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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 197

disease mimics [15,19,20] . Therefore, careful consideration should be given to whether invasive investigation is
justified at this stage. Later incorporation of muscle biopsy may be relevant for evaluation and functional
[4]
validation of identified novel variants , in cases where definitive genetic diagnosis is not forthcoming, for
[89]
investigation using more disease-relevant post-mitotic tissues, including to interrogate mtDNA deletions
and/or histological and biochemical evidence of mitochondrial disease in the absence of genetic diagnosis.

THE ROLE OF SERUM BIOMARKERS
The addition of sensitive and specific serum biomarkers to the initial evaluation may aid stratification
of genetic testing. Traditional and commonly tested serum biomarkers of mitochondrial disease include
lactate, pyruvate, their ratio, and CK. However, results may vary substantially, depending on factors
[90]
including activity, diet and sample handling and they lack sufficient sensitivity and specificity for clinical
[91]
utility in mitochondrial disease . Recently, more sensitive and specific serum biomarkers have been
identified, although there remains scope for improvement.

Elevated levels of fibroblast growth factor-21 (FGF-21) have been demonstrated in people with muscle-
manifesting mitochondrial diseases, compared to non-mitochondrial disease and healthy controls [91-93] .
Further research indicates FGF-21 levels best correlate with defects of mitochondrial translation and may
[93]
be normal in defects of respiratory chain complexes or their assembly factors . More recent functional
studies of mitochondrial myopathy in human and mouse models demonstrate the crucial role of FGF-21 in
mt
the integrated mitochondrial stress response (ISR ), activating the systemic stress response and inducing
systemic metabolic consequences . However, FGF-21 levels can also be elevated in non-mitochondrial
[94]
diseases, including some non-mitochondrial myopathies, cancer, obesity, renal disease, diabetes and liver
disease , limiting diagnostic utility independent of clinical context.
[90]

The elevation of growth differentiation factor 15 (GDF-15) was identified in Thymidine Kinase (TK2)-
[95]
related mitochondrial disease . It was further evaluated in patient cohorts with mitochondrial and
non-mitochondrial diseases [96-100] , with some suggestion that GDF-15 levels may correlate with disease
[97]
severity . Davis and colleagues demonstrated improved diagnostic sensitivity and a higher diagnostic
odds ratio for GDF-15 compared to FGF-21, noting that GDF-15 was potentially more broadly applicable
[96]
than FGF-21 . This was followed by suggestion of better correlation with mitochondrial translation
[90]
and mtDNA maintenance defects . GDF-15 may also be elevated, albeit to a lesser degree, in non-
mitochondrial muscle and metabolic diseases, pregnancy, diabetes, cancer, liver fibrosis and cardiovascular
disease [90,101] , and may reflect oxidative stress [101] .

Both FGF-21 and GDF-15 are non-invasive serum assays, and although not independently diagnostic ,
[101]
[99]
offer superior utility to classical biomarkers . They therefore complement clinical evaluation and can
better inform decision making on subsequent costly tests such as NGS, whilst noting clinically relevant
limitations.

WHICH SEQUENCING APPROACH?
Although targeted NGS panels achieved early advances in genetic diagnosis, there are clear benefits of WES
or WGS approaches. Both generate vastly more data and demand upfront resources for analysis, although
costs are rapidly decreasing, and can simultaneously analyse mtDNA, identify novel disease genes and
variants, as well as monogenic phenocopies.

WES has demonstrated increased diagnostic yield in mitochondrial disease studies as outlined above [15-23] ,
although it has frequently been utilised only for nuclear genome analysis following dedicated mtDNA
genome sequencing. Off-target WES reads sufficiently capture mtDNA to assemble a mitochondrial
[75]
genome [102] and analyse mtDNA variants with reasonable precision , owing to the abundance of mtDNA