GAA Repeat Expansion Research Articles

Activating frataxin expression by single-stranded siRNAs targeting the GAA repeat expansion

Friedreich's ataxia (FRDA) is an incurable neurodegenerative disorder caused by reduced expression of the mitochondrial protein frataxin (FXN). The genetic cause of the disease is an expanded GAA repeat within the FXN gene. Agents that increase expression of FXN protein are a potential approach to therapy. We previously described anti-trinucleotide GAA duplex RNAs (dsRNAs) and antisense oligonucleotides (ASOs) that activate FXN protein expression in multiple patient derived cell lines. Here we test two distinct series of compounds for their ability to increase FXN expression. ASOs with butane linkers showed low potency, which is consistent with the low Tm values and suggesting that flexible conformation impairs activity. By contrast, single-stranded siRNAs (ss-siRNAs) that combine the strengths of dsRNA and ASO approaches had nanomolar potencies. ss-siRNAs provide an additional option for developing nucleic acid therapeutics to treat FRDA.

Novel Nrf2-Inducer Prevents Mitochondrial Defects and Oxidative Stress in Friedreich's Ataxia Models.

Friedreich’s Ataxia (FRDA) is an autosomal recessive neurodegenerative disorder, affecting dorsal root ganglia (DRG), cerebellar dentate nuclei and heart. It is caused by a GAA repeat expansion mutation within the frataxin gene (FXN). This impedes FXN transcription resulting in a progressive decrease of the mitochondrial protein, frataxin. Increased oxidative stress leading to a chronic depletion of endogenous antioxidants affects the survival of the cells and causes neurodegeneration. In particular, cerebellar granule neurons (CGNs) show a significant increase of reactive oxygen species (ROS), lipid peroxidation and lower level of reduced glutathione (GSH). In FRDA, one of the major pathways of oxidant scavengers, the Nrf2 antioxidant pathway, is defective. Previous studies on FRDA-like CGNs showed that the reduced level of frataxin and the oxidative stress induce mitochondrial impairments. By triggering the Nrf2 endogenous pathway pharmacologically we determined whether this could promote mitochondrial fitness and counteract oxidative stress. In this work, we sought to investigate the beneficial effect of a promising Nrf2-inducer, omaveloxolone (omav), in CGNs from two FRDA mouse models, KIKO and YG8R, and human fibroblasts from patients. We found that CGNs from both KIKO and YG8R presented Complex I deficiency and that omav was able to restore substrate availability and Complex I activity. This was also confirmed in human primary fibroblasts from FRDA patients. Although fibroblasts are not the major tissue affected, we found that they show significant differences recapitulating the disease; this is therefore an important tool to investigate patients’ pathophysiology. Interestingly, we found that patient fibroblasts had an increased level of endogenous lipid peroxidation and mitochondrial ROS (mROS), and lower GSH at rest. Omav was able to reverse this phenotype, protecting the cells against oxidative stress. By stimulating the cells with hydrogen peroxide (H2O2) and looking for potential mitochondrial pathophysiology, we found that fibroblasts could not maintain their mitochondrial membrane potential (ΔΨm). Remarkably, omav was protective to mitochondrial depolarization, promoting mitochondrial respiration and preventing cell death. Our results show that omav promotes Complex I activity and protect cells from oxidative stress. Omav could, therefore, be used as a novel therapeutic drug to ameliorate the pathophysiology of FRDA.

Genetic testing for clinically suspected spinocerebellar ataxias: report from a tertiary referral centre in India

Spinocerebellar ataxias (SCAs) are a heterogeneous group of neurodegenerative syndromes, characterized by a wide range of muscular weakness and motor deficits, caused due to cerebellar degeneration. The prevalence of the syndromes of SCA varies across the world and is known to be linked to the instability of trinucleotide repeats within the high-end normal alleles, along with susceptible haplotype. We estimated sizes of the CAG or GAA repeat expansions at the SCA1, SCA2, SCA3, SCA12 and frataxin loci among 864 referrals of subjects to genetic counselling and testing (GCAT) clinic, National Institute of Mental Health and Neurosciences, Bengaluru, India, with suspected SCA. The most frequent mutations detected were SCA1 (n = 100 (11.6%)) and SCA2 (n = 98 (11.3%)) followed by SCA3 (n = 40 (4.6%)), FRDA (n = 20 (2.3%)) and SCA12 (n = 8 (0.9%)).

Activation of Frataxin Protein Expression by Antisense Oligonucleotides Targeting the Mutant Expanded Repeat.

Friedreich's Ataxia (FA) is an inherited neurologic disorder caused by an expanded GAA repeat within intron 1 of the frataxin (FXN) gene that reduces expression of FXN protein. Agents that increase expression of FXN have the potential to alleviate the disease. We previously reported that duplex RNAs (dsRNAs) and antisense oligonucleotides (ASOs) complementary to the GAA repeat could enhance expression of FXN protein. We now explore the potential of a diverse group of chemically modified dsRNAs and ASOs to define the breadth of repeat-targeted synthetic nucleic acids as a platform for therapeutic development for FA. ASOs and dsRNAs can activate FXN protein expression in FA patient-derived cell lines that possess varied numbers of GAA repeats. Increased FXN protein expression was achieved by ASOs incorporating diverse chemical modifications with low nanomolar potencies, suggesting substantial flexibility in choosing compounds for further chemical optimization and animal studies. Our data encourage further development of ASOs as agents to treat FA.

Somatic instability of the expanded GAA repeats in Friedreich's ataxia.

Friedreich’s ataxia (FRDA) is a genetic neurodegenerative disorder caused by transcriptional silencing of the frataxin gene (FXN) due to expansions of GAA repeats in intron 1. FRDA manifests with multiple symptoms, which may include ataxia, cardiomyopathy and diabetes mellitus. Expanded GAA tracts are genetically unstable, exhibiting both expansions and contractions. GAA length correlates with severity of FRDA symptoms and inversely with age of onset. Thus, tissue-specific somatic instability of long GAA repeats may be implicated in the development of symptoms and disease progression. Herein, we determined the extent of somatic instability of the GAA repeats in heart, cerebral cortex, spinal cord, cerebellar cortex, and pancreatic tissues from 15 FRDA patients. Results demonstrate differences in the lengths of the expanded GAAs among different tissues, with significantly longer GAA tracts detected in heart and pancreas than in other tissues. The expansion bias detected in heart and pancreas may contribute to disease onset and progression, making the mechanism of somatic instability an important target for therapy. Additionally, we detected significant differences in GAA tract lengths between lymphocytes and fibroblast pairs derived from 16 FRDA patients, with longer GAA tracts present in the lymphocytes. This result urges caution in direct comparisons of data obtained in these frequently used FRDA models. Furthermore, we conducted a longitudinal analysis of the GAA repeat length in lymphocytes collected over a span of 7–9 years and demonstrated progressive expansions of the GAAs with maximum gain of approximately 9 repeats per year. Continuous GAA expansions throughout the patient’s lifespan, as observed in FRDA lymphocytes, should be considered in clinical trial designs and data interpretation.

Selected missense mutations impair frataxin processing in Friedreich ataxia.

ObjectiveFrataxin (FXN) is a highly conserved mitochondrial protein. Reduced FXN levels cause Friedreich ataxia, a recessive neurodegenerative disease. Typical patients carry GAA repeat expansions on both alleles, while a subgroup of patients carry a missense mutation on one allele and a GAA repeat expansion on the other. Here, we report that selected disease‐related FXN missense mutations impair FXN localization, interaction with mitochondria processing peptidase, and processing.MethodsImmunocytochemical studies and subcellular fractionation were performed to study FXN import into the mitochondria and examine the mechanism by which mutations impair FXN processing. Coimmunoprecipitation was performed to study the interaction between FXN and mitochondrial processing peptidase. A proteasome inhibitor was used to model traditional therapeutic strategies. In addition, clinical profiles of subjects with and without point mutations were compared in a large natural history study.Results FXNI 154F and FXNG 130V missense mutations decrease FXN 81–210 levels compared with FXNWT, FXNR 165C, and FXNW 155R, but do not block its association with mitochondria. FXNI 154F and FXNG 130V also impair FXN maturation and enhance the binding between FXN 42–210 and mitochondria processing peptidase. Furthermore, blocking proteosomal degradation does not increase FXN 81–210 levels. Additionally, impaired FXN processing also occurs in fibroblasts from patients with FXNG 130V. Finally, clinical data from patients with FXNG 130V and FXNI 154F mutations demonstrates a lower severity compared with other individuals with Friedreich ataxia.InterpretationThese data suggest that the effects on processing associated with FXNG 130V and FXNI 154F mutations lead to higher levels of partially processed FXN, which may contribute to the milder clinical phenotypes in these patients.

Liver Growth Factor (LGF) Upregulates Frataxin Protein Expression and Reduces Oxidative Stress in Friedreich's Ataxia Transgenic Mice.

Friedreich’s ataxia (FA) is a severe disorder with autosomal recessive inheritance that is caused by the abnormal expansion of GAA repeat in intron 1 of FRDA gen. This alteration leads to a partial silencing of frataxin transcription, causing a multisystem disorder disease that includes neurological and non-neurological damage. Recent studies have proven the effectiveness of neurotrophic factors in a number of neurodegenerative diseases. Therefore, we intend to determine if liver growth factor (LGF), which has a demonstrated antioxidant and neuroprotective capability, could be a useful therapy for FA. To investigate the potential therapeutic activity of LGF we used transgenic mice of the FXNtm1MknTg (FXN)YG8Pook strain. In these mice, intraperitoneal administration of LGF (1.6 μg/mouse) exerted a neuroprotective effect on neurons of the lumbar spinal cord and improved cardiac hypertrophy. Both events could be the consequence of the increment in frataxin expression induced by LGF in spinal cord (1.34-fold) and heart (1.2-fold). LGF also upregulated by 2.6-fold mitochondrial chain complex IV expression in spinal cord, while in skeletal muscle it reduced the relation oxidized glutathione/reduced glutathione. Since LGF partially restores motor coordination, we propose LGF as a novel factor that may be useful in the treatment of FA.

Disruption of Higher Order DNA Structures in Friedreich's Ataxia (GAA)n Repeats by PNA or LNA Targeting.

Expansion of (GAA)n repeats in the first intron of the Frataxin gene is associated with reduced mRNA and protein levels and the development of Friedreich’s ataxia. (GAA)n expansions form non-canonical structures, including intramolecular triplex (H-DNA), and R-loops and are associated with epigenetic modifications. With the aim of interfering with higher order H-DNA (like) DNA structures within pathological (GAA)n expansions, we examined sequence-specific interaction of peptide nucleic acid (PNA) with (GAA)n repeats of different lengths (short: n=9, medium: n=75 or long: n=115) by chemical probing of triple helical and single stranded regions. We found that a triplex structure (H-DNA) forms at GAA repeats of different lengths; however, single stranded regions were not detected within the medium size pathological repeat, suggesting the presence of a more complex structure. Furthermore, (GAA)4-PNA binding of the repeat abolished all detectable triplex DNA structures, whereas (CTT)5-PNA did not. We present evidence that (GAA)4-PNA can invade the DNA at the repeat region by binding the DNA CTT strand, thereby preventing non-canonical-DNA formation, and that triplex invasion complexes by (CTT)5-PNA form at the GAA repeats. Locked nucleic acid (LNA) oligonucleotides also inhibited triplex formation at GAA repeat expansions, and atomic force microscopy analysis showed significant relaxation of plasmid morphology in the presence of GAA-LNA. Thus, by inhibiting disease related higher order DNA structures in the Frataxin gene, such PNA and LNA oligomers may have potential for discovery of drugs aiming at recovering Frataxin expression.

Identification of novel senataxin mutations in Chinese patients with autosomal recessive cerebellar ataxias by targeted next-generation sequencing.

BackgroundAutosomal recessive cerebellar ataxias (ARCA) are a group of neurodegenerative disorders characterized by early onset of gait impairment, disturbed limb coordination, dysarthria, and eye movement abnormalities, most likely due to the degeneration of cerebellum, brainstem, and spinal cord. Despite of the rarity, ARCA are both clinically and genetically heterogeneous. To date, more than 30 culprit genes have been identified in ARCA. Unraveling the specific causative mutation in cases with ARCA remains challenging so far.MethodsThree ARCA pedigrees of Chinese ancestry were recruited. Clinical features were evaluated and peripheral blood was collected after obtaining the written inform. Laboratory examinations, brain MRI, and EMG were performed for all the affected individuals. Genomic DNA was extracted, followed by the screening of GAA repeat expansion in FXN gene to exclude Friedreich’s ataxia. Targeted next-generation sequencing combining Sanger sequencing was performed in each proband of these families.ResultsCompound heterozygous mutations, c.3190G > T (p.E1064X) and c.4883C > G (p.S1628X) of senataxin (SETX) gene were identified in one family with two affected cases. Both of the patients presented with early onset of unsteady walk, dysarthria, and diplopia. EMG test revealed decreased conduction velocity and evoked potential of both motor and sensory nerve. Moreover, elevated serum alpha-fetoprotein (AFP) and apparent cerebellar atrophy were observed. These features were typical features of ataxia with oculomotor apraxia type 2 (AOA2) and in line with the genetic results. However, no specific mutation was identified in the other two pedigrees.ConclusionsWe identified novel compound heterozygous mutations of SETX in Chinese AOA2 pedigree, which broaden the mutation spectrum of SETX. To our knowledge, this is the first report concerning Chinese AOA2 cases with SETX mutations.Electronic supplementary materialThe online version of this article (doi:10.1186/s12883-016-0696-y) contains supplementary material, which is available to authorized users.

Lentivirus-meditated frataxin gene delivery reverses genome instability in Friedreich ataxia patient and mouse model fibroblasts.

Friedreich ataxia (FRDA) is a progressive neurodegenerative disease caused by deficiency of frataxin protein, with the primary sites of pathology being the large sensory neurons of the dorsal root ganglia and the cerebellum. FRDA is also often accompanied by severe cardiomyopathy and diabetes mellitus. Frataxin is important in mitochondrial iron–sulfur cluster (ISC) biogenesis and low-frataxin expression is due to a GAA repeat expansion in intron 1 of the FXN gene. FRDA cells are genomically unstable, with increased levels of reactive oxygen species and sensitivity to oxidative stress. Here we report the identification of elevated levels of DNA double strand breaks (DSBs) in FRDA patient and YG8sR FRDA mouse model fibroblasts compared to normal fibroblasts. Using lentivirus FXN gene delivery to FRDA patient and YG8sR cells, we obtained long-term overexpression of FXN mRNA and frataxin protein levels with reduced DSB levels towards normal. Furthermore, γ-irradiation of FRDA patient and YG8sR cells revealed impaired DSB repair that was recovered on FXN gene transfer. This suggests that frataxin may be involved in DSB repair, either directly by an unknown mechanism, or indirectly via ISC biogenesis for DNA repair enzymes, which may be essential for the prevention of neurodegeneration.

The Replication of Frataxin Gene Is Assured by Activation of Dormant Origins in the Presence of a GAA-Repeat Expansion.

It is well known that DNA replication affects the stability of several trinucleotide repeats, but whether replication profiles of human loci carrying an expanded repeat differ from those of normal alleles is poorly understood in the endogenous context. We investigated this issue using cell lines from Friedreich’s ataxia patients, homozygous for a GAA-repeat expansion in intron 1 of the Frataxin gene. By interphase, FISH we found that in comparison to the normal Frataxin sequence the replication of expanded alleles is slowed or delayed. According to molecular combing, origins never fired within the normal Frataxin allele. In contrast, in mutant alleles dormant origins are recruited within the gene, causing a switch of the prevalent fork direction through the expanded repeat. Furthermore, a global modification of the replication profile, involving origin choice and a differential distribution of unidirectional forks, was observed in the surrounding 850 kb region. These data provide a wide-view of the interplay of events occurring during replication of genes carrying an expanded repeat.

Stalled DNA Replication Forks at the Endogenous GAA Repeats Drive Repeat Expansion in Friedreich’s Ataxia Cells

Friedreich's ataxia (FRDA) is caused by the expansion of GAA repeats located in the Frataxin (FXN) gene. The GAA repeats continue to expand in FRDA patients, aggravating symptoms and contributing todisease progression. The mechanism leading to repeat expansion and decreased FXN transcription remains unclear. Using single-molecule analysis of replicated DNA, we detected that expanded GAA repeats present a substantial obstacle for the replication machinery at the FXN locus in FRDA cells. Furthermore, aberrant origin activation and lack of a proper stress response to rescue the stalled forks in FRDA cells cause an increase in 3'-5' progressing forks, which could enhance repeat expansion and hinder FXN transcription by head-on collision with RNA polymerases. Treatment of FRDA cells with GAA-specific polyamides rescues DNA replication fork stalling and alleviates expansion of the GAA repeats, implicating DNA triplexes as a replication impediment and suggesting that fork stalling might be a therapeutic target for FRDA.

321. Deletion of Mutated GAA Repeats from the Intron 1 of the Frataxin Gene Using the CRISPR System Restores the Protein Expression in a Friedreich Ataxia Model

The CRISPR system is now widely used as a molecular tool to edit the genome. We used this technique in Friedreich Ataxia (FRDA), an inherited autosomal disease known to cause a decrease of the mitochondrial frataxin protein. Genetic analysis revealed a GAA repeat expansion within the intron 1 of the frataxin (FXN) gene. We used cells derived from the YG8sR mouse model where the mouse frataxin gene is knockout but contain a human FXN mutated transgene on one allele. We then deleted the GAA trinucleotide repeat using 2 specific guide RNAs (gRNAs) co-expressed with either S. pyogenes (Sp) or S. aureus (Sa) Cas9. We were able to monitored an increase up to 2-fold of frataxin mRNA and protein levels in clone cells. We also confirmed these results in vivo using DNA electroporation in the Tibialis anterior muscle of the YG8R mice. Ongoing in vivo investigation of a systemically injected AAV-DJ vector expressing the SaCas9 and 2 successful selected gRNAs in the mouse model YG8sR will hopefully provide more details answers on the efficacy of the approach and give us preliminary data to go forward for clinical trial. The deletion of the GAA repeats expansion then might be a highly valuable gene therapy approach for FRDA patients.

'Mitochondrial energy imbalance and lipid peroxidation cause cell death in Friedreich's ataxia'.

Friedreich's ataxia (FRDA) is an inherited neurodegenerative disease. The mutation consists of a GAA repeat expansion within the FXN gene, which downregulates frataxin, leading to abnormal mitochondrial iron accumulation, which may in turn cause changes in mitochondrial function. Although, many studies of FRDA patients and mouse models have been conducted in the past two decades, the role of frataxin in mitochondrial pathophysiology remains elusive. Are the mitochondrial abnormalities only a side effect of the increased accumulation of reactive iron, generating oxidative stress? Or does the progressive lack of iron-sulphur clusters (ISCs), induced by reduced frataxin, cause an inhibition of the electron transport chain complexes (CI, II and III) leading to reactive oxygen species escaping from oxidative phosphorylation reactions? To answer these crucial questions, we have characterised the mitochondrial pathophysiology of a group of disease-relevant and readily accessible neurons, cerebellar granule cells, from a validated FRDA mouse model. By using live cell imaging and biochemical techniques we were able to demonstrate that mitochondria are deregulated in neurons from the YG8R FRDA mouse model, causing a decrease in mitochondrial membrane potential (▵Ψm) due to an inhibition of Complex I, which is partially compensated by an overactivation of Complex II. This complex activity imbalance leads to ROS generation in both mitochondrial matrix and cytosol, which results in glutathione depletion and increased lipid peroxidation. Preventing this increase in lipid peroxidation, in neurons, protects against in cell death. This work describes the pathophysiological properties of the mitochondria in neurons from a FRDA mouse model and shows that lipid peroxidation could be an important target for novel therapeutic strategies in FRDA, which still lacks a cure.

Plasma circulating cell-free mitochondrial DNA in the assessment of Friedreich's ataxia

Friedreich's ataxia (FRDA) is one of the most devastating childhood onset neurodegenerative disease affecting multiple organs in the course of progression. FRDA is associated with mitochondrial dysfunction due to deficit in a nuclear encoded mitochondrial protein, frataxin. Identification of disease-specific biomarker for monitoring the severity remains to be a challenging topic. This study was aimed to identify whether circulating cell-free nuclear DNA (nDNA) and mitochondrial DNA (mtDNA) in blood plasma can be a potential biomarker for FRDA. Clinical information was assessed using International Cooperative Ataxia Rating Scale and the disease was confirmed using Long-range PCR for GAA repeat expansion within the gene encoding frataxin. The frataxin expression was measured using Western blot. Plasma nDNA and mtDNA levels were quantified by Multiplex real-time PCR. The major observation is that the levels of nDNA found to be increased, whereas mtDNA levels were reduced significantly in the plasma of FRDA patients (n=21) as compared to healthy controls (n=21). Further, plasma mtDNA levels showed high sensitivity (90%) and specificity (76%) in distinguishing from healthy controls with optimal cutoff indicated at 4.1×105GE/mL. Interestingly, a small group of follow-up patients (n=9) on intervention with, a nutrient supplement, omega-3 fatty acid (a known enhancer of mitochondrial metabolism) displayed a significant improvement in the levels of plasma mtDNA, supporting our hypothesis that plasma mtDNA can be a potential monitoring or prognosis biomarker for FRDA.

Understanding the Role of Mitochondrial Pathophysiology in Friedreich's Ataxia

Friedreich's ataxia (FRDA) is a rare inherited neurodegenerative disease. The mutation consists of a GAA repeat expansion within the FXN gene, which down regulates frataxin, leading to abnormal mitochondrial iron accumulation. Thus, may be causing changes in mitochondrial function. In the present work we aimed to describe the mitochondrial pathophysiology of cerebellar granule neurons, from a FRDA mouse models. By using functional microscopy and biochemical techniques we were able to demonstrate that mitochondria are deregulated in neurons from the FRDA mouse models, causing a decrease in mitochondrial membrane potential due to an inhibition of Complex I, which is partially compensated by an overactivation of Complex II, at least in one of the two models. This complex activity imbalance leads to ROS generation in both mitochondrial matrix and cytosol, which results in glutathione depletion and increase of lipid peroxidation. Preventing oxidative stress and in particular the increase of lipid peroxidation, in neurons, we obtained cell death protection. This work describes the pathophysiological properties of the mitochondria in FRDA mouse model neurons and shows that lipid peroxidation could be an important target for novel therapeutic strategies for this debilitating disease that still lacks a cure.

Atypical Features in a Large Turkish Family Affected with Friedreich Ataxia.

Here, we describe the clinical features of several members of the same family diagnosed with Friedreich ataxia (FRDA) and cerebral lesions, demyelinating neuropathy, and late-age onset without a significant cardiac involvement and presenting with similar symptoms, although genetic testing was negative for the GAA repeat expansion in one patient of the family. The GAA repeat expansion in the frataxin gene was shown in all of the family members except in a young female patient. MRI revealed arachnoid cysts in two patients; MRI was consistent with both cavum septum pellucidum-cavum vergae and nodular signal intensity increase in one patient. EMG showed demyelinating sensorimotor polyneuropathy in another patient. The GAA expansion-negative 11-year-old female patient had mental-motor retardation, epilepsy, and ataxia. None of the patients had significant cardiac symptoms. Description of FRDA families with different ethnic backgrounds may assist in identifying possible phenotypic and genetic features of the disease. Furthermore, the genetic heterogeneity observed in this family draws attention to the difficulty of genetic counseling in an inbred population and to the need for genotyping all affected members before delivering comprehensive genetic counseling.

A 22-Year Follow-up Study of Long-term Cardiac Outcome and Predictors of Survival in Friedreich Ataxia.

Friedreich ataxia (FRDA) is the most common genetic sensory ataxia, and myocardial involvement is a major determinant of survival. To assess FRDA survival and cardiac outcome to adapt future therapeutic trials. In a longitudinal follow-up study, all patients with genetically confirmed FRDA seen in the reference center and referred for cardiac evaluation (standard 12-lead electrocardiogram and transthoracic echocardiography) to the cardiology department were enrolled and followed up from April 27, 1990, to July 31, 2013. The setting was the French National Reference Center for Rare Diseases and the Department of Cardiology, Salpêtrière University Hospital, Paris, France. In total, 138 patients with FRDA were followed up. Among 133 patients homozygous for expanded GAA repeats, the mean (SD) age was 31 (10) years (age range, 11-62 years), with a mean (SD) age at disease onset of 16 (8) years (age range, 3-50 years) and a mean (SD) age at first wheelchair use of 26 (9) years (age range, 11-64 years). Cardiac hypertrophy was present in 57.9% (77 of 133), and electrocardiography was normal in 6.8% (9 of 133). Long-term cardiac outcome and predictors of survival in FRDA. After a mean (SD) follow-up of 10.5 (5.5) years (range, 0.6-23.0 years), the 10-year survival rate was 88.5%. In 80.0% of patients (12 of 15), death was due to cardiac causes. Predictors of survival were a shorter GAA repeat length on the smaller allele of the frataxin gene (hazard ratio [HR], 1.85; 95% CI, 1.28-2.69), left ventricular ejection fraction (HR, 0.42; 95% CI, 0.20-0.89), and left ventricular mass index (HR, 1.19; 95% CI, 1.04-1.36). Two cardiac evolutions were distinguished with a group-based trajectory model, including a low-risk cardiac group (78.6% [81 of 103] with normal ejection fraction at baseline that declined slightly over time but remained within the normal range) and a high-risk cardiac group (21.4% [22 of 103] in which the ejection fraction progressively declined during follow-up). The patients with the worse cardiac evolution had longer GAA repeats. Neurological impairment was not predictive of cardiac change over time. Survival in FRDA is determined by cardiac complications, which are dependent on the mutation (ie, the size of the expanded GAA repeat). Patients with progressive decline of the left ventricular ejection fraction had a worse prognosis. This finding demonstrates that cardiac follow-up is important in FRDA to identify individuals at risk for further cardiac complications.

Two autosomal recessive neuromuscular disorders - FRDA and LGMD2A, in a single Bulgarian pedigree

Here we report a single pedigree affected by two different recessive neuromuscular disorders, namely Friedreich ataxia (FRDA) and Limb-Girdle Muscular Dystrophy Type 2A. The molecular genetic testing showed that in this family FRDA is caused by an expanded GAA repeat in compound heterozygous state with a novel point mutation c.442C>T, p.Gln148* on the second allele of the FXN gene. This case of two siblings (male and female) is the first report of Bulgarian FRDA case carrying a point mutation in the FXN gene. The LGMD2A affected individual was found to be compound heterozygous for the two most common CAPN3 mutations for the Bulgarian population (c.550delA, p.Thr184Argfs; c.967G>T, p.Glu323*). To the best of our knowledge we are reporting the first Bulgarian family with two neurological disorders caused by mutations in genes on different chromosomes and segregating independently in the family. This single pedigree with four different mutations is an example for the high genetic heterogeneity of the Bulgarian population.

Friedreich ataxia in Norway - an epidemiological, molecular and clinical study.

BackgroundFriedreich ataxia is an autosomal recessive hereditary spinocerebellar disorder, characterized by progressive limb and gait ataxia due to proprioceptive loss, often complicated by cardiomyopathy, diabetes and skeletal deformities. Friedreich ataxia is the most common hereditary ataxia, with a reported prevalence of 1:20 000 – 1:50 000 in Central Europe. Previous reports from south Norway have found a prevalence varying from 1:100 000 – 1:1 350 000; no studies are previously done in the rest of the country.MethodsIn this cross-sectional study, Friedreich ataxia patients were identified through colleagues in neurological, pediatric and genetic departments, hospital archives searches, patients’ associations, and National Centre for Rare Disorders. All included patients, carriers and controls were investigated clinically and molecularly with genotype characterization including size determination of GAA repeat expansions and frataxin measurements. 1376 healthy blood donors were tested for GAA repeat expansion for carrier frequency analysis.ResultsTwenty-nine Friedreich ataxia patients were identified in Norway, of which 23 were ethnic Norwegian, corresponding to a prevalence of 1:176 000 and 1:191 000, respectively. The highest prevalence was seen in the north. Carrier frequency of 1:196 (95 % CI = [1:752–1:112]) was found. Homozygous GAA repeat expansions in the FXN gene were found in 27/29, while two patients were compound heterozygous with c.467 T < C, L157P and the deletion (g.120032_122808del) including exon 5a. Two additional patients were heterozygous for GAA repeat expansions only. Significant differences in the level of frataxin were found between the included patients (N = 27), carriers (N = 37) and controls (N = 27).ConclusionsIn this first thorough study of a complete national cohort of Friedreich ataxia patients, and first nation-wide study of Friedreich ataxia in Norway, the prevalence of Friedreich ataxia in Norway is lower than in Central Europe, but higher than in the last Norwegian report, and as expected from migration studies. A south–north prevalence gradient is present. Based on Hardy Weinberg’s equilibrium, the carrier frequency of 1:196 is consistent with the observed prevalence. All genotypes, and typical and atypical phenotypes were present in the Norwegian population. The patients were phenotypically similar to European cohorts. Frataxin was useful in the diagnostic work-up of heterozygous symptomatic cases.Electronic supplementary materialThe online version of this article (doi:10.1186/s13023-015-0328-4) contains supplementary material, which is available to authorized users.