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)
