title: Genetic testing in hereditary breast and ovarian cancer using massive parallel sequencing.
abstract: High throughput methods such as next generation sequencing are increasingly used in molecular diagnosis. The aim of this study was to develop a workflow for the detection of BRCA1 and BRCA2 mutations using massive parallel sequencing in a 454 GS Junior bench top sequencer. Our approach was first validated in a panel of 23 patients containing 62 unique variants that had been previously Sanger sequenced. Subsequently, 101 patients with familial breast and ovarian cancer were studied. BRCA1 and BRCA2 exon enrichment has been performed by PCR amplification using the BRCA MASTR kit (Multiplicom). Bioinformatic analysis of reads is performed with the AVA software v2.7 (Roche). In total, all 62 variants were detected resulting in a sensitivity of 100%. 71 false positives were called resulting in a specificity of 97.35%. All of them correspond to deletions located in homopolymeric stretches. The analysis of the homopolymers stretches of 6 bp or longer using the BRCA HP kit (Multiplicom) increased the specificity of the detection of BRCA1 and BRCA2 mutations to 99.99%. We show here that massive parallel pyrosequencing can be used as a diagnostic strategy to test for BRCA1 and BRCA2 mutations meeting very stringent sensitivity and specificity parameters replacing traditional Sanger sequencing with a lower cost.
authors.affiliation: Laboratorio de Genética, UDIAT-Centre Diagnòstic, Corporació Sanitària Parc Taulí Institut Universitari (UAB), Parc Taulí s/n, Sabadell, 08208 Barcelona, Spain.
authors.forename: Anna
authors.identifier: 0000-0001-7314-5962
authors.initials: A
authors.lastname: Ruiz
authors.affiliation: Unidad de Consejo Genético, Institut Oncològic del Vallès, Corporació Sanitària Parc Taulí, Parc Taulí s/n, Sabadell, 08208 Barcelona, Spain.
authors.forename: Gemma
authors.initials: G
authors.lastname: Llort
authors.affiliation: Unidad de Consejo Genético, Institut Oncològic del Vallès, Corporació Sanitària Parc Taulí, Parc Taulí s/n, Sabadell, 08208 Barcelona, Spain.
authors.forename: Carmen
authors.initials: C
authors.lastname: Yagüe
authors.affiliation: Laboratorio de Genética, UDIAT-Centre Diagnòstic, Corporació Sanitària Parc Taulí Institut Universitari (UAB), Parc Taulí s/n, Sabadell, 08208 Barcelona, Spain.
authors.forename: Neus
authors.identifier: 0000-0003-0677-240X
authors.initials: N
authors.lastname: Baena
authors.affiliation: Laboratorio de Genética, UDIAT-Centre Diagnòstic, Corporació Sanitària Parc Taulí Institut Universitari (UAB), Parc Taulí s/n, Sabadell, 08208 Barcelona, Spain.
authors.forename: Marina
authors.initials: M
authors.lastname: Viñas
authors.affiliation: Laboratorio de Genética, UDIAT-Centre Diagnòstic, Corporació Sanitària Parc Taulí Institut Universitari (UAB), Parc Taulí s/n, Sabadell, 08208 Barcelona, Spain.
authors.forename: Montse
authors.initials: M
authors.lastname: Torra
authors.affiliation: Departamento de Investigación Oncológica, Corporació Sanitària Parc Taulí Institut Universitari (UAB), Parc Taulí s/n, Sabadell, 08208 Barcelona, Spain.
authors.forename: Anna
authors.initials: A
authors.lastname: Brunet
authors.affiliation: Departamento de Oncología Médica, Corporació Sanitària Parc Taulí Institut Universitari (UAB), Parc Taulí s/n, Sabadell, 08208 Barcelona, Spain.
authors.forename: Miquel A
authors.initials: MA
authors.lastname: Seguí
authors.affiliation: Departamento de Oncología Médica, Corporació Sanitària Parc Taulí Institut Universitari (UAB), Parc Taulí s/n, Sabadell, 08208 Barcelona, Spain.
authors.forename: Eugeni
authors.initials: E
authors.lastname: Saigí
authors.affiliation: Laboratorio de Genética, UDIAT-Centre Diagnòstic, Corporació Sanitària Parc Taulí Institut Universitari (UAB), Parc Taulí s/n, Sabadell, 08208 Barcelona, Spain.
authors.forename: Miriam
authors.initials: M
authors.lastname: Guitart
mesh_terms: D019313:BRCA1 Protein / Q000235:genetics*; D024682:BRCA2 Protein / Q000235:genetics*; D001943:Breast Neoplasms / Q000235:genetics*; D004252:DNA Mutational Analysis / Q000379:methods; D005260:Female; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D009154:Mutation; D010051:Ovarian Neoplasms / Q000235:genetics*; D012680:Sensitivity and Specificity
chemical_list: D019313:BRCA1 Protein; C492913:BRCA1 protein, human; D024682:BRCA2 Protein; C551750:BRCA2 protein, human