Genome-Wide Screen for Clinically Relevant Modifiers of CAG Repeat Instability

全基因组筛选 CAG 重复不稳定性的临床相关修饰因子

• 批准号: 7754315
• 负责人: David A Mittelman
• 金额: $ 5.42万
• 依托单位: BAYLOR COLLEGE OF MEDICINE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-12-01 至 2010-10-22
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7754315
• 关键词: Affect; Alleles; Amplifiers; Ataxia; Bacteria; Behavioral; Biological Assay; Biological Models; CAG repeat; Cessation of life; Collection; Contracts; DNA; DNA Repair; DNA Repair Gene; Data; Development; Disease; Drosophila genome; Drosophila genus; Drug Delivery Systems; Etiology; Eye; Fragile X Syndrome; Gene Targeting; Gene-Modified; Genes; Genetic; Genetic Determinism; Genetic Transcription; Genome; Genomics; Goals; Human; Huntington Disease; Length; Libraries; Link; Location; Mammalian Cell; Measures; Metabolism; Methods; Modeling; Molecular; Monitor; Morphology; Motor Neurons; Mutagenesis; Myotonic Dystrophy; Neurons; Nucleotides; Pathogenesis; Pathology; Pathway interactions; Phenotype; Photoreceptors; RNA Interference; Source; System; Transgenes; Trinucleotide Repeats; Triplet Multiple Birth; Type 1 Spinocerebellar Ataxia; Work; base; clinically relevant; fly; genome wide association study; in vivo; loss of function; mutant; nervous system disorder; novel; overexpression; photoreceptor degeneration; polyglutamine; prevent; public health relevance; therapeutic target

DESCRIPTION (provided by applicant): Expansions of several triplet nucleotide repeats underlie the genetic basis for a diverse group of neurological disorders. While the pathologies of these disorders vary greatly, they share a common etiology; the instability and expansion of triplet nucleotide repeats from small repetitions, usually under 30 repeats, to far greater repetitions, anywhere from 40 to hundreds of repeats. The goal of this project is to identify novel genetic determinants that modulate the genetic instability of triplet CAG repeats. To accomplish this, an in vivo screen for triplet repeat instability will be developed using Drosophila as the model system. Using Drosophila to screen for modifiers of instability is a particularly attractive approach in that most of the Drosophila genome has been targeted either by classic mutagenesis or by in vivo siRNAs. In the first aim, a Drosophila model for Spinocerebellar ataxia 1 (SCA1), containing the full-length human ataxini gene with 82 CAG repeats, will be modified to induce a baseline instability in the CAG repeat tract. Induction of instability will be optimized by manipulation of transcription levels and genomic location of the transgene. Instability will be measured by single fly PCR and the lines with the greatest instability will be used as an assay to screen for modifiers of CAG repeat instability. The second aim will explore the possibility of developing a rapid, clinically relevant readout for the CAG repeat instability assay. Our lab has found that long CAG repeat tracts can dramatically affect the morphology of photoreceptors when expressed in the eye, and greatly impair flight when expressed in motor neurons. Molecular data from single fly PCR will be correlated to morphological or behavioral phenotypes to ascertain whether alterations to repeat length quantitatively affect these phenotypes; allowing for their use to sensitively monitor changes to the CAG repeat tract in the ataxini gene. For the final aim, a screen will be carried out with libraries of mutant flies that cover the entire genome, to identify novel contributors to CAG repeat instability. Public Health Relevance: This project aims to target a diverse group of devesting neurological disorders by tackling the common source of pathogenesis, the instability and expansion of CAG nucleotide repeats. Modifiers that enhance instability will reveal novel pathways that contribute to disease, and those that suppress instability, or specifically contract the repeats might be candidate drug targets that could exploited to prevent or delay the onset of neurological disorders caused by expanded CAG nucleotide repeats.

描述（由申请人提供）：几个三联核苷酸重复序列的扩增是一组不同神经系统疾病的遗传基础。虽然这些疾病的病理变化很大，但它们有一个共同的病因;三联核苷酸重复的不稳定性和扩增，从小的重复，通常在30个重复以下，到更大的重复，从40到数百个重复。本项目的目标是确定新的遗传决定因素，调节三重CAG重复的遗传不稳定性。为了实现这一点，在体内屏幕三重重复不稳定性将开发使用果蝇作为模型系统。使用果蝇筛选不稳定性修饰剂是一种特别有吸引力的方法，因为大多数果蝇基因组已经通过经典诱变或体内siRNA靶向。在第一个目标中，将修改包含具有82个CAG重复序列的全长人类共济失调基因的脊髓小脑共济失调1（SCA 1）的果蝇模型以诱导CAG重复序列道中的基线不稳定性。不稳定性的诱导将通过操纵转基因的转录水平和基因组定位来优化。不稳定性将通过单飞PCR测量，并且具有最大不稳定性的系将用作筛选CAG重复不稳定性的修饰剂的测定。第二个目标将探索开发一个快速的，临床相关的CAG重复不稳定性检测读出的可能性。我们的实验室已经发现，长CAG重复序列在眼睛中表达时可以显著影响光感受器的形态，并且在运动神经元中表达时会极大地损害飞行。来自单蝇PCR的分子数据将与形态学或行为表型相关，以确定重复长度的改变是否定量地影响这些表型;允许它们用于灵敏地监测共济失调基因中CAG重复序列的变化。为了最终的目标，将对覆盖整个基因组的突变果蝇文库进行筛选，以确定CAG重复不稳定性的新贡献者。 公共卫生相关性：该项目旨在通过解决发病机制的共同来源，CAG核苷酸重复序列的不稳定性和扩展，针对不同的发育性神经系统疾病。增强不稳定性的修饰物将揭示导致疾病的新途径，而那些抑制不稳定性或特异性收缩重复序列的修饰物可能是候选药物靶点，可用于预防或延迟由扩增的CAG核苷酸重复序列引起的神经系统疾病的发作。