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de Laat P.,Nijmegen Center for Mitochondrial Disorders | Smeitink J.A.M.,Nijmegen Center for Mitochondrial Disorders | Janssen M.C.H.,Nijmegen Center for Mitochondrial Disorders
Netherlands Journal of Medicine | Year: 2015

Background: Previous research has shown that dysphagia and gastrointestinal problems occur frequently in carriersof the m.3243A>G mutation; however, the exact frequency and severity have not been determined. We hypothesise that adult carriers have an increased risk for malnutrition.Methods: In this observational study we evaluated the presence of gastrointestinal problems and dysphagia in92 carriers of the m.3243A>G mutation. The severity of the general disease involvement was classified using the Newcastle Mitochondrial Disease Adult Scale (NMDAS).Gastrointestinal involvement, dysphagia and the risk for malnutrition were scored using the Gastrointestinal Symptoms Questionnaire and the Malnutrition Universal Screening Tool. Gastrointestinal symptoms and anthropometrics were compared with healthy controls.Results: Our results show that the height, weight and bodymass index (BMI) of these carriers were lower than the national average (p < 0.05). Seventy-nine carriers (86%)suffered from at least one gastrointestinal symptom,mainly flatulence or hard stools. Both frequency and severity of symptoms were significantly increased compared with reference data of healthy Dutch adults. Of the carriers, 45% reported (mostly mild) dysphagia. Solid foods cause more problems than liquids. A negative correlation between BMI and heteroplasmy levels in rinary epithelial cells (UEC) was present (Spearman correlation coefficient = - 0.319, p = 0.003).Conclusion: Dysphagia and gastrointestinal problems,especially constipation, are common symptoms in the total m.3243A>G carriers cohort and are not related to heteroplasmy levels in UEC or disease severity. The severity of gastrointestinal problems as well as overall disease severity is associated with an increased risk for malnutrition. © Van Zuiden Communications B.V. All rights reserved.


Cabrera-Orefice A.,National Autonomous University of Mexico | Ibarra-Garcia-Padilla R.,National Autonomous University of Mexico | Maldonado-Guzman R.,National Autonomous University of Mexico | Guerrero-Castillo S.,Nijmegen Center for Mitochondrial Disorders | And 5 more authors.
Journal of Bioenergetics and Biomembranes | Year: 2015

It is proposed that the Saccharomyces cerevisiae the Mitochondrial Unselective Channel (ScMUC) is tightly regulated constituting a physiological uncoupling system that prevents overproduction of reactive oxygen species (ROS). Mg2+, Ca2+ or phosphate (Pi) close ScMUC, while ATP or a high rate of oxygen consumption open it. We assessed ScMUC activity by measuring in isolated mitochondria the respiratory control, transmembrane potential (ΔΨ), swelling and production of ROS. At increasing [Pi], less [Ca2+] and/or [Mg2+] were needed to close ScMUC or increase ATP synthesis. The Ca2+-mediated closure of ScMUC was prevented by high [ATP] while the Mg2+ or Pi effect was not. When Ca2+ and Mg2+ were alternatively added or chelated, ScMUC opened and closed reversibly. Different effects of Ca2+ vs Mg2+ effects were probably due to mitochondrial Mg2+ uptake. Our results suggest that ScMUC activity is dynamically controlled by both the ATP/Pi ratio and divalent cation fluctuations. It is proposed that the reversible opening/closing of ScMUC leads to physiological uncoupling and a consequent decrease in ROS production. © 2015, Springer Science+Business Media New York.


PubMed | University of Guanajuato, National Autonomous University of Mexico, University of Washington, Nijmegen Center for Mitochondrial Disorders and University of Sao Paulo
Type: Journal Article | Journal: Journal of bioenergetics and biomembranes | Year: 2015

It is proposed that the Saccharomyces cerevisiae the Mitochondrial Unselective Channel ((Sc)MUC) is tightly regulated constituting a physiological uncoupling system that prevents overproduction of reactive oxygen species (ROS). Mg(2+), Ca(2+) or phosphate (Pi) close (Sc)MUC, while ATP or a high rate of oxygen consumption open it. We assessed (Sc)MUC activity by measuring in isolated mitochondria the respiratory control, transmembrane potential (), swelling and production of ROS. At increasing [Pi], less [Ca(2+)] and/or [Mg(2+)] were needed to close (Sc)MUC or increase ATP synthesis. The Ca(2+)-mediated closure of (Sc)MUC was prevented by high [ATP] while the Mg(2+) or Pi effect was not. When Ca(2+) and Mg(2+) were alternatively added or chelated, (Sc)MUC opened and closed reversibly. Different effects of Ca(2+) vs Mg(2+) effects were probably due to mitochondrial Mg(2+) uptake. Our results suggest that (Sc)MUC activity is dynamically controlled by both the ATP/Pi ratio and divalent cation fluctuations. It is proposed that the reversible opening/closing of (Sc)MUC leads to physiological uncoupling and a consequent decrease in ROS production.


de Almeida N.M.,Radboud University Nijmegen | Wessels H.J.C.T.,Nijmegen Center for Mitochondrial Disorders | de Graaf R.M.,Radboud University Nijmegen | Ferousi C.,Radboud University Nijmegen | And 5 more authors.
Biochimica et Biophysica Acta - Bioenergetics | Year: 2016

Electron transport, or oxidative phosphorylation, is one of the hallmarks of life. To this end, prokaryotes evolved a vast variety of protein complexes, only a small part of which have been discovered and studied. These protein complexes allow them to occupy virtually every ecological niche on Earth. Here, we applied the method of proteomics-based complexome profiling to get a better understanding of the electron transport systems of the anaerobic ammonium-oxidizing (anammox) bacteria, the N2-producing key players of the global nitrogen cycle. By this method nearly all respiratory complexes that were previously predicted from genome analysis to be involved in energy and cell carbon fixation were validated. More importantly, new and unexpected ones were discovered. We believe that complexome profiling in concert with (meta)genomics offers great opportunities to expand our knowledge on bacterial respiratory processes at a rapid and massive pace, in particular in new and thus far poorly investigated non-model and environmentally-relevant species. © 2016 Elsevier B.V.


Wortmann S.B.,Nijmegen Center for Mitochondrial Disorders | Koolen D.A.,Radboud University Nijmegen | Smeitink J.A.,Nijmegen Center for Mitochondrial Disorders | van den Heuvel L.,Nijmegen Center for Mitochondrial Disorders | Rodenburg R.J.,Nijmegen Center for Mitochondrial Disorders
Journal of Inherited Metabolic Disease | Year: 2015

Mitochondrial disorders are characterized by a broad clinical spectrum. Identical clinical signs and symptoms can be caused by mutations in different mitochondrial or nuclear genes. Vice versa, the same mutation can lead to different phenotypes. Genetic syndromes and neuromuscular disorders mimicking mitochondrial disorders further complicate the diagnostic process. Whole exome sequencing (WES) is the state of the art next generation sequencing technique to identify genetic defects in mitochondrial disorders. Until recently it has mainly been used as a research tool. In this study, the use of WES in routine diagnostics is described. The WES data of 109 patients, referred under the suspicion of a mitochondrial disorder, were examined in two steps. First, the data were filtered using a virtual gene panel of genes known to be associated with mitochondrial disease. If negative, the entire exome was examined. A molecular diagnosis was achieved in 39 % of the heterogeneous cohort, and in 57 % of the subgroup of 42 patients with the highest suspicion for a mitochondrial disease. In addition to mutations in genes known to be associated with mitochondrial disorders (e.g. TUFM, MTFMT, FBXL4), in the subgroup of patients with the lowest suspicion for a mitochondrial disorder we found mutations in several genes associated with neuromuscular disorders (e.g. SEPN1, ACTA1) and genetic syndrome (e.g. SETBP1, ARID1B). Our results show that WES technology has been successfully implemented as a state-of-the-art, molecular diagnostic test for mitochondrial disorders as well as for the mimicking disorders in daily clinical practice. It also illustrates that clinical and biochemical phenotyping is essential for successful application of WES to diagnose individual patients. © 2015, The Author(s).


PubMed | MRC Mitochondrial Biology Unit, Åbo Akademi University, Nijmegen Center for Mitochondrial Disorders and Northumbria University
Type: | Journal: Scientific reports | Year: 2015

The helicase Twinkle is indispensable for mtDNA replication in nucleoids. Previously, we showed that Twinkle is tightly membrane-associated even in the absence of mtDNA, which suggests that Twinkle is part of a membrane-attached replication platform. Here we show that this platform is a cholesterol-rich membrane structure. We fractionated mitochondrial membrane preparations on flotation gradients and show that membrane-associated nucleoids accumulate at the top of the gradient. This fraction was shown to be highly enriched in cholesterol, a lipid that is otherwise low abundant in mitochondria. In contrast, more common mitochondrial lipids, and abundant inner-membrane associated proteins concentrated in the bottom-half of these gradients. Gene silencing of ATAD3, a protein with proposed functions related to nucleoid and mitochondrial cholesterol homeostasis, modified the distribution of cholesterol and nucleoids in the gradient in an identical fashion. Both cholesterol and ATAD3 were previously shown to be enriched in ER-mitochondrial junctions, and we detect nucleoid components in biochemical isolates of these structures. Our data suggest an uncommon membrane composition that accommodates platforms for replicating mtDNA, and reconcile apparently disparate functions of ATAD3. We suggest that mtDNA replication platforms are organized in connection with ER-mitochondrial junctions, facilitated by a specialized membrane architecture involving mitochondrial cholesterol.


Rooijers K.,Netherlands Cancer Institute | Loayza-Puch F.,Netherlands Cancer Institute | Nijtmans L.G.,Nijmegen Center for Mitochondrial Disorders | Agami R.,Netherlands Cancer Institute | Agami R.,Rotterdam University
Nature Communications | Year: 2013

Mitochondria are essential cellular organelles for generation of energy and their dysfunction may cause diabetes, Parkinson's disease and multi-systemic failure marked by failure to thrive, gastrointestinal problems, lactic acidosis and early lethality. Disease-associated mitochondrial mutations often affect components of the mitochondrial translation machinery. Here we perform ribosome profiling to measure mitochondrial translation at nucleotide resolution. Using a protocol optimized for the retrieval of mitochondrial ribosome protected fragments (RPFs) we show that the size distribution of wild-type mitochondrial RPFs follows a bimodal distribution peaking at 27 and 33 nucleotides, which is distinct from the 30-nucleotide peak of nuclear RPFs. Their cross-correlation suggests generation of mitochondrial RPFs during ribosome progression. In contrast, RPFs from patient-derived mitochondria mutated in tRNA-Tryptophan are centered on tryptophan codons and reduced downstream, indicating ribosome stalling. Intriguingly, long RPFs are enriched in mutated mitochondria, suggesting they characterize stalled ribosomes. Our findings provide the first model for translation in wild-type and disease-triggering mitochondria. © 2013 Macmillan Publishers Limited.


PubMed | Nijmegen Center for Mitochondrial Disorders
Type: Journal Article | Journal: The Netherlands journal of medicine | Year: 2015

Previous research has shown that dysphagia and gastrointestinal problems occur frequently in carriers of the m.3243A>G mutation; however, the exact frequency and severity have not been determined. We hypothesise that adult carriers have an increased risk for malnutrition.In this observational study we evaluated the presence of gastrointestinal problems and dysphagia in 92 carriers of the m.3243A>G mutation. The severity of the general disease involvement was classified using the Newcastle Mitochondrial Disease Adult Scale (NMDAS). Gastrointestinal involvement, dysphagia and the risk for malnutrition were scored using the Gastrointestinal Symptoms Questionnaire and the Malnutrition Universal Screening Tool. Gastrointestinal symptoms and anthropometrics were compared with healthy controls.Our results show that the height, weight and body mass index (BMI) of these carriers were lower than the national average (p < 0.05). Seventy-nine carriers (86%) suffered from at least one gastrointestinal symptom, mainly flatulence or hard stools. Both frequency and severity of symptoms were significantly increased compared with reference data of healthy Dutch adults. Of the carriers, 45% reported (mostly mild) dysphagia. Solid foods cause more problems than liquids. A negative correlation between BMI and heteroplasmy levels in urinary epithelial cells (UEC) was present (Spearman correlation coefficient = - 0.319, p = 0.003).Dysphagia and gastrointestinal problems, especially constipation, are common symptoms in the total m.3243A>G carriers cohort and are not related to heteroplasmy levels in UEC or disease severity. The severity of gastrointestinal problems as well as overall disease severity is associated with an increased risk for malnutrition.


PubMed | Nijmegen Center for Mitochondrial Disorders
Type: Journal Article | Journal: Journal of inherited metabolic disease | Year: 2015

Mitochondrial disorders are characterized by a broad clinical spectrum. Identical clinical signs and symptoms can be caused by mutations in different mitochondrial or nuclear genes. Vice versa, the same mutation can lead to different phenotypes. Genetic syndromes and neuromuscular disorders mimicking mitochondrial disorders further complicate the diagnostic process. Whole exome sequencing (WES) is the state of the art next generation sequencing technique to identify genetic defects in mitochondrial disorders. Until recently it has mainly been used as a research tool. In this study, the use of WES in routine diagnostics is described. The WES data of 109 patients, referred under the suspicion of a mitochondrial disorder, were examined in two steps. First, the data were filtered using a virtual gene panel of genes known to be associated with mitochondrial disease. If negative, the entire exome was examined. A molecular diagnosis was achieved in 39% of the heterogeneous cohort, and in 57% of the subgroup of 42 patients with the highest suspicion for a mitochondrial disease. In addition to mutations in genes known to be associated with mitochondrial disorders (e.g. TUFM, MTFMT, FBXL4), in the subgroup of patients with the lowest suspicion for a mitochondrial disorder we found mutations in several genes associated with neuromuscular disorders (e.g. SEPN1, ACTA1) and genetic syndrome (e.g. SETBP1, ARID1B). Our results show that WES technology has been successfully implemented as a state-of-the-art, molecular diagnostic test for mitochondrial disorders as well as for the mimicking disorders in daily clinical practice. It also illustrates that clinical and biochemical phenotyping is essential for successful application of WES to diagnose individual patients.

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