Properties and determinants of GAA repeat instability

GAA 重复不稳定性的特性和决定因素

基本信息

批准号：
7555919
负责人：
SANJAY I BIDICHANDANI
金额：
$ 31.01万
依托单位：
UNIVERSITY OF OKLAHOMA HLTH SCIENCES CTR
依托单位国家：
美国
项目类别：
财政年份：
2004
资助国家：
美国
起止时间：
2004-01-01 至 2011-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7555919
关键词：
ATM deficient Address Affect Age Alleles Ataxia Ataxia Telangiectasia Autopsy Biological Assay Cell Culture Techniques Cell Line Cell Separation Cells Cerebellum Chest DNA DNA Damage DNA Double Strand Break DNA Repair Data Development Disease Disease Progression Disease model Double Effect Double Strand Break Repair Fiber Fluorescence-Activated Cell Sorting Fluorescent in Situ Hybridization Frequencies Friedreich Ataxia Gene Expression Gene Proteins Genes Genomics Goals Human Immunofluorescence Immunologic Individual Inherited Joints Label Length Lower Extremity Mammalian Cell Measurement Measures Methods Mitochondrial Proteins Mitotic Modeling Mus Mutate Nerve Degeneration Nervous system structure Neuroglia Neurons Nonhomologous DNA End Joining Pathogenesis Pathology Pathway interactions Patients Peripheral Nerves Pharmaceutical Preparations Phenotype Positioning Attribute Predisposition Prevalence Process Property Proprioception Role Sampling Screening procedure Sensory Sensory Ataxias Severities Signal Pathway Signal Transduction Site Specificity Spinal Cord Spinal Ganglia Spinocerebellar Tracts Staging System Testing Therapeutic Time Tissues Transgenic Mice Transgenic Organisms Trinucleotide Repeats Upper Extremity Western Blotting age related ataxia telangiectasia mutated protein axonal degeneration base cell type chromatin immunoprecipitation design dorsal column frataxin homologous recombination in vivo insight laser capture microdissection lymphoblastoid cell line mouse model oxidative damage progressive neurodegeneration public health relevance repaired research study response sensor single cell analysis tissue preparation vibration

项目摘要

DESCRIPTION (provided by applicant): Friedreich ataxia is the most common inherited ataxia. Friedreich ataxia patients have large expansions of a GAA triplet-repeat sequence in both copies of the FXN gene. The progressive ataxia is mainly "sensory" and is due to primary degeneration of dorsal root ganglia (DRG). The cause for the selective and progressive degeneration of the sensory DRG neurons is not known. A sensitive method called small pool PCR to analyze somatic instability of the expanded GAA triplet-repeat sequence revealed that patients have further large expansions in their DRG, which accumulated in an age-dependent fashion. Moreover, in a "humanized" transgenic mouse model carrying an expanded GAA triplet-repeat sequence within the context of the entire human FXN gene, the same age-dependent increase in prevalence of large expansions in the DRG was observed. The goal of this project is to evaluate the role of this somatic instability in Friedreich ataxia. We hypothesize that these large expansions occur specifically in neurons, and based on preliminary data, that the underlying mechanism involves DNA double-strand breaks (DSBs). We will use single-cell analysis of neurons and glial cells from DRG of two humanized transgenic mouse models to investigate what cell-type is responsible for the large expansions observed in whole-tissue preparations. DRG neurons obtained from autopsy tissue of multiple Friedreich ataxia patients will be similarly analyzed. To test the hypothesis that DSBs are associated with tissue-specific somatic instability, co-localization studies with immunofluorescence and FISH, and chromatin immunoprecipitation experiments will be used to identify DSBs at the site of the expanded GAA triplet- repeat sequence in vivo. The effect of DSBs on GAA repeat instability will be directly tested in cell culture experiments by either using repair-deficient cell lines, or treating cells from patients with DSB-inducing drugs. Finally, to test the hypothesis that the expanded GAA triplet-repeat is inordinately susceptible to DSBs, we will cross the existing mouse models for somatic instability on to a repair-deficient, ATM null background. Our data will have important implications for understanding the pathogenesis of Friedreich ataxia. Additionally, if a mechanism for the somatic instability is identified, this may yield new clues for the development of a specific therapy to slow disease progression. PUBLIC HEALTH RELEVANCE: Patients with Friedreich ataxia, the most common inherited ataxia, have expanded GAA repeat sequences in their FXN genes. Preliminary results indicate that instability of this sequence is important for the pathogenesis and progression of disease. Our experiments are designed to confirm this notion, and to determine the mechanism of this DNA instability.

描述（由申请人提供）：弗里德赖希共济失调是最常见的遗传性共济失调。 Friedreich 共济失调患者的 FXN 基因的两个拷贝中都有 GAA 三联体重复序列的大量扩展。进行性共济失调主要是“感觉性”的，是由于背根神经节（DRG）的原发性变性所致。感觉 DRG 神经元选择性和进行性变性的原因尚不清楚。一种称为小池 PCR 的敏感方法用于分析扩展的 GAA 三联体重复序列的体细胞不稳定性，结果表明患者的 DRG 进一步大幅扩展，并以年龄依赖性方式累积。此外，在整个人FXN基因范围内携带扩展的GAA三联重复序列的“人源化”转基因小鼠模型中，观察到DRG中大扩展的发生率同样随年龄而增加。该项目的目标是评估这种躯体不稳定性在弗里德赖希共济失调中的作用。我们假设这些大的扩张专门发生在神经元中，并且根据初步数据，其潜在机制涉及 DNA 双链断裂 (DSB)。我们将使用来自两个人源化转基因小鼠模型的 DRG 的神经元和神经胶质细胞的单细胞分析来研究哪种细胞类型导致在全组织制剂中观察到的大规模扩增。从多位弗里德赖希共济失调患者的尸检组织中获得的 DRG 神经元将进行类似的分析。为了检验 DSB 与组织特异性体细胞不稳定性相关的假设，将使用免疫荧光和 FISH 的共定位研究以及染色质免疫沉淀实验来识别体内扩展的 GAA 三联体重复序列位点的 DSB。 DSB 对 GAA 重复不稳定性的影响将在细胞培养实验中直接测试，方法是使用修复缺陷的细胞系，或用 DSB 诱导药物处理患者的细胞。最后，为了检验扩展的 GAA 三联体重复对 DSB 极其敏感的假设，我们将现有的体细胞不稳定性小鼠模型交叉到修复缺陷、ATM 无效背景上。我们的数据对于理解弗里德赖希共济失调的发病机制具有重要意义。此外，如果确定了体细胞不稳定的机制，这可能会为开发减缓疾病进展的特定疗法提供新线索。公共健康相关性：弗里德赖希共济失调（最常见的遗传性共济失调）患者的 FXN 基因中的 GAA 重复序列有所扩展。初步结果表明该序列的不稳定性对于疾病的发病机制和进展很重要。我们的实验旨在证实这一观点，并确定这种 DNA 不稳定的机制。