A new rapid methodological strategy to assess BRCA mutational status.
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 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.
Functional Genomic Unit, National Cancer Institute, Fondazione "G.Pascale", Via Mariano Semmola, 80131 Naples, Italy.
Emilia
E
Vuttariello
Marco
M
Borra
Celeste
C
Calise
Elvira
E
Mauriello
Stefano
S
Greggi
Aldo
A
Vecchione
Elio
E
Biffali
Gennaro
G
Chiappetta
D001943:Breast Neoplasms / Q000235:genetics; D016022:Case-Control Studies; D004252:DNA Mutational Analysis / Q000379:methods*; D017931:DNA Primers; D005260:Female; D019398:Genes, BRCA1; D024522:Genes, BRCA2; D020022:Genetic Predisposition to Disease; D006801:Humans; D009154:Mutation; D010051:Ovarian Neoplasms / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods*
D017931:DNA Primers