title: Molecular analysis of the breast cancer genes BRCA1 and BRCA2 using amplicon-based massive parallel pyrosequencing.
abstract: The aim of this study was to implement the massively parallel sequencing technology for diagnostic applications. We evaluated an amplicon-based method for the analysis of the BRCA1 and BRCA2 genes on the Roche 454 GS-FLX sequencer, to identify disease-causing mutations in breast and/or ovarian cancer patients. A first evaluation relied on the analysis of DNA fragments containing known mutations. Secondly, the entire coding regions of the BRCA1 and BRCA2 genes were interrogated in more than 400 patient samples, using a multiplex PCR-based assay. Variants were filtered on the basis of their frequency (20%) and sequencing depth (>25×). Special attention was given to sequencing accuracy in homopolymers. In the initial evaluation, all known heterozygous mutations were detected. The percentage of mutant reads ranged from 22% to 62%. For the multiplex assay, 95% sensitivity and 91% specificity were obtained. In addition, we were able to reliably distinguish mutations from noise through the analysis of the raw signal intensities in homopolymers. This work presents an evaluation of the next-generation sequencing for use in diagnostics, based on a relatively high number of samples and experiments. We anticipate that the technique would further improve, and would allow reducing the costs per analysis and the turn-around time, to benefit patients who undergo BRCA molecular testing.
authors.affiliation: Center for Human Genetics, University of Leuven, Leuven, Belgium.
authors.forename: Geneviève
authors.initials: G
authors.lastname: Michils
authors.forename: Silke
authors.initials: S
authors.lastname: Hollants
authors.forename: Luc
authors.initials: L
authors.lastname: Dehaspe
authors.forename: Jeroen
authors.initials: J
authors.lastname: Van Houdt
authors.forename: Yannick
authors.initials: Y
authors.lastname: Bidet
authors.forename: Nancy
authors.initials: N
authors.lastname: Uhrhammer
authors.forename: Yves-Jean
authors.initials: YJ
authors.lastname: Bignon
authors.forename: Joris R
authors.initials: JR
authors.lastname: Vermeesch
authors.forename: Harry
authors.initials: H
authors.lastname: Cuppens
authors.forename: Gert
authors.initials: G
authors.lastname: Matthijs
mesh_terms: D019313:BRCA1 Protein / Q000235:genetics*; D024682:BRCA2 Protein / Q000235:genetics*; D001940:Breast / Q000378:metabolism; D001943:Breast Neoplasms / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000379:methods*; D005260:Female; D005820:Genetic Testing; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D009154:Mutation; D010051:Ovarian Neoplasms / Q000235:genetics*; D010053:Ovary / Q000378:metabolism; D012680:Sensitivity and Specificity
chemical_list: D019313:BRCA1 Protein; D024682:BRCA2 Protein