Development of Analytical Tests for Detection of Pathogenicity and Identification of Molecular Defects in Unclear Genetic Variants of the DNA Mismatch Repair Gene MSH2 in Lynch Syndrome

MSH2基因失活突变导致Lynch综合征，这是一种遗传性癌症易感性。在患者中鉴定MSH2失活突变，可以在家庭成员中进行预测性诊断和靶向癌症监测。然而，在很大一部分MSH2变异中，尚不清楚它们是否失活并因此致病。这些“不确定意义的变异”（VUS）需要进行分类以确定诊断。在大多数情况下，这种分类需要在实验室对变异MSH2基因或蛋白质进行功能分析。然而，目前还没有功能测试，包括基于证据的参考限度或分类，允许将测试结果转化为致病性声明。在这个项目中，计划分析在癌症患者中发现的遗传MSH2变异。这些变异被引入到MSH2表达载体中，并在细胞培养或体外表达。然后进行功能分析，包括（例如）：表达水平（RNA和蛋白质）、蛋白质稳定性、催化活性、亚细胞定位、与蛋白质伴侣的相互作用等。除VUS外，还包括具有已知临床状态（致病性或中性）的变异作为对照，以建立参考限或确定检测的分类器。这将允许将VUS的功能结果转化为适用于临床诊断的致病性声明。在建立用于临床应用的测试系统的同时，在不同变体中发现的分子缺陷将有助于指导对MSH2蛋白功能的全面分析，包括其蛋白质相互作用表面、DNA结合模式和atp酶周期。

Inactivating mutations of the MSH2 gene cause Lynch syndrome, a heritable cancer predisposition. Identification of an inactivating mutation in MSH2 in a patient enables predictive diagnosis and targeted cancer surveillance in family members. However, in a significant portion of MSH2 variants it is unclear if they are inactivating and therefore pathogenic. These "variants of uncertain significance" (VUS) need to be classified in order to establish a diagnosis. In most cases, this classification requires a functional analyses of the variant MSH2 gene or protein in the laboratory. However, currently there are no functional tests available which comprise evidence-based reference limits or classifiers that allow translating the test result into a pathogenicity statement. In this project, it is planned to analyze genetic MSH2 variants identified in cancer patients. These variants are introduced into MSH2 expression vectors and are expressed in cell culture or in vitro. Functional analyses are then performed and comprise (for example): expression level (RNA and protein), protein stability, catalytic activity, subcellular localization, interaction with protein partners and others. Besides VUS, variants with known clinical status (pathogenic or neutral) are included as controls to establish reference limits or identify classifiers for the assays. This will allow to translate functional results of the VUS into pathogenicity statements suitable for use in clinical diagnosis.In parallel to establishing test systems for clinical applications, the molecular defects identified in the different variants will serve to guide a thourough analysis of the MSH2 protein functions, including its protein interaction surfaces, DNA binding modes and ATPase cycle.