Frataxin gene point mutations in Italian Friedreich ataxia patients

Friedreich ataxia (FRDA) is associated with a GAA-trinucleotide-repeat expansion in the first intron of the FXN gene (9q13-21), which encodes a 210-amino-acid protein named frataxin. More than 95% of patients are homozygous for 90-1,300 repeat expansion on both alleles. The remaining patients have been shown to be compound heterozygous for a GAA expansion on one allele and a micromutation on the other. The reduction of both frataxin messenger RNA (mRNA) and protein was found to be proportional to the size of the smaller GAA repeat allele. We report a clinical and molecular study of 12 families in which classical FRDA patients were heterozygous for a GAA expansion on one allele. Sequence analysis of the FXN gene allowed the identification of the second disease-causing mutation in each heterozygous patient, which makes this the second largest series of FRDA compound heterozygotes reported thus far. We have identified seven mutations, four of which are novel. Five patients carried missense mutations, whereas eight patients carried null (frameshift or nonsense) mutations. Quantitation of frataxin levels in lymphoblastoid cell lines derived from six compound heterozygous patients showed a statistically significant correlation of residual protein levels with the age at onset (r = 0.82, p < 0.05) or the GAA expansion (r = -0.76, p < 0.1). In the group of patients heterozygous for a null allele, a strong (r = -0.94, p < 0.01) correlation was observed between the size of GAA expansion and the age at onset, thus lending support to the hypothesis that the residual function of frataxin in patients' cells derive exclusively from the expanded allele.     

Malaysian siblings with friedreich ataxia and chorea: a novel deletion in the frataxin gene

BACKGROUND: Friedrich ataxia (FRDA1) is most often the result of a homozygous GAA repeat expansion in the first intron of the frataxin gene (FRDA gene). This condition is seen in individuals of European, North African, Middle Eastern and Indian descent and has not been reported in Southeast Asian populations. Approximately 4% of FRDA1 patients are compound heterozygotes. These patients have a GAA expansion on one allele and a point mutation on the other and have been reported to have an atypical phenotype. OBJECTIVE: To describe a novel dinucleotide deletion in the FRDA gene in two Malaysian siblings with FRDA1. SETTING: Tertiary referral university hospital setting. PATIENTS AND METHODS: A previously healthy 10-year-old Malaysian boy, presented with fever, lethargy, headaches, dysarthria, dysphagia, vertigo and ataxia which developed over a one week period. His neurological exam revealed evidence of dysarthria and ataxia, mild generalized weakness and choreoform movements of the tongue and hands. His reflexes were absent and Babinski sign was present bilaterally. A nine-year-old sister was found to have mild ataxia but was otherwise neurologically intact. RESULTS: Molecular genetic studies demonstrated that both siblings were compound heterozygotes with a GAA expansion on one allele and a novel dinucleotide deletion on the other allele. CONCLUSIONS: We describe a novel dinucleotide deletion in the first exon of the FRDA gene in two siblings with FRDA1. Additionally this is the first report of FRDA1 occurring in a family of southeast Asian descent, it demonstrates intrafamilial phenotypic variability, and confirms that atypical phenotypes are associated with compound heterozygosity.     

Frataxin point mutations in two patients with Friedreich's ataxia and unusual clinical features

Two patients with a progressive ataxia are presented with clinical features consistent with classic Friedreich's ataxia (FRDA), but also with features unusual for FRDA. Analysis of DNA showed that each patient is heterozygous for the expanded GAA repeat of FRDA, but carries a base change on his other frataxin allele. For one patient a non-conservative arginine to cysteine amino acid change is predicted at amino acid 165 whereas the other mutation is found at the junction of exon one and intron one. Muscle biopsy showed an absence of frataxin immunoreactivity in the patient harbouring the intronic mutation, confirming the pathological nature of the base change. These mutations extend the range of point mutations seen in FRDA, and agree with recent reports suggesting phenotypic variation in patients with FRDA harbouring point mutations in conjunction with an expanded GAA repeat.     

Friedreich's ataxia: Point mutations and clinical presentation of compound heterozygotes

Friedreich's ataxia is the most common inherited ataxia. Ninety-six percent of patients are homozygous for GAA trinucleotide repeat expansions in the first intron of the frataxin gene. The remaining cases are compound heterozygotes for a GAA expansion and a frataxin point mutation. We report here the identification of 10 novel frataxin point mutations, and the detection of a previously described mutation (G130V) in two additional families. Most truncating mutations were in exon 1. All missense mutations were in the last three exons coding for the mature frataxin protein. The clinical features of 25 patients with identified frataxin point mutations were compared with those of 196 patients homozygous for the GAA expansion. A similar phenotype resulted from truncating mutations and from missense mutations in the carboxy-terminal half of mature frataxin, suggesting that they cause a comparable loss of function. In contrast, the only two missense mutations located in the amino-terminal half of mature frataxin (D122Y and G130V) cause an atypical and milder clinical presentation (early-onset spastic gait with slow disease progression, absence of dysarthria, retained or brisk tendon reflexes, and mild or no cerebellar ataxia), suggesting that they only partially affect frataxin function. The incidence of optic disk pallor was higher in compound heterozygotes than in expansion homozygotes, which might correlate with a very low residual level of normal frataxin produced from the expanded allele. Ann Neurol 1999;45:200–206     

A novel deletion–insertion mutation identified in exon 3 of <Emphasis Type="Italic">FXN</Emphasis> in two siblings with a severe Friedreich ataxia phenotype

Friedreich ataxia (FRDA) is an autosomal recessive neurodegenerative disease most commonly caused by a GAA trinucleotide repeat expansion in the first intron of FXN, which reduces expression of the mitochondrial protein frataxin. Approximately 98% of individuals with FRDA are homozygous for GAA expansions, with the remaining 2% compound heterozygotes for a GAA expansion and a point mutation within FXN. Two siblings with early onset of symptoms experienced rapid loss of ambulation by 8 and 10 years. Diagnostic testing for FRDA demonstrated one GAA repeat expansion of 1010 repeats and one non-expanded allele. Sequencing all five exons of FXN identified a novel deletion-insertion mutation in exon 3 (c.371_376del6ins15), which results in a modified frataxin protein sequence at amino acid positions 124–127. Specifically, the amino acid sequence changes from DVSF to VHLEDT, increasing frataxin from 211 residues to 214. Using the known structure of human frataxin, a theoretical 3D model of the mutant protein was developed. In the event that the modified protein is expressed and stable, it is predicted that the acidic interface of frataxin, known to be involved in iron binding and interactions with the iron–sulphur cluster assembly factor IscU, would be impaired.     

Intrafamilial phenotypic variability in Friedreich ataxia associated with a G130V mutation in the FRDA gene

BACKGROUND: Most patients with Friedreich ataxia (FA) have a GAA trinucleotide repeat expansion in intron 1 of the FA gene (FRDA) on both arms of chromosome 9. However, some patients are compound heterozygotes and harbor a GAA expansion on one allele and a point mutation on the other. Compound heterozygous patients with FA who have a GAA expansion and a G130V mutation have been reported to have an atypical phenotype with a slow disease progression, minimal or no ataxia, or gait spasticity. OBJECTIVE: To describe intrafamilial phenotypic variability in a GAA expansion/G130V mutation compound heterozygous family with FA. SETTING: Tertiary referral university hospital setting. PATIENTS AND METHODS: A 34-year-old man presented to our hospital with a 24-year history of stiff legs and mild unsteadiness of gait. Clinical examination showed a spastic paraparesis with normal to pathologically brisk deep tendon reflexes and mild left upper limb ataxia. His 27-year-old sister presented with a slowly progressive early-onset ataxic syndrome. She had ataxia of gait, mild to severe limb ataxia, and reduced or absent deep tendon reflexes, but no evidence of spasticity on examination. RESULTS: Neurophysiologic investigations showed evidence of a sensory axonal neuropathy, and molecular genetic analysis showed that both siblings were compound heterozygotes with a GAA expansion and a G130V mutation. CONCLUSIONS: This report confirms that compound heterozygous patients with FA who have a GAA expansion and a G130V mutation may present with an ataxic phenotype and that intrafamilial phenotypic variability in these pedigrees can occur. It also emphasizes the importance of performing molecular genetic analysis for the GAA trinucleotide expansion in patients presenting with a spastic paraparesis of undetermined etiology, especially when there is neurophysiologic evidence of a sensory axonal neuropathy.     

Clinical and molecular studies in five Brazilian cases of Friedreich ataxia.

Friedreich ataxia (FRDA), the most common autosomal recessive ataxia, is caused in 94% of cases by homozygous expansions of an unstable GAA repeat localised in intron 1 of the X25 gene. We have investigated this mutation in five Brazilian patients: four with typical FRDA findings and one patient with atypical manifestations, who was considered to have some other form of cerebellar ataxia with retained reflexes. The GAA expansion was detected in all these patients. The confirmation of FRDA diagnosis in the atypical case may be pointing out, as in other reports, that clinical spectrum of Friedreich's ataxia is broader than previously recognised and includes cases with intact tendon reflexes.     

Clinical use of frataxin measurement in a patient with a novel deletion in the FXN gene

Friedreich ataxia (FRDA) is caused by a GAA expansion in the first intron of the FXN gene, which encodes frataxin. Four percent of patients harbor a point mutation on one allele and a GAA expansion on the other. We studied an Italian patient presenting with symptoms suggestive of FRDA, and carrying a single expanded 850 GAA allele. As a second diagnostic step, frataxin was measured in peripheral blood mononuclear cells, and proved to be in the pathological range (2.95 pg/μg total protein, 12.7 % of control levels). Subsequent sequencing revealed a novel deletion in exon 5a (c.572delC) which predicted a frameshift at codon 191 and a premature truncation of the protein at codon 194 (p.T191IfsX194). FXN/mRNA expression was reduced to 69.2 % of control levels. Clinical phenotype was atypical with absent dysarthria, and rapid disease progression. l-Buthionine-sulphoximine treatment of the proband’s lymphoblasts showed a severe phenotype as compared to classic FRDA.     

Limited somatic mosaicism for Friedreich's ataxia GAA triplet repeat expansions identified by small pool PCR in blood leukocytes

OBJECTIVES: Friedreich's ataxia (FRDA), the most common inherited ataxia, is associated with an unstable expansion of GAA repeats in the first intron of the frataxin gene on chromosome 9. We investigated the mosaicism of expanded alleles to elucidate the basis for genotype phenotype correlations. PATIENTS AND METHODS: We studied the instability of the GAA repeat in blood leukocytes from 45 individuals including 20 FRDA patients and 20 non-affected controls using small pool PCR combined with Southern blotting and hybridization. RESULTS: Expanded GAA repeats could be resolved into distinct alleles showing differences in length up to 1,000 triplets for an individual genome. We found a significant correlation between the size of the largest allele and the range of mosaicism. CONCLUSION: The somatic mosaicism for expanded repeats observed in FRDA patients rendered the precise measurement of allele sizes more difficult and may influence the results of studies correlating the clinical spectrum with the genotype. Following, a confidential prediction of the prognosis deduced from the repeat length is hardly possible for an individual FRDA patient.     

Very late-onset Friedreich ataxia despite large GAA triplet repeat expansions

BACKGROUND: Most patients with Friedreich ataxia (FRDA) have abnormal GAA triplet repeat expansions in both X25 genes. The size of the GAA expansion in the shorter of the 2 expanded alleles correlates significantly with parameters of clinical severity and is inversely related to the age at onset. OBJECTIVES: To describe the clinical and molecular genetic findings in a patient with very late-onset FRDA and to review the literature. PATIENT AND METHODS: A 58-year-old white woman with mild progressive gait disturbance of 15 years' duration whose examination revealed mild incoordination was analyzed for mutations in the X25 gene. A combination of long-range polymerase chain reaction and genomic Southern blot analyses were used to identify GAA expansions in intron 1 of the X25 gene. To uncover evidence of somatic variability in triplet repeat length, DNA isolated from several tissue samples was similarly analyzed. Single-strand conformational polymorphism analysis was used to screen for mutations spanning the entire coding sequence of frataxin and all intron-exon junctions of the X25 gene. RESULTS: DNA isolated from blood leukocytes revealed GAA triplet repeat expansions in both X25 genes, which were estimated to contain 835 and 1200 repeats. Similar expansions were detected in DNA isolated from lymphoblasts, fibroblasts, buccal cells, and sural nerve, with estimated mean (+/- SD) lengths of the shorter and longer expansions being 854 (+/-69) and 1283 (+/-72) triplets, respectively. A review of reported cases of late-onset Friedreich ataxia (25-39 years) and very late-onset Friedreich ataxia (> or =40 years) demonstrated that this is the first instance of a patient presenting with very late-onset FRDA despite carrying more than 800 GAA repeats in both expanded X25 alleles. CONCLUSIONS: This unique case of very late-onset FRDA highlights a limitation in our ability to accurately predict the phenotype in FRDA based solely on the size of the GAA expansion. Other genetic or environmental factors may significantly modify disease severity in FRDA.     

Molecular analysis of Friedreich's ataxia locus in the Indian population

OBJECTIVES: Friedreich's ataxia (FRDA) is an autosomal recessive neurodegenerative disorder caused by expansion of GAA repeats in the frataxin gene. We have carried out the first molecular analysis at the Friedreich's ataxia locus in the Indian population. MATERIALS AND METHODS: Three families clinically diagnosed for Friedreich's ataxia were analyzed for GAA expansion at the FRDA locus. The distribution of GAA repeats was also estimated in normal individuals of Indian origin. RESULTS: All patients clinically diagnosed for Friedreich's ataxia were found to be homozygous for GAA repeat expansion. The GAA repeat in the normal population show a bimodal distribution with 94% of alleles ranging from 7-16 repeats. CONCLUSION: Indian patients with expansion at the FRDA locus showed typical clinical features of Friedreich's ataxia. The low frequency of large normal alleles (6%) could indicate that the prevalence of this disease in the Indian population is likely to be low.     

Pathophysiogical and therapeutic progress in Friedreich ataxia

Friedreich ataxia (FRDA) is the most common hereditary autosomal recessive ataxia, but is also a multisystemic condition with frequent presence of cardiomyopathy or diabetes. It has been linked to expansion of a GAA-triplet repeat in the first intron of the FXN gene, leading to a reduced level of frataxin, a mitochondrial protein which, by controlling both iron entry and/or sulfide production, is essential to properly assemble and protect the Fe-S cluster during the initial stage of biogenesis. Several data emphasize the role of oxidative damage in FRDA, but better understanding of pathophysiological consequences of FXN mutations has led to develop animal models. Conditional knockout models recapitulate important features of the human disease but lack the genetic context, GAA repeat expansion-based knock-in and transgenic models carry a GAA repeat expansion but they only show a very mild phenotype. Cells derived from FRDA patients constitute the most relevant frataxin-deficient cell model as they carry the complete frataxin locus together with GAA repeat expansions and regulatory sequences. Induced pluripotent stem cell (iPSC)-derived neurons present a maturation delay and lower mitochondrial membrane potential, while cardiomyocytes exhibit progressive mitochondrial degeneration, with frequent dark mitochondria and proliferation/accumulation of normal mitochondria. Efforts in developing therapeutic strategies can be divided into three categories: iron chelators, antioxidants and/or stimulants of mitochondrial biogenesis, and frataxin level modifiers. A promising therapeutic strategy that is currently the subject of intense research is to directly target the heterochromatin state of the GAA repeat expansion with histone deacytelase inhibitors (HDACi) to restore frataxin levels.     

Atypical, perhaps under-recognized? An unusual phenotype of Friedreich ataxia

Friedreich ataxia (FRDA) is typically characterized by slowly progressive ataxia, depressed tendon reflexes, dysarthria, pyramidal signs, and loss of position and vibration sense with onset before 25 years. While several atypical forms of FRDA are recognized, profound vision deficit is rare. We describe here a 41-year-old man with profound vision deficit and episodic complete blindness associated with marked optic atrophy, spastic paraparesis, and sensory neuropathy without ataxia whose diagnostic evaluation revealed compound heterozygosity for two frataxin mutations, a 994 GAA repeat intronic expansion and c.389G > T (p.G130V) missense mutation. This case emphasizes that FRDA should be considered for individuals with significant vision deficit with optic atrophy and sensory neuropathy, even in the absence of ataxia. This case also raises the additional, related concern that prior studies may underestimate the frequency and varieties of variant forms of FRDA.     

Absence of aprataxin gene mutations in a Greek cohort with sporadic early onset ataxia and normal GAA triplets in frataxin gene

Phenotype of patients with the aprataxin gene mutation varies and according to previous studies, screening of aprataxin gene could be useful, once frataxin gene mutation is excluded in patients with normal GAA expansion in frataxin gene. In the present study, we sought to determine possible causative mutations in aprataxin gene (all exons and flanking intronic sequences) in 14 Greek patients with sporadic cerebellar ataxia all but one without GAA expansion in frataxin gene (1 patient was heterozygous). No detectable point mutation or deletion was found in the aprataxin gene of all the patients. Our results do not confirm the previous studies. This difference may be attributed to the different populations studied and possible different genetic background. It is still questionable whether the screening for aprataxin mutation in Greek patients’ Friedreich ataxia phenotype is of clinical importance; larger, multicenter studies are necessary to clarify this issue.     

Prolonged treatment with pimelic o-aminobenzamide HDAC inhibitors ameliorates the disease phenotype of a Friedreich ataxia mouse model

Friedreich ataxia (FRDA) is an inherited neurodegenerative disorder caused by GAA repeat expansion within the FXN gene, leading to epigenetic changes and heterochromatin-mediated gene silencing that result in a frataxin protein deficit. Histone deacetylase (HDAC) inhibitors, including pimelic o-aminobenzamide compounds 106, 109 and 136, have previously been shown to reverse FXN gene silencing in short-term studies of FRDA patient cells and a knock-in mouse model, but the functional consequences of such therapeutic intervention have thus far not been described. We have now investigated the long-term therapeutic effects of 106, 109 and 136 in our GAA repeat expansion mutation-containing YG8R FRDA mouse model. We show that there is no overt toxicity up to 5 months of treatment and there is amelioration of the FRDA-like disease phenotype. Thus, while the neurological deficits of this model are mild, 109 and 106 both produced an improvement of motor coordination, whereas 109 and 136 produced increased locomotor activity. All three compounds increased global histone H3 and H4 acetylation of brain tissue, but only 109 significantly increased acetylation of specific histone residues at the FXN locus. Effects on FXN mRNA expression in CNS tissues were modest, but 109 significantly increased frataxin protein expression in brain tissue. 109 also produced significant increases in brain aconitase enzyme activity, together with reduction of neuronal pathology of the dorsal root ganglia (DRG). Overall, these results support further assessment of HDAC inhibitors for treatment of Friedreich ataxia.     

Mapping of the second Friedreich's ataxia (FRDA2) locus to chromosome 9p23-p11: evidence for further locus heterogeneity

Friedreich's ataxia (FRDA), the most-common form of autosomal recessive ataxia, is inherited in most cases by a large expansion of a GAA triplet repeat in the first intron of the frataxin (X25) gene. Genetic heterogeneity in FRDA has been previously reported in typical FRDA families that do not link to the FRDA locus on chromosome 9q13. We report localization of a second FRDA locus (FRDA2) to chromosome 9p23-9p11, and we provide evidence for further genetic heterogeneity of the disease, in a family with the classic FRDA phenotype.     

Classical Friedreich's ataxia and its genotype.

Fourteen patients with classical features of Friedreich's ataxia (FRDA) were examined. The clinical diagnosis of FRDA was afterwards confirmed in all patients by the appropriate DNA investigation which showed markedly increased amounts of GAA repeats on both alleles of the frataxin gene. None of our patients presented with atypical features such as late-onset FRDA, FRDA with retained deep tendon reflexes or with a very slow course. Five of them are not yet confined to a wheelchair. But for 1 patient who died at age 36 years and had the largest number of GAA repeats on both alleles, there was no significant correlation between number of repeats in the shortest allele, age at onset, age at wheelchair dependence, duration of the disease and main clinical signs. All patients but 3 had between 500 and 1,050 GAA repeats. The 3 patients with, respectively, 400, 450 and 500 repeats on the shortest allele had a clinical course comparable to the other patients. Even in the case of variations in the number of repeats in the same sibship, there were only modest differences between the siblings concerning age at onset of the disease, symptoms and signs and age at wheelchair dependence. There were no qualitative differences in the main clinical features and laboratory investigations in the full-blown phase of the disorder. Molecular biology has become a major element in the diagnosis of FRDA. DNA testing for FRDA should be applied to every case of idiopathic autosomal recessive or sporadic ataxia. However, the clinical features of FRDA remain fully characteristic in many patients and keep their diagnostic value.     

Friedreich's ataxia: a clinical and genetic analysis

We report a patient with genetically confirmed Friedreich's ataxia (FRDA) who developed a previously unreported feature of a mixed sleep apnea. Initial mutation analysis, by PCR, of the parental frataxin alleles showed an apparent de novo mutation in the maternal germline. Further investigation using Southern blot analysis showed that the mother did carry an expanded mutant frataxin allele. Based upon published data, FRDA resulting from at least one allelic spontaneous expansion mutation is rare with a frequency of less than 1/1,000,000. The presence of such a mutation should be confirmed by Southern blot analysis. Our patient expands the neurological features of FRDA to include sleep apnea. The genetic analysis of the family demonstrates the importance of Southern blot analysis for accurate genotyping which, in turn, has implications for genetic counseling.     

Atypical Friedreich ataxia in patients with FXN p.R165P point mutation or comorbid hemochromatosis

BackgroundCompound heterozygosity for a trinucleotide repeat expansion and a point mutation in the FXN gene is a rare cause of Friedreich ataxia (FRDA).MethodsWe identified three Swedish FRDA patients with an FXN p.R165P missense mutation and compared their clinical features with six homozygote trinucleotide repeat expansion carriers. Patients were assessed clinically. Trinucleotide expansion length was determined and lymphocyte frataxin levels measured.Resultsp.R165P mutation carriers became wheelchair bound early, but had retained reflexes, better arm function, milder dysarthria, and were more independent in activities of daily living. One p.R165P mutation carrier developed psychosis. Frataxin levels were higher than in homozygous trinucleotide expansion patients. One patient with homozygous trinucleotide repeat expansions and comorbid hemochromatosis had more severe FRDA symptoms than his sibling without hemochromatosis.Conclusionp.R165P patients progress to a less disabling disease state than typical FRDA. Comorbid hemochromatosis may worsen FRDA symptoms through additive effects on iron metabolism.