Primary mitochondrial diseases are characterized by extensive biochemical, clinical, and genetic heterogeneity, making their analysis complex and the diagnostic process very challenging, lengthy, and frustrating. Despite rapid progress in identifying a genetic cause using next-generation sequencing, functional studies showing dysfunction in the mitochondrial respiratory chain are still required in many patients to complete the diagnosis. Current diagnostic practice involves measurement of respiratory chain enzyme activities in muscle or liver biopsies, but these assays do not provide a complete analysis of mitochondrial function and are often avoided due to the invasive nature of the biopsies. Blood sampling and skin biopsy for fibroblast culture are minimally invasive procedures. In recent years, new mitochondrial functional assays were developed in the research setting applicable to fibroblasts or even blood cells opening new opportunities, but the performance in a range of mitochondrial genetic defects and the clinical utility remain unexplored. The goal of this study is to develop functional testing methods and to establish their clinical utility for the diagnosis and confirmation of primary mitochondrial disease in minimally invasive tissue with a focus on skin fibroblasts. We hypothesize that in patients with suspected primary mitochondrial diseases an appropriate panel of tests can be developed and optimized in fibroblasts (or blood cells) with clinical robustness, good sensitivity and specificity, to allow for effective mitochondrial function testing with the strategy dependent on functional category. We will examine a series of mitochondrial functional tests for their diagnostic performance in fibroblasts of patients with known primary mitochondrial diseases, organized by functional class, and determine sensitivity and specificity. Further, we will also examine the effect of differences in tissue culture conditions on the functional test performance in fibroblasts. Environmental factors reflected in culture conditions such as amino acid abundance impact these functions with diagnostic and therapeutic implications. Multiple factors in the functionalization of mitochondrial tRNAs interact with nutritional factors such as amino acids or one-carbon folate esters. In genetic defects of mitochondrial tRNA aminoacyltransferases (ARS2), which interact with the cognate amino acid, increasing the amino acid concentration may improve functionality, whereas decreasing its concentration will exacerbate mitochondrial dysfunction. We will examine the impact of varying the cognate amino acid concentration for tRNA aminoacylation defects on functional assays. The one-carbon charging of intramitochondrial folates affects tRNA processes such as formylation of the initiator methionine by MTFMT, and post transcriptional modification of the wobble base to methyl-uridine or taurinomethyl-uridine in tRNALys and tRNALeu(UUR), which is affected by common pathogenic mutations with disease-causing functional impact. We will evaluate the impact of taurine and of modulation of folate charging by serine or formate.
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