Sarah Waller

1. Discuss the genetic basis of Friedreich’s ataxia. What is the biological function of frataxin? The genetic analysis of several loci is often requested in the investigation of patients presenting with ‘ataxia’. What are these? Why is requesting for multiple loci sometimes appropriate given that some of these conditions follow different patterns of inheritance? Sarah Waller

2. Discuss the genetic basis of Friedreich’s ataxia • Most common inherited ataxia in Europe, the Middle East, South Asia and North Africa with a carrier frequency of 1:60 – 1:00. Not documented in SE Asia, sub-Saharan Africa or among Native Americans. • Recessive disease characterised by childhood onset of slow progressive ataxia, dysarthria, muscle weakness, spasticity in the lower limbs, scoliosis, bladder dysfunction, absent lower limb reflexes, and loss of position and vibration sense. Approximately two-thirds of individuals with FRDA have cardiomyopathy; up to 30% have diabetes mellitus; and approximately 25% have an "atypical" presentation with later onset or retained tendon reflexes. • >96% of patients homozygous for expansion of GAA repeat in intron 1 of the frataxin gene (FXN); this expansion inhibits transcriptional elongation thereby reducing levels of frataxin protein. Repeat size ranges: • Normal: 5-33 • Premutation: 34-65 (uninterrupted); may expand in subsequent generations to fully penetrant alleles; alleles longer than 27 GAA triplet repeats are often interrupted by a (GAGGAA)n sequence, which is believed to stabilise the repeat. • Borderline alleles: 44-66 (uninterrupted): can be associated with late onset FA (LOFA) or very late onset FA (VLOFA) – due to somatic instability expansions in clinically relevant tissues may be greater than seen in blood samples • Full penetrance: 66-1700, usually 600-1200. Alleles with 100-300 repeats may have non-GAA interruptions and are associated with LOFA or VLOFA

3. Discuss the genetic basis of Friedreich’s ataxia • 2-4% of patients are heterozygous for the GAA repeat and for loss of function mutations in the FXN gene on the other allele. • Mutations may be non-sense, frameshift or splicing, resulting in termination of translation; missense involving the highly conserved carboxy-terminal domain; exon deletions. • Most individuals with compound heterozygote mutations for a GAA expansion and a point mutation are indistinguishable from those with homozygous expansions, apart from 2 exceptions: p.Gly130Val and p.Asp122Tyr mutations are associated with milder disease with later onset, slow progression, absence of dysarthia and retained reflexes. • Characteristic of anticipation seen with other triplet repeat disorders is not observed because Friedreich’s ataxia is inherited in an autosomal recessive manner and therefore the disease is rarely observed in more than one generation.

4. Genotype-phenotype correlations • The expanded GAA repeat shows progressive somatic instability, particularly in the dorsal root ganglia, the primary site of pathology of FRDA. This instability may explain the variable pathogenicity of borderline alleles. • The age of onset, presence of leg muscle weakness/wasting, duration until wheelchair use, and prevalence of cardiomyopathy, pes cavus, and scoliosis have all been shown to correlate with GAA expansion size. The size of the shorter of the two expanded repeat alleles shows better correlation, accounting for approximately 50% of the variation in age of onset. • Individuals with LOFA (age of onset >25 years) frequently exhibit fewer than 500 GAA repeats in at least one of the expanded alleles. Individuals with VLOFA (age of onset >40) years usually have fewer than 300 GAA repeats in at least one of the expanded alleles. • Cardiomyopathy is more frequently seen with longer GAA repeat alleles. • Diabetes mellitus or abnormal glucose tolerance does not show a clear-cut correlation with the size of the GAA expansion.

5. What is the biological function of frataxin? • Frataxin is a highly conserved 210 aa protein that is associated with the inner mitochondrial membrane and plays a role in mitochondrial iron metabolism. • Ubiquitously expressed but deficiency leads to selective loss of dorsal root ganglion neurons, myocytes & pancreatic beta cells – explains symptoms of ataxia, cardiomyopathy and diabetes mellitus in FRDA patients. • How does frataxin promote survival of these cell types? Tissues form FRDA patients show iron deposits and generalised decrease in activity of proteins that contain iron-sulphur complexes. Hypothesised to: • provide iron for heme synthesis • play pivotal role in iron-sulphur cluster formation • have anti-oxidant role by detoxifying surplus iron

6. What is the biological function of frataxin? • Iron storage • Protection against oxidative damage by limiting Fe-catalysed generation of reactive oxygen species • Storing Fe in readily available form • Iron donor in heme synthesis • Interacts with ferrochelatase, the enzyme that catalyses the final step of heme biosynthesis by inserting the ferrous ion into porphyrin

7. What is the biological function of frataxin? • Iron-sulphur clusters (ISCs). ISCs are highly conserved complexes of iron and sulphur atoms that serve as prosthetic groups for several enzymes with different functions, including energy metabolism & iron metabolism. Respiratory chain complexes I – III and aconitase have iron-sulphur cluster cores. FRDA patients and mouse models show deficiencies in ISC proteins – decreased production of cellular energy may lead to free radical formation. • Frataxin has been shown to be involved in ISC assembly through interactions with the 2 central components of this machinery – the cysteine desulphurase Nfs1 and the scaffold protein Isu. In vitro studies with the bacterial orthologue CyaY have shown that frataxin may have a role as a gatekeeper of the cluster assembly machinery.

9. The genetic analysis of several loci is often requested in the investigation of patients presenting with ‘ataxia’. What are these? • Ataxias are hereditary disorders consisting of degeneration of the cerebellum and/or spine. Cases often present with overlapping sensory and cerebellar ataxia, although one is often more evident than the other. • Autosomal dominantly inherited ataxias include the spino-cerebellar ataxias (SCAs) & dentatorubropallidoluysian atrophy (DRPLA). • Autosomal recessively inherited childhood-onset ataxias include Ataxia with oculomotor apraxia types 1 & 2 (AOA1 & AOA2), spinocerebellar atrophy with axonal neuropathy (SCAN1), ataxia with Vitamin E deficiency and ataxia telengiectasia. • Fragile X-associated tremor/ataxia syndrome (FXTAS) is a late-onset X-linked ataxic condition that may present in a similar fashion to VLOFA.

10. Why is requesting for multiple loci sometimes appropriate given that some of these conditions follow different patterns of inheritance? • Since FRDA is a childhood onset disorder it is imperative to elucidate the cause of the symptoms as quickly as possible in order to provide reproductive choice to the parents. • Somatic instability of the triplet repeats in some ataxic disorders gives rise to some variation in the phenotype. • Patterns of inheritance of severe recessive childhood-onset disorders are not always clear. • Characteristic of anticipation of dominantly inherited ataxias, whereby the triplet expansion and consequent symptoms increase with succeeding generations, may result in children displaying symptoms before parents or even grandparents.

11. Useful references • Diseases of unstable repeat expansion: mechanisms and common principles. Gatchel & Zoghbi (2005), Nature Reviews Genetics 6:743-755 • The pathogenesis of Friedreich ataxia and the structure and function of frataxin. Pandolfo & Pastore (2009) J Neurol 256 [Suppl 1]:9–17 Keywords: Friedreich’s ataxia; frataxin, triplet repeat disorder; Iron-sulphur complex; iron metabolism; ataxia;