Molecular genetic analysis of porcine von Willebrand disease: tight linkage to the von Willebrand factor locus

Abstract

von Willebrand disease (vWD), one of the most common bleeding disorders in humans, is manifested as a quantitative or qualitative defect in von Willebrand factor (vWF), an adhesive glycoprotein (GP) with critical hemostatic functions. Except for the rare severely affected patient with a gene deletion as etiology of the disease, the molecular basis for vWD is not known. We studied the molecular basis for vWD in a breeding colony of pigs with a disease closely resembling the human disorder. The porcine vWF gene is similar in size and complexity to its human counterpart, and no gross gene deletion or rearrangement was evident as the pathogenesis of porcine vWD. A restriction fragment- length polymorphism (RFLP) within the porcine vWF gene was identified with the restriction endonuclease HindIII, and 22/35 members of the pedigree were analyzed for the polymorphic site. Linkage between the vWF locus and the vWD phenotype was established with a calculated LOD score of 5.3 (1/200,000 probability by chance alone), with no crossovers identified. These findings indicate that porcine vWD is due to a molecular defect within (or near) the vWF locus, most likely representing a point mutation or small insertion/deletion within the vWF gene.

A 12.3-kb Duplication Within the VWF Gene in Pigs Affected by Von Willebrand Disease Type 3

“…The clinical signs of this disease in pigs match those in humans to a large extent and is why affected pigs provide a reliable animal model for the disease ( Nichols et al 2010 ). Though Bahou et al (1988) performed linkage analyses which indicated the location of the molecular defect for VWD within or near to the VWF gene, the causal genetic variant remained undetected. Analyzing VWF cDNA and genomic DNA of VWD-affected, VWD-carriers, and VWD-unaffected pigs, we specified the putatively causal variant as a 12.3-kb genomic tandem duplication including the exons 17 and 18.…”

“…They are phenotypically identical to VWD type 3 in humans ( Brouland et al 1999 ) and therefore are a valuable large animal model for the human disease. VWD in pigs has been localized at the VWF locus ( Bahou et al 1988 ), but the molecular mechanism and the causal genetic background remained undetected.…”

The endothelial-specific regulatory mutation, Mvwf1, is a common mouse founder allele

“…Large polymorphisms were confirmed by long-range PCR (Expand Long Template PCR System, Roche) per the manufacturer’s instructions or Southern blot as previously described (Bahou et al. 1988 ). The RIIIS/J genomic sequence assembly has been deposited at NCBI (GenBank accession number EF372924).…”

“…The integrity of the BAC inserts was confirmed by Hin dIII restriction digests, genomic PCR of 16 amplicons spanning the predicted genomic inserts (primer sets 8–23 in Supplementary Table 2), and Southern blots as previously described (Bahou et al. 1988 ).…”