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Misplaced mitochondria in a human myopathy
574
Abstracts
1. Mears, J. A., Lackner, L. L., Fang, S., Ingerman, E., Nunnari, J., and Hinshaw, J. E. (2011) Conformational changes in Dnm1 support a contractile mechanism for mitochondrial fission, Nat Struct Mol Biol 18, 20–26.
e Department of Pathology, University Hospitals of Case Medical Center, Case Western Reserve University, Cleveland, OH 44016, USA f Department of Pharmacology, Case Western Reserve University, Cleveland, OH 44016, USA g Department of Medicine, School of Medicine, Case Western Reserve University, Cleveland, OH 44016, USA
doi:10.1016/j.mito.2012.07.060
66 Regulation of superoxide dismutase 1 in intermembrane space of mitochondria Presenter: Yutaka Suzuki Yutaka Suzuki, Jan Riemer Department of Biology, University of Kaiserslautern, 67663 Kaiserslautern, Germany Reactive oxygen species (ROS) are implicated in human pathology and aging, as damaging agents and/or signaling messengers. Since a − ROS, superoxide (O ),2 is generated by the respiratory chain, mitochondrial DNA mutations and mitochondrial diseases can be causes and/or results of production of superoxide. Superoxide can be converted to molecular oxygen and hydrogen peroxide by superoxide dismutases. Superoxide dismutase 1 (SOD1) localizes to the cytosol with a minor portion targeted to the intermembrane space (IMS) of mitochondria. The activity of SOD1 is regulated by the copper chaperone for SOD1 (CCS1), which introduces copper ions and disulfide bonds to SOD1. CCS1 also localizes to cytosol and IMS. Knock out of either of those proteins in yeast and in mouse results in hypersensitivity to oxidative stress. We are interested in the mechanism of SOD1 and CCS1 targeting to the IMS and how the distribution between cytosol and IMS is regulated. In yeast, we showed that oxidation of a pair of cysteine residues has a critical role in the IMS localization of CCS1 and thereby also in providing SOD1 activity for the IMS [1]. The oxidation of CCS1 depends on Mia40, a component of a redox-regulated import machinery. Although localization of CCS1 to the IMS in mammalian cells has been reported to depend on Mia40, the cysteines that are oxidized in yeast CCS1 are not conserved in mammalian proteins. This indicates a different mechanism of CCS1 distribution in mammalian cells compared to yeast cells which we currently investigate in mechanistic detail. Our observations will contribute to a better understanding of the physiological and pathological role of superoxide in the IMS. Klöppel C*, Suzuki Y*, Kojer K, Petrungaro C, Longen S, Fiedler S, Keller S, Riemer J. (2011). Mol Biol Cell. 3749–57. doi:10.1016/j.mito.2012.07.061
68 A myopathy with retiform mitochondria Presenter: Hisashi Fujioka Hisashi Fujiokaa,b, Bernard Tandlerb,c, Laura Konczald, Mark L. Cohene, Mariana Roscab,f, Charles L. Hoppelb,f,g a Electron Microscopy Facility, Case Western Reserve University, Cleveland, OH 44016, USA b Center for Mitochondrial Diseases, School of Medicine, Case Western Reserve University, Cleveland, OH 44016, USA c Department of Biological Sciences, School of Dental Medicine, Case Western Reserve University, Cleveland, OH 44016, USA d Center for Human Genetics, Case Western Reserve University, Cleveland, OH 44016, USA
A surgical biopsy was obtained from the left quadriceps muscle of a 40-year-old male who exhibited progressive fatigue and unexplained exercise intolerance. Light microscopic examination of the specimen was unremarkable. Electron microscopic (EM) examination of the biopsy revealed an extremely unusual mitochondrial arrangement. In normal muscle, mitochondria are found in clusters beneath the sarcolemma or in rows between myofibrils. In the case in question, the muscle mitochondria were disposed in what at low magnification appeared to be a network. Some of the component organelles measured more than 19 μm in length. The cristae in these elongated organelles were for the most part obliquely oriented, and ranged from lamellar to tubular in form. The consensus among biochemists is that mitochondria form a structural network in the cytoplasm, i.e., these networks consist of but a single continuous mitochondrion that ramifies throughout the cell. This notion is based on confocal microscopy (with its limited resolution) of living cultured cells. At first blush, our case seems to fit this idea. When, however we examined, the muscle mitochondria in these putative networks using EM (with its high degree of resolution), it became apparent that long, ramifying mitochondria were joined together end-to-end and that there was no direct continuity of membranes from one organelle to another. Such “networks” were apparent only in traverse or slightly oblique sections of the myofibrils. In some cases, an element of sarcoplasmic reticulum was interposed between the ends of two closely apposed mitochondria. It is obvious that mere propinquity does not necessarily equal continuity. However this does not rule out the possibility that these organelles form a functional syncytium. Mitochondria isolated from the biopsy lost their elongated morphology to become spheroidal and were metabolically normal. It can be concluded that the muscle symptoms exhibited by our patient did not originate in defective mitochondria per se, but rather in the peculiarity of their arrangement within myofibrils. doi:10.1016/j.mito.2012.07.062
69 Misplaced mitochondria in a human myopathy Presenter: Hisashi Fujioka Hisashi Fujiokaa,b, Bernard Tandlerb,c, Laura Konczald, Mark L. Cohene, Mariana Roscab,f, Charles L. Hoppelb,f,g a Electron Microscopy Facility, Case Western Reserve University, Cleveland, OH 44016, USA b Center for Mitochondrial Diseases, School of Medicine, Case Western Reserve University, Cleveland, OH 44016, USA c Department of Biological Sciences, School of Dental Medicine, Case Western Reserve University, Cleveland, OH 44016, USA d Center for Human Genetics, University Hospitals of Case Medical Center, Case Western Reserve University, Cleveland, OH 44016, USA e Department of Pathology, University Hospitals of Case Medical Center, Case Western Reserve University, Cleveland, OH 44016, USA f Department of Pharmacology, Case Western Reserve University, Cleveland, OH 44016, USA g Department Medicine, School of Medicine, Case Western Reserve University, Cleveland, OH 44016, USA A specimen of the right quadriceps muscle was obtained by surgery from a 23-year-old female who displayed muscle weakness
Abstracts
among other symptoms. Light microscopy revealed abnormal mitochondrial distribution, consistent with primary mitochondrial disease. Electron microscopic examination of the biopsy revealed areas of typical structure, as well as areas showing morphological alterations. In the normal portions, small mitochondria participated in the formation of triads, which were situated at the Z disk; these triads consisted of paired mitochondria in close relation to elements of the sarcoplasmic reticulum (SR). In pathologically affected areas, typical triads were absent. Instead, relatively long, cylindrical mitochondria were oriented at right angles to the long axis of the myofibrils at the level where Z disks normally are present. These mitochondria measured up to 7 μm in length and usually less than 0.2 μm in width. Their cristae showed a normal range of variation. Many of these transverse mitochondria had extremely narrow segments, giving them a nodal appearance. In these narrow regions, the cristae were largely unaffected. Scattered elements of SR occasionally were present in relation to the transverse mitochondria, but rather than forming conventional triads, single SR elements were distributed unilaterally alongside the transverse mitochondria. In those myofibrils with transverse mitochondria, there were no mitochondria in the conventional interfibrillar position. Interestingly, biochemical examination of mitochondria isolated from the original biopsy revealed normal oxidative metabolism and completely normal metabolic parameters. In the face of normal mitochondrial function, it seems reasonable to suppose that the patient's muscle symptoms are based on malpositioning of the muscle mitochondria and on effacement of Z disks. The latter are sites of interaction between α-actinin, titin, and actin filaments. Because sarcomeric contraction depends on linkage to Z disks and on interplay between SR calcium, ATP, and heavy meromyosin, the aberrant morphology of the myofibrils and their attendant organelles might affect diffusion of the requisite molecules, leading to impaired contractility, hence weakness. doi:10.1016/j.mito.2012.07.063
70 Diagnostic application of measuring oxidative phosphorylation in permeabilized skin fibroblasts Presenter: Fang Ye Fang Ye, Charles L. Hoppel Center of Mitochondrial Disease, Department of Pharmacology and Medicine, Case Western Reserve University, Cleveland, OH 44106, United States The laboratory investigation in patients suspected of having a mitochondrial disease includes clinical laboratory metabolomics, genetic analyzing of mtDNA and nuclear DNA, and muscle biopsy. We asked whether cultured skin fibroblasts, a less invasive procedure, could provide a diagnostic step, possibly circumventing an open muscle biopsy in some patients. To approach this goal, we used human skin fibroblasts cultured in glucose containing medium to examine oxidative phosphorylation (OXPHOS), added to the measurement of electron transport chain (ETC) complex activities. We developed a method composed of two protocols using a high resolution respirometer (O2K, Oroboros). Protocol 1 measures oxygen consumption rates of Complexes I and II under the ADPstimulated state and maximal respiration with an uncoupler, FCCP. Following this, Complex IV rate is measured with TMPD plus ascorbate. In protocol 2, we assess fatty acid oxidation with palmitoylcarnitine and Complex III rate with duroquinol under state 3 condition, in addition to Complex III maximal respiration with FCCP. We have obtained reproducible OXPHOS data from twenty-three normal human skin fibroblast lines with multiple repeats and built
575
reference intervals for normal OXPHOS function. These data also provide validation for the analysis. To initially verify the utility of skin fibroblasts, we analyses a patient with a previously diagnosed skeletal muscle mitochondrial complex I defect. A Complex I defect was clearly identified by measuring OXPHOS in the fibroblasts. Moreover, four fibroblast lines from four patients show normal OXPHOS function which correlates with normal OXPHOS in isolated skeletal muscle mitochondria from these patients. Currently, this approach has been applied for clinical diagnosis. Fifty-two highly selected patient samples have been analyzed. We identified 24 samples with normal OXPHOS function, 15 with Complex I related defects, 5 with ADP phosphorylation system defects, 3 with fatty acid oxidation defects, 2 with Complex III related defects, and one each for defect in CoQ, pyruvate related oxidation and Complex IV. Interestingly, there are three subtypes of Complex I defects detected: 1) Decreased state 3 and maximal respiration (pyruvate, malate and glutamate) with decreased fatty acid oxidation. 2) Decreased state 3 and maximal respiration (pyruvate, malate and glutamate), but with normal fatty acid oxidation. 3) Normal state 3 oxidation (pyruvate, malate, glutamate and palmitoylcarnitine), but with decreased maximal respiration. Of diagnostic importance, this new method uncovered ADP phosphorylation defects as well as defects in substrate transport (pyruvate oxidation) and fatty acid oxidation. It is our belief that by identifying defects with a skin biopsy, we can avert an open muscle biopsy, a worthwhile endeavor. doi:10.1016/j.mito.2012.07.064
71 Targeted exome sequencing of suspected mitochondrial disorders in a hospital-based cohort Presenter: Daniel S. Lieber Daniel S. Liebera,b,c,d, Sarah E. Calvoa,b,c,d, Nancy G. Slatea,b, Shangtao Liua,b, Mark L. Borowskya, Steven G. Hershmana,b,c,d, Nina B. Golda,b, Gerard T. Berrye, David M. Muellerf, Jeremy D. Schmahmanng, Katherine B. Simsb,g, Vamsi K. Moothaa,b,c,d a Department of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114, United States b Center for Human Genetic Research, Massachusetts General Hospital, Boston, MA 02114, United States c Department of Systems Biology, Harvard Medical School, Boston, MA 02115, United States d Broad Institute of Harvard and MIT, Cambridge, MA 02141, United States e The Manton Center for Orphan Disease Research, Children's Hospital Boston, Boston, MA 02115, United States f Department of Biochemistry and Molecular Biology, Rosalind Franklin University of Medicine and Science, The Chicago Medical School, North Chicago, IL 60064, United States g Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Boston MA 02114, United States Advances in next-generation sequencing (NGS) have revolutionized genetic research and are poised to become routine tools for clinical diagnosis. However, given the difficulty of interpreting genetic variants, the utility of such tools for clinical diagnosis remains unclear, particularly for diseases that are characterized by clinical and genetic heterogeneity. We have developed a targeted “MitoExome” sequencing approach for the molecular diagnosis of suspected mitochondrial disorders in which we sequence the mitochondrial DNA (mtDNA) and the exons of ~ 1600 nuclear genes encoding the mitochondrial proteome or implicated in Mendelian disorders with phenotypic overlap to mitochondrial disease. Previously, we have shown that targeted sequencing enabled confident molecular
Abstracts
1. Mears, J. A., Lackner, L. L., Fang, S., Ingerman, E., Nunnari, J., and Hinshaw, J. E. (2011) Conformational changes in Dnm1 support a contractile mechanism for mitochondrial fission, Nat Struct Mol Biol 18, 20–26.
e Department of Pathology, University Hospitals of Case Medical Center, Case Western Reserve University, Cleveland, OH 44016, USA f Department of Pharmacology, Case Western Reserve University, Cleveland, OH 44016, USA g Department of Medicine, School of Medicine, Case Western Reserve University, Cleveland, OH 44016, USA
doi:10.1016/j.mito.2012.07.060
66 Regulation of superoxide dismutase 1 in intermembrane space of mitochondria Presenter: Yutaka Suzuki Yutaka Suzuki, Jan Riemer Department of Biology, University of Kaiserslautern, 67663 Kaiserslautern, Germany Reactive oxygen species (ROS) are implicated in human pathology and aging, as damaging agents and/or signaling messengers. Since a − ROS, superoxide (O ),2 is generated by the respiratory chain, mitochondrial DNA mutations and mitochondrial diseases can be causes and/or results of production of superoxide. Superoxide can be converted to molecular oxygen and hydrogen peroxide by superoxide dismutases. Superoxide dismutase 1 (SOD1) localizes to the cytosol with a minor portion targeted to the intermembrane space (IMS) of mitochondria. The activity of SOD1 is regulated by the copper chaperone for SOD1 (CCS1), which introduces copper ions and disulfide bonds to SOD1. CCS1 also localizes to cytosol and IMS. Knock out of either of those proteins in yeast and in mouse results in hypersensitivity to oxidative stress. We are interested in the mechanism of SOD1 and CCS1 targeting to the IMS and how the distribution between cytosol and IMS is regulated. In yeast, we showed that oxidation of a pair of cysteine residues has a critical role in the IMS localization of CCS1 and thereby also in providing SOD1 activity for the IMS [1]. The oxidation of CCS1 depends on Mia40, a component of a redox-regulated import machinery. Although localization of CCS1 to the IMS in mammalian cells has been reported to depend on Mia40, the cysteines that are oxidized in yeast CCS1 are not conserved in mammalian proteins. This indicates a different mechanism of CCS1 distribution in mammalian cells compared to yeast cells which we currently investigate in mechanistic detail. Our observations will contribute to a better understanding of the physiological and pathological role of superoxide in the IMS. Klöppel C*, Suzuki Y*, Kojer K, Petrungaro C, Longen S, Fiedler S, Keller S, Riemer J. (2011). Mol Biol Cell. 3749–57. doi:10.1016/j.mito.2012.07.061
68 A myopathy with retiform mitochondria Presenter: Hisashi Fujioka Hisashi Fujiokaa,b, Bernard Tandlerb,c, Laura Konczald, Mark L. Cohene, Mariana Roscab,f, Charles L. Hoppelb,f,g a Electron Microscopy Facility, Case Western Reserve University, Cleveland, OH 44016, USA b Center for Mitochondrial Diseases, School of Medicine, Case Western Reserve University, Cleveland, OH 44016, USA c Department of Biological Sciences, School of Dental Medicine, Case Western Reserve University, Cleveland, OH 44016, USA d Center for Human Genetics, Case Western Reserve University, Cleveland, OH 44016, USA
A surgical biopsy was obtained from the left quadriceps muscle of a 40-year-old male who exhibited progressive fatigue and unexplained exercise intolerance. Light microscopic examination of the specimen was unremarkable. Electron microscopic (EM) examination of the biopsy revealed an extremely unusual mitochondrial arrangement. In normal muscle, mitochondria are found in clusters beneath the sarcolemma or in rows between myofibrils. In the case in question, the muscle mitochondria were disposed in what at low magnification appeared to be a network. Some of the component organelles measured more than 19 μm in length. The cristae in these elongated organelles were for the most part obliquely oriented, and ranged from lamellar to tubular in form. The consensus among biochemists is that mitochondria form a structural network in the cytoplasm, i.e., these networks consist of but a single continuous mitochondrion that ramifies throughout the cell. This notion is based on confocal microscopy (with its limited resolution) of living cultured cells. At first blush, our case seems to fit this idea. When, however we examined, the muscle mitochondria in these putative networks using EM (with its high degree of resolution), it became apparent that long, ramifying mitochondria were joined together end-to-end and that there was no direct continuity of membranes from one organelle to another. Such “networks” were apparent only in traverse or slightly oblique sections of the myofibrils. In some cases, an element of sarcoplasmic reticulum was interposed between the ends of two closely apposed mitochondria. It is obvious that mere propinquity does not necessarily equal continuity. However this does not rule out the possibility that these organelles form a functional syncytium. Mitochondria isolated from the biopsy lost their elongated morphology to become spheroidal and were metabolically normal. It can be concluded that the muscle symptoms exhibited by our patient did not originate in defective mitochondria per se, but rather in the peculiarity of their arrangement within myofibrils. doi:10.1016/j.mito.2012.07.062
69 Misplaced mitochondria in a human myopathy Presenter: Hisashi Fujioka Hisashi Fujiokaa,b, Bernard Tandlerb,c, Laura Konczald, Mark L. Cohene, Mariana Roscab,f, Charles L. Hoppelb,f,g a Electron Microscopy Facility, Case Western Reserve University, Cleveland, OH 44016, USA b Center for Mitochondrial Diseases, School of Medicine, Case Western Reserve University, Cleveland, OH 44016, USA c Department of Biological Sciences, School of Dental Medicine, Case Western Reserve University, Cleveland, OH 44016, USA d Center for Human Genetics, University Hospitals of Case Medical Center, Case Western Reserve University, Cleveland, OH 44016, USA e Department of Pathology, University Hospitals of Case Medical Center, Case Western Reserve University, Cleveland, OH 44016, USA f Department of Pharmacology, Case Western Reserve University, Cleveland, OH 44016, USA g Department Medicine, School of Medicine, Case Western Reserve University, Cleveland, OH 44016, USA A specimen of the right quadriceps muscle was obtained by surgery from a 23-year-old female who displayed muscle weakness
Abstracts
among other symptoms. Light microscopy revealed abnormal mitochondrial distribution, consistent with primary mitochondrial disease. Electron microscopic examination of the biopsy revealed areas of typical structure, as well as areas showing morphological alterations. In the normal portions, small mitochondria participated in the formation of triads, which were situated at the Z disk; these triads consisted of paired mitochondria in close relation to elements of the sarcoplasmic reticulum (SR). In pathologically affected areas, typical triads were absent. Instead, relatively long, cylindrical mitochondria were oriented at right angles to the long axis of the myofibrils at the level where Z disks normally are present. These mitochondria measured up to 7 μm in length and usually less than 0.2 μm in width. Their cristae showed a normal range of variation. Many of these transverse mitochondria had extremely narrow segments, giving them a nodal appearance. In these narrow regions, the cristae were largely unaffected. Scattered elements of SR occasionally were present in relation to the transverse mitochondria, but rather than forming conventional triads, single SR elements were distributed unilaterally alongside the transverse mitochondria. In those myofibrils with transverse mitochondria, there were no mitochondria in the conventional interfibrillar position. Interestingly, biochemical examination of mitochondria isolated from the original biopsy revealed normal oxidative metabolism and completely normal metabolic parameters. In the face of normal mitochondrial function, it seems reasonable to suppose that the patient's muscle symptoms are based on malpositioning of the muscle mitochondria and on effacement of Z disks. The latter are sites of interaction between α-actinin, titin, and actin filaments. Because sarcomeric contraction depends on linkage to Z disks and on interplay between SR calcium, ATP, and heavy meromyosin, the aberrant morphology of the myofibrils and their attendant organelles might affect diffusion of the requisite molecules, leading to impaired contractility, hence weakness. doi:10.1016/j.mito.2012.07.063
70 Diagnostic application of measuring oxidative phosphorylation in permeabilized skin fibroblasts Presenter: Fang Ye Fang Ye, Charles L. Hoppel Center of Mitochondrial Disease, Department of Pharmacology and Medicine, Case Western Reserve University, Cleveland, OH 44106, United States The laboratory investigation in patients suspected of having a mitochondrial disease includes clinical laboratory metabolomics, genetic analyzing of mtDNA and nuclear DNA, and muscle biopsy. We asked whether cultured skin fibroblasts, a less invasive procedure, could provide a diagnostic step, possibly circumventing an open muscle biopsy in some patients. To approach this goal, we used human skin fibroblasts cultured in glucose containing medium to examine oxidative phosphorylation (OXPHOS), added to the measurement of electron transport chain (ETC) complex activities. We developed a method composed of two protocols using a high resolution respirometer (O2K, Oroboros). Protocol 1 measures oxygen consumption rates of Complexes I and II under the ADPstimulated state and maximal respiration with an uncoupler, FCCP. Following this, Complex IV rate is measured with TMPD plus ascorbate. In protocol 2, we assess fatty acid oxidation with palmitoylcarnitine and Complex III rate with duroquinol under state 3 condition, in addition to Complex III maximal respiration with FCCP. We have obtained reproducible OXPHOS data from twenty-three normal human skin fibroblast lines with multiple repeats and built
575
reference intervals for normal OXPHOS function. These data also provide validation for the analysis. To initially verify the utility of skin fibroblasts, we analyses a patient with a previously diagnosed skeletal muscle mitochondrial complex I defect. A Complex I defect was clearly identified by measuring OXPHOS in the fibroblasts. Moreover, four fibroblast lines from four patients show normal OXPHOS function which correlates with normal OXPHOS in isolated skeletal muscle mitochondria from these patients. Currently, this approach has been applied for clinical diagnosis. Fifty-two highly selected patient samples have been analyzed. We identified 24 samples with normal OXPHOS function, 15 with Complex I related defects, 5 with ADP phosphorylation system defects, 3 with fatty acid oxidation defects, 2 with Complex III related defects, and one each for defect in CoQ, pyruvate related oxidation and Complex IV. Interestingly, there are three subtypes of Complex I defects detected: 1) Decreased state 3 and maximal respiration (pyruvate, malate and glutamate) with decreased fatty acid oxidation. 2) Decreased state 3 and maximal respiration (pyruvate, malate and glutamate), but with normal fatty acid oxidation. 3) Normal state 3 oxidation (pyruvate, malate, glutamate and palmitoylcarnitine), but with decreased maximal respiration. Of diagnostic importance, this new method uncovered ADP phosphorylation defects as well as defects in substrate transport (pyruvate oxidation) and fatty acid oxidation. It is our belief that by identifying defects with a skin biopsy, we can avert an open muscle biopsy, a worthwhile endeavor. doi:10.1016/j.mito.2012.07.064
71 Targeted exome sequencing of suspected mitochondrial disorders in a hospital-based cohort Presenter: Daniel S. Lieber Daniel S. Liebera,b,c,d, Sarah E. Calvoa,b,c,d, Nancy G. Slatea,b, Shangtao Liua,b, Mark L. Borowskya, Steven G. Hershmana,b,c,d, Nina B. Golda,b, Gerard T. Berrye, David M. Muellerf, Jeremy D. Schmahmanng, Katherine B. Simsb,g, Vamsi K. Moothaa,b,c,d a Department of Molecular Biology, Massachusetts General Hospital, Boston, MA 02114, United States b Center for Human Genetic Research, Massachusetts General Hospital, Boston, MA 02114, United States c Department of Systems Biology, Harvard Medical School, Boston, MA 02115, United States d Broad Institute of Harvard and MIT, Cambridge, MA 02141, United States e The Manton Center for Orphan Disease Research, Children's Hospital Boston, Boston, MA 02115, United States f Department of Biochemistry and Molecular Biology, Rosalind Franklin University of Medicine and Science, The Chicago Medical School, North Chicago, IL 60064, United States g Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Boston MA 02114, United States Advances in next-generation sequencing (NGS) have revolutionized genetic research and are poised to become routine tools for clinical diagnosis. However, given the difficulty of interpreting genetic variants, the utility of such tools for clinical diagnosis remains unclear, particularly for diseases that are characterized by clinical and genetic heterogeneity. We have developed a targeted “MitoExome” sequencing approach for the molecular diagnosis of suspected mitochondrial disorders in which we sequence the mitochondrial DNA (mtDNA) and the exons of ~ 1600 nuclear genes encoding the mitochondrial proteome or implicated in Mendelian disorders with phenotypic overlap to mitochondrial disease. Previously, we have shown that targeted sequencing enabled confident molecular