Phenotypic Assessment of Cox10 Variants and their Implications for Leigh Syndrome

Phenotypic Assessment of Cox10 Variants and their Implications for Leigh Syndrome | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Short Report Phenotypic Assessment of Cox10 Variants and their Implications for Leigh Syndrome Thomas-Shadi Voges, Eun Bi Lim, Abigail MacKenzie, Kyle Mudler, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4631252/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 16 Aug, 2024 Read the published version in BMC Research Notes → Version 1 posted 8 You are reading this latest preprint version Abstract Objectives Cox10 is an enzyme required for the activity of cytochrome c oxidase. Humans who lack at least one functional copy of Cox10 have a form of Leigh Syndrome, a genetic disease that is usually fatal in infancy. As more human genomes are sequenced, new alleles are being discovered; whether or not these alleles encode functional proteins remains unclear. Thus, we set out to measure the phenotypes of many human Cox10 variants by expressing them in yeast cells. Results We successfully expressed the reference sequence and 25 variants of human Cox10 in yeast. We quantitated the ability of these variants to support growth on nonfermentable media and directly measured cytochrome c oxidase activity. 11 of these Cox10 variants supported approximately half or more the cytochrome c oxidase activity compared to the reference sequence. All of the strains containing those 11 variants also grew robustly using a nonfermentable carbon source. Cells expressing the other variants showed low cytochrome c oxidase activity and failed to grow on nonfermentable media. Saccharomyces cerevisiae Cox10 cytochrome c oxidase Leigh Syndrome Figures Figure 1 Introduction Leigh Syndrome is a rare genetic condition characterized by progressive neuromuscular defects. This disorder is highly heterogeneous, due in part to the fact that it can be caused by alterations in at least 75 different genes. All of these genetic alterations lead to mitochondrial dysfunction including reduced or eliminated capacity for oxidative phosphorylation( 1 ). Notably, loss of function in one of these genes, COX10 , leads to a severe form of Leigh Syndrome that is typically fatal in the first year of life (Table 1 ). Two patients have been reported to survive longer, albeit with significant pathology( 2 , 3 ). Table 1 Known Pathological Variants of Human COX10 M1X Lethal ( 6 ) T14I/T377I Pathogenic ( 3 ) T196K/P225L Lethal ( 7 ) N204K Lethal ( 8 ) N204D Lethal ( 9 ) G288R Lethal ( 10 ) D336V/D336G Lethal ( 7 ) D336V/D339W Pathogenic ( 2 ) P420L Lethal ( 10 ) The Cox10 protein is highly conserved in the budding yeast Saccharomyces cerevisiae ; indeed, expression of the human COX10 gene in yeast can fully restore function in a strain missing its endogenous COX10 gene( 4 ). The Cox10 enzyme is located on the mitochondrial inner membrane and catalyzes the farnesylation of heme( 5 ). This modified heme group is incorporated as an essential prosthetic group into the cytochrome c oxidase (COX) enzyme, which catalyzes the transfer of electrons from cytochrome c to molecular oxygen. Eleven different point mutations in the human COX10 gene have been described in the literature as leading to disease in humans (Table 1 ). Of course, there are many more alleles in the human population and for the majority of these we have no direct evidence as to whether or not they encode functional proteins. As of June 17, 2024, the ClinVar database lists 102 known variants of the Cox10 protein, of which nearly three-quarters are of “uncertain significance”. We have selected 25 COX10 alleles for characterization in yeast. This additional knowledge should be useful to clinicians and genetic counselors faced with patients carrying these variants. Methods Expression of human COX10 alleles in yeast A cDNA encoding the reference sequence of human Cox10 was synthesized. To allow for proper expression, this construct included 500 bp upstream and 100 bp downstream of the yeast COX10 gene. Additionally, a myc epitope tag just before the stop codon was included along with sequences to facilitate cloning. This DNA was inserted into the EcoRI site of YEplac195 ( 11 ) for expression in yeast. The resulting plasmid was either used directly or mutated using the Q5 kit (New England Biolabs) so that it encoded the various alleles. Plasmids were sequenced to confirm successful mutagenesis before being transformed( 12 ) into cox10Δ yeast for phenotypic analysis. Growth Assessment Standard yeast media and growth conditions were used throughout these experiments ( 12 ). Ten-fold serial dilutions of freshly grown yeast were plated onto rich media containing either 2% glucose or 3% glycerol and allowed to grow at 30 o C for three days before photographing. COX assays Yeast were grown overnight at 30 o C with shaking in 15 mL of rich media using 3% raffinose as a carbon source. Raffinose supports growth by cells with and without functional oxidative phosphorylation but does not suppress mitochondrial production like glucose( 13 ). Cells were washed and lysed in 500 µL cold SH buffer (0.6M sorbitol, 25 mM HEPES pH 7.4) by vortexing with glass beads for five minutes. Unlysed cells were pelleted by centrifugation at 600g at 4 o C for five minutes, twice. Mitochondria were pelleted by centrifugation at 16,000g at 4 o C for ten minutes and were resuspended in 200 µL of SH buffer ( 13 ). 20 µL of each sample was incubated with 125 µg of reduced cytochrome c in 25 mM potassium phosphate pH 6.2. Absorbance was measured at 550 nm every five seconds for one minute to calculate the rate of cytochrome c oxidation ( 14 ). Reaction rates were normalized to the amount of protein in the lysate as found by the BCA reaction. Each sample was measured at least four times. Results and Discussion We selected 25 human COX10 alleles whose functions are not clearly understood and expressed them in yeast lacking the endogenous COX10 to determine how functional the encoded proteins are. This species grows efficiently by anaerobic metabolism and therefore can survive without functional Cox10 protein when provided with a fermentable carbon source like glucose (Fig. 1 A, left column). However, carbon sources like glycerol can only be utilized aerobically and thus require a functional Cox10 enzyme. On this fuel source, cells lacking Cox10 do not survive but they thrive if they contain either the yeast or human reference sequence COX10 . Strains with certain COX10 alleles, such as S103A or V356M, survive equally as well as those with the reference sequence COX10 . Other strains, such as those with I127T or Q322P, fail to grow when cultured on glycerol (Fig. 1 A, right column), indicating that these protein variants are nonfunctional. We also directly measured the COX activity in each of these yeast strains. We found that the yeast COX10 supports slightly more COX activity than the human reference sequence COX10 , in agreement with a previously published observation( 4 ). Yeast strains with certain human COX10 alleles show activities that are close to that provided by the reference sequence while others show very low activities (Fig. 1 B). Unsurprisingly, there is a strong, general correlation between the COX activity measurements and the ability to grow on glycerol. It appears that Cox10 variants that allow approximately 50% of the reference sequence COX activity (see variants S103A, P104L and A328T) grow very efficiently on glycerol and those that allow less than 25% of the reference sequence COX activity (such as I127T and D132Y) fail to grow at all (Fig. 1 ). Among the 16 assayed alleles classified as “uncertain significance” by the ClinVar database( 15 ), we find that six (S103A, D152Y, A174T, F209L, C343R, V356M) encode functional proteins. Four alleles are classified as having “conflicting classifications of pathogenicity”; we find that three of these alleles (P104L, A328T and R431W) encode functional proteins and one (T87I) does not. T221A is classified as “likely pathogenic” and T28I is classified as “benign/likely benign”; our data disagree with these two predictions. We believe that our in vivo measurements of the activity of these alleles will provide important, actionable information for clinicians and genetic counselors who may come across patients with these alleles in their practices. The D336V and D339W alleles were selected for a slightly different reason. Pitceathly et al . have reported that a human with these two alleles survived to adulthood and that both alleles encode nonfunctional proteins( 2 ). We thought that using more sensitive enzyme assays might reveal partial activity from one or both proteins but found that both are fully nonfunctional (Fig. 1 ). Recently, a second patient with unusual COX10 alleles has been described who survived past infancy( 3 ). This patient is homozygous for two alleles, T14I and T377I. The second variant is highly similar to the T380I variant that we found to be nonfunctional in that both alterations are located in the sixth transmembrane domain( 16 ), strongly suggesting that the patient’s Cox10 protein will be nonfunctional. Thus, it remains unclear how either patient has survived when most people with nonfunctional Cox10 variants die in infancy (Table 1 ). Perhaps specific alleles of other genes found in these patients can partially suppress the loss-of-function in COX10 . Identifying these genes could potentially point to interventions to treat Leigh Syndrome patients and is an area that our laboratory is pursuing. Limitations Despite the strong evolutionary conservation in oxidative phosphorylation mechanisms, it is possible that these variants will have a different level of activity in humans compared to yeast cells. Abbreviations COX: cytochrome c oxidase Declarations Acknowledgements We thank Fatima Alauddin, Bailey Bloom, Itzel Mancilla and Javier Valle Galisteo for their assistance in producing some alleles. Authors’ Contributions SDJ designed the study and supervised the work. TSV, EBL, AM, KM, RD, NEO and SDJ performed the experiments and analyzed the resulting data. All authors read and approved the final manuscript. Funding We thank the Office of Academic Affairs at North Central College for financial support. Ethics approval and consent to participate Not applicable Consent for publication Not applicable Availability of data and materials The datasets used and analyzed during the current study are available from the corresponding author upon reasonable request. Competing interests The authors declare that they have no competing interests References Bakare AB, Lesnefsky EJ, Iyer S. Leigh Syndrome: A Tale of Two Genomes. Front Physiol. 2021;12:693734. Pitceathly RDS, Taanman JW, Rahman S, Meunier B, Sadowski M, Cirak S, et al. COX10 mutations resulting in complex multisystem mitochondrial disease that remains stable into adulthood. JAMA Neurol. 2013;70(12):1556–61. Tavasoli A, Kachuei M, Talebi S, Eghdami S. Complex mitochondrial disease caused by the mutation of COX10 in a toddler: a case-report study. Ann Med Surg (Lond). 2024;86(6):3753–6. Glerum DM, Tzagoloff A. Isolation of a human cDNA for heme A:farnesyltransferase by functional complementation of a yeast cox10 mutant. Proc Natl Acad Sci USA. 1994;91(18):8452–6. Nobrega MP, Nobrega FG, Tzagoloff A. COX10 codes for a protein homologous to the ORF1 product of Paracoccus denitrificans and is required for the synthesis of yeast cytochrome oxidase. J Biol Chem. 1990;265(24):14220–6. Coenen MJH, van den Heuvel LP, Ugalde C, Brinke M ten, Nijtmans LGJ, Trijbels FJM et al. Cytochrome c oxidase biogenesis in a patient with a mutation in COX10 gene. Annals of Neurology. 2004;56(4):560–4. Antonicka H. Mutations in COX10 result in a defect in mitochondrial heme A biosynthesis and account for multiple, early-onset clinical phenotypes associated with isolated COX deficiency. Hum Mol Genet. 2003;12(20):2693–702. Valnot I, von Kleist-Retzow JC, Barrientos A, Gorbatyuk M, Taanman JW, Mehaye B, et al. A mutation in the human heme A:farnesyltransferase gene (COX10) causes cytochrome c oxidase deficiency. Hum Mol Genet. 2000;9(8):1245–9. Tesarova M, Vondrackova A, Stufkova H, Veprekova L, Stranecky V, Berankova K, et al. Sideroblastic anemia associated with multisystem mitochondrial disorders. Pediatr Blood Cancer. 2019;66(4):e27591. Kohda M, Tokuzawa Y, Kishita Y, Nyuzuki H, Moriyama Y, Mizuno Y et al. A Comprehensive Genomic Analysis Reveals the Genetic Landscape of Mitochondrial Respiratory Chain Complex Deficiencies. Barsh GS, editor. PLoS Genet. 2016;12(1):e1005679. Gietz RD, Akio S. New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites. Gene. 1988;74(2):527–34. Amberg DC, Burke D, Strathern JN, Burke D. Methods in yeast genetics: a Cold Spring Harbor Laboratory course manual. 2005 ed. Cold Spring Harbor, N.Y: Cold Spring Harbor Laboratory Press; 2005. 230 p. Diekert K, de Kroon AI, Kispal G, Lill R. Isolation and subfractionation of mitochondria from the yeast Saccharomyces cerevisiae. Methods Cell Biol. 2001;65:37–51. Taanman JW, Capaldi RA. Purification of yeast cytochrome c oxidase with a subunit composition resembling the mammalian enzyme. J Biol Chem. 1992;267(31):22481–5. National Institutes of Health. ClinVar [Internet]. [cited 2024 May 30]. https://www.ncbi.nlm.nih.gov/clinvar/ . Omasits U, Ahrens CH, Müller S, Wollscheid B. Protter: interactive protein feature visualization and integration with experimental proteomic data. Bioinformatics. 2014;30(6):884–6. Tables Table 1: Known Pathological Variants of Human COX10 M1X Lethal (6) T14I/T377I Pathogenic (3) T196K/P225L Lethal (7) N204K Lethal (8) N204D Lethal (9) G288R Lethal (10) D336V/D336G Lethal (7) D336V/D339W Pathogenic (2) P420L Lethal (10) Additional Declarations No competing interests reported. Cite Share Download PDF Status: Published Journal Publication published 16 Aug, 2024 Read the published version in BMC Research Notes → Version 1 posted Editorial decision: Revision requested 10 Jul, 2024 Reviews received at journal 04 Jul, 2024 Reviewers agreed at journal 02 Jul, 2024 Reviewers invited by journal 29 Jun, 2024 Editor invited by journal 27 Jun, 2024 Editor assigned by journal 26 Jun, 2024 Submission checks completed at journal 26 Jun, 2024 First submitted to journal 24 Jun, 2024 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-4631252","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Short Report","associatedPublications":[],"authors":[{"id":325277141,"identity":"3e982548-6782-45ba-8f11-a06b4b35711f","order_by":0,"name":"Thomas-Shadi Voges","email":"","orcid":"","institution":"North Central College","correspondingAuthor":false,"prefix":"","firstName":"Thomas-Shadi","middleName":"","lastName":"Voges","suffix":""},{"id":325277142,"identity":"f8f7f636-6720-4402-9e81-c712f100cd02","order_by":1,"name":"Eun Bi Lim","email":"","orcid":"","institution":"North Central College","correspondingAuthor":false,"prefix":"","firstName":"Eun","middleName":"Bi","lastName":"Lim","suffix":""},{"id":325277143,"identity":"79afb6fe-73e7-4b82-b686-55f3f9e67279","order_by":2,"name":"Abigail MacKenzie","email":"","orcid":"","institution":"North Central College","correspondingAuthor":false,"prefix":"","firstName":"Abigail","middleName":"","lastName":"MacKenzie","suffix":""},{"id":325277144,"identity":"68472136-4247-41ba-a5e2-7fe5b33d2912","order_by":3,"name":"Kyle Mudler","email":"","orcid":"","institution":"North Central College","correspondingAuthor":false,"prefix":"","firstName":"Kyle","middleName":"","lastName":"Mudler","suffix":""},{"id":325277145,"identity":"8a0557bc-1aa7-4388-ba93-ca245e05860f","order_by":4,"name":"Rebecca DeSouza","email":"","orcid":"","institution":"North Central College","correspondingAuthor":false,"prefix":"","firstName":"Rebecca","middleName":"","lastName":"DeSouza","suffix":""},{"id":325277146,"identity":"c3235a6e-6729-41e8-88dd-c94b64682872","order_by":5,"name":"Nmesoma E. Onyejekwe","email":"","orcid":"","institution":"North Central College","correspondingAuthor":false,"prefix":"","firstName":"Nmesoma","middleName":"E.","lastName":"Onyejekwe","suffix":""},{"id":325277147,"identity":"01e453ee-9643-4a3a-bfff-3aff3919f770","order_by":6,"name":"Stephen D. Johnston","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5ElEQVRIiWNgGAWjYFCChARmCIP5AJjiA2IJYrQAFbElAGkDBjYitDBAtfAYEKdFvj3h4ePCHXV1/NI93x58qPmTz8bAfPA2Dx4tBmceJBvPPHNYQnLO2e2GM44ZWLYxsCVb49UikZAmzdt2QMLgRu42ad4GAwM2Bh4zaXxa5GeAtdQBteQ8g2rh/4ZXC8MNsBZmkBY2mC1seLWA/cLbdlhy5ow0M8kZx4wN2JjZjC3n4HNYe07iY6DD+Pklkp9JfKiRM+Bnb3544w0+hzHwJKAJMONVDgLsBwgqGQWjYBSMghEOANL9QNvThWiIAAAAAElFTkSuQmCC","orcid":"","institution":"North Central College","correspondingAuthor":true,"prefix":"","firstName":"Stephen","middleName":"D.","lastName":"Johnston","suffix":""}],"badges":[],"createdAt":"2024-06-24 15:38:20","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-4631252/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-4631252/v1","draftVersion":[],"editorialEvents":[{"content":"https://doi.org/10.1186/s13104-024-06879-5","type":"published","date":"2024-08-16T15:57:21+00:00"}],"editorialNote":"","failedWorkflow":false,"files":[{"id":60670378,"identity":"2396d90e-ff46-4d03-bbb3-7a4a7d0521ce","added_by":"auto","created_at":"2024-07-19 10:03:22","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":715753,"visible":true,"origin":"","legend":"\u003cp\u003eActivity of human \u003cem\u003eCOX10\u003c/em\u003e variants. A) Ten-fold serial dilutions of yeast cells carrying the indicated \u003cem\u003eCOX10\u003c/em\u003e variant or an empty vector were plated on rich media with either glucose or glycerol as the carbon source. B) The same strains were lysed and COX activity was measured. The resulting specific activities were normalized to the sample containing the human \u003cem\u003eCOX10\u003c/em\u003e reference sequence. Error bars show one standard deviation.\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-4631252/v1/8663d7c795e997f76816770f.jpg"},{"id":63070899,"identity":"ace20af5-e89f-4939-ae6f-d1b3e0caf8a2","added_by":"auto","created_at":"2024-08-22 19:56:11","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":1061030,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4631252/v1/4a8653b8-c33b-4e0c-8b0d-c849ca136e3c.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Phenotypic Assessment of Cox10 Variants and their Implications for Leigh Syndrome","fulltext":[{"header":"Introduction","content":"\u003cp\u003eLeigh Syndrome is a rare genetic condition characterized by progressive neuromuscular defects. This disorder is highly heterogeneous, due in part to the fact that it can be caused by alterations in at least 75 different genes. All of these genetic alterations lead to mitochondrial dysfunction including reduced or eliminated capacity for oxidative phosphorylation(\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Notably, loss of function in one of these genes, \u003cem\u003eCOX10\u003c/em\u003e, leads to a severe form of Leigh Syndrome that is typically fatal in the first year of life (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Two patients have been reported to survive longer, albeit with significant pathology(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eKnown Pathological Variants of Human COX10\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"3\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eM1X\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLethal\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003e(\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT14I/T377I\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePathogenic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT196K/P225L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLethal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e(\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eN204K\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLethal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e(\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eN204D\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLethal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e(\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eG288R\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLethal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e(\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eD336V/D336G\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLethal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e(\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eD336V/D339W\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003ePathogenic\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eP420L\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eLethal\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e(\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe Cox10 protein is highly conserved in the budding yeast \u003cem\u003eSaccharomyces cerevisiae\u003c/em\u003e; indeed, expression of the human \u003cem\u003eCOX10\u003c/em\u003e gene in yeast can fully restore function in a strain missing its endogenous \u003cem\u003eCOX10\u003c/em\u003e gene(\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). The Cox10 enzyme is located on the mitochondrial inner membrane and catalyzes the farnesylation of heme(\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e). This modified heme group is incorporated as an essential prosthetic group into the cytochrome c oxidase (COX) enzyme, which catalyzes the transfer of electrons from cytochrome c to molecular oxygen.\u003c/p\u003e \u003cp\u003eEleven different point mutations in the human \u003cem\u003eCOX10\u003c/em\u003e gene have been described in the literature as leading to disease in humans (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Of course, there are many more alleles in the human population and for the majority of these we have no direct evidence as to whether or not they encode functional proteins. As of June 17, 2024, the ClinVar database lists 102 known variants of the Cox10 protein, of which nearly three-quarters are of \u0026ldquo;uncertain significance\u0026rdquo;. We have selected 25 \u003cem\u003eCOX10\u003c/em\u003e alleles for characterization in yeast. This additional knowledge should be useful to clinicians and genetic counselors faced with patients carrying these variants.\u003c/p\u003e"},{"header":"Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003cdiv id=\"Sec4\" class=\"Section3\"\u003e \u003ch2\u003eExpression of human COX10 alleles in yeast\u003c/h2\u003e \u003cp\u003eA cDNA encoding the reference sequence of human Cox10 was synthesized. To allow for proper expression, this construct included 500 bp upstream and 100 bp downstream of the yeast \u003cem\u003eCOX10\u003c/em\u003e gene. Additionally, a myc epitope tag just before the stop codon was included along with sequences to facilitate cloning. This DNA was inserted into the \u003cem\u003eEcoRI\u003c/em\u003e site of YEplac195 (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e) for expression in yeast. The resulting plasmid was either used directly or mutated using the Q5 kit (New England Biolabs) so that it encoded the various alleles. Plasmids were sequenced to confirm successful mutagenesis before being transformed(\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e) into \u003cem\u003ecox10Δ\u003c/em\u003e yeast for phenotypic analysis.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003eGrowth Assessment\u003c/h2\u003e \u003cp\u003eStandard yeast media and growth conditions were used throughout these experiments (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Ten-fold serial dilutions of freshly grown yeast were plated onto rich media containing either 2% glucose or 3% glycerol and allowed to grow at 30\u003csup\u003eo\u003c/sup\u003eC for three days before photographing.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section3\"\u003e \u003ch2\u003eCOX assays\u003c/h2\u003e \u003cp\u003eYeast were grown overnight at 30\u003csup\u003eo\u003c/sup\u003eC with shaking in 15 mL of rich media using 3% raffinose as a carbon source. Raffinose supports growth by cells with and without functional oxidative phosphorylation but does not suppress mitochondrial production like glucose(\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). Cells were washed and lysed in 500 \u0026micro;L cold SH buffer (0.6M sorbitol, 25 mM HEPES pH 7.4) by vortexing with glass beads for five minutes. Unlysed cells were pelleted by centrifugation at 600g at 4\u003csup\u003eo\u003c/sup\u003eC for five minutes, twice. Mitochondria were pelleted by centrifugation at 16,000g at 4\u003csup\u003eo\u003c/sup\u003eC for ten minutes and were resuspended in 200 \u0026micro;L of SH buffer (\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). 20 \u0026micro;L of each sample was incubated with 125 \u0026micro;g of reduced cytochrome c in 25 mM potassium phosphate pH 6.2. Absorbance was measured at 550 nm every five seconds for one minute to calculate the rate of cytochrome c oxidation (\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Reaction rates were normalized to the amount of protein in the lysate as found by the BCA reaction. Each sample was measured at least four times.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Results and Discussion","content":"\u003cp\u003eWe selected 25 human \u003cem\u003eCOX10\u003c/em\u003e alleles whose functions are not clearly understood and expressed them in yeast lacking the endogenous \u003cem\u003eCOX10\u003c/em\u003e to determine how functional the encoded proteins are. This species grows efficiently by anaerobic metabolism and therefore can survive without functional Cox10 protein when provided with a fermentable carbon source like glucose (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, left column). However, carbon sources like glycerol can only be utilized aerobically and thus require a functional Cox10 enzyme. On this fuel source, cells lacking Cox10 do not survive but they thrive if they contain either the yeast or human reference sequence \u003cem\u003eCOX10\u003c/em\u003e. Strains with certain \u003cem\u003eCOX10\u003c/em\u003e alleles, such as S103A or V356M, survive equally as well as those with the reference sequence \u003cem\u003eCOX10\u003c/em\u003e. Other strains, such as those with I127T or Q322P, fail to grow when cultured on glycerol (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA, right column), indicating that these protein variants are nonfunctional.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWe also directly measured the COX activity in each of these yeast strains. We found that the yeast \u003cem\u003eCOX10\u003c/em\u003e supports slightly more COX activity than the human reference sequence \u003cem\u003eCOX10\u003c/em\u003e, in agreement with a previously published observation(\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e). Yeast strains with certain human \u003cem\u003eCOX10\u003c/em\u003e alleles show activities that are close to that provided by the reference sequence while others show very low activities (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB).\u003c/p\u003e \u003cp\u003eUnsurprisingly, there is a strong, general correlation between the COX activity measurements and the ability to grow on glycerol. It appears that Cox10 variants that allow approximately 50% of the reference sequence COX activity (see variants S103A, P104L and A328T) grow very efficiently on glycerol and those that allow less than 25% of the reference sequence COX activity (such as I127T and D132Y) fail to grow at all (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eAmong the 16 assayed alleles classified as \u0026ldquo;uncertain significance\u0026rdquo; by the ClinVar database(\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e), we find that six (S103A, D152Y, A174T, F209L, C343R, V356M) encode functional proteins. Four alleles are classified as having \u0026ldquo;conflicting classifications of pathogenicity\u0026rdquo;; we find that three of these alleles (P104L, A328T and R431W) encode functional proteins and one (T87I) does not. T221A is classified as \u0026ldquo;likely pathogenic\u0026rdquo; and T28I is classified as \u0026ldquo;benign/likely benign\u0026rdquo;; our data disagree with these two predictions. We believe that our \u003cem\u003ein vivo\u003c/em\u003e measurements of the activity of these alleles will provide important, actionable information for clinicians and genetic counselors who may come across patients with these alleles in their practices.\u003c/p\u003e \u003cp\u003eThe D336V and D339W alleles were selected for a slightly different reason. Pitceathly \u003cem\u003eet al\u003c/em\u003e. have reported that a human with these two alleles survived to adulthood and that both alleles encode nonfunctional proteins(\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). We thought that using more sensitive enzyme assays might reveal partial activity from one or both proteins but found that both are fully nonfunctional (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Recently, a second patient with unusual \u003cem\u003eCOX10\u003c/em\u003e alleles has been described who survived past infancy(\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e). This patient is homozygous for two alleles, T14I and T377I. The second variant is highly similar to the T380I variant that we found to be nonfunctional in that both alterations are located in the sixth transmembrane domain(\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e), strongly suggesting that the patient\u0026rsquo;s Cox10 protein will be nonfunctional. Thus, it remains unclear how either patient has survived when most people with nonfunctional Cox10 variants die in infancy (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Perhaps specific alleles of other genes found in these patients can partially suppress the loss-of-function in \u003cem\u003eCOX10\u003c/em\u003e. Identifying these genes could potentially point to interventions to treat Leigh Syndrome patients and is an area that our laboratory is pursuing.\u003c/p\u003e"},{"header":"Limitations","content":"\u003cp\u003eDespite the strong evolutionary conservation in oxidative phosphorylation mechanisms, it is possible that these variants will have a different level of activity in humans compared to yeast cells.\u003c/p\u003e"},{"header":"Abbreviations","content":"\u003cp\u003eCOX: cytochrome c oxidase\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank Fatima Alauddin, Bailey Bloom, Itzel Mancilla and Javier Valle Galisteo for their assistance in producing some alleles. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSDJ designed the study and supervised the work. \u0026nbsp;TSV, EBL, AM, KM, RD, NEO and SDJ performed the experiments and analyzed the resulting data. \u0026nbsp;All authors read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank the Office of Academic Affairs at North Central College for financial support.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe datasets used and analyzed during the current study are available from the corresponding author upon reasonable request. \u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBakare AB, Lesnefsky EJ, Iyer S. Leigh Syndrome: A Tale of Two Genomes. Front Physiol. 2021;12:693734.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003ePitceathly RDS, Taanman JW, Rahman S, Meunier B, Sadowski M, Cirak S, et al. COX10 mutations resulting in complex multisystem mitochondrial disease that remains stable into adulthood. JAMA Neurol. 2013;70(12):1556\u0026ndash;61.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTavasoli A, Kachuei M, Talebi S, Eghdami S. Complex mitochondrial disease caused by the mutation of COX10 in a toddler: a case-report study. Ann Med Surg (Lond). 2024;86(6):3753\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGlerum DM, Tzagoloff A. Isolation of a human cDNA for heme A:farnesyltransferase by functional complementation of a yeast cox10 mutant. Proc Natl Acad Sci USA. 1994;91(18):8452\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNobrega MP, Nobrega FG, Tzagoloff A. COX10 codes for a protein homologous to the ORF1 product of Paracoccus denitrificans and is required for the synthesis of yeast cytochrome oxidase. J Biol Chem. 1990;265(24):14220\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCoenen MJH, van den Heuvel LP, Ugalde C, Brinke M ten, Nijtmans LGJ, Trijbels FJM et al. Cytochrome c oxidase biogenesis in a patient with a mutation in COX10 gene. Annals of Neurology. 2004;56(4):560\u0026ndash;4.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAntonicka H. Mutations in COX10 result in a defect in mitochondrial heme A biosynthesis and account for multiple, early-onset clinical phenotypes associated with isolated COX deficiency. Hum Mol Genet. 2003;12(20):2693\u0026ndash;702.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eValnot I, von Kleist-Retzow JC, Barrientos A, Gorbatyuk M, Taanman JW, Mehaye B, et al. A mutation in the human heme A:farnesyltransferase gene (COX10) causes cytochrome c oxidase deficiency. Hum Mol Genet. 2000;9(8):1245\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTesarova M, Vondrackova A, Stufkova H, Veprekova L, Stranecky V, Berankova K, et al. Sideroblastic anemia associated with multisystem mitochondrial disorders. Pediatr Blood Cancer. 2019;66(4):e27591.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKohda M, Tokuzawa Y, Kishita Y, Nyuzuki H, Moriyama Y, Mizuno Y et al. A Comprehensive Genomic Analysis Reveals the Genetic Landscape of Mitochondrial Respiratory Chain Complex Deficiencies. Barsh GS, editor. PLoS Genet. 2016;12(1):e1005679.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGietz RD, Akio S. New yeast-Escherichia coli shuttle vectors constructed with in vitro mutagenized yeast genes lacking six-base pair restriction sites. Gene. 1988;74(2):527\u0026ndash;34.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eAmberg DC, Burke D, Strathern JN, Burke D. Methods in yeast genetics: a Cold Spring Harbor Laboratory course manual. 2005 ed. Cold Spring Harbor, N.Y: Cold Spring Harbor Laboratory Press; 2005. 230 p.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eDiekert K, de Kroon AI, Kispal G, Lill R. Isolation and subfractionation of mitochondria from the yeast Saccharomyces cerevisiae. Methods Cell Biol. 2001;65:37\u0026ndash;51.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eTaanman JW, Capaldi RA. Purification of yeast cytochrome c oxidase with a subunit composition resembling the mammalian enzyme. J Biol Chem. 1992;267(31):22481\u0026ndash;5.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eNational Institutes of Health. ClinVar [Internet]. [cited 2024 May 30]. \u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttps://www.ncbi.nlm.nih.gov/clinvar/\u003c/span\u003e\u003cspan address=\"https://www.ncbi.nlm.nih.gov/clinvar/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eOmasits U, Ahrens CH, M\u0026uuml;ller S, Wollscheid B. Protter: interactive protein feature visualization and integration with experimental proteomic data. Bioinformatics. 2014;30(6):884\u0026ndash;6.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"},{"header":"Tables","content":"\u003ctable border=\"1\" cellspacing=\"0\" cellpadding=\"0\"\u003e\n \u003ctbody\u003e\n \u003ctr\u003e\n \u003ctd width=\"100%\" colspan=\"3\" valign=\"top\"\u003e\n \u003cp\u003e\u003cstrong\u003eTable 1:\u003c/strong\u003e Known Pathological Variants of Human \u003cem\u003eCOX10\u003c/em\u003e\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"35.49488054607509%\" valign=\"top\"\u003e\n \u003cp\u003eM1X\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.01023890784983%\" valign=\"top\"\u003e\n \u003cp\u003eLethal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"35.49488054607509%\" valign=\"top\"\u003e\n \u003cp\u003e(6)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"35.49488054607509%\" valign=\"top\"\u003e\n \u003cp\u003eT14I/T377I\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.01023890784983%\" valign=\"top\"\u003e\n \u003cp\u003ePathogenic\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"35.49488054607509%\" valign=\"top\"\u003e\n \u003cp\u003e(3)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"35.49488054607509%\" valign=\"top\"\u003e\n \u003cp\u003eT196K/P225L\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.01023890784983%\" valign=\"top\"\u003e\n \u003cp\u003eLethal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"35.49488054607509%\" valign=\"top\"\u003e\n \u003cp\u003e(7)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"35.49488054607509%\" valign=\"top\"\u003e\n \u003cp\u003eN204K\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.01023890784983%\" valign=\"top\"\u003e\n \u003cp\u003eLethal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"35.49488054607509%\" valign=\"top\"\u003e\n \u003cp\u003e(8)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"35.49488054607509%\" valign=\"top\"\u003e\n \u003cp\u003eN204D\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.01023890784983%\" valign=\"top\"\u003e\n \u003cp\u003eLethal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"35.49488054607509%\" valign=\"top\"\u003e\n \u003cp\u003e(9)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"35.49488054607509%\" valign=\"top\"\u003e\n \u003cp\u003eG288R\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.01023890784983%\" valign=\"top\"\u003e\n \u003cp\u003eLethal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"35.49488054607509%\" valign=\"top\"\u003e\n \u003cp\u003e(10)\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"35.49488054607509%\" valign=\"top\"\u003e\n \u003cp\u003eD336V/D336G\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.01023890784983%\" valign=\"top\"\u003e\n \u003cp\u003eLethal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"35.49488054607509%\" valign=\"top\"\u003e\n \u003cp\u003e(7)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"35.49488054607509%\" valign=\"top\"\u003e\n \u003cp\u003eD336V/D339W\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.01023890784983%\" valign=\"top\"\u003e\n \u003cp\u003ePathogenic\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"35.49488054607509%\" valign=\"top\"\u003e\n \u003cp\u003e(2)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003ctr\u003e\n \u003ctd width=\"35.49488054607509%\" valign=\"top\"\u003e\n \u003cp\u003eP420L\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"29.01023890784983%\" valign=\"top\"\u003e\n \u003cp\u003eLethal\u003c/p\u003e\n \u003c/td\u003e\n \u003ctd width=\"35.49488054607509%\" valign=\"top\"\u003e\n \u003cp\u003e(10)\u0026nbsp;\u003c/p\u003e\n \u003c/td\u003e\n \u003c/tr\u003e\n \u003c/tbody\u003e\n\u003c/table\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"bmc-research-notes","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"resn","sideBox":"Learn more about [BMC Research Notes](http://bmcresnotes.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/resn/default.aspx","title":"BMC Research Notes","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"Saccharomyces cerevisiae, Cox10, cytochrome c oxidase, Leigh Syndrome","lastPublishedDoi":"10.21203/rs.3.rs-4631252/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4631252/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eObjectives\u003c/h2\u003e \u003cp\u003eCox10 is an enzyme required for the activity of cytochrome c oxidase. Humans who lack at least one functional copy of Cox10 have a form of Leigh Syndrome, a genetic disease that is usually fatal in infancy. As more human genomes are sequenced, new alleles are being discovered; whether or not these alleles encode functional proteins remains unclear. Thus, we set out to measure the phenotypes of many human Cox10 variants by expressing them in yeast cells.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eWe successfully expressed the reference sequence and 25 variants of human Cox10 in yeast. We quantitated the ability of these variants to support growth on nonfermentable media and directly measured cytochrome c oxidase activity. 11 of these Cox10 variants supported approximately half or more the cytochrome c oxidase activity compared to the reference sequence. All of the strains containing those 11 variants also grew robustly using a nonfermentable carbon source. Cells expressing the other variants showed low cytochrome c oxidase activity and failed to grow on nonfermentable media.\u003c/p\u003e","manuscriptTitle":"Phenotypic Assessment of Cox10 Variants and their Implications for Leigh Syndrome","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-19 10:03:18","doi":"10.21203/rs.3.rs-4631252/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2024-07-10T15:38:21+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2024-07-04T15:29:08+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"117007187107927121940348890331273656053","date":"2024-07-02T11:03:08+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-06-29T17:50:14+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"","date":"2024-06-27T11:47:00+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-06-27T02:58:42+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2024-06-27T02:58:37+00:00","index":"","fulltext":""},{"type":"submitted","content":"BMC Research Notes","date":"2024-06-24T15:37:11+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"bmc-research-notes","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"resn","sideBox":"Learn more about [BMC Research Notes](http://bmcresnotes.biomedcentral.com)","snPcode":"","submissionUrl":"https://www.editorialmanager.com/resn/default.aspx","title":"BMC Research Notes","twitterHandle":"@BMC_series","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"em","reportingPortfolio":"BMC Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c0cdc361-2d5b-46bb-96b7-01afbc44bcb6","owner":[],"postedDate":"July 19th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[],"tags":[],"updatedAt":"2024-08-22T19:29:34+00:00","versionOfRecord":{"articleIdentity":"rs-4631252","link":"https://doi.org/10.1186/s13104-024-06879-5","journal":{"identity":"bmc-research-notes","isVorOnly":false,"title":"BMC Research Notes"},"publishedOn":"2024-08-16 15:57:21","publishedOnDateReadable":"August 16th, 2024"},"versionCreatedAt":"2024-07-19 10:03:18","video":"","vorDoi":"10.1186/s13104-024-06879-5","vorDoiUrl":"https://doi.org/10.1186/s13104-024-06879-5","workflowStages":[]},"version":"v1","identity":"rs-4631252","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4631252","identity":"rs-4631252","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}