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展是一组不同神经疾病的遗传基础。虽然这些疾病的病理差异很大，但它们有一个共同的病因：三重核苷酸重复的不稳定和扩大，从通常不到30个重复的小重复，到更大的重复，从40到数百个重复。该项目的目标是确定调节三联体CAG重复序列遗传不稳定性的新的遗传决定因素。为了实现这一点，将以果蝇为模型系统，开发一种三联体重复不稳定的体内筛选。利用果蝇来筛选不稳定的修饰物是一种特别有吸引力的方法，因为大多数果蝇基因组已经被经典的突变或体内的siRNA作为靶标。在第一个目标中，一个含有82个CAG重复的全长人类共济失调基因的脊髓小脑性共济失调1(SCA1)的果蝇模型将被修改，以在CAG重复区段诱导基线不稳定性。不稳定性的诱导将通过对转基因转录水平和基因组位置的操纵来优化。不稳定性将通过单次飞行聚合酶链式反应进行测量，最不稳定的品系将被用作筛选CAG重复不稳定的修饰物的检测方法。第二个目标将探索为CAG重复不稳定分析开发一种快速的、临床相关的读数的可能性。我们的实验室发现，长的CAG重复区段在眼睛中表达时可以极大地影响光感受器的形态，在运动神经元中表达时会极大地损害飞行。来自单次飞行聚合酶链式反应的分子数据将与形态或行为表型相关联，以确定重复长度的变化是否会定量影响这些表型；从而使它们能够用于灵敏地监测ataxini基因中CAG重复序列的变化。对于最终目标，将对覆盖整个基因组的突变果蝇文库进行筛选，以确定导致CAG重复不稳定性的新因素。 公共卫生相关性：该项目旨在通过解决致病的共同来源，CAG核苷酸重复序列的不稳定和扩大，来针对不同的破坏性神经疾病群体。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定收缩重复序列的修饰物可能成为候选药物靶点，可用于预防或延缓由扩大的CAG核苷酸重复引起的神经疾病的发病。

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.