Current Methods for BRCA1/BRCA2 molecular analysis
BRCA testing is commonly performed by direct Sanger sequencing. This method is considered the ‘gold standard’ of DNA sequencing. It is technologically reliable, widely available, and has a relatively simple workflow.
The drawbacks of Sanger sequencing are:
• limited throughput
• lower cost-effectiveness compared to next-generation sequencing (NGS)
• inability to detect large genomic rearrangements (LGRs; e.g.: large deletions/duplications)
As large deletion/duplications have been reported in the BRCA1 and BRCA2 genes, it is necessary to use alternative methods in order to detect them. The most commonly used method for analyzing LGRs is multiplex ligation-dependent probe amplification (MLPA). This technology is based on a mix of synthetic probes. Each of them is composed by two oligonucleotides which are able to bind to DNA regions in the genomic area of interest and amplify in a semiquantitative manner only if both oligonucleotides are bound and ligated. Examples of commercially available, research use-only BRCA testing MLPA probes are P002/P087 for BRCA1 and P077/P045/P090 for BRCA2 (MRC-Holland, Amsterdam, The Netherlands). MRC-Holland suggests to confirm deletions or duplications detected by a single kit using the other gene-related kits.
Otherwise, it is possible to proceed to the BRCA1/2 deletion/duplication testing by quantitative PCR (qPCR). However, although qPCR is a reliable method, a complete analysis of BRCA1 and BRCA2 genes is an intensive effort for the laboratory, given the amount of exons in BRCA1 and BRCA2.
The rapid evolution of massively parallel sequencing technology is revolutionising the management of inherited diseases. A number of early clinical studies in BRCA testing have already shown that NGS technology offers high sensitivity, specificity, rapid mutational analysis of multiple genes, and cost-effectiveness compared to current approaches, although the need for careful sequence analysis to avoid false positive results becomes a main issue, especially in case of small insertions/deletions, variant detected in repetitive regions etc. and additional parameters as coverage, rate of reads and quality reads assessment become crucial.
Recently, NGS benchtop platforms have made available gigabase-scale DNA sequencing with relatively short run times (24 h), for example MiSeq (Illumina, San Diego, CA, USA) or Ion Torrent Personal Genome Machine (PGM; Life Technologies, Carlsbad, CA, USA). The potential of high-volume analytical throughput makes NGS platforms an increasingly attractive investment for diagnostic laboratories in the clinical setting.
BRCA testing with NGS technology offers many advantages over Sanger sequencing, including the potential to detect LGRs in a single workflow, although NGS LGR detection has not been fully established in the diagnostic setting.