Detection of Genomic Variations in BRCA1 and BRCA2 Genes by Long-Range RCA and Next-Generation Sequencing

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 x 7 (in exon 22 of BRCA1) to > X 150. We detected and confirmed 100% of the mutations that predispose to the risk of cancer, together with other genoraic variations in BRCA1 and BRC42. 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. (J Mol Diagn 2012, 14.286-293; DOI: 10.1016/j.jmoldx.2012.01.013)