Next-Generation Sequencing of the BRCA1 and BRCA2 Genes for the Genetic Diagnostics of Hereditary Breast and/or Ovarian Cancer

Abstract

Genetic testing for hereditary breast and/or ovarian cancer mostly relies on laborious molecular tools that use Sanger sequencing to scan for mutations in the BRCA1 and BRCA2 genes. We explored a more efficient genetic screening strategy based on next-generation sequencing of the BRCA1 and BRCA2 genes in 210 hereditary breast and/or ovarian cancer patients. We first validated this approach in a cohort of 115 samples with previously known BRCA1 and BRCA2 mutations and polymorphisms. Genomic DNA was amplified using the Ion AmpliSeq BRCA1 and BRCA2 panel. The DNA Libraries were pooled, barcoded, and sequenced using an Ion Torrent Personal Genome Machine sequencer. The combination of different robust bioinformatics tools allowed detection of all previously known pathogenic mutations and polymorphisms in the 115 samples, without detecting spurious pathogenic calls. We then used the same assay in a discovery cohort of 95 uncharacterized hereditary breast and/or ovarian cancer patients for BRCA1 and BRCA2. In addition, we describe the allelic frequencies across 210 hereditary breast and/or ovarian cancer patients of 74 unique definitely and likely pathogenic and uncertain BRCA1 and BRCA2 variants, some of which have not been previously annotated in the public databases. Targeted next-generation sequencing is ready to substitute classic molecular methods to perform genetic testing on the BRCA1 and BRCA2 genes and provides a greater opportunity for more comprehensive testing of at-risk patients.

HBOC multi-gene panel testing: comparison of two sequencing centers

“…For example, Cybulski et al [29] used exome sequencing that offers the highest flexibility in target selection but still seems to be too expensive and not sensitive enough for routine diagnostics. In contrast, other studies focused on solely sequencing of BRCA1 and BRCA2, probably missing a relevant fraction of pathogenic mutations in other HBOC genes including TP53 and PTEN [30,31]. All 10 genes that were included in this study are known to be high-or intermediate-risk genes for HBOC [15,32,33].…”

“…The distribution of low-coverage regions is important, since cost and expenditure of time increase with the number of exons that have to be reanalyzed with a second independent method (e.g., Sanger sequencing). Some reports confirm that low-coverage regions within the target regions may occur, although additional experiments are not always required [30,35,38]. These differences may be attributable to the definition of the target region and experimental setup.…”

Information Topics of Greatest Interest for Return of Genome Sequencing Results among Women Diagnosed with Breast Cancer at a Young Age

“…Breast cancer patients who carry inherited germline mutations contributing to breast cancer risk, such as BRCA1 or BRCA2 , are at increased risk for a second primary breast cancer and other cancers (Mavaddat et al 2013); patients who are diagnosed at age 40 or younger are more likely to carry such gene mutations (Anders et al 2008; Bonadona et al 2005; Golshan et al 2006). For carriers of BRCA1 or BRCA2 , genome sequencing can be beneficial for surveillance and surgical decisions (Heemskerk-Gerritsen et al 2015; Ingham et al 2013; Riedl et al 2014; Sieh et al 2014; Trujillano et al 2015). Moreover, even for BRCA1 or BRCA2 -negative patients, genome sequencing may be beneficial in identifying other genes, such as PALB2 , CHEK2 , and ATM , that may confer an increased risk of familial breast cancer (Cybulski et al 2011; Desmond et al 2015; Janatova et al 2013; Renwick et al 2006).…”

Clinical Next-Generation Sequencing Pipeline Outperforms a Combined Approach Using Sanger Sequencing and Multiplex Ligation-Dependent Probe Amplification in Targeted Gene Panel Analysis

“…Because of the relatively large size of the BRCA1/2 genes and the allelic heterogeneity of the pathogenic mutations, clinical testing of these genes is technically complex and labor intensive and, as such, is currently restricted to screening high-risk patients as determined by their family cancer history or ethnic origin in the case of identified founder mutations. 4 Methods for mutation screening of the BRCA1 and BRCA2 genes have evolved since these genes were first characterized and testing was initiated in the mid1990s. 5 The earliest approaches to BRCA1/2 gene analysis involved indirect methods of mutation identification, such as protein truncation analysis which did not detect potentially damaging missense mutations or structural gene rearrangements.…”

“…Recent improvements to NGS technologies that facilitate massively parallel analysis of multiple genes have enabled the implementation of more cost-effective, rapid, and high-throughput methods to allow precise identification of BRCA1 and BRCA2 mutations in these high-risk patients, as well as testing of more comprehensive gene panels for cancer predisposition syndromes. 4,9,10 NGS is also capable of sensitive detection of sequence variants and may also be used for detection of copy number variants (CNVs), such as exon deletions and duplications that currently require use of MLPA or other copy number technologies.…”