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Discuss the genetic basis of Friedreich’s ataxia. What is the biological function of frataxin?

Discuss the genetic basis of Friedreich’s ataxia. What is the biological function of frataxin?. Ana Terron Kwiatkowski MRCPath I course, London 17/9/2010. Friedreich’s ataxia (FRDA). Neurodegenerative disease Most common of inherited ataxias Incidence 1:50,000

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Discuss the genetic basis of Friedreich’s ataxia. What is the biological function of frataxin?

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  1. Discuss the genetic basis of Friedreich’s ataxia. What is the biological function of frataxin? Ana Terron Kwiatkowski MRCPath I course, London 17/9/2010

  2. Friedreich’s ataxia (FRDA) • Neurodegenerative disease • Most common of inherited ataxias Incidence 1:50,000 Carrier frequency 1:60-1:90 in Caucasians • Inherited as autosomal recessive trait • Clinical features: - Onset < 20 years - Progressive gait and limb ataxia - Absence of lower limb reflexes - Dysarthria - Muscle weakness - Scoliosis - Diabetes mellitus - Cardiomyopathy (common cause of death) - Slow progression of neurological symptoms

  3. Genetic basis of FRDA • FRDA gene mapped to chromosome 9q13-21.1 (1988) • X25 Gene cloned in 1996: 7 exons (1-5a, 5b,6) • Commonest transcript from 1-5a: 210 amino acid protein = frataxin (FXN) • Alternative splicing 5b: 171 aa protein • 141-167 aa highly conserved (C.elegans, S. cerevisae) • FXN expression: heart>>> liver, skeletal muscle and pancreas spinal cord>> cerebellum >>> cerebral cortex

  4. Genetic basis of FRDA (2) • Most common mutation: GAA expansion in FXN intron 1 Normal alleles 6-34 repeats Expanded alleles 67-1700 • 96% patients homozygous • 4% compound heterozygous for GAA expansion and point mutation: slower disease progression • 17 point mutations described (2000): ms, ns, fs, exon deletion • Mutations leading to absence of frataxin – severe phenotype • FXN mRNA very low or undetectable in patients • Due to inhibition of transcription (elongation, histone hypoacetylation and methylation) • GAA expansion forms unusual DNA structure (triplexes self-association = ‘sticky DNA’) which interferes with transcription

  5. Intergenerational instability, premutations and origin of mutations • GAA repeat unstable upon transmission • Maternal: larger or smaller allele in offspring • Paternal: GAA repeat size usually decreases • Intermediate or premutation alleles prone to very large expansions in one generation- incidence of this unknown • Spontaneous expansion mutation is rare: estimated 1/1 million • Two stage expansion: meiotic and mitotic Instability occurs pre- and post-zygotically (Heterozygotes GAA repeat size in sperm < lymphocytes) • Bimodal distribution of normal size alleles: 6-12 + 14-34 repeats Expanded alleles arise from large normal alleles (common haplotype)

  6. Genotype-phenotype correlation • GAA repeat size determine Age of onset: 50% variation - size of smaller GAA allele Severity: loss-of-function disease  severity correlates with remaining activity (smaller GAA allele) Clinical features Lower repeat size: LOFA Higher repeat size: diabetes, cardiomyopathy • Repeat sizes cannot be used accurately to predict prognosis

  7. Molecular Diagnosis • Available molecular tests to detect GAA expansions (PCR, TP-PCR, Southern blotting) • Possible to screen for point mutations (4% compound heterozygotes) • Carrier testing available for relatives of FRDA and their partners • Risk for offspring – Genetic counselling • Prenatal diagnosis can be offered • Differential diagnosis: Ataxia with vitamine E deficiency (AVAD): mutations in α-tocoferol transfer protein (chrom 8)

  8. Biological function of frataxin • Human frataxin: nuclear encoded mitochondrial protein • FRDA results from mitochondrial dysfunction: mitochondrial iron accumulation  toxic free radicals (Fenton chemistry: Fe2+ + H2O2  Fe3+ + 2 OH- )  cellular damage  death • FRDA patients: - Deficient activity of iron-sulphur containing enzymes - Iron deposits in myocardium • Frataxin involved in iron efflux from mitochondria • FRDA affected tissues (neurones, heart, pancreas) - High level of expression on frataxin - Higher sensitivity to oxidative stress

  9. Consequences of frataxin deficiency • Gene expression studies in FRDA patients: - 48 differentially expressed genes associated with oxidative stress - biological evidence of mtDNA and nDNA damage  oxidative phosphorylation and ATP (impair mt function)  protein biosynthesis, signalling, transcription, DNA replication/repair, apoptosis and ubiquitination (protein degradation pathway- altered in neurological disorders) and altered immune response

  10. Therapeutic approaches • Iron chelators • Antioxidant therapy • Gene therapy • Small molecules to activate frataxin gene expression(Gottesfeld 2007; Pharmacol Ther 116:236-48; Grant et al. 2006; FEBS Lett 580: 5399-5405) • Histone deacetylase inhibitors to reverse frataxin silencing (Herman et al. 2006; Nat Chem Biol 2:551-8)

  11. The genetic analysis of several loci is often requested in the investigation of patients presenting with ‘ataxia’. What are these? Why requesting for multiple loci is sometimes appropriate given that some of these conditions follow different patterns of inheritance?

  12. Autosomal dominant cerebellar ataxias • Neurodegenerative inherited disorders • Clinical and molecular heterogeneous group - Include about 28 SCA, dentatorubral-pallidoluysian atrophy (DRPLA) and other episodic and spastic ataxias - Various degrees of cerebellar and brainstem degeneration/dysfunction - Progressive cerebellar ataxia and neurological signs - Age onset : 3rd-4th decade generally • Prevalence < 10/100,000 Regional founder affects

  13. Genetic basis of ADCA • Most ADCA caused by CAG (polyglutamine) expansion in different genes - Clinical symptoms when CAG > threshold - CAG repeat size: age of onset (50-80% effect)  severity and disease progression - Repeat sequence unstable upon transmission - Anticipation - Genes expressed ubiquitously - Disease results from ‘gain-of-function’: CAG expansion beyond threshold → altered conformation of polyglutamine tracts → insoluble intranuclear aggregates - Pathological protein accumulates in ubiquitinated neuronal intranuclear inclusions in several affected and non-affected brain structures • Molecular analysis useful to distinguish disorders which are clinically similar - predictive, prenatal diagnosis can be offered

  14. References • Delatycki et al 2000; J Med Genet 37:1-8. Friedreich ataxia: an overview. • Filla et al. 1996; Am J Hum Genet 59:554-60. • Haugen et al. 2010; PLoS Genetics 6 (1). Altered gene expression and DNA damage in peripheral blood cells from Friedreich’s ataxia patients: cellular model of pathology. • Stevanin et al. 2000; Eur J Hum Genet 8:4-18. • Bird. Gene Reviews (2009). Hereditary Ataxia Overview

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