Department of Medical Genetics, Oslo University Hospital, Oslo, Norway.
Department of Medical Genetics, Division of Child and Adolescent Health, University Hospital of North Norway, Tromsø, Norway.
Hum Genomics. 2018 Nov 20;12(1):51. doi: 10.1186/s40246-018-0183-1.
Deleterious variants in the tumour suppressor BRCA1 are known to cause hereditary breast and ovarian cancer syndrome (HBOC). Missense variants in BRCA1 pose a challenge in clinical care, as their effect on protein functionality often remains unknown. Many of the pathogenic missense variants found in BRCA1 are located in the BRCA1 C-terminal (BRCT) domains, domains that are known to be vital for key functions such as homologous recombination repair, protein-protein interactions and trans-activation (TA). We investigated the TA activity of 12 BRCA1 variants of unknown clinical significance (VUSs) located in the BRCT domains to aid in the classification of these variants.
Twelve BRCA1 VUSs were investigated using a modified version of the dual luciferase TA activity assay (TA assay) that yielded increased sensitivity and sample throughput. Variants were classified according to American College of Medical Genetics and Genomics (ACMG) criteria using TA assay results and available data. In combining our TA-assay results and available data, in accordance with the ACMG guidelines for variant classification, we proposed the following variant classifications: c.5100A>G, c.5326C>T, c.5348T>C and c.5477A>T as likely benign (class 2) variants. c.5075A>C, c.5116G>A and c.5513T>G were likely pathogenic (class 4), whereas c.5096G>A likely represents a likely pathogenic variant with moderate penetrance. Variants c.5123C>T, c.5125G>A, c.5131A>C and c.5504G>A remained classified as VUSs (class 3).
The modified TA assay provides efficient risk assessment of rare missense variants found in the BRCA1 BRCT-domains. We also report that increased post-transfection incubation time yielded a significant increase in TA assay sensitivity.
已知肿瘤抑制因子 BRCA1 中的有害变异会导致遗传性乳腺癌和卵巢癌综合征 (HBOC)。BRCA1 中的错义变异在临床护理中构成挑战，因为其对蛋白质功能的影响通常尚不清楚。在 BRCA1 中发现的许多致病性错义变异位于 BRCA1 C 末端 (BRCT) 结构域中，这些结构域对于同源重组修复、蛋白质-蛋白质相互作用和转录激活 (TA) 等关键功能至关重要。我们研究了位于 BRCT 结构域中的 12 种 BRCA1 意义不明的变异体 (VUS) 的 TA 活性，以帮助对这些变异体进行分类。
使用改良的双荧光素酶 TA 活性测定法 (TA 测定法) 研究了 12 种 BRCA1 VUS，该方法提高了灵敏度和样本通量。根据 TA 测定法结果和可用数据，根据美国医学遗传学与基因组学学院 (ACMG) 标准对变体进行分类。根据 ACMG 指南对变体分类，我们结合 TA 测定结果和可用数据，提出以下变体分类：c.5100A>G、c.5326C>T、c.5348T>C 和 c.5477A>T 为可能良性 (2 类) 变体。c.5075A>C、c.5116G>A 和 c.5513T>G 为可能致病性 (4 类)，而 c.5096G>A 可能代表具有中度外显率的可能致病性变体。变体 c.5123C>T、c.5125G>A、c.5131A>C 和 c.5504G>A 仍被归类为 VUS (3 类)。
改良的 TA 测定法为 BRCA1 BRCT 结构域中发现的罕见错义变异提供了有效的风险评估。我们还报告说，增加转染后孵育时间可显著提高 TA 测定法的灵敏度。
