20.03.15 Triplet repeat disorders - gain of function e.g. HD

Question 1

What is the origin of repeat expansion

Answer 1

• Strand slipping during DNA replication via DNA looping in actively dividing cells and or transcription mediated DNA repair pathways.
• More likely in non-dividing cells (neurons)

Question 2

What is genetic anticipation

Answer 2

Increasing severity and earlier age of onset of disease as it is inherited through consecutive generations.

Question 3

What are the two groups of triplet repeat disorders

Answer 3

• PolyQ

- RNA meditated

Question 4

What causes a polyQ disorder

Answer 4

• Gain of function due to expansion of translated repeats (polyglutamine).
• Altered protein function
• e.g. HD, SBMA, DRPLA, SCAs

Question 5

What does DRPLA stand for

Answer 5

Dentatorubral-pallidoluysian atrophy

Question 6

What are RNA mediated triplet disorders

Answer 6

• Gain of function due to expansion of non-translated repeats
• Altered RNA function
• e.g. DM1, DM2, FRAX, C9orf72, FRDA

Question 7

What common properties do polyQ disorders share

Answer 7

• Usually adult onset (unless transmitted by father, then juvenile)
• Progressive disease course, eventually fatal
• Clinical symptoms appear over a threshold number of CAG repeats (30-54)
• Strong negative correlation between number of CAGs and age of onset (longer repeats, earlier the onset)
• Repeat sequence is unstable and can increase in size during transmission (leads to anticipation)
• Gene is expressed ubiquitously
• Pathological protein accumulates in ubiquitinated neuronal intranuclear inclusions in brain.
• Threshold phenomena- high or complete penetrance once above a disease-specific repeat number

Question 8

Review of Huntington disease

Answer 8

• Prevalence= 3-10 in 100,000 in Western EU pops
• CAG repeat in exon 1 of HTT gene.
• Autosomal dominant
• Phenotype= psychiatric disturbances, chorea, progressive cognitive decline
• Average age at onset= 40yrs

Question 9

Do HD alleles expand on maternal or paternal transmission

Answer 9

• Paternal transmission

- Attributed to the increased number of meiotic divisions in spermatogenesis

Question 10

Review of SCAs

Answer 10

• Spinocerebellar ataxias
• 1-3 in 100,000 in Europe
• Autosomal dominant group of neurodegenerative diseases that affect the cerebellum
• Characterised by problems with gait and speech.
• Great clinical heterogeneity
• Some expansions remain stable when transmitted to next generation (e.g. SCA6)

Question 11

Review of DRPLA

Answer 11

• Dentatorubral-pallidoluysian atrophy
• AD, ATN1 gene
• 1 in 200,000 in Japanese (rare elsewhere)
• Clinical characteristics: ataxia, myoclonus, dementia, character changes
• Mean age of onset 31.5 yrs
• Dosage effect, homozygotes more severely affected than hets.

Question 12

Review of SBMA

Answer 12

• Spinal-bulbar muscular atrophy
• Neuromuscular disorder with progressive degeneration of lower motor neurons
• Androgen receptor gene
• X-linked only males are affected.
• Clinical features: proximal muscle weakness, fasciculations, gynecomastia, reduced fertility
• Age of neurological manifestation= 30-50 yrs
• Prevalence= 1 in 300,000 in Europeans.

Question 13

Evidence that polyQ is a gain of function disorder

Answer 13

• No other mutations foud in gene
• Expanded protein is transcribed and translated
• Critical threshold, below which the repeat is not pathogenic
• Anticipation is seen
• HD homs are clinically identical to hets
• Deletions of HTT do not cause HD
• CAG repeats in mice models cause neuropathology, but inactivation has no effect
• Ataxin 1 null mice are viable but CAG expanded mice have neurodegeneration

Question 14

What theories are there for disease mechanism in polyQ disorders

Answer 14

• Aggregation theory
• Toxic fragment hypothesis
• Transcription dysregulation hypothesis
• Cytoskeletal defects and axonal transport
• Effects on other cell types (non-neuronal)

Question 15

What is the aggregation theory in polyQ disorders

Answer 15

• Build up of toxic products produced by expanded alleles. Effects marked in neurons (cells which are long lived and non-dividing)
• Aggregates of mutant protein seen in nuclei of neurons in SCA3 and HD patients.
• However, neurodegeneration onset before aggregate formation in mice. Some human brains have no aggregates
• One study found neurons lacking inclusions had higher risk of cell death, so suggested inclusions are protective
• Inhibition of ubiquitin-proteosome system predisposes cells to polyQ toxicity, enhanced chaperone activity reduces polyQ toxicity

Question 16

What is the toxic fragment hypothesis in polyQ disorders

Answer 16

• Abnormal HTT is a substrate for proteolytic cleavage by caspases and calpains.
• Abnormal terminal fragment (containing polyQ stretch) when expressed in mice causes HD-like symptoms.
• HD protein with caspase-6 cleavage site mutation (caspase resistant) is incapable of causing neurotoxicity.

Question 17

What is the transcription dysregulation hypothesis in polyQ disorders

Answer 17

• PolyQ proteins accumulate in nucleus and inappropriately interact with transcription factors/regulators
• e.g. HTT exp interferes with CREB-binding protein mediated transcription

Question 18

What is the cytoskeletal/ axonal transport defect hypothesis in polyQ disorders

Answer 18

-Aggregation of accumulated protein disrupts axonal transport leading to neuronal dysfunction and degeneration.

Question 19

What is the hypothesis that expansions effect other cell types in polyQ disorders

Answer 19

-In HD, microglia (immune cells of CNS, mediators of neuroinflammation) are activated

Question 20

Therapeutic strategies for polyQ disorders

Answer 20

• Decrease toxic fragment formation
• Alter translocation
• Modify aggregation
• Correct protein folding
• Enhance protein degradation

Question 21

Examples of new therapies

Answer 21

• Antisense oligonucleotides= IONIS-HTT. Designed to inhibit expanded mRNA, to reduce mutant HTT protein levels
• RNA interference= adeno-associated virus (AAV) vectors expression short hairpin RNAs that reduce expression of mutation protein ataxin 1.
• Protein aggregation inhibitors= EGCG (although disease pathology is not solely due to aggregates)

Question 22

HD allele size ranges

Answer 22

• HTT gene
• Normal= <27
• Intermediate= 27-35
• Incomplete penetrance= 36-39
• Full penetrance= 40+

Question 23

SCA1 allele size ranges

Answer 23

• ATXN1 gene
• Normal= <36
• Intermediate= 36-38 (without CAT interruptions)
• Full penetrance= >38 (without CAT interruptions)

Question 24

SCA2 allele size ranges

Answer 24

• ATXN2 gene
• Normal= <32
• VUS= 32
• Reduced penetrance= 33-34
• Affected= >32

Question 25

SCA3 allele size ranges

Answer 25

• ATXN3 gene
• Normal= <45
• Intermediate= 45-59
• Full penetrance= >59

Question 26

SCA6 allele size ranges

Answer 26

• CACNA1A gene
• Normal= <19
• VUS= 19
• Full penetrance= >19

Question 27

SCA7 allele size ranges

Answer 27

• ATXN7 gene
• Normal= 19 and below
• Mutable normal= 28-33
• Reduced penetrance= 34-36
• Full penetrance= >36

Question 28

SCA17 allele size ranges

Answer 28

• TBP gene
• Normal= <41
• Reduced penetrance= 41-48
• Full penetrance= >48

Question 29

DRPLA allele size ranges

Answer 29

• ATN1 gene
• Normal= <36
• Intermediate= 36-47
• Full penetrance= >47

Question 30

SBMA allele size ranges

Answer 30

• AR gene
• Normal= <35
• VUS= 35
• Reduced penetrance= 36-37
• Full penetrance= >37

Question 31

Review of DM1

Answer 31

• Myotonic dystrophy type 1
• AD
• CTG expansion in 3’URT of DMPK gene
• Most prevalent form of muscular dystrophy= 1 in 8000 individuals
• CLinical features= myotonia, progressive muscle weakness, cataracts, testicular atrophy, cardiac conduction defects
• Typical age of onset= 20s
• Congenital form-= floppy baby, with severe respiratory distress and failure to thrive.

Question 32

Is congenital DM1 seen after maternal or paternal transmission

Answer 32

Maternal

Question 33

DM1 allele size ranges

Answer 33

• DMPK gene
• Normal= <36
• Intermediate= 36-50
• No, minimal, classical DM= >51-150
• Congenital, juvenile, classical DM = >150

Question 34

Review of DM2

Answer 34

• Complex expansion in intron 1 of CNBP gene
• (TG)n(TCTG)n(CCTG)n
• Expansion of CCTG repeat is disease causing
• Clinical features= muscle weakness, muscle pain, myotonia
• Anticipation not observed

Question 35

DM2 allele size ranges

Answer 35

• CNBP gene
• Normal= <26 (CCTG)
• Affected = 75-11,000 repeats

Question 36

What are the 3 models for the pathogenesis of DM1

Answer 36

• Haploinsufficiency
• Chromatin structure
• RNA gain of function

Question 37

Review of haploinsufficiency as a mechanism of disease in DM1

Answer 37

• Decreased levels of DMPK mRNA and protein in adult DM1 tissue
• DMPK -/- mice develop mild progressive skeletal myopathy, caridac conduction abnormalities and metabolic impairment. However only mild phenotype
• However, no DMPK point mutations identified

Question 38

Review of Chromatin structure as a mechanism of disease in DM1

Answer 38

• CTG expansion exerts an effect on chromatin structure which may silence neighboring gene DMWD and SIX5
• Reduced levels of DMWD in DM1 patients
• Doesn’t explain phenotypic similarity between DM1 and DM2

Question 39

Review of RNA gain of function as a mechanism of disease in DM1

Answer 39

• CUG repeats fold into RNA hairpins that are not exported from nucleus but accumulate in ribonuclear foci, trapping essential RNA-binding proteins (e.g. transcription factors)
• Accumulation of mutant RNAs alter RNA metabolism. Affects RNA binding proteins like MBNL1. MBNL1 knock out mice show DM1 phenotype.

Question 40

Therapies in DM1

Answer 40

• Reduce the toxicity of mutant CUG repeats. Using antisense oligonucleotides
• Normalise activities of RNA binding proteins e.g. MBNL1.
• Issues include efficient delivery to target tissues.