Repeat expansions

Question 1

What are dynamic mutations?

Answer 1

Nucleotide repeat tracts that are unstable and can expand/contract on generational transmission and causing disease when expansions are above a pathological threshold

Question 2

What is a common feature of repeat expansions?

Answer 2

ANTICIPATION
Clinical features become more severe, more frequent and occur earlier in successive generations, due to further expansion of the repeat tract

Does not happen in all repeat expansion disorders e.g. PolyA (GCN) is more stable and therefore does not show anticipation

Question 3

Example of sex specific anticipation

Answer 3

Paternal transmission of HD expansion = juvenile HD. The CAG is more unstable in spermatogenesis due to the increased no of meiotic divisions

Question 4

What is the origin of a repeat expansion?

Answer 4

REPLICATION SLIPPAGE
Meiosis or mitosis
- DNA polymerase stutters while synthesising repetitive elements
- Formation of a loop in newly synthesised DNA = addition of extra repeat units

OR unequal crossover in meiosis = expansion on 1 allele and contraction of other allele

Question 5

4 mechanisms of repeat expansion diseases

Answer 5

1. LOF - reduced transcription of gene and decreased function
2. GOF - PROTEIN toxicity from expansion of AUG-translated repeats
3. GOF - RNA toxicity through gelation and sequestration of RNA-binding proteins
4. GOF - PROTEIN toxicity from expansion of non-AUG-translated repeats

Question 6

Function of FMRP

Answer 6

RNA-binding protein. It shuttles mRNA from nucleus to areas of cells for protein assembly. Important for synapse development in brain. Also expressed in testes and ovaries

Question 7

Mechanism of Fragile X

Answer 7

CGG repeat expansion in FMR1 5’UTR resulting in hypermethylation of CpG island 250bp downstream near promoter. Results in transcriptional gene silencing and partial/complete loss of protein

Can have methylation and repeat expansion mosaicism (report of high functioning male with unmethylated full expansion)

Question 8

FRAX clinical features

Answer 8

Males: moderate-severe ID, macrocephaly, long face, large ears, joint laxity, macroorchidism
Females: variable depending on skewed X-inactivation. Normal-moderate ID

PRE-MUTATIONS: FXTAS (late onset progressive ataxia) and FXPOI (early menopause), due to toxic RNA GOF not LOF

Question 9

FRAX CGG ranges

Answer 9

NORMAL 6-50
INTERMEDIATE 46-58
PRE-MUTATION 55-200 (>90 = 90% chance of expansion into full mut range)
FULL MUTATION 200+

Also SNVs and del/dups report with no methylation defects

Question 10

AR repeat expansion example

and mechanism

Answer 10

Friedreich ataxia

Homozygous GAA repeat expansion in intron 1 of FXN (not necessarily 2 the same size)

LOF. Formation of DNA-RNA hybrids (R loops) at GAA repeats block transcriptional machinery and trigger recruitment of heterochromatic factors by repressive chromatin marks = condensation of the locus

Question 11

FXN protein function

Answer 11

FXN encodes frataxin, a mitochondrial protein that synthesises iron-sulphur clusters (ISCs) and enzymes in respiratory chain complexes 1-3

Less frataxin = less ISC = decreased iron-dependent enzyme activity = iron accumulation in mitochondria = iron mediated ROS = oxidative damage and apoptosis of tissues with high mitochondria content (heart, spinal cord, cerebral cortex)

Question 12

Why does FRDA not show anticipation?

Answer 12

AR inheritance means disease is not seen in successive generations

Size of expansion does correlate with phenotype severity and age of onset

Question 13

FRDA clinical features

Answer 13

Progressive ataxia and HCM, dysarthia, loss of position sense and paraplegia. Onset 10-15y. Death by 40y

Question 14

FRDA GAA ranges

Answer 14

NORMAL 5-33
PRE-MUT 34-65
BORDERLINE 44-66
AFFECTED 66-1700

Shortest allele associated with FRDA = 44 (with large exp on other allele)

2% have SNV+expansion

Expansions arise from large normal (12-33), usually maternal

Question 15

3x examples of:
GOF - PROTEIN toxicity from expansion of AUG-translated repeats

and mechanism

Answer 15

1. Huntington (HTT)
2. Spinocerebellar ataxia 7 (ATXN7) - extreme anticipation
3. Spinal and Bulbar Muscular Atrophy (AR) - example of pleiotropy as missense muts cause AIS infertility(decreased sensitivity to testosterone)

Poly-Q (glutamine) CAG repeat expansions

Question 16

Why are poly-Q expansions pathogenic?

Answer 16

CAG within ORF = expanded poly-Q tract in translated protein = conformational changes e.g. Beta sheets = insoluble aggregates e.g. intranuclear protein inclusions = cell proteotoxicity & degradation by UB pathway

Disease proteins are usually involved in multiple cell processes, so other dysfunctions in addition to proteotoxicity e.g. alter protein-protein interactions and localisation/stability, deregulated RNA metabolism

Question 17

3 common features of Poly-Q disorders

Answer 17

Adult onset, complete penetrance and progressively fatal

Anticipation

No other mutation types and homozygotes same as heterozygotes

Question 18

HD CAG ranges

Answer 18

NORMAL <27
INTERMEDIATE 27-35
AFFECTED (reduced penetrance) 36-39
AFFECTED (complete penetrance) >39
JUVENILE >60

Question 19

HD clinical features

Answer 19

Neuronal dysfunction and cell death causes psychiatric disturbances, motor changes e.g. chorea and cognitive decline

Average age of onset = 40y

Question 20

1 example of

GOF - RNA toxicity through gelation and sequestration of RNA-binding proteins

and mechanism

Answer 20

Myotonic dystrophy type 1. CTG expansion in DPMK 3’UTR

Long repeat RNA folds into complex structures that trap RBP (e.g. splicing regulators and TF) into RNA foci. This alters RNA processing/metabolism by decreasing RBP availability for it’s functions. Affect depends on the repeat length and expression levels of disease gene/RBP

Question 21

Mechanism of DM1

Answer 21

DPMK encodes a Ser/Thr kinase expressed in cardiac & skeletal muscle. Expanded DMPK sequesters MBNL1, an alternative splicing regulator that is important for adult isoform splicing. This leads to increased CUGBP1 mediated splicing of an embryo isoform

Question 22

DM1 CTG ranges

Answer 22

NORMAL 5-36
PRE-MUTATION 37-50
CLASSIC AFFECTED 51-150
JUVENILE >150

Presents with myotonia, distal muscle weakness, cataracts, testicular atrophy and cardiac conduction defects

Question 23

What is RAN translation?

Answer 23

Repeat associated non-AUG translation is protein synthesis occuring in multiple reading frames within non-coding repeat regions, produces homopolymeric proteins.

RAN occurs due to IRES cap-independent ribosome binding, no PIC scanning for AUG, start codon fidelity decreased e.g. ACG or GUG

Question 24

Example of

GOF - PROTEIN toxicity from RAN translation

Answer 24

ALS/FTD
GGGGCC expansion in C9orf72 accounts for 40% of familial ALS

NORMAL 5-24
AFFECTED 61-4000+

Question 25

Why do poly-Alanine disorders not show anticipation?

Origin?

Answer 25

Repeat tracts can be any combination of the 4 codons that code Alanine. Repeats are more stable in meiosis and mitosis and do no expand on transmission

GCU, GCC, GCA, GCG

Form from unequal Xover between 2 mispaired alleles

Question 26

Poly-A expansion mechanism

Answer 26

Normal poly-A tracts are flexible spacers conferring stability to 3D protein. Expansions destabilise protein, altering levels/activity, usually resulting in transcriptional deregulation and apoptosis due to proteotoxicity

Question 27

Poly-A disorder with dominant negative mechanism

Answer 27

Synpolydactyly 1

Expanded HOXD13 sequesters WT protein from cytoplasm

Question 28

Poly-A disorder with AD and AR inheritance

Answer 28

Oculopharyngeal muscular dystrophy

PABPN1

NORMAL 10aa
EXPANDED 11-17aa
(11/11 = AR inheritance, hetz=benign)

Question 29

2 technical issues for testing repeat expansions

Answer 29

High GC content different to PCR

Alignment difficult meaning large expansions cannot be accurately sized