THE TRIPLET EXPANSION DISEASES

Diabetes, Digestive, Kidney Diseases

Investigators

Linked publications & trials

Abstract

The Repeat Expansion Diseases are a group of genetic disorders that result from an increase in the number of repeats in a single tandem repeat array. These disorders include Fragile X syndrome (FXS), the most common heritable form of mental retardation, Friedreich's ataxia (FRDA), which involves motor neuron degeneration and cardiomyopathy, and Progressive myoclonus epilepsy type-1 (EPM1), a neurodegenerative disorder associated with myoclonus, cerebellar ataxia and ultimately dementia. We are interested in both the mechanism of expansion and the consequences of expansion in these disorders. We have previously shown that at physiological pHs, ionic strengths and temperatures, the FXS repeat forms hairpins and tetraplexes. We have recently shown that the FRDA repeat forms both pyrimidine:purine:pyrimidine triplexes and purine:purine:pyrimidine triplexes and that in physiological reasonable conditions, a tetraplex is the predominant structure formed by the EPM1 repeat. Our observation that the ability to form secondary structures seems to be a conserved feature of unstable DNA sequences is consistent with the idea that these structures may be responsible for expansion. Some of the biochemical properties of these structures suggest ways that these sequences may lead to expansion. For example, we have shown that tetraplexes block DNA synthesis. This may lead to expansion by causing repeated strand slippage or by the generation of double strand breaks which are recombinogenic. Some of these properties are also consistent with a role for these structures in the sequelae of expansion. For example, blocks to DNA synthesis may account for the chromosome fragility seen in FXS. We have also shown that a purine:purine:pyrimidine triplex formed in the FRDA repeat during transcription traps the RNA polymerase, causing a reduction in the production of full-length mRNA similar to what is observed in FRDA patients. However, we have previously shown that FXS alleles that have a ~100% likelihood of expansion when maternally inherited are not intrinsically prone to expansion in transgenic mice, although large deletions similar to that seen in FXS families do occur. This suggests 1) that expansion and contraction occur via different mechanisms and 2) that factors in addition to sequence/structure are also important for expansion. To identify cis-acting factors that may be important for expansion, we are in the process of generating mice containing long CGG-repeat tracts with different flanking sequences including mice containing a targeted insertion of 176 CGG-repeats into the murine homolog of the FXS gene. Potential trans-acting factors are being tested by crossing our CGG-transgenic mice with mice containing mutations in the various DNA replication, recombination and DNA damage surveillance pathways. We have also identified the transcription factors that are important for the normal regulation of the gene affected in FXS. We have shown that binding of these factors is sensitive to methylation, with binding of one of these factors being completely abolished when a small number of Cs in its binding site are methylated. This suggests that transcriptional silencing in FXS, which is associated with expansion induced promoter methylation, does not occur solely at the level of the assembly of transcriptional silent chromatin as previously thought. Rather it indicates that prevention of transcription factor binding is a crucial event in the etiology of FXS. Our data also indicate that current attempts to reactivate the gene using histone deacetylase inhibitors alone are likely to have limited efficacy.