Wit, L.E.A. de

Defective complex I assembly due to C20orf7 mutations as a new cause of Leigh syndrome (Article)

2010-08-01T00:00:00Z

Background: Leigh syndrome is an early onset, progressive, neurodegenerative disorder with developmental and motor skills regression. Characteristic magnetic resonance imaging abnormalities consist of focal bilateral lesions in the basal ganglia and/or the brainstem. The main cause is a deficiency in oxidative phosphorylation due to mutations in an mtDNA or nuclear oxidative phosphorylation gene. Methods and results: A consanguineous Moroccan family with Leigh syndrome comprise 11 children, three of which are affected. Marker analysis revealed a homozygous region of 11.5 Mb on chromosome 20, containing 111 genes. Eight possible mitochondrial candidate genes were sequenced. Patients were homozygous for an unclassified variant (p.P193L) in the cardiolipin synthase gene (CRLS1). As this variant was present in 20% of a Moroccan control population and enzyme activity was only reduced to 50%, this could not explain the rare clinical phenotype in our family. Patients were also homozygous for an amino acid substitution (p.L159F) in C20orf7, a new complex I assembly factor. Parents were heterozygous and unaffected sibs heterozygous or homozygous wild type. The mutation affects the predicted S-adenosylmethionine (SAM) dependent methyltransferase domain of C20orf7, possibly involved in methylation of NDUFB3 during the assembly process. Blue native gel electrophoresis showed an altered complex I assembly with only 30-40% of mature complex I present in patients and 70-90% in carriers. Conclusions: A new cause of Leigh syndrome can be a defect in early complex I assembly due to C20orf7 mutations.

Patients with Leber hereditary optic neuropathy fail to compensate impaired oxidative phosphorylation (Article)

2010-02-01T00:00:00Z

Ninety-five percent of Leber hereditary optic neuropathy (LHON) patients carry a mutation in one out of three mtDNA-encoded ND subunits of complex I. Penetrance is reduced and more male than female carriers are affected. To assess if a consistent biochemical phenotype is associated with LHON expression, complex I- and complex II-dependent adenosine triphosphate synthesis rates (CI-ATP, CII-ATP) were determined in digitonin-permeabilized peripheral blood mononuclear cells (PBMCs) of thirteen healthy controls and for each primary mutation of a minimum of three unrelated patients and of three unrelated carriers with normal vision and were normalized per mitochondrion (citrate synthase activity) or per cell (protein content). We found that in mitochondria, CI-ATP and CII-ATP were impaired irrespective of the primary LHON mutation and clinical expression. An increase in mitochondrial density per cell compensated for the dysfunctional mitochondria in LHON carriers but was insufficient to result in a normal biochemical phenotype in early-onset LHON patients.

Chapter 9 Reliable Assay for Measuring Complex I Activity in Human Blood Lymphocytes and Skin Fibroblasts (Article)

2009-04-06T00:00:00Z

Complex I deficiency is probably the most common enzyme defect among the group of OXPHOS disorders. To evaluate a deficiency of complex I activity, biochemical measurements based on estimation of the mitochondrial rotenone-sensitive NADH: ubiquinone oxidoreductase activity are an important tool. Skeletal muscle is the most widely used tissue to examine complex I deficiency. However, obtaining a muscle biopsy requires an invasive surgical operation. It is much easier to obtain blood lymphocytes or skin fibroblasts, and, moreover, these cells can be expanded in number by standard techniques for extensive research on complex I. On the other hand, each of these cell types has disadvantages that hinder its measurement, such as the apparent low enzyme activity of lymphocytes and the highly contaminating nonmitochondrial NADH-quinone oxidoreductase activity of fibroblasts. This chapter describes a method to assay complex I activity reliably in a minute amount of either cell type.

Correct assay of complex I activity in human skin fibroblasts by timely addition of rotenone (Article)

2008-11-01T00:00:00Z

Termination of damaged protein repair defines the occurrence of symptoms in carriers of the m.3243A>G tRNALeu mutation (Article)

2008-08-01T00:00:00Z

Background: The m.3243A>G mutation in the mitochondrial tRNALeu(UUR)gene is an example of a mutation causing a very heterogeneous phenotype. It is the most frequent cause (80%) of the MELAS syndrome (mitochondrial myopathy, encephalopathy, lactic acidosis and stroke-like episodes), but it can also lead in addition or separately to type 2 diabetes, deafness, renal tubulopathy and/or cardiomyopathy. Methods: To identify pathogenic processes induced by this mutation, we compared global gene expression levels of muscle biopsies from affected and unaffected mutation carriers with controls. Results and conclusions: Gene expression changes were relatively subtle. In the asymptomatic group 200 transcripts were upregulated and 12 were downregulated, whereas in the symptomatic group 15 transcripts were upregulated and 52 were downregulated. In the asymptomatic group, oxidative phosphorylation (OXPHOS) complex I and IV genes were induced. Protein turnover and apoptosis were elevated, most likely due to the formation of dysfunctional and reactive oxygen species (ROS) damaged proteins. These processes returned to normal in symptomatic patients. Components of the complement system were upregulated in both groups, but the strongest in the symptomatic group, which might indicate muscle regeneration - most likely, protein damage and OXPHOS dysfunction stimulate repair (protein regeneration) and metabolic adaptation (OXPHOS). In asymptomatic individuals these processes suffice to prevent the occurrence of symptoms. However, in affected individuals the repair process terminates, presumably because of excessive damage, and switches to muscle regeneration, as indicated by a stronger complement activation. This switch leaves increasingly damaged tissue in place and muscle pathology becomes manifest. Therefore, the expression of complement components might be a marker for the severity and progression of MELAS clinical course.

A simplified and reliable assay for complex I in human blood lymphocytes (Article)

2007-09-30T00:00:00Z

Complex I activity of the mitochondrial respiratory chain is difficult to measure in blood lymphocytes because of the limited access of substrates to the enzyme complex in these cells. The results of the present study show that permeabilization of human blood lymphocytes in the presence of protease inhibitors by three cycles of freeze-thawing enables reproducible detection of the rotenone-sensitive complex I activity. To that end, the water-soluble coenzyme Q10analogue CoQ1and a relatively high concentration of blood lymphocytes were combined in small quartz cuvettes so that the amount of blood needed for this assay remained low. The relationship between the initial rate of NADH oxidation by complex I and the protein concentration was quasi-linear. The fractional inhibition of the total NADH:CoQ1oxidoreductase by a saturating concentration of rotenone decreased sharply at CoQ1concentrations higher than 20 μM, which is indicative, but does not prove the involvement of a second CoQ1binding site at complex I. Since the present complex I assay requires only a small amount of blood, the functionality of this important respiratory chain complex can be assessed in an easy and reliable manner not only in adult patients but also in children suspected to have a mitochondrial disease.

Indirect evidence for a role of a subpopulation of activated neutrophils in the remodelling process after percutaneous coronary intervention. (Article)

2001-07-20T00:00:00Z

AIM: Leukocytes have been implicated in restenosis following percutaneous transluminal coronary angioplasty. We investigated the link between the activated status of circulating neutrophils and restenosis after angioplasty. METHODS AND RESULTS: The population of 108 patients with single, de novo lesions located in native coronary arteries were treated with elective balloon angioplasty (n=44) or stenting (n=64). Pre-, post-procedure and 6-month follow-up, angiograms were analysed by an independent core laboratory. Blood samples were collected immediately before treatment and the antigen CD66, which is specifically expressed by activated neutrophils, was measured. Overall, the average expression of CD66 was 6.4+/-3.6 of mean fluorescence intensity. In the stepwise linear regression model, which included biological, clinical and angiographic variables, absolute gain showed a direct association (P<0.001) with relative late loss (relative late loss=absolute late loss/pre-procedure reference diameter), whereas CD66 expression was inversely associated with relative late loss (P=0.004). CD66 expression also showed an inverse association with relative late loss in the balloon angioplasty treated patients (P=0.002, beta=-0.49). In the stent subgroup, only reference vessel diameter and acute gain were independent predictors of relative late loss. CONCLUSION: Our results confirm the beneficial role of activated neutrophils pre-procedure in the restenotic process after balloon angioplasty. The lack of a relationship between CD66 expression by neutrophils and relative late loss after stenting suggests that this leukocyte may be involved in the remodelling process.

Late Lumen Loss After Coronary Angioplasty Is Associated With the Activation Status of Circulating Phagocytes Before Treatment (Article)

1995-01-01T00:00:00Z

Background The purpose of this pilot study was to identify biological risk factors for restenosis after percutaneous transluminal coronary angioplasty (PTCA) to predict the long-term outcome of PTCA before treatment. Methods and Results To investigate whether blood granulocytes and monocytes could determine luminal renarrowing after PTCA, several characteristics of these phagocytes were assessed before angioplasty in 32 patients who underwent PTCA of one coronary artery and who had repeat angiograms at 6-month follow-up. The plasma levels of interleukin (IL)-1ß, tumor necrosis factor-, IL-6, fibrinogen, C-reactive protein, and lipoprotein(a) before angioplasty were assessed as well. We found that the expression of the membrane antigens CD64, CD66, and CD67 by granulocytes was inversely associated with the luminal renarrowing normalized for vessel size (relative loss) at 6 months after PTCA, while the production of IL-1ß by stimulated monocytes was positively associated with the relative loss. Next, these univariate predictors were corrected for the established clinical risk factors of dilation of the left anterior descending coronary artery and current smoking, which were statistically significant classic predictors in our patient group. Only the expression of CD67 did not predict late lumen loss independent of these established clinical risk factors. Multiple linear regression analysis showed that luminal renarrowing could be predicted reliably (R2=.65; P<.0001) in this patient group on the basis of the vessel dilated and only two biological risk factors that reflect the activation status of blood phagocytes, ie, the expression of CD66 by granulocytes and the production of IL-1ß by stimulated monocytes. Conclusions The results of the present study indicate that activated blood granulocytes prevent luminal renarrowing after PTCA, while activated blood monocytes promote late lumen loss. To validate this new finding, further study in an independent patient group is required.