Repp 2019 Thesis

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Repp BM (2019) Identification and verification of novel disease-causing genes and therapy options for patients with mitochondrial disorders – Focus on ACAD9. PhD Thesis 178.

» Open Access

Birgit Monica Repp (2019) PhD Thesis

Abstract: Mitochondrial diseases are a group of clinical, biochemical and genetically heterogeneous disorders that are based on a malfunction of the mitochondrial energy metabolism. In addition to the mitochondrial β-oxidation of fatty acids and the citrate cycle the re-oxidation by the aerobic hydrogen transmitting coenzymes, energy in form of ATP are formed. This reaction, also called oxidative phosphorylation (OXPHOS), takes place in the mitochondrial respiratory chain, one of five components (complex I-V) existing multi-enzyme complex. Defects in the first complex of the respiratory chain represent the most common enzymatic defect of oxidative phosphorylation.

Patients with complex I defect present a big clinical variability in symptoms ranging from mild muscle weakness in adulthood to neonatal death. The wide range of symptoms makes the finding of the right diagnosis challenging. During the last ten years Next-Generation-Sequencing (NGS) has been successfully used for the discovery of new causative genes. In this work, ACAD9 was discovered as a new assembly factor for complex I via Whole-Exome Sequencing (WES). Functional analysis by the use of lenti-viral transduction, 2D-BN-SDS-Gels, Western Blots and oxygen consumption rate (OCR) measurements confirmed the pathogenicity of the mutation. WES in five other patients with complex I or combined deficiency discovered new variants in already described disease-causing genes (BOLA3, NDUFS8, and MTFMT) and for the first time in the complex I subunit NDUFB3. Realizing, that ACAD9 mutations were found to be one of the most frequent causes of isolated complex I deficiency the genetic, clinical and biochemical data of 70 patients (of whom 29 previously unpublished) were collected and analyzed in detail. Causal variants were distributed throughout the entire gene but no obvious genotype-phenotype correlation was found. The main clinical findings were cardiomyopathy (85%), muscular weakness (75%), exercise intolerance (72%) and mild developmental delay (45%). The age of onset was in the majority of patients within the first year. These patients present the biggest subgroup (n = 52) and the group with the shortest survival (50% not surviving the first two years), suggesting the most severe course. Patients with an onset > 1 year have a better prognosis with 90% surviving ten years.

In spite of rapid progress in finding the molecular cause, in most instances no curative therapeutic options are available. In order to test potential substances (Bezafibrate, riboflavin, resveratrol and AICAR) 42 fibroblast cell lines from patients with isolated or combined complex I deficiency and three age-matched control cell lines were collected and established. A defined molecular diagnosis was present in 35 patient cell lines. After bezafibrate treatment no improvement of complex I activity in cell lines with mtDNA mutation (n = 4) was found, but in 60% of the cell lines with nuclear mutations (+15-140%). To obtain further insight about the mode of action, the effect of the bezafibrate treatment was additionally analyzed on genome-wide expression levels in a subgroup of 14 cell lines. A significant increase in expression levels of genes involved in lipid and fatty acid metabolism (ACAA2, ACADVL, CPT1A, CPT1B, ECH1), but no increase in the transcription level of genes involved in the mitochondrial biogenesis (NRF1, Tfam, PGC-1-α), was found. The largest effect was observed in a group of patients with mutations in the same gene, ACAD9 and was therefore studied more in detail. In 12 out of 17 cases the complex I activity increased significantly and five cell lines reached almost the lower level of the normal range. The increase in activity was accompanied by an increase in ACAD9 and ACADVL protein level in 6/7 patients and a larger amount of complex I assembled in supercomplexes visualized by 2D-BN/SDS-PAGE experiments (n = 4). Whole mitochondrial proteome analysis of four patients and one control (bezafibrate treated and untreated), in order to detect potential post-transcriptional effects, were performed by 2D-DIGE. Proteomics data confirmed increased expression of proteins involved in lipid and fatty acid metabolism (ACAA2, ACADVL, ACSL4) upon bezafibrate treatments. The treatment with the vitamin precursor of the flavin adenine dinucleotide (FAD) moiety, riboflavin significant increased respiratory chain complex activity in 9 out of 15 patient cell lines. In contrast to the bezafibrate treatment riboflavin did not change any protein levels. Riboflavin treatment may increase the activity and stability of the remaining ACAD9 protein and thereby leading to an increase of OCR in some patient cell lines. The positive effect in cell lines was also reported for most of the treated ACAD9 patients and is mirrored in the survival data. Patients with a disease-onset below 1year of age have a significantly better survival rate after oral riboflavin treatment (deceased n = 7/24) in contrast to untreated patients (deceased n = 16/17).

In summary, NGS coupled with functional validation of new disease alleles was successfully used to identify disease-causing variants in known and new complex I associated disease genes in six patients. Furthermore, the analysis of different treatment options support bezafibrate and riboflavin as promising treatment options for patients with complex I deficiency, especially for patients with mutations in ACAD9.


Bioblast editor: Plangger M


Labels: MiParea: Respiration, mtDNA;mt-genetics 

Stress:Mitochondrial disease  Organism: Human  Tissue;cell: HEK  Preparation: Permeabilized cells 


Coupling state: LEAK, OXPHOS  Pathway: N, S, CIV, ROX  HRR: Oxygraph-2k 

2020-07