bims-mitdis Biomed News
on Mitochondrial disorders
Issue of 2022‒05‒29
24 papers selected by
Catalina Vasilescu
University of Helsinki

  1. Mitochondrion. 2022 May 24. pii: S1567-7249(22)00044-7. [Epub ahead of print]
      Leber's hereditary optic neuropathy (LHON) is the maternal inheritance of eye disorder. LHON-linked mitochondrial DNA (mtDNA) mutations affect the ND1, ND4 or ND6 genes encoding essential subunits of complex I. However, the role of mitochondrial tRNA defects in the pathogenesis of LHON is poorly understood. In this report, Sanger sequence analysis of 22 mitochondrial tRNA genes identified 139 variants in a cohort of 811 Han Chinese probands and 485 control Chinese subjects. Among these, 32 (4 known and 28 novel/putative) tRNA variants in 69 probands may contribute to pathogenesis of LHON, as these exhibited (1) present in <1% of controls; (2) evolutionary conservation; (3) potential and significance of structural and functional modifications. Such variants may have potentially compromised structural and functional aspects in the processing of tRNAs, structure stability, tRNA charging, or codon-anticodon interactions during translation. These 32 variants presented either singly or with multiple mutations, with the primary LHON-linked ND1 3640G>A, ND4 11778G>A or ND6 14484T>C mutations in the probands. The thirty-eight pedigrees carrying only one of tRNA variants exhibited relatively low penetrances of LHON, ranging from 5.7% to 42.9%, with an average of 19%. Strikingly, the average penetrances of optic neuropathy among 33 Chinese families carrying both a known/putative tRNA variant and a primary LHON-associated mtDNA mutation were 40.1%. These findings suggested that mitochondrial tRNA variants represent a significant causative factor for LHON, accounting for 8.75% cases in this cohort. These new insights may lead to beneficial applications in the pathophysiology, disease management, and genetic counseling of LHON.
    Keywords:  Leber’s hereditary optic neuropathy; etiology; mitochondrial genetic defect; mutation; tRNA variants
  2. Nature. 2022 May 25.
      Mitochondria are epicentres of eukaryotic metabolism and bioenergetics. Pioneering efforts in recent decades have established the core protein componentry of these organelles1 and have linked their dysfunction to more than 150 distinct disorders2,3. Still, hundreds of mitochondrial proteins lack clear functions4, and the underlying genetic basis for approximately 40% of mitochondrial disorders remains unresolved5. 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 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.
  3. J Clin Invest. 2022 May 26. pii: e145660. [Epub ahead of print]
      Mitochondrial DNA (mtDNA) depletion/deletions syndromes (MDDS) encompass a clinically and etiologically heterogenous group of mitochondrial disorders due to impaired mtDNA maintenance. Among the most frequent causes of MDDS are defects in nucleoside/nucleotide metabolism, which is critical for synthesis and homeostasis of the deoxynucleoside triphosphate (dNTP) substrates of mtDNA replication. A central enzyme for generating dNTPs is ribonucleotide reductase, a critical mediator of de novo nucleotide synthesis composed of catalytic RRM1 subunits in complex with RRM2 or p53R2. Here, we report five probands from four families who presented with ptosis and ophthalmoplegia, plus other manifestations and multiple mtDNA deletions in muscle. We identified three RRM1 loss-of-function variants, including a dominant catalytic site variant (NP_001024.1: p.N427K) and two homozygous recessive variants at p.R381, which has evolutionarily conserved interactions with the specificity site. Atomistic molecular dynamics simulations indicate mechanisms by which RRM1 variants affect protein structure. Cultured primary skin fibroblasts of probands manifested mtDNA depletion under cycling conditions, indicating impaired de novo nucleotide synthesis. Fibroblasts also exhibited aberrant nucleoside diphosphate and dNTP pools and mtDNA ribonucleotide incorporation. Our data reveal primary RRM1 deficiency and, by extension, impaired de novo nucleotide synthesis are causes of MDDS.
    Keywords:  Genetic diseases; Genetics; Mitochondria; Molecular pathology
  4. J Inherit Metab Dis. 2022 May 27.
    Undiagnosed Diseases Network
      Mitochondrial complex V plays an important role in oxidative phosphorylation by catalyzing the generation of ATP. Most complex V subunits are nuclear encoded and not yet associated with recognized Mendelian disorders. Using exome sequencing, we identified a rare homozygous splice variant (c.87+3A>G) in ATP5PO, the complex V subunit which encodes the oligomycin sensitivity conferring protein, in three individuals from two unrelated families, with clinical suspicion of a mitochondrial disorder. These individuals had a similar severe infantile and often lethal multi-systemic disorder that included hypotonia, developmental delay, hypertrophic cardiomyopathy, progressive epileptic encephalopathy, progressive cerebral atrophy, and white matter abnormalities on brain MRI consistent with Leigh syndrome. cDNA studies showed a predominant shortened transcript with skipping of exon 2 and low levels of the normal full-length transcript. Fibroblasts from the affected individuals demonstrated decreased ATP5PO protein, defective assembly of complex V with greatly reduced amounts of peripheral stalk proteins, and greatly reduced complex V hydrolytic activity. Further, expression of human ATP5PO cDNA without exon 2 (hATP5PO-∆ex2) in yeast cells deleted for yATP5 (ATP5PO homolog) was unable to rescue growth on media which requires oxidative phosphorylation when compared to the wild type construct (hATP5PO-WT), indicating that exon 2 deletion leads to a non-functional protein. Collectively, our findings support the pathogenicity of the ATP5PO c.87+3A>G variant, which significantly reduces but does not eliminate complex V activity. These data along with the recent report of an affected individual with ATP5PO variants, add to the evidence that rare biallelic variants in ATP5PO result in defective complex V assembly, function and are associated with Leigh syndrome. This article is protected by copyright. All rights reserved.
    Keywords:  ATP synthase; ATP5PO; Leigh syndrome; complex V; hypertrophic cardiomyopathy; mitochondria; mitochondrial disease; seizure; splice variant
  5. Methods Mol Biol. 2022 ;2399 261-274
      Mitochondria are complex organelles with multifaceted roles in cell biology, acting as signaling hubs that implicate them in cellular physiology and pathology. Mitochondria are both the target and the origin of multiple signaling events, including redox processes and calcium signaling which are important for organellar function and homeostasis. One way to interrogate mitochondrial function is by live cell imaging. Elaborated approaches perform imaging of single mitochondrial dynamics in living cells and animals. Imaging mitochondrial signaling and function can be challenging due to the sheer number of mitochondria, and the speed, propagation, and potential short half-life of signals. Moreover, mitochondria are organized in functionally coupled interorganellar networks. Therefore, advanced analysis and postprocessing tools are needed to enable automated analysis to fully quantitate mitochondrial signaling events and decipher their complex spatiotemporal connectedness. Herein, we present a protocol for recording and automating analyses of signaling in neuronal mitochondrial networks.
    Keywords:  Computational wavelet analysis; Fluorescence microscopy; Grx1-roGFP2; Mitochondria; Mitochondrial cluster; Redox potential
  6. STAR Protoc. 2022 Jun 17. 3(2): 101401
      Mitochondrial dynamics play critical roles in both tissue homeostasis and somatic cell reprogramming. Here, we provide integrated guidance for assessing mitochondrial function and dynamics while reprogramming human fibroblasts via an integrated analysis approach. This protocol includes instructions for mitochondrial metabolic analysis in real time and flow cytometry-based assessment of mitochondrial mass and membrane potential. We also describe a protocol for quantification of mitochondrial network and key metabolites. For complete details on the use and execution of this protocol, please refer to Cha et al. (2021).
    Keywords:  Cell Biology; Cell culture; Cell-based Assays; Flow Cytometry/Mass Cytometry; Metabolism; Microscopy; Stem Cells
  7. Heliyon. 2022 May;8(5): e09353
      Mitochondrial respiratory chain (RC) function requires the stoichiometric interaction among dozens of proteins but their co-regulation has not been defined in the human brain. Here, using quantitative proteomics across three independent cohorts we systematically characterized the co-regulation patterns of mitochondrial RC proteins in the human dorsolateral prefrontal cortex (DLPFC). Whereas the abundance of RC protein subunits that physically assemble into stable complexes were correlated, indicating their co-regulation, RC assembly factors exhibited modest co-regulation. Within complex I, nuclear DNA-encoded subunits exhibited >2.5-times higher co-regulation than mitochondrial (mt)DNA-encoded subunits. Moreover, mtDNA copy number was unrelated to mtDNA-encoded subunits abundance, suggesting that mtDNA content is not limiting. Alzheimer's disease (AD) brains exhibited reduced abundance of complex I RC subunits, an effect largely driven by a 2-4% overall lower mitochondrial protein content. These findings provide foundational knowledge to identify molecular mechanisms contributing to age- and disease-related erosion of mitochondrial function in the human brain.
    Keywords:  Alzheimer disease; BLSA; Banner; Mitochondrial respiratory chain; Post-mortem brain; Proteomics; ROSMAP
  8. J Cell Biol. 2022 Jul 04. pii: e202201071. [Epub ahead of print]221(7):
      The mitochondrial unfolded protein response (UPRmt) is dedicated to promoting mitochondrial proteostasis and is linked to extreme longevity. The key regulator of this process is the transcription factor ATFS-1, which, upon UPRmt activation, is excluded from the mitochondria and enters the nucleus to regulate UPRmt genes. However, the repair proteins synthesized as a direct result of UPRmt activation must be transported into damaged mitochondria that had previously excluded ATFS-1 owing to reduced import efficiency. To address this conundrum, we analyzed the role of the import machinery when the UPRmt was induced. Using in vitro and in vivo analysis of mitochondrial proteins, we surprisingly find that mitochondrial import increases when the UPRmt is activated in an ATFS-1-dependent manner, despite reduced mitochondrial membrane potential. The import machinery is upregulated, and an intact import machinery is essential for UPRmt-mediated lifespan extension. ATFS-1 has a weak mitochondrial targeting sequence (MTS), allowing for dynamic subcellular localization during the initial stages of UPRmt activation.
  9. Autophagy. 2022 May 23.
      CLEC16A regulates mitochondrial health through mitophagy and is associated with over 20 human diseases. However, the key structural and functional regions of CLEC16A, and their relevance for human disease, remain unknown. Here, we report that a disease-associated CLEC16A variant lacks a C-terminal intrinsically disordered protein region (IDPR) that is critical for mitochondrial quality control. IDPRs comprise nearly half of the human proteome, yet their mechanistic roles in human disease are poorly understood. Using carbon detect NMR, we find that the CLEC16A C terminus lacks secondary structure, validating the presence of an IDPR. Loss of the CLEC16A C-terminal IDPR in vivo impairs mitophagy, mitochondrial function, and glucose-stimulated insulin secretion, ultimately causing glucose intolerance. Deletion of the CLEC16A C-terminal IDPR increases CLEC16A ubiquitination and degradation, thus impairing assembly of the mitophagy regulatory machinery. Importantly, CLEC16A stability is dependent on proline bias within the C-terminal IDPR, but not amino acid sequence order or charge. Together, we elucidate how an IDPR in CLEC16A regulates mitophagy and implicate pathogenic human gene variants that disrupt IDPRs as novel contributors to diabetes and other CLEC16A-associated diseases.
    Keywords:  Diabetes; NMR; insulin; mitophagy; splicing
  10. Am J Physiol Cell Physiol. 2022 May 25.
      Reactive oxygen species (ROS) are recognised both as damaging molecules and intracellular signalling entities. In addition to its role in ATP generation, the mitochondrial electron transport chain (ETC) constitutes a relevant source of mitochondrial ROS, in particular during pathological conditions. Mitochondrial ROS homeostasis depends on species- and site-dependent ROS production, their bioreactivity, diffusion, and scavenging. However, our quantitative understanding of mitochondrial ROS homeostasis has thus far been hampered by technical limitations, including lack of truly site- and/or ROS-specific reporter molecules. In this context, the use of computational models is of great value to complement and interpret empirical data, as well as to predict variables that are difficult to assess experimentally. During the last decades, various mechanistic models of ETC-mediated ROS production have been developed. Although these often-complex models have generated novel insights, their parameterisation, analysis, and integration with other computational models is not straightforward. In contrast, phenomenological (sometimes termed "minimal") models use a relatively small set of equations to describe empirical relationship(s) between ROS-related and other parameters, and generally aim to explore system behaviour and generate hypotheses for experimental validation. In this review, we first discuss ETC-linked ROS homeostasis and introduce various detailed mechanistic models. Next, we present how bioenergetic parameters (e.g. NADH/NAD+ ratio, mitochondrial membrane potential) relate to site-specific ROS production within the ETC and how these relationships can be used to design minimal models of ROS homeostasis. Finally, we illustrate how minimal models have been applied to explore pathophysiological aspects of ROS.
    Keywords:  Electron transport chain; Mathematical model; Mitochondria; Reactive oxygen species
  11. Trends Genet. 2022 May 19. pii: S0168-9525(22)00107-X. [Epub ahead of print]
      The mitochondrial genome has been difficult to manipulate because it is shielded by the organelle double membranes, preventing efficient nucleic acid entry. Moreover, mitochondrial DNA (mtDNA) recombination is not a robust system in most species. This limitation has forced investigators to rely on naturally occurring alterations to study both mitochondrial function and pathobiology. Because most pathogenic mtDNA mutations are heteroplasmic, the development of specific nucleases has allowed us to selectively eliminate mutant species. Several 'protein only' gene-editing platforms have been successfully used for this purpose. More recently, a DNA double-strand cytidine deaminase has been identified and adapted to edit mtDNA. This enzyme was also used as a component to adapt a DNA single-strand deoxyadenosine deaminase to mtDNA editing. These are major advances in our ability to precisely alter the mtDNA in animal cells.
    Keywords:  TALEN; gene editing; genetic engineering; mitochondria
  12. Methods Mol Biol. 2022 ;2399 151-170
      Data-driven research led by computational systems biology methods, encompassing bioinformatics of multiomics datasets and mathematical modeling, are critical for discovery. Herein, we describe a multiomics (metabolomics-fluxomics) approach as applied to heart function in diabetes. The methodology presented has general applicability and enables the quantification of the fluxome or set of metabolic fluxes from cytoplasmic and mitochondrial compartments in central catabolic pathways of glucose and fatty acids. Additionally, we present, for the first time, a general method to reduce the dimension of detailed kinetic, and in general stoichiometric models of metabolic networks at the steady state, to facilitate their optimization and avoid numerical problems. Representative results illustrate the powerful mechanistic insights that can be gained from this integrative and quantitative methodology.
    Keywords:  Diabetes; Fluxomics; Glucose and fatty acids catabolism; Heart; Kinetic modeling; Metabolomics
  13. Nature. 2022 May 25.
      Mitochondria generate heat due to H+ leak (IH) across their inner membrane1. IH results from the action of long-chain fatty acids on uncoupling protein 1 (UCP1) in brown fat2-6 and ADP/ATP carrier (AAC) in other tissues1,7-9, but the underlying mechanism is poorly understood. As evidence of pharmacological activators of IH through UCP1 and AAC is lacking, IH is induced by protonophores such as 2,4-dinitrophenol (DNP) and cyanide-4-(trifluoromethoxy) phenylhydrazone (FCCP)10,11. Although protonophores show potential in combating obesity, diabetes and fatty liver in animal models12-14, their clinical potential for treating human disease is limited due to indiscriminately increasing H+ conductance across all biological membranes10,11 and adverse side effects15. Here we report the direct measurement of IH induced by DNP, FCCP and other common protonophores and find that it is dependent on AAC and UCP1. Using molecular structures of AAC, we perform a computational analysis to determine the binding sites for protonophores and long-chain fatty acids, and find that they overlap with the putative ADP/ATP-binding site. We also develop a mathematical model that proposes a mechanism of uncoupler-dependent IH through AAC. Thus, common protonophoric uncouplers are synthetic activators of IH through AAC and UCP1, paving the way for the development of new and more specific activators of these two central mediators of mitochondrial bioenergetics.
  14. Mitochondrion. 2022 May 24. pii: S1567-7249(22)00043-5. [Epub ahead of print]
      Mitochondria are dynamic organelles responsible for energy production and cell metabolism. Disorders in mitochondrial function impair tissue integrity and have been implicated in multiple human diseases. Rather than constrained in host cells, mitochondria were recently found to actively travel between cells through nanotubes or extracellular vesicles. Mitochondria transportation represents a key mechanism of intercellular communication implicated in metabolic homeostasis, immune response, and stress signaling. Here we reviewed recent progress in mitochondria transfer under physiological and pathological conditions. Specifically, tumor cells imported mitochondria from adjacent cells in the microenvironment which potentially modulated cancer progression. Intercellular mitochondria trafficking also inspired therapeutic intervention of human diseases with mitochondria transplantation. Artificial mitochondria, generated through mitochondria genome engineering or mitochondria-nucleus hybridization, further advanced our understanding of mitochondrial biology and its therapeutic potential. Innovative tools and animal models of mitochondria transplantation will assist the development of new therapies for mitochondrial dysfunction-related diseases.
    Keywords:  artificial mitochondria; intercellular nanotube; microvesicle; mitochondria genome editing; mitochondria transfer; mitochondria transplantation; synthetic biology
  15. Cell Death Differ. 2022 May 25.
      Binding of the mitochondrial chaperone TRAP1 to client proteins shapes bioenergetic and proteostatic adaptations of cells, but the panel of TRAP1 clients is only partially defined. Here we show that TRAP1 interacts with F-ATP synthase, the protein complex that provides most cellular ATP. TRAP1 competes with the peptidyl-prolyl cis-trans isomerase cyclophilin D (CyPD) for binding to the oligomycin sensitivity-conferring protein (OSCP) subunit of F-ATP synthase, increasing its catalytic activity and counteracting the inhibitory effect of CyPD. Electrophysiological measurements indicate that TRAP1 directly inhibits a channel activity of purified F-ATP synthase endowed with the features of the permeability transition pore (PTP) and that it reverses PTP induction by CyPD, antagonizing PTP-dependent mitochondrial depolarization and cell death. Conversely, CyPD outcompetes the TRAP1 inhibitory effect on the channel. Our data identify TRAP1 as an F-ATP synthase regulator that can influence cell bioenergetics and survival and can be targeted in pathological conditions where these processes are dysregulated, such as cancer.
  16. Mitochondrion. 2022 May 23. pii: S1567-7249(22)00047-2. [Epub ahead of print]
      Oocytes may carry mutations in their mitochondrial DNA (mtDNA) which affect fertility and embryo development leading to hereditary diseases or rejection. Mitochondrial replacement therapies (MRTs) such as polar body transfer, spindle transfer and pronuclear transfer, aim to change dysfunctional to normal mitochondria inside oocytes and zygotes resulting in healthier offspring. Even with promising results, MRTs techniques are invasive to oocytes and may negatively affect their viability and the success of the procedure. This article shows early evidence of the use of MitoCeption, a mitochondria transfer/transplant (AMT/T) technique to possibly induce the internalization of exogenous mitochondria in a dose-dependent manner to recipient oocytes in comparison to coincubation. By using human isolated mitochondria in a mix obtained from different donors we were able to identify their mtDNA in murine oocytes by qPCR. Fluorescence microscopy showed that exogenous and transferred mitochondria (MitoTracker ® Red) by MitoCeption were internalized in oocytes and zygotes (CellTracker® Green). After maintaining mitocepted zygotes to two-cell embryos, we transferred them to subrogate female mice and obtained healthy mice pups; however, without clear evidence of the maintenance of human mtDNA in the tissues of mice pups. These early results are puzzling, and they open the path to generate more research regarding the use of MitoCeption in comparison to coincubation in order to transfer mitochondria to oocytes using less invasive procedures.
    Keywords:  MitoCeption; Mitochondria replacement therapy (MRT); coincubation; heteroplasmy; mitochondrial disease; oocytes; xenogeneic transfer/transplant; zygotes
  17. Methods Mol Biol. 2022 ;2485 111-131
      Micro-heart muscle arrays enable medium-throughput experiments to model the cardiac response to a variety of environmental and pharmaceutical effects. Here, we describe stem cell culture maintenance, methods for successful cardiac differentiation, and formation of micro-heart muscle arrays for electrophysiology and molecular biology assays.
    Keywords:  Cardiac differentiation; Electrophysiology; Induced pluripotent stem cells (iPSC); Micro-heart muscle (μHM); Pharmacology
  18. STAR Protoc. 2022 Jun 17. 3(2): 101403
      Lactate is a central metabolite in energy metabolism and is also involved in cell signaling and epigenetic regulations. Here, we describe an NADH-independent enzymatic assay allowing rapid, selective, and sensitive quantification of L-lactate down to the pmol range. We detail lactate extraction from intracellular and extracellular fractions, followed by total protein amount determination and enzymatic assay. This approach allows quantification of intracellular and extracellular L-lactate levels, validated by treating adherent and non-adherent cells with inhibitors of lactate transporters (MCT).
    Keywords:  Cell Biology; Cell culture; Metabolism; Protein Biochemistry
  19. Curr Pharm Des. 2022 May 20.
      Mitochondria regulate a range of important physiological and biochemical cellular processes including apoptotic cell death, energy production, calcium homeostasis, oxidative stress, and lipid metabolism. Given their role as the 'engines' of cells, their dysfunction is associated with a variety of disease states. Exploring the relationship between mitochondrial function and disease can reveal the mechanism(s) of drug activity and disease pathology. In this review, we summarized the methods of evaluating the structure and function of mitochondria, including the morphology, membrane fluidity, membrane potential, opening of the membrane permeability transition pore, inner membrane permeabilization, mitochondrial dynamics, mitophagy, oxidative stress, energy metabolism-related enzymes, apoptotic pathway related proteins, calcium concentration, DNA copy number, oxygen consumption, β-oxidation-related genes and proteins, cardiolipin content, and adenosine triphosphate content. We believe that the information presented in this review will help explore the pathological processes of mitochondria in the occurrence and development of diseases, as well as the activity and mechanism of drugs, and the discovery of new drugs.
    Keywords:  Disease; drug; evaluation; mechanism; method; mitochondria.
  20. Methods Mol Biol. 2022 ;2456 173-183
      A fundamental goal of systems biology is to seek a better understanding of the cell's molecular mechanisms. Experimentalists most frequently rely upon reductionist methods to isolate and analyze discrete signaling compartments, including subcellular domains, organelles, and protein-protein interactions. Among the systems-biology community, there is a growing need to integrate multiple datasets to resolve complex cellular networks. In this chapter, we share our procedures for the discovery of integrated signaling networks, across multi-proteomic data. Demonstrating these procedures, we provide an integrated analysis of the cellular proteome and extracellular (secretome) of human glioma LN229.
    Keywords:  Bioinformatics; Cell Signaling; MS/MS; Multi-omics; Multi-proteomics; Network Enrichment Analysis; Network Integration; Proteomics
  21. Metabolites. 2022 Apr 27. pii: 398. [Epub ahead of print]12(5):
      Inborn errors of metabolism (IEMs) are rare diseases caused by a defect in a single enzyme, co-factor, or transport protein. For most IEMs, no effective treatment is available and the exact disease mechanism is unknown. The application of metabolomics and, more specifically, tracer metabolomics in IEM research can help to elucidate these disease mechanisms and hence direct novel therapeutic interventions. In this review, we will describe the different approaches to metabolomics in IEM research. We will discuss the strengths and weaknesses of the different sample types that can be used (biofluids, tissues or cells from model organisms; modified cell lines; and patient fibroblasts) and when each of them is appropriate to use.
    Keywords:  inborn errors of metabolism; metabolomics; stable isotopes
  22. Methods Mol Biol. 2022 ;2485 1-13
      CRISPR-Cas9-based screening technologies enable precise, high-throughput genetic and epigenetic manipulation to study mechanisms of development and disease and identify new therapeutic targets. Here, we describe a general protocol for the generation of custom, pooled CRISPR sgRNA libraries for screening in cardiomyocyte cultures. This methodology can address a variety of lab-specific research questions in cardiomyocytes and other cell types, as the genes to be modified can be curated or whole genomes can be investigated. The use of lentiviral sgRNA delivery followed by high-throughput sequencing allows for rapid comparison and identification of candidate genes and epigenetic modifiers, which can be further validated individually or in sub-pooled libraries following screening.
    Keywords:  CRISPR; Cardiomyocyte; Genetic screen; High-throughput; Knock-out; Maturation; Proliferation; Survival
  23. Genome Med. 2022 May 24. 14(1): 56
      We describe a case series of five infants (age range: 1-90 days; 4 females and 1 male) who presented to Al Jalila Children's intensive care units (ICU) with complex multisystem disorders. Patients were Emirati, Kenyan, Jordanian, Filipino, or Pakistani. Trio rapid whole genome sequencing (rWGS) was performed on all five patients and their parents within the hospital's genomics facility. Results were returned within ~37 h from blood sample draws and were diagnostic in 3 out of 5 patients. Positive findings were a homozygous pathogenic variant in POMT1 gene causing muscular dystrophydystroglycanopathy, a mosaic tetrasomy of the short arm of chromosome 12 (12p13.33p11.1) causing Pallister-Killian syndrome, and compound heterozygous pathogenic variants in the LIPA gene causing lysosomal acid lipase deficiency and Wolman disease. The rWGS analysis provided fast and precise diagnostic findings in those 3 patients and also aided in devising better management plans for them in the intensive care setting. For example, the 3-month-old infant with pathogenic variants in the LIPA gene is now a candidate for an FDA-approved, potentially lifesaving enzyme replacement therapy (sebelipase alfa). Our case series emphasize the feasibility and utility of rWGS in pediatric intensive care setting, in a diverse population that has long been underserved in genomic services. Significant investments in local healthcare infrastructure are needed, globally, for more equitable access of genomic medicine among vulnerable patients.
  24. Cell Rep. 2022 May 24. pii: S2211-1247(22)00628-3. [Epub ahead of print]39(8): 110855
      Hypusine is a post-translational modification on eukaryotic translation initiation factor 5A (eIF5A). The last step of hypusine biosynthesis, deoxyhypusine hydroxylation, is an oxygen-dependent reaction. Here we show that deletion of the deoxyhypusine hydroxylase Lia1 compromises yeast respiration through translation downregulation of selected proteins in the respiration pathway. The translation suppression, because of the lack of deoxyhypusine hydroxylation, mainly affects translation of the N termini of the proteins, independent of the presence of proline residues but likely dependent on the interaction between the N-terminal nascent peptide and the ribosomal peptide exit tunnel. Proteomics and biochemical studies reveal that Lia1 deletion decreases N-terminal translation of proteins involved in mitochondrial respiration, oxidative stress response, and protein folding. Our work uncovers functions of the hypusine modification by considering the substrate requirement of the post-translational modification, highlights the unique challenges of translating the N termini of proteins, and reveals an oxygen-sensing mechanism in eukaryotic cells.
    Keywords:  CP: Metabolism; CP: Molecular biology; deoxyhypusine hydroxylase; hypusine; oxidative phosphorylation; oxygen sensing; translation