Identification of Mutations in the Canine von Willebrand Factor Gene Associated with Type III von Willebrand Disease

Abstract

SummaryIn 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.Parts of this paper were presented at the 39th annual meeting of the American Society of Hematology (ASH), December 5-9, San Diego, USA

DNA Testing for Type III von Willebrand Disease in Dutch Kooiker Dogs

“…After sequence analysis of this PCR product, we detected a homozygous mutation (IVS16ϩ1G→A; TGg→TGA) at the splice donor site of intron 16 of the vWF gene. This finding is consistent with that of Rieger et al 9 The mutation changes the reading frame in such a way that the newly formed codon at the normal splice junction is changed to a stop codon at amino acid position 729 in the propeptide. With such a radical mutation, the total absence of vWf:Ag in homozygously affected Kooiker dogs is readily explained.…”

“…A 501-bp fragment generated by primers DKD2 and DKD3 from another region of the cDNA was of the same size in both dogs (data not shown). Sequence analysis of the longer PCR products identified a splice site mutation (IVS16ϩ1G→A; TGgtaa→TGATAA) involving the 1st nucleotide of intron 16 (indicated in bold), resulting in the presence of 46-bp intronic DNA in the mature mRNA (data not shown), as has also been described by Rieger et al 9 The mutation changes the codon at the original splice junction (TGC) into a nonsense stop codon (TGA). This is predicted to terminate the translation process at amino acid position 729.…”

“…8 A homozygous splice site mutation at the border of exon 16 and intron 16 and a homozygous missense mutation in exon 3 of the mature von Willebrand gene in Kooiker dogs with type III von Willebrand disease was reported by others. 9 These mutations were identified by direct sequencing of the endothelial messenger RNA from affected Kooiker dogs supplied by one of us (RJS). By a different approach, we have independently found the splice site mutation in the Kooiker breed.…”

“…In the analysis of the complete coding region of the vWF gene in the affected Kooiker dogs, Rieger et al 9 found, in addition to the splice site mutation, a missense mutation (208G→A) in exon 3, causing a change of valine (V) to isoleucine (I) at amino acid position 70 in the D1 domain of the propeptide. They did not analyze the vWF gene in normal Kooiker dogs nor perform cosegregation studies in affected Kooiker dog families, leaving the significance of this mutation unresolved.…”

Phenotypic correction of von Willebrand disease type 3 blood-derived endothelial cells with lentiviral vectors expressing von Willebrand factor

“…15 Here, genomic DNA was isolated from the blood cells of the 4 VWD dogs, and genotyping revealed that all 4 dogs carried a homozygous point mutation (GϾA transition) at the first position of the donor splice site sequence of intron 16, resulting in a stop codon in the propeptide of VWF and thus identifying them as type 3 VWD dogs. 16 All animal experiments were performed in accordance with protocols approved by the Institutional Animal Care and Use Committee of the Catholic University of Leuven (Belgium).…”