A novel NDUFA1 mutation leads to a progressive mitochondrial complex I-specific neurodegenerative disease

Mitochondrial diseases have been shown to result from mutations in mitochondrial genes located in either the nuclear DNA (nDNA) or mitochondrial DNA (mtDNA). Mitochondrial OXPHOS complex I has 45 subunits encoded by 38 nuclear and 7 mitochondrial genes. Two male patients in a putative X-linked pedigree exhibiting a progressive neurodegenerative disorder and a severe muscle complex I enzyme defect were analyzed for mutations in the 38 nDNA and seven mtDNA encoded complex I subunits. The nDNA X-linked NDUFA1 gene (MWFE polypeptide) was discovered to harbor a novel missense mutation which changed a highly conserved glycine at position 32 to an arginine, shown to segregate with the disease. When this mutation was introduced into a NDUFA1 null hamster cell line, a substantial decrease in the complex I assembly and activity was observed. When the mtDNA of the patient was analyzed, potentially relevant missense mutations were observed in the complex I genes. Transmitochondrial cybrids containing the patient’s mtDNA resulted in a mild complex I deficiency. Interestingly enough, the nDNA encoded MWFE polypeptide has been shown to interact with various mtDNA encoded complex I subunits. Therefore, we hypothesize that the novel G32R mutation in NDUFA1 is causing complex I deficiency either by itself or in synergy with additional mtDNA variants.     

The pathophysiology of mitochondrial disease as modeled in the mouse

It is now clear that mitochondrial defects are associated with a plethora of clinical phenotypes in man and mouse. This is the result of the mitochondria''s central role in energy production, reactive oxygen species (ROS) biology, and apoptosis, and because the mitochondrial genome consists of roughly 1500 genes distributed across the maternal mitochondrial DNA (mtDNA) and the Mendelian nuclear DNA (nDNA). While numerous pathogenic mutations in both mtDNA and nDNA mitochondrial genes have been identified in the past 21 years, the causal role of mitochondrial dysfunction in the common metabolic and degenerative diseases, cancer, and aging is still debated. However, the development of mice harboring mitochondrial gene mutations is permitting demonstration of the direct cause-and-effect relationship between mitochondrial dysfunction and disease. Mutations in nDNA-encoded mitochondrial genes involved in energy metabolism, antioxidant defenses, apoptosis via the mitochondrial permeability transition pore (mtPTP), mitochondrial fusion, and mtDNA biogenesis have already demonstrated the phenotypic importance of mitochondrial defects. These studies are being expanded by the recent development of procedures for introducing mtDNA mutations into the mouse. These studies are providing direct proof that mtDNA mutations are sufficient by themselves to generate major clinical phenotypes. As more different mtDNA types and mtDNA gene mutations are introduced into various mouse nDNA backgrounds, the potential functional role of mtDNA variation in permitting humans and mammals to adapt to different environments and in determining their predisposition to a wide array of diseases should be definitively demonstrated.     

Diseases caused by nuclear genes affecting mtDNA stability

Diseases caused by nuclear genes that affect mitochondrial DNA (mtDNA) stability are an interesting group of mitochondrial disorders, involving both cellular genomes. In these disorders, a primary nuclear gene defect causes secondary mtDNA loss or deletion formation, which leads to tissue dysfunction. Therefore, the diseases clinically resemble those caused by mtDNA mutations, but follow a Mendelian inheritance pattern. Several clinical entities associated with multiple mtDNA deletions have been characterized, the most frequently described being autosomal dominant progressive external ophthalmoplegia (adPEO). MtDNA depletion syndrome (MDS) is a severe disease of childhood, in which tissue-specific loss of mtDNA is seen. Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) patients may have multiple mtDNA deletions and/or mtDNA depletion. Recent reports of thymidine phosphorylase mutations in MNGIE and adenine nucleotide translocator mutations in adPEO have given new insights into the mechanisms of mtDNA maintenance in mammals. The common mechanism underlying both of these gene defects could be disturbed mitochondrial nucleoside pools, the building blocks of mtDNA. Future studies on MNGIE and adPEO pathogenesis, and identification of additional gene defects in adPEO and MDS will provide further understanding about the mammalian mtDNA maintenance and the crosstalk between the nuclear and mitochondrial genomes.     

Mitochondrial DNA: Epigenetics and environment

Maintenance of the mitochondrial genome is essential for proper cellular function. For this purpose, mitochondrial DNA (mtDNA) needs to be faithfully replicated, transcribed, translated, and repaired in the face of constant onslaught from endogenous and environmental agents. Although only 13 polypeptides are encoded within mtDNA, the mitochondrial proteome comprises over 1500 proteins that are encoded by nuclear genes and translocated to the mitochondria for the purpose of maintaining mitochondrial function. Regulation of mtDNA and mitochondrial proteins by epigenetic changes and post-translational modifications facilitate crosstalk between the nucleus and the mitochondria and ultimately lead to the maintenance of cellular health and homeostasis. DNA methyl transferases have been identified in the mitochondria implicating that methylation occurs within this organelle; however, the extent to which mtDNA is methylated has been debated for many years. Mechanisms of demethylation within this organelle have also been postulated, but the exact mechanisms and their outcomes is still an active area of research. Mitochondrial dysfunction in the form of altered gene expression and ATP production, resulting from epigenetic changes, can lead to various conditions including aging-related neurodegenerative disorders, altered metabolism, changes in circadian rhythm, and cancer. Here, we provide an overview of the epigenetic regulation of mtDNA via methylation, long and short noncoding RNAs, and post-translational modifications of nucleoid proteins (as mitochondria lack histones). We also highlight the influence of xenobiotics such as airborne environmental pollutants, contamination from heavy metals, and therapeutic drugs on mtDNA methylation. Environ. Mol. Mutagen., 60:668–682, 2019. © 2019 Wiley Periodicals, Inc.     

A national perspective on prenatal testing for mitochondrial disease

Mitochondrial diseases affect >1 in 7500 live births and may be due to mutations in either mitochondrial DNA (mtDNA) or nuclear DNA (nDNA). Genetic counselling for families with mitochondrial diseases, especially those due to mtDNA mutations, provides unique and difficult challenges particularly in relation to disease transmission and prevention. We have experienced an increasing demand for prenatal diagnostic testing from families affected by mitochondrial disease since we first offered this service in 2007. We review the diagnostic records of the 62 prenatal samples (17 mtDNA and 45 nDNA) analysed since 2007, the reasons for testing, mutation investigated and the clinical outcome. Our findings indicate that prenatal testing for mitochondrial disease is reliable and informative for the nuclear and selected mtDNA mutations we have tested. Where available, the results of mtDNA heteroplasmy analyses from other family members are helpful in interpreting the prenatal mtDNA test result. This is particularly important when the mutation is rare or the mtDNA heteroplasmy is observed at intermediate levels. At least 11 cases of mitochondrial disease were prevented following prenatal testing, 3 of which were mtDNA disease. On the basis of our results, we believe that prenatal testing for mitochondrial disease is an important option for couples where appropriate genetic analyses and pre/post-test counselling can be provided.     

Morphological Findings of Extraocular Myopathy with Chronic Progressive External Ophthalmoplegia

Mitochondrial diseases are a large group of disorders resulting from mutations of nuclear DNA (nDNA) and mitochondrial DNA (mtDNA). Patients present clinically with multiple manifestations, including myopathies and multiple system disorders. Establishing a specific diagnosis often requires extensive clinical and laboratory evaluation. In this study of 2 adult patients with presumptive mitochondrial disease, the authors have identified distinctive morphological changes in medial rectus muscle biopsies that confirm the diagnosis of chronic progressive external ophthalmoplegia (CPEO). These findings demonstrate the usefulness of electron microscopy using medial rectus muscle in the diagnosis of adult patients with a slowly progressive course of mild skeletal weakness and CPEO.     

Sequence analysis of cDNAs for the human and bovine ATP synthase β subunit: mitochondrial DNA genes sustain seventeen times more mutations

We have cloned and sequenced human and bovine cDNAs for the subunit of the ATP synthase (ATP-synß), a nuclear DNA (nDNA) encoded oxidative phosphorylation (OXPHOS) gene. The two cDNAs were found to share 99% amino acid homology and 94% nucleotide homology. The evolutionary rate of ATPsynß was then compared with that of two mitochondrial DNA (mtDNA) ATP synthase genes (ATPase 6 and 8), seven other mtDNA OXPHOS genes, and a number of nuclear genes. The synonymous substitution rate for ATPsynß proved to be 1.9 × 10^–9 substitutions per site per year (substitutions × site^–1 × year^–1) (SSY). This is less than 1/2 that of the average nDNA gene, 1/12 the rate of ATPase 6 and 8, and 1/17 the rate of the average mtDNA gene. The synonymous and replacement substitution rates were used to calculate a new parameter, the selective constraint ratio. This revealed that even the most variable mtDNA protein was more constrained than the average nDNA protein. Thus, the high substitution mutation rate and strong selective constraints of mammalian mtDNA proteins suggest that mtDNA mutations may result in a disproportionately large number of human hereditary diseases of OXPHOS.     

Neuropathologic aspects of cytochrome C oxidase deficiency

Cytochrome c oxidase (COX) deficiency is an important cause of myopathy or encephalomyopathy. Considering the structural complexity of COX, its dual genetic control, and the several nuclear genes needed for its proper assembly, the phenotypic heterogeneity is not surprising. From a morphologic view point, the application of histochemistry and immunohistochemistry to the study of COX deficiency in muscle has revealed specific patterns that -we believe- are helpful both for diagnosis and for directing sequencing studies of either mitochondrial DNA (mtDNA) or nuclear DNA (nDNA) genes. Similar studies in brain have shown that patients with mutations in mtDNA appear to have different patterns of COX deficiency from patients with mutations in nDNA genes. The recent discovery of mutations in COX assembly genes coupled with the potential to generate knock-out mice with these mutations holds the promise of providing the neuropathologist with the animal models needed to study the pathogenesis of COX deficiency in brain and muscle.     

Depletion of the other genome-mitochondrial DNA depletion syndromes in humans

We present the current knowledge on the genetic and phenotypic aspects of mitochondrial DNA depletion syndromes. The human mitochondrial DNA encodes 13 of the 82 structural proteins of the mitochondrial electron transport chain. The replication and maintenance of the mtDNA require a large number of nuclear encoded enzymes and balanced nucleotide pools. Mitochondrial nucleotide synthesis is of major importance because of the constant need for nucleotides for mtDNA maintenance even in quiescent cells. As de novo enzymes are not present in the mitochondria, synthesis is accomplished via the salvage pathway. Defective mtDNA synthesis and maintenance manifest by multiple deletions or by depletion of the mitochondrial genome. Patients with multiple deletions typically present with progressive external ophthalmoplegia, ptosis and, exercise intolerance after the first decade of life. mtDNA depletion is usually an infantile disease characterized by severe muscle weakness, hepatic failure, or renal tubulopathy with fatal outcome. Linkage analysis in families with multiple mtDNA deletions reveal mutations in proteins that participate in mtDNA replication, the mitochondrial DNA polymerase gene, and the Twinkle gene, a putative mitochondrial helicase and in factors which play a role in mitochondrial nucleotide metabolism, the adenine nucleotide translocator, and the thymidine phosphorylase gene. We have recently identified mutations in an additional two essential proteins in the nucleotide salvage pathway, the mitochondrial deoxyribonucleoside kinases. The phenotype was distinctive for each gene, with hepatic failure and encephalopathy associated with mutations in the deoxyguanosine kinase gene and isolated devastating myopathy as the sole manifestation of thymidine kinase 2 deficiency. The tissue selectivity of these disorders and especially the exclusive muscle involvement in thymidine kinase 2 mutations is puzzling. The normal sequence of the remaining mtDNA copies in spite of a serious mitochondrial nucleotide imbalance is also unexpected. We propose several tissue-specific protective mechanisms and a time window, likely encompassing fetal life and even early infancy, during which nuclear nucleotide synthesis provides mitochondrial needs in all organs. We also speculate on future genes to be discovered in other phenotypes of mtDNA depletion.     

Respiratory chain complex I deficiency caused by mitochondrial DNA mutations

Defects of the mitochondrial respiratory chain are associated with a diverse spectrum of clinical phenotypes, and may be caused by mutations in either the nuclear or the mitochondrial genome (mitochondrial DNA (mtDNA)). Isolated complex I deficiency is the most common enzyme defect in mitochondrial disorders, particularly in children in whom family history is often consistent with sporadic or autosomal recessive inheritance, implicating a nuclear genetic cause. In contrast, although a number of recurrent, pathogenic mtDNA mutations have been described, historically, these have been perceived as rare causes of paediatric complex I deficiency. We reviewed the clinical and genetic findings in a large cohort of 109 paediatric patients with isolated complex I deficiency from 101 families. Pathogenic mtDNA mutations were found in 29 of 101 probands (29%), 21 in MTND subunit genes and 8 in mtDNA tRNA genes. Nuclear gene defects were inferred in 38 of 101 (38%) probands based on cell hybrid studies, mtDNA sequencing or mutation analysis (nuclear gene mutations were identified in 22 probands). Leigh or Leigh-like disease was the most common clinical presentation in both mtDNA and nuclear genetic defects. The median age at onset was higher in mtDNA patients (12 months) than in patients with a nuclear gene defect (3 months). However, considerable overlap existed, with onset varying from 0 to >60 months in both groups. Our findings confirm that pathogenic mtDNA mutations are a significant cause of complex I deficiency in children. In the absence of parental consanguinity, we recommend whole mitochondrial genome sequencing as a key approach to elucidate the underlying molecular genetic abnormality.     

The pathophysiology of mitochondrial biogenesis: towards four decades of mitochondrial DNA research

Mitochondria are with very few exceptions ubiquitous organelles in eukaryotic cells where they are essential for cell life and death. Mitochondria play a central role not only in a variety of metabolic pathways including the supply of the bulk of cellular ATP through oxidative phosphorylation (OXPHOS), but also in complex processes such as development, apoptosis, and aging. Mitochondria contain their own genome that is replicated and expressed within the organelle. It encodes 13 polypeptides all of them components of the OXPHOS system, and thus, the integrity of the mitochondrial DNA (mtDNA) is critical for cellular energy supply. In the past 12 years more than 50 point mutations and around 100 rearrangements in the mtDNA have been associated with human diseases. Also in recent years, several mutations in nuclear genes that encode structural or regulatory factors of the OXPHOS system or the mtDNA metabolism have been described. The development of increasingly powerful techniques and the use of cellular and animal models are opening new avenues in the study of mitochondrial medicine. The detailed molecular characterization of the effects produced by different mutations that cause mitochondrial cytopathies will be critical for designing rational therapeutic strategies for this group of devastating diseases.     

Mitochondria and degenerative disorders

In mammalian cells, mitochondria provide energy from aerobic metabolism. They play an important regulatory role in apoptosis, produce and detoxify free radicals, and serve as a cellular calcium buffer. Neurodegenerative disorders involving mitochondria can be divided into those caused by oxidative phosphorylation (OXPHOS) abnormalities either due to mitochondrial DNA (mtDNA) abnormalities, e.g., chronic external ophthalmoplegia, or due to nuclear mutations of OXPHOS proteins, e.g., complex I and II associated with Leigh syndrome. There are diseases caused by nuclear genes encoding non-OXPHOS mitochondrial proteins, such as frataxin in Friedreich ataxia (which is likely to play an important role in mitochondrial-cytosolic iron cycling), paraplegin (possibly a mitochondrial ATP-dependent zinc metalloprotease of the AAA-ATPases in hereditary spastic paraparesis), and possibly Wilson disease protein (an abnormal copper transporting ATP-dependent P-type ATPase associated with Wilson disease). Huntingon disease is an example of diseases with OXPHOS defects associated with mutations of nuclear genes encoding non-mitochondrial proteins such as huntingtin. There are also disorders with evidence of mitochondrial involvement that cannot as yet be assigned. These include Parkinson disease (where a complex I defect is described and free radicals are generated from dopamine metabolism), amyotrophic lateral sclerosis, and Alzheimer disease, where there is evidence to suggest mitochondrial involvement perhaps secondary to other abnormalities.     

Mitochondrial DNA mutations in human disease

The small, maternally inherited mitochondrial DNA (mtDNA) has turned out to be a Pandora's box of pathogenic mutations: 13 years into the era of "molecular mitochondrial medicine," more than 100 pathogenic point mutations and innumerable rearrangements have been associated with a striking variety of multisystemic as well as tissue-specific human diseases. After reviewing the principles of mitochondrial genetics, we consider disorders due to mutations in genes affecting mitochondrial protein synthesis and disorders due to mutations in protein-coding genes. In contrast to the remarkable progress in our understanding of etiology, pathogenesis is only partially explained by the rules of mitochondrial genetics and remains largely unclear. We review recent progress in prenatal diagnosis, epidemiology, and in the development of animal models harboring mtDNA mutations.     

Direct linkage of mitochondrial genome variation to risk factors for type 2 diabetes in conplastic strains

Recently, the relationship of mitochondrial DNA (mtDNA) variants to metabolic risk factors for diabetes and other common diseases has begun to attract increasing attention. However, progress in this area has been limited because (1) the phenotypic effects of variation in the mitochondrial genome are difficult to isolate owing to confounding variation in the nuclear genome, imprinting phenomena, and environmental factors; and (2) few animal models have been available for directly investigating the effects of mtDNA variants on complex metabolic phenotypes in vivo. Substitution of different mitochondrial genomes on the same nuclear genetic background in conplastic strains provides a way to unambiguously isolate effects of the mitochondrial genome on complex traits. Here we show that conplastic strains of rats with identical nuclear genomes but divergent mitochondrial genomes that encode amino acid differences in proteins of oxidative phosphorylation exhibit differences in major metabolic risk factors for type 2 diabetes. These results (1) provide the first direct evidence linking naturally occurring variation in the mitochondrial genome, independent of variation in the nuclear genome and other confounding factors, to inherited variation in known risk factors for type 2 diabetes; and (2) establish that spontaneous variation in the mitochondrial genome per se can promote systemic metabolic disturbances relevant to the pathogenesis of common diseases.     

Mitochondrial genotype associated with longevity and its inhibitory effect on mutagenesis

Mitochondria are not only the major site of ATP production in cells but also an important source of reactive oxygen species (ROS) under certain pathological conditions. Because mitochondrial DNA (mtDNA) in the mitochondrial matrix is exposed to ROS that leak from the respiratory chain, this extranuclear genome is prone to mutations. Therefore, the mitochondrial genome is a rich source of single nucleotide polymorphisms (SNPs) and the functional significance of SNPs in the mitochondrial genome is comparable to that of SNPs in the entire nuclear genome. To demonstrate the contribution of mitochondrial SNPs to the susceptibility to adult-onset diseases, we analyzed the mtDNA from Japanese centenarians and identified a longevity-associated mitochondrial genotype, Mt5178A. Because this genotype was demonstrated to suppress the occurrence of mtDNA mutations in the oocytes, it also would seem to decelerate the accumulation of mtDNA mutations in the somatic cells with increasing age. This genotype is likely to confer resistance to adult-onset diseases by suppressing obesity and atherosclerosis.     

Mitochondrial diseases--an expanding spectrum of disorders and affected genes

Mitochondrial diseases are a heterogeneous group of disorders caused by the impairment of the mitochondrial oxidative phosphorylation system which have been associated with various mutations of the mitochondrial DNA (mtDNA) and nuclear gene mutations. The clinical phenotypes are very diverse and the spectrum is still expanding. This review gives an overview of the principal clinical phenotypes and the molecular genetic basis of mitochondrial disorders identified so far.     

Clinical mitochondrial genetics

The last decade has been an age of enlightenment as far as mitochondrial pathology is concerned. Well established nuclear genetic diseases, such as Friedreich's ataxia,¹² Wilson disease,³ and autosomal recessive hereditary spastic paraplegia,⁴ have been shown to have a mitochondrial basis, and we are just starting to unravel the complex nuclear genetic disorders which directly cause mitochondrial dysfunction (table 1). However, in addition to the 3 billion base pair nuclear genome, each human cell typically contains thousands of copies of a small, 16.5 kb circular molecule of double stranded DNA (fig 1). Mitochondrial DNA (mtDNA) accounts for only 1% of the total cellular nucleic acid content. It encodes for 13 polypeptides which are essential for aerobic metabolism and defects of the mitochondrial genome are an important cause of human disease.⁹²⁹³ Since the characterisation of the first pathogenic mtDNA defects in 1988,⁵¹³ over 50 point mutations and well over 100 rearrangements of the mitochondrial genome have been associated with human disease⁹⁴⁹⁵ (http://www.gen.emory.edu/mitomap.html). These disorders form the focus of this article.


Keywords: mitochondrial DNA; mitochondrial disease; heteroplasmy; genetic counselling