Functional characterization of the role of distinct domains of ATM and the impact of sequence variants on the DNA damage response

ATM 不同结构域的功能特征以及序列变异对 DNA 损伤反应的影响

• 批准号: 10436183
• 负责人: PAUL R ANDREASSEN
• 金额: $ 31万
• 依托单位: CINCINNATI CHILDRENS HOSP MED CTR
• 依托单位国家: 美国
• 财政年份: 2019
• 资助国家: 美国
• 起止时间: 2019-08-20 至 2024-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10436183
• 关键词: 3-Dimensional; ATM Gene Mutation; ATM deficient; ATM function; ATM gene; Address; Apoptosis; Ataxia Telangiectasia; Ataxia Telangiectasia Patients; BRCA2 gene; Benign; Binding; Binding Sites; Biological Assay; Biological Process; Biophysics; Breast; C-terminal; CHEK2 gene; Cancer Patient; Cancer-Predisposing Gene; Catalytic Domain; Cell Cycle Arrest; Cells; Classification; ClinVar; Clinical; Codon Nucleotides; Complementary DNA; Connective Tissue Diseases; DNA; DNA Damage; DNA Double Strand Break; DNA Repair; Data; Databases; Disease; Genes; Genetic; Genetic Screening; Germ-Line Mutation; Goals; Hereditary Neoplastic Syndromes; Human; Immune System Diseases; Individual; Ionizing radiation; Knowledge; Length; Lentivirus Vector; Literature; Malignant Neoplasms; Malignant neoplasm of pancreas; Mediating; Medical Genetics; Messenger RNA; Metabolic syndrome; Missense Mutation; Mutation; NBS1 gene; Nerve Degeneration; Nucleic Acid Regulatory Sequences; Pathogenicity; Patients; Penetrance; Phenotype; Phosphotransferases; Predisposition; Prognosis; Proteins; RNA Splicing; Radiation Tolerance; Regulatory Element; Resistance; Risk; Role; Sensitivity and Specificity; Series; Signal Transduction; Source; System; Technology; Testing; Variant; Work; ataxia telangiectasia mutated protein; base; cancer risk; clinical care; clinically significant; disorder risk; expression vector; genetic approach; human disease; improved; innovation; leukemia/lymphoma; mutant; next generation sequencing; novel; prevent; protein expression; protein function; rare variant; response; segregation; three dimensional structure; treatment response; variant of unknown significance

PROJECT SUMMARY/ABSTRACT Genetic screens are now being performed for a variety of diseases, ranging from connective tissue diseases and metabolic syndromes to cancer. But the utility of these screens depends on being able to interpret the clinical significance of the variants that are identified. In particular, it is frequently difficult to determine the significance of missense variants, which are often rare. For rare variants, co-segregation studies are frequently underpowered to be useful for variant classification. Functional assays provide the most promising alternative for classifying variants of uncertain significance (VUS). Our goal, beginning with ATM, since there are >2,480 missense VUS listed for it in ClinVar, is to establish an accurate and high capacity system to functionally classify VUS. Missense variants of ATM have been identified in ataxia-telangiectasia (A-T), which is a recessive disease, and in patients predisposed to a hereditary cancer syndrome by germline mutations in ATM. ATM has a central role in regulating the cellular response to DNA double-strand breaks (DSBs), including mediating DNA damage signaling, the G2 checkpoint and cellular resistance to ionizing radiation. ATM protein expression has been problematic, in part due to large gene size and poor mRNA quality. This has greatly restricted studies to characterize the effects of ATM variants and the roles of different regions of ATM. In fact, no rigorously validated system, nor one calibrated for sensitivity and specificity, has previously been established to classify ATM VUS. We will surmount these obstacles by employing an innovative strategy for the rapid and efficient expression of full-length human ATM in ATM-deficient cells using a lentiviral vector and a codon-optimized cDNA. Importantly, by synthesizing variants in fragments of ATM which are then inserted into the expression vector, we have developed a modular approach rapid enough to evaluate ATM VUS on a large scale. In Aim 1, we will validate our novel system for characterizing ATM VUS using DSB-related assays by testing benign and pathogenic standards that have previously been defined based on clinical and genetic criteria. We will also initiate our system by characterizing 300 missense ATM VUS of the C-terminal FATKIN region, which contains the kinase domain and key regulatory elements, and which is where the most known pathogenic missense ATM variants reside. Further, we will incorporate the results of functional assays into a multifactorial analysis, along with clinical and genetic data, for robust predictions of cancer risk associated with missense ATM variants. Another important limitation to understanding the effects of variants, and the role of ATM in preventing disease, is a need to better define the roles of distinct regions of ATM, which is largely unknown. This will be addressed in Aim 2 by expressing mutants that delete regions throughout the protein. We will also test the effects of pathogenic variants on binding to the NBS1 activator and will interpret the 3-dimensional structural effects of pathogenic variants in the FATKIN region. We expect that the work proposed here will have a major clinical impact by characterizing VUS and will dramatically improve understanding of ATM function.

项目总结/摘要 基因筛选现在正在进行各种疾病，从结缔组织 疾病和代谢综合征到癌症。但这些屏幕的效用取决于能否解释 所鉴定的变异的临床意义。特别是，通常难以确定 错义变体的意义，这往往是罕见的。对于罕见的变异，共分离研究经常是 对于变异分类来说是有用的。功能测定提供了最有前途的替代方法 不确定重要性（Undercertain Significance，VUS）我们的目标，从ATM开始，因为有> 2，480个 在ClinVar中列出的错义VUS，是为了建立一个准确和高容量的系统， VUS。ATM的错义变体已经在共济失调-毛细血管扩张症（A-T）中被鉴定，这是一种隐性疾病， 以及由于ATM的生殖细胞突变而易患遗传性癌症综合征的患者。ATM有一个中央 在调节细胞对DNA双链断裂（DSB）的反应中的作用，包括介导DNA损伤 信号传导、G2检查点和细胞对电离辐射的抗性。 ATM蛋白表达一直存在问题，部分原因是基因大小大和mRNA质量差。这 极大地限制了研究，以表征ATM变体的影响和不同区域的作用， 大气压事实上，以前没有经过严格验证的系统，也没有经过灵敏度和特异性校准的系统， 建立了ATM VUS分类。我们将通过采用创新战略来克服这些障碍 使用慢病毒载体在ATM缺陷细胞中快速有效表达全长人ATM， 密码子优化的cDNA。重要的是，通过合成ATM片段中的变体，然后将其插入到 表达载体，我们已经开发了一种模块化的方法，足以快速评估ATM VUS上的大型 规模在目标1中，我们将通过以下方法验证我们使用DSB相关检测表征ATM VUS的新系统： 检测良性和致病性标准，这些标准以前是根据临床和遗传标准定义的。 我们还将通过表征C端FATKIN区域的300个错义ATM VUS来启动我们的系统， 它包含激酶结构域和关键的调控元件，这是最已知的致病性 错误的ATM变体存在。此外，我们将把功能测定的结果纳入一个多因素分析， 分析，沿着临床和遗传数据，用于与错义ATM相关的癌症风险的稳健预测 变体。理解变异影响的另一个重要限制，以及ATM在预防中的作用， 疾病，需要更好地定义ATM的不同区域的作用，这在很大程度上是未知的。这将是 通过表达缺失整个蛋白质区域的突变体来解决目标2中的问题。我们还将测试 的致病性变异结合NBS 1激活剂，并将解释三维结构的影响， FATKIN区域的致病性变体。我们预计，这里提出的工作将有一个重大的临床 通过描述VUS的特性来影响，并将大大提高对ATM功能的理解。