Toxic and nutritional factors trigger Leber hereditary optic neuropathy due to a mitochondrial tRNA mutation.

Leber hereditary optic neuropathy is a mitochondrial disease mainly due to pathologic mutations in mitochondrial genes related to the respiratory complex I of the oxidative phosphorylation system. Genetic, physiological, and environmental factors modulate the penetrance of these mutations. We report two patients suffering from this disease and harboring a m.15950G > A mutation in the mitochondrial DNA-encoded gene for the threonine transfer RNA. We also provide evidences supporting the pathogenicity of this mutation.

Pathological Features in Paediatric Patients with TK2 Deficiency.

Thymidine kinase (TK2) deficiency causes mitochondrial DNA depletion syndrome. We aimed to report the clinical, biochemical, genetic, histopathological, and ultrastructural features of a cohort of paediatric patients with TK2 deficiency. Mitochondrial DNA was isolated from muscle biopsies to assess depletions and deletions. The TK2 genes were sequenced using Sanger sequencing from genomic DNA. All muscle biopsies presented ragged red fibres (RRFs), and the prevalence was greater in younger ages, along with an increase in succinate dehydrogenase (SDH) activity and cytochrome c oxidase (COX)-negative fibres. An endomysial inflammatory infiltrate was observed in younger patients and was accompanied by an overexpression of major histocompatibility complex type I (MHC I). The immunofluorescence study for complex I and IV showed a greater number of fibres than those that were visualized by COX staining. In the ultrastructural analysis, we found three major types of mitochondrial alterations, consisting of concentrically arranged lamellar cristae, electrodense granules, and intramitochondrial vacuoles. The pathological features in the muscle showed substantial differences in the youngest patients when compared with those that had a later onset of the disease. Additional ultrastructural features are described in the muscle biopsy, such as sarcomeric de-structuration in the youngest patients with a more severe phenotype.

Mitochondrial DNA pathogenic mutations in multiple symmetric lipomatosis.

The frequency of dermatological manifestations in diseases due to mitochondrial DNA mutations is not well known, although multiple symmetric lipomatosis has been repeatedly associated to mitochondrial DNA mutations. Here, we present a patient suffering from multiple symmetric lipomatosis and other skin signs. We found a new mitochondrial DNA mutation, m.8357T>C, in the tRNALys -coding gene and, using a cybrid approach, confirmed its pathogenicity. A meta-analysis of the dermatological signs of the patient shows that they are not common in patients with confirmed mitochondrial DNA mutations and suggests that, in these cases, lipomatosis is not related to the oxidative phosphorylation dysfunction, but to an alteration of an additional function associated to particular mitochondrial tRNAs.

Oxidative Phosphorylation Dysfunction Modifies the Cell Secretome.

Mitochondrial oxidative phosphorylation disorders are extremely heterogeneous conditions. Their clinical and genetic variability makes the identification of reliable and specific biomarkers very challenging. Until now, only a few studies have focused on the effect of a defective oxidative phosphorylation functioning on the cell's secretome, although it could be a promising approach for the identification and pre-selection of potential circulating biomarkers for mitochondrial diseases. Here, we review the insights obtained from secretome studies with regard to oxidative phosphorylation dysfunction, and the biomarkers that appear, so far, to be promising to identify mitochondrial diseases. We propose two new biomarkers to be taken into account in future diagnostic trials.

Expanding the clinical phenotypes of MT-ATP6 mutations.

Mitochondrial DNA mutations at MT-ATP6 gene are relatively common in individuals suffering from striatal necrosis syndromes. These patients usually do not show apparent histochemical and/or biochemical signs of oxidative phosphorylation dysfunction. Because of this, MT-ATP6 is not typically analyzed in many other mitochondrial disorders that have not been previously associated to mutations in this gene. To correct this bias, we have performed a screening of the MT-ATP6 gene in a large collection of patients suspected of suffering different mitochondrial DNA (mtDNA) disorders. In three cases, biochemical, molecular-genetics and other analyses in patient tissues and cybrids were also carried out. We found three new pathologic mutations. Two of them in patients showing phenotypes that have not been commonly associated to mutations in the MT-ATP6 gene. These results remark the importance of sequencing the MT-ATP6 gene in patients with striatal necrosis syndromes, but also within other mitochondrial pathologies. This gene should be sequenced at least in all those patients suspected of suffering an mtDNA disorder disclosing normal results for histochemical and biochemical analyses of respiratory chain.

Mitochondrial antibiograms in personalized medicine.

Some ribosomal antibiotics used in clinical practice to fight pathogenic bacteria can provoke serious adverse drug reactions in patients. Sensitivity to the antibiotics is a multifactorial trait but the genetic variation of sensitive individuals to off-target effects of the drugs might be one of the factors contributing to this condition. Thus, the protein synthesis apparatus of mitochondria is similar to that of bacteria because of its endosymbiotic origin and, therefore, mitochondrial ribosomes are frequently unintended off-targets of these antibiotics. Because of the limitations of epidemiologic studies of pharmacogenomics, we constructed 25 transmitochondrial cell lines using platelets from individuals belonging to high-frequency European mitochondrial DNA (mtDNA) haplogroups and grew them in the absence or presence of commonly used ribosomal antibiotics. Next, we analyzed the mitochondrial synthesis of proteins and the mitochondrial oxygen consumption to ascertain whether some side effects of ribosomal drugs are due to their interaction with particular mtDNA haplogroup-defining polymorphisms. The amount of mitochondrial translation products, the p.MT-CO1/succinate dehydrogenase subunit A ratio and the ratio of respiratory complex IV quantity to citrate synthase (CS)-specific activity were significantly lower, after the treatment with linezolid, in cybrids harboring the highly frequent m.3010A allele. These results suggest that mitochondrial antibiograms should be implemented for at least the most frequent mitochondrial ribosomal RNA (rRNA) polymorphisms and combinations of polymorphisms and the most frequently used ribosomal antibiotics. In this way, we would obtain individualized barcodes for antibiotic therapy, avoid the side effects of the antibiotics and enable appropriate personalized medicine.

Transcriptomic profiling of TK2 deficient human skeletal muscle suggests a role for the p53 signalling pathway and identifies growth and differentiation factor-15 as a potential novel biomarker for mitochondrial myopathies.

BACKGROUND: Mutations in the gene encoding thymidine kinase 2 (TK2) result in the myopathic form of mitochondrial DNA depletion syndrome which is a mitochondrial encephalomyopathy presenting in children. In order to unveil some of the mechanisms involved in this pathology and to identify potential biomarkers and therapeutic targets we have investigated the gene expression profile of human skeletal muscle deficient for TK2 using cDNA microarrays. RESULTS: We have analysed the whole transcriptome of skeletal muscle from patients with TK2 mutations and compared it to normal muscle and to muscle from patients with other mitochondrial myopathies. We have identified a set of over 700 genes which are differentially expressed in TK2 deficient muscle. Bioinformatics analysis reveals important changes in muscle metabolism, in particular, in glucose and glycogen utilisation, and activation of the starvation response which affects aminoacid and lipid metabolism. We have identified those transcriptional regulators which are likely to be responsible for the observed changes in gene expression. CONCLUSION: Our data point towards the tumor suppressor p53 as the regulator at the centre of a network of genes which are responsible for a coordinated response to TK2 mutations which involves inflammation, activation of muscle cell death by apoptosis and induction of growth and differentiation factor 15 (GDF-15) in muscle and serum. We propose that GDF-15 may represent a potential novel biomarker for mitochondrial dysfunction although further studies are required.

New MT-ND1 pathologic mutation for Leber hereditary optic neuropathy.

BACKGROUND: Mutations causing Leber hereditary optic neuropathy are usually homoplasmic, show incomplete penetrance, and many of the affected positions are not well conserved through evolution. A large percentage of patients harbouring these mutations have no family history of disease. Moreover, the transfer of the mutation in the cybrid model is frequently not accompanied by the transfer of the cellular, biochemical and molecular phenotype. All these features make difficult their classification as the etiologic factors for this disease. We report a patient who exhibits typical clinical features of Leber hereditary optic neuropathy but lacks all three of the most common mitochondrial DNA mutations. METHODS: The diagnosis was made based on clinical studies. The mitochondrial DNA was completely sequenced, and the candidate mutation was analysed in more than 18 000 individuals around the world, its conservation index was estimated in more than 3100 species from protists to mammals, its position was modelled in the crystal structure of a bacteria ortholog subunit, and its functional consequences were studied in a cybrid model. RESULTS: Genetic analysis revealed an m.3472T>C transition in the MT-ND1 gene that changes a phenylalanine to leucine at position 56. Bioinformatics, molecular-genetic analysis and functional studies suggest that this transition is the etiological factor for the disorder. CONCLUSIONS: This mutation expands the spectrum of deleterious changes in mitochondrial DNA-encoded complex I polypeptides associated with this pathology and highlights the difficulties in assigning pathogenicity to new homoplasmic mutations that show incomplete penetrance in sporadic Leber hereditary optic neuropathy patients.

Identification and characterization of a new pathologic mutation in a large Leber hereditary optic neuropathy pedigree.

BACKGROUND: Most patients suffering from Leber hereditary optic neuropathy carry one of the three classic pathologic mutations, but not all individuals with these genetic alterations develop the disease. There are different risk factors that modify the penetrance of these mutations. The remaining patients carry one of a set of very rare genetic variants and, it appears that, some of the risk factors that modify the penetrance of the classical pathologic mutations may also affect the phenotype of these other rare mutations. RESULTS: We describe a large family including 95 maternally related individuals, showing 30 patients with Leber hereditary optic neuropathy. The mutation responsible for the phenotype is a novel transition, m.3734A > G, in the mitochondrial gene encoding the ND1 subunit of respiratory complex I. Molecular-genetic, biochemical and cellular studies corroborate the pathogenicity of this genetic change. CONCLUSIONS: With the study of this family, we confirm that, also for this very rare mutation, sex and age are important factors modifying penetrance. Moreover, this pedigree offers an excellent opportunity to search for other genetic or environmental factors that additionally contribute to modify penetrance.

Oxidative phosphorylation differences between mitochondrial DNA haplogroups modify the risk of Leber's hereditary optic neuropathy.

Leber's hereditary optic neuropathy is a maternally inherited optic atrophy caused by mitochondrial DNA point mutations. Previous epidemiological studies have shown that individuals from mitochondrial genetic backgrounds (haplogroups) J/Uk and H have a higher and a lower risk, respectively, of suffering this disorder. To analyze the bases of these associations at cellular and molecular levels, functional studies with cybrids provide high quality evidence. Cybrids from haplogroup J contain less mitochondrial deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) and synthesize a smaller amount of mitochondrial DNA-encoded polypeptides than those from haplogroup H. Haplogroup J cybrids also display lower oxygen consumption, mitochondrial inner membrane potential and total adenosine-5'-triphosphate (ATP) levels. Moreover, mitochondrial DNA levels correlate with many parameters of the oxidative phosphorylation system. These results suggest that the mitochondrial DNA amount determines oxidative phosphorylation capacity and, along with other recently published observations, support the possibility that mitochondrial DNA levels may be responsible for the bias of the disorder toward males, for the incomplete penetrance of mutations causing Leber's hereditary optic neuropathy and for the association of the disease with particular mitochondrial DNA haplogroups.

'Progress' renders detrimental an ancient mitochondrial DNA genetic variant.

A human mitochondrial DNA (mtDNA) transition, m.1555A>G, in the 12S rRNA gene causes non-syndromic hearing loss. However, this pathological mutation is the wild-type allele in orangutan mtDNA. Here we rule out different genetic factors as the reason for its fixation in orangutans and show that aminoglycosides negatively affect the oxidative phosphorylation function by decreasing the synthesis of mtDNA-encoded proteins and the amount and activity of respiratory complex IV. These drugs also diminish the growth rate of orangutan cells. The m.1555G nucleotide is also the wild-type allele in other mammal species and they might be at risk of suffering a mitochondrial disorder if treated with aminoglycosides. Therefore, pharmacogenomic approaches should be used to confirm this possibility. These observations are important for human health. Due to the fact that old age and high frequency are criteria widely used in mitochondrial medicine to rule out a genetic change as being a pathological mutation, our results prevent against simplistic genetic approaches that do not consider the potential effect of environmental conditions. Hence, these results suggest that some ancient and highly frequent human population polymorphisms, such as those defining mtDNA haplogroups, in mitochondrial rRNA genes can be deleterious in association with new environmental conditions. Therefore, as the discovery of ribosomal antibiotics has allowed to fight infectious diseases and this breakthrough can be considered an important scientific advance or 'progress', our results suggest that 'progress' can also have a negative counterpart and render detrimental many of these mtDNA genotypes.

Maternally inherited susceptibility to cancer.

Tumor microenvironment promotes mtDNA mutations. A number of these mutations will affect cell metabolism and increase cell survival. These mutations are positively selected and contribute to other tumor features, such as extracellular matrix remodeling and angiogenic processes, thus favoring metastases. Like somatic mutations, although with less marked effects, some mtDNA population polymorphisms will affect OXPHOS function, cell metabolism, and homeostasis. Thus, they could behave as inherited susceptibility factors for cancer. However, in addition to epidemiological evidence, other more direct clues are required. The cybrid approach can help to clarify this issue.

New mitochondrial DNA mutations in tRNA associated with three severe encephalopamyopathic phenotypes: neonatal, infantile, and childhood onset.

The reported cases showed clinical, biochemical, histopathological, and molecular features lending support to the hypothesis of a pathogenic effect of the detected mutations. Case 1 was a neonatal presentation who showed multiple mitochondrial respiratory chain enzyme defects in muscle associated with a new homoplasmic m.5514A > G transition in the tRNA(Trp) gene. Case 2 was a late infantile presentation who also showed mitochondrial respiratory chain enzyme deficiencies in muscle together with a new m.1643A > G tRNA(Val) mutation in homoplasmy. Case 3 showed a MERRF phenotype presented in childhood associated with the once previously reported m.15923A > G mutation in heteroplasmy in all the tissues studied.

Unmasking the causes of multifactorial disorders: OXPHOS differences between mitochondrial haplogroups.

Many epidemiologic studies have associated human mitochondrial haplogroups to rare mitochondrial diseases like Leber's hereditary optic neuropathy or to more common age-linked disorders such as Parkinson's disease. However, cellular, biochemical and molecular-genetic evidence that is able to explain these associations is very scarce. The etiology of multifactorial diseases is very difficult to sort out because such diseases are due to a combination of genetic and environmental factors that individually only contribute in small part to the development of the illness. Thus, the haplogroup-defining mutations might behave as susceptibility factors, but they could have only a small effect on oxidative phosphorylation (OXPHOS) function. Moreover, these effects would be highly dependent on the 'context' in which the genetic variant is acting. To homogenize this 'context' for mitochondrial DNA (mtDNA) mutations, a cellular approach is available that involves the use of what is known as 'cybrids'. By using this model, we demonstrate that mtDNA and mtRNA levels, mitochondrial protein synthesis, cytochrome oxidase activity and amount, normalized oxygen consumption, mitochondrial inner membrane potential and growth capacity are different in cybrids from the haplogroup H when compared with those of the haplogroup Uk. Thus, these inherited basal differences in OXPHOS capacity can help to explain why some individuals more quickly reach the bioenergetic threshold below which tissue symptoms appear and progress toward multifactorial disorders. Hence, some population genetic variants in mtDNA contribute to the genetic component of complex disorders. The existence of mtDNA-based OXPHOS differences opens possibilities for the existence of a new field, mitochondrial pharmacogenomics. New sequence accession nos: HM103354-HM103363.

Mitochondrial DNA depletion syndrome: new descriptions and the use of citrate synthase as a helpful tool to better characterise the patients.

Mitochondrial DNA depletion syndrome (MDS) is a clinically heterogeneous group of mitochondrial disorders characterised by a quantitative reduction of the mitochondrial DNA copy number. Three main clinical forms of MDS: myopathic, encephalomyopathic and hepatocerebral have been defined, although patients may present with other MDS associated clinical symptoms and signs that cover a wide spectrum of onset age and disease. We studied 52 paediatric individuals suspected to have MDS. These patients have been divided into three different groups, and the appropriate MDS genes have been screened according to their clinical and biochemical phenotypes. Mutational study of DGUOK, MPV17, SUCLA2, SUCLG1 and POLG allowed us to identify 3 novel mutations (c.1048G>A and c.1049G>T in SUCLA2 and c.531+4A>T in SUCLG1) and 7 already known mutations in 10 patients (8 families). Seventeen patients presented with mtDNA depletion in liver or muscle, but the cause of mtDNA depletion still remains unknown in 8 of them. When possible, we quantified mtDNA/nDNA and CS activity in the same tissue sample, providing an additional tool for the study of MDS. The ratio (mtDNA/nDNA)/CS has shed some light in the discrepant results between the mtDNA copy number and the enzymatic respiratory chain activities of some cases.

Is population frequency a useful criterion to assign pathogenicity to newly described mitochondrial DNA variants?

Population frequency has been one of the most widely used criteria to help assign pathogenicity to newly described mitochondrial DNA variants. However, after sequencing this molecule in thousands of healthy individuals, it has been observed that a very large number of genetic variants have a very low population frequency, which has raised doubts about the utility of this criterion. By analyzing the genetic variation of mitochondrial DNA-encoded genes for oxidative phosphorylation subunits in 195,983 individuals from HelixMTdb that were not sequenced based on any medical phenotype, we show that rare variants are deleterious and, along with other criteria, population frequency is still a useful criterion to assign pathogenicity to newly described variants.

Development and characterization of cell models harbouring mtDNA deletions for in vitro study of Pearson syndrome.

Pearson syndrome is a rare multisystem disease caused by single large-scale mitochondrial DNA deletions (SLSMDs). The syndrome presents early in infancy and is mainly characterised by refractory sideroblastic anaemia. Prognosis is poor and treatment is supportive, thus the development of new models for the study of Pearson syndrome and new therapy strategies is essential. In this work, we report three different cell models carrying an SLMSD: fibroblasts, transmitochondrial cybrids and induced pluripotent stem cells (iPSCs). All studied models exhibited an aberrant mitochondrial ultrastructure and defective oxidative phosphorylation system function, showing a decrease in different parameters, such as mitochondrial ATP, respiratory complex IV activity and quantity or oxygen consumption. Despite this, iPSCs harbouring 'common deletion' were able to differentiate into three germ layers. Additionally, cybrid clones only showed mitochondrial dysfunction when heteroplasmy level reached 70%. Some differences observed among models may depend on their metabolic profile; therefore, we consider that these three models are useful for the in vitro study of Pearson syndrome, as well as for testing new specific therapies. This article has an associated First Person interview with the first author of the paper.

Multicentric Standardization of Protocols for the Diagnosis of Human Mitochondrial Respiratory Chain Defects.

The quantification of mitochondrial respiratory chain (MRC) enzymatic activities is essential for diagnosis of a wide range of mitochondrial diseases, ranging from inherited defects to secondary dysfunctions. MRC lesion is frequently linked to extended cell damage through the generation of proton leak or oxidative stress, threatening organ viability and patient health. However, the intrinsic challenge of a methodological setup and the high variability in measuring MRC enzymatic activities represents a major obstacle for comparative analysis amongst institutions. To improve experimental and statistical robustness, seven Spanish centers with extensive experience in mitochondrial research and diagnosis joined to standardize common protocols for spectrophotometric MRC enzymatic measurements using minimum amounts of sample. Herein, we present the detailed protocols, reference ranges, tips and troubleshooting methods for experimental and analytical setups in different sample preparations and tissues that will allow an international standardization of common protocols for the diagnosis of MRC defects. Methodological standardization is a crucial step to obtain comparable reference ranges and international standards for laboratory assays to set the path for further diagnosis and research in the field of mitochondrial diseases.

OXPHOS toxicogenomics and Parkinson's disease.

Activities and quantities of several oxidative phosphorylation (OXPHOS) system complexes are decreased in many Parkinson's disease (PD) patients. Mutations in PD-associated nuclear genes affect OXPHOS function. Moreover, the inactivation of other nuclear genes related to mitochondrial DNA (mtDNA) replication and expression also leads to Parkinsonism. MtDNA only encodes OXPHOS subunits and the RNAs required for their expression. Mutations in mtDNA genes have also been associated with PD. Furthermore, many xenobiotics that inhibit different OXPHOS complexes provoke Parkinsonism. The binding sites for these venoms are usually mtDNA-encoded subunits. However, and despite the existence of mutations or toxicants that can cause Parkinsonism, PD only rarely results from isolated genetic or environmental factors. Combinations of nuclear and mitochondrial genetic and environmental factors have additive effects and increase the risk of PD. It is also possible that population polymorphisms in mtDNA genes, affecting interactions with different xenobiotics, may behave as susceptibility factors for developing PD only in the presence of that particular xenobiotic. Therefore, a deeper analysis of the OXPHOS function in PD is required if we want to unravel the complexities of this disorder.

[Pearson syndrome. Case report]. / Síndrome de Pearson. Reporte de un caso.

Among the etiologies of anemia in the infancy, the mitochondrial cytopathies are infrequent. Pearson syndrome is diagnosed principally during the initial stages of life and it is characterized by refractory sideroblastic anemia with vacuolization of marrow progenitor cells, exocrine pancreatic dysfunction and variable neurologic, hepatic, renal and endocrine failures. We report the case of a 14 month-old girl evaluated by a multicentric study, with clinic and molecular diagnosis of Pearson syndrome, with the 4,977-base pair common deletion of mitochondrial DNA. This entity has been associated to diverse phenotypes within the broad clinical spectrum of mitochondrial disease.