title: The molecular analysis of BRCA1 and BRCA2: Next-generation sequencing supersedes conventional approaches.
abstract: BACKGROUND
  Accurate and sensitive detection of BRCA1/2 germ-line mutations is crucial for the clinical management of women affected by breast cancer, for prevention and, notably, also for the identification of at-risk healthy relatives. The most widely used methods for BRCA1/2 molecular analysis are Sanger sequencing, and denaturing high performance liquid chromatography (dHPLC) followed by the Sanger method. However, recent findings suggest that next-generation sequencing (NGS)-based approaches may be an efficient tool for diagnostic purposes. In this context, we evaluated the effectiveness of NGS for BRCA gene analysis compared with dHPLC/Sanger sequencing.
  
  
  METHODS
  Seventy women were screened for BRCA1/2 mutations by both dHPLC/Sanger sequencing and NGS, and the data were analyzed using a bioinformatic pipeline.
  
  
  RESULTS
  Sequence data analysis showed that NGS is more sensitive in detecting BRCA1/2 variants than the conventional procedure, namely, dHPLC/Sanger.
  
  
  CONCLUSIONS
  Next-generation sequencing is more sensitive, faster, easier to use and less expensive than the conventional Sanger method. Consequently, it is a reliable procedure for the routine molecular screening of the BRCA1/2 genes.
authors.affiliation: CEINGE-Biotecnologie Avanzate, via G. Salvatore 486, 80145 Naples, Italy; Department of Molecular Medicine and Medical Biotechnologies, University of Naples Federico II, Via Pansini 5, 80131 Naples, Italy.
authors.forename: Valeria
authors.initials: V
authors.lastname: D'Argenio
authors.affiliation: CEINGE-Biotecnologie Avanzate, via G. Salvatore 486, 80145 Naples, Italy.
authors.forename: Maria Valeria
authors.initials: MV
authors.lastname: Esposito
authors.affiliation: CEINGE-Biotecnologie Avanzate, via G. Salvatore 486, 80145 Naples, Italy.
authors.forename: Antonella
authors.initials: A
authors.lastname: Telese
authors.affiliation: CEINGE-Biotecnologie Avanzate, via G. Salvatore 486, 80145 Naples, Italy; Department of Molecular Medicine and Medical Biotechnologies, University of Naples Federico II, Via Pansini 5, 80131 Naples, Italy.
authors.forename: Vincenza
authors.initials: V
authors.lastname: Precone
authors.affiliation: CEINGE-Biotecnologie Avanzate, via G. Salvatore 486, 80145 Naples, Italy.
authors.forename: Flavio
authors.initials: F
authors.lastname: Starnone
authors.affiliation: CEINGE-Biotecnologie Avanzate, via G. Salvatore 486, 80145 Naples, Italy; Department of Molecular Medicine and Medical Biotechnologies, University of Naples Federico II, Via Pansini 5, 80131 Naples, Italy.
authors.forename: Marcella
authors.initials: M
authors.lastname: Nunziato
authors.affiliation: CEINGE-Biotecnologie Avanzate, via G. Salvatore 486, 80145 Naples, Italy.
authors.forename: Piergiuseppe
authors.initials: P
authors.lastname: Cantiello
authors.affiliation: CEINGE-Biotecnologie Avanzate, via G. Salvatore 486, 80145 Naples, Italy.
authors.forename: Mariangela
authors.initials: M
authors.lastname: Iorio
authors.affiliation: CEINGE-Biotecnologie Avanzate, via G. Salvatore 486, 80145 Naples, Italy.
authors.forename: Eloisa
authors.initials: E
authors.lastname: Evangelista
authors.affiliation: Department of Senology, Istituto Nazionale Tumori - IRCCS Fondazione Pascale, Naples, Italy.
authors.forename: Massimiliano
authors.initials: M
authors.lastname: D'Aiuto
authors.affiliation: Department of Senology, Istituto Nazionale Tumori - IRCCS Fondazione Pascale, Naples, Italy.
authors.forename: Alessandra
authors.initials: A
authors.lastname: Calabrese
authors.affiliation: CEINGE-Biotecnologie Avanzate, via G. Salvatore 486, 80145 Naples, Italy; Department of Molecular Medicine and Medical Biotechnologies, University of Naples Federico II, Via Pansini 5, 80131 Naples, Italy.
authors.forename: Giulia
authors.initials: G
authors.lastname: Frisso
authors.affiliation: Department of Senology, Istituto Nazionale Tumori - IRCCS Fondazione Pascale, Naples, Italy.
authors.forename: Giuseppe
authors.initials: G
authors.lastname: D'Aiuto
authors.affiliation: CEINGE-Biotecnologie Avanzate, via G. Salvatore 486, 80145 Naples, Italy; Department of Molecular Medicine and Medical Biotechnologies, University of Naples Federico II, Via Pansini 5, 80131 Naples, Italy; IRCCS-Fondazione SDN, Naples, Italy. Electronic address: salvator@unina.it.
authors.forename: Francesco
authors.initials: F
authors.lastname: Salvatore
mesh_terms: D000328:Adult; D019313:BRCA1 Protein / Q000235:genetics*; D024682:BRCA2 Protein / Q000235:genetics*; D001943:Breast Neoplasms / Q000175:diagnosis* / Q000235:genetics; D005091:Exons; D005260:Female; D015870:Gene Expression; D015723:Gene Library; D005820:Genetic Testing; D059014:High-Throughput Nucleotide Sequencing / Q000191:economics / Q000379:methods*; D006801:Humans; D007438:Introns; D008875:Middle Aged; D060885:Multiplex Polymerase Chain Reaction; D009154:Mutation; D013997:Time Factors
chemical_list: D019313:BRCA1 Protein; C492913:BRCA1 protein, human; D024682:BRCA2 Protein; C551750:BRCA2 protein, human
keywords: BRCA1; BRCA2; Method comparison in molecular diagnostics; Molecular diagnostics; Next-generation sequencing; dHPLC