Structural analysis of the von Willebrand factor gene located on chromosome 12 is complicated by the presence of a partial unprocessed pseudogene on chromosome 22q11-13. The structures of the von Willebrand factor pseudogene and corresponding segment of the gene were determined, and methods were developed for the rapid differentiation of von Willebrand factor gene and pseudogene sequences. The pseudogene is 21-29 kilobases in length and corresponds to 12 exons (exons 23-34) of the von Willebrand factor gene. Approximately 21 kilobases of the gene and pseudogene were sequenced, including the 5' boundary of the pseudogene. The 3' boundary of the pseudogene lies within an 8-kb region corresponding to intron 34 of the gene. The presence of splice site and nonsense mutations suggests that the pseudogene cannot yield functional transcripts. The pseudogene has diverged approximately 3.1% in nucleotide sequence from the gene. This suggests a recent evolutionary origin approximately 19-29 million years ago, near the time of divergence of humans and apes from monkeys. Several repetitive sequences were identified, including 4 Alu, one Line-1, and several short simple sequence repeats. Several of these simple repeats differ in length between the gene and pseudogene and provide useful markers for distinguishing these loci. Sequence differences between the gene and pseudogene were exploited to design oligonucleotide primers for use in the polymerase chain reaction to selectivity amplify sequences corresponding to exons 23-34 from either the von Willebrand factor gene or the pseudogene. This method is useful for the analysis of gene defects in patients with von Willebrand disease, without interference from homologous sequences in the pseudogene.

von Willebrand factor (vWF) is a multimeric glycoprotein that mediates the adhesion of platelets to the subendothelium by binding to platelet glycoprotein Ib. For human vWF, this interaction can be induced in vitro by the antibiotic ristocetin or the snake venom protein botrocetin. A missense mutation, Gly-561-->Ser, was identified within the proposed glycoprotein Ib binding domain of vWF in the proband with von Willebrand disease type B, a unique variant characterized by no ristocetin-induced, but normal botrocetin-induced, binding to glycoprotein Ib. The corresponding mutant recombinant protein, rvWF(G561S), formed normal multimers and exhibited the same functional defect as the patient's plasma vWF, confirming that this mutation causes von Willebrand disease type B. These data show that botrocetin and ristocetin cofactor activities of vWF can be dissociated by a point mutation and confirm that these mediators promote vWF binding to platelets by different mechanisms. The normal botrocetin-induced binding and the defective ristocetin-induced binding of rvWF(G561S) suggest that the primary defect in von Willebrand disease type B may be a failure of normal allosteric regulation of the glycoprotein Ib binding function of vWF.

Many variants of von Willebrand disease (vWD) with qualitatively abnormal von Willebrand factor (vWF) are recognized. In vWD type IIB, the abnormal protein displays enhanced affinity for a platelet vWF receptor, the glycoprotein Ib-IX complex. 14 patients from 7 unrelated families with vWD type IIB were studied to determine the molecular basis for this phenotype. Specific oligonucleotide primers were used to amplify portions of vWF exon 28 encoding a domain that interacts with the platelet glycoprotein Ib-IX complex. Candidate missense mutations were identified for all 14 patients by DNA sequencing, allele specific oligonucleotide hybridization, and restriction endonuclease digestion. These sequence changes occur in an 11 amino acid segment within a single disulfide loop bounded by Cys(509) and Cys(695). All of these sequence changes are C -0 T transitions within CG dinucleotides. Six patients from two unrelated families were heterozygous for the encoded sequence Arg(543) -. Trp. Seven patients from four unrelated families were heterozygous for the encoded sequence Arg (545)

Southern blotting was performed with cDNA probes for the human von Willebrand factor (vWF) gene on six patients with severe type III von Willebrand's disease (vWD). A partial deletion in the 3′ end of the vWF gene was demonstrated in one individual whose parents were related and who had an alloantibody inhibitor to vWF. A resulting novel 2.0- kilobase (kb) EcoRI fragment was used for carrier detection within the patient's family, and seven carriers of this recessive trait were identified. Of the six tested, five had normal or only slightly reduced levels of vWF antigen, but with generally higher levels of factor VIII. The sixth carrier had moderately severe vWD and it is proposed that this patient is heterozygous for the defective vWF gene and a second recessive vWF defect. The novel 2.0-kb EcoRI restriction fragment was cloned and sequenced, and compared with that of the corresponding normal 4.2-kb EcoRI fragment that includes exons 41 and 42 of the vWF gene. A deletion of 2,320 base pairs (bp) which included exon 42, was identified and a novel 182-bp insert was found between the breakpoints. This insert was detected by polymerase chain reaction amplification both in the patient's DNA and in his carrier relatives.

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