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 and other coagulation factor mutations affecting thrombotic risk

Five genetic defects have been established as risk factors for venous thrombosis. Three are protein C, protein S, and antithrombin deficiencies, defects in the anticoagulant pathways of blood coagulation. Together they can be found in approximately 15% of families with inherited thrombophilia. Their laboratory diagnosis is hampered by the large genetic heterogeneity of these defects. The other two genetic risk factors, resistance to activated protein C associated with the factor V Leiden mutation and increased prothrombin associated with the prothrombin 20210 A allele, are much more prevalent and together can be found in 63% of the thrombophilia families. Because both defects are caused by a single mutation, DNA analysis is the basis of their laboratory diagnosis.     

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.     

Factor V Leiden and genetic defects of thrombophilia in childhood porencephaly

AIMS: To determine to what extent the Arg506 to Gln point mutation in the factor V gene and further genetic factors of thrombophilia affect the risk of porencephaly in neonates and infants. METHODS: The Arg506 to Gln mutation, factor V, protein C, protein S, antithrombin, antiphospholipid antibodies and lipoprotein (a) (Lp(a)) were retrospectively measured in neonates and children with porencephaly (n = 24). RESULTS: Genetic risk factors for thrombophilia were diagnosed in 16 of these 24 patients: heterozygous factor V Leiden (n = 3); protein C deficiency type I (n = 6); increased Lp (a) (n = 3); and protein S type I deficiency (n = 1). Three of the 16 infants had two genetic risk factors of thrombophilia: factor V Leiden mutation combined with increased familial Lp (a) was found in two, and factor V Leiden mutation with protein S deficiency type I in one. CONCLUSIONS: The findings indicate that deficiencies in the protein C anticoagulant pathway have an important role in the aetiology of congenital porencephaly.     

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.     

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).     

Is hormone replacement a risk factor for ischemic stroke in women with factor V Leiden mutation?

OBJECTIVE: To describe a patient with multifocal cerebral ischemia whose only identified potential risk factors were use of postmenopausal hormone replacement and heterozygosity to factor V Leiden mutation. DESIGN: A case report. SETTING: A tertiary care center. PATIENT: A 51-year-old woman taking hormone replacement (0.625 mg/d of estrogen alternating with 10 mg/d of medroxyprogesterone) presented with a generalized tonic-clonic seizure. She had persistent multifocal non-enhancing lesions on magnetic resonance imaging of the brain. A stereotactic biopsy of the brain performed to exclude gliomatosis cerebri was consistent with cerebral ischemia. An extensive evaluation to uncover the cause of stroke revealed only heterozygosity to factor V Leiden mutation. MAIN OUTCOME AND RESULTS: Hormonal replacement was discontinued and the patient had no recurrent ischemic strokes. CONCLUSIONS: Postmenopausal hormonal replacement may be a risk factor for ischemic stroke in women with the factor V Leiden mutation. Ongoing trials of hormonal replacement provide an opportunity to test this hypothesis.     

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.     

Risk of venous thromboembolism associated with a G to A transition at position 20210 in the 3'-untranslated region of the prothrombin gene

The odds ratio for the FII 20210G/A mutation in 504 patients with venous thromboembolism compared to controls was 2.0 (95% CI 1.0-4.0) and, for factor V Leiden, 5.8 (95% CI 3.3-10.3). 3/504 patients were heterozygous for both mutations. None of the patients had combined natural anticoagulant deficiency and the FII 20210G/A mutation. We conclude that the FII 20210G/A mutation is present in 2.6% of the population and the relative risk of venous thromboembolism in carriers is 2.0.     

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.     

Uterine rhabdomyosarcoma metastatic to mediastinal lymph nodes: diagnosis by transbronchial needle aspiration

An inherited tendency to hypercoagulability has been suggested as a cause of vascular thrombosis resulting in Legg-Calvé-Perthes disease (LCPD). Here we carried out an investigation of the most common inherited risk factors for hypercoagulability including the mutation in the factor V gene (factor V Leiden), the transition 20.210G-->A in the prothrombin gene, and also the homozygosity for the 677C-->T transition in the methylenetetrahydrofolate reductase gene (MTHFR). The investigation was carried out among 61 Brazilian children with LCPD, who were compared with 296 individuals from the general population. The prevalence of the factor V Leiden mutation was higher in LCPD patients than in the controls (4.9 vs. 0.7%; p = 0.03). However, no patient had the prothrombin gene variant, and no difference was found between patients and controls when homozygosity for MTHFR-T (3.2 vs. 2.6%: p = 0.64) was determined. These data suggest that in our population, the heterozygosity for factor V Leiden was the only inherited risk factor associated with the development of LCPD.     

C677T MTHFR mutation and factor V Leiden mutation in patients with TIA/minor stroke: a case-control study

A common C677T mutation in the gene for the enzyme 5,10-methylenetetrahydrofolate reductase (5,10-MTHFR) has been linked to elevated levels of homocysteine and was therefore suspected to be a candidate genetic risk factor for arterial occlusive disease. Another mutation, factor V Leiden, has been established as a common hereditary risk factor for venous thrombosis, but its role in arterial disease remains controversial. We investigated the prevalence of both the C677T MTHFR mutation and the factor V Leiden mutation in 81 patients with transient ischemic attack (TIA) or minor stroke (MS) and in 81 age- and sex-matched control subjects free from clinically manifest vascular disease. We further compared clinical and laboratory data as well as clinical course of patients carrying the factor V Leiden mutation alone or in combination with the C677T MTHFR mutation and mutation-free patients. The prevalence of the MTHFR mutation did not differ between patients and control subjects with 11.1% homozygous carriers in both groups (OR for homozygous carriers 1.0; 95% CI 0.38-2.66). However, there was a trend towards a higher prevalence of carriers of factor V Leiden in patients (12.3%) than in control subjects (4.9%) (OR 2.75; 95% CI 0.83-9.17;p=0.09). Furthermore, we found some evidence that the combined occurrence of the C677T MTHFR mutation and factor V Leiden might unfavorably affect the clinical course of the disease, but the number of respective patients was small. Larger studies with a greater number of carriers of both the C677T MTHFR mutation and factor V Leiden seem therefore warranted.     

Correlation between the functional assay for activated protein C resistance and factor V Leiden in the neonate

A factor V506 Arg-Gln mutation is the most common inherited cause of thrombophilia in adults. To date, there are no data regarding the detection of this mutation in neonatal blood or the relationship of this dysfunctional factor V to neonatal thrombosis. This study compared a modified activated protein C resistance functional assay with the PCR-based DNA assay for the factor V mutation in 115 prospectively collected umbilical cord blood samples. The incidence of activated protein C resistance in cord blood was 6%. The sensitivity and specificity of the modified assay for the factor V Leiden mutation was 100%.     

Factor V Leiden: who should be tested?

Resistance to activated protein C resulting from the genetic point mutation known as factor V Leiden is the most frequently found genetic risk factor associated with familial predisposition to venous thrombosis. Factor V Leiden is also frequent among people with nonfamilial venous thrombosis and appears to have a relatively high prevalence rate in the general population. The author comments on the findings of the first Canadian prevalence study of factor V Leiden, reported in this issue by Dr. David H. Lee and associates (see pages 285 to 289). She notes that although certain hereditary and clinical variables are known to modulate the risk of venous thrombosis in people with factor V Leiden, explanations for the relatively high prevalence of this mutation and the wide spectrum of risk associated with it are still speculative. Management guidelines for affected patients are quickly evolving but are still limited by a lack of clinical data. It is clear that further research into factor V Leiden will have considerable importance for the understanding and management of thrombotic risk.     

The factor V Leiden mutation increases the risk of venous thrombosis in patients with inflammatory bowel disease

BACKGROUND & AIMS: Thromboembolic disease is a significant cause of morbidity and mortality in patients with inflammatory bowel disease (IBD). The aim of this study was to determine the incidence and possible association of the factor V Leiden mutation with the development of thrombosis in patients with IBD. METHODS: This retrospective study included 11 patients with IBD and arterial or venous thrombosis and 51 patients with IBD and no history of thrombosis who were matched for age, sex, ethnic/racial origin, and type of IBD (controls). The presence of the factor V Leiden mutation was determined by coagulation assay and confirmed by a polymerase chain reaction method. RESULTS: Four of 11 IBD patients (36%) with thrombosis and 2 of 51 IBD controls (4%) were heterozygotes for the factor V Leiden mutation (relative risk, 14.00; 95% confidence interval, 1.55-169.25; P = 0.009, Fisher exact test). All thrombotic events in the patients with activated protein C resistance were venous with a calculated prevalence of 50% (4 of 8 patients) and a relative risk of venous thrombosis in IBD patients with factor V Leiden of 23 (95% confidence interval, 2-294; P = 0.005). CONCLUSIONS: In patients with IBD, inheritance of the factor V Leiden mutation results in a significant increased risk of venous thrombosis.     

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.     

The factor V Leiden mutation (R506Q) is not a major risk factor for migrainous cerebral infarction

The etiology of migrainous cerebral infarction is unknown, but may involve prothrombotic coagulation abnormalities. Therefore, we studied resistance to activated protein C and the presence of the Arg506Gln factor V Leiden mutation in 20 patients with migrainous cerebral infarction. Only one heterozygous carrier of the mutation was found, whereas other patients did not carry the mutation. This indicates that the factor V Leiden mutation is not a major risk factor for migrainous cerebral infarction.     

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.