A novel polymorphism in intron 1a of the human factor VII gene (G73A): study of a healthy Italian population and of 190 young survivors of myocardial infarction.

We have identified a novel polymorphism located in intron 1a of the human factor VII gene, caused by the nucleotide change G to A at position + 73. In a population of 128 healthy individuals from northern Italy, the variant A73 allele had a frequency of 0.21, whereas the frequency of the previously reported 10 bp insertion allele located at -323 in the promoter region was 0.

Pharmacokinetics of recombinant factor VIII (recombinate) using one-stage clotting and chromogenic factor VIII assay.

In a study designed to demonstrate the safety and pharmacokinetics of a recombinant factor VIII (Recombinate) manufactured in Andover, MA and Thousand Oaks, CA, two different methods of factor VIII assay (one-stage clotting and Chromogenic substrate) were compared in vivo. The study was performed in four centres in the UK: London, Oxford, Cardiff and Manchester. Two pharmacokinetic studies, at least one week apart, were performed in 30 patients with severe haemophilia A (VIII:C < 2 IU/dl).

Audit of clinical management of von Willebrand disease during 1997 at a single institution and review of treatment patterns between 1980 and 1997.

In 1997 the UK Haemophilia Centre Directors Organization published the guidelines for diagnosis and management of von Willebrand disease (vWD). The guidelines stated that desmopressin (DDAVP) should be used in type 1 and type 2N vWD, that it might be useful in other types 2 vWD and that it is ineffective in type 3 vWD. If patients are unresponsive to DDAVP or if it is contraindicated, the treatment of choice is clotting factor concentrates (CFC).

Acquired von Willebrand syndrome--report of 10 cases and review of the literature.

Acquired von Willebrand syndrome (AvWS) is a rare bleeding disorder with clinical and laboratory features closely resembling hereditary von Willebrand disease (vWD), arising in previously haemostatically normal individuals. We present a retrospective review of 10 cases with AvWS diagnosed over 17 years. The severity of the bleeding tendency varied from mild to severe forms.

Women and inherited bleeding disorders: menstrual issues.

Von Willebrand, who described a bleeder family from Aland in 1926 in whom the index patient died at the fourth menses, stated, "the trait seems to be seen among women." Menorrhagia is defined objectively as a menstrual blood loss of at least 80 mL. However, even though 5% of women aged 30 to 49 years consult their general practitioner and 12% of gynecologic referrals are for menorrhagia, the diagnosis is difficult.

Women and von Willebrand disease.

In 1926 von Willebrand made the observation that 'the trait seemed especially to be seen among women'. A pictorial bleeding assessment chart (PBAC) defines menorrhagia with a score of > 100 representing > 80 ml blood loss. A retrospective study has shown menorrhagia in 74% patients with von Willebrand disease (vWD) and vWD in 13% patients with menorrhagia.

Multiplex PCR for Detection of the Prothrombin 3'-UTR (G20210A) Polymorphism and the Factor V Leiden Mutation.

Prothrombotic evaluation of patients with a history-and in particular a family history-of venous thromboembolic disease is becoming increasingly important as our understanding of the molecular abnormalities that underlie this clinical disorder increases. A recently described G→A polymorphism at position 20210 in the 3'-untranslated region of the prothrombin gene (F2 3'-UTR) has been found to be associated with an increased risk of venous thrombotic disease. In the Leiden Thrombophilia Study (LETS), the prevalence of carriers of the 20210 A allele in the healthy population was 2.

Ectopic transcript analysis in human antithrombin deficiency.

A number of reports have demonstrated that it is possible to identify correctly spliced low-level transcripts for tissue-specific genes in a number of non-specific tissues (1-3). The number of transcripts is low (approx 1 copy every 500-1000 cells) (2), but as they are initiated at the normal mRNA start site this suggests that the normal promoters are used. Although ectopic transcript analysis has been used primarily in the study of large and complex genes, e.

Mutational analysis in antithrombin deficiency.

Human antithrombin is a single-chain glycoprotein of MW 58 kDa and the most important plasma inhibitor of the coagulation serine proteases. It is a member of the serine protease inhibitor (SERPIN) family of proteins and in common with several other members of this family, its inhibitory activity is increased many thousand-fold in the presence of heparin and other sulphated glycosaminoglycans. Type I antithrombin deficiency, i.

Screening for Candidate Mutations Causing von Willebrand's Disease (vWD).

von Willebrand factor (vWF) is a large, complex glycoprotein that exists in plasma and platelets, and is synthesized by megakaryocytes and endothelial cells. vWF plays an essential role in hemostasis in at least two ways. It is involved in platelet adhesion to the damaged vascular endothelium and also stabilizes factor VIII in plasma by acting as its carrier molecule.

Inversion mutation analysis in hemophilia a by restriction enzyme analysis and southern blotting.

Intron 22 of the factor VIII gene contains a 9.5kb region of DNA that is repeated on at least two other locations telomeric to, and at least 500 kb from, the gene. These regions are termed intron 22 homologous regions (int22h) (1) and contain the Factor VIII associated gene (F8A).

Screening for Mutations in the Human Antithrombin Gene by Hydrolink D-5000™ and MDE™ Gel Electrophoresis.

The polymerase chain reaction (PCR) provides a rapid method for generating a large amount of a defined region of DNA without recourse to cloning. However, direct sequencing of this amplified material is tedious, time-consuming, and frequently generates large amounts of normal sequence data. Based on the PCR technique, a number of screening methods for detecting DNA mutations have been developed and a number of these are described in this volume.

Detection of mutations causing hemophilia a using an in vitro coupled transcription and translation system.

Mutation detection in the factor VIII gene is complicated by the size and complexity of the gene-186 kb spanning 26 exons. The exons vary in size from 69 bp to 3106 bp and the introns from 207 bp to 32.4 kb (1).

Screening for Mutations in DNA by Single-Stranded Conformation Polymorphism (SSCP) Analysis.

A single-stranded DNA fragment may adopt several different conformations (conformers) which affect its electrophoretic mobility. The mobility of single-stranded DNA in a nondenaturing gel is dependent on both fragment length and secondary structure, which is sequence-dependent. As a result, changes in sequence may affect the conformation of the single-stranded DNA and under appropriate conditions, the differing conformations can be resolved from each other, thereby allowing an underlying sequence variation to be detected.

Detection of DNA by silver staining.

Silver stains are widely used for the detection of both proteins and nucleic acids in acrylamide gels or on various membranes. They have several advantages over conventional staining methods, including the ability to detect very small amounts of material, while avoiding the hazards of other detection systems, such as ethidium bromide or radio-isotopes.

Solid-phase sequencing of biotinylated PCR products with streptavidin-coated magnetic beads.

A novel approach to generating high-quality single-stranded DNA involves solid-phase sequencing. A biotin group is incorporated at the 5'-end of one of the amplification primers and as a result becomes incorporated into PCR product during the amplification reaction. The DNA can then be immobilized onto streptavidin-coated paramagnetic beads, simultaneously removing buffers, dNTPs, and unincorporated PCR primers.

Direct sequencing of PCR products.

Sequencing is an increasingly important part of the analysis of a polymerase chain reaction (PCR) product and is used in many areas of hemostasis. The protocols described in this chapter have been used to sequence many different PCR templates and provide high-quality, reproducible sequence data.

Amplification of DNA and RNA by PCR.

The polymerase chain reaction (PCR) has revolutionized many areas of medicine, including hemostasis. Although this volume is not devoted to PCR, many of the chapters employ the technique at some point to amplify specific DNA or RNA sequences. This chapter, therefore, provides a brief outline of the techniques and methods for the amplification of both DNA and RNA.

Isolation of DNA and RNA.

Blood samples for most coagulation tests are collected into 3.8% trisodium citrate in a ratio of 1 part anticoagulant to 9 parts blood. Whole-blood samples for DNA isolation can be stored at -50°C and the DNA prepared at a later stage.

Hemostasis : components and processes.

Hemostasis is a host defense mechanism that protects the integrity of the vascular system after tissue injury. It works in conjunction with other inflammatory, immune, and repair mechanisms to produce a coordinated response. Hemostatic systems are generally quiescent, but following tissue injury or damage these systems are rapidly activated.

The natural history of HIV disease in haemophilia.

Patients with haemophilia, particularly that due to factor VIII deficiency, have been exposed to a wide range of infective agents transmitted through blood products that have in other ways revolutionized their care. The most devastating of these transfusion transmitted infections has been the human immunodeficiency virus (HIV). AIDS in haemophilic patients was first described in 1982 and it has significantly reduced the life expectancy of these patients.

Identification and eradication of Helicobacter pylori infection in haemophilic patients.

The aim of this study was to identify and eradicate H. pylori infection in patients with haemophilia. Patients were screened for IgG antibodies against H.