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: 10255655
• 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/10255655
• 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.

目的：SDHA或SDHB的功能丧失(LOF)突变导致SDH缺乏和 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、肾细胞癌和乳腺癌。在大多数情况下， 具有这些突变的肿瘤发生在杂合胚系突变的背景下，这是 可遗传的。在已知致病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- 缺陷肿瘤在早期被发现，它们通常是可以治愈的。尽管如此，我们目前 没有能力识别有风险的患者并对他们进行适当的筛查。主 限制是我们对各种SDHA或B的功能后果缺乏了解 突变；只有引起LOF的SDHA或B变异才会导致癌症。SDHA的种系变种 和SDHB相对常见(占人口的1.5%)，但目前大多数被归类为 意义未知的变种(VU)。 计划：该提案将生成对SDHA和SDHB VU进行功能测试的新模型 在种群中可以看到，包括产生人体模型细胞系和利用深部 突变扫描方法。人体模型，可以提供强有力的证据用于 将生成变种的临床分类，并验证其是否用于测试单个变种。在……里面 此外，我们已经建立了一个酵母模型，允许对数千名 酵母菌同源基因(分别为ySdh1和ySdh2)的SDHA/B突变。使用这种酵母 结合饱和突变、功能筛选和深度测序的模型 所有SDHA和SDHB错义突变的功能后果将被描述。 方法：为了填补目前SDH缺陷细胞系的空白，我们将使用CRISPR-Cas9来产生 SDHA-和SDHB-基因敲除细胞系，并验证它们以确保它们用于确定 人类肿瘤中报告的SDHA和SDHB VUS的临床分类(目标1)。致信地址 在种群中看到的大量SDHA和SDHB变体，我们将创建 所有可能的ySdh1(目标2)和ySdh2(目标3)氨基酸变异饱和诱变 并将这些文库转化为缺乏相关内源酵母蛋白的酵母模型 (例如，在ySdh1缺失的酵母中表达的突变体ySdh1)。我们将根据SDHA/B LOF进行选择 通过在需要全功能SDH(甘油介质)的条件下培养文库来产生变异。 在深度测序之后，将进行消耗分析。功能释义 (功能正常或LOF)将通过比较计算的效果分数为每个变体制作 与胡言乱语/同义对照的那些。 临床相关性：对患有癌症的退伍军人的护理在 退伍军人事务部卫生保健总预算。据估计，超过30万名美国退伍军人 携带SDHA或SDHB种系突变。我们治愈癌症的最好机会在于及早 在SDH缺乏的肿瘤患者中进行检测，因此关键的医疗需要在于分类 SDHA和SDHB基因变异体能够识别和随后筛查高危人群 病人。确定哪些美国退伍军人有患SDH缺陷癌症的风险将 允许有针对性的、可能挽救生命的遗传咨询和增强的癌症筛查 程序。我们的研究结果也将适用于非退伍军人群体，包括 具有致病种系SDHA或SDHB突变的退伍军人的家庭成员。