Am. J. Hum. Genet. 51:850-858, 1992

Nonsense Mutations of the von Willebrand Factor Gene in Patients with von Willebrand Disease Type III and Type I Z. P. Zhang,*,t M. Lindstedt,*4t,1 G. Falk,t M. Blomback,t N. Egbergt and M. Anvret* Departments of *Clinical Genetics, tClinical Chemistry and Blood Coagulation, and tMedicine, Karolinska Hospital, Stockholm

Summary von Willebrand disease (vWD) is the most common inherited bleeding disorder in humans. The disease is caused by qualitative and quantitative abnormalities of the von Willebrand factor (vWF). Genomic DNA from 25 patients with vWD type III, the most severe form of the disease, was studied using PCR followed by restriction-enzyme analysis and direct sequencing of the products. Nonsense mutations (CGA--TGA) were detected in exons 28, 32, and 45 by screening of all the 11 CGA arginine codons of the vWF gene. Two patients were found to be homozygous and five heterozygous for the mutation. Both parents and some of the relatives of the homozygous patients carry the mutation. These are the first reported examples of homozygous point mutations associated with the severe form of vWD. In the three heterozygous probands, one of the parents carried the mutation and had vWD type I. Family studies including parents and family members with or without vWD type I indicated that these three heterozygous patients are likely to be compound heterozygous. Twenty-one individuals from these seven families with vWD type I were found to be heterozygous for the mutation.

Introduction von Willebrand factor (vWF) is a large multimeric plasma glycoprotein which plays a central role in hemostasis. It has different functional domains which interact with factor VIII, platelet glycoproteins, and collagen. It is required both as a carrier and as a stabilizer for the coagulation factor VIII in plasma (Ruggeri and Zimmerman 1987). The human vWF gene is located on the short arm of chromosome 12 (12pl2pter) and spans approximately 178 kb of DNA. It contains 52 exons, varying from 40 to 1,379 bp in length (Mancuso et al. 1989). A partial unprocessed vWF pseudogene which corresponds to the middle region of the authentic gene which encodes important functional domains of the protein has been mapped to chromosome 22 (Mancuso et al. 1991). Transcription of the gene yields a functional mRNA molecule of 8.7 kb. It encodes a precursor protein of 2,813 amino Received March 19, 1992; revision received May 20, 1992. Address for correspondence and reprints: Maria Anvret, Ph.D., Department of Clinical Genetics, Karolinska Hospital, S-10401 Stockholm, Sweden. o 1992 by The American Society of Human Genetics. All rights reserved. 0002-9297/92/5104-0019$02.00

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acids that includes a signal peptide (22 amino acids), a propeptide (741 amino acids), and the mature subunit (2,050 amino acids). von Willebrand disease (vWD) is the most common inherited bleeding disorder in humans. It is caused by qualitative and quantitative abnormalities of vWF (Ruggeri and Zimmerman 1987). Patients with vWD type I have only a mild bleeding tendency, and they account for 80% of all vWD cases. vWD type III represents a severe form of the disease, with very low to undetectable levels of vWF, and accounts for about 1% of the cases. Biochemical and DNA linkage analyses in the families investigated by us suggest that the proband (vWD type III) presents a homozygous or compound heterozygous form of vWD type I (Anvret et al. 1992). In human DNA, the CpG dinucleotides are generally predicted to be hot spots for mutations (Duncan and Miller 1980). A transition of C to T in the CGA codon for arginine results in a TGA stop codon (nonsense mutation). For example, nonsense mutations have been reported in every CGA arginine codon of the factor VIII gene in patients with hemophilia A (Tuddenham et al. 1991). So far, only two nonsense mutations in arginine codons (exons 9 and

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Nonsense Mutations of vWF Gene 45) in the vWF gene have been reported (Eikenboom et al. 1991; Lavergne et al. 1991). In this study, we have analyzed the 11 CGA arginine codons of the vWF gene by using PCR followed by restriction-enzyme analysis and direct DNA sequencing of the PCR products. Three nonsense mutations, located in exons 28, 32, and 45, were detected in the 25 vWD type III patients investigated. Both the exon 28 mutation and the exon 45 mutation create a new recognition site for the restriction enzyme DdeI. The exon 32 mutation was found by DNA sequencing. Two homozygous patients and five heterozygous (compound heterozygous) patients were found in seven families. Patients and Methods Patients

Twenty-five patients with severe vWD type III and from independent families (Bloom 1987) were investigated for the presence of nonsense mutations in the vWF gene. All the probands have a prolonged bleeding time >900 s, vWF antigen (vWF:Ag) either undetectable with electroimmunoassay (

Nonsense mutations of the von Willebrand factor gene in patients with von Willebrand disease type III and type I.

von Willebrand disease (vWD) is the most common inherited bleeding disorder in humans. The disease is caused by qualitative and quantitative abnormali...
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