Functional profiling of germline SDH variants associated with cancer susceptibility

与癌症易感性相关的种系 SDH 变异的功能分析

• 批准号: 10382367
• 负责人: Michael C Heinrich
• 金额: --
• 依托单位: PORTLAND VA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-04-01 至 2025-03-31
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10382367
• 关键词: Address; Amino Acids; Benign; Biological Assay; Budgets; CRISPR/Cas technology; Cancer Etiology; Carbon; Caring; Cell Line; Child; Classification; Clinical; Codon Nucleotides; Complement; Counseling; Coupled; DNA; DNA Sequence Alteration; Data Set; Defect; Development; Diagnosis; Early Diagnosis; Ensure; Enzymes; Family member; Foundations; Gene Mutation; General Population; Generations; Genes; Genetic; Genetic Counseling; Genetic Enhancement; Germ-Line Mutation; Glucose; Glycerol; Goals; Growth; Guidelines; Healthcare; Hereditary Disease; Heritability; Homologous Gene; Human; Human Cell Line; Impairment; Individual; Inherited; Intervention; Knock-out; Knowledge; Lead; Libraries; Life; Malignant Neoplasms; Medical; Medical Genetics; Medicine; Metabolic; Methods; Missense Mutation; Modeling; Multienzyme Complexes; Mutagenesis; Mutation; Paraganglioma; Pathogenicity; Patient Care; Patients; Pheochromocytoma; Population; Predisposition; Procedures; Proteins; Publishing; Regimen; Renal Cell Carcinoma; Reporting; Risk; Savings; Screening for cancer; Screening procedure; Source; Statistical Models; Stromal Neoplasm; Succinate Dehydrogenase; Testing; Variant; Veterans; Veterans Health Administration; Work; Yeast Model System; Yeasts; cancer risk; clinical decision-making; clinically relevant; clinically significant; complement deficiency; deep sequencing; functional loss; genetic testing; genetic variant; human model; improved; lifetime risk; loss of function; loss of function mutation; malignant breast neoplasm; mutant; mutation screening; novel; screening; testing services; tumor; variant of unknown significance; yeast protein

Objectives: Loss-of-function (LOF) mutations in SDHA or SDHB result in SDH-deficiency and increase the lifetime risk for developing a number of cancers, including GI stromal tumors, paraganglioma, pheochromocytoma, renal cell carcinoma, and breast cancer. In most cases, tumors with these mutations arise in the setting of a heterozygous germline mutation, which is heritable. In patients with a known pathogenic SDHA or B 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. Nonetheless, we currently do not have the ability to identify patients at risk and screen them appropriately. The main limitation is our lack of knowledge of the functional consequences of various SDHA or B mutations; only SDHA or B variants that cause LOF lead to cancer. Germline variants of SDHA and SDHB are relatively common (1.5% of population), however most are currently classified as variants of unknown significance (VUS). Plan: This proposal will generate novel models for functionally testing SDHA and SDHB VUS seen in the population, including generating human model cell lines and utilizing a deep mutational scanning approach. Human models, which can provide strong evidence to be used for clinical classification of variants will be generated and validated for testing individual variants. In addition, we have established a yeast model that allows functional screening of thousands of SDHA/B mutations in the yeast homolog genes (ySdh1 and ySdh2, respectively). Using this yeast model coupled with saturation mutagenesis, functional screening and deep sequencing, the functional consequences of all SDHA and SDHB missense mutations will be profiled. Methods: To fill the current void of SDH-deficient cell lines, we will use CRISPR-Cas9 to generate SDHA- and SDHB-knockout cell lines and validate them to ensure their power for determining clinical classifications for SDHA and SDHB VUS reported in human tumors (Aim 1). To address the large number of SDHA and SDHB variants seen in the population, we will create libraries of all possible ySdh1, (Aim 2) and ySdh2 (Aim 3) amino-acid variants by saturation mutagenesis and transform these libraries into yeast models deficient in the relevant endogenous yeast protein (e.g. mutant ySdh1 expressed in ySdh1-deleted yeast). We will select against SDHA/B LOF variants by growing the libraries under conditions requiring fully functional SDH (glycerol media). 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. Clinical Relevance: The care of veterans with cancer represents a significant portion of the overall Veterans Affairs Health Care budget. It is estimated that more than 300,000 US veterans harbor an SDHA or SDHB germline mutation. 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 SDHA and SDHB genetic variants to enable identification and subsequent screening of at-risk patients. Identifying which US Veterans are at risk of developing an SDH-deficient cancer will allow focused and potentially life-saving genetic counseling and enhanced cancer screening procedures. The results from our study will also be relevant to non-veteran populations, including family members of Veterans with pathogenic germline SDHA or SDHB mutations.

目标：SDHA或SDHB中的功能丧失（LOF）突变导致SDH缺陷和 增加终身出现多种癌症的风险，包括胃肠道基质肿瘤， paraganglioma，嗜铬细胞瘤，肾细胞癌和乳腺癌。在大多数情况下， 具有这些突变的肿瘤是在杂合种系突变的情况下出现的 遗传。在患有已知病原SDHA或B突变的患者中，遗传咨询（筛查 其他家庭成员）和增强的癌症筛查程序。当SDH- 在早期发现缺乏肿瘤，通常可以治愈。尽管如此，我们目前 没有能力识别有风险的患者并适当筛查他们。主 限制是我们缺乏对各种SDHA或B的功能后果的了解 突变；只有导致LOF导致癌症的SDHA或B变体。 SDHA的种系变体 SDHB和SDHB相对普遍（占人口的1.5％），但是大多数目前被归类为 未知意义的变体（VUS）。 计划：该建议将生成用于在功能上测试SDHA和SDHB VUS的新型模型 在人群中看到，包括生成人类模型细胞系和使用深度 突变扫描方法。人类模型，可以提供有力的证据 将生成和验证变体的临床分类，以测试单个变体。在 此外，我们建立了一个酵母模型，该模型允许筛选数千个 酵母同源基因中的SDHA/B突变（分别为YSDH1和YSDH2）。使用此酵母 模型结合饱和诱变，功能筛选和深度测序， 所有SDHA和SDHB错义突变的功能后果将得到介绍。 方法：为了填充缺陷SDH缺陷细胞系的当前空隙，我们将使用CRISPR-CAS9生成 SDHA-和SDHB-KNOCKOUT单元线并验证它们以确保其确定能力 SDHA和SDHB VU的临床分类在人类肿瘤中报道（AIM 1）。解决 在人群中看到的大量SDHA和SDHB变体，我们将创建 所有可能的YSDH1，（AIM 2）和YSDH2（AIM 3）氨基酸变体通过饱和诱变 并将这些库转换为缺乏相关内源酵母蛋白缺乏的酵母模型 （例如，在YSDH1删除的酵母中表达的突变体YSDH1）。我们将选择反对SDHA/B LOF 通过在需要功能齐全的SDH（甘油培养基）的条件下种植库来种植库。 进行深度测序后，将进行耗竭分析。功能解释 （在功能上正常或LOF）将通过比较计算的效果分数来制作每个变体 具有胡说八道/同义控件的那些。 临床相关性：癌症退伍军人的护理代表着很大一部分 整体退伍军人事务保健预算。据估计，超过30万美国退伍军人 携带SDHA或SDHB种系突变。我们最好的癌症治愈机会在早期 SDH缺陷型肿瘤患者的检测，因此关键的医学需求在于分类 SDHA和SDHB遗传变异以识别和随后筛查处于危险中 患者。确定哪些美国退伍军人有患有SDH缺陷癌症的风险 允许集中且潜在的挽救生命的遗传咨询并增强癌症筛查 程序。我们研究的结果也将与非退伍军人人口有关，包括 具有致病性系SDHA或SDHB突变的退伍军人家庭成员。