Sequencing-based Functional Assays for Classification of BRCA2 Variants in Mouse ES Cells

Sequencing of genes, such as BRCA1 and BRCA2, is recommended for individuals with a personal or family history of early onset and/or bilateral breast and/or ovarian cancer, or a history of male breast cancer. Such sequencing efforts have resulted in the identification of more than 6000 BRCA2 variants. The functional significance of most variants remains unknown; consequently, they are called variants of uncertain clinical significance (VUS). We have previously developed mouse embryonic stem cell (mESC)-based assays for functional classification of BRCA2 variants. While the assays are reliable, the experimental process is time-consuming. This has impeded the number of variants that can be examined at a time. To overcome this, we have now developed a next-generation sequencing (NGS)-based approach for functional evaluation of BRCA2 variants. In this approach, instead of using clonogenic or XTT-based cell survival assays, pools of mESC expressing 25-30 BRCA2 variants from a given exon are analyzed by NGS. We have also developed a statistical model that utilizes the NGS data to determine the probability of impact on function (PIF) for these variants. While CRISPR/Cas9-based high throughput methods have resulted in the functional characterization of thousands of variants, only a fraction of the variants are included in ClinVar. In contrast, our targeted approach, which involves generating specific variants, resulted in the functional evaluation of 223 variants that are all listed in ClinVar. Our functional classification of BRCA2 variants is concordant with the classification reported in ClinVar or reported by other well-established assays. Overall design: Saturation genome editing (SGE) coupled with drug sensitivity assays of BRCA2

针对具有早发性乳腺癌/卵巢癌、双侧乳腺癌/卵巢癌个人或家族病史，或男性乳腺癌病史的个体，推荐对BRCA1、BRCA2等基因进行测序。此类测序工作已累计鉴定出超过6000种BRCA2变异体。其中绝大多数变异体的临床功能意义尚不明确，因此被称为临床意义未明变异体（variants of uncertain clinical significance, VUS）。我们此前开发了基于小鼠胚胎干细胞（mouse embryonic stem cell, mESC）的BRCA2变异体功能分类检测方法。尽管该检测方法可靠性优异，但实验流程耗时较长，限制了单次可检测的变异体数量。为解决这一问题，本研究开发了一种基于下一代测序（next-generation sequencing, NGS）的BRCA2变异体功能评估新方法。该方法不再使用集落形成或基于XTT的细胞存活检测手段，而是通过NGS分析携带某一外显子上25至30种BRCA2变异体的小鼠胚胎干细胞混合池。我们还开发了一款统计模型，可利用NGS数据计算这些变异体的功能影响概率（probability of impact on function, PIF）。尽管基于CRISPR/Cas9的高通量方法已实现数千种变异体的功能鉴定，但仅有少数变异体被收录于ClinVar数据库。与之形成鲜明对比的是，本研究通过靶向性策略——即针对性生成特定变异体——完成了223种均已收录于ClinVar的BRCA2变异体的功能评估。我们对BRCA2变异体的功能分类结果与ClinVar中已报道的分类，或其他成熟检测方法的结果均保持一致。整体实验设计：饱和基因组编辑（saturation genome editing, SGE）联合BRCA2药物敏感性检测