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: 10166885
• 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 2023-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10166885
• 关键词: 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; 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.

项目概要/摘要 现在正在对多种疾病进行基因筛查，包括结缔组织疾病 疾病和癌症的代谢综合征。但这些屏幕的实用性取决于能否解释 所识别的变异的临床意义。特别是，通常很难确定 错义变异的重要性，这种变异通常很少见。对于罕见变异，共分离研究经常进行 动力不足，无法用于变体分类。功能测定提供了最有希望的替代方案 用于对不确定意义的变体（VUS）进行分类。我们的目标是从 ATM 开始，因为有 >2,480 ClinVar中列出的错义VUS，是为了建立一个准确且高容量的系统来进行功能分类 VUS。 ATM 的错义变异已在共济失调毛细血管扩张症 (A-T) 中被发现，这是一种隐性疾病， 以及因 ATM 种系突变而易患遗传性癌症综合征的患者。 ATM机有一个中央 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抵抗力。 ATM 蛋白表达一直存在问题，部分原因是基因大小较大且 mRNA 质量较差。这 描述 ATM 变异的影响和不同区域的作用的研究受到很大限制 自动提款机。事实上，之前还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。 已建立对 ATM VUS 进行分类。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地表达全长人类 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 作为表达载体，我们开发了一种模块化方法，足以快速评估大型 ATM VUS 规模。在目标 1 中，我们将通过 DSB 相关分析来验证我们用于表征 ATM VUS 的新系统： 测试先前根据临床和遗传标准定义的良性和致病性标准。 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， 其中包含激酶结构域和关键调控元件，也是最已知的致病因子所在 存在错义 ATM 变体。此外，我们将把功能测定的结果纳入多因素分析中。 分析以及临床和遗传数据，用于与错义 ATM 相关的癌症风险的可靠预测 变种。了解变异的影响以及 ATM 在预防中的作用的另一个重要限制 疾病，需要更好地定义 ATM 不同区域的作用，而这在很大程度上是未知的。这将是 通过表达删除整个蛋白质区域的突变体来解决目标 2 中的问题。我们也会测试一下效果 结合 NBS1 激活剂的致病变异，并将解释 3 维结构效应 FATKIN 区域的致病性变异。我们预计这里提出的工作将具有重大的临床意义 通过描述 VUS 的影响，将极大地提高对 ATM 功能的理解。