19.03.14 Friedreich's ataxia (LoF)

Question 1

What is a repeat expansion disorder?

Answer 1

• Expansion of nucleotide STRs that are polymorphic
• STRs are 3 nucleotides or more
• Exceed the threshold, and they become unstable (increase or decrease) through generations (dynamic mutations)
• Expand greater then cut-off and you get a phenotype
• Larger expansion = more severe phenotype (anticipation)

Question 2

How do repeat expansion disorders occur?

Answer 2

• At DNA replication, STRs expand or contract during meiosis due to slippage of the replisome on the template DNA

Question 3

Can they be in coding and non-coding DNA?

Answer 3

Yes

• HD, SCAs and SBMA are found in coding regions
• FRAX, DM1 and FA are found in non-coding regions

Question 4

Examples of GoF repeat disorders

Answer 4

HD and SCas

- CAG repeat expansions result in polyglutamine aggregates

Question 5

Examples of LoF repeat disorders

Answer 5

FRAX, FRAXE and FA

• Repeat expansions/point mutations result in reduced or absence of gene product or its function
• FRAXE is a mild to mod MR syndrome - caused by silencing of FMR2 gene on Xq28 by CCG expansion or deletion

Question 6

FA - symptoms, life expectancy,

Answer 6

• FA is the most common autosomal recessive ataxia
• Progressive ataxia with mean age of onset between 10 and 15, usually before age 25, and hypertrophic cardiomyopathy
• Most become paraplegic and require a wheelchair
• Death at 37 yrs
• Common symptoms include o Dysarthia (slow, slurred speech caused by paralysis, weakness, or inability to coordinate the muscles of the mouth), muscle weakness, ataxia, cardiomyopathy, scoliosis.
• 25% present later in life or milder symptoms
• Disease causes degeneration of posterior columns in spinal cord, lose sensory neurons in dorsal root ganglia, and later degeneration of cerebellar cortex

Question 7

Genetics of FA

Answer 7

• AR (unlike other repeat expansions)
• 98% caused by HOM GAA expansion in intron 1 of the frataxin gene (FXN) at 9q13
• N.B. sizes of repeats on either allele can be different but still called HOM
• Expansion causes FXN to be defective, so get deficiency of frataxin (mitochondrial protein). Normally frataxin binds iron and is required for synthesis of iron-sulphur clusters. So with no frataxin you don’t produce the enzymes required for respiratory chain complexes
• Deficiency of frataxin causes all clinical symptoms
• Remaining 2% have expansion and point mutation. whole range of mutations identified, and severity us due to length of expansion on other alleles
• Most common PM is p.Gly130Val - this with expansion in trans causes later onset, slower progression, no cardiomyopathy.
• No cases of two PMs have been reported - ?lethal. FXN null mice are lethal in utero - Phenoytpe due to FXN deficiency and not FXN knockout

Question 8

What are the repeat categories in FA?

Answer 8

• Normal - 5-33 GAA repeats (most are <12, over 27 is rare, 9 rpts is the most common)
• Premutation - 34-65 GAA repeats (very rare, <1% of alleles). No associated with FA but may expand further. - - Also get GAGGAA interruptions thought to stabilise them and prevant expansion.
• Borderline - 44-66 GAA repeats. 44 is smallest expansion recorded associated with disease.
• Full penentrance - 66-~1700 GAA repeats. Ones with interruptions are usually shorter and associated with later onset.

Question 9

Does expansion risk change if mat or pat inherited?

Answer 9

Yes.

• Maternal transmission is associated with expansions and contractions
• Paternal transmission is associated primarily, but not exclusively, with contractions
• There is a strong contraction bias among longer expansions (>500 repeats).

Question 10

Genotype/phenotype correlation

Answer 10

• Full penetrance
• Wide size range means variability in symptoms (onset can vary from 5 to 50)
• some missense variants can be milder
• shorter rpts = later onset, slower progression, milder
• size of shorter rpt correlates better with age of onset
• late onset (>25 years) - normally have <500 rpt in one allele
• Very late onset (>40 years) - usually have <300 rpts in one allele
• less FRDA activity = more severe phenotype
• Although can’t precisely predict correlation, as genetic background has an affect), somatic mosaicism of expansion, aberrant splicing, altered expression of different isoforms.

Question 11

Testing strategy for FA

Answer 11

• Offer diagnostic, carrier and prenatal testing
  1) Long range PCR flanking repeat region and TP PCR - if 2 alleles - NMD.
  2) Long range PCR is also ran out on a gel to see larger expansions that are too big for the size standard
  3) Can seq region if only one expanded allele found (however there is a high carrier rate in pop)
  4) MLPA can also be used to detect exon del/dup

Question 12

Molecular pathogenesis

Answer 12

• Get formation of non-B DNA structures which block the transcriptional machinery from moving along the DNA template (blocks elongation and inhibits transcription).
• RNA binds to DNA template behind machinery forming DNA-RNA hybrid structures (called ‘R-loops’). These form over the GAA repeat and block FXN elongation and transcription.

Question 13

Frataxin protein function

Answer 13

• Nuclear MIT protein and localizes to MIT matrix
• Well conserved across species and ubiquitously expressed in low levels (higher levels in affected tissues)
• Function is in iron homeostasis - biogenesis of iron-sulphur, iron-trafficking in MIT and as an iron chaperone.
• Iron is important for respiratory chain complexes, so frataxin has direct effect on MIT function and respiration.
• Loss of frataxin - reduces iron cluster synthesis and assembly, reduces activity of enzymes reliant on iron and get iron accumulation in MIT (form iron cystallite aggregates).
• Increased Iron is toxic - generates reactive oxygen species - get tissue degeneration
• Affects tissues rich in MIT (heart, neuronal cells in CNS and PNS)

Question 14

Therapeutic options for FA

Answer 14

• No effective therapies to slow down deterioration
• Carriers have no symptoms, so need drugs to increase FXN activity to carrier level
• Most drugs focus on increasing transcription (HDCAi - histone deacetylase inhibitor) to get more FXN mRNA upstream and downstream of expansion.
• Also have interferon gamma trials in children which look promising - aim of this is to upregulate levels of FXN in all cell types
• Antioxidants (co-enzyme Q10) and iron transport molecules (Deferiprone) are also being investigated as possible treatments.
• co-enzyme Q10 - in MIT membrane and facilitates electron transport with MIT - shown to improve ataxia score but not enough studies completed yet
• Deferiprone - iron chelator that moves iron around MIT, reduces oxidative stress and improves cardiac hypertrophy.