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：
8091715
负责人：
SANJAY I BIDICHANDANI
金额：
$ 3.7万
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 2010-12-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8091715
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 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 Names 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 Principal Investigator Process Property Proprioception Research Research Personnel Resource Sharing 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 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 programs progressive neurodegeneration public health relevance repaired research study response single cell analysis single molecule 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）：弗里德里希共济失调是最常见的遗传性共济失调。 Friedreich共济失调患者在FXN基因的两个副本中都具有GAA三胞胎重复序列的大量扩张。进行性共济失调主要是“感觉”，是由于背根神经节（DRG）的一级变性。尚不清楚感觉性DRG神经元的选择性和进行性变性的原因。一种称为小池PCR的敏感方法，用于分析扩展的GAA三胞胎重复序列的体细胞不稳定性，发现患者的DRG中有进一步的扩张，这种扩张以年龄依赖性的方式积累。此外，在整个人类FXN基因的背景下携带扩展的GAA三重态重复序列的“人性化”转基因小鼠模型中，观察到DRG中大型扩张的患病率相同。该项目的目的是评估这种躯体不稳定性在弗里德里希共济失调中的作用。我们假设这些大型扩展是在神经元中特别发生的，并且基于初步数据，即潜在的机制涉及DNA双链断裂（DSB）。我们将使用来自两个人源性转基因小鼠模型的DRG的神经元和神经胶细胞的单细胞分析，以研究哪些细胞类型负责全组织制剂中观察到的大型扩张。将类似地分析从多个Friedreich共济失调患者的尸检组织获得的DRG神经元。为了测试DSB与组织特异性体细胞不稳定性相关的假设，将使用免疫荧光和鱼类的共定位研究，以及染色质免疫沉淀实验将用于鉴定在VIV中扩展的GAA GAA三胞胎重复序列的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，或者使用修复缺陷的细胞系或从DSB诱导药物的患者中处理细胞。最后，为了测试扩展的GAA三胞胎重复的假设非常容易受到DSB的影响，我们将越过现有的鼠标模型，以使体细胞不稳定性达到修复缺陷的ATM无效背景。我们的数据将对理解Friedreich共济失调的发病机理具有重要意义。此外，如果确定了躯体不稳定性的机制，这可能会为开发特定疗法以减慢疾病进展而产生新的线索。公共卫生相关性：最常见的遗传性共济失调的弗里德里希共济失调患者在其FXN基因中扩大了GAA重复序列。初步结果表明，该序列的不稳定性对于疾病的发病机理和进展很重要。我们的实验旨在确认这一概念，并确定该DNA不稳定性的机制。