Smeets, H.J.

Physical activity is the key determinant of skeletal muscle mitochondrial function in type 2 diabetes (Article)

2012-09-01T00:00:00Z

Context: Conflicting data exist on mitochondrial function and physical activity in type 2 diabetes mellitus (T2DM) development. Objective: The aim was to assess mitochondrial function at different stages during T2DM development in combination with physical exercise in longstanding T2DM patients. Design and Methods: We performed cross-sectional analysis of skeletal muscle from 12 prediabetic 11 longstanding T2DM male subjects and 12 male controls matched by age and body mass index. Intervention: One-year intrasubject controlled supervised exercise training intervention was done in longstanding T2DM patients. Main Outcome Measurements: Extensive ex vivo analyses of mitochondrial quality, quantity, and function were collected and combined with global gene expression analysis and in vivo ATP production capacity after 1 yr of training. Results: Mitochondrial density, complex I activity, and the expression of Krebs cycle and oxidative phosphorylation system-related genes were lower in longstanding T2DM subjects but not in prediabetic subjects compared with controls. This indicated a reduced capacity to generate ATP in longstanding T2DM patients only. Gene expression analysis in prediabetic subjects suggested a switch from carbohydrate toward lipid as an energy source. One year of exercise training raised in vivo skeletal muscle ATP production capacity by 21 ± 2% with an increased trend in mitochondrial density and complex I activity. In addition, expression levels of β-oxidation, Krebs cycle, and oxidative phosphorylation system-related genes were higher after exercise training. Conclusions: Mitochondrial dysfunction is apparent only in inactive longstanding T2DM patients, which suggests that mitochondrial function and insulin resistance do not depend on each other. Prolonged exercise training can, at least partly, reverse the mitochondrial impairments associated with the longstanding diabetic state. Copyright

Defective NDUFA9 as a novel cause of neonatally fatal complex I disease (Article)

2012-01-01T00:00:00Z

Background: Mitochondrial disorders are associated with abnormalities of the oxidative phosphorylation (OXPHOS) system and cause significant morbidity and mortality in the population. The extensive clinical and genetic heterogeneity of these disorders due to a broad variety of mutations in several hundreds of candidate genes, encoded by either the mitochondrial DNA (mtDNA) or nuclear DNA (nDNA), impedes a straightforward genetic diagnosis. A new disease gene is presented here, identified in a single Kurdish patient born from consanguineous parents with neonatally fatal Leigh syndrome and complex I deficiency. Methods and results: Using homozygosity mapping and subsequent positional candidate gene analysis, a total region of 255.8 Mb containing 136 possible mitochondrial genes was identified. A pathogenic mutation was found in the complex I subunit encoding the NDUFA9 gene, changing a highly conserved arginine at position 321 to proline. This is the first diseasecausing mutation ever reported for NDUFA9. Complex I activity was restored in fibroblasts of the patient by lentiviral transduction with wild type but not mutant NDUFA9, confirming that the mutation causes the complex I deficiency and related disease. Conclusions: The data show that homozygosity mapping and candidate gene analysis remain an efficient way to detect mutations even in small consanguineous pedigrees with OXPHOS deficiency, especially when the enzyme deficiency in fibroblasts allows appropriate candidate gene selection and functional complementation.

Riboflavin-responsive oxidative phosphorylation complex i deficiency caused by defective ACAD9: New function for an old gene (Article)

2011-01-01T00:00:00Z

Mitochondrial complex I deficiency is the most common oxidative phosphorylation defect. Mutations have been detected in mitochondrial and nuclear genes, but the genetics of many patients remain unresolved and new genes are probably involved. In a consanguineous family, patients presented easy fatigability, exercise intolerance and lactic acidosis in blood from early childhood. In muscle, subsarcolemmal mitochondrial proliferation and a severe complex I deficiency were observed. Exercise intolerance and complex I activity was improved by a supplement of riboflavin at high dosage. Homozygosity mapping revealed a candidate region on chromosome three containing six mitochondria-related genes. Four genes were screened for mutations and a homozygous substitution was identified in ACAD9 (c.1594C>T), changing the highly conserved arginine-532 into tryptophan. This mutation was absent in 188 ethnically matched controls. Protein modelling suggested a functional effect due to the loss of a stabilizing hydrogen bond in an α-helix and a local flexibility change. To test whether the ACAD9 mutation caused the complex I deficiency, we transduced fibroblasts of patients with wild-type and mutant ACAD9. Wild-type, but not mutant, ACAD9 restored complex I activity. An unrelated patient with the same phenotype was compound heterozygous for c.380G>A and c.1405C>T, changing arginine-127 into glutamine and arginine-469 into tryptophan, respectively. These amino acids were highly conserved and the substitutions were not present in controls, making them very probably pathogenic. Our data support a new function for ACAD9 in complex I function, making this gene an important new candidate for patients with complex I deficiency, which could be improved by riboflavin treatment.

Functional annotation of heart enriched mitochondrial genes GBAS and CHCHD10 through guilt by association (Article)

2010-11-12T00:00:00Z

Despite the mitochondria ubiquitous nature many of their components display divergences in their expression profile across different tissues. Using the bioinformatics-approach of guilt by association (GBA) we exploited these variations to predict the function of two so far poorly annotated genes: Coiled-coil-helix-coiled-coil-helix domain containing 10 (CHCHD10) and glioblastoma amplified sequence (GBAS). We predicted both genes to be involved in oxidative phosphorylation. Through in vitro experiments using gene-knockdown we could indeed confirm this and furthermore we asserted CHCHD10 to play a role in complex IV activity.

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.

The unfolding clinical spectrum of POLG mutations (Article)

2009-11-01T00:00:00Z

Background: Mutations in the DNA polymerase-γ (POLG) gene are a major cause of clinically heterogeneous mitochondrial diseases, associated with mtDNA depletion and multiple deletions. Objective: To determine the spectrum of POLG mutations in our Dutch patient cohort, to evaluate the pathogenicity of novel mutations, and to establish genotype-phenotype correlations. Results: The authors identified 64 predominantly recessive mutations in 37 patients from a total of 232 patients, consisting of 23 different mutations. The substitution p.A467T was most frequently observed (n = 23), but was as frequent in childhood cases as in adult cases. Five new pathogenic recessive mutations, p.Lys925ArgfsX42, p.R275X, p.G426S, p.A804T and p.R869Q were identified. The known dominant chronic progressive external ophthalmoplegia (CPEO) mutation p.R943H was for the first time associated with premature ovarian failure as well. In 19 patients the authors identified only a single recessive mutation, or a sequence variant with unclear clinical significance. The data substantiate earlier observations that in POLG patients a fatal status epilepticus and liver failure can be triggered by sodium valproate. It is therefore important to exclude POLG mutations before administering this treatment. Conclusion: The clinical features of the patient are the most important features to select putative POLG mutation carriers and not the presence of mtDNA deletions or OXPHOS (oxidative phosphorylation) activity. The authors conclude that POLG mutations are an important cause of heterogeneous mitochondrial pathology and that more accurate genotype-phenotype correlations allow a more rapid genetic diagnosis and improved prognosis for mutation carriers.

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.

Clinical expression of leber hereditary optic neuropathy is affected by the mitochondrial DNA-haplogroup background (Article)

2007-08-01T00:00:00Z

Leber hereditary optic neuropathy (LHON) is due primarily to one of three common point mutations of mitochondrial DNA (mtDNA), but the incomplete penetrance implicates additional genetic or environmental factors in the pathophysiology of the disorder. Both the 11778G→A and 14484T→C LHON mutations are preferentially found on a specific mtDNA genetic background, but 3460G→A is not. However, there is no clear evidence that any background influences clinical penetrance in any of these mutations. By studying 3,613 subjects from 159 LHON-affected pedigrees, we show that the risk of visual failure is greater when the 11778G→A or 14484T→C mutations are present in specific subgroups of haplogroup J (J2 for 11778G→A and J1 for 14484T→C) and when the 3460G→A mutation is present in haplogroup K. By contrast, the risk of visual failure is significantly less when 11778G→A occurs in haplogroup H. Substitutions on MTCYB provide an explanation for these findings, which demonstrate that common genetic variants have a marked effect on the expression of an ostensibly monogenic mtDNA disorder.

A MELAS-associated ND1 mutation causing Leber hereditary optic neuropathy and spastic dystonia (Article)

2007-06-01T00:00:00Z

Objective: To report a novel mutation that is associated with Leber hereditary optic neuropathy (LHON) within the same family affected by spastic dystonia. Design: Leber hereditary optic neuropathy is a mitochondrial disorder characterized by isolated central visual loss. Of patients with LHON, 95% carry a mutation in 1 of 3 mitochondrial DNA-encoded complex I genes. The complete mitochondrial DNA was screened for mutations in a patient with LHON without 1 of these 3 primary mutations. The heteroplasmy level and biochemical consequence of the mutation were determined. Results: A pathogenic 3697G>A/ND1 mutation was detected and seemed associated with an isolated complex I deficiency. This family has similar clinical characteristics as the previously described families with LHON and dystonia with an ND6 mutation. Conclusions: The 3697G>A/ND1 mitochondrial DNA mutation causes the LHON and spastic dystonia phenotype in the same family. This mutation can also cause MELAS syndrome (which encompasses mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke), and other genetic factors may contribute to the clinical expression.

mtDNA point mutations are present at various levels of heteroplasmy in human oocytes. (Article)

2007-03-01T00:00:00Z

Little is known about the load of mutations and polymorphisms in the mitochondrial DNA (mtDNA) of human oocytes and the possible effect these mutations may have during life. To investigate this, we optimised at the single cell level the recently developed method to screen the entire mtDNA for mainly heteroplasmic mutations by denaturing high performance liquid chromatography analysis. This method is sensitive (approximately 1% heteroplasmy detectable), specific and rapid. The entire mtDNA of 26 oocytes of 13 women was screened by this method. Ten different heteroplasmic mutations, of which only one was located in the D-loop and two were observed twice, were detected in seven oocytes with mutation loads ranging from <5% to 50%. From eight women >1 oocyte was received and in four of them heteroplasmic differences between oocytes of the same woman were observed. In one of these four, two homoplasmic D-loop variants were also detected. Additionally, four oocytes of a single woman were sequenced using the MitoChip (which lacks the D-loop region), but all sequences were identical. It is concluded that heteroplasmic mtDNA mutations are common in oocytes and that, depending on the position and mutation load, they might increase the risk of developing OXPHOS disease early or later in life.

Cardiac Involvement in Adults With m.3243A>G MELAS Gene Mutation (Article)

2007-01-15T00:00:00Z

Cardiac data in adults with mitochondrial encephalomyopathy, lactic acidosis, and strokelike episodes (MELAS syndrome) or asymptomatic gene carriers with the mitochondrial deoxyribonucleic acid adenine-to-guanine point mutation at nucleotide pair 3243 are scarce. Twelve subjects (mean age 35 ± 13 years), 8 with MELAS syndrome (patients) and 4 asymptomatic gene carriers (carriers), were enrolled in the study. Each subject underwent electrocardiography, exercise testing, Holter monitoring, echocardiography, and genetic and biochemical analysis for respiratory chain enzyme activity (complex I rest activity) in skeletal muscle. On electrocardiography and Holter monitoring, none of the subjects had evidence of preexcitation, cardiac arrhythmias, or conduction abnormalities. Patients had significantly lower (42 ± 17% from normal vs 103 ± 14%, p <0.02) exercise tolerance. All but 1 of the patients and none of the gene carriers had ragged red fibers on muscle biopsy. The mean percentage of gene mutation in skeletal muscle tended to be higher in patients (53 ± 19%, range 19% to 73%) compared with carriers (33 ± 20%, range 15% to 62%). Mean complex I rest activity in patients (36 ± 18%, range 10% to 58%) was significantly (p <0.01) lower compared with carriers (120 ± 60%, range 72% to 205%). Left ventricular (LV) abnormalities were confined to patients with MELAS syndrome. Two patients had LV hypertrophy, 5 had LV systolic abnormalities, and 5 had LV diastolic dysfunction. Apart from 1 patient with an isolated LV diastolic abnormality, all patients with LV abnormalities had ragged red fibers. Patients with abnormal systolic LV function had a trend toward a higher percentage of mutated skeletal muscle (59.7 ± 10.7% vs 35.8 ± 21.3%, p <0.10) and significantly lower complex I rest activity (26.7 ± 14.0% vs 97.8% ± 57.9, p <0.01). In conclusion, none of the MELAS gene carriers had cardiac abnormalities, whereas most patients with the MELAS phenotype, particularly those with ragged red fibers, had LV involvement.