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

描述（由申请人提供）：几个三联体核苷酸重复序列的扩增是多种神经系统疾病的遗传基础。虽然这些疾病的病理差异很大，但它们有一个共同的病因；三联体核苷酸重复的不稳定性和扩展，从小的重复，通常少于30次重复，到更大的重复，从40到数百次重复。这个项目的目标是确定新的遗传决定因素，调节CAG重复三联体的遗传不稳定性。为了实现这一目标，三联体重复不稳定性的体内筛选将以果蝇为模型系统开发。利用果蝇筛选不稳定性修饰因子是一种特别有吸引力的方法，因为大多数果蝇基因组都是通过经典突变或体内sirna靶向的。在第一个目标中，将对含有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 （SCA1）果蝇模型进行修饰，以诱导CAG重复序列的基线不稳定性。不稳定性的诱导将通过操纵转录水平和转基因的基因组位置来优化。不稳定性将通过单蝇PCR来测量，不稳定性最大的系将被用作筛选CAG重复不稳定性修饰剂的实验。第二个目标将探索开发CAG重复不稳定性测定的快速、临床相关读数的可能性。我们的实验室发现，长CAG重复束在眼睛中表达时可以显著影响光感受器的形态，在运动神经元中表达时可以极大地损害飞行。来自单蝇PCR的分子数据将与形态学或行为表型相关联，以确定重复长度的改变是否定量地影响这些表型；允许它们用于敏感地监测ataxini基因中CAG重复束的变化。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。

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.