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.

Pyruvate and uridine rescue the metabolic profile of OXPHOS dysfunction

“…Seventy-six percent (13/17) defects were encoded for by nuclear genes, 24% (4/17) by mtDNA. The phenotypic diagnosis of OXPHOS dysfunction in these fibroblast cell lines was previously characterised through enzymology and oxygraphy [ 11 ]. We included the PDH cell lines as a comparison group with non-OXPHOS related mitochondrial dysfunction.…”

“…Oxygraphy was performed as described previously [ 11 ] using the Oxygraph O2K from Oroboros instruments accordingly to previously published resources with a technical n of ≥3 [ 11 , 14 ]. Briefly, fibroblasts were harvested by trypsinisation from a T175 flask at a density of 80%, washed in PBS (phosphate buffered saline) and resuspended in Miro5 buffer to 20 million cells/mL.…”

“…Here, we applied tracing of U– 13 C-labeled glucose and U– 13 C-glutamine to determine the metabolic profile of OXPHOS dysfunction in genetic and pharmacological models of PMD [ [11] , [12] , [13] ]. Our results reveal a distinct and universal signature of OXPHOS deficiency with as main feature the depletion of aspartic acid.…”

Enhanced cGAS-STING–dependent interferon signaling associated with mutations in ATAD3A

“…Within the first year of life, she developed feeding difficulties, with relatively rapid feeding noted to easily promote vomiting, eventually necessitating the placement of a gastrostomy at 3 yr of age. Respiratory chain enzyme analysis in fibroblasts was normal ( Bird et al, 2019 ). From age 2 yr, she developed a progressive hardening of the skin, a change in facial appearance due to loss of fat tissue, and areas of panniculitis involving the lower limbs.…”

Enhanced cGAS-STING-dependent interferon signaling associated with mutations in ATAD3A

“…Within the first year of life she developed feeding difficulties, with relatively rapid feeding noted to easily promote vomiting, eventually necessitating the placement of a gastrostomy at three years of age. Respiratory chain enzyme analysis in fibroblasts was normal (Bird et al, 2019). From age two years she developed a progressive hardening of the skin, and a change in facial appearance due to loss of fat tissue, and areas of panniculitis involving the lower limbs.…”