title: Simultaneous detection of BRCA mutations and large genomic rearrangements in germline DNA and FFPE tumor samples.
abstract: The development of breast and ovarian cancer is strongly connected to the inactivation of the BRCA1 and BRCA2 genes by different germline and somatic alterations, and their diagnosis has great significance in targeted tumor therapy, since recently approved PARP inhibitors show high efficiency in the treatment of BRCA-deficient tumors. This raises the need for new diagnostic methods that are capable of performing an integrative mutation analysis of the BRCA genes not only from germline DNA but also from formalin-fixed and paraffin-embedded (FFPE) tumor samples. Here we describe the development of such a methodology based on next-generation sequencing and a new bioinformatics software for data analysis. The diagnostic method was initially developed on an Illumina MiSeq NGS platform using germline-mutated stem cell lines and then adapted for the Ion Torrent PGM NGS platform as well. We also investigated the usability of NGS coverage data for the detection of copy number variations and exon deletions as a replacement of the conventional MLPA technique. Finally, we tested the developed workflow on FFPE samples from breast and ovarian cancer patients. Our method meets the sensitivity and specificity requirements for the genetic diagnosis of breast and ovarian cancers both from germline and FFPE samples.
authors.affiliation: Institute of Genetics, Biological Research Centre of the Hungarian Academy of Sciences, Szeged 6726, Hungary.
authors.forename: Márton Zsolt
authors.lastname: Enyedi
authors.affiliation: Delta Bio 2000 Ltd., Szeged 6726, Hungary.
authors.forename: Gábor
authors.initials: G
authors.lastname: Jaksa
authors.affiliation: Delta Bio 2000 Ltd., Szeged 6726, Hungary.
authors.forename: Lajos
authors.initials: L
authors.lastname: Pintér
authors.affiliation: Department of Pathology, Faculty of Medicine, University of Szeged, Szeged 6720, Hungary.
authors.forename: Farkas
authors.initials: F
authors.lastname: Sükösd
authors.affiliation: Delta Bio 2000 Ltd., Szeged 6726, Hungary.
authors.forename: Zoltán
authors.initials: Z
authors.lastname: Gyuris
authors.affiliation: Delta Bio 2000 Ltd., Szeged 6726, Hungary.|Department of Pathology, Faculty of Medicine, University of Szeged, Szeged 6720, Hungary.
authors.forename: Adrienn
authors.initials: A
authors.lastname: Hajdu
authors.affiliation: Delta Bio 2000 Ltd., Szeged 6726, Hungary.
authors.forename: Erika
authors.initials: E
authors.lastname: Határvölgyi
authors.affiliation: Delta Bio 2000 Ltd., Szeged 6726, Hungary.
authors.forename: Katalin
authors.initials: K
authors.lastname: Priskin
authors.affiliation: Institute of Genetics, Biological Research Centre of the Hungarian Academy of Sciences, Szeged 6726, Hungary.
authors.forename: Lajos
authors.initials: L
authors.lastname: Haracska
mesh_terms: D019313:BRCA1 Protein / Q000235:genetics*; D024682:BRCA2 Protein / Q000235:genetics*; D001943:Breast Neoplasms / Q000175:diagnosis / Q000235:genetics; D019295:Computational Biology; D004247:DNA / Q000235:genetics; D004252:DNA Mutational Analysis; D005260:Female; D005557:Formaldehyde; D015321:Gene Rearrangement; D015894:Genome, Human; D018095:Germ-Line Mutation; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D009154:Mutation; D010051:Ovarian Neoplasms / Q000175:diagnosis / Q000235:genetics; D010232:Paraffin; D013234:Stem Cells / Q000378:metabolism
chemical_list: D019313:BRCA1 Protein; C492913:BRCA1 protein, human; D024682:BRCA2 Protein; C551750:BRCA2 protein, human; D005557:Formaldehyde; D010232:Paraffin; D004247:DNA
keywords: BRCA1-BRCA2; FFPE; NGS; germline; multiplex PCR