Transcript Snímek 1

 Nemoci spojené s expanzí
mikrosatelitních repetitivních
sekvencí – myotonická dystrofie
typu 1
 Kongenitální myotonie a mutace
v genu CLCN1
Repeat sequences in the human genome
 Half of the human genome consists of repetitive DNA,
significant proportion is organized in tandem arrays (copy
number variation).
• Repeat unit sizes 1- 4 nucleotides and spanning less than
100 bp are typically defined as microsatellite repeats.
• Repeat unit sizes 10 - 40 nucleotides covering several
hundreds of bp are referred to as minisatellite repeats.
• The term midisatellite repeats has been proposed for loci
containing repeat units of 40 - 100 nucleotides that can extend
over distances of 250–500 kb.
• Macrosatellite repeats are the largest class of repeat arrays
with unit sizes of >100 nucleotides but which are typically
much larger and can span hundreds of kb of DNA.
Microsatelite repeats and diseases
Genome Res. 2008 18: 1011-1019
Microsatelite repeats and diseases
FMR1, 5´UTR,
CGG repeats
• FXS: >200
• FXTAS: 55-200
• Normal: 5-54
• Repeat expansions can occur in 5′UTRs, coding regions, introns, 3′UTRs.
• Repeat lengths in introns, 3′UTRs, and 5′UTRs can become much larger
than in coding regions.
• Premutation alleles do not show usually disease symptoms, but can expand
in the next generation.
• As repeats get longer, symptoms are seen at an earlier age and are more
severe.
Spinocerebellar ataxias (SCA12), Spinal Bulbar Muscular Atrophy (SBMA), myotonic dystrophy (DM)
Myotonic dystrophy type 1; DM1
DM1 is caused by an expansion
of the CTG repeat in the 3’UTR of
the dystrophia myotonica protein
kinase gene (DMPK, 19q13.3),
AD inheritance
• Individuals with 5 to 37 repeats are unaffected.
• Individuals with 38-50 repeats carry the premutation. These individuals
are asymptomatic. However, these repeats are unstable and can expand
during meiosis. As a result, such individuals are at risk of having affected
children.
• ~ 50 to 150 repeats are consistent with the mild adult-onset form of DM1,
~100 to 1000 repeats are consistent with the classic adult or childhood
onset form of DM1,
> 1000 repeats are consistent with the congenital form of DM1 and often
result in severe neonatal complications.
Myotonic dystrophy type 1; DM1
• Patients with 50–150 CTG repeats
(mild adult onset form of DM1) may
develop cataract, diabetes, myotonia,
mild muscle weakness.
• Patients with 100–1000 CTG
repeats (the classic form of DM1)
are affected earlier and more severely.
• CTG repeat size above 1000 is
associated with the congenital form
of DM1, which may be fatal due to
respiratory failure. Feeding difficulties,
muscle weakness, foot deformity, and
cognitive impairments are present in
surviving infants.
Myotonic dystrophy type 1; DM1
• The expanded CTG repeat ‘dynamic’ mutation - the
number of repeats tends to
increase in size over
generations.
• Expansion of the CTG repeats
commonly occurs during
meiosis. As a result, children of
affected individuals tend to have
severe symptoms and earlier
onset than their parents.
Myotonic dystrophy type 2; DM2
DM2 (also known as proximal
myotonic myopathy [PROMM])
is caused by an expansion of
the CCTG repeat in the first
intron of the zinc finger 9 gene
(ZNF9, 3q21), AD inheritance.
• Unaffected individuals have less than 24 repeats.
• Affected individuals have between 75 and 11000 repeats.
• The repeat structure in DM2 is more complex than the triplet repeat seen in DM1. The
normal repeat structure is (TG)12-26(TCTG)7-12(CCTG)3-9(g/tCTG)0-4(CCTG)4-15.
• Individuals with 22-33 uninterrupted CCTG repeats carry a premutation. These
individuals are asymptomatic. However, these repeats are unstable and very likely to expand
during meiosis (risk of having affected children).
• The minimum pathogenic length of the expanded region appears to be 75 uninterrupted
CCTG repeats. Repeat counts can increase to over 11000 in affected individuals, with a mean
repeat length of ~5000 repeats. The expanded region has been shown to display an even
greater instability than the DM1 mutation.
• Unlike DM1, the length of the DM2 expansion does not appear to correlate
significantly with the age of onset or severity of disease symptoms.
Pathogenesis of DM1 and DM2
• Nuclei of cells of DM1/DM2 patients → expression
of genes containing CTG/CCTG repeat expansions
→ nuclear foci containing RNA with
expanded CUG/CCUG repeats → capture
of RNA binding proteins (proteins
regulating mRNA splicing) – muscleblind
(MBNL) protein and others →
misregulation of splicing of certain genes:
chloride channel 1 (CLCN1), insulin
receptor (IR), cardiac troponin T,
skeletal troponin T, and others.
• Insulin receptor and chloride ion channel premRNAs are misspliced in DM patients → the
symptoms of insulin resistance and myotonia.
In these two images of the same muscle precursor cell, the top image shows the location of the Mbnl1 splicing
factor (green) and the bottom image shows the location of RNA repeats (red) inside the cell nucleus (blue). The
white arrows point to two large foci in the cell nucleus where Mbnl1 is sequestered with RNA.
Pathogenesis of DM1 and DM2
Y. Kino, Nucleic Acids Res 2009
Model of CLCN1 splicing regulation. Exon splicing enhancer (ESE) is
located at the 5′ end of exon 7A. MBNL1 represses exon 7A inclusion
through inhibiting ESE. RNAs carrying expanded CUG/CCUG repeats
deplete MBNL1 protein, resulting in the facilitation of exon 7A
inclusion.
J Gen Physiol
2007;129:7994
Proper CLCN1 pre-mRNA splicing in normal skeletal muscle is regulated by MBNL1
protein. Depletion of MBNL1 proteins results in inclusion of additional exons (e.g.,
exon 7a) containing premature termination codons. Aberrantly spliced CLCN1
transcripts are exported from the nucleus, degraded through the nonsense-mediated
decay pathway, and/or produce truncated proteins. These effects result in a
dramatic reduction in the number of functional ClC-1 channel.
Toxic RNAs have myriad downstream effects on cellular metabolism.
• Expression of repeat-containing RNAs can induce hypermethylation and
heterochromatinization of the neighboring DNA (FRAXA) (1).
• The double-stranded hairpin structure formed by the repeat RNAs can
sequester RNA-binding proteins such as MBNL1 (2). This leads to altered
splicing of MBNL1 target RNAs (3).
• In addition, in some cases kinase pathways are activated through unknown
mechanisms, leading to aberrant phosphorylation and localization of
CUGBP1 (4). This also has impact on splice site choice and perhaps on
decay and translation of CUGBP1 target messenger RNAs (mRNAs) (5 and
6).
• The toxic RNA can be cleaved by the Dicer protein to generate siRNAs that
may inhibit expression of genes containing complementary repeats (7). This
siRNA pathway can also induce silencing of the toxic repeat-containing gene
(8).
• Finally, when CAG repeats lie within a coding region they can encode
polyglutamine, which also has toxic effects on the cell (9).
A.M. Dickson 2010
Muscleblind (MBNL1) sequestration
• MBNL1 is a RNA-binding protein (binding to hairpins formed by CAG,
CUG, CCUG), sequestration of MBNL1 is strongly implicated in disease
presentation.
• MBNL1 localization to the RNA foci sequesters MBNL1 away from its
normal targets and this leads to disease symptoms. Several mRNAs
whose splicing is regulated by MBNL1 exhibit aberrant splicing in DM.
• MBNL1 knockout mouse model reproduces much of the pathology as seen
in DM mouse models. Overexpression of MBNL1 in a DM mouse model is
sufficient to ameliorate both splicing defects and disease symptoms.
RNA interference
• In DM1 cells, CUG-containing RNAs form hairpin structures, which can be
cleaved by the RNA interference (RNAi) machinery to generate small
interference RNAs (siRNAs).
• These siRNAs are capable of binding complementary sequences in target
mRNAs, possibly interfering with their expression and contributing to
disease pathogenesis.
G. Sicot, Human Molecular Genetics, 2011
- The expanded gene is transcribed into sense and
anti-sense transcripts.
- Sense and anti-sense transcripts might be
connected with repeat-associated non-ATG (RAN)
translation in all possible reading frames
generating homopolymers
- iRNA pathways might be activated by the
processing of dsRNA structures, which can result
from the folding of CUG-containing transcripts into
hairpin structures, or from the hybridization of
complementary sense and anti-sense transcripts.
Three disease mechanisms for microsatellite expansion disorders.
Expanded microsatellite repeats have been traditionally classified as either coding
disorders or non-coding disorders that give rise to protein gain-of-function or loss-offunction or RNA toxicity mechanisms. For traditional ‘coding’ disorders, the repeat
expansion is translated as part of a larger open-reading frame (ORF) and results in the
expression of a mutant protein that disrupts normal cellular function and induces toxicity.
For example Huntington’s disease (HD), a late-onset neurodegenerative disorder, is
caused by a CAG expansion within the first exon of huntingtin gene that is translated as a
polyglutamine tract in the huntingtin protein, HTT. For traditional ‘non-coding’ disorders
(blue), the repeat expansion remains in the RNA transcript, accumulates as RNA foci that
sequester RNA binding proteins and lead to a loss of their normal function. For example,
in myotonic dystrophy, CUG(G) expanded RNA transcripts sequester MBNL proteins from
their normal splicing targets leading to a MBNL loss-of-function and alternative splicing
dysregulation. The recent discovery of repeat associated non-ATG (RAN) translation adds
a third pathway for disease. RNA transcripts from both ‘non-coding’ and ‘coding’ disorders
may undergo RAN translation. Once in the cytoplasm, these transcripts are capable of
producing proteins in all three reading frames, which may contribute to cellular
toxicity/stress. Depending upon the flanking sequences, each of these RAN proteins will
have a distinct expanded peptide repeats (colored boxes) and unique different C-terminal
regions (f1, f2 and f3). If the repeat is also within an ATG-initiated open-reading frame,
this ATG-initiated protein will share the expanded peptide repeat and C-terminal region
with one of the RAN proteins but will have an additional N-terminal region. Further
complexity is added by fact that many expansion mutations are bidirectionally transcribed,
which doubles the number of distinct RAN proteins that may be produced.
J.D. Cleary, Current Opinion in Genetics & Development 2014
Molecular pathogenesis of DM1: mechanisms of RNA toxicity, spliceopathy,
deregulation of gene expression and proteotoxicity.
• The expanded DMPK gene is transcribed into sense and anti-sense
transcripts.
• CUG-containing DMPK transcripts form alternative RNA structures and
accumulate in the nucleus of DM1 cells, resulting in reduced DMPK protein
levels.
• Loss of function of MBNL1, through sequestration into RNA foci,
deregulates alternative splicing.
• CELF1 (CUGBP/Elav-like family member 1) hyperphosphorylation and
upregulation affect alternative splicing and translation efficiency.
• Leaching of transcription factors (TF) by expanded DMPK transcripts may
mediate changes in gene expression.
• iRNA pathways might be activated by the processing of dsRNA structures,
which can result from the folding of CUG-containing transcripts into hairpin
structures, or from the hybridization of complementary sense and antisense DMPK transcripts.
• Both sense and anti-sense DMPK transcripts might be connected with
repeat-associated non-ATG (RAN) translation in all possible reading
frames generating homopolymers, which might be deleterious to the cell.
Repeat-associated non-ATG (RAN) translation
• Repeat-associated non-ATG (RAN)
translation occurs independently of
an ATG initiation codon in all
reading frames. RAN translation
occurs across long, hairpin-forming
repeats which affect translational
initiation.
• RNA translation on sense CUGcontaining transcript s produce
polyleucine (CUG), polycysteine
(UGC) and polyalanine (GCU) tracts.
• RAN translation on antisense CAGcontaining transcripts produce
polyglutamine (CAG), polyserine
(AGC) and polyalanine (GCA)
homopolymeric peptides.
• These toxic homopolymeric
proteins are considered in
pathogenic models of microsatellite
disorders.
Warner JP, J Med Genet 1996
Repeat primed PCR.
Stippled box represents
(CAG)n repeat. F shows 5'
fluoresceinated primer.
(A) For large alleles exceeding 100
CAG the PCR using flanking
primers P1 and P2 fails to give a
product.
(B) In the early amplification cycles
primer P4 (the repeat specific 3'
terminus) binds at multiple sites
within CAG alleles giving rise to a
mixture of products. Specificity is
dictated by P1. A 10:1 molar ratio of
P3 to P4 ensures that primer P4 is
exhausted in the early amplification
cycles.
(C) The primer P3 amplifies
from the end of products from
previous amplification rounds. A
long extension time is used
to allow complete extension of the
larger sized products within the
PCR product mixture.
Electrophoreogram of PCR
(primers P1 + P2)
The axis shows migration time in
minutes. CAG allele sizes shown
with the arrows. (A) Trace
obtained from a heterozygous
normal subject.
(B) Trace obtained from a
heterozygous
subject with a small expansion.
(C) Trace obtained from a patient
with myotonic dystrophy and an
expanded allele size of >4 kb as
determined by Southern blot
analysis.
The larger allele fails to amplify.
Warner JP, J Med Genet 1996
Electrophoreogram of repeat-primed
PCR (P1+P3+P4)
The axis shows migration time in
minutes. CAG allele sizes shown with
the arrows. The same people typed in
fig 2 were retyped. Note the
characteristic ladder with a 3 bp
periodicity.
(A) Both alleles give peaks and all the
intermediate priming sites give peaks.
There is a slight continuation of the
pattern beyond the maximum allele size
owing to mispriming at the end of the
repeat. This usually extends for less
than 30 bp.
(B) Both alleles give peaks as in (A).
(C) The ladder shows the presence of a
large CAG allele undetectable using
flanking primers.
Warner JP, J Med Genet 1996
Close up of traces shown
in fig 3. The axis shows
migration time in minutes.
CAG allele sizes shown
with the arrows.
(A) Detail from trace shown
in fig 3B.
(B) Detail from trace shown
in fig 3C.
Warner JP, J Med Genet 1996
• Restrikční štěpení DNA.
• Elektroforetické rozdělení
naštěpené DNA v
agarózovém gelu
Southern blot:
• 0,25 M HCl - depurinace
DNA
• 0,5M NaOH - denaturace
DNA, rozštěpení cukrfosfátové vazby v místě
depurinace (účinnější přenos
DNA z gelu na membránu).
• Alkalický přenos DNA na
membránu v 0.5 M NaOH
(vazba negativně nabité
DNA k pozitivně nabité
membráně), různé možnosti.
Myotonia congenita (MC)
Mutations in the genes coding skeletal muscle chloride channel 1
(CLCN1) and alpha subunit of voltage-gated sodium channel 4 (SCN4A)
CLCN1:
SCN4A:
• chromosome 7q35
• 23 exons
• mutations associated with
recesive and/or dominant
inheritance
• chromosome 17q23
• 24 exons
• mutations associated with
dominant inheritance
Sato et al. (2001) and Yu et al. (2004).
Rayan et al. (2013)
Myotonia congenita (MC); CLCN1 protein
CLCN1 exists as homodimer with each individual subunit forming Cl pore.
• The heterozygous situation - mixture of 25% WT channels,
25% channels carrying two mutant subunits, and 50%
channels carrying one WT and one mutant subunit.
• Dominant MC is due to dominant-negative effects of the
mutant subunit on the WT subunit.
Myotonia congenita (MC); CLCN1 mutations
• PTC mutations - recessive MC
• Missense mutations recessive or dominant MC.
• p.(Glu291Lys) is inherited
recessively; reversing the
charge is expected to have
drastic effects – misfolding and
degradation of the protein,
inability to create the dimer
structure.
• p.(Glu291Asp) is inherited
dominantly; conserving the
negative charge by replacing
glutamate with aspartate – subtle
effect on the protein structure.
Glutamate
Lysine
Aspartate
Myotonia congenita (MC); CLCN1 mutations
• A homology model of the human dimeric ClC-1 channel on the basis of
known crystallographic structure.
• Identification of AAs which form the dimer interface or the Cl- ion pathway.
• A search for mutations of AAs forming the dimer interface or the Cl- ion
pathway.
• A assesment of the correlation between the localisation of a mutation and
the type of MC (recessive/dominant).
• In case of mutations localised in the dimer interface, the correlation
between the localisation of a mutation and the dominant MC was
observed.
Réblová K. (2013)
Cederholm et al. (2010)