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Project

Development of a novel diagnotic assay for the biochemical diagnosis of mitochondrial disorders in vital tissue (MITOCHONDRIAL)

Primary mitochondrial disorders are characterized by aberrancies in normal mitochondrial functioning due to mutations in either nuclear or mitochondrial DNA-encoded genes. The estimated prevalence is at least 1 in 5.000 births, suggesting that there are at least 1.200 individuals in Flanders with an inherited mitochondrial disorder. However, less than 250 patients in Flanders have a diagnosis. We follow approximately 100 patients with a suspected or genetically confirmed mitochondrial disorder at the Metabolic Center UZ Leuven. The major discrepancy between the number of patients with a diagnosis in Flanders and the estimated prevalence can at least in part be attributed to the lack of readily available sensitive techniques for the diagnosis in Flanders.

The current state-of-the-art technique for biochemical diagnosis of mitochondrial disorders is the measurement of the bioenergetic potential in isolated mitochondria from fresh tissue biopsies. Indeed, approximately 25% of patients suspected of a mitochondrial disease have abnormalities in fresh tissue while no defect in complex I-V and the pyruvate dehydrogenase complexes can be found in frozen tissue [1]. Neither of these techniques is available for diagnostic purposes in Flanders (and Belgium) and the major drawbacks of the technique on vital samples is that it is very expensive and requires 300 mg of tissue. The cost, directly paid by the patient, is more than €500 in Nijmegen, the Netherlands (closest center performing the analysis) and an (invasive) open muscle biopsy is required to collect 300 mg of tissue. In contrast, the public health care system (RIZIV) only provides a reimbursement of €46/sample. This emphasizes the need to a develop novel sensitive diagnostic assay that measures the bioenergetic potential of vital isolated mitochondria using limited sample amounts at a lower cost.

Therefore, the aim of this project is to develop a novel assay for the biochemical diagnosis of mitochondrial disorders in isolated mitochondria from fresh tissue biopsies from patients using High Resolution Respirometry (HRR). The novel assay would require less than 10 mg of fresh tissue at a cost of less than €100/sample. The lower cost will allow us to offer the test within the public health care reimbursement and 10 mg of tissue (muscle, liver) can be obtained via a needle biopsy or via transjugular biopsy of liver. Furthermore, vital patient-derived fibroblast cultures (1.000.000 cells) will also be studied as an alternative given the non-invasive nature of sampling (skin biopsy). Despite false-negative results using conventional techniques which explain why testing fibroblasts is currently not a method of choice, fibroblast cultures have proven to be useful for diagnostic purposes. We will determine whether HRR can improve predictive value for fibroblast cultures. HRR has been used for research purposes to study mitochondrial function, but not as a diagnostic tool for inherited mitochondrial disorders. The technique is currently being used in our lab for research purposes and has been successfully applied to distinguish fibroblasts from controls and patients with a genetically confirmed mitochondrial disease (unpublished data). In order to develop a diagnostic assay which requires less than 10 mg of fresh tissue at a lower cost (less than €100/sample), as is the case for our HRR assay, we need to 1) establish reference ranges using tissue biopsies from disease controls, 2) confirm the sensitivity in patients with genetically confirmed mitochondrial disorders and 3) prospectively validate the technique in patients suspected of mitochondrial disease. Taken together, the successful execution of this project will lead to the development of a novel diagnostic assay that allows sensitive biochemical diagnosis of inherited mitochondrial disorder in Flanders.

Date:1 Jan 2017 →  30 Sep 2019
Keywords:diagnotic assay, biochemical diagnosis, mitochondrial disorders, vital tissue
Disciplines:Cardiac and vascular medicine