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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

基本信息

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
项目类别：
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

项目摘要

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sdhh缺乏症和