Biswas Kajal, Lipton Gary B, Stauffer Stacey, Sullivan Teresa, Cleveland Linda, Southon Eileen, Reid Susan, Magidson Valentin, Iversen Edwin S, Sharan Shyam K
Mouse Cancer Genetics Program, Center for Cancer Research, National Cancer Institute, Frederick, MD, 21702, USA.
Department of Statistical Science, Duke University, Durham, NC, 27708, USA.
NPJ Genom Med. 2020 Dec 8;5(1):52. doi: 10.1038/s41525-020-00158-5.
Sequencing-based genetic tests to identify individuals at increased risk of hereditary breast and ovarian cancers have resulted in the identification of more than 40,000 sequence variants of BRCA1 and BRCA2. A majority of these variants are considered to be variants of uncertain significance (VUS) because their impact on disease risk remains unknown, largely due to lack of sufficient familial linkage and epidemiological data. Several assays have been developed to examine the effect of VUS on protein function, which can be used to assess their impact on cancer susceptibility. In this study, we report the functional characterization of 88 BRCA2 variants, including several previously uncharacterized variants, using a well-established mouse embryonic stem cell (mESC)-based assay. We have examined their ability to rescue the lethality of Brca2 null mESC as well as sensitivity to six DNA damaging agents including ionizing radiation and a PARP inhibitor. We have also examined the impact of BRCA2 variants on splicing. In addition, we have developed a computational model to determine the probability of impact on function of the variants that can be used for risk assessment. In contrast to the previous VarCall models that are based on a single functional assay, we have developed a new platform to analyze the data from multiple functional assays separately and in combination. We have validated our VarCall models using 12 known pathogenic and 10 neutral variants and demonstrated their usefulness in determining the pathogenicity of BRCA2 variants that are listed as VUS or as variants with conflicting functional interpretation.
用于识别遗传性乳腺癌和卵巢癌风险增加个体的基于测序的基因检测，已鉴定出超过40000个BRCA1和BRCA2的序列变异。这些变异中的大多数被认为是意义未明的变异（VUS），因为它们对疾病风险的影响尚不清楚，这主要是由于缺乏足够的家族连锁和流行病学数据。已经开发了几种检测方法来研究VUS对蛋白质功能的影响，这些方法可用于评估它们对癌症易感性的影响。在本研究中，我们使用一种成熟的基于小鼠胚胎干细胞（mESC）的检测方法，报告了88个BRCA2变异的功能特征，包括几个以前未表征的变异。我们研究了它们挽救Brca2基因敲除mESC致死性的能力以及对六种DNA损伤剂（包括电离辐射和一种PARP抑制剂）的敏感性。我们还研究了BRCA2变异对剪接的影响。此外，我们开发了一个计算模型来确定变异对功能影响的概率，该模型可用于风险评估。与以前基于单一功能检测的VarCall模型不同，我们开发了一个新平台，分别并结合分析来自多种功能检测的数据。我们使用12个已知的致病变异和10个中性变异验证了我们的VarCall模型，并证明了它们在确定列为VUS或功能解释相互矛盾的BRCA2变异致病性方面的有用性。
