Francesco Bernardi

Mutations in GJB6 cause nonsyndromic autosomal dominant deafness at DFNA3 locus.

factors1. Mutations in the connexin26 gene (GJB2), located on 13q12, are responsible for non-syndromic recessive and dominant forms of deafness2–4. Connexin-31 and connexin-32 have also been implicated in deafness5,6. The identification of deaf families linked to 13q12 but negative for mutations in GJB2 (ref. 7) suggested the presence of other deafness genes in this region. Recently, the mouse connexin-30 gene (Gjb6), which is expressed in cochlea, has been mapped to a region with syntenic homology to human chromosome 13q12 (refs 8,9). To verify if human GJB6 is involved in deafness, we cloned a 1,799-bp cDNA fragment containing an ORF of 261 amino acids (EMBL HSA005585). CX30 protein has a structure similar to that of other connexins10 and shares 93% homology with mouse Cx30 and 76% identity with human CX26. GJB6 is not interrupted by introns and maps to chromosome 13q12, approximately 800 kb centromeric to GJB2. SSCP mutational analysis in 198 deaf patients, including 38 families linked to 13q12, revealed a threonine-to-methionine change at position 5 (T5M) in an Italian family affected by bilateral middle/ high-frequency hearing loss (Fig. 1a–c). Audiograms in T5M family members showed a 20–50-dB decrease at frequencies of 2,000–8,000 Hz (I-2), a progressive impaired threshold above 500 Hz (II-1) and a profound sensorineural deafness (II-2). This variability of hearing impairment can be explained by a different expressivity of the disease, which is almost the rule for dominant deafness. Northern blots, RT-PCR and in situ hybridization on mouse embryos revealed Gjb6 expression in trachea, thyroid, thymus, brain and cochlea, confirming reported expression patterns (refs 8,9,11). The threonine residue at position 5 is evolutionarily conserved and also present in human connexin 26 (Fig. 1d). The T5M substitution abolishes a hydrophilic residue possibly involved in interor Mutations in GJB6 cause nonsyndromic autosomal dominant deafness at DFNA3 locus correspondence

Predictive Value of D-Dimer Test for Recurrent Venous Thromboembolism After Anticoagulation Withdrawal in Subjects With a Previous Idiopathic Event and in Carriers of Congenital Thrombophilia

Background We have shown that normal D‐dimer levels obtained after the discontinuation of oral anticoagulant treatment (OAT) has a high negative predictive value for recurrent venous thromboembolism (VTE). The aim of the present study was to assess the predictive value of D‐dimer for recurrent VTE in subjects with a previous unprovoked event who are either carriers of inherited thrombophilia or not. Methods and Results We prospectively evaluated 599 patients (301 males) with a previous VTE episode. They were repeatedly examined for D‐dimer levels after OAT withdrawal and were screened for inherited thrombophilic alterations. Alterations were detected in 130 patients (21.7%), factor V Leiden (70 patients; 2 of whom were homozygotes) and prothrombin mutation (38 patients) were the most prevalent ones. Recurrent events were recorded in 58 subjects (9.7%) during a follow‐up of 870.7 patient‐years. Altered D‐dimer levels at 1 month after OAT withdrawal were associated with a higher rate of subsequent recurrence in all subjects investigated, especially in those with an unprovoked qualifying VTE event (hazard ratio, 2.43; 95% confidence interval, 1.18 to 4.61) and in those with thrombophilia (hazard ratio, 8.34; 95% confidence interval, 2.72 to 17.43). The higher relative risk for recurrence of altered D‐dimer was confirmed by multivariate analysis after adjustment for other risk factors. The negative predictive value of D‐dimer was 92.9% and 95.8% in subjects with an unprovoked qualifying event or with thrombophilia, respectively. Conclusions D‐dimer levels measured 1 month after OAT withdrawal have a high negative predictive value for recurrence in subjects with unprovoked VTE who are either carriers or not carriers of congenital thrombophilia. (Circulation. 2003;108:313‐318.)

Polymorphisms in the factor VII gene and the risk of myocardial infarction in patients with coronary artery disease

BACKGROUND High plasma levels of coagulation factor VII have been suggested to be predictors of death due to coronary artery disease. Since polymorphisms in the factor VII gene contribute to variations in factor VII levels, such polymorphisms may be associated with the risk of myocardial infarction, which is precipitated by thrombosis. METHODS We studied a total of 444 patients, 311 of whom had severe, angiographically documented coronary atherosclerosis. Of these 311 patients, 175 had documentation of a previous myocardial infarction. As a control group, 133 patients with normal coronary arteriograms were also included. We measured the levels of activated factor VII and assessed three polymorphisms in the factor VII gene, one involving the promoter (A1 and A2 alleles), one involving the catalytic region (R353Q), and one involving intron 7. RESULTS Each of the polymorphisms influenced factor VII levels. Patients with the A2A2 and QQ genotypes had the lowest levels of activated factor VII (66 percent and 72 percent lower, respectively, than the levels in patients with the wild-type genotypes). The frequencies of the various genotypes in the patients free of coronary artery disease were similar to those in the entire population of patients with coronary artery disease. In the latter group, there were significantly more heterozygotes and homozygotes for the A2 and Q alleles among those who had not had a myocardial infarction than among those who had had an infarction (P=0.008 for the presence of the promoter polymorphism and P=0.01 for the presence of the R353Q polymorphism by chi-square analysis). The adjusted odds ratio for myocardial infarction among the patients with the A1A2 or RQ genotype was 0.47 (95 percent confidence interval, 0.27 to 0.81). CONCLUSIONS Our findings suggest that certain factor VII genotypes have a role in protection against myocardial infarction. This may explain why some patients do not have myocardial infarction despite the presence of severe coronary atherosclerosis.

No evidence of association between prothrombotic gene polymorphisms and the development of acute myocardial infarction at a young age

Background—We investigated the association between 9 polymorphisms of genes encoding hemostasis factors and myocardial infarction in a large sample of young patients chosen because they have less coronary atherosclerosis than older patients, and thus their disease is more likely to be related to a genetic predisposition to a prothrombotic state. Methods and Results—This nationwide case-control study involved 1210 patients who had survived a first myocardial infarction at an age of <45 years who underwent coronary arteriography in 125 coronary care units and 1210 healthy subjects matched for age, sex, and geographical origin. None of the 9 polymorphisms of genes encoding proteins involved in coagulation (G-455A &bgr;-fibrinogen: OR, 1.0; CI, 0.8 to 1.2; G1691A factor V: OR, 1.1; CI, 0.6 to 2.1; G20210A factor II: OR, 1.0; CI, 0.5 to 1.9; and G10976A factor VII: OR, 1.0; CI, 0.8 to 1.3), platelet function (C807T glycoprotein Ia: OR, 1.1; CI, 0.9 to 1.3; and C1565T glycoprotein IIIa: OR, 0.9; CI, 0.8 to 1.2), fibrinolysis (G185T factor XIII: OR, 1.2; CI, 0.9 to 1.6; and 4G/5G plasminogen activator inhibitor type 1: OR, 0.9; CI, 0.7 to 1.2), or homocysteine metabolism (C677T methylenetetrahydrofolate reductase: OR, 0.9; CI, 0.8 to 1.1) were associated with an increased or decreased risk of myocardial infarction. Conclusions—This study provides no evidence supporting an association between 9 polymorphisms of genes encoding proteins involved in hemostasis and the occurrence of premature myocardial infarction or protection against it.

Resistance to activated protein C in healthy women taking oral contraceptives.

Summary. Resistance to activated protein C (APC) is at present considered the most frequent laboratory abnormality in patients with deep‐vein thrombosis. An increased risk for venous thrombosis is associated to the use of oral contraceptives (OC).

Factor VII Deficiency

The complex formed between the procoagulant serine protease activated factor VII (FVII) and the membrane protein tissue factor, exposed on the vascular lumen upon injury, triggers the initiation of blood clotting. This review describes the clinical picture of FVII deficiency and provides information on diagnosis and management of the disease. FVII deficiency, the most common among the rare congenital coagulation disorders, is transmitted with autosomal recessive inheritance. Clinical phenotypes range from asymptomatic condition, even in homozygotes, to severe disease characterized by life-threatening and disabling symptoms (central nervous system and gastrointestinal bleeding and hemarthrosis), with early age of presentation and the need for prophylaxis. In females, menorrhagia is prevalent and affects two thirds of the patients of fertile age. Although FVII gene mutations are extremely heterogeneous, several recurrent mutations have been reported, a few of them relatively frequent. The study of genotype-phenotype relationships indicates that modifier (environmental and/or inherited) components modulate expressivity of FVII deficiency, as reflected by patients with identical FVII mutations and discordant clinical phenotypes. Several treatment options are available for FVII deficiency: the most effective are plasma-derived FVII concentrates and recombinant activated FVII (rFVIIa). Treatment-related side effects are rare.

A polymorphism in the 5′ region of coagulation factor VII gene (F7) caused by an inserted decanucleotide

We describe a polymorphism in the 5′ region of the coagulation factor VII (FVII) gene, originating from a decanucleotide (CCTATATCCT) insert present in the less frequent allele. This marker can be detected by restriction analysis of polymerase chain reaction products.

Thrombosis in inherited factor VII deficiency.

Summary.  Thrombosis in congenital factor (F) VII deficiency was investigated through extensive phenotypic and molecular‐genetic studies. Patients with a history of thrombosis among 514 entries in the FVII Deficiency Study Group database were evaluated. Thrombotic events were arterial in one case, disseminated intravascular coagulation in another and venous in seven. Gene mutations were characterized in eight patients: three were homozygous, three compound heterozygous and two heterozygous. FXa and IIa generation assays were consistent with the genetic lesions. One patient was heterozygous for the FV Leiden and one for the FIIG20210A mutation. In seven patients, surgical interventions and/or replacement therapies had a close temporal relationship with thrombosis, while in the remaining, events were apparently spontaneous. Thromboses were not associated with any specific age, phenotype, mutation zygosity or thrombophilic abnormalities. In particular, severe FVII deficiency did not seem to offer protection from strong thrombosis risk factors such as surgery and replacement therapy.

Daily and Circadian Rhythms of Tissue Factor Pathway Inhibitor and Factor VII Activity

Objective—Diurnal variations in levels of factor VII (FVII), FVIII, proteins C and S, antithrombin, plasminogen activator inhibitor-1, prothrombin fragment F1+2, and D-dimers in healthy humans point to the existence of circadian rhythms of coagulation factors. We sought for temporal fluctuations of tissue factor pathway inhibitor (TFPI) activity in human and mouse plasma. Methods and Results—TFPI activity showed significant daily variations with highest levels in the morning in healthy men (+11%) and in mice at the light-to-dark transition (+63%), the beginning of the physically active period. Variations in FVII activity paralleled those in TFPI. In mice, the feeding schedule had a strong impact on these rhythms. Although restricted feeding and fasting shifted the peak of TFPI, the FVII peak disappeared. Investigation of temporal fluctuations in constant darkness indicated the existence of daily rhythms for TFPI and of true circadian rhythms for FVII. Conclusions—For the first time, we report, both in humans and mice, temporal variations in TFPI activity. The coherent variations in FVII and TFPI activity could interplay to maintain the coagulation equilibrium. The chronobiological patterns should be considered to analyze activity levels of these factors. Moreover, the mouse model could be exploited to investigate modifiers of coagulation rhythms potentially associated to morning peaks of cardiovascular events.

Stimulation of P2 (P2X7) receptors in human dendritic cells induces the release of tissue factor-bearing microparticles

Receptors for extracellular nucleotides are the focus of increasing attention for their ability to cause release of plasma membrane vesicles (micropar‐ticles, MPs). Here, we show that monocyte‐derived human dendritic cells (DCs) stimulated with a P2X7 receptor (P2X7R) agonist undergo a large release of MPs endowed with procoagulant activity. Functional and Western blot studies revealed that MPs contain the membrane‐bound form of tissue factor (TF), a glycop‐rotein acting as essential cofactor of activated factor VII and triggering blood coagulation. Quiescent DCs express the membrane‐bound (full length), as well as truncated alternatively spliced TF forms. DC reactivity to anti‐TF Abs disappeared almost completely on stimulation with ATP or benzoyl ATP (BzATP), as shown by immunoblot and confocal microscopy analysis. Concurrently, TF reactivity and activity appeared in the vesicular fraction, indicating that MPs are important carriers for the dissemination of full‐length TF form. Activity of MP‐bound TF, comparable to that of relipidated recombinant TF, was dose dependently inhibited by the addition of a specific anti‐human TF antibody. We infer that a large fraction of this protein, and its procoagulant potential, are “deliverable” after physiological or pathological stimuli. These findings might have implications for triggering and propagating coagulation in healthy and atherosclerotic vessels.—MarcelloBaroni, CinziaPizzirani, MirkoPinotti, DavideFerrari, ElenaAdinolfi, SaraCalzavarini, PierpaoloCaruso, FrancescoBernardi, FrancescoDi Virgilio. Stimulation of P2 (P2X7) receptors in human dendritic cells induces the release of tissue factor‐bearing microparticles. FASEB J. 21, 1926–1933 (2007)