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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

基本信息

批准号：
10447686
负责人：
Michael C Heinrich
金额：
$ 14.05万
依托单位：
OREGON HEALTH & SCIENCE UNIVERSITY
依托单位国家：
美国
项目类别：
财政年份：
2021
资助国家：
美国
起止时间：
2021-07-08 至 2024-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10447686
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 Persons 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

项目摘要

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.

目的：琥珀酸脱氢酶亚单位(SDHx)基因功能丧失(LOF)突变导致 SDH缺乏并增加患多种癌症的终生风险，包括胃肠道间质瘤，副神经节瘤、嗜铬细胞瘤和肾癌。已知的LOF/病原性LOF患者生殖系SDHx突变、遗传咨询(筛查其他家庭成员)和增强的癌症说明了筛查程序。当SDH缺乏的肿瘤在早期被发现时，它们通常是是可以治愈的。然而，3.2%的人群携带着意义未知的生殖系SDHx错义变体 (VUS，未知致病或良性)。因此，我们目前没有能力识别许多由于我们缺乏对功能性疾病的了解，有风险的患者应进行适当的筛查生殖系SDHx vus的后果。计划：该提案将使用深度突变扫描(DMS)方法对SDHA VU进行功能性测试我们培育的新型SDHA缺失细胞系。我们将通过配对DMS来识别所有LOF SDHA变体用我们实验室开发的负代谢选择方法。以确定此方法是否扩展为了测试其他SDHx基因(SDHB、C、D)的VU，我们将生成这些个体的敲除模型基因和评估在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的证据变种。方法：为了解决在人群中看到的大量SDHA变体，我们将创建所有通过饱和突变获得可能的SDHA氨基酸变异，并利用该文库来补充我们的SDHA- 缺乏细胞系。我们将通过在代谢物耗尽的情况下培养文库来筛选SDHA LOF变体需要全功能SDH(-丙酮酸、-天冬氨酸)的条件。在深度测序之后，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 通过将计算的效果分数与无意义/同义对照的分数进行比较(目标1)。要填满水流在没有SDH缺陷细胞系的情况下，我们将使用CRISPR/Cas9产生SDHB-、SDHC-和SDHD-基因敲除并验证它们的有效性。使用代谢物耗竭介质的阴性选择将被应用于这些确定其在未来DMS实验中的效用的新模型(目标2)。临床意义：我们治愈癌症的最好机会在于及早发现SDH缺陷患者因此，关键的医疗需要在于对SDHx基因变体进行分类，以便能够识别和随后对高危患者进行筛查。

项目成果

期刊论文数量（14）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Ripretinib versus sunitinib in gastrointestinal stromal tumor: ctDNA biomarker analysis of the phase 3 INTRIGUE trial.

Ripretinib 与舒尼替尼治疗胃肠道间质瘤：3 期 INTRIGUE 试验的 ctDNA 生物标志物分析。

DOI：
10.1038/s41591-023-02734-5
发表时间：
2024
期刊：
Nature medicine
影响因子：
82.9
作者：
Heinrich,MichaelC;Jones,RobinL;George,Suzanne;Gelderblom,Hans;Schöffski,Patrick;vonMehren,Margaret;Zalcberg,JohnR;Kang,Yoon-Koo;Razak,AlbiruniAbdul;Trent,Jonathan;Attia,Steven;LeCesne,Axel;Siontis,BrittanyL;Goldstein,Davi
通讯作者：
Goldstein,Davi

TKI Treatment Sequencing in Advanced Gastrointestinal Stromal Tumors.

晚期胃肠道基质肿瘤中的TKI治疗测序。

DOI：
10.1007/s40265-022-01820-1
发表时间：
2023-01
期刊：
DRUGS
影响因子：
11.5
作者：
Khosroyani, Homma M.;Klug, Lillian R.;Heinrich, Michael C.
通讯作者：
Heinrich, Michael C.

ASO Visual Abstract: Ten-Year Survivorship in Patients with Metastatic Gastrointestinal Stromal Tumors.

ASO 视觉摘要：转移性胃肠道间质瘤患者的十年生存率。

DOI：
发表时间：
2022
期刊：
Annals of surgical oncology
影响因子：
3.7
作者：
Sutton,ThomasL;Walker,BrettS;Billingsley,KevinG;Corless,ChristopherL;Sheppard,BrettC;Heinrich,MichaelC;Mayo,SkyeC
通讯作者：
Mayo,SkyeC

Ten-Year Survivorship in Patients with Metastatic Gastrointestinal Stromal Tumors.