Department of Clinical Genetics, Odense University Hospital, Soenderboulevard 29, 5000 Odense C, Denmark.
Breast Cancer Res Treat. 2012 Apr;132(3):1009-23. doi: 10.1007/s10549-011-1674-0. Epub 2011 Jul 19.
Mutations in BRCA1 and BRCA2 predispose carriers to early onset breast and ovarian cancer. A common problem in clinical genetic testing is interpretation of variants with unknown clinical significance. The Evidence-based Network for the Interpretation of Germline Mutant Alleles (ENIGMA) consortium was initiated to evaluate and implement strategies to characterize the clinical significance of BRCA1 and BRCA2 variants. As an initial project of the ENIGMA Splicing Working Group, we report splicing and multifactorial likelihood analysis of 25 BRCA1 and BRCA2 variants from seven different laboratories. Splicing analysis was performed by reverse transcriptase PCR or mini gene assay, and sequencing to identify aberrant transcripts. The findings were compared to bioinformatic predictions using four programs. The posterior probability of pathogenicity was estimated using multifactorial likelihood analysis, including co-occurrence with a deleterious mutation, segregation and/or report of family history. Abnormal splicing patterns expected to lead to a non-functional protein were observed for 7 variants (BRCA1 c.441+2T>A, c.4184_4185+2del, c.4357+1G>A, c.4987-2A>G, c.5074G>C, BRCA2 c.316+5G>A, and c.8754+3G>C). Combined interpretation of splicing and multifactorial analysis classified an initiation codon variant (BRCA2 c.3G>A) as likely pathogenic, uncertain clinical significance for 7 variants, and indicated low clinical significance or unlikely pathogenicity for another 10 variants. Bioinformatic tools predicted disruption of consensus donor or acceptor sites with high sensitivity, but cryptic site usage was predicted with low specificity, supporting the value of RNA-based assays. The findings also provide further evidence that clinical RNA-based assays should be extended from analysis of invariant dinucleotides to routinely include all variants located within the donor and acceptor consensus splicing sites. Importantly, this study demonstrates the added value of collaboration between laboratories, and across disciplines, to collate and interpret information from clinical testing laboratories to consolidate patient management.
BRCA1 和 BRCA2 基因突变使携带者易患早发性乳腺癌和卵巢癌。在临床基因检测中，一个常见的问题是解释具有未知临床意义的变异。启动了基于证据的种系突变等位基因解释网络（ENIGMA）联盟，以评估和实施策略来描述 BRCA1 和 BRCA2 变异的临床意义。作为 ENIGMA 剪接工作组的初始项目，我们报告了来自七个不同实验室的 25 个 BRCA1 和 BRCA2 变体的剪接和多因素似然分析。通过逆转录酶 PCR 或微型基因测定进行剪接分析，并进行测序以鉴定异常转录本。将发现与使用四个程序的生物信息学预测进行比较。使用多因素似然分析估计致病性的后验概率，包括与有害突变的共发生、分离和/或家族史报告。观察到 7 个变体（BRCA1 c.441+2T>A、c.4184_4185+2del、c.4357+1G>A、c.4987-2A>G、c.5074G>C、BRCA2 c.316+5G>A 和 c.8754+3G>C）存在导致无功能蛋白的异常剪接模式。剪接和多因素分析的综合解释将起始密码子变体（BRCA2 c.3G>A）归类为可能致病性，7 个变体具有不确定的临床意义，另外 10 个变体提示临床意义低或不太可能致病性。生物信息学工具预测高灵敏度破坏共识供体或受体位点，但预测隐匿性位点使用的特异性低，支持 RNA 为基础的检测的价值。这些发现还进一步证明，从分析不变二核苷酸扩展到常规包括供体和受体剪接位点内的所有变体，应将临床 RNA 为基础的检测扩展到分析。重要的是，这项研究表明实验室之间以及跨学科合作的附加价值，以整理和解释临床检测实验室的信息，以整合患者管理。
