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: 10618794
• 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/10618794
• 关键词: Address; Amino Acids; Benign; Biological Assay; Budgets; CRISPR/Cas technology; Cancer Etiology; Carbon; Caring; Categories; 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; Heterozygote; 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; Recommendation; Regimen; Renal Cell Carcinoma; Reporting; Risk; Screening for cancer; Screening procedure; Source; Statistical Models; Stromal Neoplasm; Succinate Dehydrogenase; Technology; 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 的功能丧失 (LOF) 突变导致 SDH 缺陷和 增加终生患多种癌症的风险，包括胃肠道间质瘤， 副神经节瘤、嗜铬细胞瘤、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，即 遗传。对于已知致病性 SDHA 或 B 突变的患者，进行遗传咨询（以筛查 指出了其他家庭成员）和加强的癌症筛查程序。当SDH- 缺陷肿瘤在早期被发现，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。主要 局限性是我们缺乏对各种 SDHA 或 B 的功能后果的了解 突变；只有导致 LOF 的 SDHA 或 B 变体才会导致癌症。 SDHA 种系变异 和 SDHB 相对常见（占人口的 1.5%），但目前大多数被归类为 意义不明的变异（VUS）。 计划：该提案将生成用于功能测试 SDHA 和 SDHB VUS 的新模型 在人群中看到的，包括生成人类模型细胞系和利用深层 突变扫描方法。人体模型，可以提供强有力的证据用于 将生成并验证变异的临床分类，以测试各个变异。在 此外，我们还建立了一个酵母模型，可以对数千种酵母进行功能筛选 酵母同源基因（分别为 ySdh1 和 ySdh2）中的 SDHA/B 突变。使用这种酵母 模型结合饱和诱变、功能筛选和深度测序， 将分析所有 SDHA 和 SDHB 错义突变的功能后果。 方法：为了填补目前 SDH 缺陷细胞系的空白，我们将使用 CRISPR-Cas9 生成 SDHA 和 SDHB 敲除细胞系并对其进行验证以确保其确定能力 人类肿瘤中报告的 SDHA 和 SDHB VUS 的临床分类（目标 1）。致地址 由于人群中存在大量 SDHA 和 SDHB 变体，我们将创建以下库 通过饱和诱变产生所有可能的 ySdh1、（目标 2）和 ySdh2（目标 3）氨基酸变体 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 ySdh1）。我们将选择 SDHA/B LOF 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体。 深度测序后，将进行耗尽分析。功能解释 （功能正常或 LOF）将通过比较计算的效果分数为每个变体进行 与那些无意义/同义的控件。 临床相关性：患有癌症的退伍军人的护理占了很大一部分 退伍军人事务部医疗保健总体预算。据估计，超过30万名美国退伍军人 含有 SDHA 或 SDHB 种系突变。治愈癌症的最佳机会在于早期 SDH 缺陷肿瘤患者的检测，因此关键的医疗需求在于分类 SDHA 和 SDHB 遗传变异可实现高危人群的识别和后续筛查 患者。确定哪些美国退伍军人有患 SDH 缺陷型癌症的风险将 允许进行有针对性的、可能挽救生命的遗传咨询和加强癌症筛查 程序。我们的研究结果也与非退伍军人群体相关，包括 具有致病性种系 SDHA 或 SDHB 突变的退伍军人家庭成员。