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Page 190 Watson et al. J Transl Genet Genom 2020;4:188-202 I http://dx.doi.org/10.20517/jtgg.2020.31

AN HISTORICAL PERSPECTIVE ON THE APPROACH TO DIAGNOSIS
Techniques for diagnosing mitochondrial disease have significantly advanced since Ernster and colleagues
[27]
described the enzyme activity of skeletal muscle mitochondria in 1959 , paving the way for their
[28]
identification of Luft’s first reported case of mitochondrial disease . Subsequent development of the
[29]
modified Gomori trichrome stain allowed rapid identification of “ragged red” fibres on muscle biopsy,
the first pathologic hallmark of mitochondrial disease. Clinical-histological descriptions of mitochondrial
diseases ensued, and early diagnostic criteria were based on recognising a constellation of features
comprising a clinical syndrome - such as mitochondrial encephalomyopathy with lactic acidosis and
stroke-like episodes (MELAS) - combined with biochemical and/or histopathological evidence from
muscle tissues [30-36] . This approach resulted in biases towards identified disease syndromes, and led to
[37]
underdiagnosis of those with non-classical symptoms .
[38]
The mitochondrial genome was sequenced in its entirety in 1981 and the first two reports of genetic
causes of mitochondrial diseases were published in 1988 [39,40] . Shortly afterward, the m.3243A>G mutation
was identified as the (most common) cause for the MELAS syndrome . Since this discovery, more than
[41]
300 pathogenic mtDNA point mutations, deletions and rearrangements have been reported, involving
almost all 37 mtDNA-encoded genes [1,42] . Although the nuclear genome encodes a vastly greater proportion
[43]
of the mitochondrial proteome (~1200 genes) , including over 320 genes implicated in disease to
date [1,3,4,44-46] , causative nuclear gene involvement was only definitively established some years after the
first mtDNA mutations were identified [47,48] . Nuclear disease plays a significantly greater role than initially
appreciated, accounting for the majority of childhood-onset disease and a substantial proportion of
[16]
[5]
adult-onset mitochondrial disease . Early tools for genetic diagnosis were limited, testing one or a small
panel of common mtDNA point mutations, with poor sensitivity for heteroplasmy below ~30%-50% by
[42]
Sanger sequencing . Despite limitations, the advent of genetic diagnosis permitted greater appreciation
of the broad spectrum and phenotypic variability associated with specific mutations and mitochondrial
diseases in general [49-52] , which has continued to expand alongside improving sequencing techniques.

Technology to facilitate routine clinical genetic diagnosis did not become readily available until the mid-
2000s and, historically, relied on sequential Sanger sequencing of clinically prioritised individual genes
[42]
- a costly, laborious and limited approach, which necessarily biased towards known genotype-phenotype
correlations. Consequently, definitive genetic diagnosis was difficult to achieve and molecular diagnosis
[53]
rates remained low , with clinical and biochemical characterisation of greatest utility (albeit imperfect) in
confirming or excluding mitochondrial disease. This embedded a “biopsy first” approach, with the clinical
and biochemical phenotype guiding targeted genetic testing [4,54] . The advent of powerful, high-throughput,
[55]
NGS technologies enabling simultaneous interrogation of many, or all genes , has transformed genetic
diagnosis. Consequently, the genetic landscape of mitochondrial diseases - and understanding of
mitochondrial biology - has expanded rapidly over the last two decades, challenging established aetiological
concepts of disease and clinical diagnostic approaches.

LIMITATIONS OF THE TRADITIONAL FUNCTION TO GENE APPROACH
Numerous iterations of a diagnostic algorithm for mitochondrial diseases have been proposed. Although
the complexity of the disease has precluded consensus, common is a “function to gene” approach centred
on muscle biopsy: combining clinical features with biochemical and enzymatic characterisation from
muscle biopsy to guide targeted genetic testing. However, there are significant limitations of this approach,
prompting calls for a paradigm shift to a “genetics first” approach followed by functional validation. [54,56,57]
Figure 1 provides a comparative summary of these approaches.

Muscle biopsy can be a helpful diagnostic tool, demonstrating histological and - often more sensitive -
ultrastructural changes, as well as providing biochemical and enzymatic information. Muscle tissue may