Trujillano Daniel, Weiss Maximilian E R, Schneider Juliane, Köster Julia, Papachristos Efstathios B, Saviouk Viatcheslav, Zakharkina Tetyana, Nahavandi Nahid, Kovacevic Lejla, Rolfs Arndt
Centogene AG, Rostock, Germany.
Centogene AG, Rostock, Germany.
J Mol Diagn. 2015 Mar;17(2):162-70. doi: 10.1016/j.jmoldx.2014.11.004. Epub 2014 Dec 31.
Genetic testing for hereditary breast and/or ovarian cancer mostly relies on laborious molecular tools that use Sanger sequencing to scan for mutations in the BRCA1 and BRCA2 genes. We explored a more efficient genetic screening strategy based on next-generation sequencing of the BRCA1 and BRCA2 genes in 210 hereditary breast and/or ovarian cancer patients. We first validated this approach in a cohort of 115 samples with previously known BRCA1 and BRCA2 mutations and polymorphisms. Genomic DNA was amplified using the Ion AmpliSeq BRCA1 and BRCA2 panel. The DNA Libraries were pooled, barcoded, and sequenced using an Ion Torrent Personal Genome Machine sequencer. The combination of different robust bioinformatics tools allowed detection of all previously known pathogenic mutations and polymorphisms in the 115 samples, without detecting spurious pathogenic calls. We then used the same assay in a discovery cohort of 95 uncharacterized hereditary breast and/or ovarian cancer patients for BRCA1 and BRCA2. In addition, we describe the allelic frequencies across 210 hereditary breast and/or ovarian cancer patients of 74 unique definitely and likely pathogenic and uncertain BRCA1 and BRCA2 variants, some of which have not been previously annotated in the public databases. Targeted next-generation sequencing is ready to substitute classic molecular methods to perform genetic testing on the BRCA1 and BRCA2 genes and provides a greater opportunity for more comprehensive testing of at-risk patients.
遗传性乳腺癌和/或卵巢癌的基因检测大多依赖于费力的分子工具，这些工具使用桑格测序法来扫描BRCA1和BRCA2基因中的突变。我们在210例遗传性乳腺癌和/或卵巢癌患者中，基于BRCA1和BRCA2基因的新一代测序探索了一种更有效的基因筛查策略。我们首先在一组115个已知BRCA1和BRCA2突变及多态性的样本中验证了这种方法。使用Ion AmpliSeq BRCA1和BRCA2检测板扩增基因组DNA。将DNA文库混合、添加条形码，并使用Ion Torrent个人基因组测序仪进行测序。不同强大生物信息学工具的组合能够检测出115个样本中所有先前已知的致病突变和多态性，且未检测到假阳性致病结果。然后，我们在95例未明确诊断的遗传性乳腺癌和/或卵巢癌患者的发现队列中对BRCA1和BRCA2使用相同的检测方法。此外，我们描述了210例遗传性乳腺癌和/或卵巢癌患者中74种独特的明确和可能致病以及不确定的BRCA1和BRCA2变异的等位基因频率，其中一些变异此前未在公共数据库中注释。靶向新一代测序已准备好替代经典分子方法对BRCA1和BRCA2基因进行基因检测，并为对高危患者进行更全面的检测提供了更大的机会。
