Enhanced mismatch mutation analysis: a new high throughput screening method for simultaneous detection of point mutation and gross gene rearrangement designed for BRCA1 and BRCA2 genes.

Abstract

Abstract Abstract #1102 Background : Recent studies showed that large genetic rearrangements (duplication or deletion of one or several exons) are of great importance in genetic diseases. Only few methods such as QMPSF and MLPA fit a routine use for these large deletions and duplications. They have to be performed additionally to a point mutation detection method (usually sequencing or dHPLC), which is labor intensive, time consuming and expensive. We present here a method called Enhanced Mismatch Mutation Analysis (EMMA) based on heteroduplex analysis that offers great sensitivity for point mutation detection and simultaneously detection of gross gene rearrangement by capillary electrophoresis.
 Material and Methods : EMMA is able to detect point mutations in PCR fragment ranging from 200 bp to 550 bp. It is thus possible to perform size multiplexing and analyze up to 6 fragments in the same run. PCR multiplex fragments of different sizes are prepared by semi-quantitative PCR. Separations are performed in multi-capillary electrophoresis. Poly(acrylamide-g-poly(dimethylacrylamide)) is used as a self coating sieving matrix; this separation matrix contains also additives like free nucleotides for a better sensitivity. One of the fragment is chosen as a reference for peak area normalization.
 Results : A series of 30 patients has been screened for BRCA1 and BRCA2 genes simultaneously by EMMA and the classical package dHPLC-QMPSF. All mutations and polymorphism detected by dHPLC have been detected by EMMA and new polymorphisms have been characterized for those genes. Furthermore, we will present the whole results of the positive mutation controls of BRCA1 and BRCA2 of the Institut Bergonie molecular genetic lab database screened by EMMA.
 Discussion : Using a unique condition of capillary electrophoresis separation for a lower number of PCR reaction for each gene (9 for BRCA1 and 15 for BRCA2), EMMA demonstrates a 5 times saving of lab time for technician, and displays a large easiness to use it in a routine way for molecular diagnosis. Finally this technology will benefit of a grant of French Ministry of Industry to be spread in molecular lab of Comprehensive Cancer Centers. Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 1102.