Genotype and phenotype analysis of Friedreich's ataxia compound heterozygous patients

Friedreich's ataxia is caused by mutations in the FRDA gene that encodes frataxin, a nuclear-encoded mitochondrial protein. Most patients are homozygous for the expansion of a GAA triplet repeat within the FRDA gene, but a few patients show compound heterozygosity for a point mutation and the GAA-repeat expansion. We analyzed DNA samples from a cohort of 241 patients with autosomal recessive or isolated spinocerebellar ataxia for the GAA triplet expansion. Patients heterozygous for the GAA expansion were screened for point mutations within the FRDA coding region. Molecular analyses included the single-strand conformation polymorphism analysis, direct sequencing, and linkage analysis with FRDA locus flanking markers. Seven compound heterozygous patients were identified. In four patients, a point mutation that predicts a truncated frataxin was detected. Three of them associated classic early-onset Friedreich's ataxia with an expanded GAA allele greater than 800 repeats. The other patient associated late-onset disease at the age of 29 years with a 350-GAA repeat expansion. In two patients manifesting the classical phenotype, no changes were observed by single-strand conformation polymorphism (SSCP) analysis. Linkage analysis in a family with two children affected by an ataxic syndrome, one of them showing heterozygosity for the GAA expansion, confirmed no linkage to the FRDA locus. Most point mutations in compound heterozygous Friedreich's ataxia patients are null mutations. In the present patients, clinical phenotype seems to be related to the GAA repeat number in the expanded allele. Complete molecular definition in these patients is required for clinical diagnosis and genetic counseling.     

G130V, a common FRDA point mutation, appears to have arisen from a common founder

Friedreich ataxia (FRDA) is the most common inherited ataxia. About 98% of mutant alleles have an expansion of a GAA trinucleotide repeat in intron 1 of the affected gene, FRDA. The other 2% are point mutations. Of the 17 point mutations so far described, three appear to be more common. One of these is the G130V mutation in exon 4 of FRDA. G130V, when present with an expanded GAA repeat on the other allele, is associated with an atypical FRDA phenotype. Haplotype analysis was undertaken on the four families who have been described with this mutation. The results suggest a common founder for this mutation. Although marked differences in extragenic marker haplotypes were seen in one family, similar intragenic haplotyping suggests the same mutation founder for this family with the differences explicable by two recombination events.     

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.     

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.     

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.     

Molecular analysis of the androgen receptor gene in 52 patients with complete or partial androgen insensitivity syndrome: a collaborative study.

In patients with androgen insensitivity syndrome (AIS), RFLP study of the androgen receptor gene made it possible to analyze whether deletions or mutations could be responsible for abnormalities in androgen responsiveness. We studied RFLPs of DNA from 25 46,XY patients with partial AIS (PAIS), defined as a concentration of androgen receptor in genital-skin fibroblasts less than 340 fmol/mg DNA, and DNA from 27 46,XY patients with complete AIS (CAIS) with no detectable androgen receptor site. DNA samples were digested with BamHI, EcoRI, HindIII and TaqI restriction enzymes and hybridized with three cDNA probes covering the three domains of the androgen receptor. When we had the maternal and an unaffected brother's DNA, we analyzed the two androgen receptor gene polymorphisms described, the HindIII and the exon 1 CAG repeat polymorphisms, in order to distinguish the two maternal X chromosomes, and to detect carriers of AIS. We did not find any large deletion among the 52 patients. We observed a heterozygous mother in 3 of 14 families studied with the HindIII polymorphism, and in 12 of 25 families using the exon 1 CAG repeat polymorphism. This study suggests that in AIS, abnormalities in androgen receptor response could be related to point mutations or microdeletions rather than to gross structural alterations of the androgen receptor gene. Furthermore, unless the point mutation has been described, exon 1 and HindIII polymorphism studies would enable the identification of carriers in 50% of families, and the prenatal diagnosis of AIS.     

Nonsense mutations of the von Willebrand factor gene in patients with von Willebrand disease type III and type I.

von Willebrand disease (vWD) is the most common inherited bleeding disorder in humans. The disease is caused by qualitative and quantitative abnormalities of the von Willebrand factor (vWF). Genomic DNA from 25 patients with vWD type III, the most severe form of the disease, was studied using PCR followed by restriction-enzyme analysis and direct sequencing of the products. Nonsense mutations (CGA----TGA) were detected in exons 28, 32, and 45 by screening of all the 11 CGA arginine codons of the vWF gene. Two patients were found to be homozygous and five heterozygous for the mutation. Both parents and some of the relatives of the homozygous patients carry the mutation. These are the first reported examples of homozygous point mutations associated with the severe form of vWD. In the three heterozygous probands, one of the parents carried the mutation and had vWD type I. Family studies including parents and family members with or without vWD type I indicated that these three heterozygous patients are likely to be compound heterozygous. Twenty-one individuals from these seven families with vWD type I were found to be heterozygous for the mutation.     

Recurrence of the T666M calcium channel CACNA1A gene mutation in familial hemiplegic migraine with progressive cerebellar ataxia.

Familial hemiplegic migraine (HM) is an autosomal dominant migraine with aura. In 20% of HM families, HM is associated with a mild permanent cerebellar ataxia (PCA). The CACNA1A gene encoding the alpha1A subunit of P/Q-type voltage-gated calcium channels is involved in 50% of unselected HM families and in all families with HM/PCA. Four CACNA1A missense mutations have been identified in HM: two in pure HM and two in HM/PCA. Different CACNA1A mutations have been identified in other autosomal dominant conditions: mutations leading to a truncated protein in episodic ataxia type 2 (EA2), small expansions of a CAG trinucleotide in spinocerebellar ataxia type 6 and also in three families with EA2 features, and, finally, a missense mutation in a single family suffering from episodic ataxia and severe progressive PCA. We screened 16 families and 3 nonfamilial case patients affected by HM/PCA for specific CACNA1A mutations and found nine families and one nonfamilial case with the same T666M mutation, one new mutation (D715E) in one family, and no CAG repeat expansion. Both T666M and D715E substitutions were absent in 12 probands belonging to pure HM families whose disease appears to be linked to CACNA1A. Finally, haplotyping with neighboring markers suggested that T666M arose through recurrent mutational events. These data could indicate that the PCA observed in 20% of HM families results from specific pathophysiologic mechanisms.     

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.     

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     

Friedreich's ataxia variants I154F and W155R diminish frataxin-based activation of the iron-sulfur cluster assembly complex

Friedreich's ataxia (FRDA) is a progressive neurodegenerative disease that has been linked to defects in the protein frataxin (Fxn). Most FRDA patients have a GAA expansion in the first intron of their Fxn gene that decreases protein expression. Some FRDA patients have a GAA expansion on one allele and a missense mutation on the other allele. Few functional details are known for the ～15 different missense mutations identified in FRDA patients. Here in vitro evidence is presented that indicates the FRDA I154F and W155R variants bind more weakly to the complex of Nfs1, Isd11, and Isu2 and thereby are defective in forming the four-component SDUF complex that constitutes the core of the Fe-S cluster assembly machine. The binding affinities follow the trend Fxn ～ I154F > W155F > W155A ～ W155R. The Fxn variants also have diminished ability to function as part of the SDUF complex to stimulate the cysteine desulfurase reaction and facilitate Fe-S cluster assembly. Four crystal structures, including the first for a FRDA variant, reveal specific rearrangements associated with the loss of function and lead to a model for Fxn-based activation of the Fe-S cluster assembly complex. Importantly, the weaker binding and lower activity for FRDA variants correlate with the severity of disease progression. Together, these results suggest that Fxn facilitates sulfur transfer from Nfs1 to Isu2 and that these in vitro assays are sensitive and appropriate for deciphering functional defects and mechanistic details for human Fe-S cluster biosynthesis.     

Marked Phenotypic Heterogeneity Associated with Expansion of a CAG Repeat Sequence at the Spinocerebellar Ataxia 3/Machado-Joseph Disease Locus

The spinocerebellar ataxia 3 locus (SCA3) for type I autosomal dominant cerebellar ataxia (ADCA type I), a clinically and genetically heterogeneous group of neuro-degenerative disorders, has been mapped to chromosome 14q32.1. ADCA type I patients from families segregating SCA3 share clinical features in common with those with Machado-Joseph disease (MJD), the gene of which maps to the same region. We show here that the disease gene segregating in each of three French ADCA type I kindreds and in a French family with neuropatho-logical findings suggesting the ataxochoreic form of dentatorubropallidoluysian atrophy carries an expanded CAG repeat sequence located at the same locus as that for MJD. Analysis of the mutation in these families shows a strong negative correlation between size of the expanded CAG repeat and age at onset of clinical disease. Instability of the expanded triplet repeat was not found to be affected by sex of the parent transmitting the mutation. Evidence was found for somatic and gonadal mosaicism for alleles carrying expanded trinucleotide repeats.     

The First Cellular Models Based on Frataxin Missense Mutations That Reproduce Spontaneously the Defects Associated with Friedreich Ataxia

Background

Friedreich ataxia (FRDA), the most common form of recessive ataxia, is due to reduced levels of frataxin, a highly conserved mitochondrial iron-chaperone involved in iron-sulfur cluster (ISC) biogenesis. Most patients are homozygous for a (GAA)_n expansion within the first intron of the frataxin gene. A few patients, either with typical or atypical clinical presentation, are compound heterozygous for the GAA expansion and a micromutation.

Methodology

We have developed a new strategy to generate murine cellular models for FRDA: cell lines carrying a frataxin conditional allele were used in combination with an EGFP-Cre recombinase to create murine cellular models depleted for endogenous frataxin and expressing missense-mutated human frataxin. We showed that complete absence of murine frataxin in fibroblasts inhibits cell division and leads to cell death. This lethal phenotype was rescued through transgenic expression of human wild type as well as mutant (hFXN^G130V and hFXN^I154F) frataxin. Interestingly, cells expressing the mutated frataxin presented a FRDA-like biochemical phenotype. Though both mutations affected mitochondrial ISC enzymes activities and mitochondria ultrastructure, the hFXN^I154F mutant presented a more severe phenotype with affected cytosolic and nuclear ISC enzyme activities, mitochondrial iron accumulation and an increased sensitivity to oxidative stress. The differential phenotype correlates with disease severity observed in FRDA patients.

Conclusions

These new cellular models, which are the first to spontaneously reproduce all the biochemical phenotypes associated with FRDA, are important tools to gain new insights into the in vivo consequences of pathological missense mutations as well as for large-scale pharmacological screening aimed at compensating frataxin deficiency.     

A common point mutation in the tyrosine hydroxylase gene in autosomal recessive L-DOPA-responsive dystonia in the Dutch population

This report concerns one new mutation in the tyrosine hydroxylase (TH) gene in three patients originating from three unrelated Dutch families with autosomal recessive L-DOPA-responsive dystonia (DRD). In this study, all exons of the TH gene were amplified by the polymerase chain reaction and subjected to analyses by single-strand conformation polymorphism. An aberrant migration pattern was observed for exon 6 of the TH gene in all patients. Direct sequencing of the coding region of exon 6 revealed the presence of one novel missense mutation. An a698g transition resulted in the substitution of the evolutionary conserved arginine 233 by a histidine (R233H). All patients were homozygous for the mutation. This new mutation in the TH gene was confirmed by restriction enzyme analysis with the restriction enzyme HhaI. Thus, a high proportion of defective TH alleles may be R233H in The Netherlands. Received: 25 July 1997 / Accepted: 10 February 1998