[The document of chemical risk assessment: experiences in small companies]. / Il documento di valutazione del rischio chimico: esperienze nelle piccole imprese.

Were examined 60 documents of chemical risk assessment of companies from 19 municipalities in the provinces of Siena and Grosseto. The review of evaluations was conducted initially by checking the internal coherence of the documents. What emerged was subsequently analyzed together with the staff who performed the inspection. Although in 12 companies has been declared the presence of carcinogens, in only 5 the evaluation of exposure have been done using measures, others have used models or other techniques. Overall assessment showed that only 8 out of 60 (13.3%) were wrote correctly.

Abnormal propeptide processing resulting in the presence of two abnormal species of protein C in plasma: characterization of the dysfunctional protein C Padua3 (protein C(R-1L/propeptide)).

A heterozygous G-->T transversion at position 1388 of the protein C (PC) gene which predicted the substitution of Arg(-1) to a Leu (PC(R-1L)) was identified in a thrombophilic patient. The PC(R-1L) was purified from the patient's plasma by immunoaffinity chromatography using Ca++-independent and Ca++-dependent monoclonal antibodies. NH2-terminal sequencing of the light chain of PC(R-1L) revealed two amino acid sequences: one was identical to the complete propeptide sequence of PC, while the other matched the normal PC light chain sequence elongated by one amino acid (Leucine at position 1). Activated PC(R-1L/propeptide) exhibited normal amidolytic and impaired anticoagulant activity. Thus, the substitution of a Leu for an Arg at position -1 of PC shifts the propeptidase cleavage site by one amino acid. In addition, in PC(R-1L/propeptide) the propeptide cleavage at Lys(-2) is less efficient since approximately 60% of PC variant molecules present in patient's plasma retained the entire propeptide. Our findings suggest that depending on the specific amino acid substitution at position-1, PC can be secreted in plasma containing the entire propeptide attached to the light chain. Impaired interaction of elongated APC molecules with a membrane-surface and/or factor Va which is the physiological substrate for APC, is manifested in vivo by thrombophilia.

The risk of fetal loss in family members of probands with factor V Leiden mutation.

In order to investigate the risk of fetal loss in carriers of factor V Leiden who are family members of probands with this mutation, we performed a retrospective cohort study including 109 women who had been pregnant at least once and were family members of 61 probands with venous thromboembolism and a single identified factor V Leiden mutation. The rate of pregnancies ending in unexplained fetal loss, early miscarriage, late miscarriage or stillbirth in women with the factor V Leiden was compared with that of women with normal genotype. In the 65 women who were carriers of factor V Leiden 31 of the 191 pregnancies (16.2% per pregnancy) resulted in unexplained fetal loss, as compared to 13 of the 121 pregnancies (10.7% per pregnancy) in the 44 non-carriers (relative risk, 1.5; 95% CI, 0.8-3.2). After the first trimester of pregnancy, 25 pregnancies (13.1% per pregnancy) among carriers of factor V Leiden ended in fetal loss, as compared to 7 (5.8% per pregnancy) among females with normal genotype (relative risk, 2.3; 95% CI, 1.01 to 5.1). We conclude that carriers of factor V Leiden who are family members of probands with this mutation have a statistically significant and clinically important risk of late miscarriage or stillbirth. Studies addressing the benefit-to-risk ratio of adopting routinary thromboprophylactic measures following the first trimester of pregnancy in these women are strongly indicated.

A new factor X defect (factor X Padua 3): a compound heterozygous between true deficiency (Gly(380)-->Arg) and an abnormality (Ser(334)-->Pro).

We report a novel mutation in Factor X (FX) gene which results in a phenotype without any bleeding tendency. The proband has been found to be a compound heterozygote between a novel FX true deficiency (Gly(380)-->Arg) and a previously reported dysfunctional mutation Ser(334)-->Pro (FX Marsiglia). Prothrombin time (PT) and partial thromboplastin time (PTT) were moderately prolonged and were fully corrected by the addition of normal serum. Her FX activity level varied between 8% and 19% of normal according to the method used whereas the FX antigen level was 40% of the normal control value. All the exons and intron/exon junctions of the FX gene were studied using a combined approach of polymerase chain reaction and conformation sensitive gel electrophoresis. A transversion G to A in exon 8 resulting in the replacement of Gly380 by Arg was found in the proband, in the father and in a proband's brother, whereas heterozygous FX Marsiglia was present in the proband's mother and her sister. Gly380 is strictly linked to Ser379, a component of the catalytic triad. The substitution of Gly for Arg causes the introduction of a large charged amino acid which could affect the catalytic function of FX leading to secretion problem, accounting for the cross-reactive material (CRM) negative phenotype.

Clinical and laboratory expression of associated thrombophilic conditions (homozygous/heterozygous factor V Leiden mutation and heterozygous prothrombin variant 20210A) in an Italian family.

Prothrombin variant 20210A is maintained to be a mild risk factor for venous thromboembolism (VTE). The association of this defect with other inherited thrombophilic conditions may result in an increased risk of developing VTE. In this article, a family is described in which prothrombin variant was associated with either homozygous or heterozygous factor V Leiden (FV Leiden) mutation. All family members except the proband were asymptomatic despite the presence and the severity of the underlying genetic defect(s). The proband, who carried homozygous FV Leiden mutation and heterozygous prothrombin variant, experienced recurrent VTE during pregnancies, whereas one brother, with the same defect, was asymptomatic. Mean prothrombin antigen and activity levels were higher in carriers of the prothrombin variant as compared with noncarriers. Thrombin generation was assessed in family members, in carriers of prothrombin variant or homozygous FV Leiden mutation and in a control group. Most of the family members presented with increased prothrombin fragment 1+2 levels possibly because of the presence of the FV Leiden mutation. Although it is conceivable that the co-inheritance of prothrombin variant and FV Leiden mutation may increase the risk of VTE, patients with these combined defects may remain asymptomatic. It is likely that acquired triggering conditions play a major role in determining VTE in carriers of a mild genetic predisposition. This has to be taken into account when recommendation for thromboprophylaxis is given.

Asymptomatic homozygous nt 20210 G to A prothrombin polymorphism in two blood donors belonging to two different kindreds.

The nucleotide (nt) 20210 G to A prothrombin polymorphism has been associated with an increased incidence of thrombosis, particularly venous thrombosis. The increased incidence of thrombosis resulted from an increase in prothrombin levels. Two homozygous patients with this abnormality were found to be completely asymptomatic. In one of the two cases there was no thrombosis even after the additional risk factor of oral contraceptive therapy for several months. Prothrombin activity and antigen as assayed by several methods were at the upper limits of normalcy in one case and slightly elevated in the other. The two cases described in this article do not rule out the possibility that this prothrombin abnormality be associated, given special acquired circumstances, with thrombosis. However, they indicate that extreme caution is needed to avoid an inaccurate conclusion. The fact that homozygous patients may remain asymptomatic indicates that the prothrombotic defect is very mild, if any.

Prothrombin antigen levels in symptomatic and asymptomatic carriers of the 20210A prothrombin variant.

A recently discovered variant in the prothrombin gene (20210A) has been found in approximately 5-10% of patients with venous thromboembolism. It has been shown that patients with this variant present with high levels of prothrombin in plasma and this is maintained to be the most likely mechanism by which the risk of thrombosis is increased. We have evaluated prothrombin antigen levels in 50 carriers of the 20210A allele and compared with non-carriers. 327 subjects were subdivided according to deficiency status and previous thrombosis. 30 symptomatic and 20 asymptomatic carriers had increased mean prothrombin levels as compared to symptomatic (n=178) or asymptomatic (n=99) non-carriers. The percentage of subjects with prothrombin levels above cut-off values of 1.15u/ml or 1.30u/ml was significantly higher in carriers of the prothrombin variant as compared to non-carriers, regardless of a previous thrombosis. However, among non-carriers the percentage of those with prothrombin levels above cut-off values was significantly higher in the group of symptomatic as compared to asymptomatic individuals. In conclusion, increased prothrombin antigen levels, as detected by a specific ELISA, were found among 50 symptomatic and asymptomatic carriers of the 20210A prothrombin variant as well as among a large group of symptomatic non-carriers. The data are in agreement with those found by using functional tests for the determination of prothrombin levels in these patients.

Compound heterozygous protein C deficiency resulting in the presence of only the beta-form of protein C in plasma.

About 30% of human plasma protein C (PC) is of lower molecular weight than the predominant alpha-form. The minor beta-form arises as a consequence of the lack of glycosylation at Asn329. Although the functional role of Asn329 has been investigated by in vitro mutagenesis, until now no naturally occurring mutations have been reported at this site. We describe here the case of two identical twin sisters compound heterozygous for two novel PC mutations: Cys78-->Stop inherited from the maternal side and Asn329-->Thr inherited from the paternal side, associated with the presence of only the beta-form of PC in plasma. The Cys78-->Stop substitution is predicted to abolish PC synthesis from one allele, whereas the Asn329-->Thr substitution results in the reduced synthesis of a beta PC variant with decreased functional activity. PCN329T from the two monovular twin sisters was purified and its active form APCN329T was assessed for its ability to inactivate factor Va. Whereas no differences were observed between the activation rates of normal PC and PCN329T, APCN329T inactivated human factor Va with a rate slower than the normal APC. This is the first report of a PC defect involving glycosylation of the molecule. This defect results in the presence of only the beta-form of PC in human plasma and is responsible for the reduced anticoagulant activity observed.

Factor V Leiden mutation and the risk of venous thromboembolism in pregnant women.

BACKGROUND AND OBJECTIVES: In this retrospective, single center, cohort study we assessed the risk of pregnancy-related venous thromboembolism (VTE) in women belonging to a large number of families identified because of a symptomatic proband with single identified factor V Leiden mutation. DESIGN AND METHODS: Female family members who had experienced at least one full-term pregnancy were enrolled in the study. Two hundred and seventy pregnancies occurred in 105 carriers and 215 pregnancies in 81 non-carriers of factor V Leiden mutation. RESULTS: The frequency of VTE was 6.4% for heterozygous, 16.7% for homozygous, 20% for double heterozygous carriers of thrombophilic defects, and 1.2% for non-carriers. The majority of VTE events related to pregnancy occurred in the post-partum period. The relative risks of developing pregnancy-related VTE in women who were carriers of heterozygous and homozygous (or combined heterozygous) factor V Leiden mutation as compared to non-carriers were 5.3 (95% CI, 0.6 to 43.9) and 15.4 (95% CI, 1.4 to 164), respectively. INTERPRETATION AND CONCLUSIONS: Factor V Leiden mutation is a risk factor for pregnancy-related VTE, especially in its homozygous form and in combination with other thrombophilic abnormalities. Screening of families with this mutation might be useful for women of fertile age, as they may take advantage from thromboprophylaxis during pregnancy and the post-partum period.