Lecture 8: Repeat Expansion Disorders I

Question 1

What is genetic anticipation?

Answer 1

When a disease has earlier onset and increasing severity in later generations

Question 2

What is the Sherman paradox?

Answer 2

An example of genetic anticipation where it was observed that there were increased number of individuals with symptoms of Fragile X Syndrome (FXS) in later generations.

Question 3

What is the molecular basis of genetic anticipation?

Answer 3

Expanded number of microsatellite repeats (Short tandem repeats of up to around 9-10 nucleotides)

Question 4

What type of microsatellite repeat is most commonly expanded in REDs?

Answer 4

a triplicate repeat sequence

Question 5

What is the basic cause for Fragile X Syndrome?

Answer 5

Expanded number of CGG repeats in the 5’ untranslated region (UTR) of the FMR1 gene

Question 6

What is the full name of the gene lost in FXS?

Answer 6

Fragile X Messenger Ribonucleoprotein 1 (FMR1)

Question 7

What is the product of the FMR1 gene?

Answer 7

FMRP

Question 8

True or false: expanded repeats are stable?

Answer 8

False: they are unstable

Question 9

What is dynamic mutation?

Answer 9

Since expanded repeats are unstable, they can expand and contract on parental transmission and in somatic tissues (somatic instability) = this is called dynamic mutation

Question 10

True or false: Dynamic mutation can be inherited in a typical Mendelian fashion?

Answer 10

False

Question 11

True or false: REDs can only occur in coding exons of genes to have an affect?

Answer 11

False: they can occur in:
- UTRs (E.g. FXS)
- Introns (E.g. Friedreich Ataxia)
- Exons (E.g. Huntington’s Disease)

Question 12

How many human REDs have been identified?

Answer 12

over 50

Question 13

How many different sequence repeats have been identified associated with REDs?

Answer 13

13 (so not all microsatellite repeats are associated with REDs)

Question 14

How does the number of repeats correlate with (1) disease severity and (2) age of onset

Answer 14

(1) positively correlates with disease severity

(2) negatively correlates with age of onset

Question 15

What are the different modes of inheritance of REDs?
(there are 3)

Answer 15

• autosomal dominant
• autosomal recessive
• X-linked

Question 16

What are the 4 mechanisms of disease for REDs?

Answer 16

1. Expansion of non-coding repeats - leading to loss of function (lof) of gene containing the repeat
2. Expansions of CAG coding repeats leading to gain of function and production of abnormal proteins containing expanded polyglutamine (PolyQ) fragments
3. Expansions resulting in gain of function RNA containing an expanded repeat
4. Expansions resulting in gain of function Repeat Associated Non-ATG (RAN) translation of repeat containing RNA to produce toxic peptides

Question 17

REDs that involve a loss of function are inherited in which manner?

Answer 17

autosomal recessive
(or X-linked dominant if affecting sex chromosomes - such as FXS)

Question 18

REDs that involve a gain of function are inherited in which manner?

Answer 18

Dominant inheritance

Question 19

True or false: disease associated with each RED will only follow one of the four mechanisms?

Answer 19

False: the mechanisms are not mutually exclusive
(there are contributing factors from more than one mechanism in some REDs, E.g. HD)

Question 20

What does it mean that FXS is X-linked dominant?

Answer 20

Increased severity in males, females are always manifesting carriers that exhibit symptoms of the disease but typically more mildly due to random X-inactivation

Question 21

What is the commonest myogenetic cause of autism?

Answer 21

FXS

Question 22

Give 2 physical and 3 medical manifestations associated with FXS

Answer 22

Physical:
1. mild abnormal facial features (sucken eyes, large ears)
2. macroorchidism (increased testicular volume)

Medical:
1. Seizures
2. Mitral valve prolapse
3. GI problems
4. Otitis media (middle ear infections)

Question 23

What is the locus and cytogenetic nomenclature of the ‘fragile site’ associated with FXS?

Answer 23

Locus = Xq27.3
Cytogenetic nomenclature = FRAXA

Question 24

How many CGG repeats are present in the FMR1 gene associated with:
1) normal/stable repeat
2) intermediate repeat
3) Premutation repeat
4) Full FRAX mutation

Answer 24

1) normal /stable = 6-44 repeats (average 30 repeats)

2) Intermediate = 45-54 repeats (at this stage the repeat number is less stable and more susceptible to dynamic mutation but no disease associated yet)

3) Premutation = 55-200 repeats (may have symptoms related to fragile X associated disorders such as FXTAS and FXPOI)

4) Full FRAX mutation = 200 to more than 4000 repeats

Question 25

What term is used to describe females with premutation in FXS?

Answer 25

Carrier

Question 26

What is the difference between a premutation carrier and a manifesting carrier of X-linked disorders?

Answer 26

A premutation carrier does not have the full mutation that causes the disease A manifesting carrier has the full disease causing mutation

Question 27

Why does severity of FXS vary more in females?

Answer 27

Dependent on the number of repeats as well as X-inactivation bias

Question 28

FMR1 mutation has maternal ___(1)___ bias and some paternal ___(2)___ bias

Answer 28

(1) expansion (2) contraction

Question 29

Explain how normal CGG repeat number allows protein FMRP to be produced

Answer 29

normal mRNA produced that is translated to FMRP in embryonic stem cells and differentiated cells - the DNA is in active euchromatin form characterised by unmethylated DNA and H3K9Ac (acetylation of Lys9 on histone 3) and H3K4Me (Methylation of Lys4 on histone 3)

Question 30

Explain how >200 CGG repeats in FXS prevent protein FMRP production

Answer 30

In embryonic stem cells, FMR1 is initially transcribed (mRNA has expanded repeats) and translated but during differentiation the expanded mRNA initiates silencing. During differentiation of neurons, the expanded mRNA interacts back with the DNA, resulting in methylation of DNA (MeCpG) and histones (H3K9Me2) to sequester DNA into heterochromatin and prevent FMRP expression

Question 31

How does loss of FMRP function in a normal neuron?

Answer 31

FMRP normally localises to postsynaptic spaces of dendritic spines and transports target mRNAs in and out of the nucleus. When phosphorylated = FMRP binds to and represses translation of target dendritic mRNAs. Synaptic signals result in de-phosphorylation of FMRP to remove repression and allow synthesis of key synaptic plasticity proteins that play important roles in memory and learning.

Question 32

How does loss of FMRP lead to the FXS disorder?

Answer 32

No FMRP means there is no repression of translation of transcripts normally targeted by FMRP so proteins are abnormally and excessively expressed leading to the symptoms associated with Fragile X

Question 33

How many distinct disorders are due to CAG repeat expansion in protein-coding regions?

Answer 33

nine

Question 34

What type of disorders result from expansions in coding repeats (CAG)?

Answer 34

Dominant, gain of function disorders

Question 35

Give an example of a disorder that results from expansions in coding repeats (CAG)?

Answer 35

Huntington disease

Question 36

What is Huntington disease characterised by?

Answer 36

Neuronal degeneration

Question 37

Give 3 clinical features of huntington disease

Answer 37

1. progressive, selective neural cell death 2. Choreic movements 3. Dementia

Question 38

What is the genetic basis for HD?

Answer 38

Expansion of CAG trinucleotide microsatellite repeat within the HTT gene beginning at codon 18 in exon 1 resulting in polyQ residues in the htt protein

Question 39

What is the name of the gene affected in Huntingon Disease?

Answer 39

HTT on chromosome 4

Question 40

What is the product of the HTT gene?

Answer 40

htt (hungtintin protein)

Question 41

How many CAG repeats are present in the HTT gene associated with: 1) normal allele 2) mutable normal allele (intermediate) 3) HD mutated allele with reduced penetrance 4) HD allele

Answer 41

1) normal allele = 11-26 CAG repeats 2) mutable normal allele =27-35 repeats (prone to dynamic mutation) 3) HD allele with reduced penetrance = 36-39 repeats (some people will have disease depending on other genetic factors) 4) HD allele = >39- around 250 repeats (lower number of repeats = later onset, less severe symptoms)

Question 42

Describe the expansion bias in HD?

Answer 42

Paternal expansion bias of more than 7 CAG repeats as goes down paternal line

Question 43

What can be used to estimate the repeat numbers in HD?

Answer 43

A PCR assay - PCR primers flank repeat containing region to amplify repeat - products separated and size of band corresponds to number of CAG repeats (i.e. bigger band = more repeats)

Question 44

What is the structure and function of wild type huntingtin protein?

Answer 44

Huntingtin mainly consists of HEAT repeats (Huntingtin, Elongation factor 3, a protein subunit of protein phosphatase 2A, and TOR1) - it is widely expressed but has highest levels in neurons of CNS It acts as a scaffold protein to coordinate other protein complexes Also acts as a transcriptional regulator

Question 45

Describe the pathogenic cellular mechanisms in Huntington disease

Answer 45

N-terminal polyQ-containing fragments oligomerise, aggregate and form cytoplasmic inclusions --> these oligomers, aggregates and inclusions impair proteostasis network, synaptic function, axonal transport and mitochondria. - N-terminal polyQ fragments also translocate to the nucleus where they form intranuclear inclusions that impair gene transcription

Question 46

can HD be only caused by gain of function of Htt protein associated with toxicity of expanded polyQ tract?

Answer 46

No, model systems where CAG repeats are interrupted by CAA repeats (which also encode glutamine) decreased cellular toxicity even though the number of glutamines were the same --> this means age of onset is better predicted by the length of uninterrupted CAG repeats rather than number of Qs in huntingtin protein