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Fragile X syndrome
X linked disease, 1:4000 in males
Clinical manifestations
• Cognitive difficulties
• Attention and behavioral problems
• Macro-orchidism
• Mild facial dysmorphologies
• Connective tissue abnormalities
• Anticipation
Fragile X chromosomes
Fragile X chromosomes
FRAXA- rare folate-sensitive fragile
sites: mutation stages
• At the loci of fragile sites there are naturally occurring
polymorphisms of the number of copies of tandem
repeats of the trinucleotide repeat CGG.
• The fragile site is seen cytogenetically
• The gene associated with the repeat is apparently
normally expressed.
• Beyond the premutation is the full mutation where the
fragile site is seen and the relevant gene is
transcriptionally silenced.
• Normal chromosomes 5-55 copies of CGG
• Premutation ~55-230 copies of CGG
• Full mutation >~230 copies of CGG
The mutation - (CGG)n
• In exon 1 - 5’ untranslated region.
• Transmitting males
• Normal alleles - 6-53, full mutation >230
repeats,
• However there is a gray zone
• Punctuations by AGGs at the 3’ end: (CGG)8-15
AGG (CGG)9-13 AGG(CGG)x
• In very unstable alleles the 2 AGG are lost
• 33 or less uninterrupted CGGs - stability,
• >39 uninterrupted CGGs - instability
The FMR protein (FMRP)
• The FMRP protein probably forms
ribonucleoprotein (RNP) complexes, has 3 RNAbinding motifs, a RGG-box and 2 KH-domains.
• Has a key role in regulation of translation.
• Binds a subset of brain mRNAs including its
own.
• In brain, it shuttles a subset of mRNA to
the dendritic spines.
•Absence of the protein (deletion) cause
immature dendritic spine morphology.
Fragile X syndrome - consequences of
expansion
• Methylation of the C (CGG) - due to
mispaired Cs in secondary structures, are
templates for methylation.
• Methylation of the promoter is accompanied
(reason unknown), leading to lack of
transcription initiation.
Disease mechanism -protein loss of
function
Repeat expansion diseases
•trinucleotide diseases: CGG, CAG and
CTG
•tetranucleotide - MD type 2, CCTG in
intron 1.
•pentanucletodie - SCA10, ATTCT up
to 4,500 repeats in intron 1.
Repeat location
Coding disorders
Diseases with a CAG expansion within the
coding region, produces an enlarged
polyglutamine tract Huntigngton, Kennedy,
Spinocerebellar ataxia type 1…) .
Non coding disorders
1. Untranslated 5’ (Fragile X,syndrome,
Spinocerebellar Ataxia type 12..)
2. Untranslated 3’ (myotonic dystrophy)
3. Intron (Friedreich ataxia, )
Features of Diseases caused by repeat instability
 40 neurodegenerative and neuromuscular diseases
are caused by gene-specific instability of certain
repeat tracts.
 Most diseases are caused by triple repeat
expansion. Some by longer repeated unit.
 The common triplets are: (CTG)* (CAG) found in at
least 14 diseases, (CCG)*(CGG), GCG*CGC, GAA.
 Not all possible triplets are found to be expanded in
these diseases. Only those that have the ability to
form unusual DNA structures.
CAG expansion in the coding sequence
• Huntington’s disease
• Spinocerebellar ataxias
• Kennedy’s disease
Gain of function at the protein level
• The most common trinucleotide repeat causing disease by
altering protein physiology is the (CAG)n.
• (CAG)n in the coding region of a gene. Although expansion
sizes, structures, cellular localization and functions of the
resulting proteins differ, all (CAG)n-induced diseases are
neurodegenerative disorders.
• All disorders are associated with neuronal aggregates that
contain the disease-causing gene product.
• PolyQ stretches have an inherent ability to aggregate.
Apart from binding to many other proteins, glutamine also
shows self-interaction.
• Once polyQ stretches exceed a certain length, they
are no longer soluble and form aggregates.
Gain of function at the protein level
• The threshold length, above which in vitro
aggregation takes place, is strikingly similar to the
threshold that causes disease, (40 CAGs).
• Although it is tempting to hold the aggregates
responsible for the development of disease, some
evidence exists that the large aggregates are not
the primary cause of cell toxicity.
Features of Diseases caused by repeat instability
• Dynamic
• Repeat tract length – correlates with age of onset
and disease severity.
• Anticipation - The number of inherited repeats
increases significantly from generation to
generation causing earlier onset and faster
progression
• Longer repeats – more likely to undergo expansion.
• In many diseases (CAG repeats, glutamine) there
is a late onset.
Hypothesis
• The extended glutamine portion has a
gain of toxic function which leads to a
cumulative damage in the affected
cells, possibly in the form of
glutamine aggregate formation.
• A new mechanism was recently
suggested.
Kaplan et al, 2007
Kaplan et al, 2007
A universal mechanism
• Onset and progression of the disease are
determined by the rate of expansion of the
tri-nucleotide repeat in certain cells in the
patient’s body.
• The disease manifests when the trinucleotide
repeat expands beyond a certain threshold in a
sufficient number of these cells and
progresses as more and more cells do so.
Kaplan et al, 2007
Friedreich’s Ataxia
• The most common heritable form of
ataxia associated with progressive gait and
limb ataxia
• Degeneration of large sensory neurons
• Death is due to cardiac failure
•Autosomal recessive,thus no anticipation
• FRDA gene encoded frataxin
• In FRDA disease the protein levels are
severely reduced causing mitochondrial
dysfunction
Friedreich’s Ataxia
• The disease is caused by GAA-CTT
repeat expansion
• The repeat is located in intron 1,
within an Alu sequences of the FRDA
gene
• Normal alleles 7-34 repeats
• Disease causing >100 repeats
• GAG interruptions
• Very low mRNA levels
Model
• Transcription-coupled triplex formation
• The triplex blocks progress by RNA
polymerase
• No transcripts are produced
• No protein
• Loss of function
Disease Mechanisms
• Loss of function at the protein level
(fragile X)
• Gain-of-function at the protein level
(Huntington)
• Loss of function at the RNA level
(Friedreich’s Ataxia(
• RNA gain of function (DM)
Myotonic dystrophy type 1
• Autosomal Dominant inheritance
• The most common form is adult muscular
dystrophy, 1:8000 births
• A wide spectrum of clinical phenotype:
effecting the skeletal muscles (myotonia),
eye, heart, endocrine systems,and more
• CTG in the 3’ untranslated region of the
DMPK gene in 98% of the cases
• The remaining 2% are DM type 2 and
found to carry a CCTG expansion in the
ZNF9 gene.
Myotonic dystrophy type 1
• 5-27 repeats in normal alleles
• Several thousands (>3,000) in patients
• Variable phenotype and the anticipation are corrleated
with the number of expanded repeats
• The DMPK protein is a serin threonin protein kinase
expressed exclusively in muscle and heart
•Autophosphorylation and phosphorylation of Histon H1 and
many other proteins such as myosin phosphatase.
• DMPK phoshorylation inhibits the mypt1 activity -> high
levels of the phos protein -> Ca+ sensitization of smooth
muscles and cytoskeleton changes in non-muscle cells.
Myotonic dystrophy type 1
• The mutation cause dysfunction of of DMPK activity.
• Suggestions: haploinsufficiency
• However, mouse models did not support it:
1. Heterozygous and homozygous DMPK knockout mice
showed no myotonia (the major symptom) nor catarct
characteristics of DM1.
2. However, these mice developed late-onset skeletal
myopathy and altered calcium ion homeostasis.
3. Cardiac phenotype
Conclusion: haploinsufficiency (loss of function) plays a role
but inactivation of the DMPK alone cannot be responsible
for the complete DM1 phenotype.
Myotonic dystrophy type 1
• The expanded repeat containing mRNAs were retained in
nuclear foci.
• This is thought to occur because expanded RNA (CUG)n
molecules.
• Form secondary structures such as hairpins.
• In the foci, RNA molecules sequester RNA-binding
proteins (RNA-BPs), which bind specifically to (CUG)n
(CUG-binding proteins (CUG-BPs).
• Since mRNA processing, including splicing, is normally
regulated by a dynamic complex of RNABPs, the function
of these proteins in the presence of expanded (CUG)n was
investigated.
Myotonic dystrophy type 1
• MBNL1 is a specific CUG-BP.
• Proteins that are essential in terminal
differentiation of muscle and
photoreceptor cells.
• It accumulates in the nuclear foci in DM1
cells, so that MBNL1 cannot exert its
normal function during a critical period of
cell differentiation.