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

Background: The aim of this study was to assess the ability of a Next Generation Sequencing (NGS)-based approach in the detection of BRCA1/2 Large Genomic Rearrangements (LGRs). Methods: We investigated 287 consecutive unrelated Italian women affected by breast or ovarian cancer. A NGS pipeline with a reliable Copy number Variation (CNV) prediction algorithm was applied. In addition, all samples were 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 identified 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, BRCA2 exon 25 deletion, not revealed by NGS CNV prediction algorithm, was detected in 8 unrelated women as false positive result. Sequencing analysis 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 in BRCA1/2 genes was the 12% of all disease-causing mutations detected in our patients. In particular, BRCA1 rearrangements were the 14.5% of all BRCA1 causing variants identified. Differently, BRCA2 large 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 the sensitivity and specificity parameters required on the BRCA1/2 LGRs detection. The workflow represents a robust and easy-to-use method for full BRCA1/2 screening, which can be easily implemented in routine diagnostic testing.