title: Next-generation sequencing for the diagnosis of hereditary breast and ovarian cancer using genomic capture targeting multiple candidate genes.
abstract: To optimize the molecular diagnosis of hereditary breast and ovarian cancer (HBOC), we developed a next-generation sequencing (NGS)-based screening based on the capture of a panel of genes involved, or suspected to be involved in HBOC, on pooling of indexed DNA and on paired-end sequencing in an Illumina GAIIx platform, followed by confirmation by Sanger sequencing or MLPA/QMPSF. The bioinformatic pipeline included CASAVA, NextGENe, CNVseq and Alamut-HT. We validated this procedure by the analysis of 59 patients' DNAs harbouring SNVs, indels or large genomic rearrangements of BRCA1 or BRCA2. We also conducted a blind study in 168 patients comparing NGS versus Sanger sequencing or MLPA analyses of BRCA1 and BRCA2. All mutations detected by conventional procedures were detected by NGS. We then screened, using three different versions of the capture set, a large series of 708 consecutive patients. We detected in these patients 69 germline deleterious alterations within BRCA1 and BRCA2, and 4 TP53 mutations in 468 patients also tested for this gene. We also found 36 variations inducing either a premature codon stop or a splicing defect among other genes: 5/708 in CHEK2, 3/708 in RAD51C, 1/708 in RAD50, 7/708 in PALB2, 3/708 in MRE11A, 5/708 in ATM, 3/708 in NBS1, 1/708 in CDH1, 3/468 in MSH2, 2/468 in PMS2, 1/708 in BARD1, 1/468 in PMS1 and 1/468 in MLH3. These results demonstrate the efficiency of NGS in performing molecular diagnosis of HBOC. Detection of mutations within other genes than BRCA1 and BRCA2 highlights the genetic heterogeneity of HBOC.
authors.affiliation: 1] Department of Cancer Biology and Genetics, CLCC François Baclesse, Caen, France [2] Inserm U1079, Rouen, France.
authors.forename: Laurent
authors.initials: L
authors.lastname: Castéra
authors.affiliation: 1] Department of Cancer Biology and Genetics, CLCC François Baclesse, Caen, France [2] Inserm U1079, Rouen, France [3] Department of Biochemistry and Toxicology, Caen University, Caen, France.
authors.forename: Sophie
authors.initials: S
authors.lastname: Krieger
authors.affiliation: Department of Cancer Biology and Genetics, CLCC François Baclesse, Caen, France.
authors.forename: Antoine
authors.initials: A
authors.lastname: Rousselin
authors.affiliation: Department of Cancer Biology and Genetics, CLCC François Baclesse, Caen, France.
authors.forename: Angélina
authors.initials: A
authors.lastname: Legros
authors.affiliation: Department of Cancer Biology and Genetics, CLCC François Baclesse, Caen, France.
authors.forename: Jean-Jacques
authors.initials: JJ
authors.lastname: Baumann
authors.affiliation: Department of Cancer Biology and Genetics, CLCC François Baclesse, Caen, France.
authors.forename: Olivia
authors.initials: O
authors.lastname: Bruet
authors.affiliation: Department of Cancer Biology and Genetics, CLCC François Baclesse, Caen, France.
authors.forename: Baptiste
authors.initials: B
authors.lastname: Brault
authors.affiliation: Department of Cancer Biology and Genetics, CLCC François Baclesse, Caen, France.
authors.forename: Robin
authors.initials: R
authors.lastname: Fouillet
authors.affiliation: Department of Cancer Biology and Genetics, CLCC François Baclesse, Caen, France.
authors.forename: Nicolas
authors.initials: N
authors.lastname: Goardon
authors.affiliation: Department of Cancer Biology and Genetics, CLCC François Baclesse, Caen, France.
authors.forename: Olivier
authors.initials: O
authors.lastname: Letac
authors.affiliation: 1] Inserm U1079, Rouen, France [2] Department of Genetics, University Hospital, Rouen, France.
authors.forename: Stéphanie
authors.initials: S
authors.lastname: Baert-Desurmont
authors.affiliation: 1] Inserm U1079, Rouen, France [2] Department of Genetics, University Hospital, Rouen, France.
authors.forename: Julie
authors.initials: J
authors.lastname: Tinat
authors.affiliation: Department of Genetics, University Hospital, Fort-de-France, France.
authors.forename: Odile
authors.initials: O
authors.lastname: Bera
authors.affiliation: Department of Genetics, University Hospital, Rennes, France.
authors.forename: Catherine
authors.initials: C
authors.lastname: Dugast
authors.affiliation: Department of Genetics, CLCC François Baclesse, Caen, France.
authors.forename: Pascaline
authors.initials: P
authors.lastname: Berthet
authors.affiliation: Department of Genetics, CLCC François Baclesse, Caen, France.
authors.forename: Florence
authors.initials: F
authors.lastname: Polycarpe
authors.affiliation: Department of Genetics, Jacques Monod Hospital, Le Havre, France.
authors.forename: Valérie
authors.initials: V
authors.lastname: Layet
authors.affiliation: 1] Department of Cancer Biology and Genetics, CLCC François Baclesse, Caen, France [2] Inserm U1079, Rouen, France.
authors.forename: Agnes
authors.initials: A
authors.lastname: Hardouin
authors.affiliation: 1] Inserm U1079, Rouen, France [2] Department of Genetics, University Hospital, Rouen, France [3] Rouen University, IRIB, Rouen, France.
authors.forename: Thierry
authors.initials: T
authors.lastname: Frébourg
authors.affiliation: 1] Department of Cancer Biology and Genetics, CLCC François Baclesse, Caen, France [2] Inserm U1079, Rouen, France.
authors.forename: Dominique
authors.initials: D
authors.lastname: Vaur
mesh_terms: D000328:Adult; D000368:Aged; D019313:BRCA1 Protein / Q000235:genetics / Q000378:metabolism; D024682:BRCA2 Protein / Q000235:genetics / Q000378:metabolism; D001943:Breast Neoplasms / Q000175:diagnosis / Q000235:genetics; D018567:Breast Neoplasms, Male / Q000175:diagnosis / Q000235:genetics*; D016022:Case-Control Studies; D019295:Computational Biology; D005260:Female; D015321:Gene Rearrangement; D020022:Genetic Predisposition to Disease; D005820:Genetic Testing; D023281:Genomics / Q000379:methods*; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D008297:Male; D008875:Middle Aged; D009154:Mutation; D010051:Ovarian Neoplasms / Q000175:diagnosis / Q000235:genetics*; D015203:Reproducibility of Results; D016159:Tumor Suppressor Protein p53 / Q000235:genetics / Q000378:metabolism
chemical_list: D019313:BRCA1 Protein; C492913:BRCA1 protein, human; D024682:BRCA2 Protein; C551750:BRCA2 protein, human; C495901:TP53 protein, human; D016159:Tumor Suppressor Protein p53