This document discusses autosomal disorders of mitochondrial DNA maintenance. It notes that these disorders involve defects in both the nuclear and mitochondrial genomes that can lead to tissue-specific oxidative phosphorylation defects and disease symptoms. Key points include: 1) Mutations in the POLG gene are a major cause of these disorders and can result in a broad spectrum of conditions from mild PEO to Alpers syndrome. Most POLG mutations are autosomal recessive. 2) Mutations in the PEO1 gene are a major cause of autosomal dominant PEO. 3) Mutations in the ANT1 gene are a relatively rare cause of autosomal dominant PEO. Two case studies are described to illustrate POL

1. Mitochondrial DNA maintenance
disorders
Carl Fratter
Oxford Medical Genetics Labs

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)

5. Disorders associated with
mtDNA depletion:
• Alpers syndrome
– POLG (2004)
• Hepatocerebral form
– DGUOK (2002)
– MPV17 (2006)
– PEO1 (2007)
• Encephalomyopathic form
– SUCLA2 (2005)
– RRM2B (2007)
• Myopathic form
– TK2 (2001)
[All autosomal recessive]

6. MtDNA Replication

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:

12. Case 1: AD
Single
Del
Ctrl
Normal
Ctrl
16.6 kb
Normal fragment
8.6 kb fragment
(8 kb deletion)
Average
Exposure
Time
Long
Exposure
Time
11.6 kb
fragment
Mult
Del
Ctrl
AD

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