18.03.14 Triplet repeat LOF FRDA

Question 1

What is the effect of a loss of function mutation?

Answer 1

Results in reduced or absence of gene product or its function.

Question 2

Give two examples of loss of function triplet repeat disorders.

Answer 2

1) Fragile X syndrome

2) Friedreich’s ataxia

Question 3

Give a brief overview of FRDA including its incidence and carrier frequency.

Answer 3

1) It is the most common inherited ataxia in Europe, the Middle East, South Asia (Indian subcontinent) and North Africa;
2) FRDA is the most common autosomal recessive ataxia.
3) Prevalence of FRDA ~1/50,000 Caucasians
4) Carrier freq 1/50-1/100 in peoples of European, North African, Middle Eastern, and Indian origin.
5) It is a multi-systemic degenerative disease characterised by progressive ataxia with mean age of onset between 10 and 15, usually before age 25, and hypertrophic cardiomyopathy.
6) Nearly all patients become paraplegic and require a wheelchair; the average time from symptom onset to wheelchair dependence is ten years.
7) Mean age of death is 37.5 years and the causes of death can be: cardiac dysfunction (59%), probable cardiac dysfunction (3.3%), noncardiac involvement (27.9%) and unknown (9.8%).

Question 4

Give four features of FRDA.

Answer 4

Dysarthia (slow, slurred speech caused by paralysis, weakness, or inability to coordinate the muscles of the mouth)

Muscle weakness (absence of muscle stretch reflexes)

Gait, limb and truncal ataxia

Loss of position and vibration sense

Diminished tendon reflexes

Cardiomyopathy (approx two thirds)

Diabetes mellitus (30%)

Scoliosis

Bladder dysfunction

Other skeletal abnormalities

Affected vision/ abnormalities of eye movements

Question 5

What is the presentation of atypical FRDA?

Answer 5

● ~25% of FRDA patients have an atypical presentation with later onset (late onset or very late onset FRDA, LOFA / VLOFA, respectively) or less severe presentation. The proportion of this group of patients has greatly expanded since the advent of molecular testing in 1996, prior to which many such cases would have been undetected.

Question 6

Which neuropathological changes are associated with FRDA?

Answer 6

1. Degeneration of posterior columns of the spinal cord
2. Loss of large primary sensory neurons in the dorsal root ganglia (DRG)
3. Mild, late onset degeneration of the cerebellar cortex

Question 7

What is the inheritance pattern of FRDA?

Answer 7

AR

Question 8

What is the common genetic mutation in patients with FRDA?

Answer 8

~98% of FRDA patients are homozygous for an expansion of a GAA repeat in intron 1 of the frataxin gene (FXN) at 9q12.1

Question 9

What is the molecular effect of a pathogenic expansion in the FXN gene?

Answer 9

The expansion results in defective transcription of the FXN gene, leading to deficiency of frataxin, a small (210 aa) mitochondrial protein.

Frataxin binds iron and is required for the synthesis of iron-sulphur clusters and, thereby, for the synthesis of enzymes in the respiratory chain complexes I – III and aconitase.

Deficiency of frataxin, a small mitochondrial protein, is responsible for all clinical and morphological manifestations of FRDA [Causes a transcriptional defect].

Question 10

2% of FRDA patients do not have a pathogenic expansion, what is the mutation underlying disease in these cases?

Answer 10

~2% of patients have an expansion on one allele and a point mutation/exonic deletion on the other.

Nonsense, missense, frameshift, and splicing defect mutations have all been identified.

Clinical phenotype is related to the length of the expansion.

Question 11

What is the most frequent point mutation seen in FRDA patients?

Answer 11

p.(Gly130Val)

Patients with this mutation and an expansion in trans have, phenotypically, a later age of onset, slower disease progression, marked lower limb spasticity and absence of dysarthria and cardiomyopathy, compared to those homozygous for the expansion.

I154F interferes with Fe/S protein cluster interactions.

Question 12

What may explain the lack of affected FRDA patients with point mutations detected on both alleles?

Answer 12

Could be due to prenatal lethality as frataxin null mice die in utero, suggesting that FRDA is caused by a deficiency, rather than a complete lack of frataxin. Truncating mutations are the most common point mutations.

Question 13

What are the different classes of FXN repeat expansions?

Answer 13

1) Normal 5-33 repeats
2) Premutation 34-65
3) Borderline 44-66
4) Full penetrance 66-~1700

Question 14

Why is there overlap between the premutation and borderline repeat expansion classes in FRDA?

Answer 14

due to the rarity with which they occur, the exact demarcation between normal and full penetrance alleles has not been clearly determined

Question 15

Describe the bimodal distribution of FXN normal alleles.

Answer 15

80-85% <12 repeats (small normal; SN)

15% 12-33 repeats (large normal; LN)

Normal alleles with >27 repeats are rare

Question 16

Describe premutation FXN expansion alleles.

Answer 16

34-65

Likely to account for <1% FXN alleles

Not associated with FRDA but may expand during intergenerational transmission, resulting in disease-causing alleles in offspring

Interruptions in these alleles, typically (GAGGAA)n are thought to stabilise them and prevent expansion into abnormal range

Question 17

Describe borderline FXN expansion alleles.

Answer 17

44-66

The shortest allele reported to be associated with FRDA is 44 uninterrupted GAA repeats (Sharma et al. 2004)

The 44 repeat allele was somatically unstable in the reported case, and carried a large expansion on the other allele

Question 18

Describe full penetrance FXN expansion alleles.

Answer 18

66-~1700

Uninterrupted expansions result in classic FRDA

The majority of expanded alleles contain between 600 and 1200 GAA repeats

Alleles with non-GAA interruptions (typically close to 3’ end of the repeat tract) are usually short (100-300 triplets) and are associated with (late onset FA) LOFA or (very late onset FA) VLOFA

Expansion becomes so large in full FX, chromosome compaction is disrupted and this change is visible under the microscope.

Question 19

Which parental transmission is associated with expansion and contraction of FXN alleles?

Answer 19

Both expansions and contractions of expanded alleles can occur

Maternal transmission is associated with expansions and contractions

Question 20

Which parental transmission is associated more frequently with contraction of FXN alleles? When is there a stronger contraction bias?

Answer 20

Paternal transmission is associated primarily, but not exclusively, with contractions.

There is a strong contraction bias among longer expansions (>500 repeats).

Question 21

Describe the general genotype/phenotype correlation in FRDA patients.

Answer 21

Penetrance is complete for hom/compound
hets with GAA repeat expansion/intragenic mutation.

Age of onset can varay 5-50, depending on alleles size and other factoes.

Some missense mutations have a milder phenotype.

Exonic deletions are very rare but may haven a severe, early onset phenotype

In general, shorter GAA expansion sizes are associated with later age of onset and less severe phenotype and slower disease progression (even considered a benign disease course).

Question 22

Which alleles shows greater correlation with the disease presentation?

Answer 22

The size of the shorter of the two expanded repeats shows better correlation, accounting for approximately 50% of the variation in age of onset (loss-of-function disease)

Individuals with LOFA (i.e. age of onset >25 years) frequently have <500 repeats in at least one of the expanded alleles

Individuals with VLOFA (i.e. age of onset >40 years) usually have <300 repeats in at least one of the expanded alleles

Severity also correlates with remaining FRDA activity - smaller GAA allele (loss-of-function disease)

Question 23

What are the exceptions to the general genotype-phenotype correlation?

Answer 23

Genetic background (e.g. in the Acadian population, FRDA patients have a later age of onset and much lower incidence of cardiomyopathy)

Somatic mosaicism of the GAA expansion

Other potential factors: toxic RNAs, repeat-associated proteins, aberrant splicing, altered expression of frataxin isoforms (Evans-Galea et al. 2014).

Therefore it is not possible to precisely predict clinical outcome based on genotype

Question 24

What is the front-line testing strategy for FRDA referrals?

Answer 24

Normal sizes alleles can be detected by PCR flanking the repeat region. If two alleles are detected, no further testing is required

For individuals in whom one or no allele is detected by PCR, Southern blotting or TP (triplet primed) PCR is performed.

Question 25

What additional testing can be performed for FRDA patients where zero/one expanded allele is detected and there is a strong clinical suspicion of the disease?

Answer 25

Sequence analysis , however it is important to bear in mind the relatively high carrier rate in the population (1/60 - 1/100).

Dosage analysis can be performed to detect CNV

Although historically, FA has been diagnosed with DNA-based molecular tests these technologies will miss patients with point mutations or deletions and often require additional testing

Alternatively, protein-based assay measurements of frataxin concentrations in a whole blood sample are more appropriate given deficiency of the protein is the likely causative agent of disease.

Question 26

What is the molecular pathogenesis of FRDA?

Answer 26

1) a deficiency of FXN mRNA and frataxin protein
2) caused by defects in transcription (initiation and elongation) and epigenetic changes (histone deacetylation, methylation)

Question 27

How is FXN tanscription inhibited in FRDA?

Answer 27

Formation of non-B DNA structures, such as triplexes and sticky DNA (expanded GAA alleles), which physically block the transcriptional machinery required for elongation.

DNA-RNA hybrid structures form and impede FXN transcription. These are formed during transcription when the nascent RNA hybridises to the DNA template behind the elongating RNA pol II = R-loops, formed over GAA repeats creating a roadblock for RNA pol II and promoting its termination (no further elongation).

Question 28

What is the effect on methylation of R-loop formation?

Answer 28

Increased R loop formation leads to an increase in repressive chromatin marks (H3K9Me2);

Recruitment of G9a methyltransferase is increased on expanded FXN alleles. It has therefore been proposed that formation of R loops on expanded GAA/CGG alleles act as the primary trigger for repression of expanded FXN and FRMR1 alleles, which in turn promote heterochromatin formation.

Reduced expression is associated with an increase in H3K9 methylation and decreased H3K9 acetylation.

Question 29

While in vitro studies demonstrated decreased transcription elongation of FXN via R loop formation, this effect is smaller in vivo, and there is evidence that deficient transcriptional initiation in FRDA is much more prominent, but its relationship to the expanded GAA mutation remains unclear.

Answer 29

Repressive chromatin extends from the expanded GAA in intron 1 to the upstream regions of the FXN gene, involving the FXN transcriptional start site (TSS). The major FXN transcriptional start site, normally in a nucleosome-depleted region, is rendered inaccessible by altered nucleosome positioning in FRDA

Question 30

What is the function of FXN encoded protein? Where is it expressed?

Answer 30

Frataxin: nuclear encoded mitochondrial protein - it contains mitochondrial targeting signal sequences in the 1st 20 amino acids and localises to the mitochondrial matrix. The 210 amino acid precursor is cleaved in 2 steps by the mitochondrial processing peptidase to the mature protein (aa81-210)

Function: iron homeostasis: biogenenesis of iron-sulphur clusters (ISCs), iron-trafficking in mitochondria, and as an iron chaperone. Since several complexes of the respiratory chain contain iron-sulfur clusters, frataxin has a direct impact on mitochondrial function and respiration.

Frataxin can bind multiple Fe(II) ions on exposed acidic patches and functions as an iron chaperone. It interacts with core ISC assembly proteins and has been proposed to be an iron donor that presents Fe to ISC complex scaffold proteins.

Question 31

Where is FXN expressed?

Answer 31

Ubiquitously expressed at relatively low levels and most abundant in the heart, spinal cord, cerebral and cerebellar cortex, and also expressed in liver, skeletal muscle, pancreas and dorsal root ganglia (tissues affected in FRDA).

Question 32

What is the effect of a FXN loss of function mutation on iron-sulfur clusters?

Answer 32

Loss of frataxin abrogates iron-sulfur cluster synthesis and assembly, decreasing the activity of enzymes dependent on them, and leading to iron accumulation in the mitochondria.

This negatively impacts mitochondria-rich tissues including heart cardiomyocytes and neuronal cells in the central and peripheral nervous system.

Excessive cellular iron causes cytotoxicity as a result of iron-mediated generation of reactive oxygen species (ROS). Disturbances in oxidative metabolism are associated with an increase in the production of toxic reactive oxygen species, which cause tissue degeneration

Question 33

Give a brief summary of the results of LOF FXN mutations on iron levels.

Answer 33

Frataxin deficiency causes cellular and mitochondrial iron accumulation, possibly as a consequence of suppressed utilisation of mitochondrial iron in metabolic processes such as ISC and heme biosynthesis.

Fe can’t be exported from cells as ISCs or heme, so it accumulates in mitochondria and forms iron crystallite aggregates.

This results in an increase in generation of reactive oxygen species. Frataxin deficiency is also linked to impaired activity of the nuclear factor 2-related factor 2 (Nrf2) which regulates the expression of genes involved in anti-oxidant defence, eg glutathione.

Question 34

As het FRDA carriers have a normal phenotype, it has been suggested that raising FXN to wt levels may be effective in treating FRDA patient. What therapeutic approaches are there to do this?

Answer 34

Hets are normal, therefore suggestion to increase FXN to normal levels. Either by:

Increasing transcription - class I histone deactelyase inhibitor increased FXN mRNA. However is they are non-specific.

Blocking R-loop formation by introducing an anti-GAA duplex RNA

Interferon-gamma upregulates FXN in many cell types

Question 35

Other than raising FXN levels, what other therapeutic approaches are there for FRDA patients?

Answer 35

Antioxidants and iron transport molecules e.g. Co-enzyme 10, Idebenone, Deferiprone (iron chelator).

Gene therapy - adenovirus expression of FXN in ko mouse restored Fe/S levels.