Szabo Elizabeth, Blackburn Emily, Amodeo Olivia N, Nadeau Samantha, Radecki Alexander A, Grady James P, Rath Abhijit, Heinen Christopher D
Center for Molecular Oncology, UConn Health, Farmington, Connecticut, USA.
Department of Molecular and Cell Biology, University of Connecticut, Storrs, Connecticut, USA.
Hum Mutat. 2025 Sep 6;2025:3923193. doi: 10.1155/humu/3923193. eCollection 2025.
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 or 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 or variant tested. In summary, inCAMA represents a new, calibrated assay for testing the function of virtually any or 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.
DNA错配修复（MMR）基因中意义未明的变异（VUS）会混淆疑似林奇综合征（LS）患者的诊断和治疗。为了辅助VUS的重新分类，我们开发了MMR变异的细胞内分析方法（inCAMA），并使用已知的对照变异来校准该检测方法，以便专家分类小组能够将结果作为功能证据直接应用。我们使用CRISPR基因工程技术将已知的致病和良性变异引入人类胚胎干细胞的 或 基因座，并评估它们对细胞MMR修复和损伤反应功能的影响。我们的功能检测成功区分了已知的致病和良性变异。利用这些结果，并对已知校准变异的可用致病性评分几率进行线性回归分析，我们创建了一些方程，可为未来检测的任何 或 变异生成功能性致病性评分几率。总之，inCAMA代表了一种全新的、经过校准的检测方法，可用于检测几乎任何 或 变异的功能。将检测结果直接转换为致病性评分几率，使得在VUS重新分类时可以使用任何PS3或BS3证据强度水平。
