Trinucleotide Repeat Expansion and Human Disease

1
Trinucleotide Repeat Expansion and Human Disease
Trinucleotide repeats an emerging pattern.
(2005) Nat. Rev. Genetics 6729-773 http//www.nat
ure.com/nrg/focus/repeatinstability/index.html Ev
erett CM Wood NW (2004) Trinucleotide repeats
and neurodegenerative disease. Brain 127
2385-2405 Mitas M (1997) Trinucleotide repeats
associated with human disease. Nucleic Acid Res.
25 2245-2253 Kovtun IV McMurray CT (2001)
Trinucleotide repeat expansion in haploid germ
cells by gap repair. Nat. Genetics 27 407-411
2
Anticipation/dynamic mutation Molecular
mechanisms Specific diseases
3
In certain inherited disorders symptoms become
more severe in each successive generation

• Huntingtons disease
• Spinocerebellar ataxia
• Myotonic dystrophy
• Fragile X
• Friedrichs ataxia

4
Mutations causing these diseases involve expanded
trinucleotide repeats
CTG/CAG, CGG/CCG, GAA/TTC
BUT NOT GAT/ATC, TAG/CTA, TTG/CAA
Unaffected individuals carry lt 30
repeats Minimally affected carrier/individuals
carry 50 repeats Severely affected individuals
carry 100-1000 repeats
5
Processes involved in repeat instability
Repeat expansion can take place in the germ line
or in the soma
6
Expansion mechanisms
Triplet repeats associated with human disease can
adopt hairpin conformations in vitro at
physiological salt levels and temperatures
Hairpin alignment for CTG/CAG Non-Watson-Crick
base pairs shown on right
7
Expansion is mediated by misaligned pairing of
repeats and secondary structure formation as the
duplex unpairs during-
8
Expansion during replication
9
Expansion during DNA repair
Breaks in the vicinity of a repeat allow hairpin
formation and gaps thus created are filled in by
mismatch repair
KO mice with mutations in MMR proteins (MSH2,
MSH3 and PMS2) show stabilisation and/or
contractions of CAG/CTG repeats
10
Expansion during recombination
Recombination between sequences flanking
repeats is rare in most disorders.
Loss of recombination proteins has little effect
on instability
Recombination does appear to be a major source of
instability with interrupted repeats
e.g. in 50 of de novo inherited cases
of facioscapulohumeral muscular dystrophy
11
Evidence from transgenic mice suggests most
repeat expansion in HD occurs in post-mitotic
cells in vivo
FACS sort germ cells from transgenic mice
carrying in an unstable CAG repeat. PCR check
for expansion at each stage. Limited to ST and
SZ stages.
Kovtun and McMurray 2001
12
How do expanded repeats cause disease?
Expanded repeats that cause human disease are
found in many different genic locations
13
How do expanded repeats cause disease?
Repeats in protein coding sequences - toxic
proteins
Repeats in RNA coding regions - altered RNA
function
Repeats in non-coding regions - reduced
transcription or translation
Understanding the molecular mechanisms behind the
symptoms specific to each disease will allow new
therapies to be developed
14
Huntingtons disease (HD)

• Progressive dementia
• Neuronal death and glioma
• GABAnergic efferent projection neurons lost in-
• Cortex
• Hypothalamus
• Cerebellum

15
Genetics of HD
Expansion of CAG repeat (polyglutamine) in exon 1
of huntingtin gene
Nullizygous KO mice die halfway through gestation
and hemizygotes are normal Expression of full
length protein with expanded repeats
recapitulates disease symptoms in transgenic
mice, flies and cellular models
Dominance due largely to gain of toxic function
16
Molecular mechanism
Expanded polyglutamine tracts affect 3-D
conformation Misfolded mutant protein forms
insoluble aggregates Acquires toxic function
through aberrant protein interactions
17
Models and therapies
Knock-in and inducible mouse models mimic disease
and suggest early treatment might reverse
clinical symptoms Invertebrate and single cell
models allow rapid screening of multiple test
compounds Screening for compounds that-

• Reduce aggregate formation
• Block caspase activity
• Inhibit glutamate and NDMA receptors
• Improve mitochondrial function/act as
  antioxidants
• Inhibit histone deactylases (to correct
  transcriptional dysregulation)
• Selectively inactivate the disease gene (RNAi)

18
Myotonic Dystrophy (DM1 and DM2)
Muscles fail to relax Skeletal muscle
wasting Cardiac conduction defects Cataracts En
docrine and cognitive dysfunction (DM1)
19
Genetics of DM1/2
DM1 caused by CTG repeat in 3UTR of DMPK DM2
caused by CCTG expansion in intron 1 of ZNF9

• DM1 pathogenesis could be due to-
• Haploinsufficiency for DMPK due to mRNA
  instability
• Transcriptional interference with adjacent SIX5
• Pathological effects of expanded RNA

20
Molecular mechanism
Expanded CUG or CCUG repeat-containing
transcripts form nuclear RNA foci, which recruit
muscleblind-like (MBNL) and other RNA-binding
proteins Mbnl knockout mice develop DM like eye
and muscle phenotypes
21
Models and therapies
Transgenic mice carrying a mutant human DMPK
transgene model symptoms well and cell based
models are also useful
Ribozyme based treatments that modify the
repeat-containing 3 end of the transcript are
effective in both models
Chemical compound screening in a cell based model
has identified bioflavenoids with therapeutic
activity
22
Fragile X (FRAXA)
Cognitive developmental problems Facial
dysmorphology Heart defects Abnormally long
immature dendritic spines
23
Genetics of FRAXA
Expansion of CGG repeat in 5UTR of FMR1
gene leads to haploinsufficiency for FMR1 FMR1
KO mice show cognitive problems etc. Full
mutations associated with methylation of FMR1
promoter and translational suppression due to
hairpin formation
24
Molecular mechanism
FMRP binds polyribosmes in an RNA dependent
manner, suppresses translation and localises to
dendritic spines In flies and mammals FMRP also
associates with components of the RNAi machinery
and may facilitate translational control of
selected targets
25
Models and therapies
Knockout mice, cellular and fly models available
Combining DNA demethylating agents and histone
deactylase inhibitors reactivates blocked FMR1
transcription
The KO mouse has abnormal glutamate receptor
(mGluR5) signaling and glutamate receptor
antagonist treatments correct neuroanatomical and
behavioural defects in flies
26
Friedrichs ataxia (FRDA)
Progressive gait and limb ataxia Neuronal
degeneration Diabetes Cardiac failure
27
Genetics of FRDA
Expansion of GAA/TTC repeat I intron 1 of
Frataxin gene Autosomal recessive
inheritance Triplex formation blocks RNA
polymerase progression
28
Molecular mechanism
Frataxin protein has a role in mitochondrial iron
transport and protection from free radicals by
increasing SOD activity Loss of frataxin leads
to accumulation of mitochondrial iron,
susceptibility to oxidative stress and reduced
oxidative phosphorylation
29
Models and therapies
Conditional frataxin deficient mice and yeast
YFH1 mutant models
Antioxdants and mitochondria targeted
iron-chelators being tested
Also compounds that enhance frataxin gene
expression (sodium butyrate, cisplatin, haemin)
30
Summary
More than 40 neurological, neurodegenerative and
neuromusclar disorders are linked to DNA repeat
expansion mutations
Repeat instability involves the formation of
unusual DNA Structures during DNA replication,
repair and recombination
The molecular mechanisms underlying the disorders
involve either loss of protein function or gain
of function at the protein or RNA level
Disease gene identification has lead to the
development of model systems for investigating
mechanisms and evaluating treatments