Triplet repeat disorders- chromosome.pdf

Triple repeat disorders

1. Triplet Repeat Disorders
Rajasekhar

7. Classification by Molecular Defect
•Point mutations
•Large deletions
•Trinucleotide repeat expansions
•Mitochondrial DNA mutations

8. Types of Mutations
•Single base pair substitutions
•missense, nonsense, splice site
•Deletions
•Duplications
•Inversions
•Insertions
•Repeat Expansions
Fourteen genetic neurodegenerative diseases and three fragile
sites have been associated with the expansion of (CTG)n
•(CAG)n , (CGG)n • (CCG)n , or (GAA)n • (TTC)n repeat
tracts.

9. Trinucleotide repeats in human genetic
disease

10. Contrasting Features of TRDs
Huntington
disease
AD CAG Coding region
(exon 1)
Fragile X
syndrome
X-linked CGG 5’-untranslated
region
Myotonic
dystrophy
AD CTG 3’-untranslated
region
Friedreich
ataxia
AR GAA Intron

11. Unusual Aspects of Inheritance of TRDs
•Anticipation: tendency toward earlier age of onset and/or greater
severity in each subsequent generation, due to progressive
expansion of the repeat length.
•Parent-of-origin effects
•Skewed X-inactivation
•Methylation effects
•Incomplete penetrance
•Variable expressivity
•Premutation alleles: asymptomatic, but unstable, with a tendency
to expand in the next generation

13. Trinucleotide Repeat Disorder

•Definition: a class of clinically heterogenous disorders,
defined by the presence of an abnormal and unstable
expansion of DNA—triplet repeats—in the mutant gene,
with up to 200 copies of the repeated trinucleotide
•Examples Hereditary—CAG repeats (Huntington’s
disease, Kennedy’s disease), CGG (fragile X syndrome),
GCT (myotonic dystrophy, spinal and bulbar muscle
atrophy); Acquired—some forms of colorectal CA

14. Trinucleotide repeat: a type of short tandem repeat
• The size of repeat region varies between individuals and is
polymorphic in normal individuals
• For some trinucleotide repeats, when the number of repeats exceeds
a certain threshold, a neurological disease results
7 repeats
8 repeats
CAG

15. Why Know About
Trinucleotide Repeat Disorders?
•Over 17 genetic different disorders that cause a significant proportion
of inherited neurological disease in adults
•Molecular diagnosis is available for diagnostic confirmation,
predictive testing, prenatal testing, preconception testing, and
preimplantation diagnosis.
•Genetic counseling issues are complex and important to understand as
are related ethical issues

16. Timeline of Gene Discoveries for
Trinucleotide Repeat Disorders
•1991Fragile X MR syndrome, SBMA
•1992Myotonic dystrophy
•1993Huntington disease, SCA1, FRAXE, DRPLA/HR
•1994Machado-Joseph disease/SCA3
•1996SCA2, Friedreich ataxia, SCA6
•1997SCA7
•1999SCA10, SCA5, SCA4
•2000sOther SCAs, Psychiatric disorders?

17. Major Features of Most
Trinucleotide Repeat Disorders
•Neurological/cognitive symptoms
•Many are autosomal dominant with variable expression (exceptions:
Friedreich ataxia (recessive); Spinobulbar muscular atrophy, Fragile X
syndrome, FRAXE MR - X-linked recessive)
•Later age of onset (exceptions: congenital myotonic dystrophy,
Fragile X syndrome, FRAXE)
•Meiotic and mitotic instability with some degree of anticipation in
many of the conditions

18. •Friedreich Ataxia:
–Autosomal recessive progressive neurological disorder, onset < 25 years with ataxia
due to expansion of GAA repeat in intron of FRATAXIN gene
•Spinocerebellar Ataxia (many types):
–Autosomal dominant progressive neurological disorder characterized by ataxia usually
in the 3rd or 4th decade due to expanded CAG repeat in coding exons of SCA genes
•Myotonic Dystrophy:
–Autosomal dominant progressive neurological disorder with variable expression and
anticipation due to expansion of 3’ UTR region of DMPK gene
•Fragile X syndrome:
–X-linked recessive mental retardation syndrome due to expansion of CGG repeat in 5’
UTR region of FRAXA gene
Anticipation is term that is used to describe the worsening of the
clinical phenotype with each successive generation. This results in
symptoms being manifested at an earlier age of onset, as well as with
more severe symptoms, in each successive generation.
Anticipation

19. 5’ UTR exon intron exon intron exon 3’ UTR
CGG

**FRAXA
FRAXE
GAA

FA
CAG

**HD
SCAs
SMBA
DRLP
A
CTG

MD
TRD can involve expansions of various repeats in coding
and non-coding regions of the gene

20. Anticipation, Meiotic Expansion, and Parent of Origin
Effects for Different Disorders
•HD paternal > maternal - mild
•SCA1paternal > maternal -mild
•DRPLApaternal > maternal - mild
•SMBApaternal > maternal - mild
•DMmaternal >>> paternal - significant
•FRAXAmaternal >>> paternal - significant
C
A
G

21. CAG (Glutamine) TRD
•CAG expansion leads to gain of function “neurotoxic” mutation
•Demonstrate that age of onset, rapidity of progression, and severity of
disorder correlate with increasing repeat size
•Associated with normal alleles of 5-34 repeats and disease alleles of
40-100 repeats with an unstable intermediate repeat range
•CAG codes for an amino acid called glutamine, there are eight TRDs
(DRPLA, HD, SBMA, SCA1, SCA1 SCA2 SCA3 SCA6 SCA7 are
collectively known as polyglutamine diseases: All these TRDs are
characterized by a progressive degeneration of nerve cells in certain
parts of the body.

22. Pathophysiology of
Expanded Polyglutamine Tracts
•Knock out mice lack the neurological phenotype
•Heterozygous transgenic mutant mice with expanded polyglutamine
tracts exhibit neurological phenotype
•Cell loss is an apoptotic event
•The ‘toxic fragment’ hypothesis, where proteins are cleaved into a short
toxic fragments with polyglutamine tracts that aggregate in the nucleus,
has been raised
•Association of CAG expansions with GAPDH suggests further roles for
regulation of cellular metabolism
•Key events regulating specificity of neuronal loss not understood

23. FMR1 in FRAXA Mental Retardation
•17 exon FMR1 gene cloned in 1991
•Highest FMR1 expression in neurons
and spermatogonia
•FMR1P associates with translating
ribososmes and is involved in
nucleocytoplasmic shuttling
•Approximate repeat ranges:
•6-45 CGGs (0-3 AGGs) Unmethylated, Stable, Normal
•46-60 CGGs (0-2 AGGs) Unmethylated, +/- Instability, Normal
•60-200 CGGs Unmethylated, Premutation - Unstable, Normal
•> 200 CGGs -Methylated, no FMR1, Unstable, Affected

24. Myotonic Dystrophy
•Common adult-onset muscular
dystrophy (1 in 8000 in Caucasians,
1 in 475 in Quebec
•Autosomal dominant - 19q13.2-.3
•Expansion of 3’ UTR CTG repeat in 15 exon myotonic dystrophy
protein kinase gene (DMPK)
–5 - 37normal
–50 - 90mild - cataract, balding, limited muscle involvement, >
50y 90 - 1000 classic muscle weakness, myotonia, cataracts,
onset 20-30y
–> 1000often congenital, hypotonia, developmental delays
•Instability of repeats - 10% expansion, 3% contraction.
•Congenital DM always due to maternal expansion

25. Friedreich’s Ataxia
•Most common inherited ataxia
•2-4/100,000 carrier frequency 1/60-1/100
•Onset range 4-40yrs
•Usually causes death by 4
th
and 5
th
decade
•96% of patients homozygous GAA expansion
•4% have one expanded allele and a loss of function point mutation in
other allele
•Frataxin is a highly conserved protein located in the inner
mitochondrial membrane.
•Decreased frataxin levels impairs biosynthesis of Fe-S cluster
containing protein causing decreased production of cellular energy
and increased free radical production.

26. Friedreich’s Ataxia
•FXN gene located on 9q21.11
•Friedreich ataxia results from an increased number of copies
(expansion) of the GAA trinucleotide repeat in the FXN gene. In
people with this condition, the GAA segment is abnormally
repeated 66 to more than 1,000 times. The length of the GAA
trinucleotide repeat appears to be related to the age at which the
symptoms of Friedreich ataxia appear. People with GAA segments
repeated fewer than 300 times tend to have a later appearance of
symptoms (after age 25) than those with larger GAA trinucleotide
repeats

27. Huntington Disease
•Average age of onset 40 years (range 2- >80 years); Progression over 10-25
years.
•Movement disorder: choreic movements, twitching, balance problems,
tracking problems, slowing of voluntary movement. In juvenile HD and in
late stages of HD, rigidity and dystonia.
•Cognitive dysfunction: problem solving, cognitive flexibility, short term
memory, visuospatial functioning; progression to a global subcortical
dementia
•Personality changes: depression, apathy, irritability, impulsive behavior,
affective disorders, rarely psychoses, increased alcohol use in early stages,
increased suicide rate

28. Huntington Disease

29. Huntington gene and CAG repeats
•Huntingtin gene on 4p16.3 cloned in 1993
•Disease mutation - CAG expansion in exon 1
–Repeat numberOutcome
–10-28:normal, no transmission of HD
–29-35:normal, paternal meiotic instability
–36-39:reduced penetrance (25%: 36 repeats, 90%: 39 repeats)
–40-100+:will develop HD if person lives long enough
•Increased meiotic instability in males - Paternal transmission of
expanded allele associated with over 3/4 of juvenile disease
• Encodes 348 kD huntingtin protein which is a target for caspase 3, a
protease associated with neuronal apoptosis

30. Pathology of Huntington Disease
•Brain atrophy involving caudate nucleus and putamen with loss of
striatal neurons and secondary atrophy of globus pallidus
•Dilation of lateral and third ventricles
•Additional atrophy throughout cortex, especially frontal and parietal
lobes
•Loss of small neurons precedes
larger neurons with neurons utilizing GABA
and enkephalin or substance P preferentially
•Fibrillary gliosis

31. Potential Roles of Huntingtin
•A handful of huntintin interacting proteins have been described and
suggest additional roles for the protein:
•HIP1 (homologous to yeast gene with cytoskeletal functions) with
affinity to normal sized tracts
•HIP2 which encodes an ubiquitin conjugating enzyme,
•HAP1 has affinity to larger polyglutamine tracts
•GADPH has direct affinity for polyglutamine tracts and is involved in
several key cellular functions
•EGF receptor complex where huntingtin binds to SH3 domains of
Grb2 and Ras-GAP suggesting some role in the EGF signaling
pathway

33. Molecular Detection of Trinucleotide Repeats:
Determine the size of the repeat
•‘Short’ Repeats (eg. HD):
–PCR based typing of alleles using primers directly
flanking repeat region - With appropriate controls and
size marker the size can be determined accurately
•Long repeats:determine size of the repeat
–PCR and Southern blot analysis
•Methlylation status (e.g. ,Fragile X)
•Immunoassays

34. Dentatorubropallidoluysian Atrophy
•DRPLA affects both the mind and body. It is characterized by abrupt muscle
jerking, involuntary movements, and eventual dementia.
•Both men and women equally affected including in young age.
•DRPLA gene lies on 12, and codes for atrophin-1 protein and is also named
“DRPLA”. Atrophin-1 is involved in the pathway that helps insulin take
effect in the body’s cells. Since insulin helps determine how cells utilize
their energy, it is essential that this pathway work smoothly so that cells can
function efficiently. If there is a kink in the plan, it could spell disaster for an
affected nerve cell.
•Typically, in asymptomatic individuals there are between 6 and 35 copies of
CAG in the DRPLA allele. In a person with the disease, however, the allele
has anywhere between 49 and 88 copies.

35. Spino-bulbar Muscular Atrophy
•SBMA occurs predominantly in males and is characterized by
weakness and atrophy of the proximal muscles. Difficulties with
swallowing and articulating speech are also common symptoms of
SBMA.
•The gene involved in SBMA is called the Androgen Receptor (AR)
gene is located on the X. In its normal state, then, the AR protein
helps cells carry out the instructions contained within DNA.
However, in people with SBMA, the protein has extra glutamines,
resulting from the extra CAGs in the AR gene. The extra glutamines
create an altered form of the AR protein that does not perform its
actions in the same way as the normal AR.
• Typically, in asymptomatic individuals there are between 9 and 36
copies of CAG in the AR allele. In a person with the disease,
however, the allele has anywhere between 38 and 62 copies
Kennedy diseases : Spinal cord and bulbar (part of brain)

36. Spinal Muscular Atrophy
•SMA was described independently by Werdnig and Hoffman in
1891. Werdnig described the condition as “neurogenic dystrophy
and Hoffman established the spinal nature of the disease.
•Spinal Muscular Atrophy (SMA) is a Motor Neuron Disease. It is
caused by the mutation of the Survival of Motor Neuron (SMN)
gene located on 5q13. It occurs due to the loss of motor neurons
within the spinal cord and brain. It results in the progressive
wasting away of muscles (atrophy) and muscle weakness. SMA can
affect people of all ages, races or genders, but the majority of cases
occur in infancy or childhood.

37. Type I spinal muscular atrophy (also called Werdnig-Hoffman
disease) is a severe form of the disorder that is evident at birth or within
the first few months of life. Affected infants are developmentally
delayed; most are unable to support their head or sit unassisted.
Children with this type have breathing and swallowing problems that
may lead to choking or gagging.
Type II spinal muscular atrophy is characterized by muscle weakness
that develops in children between ages 6 and 12 months. Children with
type II can sit without support, although they may need help getting to
a seated position. Individuals with this type of spinal muscular atrophy
cannot stand or walk unaided.

38. Type III spinal muscular atrophy (also called Kugelberg-Welander
disease or juvenile type) has milder features that typically develop
between early childhood and adolescence. Individuals with type III
spinal muscular atrophy can stand and walk unaided, but walking and
climbing stairs may become increasingly difficult. Many affected
individuals will require wheelchair assistance later in life.
The signs and symptoms of type IV spinal muscular atrophy often
occur after age 30. Affected individuals usually experience mild to
moderate muscle weakness, tremor, twitching, or mild breathing
problems. Typically, only muscles close to the centre of the body
(proximal muscles), such as the upper arms and legs, are affected in
type IV spinal muscular atrophy.

40. TRDs in Humans

41. Summary
•Trinucleotide repeat disorders account for a large proportion of
inherited neurological and mental retardation conditions
•Sensitive and specific DNA based diagnosis may be used for
diagnostic, predictive, and prenatal testing if desired
•Genetic counseling and education is useful for at-risk individuals to
make informed choice about testing options
•Huntington Disease is an autosomal dominant later onset progressive
neurodegenerative disorder due to expansion of a CAG repeat in
coding region
•Guidelines for predictive and prenatal HD testing are well-established
and serve as prototype for predictive testing for adult onset conditions.

42. These two proteins
are present only in
the brain, and this
finding could
explain why HD
only affects the
brain even though
the huntingtin
protein is present
throughout the body.
The number of CAG
repeats in the
huntington gene
determines how the
huntingtin protein
interacts with HIP-1
and HAP-1. As repeat
numbers increase,
huntingtin binds less
to HIP-1 and more to
HAP-1


Various experiments have revealed that the huntingtin protein interacts with two proteins:
huntingtin’s interactor protein (HIP-1) and huntingtin’s associated protein (HAP-1).