Pluripotent stem cells as a novel model to test hereditary cancer variants

描述(申请人提供)：林奇综合征(LS)是一种遗传性疾病，易使患者患上结直肠癌、子宫内膜癌、卵巢癌和其他癌症。LS的明确诊断依赖于检测到其中一个DNA错配修复(MMR)基因中有害的生殖系突变。然而，当测序显示一种变异，如错义突变，其对基因功能的影响并不立即明显时，临床医生和遗传顾问就会遇到问题。这些不确定意义的变种(VU)不能用于确认或排除LS，从而导致对如何管理患者及其家人的不确定性。实验室研究确定VU是否扰乱蛋白质功能，已成为确定其与疾病相关性的重要策略。研究VUS在人类细胞中的功能是确定其对MMR功能影响的最彻底的途径，然而，这需要开发合适的细胞培养模型。我们假设人类多能干细胞(PSC)可以作为测试MMR基因变异的功能效应的理想模型系统。PSCs是不朽的，这使它们成为实验室中实用的模型系统，但没有转化，因此它们可能更接近于变异蛋白首先影响LS患者早期转化事件的环境 细胞。此外，它们是多能性的，这意味着它们可以分化为多种细胞类型，以检查细胞类型的特定效应。我们的目标是测试PSCs是否可以作为一种有效的模型系统，通过利用先进的技术在这些细胞中进行遗传操作来测试遗传变异的功能。这一建议的目的包括：1)研究不同PSC系中的MMR通路以及从这些细胞分化而来的细胞类型。我们将测试MMR蛋白的表达，G/T错配的修复，以及激活细胞周期检查点和/或凋亡的能力，以应对不同的DNA损伤剂。2)检查MMR基因MSH2可能与癌症相关的变异是否会扰乱PSCs的细胞修复和反应功能。我们将进行TALEN介导的针对内源性MSH2基因座的基因打靶，以创建表达不同MSH2 VU的非转化细胞培养模型。这些细胞将在不同的分化阶段进行研究，包括AS 检测VUS对DNA修复、细胞周期检查点和细胞死亡信号功能的影响。这项建议将使我们能够建立一个新的细胞培养模型系统，用于帮助确定不同MMR基因VU的临床意义，同时还可以提高对MMR途径的整体理解，使所有MMR缺陷癌症患者受益。

DESCRIPTION (provided by applicant): Lynch syndrome (LS) is a hereditary disease that predisposes patients to colorectal, endometrial, ovarian and other cancers. Definitive diagnosis of LS depends on detection of a deleterious, germline mutation in one of the DNA mismatch repair (MMR) genes. A problem arises for clinicians and genetic counselors, however, when the sequencing reveals a variation, such as a missense mutation, whose effect on gene function is not immediately obvious. These variants of uncertain significance (VUS) cannot be used to confirm nor rule out LS resulting in uncertainty about how to manage the patient and their family members. Laboratory studies to determine whether a VUS disrupts protein function has become an important strategy for determining their relevance to disease. Studying the function of a VUS in human cells is the most thorough approach to determining its effects on MMR function, however, this requires development of the proper cell culture model. We hypothesize that human pluripotent stem cells (PSC) can serve as an ideal model system for testing the functional effects of MMR gene variants. PSCs are immortal, which makes them a practical model system in the laboratory, yet non-transformed, thus they may more closely resemble the environment in which the variant protein first influences early transformation events in LS patient cells. In addition, they are pluripotent which means they can be differentiated into multiple cell types to examine cell-type specific effects. Our goal is to test whether PSCs can serve as an effective model system for testing the function of genetic variants by utilizing advanced techniques for genetic manipulation in these cells. The aims of this proposal include: 1) Examining the MMR pathway in various PSC lines and differentiated cell types derived from these cells. We will test for MMR protein expression, repair of G/T mismatches and ability to activate cell cycle checkpoints and/or apoptosis in response to different DNA damaging agents. 2) Examining whether likely cancer- associated variants of the MMR gene MSH2 disrupt cellular repair and response functions in PSCs. We will perform TALEN-mediated gene targeting at the endogenous MSH2 gene locus to create non-transformed cell culture models expressing different MSH2 VUS. These cells will be studied at different stages of differentiation including as intestinal epithelial-like cells to examine the effects of the VUS on DNA repair, cell cycle checkpoint and cell death signaling functions. This proposal will allow us to establish a new cell culture model system that can be used to help define the clinical significance of different MMR gene VUS, while also improving the overall understanding of the MMR pathway to benefit all patients suffering from MMR-defective cancers.