Definitive investigations have become more complex and specialized. However, clinical clues can point towards a mitochondrial disorder and fairly simple tests support the diagnosis sufficiently to proceed to specific investigations of mitochondrial function.

To a certain extent there is a relationship between the type of disease and the site of metabolic defect along the pathway from the inner mitochondrial membrane to the termination of the respiratory chain, but there is considerable heterogeneity.

It has long been known that most of the genes responsible for mitochondrial function are nuclear rather than mitochondrial genes, and recently some of these nuclear genes have become much more accessible to analysis. The most important of these is POLG or POLG1, that is, mitochondrial DNA polymerase-gamma.

A major way by which mutations in POLG1 affect mitochondrial function is by inducing mitochondrial depletion. Such mitochondrial depletion may be either generalized or organ specific. Other genes of recent interest include Twinkle, DGUOK and the gene for thymidine kinase 2.

Myopathy with fatigue (various mt tRNA mutations)

• 'Progressive neuronal degeneration of childhood' Alpers disease - with hemiclonic and focal myoclonic seizures often with epilepsia partialis continua, associated with developmental regression and cerebral atrophy ± terminal liver failure (POLG1).
• Leigh disease: regression with hypotonia, oculomotor and respiratory disturbances, with symmetrical lesions on imaging in basal ganglia, thalami, substantia nigra, red nuclei, cerebellum and commonly also in periaqueductal grey matter and spinal cord, and often COX (COX = cytochrome C oxidase = complex IV of the respiratory' chain) deficiency in muscle (genetically heterogeneous: many mitochondrial mutations, especially ATPase6 and genes for tRNA, nuclear genes, especially SURF1 - codes for assembly factor for COX - and genes for Complex 1, but only rarely POLG1).
• Leigh-like syndrome
  • similar to Leigh but oculomotor and respiratory impairment usually absent, more variable neurology (genes as in Leigh)
  • Episodic, static or progressive ataxia ± other neurological deficits ± cerebellar lactate peak on H-MRS (POLG1, mtDNA depletion, ubiquinone deficiency).
  • Chronic encephalomyopathy of childhood; fatigue, pigmentary retinopathy, oculomotor disturbance, sensorineural deafness, ataxia, pyramidal signs, regression, including NARP (mtDNA T8993G or C in complex V = ATPase6).
• Kearns-Sayre syndrome
  • ptosis and ophthalmoplegia, pigmentary retinopathy, heart block, short stature (large mitochondrial rearrangements).
• Pearson syndrome: infantile-onset pancreatic disorder, sideroblastic anaemia, later Kearns-Sayre (large mitochondrial deletion).
• MELAS - mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes: severe migraines, partial epileptic seizures, hemipareses, and cerebral lesions on imaging which superficially look as if they were ischaemic but do not correspond to known vascular territories (mtDNA A3243C, or T3271C).
• MERRF - myoclonic epilepsy with ragged red fibres: myoclonic epileptic seizures with repetitive myoclonus and progressive ataxia and regression in late childhood (mtDNA A8344G).
• MNGIE-mitochondrial neurogastrointestinal encephalomyopathy (nDNA thymidine phosphorylase).
• LHON - Leber hereditary optic neuropathy (mtDNA C3460A, C11778A, T14484C in complex 1 subunits).
• Movement disoiders and various semiologies including paroxysmal dystonia (PDH deficiency, but also mt tRNA and LHON mutations).
• Guillain-Barre type acute polyneuropathy with atypical features including normal GSF protein but increased lactate, with or without recurrence (El a mutations in PDH complex).
• Infantile spinocerebellar ataxia with axonal neuropathy (perhaps Twinkle and twinky.
• Nonspecific combination of static or progressive neurodevelopmental disorder with paroxysmal events such as epileptic seizures and migraine, and evidence of more than one component of the nervous system (POLG1 especially).

• Because these disorders of oxidative phosphorylation (OXPHOS) result in impaired aerobic energy metabolism, primary indicators are increased blood and CSF lactates.
• However, normal lactates do not exclude a mitochondrial disorder, especially in POLG1

Combinations of the following investigations may give clues, depending on the systems involved:

• EEG polyspikes mini-bursts on rhythmic slow activity
• Slow nerve conduction velocity
• CT: calcification of the basal ganglia
• MRI: altered signal in basal ganglia structures (especially globus pallidus), substantia nigra, red nuclei, periaqueductal grey matter, alterations in white matter of centrum semiovale cerebellum
• Neuronal migration disorder in some, e.g. perisylvian polymicrogyria
• MR spectroscopy
• Lacate peak on proton MRS in basal ganglia
• Lactate peak on H-MRS in cerebellum - especially when ataxia
• CSF: increased protein (increased albumin).
• ECG: cardiac conduction defect including heart block and Wolff-Parkinson-White syndrome
• Haematology: marrow suppression
• Blood biochemistry
  • Low albumin
  • Increased alanine and proline
  • Raised transaminases
• Urine biochemistry
  • Generalized aminoaciduria
  • Impaired phosphate reabsorption
  • Krebs cycle organic acids (e.g. fumarate, succinate)

Special mitochondrial investigations

• The combination of clinical and investigation results may point not only to a mitochondrial disorder but also to a specific phenotype.
• Phenotypes such as MELAS, MERRFor Kearns-Sayre relate to a defect of the mitochondrial genome, whereas phenotypes such as Alpers-Huttenlocher would point towards a nuclear mitochondrial gene, in particular polymerase gamma (POLG1) or the mitochondrial DNA helicase Twinkle.

If the clinical picture points to a deficiency in pyruvate dehydrogenase (PDH) such as neonatal encephalopathy with lactic acidosis and absent, hypoplastic or dysplastic corups callosum or in the older child progressive dystonia with altered signal in globus pallidus on imaging, PDH activity may be measured in cultured skin fibroblasts.

While the clinical phenotype suggesting mitochondrial depletion will have pointed towards studies of POLG1 and Twinkle as indicated above, or when there is a less demined but persuasive suggestion of mitochondrial dysfunction, then the next stage is study of muscle and possibly liver.

Histological examination of muscle biopsy may show ragged red fibres on Gomori stain, lipid droplets and abnormal staining for cytochrome c oxidase. Mitochondria may be abnormal on electronmicroscopy. functional studies of all components of the mitochondrial respiratory chain will be undertaken on fresh specimens.

Liver biopsy with functional studies may be needed when the muscle biopsy studies are negative despite pointers to a mitochondrial disorder.

While until recently it has been recommended that CoQ10 be estimated in fresh muscle, while cell (or fibroblast) CoQ10 may be a better way of determining ubiquinone deficiency.

Diagnostic confusion is not infrequent between mitochondrial disorders and other conditions. There may be 'pseudo-' or secondary mitochondrial deficiencies, but also true mitochondrial disorders may masquerade as for instance monoamine neurotransmitter conditions.