Repeat Expansion Disorders Flashcards
What were some early disorders with generational differences in phenotype?
1905 - Nettleship
= children with ‘certain degenerative disorders’ show symptoms earlier than parents
1918 - Fleisher
= myotonic dystrophy
= increased expressivity - showed worsening severity, earlier onset with each succeeding generation
(can also be more individuals affected = penetrance)
= Genetic Anticipation
= earlier onset, increasing severity in later generations (e.g. myotonic dystrophy)
= increased numbers of individuals with symptoms in later generations
(e.g. fragile X syndrome)
= sherman paradox
What is the molecular basis for genetic anticipation?
= expanded numbers of MICROSATELLITE repeats
1991 - cause of Fragile X Syndrome discovered
= expanded number of CGG repeats in the 5’ untranslated region of FMR1 gene
(FMR1 - now called Fragile X Messenger Ribonucleoprotein 1)
1991 - cause of SBMA (spinal and bulbar muscular atrophy) determined
= expanded number of CAG repeats in coding region of androgen receptor gene
Expanded repeats are unstable
= numbers of repeats can expand on parental transmission and in some somatic tissue
= dynamic mutation
= expanded numbers of repeats in later generations explains genetic anticipation + sherman’s paradox
What are some key general facts about repeat expansion disorders?
= >50 human REDs identified
(each with expanded number of repeats at particular locus characteristic of specific RED)
= 13 different sequence repeats associated with REDs
= number of repeats positively correlates with disease severity, negatively correlates with age on onset
= expansion of one disease causing repeat does NOT promote expansion of other repeats in patient’s genome
= REDs can be autosomal dominant, autosomal recessive or X-lined
= for each RED = threshold number of repeats below which repeats are stably inherited, above which repeats show intergenerational and somatic instability
= DYNAMIC MUTATION
= repeats can also contract
What are the 4 mechanisms of disease for REDs?
(not mutually exclusive)
- Expansion of non-coding repeats
(leading to loss of function of gene containing repeat)
= loss of function - recessive inheritance for autosomal expansions
e.g. Friedrich’s ataxia (GAA/TTC expansion in first intron of FXN) = autosomal recessive
e.g. Fragile X (CGG/CCG expansion in 5’ UTR of FMR1) = X-linked dominant
- Expansions of CAG coding repeats
(leading to gain of function and production of abnormal protein containing an expanded polyglutamine tract)
= dominant inheritance
= e.g. Huntington’s Disease
= e.g. Kennedy’s / SBMA
- Expansions resulting in gain of function of RNA containing an expanded repeat
= dominant inheritance
= e.g. Myotonic dystrophy type 1 + 2
= e.g. FXTAS - fragile x associated tremor ataxia syndrome
= e.g. FXPOI - fragile x associated premature ovarian insufficiency
- Expansions resulting in Repeat Associated, Non-ATG (RAN) translation of repeat containing RNA
(leading to production of toxic peptides)
= gain of function
= dominant inheritance
= e.g. ALS/FTLD (C9orf72)
= e.g. DM1
= e.g. FXTAS
= e.g. HS
What is Fragile X Syndrome?
= X-linked (dominant)
= most common cause of inherited intellectual disability (range of severity, average IQ-40)
= increased severity in males, relatively mild in females (random X-inactivation)
= prevalence = 1 in 4000-7000
= most common monogenetic cause of autism
physical manifestations:
= mild abnormal facial features (sunken eyes, arched palate, large ears)
= macroorchidism (enlarged testes)
medical problems:
= otitis media
= seizures
= mitral valve prolapse
= GI problems
What is the ‘fragile site’ FXS is associated with?
= FRAXA on the X-chromosome (Xq27.3)
= fragile site can be visualised as a gap in staining of X chromosome in metaphase spreads
(from cells grown under conditions of replicative stress)
FRAXA site
= CGG repeat expansion in 5’ UTR of FMR1 gene on X chromosome
= stable repeat no 6-44 (average = 30)
= intermediate repeat 45-54 (grey zone)
= premutation repeat 55-200 (associated with FXTAS and FXPOI)
= full FRAX mutation 200->400 CGG repeats
gene affected: FMR1
How does genetic anticipation affect a Fragile X family pedigree?
= repeat size increases through generations
(as do severity of symptoms)
= females with premutation repeat levels = termed carriers
= FMR1 mutation may have a maternal expansion bias
= females can have FXS
(severity of symptoms depends on X-inactivation bias)
What is the epigenetic mechanism involved in FXS?
= FMR1 expanded mRNA silences the FMR1 locus through an epigenetic mechanism
<40 CGG repeats
= FMRP produced in hESCs and differentiated cells
= active euchromatin characterised by H3K9Ac, H3K4Me
= DNA unmethylated
> 200 CGG repeats
= hESCs FMR1 transcribed and translated
= during differentiation expanded FMR1 mRNA initiates silencing
= differentiated neurons >200 CGG, repressive (inactive) heterochromatin MeCpG and H3K9ME2
= no FMRP expression
= expanded FMR1 mRNA
(increases repeat number in transcript, interacts with expanded repeats in DNA genome = gene silencing)
What is FXS caused by?
= loss of Fragile X Messenger Ribonucleoprotein 1 (FMR1)
= FMR1 normally localises to postsynaptic spaces of dendritic spines
= shuttles in and out of nucleus transporting target mRNAs
= phosphorylated FMRP binds to and represses translation of ~400 target dendritic mRNAs
= on receipt of synaptic signals = FMRP is de-phosphorylated = no longer represses translation
= allowing synthesis of key synaptic plasticity proteins
= in FXS = there is repression of translation of transcripts normally regulated by FMRP
= leading to the symptoms associated with FXS
What is an example of a disease caused by CAG repeat expansion in protein-coding region?
= 9 distinct diseases
(dependent on repeat length , intrinsic function of protein)
e.g. Dominant gain of function disorder = Huntington’s Disease (HD)
= characterised by neuronal degeneraton
= clinical features = progressive, selective (localised), neural cell death associated with choreic (writhing dance-like) movements and dementia
What is the genetic basis for HD?
= expansion of CAG trinucleotide repeat - encoding glutamine (Q)
= gene affected - HTT (IT15) 4p16.3 = encodes 3144 aa(23Q) huntingtin (htt) protein
= CAG repeat region begins at codon 18 in exon 1
= normal allele 11-26 CAG repeats (11-26 Q residues)
= mutable normal allele 27-35 CAG repeats
= HD allele with reduced penetrance 36-39 CAG
= HD allele >39-~250 repeats
= longer repeat length = associated with earlier onset, more sever symptoms
= paternal expansion bias >7 CAG
= increased somatic instability associated with earlier onset
How can CAG repeat numbers be estimated?
= using PCR assay
= PCR primers flank repeat containing region
= size of PCR product dependent on number of CAG repeats
What is the structure of Huntingtin protein?
= mainly consists of HEAT repeats
(Huntingtin Elongation factor 3) = a subunit of protein phosphatase 2A and TOR1
(widely expressed, highest levels in neurons of CNS)
= WT protein acts as:
= scaffold to coordinate complexes of other proteins
= transcriptional regulator
Huntingtin fragments
= HTT
= PRD (proline rich domain)
= PolyQ (polyglutamine)
What are the cellular mechanisms in Huntington disease?
N-terminal polyQ protein fragments
= translocate to the nucleus
= form intranuclear inclusions
= impair gene transcription (e.g. of BDNF)
N-terminal polyQ containing fragments
= oligomerise, aggregate
= form cytoplasmic inclusions
Oligomers, aggregates and inclusions impair:
= proteostasis network
= synaptic function
= axonal transport
= mitochondria
(+more)
Why is the cause of HD more than just the gain of function Htt protein?
Model systems where CAG (Q) repeats are interrupted by CAA (Q) repeats
= have decreased cellular toxicity
= even though there is the same number of glutamines (Q) expressed in protein
=HD age of onset better predicted by length of uninterrupted CAG repeats
(rather than number of Qs in encoded protein)
Possible explanation:
= RNA gain of function contributes to HD
= BUT reduced formation of toxic RNAs with interruption to CAG repeats
= somatic instability predicts age of onset - repeats with interruptions more stable than uninterrupted repeats
What is an example of an RED caused by expansions resulting in gain of RNA function?
= DM (dystrophia myotonica) myotonic dystrophy
= second most common type of muscular dystrophy
= dominant inheritance pattern
= affects 1 in 8500
= two forms : DM1 and DM2
(repeat expansions in different genes)
(BUT have common pathogenic mechanism by RNA gain of function)
Multisystemic disorder with varied clinical features:
= myotonia (impaired muscle relaxation)
= muscle wasting
= insulin resistance
= cardiac conduction defects
= cataracts
= cognitive dysfunction
What are the key features in DM1?
= results from expansion of CTG repeats in 3’ UTR of DMPK gene
= normal repeat length 5-37
= premutation 37-50
= pathogenic 50-1000
= congenital form >100 repeats
= maternal expansion bias
= extensive somatic instability in proliferative tissue particularly muscle
= severity of disease correlates with repeat length
= age of onset has inverse correlation with repeat length
What are the key features of DM2?
= CCTG repeats in intron 1 of ZNF9/CNBP gene
= normal repeat length <30
= premutation 31-74
= pathogenic 75-11,000
What is a mechanism of disease associated RNA gain of function?
= sequestration of splicing proteins
= DNA containing expanded repeats transcribed to generate RNA with expanded repeats
= repeat containing RNA forms imperfect hairpins
Abberrant splicing leads to symptoms:
= insulin receptor = insulin resistance
= chloride channel = mytonia
= cardiac troponin T = cardiac abnormalities
= unidentified genes = cataracts, testicular failure
What are the different diseases associated with variation in repeat length in FMR1?
one gene:
= FMR1
3/4 distinct repeat length ranges:
= 6-54 = normal (stable) / grey zone
= 55-200 = ‘premutation’ in context of FXS (unstable expanded repeat)
= 200-4000 = unstable expanded repeat - FXS
3 different diseases result:
= Fragile X syndrome - repeat length 200-4000
= Fragile X-associated tremor/ataxia syndrome (FXTAS) - 55-200 premutation repeat (40% males, 8% females)
(can be late onset >50 years - neuropsychiatric degenerative disorder)
= Fragile X associated Premature ovarian insufficiency (FXPOI) - 25% of female carrying premutation repeat 55-200
How does variation in repeat length in FMR1 lead to different conditions?
2 different disease mechanisms:
Full FRAXA mutation
= silencing of FMR1 transcription and consequent lack of FMR1 protein
= associated with Fragile X syndrome
Pre-mutation
= 55-200 repeats
= leads to increased transcription (up to 8x level of normal RNA)
= associated with FXTAS, FXPOI
Increased levels of repeat containing RNA
= sequesters several RNA binding proteins (cfDM)
= results in dysregulation of proteins whose expressed is usually regulated by those RNA binding proteins
= may lead to FXTAS / FXPOI
How does RAN translation of repeat containing RNA lead to production of toxic peptides?
Repeat expansion transcribed in 2 directions:
Translation initiated
= without ATG initiation codon
= on both transcripts within repeat region in all 3 reading frames
= generates peptides with repeated amino acid sequences = toxic peptides
(gain of function - dominant inheritance)
What is an example of disease caused by RAN translation of repeat containing RNA?
= ALS (amyotrophic lateral sclerosis) (MND)
= expanded GGGGCC repeats in first intron of C9orf72 causes autosomal dominant ALS
3 different contributory mechanisms:
- Haploinsufficiency
- RNA toxicity
= sequestered RNA binding proteins
= stop normal function - Toxic RAN peptides
= translated in both directions = many reading frames = alternative a.a. = toxic
What are the mechanisms that promote repeat instability?
Only some sequence repeats show instability
For stable repeats:
= repeats are stable if number of uninterrupted repeats totals less than ~100-150bp, ~30-50 trinucleotide repeats
Two types of normal alleles common at RED loci:
= short-normal alleles (e.g. 19 CAG HD)
= long-normal alleles - longer repetitive runs with stabilising interruptions (e.g. AGG within CGGn runs in FMR1 gene)
For unstable repeats:
= above ~100-150bp of repeat sequence show intergenerational instability - dynamic mutation
(both expansion and contraction)
Expanded repeat sequences may show somatic instability (in dividing and non-dividing cells)
What makes microsatellite repeats associated with REDs unstable?
= all can form unusual non-B DNA structures
(e.g. imperfect hairpins)
= alternative DNA structures are difficult to replicate and transcribe
How does DNA replication lead to repeat instability in dividing cells?
Hairpins have different levels of stability
(e.g. CTG hairpin more stable than CAG hairpin)
Replication of dsDNA is asymmetric
= leading and lagging strands
Structure prone sequence on lagging strand = promotes instability
Contractions
= more structure prone sequence on lagging strand template
= replication machinery skips hairpin on lagging strand template
Expansions
= replication fork stalling, reversal and restart
= unwinds hairpin
= reforms replication fork
= hairpin on newly synthesised strand
(OIZ = Okazaki initiation zone = single-stranded during replication)
What are the replication mechanisms for repeat expansion that account for genetic features at a molecular level in dividing cells?
Secondary structures more likely to form on lagging-strand template
= as OIZ is single stranded for extended perioid
Secondary structures more likely as length of the repetitive run increases
Repeats more unstable when more structure prone strand is on the lagging strand template
Genetic anticipation explained by consecutive replication stalls and restarts within longer repetitive runs
= progressively increasing their instability
How does a change of replication context impact on repeat stability?
= ‘Ori switch’
In stable repeat
= replication origins ‘x and y’ active
= repeats replicated from origin X
= structure prone repeat on leading strand template
= continuous replications
= repeats are stable
Ori Switch
= origin ‘x’ is inactivated
= repeats replicated from origin ‘y’
= structure prone repeat is now on lagging strand template
= discontinuous replication
= template transiently single-stranded
= promotes secondary structure formation
= unstable repeats
How do active DNA repair pathways promote repeat instability in dividing and non-dividing somatic cells?
= presence of mismatch repair proteins PROMOTES repeat instability
= proposed mechanism of instability has role for MutSβ in stabilising DNA structures:
Toxic oxidation cycle
= mechanism for repeat instability in HD in terminally differentiated neuronal tissue
Base excision repair
= glucosylase removes base
= AP endonuclease nicks DNA strand
DNA pol δ
= initiates repair synthesis
= displacing repeat sequence which forms hairpin
= MutSβ stabilises hairpin
(blocks access by Fen1 flap endonuclease)
MutSβ
= homolog of E.coli MutS protein
= normal role is to promote repair of mismatches in regular B form DNA
Incorporation of hairpin leads to repeat expansion
How do transcription and DNA repair promote repeat instability in dividing and non-dividing cells?
- DNA unwinding during transcription promotes formation of slipped strand structure
- Hairpin stabilised by mismatch repair protein MutSβ binding
- Next round of transcription = RNA pol II blocked at hairpin
- Stalled RNA Pol II recruits NER proteins to promote transcription coupled repair (TC-NER)
- NER may cut at two points to excise hairpin
= leading to CONTRACTION after further round of replication
OR
incomplete NER
= can lead to expansion on further round of repair/replication
What happens to repeat instability during intergenerational transmission? How is it influenced?
= Repeat numbers in REDs may dramatically expand or contract when transmitted between generation
Influenced by:
Parental age
= e.g. CTG (DM1) directly correlates with parental age
= indicates repeat instability precedes fertilisation
= longer lifetime provides more opportunity for instability
Parental Sex
= e.g CGG repeats in FXS maternal expansion, paternal contraction bias
= e.g. CAG repeats in HD paternal bias
= differences in duration of spermatogenesis, oogenesis
= counterselection for expansions differs in sperm and oocytes
= differential DNA repair during spermatogenesis and oogenesis
= differential pattern of origin firing
= chromatin status in oocytes vs sperm
Give a summary of the 4 mechanisms of disease in REDs
