Mitochondrial biomarkers and biomarkers of mitochondrial disease
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Background: Biomarkers are objective and reproducible tools that are essential for making the diagnosis and following disease development in clinical practice. Mitochondria are multifunctional organelles and the major site of cellular energy production. Primary mitochondrial disorders are a group of heterogeneous conditions characterised by impaired energy metabolism; however, mitochondrial dysfunction is also a feature of many other disease processes, particularly age-related neurodegenerative disorders. Thus, mitochondrial dysfunction can be both a primary disease manifestation or a secondary consequence of another disease process. Common for both is the lack of biomarkers that are robust enough to use in the clinical follow-up of patients. Objective: To evaluate new biomarkers and new ways of using known biomarkers for the diagnosis and follow up of primary mitochondrial disorders and neurological diseases in which mitochondria or mitochondrial dysfunction may play a role. Materials and methods: The study population in paper I were newly diagnosed patients with relapsing-remitting multiple sclerosis (MS) and an age and gender matched control group. In paper II and III the cohort constituted of patients with known, genetically confirmed primary mitochondrial disease. Paper II was an international collaborative study that included a cohort of patients with large-scale single mitochondrial DNA (mtDNA) deletions, while in paper III, the study cohort were Norwegian patients with mitochondrial disease caused by known nuclear and mtDNA mutations. Cell-free mtDNA in cerebrospinal fluid was investigated in paper I, products from long-range PCR in urine in paper II and serum levels of fibroblast growth factor 21 (FGF-21), growth and differentiation factor 15 (GDF-15) and neurofilament light chain (NF-L) were investigated in paper III. Results: We found that the levels of cell-free mtDNA in cerebrospinal fluid were elevated in patients with relapsing-remitting multiple sclerosis relative to controls, and that there was an inverse correlation between level of free mtDNA and disease duration. In our studies using urine sediment cells, we found it was possible to identify large-scale single mtDNA deletions in urine in 80% of cases. In addition, this source of material also allowed us to confirm breakpoints, heteroplasmy levels and deletion size in the DNA extracted from urinary sediment cells. We found that using a combination of serum biomarkers FGF-21, GDF-15 and NF-L, we could differentiate between subgroups of patients with mitochondrial disease. The levels of NF-L increased with the degree of central nervous system involvement and this was especially seen in patients with multisystemic and complex disease. In contrast, FGF-21 and GDF-15 were not significantly elevated in these patients, but markedly increased in those with primary myopathies. Conclusions: These three studies provide novel information concerning the use of biomarkers in the diagnosis and management of mitochondrial disease and the use of mitochondrial biomarkers in diseases where the primary process is thought to be inflammatory. Our findings in MS suggest that mitochondria play a role in the early disease pathogenesis. In addition, we found that changes in the level of mtDNA in cerebrospinal fluid correlated with disease activity. While this could potentially provide a novel marker for early disease activity, the need for lumbar puncture would limit its applicability. We found that urine provides a readily available source of mtDNA for diagnostic screening of patients suspected clinically of having a single mtDNA deletion. These findings should help reduce the need for invasive muscle biopsy. Where diagnosis is less clear, but strong suspicion of mitochondrial disease, the combined use of FGF-21, GDF-15 and NF-L provides diagnostic information, insight into differential tissue involvement and the possibility of following disease activity.
Has partsPaper I: Varhaug KN, Vedeler CA, Myhr KM, Aarseth JH, Tzoulis C, Bindoff LA. Increased levels of cell-free mitochondrial DNA in the cerebrospinal fluid of patients with multiple sclerosis. Mitochondrion. 2017 May 1;34:32-5. The article is available in the thesis file. The article is also available at: https://doi.org/10.1016/j.mito.2016.12.003
Paper II: Varhaug KN, Nido GS, de Coo I, Isohanni P, Suomalainen A, Tzoulis C, Knappskog P, Bindoff LA. Using urine to diagnose large‐scale mtDNA deletions in adult patients. Annals of Clinical and Translational Neurology. 2020 Aug;7(8):1318-26. The article is available at: https://hdl.handle.net/11250/2739527
Paper III: Varhaug KN, Hikmat O, Nakkestad HL, Vedeler CA, Bindoff LA. Serum biomarkers in primary mitochondrial disorders. Brain Communications. 2021;3(1):fcaa222. The article is available in the thesis file. The article is also available at: https://doi.org/10.1093/braincomms/fcaa222