Saturation genome editing of 11 codons and exon 13 of BRCA2 coupled with chemotherapeutic drug response accurately determines pathogenicity of variants.

IF 4.5 2区 生物学 Q1 Agricultural and Biological Sciences PLoS Genetics Pub Date : 2023-09-15 eCollection Date: 2023-09-01 DOI:10.1371/journal.pgen.1010940
Sounak Sahu, Teresa L Sullivan, Alexander Y Mitrophanov, Mélissa Galloux, Darryl Nousome, Eileen Southon, Dylan Caylor, Arun Prakash Mishra, Christine N Evans, Michelle E Clapp, Sandra Burkett, Tyler Malys, Raj Chari, Kajal Biswas, Shyam K Sharan
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Abstract

The unknown pathogenicity of a significant number of variants found in cancer-related genes is attributed to limited epidemiological data, resulting in their classification as variant of uncertain significance (VUS). To date, Breast Cancer gene-2 (BRCA2) has the highest number of VUSs, which has necessitated the development of several robust functional assays to determine their functional significance. Here we report the use of a humanized-mouse embryonic stem cell (mESC) line expressing a single copy of the human BRCA2 for a CRISPR-Cas9-based high-throughput functional assay. As a proof-of-principle, we have saturated 11 codons encoded by BRCA2 exons 3, 18, 19 and all possible single-nucleotide variants in exon 13 and multiplexed these variants for their functional categorization. Specifically, we used a pool of 180-mer single-stranded donor DNA to generate all possible combination of variants. Using a high throughput sequencing-based approach, we show a significant drop in the frequency of non-functional variants, whereas functional variants are enriched in the pool of the cells. We further demonstrate the response of these variants to the DNA-damaging agents, cisplatin and olaparib, allowing us to use cellular survival and drug response as parameters for variant classification. Using this approach, we have categorized 599 BRCA2 variants including 93-single nucleotide variants (SNVs) across the 11 codons, of which 28 are reported in ClinVar. We also functionally categorized 252 SNVs from exon 13 into 188 functional and 60 non-functional variants, demonstrating that saturation genome editing (SGE) coupled with drug sensitivity assays can enhance functional annotation of BRCA2 VUS.

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BRCA2的11个密码子和外显子13的饱和基因组编辑与化疗药物反应相结合,准确地确定了变体的致病性。
在癌症相关基因中发现的大量变体的致病性未知,这归因于有限的流行病学数据,导致它们被分类为意义不确定的变体(VUS)。到目前为止,癌症基因-2(BRCA2)具有最高数量的VUS,这就需要开发几种强大的功能测定来确定其功能意义。在此,我们报道了使用表达人BRCA2单拷贝的人源化小鼠胚胎干细胞(mESC)系进行基于CRISPR-Cas9的高通量功能测定。作为原理的证明,我们已经饱和了BRCA2外显子3、18、19编码的11个密码子,以及外显子13中所有可能的单核苷酸变体,并对这些变体进行了多重编码以进行功能分类。具体来说,我们使用了一个180聚体单链供体DNA库来产生所有可能的变体组合。使用基于高通量测序的方法,我们发现非功能性变体的频率显著下降,而功能性变体在细胞池中富集。我们进一步证明了这些变体对DNA损伤剂顺铂和奥拉帕尼的反应,使我们能够使用细胞存活率和药物反应作为变体分类的参数。使用这种方法,我们对599种BRCA2变体进行了分类,包括11个密码子中的93种单核苷酸变体(SNV),其中28种在ClinVar中报道。我们还将来自外显子13的252个SNV功能性分类为188个功能性变体和60个非功能性变体,证明饱和基因组编辑(SGE)与药物敏感性分析相结合可以增强BRCA2 VUS的功能注释。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
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来源期刊
PLoS Genetics
PLoS Genetics 生物-遗传学
CiteScore
8.10
自引率
2.20%
发文量
438
审稿时长
1 months
期刊介绍: PLOS Genetics is run by an international Editorial Board, headed by the Editors-in-Chief, Greg Barsh (HudsonAlpha Institute of Biotechnology, and Stanford University School of Medicine) and Greg Copenhaver (The University of North Carolina at Chapel Hill). Articles published in PLOS Genetics are archived in PubMed Central and cited in PubMed.
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