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Mitochondria are epicentres of eukaryotic metabolism and bioenergetics. Pioneering efforts in recent decades have established the core protein componentry of these organelles(1) and have linked their dysfunction to more than 150 distinct disorders(2,3). Still, hundreds of mitochondrial proteins lack clear functions(4), and the underlying genetic basis for approximately 40\% of mitochondrial disorders remains unresolved(5). Here, to establish a more complete functional compendium of human mitochondrial proteins, we profiled more than 200 CRISPR-mediated HAP1 cell knockout lines using mass spectrometry-based multiomics analyses. This effort generated approximately 8.3 million distinct biomolecule measurements, providing a deep survey of the cellular responses to mitochondrial perturbations and laying a foundation for mechanistic investigations into protein function. Guided by these data, we discovered that PIGY upstream open reading frame (PYURF) is an S-adenosylmethionine-dependent methyltransferase chaperone that supports both complex I assembly and coenzyme Q biosynthesis and is disrupted in a previously unresolved multisystemic mitochondrial disorder. We further linked the putative zinc transporter SLC30A9 to mitochondrial ribosomes and OxPhos integrity and established RAB5IF as the second gene harbouring pathogenic variants that cause cerebrofaciothoracic dysplasia. Our data, which can be explored through the interactive online MITOMICS.app resource, suggest biological roles for many other orphan mitochondrial proteins that still lack robust functional characterization and define a rich cell signature of mitochondrial dysfunction that can support the genetic diagnosis of mitochondrial diseases.

Genetic processes require the activity of multiple topoisomerases, essential enzymes that remove topological tension and intermolecular linkages in DNA. We have investigated the subcellular localisation and activity of the six human topoisomerases with a view to understanding the topological maintenance of human mitochondrial DNA. Our results indicate that mitochondria contain two topoisomerases, TOP1MT and TOP3A. Using molecular, genomic and biochemical methods we find that both proteins contribute to mtDNA replication, in addition to the decatenation role of TOP3A, and that TOP1MT is stimulated by mtSSB. Loss of TOP3A or TOP1MT also dysregulates mitochondrial gene expression, and both proteins promote transcription elongation in vitro. We find no evidence for TOP2 localisation to mitochondria, and TOP2B knockout does not affect mtDNA maintenance or expression. Our results suggest a division of labour between TOP3A and TOP1MT in mtDNA topology control that is required for the proper maintenance and expression of human mtDNA.

Children identified with developmental disabilities or delays (DD) are increasingly being educated in inclusive mainstream classroom environments. At the kindergarten level, there have been pedagog...

Policies related to the inclusion of children with disabilities in mainstream classrooms have led to questions regarding how teachers can help cultivate inclusive learning communities where all children are supported and valued. In play-based kindergarten programs, teachers are tasked with ensuring goals for children's learning and development are cultivated in play. However, debates persist regarding the optimal role of the teacher in play and how to meaningfully support the play of children with disabilities. The current multiple case study explored the perspectives and approaches of three kindergarten teachers who highly valued, and strived to enable, participation and inclusion in play-based learning, referred to here as enactors. A minimum of three hours of observation were conducted in each classroom in the fall, and semi-structured teacher interviews were conducted in the fall and spring of the school year. Enactors shared some common themes related to implementing play-based learning to promote inclusion, including a balance of child agency and teacher guidance, involvement that is child-centred and flexible, and the importance of supporting social interactions in play. These views informed both common and unique practices observed in play, including one-on-one conversations, supporting small groups, becoming an active play partner, and collaboratively addressing problems that arose in play. These results illustrate ways enactors gave meaning to the concept of inclusion through their play practices, providing salient examples of play alongside teachers' craft knowledge to help support inclusive play-based learning practices going forward.

Leucine zipper-EF-hand containing transmembrane protein 1 (LETM1) encodes an inner mitochondrial membrane protein with an osmoregulatory function controlling mitochondrial volume and ion homeostasis. The putative association of LETM1 with a human disease was initially suggested in Wolf-Hirschhorn syndrome, a disorder that results from de novo monoallelic deletion of chromosome 4p16.3, a region encompassing LETM1. Utilizing exome sequencing and international gene-matching efforts, we have identified 18 affected individuals from 11 unrelated families harboring ultra-rare bi-allelic missense and loss-of-function LETM1 variants and clinical presentations highly suggestive of mitochondrial disease. These manifested as a spectrum of predominantly infantile-onset (14/18, 78%) and variably progressive neurological, metabolic, and dysmorphic symptoms, plus multiple organ dysfunction associated with neurodegeneration. The common features included respiratory chain complex deficiencies (100%), global developmental delay (94%), optic atrophy (83%), sensorineural hearing loss (78%), and cerebellar ataxia (78%) followed by epilepsy (67%), spasticity (53%), and myopathy (50%). Other features included bilateral cataracts (42%), cardiomyopathy (36%), and diabetes (27%). To better understand the pathogenic mechanism of the identified LETM1 variants, we performed biochemical and morphological studies on mitochondrial K+/H+ exchange activity, proteins, and shape in proband-derived fibroblasts and muscles and in Saccharomyces cerevisiae, which is an important model organism for mitochondrial osmotic regulation. Our results demonstrate that bi-allelic LETM1 variants are associated with defective mitochondrial K+ efflux, swollen mitochondrial matrix structures, and loss of important mitochondrial oxidative phosphorylation protein components, thus highlighting the implication of perturbed mitochondrial osmoregulation caused by LETM1 variants in neurological and mitochondrial pathologies. This research was supported using resources of the Core Facility Cell Imaging and Ultrastructure Research, University of Vienna, a member of the Vienna Life-Science Instruments (VLSI) and the VetCore Facility (Imaging) of the University of Veterinary Medicine Vienna. We acknowledge International Centre for Genomic Medicine in Neuromuscular Diseases. This research was funded in part, by the Wellcome Trust (WT093205MA, WT104033AIA, and the Synaptopathies Strategic Award, 165908). This study was funded by the Medical Research Council (MR/S01165X/1, MR/S005021/1, G0601943), The National Institute for Health Research University College London Hospitals Biomedical Research Centre, Rosetrees Trust, Ataxia UK, Multiple System Atrophy Trust, Brain Research United Kingdom, Sparks Great Ormond Street Hospital Charity, Muscular Dystrophy United Kingdom (MDUK), Muscular Dystrophy Association (MDA USA) and Senior Non-Clinical Fellow ship to A. Spinazzola, (MC_PC_13029). K.N. and S.E.M.M. were supported by the Austrian Science Funds FWF-P29077 and P31471. A. Spinazzola receives support also from The Lily Foun dation and Brain Research UK. R.K. was supported by European Academy of Neurology Research Training Fellowship and Rosetrees Trust PhD Plus award (PhD2022\100042). info:eu-repo/semantics/publishedVersion

Kindergarten teachers are confronted with an evolving context for teaching and assessment. Recent reforms throughout the world have resulted in two fundamental shifts: (a) a growing emphasi...

Kindergarten teachers face the challenge of integrating contemporary assessment practices with play-based pedagogy. The current study addresses this challenge by presenting a kindergarten assessment framework rooted in theory and current classroom practices, based on teacher interview and observational data collected in 20 kindergarten classrooms. Ten teachers subsequently participated in extended observations and video elicitation interviews. Results uncovered seven different assessment pathways by which teachers mobilized learning goals through play pedagogies and assessment. Based on these pathways, a comprehensive assessment framework was developed underscoring the cyclical relation between student learning goals, types of play, and assessment contexts and practices. This framework supports teachers’ negotiation and integration of assessment practices with play-based pedagogies to promote both academic and developmental learning goals.

Interactions between the products of the nuclear and mitochondrial genomes are critical for the function of most eukaryotic cells. Recently the introduction of mitochondrial replacement therapy has raised the question of incompatibilities between mitochondrial and nuclear variants, and their potential influence on the genetic makeup of human populations. Such interactions could also contribute to the variability of the penetrance of pathogenic DNA variants. This led us to investigate the frequencies of combinations of nuclear and mitochondrial SNP alleles (mitonuclear combinations) in healthy individuals (n=5375) and in a cohort of patients with Parkinson’s disease (PD, n=2210). In the unaffected population, we were not able to find associations between nuclear and mitochondrial variants with a false discovery rate below 0.05 after accounting for multiple testing (i.e., the number of combinations examined). However, in the PD cohort, five combinations surpassed this threshold. Next, combining both cohorts, we investigated whether these associations were modulated by disease status. All five combinations were significant (p−3 for all tests).These combinations also showed significant evidence for an effect of the interaction between the mitochondrial and nuclear variants on disease risk. Their nuclear components mapped to TBCA, NIBAN3 and GLT25D1 and an uncharacterised intergenic region. In summary, starting from a single cohort design we identified combinations of nuclear and mitochondrial variants affecting PD disease risk.

As school authorities strive toward inclusive models of education for children with neurodevelopmental delay and disability (NDD), many kindergarten curricula have mandated pedagogy centered on learning through play. Children with NDD tend to experience greater social isolation and lower rates of social play engagement compared to typically developing peers. Consequently, issues related to social participation and inclusion may be particularly salient in play-based kindergarten classrooms. The current qualitative study explored how eight kindergarten teachers in Ontario, Canada conceptualized and promoted inclusion in play for children with NDD. Classroom observation and teacher interviews were conducted with a focus on the teacher’s role in play. Teachers endorsed the use of several indirect (i.e., environmental) strategies to promote social participation, alongside proactive teacher support in play. Teachers who shared multiple aspects of an interventionist viewpoint toward disability, and identified the social benefits of inclusion in play for children with NDD, tended to provide more proactive support to all children in play. Teachers also provided reactive support in play to address emerging social conflict. Implications for fostering the meaningful inclusion of children with NDD in play-based learning are discussed.

Thyroid hormones are regarded as the major controllers of metabolic rate and oxygen consumption in mammals. Although it has been demonstrated that thyroid hormone supplementation improves bovine embryo development in vitro, the cellular mechanisms underlying these effects are so far unknown. In this study, we investigated the role of thyroid hormone in development of human preimplantation embryos. Embryos were cultured in the presence or absence of 10−7 M triiodothyronine (T3) till blastocyst stage. Inner cell mass (ICM) and trophectoderm (TE) were separated mechanically and subjected to RNAseq or quantification of mitochondrial DNA copy number. Analyses were performed using DESeq (v1.16.0 on R v3.1.3), MeV4.9 and MitoMiner 4.0v2018 JUN platforms. We found that the exposure of human preimplantation embryos to T3 had a profound impact on nuclear gene transcription only in the cells of ICM (1178 regulated genes—10.5% of 11 196 expressed genes) and almost no effect on cells of TE (38 regulated genes—0.3% of expressed genes). The analyses suggest that T3 induces in ICM a shift in ribosome and oxidative phosphorylation activity, as the upregulated genes are contributing to the composition and organization of the respiratory chain and associated cofactors involved in mitoribosome assembly and stability. Furthermore, a number of genes affecting the citric acid cycle energy production have reduced expression. Our findings might explain why thyroid disorders in women have been associated with reduced fertility and adverse pregnancy outcome. Our data also raise a possibility that supplementation of culture media with T3 may improve outcomes for women undergoing in vitro fertilization., Embryos cultured in the presence or absence of triiodothyronine (T3) till blastocyst stage upregulate genes contributing to the composition and organization of the respiratory chain and associated cofactors involved in mitoribosome assembly and stability. Furthermore, a number of genes affecting the citric acid cycle energy production have reduced expression.

Intracellular heterogeneity contributes significantly to cellular physiology and, in a number of debilitating diseases, cellular pathophysiology. This is greatly influenced by distinct organelle populations and to understand the aetiology of disease, it is important to have tools able to isolate and differentially analyse organelles from precise location within tissues. Here, we report the development of a subcellular biopsy technology that facilitates the isolation of organelles, such as mitochondria, from human tissue. We compared the subcellular biopsy technology to laser capture microdissection (LCM) that is the state-of-the-art technique for the isolation of cells from their surrounding tissues. We demonstrate an operational limit of 20 µm for LCM and then, for the first time in human tissue, show that subcellular biopsy can be used to isolate mitochondria beyond this limit.

Funder: Fight for Sight UK; doi: http://dx.doi.org/10.13039/501100000615, Funder: Wellcome Trust; doi: http://dx.doi.org/10.13039/100004440, Wolfram syndrome (WS) is an ultra-rare progressive neurodegenerative disorder defined by early-onset diabetes mellitus and optic atrophy. The majority of patients harbour recessive mutations in the WFS1 gene, which encodes for Wolframin, a transmembrane endoplasmic reticulum protein. There is limited availability of human ocular and brain tissues, and there are few animal models for WS that replicate the neuropathology and clinical phenotype seen in this disorder. We, therefore, characterised two wfs1 zebrafish knockout models harbouring nonsense wfs1a and wfs1b mutations. Both homozygous mutant wfs1a−/− and wfs1b−/− embryos showed significant morphological abnormalities in early development. The wfs1b−/− zebrafish exhibited a more pronounced neurodegenerative phenotype with delayed neuronal development, progressive loss of retinal ganglion cells and clear evidence of visual dysfunction on functional testing. At 12 months of age, wfs1b−/− zebrafish had a significantly lower RGC density per 100 μm2 (mean ± standard deviation; 19 ± 1.7) compared with wild-type (WT) zebrafish (25 ± 2.3, p < 0.001). The optokinetic response for wfs1b−/− zebrafish was significantly reduced at 8 and 16 rpm testing speeds at both 4 and 12 months of age compared with WT zebrafish. An upregulation of the unfolded protein response was observed in mutant zebrafish indicative of increased endoplasmic reticulum stress. Mutant wfs1b−/− zebrafish exhibit some of the key features seen in patients with WS, providing a versatile and cost-effective in vivo model that can be used to further investigate the underlying pathophysiology of WS and potential therapeutic interventions.

The SLC25A26 gene encodes a mitochondrial inner membrane carrier that transports S-adenosylmethionine (SAM) into the mitochondrial matrix in exchange for S-adenosylhomocysteine (SAH). SAM is the predominant methyl-group donor for most cellular methylation processes, of which SAH is produced as a by-product. Pathogenic, biallelic SLC25A26 variants are a recognized cause of mitochondrial disease in children, with a severe neonatal onset caused by decreased SAM transport activity. Here, we describe two, unrelated adult cases, one of whom presented with recurrent episodes of severe abdominal pain and metabolic decompensation with lactic acidosis. Both patients had exercise intolerance and mitochondrial myopathy associated with biallelic variants in SLC25A26, which led to marked respiratory chain deficiencies and mitochondrial histopathological abnormalities in skeletal muscle that are comparable to those previously described in early-onset cases. We demonstrate using both mouse and fruit fly models that impairment of SAH, rather than SAM, transport across the mitochondrial membrane is likely the cause of this milder, late-onset phenotype. Our findings associate a novel pathomechanism with a known disease-causing protein and highlight the quests of precision medicine in optimizing diagnosis, therapeutic intervention and prognosis.

BackgroundThe spectrum of mitochondrial disease is genetically and phenotypically diverse, resulting from pathogenic variants in over 400 genes, with aerobic energy metabolism defects as a common denominator. Such heterogeneity poses a significant challenge in making an accurate diagnosis, critical for precision medicine.MethodsIn an international collaboration initiated by the European Network for Mitochondrial Diseases (GENOMIT) we recruited 2,023 pediatric patients at 11 specialist referral centers between October 2010 and January 2021, accumulating exome sequencing and HPO-encoded phenotype data. An exome-wide search for variants in known and potential novel disease genes, complemented by functional studies, followed ACMG guidelines.Results1,109 cases (55%) received a molecular diagnosis, of which one fifth have potential disease-modifying treatments (236/1,109, 21%). Functional studies enabled diagnostic uplift from 36% to 55% and discovery of 62 novel disease genes. Pathogenic variants were identified within genes encoding mitochondrial proteins or RNAs in 801 cases (72%), while, given extensive phenotype overlap, the remainder involved proteins targeted to other cellular compartments. To delineate genotype-phenotype associations, our data was complemented with registry and literature data to develop “GENOMITexplorer”, an open access resource detailing patient- (n=3,940), gene- (n=427), and variant-level (n=1,492) associations (prokischlab.github.io/GENOMITexplorer/).ConclusionsReaching a molecular diagnosis was essential for implementation of precision medicine and clinical trial eligibility, underlining the need for genome-wide screening given inability to accurately define mitochondrial diseases clinically. Key to diagnostic success were functional studies, encouraging early acquisition of patient- derived tissues and routine integration of high-throughput functional data to improve patient care by uplifting diagnostic rate.

Increasing opportunities for children’s outdoor play is emerging as a priority for multiple sectors given the associated developmental benefits. This study examined the commonalities of multi-secto...

Transport And Golgi Organization protein 2 (TANGO2) deficiency has recently been identified as a rare metabolic disorder with a distinct clinical and biochemical phenotype of recurrent metabolic crises, hypoglycemia, lactic acidosis, rhabdomyolysis, arrhythmias, and encephalopathy with cognitive decline. We report nine subjects from seven independent families, and we studied muscle histology, respiratory chain enzyme activities in skeletal muscle and proteomic signature of fibroblasts. All nine subjects carried autosomal recessive TANGO2 mutations. Two carried the reported deletion of exons 3 to 9, one homozygous, one heterozygous with a 22q11.21 microdeletion inherited in trans. The other subjects carried three novel homozygous (c.262C>T/p.Arg88*; c.220A>C/p.Thr74Pro; c.380+1G>A), and two further novel heterozygous (c.6_9del/p.Phe6del); c.11‐13delTCT/p.Phe5del mutations. Immunoblot analysis detected a significant decrease of TANGO2 protein. Muscle histology showed mild variation of fiber diameter, no ragged‐red/cytochrome c oxidase‐negative fibers and a defect of multiple respiratory chain enzymes and coenzyme Q10 (CoQ10) in two cases, suggesting a possible secondary defect of oxidative phosphorylation. Proteomic analysis in fibroblasts revealed significant changes in components of the mitochondrial fatty acid oxidation, plasma membrane, endoplasmic reticulum‐Golgi network and secretory pathways. Clinical presentation of TANGO2 mutations is homogeneous and clinically recognizable. The hemizygous mutations in two patients suggest that some mutations leading to allele loss are difficult to detect. A combined defect of the respiratory chain enzymes and CoQ10 with altered levels of several membrane proteins provides molecular insights into the underlying pathophysiology and may guide rational new therapeutic interventions., Agència de Gestió d'Ajuts Universitaris i de Recerca (AGAUR), Grant/Award Number: 2014: SGR 393; Association Française contre les Myopathies (FR), Grant/Award Number: 21644; CERCA Programme/ Generalitat de Catalunya, the Hesperia Foundation, the Secretariat for Universities and Research of the Ministry of Business and Knowledge of the Government of Catalonia, Grant/Award Number: [2017SGR1206]; European Research Council, Grant/Award Number: 309548; FEuropean Union Seventh Framework Programme, Grant/Award Number: (FP7/2007‐2013); Instituto de la Marató de TV3, Grant/Award Number: 345/C/2014rm Care (CA); Instituto de Salud Carlos III, Grant/Award Number: grants PI17‐01286, PI17/00109, PI16/00579, PI16/01048, PI14/00581 and CP09/00011; Medical Research Council, Grant/Award Number: MR/N025431/1; Mitochondrial Disease Patient Cohort (UK) , Grant/Award Number: (G0800674); National Institute for Health Research (NIHR) doctoral fellowship, Grant/Award Number: NIHR‐HCS‐D12‐03‐04; Newton Fund, Grant/Award Number: MR/N027302/1; Wellcome Centre for Mitochondrial Research, Grant/Award Number: (203105/Z/16/Z); Wellcome Investigator fund, Grant/Award Number: 109915/Z/15/Z; Wellcome Trust Pathfinder Scheme, Grant/Award Number: 201064/Z/16/Z

Guided play, a balanced approach to involvement in play that includes child- and adult-direction in learning activities, holds great promise for children’s effective and engaged learning in...

Mitochondrial disease is hugely diverse with respect to associated clinical presentations and underlying genetic causes, with pathogenic variants in over 300 disease genes currently described. Approximately half of these have been discovered in the last decade due to the increasingly widespread application of next generation sequencing technologies, in particular unbiased, whole exome—and latterly, whole genome sequencing. These technologies allow more genetic data to be collected from patients with mitochondrial disorders, continually improving the diagnostic success rate in a clinical setting. Despite these significant advances, some patients still remain without a definitive genetic diagnosis. Large datasets containing many variants of unknown significance have become a major challenge with next generation sequencing strategies and these require significant functional validation to confirm pathogenicity. This interface between diagnostics and research is critical in continuing to expand the list of known pathogenic variants and concomitantly enhance our knowledge of mitochondrial biology. The increasing use of whole exome sequencing, whole genome sequencing and other “omics” techniques such as transcriptomics and proteomics will generate even more data and allow further interrogation and validation of genetic causes, including those outside of coding regions. This will improve diagnostic yields still further and emphasizes the integral role that functional assessment of variant causality plays in this process—the overarching focus of this review.

ContextThe standards-based movement in U.S. public education has reached as far as kindergarten. Early primary teachers are increasingly required to teach academic standards in core subject areas, while engaging in increased levels of student assessment. In kindergarten, this growing emphasis on academic standards and student assessment is expected to operate alongside longstanding social and personal developmental expectations. However, recent research has identified a significant tension as teachers endeavor to negotiate a balance between traditional developmental programming and new standards-based academic curricula.PurposeThe purpose of this scoping review is to synthesize research related to three kindergarten traditions—Reggio Emilia, Waldorf, and Montessori—to develop a common understanding of key tenets for kindergarten assessment that can inform policy and practice in public education contexts.Research DesignA scoping review methodology was used to analyze research on assessment practices native to three kindergarten traditions—Reggio Emilia, Waldorf, and Montessori. This methodology followed a five-stage framework: (a) identifying the research question, (b) identifying relevant studies, (c) study selection, (d) charting the data, and (e) summarizing and reporting the results. Guiding the collection of articles was the following research question: “What does the extant literature on practices native to the three focal kindergarten traditions tell us about the assessment of kindergarten (4–6-year-olds) students’ learning?” In total, 80 texts satisfied the inclusion criteria across all traditions and were included in this study.ConclusionsEmpirical and non-empirical literature pertaining to each tradition were analyzed and considered in relation to their potential contribution to public education. In comparing across traditions, differences were evident based on their (a) assessment discourses and purposes, (b) reference systems, (c) assessment methods, and (d) uses of assessment information. However, the three traditions also maintained key commonalities leading to the identification of core tenets for kindergarten assessment. Specifically, three core priorities for kindergarten assessment were identified: (a) a commitment to child-centered and developmentally appropriate teaching, (b) a continuous embedded formative assessment approach, and (c) the use of multiple methods for gaining assessment information. In addition to core priority areas, results from this study suggest consistent processes that facilitate assessment practices at the kindergarten level. These four iterative processes are: (a) participation in teaching and learning, (b) reconstruction of teaching and learning, (c) engagement in assessment dialogues, and (d) integration of feedback for enhanced teaching and learning.

There is rising concern in research and practice to improve early years mathematics education as initial mathematical mastery sets the foundation for future academic success. In response to these concerns, this study used observational data from 20 kindergarten classrooms to understand current pedagogies teachers use for mathematics. In particular, our study is situated in kindergarten classrooms that promote play as a dominant pedagogical approach as research and policies endorse play as an effective teaching strategy for early learners. However, research also suggests that teachers express challenges implementing novel pedagogies, such as play, and use various forms of instruction when teaching math. Results showed teachers used four mathematics pedagogies (free play, guided play, teacher-directed play, and direct instruction). The enactment of these pedagogies gave rise to three classroom pedagogical orientations: child-controlled, shared-control, or teacher-controlled math contexts, indicating that combinations of play and direct instruction pedagogies are used to teach math in kindergarten.

Autophagy is the major intracellular degradation route in mammalian cells. Systemic ablation of core autophagy-related (ATG) genes in mice leads to embryonic or perinatal lethality, and conditional models show neurodegeneration. Impaired autophagy has been associated with a range of complex human diseases, yet congenital autophagy disorders are rare.We performed a genetic, clinical, and neuroimaging analysis involving five families. Mechanistic investigations were conducted with the use of patient-derived fibroblasts, skeletal muscle-biopsy specimens, mouse embryonic fibroblasts, and yeast.We found deleterious, recessive variants in human ATG7, a core autophagy-related gene encoding a protein that is indispensable to classical degradative autophagy. Twelve patients from five families with distinct ATG7 variants had complex neurodevelopmental disorders with brain, muscle, and endocrine involvement. Patients had abnormalities of the cerebellum and corpus callosum and various degrees of facial dysmorphism. These patients have survived with impaired autophagic flux arising from a diminishment or absence of ATG7 protein. Although autophagic sequestration was markedly reduced, evidence of basal autophagy was readily identified in fibroblasts and skeletal muscle with loss of ATG7. Complementation of different model systems by deleterious ATG7 variants resulted in poor or absent autophagic function as compared with the reintroduction of wild-type ATG7.We identified several patients with a neurodevelopmental disorder who have survived with a severe loss or complete absence of ATG7, an essential effector enzyme for autophagy without a known functional paralogue. (Funded by the Wellcome Centre for Mitochondrial Research and others.).

In recent years, next-generation sequencing (NGS) has become a powerful tool for studying both inherited and somatic heteroplasmic mitochondrial DNA (mtDNA) variation. NGS has proved particularly powerful when combined with single-cell isolation techniques, allowing the investigation of low-level heteroplasmic variants both between cells and within tissues. Nevertheless, there remain significant challenges, especially around the selective enrichment of mtDNA from total cellular DNA and the avoidance of nuclear pseudogenes. This chapter summarizes the techniques needed to enrich, amplify, sequence, and analyse mtDNA using NGS .

Supplemental Digital Content is available in the text., Background: Approximately 25% of patients with pulmonary arterial hypertension (PAH) have been found to harbor rare mutations in disease-causing genes. To identify missing heritability in PAH, we integrated deep phenotyping with whole-genome sequencing data using Bayesian statistics. Methods: We analyzed 13 037 participants enrolled in the NBR study (NIHR BioResource—Rare Diseases), of which 1148 were recruited to the PAH domain. To test for genetic associations between genes and selected phenotypes of pulmonary hypertension, we used the Bayesian rare variant association method BeviMed. Results: Heterozygous, high impact, likely loss-of-function variants in the kinase insert domain receptor (KDR) gene were strongly associated with significantly reduced transfer coefficient for carbon monoxide (posterior probability=0.989) and older age at diagnosis (posterior probability=0.912). We also provide evidence for familial segregation of a rare nonsense KDR variant with these phenotypes. On computed tomographic imaging of the lungs, a range of parenchymal abnormalities were observed in the 5 patients harboring these predicted deleterious variants in KDR. Four additional PAH cases with rare likely loss-of-function variants in KDR were independently identified in the US PAH Biobank cohort with similar phenotypic characteristics. Conclusions: The Bayesian inference approach allowed us to independently validate KDR, which encodes for the VEGFR2 (vascular endothelial growth factor receptor 2), as a novel PAH candidate gene. Furthermore, this approach specifically associated high impact likely loss-of-function variants in the genetically constrained gene with distinct phenotypes. These findings provide evidence for KDR being a clinically actionable PAH gene and further support the central role of the vascular endothelium in the pathobiology of PAH.

Oxidative phosphorylation (OXPHOS) defects caused by somatic mitochondrial DNA (mtDNA) mutations increase with age in human colorectal epithelium and are prevalent in colorectal tumours, but whether they actively contribute to tumorigenesis remains unknown. Here we demonstrate that mtDNA mutations causing OXPHOS defects are enriched during the human adenoma/carcinoma sequence, suggesting they may confer a metabolic advantage. To test this we deleted the tumour suppressor Apc in OXPHOS deficient intestinal stem cells in mice. The resulting tumours were larger than in control mice due to accelerated cell proliferation and reduced apoptosis. We show that both normal crypts and tumours undergo metabolic remodelling in response to OXPHOS deficiency by upregulating the de novo serine synthesis pathway (SSP). Moreover, normal human colonic crypts upregulate the SSP in response to OXPHOS deficiency prior to tumorigenesis. Our data show that age-associated OXPHOS deficiency causes metabolic remodelling that can functionally contribute to accelerated intestinal cancer development.

Reversible infantile respiratory chain deficiency (RIRCD) is a rare mitochondrial myopathy leading to severe metabolic disturbances in infants, which recover spontaneously after 6‐months of age. RIRCD is associated with the homoplasmic m.14674T>C mitochondrial DNA mutation; however, only ~ 1/100 carriers develop the disease. We studied 27 affected and 15 unaffected individuals from 19 families and found additional heterozygous mutations in nuclear genes interacting with mt‐tRNAGlu including EARS2 and TRMU in the majority of affected individuals, but not in healthy carriers of m.14674T>C, supporting a digenic inheritance. Our transcriptomic and proteomic analysis of patient muscle suggests a stepwise mechanism where first, the integrated stress response associated with increased FGF21 and GDF15 expression enhances the metabolism modulated by serine biosynthesis, one carbon metabolism, TCA lipid oxidation and amino acid availability, while in the second step mTOR activation leads to increased mitochondrial biogenesis. Our data suggest that the spontaneous recovery in infants with digenic mutations may be modulated by the above described changes. Similar mechanisms may explain the variable penetrance and tissue specificity of other mtDNA mutations and highlight the potential role of amino acids in improving mitochondrial disease., Heterozygous mutations in nuclear genes interacting with mt‐tRNAGlu induce the integrated stress response and metabolic rearrangements, reducing penetrance and promoting spontaneous recovery in a rare mitochondrial myopathy.