Lecture 3

Question 1

Repeat expansion Disorders:

Answer 1

• Diseases caused by expansion of repetitive DNA sequences
• Initially triplet repeat/trinucleotide repeat expansions (FXS, SBMA, DM, HD etc.)
• Penta- (SCA10 & SCA31), hexa- (SCA36 & ALS/FTD) & more complex (EPM1) pathological expansions exist

Question 2

Repeat expansion disorders are…

Answer 2

Most autosomal dominant and GOF- \Look at diarma

Question 3

What are the two main types of effect of mutations ( at what level) ?

Answer 3

Protein or RNA

Question 4

Disease causing expansion

Answer 4

can occur throughout gene

Question 5

Fragile X syndrome –

Answer 5

• X-linked condition
- fully penetrant males
- ~50% penetrance females
• Long face, protruding ears, low muscle tone, macroorchidism(large testicles)
• Learning disabilities – normal IQ to severe mental retardation
• Cytogenetic abnormality 1969
• FMR1 cloned 1991

Question 6

FMRP-

Answer 6

• FXS caused by loss of FMRP function
• RNA-binding protein
• Associated with polyribosomes & mRNA granules transported to synapses and translation response to neural activity
• Functions as translational repressor, stalls protein synthesis during mRNP transport
• Important for LTP, learning & memory

Question 7

Long expansion in FMRP causes what ??

Answer 7

Fragile X syndrome

Question 8

Different lengths expansions

Answer 8

Give different diseases

Question 9

FXTAS-

Answer 9

• 55-200 CGG repeats in FMR1
• Late onset, >50
• Intention tremor & ataxia
• White matter lesions middle cerebellar peduncle
• Ubiquitinated inclusions
• Normal FMRP levels → RNA toxicity
Protein still made inspite of long repeats GOF via RNA toxicity whereas fragile X is LOF

Question 10

What is the effect of really long repeats ?

Answer 10

Interfere with the transcription and translation

- LOF no protein e.g Fragile X syndrome

Question 11

Polyglutamine disorders-

Answer 11

• CAG triplet repeat expansion disorders
• 1991 - spinobulbar muscular atrophy (SBMA) or Kennedy’s disease
• 1993 - Huntington disease (HD) high prevalence in caucasions
• 1994 - Dentatorubral-pallidoluysian atrophy (DRPLA) Japanese equivalent of Huntingtons – low prevalence in the uk
• 1993-2001 - spinocerebellar ataxia (SCA) types 1,2,3,6,7 and 17 ( now over 50)

Question 12

Common features of polyglutamine diseases-

Answer 12

• Autosomal dominant inheritance (except SBMA – X-linked recessive)
• Genetic anticipation (next slide)
• Inverse relationship between CAG repeat length and age of onset
• Toxic “gain of function” mechanism
• Protein accumulation and aggregation
Repeat gets longer age of onset gets earlier

Question 13

Polyglutamine diseases show genetic anticipation-

Answer 13

• Age of onset becomes earlier in successive generations
• Mainly associated with paternal transmission
• Explained by inter-generational repeat expansions during spermatogenesis
As repeat get longer during generations the age of onset gets longer

Question 14

Huntington disease (HD)-

Answer 14

• Huntington’s chorea described in 1872 by George Huntington
• Hereditary progressive neurodegenerative disorder
• Fatal - death usually 15-20 y after onset
• Frequency 4-10/100,000
• Autosomal dominant inheritance (before mendels work)
• Penetrance nearly 100%- very likely for disease to manifest if you have a long enough life span

Question 15

HD Pathology-

Answer 15

• Severe atrophy of the striatum
• Cortical atrophy – overall reduction in brain size
• Loss of GABAergic medium-sized spiny striatal neurones (MSN)
• Juvenile cases more severe - widespread neuronal loss ( in cerebellar)
• Astrogliosis and microglial activation

Question 16

Neuronal loss in the Striatum
Caudate nucleus doesn’t bulge into internal capsule/lateral ventricle
Huge neuronal loss in the caudate
are pathological signs of what disease ??

Answer 16

Huntingtons

Question 17

HD geen and mutation

Answer 17

Tend to occur through paternal transmission

Question 18

Toxic gain of function mechanism-

Answer 18

• Htt knockout embryonic lethal E7.5
• +/- mice normal – not haploinsufficiency
• Rescue embryonic lethality by breeding +/- mice with 72Q YAC transgenics
- 72Q/- mice develop normally, show HD-like symptoms as adults

Question 19

evidence for GOF

Answer 19

Yac can rescue embryonic lethal phenotype –

Question 20

HTT aggreagation

Answer 20

Htt protein aggregation in HD cortex-
Aggregates detected with anti-huntingtin or anti-ubiquitin antibodies
Range of nuclear and cytoplasmic pathology
No direct correlation between aggregation and neuronal

Question 21

Protein sequestration-

Answer 21

• Many proteins (especially transcription factors) have long (15-42) non-pathological polyglutamine tracts
• Proteins with non-pathological polyglutamine tracts (e.g. TBP & CBP) can be sequestered into mutant polyglutamine aggregates

Question 22

Transcriptional dysregulation in HD-

Answer 22

• CREB-binding protein (CBP) - transcriptional co-activator
• Present limiting amounts in cells
• Possesses histone acetyltransferase (HAT) activity
• Decreased histone acetylation & global dysregulation of transcription seen in HD

Question 23

Histone deacetylase (HDAC) inhibitors as potential therapeutic agents in what disease ??

Answer 23

HD

-Silence genes using HDAC inhibitors

Question 24

SBMA-

Answer 24

• Adult onset neuromuscular disease
• Muscle weakness and wasting especially in the extremities, face and throat
• Speech & swallowing difficulties, muscle cramps
• Degeneration of spinal and bulbar motor neurones
• Normal lifespan unlike ALS
• CAG repeat expansion in androgen receptor
• X-linked recessive – why? Whereas all others autosomal dominant ?!

Question 25

Examples of neurodegenerative & neuromuscular disorders caused by non-coding repeat expansions

Answer 25

• Myotonic dystrophy • Fragile X-associated tremor/ataxia syndrome – 60-200 CGG in 5’-UTR of FMR1 • Spinocerebellar ataxia 8, 10, 12, 31 & 36 • ALS/FTD – intronic GGGGCC in C9ORF72 Pathological non-coding repeat expansions tend to be longer than disease-causing coding repeat expansions

Question 26

Myotonic dystrophy-

Answer 26

• Autosomal dominant neuromuscular disease • Multisystemic – muscle, cardiac, cognitive, behavioural, eye, endocrine symptoms • 1:20,000 births • Congenital, childhood & adult forms – dependent on repeat length Longer repeat length earlier onset

Question 27

Myotonic dystrophy genetics-

Answer 27

* DM1 CTG expansion in 3’-UTR of DMPK * DM2 CCTG expansion in 1st intron of CNBP/ZNF9 * DM1 more common * Genetic anticipation seen in DM1, mainly maternal transmission

Question 28

Mechanisms of non-coding repeat toxicity-

Answer 28

``` • RNA foci/protein sequestration - splicing (DM) - nucleocytoplasmic transport (ALS/FTD) • Repeat-associated non-AUG translation (RANT) • Haploinsufficiency ```

Question 29

Myotonic dystrophy is an example of what opathy ?

Answer 29

Spliceopathy

Question 30

Experiment to show why SBMA is X linked ?

Answer 30

Overexp 97 glutamine expressed phenotypes Early on off spring maintained weight other did not Severely affected males Aggregated proteins – stuck in the well Aggregated in nucleus- pathologically different in males then females Castration – removal of testosterone phenotype is rescued Dragging feet but better after castration Sham operated protein accumulation Castration – ameliorate phenotype Testosterone induces symptoms and pathology in 97Q females All phenotypes shown in males induced in females

Question 31

SBMA

Answer 31

Testosterone reduction prevents phenotypic expression in a transgenic mouse model of spinal and bulbar muscular atrophy