A. D'Orazio

Polymorphisms in the Coagulation Factor VII Gene and the Risk of Myocardial Infarction

BACKGROUND High blood levels of coagulation factor VII are associated with a risk of ischemic vascular disease. Although factor VII levels may be genetically determined, the relation between genetic polymorphisms of factor VII, factor VII blood levels, and the risk of myocardial infarction has not been established. METHODS We performed a case-control study of 165 patients with familial myocardial infarction (mean [+/-SD] age, 55+/-9 years) and 225 controls without a personal or family history of cardiovascular disease (mean age, 56+/-8 years). The polymorphisms involving R353Q and hypervariable region 4 of the factor VII gene were studied. Factor VII clotting activity and antigen levels were also measured. RESULTS Patients with the QQ or H7H7 genotype had a decreased risk of myocardial infarction (odds ratios, 0.08 [95 percent confidence interval, 0.01 to 0.9] and 0.22 [95 percent confidence interval, 0.08 to 0.63], respectively). For the R353Q polymorphism, the RR genotype was associated with the highest risk, followed by the RQ genotype and then by the QQ genotype (P<0.001). For the polymorphism involving hypervariable region 4, the combined H7H5 and H6H5 genotypes were associated with the highest risk, followed in descending order by the H6H6, H6H7, and H7H7 genotypes (P<0.001). Patients with the QQ or H7H7 genotype had lower levels of both factor VII antigen and factor VII clotting activity than those with the RR or H6H6 genotype. Patients with the lowest level of factor VII clotting activity had a lower risk of myocardial infarction than those with the highest level (odds ratio, 0.13; 95 percent confidence interval, 0.05 to 0.34). CONCLUSIONS Our findings suggest that certain polymorphisms of the factor VII gene may influence the risk of myocardial infarction. It is possible that this effect may be mediated by alterations in factor VII levels.

Genetic Variation in the Renin–Angiotensin System and Abdominal Adiposity in Men: The Olivetti Prospective Heart Study

Context Angiotensinogen (AGT), angiotensin-converting enzyme (ACE), and angiotensin II receptor type I (AT2R1) are expressed in adipose tissue, but their role in obesity is unknown. Common polymorphisms involve the ACE gene on chromosome 17 (ACE I/D), the AGT gene on chromosome 1, and the AT2R1 receptor gene on chromosome 3. Contribution Among 959 adult Italian men, DD homozygosity in the ACE gene was associated with overweight (odds ratio, 1.82 [95% CI, 1.16 to 2.87]) and abdominal obesity (odds ratio, 1.76 [CI, 1.06 to 2.90]) compared with the genotypes I/D and II. It was also associated with increases in weight over 20 years. Polymorphisms of AGT and AT2R1 were unrelated to measures of body size. Implications These results suggest that the reninangiotensin system plays a role in the development of obesity. The Editors Human obesity is caused by the interaction of genetic predisposition and many environmental and lifestyle factors (1). Although the chromosomal location of a few putative major genes for human obesity has been identified (2-5), the presence of a greater number of minor genes involved in the process of adipogenesis or in the regulation of adipocyte metabolism probably engenders susceptibility to obesity (6). Polymorphism in several obesity candidate genes has been the subject of intensive investigation, but little attention has been paid to the genes encoding for components of the reninangiotensin system. The products of these genes (angiotensinogen [AGT], angiotensin-converting enzyme [ACE], and angiotensin II type 1 [AT2R1] and type II [AT2R2] receptors) are expressed in the adipose tissue in animal models as well as in humans (7-10). Recent experimental studies suggest that adipose tissue in the reninangiotensin system plays a role in adipocyte growth and differentiation through angiotensin II (11, 12). In addition, epidemiologic studies have reported associations between AGT plasma levels (13, 14), plasma renin activity (15, 16), plasma ACE activity (17), and body mass index (BMI). We investigated the relationship of overweight, obesity, and body fat distribution to three common polymorphisms of the reninangiotensin system: intron 16 of the ACE gene on chromosome 17 (18), the M235T polymorphism of the AGT gene in exon 2 on chromosome 1 (19), and the A-to-C polymorphism in the 3-untranslated region at nucleotide 1166 of the AT2R1 gene on chromosome 3 (20). Because the reninangiotensin system plays a fundamental role in blood pressure regulation and in vascular and cardiac modifications, these polymorphisms have been studied with regard to hypertension and cardiovascular disease. Associations have been reported between the AGT M235T and the AT2R1 A1166C variants and hypertension (19-24), as well as among ACE I/D polymorphism, insulin sensitivity (25-27), and the risk for coronary heart and cerebrovascular disease (28, 29). In adult men who attended the 19941995 follow-up examination of the Olivetti Prospective Heart Study, we examined associations between these polymorphic variants and overweight or obesity, body fat distribution, and related metabolic and hemodynamic variables. We also reported longitudinal findings for a subset of participants who were first examined in 1975 and had been followed for 20 years. Methods Study Sample and Procedures We used the DNA bank and the database of the Olivetti Prospective Heart Study, an epidemiologic investigation of cardiovascular risk factors in men working at the Olivetti factories in southern Italy. The procedures of the Olivetti Prospective Heart Study, which began in 1975, have been described in detail elsewhere (30). Between May 1994 and December 1995, we examined 1075 men 25 to 75 years of age. The participants were seen in the morning, after fasting, in a quiet room at the medical center of the Olivetti factories in Pozzuoli and Marcianise, suburbs of Naples, Italy. We obtained anthropometric measurements, performed blood tests, and administered a fixed-sequence questionnaire that assessed demographic information and medical history. Genotyping of the three polymorphisms of the reninangiotensin system was possible in 959 participants. A group of 457 men seen at the 19941995 follow-up visit of the Olivetti Prospective Heart Study had also been examined in 1975; of these, 143 reported being under dietary restriction for various reasons at follow-up examination. Since the Olivetti Prospective Heart Study aims to evaluate spontaneous changes in body mass and blood pressure, this subgroup was excluded from the main analysis. The local ethics committee approved the study protocol, and participants gave informed consent. Anthropometric Measurements Body weight and height were measured on a standard beam-balance scale with an attached ruler. Body weight was measured to the nearest 0.1 kg, and height was measured to the nearest 1 cm; participants wore light indoor clothing and no shoes. Body mass index was calculated as weight in kilograms divided by the square of the height in meters. At the 19941995 examination, but not at the 1975 examination, waist and arm circumferences were also measured. Waist circumference was measured at the umbilicus level as participants stood erect with abdomens relaxed, arms at their sides, and feet together. After the acromion was marked with each participants arm flexed at a 90-degree angle, arm circumference was measured at the midpoint between the acromion and the olecranon with the arm relaxed and hanging just away from the side of the body. The measurements were obtained to the nearest 0.1 cm with a flexible plastic measuring tape. Overweight was defined as a BMI greater than or equal to 27 kg/m2, obesity was defined as a BMI greater than or equal to 30 kg/m2, and abdominal adiposity was defined as a waist circumference greater than 1.00 m. Blood Pressure Measurement Blood pressure was measured after the participant had been sitting upright for at least 10 minutes. Systolic and diastolic (phase V) blood pressure was measured three 2 minutes apart with a random-zero sphygmomanometer (Gelman Hawksley Ltd., Sussex, United Kingdom). The first reading for each type of pressure was discarded, and the average of the second two readings was recorded. Hypertension was defined as a systolic blood pressure 140 mm Hg or greater, a diastolic blood pressure 90 mm Hg or greater, or both, or as current use of antihypertensive drugs. Biochemical Assays After blood pressure was measured, a fasting venous blood sample was taken in the seated position without stasis. The blood specimens were immediately subjected to centrifugation and stored at 70 C until analyzed. Glucose levels were measured by using an automated method (Cobas-Mira, Roche, Italy), and serum insulin concentration was measured by using radioimmunoassay (Insuline Lisophase, Technogenetics, Milan, Italy). Insulin resistance was estimated by homeostasis model assessment using the following formula, as described by Matthews and colleagues (31): fasting serum insulin level (U/mL) fasting serum glucose level (mmol/L)/22.5. Gene Polymorphisms in the ReninAngiotensin System Genomic DNA was isolated from leukocytes with a nonenzymatic, salting-out procedure (32). The ACE I/D polymorphism in intron 16 was typed by using the method of Rigat and associates (33). To address the possibility of mistyping ID heterozygotes as DD homozygotes because of the preferential amplification of the smaller D allele, all samples typed as DD homozygotes were subjected to a second, independent polymerase chain reaction with a primer pair that permits amplification only in the presence of the I allele; this was done by using the method described by Lindpaintner and coworkers (34). The T235 allele of the ATG gene was detected by using the method of Russ and colleagues (35), and the A1166C polymorphism of the AT2R1 gene was tested as described elsewhere (36). Allelic frequencies were estimated by using gene counting, and genotype distribution was tested for HardyWeinberg equilibrium by using chi-square analysis. Statistical Analysis Analysis of variance was used to evaluate differences in quantitative variables according to genotype; analysis of covariance was performed to account for confounders. A nonparametric test (KruskalWallis) was used for variables that were not normally distributed. The interaction between the effects of gene polymorphism and age on the anthropometric indexes and blood pressure was tested by using multiple linear regression analysis. The association of categorical variables with gene polymorphisms was tested by using logistic regression analysis and is expressed as odds ratios and 95% CIs. Results are expressed as the mean SD or as the mean SE, as specified. Two-sided P values and 95% CIs were used to test the statistical significance of between-group differences. Statistical analysis was performed by using SPSS statistical software, version 10.0 (SPSS, Inc., Chicago, Illinois). Role of the Funding Source The funding source had no role in the collection, analysis, or interpretation of the data or in the decision to submit the paper for publication. Results Table 1 summarizes the main characteristics of the participants of the Olivetti Prospective Heart Study at the 19941995 examination. Nine hundred fifty-nine participants were tested for ACE, AGT, and AT2R1 polymorphism. For the ACE I/D polymorphism, 40% (n = 385) had the DD genotype, 45% (n = 431) had the ID genotype, and 15% (n = 143) had the II genotype. For the AGT polymorphism, 31% (n = 297) had the M235M genotype, 48% (n = 460) had the M235T genotype, and 21% (n = 202) had the T235T genotype. For the AT2R1 polymorphism, 54% (n = 518) had the A1166A genotype, 39% (n = 377) had the A1166C genotype, and 7% (n = 64) had the C1166C genotype. All three polymorphisms were in HardyWeinberg equilibrium, showing that the study sample excluded selection pressure for the genotypes under investigation. Table 1. Characteristics of

Bcl I Polymorphism in the Fibrinogen β-Chain Gene Is Associated With the Risk of Familial Myocardial Infarction by Increasing Plasma Fibrinogen Levels A Case-Control Study in a Sample of GISSI-2 Patients

The aim of this study was to investigate the association of the Bcl I beta-chain fibrinogen polymorphism with the risk of acute myocardial infarction (AMI) and its relationship with fibrinogen levels in the Italian population. We studied 102 AMI patients, selected within the framework of the GISSI-2 trial, who had a familial history of arterial thrombosis (at least one first-degree relative suffering from AMI or stroke before 65 years) and 173 control subjects (with neither AMI nor personal or familial history of arterial thrombosis). All subjects were Italian. Patients showed fibrinogen levels higher than control subjects. There was a highly significant difference in allele frequency in cases versus control subjects, the B2 allele frequencies being respectively 0.28 versus 0.17 (P = .002). In multivariate analysis, adjusted for sex, age, smoking habits, and history of hyperlipidemia, hypertension, or diabetes, the (B1B2 + B2B2) genotype was associated with a higher risk of AMI (odds ratio 2.4, 95% confidence interval, 1.2 to 4.6). The Bcl I genotype was also associated with fibrinogen levels, independently of gender and smoking habits, the (B1B2 + B2B2) subjects showing the highest levels in both cases and control subjects. The difference in fibrinogen levels between cases and control subjects was significantly influenced by the genotype (significant interaction, P = .042). The B2 allele of the Bcl I polymorphism in the beta-chain of the fibrinogen gene is a new factor associated with the risk of familial AMI through its association with fibrinogen levels. These data provide evidence for a causal role of fibrinogen in familial AMI.

The Decanucleotide Insertion/Deletion Polymorphism in the Promoter Region of the Coagulation Factor VII Gene and the Risk of Familial Myocardial Infarction

Recently, an association has been found between factor VII polymorphisms and the risk of familial myocardial infarction. To obtain a thorough evaluation of the influence of factor VII gene on the risk of myocardial infarction, we extended our analysis to the role of a decanucleotide insertion/deletion functional polymorphism (-323 0/10-bp) in the promoter region of factor VII and to possible interactions with the HVR4 intron polymorphism. We performed a case-control study of 176 patients with myocardial infarction, over 45 years, who had a familial history of arterial thrombosis and 227 control subjects without a personal or family history of cardiovascular disease. The frequency of the rare allele of 10 bp was lower in cases (0.14 95% CI, 0.10-0.17) than in controls (0.19 95% CI, 0.16-0.23; chi(2)=4.7, p=0.03). Allowing for Hardy-Weinberg equilibrium in controls and testing for association under restricted maximisation, there was a significant difference in genotype frequency between cases and controls (p=0.02). Carriers of the 10-bp allele had an odds ratio for myocardial infarction of 0.65 (95% CI, 0.37-1.12), in multivariate logistic regression analysis. Combination analysis of -323 0/10-bp and HVR4 polymorphisms shows half reduction in the risk of myocardial infarction in comparison with the reference group for all the other groups, suggesting that there was no additivity between the effect of the 10-bp and the H7 alleles. Our findings suggest that the promoter polymorphism of factor VII gene may influence the risk of familial myocardial infarction.

A polymorphic cluster in the 5' region of the human coagulation factor VII gene : Detection, frequency, and linkage disequilibrium

Human coagulation factor VII (FVII) gene contains in the proximal part of the promoter three polymorphisms: two nucleotide substitutions (at position –122, –401) and a decanucleotide insertion (at position –323) [1,2]. Recently, differences in FVII antigen level and coagulation activity have been reported in plasma of individuals carrying the –323 insertion versus non carriers [3,4]. Moreover, a reporter gene construct carrying the –323 insertion allele showed in cell transection studies 33’i%o less activity compared with the construct not carrying the insertion [1].Thus, the presence of polymorphisms in the regulatory part of the gene may influence its transcription; the study of distribution and linkage disequilibrium of these polymorphisms in the population becomes therefore important, considering that changes in plasma levels of coagulation factors can be risk factors for cardiovascular diseases [5,6]. While the –323 insertion has been characterized in a population study [2], the other two polymor-

Alu-repeat polymorphism in the tissue-type plasminogen activator (t-PA) gene, t-PA levels and risk of familial myocardial infarction (MI)

Several authors have suggested that increased t-PA antigen levels were associated to thrombotic events in coronary, cerebral and peripheral arteries. Baseline and stimulated levels of t-PA in plasma appear highly heritable, therefore, it could be of interest to evaluate the role of genetic variations in the t-PA locus in determining its plasma levels and its association with a parental history of thrombosis. We studied an insertion deletion polymorphism located in the first Alu sequence in intron h of the t-PA gene, in a sample of the population, including 327 subjects (202 M,125 F, aged 20-78 years) from all over Italy. Genotype analysis was also performed on 114 patients with MI and at least one first degree relative affected by MI or stroke before 65 years compared with 145 controls, aged over 45 years. The genotype distribution in all groups were in Hardy-Weinberg equilibrium. The I and D allele frequencies in the Italian sample were 0.55 and 0.45 respectively. There were no differences in genotype distribution between MI patients with familial history of thrombosis and controls (29 and 31%, 51 and 50%, 20 and 20% for I/I, I/D and D/D, respectively in cases and controls). There was no significant interaction of sex or age on the association between t-PA polymorphism and familial MI. The levels of t-PA and PAI-1 (both antigen and activity) were not differently distributed among the t-PA genotypes in the healthy Italian population sample. However, an association between t-PA polymorphisms and PAI-1 antigen was found in patients with MI. Patients carrying the genotype I/I showed the higher levels of PAI-1 antigen (31 ± 17 in I/I vs. 20 ± 15 and 21 ± 14, respectively in I/D and D/D, P < 0.01). These data do not support a role for the Alu repeat polymorphism of t-PA gene in determining the blood levels of t-PA and the risk of familial MI in the Italian population. The relation found between t-PA polymorphism and PAI-1 antigen levels in Italian patients should be further clarified.

4G/5G PAI-1 Promoter Polymorphism and Acute-Phase Levels of PAI-1 Following Coronary Bypass Surgery: A Prospective Study

AbstractBackground and objective: The 4G/5G plasminogen activator inhibitor-1 (PAI-1) promoter polymorphism has been associated with basal PAI-1 levels, with ischemic heart disease, and with adverse prognosis in critically ill patients. We hypothesized it might also influence the acute-phase levels of PAI-1 following coronary bypass surgery. Methods: In 111 consecutive patients undergoing elective coronary bypass surgery, 4G/5G genotyping and serial plasma PAI-1 activity and antigen levels were prospectively measured before surgery, daily up to 72 h, and at discharge. The inflammatory reaction was additionally assessed by white cell count, fibrinogen, interleukin-6, and C-reactive protein levels. Results: PAI-1 activity and antigen concentrations increased approximately two-fold after surgery, peaking at 48 hours. Carriers of the 4G-allele, compared with 5G/5G homozygotes, showed approximately 20% higher PAI-1 activity and antigen both preoperatively (P = 0.007 and P = 0.035) and after surgery. White cell count, fibrinogen, interleukin-6, and C-reactive protein values did not differ significantly according to genotypic groups. In multivariate analysis, the 4G/5G genotype was the only significant modulator of postoperative PAI-1 activity (P = 0.003) and the main significant modulator of postoperative PAI-1 antigen (P = 0.013). No significant interaction was found between the effects of time and genotype on postoperative PAI-1. This indicates that the association between 4G/5G and acute-phase PAI-1 levels is secondary to the genotype-related difference of baseline PAI-1. Conclusions: Postoperative PAI-1 concentrations of patients undergoing elective coronary bypass surgery are higher in carriers of the 4G-allele than in 5G/5G homozygotes as a result of higher baseline values. Knowledge of 4G/5G status may be useful to predict acute-phase PAI-1 concentrations.

Decrease in urokinase-type plasminogen activator (u-PA) levels in patients with non-insulin dependent diabetes mellitus

Summary The objective of the study was to investigate the activity and antigen levels of u-PA in non-insulin dependent diabetes mellitus (NIDDM) patients, and their changes after venous stasis. A cross-sectional case-control study was carried out in the out-patient diabetology clinical unit of the General Hospital in Pescara, Italy. Fifty-nine unselected NIDDM patients (23 females and 36 males; mean age 63±10, with diabetes definition by WHO criteria) were studied. A group of 50 subjects, age matched (age 58±9, 42 males, 8 females), without a history of diabetes, were consecutively recruited among healthy relatives of patients admitted to the hospital for surgical interventions to serve as a control group. The levels of both u-PA activity (scu-PA) and antigen (u-PA Ag) were significantly lower in NIDDM patients compared to controls. In contrast, t-PA, PAI-1 antigen and PAI activity levels were increased in NIDDM patients. There was no change in u-PA levels (both activity and antigen) after venous stasis, while t-PA levels were significantly increased. There was no difference in u-PA (both activity and antigen) and in t-PA levels between male and female patients. However, fibrinogen levels were lower and PAI-1, both antigen and activity, was higher in men than in women. No difference was found between patients with and without macrovascular complications in the parametres studied. Besides changes in t-PA and PAI-1 levels, NIDDM is also characterized by a decrease in u-PA activity and antigen levels. Such impairment of the fibrinolytic system, together with the increase in fibrinogen levels, could contribute to the prothrombotic condition peculiar to these patients.