A comparison of BRCA1 mutation analysis by direct sequencing, SSCP and DHPLC

The most sensitive screening technique for genes that predispose patients f or particular cancers is direct sequencing. However, sequencing of complex genes is technically demanding, costly and time-consuming. We have tested a lternate screening techniques to find a fast sensitive method for detecting alterations of DNA in the large BRCA1 gene prior to sequencing. Sequencing of this gene is particularly arduous because it lacks clearly defined muta tion sites. The single-strand conformation polymorphism (SSCP) technique is one of the most frequently used pre-screening methods but its sensitivity and efficiency is not completely satisfying. We have compared the SSCP assa y with a newly developed technique called denaturing high performance liqui d chromatography (DHPLC) to screen the BRCA1gene. We studied 23 patients at high risk for early onset breast and ovarian cancer and four controls. In these patients, a total of 113 fragments with sequence variations in the BR CA1 gene could be identified. The DHPLC technique resolved 100% of the DNA alterations that were observed in cycle sequencing. In contrast, mutation a nalysis by SSCP accounted for 94% of the detected variations. In addition, DHPLC screening allowed us to discriminate between different alterations in a single fragment, because of the characteristic elution profiles of the D NA molecules. Polymorphisms that were present in our samples could be predi cted by means of DHPLC testing independently of sequence analysis. We concl ude that DHPLC is a highly potent screening method for genetic analyses. It is highly sensitive, efficient and economical and can be automated.