation-specific oligonucleotide probes. Functional APC resistance was present in 93 subjects (9%). FV Leiden mutation was found in 87 of 93 subjects with APC resistance by PCR method. The FV Leiden carrier frequency was found to be 8.4% (87/1030). Seventy-six individuals were heterozygous (7.3%), and 11 were homozygous (1.06%). Among the 87 subjects with FV Leiden mutation, 45 subjects had MTHFR C677T gene mutation (7 homozygous, 38 heterozygous) and 4 subjects had heterozygote prothrombin G20210A gene mutation. A combination of FV Leiden and prothrombin G20210A and MTHFR C677T gene mutation was detected in 3 subjects. The results indicate that FV Leiden prevalence is quite high and coexistence of FV Leiden with other hereditary causes of thrombosis such as prothrombin G20210A mutation and MTHFR enzyme defect is not rare in healthy population of Aegean region of Turkey.     

The activated protein C (APC)-resistant phenotype of APC cleavage site mutants of recombinant factor V in a reconstituted plasma model.

Recently, new missense mutations in the activated protein C (APC) cleavage sites of human factor V (FV) distinct from the R506Q (FV Leiden) mutation have been reported. These mutations affect the APC cleavage site at arginine (Arg) 306 in the heavy chain of activated FV. Whether these mutations result in APC resistance and are associated with a risk of thrombosis is not clear. The main objective of the present study was to identify the APC-resistant phenotype of FV molecules with different mutations in APC cleavage sites. To study this, recombinant FV mutants were reconstituted in FV-deficient plasma, after which normalized APC-sensitivity ratios (n-APC-SRs) were measured in activated partial thromboplastin time-based and Russell's Viper Venom time-based APC-resistance tests. The mutations introduced in FV were R306G, R306T, R506Q, R679A and combinations of these mutations. Based on the APC-sensitivity ratios, we conclude that the naturally occurring mutations at Arg306 (i.e. FV HongKong and FV Cambridge) result in a mildly reduced sensitivity for APC (n-APC-SR, 0.74-0.87), whereas much lower values (n-APC-SR, 0.41-0.51) are obtained for the mutation at Arg506 (FV Leiden). No effect on the n-APC-SR was observed for the recombinant FV mutant containing the single Ala679 mutation. Because reduced sensitivity for APC, not due to FV Leiden, is a risk factor for venous thrombosis, these data suggest that mutations at Arg306 might be associated with a mild risk of venous thrombosis.     

Low risk of venous thrombosis in two families with combined type I plasminogen deficiency and factor V R506Q mutation

Two families with type I plasminogen deficiency and APC resistance are reported. The proposita of family A suffered from ischemic stroke when taking estrogen-progesterone-containing oral contraceptive. Several hemostatic challenges in the past (ovariectomy, appendectomy, and two pregnancies) were without thrombosis. Plasminogen activity and antigen (60 and 58%, normal range 72–136 and 69–135%, respectively) were reduced, and an increased APC resistance (APC-SR = 1.55; normal range 1.8–3.00) associated with G → A change at 1,691 nucleotide position in exon 10 of FV gene (FV Leiden) was observed. The asymptomatic son had isolated plasminogen deficiency (activity 57% and antigen 60%) whereas the asymptomatic daughter had isolated APC resistance (APC-SR = 1.61) and FV Leiden mutation. The proposita of family B, referred for superficial thrombophlebitis, had low plasminogen levels (activity 55% and antigen 53%) and APC resistance (APC-SR = 1.5) whereas the asymptomatic mother and the brother had isolated APC resistance (APC-SR = 1.62 and 1.8, respectively) and the asymptomatic father isolated plasminogen deficiency (activity 61% and antigen 62%). These data suggest that the combination of plasminogen deficiency and APC resistance probably does not significantly increase the risk of venous thrombosis. However, larger experience with additional cases is needed to definitely assess the magnitude of thrombotic risk in these families. Am. J. Hematol. 57:344–347, 1998. © 1998 Wiley-Liss, Inc.     

Impaired APC cofactor activity of factor V plays a major role in the APC resistance associated with the factor V Leiden (R506Q) and R2 (H1299R) mutations

Activated protein C (APC) resistance is a major risk factor for venous thrombosis. Factor V (FV) gene mutations like FV(Leiden) (R506Q) and FV(R2) (H1299R) may cause APC resistance either by reducing the susceptibility of FVa to APC-mediated inactivation or by interfering with the cofactor activity of FV in APC-catalyzed FVIIIa inactivation. We quantified the APC cofactor activity expressed by FV(Leiden) and FV(R2) and determined the relative contributions of reduced susceptibility and impaired APC cofactor activity to the APC resistance associated with these mutations. Plasmas containing varying concentrations of normal FV, FV(Leiden), or FV(R2) were assayed with an APC resistance assay that specifically measures the APC cofactor activity of FV in FVIIIa inactivation, and with the activated partial thromboplastin time (aPTT)-based assay, which probes both the susceptibility and APC cofactor components. FV(R2) expressed 73% of the APC cofactor activity of normal FV, whereas FV(Leiden) exhibited no cofactor activity in FVIIIa inactivation. Poor susceptibility to APC and impaired APC cofactor activity contributed equally to FV(Leiden)-associated APC resistance, whereas FV(R2)-associated APC resistance was entirely due to the reduced APC cofactor activity of FV(R2). Thrombin generation assays confirmed the importance of the anticoagulant activity of FV and indicated that FV(Leiden) homozygotes are exposed to a higher thrombotic risk than heterozygotes because their plasma lacks normal FV acting as an anticoagulant protein.     

Activated protein C resistance as an additional risk factor for thrombosis in protein C-deficient families

Heterozygous protein C deficiency is associated with an increased risk for thrombosis. This association is restricted to a minority of protein C-deficient families, which have been defined as clinically dominant protein C-deficient. In contrast, in the clinically recessive protein C- deficient families, only the homozygous family members are (severely) affected. One possible explanation for this difference in thrombotic risk between families may be the presence of a second hereditary risk factor. A good candidate for this second risk factor is the recently identified resistance to activated protein C (APC). APC resistance, which is associated with a mutation in the FV gene (FV Leiden), is a common and strong risk factor for thrombosis. We show here that the prevalence of the FV Leiden mutation is high among symptomatic protein C-deficient probands (19%). In 6 clinically dominant protein C- deficient families, the segregation of the FV Leiden mutation and the protein C gene mutation was studied. A thrombotic episode had been experienced by 73% of the family members having both the protein C gene mutation and the FV Leiden mutation. In contrast, respectively, 31% and 13% of the family members having either the protein C gene mutation or the FV Leiden mutation had experienced a thrombotic episode. Moreover, the result of a two locus linkage analysis support the assumption that the FV gene and the protein C gene are the two trait loci responsible for the thrombophilia. These results indicate that carriers of both gene defects have an increased risk for thrombosis compared with related carriers of the single defect.     

Factor V Leiden: a disorder of factor V anticoagulant function

PURPOSE OF REVIEW: Activated protein C (APC) resistance, which is often associated with the factor V R506Q (FV Leiden) mutation, is a common risk factor for venous thrombosis. Study of the mechanism of APC resistance has revealed that coagulation FV stimulates the APC-catalysed inactivation of FVIIIa, and that this anticoagulant function of FV is impaired in FV Leiden. The present review covers the discovery, the physiological significance and the structural requirements of the APC-cofactor activity of FV. RECENT FINDINGS: Recent in vitro and in vivo experiments indicate that the anticoagulant activity of FV is physiologically relevant and that FV plays a major role in the maintenance of the haemostatic balance. Quantitative and functional defects of the APC-cofactor activity of FV lead to increased thrombin generation and are associated with a prothrombotic state. Although the structural requirements for the expression of the APC-cofactor activity of FV are now beginning to be unravelled, the underlying molecular mechanism remains elusive. SUMMARY: The APC-cofactor activity of FV and its impairment in FV Leiden can explain the different thrombosis risks associated with heterozygosity, homozygosity and pseudo-homozygosity for FV Leiden. Elucidation of the molecular mechanism of the anticoagulant function of factor V may provide novel targets for the design of antithrombotic drugs.     

Molecular analysis of factor V Leiden, factor V Hong Kong, factor II G20210A, methylenetetrahydrofolate reductase C677T, and A1298C mutations related to Turkish thrombosis patients.

Inherited gene disorders related to the hemostatic system have been documented as risk factors for thrombosis. The roles of factor V Hong Kong (FV Hong Kong), factor V Leiden (FV Leiden), factor II G20210A (FII G20210A), methylenetetrahydrofolate reductase (MTHFR) C677T, and MTHFR A1298C mutations in Turkish patients with thrombosis (270 patients) compared with healthy controls (114 subjects) were evaluated. Polymerase chain reaction-based restriction enzyme analysis was carried out to screen these mutations, and single-strand conformation analysis was established to identify variations using the primers selected for restriction enzyme analysis studies. As a result, a significant relationship was determined among FV Leiden, FII G20210A, and thrombosis. The FV Hong Kong mutation was observed in only 2 patients with pulmonary vein thrombosis who are FV Leiden/FV Hong Kong compound heterozygous for FV gene. MTHFR C677T and A1298C were equally distributed in the patient group compared with the control group. All named mutations were also identified with single-strand conformation analysis, but a new variant/polymorphism during studies was not found. Because some inherited abnormalities are associated with thromboembolic disorders, determining the mutations and gene-to-gene interactions in patients with thrombosis history has a great impact on diagnosis and treatment of these diseases.     

The factor V (FV) gene ASP79HIS polymorphism modulates FV plasma levels and affects the activated protein C resistance phenotype in presence of the FV Leiden mutation

BACKGROUND AND OBJECTIVES: In carriers of the factor V (FV) Leiden mutation, different trans-acting gene variants (HR2 haplotype and FV Cambridge mutation) affect activated protein C (APC) sensitivity. Among a series of FV gene variants characterized, the Asp79His polymorphism appeared to be a good candidate for the modulation of FV activity. DESIGN AND METHODS: In a group of 150 apparently healthy subjects without the FV Leiden mutation and in 55 apparently healthy subjects with mutation, genotypes of the Asp79His polymorphism and of the HR haplotype were characterized and plasma levels of FV coagulant activity and APC ratios evaluated. RESULTS: In the group without the FV Leiden mutation, 16 subjects (10.7%) carried the His 79 allele and 15 subjects (10.0%) the HR2 haplotype. Two of them carried both gene variants. As compared to FV activity levels in non-carriers (106.4+18.5%), values were lower in subjects with the His79 allele (95.2+25.2%; p=0.025) and in those with the HR2 haplotype (93.7+16.2%; p =0.007). FV activity levels were further reduced in carriers of both FV gene variants (78.7+3.3%; p =0.009). APC values were similar among individuals carrying different FV genotypes. In the group with the FV Leiden mutation, APC ratios were lower in subjects carrying the His 79 allele (0.63; p =0.008) or the HR2 haplotype (0.63; p =0.026) than in subjects without (0.69) reflecting FV activity values. INTERPRETATION AND CONCLUSIONS: Present data suggest that carriership of the His79 allele modulate plasma levels of FV coagulant activity and, in subjects carrying the FV Leiden mutation, affects APC sensitivity.     

Influence of three potential genetic risk factors for thrombosis in 43 families carrying the factor V Arg 506 to Gln mutation

The factor V (FV) Arg 506 to Gln mutation is the most common abnormality observed in familial thrombophilia. Many studies have shown that its clinical expression differs among families and among carriers. Some thrombotic patients carry an additional genetic risk factor such as protein C, protein S or antithrombin deficiency. We sought to identify other genetic risk factors potentially favouring expression of the thrombotic phenotype in 370 members of 43 families with the FV Arg 506 to Gln mutation. We analysed three candidate polymorphisms in genes involved in the PC anticoagulant pathway, consisting of two polymorphic sites in the 5' non-transcribed region of the PC gene, -1654 C/T and -1641 A/G, with three known combinations (TA, CA and CG) that influence the protein C plasma level; one polymorphic site (4070 A/G) in exon 13 of the FV gene, which influences the plasma factor V concentration, and one polymorphic site (677 C/T) in the methylenetetrahydrofolate reductase gene, which is often associated with moderate hyperhomocysteinaemia. The distribution of these different polymorphisms was similar in patients with a history of thrombosis and those who remained asymptomatic, ruling out the possibility that each of these polymorphisms alone can play a role in the onset of thrombosis in carriers of the FV Arg 506 to Gln mutation.     

Association between adverse pregnancy outcomes and maternal factor V G1691A (Leiden) and prothrombin G20210A genotypes in women with a history of recurrent idiopathic miscarriages

Thrombophilia was implicated in the development of pregnancy complications, including recurrent idiopathic pregnancy loss, and is aggravated in women who are carriers of factor V G1691A (FV Leiden) and prothrombin (PRT) G20210A single-nucleotide polymorphisms (SNPs). Previous studies examined the role of FV-Leiden and PRT G20210A in recurrent pregnancy loss with conflicting results. Here we examined the prevalence of FV Leiden and PRT G20210A SNPs, in 200 women with 3 or more consecutive early (n = 87), late (n = 41), or early-late (n = 72) recurrent pregnancy losses, and 200 age-matched fertile parous control women. APC resistance (APCR) was detected functionally (measuring the activated clotting time triggered by activated factor X in presence of a fixed amount of purified APC), and FV-Leiden and PRT G20210A genotypes were assessed by PCR. The frequency of the mutant FV (0.1400 vs. 0.0276; P < 0.001) but not PRT 20210 (0.0100 vs. 0.0225; P = 0.159) allele was higher in patients than controls, respectively. APC resistance with factor V Leiden was seen in 27% of patients compared to 11.5% of controls, while APC resistance without factor V Leiden was seen in 12.5% of patients compared to 9.5% of controls. Regression analysis demonstrated that the significant predictors for early abortion was FV Leiden; those for late abortion were oral contraceptive, APCR, and FV Leiden; and predictors for early-late abortions were oral contraceptives, obesity, FV Leiden, and smoking. APC resistance and FV Leiden, as well as combination of both, are common thrombotic defects seen in women with idiopathic recurrent pregnancy loss, thus testing for these is recommended in women who have experienced recurrent miscarriages.     

Pseudohomozygosity for activated protein C resistance is a risk factor for venous thrombosis

Pseudohomozygosity for activated protein C resistance (APC-r) is a rare condition due to the association of heterozygous FV Leiden mutation and partial type I FV deficiency. To assess the risk of venous thromboembolism in these subjects, seven families including 11 pseudohomozygotes and 45 relatives were examined. Among the relatives, 16 were heterozygous FV Leiden carriers, nine showed partial FV deficiency and 20 no abnormalities. Deep vein thrombosis occurred in 4/11 (36.3%) pseudohomozygous patients versus 6/16 (37. 4%) FV Leiden carriers and 1/20 (5%) normal relatives. Pseudohomozygotes and FV Leiden carriers had a significantly increased risk of venous thrombosis in comparison to normal relatives (RR 8.8 and 5.7, respectively). There was no difference between the thrombotic risk of pseudohomozygous subjects and of FV Leiden carriers (RR 1.6, 95% CI 0.43-5.7). Furthermore, there was no difference in thrombosis-free survival between pseudohomozygotes and 45 consecutive FV Leiden heterozygous outpatients, suggesting that a referral bias may explain the apparent younger age of thrombosis in the pseudohomozygotes in comparison to relatives with FV Leiden heterozygosity (27 years v 54 years; P = 0.01). Pseudohomozygosity for APC resistance carries a significantly higher risk for venous thromboembolism in comparison to normal subjects, but probably not in comparison to heterozygous FV Leiden carriers.     

Coexistence of factor V G1691A and factor II G20210A gene mutations in a thrombotic family is associated with recurrence and early onset of venous thrombosis.

Two G-to-A mutations at positions 1691 of the factor V (FV) gene and 20210 of the prothrombin (FII) gene have been associated with an increased risk of venous thromboembolism. We report a thrombosis-prone family in which one subject--the propositus who exhibited combined heterozygous FV G1691A and FII G20210A mutations--showed spontaneous and early clinical onset (at 23 years), recurrences of deep-vein thrombosis and pulmonary embolism. His asymptomatic father carried the FII G20210A substitution and his mother, characterized by an isolated thrombotic episode on occasion of surgery (at 48 years), carried the FV G1691A substitution. In the maternal lineage, one of the propositus' uncles had thrombosis on occasion of a bone fracture (at 65 years) despite the absence of known prothrombotic defects. A sister of the propositus carried the FII G20210A and the brother the FV G1691A mutation. They have been asymptomatic until now. The propositus' two children, 20 and 16 years old, both carry the FV G1691A substitution and have been asymptomatic until now. The plasma levels of FII were higher in carriers of the FII G20210A allele if compared with noncarriers, and the activated protein C resistance phenotype, associated with the FV Leiden mutation, showed a complete correlation with the FV G1691A mutation. Despite the very limited number of thrombotic cases involved in this survey, which does not allow statistically sound conclusions, the data obtained from this family suggest that the synergy of inherited factors and transient risk conditions could play a key role in the occurrence of thrombotic accidents.     

Gene analysis and prenatal diagnosis for two families of congenital factor V deficiency

Summary. We studied two families in which the probands had severe bleeding tendency and showed low plasma levels of coagulation factor V (FV) antigen and activity. Sequence analysis of the FV gene on proband 1 demonstrated a novel G16088C homozygous missense mutation in exon 3 resulting in an Asp 68 to His substitution and on proband 2, a C69969T homozygous missense mutation in exon 23 leading to Gly2079Val. The parents of both families were each heterozygous for the corresponding FV gene defect. During their second pregnancy, the two families requested prenatal diagnosis. Chorionic villi were analysed at 12 weeks of gestation and cord blood samples were tested at 22 weeks. Microsatellite analysis performed in family 1 showed that the foetus sample was not contaminated by maternal tissue. The foetus 1 was found to be heterozygous for the familiar G16088C mutation with lower FV activity in the cord blood; the foetus 2 was a normal one. The diagnosis was confirmed after the birth. This is the first report of prenatal diagnosis for FV deficiency.     

Use of a generally applicable tissue factor--dependent factor V assay to detect activated protein C-resistant factor Va in patients receiving warfarin and in patients with a lupus anticoagulant

The original activated partial thromboplastin time-based assay for activated protein C (APC)-resistant factor Va (FVa) requires carefully prepared fresh plasma and cannot be used in patients receiving warfarin or in patients with antiphospholipid antibodies. A new test is described here that circumvents these limitations and distinguishes without overlap heterozygotes for APC-resistant FVa from persons with normal FV. A diluted test plasma is incubated with an FV-deficient substrate plasma and tissue factor and then clotted with Ca2+ or Ca2+ plus APC. Test results are independent of the FV level or the dilution of the test plasma used. Of 39 controls, 37 gave normal results. Two controls (5%) gave results indicative of APC resistant FVa and on DNA analysis were found to be heterozygous for FV R506Q. Twenty of 21 randomly selected patients receiving warfarin gave normal results. In the single patient with abnormal results, heterozygous FV R506Q was confirmed by DNA analysis. Two of 15 patients with protein S deficiency and 5 of 29 patients with a lupus anticoagulant had abnormal results. APC resistance caused by FV R506Q was confirmed in the five of these seven patients available for DNA analysis. APC-resistant FVa was also detected in 10 of 21 (46%) stored plasma from unrelated patients with venous thrombosis and negative earlier evaluation for a lupus anticoagulant or a deficiency of protein C, protein S, or antithrombin, which confirms a high incidence of this defect among patients with venous thrombosis.     

Method-dependent influence of certain polymorphisms in the factor V B-domain on the response to activated protein C.

The factor V (FV) B-domain is extremely important to the cofactor function of native FV for activated protein C (APC) in the inactivation of factor VIII (FVIII). In a previous study, we found that in the B-domain coding portion of DNA, the polymorphisms at nucleotide positions 2391, 2663, 2684, and 2863 were associated. In the major allele, all bases are A (A allele) and those in the minor allele are G (G allele). This study concerns itself with the question of whether or not there are differences in the APC response between the A allele and the G allele in plasma samples from persons without the FV Leiden. The APC ratios of homozygous carriers of the major A allele and the minor G allele do not differentiate themselves in classical activated partial thromboplastin time-based assays. In contrast, a test based on the deactivation of FVIII in the tenase complex in homozygous carriers of the minor G allele showed significantly lower APC ratios (P = 0.001) in comparison with the major A allele. The results of the investigation after modification of the test indicate that mutative changes in the B-domain apparently influence the interaction among phospholipids, APC, FV, and protein S. An increase in FVIII through the introduction of the FVIII concentrate Kogenate to the plasma samples was associated with a drop in the APC ratios of both genotypes. After defining 59 age- and sex-based matched pairs without the FV Leiden, the observed frequency of the minor G allele was higher in the non-thrombotic group (33.0%) than in the thrombotic group (22.8%). However, the difference did not reach the level of significance (odds ratio, 0.53; 95% confidence interval, 0.26-1.12). It does, nevertheless, appear possible that a homozygous condition for the minor allele in combination with a defect known to be associated with thrombophilia represents an additional thrombogenetic risk factor.     

Coinheritance of Factor V (FV) Leiden enhances thrombin formation and is associated with a mild bleeding phenotype in patients homozygous for the FVII 9726+5G>A (FVII Lazio) mutation

We investigated the role of thrombophilic mutations as possible modifiers of the clinical phenotype in severe factor VII (FVII) deficiency. Among 7 patients homozygous for a cross-reacting material-negative (CRM-) FVII defect (9726+5G>A, FVII Lazio), the only asymptomatic individual carried FV Leiden. Differential modulation of FVII levels by intragenic polymorphisms was excluded by a FVII to factor X (FX) gene haplotype analysis. The coagulation efficiency in the FV Leiden carrier and a noncarrier was evaluated by measuring FXa, FVa, and thrombin generation after extrinsic activation of plasma in the absence and presence of activated protein C (APC). In both patients coagulation factor activation was much slower and resulted in significantly lower amounts of FXa and thrombin than in a normal control. However, more FXa and thrombin were formed in the plasma of the patient carrying FV Leiden than in the noncarrier, especially in the presence of APC. These results were confirmed in FV-FVII doubly deficient plasma reconstituted with purified normal FV or FV Leiden. The difference in thrombin generation between plasmas reconstituted with normal FV or FV Leiden gradually decreased at increasing FVII concentration. We conclude that coinheritance of FV Leiden increases thrombin formation and can improve the clinical phenotype in patients with severe FVII deficiency.     

Molecular characterization of 3 factor V mutations, R2174L, V1813M, and a 5-bp deletion, that cause factor V deficiency

We identified 3 mutations in the factor V (FV) gene (F5) associated with FV deficiency in 3 unrelated Japanese patients. Patient 1 had severe bleeding symptoms (plasma FV activity, <1%; FV antigen, 9%) and was a compound heterozygote for a novel 5-bp deletion in exon 22 and the V1813M mutation. Patient 2 had moderate bleeding symptoms (plasma FV activity, <1%; FV antigen, 4%) and was homozygous for the V1813M mutation. Patient 3 had very mild symptoms (plasma FV activity, 1%; FV antigen, 5%) and was homozygous for the novel R2174L mutation. A study of recombinant protein expression revealed that the FV coagulant-specific activities in conditioned media for the FV-R2174L and FV-V1813M mutants were reduced to approximately 40% and 28% of wild-type FV, respectively. The amounts of FV-R2174L protein and messenger RNA in the platelets of patient 3 were similar to those of healthy subjects; however, the amount of FV-V1813M protein in patient 2 was decreased. Our data suggest that the severity of the bleeding tendency in patients with FV deficiency is correlated not only with plasma FV activity but also with the amount of FV protein in the platelets.     

The factor V HR2 haplotype: prevalence and association of the A4070G and A6755G polymorphisms.

Recently, a polymorphism was identified in exon 25 of the factor V gene that is possibly a functional candidate for the HR2 haplotype. This haplotype is characterized by a single base substitution named R2 (A4070G) in the B domain of the protein. A mutation (A6755G; 2194Asp-->Gly) located near the C terminus has been hypothesized to influence protein folding and glycosylation, and might be responsible for the shift in factor V isoform (FV1 / FV2) ratio. This study investigated the prevalence of these two factor V HR2 haplotype polymorphisms in a cohort of normal blood donors, patients with osteoarthritis and women with complications during pregnancy, and in families of factor V Leiden individuals. A high allele frequency for the two polymorphisms was found in the blood donor group (6.2% R2, 5.6% A6755G). No significant difference in allele frequency was observed in the clinical groups (obstetric complications and osteoarthritis, 4.1-4.9% for the two polymorphisms) when compared with that of healthy blood donors. We confirm that the factor V A6755G polymorphism shows strong linkage to the R2 allele, although it is not exclusively inherited with the exon 13 A4070G variant and can occur independently.     

Characterization of an apparently synonymous F5 mutation causing aberrant splicing and factor V deficiency

Coagulation factor V (FV) deficiency is a rare autosomal recessive bleeding disorder. We investigated a patient with severe FV deficiency (FV:C < 3%) and moderate bleeding symptoms. Thrombin generation experiments showed residual FV expression in the patient's plasma, which was quantified as 0.7 ± 0.3% by a sensitive prothrombinase‐based assay. F5 gene sequencing identified a novel missense mutation in exon 4 (c.578G>C, p.Cys193Ser), predicting the abolition of a conserved disulphide bridge, and an apparently synonymous variant in exon 8 (c.1281C>G). The observation that half of the patient's F5 mRNA lacked the last 18 nucleotides of exon 8 prompted us to re‐evaluate the c.1281C>G variant for its possible effects on splicing. Bioinformatics sequence analysis predicted that this transversion would activate a cryptic donor splice site and abolish an exonic splicing enhancer. Characterization in a F5 minigene model confirmed that the c.1281C>G variant was responsible for the patient's splicing defect, which could be partially corrected by a mutation‐specific morpholino antisense oligonucleotide. The aberrantly spliced F5 mRNA, whose stability was similar to that of the normal mRNA, encoded a putative FV mutant lacking amino acids 427–432. Expression in COS‐1 cells indicated that the mutant protein is poorly secreted and not functional. In conclusion, the c.1281C>G mutation, which was predicted to be translationally silent and hence neutral, causes FV deficiency by impairing pre‐mRNA splicing. This finding underscores the importance of cDNA analysis for the correct assessment of exonic mutations.