Trinucleotide repeats (TNRs)

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Trinucleotide repeats (TNRs)

• Dr. Derakhshandeh, PhD

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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

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Trinucleotide repeats

• TNRs undergo high frequency mutagenesis
• To understand better the molecular mechanisms of
  TNR instability in cultured cells

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• A new genetic assay was created using a shuttle
  vector
• The shuttle vector contains a promoter-TNR-reporte
  r gene construct whose expression is dependent on
  TNR length.

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• The vector harbors the SV40 ori
• (CAGCTG)2533

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• 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

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Expanded polyglutamine induced cell death

• the expanded polyglutamine
• responsible for degeneration of neurons
• Insertion of polyglutamine coding region into a
  nonpathogenic gene in nerve cell
• death in transgenic mice (Ordway, J.M. et. al.
  1997)
• an expanded polyglutamine tract
• induced cell death in culture cells (Bok KS, et
  al.1999)

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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.

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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

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(spinocerebellar ataxia 3)
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Repeats in non-coding sequences
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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

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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

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• Fragile X syndrome

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Fragile X Syndrome
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Fragile X syndrome

• Fragile X syndrome (FRAXA)
• Fragile XE MR (FRAXE)
• 1 in 2000 boys1 in 4000 in birth
• are estimated to be affected

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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
• Its associated with mental retardation, but it
  is less frequent and lacks a consistent phenotype
  

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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 hypermethylated, which resultabsence of FMRP
(lane F)
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Fragile X syndrome (FRAXA)

• expansion of a polymorphic (CGG)n repeat in the
  5'-untranslated region (UTR)
• gt 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)

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Repeats in non-coding sequences
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Sequence of the 5'-UTR region of the FMR1 gene
Sequence of the 5'-UTR region of the FMR1 gene
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Fragile X syndrome (FRAXA)

• Mental retardation
• Macroorchidism
• Some dysmorphic features
• Hyperactivity

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Fragile X Syndrome
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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)

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• Friedreich ataxia (FRDA)

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Ch 9
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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

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(No Transcript)
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Friedreich ataxia

• FRDA is caused by a large intronic GAA repeat
  expansion
• located on chromosome 9 (GeneX25/Protein
  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

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Repeats in non-coding sequences
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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

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Models of structures that may mediate mRNA
synthesis and DNA replication inhibition by
GAATTC repeats
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in FRDA patients

• (GAA.TTC) (gt 59 repeats)
• the lengths of (GAA.TTC) (gt 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 Apr3(4)465-75.

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• 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

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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 ironsulfur-dependent enzyme
  deficiencies
• increased sensitivity to oxidative stress
• Frataxin
• hypersensitivity to iron and H2O2 stress

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Frataxin insufficiency

• frataxin insufficiency may result in abnormal
  ironsulfur homeostasis
• toxic side-effect of accumulated iron
• mitochondrial dysfunction
• free radical production
• oxidative stress
• cellular degeneration
• Wong, A, et al. Hum. Mol. Genet., 8, 425430
  (1999)

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• Myotonic dystrophy (DM)

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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).

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(No Transcript)
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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, 13631368

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Repeats in non-coding sequences
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• Spinocerebellar ataxia type 8 (SCBA8)

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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 (110250 repeats)

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(No Transcript)
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Repeats in non-coding sequences
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• Parkinsonism
• (PD)

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SCA-2 and SCA-3 repeats in Parkinsonism

• expansion of triplet repeats encoding
  polyglutamine (polyQ) tracts

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• POLYGLUTAMINE DISEASES

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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

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POLYGLUTAMINE DISEASES

• different polyglutamine diseases have little in
  common
• the length of the expansion gt 3540
• the greater the number of glutamine repeats in a
  protein
• the earlier the onset of disease and the more
  severe the symptoms

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Expansion disorders

• Many major neurodegenerative diseases
• Alzheimer's disease
• Parkinson's disease
• Huntington Disease

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• Alzheimer's disease

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Alzheimer's disease

• various types of familial Alzheimer's disease
  (AD) genes
• mutants of amyloid precursor protein (APP)
• polyglutamine repeat Q79

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• Huntington's disease

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Huntington's disease

• inherited as a autosomal dominant
• a polymorphic CAG repeat tract in exon 1, which
  is 35 units in length

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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

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HD CAG size determines ATP/ADP in
lymphoblastoid cells
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A Polymorphic Trinucleotide Repeat at DXS8170 in
the Critical Region of X-Linked Retinitis
Pigmentosa Locus RP3 at Xp21.1
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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 ß-helix.

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Q37 chain under conditions in which it adopts
ß-strand topologies
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Summary

• Since the identification in 1991 of repeat
  instability as a disease-causing mutation,
  gene-specific 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
  tissue-specific somatic instability occurs.
• There are unique and common effectors for the
  instability of different repeat sequences,
  although each disease or locus is unique.