A New Rapid Methodological Strategy to Assess BRCA Mutational Status
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RESEARCH
A New Rapid Methodological Strategy to Assess BRCA Mutational Status Emilia Vuttariello • Marco Borra • Celeste Calise • Elvira Mauriello • Stefano Greggi • Aldo Vecchione Elio Biffali • Gennaro Chiappetta
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Published online: 26 January 2013 Ó The Author(s) 2013. This article is published with open access at Springerlink.com
Abstract Hereditary cancers account for approximately 10 % of breast and ovarian cancers. Mutations of the BRCA1 and BRCA2 genes, encoding two proteins involved in DNA repair, underlie most cases of such hereditary cancers. Women with BRCA mutations develop breast cancer in 50–80 % of cases and ovarian cancer in 10–40 % of cases. Assessing BRCA mutational status is needed to direct the clinical management of women with predisposition to these hereditary cancers. However, BRCA screening constitutes a bottleneck in terms of costs and time to deliver results. We developed a PCR-based assay using 73 primer pairs covering the entire coding regions of BRCA1 and BRCA2. PCR primers, containing at the 5’ end the universal M13 primer sequences, were pre-spotted in 96-well plates. Following PCR, direct sequencing was performed using M13 primers, allowing to standardize the conditions. PCR amplification and sequencing were successful for each amplicon. We tested and validated the assay on 10 known gDNAs from patients with Hereditary breast and ovarian cancer (HBOC). Our
E. Vuttariello C. Calise G. Chiappetta (&) Functional Genomic Unit, National Cancer Institute, Fondazione ‘‘G.Pascale’’, Via Mariano Semmola, 80131 Naples, Italy e-mail: [email protected] M. Borra E. Mauriello E. Biffali Molecular Biology Service, Stazione Zoologica ‘‘A. Dohrn’’, Villa Comunale, 80121 Naples, Italy S. Greggi Gynecologic Oncology Unit, Cancer Institute, Fondazione ‘‘G.Pascale’’, Via Mariano Semmola, 80131 Naples, Italy A. Vecchione National Cancer Institute, Fondazione ‘‘G.Pascale’’, Via Mariano Semmola, 80131 Naples, Italy
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strategy is a promising time and cost-effective method to detect BRCA mutations in the clinical setting, which is essential to formulate a personalized therapy for patients with HBOC. Keywords BRCA1 BRCA2 Hereditary breast and ovarian cancer (HBOC) Genetic testing Direct sequencing
Introduction In this study we present a methodology for the direct sequencing of BRCA genes through a simple workflow implementable in a diagnostic lab. Breast and ovarian cancer are the leading cause of cancer death in women worldwide. Most tumors are considered sporadic, whereas the remaining 5–10 % is inherited as autosomal dominant disease and defined as Hereditary breast and ovarian cancer (HBOC) [1]. BRCA1 and BRCA2 were identified in the early 90s as the genes that confer a higher risk of developing this hereditary cancer syndrome [2, 3]. Patients with HBOC, differently from those with the sporadic type of cancer, are characterized by a young age of onset and the presence in the family of numerous cases of cancer, not only of breast cancer but also ovarian and/or cancer affect
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