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Mitochondria are the main site of cellular adenosine triphosphate (ATP) generation which is achieved by a series of multi-subunit complexes and electron carriers which together create the oxidative phosphorylation system (OXPHOS). Whenever a defect in any of the numerous mitochondrial pathways occurs it is commonly referred to as a mitochondrial disorder. Mitochondrial disorders are a heterogeneous group of disorders characterised by impaired energy production and include a wide range of defects of either mitochondrial DNA (mtDNA) or nuclear DNA (nDNA) encoded proteins. In cases of dysfunction in the respiratory chain (complex I to IV) it is known to be a respiratory chain deficiency (RCD) which presents a huge challenge for routine diagnosis largely due to the lack of a specific and sensitive biomarker(s). One sure way of confirming the suspicion of a RCD is by performing enzyme analysis on a muscle sample obtained through a biopsy. However, due to the lack of theatre time available to clinicians and the relative large number of false positive patients that are being selected for biopsies, it was decided to develop a biosignature to limit the number of false positive patients from the diagnostic workflow. An untargeted liquid chromatography mass spectrometry (LC-MS) metabolomics approach was used to investigate RCDs in children from South Africa. Sample preparation, a liquid chromatography time-of-flight mass spectrometry method and data processing methods were standardised. Furthermore the developed methodology made use of reverse phase chromatography in conjunction with positive electrospray ionisation (ESI) and a hydrophilic interaction chromatography (HILIC) in negative electrospray ionisation. Urine samples of 61 patients representing three different experimental groups were analysed. The three experimental groups comprised of patients with respiratory chain deficiencies, clinical referred controls (CRC) and patients suffering from various neuromuscular disorders (NMD). After a variety of data mining steps and statistical analysis a list of 12 features were compiled with the ability to distinguish between patients with RCDs and CRCs. The proposed signature was also tested on the neuromuscular disorder group, but this result indicated that the biosignature performed better when used to differentiate between patients with RCDs and CRCs, since the model was designed with this purpose. An alternative validation study is required to identify the features found with this proposed biosignature, to ensure that this biosignature can be practically implemented as a non-invasive screening method. / MSc (Chemistry), North-West University, Potchefstroom Campus, 2014

Mitochondria are the main site of cellular adenosine triphosphate (ATP) generation which is achieved by a series of multi-subunit complexes and electron carriers which together create the oxidative phosphorylation system (OXPHOS). Whenever a defect in any of the numerous mitochondrial pathways occurs it is commonly referred to as a mitochondrial disorder. Mitochondrial disorders are a heterogeneous group of disorders characterised by impaired energy production and include a wide range of defects of either mitochondrial DNA (mtDNA) or nuclear DNA (nDNA) encoded proteins. In cases of dysfunction in the respiratory chain (complex I to IV) it is known to be a respiratory chain deficiency (RCD) which presents a huge challenge for routine diagnosis largely due to the lack of a specific and sensitive biomarker(s). One sure way of confirming the suspicion of a RCD is by performing enzyme analysis on a muscle sample obtained through a biopsy. However, due to the lack of theatre time available to clinicians and the relative large number of false positive patients that are being selected for biopsies, it was decided to develop a biosignature to limit the number of false positive patients from the diagnostic workflow. An untargeted liquid chromatography mass spectrometry (LC-MS) metabolomics approach was used to investigate RCDs in children from South Africa. Sample preparation, a liquid chromatography time-of-flight mass spectrometry method and data processing methods were standardised. Furthermore the developed methodology made use of reverse phase chromatography in conjunction with positive electrospray ionisation (ESI) and a hydrophilic interaction chromatography (HILIC) in negative electrospray ionisation. Urine samples of 61 patients representing three different experimental groups were analysed. The three experimental groups comprised of patients with respiratory chain deficiencies, clinical referred controls (CRC) and patients suffering from various neuromuscular disorders (NMD). After a variety of data mining steps and statistical analysis a list of 12 features were compiled with the ability to distinguish between patients with RCDs and CRCs. The proposed signature was also tested on the neuromuscular disorder group, but this result indicated that the biosignature performed better when used to differentiate between patients with RCDs and CRCs, since the model was designed with this purpose. An alternative validation study is required to identify the features found with this proposed biosignature, to ensure that this biosignature can be practically implemented as a non-invasive screening method. / MSc (Chemistry), North-West University, Potchefstroom Campus, 2014

No / Accumulation of large-scale mitochondrial DNA (mtDNA) deletions and chronic, subclinical inflammation are concomitant during skin aging, thus raising the question of a causal link. To approach this, we generated mice expressing a mutant mitochondrial helicase (K320E-TWINKLE) in the epidermis to accelerate the accumulation of mtDNA deletions in this skin compartment. Mice displayed low amounts of large-scale deletions and a dramatic depletion of mtDNA in the epidermis and showed macroscopic signs of severe skin inflammation. The mtDNA alterations led to an imbalanced stoichiometry of mitochondrial respiratory chain complexes, inducing a unique combination of cytokine expression, causing a severe inflammatory phenotype, with massive immune cell infiltrates already before birth. Altogether, these data unraveled a previously unknown link between an imbalanced stoichiometry of the mitochondrial respiratory chain complexes and skin inflammation and suggest that severe respiratory chain dysfunction, as observed in few cells leading to a mosaic in aged tissues, might be involved in the development of chronic subclinical inflammation. / Deutsche Forschungsgemeinschaft (Wi 889/6-3 to RJW, SFB 829 A14 to RJW, Cologne Excellence Cluster on Cellular Stress Responses in Aging-associated Diseases–CECAD to RJW, BR2304/9-1 to BB, and SFB 829 A1, A5, and Z2 to CMN) and the Center of Molecular Medicine Cologne of the Medical Faculty (CMMC, to RJW)

CoenzymeQ10 (CoQ10) functions as an electron carrier in mitochondria transporting electrons from CI and CII to CIII in the respiratory chain (RC) for normal cellular energy (ATP) production. Mutations in genes of a complicated and not yet well understood CoQ10 biosynthesis cause primary CoQ10 deficiency, a rare autosomal recessive mitochondrial disorder (MD) with diverse heterogeneous clinical phenotypes. Although the major function of CoQ10 is to serve as electron transfer molecule it furthermore possesses multiple metabolic functions which can result in secondary CoQ10 deficiency. Five main clinical phenotypes are associated with CoQ10 deficiency although distinct genotype-phenotype associations are still absent due to the limited molecular genetic diagnoses of most reported CoQ10 deficiency cases. A correlation was found between reduced levels of CoQ10 in muscle tissue and deficient CII + III RC enzyme activities in a South African patient cohort, the current indicators for potential CoQ10 deficiency. The aim of the study was therefore to identify nuclear-encoded mutations in genes associated with CoQ10 deficiencies in a cohort of South African patients diagnosed with respiratory chain deficiencies (RCDs). A high throughput target enrichment strategy was performed in order to identify previously reported and/or possible novel CoQ10-assosciated disease-causing variants using Ion Torrent next generation sequencing (NGS) and an in-house developed bioinformatics pipeline. The data obtained were compared to clinical presentations of the patients to interpret the results of the identified variants considered to be possibly pathogenic. Targeted genes associated with primary CoQ10- and secondary CoQ10 deficiency was successfully sequenced in 24 patients, identifying 16 possible disease-causing variants. Of these variants three compound heterozygous variants were identified in three patients in genes ETFDH, COQ6 and COQ7, which were considered to be pathogenic according to the available data provided. Further validation of these three variants supported its pathogenicity in at least two of these variants (ETFDH and COQ6). In conclusion: This study contributed to better understanding the aetiology of a South African cohort of patients diagnosed with MDs. It also highlighted the valuable role of NGS for such investigations, and furthermore identified areas in the biochemical and molecular diagnostic strategy where improvements could be made in the future. / MSc (Biochemistry), North-West University, Potchefstroom Campus, 2015

CoenzymeQ10 (CoQ10) functions as an electron carrier in mitochondria transporting electrons from CI and CII to CIII in the respiratory chain (RC) for normal cellular energy (ATP) production. Mutations in genes of a complicated and not yet well understood CoQ10 biosynthesis cause primary CoQ10 deficiency, a rare autosomal recessive mitochondrial disorder (MD) with diverse heterogeneous clinical phenotypes. Although the major function of CoQ10 is to serve as electron transfer molecule it furthermore possesses multiple metabolic functions which can result in secondary CoQ10 deficiency. Five main clinical phenotypes are associated with CoQ10 deficiency although distinct genotype-phenotype associations are still absent due to the limited molecular genetic diagnoses of most reported CoQ10 deficiency cases. A correlation was found between reduced levels of CoQ10 in muscle tissue and deficient CII + III RC enzyme activities in a South African patient cohort, the current indicators for potential CoQ10 deficiency. The aim of the study was therefore to identify nuclear-encoded mutations in genes associated with CoQ10 deficiencies in a cohort of South African patients diagnosed with respiratory chain deficiencies (RCDs). A high throughput target enrichment strategy was performed in order to identify previously reported and/or possible novel CoQ10-assosciated disease-causing variants using Ion Torrent next generation sequencing (NGS) and an in-house developed bioinformatics pipeline. The data obtained were compared to clinical presentations of the patients to interpret the results of the identified variants considered to be possibly pathogenic. Targeted genes associated with primary CoQ10- and secondary CoQ10 deficiency was successfully sequenced in 24 patients, identifying 16 possible disease-causing variants. Of these variants three compound heterozygous variants were identified in three patients in genes ETFDH, COQ6 and COQ7, which were considered to be pathogenic according to the available data provided. Further validation of these three variants supported its pathogenicity in at least two of these variants (ETFDH and COQ6). In conclusion: This study contributed to better understanding the aetiology of a South African cohort of patients diagnosed with MDs. It also highlighted the valuable role of NGS for such investigations, and furthermore identified areas in the biochemical and molecular diagnostic strategy where improvements could be made in the future. / MSc (Biochemistry), North-West University, Potchefstroom Campus, 2015

No description available.