Our understanding of mitochondrial biochemistry and genetics has important implications for the investigation of suspected mitochondrial disease. In patients with a clearly defined clinical syndrome it may be possible to confirm the diagnosis with a simple molecular genetic test carried out on DNA extracted from blood. A good example of this is Leber hereditary optic neuropathy, where over 97% of cases are caused by one three defined mtDNA point mutations that are usually homoplasmic in blood.82 A similar approach may also be possible for nuclear genetic mitochondrial disorders (see table 1, although most of these genetic tests are still within the realms of research and are not part of a routine diagnostic service). Investigating the remaining patients is more complex, partly because many disorders may mimic mitochondrial disease and also because there is no one single test that will prove or disprove whether a patient has a mitochondrial disorder. Many different genetic defects in both mitochondrial and nuclear DNA can cause similar neurological disorders, so rather than carry out a series of random genetic tests, it is better to approach the problem systematically to identify and characterise the underlying metabolic defect.
Heteroplasmy is the main problem when investigating mtDNA disorders. Pathogenic mtDNA mutations may not be detectable in blood using conventional techniques, and, almost counterintuitively, direct sequencing of mtDNA is the least robust technique of all. This means that a negative blood test result does not exclude a particular genetic diagnosis (for example, see Chinnery et al, 199783). If mitochondrial disease is suspected and the blood DNA tests are negative, the patient should have a muscle biopsy (usually the first choice in adults) or a skin biopsy (usually the first choice in children). Urine sediment, and to a lesser degree hair follicles, are excellent sources for non-invasive mtDNA testing.84
Fresh muscle can be analysed histologically and histochemically for evidence of mitochondrial disease. Characteristic features include ragged red fibres which can be seen with the Gomori-trichrome stain85 or with succinate dehydrogenase histochemistry.86 The ragged red appearance is caused by the subsarcolemmal accumulation of mitochondria and is thought to be a response to metabolic stress within a diseased muscle cell.87 There may also be a reduction in cytochrome c oxidase activity (COX, complex IV) either within some of the fibres (a mosaic defect, suggestive of a mtDNA disorder,88 but see Sasarman et al, 200289) or affecting all the fibres within the entire biopsy (suggesting a nuclear genetic defect).
Specialist centres carry out measurements of the individual respiratory chain complexes, which may also provide a clue to the underlying genetic defect. These can be done on fresh muscle or cultured fibroblasts grown from a skin biopsy. If a single complex is deficient, this points to a genetic defect in the relevant coding region of mtDNA or nuclear DNA, or a gene involved in the assembly of that particular complex. If there are multiple complex defects, that suggests a generalised defect of protein synthesis, and an underlying mtDNA defect involving a tRNA gene (including deletions that remove tRNA genes), or perhaps a nuclear gene defect with secondary effects on mtDNA. It is worth remembering that mitochondrial biochemical tests carried out on muscle and fibroblasts in the laboratory measure mitochondrial function under optimal conditions. It is therefore possible that there is a functional defect of mitochondrial metabolism that is not detectable in the laboratory (mutations in the ATPase 6 gene causing NARP (neurogenic weakness with ataxia and retinitis pigmentosa) are a good example of this pitfall). Evidence of impaired mitochondrial function may only be apparent on clinical testing using techniques such as exercise testing with lactate measurements,90 magnetic resonance spectroscopy,91 or infrared spectroscopy.92
A structured approach to investigation allows targeted genetic analysis, which often means a Southern blot of muscle mtDNA looking for a mtDNA rearrangement, a series of allele specific assays looking for common point mutations of mtDNA or nuclear DNA, and direct sequencing of the relevant genes. A significant proportion of adults have rare or unique mtDNA defects (that is, "private" mutations). These are identified by mtDNA sequencing which should also be carried out on DNA extracted from muscle (but with the caveat noted above).
Proving a mtDNA mutation is pathogenic
mtDNA is highly polymorphic, with any two individuals differing at up to 60 base pairs (see the mtDNA sequence databases in table 4). The variation is so great that it is not unusual to find unique base changes in control individuals. This presents a particular problem when investigating patients with suspected mtDNA disorders—when is the base change a neutral polymorphism and when is it pathogenic?
Five "canonical" criteria suggest that a novel base change is pathogenic
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- The mutation must not be a known polymorphism (as described on one of the established sequence data bases, see table 4).
- The base change must affect a site that has been conserved during evolution. If the site is conserved across species then it implies that it is functionally important, and a mutation at this site is likely to be deleterious. The mutation must also be in a region that is functionally important. This essentially means anywhere in the tRNA genes, certain regions of the rRNA genes, or causing an amino acid change in the protein encoding genes.
- Deleterious mutations are usually (but not exclusively) heteroplasmic. This implies that the mutation occurred recently and it has not had time to "fix" in the female line, or that there has been selection against fixation acting at the level of the organism.
- The mutation segregates with the disease clinically. For heteroplasmic mutations this means that severely affected individuals have a high percentage level of mutated mtDNA, and unaffected individuals have a lower percentage level of mutated mtDNA.
- The mutation segregates with the disease biochemically. This is usually achieved by single cell mtDNA analysis.93 Individual muscle fibres are microdissected from thick cross sections of muscle and the percentage level of mutated mtDNA is measured in histochemically normal and abnormal muscle fibres (either because they are ragged red or COX deficient). For a pathogenic mutation, the percentage level of mutated mtDNA will be higher in the pathologically abnormal fibres.
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