RAPID MUTATION SCREENING IN TYPE 2A VON WILLEBRANDS DISEASE USING UNIVERSAL HETERODUPLEX GENERATORS

Patients with type 2A von Willebrand's disease (VWD) commonly have mis sense mutations in the A2 domain of the von Willebrand factor (VWF) pr otein. This domain is encoded by the 3' region of VWF gene exon 28 and the large majority of patients have heterozygous mutations clustered in the sequence between codons 742 and 909. We describe a DNA-based di agnostic technique which enables at least 10 previously described muta tions to be rapidly identified. The method involves polymerase chain r eaction (PCR) amplification of two exon 28 gene segments between codon s 717-788 and 803-893, respectively. Each fragment is then hybridized with a synthetic complementary DNA molecule of similar size, termed a Universal Heteroduplex Generator (UHG). The UHG contains base deletion s contiguous to the sites of known mutations and, following hybridizat ion, allele-specific heteroduplexes are generated which can be detecte d by simple polyacrylamide gel electrophoresis and ethidium bromide st aining. A small panel of UHG molecules covering the 3' region of exon 28 should enable the large majority of type 2A VWD patients to be rapi dly diagnosed by genotype.