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）：多种神经系统疾病的遗传基础的几个三重核苷酸重复的扩展。尽管这些疾病的病理差异很大，但它们具有共同的病因。三胞胎核苷酸重复重复的不稳定性和扩展，通常在30次以下重复到更大的重复，从40到数百个重复次数。该项目的目的是确定调节三胞胎CAG重复遗传不稳定性的新型遗传决定因素。为此，将使用果蝇作为模型系统来开发三胞胎重复不稳定性的体内屏幕。使用果蝇筛选不稳定性的修饰者是一种特别有吸引力的方法，因为大多数果蝇基因组都是经典诱变或体内siRNA的靶向。在第一个目的中，将修改一个包含具有82个CAG重复序列的全长人（SCA1）1（SCA1）1（SCA1）的果蝇模型，以诱导CAG重复区域的基线不稳定性。通过操纵转录水平和转基因的基因组位置，将优化不稳定性的诱导。不稳定性将通过单飞PCR来衡量，并且最大不稳定性的线将用作筛选CAG重复不稳定性修饰符的测定法。第二个目标将探讨为CAG重复不稳定性测定而开发快速，临床相关的读数的可能性。我们的实验室发现，在眼睛中表达的长CAG重复道可以显着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。来自单个蝇PCR的分子数据将与形态学或行为表型相关，以确定重复长度的变化是否在定量上影响这些表型；允许它们使用敏感地监视对acaxini基因中CAG重复道的变化。对于最终目标，将使用覆盖整个基因组的突变蝇库进行屏幕，以识别CAG重复不稳定性的新贡献者。 公共卫生相关性：该项目旨在通过应对常见的发病机理，CAG核苷酸重复序列的不稳定性和扩展来瞄准各种各样的神经系统疾病。增强不稳定性的修饰符将揭示出有助于疾病的新型途径，而那些抑制不稳定性或专门收缩的途径可能是可以利用的药物靶标，可以预防或延迟由CAG核苷酸重复量扩大引起的神经系统疾病的发作。