Hereditary Cancer Variants of Uncertain Significance in Stem Cells

干细胞中意义不确定的遗传性癌症变异

• 批准号: 10640059
• 负责人: Christopher D. Heinen
• 金额: $ 35.43万
• 依托单位: UNIVERSITY OF CONNECTICUT SCH OF MED/DNT
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-04-04 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10640059
• 关键词: Affect; Amino Acids; Apoptotic; Biochemical; Biological Assay; Biological Models; CRISPR/Cas technology; Cancer Etiology; Cancer Patient; Cancer cell line; Cell Culture Techniques; Cell Differentiation process; Cell Survival; Cell physiology; Cells; Cessation of life; Classification; Classification Scheme; Colon; Colon Carcinoma; Colorectal; Colorectal Cancer; DNA; DNA Damage; DNA sequencing; Defect; Detection; Development; Diagnosis; Disease; Endometrial; Engineering; Environment; Event; Failure; Family; Family member; Frustration; Gene Expression; Gene Targeting; Genes; Genetic; Genome Stability; Genomic Instability; Germ-Line Mutation; Goals; Guidelines; Hereditary Disease; Hereditary Malignant Neoplasm; Hereditary Nonpolyposis Colorectal Neoplasms; Human; In Vitro; Individual; Induction of Apoptosis; Inherited; Intestines; Label; Laboratories; Laboratory Study; MLH1 gene; MSH2 gene; MSH6 gene; Malignant Neoplasms; Masks; Measures; Mediating; Microsatellite Instability; Mismatch Repair; Missense Mutation; Modeling; Mutation; Nature; Organoids; Ovarian; Pathogenesis; Pathogenicity; Pathway interactions; Patients; Phenotype; Physicians; Pluripotent Stem Cells; Population; Proteins; Risk; Testing; Time; Transgenes; Uncertainty; Variant; Woman; cell immortalization; cell injury; cell type; colon cancer patients; gene function; gene repair; genetic counselor; genetic variant; genomic locus; human pluripotent stem cell; human stem cells; men; prevent; protein expression; protein function; repair function; repaired; response; senescence; stem cells; variant of unknown significance

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.

项目概要 林奇综合征 (LS) 是一种遗传性疾病，使患者易患结直肠癌、子宫内膜癌、卵巢癌和 其他癌症。 LS 的最终诊断取决于检测到以下任一基因中的有害种系突变： DNA错配修复（MMR）基因。然而，临床医生和遗传咨询师遇到了一个问题： 当测序揭示出一种变异，例如错义突变，其对基因功能的影响不是 立即显而易见。这些意义不确定的变体 (VUS) 不能用于确认或排除 LS 导致不确定如何管理患者及其家人。实验室研究 确定 VUS 是否会破坏蛋白质功能已成为治疗策略的重要组成部分 确定它们与疾病的相关性。研究VUS在人体细胞中的功能是最彻底的 确定其对 MMR 功能影响的方法。我们提出，人类文化的进步 多能干细胞 (PSC) 以及 CRISPR-Cas9 介导的基因编辑技术的发展提供了 用于测试 MMR 基因变异功能效果的理想模型系统。 PSC 是不朽的，这使得 它们是实验室中的实用模型系统，但未经过改造，因此它们可能更类似于 变异蛋白首先影响 LS 患者细胞早期转化事件的环境。在 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来检查细胞 类型特定效果。由于 LS 患者主要受结直肠癌影响，我们将进一步检查亚组 源自 PSC 的人结肠类器官 (HCO) 中的 VUS。我们的目标是进行大规模的 根据以下目标对 MMR 基因 MSH2、MSH6 和 MLH1 中的 VUS 进行筛选： 1) 检查是否 MMR 基因中与癌症相关的变异会破坏 PSC 中的细胞修复和反应功能。我们将 在内源基因位点上进行 CRISPR-Cas9 介导的基因打靶，以创建非转化细胞 表达不同VUS的文化模型。 2) 对于那些具有中等 MMR 效应的表达 VUS 的 PSC， 我们将细胞分化为 HCO，并检查它们诱导细胞凋亡或衰老反应的能力 对肠道细胞造成损害。 3) 对于仍然显示不确定的中间功能的 VUS HCO，我们将 检查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 类型/变异 HCO 来评估变异表达细胞是否存在生存优势。这 该提案将有助于对大量MMR基因VUS进行疾病意义分类，这可以 被世界各地的临床医生和遗传咨询师用来协助管理和预防癌症 患者及其家人。