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，患者易患结直肠癌、子宫内膜癌、卵巢癌和其他癌症。LS的连续诊断依赖于检测DNA错配修复（MMR）基因中的一个有害的种系突变。然而，当测序揭示了一个变异，如错义突变，其对基因功能的影响并不立即明显时，临床医生和遗传咨询师就会遇到一个问题。这些意义不确定的变异（VUS）不能用于确认或排除LS，从而导致如何管理患者及其家庭成员的不确定性。确定VUS是否破坏蛋白质功能的实验室研究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功能影响的最彻底方法，然而，这需要开发适当的细胞培养模型。我们假设，人类多能干细胞（PSC）可以作为一个理想的模型系统，用于测试MMR基因变异的功能影响。PSC是永生的，这使得它们成为实验室中的实用模型系统，但未转化，因此它们可能更接近于变体蛋白首先影响LS患者中的早期转化事件的环境 细胞此外，它们是多能的，这意味着它们可以分化成多种细胞类型，以检查细胞类型特异性效应。我们的目标是测试PSC是否可以作为一个有效的模型系统，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。该提议的目的包括：1）检查各种PSC系和源自这些细胞的分化细胞类型中的MMR途径。我们将测试MMR蛋白表达，G/T错配的修复和激活细胞周期检查点和/或细胞凋亡的能力，以响应不同的DNA损伤剂。2)检查MMR基因MSH 2的可能的癌症相关变体是否破坏PSC中的细胞修复和反应功能。我们将在内源性MSH 2基因位点进行TALEN介导的基因靶向，以创建表达不同MSH 2 VUS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 肠上皮样细胞，以检查VUS对DNA修复、细胞周期检查点和细胞死亡信号传导功能的影响。该提案将使我们能够建立一种新的细胞培养模型系统，可用于帮助定义不同MMR基因VUS的临床意义，同时还可以提高对MMR通路的整体理解，以使所有患有MMR缺陷癌症的患者受益。

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.