FRCPath Part 1 Self Help Session

1. FRCPath Part 1 Self Help Session Trinucleotide Repeat Disorders Helene Schlecht

2. Various pathogenic mechanisms underlie trinucleotide repeat diseases. With examples discuss how the following mechanisms can cause trinucleotide repeat disease: • Loss of function • RNA gain of function • Protein gain of function

3. Introduction • Dynamic trinucleotide mutations are short tandem repeat polymorphisms that above a certain size become unstable. • Mechanism of repeat expansion. • Unclear - Related to parent of origin and timing of expansion during development. • Possible models: loop formation during base excision repair/ nucleotide excision repair; Slippage model; Replication involving lagging strand. McMurray 2010

4. Introduction • Three pathogenic mechanisms underlying trinucleotide repeat disorders: • Loss of function • RNA gain of function • Protein gain of function

5. Loss of function • Features – no protein function, other types of mutations have same effect; non coding. • Examples FRAXA, FRAXE and Friedreich ataxia (FRDA)

6. Loss of function • FRAXA • Fragile X syndrome one of most common forms of inherited mental retardation. • X-linked dominant with reduced penetrance (80% males, 30% females). • Phenotype mild to severe mental retardation, mildly abnormal facial features. • Associated with fragile site A – Xq27.3 • CGG repeat in 5’UTR of FMR1 gene • AGG interuptions • Normal range 7-~60 • Pre mutation ~60-200 – unmethylated, but unstable. • Full mutations >200 repeats – abnormally hypermethylated.

7. Loss of function • FRAXA – cont. • The expansion causes the CpG island within the promoter region as well as the repeat to become methylated and histone acetylation decreases – leads to transcriptional silencing. • FMR1 gene is widely expressed in human tissues. • FMR1 protein has domains that suggest it’s an RNA-binding protein, which is critical for its function. • Highly expressed in brain neurons and regulates mRNA translation in dendrites, thereby modulates synaptic function. So reduced FMR1 causes mental retardation.

8. Loss of function Model for FMRP function: A, FMRP dimerizes in cytoplasm, enter nucleus and assembles into mRNP. Transports mRNA into cytoplasm. B, In fragile X patients, get abnormal regulation, localisation or abundance of mRNA.

9. Loss of function • FRAXE • CCG repeat in 5’UTR of FMR2 gene • Full mutations >200 repeats • Similar mechanism to FRAXA • FMR2 protein is thought to act as a transcription factor, but cellular function and role in disease still unknown. • FRDA Schmucker 2010 • Neurodegenerative disorder. • GAA repeat in intron 1 of FDRA gene. • Recessive • Pre mutation – 34-100; Full mutation 66-1700 • Expansion decreases mRNA levels. 2 models: • Non-B DNA conformation and/or heterochromatin-mediated gene silencing. • Frataxin involved in iron homeostasis. • Frataxin deficiency disrupts iron-sulphur enzymes, mitochondrial iron overload causing iron dysregulation and increased sensitivity to oxidative stress.

10. RNA Gain of function • Features – no other types of mutations; repeated allele is transcribed, but not translated; non coding. • Examples DM1, DM2, FXTAS, SCA8, 10 and 12 • Myotonic Dystrophy (DM1/2) • DM1 - CTG repeat in 3’UTR of DMPK. • DM2 – CCTG repeat in intron 1 of zinc finger protein 9 (ZNF9). • Two functionally different genes, but same toxic RNA gain of function mechanism.

11. RNA Gain of function • DM1/2 toxic RNA gain of function mechanism • Almost all of expanded mRNA retained in nuclear foci, due to secondary structures (eg hairpins). • Expanded mRNA can sequester RNA-binding proteins – bind to the (CUG)n (CUG-BP1 and MBNL1). • RNA binding proteins regulate mRNA processing (eg splicing). • DMPK and ZNF9 splicing is normal, but thought expansion causes aberrant expression of other transcripts, normally regulated by CUG-BPs.

12. RNA Gain of function • Fragile X-associated tremor/ataxia syndrome (FXTAS) • CGG pre mutation carriers (55-200 repeats) in 5’UTR of FMR1. (20% female pre mutation carriers develop premature ovarian failure). • mRNA levels are elevated, but protein levels close to normal – so toxic RNA. • Proposed similar mechanism to DM. • Clear difference with DM is DMPK and ZNF9 normal transcript levels. • ? Larger DM repeats, so more short repeats in FXTAS has same effect. • SCA 8, 10 & 12 • Repeats are in non-coding regions. Mechanism unknown thought to be RNA gain of function.

13. Protein Gain of function • Features • No other types of mutations • Coding regions • Altered protein by CAG repeat – polyglutamine tract • Neurodegenerative disorders. • Associated with neuronal aggregates containing expanded gene product. • PolyQ tracts become insoluble and form aggregates. • Repeat size threshold similar between disease (~35-40rps). • Examples: Huntington’s disease, Kennedy’s disease (SBMA), DRPLA, SCA 1, 2, 3, 6, 7 & 17

14. Protein Gain of function • Huntington’s disease • Chorea, incoordination, motor impersistence. • CAG repeat at N terminus of Huntingtin protein. • Protein function – precise function unknown, but does have role in vesicular transport, cytoskeletal anchoring and clathrin-mediated endocytosis. • Loss of function may account for some of pathogenesis in HD, most thought to be protein gain of function. • Mechanism of how inclusions lead to pathology unclear – affects transcription regulation, apoptosis, mitochondrial function, tumour suppression, vesicular and neurotransmitter release and axonal transport.

15. Protein Gain of function • Kennedy’s disease • Weakness and atrophy of proximal muscles. Difficulties swallowing and articulating speech. • CAG repeat in androgen receptor gene on X chromosome. • Androgen receptor regulates transcription of hormone-responsive genes upon androgen binding. • Expansion causes decrease in protein expression and alter transcriptional activation – causes androgen insensitivity in SBMA patients. • Motor neuron dysfunction due to toxic protein gain of function.

16. Protein Gain of function • Spinocerebellar ataxias • Expanded CAG repeats seen in SCA 1, 2, 3, 6, 7 and 17. • All SCA gene are different: Ataxin 1-3 and 7, subunit of CACNA1A (SCA 6), TBP (SCA 17). • Disease pathways unclear. • Cannot rule out some polyQ diseases have loss of function effect on pathogenicity.

17. Keywords • Trinucleotide repeat • Dynamic mutation • Short tandem repeat polymorphisms • Loss of function – absence of transcription • RNA gain of function – toxic RNA – splicing • Protein gain of function – neuronal aggregates.

18. References • McMurray 2010 – mechanism expansion • Brouwer 2009 – loss of function, gain of function • Jin 2000 – Fragile X • Schmucker 2010 – FRDA • Walker 2007 – HD