Human Molecular Genetics Advance Access published online on May 3, 2007
Human Molecular Genetics, doi:10.1093/hmg/ddm090
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Defects in Maintenance of Mitochondrial DNA are Associated with Intramitochondrial Nucleotide Imbalances
1 Mitochondrial Genetics Group, Nuffield Department of Obstetrics and Gynaecology, Level 3, Women's Centre, The John Radcliffe Hospital, Oxford 2 Laboratoire de Biochimie 1, APHP, hopital de Bicetre, Le Kremlin-Bicetre, France 3 Division of Molecular Neurogenetics, National Neurological Institute "C. Besta", Milano 20126, Italy 4 Biomedicum-Helsinki, Programme of Neurosciences, Helsinki, Finland 5 Centre d'Investigacions en Bioquimica i Biologia Molecular, Hospital Vall d'Hebron, Barcelona, Spain 6 Mitochondrial and Metabolic Disease Center, University of California, San Diego, CA USA
* Corresponding author: Mitochondrial Genetics Group, Nuffield Department of Obstetrics and Gynaecology, Level 3, Women's Centre, The John Radcliffe Hospital, Oxford, OX3 9DU, UK. Phone (44) 1865 221067 Fax (44) 1865 769141 joanna.poulton{at}obs-gyn.ox.ac.uk
Received January 8, 2007; Revised April 1, 2007; Accepted April 1, 2007
Defects mtDNA maintenance range from fatal multisystem childhood diseases like Alpers syndrome, to milder diseases in adults, including mitochondrial DNA depletion syndromes (MDS) and familial Progressive External Ophthalmoplegia (AdPEO). Most are associated with defects in genes involved in mitochondrial deoxynucleotide metabolism or utilisation, such as mutations in thymidine kinase 2 (TK2) as well as the mtDNA replicative helicase Twinkle and gamma polymerase (POLG). We have developed an in vitro system to measure incorporation of radiolabelled dNTPs into mitochondria of saponin permabilized cells. We used this to compare the rates of mtDNA synthesis in cells from 12 patients with diseases of mtDNA maintenance. We observed reduced incorporation of exogenous 
32P-dTTP in fibroblasts from a patient with Alpers Syndrome associated with the A467T substitution in POLG, a patient with dGK mutations, and a patient with mtDNA depletion of unknown origin compared to controls. However, incorporation of 32P -dTTP relative to either cell doubling time or 32P-dCTP incorporation was increased in patients with thymidine kinase deficiency or PEO as the result of TWINKLE mutations compared with controls. The specific activity of newly synthesized mtDNA depends on the size of the endogenous pool diluting the exogenous labelled nucleotide. Our result is consistent with a deficiency in the intra-mitochondrial pool of dTTP relative to dCTP in cells from patients with TK2 deficiency and TWINKLE mutations. Such DNA precursor asymmetry could cause pausing of the replication complex and hence exacerbate the propensity for age-related mtDNA mutations. Because deviations from the normal concentrations of dNTPs are known to be mutagenic, we suggest that intra-mitochondrial nucleotide imbalance could underlie the multiple mtDNA mutations observed in these patients.
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