Identification of specific BRCA1 and BRCA2 variants by DHPLC

Denaturing high performance liquid chromatography (DHPLC) is generating inc reasing interest in clinical genetics as a reliable tool for the analysis o f genetic alterations. In the work presented here our intentions were to op timize primer design and DHPLC analysis conditions for a qualitative detect ion of BRCA1 and BRCA2 variations. The BRCA1 and BRAC2 genes display a high proportion of polymorphisms. Sequencing efforts geared towards the distinc tion of tumor-related mutations and benign variants still remain time-consu ming and expensive. DHPLC elution profiles, however, permit the correlation of a characteristic chromatographic profile with a specific sequence alter ation. In this study we evaluate the sensitivity of DHPLC for the identific ation of unique polymorphisms, which are frequent in the Caucasian populati on, in lieu of sequence analysis. The complete BRCA1 gene and parts of BRCA 2 were examined. In the case of BRCA1, 431 out of 432 heterozygotes were id entified correctly. Tn addition, 18 new profiles were identified which had not been detected previously in our studies and which represented new mutat ions or rare polymorphisms. For BRCA2, 135 out of 137 simple sequence varia nts were classified correctly. In addition, six new profiles were identifie d which represented new mutations or rare polymorphisms. Hum Mutat 16:345-3 53, 2000. (C) 2000 Wiley-Liss, Inc.