A High-Throughput Functional Complementation Assay for Classification of BRCA1 Missense Variants
A High-Throughput Functional Complementation Assay for Classification of BRCA1 Missense Variants
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DOI：
10.1158/2159-8290.cd-13-0094
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发表时间：
2013-10-01
期刊：
影响因子：
28.2
通讯作者：
Jonkers, Jos
中科院分区：
文献类型：
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作者：
Bouwman, Peter;van der Gulden, Hanneke;Jonkers, Jos
BRCA1和BRCA2突变占遗传性乳腺癌和卵巢癌的大部分，因此在早发性乳腺癌患者中常规进行这两个基因的序列分析。除了明确破坏蛋白质功能或已知增加癌症风险的突变外，还发现了大量不确定意义的序列变异（VUS）。尽管已经描述了几种BRCA1 VUSs的功能分析，但到目前为止，还不可能在全长蛋白的背景下进行高通量分析。我们开发了一种相对快速和简单的基于dna的功能检测方法，根据BRCA1 VUSs在功能上补充BRCA1缺陷小鼠胚胎干细胞的能力对其进行分类。使用这种方法，我们分析了74种未分类的BRCA1错义突变，其中所有预测的致病变异都局限于BRCA1 RING和BRCT结构域。意义：BRCA1 VUSs常见于遗传性乳腺癌或卵巢癌患者，是临床遗传学家面临的一个严重问题。本文描述了一种可靠的高通量测定方法的产生、验证和应用，用于不确定意义的BRCA1序列变异的功能分类。(c) 2013年aacr。
Mutations in BRCA1 and BRCA2 account for the majority of hereditary breast and ovarian cancers, and therefore sequence analysis of both genes is routinely conducted in patients with early-onset breast cancer. Besides mutations that clearly abolish protein function or are known to increase cancer risk, a large number of sequence variants of uncertain significance (VUS) have been identified. Although several functional assays for BRCA1 VUSs have been described, thus far it has not been possible to conduct a high-throughput analysis in the context of the full-length protein. We have developed a relatively fast and easy cDNA-based functional assay to classify BRCA1 VUSs based on their ability to functionally complement BRCA1-deficient mouse embryonic stem cells. Using this assay, we have analyzed 74 unclassified BRCA1 missense mutants for which all predicted pathogenic variants are confined to the BRCA1 RING and BRCT domains.SIGNIFICANCE: BRCA1 VUSs are frequently found in patients with hereditary breast or ovarian cancer and present a serious problem for clinical geneticists. This article describes the generation, validation, and application of a reliable high-throughput assay for the functional classification of BRCA1 sequence variants of uncertain significance. (C) 2013 AACR.