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Page 192 Watson et al. J Transl Genet Genom 2020;4:188-202 I http://dx.doi.org/10.20517/jtgg.2020.31

Clinical characterisation remains imperative - not least to identify and proactively manage organ
involvement - with certain phenotypes indicative of a specific or restricted genotype, particularly amongst
“classical” mtDNA-based syndromes, such as MELAS. However, even where a defined syndrome is present,
[52]
genetic heterogeneity is common (e.g., Leigh Syndrome ). More often, clinical features do not neatly
fit a specific clinical syndrome and presentations can be heterogeneous, with poor phenotype-genotype
correlation and therefore, low predictive value for specific genetic diagnosis [61,70] . Muscle biopsy findings do
[71]
not reliably predict specific genetic aetiologies either . Accordingly, the traditional biopsy-first approach,
followed by Sanger sequencing of clinically prioritised individual genes has been estimated to achieve
[53]
genetic diagnosis in only approximately 11% of patients overall . Further, incremental sequencing costs
can exceed the costs of WES with targeted panel analysis whilst the iterative process can prolong the
[53]
diagnostic odyssey for individuals and reinforce diagnostic bias [14,37] .

THE IMPACT AND CHALLENGES OF EVOLVING NGS TECHNOLOGIES
Targeted nuclear gene panels incrementally improve diagnosis compared to traditional single-gene
Sanger sequencing, with rates reported between 6%-37% after mtDNA sequencing and dependent on the
selected gene set and patient group, as summarised in Table 1 [22,23,53,61,71-74] . However, the vast majority of
patients remain undiagnosed. This approach focuses on commonly known disease genes and mutations,
contributing less to the collective understanding of mitochondrial biology and disease. Unsurprisingly
then, approaches utilising WES combined with mtDNA sequencing (either in advance, or incorporated
into WES [75-77] ) have further improved genetic diagnosis rates to between 35%-68%, depending on the
selected patient group, as summarised in Table 2 [15-21,23] . In one study, 31% of cases resolved through WES
[15]
would have been missed using contemporaneous MitoCarta-based panels . These results have included
many novel disease genes and mutations, (43%-51% of cases in two paediatric studies [16,17] ), thus expanding
genotypic heterogeneity, whilst demonstrating greater phenotypic heterogeneity of known disease
genes [3,23,78] , highlighting the shortcomings of candidate gene approaches.

NGS technologies have dramatically accelerated the identification of novel mitochondrial disease genes
and mutations, with around 15-20 new genes discovered annually over the past decade and more than
350 genes across the nuclear and mitochondrial genome implicated in disease [3,4,46] . Identification and
functional validation of novel gene and mutation candidates have in turn provided novel insights into
mitochondrial structure, function, dynamics, and mechanisms of disease [23,79] . Even early studies evaluating
NGS technologies recognised their potential to revolutionise the diagnostic process for heterogeneous
[42]
disorders, such as mitochondrial disease . WES or WGS are already resolving many outstanding
[21]
challenges associated with mitochondrial disease genetics, in turn improving patient care . However, a
distinction must be drawn between routine clinical genetic testing and the ongoing interchange between
research and genetic diagnosis. The latter is critical for expanding the list of known pathogenic variants,
improving the understanding of mitochondrial biology and disease, enabling refinement of the diagnostic
pipeline and enhancing the routine interpretation of genetic variants. This is necessary to positively impact
the evolution of genetic diagnosis from here, as well as inform clinical management, family planning and
potential therapeutic avenues.

A further important benefit of comprehensive, non-targeted sequencing is the identification of pathogenic
non-mitochondrial disease variants: mimics and phenocopies, especially neurological disorders and
neuromuscular diseases, amongst other monogenic disorders, providing definitive genetic diagnosis,
and, at times, important therapeutic options . Depending on the cohort selection criteria for WES
[56]
studies, proportions of (solved) cases attributable to mitochondrial diseases range from 25%-89% [15,17,19-21]
underscoring the clinically important overlap with other monogenic disorders. Given the broad range of
overlapping disorders to be considered, necessitating multiple sequential panels, the additional cost of
exome sequencing is rapidly negated - and costs continue to decrease. In contrast to targeted gene panels,