Functional profiling of germline SDH variants associated with cancer susceptibility

目的：SDHA或SDHB的功能丧失（LOF）突变导致SDH缺陷， 增加患上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出现在杂合生殖系突变的情况下， 遗传的在已知致病性SDHA或B突变的患者中， 其他家庭成员）和增强的癌症筛查程序。当SDH- 缺陷肿瘤在早期阶段被检测到，它们通常是可治愈的。尽管如此，我们目前 没有能力识别有风险的患者并对其进行适当的筛查。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;只有引起LOF的SDHA或B变体才能导致癌症。SDHA的种系变体 和SDHB相对常见（占人口的1.5%），但目前大多数被归类为 未知意义的变异体（Variants of Unknown Significance，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，包括产生人类模型细胞系和利用深 突变扫描法人体模型，可以提供强有力的证据，用于 将生成变体的临床分类并验证用于测试个体变体。在 此外，我们已经建立了一个酵母模型，允许功能筛选数千个 酵母同源基因中的SDHA/B突变（分别为ySdh 1和ySdh 2）。用这种酵母 结合饱和诱变、功能筛选和深度测序， 将对所有SDHA和SDHB错义突变的功能后果进行分析。 方法：为了填补目前缺乏SDH细胞系的空白，我们将使用CRISPR-Cas9来产生 SDHA和SDHB敲除细胞系，并对其进行验证，以确保其确定 在人类肿瘤中报道的SDHA和SDHB VUS的临床分类（目的1）。解决 由于在人群中发现了大量的SDHA和SDHB变异体，我们将创建 通过饱和诱变法测定所有可能的ySdh 1（Aim 2）和ySdh 2（Aim 3）氨基酸变体 并将这些文库转化为缺乏相关内源性酵母蛋白的酵母模型 (e.g.在缺失ySdh 1的酵母中表达的突变体ySdh 1）。我们将选择SDHA/B LOF 通过在需要完全功能性SDH（甘油培养基）的条件下生长文库来扩增变体。 深度测序后，将进行耗竭分析。功能解释 通过比较计算的效应评分，对每个变体进行功能正常或LOF评估 与无意义/同义对照的那些。 临床相关性：癌症退伍军人的护理代表了癌症治疗的重要组成部分。 退伍军人事务部的整体医疗预算。据估计，超过30万美国退伍军人 携带SDHA或SDHB种系突变。我们治愈癌症的最好机会在于早期 因此，关键的医疗需求在于对患有SDH缺陷型肿瘤的患者进行分类 SDHA和SDHB遗传变异，以实现识别和随后的筛查风险 患者确定哪些美国退伍军人有患上缺乏SDH的癌症的风险， 允许重点和潜在的挽救生命的遗传咨询和加强癌症筛查 程序.我们的研究结果也将与非退伍军人群体相关，包括 具有致病性种系SDHA或SDHB突变的退伍军人的家庭成员。

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.