Hereditary Cancer Variants of Uncertain Significance in Stem Cells

项目摘要 林奇综合征(LS)是一种遗传性疾病，患者易患结直肠、子宫内膜、卵巢和 其他癌症。LS的确诊取决于检测到一种有害的生殖系突变 DNA错配修复(MMR)基因。然而，临床医生和遗传咨询师面临着一个问题， 当测序显示一个变异，如错义突变，其对基因功能的影响不是 一目了然。这些不确定意义的变种(VU)不能用于确认或排除LS 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 确定它们与疾病的相关性。研究VU在人类细胞中的功能是最彻底的 确定其对MMR功能影响的方法。我们认为人类文化的进步 随着CRISPR-Cas9介导的基因编辑的发展，多能干细胞(PSC)提供了一种 测试MMR基因变异的功能效应的理想模型系统。PSC是不朽的，这使得 它们在实验室中是一个实用的模型系统，但没有经过改造，因此它们可能更接近于 变异蛋白首先影响LS患者细胞早期转化事件的环境。在……里面 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- 键入特定效果。由于LS患者主要受结直肠癌的影响，我们将进一步检查亚型 人结肠类器官(HCOs)中VUS的含量。我们的目标是表演一场大规模的 在MMR基因MSH2、MSH6和MLH1中筛选VUS的目的如下：1)检测 MMR基因中与癌症相关的变异破坏了PSCs的细胞修复和反应功能。我们会 进行CRISPR-Cas9介导的针对内源基因座的基因打靶以创建未转化细胞 表达不同VU的文化模型。2)对于那些具有中等MMR效应的表达VUS的PSC， 我们将把细胞分化为HCOs，并检测它们诱导凋亡或衰老反应的能力。 对肠道细胞造成损害。3)对于仍显示不确定的中间功能的VU HCO，我们将 研究对基因组稳定性和细胞存活的长期影响，包括通过使用混合野生- 分型/变异型HCOs，以评估变异型表达细胞是否具有生存优势。这 该建议将有助于对大量MMR基因vus进行疾病意义分类，这些基因可以 被世界各地的临床医生和遗传咨询师用来帮助管理和预防癌症 病人和他们的家人。

Project Summary 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 one important part of a 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. We propose that advances in the culture of human pluripotent stem cells (PSC) along with the development of CRISPR-Cas9 mediated gene editing provide 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. As LS patients are mostly affected by colorectal cancer, we will further examine subsets of VUS in human colonic organoids (HCOs) derived from the PSCs. Our goal is to perform a large-scale screen of VUS in the MMR genes MSH2, MSH6 and MLH1 under the following aims: 1) Examining whether cancer-associated variants in the MMR genes disrupt cellular repair and response functions in PSCs. We will perform CRISPR-Cas9-mediated gene targeting at the endogenous gene loci to create non-transformed cell culture models expressing different VUS. 2) For those VUS-expressing PSCs with intermediate MMR effects, we will differentiate the cells into HCOs and examine their ability to induce an apoptotic or senescent response to damage in intestinal cells. 3) For VUS HCOs that still display inconclusive, intermediate function, we will examine longer-term effects on genome stability and cell survival including through the use of mixed wild- type/variant HCOs to assess whether a survival advantage exists for the variant-expressing cells. This proposal will assist in the disease significance classification for a large number of MMR gene VUS which can be used by clinicians and genetic counselors world-wide to assist in managing and preventing cancer in patients and their families.