title: Detection of genomic variations in BRCA1 and BRCA2 genes by long-range PCR and next-generation sequencing.
abstract: Advances in sequencing technologies, such as next-generation sequencing (NGS), represent an opportunity to perform genetic testing in a clinical scenario. In this study, we developed and tested a method for the detection of mutations in the large BRCA1 and BRCA2 tumor suppressor genes, using long-range PCR (LR-PCR) and NGS, in samples from individuals with a personal and/or family history of breast and/or ovarian cancer. Eleven LR-PCR fragments, between 3000 and 15,300 bp, containing all coding exons and flanking splice junctions of BRCA1 and BRCA2, were obtained from DNA samples of five individuals carrying mutations in either BRCA1 or BRCA2. Libraries for NGS were prepared using an enzymatic (Nextera technology) method. We analyzed five individual samples in parallel by NGS and obtained complete coverage of all LR-PCR fragments, with an average coding sequence depth for each nucleotide of >30 reads, running from ×7 (in exon 22 of BRCA1) to >×150. We detected and confirmed 100% of the mutations that predispose to the risk of cancer, together with other genomic variations in BRCA1 and BRCA2. Our approach demonstrates that genomic LR-PCR, together with NGS, using the GS Junior 454 System platform, is an effective method for patient sample analysis of BRCA1 and BRCA2 genes. In addition, this method could be performed in regular molecular genetics laboratories.
authors.affiliation: Molecular Genetics Unit, Hospital of Terrassa, Terrassa, Spain.
authors.forename: Imma
authors.initials: I
authors.lastname: Hernan
authors.forename: Emma
authors.initials: E
authors.lastname: Borràs
authors.forename: Miguel
authors.initials: M
authors.lastname: de Sousa Dias
authors.forename: María José
authors.initials: MJ
authors.lastname: Gamundi
authors.forename: Begoña
authors.initials: B
authors.lastname: Mañé
authors.forename: Gemma
authors.initials: G
authors.lastname: Llort
authors.forename: José A G
authors.initials: JA
authors.lastname: Agúndez
authors.forename: Miguel
authors.initials: M
authors.lastname: Blanca
authors.forename: Miguel
authors.initials: M
authors.lastname: Carballo
mesh_terms: D019313:BRCA1 Protein / Q000235:genetics; D024682:BRCA2 Protein / Q000235:genetics; D001483:Base Sequence; D001943:Breast Neoplasms / Q000175:diagnosis* / Q000235:genetics; D058893:DNA Barcoding, Taxonomic / Q000379:methods; D004252:DNA Mutational Analysis / Q000379:methods*; D005260:Female; D019398:Genes, BRCA1; D024522:Genes, BRCA2; D005820:Genetic Testing; D014644:Genetic Variation; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D009154:Mutation; D021141:Nucleic Acid Amplification Techniques; D010051:Ovarian Neoplasms / Q000175:diagnosis* / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods
chemical_list: D019313:BRCA1 Protein; C492913:BRCA1 protein, human; D024682:BRCA2 Protein; C551750:BRCA2 protein, human