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Diagnosis of mitochondrial DNA depletion syndromes
  1. Shamima Rahman1,2,3,
  2. Joanna Poulton4,5
  1. 1
    Mitochondrial Research Group, UCL Institute of Child Health, London, UK
  2. 2
    MRC Centre for Neuromuscular Diseases, National Hospital for Neurology, Queen Square, London, UK
  3. 3
    Metabolic Unit, Great Ormond Street Hospital, London, UK
  4. 4
    Nuffield Department of Obstetrics and Gynaecology, University of Oxford, Oxford, UK
  5. 5
    Dubowitz Neuromuscular Centre, Great Ormond Street Hospital, London, UK
  1. Professor Joanna Poulton, Nuffield Department of Obstetrics and Gynaecology, Level 3, The Women’s Centre, John Radcliffe Hospital, Headington, Oxford OX3 9DU, UK; Joanna.Poulton{at}

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Mitochondrial DNA (mtDNA) depletion syndromes (MDDS) are a group of clinically heterogeneous autosomal recessive disorders characterised by a severe quantitative reduction of total mtDNA, the genetic material present exclusively within mitochondria. mtDNA is a 16.5 kb circular genome, encoding 13 subunits of the respiratory chain and 24 RNA molecules necessary for the intramitochondrial translation of these 13 proteins. Unlike nuclear DNA, where every cell contains two copies of each gene (one copy from each parent), mtDNA is a multicopy genome, each cell containing thousands of copies. mtDNA depletion has been defined as a residual mtDNA copy number of <30% compared with age-matched controls,1 2 but mtDNA levels are often <10%, and sometimes as little as 1–2%, of controls, particularly in the hepatocerebral form of the disease. Recognised clinical presentations of MDDS include early-onset hepatocerebral disease overlapping with Alpers syndrome, isolated myopathy, encephalomyopathy and the MNGIE (mitochondrial neurogastrointestinal encephalomyopathy) syndrome (table 1).

Table 1 The mitochondrial DNA (mtDNA) depletion syndromes: genes, phenotypes and pathogenic mechanisms

MDDS may be caused by recessive defects in proteins involved in mtDNA replication (making copies of mtDNA) or in proteins that synthesise deoxyribonucleoside triphosphates (dNTPs) for incorporation into mtDNA (table 1). Both types of defect are likely to cause stalling of the replication complex,3 a mechanism that is also important in the generation of multiple mtDNA deletions, another defect of mtDNA maintenance. Alpers syndrome and related hepatocerebral disorders constitute the most common subgroup of MDDS, caused by mutations in the POLG gene encoding the catalytic subunit of the mitochondrial DNA polymerase γ,4 5 the enzyme responsible for mtDNA replication. Alpers syndrome is characterised by intractable epilepsy and liver dysfunction, but either problem can present in isolation. In patients with POLG mutations and Alpers syndrome, the degree of tissue mtDNA depletion has some correlation with the clinical severity.6 As well as profound tissue depletion of mtDNA, the most severely affected may have mosaic mtDNA depletion in fibroblast cultures, a diagnostic clue that may be useful even posthumously in ∼20% of patients with MDDS, including POLG and DGUOK mutants.7 Not surprisingly, the mutations in these patients occur in the most important parts of POLG, namely the catalytic domains. Patients with mutations only in non-catalytic (linker) domains are likely to present later, with primarily neurological rather than liver disease and much less, if any, mtDNA depletion.6

mtDNA synthesis occurs throughout the cell cycle and is dependent on both salvage of nucleosides to provide a constant supply of dNTPs and, during replication of nuclear DNA, import into the mitochondria of nucleotides synthesised de novo in the cytoplasm. The enzymes catalysing the first steps of the mitochondrial nucleoside salvage pathway are deoxyguanosine kinase (dGK) and thymidine kinase 2, and mutations in the genes encoding both these enzymes have been implicated in the pathogenesis of MDDS.8 9 More than 80 affected patients from ∼50 families with DGUOK mutations have previously been reported.8 1022 In this issue of the journal, Lee and colleagues report a new family with DGUOK mutations and the new technology that was used to diagnose it.23 Their family has the classical phenotype of dGK deficiency, which typically presents soon after birth with early-onset progressive liver disease, usually associated with neurological dysfunction (hypotonia, nystagmus and psychomotor retardation) by the age of 3 months. Peripheral neuropathy and renal tubulopathy have been reported in occasional patients. In most cases, the course is of rapidly progressive liver disease and neurological deterioration, with death occurring by the age of 12 months or shortly after.

Perspective on the paper by Lee et al (see p 55)

Establishing a diagnosis of MDDS can be difficult. Clues to diagnosis include observation of mitochondrial proliferation (ragged red fibres and/or fibres staining intensely positive for succinate dehydrogenase) on muscle histology, which is unusual in other mitochondrial disorders of childhood onset. However, muscle histology may be normal in MDDS cases with a purely hepatic presentation. Biochemical investigations may also be helpful in suggesting a diagnosis of MDDS, which should be suspected when there are multiple respiratory chain defects involving two or more of complexes I, III, IV and V and typically sparing complex II, the subunits of which are entirely encoded by nuclear genes.24 Other documented causes of multiple respiratory chain defects with sparing of complex II include mtDNA RNA mutations (including large-scale rearrangements of mtDNA) and nuclear-encoded defects of mitochondrial translation. It should be noted that isolated deficiency of complex IV or I may precede multiple respiratory chain defects early in the disease course of some cases of MDDS.24 MDDS is confirmed by demonstrating a severe reduction of mtDNA content compared with age-specific control values, using Southern or dot blot25 or quantitative real-time PCR analysis.2 However, mtDNA copy number may be normal in unaffected tissues (including muscle, blood and fibroblasts) in some patients with documented DGUOK mutations, and so liver biopsy may be necessary to confirm the diagnosis.

Liver biopsy is potentially hazardous and is contraindicated in the context of liver failure with severe coagulopathy, because of the risk of life-threatening haemorrhage. There is therefore increasing pressure to proceed to direct sequence analysis of the DGUOK gene early in the diagnostic process, as a molecular diagnosis of dGK deficiency may be regarded as a useful prognostic indicator in patients in whom liver transplantation is being considered. The cases of nine patients with DGUOK mutations who underwent hepatic transplantation have been reported.11 13 14 1719 22 Six of these infants died, so a molecular diagnosis of dGK mutation may lead to a decision not to transplant, particularly if there is evidence of neurological involvement. Sequence analysis of DGUOK alone is likely to yield a genetic diagnosis in fewer than 20% of cases of hepatocerebral MDDS.11 21 In the remaining cases, responsible mutations may be in the POLG (most commonly), MPV17 or PEO1 genes (table 1), or in as yet unknown genes. An ideal diagnostic strategy for hepatocerebral MDDS thus needs to include sequence analysis of POLG, MPV17 and PEO1, in addition to DGUOK. Such extensive sequence analysis of multiple candidate genes is currently not available on a diagnostic basis in most centres.

In the study reported in this issue, Lee et al used new technology to confirm mtDNA depletion and identify the causative mutations in a single step.23 The new technology uses the technique of comparative genomic hybridisation (CGH).26 This method is widely used for performing detailed cytogenetics at a resolution that cannot be achieved by conventional microscopy. It can investigate the number of copies present for every region of the whole of the human genome using either a chromosomal spread of a control human cell or synthetic oligonucleotides to represent all or some of the sequences present in the human genome. In this case, the array was specifically designed to cover mtDNA and 130 nuclear genes that are relevant to it. The sequences present in the patient’s DNA are compared with those present in DNA from a control. This is done by labelling the patient DNA in one colour (for example, red) and the control DNA in another colour (for example, green) and hybridising them both to the array of sequences representing the entire human genome. If the patient has a normal karyotype, then the detection array will be uniformly yellow. If the patient has a triplication, then a series of oligonucleotide probes or a region of one chromosome will appear red. Conversely, if the patient has a deletion, as in the case report of Lee et al, that series of probes or chromosomal region will appear green. This method is used routinely by cytogenetic laboratories for cancer genetics and for karyotyping children with developmental delay or dysmorphic syndromes. It is not able to pick up subtle changes in DNA sequence, tiny DNA losses or reciprocal translocations that do not result in gain or loss of DNA. In this paper, Lee et al found reduced mtDNA levels, and identified a causative deletion in one DGUOK allele. However, to find the second DGUOK mutation, they were obliged to sequence the DGUOK gene.

Array CGH is a costly technique which cannot be performed in all patients with suspected MDDS on a routine basis in most centres. Other diagnostic tests are clearly needed. Metabolite assays are helpful in the diagnosis of MNGIE syndrome. In this disorder, characteristic increases in thymidine and deoxyuridine are observed in plasma and urine, and the diagnosis can be confirmed by assay of thymidine phosphorylase activity in platelets.27 Characteristic metabolites are also observed in patients with MDDS caused by mutations in the SUCLG1 and SUCLA2 genes encoding the α and β subunits of succinyl-CoA ligase.2831 In these disorders, raised succinyl-CoA inhibits methylmalonyl-CoA mutase, resulting in increased plasma and urine methylmalonic acid concentrations (although concentrations may be only slightly increased).

The amino acid, tyrosine, may potentially be a useful metabolite in dGK deficiency. Raised tyrosine concentrations have previously been reported in seven patients with DGUOK mutations, including increases in newborn screening samples in five.19 22 The mechanism of hypertyrosinaemia in dGK deficiency is not clear. It may be a non-specific effect of liver disease, but the very early onset (including detection in newborn screening samples taken on day 1 of life in several cases) and lack of reports of increased tyrosine in other forms of hepatocerebral MDDS raises suspicion that an accumulated metabolite may be inhibiting a step in the tyrosine-degradation pathway, such as 4-hydroxyphenylpyruvate dioxygenase or fumarylacetoacetase. Furthermore, the observation of hepatic tumours in two patients with dGK deficiency and raised tyrosine concentrations19 is intriguing, as hepatocellular carcinoma is a common feature of untreated tyrosinaemia type I (fumarylacetoacetase deficiency).

In conclusion, the work by Lee and colleagues suggests a novel (although costly) way of investigating suspected mitochondrial disease using array CGH. The confirmation of raised plasma tyrosine concentrations in affected patients with DGUOK mutations may provide a metabolic clue for early detection of these patients, allowing appropriate direction of molecular investigations and (possibly) early intervention with nucleoside therapy,7 or hepatic transplantation in cases without evidence of neurological impairment.


We acknowledge our funding bodies: the Medical Research Council, the Wellcome Trust, SPARKS, the Angus Memorial Mitochondrial Fund and the Great Ormond Street Hospital and Institute of Child Health Science Development Initiative. SR is a DH/HEFCE senior clinical lecturer. We thank Professor Peter Clayton for helpful discussions.


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