Novel models for functional profiling of germline SDH variants associated with cancer

与癌症相关的种系 SDH 变异功能分析的新模型

• 批准号: 10290625
• 负责人: Michael C Heinrich
• 金额: $ 18万
• 依托单位: OREGON HEALTH & SCIENCE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-07-08 至 2023-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10290625
• 关键词: Address; American; Amino Acids; Aspartic Acid; Benign; Biological Assay; Biological Models; CRISPR/Cas technology; Cell Line; Cell model; Classification; Clinical; Complement; Complex; Coupled; DNA Sequence Alteration; Data; Defect; Early Diagnosis; Enzymes; Family member; Frequencies; Functional disorder; Future; Gene Frequency; General Population; Genes; Genetic; Genetic Counseling; Genome; Genomics; Guidelines; Hereditary Disease; Human; Human Cell Line; Individual; Inherited; Intervention; Joints; Knock-out; Knowledge; Laboratories; Libraries; Life; Malignant Neoplasms; Medical; Medical Genetics; Metabolic; Methods; Modeling; Multienzyme Complexes; Mutagenesis; Mutation; Paraganglioma; Pathogenicity; Patients; Pheochromocytoma; Population; Pyruvate; Renal Cell Carcinoma; Reporting; Resectable; Risk; Savings; Screening for cancer; Screening procedure; Statistical Data Interpretation; Stromal Neoplasm; Succinate Dehydrogenase; Susceptibility Gene; Syndrome; Testing; Variant; cancer predisposition; cancer risk; clinical decision-making; clinically relevant; clinically significant; deep sequencing; enzyme activity; experimental study; genetic counselor; genetic testing; genetic variant; human model; improved; lifetime risk; loss of function; medical schools; molecular pathology; mutation screening; novel; screening; tumor; variant of unknown significance

Objectives: Loss-of-function (LOF) mutations in succinate dehydrogenase subunit (SDHx) genes result in SDH-deficiency and increase the lifetime risk for developing a number of cancers, including GI stromal tumors, paraganglioma, pheochromocytoma, and renal cell carcinoma. In patients with a known LOF/pathogenic germline SDHx mutation, genetic counseling (to screen other family members) and enhanced cancer screening procedures are indicated. When SDH-deficient tumors are detected at early stages, they are often curable. However, 3.2% of the population carry germline SDHx missense variants of unknown significance (VUS, not known to be pathogenic or benign). Therefore, we currently do not have the ability to identify many patients at risk and screen them appropriately because of our lack of knowledge of the functional consequences of germline SDHx VUS. Plan: This proposal will functionally test SDHA VUS utilizing a deep mutational scanning (DMS) approach in a novel SDHA-deficient cell line that we have generated. We will identify all LOF SDHA variants by pairing DMS with a negative metabolic selection method developed in our laboratory. To determine if this approach extends to test VUS of the other SDHx genes (SDHB, C, D), we will generate knockout models of these individual genes and asses negative metabolic selection in each cell line. Human models, which can provide strong evidence to be used for clinical variant classification will be generated and validated for testing individual variants. Methods: To address the large number of SDHA variants seen in the population, we will create libraries of all possible SDHA amino-acid variants by saturation mutagenesis and use this library to complement our SDHA- deficient cell line. We will select against SDHA LOF variants by growing the libraries under metabolite-depleted conditions requiring fully functional SDH (-pyruvate, -aspartic acid). Following deep sequencing, depletion analysis will be performed. Functional interpretations (functionally normal or LOF) will be made for each variant by comparing calculated effect scores with those of nonsense/ synonymous controls (Aim 1). To fill the current void of SDH-deficient cell lines, we will use CRISPR/Cas9 to generate SDHB-, SDHC-, and SDHD-knockout cell lines and validate them. Negative selection using metabolite-depleted medium will be applied to these novel models to determine their utility for future DMS experiments (Aim 2). Clinical Relevance: Our best chance of a cancer cure lies in early detection in patients with SDH-deficient tumors, thus the critical medical need lies in classifying SDHx genetic variants to enable identification and subsequent screening of at-risk patients.

目标：琥珀酸脱氢酶亚基 (SDHx) 基因的功能丧失 (LOF) 突变导致 SDH 缺乏会增加终生罹患多种癌症的风险，包括胃肠道间质瘤、 副神经节瘤、嗜铬细胞瘤和肾细胞癌。对于已知 LOF/致病性的患者 种系 SDHx 突变、遗传咨询（筛选其他家庭成员）和增强癌症 指出了筛选程序。当 SDH 缺陷的肿瘤在早期被发现时，通常会发现 可治愈的。然而，3.2% 的人群携带意义不明的种系 SDHx 错义变异 （VUS，未知是致病性还是良性）。因此，我们目前还没有能力识别许多 由于我们缺乏对功能的了解，因此对处于危险中的患者进行了适当的筛查 种系 SDHx VUS 的后果。 计划：该提案将利用深度突变扫描 (DMS) 方法对 SDHA VUS 进行功能测试 我们生成的新型 SDHA 缺陷细胞系。我们将通过配对 DMS 来识别所有 LOF SDHA 变体 使用我们实验室开发的负代谢选择方法。确定该方法是否可扩展 为了测试其他 SDHx 基因（SDHB、C、D）的 VUS，我们将生成这些个体的敲除模型 基因并评估每个细胞系中的负代谢选择。人体模型，可以提供强大的 将生成用于临床变异分类的证据，并验证用于测试个体 变种。 方法：为了解决人群中发现的大量 SDHA 变体，我们将创建所有的库 通过饱和诱变可能的 SDHA 氨基酸变体，并使用该库来补充我们的 SDHA- 缺陷细胞系。我们将通过在代谢物耗尽的条件下培养文库来选择 SDHA LOF 变体 需要全功能 SDH（-丙酮酸、-天冬氨酸）的条件。深度测序后，耗尽 将进行分析。将对每个变体进行功能解释（功能正常或 LOF） 通过将计算出的效果分数与无意义/同义对照的效果分数进行比较（目标 1）。来填充当前的 由于没有 SDH 缺陷细胞系，我们将使用 CRISPR/Cas9 生成 SDHB、SDHC 和 SDHD 敲除细胞系 细胞系并验证它们。使用代谢物耗尽培养基的负选择将应用于这些 新颖的模型来确定它们对未来 DMS 实验的效用（目标 2）。 临床相关性：治愈癌症的最佳机会在于早期发现 SDH 缺陷患者 肿瘤，因此关键的医疗需求在于对 SDHx 遗传变异进行分类，以便能够识别和 随后对高危患者进行筛查。