Fattore VII
E' la proteina plasmatica che, interagendo con il Fattore Tissutale (Fattore III), è iniziatore delle reazioni enzimatiche della coagulazione.
Blood. 2009 Jun 18;113(25):6461-4. Epub 2009 Apr 22.
Rescue of coagulation factor VII function by the U1+5A snRNA.
Pinotti M, Balestra D, Rizzotto L, Maestri I, Pagani F, Bernardi F.
Department of Biochemistry, University of Ferrara, Italy. pnm@unife.it
Abstract
Our previous studies with genomic minigenes have demonstrated that an engineered small nuclear RNA-U1 (U1+5a) partially rescued coagulation factor VII (FVII) mRNA processing impaired by the 9726+5G>A mutation. Here, to evaluate the U1+5a effects on FVII function, we devised a full-length FVII splicing-competent construct (pSCFVII-wt). This construct drove in COS-1 cells the synthesis of properly processed FVII transcripts and of secreted functional FVII (23 +/- 4 ng/mL), which were virtually undetectable upon introduction of the 9726+5G>A mutation (pSCFVII-9726+5a). Cotransfection of pSCFVII-9726+5a with pU1+5a resulted in a partial rescue of FVII splicing and protein biosynthesis. The level increase in medium was dose dependent and, with a molar excess (1.5x) of pU1+5a, reached 9.5% plus or minus 3.2% (5.0 +/- 2.8 ng/mL) of FVII-wt coagulant activity. These data provide the first insights into the U1-snRNA-mediated rescue of donor splice sites at protein level, thus further highlighting its therapeutic implications in bleeding disorders, which would benefit even from tiny increase of functional levels.
PMID: 19387004 [PubMed - indexed for MEDLINE]
Semin Thromb Hemost. 2009 Jun;35(4):400-6. Epub 2009 Jul 13.
Factor VII Deficiency.
Dipartimento di Medicina Interna e Sanità Pubblica, Università dell'Aquila, L'Aquila 67010, Italy. mariani@cc.univaq.it
Comment in:
Abstract
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.
PMID: 19598068 [PubMed - indexed for MEDLINE]
J Thromb Haemost. 2009 Feb 25. [Epub ahead of print]
Major differences in bleeding symptoms between factor VII deficiency and haemophilia B.
Bernardi F, Dolce A, Pinotti M, Shapiro AD, Santagostino E, Peyvandi F, Batorova A, Lapecorella M, Schved JF, Ingerslev J, Mariani G; for the International Factor VII Deficiency Study Group.
Department of Biochemistry and Molecular Biology, University of Ferrara, Ferrara, Italy.
Summary Background: The autosomally-inherited factor VII (FVII) deficiency and the X-linked haemophilia B offer an attractive model to investigate whether reduced levels of FVII and factor IX (FIX), acting in the initiation and amplification of coagulation respectively, influence haemostasis to a different extent in relation to age and bleeding site. Methods: Haemophilia B patients (n=296) and FVII deficient males (n=109) were compared for FVII/FIX clotting activity, F7/F9 genotypes and clinical phenotypes in a retrospective, multi-centre, cohort study. Results: Major clinical differences between diseases were observed. Bleeding occurred earlier in haemophilia B (median age 2.0 years, IR 0.9-5.0) than in FVII deficiency (5.2 years, IR 1.9-15.5) and the bleeding-free survival in FVII deficiency was similar to that observed in "mild" haemophilia B (p= 0.96). The most frequent disease-presenting symptoms in haemophilia B (haematomas and oral bleeding) differed from those in FVII deficiency (epistaxis and central nervous system bleeding). Differences were confirmed by analysis of FVII deficient women. Conclusions: Our data support the notion that low FVII levels sustain haemostasis better than similarly reduced FIX levels. On the other hand, minute amounts of FVII, differently from FIX, are needed to prevent fatal bleeding, as indicated by the rarity of null mutations and the associated life-threatening symptoms in FVII deficiency, which contributes to shape clinical differences between diseases in the lowest factor level range. Differences between diseases are only partially explained by mutational patterns and could pertain to the specific roles of FVII and FIX in coagulation phases and to vascular bed-specific components.
PMID: 19245420 [PubMed - as supplied by publisher]
Blood. 2008 Mar 1;111(5):2681-4. Epub 2007 Dec 21.
U1-snRNA-mediated rescue of mRNA processing in severe factor VII deficiency.
Pinotti M, Rizzotto L, Balestra D, Lewandowska MA, Cavallari N, Marchetti G, Bernardi F, Pagani F.
Department of Biochemistry and Molecular Biology, University of Ferrara, Via Fossato di Mortara 74, Ferrara, Italy. pmm@unife.it
Small nuclear U1-RNAs (snRNAs), the spliceosome components selectively recognizing donor splice sites (5'ss), were engineered to restore correct mRNA processing in a cellular model of severe coagulation factor VII (FVII) deficiency, caused by the IVS7 9726 + 5g/a change. Three U1-snRNAs, complementary to the mutated 5'ss (U1 + 5a) or to neighboring sequences were expressed with FVII minigenes in a hepatoma cell line. The U1-snRNAs reduced from 80% to 40% the exon 7 skipping, thus increasing exon definition. The U1 + 5a construct also dramatically increased recognition of the correct 5'ss over the 37-bp downstream cryptic site preferentially activated by the mutation, thus inducing appreciable synthesis of normal transcripts (from barely detectable to 50%). This effect, which was dose-dependent, clearly demonstrated that impaired recognition by the U1-snRNA was the mechanism responsible for FVII deficiency. These findings suggest compensatory U1-snRNAs as therapeutic tools in coagulation factor deficiencies caused by mutations at 5'ss, a frequent cause of severe defects.
PMID: 18156490 [PubMed - indexed for MEDLINE]
FASEB J. 2007 Jun;21(8):1926-33. Epub 2007 Feb 21.
Stimulation of P2 (P2X7) receptors in human dendritic cells induces the release of tissue factor-bearing microparticles.
Baroni M, Pizzirani C, Pinotti M, Ferrari D, Adinolfi E, Calzavarini S, Caruso P, Bernardi F, Di Virgilio F.
Department of Biochemistry, University of Ferrara, Ferrara, Italy.
Receptors for extracellular nucleotides are the focus of increasing attention for their ability to cause release of plasma membrane vesicles (microparticles, 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 glycoprotein 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.
PMID: 17314141 [PubMed - indexed for MEDLINE]
Mol Med. 2006 Jul-Aug;12(7-8):137-42.
Intracellular evaluation of ER targeting elucidates a mild form of inherited coagulation deficiency.
Rizzotto L, Pinotti M, Pinton P, Rizzuto R, Bernardi F.
Department of Biochemistry and Molecular Biology, University of Ferrara, Via Fossato di Mortara 74, Ferrara, Italy.
Missense mutations reduce protein levels through several molecular mechanisms. Among them, altered targeting to endoplasmic reticulum (ER) and its relationship with clinical phenotypes in patients have been poorly investigated. To address this point, we studied the prepeptide mutations (L-48P, L-42P) associated with mild deficiency of factor VII (FVII), the serine-protease triggering blood coagulation. Mutations were introduced into the native FVII to evaluate secreted and intracellular protein levels, and into a chimeric FVII-GFP to study ER targeting in living cells. In conditioned medium from stably or transiently transfected cells, expression levels of the -48PFVII (9% and 55%, respectively) and particularly those of the -42PFVII (2% and 12%) were decreased compared with those of WtFVII, indicating the causative nature of mutations. Markedly reduced protein levels were observed in cell organelles for -48PFVII (10.5 +/- 4.9 ng/mL; Wt-FVII, 130 +/- 43.4 ng/mL) and -42PFVII (approximately 5 ng/mL), thus suggesting impaired ER targeting. Fluorescence of the -48PFVII-GFP and -42PFVII-GFP was diffuse, covered the nucleus, and declined upon plasma membrane permeabilization with digitonin, which demonstrated mislocalization of variants in the cytosol. Noticeably, the residual fluorescence of -48PFVII-GFP (10%) and -42PFVII-GFP (20%) in organelles was fairly compatible with FVII levels in patients' plasma. The studies with the native and chimeric proteins indicated that both prepeptide mutations were associated with residual expression of normal FVII, which explained the mild form of FVII deficiency in patients. This approach, extendable to other coagulation serine proteases, clearly contributed to elucidate the relationship of genotype with plasma and clinical phenotype.
PMID: 17088945 [PubMed - indexed for MEDLINE]
PMCID: PMC1626593
J Thromb Haemost. 2006 Jun;4(6):1308-14.
Intracellular readthrough of nonsense mutations by aminoglycosides in coagulation factor VII.
Pinotti M, Rizzotto L, Pinton P, Ferraresi P, Chuansumrit A, Charoenkwan P, Marchetti G, Rizzuto R, Mariani G, Bernardi F; International Factor VII Deficiency Study Group.
Department of Biochemistry and Molecular Biology, University of Ferrara, Ferrara, Italy.
BACKGROUND: Nonsense mutations in coagulation factor (F) VII potentially cause a lethal hemorrhagic diathesis. Readthrough of nonsense mutations by aminoglycosides has been studied in a few human disease models with variable results. OBJECTIVES: We investigated the K316X and W364X FVII mutations, associated with intracranial hemorrhage, and their correction by aminoglycosides. The rare nonsense mutations in FVII represent favorite models to test this strategy, because even tiny increases in the amount of functional full-length protein in patients could ameliorate hemorrhagic phenotypes. RESULTS: A FVII-green fluorescent protein (GFP) chimaera provided us with a fluorescent model of FVII expression in living cells. Appreciable fluorescence in cells transfected with nonsense FVII-GFP mutants was detected upon geneticin treatment, thus demonstrating suppression of premature translation termination. To investigate the rescue of FVII function, nonsense variants of the native FVII without GFP (p316X-FVII and p364X-FVII) were transfected and found to secrete low amounts of FVII (approximately 1% of Wt-FVII activity), thus suggesting a spontaneous stop codon readthrough. Geneticin treatment of cells resulted in a significant and dose-dependent increase of secreted FVII molecules (p316X-FVII, 24 +/- 12 ng mL(-1), 3.6 +/- 0.8% of Wt-FVII activity; p364X-FVII, 26 +/- 10 ng mL(-1), 3.7+/-0.6%) characterized by reduced specific activity, thus indicating the synthesis of dysfunctional proteins. Similar results were observed with gentamicin, a commonly used aminoglycoside of potential interest for patient treatment. CONCLUSIONS: Our approach, extendable to other coagulation factors, represents an effective tool for a systematic study of the effects of aminoglycosides and neighboring sequences on nonsense codon readthrough. These results provide the rationale for a mutation-specific therapeutic approach in FVII deficiency.
PMID: 16706976 [PubMed - indexed for MEDLINE]
Thromb Haemost. 2005 Mar;93(3):481-7.
Clinical phenotypes and factor VII genotype in congenital factor VII deficiency.
Mariani G, Herrmann FH, Dolce A, Batorova A, Etro D, Peyvandi F, Wulff K, Schved JF, Auerswald G, Ingerslev J, Bernardi F; International Factor VII Deficiency Study Group.
Dipartimento di Medicina Interna e Sanità Pubblica, Università de L'Aquila, Italy.
To investigate the relationship between clinical phenotype, clotting activity (FVIIc) and FVII genotype, a multi-center study of factor VII (FVII) congenital deficiency with centralized genotyping and specific functional assays was carried out. FVII mutations characterized in patients (n=313) were extremely heterogeneous (103 different, 22 novel). Clinical phenotypes ranged from asymptomatic condition, including 15 homozygotes and 14 double heterozygotes, to patients with a severe disease characterized by life-threatening and disabling symptoms (CNS, GI bleeding and hemarthrosis) strongly associated with an early age of presentation. Based on type and number of symptoms we classified 90 'severe' (median FVIIc 1.4%, IQR [Interquartile Range] 0.9-3.8), 83 'moderate' (FVIIc 3%, IQR 1-21.7), and 140 'mild' bleeders (FVIIc 14%, IQR 3-31). The significantly different FVIIc levels, and the decreasing prevalence of homozygotes or double heterozygotes among severe (98%), moderate (84%) and mild (56%) bleeders, further support our classification. The excess of females among moderate bleeders (female/male ratio = 2.6) is attributable to menorrhagia. There was no evidence for modulation of clinical features by frequent functional polymorphisms. Homozygotes for the same mutation (Ala294Val; 11125delC) with similar FVIIc and FXa generation levels, showed striking differences in clinical phenotypes. Our study depicts the ample clinical picture of this rare disorder, proposes a severity classification and provides arguments for the early management of the disease in the severe cases. Genotype-phenotype relationships indicate the presence of major environmental and/or extragenic components modulating expressivity of FVII deficiency.
PMID: 15735798 [PubMed - in process]
Haematologica. 2004 Dec;89(12):1504-9.
Characterization of mild coagulation factor VII deficiency: activity and clearance of the Arg315Trp and Arg315Lys variants in the Cys310-Cys329 loop (c170s).
Furlan Freguia C, Toso R, Pollak ES, Arruda VR, Pinotti M, Bernardi F.
Department of Biochemistry and Molecular Biology, University of Ferrara, Italy.
BACKGROUND AND OBJECTIVES: Arginine 315 in factor VII (FVII) belongs to a solvent-exposed loop involved in direct interaction with the co-factor (tissue factor, TF), in transmission of TF-induced effects and potentially in FVIIa inactivation. Natural FVII variants at position 315 provide peculiar models for structure-function studies. DESIGN AND METHODS: We characterized a mild coagulation FVII deficiency associated with reduced FVII activity (26%) and antigen (67%). Mutations were searched by FVII gene sequencing. FVII variants were created by mutagenesis of FVII cDNA and characterized through expression in HEK293 cells followed by functional studies. FVII antigen in media was estimated by immunoassay while FVII activity was assessed by prothrombin-time based and FXa generation assays. FVII variants were injected into mice to investigate their recovery and half-life. One-way ANOVA was used to test statistical significance. RESULTS: The patient was double heterozygous for a novel R315W mutation and for the R304Q substitution (FVII Padua) previously demonstrated to impair TF binding. The recombinant 315W-FVII was normally expressed in medium but showed a markedly reduced coagulant function (52%) and activity towards factor X (FX) in plasma (34%). Moreover, the 315W-FVII showed significantly decreased recovery of the protein (20%) and a slightly shorter half-life (8.6 min) as compared to wt-FVII (50% and 10.7 min). We also studied the conservative R315K change that was responsible for low recovery (20%) and a decreased half-life (7 min) of a FVII variant with virtually normal FVII antigen and activity levels. INTERPRETATION AND CONCLUSIONS: These findings suggest a dual role of R315 for FVII function and clearance, and indicate that substitutions at this position have appreciable effects on human FVII biology, compatible with residual FVII function and thus with mild FVII deficiency.
PMID: 15590402 [PubMed - indexed for MEDLINE]
Haemophilia. 2004 Oct;10 Suppl 4:180-3.
Clinical picture and management of congenital factor VII deficiency.
Mariani G, Dolce A, Marchetti G, Bernardi F.
Department of Internal Medicine and Public Health, University of L'Aquila, L'Aquila I-6F100, Italy. gmariani@cc.univaq.it
In patients with congenital FVII deficiency, bleeding manifestations and clinical presentation vary widely, ranging from asymptomatic subjects to patients with haemorrhages that may cause important handicaps. Owing to menorrhagia, which occurs in about two-thirds of women of fertile age, bleeding is more frequent in women than in men. Gum bleeding and easy bruising are also more frequent in females. FVII:C levels are not a good predictor of bleeding tendency as there is a wide overlap between bleeders and asymptomatic patients. We propose a three-grade system of classification based on clinical considerations. Therapy for congenital FVIII bleeding is discussed, with the advantages and disadvantages of each treatment, and the suggested single dose given.
PMID: 15479395 [PubMed - indexed for MEDLINE]
Haemophilia. 2004 Oct;10 Suppl 4:177-9.
How to evaluate phenotype-genotype relationship in rare coagulation haemorrhagic disorders: examples from FVII deficiency.
Bernardi F, Marchetti G, Dolce A, Mariani G.
Department of Biochemistry and Molecular Biology, University of Ferrara, 44100 Ferrara, Italy. ber@dns.unife.it
The study of the molecular pathogenesis of several single-gene disorders, such as coagulation-factor deficiencies, has revealed the variability of phenotypic expression, even of the same mutations in single genes. These studies underline the complexity of research dealing with the definition of the molecular bases of disorders. Sequence variations provide only the starting point to define pathological genotype-phenotype relationships.
PMID: 15479394 [PubMed - indexed for MEDLINE]
J Thromb Haemost. J Thromb Haemost. 2003 Oct;1(10):2153-8.
Thrombosis in inherited factor VII deficiency.
Mariani G, Herrmann FH, Schulman S, Batorova A, Wulff K, Etro D, Dolce A, Auerswald G, Astermark J, Schved JF, Ingerslev J, Bernardi F; International Factor VII Deficiency Study Group.
Cattedra e Divisione di Ematologia, Università di Palermo, Palermo University Hospital, Via del Vespro 127, 90127 Palermo, Italy. guglielmo.mariani@tin.it
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.
PMID: 14521598 [PubMed - indexed for MEDLINE]
Mariani G, Herrmann FH, Schulman S, Batorova A, Wulff K, Etro D, Dolce A, Auerswald G, Astermark J, Schved JF, Ingerslev J, Bernardi F; International Factor VII Deficiency Study Group.
Cattedra e Divisione di Ematologia, Università di Palermo, Palermo University Hospital, Via del Vespro 127, 90127 Palermo, Italy. guglielmo.mariani@tin.it
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.
PMID: 14521598 [PubMed - indexed for MEDLINE]
Biochem J. 2003 Feb 1;369(Pt 3):563-71.
Factor VII mutant V154G models a zymogen-like form of factor VIIa.
Toso R, Bernardi F, Tidd T, Pinotti M, Camire RM, Marchetti G, High KA, Pollak ES.
Department of Biochemistry and Molecular Biology, University of Ferrara, Via Luigi Borsari, 46 Ferrara 44100, Italy. r-toso@hotmail.com
Proteolytic cleavage of the peptide bond between Arg(152) and Ile(153) converts the procoagulant protein Factor VII (FVII) to an activated two-chain form (FVIIa). The formation of a salt bridge between Ile(153) and Asp(343) drives the conversion of FVIIa from being zymogen-like to the active form. In the present paper, we describe the novel FVII mutant V154G (Val(154)-->Gly mutation; residue 17 in the chymotrypsin numbering system), found in three FVII-deficient patients, which models a zymogen-like form of FVIIa. Recombinant V154G FVIIa, although normally cleaved, shows markedly reduced activity towards peptidyl substrate and undetectable activity towards macromolecular substrates. Susceptibility of Ile(153) to chemical modification, in either the presence or the absence of tissue factor (TF), suggests that the reduced V154G FVIIa activity is caused by impaired salt-bridge formation, thus resulting in a zymogen-like FVIIa form. The TF-mediated protection from chemical modification of V154A indicated that Gly(154) is responsible for this peculiar feature, and suggests that this region, proximal to the heavy chain N-terminus, is directly involved in the conversion of FVII into FVIIa. V154G FVII was exploited to study the FVII-TF interaction, together with three additional FVII variants that were expressed to serve as models for different FVII forms. The comparison of binding affinities of full-length TF after relipidation in L-alpha-phosphatidylcholine for the zymogen FVII (Arg(152)-->Gln, K (d)=1.04+/-0.27 nM), inactive FVIIa (Ser(344)-->Ala, K (d)=0.27+/-0.06 nM) and a zymogen-like FVIIa (V154G, K (d)=1.15+/-0.16 nM) supports the hypothesis that preferential binding of TF to active FVIIa is insufficient to drive the 10(5)-fold enhancement of FVIIa activity. In addition, the inability of V154G FVIIa to accommodate an inhibitor in the active site, indicating an improperly shaped specificity pocket, would explain the low activity of the zymogen-like form of FVIIa, which is predominant in the absence of TF.
PMID: 12358603 [PubMed - indexed for MEDLINE]
Toso R, Bernardi F, Tidd T, Pinotti M, Camire RM, Marchetti G, High KA, Pollak ES.
Department of Biochemistry and Molecular Biology, University of Ferrara, Via Luigi Borsari, 46 Ferrara 44100, Italy. r-toso@hotmail.com
Proteolytic cleavage of the peptide bond between Arg(152) and Ile(153) converts the procoagulant protein Factor VII (FVII) to an activated two-chain form (FVIIa). The formation of a salt bridge between Ile(153) and Asp(343) drives the conversion of FVIIa from being zymogen-like to the active form. In the present paper, we describe the novel FVII mutant V154G (Val(154)-->Gly mutation; residue 17 in the chymotrypsin numbering system), found in three FVII-deficient patients, which models a zymogen-like form of FVIIa. Recombinant V154G FVIIa, although normally cleaved, shows markedly reduced activity towards peptidyl substrate and undetectable activity towards macromolecular substrates. Susceptibility of Ile(153) to chemical modification, in either the presence or the absence of tissue factor (TF), suggests that the reduced V154G FVIIa activity is caused by impaired salt-bridge formation, thus resulting in a zymogen-like FVIIa form. The TF-mediated protection from chemical modification of V154A indicated that Gly(154) is responsible for this peculiar feature, and suggests that this region, proximal to the heavy chain N-terminus, is directly involved in the conversion of FVII into FVIIa. V154G FVII was exploited to study the FVII-TF interaction, together with three additional FVII variants that were expressed to serve as models for different FVII forms. The comparison of binding affinities of full-length TF after relipidation in L-alpha-phosphatidylcholine for the zymogen FVII (Arg(152)-->Gln, K (d)=1.04+/-0.27 nM), inactive FVIIa (Ser(344)-->Ala, K (d)=0.27+/-0.06 nM) and a zymogen-like FVIIa (V154G, K (d)=1.15+/-0.16 nM) supports the hypothesis that preferential binding of TF to active FVIIa is insufficient to drive the 10(5)-fold enhancement of FVIIa activity. In addition, the inability of V154G FVIIa to accommodate an inhibitor in the active site, indicating an improperly shaped specificity pocket, would explain the low activity of the zymogen-like form of FVIIa, which is predominant in the absence of TF.
PMID: 12358603 [PubMed - indexed for MEDLINE]
PMCID: PMC1223097
Thromb Haemost. 2002 Nov;88(5):763-7.
Polymorphic changes in the 5' flanking region of factor VII have a combined effect on promoter strength.
Kudaravalli R, Tidd T, Pinotti M, Ratti A, Santacroce R, Margaglione M, Dallapiccola B, Bernardi F, Fortina P, Devoto M, Pollak ES.
The Children's Hospital of Philadelphia, PA, USA.
Polymorphic differences in the 5' flanking region of the gene encoding procoagulant protein Factor VII (FVII) are associated with variations in FVII coagulant activity (FVII:C) and FVII antigen (FVII:Ag) levels. A decanucleotide insert polymorphism (CCTATATCCT) at 323 bp upstream of the start site of translation correlates with a decrease of approximately 20% FVII: C levels per allele containing this insert. However, linkage disequilibrium of the decanucleotide polymorphism with two single nucleotide polymorphisms (SNPs) at -122 and -401 have made it difficult to pinpoint the functional role, if any, of these genetic changes in lowering FVII levels. In vitro reporter gene studies in HepG2 cells analyzing the 8 possible combinations of polymorphic sites at -401, -323, and -122 reveal the necessity of the presence of the three concurrent polymorphic changes to maximally decrease promoter strength. In addition, these in vitro results are supported by in vivo studies in 89 individuals of African heritage, 34% of whom display a new haplotype that shows the polymorphic changes at -323 and -401 but lacks the change at -122.
PMID: 12428091 [PubMed - indexed for MEDLINE]
Biochem J. 2002 Apr 15;363(Pt 2):411-6.
A frequent human coagulation Factor VII mutation (A294V, c152) in loop 140s affects the interaction with activators, tissue factor and substrates.
Toso R, Pinotti M, High KA, Pollak ES, Bernardi F.
Department of Biochemistry and Molecular Biology, University of Ferrara, Via Borsari, 46 Ferrara 44100, Italy. r_toso@hotmail.com
Activated Factor VII (FVIIa) is a vitamin-K-dependent serine protease that initiates blood clotting after interacting with its cofactor tissue factor (TF). The complex FVIIa-TF is responsible for the activation of Factor IX (FIX) and Factor X (FX), leading ultimately to the formation of a stable fibrin clot. Activated FX (FXa), a product of FVIIa enzymic activity, is also the most efficient activator of zymogen FVII. Interactions of FVII/FVIIa with its activators, cofactor and substrates have been investigated extensively to define contact regions and residues involved in the formation of the complexes. Site-directed mutagenesis and inhibition assays led to the identification of sites removed from the FVIIa active site that influence binding specificity and affinity of the enzyme. In this study we report the characterization of a frequent naturally occurring human FVII mutant, A294V (residue 152 in the chymotrypsin numbering system), located in loop 140s. This region undergoes major rearrangements after FVII activation and is relevant to the development of substrate specificity. FVII A294V shows delayed activation by FXa as well as reduced activity towards peptidyl and macromolecular substrates without impairing the catalytic efficiency of the triad. Also, the interaction of this FVII variant with TF was altered, suggesting that this residue, and more likely loop 140s, plays a pivotal role not only in the recognition of FX by the FVIIa-TF complex, but also in the interaction of FVII with both its activators and cofactor TF.
PMID: 11931672 [PubMed - indexed for MEDLINE]
PMCID: PMC1222493
Blood. 2002 Feb 15;99(4):1495-7.
Residual factor VII activity and different hemorrhagic phenotypes in CRM(+) factor VII deficiencies (Gly331Ser and Gly283Ser).
Pinotti M, Etro D, Bindini D, Papa ML, Rodorigo G, Rocino A, Mariani G, Ciavarella N, Bernardi F.
Dipartimento di Biochimica e Biologia Molecolare-CIBF, University of Ferrara, Italy.
Two cross-reacting material-positive (CRM(+)) factor VII (FVII) mutations, associated with similar reductions in coagulant activity (2.5%) but with mild to asymptomatic (Gly331Ser, c184 [in chymotrypsin numbering]) or severe (Gly283Ser, c140) hemorrhagic phenotypes, were investigated. The affected glycines belong to structurally conserved regions in the c184 through c193 and c140s activation domain loops, respectively. The natural mutants 331Ser-FVII and 283Ser-FVII were expressed, and in addition 331Ala-FVII and 283Ala-FVII were expressed because 3 functional serine-proteases bear alanine at these positions. The 331Ser-FVII, present in several asymptomatic subjects, showed detectable factor Xa generation activity in patient plasma (0.7% +/- 0.2%) and in reconstituted system with the recombinant molecules (2.7% +/- 1.1%). The reduced activity of recombinant 283Ala-FVII (7.2% +/- 2.2%) indicates that the full function of FVII requires glycine at this position, and the undetectable activity of 283Ser-FVII suggests that the oxydrile group of Ser283 participates in causing severe CRM(+) deficiency. Furthermore, in a plasma system with limiting thromboplastin concentration, 283Ser-FVII inhibited wild-type FVIIa activity in a dose-dependent manner.
PMID: 11830508 [PubMed - indexed for MEDLINE]
Blood. 2000 Jun 1;95(11):3423-8.
Modulation of factor VII levels by intron 7 polymorphisms: population and in vitro studies.
Pinotti M, Toso R, Girelli D, Bindini D, Ferraresi P, Papa ML, Corrocher R, Marchetti G, Bernardi F.
Dipartimento di Biochimica e Biologia Molecolare-CIBF, Università di Ferrara, Ferrara, Italy.
Previous studies have established that factor VII gene (F7) polymorphisms (5'F7 and R353Q) contribute about one-third of factor VII (FVII) level variation in plasma. However, F7 genotyping in patients with cardiovascular disease has produced conflicting results. Population and expression studies were used to investigate the role of intron 7 (IVS7 ) polymorphisms, including repeat and sequence variations, in controlling activated FVII (FVIIa) and antigen (FVIIag) levels. Genotype-phenotype studies performed in 438 Italian subjects suggested a positive relation between the IVS7 repeat number and FVII levels. The lowest values were associated with the IVS7 + 7G allele. The screening of 52 patients with mild FVII deficiency showed an 8-fold increase in frequency (8%) of this allele, and among heterozygotes for identical mutations, lower FVII levels were observed in the IVS7 + 7G carriers. This frequent genetic component participates in the phenotypic heterogeneity of FVII deficiency. The evaluation of the individual contribution of polymorphisms was assisted by the expression of each IVS7 variant, as a minigene, in eukaryotic cells. The novel quantitative analysis revealed that higher numbers of repeats were associated with higher mRNA expression levels and that the IVS7 + 7G allele, previously defined as a functionally silent polymorphism, was responsible for the lowest relative mRNA expression. Taken together, these findings indicate that the IVS7 polymorphisms contribute to the plasmatic variance of FVII levels via differential efficiency of mRNA splicing. These studies provide further elements to understand the control of FVII levels, which could be of importance to ensure the hemostatic balance under pathologic conditions.
PMID: 10828024 [PubMed - indexed for MEDLINE]
Arterioscler Thromb Vasc Biol. 1999 Aug;19(8):2024-8.
Oral contraceptives highlight the genotype-specific association between serum phospholipids and activated factor VII.
Mariani G, Conard J, Bernardi F, Bertina R, Garcia VV, Prydz H, Samama M, Sandset PM, Puopolo M, Ciarla MV, Poso R, Di Nucci GD, Ceci F, Marchetti G.
Hematology and Bone Marrow Transplantation Unit, University Hospital, Palermo, Italy. mariangu@tin.it
The present analysis was undertaken to study the effect of oral contraceptive (OC) use on activated factor VII (FVIIa) in subjects characterized by FVII genotypes, with the further aim of evaluating the role of lipids in this pharmacological interaction. In OC users (n=42) and nonusers (n=130) of comparable age, we examined the FVII phenotypic variables (FVII coagulant activity [FVIIc], FVII antigen, and FVIIa), FVII genotypes (the 353R/Q and 5'F7 polymorphisms analyzed in combination; alleles M1/M2 and A1/A2, respectively), and a number of lipid and lipoprotein parameters: serum concentrations of total cholesterol (chol), low density lipoprotein and high density lipoprotein-chol, triglycerides, phospholipids (PhLs), apolipoprotein A1, and lipoprotein(a). PhLs, triglycerides, apolipoprotein A1, chol, FVII antigen, FVIIc, and high density lipoprotein-chol levels were shown to be statistically higher in users than nonusers. FVII levels, particularly those of FVIIa and FVIIc, were much higher in homozygotes for the A1 and M1 alleles (A11 M11), especially in OC users. A strong association was found between PhL and FVIIa: in the multiple regression analysis, women taking OCs who had elevated PhL concentrations also had very high levels of FVIIa, but only if their genotype was A11 M11. These results indicate that the increased FVII levels in OC users depend on the FVII genotype and that high PhL concentrations predict very high levels of FVIIa and FVIIc.
PMID: 10446088 [PubMed - indexed for MEDLINE]
Haematologica. 1999 Jul;84(7):620-6.
Serum phospholipids are the main environmental determinants of activated factor VII in the most common FVII genotype. European Union Concerted Action "Clotart".
Mariani G, Bernardi F, Bertina R, Vicente VV, Prydz H, Samama M, Sandset PM, Di Nucci GD, Testa MG, Bendz B, Chiarotti F, Ciarla MV, Strom R.
Hematology and BMT Unit, University Hospital, via del Vespro 129, 90127 Palermo, Italy. mariangu@tin.it
BACKGROUND AND OBJECTIVE: Numerous studies have emphasized the role of triglyceride-rich lipoproteins and of Factor VII (FVII) polymorphisms in determining levels of FVII activity. DESIGN AND METHODS: This study was undertaken to evaluate the role of other lipid fractions and the interaction between lipids and FVII in subjects with recognised genotypes. Volunteer subjects (n=459) from 5 European countries were studied. Blood samples were drawn irrespective of the time of day or fasting status. Levels of FVII activity (FVIIc), activated FVII (FVIIa) and FVII antigen (FVIIAg) were evaluated with reference to a number of lipid parameters (HDL-, LDL- and total cholesterol, triglycerides, phospholipids, lipoprotein(a), and apoliproptein A1). The two most common FVII polymorphisms were analyzed in combination (353R/Q and 5'F7; alleles M1/M2 and A1/A2, respectively). RESULTS: Homozygotes for the A1 and M1 alleles (M11/A11) had significantly higher FVII levels. At multiple regression analysis the strongest predictor of FVIIa and FVIIc was the concentration of phospholipids. This interaction was confined to the A11M11 genotype subjects. INTERPRETATION AND CONCLUSIONS: These data indicate that lipids contribute mainly to FVIIa levels through their phospholipid content, and that the degree of this contribution is strictly dependent on FVII genotypes.
PMID: 10406904 [PubMed - indexed for MEDLINE]
Blood. 1998 Sep 1;92(5):1646-51.
Molecular mechanisms of FVII deficiency: expression of mutations clustered in the IVS7 donor splice site of factor VII gene.
Pinotti M, Toso R, Redaelli R, Berrettini M, Marchetti G, Bernardi F.
Dipartimento di Biochimica e Biologia Molecolare - CIBF, Sezione SBPGU, Università di Ferrara, Ferrara; the Divisione di Ematologia, Ospedale Niguarda, Milano, Italy.
In three Italian patients, two point mutations and a short deletion were found in the intron 7 of factor VII gene, clustered in the donor splice site and located in the first of several repeats. The mutation 9726+5G-->A, the most frequent cause of symptomatic factor VII deficiency in Italy, as well as the deletion (9729del4) gave rise in expression studies to abnormally spliced transcripts, which were exclusively produced from the cryptic site in the second repeat. The insertion in the mature mRNA of the first intronic repeat caused (9726+5G-->A) a reading frameshift, abolishing most of the factor VII catalytic domain, or produced (9729del4), an altered factor with 11 additional residues, the activity of which was not detectable in the cell medium after mutagenesis and expression studies. Studies of factor VII ectopic mRNA from leukocytes and expression studies indicated that the deleted gene produced 30% of normally spliced transcript. Differently, the 9726+5G-->A mutation permitted a very low level (0.2% to 1%) of correct splicing to occur, which could be of great importance to prevent the onset, in the homozygous patients, of most of the life-threatening bleeding symptoms. The 9726+7A-->G mutation was found to be a rare and functionally silent polymorphism. These findings, which provide further evidence of the interplay of sequence and position in the 5' splice site selection, throw light on the heterogeneous molecular bases and clinical phenotypes of FVII deficiency. Copyright 1998 by The American Society of Hematology.
PMID: 9716592 [PubMed - indexed for MEDLINE]
Blood Coagul Fibrinolysis. 1998 Mar;9 Suppl 1:S83-8.
Molecular and clinical aspects of factor VII deficiency.
Mariani G, Lo Coco L, Bernardi F, Pinotti M.
Haematology and Bone Marrow Transplantation Unit, University of Palermo Medical School, Italy. mariangu@tin.it
Factor VII (FVII) plays an important role in the initiation of blood coagulation, forming a complex with tissue factor (TF) which activates FIX and FX and FVII zymogen. FVII deficiency displays considerable phenotypic and molecular heterogeneity and there are inconsistencies between the clinical picture observed and the underlying clotting and molecular defects. We have reviewed the data available in the literature on FVII-deficient patients. Clinically, cases range from asymptomatic to patients with severe haemorrhagic tendencies. Asymptomatic patients typically have FVII activity levels of >20% and are heterozygotes, double heterozygotes or homozygotes. Mild FVII-deficient patients, with FVII activity levels >2%, may be double heterozygotes or homozygotes for FVII gene missense mutations. Undetectable FVII levels in severely affected patients are often due to severe gene defects such as frameshifts or mutations affecting the splice sites. The analysis of structure-function relationships in FVII deficiency is difficult due to the complexity of the interactions involving FVII. Also, assays using different reagents may give different results with a given plasma sample, and are not very accurate at low levels of FVII which, although relatively low, may be clinically significant, adding complexity to the analysis of FVII deficiency. The sensitivity of our methods for phenotypic evaluation of FVII deficiency remains inadequate.
PMID: 9819034 [PubMed - indexed for MEDLINE]
Arterioscler Thromb Vasc Biol. 1997 Nov;17(11):2548-53.
Contribution of factor VII genotype to activated FVII levels. Differences in genotype frequencies between northern and southern European populations.
Bernardi F, Arcieri P, Bertina RM, Chiarotti F, Corral J, Pinotti M, Prydz H, Samama M, Sandset PM, Strom R, Garcia VV, Mariani G.
Dipartimento di Biochimica e Biologia Molecolare, Università degli Studi di Ferrara, Italy. Ber@dns.unife.it
The relationship between coagulation factor VII (FVII) levels in plasma and FVII genotypes, determined by three polymorphisms (5'F7, IVS7, and 353R/Q), were studied in 500 control subjects enrolled in European multicenter study. The selection of particular FVII genotypes and the analysis of variance clearly indicated the independent contribution of a single 5'F7 insertion (A2) or 353Q (M2) allele to lowering plasma levels of activated FVII (FVIIa) (by a mean 25%). The M2 allele alone was found to make a major contribution to the genetically determined component of the FVIIa levels. Genotypes associated with low FVII levels were significantly rarer in the northern part of Europe (Oslo) than in the southern part (Rome, Murcia). The contribution made by the FVII genotype to the total variance of FVIIa levels was higher (30%) than that made to either FVII activity (25%) or FVII antigen (12%). Subjects with different FVII genotypes showed up to fivefold differences in mean FVIIa values, thus allowing attribution of a substantial part of the considerable interindividual variation to genetic variation, which may be of assistance in the interpretation of FVIIa levels on an individual basis. When FVII levels were adjusted by age and by triglyceride levels, the contribution of FVII genotypes to the FVII phenotypic variance was virtually unchanged. Taken together, these data indicate that in healthy control subjects the FVII genotype is a major predictor of plasma FVIIa levels and would support further study on the role of FVII genetic components in the development of cardiovascular disease.
PMID: 9409226 [PubMed - indexed for MEDLINE]
Blood Coagul Fibrinolysis. 1996 Mar;7(2):114-7.
Plasma factor VII levels are influenced by a polymorphism in the promoter region of the FVII gene.
Sacchi E, Tagliabue L, Scoglio R, Baroncini C, Coppola R, Bernardi F, Mannucci PM.
Angelo Bianchi Bonomi Hemophilia and Thrombosis Center, IRCCS Maggiore Hospital, Milan, Italy.
Genetic factors play a role in determining the variability of plasma factor VII (FVII) levels in healthy individuals. There is also evidence that high serum lipids are associated with high FVII levels in plasma. In the promoter region of the human FVII a DNA polymorphism has been described, originating from a decanucleotide insert present in the less frequent allele. This biallelic system, reflecting the absence (AA) or presence (Aa) of the decanucleotide, can be detected by a DNA enzyme immunoassay of PCR products. We evaluated the association between the polymorphic alleles and the levels of FVII:Ag and FVII:C in 100 healthy individuals and in 19 hypertriglyceridemic individuals. Among healthy individuals, mean FVII:Ag and FVII:C levels of those with the homozygous genotype (A/A; mean FVII:Ag 112%, mean FVII:C 109%) were significantly higher (P < 0.001) than the mean levels of those with the heterozygous genotype (A/a, mean FVII:Ag 80%, mean FVII:C 90%; P < 0.001). Similar genotype-associated differences for FVII:Ag and FVII:C were found in individuals with triglycerides above 250 mg/dl (P < 0.05). FVII:C and FVII:Ag levels were positively related to triglycerides only in individuals without the insert (P < 0.01); there was no significant relationship in those carrying the allele with the insert (A/a; P = 0.43 and 0.08). Our findings of genotype-associated differences in FVII levels and interactions with triglycerides are similar to those obtained with the amino acid dimorphism at position 353 of the factor VII protein.
PMID: 8735799 [PubMed - indexed for MEDLINE]
Arterioscler Thromb Vasc Biol. 1996 Jan;16(1):72-6.
Factor VII gene polymorphisms contribute about one third of the factor VII level variation in plasma.
Bernardi F, Marchetti G, Pinotti M, Arcieri P, Baroncini C, Papacchini M, Zepponi E, Ursicino N, Chiarotti F, Mariani G.
Dipartimento di Biochimica e Biologia Moleculare, Università di Ferrara, Italia.
To assess the role of genetic variation in determining factor VII (FVII) activity and antigen levels we studied a polymorphism located in the 5' region of the gene (5'F7), an intronic mutation (IVS7), and the 353Arg-Gln polymorphism. All the polymorphisms, which showed strong allelic association, analyzed separately or in combination by the one-way analysis of variance, were associated with significantly different FVII levels. The 5'F7 and 353Arg-Gln polymorphic systems, which have very similar allele frequencies, contributed to a similar extent to the total phenotypic variance, whereas the contribution of the IVS7 polymorphism was lower. Genetic variation at the FVII locus, evaluated on combined genotypes, accounted for up to 40% of the phenotype FVII variance. As also shown by the two-way analysis of variance, the use of two out of three markers is advisable, and since the 5'F7 polymorphism can be screened by a simple immunoassay, it should be preferred for population-based studies. No substantial differences between FVII activity and FVII antigen levels were found, thus suggesting that the variation was due to biosynthesis- or stability-mediated mechanisms. The genetic control of FVII levels described in this study plays an important role in determining plasma FVII level variability, which may influence the hemostatic balance.
PMID: 8548429 [PubMed - indexed for MEDLINE]
Hum Mutat. 1996;8(2):108-15.
Mutation pattern in clinically asymptomatic coagulation factor VII deficiency.
Bernardi F, Castaman G, Pinotti M, Ferraresi P, Di Iasio MG, Lunghi B, Rodeghiero F, Marchetti G.
Dipartimento di Biochimica e Biologia Molecolare, Universitá di Ferrara, Italy.
A total of 122 subjects, referred after presurgery screening or checkup for prolonged prothrombin time, were characterized for the presence of coagulation factor VII deficiency. Fourteen subjects carried a partial and asymptomatic deficiency, and in half of them dysfunctional molecules were detected in plasma. In nine subjects we found five missense mutations differing from those previously found in factor VII deficient patients. The others were homozygous for a common polymorphism (R353Q) that affects factor VII levels. A new codon dimorphism (A330) was also found in exon 8. Four mutations (R223W, M298I, R304Q, and R353Q) located at FVII-specific residues point out protein regions that are important for coagulation factor evolution, and two mutations (G342E and E265K) affect generic or partially generic residues. The newly reported mutations were combined with those we previously found, totalling 17 independent mutations responsible for FVII deficiency in 27 Italian pedigrees. We observed several similarities with the mutation pattern determined in factor IX, which include a high percentage of transitions at CpG doublets, the presence of hot spot sites affected by multiple substitutions, and of several topologically equivalent mutations.
PMID: 8844208 [PubMed - indexed for MEDLINE]
Br J Haematol. 1994 Mar;86(3):610-8.
Molecular defects in CRM+ factor VII deficiencies: modelling of missense mutations in the catalytic domain of FVII.
Bernardi F, Liney DL, Patracchini P, Gemmati D, Legnani C, Arcieri P, Pinotti M, Redaelli R, Ballerini G, Pemberton S, et al.
Centro Studi Biochimici delle Patologie del Genoma Umano-Istituto di Chimica Biologica, Ferrara, Italy.
The molecular defects causing CRM+ factor VII deficiency were investigated in seven unrelated subjects and several members of their families. Four missense mutations located in the catalytic domain of factor VII were found. The previously reported 304Arg-->Gln substitution was present in the homozygous and heterozygous forms, with different polymorphic haplotypes, thus demonstrating that it is recurrent and frequent in the Italian population. The 310Cys-->Phe substitution was found in the homozygous form and in the compound heterozygous condition with the nonsense mutation 356Trp-->stop. Two missense mutations, 298Met-->Ile and 342Gly-->Arg, were found in the homozygous and in the heterozygous condition respectively. Molecular heterogeneity was further increased by finding of the 353Arg-->Gln polymorphism in the doubly heterozygous condition with the 304 and 342 mutations. Plausible explanations for loss of FVII function were found by inspecting a model of the serine protease domain of factor VIIa. Inefficient activation of the catalytic site is predicted for 298Met-->Ile. 342Gly-->Arg would directly distort the geometry of the 'oxyanion hole' preventing formation of a substrate enzyme intermediate. 310Cys-->Phe is predicted to have an adverse effect on tissue factor interaction. These mutations point to important regions of the factor VII molecule.
PMID: 8043443 [PubMed - indexed for MEDLINE]
Hum Genet. 1993 Nov;92(5):446-50.
Molecular analysis of factor VII deficiency in Italy: a frequent mutation (FVII Lazio) in a repeated intronic region.
Bernardi F, Patracchini P, Gemmati D, Ferrati M, Arcieri P, Papacchini M, Redaelli R, Baudo F, Mariani G, Marchetti G.
Centro Studi Biochimici delle Patologie del Genoma Umano, Istituto di Chimica Biologica, Università di Ferrara, Italy.
Molecular defects and polymorphic haplotypes of coagulation factor VII gene were studied in eight unrelated Italian subjects with factor VII deficiency, seven having the factor VII- variant, one the factor VIIR variant. An intron 7 mutation, which alters the consensus donor splice site sequence, was found in six subjects. The presence of the founder effect is suggested by their common geographical origin (a mountain area in the Lazio region) and by the identical polymorphic haplotype underlying the mutation. A different mutation, also located in the 5' monomer of the repeated intron 7 sequence, was found in the heterozygous condition in a subject from Northern Italy. New polymorphic alleles were detected in the repeated intron 7 region in subjects from Eastern Africa. Two missense mutations in codon 97 (Gly-->Cys, Gly-->Ser), the first found in the compound heterozygous condition with the frequent intron 7 mutation, suggest the presence of a hot spot mutation site in the second epidermal growth factor domain. Two neutral dimorphisms at codon 333Ser and 115His were detected, the last in linkage disequilibrium with the 353Arg/Gln polymorphism, and showing differences in frequency in the FVII deficient and control subjects.
PMID: 8244334 [PubMed - indexed for MEDLINE]
Hum Genet. 1993 Jan;90(5):575-6.
A polymorphism in the 5' region of coagulation factor VII gene (F7) caused by an inserted decanucleotide.
Marchetti G, Patracchini P, Papacchini M, Ferrati M, Bernardi F.
Centro di Studi Biochimici delle Patologie del Genoma Umano, Università di Ferrara, Italy.
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.
PMID: 8381388 [PubMed - indexed for MEDLINE]
Hum Genet. 1992 Jul;89(5):497-502.
Detection of two missense mutations and characterization of a repeat polymorphism in the factor VII gene (F7).
Marchetti G, Patracchini P, Gemmati D, DeRosa V, Pinotti M, Rodorigo G, Casonato A, Girolami A, Bernardi F.
Centro Studi Biochimici delle Patologie del Genoma Umano, Instituto di Chimica Biologica, Ferrara, Italy.
The 3' portion of the coagulation factor VII gene, containing the activation and serine protease domains, was investigated in four subjects with factor VII deficiency by temperature gradient gel electrophoresis and sequencing of polymerase chain reaction (PCR) products. Molecules displaying an altered melting behaviour were detected in three subjects, and direct sequencing showed two mutations. A G-to-T transversion causing a missense mutation, Cys-310 to Phe, suppresses a disulphide bond conserved in the catalytic domain of all serine proteases. This mutation, which in the homozygous form causes a severe reduction in protease activity (4%), was found in two patients from different Italian regions. A G-to-A transition, which gives rise to a missense mutation, Arg-304 to Gln, and is associated with the factor VII padua variant, was found in the heterozygous form in a subject also affected by von Willebrand disease. Two polymorphic alleles, which differ in one repeat monomer element, were precisely mapped in a region spanning the exon-intron 7 border of the factor VII gene and studied in families with factor VII or X deficiency.
PMID: 1634227 [PubMed - indexed for MEDLINE]
Major differences in bleeding symptoms between factor VII deficiency and haemophilia B.
Bernardi F, Dolce A, Pinotti M, Shapiro AD, Santagostino E, Peyvandi F, Batorova A, Lapecorella M, Schved JF, Ingerslev J, Mariani G; for the International Factor VII Deficiency Study Group.
Department of Biochemistry and Molecular Biology, University of Ferrara, Ferrara, Italy.
Summary Background: The autosomally-inherited factor VII (FVII) deficiency and the X-linked haemophilia B offer an attractive model to investigate whether reduced levels of FVII and factor IX (FIX), acting in the initiation and amplification of coagulation respectively, influence haemostasis to a different extent in relation to age and bleeding site. Methods: Haemophilia B patients (n=296) and FVII deficient males (n=109) were compared for FVII/FIX clotting activity, F7/F9 genotypes and clinical phenotypes in a retrospective, multi-centre, cohort study. Results: Major clinical differences between diseases were observed. Bleeding occurred earlier in haemophilia B (median age 2.0 years, IR 0.9-5.0) than in FVII deficiency (5.2 years, IR 1.9-15.5) and the bleeding-free survival in FVII deficiency was similar to that observed in "mild" haemophilia B (p= 0.96). The most frequent disease-presenting symptoms in haemophilia B (haematomas and oral bleeding) differed from those in FVII deficiency (epistaxis and central nervous system bleeding). Differences were confirmed by analysis of FVII deficient women. Conclusions: Our data support the notion that low FVII levels sustain haemostasis better than similarly reduced FIX levels. On the other hand, minute amounts of FVII, differently from FIX, are needed to prevent fatal bleeding, as indicated by the rarity of null mutations and the associated life-threatening symptoms in FVII deficiency, which contributes to shape clinical differences between diseases in the lowest factor level range. Differences between diseases are only partially explained by mutational patterns and could pertain to the specific roles of FVII and FIX in coagulation phases and to vascular bed-specific components.
PMID: 19245420 [PubMed - as supplied by publisher]
