Download Trinucleotide repeats (TNRs)

Trinucleotide repeats
(TNRs)
Dr. Derakhshandeh, PhD
INTRODUCTION
Trinucleotide repeats (TNRs) are microsatellite
sequences
Disease-causing repeat instability is an
important and unique form of mutation
linked to more than 40 neurological,
neurodegenerative and neuromuscular
disorders.
I.g. Huntington's disease, myotonic dystrophy
and fragile X syndrome
Trinucleotide repeats
TNRs undergo high frequency mutagenesis
To understand better the molecular
mechanisms of TNR instability in cultured
cells
A new genetic assay was created using a shuttle
vector
The shuttle vector contains a promoter-TNRreporter gene construct whose expression is
dependent on TNR length.
The vector harbors the SV40 ori
(CAG•CTG)25–33
The shuttle vector is propagated in
cultured cells
It recovered and analyzed in yeast using
selection for reporter gene expression.
Richard Pelletier, Nucleic Acids Research 2005 33(17):5667-5676
Disorders caused by
trinucleotide repeat
First: the mutant repeats show both somatic and
germline instability
Secondly:
an earlier age of onset
and increasing severity of phenotype in subsequent
generations (anticipation)
Finally, the parental origin of the disease allele
can often influence anticipation
with paternal transmissions carrying a greater risk of
expansion for many of these disorders.
Category of the trinucleotide
repeat (based on the relative location)
first subclass:
–Repeats in non-coding sequences:
For six diseases
second subclass:
–Exonic (CAG)n repeats
code for polyglutamine tracts
Repeats in non-coding sequences
NON-CODING TRINUCLEOTIDE
REPEAT DISORDERS
Large and variable repeat expansions that
result in multiple tissue:
– dysfunction
– degeneration
Phenotypic manifestations within a
disease are variable
– Degree of somatic heterogeneity
Pre-mutations
The larger mutations often are transmitted from
a small pool of clinically silent intermediate size
expansions
CGG, GCC, GAA, CTG and CAG
particular trinucleotide sequence +
its location with respect to a gene
– Important defining factors in dictating the unique
mechanism of pathogenesis for each disease
Fragile X syndrome
Fragile X Syndrome
Fragile X syndrome
Fragile X syndrome (FRAXA)
Fragile XE MR (FRAXE)
1 in 2000 boys
1 in 4000 girls
are estimated to be affected
Fragile X Syndrome
most common inherited form of familial mental
retardation
(CGG)n trinucleotide expansion in the FMR1 gene
leading to the typical Martin-Bell phenotype
Clinical features vary depending on age
Expansion of a (CCG)n repeat in the FMR2 gene
corresponds to the FRAXE fragile site
It lies distal to FRAXA
It’s associated with mental retardation, but it is
less frequent and lacks a consistent phenotype
The transcription of the FMR1 gene of normal and premutation alleles. Both alleles
are translated into FMRP, which is demonstrated by Western blotting (lane N and P)
The full mutation allele is hypermethylatedthereforetranscribed, which resultabsenc
of FMRP (lane F)
Repeats in non-coding sequences
Sequence of the 5'-UTR region of the FMR1 gene
Sequence of the 5'-UTR region of the FMR1 gene
Fragile X syndrome (FRAXA)
Mental retardation
Macroorchidism
Some dysmorphic features
Hyperactivity
Fragile X
Syndrome
Fragile X syndrome (FRAXA)
expansion of a polymorphic (CGG)n repeat in
the 5'-untranslated region (UTR)
> 230 trinucleotides
hypermethylation together with a CpG island
within the FMR1 promoter region
transcriptional silencing of the FMR1 gene
reduced FMR1 transcription and loss of gene
product (FMRP)
Fragile XE MR (FRAXE)
mild mental retardation
variable behavior abnormalities
expansion of a polymorphic (GCC)n repeat
in the promoter region of the FMR2 gene
the expanded repeats are hypermethylated
leading to transcriptional silencing of FMR2
subsequent loss of gene product (FMR2)
Friedreich ataxia
(FRDA)
Friedreich ataxia (FRDA)
autosomal recessive
the only triplet repeat disorder
that does not show anticipation
Ataxia (loss of voluntary muscular
coordination)
Diminished reflexes
Cardiomyopathy (heart enlargement)
Diabetes
Degeneration in the spinal cord
Friedreich ataxia
FRDA is caused by a large intronic GAA repeat
expansion
located on chromosome 9 (Gene:X25/Potein:
frataxin)
which leads to reduced gene expression
The expanded AT-rich sequence most probably
causes
self-association of the GAA/TTC tract, which
stabilizes the DNA in a triplex structure
Repeats in non-coding sequences
FRDA & triplex structure
A novel DNA structure
sticky DNA
lengths of (GAA.TTC)n
in intron 1 of the frataxin gene of
Friedreich's ataxia patients
Sticky DNA is formed by the association of
two purine.purine.pyrimidine (R.R.Y)
triplexes
in negatively supercoiled plasmids at
neutral pH
Models of
structures
that may
mediate
mRNA
synthesis
and DNA
replication
inhibition
by
GAA·TTC
repeats
in FRDA patients
(GAA.TTC) (> 59 repeats)
– the lengths of (GAA.TTC) (> 59 repeats)
– inhibit transcription in vivo and in vitro
– adopt the sticky conformation
(GAAGGA.TCCTTC)65
–
–
–
–
found in intron 1
does not form sticky DNA
does not inhibit transcription
or associate with the disease
Sakamoto,et al. MMol Cell. 1999 Apr;3(4):465-75.
frataxin is found in the mitochondria of
humans
we do not yet know its function
there is a very similar protein in yeast,
YFH1,
YFH1 is involved in controlling:
– iron levels
– and respiratory function
Frataxin and YFH1 are so similar, studying
YFH1 may help us understand the role of
frataxin in FRDA
Reduced X25 mRNA
decreases frataxin levels
a partial loss of frataxin function
Disruption of the yeast X25 homolog
(YFH1):
– abnormal accumulation of mitochondrial iron
– loss of mtDNA
– multiple iron–sulfur-dependent enzyme
deficiencies
– increased sensitivity to oxidative stress
Frataxin :
– hypersensitivity to iron and H2O2 stress
Frataxin insufficiency
frataxin insufficiency may result in
abnormal iron–sulfur homeostasis
mitochondrial dysfunction
free radical production
oxidative stress
cellular degeneration
Wong, A, et al. Hum. Mol. Genet., 8, 425–430 (1999)
Myotonic dystrophy
(DM)
Myotonic dystrophy (DM)
multisystem disorder
highly variable phenotypes
Anticipation
Myotonia
muscle weakness
Developmental abnormalities
mental handicap
Hypotonia
respiratory distress are often evident in the more
severe congenital myotonic dystrophy (CDM).
DM
CTG trinucleotide repeat
in the 3'-UTR of the protein kinase gene,
DMPK
The CTG repeat is located within the
promoter of a upstream homeobox gene
Loss of function of either or both of these
proteins could contribute to some of the
features in DM
Korade-Mirnics, Z. et al. (1998) Nucleic Acids Res., 26,
1363–1368
Repeats in non-coding sequences
Spinocerebellar ataxia type 8
(SCBA8)
Spinocerebellar ataxia type 8
(SCBA8)
progressive ataxia
with cerebellar atrophy
decreased brisk reflexes
SCA8 is expressed primarily in the
brain
is caused by an expanded CTG
repeat in its 3'-terminal exon (~110–250
repeats)
Repeats in non-coding sequences
Parkinsonism
(PD)
SCA-2 and SCA-3 repeats in
Parkinsonism
expansion of triplet repeats
encoding polyglutamine
(polyQ) tracts
POLYGLUTAMINE
DISEASES
POLYGLUTAMINE DISEASES
have repeat expansions that are
much smaller in size and variation
characterized by progressive
neuronal dysfunction
begins in mid-life and results in
severe neurodegeneration
POLYGLUTAMINE DISEASES
different polyglutamine diseases have
little in common:
–the length of the expansion > 35–40
–the greater the number of glutamine
repeats in a protein
the earlier the onset of disease and
the more severe the symptoms
Expansion disorders
Many major neurodegenerative
diseases:
Alzheimer's disease
Parkinson's disease
Huntington Disease
Alzheimer's disease
Alzheimer's disease
various types of familial Alzheimer's
disease (AD) genes
mutants of amyloid precursor protein
(APP)
polyglutamine repeat Q79
Huntington's disease
Huntington's disease
inherited as a autosomal dominant
a polymorphic CAG repeat tract in
exon 1, which is 35 units in length
Huntingtin in mitochondrial energy
metabolism
HD CAG size determines [ATP/ADP] in
lymphoblastoid cells
HD CAG repeat implicates a dominant property
of huntingtin in mitochondrial energy metabolism
Ihn Sik Seong, et al.Human Molecular Genetics 2005 14(19):2871-2880
HD CAG size determines [ATP/ADP]
in lymphoblastoid cells
A Polymorphic Trinucleotide Repeat at DXS8170 in
the Critical Region of X-Linked Retinitis Pigmentosa
Locus RP3 at Xp21.1
possible mechanism of cell death
the abnormally long sequence of
glutamines acquires a shape that prevents
the host protein from folding into its proper
shape.
if, the length of polyglutamine repeats is
longer than the critical value found in
disease, it acquires a specific shape called
a β-helix.
Q37
chain
under
conditi
ons in
which
it
adopts
βstrand
topolo
gies
Summary
Since the identification in 1991 of repeat
instability as a disease-causing mutation, genespecific repeat instability is now known to be the
mutational cause of at least 40 neurological,
neurodegenerative and neuromuscular
diseases.
Both germline (parent-to-offspring) and tissuespecific somatic instability occurs.
There are unique and common effectors for the
instability of different repeat sequences,
although each disease or locus is unique.