A highly sensitive, fast, and economical technique for mutation analysis in hereditary breast and ovarian cancers

Mutation analysis of complex genes without hotspots for sequence variations , such as BRCA1, is time-consuming and expensive. Of all currently availabl e methods, direct sequencing has the highest sensitivity, but also the high est costs. Other techniques, such as SSCP, DGGE, and PTT, are more economic al but, depending on the experience of the investigator, have at best a sen sitivity of 90%. We investigated in a prospective study the feasibility and accuracy of the DHPLC technique. We present the application of the DHPLC p rotocol for BRCA1 mutation detection on a HPLC device from Bio-Tek Kontron Instruments (Neufahrn, Germany). DNA from 46 women with hereditary breast a nd ovarian cancer undergoing genetic testing for BRCA1 mutations were teste d. Of 1,518 amplicons analyzed by DHPLC, corresponding to 33 fragments span ning the entire BRCA1 gene, 626 were also directly sequenced, The compariso n demonstrated that DHPLC detected all alterations found by direct sequenci ng. No false-positive signals were seen in cases of homozygous sequences. F urther, no false-negative results were ever obtained in women with mutation s or polymorphisms, or both. In cases of known genetic variations, the natu re of the alterations could be predicted by DHPLC, We also compared differe nt separation matrices. Up to about 500 injections, no significant differen ces in sensitivity could be observed between poly(styrene divinylbenzene) a nd end-capped silica based columns. However, after more than 500 injections , the resolution of hetero- from homoduplex deteriorated rapidly on silica columns. Hum Mutat 14:333-339, 1999. (C) 1999 Wiley-Liss, Inc.