title: Long-range PCR and next-generation sequencing of BRCA1 and BRCA2 in breast cancer.
abstract: Individuals and families carrying mutations in BRCA1 and BRCA2 (BRCA1/2) have a markedly elevated risk of developing breast and ovarian cancers. The first-generation of BRCA1/2 mutation analysis targeted only the coding exons and has implicated protein-truncating mutations (indel, nonsense) in BRCA1/2 inactivation. Recently, heritable breast cancers have also been attributed to other exonic mutations (missense, silent) and mutations in introns and untranslated regions. However, analysis of these alterations has been prohibitively laborious and cost intensive, and the proportion of cases carrying mutations in unscreened regions of BRCA1/2 and other predisposition genes is unknown. We have developed and validated a next-generation sequencing (NGS) approach for BRCA1/2 mutation analysis by applying long-range PCR and deep sequencing. Genomic DNA from familial breast cancer patients (N = 12) were screened and NGS successfully identified all 19 distinct (51 total) BRCA1 and 35 distinct (63 total) BRCA2 sequence alterations detectable by the Sanger sequencing, with no false-negative or positive results. In addition, we report the robust detection of variants from introns and untranslated regions. These results illustrate that NGS can provide comprehensive genetic information more quickly, accurately, and at a lower cost than conventional approaches, and we propose NGS to be a more effective method for BRCA1/2 mutational analysis. Advances in NGS will play an important role in enabling molecular diagnostics and personalized treatment of breast and ovarian cancers.
authors.affiliation: Fred A Litwin Centre for Cancer Genetics, Samuel Lunenfeld Research Institute, Mount Sinai Hospital, University of Toronto, Toronto, Ontario, Canada. ozcelik@lunenfeld.ca
authors.forename: Hilmi
authors.initials: H
authors.lastname: Ozcelik
authors.forename: Xuejiang
authors.initials: X
authors.lastname: Shi
authors.forename: Martin C
authors.initials: MC
authors.lastname: Chang
authors.forename: Eric
authors.initials: E
authors.lastname: Tram
authors.forename: Matt
authors.initials: M
authors.lastname: Vlasschaert
authors.forename: Nando
authors.initials: N
authors.lastname: Di Nicola
authors.forename: Anna
authors.initials: A
authors.lastname: Kiselova
authors.forename: Denise
authors.initials: D
authors.lastname: Yee
authors.forename: Aaron
authors.initials: A
authors.lastname: Goldman
authors.forename: Mark
authors.initials: M
authors.lastname: Dowar
authors.forename: Balram
authors.initials: B
authors.lastname: Sukhu
authors.forename: Rita
authors.initials: R
authors.lastname: Kandel
authors.forename: Kathy
authors.initials: K
authors.lastname: Siminovitch
mesh_terms: D001943:Breast Neoplasms / Q000175:diagnosis / Q000235:genetics*; D005260:Female; D019398:Genes, BRCA1; D024522:Genes, BRCA2; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D009154:Mutation; D016133:Polymerase Chain Reaction; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity