A Cell-Based Functional Assay Calibrated for Analysis of MSH6 and MSH2 Mismatch Repair Gene Variants

IF 3.7 2区 医学 Q2 GENETICS & HEREDITY
Elizabeth Szabo, Emily Blackburn, Olivia N. Amodeo, Samantha Nadeau, Alexander A. Radecki, James P. Grady, Abhijit Rath, Christopher D. Heinen
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Abstract

Variants of uncertain significance (VUS) in the DNA mismatch repair (MMR) genes can confound the diagnosis and treatment of suspected Lynch syndrome (LS) patients. To aid the reclassification of VUS, we developed the in cellulo analysis of MMR variants (inCAMA) and used known control variants to calibrate this assay such that results can be readily applied as functional evidence by expert classification panels. We used CRISPR gene engineering to introduce known pathogenic and benign variants into the MSH6 or MSH2 loci in human embryonic stem cells and assessed their effects on cellular MMR repair and damage response functions. Our functional assay successfully discerned known pathogenic and benign variants. Using these results and performing a linear regression analysis with available odds of pathogenicity scores for the known calibration variants, we created equations that can generate a functional odds of pathogenicity score for any future MSH6 or MSH2 variant tested. In summary, inCAMA represents a new, calibrated assay for testing the function of virtually any MSH6 or MSH2 variant. The conversion of assay results directly into odds of pathogenicity scores makes it possible to use any PS3 or BS3 evidence strength level toward the reclassification of VUS.

Abstract Image

MSH6和MSH2错配修复基因变异的细胞功能分析校准
DNA错配修复(MMR)基因中的不确定意义变异(VUS)可能会混淆疑似Lynch综合征(LS)患者的诊断和治疗。为了帮助VUS的重新分类,我们开发了MMR变异的细胞内分析(inCAMA),并使用已知的对照变异来校准该分析,以便结果可以很容易地作为专家分类小组的功能证据。我们使用CRISPR基因工程将已知的致病和良性变异引入人胚胎干细胞的MSH6或MSH2位点,并评估它们对细胞MMR修复和损伤反应功能的影响。我们的功能分析成功地识别了已知的致病和良性变异。利用这些结果,并对已知校准变体的现有致病性分数进行线性回归分析,我们创建了可以为任何未来测试的MSH6或MSH2变体生成功能性致病性分数的方程。总之,inCAMA代表了一种新的、校准的检测方法,几乎可以检测任何MSH6或MSH2变异的功能。将检测结果直接转换为致病几率评分,可以使用任何PS3或BS3证据强度水平对VUS进行重新分类。
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来源期刊
Human Mutation
Human Mutation 医学-遗传学
CiteScore
8.40
自引率
5.10%
发文量
190
审稿时长
2 months
期刊介绍: Human Mutation is a peer-reviewed journal that offers publication of original Research Articles, Methods, Mutation Updates, Reviews, Database Articles, Rapid Communications, and Letters on broad aspects of mutation research in humans. Reports of novel DNA variations and their phenotypic consequences, reports of SNPs demonstrated as valuable for genomic analysis, descriptions of new molecular detection methods, and novel approaches to clinical diagnosis are welcomed. Novel reports of gene organization at the genomic level, reported in the context of mutation investigation, may be considered. The journal provides a unique forum for the exchange of ideas, methods, and applications of interest to molecular, human, and medical geneticists in academic, industrial, and clinical research settings worldwide.
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