The GAA Triplet-Repeat Expansion in Friedreich Ataxia Interferes with Transcription and May Be Associated with an Unusual DNA Structure

Department of Neurology, Baylor College of Medicine, Houston, TX 77030, USA.
The American Journal of Human Genetics (Impact Factor: 10.93). 02/1998; 62(1):111-21. DOI: 10.1086/301680
Source: PubMed


Friedreich ataxia (FRDA), an autosomal recessive, neurodegenerative disease is the most common inherited ataxia. The vast majority of patients are homozygous for an abnormal expansion of a polymorphic GAA triplet repeat in the first intron of the X25 gene, which encodes a mitochondrial protein, frataxin. Cellular degeneration in FRDA may be caused by mitochondrial dysfunction, possibly due to abnormal iron accumulation, as observed in yeast cells deficient for a frataxin homologue. Using RNase protection assays, we have shown that patients homozygous for the expansion have a marked deficiency of mature X25 mRNA. The mechanism(s) by which the intronic GAA triplet expansion results in this reduction of X25 mRNA is presently unknown. No evidence was found for abnormal splicing of the expanded intron 1. Using cloned repeat sequences from FRDA patients, we show that the GAA repeat per se interferes with in vitro transcription in a length-dependent manner, with both prokaryotic and eukaryotic enzymes. This interference was most pronounced in the physiological orientation of transcription, when synthesis of the GAA-rich transcript was attempted. These results are consistent with the observed negative correlation between triplet-repeat length and the age at onset of disease. Using in vitro chemical probing strategies, we also show that the GAA triplet repeat adopts an unusual DNA structure, demonstrated by hyperreactivity to osmium tetroxide, hydroxylamine, and diethyl pyrocarbonate. These results raise the possibility that the GAA triplet-repeat expansion may result in an unusual yet stable DNA structure that interferes with transcription, ultimately leading to a cellular deficiency of frataxin.

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Available from: Tetsuo Ashizawa, Jul 31, 2014
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    • "To determine YG8sR GAA repeat purity, MboII digestion of GAA PCR products and subsequent agarose gel electrophoresis was carried out as previously described (Holloway et al., 2011). To sequence the YG8sR GAA repeat, genomic DNA was extracted from fibroblast cells using the DNeasy Blood and Tissue kit (Qiagen), PCR amplification was performed on 50 ng of DNA using Q5 High Fidelity DNA polymerase (NEB) and primers 104F and 629R as previously described (Bidichandani et al., 1998), and Sanger sequencing was carried out with primer 629R. "
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    ABSTRACT: Friedreich ataxia (FRDA) is an autosomal recessive neurodegenerative disorder caused by a GAA repeat expansion mutation within intron 1 of the FXN gene, resulting in reduced levels of frataxin protein. We have previously reported the generation of human FXN yeast artificial chromosome (YAC) transgenic FRDA mouse models containing 90-190 GAA repeats, but the presence of multiple GAA repeats within these mice is considered suboptimal. We now describe the cellular, molecular and behavioural characterisation of a newly developed YAC transgenic FRDA mouse model, designated YG8sR, which we have shown by DNA sequencing to contain a single pure GAA repeat expansion. The founder YG8sR mouse contained 120 GAA repeats, but due to intergenerational expansion we have now established a colony of YG8sR mice that contain approximately 200 GAA repeats. We show that YG8sR mice have a single copy of the FXN transgene, which is integrated at a single site as confirmed by fluorescence in situ hybridisation (FISH) analysis of metaphase and interphase chromosomes. We have identified significant behavioural deficits, together with a degree of glucose intolerance and insulin hypersensitivity, in YG8sR FRDA mice compared to Y47R and wild-type (WT) control mice. We have also detected increased somatic GAA repeat instability in the brain and cerebellum of YG8sR mice, together with significantly reduced expression of FXN, FAST-1 and frataxin and reduced aconitase activity compared to Y47R mice. Furthermore, we have confirmed the presence of pathological vacuoles within neurons of the dorsal root ganglia (DRG) of YG8sR mice. These novel GAA repeat expansion-based YAC transgenic FRDA mice, which exhibit progressive FRDA-like pathology, represent an excellent model for the investigation of FRDA disease mechanisms and therapy.
    Full-text · Article · Feb 2015 · Disease Models and Mechanisms
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    • "Pathogenic GAA expansion alleles are in the size range of 60 to more than 1300 repeats. The presence of a GAA repeat expansion results in the inhibition of FXN gene expression, reduced levels of full length FXN transcript and an insufficiency of the mitochondrial protein frataxin [2]–[5]. "
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    ABSTRACT: Friedreich ataxia (FRDA) is an autosomal recessive disorder characterized by neurodegeneration and cardiomyopathy. The presence of a GAA trinucleotide repeat expansion in the first intron of the FXN gene results in the inhibition of gene expression and an insufficiency of the mitochondrial protein frataxin. We previously generated BAC-based transgenic mice containing an FXN-EGFP genomic reporter construct in which the EGFP gene is fused in-frame immediately following the final codon of exon 5a of the human FXN gene. These transgenic mice were mated with mice heterozygous for a knockout mutation of the murine Fxn gene, to generate mice homozygous for the Fxn knockout mutation and hemizygous or homozygous for the human transgene. Rescue of the embryonic lethality that is associated with homozygosity for the Fxn knockout mutation was observed. Rescue mice displayed normal behavioral and histological parameters with normal viability, fertility and life span and without any signs of aberrant phenotype. Immunoblotting demonstrated the production of full-length frataxin-EGFP fusion protein that appears to act as a bifunctional hybrid protein. This study shows frataxin replacement may be a viable therapeutic option. Further, these mice should provide a useful resource for the study of human FXN gene expression, frataxin function, the evaluation of pharmacologic inducers of FXN expression in a whole-animal model and provide a useful source of cells for stem cell transplantation studies.
    Full-text · Article · Mar 2014 · PLoS ONE
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    • "Expanded alleles lead to the inhibition of FXN expression resulting in decreased levels of the encoded protein, frataxin [10] [11]. The transcriptional repression of the FXN gene induced by the GAA expansion is due to arrest of RNA polymerase II progression and to heterochromatinmediated gene silencing [12] [13] [14] [15] [16]. Frataxin is a mitochondrial protein involved in cellular iron use and maintenance of the redox status [4]. "
    Dataset: NNOS 2013

    Full-text · Dataset · Nov 2013
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