Post on 02-Jan-2016
Trinucleotide repeats Trinucleotide repeats (TNRs)(TNRs)
Dr. Derakhshandeh, PhDDr. Derakhshandeh, PhD
INTRODUCTIONINTRODUCTION
Trinucleotide repeats (TNRs) are microsatellite Trinucleotide repeats (TNRs) are microsatellite sequencessequences
Disease-causing repeat instability is an Disease-causing repeat instability is an important and unique form of mutationimportant and unique form of mutation
linked to more than 40 neurological, linked to more than 40 neurological, neurodegenerative and neuromuscular neurodegenerative and neuromuscular disorders. disorders.
I.g. Huntington's disease, myotonic dystrophy I.g. Huntington's disease, myotonic dystrophy and fragile X syndromeand fragile X syndrome
Trinucleotide repeatsTrinucleotide repeats
TNRs undergo high frequency mutagenesisTNRs undergo high frequency mutagenesis
To understand better the molecular To understand better the molecular mechanisms of TNR instability in cultured mechanisms of TNR instability in cultured cellscells
A new genetic assay was created using a shuttle A new genetic assay was created using a shuttle vectorvector
The shuttle vector contains a promoter-TNR-The shuttle vector contains a promoter-TNR-reporter gene construct whose expression is reporter gene construct whose expression is dependent on TNR length.dependent on TNR length.
The vector harbors the SV40 The vector harbors the SV40 oriori
(CAG•CTG)25–33 (CAG•CTG)25–33
The shuttle vector is propagated in The shuttle vector is propagated in cultured cellscultured cells
It recovered and analyzed in yeast using It recovered and analyzed in yeast using selection for reporter gene expression. selection for reporter gene expression.
Richard Pelletier, Nucleic Acids Research 2005 33(17):5667-5676Richard Pelletier, Nucleic Acids Research 2005 33(17):5667-5676
Disorders caused by Disorders caused by trinucleotide repeattrinucleotide repeat
First: the mutant repeats show both somatic and First: the mutant repeats show both somatic and germline instabilitygermline instability
Secondly:Secondly: an earlier age of onsetan earlier age of onset and increasing severity of phenotype in subsequent and increasing severity of phenotype in subsequent generations (anticipation) generations (anticipation)
Finally, the parental origin of the disease allele Finally, the parental origin of the disease allele can often influence anticipation can often influence anticipation
with paternal transmissions carrying a greater risk of with paternal transmissions carrying a greater risk of expansion for many of these disorders. expansion for many of these disorders.
Category of the trinucleotide Category of the trinucleotide repeat repeat (based on the relative location)(based on the relative location)
first subclass:first subclass:
–Repeats in non-coding sequences:Repeats in non-coding sequences:
For six diseasesFor six diseases
second subclass:second subclass:
–Exonic (CAG)n repeats Exonic (CAG)n repeats
code for polyglutamine tracts code for polyglutamine tracts
Repeats in non-coding sequencesRepeats in non-coding sequences
NON-CODING TRINUCLEOTIDE NON-CODING TRINUCLEOTIDE REPEAT DISORDERSREPEAT DISORDERS
Large and variable repeat expansions that Large and variable repeat expansions that result in multiple tissue:result in multiple tissue:– dysfunctiondysfunction– degeneration degeneration
Phenotypic manifestations within a Phenotypic manifestations within a disease are variabledisease are variable– Degree of somatic heterogeneityDegree of somatic heterogeneity
Pre-mutationsPre-mutationsThe larger mutations often are transmitted from The larger mutations often are transmitted from a small pool of clinically silent intermediate size a small pool of clinically silent intermediate size expansionsexpansions
CGG, GCC, GAA, CTG and CAGCGG, GCC, GAA, CTG and CAG
particular trinucleotide sequence particular trinucleotide sequence ++ its location with respect to a geneits location with respect to a gene
– Important defining factors in dictating the unique Important defining factors in dictating the unique mechanism of pathogenesis for each disease mechanism of pathogenesis for each disease
Fragile X syndromeFragile X syndrome
Fragile X Fragile X SyndromeSyndrome
Fragile X syndromeFragile X syndrome
Fragile X syndrome (FRAXA)Fragile X syndrome (FRAXA)
Fragile XE MR (FRAXE)Fragile XE MR (FRAXE)
1 in 2000 boys1 in 2000 boys
1 in 4000 girls1 in 4000 girlsare estimated to be affectedare estimated to be affected
Fragile X SyndromeFragile X Syndromemost common inherited form of familial mental most common inherited form of familial mental retardationretardation
(CGG)n trinucleotide expansion in the FMR1 gene (CGG)n trinucleotide expansion in the FMR1 gene leading to the typical Martin-Bell phenotypeleading to the typical Martin-Bell phenotype
Clinical features vary depending on ageClinical features vary depending on age
Expansion of a (CCG)n repeat in the FMR2 gene Expansion of a (CCG)n repeat in the FMR2 gene corresponds to the FRAXE fragile site corresponds to the FRAXE fragile site
ItIt lies distal to FRAXA lies distal to FRAXA
It’sIt’s associated with mental retardation, but it is associated with mental retardation, but it is less frequent and lacks a consistent phenotype less frequent and lacks a consistent phenotype
The transcription of the FMR1 gene of normal and premutation alleles. Both alleles 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). are translated into FMRP, which is demonstrated by Western blotting (lane N and P). The full mutation allele is hypermethylatedthereforetranscribed, which resultabsence The full mutation allele is hypermethylatedthereforetranscribed, which resultabsence
of FMRP (lane F)of FMRP (lane F)
Repeats in non-coding sequencesRepeats in non-coding sequences
Sequence of the 5'-UTR region of the Sequence of the 5'-UTR region of the FMR1FMR1 gene gene
Sequence of the 5'-UTR region of the FMR1 gene
Fragile X syndrome (FRAXA)Fragile X syndrome (FRAXA)
Mental retardationMental retardation
MacroorchidismMacroorchidism
Some dysmorphic featuresSome dysmorphic features
HyperactivityHyperactivity
Fragile X Fragile X SyndromeSyndrome
Fragile X syndrome (FRAXA)Fragile X syndrome (FRAXA)expansion of a polymorphic (CGG)n repeat in expansion of a polymorphic (CGG)n repeat in the 5'-untranslated region (UTR)the 5'-untranslated region (UTR)
> 230 trinucleotides > 230 trinucleotides
hypermethylation together with a CpG island hypermethylation together with a CpG island within the within the FMR1FMR1 promoter region promoter region
transcriptional silencing of the transcriptional silencing of the FMR1FMR1 gene gene
reduced reduced FMR1 FMR1 transcription and loss of gene transcription and loss of gene product (FMRP) product (FMRP)
Fragile XE MR (FRAXE)Fragile XE MR (FRAXE)
mild mental retardationmild mental retardation
variable behavior abnormalities variable behavior abnormalities
expansion of a polymorphic (GCC)n repeat expansion of a polymorphic (GCC)n repeat
in the promoter region of the in the promoter region of the FMR2FMR2 gene gene
the expanded repeats are hypermethylatedthe expanded repeats are hypermethylated
leading to transcriptional silencing of leading to transcriptional silencing of FMR2FMR2
subsequent loss of gene product (FMR2) subsequent loss of gene product (FMR2)
Friedreich ataxia Friedreich ataxia (FRDA)(FRDA)
Friedreich ataxia (FRDA)Friedreich ataxia (FRDA)
autosomal recessive autosomal recessive the only triplet repeat disorder the only triplet repeat disorder that does not show anticipationthat does not show anticipationAtaxia (Ataxia (loss of voluntary muscular loss of voluntary muscular coordination) coordination)
Diminished reflexesDiminished reflexesCardiomyopathy (Cardiomyopathy (heart enlargement)heart enlargement)
Diabetes Diabetes Degeneration in the spinal cord Degeneration in the spinal cord
Friedreich ataxiaFriedreich ataxia
FRDA is caused by a large intronic GAA repeat FRDA is caused by a large intronic GAA repeat expansion expansion
located on chromosome 9 (Gene:X25/Potein: located on chromosome 9 (Gene:X25/Potein: frataxinfrataxin))
which leads to reduced which leads to reduced genegene expression expression
The expanded AT-rich sequence most probably The expanded AT-rich sequence most probably causes causes
self-association of the GAA/TTC tract, which self-association of the GAA/TTC tract, which stabilizes the DNA in a triplex structure stabilizes the DNA in a triplex structure
Repeats in non-coding sequencesRepeats in non-coding sequences
FRDA & triplex structure FRDA & triplex structure A novel DNA structureA novel DNA structuresticky DNAsticky DNAlengths of (GAA.TTC)n lengths of (GAA.TTC)n in intron 1 of the frataxin gene of in intron 1 of the frataxin gene of Friedreich's ataxia patientsFriedreich's ataxia patientsSticky DNA is formed by the association of Sticky DNA is formed by the association of two purine.purine.pyrimidine (R.R.Y) two purine.purine.pyrimidine (R.R.Y) triplexes triplexes in negatively supercoiled plasmids at in negatively supercoiled plasmids at neutral pH neutral pH
Models of Models of structures structures that may that may mediate mediate mRNA mRNA synthesis synthesis and DNA and DNA replication replication inhibition inhibition by by GAA·TTC GAA·TTC
repeatsrepeats
in FRDA patientsin FRDA patients(GAA.TTC) (> 59 repeats)(GAA.TTC) (> 59 repeats)– the lengths of (GAA.TTC) (> 59 repeats) the lengths of (GAA.TTC) (> 59 repeats) – inhibit transcription in vivo and in vitro inhibit transcription in vivo and in vitro – adopt the sticky conformation adopt the sticky conformation
(GAAGGA.TCCTTC)65(GAAGGA.TCCTTC)65– found in intron 1found in intron 1– does not form sticky DNA does not form sticky DNA – does not inhibit transcriptiondoes not inhibit transcription– or associate with the disease or associate with the disease
Sakamoto,et al. MMol Cell. 1999 Apr;3(4):465-75Sakamoto,et al. MMol Cell. 1999 Apr;3(4):465-75..
frataxin is found in the mitochondria of frataxin is found in the mitochondria of humanshumanswe do not yet know its functionwe do not yet know its function there is a very similar protein in yeast, there is a very similar protein in yeast, YFH1, YFH1, YFH1 is involved in controlling:YFH1 is involved in controlling:– iron levelsiron levels– and respiratory functionand respiratory function
Frataxin and YFH1 are so similar, studying Frataxin and YFH1 are so similar, studying YFH1 may help us understand the role of YFH1 may help us understand the role of frataxin in FRDA frataxin in FRDA
Reduced Reduced X25X25 mRNA mRNA
decreases frataxin levelsdecreases frataxin levelsa partial loss of frataxin function a partial loss of frataxin function Disruption of the yeast Disruption of the yeast X25X25 homolog homolog (YFH1):(YFH1):– abnormal accumulation of mitochondrial ironabnormal accumulation of mitochondrial iron– loss of mtDNAloss of mtDNA– multiple iron–sulfur-dependent enzyme multiple iron–sulfur-dependent enzyme
deficiencies deficiencies – increased sensitivity to oxidative stress increased sensitivity to oxidative stress
Frataxin : Frataxin : – hypersensitivity to iron and H2O2 stress hypersensitivity to iron and H2O2 stress
Frataxin insufficiencyFrataxin insufficiency
frataxin insufficiency may result in frataxin insufficiency may result in abnormal iron–sulfur homeostasis abnormal iron–sulfur homeostasis
mitochondrial dysfunctionmitochondrial dysfunction
free radical productionfree radical production
oxidative stressoxidative stress
cellular degenerationcellular degeneration
Wong, A, et al. Wong, A, et al. Hum. Mol. Genet.Hum. Mol. Genet., , 88, 425–430 (1999) , 425–430 (1999)
Myotonic dystrophy Myotonic dystrophy (DM)(DM)
Myotonic dystrophy (DM)Myotonic dystrophy (DM)
multisystem disordermultisystem disorder
highly variable phenotypeshighly variable phenotypes
AnticipationAnticipation
MyotoniaMyotonia
muscle weakness muscle weakness
Developmental abnormalitiesDevelopmental abnormalities
mental handicapmental handicap
HypotoniaHypotonia
respiratory distress are often evident in the more respiratory distress are often evident in the more severe congenital myo tonic dystrophy (CDM). severe congenital myo tonic dystrophy (CDM).
DMDMCTG trinucleotide repeatCTG trinucleotide repeat
in the 3'-UTR of the protein kinase gene, in the 3'-UTR of the protein kinase gene, DMPKDMPK
The CTG repeat is located within the The CTG repeat is located within the promoter of a upstream homeobox gene promoter of a upstream homeobox gene
Loss of function of either or both of these Loss of function of either or both of these proteins could contribute to some of the proteins could contribute to some of the features in DMfeatures in DM
Korade-Mirnics, Z. et al. (1998) Korade-Mirnics, Z. et al. (1998) Nucleic Acids Res.Nucleic Acids Res., , 2626, , 1363–1368 1363–1368
Repeats in non-coding sequencesRepeats in non-coding sequences
Spinocerebellar ataxia type 8 Spinocerebellar ataxia type 8 (SCBA8)(SCBA8)
Spinocerebellar ataxia type 8 Spinocerebellar ataxia type 8 (SCBA8)(SCBA8)
progressive ataxia progressive ataxia
with cerebellar atrophywith cerebellar atrophy
decreased brisk reflexesdecreased brisk reflexes
SCA8SCA8 is expressed primarily in the is expressed primarily in the brainbrain
is caused by an expanded CTG is caused by an expanded CTG repeat in its 3'-terminal exonrepeat in its 3'-terminal exon (~110–250 (~110–250 repeats)repeats)
Repeats in non-coding sequencesRepeats in non-coding sequences
ParkinsonismParkinsonism
(PD)(PD)
SCA-2 and SCA-3 repeats in SCA-2 and SCA-3 repeats in ParkinsonismParkinsonism
expansion of triplet repeats expansion of triplet repeats encoding polyglutamine encoding polyglutamine (polyQ) tracts (polyQ) tracts
POLYGLUTAMINE POLYGLUTAMINE DISEASESDISEASES
POLYGLUTAMINE DISEASESPOLYGLUTAMINE DISEASES
have repeat expansions that are have repeat expansions that are much smaller in size and variation much smaller in size and variation
characterized by progressive characterized by progressive neuronal dysfunction neuronal dysfunction
begins in mid-life and results in begins in mid-life and results in severe neurodegeneration severe neurodegeneration
POLYGLUTAMINE DISEASESPOLYGLUTAMINE DISEASES
different polyglutamine diseases have different polyglutamine diseases have little in common:little in common:
–the length of the expansion > 35–40the length of the expansion > 35–40–the greater the number of glutamine the greater the number of glutamine
repeats in a proteinrepeats in a proteinthe earlier the onset of disease and the earlier the onset of disease and the more severe the symptomsthe more severe the symptoms
Expansion disordersExpansion disordersMany major neurodegenerative Many major neurodegenerative diseases:diseases:
Alzheimer's diseaseAlzheimer's disease
Parkinson's diseaseParkinson's disease
Huntington DiseaseHuntington Disease
Alzheimer's diseaseAlzheimer's disease
Alzheimer's diseaseAlzheimer's diseasevarious types of familial Alzheimer's various types of familial Alzheimer's disease (AD) genes disease (AD) genes
mutants of amyloid precursor protein mutants of amyloid precursor protein (APP)(APP)
polyglutaminepolyglutamine repeat Q79 repeat Q79
Huntington's diseaseHuntington's disease
Huntington's diseaseHuntington's disease
inherited as a autosomal dominant inherited as a autosomal dominant
a polymorphic CAG repeat tract in a polymorphic CAG repeat tract in exon 1, which is 35 units in length exon 1, which is 35 units in length
Huntingtin in mitochondrial energy Huntingtin in mitochondrial energy metabolismmetabolism
HD CAG size determines [ATP/ADP] in HD CAG size determines [ATP/ADP] in lymphoblastoid cellslymphoblastoid cells
HD CAG repeat implicates a dominant property HD CAG repeat implicates a dominant property of huntingtin in mitochondrial energy metabolismof huntingtin in mitochondrial energy metabolism
Ihn Sik Seong, et al.Human Molecular Genetics 2005 14(19):2871-2880Ihn Sik Seong, et al.Human Molecular Genetics 2005 14(19):2871-2880
HDHD CAG size determines [ATP/ADP] CAG size determines [ATP/ADP] in lymphoblastoid cellsin lymphoblastoid cells
A Polymorphic Trinucleotide Repeat at A Polymorphic Trinucleotide Repeat at DXS8170DXS8170 in in the Critical Region of X-Linked Retinitis Pigmentosa the Critical Region of X-Linked Retinitis Pigmentosa
Locus Locus RP3RP3 at Xp21.1 at Xp21.1
possible mechanism of cell deathpossible mechanism of cell death
the abnormally long sequence of the abnormally long sequence of glutamines acquires a shape that prevents glutamines acquires a shape that prevents the host protein from folding into its proper the host protein from folding into its proper shape. shape.
if, the length of polyglutamine repeats is if, the length of polyglutamine repeats is longer than the critical value found in longer than the critical value found in disease, it acquires a specific shape called disease, it acquires a specific shape called a β-helix. a β-helix.
QQ37 37 chain chain under under conditiconditions in ons in which which
it it adopts adopts
β-β-strand strand topolotopolo
giesgies
SummarySummarySince the identification in 1991 of repeat Since the identification in 1991 of repeat instability as a disease-causing mutation, gene-instability as a disease-causing mutation, gene-specific repeat instability is now known to be the specific repeat instability is now known to be the mutational cause of at least 40 neurological, mutational cause of at least 40 neurological, neurodegenerative and neuromuscular neurodegenerative and neuromuscular diseases. diseases.
Both germline (parent-to-offspring) and tissue-Both germline (parent-to-offspring) and tissue-specific somatic instability occurs. specific somatic instability occurs.
There are unique and common effectors for the There are unique and common effectors for the instability of different repeat sequences, instability of different repeat sequences, although each disease or locus is unique. although each disease or locus is unique.