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: 8652473
• 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 2016-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8652473
• 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; 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）：