L8, Repeat Expansion Disorders

Question 1

Definition and rough basis of genetic anticipation:

Answer 1

• Earlier onset, increasing severity in later generations (e.g. myotonic dystrophy)
• Increased numbers of individuals with symptoms in later generations (e.g. Fragile X syndrome)
• Based upon expanded numbers of unstable microsatellite repeats (Short tandem repeat of up to 9nts)

Question 2

Define dynamic mutation:

Answer 2

• Unstable repeats that can expand on parental transmission and in somatic tissue
• Basis for genetic anticipation

Question 3

Key examples of repeat expansion disorders with implicated gene region:

Answer 3

• Huntington’s disease (coding exon)
• Fragile X; FRAXA or FXTAS (5’UTR)
• SBMA/Spinal and Bulbar Muscular Atrophy (coding exon)
• DM/ Myotonic dystrophy (Type 1: 3’UTR)

Question 4

How many human REDs are there currently:

Answer 4

• Over 50, with 13 different sequence repeats associated

Question 5

Characteristics of repeat expansion disorders:

Answer 5

• Generally, number of repeats positively correlates with disease severity, negatively correlates with age of onset
• Expansion of one disease causing repeat does not interfere with other repeat in patient’s genome
• Can be AD, AR or X-linked

Question 6

Mechanisms for REDs (x4):

Answer 6

1. Expansion of non-coding repeats leading to LOF of gene containing repeat -> AR
2. Expansion of CAG coding repeats leading to GOF and production of abnormal protein containing and expanded polyglutamine tract -> AD
3. Expansions resulting in GOF of RNA containing an expanded repeat -> AD
4. Expansions resulting in repeat associated, non-ATG (RAN) translation of repeat containing RNA leading to production of toxic peptides -> AD

Question 7

Fragile X characeristics:

Answer 7

• Commonest cause of inherited intellectual disability
• Increased severity in males, relatively mild in females (random X-inactivation)
• Prevalence of FXS: 1 in 4000 to 1 in 7000
• Autism common
• Mild abnormal facial features (sunken eyes, arched palate, large ears, macroorchidism)
• Otitis media, seizures, mitral valve prolapse, GI problems

Question 8

FRAXA site:

Answer 8

• Xq27.3
• First fragile site described on X chromosome
• CGG repeat expansion in 5’ UTR of FMR1 gene on X chromosome

Question 9

Thresholds for FXS:

Answer 9

• Stable: 6-44 repeats
• IM: 44-54 repeats
• Premutation: 55-200 repeats (associated with FXTAS, FXPOI)
• Full FRAX mutation: 200 to >4000 repeats

Question 10

Epigenetic mechanism of FMR1: (transcription under normal conditions vs FXS)

Answer 10

• if <40 CGG i.e. normal: FMRP produced in hESCs and differentiated cells (active euchromatin; DNA unmethylated) -> transcribed
• if >200 CGG, hESCs FMR1 transcribed and translated -> expanded FMR1 mRNA initiates silencing (>200 CGG, repressive heterochromatin, MeCpG and H3K9Me2 -> No FMRP expression (the transcript interacts back with the DNA to silence/methylate it)

Question 11

Role of FMRP:

Answer 11

• 71 kDa protein
• Normally localises to postsynaptic spaces of dendritic spines
• Shuttles in and out of the nucleus transporting target mRNAs
• Phosphorylated FMRP binds to and represses translation of ~400 target dendritic mRNAs (normal conditions)
• On receipt of synaptic signals, FMRP is de-phosphorylated. It no longer represses translation and allows synthesis of key synaptic plasticity proteins
• See FC

Question 12

Huntington’s disease: Characteristics

Answer 12

• Characterised by neuronal degeneration (most common monogenic cause)
• Clinical features include progressive, selective neural cell death associated with choreic (writhing, dance-like) movements and dementia

Question 13

What repeat sequence causes HD? Gene affected? Threshold repeats for disease:

Answer 13

• Expansion of CAG tri-nt repeat encoding glutamine (Q) in exon 1
• Affects HTT gene (4p16.3)
• Normal allele: 11-26 CAG repeats
• Mutable normal allele: 27-35 CAG repeats
• HD allele (reduced penetrance): 36-39
• HD allele: 39 to ~250 CAG repeats
• Paternal expression bias >7 CAG

Question 14

How can HD repeat numbers be estimated?

Answer 14

• PCR assay
• PCR primers flank repeat containing region -> size of PCR product is dependent on number of CAG repeats

Question 15

Htt structure; wild type protein

Answer 15

• Protein mainly consists of HEAT repeats (Huntingtin Elongation factor 3; subunit of protein phosphatase 2A and TOR1
• Widely expressed with highest levels in neurons of the CNS
• The wild type protein acts as a scaffold to coordinate complexes of other proteins
• Also acts as a transcriptional regulator

Question 16

How does the expanded glutamine tract cause HD?

Answer 16

• Causes dysfunction and death of neurons
• N-terminal polyA protein fragments translocate to the nucleus, form intranuclear inclusions and impair gene transcription e.g. of BDNF
• Outside the nucleus, these fragments can also oligomerise, aggregate and form cytoplasmic inclusions -> impairment of proteostasis network as well as synaptic function, axonal transport and mitochondria

Question 17

What is the cause of SBMA aka Kennedy’s disease?

Answer 17

• Expanded CAG repeats in coding region of androgen receptor (AR) gene