Prothrombin antigen levels in symptomatic and asymptomatic carriers of the 20210A prothrombin variant

A recently discovered variant in the prothrombin gene (20210A) has been found in approximately 5-10% of patients with venous thromboembolism. It has been shown that patients with this variant present with high levels of prothrombin in plasma and this is maintained to be the most likely mechanism by which the risk of thrombosis is increased. We have evaluated prothrombin antigen levels in 50 carriers of the 20210A allele and compared with non-carriers. 327 subjects were subdivided according to deficiency status and previous thrombosis. 30 symptomatic and 20 asymptomatic carriers had increased mean prothrombin levels as compared to symptomatic (n=178) or asymptomatic (n=99) non-carriers. The percentage of subjects with prothrombin levels above cut-off values of 1.15u/ml or 1.30u/ml was significantly higher in carriers of the prothrombin variant as compared to non-carriers, regardless of a previous thrombosis. However, among non-carriers the percentage of those with prothrombin levels above cut-off values was significantly higher in the group of symptomatic as compared to asymptomatic individuals. In conclusion, increased prothrombin antigen levels, as detected by a specific ELISA, were found among 50 symptomatic and asymptomatic carriers of the 20210A prothrombin variant as well as among a large group of symptomatic non-carriers. The data are in agreement with those found by using functional tests for the determination of prothrombin levels in these patients.     

The prothrombin gene G20210A variant: prevalence in a U.K. anticoagulant clinic population

We have investigated the prevalence of a recently reported genetic variation in the prothrombin gene (G20210A) in patients with an objectively confirmed history of venous thrombosis, 12/219 patients (5.5%) were found to be heterozygous carriers of the 20210A allele. The incidence of the 20210A allele in a group of 164 healthy controls was 1.2% (allele frequency 0.61%, 95% CI 0.08-2.19). When patients with a known alternative hereditary risk factor for venous thrombosis (factor V Leiden mutation or deficiency of antithrombin, protein C or protein S) were excluded, the G20210A variant was found to increase the risk for venous thrombosis by approximately 5-fold (odds ratio 5.4, 95% CI 1.16-25.0). This prothrombin gene sequence variation adds further to the list of recognized genetic risk factors for thrombophilia.     

Factor V Leiden (G1691A), the prothrombin 3'-untranslated region variant (G20210A) and thermolabile methylenetetrahydrofolate reductase (C677T): a single genetic test genotypes all three loci--determination of frequencies in the S. Wales population of the UK

Simultaneous genetic diagnosis of factor V (FV) Leiden (G1691A), the prothrombin variant (G20210A) and the thermolabile methylenetetrahydrofolate reductase (MTHFR) variant (C677T) has been achieved using multiplex heteroduplex analysis. All three loci are amplified in a single polymerase chain reaction (PCR) containing test DNA and three heteroduplex generators, respectively detecting the three nucleotide substitutions. After PCR, the products are analysed directly without further manipulation and the resulting heteroduplex profiles permit straightforward interpretation of the respective genotypes. The multiplex test has been used to assess the prevalence and allele frequency of each of the three nucleotide substitutions in 300 individuals (150 males and 150 females) from the local (S. Wales) population. A prevalence of 8% and an allele frequency of 0.040 +/- 0.015 (95% confidence interval) was obtained for FV Leiden; the prothrombin variant showed a prevalence of 1% and an allele frequency of 0.007 +/- 0.006 (95% confidence interval); the MTHFR mutation showed a prevalence of 60% and an allele frequency of 0.377 +/- 0.039 (95% confidence interval). This method is applicable to investigation of large cohorts of patients with arterial or venous thrombotic disease.     

Screening methods in genetic diagnosis of hereditary protein C deficiency

Genomic analysis and detailed blood coagulation examinations of 22 family members of 18 families with repeatedly low protein C activity have been performed. Blood coagulation examinations: INR, fibrinogen, plasminogen, alpha-2-antiplasmin, lupus anticoagulant, APC resistance test, protein C activity and antigen, protein S activity and antithrombin activity. Genetic examinations: the presence of FII G20210A alle and FV:Q506 Leiden mutation were examined and for the mutation screening in the protein C gene combination of polymerase chain reaction (PCR) with denaturing gradient gelelectrophoresis (DGGE) or with single-strand conformation polymorphism (SSCP) analysis has been performed. The amplified DNA fragments with aberrant migration during DGGE and SSCP analysis were sequenced. Nine family members of seven families were identified carrying mutations in the protein C gene: one nonsense mutation in exon VII (Arg 157-Stop), two types of missense mutations in four patients in exon IXA (230 Arg-Lys, 254 Thr-Ile, the latter is a new mutation, Protein C Pécs), one missense mutation in two patients in exon IXB (325 Val-Ala), one missense mutation in exon IXC (359 Asp-Asn) and a rare frameshift deletion in exon IXC (364 Met-Trp, 378 Stop). Nine families were evaluated carrying no mutation in their protein C gene, but other genetic or blood coagulation disturbances have been identified, eight of them had borderline decrease in their protein C activity (60-70%). The presence of FV:Q506 mutation could be diagnosed in eight families (in 3 cases homozygous, in 5 cases heterozygous form), among them combination of the defects could be proved in three of the eight families: FV:Q506 Leiden mutation with antiphospholipoid antibodies in 2 families and the presence of Leiden mutation with prothrombin gene mutation in 1 family. Protein S deficiency in combination with prothrombin gene mutation has been identified in 1 family. There were 2 families where no genetic or blood coagulation alterations could be detected in the background of the repeatedly low protein C activity. Large deletions or insertions which are not detectable by our screening methods could not be excluded in these families and therefore sequencing of the total protein C gene had been performed with negative results. According to the literature and our experience the screening methods that were administered in this study are suitable for the detection of mutations in the protein C gene.     

Clinical studies and thrombin generation in patients homozygous or heterozygous for the G20210A mutation in the prothrombin gene

A genetic variation in the prothrombin gene, the G-->A transition at nucleotide 20210, is a risk factor for venous thrombosis in heterozygotes and is associated with increased prothrombin activity. The homozygous phenotype and the extent of thrombin generation in heterozygous and homozygous subjects are unknown. We investigated a family that included 2 homozygous and 5 heterozygous carriers of the 20210 A allele. The homozygous propositus and his presumably heterozygous father suffered from deep-vein thrombosis. His presumably heterozygous mother and his homozygous sister had recurrent phlebitis at a young age. The remaining 5 affected family members are still asymptomatic. We studied thrombin generation in the family and in 22 unrelated carriers of the 20210 A allele by measuring (1) prothrombin fragment F1+2 (F1+2) as an index of ongoing thrombin generation and (2) the endogenous thrombin potential (ETP) as an index of the possible thrombin-forming capacity. Their F1+2 levels were not different from those of age-matched controls, and thus, ongoing hemostatic system activation was not detectable. A significantly increased ETP was found in the heterozygous carriers of the 20210A allele compared with the controls (527.8+/-114.9 versus 387+/-50.1 nmol/L x min, P<0.0001). In the 2 homozygotes, the ETP was almost twice (639 and 751 nmol/L x min, respectively) as high as in the controls. We conclude that homozygosity for the G20210A mutation in the prothrombin gene is associated with a severe, albeit more benign, thrombotic diathesis compared with homozygosity for deficiencies of antithrombin, protein C, or protein S. In carriers of the 20210 A allele, the pathomechanisms leading to thrombosis should be sought in the higher amounts of thrombin that may be formed once thrombin generation is triggered, rather than in ongoing thrombin generation in vivo.     

CT pulmonary angiography. Stay tuned

It is well established that genetic disorders interact with environmental factors to cause thrombotic diseases. Therefore, antithrombin, protein C, protein S deficiencies and the more recently described factor V Leiden and prothrombin mutations are currently been investigated to explain some thrombophilic states. We report the case of a 63-year-old man who developed two transient ischemic attacks and two years later an extensive femoro-iliac venous thrombosis. He was genotyped as FV R506Q negative and FII G20210A positive in homozygous state (FII 20210AA).     

Axonal regeneration through muscle autografts submitted to local anaesthetic pretreatment

Over a 3 year period the R506Q mutation in the factor V (FV) FV:Q506 gene, FV, factor XII (FXII), prothrombin, protein C, protein S, antithrombin, heparin cofactor II, anticardiolipin antibodies and lipoprotein (a) (Lp(a)) were measured in 32 infants and children with sinus thrombosis. Heterozygous FV:Q506 (n=5), homozygous FV:Q506 (n=2), homozygous FXII deficiency (n=1), protein C deficiency type I (n=5), protein C deficiency type II (n=1), antithrombin deficiency type I (n=1) increased Lp (a) (n=5), activated protein C-resistance without mutation in the FV gene (n=2), and increased anticardiolipin IgG antibodies (n=2) were diagnosed in the children investigated. In a further two patients we found combinations of increased Lp(a) with moderate hyperhomocystinaemia and heterozygous plasminogen deficiency with heterozygous FXII deficiency. In addition, increased anticardiolipin IgG antibodies were found in combination with heterozygous FV:Q506 (n=1) and protein C type I deficiency (n=2) respectively. Out of 32 patients with venous sinus thrombosis, 3 showed additional peripheral venous vascular occlusion. Contributing factors were present in 31 out of 32 patients investigated. Family members of 10 affected children had suffered from venous thrombo-embolism prior to the study. CONCLUSION: Our data suggest that additional contributing factors may promote manifestation of cerebral venous sinus thrombosis in infants and children with an inherited prothrombotic state. Further prospective studies are required to evaluate their potential role as "triggering" agents.     

A common genetic variation in the 3'-untranslated region of the prothrombin gene is associated with elevated plasma prothrombin levels and an increase in venous thrombosis

We have examined the prothrombin gene as a candidate gene for venous thrombosis in selected patients with a documented familial history of venous thrombophilia. All the exons and the 5'- and 3'-UT region of the prothrombin gene were analyzed by polymerase chain reaction and direct sequencing in 28 probands. Except for known polymorphic sites, no deviations were found in the coding regions and the 5'-UT region. Only one nucleotide change (a G to A transition) at position 20210 was identified in the sequence of the 3'-UT region. Eighteen percent of the patients had the 20210 AG genotype, as compared with 1% of a group of healthy controls (100 subjects). In a population-based case-control study, the 20210 A allele was identified as a common allele (allele frequency, 1.2%; 95% confidence interval, 0.5% to 1.8%), which increased the risk of venous thrombosis almost threefold {odds ratio, 2.8; 95% confidence interval, 1.4 to 5.6}. The risk of thrombosis increased for all ages and both sexes. An association was found between the presence of the 20210 A allele and elevated prothrombin levels. Most individuals (87%) with the 20210 A allele are in the highest quartile of plasma prothrombin levels (> 1.15 U/mL). Elevated prothrombin itself also was found to be a risk factor for venous thrombosis.     

Geographic distribution of the 20210 G to A prothrombin variant

A variant in prothrombin (clotting factor II), a G to A transition at nucleotide position 20210, has recently been shown to be associated with the prothrombin plasma levels and the risk of both venous and arterial thrombosis. The purpose of this study was to investigate the prevalence of carriership of this mutation in various populations. We combined data from 11 centres in nine countries, where tests for this mutation had been performed in groups representing the general population. We calculated an overall prevalence estimate, by a precision-weighted method, and, since the distribution of the prevalences did not appear homogeneous, by an unweighted average of the prevalences. We examined differences in the prevalences by geographical location and ethnic background as a possible explanation for the heterogeneity. Among a total of 5527 individuals who had been tested, 111 heterozygous carriers of the 20210A mutation were found. The prevalence estimates varied from 0.7 to 4.0 between the centres. The overall prevalence estimate was 2.0 percent (CI95 1.4-2.6%). The variation around the summary estimate appeared more than was expected by chance alone, and this heterogeneity could be explained by geographic differences. In southern Europe, the prevalence was 3.0 percent (CI95 2.3 to 3.7%), nearly twice as high as the prevalence in northern Europe (1.7%, CI95 1.3 to 2.2%). The prothrombin variant appeared very rare in individuals from Asian and African descent. The 20210A prothrombin variant is a common abnormality, with a prevalence of carriership between one and four percent. It is more common in southern than in northern Europe. Since this distribution within Europe is very different to that of another prothrombotic mutation (factor V Leiden or factor V R506Q), founder effects are the most likely explanation for the geographical distribution of both mutations.     

Rapid multiplex analysis for the factor V Leiden and prothrombin G20210A mutations associated with hereditary thrombophilia

Thromboembolic episodes are common events and affect approximately one in 1,000 persons annually. Pulmonary embolism alone accounts for 50,000 to 100,000 deaths per year in the United States with > 50% of those being elderly persons. Resistance to activated protein C is the most common inherited disorder associated with hereditary thrombophilia. A missense mutation has been identified in the gene coding for coagulation factor V (codon 506) which renders this procoagulant factor resistant to inactivation by activated protein C resulting in an increased risk for venous thrombosis. Recently, a second polymorphism was identified in the prothrombin gene (factor II) which is also associated with increased risk for venous thrombosis. Because of the high prevalence of these two mutations in the general population as well as in specific patient populations, the ability readily to detect these two mutations must be feasible. In this study, we evaluated 303 patients for the prothrombin mutatin (G20210A) which were previously tested for the factor V mutation using established polymerase chain reaction-mediated restriction fragment length polymorphism assays. In these patients, 30 (9.9%) were found to be heterozygous for the factor V Leiden mutation with no homozygous mutants identified. Twenty individuals (6.6%) were heterozygous for the prothrombin G20210A mutation, and we identified two individuals (0.66%) who were homozygous for the 20210A allele. Of the total 303 individuals screened, two were double heterozygotes for both the factor V Leiden and the prothrombin gene mutations. We also describe a multiplex polymerase chain reaction-mediated restriction fragment length polymorphism assay for detecting both mutations in a single-tube double-enzyme digestion reaction making identification of these two mutations easily achievable.     

The heterozygous 20210 G/A prothrombin genotype is associated with early venous thrombosis in inherited thrombophilias and is not increased in frequency in artery disease

A genetic variation in the 3'-untranslated region of the prothrombin mRNA (20210 G/A) has recently been reported to be associated with elevated plasma prothrombin levels and with an increased incidence of venous thrombosis. We determined the frequency of this mutation, the detection of which was improved by allele-specific amplification of exon 14 and by denaturing gradients (denaturing gradient gel electrophoresis), in cohorts of patients affected by venous thrombosis (n = 132) or by coronary or cerebrovascular diseases (n = 195) and in normal subjects from various populations. An overlapping frequency of the heterozygous genotype (4%) was found in normal subjects from Italy and Cyprus, and no carrier was detected in 40 subjects of Indian or Somali origin. The 20210 GA heterozygous genotype was not increased in frequency in patients with arterial disease. In contrast, the GA genotype was associated (P = .007) with venous thrombosis both in simple heterozygotes (16%) with a family history of thrombosis as well as in double heterozygotes (14%) for other known thrombophilic defects. A synergic interaction between the prothrombin 20210 GA genotype and the factor V Leiden mutation, both potentially affecting the prothrombinase complex, was suggested by the early onset of thrombosis (median age 22 years) in doubly heterozygous patients. The association of the 20210 A allele with higher prothrombin levels was confirmed in the Italian population. However, the prothrombin assay does not allow an efficient preselection of patients for the DNA analysis.     

Genetic susceptibility to pregnancy-related venous thromboembolism: roles of factor V Leiden, prothrombin G20210A, and methylenetetrahydrofolate reductase C677T mutations

OBJECTIVE: This study's objective was to evaluate the association between venous thromboembolism during pregnancy and the postpartum period and the factor V Arg 506 Gln (factor V Leiden), the prothrombin G20210A, and methylenetetrahydrofolate reductase C677T polymorphisms. STUDY DESIGN: In this case-control study 42 case patients and 213 control subjects (parous age-matched women without history of thrombosis) were genotyped for all the polymorphisms. Moreover, antiphospholipid antibodies and protein C, protein S, and antithrombin III deficiencies were investigated in each case. RESULTS: Ten case patients (23.8%) and 4 control subjects (1.9%; odds ratio 16.3, 95% confidence interval 4.8-54.9) carried the factor V Leiden mutation; 13 case patients (31.0%) and 9 control subjects (4.2%; odds ratio 10.2, 95% confidence interval 4.0-25.9) were carriers of the prothrombin G20210A allele. Finally, 12 case patients (28.6%) and 34 control subjects (16.0%; odds ratio 2.1, 95% confidence interval 1.0-4.5) were homozygotes for methylenetetrahydrofolate reductase C677T. Overall, mutations were found in 25 case patients (59.5%) and 47 control patients (22.2%; odds ratio 5.2, 95% confidence interval 4.9-19.6). One patient carried the antithrombin III deficiency and 1 the protein S deficiency, whereas 2 women had a primary antiphospholipid syndrome. CONCLUSIONS: The significant risk estimates of having a pregnancy-related venous thromboembolism in the presence of the prothrombotic genetic risk factors analyzed suggest to screen for these mutations women with a personal history of thromboembolic events during pregnancy or the postpartum period.     

Anticoagulant response to Agkistrodon contortrix venom (ACV test): a new global test to screen for defects in the anticoagulant protein C pathway

As specific assays used to identify defects in the protein C (PC) anticoagulant pathway are laborious and expensive, we describe here a global test to screen for these defects. This assay is expressed as the ratio of two activated partial thromboplastin times, one in the absence and one in the presence of 0,125 U/ml of the PC activator of Agkistrodon contortrix venom (ACV). Eight of the 168 healthy volunteers of the control group exhibited an ACV ratio below the lower normal limit of 3.37 [6 subjects with the mutation Arg 506 to Gln in their factor V gene (FV R506Q) and one with PS deficiency]. 128 patients who have had at least one episode of deep-vein thrombosis were retrospectively studied. All patients carrying FV Q506R (n = 48), PC deficiency (n = 14) or combined defects, i.e. FV Q506R and PC deficiency (n = 4) or FV Q506R and PS deficiency (n = 3), had ACV ratios < 3.37. PS deficient plasmas (n = 20) exhibited ACV ratios which overlapped normal range. ACV ratios of one out of seven patients with antithrombin deficiency, and 10% of patients without identified defect in the PC anticoagulant pathway (n = 30) were < 3.37. An ACV ratio raised to 3.70 could lead to a test identifying all patients with a defect in the PC anticoagulant pathway.     

Prevalence of the prothrombin 20210 G-to-A variant in blacks: infants, patients with venous thrombosis, patients with myocardial infarction, and control subjects

A genetic variation in the prothrombin gene is located in the 3-untranslated region at position 20210 where a G-->A transition occurs. The prevalence of the mutation is 1% to 2% in white populations, and the mutation is associated with an increased risk of venous thrombosis and myocardial infarction. We report the prevalence of the A allele in blacks at birth; in black control subjects with no history of heart attack, stroke, or blood clots; in black patients with venous thrombosis; and in black patients with myocardial infarction. Among 318 infants, the prevalence of the A allele was 0.2% (1 heterozygote), with an exact, one-sided upper 95% confidence limit of 0.7%. Among 185 control subjects the variant was absent, yielding an exact, one-sided upper 95% confidence limit of 0.8% for the A allele. The heterozygous genotype was found in 2 of 91 subjects with deep vein thrombosis and in none of 123 subjects with myocardial infarction. The very low prevalence of the A allele indicates that the prothrombin variant is not a major cause of venous thrombosis or myocardial infarction in blacks.     

Resistance to activated protein C caused by the factor VR506Q mutation is a common risk factor for venous thrombosis

In 1993, inherited resistance to activated protein C (APC) was described as a novel risk factor for venous thrombosis. APC-resistance is present in 20-60% of venous thrombosis cases. It is caused by a single point mutation in the factor V gene which substitutes arginine (R) at position 506 with a glutamine (Q). The mutation is common in Caucasians with up to 15% prevalence in the population, whereas it is not found among other human races. Mutated factor V (FVR506Q, FV:Q506 or FV Leiden) is partially resistant to APC which results in a hypercoagulable state conferring a life-long increased risk of thrombosis. Individuals having FV:Q506 combined with other anticoagulant defects have a high risk of thrombosis, and it is now generally accepted that severe thrombophilia is a multigenetic disease. Easy functional and genetic tests for inherited APC-resistance will profoundly influence the development of prophylactic regimens and hopefully result in a decreased incidence of thrombosis.     

Oral contraceptives enhance the risk of clinical manifestation of venous thrombosis at a young age in females homozygous for factor V Leiden

In 29 patients (17 females) homozygous Arg 506 Gln mutation (FV Leiden) was identified. 25 had been investigated because of venous thromboembolism (VTE); four asymptomatic patients were found during family studies. The first VTE had occurred significantly earlier in females (median age [m] 26 years, range 17-49) than in males (m=38 years, range 21-82) (P=0.01). 12 females (80%) had taken oral contraceptives (OC, estrogen content 0.02-0.1 mg) for 6-150 months prior to thrombosis. Further triggering conditions in females were hormone replacement (n=1) and pregnancy (n=2). In 8/10 males the first VTE had occurred spontaneously--in two after surgery. The sites of VTE were deep vein thrombosis, pulmonary embolism, caval vein thrombosis and superficial thrombophlebitis. From our data we conclude that OC medication is the most important precipitating factor for VTE in females with homozygous FV Leiden.     

Multigenic thrombophilia: genetic anomaly of factor II and mutation of factor V Leiden. Study in a French family

BACKGROUND: A genetic variation of the prothrombin (factor II) gene, a G to A transition at nucleotide position 20210, was recently found in patients with familial thrombophilia (predisposition to venous thrombosis). It seems to be frequent in patients with the factor V Leiden mutation. We report a family with the factor V Leiden and/or the genetic variation of prothrombin in 3 members. CASE REPORT: The patient had repeated episodes of deep vein thromboses starting at the age of 30 during the 4th pregnancy. She is a heterozygous carrier of both the factor V Leiden nutation and the prothrombin mutation 20210 A. She has 4 asymptomatic children, aged 28 to 32 and 3 of them have been explored: one son has the prothrombin mutation, one daughter the factor V Leiden and one has none of them. DISCUSSION: This case report illustrates the polygenic nature of thrombophilia which may explain the heterogeneity of clinical expression observed in isolated congenital abnormalities, especially in factor V Leiden mutation.     

A single genetic origin for the common prothrombotic G20210A polymorphism in the prothrombin gene

The polymorphism G20210A in the 3' untranslated region of the prothrombin gene is associated with an increased level of factor II activity and confers a twofold to fivefold increase in the risk for venous thromboembolism. Among Caucasian populations, the prevalence of factor II G20210A heterozygotes is 1% to 6%, whereas in non-Caucasian populations it is very rare or absent. The aim of the present study was to discern whether factor II G20210A originated from a single or recurrent mutational events. Allele frequencies of four dimorphisms spanning 16 of 21 kb of the factor II gene were determined in 133 unrelated Caucasian subjects of Jewish, Austrian, and French origins who bore factor II G20210A (10 homozygotes and 123 heterozygotes) and 110 Caucasian controls. Remarkable differences in the allele frequencies for each dimorphism were observed between the study groups (P = .0007 or less), indicating strong linkage disequilibrium and suggesting a founder effect. Indeed, a founder haplotype was present in 68% of 20210A mutant alleles and only in 34% of 20210G normal alleles (P < .0001). These data strongly support a single origin for factor II G20210A that probably occurred after the divergence of Africans from non-Africans and of Caucasoid from Mongoloid subpopulations.