Mitochondrial disorders are inherited metabolic conditions caused by pathogenic variants in genes affecting oxidative phosphorylation and ATP synthesis. They are a heterogeneous group of disorders with an incidence of 1 in 5000. They can affect a single organ or multiple systems, with age of onset from prenatal to old age. Organs and systems with high energy requirements such as the brain, peripheral nerves, muscles, heart, eyes, and ears tend to be affected. Mitochondrial disorders can be caused by pathogenic variants in the mitochondrial DNA (mtDNA), which is solely maternally inherited, or by pathogenic variants in more than 300 nuclear genes and can follow any inheritance pattern. mtDNA variants can be homoplasmic (present in all copies of the mtDNA) or heteroplasmic (present in a proportion of the mtDNA).

Diagnosis
Mitochondrial disorders are difficult to diagnose because they are individually rare, and the clinical features overlap with other common or rare disorders. Whole genome sequencing (WGS) offers a promising approach. We analysed 319 families with suspected mitochondrial disorders who were undiagnosed following standard NHS testing and had short read WGS through the 100,000 Genomes Project. Nuclear variants were prioritised based on gene panels, ClinVar pathogenic/likely pathogenic variants and the top ten prioritised variants from Exomiser. mtDNA variants were called using an in-house pipeline and compared to a list of pathogenic variants. Copy number variants and short tandem repeats for 13 neurological disorders were also analysed. Likely causative variants were classified using American College of Medical Genetics (ACMG) guidelines. We found a definite or probable diagnosis in 98/139 (31%). Interestingly, ~60% of diagnoses were non-mitochondrial disorders including developmental disorders with intellectual disability, epileptic encephalopathies, myopathies, other metabolic disorders and leukodystrophies. These would have been missed if a targeted approach was taken, and some have specific treatments.

Whole genome sequencing projects also offer an opportunity to study nuclear mitochondrial DNA segments (NUMTs) in much greater detail than previously possible. NUMTs are copies of all or part of the mtDNA inserted in the nuclear genome. Wei Wei developed a pipeline for identifying NUMTs from short read WGS using split reads and junction reads. We investigated whether NUMT insertion is a cause of rare disease or cancer in the 100,000 genomes project, using gene lists from PanelApp and the COSMIC database. We did not find evidence of rare disorders caused by germline NUMT insertion, but we did find a probably causative somatic insertion in a myxoid liposarcoma.

Natural history
Natural history studies tend to be small and recruit participants from specialist clinics. Working together with the National Congenital Anomalies and Rare Diseases Registration Service (NCARDRS), we have established a national registry of individuals with genetically confirmed mitochondrial disorders. The registry contains 1134 affected individuals resident in England (48% male, 15% children). 73% have mtDNA diagnoses, most commonly m.3243A>G, Leber Hereditary Optic Neuropathy, and large scale mtDNA rearrangements. The commonest nuclear genetic diagnoses were SPG7, dominant optic atrophy (OPA1) and autosomal recessive POLG. We have linked to Hospital Episode Statistics, death certificates and the cancer registry. We present survival analysis, causes of death and cancer standardised incidence ratios.

Treatment
Mitochondrial disorders can cause early death or lifelong disability and currently have no cure. One approach to treatment is to induce mitochondrial biogenesis. Nicotinamide Riboside (NR) is an NAD+ booster which showed promising results in two mouse models of mitochondrial disorders. We describe an open label study to test the effect of NR for four weeks in eight individuals with m.3243A>G, m.3243A>T or large scale mtDNA deletions. NR was safe and well tolerated. There was no improvement in six-minute walk test distance, grip strength, SF-36 questionnaire results or phosphocreatine recovery time after exercise on 31P-MRS. There was a marginally significant improvement in the timed up and go test. Muscle biopsy results showed that the citrate synthase and mtDNA copy number (which are measures of mitochondrial biogenesis) were not significantly different after 4 weeks of NR but increased expression was observed for 16 mitochondrial genes.

We have explored genotypes and phenotypes in mitochondrial disorders considering diagnosis, natural history, and a possible treatment.