DNA Repair Function Scores for 2172 Variants in the BRCA1 Amino-Terminus

Single nucleotide variants are the most frequent type of sequence changes detected in the genome and these are frequently variants of uncertain significance (VUS). VUS are changes in DNA for which disease risk association is unknown. Thus, methods that classify the functional impact of a VUS can be used as evidence for variant interpretation. In the case of the breast and ovarian cancer specific tumor suppressor protein, BRCA1, pathogenic missense variants frequently score as loss of function in an assay for homology-directed repair (HDR) of DNA double-strand breaks. We previously published functional results using a multiplexed assay for 1056 amino acid substitutions residues 2-192 in the amino terminus of BRCA1. In this study, we have re-assessed the data from this multiplexed assay using an improved analysis pipeline. These new analysis methods yield functional scores for more variants in the first 192 amino acids of BRCA1, plus we report new results for BRCA1 amino acid residues 193-302. We now present the functional classification of 2172 BRCA1 variants in BRCA1 residues 2-302 using the multiplexed HDR assay. Comparison of the functional determinations of the missense variants with clinically known benign or pathogenic variants indicated 93% sensitivity and 100% specificity for this assay. The results from BRCA1 variants tested in this assay are a resource for clinical geneticists for evidence to evaluate VUS in BRCA1. Overall design: In this study, we have re-analyzed the BRCA1 amino-terminus (DOI: 10.1016/j.ajhg.2018.07.016) for the functional impact of single missense substitutions using an updated analysis protocol. The samples provided below are the sequencing data for BRCA1 amino acid residues 193-302, as well as the barcode map (.tsv) used to map the variants.

单核苷酸变异是基因组中最常见的序列改变类型，此类变异多为意义未明变异（variants of uncertain significance, VUS）。VUS是指与疾病风险关联尚不明确的DNA序列改变。因此，可用于分类VUS功能影响的方法，可作为变异解读的佐证依据。针对乳腺与卵巢癌特异性抑癌蛋白BRCA1而言，致病变异中的错义变异在DNA双链断裂同源定向修复（homology-directed repair, HDR）检测中常表现为功能丧失。本团队此前曾针对BRCA1氨基末端2-192位氨基酸残基的1056个错义替换变异，通过多重检测实验发表了相关功能研究结果。

本研究通过优化后的分析流程，对该多重检测实验的原始数据进行了重新评估。新的分析方法可为BRCA1前192位氨基酸区域内更多变异提供功能评分，同时本研究还报道了BRCA1 193-302位氨基酸残基的全新功能结果。本研究依托该多重HDR检测技术，完成了BRCA1 2-302位氨基酸残基区域内共计2172个变异的功能分类。将错义变异的功能判定结果与临床已知的良性或致病变异进行比对后显示，该检测的灵敏度达93%，特异性达100%。本实验检测得到的BRCA1变异结果，可为临床遗传学家评估BRCA1基因座上的VUS提供重要佐证依据。

整体实验设计：本研究通过更新后的分析流程，重新分析了BRCA1氨基末端区域（DOI: 10.1016/j.ajhg.2018.07.016）的单错义替换功能影响。下文提供的样本包括BRCA1 193-302位氨基酸残基的测序数据，以及用于变异映射的条码映射表（.tsv格式）。