Identification of three candidate mutations causing type IIA von Willebrand disease using a rapid, nonradioactive, allele-specific hybridization method

Abstract

Type IIA von Willebrand disease (vWD), the most common type II vWD variant, is characterized by decreased binding of von Willebrand factor (vWF) to platelet glycoprotein Ib (Gplb) and by a decrease in large and intermediate vWF multimers. Mutations reported to cause vWD type IIA are clustered within the A2 domain of vWF, which is encoded by exon 28. Genomic DNA from affected members of 12 unrelated families with type IIA vWD were screened for these mutations by a rapid, nonradioactive, allele-specific oligonucleotide (ASO) hybridization method. Oligonucleotides containing each of eight mutations were cross-linked onto a nylon membrane by UV irradiation. A fragment of vWF exon 28 was amplified from peripheral blood leukocyte DNA using biotinylated primers and hybridized to the immobilized oligonucleotides. Positive signals were detected with an avidin-alkaline phosphatase conjugate and chemiluminescent substrate. Thus, in a single hybridization reaction, a patient sample could be analyzed for a large number of mutations simultaneously. Polymerase chain reaction (PCR) products from four patients did not contain any of the tested mutations and therefore were sequenced. Three additional candidate missense mutations, two of them novel, were identified: Arg(834)-->Gln in one patient, Gly(846)-->Arg in one patient, and Val(902)-->Glu in three ostensibly unrelated patients. By ASO hybridization, the mutations were confirmed in the affected patients and excluded in unaffected relatives and 50 normal controls. In one family, the Val(902)-->Glu mutation was shown to be a de novo mutation. This rapid screening method is applicable to other subtypes of vWD for which mutations have been identified.

Identification of Type 2 von Willebrand Disease in Previously Diagnosed Type 1 Patients: a Reappraisal Using Phenotypes, Genotypes and Molecular Modelling

“…A 936 Table 1 Phenotypic and genotypic data on the 17 kindreds with a discrepant VWF: activity/VWF: antigen ratio bp fragment of the 5' region of the exon 28 that encoded for the VWF-A1 domain was amplified by polymerase chain reaction (PCR). The primer sequences were 5'-TGGGAATATGGAAGTGCATTG-3' and 5'-CCGATCC-TTCCAGGACGAAC-3' (18). The PCR products were purified using the QIAmp DNA purification kit (Qiagen Ltd.).…”

A Novel Mutation Glyl672→Arg in Type 2A and a Homozygous Mutation in Type 2B von Willebrand Disease

“…The G(5075)->A, detected in patient T5 with type 2A vWD, is a transition substituting amino acid Gly 1609 with Arg. Although the same transition has been found in Caucasians (20,21), there is no proof that this transition is a cause of type 2A vWD since expression experi ments have not been performed. The sequence of this mutant was con sistent with that of the pseudogene.…”

“…Of the transitions identified in the present study, the R1597W detected in patients T2 and T3 is the most frequent among reported type 2A vWD transitions (8,(16)(17)(18) and belongs to group II (14,15). The amino acid substitution R1597Q, detected in patient T4 with type 2A vWD, has also been identified else where (8,19,20), but no expression studies have been conducted. The G(5075)->A, detected in patient T5 with type 2A vWD, is a transition substituting amino acid Gly 1609 with Arg.…”