权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

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

基本信息

批准号：
9796835
负责人：
PAUL R ANDREASSEN
金额：
$ 32.48万
依托单位：
CINCINNATI CHILDRENS HOSP MED CTR
依托单位国家：
美国
项目类别：
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/9796835
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 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 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 outcome forecast prevent protein expression protein function rare variant response segregation three dimensional structure variant of unknown significance

项目摘要

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/文摘