bims-mitdis Biomed News
on Mitochondrial disorders
Issue of 2022–04–17
forty-two papers selected by
Catalina Vasilescu, University of Helsinki



  1. Methods Mol Biol. 2022 ;2431 409-416
      Mitochondria are essential organelles that generate energy and play vital roles in cellular metabolism. The small circular mitochondrial genome encodes key components of the mitochondrial respiratory apparatus. Depletion of, or mutations in mitochondrial DNA (mtDNA) cause mitochondrial dysfunction and disease. mtDNA is packaged into nucleoids, which are transported throughout the cell within mitochondria. Efficient transport of nucleoids is essential in neurons, where mitochondrial function is required locally at synapses. Here I describe methods for visualization of nucleoids in Drosophila neurons using a GFP fusion of the mitochondrial transcription factor TFAM. TFAM-GFP, together with mCherry-labeled mitochondria, was used to visualize nucleoids in fixed larval segmental nerves. I also describe how these tools can be used for live imaging of nucleoid dynamics. Using Drosophila as a model system, these methods will enable further characterization and analysis of nucleoid dynamics in neurons.
    Keywords:  Drosophila; Live imaging; Mitochondrial DNA; Nucleoid; TFAM
    DOI:  https://doi.org/10.1007/978-1-0716-1990-2_21
  2. Front Cell Dev Biol. 2022 ;10 858286
      The Mitofusin 2 protein (MFN2), encoded by the MFN2 gene, was first described for its role in mediating mitochondrial fusion. However, MFN2 is now recognized to play additional roles in mitochondrial autophagy (mitophagy), mitochondrial motility, lipid transfer, and as a tether to other organelles including the endoplasmic reticulum (ER) and lipid droplets. The tethering role of MFN2 is an important mediator of mitochondrial-ER contact sites (MERCs), which themselves have many important functions that regulate mitochondria, including calcium homeostasis and lipid metabolism. Exemplifying the importance of MFN2, pathogenic variants in MFN2 are established to cause the peripheral neuropathy Charcot-Marie-Tooth Disease Subtype 2A (CMT2A). However, the mechanistic basis for disease is not clear. Moreover, additional pathogenic phenotypes such as lipomatosis, distal myopathy, optic atrophy, and hearing loss, can also sometimes be present in patients with CMT2A. Given these variable patient phenotypes, and the many cellular roles played by MFN2, the mechanistic underpinnings of the cellular impairments by which MFN2 dysfunction leads to disease are likely to be complex. Here, we will review what is known about the various functions of MFN2 that are impaired by pathogenic variants causing CMT2A, with a specific emphasis on the ties between MFN2 variants and MERCs.
    Keywords:  CMT2A; MFN2; lipid homeostasis; mitochondria; mitochondrial dynamics; mitochondrial endoplasmic reticulum contact sites; mitophagy; mtDNA
    DOI:  https://doi.org/10.3389/fcell.2022.858286
  3. Methods Mol Biol. 2022 ;2431 291-310
      Mitochondria are highly dynamic organelles which form intricate networks with complex dynamics. Mitochondrial transport and distribution are essential to ensure proper cell function, especially in cells with an extremely polarised morphology such as neurons. A layer of complexity is added when considering mitochondria have their own genome, packaged into nucleoids. Major mitochondrial morphological transitions, for example mitochondrial division, often occur in conjunction with mitochondrial DNA (mtDNA) replication and changes in the dynamic behaviour of the nucleoids. However, the relationship between mtDNA dynamics and mitochondrial motility in the processes of neurons has been largely overlooked. In this chapter, we describe a method for live imaging of mitochondria and nucleoids in differentiated SH-SY5Y cells by instant structured illumination microscopy (iSIM). We also include a detailed protocol for the differentiation of SH-SY5Y cells into cells with a pronounced neuronal-like morphology and show examples of coordinated mitochondrial and nucleoid motility in the long processes of these cells.
    Keywords:  Axonal transport; Instant structured illumination microscopy (iSIM); Mitochondria; Mitochondrial DNA; Mitochondrial fission; Neuronal differentiation; Nucleoids; SH-SY5Y cells; Superresolution
    DOI:  https://doi.org/10.1007/978-1-0716-1990-2_15
  4. Methods Mol Biol. 2022 ;2431 533-546
      Intracellular trafficking of organelles driven by molecular motors underlies essential cellular processes. Mitochondria, the powerhouses of the cell, are one of the major cargoes of molecular motors. Efficient distribution of mitochondria ensures cellular fitness while defects in this process contribute to severe pathologies, such as neurodegenerative diseases. Reconstitution of the mitochondrial microtubule-based transport in vitro in a bottom-up approach provides a powerful tool to investigate the mitochondrial trafficking machinery in a controlled environment in the absence of complex intracellular interactions. In this chapter, we describe the procedures for achieving such reconstitution of mitochondrial transport.
    Keywords:  Adaptor proteins; Interference reflection microscopy; Kinesin-1; Mitochondria; Molecular motors; Motility assay; TIRF microscopy; TRAK
    DOI:  https://doi.org/10.1007/978-1-0716-1990-2_28
  5. Pediatr Int. 2022 Jan;64(1): e15143
      
    Keywords:   SLC25A26 ; L-carnitine; mitochondrial cardiomyopathy; mitochondrial diseases; neonates
    DOI:  https://doi.org/10.1111/ped.15143
  6. Bioinformatics. 2022 Apr 12. pii: btac216. [Epub ahead of print]
       SUMMARY: We present MitoVisualize, a new tool for analysis of the human mitochondrial DNA (mtDNA). MitoVisualize enables visualization of: (1) the position and effect of variants in mitochondrial transfer RNA (tRNA) and ribosomal RNA (rRNA) secondary structures alongside curated variant annotations, (2) data across RNA structures, such as to show all positions with disease-associated variants or with post-transcriptional modifications, and (3) the position of a base, gene or region in the circular mtDNA map, such as to show the location of a large deletion. All visualizations can be easily downloaded as figures for reuse. MitoVisualize can be useful for anyone interested in exploring mtDNA variation, though is designed to facilitate mtDNA variant interpretation in particular.
    AVAILABILITY AND IMPLEMENTATION: MitoVisualize can be accessed via https://www.mitovisualize.org/. The source code is available at https://github.com/leklab/mito_visualize/.
    SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
    DOI:  https://doi.org/10.1093/bioinformatics/btac216
  7. Trends Biochem Sci. 2022 Apr 06. pii: S0968-0004(22)00067-6. [Epub ahead of print]
      Age-associated changes in mitochondria are closely involved in aging. Apart from the established roles in bioenergetics and biosynthesis, mitochondria are signaling organelles that communicate their fitness to the nucleus, triggering transcriptional programs to adapt homeostasis stress that is essential for organismal health and aging. Emerging studies revealed that mitochondrial-to-nuclear (mito-nuclear) communication via altered levels of mitochondrial metabolites or stress signals causes various epigenetic changes, facilitating efforts to maintain homeostasis and affect aging. Here, we summarize recent studies on the mechanisms by which mito-nuclear communication modulates epigenomes and their effects on regulating the aging process. Insights into understanding how mitochondrial metabolites serve as prolongevity signals and how aging affects this communication will help us develop interventions to promote longevity and health.
    Keywords:  UPR(mt); aging; epigenetic regulation; longevity; mitochondrial metabolites; mitochondrial–nuclear communication
    DOI:  https://doi.org/10.1016/j.tibs.2022.03.008
  8. Int J Mol Sci. 2022 Mar 29. pii: 3738. [Epub ahead of print]23(7):
      Mitochondria are the most complex intracellular organelles, their function combining energy production for survival and apoptosis facilitation for death. Such a multivariate physiology is structurally and functionally reflected upon their membrane configuration and lipid composition. Mitochondrial double membrane lipids, with cardiolipin as the protagonist, show an impressive level of complexity that is mandatory for maintenance of mitochondrial health and protection from apoptosis. Given that lipidomics is an emerging field in cancer research and that mitochondria are the organelles with the most important role in malignant maintenance knowledge of the mitochondrial membrane, lipid physiology in health is mandatory. In this review, we will thus describe the delicate nature of the healthy mitochondrial double membrane and its role in apoptosis. Emphasis will be given on mitochondrial membrane lipids and the changes that they undergo during apoptosis induction and progression.
    Keywords:  apoptosis; cardiolipin; ceramide; cytochrome C; double membrane; mitochondria; mitochondrial lipids
    DOI:  https://doi.org/10.3390/ijms23073738
  9. EMBO J. 2022 Apr 12. e109390
      Mitophagy removes defective mitochondria via lysosomal elimination. Increased mitophagy coincides with metabolic reprogramming, yet it remains unknown whether mitophagy is a cause or consequence of such state changes. The signalling pathways that integrate with mitophagy to sustain cell and tissue integrity also remain poorly defined. We performed temporal metabolomics on mammalian cells treated with deferiprone, a therapeutic iron chelator that stimulates PINK1/PARKIN-independent mitophagy. Iron depletion profoundly rewired the metabolome, hallmarked by remodelling of lipid metabolism within minutes of treatment. DGAT1-dependent lipid droplet biosynthesis occurred several hours before mitochondrial clearance, with lipid droplets bordering mitochondria upon iron chelation. We demonstrate that DGAT1 inhibition restricts mitophagy in vitro, with impaired lysosomal homeostasis and cell viability. Importantly, genetic depletion of DGAT1 in vivo significantly impaired neuronal mitophagy and locomotor function in Drosophila. Our data define iron depletion as a potent signal that rapidly reshapes metabolism and establishes an unexpected synergy between lipid homeostasis and mitophagy that safeguards cell and tissue integrity.
    Keywords:  DGAT1; iron; lipid droplet; metabolism; mitophagy
    DOI:  https://doi.org/10.15252/embj.2021109390
  10. J Inherit Metab Dis. 2022 Apr 11.
      Mitochondrial trifunctional protein (MTP) is involved in long-chain fatty acid β-oxidation (lcFAO). Deficiency of one or more of the enzyme activities as catalyzed by MTP causes generalized MTP deficiency (MTPD), long-chain hydroxyacyl-CoA dehydrogenase deficiency (LCHADD), or long-chain ketoacyl-CoA thiolase deficiency (LCKATD). When genetic variants result in thermo-sensitive enzymes, increased body temperature (e.g. fever) can reduce enzyme activity and be a risk factor for clinical decompensation. This is the first description of five patients with a thermo-sensitive MTP deficiency. Clinical and genetic information was obtained from clinical files. Measurement of LCHAD and LCKAT activities, lcFAO-flux studies and palmitate loading tests were performed in skin fibroblasts cultured at 37°C and 40°C. In all patients (four MTPD, one LCKATD), disease manifested during childhood (manifestation age: 2-10 years) with myopathic symptoms triggered by fever or exercise. In four patients, signs of retinopathy or neuropathy were present. Plasma long-chain acylcarnitines were normal or slightly increased. HADHB variants were identified (at age: 6-18 years) by whole exome sequencing or gene panel analyses. At 37°C, LCHAD and LCKAT activities were mildly impaired and lcFAO-fluxes were normal. Remarkably, enzyme activities and lcFAO-fluxes were markedly diminished at 40°C. Preventive (dietary) measures improved symptoms for most. All patients with thermo-sensitive MTP deficiency had a long diagnostic trajectory and both genetic and enzymatic testing were required for diagnosis. The frequent absence of characteristic acylcarnitine abnormalities poses a risk for a diagnostic delay. Given the positive treatment effects, upfront genetic screening may be beneficial to enhance early recognition. Positive treatment effects emphasize the need for early recognition of thermo-sensitive MTP deficiency, but the frequent absence of characteristic acylcarnitine abnormalities poses a risk for a diagnostic delay and shows the importance of both genetic and enzymatic testing for a proper diagnosis. M.S., S.F., S.A.F. and G.V. were involved in acquisition of data, interpretation of data and drafting of the manuscript. S.F., M.D., M.E. and J.R. were involved in the biochemical analysis for all patients. N.D., C.H.O, L.R., L.K.D., H.H.H., S.O., D.C. and S.J.M. were involved in diagnostics and long-term care of patients. All authors were involved in the interpretation of data and reviewing and editing the manuscript. M.S. and S.F. take responsibility for the collection of data, the interpretation and publication. All authors have given final approval of the version to be published. Marit Schwantje, Merel Ebberink, Mirjam Doolaard, Jos Ruiter, Sabine Fuchs, Niklas Darin Carola Hedberg-Oldfors, Luc Régal, Laura Donker Kaat, Hidde Huidekoper, Simon Olpin, Duncan Cole, Stuart Moat, Gepke Visser and Sacha Ferdinandusse declare to have no potential conflicts of interests. None of the authors have accepted reimbursements, fees, funds, or salaries from an organization that may in any way gain or lose financially from the results reported in this manuscript. None of the authors have any competing interests regarding relevant financial activities outside the submitted work, intellectual property or any other relationships. This research was funded by Metakids and PNO zorg. The content of the article has not been influenced by the sponsor. Ethical approval was not required for this study, as the paper does not report on primary research but on a retrospective chart study. All data were collected as part of routine diagnostics and treatment. All procedures followed were in accordance with the ethical standards of the responsible committee on human experimentation (institutional and national) and with the Helsinki Declaration of 1975, as revised in 2000. Informed consent for publication was obtained from all patients and/or their guardians. This work was generated within the "United for Metabolic Diseases (UMD)" and the "European Reference Network for Hereditary Metabolic Disorders (MetaBERN)". The authors are grateful for their efforts to improve care for patients with (genetic) metabolic diseases. We thank Conny Dekker and Heleen te Brinke for their technical assistance on the immunoblot analysis.
    Keywords:  Long-chain fatty acid oxidation disorders; long-chain ketoacyl-CoA thiolase deficiency; mitochondrial trifunctional protein complex; mitochondrial trifunctional protein deficiency; myopathy; thermo-sensitivity
    DOI:  https://doi.org/10.1002/jimd.12503
  11. Nat Cell Biol. 2022 Apr 13.
      D-2-Hydroxyglutarate (D-2HG) is an α-ketoglutarate-derived mitochondrial metabolite that causes D-2-hydroxyglutaric aciduria, a devastating developmental disorder. How D-2HG adversely affects mitochondria is largely unknown. Here, we report that in Caenorhabditis elegans, loss of the D-2HG dehydrogenase DHGD-1 causes D-2HG accumulation and mitochondrial damage. The excess D-2HG leads to a build-up of 3-hydroxypropionate (3-HP), a toxic metabolite in mitochondrial propionate oxidation, by inhibiting the 3-HP dehydrogenase HPHD-1. We demonstrate that 3-HP binds the MICOS subunit MIC60 (encoded by immt-1) and inhibits its membrane-binding and membrane-shaping activities. We further reveal that dietary and gut bacteria affect mitochondrial health by modulating the host production of 3-HP. These findings identify a feedback loop that links the toxic effects of D-2HG and 3-HP on mitochondria, thus providing important mechanistic insights into human diseases related to D-2HG and 3-HP.
    DOI:  https://doi.org/10.1038/s41556-022-00883-2
  12. Methods Mol Biol. 2022 ;2431 385-407
      Precise distribution of mitochondria is essential for maintaining neuronal homeostasis. Although detailed mechanisms governing the transport of mitochondria have emerged, it is still poorly understood how the regulation of transport is coordinated in space and time within the physiological context of an organism. How alteration in mitochondrial functionality may trigger changes in organellar dynamics also remains unclear in this context. Therefore, the use of genetically encoded tools to perturb mitochondrial functionality in real time would be desirable. Here we describe methods to interfere with mitochondrial function with high spatiotemporal precision with the use of photosensitizers in vivo in the intact wing nerve of adult Drosophila. We also provide details on how to visualize the transport of mitochondria and to improve the quality of the imaging to attain super-resolution in this tissue.
    Keywords:  Axonal transport; Drosophila; Intravital imaging; KillerRed; Mitochondria; Neurons; Reactive oxygen species (ROS); Super-resolution radial fluctuations (SRRF); SuperNova
    DOI:  https://doi.org/10.1007/978-1-0716-1990-2_20
  13. Commun Biol. 2022 Apr 12. 5(1): 349
      Protein O-GlcNAcylation is increasingly recognized as an important cellular regulatory mechanism, in multiple organs including the heart. However, the mechanisms leading to O-GlcNAcylation in mitochondria and the consequences on their function remain poorly understood. In this study, we use an in vitro reconstitution assay to characterize the intra-mitochondrial O-GlcNAc system without potential cytoplasmic confounding effects. We compare the O-GlcNAcylome of isolated cardiac mitochondria with that of mitochondria acutely exposed to NButGT, a specific inhibitor of glycoside hydrolase. Amongst the 409 O-GlcNAcylated mitochondrial proteins identified, 191 display increased O-GlcNAcylation in response to NButGT. This is associated with enhanced Complex I (CI) activity, increased maximal respiration in presence of pyruvate-malate, and a striking reduction of mitochondrial ROS release, which could be related to O-GlcNAcylation of specific subunits of ETC complexes (CI, CIII) and TCA cycle enzymes. In conclusion, our work underlines the existence of a dynamic mitochondrial O-GlcNAcylation system capable of rapidly modifying mitochondrial function.
    DOI:  https://doi.org/10.1038/s42003-022-03282-3
  14. Front Oncol. 2022 ;12 865686
      Voltage-gated potassium channels control neuronal excitability and cardiac action potentials. In addition, these proteins are involved in a myriad of cellular processes. The potassium channel Kv1.3 plays an essential role in the immune response mediated by leukocytes. Kv1.3 is functional both at the plasma membrane and the inner mitochondrial membrane. Plasma membrane Kv1.3 mediates cellular activation and proliferation, whereas mitochondrial Kv1.3 participates in cell survival and apoptosis. Therefore, this protein emerges as an important target in cancer therapies. Several forward-traffic motifs target the channel to the plasma membrane in a COPII-dependent manner. However, the mitochondrial import pathway for Kv1.3 is largely unknown. Here, we deciphered the mitochondrial routing of the mitoKv1.3 channel. Kv1.3 uses the TIM23 complex to translocate to the inner mitochondrial membrane. This mechanism is unconventional because the channel is a multimembrane spanning protein without a defined N-terminal presequence. We found that transmembrane domains cooperatively mediate Kv1.3 mitochondrial targeting and identified the cytosolic HSP70/HSP90 chaperone complex as a key regulator of the process. Our results provide insights into the mechanisms mediating the localization of Kv1.3 to mitochondrial membranes, further extending the knowledge of ion channel biogenesis and turnover in mitochondria.
    Keywords:  TIM-TOM complex; apoptosis; cancer; mitochondria; potassium channels
    DOI:  https://doi.org/10.3389/fonc.2022.865686
  15. Front Neurol. 2022 ;13 863047
       Introduction: Verbal auditory agnosia is rarely caused by mitochondrial encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) syndrome. Lactate acidosis, which is the adverse effect of metformin, has proposed links to mitochondrial dysfunction and may trigger clinical features of mitochondrial diseases.
    Case Presentation: A 43-year-old right-handed man presented to our emergency department with acute onset fever and headache accompanied by impaired hearing comprehension. He could communicate well through handwritten notes but could not understand what others were saying. He had been diagnosed as having diabetes mellitus 2 months prior to this event. Vildagliptin 100 mg/day and metformin 1,700 mg/day were prescribed for glucose control. Laboratory tests revealed elevated lactate levels in serum and cerebrospinal fluid of the patient. Brain MRI disclosed bilateral temporal lesions. Acute encephalitis with temporal involved was initially diagnosed and acyclovir was given empirically. However, follow-up MRI after acyclovir treatment revealed a progression of prior lesions. Further mitochondrial genome analysis revealed a mitochondrial DNA point mutation at position 3,243 (m.3243A > G) with 25% heteroplasmy, which is compatible with MELAS. His clinical symptoms and serum lactate levels were improved after discontinuing the metformin use.
    Conclusions: To our knowledge, this is the first report of a patient having late-onset MELAS syndrome that manifested as acute verbal auditory agnosia, which was identified after the patient began using metformin. Metformin is known to inhibit mitochondrial function and could trigger clinical features of MELAS syndrome. We encourage clinicians to maintain a high level of awareness that diabetes mellitus can be caused by mitochondrial disease and to exercise caution in the prescription of metformin.
    Keywords:  MELAS; auditory agnosia; case report; metformin; pure word deafness
    DOI:  https://doi.org/10.3389/fneur.2022.863047
  16. J Clin Invest. 2022 Apr 15. pii: e157560. [Epub ahead of print]132(8): 1-5
      RASopathies are a family of rare autosomal dominant disorders that affect the canonical Ras/MAPK signaling pathway and manifest as neurodevelopmental systemic syndromes, including Costello syndrome (CS). In this issue of the JCI, Dard et al. describe the molecular determinants of CS using a myriad of genetically modified models, including mice expressing HRAS p.G12S, patient-derived skin fibroblasts, hiPSC-derived human cardiomyocytes, an HRAS p.G12V zebrafish model, and human lentivirally induced fibroblasts overexpressing HRAS p.G12S or HRAS p.G12A. Mitochondrial proteostasis and oxidative phosphorylation were altered in CS, and inhibition of the AMPK signaling pathway mediated bioenergetic changes. Importantly, the pharmacological induction of this pathway restored cardiac function and reduced the developmental defects associated with CS. These findings identify a role for altered bioenergetics and provide insights into more effective treatment strategies for patients with RASopathies.
    DOI:  https://doi.org/10.1172/JCI157560
  17. Elife. 2022 Apr 11. pii: e76557. [Epub ahead of print]11
      High frequencies of mutant mitochondrial DNA (mtDNA) in human cells lead to cellular defects that are associated with aging and disease. Yet much remains to be understood about the dynamics of the generation of mutant mtDNAs and their relative replicative fitness that informs their fate within cells and tissues. To address this, we utilize long-read single-molecule sequencing to track mutational trajectories of mtDNA in the model organism Saccharomyces cerevisiae. This model has numerous advantages over mammalian systems due to its much larger mtDNA and ease of artificially competing mutant and wild-type mtDNA copies in cells. We show a previously unseen pattern that constrains subsequent excision events in mtDNA fragmentation in yeast. We also provide evidence for the generation of rare and contentious non-periodic mtDNA structures that lead to persistent diversity within individual cells. Finally, we show that measurements of relative fitness of mtDNA fit a phenomenological model that highlights important biophysical parameters governing mtDNA fitness. Altogether, our study provides techniques and insights into the dynamics of large structural changes in genomes that we show are applicable to more complex organisms like humans.
    Keywords:  S. cerevisiae; computational biology; genetics; genomics; systems biology
    DOI:  https://doi.org/10.7554/eLife.76557
  18. STAR Protoc. 2022 Jun 17. 3(2): 101275
      Cardiac fibroblasts (CFBs) are a key therapeutic target due to their supportive roles during heart development and response to injury and disease. Here, we describe a robust protocol to differentiate human pluripotent stem cells (hPSCs) into CFBs through an epicardial intermediate. We discuss in detail the characterization of the resulting epicardial-derived fibroblasts (EpiC-FBs) using immunofluorescence microscopy, flow cytometry, and qPCR. We anticipate that these EpiC-FBs can be applied to drug testing, disease modeling, and tissue engineering. For complete details on the use and execution of this protocol, please refer to Bao et al. (2016), Floy et al. (2021), and Lian et al. (2015).
    Keywords:  Cell Biology; Cell Differentiation; Developmental biology; Flow Cytometry/Mass Cytometry; Microscopy; Molecular Biology; Stem Cells
    DOI:  https://doi.org/10.1016/j.xpro.2022.101275
  19. Science. 2022 Apr 15. 376(6590): eabf8271
      Nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) remain without effective therapies. The mechanistic target of rapamycin complex 1 (mTORC1) pathway is a potential therapeutic target, but conflicting interpretations have been proposed for how mTORC1 controls lipid homeostasis. We show that selective inhibition of mTORC1 signaling in mice, through deletion of the RagC/D guanosine triphosphatase-activating protein folliculin (FLCN), promotes activation of transcription factor E3 (TFE3) in the liver without affecting other mTORC1 targets and protects against NAFLD and NASH. Disease protection is mediated by TFE3, which both induces lipid consumption and suppresses anabolic lipogenesis. TFE3 inhibits lipogenesis by suppressing proteolytic processing and activation of sterol regulatory element-binding protein-1c (SREBP-1c) and by interacting with SREBP-1c on chromatin. Our data reconcile previously conflicting studies and identify selective inhibition of mTORC1 as a potential approach to treat NASH and NAFLD.
    DOI:  https://doi.org/10.1126/science.abf8271
  20. Sci Rep. 2022 Apr 13. 12(1): 6181
      Charcot-Marie-Tooth disease type 2A (CMT2A) is a rare inherited axonal neuropathy caused by mutations in MFN2 gene, which encodes Mitofusin 2, a transmembrane protein of the outer mitochondrial membrane. We performed a cross-sectional analysis on thirteen patients carrying mutations in MFN2, from ten families, describing their clinical and genetic characteristics. Evaluated patients presented a variable age of onset and a wide phenotypic spectrum, with most patients presenting a severe phenotype. A novel heterozygous missense variant was detected, p.K357E. It is located at a highly conserved position and predicted as pathogenic by in silico tools. At a clinical level, the p.K357E carrier shows a severe sensorimotor axonal neuropathy. In conclusion, our work expands the genetic spectrum of CMT2A, disclosing a novel mutation and its related clinical effect, and provides a detailed description of the clinical features of a cohort of patients with MFN2 mutations. Obtaining a precise genetic diagnosis in affected families is crucial both for family planning and prenatal diagnosis, and in a therapeutic perspective, as we are entering the era of personalized therapy for genetic diseases.
    DOI:  https://doi.org/10.1038/s41598-022-10220-0
  21. Cell Rep. 2022 Apr 12. pii: S2211-1247(22)00387-4. [Epub ahead of print]39(2): 110635
      Circadian genes such as Clock, Bmal1, Cryptochrome1/2, and Period1/2/3 constitute the precise circadian system. ClockΔ19 is a commonly used mouse model harboring a circadian clock gene mutation, which lacks the EXON-19-encoded 51 amino acids. Previous reports have shown that ClockΔ19 mice have severe metabolic abnormalities. Here, we report that the mitochondria of ClockΔ19 mice exhibit excessive fission and dysfunction. We also demonstrate that CLOCK binds to the RNA-binding protein PUF60 through its EXON 19. Further, we find that PUF60 directly maintains mitochondrial homeostasis through regulating Drp1 mRNA stability, while the association with CLOCK can competitively inhibit this function. In ClockΔ19 mice, CLOCKΔ19 releases PUF60, leading to enhanced Drp1 mRNA stability and persistent mitochondrial fission. Our results reveal a direct post-transcriptional role of CLOCK in regulating mitochondrial homeostasis via Drp1 mRNA stability and that the loss of EXON 19 of CLOCK in ClockΔ19 mice leads to severe mitochondrial homeostasis disorders.
    Keywords:  CP: Metabolism; CP: Molecular biology; Clock; Drp1; PUF60; mRNA stability; mitochondrial fission
    DOI:  https://doi.org/10.1016/j.celrep.2022.110635
  22. Elife. 2022 Apr 11. pii: e74335. [Epub ahead of print]11
      
    Background: Master athletes prove that preserving a high level of physical function up to very late in life is possible, but the mechanisms responsible for their high function remain unclear.
    Methods: We performed muscle biopsies in 15 octogenarian world class track and field masters athletes (MA) and 14 non-athlete age/sex-matched controls (NA) to provide insights into mechanisms for preserving function in advanced age. Muscle samples were assessed for respiratory compromised fibers, mtDNA copy number, and proteomics by liquid-chromatography mass spectrometry.
    Results: MA exhibited markedly better performance on clinical function tests and greater cross-sectional area of the vastus lateralis muscle. Proteomics analysis revealed marked differences, where most of the ~800 differentially represented proteins in MA versus NA pertained to mitochondria structure/function such as electron transport capacity (ETC), cristae formation, mitochondrial biogenesis, and mtDNA-encoded proteins. In contrast, proteins from the spliceosome complex and nuclear pore were downregulated in MA. Consistent with proteomics data, MA had fewer respiratory compromised fibers, higher mtDNA copy number, and an increased protein ratio of the cristae-bound ETC subunits relative to the outer mitochondrial membrane protein voltage dependent anion channel. There was a substantial overlap of proteins overrepresented in MA versus NA with proteins that decline with aging and which are higher in physically active than sedentary individuals. However, we also found 176 proteins related to mitochondria that are uniquely differentially expressed in MA.
    Conclusions: We conclude that high function in advanced age is associated with preserving mitochondrial structure/function proteins, with under-representation of proteins involved in the spliceosome and nuclear pore complex. Whereas many of these differences in MA appear related to their physical activity habits, others may reflect unique biological (e.g., gene, environment) mechanisms that preserve muscle integrity and function with aging.
    Funding: Funding for this study was provided by operating grants from the Canadian Institutes of Health Research (MOP 84408 to TT and MOP 125986 to RTH). Supported in part by the Intramural Research Program of the National Institute on Aging, NIH, Baltimore, MD, United States.
    Keywords:  epidemiology; global health; human
    DOI:  https://doi.org/10.7554/eLife.74335
  23. Nat Genet. 2022 Apr;54(4): 518-525
      Typical genotyping workflows map reads to a reference genome before identifying genetic variants. Generating such alignments introduces reference biases and comes with substantial computational burden. Furthermore, short-read lengths limit the ability to characterize repetitive genomic regions, which are particularly challenging for fast k-mer-based genotypers. In the present study, we propose a new algorithm, PanGenie, that leverages a haplotype-resolved pangenome reference together with k-mer counts from short-read sequencing data to genotype a wide spectrum of genetic variation-a process we refer to as genome inference. Compared with mapping-based approaches, PanGenie is more than 4 times faster at 30-fold coverage and achieves better genotype concordances for almost all variant types and coverages tested. Improvements are especially pronounced for large insertions (≥50 bp) and variants in repetitive regions, enabling the inclusion of these classes of variants in genome-wide association studies. PanGenie efficiently leverages the increasing amount of haplotype-resolved assemblies to unravel the functional impact of previously inaccessible variants while being faster compared with alignment-based workflows.
    DOI:  https://doi.org/10.1038/s41588-022-01043-w
  24. Geroscience. 2022 Apr 13.
      We analyzed the effects of aging on protein abundance and acetylation, as well as the ability of the mitochondrial-targeted drugs elamipretide (SS-31) and nicotinamide mononucleotide (NMN) to reverse aging-associated changes in mouse hearts. Both drugs had a modest effect on restoring the abundance and acetylation of proteins that are altered with age, while also inducing additional changes. Age-related increases in protein acetylation were predominantly in mitochondrial pathways such as mitochondrial dysfunction, oxidative phosphorylation, and TCA cycle signaling. We further assessed how these age-related changes associated with diastolic function (Ea/Aa) and systolic function (fractional shortening under higher workload) measurements from echocardiography. These results identify a subset of protein abundance and acetylation changes in muscle, mitochondrial, and structural proteins that appear to be essential in regulating diastolic function in old hearts.
    Keywords:  Acetylomics; Aging; Elamipretide; Heart; Mitochondria; NMN; Proteomics; SS-31
    DOI:  https://doi.org/10.1007/s11357-022-00564-w
  25. Neurology. 2022 Apr 15. pii: 10.1212/WNL.0000000000200299. [Epub ahead of print]
       OBJECTIVE: Stroke management in the context of primary mitochondrial disease is clinically challenging and the best treatment options for patients with stroke-like episodes remain uncertain. We sought to perform a systematic review on the safety and efficacy of L-arginine use in the acute and prophylactic management of stroke-like episodes in patients with mitochondrial disease.
    METHODS: The systematic review was registered in PROSPERO (CRD42020181230). We searched six databases from inception - 15/01/2021: MEDLINE, Embase, Scopus, Web of Science, CINAHL and ClinicalTrials.gov. Original articles and registered trials available, in English, reporting L-arginine use in the acute or prophylactic management of stroke-like episodes in patients with genetically confirmed mitochondrial disease were eligible for inclusion.Data on safety and treatment response were extracted and summarized by multiple observers. Risk of bias was assessed by the methodological quality of case reports, case series and a risk-of-bias checklist for non-randomized studies. Quality of evidence was synthesized using the Oxford Centre for Evidence-Based Medicine Levels of Evidence and Grade of Recommendations. The predetermined main outcome measures were clinical response to L-arginine treatment, adverse events, withdrawals, and deaths (on treatment and/or during follow up), as defined by the author.
    RESULTS: Thirty-seven articles met inclusion [0=randomised controlled trials (RCTs); 3 open-label; 1 retrospective cohort; 33 case reports/case series] (N = 91 patients; 86% m.3243A>G). In the case reports, 54% of patients reported a positive clinical response to acute L-arginine, of which 40% were concomitantly treated with AEDs. Improved headache at 24-hours was the greatest reported benefit in response to IV L-arginine in the open-label trials (31/39, 79%). Of 15/48 patients (31%) who positively responded to prophylactic L-arginine, AEDs were either used (7/15) or unreported (8/15). Moderate adverse events were reported in the follow-up of both IV and oral L-arginine treatment and 11 patients (12%) died during follow up or while on prophylactic treatment.
    CONCLUSION: The available evidence is of poor methodological quality and classified as Level 5. IV and/or oral L-arginine confers no demonstrable clinical benefit in either the acute or prophylactic treatment of MELAS, with more robust controlled trials required to assess its efficacy and safety profile.
    DOI:  https://doi.org/10.1212/WNL.0000000000200299
  26. Signal Transduct Target Ther. 2022 Apr 15. 7(1): 103
      Dynamic change of mitochondrial morphology and distribution along neuronal branches are essential for neural circuitry formation and synaptic efficacy. However, the underlying mechanism remains elusive. We show here that Pink1 knockout (KO) mice display defective dendritic spine maturation, reduced axonal synaptic vesicles, abnormal synaptic connection, and attenuated long-term synaptic potentiation (LTP). Drp1 activation via S616 phosphorylation rescues deficits of spine maturation in Pink1 KO neurons. Notably, mice harboring a knockin (KI) phosphor-null Drp1S616A recapitulate spine immaturity and synaptic abnormality identified in Pink1 KO mice. Chemical LTP (cLTP) induces Drp1S616 phosphorylation in a PINK1-dependent manner. Moreover, phosphor-mimetic Drp1S616D restores reduced dendritic spine localization of mitochondria in Pink1 KO neurons. Together, this study provides the first in vivo evidence of functional regulation of Drp1 by phosphorylation and suggests that PINK1-Drp1S616 phosphorylation coupling is essential for convergence between mitochondrial dynamics and neural circuitry formation and refinement.
    DOI:  https://doi.org/10.1038/s41392-022-00933-z
  27. Cells. 2022 Apr 06. pii: 1246. [Epub ahead of print]11(7):
      Little is known about the early pathogenic events by which mutant superoxide dismutase 1 (SOD1) causes amyotrophic lateral sclerosis (ALS). This lack of mechanistic understanding is a major barrier to the development and evaluation of efficient therapies. Although protein aggregation is known to be involved, it is not understood how mutant SOD1 causes degeneration of motoneurons (MNs). Previous research has relied heavily on the overexpression of mutant SOD1, but the clinical relevance of SOD1 overexpression models remains questionable. We used a human induced pluripotent stem cell (iPSC) model of spinal MNs and three different endogenous ALS-associated SOD1 mutations (D90Ahom, R115Ghet or A4Vhet) to investigate early cellular disturbances in MNs. Although enhanced misfolding and aggregation of SOD1 was induced by proteasome inhibition, it was not affected by activation of the stress granule pathway. Interestingly, we identified loss of mitochondrial, but not lysosomal, integrity as the earliest common pathological phenotype, which preceded elevated levels of insoluble, aggregated SOD1. A super-elongated mitochondrial morphology with impaired inner mitochondrial membrane potential was a unifying feature in mutant SOD1 iPSC-derived MNs. Impaired mitochondrial integrity was most prominent in mutant D90Ahom MNs, whereas both soluble disordered and detergent-resistant misfolded SOD1 was more prominent in R115Ghet and A4Vhet mutant lines. Taking advantage of patient-specific models of SOD1-ALS in vitro, our data suggest that mitochondrial dysfunction is one of the first crucial steps in the pathogenic cascade that leads to SOD1-ALS and also highlights the need for individualized medical approaches for SOD1-ALS.
    Keywords:  ALS1; SOD1; axonal trafficking; live cell imaging; mitochondria
    DOI:  https://doi.org/10.3390/cells11071246
  28. J Biol Chem. 2022 Apr 09. pii: S0021-9258(22)00366-0. [Epub ahead of print] 101926
      Skeletal muscle dynamically regulates systemic nutrient homeostasis through transcriptional adaptations to physiological cues. In response to changes in the metabolic environment (e.g., alterations in circulating glucose or lipid levels), networks of transcription factors and co-regulators are recruited to specific genomic loci to fine-tune homeostatic gene regulation. Elucidating these mechanisms is of particular interest as these gene regulatory pathways can serve as potential targets to treat metabolic disease. The zinc-finger transcription factor Krüppel-like factor 15 (KLF15) is a critical regulator of metabolic homeostasis, however its genome-wide distribution in skeletal muscle has not been previously identified. Here, we characterize the KLF15 cistrome in vivo in skeletal muscle and find that the majority of KLF15 binding is localized to distal intergenic regions and associated with genes related to circadian rhythmicity and lipid metabolism. We also identify critical interdependence between KLF15 and the nuclear receptor PPARδ in the regulation of lipid metabolic gene programs. We further demonstrate that KLF15 and PPARδ co-localize genome-wide, physically interact, and are dependent on one another to exert their transcriptional effects on target genes. These findings reveal that skeletal muscle KLF15 plays a critical role in metabolic adaptation through its direct actions on target genes and interactions with other nodal transcription factors such as PPARδ.
    Keywords:  Kruppel-like factor (KLF); energy metabolism; metabolism; nuclear receptors; peroxisome proliferator-activated receptors; skeletal muscle; transcription; transcription factors
    DOI:  https://doi.org/10.1016/j.jbc.2022.101926
  29. Int J Mol Sci. 2022 Mar 26. pii: 3650. [Epub ahead of print]23(7):
      Given the popularity of ketogenic diets, their potential long-term consequences deserve to be more carefully monitored. Mitochondrially derived formate has a critical role in mammalian one-carbon (1C) metabolism and development. The glycine cleavage system (GCS) accounts for another substantial source for mitochondrially derived 1C units.
    OBJECTIVE: We investigated how the ketogenic state modulates mitochondrial formate generation and partitioning of 1C metabolic fluxes.
    DESIGN: HepG2 cells treated with physiological doses (1 mM and 10 mM) of β-hydroxybutyrate (βHB) were utilized as the in vitro ketogenic model. Eight-week male C57BL/6JNarl mice received either a medium-chain fatty-acid-enriched ketogenic diet (MCT-KD) or a control diet AIN 93M for 8 weeks. Stable isotopic labeling experiments were conducted.
    RESULTS AND CONCLUSIONS: MCT-KD is effective in weight and fat loss. Deoxythymidine (dTMP) synthesis from the mitochondrial GCS-derived formate was significantly suppressed by βHB and consumption of MCT-KD. Consistently, plasma formate concentrations, as well as the metabolic fluxes from serine and glycine, were suppressed by MCT-KD. MCT-KD also decreased the fractional contribution of mitochondrially derived formate in methionine synthesis from serine. With the worldwide application, people and medical professionals should be more aware of the potential metabolic perturbations when practicing a long-term ketogenic diet.
    Keywords:  glycine cleavage system; ketogenic diet; medium-chain triglycerides; mitochondrial formate production; one-carbon metabolism; stable isotopic labeling experiments
    DOI:  https://doi.org/10.3390/ijms23073650
  30. Eur J Med Genet. 2022 Apr 06. pii: S1769-7212(22)00062-3. [Epub ahead of print]65(6): 104481
      Neurodevelopmental disorder with cardiomyopathy, spasticity, and brain abnormalities (NEDCASB; MIM# 619121) is a recently described metabolic disorder with characteristic features of mild dysmorphism, intellectual disability, spasticity, peripheral neuropathy, cardiomyopathy, and thin corpus callosum. Biallelic variants in SHMT2 (MIM 138450), encoding mitochondrial serine hydroxymethyltransferase enzyme, have been recently linked to this disorder. Till now, a total of seven variants including six missense and one deletion-insertion has been reported in SHMT2. We hereby report an additional individual with novel homozygous missense variant c.1133A > G in SHMT2 (NM_005412.6) identified by exome sequencing and review the phenotype and genotype of the previously reported individuals with NEDCASB.
    Keywords:  Cardiomyopathy; Mitochondria; SHMT2
    DOI:  https://doi.org/10.1016/j.ejmg.2022.104481
  31. Nat Commun. 2022 Apr 13. 13(1): 1991
      Estrogen-related receptors (ERR) α and γ were shown recently to serve as regulators of cardiac maturation, yet the underlying mechanisms have not been delineated. Herein, we find that ERR signaling is necessary for induction of genes involved in mitochondrial and cardiac-specific contractile processes during human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) differentiation. Genomic interrogation studies demonstrate that ERRγ occupies many cardiomyocyte enhancers/super-enhancers, often co-localizing with the cardiogenic factor GATA4. ERRγ interacts with GATA4 to cooperatively activate transcription of targets involved in cardiomyocyte-specific processes such as contractile function, whereas ERRγ-mediated control of metabolic genes occurs independent of GATA4. Both mechanisms require the transcriptional coregulator PGC-1α. A disease-causing GATA4 mutation is shown to diminish PGC-1α/ERR/GATA4 cooperativity and expression of ERR target genes are downregulated in human heart failure samples suggesting that dysregulation of this circuitry may contribute to congenital and acquired forms of heart failure.
    DOI:  https://doi.org/10.1038/s41467-022-29733-3
  32. J Hepatol. 2022 Apr 11. pii: S0168-8278(22)00226-4. [Epub ahead of print]
       BACKGROUND & AIMS: Mitochondrial dysfunction is considered a pathogenic linker in the development of non-alcoholic steatohepatitis (NASH). Inappropriate mitochondrial protein-quality control, possibly induced due to insufficiency of the mitochondrial matrix caseinolytic protease P (ClpP), can potentially cause mitochondrial dysfunction. Herein, we aimed to investigate hepatic ClpP levels in a diet-induced model of NASH and determine whether supplementation of ClpP can ameliorate the diet-induced NASH.
    METHODS: NASH was induced by a high fat/high fructose (HF/HFr) diet in C57BL/6J mice. Stress/inflammatory signals were induced in mouse primary hepatocytes (MPHs) by treatment with palmitate/oleate (PA/OA). ClpP levels in hepatocytes were reduced using the RNAi-mediated gene knockdown technique but increased through the viral transduction of ClpP. ClpP activation was induced by administering a chemical activator of ClpP.
    RESULTS: Hepatic ClpP protein levels in C57BL/6J mice fed a HF/HFr diet were lower than the levels in those fed a normal chow diet. PA/OA treatment also decreased the ClpP protein levels in MPHs. Overexpression or activation of ClpP reversed the PA/OA-induced mitochondrial dysfunction and stress/inflammatory signal activation in MPHs, whereas ClpP knockdown induced mitochondrial dysfunction and stress/inflammatory signals in these cells. On the other hand, ClpP overexpression or activation improved HF/HFr-induced NASH characteristics such as hepatic steatosis, inflammation, fibrosis, and injury in the C57BL/6J mice, whereas ClpP knockdown further augmented steatohepatitis in mice fed a HF/HFr diet.
    CONCLUSIONS: Reduced ClpP expression and subsequent mitochondrial dysfunction are key to the development of diet-induced NASH. ClpP supplementation through viral transduction or chemical activation represents potential therapeutic strategies for preventing diet-induced NASH.
    Keywords:  inflammation; mitochondrial dysfunction; proteostasis; steatohepatitis
    DOI:  https://doi.org/10.1016/j.jhep.2022.03.034
  33. Nat Commun. 2022 Apr 14. 13(1): 1998
      Cell type assignment is a major challenge for all types of high throughput single cell data. In many cases such assignment requires the repeated manual use of external and complementary data sources. To improve the ability to uniformly assign cell types across large consortia, platforms and modalities, we developed Cellar, a software tool that provides interactive support to all the different steps involved in the assignment and dataset comparison process. We discuss the different methods implemented by Cellar, how these can be used with different data types, how to combine complementary data types and how to analyze and visualize spatial data. We demonstrate the advantages of Cellar by using it to annotate several HuBMAP datasets from multi-omics single-cell sequencing and spatial proteomics studies. Cellar is open-source and includes several annotated HuBMAP datasets.
    DOI:  https://doi.org/10.1038/s41467-022-29744-0
  34. Mol Genet Metab. 2022 Mar 25. pii: S1096-7192(22)00179-2. [Epub ahead of print]
      Very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency has been a target of expanded newborn screening (ENBS) using tandem mass spectrometry in Japan. Since the implementation of ENBS, a number of novel ACADVL variants responsible for VLCAD deficiency have been identified. In this study, genotypic differences in Japanese patients with VLCAD deficiency were investigated before and after ENBS. The ACADVL variants in 61 subjects identified through ENBS (ENBS group) and in 40 patients who subsequently developed clinical symptoms without undergoing ENBS (pre-ENBS group) were compared. Subjects in the ENBS group underwent genetic testing and/or VLCAD enzyme activity measurements. Patients in the pre-ENBS group were stratified into three clinical phenotypes and underwent genetic testing. This study revealed that the variants p.K264E, p.K382Q and c.996dupT were found in both groups, but their frequencies were lower in the ENBS group (5.2%, 3.1% and 4.2%, respectively) than in the pre-ENBS group (16.5%, 12.7% and 10.1%, respectively). In addition, p.C607S, p.T409M, p.M478I, p.G289R, p.C237R, p.T260M, and p.R229* were exclusively identified in the ENBS group. Among these variants, p.C607S exhibited the highest frequency (18.8%). The patients who were heterozygous for p.C607S demonstrated 7-42% of control enzyme activity. p.C607S is suspected to be unique to Japanese individuals. According to a comparison of enzyme activity, patients with the p.C607S variant may exhibit higher enzyme activity than those with the p.A416T, p.A180T, p.R450H, and p.K264E variants, which are responsible for the myopathic form of the disease. The VLCAD deficiency genotypes have changed since the initiation of ENBS in Japan.
    Keywords:  Common variant; Enzyme activity; Genotype-phenotype correlation; Newborn screening; Very long-chain acyl-CoA dehydrogenase (VLCAD) deficiency; p.C607S
    DOI:  https://doi.org/10.1016/j.ymgme.2022.03.009
  35. Cell. 2022 Apr 14. pii: S0092-8674(22)00337-3. [Epub ahead of print]185(8): 1444-1444.e1
      The peroxisome proliferator-activated receptor γ coactivator-1α (Ppargc1a) gene encodes several PGC-1α isoforms that regulate mitochondrial bioenergetics and cellular adaptive processes. Expressing specific PGC-1α isoforms in mice can confer protection in different disease models. This SnapShot summarizes how regulation of Ppargc1a transcription, splicing, translation, protein stability, and activity underlies its multifaceted functions. To view this SnapShot, open or download the PDF.
    DOI:  https://doi.org/10.1016/j.cell.2022.03.027
  36. Cell Chem Biol. 2022 Apr 09. pii: S2451-9456(22)00129-5. [Epub ahead of print]
      A critical step in repurposing the cellular translation machinery for the synthesis of polymeric products is the acylation of transfer RNA (tRNA) with unnatural monomers. Toward this goal, flexizymes, ribozymes capable of aminoacylation, have emerged as a uniquely adept tool for charging tRNA with ever increasingly diverse substrates. In this review, we present a library of monomer substrates that have been tested for tRNA acylation with the flexizyme system. From this mile-high view, we provide insights for understanding the chemical factors that influence flexizyme-mediated tRNA acylation. We conclude that flexizymes are primitive esterification catalysts that display a modest binding affinity to the monomer's aromatic recognition element. Together, these robust, yet flexible, flexizyme systems provide researchers with unprecedented access for preparing unnatural acyl-tRNA and the opportunity to repurpose the translation machinery for the synthesis of novel biologically derived structures beyond native proteins and peptides.
    Keywords:  aminoacylation; flexizyme; genetic code; ribozymes; tRNA; translation
    DOI:  https://doi.org/10.1016/j.chembiol.2022.03.012
  37. Cell Metab. 2022 Apr 09. pii: S1550-4131(22)00125-5. [Epub ahead of print]
      We investigate the extent to which human genetic data are incorporated into studies that hypothesize novel links between genes and metabolic disease. To lower the barriers to using genetic data, we present an approach to enable researchers to evaluate human genetic support for experimentally determined hypotheses.
    DOI:  https://doi.org/10.1016/j.cmet.2022.03.011
  38. Hum Mutat. 2022 Apr 14.
      To avoid acquired variants found in the blood, cultured skin fibroblasts are a recommended DNA source for germline genetic testing in patients with hematologic disorders, but data are lacking regarding practicality and limitations. We conducted a retrospective cohort study of 350 subjects with hematologic disorders who underwent skin fibroblast culture for germline genetic testing. We analyzed next-generation sequencing data from the targeted capture of 144 inherited cancer and bonemarrow failure genes to identify variants at heterozygous and subclonal variant allele frequencies. Sixteen (5%) biopsies failed to culture. Culture failure was more likely in samples with delays in culture initiation (OR = 4.3; p < 0.01) or a pathogenic variant in a telomere gene (OR = 42.6; p < 0.01). Median culture time was 28 days (IQR 22-29 days). Culture time was longer for subjects with prior allogeneic stem cell transplantation (+10.7%; p = 0.02) and shorter in subjects with a heterozygous pathogenic variant (-11.9%; p < 0.01), larger biopsy size (-10.6%; p < 0.01), or lymphoid malignancy (-8.4%; p < 0.01). Subclonal variants were identified in 10 (4%) and confirmed in five (56%) of eight with alternate samples available. Subclonal and discordant variants illustrate that germline testing from cultured skin fibroblasts requires phenotypic correlation and, in rare cases, follow-up studies for optimal interpretation.
    Keywords:  germline genetics; inherited; leukemia; lymphoma; myelodysplastic syndrome; skin fibroblasts
    DOI:  https://doi.org/10.1002/humu.24374
  39. Front Mol Biosci. 2022 ;9 714008
      Inefficient differentiation and insufficient maturation are barriers to the application of human pluripotent stem cell (hPSC)-derived cardiomyocytes (CMs) for research and therapy. Great strides have been made to the former, and multiple groups have reported cardiac differentiation protocol that can generate hPSC-CMs at high efficiency. Although many such protocols are based on the modulation of the WNT signaling pathway, they differ in their timing and in the WNT inhibitors used. Little is currently known about whether and how conditions of differentiation affect cardiac maturation. Here we adapted multiple cardiac differentiation protocols to improve cost-effectiveness and consistency, and compared the properties of the hPSC-CMs generated. Our results showed that the schedule of differentiation, but not the choice of WNT inhibitors, was a critical determinant not only of differentiation efficiency, which was expected, but also CM maturation. Among cultures with comparable purity, hPSC-CMs generated with different differentiation schedules vary in the expression of genes which are important for developmental maturation, and in their structural, metabolic, calcium transient and proliferative properties. In summary, we demonstrated that simple changes in the schedule of cardiac differentiation could promote maturation. To this end, we have optimized a cardiac differentiation protocol that can simultaneously achieve high differentiation efficiency and enhanced developmental maturation. Our findings would advance the production of hPSC-CMs for research and therapy.
    Keywords:  cardiac differentiation; cardiac maturation; hPSC-CMs; human pluripotent stem cell derived cardiomyocytes; mitochondria; wnt signalling pathway
    DOI:  https://doi.org/10.3389/fmolb.2022.714008
  40. Eur Urol Focus. 2022 Apr 11. pii: S2405-4569(22)00072-4. [Epub ahead of print]
      Genetic testing is a critical tool in the medical management of disease; however, for variants of uncertain significance there is insufficient evidence to prove a connection between the variant and disease and they should not be used as a basis for clinical decisions.
    DOI:  https://doi.org/10.1016/j.euf.2022.03.018