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 3 222 NAPD EMAD 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 PD EMAD [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.R627W Exon 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