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Summary Background Type 3 von Willebrand disease (VWD) is the most severe form of the disease and is classically inherited in an autosomal recessive fashion. Objectives The aim of the current study was to investigate the molecular pathogenesis of a Canadian cohort of type 3 VWD patients. Patients/Methods 34 families comprised of 100 individuals were investigated. Phenotypic data, including bleeding scores (BS), von Willebrand factor (VWF) laboratory values, and anti-VWF inhibitor status were included as well as sequence analysis. Results We identified 31 different mutations (20 novel): 8 frameshift, 5 splice site, 9 nonsense, 1 gene conversion, 6 missense, and 2 partial gene deletion mutations. The majority of mutations identified were in the propeptide (42%); index cases (IC) with these mutations exhibited more severe bleeding (BS=22) than those with mutations elsewhere in VWF (BS=13). 62 of 68 (91%) mutant alleles were identified. Twenty-nine IC (85%) had a VWF null genotype identified; 17 homozygous, 12 compound heterozygous. In five IC (15%), two mutant VWF alleles were not identified to explain the type 3 VWD phenotype. In four ICs only one mutant VWF allele was identified and in one IC no mutant VWF alleles were identified. Conclusions We have investigated the molecular pathogenesis of a Canadian cohort of type 3 VWD patients. Obligate carriers are not phenotypically silent in the Canadian population; 48% have been diagnosed with type 1 VWD. In ~50% of families in this study the inheritance pattern for type 3 VWD is co-dominant and not recessive.

Summary. Background: von Willebrand disease (VWD) is the most common bleeding disorder known in humans, with type 1 VWD representing the majority of cases. Unlike the other variant forms of VWD, type 1 disease represents a complex genetic trait, influenced by both genetic and environmental factors. Aim: To evaluate the contribution of the von Willebrand factor (VWF) and ABO blood group loci to the type 1 VWD phenotype, and to assess the potential for locus heterogeneity in this condition, we have performed genetic linkage and association studies on a large, unselected type 1 VWD population. Method: We initially collected samples from 194 Canadian type 1 VWD families for analysis. After the exclusion of families found to have either type 2 or type 3 VWD, and pedigrees with samples from single generations, linkage and association analysis was performed on 155 type 1 VWD families. Results and conclusion: The linkage study has shown a low heterogeneity LOD score of 2.13 with the proportion of families linked to the VWF gene estimated to be 0.41. Linkage was not detected to the ABO locus in this type 1 VWD population. In the family-based association test, significant association was found between the type 1 VWD phenotype, the quantitative traits, VWF:Ag, VWF:RCo, and FVIII:C and the ABO ÔOÕ and ÔAÕ alleles and the VWF codon 1584 variant. There was also weak association with the )1185 promoter polymorphism and VWF:Ag, VWF:RCo, and FVIII:C plasma levels. These studies provide further evidence to support the role for genetic loci other than VWF and ABO in the pathogenesis of type 1 VWD.

Canine hemophilia A closely mimics the human disease and has been used previously in the development of factor VIII (FVIII) protein replacement products. FVIIIdeficient dogs were studied to evaluate an in vivo gene therapy approach using an E1/E2a/E3-deficient adenoviral vector encoding canine FVIII. Results demonstrated a high level of expression of the canine protein and complete phenotypic correction of the coagulation defect in all 4 treated animals. However, FVIII expression was short-term, lasting 5 to 10 days following vector infusion. All 4 dogs displayed a biphasic liver toxicity, a transient drop in platelets, and development of anticanine FVIII antibody. Canine FVIII inhibitor development was transient in 2 of the 4 treated animals. These data demonstrate that systemic delivery of attenuated adenoviral vectors resulted in liver toxicity and hematologic changes. Therefore, the development of further attenuated adenoviral vectors encoding canine FVIII will be required to improve vector safety and reduce the risk of immunologic sequelae, and may allow achievement of sustained phenotypic correction of canine hemophilia A. IntroductionHemophilia A is a severe, X-linked bleeding disorder caused by a deficiency of blood coagulation factor VIII (FVIII). Hemophilia A has an incidence approaching 1 in 4000 males in all populations, 1 and in its severe form, is a life-threatening, crippling disease. Infusion of plasma-derived or recombinant FVIII protein in response to bleeding crises is currently the most widely accepted therapy 1 and has dramatically increased the life expectancy and quality of life for many patients with hemophilia. However, the high cost and short supply of FVIII replacement products has resulted in their availability being limited to less than 10% of the world's hemophilic population.Gene therapy for hemophilia A would provide prophylactic expression of FVIII and correction of the coagulation defect. Considerable progress has been made recently in the development of adenoviral vector-mediated gene therapy for hemophilia A. 2,3 Potent adenoviral vectors encoding a human FVIII complementary DNA (cDNA) have been developed that mediated expression of physiologic levels of FVIII in mice, 4-7 monkeys, 8 and dogs, 9 and sustained human FVIII expression in normal 5 and hemophilic mice. 7 Treatment of hemophilic mice and dogs resulted in human FVIII expression and complete phenotypic correction, verifying the feasibility of adenoviral vector administration for the treatment of hemophilia A. 7,[9][10][11] Expression in the hemophilic mice was sustained for at least 1 year, 7,11 whereas the duration of expression in the hemophilic dogs was short-term, limited by a rapid antibody response to the human FVIII protein. 9 Canine hemophilia A was first described 50 years ago, 12,13 and FVIII-deficient dogs have been used to support the development of FVIII pharmaceutical products. [14][15][16][17][18][19] However, human FVIII is highly immunogenic in dogs when the protein is delivered intravenously 20 or v...