An elusive expansion at the FRDA locus

1
An elusive expansion at the FRDA locus
Claire Healey, Andrew Purvis, Mohammed Kiron
Kibria, Kara Gaffing, Fiona Coyne Roger
Mountford Cheshire and Merseyside Regional
Molecular Genetics Laboratory, Liverpool Womens
Hospital
2
Presentation Overview

• Introduction
• Friedreich ataxia
• Clinical symptoms
• Molecular pathology
• Case 1
• Diagnostic referral
• CAG repeat expansion testing
• Unusual TP-PCR result
• Case 2
• Diagnostic referral
• Premutation plus GAA repeat expansion within the
  disease-causing size range
• Case 3
• Carrier testing

3
Friedreich Ataxia (FRDA)

• Autosomal recessive neurodegenerative disorder
• Affects the spinal column and cerebellum
• Slowly progressive ataxia of the gait limbs
• Onset 10 15 years of age
• Associated with
• Muscle weakness
• Spasticity in the lower limbs
• Absent lower limb reflexes
• Dysarthria
• Scoliosis
• Pes cavus
• Bladder dysfunction
• Loss of position and vibration sense

4
FRDA

• Additional clinical symptoms
• 30
• Hypertrophic non-obstructive
• cardiomyopathy
• 10-25
• Optic atrophy
• Deafness
• Glucose intolerance
• or
• Diabetes mellitus
• 25
• Atypical presentation
• Later age of onset
• Retained tendon reflexes
• or

5
Genetics of FRDA

• Incidence of 2-4 per 100,000 Europe, N.
  Africa, Middle East S. Asia
• Carrier frequency of 1100
• FRDA gene (Frataxin or X25) indentified in 1996
• Expansion of GAA triplet repeat within intron 1
  98 mutations

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• Normal alleles 5-33 GAA repeats
• Alleles gt 27 repeats rare
• Premutation alleles 34-65 GAA repeats
• Expanded alleles gt 66 GAA repeats
• Some alleles have interrupted sequences
• GAAGGA or GAGGAA

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Genetics of FRDA

• Incidence of 2-4 per 100,000 Europe, N.
  Africa, Middle East S. Asia
• Carrier frequency of 1100
• FRDA gene (Frataxin or X25) indentified in 1996
• 98 mutations expansion of GAA triplet repeat
  within intron 1

5a
1
4
2
3
? 106
? 165
? 182
? 1

• 1-2 FRDA patients GAA expansion plus
  inactivating mutation,
• (nonsense, splicing, frameshift or
  missense)
• Homozygous expansion compound heterozygous
  patients
• clinically indistinguishable
• Patients with missense mutations near the
  carboxy-terminus have atypically
• mild FRDA
• No patients have been described with two
  identified
• point mutations

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Molecular Genetic Testing

• Detection of GAA repeats
• Current testing strategy
• F-PCR across repeat region with FAM-labelled
  primers

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Molecular Genetic Testing

• Detection of GAA repeats
• Current testing strategy
• F-PCR across repeat region with FAM-labelled
  primers

n/n (8/29 repeats)
n/?
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Molecular Genetic Testing

• Detection of GAA repeats
• Current testing strategy
• F-PCR across repeat region with FAM-labelled
  primers
• Triplet-prime PCR

n
E
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Case 1

• Diagnostic referral
• Expansion point mutation analysis requested
• Institute of Neurology
• GAA repeat flanking PCR
• TP-PCR
• Clinical details
• 52 year old female
• No further details avaliable

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Case 1

• F-PCR

8 repeats
Patient 1.
31 rpt control 2.
Expansion control 3.
Hom Het normal controls 4. 5.
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Molecular Genetic Testing
Triplet-prime PCR
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Molecular Genetic Testing
Triplet-prime PCR
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Molecular Genetic Testing
Triplet-prime PCR
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cttcttcttcttcttcttcttcttctt
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Molecular Genetic Testing
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Case 1

• TP-PCR

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Case 1

• Modified TP-PCR

Primers FATP-P3-F-FAM FATP-P1-R FATP-P4-F GAA
Int FATP-P4-F GAG Int
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Case 1

• Southern Blot

EcoRV FA3PEx1
Patient Normal E/E n/E 1.
2. 3. 4.
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Case 1

• ? Clinical Significance
• Long GAA repeats tracts form abnormal sticky
  triplex DNA structures

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Case 1

• ? Clinical Significance
• Long GAA repeats tracts form abnormal sticky
  triplex DNA structures
• Inhibit transcription reduced Frataxin protein
• Interrupted alleles
• Triplexes less likely to form
• Not predicted to inhibit transcription of
  Frataxin to the same extent as pure GAA repeats
• Shorter in length (equivalent to alleles of
  100-300 triplets)
• May be associated with late on-set disease
• (GAGGAA)n (GAAAGAA)n interruptions may
  stabilise premutation alleles
• May prevent expansion into abnormal size range
• Clear guidelines regarding the implications of
  these interruptions and their clinical
  significance have not been established

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Case 1

• ? Clinical Significance
• Patient
• 1 normal allele
• 1 interrupted allele
• No further mutations identified on sequence
  analysis
• Unlikely to be affected with FA
• ? chance finding unrelated to the patients
  symptoms
• Further work
• Sequence interrupted allele
• Detection of interrupted
• May be difficult using standard TP-PCR
• Requires contiguous run of GAA repeats

22
Case 2

• Diagnostic referral
• 53 year old female
• Progressive cerebellar degeneration
• F-PCR analysis identified an allele within the
  premutation range (38 rpts)
• TP-PCR analysis detected the presence of an
  expansion

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Case 2

• Southern blot analysis
• Confirmed presence of an allele in the
  premutation size range an expanded allele in
  the affected size range

EcoRV FA3PEx1
Patient Normal E/E n/E 1. 2.
3. 4.
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Case 2

• ? Clinical Significance
• Patient
• 1 allele within premutation size range
• 1 allele within affected size range
• Identified in peripheral lymphocytes
• Premutation alleles
• Not thought to affect transcription of the
  Frataxin gene
• Not thought to be pathogenic
• May show somatic instability
• ? if a significant proportion of such alleles
  expand into the affected size range in
  appropriate tissues, this may lead to atypical
  disease
• Increases the likelihood of a diagnosis of FA
• Further work
• Testing of other tissue types
• Family studies

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Case 3

• Diagnostic referral
• 10 year old child
• Progressive ataxia, weakness, deteriorating motor
  skills, cerebellar dysfunction
• Two GAA repeat expansions
• Mother identified as a carrier using standard
  testing strategy
• Southern blot analysis
• EcoRV
• 
  FA3PEx1

1
7 8
23 Kb -
9.4 Kb -
6.5 Kb -
4.3 Kb -
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Case 3

• Diagnostic referral
• 10 year old child
• Progressive ataxia, weakness, deteriorating motor
  skills, cerebellar dysfunction
• Mother identified as a carrier using standard
  testing strategy
• Modified TP-PCR Assay
• Different locus specific P1-primer
  

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Case 3

• DNA sequencing
• Primers flanking the standard P1 priming site
• 30bp deletion
• Covering the whole of the standard TP-PCR P1
  priming site in the patients father and the
  affected child
• Deletion present on the same allele as the
  expansion
• Explains why the expansion in the patients
  father could not be detected using standard
  TP-PCR
• Summary
• Samples harbouring such a deletion would give
  results consistent with homozygosity for the same
  size normal allele using these assays
• Deletion would not be detected - potentially an
  expansion could be missed
• 115 FA referrals with 1 allele in the normal
  range and no TP-PCR expansion were tested for the
  presence of this deletion
• No further deletions were identified in this
  cohort
• Likely that such a deletion is either very
  uncommon or private to this family

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Acknowledgements

• All within the molecular genetics laboratory
• Andrew Purvis
• Mohammed Kiron Kibria
• Kara Gaffing
• Fiona Coyne
• Roger Mountford

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Thank-you for listening