A low-cost next-generation sequencing (NGS) approach to detecting a full spectrum of BRCA1 and BRCA2 mutations in individual breast cancers could help speed the identification of new disease-related alterations, make routine clinical genotyping feasible in breast cancer patients and at-risk individuals, and help ensure patients are prescribed the most effective therapies, scientists claim. Existing sequencing approaches to analyzing BRCA1 and BRCA2 mutations are prohibitively laborious and expensive for routine use, and have focused primarily on the identification of protein-truncating mutations in coding exons. This means that other types of exonic changes and alterations in introns and untranslated regions of the two genes have remained largely unexplored, even though recent research indicates such mutations may play a role in heritable breast cancer. As a result, the proportion of breast cancer patients carrying tumor-relevant mutations in unscreened regions of BRCA1/2 or other predisposition genes remains unknown.
A Canadian team led by researchers at the University of Toronto’s Mount Sinai Hospital now reports on the development and validation of approach to sequencing BRCA1 and 2 that uses long-range PCR to generate BRCA1/2 amplicons for analysis using NGS technology. In contrast with conventional screening methods that target just the BRCA1 and BRCA2 exons, the deep sequencing approach can screen the entire genomic region, including introns and untranslated regions.
K. Siminovitch, M.D., et al tested the technology to screen genomic DNA from 12 familial breast cancer patients for alterations in the BRCA1 and BRCA2 genes. The results showed that, as well as picking out all the genetic changes that were identified by Sanger sequencing, the NGS approach detected variants in introns and untranslated regions. Importantly, the analyses were carried out cheaply and with a turnaround time of just 12 days. And despite the small sample size, the data showed a large variability in the number, type, and frequency of variants identified from the patients.
The Toronto-based team report this in the Journal of Molecular Diagnostics in a paper titled “Long-range PCR and next-generation sequencing of BRCA1 and BRCA2 in breast cancer.” Lead author H. Ozcelik, Ph.D., says one of the key advantages of an approach that applies long-range PCR is the ability to detect large genomic duplications, deletions, and insertions.
“When combined with next-generation sequencing, long-range PCR can be a powerful tool in the detection of BRCA variants in the clinical setting,” he states. “Our challenge now is to establish analytical methods that systematically investigate this more comprehensive data in order to provide better risk information for clinical management of the disease. Given the extensive level of genetic information acquired from each patient, profiles can be constructed in breast cancer patients compared to population controls to produce a more effective means of generating BRCA1/2-associated risk to the individuals and their families.”