Giuseppe Cappucci

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

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.

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.

Abnormally high circulation levels of tissue plasminogen activator and plasminogen activator inhibitor-1 in patients with a history of ischemic stroke.

We evaluated 106 subjects with and 109 subjects without a history of ischemic stroke. All were attending a metabolic ward. The two groups were compared for major risk factors for ischemic events. A positive family history for ischemic complications of atherosclerosis was more common in subjects with a history of stroke than in those without; moreover, plasma levels of plasminogen activator inhibitor-1 (PAI-1) and tissue-type plasminogen activator (TPA) were higher in patients with documented previous events. A strong positive significant correlation was found between TPA and PAI-1 levels, and an interaction between age and TPA was observed when the sample was stratified according to ages being above or below 70 years. When the patient population was analyzed according to the number of ischemic events, it was found that 62 of the 106 subjects with a history of stroke had experienced more than one ischemic event. Under these conditions, the levels of TPA and PAI-1 still correlated with the occurrence of previous ischemic episodes. As in the whole patient sample, TPA was the strongest discriminator. We conclude that in subjects attending a metabolic ward, TPA and PAI-1 levels consistently help identify subjects with a history of cerebral ischemic episodes and that TPA is the strongest discriminator.

Apolipoprotein E polymorphisms in age-related macular degeneration in an Italian population.

Objective: Apolipoprotein E (apoE) is an important regulator of cholesterol and lipid transport during compensatory synaptogenesis. Our purpose was to investigate the role of apoE gene polymorphisms in Italian patients with age-related macular degeneration (AMD). Methods: We used the polymerase chain reaction technique to analyze apoE genotypes in 87 patients with AMD, in 47 age-matched controls and in 1,287 individuals from a general reference population. Results: The frequency of allele Ε4 carriers was significantly higher (p = 0.002) in the general population than in AMD patients, while the frequency of allele Ε2 was higher in the patients (p = 0.069) with an increased risk for AMD in the patients versus the population-based controls (odds ratio = 1.7; 95% confidence interval: 1.0–2.9). Allele Ε4 was associated with a decreased risk for AMD in the patients versus the population-based controls (odds ratio = 0.3; 95% confidence interval: 0.1–0.8). Clinical Relevance: These data suggest that apoE testing may represent a tool for the evaluation of the relative risk of AMD. Consequently, a preventive strategy can be initiated at an early stage of the disorder. Conclusion: The apoE gene polymorphism showed a significant association with the risk of AMD. The lower frequency of the Ε4 allele in AMD patients suggests that the apoE gene could play a protective role in the pathogenesis of the disease. In contrast, the Ε2 allele was found associated with a slightly increased risk of AMD, although we did not find a statistically significant effect.

Anticardiolipin antibodies in patients with liver disease.

Objective:Our aim was to test the hypothesis that anticardiolipin antibodies (aCL) may cause an antiphospholipid syndrome and thrombotic events in patients with liver disease.Methods:aCL were measured in 116 healthy controls and 372 patients with liver disease of different stage and etiology: 136 cases secondary to hepatitis C virus (HCV) infection, 139 due to hepatitis B virus (HBV) infection, 69 with alcoholic liver damage, and 28 cryptogenic in origin. Prior thrombotic events were recorded. The results were related to age, gender, stage, severity, and etiology of the liver disease, as well as to the occurrence of organ- and nonorgan-specific autoantibodies.Results:aCL were positive in 4.4% of controls and in 18.8% of patients (p < 0.0002). Patients with aCL were more frequently men with an advanced cirrhosis and simultaneous occurrence of anti–smooth-muscle antibodies (ASMA) in serum (p < 0.0006); their liver damage was often secondary to HBV (37.3%) or alcohol abuse (18.5%). At conditional logistic regression analysis, only the presence of ASMA (odds ratio [OR] = 3.02, 95% confidence interval [CI] 1.7–5.5, p= 0.0003), HBV (OR = 3.4, 95% CI 1.6–7.2, p= 0.0013), or alcoholic liver disease (OR = 5.3, 95% CI 2.3–12.2, p= 0.0001) were independently associated with aCL. Thrombosis was encountered in 24 patients (6.4%). At conditional logistic regression analysis, thrombosis was significantly associated with advanced age (OR = 1.07, 95% CI 1.0–1.1, p= 0.0094), development of hepatocellular carcinoma (OR = 17.8, 95% CI 1.6–196.0, p= 0.01), HBV etiology (OR = 6.3, 95% CI, 1.6–24.6, p= 0.0076), or cryptogenic liver disease (OR = 54.8, 95% CI 5–599.9, p= 0.001). Of the five patients with newly documented portal thrombosis during the follow-up, only one tested positive for aCL.Conclusion:In patients with nonautoimmune liver disease, aCL production is an epiphenomenon of the liver damage and is not associated with thrombotic complications. These data do not support the hypothesis that HCV is a cause of the antiphospholipid syndrome.

Deletion Polymorphism in the Angiotensin-Converting Enzyme Gene in Patients With a History of Ischemic Stroke

We evaluated the genotypes of the angiotensin-converting enzyme (ACE) gene in 101 subjects with and 109 subjects without a history of ischemic stroke. All were attending a metabolic ward. The two groups were compared for major risk factors for ischemic events. Genotypes were determined by polymerase chain reaction with oligonucleotide primers flanking the polymorphic region in intron 16 of the ACE gene. Deletion polymorphism of the ACE gene (DD genotype) was shown to be more common in subjects with a history of stroke than in those without (relative risk, 1.76; confidence intervals, 1.02 to 3.05). A positive family history for ischemic complications of atherosclerosis was also more common in subjects with documented events (relative risk, 1.99; confidence intervals, 1.10 to 3.59). DD genotype and a positive family history were strong independent discriminators of cerebral ischemia. Plasma levels of tissue-type plasminogen activator (TPA) and plasminogen activator inhibitor-1 help identify subjects with a history of cerebral ischemic episodes. When such fibrinolytic variables were included in the analysis, the DD genotype still strongly and independently discriminated subjects with a stroke history and significantly interacted with TPA levels > 10 ng/mL in such identification. We conclude that in subjects attending a metabolic ward, homozygosity for a deletion polymorphism of the ACE gene consistently discriminates subjects with a stroke history. Interaction with TPA improves such identification.

Plasminogen Activator Inhibitor-1 (PAI-1) Antigen Plasma Levels in Subjects Attending a Metabolic Ward: Relation to Polymorphisms of PAI-1 and Angiontensin Converting Enzyme (ACE) Genes

Plasminogen activator inhibitor 1 (PAI-1) is a determinant of vascular events. Subjects in metabolic wards are at high risk for these events. The renin-angiotensin system modulates plasma PAI-1 levels. An insertion (4G)/deletion (5G) polymorphism is involved in the regulation of the circulating levels of PAI-1. We have evaluated the levels of plasma PAI-1 in 208 individuals from our metabolic ward and correlated these levels with the 4G/5G genotype as well as with a genotype (homozygosity for a deletion polymorphism, DD genotype) of the angiotensin-converting enzyme (ACE) gene. Homozygosity for the insertion genotype (5G/5G) was associated with PAI-1 levels lower than those associated with the deletion genotype (4G/4G) (26.2x/:1.6 versus 33.7x/:1.7 ng/mL, P = .036). Plasma PAI-1 levels appeared to depend on the genotype (P = .014) as much as on age (P = .044), t-PA (P = .0001), or triglyceride levels (P = .005). The association between triglycerides and PAI-1 was significant in subjects carrying the 4G/4G and the 4G/5G genotypes (P = .013 and .036, respectively) but not in those with the 5G/5G genotype. When stratified according to PAI-1 and ACE genotypes, individuals homozygous for both deletions (4G/4G-DD genotypes) exhibited higher plasma PAI-1 levels compared with those of individuals without such homozygosities. However, this difference did not reach statistical significance. We conclude that in a group of subjects from a metabolic ward, a 4G/5G polymorphism of the PAI-1 gene exerts effects on plasma PAI-1 antigen levels comparable to those of established determinants. The association between triglycerides and plasma PAI-1 levels is genotype dependent. A trend to a positive interaction between ACE DD and PAI-1 4G/4G in the regulation of circulating plasma PAI-1 levels is present in this setting.