Giovanna Marchetti

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.

Risks factors for highly unstable response to oral anticoagulation: a case-control study

The factors associated with persistent instability of oral anticoagulant treatment (OAT) were investigated in a case‐control study. The most unstable patients from 35 Italian anticoagulation clinics were matched with stable controls, for gender, age and OAT indication. Socio‐demographic data, medical history, dietary and life habits, cytochrome P450 CYP2C9 variants, blood cell count, liver and renal functions were investigated. An ‘Abbreviated Mental Test’ (AMT) and a questionnaire to assess patient compliance to, and comprehension of, OAT indications and mechanisms were administered. An International Normalized Ratio (INR) above 4·5 was more frequently found in cases (n = 77) than controls (n = 80) (12·3% vs. 0·4%; P < 0·0001). The odds ratio for instability was significantly higher for: people who worked versus pensioners, acenocoumarol versus warfarin, and an insufficient score in the AMT and/or in the questionnaire. Cytochrome P450 CYP2C9 variants *1/*3 or *2/*3 or *3/*3 were more frequent among cases than controls (29·9% vs.15·0%; P = 0·042). No differences were observed as regards the other variables. In conclusion, we found that high intra‐individual variability in OAT control was multifactorial, but poor OAT comprehension was prevalent.

Detection of two missense mutations and characterization of a repeat polymorphism in the factor VII gene (F7)

SummaryThe 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.

Polymorphisms at LDLR locus may be associated with coronary artery disease through modulation of coagulation factor VIII activity and independently from lipid profile.

High levels of coagulation factor VIII (FVIII) have been associated with cardiovascular disease. Low-density lipoprotein receptor (LDLR) has been recently demonstrated to contribute to FVIII clearance from plasma. The aim of this study was to evaluate 3 single nucleotide polymorphisms in SMARCA4-LDLR gene locus (rs1122608, rs2228671, and rs688) and FVIII coagulant activity (FVIII:c) in subjects with (n = 692) or without (n = 291) angiographically confirmed coronary artery disease (CAD). High FVIII:c levels were an independent risk factor for CAD. The rs688 and rs2228671 genotypes were predictors of FVIII:c with T alleles associated with higher FVIII:c levels. The rs2228671T allele was associated also with reduced total and LDL-cholesterol levels. With respect to the risk of CAD, no association was found for rs2228671. Consistently with higher FVIII:c levels, the rs688T allele was associated with CAD, whereas, consistently with a favorable lipid profile, the rs1122608T allele was associated with a decreased CAD prevalence. After adjustment for classic cardiovascular risk factors, including plasma lipids, rs688 remained associated with CAD (OR for T carriers: 1.67 with 95% confidence interval, 1.10-2.54). Haplotype analysis confirmed such results. Our data suggest that polymorphisms at LDLR locus modulate FVIII:c levels and may be associated with CAD risk independently from plasma lipids.

Molecular defects in CRM+ factor VII deficiencies: modelling of missense mutations in the catalytic domain of FVII

Summary. The molecular defects causing CRM+ factor VII deficiency were investigated in seven unrelated subjects and several members of their families.

Molecular analysis of factor VII deficiency in Italy: a frequent mutation (FVII Lazio) in a repeated intronic region

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.

Intracellular readthrough of nonsense mutations by aminoglycosides in coagulation factor VII

Summary.  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 (∼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.

Clinical picture and management of congenital factor VII deficiency.

Summary.  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.

Sublocalization of von Willebrand factor pseudogene to 22q11.22-q11.23 by in situ hybridization in a 46,X,t(X;22)(pter;q11.21) translocation

SummaryThe von Willebrand factor pseudogene, previously mapped to chromosome 22, was sublocalized by in situ hybridization using as probe a von Willebrand factor cDNA fragment completely contained in the pseudogenic region. Chromosome spreads were from a patient carrying a unique balanced de novo translocation 46,X,t(X;22)(pter;q11.21). Silver grain analysis indicated that the human von Willebrand factor pseudogene is located on 22q11.22–q11.23, a region relevant for several somatic and constitutional chromosomal alterations.

Von Willebrand disease investigated by two novel RFLPs

Two partial cDNAs for von Willebrand factor (vWF) were used to investigate gene lesions and restriction fragment length polymorphisms (RFLPs) in vW disease (vWd) and normal controls. No gene alteration was detected but two TaqI RFLPs, likely to be intronic and originating from point mutations, were found in the 3’ part of vWF gene. The two TaqI RFLPs, identified by the same probe, are informative in approximately 50% of the subjects. Used in combination with two other known RFLPs, they define several haplotypes similarly distributed in vWd and normals. Linkage disequilibrium between loci identified by the RFLPs is present.