Elvira Grandone

Aspirin for Preventing the Recurrence of Venous Thromboembolism

BACKGROUND About 20% of patients with unprovoked venous thromboembolism have a recurrence within 2 years after the withdrawal of oral anticoagulant therapy. Extending anticoagulation prevents recurrences but is associated with increased bleeding. The benefit of aspirin for the prevention of recurrent venous thromboembolism is unknown. METHODS In this multicenter, investigator-initiated, double-blind study, patients with first-ever unprovoked venous thromboembolism who had completed 6 to 18 months of oral anticoagulant treatment were randomly assigned to aspirin, 100 mg daily, or placebo for 2 years, with the option of extending the study treatment. The primary efficacy outcome was recurrence of venous thromboembolism, and major bleeding was the primary safety outcome. RESULTS Venous thromboembolism recurred in 28 of the 205 patients who received aspirin and in 43 of the 197 patients who received placebo (6.6% vs. 11.2% per year; hazard ratio, 0.58; 95% confidence interval [CI], 0.36 to 0.93) (median study period, 24.6 months). During a median treatment period of 23.9 months, 23 patients taking aspirin and 39 taking placebo had a recurrence (5.9% vs. 11.0% per year; hazard ratio, 0.55; 95% CI, 0.33 to 0.92). One patient in each treatment group had a major bleeding episode. Adverse events were similar in the two groups. CONCLUSIONS Aspirin reduced the risk of recurrence when given to patients with unprovoked venous thromboembolism who had discontinued anticoagulant treatment, with no apparent increase in the risk of major bleeding. (Funded by the University of Perugia and others; WARFASA ClinicalTrials.gov number, NCT00222677.).

Meta-Analysis of Genome-Wide Association Studies in Celiac Disease and Rheumatoid Arthritis Identifies Fourteen Non-HLA Shared Loci

Epidemiology and candidate gene studies indicate a shared genetic basis for celiac disease (CD) and rheumatoid arthritis (RA), but the extent of this sharing has not been systematically explored. Previous studies demonstrate that 6 of the established non-HLA CD and RA risk loci (out of 26 loci for each disease) are shared between both diseases. We hypothesized that there are additional shared risk alleles and that combining genome-wide association study (GWAS) data from each disease would increase power to identify these shared risk alleles. We performed a meta-analysis of two published GWAS on CD (4,533 cases and 10,750 controls) and RA (5,539 cases and 17,231 controls). After genotyping the top associated SNPs in 2,169 CD cases and 2,255 controls, and 2,845 RA cases and 4,944 controls, 8 additional SNPs demonstrated P<5×10−8 in a combined analysis of all 50,266 samples, including four SNPs that have not been previously confirmed in either disease: rs10892279 near the DDX6 gene (Pcombined = 1.2×10−12), rs864537 near CD247 (Pcombined = 2.2×10−11), rs2298428 near UBE2L3 (Pcombined = 2.5×10−10), and rs11203203 near UBASH3A (Pcombined = 1.1×10−8). We also confirmed that 4 gene loci previously established in either CD or RA are associated with the other autoimmune disease at combined P<5×10−8 (SH2B3, 8q24, STAT4, and TRAF1-C5). From the 14 shared gene loci, 7 SNPs showed a genome-wide significant effect on expression of one or more transcripts in the linkage disequilibrium (LD) block around the SNP. These associations implicate antigen presentation and T-cell activation as a shared mechanism of disease pathogenesis and underscore the utility of cross-disease meta-analysis for identification of genetic risk factors with pleiotropic effects between two clinically distinct diseases.

Increased Risk for Venous Thrombosis in Carriers of the Prothrombin G→A20210 Gene Variant

Venous thrombosis is the third most common cardiovascular disorder after ischemic heart disease and stroke [1]. In addition to circumstantial risk factors (such as surgery, pregnancy, or immobilization), genetic abnormalities leading to hypercoagulability and to thrombophilia have been found in patients with thromboembolic disease [2]. Among patients from different ethnic populations, a common mutation in the gene of factor V (factor V Leiden mutation) [3] has been found in up to 60% of cases of familial thrombophilia [4]. Although the factor V Leiden mutation is a major risk factor for the development of venous thrombosis, many thrombotic events have an unclear pathogenesis. A common mutation, a GA transition at nucleotide position 20210 in the prothrombin gene locus, has recently been described [5]. The A allele is associated with higher plasma prothrombin levels, and in one study [5], carriers of this allele had a 2.8-fold increased risk for venous thrombosis compared with persons homozygous for the G allele. We sought to determine the presence of this mutation in 281 patients with venous thrombosis and investigated whether the presence of the prothrombin A (20210) allele was an independent, inherited risk factor for venous thrombosis. Methods After approval by the local ethics committees, the study was conducted according to the principles of the Declaration of Helsinki. All participants provided informed consent. Patients Persons with documented venous thrombosis who had been referred to one of two thrombosis centers (the IRCCS Casa Sollievo della Sofferenza in San Giovanni Rotondo, Italy, and the Ospedale A. Cardarelli in Naples, Italy) were investigated. Deep venous thrombosis was confirmed by phlebography or ultrasonography. Pulmonary embolism was diagnosed by angiography or ventilation-perfusion lung scanning. By using a previously validated questionnaire based on the World Health Organization questionnaire for cardiovascular disease [6], specially trained staff obtained a complete clinical summary from all patients, with emphasis on personal history of circumstantial risk factors for venous thromboembolism (surgery, immobilization, pregnancy, postpartum, trauma, use of oral contraceptives, varicose veins, and cancer) and for arterial thrombosis. Controls While patients were being recruited, apparently healthy persons were also randomly selected from a healthy population in southern Italy. None of these persons had been exposed to circumstantial risk factors for 8 weeks before the visit or had a history of venous thromboembolism, as determined by using a structured questionnaire validated for the retrospective diagnosis of venous thrombosis [7]. As was done with patients, trained staff obtained a detailed clinical summary from all controls, with emphasis on personal and family history for angina pectoris, myocardial infarction, ischemic stroke, and peripheral arterial disease [6]. Blood Collection and Coagulation Tests Blood samples were collected into vacuum tubes that contained 3.8% trisodium citrate as an anticoagulant. Samples were subjected to centrifugation at 2000 g for 15 minutes to obtain platelet-poor plasma, which was frozen and stored at 70C until assays were performed. Antiphospholipid antibodies (lupus anticoagulant and IgG anticardiolipin antibodies [measured by enzyme-linked immunosorbent assay, Byk Gulden, Italy]); antithrombin III, protein C, and amidolytic and immunologic protein S antigen (Behering, Marburg, Germany); and total and free protein S antigen (measured by enzyme-linked immunosorbent assay [Diagnostica Stago, Asnieres, France]) were measured in all patients [8, 9]. A thromboplastin-based assay on factor II-deficient plasma (Diagnostica Stago) was performed to measure prothrombin activity in 157 controls (72 men and 85 women) who were indistinguishable from other controls in age, sex, and social status (occupation). Results were expressed as the percentage of the amount of prothrombin in pooled normal plasma (arbitrarily designated as 100%). Clotting assays were performed on a KC4 Amelung coagulometer (Amelung, Austria). Extraction and Analysis of DNA Standard protocols were used to extract DNA from peripheral blood leukocytes [6]. The presence of factor V Leiden mutation was detected as described elsewhere [10], with some modifications [11]. The presence of the GA mutation of the prothrombin gene was tested according to the method of Poort and colleagues [5]. Statistical Analysis All analyses were done by using the Statistical Package for Social Science, version 6.1 for Macintosh (SPS, Inc., Chicago, Illinois). Differences in means and proportions were tested by using the Mann-Whitney U-test or the Kruskal-Wallis one-way analysis of variance and chi-square statistic or the Fisher exact test, as appropriate. We performed all analyses after grouping homozygous and heterozygous carriers of the prothrombin mutation and factor V Leiden mutation. Prevalence odds ratios (ORs), considered as the prevalence of existing disease, and 95% CIs were calculated by the normal approximation. If np(1 p) was small (<5), we used the exact method to calculate CIs (n = total number of participants, p = proportion of participants with a specific condition, and 1 p = proportion of participants without that condition). Adjusted odds ratios and their 95% CIs were calculated by logistic regression models that controlled for age, sex, presence of arterial thrombotic episodes, and factor V Leiden mutation. For all tests, a P value of 0.05 or less was considered statistically significant. Results Between May 1996 and December 1997, 348 patients (151 men and 197 women) with documented venous thrombosis were investigated. Sixty-seven patients (19.25%) had had at least one previous thrombotic events. Because these 67 patients are likely to have a greater susceptibility to thromboembolic episodes, we excluded them from the analysis. Thus, we analyzed 281 patients (128 men and 153 women; age range, 3 to 81 years). The median age at the time of the first thrombotic episode was 38.0 years (range, 16 to 81 years) for men and 35.0 years (range, 3 to 77 years) for women. The presenting thrombotic episode was deep venous thrombosis in one leg in 234 patients (106 men and 128 women; age range, 16 to 81 years), thrombosis of the upper extremities in 27 patients (13 men and 14 women; age range, 3 to 72 years), and isolated mesenteric vein thrombosis in 20 patients (age range, 17 to 70 years; 9 men and 11 women). Forty-five patients (17 men and 28 women) who had had deep venous thrombosis had also experienced an episode of pulmonary embolism. The control group consisted of 850 randomly selected, apparently healthy persons (388 men and 462 women; median age, 36.0 years [range, 22 to 66 years]). All patients and controls were white and were from the same region. The clinical characteristics of patients and controls are shown in Table 1. Table 1. Clinical Characteristics of Study Participants Forty patients (14.23% [95% CI, 10.14% to 18.32%]) carried the prothrombin GA20210 mutation; 35 were heterozygotes and 5 were homozygotes. Thirty-nine controls (4.59% [CI, 3.17% to 6.01%]) (P < 0.001) were heterozygotes, and none were homozygotes. The A20210 allele was noted in 8.01% of patients (CI, 5.77% to 10.25%) and 2.29% of controls (CI, 1.58% to 3.00%) (P < 0.001). The distribution of genotypes did not significantly differ from distributions predicted from the Hardy-Weinberg equilibrium in patients (P = 0.151) or controls (P > 0.2) and closely resembled those reported elsewhere [5]. Mean (SD) prothrombin activity was 99.33% 16.30% in the 150 patients without the prothrombin A20210 allele and 125.20% 7.69% in the 7 controls with the allele (Mann-Whitney U-test, P < 0.001). Fifty-one patients carried the factor V Leiden mutation (18.15% [CI, 13.64% to 22.66%]); 50 were heterozygotes and 1 was a homozygote. Forty-three controls carried this mutation (5.06% [CI, 3.59% to 6.53%]; P < 0.001); all were heterozygotes. Crude odds ratios for venous thrombosis associated with the presence of the prothrombin GA20210 or factor V Leiden mutation are reported in Table 1. We analyzed the association between the two gene variants and a history of venous thrombosis by stratifying patients and controls according to the presence of one or two gene variants (Table 2). The increased risk (odds ratio, 2.51) associated with the prothrombin GA20210 mutation was clearly unrelated to the overrepresentation of the factor V Leiden mutation; the coexistence of both mutations was seen only in patients (n = 14; 4.98% [CI, 2.44% to 7.52%]). The estimated risk associated with the prothrombin GA20210 mutation was similar when we excluded carriers of inherited defects of natural anticoagulants, patients with antiphospholipid antibodies, and patients exposed to circumstantial risk factors for venous thromboembolism. However, the risk for venous thrombosis associated with the prothrombin GA20210 mutation approached statistical significance only in the subset of patients with additional risk factors (Table 2). The distribution of the prothrombin GA20210 gene variant was similar in patients with and those without additional risk factors (P = 0.171). The prevalence of the factor V Leiden mutation did not differ between patients with and those without risk factors. The median age at the time of the first thrombotic episode was 38.0 years (range, 10 to 67 years) in the 26 patients with the prothrombin GA20210 mutation and 39.0 years (range, 16 to 65 years) in the 37 patients carrying the factor V Leiden mutation. The median age was 37.0 years (range, 3 to 81 years) in the 204 patients without either mutation and 31.5 years (range, 16 to 49 years) in the 14 patients carrying both mutations (Kruskal-Wallis test, P = 0.129). Table 2. Risk Estimate in All Study Participants and after Stratification by Risk Factors for Venous Thrombosis according to Presence of Prothro

Prognostic factors in noncirrhotic patients with splanchnic vein thromboses.

OBJECTIVES AND METHODS:Splanchnic vein thrombosis (SVT), not associated with cancer or liver cirrhosis, is a rare event and scanty data are available on its natural history, long-term prognosis, and treatment. In this study 121 SVT patients consecutively seen from January 1998 to December 2005 were included and 95 of them were followed up for a median time of 41 months. Screening for thrombophilic factors was performed in 104 patients. New thrombotic or bleeding episodes were registered and anticoagulant therapy was performed according to preestablished criteria.RESULTS:SVT was an incidental finding in 34 (28.1%) patients; 34 (28.1%) presented with abdominal infarction; 39 (32.2%) had bowel ischemia or acute portal vein thrombosis; 14 (11.6%) had bleeding from portal hypertensive sources. Survival rates at 1, 3, and 7 yr were 95%, 93.3%, and 89.6%, respectively; 87.5% of deaths occurred at onset of SVT as complications of intestinal infarction. Patients with isolated portal vein thromboses had symptoms and intestinal infarction in 16/41 (39%) and 0/41 (0%) of the cases, respectively, whereas superior mesenteric vein thromboses, isolated or not, were associated with symptoms and intestinal infarction in 69/75 (92%) and 34/75 (45%), respectively.During the follow-up 14 (14.7%) suffered from 39 episodes of gastrointestinal bleeding with no deaths. A previous gastrointestinal bleed was associated with new hemorrhagic events during follow-up.New venous thrombotic episodes occurred in 10 of 95 patients (10.5%), of which 73% were in the splanchnic area. Seven out of these 10 patients had a chronic myeloproliferative disease (MPD) and none was on anticoagulation.CONCLUSIONS:Anticoagulant therapy was effective to obtain recanalization of acute SVT in 45.4% of patients and preserved patients from recurrent thrombosis when given lifelong.

The JAK2 V617F mutation frequently occurs in patients with portal and mesenteric venous thrombosis

Summary.  Background: Myeloproliferative disorders (MPDs) represent a risk factor for thrombosis in the portal, mesenteric, and hepatic districts. Objective: We aimed to assess whether the Janus kinase 2 (JAK2) V617F mutation, an acquired mutation that occurs in MPD patients, is a risk factor for portal and mesenteric venous thrombosis (PMVT) independently of the presence of overt MPDs. Patients and methods: The medical histories of 99 patients presenting with PMVT were obtained. The presence of the JAK2 V617F and VHL 598C > T mutations was determined by polymerase chain reaction followed by restriction enzyme analysis and direct cycle sequence analysis. Results: Over a 10‐year period of observation, of the 99 patients presenting with PMVT, the JAK2 V617F mutation was detected in heterozygous state in 17 individuals [17.2%; 95% confidence interval (95% CI) 10.9–25.9]. None of the patients presenting with the JAK2 V617F mutation carried an inherited thrombophilic risk factor. Seven patients with (43.8%; 95% CI 19.8–70.1) and two without (2.4%; 95% CI 0.3–8.4) the JAK2 V617F mutation had a diagnosis of MPD at the occurrence of the venous thrombotic event. After a median follow‐up of 41 months (range 3–114 months), three out of the 10 patients carrying the JAK2 V617F mutation were then diagnosed as having idiopathic myelofibrosis (n = 2) or polycythemia vera (n = 1), whereas in seven patients a MPD was not detected. Two of the 83 patients without the JAK2 V617F mutation went on to develop MPDs. Conclusions: Determination of the JAK2 V617F mutation may contribute to the search for genetic determinants of PMVT and may be useful to recognize patients who should be carefully observed for the subsequent development of overt MPDs.

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

OBJECTIVE This studys 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.

PAI-1 plasma levels in a general population without clinical evidence of atherosclerosis: relation to environmental and genetic determinants.

Plasminogen activator inhibitor-1 (PAI-1) plasma levels have been consistently related to a polymorphism (4G/5G) of the PAI-1 gene. The renin-angiotensin pathway plays a role in the regulation of PAI-1 plasma levels. An insertion (I)/deletion (D) polymorphism of the angiotensin-converting enzyme (ACE) gene has been related to plasma and cellular ACE levels. In 1032 employees (446 men and 586 women; 22 to 66 years old) of a hospital in southern Italy, we investigated the association between PAI-1 4G/5G and the ACE I/D gene variants and plasma PAI-1 antigen levels. None of the individuals enrolled had clinical evidence of atherosclerosis. In univariate analysis, PAI-1 levels were significantly higher in men (P<.001), alcohol drinkers (P<.001), smokers (P=.009), and homozygotes for the PAI-1 gene deletion allele (4G/4G) (P=.012). Multivariate analysis documented the independent effect on PAI-1 plasma levels of body mass index (P<.001), triglycerides (P<.001), sex (P<.001), PAI-1 4G/5G polymorphism (P=.019), smoking habit (P=.041), and ACE I/D genotype (P=.042). Thus, in addition to the markers of insulin resistance and smoking habit, gene variants of PAI-1 and ACE account for a significant portion of the between-individual variability of circulating PAI-1 antigen concentrations in a general population without clinical evidence of atherosclerosis.

Inherited Prothrombotic Conditions and Premature Ischemic Stroke: Sex Difference in the Association With Factor V Leiden

At a young age, ischemic stroke is an uncommon event in which prothrombotic factors are likely to play an important role. In 202 referred cases, 105 men and 97 women, median age 39 years (range, 3 to 50), with a history of ischemic stroke and in 1036 age frequency-matched apparently healthy individuals from the same ethnic background, we have investigated whether inherited prothrombotic conditions increase the risk of ischemic stroke. Neither abnormal plasma levels of natural anticoagulants and fibrinogen nor significant increase of the prothrombin A20210 allele was found in stroke cases compared with controls. Hypertension (odds ratio [OR], 22.61), male sex (OR, 2.30), smoking (OR, 2.78) and alcohol habits (OR, 0.14), a personal history of venous thromboembolism (OR, 4.53), a family history of stroke (OR, 1.93), high circulating levels of fibrinogen (P=0.0190), and total cholesterol (P=0.101) were all independently associated with ischemic stroke. Compared with noncarriers, carriers of the factor V (FV) Leiden mutation (OR, 2.56), and to a lesser extent, of the methylenetetrahydrofolate reductase (MTHFR) TT genotype (OR, 1.60), had an independent higher estimated risk of having a history of ischemic stroke. The relationship with the FV Leiden mutation was greater in women (OR, 3.95). Thus, in addition to established determinants, FV Leiden mutation is independently associated with the occurrence of ischemic stroke in this setting. The greater association in women suggests the possibility of an interaction of this genotype with female hormones.

The PAI-1 Gene Locus 4G/5G Polymorphism Is Associated With a Family History of Coronary Artery Disease

A family history of ischemic events is a major determinant of coronary artery disease (CAD). Plasma levels of plasminogen activator inhibitor 1 (PAI-1) modulate this risk. A deletion/insertion polymorphism within the PAI-1 locus (4G/5G) affects the expression of this gene. We investigated the relationship between the PAI-1 4G/5G polymorphism in 1179 healthy employees of our institution and the occurrence of CAD in their first-degree relatives. A family history of documented ischemic coronary disease was assessed by a modified WHO questionnaire. The PAI-1 4G/5G polymorphism was evaluated by polymerase chain reaction and endonuclease digestion. The group with a first-degree relative who had suffered from a coronary ischemic episode had a higher number of homozygotes for the deleted allele (4G/4G) of the PAI-1 gene compared with subjects without such a family history (odds ratio [OR] = 1.62, 95% confidence interval [CI]=1.17 to 2.25; P=.005). The frequency of the 4G allele was abnormally high as well (OR=1.29, 95% CI=1.04 to 1.60; P=.025). The individuals with a positive family history were older (P<.001) and exhibited a higher body mass index (P=.033) and total cholesterol levels (P<.001) than those without. In a multiple logistic regression analysis, age (P=.006) and PAI-1 4G/4G (P=.024) independently contributed to a family history of coronary heart disease, with 4G/4G carriers exhibiting a more frequent family history of CAD (OR=1.60). The PAI-1 4G/5G polymorphism to some extent thus accounts for the risk of CAD related to a family history for such an event. These findings support the hypothesis that the 4G variant is a transmissible coronary risk factor.

Prevalence of Apolipoprotein E Alleles in Healthy Subjects and Survivors of Ischemic Stroke An Italian Case-Control Study

BACKGROUND AND PURPOSE The epsilon4 allele of the apolipoprotein E (apoE) has been related to the occurrence of myocardial infarction, but its association with ischemic stroke is controversial. We have evaluated the relation between apoE alleles and the occurrence of cerebrovascular ischemia. METHODS The apoE epsilon genotypes of 100 patients with a documented history of ischemic stroke without clinically apparent dementia (stroke+) and 108 subjects without such history (stroke-) were determined. The relative frequency of the apoE alleles and genotypes was estimated in 398 healthy subjects aged < 40 years from the same ethnic background. RESULTS The frequency of the apoE epsilon4 allele in stroke+ (0.18 [95% CI, 0.12 to 0.25]) was higher than in stroke- (0.07 [95% CI, 0.03 to 0.12]; P<.001) or in healthy subjects (0.09 [95% CI, 0.07 to 0.12]; P<.001). Carriers of the epsilon4 allele differed between stroke+ (0.30 [95% CI, 0.19 to 0.42]) and stroke- (0.12 [95% CI, 0.5 to 0.22]; P=.004) or healthy subjects (0.16 [95% CI; 0.12 to 0.22]; P=.015). Accordingly, epsilon3/epsilon3 homozygotes were less frequent in stroke+ (0.59 [95% CI, 0.45 to 0.71]) than in stroke- (0.72 [95% CI, 0.59 to 0.82]; P=.063) or in healthy subjects (0.73 [95% CI, 0.67 to 0.78]; P=.01). In a multiple logistic regression analysis, age (P<.03), positive family history (P<.04) and apoE (P<.002) independently contributed to a stroke history, with epsilon4 carriers exhibiting a higher estimated risk (odds ratio, 5.05). CONCLUSIONS Our data show an association between apoE gene and a personal history of ischemic stroke and support the possibility that the apoE gene is a susceptibility locus for the risk of cerebrovascular ischemic disease.