A comprehensive BRCA1/2 NGS pipeline for an immediate Copy Number Variation (CNV) detection in breast and ovarian cancer molecular diagnosis

ABSTRACTBackground:The aim of this study was to assess the ability of a Next Generation Sequencing (NGS)-based ap-proach in the detection ofBRCA1/2Large Genomic Rearrangements (LGRs).Methods:We investigated 287 consecutive unrelated Italian women affected by breast or ovarian cancer. A NGSpipeline with a reliable Copy number Variation (CNV) prediction algorithm was applied. In addition, all sampleswere investigated using MAQ (Multiplex Amplicon Quantification) assay, while the MLPA (Multiplex Ligation-dependent Probe Amplification) technique was used as alternative method if necessary.Results:Small pathogenic variants were identified in 80 samples. During NGS analysis, 11 samples were iden-tified by Amplicon Suite Software (SmartSeq, Novara, Italy) as positive for large deletions or duplications.However, MAQ assay provided positive results in 19 patients. In particular,BRCA2exon 25 deletion, not re-vealed by NGS CNV prediction algorithm, was detected in 8 unrelated women as false positive result. Sequencinganalysis showed IVS24-113T/G (c.9257-113T > G) variant in 6 of these 8 patients, while the novel IVS24-129G > A (c.9257-129G > A) variant was detected in only two samples.Discussion:The prevalence of large rearrangements inBRCA1/2genes was the 12% of all disease-causing mu-tations detected in our patients. In particular,BRCA1rearrangements were the 14.5% of allBRCA1causingvariants identified. Differently,BRCA2large deletions were only the 6.9% of all mutations occurring in this gene.While MAQ assay showed 2,8% of false positive results, our integrative NGS-based approach fully satisfied thesensitivity and specificity parameters required on theBRCA1/2LGRs detection. The workflow represents arobust and easy-to-use method for fullBRCA1/2screening, which can be easily implemented in routine dia-gnostic testing