{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,3]],"date-time":"2026-10-03T22:29:13Z","timestamp":1791066553947,"version":"4.1.0"},"update-to":[{"DOI":"10.12688\/f1000research.53230.1","type":"new_version","label":"New version","source":"publisher","updated":{"date-parts":[[2022,9,2]],"date-time":"2022-09-02T00:00:00Z","timestamp":1662076800000}}],"reference-count":113,"publisher":"F1000 Research Ltd","license":[{"start":{"date-parts":[[2022,9,2]],"date-time":"2022-09-02T00:00:00Z","timestamp":1662076800000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/501100000024","name":"Canadian Institutes of Health Research","doi-asserted-by":"crossref","id":[{"id":"10.13039\/501100000024","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":["f1000research.com"],"crossmark-restriction":false},"short-container-title":["F1000Res"],"abstract":"<ns3:p>\n                    <ns3:bold>Background:<\/ns3:bold>\n                    Pathogenic variants in\n                    <ns3:italic>MFN2<\/ns3:italic>\n                    cause Charcot-Marie-Tooth disease (CMT) type 2A (CMT2A) and are the leading cause of the axonal subtypes of CMT. CMT2A is characterized by predominantly distal motor weakness and muscle atrophy, with highly variable severity and onset age. Notably, some\n                    <ns3:italic>MFN2<\/ns3:italic>\n                    variants can also lead to other phenotypes such as optic atrophy, hearing loss and lipodystrophy. Despite the clear link between\n                    <ns3:italic>MFN2<\/ns3:italic>\n                    and CMT2A, our mechanistic understanding of how dysfunction of the MFN2 protein causes human disease pathologies remains incomplete. This lack of understanding is due in part to the multiple cellular roles of MFN2. Though initially characterized for its role in mediating mitochondrial fusion, MFN2 also plays important roles in mediating interactions between mitochondria and other organelles, such as the endoplasmic reticulum and lipid droplets. Additionally, MFN2 is also important for mitochondrial transport, mitochondrial autophagy, and has even been implicated in lipid transfer. Though over 100 pathogenic\n                    <ns3:italic>MFN2<\/ns3:italic>\n                    variants have been described to date, only a few have been characterized functionally, and even then, often only for one or two functions.\n                  <\/ns3:p>\n                  <ns3:p>\n                    <ns3:bold>Method:<\/ns3:bold>\n                    Several MFN2-mediated functions were characterized in fibroblast cells from a patient presenting with cerebellar ataxia, deafness, blindness, and diffuse cerebral and cerebellar atrophy, who harbours a novel homozygous MFN2 variant, D414V, which is found in a region of the HR1 domain of MFN2 where few pathogenic variants occur.\n                  <\/ns3:p>\n                  <ns3:p>\n                    <ns3:bold>Results:<\/ns3:bold>\n                    We found evidence for impairment of several MFN2-mediated functions. Consistent with reduced mitochondrial fusion, patient fibroblasts exhibited more fragmented mitochondrial networks and had reduced mtDNA copy number. Additionally, patient fibroblasts had reduced oxygen consumption, fewer mitochondrial-ER contacts, and altered lipid droplets that displayed an unusual perinuclear distribution.\n                  <\/ns3:p>\n                  <ns3:p>\n                    <ns3:bold>Conclusion:<\/ns3:bold>\n                    Overall, this work characterizes D414V as a novel variant in MFN2 and expands the phenotypic presentation of\n                    <ns3:italic>MFN2<\/ns3:italic>\n                    variants to include cerebellar ataxia.\n                  <\/ns3:p>","DOI":"10.12688\/f1000research.53230.2","type":"journal-article","created":{"date-parts":[[2022,9,2]],"date-time":"2022-09-02T11:20:08Z","timestamp":1662117608000},"page":"606","update-policy":"https:\/\/doi.org\/10.12688\/f1000research.crossmark-policy","source":"Crossref","is-referenced-by-count":13,"title":["Characterization of a novel variant in the HR1 domain of MFN2 in a patient with ataxia, optic atrophy and sensorineural hearing loss"],"prefix":"10.12688","volume":"10","author":[{"given":"Govinda","family":"Sharma","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mashiat","family":"Zaman","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rasha","family":"Sabouny","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Matthew","family":"Joel","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-3957-0647","authenticated-orcid":false,"given":"Kristina","family":"Martens","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Davide","family":"Martino","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"A.P. 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Feely","year":"2011","journal-title":"Neurology."},{"key":"ref9","doi-asserted-by":"publisher","first-page":"4914","DOI":"10.1038\/s41467-019-12912-0","article-title":"Structural insights of human mitofusin-2 into mitochondrial fusion and CMT2A onset.","volume":"10","author":"Y Li","year":"2019","journal-title":"Nat Commun."},{"key":"ref10","doi-asserted-by":"publisher","first-page":"858-862","DOI":"10.1126\/science.1099793","article-title":"Structural Basis of Mitochondrial Tethering by Mitofusin Complexes.","volume":"305","author":"T Koshiba","year":"2004","journal-title":"Science."},{"key":"ref11","doi-asserted-by":"publisher","first-page":"372-376","DOI":"10.1038\/nature21077","article-title":"MFN1 structures reveal nucleotide-triggered dimerization critical for mitochondrial fusion.","volume":"542","author":"Y Cao","year":"2017","journal-title":"Nature."},{"key":"ref12","doi-asserted-by":"publisher","first-page":"621-629","DOI":"10.1083\/jcb.201609019","article-title":"Structures 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Wolf","year":"2019","journal-title":"Cells."},{"key":"ref38","doi-asserted-by":"publisher","first-page":"1782-1800","DOI":"10.1093\/hmg\/ddz008","article-title":"MFN2 mutations in Charcot\u2013Marie\u2013Tooth disease alter mitochondria-associated ER membrane function but do not impair bioenergetics.","volume":"28","author":"D Larrea","year":"2019","journal-title":"Hum Mol Genet."},{"key":"ref39","doi-asserted-by":"publisher","first-page":"e45241","DOI":"10.15252\/embr.201745241","article-title":"Mitofusin gain and loss of function drive pathogenesis in Drosophila models of CMT2A neuropathy.","volume":"19","author":"N El Fissi","year":"2018","journal-title":"EMBO Rep."},{"key":"ref40","doi-asserted-by":"publisher","first-page":"365-369","DOI":"10.1111\/jns.12192","article-title":"Increased mitochondrial fusion in a autosomal recessive CMT2A family with mitochondrial GTPase mitofusin 2 mutations.","volume":"21","author":"P Codron","year":"2016","journal-title":"J Peripher Nerv 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Rocha","year":"2017","journal-title":"Elife."},{"key":"ref44","doi-asserted-by":"publisher","first-page":"152-164","DOI":"10.1111\/jns5.12066","article-title":"Characterization of the mitofusin 2 R94W mutation in a knock-in mouse model.","volume":"19","author":"A Strickland","year":"2014","journal-title":"J Peripher Nerv Syst."},{"key":"ref45","doi-asserted-by":"publisher","first-page":"245-256","DOI":"10.1007\/s00401-012-1036-y","article-title":"Mitofusin 2 mutations affect mitochondrial function by mitochondrial DNA depletion.","volume":"125","author":"S Vielhaber","year":"2013","journal-title":"Acta Neuropathol."},{"key":"ref46","doi-asserted-by":"publisher","first-page":"e219-e219","DOI":"10.1093\/brain\/aws049","article-title":"MFN2 mutations cause compensatory mitochondrial DNA proliferation.","volume":"135","author":"K Sitarz","year":"2012","journal-title":"Brain."},{"key":"ref47","doi-asserted-by":"publisher","first-page":"23-34","DOI":"10.1093\/brain\/awr323","article-title":"The MFN2 gene is responsible for mitochondrial DNA instability and optic atrophy \u2018plus\u2019 phenotype.","volume":"135","author":"C Rouzier","year":"2012","journal-title":"Brain."},{"key":"ref48","doi-asserted-by":"publisher","first-page":"1678-1681","DOI":"10.1212\/01.wnl.0000311275.89032.22","article-title":"Severe early-onset axonal neuropathy with homozygous and compound heterozygous MFN2 mutations.","volume":"70","author":"G Nicholson","year":"2008","journal-title":"Neurology."},{"key":"ref49","doi-asserted-by":"publisher","first-page":"23-34","DOI":"10.1093\/brain\/awr323","article-title":"The MFN2 gene is responsible for mitochondrial DNA instability and optic atrophy \u2018plus\u2019 phenotype.","volume":"135","author":"C Rouzier","year":"2011","journal-title":"Brain."},{"key":"ref50","doi-asserted-by":"publisher","first-page":"2103-2118","DOI":"10.1093\/brain\/awl174","article-title":"Early onset severe and late-onset mild Charcot-Marie-Tooth disease with mitofusin 2 (MFN2) mutations.","volume":"129","author":"K Chung","year":"2006","journal-title":"Brain."},{"key":"ref51","doi-asserted-by":"publisher","first-page":"1420-1435","DOI":"10.1016\/j.jacl.2018.07.009","article-title":"MFN2-associated lipomatosis: Clinical spectrum and impact on adipose tissue.","volume":"12","author":"E Capel","year":"2018","journal-title":"J Clin Lipidol."},{"key":"ref52","doi-asserted-by":"publisher","first-page":"5109-5114","DOI":"10.1093\/hmg\/ddv229","article-title":"Homozygous mutations in MFN2 cause multiple symmetric lipomatosis associated with neuropathy.","volume":"24","author":"S Sawyer","year":"2015","journal-title":"Hum Mol 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Med."},{"key":"ref60","doi-asserted-by":"publisher","DOI":"10.3791\/54899","article-title":"Study of endoplasmic reticulum and mitochondria interactions by in situ proximity ligation assay in fixed cells.","author":"E Tubbs","year":"2016","journal-title":"J Vis Exp."},{"key":"ref61","doi-asserted-by":"publisher","first-page":"676-682","DOI":"10.1038\/nmeth.2019","article-title":"Fiji: an open-source platform for biological-image analysis.","volume":"9","author":"J Schindelin","year":"2012","journal-title":"Nat Methods."},{"key":"ref62","doi-asserted-by":"publisher","first-page":"2723-2734","DOI":"10.1172\/JCI31604","article-title":"Ataxia-telangiectasia mutated kinase regulates ribonucleotide reductase and mitochondrial homeostasis.","volume":"117","author":"J Eaton","year":"2007","journal-title":"J Clin Invest."},{"key":"ref63","doi-asserted-by":"publisher","first-page":"402-408","DOI":"10.1006\/meth.2001.1262","article-title":"Analysis of relative gene expression data using real-time 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Alliance."},{"key":"ref70","article-title":"HGMD."},{"key":"ref71","doi-asserted-by":"publisher","first-page":"2093-2102","DOI":"10.1093\/brain\/awl126","article-title":"MFN2 mutation distribution and genotype\/phenotype correlation in Charcot\u2013Marie\u2013Tooth type 2.","volume":"129","author":"K Verhoeven","year":"2006","journal-title":"Brain."},{"key":"ref72","doi-asserted-by":"publisher","first-page":"2664-2676","DOI":"10.1093\/brain\/awr184","article-title":"Genetic spectrum of hereditary neuropathies with onset in the first year of life.","volume":"134","author":"J Baets","year":"2011","journal-title":"Brain: J Neurol."},{"key":"ref73","doi-asserted-by":"publisher","first-page":"594-598","DOI":"10.1111\/cge.12432","article-title":"A cohort study of MFN2 mutations and phenotypic spectrums in Charcot-Marie-Tooth disease 2A patients.","volume":"87","author":"B Choi","year":"2015","journal-title":"Clin Genet."},{"key":"ref74","doi-asserted-by":"publisher","first-page":"276-281","DOI":"10.1002\/ana.20797","article-title":"Axonal neuropathy with optic atrophy is caused by mutations in mitofusin 2.","volume":"59","author":"S Z\u00fcchner","year":"2006","journal-title":"Ann Neurol."},{"key":"ref75","doi-asserted-by":"publisher","first-page":"471-475","DOI":"10.1126\/science.1231031","article-title":"PINK1-phosphorylated mitofusin 2 is a Parkin receptor for culling damaged mitochondria.","volume":"340","author":"Y Chen","year":"2013","journal-title":"Science."},{"key":"ref76","doi-asserted-by":"publisher","first-page":"245-256","DOI":"10.1007\/s00401-012-1036-y","article-title":"Mitofusin 2 mutations affect mitochondrial function by mitochondrial DNA depletion.","volume":"125","author":"S Vielhaber","year":"2013","journal-title":"Acta Neuropathol."},{"key":"ref77","doi-asserted-by":"publisher","first-page":"157-168","DOI":"10.1091\/mbc.E20-09-0605","article-title":"Identification of new OPA1 cleavage site reveals that short isoforms regulate mitochondrial fusion.","volume":"32","author":"R Wang","year":"2021","journal-title":"Mol Biol Cell."},{"key":"ref78","doi-asserted-by":"publisher","first-page":"5670-5681","DOI":"10.1074\/jbc.M608168200","article-title":"Identification of a novel mitochondrial complex containing mitofusin 2 and stomatin-like protein 2.","volume":"282","author":"P H\u00e1jek","year":"2007","journal-title":"J Biol Chem."},{"key":"ref79","doi-asserted-by":"publisher","first-page":"1844-1856","DOI":"10.15252\/embr.201642698","article-title":"The membrane scaffold SLP2 anchors a proteolytic hub in mitochondria containing PARL and the i-AAA protease YME1L.","volume":"17","author":"T Wai","year":"2016","journal-title":"EMBO Rep."},{"key":"ref80","doi-asserted-by":"publisher","first-page":"1589-1600","DOI":"10.1038\/emboj.2009.89","article-title":"SLP-2 is required for stress-induced mitochondrial hyperfusion.","volume":"28","author":"D Tondera","year":"2009","journal-title":"EMBO J."},{"key":"ref81","doi-asserted-by":"publisher","first-page":"16017-16022","DOI":"10.1073\/pnas.1408061111","article-title":"A Mitofusin-2-dependent inactivating cleavage of Opa1 links changes in mitochondria cristae and ER contacts in the postprandial liver.","volume":"111","author":"A Sood","year":"2014","journal-title":"Proc Natl Acad Sci U S A."},{"key":"ref82","doi-asserted-by":"publisher","first-page":"3525-3532","DOI":"10.1091\/mbc.e09-03-0252","article-title":"Mitofusins and OPA1 mediate sequential steps in mitochondrial membrane fusion.","volume":"20","author":"Z Song","year":"2009","journal-title":"Mol Biol Cell."},{"key":"ref83","doi-asserted-by":"publisher","first-page":"303-311","DOI":"10.1083\/jcb.200209015","article-title":"The intramitochondrial dynamin-related GTPase, Mgm1p, is a component of a protein complex that mediates mitochondrial fusion.","volume":"160","author":"E Wong","year":"2003","journal-title":"J Cell Biol."},{"key":"ref84","doi-asserted-by":"publisher","first-page":"2342-2356","DOI":"10.1091\/mbc.e02-12-0788","article-title":"Mgm1p, a dynamin-related GTPase, is essential for fusion of the mitochondrial outer membrane.","volume":"14","author":"H Sesaki","year":"2003","journal-title":"Mol Biol Cell."},{"key":"ref85","doi-asserted-by":"publisher","first-page":"36-48","DOI":"10.1016\/j.biocel.2014.05.041","article-title":"A proteomic screen with Drosophila Opa1-like identifies Hsc70-5\/Mortalin as a regulator of mitochondrial morphology and cellular homeostasis.","volume":"54","author":"S Banerjee","year":"2014","journal-title":"Int J Biochem Cell Biol."},{"key":"ref86","doi-asserted-by":"publisher","first-page":"315-325","DOI":"10.1042\/BC20070110","article-title":"Metalloprotease-mediated OPA1 processing is modulated by the mitochondrial membrane potential.","volume":"100","author":"O Guillery","year":"2008","journal-title":"Biology of the Cell."},{"key":"ref87","doi-asserted-by":"publisher","first-page":"2328-2337","DOI":"10.1073\/pnas.1810932116","article-title":"Altered interplay between endoplasmic reticulum and mitochondria in Charcot\u2013Marie\u2013Tooth type 2A neuropathy.","volume":"116","author":"N Bernard-Marissal","year":"2019","journal-title":"Proc Natl Acad Sci U S A."},{"key":"ref88","doi-asserted-by":"publisher","first-page":"405-414","DOI":"10.1083\/jcb.200611080","article-title":"Complementation between mouse Mfn1 and Mfn2 protects mitochondrial fusion defects caused by CMT2A disease mutations.","volume":"176","author":"S Detmer","year":"2007","journal-title":"J Cell Biol."},{"key":"ref89","doi-asserted-by":"crossref","DOI":"10.1242\/jcs.258944","article-title":"The role of mitochondrial dynamics in mtDNA maintenance.","volume":"134","author":"R Sabouny","year":"2021","journal-title":"J Cell Sci."},{"key":"ref90","doi-asserted-by":"publisher","first-page":"2250-2251","DOI":"10.1080\/15548627.2016.1215383","article-title":"When MFN2 (mitofusin 2) met autophagy: A new age for old muscles.","volume":"12","author":"D Sebasti\u00e1n","year":"2016","journal-title":"Autophagy."},{"key":"ref91","doi-asserted-by":"publisher","first-page":"131-142","DOI":"10.1212\/WNL.0000000000003491","article-title":"Dysregulated mitophagy and mitochondrial organization in optic atrophy due to OPA1 mutations.","volume":"88","author":"C Liao","year":"2017","journal-title":"Neurology."},{"key":"ref92","doi-asserted-by":"crossref","DOI":"10.15252\/emmm.201911659","article-title":"FBXL4 deficiency increases mitochondrial removal by autophagy.","volume":"12","author":"D Alsina","year":"2020","journal-title":"EMBO Mol Med."},{"key":"ref93","doi-asserted-by":"publisher","first-page":"1013-1031","DOI":"10.1007\/s00401-019-02059-z","article-title":"MSTO1 mutations cause mtDNA depletion, manifesting as muscular dystrophy with cerebellar involvement.","volume":"138","author":"S Donkervoort","year":"2019","journal-title":"Acta Neuropathol."},{"key":"ref94","doi-asserted-by":"publisher","first-page":"13534-13539","DOI":"10.1073\/pnas.1109263108","article-title":"Super-resolution microscopy reveals that mammalian mitochondrial nucleoids have a uniform size and frequently contain a single copy of mtDNA.","volume":"108","author":"C Kukat","year":"2011","journal-title":"Proc Natl Acad Sci U S A."},{"key":"ref95","doi-asserted-by":"publisher","first-page":"11863-11868","DOI":"10.1073\/pnas.1301951110","article-title":"Dynamics of nucleoid structure regulated by mitochondrial fission contributes to cristae reformation and release of cytochrome c.","volume":"110","author":"R Ban-Ishihara","year":"2013","journal-title":"Proc Natl Acad Sci U S A."},{"key":"ref96","doi-asserted-by":"publisher","first-page":"211-223","DOI":"10.1128\/MCB.01054-14","article-title":"Dynamics of Mitochondrial DNA Nucleoids Regulated by Mitochondrial Fission Is Essential for Maintenance of Homogeneously Active Mitochondria during Neonatal Heart Development.","volume":"35","author":"T Ishihara","year":"2015","journal-title":"Mol Cell Biol."},{"key":"ref97","doi-asserted-by":"publisher","first-page":"e223-e223","DOI":"10.1093\/brain\/aws111","article-title":"MFN2, a new gene responsible for mitochondrial DNA depletion.","volume":"135","author":"F Renaldo","year":"2012","journal-title":"Brain."},{"key":"ref98","doi-asserted-by":"publisher","first-page":"17190-17197","DOI":"10.1074\/jbc.M212754200","article-title":"Mitofusin-2 Determines Mitochondrial Network Architecture and Mitochondrial Metabolism: A novel regulatory mechanism altered in obesity.","volume":"278","author":"D Bach","year":"2003","journal-title":"J Biol Chem."},{"key":"ref99","doi-asserted-by":"publisher","first-page":"462523","DOI":"10.1101\/462523","article-title":"CMT disease 2A and demyelination decouple ATP and ROS production by axonal mitochondria.","author":"G van Hameren","year":"2018","journal-title":"bioRxiv."},{"key":"ref100","doi-asserted-by":"publisher","first-page":"1405-1415","DOI":"10.1093\/hmg\/ddi149","article-title":"The Charcot\u2013Marie\u2013Tooth type 2A gene product, Mfn2, up-regulates fuel oxidation through expression of OXPHOS system.","volume":"14","author":"S Pich","year":"2005","journal-title":"Hum Mol Genet."},{"key":"ref101","doi-asserted-by":"publisher","first-page":"E1208-E1221","DOI":"10.1152\/ajpendo.00546.2012","article-title":"A form of mitofusin 2 (Mfn2) lacking the transmembrane domains and the COOH-terminal end stimulates metabolism in muscle and liver cells.","volume":"305","author":"J Segal\u00e9s","year":"2013","journal-title":"Am J Physiol Endocrinol Metab."},{"key":"ref102","doi-asserted-by":"publisher","first-page":"3741-3753","DOI":"10.1093\/hmg\/ddw220","article-title":"Loss-of-function mutations in the SIGMAR1 gene cause distal hereditary motor neuropathy by impairing ER-mitochondria tethering and Ca2+ signalling.","volume":"25","author":"E Gregianin","year":"2016","journal-title":"Hum Mol Genet."},{"key":"ref103","doi-asserted-by":"publisher","first-page":"615-627","DOI":"10.1093\/hmg\/ddy352","article-title":"Sensory neuropathy-causing mutations in ATL3 affect ER\u2013mitochondria contact sites and impair axonal mitochondrial distribution.","volume":"28","author":"M Krols","year":"2018","journal-title":"Hum Mol Genet."},{"key":"ref104","doi-asserted-by":"publisher","DOI":"10.3389\/fnmol.2018.00038","article-title":"Mitofusin-Dependent ER Stress Triggers Glial Dysfunction and Nervous System Degeneration in a Drosophila Model of Friedreich\u2019s Ataxia.","volume":"11","author":"O Edenharter","year":"2018","journal-title":"Front Mol Neurosci."},{"key":"ref105","doi-asserted-by":"publisher","first-page":"342-348","DOI":"10.1016\/j.expneurol.2012.11.001","article-title":"Endoplasmic reticulum stress contributes to prediabetic peripheral neuropathy.","volume":"247","author":"S Lupachyk","year":"2013","journal-title":"Exp Neurol."},{"key":"ref106","doi-asserted-by":"publisher","first-page":"765-772","DOI":"10.1016\/j.jnutbio.2014.03.007","article-title":"Proanthocyanidins protect against early diabetic peripheral neuropathy by modulating endoplasmic reticulum stress.","volume":"25","author":"Y Ding","year":"2014","journal-title":"J Nutr Biochem."},{"key":"ref107","doi-asserted-by":"publisher","first-page":"g459","DOI":"10.1136\/bmj.g459","article-title":"Chronic and slowly progressive weakness of the legs and hands.","volume":"348","author":"H Nightingale","year":"2014","journal-title":"BMJ (Clinical research ed.)."},{"key":"ref108","doi-asserted-by":"publisher","first-page":"43-56","DOI":"10.1016\/j.phrs.2018.09.006","article-title":"Regulation of ER-mitochondria contacts by Parkin via Mfn2.","volume":"138","author":"V Basso","year":"2018","journal-title":"Pharmacol Res."},{"key":"ref109","doi-asserted-by":"publisher","first-page":"827-835","DOI":"10.1016\/j.cmet.2019.02.011","article-title":"Mitochondria Bound to Lipid Droplets: Where Mitochondrial Dynamics Regulate Lipid Storage and Utilization.","volume":"29","author":"I Benador","year":"2019","journal-title":"Cell Metab."},{"key":"ref110","doi-asserted-by":"publisher","DOI":"10.1016\/j.semcdb.2020.04.013","article-title":"The biology of lipid droplet-bound mitochondria.","author":"M Veliova","year":"2020","journal-title":"Semin Cell Dev Biol."},{"key":"ref111","doi-asserted-by":"publisher","first-page":"2849-2858","DOI":"10.1016\/j.celrep.2019.02.039","article-title":"Mitofusin 2 in Mature Adipocytes Controls Adiposity and Body Weight.","volume":"26","author":"G Mancini","year":"2019","journal-title":"Cell Rep."},{"key":"ref112","doi-asserted-by":"publisher","first-page":"1039-1040","DOI":"10.15252\/embr.201744341","article-title":"Let\u2019s burn whatever you have: mitofusin 2 metabolically re-wires brown adipose tissue.","volume":"18","author":"M Scheideler","year":"2017","journal-title":"EMBO Rep."},{"key":"ref113","doi-asserted-by":"publisher","first-page":"e66837-e66837","DOI":"10.1371\/journal.pone.0066837","article-title":"Dynamics and molecular determinants of cytoplasmic lipid droplet clustering and dispersion.","volume":"8","author":"D Orlicky","year":"2013","journal-title":"PLoS One."}],"container-title":["F1000Research"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/f1000research.com\/articles\/10-606\/v2\/xml","content-type":"application\/xml","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/f1000research.com\/articles\/10-606\/v2\/pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/f1000research.com\/articles\/10-606\/v2\/iparadigms","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2024,1,24]],"date-time":"2024-01-24T12:25:17Z","timestamp":1706099117000},"score":1,"resource":{"primary":{"URL":"https:\/\/f1000research.com\/articles\/10-606\/v2"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2022,9,2]]},"references-count":113,"URL":"https:\/\/doi.org\/10.12688\/f1000research.53230.2","relation":{"has-review":[{"id-type":"doi","id":"10.5256\/f1000research.136924.r149306","asserted-by":"subject"},{"id-type":"doi","id":"10.5256\/f1000research.136924.r218485","asserted-by":"subject"},{"id-type":"doi","id":"10.5256\/f1000research.136924.r218485","asserted-by":"object"},{"id-type":"doi","id":"10.5256\/f1000research.136924.r149306","asserted-by":"object"}]},"ISSN":["2046-1402"],"issn-type":[{"value":"2046-1402","type":"electronic"}],"subject":[],"published":{"date-parts":[[2022,9,2]]},"assertion":[{"value":"Indexed","URL":"https:\/\/f1000research.com\/articles\/10-606\/v2#article-reports","order":0,"name":"referee-status","label":"Referee status","group":{"name":"current-referee-status","label":"Current Referee Status"}},{"value":"10.5256\/f1000research.56591.r89928, Joanna Poulton, Nynke van Polanen, Nuffield Department of Women's and Reproductive Health, University of Oxford, Oxford, 12 Aug 2021, version 1, 2 approved with reservations","URL":"https:\/\/f1000research.com\/articles\/10-606\/v1#referee-response-89928","order":0,"name":"referee-response-89928","label":"Referee Report","group":{"name":"article-reports","label":"Article Reports"}},{"value":"<b>Timothy Shutt<\/b>; \n<i>Posted: 30 Jul 2022<\/i>; \n<i>&gt;We would like to thank the reviewers for their interest in the manuscript and their suggestions, which we have taken to heart and have addressed to the best of our ability. We would also like to apologize for the extended period of time for the revisions. The first author left the lab shortly after the paper was initially submitted, and it took some time to get a new person up and running on the project after the reviews were all received. In addition to several clarifications in the text, the revised version of the manuscript contains new data showing alterations to the expression of mitochondrial fusion proteins, as well as new mtDNA copy number data normalized to B-tubulin. We hope that the revisions answer the questions and issues raised by the reviewers. Below are the specific point by point response to the reviews.<\/i>  Reviewer 1  Thank you for asking me to review this paper characterizing a mutation in MFN2 that results in an unusual phenotype. I think that this paper is ready for indexing provided they can confirm some additional details including the number of controls studied.  \n<b>Main points:<\/b>  1. Neither the methods nor the results clearly spell out the N for each experiment. Figure 2B suggests that data was generated from 24 control and 27 patient fibroblasts.&nbsp; How many controls were used?&nbsp;  \n<i>&gt;We have updated the figure legends to indicate the number of cells used for analysis in each experiment. Furthermore, all of the images analyzed are included in the extended data, should anyone wish to reanalyze the data.<\/i>  The study would not be ready for publication if they only used one control.&nbsp; \n<i>&gt;While we only used a single control line for these experiments, our previously published work has shown that this control line behaves similar to other control lines (PMID: 31231018). We have added the following text to make this clear to the reader:  \u201cAs a control fibroblast line, we used an age-matched cell line that we have shown previously to behave similar to other controls fibroblast lines with respect to mitochondrial function (PMID: 31231018).\u201d<\/i>  They should state how many repetitions of each of the experiments there were (their single statement, \u201c\u2026three independent biological replicates\u201d on page 6 is not sufficiently detailed).&nbsp;These additional data would provide important support for the findings.  \n<i>&gt;We have clarified the text in the methods as follows:  \u201cFor all of the image analysis in this manuscript, the data shown in the figures are from a single biological replicate with three technical replicates (e.g. images used for analysis were gathered from three coverslips processed in parallel). Experiments were repeated independently at least twice, for a total of three biological replicates, and all reported trends were consistent with the representative data reported in the figures.\u201d<\/i>  Even though the findings are relevant and interesting, that only one patient has been used should be stated more clearly along with the need to study more patients\/relative with the novel D414V MFN2 variant.  \n<i>&gt;We agree that more patients with the D414V variant would strengthen the study, unfortunately we are only aware of this single case with this clinical presentation. Other family members were not available for investigation and there was no family history of similar disease. We have clarified the text to highlight the fact that we have only one patient with the following addition to the discussion:  \u201cUnfortunately, the D414V variant has not been described to date in any other patients. As such, we only have a single patient cell line in which to study this variant.\u201d<\/i>  2.&nbsp;The loss of mitochondria ER contacts (MERCs) is very beautiful and interesting.&nbsp; This may well be linked to the slight reduction in mitochondrial DNA copy number and in respiratory chain function. The authors discuss the hypothesis that mtDNA replication is impaired in MFN2 because of the effect of excessive mitochondrial fragmentation on the distribution of the replication machinery (ref 19).&nbsp; However, they barely discuss two other factors that are relevant to mtDNA replication: mitochondrial dynamics affect both nucleoid division and mitophagy (and hence mtDNA turnover).&nbsp; Interactions with the ER are thought to license mtDNA replication as well as initiating nucleoid division (ref 30) so having a reduced MERC area, and potentially having a smaller midzone (Kleele\n<i>&nbsp;et al.&nbsp;<\/i>(2021\n<sup>1<\/sup>)) could have an important effect. Impaired mitochondrial fusion may increase (Liao&nbsp;\n<i>et al<\/i>. (2017\n<sup>2<\/sup>)) or decrease (Sebasti&aacute;n\n<i>&nbsp;et al<\/i>.(2016\n<sup>3<\/sup>)) mitophagy in impaired MFN2 expression. Increased mitophagy could decrease mtDNA copy number and slow recovery from mtDNA depletion (ref 19).  \n<i>&gt;As the initial discussion was already somewhat lengthy, we had not gone into details on these other possibilities. However, in light of the new data in the revised manuscript, which suggest that mtDNA copy number may be reduced in part by reduced levels of mitochondria, we have amended the discussion to further discuss other factors linking fusion to altered mtDNA.<\/i>  3. The decrease in lipid droplets is interesting and I think novel in MFN2 mutants. Examining the distribution of lipid droplets in the form of distance to the nucleus is also a nice addition to the data. They should mention that (i) the exact molecular mechanisms how MFN2 maintains lipid homeostasis is unknown (ii) perilipin 1 much more abundant in adipose tissue (ref 34) than fibroblasts. From the discussion it is not clear how perilipins can explain the altered distribution found in the fibroblasts in the present paper. The lipid droplets in figure 5 could follow either (i) the mitochondrial network which seems also more perinuclear in the representative figure or (ii) the microtubular network which could be clarified by drug treatments such as nocodazole.  \n<i>&gt;We appreciate the comments from the reviewer regarding the interesting aspects of lipid droplet alterations. We agree that there is certainly much more to learn in this regard, however, we believe that it is beyond the scope of the current investigation. As suggested, we included the following statements in our discussion:  \u201cThough it should be noted that perilipin 1 is more abundant in adipose tissue that in fibroblasts (34).\u201d  \u201c\u2026 it is clear that the roles of MFN2 in maintaining lipid homeostasis are important, though the exact molecular mechanisms remain undefined\u201d<\/i>  4. Was the number of mitochondrial nucleoids or lipid droplets corrected for the cell size? \n<i>&gt;No, we did not correct for cell size, as we did not have a marker for the plasma membrane. Furthermore, the surface area covered by the cell does not necessarily correlate to the cell size if the cells do not attach and spread out to the same extent. <\/i>  Did the authors also measure the size of the lipid droplets in these cells?  \n<i>&gt;We did not measure the size of the lipid droplets, as the image processing required to visualize individual lipid droplets did not preserve the size information, though it is still reliable to quantify the number and distribution of the lipid droplets. At least by eye, the lipid droplet size does not appear to change between the cell lines.<\/i>  5. The patient\u2019s presentation is interesting: impaired vision, poor motor function and deafness. Of these, unusual features included early loss of vision, cerebellar ataxia, and brain MRI abnormalities.&nbsp; However, some basic clinical details are missing.  a) No segregation analysis is presented, presumably his parents may be no longer available. It would be useful to support recessive inheritance by identifying an asymptomatic individual in the pedigree who is heterozygous for the same mutation.  \n<i>&gt;Indeed, the patient\u2019s parents are not available for research, which is unfortunately common when investigating patients in adulthood. The suggestion to study heterozygous family members is of interest and we would have pursued this if other family members were available for research. Participation in research was offered to his sister who lives in the same city (who would have had a 50% chance of being a heterozygous carrier of this variant) but she declined participation.&nbsp;<\/i>  b)&nbsp; Analysis of skeletal muscle for mitochondrial DNA copy number and variable deletions.&nbsp; Muscle is more relevant than these data that they present in fibroblasts, and should be available because the patient had a muscle biopsy.  \n<i>&gt;Unfortunately, the muscle tissue was not available for mtDNA copy number analysis, as the patient\u2019s muscle biopsy was done when he lived in a different country.<\/i>  6. The authors present a nice visualization of mitochondrial fusion. As MFN1 is also important in mitochondrial fusion and MFN2 might interact with OPA1, do the authors have any data in changes of MFN1 and OPA1 levels in this patient? \n<i>&gt;At the reviewer\u2019s suggestion, we investigated the levels of MFN1 and OPA1 in our patient fibroblasts, and discovered some interesting differences (Fig 1E). Notably, we see decreased protein levels for both MFN1 and MFN2, as well as several other mitochondrial proteins, suggesting that overall mitochondrial abundance is reduced in the patient cells. In addition, we see unexpected changes to the abundance of OPA1 protein isoforms, which to our knowledge have not been noted for other pathogenic MFN2 variants. These new data and a discussion of these novel results has been added to the revised manuscript.<\/i>  \n<b>Minor points:<\/b>  A cartoon to illustrate the main findings would be welcome.&nbsp;  \n<i>&gt;We decided against including a cartoon figure, as it is not simple to illustrate the various findings of this manuscript in a straightforward figure that is easy to interpret, and did not feel it would add significantly to the manuscript.<\/i> &nbsp; Sentence on page 4, paragraph 4, line 3, please rephrase (\u201cCentral nervous \u2026 of cases\u201d)  \n<i>&gt;We have revised the text to make the meaning clearer, as follows: <\/i>  \n<i>\u201cCentral nervous system involvement is also rarely described (50), with periventricular and subcortical white matter lesions in 8 out of 21 patients from one series. A small minority of cases had transient neurological deficits such as dysarthria or paraesthesiae.\u201d<\/i>  Please state the glucose content of the medium during cell maintenance.  \n<i>&gt;&nbsp;The medium contains low glucose (5.56mM).<\/i> &nbsp; Page 5, third paragraph, line 9, goat anti-rabbit, should be goat anti-mouse.  \n<i>&gt;&nbsp;Corrected<\/i> &nbsp; Please state the company of the DAPI labelling.  \n<i>&gt;The DAPI used in this study is included in the Prolong Glass Antifade Mountant with NucBlue Stain from Fisher Scientific (Thermo Scientific P36983). In this case, the NucBlue in the commercial description is DAPI.<\/i> &nbsp; Please state which program is used to do the statistical testing and if a p-value of &lt;0.05 was considered significant.  \n<i>&gt;All statistical analyses were performed using Prism, and yes, for all analyses the significant differences indicate p&lt;0.05.<\/i>","URL":"https:\/\/f1000research.com\/articles\/10-606\/v2#referee-comment-8588","order":1,"name":"referee-comment-8588","label":"Referee Comment","group":{"name":"article-reports","label":"Article Reports"}},{"value":"10.5256\/f1000research.136924.r149306, Daniele Ghezzi, Alessia Nasca, Unit of Medical Genetics and Neurogenetics, Fondazione IRCCS Istituto Neurologico Carlo Besta, Milan, 06 Sep 2022, version 2, indexed","URL":"https:\/\/f1000research.com\/articles\/10-606\/v2#referee-response-149306","order":2,"name":"referee-response-149306","label":"Referee Report","group":{"name":"article-reports","label":"Article Reports"}},{"value":"10.5256\/f1000research.136924.r218485, Antonietta Franco, University of Washington, Seattle, Washington, 24 Jan 2024, version 2, indexed","URL":"https:\/\/f1000research.com\/articles\/10-606\/v2#referee-response-218485","order":3,"name":"referee-response-218485","label":"Referee Report","group":{"name":"article-reports","label":"Article Reports"}},{"value":"Canadian Institutes of Health Research. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.","order":4,"name":"grant-information","label":"Grant Information"},{"value":"This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.","order":0,"name":"copyright-info","label":"Copyright"}]}}