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: 10379237
• 负责人: Christopher D. Heinen
• 金额: $ 9.69万
• 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/10379237
• 关键词: Affect; Amino Acids; Apoptosis; 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; Colorectal; Colorectal Cancer; DNA; DNA Damage; DNA sequencing; Defect; Detection; Development; Diagnosis; Disease; Endometrial; Engineering; Environment; Event; Failure; Family; Family member; 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; 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; Problem Solving; Proteins; Risk; Testing; Time; Transgenes; Uncertainty; Variant; Woman; 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.

项目摘要 Lynch综合征（LS）是一种遗传性疾病，患者易患结直肠、子宫内膜、卵巢和子宫内膜癌。 其他癌症。LS的连续诊断依赖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的一个问题是， 当测序揭示了一个变异，如错义突变，其对基因功能的影响不是 立即明显。这些不确定意义的变异（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庭成员。实验室研究， 确定VUS是否破坏蛋白质功能已成为一种策略的重要组成部分， 确定它们与疾病的相关性。研究VUS在人类细胞中的功能是最彻底的 确定其对MMR功能的影响的方法。我们认为人类文化的进步 多能干细胞（PSC）沿着CRISPR-Cas9介导的基因编辑的发展， 理想的模型系统，用于测试MMR基因变异体的功能效应。PSC是不朽的，这使得 它们是实验室中的实际模型系统，但未转化，因此它们可能更接近于 变异蛋白首先影响LS患者细胞中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，以检查细胞， 类型的具体影响。由于LS患者大多受结直肠癌的影响，我们将进一步检查亚群 VUS在来源于PSC的人结肠类器官（HCO）中的表达。我们的目标是进行大规模的 在MMR基因MSH 2、MSH 6和MLH 1中筛选VUS，目的如下：1）检查是否 MMR基因中的癌症相关变体破坏PSC中的细胞修复和应答功能。我们将 在内源基因座处进行CRISPR-Cas9介导的基因靶向以产生非转化细胞， 表达不同VUS的培养模型。2)对于那些具有中等MMR效应的表达VUS的PSC， 我们将使细胞分化为HCOs，并检测它们诱导凋亡或衰老反应的能力 对肠道细胞的损害。3)对于仍然显示不确定的中间功能的VUS 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型， 类型/变体HCO，以评估表达变体的细胞是否存在存活优势。这 该提案将有助于对大量MMR基因VUS进行疾病意义分类， 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， 患者及其家属。