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Watson et al. J Transl Genet Genom 2020;4:188-202 I http://dx.doi.org/10.20517/jtgg.2020.31 Page 195

A PROPOSED NEW APPROACH
We suggest a genetics first diagnostic approach given the technical suitability of NGS for mitochondrial
[26]
disease genetics and the expanding capability to reliably identify and call mitochondrial disease variants .
A genetics first diagnostic approach is also advocated for by others [56,57] and a proposed process is outlined
[1]
in Figure 2 (adapted from Davis et al. ).
The first stage aims to stratify the population for testing by answering two questions: (1) “is mitochondrial
disease likely?” and, if so, (2) “is there a distinctive phenotype indicative of the genotype?” to inform the
most appropriate genetic testing strategy. The next stage focuses on molecular diagnosis - either identifying
a known pathogenic mutation, validating a novel mitochondrial-disease causing variant, or identifying a
genetic phenocopy.

A careful and comprehensive history, including inheritance pattern where possible, together with
comprehensive clinical examination, enables accurate clinical phenotyping and should be combined with
tailored initial investigations to characterise organ involvement and form an initial clinical estimate of the
likelihood of mitochondrial disease. Routine laboratory investigations, including those aimed at excluding
infective or inflammatory processes and other mimics, should be undertaken alongside specific evaluation
of serum lactate and pyruvate, creatine kinase (CK) and a urinary metabolic screen: indicators of disease
but with limited sensitivity and specificity [54,66] . In adults, neuroimaging typically includes MRI of the brain
(ideally with MR spectroscopy of CSF), and may demonstrate characteristic or non-specific patterns, or be
normal [66,84-86] . Electroencephalogram, nerve conduction studies and electromyography may complement
the initial clinical evaluation. Cardiac evaluation with electrocardiogram, 24-hour holter monitor
and echocardiogram is critical to evaluate potentially life-threatening organ involvement and bedside
ophthalmological examination may be augmented by retinal photography, or formal ophthalmological
evaluation where appropriate.

The incorporation of biomarkers may aid clinical stratification (discussed below). If initial clinical
evaluation and investigations are equivocal and/or biomarkers are negative, further supportive evidence
for disease should be sought, prior to initiating comprehensive genetic testing. For example, the yield from
[87]
detailed ophthalmological evaluation is high , with findings often specific for mitochondrial disease,
whereas other investigations, such as GI motility, although predictive of a positive genetic diagnosis when
[88]
present, are less specific for mitochondrial diseases or a particular genetic culprit .

The combination of suggestive clinical features, inheritance and initial investigations, together with positive
biomarkers, should prompt the clinician to progress to genetic evaluation. Where a classical phenotype
suggests a deletion syndrome, or one of a restricted group of causative genes or mutations, established
targeted sequencing approaches in an appropriate tissue source (deletions often require uroepithelium or
muscle) are readily available, rapid and cost-effective. If targeted sequencing returns negative, and in the
many instances where a specific genetic cause or candidate is not able to be proposed, a comprehensive
sequencing approach encompassing all potentially causative genes should be considered (discussed further
below).

If, after comprehensive bigenomic sequencing, a genetic diagnosis still cannot be established, a review of
the clinical presentation, consideration for further investigations - including muscle biopsy for biochemical
and enzymatic studies, and genetics (in post-mitotic tissue) - and a periodic review of genetic data should
be undertaken, as bioinformatics pipelines, variant analysis and the catalogue of known disease genes and
pathogenic mutations are rapidly evolving.

With this proposed approach, muscle biopsy is not omitted entirely. Rather, it is selectively utilised to
achieve specific end-points. Scenarios where early incorporation of muscle biopsy may be relevant include