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  • Review Article
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Applying genomic and transcriptomic advances to mitochondrial medicine

Nature Reviews Neurology volume 17, pages 215–230 (2021)Cite this article

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Abstract

Next-generation sequencing (NGS) has increased our understanding of the molecular basis of many primary mitochondrial diseases (PMDs). Despite this progress, many patients with suspected PMD remain without a genetic diagnosis, which restricts their access to in-depth genetic counselling, reproductive options and clinical trials, in addition to hampering efforts to understand the underlying disease mechanisms. Although they represent a considerable improvement over their predecessors, current methods for sequencing the mitochondrial and nuclear genomes have important limitations, and molecular diagnostic techniques are often manual and time consuming. However, recent advances in genomics and transcriptomics offer realistic solutions to these challenges. In this Review, we discuss the current genetic testing approach for PMDs and the opportunities that exist for increased use of whole-genome NGS of nuclear and mitochondrial DNA (mtDNA) in the clinical environment. We consider the possible role for long-read approaches in sequencing of mtDNA and in the identification of novel nuclear genomic causes of PMDs. We examine the expanding applications of RNA sequencing, including the detection of cryptic variants that affect splicing and gene expression and the interpretation of rare and novel mitochondrial transfer RNA variants.

Key points

  • At present, the diagnosis of primary mitochondrial disease is a multistep process often involving a number of time-consuming and highly manual molecular techniques.

  • Early whole-genome sequencing of blood, analysing both mitochondrial and nuclear DNA, is likely to improve diagnostic efficiency in some people with mitochondrial disease.

  • In future, the application of long-read sequencing to mitochondrial DNA could build on the advances made by next-generation sequencing to further enhance coverage, and enable the identification of large-scale rearrangements and point mutations in a single test.

  • As with other rare diseases, whole-genome long-read sequencing might provide the next diagnostic uplift given its ability to identify structural variants, short tandem repeat variants and epigenetic modifications, and to phase compound heterozygous variants.

  • Mitochondrial medicine is poised to benefit substantially from the increasing use of RNA sequencing of tissue samples; advances in pre-processing and sequencing of transfer RNA are enabling new insights into this molecule, which plays an outsized role in the aetiology of mitochondrial diseases.

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Fig. 1: Summary of PMD symptoms.
Fig. 2: Approach to finding a genetic diagnosis in the many patients with suspected PMD in whom targeted testing is not feasible.
Fig. 3: Single-molecule real-time sequencing and nanopore sequencing.
Fig. 4: The role of long-read sequencing in mitochondrial medicine.
Fig. 5: The role of RNA sequencing in mitochondrial medicine.
Fig. 6: Sequencing of transfer RNA in mitochondrial medicine.

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Acknowledgements

The University College London Hospitals/University College London Queen Square Institute of Neurology sequencing facility receives a proportion of funding from the Department of Health’s National Institute for Health Research Biomedical Research Centres funding scheme. The clinical and diagnostic ‘Rare Mitochondrial Disorders’ Service in London is funded by the UK NHS Highly Specialized Commissioners. The work of R.D.S.P. is supported by a Medical Research Council Clinician Scientist Fellowship (MR/S002065/1). J.V. holds a fellowship from the Health Education England Genomics Education Programme. All authors are supported by a Medical Research Council strategic award to establish an International Centre for Genomic Medicine in Neuromuscular Diseases (ICGNMD) (MR/S005021/1). The authors are grateful for the feedback on the manuscript provided by L. Wilson, research manager for the ICGNMD, and for the expert input from R. Labrum and C. Woodward, Neurogenetics Unit, The National Hospital for Neurology and Neurosurgery, London.

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Authors and Affiliations

  1. Department of Neuromuscular Diseases, UCL Queen Square Institute of Neurology and The National Hospital for Neurology and Neurosurgery, London, UK

    William L. Macken, Jana Vandrovcova, Michael G. Hanna & Robert D. S. Pitceathly

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  1. William L. Macken
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All authors contributed equally to the conceptualization and writing of the paper.

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Correspondence to Robert D. S. Pitceathly.

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Supplementary information

Glossary

Polycistronic transcript

A transcript that contains the code for more than one polypeptide.

mtDNA maintenance

Continuous re-synthesis of mtDNA by a nuclear-encoded replication apparatus supported by a sustained pool of mitochondrial nucleotides.

Postmitotic tissue

Tissues, such as muscle, that are terminally differentiated and no longer replicate, and therefore are more likely to retain pathogenic mtDNA variants than mitotic tissue.

Deep sequencing

Sequencing a DNA locus many more times than in standard NGS, enabling low levels of alternative alleles (heteroplasmy and mosaicism) to be identified.

Next-generation sequencing

(NGS). A process by which DNA is fragmented into short molecules and denatured; millions of sequencing reactions (addition of fluorescence-labelled nucleotides to form a complementary strand) then occur concurrently and the short sequences or ‘reads’ generated are mapped to a reference genome.

Short reads

The fragments of genetic sequence generated in NGS; typically ~150 bp in length.

Structural variants

(SVs). Large genetic variants such as copy number variants (deletions and duplications), inversions, and translocations, typically >1,000 bp.

Phase

The homologous chromosome of origin (either maternal or paternal).

Epigenetic modifications

Chemical modifications to DNA or the histone molecules around which DNA is packaged; they do not change the genetic code, but can alter gene expression.

Whole-exome trio

Sequencing and comparison of the coding DNA of an affected proband and the proband’s unaffected parents.

Restriction fragment length polymorphism

Differences between individuals in the length of DNA fragments produced by restriction enzymes; the presence of a mutation can create or remove a restriction site.

mtDNA large-scale rearrangement

A rearrangement, typically a deletion and/or duplication, of >1,000 bp in mitochondrial DNA.

Long-range PCR

PCR amplification of mtDNA as one or two fragments using specialized polymerase; traditional PCR amplifies shorter fragments of DNA.

Coverage

Refers to the adequate sequencing of a locus; targeted sequencing can have poor uniformity of coverage.

GC content

Proportion of bases that are guanine–cytosine.

Phenocopies

Diseases with clinical presentations that overlap substantially with the disease of interest.

De novo assembly

Assembly of reads into a continuous sequence without the need to align them against a reference sequence.

Homopolymeric regions

Sequences of DNA comprising identical repeated units of sequence.

Contigs

Consensus sequences comprising overlapping short sequence reads.

Imprinted genes

Genes that are expressed from only one parental origin; the silenced parental copy is said to be ‘imprinted’.

Adaptor ligation

A short synthetic DNA molecule added to the end of the DNA fragment to enable sequencing of that fragment.

Anticodon

The three-nucleotide sequence in tRNA, which is complementary to a codon in mRNA.

Wobble position

The third nucleotide in the anticodon; the Watson–Crick base pairing here is less specific than usual and atypical pairing can occur.

Cybrids

Cell lines created by fusing an enucleated cell (only containing mtDNA) with a nucleated cell, which can contain nDNA and mtDNA or be modified to contain only nDNA.

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Macken, W.L., Vandrovcova, J., Hanna, M.G. et al. Applying genomic and transcriptomic advances to mitochondrial medicine. Nat Rev Neurol 17, 215–230 (2021). https://doi.org/10.1038/s41582-021-00455-2

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