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.

Public Health Relevance

Expansions of short repeated DNA microsatellite sequences in the DMPK gene result in myotonic muscular dystrophy type 1 (DM1). To gain insight into the mechanisms of microsatellite expansion in DM1 and other neurological diseases we have engineered human cells in which different lengths of DMPK microsatellite repeat DNA have been inserted at a unique chromosomal location and have shown that decreased expression of specific genes promotes DMPK microsatellite instability. Our work will result in the identification of genes that contribute to microsatellite expansion and may therefore have clinical prognostic value, in addition to revealing molecular mechanisms of microsatellite instability.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
1R01GM099874-01
Application #
8218826
Study Section
Special Emphasis Panel (ZRG1-GGG-E (91))
Program Officer
Krasnewich, Donna M
Project Start
2012-05-01
Project End
2016-04-30
Budget Start
2012-05-01
Budget End
2013-04-30
Support Year
1
Fiscal Year
2012
Total Cost
$277,400
Indirect Cost
$87,400
Name
Wright State University
Department
Biochemistry
Type
Schools of Medicine
DUNS #
047814256
City
Dayton
State
OH
Country
United States
Zip Code
45435