Zoe Allen MRCPath Part 1 Course 04.09.09

1. Describe how identification of triplet repeat mutations has improved our understanding of the genetics and clinical expression of some of the inherited neurological disorders Zoe Allen MRCPath Part 1 Course 04.09.09

2. Identification of triplet repeat disorders 1918 – Clinicans suspected anticipation in myotonic dystrophy 1968 – Dr Kennedy clinically defined a disorder known as Kennedy’s disease (spinal and bulbar muscular atrophy) 1991 – Genetic cause of SBMA mapped, led to breakthrough for other triplet repeat diseases to be identified 1991 – At same time, independent group found FraxA CGG repeat 1992 – Myotonic dystrophy CTG repeat identified Since then, over 25 different triplet repeat disorders have been identified

3. Lessons from 1991 • Novel mutation characteristics – mutations are ‘dynamic’ • Analysis of families segregating with FraxA showed 3 distinct classes of alleles • Normal size range • Disease size range • “Intermediate” size range • Repeats show tendency to expand through generations • Sex of person transmitting allele determines whether it will expand • Triplet repeats can occur in both coding and non-coding regions • Class 1 – repeats outside coding sequences • Class 2 – repeats inside coding sequences

6. Polyglutamine expansion diseases • Chronic progressive neurodegenerative diseases • Unstable expansions of CAG polyglutamine tracts in coding region • Gain of function mutation – toxic to neurons How do we know gain of function? Loss of function of AR gene = androgen insensitivity or testicular feminization syndrome. No weakness or motor neuron degeneration Polyglutamine tract in AR does not affect protein’s ability to transactivate further genes (e.g. CREB binding protein) HD – individuals with deletion of huntingtin gene do not show HD or ataxia Transgenic mice and flies expressing expanded genes (huntingtin, ataxin-1 etc) do show altered phenotypes corresponding to human diseases PolyQ expansion must cause gain of function making it toxic to neurons

7. Gain of function toxicity • 2 hypotheses • PolyQ tract is substrate for transglutaminase enzyme • Enzyme cross-links polyQ tract to other proteins containing lysyl groups • = formation of aggregates • Two antiparallel PolyQ repeat strands can be linked together by hydrogen bonds • Undergo multimerization and aggregation by polar zipper formation • Aggregation into inclusion bodies is neither necessary nor sufficient for cellular dysfunction • ? Aggregates stop cells from interacting with other cellular factors etc and doing their ‘normal’ job

8. Huntington Disease • Movement disorder – chorea, rigidity • Cognitive dysfunction - dementia • Psychiatric disturbance • CAG repeat expansion in exon 1 of the huntingtin gene (chromosome 4) • Normal <36 repeats • Incomplete penetrance 36-39 repeats • Affected >39 repeats • Autosomal dominant • Incidence 1/10,000 • CAG repeat length inversely correlates with age of onset

9. Juvenile HD • Onset before age 20 • Accounts for less than 10% all HD cases • Usually transmitted from an affected father • Large CAG repeats (>60) • Shows rigidity and seizures but no chorea • Commonly demonstrates anticipation only on paternal transmission • However in 2005 a report of maternal transmission was noted • Mother had symptoms age 18 • Daughter had symptoms age 3, died age 7

10. Pathogenic pathway of mutant htt

11. Friedreich Ataxia • Homozygous GAA repeat in intron 1 of FRDA gene =98% • Point mutation + GAA repeat = 2% • Autosomal recessive inheritance • Most common of inherited ataxia syndromes • Carrier rate – 1/60  1/110 depending on study • Normal = 5-33 repeats • Premutation = 34-65 • Affected = 66 – 1000+ pure GAA repeats • Alleles longer than 27 repeats can be interrupted by (GAGGAA)n seq – stabilize PM alleles and prevent expansion

12. Friedreich Ataxia Symptoms • Progressive gait and limb ataxia • Absent lower limb reflexes • Loss of sensory perception • Cardiomyopathy – commenst cause of death • Diabetes mellitus • Scoliosis • Foot deformity – pes cavus Transmission • Maternal transmission can give larger or smaller alleles in offspring • Paternal transmission – GAA repeat size nearly always decreases • Size of triplet repeat influences direction of instability • Smaller alleles prone to increase • Larger alleles tend to contract Anticipation • AR so unlikely to appear in 2 generations

13. Loss of function • Deficiency of mitochondrial protein frataxin • Directly proportional to length of expanded GAA • Matches clinical severity • GAA repeat – transcriptional silencing of FRDA gene • Heterochromatin formation in sequence flanking GAA repeat • GAA repeat adopts abnormal DNA structure that interferes with transcription • Missense mutations – frataxin highly conserved • Frataxin required for formation of iron-sulphur clusters and synthesis of enzymes in respiratory chain complexes • Deficiency  • Misdistribution of mitochondrial iron • Reduced mitochondrial function and increased oxidative damage • Individuals show deficient ATP production

14. FXTAS • Associated with premutation of FRAXA locus in males • CGG repeat in 5’UTR of FMR1 gene (X chromosome) • 55-200 repeats = PM • Progressive ataxia and intention tremor • Neuropathological changes • Degeneration of cerebellum • Ubiquitin positive intranuclear inclusions in neurons and glia • RNA gain of function disorder • PM individuals show increased expression of FMR1 mRNA • FMR1 protein levels reduced despite increased mRNA • Immunostaining of FXTAS brain tissue shows intranuclear inclusions • More CGG repeats = more inclusions

15. Huntington Disease Autosomal dominant Toxic gain of function mutation CAG in coding region Expansion paternally biased Larger repeat = earlier age of onset Anticipation observed Intranuclear inclusions Friedreich Ataxia Autosomal recessive Loss of function GAA in non-coding region Expansion maternally biased Larger repeat = earlier age of onset No anticipation seen as recessive Comparison FXTAS X-linked dominant RNA gain of function GCC in non-coding region Expansion maternally biased Premutation alleles only No anticipation Intranuclear inclusions

16. Lessons learnt Dynamic mutations Several different mechanisms of pathogenicity Anticipation Parental transmission bias common Alleles can also contract (FRDA) Conclusions