Functional Genomic Unit, National Cancer Institute, Fondazione "G.Pascale", Via Mariano Semmola, 80131 Naples, Italy.
Mol Biotechnol. 2013 Jul;54(3):954-60. doi: 10.1007/s12033-012-9646-0.
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.
遗传性癌症约占乳腺癌和卵巢癌的 10%。BRCA1 和 BRCA2 基因的突变，这两个基因编码参与 DNA 修复的两种蛋白质，是大多数此类遗传性癌症的基础。BRCA 突变的女性在 50-80%的情况下会发展为乳腺癌，在 10-40%的情况下会发展为卵巢癌。评估 BRCA 突变状态对于指导有这些遗传性癌症倾向的女性的临床管理是必要的。然而，BRCA 筛查在成本和获得结果的时间方面构成了一个瓶颈。我们开发了一种基于 PCR 的检测方法，使用 73 对引物覆盖 BRCA1 和 BRCA2 的整个编码区。PCR 引物在 96 孔板中预先点样，在 5' 端包含通用的 M13 引物序列。PCR 后，使用 M13 引物直接进行测序，允许标准化条件。每个扩增子的 PCR 扩增和测序都成功。我们在 10 名遗传性乳腺癌和卵巢癌 (HBOC) 患者的已知 gDNA 上测试和验证了该检测方法。我们的策略是一种有前途的、节省时间和成本的方法，可以在临床环境中检测 BRCA 突变，这对于为 HBOC 患者制定个性化治疗方案至关重要。
