Genetic polymorphism of 5,10-MTHFR reductase gene in offspring of patients with myocardial infarction.

A family history of myocardial infarction is a major determinant of ischemic disease. A C->T677 polymorphism in the methylenetetrahydrofolate reductase (MTHFR) gene has been identified as a cause of mild hyperhomocysteinemia, a risk factor for arterial thrombosis. We have investigated the relationship between the MTHFR TT genotype and a family history of myocardial infarction in a cohort of 982 apparently healthy individuals. Subjects whose first-degree relatives suffered from a myocardial infarction, showed raised median age (p <0.001), total cholesterol (p <0.001) and plasma fibrinogen (p = 0.023) and a higher than normal frequency of C-reactive protein levels >0.33 mg/dl (p = 0.012). Moreover, when compared to subjects without such family history, a higher number of homozygotes for the T allele of the MTHFR gene (p = 0.027), and of the 4G allele of the plasminogen activator inhibitor-1 gene (p = 0.002) was found in the subsetting of the offspring of patients with myocardial infarction. In a multiple logistic regression analysis, age (OR 1.02 [95%-CI: 1.00-1.05]), total cholesterol (OR 1.40 [95%-CI: 1.14-1.71]), C-reactive protein levels >0.33 mg/l (OR: 1.87 [95%-CI: 1.10-3.20]), plasminogen activator inhibitor-1 4G/4G (OR: 1.84 [95%-CI: 1.27-2.66]), and MTHFR TT genotype (OR 1.62 [95%-CI: 1.08-2.42]), were all associated with a family history of myocardial infarction. Thus, the MTHFR TT genotype independently accounts for the risk of a family history for myocardial infarction in the present setting.

Fibrinogen plasma levels in an apparently healthy general population--relation to environmental and genetic determinants.

Elevated fibrinogen levels are an independent risk factor for cardiovascular ischemic disease. We investigated the relationship between cardiovascular ischemic risk factors, the fibrinogen Bbeta-chain G/A(-455) polymorphism and plasma fibrinogen levels in 989 apparently healthy subjects. Fibrinogen values were higher in subjects with C reactive protein (C-RP) >0.33 mg/dl, BMI >23.9 kg/m2, total cholesterol >4.84 mmol/l, triglycerides > 1.02 mmol/l, PAI-1 antigen >12.2 ng/ml, carriers of the A allele, first-degree relative history of coronary artery disease, or consuming >10 cigarettes per day (p<0.01). Men and ethanol drinkers showed lower plasma fibrinogen levels (p<0.01). The multivariate analysis confirmed the independent effect of C-RP, age, BMI, total cholesterol, gender, PAI-1, -455 G/A polymorphism, (p<0.05). BMI, total cholesterol, PAI-1, alcohol and smoking habit raised with the increase of age and differed between sexes. The A(-455) allele increasing effect was significant in women, especially in subjects aged <30 years, and in men aged <43 years. These results indicate that environmental factors contributed to a larger extent to fibrinogen variability, whereas the A(-455) allele was associated with a steeper increase in younger age quartiles.

Factor V Leiden is associated with repeated and recurrent unexplained fetal losses.

Activated protein C resistance (APCR) is responsible for most cases of familial thrombosis. The factor V missense mutation Arg506 > Gln (FV Leiden) has been recognized as the commonest cause of this condition. Recently, it has been suggested that APCR is associated with second trimester fetal loss. We investigated the distribution of FV Leiden in a sample (n = 43) of Caucasian women with a history of two or more unexplained fetal losses. A group (n = 118) of parous women with uneventful pregnancies from the same ethnical background served as control. We found the mutation in 7 cases (16.28%) and 5 controls (4.24%; p = 0.011). A statistically significant difference between women with only early fetal loss vs those with late events (p = 0.04) was observed. Our data demonstrate a strong association between FV Leiden and fetal loss. Furthermore, they indicate that late events are more common in these patients.

A frameshift mutation in the human fibrinogen Aalpha-chain gene (Aalpha(499)Ala frameshift stop) leading to dysfibrinogen San Giovanni Rotondo.

We have investigated a 53-yr-old asymptomatic white man with decreased functional, but not immunologic, fibrinogen plasma levels together with prolonged thrombin and reptilase times, detected through routine coagulation studies prior to a surgical procedure. A new heterozygous single nucleotide deletion (C) at position Ala499 within the Aalpha-chain gene was identified, which predicted changes of the corresponding amino acids encoded by the subsequent portion of the exon V and the appearance of a premature stop codon at position 518 (Aalpha[499]Ala frameshift stop). The new dysfunctional fibrinogen, San Giovanni Rotondo variant, was confirmed in vivo by SDS-PAGE analysis of HPLC-purified fibrinogen chains. Mass spectrum examination of the abnormal HPLC-purified peak gave an estimated mass (56,088 Da) similar to that predicted by DNA analysis of the mutated Aalpha-chain gene (56,088 Da) and, after tryptic digestion, the truncated Aalpha-chain was shown only in the propositus, who also carried normal Aalpha-chain. In addition, mass spectrum analysis of the tryptic digest of the abnormal chain confirmed the presence of a new and unpaired cysteine at the last position that was predicted to form a disulfide bridge with human serum albumin. Immuno-blot analysis confirmed that fibrinogen San Giovanni Rotondo variant, but not normal fibrinogen. contained substantial amounts of albumin. Present findings confirm that truncated Aalpha-chain lacking part of the terminal domain may be incorporated into mature fibrinogen molecules and normally secreted in the bloodstream.

Raised plasma fibrinogen concentrations in subjects attending a metabolic ward--relation to family history and vascular risk factors.

We have evaluated plasma fibrinogen levels in 171 subjects attending a metabolic ward. As in the general population, a significant difference in plasma fibrinogen concentrations (p < 0.05) was found between subjects with diabetes mellitus or hypertension and those without. However, fibrinogen was also abnormally high (p < 0.05) when evaluated according to the presence of a family history of ischemic complications of atherosclerosis (p < 0.05). In this setting, fibrinogen correlated with diabetes mellitus or hypertension as well as with familial risk, and the latter interacted with hypertension (p < 0.05) in accounting for plasma fibrinogen. The relationships between certain fibrinogen genotypes and familial risk have then been evaluated. Analysis of a locus (1.3 kb, HAE III digestion) of the promoter region of the B beta fibrinogen gene, identified a polymorphic cutting site. The allele with the alternative restriction site (H1) was associated with mean fibrinogen levels which were 0.1-0.3 g/l lower than those associated with the other allele (H2). This difference was not statistically significant. No obvious association was found between the familial risk and the presence of the H2 allele. We conclude that in a group of subjects from a metabolic ward, a positive family history for ischemic complications of atherosclerosis is consistently associated with high plasma fibrinogen levels. Interaction with hypertension significantly strengthens the association.

The methylenetetrahydrofolate reductase TT677 genotype is associated with venous thrombosis independently of the coexistence of the FV Leiden and the prothrombin A20210 mutation.

A polymorphism, C-->T677, in the methylenetetrahydrofolate reductase (MTHFR) gene has been identified as a cause of mild hyperhomocysteinemia, a risk factor for venous thrombosis. We have investigated the frequency of the TT genotype in 277 consecutive patients with confirmed deep venous thrombosis and 431 healthy subjects. The TT MTHFR genotype was more frequent in patients than in controls (25.6% vs. 18.1%; p = 0.016). The risk of thrombosis among carriers of this genotype was significantly increased [odds ratio: 1.6 (95% CI: 1.1-2.3)]. The estimated risk associated with the TT genotype was 2.0 (95% CI: 1.3-3.1) in subjects with (n = 122), and 1.3 (95% CI: 0.8-2.0) in those without (n = 155) predisposing (hereditary, acquired or circumstantial) risk factors for venous thrombosis. Factor V Leiden and prothrombin G-->A20210 are known risk factors for venous thrombosis. After stratification for FV Leiden and prothrombin A20210 mutations, a significant association was also observed. After adjustment for sex, FV Leiden and prothrombin A20210 mutation, the estimated risk of venous thrombosis among carriers of the TT MTHFR genotype was 1.7 (95% CI: 1.2-2.6). The TT MTHFR genotype is independently associated with venous thrombosis, mainly among individuals with a high risk profile.

Identification of fetal gender in maternal blood is a helpful tool in the prenatal diagnosis of haemophilia.

Fetal DNA identification in maternal circulation has provided a new approach for non-invasive prenatal diagnosis. However, fetal DNA can persist in maternal blood long after the delivery, severely hampering this possibility. We addressed the issue of fetal DNA persistence in maternal blood. Thus, we investigated cell-free fetal DNA as a reliable approach in prenatal diagnosis of haemophilia. Forty non-pregnant women, who had had at least a male fetus, 29 control pregnant women, and 14 pregnant women, carriers of hemophilia A or B. The assessment of Y-chromosomal sequences was performed by analysing SRY and amelogenin genes using PCR-based techniques. A protocol consisting of double centrifugation at full speed followed by plasma filtration hampered the detection of Y chromosome-specific sequence in non-pregnant women. In 29 control pregnant women, blinded determination of fetal sex confirmed the specificity and sensitivity of the method applied. In 14 pregnant carriers of hemophilia, the investigation revealed a male fetus in nine pregnancies. Excluding the three cases in which a spontaneous miscarriage occurred, the sensitivity and specificity of fetal sex prediction by SRY and amelogenin gene analyses were both 100% as compared with the invasive approach and the fetal sex outcome at birth (six males and five females). Because of its high accuracy in prediction, fetal gender determination with cell-free fetal DNA in maternal plasma may be a useful tool in prenatal diagnosis of haemophilia allowing for the avoidance of invasive procedures for female fetuses.

A comprehensive on-line digestion-liquid chromatography/mass spectrometry/collision-induced dissociation mass spectrometry approach for the characterization of human fibrinogen.

An automatic on-line digestion-liquid chromatography/mass spectrometry/collision-induced dissociation mass spectrometry (LC-MS/CID-MS) protocol has been developed for detection of errors in the biosynthesis of human fibrinogen, such as amino acid (AA) mismatch or incorrect post-translational modification (PTM). Using on-line digestion on an immobilized-enzyme column, the reaction time is significantly reduced (less than 20 min) and the entire approach is suitable for automation. The two-loop MS experiments (full-scan acquisition and sugar moieties monitoring by SIM) allow checking both the correct AA mapping via the peptides generated by the digestion of the PTM. Since the protocol was designed for application on a routine basis, as a proof-of-concept detection of a rare case of 'abnormal' fibrinogen has been demonstrated. The advantage of the proposed approach is exemplified by the fact that the DNA sequence information for the case investigated had not shown any evidence of the abnormality.

C-reactive protein in offspring is associated with the occurrence of myocardial infarction in first-degree relatives.

The relevance of elevated levels of C-reactive protein (CRP) in cardiovascular disease is gaining increasing recognition. A family history of coronary artery disease is a major determinant of coronary artery disease in the offspring. In a cohort of 1048 individuals without clinical evidence of atherosclerosis, we investigated the relationships between CRP levels and a family history of myocardial infarction. We measured CRP, fibrinogen, plasminogen activator inhibitor-1, total cholesterol, triglycerides, and some genetic polymorphisms: plasminogen activator inhibitor-1 (4G/5G), fibrinogen (Bbeta-chain G-->A(-455)), and angiotensin-converting enzyme insertion/deletion (I/D). Clinical data were collected by a World Health Organization-modified questionnaire for cardiovascular disease. When compared with subjects without first-degree relatives who had suffered a myocardial infarction (n=867), subjects with such first-degree relatives (n=181) were older (P=0.001), more often hypertensive (P<0. 001), and homozygous for the 4G allele (4G/4G) of the plasminogen activator inhibitor-1 gene (P=0.003). In addition, they had a higher body mass index (P=0.036), raised plasma fibrinogen (P<0.007) and total cholesterol (P<0.001) concentrations, and CRP levels >0.33 mg/L (P=0.005). In a multiple logistic regression analysis, age (odds ratio [OR] 1.03, 95% confidence interval [95% CI] 1.01 to 1. 05), total cholesterol (OR 1.35, 95% CI 1.11 to 1.65), plasminogen activator inhibitor-1 4G/4G (OR 1.72, 95% CI 1.20 to 2.45), and CRP levels >0.33 mg/L (OR 1.75, 95% CI 1.05 to 2.91) were all independently associated with a positive family history of myocardial infarction. We therefore conclude that raised levels of CRP independently identify the offspring of patients with a myocardial infarction.

Fibrinogen and mechanisms of thrombosis. A difficult link.

Epidemiological observations indicate that high plasma fibrinogen levels are strongly correlated to the frequency of two major thrombotic complications of atherosclerosis: stroke and myocardial infarction. Thrombosis is increasingly recognized as a central mechanism in stroke and myocardial infarction, and fibrinogen is involved in events thought to play a major role in thrombosis. Therefore, elucidation of the relationship between fibrinogen and thrombosis may strengthen the predictive value of this protein and define new interventions against stroke and myocardial infarction. In addition, advances in the understanding of the atherogenic potential of several risk factors of coronary heart disease took advantage of information emerging from the measurement of the factor in population-based studies. Thus, it is conceivable that measuring plasma fibrinogen to predict stroke and myocardial infarction is a major direction to be followed to gain insight into the thrombogenic potential on this protein and inspire new strategies against thrombotic complications of atherosclerosis.

Determining sulfur-containing amino acids by capillary electrophoresis: a fast novel method for total homocyst(e)ine human plasma.

A high-performance capillary electrophoresis (HPCE) method based on laser-induced fluorescence detection is presented here. It enables the determination of sulfur-containing amino acids within 15 min. Fluorescence of sulfur-containing amino acids in plasma is linear over a range of 50-150 micromol/L for L-methionine, 5-100 micromol/L for L-homocysteine, and 50-200 micromol/L for L-cysteine. For homocysteine, we were able to detect 1 fmol injected, equivalent to a plasma concentration of 10 nmol/L. A similar sensitivity is present for cysteine, an even lower one being found for methionine. The intra- and interassay relative standard deviations are < 1%. High-performance liquid chromatography (HPLC) methods are commonly employed for quantifying blood concentrations of sulfur-containing amino acids. A comparative analysis of HPCE and HPLC quantitation of homocysteine has been carried out in 61 blood samples. Plasma concentrations measured by HPCE were in good agreement with those obtained employing an HPLC-based method, a satisfactory correlation being observed between the concentrations obtained by the two methods (r= 0.9972). Thus, the HPCE-based procedure presented here for the measurement of sulfur-containing amino acids in plasma is a simple, fast, accurate, and very sensitive method, suitable for routine determinations in clinical studies.

Age and homocysteine plasma levels are risk factors for thrombotic complications after ovarian stimulation.

BACKGROUND: The magnitude of thrombotic risk during ovarian stimulation cycles is not known. We calculated the magnitude of thrombotic risk in a cohort of women starting a new cycle of ovarian stimulation and investigated the role of inherited and acquired thrombophilia for these events. METHODS: This is an observational study involving outpatients of a clinical research centre. Consecutive women undergoing ovarian stimulation (n = 305) were enrolled. Blood samples for studying inherited and acquired thrombophilia were obtained > or = 2 months after the last cycle of treatment. Odds ratios (OR) and confidence intervals (CI) were determined for markers significantly associated with thrombotic events. Blood samples were analysed for inherited and acquired causes of thrombophilia (antithrombin, protein C, protein S, antiphospholipid antibodies, the Factor V Leiden and FIIA20210 mutations, the TT677 MTHFR genotype, and homocysteine plasma levels). RESULTS: Thrombotic events were observed in 4/747 cycles of ovarian stimulation, with a prevalence of 0.5%, corresponding to 1.6 per 100 000 cycles/woman. Age > or = 39 years and homocysteine plasma levels above the 97.5 percentile were significantly associated with thrombotic events during IVF [OR 15.2 (95% CI 2.0-115.0) and 14.4 (1.5-141.3) respectively]. CONCLUSIONS: Age > or = 39 years and mild hyperhomocysteinaemia are strongly associated with the occurrence of thrombotic events during IVF.

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.

In vitro inhibition by defibrotide of monocyte superoxide anion generation: a possible mechanism for the antithrombotic effect of a polydeoxyribonucleotide-derived drug.

In an attempt to elucidate the antithrombotic potential of defibrotide (D) we have evaluated several functions of monocytes from 7 healthy subjects before and after in vitro incubation of the cells with increasing concentrations of this drug. At concentrations as high as 40 micrograms/ml, D hardly affected the expression of both the procoagulant activity of monocytes and the formation of superoxide anion in response to 1 mg/ml zymosan (STZ). In contrast, at concentrations that may be achieved in vivo following the administration of the drug (5-20 micrograms/ml), D impaired in a dose-dependent manner (p less than 0.05) the generation of O-2 in response to N-formyl-L-methionyl-L-leucyl-L-phenylalanine (FMLP, 1 microM) or calcium ionophore A23187 (10 microM). Regardless of the agonist employed, at concentrations between 1 and 5 mM, extracellular Ca2+ had little effect on the impairment of superoxide anion generation by D. In contrast, the inhibitory effect was time-dependent, the maximum impairment (greater than 30%) being observed when the cells were preincubated with the drug for 20 h. These data support the concept that the antithrombotic potential of D involves the ability of the drug to affect the generation of free radicals by leukocytes and suggest that future in vivo studies for the evaluation of the activity of D should take into account the role of monocytes in hemostasis and thrombosis.

Cu/Zn superoxide dismutase in patients with non-familial Alzheimer's disease.

Chromosome 21 contains genes whose altered expression has long been associated with Down's syndrome and whose altered structure with some cases of Alzheimer's disease (AD). The gene for the Cu/Zn superoxide dismutase enzyme (SOD-1), a key enzyme in the metabolism of oxygen free radicals, is located on the distal portion of chromosome 21. Due to the triplication of the SOD-1 gene, patients with Down's syndrome have an almost 50% increase in their SOD activity. On the other hand, almost 25% of the patients with Down's syndrome over 40 years of age develop progressive dementia, with clinical symptoms of AD. Therefore, we decided to evaluate whether abnormalities in the production of free radicals could be detected in blood cells from AD patients, and whether they correlated with molecular variations in the Cu/Zn SOD-1 gene. Superoxide anion production was evaluated spectrophotometrically in suspensions of monocytes from 9 sporadic AD patients, and from 9 aged-matched apparently normal controls. After stimulation with increasing concentrations of n-formyl-methionyl-leucyl-phenylalanine (fMLP) or Ca ionophore A23187, monocyte free radical generation was quantitatively and qualitatively normal. Furthermore, restriction fragment length polymorphism (RFLP) analysis of leukocyte DNA digested with a variety of enzymes, gave comparable results in patients and controls. Our data support the possibility that in addition to the generation of free radicals, other directions should be explored to elucidate the mechanisms of dementia in AD.

Plasma lipoprotein(a) levels in subjects attending a metabolic ward. Discrimination between individuals with and without a history of ischemic stroke.

In this cross-sectional study we compared the abilities of lipoprotein(a) [Lp(a)], plasminogen activator inhibitor-1 (PAI-1), and tissue plasminogen activator (TPA) to discriminate between individuals with and without a history of stroke from among subjects in a metabolic ward. A total of 210 subjects (108 men and 102 women; mean age, 63.8 years; range, 31 to 86 years) provided plasma and DNA samples for the study. Of these, 51 men and 50 women had a history of ischemic stroke. The 109 subjects without a history of stroke were compared with those with such a history for major risk factors for ischemic events. Mean plasma TPA and PAI-1 levels significantly (P < .001) discriminated among subjects younger than 70 years with a history of stroke. The mean plasma Lp(a) level of stroke subjects (21.9 mg/dL) did not differ significantly from that of control subjects (15.2 mg/dL). However, among individuals < 70 years old, Lp(a) plasma levels > 50 mg/dL were more common among stroke patients (8 with versus 1 without, P < .01 by chi 2 test). A molecular variation in the 5' flanking region of the apo(a) gene that has been related to elevated Lp(a) plasma levels (G/A-914) was not strongly correlated with circulating levels of Lp(a), nor did Lp(a) levels correlate with a polymorphism of the apo(a) gene (G/A-21), which is strongly linked (P < .001) to the G/A-914 variation. In this setting, the relation between Lp(a) and cerebral ischemia appears to be limited to individuals below 70 years with elevated (> 50 mg/dL) plasma levels of the lipoprotein.