In humans, type III von Willebrand disease is caused by deletions or nonsense mutations. In dogs, the underlying genetic defects have not been determined yet. We searched for the genetic defect in four related type III deficient Dutch Kooiker dogs obtained from one breeder. Mutation analysis was performed with total RNA isolated from platelets or whole blood. The complete coding region of the vWf gene was amplified by RT-PCR and sequenced by the cycle sequencing technique. Two homozygous mutations were found, a G-->A transition at the first position of the donor splice site sequence of intron 16 (TGgtaagt-->TGataagt) and a missense mutation at nt 208 (G-->A) (1). The splice site defect resulted in the generation of a transcript containing 46bp of intron sequence and a stop codon at amino acid position 729 in the propeptide region of the vWf protein. This mutation seems to be causative for the type III phenotype. The effect of the missense mutation in exon 3 which causes a change of Val to Ile on the vWD phenotype is unclear. Probably, this transition represents a polymorphism occurring in Dutch Kooiker dogs. Both mutations were not present in 5 healthy mongrel dogs.
"The complete cDNA sequence of canine VWF predicts a protein of 2,813 amino acids and thus is identical in the number of amino acids to human VWF (Figure 2) [36, 37]. The full-length human and canine VWF are 87.1% identical at the nucleotide level and 86.2% at the protein level, with an additional 6% conservative substitutions. "
[Show abstract][Hide abstract] ABSTRACT: Use of animal models of inherited and induced von Willebrand factor (VWF) deficiency continues to advance the knowledge of VWF-related diseases: von Willebrand disease (VWD), thrombotic thrombocytopenic purpura (TTP), and coronary artery thrombosis. First, in humans, pigs, and dogs, VWF is essential for normal hemostasis; without VWF bleeding events are severe and can be fatal. Second, the ADAMTS13 cleavage site is preserved in all three species suggesting all use this mechanism for normal VWF multimer processing and that all are susceptible to TTP when ADAMTS13 function is reduced. Third, while the role of VWF in atherogenesis is debated, arterial thrombosis complicating atherosclerosis appears to be VWF-dependent. The differences in the VWF gene and protein between humans, pigs, and dogs are relatively few but important to consider in the design of VWF-focused experiments. These homologies and differences are reviewed in detail and their implications for research projects are discussed. The current status of porcine and canine VWD are also reviewed as well as their potential role in future studies of VWF-related disorders of hemostasis and thrombosis.
"Robert Montgomery's team has reported the cDNA sequence for canine VWF (Haberichter et al. 2000) and found that the human and canine VWF are 87.1% and 86.2 % identical at the nucleotide and protein levels, respectively. This cDNA and reports from others (Rieger et al. 1998) enabled us, in collaboration with "
[Show abstract][Hide abstract] ABSTRACT: Dogs with hemophilia A, hemophilia B, von Willebrand disease (VWD), and factor VII deficiency faithfully recapitulate the severe bleeding phenotype that occurs in humans with these disorders. The first rational approach to diagnosing these bleeding disorders became possible with the development of reliable assays in the 1940s through research that used these dogs. For the next 60 years, treatment consisted of replacement of the associated missing or dysfunctional protein, first with plasma-derived products and subsequently with recombinant products. Research has consistently shown that replacement products that are safe and efficacious in these dogs prove to be safe and efficacious in humans. But these highly effective products require repeated administration and are limited in supply and expensive; in addition, plasma-derived products have transmitted bloodborne pathogens. Recombinant proteins have all but eliminated inadvertent transmission of bloodborne pathogens, but the other limitations persist. Thus, gene therapy is an attractive alternative strategy in these monogenic disorders and has been actively pursued since the early 1990s. To date, several modalities of gene transfer in canine hemophilia have proven to be safe, produced easily detectable levels of transgene products in plasma that have persisted for years in association with reduced bleeding, and correctly predicted the vector dose required in a human hemophilia B liver-based trial. Very recently, however, researchers have identified an immune response to adeno-associated viral gene transfer vector capsid proteins in a human liver-based trial that was not present in preclinical testing in rodents, dogs, or nonhuman primates. This article provides a review of the strengths and limitations of canine hemophilia, VWD, and factor VII deficiency models and of their historical and current role in the development of improved therapy for humans with these inherited bleeding disorders.
ILAR journal / National Research Council, Institute of Laboratory Animal Resources 02/2009; 50(2):144-67. DOI:10.1093/ilar.50.2.144 · 2.39 Impact Factor
"Due to the technical challenge of constructing viral vectors encoding VWF, the initial goal of this study was to use the technique of chimeraplasty for the treatment of VWD. As an animal model, we wanted to use Dutch Kooiker dogs suffering from VWD type 3 where the point mutation (G to A transition) at the first position of the donor splice site sequence of intron 16, responsible for the VWD type 3 phenotype, has been identified . An earlier attempt to correct a point mutation in the VWF gene in a lymphoblastoid cell line by chimeraplasty was unsuccessful however, possibly "
[Show abstract][Hide abstract] ABSTRACT: Chimeraplasty or the use of chimeric RNA/DNA oligonucleotides (RDOs) to correct single-base mutations emerged in the field of gene therapy with reported base pair conversions of up to 40%. We investigated the applicability of chimeraplasty to correct a point mutation in the von Willebrand Factor (VWF) gene resulting in a von Willebrand Disease (VWD) type 3 phenotype. Although we have access to VWD type 3 dogs, we used wild type endothelial cells for in vitro studies, as isolation of endothelial cells from VWD type 3 dogs is not straightforward due to the bleeding diathesis. RDOs to convert the wild type VWF gene into VWD type 3 gDNA were constructed and used in various transfection conditions. However, no gene conversion could be detected either in the RNA or in the DNA isolated from transfected cells, not even with the sensitive colony hybridisation technique, despite the presence of RDOs in the cell nucleus. On the other hand, sequence analysis of isolated DNA of transfected cells did reveal the presence of VWF type 3 DNA. However, this apparent conversion is very likely not the result of RDO-mediated nucleotide conversion as the same VWF type 3 DNA sequence was also detected in negative control experiments where no RDO was used. Our negative results are in line with the emerging reports of chimeraplasty failure and can contribute to the call for an international "chimeraplasty consortium" with free exchange of results to clarify the controversy about the applicability of the RDO-mediated base conversion.
Thrombosis Research 02/2007; 119(1):93-104. DOI:10.1016/j.thromres.2005.12.009 · 2.45 Impact Factor
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