Mitochondrial DNA maintenance disorders

Carl FratterOxford Medical Genetics Labs

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

Autosomal disorders of mitochondrial DNA maintenance

Normal

Depleted

MultipleDeletions

mtDNA

tissue-specific oxidative phosphorylation defects

disease symptoms

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)

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]

MtDNA Replication

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

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

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.

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.

Case 1: AD

3

22 2

NA PDEM AD

PEO, ptosis

Mild symptoms of mito myopathy

Key:

Case 1: AD

SingleDelCtrl

NormalCtrl

16.6 kbNormal fragment

8.6 kb fragment(8 kb deletion)

AverageExposureTime

LongExposureTime

11.6 kbfragment

MultDelCtrl

AD

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]

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…..

Case 2: SO – DNA results

SO

Normal

SO

Normal

c.2740A>C; p.T914P

c.1879C>T; p.R627WExon 10

Exon 18

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

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

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