title: Liver mtDNA content increases during development: a comparison of methods and the importance of age- and tissue-specific controls for the diagnosis of mtDNA depletion.
abstract: BACKGROUND
  The quantitative loss of mitochondrial DNA (mtDNA) known as mtDNA depletion, often gives rise to liver disease. The diagnosis of mtDNA depletion syndrome is frequently imprecise, both for technical reasons and because of the lack of established age-adjusted normal ranges. We aimed to refine quantitative methods for diagnosing the hepatic type of mtDNA depletion syndrome, firstly by establishing an age-matched reference range for mitochondrial to nuclear DNA ratio (henceforth "mtDNA content") and secondly by investigating mtDNA in fibroblasts.
  
  
  METHODS
  By comparing realtime PCR with an established method for quantifying mtDNA content we established a reference range for young children using biopsy and post-mortem material from patients <15 years. In addition, we investigated the arrangement of mtDNA in nucleoids from fibroblasts using fluorescence microscopy.
  
  
  RESULTS
  Both methods showed that the mtDNA content of liver increases rapidly over the perinatal period. In a patient whose liver mtDNA content fell, but remained within the reference range, early investigation and age-matched controls were essential, as we found a progressive increase in muscle mtDNA copy number, respiratory chain activity and muscle power with age. In three further patients, fluorescence microscopy of the fibroblasts proved diagnostic. In one case a movement disorder was an important pointer.
  
  
  CONCLUSIONS
  These cases highlight the (i) need for comparing mtDNA copy number data generated from patients to DNA isolated from an age-matched normal range from the tissue of interest and (ii) the utility of mtDNA staining with PicoGreen as a method to detect aberrant nucleoid morphology in mtDNA depletion patient fibroblast lines when affected tissues are not available for measuring mtDNA copy number.
authors.affiliation: University of Oxford, Nuffield Department of Obstetrics and Gynaecology, The Womens Centre, John Radcliffe Hospital, Oxford OX3 9DU, UK.
authors.forename: Karl J
authors.initials: KJ
authors.lastname: Morten
authors.forename: Neil
authors.initials: N
authors.lastname: Ashley
authors.forename: Frits
authors.initials: F
authors.lastname: Wijburg
authors.forename: Nedim
authors.initials: N
authors.lastname: Hadzic
authors.forename: Jeremy
authors.initials: J
authors.lastname: Parr
authors.forename: Sandeep
authors.initials: S
authors.lastname: Jayawant
authors.forename: Susan
authors.initials: S
authors.lastname: Adams
authors.forename: Laurence
authors.initials: L
authors.lastname: Bindoff
authors.forename: Henk D
authors.initials: HD
authors.lastname: Bakker
authors.forename: Giorgina
authors.initials: G
authors.lastname: Mieli-Vergani
authors.forename: Massimo
authors.initials: M
authors.lastname: Zeviani
authors.forename: Joanna
authors.initials: J
authors.lastname: Poulton
mesh_terms: D000367:Age Factors; D002675:Child, Preschool; D004272:DNA, Mitochondrial / Q000378:metabolism*; D017809:Fatal Outcome; D005347:Fibroblasts / Q000166:cytology; D006801:Humans; D015151:Immunoblotting; D007223:Infant; D008099:Liver / Q000254:growth & development* / Q000378:metabolism*; D008107:Liver Diseases / Q000175:diagnosis / Q000378:metabolism*; D008297:Male; D028361:Mitochondrial Diseases / Q000175:diagnosis*; D009930:Organic Chemicals; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D004272:DNA, Mitochondrial; D009930:Organic Chemicals; C099476:PicoGreen