A reliable method for the detection of BRCA1 and BRCA2 mutations in fixed tumour tissue utilising multiplex PCR-based targeted next generation sequencing.
A reliable method for the detection of BRCA1 and BRCA2 mutations in fixed tumour tissue utilising multiplex PCR-based targeted next generation sequencing.
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DOI：
10.1186/s12907-015-0004-6
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发表时间：
2015
影响因子：
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通讯作者：
Mills J
中科院分区：
文献类型：
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作者：
Ellison G;Huang S;Carr H;Wallace A;Ahdesmaki M;Bhaskar S;Mills J
BRCA1或BRCA2的生殖系突变导致一生中患卵巢癌或乳腺癌的概率很高。这些基因也可能参与非遗传性肿瘤的发生，因为在一些此类癌症中发现了体细胞BRCA1/2致病性变异。由于体细胞BRCA致病性变异患者可能受益于聚ADP核糖聚合酶抑制剂治疗，因此能够检测常规可用肿瘤样本中的体细胞变化非常重要。此类样本通常是福尔马林固定石蜡包埋（FFPE）组织，其中提取的DNA往往高度碎片化且数量有限，这使得对BRCA1和BRCA2等大基因的分析具有挑战性。由于存在正常组织，体细胞变化可能仅在样本的一部分中明显，这使得分析更加困难。
我们研究了使用下一代测序方法分析从FFPE卵巢和乳腺肿瘤组织中提取的DNA以识别BRCA1/BRCA2中重要DNA变异的可行性，这些方法足够灵敏以检测低水平突变，多重扩增以减少所需的DNA量，并且具有短的扩增子设计。研究了两种具有不同设计且仅针对BRCA1/2外显子的GeneRead DNAseq靶向外显子富集试剂盒以及Ion AmpliSeq BRCA社区试剂盒的实用性，然后使用TruSeq DNA无PCR高通量样本制备试剂盒进行文库制备和接头连接，并在MiSeq上进行NGS分析。
使用GeneRead方法，我们成功分析了超过76%的样本，BRCA1/2编码区域的覆盖率>95%，平均读取深度>1000倍。在可能的情况下，通过桑格测序或重复对所有识别出的突变进行了确认，以消除由于FFPE材料中的假象导致假阳性结果的风险。混合实验表明，如果样本中>10%存在BRCA1/2变异，则可以检测到。使用Ion AmpliSeq BRCA试剂盒对一个样本子集进行了评估，对于一部分样本实现了>99%的覆盖率和足够的读取深度。
在固定组织中检测BRCA1/2变异是可行的，并且可以前瞻性地进行，以便为卵巢癌或乳腺癌患者做出最佳治疗决策。
本文的在线版本（doi:10.1186/s12907 - 2015 - 0004 - 6）包含补充材料，授权用户可获取。
Germline mutations in BRCA1 or BRCA2 lead to a high lifetime probability of developing ovarian or breast cancer. These genes can also be involved in the development of non-hereditary tumours as somatic BRCA1/2 pathogenic variants are found in some of these cancers. Since patients with somatic BRCA pathogenic variants may benefit from treatment with poly ADP ribose polymerase inhibitors, it is important to be able to test for somatic changes in routinely available tumour samples. Such samples are typically formalin-fixed paraffin-embedded (FFPE) tissue, where the extracted DNA tends to be highly fragmented and of limited quantity, making analysis of large genes such as BRCA1 and BRCA2 challenging. This is made more difficult as somatic changes may be evident in only part of the sample, due to the presence of normal tissue. We examined the feasibility of analysing DNA extracted from FFPE ovarian and breast tumour tissue to identify significant DNA variants in BRCA1/ BRCA2 using next generation sequencing methods that were sensitive enough to detect low level mutations, multiplexed to reduce the amount of DNA required and had short amplicon design. The utility of two GeneRead DNAseq Targeted Exon Enrichment Panels with different designs targeting only BRCA1/2 exons, and the Ion AmpliSeq BRCA community panel, followed by library preparation and adaptor ligation using the TruSeq DNA PCR-Free HT Sample Preparation Kit and NGS analysis on the MiSeq were investigated. Using the GeneRead method, we successfully analysed over 76% of samples, with >95% coverage of BRCA1/2 coding regions and a mean average read depth of >1000-fold. All mutations identified were confirmed where possible by Sanger sequencing or replication to eliminate the risk of false positive results due to artefacts within FFPE material. Admixture experiments demonstrated that BRCA1/2 variants could be detected if present in >10% of the sample. A sample subset was evaluated using the Ion AmpliSeq BRCA panel, achieving >99% coverage and sufficient read depth for a proportion of the samples. Detection of BRCA1/2 variants in fixed tissue is feasible, and could be performed prospectively to facilitate optimum treatment decisions for ovarian or breast cancer patients. The online version of this article (doi:10.1186/s12907-015-0004-6) contains supplementary material, which is available to authorized users.