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Diagnostic Strategy for Analytical Scanning of BRCA1 Gene by Fluorescence-assisted Mismatch Analysis Using Large, Bifluorescently Labeled Amplicons¹

Unité Immunogénétique et Unité/Institut National de la Santé et de la Recherche Médicale (INSERM) 276, Institut Pasteur, Paris, France [E. R., T. M., M. T.]; Department of Experimental Medicine, University of L’Aquila, L’Aquila, Italy [E. R., P. M., C. F., S. M.]; Service de Génétique Oncologique/INSERM CRI 9703, Institut Paoli Calmettes, Marseille, France [H. S.]; Unité de Génétique Oncologique, Institut Curie, Paris, France [D. S-L.]; Department Experimental Medicine and Pathology, University "La Sapienza," Rome, Italy [A. G.] and Neuromed Institute, Pozzilli, Isernia, Italy [A. G.]

The aim of this study was to develop a protocol for reliable, sensitive, and cost-effective mutation scanning of the BRCA1 gene, based on a modification of fluorescence-assisted mismatch analysis. The main features of this method are: (a) robust PCR amplification and strandspecific labeling of 25 large amplicons using uniform conditions and universal fluorescent primers; and (b) sensitive characterization of the position of sequence changes. The diagnostic accuracy of this method was tested by scanning the large exon 11 in 12 DNA samples with reported mutations. In a blind test, specific patterns of fluorescence profiles were obtained, and all were attributed correctly, without sequencing, to each mutation or polymorphism. Seven breast/ovarian cancer families with high probability of BRCA1-related predisposition were screened. Three truncating mutations (of which one was novel and three were missense changes, including two novel ones) were detected. The three missense mutations affect the highly conserved BRCT domain. Scanning by FAMA appears to be free of biases for particular types of sequence changes—except for exon deletions/duplications, which cannot be detected by conventional PCR-based methods—and allows substantial savings in the number of sequencing reactions and in the time invested in their interpretation. Therefore, it lends itself to screening structurally complex loci in the diagnostic context and in other fields of genetic analysis.

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