Linkage disequilibrium and haplotype analysis in German Friedreich ataxia families

The main mutation causing Friedreich ataxia (FRDA) is the expansion of a GAA repeat localized within the intron between exon 1 and exon 2 of the gene X25. This expansion has been observed in 98% of FRDA chromosomes. To analyze frequencies of markers tightly linked to the Friedreich ataxia gene and to investigate wheter a limited number of ancestral chromosomes are shared by German FRDA families, a detailed analysis employing nine polymorphic markers was performed. We found strong linkage disequilibria and association of FRDA expansions with a few haplotypes. FRDA haplotypes differ significantly from control haplotypes. Our results confirm that GAA repeat expansions in intron 1 of the frataxin gene are limited to a few chromosomes and indicate an obvious founder effect in German patients. Based on these analyses, we estimate a minimum age of the mutation of 107 generations.     

Two New Pimelic Diphenylamide HDAC Inhibitors Induce Sustained Frataxin Upregulation in Cells from Friedreich's Ataxia Patients and in a Mouse Model

Background

Friedreich''s ataxia (FRDA), the most common recessive ataxia in Caucasians, is due to severely reduced levels of frataxin, a highly conserved protein, that result from a large GAA triplet repeat expansion within the first intron of the frataxin gene (FXN). Typical marks of heterochromatin are found near the expanded GAA repeat in FRDA patient cells and mouse models. Histone deacetylase inhibitors (HDACIs) with a pimelic diphenylamide structure and HDAC3 specificity can decondense the chromatin structure at the FXN gene and restore frataxin levels in cells from FRDA patients and in a GAA repeat based FRDA mouse model, KIKI, providing an appealing approach for FRDA therapeutics.

Methodology/Principal Findings

In an effort to further improve the pharmacological profile of pimelic diphenylamide HDACIs as potential therapeutics for FRDA, we synthesized additional compounds with this basic structure and screened them for HDAC3 specificity. We characterized two of these compounds, 136 and 109, in FRDA patients'' peripheral blood lymphocytes and in the KIKI mouse model. We tested their ability to upregulate frataxin at a range of concentrations in order to determine a minimal effective dose. We then determined in both systems the duration of effect of these drugs on frataxin mRNA and protein, and on total and local histone acetylation. The effects of these compounds exceeded the time of direct exposure in both systems.

Conclusions/Significance

Our results support the pre-clinical development of a therapeutic approach based on pimelic diphenylamide HDACIs for FRDA and provide information for the design of future human trials of these drugs, suggesting an intermittent administration of the drug.     

Skin fibroblast metabolomic profiling reveals that lipid dysfunction predicts the severity of Friedreich’s ataxia

Friedreich’s ataxia (FRDA) is an autosomal recessive neurodegenerative disorder caused by a triplet guanine-adenine-adenine (GAA) repeat expansion in intron 1 of the FXN gene, which leads to decreased levels of the frataxin protein. Frataxin is involved in the formation of iron-sulfur (Fe-S) cluster prosthetic groups for various metabolic enzymes. To provide a better understanding of the metabolic status of patients with FRDA, here we used patient-derived fibroblast cells as a surrogate tissue for metabolic and lipidomic profiling by liquid chromatography-high resolution mass spectrometry. We found elevated HMG-CoA and β-hydroxybutyrate-CoA levels, implying dysregulated fatty acid oxidation, which was further demonstrated by elevated acyl-carnitine levels. Lipidomic profiling identified dysregulated levels of several lipid classes in FRDA fibroblast cells when compared with non-FRDA fibroblast cells. For example, levels of several ceramides were significantly increased in FRDA fibroblast cells; these results positively correlated with the GAA repeat length and negatively correlated with the frataxin protein levels. Furthermore, stable isotope tracing experiments indicated increased ceramide synthesis, especially for long-chain fatty acid-ceramides, in FRDA fibroblast cells compared with ceramide synthesis in healthy control fibroblast cells. In addition, PUFA-containing triglycerides and phosphatidylglycerols were enriched in FRDA fibroblast cells and negatively correlated with frataxin levels, suggesting lipid remodeling as a result of FXN deficiency. Altogether, we demonstrate patient-derived fibroblast cells exhibited dysregulated metabolic capabilities, and their lipid dysfunction predicted the severity of FRDA, making them a useful surrogate to study the metabolic status in FRDA.     

Pharmacological Screening Using an FXN-EGFP Cellular Genomic Reporter Assay for the Therapy of Friedreich Ataxia

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. There is a correlation between expansion length, the amount of residual frataxin and the severity of disease. As the coding sequence is unaltered, pharmacological up-regulation of FXN expression may restore frataxin to therapeutic levels. To facilitate screening of compounds that modulate FXN expression in a physiologically relevant manner, we established a cellular genomic reporter assay consisting of a stable human cell line containing an FXN-EGFP fusion construct, in which the EGFP gene is fused in-frame with the entire normal human FXN gene present on a BAC clone. The cell line was used to establish a fluorometric cellular assay for use in high throughput screening (HTS) procedures. A small chemical library containing FDA-approved compounds and natural extracts was screened and analyzed. Compound hits identified by HTS were further evaluated by flow cytometry in the cellular genomic reporter assay. The effects on FXN mRNA and frataxin protein levels were measured in lymphoblast and fibroblast cell lines derived from individuals with FRDA and in a humanized GAA repeat expansion mouse model of FRDA. Compounds that were established to increase FXN gene expression and frataxin levels included several anti-cancer agents, the iron-chelator deferiprone and the phytoalexin resveratrol.     

Base Excision Repair of Chemotherapeutically-Induced Alkylated DNA Damage Predominantly Causes Contractions of Expanded GAA Repeats Associated with Friedreich's Ataxia

Expansion of GAA·TTC repeats within the first intron of the frataxin gene is the cause of Friedreich''s ataxia (FRDA), an autosomal recessive neurodegenerative disorder. However, no effective treatment for the disease has been developed as yet. In this study, we explored a possibility of shortening expanded GAA repeats associated with FRDA through chemotherapeutically-induced DNA base lesions and subsequent base excision repair (BER). We provide the first evidence that alkylated DNA damage induced by temozolomide, a chemotherapeutic DNA damaging agent can induce massive GAA repeat contractions/deletions, but only limited expansions in FRDA patient lymphoblasts. We showed that temozolomide-induced GAA repeat instability was mediated by BER. Further characterization of BER of an abasic site in the context of (GAA)₂₀ repeats indicates that the lesion mainly resulted in a large deletion of 8 repeats along with small expansions. This was because temozolomide-induced single-stranded breaks initially led to DNA slippage and the formation of a small GAA repeat loop in the upstream region of the damaged strand and a small TTC loop on the template strand. This allowed limited pol β DNA synthesis and the formation of a short 5''-GAA repeat flap that was cleaved by FEN1, thereby leading to small repeat expansions. At a later stage of BER, the small template loop expanded into a large template loop that resulted in the formation of a long 5''-GAA repeat flap. Pol β then performed limited DNA synthesis to bypass the loop, and FEN1 removed the long repeat flap ultimately causing a large repeat deletion. Our study indicates that chemotherapeutically-induced alkylated DNA damage can induce large contractions/deletions of expanded GAA repeats through BER in FRDA patient cells. This further suggests the potential of developing chemotherapeutic alkylating agents to shorten expanded GAA repeats for treatment of FRDA.     

DNA triplex structures in neurodegenerative disorder, Friedreich’s ataxia

It is now established that a small fraction of genomic DNA does adopt the non-canonical B-DNA structure or 'unusual' DNA structure. The unusual DNA structures like DNA-hairpin, cruciform, Z-DNA, triplex and tetraplex are represented as hotspots of chromosomal breaks, homologous recombination and gross chromosomal rearrangements since they are prone to the structural alterations. Friedreich's ataxia (FRDA), the autosomal recessive degenerative disorder of nervous and muscles tissue, is caused by the massive expansion of (GAA) repeats that occur in the first intron of Frataxin gene X25 on chromosome 9q13-q21.1. The purine strand of the DNA in the expanded (GAA) repeat region folds back to form the (R.R*Y) type of triplex, which further inhibits the frataxin gene expression, and this clearly suggests that the shape of DNA is the determining factor in the cellular function. FRDA is the only disease known so far to be associated with DNA triplex. Structural characterization of GAA-containing DNA triplexes using some simple biophysical methods like UV melting, UV absorption, circular dichroic spectroscopy and electrophoretic mobility shift assay are discussed. Further, the clinical aspects and genetic analysis of FRDA patients who carry (GAA) repeat expansions are presented. The potential of some small molecules that do not favour the DNA triplex formation as therapeutics for FRDA are also briefly discussed.     

A novel GAA-repeat-expansion-based mouse model of Friedreich’s ataxia

Friedreich’s 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 ~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 with control Y47R and wild-type (WT) 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 with 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.KEY WORDS: GAA repeat, Friedreich’s ataxia, FRDA, YG8sR, Mouse model     

Dissecting the epidemiology of a trinucleotide repeat disease – example of FRDA in Finland

Friedreich ataxia (FRDA) is associated with the expansion of a GAA trinucleotide repeat in the first intron of the frataxin (X25) gene. Worldwide it is considered to be the most common form of hereditary ataxia, but it is infrequently encountered in Finland. We have performed the first epidemiological study on the frequency of FRDA in Finland by combining results from a nationwide clinical survey and a molecular carrier testing study. Haplotype analysis was performed for the Finnish FRDA patients and the distribution of frataxin gene GAA repeats was analyzed in controls. In the general population of Finland, the carrier frequency was only 1 in 500, corresponding to a birth incidence of 1 in 10(6). In the more sparsely populated Northern Finland the carrier frequency was five times higher and also four out of the seven Finnish FRDA patients originated from this region. Haplotype analysis revealed the major universal risk haplotype in all the investigated patients. Alleles in the uppermost end of the normal variation (28-36 GAA) were totally missing in the Finnish population. The relative enrichment of the FRDA mutation in the north probably dates back to the internal migration movement and inhabitation of northern Finland in the 1500s. Breaking down the epidemiology of FRDA into clinical and molecular components brings along the possibility of providing more reliable and population-based genetic counseling and recurrence risk estimations.     

Frataxin knockin mouse

Friedreich ataxia is the consequence of frataxin deficiency, most often caused by a GAA repeat expansion in intron 1 of the corresponding gene. Frataxin is a mitochondrial protein involved in iron homeostasis. As an attempt to generate a mouse model of the disease, we introduced a (GAA)(230) repeat within the mouse frataxin gene by homologous recombination. GAA repeat knockin mice were crossed with frataxin knockout mice to obtain double heterozygous mice expressing 25-36% of wild-type frataxin levels. These mice were viable and did not develop anomalies of motor coordination, iron metabolism or response to iron loading. Repeats were meiotically and mitotically stable.