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缺乏症状并增加了发展多种癌症的终身风险，包括GI基质肿瘤， paraganglioma，嗜铬细胞瘤和肾细胞癌。在患有已知LOF/病原体的患者中种系SDHX突变，遗传咨询（筛查其他家庭成员）并增强癌症指示筛选程序。当在早期检测到SDH缺陷肿瘤时，它们通常是可以治愈。但是，3.2％的人口具有未知意义的种系SDHX错义变体（VUS，不知道是致病性或良性的）。因此，我们目前没有能力识别许多由于我们缺乏对功能的知识，有风险的患者适当地筛选他们种系SDHX VUS的后果。计划：该提案将在功能上测试SDHA VUS，利用深度突变扫描（DMS）方法我们已经生成的新型SDHA缺陷细胞系。我们将通过配对DMS来识别所有LOF SDHA变体我们实验室开发了一种负代谢选择方法。确定这种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驴子在每个细胞系中都有负代谢选择。可以提供强大的人类模型用于临床变体分类的证据将被生成并验证用于测试个体变体。方法：要解决人群中看到的大量SDHA变体，我们将创建所有人的库可能通过饱和诱变而可能的SDHA氨基酸变体，并使用此库来补充我们的SDHA- 不足的单元线。我们将通过在代谢物下种植库来选择SDHA LOF变体需要功能齐全的SDH（ - 丙酮酸， - 天冬氨酸）的条件。进行深度测序后，耗尽将进行分析。将为每个变体做出功能解释（功能正常或LOF）通过将计算出的效应分数与胡说八道/同义控件的分数进行比较（AIM 1）。填充电流缺乏SDH缺陷细胞系，我们将使用CRISPR/CAS9生成SDHB-，SDHC-和SDHD-KNOCKOUT 细胞线并验证它们。使用代谢物耗尽培养基的负选择将应用于这些培养基用于确定未来DMS实验的实用性的新型模型（AIM 2）。临床相关性：我们最好的癌症治疗机会在于SDH缺陷患者的早期检测肿瘤，因此，关键医学需求在于对SDHX遗传变异的分类，以实现鉴定和随后筛查处于风险的患者。

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）

TKI Treatment Sequencing in Advanced Gastrointestinal Stromal Tumors.

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.

DOI：
10.1245/s10434-022-12063-5
发表时间：
2022-10
期刊：
Annals of surgical oncology
影响因子：
3.7
作者：
通讯作者：

Paraganglioma of the Head and Neck: A Review.

头颈副神经节瘤：回顾。

DOI：
10.1016/j.eprac.2022.10.002
发表时间：
2023
期刊：
Endocrine practice : official journal of the American College of Endocrinology and the American Association of Clinical Endocrinologists
影响因子：
0
作者：
Sandow,Lyndsey;Thawani,Rajat;Kim,MyungSun;Heinrich,MichaelC
通讯作者：
Heinrich,MichaelC

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