Download An elusive expansion at the FRDA locus

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 Women’s Hospital
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;
• GAA repeat expansion undetected using standard analysis
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
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
 Unusually slow disease progression
Genetics of FRDA
•
•
Incidence of 2-4 per 100,000 – Europe, N. Africa, Middle East & S. Asia
Carrier frequency of ~ 1:100
•
FRDA gene (Frataxin or X25) indentified in 1996:
1.
Expansion of GAA triplet repeat within intron 1 = 98% mutations
1
2
3

aaaaaaaaaaaaaaagaagaag
aagaagaagaagaaaataaaga
Normal alleles: 5-33 GAA repeats;
 Alleles > 27 repeats rare;
 Premutation alleles: 34-65 GAA repeats;
 Expanded alleles: > 66 GAA repeats


Some alleles have interrupted sequences:
GAAGGA or GAGGAA
4
5a
Genetics of FRDA
•
•
Incidence of 2-4 per 100,000 – Europe, N. Africa, Middle East & S. Asia
Carrier frequency of 1:100
•
FRDA gene (Frataxin or X25) indentified in 1996:
1.
98% mutations = expansion of GAA triplet repeat within intron 1
1
2
3
4
5a




1
106
165
182
2.
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
Molecular Genetic Testing
Detection of GAA repeats:
•
Current testing strategy:
a)
F-PCR across repeat region with FAM-labelled primers
Molecular Genetic Testing
Detection of GAA repeats:
•
Current testing strategy:
a)
F-PCR across repeat region with FAM-labelled primers
n/n (8/29 repeats)
n/?
Molecular Genetic Testing
Detection of GAA repeats:
•
Current testing strategy:
a)
b)
F-PCR across repeat region with FAM-labelled primers;
Triplet-prime PCR
n
E
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
Case 1
F-PCR:
8 repeats
Patient
1.
31 rpt control
2.
Expansion control
3.
Hom & Het normal controls
4. & 5.
Molecular Genetic Testing
Triplet-prime PCR:
gaagaagaagaagaagaagaa
cttcttcttcttcttcttcttcttctt
Molecular Genetic Testing
Triplet-prime PCR:
gaagaagaagaagaagaagaa
cttcttcttcttcttcttcttcttctt
Molecular Genetic Testing
Triplet-prime PCR:
gaagaagaagaagaagaagaa
cttcttcttcttcttcttcttcttctt
Molecular Genetic Testing
gaagaagaagaagaagaagaa
cttcttcttcttcttcttcttcttctt
gaagaagaagaagaagaagaa
gaagaagaagaagaagaagaa
gaagaagaagaagaagaagaa
Case 1
TP-PCR:
Case 1
Modified TP-PCR:
Primers:
FATP-P3-F-FAM
FATP-P1-R
FATP-P4-F GAA Int + FATP-P4-F GAG Int
Case 1
Southern Blot:
EcoRV
FA3PEx1
Patient
Normal
E/E
n/E
1.
2.
3.
4.
Case 1
? Clinical Significance:
•
Long GAA repeats tracts form abnormal ‘sticky’ triplex DNA structures;
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
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 patient’s symptoms
•
Further work:

•
Sequence interrupted allele
Detection of interrupted:


May be difficult using standard TP-PCR;
Requires contiguous run of GAA repeats
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
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.
4.
2.
3.
Case 2
? Clinical Significance:
•
Patient:



•
Premutation alleles:



•
1 allele within premutation size range;
1 allele within affected size range;
Identified in peripheral lymphocytes
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
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:
23 Kb -
9.4 Kb -
EcoRV
FA3PEx1
6.5 Kb -
4.3 Kb -
1
7 8
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;
No expansion detected
Standard TP-PCR
Modified TP-PCR
Mother
Father
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 patient’s
father and the affected child;


Deletion present on the same allele as the expansion;
Explains
Motherwhy the expansion in the patient’s father could not be detected
using standard TP-PCR
Summary:


Samples harbouring such a deletion would give results consistent with
homozygosity
Fatherfor 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
testedchild
for the presence of this deletionBreak point
Affected
No further deletions were identified in this cohort
Likely that such a deletion is either very uncommon or private to this family
Acknowledgements
All within the molecular genetics laboratory
Andrew Purvis
Mohammed Kiron Kibria
Kara Gaffing
Fiona Coyne
Roger Mountford
Thank-you for listening