Mitochondrial diseases are clinically and genetically heterogeneous. These diseases were initially described a little over three decades ago. Limited diagnostic tools created disease descriptions based on clinical, biochemical analytes, neuroimaging, and muscle biopsy findings. This diagnostic mechanism continued to evolve detection of inherited oxidative phosphorylation disorders and expanded discovery of mitochondrial physiology over the next two decades. Limited genetic testing hampered the definitive diagnostic identification and breadth of diseases. Over the last decade, the development and incorporation of massive parallel sequencing has identified approximately 300 genes involved in mitochondrial disease. Gene testing has enlarged our understanding of how genetic defects lead to cellular dysfunction and disease. These findings have expanded the understanding of how mechanisms of mitochondrial physiology can induce dysfunction and disease, but the complete collection of disease-causing gene variants remains incomplete. This article reviews the developments in disease gene discovery and the incorporation of gene findings with mitochondrial physiology. This understanding is critical to the development of targeted therapies.

Riboflavin, known as vitamin B2, a water-soluble vitamin, is an essential nutrient in vertebrates, hence adequate dietary intake is imperative. Riboflavin plays a role in a variety of metabolic pathways, serving primarily as an integral component of its crucial biologically active forms, the flavocoenzymes flavin adenine dinucleotide and flavin mononucleotide. These flavocoenzymes ensure the functionality of numerous flavoproteins including dehydrogenases, oxidases, monooxygenases, and reductases, which play pivotal roles in mitochondrial electron transport chain, β-oxidation of fatty acids, redox homeostasis, citric acid cycle, branched-chain amino acid catabolism, chromatin remodeling, DNA repair, protein folding, and apoptosis. Unsurprisingly, impairment of flavin homeostasis in humans has been linked to various diseases including neuromuscular and neurological disorders, abnormal fetal development, and cardiovascular diseases. This review presents an overview of riboflavin metabolism, its role in mitochondrial function, primary and secondary flavocoenzyme defects associated with mitochondrial dysfunction, and the role of riboflavin supplementation in these conditions.

Mitochondrial disorders (MD) include a large group of maternally inherited, autosomal dominant, or recessive genetic syndromes caused by mitochondrial dysfunction. MD can be diagnosed at any age and many of them show a multisystem presentation with variable combinations of symptoms. Given the important role of mitochondria in neuronal homeostasis, neurological manifestations, including movement disorders, can accompany MD. Movement disorders (MoD), either hypo- or hyperkinetic type, are reported in MD, but the real incidence and a detailed characterization of these features are not addressed in population-based studies. Dystonia, usually in the context of Leigh syndrome, is the main extrapyramidal movement disorder in pediatric MD patients; whereas parkinsonism is the most prevalent hypokinetic disorder in adult MD patients. Ataxia is a common feature in MD, in both the pediatric and adult MD populations. Other MoD, such as myoclonus, chorea, or tremor, may also occur in MD. MoD manifest more frequently in the context of a complex phenotype but rarely can be isolated. From a genetic point of view, MoD are described in patients with either mutations in mtDNA or in nuclear genes related to mitochondria, and the same gene can be associated with different types of MoD. Recent studies demonstrate that the dopaminergic nigrostriatal system is very vulnerable to mitochondrial dysfunction and defects of mtDNA maintenance are frequently associated with a nigrostriatal degeneration, which may explain the pathophysiological mechanism. Therapeutic interventions for MoD in MD do not differ from treatment options used for MoD with different etiopathological background. Some forms benefit from specific treatments, e.g., primary Coenzyme Q10 deficiencies. Newer therapeutic strategies have been pursued which act on different mechanisms of mitochondrial dysfunction, but clinical trials are warranted to improve the management of MD patients.

Mitochondrial diseases are multi-systemic, heterogeneous groups of diseases that are associated with various neuromuscular problems, cardiovascular disorders, metabolic syndrome, cancer, and obesity. Mitochondrial diseases are due to mutations in mitochondrial DNA or nuclear DNA that can affect the assembly of the mitochondrial components and mitochondrial function. Typically, mitochondrial diseases can be inherited through an autosomal dominant, autosomal recessive or X-linked pattern of inheritance. To date, there are more than 100 mitochondrial diseases identified. However, clinical phenotype heterogeneity is a huge problem for the diagnosis of mitochondrial diseases, as patients with the same mutations exhibit different clinical symptoms. Also, the heteroplasmy/homoplasmy conditions complicate the diagnosis process. Here, in this review, we discuss these challenges and problems in mitochondrial disease diagnosis, focusing on the mutational profile of both primary and secondary mitochondrial diseases. We also review the utilization of next-generation technology and multi-omics strategy to improve the diagnosis. The discussion addresses the current evidence of those applications and the challenges that need some improvement for better diagnosis yield.

Mitochondrial diseases collectively represent the most common cause of inherited metabolic disease. They are estimated to affect at least 1 in 8,000 adults and at least 1 in 250 adults carry a disease-causing genetic mutation. They comprise a heterogeneous group of disorders caused by mutations in either the nuclear or mitochondrial genome, which ultimately result in dysfunction of the critical cellular energy producing mitochondrial respiratory chain. Owing to the key role of mitochondria in energy production, mitochondrial disorders predominantly manifest in tissues with high metabolic demand. However, they demonstrate significant phenotypic and genotypic variability, often rendering the diagnostic process protracted and challenging. Since Luft’s first description of mitochondrial disease nearly 60 years ago, substantial evolution in diagnostic techniques have simultaneously improved the diagnosis and understanding of mitochondrial disease and biology, but the standard diagnostic approach has failed to evolve at the same pace. Although sequencing technologies and analysis for the diagnosis of mitochondrial disease continue to evolve, advances to date, our expanding understanding of mitochondrial diseases and the increasing affordability of these new technologies justify a paradigm shift in the diagnostic approach. We review the progression, impact and challenges of diagnosing mitochondrial diseases and propose a minimally invasive “genetics first” approach incorporating stratification using non-invasive biomarkers, followed by non-targeted next-generation sequencing, such as whole genome sequencing. Such an approach may improve diagnostic yield and streamline diagnosis, leaving invasive investigations to address diagnostic challenges and functional validation of novel variants.

In eukaryotic cells, mitochondria perform the essential function of producing cellular energy in the form of ATP via the oxidative phosphorylation system. This system is composed of 5 multimeric protein complexes of which 13 protein subunits are encoded by the mitochondrial genome: Complex I (7 subunits), Complex III (1 subunit), Complex IV (3 subunits), and Complex V (2 subunits). Effective mitochondrial translation is necessary to produce the protein subunits encoded by the mitochondrial genome (mtDNA). Defects in mitochondrial translation are known to cause a wide variety of clinical disease in humans with high-energy consuming organs generally most prominently affected. Here, we review several classes of disease resulting from defective mitochondrial translation including disorders with mitochondrial tRNA mutations, mitochondrial aminoacyl-tRNA synthetase disorders, mitochondrial rRNA mutations, and mitochondrial ribosomal protein disorders.

Aim: The North American Mitochondrial Disease Consortium (NAMDC) comprises a network of 17 clinical centers with a mission to conduct translational research on mitochondrial diseases. NAMDC is a part of the Rare Disease Clinical Research Network (RDCRN) and is funded by the National Institutes of Health. To foster its mission, NAMDC has implemented a comprehensive Mitochondrial Disease Clinical Registry (hereafter NAMDC Registry), collected biosamples deposited into the NAMDC Biorepository, defined phenotypes and genotypes of specific disorders, collected natural history data, identified outcome measures, characterized safety and long-term toxicity and efficacy of promising therapies, and trained young investigators interested in patient-oriented research in mitochondrial disease.

Methods: Research conducted by NAMDC is built on the foundation of the Clinical Registry. Data within the registry are encrypted and maintained in a centralized database at Columbia University Medical Center. In addition to clinical data, NAMDC has established a mitochondrial disease biorepository, collecting DNA, plasma, cell, and tissue samples. Specimens are assigned coded identifiers in compliance with all relevant regulatory entities and with emerging NIH guidelines for biorepositories. NAMDC funds two pilot projects each year. Pilot grants are small grants typically supporting an early stage concept to obtain preliminary data. Pilot grants must enhance and address major issues in mitochondrial medicine and specific areas of need for the field and for the successful outcome of NAMDC. The grant selection process is facilitated by input from multiple stakeholders including patient organizations and the strategic leadership of NAMDC. To train new mitochondrial disease investigators, NAMDC has established a Fellowship Program which offers a unique training opportunity to senior postdoctoral clinical fellows. The fellowship includes a 6-month period of intensive training in clinical trial methodology through the Clinical Research Enhancement through Supplemental Training program and equivalent programs at the other sites, along with rotations up to 3 months each to two additional consortium sites where a rich and varied training experience is provided. Nine core educational sites participate in this training program, each offering a summer grant program in mitochondrial medicine funded by our NAMDC partner the United Mitochondrial Disease Foundation (www.umdf.org). All clinical research in NAMDC depends on the participation of mitochondrial disease patients. Since individual mitochondrial disorders are often extremely rare, major communication and recruitment efforts are required. Therefore, NAMDC has forged a very close partnership with the premier patient advocacy group for mitochondrial diseases in North America, the United Mitochondrial Disease Foundation (UMDF).

Results: The NAMDC Registry has confirmed the clinical and genetical heterogeneity of mitochondrial diseases due to primary mutations in mitochondrial DNA or nuclear DNA. During the 8 years of this NIH-U54 grant, this consortium, acting in close collaboration with a patient advocacy group, the UMDF, has effectively addressed these complex diseases. NAMDC has expanded a powerful patient registry with more than 1600 patients enrolled to date, a website for education and recruitment of patients (www.namdc.org), a NAMDC biorepository housed at the Mayo Clinic in Rochester, MN, and essential diagnostic guidelines for consensus research. In addition, eight clinical studies have been initiated and the NAMDC fellowship program has been actively training the next generation of mitochondrial disease clinical investigators, of which six have completed the program and remain actively involved in mitochondrial disease research.

Conclusion: The NAMDC Patient Registry and Biorepository is actively facilitating mitochondrial disease research, and accelerating progress in the understanding and treatment of mitochondrial diseases.

Primary coenzyme Q₁₀ (CoQ₁₀) deficiency encompasses a subset of mitochondrial diseases caused by mutations affecting proteins involved in the CoQ₁₀ biosynthetic pathway. One of the most frequent clinical syndromes associated with primary CoQ₁₀ deficiency is the severe infantile multisystemic form, which, until recently, was underdiagnosed. In the last few years, the availability of genetic screening through whole exome sequencing and whole genome sequencing has enabled molecular diagnosis in a growing number of patients with this syndrome and has revealed new disease phenotypes and molecular defects in CoQ₁₀ biosynthetic pathway genes. Early genetic screening can rapidly and non-invasively diagnose primary CoQ₁₀ deficiencies. Early diagnosis is particularly important in cases of CoQ₁₀ deficient steroid-resistant nephrotic syndrome, which frequently improves with treatment. In contrast, the infantile multisystemic forms of CoQ₁₀ deficiency, particularly when manifesting with encephalopathy, present therapeutic challenges, due to poor responses to CoQ₁₀ supplementation. Administration of CoQ₁₀ biosynthetic intermediate compounds is a promising alternative to CoQ₁₀; however, further pre-clinical studies are needed to establish their safety and efficacy, as well as to elucidate the mechanism of actions of the intermediates. Here, we review the molecular defects causes of the multisystemic infantile phenotype of primary CoQ₁₀ deficiency, genotype-phenotype correlations, and recent therapeutic advances.

Electron paramagnetic resonance spectroscopy (EPR) is an analytical technique that, uniquely, can be used to directly interrogate flash-frozen tissue. Quantitative information on the thermodynamic potential of the mitochondrion to synthesize ATP, and the extent of reactive oxygen species-mediated oxidative stress on the mitochondrion and the cell at large, can be obtained. A compromised ability to synthesize ATP and oxidative stress are two of the characteristic sequelae of mitochondrial disease and therapeutic approaches may differ widely depending on which of these is dominant. EPR, therefore, has a role to play in the characterization, diagnosis, and ongoing evaluation of therapies for mitochondrial disease in human patients and model systems. An introduction to mitochondrial disease is followed by a description of EPR, a summary of the EPR signals that can be expected from tissue samples, sample preparation and analytical methods, and a case study in which EPR and complementary techniques were employed on a rat model to study human mitochondrial disease.

Mitochondrial neurogastrointestinal encephalomyopathy (MNGIE) is an ultra-rare disease caused by mutations in TYMP, the gene encoding for the enzyme thymidine phosphorylase. The resulting enzyme deficiency leads to a systemic accumulation of thymidine and 2’-deoxyuridine and ultimately mitochondrial failure due to a progressive acquisition of secondary mitochondrial DNA (mtDNA) mutations and mtDNA depletion. MNGIE is characterised by gastrointestinal dysmotility, cachexia, peripheral neuropathy, ophthalmoplegia, ptosis and leukoencephalopathy. The disease is progressively degenerative and leads to death at an average age of 37.6 years. Patients invariably encounter misdiagnoses, diagnostic delays, and non-specific clinical management. Despite its rarity, MNGIE has invoked much interest in the development of therapeutic strategies, mainly because it is one of the few mitochondrial disorders where the molecular abnormality is metabolically and physically accessible to manipulation. This review provides a résumé of the current diagnosis and treatment approaches and aims to increase the clinical awareness of MNGIE and thereby facilitate early diagnosis and timely access to treatments, before the development of untreatable and irreversible organ damage.