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.

项目摘要/摘要 目前正在对从结缔组织到结缔组织的各种疾病进行基因筛查 疾病和代谢综合征到癌症。但这些屏幕的效用取决于是否能够解释 已确定的变异的临床意义。特别是，经常很难确定 错义变体的重要性，这种变体通常很少见。对于罕见的变异，共同分离研究经常是 能力不足，不能用于不同的分类。功能分析提供了最有希望的替代方案 用于对不确定意义的变体(VU)进行分类。我们的目标是从ATM开始，因为有2,480个 在ClinVar中为其列出的误解VU，是为了建立一个准确的、大容量的系统来进行功能分类 沃斯。在共济失调-毛细血管扩张症(A-T)中发现了ATM的错义变体，这是一种隐性疾病， 以及通过ATM胚系突变而易患遗传性癌症综合征的患者。自动取款机有一个中央 在调节细胞对DNA双链断裂(DSB)反应中的作用，包括介导DNA损伤 信号、G2检查点和细胞对电离辐射的抵抗。 ATM蛋白的表达一直存在问题，部分原因是基因大小和信使核糖核酸质量差。这 极大地限制了研究ATM变异的影响和不同区域的作用 自动取款机。事实上，没有经过严格验证的系统，也没有经过灵敏度和特异度校准的系统 已建立对自动柜员机VU进行分类。我们将通过采取创新战略来克服这些障碍 为了利用慢病毒载体在ATM缺陷细胞中快速有效地表达全长人ATM 密码子优化的cDNA.重要的是，通过在ATM片段中合成变体，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，足够快地在大范围内评估ATM VU 比例。在目标1中，我们将验证我们的新系统，用于使用与DSB相关的分析来表征ATM VU 检测之前根据临床和遗传标准定义的良性和致病标准。 我们还将通过表征C-末端Fatkin区域的300个错义ATM VU来启动我们的系统， 它包含激酶结构域和关键调控元件，也是最已知的致病因素 错误的ATM变体仍然存在。此外，我们将把功能分析的结果合并到多因素分析中 与临床和遗传数据一起分析与错义ATM相关的癌症风险的可靠预测 变种。理解变异的影响以及ATM在预防中的作用的另一个重要限制 疾病，是需要更好地定义ATM不同区域的作用，这在很大程度上是未知的。这将是 在目标2中，通过表达删除整个蛋白质区域的突变体来解决。我们还将测试其效果 将解释致病变异体与NBS1激活剂结合的三维结构效应 Fatkin区的致病变异。我们希望在这里提出的工作将有一个重大的临床 通过描述VU的特性产生影响，并将极大地提高对ATM功能的理解。