European Journal of Human Genetics - Abstract of article: Next-generation sequencing for the diagnosis of hereditary breast and ovarian cancer using genomic capture targeting multiple candidate genes

European Journal of Human Genetics - Abstract of article: Next-generation sequencing for the diagnosis of hereditary breast and ovarian cancer using genomic capture targeting multiple candidate genes

Article

European Journal of Human Genetics advance online publication 19 February 2014; doi: 10.1038/ejhg.2014.16

Next-generation sequencing for the diagnosis of hereditary breast and ovarian cancer using genomic capture targeting multiple candidate genes

Laurent Castéra^1,2, Sophie Krieger^1,2,3, Antoine Rousselin¹, Angélina Legros¹, Jean-Jacques Baumann¹, Olivia Bruet¹, Baptiste Brault¹, Robin Fouillet¹, Nicolas Goardon¹, Olivier Letac¹, Stéphanie Baert-Desurmont^2,4, Julie Tinat^2,4, Odile Bera⁵, Catherine Dugast⁶, Pascaline Berthet⁷, Florence Polycarpe⁷, Valérie Layet⁸, Agnes Hardouin^1,2, Thierry Frébourg^2,4,9 and Dominique Vaur^1,2

1. ¹Department of Cancer Biology and Genetics, CLCC François Baclesse, Caen, France
2. ²Inserm U1079, Rouen, France
3. ³Department of Biochemistry and Toxicology, Caen University, Caen, France
4. ⁴Department of Genetics, University Hospital, Rouen, France
5. ⁵Department of Genetics, University Hospital, Fort-de-France, France
6. ⁶Department of Genetics, University Hospital, Rennes, France
7. ⁷Department of Genetics, CLCC François Baclesse, Caen, France
8. ⁸Department of Genetics, Jacques Monod Hospital, Le Havre, France
9. ⁹Rouen University, IRIB, Rouen, France

Correspondence: Dr D Vaur, Centre François Baclesse, Laboratoire de Biologie et de Génétique du Cancer; 3, avenue du général Harris, Caen 14076 Cedex 05, France. Tel: +33 231 45 50 54; Fax: +33 2 31 45 50 53; E-mail: d.vaur@baclesse.fr

Received 7 October 2013; Revised 15 January 2014; Accepted 16 January 2014
Advance online publication 19 February 2014

Topof page

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 BRCA1and 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 inPMS1 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.

Keywords: 

NGS; breast; ovary; cancer; BRCA1; BRCA2