2. Autosomal disorders of mitochondrial
DNA maintenance
• Unique group of disorders involving defects in both of the
genomes within human cells
– Primary nuclear gene defect in a gene that affects mitochondrial
DNA replication
– Secondary mitochondrial DNA defect – tissue-specific
3. Autosomal disorders of mitochondrial
DNA maintenance
Normal
Depleted
Multiple
Deletions
mtDNA
→ tissue-specific oxidative phosphorylation defects
→ disease symptoms
4. Disorders associated with multiple
mtDNA deletions:
• Progressive external ophthalmoplegia with mitochondrial DNA
deletions
– Autosomal dominant
• PEOA1 – POLG (2001)
• PEOA2 – ANT1 (2000)
• PEOA3 – Twinkle (PEO1) (2001)
• PEOA4 – POLG2 (2006)
– Autosomal recessive
• PEOB1 – POLG (2001)
[note: POLG can cause AD or AR disease; any given mutation is
either associated with AD or AR disease]
• Other:
– MIRAS – POLG (2005)
– SANDO – POLG (2003)
– MNGIE – ECGF1 (thymidine phosphorylase) (1999)
– MNGIE without leukoencephalopathy – POLG (2003)
– Optic Atrophy ‘plus’ – OPA1 (2007)
7. Diagnosis of autosomal disorders of
mtDNA maintenance
• 2 complementary approaches -
Analysis of secondary mitochondrial DNA defects:
• Multiple mtDNA deletions:
– Testing of muscle DNA
– Long range PCR
– Southern blotting
• MtDNA depletion:
– Testing of muscle or liver DNA
– Real-time PCR assay to compare mtDNA copy number to that for an
autosomal nuclear gene
– Results are compared to normal controls
BUT availability of affected tissue can be a problem
8. Diagnosis of autosomal disorders of
mtDNA maintenance
Analysis of primary nuclear gene defects:
• Any DNA sample is suitable
• POLG analysis:
– Restriction digest PCR analysis for 3 particularly common POLG
mutations: p.A467T, p.W748S, p.G848S.
– If appropriate, DNA sequencing of the entire coding region of POLG is
undertaken
• PEO1 (Twinkle) analysis
– DNA sequencing of part of coding region
• ANT1 analysis
– DNA sequencing of coding region
9. Overview of Results
• Mutations in the POLG gene are a major cause of
autosomal disorders of mtDNA maintenance, accounting
for 25% of patients with PEO with mtDNA deletions and
67% of patients with a possible diagnosis of Alpers
syndrome in our cohort.
• Most POLG gene mutations are associated with
recessive disease, and there are several common
founder mutations.
• There appear to be genotype:phenotype correlations
associated with some POLG mutations.
10. Overview of Results
• Mutations in the PEO1 gene also account for a
significant proportion (18%) of PEO with mtDNA
deletions in our cohort.
• Mutation screening of ANT1 recently introduced as a
service:
– Mutations identified in 1 out of 23 patients with PEO with mtDNA
deletions and no mutation identified in POLG or PEO1
– Therefore, mutations in ANT1 appear to be a relatively rare
cause of PEO
• For POLG, PEO1 and ANT1, the vast majority of
mutations are missense changes.
11. Case 1: AD
3
22 2
NA PDEM AD
PEO, ptosis
Mild symptoms of
mito myopathy
Key:
13. Case 1: AD
NA PDEM AD
[R227W]+
[T251I;P587L]
[T251I;P587L]+
[T251I;P587L]
Inferred
[T251I;P587L] het
Inferred
[R227W]+[T251I;P587L]
[R227W]+
[T251I;P587L]
[R227W]+
[T251I;P587L]
14. Case 2: SO
• Patient SO, died aged 1 year, movement disorder,
hypotonia, abnormal liver function ⇒ possible diagnosis
of Alpers syndrome
• MtDNA depletion in liver identified prior to reports of
POLG mutations in Alpers
• Subsequently, POLG testing initiated…..
15. Case 2: SO – DNA results
SO
Normal
SO
Normal
c.2740A>C; p.T914P
c.1879C>T; p.R627WExon 10
Exon 18
16. Case 2: SO – DNA results (contd)
• p.T914P & p.R627W are previously reported mutations
• Compound heterozygosity confirmed by testing the
parents
• Can offer prenatal diagnosis – CVS planned in the next
few weeks
17. Summary
• Mutations in the POLG gene are a major cause of
autosomal disorders of mtDNA maintenance, and lead to
a broad spectrum of disorders (from mild PEO to Alpers)
– Mainly autosomal recessive
– Common founder mutations
• Mutations in the PEO1 gene are a major cause of
autosomal dominant PEO
• Mutations in the ANT1 gene are a relatively rare cause of
autosomal dominant PEO
18. Acknowledgements
• Molecular Genetics Lab, The Churchill:
– Conrad Smith
– Julie Evans
– Anthony O’Rourke
– Iain Dow
– Helen Lord
– Anneke Seller
• NDOG, John Radcliffe Hospital:
– Prof Jo Poulton
