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Mitochondrial transcriptome in TEFM patients a Transcriptome-wide RNA-Seq analysis showing the effects of TEFM loss in skeletal muscle of patient 2 (P2) on mtRNA transcripts. A map of mtDNA showing the change in sequence read coverage (log2 fold change between patient 2 and two healthy controls) on both strands. b Quantification of the difference in reads per gene between skeletal muscle from P2 and from two healthy controls (n = 3 technical replicates) for mt-mRNA derived reads for both strands, from proximal to distal from the promoter and c reads corresponding to mt-tRNA for both strands in the order they are transcribed (n = 3 technical replicates) d Transcriptome-wide RNA-Seq analysis of the effects of TEFM loss in fibroblast cells of P2 on mtRNA transcripts. A map of mtDNA showing the change in sequence read coverage (log2 fold change between P2 and fibroblast cells from healthy controls) on both strands. e Quantification of the differences in number of reads per gene between fibroblasts from P2 and from two healthy controls (n = 3 technical replicates) for mt-mRNA reads and f reads corresponding to mt-tRNA fragments in the order they are transcribed from the promoter (n = 3 technical replicates). All trend lines were fitted by linear regression. Source data are provided as a Source Data file.
Source publication
Mutations in the mitochondrial or nuclear genomes are associated with a diverse group of human disorders characterized by impaired mitochondrial respiration. Within this group, an increasing number of mutations have been identified in nuclear genes involved in mitochondrial RNA biology. The TEFM gene encodes the mitochondrial transcription elongati...
Citations
... Exome sequencing (ES) or custom clinical ES (CES) were performed in different centers using genomic DNA and published procedures. 17,18 Variants were confirmed by Sanger sequencing. ...
Objective
COASY, the gene encoding the bifunctional enzyme CoA synthase, which catalyzes the last two reactions of cellular de novo coenzyme A (CoA) biosynthesis, has been linked to two exceedingly rare autosomal recessive disorders, such as COASY protein‐associated neurodegeneration (CoPAN), a form of neurodegeneration with brain iron accumulation (NBIA), and pontocerebellar hypoplasia type 12 (PCH12). We aimed to expand the phenotypic spectrum and gain insights into the pathogenesis of COASY‐related disorders.
Methods
Patients were identified through targeted or exome sequencing. To unravel the molecular mechanisms of disease, RNA sequencing, bioenergetic analysis, and quantification of critical proteins were performed on fibroblasts.
Results
We identified five new individuals harboring novel COASY variants. While one case exhibited classical CoPAN features, the others displayed atypical symptoms such as deafness, language and autism spectrum disorders, brain atrophy, and microcephaly. All patients experienced epilepsy, highlighting its potential frequency in COASY‐related disorders. Fibroblast transcriptomic profiling unveiled dysregulated expression in genes associated with mitochondrial respiration, responses to oxidative stress, transmembrane transport, various cellular signaling pathways, and protein translation, modification, and trafficking. Bioenergetic analysis revealed impaired mitochondrial oxygen consumption in COASY fibroblasts. Despite comparable total CoA levels to control cells, the amounts of mitochondrial 4′‐phosphopantetheinylated proteins were significantly reduced in COASY patients.
Interpretation
These results not only extend the clinical phenotype associated with COASY variants but also suggest a continuum between CoPAN and PCH12. The intricate interplay of altered cellular processes and signaling pathways provides valuable insights for further research into the pathogenesis of COASY‐associated diseases.
... There is no mechanism to induce expression of specific transcripts of individual oxidative phosphorylation subunits or tRNAs and rRNAs. Generally, impairments to transcription initiation and elongation reduce the abundance of mRNAs, tRNAs and rRNA and will lead to an overall decrease in the synthesis of individual subunits of the oxidative phosphorylation complexes and their steady-state abundance in the membrane [6,7]. However, another class of errors that occur during transcription as well in the posttranscriptional maturation steps can alter the reading frame of the genetic template and induce aberrations in protein synthesis on mitochondrial ribosomes, in particular misfolding of the nascent chain. ...
... TEFM is a mitochondrial transcription elongation factor that interacts with POLRMT to ensure the long polycistronic transcripts are generated [10][11][12]. Whereas pathogenic variants in TEFM impair the ability to synthesize RNA molecules distal from the promoters [7], the processivity facilitated by TEFM can also introduce mutations during transcription [8]. While the conventional thinking is that in a healthy wild type setting the full polycistronic transcripts are generated regularly from the light and heavy strand promoters of the genome, this view does not consider low-level processivity mistakes whereby the POLRMT-TEFM complex disassociates from the genome template and generates a partial transcript. ...
Human mitochondrial DNA is one of the most simplified cellular genomes and facilitates compartmentalized gene expression. Within the organelle, there is no physical barrier to separate transcription and translation, nor is there evidence that quality control surveillance pathways are active to prevent translation on faulty mRNA transcripts. Mitochondrial ribosomes synthesize 13 hydrophobic proteins that require co-translational insertion into the inner membrane of the organelle. To maintain the integrity of the inner membrane, which is essential for organelle function, requires responsive quality control mechanisms to recognize aberrations in protein synthesis. In this review, we explore how defects in mitochondrial protein synthesis can arise due to the culmination of inherent mistakes that occur throughout the steps of gene expression. In turn, we examine the stepwise series of quality control processes that are needed to eliminate any mistakes that would perturb organelle homeostasis. We aim to provide an integrated view on the quality control mechanisms of mitochondrial protein synthesis and to identify promising avenues for future research.
... Its important role in the regulation of expression of mitochondrial genes has also pathophysiological consequences, as its increased expression promotes tumour growth and metastases and predicts poor prognosis and survival of hepatocellular carcinomas and glioma patients [53][54][55]. Moreover, TEFM variants cause early onset of neurological diseases such as mitochondrial encephalomyopathy, epilepsy and others [56]. ...
Proteins from the Bcl-2 family play an essential role in the regulation of apoptosis. However, they also possess cell death-unrelated activities that are less well understood. This prompted us to study apoptosis-unrelated activities of the Bax and Bak, pro-apoptotic members of the Bcl-2 family. We prepared Bax/Bak-deficient human cancer cells of different origin and found that while respiration in the glioblastoma U87 Bax/Bak-deficient cells was greatly enhanced, respiration of Bax/Bak-deficient B lymphoma HBL-2 cells was slightly suppressed. Bax/Bak-deficient U87 cells also proliferated faster in culture, formed tumours more rapidly in mice, and showed modulation of metabolism with a considerably increased NAD⁺/NADH ratio. Follow-up analyses documented increased/decreased expression of mitochondria-encoded subunits of respiratory complexes and stabilization/destabilization of the mitochondrial transcription elongation factor TEFM in Bax/Bak-deficient U87 and HBL-2 cells, respectively. TEFM downregulation using shRNAs attenuated mitochondrial respiration in Bax/Bak-deficient U87 as well as in parental HBL-2 cells. We propose that (post)translational regulation of TEFM levels in Bax/Bak-deficient cells modulates levels of subunits of mitochondrial respiratory complexes that, in turn, contribute to respiration and the accompanying changes in metabolism and proliferation in these cells.
... Its important role in the regulation of expression of mitochondrial genes has also pathophysiological consequences, as its increased expression promotes tumour growth and metastases and predicts poor prognosis and survival of hepatocellular carcinomas and glioma patients [53][54][55]. Moreover, TEFM variants cause early onset of neurological diseases such as mitochondrial encephalomyopathy, epilepsy and others [56]. ...
Proteins from the Bcl-2 family play an essential role in regulation of apoptosis. However, they also possess cell death-unrelated activities that are less well understood. This prompted us to study apoptosis-unrelated activities of the Bax and Bak, pro-apoptotic members of the Bcl-2 family. We prepared Bax/Bak-deficient human cancer cells of different origin and found that while respiration in the glioblastoma U87 Bax/Bak-deficient cells was greatly enhanced, respiration of Bax/Bak-deficient B lymphoma HBL-2 cells was slightly suppressed. Bax/Bak-deficient U87 cells also proliferated faster in culture, formed tumours more rapidly in mice, and showed modulation of metabolism with considerably increased NAD ⁺ /NADH ratio. Follow-up analyses documented increased/decreased expression of mitochondria-encoded subunits of respiratory complexes and stabilization/destabilization of the mitochondrial transcription elongation factor TEFM in Bax/Bak-deficient U87 and HBL-2 cells, respectively. We propose that (post)translational regulation of TEFM levels in Bax/Bak-deficient cells modulates levels of subunits of mitochondrial respiratory complexes that, in turn, contribute to respiration and the accompanying changes in metabolism and proliferation in these cells.
... Impaired mitochondria can result in the increased production of ROS and lead to a reduction in axon outgrowth [13,17]. The role of mitochondria in axon outgrowth is particularly relevant as both mitochondrial CMS zebrafish models, TEFM and SLC25A1, have been shown to display abnormal axon outgrowth [18,19]. ...
... Therefore, it may be beneficial to study neuromuscular transmission in patients with CHCHD10 mutations, especially those diagnosed with mitochondrial myopathy or motor neuropathy. The identification of an NMJ defect in patients may provide novel therapeutic options, such as salbutamol or other drugs targeting the NMJ [19]. ...
... While a diagnosis of myasthenia gravis can frequently be excluded, the line between some subtypes of CMS and mitochondrial myopathy is less obvious. Recently, two nuclear encoded mitochondria-associated genes were identified as causative for CMS-SLC25A1 [18,[84][85][86][87] and TEFM [19]. Notably, it is only specific "milder" genetic mutations to SLC25A1 and TEFM that cause CMS; other previously identified mutations are associated with more severe and classical mitochondrial disease. ...
Congenital myasthenic syndromes (CMS) are a group of rare, neuromuscular disorders that usually present in childhood or infancy. While the phenotypic presentation of these disorders is diverse, the unifying feature is a pathomechanism that disrupts neuromuscular transmission. Recently, two mitochondrial genes—SLC25A1 and TEFM—have been reported in patients with suspected CMS, prompting a discussion about the role of mitochondria at the neuromuscular junction (NMJ). Mitochondrial disease and CMS can present with similar symptoms, and potentially one in four patients with mitochondrial myopathy exhibit NMJ defects. This review highlights research indicating the prominent roles of mitochondria at both the pre- and postsynapse, demonstrating the potential for mitochondrial involvement in neuromuscular transmission defects. We propose the establishment of a novel subcategorization for CMS—mitochondrial CMS, due to unifying clinical features and the potential for mitochondrial defects to impede transmission at the pre- and postsynapse. Finally, we highlight the potential of targeting the neuromuscular transmission in mitochondrial disease to improve patient outcomes.
Mitochondria are hubs of metabolic activity with a major role in ATP conversion by oxidative phosphorylation (OXPHOS). The mammalian mitochondrial genome encodes 11 mRNAs encoding 13 OXPHOS proteins along with 2 rRNAs and 22 tRNAs, that facilitate their translation on mitoribosomes. Maintaining the internal production of core OXPHOS subunits requires modulation of the mitochondrial capacity to match the cellular requirements and correct insertion of particularly hydrophobic proteins into the inner mitochondrial membrane. The mitochondrial translation system is essential for energy production and defects result in severe, phenotypically diverse diseases, including mitochondrial diseases that typically affect postmitotic tissues with high metabolic demands. Understanding the complex mechanisms that underlie the pathologies of diseases involving impaired mitochondrial translation is key to tailoring specific treatments and effectively targeting the affected organs. Disease mutations have provided a fundamental, yet limited, understanding of mitochondrial protein synthesis, since effective modification of the mitochondrial genome has proven challenging. However, advances in next generation sequencing, cryoelectron microscopy, and multi-omic technologies have revealed unexpected and unusual features of the mitochondrial protein synthesis machinery in the last decade. Genome editing tools have generated unique models that have accelerated our mechanistic understanding of mitochondrial translation and its physiological importance. Here we review the most recent mouse models of disease pathogenesis caused by defects in mitochondrial protein synthesis and discuss their value for preclinical research and therapeutic development.
Congenital myasthenic syndromes (CMS) are a rare group of inherited disorders caused by gene defects associated with the neuromuscular junction and potentially treatable with commonly available medications such as acetylcholinesterase inhibitors and β2 adrenergic receptor agonists. In this study, we identified and genetically characterized the largest cohort of CMS patients from India to date.
Genetic testing of clinically suspected patients evaluated in a South Indian hospital during the period 2014–19 was carried out by standard diagnostic gene panel testing or using a two-step method that included hotspot screening followed by whole-exome sequencing. In total, 156 genetically diagnosed patients (141 families) were characterized and the mutational spectrum and genotype-phenotype correlation described. Overall, 87 males and 69 females were evaluated, with the age of onset ranging from congenital to fourth decade (mean 6.6 ± 9.8 years). The mean age at diagnosis was 19 ± 12.8 (1–56 years), with a mean diagnostic delay of 12.5 ± 9.9 (0–49 years). Disease-causing variants in 17 CMS-associated genes were identified in 132 families (93.6%), while in nine families (6.4%), variants in genes not associated with CMS were found. Overall, postsynaptic defects were most common (62.4%), followed by glycosylation defects (21.3%), synaptic basal lamina genes (4.3%) and presynaptic defects (2.8%). Other genes found to cause neuromuscular junction defects (DES, TEFM) in our cohort accounted for 2.8%.
Among the individual CMS genes, the most commonly affected gene was CHRNE (39.4%), followed by DOK7 (14.4%), DPAGT1 (9.8%), GFPT1 (7.6%), MUSK (6.1%), GMPPB (5.3%) and COLQ (4.5%). We identified 22 recurrent variants in this study, out of which eight were found to be geographically specific to the Indian subcontinent. Apart from the known common CHRNE variants p.E443Kfs*64 (11.4%) and DOK7 p.A378Sfs*30 (9.3%), we identified seven novel recurrent variants specific to this cohort, including DPAGT1 p.T380I and DES c.1023+5G>A, for which founder haplotypes are suspected.
This study highlights the geographic differences in the frequencies of various causative CMS genes and underlines the increasing significance of glycosylation genes (DPAGT1, GFPT1 and GMPPB) as a cause of neuromuscular junction defects. Myopathy and muscular dystrophy genes such as GMPPB and DES, presenting as gradually progressive limb girdle CMS, expand the phenotypic spectrum. The novel genes MACF1 and TEFM identified in this cohort add to the expanding list of genes with new mechanisms causing neuromuscular junction defects.
