Search

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. [ABSTRACT FROM AUTHOR]

Abstract: Background: The development of MALDI Imaging Mass Spectrometry is a promising technique in the investigation of biological molecular repertoire. We have pursued an analytical assessment of this technique in its application to proteome analysis. Methods: A specific statistical method of analysis has been developed to enable data processing in the absence of internal standards, by defining similarity scores. Results: The investigated linear mode MALDI-TOF set-up allows to obtain data variations comprised within the 30% of variation when assaying tissues samples from the same animal, while the 60% of variation was highlighted in the inter-mice series assaying syngenic animals. Conclusions: This analytical assessment represents the first step of a process that should validate the utilisation of this technique in the clinical practice. [Copyright &y& Elsevier]