Massively Parallel Sequencing (MPS) pipelines are widely used in molecular diagnostics providing both qualitative and quantitative information from target sequences. Several type of genomic variations could be detected by means of MPS analysis including single nucleotide variants (SNVs), small insertions or deletions (indels), copy number variations (CNVs) and large genomic rearrangements (LGRs). In this study a novel lightweight software, namely Copy Number eValuation (CNeV), was developed to predict both CNV and LGRs in BRCA1/2 genes from Hereditary Breast and Ovarian Cancer (HBOC) selected population. Furthermore, inhouse High Resolution Melting Analysis (HRMA) protocol was performed to confirm in silico results.
Amplicon library were generated with Multiplicom BRCA MASTR Dx CE-IVD kit and assayed on Illumina/MiSeq platform following manufactures’ instructions. Coverage data were produced and analyzed with CNeV software to predict quantitative status of germ-line BRCA1/2 exons. Custom HRMA strategy was then performed to confirm positive predictions of CNV status using Albumin amplicons as a reference on a Roche LightCycler® 480 Real-Time PCR System (Roche Diagnostics). MLPA analysis was used as a confirmatory test for all samples.
A training set of 62 genotyped samples was used to validate the CNeV algorithm, all the 6 positive samples were detected (100 % sensitivity), while 14/56 negative samples were false positive (75 % specificity). All the positive results of CNeV algorithm were also confirmed by custom HRMA.
Large genomic rearrangements have recently been identified in HBOC families and account for a small but still significant proportion of cases. In fact, about 90 and 20 LGRs are reported as pathogenic variants in BRCA1 and BRCA2 carriers, respectively. Thus, structural variants must be included for a valid and complete molecular diagnostic workflow. In this scenario, we estimate that in silico analyses will provide an important reduction of costs and time instead of common “wet” quantitative ones. Finally, CNeV software coupled with amplicon-targeted custom HRMA assay was able to drastically reduce the number of MLPA test in the diagnostic routine workflow. Our preliminary results reveal that this strategy could strongly reduces the costs and time for BRCA testing by about 74 %.
for more information on the BRCA MASTR Dx.
Authors: G.L. Scaglione, E. De Paolis, A. Minucci, E.D. Capoluongo
Laboratory of Clinical Molecular and Personalized Diagnostics, Institute of Biochemistry and Clinical Biochemistry, Catholic University, Rome, Italy (Italy)