Oxygraphy Versus Enzymology for the Biochemical Diagnosis of Primary Mitochondrial Disease

Abstract

Primary mitochondrial disease (PMD) is a large group of genetic disorders directly affecting mitochondrial function. Although next generation sequencing technologies have revolutionized the diagnosis of these disorders, biochemical tests remain essential and functional confirmation of the critical genetic diagnosis. While enzymological testing of the mitochondrial oxidative phosphorylation (OXPHOS) complexes remains the gold standard, oxygraphy could offer several advantages. To this end, we compared the diagnostic performance of both techniques in a cohort of 34 genetically defined PMD patient fibroblast cell lines. We observed that oxygraphy slightly outperformed enzymology for sensitivity (79 ± 17% versus 68 ± 15%, mean and 95% CI), and had a better discriminatory power, identifying 58 ± 17% versus 35 ± 17% as "very likely" for oxygraphy and enzymology, respectively. The techniques did, however, offer synergistic diagnostic prediction, as the sensitivity rose to 88 ± 11% when considered together. Similarly, the techniques offered varying defect specific information, such as the ability of enzymology to identify isolated OXPHOS deficiencies, while oxygraphy pinpointed PDHC mutations and captured POLG mutations that were otherwise missed by enzymology. In summary, oxygraphy provides useful information for the diagnosis of PMD, and should be considered in conjunction with enzymology for the diagnosis of PMD.

Keywords: Primary mitochondrial disease (PMD); diagnostics; enzymology; oxidative phosphorylation (OXPHOS); oxygraphy; respiration.

Figure 1

Mutational mapping. Genetic changes (red) of primary mitochondrial disease (PMD) patients reported in this study mapping to the mitochondrial systems of: primary OXPHOS, TCA cycle connections, the mtDNA system, and structural components. Abbreviations: OXPHOS CI–V, oxidative phosphorylation complexes I–V; TCA, tricarboxylic acid cycle.

Figure 2

Enzymology in control and PMD patient fibroblast cell lines. Control and PMD patient fibroblasts were measured CS and respiratory chain complex I–IV (CI–IV) activity by spectrophotometric methods. Results are presented as either (a) raw rates, or (b) relative to CS activity. Median is displayed for controls with error bars showing the 1.25th and 98.75th percentiles of the reference range, and the green shading region shows the range. Each data point represents the average of each patient or control from ≥ 2 technical replicates Abbreviations: CI–IV, respiratory chain complexes I–IV; CS, citrate synthase.

Figure 3

Oxygraphy testing in control and PMD patient fibroblast cells. (a) Representative trace from control fibroblasts, with substrates and inhibitors injected as described. Blue line indicates the oxygen partial pressure in the chamber, and the red line indicates the inverted rate of change of the blue line (rate of oxygen consumption). (b) Resting, coupled, and uncoupled rates of respiration. (c) Ratios and calculated values from data in panel (b). Median is displayed for controls with error bars showing the 1.25th and 98.75th percentiles of the reference range, and the green shading region shows the range. Each data point represents the average of each patient or control from ≥ 3 technical replicates. Abbreviations: As, ascorbate; CI–IV, respiratory chain complexes I–IV; CCR, coupling control ratio (maximal uncoupled activity over maximal coupled activity; Cyt C, cytochrome C; Dig, digitonin; Gp, Glycerophosphate; G, glutamate; M, malate; Py, pyruvate; Rot, rotenone; S, succinate.

Figure 4

Disease prediction and sensitivity for enzymology, oxygraphy, or combined methods for detecting PMD as presented (a) in a table, or (b) through the plotting of the combined positive Z scores for each test and combined value. Median is displayed for controls with error bars showing the 1.25th and 98.75th percentiles of the reference range, and the green shading region shows the range. Abbreviations: CI–CIV, respiratory chain complexes I–IV.

Figure 5

Diagnostic guide to interpreting enzymology and oxygraphy results in the absence of a definitive genetic result. Next generation sequencing or targeted genetic mitochondrial panels can provide a definitive diagnosis of mitochondrial disease and should alleviate the need for further biochemical testing. In cases of non-definitive diagnostic results, biochemical testing is required. In this regard, oxygraphy and enzymology would ideally be deployed synergistically for the optimal chance of detecting dysfunction in fibroblasts, and for the identification of the type of underlying defect.