M. S. Enayat

Clinical and laboratory evaluation of the treatment of von Willebrand's disease patients with heat-treated factor VIII concentrate (BPL 8Y)

Adequate rises of vWF:activity and satisfactory haemostasis have been obtained in six von Willebrand patients treated with heated 8Y factor VIII concentrate, despite multimeric analysis showing the concentrate to have an alteration in the proportions of the different vWF:Ag multimers including slight loss of the highest molecular weight multimers and an abnormal triplet. As heat‐treated concentrates reduce the risk of transmitting blood borne viruses 8Y concentrate should be considered as a possible first line treatment of vWD patients in the U.K. if they do not have mild forms of type I von Willebrands disease which can be treated with desmopressin (DDAVP) infusions.

Distinguishing between type 2B and pseudo-von Willebrand disease and its clinical importance

Pseudo‐von Willebrand disease (p‐VWD) and type 2B von Willebrand disease (VWD) have similar phenotypic parameters and clinical symptoms, but different aetiologies. Fourteen individuals from five families with a historical diagnosis of type 2B VWD but with no mutation in the von Willebrand factor gene were re‐investigated for the possibility of p‐VWD, using platelet aggregation in the presence of cryoprecipitate. p‐VWD was confirmed by targeted DNA sequencing of the glycoprotein Ibα gene, identifying a heterozygous Glycine 233 Valine substitution. This study suggests that p‐VWD may be under diagnosed, and that platelet aggregation in the presence of cryoprecipitate is useful in differentiating this disorder from type 2B VWD.

Site and type of mutations in the factor VIII gene in patients and carriers of haemophilia A

Haemophilia A is an X‐linked bleeding disorder caused by reduced or absent FVIII (FVIII) protein caused by mutations in the FVIII gene. We have used Southern blotting and chemical mismatch analysis (CMA) to identify the mutations causing haemophilia A in 59 local or referred patients or carriers of haemophilia A. Southern blot analysis of 87 families with FVIII : C < 5% identified 31 as positive for the intron 22 inversion. Analysis of 19 of the inversion‐negative families and a further nine families with mild or moderate haemophilia A by CMA resulted in the identification of a heterogeneous spectrum of mutations in the FVIII gene comprising 21 single base‐pair substitutions and nine deletions. Seventeen of the base‐pair substitutions are missense, two nonsense, and two are splice‐site mutations. Two patients were found to have compound mutations with two mutations identified on a single X chromosome. Six of the point mutations and six of the deletions have not been reported previously in the haemophilia A mutation database. Unusually, a missense mutation, as well as deletion and splice‐site mutations, was found to be associated with exon‐skipping events.

A novel alanine or threonine 789 to proline mutation causing type 2N von Willebrand’s disease when inherited homozygously or heterozygously with arginine 854 to glutamine mutation

regulation of F9 in haemophilia B Leyden patients. In addition, Crossley et al. showed that patients with haemophilia B Brandenburg (caused by a )26 mutation, which disrupts an ARE in the promoter region) did not show postpubertal increases of plasma levels of FIX [4]. Transgenic mice studies demonstrated that puberty-onset amelioration is not specifically dependent on the male sex hormone surge during puberty [5]. The age-related stability element (ASE) of the ASE/ age-related increase element (AIE)-mediated genetic mechanism seemed to play an important role in the increase in FIX levels. Furthermore, in both male and female mice, growth hormone appeared to be directly responsible for puberty-onset recovery of FIX production. Upon removal of the pituitary organ in male and female transgenic mice, serum levels of FIX fell rapidly and intraperitoneal administration of growth hormone restored FIX levels after 4–8 days. In women, the sex hormone that dominates female development is estradiol. Depending on which mechanism testosterone or growth hormone plays the most important role in human, FIX levels might rise after puberty in female carriers of haemophilia B Leyden. Future longitudinal studies will be necessary to unravel this question.

Mutation Analysis in F9 Gene of 17 Families with Haemophilia B from Iran

Summary.  Seventeen haemophilia B families from Iran were investigated to determine the causative mutation. All the essential regions of the F9 gene were initially screened by conformational sensitive gel electrophoresis and exons with band shift were sequenced. Seven of the 15 mutations identified in these families were novel mutations. The mutations were authenticated in nine families as other affected members or heterozygous female carriers were available for verification.

2B or not 2B? Differential identification of type 2B, versus pseudo-von Willebrand disease – response to Favarolo

We are grateful to Dr Favarolo for commenting so fully on our recent publication (Enayat et al., 2006). Because our laboratory was in the fortunate position of being able to carry out molecular investigation of von Willebrand disease (VWD), we began to question established diagnoses of kindreds attending our local haemostasis units in the west Midlands. Once a diagnostic label is attached, people are reticent to challenge this, particularly if the condition only produces symptoms infrequently. The diagnosis and classification of VWD requires the use of many tests and it is important that the initial tests include the best available to avoid misdiagnosis, hence our proposal to include cryoprecipitate-induced platelet aggregation to screen for pseudo-VWD. We will also be gathering data on other VWD types prospectively. We are interested in Dr Favarolo’s suggested alternative assays and those of Caron et al (2006). With regard to other specific questions:

Independent occurrence of the novel Arg2163 to His mutation in the factor VIII gene in three unrelated families with haemophilia A with different phenotypes

Using chemical mismatch analysis or denaturing gradient gel electrophoresis followed by nucleotide sequencing, we have identified the same G6545A mutation leading to an Arg 2163 His substitution in the factor VIII gene of three haemophiliacs from unrelated families. One of the affected individuals has severe haemophilia, while the other two are moderately severe. While we cannot exclude the possibility that these differences in phenotype arise from differences in VIII:C assay methods, other studies have also identified different clinical phenotypes in individuals with the same mutations, and suggested that they may arise from extragenic factors that affect or modify gene expression or protein function. The G6545A mutation occurs at a CG dinucleotide which is a known mutation hotspot, and which may explain the independent occurrence in unrelated families. Hum Mutat 11:334, 1998.