Denaturing High-Performance Liquid Chromatography Detects Reliably BRCA1 and BRCA2 Mutations

• Published in: Genomics (San Diego, Calif.) Calif.), 1999-12, Vol.62 (3), p.369-376
• Main Authors: Wagner, Teresa, Stoppa-Lyonnet, Dominique, Fleischmann, Elisabeth, Muhr, Daniela, Pagès, Sabine, Sandberg, Therese, Caux, Virginie, Moeslinger, Regina, Langbauer, Gudrun, Borg, Ake, Oefner, Peter
• Format: Article
• Language: English
• Subjects: Biological and medical sciences; BRCA1 gene; BRCA1 Protein - genetics; BRCA2 gene; BRCA2 Protein; Breast Neoplasms - genetics; Chromatography, High Pressure Liquid - methods; Denaturing high-performance liquid chromatography; Diverse techniques; Electrophoresis, Polyacrylamide Gel; Evaluation Studies as Topic; Female; Fundamental and applied biological sciences. Psychology; Genetic Testing - economics; Genetic Testing - methods; Gynecology. Andrology. Obstetrics; Humans; Mammary gland diseases; Medical sciences; Molecular and cellular biology; Mutation; Neoplasm Proteins - genetics; Nucleic Acid Heteroduplexes - genetics; Ovarian Neoplasms - genetics; Reproducibility of Results; Sensitivity and Specificity; Sequence Analysis, DNA - methods; Transcription Factors - genetics; Tumors
• ISSN: 0888-7543; 1089-8646
• DOI: 10.1006/geno.1999.6026

Denaturing high-performance liquid chromatography (DHPLC) is a recently developed method of comparative sequencing based upon heteroduplex detection. To assess the reliability of this method, 180 different mutations (54 deletions, 12 insertions, and 117 single base substitutions) in BRCA1 and BRCA2 were tested. Second, 25 index individuals with complete DHPLC analysis of BRCA1 were reanalyzed by dye-terminator sequencing. Third, 41 index individuals were analyzed concomitantly by both DGGE and DHPLC. Of the 180 different BRCA1 and BRCA2 mutations, 179 showed heterozygous DHPLC elution profiles. Dye-terminator sequencing of the entire BRCA1 gene, including 5592 bp of coding sequence and 5206 bp of flanking noncoding sequence, in 25 index individuals did not reveal additional variants missed by DHPLC. The concomitant analysis of 41 index cases showed that 4 probably disease-associated mutations were identified by DHPLC while only 3 of those 4 sites were detected by denaturing gradient gel electrophoresis. We conclude that DHPLC is a sensitive and cost-effective method for the screening of BRCA1 and BRCA2.