Rapid detection of copy number variations and point mutations in BRCA1/2 genes using a single workflow by ion semiconductor sequencing pipeline

Abstract

Molecular analysis of BRCA1 (MIM# 604370) and BRCA2 (MIM #600185) genes is essential for familial breast and ovarian cancer prevention and treatment. An efficient, rapid, cost-effective accurate strategy for the detection of pathogenic variants is crucial. Mutations detection of BRCA1/2 genes includes screening for single nucleotide variants (SNVs), small insertions or deletions (indels), and Copy Number Variations (CNVs). Sanger sequencing is unable to identify CNVs and therefore Multiplex Ligation Probe amplification (MLPA) or Multiplex Amplicon Quantification (MAQ) is used to complete the BRCA1/2 genes analysis. The rapid evolution of Next Generation Sequencing (NGS) technologies allows the search for point mutations and CNVs with a single platform and workflow. In this study we test the possibilities of NGS technology to simultaneously detect point mutations and CNVs in BRCA1/2 genes, using the OncomineTM BRCA Research Assay on Personal Genome Machine (PGM) Platform with Ion Reporter Software for sequencing data analysis (Thermo Fisher Scientific). Comparison between the NGS-CNVs, MLPA and MAQ results shows how the NGS approach is the most complete and fast method for the simultaneous detection of all BRCA mutations, avoiding the usual time consuming multistep approach in the routine diagnostic testing of hereditary breast and ovarian cancers.

Screening of BRCA1/2 genes mutations and copy number variations in patients with high risk for hereditary breast and ovarian cancer syndrome (HBOC)

“…Copy number variation in both genes was reported by Wen-Ming Cao et al [ 35 ] with a percentage of 16.1% (5/31). While, other studies state that BRCA1/2 CNVs for HBOC patients is rarely found [ 64 , 65 ]. More global studies are required to define the implication of BRCA1/2 CNVs in HBOC.…”

Germline copy number variations in BRCA1/2 negative families: Role in the molecular etiology of hereditary breast cancer in Tunisia

“…Pathogenic CNVs are more frequent in BRCA1 than BRCA2 and reach respectively 27% and 8% of BRCA genetic variations. This may be explained by the higher number of Alu sequences in BRCA1 , and also by the homologous recombination events that occur between BRCA1 and its pseudogene [ 10 , 11 ]. The highest contribution of BRCA1 CNVs was reported in the Dutch population in which 27% to 36% of all germline BRCA1 mutations are CNVs [ 6 ].…”

Applications of Next Generation Sequencing to the Analysis of Familial Breast/Ovarian Cancer

“…The advent of NGS technologies made it possible to perform multi-gene massive sequencing, with very sensitive detection of gene variants, even complex, and copy number variations as well [62,63]. Several works [64][65][66][67] demonstrated the advantage of using NGS with respect to traditional methods for BRCA variant identification in terms of sensitivity, cost and time reduction. Importantly, the latter is very relevant for therapy decision-making, in consideration of the possible use of anti-PARP drugs for ovarian and breast cancer treatment [68][69][70].…”

“…Some reports [66,79,80] described simultaneous NGS sequencing data collection and identification of BRCA rearrangements, simply by evaluating the presence of copy number variations (CNVs). This provides a fast and useful global first-step analysis that leads to more in-depth confirmatory investigation on positive samples by complementary tests, such as MLPA.…”