Diagnostic Algorithm in Mitochondrial Disease

The diagnosis of mitochondrial disease requires a multidisciplinary approach that combines clinical characteristics, histopathological, biochemical and genetic investigations, for which a diagnostic algorithm has been devised (Figure 1.20) (McFarland et al., 2010).

Despite the vast clinical heterogeneity of mitochondrial diseases, precedent exists for which targeted mtDNA or nuclear gene screening can be performed based on clinical features alone. For example, visual failure in a young adult may prompt targeted sequencing of the three LHON mtDNA mutations (Yu-Wai-Man et al., 2003), while the classic MELAS phenotype evokes screening for the common m.3243A>G mutation (Nesbitt et al., 2013).

Nonetheless, paramount to the diagnosis of mitochondrial disease for almost all patients is the requirement for clinically affected tissue samples, which is often skeletal muscle but can also include liver, brain or cardiac tissue if available. Available tissue can be subject to

Figure 1.20 Current Diagnostic Algorithm for Investigating Mitochondrial Disease. Despite the increasing number of mtDNA and nuclear gene mutations associated with mitochondrial disease and advances in investigative tools and techniques, the diagnostic algorithm for the investigation of

Histopathology

Two common stains used are haematoxylin and eosin (H&E) and modified Gomori trichrome. H&E is a general stain for observing muscle fibre morphological abnormalities (Figure 1.21A). Modified Gomori trichrome stain is used to detect the presence of ‘ragged red fibres’ (Gomori, 1950; Engel and Cunningham, 1963), characterised by the abnormal

subsarcolemmal accumulation of mitochondria, which indicates mitochondrial proliferation.

The standard histochemical assay used is sequential cytochrome c oxidase/succinate

dehydrogenase (COX-SDH) histochemistry (Figure 1.21A) (Old and Johnson, 1989; Sciacco and Bonilla, 1996), which can visualise RC activities for complex IV and II. Global or mosaic patterns of COX-deficiency can be visualised by the presence of blue fibres, which can occur due to mtDNA defects or nuclear mutations. A mosaic pattern of COX-deficiency is typically associated with mtDNA mutations, often due to heteroplasmy and the multi-copy nature of mtDNA. However, COX-deficient fibres are also noted in other neuromuscular diseases and can be observed as part of the normal ageing process (Greaves et al., 2011; Vincent et al., 2016).

The disadvantage of sequential COX-SDH histochemistry is that it does accommodate activities of the other RC complexes, particularly complex I which is the one of the most commonly affected enzymes of the OXPHOS system (Loeffen et al., 2000). Recently, a novel quadruple immunofluorescence assay has been developed for the quantification of complex I and IV abundance in individual skeletal muscle fibres using monoclonal antibodies for NDUFB8 and MT-COI, together with VDAC1/porin as a mitochondrial mass marker and laminin as a marker of myofibre boundaries (Figure 1.21B) (Rocha et al., 2015). It has been demonstrated as accurate and reproducible in a large proportion of muscle fibres from range mitochondrial disease patients with mtDNA and nuclear genetic defects, thus showing promise in aiding the diagnostic process.

Figure 1.21 Histopathological Techniques in the Diagnosis of Mitochondrial Disease. Panel demonstrating sections of (A) current standard histopathology techniques in skeletal muscle from a patient harbouring a single large-scale mtDNA deletion, which show ragged red fibres and COX- deficient fibres; (i) H&E and (ii) modified Gomori trichrome stain, (iii) COX, (iv) SDH and (v) sequential (merged) COX-SDH reactions. Panel demonstrating sections from the same patient subjected to the novel quadruple immunofluorescence assay for the quantification of complex I and IV protein abundance; (i) laminin is used as a marker of myofibre boundaries, quantification of (ii) NDUFB8 (complex I), (iii) MT-COI (complex IV) and (iv) VDAC1/porin, plus (v) a merged image. Adopted and amended from Alston et al. (2017).

Biochemistry

In vitro spectrophotometric biochemical assays for measuring the activity of each individual

RC complex in muscle relative to activity of the matrix enzyme citrate synthase in frozen muscle is an essential diagnostic method, particularly in suspected early-onset mitochondrial disorders (Kirby et al., 2007). However, assays require a significant quantity of muscle (>50mg) and complex V cannot be reliably measured in frozen muscle.

Genetic and Molecular Studies

Genetic investigations and the order of sequenced mutations or genes is dependent upon the clinical, histochemical and biochemical findings. Rapid whole mitochondrial genome sequencing using next generation sequencing (NGS) that can also accurately measure

heteroplasmy may be considered in a significant proportion of patients following exclusion of common mtDNA mutations (Tang et al., 2013). Targeted Sanger sequencing of a subset of known causative nuclear genes is also routinely performed following exclusion of mtDNA mutations. This includes mutational screening of nuclear genes involved mtDNA maintenance or mitochondrial translation in patients with multiple RC deficiency in affected tissues, or RC enzyme complex subunits and assembly factors for patients with isolated RC deficiency. Segregation studies are also integral to confirming maternal inheritance of a causative mtDNA mutation and the carrier status of unaffected and affected relatives of the proband. This is not always feasible however, especially in late-onset patients where access to DNA from parents or relatives is frequently a challenge.

Regarding mtDNA maintenance disorders, quantitative real-time PCR of muscle DNA is used to determine relative mtDNA copy number (Dimmock et al., 2010; Venegas and Halberg, 2012). Single large-scale mtDNA deletion and multiple mtDNA deletions are typically detected by long range PCR or occasionally be Southern blotting (Figure 3.1C and D). The presence of multiple mtDNA deletions, depletion or both would allow a mutational screening of a subset of known causative nuclear genes associated with mtDNA maintenance disorders. (El-Hattab and Scaglia, 2013; Ahmed et al., 2015).

1.7 Applications of DNA Sequencing and the Emergence of Next Generation Sequencing (NGS) Technologies

The advance of human society has been greatly enriched by the ability to sequence the genetic code, comprising of just four dNTPs, that dictates the vast diversity of all life on Earth. The

power of DNA sequencing has led to landmark discoveries and outcomes, including the discovery of non-coding RNAs (Eddy, 2001), estimation of evolutionary trees (Felsenstein, 1981) and of course, sequencing of the mitochondrial genome (Anderson et al., 1981).