Table of Contents Author Guidelines Submit a Manuscript
BioMed Research International
Volume 2014, Article ID 542541, 8 pages
http://dx.doi.org/10.1155/2014/542541
Research Article

Genetic Testing in Hereditary Breast and Ovarian Cancer Using Massive Parallel Sequencing

1Laboratorio de Genética, UDIAT-Centre Diagnòstic, Corporació Sanitària Parc Taulí Institut Universitari (UAB), Parc Taulí s/n, Sabadell, 08208 Barcelona, Spain
2Unidad de Consejo Genético, Institut Oncològic del Vallès, Corporació Sanitària Parc Taulí, Parc Taulí s/n, Sabadell, 08208 Barcelona, Spain
3Departamento de Investigación Oncológica, Corporació Sanitària Parc Taulí Institut Universitari (UAB), Parc Taulí s/n, Sabadell, 08208 Barcelona, Spain
4Departamento de Oncología Médica, Corporació Sanitària Parc Taulí Institut Universitari (UAB), Parc Taulí s/n, Sabadell, 08208 Barcelona, Spain

Received 27 February 2014; Accepted 5 June 2014; Published 26 June 2014

Academic Editor: Ozgur Cogulu

Copyright © 2014 Anna Ruiz et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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.