Second-site genetic modifiers of CTG/CAG microsatellite stability

CTG/CAG 微卫星稳定性的第二位点遗传修饰剂

• 批准号: 8870378
• 负责人: Michael LEFFAK
• 金额: $ 27.74万
• 依托单位: WRIGHT STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2012
• 资助国家: 美国
• 起止时间: 2012-05-01 至 2017-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8870378
• 关键词: 3' Untranslated Regions; Affect; Age of Onset; Alleles; Biological Assay; Biological Models; C-terminal; CAG repeat; Candidate Disease Gene; Cell Culture System; Cell Line; Cells; Characteristics; Child; Chromatin; Clinical; Colon Carcinoma; Cultured Cells; Custom; DNA; DNA Damage; DNA Repair; DNA Sequence; DNA biosynthesis; DNA-Directed DNA Polymerase; Data; Dependence; Diagnosis; Disease; Environment; Family; Fluorescence; Gene Expression; Gene Family; Gene Targeting; Genes; Genetic; Genome; Genome Stability; Genomic Instability; Goals; Hela Cells; Human; Human Engineering; Huntington Disease; Individual; Inherited; Knock-in Mouse; Length; Libraries; Life; Link; Location; Metabolic; Metabolic Pathway; Metabolism; Microsatellite Instability; Microsatellite Repeats; Mismatch Repair; Modeling; Molecular; Monitor; Mosaicism; Muscular Dystrophies; Mutation; Myotonic Dystrophy; Neurodegenerative Disorders; Ontology; Organ; Other Genetics; Parents; Pathway interactions; Patients; Penetrance; Phenotype; Physicians; Plasmids; Proteins; RNA Interference; Recurrence; Replication Origin; Risk; Risk Factors; Severity of illness; Signal Transduction; Site; Skeletal Muscle; Symptoms; System; Technology; Testing; Time; Tissues; Transcript; Transfection; Treatment Protocols; Trinucleotide Repeats; Work; Zinc Fingers; base; c-myc Genes; chemotherapy; human embryonic stem cell; in vivo; insight; malignant breast neoplasm; nervous system disorder; nuclease; outcome forecast; prognostic value; screening; sensor; small hairpin RNA

DESCRIPTION (provided by applicant): Genomic instability of simple DNA sequence repeats (DNA microsatellites) is the cause of more than 30 human neurological diseases. In the case of myotonic dystrophy type 1 (DM1), an autosomal dominant disease of skeletal muscle with multiple phenotypes in other organs, the molecular trigger of disease is an increase in the number of CTG/CAG trinucleotide repeats in the 3' UTR of the DMPK gene. Dramatically increased CTG/CAG copy numbers can occur intergenerationally, or more modest expansions can occur somatically throughout life and differ substantially between tissues. The extent of CTG/CAG expansion is linked to increased disease severity and earlier age of onset of symptoms, but the penetrance of DM1 varies widely. This suggests that other genetic loci (second site genes) significantly affect the stability of CTG/CAG repeats in trans. The goal of this project is to characterize second site genes that contribute to CTG/CAG microsatellite instability. We will take a candidate gene approach to carry out the Aims of this work: (i) to identify genes required for the instability of DMPK (CTG/CAG) repeats by shRNA knockdown, small pool PCR and PAGE; (ii) to determine the effect of gene knockdown on the time course and length dependence of CTG/CAG instability, and effects on other disease-related microsatellites; (iii) to identify DNA hairpin pathways of CTG/CAG instability in vivo. The long term goals of this work are to understand the mechanistic basis for human CTG/CAG microsatellite instability in vivo, and the correlation of second site gene expression levels with DM1 phenotypes. We have engineered human cultured cells in which different lengths of DMPK CTG/CAG microsatellite repeat DNA have been inserted at a unique chromosomal location; this model assay system mimics the CTG/CAG instability observed in DM1 patient cells. Importantly, we have shown that knockdown of second site genes in DNA metabolic pathways promotes CTG/CAG repeat instability. The first result of this project will be the compilation of a list of genes whose expression levels could be used to predict CTG/CAG instability in a family, or in specific tissues of a patient. We will also perform molecular characterization of the effect of second site gene knockdown on the formation of unstable DNA hairpin intermediates in vivo, the rate of instability, the instability of pre-mutation CTG/CAG repeats, and the effects of these genetic modifiers on the stability of other microsatellites. The clinical value of these risk factors would include the prognosis of sporadic symptoms that are difficult to predict by periodic screening, and the individualization of treatment regimens. Similar tests of gene expression levels are currently in use by more than 7500 physicians and 90,000 patients to predict chemotherapy benefit and disease recurrence in breast and colon cancer. Our data generated thus far using this assay system as a sensor of DNA metabolism show that the identification of genes involved in CTG/CAG repeat instability will give insight into the basic mechanisms of genome stability and microsatellite expansion in multiple neurodegenerative disorders.

描述（由申请人提供）： 简单 DNA 序列重复（DNA 微卫星）的基因组不稳定性是 30 多种人类神经系统疾病的原因。强直性肌营养不良 1 型 (DM1) 是一种常染色体显性遗传的骨骼肌疾病，在其他器官中具有多种表型，疾病的分子触发因素是 DMPK 基因 3' UTR 中 CTG/CAG 三核苷酸重复次数的增加。 CTG/CAG 拷贝数的显着增加可以在代际间发生，或者更温和的扩增可以在整个生命过程中发生，并且在组织之间存在显着差异。 CTG/CAG 扩张的程度与疾病严重程度的增加和症状出现的年龄较早有关，但 DM1 的外显率差异很大。这表明其他基因位点（第二位点基因）显着影响反式 CTG/CAG 重复序列的稳定性。该项目的目标是表征导致 CTG/CAG 微卫星不稳定性的第二位点基因。我们将采用候选基因方法来实现这项工作的目标：（i）通过shRNA敲低、小池PCR和PAGE来鉴定DMPK（CTG/CAG）重复不稳定所需的基因； (ii) 确定基因敲低对 CTG/CAG 不稳定性的时间过程和长度依赖性的影响，以及对其他疾病相关微卫星的影响； (iii) 鉴定体内 CTG/CAG 不稳定性的 DNA 发夹途径。这项工作的长期目标是了解人类 CTG/CAG 体内微卫星不稳定性的机制基础，以及第二位点基因表达水平与 DM1 表型的相关性。我们设计了人类培养细胞，其中不同长度的 DMPK CTG/CAG 微卫星重复 DNA 已插入到独特的染色体位置；该模型分析系统模拟了在 DM1 患者细胞中观察到的 CTG/CAG 不稳定性。重要的是，我们已经证明 DNA 代谢途径中第二位点基因的敲除会促进 CTG/CAG 重复的不稳定性。该项目的第一个结果将是汇编一系列基因，这些基因的表达水平可用于预测家族或患者特定组织中的 CTG/CAG 不稳定性。我们还将对第二位点基因敲低对体内不稳定DNA发夹中间体形成的影响、不稳定率、突变前CTG/CAG重复序列的不稳定性以及这些遗传修饰剂对其他微卫星稳定性的影响进行分子表征。这些危险因素的临床价值包括难以通过定期筛查预测的散发症状的预后以及治疗方案的个体化。目前，超过 7500 名医生和 90,000 名患者正在使用类似的基因表达水平测试来预测乳腺癌和结肠癌的化疗效果和疾病复发。迄今为止，我们使用该检测系统作为 DNA 代谢传感器生成的数据表明，鉴定与 CTG/CAG 重复不稳定性相关的基因将有助于深入了解多种神经退行性疾病中基因组稳定性和微卫星扩展的基本机制。