Somatic instability of the expanded GAA triplet-repeat sequence in Friedreich ataxia progresses throughout life

Friedreich ataxia (FRDA) patients are homozygous for expanded GAA triplet-repeat alleles in the FXN gene. Primary neurodegeneration involving the dorsal root ganglia (DRG) results in progressive ataxia. While it is known that DRG are inherently sensitive to frataxin deficiency, recent observations also indicate that they show age-dependent, further expansion of the GAA triplet-repeat mutation. Whether somatic instability is progressive has not been systematically investigated in FRDA patients. "Small-pool" PCR analysis of approximately 2300 individual molecules from tissues of an 18-week fetus homozygous for expanded alleles revealed very low levels of instability compared with adult-derived tissues (4.2% versus 30.6%, p<0.0001). Mutation load in blood samples from multiple patients and carriers increased significantly with age, ranging from 7.5% at 18-weeks gestation to 78.7% at 49 years of age (R=0.91; p=0.0001). Therefore, somatic instability in FRDA occurs mostly after early embryonic development and progresses throughout life, lending further support to the role of postnatal somatic instability in disease pathogenesis.     

Structure-function analysis of Friedreich's ataxia mutants reveals determinants of frataxin binding and activation of the Fe-S assembly complex

Friedreich's ataxia (FRDA) is a progressive neurodegenerative disease associated with the loss of function of the protein frataxin (FXN) that results from low FXN levels due to a GAA triplet repeat expansion or, occasionally, from missense mutations in the FXN gene. Here biochemical and structural properties of FXN variants, including three FRDA missense mutations (N146K, Q148R, and R165C) and three related mutants (N146A, Q148G, and Q153A), were determined in an effort to understand the structural basis for the loss of function. In vitro assays revealed that although the three FRDA missense mutations exhibited similar losses of cysteine desulfurase and Fe-S cluster assembly activities, the causes for these activation defects were distinct. The R165C variant exhibited a k(cat)/K(M) higher than that of native FXN but weak binding to the NFS1, ISD11, and ISCU2 (SDU) complex, whereas the Q148R variant exhibited the lowest k(cat)/K(M) of the six tested FXN variants and only a modest binding deficiency. The order of the FXN binding affinities for the SDU Fe-S assembly complex was as follows: FXN > Q148R > N146A > Q148G > N146K > Q153A > R165C. Four different classes of FXN variants were identified on the basis of their biochemical properties. Together, these structure-function studies reveal determinants for the binding and allosteric activation of the Fe-S assembly complex and provide insight into how FRDA missense mutations are functionally compromised.     

Novel Frataxin Isoforms May Contribute to the Pathological Mechanism of Friedreich Ataxia

Friedreich ataxia (FRDA) is an inherited neurodegenerative disease caused by frataxin (FXN) deficiency. The nervous system and heart are the most severely affected tissues. However, highly mitochondria-dependent tissues, such as kidney and liver, are not obviously affected, although the abundance of FXN is normally high in these tissues. In this study we have revealed two novel FXN isoforms (II and III), which are specifically expressed in affected cerebellum and heart tissues, respectively, and are functional in vitro and in vivo. Increasing the abundance of the heart-specific isoform III significantly increased the mitochondrial aconitase activity, while over-expression of the cerebellum-specific isoform II protected against oxidative damage of Fe-S cluster-containing aconitase. Further, we observed that the protein level of isoform III decreased in FRDA patient heart, while the mRNA level of isoform II decreased more in FRDA patient cerebellum compared to total FXN mRNA. Our novel findings are highly relevant to understanding the mechanism of tissue-specific pathology in FRDA.     

GAA repeat instability in Friedreich ataxia YAC transgenic mice

Friedreich ataxia (FRDA) is primarily caused by an unstable GAA repeat-expansion mutation within intron 1 of the FRDA gene. However, the exact mechanisms leading to this expansion and its consequences are not fully understood. To study the dynamics of this mutation, we have generated two lines of human FRDA YAC transgenic mice that contain GAA repeat expansions within the appropriate genomic context. We have detected intergenerational instability and age-related somatic instability in both lines, with pronounced expansions found in the cerebellum. The dynamic nature of our transgenic GAA repeats is comparable with previous FRDA patient somatic tissue data. However, there is a difference between our FRDA YAC transgenic mice and other trinucleotide-repeat mouse models, which do not show pronounced repeat instability in the cerebellum. This represents the first mouse model of FRDA GAA repeat instability that will help to dissect the mechanism of this repeat.     

Chromosome fragility at GAA tracts in yeast depends on repeat orientation and requires mismatch repair

Expansion of triplex-forming GAA/TTC repeats in the first intron of FXN gene results in Friedreich's ataxia. Besides FXN, there are a number of other polymorphic GAA/TTC loci in the human genome where the size variations thus far have been considered to be a neutral event. Using yeast as a model system, we demonstrate that expanded GAA/TTC repeats represent a threat to eukaryotic genome integrity by triggering double-strand breaks and gross chromosomal rearrangements. The fragility potential strongly depends on the length of the tracts and orientation of the repeats relative to the replication origin, which correlates with their propensity to adopt triplex structure and to block replication progression. We show that fragility is mediated by mismatch repair machinery and requires the MutSbeta and endonuclease activity of MutLalpha. We suggest that the mechanism of GAA/TTC-induced chromosomal aberrations defined in yeast can also operate in human carriers with expanded tracts.     

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.     

Expansion of GAA triplet repeats in the human genome: unique origin of the FRDA mutation at the center of an Alu

Friedreich ataxia is caused by expansion of a GAA triplet repeat (GAA-TR) in the FRDA gene. Normal alleles contain <30 triplets, and disease-causing expansions (66-1700 triplets) arise via hyperexpansion of premutations (30-65 triplets). To gain insight into GAA-TR instability we analyzed all triplet repeats in the human genome. We identified 988 (GAA)(8+) repeats, 291 with >or=20 triplets, including 29 potential premutations (30-62 triplets). Most other triplet repeats were restricted to <20 triplets. We estimated the expected frequency of (GAA)(6+) repeats to be negligible, further indicating that GAA-TRs have undergone significant expansion. Eighty-nine percent of (GAA)(8+) sequences map within G/A islands, and 58% map within the poly(A) tails of Alu elements. Only two other (GAA)(8+) sequences shared the central Alu location seen at the FRDA locus. One showed allelic variation, including expansions analogous to short Friedreich ataxia mutations. Our data demonstrate that GAA-TRs have expanded throughout primate evolution with the generation of potential premutation alleles at multiple loci.     

Conformational stability of human frataxin and effect of Friedreich's ataxia-related mutations on protein folding

The neurodegenerative disorder FRDA (Friedreich's ataxia) results from a deficiency in frataxin, a putative iron chaperone, and is due to the presence of a high number of GAA repeats in the coding regions of both alleles of the frataxin gene, which impair protein expression. However, some FRDA patients are heterozygous for this triplet expansion and contain a deleterious point mutation on the other allele. In the present study, we investigated whether two particular FRDA-associated frataxin mutants, I154F and W155R, result in unfolded protein as a consequence of a severe structural modification. A detailed comparison of the conformational properties of the wild-type and mutant proteins combining biophysical and biochemical methodologies was undertaken. We show that the FRDA mutants retain the native fold under physiological conditions, but are differentially destabilized as reflected both by their reduced thermodynamic stability and a higher tendency towards proteolytic digestion. The I154F mutant has the strongest effect on fold stability as expected from the fact that the mutated residue contributes to the hydrophobic core formation. Functionally, the iron-binding properties of the mutant frataxins are found to be partly impaired. The apparently paradoxical situation of having clinically aggressive frataxin variants which are folded and are only significantly less stable than the wild-type form in a given adverse physiological stress condition is discussed and contextualized in terms of a mechanism determining the pathology of FRDA heterozygous.     

Blood cells from Friedreich ataxia patients harbor frataxin deficiency without a loss of mitochondrial function

Friedreich ataxia (FRDA) is an autosomal recessive neurodegenerative disorder caused by GAA triplet expansions or point mutations in the FXN gene on chromosome 9q13. The gene product called frataxin, a mitochondrial protein that is severely reduced in FRDA patients, leads to mitochondrial iron accumulation, Fe-S cluster deficiency and oxidative damage. The tissue specificity of this mitochondrial disease is complex and poorly understood. While frataxin is ubiquitously expressed, the cellular phenotype is most severe in neurons and cardiomyocytes. Here, we conducted comprehensive proteomic, metabolic and functional studies to determine whether subclinical abnormalities exist in mitochondria of blood cells from FRDA patients. Frataxin protein levels were significantly decreased in platelets and peripheral blood mononuclear cells from FRDA patients. Furthermore, the most significant differences associated with frataxin deficiency in FRDA blood cell mitochondria were the decrease of two mitochondrial heat shock proteins. We did not observe profound changes in frataxin-targeted mitochondrial proteins or mitochondrial functions or an increase of apoptosis in peripheral blood cells, suggesting that functional defects in these mitochondria are not readily apparent under resting conditions in these cells.     

Origin and instability of GAA repeats: insights from Alu elements

Expansion of GAA repeats in the intron of the frataxin gene is involved in the autosomal recessive Friedreich's ataxia (FRDA). The GAA repeats arise from a stretch of adenine residues of an Alu element. These repeats have a size ranging from 7- 38 in the normal population, and expand to thousands in the affected individuals. The mechanism of origin of GAA repeats, their polymorphism and stability are not well understood. In this study, we have carried out an extensive analysis of GAA repeats at several loci in the humans. This analysis indicates the association of a majority of GAA repeats with the 3' end of an "A" stretch present in the Alu repeats. Further, the prevalence of GAA repeats correlates with the evolutionary age of Alu subfamilies as well as with their relative frequency in the genome. Our study on GAA repeat polymorphism at some loci in the normal population reveals that the length of the GAA repeats is determined by the relative length of the flanking A stretch. Based on these observations, a possible mechanism for origin of GAA repeats and modulatory effects of flanking sequences on repeat instability mediated by DNA triplex is proposed.