Mitochondrial myopathies and encephalomyopathies

Defects of mitochondrial metabolism result in a wide variety of human disorders, which can present at any time from infancy to late adulthood and involve virtually any tissue either alone or in combination. Abnormalities of the electron transport and oxidative phosphorylation (OXPHOS) system are probably the most common cause of mitochondrial diseases. Thirteen of the protein subunits of OXPHOS are encoded by mitochondrial DNA (mtDNA) and mutations of this genome are important causes of OXPHOS deficiency. The link between genotype and phenotype with respect to mtDNA mutations is not clear: the same mutation may result in a variety of phenotypes, and the same phenotype may be seen with a variety of different mtDNA mutations. The pathogenesis of mtDNA mutations is unclear although OXPHOS and ATP deficiency, and free radical generation, are thought to contribute to tissue dysfunction. There is now strong evidence for mitochondrial dysfunction in neurodegenerative disorders. In some cases, e.g. Friedreich's ataxia, hereditary spastic paraplegia, this is a result of a mutation of a nuclear gene encoding a mitochondrial protein, whilst in others, e.g. Huntington's disease, amyotrophic lateral sclerosis, the OXPHOS defect is secondary to events induced by a mutation in a nuclear gene encoding a non-mitochondrial protein. In yet a third group, e.g. Parkinson's disease, Alzheimer's disease, the relationship of the mitochondrial defect to aetiology and pathogenesis is unclear.     

Genetic defects in the oxidative phosphorylation (OXPHOS) system

The oxidative phosphorylation (OXPHOS) system consists of five multiprotein complexes and two mobile electron carriers embedded in the lipid bilayer of the mitochondrial inner membrane. With the exception of complex II and the mobile carriers, the other parts of the OXPHOS system are under dual genetic control. Due to this bigenomic control, the inheritance of OXPHOS system defects is either maternal, in the case of mitochondrial DNA mutations, autosomal or X-linked, in the case of nuclear gene defects. In this review, our current genetic understanding of OXPHOS system enzyme deficiencies will be summarized, and future directions that the field might take to unravel so-far genetically unresolved OXPHOS system enzyme deficiencies will be described, with special emphasis on complex I biogenesis.     

Mitochondrial disorders. A diagnostic challenge in clinical chemistry.

Mitochondria play a pivotal role in cellular metabolism and in energy production in particular. Defects in structure or function of mitochondria, mainly involving the oxidative phosphorylation (OXPHOS), mitochondrial biogenesis and other metabolic pathways, have been shown to be associated with a wide spectrum of clinical phenotypes. The ubiquitous nature of mitochondria and their unique genetic features contribute to the clinical, biochemical and genetic heterogeneity of mitochondrial diseases. We will focus on the recent advances in the field of mitochondrial disorders and their consequences for an advanced clinical and genetic diagnostics. In addition, an overview on recently identified genetic defects and their pathogenic molecular mechanisms will be given.     

Measurement of the energy-generating capacity of human muscle mitochondria: diagnostic procedure and application to human pathology

BACKGROUND: Diagnosis of mitochondrial disorders usually requires a muscle biopsy to examine mitochondrial function. We describe our diagnostic procedure and results for 29 patients with mitochondrial disorders. METHODS: Muscle biopsies were from 43 healthy individuals and 29 patients with defects in one of the oxidative phosphorylation (OXPHOS) complexes, the pyruvate dehydrogenase complex (PDHc), or the adenine nucleotide translocator (ANT). Homogenized muscle samples were used to determine the oxidation rates of radiolabeled pyruvate, malate, and succinate in the absence or presence of various acetyl Co-A donors and acceptors, as well as specific inhibitors of tricarboxylic acid cycle or OXPHOS enzymes. We determined the rate of ATP production from oxidation of pyruvate. RESULTS: Each defect in the energy-generating system produced a specific combination of substrate oxidation impairments. PDHc deficiencies decreased substrate oxidation reactions containing pyruvate. Defects in complexes I, III, and IV decreased oxidation of pyruvate plus malate, with normal to mildly diminished oxidation of pyruvate plus carnitine. In complex V defects, pyruvate oxidation improved by addition of carbonyl cyanide 3-chlorophenyl hydrazone, whereas other oxidation rates were decreased. In most patients, ATP production was decreased. CONCLUSION: The proposed method can be successfully applied to the diagnosis of defects in PDHc, OXPHOS complexes, and ANT.     

Renal function and mitochondrial cytopathy (MC): more questions than answers?

Our knowledge of mitochondrial biology has advanced significantly in the last 10 years. The effects of mitochondrial dysfunction or cytopathy (MC) on the heart and neuromuscular system are well known, and its involvement in the pathophysiology of several common clinical disorders such as diabetes, hyperlipidaemia and hypertension, is just beginning to emerge; however, its contribution to renal disease has received much less attention, and the available literature raises some interesting questions: Why do children with MC commonly present with a renal phenotype that is often quite different from adults? How does a mutation in mitochondrial DNA (mtDNA) lead to disease at the cellular level, and how can a single mtDNA point mutation result in such a variety of renal- and non-renal phenotypes in isolation or combined? Why are some regions of the nephron seemingly more sensitive to mitochondrial dysfunction and damage by mitochondrial toxins? Perhaps most important of all, what can be done to diagnose and treat MC, now and in the future? In this review we summarize our current understanding of the relationship between mitochondrial biology, renal physiology and clinical nephrology, in an attempt to try to answer some of these questions. Although MC is usually considered a rare defect, it is almost certainly under-diagnosed. A greater awareness and understanding of kidney involvement in MC might lead to new treatment strategies for diseases in which mitochondrial dysfunction is secondary to toxic or ischaemic injury, rather than to an underlying genetic mutation.     

A mitochondrial bioenergetic etiology of disease

The classical Mendelian genetic perspective has failed to adequately explain the biology and genetics of common metabolic and degenerative diseases. This is because these diseases are primarily systemic bioenergetic diseases, and the most important energy genes are located in the cytoplasmic mitochondrial DNA (mtDNA). Therefore, to understand these “complex” diseases, we must investigate their bioenergetic pathophysiology and consider the genetics of the thousands of copies of maternally inherited mtDNA, the more than 1,000 nuclear DNA (nDNA) bioenergetic genes, and the epigenomic and signal transduction systems that coordinate these dispersed elements of the mitochondrial genome.     

Molecular mechanisms of mitochondrial diabetes (MIDD)

Mitochondria provide cells with most of the energy in the form of adenosine triphosphate (ATP). Mitochondria are complex organelles encoded both by nuclear and mtDNA. Only a few mitochondrial components are encoded by mtDNA, most of the mt-proteins are nuclear DNA encoded. Remarkably, the majority of the known mutations leading to a mitochondrial disease have been identified in mtDNA rather than in nuclear DNA. In general, the idea is that these pathogenic mutations in mtDNA affect energy supply leading to a disease state. Remarkably, different mtDNA mutations can associate with distinct disease states, a situation that is difficult to reconcile with the idea that a reduced ATP production is the sole pathogenic factor. This review deals with emerging insight into the mechanism by which the A3243G mutation in the mitochondrial tRNA (Leu, UUR) gene associates with diabetes as major clinical expression. A decrease in glucose-induced insulin secretion by pancreatic beta-cells and a premature aging of these cells seem to be the main process by which this mutation causes diabetes. The underlying mechanisms and variability in clinical presentation are discussed.     

Quantification of mitochondrial DNA deletion,depletion, and overreplication: application to diagnosis

BACKGROUND: Many mitochondrial pathologies are quantitative disorders related to tissue-specific deletion, depletion, or overreplication of mitochondrial DNA (mtDNA). We developed an assay for the determination of mtDNA copy number by real-time quantitative PCR for the molecular diagnosis of such alterations. METHODS: To determine altered mtDNA copy number in muscle from nine patients with single or multiple mtDNA deletions, we generated calibration curves from serial dilutions of cloned mtDNA probes specific to four different mitochondrial genes encoding either ribosomal (16S) or messenger (ND2, ND5, and ATPase6) RNAs, localized in different regions of the mtDNA sequence. This method was compared with quantification of radioactive signals from Southern-blot analysis. We also determined the mitochondrial-to-nuclear DNA ratio in muscle, liver, and cultured fibroblasts from a patient with mtDNA depletion and in liver from two patients with mtDNA overreplication. RESULTS: Both methods quantified 5-76% of deleted mtDNA in muscle, 59-97% of mtDNA depletion in the tissues, and 1.7- to 4.1-fold mtDNA overreplication in liver. The data obtained were concordant, with a linear correlation coefficient (r(2)) between the two methods of 0.94, and indicated that quantitative PCR has a higher sensitivity than Southern-blot analysis. CONCLUSIONS: Real-time quantitative PCR can determine the copy number of either deleted or full-length mtDNA in patients with mitochondrial diseases and has advantages over classic Southern-blot analysis.     

Mitochondrial DNA analysis in clinical laboratory diagnostics

Mitochondrial disorders are increasingly being diagnosed, especially among patients with multiple, seemingly unrelated, neuromuscular and multi-sytem disorders. The genetics are complex, in particular as the primary mutation can be either on the nuclear or the mitochondrial DNA (mtDNA). mtDNA mutations are often maternally inherited, but can be sporadic or secondary to autosomally inherited mutations in nuclear genes that regulate mtDNA biosynthesis. mtDNA mutations demonstrate extreme variable expressivity in terms of clinical manifestations and severity, even within a family. Disease is often episodic. Several well-defined clinical syndromes associated with specific mutations are described, yet the genotype-phenotype correlation is fair at best and most patients do not fit within any defined syndrome and have rare or novel mutations. In most patients, mutant and wild-type mtDNA coexist ("heteroplasmy"), although homoplasmic mtDNA mutations also are known. "Standard" mtDNA clinical diagnostics usually consists of a PCR-based assay to detect a small number of relatively common point mutations and Southern blotting (or PCR) for large (>500 bp) rearrangements. In selected cases testing negative, additional analyses can include real-time PCR for mtDNA depletion, and full mtDNA genome screening for the detection of rare and novel point mutations by a variety of methods. Prenatal diagnosis is problematic in most cases.     

Maternally transmitted antigens are codominantly expressed by mouse cells containing two kinds of mitochondrial DNA

Mtf, a cytoplasmic, probably mitochondrial factor, controls Mta polymorphism. We tested for dominance between two forms of Mtf to determine whether Mta is controlled by positive or negative genetic mechanisms. We fused Mtf-disparate cells containing distinct mtDNA markers and selected for hybrids containing both. Such mtDNA heteroplasmons codominantly and stably express alternative Mta antigens. Stable codominance excludes negative genetic mechanisms as well as a model of induced nuclear compensation, and implies Mtf controls Mta expression through a positive genetic mechanism.     

Simultaneous detection and quantification of mitochondrial DNA deletion(s), depletion, and over-replication in patients with mitochondrial disease

Heterogeneous clinical expression of mitochondrial DNA (mtDNA) disorders depends on both qualitative and quantitative changes in mtDNA. We developed a sensitive and effective method that simultaneously detects mtDNA deletion(s) and quantifies total mtDNA content. The percentage of deletions and mtDNA content of 19 patients with single or multiple deletions were analyzed by real-time quantitative polymerase chain reaction (real-time qPCR) using TaqMan probes specific for mtDNA (tRNA leu(UUR), ND4, ATPase8, and D-loop regions) and nuclear DNA (AIB1, beta-2-microglobulin, and beta-actin). The proportion of deletion mutants determined by real-time qPCR was consistent with that determined by Southern analysis. Most patients with mtDNA deletions also demonstrated compensatory mtDNA over-replication. Multiple mtDNA deletions that were not detectable by Southern analysis due to low percentage of each deletion molecule were readily detected and quantified by real-time qPCR. Furthermore, 12 patients with clinical features and abnormal biochemical/histopathological results consistent with mitochondrial respiratory chain disorders without identified mtDNA mutations had either substantially depleted or significantly over-replicated mtDNA content, supporting the diagnosis of mitochondrial disease. Our results demonstrate that both qualitative and quantitative analyses are important in molecular diagnosis of mitochondrial diseases. The presence of deletion(s) and mtDNA depletion or compensatory over-replication can be determined simultaneously by real-time qPCR.     

Molecular mechanisms of mitochondrial diabetes (MIDD)

Mitochondria provide cells with most of the energy in the form of adenosine triphosphate (ATP). Mitochondria are complex organelles encoded both by nuclear and mtDNA. Only a few mitochondrial components are encoded by mtDNA, most of the mt‐proteins are nuclear DNA encoded. Remarkably, the majority of the known mutations leading to a mitochondrial disease have been identified in mtDNA rather than in nuclear DNA. In general, the idea is that these pathogenic mutations in mtDNA affect energy supply leading to a disease state. Remarkably, different mtDNA mutations can associate with distinct disease states, a situation that is difficult to reconcile with the idea that a reduced ATP production is the sole pathogenic factor. This review deals with emerging insight into the mechanism by which the A3243G mutation in the mitochondrial tRNA (Leu, UUR) gene associates with diabetes as major clinical expression. A decrease in glucose‐induced insulin secretion by pancreatic beta‐cells and a premature aging of these cells seem to be the main process by which this mutation causes diabetes. The underlying mechanisms and variability in clinical presentation are discussed.     

Activities of mitochondrial oxidative phosphorylation enzymes in cultured amniocytes

Amniocytes represent a population of foetal cells that can be used for prenatal diagnosis in families with suspected mitochondrial oxidative phosphorylation (OXPHOS) defects. In this paper, we present a complex protocol for evaluation of the function of mitochondrial OXPHOS enzymes in cultured amniocytes using three independent and complementary methods: (a) spectrophotometry as a tool for determination of the capacities of mitochondrial respiratory-chain enzymes (NADH ubiquinone oxidoreductase, succinate- and glycerophosphate cytochrome c reductase, cytochrome c oxidase and citrate synthase); (b) polarography as a tool for the evaluation of mitochondrial OXPHOS enzyme functions in situ using digitonin-permeabilised amniocytes (rotenone-sensitive oxidation of pyruvate+malate, antimycin A-sensitive oxidation of succinate, KCN-sensitive oxidation of cytochrome c, ADP-activated substrate oxidation) and (c) cytofluorometric determination of tetramethyl rhodamine methyl ester (TMRM) fluorescence in digitonin-permeabilised amniocytes as a sensitive way to determine the mitochondrial membrane potential under steady-state conditions (state 4 with succinate). These protocols are presented together with reference control values using 9-22 independent cultures of amniocytes.     

Septic patients with mitochondrial DNA haplogroup JT have higher respiratory complex IV activity and survival rate

ObjectiveThe influence of mitochondrial deoxyribonucleic acid (mtDNA) haplogroup or oxidative phosphorylation system (OXPHOS) function on survival of septic patients has been scarcely studied. However, the association between mtDNA haplogroup, OXPHOS capacity at diagnosis of severe sepsis, and survival has been not previously reported, and that was the objective of the present study.MethodsThis was a prospective, multicenter, observational study. Blood samples from 198 patients at diagnosis of severe sepsis were analyzed to determine mtDNA haplogroup and platelet respiratory complex IV (CIV) specific activity. The end point of the study was 30-day survival.ResultsSeptic patients with mtDNA haplogroup JT showed higher 30-day survival than those with mtDNA haplogroup non-JT (31/38 [81.6%] vs 99/160 [61.9%]; P= .02). Septic patients with mtDNA haplogroup JT showed higher platelet CIV specific activity than those with mtDNA haplogroup non-JT (P= .002).ConclusionsThe main novel finding of our study, including the largest series providing data on platelet CIV specific activity according to mtDNA haplogroup in severe septic patients, was that those with mtDNA haplogroup JT showed higher survival and higher platelet CIV specific activity at diagnosis of severe sepsis than patients with mtDNA haplogroup non-JT.     

Detection and quantification of heteroplasmic mutant mitochondrial DNA by real-time amplification refractory mutation system quantitative PCR analysis: a single-step approach

BACKGROUND: The A3243G mitochondrial tRNA leu(UUR) point mutation causes mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome, the most common mitochondrial DNA (mtDNA) disorder, and is also found in patients with maternally inherited diabetes and deafness syndrome (MIDD). To correlate disease manifestation with mutation loads, it is necessary to measure the percentage of the A3243G mtDNA mutation. METHODS: To reliably quantify low proportions of the mutant mtDNA, we developed a real-time amplification refractory mutation system quantitative PCR (ARMS-qPCR) assay. We validated the method with experimental samples containing known proportions of mutant A3243G mtDNA generated by mixing known amounts of cloned plasmid DNA containing either the wild-type or the mutant sequences. RESULTS: A correlation coefficient of 0.9995 between the expected and observed values for the proportions of mutant A3243G in the experimental samples was found. Evaluation of a total of 36 patient DNA samples demonstrated consistent results between PCR-restriction fragment length polymorphism (RFLP) analysis and real-time ARMS-qPCR. However, the latter method was much more sensitive for detecting low percentages of mutant heteroplasmy. Three samples contained allele-specific oligonucleotide-detectable but RFLP-undetectable mutations. CONCLUSIONS: The real-time ARMS-qPCR method provides rapid, reliable, one-step quantitative detection of heteroplasmic mutant mtDNA.     

Techniques and pitfalls in the detection of pathogenic mitochondrial DNA mutations

Mutations in the mitochondrial DNA (mtDNA) are now recognized as major contributors to human pathologies and possibly to normal aging. A large number of rearrangements and point mutations in protein coding and tRNA genes have been identified in patients with mitochondrial disorders. In this review, we discuss genotype-phenotype correlations in mitochondrial diseases and common techniques used to identify pathogenic mtDNA mutations in human tissues. Although most of these approaches employ standard molecular biology tools, the co-existence of wild-type and mutated mtDNA (mtDNA heteroplasmy) in diseased tissues complicates both the detection and accurate determination of the size of the mutated fractions. To address these problems, novel approaches were developed and are discussed in this review.     

Drug-induced liver injury through mitochondrial dysfunction: mechanisms and detection during preclinical safety studies

Mitochondrial dysfunction is a major mechanism whereby drugs can induce liver injury and other serious side effects such as lactic acidosis and rhabdomyolysis in some patients. By severely altering mitochondrial function in the liver, drugs can induce microvesicular steatosis, a potentially severe lesion that can be associated with profound hypoglycaemia and encephalopathy. They can also trigger hepatic necrosis and/or apoptosis, causing cytolytic hepatitis, which can evolve into liver failure. Milder mitochondrial dysfunction, sometimes combined with an inhibition of triglyceride egress from the liver, can induce macrovacuolar steatosis, a benign lesion in the short term. However, in the long term this lesion can evolve in some individuals towards steatohepatitis, which itself can progress to extensive fibrosis and cirrhosis. As liver injury caused by mitochondrial dysfunction can induce the premature end of clinical trials, or drug withdrawal after marketing, it should be detected during the preclinical safety studies. Several in vitro and in vivo investigations can be performed to determine if newly developed drugs disturb mitochondrial fatty acid oxidation (FAO) and the oxidative phosphorylation (OXPHOS) process, deplete hepatic mitochondrial DNA (mtDNA), or trigger the opening of the mitochondrial permeability transition (MPT) pore. As drugs can be deleterious for hepatic mitochondria in some individuals but not in others, it may also be important to use novel animal models with underlying mitochondrial and/or metabolic abnormalities. This could help us to better predict idiosyncratic liver injury caused by drug-induced mitochondrial dysfunction.     

Mitochondrial dysfunction and migraine: evidence and hypotheses

The molecular basis of migraine is still not completely understood. An impairment of mitochondrial oxidative metabolism might play a role in the pathophysiology of this disease, by influencing neuronal information processing. Biochemical assays of platelets and muscle biopsies performed in migraine sufferers have shown a decreased activity of the respiratory chain enzymes. Studies with phosphorus magnetic resonance spectroscopy ((31)P-MRS) have demonstrated an impairment of the brain oxidative energy metabolism both during and between migraine attacks. However, molecular genetic studies have not detected specific mitochondrial DNA (mtDNA) mutations in patients with migraine, although other studies suggest that particular genetic markers (i.e. neutral polymorphisms or secondary mtDNA mutations) might be present in some migraine sufferers. Further studies are still needed to clarify if migraine is associated with unidentified mutations on the mtDNA or on nuclear genes that code mitochondrial proteins. In this paper, we review morphological, biochemical, imaging and genetic studies which bear on the hypothesis that migraine may be related to mitochondrial dysfunction at least in some individuals.