[Mutation detection enhancement (MDE) gel electrophoresis method for analysis of the von Willebrand factor gene]

We conducted electrophoresis using Mutation Detection Enhancement (MDETM) gel (AT Biochem) on the von Willebrand factor (vWF) gene from three unrelated patients with type 2A von Willebrand disease and one type 3 patient whose point mutations we had identified earlier. The mutations were located on the 3'region of exon 28 in the vWF gene in which three known polymorphisms were included. To eliminate any influence of the polymorphisms, 671bp including two AluI sites amplified by polymerase chain reaction (PCR) from the region of the vWF gene was digested with the restriction enzyme AluI and three fragments (283, 148, and 240bp) were obtained. Three polymorphisms were contained in the 283bp fragment and four mutations were found in the remaining fragments. Following MDETM gel electrophoresis, three of the four cases showed heteroduplex formations. One mutation was not detected, although the mutation was found to be heterozygous by the restriction analysis of MspI. Failure to detect this mutation may be attributed to the fact that it was located on the extreme end of the 148bp fragment of the 5' region. The results of this study suggest the MDETM gel electrophoresis method is useful in detecting gene mutations or polymorphisms in heterozygous subjects. Furthermore, this technique is more convenient than other methods for a base mismatch detection since specific apparatus or radioisotopes are not required.