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Abstract Neuroferritinopathy is a disorder of neurodegeneration with brain iron accumulation that has no proven disease‐modifying treatments. Clinical trials require biomarkers of iron deposition. We examined brain iron accumulation in one presymptomatic FTL mutation carrier, two individuals with neuroferritinopathy and one healthy control using ultra‐high‐field 7T MRI. There was increased magnetic susceptibility, suggestive of iron deposition, in superficial and deep gray matter in both presymptomatic and symptomatic neuroferritinopathy. Cavitation of the putamen and globus pallidus increased with disease stage and at follow up. The widespread brain iron deposition in presymptomatic and early disease provides an opportunity for monitoring disease‐modifying intervention.

This article has been retracted. Please see the Retraction Notice for more detail: https://doi.org/10.1186/s13063-021-05965-4.

Abstract To understand neurological complications of COVID-19 better both acutely and for recovery, we measured markers of brain injury, inflammatory mediators, and autoantibodies in 203 hospitalised participants; 111 with acute sera (1–11 days post-admission) and 92 convalescent sera (56 with COVID-19-associated neurological diagnoses). Here we show that compared to 60 uninfected controls, tTau, GFAP, NfL, and UCH-L1 are increased with COVID-19 infection at acute timepoints and NfL and GFAP are significantly higher in participants with neurological complications. Inflammatory mediators (IL-6, IL-12p40, HGF, M-CSF, CCL2, and IL-1RA) are associated with both altered consciousness and markers of brain injury. Autoantibodies are more common in COVID-19 than controls and some (including against MYL7, UCH-L1, and GRIN3B) are more frequent with altered consciousness. Additionally, convalescent participants with neurological complications show elevated GFAP and NfL, unrelated to attenuated systemic inflammatory mediators and to autoantibody responses. Overall, neurological complications of COVID-19 are associated with evidence of neuroglial injury in both acute and late disease and these correlate with dysregulated innate and adaptive immune responses acutely.

BackgroundLeber Hereditary Optic Neuropathy (LHON) is the most common inherited mitochondrial disease characterized by bilateral, painless, subacute visual loss with a peak age of onset in the second to third decade. Historically, LHON was thought to be exclusively maternally inherited due to mutations in mitochondrial DNA (mtDNA); however, recent studies have identified an autosomal recessive form of LHON (arLHON) caused by point mutations in the nuclear gene, DNAJC30.Case PresentationsIn this study, we report the cases of three Eastern European individuals presenting with bilateral painless visual loss, one of whom was also exhibiting motor symptoms. After a several-year-long diagnostic journey, all three patients were found to carry the homozygous c.152A>G (p.Tyr51Cys) mutation in DNAJC30. This has been identified as the most common arLHON pathogenic variant and has been shown to exhibit a significant founder effect amongst Eastern European individuals.ConclusionThis finding adds to the growing cohort of patients with arLHON and demonstrates the importance of DNAJC30 screening in patients with molecularly undiagnosed LHON, particularly in Eastern European individuals. It is of heightened translational significance as patients diagnosed with arLHON exhibit a better prognosis and response to therapeutic treatment with the co-enzyme Q10 analog idebenone.

Traumatic brain injury and aneurysmal subarachnoid haemorrhage are a major cause of morbidity and mortality worldwide. Treatment options remain limited and are hampered by our understanding of the cellular and molecular mechanisms, including the inflammatory response observed in the brain. Mitochondrial DNA (mtDNA) has been shown to activate an innate inflammatory response by acting as a damage-associated molecular pattern (DAMP). Here, we show raised circulating cell-free (ccf) mtDNA levels in both cerebrospinal fluid (CSF) and serum within 48?h of brain injury. CSF ccf-mtDNA levels correlated with clinical severity and the interleukin-6 cytokine response. These findings support the use of ccf-mtDNA as a biomarker after acute brain injury linked to the inflammatory disease mechanism.

Abstract Background The COVID-19 pandemic has presented unique challenges for rapidly designing, initiating, and delivering therapeutic clinical trials. PRINCIPLE (Platform Randomised Trial of Treatments in the Community for Epidemic and Pandemic Illnesses) is the UK national platform investigating repurposed therapies for COVID-19 treatment of older people in the community at high risk of complications. Standard methods of patient recruitment were failing to meet the required pace and scale of enrolment. This paper describes the development and appraisal of a near real-time, data-driven, ethical approach for enhancing recruitment in community care by contacting people with a recent COVID-19 positive test result from the central NHS Test and Trace service within approximately 24–48 h of their test result. Methods A multi-disciplinary team was formed to solve the technical, ethical, public perception, logistical and information governance issues required to provide a near-real time (approximately within 24–48 h of receiving a positive test) feed of potential trial participants from test result data to the research team. PRINCIPLE was also given unique access to the Summary Care Record (SCR) to ensure safe prescribing, and to enable the trial team to quickly and safely bring consented patients into the trial. A survey of the public was used to understand public perceptions of the use of test data for this proposed methodology. Results Prior to establishing the data service, PRINCIPLE registered on average 87 participants per week. This increased by up to 87 additional people registered per week from the test data, contributing to an increase from 1013 recruits to PRINCIPLE at the start of October 2020 to 2802 recruits by 20 December 2020. Whilst procedural caveats were identified by the public consultation, out of 2639 people contacted by PRINCIPLE following a positive test result, no one raised a concern about being approached. Conclusions This paper describes a novel approach to using near-real time NHS operational data to recruit community-based patients within a few days of presentation with acute illness. This approach increased recruitment and reduced time between positive test and randomisation, allowing more rapid evaluation of treatments and increased safety for participants. End-to-end public and patient involvement in the design of the approach provided evidence to inform information governance decisions. Trial registration PRINCIPLE is funded by UK Research and Innovation and the Department of Health and Social Care through the National Institute for Health Research. EudraCT number: 2020-001209-22 . 26/03/2020 ISRCTN registry: ISRCTN86534580 . 20/03/2020 REC number: 20/SC/058 IRAS number: 281958

IntroductionWhile complement is a contributor to disease severity in severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections, all three complement pathways might be activated by the virus. Lectin pathway activation occurs through different pattern recognition molecules, including mannan binding lectin (MBL), a protein shown to interact with SARS-CoV-2 proteins. However, the exact role of lectin pathway activation and its key pattern recognition molecule MBL in COVID-19 is still not fully understood.MethodsWe therefore investigated activation of the lectin pathway in two independent cohorts of SARS-CoV-2 infected patients, while also analysing MBL protein levels and potential effects of the six major single nucleotide polymorphisms (SNPs) found in the MBL2 gene on COVID-19 severity and outcome.ResultsWe show that the lectin pathway is activated in acute COVID-19, indicated by the correlation between complement activation product levels of the MASP-1/C1-INH complex (p=0.0011) and C4d (p

Mitochondria play a key role in cell homeostasis as a major source of intracellular energy (adenosine triphosphate), and as metabolic hubs regulating many canonical cell processes. Mitochondrial dysfunction has been widely documented in many common diseases, and genetic studies point towards a causal role in the pathogenesis of specific late-onset disorder. Together this makes targeting mitochondrial genes an attractive strategy for precision medicine. However, the genetics of mitochondrial biogenesis is complex, with over 1,100 candidate genes found in two different genomes: the nuclear DNA and mitochondrial DNA (mtDNA). Here, we review the current evidence associating mitochondrial genetic variants with distinct clinical phenotypes, with some having clear therapeutic implications. The strongest evidence has emerged through the investigation of rare inherited mitochondrial disorders, but genome-wide association studies also implicate mtDNA variants in the risk of developing common diseases, opening to door for the incorporation of mitochondrial genetic variant analysis in population disease risk stratification.

The most commonly used markers to assess complement activation are split products that are produced through activation of all three pathways and are located downstream of C3. In contrast, C4d derives from the cleavage of C4 and indicates either classical (CP) or lectin pathway (LP) activation. Although C4d is perfectly able to distinguish between CP/LP and alternative pathway (AP) activation, no well-established markers are available to differentiate between early CP and LP activation. Active enzymes of both pathways (C1s/C1r for the CP, MASP-1/MASP-2 for the LP) are regulated by C1 esterase inhibitor (C1-INH) through the formation of covalent complexes. Aim of this study was to develop validated immunoassays detecting C1s/C1-INH and MASP-1/C1-INH complex levels. Measurement of the complexes reveals information about the involvement of the respective pathways in complement-mediated diseases. Two sandwich ELISAs detecting C1s/C1-INH and MASP-1/C1-INH complex were developed and tested thoroughly, and it was investigated whether C1s/C1-INH and MASP-1/C1-INH complexes could serve as markers for either early CP or LP activation. In addition, a reference range for these complexes in healthy adults was defined, and the assays were clinically validated utilizing samples of 414 COVID-19 patients and 96 healthy controls. The immunoassays can reliably measure C1s/C1-INH and MASP-1/C1-INH complex concentrations in EDTA plasma from healthy and diseased individuals. Both complex levels are increased in serum when activated with zymosan, making them suitable markers for early classical and early lectin pathway activation. Furthermore, measurements of C1-INH complexes in 96 healthy adults showed normally distributed C1s/C1-INH complex levels with a physiological concentration of 1846 ± 1060 ng/mL (mean ± 2SD) and right-skewed distribution of MASP-1/C1-INH complex levels with a median concentration of 36.9 (13.18 - 87.89) ng/mL (2.5-97.5 percentile range), while levels of both complexes were increased in COVID-19 patients (p

Here the authors describe high depth mitochondrial DNA (mtDNA) sequence analysis of 146 human induced pluripotent stem cell (hiPSC) lines as well as single cell RNA-seq (scRNAseq) of hiPSCs undergoing differentiation from 125 donors; reporting mtDNA diversity and some variants favoured after reprogramming.

Mitochondrial DNA (mtDNA) depletion/deletions syndromes (MDDS) encompass a clinically and etiologically heterogenous group of mitochondrial disorders caused by impaired mtDNA maintenance. Among the most frequent causes of MDDS are defects in nucleoside/nucleotide metabolism, which is critical for synthesis and homeostasis of the deoxynucleoside triphosphate (dNTP) substrates of mtDNA replication. A central enzyme for generating dNTPs is ribonucleotide reductase, a critical mediator of de novo nucleotide synthesis composed of catalytic RRM1 subunits in complex with RRM2 or p53R2. Here, we report 5 probands from 4 families who presented with ptosis and ophthalmoplegia as well as other clinical manifestations and multiple mtDNA deletions in muscle. We identified 3 RRM1 loss-of-function variants, including a dominant catalytic site variant (NP_001024.1: p.N427K) and 2 homozygous recessive variants at p.R381, which has evolutionarily conserved interactions with the specificity site. Atomistic molecular dynamics simulations indicate mechanisms by which RRM1 variants affect protein structure. Cultured primary skin fibroblasts of probands manifested mtDNA depletion under cycling conditions, indicating impaired de novo nucleotide synthesis. Fibroblasts also exhibited aberrant nucleoside diphosphate and dNTP pools and mtDNA ribonucleotide incorporation. Our data reveal that primary RRM1 deficiency and, by extension, impaired de novo nucleotide synthesis are causes of MDDS.

Using an in vitro culture system, Pezet et al. studied the influence of oxygen on the mitochondrial DNA (mtDNA) in primordial germ cell-like cells (PGCLCs) in vitro. Low oxygen levels resembling in vivo reduced the cell mtDNA content causing a genetic bottleneck and the segregation of different mtDNA genotypes.

The competitive dynamics of mitochondrial haplotypes juxtaposed within the same cell are poorly studied. Here the authors show, in the context of a transmissible cancer, that one haplotype has recurrently entered cancer cells by horizontal transfer and appears to have a ‘selfish’ selective advantage.

Recent evidence has questioned the dogma of strict maternal transmission of mitochondrial DNA (mtDNA) in humans. Wei et al. saw no evidence of paternal transmission of mtDNA in 11,035 human trios, and show that nuclear-mitochondrial segments (NUMTs) can give the impression of paternal mtDNA transmission, but are actually inherited through the nuclear genome.

Summary: Clotting Factor V (FV) is primarily synthesized in the liver and when cleaved by thrombin forms pro-coagulant Factor Va (FVa). Using whole blood RNAseq and scRNAseq of peripheral blood mononuclear cells, we find that FV mRNA is expressed in leukocytes, and identify neutrophils, monocytes, and T regulatory cells as sources of increased FV in hospitalized patients with COVID-19. Proteomic analysis confirms increased FV in circulating neutrophils in severe COVID-19, and immunofluorescence microscopy identifies FV in lung-infiltrating leukocytes in COVID-19 lung disease. Increased leukocyte FV expression in severe disease correlates with T-cell lymphopenia. Both plasma-derived and a cleavage resistant recombinant FV, but not thrombin cleaved FVa, suppress T-cell proliferation in vitro. Anticoagulants that reduce FV conversion to FVa, including heparin, may have the unintended consequence of suppressing the adaptive immune system.

The role of mitochondrial DNA mutations in organismal fitness and lifespan have been studied in mitochondrial mutator models with varying results. Here, the authors generate a new APOBEC1 expression-based Drosophila mutator model and show that it has limited mitochondrial function and reduced lifespan.

Abstract Background Mitochondrial DNA copy number (mtDNA CN) exhibits interindividual and intercellular variation, but few genome-wide association studies (GWAS) of directly assayed mtDNA CN exist. We undertook a GWAS of qPCR-assayed mtDNA CN in the Avon Longitudinal Study of Parents and Children (ALSPAC) and the UK Blood Service (UKBS) cohort. After validating and harmonising data, 5461 ALSPAC mothers (16–43 years at mtDNA CN assay) and 1338 UKBS females (17–69 years) were included in a meta-analysis. Sensitivity analyses restricted to females with white cell-extracted DNA and adjusted for estimated or assayed cell proportions. Associations were also explored in ALSPAC children and UKBS males. Results A neutrophil-associated locus approached genome-wide significance (rs709591 [MED24], β (change in SD units of mtDNA CN per allele) [SE] − 0.084 [0.016], p = 1.54e−07) in the main meta-analysis of adult females. This association was concordant in magnitude and direction in UKBS males and ALSPAC neonates. SNPs in and around ABHD8 were associated with mtDNA CN in ALSPAC neonates (rs10424198, β [SE] 0.262 [0.034], p = 1.40e−14), but not other study groups. In a meta-analysis of unrelated individuals (N = 11,253), we replicated a published association in TFAM (β [SE] 0.046 [0.017], p = 0.006), with an effect size much smaller than that observed in the replication analysis of a previous in silico GWAS. Conclusions In a hypothesis-generating GWAS, we confirm an association between TFAM and mtDNA CN and present putative loci requiring replication in much larger samples. We discuss the limitations of our work, in terms of measurement error and cellular heterogeneity, and highlight the need for larger studies to better understand nuclear genomic control of mtDNA copy number.

Similar to cancers, somatic mutations might lead to neurodegenerative diseases. Here, the authors perform ultra-deep sequencing of 102 genes in 173 adult human brains, detect somatic mutations in 54 brains, and develop a mathematical model to estimate the frequency of mutated foci in human brains.

Mutations in the m.13094T>C MT-ND5 gene have been previously described in three cases of Leigh Syndrome (LS). In this retrospective, international cohort study we identified 20 clinically affected individuals (13 families) and four asymptomatic carriers. Ten patients were deceased at the time of analysis (median age of death was 10 years (range: 5·4 months−37 years, IQR = 17·9 years). Nine patients manifested with LS, one with mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS), and one with Leber hereditary optic neuropathy. The remaining nine patients presented with either overlapping syndromes or isolated neurological symptoms. Mitochondrial respiratory chain activity analysis was normal in five out of ten muscle biopsies. We confirmed maternal inheritance in six families, and demonstrated marked variability in tissue segregation, and phenotypic expression at relatively low blood mutant loads. Neuropathological studies of two patients manifesting with LS/MELAS showed prominent capillary proliferation, microvacuolation and severe neuronal cell loss in the brainstem and cerebellum, with conspicuous absence of basal ganglia involvement. These findings suggest that whole mtDNA genome sequencing should be considered in patients with suspected mitochondrial disease presenting with complex neurological manifestations, which would identify over 300 known pathogenic variants including the m.13094T>C. Keywords: Mitochondrial encephalomyopathy, Lactic acidosis and stroke-like episodes (MELAS), Leigh syndrome (LS), Mitochondrial DNA, Heteroplasmy, Neuropathology

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

mtDNA is transmitted through the maternal line and its sequence variability, which is population specific, is assumed to be phenotypically neutral. However, several studies have shown associations between the variants defining some genetic backgrounds and the susceptibility to several pathogenic phenotypes, including neurodegenerative diseases. Many of these studies have found that some of these variants impact many of these phenotypes, including the ones defining the Caucasian haplogroups H, J, and Uk, while others, such as the ones defining the T haplogroup, have phenotype specific associations. In this review, we will focus on those that have shown a pleiotropic effect in population studies in neurological diseases. We will also explore their bioenergetic and genomic characteristics in order to provide an insight into the role of these variants in disease. Given the importance of mitochondrial population variants in neurodegenerative diseases a deeper analysis of their effects might unravel new mechanisms of disease and help design new strategies for successful treatments.

In order to limit the adverse effects of excessive inflammation, anti-inflammatory responses are stimulated at an early stage of an infection, but during sepsis these can lead to deactivation of immune cells including monocytes. In addition, there is emerging evidence that the up-regulation of mitochondrial quality control mechanisms, including mitochondrial biogenesis and mitophagy, is important during the recovery from sepsis and inflammation. We aimed to describe the relationship between the compensatory immune and mitochondrial responses that are triggered following exposure to an inflammatory stimulus in human monocytic cells. Incubation with lipopolysaccharide resulted in a change in the immune phenotype of THP-1 cells consistent with the induction of endotoxin tolerance, similar to that seen in deactivated septic monocytes. After exposure to LPS there was also early evidence of oxidative stress, which resolved in association with the induction of antioxidant defenses and the stimulation of mitochondrial degradation through mitophagy. This was compensated by a parallel up-regulation of mitochondrial biogenesis that resulted in an overall increase in mitochondrial respiratory activity. These observations improve our understanding of the normal homeostatic responses that limit the adverse cellular effects of unregulated inflammation, and which may become ineffective when an infection causes sepsis.

This study sought evidence for a specific cerebellar contribution to cognition by characterising the cognitive phenotype of Spinocerebellar Ataxia Type 6 (SCA-6); an autosomal dominant genetic disease which causes a highly specific late-onset cerebellar degeneration. A comprehensive neuropsychological assessment was administered to 27 patients with genetically confirmed SCA-6. General intellectual ability, memory and executive function were examined using internationally standardised tests (Wechsler Adult Intelligence Scale-III, Wechsler Memory Scale-III, Delis and Kaplan Executive Function System, Brixton Spatial Anticipation test). The patient group showed no evidence of intellectual or memory decline. However, tests of executive function involving skills of cognitive flexibility, inhibition of response and verbal reasoning and abstraction demonstrated significant impairment at the group level with large effect sizes. The results demonstrate an executive deficit due to SCA-6 that can be conceptualised as parallel to the motor difficulties suffered by these patients: the data support a role for the cerebellum in the regulation and coordination of cognitive, as well as motor processes that is relevant to individual performance.

Neuroferritinopathy is a progressive potentially treatable adult-onset movement disorder caused by mutations in the ferritin light chain gene (FTL1). Features overlap with common extrapyramidal disorders: idiopathic torsion dystonia, idiopathic Parkinson's disease and Huntington's disease, but the phenotype and natural history have not been defined. We studied a genetically homogeneous group of 41 subjects with the 460InsA mutation in FTL1, documenting the presentation, clinical course, biochemistry and neuroimaging. The mean age of onset was 39.4 years (SD = 13.3, range 13–63), beginning with chorea in 50%, focal lower limb dystonia in 42.5% and parkinsonism in 7.5%. The majority reported a family history of a movement disorder often misdiagnosed as Huntington's disease. The disease progressed relentlessly, becoming generalized over a 5–10 year period, eventually leading to aphonia, dysphagia and severe motor disability with subcortical/frontal cognitive dysfunction as a late feature. A characteristic action-specific facial dystonia was common (65%), and in 63% there was asymmetry throughout the disease course. Serum ferritin levels were low in the majority of males and post-menopausal females, but within normal limits for pre-menopausal females. MR brain imaging was abnormal on all affected individuals and one presymptomatic carrier. In conclusion, isolated parkinsonism is unusual in neuroferritinopathy, and unlike Huntington's disease, cognitive changes are absent or subtle in the early stages. Depressed serum ferritin is common and provides a useful screening test in routine practice, and gradient echo brain MRI will identify all symptomatic cases. [ABSTRACT FROM AUTHOR]

We describe a patient with chronic progressive external ophthalmoplegia (CPEO) due to a deletion of mitochondrial DNA (mtDNA) who had electromyographic evidence of myotonic discharges. Myotonia has not previously been described in association with mitochondrial disease and this report extends the known phenotypic expression of these disorders. Muscle Nerve 28: 757–759, 2003 [ABSTRACT FROM AUTHOR]

Background We aimed to determine whether sodium valproate (VPA) should be contraindicated in all mitochondrial diseases, due to known VPA-induced severe hepatotoxicity in some mitochondrial diseases.Methods We systematically reviewed the published literature for mitochondrial DNA (mtDNA) and common nuclear genotypes of mitochondrial diseases using PubMed, Ovid Embase, Ovid Medline and MitoPhen databases. We extracted patient-level data from peer-reviewed articles, published until July 2022, using the Human Phenotype Ontology to manually code clinical presentations for 156 patients with genetic diagnoses from 90 publications.Results There were no fatal adverse drug reactions (ADRs) in the mtDNA disease group (35 patients), and only 1 out of 54 patients with a non-POLG mitochondrial disease developed acute liver failure. There were fatal outcomes in 53/102 (52%) POLG VPA-exposed patients who all harboured recessive mutations.Conclusions Our findings confirm the high risk of severe ADRs in any patient with recessive POLG variants irrespective of the phenotype, and therefore recommend that VPA is contraindicated in this group. However, there was limited evidence of toxicity to support a similar recommendation in other genotypes of mitochondrial diseases.

Mutations in nuclear genes involved in mitochondrial DNA (mtDNA) maintenance cause a wide range of clinical phenotypes associated with the secondary accumulation of multiple mtDNA deletions in affected tissues. The majority of families with autosomal dominant progressive external ophthalmoplegia (PEO) harbour mutations in genes encoding one of three well-characterized proteins—polγ, Twinkle or Ant 1. Here we show that a heterozygous mis-sense mutation in OPA1 leads to multiple mtDNA deletions in skeletal muscle and a mosaic defect of cytochrome c oxidase (COX). The disorder presented with visual failure and optic atrophy in childhood, followed by PEO, ataxia, deafness and a sensory-motor neuropathy in adult life. COX-deficient skeletal muscle fibres contained supra-threshold levels of multiple mtDNA deletions, and genetic linkage, sequencing and expression analysis excluded POLG1, PEO1 and SLC25A4, the gene encoding Ant 1, as the cause. This demonstrates the importance of OPA1 in mtDNA maintenance, and implicates OPA1 in diseases associated with secondary defects of mtDNA. [ABSTRACT FROM AUTHOR]

Mammalian mitochondrial DNA (mtDNA) is inherited uniparentally through the female germline without undergoing recombination. This poses a major problem as deleterious mtDNA mutations must be eliminated to avoid a mutational meltdown over generations. At least two mechanisms that can decrease the mutation load during maternal transmission are operational: a stochastic bottleneck for mtDNA transmission from mother to child, and a directed purifying selection against transmission of deleterious mtDNA mutations. However, the molecular mechanisms controlling these processes remain unknown. In this study, we systematically tested whether decreased autophagy contributes to purifying selection by crossing the C5024T mouse model harbouring a single pathogenic heteroplasmic mutation in the tRNAAla gene of the mtDNA with different autophagy-deficient mouse models, including knockouts of Parkin, Bcl2l13, Ulk1, and Ulk2. Our study reveals a statistically robust effect of knockout of Bcl2l13 on the selection process, and weaker evidence for the effect of Ulk1 and potentially Ulk2, while no statistically significant impact is seen for knockout of Parkin. This points at distinctive roles of these players in germline purifying selection. Overall, our approach provides a framework for investigating the roles of other important factors involved in the enigmatic process of purifying selection and guides further investigations for the role of BCL2L13 in the elimination of non-synonymous mutations in protein-coding genes.

Mitochondria are implicated in the pathogenesis of cardiovascular diseases (CVDs) but the reasons for this are not well understood. Maternally-inherited population variants of mitochondrial DNA (mtDNA) which affect all mtDNA molecules (homoplasmic) are associated with cardiometabolic traits and the risk of developing cardiovascular disease. However, it is not known whether mtDNA mutations only affecting a proportion of mtDNA molecules (heteroplasmic) also play a role. To address this question, we performed a high-depth (~1000-fold) mtDNA sequencing of blood DNA in 1,399 individuals with hypertension (HTN), 1,946 with ischemic heart disease (IHD), 2,146 with ischemic stroke (IS), and 723 healthy controls. We show that the per individual burden of heteroplasmic single nucleotide variants (mtSNVs) increases with age. The age-effect was stronger for low-level heteroplasmies (heteroplasmic fraction, HF, 5-10%), likely reflecting acquired somatic events based on trinucleotide mutational signatures. After correcting for age and other confounders, intermediate heteroplasmies (HF 10-95%) were more common in hypertension, particularly involving non-synonymous variants altering the amino acid sequence of essential respiratory chain proteins. These findings raise the possibility that heteroplasmic mtSNVs play a role in the pathophysiology of hypertension.

Abstract Mitochondrial disorders affect 1/5,000 and have no cure. Inducing mitochondrial biogenesis with bezafibrate improves mitochondrial function in animal models, but there are no comparable human studies. We performed an open‐label observational experimental medicine study of six patients with mitochondrial myopathy caused by the m.3243A>G MTTL1 mutation. Our primary aim was to determine the effects of bezafibrate on mitochondrial metabolism, whilst providing preliminary evidence of safety and efficacy using biomarkers. The participants received 600–1,200 mg bezafibrate daily for 12 weeks. There were no clinically significant adverse events, and liver function was not affected. We detected a reduction in the number of complex IV‐immunodeficient muscle fibres and improved cardiac function. However, this was accompanied by an increase in serum biomarkers of mitochondrial disease, including fibroblast growth factor 21 (FGF‐21), growth and differentiation factor 15 (GDF‐15), plus dysregulation of fatty acid and amino acid metabolism. Thus, although potentially beneficial in short term, inducing mitochondrial biogenesis with bezafibrate altered the metabolomic signature of mitochondrial disease, raising concerns about long‐term sequelae.

[This corrects the article DOI: 10.1371/journal.pgen.1007126.].

Abstract Ten years ago, there was an emerging view that the molecular basis for adult mitochondrial disorders was largely known and that the clinical phenotypes had been well described. Nothing could have been further from the truth. The establishment of large cohorts of patients has revealed new aspects of the clinical presentation that were not previously appreciated. Over time, this approach is starting to provide an accurate understanding of the natural history of mitochondrial disease in adults. Advances in molecular diagnostics, underpinned by next generation sequencing technology, have identified novel molecular mechanisms. Recently described mitochondrial disease phenotypes have disparate causes, and yet share common mechanistic themes. In particular, disorders of mtDNA maintenance have emerged as a major cause of mitochondrial disease in adults. Progressive mtDNA depletion and the accumulation of mtDNA mutations explain some of the clinical features, but the genetic and cellular processes responsible for the mtDNA abnormalities are not entirely clear in each instance. Unfortunately, apart from a few specific examples, treatments for adult mitochondrial disease have not been forthcoming. However, the establishment of international consortia, and the first multinational randomised controlled trial, have paved the way for major progress in the near future, underpinned by growing interest from the pharmaceutical industry. Adult mitochondrial medicine is, therefore, in its infancy, and the challenge is to harness the new understanding of its molecular and cellular basis to develop treatments of real benefit to patients.

Inherited mitochondrial DNA (mtDNA) mutations have emerged as a common cause of human disease, with mutations occurring multiple times in the world population. The clinical presentation of three pathogenic mtDNA mutations is strongly associated with a background mtDNA haplogroup, but it is not clear whether this is limited to a handful of examples or is a more general phenomenon. To address this, we determined the characteristics of 30,506 mtDNA sequences sampled globally. After performing several quality control steps, we ascribed an established pathogenicity score to the major alleles for each sequence. The mean pathogenicity score for known disease-causing mutations was significantly different between mtDNA macro-haplogroups. Several mutations were observed across all haplogroup backgrounds, whereas others were only observed on specific clades. In some instances this reflected a founder effect, but in others, the mutation recurred but only within the same phylogenetic cluster. Sequence diversity estimates showed that disease-causing mutations were more frequent on young sequences, and genomes with two or more disease-causing mutations were more common than expected by chance. These findings implicate the mtDNA background more generally in recurrent mutation events that have been purified through natural selection in older populations. This provides an explanation for the low frequency of mtDNA disease reported in specific ethnic groups.

Tubulointerstitial kidney disease is an important cause of progressive renal failure whose aetiology is incompletely understood. We analysed a large pedigree with maternally inherited tubulointerstitial kidney disease and identified a homoplasmic substitution in the control region of the mitochondrial genome (m.547A>T). While mutations in mtDNA coding sequence are a well recognised cause of disease affecting multiple organs, mutations in the control region have never been shown to cause disease. Strikingly, our patients did not have classical features of mitochondrial disease. Patient fibroblasts showed reduced levels of mitochondrial tRNAPhe, tRNALeu1 and reduced mitochondrial protein translation and respiration. Mitochondrial transfer demonstrated mitochondrial transmission of the defect and in vitro assays showed reduced activity of the heavy strand promoter. We also identified further kindreds with the same phenotype carrying a homoplasmic mutation in mitochondrial tRNAPhe (m.616T>C). Thus mutations in mitochondrial DNA can cause maternally inherited renal disease, likely mediated through reduced function of mitochondrial tRNAPhe.

Recent reports have questioned the accepted dogma that mammalian mitochondrial DNA (mtDNA) is strictly maternally inherited. In humans, the argument hinges on detecting a signature of inter-molecular recombination in mtDNA sequences sampled at the population level, inferring a paternal source for the mixed haplotypes. However, interpreting these data is fraught with difficulty, and direct experimental evidence is lacking. Using extreme-high depth mtDNA re-sequencing up to ~1.2 million-fold coverage, we find no evidence that paternal mtDNA haplotypes are transmitted to offspring in humans, thus excluding a simple dilution mechanism for uniparental transmission of mtDNA present in all healthy individuals. Our findings indicate that an active mechanism eliminates paternal mtDNA which likely acts at the molecular level.

The accuracy of mitochondrial protein synthesis is dependent on the coordinated action of nuclear-encoded mitochondrial aminoacyl-tRNA synthetases (mtARSs) and the mitochondrial DNA-encoded tRNAs. The recent advances in whole-exome sequencing have revealed the importance of the mtARS proteins for mitochondrial pathophysiology since nearly every nuclear gene for mtARS (out of 19) is now recognized as a disease gene for mitochondrial disease. Typically, defects in each mtARS have been identified in one tissue-specific disease, most commonly affecting the brain, or in one syndrome. However, mutations in the AARS2 gene for mitochondrial alanyl-tRNA synthetase (mtAlaRS) have been reported both in patients with infantile-onset cardiomyopathy and in patients with childhood to adulthood-onset leukoencephalopathy. We present here an investigation of the effects of the described mutations on the structure of the synthetase, in an effort to understand the tissue-specific outcomes of the different mutations.The mtAlaRS differs from the other mtARSs because in addition to the aminoacylation domain, it has a conserved editing domain for deacylating tRNAs that have been mischarged with incorrect amino acids. We show that the cardiomyopathy phenotype results from a single allele, causing an amino acid change p.R592W in the editing domain of AARS2, whereas the leukodystrophy mutations are located in other domains of the synthetase. Nevertheless, our structural analysis predicts that all mutations reduce the aminoacylation activity of the synthetase, because all mtAlaRS domains contribute to tRNA binding for aminoacylation. According to our model, the cardiomyopathy mutations severely compromise aminoacylation whereas partial activity is retained by the mutation combinations found in the leukodystrophy patients. These predictions provide a hypothesis for the molecular basis of the distinct tissue-specific phenotypic outcomes.

Recent sequencing studies have extensively explored the somatic alterations present in the nuclear genomes of cancers. Although mitochondria control energy metabolism and apoptosis, the origins and impact of cancer-associated mutations in mtDNA are unclear. In this study, we analyzed somatic alterations in mtDNA from 1675 tumors. We identified 1907 somatic substitutions, which exhibited dramatic replicative strand bias, predominantly C > T and A > G on the mitochondrial heavy strand. This strand-asymmetric signature differs from those found in nuclear cancer genomes but matches the inferred germline process shaping primate mtDNA sequence content. A number of mtDNA mutations showed considerable heterogeneity across tumor types. Missense mutations were selectively neutral and often gradually drifted towards homoplasmy over time. In contrast, mutations resulting in protein truncation undergo negative selection and were almost exclusively heteroplasmic. Our findings indicate that the endogenous mutational mechanism has far greater impact than any other external mutagens in mitochondria and is fundamentally linked to mtDNA replication.

Identifying the genetic basis for mitochondrial diseases is technically challenging given the size of the mitochondrial proteome and the heterogeneity of disease presentations. Using next-generation exome sequencing, we identified in a patient with severe combined mitochondrial respiratory chain defects and corresponding perturbation in mitochondrial protein synthesis, a homozygous p.Arg323Gln mutation in TRIT1. This gene encodes human tRNA isopentenyltransferase, which is responsible for i6A37 modification of the anticodon loops of a small subset of cytosolic and mitochondrial tRNAs. Deficiency of i6A37 was previously shown in yeast to decrease translational efficiency and fidelity in a codon-specific manner. Modelling of the p.Arg323Gln mutation on the co-crystal structure of the homologous yeast isopentenyltransferase bound to a substrate tRNA, indicates that it is one of a series of adjacent basic side chains that interact with the tRNA backbone of the anticodon stem, somewhat removed from the catalytic center. We show that patient cells bearing the p.Arg323Gln TRIT1 mutation are severely deficient in i6A37 in both cytosolic and mitochondrial tRNAs. Complete complementation of the i6A37 deficiency of both cytosolic and mitochondrial tRNAs was achieved by transduction of patient fibroblasts with wild-type TRIT1. Moreover, we show that a previously-reported pathogenic m.7480A>G mt-tRNASer(UCN) mutation in the anticodon loop sequence A36A37A38 recognised by TRIT1 causes a loss of i6A37 modification. These data demonstrate that deficiencies of i6A37 tRNA modification should be considered a potential mechanism of human disease caused by both nuclear gene and mitochondrial DNA mutations while providing insight into the structure and function of TRIT1 in the modification of cytosolic and mitochondrial tRNAs.

Mitochondrial DNA (mtDNA) is highly polymorphic at the population level, and specific mtDNA variants affect mitochondrial function. With emerging evidence that mitochondrial mechanisms are central to common human diseases, it is plausible that mtDNA variants contribute to the "missing heritability" of several complex traits. Given the central role of mtDNA genes in oxidative phosphorylation, the same genetic variants would be expected to alter the risk of developing several different disorders, but this has not been shown to date. Here we studied 38,638 individuals with 11 major diseases, and 17,483 healthy controls. Imputing missing variants from 7,729 complete mitochondrial genomes, we captured 40.41% of European mtDNA variation. We show that mtDNA variants modifying the risk of developing one disease also modify the risk of developing other diseases, thus providing independent replication of a disease association in different case and control cohorts. High-risk alleles were more common than protective alleles, indicating that mtDNA is not at equilibrium in the human population, and that recent mutations interact with nuclear loci to modify the risk of developing multiple common diseases.